Date of Award

January 2019

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Michael Girardi

Abstract

The most common types of cancer in the world are keratinocyte-derived cutaneous carcinomas, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Although the gold standard for treatment is surgical excision, this is not always feasible for some patients who may have extensive disease or are not surgical candidates. Our aim was to develop a non-surgical, localized treatment for cutaneous neoplasms like squamous cell carcinoma (SCC) by using biodegradable nanoparticles (NPs) composed of poly-lactic acid hyperbranched polyglycerol polymers, which can be biochemically rendered to be non-adhesive (NNPs) or bioadhesive (BNPs), to deliver potent anti-tumor drugs such as camptothecin (CPT) to tumors. Previous studies have demonstrated that this unique nanoparticle coating enhances drug delivery with improved solubility and bioavailability within solid tumors, such as glioblastoma and uterine carcinoma. Early investigation of nanoparticle delivery of chemotherapy to treat cutaneous malignancies with in vitro studies using murine squamous cell carcinoma cell lines showed enhanced association with BNPs and NNPs by flow cytometry and confocal microscopy. This project is a continuation of these previous studies, investigating the in vivo efficacy of NP-encapsulated anti-tumor drugs like CPT and immune-modulating agents like Toll-like receptors (TLRs) ligands in the localized treatment of squamous cell carcinoma (SCC) in mice, in addition to further characterizing the mechanism of action of these drug-encapsulating particles in vitro. First, we confirmed the mechanism of action of these nanoparticles by demonstrating internalization of dye-loaded BNPs and NNPs in a murine PDV squamous cell carcinoma line with confocal microscopy, showing enhanced uptake with BNPs compared the NNPs. The rate of release of drug from these particles at physiologic conditions were assessed, revealing a fast peak release within 12 hours of drug administration and reaching nadir by 48 hours. Next, injection of dye-loaded NPs into PDV SCC tumors in mice resulted in widespread intratumoral distribution. Having established the ability of dye-loaded NNPs and BNPs to associate with and be taken up by SCC cells and tumors, we then assessed the potential therapeutic effects of NNP-encapsulated CPT (NNP-CPT) and BNP-encapsulated CPT (BNP-CPT) on tumors in vivo. Using subdermally transplanted SCC tumors in B6TCRβ-/- mice and wildtype B6 mice (WT), we designed several different experiments to determine the ideal drug concentrations and frequency of injections for maximum tumor resolution with NP encapsulated CPT. In TCRβ-/- mice, we started with weekly injections of 0.5mg CPT/100 µl of BNP-CPTs and 100 µl of PBS as control. Subsequent studies evolved to compare BNP-CPT, NNP-CPT, free CPT in IL (IL-CPT), and control vehicle (IL) treated tumors using external measurements of tumor growth and histology to determine the effect of NP-delivery of CPT. These experiments showed moderate advantages in delaying tumor progression, decreasing tumor burden and increasing necrosis within tumors using nanoparticle encapsulated CPT treatment. However, working in an immune-deficient model resulted in poor healing wounds, which prompted us to test NP-encapsulated CPT in transplanted SCC tumors in WT mice. Though wound healing was significantly improved in this model, ultimately, monotherapy with NP-encapsulated CPT was unsuccessful in decreasing tumor progression or burden. This led us to pursue immune modulating agents in WT mice to enhance the effect of NP-encapsulated CPT treatment. A small pilot study testing the combination of CPT-loaded NPs with a TLR9 agonist as weekly injections for 2 weeks showed promise in complete tumor resolution, though was inferior to monotherapy with TLR9 agonist alone. Together, these results demonstrate the potential for effective treatment of cutaneous carcinomas like SCC using biodegradable nanoparticle delivery of antitumor agents like chemotherapy and immune modulation. Next steps will be to study NP-encapsulated CPT treatment in a spontaneous SCC model system, which may better reflect the SCC tumor environment and structure seen in human skin cancers. Future studies should also aim to investigate the use of nanoparticles for delivering combinations of chemotherapy and immune modulating agents.

Comments

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