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Amino-Acid-Derived Anionic Polyacrylamides with Tailored Hydrophobicity–Physicochemical Properties and Cellular Interactions
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  • Jonas De Breuck
    Jonas De Breuck
    Macromolecular Chemistry, University of Bayreuth, Universitätsstraße 30, 95447 Bayreuth, Germany
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  • Michael Streiber
    Michael Streiber
    Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Humboldtstrasse 10, 07743 Jena, Germany
    Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, 07743 Jena, Germany
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  • Michael Ringleb
    Michael Ringleb
    Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Humboldtstrasse 10, 07743 Jena, Germany
    Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, 07743 Jena, Germany
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    Orcidhttps://orcid.org/0000-0002-7320-8529
  • Dennis Schröder
    Dennis Schröder
    Macromolecular Chemistry, University of Bayreuth, Universitätsstraße 30, 95447 Bayreuth, Germany
    Bavarian Polymer Institute, Universitätsstraße 30, 95447 Bayreuth, Germany
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  • Natascha Herzog
    Natascha Herzog
    Macromolecular Chemistry, University of Bayreuth, Universitätsstraße 30, 95447 Bayreuth, Germany
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  • Ulrich S. Schubert
    Ulrich S. Schubert
    Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Humboldtstrasse 10, 07743 Jena, Germany
    Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, 07743 Jena, Germany
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    Orcidhttps://orcid.org/0000-0003-4978-4670
  • Stefan Zechel
    Stefan Zechel
    Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Humboldtstrasse 10, 07743 Jena, Germany
    Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, 07743 Jena, Germany
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  • Anja Traeger
    Anja Traeger
    Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Humboldtstrasse 10, 07743 Jena, Germany
    Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, 07743 Jena, Germany
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    Orcidhttps://orcid.org/0000-0001-7734-2293
  • Meike N. Leiske*
    Meike N. Leiske
    Macromolecular Chemistry, University of Bayreuth, Universitätsstraße 30, 95447 Bayreuth, Germany
    Bavarian Polymer Institute, Universitätsstraße 30, 95447 Bayreuth, Germany
    *mail: [email protected]
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    Orcidhttps://orcid.org/0000-0002-8525-2324
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ACS Polymers Au

Cite this: ACS Polym. Au 2024, 4, 3, 222–234
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https://doi.org/10.1021/acspolymersau.3c00048
Published March 12, 2024

Copyright © 2024 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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Polyanions can internalize into cells via endocytosis without any cell disruption and are therefore interesting materials for biomedical applications. In this study, amino-acid-derived polyanions with different alkyl side-chains are synthesized via postpolymerization modification of poly(pentafluorophenyl acrylate), which is synthesized via reversible addition–fragmentation chain-transfer (RAFT) polymerization, to obtain polyanions with tailored hydrophobicity and alkyl branching. The success of the reaction is verified by size-exclusion chromatography, NMR spectroscopy, and infrared spectroscopy. The hydrophobicity, surface charge, and pH dependence are investigated in detail by titrations, high-performance liquid chromatography, and partition coefficient measurements. Remarkably, the determined pKa-values for all synthesized polyanions are very similar to those of poly(acrylic acid) (pKa = 4.5), despite detectable differences in hydrophobicity. Interactions between amino-acid-derived polyanions with L929 fibroblasts reveal very slow cell association as well as accumulation of polymers in the cell membrane. Notably, the more hydrophobic amino-acid-derived polyanions show higher cell association. Our results emphasize the importance of macromolecular engineering toward ideal charge and hydrophobicity for polymer association with cell membranes and internalization. This study further highlights the potential of amino-acid-derived polymers and the diversity they provide for tailoring properties toward drug delivery applications.

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1. Introduction

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During the last decades, the application of (polymeric) nanocarriers became an essential asset for drug delivery, therapeutic, and diagnostic systems. (1) To increase the therapeutic efficiency, it is important not only to repress undesired interactions with cells and tissue but also to induce a targetability to specific cells. (2) Recently, significant effort was made to incorporate tissue-targeting motifs into polymeric nanocarriers to increase their specificity. (3−6) Their performance was shown to highly depend on several physiochemical properties of the nanocarriers, such as its shape, size, charge, and hydrophobicity. (7)
The intracellular delivery of therapeutic biomacromolecules such as nucleic acids or peptides is of critical importance for their therapeutic efficiency. Owing to their size, charge, and inherent instability, biomacromolecules often need to be encapsulated for more stability and enhanced cellular uptake via endocytic pathways for intracellular delivery of drugs as they are impermeable to the cell membrane. After membrane engulfment, referred to as endocytosis, a rapid release of the therapeutics (i.e., endosomal escape) is required to minimize degradation and maximize therapeutic efficiency. (8) Over the years, various pH-responsive materials have been investigated regarding their endosomal escape property. pH-responsive polymers are protonated during endosomal maturation, as the pH value of the extracellular physiological environment (bloodstream: pH = 7.4) differs from the endosomal pH value (5.0–6.8). Biocompatible, biodegradable, and amphiphilic polypeptides have shown a pH-dependent membrane-permeabilizing capability due to their pH-responsive amines or carboxylic acid groups. (9) Besides, synthetic alternatives, which are often more cost-effective compared with peptides, have attracted attention in the past. (10−12) pH-responsive polymers are polyelectrolytes and can carry anionic (polyanions) or cationic (polycation) dissociable groups. Polycations (i.e., poly(ethylene imine)s) are known for their buffer capacity and protonation inside endosomal compartments, which is linked to its potential to cross these after acidification resulting in osmotic swelling, also discussed as the proton sponge effect. (13) Furthermore, strong interactions with the endolysosomal membrane have been described to facilitate the release of therapeutics by pore formation. (14) Here, a slightly acidic pKa-value of the polymeric amine is desirable in order to promote endosomal membrane interaction under acidic conditions, but at the same time to minimize interactions with the cytoplasmic membrane to improve the compatibility of the polycations. (10) This high membrane interaction further renders them suitable for applications as antimicrobial polymers. (15,16)
In contrast, polyanions are often formed of polycarboxylates, that are negatively charged at the physiological pH value of blood (pH = 7.4) and, consequently, do not penetrate negatively charged cell membranes due to repulsive interactions. (8) However, their pH-induced protonation and loss/reduction of anionic character can cause endosomal membrane disruption. To this end, the pH response of polyanions (i.e., the pKa-value) often takes place at physiologically relevant pH values (i.e., pH = 4–6), (8) making them an attractive alternative to polycations, which often feature a higher pKa-value outside the physiological range (i.e., pH = 7–9). (17) Below the pKa, the increased membrane interaction of polyanions is attributed to less ionic repulsion between polymers and the cell membrane, which is also facilitated by their increased hydrophobicity in the nonionic state. Consequently, the pH-dependent endosomolytic activity of polyanions is generally considered less harmful, as they are unlikely to be internalized into cells via translocation of the synthetic bilayer, which often causes cell disruption. (18) When lipid membranes are faced with polyelectrolytes, which also contain hydrophobic groups, these are often incorporated into the bilayer instead of self-association. (12) Here, the additional hydrophobic forces were shown to be critical to enable the binding of anionic polymer to neutral and even negatively charged lipid bilayers. (19) Conversely, it was previously shown that excessive polymer hydrophobicity can lead to cell toxicity at a neutral pH value. (20) This is probably caused by the globule configuration adopted by the polymer in the solution. While polyanions seem to be promising materials for intracellular drug delivery, little is known about the potential to manipulate these polymers and they need to be evaluated more carefully.
It was already shown that polyelectrolytes with different polymer backbones (e.g., acrylate, methacrylate, acrylamide, methacrylamide) incorporating large hydrophobic moieties as well as using branched structures increased the interaction with lipid membranes. (21−23) However, to optimize the performance of polyanions and particularly polycarboxylates, their physicochemical properties should be molecularly engineered by the development of tailored, functional polymers with an optimal hydrophilic–lipophilic balance (HLB). Investigations regarding protonation and hydrophobicity of polyanions with tailored structures are needed for the identification of structure–property relationships.
Functional polymers are often prepared from the respective monomers. (24−26) However, if the synthesis of a target polymer to a certain molar mass with narrow dispersity is hampered and/or its corresponding monomer is difficult if not impossible to synthesize, postpolymerization modification (PPM) of reactive prepolymers is the method of choice. (27,28) Some examples are radical thiol–ene, (29) nucleophilic thiol–ene modification, (30) and aminolysis modification with functional polymers. (31,32)
In this context, polymers with activated ester side chains, such as N-acryloxysuccinimide (NAS) and pentafluorophenyl acrylate (PFPA), are widely used. Reports have shown that activated ester polymers such as poly(N-acryloxysuccinimide) (PNAS) and poly(pentafluorophenyl) acrylate (PPFPA) react fast and quantitively with primary or secondary amines resulting in the corresponding poly(acrylamide) (P(AAm)) derivatives. (33,34) While among activated ester monomers, NAS is the oldest and the most regularly employed one, (35,36) polymers derived from PFPA commonly feature a higher hydrolytic stability and enhanced solubility in a wider range of organic solvents. (34) Previously, PNAS and PPFPA were already employed to synthesize various polymer architectures, such as activated polymer brushes (32,37) and nano(hydro)gels. (38) The modification of activated ester polymers with cross-linking bis-amines, (39) amino-modified chromophores, (40,41) amino-sugars, (31) and alkylated amines (42) has been shown. They have further been modified with small peptides. (43−46) However, the modification with amino acids is less explored.
Instead, vinylic polymers with amino acids in the side chains were often retrieved by radical polymerization. (47) Side-chain amino-acid-functionalized poly(meth)acrylates (P(M)A) or poly(meth)acrylamides (P(M)AAm) were obtained from esterified amino acids. The esterification was performed by the Steglich esterification reaction of 2-hydroxyethyl (meth)acrylates or (meth)acrylamides, respectively, with the carboxylic acid moiety of the α-amine-protected amino acid, yielding cationic or zwitterionic polymers. (17,21,48−50) Alternatively, side-chain amino-acid-functionalized P(M)A or P(M)AAm was previously synthesized from the amino-acid-derived vinyl monomers. In general, the monomers were synthesized by the reaction of the carboxylic acid or the amine group of the (protected) amino acid with an activated vinyl compound (e.g., (meth)acryloyl chloride). (3,51,52)
The aim of this study was to synthesize various amino-acid-derived anionic polyacrylamides (P(AA–OH-AAm)) with carboxylic acids in the side chain and tailored hydrophilicity. These polyanions were obtained by the modification of the same PPFPA precursor to obtain fast and quantitative formation of P(AA–OH-AAm). Modification with different amino acids was performed to investigate the correlation between the alkyl side-chain (length and branching) and the physicochemical properties of the polymer. Additionally, we expected the synthesized polyanions to show low-fouling properties due to their peptide-like moieties, enabling applications in the field of protein interactions and delivery materials. Therefore, an investigation was conducted to assess whether different physicochemical properties of the synthesized P(AA–OH-AAm) influence their interactions with proteins and cells to get an initial understanding of their potential applications in a biological environment.

2. Results and Discussion

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2.1. Synthesis and Characterization of Amino-Acid-Derived Polyanions

Neutral α-amino acids with different alkyl substituents in α-position served as starting material for the development of P(AA–OH-AAm) with tailored hydrophilicity. The screened P(AA–OH-AAm) were derived from glycine (Gly), alanine (Ala), aminobutyric acid (Abu), norvaline (Nva), valine (Val), iso-leucine (Ile), and leucine (Leu), which were all used in their L-stereospecific form. This library allowed for a comparison of the effect of increasing hydrophobicity (Gly < Ala < Abu < Nva) as well as the effect of side-chain branching (Scheme 1). To ensure high comparability of these polymers and eliminate effects caused by polymer length and dispersity, it was chosen to prepare them via a PPM approach.

Scheme 1

Scheme 1. Schematic Representation of the Synthesis of Carboxylated Polyacrylamides with Tailored Hydrophobicity via PPM of PPFPA with Indicated Amino Acids, Yielding Anionic Polymers with Varying Alkyl Substituentsa
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aPlease note that Gly does not feature a stereo center.

P(AA–OH-AAm) were synthesized by the active ester approach employing the polymerization of PFPA according to Zerdan et al. (53) Reversible addition–fragmentation chain-transfer (RAFT) polymerization proceeded in a controlled manner using a monomer to chain-transfer agent (CTA) ratio of 257 yielding PPFPA (Mn,app(PPFPA) = 33,900 g mol–1) with a narrow dispersity (Đ = 1.32) according to size-exclusion chromatography (SEC) measurements (Table 1 and Figure S1). The proton nuclear magnetic resonance (1H NMR) spectrum of PPFPA showed characteristic signals from the backbone protons around δ = 2.0 and 3.0 ppm (Figure 1B). Complementary 19F NMR analysis (Figure S2A) showed the expected three signals at δ = −153, – 157, and −163 ppm, which were attributed to the PFP group. The absence of hydrolytic poly(acrylic acid) (PAA) byproducts was confirmed by 13C NMR spectroscopy (Figure S2B). Here, only one type of carbonyl signal was present at δ = 170 ppm, suggesting the absence of hydrolyzed PAA groups in the polymer, which would generally reveal a second carbonyl signal around δ = 180 ppm. In addition, FTIR spectroscopy (Figure S5B) did not show the characteristic PAA band at v~ = 1738 cm–1. (54)

Figure 1

Figure 1. (A) FTIR spectra and (B) 1H NMR spectra (300 MHz) of poly(pentafluorophenyl acrylate) (green, CDCl3) and P(Nva-OH-AAm) (blue, d-DPBS).

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PPM of PPFPA was performed by aminolysis with different amino acids comprising varying alkyl substituents in R-position (Scheme 1). A particular challenge was the poor solubility of amino acids in organic solvents. (55,56) Eventually, dimethylformamide (DMF) was chosen as it is regularly employed for PPM of PPFPA, (34) and was found to suspend all amino acids used in this study well. It was further assumed that DMF slightly dissolves amino acids at elevated temperatures. (57) The reaction was performed at 40 °C for 72 h to allow adequate solvation and consumption of the suspended amino acids, leading to a continuous reaction. Hence, PPM of homopolymers with Gly, Ala, Abu, Nva, Val, Ile, and Leu proceeded with the quantitative conversion of the activated ester functional groups yielding the corresponding polyacrylamides P(Gly-OH-AAm), P(Ala-OH-AAm), P(Abu–OH-AAm), P(Nva-OH-AAm), P(Val-OH-AAm), P(Ile-OH-AAm), and P(Leu-OH-AAm), respectively. The success of each synthetic step was verified via 1H-, 19F-NMR, and FTIR spectroscopy.
1H NMR spectra of all amino-acid-modified polymers (Figure S3), and exemplarily P(Nva-OH-AAm) in Figure 1B, showed the appearance of a new signal at δ = 4.0 ppm attributed to the C–H adjacent to the newly formed amide bond. Moreover, predominant peaks of CH3-groups appeared in the spectra (δ ≈ 0.0–2.0 ppm) of amino-acid-derived polymers with long linear alkyl substituents (i.e., Nva) or branched alkyl substituents (i.e., Val, Ile, and Leu). The absence of any peak in 19F NMR spectra (Figure S4) of the AA-side-chain polymer indicated a full conversion of active esters in all PPM reactions.
Additionally, as an example, the FTIR spectra of PPFPA and (P(Nva-OH-AAm) are shown in Figure 1A while the other FTIR spectra are depicted in Figure S5A. Here, the conversion of functional groups is also shown by a shift in absorption band from a carbonyl adjacent to PFPP at v~ = 1780 cm–1 to a carbonyl group in an amide bond at v~ = 1550 cm–1. (58) The presence of the carboxylic acid functional group in the polyanions can also be distinguished by its C═O symmetric stretch at v~ = 1410 cm–1. The absence of PAA groups, which may occur as a hydrolytic byproduct, was further suggested by the absence of the characteristic band at v~ = 1738 cm–1 (Figure S5B). (54)
SEC measurements (Figure 2 and Table 1) in 0.07 M aqueous Na2HPO4 as eluent were performed to verify the integrity of the polymers during the PPM process.

Figure 2

Figure 2. Size-exclusion chromatograms (0.07 M aq. Na2HPO4) of P(AA–OH-Aam). (A) Effect of increasing alkyl chain length. (B) Effect of alkyl branching. (C) Effect of alkyl branching position.

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Table 1. Properties of PPFPA and Different Amino-Acid-Functionalized Polymers
polymer nameMa [g mol–1]rel. 6-AF by emissionbMn,appc [g mol–1]ĐcpKadZPe [mV]
PPFPA238.11n.a.33,900f1.32fn.a.n.a.
P(Gly-OH-AAm)129.121.0011,8001.735.0–26.6 ± 1.9
P(Ala-OH-AAm)143.141.9416,3001.795.5–41.9 ± 1.0
P(Abu–OH-AAm)157.171.0622,2001.545.1–37.8 ± 2.3
P(Nva-OH-AAm)171.22.3913,8002.775.0–31.1 ± 3.3
P(Val-OH-AAm)171.21.9422,8001.434.8–38.4 ± 2.2
P(Ile-OH-AAm)185.221.2824,9001.755.0–31.6 ± 1.9
P(Leu-OH-AAm)185.221.2319,6001.485.1–37.1 ± 2.2
a

Molar mass of repeating unit (RU).

b

Fluorescence measurements (λex = 450 nm, λem = 517 nm) in DPBS (1.0 mg mL–1). Values correspond to 6AF-labeled polymers.

c

SEC in 0.07 M aq. Na2HPO4 (standard: PMA Na salt).

d

Titration of a 5 mg mL–1 polymer solution with 0.1 M HCl. pKa-value refers to carboxylic acid function.

e

ELS measurements in DPBS (2.0 mg mL–1).

f

SEC in DMAc + 0.21 w% LiCl (standard: PMMA). n.a. not applicable.

The results revealed an increased Đ (Table 1), potentially related to the more polar eluent and high density of charged groups in the polymers which can cause hydrogen bonding and ionic interactions with the stationary phase leading to broader peaks and longer retention times. (59) Furthermore, the slight high-molar mass shoulder was visible in some polymers, indicating minor chain coupling, which may occur during aminolysis. It was observed that P(AA–OH-AAm) with increasing alkyl chain show shorter elution times (Figure 2A) as can be expected due to the higher molar mass and hydrodynamic volume. Interestingly, branching and the position of branching points also influenced the elution time due to altered sterical hindrance. Figure 2B shows that Val-functionalized polymers (branched alkyl substituent) feature a later elution time, meaning smaller size, compared with Nva-functionalized polymers (linear alkyl substituent). We attribute this apparent smaller size of P(Val-OH-AAm) to their decreased sterical hindrance and more compact alignment of polymers with short-chain branching. A similar trend was observed for polymers modified with P(Ile-OH-AAm) (long-chain branching) and P(Leu-OH-AAm) (terminal short-chain branching) as shown in Figure 2C.
To allow for more in-depth materials characterization as well as biological evaluation, fluorescently labeled P(AA–OH-AAm) were further prepared. Here, a controlled number of fluorescent dye molecules, namely, 6-aminofluorescein (6-AF), was incorporated into the PPFPA prepolymer side chain prior to PPM with amino acids. This method allowed to achieve a similar labeling efficiency of the polymers as was determined by fluorescence spectroscopy measurements (Table 1). The resulting fluorescently labeled P(AA–OH-AAm) will further be referred to as P(AA–OH-AAm)-6AF. The successful dye labeling was verified by the overlay of SEC traces obtained by the RI and the UV detector (λ = 490 nm; Figure S9). The absence of other signals of the UV trace indicated the absence of unbound dye.

2.2. pH-Responsiveness and Hydrophobicity

Next, the physiochemical properties and in vitro properties of the homopolymers were characterized to investigate the influence of the length and branching of alkyl groups in the side chain of polycarboxylates. Acid–base titration was utilized to gain information about the pKa-values of P(AA–OH-AAm) polymers (Figures S6 and S7). It was found that all P(AA–OH-AAm) possessed pKa-values between pH = 4.8 and 5.5 attributed to the carboxylic acid in the side chain while some also showed less pronounced pKa-value around pH ≈ 8, which might be caused by intramolecular interactions with the amide function. The pKa-values of the carboxylic acids are similar to the pKa-value of poly(acrylic acid) (pKa = 4.5). (60) An effect of the alkyl substituent was not observed. ζ Potential measurements of P(AA–OH-AAm) in DPBS (pH = 7.4) verified the presence of a negative surface charge at the physiological pH of the bloodstream (Figure S8A and Table S2). Dynamic light scattering (DLS) measurements in DPBS were performed to exclude aggregation, which could lead to an altered uptake profile. At a polymer concentration of 2 mg mL–1, all P(AA–OH-AAm) revealed a number size distribution of around 10 nm diameter (Figure S8B and Table S1), indicating the absence of larger polymer aggregates in buffered systems.
To assess the extent of influence of the alkyl substituent on the hydrophilicity of P(AA–OH-AAm), high-performance liquid chromatography (HPLC) measurements of all P(AA–OH-AAm)-6AF were performed on a reversed-phase column with an acetonitrile/water gradient. The aqueous phase was acidified with 0.1v% trifluoroacetic acid (TFA) to ensure protonation of the carboxylic acid groups. The polymers showed different retention times related to the hydrophobicity of the alkyl substituent in the side chain (Figure 3A). It was observed that with increasing linear alkyl substituent (Gly < Ala < Abu < Nva), the retention time increased due to the stronger interaction with the column confirming increased hydrophobicity. However, it was also observed that side-chain alkyl branching (i.e., Nva and Val, Ile and Leu) did not affect the hydrophobicity of P(AA–OH-Aam).

Figure 3

Figure 3. Hydrophilicity of P(AA–OH-AAm). (A) HPLC chromatograms (acetonitrile/water gradient, 0.1v% TFA). Spectra show the absolute fluorescence intensity of eluting 6AF-labeled polymers (λex = 480 nm; λem = 520 nm). (B) pH-dependent relative hydrophilicity of 6AF-labeled polymers. Columns show the reduction of fluorescence of a 0.1 mg mL–1 aqueous solution at the indicated pH value after treatment with CHCl3 compared with the fluorescence of the sample prior to treatment with CHCl3. For normalized fluorescence graphs, please refer to Figures S12 and S13.

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These results were further supported by partition coefficient (PC) measurements of P(AA–OH-AAm)-6AF at pH = 7 (Figure 3B), where the decrease of fluorescence of an aqueous polymer solution upon treatment with a hydrophobic CHCl3 phase was quantified by fluorescence spectroscopy measurements. Here, the fluorescence reduction followed the same increasing trend with increasing alkyl chain while revealing similar values for structural isomers. Additionally, the PC of P(AA–OH-AAm) was determined at pH = 4 to gain information about the increase in hydrophobicity at lower pH values. The results in Figure 3B confirmed the expected increased hydrophobicity of carboxylated polymers at pH = 4 compared with pH = 7. In addition, the increase was found to be more drastic for polymers that contained more hydrophobic amino acids in the side chain.

2.3. Protein Fouling of Amino-Acid-Derived Polyanions

To gain important information about the protein fouling of P(AA–OH-AAm), we studied the interaction of two representatives, namely, P(Gly-OH-AAm) and P(Nva-OH-AAm), with bovine serum albumin (BSA) and lysozyme. Here, BSA was chosen as a negatively charged model protein, while lysozyme represented a positively charged model protein. (61) Fouling was analyzed by DLS measurements (Figures 4 and S14 and Table S3), which provided information about the formation of aggregates over time. Owing to the negative charge of P(AA–OH-AAm), a stronger interaction with lysozyme than with BSA was expected. Indeed, mixtures of BSA and P(Nva-OH-AAm) only revealed a minor formation of aggregates, as could be concluded from stable particle diameters and count rates (Figure 4). The aggregation of P(Gly-OH-AAm) with BSA appeared to be slightly increased, which might be caused by increased exposure of the hydrophobic polymer backbone. However, mixtures of P(Nva-OH-AAm) and lysozyme indicated the formation of large nanoparticles (d ≈ 700 nm) over the course of 4 h, which remained stable up to 24 h. The formation of aggregates was further suggested by a decrease of the mean count rate over time. Lysozyme interacts as well with P(Gly-OH-AAm), leading to, however, smaller aggregates (d ≈ 150 nm). Thus, the interaction with proteins depends on both, the protein and polymer characteristics, showing stronger interactions with cationic proteins. However, polyanions, in particular sulfated and carboxylated polysaccharides, are known for their good modulation of blood coagulation. (62)

Figure 4

Figure 4. Interaction of P(Nva-OH-AAm) with BSA and Lysozyme. P(Nva-OH-AAm) (c = 0.5 mg mL–1) and indicated proteins (c = 0.5 mg mL–1) were incubated in DPBS at 37 °C for indicated durations. Number mean diameter (black) and mean count rate (gray) were analyzed by DLS measurements at 37 °C. Values (scatter) represent the mean and SD of five measurements with three runs each. Dashed lines do not represent measured values.

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2.4. Interaction of Amino-Acid-Derived Polyanions with Cells

Following the in-depth examination of the obtained polymers, they were tested for their properties in biological systems. As such, the first test should provide information about their cytocompatibility. The cytocompatibility of all P(AA–OH-AAm) was studied by an MTT assay (Figure S15), showing that all polymers were well tolerated by L929 mouse fibroblasts at concentrations up to 0.1 mg mL–1 (for 24 h). Additionally, no hemolytic activity at pH 7.4 or 6 and no erythrocyte aggregation were detected at concentrations per repeating unit (cRU) = 1 mM (Figures S16 and S19).
Previously, Duvall and co-workers have screened a library of copolymers with acrylic acid moieties and different hydrophobic alkyl acrylamide units of varying ratios aiming to find an ideal ionic–hydrophobic balance for the modification of cell membranes. (63) Their copolymers further showed some pH-dependent activity. In contrast, the amino-acid-derived anionic polymers of the current study did not reveal any hemolytic activity or erythrocyte aggregation (Figures S16 and S19), emphasizing a favorable balance between anionic character and hydrophobicity. These results further indicated that no membrane disruption and, consequently, no harm to the cells were expected for subsequent cell association experiments.
In previous studies, carboxylated polymers have already shown unique cell association properties. For example, Kempe and co-workers as well as Thayumanavan and colleagues reported the passive diffusion of Cy5-labeled anionic polymers into cells, resulting in mitochondrial colocalization. (7,64) These previous studies also highlighted the importance of the dye for the phenomenon. To avoid passive diffusion, 6AF-labeled polymers were used in the current study. In a preliminary experiment (Figure S20), different concentrations were investigated, in which cRU = 1 mM (cpolymer = 4 μM) was determined to be the optimal concentration for subsequent more detailed investigations.
Next, the time-dependent association of polymers with L929 mouse fibroblasts was studied by flow cytometry. It was found that for all seven P(AA–OH-AAm)-6AF, both the mean fluorescence intensity (MFI) and the number of 6AF-positive cells increased over time (Figure 5A,B). Here, polymers with more hydrophobic amino acids (i.e., Nva, Val, Ile, and Leu) showed high cell association with ≥70% 6AF-positive cells after 1 h of incubation. Polymers with more hydrophilic amino acids (i.e., Gly, Ala, and Abu) associated slower to L929 mouse fibroblasts while following an association trend, which is opposite to their hydrophobicity (Gly > Ala > Abu). Based on the results of the protein fouling, one can assume that P(Gly-OH-AAm) revealed some hydrophobic character due to the accessibility of the hydrophobic polymer backbone. Consequently, the polymers follow different association pathways.

Figure 5

Figure 5. Cellular association of different P(AA–OH-AAm)-6AF determined via flow cytometry measurements. Polymer concentration: 1 mMRU, 50,000 cells seeded in 250 μL in a 48 well plate. Normalized MFI was calculated by eq 3. (A, B) Association of polymers with L929 mouse fibroblasts in DMEM supplemented with 10% FBS. Incubation for indicated time points at 37 °C. (C) Incubation at 37 or 4 °C in DMEM supplemented with 10% FBS for 4 h. Reduction was calculated by eq 4. Statistical analysis (one-way ANOVA with Tukey test) was performed for 4 h incubation time (A, B) to compare the association efficiency of the different P(AA–OH-AAm)-6AF. For temperature-dependent experiments (C), the statistical analysis compared the association at 37 °C versus 4 °C per polymer type. *Indicates a significance of p < 0.5. All other comparisons were not statistically significant at p < 0.5.

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Several studies have shown that polymer association or uptake which follows an active pathway, such as endocytosis or macropinocytosis, can be suppressed by lowering the incubation temperature from 37 to 4 °C. Surprisingly, neither the number of 6AF-positive cells nor the MFI dropped significantly when the cells were incubated at a lower temperature (Figure 5C), suggesting the absence of active polymer uptake.
To further investigate the interaction between P(AA–OH-AAm) with cells, L929 fibroblasts were incubated with P(Nva-OH-AAm)-6AF and imaged via confocal laser scanning microscopy (CLSM). We specifically chose this polymer based on its hydrophilic–lipophilic balance (HLB) of 10.05, which was calculated after Griffin. (65) In contrast, P(Gly-OH-AAm) possessed an HLB value of 12.83 and could consequently be considered a detergent. A previous study has found that a series of Triton X, commonly applied membrane-permeabilizing agents, possessed altered membrane-disrupting activity depending on their HLB value, which revealed a maximum at an HLB of 12–13 (Triton X-100), while at an HLB of 10, the activity was negligibly low. (66) Other earlier studies have further described the potential to remove phospholipids from cell membranes by anionic detergents such as Triton X-100 or sodium dodecyl sulfate. (67) In particular, the solubilization process relies on the formation of mixed micelles of the detergent and the phospholipids. (68,69) These properties were further suggested after testing the emulsifying properties of P(AA–OH-AAm) with linear alkyl substituents (see the SI for further information).
After 4 h of incubation, a strong colocalization of P(Nva-OH-AAm)-6AF with the cell membrane was observed and no intracellular 6AF signal was detectable (Figure 6). These imaging results provided further clarification on the temperature study as polymer association to cell membranes is mostly a passive process.

Figure 6

Figure 6. Cellular uptake of P(Nva-OH-AAm)-6AF into L929 mouse fibroblasts visualized by CLSM. Cells were incubated with polymers at 37 °C for indicated times. Cyan: Hoechst 33342/nucleus. Red: CellMask Plasma Membrane Stain/membrane. Blue: 6AF/polymer.

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To further verify the compatibility and uptake mechanism, L929 mouse fibroblasts were incubated with P(Nva-OH-AAm)-6AF for 24 h. Subsequently, intracellular spot-like fluorescence signals of 6AF were visible, indicating a vesicular localization and, consequently, active albeit very slow polymer internalization. Acute cell death was not observed. Previous studies showed an intracellular mitochondrial colocalization of carboxylated Cy5-labeled polymers after 4 h (passive diffusion). (7) In addition, our previous reports revealed a lysosomal colocalization of glutamic-acid-derived polymers after 4 h (active uptake). (49) However, in the current study, the polymer uptake appeared to be slower compared with these previous reports. In particular, the slow and strong accumulation of anionic polymers at the cell membrane appears to be a rare occurrence in literature. We assume that the combination of anionic and hydrophobic features in our polymers favors this accumulation.
L929 fibroblasts were used as a well-studied eukaryotic model cell line. As such, the cell membrane consists of a phospholipid bilayer, which is characterized by its lipophilic center and anionic outer lipid head groups. Hydrophobic, nonionic polymers are known to interact strongly with these membranes, (70) while anionic polymers generally show very low association. (8) It is assumed that the use of anionic, hydrophobic polymers which may possess both properties leads to the phenomenon of slow membrane integration. In addition, the increasing alkyl chain length inversely correlates with the charge density and, thus, electrostatic repulsion further influencing its accessibility. Therefore, it is important to tune the length of the alkyl chain to find an optimum between hydrophobicity and charge density. Furthermore, we assume that the biomimicking motifs in our amino-acid-derived polymers contributed to the incorporation into the cell membrane. Future studies will focus on a detailed understanding of the cell association of anionic, amino-acid-derived polymers as well as their exploitation for cell-membrane modifications.

3. Conclusions

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P(AA–OH-AAm) with different alkyl substituents were successfully synthesized via PPM of PPFPA which was verified via 1H-, 19F-NMR, and FTIR spectroscopy. Characterization of the physicochemical properties revealed that the alkyl chain did not show any relation between the side chain and the pKa-value of the carboxylic acid as all were in a similar range of poly(acrylic acid). This emphasizes the complicated nature of the relationship between hydrophobicity and protonation of polyelectrolytes and shows that there are no simple correlations in this complex interplay. ζ-Potential and DLS measurements in DPBS confirmed that the surface charge and no self-assembly occurred. HPLC measurements confirmed the hypothesis that the alkyl chain can tailor the side chain hydrophobicity resulting in increased hydrophobicity for longer alkyl chains. Interestingly, branching in the alkyl chain did not affect the hydrophobicity of P(AA–OH-AAm). The results were supported by PC measurements at pH = 7. Additionally, PC measurement at pH = 4 confirmed the expected increase in hydrophobicity where it was found more drastic for the more hydrophobic P(AA–OH-AAm). All P(AA–OH-AAm) were tolerated by cells up to concentrations of 0.1 mg mL–1. Time-dependent association with L929 fibroblast revealed higher association of more hydrophobic P(AA–OH-AAm), namely, P(Nva-OH-AAm), P(Val-OH-AAm), P(Ile-OH-AAm), and P(Leu-OH-AAm). CLSM measurements revealed that P(Nva-OH-AAm) accumulated at the membrane and was internalized very slowly. Further studies will aim for a more detailed understanding of the cell association of anionic P(AA–OH-AAm) and their molecular engineering to tailor the pH response and membrane interactions further.

4. Experimental Part

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4.1. Materials and Instrumentation

4.1.1. Materials

Pentafluorophenyl acrylate (PFPA) was purchased from abcr with 95% purity. 2-(Dodecylthiocarbonothioylthio)-2-methylpropionic acid (DTCTTMPA) (98% purity), Triton X-100, and MEHQ inhibitor remover were purchased from Sigma-Aldrich. The initiator 2,2′-azobis(2,4-dimethylvaleronitril) (V65) was purchased from Wako Chemicals with 98% purity. Acetonitrile was purchased from Fisher Scientific as an extra dry solvent with a purity of 99.9%. THF (stabilized with 0.025% butylated hydroxytoluene) for dialysis was purchased from Fisher Scientific in analytical reagent grade. Dimethylformamide (DMF) was purchased from Fisher Scientific. Amino acids were purchased from Carbolution. 6-Aminofluoresceine (6-AF) was obtained from BLDPharm. Thiazolyl blue tetrazolium bromide (MTT) was obtained from TCI Chemicals. Dulbecco's phosphate buffered saline (DPBS) was purchased from VWR. Dulbecco’s Modified Eagle Medium (DMEM) Low Glucose, Fetal Bovine Serum Advanced, Penicillin/Streptomycin (Pen/Strep), and Dulbecco’s PBS were purchased from Capricorn Scientific. Polyethylene imine, branched, Mw 10,000 (bPEI 10,000) was purchased from Polysciences Inc. Bovine serum albumin (BSA) and lysozyme were obtained as lyophilized powders from VWR.

4.1.2. Proton Nuclear Resonance (1H NMR) and Fluorine Nuclear Resonance (19F-NMR) Spectroscopy

1H- and 19F-NMR spectra of PPFPA were recorded on a Bruker Avance Neo Nanobay (300 MHz) spectrometer equipped with a 1H, 13C, 19F, and 31P-BBO probe at room temperature. 13C-NMR spectra of PPFPA were recorded on a Bruker Avance IV (NEO) (500 MHz) spectrometer equipped with a 1H, 13C, and 19F probe at room temperature. Chemical shifts are given in parts per million (ppm or δ-scale) relative to deuterated chloroform or water as indicated in the spectra. Amino-acid-modified polymers were analyzed from deuterated DPBS (d-DPBS), which was prepared by lyophilization of commercial DPBS and subsequent addition of D2O at an equal volume.
1H- and 19F-NMR spectra of amino-acid-derived polyacrylamides were recorded on a Bruker Avance 300 (300 MHz) spectrometer equipped with a BACS-120 autosampler and a 1H, 13C, 19F, and 31P-BBO probe at room temperature.

4.1.3. Size-Exclusion Chromatography (SEC)

SEC measurements of PPFPA were performed on the following setup: Agilent 1200 series, PSS (degasser), G1310A (pump), G1329A (autosampler), Techlab (oven), G7162A (RI detector), G1315D (diode array detector), and PSS GRAM 30 Å guard column and PSS GRAM 1000 Å (10 μm particle size) (column set), dimethylacetamide (DMAc) with 0.21 w% of lithium chloride as additive as eluent at 1 mL min–1 at 40 °C, poly(methyl methacrylate) (PMMA) (standard).
Aqueous SEC measurement was performed on an instrument consisting of a column set with a Suprema precolumn (particle size = 5 μm) and three Suprema main columns (particle size = 5 μm, 1 Å × 30 Å; 2 Å × 1000 Å) with separation range from 100 to 1,000,000 Da (PSS, Mainz, Germany) together with a variable wavelength detector (1200 Series, Agilent Technologies). As solvent, 0.07 M aqueous Na2HPO4 was used (for dissolving polymer and as eluting solvent) with a flow rate of 0.8 mL min–1 and the columns were maintained at room temperature. As internal standard, ethylene glycol (HPLC grade) was used. The calibration was done with narrowly distributed poly(methacrylic acid) sodium salt homopolymers (PMA Na salt; PSS calibration kit). An injection volume of 60 μL was used for the measurements. The samples were dissolved with a concentration of 2 mg mL–1 and filtered through a 0.22 μm PTFE Nylon filter before analysis. The UV detector was set to λ = 490 nm for measurements of 6AF-labeled polymers.
Reported molar masses of polymers in this study are based on the indicated standards and denoted as Mn,app.

4.1.4. High-Performance Liquid Chromatography (HPLC)

HPLC measurements were conducted with a Jupiter 5 μm C18 300 Å LC column (250 mm × 4.6 mm). The run time was 35 min. A mixture of 0.1 v/v% TFA in water and acetonitrile served as eluent (Table 2). The fluorescence detected was set to λex = 480 nm and λem = 520 nm with 1× gain. The ELSD detector was set to λ = 215 nm.
Table 2. Composition of HPLC Eluent at Indicated Time Points
time [min]fraction of 0.1 v/v% aqueous TFA [%]fraction of acetonitrile [%]
098.02.0
550.050.0
100.0100.0
170.0100.0
2598.02.0
3598.02.0

4.1.5. UV-Vis Spectroscopy

Absorption measurements were performed on a Jasco Spectrometer V-670 in the range of 250–500 nm (scan speed 400 nm min–1) at 25 °C using a quartz glass cuvette (D = 10 mm). Baseline correction was carried out by measuring a blank sample with deionized water before measurements.

4.1.6. Fluorescence Spectroscopy

Fluorescence emission (λem = 517 nm) curves were measured on a Shimadzu RF 5301 PC spectro-fluorophotometer at an excitation wavelength of 450 nm using a quartz glass cuvette (D = 10 mm). Baseline correction was carried out by a background measurement prior to the sample measurement.

4.1.7. Dynamic Light Scattering (DLS)

DLS was measured on a Zetasizer Nano-ZS Malvern apparatus (Malvern Instruments Ltd.) using disposable cuvettes. The excitation light source was a He–Ne laser at 633 nm and the intensity of the scattered light was measured at an angle of 173°. This method measures the rate of intensity fluctuation, and the size of the particles is determined through the Stokes–Einstein equation. Results are based on the material’s properties of polystyrene latexes through the settings of the instrument. The concentration of the polymer solution was 2 mg mL–1 (in DPBS) in all cases. Details regarding size results as well as count rates can be found in Table S1.

4.1.8. Electrophoretic Light Scattering (ELS)

ELS was used to measure the zeta potential (ζ). The measurement was also performed on a Zetasizer Nano-ZS by applying laser Doppler velocimetry. For each measurement, 20 runs were carried out using the slow-field reversal and the fast-field reversal mode at 150 V. Each experiment was performed in triplicates at 25 °C. The zeta potential was calculated from the electrophoretic mobility (μ) according to the Henry equation. Henry coefficient f(ka) was calculated according to Ohshima. (71) The concentration of the polymer solution was 2 mg mL–1 (in DPBS) in all cases. Details regarding charge results as well as count rates can be found in Table S2.

4.1.9. Fourier-Transform Infrared Spectroscopy (FTIR)

IR spectra were recorded from solids on a PerkinElmer Spectrum 100 FTIR spectrometer in attenuated total reflection (ATR) mode.

4.2. Synthesis and Characterization

4.2.1. Poly(pentafluorophenyl) Acrylate (PPFPA)

Poly(pentafluorophenyl) acrylate was prepared utilizing a modified procedure originally described by Zerdan et al. (53)
Ten grams of pentafluorophenyl acrylate (PFPA) was transferred into a 10 mL vial and stirred with inhibitor remover for 49 min. Meanwhile, a 25 mL round-bottom flask equipped with a rare earth stirring bar was heated with a heat gun. The atmosphere inside the flask was converted into an argon atmosphere by three cycles of applying a vacuum and subsequently filling the flask with argon. Afterward,12 mL (9.4 g) of anhydrous acetonitrile was transferred to the flask. 55 mg (0.151 mmol) of the chain-transfer agent (CTA) 2-(dodecylthiocarbonothyolthio)-2-methylpropionic acid (DTCTTMPA) was added to the reaction flask and the solution was stirred until all DTCTTMPA was dissolved. Afterward, the monomer was separated from the inhibitor remover utilizing a needle with a smaller diameter than the diameter of the inhibitor remover beads. 9.21 g (38.69 mmol) of the monomer PFPA was added to the flask to achieve a ratio of monomer:CTA of 257:1. A solution of V65 in acetonitrile (c = 10 mg mL–1) was then prepared and 937 μL (0.038 mmol) of the solution was added to the flask and it was sealed with a rubber septum. The final monomer:CTA:initiator ratio was 257:1:0.25. The mixture was stirred for 2 min to homogenize the solution before it was purged with argon for 15 min. The yellow mixture was then transferred into a preheated oil bath thermostated at 60 °C and stirred for 17 h. The mixture was subsequently transferred to a dialysis tubing (Spectrum Spectra/Por Cellulose Ester, MWCO: 8 kDa), and dialysis against THF was performed for 3 days with daily solvent change. Then, the solvent was removed under reduced pressure and the polymer was dried in a vacuum oven at 60 °C for 1 day. The product was obtained as a yellow solid. Yield: 1.97 g (21%).
The DP of the resulting polymer was calculated by end-group analysis, using the signal of the CTA’s terminal CH3 group at δ = 0.91 ppm and the CH group of the polymer backbone at δ = 3.12 ppm, suggesting a DP of 132.
SEC (DMAc + 0.21 w% LiCl; PMMA-standard): Mn,app = 33,900 g mol–1; Đ = 1.32.

4.2.2. Postpolymerization Modification of PPFPA with Aliphatic Amino Acids

The postpolymerization modification of PPFPA is exemplarily described for reactions with Gly-yielding P(Gly-OH-AAm). Quantities and moles are given relative to the single repeating unit PFPA of the polymer and can be found in Table 3.
Table 3. Quantities of Amino Acids Used for PPM of PPFPA
amino acidn [mmol]m [mg]
Gly1.2695
Ala112
Abu130
Nva148
Val148
Ile165
Leu165
In a 5 mL reaction vessel (Biotage), PPFPA (100 mg, 0.42 mmol, 1.00 equiv) was dissolved in anhydrous DMF (5 mL). 250 μL of triethylamine was added. After that, Gly (95 mg, 1.26 mmol, 3 equiv) was added, the reaction vial was closed with a septum, and the suspension was stirred at 40 °C for 72 h. Then, the solid was filtered off and the remaining solution was dialyzed (RC, MWCO 3.5 kDa, SpectraPor) against 1 L of deionized water for 3 days with daily water change. Upon lyophilization, the product was yielded as an off-white to yellowish powder. Polymers were analyzed via 1H NMR, 19F-NMR, FTIR spectroscopy, and aqueous SEC. 1H NMR showing C–H adjacent to an amide bond (δ = 4.0 ppm) and FTIR showing a shift in absorption from a carbonyl adjacent to PFP at v~ = 1780 cm–1 toward a carbonyl group in an amide bond v~ = 1550 cm–1 both confirmed the conversion of the reaction. NMR and FTIR spectra can be found in the Supporting Information. Molar mass and dispersity from SEC measurements can be found in Table 1.

4.2.3. Postpolymerization Modification of PPFPA with 6-AF and Aliphatic Amino Acids

The postpolymerization modification of PPFPA is exemplarily described for reactions with Gly-yielding P(Gly-OH-AAm)-6AF. Quantities and moles are given relative to the single repeating unit PFPA of the polymer and can be found in Table 3.
In a 5 mL reaction vessel (Biotage), PPFPA (100 mg, 0.42 mmol, 1.00 equiv) was dissolved in anhydrous DMF (5 mL). Twenty-five μL triethylamine was added. After that, 6-AF (1 mg, 0.004 mmol, 0.01 equiv) was added and the reaction vessel was closed with a septum. The reaction mixture was stirred in the dark at 40 °C for 24 h. Then, 225 μL of triethylamine and Gly (95 mg, 1.26 mmol, 3 equiv) were added, the reaction vial was closed with a septum, and the suspension was stirred at 40 °C for 72 h. Then, the solid was filtered off and the remaining solution was dialyzed (RC, MWCO 3.5 kDa, SpectraPor) against 1 L of deionized water for 3 days with daily water changes. Upon lyophilization, the product was yielded as a yellow to greenish powder. Yields were determined gravimetrically. The labeling efficiency (LE) was determined by UV–vis measurements in DPBS according to the calibration of pure 6-AF in DPBS (Figure S10).
Yields (% assuming quantitative amino acid modification) and LE:
P(Gly-OH-AAm)-6AF: 19 mg (35%); LE: 11%
P(Ala-OH-AAm)-6AF: 48 mg (80%); LE: 14%
P(Abu–OH-AAm)-6AF: 47 mg (71%); LE: 8%
P(Nva-OH-AAm)-6AF: 45 mg (63%); LE: 12%
P(Val-OH-AAm)-6AF: 45 mg (63%); LE: 13%
P(Ile-OH-AAm)-6AF: 48 mg (62%); LE: 10%
P(Leu-OH-AAm)-6AF: 52 mg (67%); LE 9%
Polymers were analyzed by aqueous SEC (0.07 M aq. Na2HPO4 (standard: PMA Na salt)). Elugrams can be found in Figure S9.
P(Gly-OH-AAm)-6AF: Mn,app = 11,500 g mol–1; Đ = 1.84.
P(Ala-OH-AAm)-6AF: Mn,app = 14,200 g mol–1; Đ = 1.70.
P(Abu–OH-AAm)-6AF: Mn,app = 13,800 g mol–1; Đ = 1.70.
P(Nva-OH-AAm)-6AF: Mn,app = 13,400 g mol–1; Đ = 1.62.
P(Val-OH-AAm)-6AF: Mn,app = 14,200 g mol–1; Đ = 1.57.
P(Ile-OH-AAm)-6AF: Mn,app = 16,300 g mol–1; Đ = 1.55.
P(Leu-OH-AAm)-6AF: Mn,app = 10,500 g mol–1; Đ = 2.11.

4.2.4. pH Response

The pH response of the different polymers was investigated by acidic titration. A 5 mg mL–1 solution of the polymer in deionized water was prepared and 20 μL of 1.0 M aq. NaOH was added to obtain a starting pH value >11. Subsequently, the solution was titrated with 0.1 M aq. HCl until a pH value <3 was reached. Measurements were done with a Mettler Toledo digital pH meter. The pKa was calculated from the first derivation of the titration curve according to our previous work. (72)

4.2.5. Hydrophilic/Hydrophobic Ratio

The procedure was modified from the literature. (21) A known concentration of amino-acid-functionalized polymers (1 or 0.1 mg mL–1 as indicated) was dissolved in deionized water (diH2O) of indicated pH value or DPBS. To ensure precise pH values, 200 μL of a 10-fold stock solution of the respective polymer was diluted with diH2O to a total volume of 1.5 mL. The pH value was adjusted by the addition of an aqueous 0.1 M HCl or NaOH solution. After the desired pH value was reached, diH2O was added to reach a final volume of 2.0 mL. The fluorescence of this solution was measured. Then, an equal volume of chloroform was added, and the solution was vortexed for 1 min at maximum speed. The 0.1 mg mL–1 solution was allowed to stand for 15 min (or 24 h in the case of 1 mg mL–1 solutions) to separate the aqueous and the organic layer. A sample was carefully taken from each layer and analyzed via fluorescence measurements (λex = 450 nm, λem = 517 nm). To obtain the relative fluorescence, the raw results were normalized by the peak maximum of the emission trace of the respective polymer observed before the addition of chloroform (eq 1). The results show the normalized fluorescence intensity.
relativefluorescenceemission=intensityafterchloroformtreatmentintensitybeforechloroformtreatment
(1)
The hydrophobic–lipophilic balance (HLB) of polymers was predicted with MarvinSketch 23.14 using the Griffin method. (65)

4.2.6. Protein Fouling

Protein fouling of P(Gly-OH-AAm) and P(Nva-OH-AAm) with BSA and lysozyme, respectively, was analyzed by DLS measurements.
Stock solutions of the polymers and the proteins in DPBS were prepared (c = 1 mg mL–1) and mixed in a ratio of 1:1, yielding concentrations of c = 0.5 mg mL–1. The samples were measured immediately after mixing (t = 0 h) and at indicated time points (i.e., 0.5, 1, 2, 4, and 24 h). The time points indicate the time interval in-between measurements at which the samples were incubated at 37 °C while shaking at 100 rpm. Measurements were conducted at 37 °C with five measurements and three runs each. After the measurement, samples were placed back into the incubator immediately.

4.2.7. Statistical Analysis

All data plotted with error bars were expressed as means with standard deviation. p-Values were generated by analyzing data with a one-way ANOVA and Turkey test using OriginLab.

4.3. Biological Evaluation

4.3.1. Cell Culture

L929 cells were cultured in low-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U mL–1 penicillin, and 100 μg mL–1 streptomycin. The cells were incubated at 37 °C using 5% CO2 in a humidified incubator.

4.3.2. Cell Viability Assay

The cell viability assay was slightly modified from our previous procedure. (2) Cells were cultured as described above. For the cell viability assay, cells (104 per well) were seeded in 96 well plates and allowed to adhere overnight. No cells were seeded in the outer wells. The media was subsequently removed and replaced by fresh polymer-containing media. Then, the cells were incubated at 37 °C for an additional 24 h. After that, the media was removed, the cells were washed with 100 μL of DPBS, and then fresh media containing thiazolyl blue tetrazolium bromide (MTT) (concentration: 1 mg mL–1) was added (100 μL/well). Note: MTT (50 mg) was dissolved in 10 mL of sterile PBS, filtrated (membrane, 0.22 μm), and 1 to 5 diluted in culture medium prior to use in this assay. After incubation for 3 h at 37 °C, 100 μL of i-PrOH was added to each well and the plates were gently shaken in the dark for 15 min to dissolve the formazan crystals. Quantification was done by measuring the absorbance at λ = 580 nm using a microplate reader (Genios Pro, Tecan). Untreated cells on the same plate served as negative control (100% viability), cells treated with 20% DMSO as positive control (0% viability), and wells without cells as background. Experiments were formed in triplicates on three different plates. Relative cell viability was calculated by using eq 2.
%cellviability=Abs.sample−Abs.backgroundAbs.negativecontrol−Abs.background×100
(2)

4.3.3. Cell Association via Flow Cytometry

4.3.3.1. Cell Association Assay I: Concentration-Dependent Polymer Association with L929
The cell association assay is adapted from Leiske et al. (21) The L929 cells were cultured at 37 °C in a humidified 5% (v/v) CO2 atmosphere in DMEM Low Glucose (1 g L–1). The media was supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U mL–1 penicillin, and 100 μg mL–1 streptomycin (culture medium). For the experiments, 2 × 105 cells mL–1 were seeded in 250 μL of culture medium in a 48 well plate and cultivated for 24 h until treatment. One hour before the treatment, the medium was changed to 225 μL of fresh culture medium. The polymers were diluted in DPBS to a concentration of cRU = 0.5, 1.0, 5.0, and 10 mM. Cells were treated with 25 μL of respective polymer to receive final concentrations of cRU = 0.05, 0.10, 0.50, and 1.00 mM. Cells were additionally treated with 25 μL of DPBS as negative control (NC). After 4 h of incubation at 37 °C, 5% (v/v) CO2, the supernatant was discarded. The cells were immediately detached with trypsin-EDTA, resuspended in PBS, and analyzed by flow cytometry (cytoFLEX (Beckman Coulter)). A minimum of 10,000 single cells were measured and analyzed by forward and sideward scatter (FSC/SSC). Fluorescence was measured at λex = 488 nm with a 510/20 nm bandpass filter (GFP channel). Positive cells were identified by gating against NC. A detailed gating strategy is provided in Figure S17.
The normalized MFI was calculated using eq 3.
normalizedMFI[a.u.]=MFIsampleMFINC×1rfluorescencesample
(3)
4.3.3.2. Cell Association Assay II: Time- and Temperature-Dependent Polymer Association with L929
L929 cells were seeded as described before and cultivated for 24 h until treatment. One hour before the treatment, the medium got changed to 225 μL of fresh culture medium. The polymers were diluted in DPBS to a concentration of cRU = 10 mM. Cells were treated with 25 μL of the respective polymer at different time points to achieve final incubation times of 0.5, 1.0, 2.0, and 4.0 h at 37 °C, 5% (v/v) CO2 with cRU = 1.0 mM. Cells were additionally treated with 25 μL of DPBS as NC. An additional 48 well plate was treated in parallel at 4 °C (refrigerator) for 4 h. After incubation, the cells were measured as described before. A detailed gating strategy is provided in Figure S18.
The normalized MFI was calculated using eq 3. The temperature-dependent reduction of cell association was calculated using eq 4.
reduction[%]=1−MFI4°C(normalized)MFI37°C(normalized)×100%
(4)

4.3.4. Confocal Laser-Scanning Microscopy (CLSM)

The assay is adapted from Richter et al. (73) For cell association studies, cells were seeded as described before in 500 μL of culture medium in 4 well glass-bottomed dishes (Greiner Bio-One, Cat# 627860) and cultivated for 24 h until treatment. One hour prior to treatment, the medium got changed to 450 μL of fresh culture medium. The polymers were diluted in DPBS to a concentration of 10 mM (RU). Cells were treated with 50 μL of the polymer to achieve a final concentration of 1.0 mM (RU) and incubated for 4 or 24 h at 37 °C in humidified 5% (v/v) CO2. To stain nuclei, Hoechst 33342 (Invitrogen, Cat# H3570) was added (1:1000). To stain cellular membrane, CellMask Plasma Membrane Stain (Invitrogen, Cat# C10046) was added (1:1000). After further incubation of 10 min, the medium was replaced by 500 μL of fresh medium. Live cell imaging was performed using a LSM880, Elyra PS.1 system (Zeiss) applying laser for excitation at 405 nm (1.0%) (emission filter 410–484 nm, detection of Hoechst), 488 nm (2.0%) (emission filter 490–579 nm, detection of fluorescein), and 633 nm (1.5%) (emission filter 638–759 nm, detection of CellMask). To avoid cross-talk between the dyes, three different tracks were used. Images were acquired using the ZEN software, version 2.3 SP1 (Zeiss). The experiments were performed at least twice. All images were processed with ZEN software, version 3.7 (ZEN lite) (Zeiss). The same values were applied to all images.

4.3.5. Hemoglobin Release and Erythrocyte Aggregation

The erythrocyte aggregation and hemoglobin release assay were adapted from Richter et al. (73) The interaction of polymers with cellular membranes was detected by measuring the release of hemoglobin from erythrocytes due to potential membrane lysis. The blood, collected in tubes with citrate, was provided by the Department of Transfusion Medicine of the University Hospital, Jena. The experimental protocols used therein were approved by the ethics committee of the Jena University Hospital (2021–2058-Material). Blood from at least three different human donors was used. To isolate erythrocytes, the blood was centrifuged at 4500g for 5 min. The pellet was washed three times with DPBS. 500 μL of aliquots of erythrocytes were mixed with 500 μL of polymer solution in DPBS to receive a final concentration of cRU = 1 mM (Test solution). To determine aggregation, 100 μL of the respective test solutions were transferred to a 96 well plate in three technical replicates. bPEI 10000 was used as a positive control (50 μg mL–1) and only DPBS as NC. After incubation at 37 °C for 2 h, the absorbance was measured at λ = 645 nm. The aggregation rate was calculated by eq 5.
aggregationrate=absorbanceNCabsorbancesample
(5)
Exemplary microscopy images of aggregation are provided in Figure S19.
In parallel, the remaining 700 μL of test solutions was incubated for 1 h at 37 °C. To determine hemoglobin release, the tubes were centrifuged at 2400g for 5 min and the supernatant was transferred to a 96 well plate in three technical replicates. To measure the hemolytic effect of the polymers, the absorbance was measured at λ = 544 with λ = 630 nm as reference bandwidth. As a positive control, 1% Triton X-100 (T-X) was used (100% hemolysis) and pure DPBS was used as NC. The hemolysis (%) was calculated using eq 6.
hemolysis(%)=absorbancesampleabsorbanceT‐X×100
(6)
Values below 2% hemolysis are classified as nonhemolytic, values between 2 and 5% are slightly hemolytic, and values above 5% are hemolytic.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acspolymersau.3c00048.

  • The Supporting Information is available free of charge and contains further analysis of the materials in this study, including 1H and 19F-NMR spectra, fluorescence spectra, FTIR spectra, SEC data, cell viability data, cell association, and uptake data (PDF)

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Author Information

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  • Corresponding Author
  • Authors
    • Jonas De Breuck - Macromolecular Chemistry, University of Bayreuth, Universitätsstraße 30, 95447 Bayreuth, Germany
    • Michael Streiber - Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Humboldtstrasse 10, 07743 Jena, Germany;  Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, 07743 Jena, Germany
    • Michael Ringleb - Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Humboldtstrasse 10, 07743 Jena, Germany;  Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, 07743 Jena, Germany;  Orcidhttps://orcid.org/0000-0002-7320-8529
    • Dennis Schröder - Macromolecular Chemistry, University of Bayreuth, Universitätsstraße 30, 95447 Bayreuth, Germany;  Bavarian Polymer Institute, Universitätsstraße 30, 95447 Bayreuth, Germany
    • Natascha Herzog - Macromolecular Chemistry, University of Bayreuth, Universitätsstraße 30, 95447 Bayreuth, Germany
    • Ulrich S. Schubert - Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Humboldtstrasse 10, 07743 Jena, Germany;  Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, 07743 Jena, Germany;  Orcidhttps://orcid.org/0000-0003-4978-4670
    • Stefan Zechel - Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Humboldtstrasse 10, 07743 Jena, Germany;  Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, 07743 Jena, Germany
    • Anja Traeger - Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Humboldtstrasse 10, 07743 Jena, Germany;  Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, 07743 Jena, Germany;  Orcidhttps://orcid.org/0000-0001-7734-2293
  • Author Contributions

    M.S. and M.R. contributed equally to this work. The primary data are available. CRediT: Jonas De Breuck data curation, formal analysis, methodology, writing-original draft; Michael Streiber data curation, formal analysis, methodology, writing-review & editing; Michael Ringleb data curation, formal analysis, methodology, writing-review & editing; Dennis Schröder data curation, formal analysis, writing-review & editing; Natascha Herzog data curation, formal analysis; Ulrich S. Schubert funding acquisition, supervision, writing-review & editing; Stefan Zechel project administration, supervision, writing-review & editing; Anja Traeger conceptualization, funding acquisition, project administration, supervision, writing-review & editing; Meike N. Leiske conceptualization, funding acquisition, supervision, writing-original draft.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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The authors would like to acknowledge Prof Ruth Freitag, Prof Andreas Greiner, Prof Hans-Werner Schmidt, Prof Thomas Scheibel, and Prof Georg Papastavrou for providing access to their laboratories and equipment. The authors thank Dr Martin Humenik for his help with high-performance liquid chromatography measurements. The authors would like to thank Rika Schneider for conducting size-exclusion chromatography measurements, Martina Fried for assistance with polymer titrations, and Carolin Kellner for support with hemolysis and erythrocyte aggregation assays. M.N.L. acknowledges the financial support from the “Fonds der Chemischen Industrie im Verband der Chemischen Industrie.” The authors gratefully acknowledge the Bundesministerium für Bildung und Forschung (BMBF, Germany, #13XP5034A PolyBioMik), the DFG Projects PolyTarget (SFB 1278, project B01, C06, project ID: 316213987) as well as the “Thüringer Aufbaubank (TAB)″ (2021 FGI 0005), and the “Europäischer Fond für regionale Entwicklung (EFRE)″ (2018FGI0025) for funding of flow cytometry devices at the Jena Center for Soft Matter (JCSM). This work was performed within the Joint Lab for Polymers Jena-Bayreuth.

References

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    Static or dynamic light scattering measurements were performed in parallel, on dil. mixts. of DPPC/DPPA vesicles (typical radius 60 nm) and hydrophobically modified polymers. This technique gave evidence of the slow kinetics involved in both the reorganization of an adsorbed polymer layer and the membrane breakage. Hours, or sometimes days, were required in order to follow the variation of both the hydrodynamic radius and the scattering intensity at intermediate stages. Images of the intermediate species were collected using freeze-fracture electron microscopy (FFEM). Comparison of different polymers (of varying mol. wt. or structure) revealed the prime importance of hydrophobicity on the disruption of membranes. Although the presence of a few percent of pendant alkyl chains along the polymer backbone induced adsorption to membranes, only the assocn. with the more hydrophobic ones (>25 mol% of pendant octyl groups) resulted in small mixed objects of micellar size (radius about 10 nm). The drop of the mean radius of intermediate structures formed upon the vesicle breakage was also sensitive to temp. A tentative mechanism was proposed on the basis of kinetics and FFEM studies. (c) 2001 Academic Press.
  20. 20
    Yessine, M.-A.; Lafleur, M.; Meier, C.; Petereit, H.-U.; Leroux, J.-C. Characterization of the Membrane-Destabilizing Properties of Different PH-Sensitive Methacrylic Acid Copolymers. Biochim. Biophys. Acta, Biomembr. 2003, 1613 (1–2), 28– 38,  DOI: 10.1016/S0005-2736(03)00137-8
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    Characterization of the membrane-destabilizing properties of different pH-sensitive methacrylic acid copolymers
    Yessine, Marie-Andree; Lafleur, Michel; Meier, Christian; Petereit, Hans-Ulrich; Leroux, Jean-Christophe
    Biochimica et Biophysica Acta, Biomembranes (2003), 1613 (1-2), 28-38CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
    The intracellular delivery of active biomacromols. from endosomes into the cytoplasm generally requires a membrane-disrupting agent. Since endosomes have a slightly acidic pH, anionic carboxylated polymers could be potentially useful for this purpose since they can destabilize membrane bilayers by pH-triggered conformational change. In this study, five different pH-sensitive methacrylic acid (MAA) copolymers were characterized with respect to their physicochem. and membrane lytic properties as a function of pH. pH-dependent conformational changes were studied in aq. soln. by turbidimetry and spectrofluorimetry. The hydrophobic domains that formed upon a decrease in pH were found to be dependent on copolymer's compn. Hemolysis and cytotoxicity assays demonstrated that the presence of the hydrophobic Et acrylate monomer and/or sufficient protonation of the carboxylic acid groups were important parameters for efficient membrane destabilization. Excessive copolymer hydrophobicity was not assocd. with membrane destabilization, but resulted in high macrophage cytotoxicity. Overall, this study gave more insights into the structure-activity relationship of MAA copolymers with membrane bilayers. Gaining knowledge of modulation of the physicochem. properties of copolymers and the optimization of copolymer-lipid interactions may lead to the elaboration of much more efficient drug delivery systems.
  21. 21
    Leiske, M. N.; De Geest, B. G.; Hoogenboom, R. Impact of the Polymer Backbone Chemistry on Interactions of Amino-Acid-Derived Zwitterionic Polymers with Cells. Bioact. Mater. 2023, 24 (January), 524– 534,  DOI: 10.1016/j.bioactmat.2023.01.005
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    21
    Impact of the polymer backbone chemistry on interactions of amino-acid-derived zwitterionic polymers with cells
    Leiske, Meike N.; De Geest, Bruno G.; Hoogenboom, Richard
    Bioactive Materials (2023), 24 (), 524-534CODEN: BMIAD4; ISSN:2452-199X. (KeAi Communications Co., Ltd.)
    Zwitterionic polymers are known to interact with cells and have been shown to reveal cancer cell specificity. In this work, the importance of the chem. of the polymer backbone for the cellular specificity of amino-acid-derived polyzwitterions is demonstrated. A series of glutamic acid (Glu)-based vinyl monomers (i.e., an acrylate, a methacrylate, an acrylamide, and a methacrylamide) were prepd. and used for reversible addn.-fragmentation chain-transfer (RAFT) polymn., yielding defined polymers with narrow size distribution (ETH < 1.3). All Glu-functionalised, zwitterionic polymers revealed high cytocompatibility; however, differences in cellular assocn. and specificity were obsd. In particular, the methacrylamide-derived polymers showed high assocn. with both, breast cancer cells and non-cancerous dendritic cells and, consequently, lack specificity. In contrast, high specificity to only breast cancer cells was obsd. for polyacrylates, -methacrylates, and -acrylamides. Detailed anal. of the polymers revealed differences in hydrophobicity, zeta potential, and potential side chain hydrolysis, which are impacted by the polymer backbone and might be responsible for the altered the cell assocn. of these polymers. It is shown that a slightly neg. net charge is preferred over a neutral charge to retain cell specificity. This was also confirmed by assocn. expts. in the presence of competitive amino acid transporter substrates. The affinity of slightly neg. charged Glu-derived polymers to the xCT Glu/cystine cell membrane antiporter was found to be higher than that of neutrally charged polymers. Our results emphasize the importance of the polymer backbone for the design of cell-specific polymers. This study further highlights the potential to tailor amino-acid-derived zwitterionic materials beyond their side chain functionality.
  22. 22
    Wang, S.; Chen, R. PH-Responsive, Lysine-Based, Hyperbranched Polymers Mimicking Endosomolytic Cell-Penetrating Peptides for Efficient Intracellular Delivery. Chem. Mater. 2017, 29 (14), 5806– 5815,  DOI: 10.1021/acs.chemmater.7b00054
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    22
    pH-Responsive, Lysine-Based, Hyperbranched Polymers Mimicking Endosomolytic Cell-Penetrating Peptides for Efficient Intracellular Delivery
    Wang, Shiqi; Chen, Rongjun
    Chemistry of Materials (2017), 29 (14), 5806-5815CODEN: CMATEX; ISSN:0897-4756. (American Chemical Society)
    The insufficient delivery of biomacromol. therapeutic agents into the cytoplasm of mammalian cells remains a major barrier to their pharmaceutical applications. Cell-penetrating peptides (CPPs) are considered as potential carriers for cytoplasmic delivery of macromol. drugs. However, due to the pos. charge of most CPPs, strong nonspecific cell membrane bindings may lead to relatively high toxicity. In this study, we report a series of anionic, CPP-mimicking, lysine-based hyperbranched polymers, which caused complete membrane disruption at late endosomal pH while remaining nonlytic at physiol. pH. The pH-responsive conformational alterations and the multivalency effect of the hyperbranched structures were demonstrated to effectively facilitate their interaction with cell membranes, thus leading to significantly enhanced membrane-lytic activity compared with their linear counterpart. The unique structures and pH-responsive cell-penetrating abilities make the novel hyperbranched polymers promising candidates for cytoplasmic delivery of biomacromol. payloads.
  23. 23
    Chen, S.; Wang, S.; Kopytynski, M.; Bachelet, M.; Chen, R. Membrane-Anchoring, Comb-Like Pseudopeptides for Efficient, PH-Mediated Membrane Destabilization and Intracellular Delivery. ACS Appl. Mater. Interfaces 2017, 9 (9), 8021– 8029,  DOI: 10.1021/acsami.7b00498
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    23
    Membrane-Anchoring, Comb-Like Pseudopeptides for Efficient, pH-Mediated Membrane Destabilization and Intracellular Delivery
    Chen, Siyuan; Wang, Shiqi; Kopytynski, Michal; Bachelet, Marie; Chen, Rongjun
    ACS Applied Materials & Interfaces (2017), 9 (9), 8021-8029CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)
    Endosomal release has been identified as a rate-limiting step for intracellular delivery of therapeutic agents, in particular macromol. drugs. Herein, we report a series of synthetic pH-responsive, membrane-anchoring polymers exhibiting dramatic endosomolytic activity for efficient intracellular delivery. The comb-like pseudopeptidic polymers were synthesized by grafting different amts. of decylamine (NDA), which act as hydrophobic membrane anchors, onto the pendant carboxylic acid groups of a pseudopeptide, poly(L-lysine iso-phthalimide). The effects of the hydrophobic relatively long alkyl side chains on aq. soln. properties, cell membrane destabilization activity, and in-vitro cytotoxicity were investigated. The optimal polymer contg. 18 mol % NDA exhibited limited hemolysis at pH 7.4 but induced nearly complete membrane destabilization at endosomal pH within only 20 min. The mechanistic investigation of membrane destabilization suggests the polymer-mediated pore formation. It has been demonstrated that the polymer with hydrophobic side chains displayed a considerable endosomolytic ability to release endocytosed materials into the cytoplasm of various cell lines, which is of crit. importance for intracellular drug delivery applications.
  24. 24
    Lehnen, A.-C.; Gurke, J.; Bapolisi, A. M.; Reifarth, M.; Bekir, M.; Hartlieb, M. Xanthate-Supported Photo-Iniferter (XPI)-RAFT Polymerization: Facile and Rapid Access to Complex Macromolecules. Chem. Sci. 2023, 14 (3), 593– 603,  DOI: 10.1039/D2SC05197D
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    24
    Xanthate-supported photo-iniferter (XPI)-RAFT polymerization: facile and rapid access to complex macromolecules
    Lehnen, Anne-Catherine; Gurke, Johannes; Bapolisi, Alain M.; Reifarth, Martin; Bekir, Marek; Hartlieb, Matthias
    Chemical Science (2023), 14 (3), 593-603CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    Xanthate-supported photo-iniferter (XPI)-reversible addn.-fragmentation chain-transfer (RAFT) polymn. is introduced as a fast and versatile photo-polymn. strategy. Small amts. of xanthate are added to conventional RAFT polymns. to act as a photo-iniferter under light irradn. Radical exchange is facilitated by the main CTA ensuring control over the mol. wt. distribution, while xanthate enables an efficient photo-(re)activation. The photo-active moiety is thus introduced into the polymer as an end group, which makes chain extension of the produced polymers possible directly by irradn. This is in sharp contrast to conventional photo-initiators, or photo electron transfer (PET)-RAFT polymns., where radical generation depends on the added small mols. In contrast to regular photo-iniferter-RAFT polymn., photo-activation is decoupled from polymn. control, rendering XPI-RAFT an elegant tool for the fabrication of defined and complex macromols. The method is oxygen tolerant and robust and was used to perform screenings in a well-plate format, and it was even possible to produce multiblock copolymers in a coffee mug under open-to-air conditions. XPI-RAFT does not rely on highly specialized equipment and qualifies as a universal tool for the straightforward synthesis of complex macromols. The method is user-friendly and broadens the scope of what can be achieved with photo-polymn. techniques.
  25. 25
    Martin, L.; Gody, G.; Perrier, S. Preparation of Complex Multiblock Copolymers via Aqueous RAFT Polymerization at Room Temperature. Polym. Chem. 2015, 6 (27), 4875– 4886,  DOI: 10.1039/C5PY00478K
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    Preparation of complex multiblock copolymers via aqueous RAFT polymerization at room temperature
    Martin, Liam; Gody, Guillaume; Perrier, Sebastien
    Polymer Chemistry (2015), 6 (27), 4875-4886CODEN: PCOHC2; ISSN:1759-9962. (Royal Society of Chemistry)
    We describe a simple new approach towards complex multiblock copolymer architectures at ambient temps. Using the redox pair TBHP/AsAc for initiation, aq. reversible addn.-fragmentation chain transfer (RAFT) polymn. is used to develop a one-pot sequential monomer addn. process that yields high order multiblock copolymers at 25 °C. Working at ambient temp. permits the polymn. of monomers yielding polymers with lower crit. soln. temp. (LCST) and reduces the risk of side reactions by chain transfer. Our approach is initially demonstrated with the prepn. of well-defined low and high d.p. (DP) poly(4-acryloylmorpholine) (PNAM) and poly(2-hydroxyethyl acrylate) (PHEA) multiblock homopolymers with D under 1.35. To highlight the potential of our approach, more challenging multiblock copolymers were prepd.: a pentablock copolymer of high DP (an av. of 100 per block) including low LCST blocks of poly(N-isopropylacrylamide) (PNIPAM) and poly(N,N-diethylacrylamide) (PDEA), two polyacrylate multiblock copolymers (DP of 10 per block) using a range of different functional acrylate monomers, and a heptablock copolymer (DP 10 per block) consisting of both polyacrylate and polyacrylamido blocks, all with a final dispersity of around 1.3.
  26. 26
    Mori, H.; Endo, T. Amino-Acid-Based Block Copolymers by RAFT Polymerization. Macromol. Rapid Commun. 2012, 33 (13), 1090– 1107,  DOI: 10.1002/marc.201100887
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    Amino-Acid-Based Block Copolymers by RAFT Polymerization
    Mori, Hideharu; Endo, Takeshi
    Macromolecular Rapid Communications (2012), 33 (13), 1090-1107CODEN: MRCOE3; ISSN:1022-1336. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. This review summarizes recent advances in the design and synthesis of amino-acid-based block copolymers by reversible addn.-fragmentation chain transfer (RAFT) polymn. of amino-acid-bearing monomers. We will mainly focus on stimuli-responsive block copolymers, such as pH-, thermo-, and dual-stimuli-responsive block copolymers, and self-assembled block copolymers, including amphiphilic and double-hydrophilic block copolymers having tunable chiroptical properties. We will also highlight recent results in RAFT synthesis of amino-acid-based copolymers having various properties, such as catalytic and optoelectronic properties, crosslinked block copolymer micelles, unimol. micelles, and org.-inorg. hybrids.
  27. 27
    Leiske, M. N.; Singha, R.; Jana, S.; De Geest, B. G.; Hoogenboom, R. Amidation of Methyl Ester-Functionalised Poly(2-Oxazoline)s as a Powerful Tool to Create Dual PH- and Temperature-Responsive Polymers as Potential Drug Delivery Systems. Polym. Chem. 2023, 14 (17), 2034– 2044,  DOI: 10.1039/D3PY00050H
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    27
    Amidation of methyl ester-functionalised poly(2-oxazoline)s as a powerful tool to create dual pH- and temperature-responsive polymers as potential drug delivery systems
    Leiske, Meike N.; Singha, Ronak; Jana, Somdeb; De Geest, Bruno G.; Hoogenboom, Richard
    Polymer Chemistry (2023), 14 (17), 2034-2044CODEN: PCOHC2; ISSN:1759-9962. (Royal Society of Chemistry)
    In the past two decades, thermoresponsive polymers based on tertiary amine groups have been studied extensively as a class of dual-responsive polymers. In particular, their temp.-dependent phase transition can be further modulated by the pH value of the environment, rendering them interesting for applications in the biomedical area. In this contribution, we report methyl-ester-functionalised poly(2-alkyl-2-oxazoline)s (PAOx) as a versatile platform for the synthesis of tertiary-amine-functionalised PAOx via straightforward post-polymn. amidation. The resulting polymers were investigated regarding their stimuli-responsiveness to both, pH value and temp. by turbidimetry measurements. Dynamic light scattering further confirmed the formation of polymeric nanoparticles upon phase sepn. of block copolymers comprising a responsive polymer block and poly(2-methyl-2-oxazoline) as permanently hydrophilic block. Furthermore, a hydrophobic Rhodamine B deriv. was used as model cargo and was found to induce the formation of stable nanoparticles in biol. media beyond the responsiveness to pH and temp. of these polymers. Treatment of MDA-MB-231 breast cancer cells with such nanoparticles contg. both, the Rhodamine B octadecyl-deriv. and paclitaxel, suppressed their proliferation sufficiently in vitro. Altogether, PAOx with tertiary amines are presented as versatile materials with interesting characteristics and potential applications in the area of polymeric drug delivery.
  28. 28
    Das, A.; Theato, P. Activated Ester Containing Polymers: Opportunities and Challenges for the Design of Functional Macromolecules. Chem. Rev. 2016, 116 (3), 1434– 1495,  DOI: 10.1021/acs.chemrev.5b00291
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    Activated Ester Containing Polymers: Opportunities and Challenges for the Design of Functional Macromolecules
    Das, Anindita; Theato, Patrick
    Chemical Reviews (Washington, DC, United States) (2016), 116 (3), 1434-1495CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. The objective of this review is to provide the readers with an extensive overview of the advancement made in the area of polymeric activated ester chem. in designing precisely defined functionally diverse polymer structures, starting from utilizing classical polymn. techniques to state-of-the-art controlled polymn. methods and to highlight their potential applications in various interdisciplinary research areas. This review is divided into two parts. The first part will emphasize the development of activated ester chem. in constructing different reactive polymer structures where detailed information will be provided on various activated ester monomers being used under different polymn. techniques, and their advantages and drawbacks will be discussed explicitly. In the second part, focus will be on the versatility and fidelity of this chem. in fabricating numerous functional materials and their possible opportunities in different areas of application ranging from biomedical sciences to materials chem.
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    Singha, N. K.; Gibson, M. I.; Koiry, B. P.; Danial, M.; Klok, H. A. Side-Chain Peptide-Synthetic Polymer Conjugates via Tandem “Ester-Amide/Thiol-Ene” Post-Polymerization Modification of Poly(Pentafluorophenyl Methacrylate) Obtained Using ATRP. Biomacromolecules 2011, 12 (8), 2908– 2913,  DOI: 10.1021/bm200469a
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    29
    Side-Chain Peptide-Synthetic Polymer Conjugates via Tandem "Ester-Amide/Thiol-Ene" Post-Polymerization Modification of Poly(pentafluorophenyl methacrylate) Obtained Using ATRP
    Singha, Nikhil K.; Gibson, Matthew I.; Koiry, Bishnu P.; Danial, Maarten; Klok, Harm-Anton
    Biomacromolecules (2011), 12 (8), 2908-2913CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
    Herein the concept of tandem postpolymn. modification as a versatile route to synthesize well-defined, highly functionalized polymers is introduced. Poly(pentafluorophenyl methacrylate) obtained by atom transfer radical polymn. was first modified with allylamine, which displaces the active ester to give well-defined polymers with pendant alkene groups, which are difficult to obtain by direct (radical) polymn. of allylic-functional monomers. The produced poly(allylmethacrylamide) was modified by a second postpolymn. modification reaction with a thiol-terminated peptide (CVPGVG) using AIBN as the radical source. NMR, IR, and SEC demonstrated successful conjugation onto the polymer to give a polymer-peptide hybrid material. This versatile strategy should extend the scope of controlled radical polymn. and "click"-type reactions.
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    Gok, O.; Kosif, I.; Dispinar, T.; Gevrek, T. N.; Sanyal, R.; Sanyal, A. Design and Synthesis of Water-Soluble Multifunctionalizable Thiol-Reactive Polymeric Supports for Cellular Targeting. Bioconjugate Chem. 2015, 26 (8), 1550– 1560,  DOI: 10.1021/acs.bioconjchem.5b00182
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    Design and synthesis of water-soluble multifunctionalizable thiol-reactive polymeric supports for cellular targeting
    Gok, Ozgul; Kosif, Irem; Dispinar, Tugba; Gevrek, Tugce Nihal; Sanyal, Rana; Sanyal, Amitav
    Bioconjugate Chemistry (2015), 26 (8), 1550-1560CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    Design and synthesis of novel water-sol. polymers bearing reactive side chains are actively pursued due to their increasing demand in areas such as bioconjugation and drug delivery. This study reports the fabrication of poly(ethylene glycol) methacrylate based thiol-reactive water-sol. polymeric supports that can serve as targeted drug delivery vehicles. Thiol-reactive maleimide units were incorporated into polymers as side chains by use of a furan-protected maleimide contg. monomer. Atom transfer radical polymn. (ATRP) was employed to obtain a family of well-defined copolymers with narrow mol. wt. distributions. After the polymn., the maleimide groups were activated to their reactive form, ready for conjugation with thiol-contg. mols. Efficient functionalization of the maleimide moieties was demonstrated by conjugation of a tripeptide glutathione under mild and reagent-free aq. conditions. Addnl., hydrophobic thiol-contg. dye (Bodipy-SH) and a cyclic peptide-based targeting group (cRGDfC) were sequentially appended onto the maleimide bearing polymers to demonstrate their efficient multifunctionalization. The conjugates were utilized for in vitro expts. over both cancerous and healthy breast cell lines. Obtained results demonstrate that the conjugates were nontoxic, and displayed efficient cellular uptake. The presence of the peptide based targeting group had a clear effect on increasing the uptake of the dye-conjugated polymers into cells when compared to the construct devoid of the peptide. Overall, the facile synthesis and highly efficient multifunctionalization of maleimide-contg. thiol-reactive copolymers offer a novel and attractive class of polyethylene glycol-based water-sol. supports for drug delivery.
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    Boyer, C.; Davis, T. P. One- Pot Synthesis and Biofunctionalization of Glycopolymers via RAFT Polymerization and Thiol–Ene Reactions. Chem. Commun. 2009, (40), 6029– 6031,  DOI: 10.1039/b910296e
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    One- pot synthesis and biofunctionalization of glycopolymers via RAFT polymerization and thiol-ene reactions
    Boyer, Cyrille; Davis, Thomas P.
    Chemical Communications (Cambridge, United Kingdom) (2009), (40), 6029-6031CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    A modification of a polymeric activated ester with sugar-amines and a simultaneous aminolysis-thiol-ene reaction involving the RAFT end-groups yield biotin functionalized glycopolymer in one-pot.
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    Murata, H.; Prucker, O.; Rühe, J. Synthesis of Functionalized Polymer Monolayers from Active Ester Brushes. Macromolecules 2007, 40 (15), 5497– 5503,  DOI: 10.1021/ma0624550
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    Synthesis of Functionalized Polymer Monolayers from Active Ester Brushes
    Murata, Hironobu; Prucker, Oswald; Ruehe, Juergen
    Macromolecules (Washington, DC, United States) (2007), 40 (15), 5497-5503CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
    The synthesis of densely grafted monolayers of polymers carrying a wide spectrum of functional groups is described. To this surface-attached chains with active ester groups were generated through surface initiated polymn. of N-methacryloyl-β-alanine N'-oxysuccinimide ester (MAC2AE) using a self-assembled monolayer of an azo initiator on the surfaces of silicon oxide substrates. The layer thickness of the polymer monolayers can be easily controlled through the adjustment 15-crown of the monomer concn. during polymn. The aminolysis of the active ester groups within the brush with various n-alkylamines (C3-C18) was studied by IR and surface plasmon spectroscopy. Amines carrying addnl. functional groups such as amino acids, sugars, dyes, crown ethers, and amino-terminated polymers such as poly(ethylene glycol) derivs. can be used to incorporate many different functional moieties into the polymers.
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    Moog, K. E.; Barz, M.; Bartneck, M.; Beceren-Braun, F.; Mohr, N.; Wu, Z.; Braun, L.; Dernedde, J.; Liehn, E. A.; Tacke, F.; Lammers, T.; Kunz, H.; Zentel, R. Polymeric Selectin Ligands Mimicking Complex Carbohydrates: From Selectin Binders to Modifiers of Macrophage Migration. Angew. Chemie - Int. Ed. 2017, 56 (5), 1416– 1421,  DOI: 10.1002/anie.201610395
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    Polymeric Selectin Ligands Mimicking Complex Carbohydrates: From Selectin Binders to Modifiers of Macrophage Migration
    Moog, Kai E.; Barz, Matthias; Bartneck, Matthias; Beceren-Braun, Figen; Mohr, Nicole; Wu, Zhuojun; Braun, Lydia; Dernedde, Jens; Liehn, Elisa A.; Tacke, Frank; Lammers, Twan; Kunz, Horst; Zentel, Rudolf
    Angewandte Chemie, International Edition (2017), 56 (5), 1416-1421CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Novel polymeric cell adhesion inhibitors were developed in which the selectin tetrasaccharide sialyl-LewisX (SLeX) is multivalently presented on a biocompatible poly(2-hydroxypropyl)methacrylamide (PHPMA) backbone either alone (P1) or in combination with O-sulfated tyramine side chains (P2). For comparison, corresponding polymeric glycomimetics were prepd. in which the crucial "single carbohydrate" substructures fucose, galactose, and sialic acid side chains were randomly linked to the PHPMA backbone (P3 or P4 (O-sulfated tyramine)). All polymers have an identical d.p., as they are derived from the same precursor polymer. Binding assays to selectins, to activated endothelial cells, and to macrophages show that polyHPMA with SLeX is an excellent binder to E-, L-, and P-selectins. However, mimetic P4 can also achieve close to comparable binding affinities in in vitro measurements and surprisingly, it also significantly inhibits the migration of macrophages; this provides new perspectives for the therapy of severe inflammatory diseases.
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    Eberhardt, M.; Mruk, R.; Zentel, R.; Théato, P. Synthesis of Pentafluorophenyl(Meth)Acrylate Polymers: New Precursor Polymers for the Synthesis of Multifunctional Materials. Eur. Polym. J. 2005, 41 (7), 1569– 1575,  DOI: 10.1016/j.eurpolymj.2005.01.025
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    Synthesis of pentafluorophenyl (meth)acrylate polymers: New precursor polymers for the synthesis of multifunctional materials
    Eberhardt, Marc; Mruk, Ralf; Zentel, Rudolf; Theato, Patrick
    European Polymer Journal (2005), 41 (7), 1569-1575CODEN: EUPJAG; ISSN:0014-3057. (Elsevier B.V.)
    Pentafluorophenyl acrylate and -methacrylate were polymd. using AIBN as a thermal initiator. The obtained polymers were sol. polymeric active esters that could be used for the prepn. of multifunctional polymers. The reactivity of poly(pentafluorophenyl acrylate) and poly(pentafluorophenyl methacrylate) towards primary and secondary amines, as well as alcs. was investigated in a quant. way. Both poly(active esters) reacted satisfactorily with aliph. primary and secondary amines but only low conversion was found in the case of arom. amines. Conversions of only 30% were reached when poly(pentafluorophenyl acrylate) was treated with one equiv. of alc. under base catalysis. In time resolved FT-IR studies the rate consts. of the polymer analogous reactions were detd.
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    Batz, H. G.; Franzmann, G.; Ringsdorf, H. Pharmakologisch Aktive Polymere, 5. Modellreaktionen Zur Umsetzung von Pharmaka Und Enzymen Mit Monomeren Und Polymeren Reaktiven Estern. Die Makromol. Chemie 1973, 172 (1), 27– 47,  DOI: 10.1002/macp.1973.021720103
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    Ferruti, P.; Bettelli, A.; Feré, A. High Polymers of Acrylic and Methacrylic Esters of N-Hydroxysuccinimide as Polyacrylamide and Polymethacrylamide Precursors. Polymer Guildf 1972, 13 (10), 462– 464,  DOI: 10.1016/0032-3861(72)90084-5
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    Choi, J.; Schattling, P.; Jochum, F. D.; Pyun, J.; Char, K.; Theato, P. Functionalization and Patterning of Reactive Polymer Brushes Based on Surface Reversible Addition and Fragmentation Chain Transfer Polymerization. J. Polym. Sci., Part A: Polym. Chem. 2012, 50 (19), 4010– 4018,  DOI: 10.1002/pola.26200
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    Functionalization and patterning of reactive polymer brushes based on surface reversible addition and fragmentation chain transfer polymerization
    Choi, Jiyeon; Schattling, Philipp; Jochum, Florian D.; Pyun, Jeffrey; Char, Kookheon; Theato, Patrick
    Journal of Polymer Science, Part A: Polymer Chemistry (2012), 50 (19), 4010-4018, S4010/1-S4010/3CODEN: JPACEC; ISSN:0887-624X. (John Wiley & Sons, Inc.)
    We present the synthesis of reactive polymer brushes prepd. by surface reversible addn.-fragmentation chain transfer polymn. of pentafluorophenyl acrylate. The reactive ester moieties can be used to functionalize the polymer brush film with virtually any functionality by simple post-polymn. modification with amines. Dithiobenzoic acid benzyl-(4-ethyltrimethoxylsilyl) ester was used as the surface chain transfer agent (S-CTA) and the anchoring group onto the silicon substrates. Reactive polymer brushes with adjustable mol. wt., high grafting d., and conformal coverage through the grafting-from approach were obtained. Subsequently, the reactive polymer brushes were converted with amino-spiropyrans resulting in reversible light-responsive polymer brush films. The wetting behavior could be altered by irradn. with UV or visible light. Furthermore, a patterned surface of polymer brushes was obtained using a lithog. technique. UV irradn. of the S-CTA-modified substrates leads to a selective degrdn. of S-CTA in the exposed areas and gives patterned activated polymer brushes after a subsequent RAFT polymn. step. Conversion of the patterned polymer brushes with 5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid resulted in patterned fluorescent polymer brush films. The utilization of reactive polymer brushes offers an easy approach in the fabrication of highly functional brushes, even for functionalities whose introduction is limited by other strategies. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012.
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    Kockelmann, J.; Zentel, R.; Nuhn, L. Post-Polymerization Modifications to Prepare Biomedical Nanocarriers with Varying Internal Structures, Their Properties and Impact on Protein Corona Formation. Macromol. Chem. Phys. 2023, 224, 2300199  DOI: 10.1002/macp.202300199
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    Post-Polymerization Modifications to Prepare Biomedical Nanocarriers with Varying Internal Structures, their Properties and Impact on Protein Corona Formation
    Kockelmann, Johannes; Zentel, Rudolf; Nuhn, Lutz
    Macromolecular Chemistry and Physics (2023), 224 (24), 2300199CODEN: MCHPES; ISSN:1022-1352. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. The reactive ester approach provides access to various types of drug delivery systems. Either amphiphilic block copolymer micelles with hydrophobic cores can be generated for encapsulation of hydrophobic drugs, or they are (reversibly) crosslinked by polar mols. into nano(hydro)gel particles affording hydrophilic cores and coronas. Beyond short oligonucleotides complexation or covalent drug conjugation inside the core, a surface functionalization with targeting units is further possible to address a large variety of drug delivery scenarios. Interestingly, the reactive ester approach can thereby not only govern the nanocarriers inner structure and surface property, but at the same time also provide strategies to prevent protein corona formation. These features are summarized in this article and underline the concept of reactive ester macromols. as beneficial tool for assisting in drug delivery.
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    De Coen, R.; Vanparijs, N.; Risseeuw, M. D. P.; Lybaert, L.; Louage, B.; De Koker, S.; Kumar, V.; Grooten, J.; Taylor, L.; Ayres, N.; Van Calenbergh, S.; Nuhn, L.; De Geest, B. G. PH-Degradable Mannosylated Nanogels for Dendritic Cell Targeting. Biomacromolecules 2016, 17 (7), 2479– 2488,  DOI: 10.1021/acs.biomac.6b00685
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    pH-Degradable Mannosylated Nanogels for Dendritic Cell Targeting
    De Coen, Ruben; Vanparijs, Nane; Risseeuw, Martijn D. P.; Lybaert, Lien; Louage, Benoit; De Koker, Stefaan; Kumar, Vimal; Grooten, Johan; Taylor, Leeanne; Ayres, Neil; Van Calenbergh, Serge; Nuhn, Lutz; De Geest, Bruno G.
    Biomacromolecules (2016), 17 (7), 2479-2488CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
    We report on the design of glycosylated nanogels via core-crosslinking of amphiphilic non-water-sol. block copolymers composed of an acetylated glycosylated block and a pentafluorophenyl (PFP) activated ester block prepd. by reversible addn.-fragmentation (RAFT) polymn. Self-assembly, pH-sensitive core-crosslinking, and removal of remaining PFP esters and protecting groups are achieved in one pot and yield fully hydrated sub-100 nm nanogels. Using cell subsets that exhibit high and low expression of the mannose receptor (MR) under conditions that suppress active endocytosis, we show that mannosylated but not galactosylated nanogels can efficiently target the MR that is expressed on the cell surface of primary dendritic cells (DCs). These nanogels hold promise for immunol. applications involving DCs and macrophage subsets.
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    Duret, D.; Haftek-Terreau, Z.; Carretier, M.; Ladavière, C.; Charreyre, M.-T.; Favier, A. Fluorescent RAFT Polymers Bearing a Nitrilotriacetic Acid (NTA) Ligand at the α-Chain-End for the Site-Specific Labeling of Histidine-Tagged Proteins. Polym. Chem. 2017, 8 (10), 1611– 1615,  DOI: 10.1039/C6PY02222G
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    Fluorescent RAFT polymers bearing a nitrilotriacetic acid (NTA) ligand at the α-chain-end for the site-specific labeling of histidine-tagged proteins
    Duret, Damien; Haftek-Terreau, Zofia; Carretier, Matthieu; Ladaviere, Catherine; Charreyre, Marie-Therese; Favier, Arnaud
    Polymer Chemistry (2017), 8 (10), 1611-1615CODEN: PCOHC2; ISSN:1759-9962. (Royal Society of Chemistry)
    Site-specific labeling of proteins is a valuable tool to study biol. processes. We show that it can be achieved using fluorescent polymer probes synthesized by RAFT polymn. Thanks to an original control agent functionalized with a nitrilotriacetic acid (NTA) ligand, an α-NTA polymer probe was prepd. to selectively label histidine-tagged recombinant proteins.
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    Cepraga, C.; Gallavardin, T.; Marotte, S.; Lanoë, P. H.; Mulatier, J. C.; Lerouge, F.; Parola, S.; Lindgren, M.; Baldeck, P. L.; Marvel, J.; Maury, O.; Monnereau, C.; Favier, A.; Andraud, C.; Leverrier, Y.; Charreyre, M. T. Biocompatible Well-Defined Chromophore-Polymer Conjugates for Photodynamic Therapy and Two-Photon Imaging. Polym. Chem. 2013, 4 (1), 61– 67,  DOI: 10.1039/C2PY20565C
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    Biocompatible well-defined chromophore-polymer conjugates for photodynamic therapy and two-photon imaging
    Cepraga, Cristina; Gallavardin, Thibault; Marotte, Sophie; Lanoe, Pierre-Henri; Mulatier, Jean-Christophe; Lerouge, Frederic; Parola, Stephane; Lindgren, Mikael; Baldeck, Patrice L.; Marvel, Jacqueline; Maury, Olivier; Monnereau, Cyrille; Favier, Arnaud; Andraud, Chantal; Leverrier, Yann; Charreyre, Marie-Therese
    Polymer Chemistry (2013), 4 (1), 61-67CODEN: PCOHC2; ISSN:1759-9962. (Royal Society of Chemistry)
    A versatile approach is introduced for the synthesis of well-defined, biocompatible conjugates combining two-photon chromophores and hydrophilic multifunctional polymers synthesized by RAFT controlled radical polymn. As an illustration, two different classes of conjugates carrying multiple fluorophores (based on an anthracene moiety, Anth) or photosensitizers (based on a dibromobenzene moiety, DBB) along the polymer chain were elaborated for bioimaging and photodynamic therapy (PDT) applications, resp. In both cases, the polymer greatly improved the soly. in biorelevant media as well as the cell uptake. Anth conjugates provided high quality fluorescence microscopy images using both one- and two-photon excitation. DBB conjugates potently induced the death of cancer cells upon photoactivation.
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    Engler, A. C.; Chan, J. M. W.; Coady, D. J.; O’Brien, J. M.; Sardon, H.; Nelson, A.; Sanders, D. P.; Yang, Y. Y.; Hedrick, J. L. Accessing New Materials through Polymerization and Modification of a Polycarbonate with a Pendant Activated Ester. Macromolecules 2013, 46 (4), 1283– 1290,  DOI: 10.1021/ma4001258
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    Accessing New Materials through Polymerization and Modification of a Polycarbonate with a Pendant Activated Ester
    Engler, Amanda C.; Chan, Julian M. W.; Coady, Daniel J.; O'Brien, Jeannette M.; Sardon, Haritz; Nelson, Alshakim; Sanders, Daniel P.; Yang, Yi Yan; Hedrick, James L.
    Macromolecules (Washington, DC, United States) (2013), 46 (4), 1283-1290CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
    Functionalized polycarbonates were synthesized by organocatalytic ring-opening polymn. (ROP) of a cyclic monomer with a pendant activated ester (MTC-OC6F5) followed by a postpolymn. modification with both small mols. and macromols. Controlled ROP to form homopolymers and diblock copolymers was realized using catalytic quantities of triflic acid. For the homopolymers, a linear relationship between [M]0/[I]0 and mol. wt. (by GPC) demonstrated the living nature of the polymn. Poly(MTC-OC6F5) was functionalized under mild reaction conditions with a variety of amines to obtain polymers with pendant primary, secondary, and tertiary amides. Graft polymers with a high grafting d. of over 87% were synthesized using amine-terminated poly(ethylene glycol) of two different mol. wts. (2 and 5 kDa). The prepn. of poly(MTC-OC6F5) provides a means of accessing a wide range of functional polycarbonates with minimal synthetic steps. This new methodol. for the formation of functionalized polycarbonates provides a simple and versatile platform for the synthesis of new and innovative materials.
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    Smith, E.; Bai, J.; Oxenford, C.; Yang, J.; Somayaji, R.; Uludag, H. Conjugation of Arginine-Glycine-Aspartic Acid Peptides to Thermoreversible N-Isopropylacrylamide Polymers. J. Polym. Sci., Part A: Polym. Chem. 2003, 41 (24), 3989– 4000,  DOI: 10.1002/pola.10965
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    Conjugation of arginine-glycine-aspartic acid peptides to thermoreversible N-isopropylacrylamide polymers
    Smith, Erin; Bai, Jiang; Oxenford, Cassie; Yang, Jennifer; Somayaji, Ranjani; Uludag, Hasan
    Journal of Polymer Science, Part A: Polymer Chemistry (2003), 41 (24), 3989-4000CODEN: JPACEC; ISSN:0887-624X. (John Wiley & Sons, Inc.)
    Thermoreversible polymeric biomaterials are finding increased acceptance in tissue engineering applications. One drawback of the polymers is their synthetic nature, which does not allow direct interaction of mammalian cells with the polymers. This limitation may be alleviated by grafting arginine-glycine-aspartic acid (RGD) contg. peptides onto the polymer backbone to facilitate interactions with cell-surface integrins. Toward this goal, N-isopropylacrylamide (NiPAM)-based thermoreversible polymers contg. amine-reactive N-acryloxysuccinimide (NASI) groups were synthesized. Conjugation of RGD-contg. peptides to polymers was demonstrated with 1H NMR spectroscopy and reverse-phase high-pressure liq. chromatog. The conjugation reaction was optimal at 4 °C and pH of 8.0, and increased with the increasing NASI content of polymers. With a peptide grafting ratio of 0.25 mol %, there was no significant change in the lower crit. soln. temp. of the polymers. Finally, the NASI-contg. polymers, cast as films, on tissue culture polystyrene, were shown to conjugate to RGD-contg. peptides and support C2C12 cell attachment. We conclude that NASI-contg. thermoreversible polymers are amenable for grafting biomimetic peptides to impart cell adhesiveness to the polymers.
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    Baessler, K. A.; Lee, Y.; Sampson, N. S. B1 Integrin Is an Adhesion Protein for Sperm Binding To Eggs. ACS Chem. Biol. 2009, 4 (5), 357– 366,  DOI: 10.1021/cb900013d
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    β1 Integrin Is an Adhesion Protein for Sperm Binding to Eggs
    Baessler, Keith A.; Lee, Younjoo; Sampson, Nicole S.
    ACS Chemical Biology (2009), 4 (5), 357-366CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    We investigated the role of β1 integrin in mammalian fertilization and the mode of inhibition of fertilinβ-derived polymers. We detd. that polymers displaying the Glu-Cys-Asp peptide from the fertilinβ disintegrin domain mediate inhibition of mammalian fertilization through a β1 integrin receptor on the egg surface. Inhibition of fertilization is a consequence of competition with sperm binding to the cell surface, not activation of an egg-signaling pathway. The presence of the β1 integrin on the egg surface increases the rate of sperm attachment but does not alter the total no. of sperm that can attach or fuse to the egg. We conclude that the presence of β1 integrin enhances the initial adhesion of sperm to the egg plasma membrane and that subsequent attachment and fusion are mediated by addnl. egg and sperm proteins present in the β1 integrin complex. Therefore, the mechanisms by which sperm fertilize wild-type and β1 knockout eggs are different.
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    Börner, H. G.; Sütterlin, R. I.; Theato, P.; Wiss, K. T. Topology-Dependent Swichability of Peptide Secondary Structures in Bioconjugates with Complex Architectures. Macromol. Rapid Commun. 2014, 35 (2), 180– 185,  DOI: 10.1002/marc.201300808
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    Topology-dependent swichability of peptide secondary structures in bioconjugates with complex architectures
    Borner Hans G; Sutterlin Romina I; Theato Patrick; Wiss Kerstin T; Theato Patrick
    Macromolecular rapid communications (2014), 35 (2), 180-185 ISSN:.
    Peptide sequences, which exhibit a reversible pH-responsive coil to α-helix secondary structure transition, are conjugated to polymer precursors to yield linear AB and graft ABA peptide-poly(ethylene oxide) conjugates. While the PEO B-block is comparable, the conjugates differ in topologies of the peptide bearing A-blocks. The influences of topology on the structure transitions in the peptide segments are investigated, comparing linear AB-bioconjugates with graft ABA-bioconjugates having multiple peptide segments combined in star or pom-pom topologies.
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    Nuhn, L.; Hartmann, S.; Palitzsch, B.; Gerlitzki, B.; Schmitt, E.; Zentel, R.; Kunz, H. Water-Soluble Polymers Coupled with Glycopeptide Antigens and T-Cell Epitopes as Potential Antitumor Vaccines. Angew. Chem., Int. Ed. 2013, 52 (40), 10652– 10656,  DOI: 10.1002/anie.201304212
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    Water-soluble polymers coupled with glycopeptide antigens and T-cell epitopes as potential antitumor vaccines
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    Angewandte Chemie, International Edition (2013), 52 (40), 10652-10656CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    First examples of nanosized polymer-linked vaccines have been synthesized by coupling tumor-assocd. MUC1 glycopeptides and T-cell epitope peptides to water-sol. methacrylamide polymers through the application of active ester aminolysis and Huisgen cycloaddn. as orthogonal conjugation methods. The attachment of the tetanus toxoid T-cell epitope P2 onto the hydrophilic polymer vaccines causes their self-assembly to micelle-like nanoobjects. The novel vaccine concept was extended to block copolymers consisting of hydrophilic and hydrophobic domains, in which the latter reinforce the formation of nanoparticles as well. The novel polymer-based glycopeptide vaccines induce significant MHC-II-mediated immune reactions in mice and elicit IgG antibodies, which recognize MCF-7 breast tumor cells. In particular, the block copolymer contg. addnl. nanostructure-promoting domains induced antibodies that exhibit high affinity to the tumor cells. Considering the numerous degrees of freedom for structure modification, this novel vaccine concept may open up new ways for the construction of efficient immunotherapeutics, for example antitumor vaccines.
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    Leiske, M. N.; Kempe, K. A Guideline for the Synthesis of Amino-Acid-Functionalized Monomers and Their Polymerizations. Macromol. Rapid Commun. 2022, 43 (2), 1– 24,  DOI: 10.1002/marc.202100615
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    Maity, T.; Paul, S.; De, P. Side-Chain Amino Acid-Based Macromolecular Architectures. J. Macromol. Sci. Part A 2023, 60 (1), 2– 17,  DOI: 10.1080/10601325.2023.2169158
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    Side-chain amino acid-based macromolecular architectures
    Maity, Tanmoy; Paul, Soumya; De, Priyadarsi
    Journal of Macromolecular Science, Part A: Pure and Applied Chemistry (2023), 60 (1), 2-17CODEN: JSPCE6; ISSN:1060-1325. (Taylor & Francis, Inc.)
    A review. Amino acids, the natural building blocks of life, are highly functional small mols. with chirality and important catalytic properties. Imparting the exquisite properties of amino acids into the macromols. allows us to prep. unique, well-defined, biocompatible, functional, and tailorable materials. They exhibit stimuli responsiveness, tunable optical characteristics, enhanced thermal, mech., and biol. properties with desired cell-materials interactions. Considering the salient features and diverse applications of side-chain amino acid-contg. polymers, in this feature article, we present a concise highlight on the recent trends (2015-present) of the synthesis and utilization of pendant amino acid-based polymeric architectures. This article covers the synthesis of different classes of side-chain amino acid-derived polymeric architectures, and their recent utilization in drug and gene delivery, antibacterial activity, anti-fouling activity, heavy metal ion sensing, tissue engineering, etc. We hope that this study will provide a comprehensive outline of key aspects assocd. with the construction of functional 'bio-inspired' polymeric materials with desired characteristics, and their enhanced potential applications.
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    Leiske, M. N.; Mazrad, Z. A. I.; Zelcak, A.; Wahi, K.; Davis, T. P.; McCarroll, J. A.; Holst, J.; Kempe, K. Zwitterionic Amino Acid-Derived Polyacrylates as Smart Materials Exhibiting Cellular Specificity and Therapeutic Activity. Biomacromolecules 2022, 23 (6), 2374– 2387,  DOI: 10.1021/acs.biomac.2c00143
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    Zwitterionic Amino Acid-Derived Polyacrylates as Smart Materials Exhibiting Cellular Specificity and Therapeutic Activity
    Leiske, Meike N.; Mazrad, Zihnil. A. I.; Zelcak, Aykut; Wahi, Kanu; Davis, Thomas P.; McCarroll, Joshua A.; Holst, Jeff; Kempe, Kristian
    Biomacromolecules (2022), 23 (6), 2374-2387CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
    The synthesis of new amino acid-contg., cell-specific, therapeutically active polymers is presented. Amino acids served as starting material for the prepn. of tailored polymers with different amino acids in the side chain. The reversible addn.-fragmentation chain-transfer (RAFT) polymn. of acrylate monomers yielded polymers of narrow size distribution (D ≤ 1.3). In particular, glutamate (Glu)-functionalized, zwitterionic polymers revealed a high degree of cytocompatibility and cellular specificity, i.e., showing assocn. to different cancer cell lines, but not with nontumor fibroblasts. Energy-dependent uptake mechanisms were confirmed by means of temp.-dependent cellular uptake expts. as well as localization of the polymers in cellular lysosomes detd. by confocal laser scanning microscopy (CLSM). The amino acid receptor antagonist O-benzyl-L-serine (BzlSer) was chosen as an active ingredient for the design of therapeutic copolymers. RAFT copolymn. of Glu acrylate and BzlSer acrylate resulted in tailored macromols. with distinct monomer ratios. The targeted, cytotoxic activity of copolymers was demonstrated by means of multiday in vitro cell viability assays. To this end, polymers with 25 mol % BzlSer content showed cytotoxicity against cancer cells, while leaving fibroblasts unaffected over a period of 3 days. Our results emphasize the importance of biol. derived materials to be included in synthetic polymers and the potential of zwitterionic, amino acid-derived materials for cellular targeting. Furthermore, it highlights that the fine balance between cellular specificity and unspecific cytotoxicity can be tailored by monomer ratios within a copolymer.
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    Fujii, S.; Sakurai, K. Zwitterionic Amino Acid Polymer-Grafted Core-Crosslinked Particle toward Tumor Delivery. Biomacromolecules 2022, 23 (9), 3968– 3977,  DOI: 10.1021/acs.biomac.2c00803
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    Zwitterionic Amino Acid Polymer-Grafted Core-Crosslinked Particle toward Tumor Delivery
    Fujii, Shota; Sakurai, Kazuo
    Biomacromolecules (2022), 23 (9), 3968-3977CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
    Zwitterionic amino acid polymers (ZAPs) exhibit biocompatibility and recognition capability for amino acid transporters (AATs) overexpressed on cancer cells. They are potential cancer-targeting ligands in nanoparticle-based nanomedicines utilized in cancer chemotherapy. Here, a poly(glutamine methacrylate) (pGlnMA)-grafted core-crosslinked particle (pGlnMA-CCP) is prepd. through the formation of nanoemulsions stabilized using amphiphilic block copolymers comprising pGlnMA as the hydrophilic block. The chain conformation of the grafted polymer and the particle structure of pGlnMA-CCP are precisely elucidated by dynamic light scattering, X-ray scattering, and transmission electron microscopy. pGlnMA-CCP demonstrates active cellular uptake and deep penetration behaviors for cancer cells and spheroids, resp., via an AAT-mediated mechanism. The in vivo pharmacokinetics of pGlnMA-CCP is practically comparable to those of a CCP covered with poly(polyethylene glycol methacrylate) (pPEGMA), which inhibits protein adsorption and prolongs blood retention, implying that the biocompatible properties of pGlnMA are similar to those of pPEGMA. Furthermore, pGlnMA-CCP accumulates in cancer tissues at a higher level than that of pPEGMA systems. The results demonstrate that the properties of cancer targetability, tumor permeability, efficient tumor accumulation, and biocompatibility can be obtained by grafting pGlnMA onto nanoparticles, suggesting a high potential of pGlnMA as a ligand for cancer-targeting nanomedicines.
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    Koyama, E.; Sanda, F.; Endo, T. Syntheses and Radical Polymerizations of Methacrylamides Derived from Optically Active Amino Alcohols. Macromol. Chem. Phys. 1997, 198 (11), 3699– 3707,  DOI: 10.1002/macp.1997.021981130
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    Syntheses and radical polymerizations of methacrylamides derived from optically active amino alcohols
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    Macromolecular Chemistry and Physics (1997), 198 (11), 3699-3707CODEN: MCHPES; ISSN:1022-1352. (Huethig & Wepf Verlag)
    Syntheses and radical polymns. of methacrylamides derived from optically active amino alcs. were carried out. The monomers were prepd. by condensation of optically active amino alcs. and methacrylic acid using 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride (EDC.HCl). Radical polymns. of the monomers were carried out in the presence of 2,2-azoisobutyronitrile (AIBN) (3 mol.-%) in MeOH and DMF as solvents, resulting with good yields in polymers with ‾Mn of 17,800-81,000. The monomer reactivity ratios of N-(1-hydroxy-4-methyl-2-pentyl)methacrylamide (MA-Lol) and Me methacrylate (MMA) in the copolymn. were estd. to be 0.50 and 2.38, resp. The lower reactivity of MA-Lol in comparison with MMA is similar to common methacrylamide derivs. The hydrophilicity of the polymers was evaluated by the heat of fusion of water using differential scanning calorimetry (DSC). The detd. order poly(MA-Aol) > poly(MA-Lol) > poly(MA-Fol) reflects the bulkiness of the side chains.
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    Yaşayan, G.; Redhead, M.; Magnusson, J. P.; Spain, S. G.; Allen, S.; Davies, M.; Alexander, C.; Fernández-Trillo, F. Well-Defined Polymeric Vesicles with High Stability and Modulation of Cell Uptake by a Simple Coating Protocol. Polym. Chem. 2012, 3 (9), 2596– 2604,  DOI: 10.1039/c2py20352a
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    Polymer Chemistry (2012), 3 (9), 2596-2604CODEN: PCOHC2; ISSN:1759-9962. (Royal Society of Chemistry)
    Amphiphilic polymers have been synthesized by controlled free radical polymn. techniques. These polymers self-assemble into well-defined vesicles in aq. conditions, enabling encapsulation of a model hydrophilic mol. The polymeric vesicles show high stability against a range of aq. conditions with marginal release of cargo, even in the presence of known cell-membrane disruptive polymers such as branched poly(ethyleneimine) (b-PEI). This stability allows for inversion of the surface charge of the polymeric vesicles by a simple coating protocol leading to an enhanced uptake by mammalian cells.
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    Bou Zerdan, R.; Geng, Z.; Narupai, B.; Diaz, Y. J.; Bates, M. W.; Laitar, D. S.; Souvagya, B.; Van Dyk, A. K.; Hawker, C. J. Efficient Synthesis of Branched Poly(Acrylic Acid) Derivatives via Postpolymerization Modification. J. Polym. Sci. 2020, 58 (14), 1989– 1997,  DOI: 10.1002/pol.20200287
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    The utility of pentafluorophenyl esters for the selective introduction of functional units and branch points in well-defined poly(acrylic acid) (PAA) derivs. is demonstrated using a combination of controlled radical polymn. and postpolymn. modification. Reversible addn.-fragmentation chain transfer enables the synthesis of well-defined copolymers-poly(pentafluorophenyl acrylate-co-tert-Bu acrylate)-with the active ester repeat units serving as attachment points for reaction with primary amines, specifically tris(2-(t-butoxycarbonyl)ethyl)methyl amine (Behera's amine). Deprotection using trifluoroacetic acid removes both the backbone and side chain t-Bu esters to give a series of branched PAA derivs. contg. novel tricarboxylic acid side chains that are well suited to complexation and multidentate interactions. Surprisingly, the active ester homopolymer is shown to have the highest reactivity with Behera's amine when compared to copolymers with lower incorporation of pentafluorophenyl esters, suggesting an intriguing interplay of neighboring group effects and steric interactions. The ability to tune the efficiency of postpolymn. modification gives a library of PAA derivs.
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    The chem. reactions between poly(acrylic acid) and divalent metal oxides (metal = Ca, Cu, Mg, Zn) were studied by FTIR spectroscopy. There was clear evidence of chem. bonding between the polymer and the metals as a result of their interaction. From the energy difference between the sym. and the asym. bands of the various possible structures, monodentate metal-carboxylate bonding was the most probable in all cases. Also, from the anal. of the corresponding molar relations, the particle size of the oxides played an important role in the reaction.
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    Roy, S.; Mahali, K.; Mondal, S.; Dolui, B. K. Thermodynamics of DL-Alanine Solvation in Water-Dimethylsulfoxide Mixtures at 298.15 K. Russ. J. Phys. Chem. A 2015, 89 (4), 654– 662,  DOI: 10.1134/S0036024415040226
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    Roy, S.; Mahali, K.; Mondal, S.; Dolui, B. K.
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    In this study we mainly discuss the transfer Gibbs free energy ΔG0t(i) and ΔS0t(i)entropy of DL-alanine at 298.15 K and consequently the involved chem. transfer free energy (ΔG0t,ch(i)) and entropy (TΔS0t,ch(i)) in aq. mixts. of dimethylsulfoxide are discussed to clarify the solvation chem. of DL-alanine. For the evaluation of these energy terms, soly. of this amino acid has been measured by formol titrimetry at five equidistant temps. i.e., from 288.15 to 308.15 K in different compn. of this mixed solvent system. The various solvent parameters as well as thermodn. parameters like molar volume, d., dipole moment and solvent diam. of this solvent system have also been reported here. The chem. effects of the transfer Gibbs energies (ΔG0t,ch(i)) and entropies of transfer (TΔS0t,ch(i)) have been obtained after elimination of cavity effect and dipole-dipole interaction effects from the total transfer energies. Here the chem. contribution of transfer energetics of DL-alanine is mainly guided by the composite effects of increased dispersion interaction, basicity effect and decreased acidity, hydrogen bonding effects, hydrophilic hydration and hydrophobic hydration of aq. DMSO mixts. as compared to that of ref. solvent, water.
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    The effects of various solvents and pH on the solys. of glycine, L-alanine, L-valine, L-phenylalanine, and DL-aminooctanoic acid were studied in a series of pure aq. and alc. solns. The aq. soly. was inversely proportional to the size of the nonpolar portion of the mol. A low nonaq. soly. was due to a dominance of the amino acid by the charged α-amino carboxylic acid portion of the mol. In aq. and alc. solns., an isoelec. band of min. soly. was formed. A distinct increase in soly. proportional to the addn. of acid or base was seen as the pH exceeded the limits of the isoelec. band. In the alc. solvent systems studied, the addn. of either acid or base produced a greater divergence from the isoelec. pH than would be seen in a pure aq. system.
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    Mahali, K.; Roy, S.; Dolui, B. K. Solubility and Solvation Thermodynamics of a Series of Homologous α-Amino Acids in Nonaqueous Binary Mixtures of Ethylene Glycol and Dimethyl Sulfoxide. J. Chem. Eng. Data 2015, 60 (5), 1233– 1241,  DOI: 10.1021/je5007899
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    In this article the std. free energies (ΔG0t(i)) and entropies (ΔS0t(i)) of transfer of four homologous α-amino acids including glycine (Gly), DL-alanine (DL-Ala), DL-α-amino butyric acid (DL-Aba.) and DL-nor-valine (DL-n-Val) from ethylene glycol (EG) to nonaq. mixts. of ethylene glycol and DMSO (DMSO) at 298.15 K are reported. The Gibbs energies of solns. were detd. from soly. measurements of each amino acid at different temps., i.e., from 288.15 to 308.15 K by "formol titrimetry". The chem. parts of free energies (ΔG0t,ch(i)) and entropies (TΔS0t,ch(i)) of transfer of the homologous α-amino acids were computed by subtracting the cavity effects and dipole-dipole interaction effects from the total transfer energies. The characteristics of the solvation thermodn. of α-amino acids in EG-DMF and EG-ACN mixed solvent systems studied earlier are also discussed here for comparison.
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    Two different series of polyacrylamides contg. different amts. of salicylideneaniline moieties have been synthesized via a double polymer analogous reaction of poly(pentafluorophenyl acrylate) (PPFPA). All copolymers were designed to exhibit a lower crit. soln. temp. (LCST) in aq. soln., which was dependent on (i) the amt. of incorporated chromophoric salicylideneaniline groups and (ii) the isomerization state of the resp. salicylideneaniline group. Higher LCST values were measured for UV-irradiated solns. of the copolymers in comparison to the nonirradiated copolymer solns. A max. difference in the LCST of up to 13 °C was found for poly(N-cyclopropylacrylamide) copolymer contg. 15.0 mol % of salicylideneaniline groups. Within this temp. range, a reversible soly. change of the copolymer could be induced by irradn. with light.
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    Tailor-Made Protein Corona Formation on Polystyrene Microparticles and its Effect on Epithelial Cell Uptake
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    ACS Applied Materials & Interfaces (2022), 14 (41), 47277-47287CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)
    Microplastic particles are pollutants in the environment with a potential impact on ecol. and human health. As soon as microplastic particles get in contact with complex (biol.) environments, they will be covered by an eco- and/or protein corona. In this contribution, protein corona formation was conducted under defined lab. conditions on polystyrene (PS) microparticles to investigate the influence on surface properties, protein corona evolution, particle-cell interactions, and uptake in two murine epithelial cells. To direct protein corona formation, PS particles were preincubated with five model proteins, namely, bovine serum albumin (BSA), myoglobin, β-lactoglobulin, lysozyme, and fibrinogen. Subsequently, the single-protein-coated particles were incubated in a cell culture medium contg. a cocktail of serum proteins to analyze changes in the protein corona profile as well as in the binding kinetics of the model proteins. Therein, we could show that the precoating step has a crit. impact on the final compn. of the protein corona. Yet, since proteins building the primary corona were still detectable after addnl. incubations in a protein-contg. medium, backtracking of the particle's history is possible. Interestingly, whereas the precoating history significantly disturbs particle-cell interactions (PCIs), the cellular response (i.e., metabolic activity, MTT assay) stays unaffected. Of note, lysozyme precoating revealed one of the highest rates in PCI for both epithelial cell lines. Taken together, we could show that particle history has a significant impact on protein corona formation and subsequently on the interaction of particles with murine intestinal epithelial-like cells. However, as this study was limited to one cell type, further work is needed to assess if these observations can be generalized to other cell types.
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    A review. Thrombosis is a leading cause of death worldwide, necessitating the search for more effective and safer treatments. Current parenteral anticoagulant medications indicated for the prevention and treatment of thrombosis include polyanionic heparins. Despite their well-established status, these medications have drawbacks and fail to meet the criteria for an ideal anticoagulant. The predictability and control of heparin's biol. activity pose challenges due to the chaotic structure of this polymer, characterized by a mixt. of larger and smaller mols. with a loosely defined linear architecture. Consequently, cases of heparin overdose are common. Protamine sulfate can halt bleeding caused by unfractionated heparin but concurrently elicits significant adverse effects. Although novel antidotes for anticoagulants have been developed, bleeding during anticoagulation therapy remains a concern. Over the past two decades, research into heparin mimetics has witnessed a significant surge. This intensification is underscored by statistical evidence indicating an increase in thrombotic episodes, highlighting the necessity for new anticoagulant alternatives. This review outlines the efforts to replace heparins and their antidotes with block copolymers. We have concd. on studies involving well-defined synthetic anionic and cationic block copolymers, which are obtained by free radical polymn. techniques. Block copolymers comprised of anionic poly(sodium 4-styrenesulfonate) or poly(2-acryloylamido-2-methylpropanesulfonic acid) blocks and a neutral poly(ethylene glycol) block seem to be the most promising candidates for future anticoagulants. Complementary polycationic antidotes to regulate anticoagulant activity have also been proposed; however, further research focusing on structure-activity relationships and safety is necessary to confirm their utility.
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    Dailing, E. A.; Kilchrist, K. V.; Tierney, J. W.; Fletcher, R. B.; Evans, B. C.; Duvall, C. L. Modifying Cell Membranes with Anionic Polymer Amphiphiles Potentiates Intracellular Delivery of Cationic Peptides. ACS Appl. Mater. Interfaces 2020, 12 (45), 50222– 50235,  DOI: 10.1021/acsami.0c13304
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    Modifying Cell Membranes with Anionic Polymer Amphiphiles Potentiates Intracellular Delivery of Cationic Peptides
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    ACS Applied Materials & Interfaces (2020), 12 (45), 50222-50235CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)
    Rapid, facile, and noncovalent cell membrane modification with alkyl-grafted anionic polymers was sought as an approach to enhance intracellular delivery and bioactivity of cationic peptides. We synthesized a library of acrylic acid-based copolymers contg. varying amts. of an amine-reactive pentafluorophenyl acrylate monomer followed by postpolymn. modification with a series of alkyl amines to afford precise control over the length and d. of aliph. alkyl side chains. This synthetic strategy enabled systematic investigation of the effect of the polymer structure on membrane binding, potentiation of peptide cell uptake, pH-dependent disruption of lipid bilayers for endosome escape, and intracellular bioavailability. A subset of these polymers exhibited pKa of ~ 6.8, which facilitated stable membrane assocn. at physiol. pH and rapid, pH-dependent endosomal disruption upon endocytosis as quantified in Galectin-8-YFP reporter cells. Cationic cell penetrating peptide (CPP) uptake was enhanced up to 15-fold in vascular smooth muscle cells in vitro when peptide treatment was preceded by a 30-min pretreatment with lead candidate polymers. We also designed and implemented a new and highly sensitive assay for measuring the intracellular bioavailability of CPPs based on the NanoLuciferase (NanoLuc) technol. previously developed for measuring intracellular protein-protein interactions. Using this split luciferase class of assay, polymer pretreatment enhanced intracellular delivery of the CPP-modified HiBiT peptide up to 30-fold relative to CPP-HiBiT without polymer pretreatment (p < 0.05). The overall structural analyses show that polymers contg. 50:50 or 70:30 molar ratios of carboxyl groups to alkyl side chains of 6-8 carbons maximized peptide uptake, pH-dependent membrane disruption, and intracellular bioavailability and that this potentiation effect was maximized by pairing with CPPs with high cationic charge d. These results demonstrate a rapid, mild method for polymer modification of cell surfaces to potentiate intracellular delivery, endosome escape, and bioactivity of cationic peptides.
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    Richter, F.; Leer, K.; Martin, L.; Mapfumo, P.; Solomun, J. I.; Kuchenbrod, M. T.; Hoeppener, S.; Brendel, J. C.; Traeger, A. The Impact of Anionic Polymers on Gene Delivery: How Composition and Assembly Help Evading the Toxicity-Efficiency Dilemma. J. Nanobiotechnol. 2021, 19 (1), 292,  DOI: 10.1186/s12951-021-00994-2
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    The impact of anionic polymers on gene delivery: how composition and assembly help evading the toxicity-efficiency dilemma
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    Cationic polymers have been widely studied for non-viral gene delivery due to their ability to bind genetic material and to interact with cellular membranes. However, their charged nature carries the risk of increased cytotoxicity and interaction with serum proteins, limiting their potential in vivo application. Therefore, hydrophilic or anionic shielding polymers are applied to counteract these effects. Herein, a series of micelle-forming and micelle-shielding polymers were synthesized via RAFT polymn. The copolymer poly[(Bu acrylate)-b-(2-(di-Me amino)ethyl acrylamide)] (P(nBA-b-DMAEAm)) was assembled into cationic micelles and different shielding polymers were applied, i.e., poly(acrylic acid) (PAA), poly(4-acryloyl morpholine) (PNAM) or P(NAM-b-AA) block copolymer. These systems were compared to a triblock terpolymer micelle comprising PAA as the middle block. The assemblies were investigated regarding their morphol., interaction with pDNA, cytotoxicity, transfection efficiency, polyplex uptake and endosomal escape. The naked cationic micelle exhibited superior transfection efficiency, but increased cytotoxicity. The addn. of shielding polymers led to reduced toxicity. In particular, the triblock terpolymer micelle convinced with high cell viability and no significant loss in efficiency. The highest shielding effect was achieved by layering micelles with P(NAM-b-AA) supporting the colloidal stability at neutral zeta potential and completely restoring cell viability while maintaining moderate transfection efficiencies. The high potential of this micelle-layer-combination for gene delivery was illustrated for the first time.

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  • Abstract

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    Scheme 1

    Scheme 1. Schematic Representation of the Synthesis of Carboxylated Polyacrylamides with Tailored Hydrophobicity via PPM of PPFPA with Indicated Amino Acids, Yielding Anionic Polymers with Varying Alkyl Substituentsa
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    aPlease note that Gly does not feature a stereo center.

    Figure 1

    Figure 1. (A) FTIR spectra and (B) 1H NMR spectra (300 MHz) of poly(pentafluorophenyl acrylate) (green, CDCl3) and P(Nva-OH-AAm) (blue, d-DPBS).

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    Figure 2

    Figure 2. Size-exclusion chromatograms (0.07 M aq. Na2HPO4) of P(AA–OH-Aam). (A) Effect of increasing alkyl chain length. (B) Effect of alkyl branching. (C) Effect of alkyl branching position.

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    Figure 3

    Figure 3. Hydrophilicity of P(AA–OH-AAm). (A) HPLC chromatograms (acetonitrile/water gradient, 0.1v% TFA). Spectra show the absolute fluorescence intensity of eluting 6AF-labeled polymers (λex = 480 nm; λem = 520 nm). (B) pH-dependent relative hydrophilicity of 6AF-labeled polymers. Columns show the reduction of fluorescence of a 0.1 mg mL–1 aqueous solution at the indicated pH value after treatment with CHCl3 compared with the fluorescence of the sample prior to treatment with CHCl3. For normalized fluorescence graphs, please refer to Figures S12 and S13.

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    Figure 4

    Figure 4. Interaction of P(Nva-OH-AAm) with BSA and Lysozyme. P(Nva-OH-AAm) (c = 0.5 mg mL–1) and indicated proteins (c = 0.5 mg mL–1) were incubated in DPBS at 37 °C for indicated durations. Number mean diameter (black) and mean count rate (gray) were analyzed by DLS measurements at 37 °C. Values (scatter) represent the mean and SD of five measurements with three runs each. Dashed lines do not represent measured values.

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    Figure 5

    Figure 5. Cellular association of different P(AA–OH-AAm)-6AF determined via flow cytometry measurements. Polymer concentration: 1 mMRU, 50,000 cells seeded in 250 μL in a 48 well plate. Normalized MFI was calculated by eq 3. (A, B) Association of polymers with L929 mouse fibroblasts in DMEM supplemented with 10% FBS. Incubation for indicated time points at 37 °C. (C) Incubation at 37 or 4 °C in DMEM supplemented with 10% FBS for 4 h. Reduction was calculated by eq 4. Statistical analysis (one-way ANOVA with Tukey test) was performed for 4 h incubation time (A, B) to compare the association efficiency of the different P(AA–OH-AAm)-6AF. For temperature-dependent experiments (C), the statistical analysis compared the association at 37 °C versus 4 °C per polymer type. *Indicates a significance of p < 0.5. All other comparisons were not statistically significant at p < 0.5.

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    Figure 6

    Figure 6. Cellular uptake of P(Nva-OH-AAm)-6AF into L929 mouse fibroblasts visualized by CLSM. Cells were incubated with polymers at 37 °C for indicated times. Cyan: Hoechst 33342/nucleus. Red: CellMask Plasma Membrane Stain/membrane. Blue: 6AF/polymer.

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  • References

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      A review. Depending on the context, nanotechnologies developed as nanomedicines (nanosized therapeutics and imaging agents) are presented as either a remarkable technol. revolution already capable of delivering new diagnostics, treatments for unmanageable diseases, and opportunities for tissue repair or highly dangerous nanoparticles, nanorobots, or nanoelectronic devices that will wreak havoc in the body. The truth lies firmly between these two extremes. Rational design of "nanomedicines" began almost half a century ago, and >40 products have completed the complex journey from lab to routine clin. use. Here we critically review both nanomedicines in clin. use and emerging nanosized drugs, drug delivery systems, imaging agents, and theranostics with unique properties that promise much for the future. Key factors relevant to the design of practical nanomedicines and the regulatory mechanisms designed to ensure safe and timely realization of healthcare benefits are discussed.
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      The use of nanoparticles as drug carriers has provided a powerful weapon in the fight against cancer. These nanocarriers are able to transport drugs that exhibit very different nature such as lipophilic or hydrophilic drugs and big macromols. as proteins or RNA. Moreover, the external surface of these carriers can be decorated with different moieties with high affinity for specific membrane receptors of the tumoral cells to direct their action specifically to the malignant cells. The selectivity improvement yielded by these nanocarriers provided a significative enhancement in the efficacy of the transported drug, while the apparition of side effects in the host was reduced. Addnl., it is possible to incorporate targeting moieties selective for organelles of the cell, which improves even more the effect of the transported agents. In the last years, more sophisticated strategies such as the use of switchable, hierarchical or double targeting strategies have been proposed for overcoming some of the limitations of conventional targeting strategies. In this review, recent advances in the development of targeted nanoparticles will be described with the aim to present the current state of the art of this technol. and its huge potential in the oncol. field.
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      We synthesized lysine- and phenylalanine-based zwitterionic amino acid polymers (ZAPs) and evaluated their recognition capability for amino acid transporters (AATs), which are overexpressed on cancer cells as compared with normal cells. This study clearly demonstrates that ZAPs are internalized via AAT-mediated endocytosis with no cytotoxicity owing to their zwitterionic amino acid nature.
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      Biomaterials (2019), 217 (), 119299CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
      A review. Charge-convertible polymers are a class of intelligent polymers that can convert their charges in response to a certain stimulus in their environment. This unique property endows charge-convertible polymer-based biomaterials with great advantages in the treatment of disease. Drug-loaded charge-convertible polymeric nanoparticles have the ability to target tumor cells by converting their surface charges from neg. or neutral to pos. at the tumor site. In addn., charge-convertible polymeric biomaterials can form complexes with neg. charged therapeutic agents and release them through charge conversion at the desired time and site. In this review, the properties of charge-convertible polymers and their applications in the treatment of cancer and stroke are covered. More importantly, the limitations and perspectives of charge-convertible polymeric biomaterials in future clin. applications are discussed.
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      Design considerations for tumor-targeted nanoparticles
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      Nature Nanotechnology (2010), 5 (1), 42-47CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
      Inorg./org. hybrid nanoparticles are potentially useful in biomedicine, but to avoid non-specific background fluorescence and long-term toxicity, they need to be cleared from the body within a reasonable timescale. Previously, we have shown that rigid spherical nanoparticles such as quantum dots can be cleared by the kidneys if they have a hydrodynamic diam. of approx. 5.5 nm and a zwitterionic surface charge. Here, we show that quantum dots functionalized with high-affinity small-mol. ligands that target tumors can also be cleared by the kidneys if their hydrodynamic diam. is less than this value, which sets an upper limit of 5-10 ligands per quantum dot for renal clearance. Animal models of prostate cancer and melanoma show receptor-specific imaging and renal clearance within 4 h post-injection. This study suggests a set of design rules for the clin. translation of targeted nanoparticles that can be eliminated through the kidneys.
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      Theodorou, I.; Anilkumar, P.; Lelandais, B.; Clarisse, D.; Doerflinger, A.; Gravel, E.; Ducongé, F.; Doris, E. Stable and Compact Zwitterionic Polydiacetylene Micelles with Tumor-Targeting Properties. Chem. Commun. 2015, 51 (80), 14937– 14940,  DOI: 10.1039/C5CC05333A
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      Stable and compact zwitterionic polydiacetylene micelles with tumor-targeting properties
      Theodorou, Ioanna; Anilkumar, Parambath; Lelandais, Benoit; Clarisse, Damien; Doerflinger, Anaelle; Gravel, Edmond; Duconge, Frederic; Doris, Eric
      Chemical Communications (Cambridge, United Kingdom) (2015), 51 (80), 14937-14940CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      Compact polymd. polydiacetylene-micelles with "stealth" zwitterionic surface coating were assembled and tested in a murine xenograft model of breast cancer. In vivo fluorescence imaging indicated accumulation in the tumor area and histol. studies revealed predominant uptake of the micelles at the margins of the tumor, thereby allowing the delineation of its vol.
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      Mahmoud, A. M.; de Jongh, P. A. J. M.; Briere, S.; Chen, M.; Nowell, C. J.; Johnston, A. P. R.; Davis, T. P.; Haddleton, D. M.; Kempe, K. Carboxylated Cy5-Labeled Comb Polymers Passively Diffuse the Cell Membrane and Target Mitochondria. ACS Appl. Mater. Interfaces 2019, 11 (34), 31302– 31310,  DOI: 10.1021/acsami.9b09395
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      Carboxylated Cy5-Labeled Comb Polymers Passively Diffuse the Cell Membrane and Target Mitochondria
      Mahmoud, Ayaat M.; de Jongh, Patrick A. J. M.; Briere, Sibylle; Chen, Moore; Nowell, Cameron J.; Johnston, Angus P. R.; Davis, Thomas P.; Haddleton, David M.; Kempe, Kristian
      ACS Applied Materials & Interfaces (2019), 11 (34), 31302-31310CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)
      A detailed understanding of the cellular uptake and trafficking of nanomaterials is essential for the design of "smart" intracellular drug delivery vehicles. Typically, cellular interactions can be tailored by endowing materials with specific properties, for example, through the introduction of charges or targeting groups. In this study, water-sol. carboxylated N-acylated poly(amino ester)-based comb polymers of different d.p. and side-chain modification were synthesized via a combination of spontaneous zwitterionic copolymn. and redox-initiated reversible addn.-fragmentation chain-transfer polymn. and fully characterized by 1H NMR spectroscopy and size exclusion chromatog. The comb polymers showed no cell toxicity against NIH/3T3 and N27 cell lines nor hemolysis. Detailed cellular assocn. and uptake studies by flow cytometry and confocal laser scanning microscopy (CLSM) revealed that the carboxylated polymers were capable of passively diffusing cell membranes and targeting mitochondria. The interplay of pendant carboxylic acids of the comb polymers and the Cy5-label was identified as major driving force for this behavior, which was demonstrated to be applicable in NIH/3T3 and N27 cell lines. Blocking of the carboxylic acids through modification with 2-methoxyethylamine and poly(2-ethyl-2-oxazoline) or replacement of the dye label with a different dye (e.g., fluorescein) resulted in an alteration of the cellular uptake mechanism toward endocytosis as demonstrated by CLSM. In contrast, partial modification of the carboxylic acid groups allowed to retain the cellular interaction, hence, rendering these comb polymers a highly functional mitochondria targeted carrier platform for future drug delivery applications and imaging purposes.
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      Wang, S. PH-Responsive Amphiphilic Carboxylate Polymers: Design and Potential for Endosomal Escape. Front. Chem. 2021, 9 (March), 1– 8,  DOI: 10.3389/fchem.2021.645297
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      Li, M.; Zhang, W.; Li, J.; Qi, Y.; Peng, C.; Wang, N.; Fan, H.; Li, Y. Zwitterionic Polymers: Addressing the Barriers for Drug Delivery. Chin. Chem. Lett. 2023, 34 (11), 108177  DOI: 10.1016/j.cclet.2023.108177
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      Zwitterionic polymers: Addressing the barriers for drug delivery
      Li, Muzi; Zhang, Wen; Li, Jiaxin; Qi, Yinghe; Peng, Chen; Wang, Nan; Fan, Huili; Li, Yan
      Chinese Chemical Letters (2023), 34 (11), 108177CODEN: CCLEE7; ISSN:1001-8417. (Elsevier B.V.)
      A review. Nanocarriers play an important role in drug delivery for disease treatment. However, nanocarriers face a series of physiol. barriers after administration such as blood clearance, nonspecific tissue/cell localization, poor cellular uptake, and endosome trapping. These physiol. barriers seriously reduce the accumulation of drugs in target action site, which results in poor therapeutic efficiency. Although polyethylene glycol (PEG) can increase the blood circulation time of nanocarriers, its application is limited due to the "PEG dilemma". Zwitterionic polymers have been emerging as an appealing alternative to PEG owing to their excellent performance in resisting nonspecific protein adsorption. Importantly, the diverse structures bring functional versatility to zwitterionic polymers beyond nonfouling. This review focuses on the structures and characters of zwitterionic polymers, and will discuss and summarize the application of zwitterionic polymers for drug delivery. We will highlight the strategies of zwitterionic polymers to address the physiol. barriers during drug delivery. Finally, we will give some suggestions that can be utilized for the development of zwitterionic polymers for drug delivery. This review will also provide an outlook for this field. Our aim is to provide a comprehensive and systemic review on the application of zwitterionic polymers for drug delivery and promote the development of zwitterionic polymers.
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      Monnery, B. D. Polycation-Mediated Transfection: Mechanisms of Internalization and Intracellular Trafficking. Biomacromolecules 2021, 22 (10), 4060– 4083,  DOI: 10.1021/acs.biomac.1c00697
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      Polycation-Mediated Transfection: Mechanisms of Internalization and Intracellular Trafficking
      Monnery, Bryn D.
      Biomacromolecules (2021), 22 (10), 4060-4083CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      Polyplex-mediated gene transfection is now in its' fourth decade of serious research, but the promise of polyplex-mediated gene therapy has yet to fully materialize. Only approx. one in a million applied plasmids actually expresses. A large part of this is due to an incomplete understanding of the mechanism of polyplex transfection. There is an assumption that internalization must follow a canonical mechanism of receptor mediated endocytosis. Herein, we present arguments that untargeted (and most targeted) polyplexes do not utilize these routes. By incorporating knowledge of syndecan-polyplex interactions, we can show that syndecans are the "target" for polyplexes. Further, it is known that free polycations (which disrupt cell-membranes by acid-catalyzed hydrolysis of phospholipid esters) are necessary for (untargeted) endocytosis. This can be incorporated into the model to produce a novel mechanism of endocytosis, which fits the obsd. phenomenol. After membrane translocation, polyplex contg. vesicles reach the endosome after diffusing through the actin mesh below the cell membrane. From there, they are acidified and trafficked toward the lysosome. Some polyplexes are capable of escaping the endosome and unpacking, while others are not. Herein, it is argued that for some polycations, as acidification proceeds the polyplexes excluding free polycations, which disrupt the endosomal membrane by acid-catalyzed hydrolysis, allowing the polyplex to escape. The polyplex's internal charge ratio is now insufficient for stability and it releases plasmids which diffuse to the nucleus. A small proportion of these plasmids diffuse through the nuclear pore complex (NPC), with aggregation being the major cause of loss. Those plasmids that have diffused through the NPC will also aggregate, and this appears to be the reason such a small proportion of nuclear plasmids express mRNA. Thus, the structural features which promote unpacking in the endosome and allow for endosomal escape can be detd., and better polycations can be designed.
    11. 11
      Agarwal, S.; Zhang, Y.; Maji, S.; Greiner, A. PDMAEMA Based Gene Delivery Materials. Mater. Today 2012, 15 (9), 388– 393,  DOI: 10.1016/S1369-7021(12)70165-7
      11
      PDMAEMA based gene delivery materials
      Agarwal, Seema; Zhang, Yi; Maji, Samarendra; Greiner, Andreas
      Materials Today (Oxford, United Kingdom) (2012), 15 (9), 388-393CODEN: MTOUAN; ISSN:1369-7021. (Elsevier Ltd.)
      A review. Gene transfection is the transfer of genetic material like DNA into cells. Cationic polymers which form nanocomplexes with DNA, so-called non-viral gene vectors, are a highly promising platform for efficient gene transfection. Despite intensive research efforts and some of the on-going clin. trials on gene transfection, none of the existing cationic polymer systems are generally acceptable for human gene therapy. Since the process of gene transfection is complex and puts different challenges and demands on the delivery system, there is a strong requirement for the design and development of a multifunctional system in a simple way. This review will discuss recent efforts in design, synthesis, and performance of poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) nanocomplexes with DNA.
    12. 12
      Yessine, M. Membrane-Destabilizing Polyanions: Interaction with Lipid Bilayers and Endosomal Escape of Biomacromolecules. Adv. Drug Delivery Rev. 2004, 56 (7), 999– 1021,  DOI: 10.1016/j.addr.2003.10.039
      12
      Membrane-destabilizing polyanions: interaction with lipid bilayers and endosomal escape of biomacromolecules
      Yessine, Marie-Andree; Leroux, Jean-Christophe
      Advanced Drug Delivery Reviews (2004), 56 (7), 999-1021CODEN: ADDREP; ISSN:0169-409X. (Elsevier Science B.V.)
      A review. Water-sol. synthetic polyanions are employed nowadays in a multitude of industrial and biomedical applications and are studied extensively as simplified models of natural polyelectrolytes. The most interesting feature of carboxylated polymers is undoubtedly their ability to undergo coil-to-globule conformational change upon a decrease in pH of the surrounding environment. Over the years, scientists have gained better insights into the conformational behavior of these polymers in soln. and in the presence of membrane bilayers. In addn., when used as protein models, anionic polyelectrolytes can provide valuable information on physiol. processes such as domain formation in biol. membranes. Recently, polyanions have been evaluated as part of drug delivery systems, either as complexes/conjugates with biomols., or in the prepn. of pH-sensitive liposomal formulations. This article reviews the fundamental and practical aspects of pH-responsive synthetic polyanions in drug delivery. The pH-dependent conformational behavior of these polymers in aq. soln. is described in detail using poly(methacrylic acid) as the model polymer. Since binding to cellular membranes is a fundamental issue in understanding the mechanism of action of polyanions in cytoplasmic drug delivery, studies characterizing their interactions with phospholipid bilayers at neutral as well as at acidic pH are reviewed. Finally, pH-responsive delivery systems based on these polymers are described. As the conformational properties of pH-sensitive polyanions can be easily modulated by carefully adjusting their compn., such formulations may represent an attractive strategy to improve the escape of active biomols. from acidic endosomal compartments.
    13. 13
      Behr, J.-P. The Proton Sponge: A Trick to Enter Cells the Viruses Did Not Exploit. Chimia Aarau 1997, 51 (1–2), 34,  DOI: 10.2533/chimia.1997.34
      13
      The proton sponge. A trick to enter cells the viruses did not exploit
      Behr, Jean Paul
      Chimia (1997), 51 (1/2), 34-36CODEN: CHIMAD; ISSN:0009-4293. (Neue Schweizerische Chemische Gesellschaft)
      A review with 14 refs. on transfection and gene transfer mechanisms. Several non-permanent polycations possessing substantial buffering capacity below physiol. pH, such as lipopolyamines and polyethylenimines, are efficient transfection agents per se, i.e. without the addn. of lysosomotropic bases, or cell targeting, or membrane disruption agents. These vectors deliver genes as well as oligonucleotides both in vitro and in vivo. Their efficiency may rely on extensive endosome swelling and rupture that provides an escape mechanism for the polycation/DNA particles.
    14. 14
      Bus, T.; Traeger, A.; Schubert, U. S. The Great Escape: How Cationic Polyplexes Overcome the Endosomal Barrier. J. Mater. Chem. B 2018, 6 (43), 6904– 6918,  DOI: 10.1039/C8TB00967H
      14
      The great escape: how cationic polyplexes overcome the endosomal barrier
      Bus, Tanja; Traeger, Anja; Schubert, Ulrich S.
      Journal of Materials Chemistry B: Materials for Biology and Medicine (2018), 6 (43), 6904-6918CODEN: JMCBDV; ISSN:2050-7518. (Royal Society of Chemistry)
      A review. The targeted and efficiency-oriented delivery of (therapeutic) nucleic acids raises hope for successful gene therapy, i.e., for the local and individual treatment of acquired and inherited genetic disorders. Despite promising achievements in the field of polymer-mediated gene delivery, the efficiency of the non-viral vectors remains orders of magnitude lower than viral-mediated ones. Several obstacles on the mol. and cellular level along the gene delivery process were identified, starting from the design and formulation of the nano-sized carriers up to the targeted release to their site of action. In particular, the efficient escape from endo-lysosomal compartments was demonstrated to be a major barrier and its exact mechanism still remains unclear. Different hypotheses and theories of the endosomal escape were postulated. The most popular one is the so-called "proton sponge" hypothesis, claiming an escape by rupture of the endosome through osmotic swelling. It was the first effort to explain the excellent transfection efficiency of poly(ethylene imine). Moreover, it was thought that a unique mechanism based on the ability to capture protons and to buffer the endosomal pH is the basis of endosomal escape. Recent theories deal with the direct interaction of the cationic polyplex or free polymer with the exoplasmic lipid leaflet causing membrane destabilization, permeability or polymer-supported nanoscale hole formation. Both escape strategies are more related to viral-mediated escape compared to the "proton sponge" effect. This review addresses the different endosomal release theories and highlights their key mechanism.
    15. 15
      Braksch, C. P.; Lehnen, A. C.; Bapolisi, A. M.; Gurke, J.; De Breuck, J.; Leiske, M. N.; Hartlieb, M. Staphylococcus Aureus Selective Antimicrobial Polymers Based on an Arginine-Derived Monomer. J. Polym. Sci. 2024, 62 (1), 132– 145,  DOI: 10.1002/pol.20230591
      15
      Staphylococcus aureus selective antimicrobial polymers based on an arginine-derived monomer
      Braksch, Cevin P.; Lehnen, Anne-Catherine; Bapolisi, Alain M.; Gurke, Johannes; De Breuck, Jonas; Leiske, Meike N.; Hartlieb, Matthias
      Journal of Polymer Science (Hoboken, NJ, United States) (2024), 62 (1), 132-145CODEN: JPSHBC; ISSN:2642-4169. (John Wiley & Sons, Inc.)
      The continuous spread of resistance genes among bacterial populations, in particular in hospital setting, threatens the advances of our medical systems and already costs more than 1 million lives per yr. Antimicrobial polymers (APs) are a promising type of materials to counteract these developments, as they are non-susceptible toward resistance development, and even target resistant bacteria. We herein show the synthesis of an arginine-contg. monomer and its polymn. via photo-induced reversible addn.-fragmentation chain-transfer polymn. The synthesis is straightforward, and the macromols. possess two charged functions (i.e., guanidinium and ammonium) per repeating unit after deprotection. Assessment of the biol. activity reveals that polymers inhibit the growth of methicillin-resistant Staphylococcus aureus (MRSA) while being relatively inactive against tested Gram-neg. strains. Surprisingly, among the tested polymers, the homopolymer of the arginine-derived monomer is the best-performing material, even though it possesses no notable hydrophobic units. Toxicity tests against red blood cells and other mammalian cells show a good biocompatibility of polymers, leading to an overall excellent selectivity. Using membrane models, the mechanism of action was found to be membrane disruption. The good selectivity of the herein presented polymer for MRSA makes them promising materials for the development of therapeutic agents against such infections.
    16. 16
      Lehnen, A. C.; Kogikoski, S.; Stensitzki, T.; AlSawaf, A.; Bapolisi, A. M.; Wolff, M.; De Breuck, J.; Müller-Werkmeister, H. M.; Chiantia, S.; Bald, I.; Leiske, M. N.; Hartlieb, M. Anisotropy in Antimicrobial Bottle Brush Copolymers and Its Influence on Biological Activity. Adv. Funct. Mater. 2023, 2312651  DOI: 10.1002/adfm.202312651
      There is no corresponding record for this reference.
    17. 17
      Nayak, K.; Ghosh, P.; Khan, M. E. H.; De, P. Side-Chain Amino-Acid-Based Polymers: Self-Assembly and Bioapplications. Polym. Int. 2022, 71 (4), 411– 425,  DOI: 10.1002/pi.6278
      17
      Side-chain amino-acid-based polymers: self-assembly and bioapplications
      Nayak, Kasturee; Ghosh, Pooja; Khan, Md Ezaz Hasan; De, Priyadarsi
      Polymer International (2022), 71 (4), 411-425CODEN: PLYIEI; ISSN:0959-8103. (John Wiley & Sons Ltd.)
      A review. The design and development of side-chain amino-acid-based polymeric nanostructures have attracted a significant research interest as they bring a remarkable revolution in various territories of the biomedical field. The incorporation of amino acid moieties into the side chain of synthetic polymeric scaffolds exhibits several beneficial properties like aq. soly., chiral recognition, high biocompatibility, stimuli responsiveness, antifouling properties, as well as their capability to form higher ordered self-assembled architectures. Considering the important features and widespread applications of side-chain amino-acid-contg. polymers, here we shed light on the self-assembled characteristics of side-chain amino-acid-contg. polymers and their implications. The primary aim of this review article is to highlight the recent achievements of this bright area of research. We describe the numerous aspects of side-chain amino-acid-contg. polymers focusing mainly on self-assembly properties and the biomedical applications, which include drug and gene delivery, antimicrobial activity, antifouling coating, wound healing, tissue engineering etc. 2021 Society of Industrial Chem.
    18. 18
      Kauffman, W. B.; Fuselier, T.; He, J.; Wimley, W. C. Mechanism Matters: A Taxonomy of Cell Penetrating Peptides. Trends Biochem. Sci. 2015, 40 (12), 749– 764,  DOI: 10.1016/j.tibs.2015.10.004
      18
      Mechanism Matters: A Taxonomy of Cell Penetrating Peptides
      Kauffman, W. Berkeley; Fuselier, Taylor; He, Jing; Wimley, William C.
      Trends in Biochemical Sciences (2015), 40 (12), 749-764CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Ltd.)
      A review. The permeability barrier imposed by cellular membranes limits the access of exogenous compds. to the interior of cells. Researchers and patients alike would benefit from efficient methods for intracellular delivery of a wide range of membrane-impermeant mols., including biochem. active small mols., imaging agents, peptides, peptide nucleic acids, proteins, RNA, DNA, and nanoparticles. There has been a sustained effort to exploit cell penetrating peptides (CPPs) for the delivery of such useful cargoes in vitro and in vivo because of their biocompatibility, ease of synthesis, and controllable phys. chem. Here, we discuss the many mechanisms by which CPPs can function, and describe a taxonomy of mechanisms that could be help organize future efforts in the field.
    19. 19
      Ladavière, C.; Toustou, M.; Gulik-Krzywicki, T.; Tribet, C. Slow Reorganization of Small Phosphatidylcholine Vesicles upon Adsorption of Amphiphilic Polymers. J. Colloid Interface Sci. 2001, 241 (1), 178– 187,  DOI: 10.1006/jcis.2001.7675
      19
      Slow Reorganization of Small Phosphatidylcholine Vesicles upon Adsorption of Amphiphilic Polymers
      Ladaviere, C.; Toustou, M.; Gulik-Krzywicki, T.; Tribet, C.
      Journal of Colloid and Interface Science (2001), 241 (1), 178-187CODEN: JCISA5; ISSN:0021-9797. (Academic Press)
      Static or dynamic light scattering measurements were performed in parallel, on dil. mixts. of DPPC/DPPA vesicles (typical radius 60 nm) and hydrophobically modified polymers. This technique gave evidence of the slow kinetics involved in both the reorganization of an adsorbed polymer layer and the membrane breakage. Hours, or sometimes days, were required in order to follow the variation of both the hydrodynamic radius and the scattering intensity at intermediate stages. Images of the intermediate species were collected using freeze-fracture electron microscopy (FFEM). Comparison of different polymers (of varying mol. wt. or structure) revealed the prime importance of hydrophobicity on the disruption of membranes. Although the presence of a few percent of pendant alkyl chains along the polymer backbone induced adsorption to membranes, only the assocn. with the more hydrophobic ones (>25 mol% of pendant octyl groups) resulted in small mixed objects of micellar size (radius about 10 nm). The drop of the mean radius of intermediate structures formed upon the vesicle breakage was also sensitive to temp. A tentative mechanism was proposed on the basis of kinetics and FFEM studies. (c) 2001 Academic Press.
    20. 20
      Yessine, M.-A.; Lafleur, M.; Meier, C.; Petereit, H.-U.; Leroux, J.-C. Characterization of the Membrane-Destabilizing Properties of Different PH-Sensitive Methacrylic Acid Copolymers. Biochim. Biophys. Acta, Biomembr. 2003, 1613 (1–2), 28– 38,  DOI: 10.1016/S0005-2736(03)00137-8
      20
      Characterization of the membrane-destabilizing properties of different pH-sensitive methacrylic acid copolymers
      Yessine, Marie-Andree; Lafleur, Michel; Meier, Christian; Petereit, Hans-Ulrich; Leroux, Jean-Christophe
      Biochimica et Biophysica Acta, Biomembranes (2003), 1613 (1-2), 28-38CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
      The intracellular delivery of active biomacromols. from endosomes into the cytoplasm generally requires a membrane-disrupting agent. Since endosomes have a slightly acidic pH, anionic carboxylated polymers could be potentially useful for this purpose since they can destabilize membrane bilayers by pH-triggered conformational change. In this study, five different pH-sensitive methacrylic acid (MAA) copolymers were characterized with respect to their physicochem. and membrane lytic properties as a function of pH. pH-dependent conformational changes were studied in aq. soln. by turbidimetry and spectrofluorimetry. The hydrophobic domains that formed upon a decrease in pH were found to be dependent on copolymer's compn. Hemolysis and cytotoxicity assays demonstrated that the presence of the hydrophobic Et acrylate monomer and/or sufficient protonation of the carboxylic acid groups were important parameters for efficient membrane destabilization. Excessive copolymer hydrophobicity was not assocd. with membrane destabilization, but resulted in high macrophage cytotoxicity. Overall, this study gave more insights into the structure-activity relationship of MAA copolymers with membrane bilayers. Gaining knowledge of modulation of the physicochem. properties of copolymers and the optimization of copolymer-lipid interactions may lead to the elaboration of much more efficient drug delivery systems.
    21. 21
      Leiske, M. N.; De Geest, B. G.; Hoogenboom, R. Impact of the Polymer Backbone Chemistry on Interactions of Amino-Acid-Derived Zwitterionic Polymers with Cells. Bioact. Mater. 2023, 24 (January), 524– 534,  DOI: 10.1016/j.bioactmat.2023.01.005
      21
      Impact of the polymer backbone chemistry on interactions of amino-acid-derived zwitterionic polymers with cells
      Leiske, Meike N.; De Geest, Bruno G.; Hoogenboom, Richard
      Bioactive Materials (2023), 24 (), 524-534CODEN: BMIAD4; ISSN:2452-199X. (KeAi Communications Co., Ltd.)
      Zwitterionic polymers are known to interact with cells and have been shown to reveal cancer cell specificity. In this work, the importance of the chem. of the polymer backbone for the cellular specificity of amino-acid-derived polyzwitterions is demonstrated. A series of glutamic acid (Glu)-based vinyl monomers (i.e., an acrylate, a methacrylate, an acrylamide, and a methacrylamide) were prepd. and used for reversible addn.-fragmentation chain-transfer (RAFT) polymn., yielding defined polymers with narrow size distribution (ETH < 1.3). All Glu-functionalised, zwitterionic polymers revealed high cytocompatibility; however, differences in cellular assocn. and specificity were obsd. In particular, the methacrylamide-derived polymers showed high assocn. with both, breast cancer cells and non-cancerous dendritic cells and, consequently, lack specificity. In contrast, high specificity to only breast cancer cells was obsd. for polyacrylates, -methacrylates, and -acrylamides. Detailed anal. of the polymers revealed differences in hydrophobicity, zeta potential, and potential side chain hydrolysis, which are impacted by the polymer backbone and might be responsible for the altered the cell assocn. of these polymers. It is shown that a slightly neg. net charge is preferred over a neutral charge to retain cell specificity. This was also confirmed by assocn. expts. in the presence of competitive amino acid transporter substrates. The affinity of slightly neg. charged Glu-derived polymers to the xCT Glu/cystine cell membrane antiporter was found to be higher than that of neutrally charged polymers. Our results emphasize the importance of the polymer backbone for the design of cell-specific polymers. This study further highlights the potential to tailor amino-acid-derived zwitterionic materials beyond their side chain functionality.
    22. 22
      Wang, S.; Chen, R. PH-Responsive, Lysine-Based, Hyperbranched Polymers Mimicking Endosomolytic Cell-Penetrating Peptides for Efficient Intracellular Delivery. Chem. Mater. 2017, 29 (14), 5806– 5815,  DOI: 10.1021/acs.chemmater.7b00054
      22
      pH-Responsive, Lysine-Based, Hyperbranched Polymers Mimicking Endosomolytic Cell-Penetrating Peptides for Efficient Intracellular Delivery
      Wang, Shiqi; Chen, Rongjun
      Chemistry of Materials (2017), 29 (14), 5806-5815CODEN: CMATEX; ISSN:0897-4756. (American Chemical Society)
      The insufficient delivery of biomacromol. therapeutic agents into the cytoplasm of mammalian cells remains a major barrier to their pharmaceutical applications. Cell-penetrating peptides (CPPs) are considered as potential carriers for cytoplasmic delivery of macromol. drugs. However, due to the pos. charge of most CPPs, strong nonspecific cell membrane bindings may lead to relatively high toxicity. In this study, we report a series of anionic, CPP-mimicking, lysine-based hyperbranched polymers, which caused complete membrane disruption at late endosomal pH while remaining nonlytic at physiol. pH. The pH-responsive conformational alterations and the multivalency effect of the hyperbranched structures were demonstrated to effectively facilitate their interaction with cell membranes, thus leading to significantly enhanced membrane-lytic activity compared with their linear counterpart. The unique structures and pH-responsive cell-penetrating abilities make the novel hyperbranched polymers promising candidates for cytoplasmic delivery of biomacromol. payloads.
    23. 23
      Chen, S.; Wang, S.; Kopytynski, M.; Bachelet, M.; Chen, R. Membrane-Anchoring, Comb-Like Pseudopeptides for Efficient, PH-Mediated Membrane Destabilization and Intracellular Delivery. ACS Appl. Mater. Interfaces 2017, 9 (9), 8021– 8029,  DOI: 10.1021/acsami.7b00498
      23
      Membrane-Anchoring, Comb-Like Pseudopeptides for Efficient, pH-Mediated Membrane Destabilization and Intracellular Delivery
      Chen, Siyuan; Wang, Shiqi; Kopytynski, Michal; Bachelet, Marie; Chen, Rongjun
      ACS Applied Materials & Interfaces (2017), 9 (9), 8021-8029CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)
      Endosomal release has been identified as a rate-limiting step for intracellular delivery of therapeutic agents, in particular macromol. drugs. Herein, we report a series of synthetic pH-responsive, membrane-anchoring polymers exhibiting dramatic endosomolytic activity for efficient intracellular delivery. The comb-like pseudopeptidic polymers were synthesized by grafting different amts. of decylamine (NDA), which act as hydrophobic membrane anchors, onto the pendant carboxylic acid groups of a pseudopeptide, poly(L-lysine iso-phthalimide). The effects of the hydrophobic relatively long alkyl side chains on aq. soln. properties, cell membrane destabilization activity, and in-vitro cytotoxicity were investigated. The optimal polymer contg. 18 mol % NDA exhibited limited hemolysis at pH 7.4 but induced nearly complete membrane destabilization at endosomal pH within only 20 min. The mechanistic investigation of membrane destabilization suggests the polymer-mediated pore formation. It has been demonstrated that the polymer with hydrophobic side chains displayed a considerable endosomolytic ability to release endocytosed materials into the cytoplasm of various cell lines, which is of crit. importance for intracellular drug delivery applications.
    24. 24
      Lehnen, A.-C.; Gurke, J.; Bapolisi, A. M.; Reifarth, M.; Bekir, M.; Hartlieb, M. Xanthate-Supported Photo-Iniferter (XPI)-RAFT Polymerization: Facile and Rapid Access to Complex Macromolecules. Chem. Sci. 2023, 14 (3), 593– 603,  DOI: 10.1039/D2SC05197D
      24
      Xanthate-supported photo-iniferter (XPI)-RAFT polymerization: facile and rapid access to complex macromolecules
      Lehnen, Anne-Catherine; Gurke, Johannes; Bapolisi, Alain M.; Reifarth, Martin; Bekir, Marek; Hartlieb, Matthias
      Chemical Science (2023), 14 (3), 593-603CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      Xanthate-supported photo-iniferter (XPI)-reversible addn.-fragmentation chain-transfer (RAFT) polymn. is introduced as a fast and versatile photo-polymn. strategy. Small amts. of xanthate are added to conventional RAFT polymns. to act as a photo-iniferter under light irradn. Radical exchange is facilitated by the main CTA ensuring control over the mol. wt. distribution, while xanthate enables an efficient photo-(re)activation. The photo-active moiety is thus introduced into the polymer as an end group, which makes chain extension of the produced polymers possible directly by irradn. This is in sharp contrast to conventional photo-initiators, or photo electron transfer (PET)-RAFT polymns., where radical generation depends on the added small mols. In contrast to regular photo-iniferter-RAFT polymn., photo-activation is decoupled from polymn. control, rendering XPI-RAFT an elegant tool for the fabrication of defined and complex macromols. The method is oxygen tolerant and robust and was used to perform screenings in a well-plate format, and it was even possible to produce multiblock copolymers in a coffee mug under open-to-air conditions. XPI-RAFT does not rely on highly specialized equipment and qualifies as a universal tool for the straightforward synthesis of complex macromols. The method is user-friendly and broadens the scope of what can be achieved with photo-polymn. techniques.
    25. 25
      Martin, L.; Gody, G.; Perrier, S. Preparation of Complex Multiblock Copolymers via Aqueous RAFT Polymerization at Room Temperature. Polym. Chem. 2015, 6 (27), 4875– 4886,  DOI: 10.1039/C5PY00478K
      25
      Preparation of complex multiblock copolymers via aqueous RAFT polymerization at room temperature
      Martin, Liam; Gody, Guillaume; Perrier, Sebastien
      Polymer Chemistry (2015), 6 (27), 4875-4886CODEN: PCOHC2; ISSN:1759-9962. (Royal Society of Chemistry)
      We describe a simple new approach towards complex multiblock copolymer architectures at ambient temps. Using the redox pair TBHP/AsAc for initiation, aq. reversible addn.-fragmentation chain transfer (RAFT) polymn. is used to develop a one-pot sequential monomer addn. process that yields high order multiblock copolymers at 25 °C. Working at ambient temp. permits the polymn. of monomers yielding polymers with lower crit. soln. temp. (LCST) and reduces the risk of side reactions by chain transfer. Our approach is initially demonstrated with the prepn. of well-defined low and high d.p. (DP) poly(4-acryloylmorpholine) (PNAM) and poly(2-hydroxyethyl acrylate) (PHEA) multiblock homopolymers with D under 1.35. To highlight the potential of our approach, more challenging multiblock copolymers were prepd.: a pentablock copolymer of high DP (an av. of 100 per block) including low LCST blocks of poly(N-isopropylacrylamide) (PNIPAM) and poly(N,N-diethylacrylamide) (PDEA), two polyacrylate multiblock copolymers (DP of 10 per block) using a range of different functional acrylate monomers, and a heptablock copolymer (DP 10 per block) consisting of both polyacrylate and polyacrylamido blocks, all with a final dispersity of around 1.3.
    26. 26
      Mori, H.; Endo, T. Amino-Acid-Based Block Copolymers by RAFT Polymerization. Macromol. Rapid Commun. 2012, 33 (13), 1090– 1107,  DOI: 10.1002/marc.201100887
      26
      Amino-Acid-Based Block Copolymers by RAFT Polymerization
      Mori, Hideharu; Endo, Takeshi
      Macromolecular Rapid Communications (2012), 33 (13), 1090-1107CODEN: MRCOE3; ISSN:1022-1336. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. This review summarizes recent advances in the design and synthesis of amino-acid-based block copolymers by reversible addn.-fragmentation chain transfer (RAFT) polymn. of amino-acid-bearing monomers. We will mainly focus on stimuli-responsive block copolymers, such as pH-, thermo-, and dual-stimuli-responsive block copolymers, and self-assembled block copolymers, including amphiphilic and double-hydrophilic block copolymers having tunable chiroptical properties. We will also highlight recent results in RAFT synthesis of amino-acid-based copolymers having various properties, such as catalytic and optoelectronic properties, crosslinked block copolymer micelles, unimol. micelles, and org.-inorg. hybrids.
    27. 27
      Leiske, M. N.; Singha, R.; Jana, S.; De Geest, B. G.; Hoogenboom, R. Amidation of Methyl Ester-Functionalised Poly(2-Oxazoline)s as a Powerful Tool to Create Dual PH- and Temperature-Responsive Polymers as Potential Drug Delivery Systems. Polym. Chem. 2023, 14 (17), 2034– 2044,  DOI: 10.1039/D3PY00050H
      27
      Amidation of methyl ester-functionalised poly(2-oxazoline)s as a powerful tool to create dual pH- and temperature-responsive polymers as potential drug delivery systems
      Leiske, Meike N.; Singha, Ronak; Jana, Somdeb; De Geest, Bruno G.; Hoogenboom, Richard
      Polymer Chemistry (2023), 14 (17), 2034-2044CODEN: PCOHC2; ISSN:1759-9962. (Royal Society of Chemistry)
      In the past two decades, thermoresponsive polymers based on tertiary amine groups have been studied extensively as a class of dual-responsive polymers. In particular, their temp.-dependent phase transition can be further modulated by the pH value of the environment, rendering them interesting for applications in the biomedical area. In this contribution, we report methyl-ester-functionalised poly(2-alkyl-2-oxazoline)s (PAOx) as a versatile platform for the synthesis of tertiary-amine-functionalised PAOx via straightforward post-polymn. amidation. The resulting polymers were investigated regarding their stimuli-responsiveness to both, pH value and temp. by turbidimetry measurements. Dynamic light scattering further confirmed the formation of polymeric nanoparticles upon phase sepn. of block copolymers comprising a responsive polymer block and poly(2-methyl-2-oxazoline) as permanently hydrophilic block. Furthermore, a hydrophobic Rhodamine B deriv. was used as model cargo and was found to induce the formation of stable nanoparticles in biol. media beyond the responsiveness to pH and temp. of these polymers. Treatment of MDA-MB-231 breast cancer cells with such nanoparticles contg. both, the Rhodamine B octadecyl-deriv. and paclitaxel, suppressed their proliferation sufficiently in vitro. Altogether, PAOx with tertiary amines are presented as versatile materials with interesting characteristics and potential applications in the area of polymeric drug delivery.
    28. 28
      Das, A.; Theato, P. Activated Ester Containing Polymers: Opportunities and Challenges for the Design of Functional Macromolecules. Chem. Rev. 2016, 116 (3), 1434– 1495,  DOI: 10.1021/acs.chemrev.5b00291
      28
      Activated Ester Containing Polymers: Opportunities and Challenges for the Design of Functional Macromolecules
      Das, Anindita; Theato, Patrick
      Chemical Reviews (Washington, DC, United States) (2016), 116 (3), 1434-1495CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. The objective of this review is to provide the readers with an extensive overview of the advancement made in the area of polymeric activated ester chem. in designing precisely defined functionally diverse polymer structures, starting from utilizing classical polymn. techniques to state-of-the-art controlled polymn. methods and to highlight their potential applications in various interdisciplinary research areas. This review is divided into two parts. The first part will emphasize the development of activated ester chem. in constructing different reactive polymer structures where detailed information will be provided on various activated ester monomers being used under different polymn. techniques, and their advantages and drawbacks will be discussed explicitly. In the second part, focus will be on the versatility and fidelity of this chem. in fabricating numerous functional materials and their possible opportunities in different areas of application ranging from biomedical sciences to materials chem.
    29. 29
      Singha, N. K.; Gibson, M. I.; Koiry, B. P.; Danial, M.; Klok, H. A. Side-Chain Peptide-Synthetic Polymer Conjugates via Tandem “Ester-Amide/Thiol-Ene” Post-Polymerization Modification of Poly(Pentafluorophenyl Methacrylate) Obtained Using ATRP. Biomacromolecules 2011, 12 (8), 2908– 2913,  DOI: 10.1021/bm200469a
      29
      Side-Chain Peptide-Synthetic Polymer Conjugates via Tandem "Ester-Amide/Thiol-Ene" Post-Polymerization Modification of Poly(pentafluorophenyl methacrylate) Obtained Using ATRP
      Singha, Nikhil K.; Gibson, Matthew I.; Koiry, Bishnu P.; Danial, Maarten; Klok, Harm-Anton
      Biomacromolecules (2011), 12 (8), 2908-2913CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      Herein the concept of tandem postpolymn. modification as a versatile route to synthesize well-defined, highly functionalized polymers is introduced. Poly(pentafluorophenyl methacrylate) obtained by atom transfer radical polymn. was first modified with allylamine, which displaces the active ester to give well-defined polymers with pendant alkene groups, which are difficult to obtain by direct (radical) polymn. of allylic-functional monomers. The produced poly(allylmethacrylamide) was modified by a second postpolymn. modification reaction with a thiol-terminated peptide (CVPGVG) using AIBN as the radical source. NMR, IR, and SEC demonstrated successful conjugation onto the polymer to give a polymer-peptide hybrid material. This versatile strategy should extend the scope of controlled radical polymn. and "click"-type reactions.
    30. 30
      Gok, O.; Kosif, I.; Dispinar, T.; Gevrek, T. N.; Sanyal, R.; Sanyal, A. Design and Synthesis of Water-Soluble Multifunctionalizable Thiol-Reactive Polymeric Supports for Cellular Targeting. Bioconjugate Chem. 2015, 26 (8), 1550– 1560,  DOI: 10.1021/acs.bioconjchem.5b00182
      30
      Design and synthesis of water-soluble multifunctionalizable thiol-reactive polymeric supports for cellular targeting
      Gok, Ozgul; Kosif, Irem; Dispinar, Tugba; Gevrek, Tugce Nihal; Sanyal, Rana; Sanyal, Amitav
      Bioconjugate Chemistry (2015), 26 (8), 1550-1560CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      Design and synthesis of novel water-sol. polymers bearing reactive side chains are actively pursued due to their increasing demand in areas such as bioconjugation and drug delivery. This study reports the fabrication of poly(ethylene glycol) methacrylate based thiol-reactive water-sol. polymeric supports that can serve as targeted drug delivery vehicles. Thiol-reactive maleimide units were incorporated into polymers as side chains by use of a furan-protected maleimide contg. monomer. Atom transfer radical polymn. (ATRP) was employed to obtain a family of well-defined copolymers with narrow mol. wt. distributions. After the polymn., the maleimide groups were activated to their reactive form, ready for conjugation with thiol-contg. mols. Efficient functionalization of the maleimide moieties was demonstrated by conjugation of a tripeptide glutathione under mild and reagent-free aq. conditions. Addnl., hydrophobic thiol-contg. dye (Bodipy-SH) and a cyclic peptide-based targeting group (cRGDfC) were sequentially appended onto the maleimide bearing polymers to demonstrate their efficient multifunctionalization. The conjugates were utilized for in vitro expts. over both cancerous and healthy breast cell lines. Obtained results demonstrate that the conjugates were nontoxic, and displayed efficient cellular uptake. The presence of the peptide based targeting group had a clear effect on increasing the uptake of the dye-conjugated polymers into cells when compared to the construct devoid of the peptide. Overall, the facile synthesis and highly efficient multifunctionalization of maleimide-contg. thiol-reactive copolymers offer a novel and attractive class of polyethylene glycol-based water-sol. supports for drug delivery.
    31. 31
      Boyer, C.; Davis, T. P. One- Pot Synthesis and Biofunctionalization of Glycopolymers via RAFT Polymerization and Thiol–Ene Reactions. Chem. Commun. 2009, (40), 6029– 6031,  DOI: 10.1039/b910296e
      31
      One- pot synthesis and biofunctionalization of glycopolymers via RAFT polymerization and thiol-ene reactions
      Boyer, Cyrille; Davis, Thomas P.
      Chemical Communications (Cambridge, United Kingdom) (2009), (40), 6029-6031CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      A modification of a polymeric activated ester with sugar-amines and a simultaneous aminolysis-thiol-ene reaction involving the RAFT end-groups yield biotin functionalized glycopolymer in one-pot.
    32. 32
      Murata, H.; Prucker, O.; Rühe, J. Synthesis of Functionalized Polymer Monolayers from Active Ester Brushes. Macromolecules 2007, 40 (15), 5497– 5503,  DOI: 10.1021/ma0624550
      32
      Synthesis of Functionalized Polymer Monolayers from Active Ester Brushes
      Murata, Hironobu; Prucker, Oswald; Ruehe, Juergen
      Macromolecules (Washington, DC, United States) (2007), 40 (15), 5497-5503CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
      The synthesis of densely grafted monolayers of polymers carrying a wide spectrum of functional groups is described. To this surface-attached chains with active ester groups were generated through surface initiated polymn. of N-methacryloyl-β-alanine N'-oxysuccinimide ester (MAC2AE) using a self-assembled monolayer of an azo initiator on the surfaces of silicon oxide substrates. The layer thickness of the polymer monolayers can be easily controlled through the adjustment 15-crown of the monomer concn. during polymn. The aminolysis of the active ester groups within the brush with various n-alkylamines (C3-C18) was studied by IR and surface plasmon spectroscopy. Amines carrying addnl. functional groups such as amino acids, sugars, dyes, crown ethers, and amino-terminated polymers such as poly(ethylene glycol) derivs. can be used to incorporate many different functional moieties into the polymers.
    33. 33
      Moog, K. E.; Barz, M.; Bartneck, M.; Beceren-Braun, F.; Mohr, N.; Wu, Z.; Braun, L.; Dernedde, J.; Liehn, E. A.; Tacke, F.; Lammers, T.; Kunz, H.; Zentel, R. Polymeric Selectin Ligands Mimicking Complex Carbohydrates: From Selectin Binders to Modifiers of Macrophage Migration. Angew. Chemie - Int. Ed. 2017, 56 (5), 1416– 1421,  DOI: 10.1002/anie.201610395
      33
      Polymeric Selectin Ligands Mimicking Complex Carbohydrates: From Selectin Binders to Modifiers of Macrophage Migration
      Moog, Kai E.; Barz, Matthias; Bartneck, Matthias; Beceren-Braun, Figen; Mohr, Nicole; Wu, Zhuojun; Braun, Lydia; Dernedde, Jens; Liehn, Elisa A.; Tacke, Frank; Lammers, Twan; Kunz, Horst; Zentel, Rudolf
      Angewandte Chemie, International Edition (2017), 56 (5), 1416-1421CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Novel polymeric cell adhesion inhibitors were developed in which the selectin tetrasaccharide sialyl-LewisX (SLeX) is multivalently presented on a biocompatible poly(2-hydroxypropyl)methacrylamide (PHPMA) backbone either alone (P1) or in combination with O-sulfated tyramine side chains (P2). For comparison, corresponding polymeric glycomimetics were prepd. in which the crucial "single carbohydrate" substructures fucose, galactose, and sialic acid side chains were randomly linked to the PHPMA backbone (P3 or P4 (O-sulfated tyramine)). All polymers have an identical d.p., as they are derived from the same precursor polymer. Binding assays to selectins, to activated endothelial cells, and to macrophages show that polyHPMA with SLeX is an excellent binder to E-, L-, and P-selectins. However, mimetic P4 can also achieve close to comparable binding affinities in in vitro measurements and surprisingly, it also significantly inhibits the migration of macrophages; this provides new perspectives for the therapy of severe inflammatory diseases.
    34. 34
      Eberhardt, M.; Mruk, R.; Zentel, R.; Théato, P. Synthesis of Pentafluorophenyl(Meth)Acrylate Polymers: New Precursor Polymers for the Synthesis of Multifunctional Materials. Eur. Polym. J. 2005, 41 (7), 1569– 1575,  DOI: 10.1016/j.eurpolymj.2005.01.025
      34
      Synthesis of pentafluorophenyl (meth)acrylate polymers: New precursor polymers for the synthesis of multifunctional materials
      Eberhardt, Marc; Mruk, Ralf; Zentel, Rudolf; Theato, Patrick
      European Polymer Journal (2005), 41 (7), 1569-1575CODEN: EUPJAG; ISSN:0014-3057. (Elsevier B.V.)
      Pentafluorophenyl acrylate and -methacrylate were polymd. using AIBN as a thermal initiator. The obtained polymers were sol. polymeric active esters that could be used for the prepn. of multifunctional polymers. The reactivity of poly(pentafluorophenyl acrylate) and poly(pentafluorophenyl methacrylate) towards primary and secondary amines, as well as alcs. was investigated in a quant. way. Both poly(active esters) reacted satisfactorily with aliph. primary and secondary amines but only low conversion was found in the case of arom. amines. Conversions of only 30% were reached when poly(pentafluorophenyl acrylate) was treated with one equiv. of alc. under base catalysis. In time resolved FT-IR studies the rate consts. of the polymer analogous reactions were detd.
    35. 35
      Batz, H. G.; Franzmann, G.; Ringsdorf, H. Pharmakologisch Aktive Polymere, 5. Modellreaktionen Zur Umsetzung von Pharmaka Und Enzymen Mit Monomeren Und Polymeren Reaktiven Estern. Die Makromol. Chemie 1973, 172 (1), 27– 47,  DOI: 10.1002/macp.1973.021720103
      There is no corresponding record for this reference.
    36. 36
      Ferruti, P.; Bettelli, A.; Feré, A. High Polymers of Acrylic and Methacrylic Esters of N-Hydroxysuccinimide as Polyacrylamide and Polymethacrylamide Precursors. Polymer Guildf 1972, 13 (10), 462– 464,  DOI: 10.1016/0032-3861(72)90084-5
      There is no corresponding record for this reference.
    37. 37
      Choi, J.; Schattling, P.; Jochum, F. D.; Pyun, J.; Char, K.; Theato, P. Functionalization and Patterning of Reactive Polymer Brushes Based on Surface Reversible Addition and Fragmentation Chain Transfer Polymerization. J. Polym. Sci., Part A: Polym. Chem. 2012, 50 (19), 4010– 4018,  DOI: 10.1002/pola.26200
      37
      Functionalization and patterning of reactive polymer brushes based on surface reversible addition and fragmentation chain transfer polymerization
      Choi, Jiyeon; Schattling, Philipp; Jochum, Florian D.; Pyun, Jeffrey; Char, Kookheon; Theato, Patrick
      Journal of Polymer Science, Part A: Polymer Chemistry (2012), 50 (19), 4010-4018, S4010/1-S4010/3CODEN: JPACEC; ISSN:0887-624X. (John Wiley & Sons, Inc.)
      We present the synthesis of reactive polymer brushes prepd. by surface reversible addn.-fragmentation chain transfer polymn. of pentafluorophenyl acrylate. The reactive ester moieties can be used to functionalize the polymer brush film with virtually any functionality by simple post-polymn. modification with amines. Dithiobenzoic acid benzyl-(4-ethyltrimethoxylsilyl) ester was used as the surface chain transfer agent (S-CTA) and the anchoring group onto the silicon substrates. Reactive polymer brushes with adjustable mol. wt., high grafting d., and conformal coverage through the grafting-from approach were obtained. Subsequently, the reactive polymer brushes were converted with amino-spiropyrans resulting in reversible light-responsive polymer brush films. The wetting behavior could be altered by irradn. with UV or visible light. Furthermore, a patterned surface of polymer brushes was obtained using a lithog. technique. UV irradn. of the S-CTA-modified substrates leads to a selective degrdn. of S-CTA in the exposed areas and gives patterned activated polymer brushes after a subsequent RAFT polymn. step. Conversion of the patterned polymer brushes with 5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid resulted in patterned fluorescent polymer brush films. The utilization of reactive polymer brushes offers an easy approach in the fabrication of highly functional brushes, even for functionalities whose introduction is limited by other strategies. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012.
    38. 38
      Kockelmann, J.; Zentel, R.; Nuhn, L. Post-Polymerization Modifications to Prepare Biomedical Nanocarriers with Varying Internal Structures, Their Properties and Impact on Protein Corona Formation. Macromol. Chem. Phys. 2023, 224, 2300199  DOI: 10.1002/macp.202300199
      38
      Post-Polymerization Modifications to Prepare Biomedical Nanocarriers with Varying Internal Structures, their Properties and Impact on Protein Corona Formation
      Kockelmann, Johannes; Zentel, Rudolf; Nuhn, Lutz
      Macromolecular Chemistry and Physics (2023), 224 (24), 2300199CODEN: MCHPES; ISSN:1022-1352. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. The reactive ester approach provides access to various types of drug delivery systems. Either amphiphilic block copolymer micelles with hydrophobic cores can be generated for encapsulation of hydrophobic drugs, or they are (reversibly) crosslinked by polar mols. into nano(hydro)gel particles affording hydrophilic cores and coronas. Beyond short oligonucleotides complexation or covalent drug conjugation inside the core, a surface functionalization with targeting units is further possible to address a large variety of drug delivery scenarios. Interestingly, the reactive ester approach can thereby not only govern the nanocarriers inner structure and surface property, but at the same time also provide strategies to prevent protein corona formation. These features are summarized in this article and underline the concept of reactive ester macromols. as beneficial tool for assisting in drug delivery.
    39. 39
      De Coen, R.; Vanparijs, N.; Risseeuw, M. D. P.; Lybaert, L.; Louage, B.; De Koker, S.; Kumar, V.; Grooten, J.; Taylor, L.; Ayres, N.; Van Calenbergh, S.; Nuhn, L.; De Geest, B. G. PH-Degradable Mannosylated Nanogels for Dendritic Cell Targeting. Biomacromolecules 2016, 17 (7), 2479– 2488,  DOI: 10.1021/acs.biomac.6b00685
      39
      pH-Degradable Mannosylated Nanogels for Dendritic Cell Targeting
      De Coen, Ruben; Vanparijs, Nane; Risseeuw, Martijn D. P.; Lybaert, Lien; Louage, Benoit; De Koker, Stefaan; Kumar, Vimal; Grooten, Johan; Taylor, Leeanne; Ayres, Neil; Van Calenbergh, Serge; Nuhn, Lutz; De Geest, Bruno G.
      Biomacromolecules (2016), 17 (7), 2479-2488CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      We report on the design of glycosylated nanogels via core-crosslinking of amphiphilic non-water-sol. block copolymers composed of an acetylated glycosylated block and a pentafluorophenyl (PFP) activated ester block prepd. by reversible addn.-fragmentation (RAFT) polymn. Self-assembly, pH-sensitive core-crosslinking, and removal of remaining PFP esters and protecting groups are achieved in one pot and yield fully hydrated sub-100 nm nanogels. Using cell subsets that exhibit high and low expression of the mannose receptor (MR) under conditions that suppress active endocytosis, we show that mannosylated but not galactosylated nanogels can efficiently target the MR that is expressed on the cell surface of primary dendritic cells (DCs). These nanogels hold promise for immunol. applications involving DCs and macrophage subsets.
    40. 40
      Duret, D.; Haftek-Terreau, Z.; Carretier, M.; Ladavière, C.; Charreyre, M.-T.; Favier, A. Fluorescent RAFT Polymers Bearing a Nitrilotriacetic Acid (NTA) Ligand at the α-Chain-End for the Site-Specific Labeling of Histidine-Tagged Proteins. Polym. Chem. 2017, 8 (10), 1611– 1615,  DOI: 10.1039/C6PY02222G
      40
      Fluorescent RAFT polymers bearing a nitrilotriacetic acid (NTA) ligand at the α-chain-end for the site-specific labeling of histidine-tagged proteins
      Duret, Damien; Haftek-Terreau, Zofia; Carretier, Matthieu; Ladaviere, Catherine; Charreyre, Marie-Therese; Favier, Arnaud
      Polymer Chemistry (2017), 8 (10), 1611-1615CODEN: PCOHC2; ISSN:1759-9962. (Royal Society of Chemistry)
      Site-specific labeling of proteins is a valuable tool to study biol. processes. We show that it can be achieved using fluorescent polymer probes synthesized by RAFT polymn. Thanks to an original control agent functionalized with a nitrilotriacetic acid (NTA) ligand, an α-NTA polymer probe was prepd. to selectively label histidine-tagged recombinant proteins.
    41. 41
      Cepraga, C.; Gallavardin, T.; Marotte, S.; Lanoë, P. H.; Mulatier, J. C.; Lerouge, F.; Parola, S.; Lindgren, M.; Baldeck, P. L.; Marvel, J.; Maury, O.; Monnereau, C.; Favier, A.; Andraud, C.; Leverrier, Y.; Charreyre, M. T. Biocompatible Well-Defined Chromophore-Polymer Conjugates for Photodynamic Therapy and Two-Photon Imaging. Polym. Chem. 2013, 4 (1), 61– 67,  DOI: 10.1039/C2PY20565C
      41
      Biocompatible well-defined chromophore-polymer conjugates for photodynamic therapy and two-photon imaging
      Cepraga, Cristina; Gallavardin, Thibault; Marotte, Sophie; Lanoe, Pierre-Henri; Mulatier, Jean-Christophe; Lerouge, Frederic; Parola, Stephane; Lindgren, Mikael; Baldeck, Patrice L.; Marvel, Jacqueline; Maury, Olivier; Monnereau, Cyrille; Favier, Arnaud; Andraud, Chantal; Leverrier, Yann; Charreyre, Marie-Therese
      Polymer Chemistry (2013), 4 (1), 61-67CODEN: PCOHC2; ISSN:1759-9962. (Royal Society of Chemistry)
      A versatile approach is introduced for the synthesis of well-defined, biocompatible conjugates combining two-photon chromophores and hydrophilic multifunctional polymers synthesized by RAFT controlled radical polymn. As an illustration, two different classes of conjugates carrying multiple fluorophores (based on an anthracene moiety, Anth) or photosensitizers (based on a dibromobenzene moiety, DBB) along the polymer chain were elaborated for bioimaging and photodynamic therapy (PDT) applications, resp. In both cases, the polymer greatly improved the soly. in biorelevant media as well as the cell uptake. Anth conjugates provided high quality fluorescence microscopy images using both one- and two-photon excitation. DBB conjugates potently induced the death of cancer cells upon photoactivation.
    42. 42
      Engler, A. C.; Chan, J. M. W.; Coady, D. J.; O’Brien, J. M.; Sardon, H.; Nelson, A.; Sanders, D. P.; Yang, Y. Y.; Hedrick, J. L. Accessing New Materials through Polymerization and Modification of a Polycarbonate with a Pendant Activated Ester. Macromolecules 2013, 46 (4), 1283– 1290,  DOI: 10.1021/ma4001258
      42
      Accessing New Materials through Polymerization and Modification of a Polycarbonate with a Pendant Activated Ester
      Engler, Amanda C.; Chan, Julian M. W.; Coady, Daniel J.; O'Brien, Jeannette M.; Sardon, Haritz; Nelson, Alshakim; Sanders, Daniel P.; Yang, Yi Yan; Hedrick, James L.
      Macromolecules (Washington, DC, United States) (2013), 46 (4), 1283-1290CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
      Functionalized polycarbonates were synthesized by organocatalytic ring-opening polymn. (ROP) of a cyclic monomer with a pendant activated ester (MTC-OC6F5) followed by a postpolymn. modification with both small mols. and macromols. Controlled ROP to form homopolymers and diblock copolymers was realized using catalytic quantities of triflic acid. For the homopolymers, a linear relationship between [M]0/[I]0 and mol. wt. (by GPC) demonstrated the living nature of the polymn. Poly(MTC-OC6F5) was functionalized under mild reaction conditions with a variety of amines to obtain polymers with pendant primary, secondary, and tertiary amides. Graft polymers with a high grafting d. of over 87% were synthesized using amine-terminated poly(ethylene glycol) of two different mol. wts. (2 and 5 kDa). The prepn. of poly(MTC-OC6F5) provides a means of accessing a wide range of functional polycarbonates with minimal synthetic steps. This new methodol. for the formation of functionalized polycarbonates provides a simple and versatile platform for the synthesis of new and innovative materials.
    43. 43
      Smith, E.; Bai, J.; Oxenford, C.; Yang, J.; Somayaji, R.; Uludag, H. Conjugation of Arginine-Glycine-Aspartic Acid Peptides to Thermoreversible N-Isopropylacrylamide Polymers. J. Polym. Sci., Part A: Polym. Chem. 2003, 41 (24), 3989– 4000,  DOI: 10.1002/pola.10965
      43
      Conjugation of arginine-glycine-aspartic acid peptides to thermoreversible N-isopropylacrylamide polymers
      Smith, Erin; Bai, Jiang; Oxenford, Cassie; Yang, Jennifer; Somayaji, Ranjani; Uludag, Hasan
      Journal of Polymer Science, Part A: Polymer Chemistry (2003), 41 (24), 3989-4000CODEN: JPACEC; ISSN:0887-624X. (John Wiley & Sons, Inc.)
      Thermoreversible polymeric biomaterials are finding increased acceptance in tissue engineering applications. One drawback of the polymers is their synthetic nature, which does not allow direct interaction of mammalian cells with the polymers. This limitation may be alleviated by grafting arginine-glycine-aspartic acid (RGD) contg. peptides onto the polymer backbone to facilitate interactions with cell-surface integrins. Toward this goal, N-isopropylacrylamide (NiPAM)-based thermoreversible polymers contg. amine-reactive N-acryloxysuccinimide (NASI) groups were synthesized. Conjugation of RGD-contg. peptides to polymers was demonstrated with 1H NMR spectroscopy and reverse-phase high-pressure liq. chromatog. The conjugation reaction was optimal at 4 °C and pH of 8.0, and increased with the increasing NASI content of polymers. With a peptide grafting ratio of 0.25 mol %, there was no significant change in the lower crit. soln. temp. of the polymers. Finally, the NASI-contg. polymers, cast as films, on tissue culture polystyrene, were shown to conjugate to RGD-contg. peptides and support C2C12 cell attachment. We conclude that NASI-contg. thermoreversible polymers are amenable for grafting biomimetic peptides to impart cell adhesiveness to the polymers.
    44. 44
      Baessler, K. A.; Lee, Y.; Sampson, N. S. B1 Integrin Is an Adhesion Protein for Sperm Binding To Eggs. ACS Chem. Biol. 2009, 4 (5), 357– 366,  DOI: 10.1021/cb900013d
      44
      β1 Integrin Is an Adhesion Protein for Sperm Binding to Eggs
      Baessler, Keith A.; Lee, Younjoo; Sampson, Nicole S.
      ACS Chemical Biology (2009), 4 (5), 357-366CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      We investigated the role of β1 integrin in mammalian fertilization and the mode of inhibition of fertilinβ-derived polymers. We detd. that polymers displaying the Glu-Cys-Asp peptide from the fertilinβ disintegrin domain mediate inhibition of mammalian fertilization through a β1 integrin receptor on the egg surface. Inhibition of fertilization is a consequence of competition with sperm binding to the cell surface, not activation of an egg-signaling pathway. The presence of the β1 integrin on the egg surface increases the rate of sperm attachment but does not alter the total no. of sperm that can attach or fuse to the egg. We conclude that the presence of β1 integrin enhances the initial adhesion of sperm to the egg plasma membrane and that subsequent attachment and fusion are mediated by addnl. egg and sperm proteins present in the β1 integrin complex. Therefore, the mechanisms by which sperm fertilize wild-type and β1 knockout eggs are different.
    45. 45
      Börner, H. G.; Sütterlin, R. I.; Theato, P.; Wiss, K. T. Topology-Dependent Swichability of Peptide Secondary Structures in Bioconjugates with Complex Architectures. Macromol. Rapid Commun. 2014, 35 (2), 180– 185,  DOI: 10.1002/marc.201300808
      45
      Topology-dependent swichability of peptide secondary structures in bioconjugates with complex architectures
      Borner Hans G; Sutterlin Romina I; Theato Patrick; Wiss Kerstin T; Theato Patrick
      Macromolecular rapid communications (2014), 35 (2), 180-185 ISSN:.
      Peptide sequences, which exhibit a reversible pH-responsive coil to α-helix secondary structure transition, are conjugated to polymer precursors to yield linear AB and graft ABA peptide-poly(ethylene oxide) conjugates. While the PEO B-block is comparable, the conjugates differ in topologies of the peptide bearing A-blocks. The influences of topology on the structure transitions in the peptide segments are investigated, comparing linear AB-bioconjugates with graft ABA-bioconjugates having multiple peptide segments combined in star or pom-pom topologies.
    46. 46
      Nuhn, L.; Hartmann, S.; Palitzsch, B.; Gerlitzki, B.; Schmitt, E.; Zentel, R.; Kunz, H. Water-Soluble Polymers Coupled with Glycopeptide Antigens and T-Cell Epitopes as Potential Antitumor Vaccines. Angew. Chem., Int. Ed. 2013, 52 (40), 10652– 10656,  DOI: 10.1002/anie.201304212
      46
      Water-soluble polymers coupled with glycopeptide antigens and T-cell epitopes as potential antitumor vaccines
      Nuhn, Lutz; Hartmann, Sebastian; Palitzsch, Bjoern; Gerlitzki, Bastian; Schmitt, Edgar; Zentel, Rudolf; Kunz, Horst
      Angewandte Chemie, International Edition (2013), 52 (40), 10652-10656CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      First examples of nanosized polymer-linked vaccines have been synthesized by coupling tumor-assocd. MUC1 glycopeptides and T-cell epitope peptides to water-sol. methacrylamide polymers through the application of active ester aminolysis and Huisgen cycloaddn. as orthogonal conjugation methods. The attachment of the tetanus toxoid T-cell epitope P2 onto the hydrophilic polymer vaccines causes their self-assembly to micelle-like nanoobjects. The novel vaccine concept was extended to block copolymers consisting of hydrophilic and hydrophobic domains, in which the latter reinforce the formation of nanoparticles as well. The novel polymer-based glycopeptide vaccines induce significant MHC-II-mediated immune reactions in mice and elicit IgG antibodies, which recognize MCF-7 breast tumor cells. In particular, the block copolymer contg. addnl. nanostructure-promoting domains induced antibodies that exhibit high affinity to the tumor cells. Considering the numerous degrees of freedom for structure modification, this novel vaccine concept may open up new ways for the construction of efficient immunotherapeutics, for example antitumor vaccines.
    47. 47
      Leiske, M. N.; Kempe, K. A Guideline for the Synthesis of Amino-Acid-Functionalized Monomers and Their Polymerizations. Macromol. Rapid Commun. 2022, 43 (2), 1– 24,  DOI: 10.1002/marc.202100615
      There is no corresponding record for this reference.
    48. 48
      Maity, T.; Paul, S.; De, P. Side-Chain Amino Acid-Based Macromolecular Architectures. J. Macromol. Sci. Part A 2023, 60 (1), 2– 17,  DOI: 10.1080/10601325.2023.2169158
      48
      Side-chain amino acid-based macromolecular architectures
      Maity, Tanmoy; Paul, Soumya; De, Priyadarsi
      Journal of Macromolecular Science, Part A: Pure and Applied Chemistry (2023), 60 (1), 2-17CODEN: JSPCE6; ISSN:1060-1325. (Taylor & Francis, Inc.)
      A review. Amino acids, the natural building blocks of life, are highly functional small mols. with chirality and important catalytic properties. Imparting the exquisite properties of amino acids into the macromols. allows us to prep. unique, well-defined, biocompatible, functional, and tailorable materials. They exhibit stimuli responsiveness, tunable optical characteristics, enhanced thermal, mech., and biol. properties with desired cell-materials interactions. Considering the salient features and diverse applications of side-chain amino acid-contg. polymers, in this feature article, we present a concise highlight on the recent trends (2015-present) of the synthesis and utilization of pendant amino acid-based polymeric architectures. This article covers the synthesis of different classes of side-chain amino acid-derived polymeric architectures, and their recent utilization in drug and gene delivery, antibacterial activity, anti-fouling activity, heavy metal ion sensing, tissue engineering, etc. We hope that this study will provide a comprehensive outline of key aspects assocd. with the construction of functional 'bio-inspired' polymeric materials with desired characteristics, and their enhanced potential applications.
    49. 49
      Leiske, M. N.; Mazrad, Z. A. I.; Zelcak, A.; Wahi, K.; Davis, T. P.; McCarroll, J. A.; Holst, J.; Kempe, K. Zwitterionic Amino Acid-Derived Polyacrylates as Smart Materials Exhibiting Cellular Specificity and Therapeutic Activity. Biomacromolecules 2022, 23 (6), 2374– 2387,  DOI: 10.1021/acs.biomac.2c00143
      49
      Zwitterionic Amino Acid-Derived Polyacrylates as Smart Materials Exhibiting Cellular Specificity and Therapeutic Activity
      Leiske, Meike N.; Mazrad, Zihnil. A. I.; Zelcak, Aykut; Wahi, Kanu; Davis, Thomas P.; McCarroll, Joshua A.; Holst, Jeff; Kempe, Kristian
      Biomacromolecules (2022), 23 (6), 2374-2387CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      The synthesis of new amino acid-contg., cell-specific, therapeutically active polymers is presented. Amino acids served as starting material for the prepn. of tailored polymers with different amino acids in the side chain. The reversible addn.-fragmentation chain-transfer (RAFT) polymn. of acrylate monomers yielded polymers of narrow size distribution (D ≤ 1.3). In particular, glutamate (Glu)-functionalized, zwitterionic polymers revealed a high degree of cytocompatibility and cellular specificity, i.e., showing assocn. to different cancer cell lines, but not with nontumor fibroblasts. Energy-dependent uptake mechanisms were confirmed by means of temp.-dependent cellular uptake expts. as well as localization of the polymers in cellular lysosomes detd. by confocal laser scanning microscopy (CLSM). The amino acid receptor antagonist O-benzyl-L-serine (BzlSer) was chosen as an active ingredient for the design of therapeutic copolymers. RAFT copolymn. of Glu acrylate and BzlSer acrylate resulted in tailored macromols. with distinct monomer ratios. The targeted, cytotoxic activity of copolymers was demonstrated by means of multiday in vitro cell viability assays. To this end, polymers with 25 mol % BzlSer content showed cytotoxicity against cancer cells, while leaving fibroblasts unaffected over a period of 3 days. Our results emphasize the importance of biol. derived materials to be included in synthetic polymers and the potential of zwitterionic, amino acid-derived materials for cellular targeting. Furthermore, it highlights that the fine balance between cellular specificity and unspecific cytotoxicity can be tailored by monomer ratios within a copolymer.
    50. 50
      Fujii, S.; Sakurai, K. Zwitterionic Amino Acid Polymer-Grafted Core-Crosslinked Particle toward Tumor Delivery. Biomacromolecules 2022, 23 (9), 3968– 3977,  DOI: 10.1021/acs.biomac.2c00803
      50
      Zwitterionic Amino Acid Polymer-Grafted Core-Crosslinked Particle toward Tumor Delivery
      Fujii, Shota; Sakurai, Kazuo
      Biomacromolecules (2022), 23 (9), 3968-3977CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      Zwitterionic amino acid polymers (ZAPs) exhibit biocompatibility and recognition capability for amino acid transporters (AATs) overexpressed on cancer cells. They are potential cancer-targeting ligands in nanoparticle-based nanomedicines utilized in cancer chemotherapy. Here, a poly(glutamine methacrylate) (pGlnMA)-grafted core-crosslinked particle (pGlnMA-CCP) is prepd. through the formation of nanoemulsions stabilized using amphiphilic block copolymers comprising pGlnMA as the hydrophilic block. The chain conformation of the grafted polymer and the particle structure of pGlnMA-CCP are precisely elucidated by dynamic light scattering, X-ray scattering, and transmission electron microscopy. pGlnMA-CCP demonstrates active cellular uptake and deep penetration behaviors for cancer cells and spheroids, resp., via an AAT-mediated mechanism. The in vivo pharmacokinetics of pGlnMA-CCP is practically comparable to those of a CCP covered with poly(polyethylene glycol methacrylate) (pPEGMA), which inhibits protein adsorption and prolongs blood retention, implying that the biocompatible properties of pGlnMA are similar to those of pPEGMA. Furthermore, pGlnMA-CCP accumulates in cancer tissues at a higher level than that of pPEGMA systems. The results demonstrate that the properties of cancer targetability, tumor permeability, efficient tumor accumulation, and biocompatibility can be obtained by grafting pGlnMA onto nanoparticles, suggesting a high potential of pGlnMA as a ligand for cancer-targeting nanomedicines.
    51. 51
      Koyama, E.; Sanda, F.; Endo, T. Syntheses and Radical Polymerizations of Methacrylamides Derived from Optically Active Amino Alcohols. Macromol. Chem. Phys. 1997, 198 (11), 3699– 3707,  DOI: 10.1002/macp.1997.021981130
      51
      Syntheses and radical polymerizations of methacrylamides derived from optically active amino alcohols
      Koyama, Emiko; Sanda, Fumio; Endo, Takeshi
      Macromolecular Chemistry and Physics (1997), 198 (11), 3699-3707CODEN: MCHPES; ISSN:1022-1352. (Huethig & Wepf Verlag)
      Syntheses and radical polymns. of methacrylamides derived from optically active amino alcs. were carried out. The monomers were prepd. by condensation of optically active amino alcs. and methacrylic acid using 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride (EDC.HCl). Radical polymns. of the monomers were carried out in the presence of 2,2-azoisobutyronitrile (AIBN) (3 mol.-%) in MeOH and DMF as solvents, resulting with good yields in polymers with ‾Mn of 17,800-81,000. The monomer reactivity ratios of N-(1-hydroxy-4-methyl-2-pentyl)methacrylamide (MA-Lol) and Me methacrylate (MMA) in the copolymn. were estd. to be 0.50 and 2.38, resp. The lower reactivity of MA-Lol in comparison with MMA is similar to common methacrylamide derivs. The hydrophilicity of the polymers was evaluated by the heat of fusion of water using differential scanning calorimetry (DSC). The detd. order poly(MA-Aol) > poly(MA-Lol) > poly(MA-Fol) reflects the bulkiness of the side chains.
    52. 52
      Yaşayan, G.; Redhead, M.; Magnusson, J. P.; Spain, S. G.; Allen, S.; Davies, M.; Alexander, C.; Fernández-Trillo, F. Well-Defined Polymeric Vesicles with High Stability and Modulation of Cell Uptake by a Simple Coating Protocol. Polym. Chem. 2012, 3 (9), 2596– 2604,  DOI: 10.1039/c2py20352a
      52
      Well-defined polymeric vesicles with high stability and modulation of cell uptake by a simple coating protocol
      Yasayan, Gokcen; Redhead, Martin; Magnusson, Johannes P.; Spain, Sebastian G.; Allen, Stephanie; Davies, Martyn; Alexander, Cameron; Fernandez-Trillo, Francisco
      Polymer Chemistry (2012), 3 (9), 2596-2604CODEN: PCOHC2; ISSN:1759-9962. (Royal Society of Chemistry)
      Amphiphilic polymers have been synthesized by controlled free radical polymn. techniques. These polymers self-assemble into well-defined vesicles in aq. conditions, enabling encapsulation of a model hydrophilic mol. The polymeric vesicles show high stability against a range of aq. conditions with marginal release of cargo, even in the presence of known cell-membrane disruptive polymers such as branched poly(ethyleneimine) (b-PEI). This stability allows for inversion of the surface charge of the polymeric vesicles by a simple coating protocol leading to an enhanced uptake by mammalian cells.
    53. 53
      Bou Zerdan, R.; Geng, Z.; Narupai, B.; Diaz, Y. J.; Bates, M. W.; Laitar, D. S.; Souvagya, B.; Van Dyk, A. K.; Hawker, C. J. Efficient Synthesis of Branched Poly(Acrylic Acid) Derivatives via Postpolymerization Modification. J. Polym. Sci. 2020, 58 (14), 1989– 1997,  DOI: 10.1002/pol.20200287
      53
      Efficient synthesis of branched poly(acrylic acid) derivatives via postpolymerization modification
      Bou Zerdan, Raghida; Geng, Zhishuai; Narupai, Benjaporn; Diaz, Yvonne J.; Bates, Morgan W.; Laitar, David S.; Souvagya, Biswas; Van Dyk, Antony K.; Hawker, Craig J.
      Journal of Polymer Science (Hoboken, NJ, United States) (2020), 58 (14), 1989-1997CODEN: JPSHBC; ISSN:2642-4169. (John Wiley & Sons, Inc.)
      The utility of pentafluorophenyl esters for the selective introduction of functional units and branch points in well-defined poly(acrylic acid) (PAA) derivs. is demonstrated using a combination of controlled radical polymn. and postpolymn. modification. Reversible addn.-fragmentation chain transfer enables the synthesis of well-defined copolymers-poly(pentafluorophenyl acrylate-co-tert-Bu acrylate)-with the active ester repeat units serving as attachment points for reaction with primary amines, specifically tris(2-(t-butoxycarbonyl)ethyl)methyl amine (Behera's amine). Deprotection using trifluoroacetic acid removes both the backbone and side chain t-Bu esters to give a series of branched PAA derivs. contg. novel tricarboxylic acid side chains that are well suited to complexation and multidentate interactions. Surprisingly, the active ester homopolymer is shown to have the highest reactivity with Behera's amine when compared to copolymers with lower incorporation of pentafluorophenyl esters, suggesting an intriguing interplay of neighboring group effects and steric interactions. The ability to tune the efficiency of postpolymn. modification gives a library of PAA derivs.
    54. 54
      Hu, H.; Saniger, J.; Garcia-Alejandre, J.; Castaiio, V. M. Fourier Transform Infrared Spectroscopy Studies of the Reaction between Polyacrylic Acid and Metal Oxides. Mater. Lett. 1991, 12, 281– 285,  DOI: 10.1016/0167-577X(91)90014-W
      54
      Fourier transform infrared spectroscopy studies of the reaction between polyacrylic acid and metal oxides
      Hu, H.; Saniger, J.; Garcia-Alejandre, J.; Castano, V. M.
      Materials Letters (1991), 12 (4), 281-5CODEN: MLETDJ; ISSN:0167-577X.
      The chem. reactions between poly(acrylic acid) and divalent metal oxides (metal = Ca, Cu, Mg, Zn) were studied by FTIR spectroscopy. There was clear evidence of chem. bonding between the polymer and the metals as a result of their interaction. From the energy difference between the sym. and the asym. bands of the various possible structures, monodentate metal-carboxylate bonding was the most probable in all cases. Also, from the anal. of the corresponding molar relations, the particle size of the oxides played an important role in the reaction.
    55. 55
      Roy, S.; Mahali, K.; Mondal, S.; Dolui, B. K. Thermodynamics of DL-Alanine Solvation in Water-Dimethylsulfoxide Mixtures at 298.15 K. Russ. J. Phys. Chem. A 2015, 89 (4), 654– 662,  DOI: 10.1134/S0036024415040226
      55
      Thermodynamics of DL-alanine solvation in water-dimethylsulfoxide mixtures at 298.15 K
      Roy, S.; Mahali, K.; Mondal, S.; Dolui, B. K.
      Russian Journal of Physical Chemistry A (2015), 89 (4), 654-662CODEN: RJPCBS; ISSN:0036-0244. (SP MAIK Nauka/Interperiodica)
      In this study we mainly discuss the transfer Gibbs free energy ΔG0t(i) and ΔS0t(i)entropy of DL-alanine at 298.15 K and consequently the involved chem. transfer free energy (ΔG0t,ch(i)) and entropy (TΔS0t,ch(i)) in aq. mixts. of dimethylsulfoxide are discussed to clarify the solvation chem. of DL-alanine. For the evaluation of these energy terms, soly. of this amino acid has been measured by formol titrimetry at five equidistant temps. i.e., from 288.15 to 308.15 K in different compn. of this mixed solvent system. The various solvent parameters as well as thermodn. parameters like molar volume, d., dipole moment and solvent diam. of this solvent system have also been reported here. The chem. effects of the transfer Gibbs energies (ΔG0t,ch(i)) and entropies of transfer (TΔS0t,ch(i)) have been obtained after elimination of cavity effect and dipole-dipole interaction effects from the total transfer energies. Here the chem. contribution of transfer energetics of DL-alanine is mainly guided by the composite effects of increased dispersion interaction, basicity effect and decreased acidity, hydrogen bonding effects, hydrophilic hydration and hydrophobic hydration of aq. DMSO mixts. as compared to that of ref. solvent, water.
    56. 56
      Needham, T. E.; Paruta, A. N.; Gerraughty, R. J. Solubility of Amino Acids in Pure Solvent Systems. J. Pharm. Sci. 1971, 60 (4), 565– 567,  DOI: 10.1002/jps.2600600410
      56
      Solubility of amino acids in pure solvent systems
      Needham, T. E., Jr.; Paruta, Anthony N.; Gerraughty, Robert J.
      Journal of Pharmaceutical Sciences (1971), 60 (4), 565-7CODEN: JPMSAE; ISSN:0022-3549.
      The effects of various solvents and pH on the solys. of glycine, L-alanine, L-valine, L-phenylalanine, and DL-aminooctanoic acid were studied in a series of pure aq. and alc. solns. The aq. soly. was inversely proportional to the size of the nonpolar portion of the mol. A low nonaq. soly. was due to a dominance of the amino acid by the charged α-amino carboxylic acid portion of the mol. In aq. and alc. solns., an isoelec. band of min. soly. was formed. A distinct increase in soly. proportional to the addn. of acid or base was seen as the pH exceeded the limits of the isoelec. band. In the alc. solvent systems studied, the addn. of either acid or base produced a greater divergence from the isoelec. pH than would be seen in a pure aq. system.
    57. 57
      Mahali, K.; Roy, S.; Dolui, B. K. Solubility and Solvation Thermodynamics of a Series of Homologous α-Amino Acids in Nonaqueous Binary Mixtures of Ethylene Glycol and Dimethyl Sulfoxide. J. Chem. Eng. Data 2015, 60 (5), 1233– 1241,  DOI: 10.1021/je5007899
      57
      Solubility and Solvation Thermodynamics of a Series of Homologous α-Amino Acids in Nonaqueous Binary Mixtures of Ethylene Glycol and Dimethyl Sulfoxide
      Mahali, Kalachand; Roy, Sanjay; Dolui, Bijoy Krishna
      Journal of Chemical & Engineering Data (2015), 60 (5), 1233-1241CODEN: JCEAAX; ISSN:0021-9568. (American Chemical Society)
      In this article the std. free energies (ΔG0t(i)) and entropies (ΔS0t(i)) of transfer of four homologous α-amino acids including glycine (Gly), DL-alanine (DL-Ala), DL-α-amino butyric acid (DL-Aba.) and DL-nor-valine (DL-n-Val) from ethylene glycol (EG) to nonaq. mixts. of ethylene glycol and DMSO (DMSO) at 298.15 K are reported. The Gibbs energies of solns. were detd. from soly. measurements of each amino acid at different temps., i.e., from 288.15 to 308.15 K by "formol titrimetry". The chem. parts of free energies (ΔG0t,ch(i)) and entropies (TΔS0t,ch(i)) of transfer of the homologous α-amino acids were computed by subtracting the cavity effects and dipole-dipole interaction effects from the total transfer energies. The characteristics of the solvation thermodn. of α-amino acids in EG-DMF and EG-ACN mixed solvent systems studied earlier are also discussed here for comparison.
    58. 58
      Jochum, F. D.; Theato, P. Temperature- and Light-Responsive Polyacrylamides Prepared by a Double Polymer Analogous Reaction of Activated Ester Polymers. Macromolecules 2009, 42 (16), 5941– 5945,  DOI: 10.1021/ma900945s
      58
      Temperature- and Light-Responsive Polyacrylamides Prepared by a Double Polymer Analogous Reaction of Activated Ester Polymers
      Jochum, Florian D.; Theato, Patrick
      Macromolecules (Washington, DC, United States) (2009), 42 (16), 5941-5945CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
      Two different series of polyacrylamides contg. different amts. of salicylideneaniline moieties have been synthesized via a double polymer analogous reaction of poly(pentafluorophenyl acrylate) (PPFPA). All copolymers were designed to exhibit a lower crit. soln. temp. (LCST) in aq. soln., which was dependent on (i) the amt. of incorporated chromophoric salicylideneaniline groups and (ii) the isomerization state of the resp. salicylideneaniline group. Higher LCST values were measured for UV-irradiated solns. of the copolymers in comparison to the nonirradiated copolymer solns. A max. difference in the LCST of up to 13 °C was found for poly(N-cyclopropylacrylamide) copolymer contg. 15.0 mol % of salicylideneaniline groups. Within this temp. range, a reversible soly. change of the copolymer could be induced by irradn. with light.
    59. 59
      Chiantore, O. Polymer-Substrate Interactions in Size Exclusion Chromatography with Silica Gels and Pure Solvents. J. Liq. Chromatogr. 1984, 7 (1), 1– 11,  DOI: 10.1080/01483918408073946
      59
      Polymer-substrate interactions in size exclusion chromatography with silica gels and pure solvents
      Chiantore, Oscar
      Journal of Liquid Chromatography (1984), 7 (1), 1-11CODEN: JLCHD8; ISSN:0148-3919.
      The retention vols. of polystyrene [9003-53-6], poly(α-methylstyrene) [25014-31-7], and poly(Me methacrylate) [9011-14-7] mols. eluted with different pure solvents on silica gel size-exclusion chromatog. columns were measured. Departures from the universal calibration plot due to polymer adsorption onto the silica gel substrate are found in some solvent systems, when the polymer-gel interactions overcome the solvent-gel ones. The competition between the interactions developed in the systems is adequately expressed through the soly. parameters of the polymers and of the solvents.
    60. 60
      Echeverria, C.; Peppas, N. A.; Mijangos, C. Novel Strategy for the Determination of UCST-like Microgels Network Structure: Effect on Swelling Behavior and Rheology. Soft Matter 2012, 8 (2), 337– 346,  DOI: 10.1039/C1SM06489D
      60
      Novel strategy for the determination of UCST-like microgels network structure: effect on swelling behavior and rheology
      Echeverria, Coro; Peppas, Nicholas A.; Mijangos, Carmen
      Soft Matter (2012), 8 (2), 337-346CODEN: SMOABF; ISSN:1744-683X. (Royal Society of Chemistry)
      UCST-type interpenetrated and random copolymer microgels of polyacrylamide and poly(acrylic acid) were obtained via inverse emulsion polymn. method. The morphol. of the microgels was detd. by SEM and at. force microscopy (AFM). Dynamic light scattering was used to study both the swelling behavior as a function of temp. and pH and the particle size distribution of the system. Concerning the structural characterization, a combination of the equil. swelling theory (Peppas-Merrill equation) and AFM technique was used to det. the mesh size of microgels. An oscillatory rheometer was used to study the viscoelastic properties. The moduli, G' and G'', of the microgel dispersions suggested a solid-like behavior and structure formation. Scaling theory was applied to describe the structure formation (clustering) and the fractal dimension. The influence of compn. and type of microgel, random or interpenetrated, was discussed in the above mentioned properties and behaviors.
    61. 61
      Jasinski, J.; Wilde, M. V.; Voelkl, M.; Jérôme, V.; Fröhlich, T.; Freitag, R.; Scheibel, T. Tailor-Made Protein Corona Formation on Polystyrene Microparticles and Its Effect on Epithelial Cell Uptake. ACS Appl. Mater. Interfaces 2022, 14 (41), 47277– 47287,  DOI: 10.1021/ACSAMI.2C13987/SUPPL_FILE/AM2C13987_SI_001.PDF
      61
      Tailor-Made Protein Corona Formation on Polystyrene Microparticles and its Effect on Epithelial Cell Uptake
      Jasinski, Julia; Wilde, Magdalena V.; Voelkl, Matthias; Jerome, Valerie; Froehlich, Thomas; Freitag, Ruth; Scheibel, Thomas
      ACS Applied Materials & Interfaces (2022), 14 (41), 47277-47287CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)
      Microplastic particles are pollutants in the environment with a potential impact on ecol. and human health. As soon as microplastic particles get in contact with complex (biol.) environments, they will be covered by an eco- and/or protein corona. In this contribution, protein corona formation was conducted under defined lab. conditions on polystyrene (PS) microparticles to investigate the influence on surface properties, protein corona evolution, particle-cell interactions, and uptake in two murine epithelial cells. To direct protein corona formation, PS particles were preincubated with five model proteins, namely, bovine serum albumin (BSA), myoglobin, β-lactoglobulin, lysozyme, and fibrinogen. Subsequently, the single-protein-coated particles were incubated in a cell culture medium contg. a cocktail of serum proteins to analyze changes in the protein corona profile as well as in the binding kinetics of the model proteins. Therein, we could show that the precoating step has a crit. impact on the final compn. of the protein corona. Yet, since proteins building the primary corona were still detectable after addnl. incubations in a protein-contg. medium, backtracking of the particle's history is possible. Interestingly, whereas the precoating history significantly disturbs particle-cell interactions (PCIs), the cellular response (i.e., metabolic activity, MTT assay) stays unaffected. Of note, lysozyme precoating revealed one of the highest rates in PCI for both epithelial cell lines. Taken together, we could show that particle history has a significant impact on protein corona formation and subsequently on the interaction of particles with murine intestinal epithelial-like cells. However, as this study was limited to one cell type, further work is needed to assess if these observations can be generalized to other cell types.
    62. 62
      Swieton, J.; Kaminski, K.; Miklosz, J.; Mogielnicki, A.; Kalaska, B. Anionic and Cationic Block Copolymers as Promising Modulators of Blood Coagulation. Eur. Polym. J. 2023, 199, 112452  DOI: 10.1016/j.eurpolymj.2023.112452
      62
      Anionic and cationic block copolymers as promising modulators of blood coagulation
      Swieton, Justyna; Kaminski, Kamil; Miklosz, Joanna; Mogielnicki, Andrzej; Kalaska, Bartlomiej
      European Polymer Journal (2023), 199 (), 112452CODEN: EUPJAG; ISSN:0014-3057. (Elsevier Ltd.)
      A review. Thrombosis is a leading cause of death worldwide, necessitating the search for more effective and safer treatments. Current parenteral anticoagulant medications indicated for the prevention and treatment of thrombosis include polyanionic heparins. Despite their well-established status, these medications have drawbacks and fail to meet the criteria for an ideal anticoagulant. The predictability and control of heparin's biol. activity pose challenges due to the chaotic structure of this polymer, characterized by a mixt. of larger and smaller mols. with a loosely defined linear architecture. Consequently, cases of heparin overdose are common. Protamine sulfate can halt bleeding caused by unfractionated heparin but concurrently elicits significant adverse effects. Although novel antidotes for anticoagulants have been developed, bleeding during anticoagulation therapy remains a concern. Over the past two decades, research into heparin mimetics has witnessed a significant surge. This intensification is underscored by statistical evidence indicating an increase in thrombotic episodes, highlighting the necessity for new anticoagulant alternatives. This review outlines the efforts to replace heparins and their antidotes with block copolymers. We have concd. on studies involving well-defined synthetic anionic and cationic block copolymers, which are obtained by free radical polymn. techniques. Block copolymers comprised of anionic poly(sodium 4-styrenesulfonate) or poly(2-acryloylamido-2-methylpropanesulfonic acid) blocks and a neutral poly(ethylene glycol) block seem to be the most promising candidates for future anticoagulants. Complementary polycationic antidotes to regulate anticoagulant activity have also been proposed; however, further research focusing on structure-activity relationships and safety is necessary to confirm their utility.
    63. 63
      Dailing, E. A.; Kilchrist, K. V.; Tierney, J. W.; Fletcher, R. B.; Evans, B. C.; Duvall, C. L. Modifying Cell Membranes with Anionic Polymer Amphiphiles Potentiates Intracellular Delivery of Cationic Peptides. ACS Appl. Mater. Interfaces 2020, 12 (45), 50222– 50235,  DOI: 10.1021/acsami.0c13304
      63
      Modifying Cell Membranes with Anionic Polymer Amphiphiles Potentiates Intracellular Delivery of Cationic Peptides
      Dailing, Eric A.; Kilchrist, Kameron V.; Tierney, J. William; Fletcher, R. Brock; Evans, Brian C.; Duvall, Craig L.
      ACS Applied Materials & Interfaces (2020), 12 (45), 50222-50235CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)
      Rapid, facile, and noncovalent cell membrane modification with alkyl-grafted anionic polymers was sought as an approach to enhance intracellular delivery and bioactivity of cationic peptides. We synthesized a library of acrylic acid-based copolymers contg. varying amts. of an amine-reactive pentafluorophenyl acrylate monomer followed by postpolymn. modification with a series of alkyl amines to afford precise control over the length and d. of aliph. alkyl side chains. This synthetic strategy enabled systematic investigation of the effect of the polymer structure on membrane binding, potentiation of peptide cell uptake, pH-dependent disruption of lipid bilayers for endosome escape, and intracellular bioavailability. A subset of these polymers exhibited pKa of ~ 6.8, which facilitated stable membrane assocn. at physiol. pH and rapid, pH-dependent endosomal disruption upon endocytosis as quantified in Galectin-8-YFP reporter cells. Cationic cell penetrating peptide (CPP) uptake was enhanced up to 15-fold in vascular smooth muscle cells in vitro when peptide treatment was preceded by a 30-min pretreatment with lead candidate polymers. We also designed and implemented a new and highly sensitive assay for measuring the intracellular bioavailability of CPPs based on the NanoLuciferase (NanoLuc) technol. previously developed for measuring intracellular protein-protein interactions. Using this split luciferase class of assay, polymer pretreatment enhanced intracellular delivery of the CPP-modified HiBiT peptide up to 30-fold relative to CPP-HiBiT without polymer pretreatment (p < 0.05). The overall structural analyses show that polymers contg. 50:50 or 70:30 molar ratios of carboxyl groups to alkyl side chains of 6-8 carbons maximized peptide uptake, pH-dependent membrane disruption, and intracellular bioavailability and that this potentiation effect was maximized by pairing with CPPs with high cationic charge d. These results demonstrate a rapid, mild method for polymer modification of cell surfaces to potentiate intracellular delivery, endosome escape, and bioactivity of cationic peptides.
    64. 64
      Jiang, Z.; Liu, H.; He, H.; Yadava, N.; Chambers, J. J.; Thayumanavan, S. Anionic Polymers Promote Mitochondrial Targeting of Delocalized Lipophilic Cations. Bioconjugate Chem. 2020, 31 (5), 1344– 1353,  DOI: 10.1021/acs.bioconjchem.0c00079
      64
      Anionic Polymers Promote Mitochondrial Targeting of Delocalized Lipophilic Cations
      Jiang, Ziwen; Liu, Hongxu; He, Huan; Yadava, Nagendra; Chambers, James J.; Thayumanavan, S.
      Bioconjugate Chemistry (2020), 31 (5), 1344-1353CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      Mitochondria are therapeutic targets in many diseases including cancer, metabolic disorders, and neurodegenerative diseases. Therefore, strategies to deliver therapeutics of interest to mitochondria are important for therapeutic development. As delocalized lipophilic cations (DLCs) preferentially accumulate in mitochondria, DLC-conjugation has been utilized to facilitate therapeutic delivery systems with mitochondrial targeting capability. Here we report that upon DLC-conjugation, anionic polymers exhibit significantly improved mitochondrial targeting when compared to cationic polymers and charge-neutral polymers. Considering that the cell membrane generally bears a net neg. charge, the obsd. phenomenon is unexpected. Notably, the DLC-conjugated anionic polymers circumvent endosomal entrapment. The rapid mitochondrial accumulation of DLC-conjugated anionic polymers is likely a membrane-potential-driven process, along with the involvement of the mitochondrial pyruvate carrier. Moreover, the structural variations on the side chain of DLC-conjugated anionic polymers do not compromise the overall mitochondrial targeting capability, widely extending the applicability of anionic macromols. in therapeutic delivery systems.
    65. 65
      Griffin, W. C. Classification of Surface-Active Agents by “HLB. J. Cosmet. Sci. 1949, 1, 311– 326
      There is no corresponding record for this reference.
    66. 66
      Egan, R. W. Hydrophile-Lipophile Balance and Critical Micelle Concentration as Key Factors Influencing Surfactant Disruption of Mitochondrial Membranes. J. Biol. Chem. 1976, 251 (14), 4442– 4447,  DOI: 10.1016/S0021-9258(17)33316-1
      66
      Hydrophile-lipophile balance and critical micelle concentration as key factors influencing surfactant disruption of mitochondrial membranes
      Egan, Robert W.; Jones, Marjorie A.; Lehninger, Albert L.
      Journal of Biological Chemistry (1976), 251 (14), 4442-7CODEN: JBCHA3; ISSN:0021-9258.
      A systematic approach is described for the selection of surfactants for disrupting biol. membranes, for solubilizing their components, and for removing the surfactant by dialysis. The 2 relevant surfactant parameters were the crit. micelle concn. (CMC) and the hydrophile-lipophile balance (HLB). Rat liver mitochondria were treated with 2 series of nonionic surfactants, and the extent of extn. of total protein, total lipid, and 6 enzymes was detd. Within the homologous series of Triton surfactants, max. protein and phospholipid extn. occurred at HLB values between 12.5 and 13.5. In addn., a single surfactant species solubilized more protein than a mixt. of surfactants with the same mean HLB values. In order to examine independently the effect of CMC and HLB on protein extn., a specialty surfactant, S10-7, was prepd. and compared with its structurally similar analog, Brij 56. Above a concn. of 0.35%, both Brij 56 and S10-7 extd. ∼70% of the mitochondrial protein. Hence, for optimum extn. of mitochondrial protein and lipids, the HLB must be ∼13, and the surfactant concn. must be above the CMC. The S10-7 dialyzed almost as rapidly as cholate and far more rapidly than Brij 58 and Triton X-100. It therefore possesses the 2 most desirable surfactant properties for disruption of membranes, a high CMC for rapid dialysis and an HLB value of 13.2.
    67. 67
      Rideal, E.; Taylor, F. H. On Haemolysis by Anionic Detergents. Proc. R. Soc. London, Ser. B 1957, 146 (923), 225– 241,  DOI: 10.1098/rspb.1957.0007
      67
      Hemolysis by anionic detergents
      Rideal, Eric C.; Taylor, F. H.
      Proceedings of the Royal Society of London, Series B: Biological Sciences (1957), 146 (), 225-41CODEN: PRLBA4; ISSN:0962-8452.
      Synthetic anionic detergents produce hemolysis of human erythrocytes rapidly by action on free phospholipide in the cell wall and slowly by a process in several stages apparently involving breakdown of a lipoprotein in the cell wall. 23 references.
    68. 68
      Manaargadoo-Catin, M.; Ali-Cherif, A.; Pougnas, J.-L.; Perrin, C. Hemolysis by Surfactants─A Review. Adv. Colloid Interface Sci. 2016, 228, 1– 16,  DOI: 10.1016/j.cis.2015.10.011
      68
      Hemolysis by surfactants - A review
      Manaargadoo-Catin, Magalie; Ali-Cherif, Anais; Pougnas, Jean-Luc; Perrin, Catherine
      Advances in Colloid and Interface Science (2016), 228 (), 1-16CODEN: ACISB9; ISSN:0001-8686. (Elsevier B.V.)
      An overview of the use of surfactants for erythrocyte lysis and their cell membrane action mechanisms is given. Erythrocyte membrane characteristics and its assocn. with the cell cytoskeleton are presented in order to complete understanding of the erythrocyte membrane distortion. Cell homeostasis disturbances caused by surfactants might induce changes starting from shape modification to cell lysis. Two main mechanisms are hypothesized in literature which are osmotic lysis and lysis by solubilization even if the boundary between them is not clearly defined. Another specific mechanism based on the formation of membrane pores is suggested in the particular case of saponins. The lytic potency of a surfactant is related to its affinity for the membrane and the modification of the lipid membrane curvature. This is to be related to the surfactant shape defined by its hydrophobic and hydrophilic moieties but also by exptl. conditions. As a consequence, prediction of the hemolytic potency of a given surfactant is challenging. Several studies are focused on the relation between surfactant erythrolytic potency and their physico-chem. parameters such as the crit. micellar concn. (CMC), the hydrophile-lipophile balance (HLB), the surfactant membrane/water partition coeff. (K) or the packing parameter (P). The CMC is one of the most important factors considered even if a lytic activity cut-off effect points out that the only consideration of CMC not enough predictive. The relation K.CMC must be considered in addn. to the CMC to predict the surfactant lytic capacity within the same family of non ionic surfactant. Those surfactant structure/lytic activity studies demonstrate the requirement to take into account a combination of physico-chem. parameters to understand and foresee surfactant lytic potency.
    69. 69
      Lichtenberg, D.; Robson, R. J.; Dennis, E. A. Solubilization of Phospholipids by Detergents Structural and Kinetic Aspects. Biochim. Biophys. Acta, Rev. Biomembr. 1983, 737 (2), 285– 304,  DOI: 10.1016/0304-4157(83)90004-7
      69
      Solubilization of phospholipids by detergents. Structural and kinetic aspects
      Lichtenberg, Dov; Robson, Robert J.; Dennis, Edward A.
      Biochimica et Biophysica Acta, Reviews on Biomembranes (1983), 737 (2), 285-304CODEN: BRBMC5; ISSN:0304-4157.
      A review with 139 refs.
    70. 70
      Bochicchio, D.; Panizon, E.; Monticelli, L.; Rossi, G. Interaction of Hydrophobic Polymers with Model Lipid Bilayers. Sci. Rep. 2017, 7 (1), 6357  DOI: 10.1038/s41598-017-06668-0
      70
      Interaction of hydrophobic polymers with model lipid bilayers
      Bochicchio D; Panizon E; Rossi G; Monticelli L
      Scientific reports (2017), 7 (1), 6357 ISSN:.
      The interaction of nanoscale synthetic materials with cell membranes is one of the key steps determining nanomaterials' toxicity. Here we use molecular simulations, with atomistic and coarse-grained resolution, to investigate the interaction of three hydrophobic polymers with model lipid membranes. Polymer nanoparticles made of polyethylene (PE), polypropylene (PP) and polystyrene with size up to 7 nm enter easily POPC lipid membranes, localizing to the membrane hydrophobic core. For all three materials, solid polymeric nanoparticles become essentially liquid within the membrane at room temperature. Still, their behavior in the membrane core is not the same: PP and PS disperse in the core of the bilayer, while PE shows a tendency to aggregate. We also examined the interaction of the polymers with heterogeneous membranes, consisting of a ternary lipid mixture exhibiting liquid-ordered/liquid-disordered phase separation. The behavior of the three polymers is markedly different: PP disfavors lipid phase separation, PS stabilizes it, and PE modifies the topology of the phase boundaries and causes cholesterol depletion from the liquid ordered phase. Our results show that different hydrophobic polymers have major effects on the properties of lipid membranes, calling for further investigations on model systems and cell membranes.
    71. 71
      Ohshima, H. A Simple Expression for Henry’s Function for the Retardation Effect in Electrophoresis of Spherical Colloidal Particles. J. Colloid Interface Sci. 1994, 168 (1), 269– 271,  DOI: 10.1006/jcis.1994.1419
      71
      A simple expression of Henry's function for the retardation effect in electrophoresis of spherical colloidal particles
      Ohshima, Hiroyuki
      Journal of Colloid and Interface Science (1994), 168 (1), 269-71CODEN: JCISA5; ISSN:0021-9797.
      A simple approx. expression is proposed for the Henry function for the retardation effect on the electrophoresis of spherical colloidal particles with relative errors < 1%. An approx. expression for infinitely long cylindrical particles is also given.
    72. 72
      Leiske, M. N.; Walker, J. A.; Zia, A.; Fletcher, N. L.; Thurecht, K. J.; Davis, T. P.; Kempe, K. Synthesis of Biscarboxylic Acid Functionalised EDTA Mimicking Polymers and Their Ability to Form Zr(Iv) Chelation Mediated Nanostructures. Polym. Chem. 2020, 11 (16), 2799– 2810,  DOI: 10.1039/D0PY00304B
      72
      Synthesis of biscarboxylic acid functionalised EDTA mimicking polymers and their ability to form Zr(IV) chelation mediated nanostructures
      Leiske, Meike N.; Walker, Julia A.; Zia, Aadarash; Fletcher, Nicholas L.; Thurecht, Kristofer J.; Davis, Thomas P.; Kempe, Kristian
      Polymer Chemistry (2020), 11 (16), 2799-2810CODEN: PCOHC2; ISSN:1759-9962. (Royal Society of Chemistry)
      In an effort to design novel chelating polymers, polyacrylates with biscarboxylic acid contg. repeating units were synthesized by reversible addn.-fragmentation chain-transfer (RAFT) polymn. of a new EDTA mimicking monomer bearing two carboxylic acids bridged by a tertiary amine group. The monomer presented is accessible in a simple two step strategy and can be polymerised in a controlled fashion enabling the synthesis of defined homopolymers (D < 1.3) and the chain extension of a N-acryloyl morpholine chain transfer agent (CTA) to afford block copolymers of tailored compn. Upon deprotection of the carboxylic acid groups, the polymers showed similar pKa values as EDTA and were able to efficiently complex Zr(IV) in aq. soln. Specifically, complexation studies using block copolymers revealed the formation of defined nanostructures, which were stable in serum contg. cell media for at least 24 h. Their negligible cell toxicity and ability to encapsulate 89Zr render the polymers and nanostructures presented suitable candidates for future diagnostic and radiotherapeutic applications in biomedicine.
    73. 73
      Richter, F.; Leer, K.; Martin, L.; Mapfumo, P.; Solomun, J. I.; Kuchenbrod, M. T.; Hoeppener, S.; Brendel, J. C.; Traeger, A. The Impact of Anionic Polymers on Gene Delivery: How Composition and Assembly Help Evading the Toxicity-Efficiency Dilemma. J. Nanobiotechnol. 2021, 19 (1), 292,  DOI: 10.1186/s12951-021-00994-2
      73
      The impact of anionic polymers on gene delivery: how composition and assembly help evading the toxicity-efficiency dilemma
      Richter, Friederike; Leer, Katharina; Martin, Liam; Mapfumo, Prosper; Solomun, Jana I.; Kuchenbrod, Maren T.; Hoeppener, Stephanie; Brendel, Johannes C.; Traeger, Anja
      Journal of Nanobiotechnology (2021), 19 (1), 292CODEN: JNOAAO; ISSN:1477-3155. (BioMed Central Ltd.)
      Cationic polymers have been widely studied for non-viral gene delivery due to their ability to bind genetic material and to interact with cellular membranes. However, their charged nature carries the risk of increased cytotoxicity and interaction with serum proteins, limiting their potential in vivo application. Therefore, hydrophilic or anionic shielding polymers are applied to counteract these effects. Herein, a series of micelle-forming and micelle-shielding polymers were synthesized via RAFT polymn. The copolymer poly[(Bu acrylate)-b-(2-(di-Me amino)ethyl acrylamide)] (P(nBA-b-DMAEAm)) was assembled into cationic micelles and different shielding polymers were applied, i.e., poly(acrylic acid) (PAA), poly(4-acryloyl morpholine) (PNAM) or P(NAM-b-AA) block copolymer. These systems were compared to a triblock terpolymer micelle comprising PAA as the middle block. The assemblies were investigated regarding their morphol., interaction with pDNA, cytotoxicity, transfection efficiency, polyplex uptake and endosomal escape. The naked cationic micelle exhibited superior transfection efficiency, but increased cytotoxicity. The addn. of shielding polymers led to reduced toxicity. In particular, the triblock terpolymer micelle convinced with high cell viability and no significant loss in efficiency. The highest shielding effect was achieved by layering micelles with P(NAM-b-AA) supporting the colloidal stability at neutral zeta potential and completely restoring cell viability while maintaining moderate transfection efficiencies. The high potential of this micelle-layer-combination for gene delivery was illustrated for the first time.

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