Track: Discovery and Basic Research - Pharmaceutics - Molecular Biopharmaceutics
Category: Poster Abstract
Using Allosteric Peptides to Improve Selective Nanoparticle Uptake in CXCR4-Expressing Cells
Purpose: CXCR4 is a G protein-coupled receptor (GPCR) that regulates essential biological processes, such as proliferation, migration, chemotaxis, and survival. Its overexpression is associated with a broad spectrum of diseases, including but not limited to HIV and cancer. Orthosteric and allosteric ligands are potential agents that can modulate CXCR4-mediated responses to therapeutic advantage. Interestingly, allosteric modulators bind to receptors at sites distinct from the orthosteric binding site, have the potential to preserve the function of CXCR4, and minimize adverse effects expected from using orthosteric ligands. Allosteric modulators can be conjugated to the surface of drug-loaded nanoparticles (NPs) to help NPs be internalized upon receptor binding and efficiently deliver drugs within the target cells while sparing normal cells. This work will investigate the effects of allosteric ligands on CXCR4 signaling pathways as well as the potential to improve NP uptake through this receptor pathway. Methods: Four peptide candidates and SDF-1ɑ, a physiological ligand, will be evaluated for CXCR4 allosterism. β-arrestin complementation (i.e., CXCR4 interaction with β-arrestin 2) was assessd on MDA-MB-231 cells expressing a click beetle luciferase. Calcium mobilization will be studied using the Fluo-4 NW assay kit (Sigma-Aldrich) in CCRF-CEM cells. A Genios (Tecan) microplate reader will be used to assess bioluminescence and fluorescence intensities as markers of β-arrestin complementation and calcium mobilization. Flash Nanoprecipitation will prepare ligand-conjugated NPs using polycaprolactone-b-polyethylene glycol (PCL-b-PEG) diblock copolymer. NPs will be characterized by dynamic light scattering and field emission scanning electron microscopy. Cellular uptake of NPs will be measured and visualized by flow cytometry and confocal microscopy, respectively. Results: Based on the published literature, we expect that RSVM peptide, a 17-mer peptide derived from SDF-1ɑ, will show a greater increase in β-arrestin complementation than other peptide candidates. For calcium mobilization and chemotaxis, only 4DV3, out of four peptide candidates, is expected to show a higher response in the presence of SDF-1ɑ. None of the three other peptides have been shown to phosphorylate ERK1/2, a signaling pathway responsible for chemotaxis and proliferation. PCL-b-PEG NPs are expected to have sizes in the range of 150-250 nm with a unimodal distribution and low polydispersity index, with ligand-conjugated NPs diameters slightly larger than plain NPs. Barring other factors, RSVM peptide-conjugated NPs should have the most significant cellular uptake in CXCR4-expressing cells. Conclusion: CXCR4 allosteric peptides and NPs have been successfully prepared. We hope to show that allosteric ligands that potentiate receptor internalization upon binding can be utilized in more efficient NP delivery strategies. Upon completion of the current studies, CXCR4-targeted NPs loaded with an anti-cancer drug in the core will be further explored for in vitro drug release and cell viability studies. These results can be applied to developing more precisely targeted NP formulations to treat a variety of cancers to achieve greater efficacy and less toxicity. References: Sachpatzidis A, Benton BK, Manfredi JP, et al. Identification of allosteric peptide agonists of CXCR4. J Biol Chem. 2003;278(2):896-907. Epub 2002/11/06. doi: 10.1074/jbc.M204667200. Ehrlich A, Ray P, Luker KE, et al. Allosteric peptide regulators of chemokine receptors CXCR4 and CXCR7. Biochem Pharmacol. 2013;86(9):1263-1271. Doi: 10.1016/j.bcp.2013.08.019. Lee IH, Palombo MS, Zhang X, et al. Design and evaluation of a CXCR4 targeting peptide 4DV3 as an HIV entry inhibitor and a ligand for targeted drug delivery. Eur J Pharm Biopharm 2019;138:11-22.