Biodegradable and pH-sensitive hydrogels for potential colon-specific drug delivery: characterization and in vitro release studies

A novel pH-sensitive and biodegradable composite hydrogel, based on a methacrylated and succinic derivative of dextran, named Dex-MA-SA, and a methacrylated and succinic derivative of alpha,beta-poly( N-2-hydroxyethyl)- DL-aspartamide (PHEA), named PHM-SA, was produced by photocross-linking. The goal was to obtain a colon-specific drug delivery system, exploiting both the pH-sensitive behavior and the colon-specific degradability. The hydrogel prepared with a suitable ratio between the polysaccharide and the polyaminoacid was characterized regarding its swelling behavior in gastrointestinal simulated conditions, chemical and enzymatic degradability, interaction with mucin, and cell compatibility on CaCo-2 cells. Moreover, 2-methoxyestradiol was chosen as a model of anticancer drug and release studies, were performed in the absence or in the presence of dextranase and esterase. The obtained hydrogel, due to its pH-sensitive swelling and enzymatic degradability, together with mucoadhesion and cell compatibility, could be potentially useful as system for the oral treatment of colonic cancer.     

Synthesis and degradation test of hyaluronic acid hydrogels

Hyaluronic acid (HA) hydrogels prepared with three different crosslinking reagents were assessed by in vitro and in vivo degradation tests for various tissue engineering applications. Adipic acid dihydrazide grafted HA (HA-ADH) was synthesized and used for the preparation of methacrylated HA (HA-MA) with methacrylic anhydride and thiolated HA (HA-SH) with Traut's reagent (imminothiolane). (1)H NMR analysis showed that the degrees of HA-ADH, HA-MA, and HA-SH modification were 69, 29, and 56 mol%, respectively. HA-ADH hydrogel was prepared by the crosslinking with bis(sulfosuccinimidyl) suberate (BS(3)), HA-MA hydrogel with dithiothreitol (DTT) by Michael addition, and HA-SH hydrogel with sodium tetrathionate by disulfide bond formation. According to in vitro degradation tests, HA-SH hydrogel was degraded very fast, compared to HA-ADH and HA-MA hydrogels. HA-ADH hydrogel was degraded slightly faster than HA-MA hydrogel. Based on these results, HA-MA hydrogels and HA-SH hydrogels were implanted in the back of SD rats and their degradation was assessed according to the pre-determined time schedule. As expected from the in vitro degradation test results, HA-SH hydrogel was in vivo degraded completely only in 2 weeks, whereas HA-MA hydrogels were degraded only partially even in 29 days. The degradation rate of HA hydrogels were thought to be controlled by changing the crosslinking reagents and the functional group of HA derivatives. In addition, the state of HA hydrogel was another factor in controlling the degradation rate. Dried HA hydrogel at 37 degrees C for a day resulted in relatively slow degradation compared to the bulk HA hydrogel. There was no adverse effect during the in vivo tests.     

Synergistic effect of TGFbeta-3 on chondrogenic differentiation of rabbit chondrocytes in thermo-reversible hydrogel constructs blended with hyaluronic acid by in vivo test

In this study, a hydrogel composite, based on the thermo-reversible hydrogel of p(NiPAAm-co-AAc) and hyaluronic acid (HA) was used as an injectable cell and growth factor carrier for cartilage tissue engineering applications. Rabbit chondrocytes were embedded in blended hydrogel composites co-encapsulated with the transforming growth factor beta-3 (TGFbeta-3). The blended hydrogel with the embedded chondrocytes and HA co-encapsulating unloaded growth factors and those with the thermo-reversible hydrogel were used as the controls to examine the effects of TGFbeta-3 on neocartilage formation. The blended hydrogel loaded with TGFbeta-3 embedded with chondrocytes were injected subcutaneously into the nude mice. The mice were monitored for 8 weeks after the injection. Both the differentiation and level of cartilage-specific ECM production were significantly higher in the presence of HA and growth factor than in the control without the growth factor. The level of cartilage associated ECM proteins was examined by immunohistochemical staining (collagen types II and X) as well as by Safranin-O and Alcian blue (GAG) staining. The results showed the potential application of blended hydrogel mixed with the growth factor to neocartilage formation.     

Dielectric properties of alginate beads and bound water relaxation studied by electrorotation

The electrical and dielectric properties of Ba²⁺ and Ca²⁺ cross‐linked alginate hydrogel beads were studied by means of single‐particle electrorotation. The use of microstructured electrodes allowed the measurements to be performed over a wide range of medium conductivity from about 5 mS/m to 1 S/m. Within a conductivity range, the beads exhibited measurable electrorotation response at frequencies above 0.2 MHz with two well‐resolved co‐ and antifield peaks. With increasing medium conductivity, both peaks shifted toward higher frequency and their magnitudes decreased greatly. The results were analyzed using various dielectric models that consider the beads as homogeneous spheres with conductive loss and allow the complex rotational behavior of beads to be explained in terms of conductivity and permittivity of the hydrogel. The rotation spectra could be fitted very accurately by assuming (a) a linear relationship between the internal hydrogel conductivity and the medium conductivity, and (b) a broad internal dispersion of the hydrogel centered between 20 and 40 MHz. We attribute this dispersion to the relaxation of water bound to the polysaccharide matrix of the beads. The dielectric characterization of alginate hydrogels is of enormous interest for biotechnology and medicine, where alginate beads are widely used for immobilization of cells and enzymes, for drug delivery, and as microcarriers for cell cultivation. © 1999 John Wiley & Sons, Inc. Biopoly 50: 227–237, 1999     

Reticulated hyaluronan hydrogels: a model for examining cancer cell invasion in 3D.

The extracellular polysaccharide hyaluronan (HA) controls cell migration, differentiation and proliferation, and contributes to the invasiveness of human cancers. The roles of HA cell surface receptors and hyaluronidases (HAses) in this process are still controversial. In order to investigate their involvement in cancer pathogenesis, we developed a reticulated HA hydrogel, a three-dimensional matrix in which cells can invade and grow. We have studied thirteen cell lines, from primary tumors or metastases, that migrated into the HA hydrogel and proliferated giving rise to clusters and colonies. The number of colonies, which reflects tumor cell invasiveness, ranged from 7 to 193 after 5 days of culture. Invasion was dependent on the production of HAse as well as other factors. Optimal colonization occurred when cells released HAse, lacked HA-binding sites and did not secrete HA. Moreover, we describe for the first time a HAse activity at physiological pH that may be responding to the confinement of the enzyme in a three-dimensional structure. We show here that this reticulated matrix provides a three-dimensional model for investigating mechanisms involved in malignant invasion.     

Collagens, stromal cell-derived factor-1alpha and basic fibroblast growth factor increase cancer cell invasiveness in a hyaluronan hydrogel

Abstract.   Objective : Beyond to control of cell migration, differentiation and proliferation, the extracellular matrix (ECM) also contributes to invasiveness of human cancers. As the roles of hyaluronan (HA) and collagens in this process are still controversial, we have investigated their involvement in cancer pathogenesis. Materials and methods : With this aim in view, we developed a three-dimensional matrix, as reticulate HA hydrogel alone or coated with different collagens, in which cells could invade and grow. Results : We show that cancer cells, which were non-invasive in a single HA hydrogel, acquired this capacity in the concomitant presence of type I or III collagens. Both types of ECM compound, HA and collagens, possess the capacity to stimulate production of metalloprotease-2, recognized otherwise as a factor for poor cancer prognosis. HA-provoked cellular invasiveness resulted from CD44-mediated increase in cytosolic [Ca²⁺] and its subsequent hydrolysis due to ADAM (a disintegrin and metalloprotease) proteolytic activity. Interestingly, this mechanism seemed to be absent in non-invasive cancer cell lines. Conclusion : Furthermore, using basic fibroblast growth factor and stromal cell-derived factor-1α, we also show that this three-dimensional reticulate matrix may be considered as a valuable model to study chemokinetic and chemotactic potentials of factors present in tumour stroma.     

Disulfide cross-linked hyaluronan hydrogels

A new disulfide cross-linking strategy was developed to prepare hyaluronic acid (HA) hydrogel from thiol-modified HA. First, dithiobis(propanoic dihydrazide) (DTP) and dithiobis(butyric dihydrazide) (DTB) were synthesized and then coupled to HA with carbodiimide chemistry. Next, disulfide bonds of the initially formed gel were reduced using dithiothreitol (DTT) to give, after exhaustive dialysis, the corresponding thiol-modified macromolecular derivatives HA-DTPH and HA-DTBH. The degree of substitution of HA-DTPH and HA-DTBH could be controlled from 20% to 70% of available glucuronate carboxylic acid groups. The pK(a) values of the HA-thiol derivatives were determined spectrophotometrically to be pK(a) = 8.87 (HA-DTPH) and pK(a) = 9.01 (HA-DTBH). The thiol groups could be oxidized in air to reform disulfide linkages, which resulted in HA-DTPH and HA-DTBH hydrogel films. Further oxidation of these hydrogels with dilute H(2)O(2) created additional cross-links and afforded poorly swellable films. The disulfide cross-linking was reversible, and films could be again reduced to sols with DTT. Release of blue dextran from cross-linked films was used as a model for drug release. The rapid gelation of the HA-DTPH solution under physiological conditions was also achieved, which demonstrated the capacity for in situ cell encapsulation. Thus, L-929 murine fibroblasts were encapsulated in HA-DTPH hydrogel; these cells remained viable and proliferated during 3 days of culture in vitro.     

An in situ collagen-HA hydrogel system promotes survival and preserves the proangiogenic secretion of hiPSC-derived vascular smooth muscle cells

Human-induced pluripotent stem cell-derived vascular smooth muscle cells (hiPSC-VSMCs) with proangiogenic properties have huge therapeutic potential. While hiPSC-VSMCs have already been utilized for wound healing using a biomimetic collagen scaffold, an in situ forming hydrogel mimicking the native environment of skin offers the promise of hiPSC-VSMC mediated repair and regeneration. Herein, the impact of a collagen type-I-hyaluronic acid (HA) in situ hydrogel cross-linked using a polyethylene glycol-based cross-linker on hiPSC-VSMCs viability and proangiogenic paracrine secretion was investigated. Our study demonstrated increases in cell viability, maintenance of phenotype and proangiogenic growth factor secretion, and proangiogenic activity in response to the conditioned medium. The optimally cross-linked and functionalized collagen type-I/HA hydrogel system developed in this study shows promise as an in situ hiPSC-VSMC carrier system for wound regeneration.     

Hyaluronan (HA) Interacting Proteins RHAMM and Hyaluronidase Impact Prostate Cancer Cell Behavior and Invadopodia Formation in 3D HA-Based Hydrogels

To study the individual functions of hyaluronan interacting proteins in prostate cancer (PCa) motility through connective tissues, we developed a novel three-dimensional (3D) hyaluronic acid (HA) hydrogel assay that provides a flexible, quantifiable, and physiologically relevant alternative to current methods. Invasion in this system reflects the prevalence of HA in connective tissues and its role in the promotion of cancer cell motility and tissue invasion, making the system ideal to study invasion through bone marrow or other HA-rich connective tissues. The bio-compatible cross-linking process we used allows for direct encapsulation of cancer cells within the gel where they adopt a distinct, cluster-like morphology. Metastatic PCa cells in these hydrogels develop fingerlike structures, “invadopodia”, consistent with their invasive properties. The number of invadopodia, as well as cluster size, shape, and convergence, can provide a quantifiable measure of invasive potential. Among candidate hyaluronan interacting proteins that could be responsible for the behavior we observed, we found that culture in the HA hydrogel triggers invasive PCa cells to differentially express and localize receptor for hyaluronan mediated motility (RHAMM)/CD168 which, in the absence of CD44, appears to contribute to PCa motility and invasion by interacting with the HA hydrogel components. PCa cell invasion through the HA hydrogel also was found to depend on the activity of hyaluronidases. Studies shown here reveal that while hyaluronidase activity is necessary for invadopodia and inter-connecting cluster formation, activity alone is not sufficient for acquisition of invasiveness to occur. We therefore suggest that development of invasive behavior in 3D HA-based systems requires development of additional cellular features, such as activation of motility associated pathways that regulate formation of invadopodia. Thus, we report development of a 3D system amenable to dissection of biological processes associated with cancer cell motility through HA-rich connective tissues.     

Facile synthesis and characterization of disulfide-cross-linked hyaluronic acid hydrogels for protein delivery and cell encapsulation

Injectable hyaluronic acid (HA) hydrogels cross-linked via disulfide bond are synthesized using a thiol-disulfide exchange reaction. The production of small-molecule reaction product, pyridine-2-thione, allows the hydrogel formation process to be monitored quantitatively in real-time by UV spectroscopy. Rheological tests show that the hydrogels formed within minutes at 37 °C. Mechanical properties and equilibrium swelling degree of the hydrogels can be controlled by varying the ratio of HA pyridyl disulfide and macro-cross-linker PEG-dithiol. Degradation of the hydrogels was achieved both enzymatically and chemically by disulfide reduction with distinctly different kinetics and profiles. In the presence of hyaluronidase, hydrogel mass loss over time was linear and the degradation was faster at higher enzyme concentrations, suggesting surface-limited degradation. The kinetics of hydrogel erosion by glutathione was not linear, nor did the erosion rate correlate linearly with glutathione concentration, suggesting a bulk erosion mechanism. A cysteine-containing chemokine, stromal cell-derived factor 1α, was successfully encapsulated in the hydrogel and released in vitro without chemical alteration. Several different cell types, including fibroblasts, endothelial cells, and mesenchymal stem cells, were successfully encapsulated in the hydrogels with high cell viability during and after the encapsulation process. Substantial cell viability in the hydrogels was maintained up to 7 days in culture despite the lack of adhesion between the HA matrix and the cells. The facile synthesis of disulfide-cross-linked, dual-responsive degradable HA hydrogels may enable further development of bioactive matrices potentially suitable for tissue engineering and drug delivery applications.     

Microinjected fluorescent polystyrene beads exhibit saltatory motion in tissue culture cells

Microinjected 0.26-micron fluorescent, carboxylated microspheres were found to display classical saltatory motion in tissue culture cells. The movement of a given particle was characterized by a discontinuous velocity distribution and was unaffected by the activity of adjacent particles. The microspheres were translocated at velocities of up to 4.7 micron/s and sometimes exhibited path lengths greater than 20 micron for a single saltation . The number of beads injected into a cell could range from a few to over 500 with no effect on the cell's ability to transport them. Neither covalent cross-linking nor preincubation of the polystyrene beads with various proteins inhibited the saltatory motion of the injected particles. The motion of the injected beads in cultured cells was reversibly inhibited by the microtubule poison nocodazole, under conditions in which actin-rich, nitrobenzoxadiazol - phallacidin -staining structures remain intact. Whole-cell high voltage electron microscopy of microinjected cells that were known to be moving the fluorescent microspheres revealed that the beads were embedded in the cytoplasmic matrix and did not appear to be membrane bound. The enhanced detectability of the fluorescent particles over endogenous organelles and the ability to modify the surfaces of the beads before injection may enable more detailed studies on the mechanism of saltatory particle motion.     

Compatibility of cells of the nervous system with structured biodegradable chitosan-based hydrogel matrices

Hydrogel matrices for cell cultivation have been generated by two-photon laser polymerization of unsaturated chitosan derivatives and methacrylated hyaluronic acid. The adhesive and toxic properties of the matrices have been assessed, and the matrices have been shown to have a good compatibility with primary hippocampal cell cultures. The formation of morphologically normal neural networks by cells of the nervous system cultured on the surface of hydrogel matrices has been observed. The metabolic status of dissociated hippocampal cells cultured on the matrices was similar to that of the control cultures, as shown by the results of MTT reductase activity assay. Thus, matrices based on unsaturated polysaccharide derivatives crosslinked by laser irradiation showed good compatibility with differentiated cells of the nervous system and considerable potential for use in neurotransplantation.     

Calcium alginate beads as a slow-release system for delivering angiogenic molecules in vivo and in vitro.

A method previously used in this laboratory for entrapment of tumor cells in alginate beads has been extended to provide a slow release delivery system for growth factors with known in vivo angiogenic activity. Protein growth factors were entrapped in alginate beads in amounts sufficient to cause incorporation of 3H-thymidine by COMMA-D cells in vitro, and in vivo neovascularization when injected subcutaneously into Balb/c mice. Entrapment of 125I-labelled growth factors showed that the amount of molecule entrapped in alginate beads may vary with the charge of the molecule. In vitro cell proliferation studies showed that entrapment in alginate beads may provide a slow-release system or a stabilizing environment for the protein. In some cases biological activity of the growth factor in solution was increased by the presence of control alginate beads. When alginate-entrapped growth factors were injected into Balb/c mice, induction of new blood vessels could be monitored qualitatively by macroscopic photography and assessed quantitatively by measuring the pooling of radiolabelled red blood cells at the experimental site. Subcutaneous injection of purified angiogenic factors not entrapped in alginate beads did not cause neovascularization. Diffusion of 125I-labelled growth factors from alginate beads in the animal showed that release in vivo may depend on the charge of the protein molecule. These results indicate that injection of purified molecules entrapped in alginate beads provides an effective localized and slow-release delivery of biologically active molecules. This delivery system may extend the time of effectiveness of biologically active molecules in vivo compared to direct injection without alginate entrapment. The method of entrapment and injection has potential for identifying active factors in tumor-induced angiogenesis and testing new compounds as modulators of neovascularization.     

Immobilization of Coleus blumei plant cells in temperature-sensitive hydrogel

Plant cell culture of Coleus blumei when entrapped in temperature-sensitive hydrogel beads [poly(N-isopropylacrylamide) containing 0.5% alginate], retained viability during the three weeks culture. Test also indicated that the hydrogel beads (containing 0.5% alginate) with immobilized cells were still temperature sensitive in aqueous solution.     

Research on the Long Time Swelling Properties of Poly (vinyl alcohol)/Hydroxylapatite Composite Hydrogel

Poly(vinyl alcohol)/hydroxylapatite(PVA/HA)composite hydrogel was prepared by repeated freezing and thawing.Thewater loss properties of the resultant hydrogel were investigated by using optical microscope.Long time immersion tests ofPVA/HA composite hydrogel were carried out in the diluted calf serum solution to study the change laws of swelling propertieswith the freezing-thawing cycles and HA content.The micro-morphologies of PVA/HA composite hydrogel after long timeimmersion were observed by means of the high-accuracy 3D profiler.The results show that the swelling process of PVA/HAcomposite hydrogel is the converse process of its water loss.Long time swelling ratio curves of PVA/HA composite hydrogel inthe calf serum solution are manifested as four stages of quick increase,decrease,slow decrease and stable balance,and itsequilibrium swelling ratio decreases with the increase of freezing-thawing cycles and HA content.It is revealed that the networkstructure of the composite hydrogel immersed for a long period is significantly improved with the increase of HA content.Perfect network structures of PVA/HA composite hydrogel as well as full and equilibrium tissues after swelling equilibrium areobtained when the HA content is 3% and the number of freezing-thawing cycles is 7.     

Bacterial cellulose-chitosan composite hydrogel beads for enzyme immobilization

In this work, we report the preparation of bacterial cellulose (BC)-chitosan composite hydrogel beads by co-dissolution of BC and chitosan in 1-ethyl-3-methylimidazolium acetate and subsequent reconstitution with distilled water. The BC-chitosan hydrogel beads were used as enzyme supports for immobilizing Candida rugosa lipase by physical adsorption and covalent cross-linking. BC-chitosan hydrogel beads immobilized lipase more efficiently than microcrystalline cellulose (MCC)-chitosan hydrogel beads. The amount of protein adsorbed onto BCchitosan beads was 3.9 times higher than that adsorbed onto MCC-chitosan beads, and the catalytic activity of lipase was 1.9 times higher on the BC-chitosan beads. The lipase showed the highest thermal and operational stability when covalently cross-linked on BC-chitosan hydrogel beads. The half-life time of the lipase cross-linked on BC-chitosan bead at 60°C was 22.7 times higher than that of free lipase. Owing to their inherent biocompatibility and biodegradability, the BC-chitosan composite hydrogel beads described here could be used to immobilize proteins for various biomedical, environmental, and biocatalytic applications.     

Gradients with depth in electrospun fibrous scaffolds for directed cell behavior

A major obstacle in creating viable tissue-engineered constructs using electrospinning is the lack of complete cellularization and vascularization due to the limited porosity in these densely packed fibrous scaffolds. One potential approach to circumvent this issue is the use of various gradients of chemical and biophysical cues to drive the infiltration of cells into these structures. Toward this goal, this study focused on creating durotactic (mechanical) and haptotactic (adhesive) gradients through the thickness of electrospun hyaluronic acid (HA) scaffolds using a unique, yet simple, modification of common electrospinning protocols. Specifically, both mechanical (via cross-linking: ranging from 27-100% modified methacrylated HA, MeHA) and adhesive (via inclusion of the adhesive peptide RGD: 0-3 mM RGD) gradients were each fabricated by mixing two solutions (one ramping up, one ramping down) prior to electrospinning and fiber collection. Gradient formation was verified by fluorescence microscopy, FTIR, atomic force microscopy, and cellular morphology assessment of scaffolds at different points of collection (i.e., with scaffold thickness). To test further the functionality of gradient scaffolds, chick aortic arch explants were cultured on adhesive gradient scaffolds for 7 days, and low RGD-high RGD gradient scaffolds showed significantly greater cell infiltration compared with high RGD-low RGD gradients and uniform high RGD or uniform low RGD control scaffolds. In addition to enhanced infiltration, this approach could be used to fabricate graded tissue structures, such as those that occur at interfaces.     

Production of polyclonal antibodies against peptide antigens using polystyrene beads as a carrier

We describe a method for producing polyclonal antibody (PAb) against peptide antigen (the epitope region of cytochromes P450 1A2 and 3A4, HA tag, FLAG tag, and c-myc tag) using a polymer bead as a carrier. Carboxylated polystyrene beads (0.05, 0.5, 1, and 2 μm diam) were conjugated with the peptide, which is known as an epitope region, using a chemical cross-linker and injected into rabbits with adjuvant. By immunizing the animals intradermally twice at two week intervals, an immunoblot assay with anti-sera showed the efficient generation of the PAb.     

Swelling induced detachment of chondrocytes using RGD-modified poly(N-isopropylacrylamide) hydrogel beads

Thermally sensitive poly(N-isopropylacrylamide, NIPAAm) hydrogel beads conjugated with a cell adhesive motif, GRGDY, were prepared and utilized as cell culture substrate for chondrocytes. They were produced to be uniform in size and distribution by using calcium alginate as a temporal mold. The RGD moieties were introduced, in a spatially selective manner, to the surface of the beads by conjugating GRGDY under the precollapsed state at a higher temperature above the lower critical solution temperature (LCST). These RGD-conjugated polyNIPAAm beads demonstrated a reversible swelling and deswelling behavior around the LCST, which enabled the chondrocytes attached on the surface of collapsed beads at 37 degrees C to readily detach when the temperature was shifted below 37 degrees C. The cell detachment percentage was largely affected by the temperature-dependent reswelling extent of the collapsed RGD-modified beads.     

Three-dimensional biomimetic hyaluronic acid hydrogels to investigate glioblastoma stem cell behaviors

Glioblastoma multiforme (GBM) is the deadliest form of primary brain tumor. GBM tumors are highly heterogeneous, being composed of tumor cells as well as glioblastoma stem cells (GSCs) that contribute to drug resistance and tumor recurrence following treatment. To develop therapeutic strategies, an improved understanding of GSC behavior in their microenvironment is critical. Herein, we have employed three-dimensional (3D) hyaluronic acid (HA) hydrogels that allow the incorporation of brain microenvironmental cues to investigate GSC behavior. U87 cell line and patient-derived D456 cells were cultured as suspension cultures (serum-free) and adherently (in the presence of serum) and were then encapsulated in HA hydrogels. We observed that all the seeded single cells expanded and formed spheres, and the size of the spheres increased with time. Increasing the initial cell seeding density of cells influenced the sphere size distribution. Interestingly, clonal expansion of serum-free grown tumor cells in HA hydrogels was observed. Also, stemness marker expression of serum and/or serum-free grown cells was altered when cultured in HA hydrogels. Finally, we demonstrated that HA hydrogels can support long-term GSC culture (up to 60 days) with retention of stemness markers. Overall, such biomimetic culture systems could further our understanding of the microenvironmental regulation of GSC phenotypes.