Cationic Polymer Nanoparticles-Mediated Delivery of miR-124 Impairs Tumorigenicity of Prostate Cancer Cells

Abstract

MicroRNAs (miRNAs) play a pivotal role in regulating the expression of genes involved in tumor development, invasion, and metastasis. In particular, microRNA-124 (miR-124) modulates the expression of carnitine palmitoyltransferase 1A (CPT1A) at the post-transcriptional level, impairing the ability of androgen-independent prostate cancer (PC3) cells to completely metabolize lipid substrates. However, the clinical translation of miRNAs requires the development of effective and safe delivery systems able to protect nucleic acids from degradation. Herein, biodegradable polyethyleneimine-functionalized polyhydroxybutyrate nanoparticles (PHB-PEI NPs) were prepared by aminolysis and used as cationic non-viral vectors to complex and deliver miR-124 in PC3 cells. Notably, the PHB-PEI NPs/miRNA complex effectively protected miR-124 from RNAse degradation, resulting in a 30% increase in delivery efficiency in PC3 cells compared to a commercial transfection agent (Lipofectamine RNAiMAX). Furthermore, the NPs-delivered miR-124 successfully impaired hallmarks of tumorigenicity, such as cell proliferation, motility, and colony formation, through CPT1A modulation. These results demonstrate that the use of PHB-PEI NPs represents a suitable and convenient strategy to develop novel nanomaterials with excellent biocompatibility and high transfection efficiency for cancer therapy.

Keywords: aminolysis; gene delivery; miR-124; polyethyleneimine (PEI); polyhydroxybutyrate (PHB); polymer nanoparticles; prostate cancer.

Scheme 1

Preparation route of polyethyleneimine-functionalized PHB nanoparticles (PHB-PEI NPs) by nanoprecipitation and aminolysis.

Figure 1

Size, morphological, chemical characterization, and cytotoxicity of PHB based NPs. (a) Size distribution and (b) zeta potential of PHB and PHB-PEI NPs. TEM and SEM images of: (c, e) PHB, and (d, f) PHB-PEI NPs. (g) FTIR-ATR spectra of PHB and PHB-PEI NPs along with difference spectra. (h, i) In vitro cytotoxicity of PHB-PEI NPs. Cytotoxicity was determined in PC3 cells after 6, 24, 48, and 72 h of incubation with varying concentrations of PHB-PEI NPs using: (h) the Cell Counting Kit-8 (CCK-8) assay, and (i) the Lactate Dehydrogenase (LDH) assay. Untreated cells were used as control. Data are presented as the mean ± SD for three independent measurements.

Figure 2

Characterization of PHB-PEI NPs/miR-124 complexes. (a) Electrophoretic mobility of miR-124 NPs at different N/P ratios. (b) miRNA-124 release profile evaluated by qRT-PCR analysis for 24 h in growth medium. Free miR-124 was used as control. (c) Average hydrodynamic diameter and (d) zeta potential of miR-124 NPs at different N/P ratios (mean ± SD, n = 6). Statistically significant variations: ### p < 0.001 N/P 10:1 versus N/P 5:1, N/P 1:1, and free miR-124.

Figure 3

Intracellular delivery efficiency of PHB-PEI NPs/miR-124 complexes. (a) PC3 uptake of miR-124 NPs analyzed by Fluorescent Activated Cell Sorting (FACS). To facilitate observations, miR-124 was labeled with the Cy5 fluorescent probe (red) to form Cy5-miR-124-NPs at different N/P ratios (1:1 to 10:1). Lipofectamine RNAiMAX (iMAX) was used as a control. Cells were incubated with fluorescent nanocomplexes for 4 h, washed with PBS, and incubated for another 20 h prior to analysis by flow cytometer (mean ± SD, n = 6). Statistically significant variations: *** p < 0.001 N/P 10:1 versus N/P 5:1, N/P 1:1, and iMAX. (b) Fluorescence images of PC3 cells incubated with Cy5-miR-124-NPs (N/P ratio 10:1) or Cy5-miR-124-iMAX for 4 h. Magnification 40×, scale bar is 50 μm.

Figure 4

Expression of CPT1A in the PC3 cells in the presence of nanocomplexes. Cells were cultured for 24 h after transfection with miR-124-NPs or miR-NC-NPs at a 10:1 N/P ratio. The untreated cells were used as control (CTL). (a) miRNA target level (CPT1A) was quantified by quantitative real-time PCR (qPCR) and normalized to actin (ACTB) as a housekeeping gene. The comparative cycle threshold (CT) method (2^−∆∆Ct) was applied to calculate relative differences in qPCR results. (b) Western blot analysis of CPT1A protein was performed on total PC3 protein fraction. The protein expression was normalized to the housekeeping protein ACTB. The bars represent the mean ± SD, n = 3. Statistically significant variations: §§§ p < 0.001 miRNA versus miR-NC and CTL. (c) Representative fluorescent images of CPT1A expression. Immunofluorescence stain was performed with a FITC-labeled secondary antibody and counterstained with DAPI. Magnification 40×, scale bar is 50 μm.

Figure 5

Influence of forced expression of miR-124 on PC3 proliferation, migration, and invasion. (a) Cell proliferation of PC3 cells was determined by CCK8 assay after 12, 24, and 48 h. (b) Colony formation assay was performed after 14 days of culture. For quantification, colonies with at least 50 cells were considered. Representative micrographs were obtained using phase contrast microscope after staining with crystal violet. (c) Wound-healing assay performed on transfected PC3 cell, and wound closure rate measured by detecting the closure distance after 24 h. Representative micrographs of the cell migration (top) and quantification (bottom) from three independent experiments are presented. (d) Transwell invasion assay with Matrigel performed in miR-124-NPs or miR-NC-NPs-transfected PC3 cells after 24 h. Five random fields in each well were counted under a microscope. The bars represent the mean ± SD, n = 3. Statistically significant variations: ## p < 0.01, miRNA versus miR-NC.