Inhibition of MDR1 gene expression and enhancing cellular uptake for effective colon cancer treatment using dual-surface-functionalized nanoparticles

Abstract

Nanomedicine options for colon cancer therapy have been limited by the lack of suitable carriers capable of delivering sufficient drug into tumors to cause lethal toxicity. To circumvent this limitation, we fabricated a camptothecin (CPT)-loaded poly(lactic-co-glycolic acid) nanoparticle (NP) with dual-surface functionalization-Pluronic F127 and chitosan-for inhibiting multi-drug resistant gene 1 (MDR1) expression and enhancing tumor uptake. The resultant spherical NPs-P/C had a desirable particle size (∼268 nm), slightly positive zeta-potential, and the ability to efficiently down-regulate the expression of MDR1. In vitro cytotoxicity tests revealed that the 24 and 48 h IC50 values of NPs-P/C1 were 2.03 and 0.67 μm, respectively, which were much lower than those for free CPT and other NPs. Interestingly, NPs-P/C1 showed the highest cellular uptake efficiency (approximately 85.5%) among the different drug formulations. Most importantly, treatment of colon tumor-bearing mice with various drug formulations confirmed that the introduction of Pluronic F127 and chitosan to the NP surface significantly enhanced the therapeutic efficacy of CPT, induced tumor cell apoptosis, and reduced systemic toxicity. Collectively, these findings suggest that our one-step-fabricated, dual-surface-functionalized NPs may hold promise as a readily scalable and effective drug carrier with clinical potential in colon cancer therapy.

Keywords: Chemotherapy; Colon cancer; Enhanced cellular uptake; Multidrug resistant gene 1; Nanoparticles; Surface functionalization.

Figure 1

Preparation of dual-surface–functionalized NPs using a one-step fabrication process. (a) Schematic illustration of the preparation of NPs-P/C, and cellular uptake and release of CPT into the nucleus. (b) Representative SEM, TEM, size distribution, and zeta-potential profiles of NPs-P and NPs-P/C1.

Figure 2

In vitro cumulative release of CPT from different NPs in PBS (pH 7.4) at 37 °C. Data are presented as means ± S.E.M. (n = 3).

Figure 3

In vitro cytotoxicity of free CPT and various NPs against Colon-26 cells after incubating for 24 h (a), and 48 h (b) based on MTT assays. Triton X-100 (0.1%) was used as a positive control to produce a maximum cell death rate (100%), whereas cell culture medium was used as a negative control (death rate defined as 0%). Cytotoxicity is given as the percentage of viable cells remaining after treatment. Each point represents the mean ± S.E.M. (n = 5).

Figure 4

In vitro cytotoxicity of free CPT and NPs-P/C1 against Caco2-BBE cells based on ECIS technology. (a) ECIS was used to determine cell viability in real time during an extended exposure to an NP suspension (CPT, 10 µM). As controls, ECIS was also performed on untreated cells or cells treated with Triton X-100 (0.1%) in RPMI-1640 medium. (b) Morphology of Caco2-BBE cells grown for 38 h in 8-well ECIS dishes.

Figure 5

The apoptosis profiles of Colon-26 cells following treatment with free CPT or NPs-P/C1 (CPT, 10 µM). The percentages of cell states are indicated in each quadrant. Lower left, viable cells; lower right, early apoptotic cells; upper left, necrotic cells; upper right, late apoptotic cells. Data are presented as means ± S.E.M. (n = 3).

Figure 6

In vitro inhibition of the expression of the MDR1 gene by NPs-P/C1 in Colon-26 cells at 8 h (a) and 16 h (b). Each point represents the mean ± S.E.M. (n = 3). Statistical significance was assessed using Student’s t-test (*P < 0.05 and **P < 0.01).

Figure 7

Fluorescence images showing cellular uptake of free CPT and NPs-P/C1 in Colon-26 cells after treatment with different CPT concentrations (0, 50 and 100 µM) for 3 h. Scale bar = 10 µm.

Figure 8

Quantification of cellular uptake of free CPT and various NPs by Colon-26 cells. (a) Flow cytometry histogram profiles of fluorescence intensity for cells treated with free CPT or NPs (CPT, 25 µM) for 5 h. Percentage of CPT-containing Colon-26 cells (b) and quantification of CPT fluorescent intensity in Colon-26 cells (c) after treatment with free CPT or NPs (CPT, 25 µM) at different time points (1, 3, and 5 h). Each point represents the mean ± S.E.M. (n = 3). Statistical significance was assessed using Student’s t-test (*P < 0.05 and **P < 0.01).

Figure 9

Anti-cancer effects of free CPT, NPs-PVA, NPs-P, and NPs-P/C1 in vivo. (a) Changes in body weight in different treatment groups. Mouse body weight was normalized to day 0 body weight (expressed as a percentage) and is depicted as the mean of each treatment group ± S.E.M. (n = 5). (b) Tumor growth profiles in different groups during treatment with free CPT or various NPs. Arrows indicate the days of drug injections. Each point represents the mean ± S.E.M. (n = 5). (c) Tumor weights in each treatment group at the study end point. Each point represents the mean ± S.E.M. (n = 5). Statistical significance was assessed using non-parametric Mann-Whitney test (*P < 0.05 and **P < 0.01). (d) H&E staining of tumor tissues from mice treated with free CPT or various NPs for12 days. Scale bar = 20 µm.

Figure 10

Measurement of apoptosis. (a) Representative images of double-fluorescence labeling of DAPI nuclear staining (blue) and TUNEL staining (green) in tumors after a 12-d treatment. Scale bar = 20 µm. (b) Percentages of TUNEL-positive cells in tumor sections of PBS-, free CPT-, and NP-treated groups. Each point represents the mean ± S.E.M. (n = 3). Statistical significance was assessed using non-parametric Mann-Whitney test (*P < 0.05 and **P < 0.01).

Figure 11

H&E staining of sections of major organ tissues obtained from tumor-bearing mice after a 12-d treatment with PBS, free CPT, or different NPs. Heart, liver, spleen, lung, and kidney were harvested on day 20 after tumor transplantation. Scale bar = 20 µm.

Figure 12

Biodistribution of CPT at 4, 24, and 48 h after intravenous injection of free CPT or NPs-P/C1 into colon tumor-bearing mice. Each point represents the mean ± S.E.M. (n = 3). Statistical significance was assessed using non-parametric Mann-Whitney test.