Multifunctional Nanoparticle Platforms for Nucleic Acid Delivery, Cancer Therapy, and Immune Cell Engineering

Abstract

Cancer remains a leading cause of morbidity and mortality worldwide despite substantial advances in surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy. Many tumors develop mechanisms that enable therapeutic resistance, immune evasion, and disease recurrence, limiting the long-term effectiveness of conventional treatments. Gene therapy and immune-cell engineering have emerged as promising strategies for addressing these challenges because they enable precise modulation of cellular behavior and therapeutic protein expression. However, the successful clinical application of nucleic-acid therapeutics remains limited by poor stability, rapid degradation, inefficient cellular uptake, restricted intracellular trafficking, and inadequate target specificity.Nanoparticles offer a versatile platform for overcoming these barriers. Through careful control of size, surface chemistry, composition, and functional ligands, nanoparticle systems can protect therapeutic cargos, improve pharmacokinetics, facilitate intracellular delivery, and enable cell-specific targeting. Polymeric and inorganic nanoparticles are particularly attractive because they can integrate multiple complementary functions within a single platform, including cargo condensation, serum stabilization, endosomal escape, controlled release, imaging capability, and receptor-mediated targeting. This dissertation investigates the design, optimization, and application of multifunctional nanoparticle platforms for cancer therapy and immune-cell programming. Four complementary nanoparticle systems were developed to address major challenges in nucleic-acid delivery and cancer immunotherapy. The first study examined iron oxide nanoparticle-based mRNA delivery systems for hard-to-transfect cancer cells. Iron oxide cores with different surface chemistries were evaluated to determine how nanoparticle composition influences mRNA condensation, intracellular trafficking, endosomal escape, cargo release, transfection efficiency, and biocompatibility. The results demonstrated that optimized iron oxide nanocarriers significantly enhanced mRNA delivery and gene expression while maintaining favorable cytocompatibility, establishing important design principles for nonviral mRNA delivery. The second study developed a chitosan-based nanoparticle platform for the co-delivery of paclitaxel, a chemotherapeutic agent, and polyinosinic-polycytidylic acid (Poly IC), an immunostimulatory nucleic-acid analogue. By integrating chemotherapy and immune activation within a single formulation, the nanoparticles simultaneously promoted direct tumor-cell killing and dendritic-cell activation. The platform demonstrated efficient cellular uptake, sustained drug release, potent cytotoxic effects against multiple cancer cell types, and robust induction of immune-stimulatory responses, illustrating the potential of multifunctional nanoparticles for chemoimmunotherapy. The third study focused on targeted mRNA delivery to T lymphocytes for chimeric antigen receptor (CAR) T-cell engineering. An antibody-coated polymeric nanoparticle platform composed of perfluorinated polyethyleneimine, PEGylated polyethyleneimine, heparin, and anti-CD3 antibodies was developed to selectively target T cells. The nanoparticles achieved efficient mRNA encapsulation, cellular uptake, and intracellular delivery, resulting in transient CAR expression and antigen-dependent T-cell activation. This approach offers a nonviral alternative to conventional ex vivo CAR-T manufacturing and demonstrates the feasibility of direct immune-cell programming through targeted nanoparticle delivery in vivo. The fourth study extended this platform to macrophage engineering for cancer immunotherapy. Macrophage-targeted nanoparticles functionalized with anti-F4/80 antibodies were designed to deliver CAR-encoding mRNA and generate CAR-macrophages in situ. The engineered macrophages exhibited enhanced tumor-cell recognition, antigen-specific phagocytosis, and pro-inflammatory activation. In vivo administration enabled targeted macrophage transfection and demonstrated significant antitumor activity in a syngeneic breast cancer model, highlighting the potential of nanoparticle-mediated immune-cell programming for the treatment of solid tumors. Collectively, these studies establish a versatile framework for the rational design of multifunctional nanoparticle systems capable of overcoming critical barriers to nucleic-acid delivery. The work demonstrates the progression of nanoparticle technology from cancer-cell transfection and chemoimmunotherapy to targeted in vivo immune-cell engineering. The design principles and therapeutic strategies developed in this dissertation may facilitate the translation of next-generation gene therapies and immunotherapies for cancer treatment.