Nanoparticle-mediated combination chemotherapy and photodynamic therapy overcomes tumor drug resistance
Graphical abstract
Nanoparticle-mediated combination chemotherapy and PDT results in enhanced tumor cell apoptosis and inhibition of cell proliferation. In addition to enabling cell kill through generation of reactive oxygen species (PDT), methylene blue also inhibits P-glycoprotein mediated doxorubicin efflux.
Introduction
Development of drug resistance significantly limits the success of chemotherapy in cancer patients and contributes to cancer recurrence and high mortality rates [1]. Tumor cells utilize specific mechanisms to survive toxic concentrations of anticancer agents. Overexpression of key proteins such as Bcl-2 and Mdm2 [2], Hsp70 [3], and mutations in TP53 [4] have been correlated to drug resistance. In addition, overexpression of drug efflux transporters like P-glycoprotein (P-gp) [5] and sequestration of drug in acidic intracellular organelles [6] or in the acidic tumor microenvironment [7] reduce availability of the drug at its site of action inside the tumor cell. The multi-factorial nature of drug resistance warrants therapies that employ multiple mechanisms to kill the tumor cell.
Photodynamic therapy (PDT) is based on the concept that photosensitizers, when exposed to light of specific wavelength, generate cytotoxic reactive oxygen species (ROS) capable of killing tumor cells [8]. In addition to their photodynamic properties, photosensitizers like methylene blue also inhibit P-gp-mediated efflux of anticancer drugs [9]. At the cellular level, this results in increased availability of the anticancer drug at its site of action. PDT has also been shown to damage tumor vasculature through direct effects on vascular endothelial cells [10]. Another important advantage of combining PDT with chemotherapy is the potential for inducing antitumor immunity [11]. Based on these multiple mechanisms, we rationalized that methylene blue-mediated PDT could significantly enhance the cytotoxicity of drugs like doxorubicin in drug-refractory cancers.
A major issue with chemotherapy and PDT is the decreased therapeutic efficacy and increased toxicity associated with the non-specific accumulation of therapeutic agents in non-target tissues. For example, doxorubicin causes cardiotoxicity due its accumulation in the heart [12]. Similarly, clinical use of methylene blue in PDT has been limited because of its extensive accumulation and inactivation in erythrocytes and endothelial cells [13], [14]. Encapsulation in a carrier like nanoparticles can significantly improve the accumulation of the therapeutic agent in the target tumor tissue through ‘Enhanced Permeation and Retention’ effect [15]. Further, encapsulation in nanoparticles can protect the drugs from inactivation in the biological environment [16], [17]. We have recently reported the fabrication of a novel surfactant-polymer nanoparticle delivery system suitable for efficient cellular delivery of charged, polar drugs like methylene blue and doxorubicin [18], [19]. These nanoparticles are fabricated from an anionic surfactant, Aerosol-OT™ (AOT), a pharmaceutical excipient [20], and a naturally occurring polysaccharide polymer, sodium alginate [21]. Our previous studies show that, in addition to improving the cellular delivery of encapsulated drugs, AOT-alginate nanoparticles significantly improve the ROS yield of photosensitizers like methylene blue [22]. In the current study, using AOT-alginate nanoparticles as a carrier, we investigated the efficacy of combination PDT and chemotherapy in a mouse drug-resistant mammary adenocarcinoma tumor model, and demonstrated that combining PDT with chemotherapy could overcome drug resistance by invoking multiple anticancer mechanisms.
Section snippets
Materials
Methylene blue, doxorubicin, ammonium acetate, sodium alginate, polyvinyl alcohol, and calcium chloride were purchased from Sigma-Aldrich (St. Louis, MO). AOT, acetonitrile, methanol, methylene chloride, and sodium acetate were purchased from Fisher Scientific (Chicago, IL). RPMI 1640 cell culture medium and phosphate buffered saline (PBS) were purchased from Invitrogen (Carlsbad, CA). JC tumor cells were purchased from American Type Culture Collection (Manassas, VA).
Nanoparticle formulation and characterization
Nanoparticles loaded with
Nanoparticle characterization
AFM studies indicated that nanoparticles loaded with both doxorubicin and methylene blue had a near spherical morphology (Fig. 1), with a number-average lateral diameter of 40 ± 7 nm. Dynamic light scattering (DLS) studies showed that nanoparticles had a number-average diameter ~ 73 nm. The difference in particle size measurement between AFM and DLS studies could be attributed to the fact that DLS measures diameter of hydrated particles while AFM measures the size of dry particles. Zeta potential
Discussion
Clinical efficacy of many anticancer drugs is limited by the development of drug resistance [26]. Distribution of the drug to non-target tissues, reduced drug accumulation in the tumor tissue, and poor penetration of the drug into the tumor cell contribute to reduced anticancer efficacy. In our studies, doxorubicin, either free or encapsulated in nanoparticles, was not effective against JC tumors. This is in agreement with previous studies, which have shown that JC tumor cells overexpress P-gp,
Acknowledgements
The funding was from the Presidential Research Enhancement Program, Wayne State University. We thank Prof. Guangzhao Mao and Dr. Hitesh Handa for assistance with AFM characterization of nanoparticles.
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