A major complication in the replacement therapy of Factor FVIII (FVIII) for Hemophilia A (HA) is the development of unwanted immune responses. Previous studies from our lab have shown that pretreatment of FVIII in the presence of Phosphatidylserine (PS) resulted in hyporesponsiveness to subsequent administration of FVIII alone, due to the ability of PS to convert an immunogen to a tolerogen. We investigated the importance of biophysical properties of PS liposomes on its ability to convert an immunogen to a tolerogen. PS particles were prepared differing in size, protein-lipid topology, lamellarity and % association to FVIII keeping the composition of the particle same. PS particles were prepared in two different sizes with differing biophysical properties, smaller particles in the nanometer range (200 nm) and larger size particles in the micron range (2um). HA animals treated with both the nanometer and micron size PS particle showed a significant reduction in anti-FVIII antibody titers when compared to animals receiving free FVIII alone. Upon rechallenge with free FVIII animals that received FVIII along with the nanometer size particle continued to show reduced antibody responses. Animals receiving the micron size particle showed a slight increase in titers although they remained significantly lower than the free FVIII treated group. Upon culture with bone marrow dendritic cells (BMDCs), the nanometer size particle showed a reduction in CD40 expression and an increase in TGF-β cytokine production; which was not observed with the micron size particle. These results show that biophysical properties of PS play an important role in tolerance.