Water-soluble polymers for targeted drug delivery to human squamous carcinoma of head and neck.

Human squamous cell carcinoma of the head and neck (SCCHN) is characterized by over expression of a tumor cell surface-specific receptor namely Hsp47/CBP2 that makes it a favorable candidate for targeted delivery of anticancer drugs. Several synthetic peptides have been identified as effective ligands for binding to CBP2. The purpose of this study is to investigate the potential of water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (Dox) conjugates containing a Hsp47/CBP2 binding peptide sequence, namely WHYPWFQNWAMA for targeted delivery to SCCHN. An HPMA copolymer containing Dox and CBP2 targeting peptide conjugated via lysosomally degradable glycylphenylalanylleucylglycine (GFLG) spacer was synthesized by free radical precipitation copolymerization. A control polymer without targeting moiety was also synthesized. The conjugates were characterized for drug content, peptide content, molecular weight and molecular weight distribution. The uptake of polymeric conjugates by both drug resistant and drug sensitive SCCHN cells were determined in vitro by flow cytometry using FACS scan analysis. Cytotoxicity of the conjugates towards drug sensitive as well as multidrug resistant SCCHN cells were evaluated by a clonal survival assay and compared to free Dox. The cytotoxicity of the free peptide was similarly evaluated. The internalization and subcellular fate of the conjugates in drug sensitive SCCHN cells was monitored using confocal microscopy. The new targetable copolymer contained 0.16 mmole peptide/g polymer. Studies on drug sensitive SCCHN cells demonstrated lesser uptake of both targeted and non-targeted conjugates compared to free Dox suggesting a slower endocytic mechanism of uptake for the conjugates as opposed to rapid diffusion of free Dox. At higher Dox equivalent concentrations (>20 microM) the targeted conjugate showed significantly higher uptake (p < or = 0.028) than the non-targeted conjugate. The uptake of the targeted conjugate was inhibited in the presence of an anti Hsp47 antibody suggesting the involvement of active receptor mediated endocytosis in cell entry of the conjugate. Compared to free Dox, the targeted and non-targeted conjugates caused marginally lower inhibition (p < or = 0.01) of the drug sensitive SCCHN cells. In contrast, the same conjugates showed significantly higher uptake (p < or = 0.004) by drug resistant SCCHN cells and caused significantly higher inhibition (p < or = 0.02) of drug resistant SCCHN cells when compared to free Dox. Results suggest that the polymeric conjugates were able to overcome drug resistance. Confocal microscopy studies demonstrated the uptake of the polymeric conjugates, followed by internalization, intralysosomal localization and subsequent release of Dox. HPMA copolymer-Dox-peptide conjugates targeted to SCCHN cells were able to overcome drug resistance and increase efficacy in vitro. The results suggest that targetable polymeric conjugates have potential to improve systemic head and neck cancer chemotherapy by increasing tumor localization and reducing dose-limiting toxicity.