Reversal of doxorubicin-resistance by multifunctional nanoparticles in MCF-7/ADR cells

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Abstract

The efficacy of many chemotherapeutic agents is reduced in cells that have developed multiple drug resistance (MDR). To address this important problem, a biodegradable polymer was coupled to a photosensitizer and the resulting photosensitizer-nanoparticles were loaded with the chemotherapeutic agent doxorubicin. The combination of photosensitizer and chemotherapeutic agent had a synergistic action on a doxorubicin-resistant breast cancer MCF-7 cell line. To increase the effectiveness of this combination, d-alpha-tocopheryl poly(ethylene glycol) 1000 succinate (TPGS), an inhibitor of the multidrug transporter overproduced in these resistant cells, was added during the formation of the nanoparticles. The insertion of TPGS decreased the P-glycoprotein activity, increased the intracellular accumulation doxorubicin, and also increased the therapeutic efficacy of the resulting nanoparticles. Both TPGS and irradiation of the photoreactive nanoparticles caused doxorubicin to move from the cytoplasm to the nucleus. This combination of photodynamic activity in a powerful nanocarrier loaded with the chemotherapeutic agent doxorubicin can be used to deliver two types of cancer therapy simultaneously, and the addition of TPGS can further enhance the entry of doxorubicin into the nucleus. Therefore, this innovative delivery system can act as a potential nanomedicine for both drug-sensitive and drug-resistant cancer therapy.

Introduction

Nanotechnology has provided a platform for functionalized drug delivery. In applications of this technique, a number of nanomaterials have been investigated as potential carriers for hydrophobic drugs. Because enhanced permeability and retention of these nanoparticles is seen in tumor tissue compared to that in normal tissue, nano-sized polymer–drug conjugates may preferentially accumulate in the tumor microenvironment over time [1], [2], [3]. Several nano-sized delivery systems have recently been developed with dendrimers, nanoparticles, and micelles [4], [5], [6], [7], [8], along with bifunctionalized micelles that include chemotherapeutic agents and photosensitizers, which produce a synergistic combination [9], [10].

However, the potential effects of many chemotherapeutic agents are undermined by the presence of multiple drug resistance (MDR). One well studied mechanism of MDR involves over-expression of efflux pumps, such as, the ABC-transporter family P-glycoprotein (P-gp), on the cell surface, which transport chemotherapeutic agents out of the cells [11] and prevent their intracellular accumulation [12], [13]. P-gp, which can transport a wide range of structurally and functionally unrelated cytotoxic drugs, e.g., doxorubicin (Dox), etoposide, paclitaxel, and vincristine out of tumor cells [14], [15], is expressed in approximately 40% of breast cancer tumors; and such tumors are three times less likely to respond to chemotherapy than those that do not express P-gp [16].

Systems that cause sequestration of drugs within discrete cytoplasmic organelles and prevent their entry into the nucleus may also contribute to resistance to chemotherapeutic drugs. Doxorubicin, an anthracycline that is active in the nucleus, is commonly used to treat many types of cancer [17], [18]; but in Dox-resistant breast cancer cells, for example, it is sequestered almost exclusively in cytoplasmic organelles [19], [20], [21].

It was hypothesized that an agent that combined multiple strategies for overcoming drug resistance could improve the efficacy of currently available chemotherapeutic agents. To this end, the molecules of d-alpha-tocopheryl poly(ethylene glycol) 1000 succinate (TPGS), containing a lipophilic nonpolar head and a hydrophilic tail, that is a surfactant with high emulsification efficiency and a P-gp efflux inhibitor [22], [23], [24] were incorporated into designed 4-armed porphyrin-polylactide nanoparticles (PPLA-NPs). TPGS is one of the several inhibitors of P-gp. The PPLA-NPs are capable of photochemical internalization (PCI), which is carried out by irradiating a photosensitizer that is delivered to the membranes of endosomes/lysosomes by the nanoparticles [25], [26]. The membranes rupture after irradiation with a specific wavelength; and drugs loaded within the nanoparticles can then be released, bypassing the sequestration that causes drug resistance [27]. In this study, the efficacy of Dox delivered by these multifunctional nanoparticles against MDR breast cancer cells was tested.

Section snippets

Chemicals and drugs

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), and DMSO (dimethylsulfoxide), and tetrahydrofuran (THF) were obtained from Sigma (Sigma-Aldrich, Germany and USA). Doxorubicin was obtained from Calbiochem (Calbiochem, China). Sodium phosphate dibasic (Na2HPO4) and sodium chloride (NaCl) were obtained from Tedia (Tedia, Fairfield, OH, USA); potassium chloride (KCl) and potassium dihydrogenphosphate (KH2PO4) from Showa (Showa, Japan); sodium bicarbonate (NaHCO3) from Scharlau

Characterization of PPLA-NPs

The structure of the 4-armed PPLA is shown in Fig. 1. The molecular weight of each PLA chain on PPLA, obtained from the 1H NMR analysis, was 13,140 and the polydispersity, obtained from GPC, was 1.66 (Mw/Mn: 23,600/14,200). Thus, the hydroxyl-terminated porphyrin can successfully act as an initiator of ROP for lactide. For nanoparticle formation, PPLA-NPs without TPGS coating had a diameter of around 78 nm, as determined by DLS.

Physical characterization of TPGS-coated PPLA-NPs and Dox-PPLA-NPs

The TPGS-coated PPLA-NPs exhibited a smaller particle size (71 nm, by

Discussion

The objective of this study was to test whether combining multiple strategies for overcoming drug resistance could improve the efficacy of currently available chemotherapeutic agents. The cell-killing effects were tested for the combined chemotherapy (Dox), photochemical internalization (PCI), nanoparticle delivery, and inhibition of P-gp; and the results showed that the combination of Dox loaded into TPGS-coated PPLA-NPs plus light was more cytotoxic to drug-resistant breast cancer cells than

Conclusion

In summary, a powerful nanocarrier with photodynamic activity (4-armed porphyrin-PLA) has been developed, the resulting nanoparticles loaded with Dox, and this combination used to deliver two types of cancer therapy simultaneously. This combination of chemotherapy and phototherapy has synergistic effects as evaluated by median effect analysis. TPGS can be added to block drug efflux in Dox-resistant cells, and both TPGS and irradiation of the photoreactive nanoparticles increase the

Acknowledgment

This research was supported by grants from National Health Research Institutes (NHRI-EX99-9833EI) and National Science Council of the Republic of China (NSC 97-2113-M-005-002-).

References (40)

  • D. Missirlis et al.

    Doxorubicin encapsulation and diffusional release from stable, polymeric, hydrogel nanoparticles

    European Journal of Pharmaceutical Sciences

    (2006)
  • E.K. Park et al.

    Folate-conjugated methoxy poly(ethylene glycol)/poly(epsilon-caprolactone) amphiphilic block copolymeric micelles for tumor-targeted drug delivery

    J. Control. Release

    (2005)
  • T. Mosmann

    Rapid colorimetric assay for cellular growth and survival — application to proliferation and cyto-toxicity assays

    J. Immunol. Methods

    (1983)
  • T.C. Chou et al.

    Quantitative analysis of dose-effect relationships: the combined effects of multiple-drugs or enzyme-inhibitors

    Adv. Enzyme Regul.

    (1984)
  • Y. Bae et al.

    Mixed polymeric micelles for combination cancer chemotherapy through the concurrent delivery of multiple chemotherapeutic agents

    J. Control. Release

    (2007)
  • M. Solazzo et al.

    P-gp localization in mitochondria and its functional characterization in multiple drug-resistant cell lines

    Experimental Cell Research

    (2006)
  • J.J. Yu et al.

    Bio-distribution and anti-tumor efficacy of PEG/PLA nano particles loaded doxorubicin

    Journal of Drug Targeting

    (2007)
  • P.S. Lai et al.

    Enhanced cytotoxicity of saporin by polyamidoamine dendrimer conjugation and photochemical internalization

    Journal of Biomedical Materials Research Part A

    (2008)
  • M.J. Shieh et al.

    Reduced skin photosensitivity with meta-tetra(hydroxyphenyl) chlorin-loaded micelles based on a poly(2-ethyl-2-oxazoline)-b-poly(D, L-lactide) diblock copolymer in vivo

    Molecular Pharmaceutics

    (2010)
  • M.M. Gottesman et al.

    Biochemistry of multidrug-resistance mediated by the multidrug transporter

    Annual Review of Biochemistry

    (1993)
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    Both authors contributed equally to this work.

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