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

Abstract

Tumor drug resistance significantly limits the success of chemotherapy in the clinic. Tumor cells utilize multiple mechanisms to prevent the accumulation of anticancer drugs at their intracellular site of action. In this study, we investigated the anticancer efficacy of doxorubicin in combination with photodynamic therapy using methylene blue in a drug-resistant mouse tumor model. Surfactant-polymer hybrid nanoparticles formulated using an anionic surfactant, Aerosol-OT (AOT), and a naturally occurring polysaccharide polymer, sodium alginate, were used for synchronized delivery of the two drugs. Balb/c mice bearing syngeneic JC tumors (mammary adenocarcinoma) were used as a drug-resistant tumor model. Nanoparticle-mediated combination therapy significantly inhibited tumor growth and improved animal survival. Nanoparticle-mediated combination treatment resulted in enhanced tumor accumulation of both doxorubicin and methylene blue, significant inhibition of tumor cell proliferation, and increased induction of apoptosis. These data suggest that nanoparticle-mediated combination chemotherapy and photodynamic therapy using doxorubicin and methylene blue has significant therapeutic potential against drug-resistant tumors.

Fig. 1

A height image of nanoparticles loaded with doxorubicin and methylene blue acquired by tapping-mode atomic force microscopy in air.

Fig. 2

(A) Tumor growth inhibition curve and (B) Kaplan–Meier survival plot of Balb/c mice bearing subcutaneous JC tumor. Mice received empty nanoparticles plus light exposure (Blank NP/PDT), methylene blue nanoparticles plus light exposure (M-NP/PDT), doxorubicin nanoparticles plus light exposure (D-NP/PDT), nanoparticles loaded with doxorubicin and methylene blue without light exposure (D-M-NP), nanoparticles loaded with methylene blue and doxorubicin plus light exposure (D-M-NP/PDT), doxorubicin and methylene blue in solution plus light exposure (D-M-soln/PDT), or just the vehicle plus light exposure (vehicle). Tumors were measured and animal survival were monitored every 3rd day until day 30 or tumor volume ≥2000 mm³. Data as mean±SD (n=7–10 mice).

Fig. 3

PCNA staining of sections from tumors that received (A) vehicle plus light exposure, (B) blank nanoparticles plus light exposure, (C) doxorubicin nanoparticles plus light exposure, (D) methylene blue nanoparticles plus light exposure, (E) doxorubicin and methylene blue in solution plus light exposure, (F) nanoparticles loaded with doxorubicin and methylene blue without light exposure, and (G) nanoparticles loaded with methylene blue and doxorubicin plus light exposure. Images were acquired at 400× magnification.

Fig. 4

TUNEL staining for different treatment groups: vehicle plus light exposure (A), blank nanoparticles plus light exposure (B), doxorubicin nanoparticles plus light exposure (C), methylene blue nanoparticles plus light exposure (D), doxorubicin and methylene blue in solution plus light exposure (E), nanoparticles loaded with doxorubicin and methylene blue without light exposure (F), and nanoparticles loaded with methylene blue and doxorubicin plus light exposure (G). Sections were imaged at 100× (I) or 400× (II).

Fig. 5

CD34 staining of sections from tumors that received (A) vehicle plus light exposure, (B) blank nanoparticles plus light exposure, (C) doxorubicin nanoparticles plus light exposure, (D) methylene blue nanoparticles plus light exposure, (E) doxorubicin and methylene blue in solution plus light exposure, (F) nanoparticles loaded with doxorubicin and methylene blue without light exposure, and (G) nanoparticles loaded with methylene blue and doxorubicin plus light exposure. Images were acquired at 400× magnification.

Fig. 6

Effect of encapsulation in nanoparticles on the biodistribution of (A) doxorubicin and (B) methylene blue. Balb/c mice bearing subcutaneous JC tumor were intravenously injected with a combination of doxorubicin and methylene blue either free in solution or encapsulated in nanoparticles. Animals were euthanized 3 h after treatment administration and tumors were collected. Doxorubicin and methylene blue concentrations in tumors and other organs were determined using LC-MS/MS and normalized to the dry organ weight. Data as mean±SD (n=3–4). *p<0.05, t-test.