Development and Evaluation of 1'-Acetoxychavicol Acetate (ACA)-Loaded Nanostructured Lipid Carriers for Prostate Cancer Therapy

Abstract

1'-acetoxychavicol acetate (ACA) extracted from the rhizomes of Alpinia conchigera Griff (Zingiberaceae) has been shown to deregulate the NF-ĸB signaling pathway and induce apoptosis-mediated cell death in many cancer types. However, ACA is a hydrophobic ester, with poor solubility in an aqueous medium, limited bioavailability, and nonspecific targeting in vivo. To address these problems, ACA was encapsulated in a nanostructured lipid carrier (NLC) anchored with plerixafor octahydrochloride (AMD3100) to promote targeted delivery towards C-X-C chemokine receptor type 4 (CXCR4)-expressing prostate cancer cells. The NLC was prepared using the melt and high sheer homogenization method, and it exhibited ideal physico-chemical properties, successful encapsulation and modification, and sustained rate of drug release. Furthermore, it demonstrated time-based and improved cellular uptake, and improved cytotoxic and anti-metastatic properties on PC-3 cells in vitro. Additionally, the in vivo animal tumor model revealed significant anti-tumor efficacy and reduction in pro-tumorigenic markers in comparison to the placebo, without affecting the weight and physiological states of the nude mice. Overall, ACA-loaded NLC with AMD3100 surface modification was successfully prepared with evidence of substantial anti-cancer efficacy. These results suggest the potential use of AMD3100-modified NLCs as a targeting carrier for cytotoxic drugs towards CXCR4-expressing cancer cells.

Keywords: 1′-acetoxychavicol acetate; AMD3100; nanostructured lipid carrier; prostate cancer; targeted delivery.

Figure 1

(A) Chemical structure of 1′-acetoxychavicol acetate (ACA) and AMD3100. (B) Schematic drawings representing blank NLC, ACA-NLC, and AMD-ACA-NLC. (C) TEM images of blank NLC, ACA-NLC, and AMD-ACA-NLC. Scale bar = 200 nm.

Figure 2

Differential Scanning Calorimetry (DSC) thermograms and Fourier-Transform Infrared Spectroscopy (FTIR) spectrum of samples. (A) Thermograms of cocoa butter-ACA physical melt, blank NLC, ACA-NLC, AMD-NLC, and AMD-ACA-NLC. The arrows point to the peak melting temperatures observed in each sample. (B) FTIR spectrum of cocoa butter, isopropyl myristate, ACA, AMD3100, and AMD-ACA-NLC. The arrows point to the characteristic peaks of each sample.

Figure 3

In vitro drug release profile of free ACA, ACA-NLC, and AMD-ACA-NLC solutions over 48 h of incubation at 37 °C. All data are shown as mean ± S.D. of three replicates.

Figure 4

Cellular uptake of C6 solution, C6-NLC and AMD-C6-NLC. (A) Representative images of C6 uptake in its free form or when encapsulated in NLC or AMD-NLC after 2, 4, 8, and 24 h of treatment. Scale bar = 50 µm. (B) Time-dependent mean fluorescence intensity of cells after different treatments. Statistically significant differences between C6 solution and AMD-C6-NLC are marked by (* p < 0.05) and (** p < 0.01). All data are shown as mean ± S.D. of three independent replicates. (C) Cellular uptake of C6-NLC and AMD-C6-NLC with endocytic inhibitors sodium azide, nystatin, chlorpromazine, and amiloride. (D) Cellular uptake of C6-NLC and AMD-C6-NLC with AMD3100 inhibition at different concentrations. Statistically significant differences between C6-NLC and AMD-C6-NLC uptake for C and D are denoted as * p < 0.05. All data are shown as mean ± S.D. of three independent replicates.

Figure 5

In vitro cytotoxic effects of treatments on PC-3 cell lines. (A) Dose-dependent cytotoxicity of ACA, ACA-NLC, and AMD-ACA-NLC on cells after 24 h of incubation. Statistically significant differences between ACA and AMD-ACA-NLC are marked with (** p < 0.01). (B) Time-dependent IC₅₀ of treatments on cells after 12, 24, and 36 h of incubation. (C) Dose-dependent cytotoxicity of blank NLC and AMD-NLC on cells after 24 h of incubation. All data are shown as mean ± S.D. of three independent replicates.

Figure 6

Migration assay conducted on PC-3 cell lines without treatment or with ACA, blank NLC, ACA-NLC, AMD-NLC, and AMD-ACA-NLC treatments. (A) Representative images of wound healing assays. Scale bar = 200 µm. (B) Percentage recovery of wounds after different treatments. Significant differences in area migrated by cells in comparison to the untreated and ACA-treated cells are marked with * p < 0.05 and ^# p < 0.05, respectively. All data are shown as mean ± S.D. of three independent replicates.

Figure 7

Invasion assay conducted on PC-3 cell lines without treatment or with ACA, blank NLC, ACA-NLC, AMD-NLC, and AMD-ACA-NLC treatments. (A) Representative images of transwell inserts. Scale bar = 50 µm. (B) Number of invaded cells per field after different treatments. Significant differences in number of invaded cells compared to the untreated are marked with * p < 0.05. All data are shown as mean ± S.D. of three independent replicates.

Figure 8

In vivo anti-tumor effects of placebo, ACA, blank NLC, ACA-NLC, AMD-NLC, and AMD-ACA-NLC treatments on NU/NU mice. (A) Percentage change in tumor volume at the end of the treatment. All data are shown as mean value ± SEM of four/five replicates per group. Statistically significant differences from placebo group are shown as * p < 0.05 and ** p < 0.1, while statistically significant differences from blank NLC and AMD-NLC are shown as ^# p < 0.05 and ^& p < 0.05, respectively. (B) Mean body weight of PC-3 induced NU/NU mice under various treatments over 28 days. All data are shown as mean value ± SD of five/six replicates per group.

Figure 9

IHC staining score of tumor markers upon treatment with placebo, ACA, blank NLC, ACA-NLC, AMD-NLC, and AMD-ACA-NLC. (A) Ki-67, (B) CXCR4, (C) p65, and (D) VEGF protein staining score are shown. Significant difference between treatments and placebo group are indicated as * p < 0.05 and ** p < 0.01. All data are shown as mean ± S.D. of three independent replicates.