Preparation and characterization of sodium hexameta phosphate cross-linked chitosan microspheres for controlled and sustained delivery of centchroman

The cross-linked microspheres using chitosan with different molecular weights and degree of deacetylation have been prepared in presence of sodium hexameta polyphosphate (SHMP) as physical cross-linker. The degree of cross-linking through electrostatic interactions in chitosan microspheres has been evaluated by varying the charge density on chitosan and varying degree of dissociation of sodium hexameta polyphosphate by solution pH. The degree of deacetylation and molecular weight of chitosan has controlled electrostatic interactions between hexameta polyphosphate anions and chitosan, which played significant role in swelling, loading and release characteristics of chitosan microspheres for centchroman. The microspheres prepared by hexameta polyphosphate anions cross-linker were compact and more hydrophobic than covalently cross-linked microspheres, which has been attributed to the participation of all amino groups of chitosan in physical cross-linking with added hexameta polyphosphate anions. The microspheres prepared under different experimental conditions have shown an initial step of burst release, which was followed by a step of controlled release for centchroman. The extent of drug release in these steps has shown dependence on properties of chitosan and degree of cross-linking between chitosan and added polyanions. The degree of swelling and release characteristics of microspheres was also studied in presence of organic and inorganic salts, which shown significant effect on controlled characteristics of microspheres due to variations in ionic strength of the medium. The initial step of drug release has followed first order kinetics and become zero order after attaining an equilibrium degree of swelling in these microspheres. The microspheres prepared using chitosan with 62% (w/w) degree of deacetylation and molecular weight of 1134 kg mol⁻¹ have shown a sustained release for centchroman for 50 h at 4% (w/w) degree of cross-linking with SHMP.     

A New Screening Method of Viral-neuraminidase Inhibitors

A new and practical method for the screening of neuraminidase inhibitors (NI) by means of the viral hemagglutination (HA)-dehemagglutination(deHA) reactions was suggested. The best conditions for the HA and deHA reactions were investigated. Existence of strong inhibition activity on the viral deHA has been recognized in the culture filtrates of some strains of actinomycetes. All of these deHA inhibitors showed NI activity that is not specified to the strain of the test viruses. About 0.25 mg/ml of the preparation obtained from the culture filtrate of the strongest actinomycetes, No. 289, inhibited the liberation of neuraminic acid from bovine submaxillary mucin by 80 HA units/ml of influenza A Fukuoka/1/70 (H₃N₂) virus up to 80%.     

Biodegradable and biocompatible synthetic saccharide-Peptide hydrogels for three-dimensional stem cell culture

Saccharide-peptide hydrogels have been developed in our laboratory as new synthetic extracellular matrices for regenerative medicine applications. In this work, we have expanded on our previously reported system and applied copolymerization of cysteine (Cys) and vinyl sulfone (VS)-functionalized saccharide-peptide polymers via Michael-type addition for encapsulation and 3D culture of cells. Specifically, our aims were to (1) develop a novel hydrogel platform, which could be applied for encapsulating and culturing mesenchymal stem cells (MSCs) in a 3D environment, (2) characterize the tunable properties of the hydrogel, specifically, degradation, mechanical, and gel network properties, and (3) determine the biocompatibility of the saccharide-peptide hydrogel material with MSCs. Hydrogel mechanical properties were tunable by varying the VS:Cys ratio (= 0.5, 1, or 2) as well as the pH (6, 7, or 8) of the cross-linking components. Stiffer gels were formed at VS:Cys = 1 and pH 6 or 7. Gels formed at pH 8 or with excess Cys (VS:Cys = 0.5) or VS (VS:Cys = 2) were significantly softer. Cross-linking pH and VS:Cys ratio also had an effect on the degradation behavior of the VS:Cys gels, with higher cross-linking pH resulting in an accelerated loss of mass. On the basis of environmental scanning electron microscopy (ESEM) analysis and fluorescence microscopy, all hydrogels appeared to exhibit porous gel networks. MSCs cultured in monolayer and exposed to soluble Cys or VS copolymers (0.1-5 mg/mL) did not exhibit measurable cytotoxicity. In addition, MSCs were cultured in 3D for up to 14 days in vitro without deleterious effects on cell viability. In summary, we have established and characterized a tunable 3D saccharide-peptide hybrid copolymer hydrogel platform for culturing MSCs. Future studies will focus on utilizing the hydrogel system for controlling the differentiation of MSCs.     

Fibre-diffraction studies of the extracellular polysaccharide from Pseudomonas elodea

X-ray fibre diffraction techniques have been used to probe the structure of the microbial polysaccharide from Pseudomonas clodca (gellan gum). The polymer adopts a threefold helical structure with an axial repeat of 0.94 nm. The polymer form weak elastic thermoreversible gels and on deacetylation forms rigid brittle gels. Deacetylation has been shown to enhance crystallinity. Equatorial reflections index onto a trigonal unit cell (a=b=1.64 nm.     

Comparison between the physicochemical behaviour of two microbial polysaccharides: RMDP17 and rhamsan.

This paper concerns the properties of two ionic polysaccharides with very close chemical structures. pH metric and conductimetric measurements showed that they behave similarly from a polyelectrolytic point of view. From optical rotation and differential scanning calorimetry (DSC) measurements, the two polymers probably adopt a double helical conformation which is destabilised by deacetylation. The main differences concern the stability of this ordered conformation and the ability of these double helices to associate to form gels. The results support a higher thermal stability of the ordered conformation for deacetylated RMDP17 (about 8 degrees C), whereas deacetylated rhamsan has a better ability to form gels.     

Thermal aggregation and ion-induced cold-gelation of bovine serum albumin

Protein cold-gelation has recently received particular attention for its relevance in bio and food technology. In this work, we report a study on bovine serum albumin cold-gelation induced by copper or zinc ions. Metal-induced cold-gelation of proteins requires two steps: during the first one, the heat treatment causes protein partial unfolding and aggregation; then, after cooling the solution to room temperature, gels are formed upon the addition of metal ions. The thermally induced behaviour has been mainly investigated through different techniques: Fourier transform infrared (FTIR) spectroscopy, circular dichroism, dynamic light scattering (DLS) and rheology. Data have shown that the aggregation process is mainly due to protein conformational changes—α-helices into β-aggregates—forming small aggregated structures with a mean diameter of about 20 nm a few minutes after heating. After metal ion addition, the viscoelastic properties of the gels have been investigated by rheological measurements. The behaviour of the elastic and viscous moduli as a function of time is discussed in terms of ion concentration and type. Our results show that: (1) the elastic behaviour depends on ion concentration and (2) at a given ion concentration, gels obtained in the presence of zinc exhibit an elastic value larger than that observed in the Cu²⁺ case. Data suggest that cold-gelation is the result of different mechanisms: the ion-mediated protein–protein interaction and the bridging effect due to the presence of divalent ions in solution.     

Oxidized chondroitin sulfate-cross-linked gelatin matrixes: a new class of hydrogels

A naturally occurring glycosaminoglycan such as chondroitin-6-sulfate was first converted in to its aldehyde derivative by periodate oxidation and used as a cross-linking agent for gelatin giving rise to a new class of hydrogels. Cross-linking was predominantly due to Schiff's base formation between the epsilon-amino groups of lysine or hydroxylysine side groups of gelatin and the aldehyde groups in oxidized chondroitin sulfate. The hydrogels were prepared from chondroitin sulfate with different degrees of oxidation and gelatin. They were characterized for degree of cross-linking, cross-linking density, equilibrium swelling, water vapor transmission rate, internal structure, and blood-compatibility. Degree of cross-linking of the gels determined by trinitrobenzene sulfonic acid assay showed that, the higher the degree of oxidation of the polysaccharide, the higher the degree of cross-linking. Examination of the internal structure by scanning electron microscopy showed that the hydrogels were highly porous in nature with interconnecting pores ranging from 50 to 200 mum. Equilibrium swelling showed that the gels retained about 90% water and did not undergo dehydration rapidly. The hydrogels were nontoxic and blood-compatible. Since an important phase of early wound healing has been shown to involve secretion of glycosaminoglycans such as chondroitin sulfate by fibroblasts which form a hydrophilic matrix suitable for remodeling during healing, this new class of hydrogels prepared from chondroitin sulfate and gelatin without employing any extraneous cross-linking agents are expected to have potential as wound dressing materials.     

Dependence of invadopodia function on collagen fiber spacing and cross-linking: computational modeling and experimental evidence

Invadopodia are subcellular organelles thought to be critical for extracellular matrix (ECM) degradation and the movement of cells through tissues. Here we examine invadopodia generation, turnover, and function in relation to two structural aspects of the ECM substrates they degrade: cross-linking and fiber density. We set up a cellular automaton computational model that simulates ECM penetration and degradation by invadopodia. Experiments with denatured collagen (gelatin) were used to calibrate the model and demonstrate the inhibitory effect of ECM cross-linking on invadopodia degradation and penetration. Incorporation of dynamic invadopodia behavior into the model amplified the effect of cross-linking on ECM degradation, and was used to model feedback from the ECM. When the model was parameterized with spatial fibrillar dimensions that closely matched the organization, in real life, of native ECM collagen into triple-helical monomers, microfibrils, and macrofibrils, little or no inhibition of invadopodia penetration was observed in simulations of sparse collagen gels, no matter how high the degree of cross-linking. Experimental validation, using live-cell imaging of invadopodia in cells plated on cross-linked gelatin, was consistent with simulations in which ECM cross-linking led to higher rates of both invadopodia retraction and formation. Analyses of invadopodia function from cells plated on cross-linked gelatin and collagen gels under standard concentrations were consistent with simulation results in which sparse collagen gels provided a weak barrier to invadopodia. These results suggest that the organization of collagen, as it may occur in stroma or in vitro collagen gels, forms gaps large enough so as to have little impact on invadopodia penetration/degradation. By contrast, dense ECM, such as gelatin or possibly basement membranes, is an effective obstacle to invadopodia penetration and degradation, particularly when cross-linked. These results provide a novel framework for further studies on ECM structure and modifications that affect invadopodia and tissue invasion by cells.     

Structural and mechanical properties of UV-photo-cross-linked poly(N-vinyl-2-pyrrolidone) hydrogels

Biocompatible poly( N-vinyl-2-pyrrolidone) (PVP) hydrogels have been produced by UV irradiation of aqueous polymer mixtures, using a high-pressure mercury lamp. The resulting materials have been characterized by a combination of experimental techniques, including rheology, small-angle neutron scattering (SANS), electron paramagnetic resonance (EPR), and pulsed gradient spin-echo nuclear magnetic resonance (PGSE-NMR), to put in evidence the relationship between the microstructural properties and the macrofunctional behavior of the gels. Viscoelastic measurements showed that UV photo-cross-linked PVP hydrogels present a strong gel mechanical behavior and viscoelastic moduli values similar to those of biological gels. The average distance between the cross-linking points of the polymer network was estimated from the hydrogels elastic modulus. However, SANS measurements showed that the network microstructure is highly inhomogeneous, presenting polymer-rich regions more densely cross-linked, surrounded by a water-rich environment. EPR and PGSE-NMR data further support the existence of these water-rich domains. Inclusion of a third component, such as glycerol, in the PVP aqueous mixture to be irradiated has been also investigated. A small amount of glycerol (<3% w/w) can be added keeping satisfactory properties of the hydrogel, while higher amounts significantly affect the cross-linking process.     

Synthesis of peptide gels for the investigation of oligopeptide-oligonucleotide interactions

Peptide gels usable as protein model systems have been synthesized by a cross-linking copolymerization of acryloyl substituted peptides with 1,4-tetramethylene dimethacrylate. A specially adapted approach to peptide synthesis allows the removal of the amino terminal Cbo group at the end of the peptide synthesis, followed by the introduction of an acryloyl group. The polymerizable peptide monomers obtained can be transferred into insoluble peptide gels by radical copoylmerization with cross-linking agents. After cleavage of the protecting groups of the side chains, these peptide gels can be used both as protein model systems for investigating peptide–oligonucleotide interaction and as sorbents for affinity chromatography. The preparation and characterization of the peptide gels Ala-Lys-Glu-Lys-Ala-OMe (I), Ala-Arg-Glu-Arg-Ala-OMe (II), Ala-Arg-Glu-Lys-Ala-OMe (III), and Ala-Arg-Ala-Lys-Ala-OMe (IV) as well as the conditions for the removal of the protecting groups is presented. Gel III contains the natural peptide sequence Arg-Glu-Lys while the other gels are analogs of this sequence.     

Self-Assembly of Polysaccharides Gives Rise to Distinct Mechanical Signatures in Marine Gels

Marine-gel biopolymers were recently visualized at the molecular level using atomic force microscopy (AFM) to reveal fine fibril-forming networks with low to high degrees of cross-linking. In this work, we use force spectroscopy to quantify the intra- and intermolecular forces within the marine-gel network. Combining force measurements, AFM imaging, and the known chemical composition of marine gels allows us to identify the microscopic origins of distinct mechanical responses. At the single-fibril level, we uncover force-extension curves that resemble those of individual polysaccharide fibrils. They exhibit entropic elasticity followed by extensions associated with chair-to-boat transitions specific to the type of polysaccharide at high forces. Surprisingly, a low degree of cross-linking leads to sawtooth patterns that we attribute to the unraveling of polysaccharide entanglements. At a high degree of cross-linking, we observe force plateaus that arise from unzipping, as well as unwinding, of helical bundles. Finally, the complex 3D network structure gives rise to force staircases of increasing height that correspond to the hierarchical peeling of fibrils away from the junction zones. In addition, we show that these diverse mechanical responses also arise in reconstituted polysaccharide gels, which highlights their dominant role in the mechanical architecture of marine gels.     

Self-Assembly of Polysaccharides Gives Rise to Distinct Mechanical Signatures in Marine Gels

Marine-gel biopolymers were recently visualized at the molecular level using atomic force microscopy (AFM) to reveal fine fibril-forming networks with low to high degrees of cross-linking. In this work, we use force spectroscopy to quantify the intra- and intermolecular forces within the marine-gel network. Combining force measurements, AFM imaging, and the known chemical composition of marine gels allows us to identify the microscopic origins of distinct mechanical responses. At the single-fibril level, we uncover force-extension curves that resemble those of individual polysaccharide fibrils. They exhibit entropic elasticity followed by extensions associated with chair-to-boat transitions specific to the type of polysaccharide at high forces. Surprisingly, a low degree of cross-linking leads to sawtooth patterns that we attribute to the unraveling of polysaccharide entanglements. At a high degree of cross-linking, we observe force plateaus that arise from unzipping, as well as unwinding, of helical bundles. Finally, the complex 3D network structure gives rise to force staircases of increasing height that correspond to the hierarchical peeling of fibrils away from the junction zones. In addition, we show that these diverse mechanical responses also arise in reconstituted polysaccharide gels, which highlights their dominant role in the mechanical architecture of marine gels.     

Electrophoresis of DNA fragments in gels from acrylamide-rich copolymers

Two polyacrylamide-rich, non-toxic, gelable copolymers have been developed to facilitate the formation of user-cast electrophoresis gels. Gel formation is accomplished with dithiothreitol as the chemical cross-linking agent. The higher molecular weight copolymer is suitable for casting gels of copolymer concentration less than or equal to 8%. Gels of 3% concentration are excellent for resolving dsDNA fragments up to approximately 3000 base pairs. Because the cross-linking chemistry is not thwarted by the presence of urea, it is also possible to cast denaturing gels with these copolymers.     

Novel hydrogels via click chemistry: synthesis and potential biomedical applications

A novel procedure for the in situ rapid chemical gelation of aqueous solutions of hyaluronan has been employed. In brief, water-soluble polysaccharide derivatives bearing side chains endowed with either azide or alkyne terminal functionality have been prepared. When the latter two types of derivatives are mixed together in aqueous solution they give rise to a 1,3-dipolar cycloaddition reaction resulting in fast gelation (in the presence of catalytic amounts of Cu(I)) at room temperature. Gel formation has been characterized rheologically and could also be followed qualitatively by means of IR spectroscopy. The resulting gels have been studied in terms of swelling properties and, in particular, NMR spectral features. Carrying out the gelation process in aqueous solutions of benzidamine and doxorubicin, respectively, the polysaccharide networks acted as drug reservoirs. The doxorubicin release resulted in well controllable acting upon the gels degree of cross-linking. Finally, formation of the click-gels using aqueous suspensions of Saccharomices cerevisiae yeast cells allowed the obtainment of scaffolds inside which cells were homogeneously distributed and smoothly adhered to the inner pores surfaces, according to SEM analysis. After 24 h about 60% of the entrapped cells exhibited proliferating activity. Click-gels prepared as detailed herein do have a number of positive features that make them, in perspective, materials of choice for drug release and tissue engineering manipulations.     

Photo-cross-linking of type I collagen gels in the presence of smooth muscle cells: mechanical properties,cell viability,and function

The effectiveness of photomediated cross-linking of type I collagen gels in the presence of rat aortic smooth muscle cells (RASMC) as a method to enhance gel mechanical properties while retaining native collagen triple helical structure and maintaining high cell viability was investigated. Collagen was chemically modified to incorporate an acrylate moiety. Collagen methacrylamide was cast into gels in the presence of a photoinitiator along with RASMC. The gels were cross-linked using visible light irradiation. Neither acrylate modification nor the cross-linking reaction altered collagen triple helical content. The cross-linking reaction, however, moved the denaturation temperature beyond the physiologic range. A twelve-fold increase in shear modulus was observed after cross-linking. Cell viability in the range of 70% (n = 4, p > 0.05) was observed in the photo-cross-linked gels. Moreover the cells were able to contract the cross-linked gel in a manner commensurate with that observed for natural type I collagen. Methacrylate-mediated photo-cross-linking is a facile route to improve mechanical properties of collagen gels in the presence of cells while maintaining high cell viability. This enhances the potential for type I collagen gels to be used as scaffolds for tissue engineering.     

Fibroblast behavior on gels of type I, III, and IV human placental collagens

Various collagens were extracted and purified from human placenta after partial pepsin digestion. We prepared type III + I (57:43), enriched type I, type III, and type IV collagens on an industrial level, and studied their biological properties with MRC5 fibroblast cells. Using the process of contraction of a hydrated collagen lattice described by Bell, we found tha the contraction rate was dependent on collagen type composition. The contraction was faster and more pronounced with pepsinized type I collagen than with pepsinized type III + I (57:43) collagen; the lowest rate was obtained with the pepsinized type III collagen. Using a new technique of collagen cross-linking, a gel was made with type IV collagen. This cross-linking procedure, based on partial oxidation of sugar residues and hydroxylysine by periodic acid, followed by neutralization, resulted in an increased number of natural cross-link bridges between oxidized and nonoxidized collagen molecules, without internal toxic residues. The fibroblasts were unable to contract type IV/IVox collagen gels. The type IV/IVox collagen gel was transparent and its amorphous ultrastructure lacked any visible striated fibrils. Fibroblast cells exhibited atypical behavior in these type IV/IVox collagen gels as evidenced by optical and electron microscopy. The penetration of fibroblasts could be measured. Fibroblasts penetrated faster in type IV/IVox collagen gels than in untreated type III + I collagen gels. The lowest rate of penetration was obtained with cross-linked type III + I gels. Fibroblast proliferation was similar on untreated or cross-linked type III + I collagen gels and slightly increased on type IV/IVox collagen gels, suggesting that this cross-linking procedure was not toxic.     

Robust cross-links in molluscan adhesive gels: testing for contributions from hydrophobic and electrostatic interactions

The cross-linking interactions that provide cohesive strength to molluscan adhesive gels were investigated. Metal-based interactions have been shown to play an important role in the glue of the slug Arion subfuscus (Draparnaud), but other types of interactions may also contribute to the glue's strength and their role has not been investigated. This study shows that treatments that normally disrupt hydrophobic or electrostatic interactions have little to no effect on the slug glue. High salt concentrations and non-ionic detergent do not affect the solubility of the proteins in the glue or the ability of the glue proteins to stiffen gels. In contrast, metal chelation markedly disrupts the gel. Experiments with gel filtration chromatography identify a 40 kDa protein that is a central component of the cross-links in the glue. This 40 kDa protein forms robust macromolecular aggregations that are stable even in the presence of high concentrations of salt, non-ionic detergent, urea or metal chelators. Metal chelation during glue secretion, however, may block some of these cross-links. Such robust, non-specific interactions in an aqueous environment are highly unusual for hydrogels and reflect an intriguing cross-linking mechanism.     

Bovine tendons. Aging and collagen cross-linking

The level of cross-linking between the polypeptide chains of the collagen molecules in bovine tendons of different ages has been assessed by measuring quantitatively through densitometry the changes in the ratios of individual cyanogen bromide peptides separated on polyacrylamide gels. An increase in the number of cross-links in mature, as compared to young, tendons correlates with a depletion in the proportion of the free, COOH-terminal peptides alpha1-CB6 and alpha2-CB3,5 and with an increase in a broad distribution of peptides moving slowly in the gels: these peptides are not seen in digests of acid-soluble collagen. Some of these peptides which are presumably cross-linked migrate more slowly than beta components and collagen alpha chains and are apparently of a higher molecular weight. No increase in cross-linked peptides is detectable beyond the age of maturation; this analysis refutes, at least in this tissue, the common presumption that progressive cross-linking occurs in collagen through an animal's lifetime.     

A study of the quaternary structure of Escherichia coli RNA polymerase using bis(imido esters).

The quaternary structure of Escherichia coli RNA polymerase has been studied by cross-linking with a periodate-cleavable bis(imido ester), N,N'-bis(2-carboximidoethyl)tartaramide dimethyl ester dihydrochloride (CETD). The cross-linked holoenzyme gives a characteristic five-band pattern after electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. The components of each band have been unambiguously identified by (a) molecular-weight measurements, (b) comparisons of cross-linking patterns of holoenzyme and core enzyme, and (c) periodate cleavage of cross-links followed by a second dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bands are (1) alphabeta and alphabeta', (2) sigmabeta and sigmabeta', (3) alphasigmabeta', (4) betabeta', and (5) sigmabetabeta'. Bands 2 and 4 are the most prominent at low reagent concentrations (up to 2.5 mM) but band 5 becomes the most prominent at higher concentrations. There are no bands corresponding to alphaalpha and alphasigma; a faint band has been tentatively identified as alphabetabeta'. Shorter bis(imido esters) are much less effective cross-linking reagents than CETD and they do not give rise to any other cross-linked species. On the basis of these observations, a model for the subunit arrangement of RNA polymerase is proposed.     

Controlling rigidity and degradation of alginate hydrogels via molecular weight distribution

The mechanical rigidity and degradation rate of hydrogels utilized as cell transplantation vehicles have been regarded as critical factors in new tissue formation. However, conventional approaches to accelerate the degradation rate of gels deteriorate their function as a mechanical support in parallel. We hypothesized that adjusting the molecular weight distribution of polymers that are hydrolytically labile but capable of forming gels would allow one to alter the degradation rate of the gels over a broad range, while limiting the range of their elastic moduli (E). We investigated this hypothesis with binary alginate hydrogels formed from both ionically and covalently cross-linked partially oxidized (1% uronic acid residues), low [molecular weight (MW) approximately 60,000 g/mol] and high MW alginates (MW approximately 120,000 g/mol) in order to examine the utility of this approach with various cross-linking strategies. Increasing the fraction of low MW alginates to 0.50 maintained a value of E similar to that for the high MW alginate gels but led to faster degradation, irrespective of the cross-linking mode. This result was attributed to a faster separation between cross-linked domains upon chain breakages for the low MW alginates, coupled with their faster chain scission than the high MW alginates. The more rapidly degrading oxidized binary hydrogels facilitated the formation of new bone tissues from transplanted bone marrow stromal cells, as compared with the nonoxidized high MW hydrogels. The results of these studies will be useful for controlling the physical properties of a broad array of hydrogel-forming polymers.