Preparation of hollow nanocapsules entrapping cationic sonosensitizer for effective sonodynamic therapy

摘要

Introduction: Sonodynamic therapy (SDT) has been developed as a novel promising noninvasive approach involving utilization of low-intensity ultrasound and sonosensitizers, which can generate reactive oxygen species (ROS) including singlet oxygen and free radicals by sonication. SDT has a potential utility for the treatment of deep cancer in comparison with other approaches using external stimuli, because ultrasound can deeply penetrate human tissues. In this study, doxorubicin (DOX) was employed as a sonosensitizer. After the accumulation into nucleus, DOX can generate ROS by sonication and the generated ROS give damage to DNA. Also, DOX derivative was synthesized for the control of intracellular distribution of sonosensitizer. Triphenyl phosphonium (TPP) moiety, which has the ability for mitochondrial targeting, was introduced to DOX for effective apoptosis derivation through the mitcondrial oxidative stress. Further, intracellular reductive-responsive nanocapsules were prepared by the introduction of disulfide bonds to self-assembled polymer vesicles of head-tail type polycations, which composed of a polyamidoamine (PAMAM) dendron head and poly(L-lysine) tail. These nanocapsules might efficiently deliver sonosensitizers to cytoplasm through the endosomal escape by buffering effect of PAMAM dendron heads and cleavage of disulfide bond in cytoplasm. Here, we evaluated the delivery of two kinds of sonosensitizers (DOX and TPP-DOX) by the entrapment into hollow nanocapsules for the application of SDT. Materials and Methods: Singlet oxygen generation with sonication was determined by using singlet oxygen sensor green (SOSG). Sonosensitizers and SOSG were dissolved in PBS (pH7.4) and solution was then sonicated. Change in fluorescent intensity at 525 ran was monitored. For evaluation of cytotoxicity and intracellular distribution, HeLa cells were seeded and grown In DMEM with 10% FBS for 24 h. Cells were incubated with varying amount of DOX, TPP-DOX, DOX@capsule and TPP-DOX@capsule for 4 h and washed by PBS(-). After 20h incubation, viability was estimated by MTT assay. When cytotoxicity with sonication was evaluated, cells were sonicated after washed. Intracellular distribution of sonosensitizers or sonosensitizer@capsules was confirmed by laser confocal microscopic observation. Results: DOX and TPP-DOX exhibited almost same singlet oxygen generation properties. For intracellular distribution and cytotoxicity, TPP-DOX displayed punctate fluorescence in cytoplasm and no toxicity, though DOX accumulated cell nucleus and showed significant toxicity at 20 mM or above. When DOX and TPP-DOX were sonicated, their cytotoxicity was enhanced resulting in decrease of viability more than 10 mM and 100 mM, respectively. In DOX@capsules and TPP-DOX@capsules, intracellular distribution of sonosensitizers showed similar tendency to free sonosensitizers. Discussion: TPP-DOX was not observed in cell nucleus but cytoplasm as punctuate fluorescence. From these results, it is considered that TPP-DOX partially accumulates mitochondria and this consideration is consistent with cytotoxicity of TPP-DOX, because DOX induces anticancer activity by the intercaration into nuclear DNA. Therefore, enhancement of cytotoxicity with sonication might derive from different mechanisms. For DOX, anticancer activity and oxidative damage to DNA by ROS was considerable and TPP-DOX may induce cell apoptosis via oxidative stress to mitochondria. Further, these sonosensitizers can be delivered to target sites by using nanocapsules, namely nanocapsules can exhibit endosomal escape by buffering effect of dendron moieties and drug release under reductive environment in cytoplasm. Conclusion: The intracellular distribution of DOX was changed by the introduction of TPP moiety, and TPP-DOX could not accumulate the nuclei. As a result of the change in intracellular distribution, the in vitro anticancer effect was remarkably different between DOX and TPP-DOX. However, the sonication could provide the decrease in cell viability for both DOX and TPP-DOX. DOX and TPP-DOX could be delivered into the cells through the entrapment into the nanocapsules prepared from head-tail type polycations.