Development of an Apolipoprotein AI Chimera for Targeted Nanodisc Drug Delivery to Breast Cancer Cells

A current challenge in cancer treatment is the ability to selectively target diseased cells with chemotherapeutics. Several promising treatments suffer this limitation and have toxic off target effects on healthy cells. Our overall objective is to generate a platform for targeted drug delivery employing self‐assembling nanodiscs comprised of phospholipids and apolipoprotein AI (apoAI). ApoAI is an exchangeable apolipoprotein located on high density lipoproteins (HDL), which promotes cholesterol efflux from macrophages and facilitates selective uptake of cholesterol by binding scavenger receptor, class B type 1 (SR‐B1), also known as the HDL receptor. We hypothesize that a chimeric protein of apoAI with MT1‐AF7p (apoAI‐AF7p), a peptide ligand that binds membrane metalloproteinase 14 (MMP14), will enhance the targeting capability of the nanodiscs to MDA‐MB‐231 breast cancer cells. MMP14 is a cell surface protein that is overexpressed in many types of cancers responsible for metastasis and linked to poor prognosis in patients. We designed a construct bearing the coding sequence for human apoAI with a hexa‐His‐tag at the N‐terminal end to facilitate protein purification by affinity chromatography, and MT1‐AF7p (HWKHLHNTKTFL) at the C‐terminal end with an intervening linker segment ([G 4 S] 3 ). The construct was designed and custom‐generated with optimized codon sequence in a pCR2.1 vector. We successfully sub‐cloned the chimeric insert into a pET20b(+) expression vector employing standard molecular biology protocols and verified the resulting plasmid through Sanger sequencing. Recombinant apoAI and apoAI‐AF7p proteins were over‐expressed in E. coli and purified using a nickel affinity chromatography. The purified proteins were visualized by 15% acrylamide SDS‐PAGE, which revealed major bands at ~ 29 kDa band for apoAI and 32 kDa band for apoAI‐AF7p consistent with their expected size difference. We obtained ~ 10 mg purified protein/L culture medium, which is sufficient to carry out structural and functional characterization. Chimeric apoAI‐AF7p nanodiscs have potential not only for targeting, but also for drug delivery since MDA‐MB‐231 cells are reported to have high expression levels of both SR‐B1 and MMP14 which mediates homing and selective uptake of the hydrophobic core of HDL respectively. Preliminary data from fluorescence microscopy indicates the presence of both these proteins as well as the LDLr. Additionally, preliminary circular dichroism data shows similar helical content in the chimera as apoA1. Our next steps are to characterize the expression of MMP14, SR‐B1, and LDLr on MDA‐MB‐231 cells via FACS, qPCR and immunoblotting as well prepare nanodiscs with the chimera. Once particle formation and protein surface expression is established, cells will be incubated with particles and we’ll assess cell binding characteristics. The chimeric nanodiscs bears potential not only for targeting breast cancer cells, but also for drug delivery since these cells are reported to have high expression levels of SRB1, which mediate uptake of the contents within the hydrophobic core. Support or Funding Information This project is supported by the National Institute of Health (NIH) under the award numbers GM105561 (VN), R25GM071638 (DK) and T34GM008074 (RM) ApoA1‐AF7p chimera design: hexa‐His tag at the N‐terminus of ApoA1 and the affinity peptide MT1‐AF7p at the C terminus. Figure 1