Biomedical applications of photo responsive lipid-based gene/drug delivery systems

Gene delivery via the nonviral route (i.e., transfection) has emerged in recent decades for biomedical applications, which provides a promising approach for elucidating gene function, genetic engineering and gene therapy for cancer and genetic diseases. The success of nonviral gene delivery highly relies on the development of efficient and biocompatible delivery vectors. Among these synthetic nanoscale vectors, liposomes and polymeric nanomaterials are excellent candidates due to the advantages of safety, easy production, minimal immunotoxicity and high transfection efficiency. It can be envisioned that once these nanocarriers have reached their desired sites, the kinetics and extent of gene release from nanocarriers plays a significant role in the therapeutic performance. The targeting capability of gene delivery systems can also be considered as another important determinant of efficacy of gene action and overall treatment outcome. Therefore, development of triggerable gene delivery systems for on-demand gene release is the subject of current and future considerations to achieve better therapeutic index of gene therapy. My PhD research is mainly focused on development of lipid-based nanocarriers where the payload release can be activated by irradiation from visible light or X-ray. By using these two triggering modalities, therapeutic effect from loaded gene and/or drug was enhanced significantly, compared with traditional liposome delivery systems. My first project focuses on gene silencing in rat PC12 cells by light-triggered liposomes. These liposomes composed of cationic and neutral lipids and a photosensitiser were utilized in asODN delivery for gene silencing. Gene silencing efficiency of the pituitary adenylyl cyclase-activating peptide (PACAP) receptor 1 (PAC1R) was enhanced by almost 40% under light irradiation compared to the non-irradiated groups. In order to assess this lightactivated gene release process, the subcellular analysis was conducted through imagingbased quantitation. Endo/lysosomal escape of antisense oligonucleotides was documented at different time points based on quantitative analysis of colocalization between fluorescently labelled DNA and endo/lysosomes. This work laid a foundation for further development of more complicated liposome delivery systems. In the second project, further modifications of these liposomes were made to deliver the larger DNA fractions, plasmid DNA (pDNA) expressing the enhanced green fluorescent protein (EGFP). Cholesterol with appropriate amount was incorporated into liposomal structure to enhance the liposome stability in physiological environment. In addition, high complexation ability of polycation molecules with the DNA molecules was also taken advantage, the designed liposome-polycation-DNA (LPD) nanocomplexes, which incorporate verteporfin (VP) in a lipid bilayer and the complex of polyethylenimine (PEI)/plasmid DNA (pDNA) encoding EGFP (polyplex) in the central cavity of the liposome. The nanocomplexes were demonstrated to obtain the light triggered release of pDNA from the liposomes upon irradiation with a near-infrared (NIR) light-emitting diode (LED) light source. The release mechanism is driven by reactive oxygen species (ROS) oxidization via the photochemical reaction from the PS, leading to the release of pDNA into the cytosol and subsequent gene transfer. Light-triggered endolysosomal escape of pDNA at different time points was confirmed by quantitative analysis of colocalization between pDNA and endolysosomes. The efficiency of this photo-induced gene transfection was demonstrated to be more than double compared to non-irradiated controls. Additionally, we observed reduced cytotoxicity of the LPD nanocomplexes compared with the polyplexes alone. We have thus shown that light-triggered and biocompatible LPD nanocomplexes enable improved control of gene delivery which will be beneficial for future gene therapies. The third part discussed my main contributions to the following work on the drug/gene delivery platforms developed by introducing verteporfin and/or gold nanoparticles into PLGA polymers or the liposomal bilayer: (1) Photodynamic therapy (PDT) by using X-ray triggered Poly (D, L-lactide-co-glycolide) (PLGA) polymer nanoconstructs (equal authorship contribution): A dual PDT system was developed that can be triggered by both red light at 690 nm and X-ray radiation. PLGA nanoparticles conjugated with folic acid (FA) and incorporating verteporfin, can generate cytotoxic singlet oxygen for cell killing effects and allowed for specific targeting to the HCT116 cancer cells which overexpress the folate receptors (FRs). (2) In vitro and in vivo enhanced gene knockdown and antitumour effect by using the X-ray triggered liposomes (second authorship contribution): The same X-ray triggered liposomes loaded with a chemotherapy drug, doxorubicin killed human colorectal cancer cells more effectively than in the absence of X-ray triggering. They have been further demonstrated better antitumor effect in the colorectal cancer in vivo, which indicates the feasibility of a synergistic effect in the course of standard radiotherapy combined with chemotherapy delivered via X-ray triggered liposomes. The future work on liposome-mediated clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease (Cas9) delivery system is summarized in the last chapter. In this study, the light-triggered liposomal gene editing systems will be investigated in human cells and zebrafish embryos. In summary, my PhD work is structured as a thesis by publication. The chapters are presented in the form of published peer-reviewed journal papers. Key words: lipid-based nanovectors; gene transfection; nonviral gene delivery; drug delivery system; light induced delivery; photochemical internalization; drug delivery; photodynamic therapy; controlled release.