B-363 Solid Lipid Nanoparticles Mediated CBLC siRNA Delivery as a Platform to Monitor and Probe Biological Functions

Abstract

Abstract Background Nanoparticles have been used as a tool for delivering therapeutics since few decades. Researchers has been focusing on advances and modifications to improve the properties of these nanoparticles such as sustained drug delivery and bioavailability. This study focuses on modifying the conventional solid lipid nanoparticles (SLN) with Arginine-Glycine-Aspartic acid tripeptide (RGD). The peptidyl link in these SLN has an ability to bind to the integrin of the target cells, allowing solid adhesion to the cells for sustained drug delivery. The use of small interfering RNA (siRNA) allow better invasion through the cell membranes, silencing the intracellular targets. This study uses Casistas B-lymphoma lineage (CBLC) siRNA as the therapeutics for the treatment of non-small cell lung cancer (NSCLC). CBL family consisting of different proteins such as CBL, CBLB, and CBLC origins from a RING finger class. They are E3 ubiquitin ligases responsible for transferring ubiquitin from E2 to E3 substrate. They function as the negative regulator of Tyrosine Kinase activity in cell cycle signaling. CBLC is abundantly present in epithelial cells and so its expression is noteworthy in lung adenocarcinoma. CBL family also possess Tyrosine Kinase Binding domain (TKB) responsible to decrease the activated Epidermal Growth Factor Receptor (aEGFR). Although, CBLC was initially known to perform the same function as other CBL family members, reports suggest that it is a demethylated target. The expression of CBLC upregulates with the treatment of NSCLC by inhibiting agents for DNA methylation. This process increases the demethylation of the CBLC promoter upregulating CBLC. This causes CBLC to compete with CBL for binding to the aEGFR leading to polyubiquitination of aEGFR. Trafficking of aEGFR in the nucleus makes it more stable progressing lung cancer. CBLC silencing allows tumor cells to be sensitized to the therapy with tyrosine kinase inhibiting agents. Methods SLN were modified with RGDP followed by the loading of CBLC siRNA. To characterize the SLN within the diameter range of 20–100 nm, Scanning Electron Microscopy (SEM) was performed. The Energy-dispersive X-ray Spectroscopy was performed to confirm the presence of Phosphorous and Nitrogen in SLN coming from lipids and RNA. Dynamic Light Scattering (DLS) was done to verify the SEM results. To measure the efficacy of the SLN, the cells were treated with control SLN vs siRNA loaded SLN. The Sybr gold assay and invitro release study was done to confirm the release of siRNA from SLN. The human lung normal cell line NL-20 as control and lung cancer cell line A549 were cultured and treated with synthesized nanoparticles for knocking down CBLC gene. The assays such as MTT, western blot, flow cytometry and mass spectrometry-based proteomics were performed to confirm the knockdown of the CBLC gene. Results The SLN characterization confirmed the diameter in the expected range. The efficacy assays confirmed the encapsulation and release of CBLC siRNA. The results from cell studies showed reduction in tumor growth and viability confirming the knockdown of CBLC gene. Conclusion The data suggests significant effects for the use of synthesized nanoparticles as a therapeutic for the treatment of NSCLC.