Unwanted immune responses against therapeutic proteins compromise treatment safety and therapeutic efficacy in patients. To mitigate these immune responses, we developed phosphatidylserine (PS)-containing lipid nanoparticles that mimic the surface properties of apoptotic cells, thereby converting an immunogen into a tolerogen. Previous studies have shown that mice prophylactically treated with PS nanoparticles- either double-chain PS or single-chain PS (LysoPS), administered intravenously or subcutaneously, suppressed humoral immune responses against therapeutic proteins such as Factor VIII (FVIII) and α-Glucosidase (GAA). However, only LysoPS successfully induced oral tolerance, whereas double-chain PS did not. This suggests that the structure of PS alters immune responses when given orally. To understand the structure of PS on the impacts on oral tolerance, we formulated dimyristoyl phosphatidylcholine (DMPC) nanoparticles containing PS variants that differed in the number of acyl chains, degree of unsaturation, and carbon chain length. These structural modifications affected PS surface exposure on the nanoparticles, which in turn modulated their tolerogenic potential. Using ovalbumin (OVA) as a model antigen, we evaluated immune responses in the presence or absence of different PS nanoparticles. Our data revealed that PS structure significantly influenced its surface exposure, which inversely correlated with OVA-specific IgG1 antibody titers and directly correlated with the frequency of T helper 3 cells (Th3). These findings highlight a structure-function relationship in PS-mediated oral tolerance, demonstrating that specific PS configurations can enhance tolerogenic outcomes by increasing PS surface exposure. This insight supports the rational design of PS-based oral immunotherapies to treat clinical conditions involving unwanted immune responses.
