Disposition of positively charged Bowman-Birk protease inhibitor conjugates in mice: influence of protein conjugate charge density and size on lung targeting.

The influence of conjugate charge density and size on the targeting of cationic Bowman-Birk inhibitor (BBI) conjugates to the lungs was studied in mice. The biodistribution of BBI, either as the native protein or in the conjugated form (conjugated to a dicationic, tetracationic, or polycationic carrier), indicated that by increasing the charge density of BBI conjugates, the lung accumulation of the conjugates administered intravenously (i.v.) can be increased. The order of lung accumulation in these studies was as follows: polycationic- > tetracationic- > dicationic-conjugated BBI > BBI. The influence of conjugate size on lung accumulation was studied in three experiments. First, the biodistribution of poly(D-lysine) carriers of equal charge density but different molecular weight demonstrated that lung accumulation of polycationic carriers increases with an increase in carrier size. Second, the biodistributions of BBI, tyramine-derivatized poly(D-lysine)3 kDa, and poly(D-lysine)3 kDa conjugated to BBI indicated that an increase in conjugate size alone is not sufficient to promote the lung accumulation of cationic BBI conjugates. Finally, the biodistribution poly(D-lysine) complexed with heparin showed that targeting of a conjugate to the lungs can be abolished by neutralizing the charge on the carrier. Collectively, data in this paper demonstrate that the carrier-mediated targeting of BBI to the lungs is dependent on (a) cationization of BBI, (b) the conjugate positive charge density, and (c) the size of the cationic conjugate if the charge density is maintained. Also, the data show the size of the conjugate alone does not make a significant impact on lung accumulation.