The Development Of Novel Hybridized Hyaluronic Acid Biomaterials For Applications In Tissue Engineering And Controlled Drug Delivery

This thesis is focused on the development of novel hybridized biomaterials for applications in tissue engineering and controlled drug delivery. Two types of materials used in my research are biopolymers and nanomaterials. Hyaluronic acid (HA), a natural polymer commonly found in extracellular matrix (ECM), was the primary polymer used in my research due to biocompatibility, biodegradability and the ease of manipulation to offer a wide range of physical and mechanical properties. Meanwhile, the nanomaterials, including Tobacco mosaic virus (TMV), gold nanorods and graphene, provide attractive properties, such as a nanoscale topography, biochemistry, high surface area, and special chemical and electrical properties. These properties could promote cell response, and allow the nanomaterials to react with a myriad of biological small molecules. Hence, in this thesis, we aimed to integrate these two types of materials to create a new type of biomaterials, and provide ideal properties that can leverage the tissue regeneration and targeted drug delivery for various treatment applications. Previously, our research group has found the intriguing effect of TMV on bone differentiation of mesenchymal stem cells (MSCs). We demonstrated that TMV could promote MSC osteogenesis via upregulating bone morphogenetic protein-2 (BMP-2) gene expression, a common gene used to enhance cartilage differentiation. This discovery combined with the clinical demand for cartilage tissue repair, which is limited by the minimum self-healing capacity of cartilage, inspired us to design a hybrid TMV scaffold in a simple injectable form, using thiol-ene “click” chemistry, to promote the MSC differentiation to cartilage. We demonstrated that cysteine-inserted TMV mutants (TMV1cys), containing thiol functional groups, could successfully crosslink to methacrylated hyaluronic acid (MeHA) polymers by thiol–ene “click” chemistry and form hydrogels under physiological conditions. The resulting hydrogels promoted in vitro chondrogenesis of MSCs by upregulating BMP-2 and enhancing collagen accumulation. In addition, incorporation of RGD-inserted TMV mutants (TMV-RGD1) in the HA hydrogels further promoted the in vitro chondrogenesis of BMSCs. Meanwhile, incorporation of gold nanorods, which provide similar size and shape as TMV, HA hydrogels showed no impact on the in vitro chondrogenesis. These results implied that the influences of nanoscale topography and biochemistry provided by TMV and TMV-RGD play critical roles in directing encapsulated MSC chondrogenesis. To better mediate new cartilage tissue formation, the physical and mechanical properties of the HA hydrogels were further optimized by varying the structures of thiol-tailored crosslinker molecules using dithiothreitol (DTT), 4-arm polyethylene glycol (PEG), and a multi-arm polyamidoamine (PAMAM) dendrimer. Chondrogenesis and osteogenesis of MSCs were highly enhanced in 4-arm PEG-crosslinked HA hydrogels, as measured by chondrogenic markers, glycosaminoglycan (GAG) and collagen accumulation, and osteogenic markers, alkaline phosphatase activity, and calcium deposition. It implied that the differentiation performance of MSCs directly correlated to the mechanical stiffness, permeability, pore size, porosity and chemistry of crosslinkers. The 4-arm PEG-crosslinked HA hydrogels seemingly mimicked the architecture of real cartilage and bone closer than other hydrogels. Aside from the application in tissue engineering, we developed a graphene oxide (GO)-hybridized HA-based hydrogel for perivascular drug delivery. The nanoscale GO was used as a novel nanocarrier for controlled drug delivery, owing to its high loading capacity of drugs resulting from the aromatic structure. HA serves as a biodegradable macroscale polymeric scaffold, making the prepared GO nanocarriers localized and stable in different microenvironments. The nanocarrier was firstly synthesized by attaching Senexin A (SNX), a kinase inhibitor and a possible anti-tumor drug, to GO via strong π–π interaction, followed by the in situ encapsulation of GO-SNX with HA-based hydrogel. The results of in vitro testing indicate high loading of SNX onto GO, and subsequent slow release of SNX within the therapeutic window. The slow release of SNX closed correlates to the loading-ratio of GO to SNX. With the in vitro results, we have demonstrated that the SNX loaded-GO hybridized HA hydrogel could be successfully attached to the decellularized scaffolds and form hydrogels under physiological condition. The hybridized materials provided a good biocompatibility and no impact on the proliferation and migration of vessel smooth muscle cells (VSMCs). More importantly, it could inhibit the dedifferentiation of VSMCs in the same manner as the SNX treatment

Injectable pH Thermo-Responsive Hydrogel Scaffold for Tumoricidal Neural Stem Cell Therapy for Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults and despite recent advances in treatment modalities, GBM remains incurable. Injectable hydrogel scaffolds are a versatile delivery system that can improve delivery of drug and cell therapeutics for GBM. In this report, we investigated an injectable nanocellulose/chitosan-based hydrogel scaffold for neural stem cell encapsulation and delivery. Hydrogels were prepared using thermogelling beta-glycerophosphate (BGP) and hydroxyethyl cellulose (HEC), chitosan (CS), and cellulose nanocrystals (CNCs). We evaluated the impact of neural stem cells on hydrogel gelation kinetics, microstructures, and degradation. Furthermore, we investigated the biomaterial effects on cell viability and functionality. We demonstrated that the incorporation of cells at densities of 1, 5 and 10 million does not significantly impact rheological and physical properties CS scaffolds. However, addition of CNCs significantly prolonged hydrogel degradation when cells were seeded at 5 and 10 million per 1 mL hydrogel. In vitro cell studies demonstrated high cell viability, release of TRAIL at therapeutic concentrations, and effective tumor cell killing within 72 h. The ability of these hydrogel scaffolds to support stem cell encapsulation and viability and maintain stem cell functionality makes them an attractive cell delivery system for local treatment of post-surgical cancers

Effects of Drug Physicochemical Properties on In-Situ Forming Implant Polymer Degradation and Drug Release Kinetics

In-situ forming implants (ISFIs) represent a simple, tunable, and biodegradable polymer-based platform for long-acting drug delivery. However, drugs with different physicochemical properties and physical states in the polymer-solvent system exhibit different drug release kinetics. Although a few limited studies have been performed attempting to elucidate these effects, a large, systematic study has not been performed until now. The purpose of this study was to characterize the in vitro drug release of 12 different small molecule drugs with differing logP and pKa values from ISFIs. Drug release was compared with polymer degradation as measured by lactic acid (LA) release and change in poly(DL-lactide-co-glycolide) (PLGA) molecular weight (MW) measured by size exclusion chromatography with multi-angle laser light scattering (SEC-MALS). Drug physical state and morphology were also measured using differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). Together, these results demonstrated that hydrophilic drugs have higher burst release at 24 h (22.8–68.4%) and complete drug release within 60 days, while hydrophobic drugs have lower burst release at 24 h (1.8–18.9%) and can sustain drug release over 60–285 days. Overall, drug logP and drug physical state in the polymer–solvent system are the most important factors when predicting the drug release rate in an ISFI for small-molecule drugs. Hydrophilic drugs exhibit high initial burst and less sustained release due to their miscibility with the aqueous phase, while hydrophobic drugs have lower initial burst and more sustained release due to their affinity for the hydrophobic PLGA. Additionally, while hydrophilic drugs seem to accelerate the degradation of PLGA, hydrophobic drugs on the other hand seem to slow down the PLGA degradation process compared with placebo ISFIs. Furthermore, drugs that were in a crystalline state within the ISFI drugs exhibited more sustained release compared with amorphous drugs

Influence of Cross-Linkers on the <i>in Vitro</i> Chondrogenesis of Mesenchymal Stem Cells in Hyaluronic Acid Hydrogels

This study aims to investigate the effect of the structures of cross-linkers on the <i>in vitro</i> chondrogenic differentiation of bone mesenchymal stem cells (BMSCs) in hyaluronic acid (HA)-based hydrogels. The hydrogels were prepared by the covalent cross-linking of methacrylated HA with different types of thiol-tailored molecules, including dithiothreitol (DTT), 4-arm poly­(ethylene glycol) (PEG), and multiarm polyamidoamine (PAMAM) dendrimer using thiol–ene “click” chemistry. The microstructure, mechanical properties, diffusivity, and degradation rates of the resultant hydrogels were controlled by the structural feature of different cross-linkers. BMSCs were then encapsulated in the resulting hydrogels and cultured in chondrogenic conditions. Overall, chondrogenic differentiation was highly enhanced in the PEG-cross-linked HA hydrogels, as measured by glycosaminoglycan (GAG) and collagen accumulation. The physical properties of hydrogels, especially the mechanical property and microarchitecture, were resulted from the structures of different cross-linkers, which subsequently modulated the fate of BMSC differentiation

Promotion of In Vitro Chondrogenesis of Mesenchymal Stem Cells Using In Situ Hyaluronic Hydrogel Functionalized with Rod-Like Viral Nanoparticles

This study focuses on the development of injectable hydrogels to mimic the cartilage microenvironment using hyaluronic acid (HA) derivatives as starting materials. Cysteine-inserted Tobacco mosaic virus (TMV) mutants (TMV1cys) could be cross-linked to methacrylated hyaluronic acid (MeHA) polymers by thiol–ene “click” chemistry and form hydrogels under physiological condition. The resulting hydrogels could promote in vitro chondrogenesis of bone marrow mesenchymal stem cells (BMSCs) significantly higher than that in the TMV-free HA hydrogels by upregulating bone morphogenetic protein-2 (BMP-2) expression and enhancing collagen accumulation

Steroid Eluting Esophageal-Targeted Drug Delivery Devices for Treatment of Eosinophilic Esophagitis

Eosinophilic esophagitis (EoE) is a chronic atopic disease that has become increasingly prevalent over the past 20 years. A first-line pharmacologic option is topical/swallowed corticosteroids, but these are adapted from asthma preparations such as fluticasone from an inhaler and yield suboptimal response rates. There are no FDA-approved medications for the treatment of EoE, and esophageal-specific drug formulations are lacking. We report the development of two novel esophageal-specific drug delivery platforms. The first is a fluticasone-eluting string that could be swallowed similar to the string test “entero-test” and used for overnight treatment, allowing for a rapid release along the entire length of esophagus. In vitro drug release studies showed a target release of 1 mg/day of fluticasone. In vivo pharmacokinetic studies were carried out after deploying the string in a porcine model, and our results showed a high local level of fluticasone in esophageal tissue persisting over 1 and 3 days, and a minimal systemic absorption in plasma. The second device is a fluticasone-eluting 3D printed ring for local and sustained release of fluticasone in the esophagus. We designed and fabricated biocompatible fluticasone-loaded rings using a top-down, Digital Light Processing (DLP) Gizmo 3D printer. We explored various strategies of drug loading into 3D printed rings, involving incorporation of drug during the print process (pre-loading) or after printing (post-loading). In vitro drug release studies of fluticasone-loaded rings (pre and post-loaded) showed that fluticasone elutes at a constant rate over a period of one month. Ex vivo pharmacokinetic studies in the porcine model also showed high tissue levels of fluticasone and both rings and strings were successfully deployed into the porcine esophagus in vivo. Given these preliminary proof-of-concept data, these devices now merit study in animal models of disease and ultimately subsequent translation to testing in humans