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Abstract
Background
Despite recent advances in cancer therapy, the treatment of bone tumors remains a major challenge. A possible underlying hypothesis, limitation, and unmet need may be the inability of therapeutics to penetrate into dense bone mineral, which can lead to poor efficacy and high toxicity, due to drug uptake in healthy organs. The development of nanostructured formulations with high affinity for bone could be an interesting approach to overcome these challenges.
Purpose
To develop a liposomal formulation with high affinity for hydroxyapatite and the ability to release doxorubicin (DOX) in an acidic environment for future application as a tool for treatment of bone metastases.
Materials and methods
Liposomes were prepared by thin-film lipid hydration, followed by extrusion and the sulfate gradient-encapsulation method. Liposomes were characterized by average diameter, ζ-potential, encapsulation percentage, X-ray diffraction, and differential scanning calorimetry. Release studies in buffer (pH 7.4 or 5), plasma, and serum, as well as hydroxyapatite-affinity in vitro analysis were performed. Cytotoxicity was evaluated by MTT assay against the MDA-MB-231 cell line, and biodistribution was assessed in bone metastasis-bearing animals.
Results
Liposomes presented suitable diameter (~170 nm), DOX encapsulation (~2 mg/mL), controlled release, and good plasma and serum stability. The existence of interactions between DOX and the lipid bilayer was proved through differential scanning calorimetry and small-angle X-ray scattering. DOX release was faster when the pH was in the range of a tumor than at physiological pH. The bone-targeted formulation showed a strong affinity for hydroxyapatite. The encapsulation of DOX did not interfere in its intrinsic cytotoxicity against the MDA-MB-231 cell line. Biodistribution studies demonstrated high affinity of this formulation for tumors and reduction of uptake in the heart.
Conclusion
These results suggest that bone-targeted pH-sensitive liposomes containing DOX can be an interesting strategy for selectively delivering this drug into bone-tumor sites, increasing its activity, and reducing DOX-related toxicity.
Acknowledgments
The authors would like to thank Fundação de Amparo à Pesquisa do Estado de Minas Gerais – FAPEMIG (REDE-40/11) and Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (306197/2014-6) for the financial support. The authors thank Dr Mateus Borba Cardoso and his staff for their competence and support during the measurements at Laboratório Nacional de Luz Síncrotron – LNLS (Campinas, Brazil). The authors also thank Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES for supporting DSF with a scholarship. PC acknowledges the National Institutes of Health for generous research and instrumentation support (R01EB009062, S10OD010650).
Author contributions
DSF was responsible for conducting and analyzing the results of the experiments performed. The following authors were responsible for conducting and analyzing the results in partnership with DSF on specified experiments: SDF and LAMF on the development and physicochemical characterization of formulations; SCAL, AM, and RMP on SAXS studies; CST on cytotoxicity studies; JDSF and RJA on synthesis and characterization of DSPE-PEG3,400-AL; and BLJPO on the stability and HA-affinity studies. ASRG and PC advised and financially supported the experiments performed at Massachusetts General Hospital, and MCO coordinated the project and financially supported the experiments performed at Universidade Federal de Minas Gerais. All authors contributed toward data analysis, drafting and critically revising the paper and agree to be accountable for all aspects of the work.
Disclosure
The authors report no conflicts of interest in this work.