Hyaluronic Acid Decorated Nanomicelles Loaded with a Potent Anticancer Flavonoid Analogue to CD44 Expressing Stem-Like Pancreatic Cancer Cells

CD44 Expressing Stem-Like Pancreatic Cancer Cells P. Kesharwani, H. O. Alsaab, S. Padhye, F. H. Sarkar, A. K. Iyer Wayne State University, Pune University Purpose Cancer stem-like cells (CSLCs) play a critical role in acquiring multidrug resistant (MDR) phenotypes. It has been well established that pancreatic cancer stem cells overexpress CD44 receptors (a target of hyaluronic acid; HA). We thus synthesized HA conjugate of copoly(styrene maleic acid) (HA-SMA) that could be engineered to form nanomicelles with a potent anticancer flavonoid analogue, 3,4-difluorobenzylidene diferuloylmethane (CDF) and evaluate its anticancer potentials against stem-like pancreatic cancer cells. Methods The HA-SMA conjugate was first synthesized by adding known amounts of HA in NaHCO3 buffer with fixed amounts of anhydrous SMA to afford the anhydride ring opening reaction of SMA with the alcohol groups of HA. Later on, SMA-CDF (non-targeted nanomicelles) and HA-SMA-CDF (targeted nanomicelles) were fabricated by self-assembly. Nanomicellar formulation was further characterized for particle size, zeta potential, morphology, stability, and in vitro release. Also, the anticancer activity of CDF loaded Nano micelles were tested against MiaPaCa-2 and AsPC-1 human pancreatic cancer cells. More specifically, triple-marker positive (CD44+/CD133+/EpCAM+) CSLCs and triple-marker-negative (CD44–/CD133–/EpCAM−) cells were isolated, cultured, and used to assess the specific targeting of HA based nanomicelles to CD44+ cells. Immunoblotting assays were performed to confirm the HASMA-CDF based targeting to CD44+ cell populations. Further, electrophoretic mobility shift assay (EMSA) was performed to investigate the effect of targeted nanomicelles in down regulating NF-κB (a marker overexpressed in tumor cells including pancreatic cancer cells). Results The engineered nanomicelles had several distinctive features. TEM indicated a smooth surface morphology and spherical geometry for the engineered targeted and non targeted nanomicelles with a size range of 71 nm and 89 nm, respectively. The micelles revealed high CDF loading with remarkable aqueous solubility. The biosafety was high due to the use of nontoxic components (i.e., hyaluronic acid and SMA). Also, potent anticancer activity was observed when the formulations were tested in human pancreatic cancer cell lines. Targeted formulation exhibited the most potent cell killing among the tested formulations. A comparable cell killing effect was also seen in AsPC-1 cells. The IC50 value for free CDF, SMA-CDF, and HA-SMA-CDF were found to be 400 nM, 245 nM, and 140 nM, respectively, in MiaPaCa-2 cells. A similar observation was noted in AsPC-1 cells, for which the IC50 values for free CDF, SMA-CDF, and HA-SMA-CDF was found to be 810 nM, 320 nM and 160 nM, respectively. Also, the HA targeted formulation had better internalization in CD44 expressing tumor cells based on receptor mediated endocytosis. Western blot analysis suggested that cells treated with HA-SMA-CDF nanomicelles displayed significant reduction of CD44 expression levels possibly due to the better uptake of targeted nanomicelles by CD44 receptor-mediated endocytosis in cells overexpressing the target. Furthermore, specific inhibition of NF-κB in CD44+ stem like cells indicate its utility for overcoming drug resistance in the treatment of most aggressive pancreatic cancer steml like cells (CSLCs).