Wang, Jun2025-01-272025-01-272025-01-272025-01-10https://hdl.handle.net/10012/21438Proteins serve as the “workers” of biochemistry, orchestrating nearly all biological functions. Functional endogenous proteins are often related to the pharmacokinetics and pharmacodynamics of drugs and nanomedicines, particularly in processes such as drug absorption, biodistribution, and metabolism. That means the innate interactions between proteins and drugs/nanoparticles exist, but the discovery and application of these interactions are underappreciated so far. By imitating the protein binding behaviors and interactions, some proteins may hold significant promise in drug and nanoparticle delivery due to their biocompatibility and functionalities. This thesis presents a methodology for engineering biomimetic protein coronas to camouflage cationic peptide/siRNA (P/si) nanocomplexes by utilizing proteins derived from the innate P/si protein corona (P/si-PC), which was also applied to the peptide-based lipid nanoparticles (pLNP). By leveraging these protein corona species, an efficient method for producing protein-bound chemotherapeutic nanoparticles in aqueous phases using microfluidic technology was developed. For cationic nanoparticles, the spontaneous nanoparticle-protein corona formation and aggregation in biofluids can trigger unexpected biological reactions. This thesis presents a biomimetic strategy for camouflaging the P/si with single or dual proteins, which exploits the unique properties of endogenous proteins and stabilizes the cationic P/si for safe and targeted delivery. An in-depth study of P/si-PC formation and protein binding was conducted. The results provided insights into the biochemical and toxicological properties of cationic nanocomplexes and the rationales for engineering biomimetic protein camouflages. Based on this, the human serum albumin (HSA) and apolipoprotein AI (Apo-AI) ranked within the top 20 abundant protein species of P/si-PC were selected to construct biomimetic HSA-dressed P/si (P/si@HSA) and dual protein (HSA and Apo-AI)-dressed P/si (P/si@HSA_AI), given that the dual-protein camouflage plays complementary roles in efficient delivery. A branched cationic cell-penetrating peptide (CPP, b-HKR) was tailored for siRNA delivery, and their nanocomplexes including the cationic P/si and biomimetic protein-dressed P/si were produced by a precise microfluidic technology. The biomimetic anionic protein camouflage greatly enhanced P/si biostability and biocompatibility, which offers a reliable strategy for overcoming the limitation of applying cationic nanoparticles in biofluids and systemic delivery. Currently, commercially applied lipid nanoparticles (LNPs) for RNA delivery, such as in siRNA and mRNA vaccines, utilize similar lipid compositions and ratios, raising the risk of unintentional patent infringement. This research attempted to engineer a novel peptide-based LNP formulation stabilized and functionalized by artificial protein corona that constitutes HSA and lipoprotein (Apo-AI; apolipoprotein E, Apo-E). The cationic peptide (b-HKR) enabled efficient siRNA condensation and reversible protein binding. Combining b-HKR and the artificial protein corona offers an alternative to the commonly used ionizable lipids, PEG-lipids, and excipients (such as sucrose), providing both pH-responsive functionality and storage stability. The in vitro results showed that the dual protein (HSA and Apo-AI) functionalized pLNP (pLNP@HSA_AI) is optimal for enhanced stability and RNAi efficacy. In contrast, single protein-functionalized pLNPs encountered a dilemma: pLNP@HSA improved stability but showed almost no RNAi efficacy, while the pLNP@AI exhibited remarkable RNAi efficacy but aggregated upon the addition of Apo-AI. The dual protein (HSA and Apo-E) functionalized pLNP (pLNP@HSA_E) also showed promise in addressing this dilemma, although the use of Apo-E is less cost-effective than Apo-AI due to its limited availability. The use of endogenous proteins, particularly albumin, for the targeted delivery of chemotherapeutics has proven practical. However, how to effectively produce the protein-bound chemotherapeutics nanoparticles in a complete aqueous phase (without the use of organic solvents) is worth pursuing to eliminate the solvent-related safety risks. In this research, the protein-bound Dox (Dox) nanoparticles were successfully produced through a one-step microfluidic mixing process in aqueous phases, in which the nanoparticle formation was instantaneously mediated by a self-assembled nano-peptide (np). The np-mediated HSA-bound Dox (D-np-HSA) and dual proteins (HSA; Apo-AI)-bound Dox (D-np-HSA-AI) nanoparticles exhibited efficient drug encapsulation and pH-triggered drug releases. In vitro cellular studies showed that the nanoparticles (D-np-HSA and D-np-HSA-AI) exhibited superior efficacy in killing tumor cells (A549 and MCF7) while being less toxic to normal cells (NIH3T3) compared to free Dox. Notably, D-np-HSA-AI was less prone to induce drug resistance, and cell lines that developed resistance to free Dox remained sensitive to D-np-HSA-AI. Besides, the results revealed that drug resistance development of A549 is associated with cellular phenotypic (size, morphology, and dividing speed) changes. Cellular (cytoplasmic and nuclear) proteomics was conducted by comparing the protein species, abundances, and relation networks of normal, Dox-induced, and nanoparticle (D-np4-HSA-AI) induced A549 cells, which aimed to provide potential protein biomarkers associated with drug resistance and druggable protein/gene targets for overcoming the drug resistance.encell-penetrating peptideprotein-bound nanoparticlessiRNAchemotherapeuticsDrug resistanceDoctoral Thesis