Tumor-Targeted Reduction-Sensitive Polymeric Mertansine Nano-Prodrug

Cancer is a worldwide devastating disease that has the leading morbidity and mortality. However, the current cancer treatment modalities, such as surgical resection, radiation therapy and chemotherapy, often cause serious adverse effects and result in low quality of life for patients. Hence, there is an urgent need to explore a safer and more efficient approach for cancer therapy. Polymer-drug conjugates have been developed as a promising nanoscale drug delivery system for cancer treatment owing to their improved drugs stability, water solubility, in vivo pharmacokinetics and bioavailability. Over decades of intensive development, more than 10 types of polymer-drug conjugates have been translated into the clinical trials, such as polyglutamic acid conjugated paclitaxel(Xyotax, Opaxio) and poly(N-(2-hydroxylpropyl) methacrylamide)-doxorubicin conjugates(PK1, PK2). The overall therapeutic effects of polymer-drug conjugates are, however, far from optimal, partly resulting from low tumor accumulation and inefficient intracellular drug release. Maytansine(DM1) is a powerful tubulin polymerization inhibitor with half-maximal inhibitory concentration(IC50) values in the picomolar range against various malignancies. The recent FDA approval of Kadcyla?(ado-trastuzumab emtansine) for the treatment of HER2-positive metastatic breast cancer inspired maytansinoid to be extensively exploited in research and development of antibody-maytansinoids conjugates(AMCs) for the selective treatment of malignancies. However, the clinical use of AMCs is challenged by their low drug content, poor stability, high cost, small scale production, and potential immunogenicity. In chapter 1, we present a brief literature overview on the current research status and challenge of polymer-drug conjugates, and recent development of multifunctional polymeric prodrug nanoparticles and combination chemotherapy.It is well noted that cytoplasm are reductive with high glutathione concentration(2-10 m M) as contrast to that in the extracellular matrix(GSH: 2-20 μM). In chapter 2, we designed and developed glutathione-sensitive hyaluronic acid-SS-mertansine prodrug(HA-SS-DM1) for targeted and efficacious treatment of CD44+ human breast tumor xenografts. Hyaluronic acid(HA) is a biodegradable, biocompatible and biological active natural polysaccharide and has shown a good targetability to many malignant cancer cells overexpressing CD44 receptors. DM1 was conjugated to HA through a reductively cleavable disulfide bond. Notably, HA-SS-DM1 had a high DM1 content of 20 wt.%. HA-SS-DM1 exhibited a superior targetability to MCF-7 cancer cells with an exceptionally low IC50 of 0.13 μg DM1 equiv./m L. The in vivo pharmacokinetic studies displayed that HA-SS-DM1 had an elimination half-life of 2.12 h. HA-SS-DM1 quickly accumulated in the MCF-7 tumor, the fluoresence intensity of which peaked at 24 h post injection, reached 8.17 %ID/g. Notably, HA-SS-DM1 displayed better tolerability with a maximum-tolerated dose(MTD) of 4 mg DM1 equiv./kg, which was 4-fold higher than free DM1(1 mg/kg). The therapeutic results demonstrated that HA-SS-DM1 effectively inhibited MCF-7 tumor growth at 800 μg DM1 equiv./kg(tumor inhibition rate: 88%) while causing reduced side effects as compared to free DM1. Glutathione-cleavable HA-SS-DM1 prodrug with excellent targetability, enhanced tolerability, and easy large-scale synthesis appears to be a promising strategy for targeted breast cancer therapy.Polymeric prodrug micelles while maintaining advantages of prodrugs, effectively protect drug molecules in its inner core, affording a high stability and reduced drug leakage. In chapter 3, we designed and developed c RGD-functionalized, reduction-sensitive micellar mertansine prodrug(c RGD-MMP) based on poly(ethylene glycol)-b-(poly(trimethylene carbonate)-graft-SSDM1)(PEG-P(TMC-g-SSDM1)) and c RGD-functionalized PEG-P(TMC-g-SSDM1). c RGD-MMP had a superb drug content of ~40 wt.% and a small size of ~45 nm. Confocal microscopy, flow cytometry and MTT assays indicated receptor-mediated uptake and high antitumor effect of c RGD-MMP in αvβ3 integrin over-expressing B16F10 melanoma cells(IC50: 0.16 μg DM1 equiv./m L). Notably, c RGD-MMP displayed a long elimination half-life of 5.25 h and a high MTD of 6 mg DM1 equiv./kg, 6-fold better than free DM1. The in vivo antitumor therapy demonstrated that c RGD-MMP could effectively inhibit B16F10 melanoma growth(Tumor inhibition rate: 97.5%), greatly improve mice survival rate(median survival time: 37 days) and diminish adverse effects as compared to free DM1 and non-targeted MMP controls. c RGD-MMP with superior stability, drug loading, and αvβ3 targetability offers an attractive alternative to AMCs for malignant tumor therapy.It is well known that MDA-MB-231 breast tumor also overexpressed αvβ3 integrins. In chapter 4, we assessed therapeutic efficacy of c RGD-MMP in MDA-MB-231 breast tumor xenografts. In vitro cell experiments revealed that c RGD-MMP exhibited superior targetability and higher antitumor effect toward MDA-MB-231 tumor cells(IC50: 0.13 μg DM1 equiv./m L) compared to non-targeting micellar mertansine prodrug(MMP). c RGD-MMP effectively accumulated in the MDA-MB-231 tumors and reached 8.09 %ID/g at 12 h post injection, which was much higher than MMP(3.32 %ID/g). The in vivo anticancer therapy studies demonstrated that c RGD-MMP effectively inhibited MDA-MB-231 breast tumor growth(tumor inhibition rate: 87.6%) and reduced adverse effects as compared to free DM1 and MMP controls. c RGD-MMP micellar mertansine prodrug exhibited strong antitumor effect against MDA-MB-231 tumor, further confirming its general applicability for αvβ3 integrin targeted cancer therapy.Combination chemotherapy with multiple-drugs could effectively slow down the mutation of cancer cells, overcome drug resistence, diminish toxic side effects and maximize therapeutic outcomes. In chapter 5, we developed a docetaxel-encapsulated, c RGD-functionalized redox-activable micellar mertansine prodrug(DTX-c RGD-MMP) for targeted and synergistic treatment of malignant B16F10 melanoma-bearing C57BL/6 mice. DTX-c RGD-MMP exhibited a small size of ca. 49 nm, high DTX and DM1 loading, low drug leakage under physiological conditions, while rapid release of both DTX and DM1 under a cytoplasmic reductive environment. Notably, MTT and flow cytometry assays showed that DTX-c RGD-MMP brought about synergistic antitumor effect to B16F10 cancer cells, with a combination index of 0.37 and an IC50 over 3 and 13-fold lower than c RGD-MMP(w/o DTX) and DTX-c RGD-Ms(w/o DM1) controls, respectively. The in vivo studies revealed that DTX-c RGD-MMP had a long circulation time and a markedly improved accumulation in B16F10 tumor as compared to the non-targeting DTX-MMP control(9.15 versus 3.13 %ID/g at 12 h post-injection). Interestingly, mice treated with DTX-c RGD-MMP showed almost complete growth inhibition of B16F10 melanoma, with tumor inhibition efficacy following an order of DTX-c RGD-MMP > DTX-MMP(w/o c RGD) > c RGD-MMP(w/o DTX) > DTX-c RGD-Ms(w/o DM1) > free DTX. In accordance, DTX-c RGD-MMP significantly improved the survival rate of B16F10 melanoma-bearing mice. Importantly, DTX-c RGD-MMP caused little adverse effects as revealed by mice body weights and histological analyses. this smart micellar mertansine prodrug provides a promising nanoplatform for emerging combinatorial cancer chemotherapy.Chapter 6 presents an overview of the thesis and provides a future perspective in the field.