Design of hybrid hydrogels with self-assembled nanogels as cross-linkers: interaction with proteins and chaperone-like activity

New hybrid hydrogels with nanogel domains were obtained by using polymerizable self-assembled nanogels as cross-linkers. Methacryloyl groups were introduced to cholesteryl group-bearing pullulan (CHP). The methacryloyl group-bearing CHPs (CHPMAs) formed nanogels by their self-association in water (R(g) = 14-17 nm). CHPMA nanogels were polymerized with 2-methacryloyloxyethyl phosphorylcholine (MPC) by radical polymerization in a semidilute aqueous solution. CHPMA nanogels acted as effective cross-linkers for gelation. TEM observation showed that the nanogel structure was retained after gelation and that the nanogels were well dispersed in the macrogel. The hybrid hydrogels showed two well-defined networks such as a nanogel intranetwork structure of less than 10 nm (physically cross-linking) and an internetwork structure of several hundred nanometers (chemically cross-linking). The immobilized nanogels retained their ability to trap and release protein (insulin was used as a model protein) by host-guest interaction of the cholesteryl group and cyclodextrin and also showed high chaperone-like activity for refolding of chemically denatured protein.     

Thiol-ene photopolymerizations provide a facile method to encapsulate proteins and maintain their bioactivity

Photoinitiated polymerization remains a robust method for fabrication of hydrogels, as these reactions allow facile spatial and temporal control of gelation and high compatibility for encapsulation of cells and biologics. The chain-growth reaction of macromolecular monomers, such as acrylated PEG and hyaluronan, is commonly used to form hydrogels, but there is growing interest in step-growth photopolymerizations, such as the thiol-ene "click" reaction, as an alternative. Thiol-ene reactions are not susceptible to oxygen inhibition and rapidly form hydrogels using low initiator concentrations. In this work, we characterize the differences in recovery of bioactive proteins when exposed to similar photoinitiation conditions during thiol-ene versus acrylate polymerizations. Following exposure to chain polymerization of acrylates, lysozyme bioactivity was approximately 50%; after step-growth thiol-ene reaction, lysozyme retained nearly 100% of its prereaction activity. Bioactive protein recovery was enhanced 1000-fold in the presence of a thiol-ene reaction, relative to recovery from solutions containing identical primary radical concentrations, but without the thiol-ene components. When the cytokine TGFβ was encapsulated in PEG hydrogels formed via the thiol-ene reaction, full protein bioactivity was preserved.     

Structure and properties of silk hydrogels

Control of silk fibroin concentration in aqueous solutions via osmotic stress was studied to assess relationships to gel formation and structural, morphological, and functional (mechanical) changes associated with this process. Environmental factors potentially important in the in vivo processing of aqueous silk fibroin were also studied to determine their contributions to this process. Gelation of silk fibroin aqueous solutions was affected by temperature, Ca(2+), pH, and poly(ethylene oxide) (PEO). Gelation time decreased with increase in protein concentration, decrease in pH, increase in temperature, addition of Ca(2+), and addition of PEO. No change of gelation time was observed with the addition of K(+). Upon gelation, a random coil structure of the silk fibroin was transformed into a beta-sheet structure. Hydrogels with fibroin concentrations >4 wt % exhibited network and spongelike structures on the basis of scanning electron microscopy. Pore sizes of the freeze-dried hydrogels were smaller as the silk fibroin concentration or gelation temperature was increased. Freeze-dried hydrogels formed in the presence of Ca(2+) exhibited larger pores as the concentration of this ion was increased. Mechanical compressive strength and modulus of the hydrogels increased with increase in protein concentration and gelation temperature. The results of these studies provide insight into the sol-gel transitions that silk fibroin undergoes in glands during aqueous processing while also providing important insight in the in vitro processing of these proteins into useful new materials.     

Cross-linking and degradation of step-growth hydrogels formed by thiol-ene photoclick chemistry

Thiol-ene photoclick hydrogels have been used for a variety of tissue engineering and controlled release applications. In this step-growth photopolymerization scheme, four-arm poly(ethylene glycol) norbornene (PEG4NB) was cross-linked with dithiol containing cross-linkers to form chemically cross-linked hydrogels. While the mechanism of thiol-ene gelation was well described in the literature, its network ideality and degradation behaviors are not well-characterized. Here, we compared the network cross-linking of thiol-ene hydrogels to Michael-type addition hydrogels and found thiol-ene hydrogels formed with faster gel points and higher degree of cross-linking. However, thiol-ene hydrogels still contained significant network nonideality, demonstrated by a high dependency of hydrogel swelling on macromer contents. In addition, the presence of ester bonds within the PEG-norbornene macromer rendered thiol-ene hydrogels hydrolytically degradable. Through validating model predictions with experimental results, we found that the hydrolytic degradation of thiol-ene hydrogels was not only governed by ester bond hydrolysis, but also affected by the degree of network cross-linking. In an attempt to manipulate network cross-linking and degradation of thiol-ene hydrogels, we incorporated peptide cross-linkers with different sequences and characterized the hydrolytic degradation of these PEG-peptide hydrogels. In addition, we incorporated a chymotrypsin-sensitive peptide as part of the cross-linkers to tune the mode of gel degradation from bulk degradation to surface erosion.     

Molecular architecture of the rod domain of the Dictyostelium gelation factor (ABP120).

The Dictyostelium discoideum gelation factor is a two-chain actin-cross-linking protein that, in addition to an N-terminal actin-binding domain, has a rod domain constructed from six tandem repeats of a 100-residue motif that has an immunoglobulin fold. To define the architecture of the rod domain of gelation factor, we have expressed in E. coli a series of constructs corresponding to different numbers of gelation factor rod repeats and have characterised them by chemical crosslinking, ultracentrifugation, column chromatography, matrix-assisted laser desorption ionisation (MALDI) mass spectrometry and NMR spectroscopy. Fragments corresponding to repeats 1-6 and 5-6 dimerise, whereas repeats 1-5 and single repeats 3 and 4 are monomeric. Repeat 6 interacts weakly and was present as monomer and dimer when analysed by analytical ultracentrifugation. Proteolytic digestion of rod5-6 resulted in the generation of two polypeptides that roughly corresponded to rod5 and part of rod6. None of these polypeptides formed dimers after chemical crosslinking. Stable dimerisation therefore appears to require repeats 5 and 6. Based on these data a model of gelation factor architecture is presented. We suggest an arrangement of the chains where only the carboxy-terminal repeats interact as was observed for filamin/ABP280, the mammalian homologue of gelation factor.     

Carbon dioxide induced silk protein gelation for biomedical applications

We present a novel method to fabricate silk fibroin hydrogels using high pressure carbon dioxide (CO(2)) as a volatile acid without the need for chemical cross-linking agents or surfactants. The simple and efficient recovery of CO(2) post processing results in a remarkably clean production method offering tremendous benefit toward materials processing for biomedical applications. Further, with this novel technique we reveal that silk protein gelation can be considerably expedited under high pressure CO(2) with the formation of extensive β-sheet structures and stable hydrogels at processing times less than 2 h. We report a significant influence of the high pressure CO(2) processing environment on silk hydrogel physical properties such as porosity, sample homogeneity, swelling behavior and compressive properties. Microstructural analysis revealed improved porosity and homogeneous composition among high pressure CO(2) specimens in comparison to the less porous and heterogeneous structures of the citric acid control gels. The swelling ratios of silk hydrogels prepared under high pressure CO(2) were significantly reduced compared to the citric acid control gels, which we attribute to enhanced physical cross-linking. Mechanical properties were found to increase significantly for the silk hydrogels prepared under high pressure CO(2), with a 2- and 3-fold increase in the compressive modulus of the 2 and 4 wt % silk hydrogels over the control gels, respectively. We adopted a semiempirical theoretical model to elucidate the mechanism of silk protein gelation demonstrated here. Mechanistically, the rate of silk protein gelation is believed to be a function of the kinetics of solution acidification from absorbed CO(2) and potentially accelerated by high pressure effects. The attractive features of the method described here include the acceleration of stable silk hydrogel formation, free of residual mineral acids or chemical cross-linkers, reducing processing complexity, and avoiding adverse biological responses, while providing direct manipulation of hydrogel physical properties for tailoring toward specific biomedical applications.     

Synthesis and characterizations of in situ cross-linkable gelatin and 4-arm-PPO-PEO hybrid hydrogels via enzymatic reaction for tissue regenerative medicine

In situ cross-linkable hybrid hydrogels composed of gelatin and 4-arm-polypropylene oxide-polyethylene oxide (Tetronic) was developed as an injectable scaffold for tissue regeneration. The gelatin was modified by hydroxyphenyl propionic acid (HPA) and the Tetronic was conjugated with tyramines (Tet-TA). The hydrogels were rapidly formed by mixing the polymer solutions containing horseradish peroxidase (HRP) and hydrogen peroxide (H(2)O(2)). The gelation time and mechanical properties of the hydrogels could be controlled by varying the HRP and H(2)O(2) concentrations. In vitro degradation study of the hybrid hydrogels was carried out using collagenase and the prolonged proteolytic degradation was obtained due to the presence of the Tetronic. Human dermal fibroblast (hDFB) was cultured in the hydrogel matrices to evaluate the cyto-compatibility. The encapsulated cells were shown to be highly viable and spread over the gel matrices, suggesting that the hybrid hydrogels have an excellent cyto-compatibility. The hydrogels were also subcutaneously injected in the back of mice and the results demonstrated that the hydrogels were rapidly formed at the injected site. From these results, we demonstrate that the in situ cross-linkable hydrogels formed by hybridization of gelatin and Tetronic via enzyme-mediated reactions hold great promise for use as injectable matrices for tissue regenerative medicine due to their tunable physico-chemical properties and excellent bioactivity.     

Controlled hydrogel formation of a recombinant spider silk protein

Due to their biocompatibility, biodegradability, and low immunogenicity, recombinant spider silk proteins have a high potential for a variety of applications when processed into morphologies such as films, capsules, beads, or hydrogels. Here, hydrogels made of the engineered and recombinantly produced spider silk protein eADF4(C16) were analyzed in detail. It has previously been shown that eADF4(C16) nanofibrils self-assemble by a mechanism of nucleation-aggregation, providing the basis of silk hydrogels. We focused on establishing a reproducible gelation process by employing different protein concentrations, chemical crosslinking, and functionalization of eADF4(C16) with fluorescein. Fluorescein strongly influenced assembly as well as the properties of the hydrogels, such as pore sizes and mechanical behavior, possibly due to its interference with packing of silk nanofibrils during hydrogel formation.     

Synthesis and gelation of copolypept(o)ides with random and block structure

Copolypept(o)ides of polysarcosine (PSar) and poly(N‐isopropyl‐L‐glutamine) (PIGA) with random and block sequence structures were synthesized by ring‐opening polymerization (ROP) of sarcosine N‐carboxyanhydrides (Sar‐NCA) and γ‐benzyl‐l ‐glutamate N‐carboxyanhydrides (BLG‐NCA) and post modification. With different distribution of Sar along the main chain, H‐bonding pattern and secondary structure of polypeptides were turned, as well as aggregation and gelation behavior. Both copolypept(o)ides formed hydrogels above their critical gelation concentrations (CGCs) without thermo‐sensitivity, which was normally reserved for PEG copolypeptides (eg, PEG‐b‐PIGA). In particular, a different mechanism from previously reported micellar percolation or fibrillar entanglement was suggested for gelation of the random copolypept(o)ide. Therefore, hydrogels from copolymers of PSar and PIGA represented a new approach to construct easy‐handling, biocompatible, biodegradable and thermo‐stable gels that could potentially be applied in biomedical fields.     

Rapid cross-linking of elastin-like polypeptides with (hydroxymethyl)phosphines in aqueous solution

In situ gelation of injectable polypeptide-based materials is attractive for minimally invasive in vivo implantation of biomaterials and tissue engineering scaffolds. We demonstrate that chemically cross-linked elastin-like polypeptide (ELP) hydrogels can be rapidly formed in aqueous solution by reacting lysine-containing ELPs with an organophosphorous cross-linker, beta-[tris(hydroxymethyl)phosphino]propionic acid (THPP) under physiological conditions. The mechanical properties of the cross-linked ELP hydrogels were largely modulated by the molar concentration of lysine residues in the ELP and the pH at which the cross-linking reaction was carried out. Fibroblasts embedded in ELP hydrogels survived the cross-linking process and were viable after in vitro culture for 3 days. DNA quantification of ELP hydrogels with encapsulated fibroblasts indicated that there was no significant difference in DNA content between day 0 and day 3 when ELP hydrogels were formed with an equimolar ratio of THPP and lysine residues of the ELPs. These results suggest that THPP cross-linking may be a biocompatible strategy for the in situ formation of cross-linked hydrogels.     

Morphology and gelation of thermosensitive xyloglucan hydrogels

Galactose modified xyloglucan is a thermally reversible hydrogel that is increasingly used in the biomedical field due to the ease of altering the gelation time and temperature by modifying the galactose removal ratio. However there is little information concerning the morphology and rheological properties of the hydrogel under physiological conditions. Differential scanning microcalorimetry (DSmicroC) showed the thermal gelation process to occur over a broad temperature range (5-50 degrees C). The rheological properties of the hydrogels were investigated as a function of concentration, temperature and ionic strength. The final elastic moduli of the hydrogels increased with increases in concentration. Isothermal rheology suggests that the gelation occurred in two distinct stages, which was influenced by the solution media. Scanning electron microscopy (SEM) was used to characterize the morphology of the xyloglucan which were thermally gelled at 37 degrees C. The resultant morphology was strongly dependent on the concentration of the hydrogel. Strong hydrogels were only obtained at 3 wt.% at 37 degrees C, and the morphology characterized by an open 3-dimensional network, comprised of thin membranes. It is proposed that the first stage of the isothermal gelation is the formation and growth of the thin membranes, followed by the formation of a three dimensional network.     

Crosslinking of Chitosan with Dialdehyde Derivatives of Nucleosides and Nucleotides. Mechanism and Comparison with Glutaraldehyde

In medical and pharmaceutical applications, chitosan is used as a component of hydrogels–macromolecular networks swollen in water. Chemical hydrogels are formed by covalent links between the crosslinking reagents and amino functionalities of chitosan. To date, the most commonly used chitosan crosslinkers are dialdehydes, such as glutaraldehyde (GA). We have developed novel GA like crosslinkers with additional functional groups–dialdehyde derivatives of uridine (oUrd) and nucleotides (oUMP and oAMP)–leading to chitosan-based biomaterials with new properties. The process of chitosan crosslinking was investigated in details and compared to crosslinking with GA. The rates of crosslinking with oUMP, oAMP, and GA were essentially the same, though much higher than in the case of oUrd. The remarkable difference in the crosslinking properties of nucleoside and nucleotide dialdehydes can be clearly attributed to the presence of the phosphate group in nucleotides that participates in the gelation process through ionic interactions with the amino groups of chitosan. Using NMR spectroscopy, we have not observed the formation of aldimine bonds. It can be concluded that the real number of crosslinks needed to cause gelation of chitosan chains may be less than 1%.     

Rheometric study of the gelation of chitosan in aqueous solution without cross-linking agent

A new process of formation of chitosan physical hydrogels in aqueous solution, without any organic solvent or cross-linking additive, was studied. The three conditions required for the physical gelation were an initial polymer concentration over C*, a critical value of the balance between hydrophilic and hydrophobic interactions, and a physicochemical perturbation responsible for a bidimensional percolating mechanism. The time necessary to reach the gel point was determined by rheometry, and gelations were compared according to different initial conditions. Thus, we investigated the influence of the polymer concentration and the degree of acetylation (DA) of chitosan on gelation. The number of junctions per unit volume at the gel point varied with the initial polymer concentration, i.e., the initial number of chain entanglements per unit volume or the number of gel precursors. The time to reach the gel point decreased with both higher DAs and concentrations. For a chitosan of DA = 36.7%, a second critical initial concentration close to 1.8% (w/w) was observed. Above this concentration, the decrease of the time to reach the gel point was higher and fewer additional junctions had to be formed to induce gelation. To optimize these physical hydrogels, to be used for cartilage regeneration, their final rheological properties were studied as a function of their degree of acetylation and their polymer concentration. Our results allowed us to define the most appropriate gel for the targeted application corresponding to a final concentration of chitosan in the gel of near 1.5% (w/w) and a DA close to 40%.     

Diepoxide-triggered conformational transition of silk fibroin: formation of hydrogels

Silk fibroin hydrogels with tunable properties could be obtained from aqueous fibroin solutions (4.2 w/v %) in a short period of time. This was achieved by the addition of ethylene glycol diglycidyl ether (EGDE) into the reaction solution. Introduction of EGDE cross-links between the fibroin molecules decreases the mobility of the chains, which triggers the conformational transition from random-coil to β-sheet structure and hence fibroin gelation. Dynamic rheological measurements conducted at 50 °C show the formation of strong to weak hydrogels depending on the pH of the reaction solution. Although EGDE attacks the amino groups of fibroin and forms interstrand cross-links, β-sheets acting as physical cross-links dominate the elasticity of the hydrogels. Mechanical response of low-modulus fibroin hydrogels formed above pH 9.7 is highly nonlinear with strong strain hardening behavior (700%) arising from the alignment of the crystallizable amino acid segments.     

Characterization of the spontaneously forming hydrogels composed of water-soluble phospholipid polymers

Spontaneously forming hydrogels composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymers, poly(MPC-co-methacrylic acid) (PMA), and poly(MPC-co-n-butyl methacrylate) (PMB) were examined. The MPC copolymer hydrogel was observed to have a spontaneous gelation property. To determine the properties of the hydrogels and why the gelation takes place, we have studied the properties of the hydrogels by scanning electron microscopy, X-ray photoelectron spectroscopy (XPS), and differential scanning calorimetry (DSC). The morphologies of the hydrogels were spongelike with a homogeneous structure. By XPS analysis in terms of the molecular distributions in the hydrogels, it was observed that a stabilization time was required for the hydrogel to undergo chain rearrangement. DSC thermograms of the hydrogels were different from their components, PMA and PMB. For the hydrogel, a crystallization peak around -30 degrees C was observed. This result indicated that some ordered structures existed in the hydrogels. To determine the role of the MPC groups, aqueous solutions of poly(methacrylic acid) (PMAc) and PMB were mixed. The mixture of PMAc-PMB turned into a sol state, and the sol state remained for a week. When the mixture was cooled, a very weak hydrogel was prepared. This result suggested that the MPC groups were the dominant unit for spontaneously forming the hydrogels.     

Synthesis and characterization of hydrogels based on grafted chitosan for the controlled drug release

Temperature and pH-responsive hydrogels based on chitosan grafted with poly acrylic acid (PAAc), poly hydroxy propyl methacrylate (PHPMA), poly (vinyl alcohol) (PVA) and gelatin were prepared for controlled drug delivery. These stimuli-responsive hydrogels were synthesized by gamma irradiation technique. The degree of gelation was over 90% and increased as chitosan, AAc and PVA content increased, while the degree of gelation decrease with the increase of gelatin content. The equilibrium swelling studies of hydrogels prepared in various conditions were carried out in an aqueous solution, and the pH sensitivity in the range of 2–9 was investigated. An increase of swelling degree with an increase in the pH was noticed and showed the highest value at pH 9. Also antibiotic drug Oxttetracycline was loaded into the hydrogels and the release studies were carried out at different pH and temperature. The in vitro release profiles of the drug showed that, the release of the drug increased as the time, temperature and pH increased and reached to maximum after 48 h at pH 9. The prepared hydrogels were characterized by using SEM, FTIR, and DSC.     

New aspects of the formation of physical hydrogels of chitosan in a hydroalcoholic medium

New aspects concerning the mechanism of formation of chitosan physical hydrogels without any cross-linking agent were studied. The gelation took place during the evaporation of a hydroalcoholic solution of chitosan. We first demonstrated that it was possible to form a physical hydrogel from a hydrochloride form of chitosan. Chromatographic methods showed that during the gel formation, when the initial concentration is over C, the critical concentration of chain entanglement, the water and acid used for the solubilization of the polymer were both eliminated. This particular situation contributed to decrease the dielectric constant of the medium and the apparent charge density of chitosan chains, thus inducing the formation of a three-dimensional network through hydrophobic interactions and hydrogen bonding. In the gelation process, this step was kinetically determining. The speed of evaporation of water and acid were determined and different initial conditions were compared. Thus, we investigated the influence of: the initial polymer concentration, the nature of the counterion and the alcohol, the temperature and the geometry of the reactor. Our results allowed us to confirm the existence of a second critical initial concentration C, from which the evaporation of water became more difficult. We suggested that C corresponded to a reorganization of the solution involving the presence of gel precursors. Then, a mechanism of formation of physical hydrogels of chitosan in a hydroalcoholic medium could be proposed. For the first time, we demonstrated that it was possible to generate physical hydrogels in the presence of various diols, which size of the carbonated chain appeared as a limiting factor for the gelation process. These physical hydrogels of chitosan are currently used in our laboratory for tissue engineering in the treatment of third degree burns with the possibility to adapt their mechanical properties from the choice of both the acid or the alcohol used.     

Characterization and in vitro evaluation of starch based hydrogels as carriers for colon specific drug delivery systems

A series of starch/methacrylic acid (MAAc) copolymer hydrogels of different compositions were synthesized using γ-rays induced polymerization and crosslinking. The effects of the preparation conditions such as the feed solution concentration, feed solution composition and irradiation dose on the gelation process of the synthesized copolymer were investigated. The swelling behavior of the starch/methacrylic acid (MAAc) copolymer hydrogels was characterized by studying the effect of the hydrogel composition on the time- and pH-dependent swelling. Swelling kinetics showed that the synthesized hydrogels possessed Fickian diffusion behavior at pH 1 and non-Fickian diffusion at pH 7 which recommend them as good candidate for colon specific drug delivery systems. The synthesized hydrogels were loaded with ketoprofen as a model drug to investigate the release behavior of the synthesized hydrogels. The results showed the ability of the hydrogels to keep the loaded drug at pH 1 and release it at pH 7. The data also showed that the release rate can be controlled by controlling the preparation conditions such as comonomer concentration and composition and irradiation dose.     

Characterization of protein release from hydrolytically degradable poly(ethylene glycol) hydrogels

We present a novel fully hydrophilic, hydrolytically degradable poly(ethylene glycol) (PEG) hydrogel suitable for soft tissue engineering and delivery of protein drugs. The gels were designed to overcome drawbacks associated with current PEG hydrogels (i.e., reaction mechanisms or degradation products that compromise protein stability): the highly selective and mild cross‐linking reaction allowed for encapsulating proteins prior to gelation without altering their secondary structure as shown by circular dichroism experiments. Further, hydrogel degradation and structure, represented by mesh size, were correlated to protein release. It was determined that polymer density had the most profound effect on protein diffusivity, followed by the polymer molecular weight, and finally by the specific chemical structure of the cross‐linker. By examining the diffusion of several model proteins, we confirmed that the protein diffusivity was dependent on protein size as smaller proteins (e.g., lysozyme) diffused faster than larger proteins (e.g., Ig). Furthermore, we demonstrated that the protein physical state was preserved upon encapsulation and subsequent release from the PEG hydrogels and contained negligible aggregation or protein–polymer adducts. These initial studies indicate that the developed PEG hydrogels are suitable for release of stable proteins in drug delivery and tissue engineering applications. Biotechnol. Bioeng. 2011; 108:197–206. © 2010 Wiley Periodicals, Inc.     

Rapid, high resolution screening of biomaterial hydrogelators by μ2rheology

A combination of sample manipulation and rheological characterization at the microscale is used to identify the gelation of poly(ethylene glycol)-heparin hydrogels over a wide range of compositions. A microfluidic device produces 50-100 droplet samples, each with a different composition. Multiple particle tracking microrheology is used to measure the rheological state of each sample. This combination requires little material and enables efficient and rapid screening of gelation conditions. The high resolution data identifies the gelation reaction percolation boundaries and a lower limit of the total hydrogelator concentration for gelation to occur, which can be used for the subsequent engineering, testing, and processing of these materials.