Targeted Nanoparticles for the Delivery of Antagomir17: New Approach for the Treatment of Chronic Lymphocytic Leukemia
Authors: Capolla Sara , Bomben Riccardo , Zorzet Sonia , Spretz Ruben , D'Agaro Tiziana , Larsen Gustavo , Pozzato Gabriele , Gattei Valter , Nunez Luis , Macor Paolo 
Published: October 2019

MicroRNAs are associated with prognosis, progression and drug resistance of Chronic Lymphocytic Leukemia (CLL). Among them, microRNA from the miR-17~92 family were demonstrated to affect CLL cells' growth and survival through the inhibition of the tumor suppressor PTEN and the proapoptotic protein Bim. Previous studies demonstrated the effect of an antagomiR17 in the reduction of leukemia cells' growth both in vitro and in vivo. However, despite these results, the in vivo clearance of antagomiR17 is strictly related to its small dimension which induced a rapid renal elimination preventing significant tumor accumulation and, as a consequence, antagomiR17 efficacy.

To overcome this efficacy problem, antagomiR17 was loaded inside biodegradable and biocompatible nanoparticles (BNPs) composed on polyethylene glycol (PEG), polycaprolactone (PCL) and polylactic acid (PLA). The advantages of using BNPs include the significant reduction of toxicity; the potential to load different types of drugs; the improved bioavailability of the payload and cell internalization. Moreover, to ensure the specific binding to cancer B cells, BNPs were conjugated to the antiCD20 antibody Rituximab, targeting leukemia B-cells. Thus, antagomiR17-loaded BNPs efficacy was evaluated by in vitro and in vivo assays.Initially, studies made by quantitative PCR demonstrated that antagomiR17 was able to reduce miR-17 expression in two leukemia cell lines, EHEB and MEC1 cells. Comprehensively, EHEB represents p53wild type patient's derived cells, which developed an indolent model of CLL, while MEC1 represents p53mut/del cells, which usually develop an aggressive model of the disease. Both cell lines were incubated with antagomiR17-loaded BNPs and a significant reduction of miR-17 expression was detected after 24 hours with a complete depletion of miR-17 after 72 hours. Cells growth was also evaluated in vitro affirming that antagomiR17 decreased the proliferation of both MEC1 and EHEB cells in comparison to non-treated cells. Our data also demonstrated that antagomiR17 maintained activity even after encapsulation inside BNPs. For what concerns in vivo studies, a localized model of B-cell malignancy was induced injecting MEC1 cells subcutaneously into SCID mice, causing the formation of a localized tumor mass at the site of cell injection after 3-4 weeks. When the tumor mass reached 300mg in weight, animals were treated once with an intraperitoneal injection of BNPs containing 26μg of antagomiR17 with or without antiCD20 antibody on the BNP surface. The tumor mass dimension and mice's survival were evaluated. Specifically, the decreased in vivo growth of MEC1 cells was more evident after a treatment with antiCD20-conjugated BNPs in respect to unconjugated BNPs (2500 vs 3500 mg), confirming again the importance of the antiCD20 antibody in BNPs homing on B cells. Furthermore, the evaluation of mice survival also confirmed the data; mice treated with antiCD20-BNPs showed an increased survival in respect to mice injected with unconjugated BNPs (37 vs 21 days).

In conclusion, our data confirms the efficacy of antiCD20 polymeric nanoparticles in the delivery of antagomiR17 into tumor B-cells. This new therapeutic approach allows to selectively restore miR17 levels and to reduce tumor cell growth, both in vitro and in vivo. Altogether, these results provide a new path for the specific delivery of small nucleic acids like miRNA, antagomiR or siRNA into tumor B-cells and offers the development of future drug combinations with standard therapeutic approach in the same targeted nanostructure.

Disclosures

Spretz: LNK Chemosolutions LLC: Employment, Equity Ownership. Larsen:LNK Chemosolutions LLC, Biotarget Inc.: Employment, Equity Ownership. Nunez:LNK Chemosolutions LLC, Biotarget Inc.: Employment, Equity Ownership.