Vertically Aligned Biointerfaces for in Vitro and in Vivo Cellular Engineering


Student thesis: Doctoral Thesis

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Award date13 Jan 2022


Genetic factors generally involve in most aspects of human diseases, including cancers, infectious, and inherited diseases. Therefore, it has been a persistent goal to develop technologies for invalidating the genome of invading pathogens, repairing pathogenic mutations/defective genes, modulating stem cells to regenerative medicine, boosting immune responses to attack tumors and improving possibilities for other therapy. Aiming at genetic factors, gene therapy is a promising technique by which gene-induced diseases can be prevented or cured. However, the delivery of gene into target cells must overcome complex cellular and tissue barriers to enable efficient gene expression without disturbing essential regulatory networks. The development of a technique that facilitates gene delivery and expression without involving complicated processes is of great value. In this thesis, we employed a micro-/nano-engineered platform that involves biological micro-/nano-needles to introduce a variety of genetic factors into various types of cells with high efficiency and minimal toxicity.

The thesis begins with an introduction to gene therapy, which focuses on the strategies in intracellular delivery, as the prerequisite of gene expression. In Chapter 2, We firstly presented a high-efficiency cellular reprogramming strategy by puncturing cells with an array of diamond nanoneedles, which was applied to temporally disrupt cell membrane in a reversible and minimum-invasive format. While induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicines, their clinical application has been hindered by the extremely low reprogramming efficiency of somatic cells. This issue is especially true for the generation of patient-specific iPSCs, as it typically requires a start with limited number of human cells. In addition, generation of patient-specific iPSCs is difficult to scale up since extensive culture and proliferation may cause harmful accumulation of mutations. The development of nanoneedle-based platform enabled high-efficiency cytoplasmic delivery of mini-intronic plasmid (MIP) vectors to initiate the conversion of human fibroblast cells to either primed iPSCs or naïve iPSCs. The nano-puncturing operation was directly performed on cells in adherent culture without any cell lift-off, and it was completed within very short time (5 min). Culturing in feeder-free medium, the treated cells achieved a reprogramming efficiency of as high as 1.17±0.28%, which was over two orders of magnitude higher than those results from common methods involving plasmid delivery. In addition, the nanoneedle arrays could be applicable to different cell input by adjust the size of nanoneedle patch without sacrificing overall operational convenience. As a result, our nanoneedle-based platforms could fully unleash the translational potential of iPSCs for clinically relevant applications.

Despite the intriguing promise of in vitro gene therapy, targeting tissue in vivo is more attractive because in this way the cells in tissue are directly manipulated without involving the cumbersome process of collecting cells from the patient and transplanting the modified cells back. Inspired by the fact that our nanoneedle platform is failed to penetrate the tissues or skins and is operated under limited conditions largely reliant on a centrifugation technique, we then established a micro-electroporation platform to exploit the in vivo gene delivery technologies for the treatment of cancers and inherited disease. In Chapter 3, we developed a novel in vivo electroporation platform which delivers DNA vaccine efficiently and controllably to skin through an electric conductive microneedle patch. As we have established the ability of nanoneedle-based transfection to improve the ex vivo transfection efficiency. In this chapter we proceeded to exploit the in vivo gene delivery technologies for the treatment of cancers and inherited disease using painless and easy-to-use transdermal micro-electroporation device. It is known that traditional microneedles supporting naked DNA delivery usually exhibited rapid clearance and low transfection efficiency. As a response, we used electric conductive microneedles as both DNA carriers and electrodes to promote cytosolic delivery of DNA vaccines for achieving the optimal immunity via in vivo electroporation. We found that applying our micro-electroporation technology, a transfection efficiency of as high as ~70% could be achieved. Additionally, high levels of dendritic cells (DCs) activation and antigen cross presentation were elicited, once a model antigen ovalbumin (OVA) plasmid was encapsulated in the micro-electroporation devices. We also demonstrated that the combined vaccine facilitated local immune activation upon electroporation through the recruitment of DCs. Interestingly, we find that our strategy can induce a robust immune response using 10 ug dose, suggesting a 10-fold dose sparing in contrast to those obtained with IM injection (100 ug dose) in a single vaccination. We further demonstrated that our micro-electroporation platform could elicit striking anti-tumor T-cell responses and significantly induce the lysis of antigen-expression cells. The additional advantages of our micro-electroporation technology include decreased cell death and tissue damage with much lower voltage required and dry-state packaging and storage of DNA vaccine at room temperature without loss of bioactivity. In this regard, our micro-electroporation technology certainly offered a platform for effective DNA delivery and immunization via a pain-less and self-administrable manner.

The thesis ends with a Chapter 4, which summarizes our development in the current study as well as the challenges, and also gives a perspective on possible directions for future investigation. In summary, we employed micro- and nanotechnology to overcome complex cellular and tissue barriers to deliver gene into the target cell to enable efficient gene expression in a noninvasive manner. Specifically, the nanoneedle was applied to penetrate the cell membrane and access the cytosolic space of living cells in a reversible and minimum-invasive manner, and the microneedle with electroporation was developed to breach both the tissue barrier (e.g., stratum corneum) and cell membrane to achieve high delivery efficiency and cell viability.

We strongly believe that our in vivo electroporation methods will open the new possibility for the treatment of a variety of diseases like cancers, both infectious and inherited ones. We also believe that the continuous development of novel delivery methods base on micro/nanotechnology will present exciting opportunities for research and technology translation and make significant contributions to clinical trials.