Synthesis, characterization, and application of a biocompatible gene delivery nanocarrier constructed from gold nanostars and a chitosan–cyclodextrin–poly(ethylene imine) graft polymer

Abstract

Gene therapy can be an efficient method to treat genetic diseases, including cancer. However, the lack of biocompatible gene delivery systems with minimal toxicity limits the clinical success of this approach. We report the synthesis of an effective gene carrier using gold nanostars (AuNSs) in combination with chitosan (CS), β-cyclodextrin (CD), and branched-poly(ethylene imine) (bPEI) as a bionanocomposite (BNC) with minimal toxicity for gene delivery applications. We synthesized and characterized the AuNS@CS–CD–bPEI (AuNS@CCP) BNC using various analytical methods. Ultraviolet-visible spectroscopy confirmed the successful synthesis of AuNSs from Au nanoseeds. Nuclear magnetic resonance proved the conjugation of CS and CD upon its multistep synthesis. Transmission electron microscopy images confirmed the median size of AuNSs as 53 nm, which increased to 70 nm for the AuNS@CCP BNC. Energy dispersive X-ray (EDX) analysis further confirmed the presence of a polymeric layer of CS–CD–bPEI on AuNSs. We also measured the zeta potential of AuNS@CCP using dynamic light scattering to check its ability to bind to nucleic acids, which was +28.2 mV. The applicability of the BNC in nucleic acid transfection was evaluated in various mammalian cell lines (HEK293T, LN308, MDA-MB-231, and CT-26) using firefly luciferase-zetagreen (FLuc-ZsGreen) reporter genes by optical imaging, and synthetic mRNA coding for SARS-CoV-2 spike protein by immunoblot analysis to prove the ability of the BNC to transfect both DNA and RNA. We also characterized the N/P ratio of the BNC with the nucleic acids to optimize cell transfection efficiency. The cytotoxicity study (MTT assay) of the BNC showed no significant toxicity when used at the optimal N/P ratio. Overall, we show that the synthesized BNC has a high potential for use as a gene delivery agent for in vivo applications, potentially using various delivery routes such as intranasal, intramuscular, intrathecal, and intraperitoneal in treating genetic diseases and for vaccines in infectious diseases.