In vitro evaluation of cytotoxicity and cellular uptake of alternating copolymers for use as drug delivery vehicles
Author(s)Miller, Michelle Teresa
Massachusetts Institute of Technology. Dept. of Chemical Engineering.
Clark K. Colton.
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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 (19F 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.(cont.) 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 125I or 3H radioactive analysis and fluorescence confocal microscopy. Microscopy images of the fluorescently labeled polymer alone demonstrated that the polymer was likely confined to vesicles within the cytoplasm and it was not found in the nucleus, but encapsulated doxorubicin was shown to be largely confined to the nucleus.(cont.) Theoretical models of polyvalent binding were employed to guide the design of the targeting polymers, however, the polymers used in this study appeared largely non-specific for the targeted cells when studied in vitro. The cellular uptake of polymer targeted with EPPT was twice that of untargeted polymer, although the difference was not statistically significant. For polymers containing folate, regardless of the amount of folate attached, the length of the spacer used, or the type of radioactive label used, the uptake did not decrease in the presence of an excess of folate, indicating a high amount of non-specific uptake for all folate-containing polymers. When all of the folate-containing polymers were used to competitively inhibit 3Hfolate, almost all inhibited the uptake by 1 or 2 orders of magnitude, suggesting that the targeted polymers bind to the FR. An in-depth study of the cell-association of these polymers clarified that polymer was taken up non -specifically in high amounts. An excess of unlabeled folate, up- or down-regulation of the FR, and cleaving the FR did not measurably affect polymer uptake, but did alter folate uptake. It was also determined that a low level of polymer does bind to the FR. The amount of surface bound polymer was much lower than the total uptake of polymer in folate-free media for each polymer concentration investigated. In addition, the amount of surface bound folate and polymer decreased when the FR was cleaved, confirming the attachment of polymer to the FR. Light scattering measurements showed that polymers that contain folate form aggregates of multiple polymers chains.(cont.) It is possible that this aggregation was allowing a portion of the folate ligands to be hidden in the aggregate and unavailable for binding to the FR, the most likely mechanistic cause of the failure of the folate-containing polymers to demonstrate targeting. The versatility of these polymer constructs allows for continued optimization of a targeting delivery system for drugs and imaging agents as lessons discovered from passed studies are incorporated into the design. Initial in vivo biodistribution studies were begun to explore the behavior of these polymers in mouse models of human cancers. The alternating copolymers used in this study do accumulate in tumors in vivo, comparable to the levels of accumulation observed in the literature. It is desirable to minimize the accumulation in other organs, while maximizing the accumulation in the tumor tissue. Therefore, this preliminary study warrants continued investigation of our polymer platform as a delivery vehicle in vitro and in vivo.
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2009.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Includes bibliographical references.
DepartmentMassachusetts Institute of Technology. Dept. of Chemical Engineering.
Massachusetts Institute of Technology