Anti-CD38 Targeted Nanoparticles for Drug Delivery in Multiple Myeloma
Authors: De La Puente Pilar , Jin Abbey , Luderer Micah John , Muz Barbara , King Justin A , Vij Ravi , Azab Abdel Kareem 
Published: October 2019

Abstract
INTRODUCTION: The main limiting factor for the treatment with chemotherapies in multiple myeloma (MM) is the lack of specificity of the drugs. We propose the use of chitosan nanoparticles (cNPS) as specific and targeted drug delivery in MM using CD38 as targeting moiety. CD38 has been used as a therapeutic target in MM; anti-CD38 monoclonal antibodies are showing promising results of selective and efficient treatment of MM in preclinical studies and in early clinical trials. Moreover, we have recently shown that while the expression of several plasma cell markers such as CD138, CD56, and CD20 changed in different stages of the disease, CD38 is constantly expressed on all forms of MM cells. We hypothesize that the anti-CD38 targeted nanoparticles (CD38-NPs) will specifically deliver therapeutic agents to MM cells, thus improve therapeutic efficacy and reduce side effects.
METHODS & RESULTS: cNPs were developed by ionic crosslinking and characterized by dynamic light scattering. A successful method to detect Bortezomib (Btz) by HPLC was developed and the encapsulation efficiency of Btz in cNPs was high (85%) after direct dispersion previous crosslinking. Stability (>30mV) and size (50nm) were not affected by drug loading and were constant for over a 2 month period at 4°C. The release kinetics of Btz from cNPs in different environments was investigated and found that our deliver system preferentially release drugs in tumor microenvironments; the tumor-like acidic pH (6.5-7.1) in MM conditioned media induced faster drug release (2.7-4.5 fold) than neutral pH representing blood and normal tissues. Then, the specificity and the kinetics of the binding of CD38-NPs to MM cells were investigated in vitro. The binding of CD38-NPs was significantly higher in MM cell lines and primary MM cells compared to normal mononuclear cells (MNCs) from peripheral blood and bone marrow. We further confirmed that CD38-NPs bind through CD38 within 2-3 hours, and the nanoparticles did not dissociate at least up to 24h after binding. Moreover, we tested the biodistribution and specificity of fluorescently labeled CD38-NPs in MM tumors models in vivo compared to the free dye and non-targeted cNPs; we found that free dye and non-targeted particles had random biodistribution in different normal tissues; while the CD38-NPs specifically bind the MM cells. Finally, the therapeutic efficacy of Btz-loaded CD38-NPs was assessed in vitro in the treatment of MM compared to free drug. Btz-loaded CD38-NPs induced significantly more killing and downregulation of pAKT in MM cell lines compared to free drug, while it did not affect MNCs. While 2.5nM free Btz induced almost no effect on MM cells, equivalent 2.5nM Btz-loaded CD38-NPs induced a profound killing of more than 50% of the MM cells. Btz-loaded CD38-NPs also led to the induction of a sub-G1 cell cycle arrest, which indicated accumulating apoptotic cells, decreased the percentage of cells in G2/M compared to free drug, and the effect was confirmed by specific signaling with reduced transition cell cycle proteins (pRb). We also found that Btz-loaded CD38-NPs significantly increased apoptosis and cell death (using Annexin/ PI assay) compared to free drug, which was confirmed by increased cleavage of caspase-3 and PARP. We also corroborated that empty (not drug loaded) CD38-NPs had no effect on survival, cell cycle, and apoptosis. Therefore, Btz-loaded CD38-NPs affected proliferation, cell cycle and apoptosis more profoundly compared to free Btz. Moreover, in vivo experiments to evaluate the efficacy and the toxicity of the Btz-loaded CD38-NPs compared with free Btz are ongoing.
CONCLUSIONS: CD38-NPs were prepared, characterized, and showed to be stable over at least 2 months after preparation. The particles had preferential Btz release in tumor-microenvironment compared to blood and normal tissue microenvironment, due to differences in acidity of the medium. CD38-NPs were shown to specifically bind to MM cells through surface CD38 receptors in vitro and in vivo. Moreover, Btz-loaded CD38-NPs reduced MM proliferation, inhibited cell cycle, and induced apoptosis more robustly than equivalent concentrations of free drug, but not in MNCs. These results confirmed that CD38-NPs specifically delivered therapeutic agents to MM cells improving therapeutic efficacy. Therefore, CD38-NPs will reduce side effects and could be used as a new drug delivery approach in myeloma.

Disclosures
De La Puente: Cellatrix LLC: Other: Co-founder. Vij:Shire: Consultancy; Takeda: Consultancy, Research Funding; Celgene: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Karyopharma: Consultancy; Bristol-Myers Squibb: Consultancy; Jazz: Consultancy; Amgen: Consultancy, Research Funding. Azab:Selexys: Research Funding; Vasculox: Research Funding; Cellatrix LLC: Other: Founder and owner; Karyopharm: Research Funding; Glycomimetics: Research Funding; Cleave Bioscience: Research Funding; Verastem: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner; Cell Works: Research Funding.