Biraja C. Dash

The influence of size and charge of chitosan/polyglutamic acid hollow spheres on cellular internalization, viability and blood compatibility

Polymeric hollow spheres can be tailored as efficient carriers of various therapeutic molecules due to their tunable properties. However, the entry of these synthetic vehicles into cells, their cell viability and blood compatibility depend on their physical and chemical properties e.g. size, surface charge. Herein, we report the effect of size and surface charge on cell viability and cellular internalization behaviour and their effect on various blood components using chitosan/polyglutamic acid hollow spheres as a model system. Negatively charged chitosan/polyglutamic acid hollow spheres of various sizes 100, 300, 500 and 1000 nm were fabricated using a template based method and covalently surface modified using linear polyethylene glycol and methoxyethanol amine to create a gradient of surface charge from negative to neutrally charged spheres respectively. The results here suggest that both size and surface charge have a significant influence on the spheres behaviour, most prominently on haemolysis, platelet activation, plasma recalcification time, cell viability and internalization over time. Additionally, cellular internalization behaviour and viability was found to vary with different cell types. These results are in agreement with those of inorganic spheres and liposomes, and can serve as guidelines for tailoring polymeric solid spheres for specific desired applications in biological and pharmaceutical fields, including the design of nanometer to submicron-sized delivery vehicles.

Tunable elastin-like polypeptide hollow sphere as a high payload and controlled delivery gene depot

Self-assembly driven processes can be utilized to produce a variety of nanostructures useful for various in vitro and in vivo applications. Characteristics such as size, stability, biocompatibility, high therapeutic loading and controlled delivery of these nanostructures are particularly crucial in relation to in vivo applications. In this study, we report the fabrication of tunable monodispersed elastin-like polypeptide (ELP) hollow spheres of 100, 300, 500 and 1000 nm by exploiting the self-assembly property and net positive charge of ELP. The microbial transglutaminase (mTGase) cross-linking provided robustness and stability to the hollow spheres while maintaining surface functional groups for further modifications. The resulting hollow spheres showed a higher loading efficiency of plasmid DNA (pDNA) by using polyplex (~70 μg pDNA/mg of hollow sphere) than that of self-assembled ELP particles and demonstrated controlled release triggered by protease and elastase. Moreover, polyplex-loaded hollow spheres showed better cell viability than polyplex alone and yielded higher luciferase expression by providing protection against endosomal degradation. Overall, the monodispersed, tunable hollow spheres with a capability of post-functionalization can provide an exciting new opportunity for use in a range of therapeutic and diagnostic applications.

Mannosylated Polyethyleneimine−Hyaluronan Nanohybrids for Targeted Gene Delivery to Macrophage-Like Cell Lines

Nonviral gene delivery systems have a number of limitations including low transfection efficiency, specificity, and cytotoxicity, especially when the target cells are macrophages. To address these issues, the hypothesis tested in this study was that mannose functionalized nanohybrids composed of synthetic and natural polymers will improve transfection efficiency, cell viability, and cell specificity in macrophages. Robust nanohybrids were designed from hyaluronic acid (HA) and branched polyethyleneimine (bPEI) using carbodiimide chemistry. The reaction product, i.e., branched polyethyleneimine-hyaluronic acid (bPEI-HA) copolymer was subsequently functionalized with mannose at the terminal end of the copolymer to obtain mannosylated-bPEI-HA (Man-bPEI-HA) copolymer. UV spectroscopy and gel retardation studies confirmed the formation of polyplexes at polymer to DNA weight ratio ≥ 2. Alamar Blue and MTT assay revealed that the cytotoxicity of the developed nanohybrids were significantly (P < 0.05) lower than that of unmodified bPEI. Mannose functionalization of these nanohybrids showed specificity for both murine and human macrophage-like cell lines RAW 264.7 and human acute monocytic leukemia cell line (THP1), respectively, with a significant level (P < 0.05) of expression of gaussia luciferase (GLuc) and green fluorescent reporter plasmids. Internalization studies indicate that a mannose mediated endocytic pathway is responsible for this higher transfection rate. These results suggest that hyaluronan-based mannosylated nanohybrids could be used as efficient carriers for targeted gene delivery to macrophages.

Implantable tissue-engineered blood vessels from human induced pluripotent stem cells

Derivation of functional vascular smooth muscle cells (VSMCs) from human induced pluripotent stem cells (hiPSCs) to generate tissue-engineered blood vessels (TEBVs) holds great potential in treating patients with vascular diseases. Herein, hiPSCs were differentiated into alpha-smooth muscle actin (α-SMA) and calponin-positive VSMCs, which were seeded onto polymer scaffolds in bioreactors for vascular tissue growth. A functional TEBV with abundant collagenous matrix and sound mechanics resulted, which contained cells largely positive for α-SMA and smooth muscle myosin heavy chain (SM-MHC). Moreover, when hiPSC-derived TEBV segments were implanted into nude rats as abdominal aorta interposition grafts, they remained unruptured and patent with active vascular remodeling, and showed no evidence of teratoma formation during a 2-week proof-of-principle study. Our studies represent the development of the first implantable TEBVs based on hiPSCs, and pave the way for developing autologous or allogeneic grafts for clinical use in patients with vascular disease.

Controlled release of plasmid DNA from hyaluronan nanoparticles.

Encapsulation of plasmid DNA (pDNA) in nanoparticulate gene delivery systems offers the possibility of control in dosing, enhanced pDNA uptake, increased resistance to nuclease degradation and sustained release of functionally active pDNA over time. Extracellular matrix based biomaterial i.e. hyaluronan (HA) was used to encapsulate pDNA (pCMV-GLuc, Gaussia Luciferase reporter plasmid DNA having CMV promoter) in submicron size particulate system. Nano size range (~400-600 nm) pDNA loaded hyaluronan nanoparticles were formulated by ionic gelation followed by the cross-linking method with high encapsulation efficiency (~75-85%). The particle preparation process was further optimized for molecular weight, cross-linking method, cross-linking time and plasmid/polymer ratio. The entrapped plasmid maintained its structural and functional integrity as revealed by agarose gel electrophoresis. The pDNA was released from the hyaluronan nanoparticles in a controlled manner over a period of one month. In vitro transfection by one-week released pDNA from nanoparticles with transfecting agent branched polyethyleneimine (bPEI) resulted in significantly higher expression levels than those in pDNA alone which demonstrated the functional bioactivity of released pDNA. For cellular localization studies, the hyaluronan nanoparticles encapsulated with FITC-dextran were incubated with adipose derived stem cells (ADSCs) and localization in the cellular environment were investigated. The results of this study illustrate that hyaluronan nanoparticles were rapidly internalized by the cells through nonspecific endocytosis and remained intact in the cytosol for up to 24 h.

Tissue-Engineered Vascular Rings from Human iPSC-Derived Smooth Muscle Cells

Summary There is an urgent need for an efficient approach to obtain a large-scale and renewable source of functional human vascular smooth muscle cells (VSMCs) to establish robust, patient-specific tissue model systems for studying the pathogenesis of vascular disease, and for developing novel therapeutic interventions. Here, we have derived a large quantity of highly enriched functional VSMCs from human induced pluripotent stem cells (hiPSC-VSMCs). Furthermore, we have engineered 3D tissue rings from hiPSC-VSMCs using a facile one-step cellular self-assembly approach. The tissue rings are mechanically robust and can be used for vascular tissue engineering and disease modeling of supravalvular aortic stenosis syndrome. Our method may serve as a model system, extendable to study other vascular proliferative diseases for drug screening. Thus, this report describes an exciting platform technology with broad utility for manufacturing cell-based tissues and materials for various biomedical applications.

Emulsion cross-linked chitosan/nanohydroxyapatite microspheres for controlled release of alendronate.

Sustained delivery of growth factors has emerged as an essential requirement for bone tissue engineering applications for the treatment of various kinds of bone defects. Chitosan (CH) has attracted particular attention for drug delivery and bone tissue engineering because of its favorable biocompatibility and biodegradability. In this study, a composite microsphere system containing CH and nanohydroxyapatite (nHA)-alendronate (AL) particles was fabricated by employing both emulsification and cross-linking strategies. The microspheres were characterized for their surface morphology, composition, size distribution, drug loading efficiency and release properties. The results showed that loading efficiency and sustained release of hydrophilic AL were significantly improved, which is ideal for locally sustained release in the bone microenvironment. In vitro osteogenic studies showed that the microspheres could enhance the osteogenic activity of rabbit adipose-derived stem cells. In conclusion, the CH/nHA-AL composite microspheres exhibit promising properties as a candidate for local treatment for bone defects.

Stem Cells and Engineered Scaffolds for Regenerative Wound Healing

The normal wound healing process involves a well-organized cascade of biological pathways and any failure in this process leads to wounds becoming chronic. Non-healing wounds are a burden on healthcare systems and set to increase with aging population and growing incidences of obesity and diabetes. Stem cell-based therapies have the potential to heal chronic wounds but have so far seen little success in the clinic. Current research has been focused on using polymeric biomaterial systems that can act as a niche for these stem cells to improve their survival and paracrine activity that would eventually promote wound healing. Furthermore, different modification strategies have been developed to improve stem cell survival and differentiation, ultimately promoting regenerative wound healing. This review focuses on advanced polymeric scaffolds that have been used to deliver stem cells and have been tested for their efficiency in preclinical animal models of wounds.