Hyaluronic acid-functionalized polymeric nanoparticles for colon cancer-targeted combination chemotherapy

Abstract

Nanoparticle (NP)-based combination chemotherapy has been proposed as an effective strategy for achieving synergistic effects and targeted drug delivery for colon cancer therapy. Here, we fabricated a series of hyaluronic acid (HA)-functionalized camptothecin (CPT)/curcumin (CUR)-loaded polymeric NPs (HA-CPT/CUR-NPs) with various weight ratios of CPT to CUR (1 : 1, 2 : 1 and 4 : 1). The resultant spherical HA-CPT/CUR-NPs had a desirable particle size (around 289 nm), relative narrow size distribution, and slightly negative zeta potential. These NPs exhibited a simultaneous sustained release profile for both drugs throughout the time frame examined. Subsequent cellular uptake experiments demonstrated that the introduction of HA to the NP surface endowed NPs with colon cancer-targeting capability and markedly increased cellular uptake efficiency compared with chitosan-coated NPs. Importantly, the combined delivery of CPT and CUR in one HA-functionalized NP exerted strong synergistic effects. HA-CPT/CUR-NP (1 : 1) showed the highest antitumor activity among the three HA-CPT/CUR-NPs, resulting in an extremely low combination index. Collectively, our findings indicate that this HA-CPT/CUR-NP can be exploited as an efficient formulation for colon cancer-targeted combination chemotherapy.

Figure 1

Particle size (a) and zeta potential (b) of various NPs. Data are presented as means ± S.E.M. (n = 3). Representative TEM images of chitosan-coated CPT/CUR-NPs (c) and HA-CPT/CUR-NPs (d). The inset highlights the TEM of an individual NP.

Figure 2

XRD patterns of free CPT (a), free CUR (b), and HA-CPT/CUR-NP (1:1) (c).

Figure 3

In vitro cumulative release of CPT and CUR from HA-CPT/CUR-NP (1:1) in PBS containing 0.5% Tween-80 at 37 °C. Data are presented as means ± S.E.M. (n = 3).

Figure 4

Cellular colocalization studies of HA-CPT/CUR-NP (1:1) in Colon-26 cells after treatment with drug concentrations of 50 or 100 μM for 3 hours. Fluorescence images show the colocalization of CPT and CUR in the cellular compartment of Colon-26 cells. Scale bar = 10 μm.

Figure 5

Quantification of cellular uptake of various NPs by Colon-26 cells. (a) Representative flow cytometry histograms of fluorescence intensity for cells treated with various NPs (50 μM CPT) for 3 hours. (b) Percentage of CPT-containing Colon-26 cells after treatment with various NPs (50 μM CPT) for 1.5 or 3 hours. Each point represents the mean ± S.E.M. (n = 3; *P < 0.05 and **P < 0.01, Student’s t-test). (c) Tissue uptake profiles of HA-CUR-NPs after co-incubating for 5 h with normal colon tissue or colon cancer tissue (red frame indicates colon tumour areas). Tissues were treated with NPs (CUR, 100 μM) and processed for fluorescence staining. Fixed cells were stained with DAPI for visualization of nuclei (purple). Scale bar represents 50 μm.

Figure 6

In vitro cytotoxicity of chitosan-coated NPs and HA-functionalized NPs against Colon-26 cells, determined by MTT assays. Cytotoxicity of CPT-loaded NPs (a) and CUR-loaded NPs (b) at different concentrations against Colon-26 cells after co-incubation for 24 or 48 hours. Triton X-100 (0.5%) 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 de?ned 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 7

In vitro cytotoxicity of HA-CPT/CUR-NP (1:1) (a), HA-CPT/CUR-NP (2:1) (b) and HA-CPT/CUR-NP (4:1) (c) against Colon-26 cells, determined by MTT assays. CI versus F_a plots for HA-CPT/CUR-NP (1:1) (d), HA-CPT/CUR-NP (2:1) (e), and HA-CPT/CUR-NP (4:1) (f).

Figure 8

In vitro cytotoxicity of various NPs against Caco2-BBE cells based on ECIS technology. ECIS was used to determine cell viability in real time during an extended exposure to an NP suspension with a total drug concentration of 25 μM. As controls, ECIS was also performed on untreated cells or cells treated with Triton X-100 (0.5%) in DMEM.

Scheme 1

Schematic illustration of the fabrication process of HA-CPT/CUR-NPs, colon cancer cell-targeted drug delivery, and the release of CPT and CUR into the cytoplasm or nucleus.