Abiraman Srinivasan

Influence of the degree of methacrylation on hyaluronic acid hydrogels properties

The properties of hyaluronic acid (HA) hydrogels having a broad range of methacrylation are presented. Increasing solubility of glycidyl methacrylate (GM) in a co-solvent mixture during the methacrylation of HA with GM was shown to produce photopolymerizable HAGM conjugates with various degree of methacrylation (DM) ranging from 14% up to 90%. Aqueous solutions of HAGM macromonomers were photocross-linked to yield hydrogels with nearly full vinyl group conversions after 10 min exposure under ultraviolet light (UV). Hydrogels were characterized by uniaxial compression and volumetric swelling measurements. Keeping the DM constant, the shear modulus was varied from 16 kPa up to 73 kPa by varying the macromonomer concentration. However, at a given macromonomer concentration while varying the DM, similarly the shear modulus varied from 22 kPa up to 65 kPa. Preliminary in-vitro cell culture studies showed that GRGDS modified HAGM hydrogels promoted similarly cell interaction at both low and high DMs, 32% and 60%, respectively. Densely cross-linked hydrogels with a high DM have been shown to be more mechanically robust while maintaining cytocompability and cell adhesion.

Synthesis, mechanical properties, biocompatibility, and biodegradation of polyurethane networks from lysine polyisocyanates

Bone defects, such as compressive fractures in the vertebral bodies, are frequently treated with acrylic bone cements (e.g., PMMA). Although these biomaterials have sufficient mechanical properties for fixing the fracture, they are non-degradable and do not remodel or integrate with host tissue. In an alternative approach, biodegradable polyurethane (PUR) networks have been synthesized that are designed to integrate with host tissue and degrade to non-cytotoxic decomposition products. PUR networks have been prepared by two-component reactive liquid molding of low-viscosity quasi-prepolymers derived from lysine polyisocyanates and poly(epsilon-caprolactone-co-DL-lactide-co-glycolide) triols. The composition, thermal transitions, and mechanical properties of the biomaterials were measured. The values of Youngs modulus ranged from 1.20-1.43 GPa, and the compressive yield strength varied from 82 to 111 MPa, which is comparable to the strength of PMMA bone cements. In vitro, the materials underwent controlled biodegradation to non-cytotoxic decomposition products, and supported the attachment and proliferation of MC3T3 cells. When cultured in osteogenic medium on the PUR networks, MC3T3 cells deposited mineralized extracellular matrix, as evidenced by von Kossa staining and tetracycline labeling. Considering the favorable mechanical and biological properties, as well as the low-viscosity of the reactive intermediates used to prepare the PUR networks, these biomaterials are potentially useful as injectable, biodegradable bone cements for fracture healing.

Nanostructured hybrid hydrogels prepared by a combination of atom transfer radical polymerization and free radical polymerization

A new method to prepare nanostructured hybrid hydrogels by incorporating well-defined poly(oligo (ethylene oxide) monomethyl ether methacrylate) (POEO(300)MA) nanogels of sizes 110-120 nm into a larger three-dimensional (3D) matrix was developed for drug delivery scaffolds for tissue engineering applications. Rhodamine B isothiocyanate-labeled dextran (RITC-Dx) or fluorescein isothiocyanate-labeled dextran (FITC-Dx)-loaded POEO(300)MA nanogels with pendant hydroxyl groups were prepared by activators generated electron transfer atom transfer radical polymerization (AGET ATRP) in cyclohexane inverse miniemulsion. Hydroxyl-containing nanogels were functionalized with methacrylated groups to generate photoreactive nanospheres. (1)H NMR spectroscopy confirmed that polymerizable nanogels were successfully incorporated covalently into 3D hyaluronic acid-glycidyl methacrylate (HAGM) hydrogels after free radical photopolymerization (FRP). The introduction of disulfide moieties into the polymerizable groups resulted in a controlled release of nanogels from cross-linked HAGM hydrogels under a reducing environment. The effect of gel hybridization on the macroscopic properties (swelling and mechanics) was studied. It is shown that swelling and nanogel content are independent of scaffold mechanics. In-vitro assays showed the nanostructured hybrid hydrogels were cytocompatible and the GRGDS (Gly-Arg-Gly-Asp-Ser) contained in the nanogel structure promoted cell-substrate interactions within 4 days of incubation. These nanostructured hydrogels have potential as an artificial extracellular matrix (ECM) impermeable to low molecular weight biomolecules and with controlled pharmaceutical release capability. Moreover, the nanogels can control drug or biomolecule delivery, while hyaluronic acid based-hydrogels can act as a macroscopic scaffold for tissue regeneration and regulator for nanogel release.

Cellular uptake of functional nanogels prepared by inverse miniemulsion ATRP with encapsulated proteins, carbohydrates, and gold nanoparticles

Atom transfer radical polymerization (ATRP) was used to produce a versatile drug delivery system capable of encapsulating a range of molecules. Inverse miniemulsion ATRP permitted the synthesis of biocompatible and uniformly cross-linked poly(ethylene oxide)-based nanogels entrapping gold nanoparticles, bovine serum albumin, rhodamine B isothiocyanate-dextran, or fluoresceine isothiocyanate-dextran. These moieties were entrapped to validate several biological outcomes and to model delivery of range of molecules. Cellular uptake of nanogels was verified by transmission electron microscopy, gel electrophoresis, Western blotting, confocal microscopy, and flow cytometry. Fluorescent colocalization of nanogels with a fluorophore-conjugated antibody for clathrin indicated clathrin-mediated endocytosis. Furthermore, internalization of nanogels either with or without GRGDS cell attachment-mediating peptides was quantified using flow cytometry. After 45 min of incubation, the uptake of unmodified FITC-Dx-loaded nanogels was 62%, whereas cellular uptake increased to >95% with the same concentration of GRGDS-modified FITC-Dx nanogels. In addition, a spheroidal coculture of human umbilical vascular endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs) validated cell endocytosis. Application of ATRP enabled the synthesis of a functionalized drug delivery system with a uniform network that is capable of encapsulating and delivering inorganic, organic, and biological molecules.

Synthesis, characterization, and in vitro cell culture viability of degradable poly(N-isopropylacrylamide-co-5,6-benzo-2-methylene-1,3-dioxepane)-based polymers and crosslinked gels

Poly(N-isopropylacrylamide-co-5,6-benzo-2-methylene-1,3-dioxepane) (poly(NIPAAm-co-BMDO)) was synthesized by atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain transfer (RAFT) polymerization. Using UV-vis spectroscopy, the lower critical solution temperature (LCST) of poly(NIPAAm) and poly(NIPAAm-co-BMDO) copolymers were measured, varying with respect to the amount of incorporated BMDO. This material is degradable and possesses a LCST above room temperature and below body temperature, making it a potential candidate for use as an injectable tissue engineering scaffold to enhance fracture repair. ATRP and RAFT enabled preparation of polymers with control over molecular weight up to M(n) = 50,000 g/mol and M(w)/M(n) < 1.2. Degradation studies were performed in basic solution and in complete Dulbeccos modified Eagle medium. The cytotoxicity of the material and its degradation products were analyzed by in vitro cell culture analyses, including cytotoxicity live/dead and CyQUANT cell proliferation assays. Crosslinked scaffolds with degradable units within the polymer backbone and at the crosslinking sites were prepared using an ester-containing diacrylate crosslinker. Furthermore, incorporation of a GRGDS peptide sequence improved cell attachment to the gels. Controlled/living radical polymerization techniques allow for precise control over macromolecular structure and are poised to become powerful tools for tissue engineering scaffold synthesis.

Preparation of cationic nanogels for nucleic acid delivery.

Cationic nanogels with site-selected functionality were designed for the delivery of nucleic acid payloads targeting numerous therapeutic applications. Functional cationic nanogels containing quaternized 2-(dimethylamino)ethyl methacrylate and a cross-linker with reducible disulfide moieties (qNG) were prepared by activators generated by electron transfer (AGET) atom transfer radical polymerization (ATRP) in an inverse miniemulsion. Polyplex formation between the qNG and nucleic acid exemplified by plasmid DNA (pDNA) and short interfering RNA (siRNA duplexes) were evaluated. The delivery of polyplexes was optimized for the delivery of pDNA and siRNA to the Drosophila Schneider 2 (S2) cell-line. The qNG/nucleic acid (i.e., siRNA and pDNA) polyplexes were found to be highly effective in their capabilities to deliver their respective payloads.

End-group effects on the properties of PEG-co-PGA hydrogels

A series of resorbable poly(ethylene glycol)-co-poly(glycolic acid) (PEG-co-PGA, 4KG5) macromonomers have been synthesized with the chemistries from three different photopolymerizable end-groups (acrylates, methacrylates and urethane methacrylates). The aim of the study is to examine the effects of the chemistry of the cross-linker group on the properties of photocross-linked hydrogels. 4KG5 hydrogels were prepared by photopolymerization with high vinyl group conversion as confirmed by (1)H nuclear magnetic resonance spectrometry using a 1D diffusion-ordered spectrometry pulse sequence. Our study reveals that the nature of end-groups in a moderately amphiphilic polymer can adjust the distribution and size of the micellar configuration in water, leading to changes in the macroscopic structure of hydrogels. By varying the chemistry of the cross-linker group (diacrylates (DA), dimethacrylates (DM) and urethane dimethacrylates (UDM)), we determined that the hydrophobicity of a single core polymer consisting of poly(glycolic acid) could be fine-tuned, leading to significant variations in the mechanical, swelling and degradation properties of the gels. In addition, the effects of cross-linker chemistry on cytotoxicity and proliferation were examined. Cytotoxicity assays showed that the three types of hydrogels (4KG5 DA, DM and UDM) were biocompatible and the introduction of RGD ligand enhanced cell adhesion. However, differences in gel properties and stability differentially affected the spreading and proliferation of myoblast C2C12 cells.

Cell-Adhesive Star Polymers Prepared by ATRP

This study presents the synthesis and evaluation of cell adhesive poly(ethylene oxide) (PEO) star polymers for potential biomedical applications. Star polymers with a size of approximately 20 nm and with relatively low polydispersities (M(w)/M(n) ≤ 1.6), containing GRGDS (Gly-Arg-Gly-Asp-Ser) segments, were prepared by atom transfer radical copolymerization of PEO methyl ether methacrylate macromonomer (MM), telechelic GRGDS-PEO-acrylate MM, and ethylene glycol dimethacrylate (EGDMA). Results from (1)H NMR spectroscopy confirmed the covalent incorporation of the peptide into the star periphery. In vitro cytotoxicity experiments showed star polymers to be cytocompatible (≥95% cell viability) and GRGDS-star hybrid hydrogels supported the attachment of MC3T3.E1 (subclone 4) cells. Hybrid hydrogels were prepared by free radical photopolymerization based on 10% (wt/v) PEO dimethacrylates M(n) = 4000 g/mol with 1% (wt/v) GRGDS-star polymers having different peptide content. Cell adhesiveness was also determined from thin film coatings prepared with GRGDS-containing star polymers on nonadherent plastic plates. After 24 h incubation, phase contrast microscopy and scanning electron microscopy (SEM) images showed uniform cell adhesion and distribution over the film containing cell-adhesive star polymers. These results confirm that incorporation of RGD ligand-binding motifs into PEO-based star polymers is required to influence substrate-cell interactions.

Synthesis, degradation and biocompatibility of tyrosine-derived polycarbonate scaffolds

Polycarbonate terpolymers consisting of desaminotyrosyl-tyrosine alkyl esters (DTR), desaminotyrosyl-tyrosine (DT), and low molecular weight blocks of poly(ethylene glycol) (PEG) are a new class of polymers that have good engineering properties while also being resorbable in vivo. This study is the first evaluation of their (i) degradation behavior, (ii) in vitro cytotoxicity, and (iii) in vivo biocompatibility. Porous, tissue engineering scaffolds were prepared by a combination of solvent casting, porogen leaching and phase separation techniques. The scaffolds (>90% porosity) displayed (i) a bimodal pore distribution with micropores of less than 20 µm and macropores between 200 and 400 µm, (ii) a highly interconnected and open pore architecture, and (iii) a highly organized microstructure where the micropores are oriented and aligned along the walls of the macropores. Molecular weight (number average, Mn) and mass loss were determined in vitro (PBS at 37 °C) for up to 28 days. All three terpolymer compositions were fast degrading and retained only 10% of their initial molecular weight after 21 days, while mass loss during the 28 days was polymer composition-dependent. In vitro biocompatibility of the polymer scaffolds was determined up to 14 days by measuring metabolic activity of MC3T3.E1 (subclone 4) pre-osteoblasts. The outcome showed no statistical difference between cells cultured in monolayer and all tested polymer scaffolds. Robust cell attachment throughout the scaffold volume was observed by confocal microscopy and SEM. The biocompatibility of resorbing scaffolds was evaluated at 12 week in a critical sized defect (CSD) rabbit calvaria model and showed only a minimal inflammatory response. Overall, the results reported here illustrate the potential utility of tyrosine-derived polycarbonate terpolymers in the design of tissue engineering scaffolds.

Photo-Cross-Linkable Thermoresponsive Star Polymers Designed for Control of Cell-Surface Interactions

Star polymers with thermoresponsive arms, consisting of 2-(2-methoxyethoxy)ethyl methacrylate (MEO₂MA) and oligo(ethylene glycol) methacrylate with ~4 ethylene oxide units (OEOMA₃₀₀, M(n) = 300), were synthesized via atom transfer radical polymerization (ATRP). 25% of the arms contained benzophenone chain-end functionality at the star periphery. A mixture of linear poly(MEO₂MA-co-OEOMA₃₀₀)-Br macroinitiators without and with benzophenone end-group macroinitiators were (MI and Bzp-MI, respectively) cross-linked with ethylene glycol dimethacrylate to form star polymers. Formation of star polymers was monitored by GPC, and the presence of benzophenone functionality in the stars was confirmed by ¹H NMR. The UV-vis spectroscopy revealed that the star polymers exhibit the low critical solution temperature (LCST) at 27 °C, slightly lower than LCST of either MI or Bzp-MI. Commercially available tissue culture grade polystyrene surface was modified by depositing a thin film of functionalized stars and UV cross-linking (λ = 365 nm). The star polymers covalently attached onto surfaces allowed a control of cell shrinkage and attachment in response to temperature changes.