Nanoparticle Design For Bone-Specific Chemotherapy and Microenvironmental Targeting In Multiple Myeloma
Authors: Reagan Michaela R , Swami Archana , Basto Pamela A , Mishima Yuji , Liu Jinhe , Glavey Siobhan , O'Callaghan Grace B , Moschetta Michele , Nagano Kenichi , Baron Roland , Roccaro Aldo M , Farokhzad Omid , Ghobrial Irene M. 
Published: October 2019

Abstract

Introduction
The bone marrow (BM) niche is known to exert a protective effect on lymphoid tumors, such as multiple myeloma (MM), where mesenchymal stem cell interactions with clonal plasma cells increase tumor proliferation and survival. However, certain cells within the BM milieu, such as mature osteoblasts and osteocytes, have demonstrated the potential to inhibit tumor growth; utilizing these cells presents a promising new anti-cancer approach. Hence, designing better methods of bone-specific delivery for both direct cancer cell treatment and indirect treatment through the modulation of bone cells may result in a potent, two-pronged anti-cancer strategy. Our work aimed to develop a novel system to target both MM and bone cells to induce greater osteogenesis and hamper tumor growth.

Methods
PEG–PLGA nanoparticles (NPs) coupled to alendronate (“bone-targeted”) or alone (“non-targeted”) were formulated and loaded with bortezomib (“BTZ-NPs”) or left empty (“BTZ-free”). NPs were characterized for their physiochemical properties, including size (using dynamic light scattering; surface charges (Zeta potential); and bone affinity (using hydroxyapatite binding). NPs were engineered with different formulation methods and those with the optimal physiochemical characteristics and drug encapsulation efficiency were used for further studies. BTZ release kinetics were analyzed using HPLC. Anti-MM effects were assessed in vitro using MTT, bioluminescence (BLI) and Annexin V/PI apoptosis flow cytometry analysis on MM1S cells. In vivo, efficacy was measured by mouse weight, BLI and survival after i.v. cancer cell injections in mice. Cellular uptake was assessed in vitro by flow cytometry and in vivo biodistribution was assessed using fluorescent whole body and fixed section imaging. Bone specificity was assessed in vitro by co-culture of bone-targeted and non-targeted NPs with bone chips or hydroxyapatite using fluorescence and TEM imaging.
In an in vivo model of myeloma treatment, female Nod/SCID beige mice were injected i.v. with 4 × 106 Luc+/GFP+ MM1S cells and, at day 21, treated with a) BTZ, b) BTZ-bone-targeted NPs, c) BTZ-non-targeted NPs or d) BTZ-free bone-targeted NPs. Using an in vivo model of pre-treatment for cancer prevention, mice were pre-treated with i.p. injections of BTZ-bone-targeted NPs and appropriate controls thrice weekly for 3 weeks. They were then injected i.v. with Luc+/GFP+ 5TGM1 or MM1S cells and monitored for BLI and survival. Static and dynamic bone histomorphometry and μCT were used to assess effects of pre-treatment on bone formation and osteolysis prevention.

Results
Our biodegradable, NPs had uniform size distribution within the range of 100 to 200 nm based on the type of formulation, with a zeta potential of ±5mV. Bone- targeted NPs showed high affinity towards bone mineral in vitro and better skeletal accumulation in vivo compared to non-targeted NPs. NPs were easily up-taken by cells in vitro, and BTZ release kinetics showed a burst followed by a sustained-release pattern over 60 hrs. BTZ-NPs induced apoptosis in MM cells in vitro. Importantly, BTZ-bone-targeted-NP pre-treated mice showed significantly less tumor burden (BLI) and longer survival than free drug or drug-free bone-targeted NPs, thus demonstrating a tumor-inhibiting effect unique to the BTZ-bone-targeted-NPs. Pre-treatment with BTZ increased bone formation in tibias and femurs, as measured by μCT of bone volume/total volume, and trabecular thickness and number, suggesting that increased bone volume may inhibit MM. In a second mouse model, both BTZ-bone-targeted NPs and BTZ-free NPs were equally able to reduce tumor growth in vivo when given after tumor formation.

Conclusion
Bone-targeted nanoparticles hold great potential for clinical applications in delivering chemotherapies to bone marrow niches, reducing off-target effects, increasing local drug concentrations, and lengthening the therapeutic window. BTZ-bone-targeted NPs are able to slow tumor growth and increase survival in mice when used as a pre-treatment. This may result, at least in part, from BTZ-induced increased bone formation. These findings indicate that BTZ-bone-targeted NPs exert a chemopreventive effect in MM in vivo, thus suggesting their potential use in the clinical setting.

Disclosures:
Basto: BIND Therapeutics: Patent licensed by BIND, Patent licensed by BIND Patents & Royalties. Farokhzad:BIND Therapeutics: Employment, Equity Ownership; Selecta Biosciences: Employment, Equity Ownership. Ghobrial:Onyx: Membership on an entity’s Board of Directors or advisory committees; BMS: Membership on an entity’s Board of Directors or advisory committees; BMS: Research Funding; Sanofi: Research Funding; Novartis: Membership on an entity’s Board of Directors or advisory committees.