In Vitro Evaluation of Cytotoxicity and Cellular Uptake of Alternating Copolymers for use as Drug Delivery Vehicles

Cancer is the collective group of diseases distinguished by uninhibited growth and spread of abnormal cells. It often results in death if the spread is not controlled. Most cancers are treated by surgery, radiation, chemotherapy, hormones, or immunotherapy. However, currently there are many issues with these forms of treatment, namely the lack of ability to consistently remove the entire tumor and the side effect of killing normal cells during the treatment process. Therefore there has been an increased interest in targeted drug delivery to tumors to specifically kill cancer cells. We have developed a highly adaptable amphiphilic alternating copolymer system that self-assembles into micelles for therapeutic delivery applications in cancer. The synthetic scheme includes the enzymatic polymerization of multifunctional linker molecules (dimethyl 5-hydroxyisopthalate) with poly(ethylene glycol). This chemoenzymatic synthesis is much faster and more convenient than an entirely chemical synthesis. Subsequent synthetic steps have been developed to attach ligands (for targeting), perfluorocarbons (F MR imaging), fluorescent dyes (NIRF imaging), and radioiodine (nuclear imaging and radioimmunotherapy) to the backbone. Attachment of hydrocarbon or perfluorocarbon sidechains provides amphiphilicity to produce the multimodal self-assembling micelles. Additionally, encapsulation procedures for chemotherapeutic agents, doxorubicin and paclitaxel, have been established. These unique alternating copolymer micelle nanoparticles were designed as delivery vehicles targeted to human cancer cells expressing the underglycosylated mucin-1 antigen, which is found on almost all epithelial cell adenocarcinomas, by use of the peptide EPPT or the folate receptor (FR) by use of folate. Development of the synthetic schemes has been coupled with in vitro toxicity experiments using various cell viability assays to minimize the toxic effect of these copolymer structures. Overall the polymers used in this study were largely non-toxic when studied in vitro. The non-toxic polymers were brought forward into drug delivery and uptake experiments. Cell death due to doxorubicin increased with encapsulation in these alternating copolymers and increased slightly more with the addition of targeting ligand to the encapsulating polymer. Encapsulating paclitaxel in polymer also increased cell death as compared to free drug. These results demonstrate that these alternating copolymers have had some success as drug delivery vehicles. Other in vitro studies included the investigation of cellular uptake by I or H radioactive analysis and fluorescence confocal microscopy. Microscopy images of the