The study of gelation kinetics and chain-relaxation properties of glutaraldehyde-cross-linked chitosan gel and their effects on microspheres preparation and drug release

The effects of gelation kinetics and chain-relaxation properties of glutaraldehyde-cross-linked chitosan gel on microspheres preparation or drug release were studied. The rate of gelation is zero order corresponding to the chitosan concentration but non-zero order corresponding to the glutaraldehyde concentration. It was suggested that the cross-linking reaction was mainly dominated by the concentration of small molecule reactant, glutaraldehyde. The relaxation of an entangled polymer chain in a gel network as a result of the swelling of cross-linked chitosan hydrogel was investigated by the stress–strain determination. The higher the cross-linking density of chitosan hydrogel, the lower the swelling ability of chitosan hydrogel due to the slower relaxation rate of polymer chain, which then results in the decreased drug-release rate.     

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.     

Effect of chemistry and morphology on the biofunctionality of self-assembling diblock copolypeptide hydrogels

Amphiphilic, diblock copolypeptides of hydrophilic lysine or glutamic acid and hydrophobic leucine or valine have been observed to self-assemble into rigid hydrogels in aqueous solution at neutral pH and very low volume fraction of polymer, > or =0.5 wt % polypeptide. Laser scanning confocal microscopy and ultra small angle neutron scattering revealed a heterogeneous microstructure with distinct domains of hydrogel matrix and pure water pores. In situ nanoscale characterization, using cryogenic transmission electron microscopy, revealed a porous, interconnected membranous network of assembled polypeptides. At concentrations of polypeptide below gelation, diblocks containing lysine were cytotoxic to cells, whereas those containing glutamic acid were noncytotoxic. At higher polypeptide concentrations, within rigid gel scaffolds, both lysine and glutamic acid based diblocks were noncytotoxic but did not support cell attachment/proliferation. The cationic chemistry observed as cytotoxic in the fluid state was essentially inert in the intact, rigid hydrogel state.     

A small-angle x-ray scattering study of alginate solution and its Sol–Gel transition by addition of divalent cations

The small-angle x-ray scattering (SAXS) technique has been applied to investigate solution and gel structures of alginate in the absence and presence of two divalent cations: Ca(II) and Cu(II). We have observed a broad maximum in the scattering curve, a characteristic of polyelectrolyte, for the purified alginate sample. The scattering maximum disappears in excess of added simple salt and shifts toward the higher angle region with increasing alginate concentration. Concentration dependence of the position and intensity of the maximum follows power law relations with exponents close to those predicted by theory. Data analysis shows an increase in correlation length ξ and cross-sectional diameter d₀, of polymer chains upon gelation and suggests that a dimeric structure is adopted in the junction zone, consistent with the “egg-box” model previously proposed. In the Ca(II)–alginate system, the molecular parameters ξ and d₀ are found to have good correlation with the macroscopic properties of gelation, such as gel point determined by viscosity measurements. However, for the Cu(II)–alginate system there is no clearly transitional behavior observed in ξ and d₀, implying that the junction zone may be replaced by a more uniformly distributed site binding of Cu(II) ions to the carboxyl groups of both mannuronate and guluronate residues, in confirmation of previous ¹³C-nmr results. © 1995 John Wiley & Sons, Inc.     

Triphasic mixture model of cell-mediated enzymatic degradation of hydrogels

One critical component of engineering living tissue equivalents is the design scaffolds (often made of hydrogels) whose degradation kinetics can match that of matrix production by cells. However, cell-mediated enzymatic degradation of a hydrogel is a highly complex and nonlinear process that is challenging to comprehend based solely on experimental observations. To address this issue, this study presents a triphasic mixture model of the enzyme-hydrogel system, which consists of a solid polymer network, water and enzyme. On the basis mixture theory, the rubber elasticity theory and the Michaelis-Menton kinetics for degradation, the model naturally incorporates a strong coupling between gel mechanical properties, the kinetics of degradation and the transport of enzyme through the gel. The model is then used to investigate the particular problem of a single spherical enzyme-producing cell, embedded in a spherical hydrogel domain, for which the governing equations can be cast within the cento-symmetric assumptions. The governing equations are subsequently solved using an implicit nonlinear finite element procedure to obtain the evolution of enzyme concentration and gel degradation through time and space. The model shows that two regimes of degradation behaviour exist, whereby degradation is dominated either by diffusion or dominated by reaction kinetics. Depending on the enzyme properties and the initial hydrogel design, the temporal and spatial changes in gel cross-linking are dramatically impacted, a feature that is likely to strongly affect new tissue development.     

Rheological and structural properties of aqueous alginate during gelation via the Ugi multicomponent condensation reaction

Turbidity, structure, and rheological features during gelation via the Ugi multicomponent condensation reaction of semidilute solutions of alginate have been investigated at different polymer and cross-linker concentrations and reaction temperatures. The gelation time of the system decreased with increasing polymer and cross-linker concentrations, and a temperature rise resulted in a faster gelation. At the gel point, a power law frequency dependence of the dynamic storage modulus (G' proportional, variant omega(n)(')) and loss modulus (G' ' proportional, variant omega(n)(' ')) was observed for all gelling systems with n' = n' ' = n. By varying the cross-linker density at a fixed polymer concentration (2.2 wt %), the power law exponent is consistent with that predicted (0.7) from the percolation model. The value of n decreases with increasing polymer concentration, whereas higher temperatures give rise to higher values of n. The elastic properties of the gels continue to grow over a long time in the postgel region, and at later stages in the gelation process, a solidlike response is observed. The turbidity of the gelling system increases as the gel evolves, and this effect is more pronounced at higher cross-linker concentration. The small-angle neutron scattering results reveal large-scale inhomogeneities of the gels, and this effect is enhanced as the cross-linker density increases. The structural, turbidity, and rheological features were found to change over an extended time after the formation of the incipient gel. It was demonstrated that temperature, polymer, and cross-linker concentrations could be utilized to tune the physical properties of the Ugi gels such as structure, transparency, and viscoelasticity.     

Triphasic mixture model of cell-mediated enzymatic degradation of hydrogels

One critical component of engineering living tissue equivalents is the design scaffolds (often made of hydrogels) whose degradation kinetics can match that of matrix production by cells. However, cell-mediated enzymatic degradation of a hydrogel is a highly complex and nonlinear process that is challenging to comprehend based solely on experimental observations. To address this issue, this study presents a triphasic mixture model of the enzyme–hydrogel system, which consists of a solid polymer network, water and enzyme. On the basis mixture theory, the rubber elasticity theory and the Michaelis–Menton kinetics for degradation, the model naturally incorporates a strong coupling between gel mechanical properties, the kinetics of degradation and the transport of enzyme through the gel. The model is then used to investigate the particular problem of a single spherical enzyme-producing cell, embedded in a spherical hydrogel domain, for which the governing equations can be cast within the cento-symmetric assumptions. The governing equations are subsequently solved using an implicit nonlinear finite element procedure to obtain the evolution of enzyme concentration and gel degradation through time and space. The model shows that two regimes of degradation behaviour exist, whereby degradation is dominated either by diffusion or dominated by reaction kinetics. Depending on the enzyme properties and the initial hydrogel design, the temporal and spatial changes in gel cross-linking are dramatically impacted, a feature that is likely to strongly affect new tissue development.     

Intramolecular disorder and its relation to mesophase structure in lipid/water mixtures.

NMR spin-half pair dipolar echo measurements are reported for the lamellar (dispersions and multibilayer stacks) and hexagonal phases of potassium palmitate/2H2O mixtures. In the lamellar Lbeta and Lgamma (gel) phases the alkyl chains are rigid and perfectly ordered, while in the lamellar Lalpha and hexagonal phases they are flexible and disordered. In particular, the measurements show that in the fluid lamellar Lalpha phase the chain is "bent" at the C9-C10 segment; but is "straight" in the hexagonal phase.     

Spin-label electron spin resonance studies of micellar dispersions of PEGs-PEs polymer-lipids

Conventional electron spin resonance (ESR) spectroscopy of different positional isomers of phosphatidylcholine spin labels (n-PCSL; n=5, 7, 10, 12, 14, and 16) has been used to study micellar dispersions made of poly(ethylene glycol)s-phosphatidylethanolamines (PEGs-PEs) polymer-lipids. Such aggregates are currently used as long circulating drug delivery systems "in vivo." We varied both the hydrocarbon chain length and the polymer size of the polymer-lipids. The dependence of the lipid-chain packing density on temperature and on label position as well as the flexibility and polarity profiles with position of chain labeling have been established for the PEGs-PEs micellar dispersions. The results show both similarity and differences either with common micellar dispersions of single chained lyso-palmitoylphosphatidylcholine (C(16)Lyso-PC) or with lamellar dispersions of double chained dipalmitoylphosphatidylcholine (DPPC). Well defined chain flexibility gradients of the same overall shape are obtained in the considered dispersions. However, the mobility of the first acyl chain segments is appreciable higher in micelles of polymer-lipids than in bilayers of DPPC and it becomes indistinguishable at the chain termini. A trend of decreasing polarity on moving toward the bilayer interior is seen in DPPC bilayers, whereas biphasic polarity profiles are obtained in micelles of polymer-lipids and C(16)Lyso-PC. Moreover, the properties of the PEGs-PEs micelles do not depend on the length of the hydrocarbon chain of the polymer-lipids but are slightly influenced by the size of the polymer.     

Mesoscopic gel at low agarose concentration in water: a dynamic light scattering study.

Previous work in our laboratory has shown that at very low agarose concentration in water gelation still occurs within mutually disconnected, high concentration regions generated by spinodal demixing. The freely diffusing particles obtained in these conditions are studied in the present work by depolarized dynamic light scattering and probe diffusion experiments. These particles are found to behave as large (in fact, mesoscopic) polymer fibers entangled in a continuously rearranged mesh with scaling parameters typical of partially flexible, neutral chains. The present results allow specifying the notion of mesoscopic gelation. They also reveal that the same symmetry-breaking mechanism that allows macroscopic gelation at polymer concentrations well below the threshold for random cross-link percolation generates additional and unexpected phenomena.     

Material properties and cytocompatibility of injectable MMP degradable poly(lactide ethylene oxide fumarate) hydrogel as a carrier for marrow stromal cells

Injectable in situ crosslinkable biomaterials seeded with multipotent progenitor cells and coupled with minimally invasive arthroscopic techniques are an attractive alternative for treating irregularly shaped osteochondral defects. An in situ crosslinkable poly(lactide-co-ethylene oxide-co-fumarate) (PLEOF) macromer has been developed with ultralow molecular weight poly(L-lactide) and poly(ethylene glycol) (PEG) units linked by fumaryl unit. The PLEOF macromer was crosslinked with the MMP-13 degradable peptide sequence QPQGLAK with acrylate end-groups or the methylene bisacrylamide (BISAM) crosslinker to form enzymatically or hydrolytically degradable hydrogels, respectively. Cell viability of the peptide crosslinker was significantly higher than that of BISAM. The relatively higher molecular weight peptide crosslinker significantly affected the water content and the rate of crosslinking (e.g., sol vs gel fraction). The addition of a small fraction of a highly reactive BISAM crosslinker to the PLEOF/peptide mixture reduced the gelation time and increased the elastic modulus while retaining enzymatic degradability of the hydrogel. Bone marrow stromal (BMS) cells were encapsulated in the peptide crosslinked PLEOF hydrogel; 84% of the encapsulated cells was viable after 1 week of incubation in osteogenic media. The encapsulated BMS cells differentiated to osteoblasts and produced a mineralized matrix, as measured by ALPase activity and calcium content. The degradation rate of the hydrogel depended on the ratio of the peptide to the BISAM crosslinker, MMP-13 concentration, and incubation time. The results demonstrate that the peptide crosslinked PLEOF hydrogel with tunable degradation characteristics is potentially useful as an injectable in situ crosslinkable carrier for bone marrow stromal cells.     

Microheterogeneity controls the rate of gelation of actin filament networks

Rapid sol-gel transitions of the actin cytoskeleton are required for many key cellular processes, including cell spreading and cell locomotion. Actin monomers assemble into semiflexible polymers that rapidly intertwine into a network, a process that in vitro takes approximately 1 min for an actin concentration of 1 mg/ml. The same actin filament network, however, takes approximately 1 h to exhibit a steady-state elasticity. We hypothesize that the slow gelation of F-actin is due to the slow establishment of a homogeneous meshwork. Using a novel method, time-resolved multiple particle tracking, which monitors the range of thermally excited displacements of microspheres imbedded in the network, we show that the increase in elasticity in a polymerizing solution of actin parallels the progressive decline of the network microheterogeneity. The rates of gelation and network homogenization slightly decrease with actin concentration and in the presence of the F-actin cross-linking proteins alpha-actinin and fascin, whereas the rate of actin polymerization increases dramatically with actin concentration. Our measurements show that the slow spatial homogenization of the actin filament network, not actin polymerization or the formation of polymer overlaps, is the rate-limiting step in the establishment of an elastic actin network and suggest that a new activity of F-actin binding proteins may be required for the rapid formation of a homogeneous stiff gel.     

Inhomogeneous alginate gel spheres: an assessment of the polymer gradients by synchrotron radiation-induced X-ray emission, magnetic resonance microimaging, and mathematical modeling

It has been previously demonstrated that calcium alginate gels prepared by dialysis often exhibit a concentration inhomogeneity being the polymer concentration considerably lower in the center of the gel than at the edges. Inhomogeneity may be a preferred structure in microcapsules due to low porosity and higher stability so that it is interesting to evaluate the polymer gradient in spherically symmetrical small alginate beads (1.0-0.7 mm diameter) obtained in different conditions. In this paper, two complementary techniques have been used to investigate this aspect. The concentration gradient of alginate has been analyzed by measuring both the spatial distribution of calcium ions in sections of alginate gel spheres, by means of x-ray fluorescence spectroscopy, and the T2 relaxation behavior on intact gel beads using magnetic resonance microimaging. The experimentally determined gradients from three-dimensional gels provide data to reevaluate the parameter estimates in the recently reported mathematical model for alginate gel formation (A. Mikkaelsen and A. Elgsaeter, Biopolymers, 1995, Vol. 36, pp. 17-41). The model may account for the gels being less inhomogeneous when nongelling sodium or magnesium ions are added during gelation.     

Rheological characterization of in situ crosslinkable hydrogels formulated from oxidized dextran and N-carboxyethyl chitosan

The gelation kinetics of an in situ gelable hydrogel formulated from oxidized dextran (Odex) and N-carboxyethyl chitosan (CEC) was investigated rheologically. Both Schiff base mediated chemical and physical crosslinking account for its rapid gelation (30-600 s) between 5 and 37 degrees C. The correlation between gelation kinetics and hydrogel properties with Odex/CEC concentration, their feed ratio, and temperature were elucidated. The gelation time determined from crossing over of storage moduli (G') and loss moduli (G' ') was in good agreement with that deduced from frequency sweeping tests according to the Winter-Chambon power law. The power law exponents for a 2% (w/v) Odex/CEC solution (ratio 5:5) at the gel point was 0.61, which is in excellent agreement with the value predicted from percolation theory (2/3). Temperature dependence of gelation time for the same hydrogel formulation is well-described by an Arrhenius plot with its apparent activation energy calculated at 51.9 kJ/mol.     

Thermoreversible protein hydrogel as cell scaffold

A thermoreversible fibrillar hydrogel has been formed from an aqueous lysozyme solution in the presence of dithiothreitol (DTT). Its physical properties and potential as a tissue engineering scaffold have been explored. Hydrogels were prepared by dissolving 3 mM protein in a 20 mM DTT/water mixture, heating to 85 degrees C and cooling at room temperature. No gel was observed for the equivalent sample without DTT. The elastic nature of the gel formed was confirmed by rheology, and the storage modulus of our gel was found to be of the same order of magnitude as for other cross-linked biopolymers. Micro differential scanning calorimetry (microDSC) experiments confirmed that the hydrogel was thermally reversible and that gelation and melting occurs through a solid-liquid-like first-order transition. Infrared spectroscopy of the hydrogel and transmission electron microscopy studies of very dilute samples revealed the presence of beta-sheet-rich fibrils that were approximately 4-6 nm in diameter and 1 mum in length. These fibrils are thought to self-assemble along their long axes to form larger fibers that become physically entangled to form the three-dimensional network observed in both cryo-scanning electron microscopy (cryo-SEM) and small-angle neutron scattering (SANS) studies. The hydrogel was subsequently cultured with 3T3 fibroblasts and cells spread extensively after 7 days and stretched actin filaments formed that were roughly parallel to each other, indicating the development of organized actin filaments in the form of stress fibers in cells.     

Towards a fully synthetic substitute of alginate: optimization of a thermal gelation/chemical cross-linking scheme ("tandem" gelation) for the production of beads and liquid-core capsules

Fully synthetic polymers were used for the preparation of hydrogel beads and capsules, in a processing scheme that, originally designed for calcium alginate, was adapted to a "tandem" process, that is the combination a physical gelation with a chemical cross-linking.The polymers feature a Tetronic backbone (tetra armed Pluronics), which exhibits a reverse thermal gelation in water solutions within a physiological range of temperatures and pHs. The polymers bear terminal reactive groups that allow for a mild, but effective chemical cross-linking. Given an appropriate temperature jump, the thermal gelation provides a hardening kinetics similar to that of alginate. With slower kinetics, the chemical cross-linking then develops an irreversible and elastic gel structure, and determines its transport properties. In the present article this process has been optimized for the production of monodisperse, high elastic, hydrogel microbeads, and liquid-core microcapsules. We also show the feasibility of the use of liquid-core microcapsules in cell encapsulation. In preliminary experiments, CHO cells have been successfully encapsulated preserving their viability during the process and after incubation. The advantages of this process are mainly in the use of synthetic polymers, which provide great flexibility in the molecular design. This, in principle, allows for a precise tailoring of mechanical and transport properties and of bioactivity of the hydrogels, and also for a precise control in material purification.     

Cytochalasin B and the structure of actin gels

We analyzed the structure of gels formed when macrophage actin-binding protein crosslinks skeletal muscle actin polymers and the effect of the fungal metabolite cytochalasin B on this structure. Measurement of the actin filament length distribution permitted calculation of the critical concentration of crosslinker theoretically required for gelation of actin polymer networks. The experimentally determined critical concentration of actin-binding protein agreed sufficiently with the theoretical to conclude that F-actin-actin-binding protein gels are networks composed of isotropically oriented filaments crosslinked at intervals. The effects of cytochalasin B on these actin networks fits this model. Cytochalasin B (1) bound to F-actin (but not to actin-binding protein), (2) decreased the length of actin filaments without increasing the quantity of monomeric actin, (3) decreased the rigidity of actin networks both in the presence and absence of crosslinking proteins and (4) increased the critical concentration of actin-binding protein required for incipient gelation by a magnitude predicted from network theory if filaments were divided and shortened by the extents observed. The effects of cytochalasin B on gelation were highly dependent on actin concentration and were inhibited by the actin-stabilizing agent phalloidin. Therefore, cytochalasin B diminishes actin gel structure by severing actin filaments at limited sites. The demonstration of gel-sol transformations in actin networks caused by limited actin filament cleavage suggests a new mechanism for the control of cytoplasmic structure.     

Viscoelastic behavior and in vivo release study of microgel dispersions with inverse thermoreversible gelation

The dispersion of microgels with two interpenetrating polymer networks of poly( N-isopropylacrylamide) and poly(acrylic acid) (PNIPAM-IPN-PAAc) has been studied for its viscoelastic behavior, biocompatibility, and in vivo release properties. The IPN microgels in water had an average hydrodynamic radius of about 85 nm at 21 degrees C, measured by dynamic light scattering method. The atomic force microscope image showed that the particles were much smaller after they were dried but remained spherical shape. The storage and loss moduli ( G' and G') of dispersions of IPN microgels were measured in the linear stress regime as functions of temperature and frequency at various polymer concentrations using a stress-controlled rheometer. For dispersions with polymer concentrations of 3.0 and 6.0 wt % above 33 degrees C, the samples behave as viscoelastic solids and the storage modulus was larger than the loss modulus over the entire frequency range. The loss tangent was measured at various frequencies as a function of temperature. The gelation temperature was determined to be 33 degrees C at the point where a frequency-independent value of the loss tangent was first observed. At pH 2.5, when heated above the gelation temperature, IPN microgels flocculate by pumping a large amount of water from the gel. When the pH value was adjusted to neutral, deprotonation of -COOH groups on PAAc made the microgel keep water even above the gelation temperature. Using an animal implantation model, the biocompatibility and drug release properties of the IPN microgel dispersion were evaluated. Fluorescein as a model drug was mixed into an aqueous microgel dispersion at ambient temperature. This drug-loaded liquid was then injected subcutaneously in Balb/C mice from Taconic Farms. The test results have shown that the IPN microgels did not adversely promote foreign body reactions in this acute implantation model and the presence of gelled microgel dispersion substantially slowed the release of fluorescein.     

Paclitaxel delivery to brain tumors from hydrogels: a computational study

Malignant gliomas are aggressive forms of primary brain tumors characterized by a poor prognosis. The most successful treatment so far is the local implantation of polymer carriers (Gliadel® wafers) for the sustained release of carmustine. To improve the effectiveness of local drug treatment, new polymer carriers and pharmacological agents are currently being investigated. Of particular interest is a set of novel thermo‐gelling polymers for the controlled release of hydrophobic drugs such as paclitaxel (e.g., OncoGel?). Herein, we use computational mass transport simulations to investigate the effectiveness of paclitaxel delivery from hydrogel‐forming polymer carriers. We found similar (within 1–2 mm) therapeutic penetration distances of paclitaxel when released from these hydrogels as compared with carmustine released from Gliadel® wafers. Effective therapeutic concentrations were maintained for >30 days for paclitaxel when released from the hydrogel as compared with 4 days for carmustine released from Gliadel® wafers. Convection in brain tissue prevented the formation of a uniform drug concentration gradient around the implant. In addition, the surface area to volume ratio of the gel is an important factor that should be considered to maintain a controlled release of paclitaxel within the degradation lifetime of the polymer matrix. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

The influence of calcium ions on fibrin polymerization

The influence of calcium ions on the polymerization induced in fibrinogen solutions by thrombin and by Reptilase has been investigated by meansof static and dynamic light scattering in combination with measurements of the release of the fibrinopeptide A. The calcium concentration was varied in the range between 0.3 and 103 calcium ions per fibrinogen molecule. The enzyme concentration was chosen sufficiently low so that it was possible to make quantitative observations as a function of time, in particular, beforethe onset of gelation. Likewise, the influence of calcium ions on the enzymatically induced polymerization of fragment X was studied. The results indicate that there are at least three mechanisms by which calcium can influence the evolution of the polymer system on the path to gelation and clotting. Which mechanism dominates depends upon the calcium concentration.