Time-dependent changes in opsonin amount associated on nanoparticles alter their hepatic uptake characteristics.

The relationship between the time-dependent change in serum proteins adsorbed on nanoparticles and their disposition to the liver was investigated by employing lecithin-coated polystyrene nanosphere with a size of 50 nm (LNS-50) as a model nanoparticle in rats. The total amount of proteins adsorbed on LNS-50 increased and the qualitative profile of serum proteins adsorbed on LNS-50 changed during the incubation with serum up to 360 min. The liver perfusion study indicated that the hepatic uptake of LNS-50 incubated with serum for 360 min was significantly larger than those of LNS-50 incubated for shorter period. It was suggested that the increase in the hepatic uptake of LNS-50 with the increase in incubation time would be ascribed mainly to the increase in the opsonin-mediated uptake by Kupffer cells. Semi-quantification of major opsonins, complement C3 (C3) and immunoglobulin G (IgG), and in vitro uptake study in primary cultured Kupffer cells demonstrated that the increase in C3 and IgG amounts adsorbed on LNS-50 was directly reflected in the increased disposition of LNS-50 to Kupffer cells. These results indicate that the amounts of opsonins associated on nanoparticles would change over time and this process would be substantially reflected in the alteration of their hepatic disposition characteristics.

Fetuin mediates hepatic uptake of negatively charged nanoparticles via scavenger receptor.

We tried to evaluate the possible involvement of fetuin in the scavenger receptors (SRs)-mediated hepatic uptake of polystyrene nanospheres with the size of 50 nm (NS-50), which has surface negative charge (zeta potential=-21.8+/-2.3 mV). The liver perfusion studies in rats revealed that the hepatic uptake of NS-50 pre-coated with fetuin (NS-50-fetuin) was significantly inhibited by poly inosinic acid (poly I), a typical inhibitor of SRs, whereas that of plain NS-50 or NS-50 pre-coated with BSA (NS-50-BSA) was not. The uptake of NS-50-fetuin by cultured Kupffer cells was also significantly inhibited by poly I, and anti-class A scavenger receptors (SR-A) antibody, suggesting that fetuin on NS-50 mediated the recognition and internalization of NS-50 by Kupffer cells and at least SR-A would be responsible for the uptake. Taken that Western blot analysis confirmed that fetuin certainly adsorbed on the surface of NS-50 after the incubation of NS-50 with serum, the results obtained in the present study indicate that fetuin would be one of the serum proteins that were substantially involved in the hepatic uptake of NS-50 via SRs.

Pre-coating with serum albumin reduces receptor-mediated hepatic disposition of polystyrene nanosphere: implications for rational design of nanoparticles.

We evaluated the in vivo disposition characteristics of polystyrene nanospheres (NS) with the particle size of 50 nm (NS-50) pre-coated with human serum albumin (HSA) after intravenous administration in rats. HSA-coated NS-50 showed much longer blood-circulating property and the hepatic uptake clearance for HSA-coated NS-50 was about 1/5 of that for NS-50. In parallel with the results obtained in the in vivo study, liver perfusion experiments also showed that the hepatic disposition of HSA-coated NS-50 was much less than that of NS-50 in the presence of serum in the perfusate. To unravel the mechanism behind the less affinity of HSA-coated NS-50 to the liver, serum proteins associated on the surface was quantitatively and qualitatively assessed. The results indicated that pre-coated HSA impaired subsequent association of serum proteins onto the surface, suggesting that the association of a given serum protein with opsonic activity might be suppressed by HSA pre-coating. From these findings, pre-coating of nanoparticles with serum albumin could be useful to prevent their rapid clearance by mononuclear phagocyte system in vivo.

Important role of serum proteins associated on the surface of particles in their hepatic disposition.

To elucidate the important factors for the difference in the hepatic disposition between polystyrene nanospheres with a size of 50 nm (NS-50) and lecithin-coated NS-50 (LNS-50), the liver perfusion studies and the in vitro uptake studies using the cultured Kupffer cells were performed. It was suggested that opsonin-mediated phagocytosis is not significantly involved in the hepatic disposition of LNS-50 in the presence of serum, whereas its involvement in the hepatic uptake of NS-50 was clearly demonstrated. Western blot analysis showed that IgG, complement C3, and fibronectin, well-known opsonins in the serum, adsorbed on the surface of NS-50 in larger amount than on the surface of LNS-50. On the other hand, serum albumin, which was suggested to function as a dysopsonin for the hepatic disposition of NS-50, was associated with both spheres almost to the same extent. These findings suggest that the hepatic disposition of LNS-50 at lower level should be ascribed to the less amount of serum opsonins associated on the surface and that the serum proteins associated with these spheres should be important as a determinant for their hepatic disposition.