Metastasis targeting: a novel approach to reach bone using zoledronate anchored nanoparticles
Vinoth Khandelwal 1,2,3
Kiran Chaudhari 3,4
Abhinesh Kumar 4
Anil Mishra 5
Jukka Monkkonen 3
Rayasa RamachandraMurthy 4
1 Slovak Academy of Sciences, Bratislava, Slovakia 2 Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy 3 University of Eastern Finland, Kuopio, Finland 4 The Maharaja Sayajirao University of Baroda, Vadodara, India 5 Institute of Nuclear Medicine and Allied Sciences, Delhi, India
|Section:||Cellular metabolism, physiology, molecular biology and genetics|
Bone metastasis targeting remains largely unexplored. Some of the bone diseases are seldom cured just because of poor distribution of drug to the bone. Zoledronic acid (ZOL) possess a strong affinity towards bone, and hence its utility in bone metastasis management makes it a perfect ligand for bone targeting. Recent studies revealed that ZOL in combination with docetaxel (DTX) showed significant synergism in the management of bone metastasis. DTX-loaded ZOL-conjugated polyethylene glycol (PEG)ylated polybutyl cyanoacrylate (PBCA) NPs (PBCA-PEG-ZOL) were prepared using the anionic polymerization technique. Physiochemical Characterization, pharmacokinetics, in vitro bone binding assay, quantitative cellular uptake, NP uptake route characterization, and cellular IPP/ApppI (isopentenyl pyrophosphate/triphosphoric acid 1-adenosin-5′-yl ester 3-(3-methylbut-3-enyl) ester) levels were performed. DTX was efficiently entrapped (75.94 ± 3.82%) in the Zol conjugated NPs, estimated by HPLC. They had discrete spherical shape, and size of around 82 nm estimated by zetasizer and transmission electron microscopy. Biodistribution studies using technetium-99m radiolabeling showed prolonged blood circulation half-life, and that the ratio of PBCA-PEG-ZOL NPs in tumor bearing bone to the normal bone was 3 fold, at any time point. Further, ZOL conjugated NPs localization in tumor bearing bone significantly increased with time and found to be 7.5 (p<0.01), 20 (p<0.001) and 155 (p<0.001) times higher after 1, 4 and 24 h respectively, as compared with unconjugated but pegylated NPs, possibly due to the remodeling of the bone lining by tumor metastasis. In vitro bone binding assay using human simulated bone-hydroxyapatite powder results confirmed that ZOL has strong binding affinity to bone, and maintained the affinity even when used as a surface ligand conjugated to NPs. It was noticed that after 4 h of treatment with BO2 cells, ZOL conjugated NPs showed two times longer residence time (measured by flow cytometry) than un-conjugated ones. PBCA-PEG-ZOL NPs showed an enhanced cytotoxic effect (MTT assay) in both BO2 and MCF-7 cell lines, due to cell cycle arrest and apoptosis (by flow cytometry). Uptake route characterization studies (by confocal microscopy & flow cytometry) with different inhibitors revealed that PBCA-PEG-ZOL NPs uptake is not entirely based upon clathrin or caveolae mediated endocytosis. PBCA-PEG-ZOL NPs blocked the mevalonate pathway and showed 7 and 5.3 times increase in IPP and ApppI production (measured by Liquid chromatography-mass spectrometry; LC-MS), in comparison to ZOL treatment, and 138 times higher than the control group in MCF-7 cell line. These results provide evidence that ZOL-conjugated NPs provide an efficient and targeted delivery system for bone metastasis. Hence, these NPs present a promising treatment in the near future for bone related diseases.
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