Anti-Invasive and Anti-Proliferative Effects of shRNA-Loaded Poly(Lactide-Co-Glycolide) Nanoparticles Following RAN Silencing in MDA-MB231 Breast Cancer Cells

Abstract

BackgroundOverexpression of the RAN GTP (RAN) gene has been shown to be linked to metastatic activity of MDA-MB231 human breast cancer cells by increasing Ras/MEK/ERK and PI3K/Akt/mTORC1 signalling. The aim of this study was to investigate the potential of polymeric nanoparticles to deliver two novel shRNA sequences, targeted against the RAN gene, to MDA-MB231 cells grown in culture and to assess their effects in a range of biological assays.MethodsBiodegradable PLGA nanoparticles, loaded with shRNA-1 and shRNA-4, were fabricated using a double emulsion solvent evaporation technique and characterised for size, zeta potential and polydispersity index before testing on the MDA-MB231 cell line in a range of assays including cell viability, migration, invasion and gene knock down.ResultsshRNA-loaded nanoparticles were successfully fabricated and delivered to MDA-MB231 cells in culture, where they effectively released their payload, causing a decrease in both cell invasion and cell migration by knocking down RAN gene expression.ConclusionResults indicate the anti-RAN shRNA-loaded nanoparticles deliver and release biological payload to MDA-MB231 cells in culture. This works paves the way for further investigations into the possible use of anti-RAN shRNA-loaded NP formulations for the treatment of breast cancer in vivo.

Nanoparticulate RNA delivery systems in cancer

“…18,[101][102][103] shRNA has proven to be a potential tool against cancer-associated perturbations in gene expression because of its ability to reduce the target gene expression through post-transcriptional gene silencing process. 6 The shRNA formation process starts in the nucleus when the primary transcript is generated by the RNA polymerase II or III promoter. It appears from the recent studies that shRNA is superior to siRNA in terms of more effective knocking down gene expression.…”

“…102,105,110 4.1 | shRNA delivery to cancer cells shRNA delivery to tumor cells relies on a drug delivery system that not only protects it from the physiochemical environment of the body but also deliver it to the specific target site. 6 Although viral mediated delivery of shRNA against tumor has been done but toxicity remained the major problem. 111 assumed that TSP driven NP can be used to deliver shRNA successfully for cancer treatment.…”

“…Several reports have demonstrated that shRNA has been used effectively to knockdown the expression of target mRNA sequences by RNA interference in cancer cells (Figures 5 and 6). 18,101‐103 shRNA has proven to be a potential tool against cancer‐associated perturbations in gene expression because of its ability to reduce the target gene expression through post‐transcriptional gene silencing process 6 . The shRNA formation process starts in the nucleus when the primary transcript is generated by the RNA polymerase II or III promoter.…”

“…The promising drug release characteristics of nanodimensional delivery systems make them a potent tool in gene therapy, where the genetic characteristics of the patient can be targeted specifically during cancer treatment 1‐4 . This concept may be possible by using suitable biotherapeutics, such as siRNA that can be encapsulated in a drug delivery vehicle and administrated to the target site without compromising their ability to deliver drug at the site of target 4‐6 . Originally the viral method of delivery was used but due to its limitations like the use of viruses in production on a large scale, toxicity and immunogenicity, the method is not preferred and a need for a new method is essential 7 (Table 1).…”

“…like the use of viruses in production on a large scale, toxicity and immunogenicity, the method is not preferred and a need for a new method is essential 7 (Table 1). 6,[8][9][10] Different types of nanoparticulate delivery systems, like lipid-based vesicles, ketals nucleoside lipid nanoparticles (NPs), cell penetrating peptides NPs, polymersomes, chitosan-based NPs have been utilized by researchers to deliver siRNA for cancer treatment. 4,5,[11][12][13][14][15] The process by which the target gene expression is reduced by siRNA is known as RNA interference (or RNAi).…”