Correlation of subcellular compartmentalization of HPMA copolymer-Mce6 conjugates with chemotherapeutic activity in human ovarian carcinoma cells

Purpose. Intracellular targets sensitive to oxidized damage generated by photodynamic therapy (PDT) utilizing N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-mesochlorin e(6) monoethylenediamine (Mce(6)) conjugates was explored to aid in the design of second-generation PDT delivery systems. Methods. Low temperature, metabolic inhibitor, and nuclear localization sequences (NLS(FITC)) were used to achieve desired subcellular localization that was evaluated by confocal analysis and subcellular fractionation. Mce(6) was bound to HPMA copolymer conjugates via non-degradable dipeptide linkers (P-GG-Mce(6), P-NLS(FITC)-GG-Mce(6)) or lysosomally degradable tetrapeptide spacers (P-GFLG-Mce(6), P-NLS(FITC)-GFLG-Mce(6)). Chemotherapeutic efficacy was assessed by the concentration that inhibited growth by 50% (IC50), cell associated drug concentration ( CAD) and confocal microscopy. Results. P-GFLG-Mce(6) possessed enhanced chemotherapeutic activity compared to P-GG-Mce(6) indicating enzymatically released Mce(6) was more active than copolymer-bound Mce(6). Lysosomes appeared less sensitive to photodamage as observed by a higher IC50. Nuclear-directed HPMA copolymer-Mce(6) conjugates (P-NLS(FITC)-GGMce(6), P-NLS( FITC)-GFLG-Mce(6)) possessed enhanced chemotherapeutic activity. However, control cationic HPMA copolymer-Mce(6) conjugates containing a scrambled NLS (P-scNLS(FITC)-GG-Mce(6)) or amino groups (P-NH2-GG-Mce(6)) also displayed increased chemotherapeutic activity. Conclusions. Nuclear delivery was observed for P-NLS( FITC)-GGMce(6) and P-NLS(FITC)-GFLG-Mce(6) indicating NLS was a feasible approach for nuclear delivery. Due to the cationic nature of NLS, increased membrane binding of PDT systems incorporating cationic nuclear targeting moieties must be addressed.