Synthesis, characterization, and evaluation of poly (D,L-lactide-co-glycolide)-based nanoformulation of miRNA-150: potential implications for pancreatic cancer therapy

Abstract

MicroRNAs are small (18-22 nucleotide long) noncoding RNAs that play important roles in biological processes through posttranscriptional regulation of gene expression. Their aberrant expression and functional significance are reported in several human malignancies, including pancreatic cancer. Recently, we identified miR-150 as a novel tumor suppressor microRNA in pancreatic cancer. Furthermore, expression of miR-150 was downregulated in the majority of tumor cases, suggesting that its restoration could serve as an effective approach for pancreatic cancer therapy. In the present study, we developed a nanoparticle-based miR-150 delivery system and tested its therapeutic efficacy in vitro. Using double emulsion solvent evaporation method, we developed a poly (D,L-lactide-co-glycolide) (PLGA)-based nanoformulation of miR-150 (miR-150-NF). Polyethyleneimine (a cationic polymer) was incorporated in PLGA matrix to increase the encapsulation of miR-150. Physical characterization of miR-150-NF demonstrated that these nanoparticles had high encapsulation efficiency (~78%) and exhibited sustained release profile. Treatment of pancreatic cancer cells with miR-150-NF led to efficient intracellular delivery of miR-150 mimics and caused significant downregulation of its target gene (MUC4) expression. Inhibition of MUC4 correlated with a concomitant decrease in the expression of its interacting partner, HER2, and repression of its downstream signaling. Furthermore, treatment of pancreatic cancer cells with miR-150-NF suppressed their growth, clonogenicity, motility, and invasion. Together, these findings suggest that PLGA-based nanoformulation could potentially serve as a safe and effective nanovector platform for miR-150 delivery to pancreatic tumor cells.

Keywords: MUC4; PLGA nanoparticles; invasion; miR-150; migration.

Figure 1

Transmission electron micrographs of PLGA nanoparticles. Notes: The transmission electron microscopic images of (A) unloaded and (B) miR-150-loaded nanoparticles. Scale bar is 1 μm. Abbreviation: PLGA, poly (D,L-lactide-co-glycolide).

Figure 2

In vitro release profile of miR-150 mimic from miR-150-NF. Notes: One milligram of miR-150-NF was incubated with 1 mL of release medium (Tris–EDTA buffer with or without 10% FBS, pH 7.4) in a rotary shaker at 100 rpm at 37°C. At 24 hour intervals, the nanoparticle suspension was centrifuged at 7,500 rpm for 10 minutes at 4°C. The amount of miR-150 in the supernatant was determined using ultraviolet spectrophotometry. The experiment was repeated three times and standard deviation was calculated. Abbreviations: EDTA, ethylenediaminetetraacetic acid; NF, nanoformulation.

Figure 3

miR-150-NF delivers miR-150 mimics to pancreatic cancer cells efficiently. Notes: Pancreatic cancer cells were seeded in six-well plates to reach 60%–70% confluence. Cells were treated with 0.1 mg of miR-150 mimics (with or without Lipofectamine [Thermo Fisher Scientific, Waltham, MA, USA]), miR-150-NF (0.05 mg [0.245 μg miR-150], 0.1 mg [0.69 μg miR-150], and 0.2 mg [1.38 μg miR-150]), blank (unloaded, 0.1 mg) PLGA nanoparticles. After 16 hours of transfection, medium was replaced with complete media and cells were further cultured for 48 hours. Expression of mature miR-150 was examined in cells by quantitative reverse-transcription-polymerase chain reaction. Comparable expression of miR-150 was observed after treatment of cells with 0.05 mg of miR-loaded NPs and miR-150 mimic (0.708 μg miR-150) with Lipofectamine, whereas significantly higher expression was observed in cells after treatment with miR-150-NF at concentrations of 0.1 mg and 0.2 mg. Results are presented as fold increase in miR-150 expression in various treatments in comparison to miRNA-150 alone. Bars represent mean ± standard deviation, n=3; *P<0.05; **P<0.001; ***P<0.0001. Abbreviations: miR, microRNA; NF, nanoformulation; NPs, nanoparticles.

Figure 4

miR-150-NF represses MUC4 expression, HER2 expression, and HER2 downstream signaling in pancreatic cancer cells. Notes: Colo-357 and HPAF cells were treated with blank NPs (0.1 mg), miR-150-NF (0.05 or 0.1 mg), or miR-150 with Lipofectamine (Thermo Fisher Scientific, Waltham, MA) for 48 hours. Immunoblotting was performed for MUC4, HER2, p-HER2, ERK1/2, pERK1/2, FAK, and pFAK. β-actin was used as a loading control. Abbreviations: lipo, Lipofectamine; NF, nanoformulation; NPs, nanoparticles; MUC4, Mucin 4; HER2, human epidermal growth factor receptor 2; pHER2, phosphorylated HER2; ERK, extracellular signal-regulated kinase; pERK, phosphorylated ERK; FAK, focal adhesion kinase; pFAK, phosphorylated FAK.

Figure 5

Treatment of pancreatic cancer cells with miR-150-NF alters the morphology of pancreatic cancer cells. Notes: Pancreatic cancer cells were seeded in six-well plates and allowed to attain 60%–70% confluence prior to miR-150-NF (0.05 or 0.1 mg/mL) treatment for 48 hours. Representative micrographs are from one of the random fields of view (magnification 100×) of cells. Abbreviation: NF, nanoformulation.

Figure 6

miR-150-NF decreases growth and clonogenicity of pancreatic cancer cells. Notes: (A) Colo-357 and HPAF cells (2×10³ per well) were seeded in 96-well plates. After 24 hours (considered as day 0), cells were treated with either miR-150-NF or blank NPs (0.05 mg each) and growth was monitored by WST-1 assay every day for next 5 days. After analysis, data were presented as relative fold-growth induction compared with growth of cells at day 0. Growth inhibition of cells treated with miR-150-NF was compared to cells treated with blank NPs on day 5 and is shown as percentage. Bars represent mean ± SD (n=3); *P<0.05; **P<0.001. (B) Pancreatic cancer cells were treated with miR-150 mimics-loaded or blank NPs and, 48 hours later, cells were trypsinized and seeded in six-well plates (1×10³ cells per well) for clonogenicity assay. After 2 weeks, colonies were stained with 0.1% crystal violet, photographed, and counted using imaging system. Data are presented as percent inhibition of clonogenic ability of miR-150-NF-treated cells as compared with their respective controls. Bars represent the mean of total number of colonies ± SD (n=3); *P<0.05. Abbreviations: miR, microRNA; NF, nanoformulation; NPs, nanoparticles; SD, standard deviation.

Figure 7

Treatment with miR-150-NF decreases the motility and invasion of pancreatic cancer cells. Notes: (A) Colo-357 and (B) HPAF cells were treated with miR-150-NF or blank NPs for 48 hours as described above. Cells were trypsinized and seeded (1×10⁶ and 2.5×10⁵ for migration and invasion, respectively) in serum-deprived media on non-coated or Matrigel-coated membranes for motility and invasion assays, respectively. Medium containing 10% FBS was added in the lower chamber as a chemoattractant and incubated for 16 hours in transwell plates. Cells that had migrated/invaded through the membrane/Matrigel to the bottom of the insert were fixed, stained, photographed using inverted phase contrast microscope, and counted in ten random view fields. Bars represent the mean ± standard deviation (n=3) of number of migrated/invaded cells per field; *P<0.05. Abbreviations: FBS, fetal bovine serum; NF, nanoformulation; NPs, nanoparticles.