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Synthesis of 19F MRI Nanotracers by Dispersion Polymerization-Induced Self-Assembly of N-(2,2,2-Trifluoroethyl)acrylamide in Water
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  • Vyshakh M. Panakkal
    Vyshakh M. Panakkal
    Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Prague 2 128 40, Czech Republic
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  • Dominik Havlicek
    Dominik Havlicek
    Department of Diagnostic and Interventional Radiology, Institute for Clinical and Experimental Medicine, Prague 140 21, Czech Republic
    Faculty of Health Studies, Technical University of Liberec, Studentská 1402/2, Liberec 461 17, Czech Republic
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    Orcidhttps://orcid.org/0000-0002-2328-2181
  • Ewa Pavlova
    Ewa Pavlova
    Institute of Macromolecular Chemistry, AS CR, Prague 6 162 06, Czech Republic
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  • Marcela Filipová
    Marcela Filipová
    Institute of Macromolecular Chemistry, AS CR, Prague 6 162 06, Czech Republic
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    Orcidhttps://orcid.org/0000-0003-3503-7973
  • Semira Bener
    Semira Bener
    Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Prague 2 128 40, Czech Republic
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    Orcidhttps://orcid.org/0000-0001-5340-068X
  • Daniel Jirak
    Daniel Jirak
    Department of Diagnostic and Interventional Radiology, Institute for Clinical and Experimental Medicine, Prague 140 21, Czech Republic
    Faculty of Health Studies, Technical University of Liberec, Studentská 1402/2, Liberec 461 17, Czech Republic
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    Orcidhttps://orcid.org/0000-0001-6834-3462
  • Ondrej Sedlacek*
    Ondrej Sedlacek
    Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Prague 2 128 40, Czech Republic
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0001-5731-2687
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Biomacromolecules

Cite this: Biomacromolecules 2022, 23, 11, 4814–4824
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https://doi.org/10.1021/acs.biomac.2c00981
Published October 17, 2022

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Abstract

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19F magnetic resonance imaging (MRI) using fluoropolymer tracers has recently emerged as a promising, non-invasive diagnostic tool in modern medicine. However, despite its potential, 19F MRI remains overlooked and underused due to the limited availability or unfavorable properties of fluorinated tracers. Herein, we report a straightforward synthetic route to highly fluorinated 19F MRI nanotracers via aqueous dispersion polymerization-induced self-assembly of a water-soluble fluorinated monomer. A polyethylene glycol-based macromolecular chain-transfer agent was extended by RAFT-mediated N-(2,2,2-trifluoroethyl)acrylamide (TFEAM) polymerization in water, providing fluorine-rich self-assembled nanoparticles in a single step. The resulting nanoparticles had different morphologies and sizes ranging from 60 to 220 nm. After optimizing their structure to maximize the magnetic relaxation of the fluorinated core, we obtained a strong 19F NMR/MRI signal in an aqueous environment. Their non-toxicity was confirmed on primary human dermal fibroblasts. Moreover, we visualized the nanoparticles by 19F MRI, both in vitro (in aqueous phantoms) and in vivo (after subcutaneous injection in mice), thus confirming their biomedical potential.

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1. Introduction

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Fluoropolymers stand out as an important class of high-performance materials with numerous commercial applications, ranging from construction materials through antiadhesive coatings and electronics to biomaterials. (1,2) Fluorinated polymers have recently shown significant potential in medicine as metal-free diagnostic tracers for 19F magnetic resonance (MR) imaging (MRI). (3,4) Although currently used clinical MRI techniques highlight the biodistribution of hydrogen nuclei (mainly from water and lipids), they suffer from a high background of omnipresent water. Conversely, 19F MRI accurately visualizes magnetically active natural fluorine atoms because there is virtually no fluorine background in the body, which enables a straightforward “hotspot” visualization of fluorinated tracers in an organism for diagnostic purposes. Furthermore, given the resonance frequency of 19F, which is very close to that of hydrogen, fluorinated tracers can be visualized on commercial MRI scanners with only minor radiofrequency coil adjustments. (5) Accordingly, spectrally resolved MR can simultaneously provide both spatial and spectroscopic data and thus more complex information and be used with various systems for its advantages over conventional imaging, including higher sensitivity. (6,7)
Notwithstanding its potential, 19F MRI is still only relatively sparsely used, mostly due to the limited availability or unfavorable properties of fluorinated tracers. On the one hand, perfluorocarbons (PFCs) and perfluoro-crown ethers (PFCEs) benefit from their high fluorine content but exhibit an extremely hydrophobic character, (8,9) thus requiring stabilization in nanoemulsions with surfactants. As a result, they display relative instability, limited biocompatibility, and suboptimal magnetic relaxations of fluorine atoms. On the other hand, fluorinated polymer materials open up countless possibilities regarding their macromolecular architecture and fluorine modifications. (3,5) In particular, water-soluble semifluorinated polymers, such as poly(N-(2-((2,2,2-trifluoroethyl)sulfinyl)ethyl)acrylamide), (10) poly(N-(2-fluoroethyl)acrylamide), (11) poly(N-(2,2-difluoroethyl)acrylamide), (12) or various statistical copolymers of fluorinated monomers with hydrophilic monomers, (13−16) show promising diagnostic potential for their excellent 19F relaxation properties and biocompatibility. However, their fluorine content is rather limited because increasing fluorine functionalization hampers polymer solubility in water and its magnetic relaxation properties. (13) Therefore, the fluoropolymer structure must be generally optimized for the maximal 19F MR signal while retaining favorable physical and biological characteristics.
As alternative tracers, self-assembled amphiphilic block copolymer nanoparticles containing a fluorinated core-forming block provide high fluorine loadings. (17,18) However, the high fluorine density within the nanoparticle core may reduce chain segment mobility and, consequently, significantly attenuate the 19F MRI signal. Nevertheless, this effect is known to be governed by the structure, hydration, and chain flexibility of the fluorinated block. For example, fluorine-rich diblock copolymers of thermoresponsive poly(N-(2,2-difluoroethyl)acrylamide) (PDFEAM) retain excellent 19F relaxation properties in water even after self-assembly upon heating above the lower critical solution temperature of PDFEAM (∼22 °C). (19) This ability to retain 19F relaxation properties can be explained by the excellent hydration of the acrylamide-based polymer backbone even in the phase-separated state. For this reason, considering its permanent hydrophobicity, we selected poly(N-(2,2,2-trifluoroethyl)acrylamide) as a hydrophobic block for the present study.
Polymerization-induced self-assembly (PISA) has recently emerged as a powerful and straightforward method for synthesizing self-assembled polymer nanoparticles. (20−25) In aqueous PISA, a water-soluble precursor block is chain extended by a hydrophobic block through direct polymerization in water, resulting in chain growth and block-copolymer self-assembly in a single step. Depending on the core-forming monomer solubility, PISA can be performed by dispersion or emulsion polymerization. In aqueous dispersion PISA, the core-forming monomers are soluble in the polymerization solvent (water), but their growing polymers become insoluble at a specific critical degree of polymerization (DP), leading to self-assembly during polymerization. Throughout PISA, the unreacted monomer is encapsulated into freshly formed nanoparticles, diffuses close to the growing chain end, and solvates the growing block, significantly boosting the polymerization rate. As such, PISA has numerous advantages over traditional methods, such as combining the synthesis and self-assembly process in a single step, shortening polymerization times, and requiring no purification. Moreover, polymer nanoparticles can be generally prepared at very high concentrations (>30 wt %) and in a wide range of morphologies. Using water as a polymerization solvent also creates an environmentally friendly path to aqueous nanoparticle dispersions. The library of PISA core-forming monomers may be still rather small, but more than a limitation this is an exciting challenge in polymer science.
In this context, the synthesis of 19F MRI nanotracers by PISA is also a promising direction with a high potential for the scalable development of advanced diagnostics. Thus far, studies on PISA of fluorinated monomers have relied on either emulsion PISA in water, which provides a low control over the polymerization process and limited access to advanced nanoparticle morphologies, (26,27) or dispersion PISA in non-aqueous solvents (i.e., alcohols), (28−30) which requires an additional step of dialysis or ultracentrifugation to transfer the nanoparticles into an aqueous environment. Whittaker and colleagues reported the synthesis of 19F MRI nanotracers by dispersion PISA of styrene in isopropanol followed by extensive dialysis to switch the dispersant to water. (31) The fluorine atoms were part of the hydrophilic shell, resulting in excellent 19F MR relaxivity, but the fluorine content was relatively low (<3 wt %), possibly limiting potential applications of such systems. To the best of our knowledge, no study on aqueous dispersion PISA of fluorinated monomers has been published to date.
Herein, we report the synthesis of 19F MRI nanotracers by aqueous dispersion PISA of N-(2,2,2-trifluoroethyl)acrylamide stabilized by a polyethylene glycol (PEG) hydrophilic block. After optimizing the reaction conditions to achieve full monomer conversion in a short time, we characterized the resulting diblock copolymer nanoparticles by NMR, size exclusion chromatography (SEC), dynamic light scattering (DLS), and transmission electron microscopy (TEM and CryoTEM). Subsequently, we thoroughly studied and optimized the 19F MR properties of the nanoparticles at 1.5 T and 4.7 T to create a robust platform for efficient diagnostic imaging. To demonstrate these properties, we performed a pilot in vivo study, where we subcutaneously administered the nanotracer to a healthy animal and monitored its distribution by 19F MRI.

2. Experimental Section

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2.1. Materials

All chemicals, including N,N′-dicyclohexylcarbodiimide (DCC) and 4-(dimethylamino)pyridine (DMAP), were purchased from Sigma-Aldrich unless stated otherwise. N-Hydroxyethyl acrylamide (HEAM) was filtered through a short pad of basic alumina before being used to remove the inhibitor. Polyethylene glycol monomethyl ether, (PEG91-OH, 4 kDa), 2,2′-azobis[2-(2-imidazolin-2-yl)propane]dichloride (VA-044) were purchased from TCI. 2-(n-Butyltrithiocarbonate) propionic acid (BTPA) (32) and N-(2,2,2-trifluoroethyl)acrylamide (TFEAM) (33) were synthesized according to literature protocols. Water was deionized with a Millipore Milli-Q water purification system.

2.2. Synthesis of PEG-BTPA macroCTA

BTPA (238 mg, 1 mmol), PEG91-OH (2 g, 0.5 mmol), and DMAP (2.4 mg, 20 μmol) were dissolved in dry dichloromethane (DCM, 20 mL). After the reaction was cooled to 4 °C, a solution of DCC (206 mg, 1 mmol) in DCM (10 mL) was added dropwise. The reaction was allowed to warm to room temperature and stirred overnight, followed by the concentration under reduced pressure and precipitation in ice-cold diethyl ether. The precipitate was filtered and dried under reduced pressure. The crude polymer was purified by gel filtration on a Sephadex LH-20 column using methanol as an eluent. The polymer-containing fractions were collected and evaporated under reduced pressure to obtain the macro CTA as a yellow powder in a 62% yield. The chain-end modification was confirmed by MALDI-TOF (Figure S1). The absence of free BTPA in the polymer sample was confirmed by SEC by UV–vis detection at 320 nm. The esterification efficiency was determined by 1H NMR spectroscopy from the integral ratios of peaks at 4.20 and 3.25 ppm and was found to be 98.6%.

2.3. Synthesis of Fluorinated Nanoparticles by Aqueous Dispersion PISA of TFEAM

Fluorinated block copolymer nanoparticles were synthesized by RAFT-mediated dispersion PISA of TFEAM in water. In a typical experiment for the synthesis of PEG91-b-PTFEAM100 (total solid content, 6 wt %), TFEAM (70 mg, 0.459 mmol), PEG-BTPA (19 mg, 4.6 μmol), VA-044 {0.3 mg, 1 μmol, [CTA]0/[VA-044]0 = 4:1}, and 1,3,5-trioxane (5 mg) internal standard were dissolved in distilled water (1.4 mL), purged with nitrogen gas, and stirred in an aluminum heating block at 50 °C for 3 h (for PTFEAM DP 50–300), respectively, and 5 h (DP 400–600). The reaction was quenched by exposure to air followed by 1H and 19F NMR analysis. Monomer conversion was determined by 1H NMR spectroscopy of the reaction mixture upon dilution with CD3OD by comparing the residual vinyl peaks at 5.5–6.5 ppm with the signal of the internal standard. To calculate the ratio of both blocks, the nanoparticles were freeze-dried, dissolved in CD3OD, and analyzed by 1H NMR. To improve the 19F MRI potential of the nanoparticles, the same protocol was used to prepare another series of copolymers by PISA of a mixture of TFEAM and HEAM (molar ratios outlined in Table 2).
Table 1. Characteristics of PEG91-b-PTFEAMx Block Copolymer Nanoparticles Synthesized by Aqueous Dispersion PISA of TFEAM with a PEG-BTPA Macro-CTAa
polymerDPTbConv.c (%)F cont.c (wt %)MnNMR,c (kg mol–1)MnSEC,d (kg mol–1)ĐdDh (nm)/PDIe
F150>9923.811.925.21.14173/0.509
F2100>9929.119.641.71.1363/0.180
F3200>9932.734.975.61.1994/0.096
F4300>9934.150.290.21.2197/0.083
F5400>9934.865.5130.51.31136/0.026
F65009835.379.2156.01.32221/0.047
a

All experiments were performed at 50 °C in water at a total solids content of 6 w/w % and [PEG-BTPA]0/[VA-044]0 = 4.

b

PTFEAM target DP defined as the ratio [PTFEAM]0/[PEG-BTPA]0.

c

Determined by 1H NMR.

d

Determined by SEC against PMMA calibration.

e

Determined by DLS in water at cpol = 1 mg mL–1.

Table 2. Characteristics of the PEG91-b-[PTFEAMx-stat-PHEAMy] Block Copolymers and Their Nanoparticles Synthesized by Aqueous Dispersion PISA of TFEAM and HEAM as Core-Forming Monomersa
polymerfHEAMbDPTcConv.d (%)F cont.d (wt %)MnNMR,d (kg mol–1)MnSEC,e (kg mol–1)ĐeDh (nm)/PDIf
F20100>9929.119.641.71.1363/0.180
F2H10.1100>9926.219.245.11.1457/0.136
F2H20.2100>9923.318.843.61.1757/0.261
F2H30.3100>9920.418.441.91.1280/0.304
F3H20.2200>9926.129.176.41.2474/0.113
F4H20.2300>9927.343.680.71.2478/0.127
F5H20.2400>9827.858.2148.31.29144/0.110
a

All experiments were performed at 50 °C in water at a total solids content of 6 w/w % and [PEG-BTPA]0/[VA-044]0 = 4.

b

Molar content of HEAM in the polymerization mixture.

c

Target DP of the core-forming block.

d

Determined by 1H NMR.

e

Determined by SEC against PMMA calibration.

f

Determined by DLS in water at cpol = 1 mg mL–1.

2.4. Polymer Characterization

SEC was used to determine the molecular weights (Mw─weight-averaged molecular weight and Mn─number-averaged molecular weight) and dispersity (Đ = Mw/Mn) of the polymers on a Watrex Streamline system equipped with a Streamline P1 Pump, a Streamline AS2 Autosampler, a Streamline CT Column Thermostat, a Streamline UV detector, and a Streamline RI detector. The separation was performed on two PLgel 5 μm mixed-D columns in a series thermostatted at 55 °C in N,N-dimethylacetamide (DMA) containing 50 mM of LiCl at an elution rate of 0.5 mL min–1. Molar masses and dispersities were calculated against narrow dispersity poly(methyl methacrylate) standards.
Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Advance MSL 400 MHz NMR spectrometer at 25 °C in CD3OD, DMSO-d6 or a mixture of H2O/D2O (95/5 v/v). Unless otherwise stated, all 19F NMR spectra were measured at cpol = 30 mg mL–1 using 20 μs pulse width, relaxation delay 8 s, acquisition time 1.5 s, and 64 scans, expressing all chemical shifts as ppm. The NMR spectra were processed using MestReNova 14.1 software, and the signal-to-noise ratios (SNRs) were calculated using the built-in MestReNova function. The Mn,NMR values were calculated by comparing the integral areas of peaks at 3.45 and 2.0 ppm.
Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) was performed on a Microflex LT MALDI-TOF MS (Bruker Daltonics) mass spectrometer. All mass spectra were recorded at an accelerating potential of 20 kV in the positive ion mode and in reflectron mode {matrix: 2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene] malononitrile (DCTB)}. Samples were applied to the MALDI plate using the dried drop method. All measurements were calibrated using methoxy poly(ethylene glycol) (PEG, Mn = 2000 Da).
Critical micelle concentration (CMC) of nanoparticles was determined by fluorescence spectrometry on a Fluorolog FL 3-22 fluorimeter (Horiba Jobin Yvon, France) after Nile red (NR) encapsulation. A stock solution of NR in methanol (5 μL) was added to each sample of a series of aqueous nanoparticle dispersions (2 mL) of different polymer concentrations to a final NR concentration of 10–6 M. After incubation for 72 h at room temperature, fluorescence was measured using an excitation wavelength λex = 550 nm. The CMC value was determined as the range at which the emission maximum of NR shifts from ∼620 nm (encapsulated NR) to ∼660 nm (free NR).
DLS measurements were used to determine the hydrodynamic diameters of the polymers in distilled water on a ZEN3600 Zetasizer Nano-ZS zeta potential analyzer (Malvern Instruments, UK). The polymer samples (cpol = 1 mg mL–1) were filtered through a 0.22 μm PTFE syringe filter before measuring. The apparent Z-averaged hydrodynamic diameter of the particles, Dh, was determined at a scattering angle of θ = 173°, and the DTS (Nano) program was used to evaluate the data.
TEM and cryo TEM (CryoTEM) observations were performed through a Tecnai G2 Spirit Twin 120 kV TEM (FEI), equipped with cryo-attachment (Gatan, cryo specimen holder) using a bright field imaging mode at an accelerating voltage of 120 kV. Aqueous solutions of the nanoparticles (3 μL, cpol = 1 mg mL–1) were dropped on a copper TEM grid coated with a thin electron transparent carbon film. Before use, the grids were treated by glow discharge (Expanded Plasma Cleaner; Harrick Plasma, USA) to hydrophilize the carbon surface. After 2 min, the excess solution was removed by touching the bottom of the grids with filtering paper to minimize oversaturation during the drying process. Additionally, the samples were negatively stained with the uranyl acetate (2 μL of 1 wt %) solution dropped onto the dried nanoparticles and removed after 30 s as described above. Lastly, the samples were left to dry completely at room temperature before observation. The CryoTEM method employed different sample preparation. Aqueous solution of nanoparticles (4 μL) was deposited on a microscopy grid covered with lacey carbon supporting films (Agae Scientific) after hydrophilization by glow discharge (performed like in the previous method). The solution excess was removed by blotting (Whatman no. 1 filter paper) for ∼1 s and then the grids were immediately plunged into liquid ethane held at −183 °C. The vitrified samples were transferred into the microscope and observed at −175 °C under the conditions described above (120 kV, bright field mode)

2.5. Magnetic Resonance Properties

Relaxometry was used to measure the 19F relaxation times of fluorinated nanoparticles in distilled water on a 1.5 T Minispec 60 MHz relaxometer (Bruker Biospin, Germany) at 37 °C equipped with a fluorine probe (resonance frequency for fluorine was 54 MHz). The T1 relaxation times were measured with the inversion recovery sequence [repetition time (TR) = 0.1–10,000 ms, recycle delay = 4 s, scans = 4, echo time (TE) = 0.05 ms, monoexponential fitting, 16 points per fitting]. The T2 relaxation times were measured with the Carr–Purcell–Meiboom–Gill (CPMG) sequence (TR = 10,000 ms, recycle delay = 2 s, scans = 8, TE = 0.05 ms, monoexponential fitting, 20,000 points per fitting).

2.6. Imaging

1H/19F MR properties of fluorinated nanoparticles were measured in water by 19F MR spectroscopy (MRS) and 19F MR imaging (MRI) on a 4.7 T (Bruker Biospec 47/20, Ettlingen, Germany) scanner at 25 °C both in aqueous phantoms (i) and in vivo (ii). The MR scanner was equipped with a 1H/19F custom-made radiofrequency surface coil. (34) (i) The T2-weighted 1H MR images were acquired for reference using a Rapid Acquisition with a Relaxation Enhancement (RARE) sequence with the following parameters: TR = 3000 ms, TE = 12 ms, effective echo time TEeff = 36 ms, turbo factor = 8, bandwidth = 34,722 Hz, spatial resolution = 0.137 × 0.137 mm2, slice thickness = 0.85 mm, number of acquisitions NA = 1, and scan time = 1 min 12 s. The fluorine phantom MRI experiment was performed using two different sequences: (1) RARE sequence was used to measure samples with different HEAM contents, with the following parameters: TR = 2000 ms, TE = 5.60 ms, TEeff = 22.40 ms, turbo factor = 10, bandwidth = 34,722 Hz, spatial resolution = 0.625 × 0.625 mm2, slice thickness = 9 mm, NA = 10–2000, and scan time = 2 m–6 h 40 min. (2) Multi-slice multi-echo sequence was used to measure F5H2 at different concentrations, with the following parameters: TR = 2000 ms, TE = 6.14, bandwidth = 34,722 Hz, spatial resolution = 0.779 × 0.779 mm2, slice thickness = 9 mm, NA = 1–100, and scan time = 4 m–7 h. The imaging experiment was performed at 25 °C in 0.5 mL Eppendorf tube phantoms containing various concentrations of nanoparticles (cpol = 5–30 mg mL–1) with the cross sections of the tubes shown in the phantom images. The 19F image was overlapped with the anatomic 1H image with spin-echo-based contrast. (ii) An in vivo measurement was performed using one healthy female BALB/c mouse as a proof of principle. The mouse was anesthetized with 5% isoflurane (Baxter, Deerfield, USA) for induction and 1.5–0.5% isoflurane for maintenance. The respiratory rate was monitored throughout the study using a trigger unit (Rapid Biomedical, Berlin, Germany). To avoid eye dryness and its potential damage, an eye cream (Ophtalmo-Septonex, Zentiva, Czech Republic) was applied before the measurement. Subcutaneous injection of F5H2 (V = 200 μL, cpol = 60 mg mL–1) in phosphate-buffered saline (PBS) was applied into the inner side of the right hind leg. On the side of the radiofrequency coil, we put the Eppendorf tube containing F5H2 (V = 200 μL, cpol = 60 mg mL–1) in PBS as a reference. For the 1H reference image, we used the same sequence parameters as in the phantom measurement. 19F MRS single-pulse sequence (TR = 2000 ms, bandwidth = 200 ppm, NA = 150, and ST = 5 min) was used to confirm the presence of the fluorine signal and to fine-tune the resonance frequency. For 19F MR imaging, we performed spectrally resolved MRI using a chemical shift imaging (CSI) sequence with the following parameters: TR = 200 ms, bandwidth = 40 ppm, spatial resolution = 2.81 × 2.83 mm2, slice thickness = 10 mm, and ST = 13 min 40 s. 19F-MR CSI and spectroscopic data were processed and analyzed using custom MATLAB (https://mathworks.com, Matlab R2021b, The MathWorks, Inc., USA) scripts.
All animal protocols were approved by the Ethics Committee of the Institute for Clinical and Experimental Medicine and the Ministry of Health of the Czech Republic (no. 58/2014) in accordance with the European Communities Council Directive (2010/63/EU).

2.7. Cytotoxicity of Fluorinated Nanoparticles

Human primary cells and culture conditions─human primary dermal fibroblasts (HFs) were kindly gifted by the Institute of Experimental Medicine, Academy of Sciences of the Czech Republic. The cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (100 U mL–1, Thermo Fisher Scientific, USA) at 37 °C under a humidified 5% CO2 atmosphere. HFs were subcultured every four days and used for experiments up to the n passage.
Cytotoxicity assay─HFs were seeded at a concentration of 8 × 103/100 μL/well on 96-well plates and cultured for 24 h at 37 °C in an incubator. Subsequently, the cells were treated with F5H2 nanoparticles in a twofold serial dilution starting from the highest final concentration (2 × 103 μg mL–1). After 72 h of incubation, 10 μL of PrestoBlue cell viability reagent (Thermo Fisher Scientific, USA) were added into the medium with cells in the presence of F5H2 nanoparticles and into the blank wells (medium with nanoparticles without cells) and incubated for 4 h 30 min at 37 °C. The fluorescence intensity of the developed dye (resorufin) was measured at an Ex/Em wavelength of 550/590 nm (bandwidth 20 nm) in the top-optic mode on a Spark multimode microplate reader (Tecan Group Ltd., Männedorf, Switzerland). The relative cell viability (%) after exposure to fluorinated nanoparticles was expressed as a percentage of viable cells after the treatment in comparison with the control set to 100%. The experiment was performed three times in sextuplicates.

3. Results and Discussion

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N-(2,2,2-Trifluoroethyl)acrylamide (TFEAM) was selected as a core-forming monomer to synthesize 19F MRI nanotracers by aqueous dispersion PISA for its solubility in water and favorable 19F MR properties. Although most semifluorinated monomers, including N-(2,2,2-trifluoroethyl)acrylate and N-(2,2,2-trifluoroethyl)methacrylate, are disqualified for their negligible solubility in water, acrylamide-containing TFEAM is soluble (estimated as 7 wt % at 50 °C) enough for aqueous dispersion PISA. Furthermore, TFEAM contains three magnetically equivalent fluorine atoms and thus provides a sole singlet in its 19F NMR spectrum. Lastly, this monomer can be easily prepared in high quantities by straightforward acrylation of trifluoroethylamine, as shown in Scheme 1.

Scheme 1

Scheme 1. Synthesis of 19F MRI Tracers by RAFT-Mediated Aqueous Dispersion PISA of TFEAM: (A) Schematic Illustration and (B) Reaction Scheme
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RAFT-mediated aqueous dispersion PISA of TFEAM was performed in distilled water using 4 kDa PEG91-BTPA as a macromolecular chain-transfer agent and VA-044 as a water-soluble initiator at a total solid concentration of 6 wt %. Even though PISA has been reported for much higher monomer concentrations, the targeted nanoparticle concentration sufficed for the intended application as a 19F MRI tracer. The polymerizations were performed at 50 °C as higher reaction temperatures (60 °C) led to macroscopic precipitation of the reaction mixture for higher target DPs (>150). Such behavior is commonly observed in aqueous PISA when using PEG-based macroCTAs, which tend to aggregate in water at elevated temperatures, as reported by Armes and colleagues. (21,35)
The polymerization kinetics measurement of DPPTFEAM 100 and 200 copolymers revealed a controlled polymerization process (Figures 1 and S2, S3). After an initial induction period (20 min for target DP 100), with a slow rate, polymerization significantly accelerated at 40 min (for DP 100), and the solution became opalescent, indicating the formation of self-assembled nanoparticles. Following polymerization kinetics, SEC showed that all polymerizations proceeded in a controlled way, with a linear increase of polymer molar mass with conversion. The SEC-based molar masses were systematically higher than the corresponding theoretical values due to the differences in the hydrodynamic sizes of our copolymers and poly(methyl methacrylate) (PMMA) SEC standards in N,N-dimethylacetamide (DMA) eluents. On the other hand, the NMR-based molar masses fit the theoretical values. Throughout the polymerization, the molar mass dispersities remained low (D̵ < 1.13), which is typical for dispersion PISA. The observed high-molecular-weight shoulders in chromatograms most probably originate from the end-to-end coupling of living macroradicals.

Figure 1

Figure 1. Aqueous dispersion PISA kinetics of TFEAM at 50 °C using PEG-BTPA macroCTA {[TFEAM]0:[PEG-BTPA]0 = 100:1}: (A) variation of monomer consumption as a function of polymerization time, (B) evolution of SEC traces during polymerization eluted with DMA/LiCl, and (C) variation of experimental number-average molar mass (Mn) and dispersity (D̵) as a function of conversion of TFEAM determined by SEC against PMMA calibration (squares), respectively NMR (triangles). The blue line represents the theoretical Mn value.

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The optimized PISA protocol was applied to synthesize a series of fluorinated copolymer nanoparticles differing in PTFEAM block length (F1-6, DP = 50–500, Table 1 and Figure 2). Longer polymerization times (3–5 h) were necessary to ensure full monomer conversions aiming to directly use the fluorinated nanoparticle dispersions as 19F MRI tracers without any purification. During the polymerization, all copolymers up to DP 500 formed nanoscale colloid dispersions without any signs of macroscopic aggregation or precipitation.

Figure 2

Figure 2. Representative 1H (A) and 19F (B) NMR spectra of PEG91-b-PTFEAM100 (F2) in CD3OD at 400 MHz and (C) transmission electron micrographs of F1–F6 nanoparticle dispersion; the scale bars represent 200 nm. Insets: physical appearance of as-prepared nanoparticle dispersions in water.

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The copolymer compositions were determined by 1H NMR after freeze-drying and dissolving in deuterated methanol and were very close to the initial monomer/macroCTA feed ratios. 19F NMR spectroscopy in CD3OD revealed a sharp singlet at −73.5 ppm corresponding to the −CF3 side-chain groups. SEC analysis confirmed that copolymer chain length increased with the TFEAM/macroCTA feed ratio, whereas dispersity remained reasonably low (Đ < 1.35) (Table 1 and Figure S4A).
The hydrodynamic diameters of the nanoparticles were measured upon dilution in water by DLS, (Figure S4B). Except for the shortest DP 50 copolymer, nanoparticle size gradually increased with chain length from 63 nm (F2, DP 100) to 221 nm (F5, DP 500), with narrow size distributions (dispersity 0.03–0.18). Nanoparticle size and morphology were also studied by TEM (Figure 2), corroborating the DLS data. The results showed a mixed morphology consisting of small spheres with worm-like micelles in the shortest DP 50 nanoparticles, which can be ascribed to the lack of proper core stabilization. Spherical morphologies were observed in nanoparticles with higher DPs (100–500). The nanoparticle diameters were, nevertheless, much larger than the calculated extended copolymer lengths. For this reason, cryoTEM measurements were performed in F2 and F6, in water, to explore the possibility of vesicular morphology (Figure S5). CryoTEM of F2 highlighted irregular, mostly donut-shaped nanoparticles. On the other hand, F6 nanoparticles showed a homogeneous cryoTEM contrast, suggesting large compound micelles rather than hollow polymersomes. (36)
19F MR imaging of PEG-b-PTFEAM micelles in water unsurprisingly provided limited information due to the restricted mobility of the core-forming fluorinated segments, thus accounting for the poor magnetic relaxation properties of the fluorine nuclei. The main 19F NMR signal can be ascribed to the traces of unreacted monomer, which disappears after ultracentrifugation leaving barely any signal in the spectrum (Figure S6). This is in sharp contrast to the excellent 19F MR properties of the homologous thermoresponsive poly(N-(2,2-difluoroethyl)acrylamide) copolymers, which were retained even after copolymer self-assembly in water. (19) Although PEG-b-PTFEAM nanoparticles lacking a 19F NMR signal may be useful for developing various stimuli-responsive “on–off” reporting systems, their core-forming block structure required optimization for the intended application as 19F MRI tracers.
To improve the 19F MRI tracing potential of our nanoparticles, we introduced a small part of the hydrophilic comonomer N-hydroxyethyl acrylamide (HEAM) into the PISA reaction mixture (Figure 3A), thereby diluting the local fluorine concentration in the core and improving chain hydration and mobility, ultimately enhancing the 19F NMR/MRI signals. Copolymers with different TFEAM/HEAM feed ratios (9:1 for F2H1, 8:2 for F2H2, and 7:3 for F2H3, respectively) and the same core DP 100 were synthesized and characterized using the aforementioned methods (Figure S7). In their 1H NMR spectra, the signals of HEAM side-chain protons overlapped with those of PEG (−CH2–OH, 3.65 ppm) and methanol (−NH–CH2–, 3.33 ppm). Therefore, the presence of HEAM was confirmed by 1H–13C HSQC 2D NMR (Figure S8), which separated the overlapped peaks by their 13C shifts.

Figure 3

Figure 3. Synthesis of PEG91-b-[PTFEAMx-stat-PHEAMy] block copolymers with a core partly hydrophilized by aqueous PISA: (A) reaction scheme and (B) TEM images of nanoparticles differing in HEAM content (FHEAM = 0–0.4) at constant core-forming block length (DP 100); the scale bars represent 200 nm. Insets: physical appearance of as-prepared nanoparticle dispersions in water.

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The measured PHEAM content was close to that of the initial monomer feed. Despite the modification, the fluorine content remained very high (20 wt % for F2H3). Stable colloidal dispersions were obtained in water at HEAM contents up to 30%. Higher hydrophilic comonomer contents (>40%) resulted in the loss of amphiphilicity, as shown by DLS, where separate sub-10 nm copolymer coils prevailed. The size of the nanoparticles did not change substantially with the HEAM content. These findings were in line with TEM experiments (Figure 3B), where a change in nanoparticle morphology was observed with the increase in HEAM content from 50 nm “donuts” (0% HEAM) to irregular-shaped separated nanoparticles of the same size (F2H3). The morphology change was confirmed by cryogenic TEM microscopy (Figure S9) for nanoparticles F2H2 and F2H3, as well.
Our 19F MR measurements confirmed the strong impact of the HEAM comonomer on the 19F MR properties of fluorinated nanoparticles. The HEAM-free nanoparticles F2 showed virtually no 19F NMR signal due to the limited relaxation of fluorine atoms. Moreover, the main signal may be ascribed to traces of unreacted monomers (−72.2 ppm) encapsulated within the micelle core, as suggested by the broad peak distribution (Figure 4A).

Figure 4

Figure 4. Partial core hydrophilization with HEAM enhances the 19F MR signal of fluorinated nanoparticles (cpol = 30 mg mL–1, DPcore = 100) in aqueous solutions. (A) Evolution of nanoparticle 19F NMR spectra (400 MHz) as a function of HEAM content; (B) variation of 19F MR relaxation times as a function of HEAM content; (C) 1H and (D) 19F RARE MRI of nanoparticles (4.7 T) with different HEAM contents; (E) overlay image of 19F MRI (red) and 1H MRI (gray); and (F) 19F MR spectra (4.7 T) of nanoparticles centered and used for MRI acquisition; inset: comparison of 19F MRI SNR values.

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Although the TFEAM monomer is detected only in trace amounts (<0.1 mol %), its superior 19F relaxation to that of the self-assembled fluoropolymer led to its dominant NMR signal. The identity of this peak was confirmed by 19F NMR measurements of the nanoparticle dispersion after the addition of additional TFEAM, which only increased peak intensity, without any change in chemical shifts. Conversely, the disassembly of the same nanoparticle dispersion by deuterated methanol led to a sharp polymer peak in the 19F spectrum, strongly exceeding the trace monomer peak, due to enhanced polymer relaxation in methanol and high fluorine content.
In aqueous solutions, the nanoparticles showed a strong 19F NMR signal only after HEAM incorporation. The nanoparticle 19F NMR signal increased rapidly with the HEAM content despite the slight decrease in fluorine content (from 29 to 20 wt %). The SNRs of the nanoparticle dispersions increased from 52 (F2H1) to 307 (F2H3) due to the enhanced magnetic relaxation of fluorinated segments. To demonstrate that the strong 19F NMR signal resulted from the polymer nanoparticle and not from the potential residual encapsulated monomer, F2H3 micelles were freeze-dried and purified from any low-molar-mass impurities by gel filtration in methanol, followed by re-assembly by nanoprecipitation. The 19F NMR spectrum of purified micelles was nearly identical to the spectrum of diluted as-obtained micelles (Figure S10).
The 19F MR relaxations of nanoparticles F2 and F2H1-3 were studied in water by MR relaxometry at both 1.5 T and 4.7 T (Figure 4B and Table S1). In general, high-intensity 19F MR images require short spin–lattice T1 relaxation times and long spin–spin T2 relaxation times. (4) While excessively long T1 relaxation times may result in inevitably long acquisition times, very short T2 times could limit the imaging to non-standard sequences with extremely short echo times (TEs), which are not routinely implemented in MR scanners.
The T1 relaxation times were measured with a single-pulse sequence using monoexponential fitting, showing reasonably low values (T1 = 350–500 ms), without significant differences between polymers. In turn, the T2 relaxation times were measured with the CPMG sequence at 4.7 T and showed a significant increase with the HEAM comonomer content from 12.2 ms (F2H1) to 40.5 ms (F2H3). Accordingly, the increase in the 19F MR signal strength with the HEAM content may be ascribed to enhanced T2 relaxation times. Even though these T2 values are lower than those commonly observed in water-soluble semifluorinated polymers, (13) they suffice to achieve a strong 19F MR signal for effective imaging thanks to the high fluorine content (>20 wt %).
The favorable 19F MR relaxation parameters of HEAM-rich copolymers allowed us to visualize nanoparticles in vitro in Eppendorf tube phantoms by 4.7 T 19F MRI using a conventional Rapid Imaging with Refocused Echoes (RARE) pulse sequence with long echo times (TE = 5.6 ms, Figures 4C–F and S12). The 19F MRI SNR of nanoparticle tracers significantly increased with the HEAM content, providing us with outstanding tracing sensitivity and with the ability to shorten the acquisition times necessary for reliable visualization. Therefore, the copolymers with a high HEAM content (F2H2 and F2H3) may be then relevant for potential in vivo tracing applications.
Micellar stability is one of the key characteristics of self-assembled nanopharmaceuticals, strongly affecting their pharmacological profile and the release of potentially encapsulated drugs. Hence, we studied the equilibrium stability of the nanoparticles by measuring their CMC, which expresses the concentration threshold above which the micelles are formed. When diluted below their CMC, the micelles disassemble into individual unimer chains. Herein, the CMC of the fluorinated nanoparticles were measured in water after encapsulating Nile red as a solvatochromic fluorescence probe (Figure S11).
The HEAM-free F2 nanoparticles (PEG91-b-PTFEAM100) showed high equilibrium stability, with CMC values ranging from 2 to 4 mg L–1, corroborating the tight micelle core packing suggested above, which leads to a poor 19F NMR signal in water. Increasing the HEAM content enhanced the 19F MRI signal but impaired nanoparticle stability due to weakened core hydrophobic interactions. For example, F2H2 showed relatively high CMC values (125–250 mg L–1), which may be detrimental to potential biomedical applications given the expected rapid blood clearance. To foster micelle stability, we increased the DP of the core-forming block from 100 (F2H2) to 400 (F5H2) while maintaining the HEAM/TFEAM molar feed ratio constant at 2:8. (37) As expected, such a chain extension had a positive effect on nanoparticle stability, and the CMC of F5H2 dropped to the range of 31–63 μg mL–1 suitable for applications. Moreover, the increased nanoparticle chain length did not attenuate the 19F NMR signal (Table S1), and the SNR values even increased from 106 (DP 100) to 286 (DP 400), reflecting the increasing fluorine content.
The increase in core block length had a major impact on the nanoparticle size and morphology (Figures 5 and S14). The hydrodynamic diameter of the nanoparticles increased from 57 (F2H2, DP 100) to 144 (F5H2, DP 400) nm, which was accompanied by a morphological transition from irregular-shaped separated nanoparticles (DP 100) to polymersomes (DP 200 and 300) and large compound micelles (DP 400). This morphological transformation may be attributed to a change in the micelle core packing parameter. Based on their favorable stability and 19F NMR characteristics, the DP 400 nanoparticles (F5H2) were selected as an optimal 19F MRI tracer for further evaluation.

Figure 5

Figure 5. (A) Intensity-weighted DLS size distributions of PEG91-b-[PTFEAMx-stat-PHEAMy] copolymer nanoparticles (x/y = 8:2) differing in core-forming block DP (100–400) in water (cpol = 1 mg mL–1) and (B) transmission electron micrograph of F5H2 nanoparticles (DP 400); the scale bar represents 200 nm.

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The aqueous dispersions of the optimized F5H2 nanoparticles showed favorable 19F MR relaxivity and MRI properties. The longitudinal relaxation time did not change significantly with the copolymer DP, remaining reasonably low (T1 = 469 ms for F5H2). Furthermore, the transverse relaxation time T2 even slightly increased to 62.5 ms for F5H2, in line with our previous 19F NMR observations. We believe that such an improvement in T2 as a function of the core DP is associated with the enhanced relaxation of fluorinated segments close to the core–shell interface of higher order nanoparticles.
The superior 19F MR relaxation of F5H2 micelles is reflected in their significantly improved 19F MRI properties (Figures 6 and S15). The nanoparticles were successfully visualized in vitro using a 4.7 T instrument at different polymer concentrations and numbers of acquisition scans. The MRI SNRs increased linearly with the polymer concentration (Figure 6C). From this correlation, the minimum concentration needed to reliably visualize the nanoparticles (SNR = 3.5) can not only be calculated but also quantitatively expresses the MRI sensitivity of our nanotracer at specific acquisition parameters. Indeed, the minimal traceable concentration decreases with the increase in acquisition time (Figure 6D).

Figure 6

Figure 6. In vitro 19F MRI/MRS properties of optimized F5H2 nanoparticles in water; (A) 19F (top), 19F (red) overlaid on 1H (colors in grayscale) RARE MRI (bottom) of F5H2 at different polymer concentrations (20 acquisition scans); the numbers 30, 20, 10, and 5 express cpol as mg mL–1. (B) 19F MR spectra (4.7 T) centered and used for MRI acquisition; (C) variation of the 19F MRI SNR ratio as a function of F5H2 concentration at different numbers of acquisition scans; and (D) minimal polymer concentrations needed to reliably visualize (SNR = 3.5) the F5H2 nanotracer at different MRI acquisition times.

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Before advancing to preliminary in vivo experiments, the non-cytotoxicity of F5H2 was confirmed by resazurin-based PrestoBlue cell viability assay upon incubation with human dermal fibroblast cells (HFs; Figure S16). After 72 h, no signs of toxicity were observed within the polymer concentration range tested (up to 2 mg mL–1). These results suggest the excellent cytocompatibility of the nanoparticles.
Given the promising results of the in vitro experiments, we performed in vivo 19F MRS and MRI experiments as a proof-of-concept study (Figure 7). For in vivo imaging, acquisition times should be reasonably short to minimize the time that the animals spend under anesthesia and between measurements to monitor metabolic processes. We subcutaneously administered the F5H2 tracer in PBS (V = 200 μL, cpol = 60 mg mL–1) into the inner side of the right hind leg of a healthy female BALB/c mouse. The target area was chosen primarily for ease of injection. In addition, the probe was only applied to the right leg, so the contralateral left leg of the mouse served as a control. At the edge of the surface coil, we placed an Eppendorf tube containing the same F5H2 solution (V = 200 μL) that we administered as a reference for the fluorine peak assignment.

Figure 7

Figure 7. In vivo 19F MRS and MRI of F5H2 nanotracer 2 h after subcutaneous injection into healthy BALB/c mouse; (A) coronal hot spot 19F MRI (left) and overlapped 1H/19F (right) images, where the F5H2 fluorine signal is highlighted in red color and (B) 19F MR CSI spectroscopic grid on a 1H MR reference scan (left); the red grid shows measured CSI voxels and the green squares represent the targeted anatomical area from which the corresponding summation spectra are displayed (right).

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After the injection, the non-localized 19F MR spectrum of the whole animal plus the reference tube showed three clearly distinguishable peaks–one corresponding to the polymer and two others assigned to the isoflurane anesthetic (Figure S17). The signal coming from the polymer was separated by a large chemical shift. After setting the precise Larmor frequency for the polymer measurements, we removed the F5H2 reference tube from the magnet without changing the position of the animal and repeated the spectroscopic measurement. The leftmost peak became significantly attenuated, whereas the other two peaks showed only negligible changes in intensity. Therefore, we confirmed that the leftmost peak (already with the resonance frequency centered at 0 ppm) reflects only the presence of the F5H2 polymer administered in vivo. The minor changes in the intensity of the remaining two peaks derive from the variable isoflurane concentration used to maintain the animal under anesthesia.
The 19F MR CSI sequence data show that the fluorine signal originates only from the injection region (Figure 7). Figure 7B shows the 19F MR CSI grid spectral view of a 1H MR image reference scan. The red grid displays CSI voxels, and the green box represents the area from which the corresponding summation of spectra is displayed. When comparing spectra from two boxes highlighted in green (left and right leg), 2 h after F5H2 administration, we confirmed the presence of F5H2 in the right leg only–the site of probe administration. No signal was detected in the area corresponding to the left leg. The high-resolution CSI matrix shows well the signal intensities of the applied polymer and corresponds to the CSI image reconstructed for the polymer frequency range only (Figure S18). Moreover, the F5H2 polymer provides a high SNR in vivo in both the spectra (SNR = 12.09) and images (SNR = 42.9) with short acquisition times (5 min for spectra, 13 min 40 s for images).
The preliminary in vivo data suggest the significant potential of our new fluorinated nanoparticles as nanotracers for medical purposes involving intramuscular or subcutaneous applications or as markers for imaging cells such as transplanted pancreatic islets labeled in vitro. (38) More detailed in vivo experiments are, however, beyond the scope of this study, which focused on PISA synthesis and preliminary 19F MRI of fluorinated nanoparticles in phantoms but will nevertheless be conducted in the near future by our research group. Finally, given the current rapid progress in the area of MRI instrumentation, the MRI sensitivity of our nanotracers may be substantially amplified by increasing magnetic field strengths and using cryogenically cooled radiofrequency coils.

4. Conclusions

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Fluorinated nanoparticles prepared by aqueous PISA of water-soluble N-(2,2,2-trifluoroethyl)acrylamide (TFEAM) using PEG-BTPA as a macroCTA show well-defined block copolymer architectures and size and morphology highly dependent on the core-forming block length. Despite their high fluorine content, their negligible 19F NMR signal in water results from the tight packing of the fluorinated hydrophobic core which leads to poor magnetic relaxation of the fluorinated segments and thus requires optimization. Increasing the hydrophilicity of their fluorinated core by incorporating a small amount of N-hydroxyethyl acrylamide (HEAM) significantly improves the 19F MRI signal of the nanoparticles. These nanotracers display good 19F MR relaxation properties and 19F MRI performance. However, micelle core hydrophilization leads to morphological changes and decreases micelle stability. Nevertheless, increasing the core-forming block length while maintaining the optimized TFEAM/HEAM ratio unchanged improves the micelle stability. Such optimized HEAM-containing nanoparticles with a lengthened core-forming block perform well as 19F MRI tracers, showing even better 19F MR relaxation properties and MRI sensitivity than low-DP copolymers on a 4.7 T instrument with magnetic fields close to those used in clinical imaging. Although the MRI sensitivity of our tracer is slightly lower than that of previously reported water-soluble semifluorinated linear polymers, it can be applied for in vivo tracing applications involving high local concentrations of nanotracers (e.g., subcutaneous administration). A more detailed in vivo study is currently underway, and the results will be published soon in a separate article. Ultimately, our nanoparticles may be used as hydrophobic drug delivery carriers combining both tracing and therapy in the same system (i.e., theranostics). Considering their straightforward synthesis, highly modular properties, and a broad range of potential applications, these polymer nanoparticles stand out as excellent materials for advanced biomedical research.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biomac.2c00981.

  • Characterization of synthesized polymers by MALDI-TOF, NMR, SED, Cryo-TEM, and MR (PDF)

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Author Information

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  • Corresponding Author
  • Authors
    • Vyshakh M. Panakkal - Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Prague 2 128 40, Czech Republic
    • Dominik Havlicek - Department of Diagnostic and Interventional Radiology, Institute for Clinical and Experimental Medicine, Prague 140 21, Czech Republic;  Faculty of Health Studies, Technical University of Liberec, Studentská 1402/2, Liberec 461 17, Czech Republic;  Orcidhttps://orcid.org/0000-0002-2328-2181
    • Ewa Pavlova - Institute of Macromolecular Chemistry, AS CR, Prague 6 162 06, Czech Republic
    • Marcela Filipová - Institute of Macromolecular Chemistry, AS CR, Prague 6 162 06, Czech Republic;  Orcidhttps://orcid.org/0000-0003-3503-7973
    • Semira Bener - Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Prague 2 128 40, Czech Republic;  Orcidhttps://orcid.org/0000-0001-5340-068X
    • Daniel Jirak - Department of Diagnostic and Interventional Radiology, Institute for Clinical and Experimental Medicine, Prague 140 21, Czech Republic;  Faculty of Health Studies, Technical University of Liberec, Studentská 1402/2, Liberec 461 17, Czech Republic;  Orcidhttps://orcid.org/0000-0001-6834-3462
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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O.S. and V.M.P. acknowledge the financial support from Czech Grant Foundation (grant Nr. 22-03102S) and Charles University Prague (grant PRIMUS/21/SCI/007). D.H. acknowledges the financial support from the Czech Grant Foundation (project number 22-02836S). D.J. acknowledges the financial support from the Ministry of Health of the Czech Republic (NU22-08-00286) and from the project National Institute for Research of Metabolic and Cardiovascular Diseases (Programme EXCELES, Project no. LX22NPO5104)─Funded by the European Union─Next Generation EU. The authors thank Dr. Carlos V. Melo for editing the manuscript.

References

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    A review. Fluorine-19 (19F) magnetic resonance (MR) mol. imaging is a promising noninvasive and quant. mol. imaging approach with intensive research due to the high sensitivity and low endogenous background signal of the 19F atom in vivo. Perfluorocarbons (PFCs) have been used as blood substitutes since 1970s. More recently, a variety of PFC nanoparticles have been designed for the detection and imaging of physiol. and pathol. changes. These mol. imaging probes have been developed to label cells, target specific epitopes in tumors, monitor the prognosis and therapy efficacy and quantitate characterization of tumors and changes in tumor microenvironment noninvasively, therefore, significantly improving the prognosis and therapy efficacy. Herein, we discuss the recent development and applications of 19F MR techniques with PFC nanoparticles in biomedicine, with particular emphasis on ligand-targeted and quant. 19F MR imaging approaches for tumor detection, oxygenation measurement, smart stimulus response and therapy efficacy monitoring, et al. nanoparticles in biomedicine, with particular emphasis on ligand-targeted and quant. 19F MR imaging approaches for tumor detection, oxygenation measurement, smart stimulus response and therapy efficacy monitoring, et al.
  9. 9
    Ruiz-Cabello, J.; Walczak, P.; Kedziorek, D. A.; Chacko, V. P.; Schmieder, A. H.; Wickline, S. A.; Lanza, G. M.; Bulte, J. W. M. In vivo “hot spot” MR imaging of neural stem cells using fluorinated nanoparticles. Magn. Reson. Med. 2008, 60, 1506– 1511,  DOI: 10.1002/mrm.21783
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    In vivo "hot spot" MR imaging of neural stem cells using fluorinated nanoparticles
    Ruiz-Cabello Jesus; Walczak Piotr; Kedziorek Dorota A; Chacko Vadappuram P; Schmieder Anna H; Wickline Samuel A; Lanza Gregory M; Bulte Jeff W M
    Magnetic resonance in medicine (2008), 60 (6), 1506-11 ISSN:.
    To optimize (19)F MR tracking of stem cells, we compared cellular internalization of cationic and anionic perfluoro-15-crown-5-ether (PFCE) nanoparticles using cell culture plates with different surface coatings. The viability and proliferation of anionic and cationic PFCE-labeled neural stem cells (NSCs) did not differ from unlabeled cells. Cationic PFCE nanoparticles ((19)F T1/T2 = 580/536 ms at 9.4 Tesla) were superior to anionic particles for intracellular fluorination. Best results were obtained with modified polystyrene culture dishes coated with both carboxylic and amino groups rather than conventional carboxyl-coated dishes. After injecting PFCE-labeled NSCs into the striatum of mouse brain, cells were readily identified in vivo by (19)F MRI without changes in signal or viability over a 2-week period after grafting. These results demonstrate that neural stem cells can be efficiently fluorinated with cationic PFCE nanoparticles without using transfection agents and visualized in vivo over prolonged periods with an MR sensitivity of approximately 140 pmol of PFCE/cell.
  10. 10
    Fu, C.; Demir, B.; Alcantara, S.; Kumar, V.; Han, F.; Kelly, H. G.; Tan, X.; Yu, Y.; Xu, W.; Zhao, J.; Zhang, C.; Peng, H.; Boyer, C.; Woodruff, T. M.; Kent, S. J.; Searles, D. J.; Whittaker, A. K. Low-fouling fluoropolymers for bioconjugation and in vivo tracking. Angew. Chem. 2020, 132, 4759– 4765,  DOI: 10.1002/ange.201914119
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    Jirak, D.; Svoboda, J.; Filipová, M.; Pop-Georgievski, O.; Sedlacek, O. Antifouling fluoropolymer-coated nanomaterials for 19F MRI. Chem. Commun. 2021, 57, 4718– 4721,  DOI: 10.1039/d1cc00642h
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    Antifouling fluoropolymer-coated nanomaterials for 19F MRI
    Jirak, Daniel; Svoboda, Jan; Filipova, Marcela; Pop-Georgievski, Ognen; Sedlacek, Ondrej
    Chemical Communications (Cambridge, United Kingdom) (2021), 57 (38), 4718-4721CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    We developed a multifunctional polymer coating for nanoparticles (NPs) that enables simultaneous detection by 19F MRI and shielding from blood plasma fouling. The coating is based on a water-sol. fluorinated poly(N-(2-fluoroethyl)acrylamide) (PFEAM) that shows high 19F MRI sensitivity, cytocompatibility and excellent antifouling properties, significantly outperforming polyethylene glycol. A proof-of-concept expt. was performed by synthesizing polymer-coated gold NPs that were successfully visualized by 19F MRI at magnetic fields close to the fields used in clin. practice. This universal approach can be used for coating and tracing of various NPs upon suitable polymer chain-end modification.
  12. 12
    Sedlacek, O.; Jirak, D.; Galisova, A.; Jager, E.; Laaser, J. E.; Lodge, T. P.; Stepanek, P.; Hruby, M. 19F Magnetic Resonance Imaging of Injectable Polymeric Implants with Multiresponsive Behavior. Chem. Mater. 2018, 30, 4892– 4896,  DOI: 10.1021/acs.chemmater.8b02115
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    19F Magnetic Resonance Imaging of Injectable Polymeric Implants with Multiresponsive Behavior
    Sedlacek, Ondrej; Jirak, Daniel; Galisova, Andrea; Jager, Eliezer; Laaser, Jennifer E.; Lodge, Timothy P.; Stepanek, Petr; Hruby, Martin
    Chemistry of Materials (2018), 30 (15), 4892-4896CODEN: CMATEX; ISSN:0897-4756. (American Chemical Society)
    Recently, magnetic resonance imaging (MRI) probes based on fluorinated compds. have emerged as highly promising contrast agents. Direct imaging of injectable thermoresponsive polymer implants by 19F MRI represents an attractive, as yet unreported tool for the straightforward and non-invasive monitoring of implant localization, size, morphol., and biodegrdn. Herein, we describe for the first time the in vivo19F MRI visualization of such implants. These were s.c. and i.m. administered as an aq. soln. to form a traceable solid implant upon the change of temp. and pH. After formation, the polymer implant was effectively visualized by 19F MRI. The kinetics of the implant degrdn. is suitable for application as injectable drug depots or in the local radiotherapy of solid tumors.
  13. 13
    Fu, C.; Zhang, C.; Peng, H.; Han, F.; Baker, C.; Wu, Y.; Ta, H.; Whittaker, A. K. Enhanced performance of polymeric 19F MRI contrast agents through incorporation of highly water-soluble monomer MSEA. Macromolecules 2018, 51, 5875– 5882,  DOI: 10.1021/acs.macromol.8b01190
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    Enhanced Performance of Polymeric 19F MRI Contrast Agents through Incorporation of Highly Water-Soluble Monomer MSEA
    Fu, Changkui; Zhang, Cheng; Peng, Hui; Han, Felicity; Baker, Carly; Wu, Yuao; Ta, Hang; Whittaker, Andrew K.
    Macromolecules (Washington, DC, United States) (2018), 51 (15), 5875-5882CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
    19F magnetic resonance imaging (MRI) is a powerful noninvasive imaging technique that shows tremendous potential for the diagnosis and monitoring of human diseases. Fluorinated compds. are commonly used as 19F MRI contrast agents to develop "hot spot" imaging. To achieve high-resoln. MR images, a high d. of 19F nuclei is required in the contrast agents. However, because of the inherent hydrophobicity of fluorinated moieties, aggregation of 19F contrast agents with high fluorine content is often obsd. in aq. soln., resulting in attenuated MR signal and low sensitivity, thus significantly limiting their further biol. applications. Here we report the synthesis and characterization of a series of polymeric 19F MRI contrast agents with high fluorine content by copolymg. the well-known fluorinated monomer 2,2,2-trifluoroethyl acrylate (TFEA) with a highly water-sol. monomer 2-(methylsulfinyl)ethyl acrylate (MSEA) using RAFT polymn. We show that these polymeric contrast agents, although with high fluorine content, display remarkable imaging performance as evidenced by preferable relaxation properties and intense in vitro/in vivo MRI signals, demonstrating the huge potential for eventual clin. applications such as MRI-guided disease diagnosis and therapy.
  14. 14
    Thurecht, K. J.; Blakey, I.; Peng, H.; Squires, O.; Hsu, S.; Alexander, C.; Whittaker, A. K. Functional Hyperbranched Polymers: Toward Targeted in Vivo 19F Magnetic Resonance Imaging Using Designed Macromolecules. J. Am. Chem. Soc. 2010, 132, 5336– 5337,  DOI: 10.1021/ja100252y
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    Functional Hyperbranched Polymers: Toward Targeted in Vivo 19F Magnetic Resonance Imaging Using Designed Macromolecules
    Thurecht, Kristofer J.; Blakey, Idriss; Peng, Hui; Squires, Oliver; Hsu, Steven; Alexander, Cameron; Whittaker, Andrew K.
    Journal of the American Chemical Society (2010), 132 (15), 5336-5337CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Accurate and early diagnosis of diseases, such as cancer, is the "holy grail" of medical imaging. Techniques. such as computed axial tomog. magnetic resonance imaging (MRI). ultrasound etc., are able to detect various diseases. but often the detection limits mean the diseases have progressed beyond acceptable recovery. Early detection requires imaging techniques with higher sensitivity, while maintaining specificity for disease states (e.g., tumors). We report on our recent efforts in developing sensitive polymeric 19F MRI contrast agents that combine controllable functionality, ability for cell targeting, and low cytotoxicity. Recent synthetic advances that facilitate intimate control over polymer structure and functionality have led to the advent of polymeric theranostics. nanomedical devices for diagnosis and treatment of diseases. Monitoring these devices in an in vivo clin. setting remains a significant scientific challenge despite some elegant attempts to develop polymeric devices or emulsions for 19F MRI. No system to date has demonstrated the ability to be both easily and universally functionalized. while exhibiting high 19F MRI sensitivity. Poor sensitivity is attributed to three main factors: poor mol. mobility. assocn. of the fluorinated segments, and low fluorine content. To overcome such issues, our approach uses hyperbranched polymers enabling high segmental mol. mobility to be achieved while maintaining high fluorine content. Controlled functionality was introduced through reversible addn.-fragmentation chain transfer (RAFT) chem., and mol. mobility was conferred via low-Tg (acrylate) and polar repeat units that are well hydrated under aq. conditions. Random branching frustrates aggregation of the fluorinated segments allowing incorporation of up to 20 mol % fluoromonomer. The "shape-persistence" of the hyperbranched mol. means that orientation of functionality, such as cell-targeting agents, can be controlled thus ensuring correct presentation for efficient biol. recognition if required.
  15. 15
    Rolfe, B. E.; Blakey, I.; Squires, O.; Peng, H.; Boase, N. R. B.; Alexander, C.; Parsons, P. G.; Boyle, G. M.; Whittaker, A. K.; Thurecht, K. J. Multimodal Polymer Nanoparticles with Combined 19F Magnetic Resonance and Optical Detection for Tunable, Targeted, Multimodal Imaging in Vivo. J. Am. Chem. Soc. 2014, 136, 2413– 2419,  DOI: 10.1021/ja410351h
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    Multimodal Polymer Nanoparticles with Combined 19F Magnetic Resonance and Optical Detection for Tunable, Targeted, Multimodal Imaging in Vivo
    Rolfe, Barbara E.; Blakey, Idriss; Squires, Oliver; Peng, Hui; Boase, Nathan R. B.; Alexander, Cameron; Parsons, Peter G.; Boyle, Glen M.; Whittaker, Andrew K.; Thurecht, Kristofer J.
    Journal of the American Chemical Society (2014), 136 (6), 2413-2419CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Understanding the complex nature of diseased tissue in vivo requires development of more advanced nanomedicines, where synthesis of multifunctional polymers combines imaging multimodality with a biocompatible, tunable, and functional nanomaterial carrier. Here the authors describe the development of polymeric nanoparticles for multimodal imaging of disease states in vivo. The nanoparticle design utilizes the abundant functionality and tunable physicochem. properties of synthetically robust polymeric systems to facilitate targeted imaging of tumors in mice. For the first time, high-resoln. 19F/1H magnetic resonance imaging is combined with sensitive and versatile fluorescence imaging in a polymeric material for in vivo detection of tumors. The authors highlight how control over the chem. during synthesis allows manipulation of nanoparticle size and function and can lead to very high targeting efficiency to B16 melanoma cells, both in vitro and in vivo. Importantly, the combination of imaging modalities within a polymeric nanoparticle provides information on the tumor mass across various size scales in vivo, from millimeters down to tens of micrometers.
  16. 16
    Sedlacek, O.; Jirak, D.; Vit, M.; Ziołkowska, N.; Janouskova, O.; Hoogenboom, R. Fluorinated water-soluble poly (2-oxazoline) s as highly sensitive 19F MRI contrast agents. Macromolecules 2020, 53, 6387– 6395,  DOI: 10.1021/acs.macromol.0c01228
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    Fluorinated Water-Soluble Poly(2-oxazoline)s as Highly Sensitive 19F MRI Contrast Agents
    Sedlacek, Ondrej; Jirak, Daniel; Vit, Martin; Ziolkowska, Natalia; Janouskova, Olga; Hoogenboom, Richard
    Macromolecules (Washington, DC, United States) (2020), 53 (15), 6387-6395CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
    Recently, 19F magnetic resonance imaging (MRI) emerged as a powerful noninvasive diagnostic tool in modern medicine. Fluorinated polymer materials represent an attractive class of MRI contrast agents (CAs) due to their structural variability and tunable properties. Herein, we describe for the first time the 19F MRI of CAs based on fluorinated water-sol. poly(2-oxazoline)s (PAOx), a polymer class with increasing popularity in biomedical sciences. A series of fluorinated PAOx with increasing fluorine content were synthesized by controlled side-chain hydrolysis of poly(2-methyl-2-oxazoline) followed by reacylation of its ethylenimine units by difluoroacetic anhydride. As the increasing fluorine content leads to the copolymer hydrophobization, their compn. was optimized for maximal 19F MRI performance while retaining good soly. in water. The magnetic properties of the water-sol. polymers were studied in vitro by 19F NMR and MRI, revealing their outstanding relaxation properties and imaging sensitivity. All CAs were found to be noncytotoxic for HeLa cells in vitro. Finally, the diagnostic potential of the new CAs was demonstrated by a successful in vivo19F MRI visualization of the selected fluorinated polymer in rats.
  17. 17
    Peng, H.; Blakey, I.; Dargaville, B.; Rasoul, F.; Rose, S.; Whittaker, A. K. Synthesis and Evaluation of Partly Fluorinated Block Copolymers as MRI Imaging Agents. Biomacromolecules 2009, 10, 374– 381,  DOI: 10.1021/bm801136m
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    Synthesis and Evaluation of Partly Fluorinated Block Copolymers as MRI Imaging Agents
    Peng, Hui; Blakey, Idriss; Dargaville, Bronwin; Rasoul, Firas; Rose, Stephen; Whittaker, Andrew K.
    Biomacromolecules (2009), 10 (2), 374-381CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
    A series of well-defined diblock copolymers of acrylic acid with partially fluorinated acrylate and methacrylate monomers were synthesized using ATRP as potential 19F MRI imaging agents. The diblock copolymers could undergo spontaneous self-assembly in mixed and aq. solvents to form stable micelles with a diam. from approx. 20-45 nm, having a fluorine-rich core that provides a strong signal for MRI examns. The obsd. MRI image intensities were related to the NMR longitudinal and transverse relaxation times, and were found to depend on polymer structure and method of micellization. Two distinct T2 relaxation times were measured; on comparison of expected MRI image intensities with those obsd. exptl., methacrylate polymers show systematically lower signal intensity than acrylate polymers. This is related to the presence of a population of nuclear spins having short T2 relaxation times that cannot be detected under high-resoln. NMR and MRI conditions.
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    Kaberov, L. I.; Kaberova, Z.; Murmiliuk, A.; Trousil, J.; Sedláček, O.; Konefal, R.; Zhigunov, A.; Pavlova, E.; Vít, M.; Jirák, D.; Hoogenboom, R.; Filippov, S. K. Fluorine-Containing Block and Gradient Copoly (2-oxazoline) s Based on 2-(3, 3, 3-Trifluoropropyl)-2-oxazoline: A Quest for the Optimal Self-Assembled Structure for 19F Imaging. Biomacromolecules 2021, 22, 2963– 2975,  DOI: 10.1021/acs.biomac.1c00367
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    Fluorine-Containing Block and Gradient Copoly(2-oxazoline)s Based on 2-(3,3,3-Trifluoropropyl)-2-oxazoline: A Quest for the Optimal Self-Assembled Structure for 19F Imaging
    Kaberov, Leonid I.; Kaberova, Zhansaya; Murmiliuk, Anastasiia; Trousil, Jiri; Sedlacek, Ondrej; Konefal, Rafal; Zhigunov, Alexander; Pavlova, Ewa; Vit, Martin; Jirak, Daniel; Hoogenboom, Richard; Filippov, Sergey K.
    Biomacromolecules (2021), 22 (7), 2963-2975CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
    The use of fluorinated contrast agents in magnetic resonance imaging (MRI) facilitates improved image quality due to the negligible amt. of endogenous fluorine atoms in the body. In this work, we present a comprehensive study of the influence of the amphiphilic polymer structure and compn. on its applicability as contrast agents in 19F MRI. Three series of novel fluorine-contg. poly(2-oxazoline) copolymers and terpolymers, hydrophilic-fluorophilic, hydrophilic-lipophilic-fluorophilic, and hydrophilic-thermoresponsive-fluorophilic, with block and gradient distributions of the fluorinated units, were synthesized. It was discovered that the CF3 in the 2-(3,3,3-trifluoropropyl)-2-oxazoline (CF3EtOx) group activated the cationic chain end, leading to faster copolymn. kinetics, whereby spontaneous monomer gradients were formed with accelerated incorporation of 2-methyl-2-oxazoline or 2-n-propyl-2-oxazoline with a gradual change to the less-nucleophilic CF3EtOx monomer. The obtained amphiphilic copolymers and terpolymers form spherical or wormlike micelles in water, which was confirmed using transmission electron microscopy (TEM), while small-angle X-ray scattering (SAXS) revealed the core-shell or core-double-shell morphologies of these nanoparticles. The core and shell sizes obey the scaling laws for starlike micelles predicted by the scaling theory. Biocompatibility studies confirm that all copolymers obtained are noncytotoxic and, at the same time, exhibit high sensitivity during in vitro 19F MRI studies. The gradient copolymers provide the best 19F MRI signal-to-noise ratio in comparison with the analog block copolymer structures, making them most promising as 19F MRI contrast agents.
  19. 19
    Kolouchova, K.; Sedlacek, O.; Jirak, D.; Babuka, D.; Blahut, J.; Kotek, J.; Vit, M.; Trousil, J.; Konefał, R.; Janouskova, O.; Podhorska, B.; Slouf, M.; Hruby, M. Self-assembled thermoresponsive polymeric nanogels for 19F MR imaging. Biomacromolecules 2018, 19, 3515– 3524,  DOI: 10.1021/acs.biomac.8b00812
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    Self-Assembled Thermoresponsive Polymeric Nanogels for 19F MR Imaging
    Kolouchova, Kristyna; Sedlacek, Ondrej; Jirak, Daniel; Babuka, David; Blahut, Jan; Kotek, Jan; Vit, Martin; Trousil, Jiri; Konefal, Rafal; Janouskova, Olga; Podhorska, Bohumila; Slouf, Miroslav; Hruby, Martin
    Biomacromolecules (2018), 19 (8), 3515-3524CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
    Magnetic resonance imaging using fluorinated contrast agents (19F MRI) enables to achive high contrast in images due to the negligible fluorine background in living tissues. In this pilot study, we developed new biocompatible, temp.-responsive, and easily synthesized polymeric nanogels contg. a sufficient concn. of magnetically equiv. fluorine atoms for 19F MRI purposes. The structure of the nanogels is based on amphiphilic copolymers contg. two blocks, a hydrophilic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) or poly(2-methyl-2-oxazoline) (PMeOx) block, and a thermoresponsive poly[N(2,2difluoroethyl)acrylamide] (PDFEA) block. The thermoresponsive properties of the PDFEA block allow us to control the process of nanogel self-assembly upon its heating in an aq. soln. Particle size depends on the copolymer compn., and the most promising copolymers with longer thermoresponsive blocks form nanogels of suitable size for angiogenesis imaging or the labeling of cells (approx. 120 nm). The in vitro19F MRI expts. reveal good sensitivity of the copolymer contrast agents, while the nanogels were proven to be noncytotoxic for several cell lines.
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    D’Agosto, F.; Rieger, J.; Lansalot, M. RAFT-mediated polymerization-induced self-assembly. Angew. Chem., Int. Ed. 2020, 59, 8368– 8392,  DOI: 10.1002/anie.201911758
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    RAFT-Mediated Polymerization-Induced Self-Assembly
    D'Agosto, Franck; Rieger, Jutta; Lansalot, Muriel
    Angewandte Chemie, International Edition (2020), 59 (22), 8368-8392CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. After a brief history that positions polymn.-induced self-assembly (PISA) in the field of polymer chem., this Review will cover the fundamentals of the PISA mechanism. Furthermore, this Review will also give an overview of some of the features and limitations of RAFT-mediated PISA in terms of the choice of the components involved, the nature of the nanoobjects that can be obtained and how the syntheses can be controlled, as well as some potential applications.
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    Warren, N. J.; Armes, S. P. Polymerization-induced self-assembly of block copolymer nano-objects via RAFT aqueous dispersion polymerization. J. Am. Chem. Soc. 2014, 136, 10174– 10185,  DOI: 10.1021/ja502843f
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    Polymerization-Induced Self-Assembly of Block Copolymer Nano-objects via RAFT Aqueous Dispersion Polymerization
    Warren, Nicholas J.; Armes, Steven P.
    Journal of the American Chemical Society (2014), 136 (29), 10174-10185CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    A review. In this Perspective, we discuss the recent development of polymn.-induced self-assembly mediated by reversible addn.-fragmentation chain transfer (RAFT) aq. dispersion polymn. This approach has quickly become a powerful and versatile technique for the synthesis of a wide range of bespoke org. diblock copolymer nano-objects of controllable size, morphol., and surface functionality. Given its potential scalability, such environmentally-friendly formulations are expected to offer many potential applications, such as novel Pickering emulsifiers, efficient microencapsulation vehicles, and sterilizable thermo-responsive hydrogels for the cost-effective long-term storage of mammalian cells.
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    Penfold, N. J.; Yeow, J.; Boyer, C.; Armes, S. P. Emerging trends in polymerization-induced self-assembly. ACS Macro Lett. 2019, 8, 1029– 1054,  DOI: 10.1021/acsmacrolett.9b00464
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    Emerging Trends in Polymerization-Induced Self-Assembly
    Penfold, Nicholas J. W.; Yeow, Jonathan; Boyer, Cyrille; Armes, Steven P.
    ACS Macro Letters (2019), 8 (8), 1029-1054CODEN: AMLCCD; ISSN:2161-1653. (American Chemical Society)
    A review. In this Perspective, we summarize recent progress in polymn.-induced self-assembly (PISA) for the rational synthesis of block copolymer nanoparticles with various morphologies. Much of the PISA literature has been based on thermally initiated reversible addn.-fragmentation chain transfer (RAFT) polymn. Herein, we pay particular attention to alternative PISA protocols, which allow the prepn. of nanoparticles with improved control over copolymer morphol. and functionality. For example, initiation based on visible light, redox chem., or enzymes enables the incorporation of sensitive monomers and fragile biomols. into block copolymer nanoparticles. Furthermore, PISA syntheses and postfunctionalization of the resulting nanoparticles (e.g., crosslinking) can be conducted sequentially without intermediate purifn. by using various external stimuli. Finally, PISA formulations have been optimized via high-throughput polymn. and recently evaluated within flow reactors for facile scale-up syntheses.
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    Le, D.; Keller, D.; Delaittre, G. Reactive and Functional Nanoobjects by Polymerization-Induced Self-Assembly. Macromol. Rapid Commun. 2019, 40, 1800551,  DOI: 10.1002/marc.201800551
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    Wan, J.; Fan, B.; Thang, S. RAFT-mediated polymerization-induced self-assembly (RAFT-PISA): current status and future directions. Chem. Sci. 2022, 13, 4192– 4224,  DOI: 10.1039/d2sc00762b
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    RAFT-mediated polymerization-induced self-assembly (RAFT-PISA): current status and future directions
    Wan, Jing; Fan, Bo; Thang, San H.
    Chemical Science (2022), 13 (15), 4192-4224CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    A review. Polymn.-induced self-assembly (PISA) combines polymn. and self-assembly in a single step with distinct efficiency that has set it apart from the conventional soln. self-assembly processes. PISA holds great promise for large-scale prodn., not only because of its efficient process for producing polymeric nano/microparticles with high solid content, but also thanks to the facile control over the particle size and morphol. Since its invention, many research groups around the world have developed new and creative approaches to broaden the scope of PISA initiations, morphologies and applications, etc. The growing interest in PISA is certainly reflected in the increasing no. of publications over the past few years, and in this review, we aim to summarize these recent advances in the emerging aspects of RAFT-mediated PISA. These include (1) non-thermal initiation processes, such as photo-, enzyme-, redox- and ultrasound-initiation; the achievements of (2) high-order structures, (3) hybrid materials and (4) stimuli-responsive nano-objects by design and adopting new monomers and new processes; (5) the efforts in the realization of upscale prodn. by utilization of high throughput technologies, and finally the (6) applications of current PISA nano-objects in different fields and (7) its future directions.
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    Cao, J.; Tan, Y.; Chen, Y.; Zhang, L.; Tan, J. Expanding the Scope of Polymerization-Induced Self-Assembly: Recent Advances and New Horizons. Macromol. Rapid Commun. 2021, 42, 2100498,  DOI: 10.1002/marc.202100498
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    Expanding the Scope of Polymerization-Induced Self-Assembly: Recent Advances and New Horizons
    Cao, Junpeng; Tan, Yingxin; Chen, Ying; Zhang, Li; Tan, Jianbo
    Macromolecular Rapid Communications (2021), 42 (23), 2100498CODEN: MRCOE3; ISSN:1022-1336. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Over the past decade or so, polymn.-induced self-assembly (PISA) has become a versatile method for rational prepn. of concd. block copolymer nanoparticles with a diverse set of morphologies. Much of the PISA literature has focused on the prepn. of well-defined linear block copolymers by using linear macromol. chain transfer agents (macro-CTAs) with high chain transfer consts. In this review, a recent process is highlighted from an unusual angle that has expanded the scope of PISA including (i) synthesis of block copolymers with nonlinear architectures (e.g., star block copolymer, branched block copolymer) by PISA, (ii) in situ synthesis of blends of polymers by PISA, and (iii) utilization of macro-CTAs with low chain transfer consts. in PISA. By highlighting these important examples, new insights into the research of PISA and future impact these methods will have on polymer and colloid synthesis are provided.
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    Czajka, A.; Armes, S. P. Time-Resolved Small-Angle X-ray Scattering Studies during Aqueous Emulsion Polymerization. J. Am. Chem. Soc. 2021, 143, 1474– 1484,  DOI: 10.1021/jacs.0c11183
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    Time-Resolved Small-Angle X-ray Scattering Studies during Aqueous Emulsion Polymerization
    Czajka, Adam; Armes, Steven P.
    Journal of the American Chemical Society (2021), 143 (3), 1474-1484CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The persulfate-initiated aq. emulsion polymn. of 2,2,2-trifluoroethyl methacrylate (TFEMA) is studied by time-resolved small-angle X-ray scattering (SAXS) at 60°C using a stirrable reaction cell. TFEMA was preferred to styrene because it offers much greater X-ray scattering contrast relative to water, which is essential for sufficient temporal resoln. The evolution in particle size is monitored by both in situ SAXS and ex situ DLS in the absence or presence of an anionic surfactant (sodium dodecyl sulfate, SDS). Post-mortem SAXS studies confirmed the formation of well-defined spherical latexes, with vol.-av. diams. of 353 ± 9 nm and 68 ± 4 nm being obtained for the surfactant-free and SDS formulations, resp. 1H NMR spectroscopy studies of the equiv. lab.-scale formulations indicated TFEMA conversions of 99% within 80 min and 93% within 60 min for the surfactant-free and SDS formulations, resp. Comparable polymn. kinetics are obsd. for the in situ SAXS expts. and the lab.-scale syntheses, with nucleation occurring after approx. 6 min in each case. After nucleation, scattering patterns are fitted using a hard sphere scattering model to det. the evolution in particle growth for both formulations. Moreover, in situ SAXS enables identification of the three main intervals (I, II, and III) that are obsd. during aq. emulsion polymn. in the presence of surfactant. These intervals are consistent with those indicated by soln. cond. and optical microscopy studies. Significant differences between the surfactant-free and SDS formulations are obsd., providing useful insights into the mechanism of emulsion polymn.
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    Czajka, A.; Liao, G.; Mykhaylyk, O. O.; Armes, S. P. In situ small-angle X-ray scattering studies during the formation of polymer/silica nanocomposite particles in aqueous solution. Chem. Sci. 2021, 12, 14288– 14300,  DOI: 10.1039/d1sc03353k
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    In situ small-angle X-ray scattering studies during the formation of polymer/silica nanocomposite particles in aqueous solution
    Czajka, A.; Liao, G.; Mykhaylyk, O. O.; Armes, S. P.
    Chemical Science (2021), 12 (42), 14288-14300CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    This study is focused on the formation of polymer/silica nanocomposite particles prepd. by the surfactant-free aq. emulsion polymn. of 2,2,2-trifluoroethyl methacrylate (TFEMA) in the presence of 19 nm glycerol-functionalized aq. silica nanoparticles using a cationic azo initiator at 60°C. The TFEMA polymn. kinetics are monitored using 1H NMR spectroscopy, while postmortem TEM anal. confirms that the final nanocomposite particles possess a well-defined core-shell morphol. Time-resolved small-angle X-ray scattering (SAXS) is used in conjunction with a stirrable reaction cell to monitor the evolution of the nanocomposite particle diam., mean silica shell thickness, mean no. of silica nanoparticles within the shell, silica aggregation efficiency and packing d. during the TFEMA polymn. Nucleation occurs after 10-15 min and the nascent particles quickly become swollen with TFEMA monomer, which leads to a relatively fast rate of polymn. Addnl. surface area is created as these initial particles grow and anionic silica nanoparticles adsorb at the particle surface to maintain a relatively high surface coverage and hence ensure colloidal stability. At high TFEMA conversion, a contiguous silica shell is formed and essentially no further adsorption of silica nanoparticles occurs. A population balance model is introduced into the SAXS model to account for the gradual incorporation of the silica nanoparticles within the nanocomposite particles. The final PTFEMA/silica nanocomposite particles are obtained at 96% TFEMA conversion after 140 min, have a vol.-av. diam. of 216 ± 9 nm and contain approx. 274 silica nanoparticles within their outer shells; a silica aggregation efficiency of 75% can be achieved for such formulations.
  28. 28
    Desnos, G.; Rubio, A.; Gomri, C.; Gravelle, M.; Ladmiral, V.; Semsarilar, M. Semi-Fluorinated Di and Triblock Copolymer Nano-Objects Prepared via RAFT Alcoholic Dispersion Polymerization (PISA). Polymers 2021, 13, 2502,  DOI: 10.3390/polym13152502
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    Semi-Fluorinated Di and Triblock Copolymer Nano-Objects Prepared via RAFT Alcoholic Dispersion Polymerization (PISA)
    Desnos, Gregoire; Rubio, Adrien; Gomri, Chaimaa; Gravelle, Mathias; Ladmiral, Vincent; Semsarilar, Mona
    Polymers (Basel, Switzerland) (2021), 13 (15), 2502CODEN: POLYCK; ISSN:2073-4360. (MDPI AG)
    A set of well-defined amphiphilic, semi-fluorinated di and triblock copolymers were synthesized via polymn.-induced self-assembly (PISA) under alc. dispersion polymn. conditions. This study investigates the influence of the length, nature and position of the solvophobic semi-fluorinated block. A poly(N,N-dimethylaminoethyl methacrylate) was used as the stabilizing block to prep. the di and tri block copolymer nano-objects via reversible addn.-fragmentation chain transfer (RAFT) controlled dispersion polymn. at 70°C in ethanol. Benzylmethacrylate (BzMA) and semi-fluorinated methacrylates and acrylates with 7 (heptafluorobutyl methacrylate (HFBMA)), 13 (heneicosafluorododecyl methacrylate (HCFDDMA)) and 21 (tridecafluorooctyl acrylate (TDFOA)) fluorine atoms were used as monomers for the core-forming blocks. The RAFT polymn. of these semi-fluorinated monomers was monitored by SEC and 1H NMR. The evolution of the self-assembled morphologies was investigated by transmission electron microscopy. The results demonstrate that the order of the blocks and the no. of fluorine atoms influence the microphase segregation of the core-forming blocks and the final morphol. of the nano-objects.
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    Huo, M.; Li, D.; Song, G.; Zhang, J.; Wu, D.; Wei, Y.; Yuan, J. Semi-Fluorinated Methacrylates: A Class of Versatile Monomers for Polymerization-Induced Self-Assembly. Macromol. Rapid Commun. 2018, 39, 1700840,  DOI: 10.1002/marc.201700840
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    Cornel, E. J.; van Meurs, S.; Smith, T.; O’Hora, P. S.; Armes, S. P. In Situ Spectroscopic Studies of Highly Transparent Nanoparticle Dispersions Enable Assessment of Trithiocarbonate Chain-End Fidelity during RAFT Dispersion Polymerization in Nonpolar Media. J. Am. Chem. Soc. 2018, 140, 12980– 12988,  DOI: 10.1021/jacs.8b07953
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    In Situ Spectroscopic Studies of Highly Transparent Nanoparticle Dispersions Enable Assessment of Trithiocarbonate Chain-End Fidelity during RAFT Dispersion Polymerization in Nonpolar Media
    Cornel, Erik J.; van Meurs, Sandra; Smith, Timothy; OHora, Paul S.; Armes, Steven P.
    Journal of the American Chemical Society (2018), 140 (40), 12980-12988CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    We report the synthesis of highly transparent poly(stearyl methacrylate)-poly(2,2,2-trifluoroethyl methacrylate) (PSMA-PTFEMA) diblock copolymer nanoparticles via polymn.-induced self-assembly (PISA) in nonpolar media at 70 °C. This was achieved by chain-extending a PSMA precursor block via reversible addn.-fragmentation chain transfer (RAFT) dispersion polymn. of TFEMA in n-tetradecane. This n-alkane has the same refractive index as the PTFEMA core-forming block at 70 °C, which ensures high light transmittance when targeting 33 nm spherical nanoparticles. Such isorefractivity enables visible absorption spectra to be recorded with minimal light scattering even at 30% wt./wt. solids. However, in situ monitoring of the trithiocarbonate RAFT end-groups during PISA requires selection of a weak n → π* band at 446 nm. Conversion of TFEMA into PTFEMA causes a contraction in the reaction soln. vol., leading to an initial increase in absorbance that enables the kinetics of polymn. to be monitored via dilatometry. At ∼98% TFEMA conversion, this 446 nm band remains const. for 2 h at 70 °C, indicating surprisingly high RAFT chain-end fidelity (and hence pseudoliving character) under monomer-starved conditions. In situ 19F NMR spectroscopy studies provide evidence for (i) the onset of micellar nucleation, (ii) solvation of the nanoparticle cores by TFEMA monomer, and (iii) surface plasticization of the nanoparticle cores by n-tetradecane at 70 °C. Finally, the kinetics of RAFT chain-end removal can be conveniently monitored by in situ visible absorption spectroscopy: addn. of excess initiator at 70 °C causes complete discoloration of the dispersion, with small-angle X-ray scattering studies confirming no change in nanoparticle morphol. under these conditions.
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    Zhao, W.; Ta, H. T.; Zhang, C.; Whittaker, A. K. Polymerization-Induced Self-Assembly (PISA)─Control over the Morphology of 19F-Containing Polymeric Nano-objects for Cell Uptake and Tracking. Biomacromolecules 2017, 18, 1145– 1156,  DOI: 10.1021/acs.biomac.6b01788
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    Polymerization-Induced Self-Assembly (PISA) - Control over the Morphology of 19F-Containing Polymeric Nano-objects for Cell Uptake and Tracking
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    Biomacromolecules (2017), 18 (4), 1145-1156CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
    Fluorine-contg. polymeric materials are receiving increasing attention as imaging probes in fluorine-19 magnetic resonance imaging (19F MRI), for example to enable quant. in vivo detection of cells. Here we describe the one-pot polymn. synthesis of 19F-contg. functional poly(oligo(ethylene glycol)) Me ether methacrylate-co-2,2,2-trifluoroethyl acrylate-b-poly(styrene-co-3-vinylbenzaldehyde) (poly(OEGA-co-TFEA)-b-poly(St-co-VBA)) copolymers as a new class of fluorinated MRI agent. A range of nanoparticle morphologies, including spheres, worm-like particles, and vesicles were formed as a consequence of polymn.-induced self-assembly (PISA). It was found that the extent of cell uptake strongly depends on the morphol. of the nano-objects, with preferable uptake for worm-like particles compared to spherical nanoparticles and vesicles. All the nano-objects have a single resonance in the 19F NMR spectrum with relatively short MRI relaxation times, which were independent of the morphol. of the nano-objects. These results confirm that these polymeric nano-objects of varied morphologies are promising as 19F MRI imaging agents for use in tracking of cells and selective MRI.
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    Lueckerath, T.; Strauch, T.; Koynov, K.; Barner-Kowollik, C.; Ng, D. Y. W.; Weil, T. DNA–Polymer Conjugates by Photoinduced RAFT Polymerization. Biomacromolecules 2019, 20, 212– 221,  DOI: 10.1021/acs.biomac.8b01328
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    DNA-Polymer Conjugates by Photoinduced RAFT Polymerization
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    Biomacromolecules (2019), 20 (1), 212-221CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
    Conventional grafting-to approaches to DNA-polymer conjugates are often limited by low reaction yields due to the sterically hindered coupling of a presynthesized polymer to DNA. The grafting-from strategy, in contrast, allows one to directly graft polymers from an initiator that is covalently attached to DNA. Herein, we report blue-light-mediated reversible addn.-fragmentation chain-transfer (Photo-RAFT) polymn. from two different RAFT agent-terminated DNA sequences using Eosin Y as the photocatalyst in combination with ascorbic acid. Three monomer families (methacrylates, acrylates and acrylamides) were successfully polymd. from DNA employing Photo-RAFT polymn. We demonstrate that the length of the grown polymer chain can be varied by altering the monomer to DNA-initiator ratio, while the self-assembly features of the DNA strands were maintained. In summary, we describe a convenient, light-mediated approach toward DNA-polymer conjugates via the grafting-from approach.
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    Bak, J. M.; Kim, K.-B.; Lee, J.-E.; Park, Y.; Yoon, S. S.; Jeong, H. M.; Lee, H.-i. Thermoresponsive fluorinated polyacrylamides with low cytotoxicity. Polym. Chem. 2013, 4, 2219– 2223,  DOI: 10.1039/c2py20747h
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    Thermoresponsive fluorinated polyacrylamides with low cytotoxicity
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    Polymer Chemistry (2013), 4 (7), 2219-2223CODEN: PCOHC2; ISSN:1759-9962. (Royal Society of Chemistry)
    Authors report the unique thermoresponsive properties of fluorinated polyacrylamides, poly[N-(2,2-difluoroethyl)acrylamide] (P2F). The soly. of fluorinated polyacrylamides in water can be easily controlled by changing the no. of fluorine atoms in N-Et groups. Moreover, we demonstrate that fluorinated polyacrylamides are less cytotoxic than poly(N-isopropylacrylamide) (PNIPAM).
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    Vít, M.; Burian, M.; Berková, Z.; Lacik, J.; Sedlacek, O.; Hoogenboom, R.; Raida, Z.; Jirak, D. A broad tuneable birdcage coil for mouse 1H/19F MR applications. J. Magn. Reson. 2021, 329, 107023,  DOI: 10.1016/j.jmr.2021.107023
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    A broad tuneable birdcage coil for mouse 1H/19F MR applications
    Vit, M.; Burian, M.; Berkova, Z.; Lacik, J.; Sedlacek, O.; Hoogenboom, R.; Raida, Z.; Jirak, D.
    Journal of Magnetic Resonance (2021), 329 (), 107023CODEN: JMARF3; ISSN:1090-7807. (Elsevier B.V.)
    In this paper, we present the design and implementation of a 1H/19F vol. coil for mouse body magnetic resonance (MR) imaging and spectroscopy using a high magnetic field (4.7 T). By changing the geometry of the coil rungs to include both nuclei for MR expts., this innovative coil can be tuned over an extremely wide range of frequency. The coil, 45 mm in diam. and 55 mm in length, consists of a 12-rung birdcage-like structure. Using two types of tuning, the coil can generate a sufficiently homogeneous B+1 electromagnetic field within a working vol. optimized for lab. mouse. The first tuning involves changing the resonance frequency over a large frequency range. The elec. capacitance between the wires can be adjusted to reflect changes in the length of the coil. The second tuning comprises a habitual tuning transformer for precise detection in a narrow band. In contrast to widely used multinuclear coils, the coil presented here features only one resonance peak and can be manipulated according to the Larmor frequencies given for 1H and 19F. The coil was successfully tested using full-wave simulations of magnetic and elec. field distributions under in vivo MR conditions.
  35. 35
    Warren, N. J.; Mykhaylyk, O. O.; Mahmood, D.; Ryan, A. J.; Armes, S. P. RAFT aqueous dispersion polymerization yields poly (ethylene glycol)-based diblock copolymer nano-objects with predictable single phase morphologies. J. Am. Chem. Soc. 2014, 136, 1023– 1033,  DOI: 10.1021/ja410593n
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    RAFT Aqueous Dispersion Polymerization Yields Poly(ethylene glycol)-Based Diblock Copolymer Nano-Objects with Predictable Single Phase Morphologies
    Warren, Nicholas J.; Mykhaylyk, Oleksandr O.; Mahmood, Daniel; Ryan, Anthony J.; Armes, Steven P.
    Journal of the American Chemical Society (2014), 136 (3), 1023-1033CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    A poly(ethylene glycol) (PEG) macromol. chain transfer agent (macro-CTA) is prepd. in high yield (>95%) with 97% dithiobenzoate chain-end functionality in a three-step synthesis starting from a monohydroxy PEG113 precursor. This PEG113-dithiobenzoate is then used for the reversible addn.-fragmentation chain transfer (RAFT) aq. dispersion polymn. of 2-hydroxypropyl methacrylate (HPMA). Polymns. conducted under optimized conditions at 50 °C led to high conversions as judged by 1H NMR spectroscopy and relatively low diblock copolymer polydispersities (Mw/Mn < 1.25) as judged by GPC. The latter technique also indicated good blocking efficiencies, since there was minimal PEG113 macro-CTA contamination. Systematic variation of the mean d.p. of the core-forming PHPMA block allowed PEG113-PHPMAx diblock copolymer spheres, worms, or vesicles to be prepd. at up to 17.5% wt./wt. solids, as judged by dynamic light scattering and transmission electron microscopy studies. Small-angle X-ray scattering (SAXS) anal. revealed that more exotic oligolamellar vesicles were obsd. at 20% wt./wt. solids when targeting highly asym. diblock compns. Detailed anal. of SAXS curves indicated that the mean no. of membranes per oligolamellar vesicle is approx. three. A PEG113-PHPMAx phase diagram was constructed to enable the reproducible targeting of pure phases, as opposed to mixed morphologies (e.g., spheres plus worms or worms plus vesicles). This new RAFT PISA formulation is expected to be important for the rational and efficient synthesis of a wide range of biocompatible, thermo-responsive PEGylated diblock copolymer nano-objects for various biomedical applications.
  36. 36
    Feng, C.; Zhu, C.; Yao, W.; Lu, G.; Li, Y.; Lv, X.; Jia, M.; Huang, X. Constructing semi-fluorinated PDEAEMA-b-PBTFVBP-b-PDEAEMA amphiphilic triblock copolymer via successive thermal step-growth cycloaddition polymerization and ATRP. Polym. Chem. 2015, 6, 7881– 7892,  DOI: 10.1039/c5py01404b
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    Constructing semi-fluorinated PDEAEMA-b-PBTFVBP-b-PDEAEMA amphiphilic triblock copolymer via successive thermal step-growth cycloaddition polymerization and ATRP
    Feng, Chun; Zhu, Chao; Yao, Wenqiang; Lu, Guolin; Li, Yongjun; Lv, Xuliang; Jia, Mingchun; Huang, Xiaoyu
    Polymer Chemistry (2015), 6 (45), 7881-7892CODEN: PCOHC2; ISSN:1759-9962. (Royal Society of Chemistry)
    A series of amphiphilic perfluorocyclobutyl-contg. ABA triblock copolymers, PDEAEMA-b-PBTFVBP-b-PDEAEMA (DEAEMA: 2-(diethylamino)ethyl methacrylate; BTFVBP: 4,4'-bis(1,2,2-trifluorovinyloxy)biphenyl), was synthesized through the site transformation strategy, combining thermal step-growth cycloaddn. polymn. of BTFVBP and atom transfer radical polymn. (ATRP) of DEAEMA. A BTFVBP trifluorovinyl aryl ether monomer was first thermally polymd. to form a semi-fluorinated perfluorocyclobutyl aryl ether-based segment, followed by end functionalization for prepg. a Br-PBTFVBP-Br macroinitiator bearing one ATRP initiating group at each end. ATRP of DEAEMA was initiated by Br-PBTFVBP-Br to afford four PDEAEMA-b-PBTFVBP-b-PDEAEMA triblock copolymers with relatively narrow mol. wt. distributions (Mw/Mn ≤ 1.42) via varying the feeding ratio of DEAEMA to the macroinitiator. The crit. micelle concn. (cmc) of the obtained amphiphilic triblock copolymers was detd. by fluorescence spectroscopy using N-phenyl-1-naphthylamine as a probe. Micellar morphologies were investigated by transmission electron microscopy. It was shown that such triblock copolymers could self-assemble into large compd. micelles, vesicles, and bowl-shaped micelles in aq. soln. with different initial water contents and compns.
  37. 37
    Sedlacek, O.; Bardoula, V.; Vuorimaa-Laukkanen, E.; Gedda, L.; Edwards, K.; Radulescu, A.; Mun, G. A.; Guo, Y.; Zhou, J.; Zhang, H.; Nardello-Rataj, V.; Filippov, S.; Hoogenboom, R. Influence of Chain Length of Gradient and Block Copoly (2-oxazoline) s on Self-Assembly and Drug Encapsulation. Small 2022, 18, 2106251,  DOI: 10.1002/smll.202106251
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    Influence of Chain Length of Gradient and Block Copoly(2-oxazoline)s on Self-Assembly and Drug Encapsulation
    Sedlacek, Ondrej; Bardoula, Valentin; Vuorimaa-Laukkanen, Elina; Gedda, Lars; Edwards, Katarina; Radulescu, Aurel; Mun, Grigoriy A.; Guo, Yong; Zhou, Junnian; Zhang, Hongbo; Nardello-Rataj, Veronique; Filippov, Sergey; Hoogenboom, Richard
    Small (2022), 18 (17), 2106251CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Amphiphilic gradient copolymers represent a promising alternative to extensively used block copolymers due to their facile one-step synthesis by statistical copolymn. of monomers of different reactivity. Herein, an in-depth anal. is provided of micelles based on amphiphilic gradient poly(2-oxazoline)s with different chain lengths to evaluate their potential for micellar drug delivery systems and compare them to the analogous diblock copolymer micelles. Size, morphol., and stability of self-assembled nanoparticles, loading of hydrophobic drug curcumin, as well as cytotoxicities of the prepd. nanoformulations are examd. using copoly(2-oxazoline)s with varying chain lengths and comonomer ratios. In addn. to several interesting differences between the two copolymer architecture classes, such as more compact self-assembled structures with faster exchange dynamics for the gradient copolymers, it is concluded that gradient copolymers provide stable curcumin nanoformulations with comparable drug loadings to block copolymer systems and benefit from more straightforward copolymer synthesis. The study demonstrates the potential of amphiphilic gradient copolymers as a versatile platform for the synthesis of new polymer therapeutics.
  38. 38
    Jirák, D.; Kríz, J.; Herynek, V.; Andersson, B.; Girman, P.; Burian, M.; Saudek, F.; Hájek, M. MRI of transplanted pancreatic islets. Magn. Reson. Med. 2004, 52, 1228– 1233,  DOI: 10.1002/mrm.20282
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    MRI of transplanted pancreatic islets
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    Magnetic resonance in medicine (2004), 52 (6), 1228-33 ISSN:0740-3194.
    A promising treatment method for type 1 diabetes mellitus is transplantation of pancreatic islets containing beta-cells. The aim of this study was to develop an MR technique to monitor the distribution and fate of transplanted pancreatic islets in an animal model. Twenty-five hundred purified and magnetically labeled islets were transplanted through the portal vein into the liver of experimental rats. The animals were scanned using a MR 4.7-T scanner. The labeled pancreatic islets were clearly visualized in the liver in both diabetic and healthy rats as hypointense areas on T2*-weighted MR images during the entire measurement period. Transmission electron microscopy confirmed the presence of iron-oxide nanoparticles inside the cells of the pancreatic islets. A significant decrease in blood glucose levels in diabetic rats was observed; normal glycemia was reached 1 week after transplantation. This study, therefore, represents a promising step toward possible clinical application in human medicine.

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  • Abstract

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    Scheme 1

    Scheme 1. Synthesis of 19F MRI Tracers by RAFT-Mediated Aqueous Dispersion PISA of TFEAM: (A) Schematic Illustration and (B) Reaction Scheme
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    Figure 1

    Figure 1. Aqueous dispersion PISA kinetics of TFEAM at 50 °C using PEG-BTPA macroCTA {[TFEAM]0:[PEG-BTPA]0 = 100:1}: (A) variation of monomer consumption as a function of polymerization time, (B) evolution of SEC traces during polymerization eluted with DMA/LiCl, and (C) variation of experimental number-average molar mass (Mn) and dispersity (D̵) as a function of conversion of TFEAM determined by SEC against PMMA calibration (squares), respectively NMR (triangles). The blue line represents the theoretical Mn value.

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    Figure 2

    Figure 2. Representative 1H (A) and 19F (B) NMR spectra of PEG91-b-PTFEAM100 (F2) in CD3OD at 400 MHz and (C) transmission electron micrographs of F1–F6 nanoparticle dispersion; the scale bars represent 200 nm. Insets: physical appearance of as-prepared nanoparticle dispersions in water.

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    Figure 3

    Figure 3. Synthesis of PEG91-b-[PTFEAMx-stat-PHEAMy] block copolymers with a core partly hydrophilized by aqueous PISA: (A) reaction scheme and (B) TEM images of nanoparticles differing in HEAM content (FHEAM = 0–0.4) at constant core-forming block length (DP 100); the scale bars represent 200 nm. Insets: physical appearance of as-prepared nanoparticle dispersions in water.

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    Figure 4

    Figure 4. Partial core hydrophilization with HEAM enhances the 19F MR signal of fluorinated nanoparticles (cpol = 30 mg mL–1, DPcore = 100) in aqueous solutions. (A) Evolution of nanoparticle 19F NMR spectra (400 MHz) as a function of HEAM content; (B) variation of 19F MR relaxation times as a function of HEAM content; (C) 1H and (D) 19F RARE MRI of nanoparticles (4.7 T) with different HEAM contents; (E) overlay image of 19F MRI (red) and 1H MRI (gray); and (F) 19F MR spectra (4.7 T) of nanoparticles centered and used for MRI acquisition; inset: comparison of 19F MRI SNR values.

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    Figure 5

    Figure 5. (A) Intensity-weighted DLS size distributions of PEG91-b-[PTFEAMx-stat-PHEAMy] copolymer nanoparticles (x/y = 8:2) differing in core-forming block DP (100–400) in water (cpol = 1 mg mL–1) and (B) transmission electron micrograph of F5H2 nanoparticles (DP 400); the scale bar represents 200 nm.

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    Figure 6

    Figure 6. In vitro 19F MRI/MRS properties of optimized F5H2 nanoparticles in water; (A) 19F (top), 19F (red) overlaid on 1H (colors in grayscale) RARE MRI (bottom) of F5H2 at different polymer concentrations (20 acquisition scans); the numbers 30, 20, 10, and 5 express cpol as mg mL–1. (B) 19F MR spectra (4.7 T) centered and used for MRI acquisition; (C) variation of the 19F MRI SNR ratio as a function of F5H2 concentration at different numbers of acquisition scans; and (D) minimal polymer concentrations needed to reliably visualize (SNR = 3.5) the F5H2 nanotracer at different MRI acquisition times.

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    Figure 7

    Figure 7. In vivo 19F MRS and MRI of F5H2 nanotracer 2 h after subcutaneous injection into healthy BALB/c mouse; (A) coronal hot spot 19F MRI (left) and overlapped 1H/19F (right) images, where the F5H2 fluorine signal is highlighted in red color and (B) 19F MR CSI spectroscopic grid on a 1H MR reference scan (left); the red grid shows measured CSI voxels and the green squares represent the targeted anatomical area from which the corresponding summation spectra are displayed (right).

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  • References

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      A review. Magnetic resonance imaging (MRI) is recognized as the most powerful clin. imaging modality due to its ability to generate detailed three-dimensional anatomical images with high spatial resoln. in a non-invasive manner without requiring harmful ionizing radiation. However, despite the wide use of these metal-based CAs, safety concerns have been raised regarding their potential toxic effects resulting from long-term in vivo accumulation. This has driven the development of org. metal-free CAs in various forms for use in MRI. Importantly, functional polymers capable of MRI via different mechanisms represent one of the most promising alternatives to current metal-based MRI CAs due to appealing features such as low toxicity, improved pharmacokinetics and biodistribution profile, and tailored structures and functionalities. Such structural and functional flexibility can enable a myriad of biomedical applications. In this review, we will highlight advances in the development of functional polymers as org. metal-free macromol. MRI CAs based on different mechanisms including polymeric nitroxide-based 1H MRI CAs, polymeric chem. exchange satn. transfer (CEST) MRI CAs, and polymeric heteronuclei-based MRI CAs. In addn., the review will address the challenges and future opportunities for these promising classes of metal-free polymeric MRI CAs.
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      Jirak, Daniel; Galisova, Andrea; Kolouchova, Kristyna; Babuka, David; Hruby, Martin
      Magnetic Resonance Materials in Physics, Biology and Medicine (2019), 32 (1), 173-185CODEN: MRBMEQ ISSN:. (Springer)
      A review. Over the last few years, the development and relevance of 19F magnetic resonance imaging (MRI) for use in clin. practice has emerged. MRI using fluorinated probes enables the achievement of a specific signal with high contrast in MRI images. However, to ensure sufficient sensitivity of 19F MRI, fluorine probes with a high content of chem. equiv. fluorine atoms are required. The majority of 19F MRI agents are perfluorocarbon emulsions, which have a broad range of applications in mol. imaging, although the content of fluorine atoms in these mols. is limited. In this review, we focus mainly on polymer probes that allow higher fluorine content and represent versatile platforms with properties tailorable to a plethora of biomedical in vivo applications. We discuss the chem. development, up to the first imaging applications, of these promising fluorine probes, including injectable polymers that form depots that are intended for possible use in cancer therapy.
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      Coloring ultrasensitive MRI with tunable metal-organic frameworks
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      Chemical Science (2021), 12 (12), 4300-4308CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      As one of the most important imaging modalities, magnetic resonance imaging still faces relatively low sensitivity to monitor low-abundance mols. A newly developed technol., hyperpolarized 129Xe magnetic resonance imaging, can boost the signal sensitivity to over 10 000-fold compared with that under conventional MRI conditions, and this technique is referred to as ultrasensitive MRI. However, there are few methods to visualize complex mixts. in this field due to the difficulty in achieving favorable "cages" to capture the signal source, namely, 129Xe atoms. Here, we proposed metal-org. frameworks as tunable nanoporous hosts to provide suitable cavities for xenon. Due to the widely dispersed spectroscopic signals, 129Xe in different MOFs was easily visualized by assigning each chem. shift to a specific color. The results illustrated that the pore size detd. the exchange rate, and the geometric structure and elemental compn. influenced the local charge experienced by xenon. We confirmed that a complex mixt. was first differentiated by specific colors in ultrasensitive MRI. The introduction of MOFs helps to overcome long-standing obstacles in ultrasensitive, multiplexed MRI.
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      Kretschmer, J.; David, T.; Dračínský, M.; Socha, O.; Jirak, D.; Vít, M.; Jurok, R.; Kuchař, M.; Císařová, I.; Polasek, M. Paramagnetic encoding of molecules. Nat. Commun. 2022, 13, 3179,  DOI: 10.1038/s41467-022-30811-9
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      Paramagnetic encoding of molecules
      Kretschmer, Jan; David, Tomas; Dracinsky, Martin; Socha, Ondrej; Jirak, Daniel; Vit, Martin; Jurok, Radek; Kuchar, Martin; Cisarova, Ivana; Polasek, Miloslav
      Nature Communications (2022), 13 (1), 3179CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)
      Abstr.: Contactless digital tags are increasingly penetrating into many areas of human activities. Digitalization of our environment requires an ever growing no. of objects to be identified and tracked with machine-readable labels. Mols. offer immense potential to serve for this purpose, but our ability to write, read, and communicate mol. code with current technol. remains limited. Here we show that magnetic patterns can be synthetically encoded into stable mol. scaffolds with paramagnetic lanthanide ions to write digital code into mols. and their mixts. Owing to the directional character of magnetic susceptibility tensors, each sequence of lanthanides built into one mol. produces a unique magnetic outcome. Multiplexing of the encoded mols. provides a high no. of codes that grows double-exponentially with the no. of available paramagnetic ions. The codes are readable by NMR in the radiofrequency (RF) spectrum, analogously to the macroscopic technol. of RF identification. A prototype mol. system capable of 16-bit (65,535 codes) encoding is presented. Future optimized systems can conceivably provide 64-bit (∼10̂19 codes) or higher encoding to cover the labeling needs in drug discovery, anti-counterfeiting and other areas.
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      Wu, L.; Liu, F.; Liu, S.; Xu, X.; Liu, Z.; Sun, X. Perfluorocarbons-based 19F magnetic resonance imaging in biomedicine. Int. J. Nanomed. 2020, 15, 7377,  DOI: 10.2147/ijn.s255084
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      Perfluorocarbons-based 19F magnetic resonance imaging in biomedicine
      Wu, Lina; Liu, Fang; Liu, Shuang; Xu, Xiuan; Liu, Zhaoxi; Sun, Xilin
      International Journal of Nanomedicine (2020), 15 (), 7377-7395CODEN: IJNNHQ; ISSN:1178-2013. (Dove Medical Press Ltd.)
      A review. Fluorine-19 (19F) magnetic resonance (MR) mol. imaging is a promising noninvasive and quant. mol. imaging approach with intensive research due to the high sensitivity and low endogenous background signal of the 19F atom in vivo. Perfluorocarbons (PFCs) have been used as blood substitutes since 1970s. More recently, a variety of PFC nanoparticles have been designed for the detection and imaging of physiol. and pathol. changes. These mol. imaging probes have been developed to label cells, target specific epitopes in tumors, monitor the prognosis and therapy efficacy and quantitate characterization of tumors and changes in tumor microenvironment noninvasively, therefore, significantly improving the prognosis and therapy efficacy. Herein, we discuss the recent development and applications of 19F MR techniques with PFC nanoparticles in biomedicine, with particular emphasis on ligand-targeted and quant. 19F MR imaging approaches for tumor detection, oxygenation measurement, smart stimulus response and therapy efficacy monitoring, et al. nanoparticles in biomedicine, with particular emphasis on ligand-targeted and quant. 19F MR imaging approaches for tumor detection, oxygenation measurement, smart stimulus response and therapy efficacy monitoring, et al.
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      Ruiz-Cabello, J.; Walczak, P.; Kedziorek, D. A.; Chacko, V. P.; Schmieder, A. H.; Wickline, S. A.; Lanza, G. M.; Bulte, J. W. M. In vivo “hot spot” MR imaging of neural stem cells using fluorinated nanoparticles. Magn. Reson. Med. 2008, 60, 1506– 1511,  DOI: 10.1002/mrm.21783
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      In vivo "hot spot" MR imaging of neural stem cells using fluorinated nanoparticles
      Ruiz-Cabello Jesus; Walczak Piotr; Kedziorek Dorota A; Chacko Vadappuram P; Schmieder Anna H; Wickline Samuel A; Lanza Gregory M; Bulte Jeff W M
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      To optimize (19)F MR tracking of stem cells, we compared cellular internalization of cationic and anionic perfluoro-15-crown-5-ether (PFCE) nanoparticles using cell culture plates with different surface coatings. The viability and proliferation of anionic and cationic PFCE-labeled neural stem cells (NSCs) did not differ from unlabeled cells. Cationic PFCE nanoparticles ((19)F T1/T2 = 580/536 ms at 9.4 Tesla) were superior to anionic particles for intracellular fluorination. Best results were obtained with modified polystyrene culture dishes coated with both carboxylic and amino groups rather than conventional carboxyl-coated dishes. After injecting PFCE-labeled NSCs into the striatum of mouse brain, cells were readily identified in vivo by (19)F MRI without changes in signal or viability over a 2-week period after grafting. These results demonstrate that neural stem cells can be efficiently fluorinated with cationic PFCE nanoparticles without using transfection agents and visualized in vivo over prolonged periods with an MR sensitivity of approximately 140 pmol of PFCE/cell.
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      Fu, C.; Demir, B.; Alcantara, S.; Kumar, V.; Han, F.; Kelly, H. G.; Tan, X.; Yu, Y.; Xu, W.; Zhao, J.; Zhang, C.; Peng, H.; Boyer, C.; Woodruff, T. M.; Kent, S. J.; Searles, D. J.; Whittaker, A. K. Low-fouling fluoropolymers for bioconjugation and in vivo tracking. Angew. Chem. 2020, 132, 4759– 4765,  DOI: 10.1002/ange.201914119
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      Jirak, D.; Svoboda, J.; Filipová, M.; Pop-Georgievski, O.; Sedlacek, O. Antifouling fluoropolymer-coated nanomaterials for 19F MRI. Chem. Commun. 2021, 57, 4718– 4721,  DOI: 10.1039/d1cc00642h
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      Antifouling fluoropolymer-coated nanomaterials for 19F MRI
      Jirak, Daniel; Svoboda, Jan; Filipova, Marcela; Pop-Georgievski, Ognen; Sedlacek, Ondrej
      Chemical Communications (Cambridge, United Kingdom) (2021), 57 (38), 4718-4721CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      We developed a multifunctional polymer coating for nanoparticles (NPs) that enables simultaneous detection by 19F MRI and shielding from blood plasma fouling. The coating is based on a water-sol. fluorinated poly(N-(2-fluoroethyl)acrylamide) (PFEAM) that shows high 19F MRI sensitivity, cytocompatibility and excellent antifouling properties, significantly outperforming polyethylene glycol. A proof-of-concept expt. was performed by synthesizing polymer-coated gold NPs that were successfully visualized by 19F MRI at magnetic fields close to the fields used in clin. practice. This universal approach can be used for coating and tracing of various NPs upon suitable polymer chain-end modification.
    12. 12
      Sedlacek, O.; Jirak, D.; Galisova, A.; Jager, E.; Laaser, J. E.; Lodge, T. P.; Stepanek, P.; Hruby, M. 19F Magnetic Resonance Imaging of Injectable Polymeric Implants with Multiresponsive Behavior. Chem. Mater. 2018, 30, 4892– 4896,  DOI: 10.1021/acs.chemmater.8b02115
      12
      19F Magnetic Resonance Imaging of Injectable Polymeric Implants with Multiresponsive Behavior
      Sedlacek, Ondrej; Jirak, Daniel; Galisova, Andrea; Jager, Eliezer; Laaser, Jennifer E.; Lodge, Timothy P.; Stepanek, Petr; Hruby, Martin
      Chemistry of Materials (2018), 30 (15), 4892-4896CODEN: CMATEX; ISSN:0897-4756. (American Chemical Society)
      Recently, magnetic resonance imaging (MRI) probes based on fluorinated compds. have emerged as highly promising contrast agents. Direct imaging of injectable thermoresponsive polymer implants by 19F MRI represents an attractive, as yet unreported tool for the straightforward and non-invasive monitoring of implant localization, size, morphol., and biodegrdn. Herein, we describe for the first time the in vivo19F MRI visualization of such implants. These were s.c. and i.m. administered as an aq. soln. to form a traceable solid implant upon the change of temp. and pH. After formation, the polymer implant was effectively visualized by 19F MRI. The kinetics of the implant degrdn. is suitable for application as injectable drug depots or in the local radiotherapy of solid tumors.
    13. 13
      Fu, C.; Zhang, C.; Peng, H.; Han, F.; Baker, C.; Wu, Y.; Ta, H.; Whittaker, A. K. Enhanced performance of polymeric 19F MRI contrast agents through incorporation of highly water-soluble monomer MSEA. Macromolecules 2018, 51, 5875– 5882,  DOI: 10.1021/acs.macromol.8b01190
      13
      Enhanced Performance of Polymeric 19F MRI Contrast Agents through Incorporation of Highly Water-Soluble Monomer MSEA
      Fu, Changkui; Zhang, Cheng; Peng, Hui; Han, Felicity; Baker, Carly; Wu, Yuao; Ta, Hang; Whittaker, Andrew K.
      Macromolecules (Washington, DC, United States) (2018), 51 (15), 5875-5882CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
      19F magnetic resonance imaging (MRI) is a powerful noninvasive imaging technique that shows tremendous potential for the diagnosis and monitoring of human diseases. Fluorinated compds. are commonly used as 19F MRI contrast agents to develop "hot spot" imaging. To achieve high-resoln. MR images, a high d. of 19F nuclei is required in the contrast agents. However, because of the inherent hydrophobicity of fluorinated moieties, aggregation of 19F contrast agents with high fluorine content is often obsd. in aq. soln., resulting in attenuated MR signal and low sensitivity, thus significantly limiting their further biol. applications. Here we report the synthesis and characterization of a series of polymeric 19F MRI contrast agents with high fluorine content by copolymg. the well-known fluorinated monomer 2,2,2-trifluoroethyl acrylate (TFEA) with a highly water-sol. monomer 2-(methylsulfinyl)ethyl acrylate (MSEA) using RAFT polymn. We show that these polymeric contrast agents, although with high fluorine content, display remarkable imaging performance as evidenced by preferable relaxation properties and intense in vitro/in vivo MRI signals, demonstrating the huge potential for eventual clin. applications such as MRI-guided disease diagnosis and therapy.
    14. 14
      Thurecht, K. J.; Blakey, I.; Peng, H.; Squires, O.; Hsu, S.; Alexander, C.; Whittaker, A. K. Functional Hyperbranched Polymers: Toward Targeted in Vivo 19F Magnetic Resonance Imaging Using Designed Macromolecules. J. Am. Chem. Soc. 2010, 132, 5336– 5337,  DOI: 10.1021/ja100252y
      14
      Functional Hyperbranched Polymers: Toward Targeted in Vivo 19F Magnetic Resonance Imaging Using Designed Macromolecules
      Thurecht, Kristofer J.; Blakey, Idriss; Peng, Hui; Squires, Oliver; Hsu, Steven; Alexander, Cameron; Whittaker, Andrew K.
      Journal of the American Chemical Society (2010), 132 (15), 5336-5337CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Accurate and early diagnosis of diseases, such as cancer, is the "holy grail" of medical imaging. Techniques. such as computed axial tomog. magnetic resonance imaging (MRI). ultrasound etc., are able to detect various diseases. but often the detection limits mean the diseases have progressed beyond acceptable recovery. Early detection requires imaging techniques with higher sensitivity, while maintaining specificity for disease states (e.g., tumors). We report on our recent efforts in developing sensitive polymeric 19F MRI contrast agents that combine controllable functionality, ability for cell targeting, and low cytotoxicity. Recent synthetic advances that facilitate intimate control over polymer structure and functionality have led to the advent of polymeric theranostics. nanomedical devices for diagnosis and treatment of diseases. Monitoring these devices in an in vivo clin. setting remains a significant scientific challenge despite some elegant attempts to develop polymeric devices or emulsions for 19F MRI. No system to date has demonstrated the ability to be both easily and universally functionalized. while exhibiting high 19F MRI sensitivity. Poor sensitivity is attributed to three main factors: poor mol. mobility. assocn. of the fluorinated segments, and low fluorine content. To overcome such issues, our approach uses hyperbranched polymers enabling high segmental mol. mobility to be achieved while maintaining high fluorine content. Controlled functionality was introduced through reversible addn.-fragmentation chain transfer (RAFT) chem., and mol. mobility was conferred via low-Tg (acrylate) and polar repeat units that are well hydrated under aq. conditions. Random branching frustrates aggregation of the fluorinated segments allowing incorporation of up to 20 mol % fluoromonomer. The "shape-persistence" of the hyperbranched mol. means that orientation of functionality, such as cell-targeting agents, can be controlled thus ensuring correct presentation for efficient biol. recognition if required.
    15. 15
      Rolfe, B. E.; Blakey, I.; Squires, O.; Peng, H.; Boase, N. R. B.; Alexander, C.; Parsons, P. G.; Boyle, G. M.; Whittaker, A. K.; Thurecht, K. J. Multimodal Polymer Nanoparticles with Combined 19F Magnetic Resonance and Optical Detection for Tunable, Targeted, Multimodal Imaging in Vivo. J. Am. Chem. Soc. 2014, 136, 2413– 2419,  DOI: 10.1021/ja410351h
      15
      Multimodal Polymer Nanoparticles with Combined 19F Magnetic Resonance and Optical Detection for Tunable, Targeted, Multimodal Imaging in Vivo
      Rolfe, Barbara E.; Blakey, Idriss; Squires, Oliver; Peng, Hui; Boase, Nathan R. B.; Alexander, Cameron; Parsons, Peter G.; Boyle, Glen M.; Whittaker, Andrew K.; Thurecht, Kristofer J.
      Journal of the American Chemical Society (2014), 136 (6), 2413-2419CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Understanding the complex nature of diseased tissue in vivo requires development of more advanced nanomedicines, where synthesis of multifunctional polymers combines imaging multimodality with a biocompatible, tunable, and functional nanomaterial carrier. Here the authors describe the development of polymeric nanoparticles for multimodal imaging of disease states in vivo. The nanoparticle design utilizes the abundant functionality and tunable physicochem. properties of synthetically robust polymeric systems to facilitate targeted imaging of tumors in mice. For the first time, high-resoln. 19F/1H magnetic resonance imaging is combined with sensitive and versatile fluorescence imaging in a polymeric material for in vivo detection of tumors. The authors highlight how control over the chem. during synthesis allows manipulation of nanoparticle size and function and can lead to very high targeting efficiency to B16 melanoma cells, both in vitro and in vivo. Importantly, the combination of imaging modalities within a polymeric nanoparticle provides information on the tumor mass across various size scales in vivo, from millimeters down to tens of micrometers.
    16. 16
      Sedlacek, O.; Jirak, D.; Vit, M.; Ziołkowska, N.; Janouskova, O.; Hoogenboom, R. Fluorinated water-soluble poly (2-oxazoline) s as highly sensitive 19F MRI contrast agents. Macromolecules 2020, 53, 6387– 6395,  DOI: 10.1021/acs.macromol.0c01228
      16
      Fluorinated Water-Soluble Poly(2-oxazoline)s as Highly Sensitive 19F MRI Contrast Agents
      Sedlacek, Ondrej; Jirak, Daniel; Vit, Martin; Ziolkowska, Natalia; Janouskova, Olga; Hoogenboom, Richard
      Macromolecules (Washington, DC, United States) (2020), 53 (15), 6387-6395CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
      Recently, 19F magnetic resonance imaging (MRI) emerged as a powerful noninvasive diagnostic tool in modern medicine. Fluorinated polymer materials represent an attractive class of MRI contrast agents (CAs) due to their structural variability and tunable properties. Herein, we describe for the first time the 19F MRI of CAs based on fluorinated water-sol. poly(2-oxazoline)s (PAOx), a polymer class with increasing popularity in biomedical sciences. A series of fluorinated PAOx with increasing fluorine content were synthesized by controlled side-chain hydrolysis of poly(2-methyl-2-oxazoline) followed by reacylation of its ethylenimine units by difluoroacetic anhydride. As the increasing fluorine content leads to the copolymer hydrophobization, their compn. was optimized for maximal 19F MRI performance while retaining good soly. in water. The magnetic properties of the water-sol. polymers were studied in vitro by 19F NMR and MRI, revealing their outstanding relaxation properties and imaging sensitivity. All CAs were found to be noncytotoxic for HeLa cells in vitro. Finally, the diagnostic potential of the new CAs was demonstrated by a successful in vivo19F MRI visualization of the selected fluorinated polymer in rats.
    17. 17
      Peng, H.; Blakey, I.; Dargaville, B.; Rasoul, F.; Rose, S.; Whittaker, A. K. Synthesis and Evaluation of Partly Fluorinated Block Copolymers as MRI Imaging Agents. Biomacromolecules 2009, 10, 374– 381,  DOI: 10.1021/bm801136m
      17
      Synthesis and Evaluation of Partly Fluorinated Block Copolymers as MRI Imaging Agents
      Peng, Hui; Blakey, Idriss; Dargaville, Bronwin; Rasoul, Firas; Rose, Stephen; Whittaker, Andrew K.
      Biomacromolecules (2009), 10 (2), 374-381CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      A series of well-defined diblock copolymers of acrylic acid with partially fluorinated acrylate and methacrylate monomers were synthesized using ATRP as potential 19F MRI imaging agents. The diblock copolymers could undergo spontaneous self-assembly in mixed and aq. solvents to form stable micelles with a diam. from approx. 20-45 nm, having a fluorine-rich core that provides a strong signal for MRI examns. The obsd. MRI image intensities were related to the NMR longitudinal and transverse relaxation times, and were found to depend on polymer structure and method of micellization. Two distinct T2 relaxation times were measured; on comparison of expected MRI image intensities with those obsd. exptl., methacrylate polymers show systematically lower signal intensity than acrylate polymers. This is related to the presence of a population of nuclear spins having short T2 relaxation times that cannot be detected under high-resoln. NMR and MRI conditions.
    18. 18
      Kaberov, L. I.; Kaberova, Z.; Murmiliuk, A.; Trousil, J.; Sedláček, O.; Konefal, R.; Zhigunov, A.; Pavlova, E.; Vít, M.; Jirák, D.; Hoogenboom, R.; Filippov, S. K. Fluorine-Containing Block and Gradient Copoly (2-oxazoline) s Based on 2-(3, 3, 3-Trifluoropropyl)-2-oxazoline: A Quest for the Optimal Self-Assembled Structure for 19F Imaging. Biomacromolecules 2021, 22, 2963– 2975,  DOI: 10.1021/acs.biomac.1c00367
      18
      Fluorine-Containing Block and Gradient Copoly(2-oxazoline)s Based on 2-(3,3,3-Trifluoropropyl)-2-oxazoline: A Quest for the Optimal Self-Assembled Structure for 19F Imaging
      Kaberov, Leonid I.; Kaberova, Zhansaya; Murmiliuk, Anastasiia; Trousil, Jiri; Sedlacek, Ondrej; Konefal, Rafal; Zhigunov, Alexander; Pavlova, Ewa; Vit, Martin; Jirak, Daniel; Hoogenboom, Richard; Filippov, Sergey K.
      Biomacromolecules (2021), 22 (7), 2963-2975CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      The use of fluorinated contrast agents in magnetic resonance imaging (MRI) facilitates improved image quality due to the negligible amt. of endogenous fluorine atoms in the body. In this work, we present a comprehensive study of the influence of the amphiphilic polymer structure and compn. on its applicability as contrast agents in 19F MRI. Three series of novel fluorine-contg. poly(2-oxazoline) copolymers and terpolymers, hydrophilic-fluorophilic, hydrophilic-lipophilic-fluorophilic, and hydrophilic-thermoresponsive-fluorophilic, with block and gradient distributions of the fluorinated units, were synthesized. It was discovered that the CF3 in the 2-(3,3,3-trifluoropropyl)-2-oxazoline (CF3EtOx) group activated the cationic chain end, leading to faster copolymn. kinetics, whereby spontaneous monomer gradients were formed with accelerated incorporation of 2-methyl-2-oxazoline or 2-n-propyl-2-oxazoline with a gradual change to the less-nucleophilic CF3EtOx monomer. The obtained amphiphilic copolymers and terpolymers form spherical or wormlike micelles in water, which was confirmed using transmission electron microscopy (TEM), while small-angle X-ray scattering (SAXS) revealed the core-shell or core-double-shell morphologies of these nanoparticles. The core and shell sizes obey the scaling laws for starlike micelles predicted by the scaling theory. Biocompatibility studies confirm that all copolymers obtained are noncytotoxic and, at the same time, exhibit high sensitivity during in vitro 19F MRI studies. The gradient copolymers provide the best 19F MRI signal-to-noise ratio in comparison with the analog block copolymer structures, making them most promising as 19F MRI contrast agents.
    19. 19
      Kolouchova, K.; Sedlacek, O.; Jirak, D.; Babuka, D.; Blahut, J.; Kotek, J.; Vit, M.; Trousil, J.; Konefał, R.; Janouskova, O.; Podhorska, B.; Slouf, M.; Hruby, M. Self-assembled thermoresponsive polymeric nanogels for 19F MR imaging. Biomacromolecules 2018, 19, 3515– 3524,  DOI: 10.1021/acs.biomac.8b00812
      19
      Self-Assembled Thermoresponsive Polymeric Nanogels for 19F MR Imaging
      Kolouchova, Kristyna; Sedlacek, Ondrej; Jirak, Daniel; Babuka, David; Blahut, Jan; Kotek, Jan; Vit, Martin; Trousil, Jiri; Konefal, Rafal; Janouskova, Olga; Podhorska, Bohumila; Slouf, Miroslav; Hruby, Martin
      Biomacromolecules (2018), 19 (8), 3515-3524CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      Magnetic resonance imaging using fluorinated contrast agents (19F MRI) enables to achive high contrast in images due to the negligible fluorine background in living tissues. In this pilot study, we developed new biocompatible, temp.-responsive, and easily synthesized polymeric nanogels contg. a sufficient concn. of magnetically equiv. fluorine atoms for 19F MRI purposes. The structure of the nanogels is based on amphiphilic copolymers contg. two blocks, a hydrophilic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) or poly(2-methyl-2-oxazoline) (PMeOx) block, and a thermoresponsive poly[N(2,2difluoroethyl)acrylamide] (PDFEA) block. The thermoresponsive properties of the PDFEA block allow us to control the process of nanogel self-assembly upon its heating in an aq. soln. Particle size depends on the copolymer compn., and the most promising copolymers with longer thermoresponsive blocks form nanogels of suitable size for angiogenesis imaging or the labeling of cells (approx. 120 nm). The in vitro19F MRI expts. reveal good sensitivity of the copolymer contrast agents, while the nanogels were proven to be noncytotoxic for several cell lines.
    20. 20
      D’Agosto, F.; Rieger, J.; Lansalot, M. RAFT-mediated polymerization-induced self-assembly. Angew. Chem., Int. Ed. 2020, 59, 8368– 8392,  DOI: 10.1002/anie.201911758
      20
      RAFT-Mediated Polymerization-Induced Self-Assembly
      D'Agosto, Franck; Rieger, Jutta; Lansalot, Muriel
      Angewandte Chemie, International Edition (2020), 59 (22), 8368-8392CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. After a brief history that positions polymn.-induced self-assembly (PISA) in the field of polymer chem., this Review will cover the fundamentals of the PISA mechanism. Furthermore, this Review will also give an overview of some of the features and limitations of RAFT-mediated PISA in terms of the choice of the components involved, the nature of the nanoobjects that can be obtained and how the syntheses can be controlled, as well as some potential applications.
    21. 21
      Warren, N. J.; Armes, S. P. Polymerization-induced self-assembly of block copolymer nano-objects via RAFT aqueous dispersion polymerization. J. Am. Chem. Soc. 2014, 136, 10174– 10185,  DOI: 10.1021/ja502843f
      21
      Polymerization-Induced Self-Assembly of Block Copolymer Nano-objects via RAFT Aqueous Dispersion Polymerization
      Warren, Nicholas J.; Armes, Steven P.
      Journal of the American Chemical Society (2014), 136 (29), 10174-10185CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A review. In this Perspective, we discuss the recent development of polymn.-induced self-assembly mediated by reversible addn.-fragmentation chain transfer (RAFT) aq. dispersion polymn. This approach has quickly become a powerful and versatile technique for the synthesis of a wide range of bespoke org. diblock copolymer nano-objects of controllable size, morphol., and surface functionality. Given its potential scalability, such environmentally-friendly formulations are expected to offer many potential applications, such as novel Pickering emulsifiers, efficient microencapsulation vehicles, and sterilizable thermo-responsive hydrogels for the cost-effective long-term storage of mammalian cells.
    22. 22
      Penfold, N. J.; Yeow, J.; Boyer, C.; Armes, S. P. Emerging trends in polymerization-induced self-assembly. ACS Macro Lett. 2019, 8, 1029– 1054,  DOI: 10.1021/acsmacrolett.9b00464
      22
      Emerging Trends in Polymerization-Induced Self-Assembly
      Penfold, Nicholas J. W.; Yeow, Jonathan; Boyer, Cyrille; Armes, Steven P.
      ACS Macro Letters (2019), 8 (8), 1029-1054CODEN: AMLCCD; ISSN:2161-1653. (American Chemical Society)
      A review. In this Perspective, we summarize recent progress in polymn.-induced self-assembly (PISA) for the rational synthesis of block copolymer nanoparticles with various morphologies. Much of the PISA literature has been based on thermally initiated reversible addn.-fragmentation chain transfer (RAFT) polymn. Herein, we pay particular attention to alternative PISA protocols, which allow the prepn. of nanoparticles with improved control over copolymer morphol. and functionality. For example, initiation based on visible light, redox chem., or enzymes enables the incorporation of sensitive monomers and fragile biomols. into block copolymer nanoparticles. Furthermore, PISA syntheses and postfunctionalization of the resulting nanoparticles (e.g., crosslinking) can be conducted sequentially without intermediate purifn. by using various external stimuli. Finally, PISA formulations have been optimized via high-throughput polymn. and recently evaluated within flow reactors for facile scale-up syntheses.
    23. 23
      Le, D.; Keller, D.; Delaittre, G. Reactive and Functional Nanoobjects by Polymerization-Induced Self-Assembly. Macromol. Rapid Commun. 2019, 40, 1800551,  DOI: 10.1002/marc.201800551
      There is no corresponding record for this reference.
    24. 24
      Wan, J.; Fan, B.; Thang, S. RAFT-mediated polymerization-induced self-assembly (RAFT-PISA): current status and future directions. Chem. Sci. 2022, 13, 4192– 4224,  DOI: 10.1039/d2sc00762b
      24
      RAFT-mediated polymerization-induced self-assembly (RAFT-PISA): current status and future directions
      Wan, Jing; Fan, Bo; Thang, San H.
      Chemical Science (2022), 13 (15), 4192-4224CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      A review. Polymn.-induced self-assembly (PISA) combines polymn. and self-assembly in a single step with distinct efficiency that has set it apart from the conventional soln. self-assembly processes. PISA holds great promise for large-scale prodn., not only because of its efficient process for producing polymeric nano/microparticles with high solid content, but also thanks to the facile control over the particle size and morphol. Since its invention, many research groups around the world have developed new and creative approaches to broaden the scope of PISA initiations, morphologies and applications, etc. The growing interest in PISA is certainly reflected in the increasing no. of publications over the past few years, and in this review, we aim to summarize these recent advances in the emerging aspects of RAFT-mediated PISA. These include (1) non-thermal initiation processes, such as photo-, enzyme-, redox- and ultrasound-initiation; the achievements of (2) high-order structures, (3) hybrid materials and (4) stimuli-responsive nano-objects by design and adopting new monomers and new processes; (5) the efforts in the realization of upscale prodn. by utilization of high throughput technologies, and finally the (6) applications of current PISA nano-objects in different fields and (7) its future directions.
    25. 25
      Cao, J.; Tan, Y.; Chen, Y.; Zhang, L.; Tan, J. Expanding the Scope of Polymerization-Induced Self-Assembly: Recent Advances and New Horizons. Macromol. Rapid Commun. 2021, 42, 2100498,  DOI: 10.1002/marc.202100498
      25
      Expanding the Scope of Polymerization-Induced Self-Assembly: Recent Advances and New Horizons
      Cao, Junpeng; Tan, Yingxin; Chen, Ying; Zhang, Li; Tan, Jianbo
      Macromolecular Rapid Communications (2021), 42 (23), 2100498CODEN: MRCOE3; ISSN:1022-1336. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. Over the past decade or so, polymn.-induced self-assembly (PISA) has become a versatile method for rational prepn. of concd. block copolymer nanoparticles with a diverse set of morphologies. Much of the PISA literature has focused on the prepn. of well-defined linear block copolymers by using linear macromol. chain transfer agents (macro-CTAs) with high chain transfer consts. In this review, a recent process is highlighted from an unusual angle that has expanded the scope of PISA including (i) synthesis of block copolymers with nonlinear architectures (e.g., star block copolymer, branched block copolymer) by PISA, (ii) in situ synthesis of blends of polymers by PISA, and (iii) utilization of macro-CTAs with low chain transfer consts. in PISA. By highlighting these important examples, new insights into the research of PISA and future impact these methods will have on polymer and colloid synthesis are provided.
    26. 26
      Czajka, A.; Armes, S. P. Time-Resolved Small-Angle X-ray Scattering Studies during Aqueous Emulsion Polymerization. J. Am. Chem. Soc. 2021, 143, 1474– 1484,  DOI: 10.1021/jacs.0c11183
      26
      Time-Resolved Small-Angle X-ray Scattering Studies during Aqueous Emulsion Polymerization
      Czajka, Adam; Armes, Steven P.
      Journal of the American Chemical Society (2021), 143 (3), 1474-1484CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The persulfate-initiated aq. emulsion polymn. of 2,2,2-trifluoroethyl methacrylate (TFEMA) is studied by time-resolved small-angle X-ray scattering (SAXS) at 60°C using a stirrable reaction cell. TFEMA was preferred to styrene because it offers much greater X-ray scattering contrast relative to water, which is essential for sufficient temporal resoln. The evolution in particle size is monitored by both in situ SAXS and ex situ DLS in the absence or presence of an anionic surfactant (sodium dodecyl sulfate, SDS). Post-mortem SAXS studies confirmed the formation of well-defined spherical latexes, with vol.-av. diams. of 353 ± 9 nm and 68 ± 4 nm being obtained for the surfactant-free and SDS formulations, resp. 1H NMR spectroscopy studies of the equiv. lab.-scale formulations indicated TFEMA conversions of 99% within 80 min and 93% within 60 min for the surfactant-free and SDS formulations, resp. Comparable polymn. kinetics are obsd. for the in situ SAXS expts. and the lab.-scale syntheses, with nucleation occurring after approx. 6 min in each case. After nucleation, scattering patterns are fitted using a hard sphere scattering model to det. the evolution in particle growth for both formulations. Moreover, in situ SAXS enables identification of the three main intervals (I, II, and III) that are obsd. during aq. emulsion polymn. in the presence of surfactant. These intervals are consistent with those indicated by soln. cond. and optical microscopy studies. Significant differences between the surfactant-free and SDS formulations are obsd., providing useful insights into the mechanism of emulsion polymn.
    27. 27
      Czajka, A.; Liao, G.; Mykhaylyk, O. O.; Armes, S. P. In situ small-angle X-ray scattering studies during the formation of polymer/silica nanocomposite particles in aqueous solution. Chem. Sci. 2021, 12, 14288– 14300,  DOI: 10.1039/d1sc03353k
      27
      In situ small-angle X-ray scattering studies during the formation of polymer/silica nanocomposite particles in aqueous solution
      Czajka, A.; Liao, G.; Mykhaylyk, O. O.; Armes, S. P.
      Chemical Science (2021), 12 (42), 14288-14300CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      This study is focused on the formation of polymer/silica nanocomposite particles prepd. by the surfactant-free aq. emulsion polymn. of 2,2,2-trifluoroethyl methacrylate (TFEMA) in the presence of 19 nm glycerol-functionalized aq. silica nanoparticles using a cationic azo initiator at 60°C. The TFEMA polymn. kinetics are monitored using 1H NMR spectroscopy, while postmortem TEM anal. confirms that the final nanocomposite particles possess a well-defined core-shell morphol. Time-resolved small-angle X-ray scattering (SAXS) is used in conjunction with a stirrable reaction cell to monitor the evolution of the nanocomposite particle diam., mean silica shell thickness, mean no. of silica nanoparticles within the shell, silica aggregation efficiency and packing d. during the TFEMA polymn. Nucleation occurs after 10-15 min and the nascent particles quickly become swollen with TFEMA monomer, which leads to a relatively fast rate of polymn. Addnl. surface area is created as these initial particles grow and anionic silica nanoparticles adsorb at the particle surface to maintain a relatively high surface coverage and hence ensure colloidal stability. At high TFEMA conversion, a contiguous silica shell is formed and essentially no further adsorption of silica nanoparticles occurs. A population balance model is introduced into the SAXS model to account for the gradual incorporation of the silica nanoparticles within the nanocomposite particles. The final PTFEMA/silica nanocomposite particles are obtained at 96% TFEMA conversion after 140 min, have a vol.-av. diam. of 216 ± 9 nm and contain approx. 274 silica nanoparticles within their outer shells; a silica aggregation efficiency of 75% can be achieved for such formulations.
    28. 28
      Desnos, G.; Rubio, A.; Gomri, C.; Gravelle, M.; Ladmiral, V.; Semsarilar, M. Semi-Fluorinated Di and Triblock Copolymer Nano-Objects Prepared via RAFT Alcoholic Dispersion Polymerization (PISA). Polymers 2021, 13, 2502,  DOI: 10.3390/polym13152502
      28
      Semi-Fluorinated Di and Triblock Copolymer Nano-Objects Prepared via RAFT Alcoholic Dispersion Polymerization (PISA)
      Desnos, Gregoire; Rubio, Adrien; Gomri, Chaimaa; Gravelle, Mathias; Ladmiral, Vincent; Semsarilar, Mona
      Polymers (Basel, Switzerland) (2021), 13 (15), 2502CODEN: POLYCK; ISSN:2073-4360. (MDPI AG)
      A set of well-defined amphiphilic, semi-fluorinated di and triblock copolymers were synthesized via polymn.-induced self-assembly (PISA) under alc. dispersion polymn. conditions. This study investigates the influence of the length, nature and position of the solvophobic semi-fluorinated block. A poly(N,N-dimethylaminoethyl methacrylate) was used as the stabilizing block to prep. the di and tri block copolymer nano-objects via reversible addn.-fragmentation chain transfer (RAFT) controlled dispersion polymn. at 70°C in ethanol. Benzylmethacrylate (BzMA) and semi-fluorinated methacrylates and acrylates with 7 (heptafluorobutyl methacrylate (HFBMA)), 13 (heneicosafluorododecyl methacrylate (HCFDDMA)) and 21 (tridecafluorooctyl acrylate (TDFOA)) fluorine atoms were used as monomers for the core-forming blocks. The RAFT polymn. of these semi-fluorinated monomers was monitored by SEC and 1H NMR. The evolution of the self-assembled morphologies was investigated by transmission electron microscopy. The results demonstrate that the order of the blocks and the no. of fluorine atoms influence the microphase segregation of the core-forming blocks and the final morphol. of the nano-objects.
    29. 29
      Huo, M.; Li, D.; Song, G.; Zhang, J.; Wu, D.; Wei, Y.; Yuan, J. Semi-Fluorinated Methacrylates: A Class of Versatile Monomers for Polymerization-Induced Self-Assembly. Macromol. Rapid Commun. 2018, 39, 1700840,  DOI: 10.1002/marc.201700840
      There is no corresponding record for this reference.
    30. 30
      Cornel, E. J.; van Meurs, S.; Smith, T.; O’Hora, P. S.; Armes, S. P. In Situ Spectroscopic Studies of Highly Transparent Nanoparticle Dispersions Enable Assessment of Trithiocarbonate Chain-End Fidelity during RAFT Dispersion Polymerization in Nonpolar Media. J. Am. Chem. Soc. 2018, 140, 12980– 12988,  DOI: 10.1021/jacs.8b07953
      30
      In Situ Spectroscopic Studies of Highly Transparent Nanoparticle Dispersions Enable Assessment of Trithiocarbonate Chain-End Fidelity during RAFT Dispersion Polymerization in Nonpolar Media
      Cornel, Erik J.; van Meurs, Sandra; Smith, Timothy; OHora, Paul S.; Armes, Steven P.
      Journal of the American Chemical Society (2018), 140 (40), 12980-12988CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      We report the synthesis of highly transparent poly(stearyl methacrylate)-poly(2,2,2-trifluoroethyl methacrylate) (PSMA-PTFEMA) diblock copolymer nanoparticles via polymn.-induced self-assembly (PISA) in nonpolar media at 70 °C. This was achieved by chain-extending a PSMA precursor block via reversible addn.-fragmentation chain transfer (RAFT) dispersion polymn. of TFEMA in n-tetradecane. This n-alkane has the same refractive index as the PTFEMA core-forming block at 70 °C, which ensures high light transmittance when targeting 33 nm spherical nanoparticles. Such isorefractivity enables visible absorption spectra to be recorded with minimal light scattering even at 30% wt./wt. solids. However, in situ monitoring of the trithiocarbonate RAFT end-groups during PISA requires selection of a weak n → π* band at 446 nm. Conversion of TFEMA into PTFEMA causes a contraction in the reaction soln. vol., leading to an initial increase in absorbance that enables the kinetics of polymn. to be monitored via dilatometry. At ∼98% TFEMA conversion, this 446 nm band remains const. for 2 h at 70 °C, indicating surprisingly high RAFT chain-end fidelity (and hence pseudoliving character) under monomer-starved conditions. In situ 19F NMR spectroscopy studies provide evidence for (i) the onset of micellar nucleation, (ii) solvation of the nanoparticle cores by TFEMA monomer, and (iii) surface plasticization of the nanoparticle cores by n-tetradecane at 70 °C. Finally, the kinetics of RAFT chain-end removal can be conveniently monitored by in situ visible absorption spectroscopy: addn. of excess initiator at 70 °C causes complete discoloration of the dispersion, with small-angle X-ray scattering studies confirming no change in nanoparticle morphol. under these conditions.
    31. 31
      Zhao, W.; Ta, H. T.; Zhang, C.; Whittaker, A. K. Polymerization-Induced Self-Assembly (PISA)─Control over the Morphology of 19F-Containing Polymeric Nano-objects for Cell Uptake and Tracking. Biomacromolecules 2017, 18, 1145– 1156,  DOI: 10.1021/acs.biomac.6b01788
      31
      Polymerization-Induced Self-Assembly (PISA) - Control over the Morphology of 19F-Containing Polymeric Nano-objects for Cell Uptake and Tracking
      Zhao, Wei; Ta, Hang T.; Zhang, Cheng; Whittaker, Andrew K.
      Biomacromolecules (2017), 18 (4), 1145-1156CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      Fluorine-contg. polymeric materials are receiving increasing attention as imaging probes in fluorine-19 magnetic resonance imaging (19F MRI), for example to enable quant. in vivo detection of cells. Here we describe the one-pot polymn. synthesis of 19F-contg. functional poly(oligo(ethylene glycol)) Me ether methacrylate-co-2,2,2-trifluoroethyl acrylate-b-poly(styrene-co-3-vinylbenzaldehyde) (poly(OEGA-co-TFEA)-b-poly(St-co-VBA)) copolymers as a new class of fluorinated MRI agent. A range of nanoparticle morphologies, including spheres, worm-like particles, and vesicles were formed as a consequence of polymn.-induced self-assembly (PISA). It was found that the extent of cell uptake strongly depends on the morphol. of the nano-objects, with preferable uptake for worm-like particles compared to spherical nanoparticles and vesicles. All the nano-objects have a single resonance in the 19F NMR spectrum with relatively short MRI relaxation times, which were independent of the morphol. of the nano-objects. These results confirm that these polymeric nano-objects of varied morphologies are promising as 19F MRI imaging agents for use in tracking of cells and selective MRI.
    32. 32
      Lueckerath, T.; Strauch, T.; Koynov, K.; Barner-Kowollik, C.; Ng, D. Y. W.; Weil, T. DNA–Polymer Conjugates by Photoinduced RAFT Polymerization. Biomacromolecules 2019, 20, 212– 221,  DOI: 10.1021/acs.biomac.8b01328
      32
      DNA-Polymer Conjugates by Photoinduced RAFT Polymerization
      Lueckerath, Thorsten; Strauch, Tina; Koynov, Kaloian; Barner-Kowollik, Christopher; Ng, David Y. W.; Weil, Tanja
      Biomacromolecules (2019), 20 (1), 212-221CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      Conventional grafting-to approaches to DNA-polymer conjugates are often limited by low reaction yields due to the sterically hindered coupling of a presynthesized polymer to DNA. The grafting-from strategy, in contrast, allows one to directly graft polymers from an initiator that is covalently attached to DNA. Herein, we report blue-light-mediated reversible addn.-fragmentation chain-transfer (Photo-RAFT) polymn. from two different RAFT agent-terminated DNA sequences using Eosin Y as the photocatalyst in combination with ascorbic acid. Three monomer families (methacrylates, acrylates and acrylamides) were successfully polymd. from DNA employing Photo-RAFT polymn. We demonstrate that the length of the grown polymer chain can be varied by altering the monomer to DNA-initiator ratio, while the self-assembly features of the DNA strands were maintained. In summary, we describe a convenient, light-mediated approach toward DNA-polymer conjugates via the grafting-from approach.
    33. 33
      Bak, J. M.; Kim, K.-B.; Lee, J.-E.; Park, Y.; Yoon, S. S.; Jeong, H. M.; Lee, H.-i. Thermoresponsive fluorinated polyacrylamides with low cytotoxicity. Polym. Chem. 2013, 4, 2219– 2223,  DOI: 10.1039/c2py20747h
      33
      Thermoresponsive fluorinated polyacrylamides with low cytotoxicity
      Bak, Jae Min; Kim, Kyung-Bin; Lee, Ji-Eun; Park, Yongjin; Yoon, Sang Sun; Jeong, Han Mo; Lee, Hyung-il
      Polymer Chemistry (2013), 4 (7), 2219-2223CODEN: PCOHC2; ISSN:1759-9962. (Royal Society of Chemistry)
      Authors report the unique thermoresponsive properties of fluorinated polyacrylamides, poly[N-(2,2-difluoroethyl)acrylamide] (P2F). The soly. of fluorinated polyacrylamides in water can be easily controlled by changing the no. of fluorine atoms in N-Et groups. Moreover, we demonstrate that fluorinated polyacrylamides are less cytotoxic than poly(N-isopropylacrylamide) (PNIPAM).
    34. 34
      Vít, M.; Burian, M.; Berková, Z.; Lacik, J.; Sedlacek, O.; Hoogenboom, R.; Raida, Z.; Jirak, D. A broad tuneable birdcage coil for mouse 1H/19F MR applications. J. Magn. Reson. 2021, 329, 107023,  DOI: 10.1016/j.jmr.2021.107023
      34
      A broad tuneable birdcage coil for mouse 1H/19F MR applications
      Vit, M.; Burian, M.; Berkova, Z.; Lacik, J.; Sedlacek, O.; Hoogenboom, R.; Raida, Z.; Jirak, D.
      Journal of Magnetic Resonance (2021), 329 (), 107023CODEN: JMARF3; ISSN:1090-7807. (Elsevier B.V.)
      In this paper, we present the design and implementation of a 1H/19F vol. coil for mouse body magnetic resonance (MR) imaging and spectroscopy using a high magnetic field (4.7 T). By changing the geometry of the coil rungs to include both nuclei for MR expts., this innovative coil can be tuned over an extremely wide range of frequency. The coil, 45 mm in diam. and 55 mm in length, consists of a 12-rung birdcage-like structure. Using two types of tuning, the coil can generate a sufficiently homogeneous B+1 electromagnetic field within a working vol. optimized for lab. mouse. The first tuning involves changing the resonance frequency over a large frequency range. The elec. capacitance between the wires can be adjusted to reflect changes in the length of the coil. The second tuning comprises a habitual tuning transformer for precise detection in a narrow band. In contrast to widely used multinuclear coils, the coil presented here features only one resonance peak and can be manipulated according to the Larmor frequencies given for 1H and 19F. The coil was successfully tested using full-wave simulations of magnetic and elec. field distributions under in vivo MR conditions.
    35. 35
      Warren, N. J.; Mykhaylyk, O. O.; Mahmood, D.; Ryan, A. J.; Armes, S. P. RAFT aqueous dispersion polymerization yields poly (ethylene glycol)-based diblock copolymer nano-objects with predictable single phase morphologies. J. Am. Chem. Soc. 2014, 136, 1023– 1033,  DOI: 10.1021/ja410593n
      35
      RAFT Aqueous Dispersion Polymerization Yields Poly(ethylene glycol)-Based Diblock Copolymer Nano-Objects with Predictable Single Phase Morphologies
      Warren, Nicholas J.; Mykhaylyk, Oleksandr O.; Mahmood, Daniel; Ryan, Anthony J.; Armes, Steven P.
      Journal of the American Chemical Society (2014), 136 (3), 1023-1033CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A poly(ethylene glycol) (PEG) macromol. chain transfer agent (macro-CTA) is prepd. in high yield (>95%) with 97% dithiobenzoate chain-end functionality in a three-step synthesis starting from a monohydroxy PEG113 precursor. This PEG113-dithiobenzoate is then used for the reversible addn.-fragmentation chain transfer (RAFT) aq. dispersion polymn. of 2-hydroxypropyl methacrylate (HPMA). Polymns. conducted under optimized conditions at 50 °C led to high conversions as judged by 1H NMR spectroscopy and relatively low diblock copolymer polydispersities (Mw/Mn < 1.25) as judged by GPC. The latter technique also indicated good blocking efficiencies, since there was minimal PEG113 macro-CTA contamination. Systematic variation of the mean d.p. of the core-forming PHPMA block allowed PEG113-PHPMAx diblock copolymer spheres, worms, or vesicles to be prepd. at up to 17.5% wt./wt. solids, as judged by dynamic light scattering and transmission electron microscopy studies. Small-angle X-ray scattering (SAXS) anal. revealed that more exotic oligolamellar vesicles were obsd. at 20% wt./wt. solids when targeting highly asym. diblock compns. Detailed anal. of SAXS curves indicated that the mean no. of membranes per oligolamellar vesicle is approx. three. A PEG113-PHPMAx phase diagram was constructed to enable the reproducible targeting of pure phases, as opposed to mixed morphologies (e.g., spheres plus worms or worms plus vesicles). This new RAFT PISA formulation is expected to be important for the rational and efficient synthesis of a wide range of biocompatible, thermo-responsive PEGylated diblock copolymer nano-objects for various biomedical applications.
    36. 36
      Feng, C.; Zhu, C.; Yao, W.; Lu, G.; Li, Y.; Lv, X.; Jia, M.; Huang, X. Constructing semi-fluorinated PDEAEMA-b-PBTFVBP-b-PDEAEMA amphiphilic triblock copolymer via successive thermal step-growth cycloaddition polymerization and ATRP. Polym. Chem. 2015, 6, 7881– 7892,  DOI: 10.1039/c5py01404b
      36
      Constructing semi-fluorinated PDEAEMA-b-PBTFVBP-b-PDEAEMA amphiphilic triblock copolymer via successive thermal step-growth cycloaddition polymerization and ATRP
      Feng, Chun; Zhu, Chao; Yao, Wenqiang; Lu, Guolin; Li, Yongjun; Lv, Xuliang; Jia, Mingchun; Huang, Xiaoyu
      Polymer Chemistry (2015), 6 (45), 7881-7892CODEN: PCOHC2; ISSN:1759-9962. (Royal Society of Chemistry)
      A series of amphiphilic perfluorocyclobutyl-contg. ABA triblock copolymers, PDEAEMA-b-PBTFVBP-b-PDEAEMA (DEAEMA: 2-(diethylamino)ethyl methacrylate; BTFVBP: 4,4'-bis(1,2,2-trifluorovinyloxy)biphenyl), was synthesized through the site transformation strategy, combining thermal step-growth cycloaddn. polymn. of BTFVBP and atom transfer radical polymn. (ATRP) of DEAEMA. A BTFVBP trifluorovinyl aryl ether monomer was first thermally polymd. to form a semi-fluorinated perfluorocyclobutyl aryl ether-based segment, followed by end functionalization for prepg. a Br-PBTFVBP-Br macroinitiator bearing one ATRP initiating group at each end. ATRP of DEAEMA was initiated by Br-PBTFVBP-Br to afford four PDEAEMA-b-PBTFVBP-b-PDEAEMA triblock copolymers with relatively narrow mol. wt. distributions (Mw/Mn ≤ 1.42) via varying the feeding ratio of DEAEMA to the macroinitiator. The crit. micelle concn. (cmc) of the obtained amphiphilic triblock copolymers was detd. by fluorescence spectroscopy using N-phenyl-1-naphthylamine as a probe. Micellar morphologies were investigated by transmission electron microscopy. It was shown that such triblock copolymers could self-assemble into large compd. micelles, vesicles, and bowl-shaped micelles in aq. soln. with different initial water contents and compns.
    37. 37
      Sedlacek, O.; Bardoula, V.; Vuorimaa-Laukkanen, E.; Gedda, L.; Edwards, K.; Radulescu, A.; Mun, G. A.; Guo, Y.; Zhou, J.; Zhang, H.; Nardello-Rataj, V.; Filippov, S.; Hoogenboom, R. Influence of Chain Length of Gradient and Block Copoly (2-oxazoline) s on Self-Assembly and Drug Encapsulation. Small 2022, 18, 2106251,  DOI: 10.1002/smll.202106251
      37
      Influence of Chain Length of Gradient and Block Copoly(2-oxazoline)s on Self-Assembly and Drug Encapsulation
      Sedlacek, Ondrej; Bardoula, Valentin; Vuorimaa-Laukkanen, Elina; Gedda, Lars; Edwards, Katarina; Radulescu, Aurel; Mun, Grigoriy A.; Guo, Yong; Zhou, Junnian; Zhang, Hongbo; Nardello-Rataj, Veronique; Filippov, Sergey; Hoogenboom, Richard
      Small (2022), 18 (17), 2106251CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Amphiphilic gradient copolymers represent a promising alternative to extensively used block copolymers due to their facile one-step synthesis by statistical copolymn. of monomers of different reactivity. Herein, an in-depth anal. is provided of micelles based on amphiphilic gradient poly(2-oxazoline)s with different chain lengths to evaluate their potential for micellar drug delivery systems and compare them to the analogous diblock copolymer micelles. Size, morphol., and stability of self-assembled nanoparticles, loading of hydrophobic drug curcumin, as well as cytotoxicities of the prepd. nanoformulations are examd. using copoly(2-oxazoline)s with varying chain lengths and comonomer ratios. In addn. to several interesting differences between the two copolymer architecture classes, such as more compact self-assembled structures with faster exchange dynamics for the gradient copolymers, it is concluded that gradient copolymers provide stable curcumin nanoformulations with comparable drug loadings to block copolymer systems and benefit from more straightforward copolymer synthesis. The study demonstrates the potential of amphiphilic gradient copolymers as a versatile platform for the synthesis of new polymer therapeutics.
    38. 38
      Jirák, D.; Kríz, J.; Herynek, V.; Andersson, B.; Girman, P.; Burian, M.; Saudek, F.; Hájek, M. MRI of transplanted pancreatic islets. Magn. Reson. Med. 2004, 52, 1228– 1233,  DOI: 10.1002/mrm.20282
      38
      MRI of transplanted pancreatic islets
      Jirak Daniel; Kriz Jan; Herynek Vit; Andersson Benita; Girman Peter; Burian Martin; Saudek Frantisek; Hajek Milan
      Magnetic resonance in medicine (2004), 52 (6), 1228-33 ISSN:0740-3194.
      A promising treatment method for type 1 diabetes mellitus is transplantation of pancreatic islets containing beta-cells. The aim of this study was to develop an MR technique to monitor the distribution and fate of transplanted pancreatic islets in an animal model. Twenty-five hundred purified and magnetically labeled islets were transplanted through the portal vein into the liver of experimental rats. The animals were scanned using a MR 4.7-T scanner. The labeled pancreatic islets were clearly visualized in the liver in both diabetic and healthy rats as hypointense areas on T2*-weighted MR images during the entire measurement period. Transmission electron microscopy confirmed the presence of iron-oxide nanoparticles inside the cells of the pancreatic islets. A significant decrease in blood glucose levels in diabetic rats was observed; normal glycemia was reached 1 week after transplantation. This study, therefore, represents a promising step toward possible clinical application in human medicine.

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    • Characterization of synthesized polymers by MALDI-TOF, NMR, SED, Cryo-TEM, and MR (PDF)


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