Binding to and internalization by cultured cells of neocarzinostatin and enhancement of its actions by conjugation with lipophilic styrene-maleic acid copolymer.

The binding of a copoly(styrene-maleic acid)-conjugated neocarzinostatin (NCS) designated as smancs (Mr 16,000), and parental NCS (Mr 12,000) to cultured cells was investigated. These drugs were labeled with fluorescein isothiocyanate which retained biological activity and were used for binding studies. The binding of these drugs to HeLa cells was dependent on time and temperature, with 2 times more drug being bound at 37 degrees C than at 0 degree C. In the presence of a 100-fold molar excess of unlabeled NCS, the binding of smancs or NCS to HeLa cells was inhibited similarly. Therefore, it was suggested that smancs binds to NCS-binding sites (receptor) of the cell surface. However, the amount of cell-bound smancs was increased about 20-fold compared with that of NCS. Scatchard plot analyses of the binding of these drugs to HeLa and WISH cells indicated that this increase was due to alterations in affinity resulting from polymer conjugation of the drug to receptor rather than to an increase in the number of drug-binding sites at the cell surface. Furthermore, when the cytotoxicity of these drugs to HeLa cells was compared, smancs needed only 5 min to achieve 50% inhibition of the control. In contrast, the same dose level of NCS required more than 90 min to achieve the same toxic effect. More rapid internalization of smancs than NCS was also elucidated under fluorescence microscopy at 37 degrees C. There was no intracellular incorporation of these drugs below 20 degrees C. These results indicated that an increased lipophilicity of smancs appears to be responsible for its increased cell surface affinity, internalization rate, and toxic effect. Concordant to this interpretation was that among various copoly(styrene-maleic acid) esters, a more hydrophobic derivative showed more internalization (butyl greater than ethyl greater than carboxylate). These results suggest the possibility of utilizing protein tailoring to augment the subcellular activity of functional proteins.