Development of a lipid-based modular drug-RNA co-delivery nanoparticle approach for breast cancer therapy

Abstract

Triple-negative breast cancer (TNBC) is a form of breast cancer characterized by the low expression of specific cell receptors, leading to the ineffectiveness of treatments targeting those receptors. While mono-chemotherapy was ineffective against TNBC, combined regimens of different chemotherapy drugs were selected as standard treatments against TNBC but still resulted low efficacy against metastatic breast cancer (MBC). Therapeutic nucleic acids, an alternative treatment, showed significant targeting ability and can overcome drug resistance. However, its stability problem and biological barriers made an appropriate carrier necessary. Therefore, drug-gene co-delivered nanoparticles have been explored to achieve synergistic therapeutic effects by combing the targeting ability of genes and the potency of drugs. Lipid-based nanoparticle, which showed good endosomal escape capability, has been explored as a carrier to co-deliver nucleic acid molecules and chemotherapy drugs to further enhance the multi-targeting ability of treatment and overcome biological barriers and drug resistance. In our previous studies, a lipid-based nanoparticle structure GT-in-DcNP enabled the co-delivery of two chemotherapy drugs and showed high efficacy and safety against TNBC. However, GT-in-DcNP structure was not designed for nucleic acid loading. To enable the loading of RNA molecules on GT-in-DcNP nanoparticle structure, layer-by-layer (LbL) approach was used. With a nanoparticle core, a layer of charged polymers, such as negatively charged RNA polymer or positively charged chitosan (Chi) polymer, could be coated on nanoparticle core through electrostatic interactions between opposite charges. On the outside of first polymer layer, another layer of polymers with opposite charges could be coated, further enhancing the biocompatibility, stability, or targeting according to the specific polymer coated. More than one layer of therapeutic agents could be loaded in LbL structure, which increased the loading capacity of therapeutic agents in lipid-based nanoparticles. This thesis research leverages on the previously developed GT-DCNP. The overarching goal of this study is developing a lipid-nanoparticle platform formulation approach to enable the RNA molecule loading with a LbL approach to enhance therapeutic efficacy against TNBC.In this study, DG-in-LNP core with different formulations were designed, prepared, and characterized. The LbL coating was carried out by electrostatic interactions between positively charged DG-in-LNP core, negatively charged nucleic acid molecules, and another layer of cationic polymer Chi. We validated that the addition of a cationic lipid (DOTAP) into DG-in-LNP core formulation was positively associated with nanoparticle average size and zeta potential, overturning the nanoparticle to positively charged structure and enabling the loading of negatively charged RNA molecules on its surface. Meanwhile, we added cholesterol into nanoparticle to stabilize its lipid structure. We also studied the impact of rehydration solvent and PTX loading on nanoparticle stability and surface charge. The formulation with the greatest stability and appropriate properties, including the size of 111.3 nm and zeta potential +15.6 mV, was used for further loading of RNA and Chi. In metastatic breast cancer cell lines, including 4T1 and MDA-MB-231, we found that the transfection efficiency of mRNA loaded in nanoparticle was low. Compared to nanoparticles formulated with mRNA, the siRNA nanoparticles had much smaller size and a more stable structure: The size of siRNA nanoparticle loaded with Chi was maintained under 150 nm, while the size of mRNA nanoparticle has exceeded 1000 nm. In MDA-MB-231 expressing luciferase reporter gene, siRNA nanoparticles with Chi loading and without were evaluated. We discovered that the loading of PTX in nanoparticle led to 20% to 40% higher luminescence signal. However, the increasing loading of Chi eliminated the difference in bioluminescence at the highest Chi loading ratio and overcome the disruption of siRNA delivery and transfection by PTX. Meanwhile, Chi also mitigated the toxicity induced by nanoparticle, enhancing the biocompatibility of nanoparticles. Nanoparticles loaded with PTX showed similar cytotoxicity as free PTX form, validating the successful release of PTX into cells. Overall, this study provides a novel approach that enables the loading of RNA molecules on stable lipid-based nanoparticle structure while enhancing the stability and targeting ability of nanoparticle.