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Insulin Crystals Grown in Short-Peptide Supramolecular Hydrogels Show Enhanced Thermal Stability and Slower Release Profile
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Biological and Medical Applications of Materials and Interfaces

Insulin Crystals Grown in Short-Peptide Supramolecular Hydrogels Show Enhanced Thermal Stability and Slower Release Profile
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  • Rafael Contreras-Montoya
    Rafael Contreras-Montoya
    Departamento de Química Orgánica, Universidad de Granada, (UGR), C. U. Fuentenueva, Avda. Severo Ochoa s/n, E-18071 Granada, Spain
    Instituto de Investigación Biosanitaria ibs.GRANADA, 18014 Granada, Spain
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    Orcidhttp://orcid.org/0000-0002-0766-004X
  • María Arredondo-Amador
    María Arredondo-Amador
    Departamento de Farmacología, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), School of Pharmacy, Instituto de Investigación Biosanitaria ibs.GRANADA, University of Granada, 18071 Granada, Spain
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  • Guillermo Escolano-Casado
    Guillermo Escolano-Casado
    Laboratorio de Estudios Cristalográficos, Instituto Andaluz de Ciencias de la Tierra (Consejo Superior de Investigaciones Científicas-UGR), Avenida de las Palmeras 4, Armilla, 18100 Granada, Spain
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  • Mari C. Mañas-Torres
    Mari C. Mañas-Torres
    Departamento de Química Orgánica, Universidad de Granada, (UGR), C. U. Fuentenueva, Avda. Severo Ochoa s/n, E-18071 Granada, Spain
    Instituto de Investigación Biosanitaria ibs.GRANADA, 18014 Granada, Spain
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  • Mercedes González
    Mercedes González
    Departamento de Farmacología, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), School of Pharmacy, Instituto de Investigación Biosanitaria ibs.GRANADA, University of Granada, 18071 Granada, Spain
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  • Mayte Conejero-Muriel
    Mayte Conejero-Muriel
    Laboratorio de Estudios Cristalográficos, Instituto Andaluz de Ciencias de la Tierra (Consejo Superior de Investigaciones Científicas-UGR), Avenida de las Palmeras 4, Armilla, 18100 Granada, Spain
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  • Vaibhav Bhatia
    Vaibhav Bhatia
    Lamark Biotech Pvt. Ltd., VIT-TBI, 632 014 Vellore, Tamil Nadu, India
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  • Juan J. Díaz-Mochón
    Juan J. Díaz-Mochón
    Departamento de Química Farmacéutica y Orgánica, Facultad de Farmacia, UGR, 18011 Granada, Spain
    Centre for Genomics and Oncological Research, Pfizer/University of Granada/Andalusian Regional Government, PTS Granada, Avenida de la Ilustración 114, 18016 Granada, Spain
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  • Olga Martínez-Augustin
    Olga Martínez-Augustin
    Departamento de Bioquímica y Biología Molecular II, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), School of Pharmacy, Instituto de Investigación Biosanitaria ibs.GRANADA, University of Granada, 18071 Granada, Spain
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    Orcidhttp://orcid.org/0000-0001-8291-3468
  • Fermín Sánchez de Medina
    Fermín Sánchez de Medina
    Departamento de Farmacología, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), School of Pharmacy, Instituto de Investigación Biosanitaria ibs.GRANADA, University of Granada, 18071 Granada, Spain
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    Orcidhttp://orcid.org/0000-0002-4516-5824
  • Modesto T. Lopez-Lopez
    Modesto T. Lopez-Lopez
    Departamento de Física Aplicada, Facultad de Ciencias, UGR, C. U. Fuentenueva, Avda. Severo Ochoa s/n, E-18071 Granada, Spain
    Instituto de Investigación Biosanitaria ibs.GRANADA, 18014 Granada, Spain
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    Orcidhttp://orcid.org/0000-0002-9068-7795
  • Francisco Conejero-Lara
    Francisco Conejero-Lara
    Departamento de Química Física, Facultad de Ciencias, UGR, C. U. Fuentenueva, Avda. Severo Ochoa s/n, E-18071 Granada, Spain
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    Orcidhttp://orcid.org/0000-0002-8282-2168
  • José A. Gavira*
    José A. Gavira
    Laboratorio de Estudios Cristalográficos, Instituto Andaluz de Ciencias de la Tierra (Consejo Superior de Investigaciones Científicas-UGR), Avenida de las Palmeras 4, Armilla, 18100 Granada, Spain
    *Email: [email protected]
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    Orcidhttp://orcid.org/0000-0002-7386-6484
  • Luis Álvarez de Cienfuegos*
    Luis Álvarez de Cienfuegos
    Departamento de Química Orgánica, Universidad de Granada, (UGR), C. U. Fuentenueva, Avda. Severo Ochoa s/n, E-18071 Granada, Spain
    Instituto de Investigación Biosanitaria ibs.GRANADA, 18014 Granada, Spain
    *Email: [email protected]
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    Orcidhttp://orcid.org/0000-0001-8910-4241
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ACS Applied Materials & Interfaces

Cite this: ACS Appl. Mater. Interfaces 2021, 13, 10, 11672–11682
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https://doi.org/10.1021/acsami.1c00639
Published March 4, 2021

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Abstract

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Protein therapeutics have a major role in medicine in that they are used to treat diverse pathologies. Their three-dimensional structures not only offer higher specificity and lower toxicity than small organic compounds but also make them less stable, limiting their in vivo half-life. Protein analogues obtained by recombinant DNA technology or by chemical modification and/or the use of drug delivery vehicles has been adopted to improve or modulate the in vivo pharmacological activity of proteins. Nevertheless, strategies to improve the shelf-life of protein pharmaceuticals have been less explored, which has challenged the preservation of their activity. Herein, we present a methodology that simultaneously increases the stability of proteins and modulates the release profile, and implement it with human insulin as a proof of concept. Two novel thermally stable insulin composite crystal formulations intended for the therapeutic treatment of diabetes are reported. These composite crystals have been obtained by crystallizing insulin in agarose and fluorenylmethoxycarbonyl-dialanine (Fmoc-AA) hydrogels. This process affords composite crystals, in which hydrogel fibers are occluded. The insulin in both crystalline formulations remains unaltered at 50 °C for 7 days. Differential scanning calorimetry, high-performance liquid chromatography, mass spectrometry, and in vivo studies have shown that insulin does not degrade after the heat treatment. The nature of the hydrogel modifies the physicochemical properties of the crystals. Crystals grown in Fmoc-AA hydrogel are more stable and have a slower dissolution rate than crystals grown in agarose. This methodology paves the way for the development of more stable protein pharmaceuticals overcoming some of the existing limitations.

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Introduction

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Thanks to the advance of the recombinant DNA technology, the number of therapeutic proteins that are used for the treatment of different diseases has increased enormously in recent years, revolutionizing the pharmaceutical industry. (1) The use of proteins for therapeutic purposes has a series of advantages in terms of specificity and safety when compared with small synthetic molecules. However, the complex structure of proteins makes them very challenging to both stabilize and preserve before administration, which translates into lower bioavailability and half-life once administered in vivo, limiting their therapeutic effect. (2) Strategies developed to extend protein in vivo half-life include protein engineering and/or chemical post-modification with polyethylene glycol, fatty acids, and polysaccharides to name just a few. (3) Although these techniques have proven effective, their success cannot be predicted and, in some cases, the modification can either reduce the basal activity of the protein or even produce adverse effects. Thus, alternative strategies that do not modify the chemical composition and tridimensional structure of the proteins have been explored. (4) Moreover, efforts have been devoted to develop systems that can exert a controlled drug release. (5)
Among the different delivery systems, microparticles, emulsions, etc., hydrogels seem to be ideal materials as they are biocompatible and biodegradable and, therefore, can be used for in vivo applications and can also be prepared under mild conditions, thus preserving the protein stability. (6) Hydrogels have been intensively studied as protein carriers that can modulate the protein release profile based on the chemical nature of the hydrogel, concentration, and type and degree of crosslinking. (7,8) In particular, injectable hydrogels have attracted a great interest due to the capacity of in vivo administration by noninvasive injection methods. (9,10) Nevertheless, most of these hydrogel systems have exclusively focused on controlling the release rate of the protein and less attention has been paid to developing hydrogels that can improve the stability of the protein, although this is the center of active research. (11,12) The preservation of a protein’s native structure is one of the main issues for many hydrogel formulations that limits their application in clinics. (6) Besides, proteins in crystalline form can show some advantages when intended for therapeutic uses, such as ease of handling, higher concentration doses per volume than their soluble format, varied dissolution rates, and even improved stability. (13,14) In this respect, herein we have developed a strategy to create novel protein delivery formulations that can simultaneously improve protein stability and modify the release profile. This strategy uses hydrogels as media for protein crystallization. When protein crystals are grown in hydrogel media, the hydrogel material is occluded inside the crystals, giving rise to composite protein crystals. (15) We have proven that owing to its inherent nature, the hydrogel is able to modify certain characteristics of the crystals, such as quality, polymorphism, etc. (16−22) Herein, we show that the hydrogel can modify the in vitro and in vivo release profiles of insulin crystals and, at the same time, enhance the stability of the protein in its crystal form. In particular, short-peptide supramolecular hydrogels (23,24) are able to stabilize insulin crystals to a higher degree, slowing down their release, compared to agarose crystals and the crystal control grown without hydrogel.

Results and Discussion

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First, we developed a novel protocol to produce homogeneous batches of small crystals with a very narrow size distribution (4 ± 1 μm) inside injectable and biocompatible hydrogels that allowed direct subcutaneous administration (Figure 1). To do so, the selection of the hydrogel was crucial since the hydrogel is responsible for (1) controlling the quality, size, and homogeneous distribution of the crystals, i.e., dissolution rates; (2) conferring improved stability to the crystals against mechanical stress and temperature; and (3) being a biocompatible and injectable matrix that can be easily reconstituted to allow homogeneous resuspension of the crystals for adequate administration. Based on our previous studies, we selected agarose, fluorenylmethoxycarbonyl-dialanine (Fmoc-AA), and Fmoc-diphenylalanine (Fmoc-FF) hydrogels since these are physical hydrogels that at lower concentrations are weak, can be taken by a syringe, and, as we have previously shown, are compatible with protein crystallization. (18,20)

Figure 1

Figure 1. Schematic chart of the preparation of composite insulin crystal formulation for in vivo administration.

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We started by studying the compatibility of insulin with the formation of the gel as a function of the gel concentration. When Fmoc-FF was mixed with insulin, it precipitated at all concentrations tested, suggesting that a strong interaction between the peptide fiber and the protein occurred. When we carried out the same experiments with Fmoc-AA, we could avoid precipitation at a lower peptide concentration of 0.2% w/v, affording very weak hydrogels in which the protein in the solution formed the expected crystals. Agarose has been more widely studied to produce protein crystals, including insulin, (22) and therefore we selected a low agarose concentration (0.05% w/v) to avoid any further influence on the crystallization process. Fmoc-AA hydrogels demonstrated a strong shear thinning behavior, which could be attributed to rupture of the chain-like network by the shear forces causing reduction in viscosity, confirming the gel nature of the media (Figure 2A). The low values of viscosity (for reference, the viscosity of water at 25 °C is 10–3 Pa·s) showed the weakness of the gel at this particular concentration, which was already observed by visual and manual exploration. To further confirm the gel-like character, we obtained the viscoelastic moduli of the sample as a function of the strain amplitude for a fixed frequency of oscillation of 1 Hz (Figure 2B). As observed, the sample showed the typical behavior of a weak gel, characterized by values of the viscoelastic moduli being approximately independent of the strain amplitude at low values of the latter (linear viscoelastic region), with the storage modulus, G′, being higher than the loss modulus, G″, by less than 1 order of magnitude. At higher values of the strain amplitude, both G′ and G″ decreased rapidly (in the nonlinear viscoelastic region), and eventually, G″ overcame G′, indicating the onset of the fluid-like behavior due to the breakage of the internal structure of the gel by the shear forces.

Figure 2

Figure 2. Viscosity (A) and viscoelastic moduli (B) values of the hydrogels (agarose: blue and Fmoc-AA: red). (C) circular dichroism (CD) spectra of Fmoc-AA. (D) Schematic representation of Fmoc-AA and agarose molecules. (E) Transmission electron microscopy (TEM) image of Fmoc-AA and (F) agarose dried gels.

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Circular dichroism (CD) showed two distinctive negative bands at 230 and 282 nm corresponding to n−π* and π–π* transitions of the Fmoc group, in agreement with previous results reported for other Fmoc derivatives (Figure 2C). Although it is reported that these peptides preferentially arrange in β-sheets, Ren and co-workers have shown that this particular peptide adopts mainly a polyproline II conformation. (25) These aggregates were directly observed by transmission electron microscopy (TEM) showing small fibers of approximately 20 and 500 nm of width and length, respectively (Figure 2E), a morphology frequently observed in this type of aggregates. (26) The length of these aggregates was significantly shortened with respect to other examples at higher concentrations, which justifies the weakness of these gels. (27) On the other hand, agarose hydrogels showed values of viscosity about 1 order of magnitude smaller than Fmoc-AA hydrogels (Figure 2A), evidencing their extremely weak nature. Nevertheless, a slight shear thinning behavior was still observed for these hydrogels, which may be interpreted as an indication of their gel nature. This gel-like character was further confirmed by the curves of G′ and G″ as a function of the shear strain amplitude, which demonstrated similar trends to those of Fmoc-AA hydrogels, although with much lower (about 3 orders of magnitude) values of the viscoelastic moduli (Figure 2B). TEM images of agarose hydrogels showed the presence of dense meshes of fiber aggregates of higher aspect ratio than those formed by Fmoc-AA (Figure 2F).
Crystallization of insulin in agarose and Fmoc-AA hydrogels, under optimized conditions, produced homogeneous batches of small crystals (4 ± 1 μm) of very narrow size distribution (Figure 3A–C). For comparison, crystals grown without hydrogels (crystal control) were also obtained (Figure S1, Supporting Information). The size of the crystals was measured manually from scanning electron microscopy (SEM) images using only crystals with well-defined edges (Figures 3D,E, and S1 Supporting Information). As shown in Figure 3A,B, the crystals presented a rhombohedral geometry corresponding to the expected and well-described R3 space group containing two Zn atoms and six insulin molecules in the asymmetric unit, as early described by Hodgkin and co-workers in 1969. (28) This polymorph, preferred for insulin hexamers, was expected since the crystallization was induced in the presence of Zn2+. (29,30) The practical absence of morphological differences between crystals grown in hydrogels and the crystal control showed that, in this case, the hydrogel media did not influence the polymorph selection. The quality of the crystals was evaluated at a molecular level by X-ray synchrotron radiation. In spite of their small sizes, the quality of the crystals was very high, being diffracted at a resolution higher than 1.2 Å. X-ray diffraction confirmed the presence of hexameric insulin units arranged in a rhombohedral crystal polymorph. The gel fibers inside the crystals do not have a crystalline pattern and therefore cannot be detected by X-ray diffraction; neither of this contributes significantly to the X-ray background scattering.

Figure 3

Figure 3. Optical microscopy images of insulin composite crystals grown in Fmoc-AA (A) and agarose (B) hydrogels. Plot of Fmoc-AA crystal size distribution (C). SEM images of Fmoc-AA crystals at 50 μm (D) and 4 μm (E) magnification.

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Having already obtained small insulin composite crystals of high quality, i.e., low density of crystal defects, and very narrow size distribution, we evaluated their physicochemical properties in order to study the influence of the hydrogel nature. The dissolution rate of both insulin composite crystals was measured and compared to that of crystal control under physiological conditions, that is, 38 °C and pH 7.0 (Figure 4A). To evaluate the dissolution rates of the isolated composite crystals, crystals were collected and cleaned from the hydrogel media. As we can observe from Figure 4A, dissolution rates at shorter times, i.e., below 10 min, were similar for all types of crystals; however, after that initial phase, the dissolution behavior followed different paths. Thus, control and agarose crystals showed an increased dissolution rate, levelling off at 30 min, with a typical concave curve of an inhomogeneous and accelerated dissolution rate (burst effect). In contrast, Fmoc-AA crystals presented a slower dissolution rate, levelling off at 120 min. In this case, the slope of the curve was less pronounced, showing a more linear increase of insulin concentration versus time. These results showed that the nature of the hydrogel inside the protein crystals modulates the dissolution rate. Insulin composite crystals obtained in Fmoc-AA hydrogels gave rise to crystals that had a slower dissolution rate. The reason why Fmoc-AA peptide fibers produce crystals of slower dissolution rate than agarose could be explained by the formation of stronger non-covalent interactions between the peptide fibers and the protein within the crystal. Fmoc-peptides, unlike agarose, can interact with proteins not only by the formation of hydrogen bonds but also by π–π interactions through the aromatic groups. (25) We have recently shown that Fmoc-FF is able to efficiently interact and solvate carbon nanotubes and lysozyme crystals, proving its capacity to form strong aromatic interactions. (31) It has also been reported that similar short peptide fibers are able to interact with proteins to different degrees. (32,33) As previously commented, the particular conformation of polyproline II adopted by Fmoc-AA in water makes the fibers more amphiphilic, displaying on their surfaces carboxylic and Fmoc- groups and, therefore, promoting interactions with the protein through both hydrogen bonds and π–π interactions. To further investigate if this interaction occurred in solution, differential scanning calorimetry (DSC) and fluorescence measurements were performed with insulin incubated in solution with Fmoc-AA gel. In both cases, the results showed that insulin in solution did not interact with the peptide fibers, suggesting that this interaction may only occur when the crystallization conditions force the protein to nucleate and form the crystals. Under these conditions, it is known that solid particles (in this case, peptide fibers) can promote protein nucleation by acting as nucleation centers.

Figure 4

Figure 4. Characterization of insulin crystals maintained at room temperature. (A) Dissolution rate of insulin crystals. (B) Differential scanning calorimetry (DSC) of insulin samples. Transition temperatures are shown at the peaks. (C) High-performance liquid chromatography (HPLC) profiles of dissolved insulin crystals.

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Next, we analyzed the resistance to thermally induced dissolution of the crystals by DSC (Figure 4B). The crystal suspensions were prediluted with crystallization buffer to 1 mg/mL of insulin concentration. The DSC thermograms of the crystals kept at room temperature (RT) showed two peaks: a small one around 40–50 °C and a bigger one at about 70 °C. Soluble insulin dissolved at 1 mg/mL in the same crystallization buffer gave rise only to the peak at 70 °C, corresponding to the thermal unfolding of insulin under the conditions of the experiments (Figure 4B). Therefore, the first small transition must correspond to the thermally induced dissolution of the insulin crystals. This peak does not occur in a second consecutive scan, as expected, since insulin does not recrystallize at 1 mg/mL under these conditions. Interestingly, crystals grown in both hydrogels were more thermally stable than the crystal control. The higher stability of Fmoc-AA crystals can be explained in the same way as the slower release profile due to stronger interactions between the protein and peptide fibers. The area under the first peak corresponds to the heat of crystal dissolution, which was similar for the two insulin composite crystals. Insulin was also more stable against thermal denaturation in the presence of Fmoc-AA, as evidenced by the higher temperature of the unfolding transition. This suggests that the interaction gain in the crystal form is somehow maintained under the experimental conditions contributing to insulin stabilization.
The integrity of insulin in the composite crystals was determined by HPLC using a diode array detector to record the UV–vis absorption spectra (Figure 4C). Samples were fractionated by HPLC using a C18 column and a gradient method. Peaks detected at 276 nm were collected and analyzed by matrix assisted laser desorption ionization-time of flight (MALDI-ToF) mass spectrometry. A calibration curve was prepared with the soluble recombinant insulin used to prepare the composite crystals by integrating the peak with a retention time of 10 min and absorbance at 276 nm (Figures S2 and S3 for crystal control, Supporting Information). A single peak at 276 nm was observed. This peak corresponded to the insulin monomer having a mass of 5808 g/mol. A Fmoc-AA gel sample prepared as for insulin crystallization was also analyzed by HPLC showing a peak absorbing at 276 nm with a retention time of 11 min. Therefore, this method was able to clearly distinguish insulin from the dipeptide. This was further demonstrated via a sample mixture analysis. Crystal samples were then dissolved, and replicates (n = 5) were analyzed. Sharp and time-resolved insulin peaks for all the crystal samples were obtained (Figure 4C). Integration of the peak at 10 min afforded a 5.0 ± 0.5 and 4.8 ± 0.4 mg/mL insulin concentration for agarose and Fmoc-AA crystals, respectively, in good correlation with the theoretical value (5 mg/mL). Mass analysis of these peaks corresponded to the mass of the insulin monomer, in agreement with the commercial insulin used to prepare the crystals (Figure S4, Supporting Information).
Next, crystal samples were evaluated by accelerated stability studies incubating the crystal’s hydrogel suspensions at 50 °C during 7 days. It is established that incubation of crystals at 50 °C for 4 days is equivalent to a room temperature stability of 2 years. (34) Insulin in solution degrades in less than 24 h under these conditions. Optical microscopy images were taken from day 1 to day 7 in order to evaluate the quality of the crystals and to observe differences. Fmoc-AA and agarose crystals kept at 50 °C for 7 days still showed good transparency and flat faces analogous to those kept at RT (Figure 5). Additionally, to confirm the quality of the crystals, SEM images were taken (Figure 5C,D). As can be seen from the images, the morphology, quality, and size of the crystals were similar to those samples kept at RT. We next tested the stability of the crystal suspensions at 60 °C. In this case, after 24 h, agarose and control crystals dissolved to the naked eye. Optical microscopy observation showed an amorphous precipitate in the agarose crystal sample. On the contrary, Fmoc-AA crystals were stable for up to 24 h without degradation, as monitored by optical microscopy. SEM images of the Fmoc-AA crystals kept for 24 h at 60 °C showed crystals of similar quality to those kept at RT. These results, in line with those obtained in the dissolution experiments and DSC, reveal the superior stabilizing effect exerted by Fmoc-AA peptide fibers on the crystals.

Figure 5

Figure 5. Characterization of insulin crystals kept at 50 °C for 7 days. Optical microscopy images of insulin crystals grown in Fmoc-AA (A) and agarose (B) and the corresponding SEM images of Fmoc-AA (C) and agarose (D). (E) HPLC profiles of dissolved insulin crystals. (F) Differential scanning calorimetry (DSC) of insulin samples. Transition temperatures are shown at the peaks.

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HPLC analysis of Fmoc-AA and agarose crystals kept at 50 °C and Fmoc-AA kept at 60 °C showed the same clean spectra as samples kept at RT (Figure 5E). The peak corresponding to the insulin monomer appeared again at 10 min. MALDI analysis of the peak confirmed the mass (5808 g/mol) of the insulin monomer. The practical absence of other peaks suggests that crystals do not suffer a significant degradation under these conditions.
DSC was used to evaluate the resistance against thermally induced dissolution of the crystals at 1 mg/mL kept at 50 °C for 7 days. Strikingly, the pretransitions corresponding to crystal dissolution were shifted to higher temperature for both hydrogels while their peak areas became larger (Figure 5F), indicating a considerable stabilization of the crystal structure by the heat treatment. Once more, the Fmoc-AA crystals were more stable than the agarose crystals but both became stabilized by the heat treatment to a similar extent. This increase in stability suggests that the interactions between proteins and protein-gel fibers within the crystal increased or became stronger after the heat treatment. Nevertheless, the clean HPLC and mass spectra of these samples show that this change is reversible and therefore the increase in stability must be due to the noncovalent interactions.
The pharmacological effect of the novel insulin composite crystals was evaluated in vivo including samples kept at 50 and 60 °C. Native insulin in soluble form was used as a reference. Human insulin shows bioactivity in rodents and, as in humans, hypoglycemic activity is related to the concentration of insulin monomers in blood. Since the insulin molecule was the same in all preparations, the hypoglycemic response is directly related to the rate of insulin release from the injection site. In this regard, our assay is pharmacokinetic in nature. First, we conducted a dose-finding study for reference insulin, on the basis of published studies, (35−38) in order to establish a dose associated with a robust, nontoxic, and reproducible decrease in the glucose plasma level in rats (data not shown). The dose selected, 16.7 μg/kg rat (or 435 mIU/kg), produced reliably a 30–40% reduction of glycemia in these conditions (Figure 6). As expected, glucose lowering started 20–30 min after administration in all cases and lasted for a little over an hour. The vehicle groups exhibited a trend toward an increase immediately after administration, which probably reflects a physiological response to the injection, as expected in conscious animals, and is blunted in the insulin-treated groups. As shown in Figure 6A, a similar hypoglycemic response was attained when the same dose of insulin was administered in the form of agarose crystals, with no discernible differences in the starting time or overall duration. Insulin control showed the same release profile as agarose crystals. In contrast, no response was observed in the case of Fmoc-AA crystals (not shown). As this lack of biological effect suggested a markedly slower rate of release to the bloodstream, larger doses, similar to those use for slow-release insulin glargine, were tested. (39) In order to obtain an effect comparable to that of reference insulin, the dose had to be increased 5-fold (i.e., 83.7 μg/kg), as shown in Figure 6B. The effect took slightly longer to set in; therefore, the sample collection pattern was changed to adapt to the different response and also to assess the possibility of a longer duration of that effect. Note that neither the empty hydrogels nor the insulin composite crystals produce any observable adverse effect in the animal during the whole treatment.

Figure 6

Figure 6. (A) Glycemia profile of agarose crystals at 20 and 50 °C. (B) Glycemia profile of Fmoc-AA crystals at 20 and 50 °C. (C) Glycemia profile of dissolved Fmoc-AA crystals kept at 50 °C and Fmoc-AA crystals kept at 60 °C. Reference insulin (black), agarose crystals (blue), and Fmoc-AA crystals (red).

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The resistance to heat of insulin crystals grown in both hydrogels was assessed by incubation of the crystals at 50 °C during 7 days (Figure 6A,B) and, in the case of Fmoc-AA crystals, also at 60 °C for 24 h (Figure 6C). The reference was clearly shown to lose biological activity in these conditions. In turn, insulin crystals grown in both hydrogels remained fully active. Our data indicate that (1) the agarose hydrogel does not affect insulin release from the injection site; (2) the Fmoc-AA hydrogel has, on the contrary, a marked slowing effect on this critical pharmacokinetic step; (3) both hydrogels fully protect insulin against the thermal challenge; and (4) neither agarose nor Fmoc-AA lowers glycemia by itself. Thus, in both instances, hydrogels may be useful to improve the stability of insulin and potentially other therapeutic proteins. As a rule, therapeutic proteins must be kept from high temperatures and handled adequately to avoid deterioration, which not only may reduce the therapeutic effect (as was the case here with reference insulin) but also may lead to enhanced immunogenicity. (14,34)
On the other hand, it is interesting to consider the different impacts of the hydrogels on the insulin monomer release, as assessed by the resulting hypoglycemic response. Fmoc-AA had a major effect at this level, as opposed to agarose, which was neutral. The slower release profile in vivo of insulin grown in Fmoc-AA hydrogels can be correlated with the slower dissolution rate in near-physiological conditions and can be justified, as commented earlier, by the stabilizing effect exerted on the crystals by this peptide. This effect could be compensated by increasing the insulin dose. With 5-fold higher levels than initially tested, the effect was comparable to the reference insulin in solution, although the effect onset tended to be somewhat slower. Even so, the duration of the effect was never consistently higher, even with higher concentrations/doses. The reason for this observation is still unclear, but it is likely that part of the insulin–gel composites release very slowly after the first 3–4 h. Such minimal input to the bloodstream insulin thus may be matched by hepatic catabolism, resulting in a lack of effect. This would explain the results obtained with the initial 16.7 μg/kg dose. To demonstrate the slow-release effect exerted by Fmoc-AA peptide over the crystals and the preservation of the biological activity of the crystals, we tested a soluble insulin formulation prepared by dissolving Fmoc-AA crystals that had been kept at 50 °C during 7 days. As can be seen in Figure 6C, the 1-fold injection produced a similar pharmacological effect in terms of hypoglycemic response and onset time as that for native insulin in soluble form used as a reference. This result showed that Fmoc-AA modifies the rate of release of insulin into the bloodstream without altering its pharmacological activity.
Finally, the toxicity of the novel insulin composite crystals was tested in two different cell lines, namely Caco 2T and IEC18 cells (Figure 7A,B), which have an intestinal epithelial phenotype. Insulin composite crystals had no effect on lactate dehydrogenase (LDH) release, an index of cytotoxicity, up to a concentration of 100 μg/mL, and produced no discernible changes in cells by phase-contrast microscopy (Figure S5, Supporting Information). Thus, these crystals are nontoxic at the concentrations attained after subcutaneous injection.

Figure 7

Figure 7. In vitro cytotoxic assay: Caco 2T (A) and IEC18 (B). Cells were submitted to increasing concentrations of Fmoc-AA hydrogel (1–1000 μg/mL) kept at 20 or 50 °C for 7 days. Toxicity was measured by LDH cytotoxicity assay following the manufacturer’s instructions.

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Conclusions

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Insulin crystals grown in agarose and Fmoc-AA hydrogels have been successfully obtained in homogeneous batches compatible with in vivo subcutaneous administration, that is, samples containing a very narrow size distribution of small crystals (less than 10 μm diameter) free of any toxic additive. The physicochemical evaluation of both composite crystals has shown that these crystals are much more stable than insulin in solution, being pharmacologically active after keeping them at 50 °C during 7 days. In addition, insulin crystals grown in Fmoc-AA hydrogels showed an enhanced thermal stability, being stable up to 60 °C during 24 h. Significantly, this increase in stability also modifies the release profile of native insulin, turning it into a slow-release one, without altering the chemical structure of the protein. These results show that the nature of the hydrogel has a major impact on the physicochemical properties of the resulting crystals. Herein we have presented two novel thermally stable insulin formulations having different release profiles using the same native insulin. The capacity of tuning and improving the physicochemical properties of therapeutic proteins by simply crystallizing them in different hydrogels offers the possibility to expand the therapeutic window of novel biopharmaceutical formulations.
Insulin has been used in this work as a proof of concept; nevertheless, we are convinced that other therapeutic proteins can also be benefited from this approach. Thus, this work anticipates the potential advantages of using protein composite crystals for therapeutic purposes.

Experimental Section

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Hydrogel Characterization

Transmission Electron Microscopy

Transmission electron microscopy of dried gels (xerogels) was conducted with a LIBRA 120 PLUS (Carl Zeiss). Hydrogels were vortexed and diluted twice with water. A drop of the fiber suspension obtained was placed on a 300-mesh copper grid and stained with uranyl acetate negative stain. The sample was dried at room temperature for 1h.

Circular Dichroism

The circular dichroism of Fmoc-AA hydrogels was recorded using the J-815 spectrophotometer of Jasco with a xenon lamp of 150 W. The mixtures were jellified into a 0.1 mm quartz cell (Hellma 0.1 mm quartz SuprasilR) using the protocols described below. Spectra were obtained from 200 to 350 nm with a 1 nm step and 0.5 s integration time per step at 25 °C. The data shown correspond to the average of 100 accumulations.

Rheological Characterization

For this, we used a Haake Mars III-controlled stress rheometer (Thermo Fisher Scientific, Waltham, MA), provided with a double cone-plate sensor of 60 mm diameter, 2° apex angle, 0.088 mm truncation, and made of titanium (sensor DC60/2° Ti L). In measurements, the gap between the double cone and the plate of the rheometer was equal to the truncation of the cone (0.088 mm), and a sample volume of 5.4 mL was required for this sensor. We measured the viscosity using a logarithmic ramp of shear rates. Separately, we obtained the storage (G′) and loss (G″) moduli of the samples by subjecting them to oscillatory shear strains of logarithmically increasing amplitude and a fixed frequency of 1 Hz. Three different samples were measured to ensure the statistical significance of the results. The mean values and standard deviations of each magnitude are provided in this work.

Crystal’s Analysis

Crystallization Protocol

Human insulin, supplied by Sigma Aldrich (ref 11376497001 ROCHE), was dissolved in 20 mM HCl and the concentration was determined spectrophotometrically at 276 nm using an extinction coefficient (ε) of 1.04 mL/(mg cm). To obtain insulin crystals, we used the batch method by following a well-established sequential protocol consisting of the mix of the mother insulin solution (final concentration: 5 mg/mL), with ZnCl2 (final: 5.0 mM), sodium citrate, pH 7.0 (final: 22.0 mM) and water to a final volume of 200 μL in a 1.5 mL Eppendorf tube. (30) To include the hydrogel, pre-warmed agarose solution at 0.5% (w/v) or Fmoc-AA dissolved in dimethyl sulfoxide (DMSO) (final concentration: 5% v/v) was added as the last step to produce the hydrogel at a final concentration of 0.05 and 0.2% w/v for agarose and Fmoc-AA, respectively. An identical protocol was used to assay the different gel concentrations initially tested. Samples were incubated at 20 °C for 24 h. For thermal stability evaluation, the samples were kept in an incubator set at 50 °C for a week or 24 h at 60 °C.

Dissolution Experiments

For the dissolution experiments, insulin crystals grown in both hydrogels were moved into the bulk solution with the help of a cat whisker and those crystals sticking to the wall were gently detached. Eppendorf tubes were then centrifuged at 11 000 rpm. The precipitant solution was replaced by PBS buffer, pH 7.4, prewarmed at 38 °C. The crystals were gently resuspended manually, and the tubes were shaken at 300 rpm and at 38 °C in a thermoshaker. Solution aliquots were taken at 2, 5, 10, 15, 20, 30, and 60 min. Tube samples were centrifuged at 11 000 rpm for 30 s, and 15 μL volume was extracted from the supernatant and diluted by adding 300 μL of water. Insulin in the solution was determined spectrophotometrically.

Differential Scanning Calorimetry

DSC experiments were carried out in a DASM-4 microcalorimeter equipped with capillary platinum cells at a scan rate of 2 °C/min. Crystal suspensions were readily diluted with crystallization buffer to a 1 mg/mL insulin concentration and immediately loaded into the calorimeter cell. The reference cell was loaded with the crystallization buffer. The samples were scanned up to 100 °C, cooled, and rescanned to check the reversibility of the thermograms. The experimental thermograms were corrected using a baseline obtained with both cells filled with crystallization buffer and normalized with the nominal insulin concentration. Then, the intrinsic heat capacity dependence was subtracted from the thermograms to give the excess heat capacity profiles.

HPLC Analysis

HPLC analyses were performed on an Agilent 1260 infinity. Analytical HPLC analyses were performed with an Agilent Poroshell 120 EC-C18, 2.7 μm, 50 mm × 4.6 mm column. Detection was by UV absorbance at 276 nm. The following eluents were used: (A) H2O + 0.1% trifluoroacetic acid (TFA); (B) MeCN + 0.1% TFA HPLC grade eluents, from 5 to 60% of MeCN in 8 min, and then from 60 to 95% of MeCN in 3 min, employed at a flow rate of 1 mL/min and filtered with a 0.2 μm filter prior to injection. The following HPLC method was used. The injection volume was 10 μL and detection wavelength was 276 nm. All experiments were conducted at 45 °C and all samples were filtered prior to injection. Crystal samples were diluted five times by adding 20 mM HCl and shaken at 1000 rpm for 45 min at 35 °C in a thermoshaker. Insulin solutions of known concentrations, 3, 8, 25, 75 and 225 μM, in 20 mM HCl were injected to obtain the calibration curve as a function of the total area of the peak (insulin peak) that absorbs at 276 nm and 10 min retention time. To quantify the insulin concentration in all HPLC experiments, the insulin peak total area was interpolated and a dilution factor of 1:5 was applied.

Mass Spectrometry

Mass spectra were recorded on a Bruker AutoFlex MALDI-ToF MS using a weight range from 1800 to 10 000 Da. Spectra were acquired for each sample in a positive ionization reflector mode (delay 270 ns, 19 kV accelerating voltage, variable laser intensity, typically more than 200 shots). Twenty microliters of sinapinic acid matrix (saturated solution of sinapinic acid in acetonitrile/0.1% TFA in water, 1:2) was mixed with 2 μL of sample. Two microliters of the resulting mixture was spotted onto the Bruker 384 stainless-steel MALDI-ToF plate for analysis in a Bruker MALDI-ToF Autoflex.

Scanning Electron Microscopy

Crystal samples were cross-linked by adding a commercial aqueous solution of glutaraldehyde 25% (v/v) (Sigma-Aldrich) to reach a final concentration of 5% (v/v). The mixture was homogenized with a pipette and allowed to cross-link by incubation at 20 °C for 24 h. The crystals were gently centrifugated, and the supernatant was removed and substituted by Milli-Q water. The process was repeated five times. Five microliters of the obtained aqueous cross-linked crystal suspension was manually extended on top of a carbon adhesive surface attached to an SEM pin stud and allowed to dry at room temperature overnight. The samples were then coated with a fine carbon layer and examined by SEM using a FEI Quanta 400 ESEM equipment. Crystal size distribution was measured from the SEM images using ImageJ 1.47 software. The final result was expressed as the average size of 100 crystals per sample. In each analyzed image, all crystals with at least one defined face were measured by taking the diagonal length of the biggest exposed face.

In Vivo Assays

All animal procedures in this study were carried out in accordance with the existing guidelines and were approved by the Animal Welfare Committee registry number CEEA 2011-354. Male Wistar rats of approximately 8 weeks obtained from Janvier Labs (Le Genest-Saint-Isle, France) were used. Animals were maintained at the Unit of Animal Research Biomedical Research Center, University of Granada, Granada, Spain, in specific pathogen-free conditions with free access to autoclaved tap water and food Harlan-Teklad 2014, Harlan Ibérica, Barcelona, Spain.

In Vivo Experiments

Rats were fasted overnight, and a sample for basal glycemia determination was obtained. Rats were then administered subcutaneously an insulin preparation or vehicle, namely, standard human insulin, agarose, and Fmoc-AA crystals, or the respective vehicle. The samples were subjected to 30 min of UV radiation for sterilization purposes. Insulin preparations were subjected to incubation at 20 or 50 °C for 1 week and 60 °C for 24 h in order to test the thermostability. Blood samples were obtained at 0.5, 1, 2, 4, 6, and 8 h after administration of Fmoc-AA crystals, or at 20, 40, 60, 90, 120, and 180 min after administration of agarose crystals, owing to the different time response (n = 10 except n = 7 for native insulin with thermal stress and n = 4 for the groups receiving gel without insulin). Blood samples were drawn from a little incision of the tip of the rat tails and the blood was spun; the plasma was stored at −80 °C until assayed for glucose levels.

Measurement of Glucose Levels

Glucose plasma levels were measured using the enzymatic kit from SpinReact (ref 1001192) following the manufacturer’s instructions.

In Vivo Data and Statistical Analysis

Samples were run at least in triplicate and the results are expressed as mean ± standard error of the mean (SEM). Differences among means were tested for statistical significance by two-way analysis of variance (ANOVA) and a posteriori Fisher’s least significant difference tests on preselected pairs. All analyses were carried out with the GraphPad Prism 6 (La Jolla, CA). Differences were considered significant at P < 0.05.

Toxicological Studies

IEC18 (passages 56–61) and Caco 2T cells (passages 65–70) were cultured in standard conditions to confluence in 96-well plates, then exposed to the composite crystals for 16 h in fresh culture medium (Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, 100 IU/mL penicillin, 0.1 mg/mL streptomycin, 25 μg/mL Fungizone (Gibco), and 2 mM l-glutamine, Sigma). LDH activity was measured in the supernatant using Pierce cytotoxicity kits (ref 88954), while cell appearance was documented with an Olympus CKX41 microscope.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsami.1c00639.

  • SEM images of all insulin crystals samples (Figure S1); HPLC calibration curve (Figure S2); HPLC analysis of control crystals (Figure S3); mass spectra of all samples (Figure S4); contrast phase microscopy of the two cell lines used to study the cytotoxicity (Figure S5) (PDF)

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Author Information

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  • Corresponding Authors
    • José A. Gavira - Laboratorio de Estudios Cristalográficos, Instituto Andaluz de Ciencias de la Tierra (Consejo Superior de Investigaciones Científicas-UGR), Avenida de las Palmeras 4, Armilla, 18100 Granada, Spain;  Orcidhttp://orcid.org/0000-0002-7386-6484;  Email: [email protected]
    • Luis Álvarez de Cienfuegos - Departamento de Química Orgánica, Universidad de Granada, (UGR), C. U. Fuentenueva, Avda. Severo Ochoa s/n, E-18071 Granada, Spain;  Instituto de Investigación Biosanitaria ibs.GRANADA, 18014 Granada, Spain;  Orcidhttp://orcid.org/0000-0001-8910-4241;  Email: [email protected]
  • Authors
    • Rafael Contreras-Montoya - Departamento de Química Orgánica, Universidad de Granada, (UGR), C. U. Fuentenueva, Avda. Severo Ochoa s/n, E-18071 Granada, Spain;  Instituto de Investigación Biosanitaria ibs.GRANADA, 18014 Granada, Spain;  Orcidhttp://orcid.org/0000-0002-0766-004X
    • María Arredondo-Amador - Departamento de Farmacología, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), School of Pharmacy, Instituto de Investigación Biosanitaria ibs.GRANADA, University of Granada, 18071 Granada, Spain
    • Guillermo Escolano-Casado - Laboratorio de Estudios Cristalográficos, Instituto Andaluz de Ciencias de la Tierra (Consejo Superior de Investigaciones Científicas-UGR), Avenida de las Palmeras 4, Armilla, 18100 Granada, Spain
    • Mari C. Mañas-Torres - Departamento de Química Orgánica, Universidad de Granada, (UGR), C. U. Fuentenueva, Avda. Severo Ochoa s/n, E-18071 Granada, Spain;  Instituto de Investigación Biosanitaria ibs.GRANADA, 18014 Granada, Spain
    • Mercedes González - Departamento de Farmacología, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), School of Pharmacy, Instituto de Investigación Biosanitaria ibs.GRANADA, University of Granada, 18071 Granada, Spain
    • Mayte Conejero-Muriel - Laboratorio de Estudios Cristalográficos, Instituto Andaluz de Ciencias de la Tierra (Consejo Superior de Investigaciones Científicas-UGR), Avenida de las Palmeras 4, Armilla, 18100 Granada, Spain
    • Vaibhav Bhatia - Lamark Biotech Pvt. Ltd., VIT-TBI, 632 014 Vellore, Tamil Nadu, India
    • Juan J. Díaz-Mochón - Departamento de Química Farmacéutica y Orgánica, Facultad de Farmacia, UGR, 18011 Granada, Spain;  Centre for Genomics and Oncological Research, Pfizer/University of Granada/Andalusian Regional Government, PTS Granada, Avenida de la Ilustración 114, 18016 Granada, Spain
    • Olga Martínez-Augustin - Departamento de Bioquímica y Biología Molecular II, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), School of Pharmacy, Instituto de Investigación Biosanitaria ibs.GRANADA, University of Granada, 18071 Granada, Spain;  Orcidhttp://orcid.org/0000-0001-8291-3468
    • Fermín Sánchez de Medina - Departamento de Farmacología, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), School of Pharmacy, Instituto de Investigación Biosanitaria ibs.GRANADA, University of Granada, 18071 Granada, Spain;  Orcidhttp://orcid.org/0000-0002-4516-5824
    • Modesto T. Lopez-Lopez - Departamento de Física Aplicada, Facultad de Ciencias, UGR, C. U. Fuentenueva, Avda. Severo Ochoa s/n, E-18071 Granada, Spain;  Instituto de Investigación Biosanitaria ibs.GRANADA, 18014 Granada, Spain;  Orcidhttp://orcid.org/0000-0002-9068-7795
    • Francisco Conejero-Lara - Departamento de Química Física, Facultad de Ciencias, UGR, C. U. Fuentenueva, Avda. Severo Ochoa s/n, E-18071 Granada, Spain;  Orcidhttp://orcid.org/0000-0002-8282-2168
  • Author Contributions

    R.C.-M. and M.A.-A. contributed equally to this work J.A.G. and L.A.C. conceived the idea and designed the crystallization experiments. O.M.-A. and F.S.M. designed the in vivo experiments. R.C.-M., G.E.-C., M.C.M.-T., and M.C.-M. did the crystallization experiments. M.T.L.-L. did the rheological experiments and interpreted the data. F.C.-L. did the DSC experiments and interpreted the data. J.J.D.-M. analyzed the HPLC and mass spectrometry data. M.A.-A. and M.G. did the in vivo experiments. O.M.-A., F.S.M., J.J.D.-M., V.B., M.T.L.-L., F.C.-L., J.A.G., and L.A.C. discussed the results and contributed to draft writing and revision. All authors read and approved the final manuscript.

  • Funding

    The research leading to these results has received funding from the “la Caixa” Banking Foundation CaixaImpulse program, EIT-Health PocPlus program, and the Ministry of Economy and Competitiveness of Spain, acknowledged through the following projects: BIO2016-74875-P, BFU2014-57736-P, AGL2014-58883-R, SAF2017-88457-R, AGL2017-85270-R, and FIS2017-85954-R co-funded by Fondo Europeo de Desarrollo Regional, ERDF, European Union, and FEDER/Junta de Andalucía-Consejería de Transformación Económica, Industria, Conocimiento y Universidades (Spain) projects P18-FR-3533, P12-FQM-2721, P12-FQM-790, CTS235, and CTS164. M.A.-A. and M.C.M.-T. were supported by fellowships from the Ministry of Education. CIBERehd is funded by Instituto de Salud Carlos III.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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The authors thank “Unidad de Excelencia Química aplicada a Biomedicina y Medioambiente” of the University of Granada. We also thank Dr. Juan José Guardia-Monteagudo for his help in performing HPLC and mass measurements. Thanks are also due to the CIC personnel of the University of Granada for technical assistance.

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    Journal of Materials Chemistry (2010), 20 (26), 5371-5375CODEN: JMACEP; ISSN:0959-9428. (Royal Society of Chemistry)
    We have developed an injectable hydrogel system composed of biodegradable polymer-phenol conjugates that are covalently crosslinked by an enzyme-mediated oxidative reaction. The oxidative reaction of hydrogen peroxide (H2O2) and horseradish peroxidase (HRP) catalyzed the crosslinking of the phenol moieties and allowed the independent tuning of the mech. strength and gelation rate. This injectable hydrogel system with an independent tuning property has great potential for controlled drug delivery as well as three dimensional (3D) cell culture and differentiation in the hydrogel.
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  10. 10
    Koshy, S. T.; Zhang, D. K. Y.; Grolman, J. M.; Stafford, A. G.; Mooney, D. J. Injectable Nanocomposite Cryogels for Versatile Protein Drug Delivery. Acta Biomater. 2018, 65, 36– 43,  DOI: 10.1016/j.actbio.2017.11.024
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    10
    Injectable nanocomposite cryogels for versatile protein drug delivery
    Koshy, Sandeep T.; Zhang, David K. Y.; Grolman, Joshua M.; Stafford, Alexander G.; Mooney, David J.
    Acta Biomaterialia (2018), 65 (), 36-43CODEN: ABCICB; ISSN:1742-7061. (Elsevier Ltd.)
    Sustained, localized protein delivery can enhance the safety and activity of protein drugs in diverse disease settings. While hydrogel systems are widely studied as vehicles for protein delivery, they often suffer from rapid release of encapsulated cargo, leading to a narrow duration of therapy, and protein cargo can be denatured by incompatibility with the hydrogel crosslinking chem. In this work, we describe injectable nanocomposite hydrogels that are capable of sustained, bioactive, release of a variety of encapsulated proteins. Injectable and porous cryogels were formed by bio-orthogonal crosslinking of alginate using tetrazine-norbornene coupling. To provide sustained release from these hydrogels, protein cargo was pre-adsorbed to charged Laponite nanoparticles that were incorporated within the walls of the cryogels. The presence of Laponite particles substantially hindered the release of a no. of proteins that otherwise showed burst release from these hydrogels. By modifying the Laponite content within the hydrogels, the kinetics of protein release could be precisely tuned. This versatile strategy to control protein release simplifies the design of hydrogel drug delivery systems. Here we present an injectable nanocomposite hydrogel for simple and versatile controlled release of therapeutic proteins. Protein release from hydrogels often requires first entrapping the protein in particles and embedding these particles within the hydrogel to allow controlled protein release. This pre-encapsulation process can be cumbersome, can damage the protein's activity, and must be optimized for each protein of interest. The strategy presented in this work simply premixes the protein with charged nanoparticles that bind strongly with the protein. These protein-laden particles are then placed within a hydrogel and slowly release the protein into the surrounding environment. Using this method, tunable release from an injectable hydrogel can be achieved for a variety of proteins. This strategy greatly simplifies the design of hydrogel systems for therapeutic protein release applications.
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  11. 11
    Ferber, S.; Behrens, A. M.; McHugh, K. J.; Rosenberg, E. M.; Linehan, A. R.; Sugarman, J. L.; Jayawardena, H. S. N.; Langer, R.; Jaklenec, A. Evaporative Cooling Hydrogel Packaging for Storing Biologics Outside of the Cold Chain. Adv. Healthcare Mater. 2018, 7, 1800220  DOI: 10.1002/adhm.201800220
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    There is no corresponding record for this reference.
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    Meis, C. M.; Salzman, E. E.; Maikawa, C. L.; Smith, A. A. A.; Mann, J. L.; Grosskopf, A. K.; Appel, E. A. Self-Assembled, Dilution-Responsive Hydrogels for Enhanced Thermal Stability of Insulin Biopharmaceuticals. ACS Biomater. Sci. Eng. 2020, 01306  DOI: 10.1021/acsbiomaterials.0c01306
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    There is no corresponding record for this reference.
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    Basu, S. K.; Govardhan, C. P.; Jung, C. W.; Margolin, A. L. Protein Crystals for the Delivery of Biopharmaceuticals. Expert Opin. Biol. Ther. 2004, 4, 301– 317,  DOI: 10.1517/14712598.4.3.301
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    Protein crystals for the delivery of biopharmaceuticals
    Basu, Sujit K.; Govardhan, Chandrika P.; Jung, Chu W.; Margolin, Alexey L.
    Expert Opinion on Biological Therapy (2004), 4 (3), 301-317CODEN: EOBTA2; ISSN:1471-2598. (Ashley Publications Ltd.)
    A review and discussion. The year 2002 marked the 20th anniversary of the first successful product of modern biotechnol., the regulatory approval of recombinant insulin for biopharmaceutical applications. Insulin is also the first cryst. protein to be approved for therapeutic use. Over the past two decades, almost 150 biopharmaceuticals have gained marketing authorization; however, insulin remains the only cryst. protein on the market. Significant research and development efforts have focused on the engineering of protein mols., efficacy testing, model development, and protein prodn. and characterization. These advances have dramatically boosted the therapeutic applications of proteins, which now include treatments against acute conditions, such as cancer, cardiovascular disease and viral disease, and chronic conditions, such as diabetes, growth hormone deficiency, haemophilia, arthritis, psoriasis and Crohn's disease. Despite these successes, many challenges normally assocd. with biopharmaceuticals, such as poor stability and limited delivery options, remain. Protein crystals have shown significant benefits in the delivery of biopharmaceuticals to achieve high concn., low viscosity formulation and controlled release protein delivery. This review discusses challenges related to the broader utilization of protein crystals in biopharmaceutical applications, as well as recent advances and valuable new directions that protein crystn.-based technologies present.
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  14. 14
    Yang, M. X.; Shenoy, B.; Disttler, M.; Patel, R.; McGrath, M.; Pechenov, S.; Margolin, A. L. Crystalline Monoclonal Antibodies for Subcutaneous Delivery. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6934– 6939,  DOI: 10.1073/pnas.1131899100
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    Crystalline monoclonal antibodies for subcutaneous delivery
    Yang, Mark X.; Shenoy, Bhami; Disttler, Matthew; Patel, Reena; McGrath, Margaret; Pechenov, Sergey; Margolin, Alexey L.
    Proceedings of the National Academy of Sciences of the United States of America (2003), 100 (12), 6934-6939CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Therapeutic applications for mAbs have increased dramatically in recent years, but the large quantities required for clin. efficacy have limited the options that might be used for administration and thus have placed certain limitations on the use of these agents. We present an approach that allows for s.c. delivery of a small vol. of a highly concd. form of mAbs. Batch crystn. of three Ab-based therapeutics, rituximab, trastuzumab, and infliximab, provided products in high yield, with no detectable alteration to these proteins and with full retention of their biol. activity in vitro. Administration s.c. of a cryst. prepn. resulted in a remarkably long pharmacokinetic serum profile and a dose-dependent inhibition of tumor growth in nude mice bearing BT-474 xenografts (human breast cancer cells) in vivo. Overall, this approach of generating high-concn., low-viscosity cryst. prepns. of therapeutic Abs should lead to improved ease of administration and patient compliance, thus providing new opportunities for the biotechnol. industry.
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  15. 15
    Lorber, B.; Sauter, C.; Théobald-Dietrich, A.; Moreno, A.; Schellenberger, P.; Robert, M. C.; Capelle, B.; Sanglier, S.; Potier, N.; Giegé, R. Crystal Growth of Proteins, Nucleic Acids, and Viruses in Gels. Prog. Biophys. Mol. Biol. 2009, 101, 13– 25,  DOI: 10.1016/j.pbiomolbio.2009.12.002
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    Crystal growth of proteins, nucleic acids, and viruses in gels
    Lorber, Bernard; Sauter, Claude; Theobald-Dietrich, Anne; Moreno, Abel; Schellenberger, Pascale; Robert, Marie-Claire; Capelle, Bernard; Sanglier, Sarah; Potier, Noelle; Giege, Richard
    Progress in Biophysics & Molecular Biology (2009), 101 (1/3), 13-25CODEN: PBIMAC; ISSN:0079-6107. (Elsevier Ltd.)
    A review. Medium-sized single crystals with perfect habits and no defect producing intense and well-resolved diffraction patterns are the dream of every protein crystallographer. Crystals of biol. macromols. possessing these characteristics can be prepd. within a medium in which mass transport is restricted to diffusion. Chem. gels (like polysiloxane) and phys. gels (such as agarose) provide such an environment and are therefore suitable for the crystn. of biol. macromols. Instructions for the prepn. of each type of gel are given to urge crystal growers to apply diffusive media for enhancing crystallog. quality of their crystals. Examples of quality enhancement achieved with silica and agarose gels are given. Results obtained with other substances forming gel-like media (such as lipidic phases and cellulose derivs.) are presented. Finally, the use of gels in combination with capillary tubes for counter-diffusion expts. is discussed. Methods and techniques implemented with proteins can also be applied to nucleic acids and nucleoprotein assemblies such as viruses.
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  16. 16
    Gavira, J. A.; Van Driessche, A. E. S.; Garcia-Ruiz, J. M. Growth of Ultrastable Protein-Silica Composite Crystals. Cryst. Growth Des. 2013, 13, 2522– 2529,  DOI: 10.1021/cg400231g
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    Growth of Ultrastable Protein-Silica Composite Crystals
    Gavira, Jose A.; Van Driessche, Alexander E. S.; Garcia-Ruiz, Juan-Ma
    Crystal Growth & Design (2013), 13 (6), 2522-2529CODEN: CGDEFU; ISSN:1528-7483. (American Chemical Society)
    Protein crystals were obtained in a wide range of silica gel concns., 2.0-22.0% (vol./vol.), using the counter-diffusion technique. The protein crystal lattice incorporates silica fibers during their growth, making the crystal appear optically translucent while maintaining the diffraction quality. The effect of the silica fibers on the nucleation and growth morphol. is discussed, and the amt. of incorporated silica matrix is quantified. The practical implications of the presence of a high hygroscope phase on the crystal properties are discussed, and the improvement of the mech. properties and stability of the crystals is shown. These reinforced protein crystals, able to include large amts. of silica, open a new range of possibilities for the characterization of protein crystals and the application in the biotechnol. industry.
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  17. 17
    Gavira, J. A.; Cera-Manjarres, A.; Ortiz, K.; Mendez, J.; Jimenez-Torres, J. A.; Patiño-Lopez, L. D.; Torres-Lugo, M. Use of Cross-Linked Poly(Ethylene Glycol)-Based Hydrogels for Protein Crystallization. Cryst. Growth Des. 2014, 14, 3239– 3248,  DOI: 10.1021/cg401668z
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    17
    Use of Cross-Linked Poly(ethylene glycol)-Based Hydrogels for Protein Crystallization
    Gavira, Jose A.; Cera-Manjarres, Andry; Ortiz, Katia; Mendez, Janet; Jimenez-Torres, Jose A.; Patino-Lopez, Luis D.; Torres-Lugo, Madeline
    Crystal Growth & Design (2014), 14 (7), 3239-3248CODEN: CGDEFU; ISSN:1528-7483. (American Chemical Society)
    Poly(ethylene glycol) (PEG) hydrogels are highly biocompatible materials extensively used for biomedical and pharmaceutical applications, controlled drug release, and tissue engineering. In this work, PEG crosslinked hydrogels, synthesized under various conditions, were used to grow lysozyme crystals by the counterdiffusion technique. Crystn. expts. were conducted using a three-layer arrangement. Results demonstrated that PEG fibers were incorporated within lysozyme crystals controlling the final crystal shape. PEG hydrogels also induced the nucleation of lysozyme crystals to a higher extent than agarose. PEG hydrogels can also be used at higher concns. (20-50% wt./wt.) as a sepn. chamber (plug) in counterdiffusion expts. In this case, PEG hydrogels control the diffusion of the crystn. agent and therefore may be used to tailor the supersatn. to fine-tune crystal size. As an example, insulin crystals were grown in 10% (wt./wt.) PEG hydrogel. The resulting crystals were of an approx. size of 500 μm.
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  18. 18
    Conejero-Muriel, M.; Contreras-Montoya, R.; Díaz-Mochón, J. J.; Álvarez de Cienfuegos, L.; Gavira, J. A. Protein Crystallization in Short-Peptide Supramolecular Hydrogels: A Versatile Strategy towards Biotechnological Composite Materials. CrystEngComm 2015, 17, 8072– 8078,  DOI: 10.1039/C5CE00850F
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    18
    Protein crystallization in short-peptide supramolecular hydrogels: a versatile strategy towards biotechnological composite materials
    Conejero-Muriel, Mayte; Contreras-Montoya, Rafael; Diaz-Mochon, Juan J.; Alvarez de Cienfuegos, Luis; Gavira, Jose A.
    CrystEngComm (2015), 17 (42), 8072-8078CODEN: CRECF4; ISSN:1466-8033. (Royal Society of Chemistry)
    Protein crystn. in hydrogels has been explored with the main purpose of facilitating the growth of high quality crystals while increasing their size to enhance their manipulation. New avenues are currently being built for the use of protein crystals as source materials to create sensors and drug delivery vehicles, to name just a few. In this sense, short-peptide supramol. hydrogels may play a crucial role in integrating protein crystals within a wider range of applications. In this article, we show that protein crystn. in short-peptide supramol. hydrogels is feasible and independent of the type of peptide that forms the hydrogel and(or) the protein, although the output is not always the same. As a general trend, it is confirmed that hydrogel fibers are always incorporated within crystals so that novel composite materials for biotechnol. applications with enhanced properties are produced.
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  19. 19
    Conejero-Muriel, M.; Gavira, J. A.; Pineda-Molina, E.; Belsom, A.; Bradley, M.; Moral, M.; García López Durán, J. D. D.; Luque González, A.; Díaz-Mochón, J. J.; Contreras-Montoya, R.; Martínez-Peragón, Á.; Cuerva, J. M.; Álvarez de Cienfuegos, L. Influence of the Chirality of Short Peptide Supramolecular Hydrogels in Protein Crystallogenesis. Chem. Commun. 2015, 51, 3862– 3865,  DOI: 10.1039/c4cc09024a
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    Influence of the chirality of short peptide supramolecular hydrogels in protein crystallogenesis
    Conejero-Muriel, Mayte; Gavira, Jose A.; Pineda-Molina, Estela; Belsom, Adam; Bradley, Mark; Moral, Monica; Duran, Juan de Dios Garcia-Lopez; Luque Gonzalez, Angelica; Diaz-Mochon, Juan J.; Contreras-Montoya, Rafael; Martinez-Peragon, Angela; Cuerva, Juan M.; Alvarez de Cienfuegos, Luis
    Chemical Communications (Cambridge, United Kingdom) (2015), 51 (18), 3862-3865CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    For the first time the influence of the chirality of the gel fibers in protein crystallogenesis has been studied. Enantiomeric hydrogels 1 and 2 were tested with model proteins lysozyme and glucose isomerase and a formamidase extd. from B. cereus. Crystn. behavior and crystal quality of these proteins in both hydrogels are presented and compared.
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  20. 20
    Escolano-Casado, G.; Contreras-Montoya, R.; Conejero-Muriel, M.; Castellví, A.; Juanhuix, J.; Lopez-Lopez, M. T.; Álvarez de Cienfuegos, L.; Gavira, J. A. Extending the Pool of Compatible Peptide Hydrogels for Protein Crystallization. Crystals 2019, 9, 244  DOI: 10.3390/cryst9050244
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    20
    Extending the pool of compatible peptide hydrogels for protein crystallization
    Escolano-Casado, Guillermo; Contreras-Montoya, Rafael; Conejero-Muriel, Mayte; Castellvi, Albert; Juanhuix, Judith; Lopez-Lopez, Modesto T.; de Cienfuegos, Luis Alvarez; Gavira, Jose A.
    Crystals (2019), 9 (5), 244CODEN: CRYSBC; ISSN:2073-4352. (MDPI AG)
    Short-peptide supramol. (SPS) hydrogels are a class of materials that have been found to be useful for (bio)technol. applications thanks to their biocompatible nature. Among the advantages reported for these peptides, their economic affordability and easy functionalization or modulation have turned them into excellent candidates for the development of functional biomaterials. It is recently demonstrated that SPS hydrogels can be used to produce high-quality protein crystals, improve their properties, or incorporate relevant materials within the crystals. In this work, it is proved that hydrogels based on methionine and tyrosine are also good candidates for growing high-quality crystals of the three model proteins: lysozyme, glucose isomerase, and thaumatin.
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  21. 21
    Contreras-Montoya, R.; Castellví, A.; Escolano-Casado, G.; Juanhuix, J.; Conejero-Muriel, M.; Lopez-Lopez, M. T.; Cuerva, J. M.; Álvarez de Cienfuegos, L.; Gavira, J. A. Enhanced Stability against Radiation Damage of Lysozyme Crystals Grown in Fmoc-CF Hydrogels. Cryst. Growth Des. 2019, 19, 4229– 4233,  DOI: 10.1021/acs.cgd.9b00131
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    21
    Enhanced Stability against Radiation Damage of Lysozyme Crystals Grown in Fmoc-CF Hydrogels
    Contreras-Montoya, Rafael; Castellvi, Albert; Escolano-Casado, Guillermo; Juanhuix, Judith; Conejero-Muriel, Mayte; Lopez-Lopez, Modesto T.; Cuerva, Juan M.; Alvarez de Cienfuegos, Luis; Gavira, Jose A.
    Crystal Growth & Design (2019), 19 (8), 4229-4233CODEN: CGDEFU; ISSN:1528-7483. (American Chemical Society)
    Lysozyme crystals grown in Fmoc-CF (Cys-Phe) hydrogels, unlike those grown in agarose, give rise to composite crystals that have an enhanced resistance against degrdn. caused by an intense exposure to X-ray irradn. Fmoc-CF dipeptide shows a clear protection of the most sensitive groups (disulfide bonds and methionines) of the protein. The protection mediated by cysteine is exerted only in its gel state since cysteine in soln. has an adverse effect. Probably, the reactive thiol groups of cysteine being locked within the rigid peptide fibers minimize cross-reactions with the proteins favoring the formation of protein crystals. Once located inside the protein crystal, the long peptide fibers are able to protect the protein against radiation damage.
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  22. 22
    Artusio, F.; Castellví, A.; Sacristán, A.; Pisano, R.; Gavira, J. A. Agarose Gel as a Medium for Growing and Tailoring Protein Crystals. Cryst. Growth Des. 2020, 20, 5564– 5571,  DOI: 10.1021/acs.cgd.0c00736
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    22
    Agarose Gel as a Medium for Growing and Tailoring Protein Crystals
    Artusio, Fiora; Castellvi, Albert; Sacristan, Anabel; Pisano, Roberto; Gavira, Jose A.
    Crystal Growth & Design (2020), 20 (8), 5564-5571CODEN: CGDEFU; ISSN:1528-7483. (American Chemical Society)
    The nucleation inducing ability of agarose gels has been exploited to study the crystn. of proteins in diffusion-dominated environments. The crystal size was successfully tuned in a wide range of gel, protein, and precipitant concns. The impact of the gel content on crystal size was independent of the specific protein, allowing the math. prediction of crystal size and pointing out the exclusivity of phys. interactions between the gel and the protein. The versatility of the technique and the fine-tuning of the nucleation flux was demonstrated by crystg. five different proteins and implementing batch and counter-diffusion crystn. In addn., the potential of agarose gel to be used not only as a growth but also as a delivery medium for serial crystallog. applications has been proven by prepg. unidimensional microcrystal slurries with 0.1% (w/v) gel. Protein crystal size was successfully tuned from a few micrometers to several hundred micrometers by performing batch crystn. in gels prepd. from variable amts. of agarose. Counter-diffusion crystn. in gel was proposed as an alternative for proteins requiring PEGs or ammonium sulfate as precipitants. The results pointed out the phys. action of agarose gels in inducing protein nucleation.
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    Tao, K.; Levin, A.; Adler-Abramovich, L.; Gazit, E. Fmoc-Modified Amino Acids and Short Peptides: Simple Bio-Inspired Building Blocks for the Fabrication of Functional Materials. Chem. Soc. Rev. 2016, 45, 3935– 3953,  DOI: 10.1039/c5cs00889a
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    23
    Fmoc-modified amino acids and short peptides: simple bio-inspired building blocks for the fabrication of functional materials
    Tao, Kai; Levin, Aviad; Adler-Abramovich, Lihi; Gazit, Ehud
    Chemical Society Reviews (2016), 45 (14), 3935-3953CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    A review. Amino acids and short peptides modified with the 9-fluorenylmethyloxycarbonyl (Fmoc) group possess eminent self-assembly features and show distinct potential for applications due to the inherent hydrophobicity and aromaticity of the Fmoc moiety which can promote the assocn. of building blocks. Given the extensive study and numerous publications in this field, it is necessary to summarize the recent progress concerning these important bio-inspired building blocks. Therefore, in this review, we explore the self-organization of this class of functional mols. from three aspects, i.e., Fmoc-modified individual amino acids, Fmoc-modified di- and tripeptides, and Fmoc-modified tetra- and pentapeptides. The relevant properties and applications related to cell cultivation, bio-templating, optical, drug delivery, catalytic, therapeutic and antibiotic properties are subsequently summarized. Finally, some existing questions impeding the development of Fmoc-modified simple biomols. are discussed, and corresponding strategies and outlooks are suggested.
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    Fleming, S.; Ulijn, R. V. Design of Nanostructures Based on Aromatic Peptide Amphiphiles. Chem. Soc. Rev. 2014, 43, 8150– 8177,  DOI: 10.1039/c4cs00247d
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    Design of nanostructures based on aromatic peptide amphiphiles
    Fleming, Scott; Ulijn, Rein V.
    Chemical Society Reviews (2014), 43 (23), 8150-8177CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    A review. Arom. peptide amphiphiles are gaining popularity as building blocks for the bottom-up fabrication of nanomaterials, including gels. These materials combine the simplicity of small mols. with the versatility of peptides, with a range of applications proposed in biomedicine, nanotechnol., food science, cosmetics, etc. Despite their simplicity, a wide range of self-assembly behaviors have been described. Due to varying conditions and protocols used, care should be taken when attempting to directly compare results from the literature. In this review, the authors rationalize the structural features which govern the self-assembly of arom. peptide amphiphiles by focusing on four segments, (i) the N-terminal arom. component, (ii) linker segment, (iii) peptide sequence, and (iv) C-terminus. It is clear that the mol. structure of these components significantly influences the self-assembly process and resultant supramol. architectures. A no. of modes of assembly have been proposed, including parallel, antiparallel, and interlocked antiparallel stacking conformations. In addn., the co-assembly arrangements of arom. peptide amphiphiles are reviewed. Overall, this review elucidates the structural trends and design rules that underpin the field of arom. peptide amphiphile assembly, paving the way to a more rational design of nanomaterials based on arom. peptide amphiphiles.
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    Mu, X.; Eckes, K. M.; Nguyen, M. M.; Suggs, L. J.; Ren, P. Experimental and Computational Studies Reveal an Alternative Supramolecular Structure for Fmoc-Dipeptide Self-Assembly. Biomacromolecules 2012, 13, 3562– 3571,  DOI: 10.1021/bm301007r
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    25
    Experimental and computational studies reveal an alternative supramolecular structure for Fmoc-dipeptide self-assembly
    Mu, Xiaojia; Eckes, Kevin M.; Nguyen, Mary M.; Suggs, Laura J.; Ren, Pengyu
    Biomacromolecules (2012), 13 (11), 3562-3571CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
    The self-assembly of fluorenylmethoxycarbonyl (Fmoc)-conjugated dialanine (Fmoc-Ala-Ala-OH) (I) mols. using combined computational and exptl. approaches. I gels were characterized using transmission electron microscopy (TEM), CD, FTIR spectroscopy, and wide-angle x-ray scattering (WAXS). Computationally, the authors simulated the assembly of I using mol. dynamics techniques. All simulations converged to a condensed fibril structure in which the Fmoc groups stacked mostly within in the center of the fibril. However, the Fmoc groups were partially exposed to water, creating an amphiphilic surface, which may be responsible for the aggregation of fibrils into nanoscale fibers obsd. in TEM. From the fibril models, radial distribution calcns. agreed with d-spacings obsd. in WAXS for the fibril diam. and π-stacking interactions. The analyses showed that dialanine, despite its short length, adopts a mainly extended polyproline II conformation. In contrast to previous hypotheses, these results indicated that β-sheet-like H-bonding was not prevalent. Rather, stacking of Fmoc groups, inter-residue H-bonding, and H-bonding with water play the important roles in stabilizing the fibril structure of supramol. assemblies of short conjugated peptides.
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  26. 26
    Smith, A. M.; Williams, R. J.; Tang, C.; Coppo, P.; Collins, R. F.; Turner, M. L.; Saiani, A.; Ulijn, R. V. Fmoc-Diphenylalanine Self Assembles to a Hydrogel via a Novel Architecture Based on π–π Interlocked β-Sheets. Adv. Mater. 2008, 20, 37– 41,  DOI: 10.1002/adma.200701221
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    Fmoc-diphenylalanine self-assembles to a hydrogel via a novel architecture based on π-π interlocked β-sheets
    Smith, Andrew M.; Williams, Richard J.; Tang, Claire; Coppo, Paolo; Collins, Richard F.; Turner, Michael L.; Saiani, Alberto; Ulijn, Rein V.
    Advanced Materials (Weinheim, Germany) (2008), 20 (1), 37-41CODEN: ADVMEW; ISSN:0935-9648. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The self-assembly of peptide hydrogelators that carry arom. substituents can be modeled by a novel nanocylindrical architecture. The proposed model suggests that the nanocylinders are formed by anti-parallel β-sheets interlocked by the π-stacking interactions of fluorenyl groups and Ph rings. This explanation is consistent with the structures obsd. in TEM and the data obtained by a variety of spectroscopic techniques.
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    Contreras-Montoya, R.; Bonhome-Espinosa, A. B.; Orte, A.; Miguel, D.; Delgado-López, J. M.; Duran, J. D. G.; Cuerva, J. M.; Lopez-Lopez, M. T.; Álvarez de Cienfuegos, L. Iron Nanoparticles-Based Supramolecular Hydrogels to Originate Anisotropic Hybrid Materials with Enhanced Mechanical Strength. Mater. Chem. Front. 2018, 2, 686– 699,  DOI: 10.1039/c7qm00573c
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    Iron nanoparticles-based supramolecular hydrogels to originate anisotropic hybrid materials with enhanced mechanical strength
    Contreras-Montoya, Rafael; Bonhome-Espinosa, Ana B.; Orte, Angel; Miguel, Delia; Delgado-Lopez, Jose M.; Duran, Juan D. G.; Cuerva, Juan M.; Lopez-Lopez, Modesto T.; Alvarez de Cienfuegos, Luis
    Materials Chemistry Frontiers (2018), 2 (4), 686-699CODEN: MCFAC5; ISSN:2052-1537. (Royal Society of Chemistry)
    Here, we report the synthesis and structural characterization of novel iron nanoparticles (FeNPs)-based short-peptide supramol. hydrogels. These hybrid hydrogels composed of Fmoc-diphenylalanine (Fmoc-FF) peptide and FeNPs were prepd. through the self-assembly of Fmoc-FF in a suspension contg. FeNPs in the presence or absence of an external magnetic field. Optical images of these hydrogels revealed the formation of column-like aggregates of FeNPs when the gels were formed in the presence of a magnetic field. Moreover, the intricate structure derived from the interwoven nature of the fiber peptides with these FeNP column-like aggregates resulted in anisotropic materials, more rigid under shear forces applied perpendicularly to the direction of the aggregates, presenting under these conditions values of G' (storage modulus) about 7 times those of the native hydrogel. To the best of our knowledge, this is the first example in which the mech. properties of peptide hydrogels were strongly enhanced due to the presence of FeNPs. A theor. model trying to explain this phenomenon is presented. Quite interesting CD, FTIR and synchrotron X-ray diffraction analyses indicated that the anti-βparallel -sheet arrangement of Fmoc-FF peptide was highly conserved in the hydrogels contg. FeNPs. Moreover, FLCS measurements showed that the diffusion of a small solute through the hydrogel network was improved in hydrogels contg. FeNPs, probably caused by the formation of preferential channels for diffusion. Taken together, our results provide a new method for the synthesis of novel hybrid Fmoc-FF-FeNPs anisotropic hydrogels with enhanced mech. strength and water-like diffusion behavior, thus easing their application in drug delivery and tissue engineering.
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    Adams, M. J.; Blundell, T. L.; Dodson, E. J.; Dodson, G. G.; Vijayan, M.; Baker, E. N.; Harding, M. M.; Hodgkin, D. C.; Rimmer, B.; Sheat, S. Structure of Rhombohedral 2 Zinc Insulin Crystals. Nature 1969, 224, 491– 495,  DOI: 10.1038/224491a0
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    Structure of rhombohedral 2 zinc insulin crystals
    Adams, Margaret Joan; Blundell, T. L.; Dodson, E. J.; Dodson, G. G.; Vijayan, M.; Baker, E. N.; Harding, M. M.; Hodgkin, D. C.; Rimmer, B.; Sheats, S.
    Nature (London, United Kingdom) (1969), 224 (5218), 491-5CODEN: NATUAS; ISSN:0028-0836.
    The 3-dimensional arrangement of pig insulin (I) was detd. in an electron d. map at 2.8 Å resolution. Heavy atoms were introduced into the rhombohedral crystal which contained 2 Zn atoms and 6 I mols./unit cell. In the electron d. map, 2 stretches of α-helix appeared. At an end of each α-helix stretch, which represented the B10 histidine, the d. was connected with 1 of the 2 peaks representing the Zn atoms on the 3-fold axis. Peptide chains and SS bonds were readily identified. All aromatic residues were well defined by individual ellipsoidal peaks. Regions with long side chains on the outside of the mol. were difficult to interpret. Two crystallographically similar independent I mols. appeared. In each mol., the B chain was wrapped around the compact A chain. The A chain was greatly folded with short stretches of almost helical conformation between residues A2-6 and A13-19. The A chain loop, A6-11, and residues A8, 9, and 10 were directedout from the surface of the mol. The SS bond, A6-11, was within the body of the mol. Both A19 and A14 tyrosines (II) appeared to be H-bonded to residues A1 and A5, resp. In the central part of the B chain, there were 3 turns of α-helix,slightly opened out at each end. Both the interchain SS bonds, B7-A7 and B19-A20, were at the ends of the helix. B1-B6 and B21-B30 were largely extended and loosely packed around the A chain. Residues A4, glutamic acid, and B29, lysine, appeared to be in contact. The closest series of contacts between 2 I mols. occurred between the extended B chains, residues B23-B28. They were arranged antiparallel and appeared to form a H-bonded pleated sheet. They surrounded the noncrystallographic 2-fold axis. An aromatic cage is formed by II B26 and phenylalanine (III) B24 and their 2-fold axis-related partners. The 2 phenyl groups at B25 formed part of the system by turning towards one another and violating the 2-fold symmetry relation. The dimer was an elongated cylinder ∼20 Å across and 40 Å long. The hexamer was a compact, oblate spheroid, formed by the coordination of 3 I dimers around the 2 Zn ions; 8.9 Å above and 8.9 Åbelow the 2-fold axis. Each Zn ion contacted the 3 B10 histidine residues and probably 3 O atoms from water. The metal coordination was 6-fold but not octahedral. The hexamer closely contacted the mols. around the 2-fold axis by the B1 III residues. A close, probably H-bonded contact occurred between the 6 glutamic acid residues B13, which enclosed a space surrounding the 3-fold axis. Many of the heavy atoms entered this space; others occupied regions on the hexamer surface, esp. at the glutamic acid residues. No heavy atoms penetrated the dimer.
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    Nanev, C. N.; Tonchev, V. D.; Hodzhaoglu, F. V. Protocol for Growing Insulin Crystals of Uniform Size. J. Cryst. Growth 2013, 375, 10– 15,  DOI: 10.1016/j.jcrysgro.2013.04.010
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    Protocol for growing insulin crystals of uniform size
    Nanev, Christo N.; Tonchev, Vesselin D.; Hodzhaoglu, Feyzim V.
    Journal of Crystal Growth (2013), 375 (), 10-15CODEN: JCRGAE; ISSN:0022-0248. (Elsevier B.V.)
    Guidelines for growing insulin crystals of a uniform size are formulated and tested exptl. A simple theor. model based on the balance of matter predicts the time evolution of the crystal size and supersatn. The time dependence of the size is checked exptl. The exptl. approach decouples crystal nucleation and growth processes according to the classical nucleation-growth-sepn. principle. The desired nucleation course was predetd. by carefully selecting the most suitable crystal nucleation parameters. Cryst. substance dispersity is predetd. during the nucleation stage of a batch crystn. process. To avert nutrition competition during the crystal growth stage, the no. d. of nucleated crystals is preset to be optimal.
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    Hodzhaoglu, F. V.; Conejero-Muriel, M.; Dimitrov, I. L.; Gavira, J. A. Optimization of the Classical Method for Nucleation and Growth of Rhombohedral Insulin Crystals by PH Titration and Screening. Bulg. Chem. Commun. 2016, 48, 29– 37
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    Contreras-Montoya, R.; Escolano, G.; Roy, S.; Lopez-Lopez, M. T.; Delgado-López, J. M.; Cuerva, J. M.; Díaz-Mochón, J. J.; Ashkenasy, N.; Gavira, J. A.; Álvarez de Cienfuegos, L. Catalytic and Electron Conducting Carbon Nanotube–Reinforced Lysozyme Crystals. Adv. Funct. Mater. 2019, 29, 1807351  DOI: 10.1002/adfm.201807351
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    Gao, Y.; Long, M. J. C.; Shi, J.; Hedstrom, L.; Xu, B. Using Supramolecular Hydrogels to Discover the Interactions between Proteins and Molecular Nanofibers of Small Molecules. Chem. Commun. 2012, 48, 8404– 8406,  DOI: 10.1039/c2cc33631f
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    Using supramolecular hydrogels to discover the interactions between proteins and molecular nanofibers of small molecules
    Gao, Yuan; Long, Marcus J. C.; Shi, Junfeng; Hedstrom, Lizbeth; Xu, Bing
    Chemical Communications (Cambridge, United Kingdom) (2012), 48 (67), 8404-8406CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    Here the authors report the first example of the use of supramol. hydrogels to discover the protein targets of aggregates of small mols.
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    Javid, N.; Roy, S.; Zelzer, M.; Yang, Z.; Sefcik, J.; Ulijn, R. V. Cooperative Self-Assembly of Peptide Gelators and Proteins. Biomacromolecules 2013, 14, 4368– 4376,  DOI: 10.1021/bm401319c
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    Cooperative Self-Assembly of Peptide Gelators and Proteins
    Javid, Nadeem; Roy, Sangita; Zelzer, Mischa; Yang, Zhimou; Sefcik, Jan; Ulijn, Rein V.
    Biomacromolecules (2013), 14 (12), 4368-4376CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
    Mol. self-assembly provides a versatile route for the prodn. of nanoscale materials for medical and technol. applications. Herein, the cooperative self-assembly of amphiphilic small mols. and proteins can have drastic effects on supramol. nanostructuring of resulting materials. The authors report that mesoscale, fractal-like clusters of proteins form at concns. that are orders of magnitude lower compared to those usually assocd. with mol. crowding at room temp. These protein clusters have pronounced effects on the mol. self-assembly of arom. peptide amphiphiles (fluorenylmethoxycarbonyl- dipeptides), resulting in a reversal of chiral organization and enhanced order through templating and binding. Moreover, the morphol. and mech. properties of the resultant nanostructured gels can be controlled by the cooperative self-assembly of peptides and protein fractal clusters, having implications for biomedical applications where proteins and peptides are both present. In addn., fundamental insights into cooperative interplay of mol. interactions and confinement by clusters of chiral macromols. is relevant to gaining understanding of the mol. mechanisms of relevance to the origin of life and development of synthetic mimics of living systems.
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    Shenoy, B.; Wang, Y.; Shan, W.; Margolin, A. L. Stability of Crystalline Proteins. Biotechnol. Bioeng. 2001, 73, 358– 369,  DOI: 10.1002/bit.1069
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    Stability of crystalline proteins
    Shenoy, Bhami; Wang, Yi; Shan, Weizhong; Margolin, Alexey L.
    Biotechnology and Bioengineering (2001), 73 (5), 358-369CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)
    By using 2 proteins, glucose oxidase and lipase, we demonstrate that dry cryst. formulations are significantly more stable than their amorphous counterparts. The results of FT-spectroscopy indicate that cryst. proteins better maintain their native conformation in accelerated stability studies. The lower tendency of cryst. proteins to aggregate is confirmed by size-exclusion chromatog. Protein crystn. may significantly improve some aspects of protein handling, and change the way biopharmaceuticals are produced, formulated, and delivered.
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    Chen, M.-C.; Ling, M.-H.; Kusuma, S. J. Poly-γ-Glutamic Acid Microneedles with a Supporting Structure Design as a Potential Tool for Transdermal Delivery of Insulin. Acta Biomater. 2015, 24, 106– 116,  DOI: 10.1016/j.actbio.2015.06.021
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    35
    Poly-γ-glutamic acid microneedles with a supporting structure design as a potential tool for transdermal delivery of insulin
    Chen, Mei-Chin; Ling, Ming-Hung; Kusuma, Setiawan Jati
    Acta Biomaterialia (2015), 24 (), 106-116CODEN: ABCICB; ISSN:1742-7061. (Elsevier Ltd.)
    Incomplete insertion is a common problem assocd. with polymer microneedles (MNs) that results in a limited drug delivery efficiency and wastage of valuable medication. This paper presents a fully insertable MN system that is composed of poly-γ-glutamic acid (γ-PGA) MNs and polyvinyl alc. (PVA)/polyvinyl pyrrolidone (PVP) supporting structures. The PVA/PVP supporting structures were designed to provide an extended length for counteracting skin deformation during insertion and mech. strength for fully inserting the MNs into the skin. When inserted into the skin, both the supporting structures and MNs can be dissolved in the skin within 4 min, thus quickly releasing the entire drug load from the MNs. To evaluate the feasibility and reproducibility of using the proposed system for treating diabetes, we administered insulin-loaded MNs to diabetic rats once daily for 2 days. The results indicated that the hypoglycemic effect in the rats receiving insulin-loaded MNs was comparable to that obsd. in rats receiving s.c. insulin injections. The relative pharmacol. availability and relative bioavailability of the insulin were in the range of 90-97%, indicating that the released insulin retained its pharmacol. activity. We obsd. no significant differences in the plasma insulin concn. profiles between the first and second administrations, confirming the stability and accuracy of using the proposed MN system for insulin delivery. These results indicated that the γ-PGA MNs contg. the supporting structure design enable complete and efficient delivery of encapsulated bioactive mols. and have great potential for the relatively rapid and convenient transdermal delivery of protein drugs. Incomplete insertion of microneedles largely limits drug delivery efficiency and wastage of valuable medication. To address this problem, we developed a fully insertable poly-glutamic acid microneedles with a supporting structure design to ensure complete and efficient delivery of encapsulated drugs. The supporting structures were designed to provide an extended length for counteracting skin compressive deformation during puncture and mech. strength for fully inserting the microneedles into the skin. When inserted into the skin, both the supporting structures and microneedles can be dissolved in the skin within 4 min, thus quickly releasing the entire drug load. This study demonstrated that the proposed microneedle system featuring this unique design allows more convenient and efficient self-administration of drugs into the skin.
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    Jabbari, N.; Asghari, M.; Ahmadian, H.; Mikaili, P. Developing a Commercial Air Ultrasonic Ceramic Transducer to Transdermal Insulin Delivery. J. Med. Signals Sens. 2015, 5, 117– 122,  DOI: 10.4103/2228-7477.157618
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    Developing a Commercial Air Ultrasonic Ceramic Transducer to Transdermal Insulin Delivery
    Jabbari Nasrollah; Asghari Mohammad Hossein; Ahmadian Hassan; Mikaili Peyman
    Journal of medical signals and sensors (2015), 5 (2), 117-22 ISSN:2228-7477.
    The application of low-frequency ultrasound for transdermal delivery of insulin is of particular public interest due to the increasing problem of diabetes. The purpose of this research was to develop an air ultrasonic ceramic transducer for transdermal insulin delivery and evaluate the possibility of applying a new portable and low-cost device for transdermal insulin delivery. Twenty-four rats were divided into four groups with six rats in each group: one control group and three experimental groups. Control group (C) did not receive any ultrasound exposure or insulin (untreated group). The second group (T1) was treated with subcutaneous insulin (Humulin(®) R, rDNA U-100, Eli Lilly and Co., Indianapolis, IN) injection (0.25 U/Kg). The third group (T2) topically received insulin, and the fourth group (T3) received insulin with ultrasound waves. All the rats were anesthetized by intraperitoneal injection of ketamin hydrochloride and xylazine hydrochloride. Blood samples were collected after anesthesia to obtain a baseline glucose level. Additional blood samples were taken every 15 min in the whole 90 min experiment. In order for comparison the changes in blood glucose levels" to " In order to compare the changes in blood glucose levels. The statistical multiple comparison (two-sided Tukey) test showed a significant difference between transdermal insulin delivery group (T2) and subcutaneous insulin injection group (T1) during 90 min experiment (P = 0.018). In addition, the difference between transdermal insulin delivery group (T2) and ultrasonic transdermal insulin delivery group (T3) was significant (P = 0.001). Results of this study demonstrated that the produced low-frequency ultrasound from this device enhanced the transdermal delivery of insulin across hairless rat skin.
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  37. 37
    Nolasco, E. L.; Zanoni, F. L.; Nunes, F. P. B.; Ferreira, S. S.; Freitas, L. A.; Silva, M. C. F.; Martins, J. O. Insulin Modulates Liver Function in a Type I Diabetes Rat Model. Cell. Physiol. Biochem. 2015, 36, 1467– 1479,  DOI: 10.1159/000430311
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    Insulin Modulates Liver Function in a Type I Diabetes Rat Model
    Nolasco, Eduardo L.; Zanoni, Fernando L.; Nunes, Fernanda P. B.; Ferreira, Sabrina S.; Freitas, Luiza A.; Silva, Mariana C. F.; Martins, Joilson O.
    Cellular Physiology and Biochemistry (2015), 36 (4), 1467-1479CODEN: CEPBEW; ISSN:1015-8987. (S. Karger AG)
    Background/Aims: Several studies have been performed to unravel the assocn. between diabetes and increased susceptibility to infection. This study aimed to investigate the effect of insulin on the local environment after cecal ligation and puncture (CLP) in rats. Methods: Diabetic (alloxan, 42 mg/kg i.v., 10 days) and non-diabetic (control) male Wistar rats were subjected to a two-puncture CLP procedure and 6 h later, the following analyses were performed:. (a) total and differential cell counts in peritoneal lavage (PeL) and bronchoalveolar lavage (BAL) fluids;. (b) quantification of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-10 and cytokine-induced neutrophil chemoattractant (CINC)-1 and CINC-2 in the PeL and BAL fluids by ELISA (ELISA);. (c) total leukocyte count using a veterinary hematol. analyzer and differential leukocyte counts on stained slides;. (d) biochem. parameters (urea, creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alk. phosphatase (ALP) by colorimetric analyses); and (e) lung, kidney, and liver morphol. analyses (hematoxylin and eosin staining). Results: Relative to controls, non-diabetic and diabetic CLP rats exhibited an increased in the concn. of IL-1β, IL-6, IL-10, CINC-1, and CINC-2 and total and neutrophil in the PeL fluid. Treatment of these animals with neutral protamine Hagedorn insulin (NPH, 1IU and 4IU, resp., s.c.), 2 h before CLP procedure, induced an increase on these cells in the PeL fluid but it did not change cytokine levels. The levels of ALT, AST, ALP, and urea were higher in diabetic CLP rats than in non-diabetic CLP rats. ALP levels were higher in diabetic sham rats than in non-diabetic sham rats. Treatment of diabetic rats with insulin completely restored ALT, AST, and ALP levels. Conclusion: These results together suggest that insulin attenuates liver dysfunction during early two-puncture CLP-induced peritoneal inflammation in diabetic rats.
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  38. 38
    Saito, S.; Thuc, L. C.; Teshima, Y.; Nakada, C.; Nishio, S.; Kondo, H.; Fukui, A.; Abe, I.; Ebata, Y.; Saikawa, T.; Moriyama, M.; Takahashi, N. Glucose Fluctuations Aggravate Cardiac Susceptibility to Ischemia/Reperfusion Injury by Modulating MicroRNAs Expression. Circ. J. 2016, 80, 186– 195,  DOI: 10.1253/circj.CJ-14-1218
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    Glucose fluctuations aggravate cardiac susceptibility to ischemia/reperfusion injury by modulating MicroRNAs expression
    Saito, Shotaro; Thuc, Luong Cong; Teshima, Yasushi; Nakada, Chisato; Nishio, Satoru; Kondo, Hidekazu; Fukui, Akira; Abe, Ichitaro; Ebata, Yuki; Saikawa, Tetsunori; Moriyama, Masatsugu; Takahashi, Naohiko
    Circulation Journal (2016), 80 (1), 186-195CODEN: CJIOBY; ISSN:1346-9843. (Japanese Circulation Society)
    Background: The influence of glucose fluctuations (GF) on cardiovascular complications of diabetes mellitus (DM) has been attracting much attention. In the present study, whether GF increase susceptibility to ischemia/reperfusion in the heart was investigated. Methods and Results: Male rats were randomly assigned to either a control, DM, and DM with GF group. DM was induced by an injection of streptozotocin, and glucose fluctuation was induced by starvation and insulin injection. One sequential program comprised 2 hypoglycemic episodes during 4 days. The isolated hearts were subjected to 20-min ischemia/30-min reperfusion. The infarct size was larger in hearts with GF than those with sustained hyperglycemia. Activities of catalase and superoxide dismutase were decreased, and expressions of NADPH oxidase and thioredoxin-interacting protein were upregulated by GF accompanied by an increase of reactive oxygen species (ROS). Swollen mitochondria with destroyed cristae were obsd. in diabetic hearts; they were further devastated by GF. Microarray anal. revealed that the expressions of microRNA (miRNA)-200c and miRNA-141 were abundant in those hearts with GF. Overexpression of miRNA-200c and miRNA-141 decreased mitochondrial superoxide dismutase and catalase activities, and increased ROS levels. Meanwhile, knockdown of miRNA-200c and miRNA- 141 significantly decreased ROS levels in cardiomyocytes exposed to GF. Conclusions: GF increased ROS generation and enhanced ischemia/reperfusion injury in the diabetic heart. Upregulated miRNA-200c and miRNA-141 may account for the increased ROS.
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    Hofmann, T.; Horstmann, G.; Stammberger, I. Evaluation of the Reproductive Toxicity and Embryotoxicity of Insulin Glargine (LANTUS) in Rats and Rabbits. Int. J. Toxicol. 2002, 21, 181– 189,  DOI: 10.1080/10915810290096315
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    Evaluation of the reproductive toxicity and embryotoxicity of insulin glargine (LANTUS) in rats and rabbits
    Hofmann, Thomas; Horstmann, Georg; Stammberger, Ingo
    International Journal of Toxicology (2002), 21 (3), 181-189CODEN: IJTOFN; ISSN:1091-5818. (Taylor & Francis Ltd.)
    Insulin glargine (LANTUS) is a new insulin analog that has a prolonged duration of action with no pronounced peak of activity, rendering it an ideal basal insulin for the treatment of diabetes. The aim of these studies was to assess the reproductive and embryotoxicity of insulin glargine. Reproductive toxicity was assessed in 25 male and 25 female Wistar rats per group treated with a daily s.c. injection of control; 1 IU/kg, 3 IU/kg, and 10 IU/kg insulin glargine; or 3 IU/kg NPH insulin in the premating and mating periods, and throughout pregnancy and lactation in the females. Embryotoxicity was assessed in 20 female rats per group injected with daily s.c. doses of control; 2 IU/kg, 6.3 IU/kg, and 20 IU/kg insulin glargine; or 6.3 IU/kg NPH insulin from the 7th to 18th day of pregnancy. Embryotoxicity was also assessed in 20 female rabbits per group treated with 0 IU/kg, 0.5 IU/kg, 1 IU/kg, and 2 IU/kg insulin glargine, or 1 IU/kg NPH insulin from the 6th to 18th day of pregnancy. The data demonstrated that, with the exception of toxicol. effects induced by hypoglycemia in response to high doses of insulin glargine and NPH insulin (including the premature dropout of female rats in the reproductive toxicity study, and increased incidence of abortions, early intrauterine deaths, and single anomalies in the rabbit embryotoxicity study), insulin glargine had no effects on reprodn., embryo-fetal development, and postnatal development in rats. Maternal and embryo-fetal toxicity in rabbits treated with middle and high doses of insulin glargine was related to the hypoglycemic effect of insulin.
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Cite this: ACS Appl. Mater. Interfaces 2021, 13, 10, 11672–11682
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  • Abstract

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    Figure 1

    Figure 1. Schematic chart of the preparation of composite insulin crystal formulation for in vivo administration.

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    Figure 2

    Figure 2. Viscosity (A) and viscoelastic moduli (B) values of the hydrogels (agarose: blue and Fmoc-AA: red). (C) circular dichroism (CD) spectra of Fmoc-AA. (D) Schematic representation of Fmoc-AA and agarose molecules. (E) Transmission electron microscopy (TEM) image of Fmoc-AA and (F) agarose dried gels.

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    Figure 3

    Figure 3. Optical microscopy images of insulin composite crystals grown in Fmoc-AA (A) and agarose (B) hydrogels. Plot of Fmoc-AA crystal size distribution (C). SEM images of Fmoc-AA crystals at 50 μm (D) and 4 μm (E) magnification.

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    Figure 4

    Figure 4. Characterization of insulin crystals maintained at room temperature. (A) Dissolution rate of insulin crystals. (B) Differential scanning calorimetry (DSC) of insulin samples. Transition temperatures are shown at the peaks. (C) High-performance liquid chromatography (HPLC) profiles of dissolved insulin crystals.

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    Figure 5

    Figure 5. Characterization of insulin crystals kept at 50 °C for 7 days. Optical microscopy images of insulin crystals grown in Fmoc-AA (A) and agarose (B) and the corresponding SEM images of Fmoc-AA (C) and agarose (D). (E) HPLC profiles of dissolved insulin crystals. (F) Differential scanning calorimetry (DSC) of insulin samples. Transition temperatures are shown at the peaks.

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    Figure 6

    Figure 6. (A) Glycemia profile of agarose crystals at 20 and 50 °C. (B) Glycemia profile of Fmoc-AA crystals at 20 and 50 °C. (C) Glycemia profile of dissolved Fmoc-AA crystals kept at 50 °C and Fmoc-AA crystals kept at 60 °C. Reference insulin (black), agarose crystals (blue), and Fmoc-AA crystals (red).

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    Figure 7

    Figure 7. In vitro cytotoxic assay: Caco 2T (A) and IEC18 (B). Cells were submitted to increasing concentrations of Fmoc-AA hydrogel (1–1000 μg/mL) kept at 20 or 50 °C for 7 days. Toxicity was measured by LDH cytotoxicity assay following the manufacturer’s instructions.

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      Basu, Sujit K.; Govardhan, Chandrika P.; Jung, Chu W.; Margolin, Alexey L.
      Expert Opinion on Biological Therapy (2004), 4 (3), 301-317CODEN: EOBTA2; ISSN:1471-2598. (Ashley Publications Ltd.)
      A review and discussion. The year 2002 marked the 20th anniversary of the first successful product of modern biotechnol., the regulatory approval of recombinant insulin for biopharmaceutical applications. Insulin is also the first cryst. protein to be approved for therapeutic use. Over the past two decades, almost 150 biopharmaceuticals have gained marketing authorization; however, insulin remains the only cryst. protein on the market. Significant research and development efforts have focused on the engineering of protein mols., efficacy testing, model development, and protein prodn. and characterization. These advances have dramatically boosted the therapeutic applications of proteins, which now include treatments against acute conditions, such as cancer, cardiovascular disease and viral disease, and chronic conditions, such as diabetes, growth hormone deficiency, haemophilia, arthritis, psoriasis and Crohn's disease. Despite these successes, many challenges normally assocd. with biopharmaceuticals, such as poor stability and limited delivery options, remain. Protein crystals have shown significant benefits in the delivery of biopharmaceuticals to achieve high concn., low viscosity formulation and controlled release protein delivery. This review discusses challenges related to the broader utilization of protein crystals in biopharmaceutical applications, as well as recent advances and valuable new directions that protein crystn.-based technologies present.
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    14. 14
      Yang, M. X.; Shenoy, B.; Disttler, M.; Patel, R.; McGrath, M.; Pechenov, S.; Margolin, A. L. Crystalline Monoclonal Antibodies for Subcutaneous Delivery. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6934– 6939,  DOI: 10.1073/pnas.1131899100
      14
      Crystalline monoclonal antibodies for subcutaneous delivery
      Yang, Mark X.; Shenoy, Bhami; Disttler, Matthew; Patel, Reena; McGrath, Margaret; Pechenov, Sergey; Margolin, Alexey L.
      Proceedings of the National Academy of Sciences of the United States of America (2003), 100 (12), 6934-6939CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Therapeutic applications for mAbs have increased dramatically in recent years, but the large quantities required for clin. efficacy have limited the options that might be used for administration and thus have placed certain limitations on the use of these agents. We present an approach that allows for s.c. delivery of a small vol. of a highly concd. form of mAbs. Batch crystn. of three Ab-based therapeutics, rituximab, trastuzumab, and infliximab, provided products in high yield, with no detectable alteration to these proteins and with full retention of their biol. activity in vitro. Administration s.c. of a cryst. prepn. resulted in a remarkably long pharmacokinetic serum profile and a dose-dependent inhibition of tumor growth in nude mice bearing BT-474 xenografts (human breast cancer cells) in vivo. Overall, this approach of generating high-concn., low-viscosity cryst. prepns. of therapeutic Abs should lead to improved ease of administration and patient compliance, thus providing new opportunities for the biotechnol. industry.
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    15. 15
      Lorber, B.; Sauter, C.; Théobald-Dietrich, A.; Moreno, A.; Schellenberger, P.; Robert, M. C.; Capelle, B.; Sanglier, S.; Potier, N.; Giegé, R. Crystal Growth of Proteins, Nucleic Acids, and Viruses in Gels. Prog. Biophys. Mol. Biol. 2009, 101, 13– 25,  DOI: 10.1016/j.pbiomolbio.2009.12.002
      15
      Crystal growth of proteins, nucleic acids, and viruses in gels
      Lorber, Bernard; Sauter, Claude; Theobald-Dietrich, Anne; Moreno, Abel; Schellenberger, Pascale; Robert, Marie-Claire; Capelle, Bernard; Sanglier, Sarah; Potier, Noelle; Giege, Richard
      Progress in Biophysics & Molecular Biology (2009), 101 (1/3), 13-25CODEN: PBIMAC; ISSN:0079-6107. (Elsevier Ltd.)
      A review. Medium-sized single crystals with perfect habits and no defect producing intense and well-resolved diffraction patterns are the dream of every protein crystallographer. Crystals of biol. macromols. possessing these characteristics can be prepd. within a medium in which mass transport is restricted to diffusion. Chem. gels (like polysiloxane) and phys. gels (such as agarose) provide such an environment and are therefore suitable for the crystn. of biol. macromols. Instructions for the prepn. of each type of gel are given to urge crystal growers to apply diffusive media for enhancing crystallog. quality of their crystals. Examples of quality enhancement achieved with silica and agarose gels are given. Results obtained with other substances forming gel-like media (such as lipidic phases and cellulose derivs.) are presented. Finally, the use of gels in combination with capillary tubes for counter-diffusion expts. is discussed. Methods and techniques implemented with proteins can also be applied to nucleic acids and nucleoprotein assemblies such as viruses.
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    16. 16
      Gavira, J. A.; Van Driessche, A. E. S.; Garcia-Ruiz, J. M. Growth of Ultrastable Protein-Silica Composite Crystals. Cryst. Growth Des. 2013, 13, 2522– 2529,  DOI: 10.1021/cg400231g
      16
      Growth of Ultrastable Protein-Silica Composite Crystals
      Gavira, Jose A.; Van Driessche, Alexander E. S.; Garcia-Ruiz, Juan-Ma
      Crystal Growth & Design (2013), 13 (6), 2522-2529CODEN: CGDEFU; ISSN:1528-7483. (American Chemical Society)
      Protein crystals were obtained in a wide range of silica gel concns., 2.0-22.0% (vol./vol.), using the counter-diffusion technique. The protein crystal lattice incorporates silica fibers during their growth, making the crystal appear optically translucent while maintaining the diffraction quality. The effect of the silica fibers on the nucleation and growth morphol. is discussed, and the amt. of incorporated silica matrix is quantified. The practical implications of the presence of a high hygroscope phase on the crystal properties are discussed, and the improvement of the mech. properties and stability of the crystals is shown. These reinforced protein crystals, able to include large amts. of silica, open a new range of possibilities for the characterization of protein crystals and the application in the biotechnol. industry.
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    17. 17
      Gavira, J. A.; Cera-Manjarres, A.; Ortiz, K.; Mendez, J.; Jimenez-Torres, J. A.; Patiño-Lopez, L. D.; Torres-Lugo, M. Use of Cross-Linked Poly(Ethylene Glycol)-Based Hydrogels for Protein Crystallization. Cryst. Growth Des. 2014, 14, 3239– 3248,  DOI: 10.1021/cg401668z
      17
      Use of Cross-Linked Poly(ethylene glycol)-Based Hydrogels for Protein Crystallization
      Gavira, Jose A.; Cera-Manjarres, Andry; Ortiz, Katia; Mendez, Janet; Jimenez-Torres, Jose A.; Patino-Lopez, Luis D.; Torres-Lugo, Madeline
      Crystal Growth & Design (2014), 14 (7), 3239-3248CODEN: CGDEFU; ISSN:1528-7483. (American Chemical Society)
      Poly(ethylene glycol) (PEG) hydrogels are highly biocompatible materials extensively used for biomedical and pharmaceutical applications, controlled drug release, and tissue engineering. In this work, PEG crosslinked hydrogels, synthesized under various conditions, were used to grow lysozyme crystals by the counterdiffusion technique. Crystn. expts. were conducted using a three-layer arrangement. Results demonstrated that PEG fibers were incorporated within lysozyme crystals controlling the final crystal shape. PEG hydrogels also induced the nucleation of lysozyme crystals to a higher extent than agarose. PEG hydrogels can also be used at higher concns. (20-50% wt./wt.) as a sepn. chamber (plug) in counterdiffusion expts. In this case, PEG hydrogels control the diffusion of the crystn. agent and therefore may be used to tailor the supersatn. to fine-tune crystal size. As an example, insulin crystals were grown in 10% (wt./wt.) PEG hydrogel. The resulting crystals were of an approx. size of 500 μm.
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    18. 18
      Conejero-Muriel, M.; Contreras-Montoya, R.; Díaz-Mochón, J. J.; Álvarez de Cienfuegos, L.; Gavira, J. A. Protein Crystallization in Short-Peptide Supramolecular Hydrogels: A Versatile Strategy towards Biotechnological Composite Materials. CrystEngComm 2015, 17, 8072– 8078,  DOI: 10.1039/C5CE00850F
      18
      Protein crystallization in short-peptide supramolecular hydrogels: a versatile strategy towards biotechnological composite materials
      Conejero-Muriel, Mayte; Contreras-Montoya, Rafael; Diaz-Mochon, Juan J.; Alvarez de Cienfuegos, Luis; Gavira, Jose A.
      CrystEngComm (2015), 17 (42), 8072-8078CODEN: CRECF4; ISSN:1466-8033. (Royal Society of Chemistry)
      Protein crystn. in hydrogels has been explored with the main purpose of facilitating the growth of high quality crystals while increasing their size to enhance their manipulation. New avenues are currently being built for the use of protein crystals as source materials to create sensors and drug delivery vehicles, to name just a few. In this sense, short-peptide supramol. hydrogels may play a crucial role in integrating protein crystals within a wider range of applications. In this article, we show that protein crystn. in short-peptide supramol. hydrogels is feasible and independent of the type of peptide that forms the hydrogel and(or) the protein, although the output is not always the same. As a general trend, it is confirmed that hydrogel fibers are always incorporated within crystals so that novel composite materials for biotechnol. applications with enhanced properties are produced.
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    19. 19
      Conejero-Muriel, M.; Gavira, J. A.; Pineda-Molina, E.; Belsom, A.; Bradley, M.; Moral, M.; García López Durán, J. D. D.; Luque González, A.; Díaz-Mochón, J. J.; Contreras-Montoya, R.; Martínez-Peragón, Á.; Cuerva, J. M.; Álvarez de Cienfuegos, L. Influence of the Chirality of Short Peptide Supramolecular Hydrogels in Protein Crystallogenesis. Chem. Commun. 2015, 51, 3862– 3865,  DOI: 10.1039/c4cc09024a
      19
      Influence of the chirality of short peptide supramolecular hydrogels in protein crystallogenesis
      Conejero-Muriel, Mayte; Gavira, Jose A.; Pineda-Molina, Estela; Belsom, Adam; Bradley, Mark; Moral, Monica; Duran, Juan de Dios Garcia-Lopez; Luque Gonzalez, Angelica; Diaz-Mochon, Juan J.; Contreras-Montoya, Rafael; Martinez-Peragon, Angela; Cuerva, Juan M.; Alvarez de Cienfuegos, Luis
      Chemical Communications (Cambridge, United Kingdom) (2015), 51 (18), 3862-3865CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      For the first time the influence of the chirality of the gel fibers in protein crystallogenesis has been studied. Enantiomeric hydrogels 1 and 2 were tested with model proteins lysozyme and glucose isomerase and a formamidase extd. from B. cereus. Crystn. behavior and crystal quality of these proteins in both hydrogels are presented and compared.
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    20. 20
      Escolano-Casado, G.; Contreras-Montoya, R.; Conejero-Muriel, M.; Castellví, A.; Juanhuix, J.; Lopez-Lopez, M. T.; Álvarez de Cienfuegos, L.; Gavira, J. A. Extending the Pool of Compatible Peptide Hydrogels for Protein Crystallization. Crystals 2019, 9, 244  DOI: 10.3390/cryst9050244
      20
      Extending the pool of compatible peptide hydrogels for protein crystallization
      Escolano-Casado, Guillermo; Contreras-Montoya, Rafael; Conejero-Muriel, Mayte; Castellvi, Albert; Juanhuix, Judith; Lopez-Lopez, Modesto T.; de Cienfuegos, Luis Alvarez; Gavira, Jose A.
      Crystals (2019), 9 (5), 244CODEN: CRYSBC; ISSN:2073-4352. (MDPI AG)
      Short-peptide supramol. (SPS) hydrogels are a class of materials that have been found to be useful for (bio)technol. applications thanks to their biocompatible nature. Among the advantages reported for these peptides, their economic affordability and easy functionalization or modulation have turned them into excellent candidates for the development of functional biomaterials. It is recently demonstrated that SPS hydrogels can be used to produce high-quality protein crystals, improve their properties, or incorporate relevant materials within the crystals. In this work, it is proved that hydrogels based on methionine and tyrosine are also good candidates for growing high-quality crystals of the three model proteins: lysozyme, glucose isomerase, and thaumatin.
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    21. 21
      Contreras-Montoya, R.; Castellví, A.; Escolano-Casado, G.; Juanhuix, J.; Conejero-Muriel, M.; Lopez-Lopez, M. T.; Cuerva, J. M.; Álvarez de Cienfuegos, L.; Gavira, J. A. Enhanced Stability against Radiation Damage of Lysozyme Crystals Grown in Fmoc-CF Hydrogels. Cryst. Growth Des. 2019, 19, 4229– 4233,  DOI: 10.1021/acs.cgd.9b00131
      21
      Enhanced Stability against Radiation Damage of Lysozyme Crystals Grown in Fmoc-CF Hydrogels
      Contreras-Montoya, Rafael; Castellvi, Albert; Escolano-Casado, Guillermo; Juanhuix, Judith; Conejero-Muriel, Mayte; Lopez-Lopez, Modesto T.; Cuerva, Juan M.; Alvarez de Cienfuegos, Luis; Gavira, Jose A.
      Crystal Growth & Design (2019), 19 (8), 4229-4233CODEN: CGDEFU; ISSN:1528-7483. (American Chemical Society)
      Lysozyme crystals grown in Fmoc-CF (Cys-Phe) hydrogels, unlike those grown in agarose, give rise to composite crystals that have an enhanced resistance against degrdn. caused by an intense exposure to X-ray irradn. Fmoc-CF dipeptide shows a clear protection of the most sensitive groups (disulfide bonds and methionines) of the protein. The protection mediated by cysteine is exerted only in its gel state since cysteine in soln. has an adverse effect. Probably, the reactive thiol groups of cysteine being locked within the rigid peptide fibers minimize cross-reactions with the proteins favoring the formation of protein crystals. Once located inside the protein crystal, the long peptide fibers are able to protect the protein against radiation damage.
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    22. 22
      Artusio, F.; Castellví, A.; Sacristán, A.; Pisano, R.; Gavira, J. A. Agarose Gel as a Medium for Growing and Tailoring Protein Crystals. Cryst. Growth Des. 2020, 20, 5564– 5571,  DOI: 10.1021/acs.cgd.0c00736
      22
      Agarose Gel as a Medium for Growing and Tailoring Protein Crystals
      Artusio, Fiora; Castellvi, Albert; Sacristan, Anabel; Pisano, Roberto; Gavira, Jose A.
      Crystal Growth & Design (2020), 20 (8), 5564-5571CODEN: CGDEFU; ISSN:1528-7483. (American Chemical Society)
      The nucleation inducing ability of agarose gels has been exploited to study the crystn. of proteins in diffusion-dominated environments. The crystal size was successfully tuned in a wide range of gel, protein, and precipitant concns. The impact of the gel content on crystal size was independent of the specific protein, allowing the math. prediction of crystal size and pointing out the exclusivity of phys. interactions between the gel and the protein. The versatility of the technique and the fine-tuning of the nucleation flux was demonstrated by crystg. five different proteins and implementing batch and counter-diffusion crystn. In addn., the potential of agarose gel to be used not only as a growth but also as a delivery medium for serial crystallog. applications has been proven by prepg. unidimensional microcrystal slurries with 0.1% (w/v) gel. Protein crystal size was successfully tuned from a few micrometers to several hundred micrometers by performing batch crystn. in gels prepd. from variable amts. of agarose. Counter-diffusion crystn. in gel was proposed as an alternative for proteins requiring PEGs or ammonium sulfate as precipitants. The results pointed out the phys. action of agarose gels in inducing protein nucleation.
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    23. 23
      Tao, K.; Levin, A.; Adler-Abramovich, L.; Gazit, E. Fmoc-Modified Amino Acids and Short Peptides: Simple Bio-Inspired Building Blocks for the Fabrication of Functional Materials. Chem. Soc. Rev. 2016, 45, 3935– 3953,  DOI: 10.1039/c5cs00889a
      23
      Fmoc-modified amino acids and short peptides: simple bio-inspired building blocks for the fabrication of functional materials
      Tao, Kai; Levin, Aviad; Adler-Abramovich, Lihi; Gazit, Ehud
      Chemical Society Reviews (2016), 45 (14), 3935-3953CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      A review. Amino acids and short peptides modified with the 9-fluorenylmethyloxycarbonyl (Fmoc) group possess eminent self-assembly features and show distinct potential for applications due to the inherent hydrophobicity and aromaticity of the Fmoc moiety which can promote the assocn. of building blocks. Given the extensive study and numerous publications in this field, it is necessary to summarize the recent progress concerning these important bio-inspired building blocks. Therefore, in this review, we explore the self-organization of this class of functional mols. from three aspects, i.e., Fmoc-modified individual amino acids, Fmoc-modified di- and tripeptides, and Fmoc-modified tetra- and pentapeptides. The relevant properties and applications related to cell cultivation, bio-templating, optical, drug delivery, catalytic, therapeutic and antibiotic properties are subsequently summarized. Finally, some existing questions impeding the development of Fmoc-modified simple biomols. are discussed, and corresponding strategies and outlooks are suggested.
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    24. 24
      Fleming, S.; Ulijn, R. V. Design of Nanostructures Based on Aromatic Peptide Amphiphiles. Chem. Soc. Rev. 2014, 43, 8150– 8177,  DOI: 10.1039/c4cs00247d
      24
      Design of nanostructures based on aromatic peptide amphiphiles
      Fleming, Scott; Ulijn, Rein V.
      Chemical Society Reviews (2014), 43 (23), 8150-8177CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      A review. Arom. peptide amphiphiles are gaining popularity as building blocks for the bottom-up fabrication of nanomaterials, including gels. These materials combine the simplicity of small mols. with the versatility of peptides, with a range of applications proposed in biomedicine, nanotechnol., food science, cosmetics, etc. Despite their simplicity, a wide range of self-assembly behaviors have been described. Due to varying conditions and protocols used, care should be taken when attempting to directly compare results from the literature. In this review, the authors rationalize the structural features which govern the self-assembly of arom. peptide amphiphiles by focusing on four segments, (i) the N-terminal arom. component, (ii) linker segment, (iii) peptide sequence, and (iv) C-terminus. It is clear that the mol. structure of these components significantly influences the self-assembly process and resultant supramol. architectures. A no. of modes of assembly have been proposed, including parallel, antiparallel, and interlocked antiparallel stacking conformations. In addn., the co-assembly arrangements of arom. peptide amphiphiles are reviewed. Overall, this review elucidates the structural trends and design rules that underpin the field of arom. peptide amphiphile assembly, paving the way to a more rational design of nanomaterials based on arom. peptide amphiphiles.
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    25. 25
      Mu, X.; Eckes, K. M.; Nguyen, M. M.; Suggs, L. J.; Ren, P. Experimental and Computational Studies Reveal an Alternative Supramolecular Structure for Fmoc-Dipeptide Self-Assembly. Biomacromolecules 2012, 13, 3562– 3571,  DOI: 10.1021/bm301007r
      25
      Experimental and computational studies reveal an alternative supramolecular structure for Fmoc-dipeptide self-assembly
      Mu, Xiaojia; Eckes, Kevin M.; Nguyen, Mary M.; Suggs, Laura J.; Ren, Pengyu
      Biomacromolecules (2012), 13 (11), 3562-3571CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      The self-assembly of fluorenylmethoxycarbonyl (Fmoc)-conjugated dialanine (Fmoc-Ala-Ala-OH) (I) mols. using combined computational and exptl. approaches. I gels were characterized using transmission electron microscopy (TEM), CD, FTIR spectroscopy, and wide-angle x-ray scattering (WAXS). Computationally, the authors simulated the assembly of I using mol. dynamics techniques. All simulations converged to a condensed fibril structure in which the Fmoc groups stacked mostly within in the center of the fibril. However, the Fmoc groups were partially exposed to water, creating an amphiphilic surface, which may be responsible for the aggregation of fibrils into nanoscale fibers obsd. in TEM. From the fibril models, radial distribution calcns. agreed with d-spacings obsd. in WAXS for the fibril diam. and π-stacking interactions. The analyses showed that dialanine, despite its short length, adopts a mainly extended polyproline II conformation. In contrast to previous hypotheses, these results indicated that β-sheet-like H-bonding was not prevalent. Rather, stacking of Fmoc groups, inter-residue H-bonding, and H-bonding with water play the important roles in stabilizing the fibril structure of supramol. assemblies of short conjugated peptides.
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    26. 26
      Smith, A. M.; Williams, R. J.; Tang, C.; Coppo, P.; Collins, R. F.; Turner, M. L.; Saiani, A.; Ulijn, R. V. Fmoc-Diphenylalanine Self Assembles to a Hydrogel via a Novel Architecture Based on π–π Interlocked β-Sheets. Adv. Mater. 2008, 20, 37– 41,  DOI: 10.1002/adma.200701221
      26
      Fmoc-diphenylalanine self-assembles to a hydrogel via a novel architecture based on π-π interlocked β-sheets
      Smith, Andrew M.; Williams, Richard J.; Tang, Claire; Coppo, Paolo; Collins, Richard F.; Turner, Michael L.; Saiani, Alberto; Ulijn, Rein V.
      Advanced Materials (Weinheim, Germany) (2008), 20 (1), 37-41CODEN: ADVMEW; ISSN:0935-9648. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The self-assembly of peptide hydrogelators that carry arom. substituents can be modeled by a novel nanocylindrical architecture. The proposed model suggests that the nanocylinders are formed by anti-parallel β-sheets interlocked by the π-stacking interactions of fluorenyl groups and Ph rings. This explanation is consistent with the structures obsd. in TEM and the data obtained by a variety of spectroscopic techniques.
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    27. 27
      Contreras-Montoya, R.; Bonhome-Espinosa, A. B.; Orte, A.; Miguel, D.; Delgado-López, J. M.; Duran, J. D. G.; Cuerva, J. M.; Lopez-Lopez, M. T.; Álvarez de Cienfuegos, L. Iron Nanoparticles-Based Supramolecular Hydrogels to Originate Anisotropic Hybrid Materials with Enhanced Mechanical Strength. Mater. Chem. Front. 2018, 2, 686– 699,  DOI: 10.1039/c7qm00573c
      27
      Iron nanoparticles-based supramolecular hydrogels to originate anisotropic hybrid materials with enhanced mechanical strength
      Contreras-Montoya, Rafael; Bonhome-Espinosa, Ana B.; Orte, Angel; Miguel, Delia; Delgado-Lopez, Jose M.; Duran, Juan D. G.; Cuerva, Juan M.; Lopez-Lopez, Modesto T.; Alvarez de Cienfuegos, Luis
      Materials Chemistry Frontiers (2018), 2 (4), 686-699CODEN: MCFAC5; ISSN:2052-1537. (Royal Society of Chemistry)
      Here, we report the synthesis and structural characterization of novel iron nanoparticles (FeNPs)-based short-peptide supramol. hydrogels. These hybrid hydrogels composed of Fmoc-diphenylalanine (Fmoc-FF) peptide and FeNPs were prepd. through the self-assembly of Fmoc-FF in a suspension contg. FeNPs in the presence or absence of an external magnetic field. Optical images of these hydrogels revealed the formation of column-like aggregates of FeNPs when the gels were formed in the presence of a magnetic field. Moreover, the intricate structure derived from the interwoven nature of the fiber peptides with these FeNP column-like aggregates resulted in anisotropic materials, more rigid under shear forces applied perpendicularly to the direction of the aggregates, presenting under these conditions values of G' (storage modulus) about 7 times those of the native hydrogel. To the best of our knowledge, this is the first example in which the mech. properties of peptide hydrogels were strongly enhanced due to the presence of FeNPs. A theor. model trying to explain this phenomenon is presented. Quite interesting CD, FTIR and synchrotron X-ray diffraction analyses indicated that the anti-βparallel -sheet arrangement of Fmoc-FF peptide was highly conserved in the hydrogels contg. FeNPs. Moreover, FLCS measurements showed that the diffusion of a small solute through the hydrogel network was improved in hydrogels contg. FeNPs, probably caused by the formation of preferential channels for diffusion. Taken together, our results provide a new method for the synthesis of novel hybrid Fmoc-FF-FeNPs anisotropic hydrogels with enhanced mech. strength and water-like diffusion behavior, thus easing their application in drug delivery and tissue engineering.
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    28. 28
      Adams, M. J.; Blundell, T. L.; Dodson, E. J.; Dodson, G. G.; Vijayan, M.; Baker, E. N.; Harding, M. M.; Hodgkin, D. C.; Rimmer, B.; Sheat, S. Structure of Rhombohedral 2 Zinc Insulin Crystals. Nature 1969, 224, 491– 495,  DOI: 10.1038/224491a0
      28
      Structure of rhombohedral 2 zinc insulin crystals
      Adams, Margaret Joan; Blundell, T. L.; Dodson, E. J.; Dodson, G. G.; Vijayan, M.; Baker, E. N.; Harding, M. M.; Hodgkin, D. C.; Rimmer, B.; Sheats, S.
      Nature (London, United Kingdom) (1969), 224 (5218), 491-5CODEN: NATUAS; ISSN:0028-0836.
      The 3-dimensional arrangement of pig insulin (I) was detd. in an electron d. map at 2.8 Å resolution. Heavy atoms were introduced into the rhombohedral crystal which contained 2 Zn atoms and 6 I mols./unit cell. In the electron d. map, 2 stretches of α-helix appeared. At an end of each α-helix stretch, which represented the B10 histidine, the d. was connected with 1 of the 2 peaks representing the Zn atoms on the 3-fold axis. Peptide chains and SS bonds were readily identified. All aromatic residues were well defined by individual ellipsoidal peaks. Regions with long side chains on the outside of the mol. were difficult to interpret. Two crystallographically similar independent I mols. appeared. In each mol., the B chain was wrapped around the compact A chain. The A chain was greatly folded with short stretches of almost helical conformation between residues A2-6 and A13-19. The A chain loop, A6-11, and residues A8, 9, and 10 were directedout from the surface of the mol. The SS bond, A6-11, was within the body of the mol. Both A19 and A14 tyrosines (II) appeared to be H-bonded to residues A1 and A5, resp. In the central part of the B chain, there were 3 turns of α-helix,slightly opened out at each end. Both the interchain SS bonds, B7-A7 and B19-A20, were at the ends of the helix. B1-B6 and B21-B30 were largely extended and loosely packed around the A chain. Residues A4, glutamic acid, and B29, lysine, appeared to be in contact. The closest series of contacts between 2 I mols. occurred between the extended B chains, residues B23-B28. They were arranged antiparallel and appeared to form a H-bonded pleated sheet. They surrounded the noncrystallographic 2-fold axis. An aromatic cage is formed by II B26 and phenylalanine (III) B24 and their 2-fold axis-related partners. The 2 phenyl groups at B25 formed part of the system by turning towards one another and violating the 2-fold symmetry relation. The dimer was an elongated cylinder ∼20 Å across and 40 Å long. The hexamer was a compact, oblate spheroid, formed by the coordination of 3 I dimers around the 2 Zn ions; 8.9 Å above and 8.9 Åbelow the 2-fold axis. Each Zn ion contacted the 3 B10 histidine residues and probably 3 O atoms from water. The metal coordination was 6-fold but not octahedral. The hexamer closely contacted the mols. around the 2-fold axis by the B1 III residues. A close, probably H-bonded contact occurred between the 6 glutamic acid residues B13, which enclosed a space surrounding the 3-fold axis. Many of the heavy atoms entered this space; others occupied regions on the hexamer surface, esp. at the glutamic acid residues. No heavy atoms penetrated the dimer.
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    29. 29
      Nanev, C. N.; Tonchev, V. D.; Hodzhaoglu, F. V. Protocol for Growing Insulin Crystals of Uniform Size. J. Cryst. Growth 2013, 375, 10– 15,  DOI: 10.1016/j.jcrysgro.2013.04.010
      29
      Protocol for growing insulin crystals of uniform size
      Nanev, Christo N.; Tonchev, Vesselin D.; Hodzhaoglu, Feyzim V.
      Journal of Crystal Growth (2013), 375 (), 10-15CODEN: JCRGAE; ISSN:0022-0248. (Elsevier B.V.)
      Guidelines for growing insulin crystals of a uniform size are formulated and tested exptl. A simple theor. model based on the balance of matter predicts the time evolution of the crystal size and supersatn. The time dependence of the size is checked exptl. The exptl. approach decouples crystal nucleation and growth processes according to the classical nucleation-growth-sepn. principle. The desired nucleation course was predetd. by carefully selecting the most suitable crystal nucleation parameters. Cryst. substance dispersity is predetd. during the nucleation stage of a batch crystn. process. To avert nutrition competition during the crystal growth stage, the no. d. of nucleated crystals is preset to be optimal.
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    30. 30
      Hodzhaoglu, F. V.; Conejero-Muriel, M.; Dimitrov, I. L.; Gavira, J. A. Optimization of the Classical Method for Nucleation and Growth of Rhombohedral Insulin Crystals by PH Titration and Screening. Bulg. Chem. Commun. 2016, 48, 29– 37
      There is no corresponding record for this reference.
    31. 31
      Contreras-Montoya, R.; Escolano, G.; Roy, S.; Lopez-Lopez, M. T.; Delgado-López, J. M.; Cuerva, J. M.; Díaz-Mochón, J. J.; Ashkenasy, N.; Gavira, J. A.; Álvarez de Cienfuegos, L. Catalytic and Electron Conducting Carbon Nanotube–Reinforced Lysozyme Crystals. Adv. Funct. Mater. 2019, 29, 1807351  DOI: 10.1002/adfm.201807351
      There is no corresponding record for this reference.
    32. 32
      Gao, Y.; Long, M. J. C.; Shi, J.; Hedstrom, L.; Xu, B. Using Supramolecular Hydrogels to Discover the Interactions between Proteins and Molecular Nanofibers of Small Molecules. Chem. Commun. 2012, 48, 8404– 8406,  DOI: 10.1039/c2cc33631f
      32
      Using supramolecular hydrogels to discover the interactions between proteins and molecular nanofibers of small molecules
      Gao, Yuan; Long, Marcus J. C.; Shi, Junfeng; Hedstrom, Lizbeth; Xu, Bing
      Chemical Communications (Cambridge, United Kingdom) (2012), 48 (67), 8404-8406CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      Here the authors report the first example of the use of supramol. hydrogels to discover the protein targets of aggregates of small mols.
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    33. 33
      Javid, N.; Roy, S.; Zelzer, M.; Yang, Z.; Sefcik, J.; Ulijn, R. V. Cooperative Self-Assembly of Peptide Gelators and Proteins. Biomacromolecules 2013, 14, 4368– 4376,  DOI: 10.1021/bm401319c
      33
      Cooperative Self-Assembly of Peptide Gelators and Proteins
      Javid, Nadeem; Roy, Sangita; Zelzer, Mischa; Yang, Zhimou; Sefcik, Jan; Ulijn, Rein V.
      Biomacromolecules (2013), 14 (12), 4368-4376CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      Mol. self-assembly provides a versatile route for the prodn. of nanoscale materials for medical and technol. applications. Herein, the cooperative self-assembly of amphiphilic small mols. and proteins can have drastic effects on supramol. nanostructuring of resulting materials. The authors report that mesoscale, fractal-like clusters of proteins form at concns. that are orders of magnitude lower compared to those usually assocd. with mol. crowding at room temp. These protein clusters have pronounced effects on the mol. self-assembly of arom. peptide amphiphiles (fluorenylmethoxycarbonyl- dipeptides), resulting in a reversal of chiral organization and enhanced order through templating and binding. Moreover, the morphol. and mech. properties of the resultant nanostructured gels can be controlled by the cooperative self-assembly of peptides and protein fractal clusters, having implications for biomedical applications where proteins and peptides are both present. In addn., fundamental insights into cooperative interplay of mol. interactions and confinement by clusters of chiral macromols. is relevant to gaining understanding of the mol. mechanisms of relevance to the origin of life and development of synthetic mimics of living systems.
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    34. 34
      Shenoy, B.; Wang, Y.; Shan, W.; Margolin, A. L. Stability of Crystalline Proteins. Biotechnol. Bioeng. 2001, 73, 358– 369,  DOI: 10.1002/bit.1069
      34
      Stability of crystalline proteins
      Shenoy, Bhami; Wang, Yi; Shan, Weizhong; Margolin, Alexey L.
      Biotechnology and Bioengineering (2001), 73 (5), 358-369CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)
      By using 2 proteins, glucose oxidase and lipase, we demonstrate that dry cryst. formulations are significantly more stable than their amorphous counterparts. The results of FT-spectroscopy indicate that cryst. proteins better maintain their native conformation in accelerated stability studies. The lower tendency of cryst. proteins to aggregate is confirmed by size-exclusion chromatog. Protein crystn. may significantly improve some aspects of protein handling, and change the way biopharmaceuticals are produced, formulated, and delivered.
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    35. 35
      Chen, M.-C.; Ling, M.-H.; Kusuma, S. J. Poly-γ-Glutamic Acid Microneedles with a Supporting Structure Design as a Potential Tool for Transdermal Delivery of Insulin. Acta Biomater. 2015, 24, 106– 116,  DOI: 10.1016/j.actbio.2015.06.021
      35
      Poly-γ-glutamic acid microneedles with a supporting structure design as a potential tool for transdermal delivery of insulin
      Chen, Mei-Chin; Ling, Ming-Hung; Kusuma, Setiawan Jati
      Acta Biomaterialia (2015), 24 (), 106-116CODEN: ABCICB; ISSN:1742-7061. (Elsevier Ltd.)
      Incomplete insertion is a common problem assocd. with polymer microneedles (MNs) that results in a limited drug delivery efficiency and wastage of valuable medication. This paper presents a fully insertable MN system that is composed of poly-γ-glutamic acid (γ-PGA) MNs and polyvinyl alc. (PVA)/polyvinyl pyrrolidone (PVP) supporting structures. The PVA/PVP supporting structures were designed to provide an extended length for counteracting skin deformation during insertion and mech. strength for fully inserting the MNs into the skin. When inserted into the skin, both the supporting structures and MNs can be dissolved in the skin within 4 min, thus quickly releasing the entire drug load from the MNs. To evaluate the feasibility and reproducibility of using the proposed system for treating diabetes, we administered insulin-loaded MNs to diabetic rats once daily for 2 days. The results indicated that the hypoglycemic effect in the rats receiving insulin-loaded MNs was comparable to that obsd. in rats receiving s.c. insulin injections. The relative pharmacol. availability and relative bioavailability of the insulin were in the range of 90-97%, indicating that the released insulin retained its pharmacol. activity. We obsd. no significant differences in the plasma insulin concn. profiles between the first and second administrations, confirming the stability and accuracy of using the proposed MN system for insulin delivery. These results indicated that the γ-PGA MNs contg. the supporting structure design enable complete and efficient delivery of encapsulated bioactive mols. and have great potential for the relatively rapid and convenient transdermal delivery of protein drugs. Incomplete insertion of microneedles largely limits drug delivery efficiency and wastage of valuable medication. To address this problem, we developed a fully insertable poly-glutamic acid microneedles with a supporting structure design to ensure complete and efficient delivery of encapsulated drugs. The supporting structures were designed to provide an extended length for counteracting skin compressive deformation during puncture and mech. strength for fully inserting the microneedles into the skin. When inserted into the skin, both the supporting structures and microneedles can be dissolved in the skin within 4 min, thus quickly releasing the entire drug load. This study demonstrated that the proposed microneedle system featuring this unique design allows more convenient and efficient self-administration of drugs into the skin.
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    36. 36
      Jabbari, N.; Asghari, M.; Ahmadian, H.; Mikaili, P. Developing a Commercial Air Ultrasonic Ceramic Transducer to Transdermal Insulin Delivery. J. Med. Signals Sens. 2015, 5, 117– 122,  DOI: 10.4103/2228-7477.157618
      36
      Developing a Commercial Air Ultrasonic Ceramic Transducer to Transdermal Insulin Delivery
      Jabbari Nasrollah; Asghari Mohammad Hossein; Ahmadian Hassan; Mikaili Peyman
      Journal of medical signals and sensors (2015), 5 (2), 117-22 ISSN:2228-7477.
      The application of low-frequency ultrasound for transdermal delivery of insulin is of particular public interest due to the increasing problem of diabetes. The purpose of this research was to develop an air ultrasonic ceramic transducer for transdermal insulin delivery and evaluate the possibility of applying a new portable and low-cost device for transdermal insulin delivery. Twenty-four rats were divided into four groups with six rats in each group: one control group and three experimental groups. Control group (C) did not receive any ultrasound exposure or insulin (untreated group). The second group (T1) was treated with subcutaneous insulin (Humulin(®) R, rDNA U-100, Eli Lilly and Co., Indianapolis, IN) injection (0.25 U/Kg). The third group (T2) topically received insulin, and the fourth group (T3) received insulin with ultrasound waves. All the rats were anesthetized by intraperitoneal injection of ketamin hydrochloride and xylazine hydrochloride. Blood samples were collected after anesthesia to obtain a baseline glucose level. Additional blood samples were taken every 15 min in the whole 90 min experiment. In order for comparison the changes in blood glucose levels" to " In order to compare the changes in blood glucose levels. The statistical multiple comparison (two-sided Tukey) test showed a significant difference between transdermal insulin delivery group (T2) and subcutaneous insulin injection group (T1) during 90 min experiment (P = 0.018). In addition, the difference between transdermal insulin delivery group (T2) and ultrasonic transdermal insulin delivery group (T3) was significant (P = 0.001). Results of this study demonstrated that the produced low-frequency ultrasound from this device enhanced the transdermal delivery of insulin across hairless rat skin.
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    37. 37
      Nolasco, E. L.; Zanoni, F. L.; Nunes, F. P. B.; Ferreira, S. S.; Freitas, L. A.; Silva, M. C. F.; Martins, J. O. Insulin Modulates Liver Function in a Type I Diabetes Rat Model. Cell. Physiol. Biochem. 2015, 36, 1467– 1479,  DOI: 10.1159/000430311
      37
      Insulin Modulates Liver Function in a Type I Diabetes Rat Model
      Nolasco, Eduardo L.; Zanoni, Fernando L.; Nunes, Fernanda P. B.; Ferreira, Sabrina S.; Freitas, Luiza A.; Silva, Mariana C. F.; Martins, Joilson O.
      Cellular Physiology and Biochemistry (2015), 36 (4), 1467-1479CODEN: CEPBEW; ISSN:1015-8987. (S. Karger AG)
      Background/Aims: Several studies have been performed to unravel the assocn. between diabetes and increased susceptibility to infection. This study aimed to investigate the effect of insulin on the local environment after cecal ligation and puncture (CLP) in rats. Methods: Diabetic (alloxan, 42 mg/kg i.v., 10 days) and non-diabetic (control) male Wistar rats were subjected to a two-puncture CLP procedure and 6 h later, the following analyses were performed:. (a) total and differential cell counts in peritoneal lavage (PeL) and bronchoalveolar lavage (BAL) fluids;. (b) quantification of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-10 and cytokine-induced neutrophil chemoattractant (CINC)-1 and CINC-2 in the PeL and BAL fluids by ELISA (ELISA);. (c) total leukocyte count using a veterinary hematol. analyzer and differential leukocyte counts on stained slides;. (d) biochem. parameters (urea, creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alk. phosphatase (ALP) by colorimetric analyses); and (e) lung, kidney, and liver morphol. analyses (hematoxylin and eosin staining). Results: Relative to controls, non-diabetic and diabetic CLP rats exhibited an increased in the concn. of IL-1β, IL-6, IL-10, CINC-1, and CINC-2 and total and neutrophil in the PeL fluid. Treatment of these animals with neutral protamine Hagedorn insulin (NPH, 1IU and 4IU, resp., s.c.), 2 h before CLP procedure, induced an increase on these cells in the PeL fluid but it did not change cytokine levels. The levels of ALT, AST, ALP, and urea were higher in diabetic CLP rats than in non-diabetic CLP rats. ALP levels were higher in diabetic sham rats than in non-diabetic sham rats. Treatment of diabetic rats with insulin completely restored ALT, AST, and ALP levels. Conclusion: These results together suggest that insulin attenuates liver dysfunction during early two-puncture CLP-induced peritoneal inflammation in diabetic rats.
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    38. 38
      Saito, S.; Thuc, L. C.; Teshima, Y.; Nakada, C.; Nishio, S.; Kondo, H.; Fukui, A.; Abe, I.; Ebata, Y.; Saikawa, T.; Moriyama, M.; Takahashi, N. Glucose Fluctuations Aggravate Cardiac Susceptibility to Ischemia/Reperfusion Injury by Modulating MicroRNAs Expression. Circ. J. 2016, 80, 186– 195,  DOI: 10.1253/circj.CJ-14-1218
      38
      Glucose fluctuations aggravate cardiac susceptibility to ischemia/reperfusion injury by modulating MicroRNAs expression
      Saito, Shotaro; Thuc, Luong Cong; Teshima, Yasushi; Nakada, Chisato; Nishio, Satoru; Kondo, Hidekazu; Fukui, Akira; Abe, Ichitaro; Ebata, Yuki; Saikawa, Tetsunori; Moriyama, Masatsugu; Takahashi, Naohiko
      Circulation Journal (2016), 80 (1), 186-195CODEN: CJIOBY; ISSN:1346-9843. (Japanese Circulation Society)
      Background: The influence of glucose fluctuations (GF) on cardiovascular complications of diabetes mellitus (DM) has been attracting much attention. In the present study, whether GF increase susceptibility to ischemia/reperfusion in the heart was investigated. Methods and Results: Male rats were randomly assigned to either a control, DM, and DM with GF group. DM was induced by an injection of streptozotocin, and glucose fluctuation was induced by starvation and insulin injection. One sequential program comprised 2 hypoglycemic episodes during 4 days. The isolated hearts were subjected to 20-min ischemia/30-min reperfusion. The infarct size was larger in hearts with GF than those with sustained hyperglycemia. Activities of catalase and superoxide dismutase were decreased, and expressions of NADPH oxidase and thioredoxin-interacting protein were upregulated by GF accompanied by an increase of reactive oxygen species (ROS). Swollen mitochondria with destroyed cristae were obsd. in diabetic hearts; they were further devastated by GF. Microarray anal. revealed that the expressions of microRNA (miRNA)-200c and miRNA-141 were abundant in those hearts with GF. Overexpression of miRNA-200c and miRNA-141 decreased mitochondrial superoxide dismutase and catalase activities, and increased ROS levels. Meanwhile, knockdown of miRNA-200c and miRNA- 141 significantly decreased ROS levels in cardiomyocytes exposed to GF. Conclusions: GF increased ROS generation and enhanced ischemia/reperfusion injury in the diabetic heart. Upregulated miRNA-200c and miRNA-141 may account for the increased ROS.
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    39. 39
      Hofmann, T.; Horstmann, G.; Stammberger, I. Evaluation of the Reproductive Toxicity and Embryotoxicity of Insulin Glargine (LANTUS) in Rats and Rabbits. Int. J. Toxicol. 2002, 21, 181– 189,  DOI: 10.1080/10915810290096315
      39
      Evaluation of the reproductive toxicity and embryotoxicity of insulin glargine (LANTUS) in rats and rabbits
      Hofmann, Thomas; Horstmann, Georg; Stammberger, Ingo
      International Journal of Toxicology (2002), 21 (3), 181-189CODEN: IJTOFN; ISSN:1091-5818. (Taylor & Francis Ltd.)
      Insulin glargine (LANTUS) is a new insulin analog that has a prolonged duration of action with no pronounced peak of activity, rendering it an ideal basal insulin for the treatment of diabetes. The aim of these studies was to assess the reproductive and embryotoxicity of insulin glargine. Reproductive toxicity was assessed in 25 male and 25 female Wistar rats per group treated with a daily s.c. injection of control; 1 IU/kg, 3 IU/kg, and 10 IU/kg insulin glargine; or 3 IU/kg NPH insulin in the premating and mating periods, and throughout pregnancy and lactation in the females. Embryotoxicity was assessed in 20 female rats per group injected with daily s.c. doses of control; 2 IU/kg, 6.3 IU/kg, and 20 IU/kg insulin glargine; or 6.3 IU/kg NPH insulin from the 7th to 18th day of pregnancy. Embryotoxicity was also assessed in 20 female rabbits per group treated with 0 IU/kg, 0.5 IU/kg, 1 IU/kg, and 2 IU/kg insulin glargine, or 1 IU/kg NPH insulin from the 6th to 18th day of pregnancy. The data demonstrated that, with the exception of toxicol. effects induced by hypoglycemia in response to high doses of insulin glargine and NPH insulin (including the premature dropout of female rats in the reproductive toxicity study, and increased incidence of abortions, early intrauterine deaths, and single anomalies in the rabbit embryotoxicity study), insulin glargine had no effects on reprodn., embryo-fetal development, and postnatal development in rats. Maternal and embryo-fetal toxicity in rabbits treated with middle and high doses of insulin glargine was related to the hypoglycemic effect of insulin.
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsami.1c00639.

    • SEM images of all insulin crystals samples (Figure S1); HPLC calibration curve (Figure S2); HPLC analysis of control crystals (Figure S3); mass spectra of all samples (Figure S4); contrast phase microscopy of the two cell lines used to study the cytotoxicity (Figure S5) (PDF)


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