Purpose: The incidence of bone diseases has been rapidly increasing with global population aging. However, most drugs intended for bone diseases do not have a high affinity for bone hydroxyapatite under physiological conditions, which limits their therapeutic potential. Therefore, development of bone targeting system is highly required for the treatment of bone diseases. Recently, it was reported that carboxylic acid rich proteins had high affinity to bone. Therefore, we hypothesized that carboxylic acid (ex. aspartic acid (Asp)) modification may be a promising approach for bone drug targeting. The aim of this study was to develop PEGylated Asp modified liposome (PEG-Asp-Lipo) as a bone-targeting carrier for the delivery of paclitaxel (PTX) and treatment of bone metastasis.
Methods: PEG-Asp-Lipo as a bone-targeting carrier of PTX was prepared by using Asp-modified 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-Asp). For the affinity to hydroxyapatite (HAP) and the pharmacokinetic study, normal, PEGylated, and Asp-modified liposomes were prepared with various lipid compositions. A HAP binding assay was performed by mixing [3H]-labeled liposomes with HAP in the solution. For the tissue distribution experiments, [3H]-labeled PEG-Asp-Lipo ([14C]-labeled PTX) was intravenously injected into mice. In vivo fluorescence images of near-infrared fluorescence-labeled PEG-Asp-Lipo were acquired using an IVIS imaging system. To evaluate the intra-bone distribution of PEG-Asp-Lipo, the distribution of FITC-labeled PEG-Asp-Lipo in the bone section was observed using a confocal laser-scanning microscope. In a bone metastatic tumor mouse model, B16-BL6/Luc cells were injected into the left ventricle of female C57BL/6 mice. 7 days and 10 days after the tumor inoculation, PEG-Asp-Lipo (PTX) was intravenously injected into mice.
Results: The affinity of Asp-Lipo to HAP increased as the concentration of DPPE-Asp increased. The bone accumulation of [3H]-labeled PEG(2)-Asp(33)-Lipo was approximately 24.6% 360 min after intravenous injection in mice, in contrast to 5.4% and 6.7% of [3H]-labeled normal Lipo and PEG(2)-Lipo, respectively (Fig. 1). Similarly, [14C]-labeled PTX encapsulated into PEG(2)-Asp(33)-Lipo predominantly accumulated in the bone. Furthermore, using an in situ imaging experiment, we observed that near-infrared fluorescence-labeled PEG(2)-Asp(33)-Lipo selectively accumulated in the bone near the joint after intravenous injection in mice (Fig. 2). We also found that FITC-labeled PEG(2)-Asp(33)-Lipo predominantly accumulated on eroded and quiescent bone surfaces. In a bone metastatic tumor mouse model, in which B16-BL6/Luc cells were injected into the left ventricle of female C57BL/6 mice, metastatic bone tumor growth was significantly inhibited by an intravenous injection of PEG(2)-Asp(33)-liposomal PTX. In contrast, PEGylated liposomal PTX hardly affected the growth of metastatic bone tumors (Fig. 3).
Conclusion: These findings indicate that bone targeting of PTX using PEG(2)-Asp(33)-Lipo is a promising approach for the treatment of bone metastasis.
Shugo Yamashita– Kobe Pharmaceutical University
Yumiko Yagi– Kyoto Pharmaceutical University
Yugo Isobe– Kyoto Pharmaceutical University
Toshiyasu Sakane– Professor, Kobe Pharmaceutical University, Kobe city, Hyogo
Akira Yamamoto– Professor, Kyoto Pharmaceutical University, Kyoto