Nanoparticle-mediated combination chemotherapy and photodynamic therapy overcomes tumor drug resistance

Tumor cells utilize multiple mechanisms to evade the cytotoxic effect of anticancer drugs. Overexpression of drug efflux transporters like P-glycoprotein (P-gp), altered tumor microenvironment, and sequestration of the drug in acidic cellular organelles are important factors that prevent the accumulation of effective cellular concentrations of anticancer drug. The hypothesis of this research is that nanoparticle-mediated combination chemotherapy and photodynamic therapy (PDT) will target multiple mechanisms of drug resistance, resulting in more effective inhibition of resistant-tumor than conventional PDT and chemotherapy. To test this hypothesis, anticancer efficacy of aerosol OT-alginate (AOT-alginate) nanoparticles loaded with a combination of doxorubicin (as a model chemotherapeutic drug) and methylene blue (as a model photosensitizer for PDT) was tested in vitro and in vivo. The objectives of the research were to (1) formulate AOT-alginate nanoparticles loaded with combination of methylene blue and doxorubicin; (2) investigate the anticancer effectiveness of nanoparticle-mediated combination therapy in vitro in drug-resistant cell lines; (3) determine the optimal treatment conditions for effective tumor growth inhibition in vivo; and (4) determine the underlying mechanisms for enhanced therapeutic efficacy. Our long-term goal is to develop an anticancer treatment modality that addresses various mechanisms of drug resistance and enables an effective therapeutic response in drug-refractory cancer.;Both doxorubicin and methylene blue could be loaded in AOT-alginate nanoparticles efficiently. Physicochemical characterization studies indicated that nanoparticles have spherical morphology and size < 100 nm in diameter with a net negative surface charge. Nanoparticles were stable and easily dispersible in buffers and serum-containing cell culture medium. Previous studies in our laboratory have established the anticancer effectiveness of nanoparticles loaded with doxorubicin; however, no information was available on the suitability of AOT-alginate nanoparticles as carriers in PDT. Initial studies, therefore, investigated the in vitro PDT effectiveness of methylene blue-loaded nanoparticles. Cytotoxicity studies indicated that AOT-alginate nanoparticles significantly enhanced and sustained the PDT efficacy of methylene blue in drug-sensitive tumor cells. Mechanistic studies suggested that greater nuclear delivery of methylene blue and enhanced reactive oxygen species (ROS) production could have contributed to the enhanced PDT efficacy of nanoparticle-encapsulated methylene blue.;The use of AOT-alginate nanoparticles for the simultaneous delivery of doxorubicin and methylene blue was then investigated in drug resistant tumor cells in vitro. Nanoparticle-mediated combination therapy resulted in a significant induction of both apoptosis and necrosis, resulting in enhanced cytotoxicity in drug-resistant tumor cells. Improvement in cytotoxicity could be correlated with enhanced intracellular and nuclear delivery of the two drugs following combination therapy. Further, nanoparticle-mediated combination therapy resulted in significant ROS production compared to single drug treatment. Based on the positive results from in vitro studies, further studies investigating the therapeutic benefit of nanoparticle-mediated combination therapy were carried out in vivo. Balb/c mice bearing syngeneic JC tumors were used as a drug-resistant tumor model. Nanoparticle-mediated combination therapy significantly inhibited tumor growth and improved animal survival. Enhanced accumulation of both doxorubicin and methylene blue in resistant tumor cells following treatment with nanoparticles resulted in significant inhibition of tumor cell proliferation and increased induction of apoptosis. In addition, nanoparticle-mediated combination therapy resulted in tumor microvessel damage.;In conclusion, nanoparticle-mediated combination chemotherapy using doxorubicin and PDT using methylene blue resulted in multiple mechanisms of tumor growth inhibition. The results of these studies thus suggest that nanoparticle-mediated combination chemotherapy and photodynamic therapy using doxorubicin and methylene blue has significant therapeutic efficacy against drug-resistant tumors.