Enhancing Proteasome-Inhibitory Activity and Specificity of Bortezomib By Endocytosis-Mediated Uptake of CD38 Targeted Nanoparticles in Multiple Myeloma

Abstract

The establishment of more effective treatments that can circumvent chemoresistance in Multiple Myeloma (MM) is a priority. Although bortezomib (BTZ) is one of the most potent proteasome inhibitors available, still possesses limitations related to dose limiting side effects. Several strategies have been developed to improve the delivery of chemotherapies to MM by targeting different moieties expressed on MM cells to nanoparticle delivery systems (NPs), which have failed mainly due to their heterogeneous expression on these cells. Our goal was to test CD38 targeted chitosan NPs as novel targeting moiety for MM to improve the potency and efficacy of BTZ in MM cells and reduce the side effects in healthy tissue. Due to its high expression on MM cells, CD38 is used as a marker for identification of MM cells, and there are several indications supporting the notion that CD38 plays significant roles in the progression of MM.We have fine-tuned the production of anti-CD38 chitosan NPs with a small size (50nm) to allow better penetration to tumors, and we showed that these NPs were stable at different storage conditions and periods. The chitosan NPs showed preferential drug release in MM tumor microenvironment compared to normal tissue microenvironment, and this release was pH dependent.The uptake of the anti-CD38 chitosan NPs to MM cells (cell lines and primary) was significantly higher than their uptake in normal cells (4-fold of BM MNCs, and 10-fold of PB MNCs) demonstrating that the anti-CD38 chitosan NPs are selective and specific to MM. BTZ-loaded anti-CD38 chitosan NPs demonstrated enhanced cytotoxicity and apoptosis on MM cells compared to similar concentration of BTZ as free drug; demonstrated by induction of more profound cell cycle arrest and apoptosis leading to cell death through inactivation of proliferative cell signaling pathways (MAPK, PI3K) and activation of pro-apoptotic pathways (PARP and Caspase-3). We also found that BTZ-loaded anti-CD38 NPs proved to be more efficacious than free BTZ and overcame CAM-DR and hypoxia-mediated drug resistance. The use of a 3DTEBM model, which recreate MM pathophysiology, corroborated the increase cytotoxicity of anti-CD38 chitosan NPs on MM cells relative to free drug and allowed to corroborated co-localization of the NPs in the MM cells.We found that BTZ-loaded NPs were significantly more potent in the proteasome activity inhibition than free BTZ and correlated with the boron content in MM cells treated with BTZ-loaded anti-CD38 NPs, which was 3-fold higher than in MM cells treated with BTZ as free drug. To confirm that the process involved in the internalization of the anti-CD38 chitosan NPs was endocytosis, we have shown that the NPs distribution in the cells co-localized with the distribution of endosomes and found that the anti-CD38 NPs were uptaken through clathrin-mediated and caveolae-mediated early endocytosis which progressed to late endocytosis and further delivery of the NPs content into the cells. Finally, we linked the internalization through endocytic pathways of anti-CD38 NPs to the enhanced proteasome activity inhibition of these NPs in MM cells. Treatment of MM cells with clathrin and caveolae-mediated early endocytosis, or late endocytosis inhibitors (but not macropinocytosis inhibitors) significantly reduced the proteasome inhibition of BTZ-loaded anti-CD38 chitosan NPs. When BTZ is administered as a free drug, it penetrated through passive diffusion into MM cells as well as in normal cells, and inhibition of the different endocytosis routes did not induce any change in its ability to inhibit the proteasome.In vivo studies showed that treatment with BTZ-loaded anti-CD38 targeted NPs (1mg/kg once a week) showed a markedly improved tumor growth inhibition, improved overall survival, and reduction of side effects compared to non-targeted NPs and free drug.In conclusion, anti-CD38 chitosan NPs showed preferential BTZ release in tumor-microenvironment, specific binding to MM cells, and an improved drug cellular uptake through endocytosis, which translated in enhanced proteasome inhibition and robust cytotoxic effect on MM cells. Furthermore, the anti-CD38 chitosan NPs specifically delivered therapeutic agents to MM cells improving therapeutic efficacy and reducing side effects in vivo. We are the first to report the enhancement of the efficacy and specificity of proteasome inhibitors due to endocytosis-driven delivery of NPs. DisclosuresDe La Puente:Cellatrix LLC: Other: Co-Founder. Vij:Celgene, Onyx, Takeda, Novartis, BMS, Sanofi, Janssen, Merck: Consultancy; Takeda, Onyx: Research Funding. Azab:Glycomimetics: Research Funding; Selexys: Research Funding; Karyopharm: Research Funding; Cellatrix LLC: Other: Founder and owner; Verastem: Research Funding; Cell Works: Research Funding; Abbvie: Research Funding; Tioma: Research Funding; Cleave Bioscience: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner.