Abstract
Gene therapy strategies can be useful for tissue engineering by modifying stem cells directly using various gene delivery carriers. Gene delivery offers a new and potentially promising treatment modality for the inherited genetic diseases or disorders. The development of delivery systems able to alter the biological profiles of therapeutic agents (viz., pDNA, siRNA, growth factors, drugs, etc.) is considered of utmost importance in biomedical research and the pharmaceutical industry. In this study, a redox- and pH-sensitive hyaluronic acid (HA) - polyethylenimine (PEI) copolymer with disulfide linkage was synthesized, characterized and examined as a potential non-viral gene vector. The physical and chemical properties of fabricated gene carrier were analyzed via 1H NMR and FT-IR for the demonstration of crosslinking of HA with PEI. TNBS assay and Ellman’s reagent were conducted for the verification of disulfide bond formation and crosslinking degree. The size, morphology and surface charge of nanoparticle were investigated by dynamic light scattering (DLS), transmission electron microscopy (TEM) and zeta potential, respectively. The ability of HA-ss-PEI conjugate to complex with plasmid DNA was observed by gel electrophoresis and the optimal N/P ratio was also determined. Fluorescent microscopy and ELISA spectroscopy were utilized to examine transfection efficiency and protein expression level of therapeutic pDNA. Finally, the ability of transfected hMSCs to differentiate into chondrocyte was investigated by Alcian blue stain and immunocytochemistry (ICC) for type II collagen. From the results, a novel redox- and pH-sensitive gene delivery nanocarrier has been successfully synthesized. The positive HA-ss-PEI conjugate complexed with negative charged plasmid DNA can be achieved via electrostatic attraction to form a stable spherical nanoparticle of 100 nm in diameter. N/P=2 possessed the optimal condition for releasing encapsulated plasmid when triggered by stimuli such as redox reaction and pH change. The nanocarrier could successfully complex with pDNA and transfect stem cells to produce specific protein for therapeutic purposes. hMSCs transfected with HA-ss-PEI/pEndo complexes could produce endostatin, an anti-angiogenic agent, and differentiate into chondrocytes after 7 days chondrogenic induction. In brief, the redox- and pH-sensitive HA-ss-PEI nanoparticles can be used as a promising non-viral gene carrier for stem cell gene therapy.