Targeted Pt(Ⅳ)prodrug-loaded Phase-transitional Nanoparticles For Precise Theranostics Against Ovarian Cancer

BackgroundOvarian cancer is the most prevalent gynecologic cancer with a high mortality rate in recent decades.At present,platinum（II）（Pt（II））-based anticancer drugs dominate the chemotherapy field of ovarian cancer.However,the patient’s quality of life has not improved significantly owing to dose-limiting toxicities,partial antitumor response,drug resistance and many other challenges.Moreover,many ovarian cancer patients have developed advanced disease at the time of diagnosis.Therefore,the development of new theranostic strategies to diagnose and overcome chemotherapy toxicities is highly desirable.In this study,multifunctional tumor-targeted nanosized ultrasound contrast agents（GSH-sensitive Pt（Ⅳ）prodrug-loaded phase-transitional nanoparticles,Pt（Ⅳ）NP-c RGD）composed of a perfluorohexane（PFH）liquid core and a hybrid lipid-polymer shell were prepared.Study strategyDesign of GSH-sensitive Pt（Ⅳ）prodrug:Cisplatin was transformed into Pt（Ⅳ）,and then was covalently attached to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy（polyethylene glycol）]（DSPE-PEG）,which enhanced not only the drug loading efficiency but also the plasma half-life.Compared with the four-coordinate centers of Pt（II）,Pt（Ⅳ）prodrug is more resistant to ligand substitution reactions and more stable in blood circulation.In addition,the two additional ligands of Pt（Ⅳ）provide a promising way to fine-tune desired biological properties,such as lipophilicity,cancer-cell targeting and improved cellular uptake.Because lipid DSPE interacts favorably with and adsorbs onto the cell membrane,it enables the prodrug to be taken up by the cells through passive diffusion,activated by reducing agents such as glutathione（GSH）,and to release the active pharmaceutical ingredient in the cytoplasm.Therefore,Pt（Ⅳ）prodrugs can improve the therapeutic effect while reducing the toxicity of chemotherapy.Design of targeted phase-transitional nanoparticles with a hybrid lipid-polymer shell:Pt（Ⅳ）NP-c RGD composed of a PFH liquid core and a hybrid lipid-polymer shell with PLGA_12k-m PEG_2k,PLGA_12k-PEG_2k-c RGD and DSPE-PEG_1k-Pt（Ⅳ）were prepared.The PFH droplets undergo phase transition when the external pressure is reduced to the vaporization pressure threshold or the temperature rises above the boiling point,causing changes in the structure and morphology of the nanoparticles for US imaging and tumor treatment.The hybrid shell remained the stable structure of the polymer shell,which can enhance the circulation time for continuous US imaging and improve therapy efficiency.In addition,the introduction of a lipid softened the outer shell and made it lighter so that it did not require much acoustic energy to promptly expand,rupture,reseal,compress,buckle,or re-spread during each acoustic cycle to enhance the ultrasonic echo signals.Therefore,nanoparticles with a hybrid lipid-polymer shell easily responded to ultrasound exposure for improved US imaging and drug delivery.Polyethylene glycol（PEG）was modified on the surface of the hybrid shell in order to escape the reticuloendothelial systems（RES）and further prolong blood-circulating duration,so that nanoparticles passively target into tumor tissue through enhanced permeability and retention effect（EPR effect）.Moreover,a cyclic Arg-Gly-Asp（c RGD）with high affinity to integrin receptor was conjugated to the outer shells of the nanoparticles to enable actively targeted accumulation within the tumor tissues.Methods and ResultsSynthesis and characterization of Pt（Ⅳ）NP-c RGD:The Pt（Ⅳ）prodrug was successfully synthesized and Pt（Ⅳ）NP-c RGD was successfully prepared.Optimal preparation methods of Pt（Ⅳ）NP-c RGD are acquired based on single factor investigation and orthogonal design test.The average size of Pt（Ⅳ）NP-c RGD was measured as 151.1±1.3 nm,the zeta potential of the Pt（Ⅳ）NP-c RGD was-5.27±0.38m V in aqueous solution.The average sizes of Pt（Ⅳ）NP-c RGD did not change significantly within 25 days at 4°C,25°C and 37°C,suggesting good storage stability.The results of serum-induced aggregation assay indicated that Pt（Ⅳ）NP-c RGD resisted the serum-induced aggregation and remained stable in the blood circulation,which resulted in prolong circulation time.Due to the phase-transition behavior of PFH,the morphology and structure of the Pt（Ⅳ）NP-c RGD expanded and collapsed under temperature rise or US stimulation.The Pt release profile in vitro showed the GSH-sensitive and US-triggered drug release of Pt（Ⅳ）NP-c RGD.In addition,Pt（Ⅳ）NP-c RGD exhibited strong echogenic signals and excellent echo persistence under US exposure in vitro.Antitumor efficacy of Pt（Ⅳ）NP-c RGD with US in vitro:The cellular uptake and cytotoxicity of Pt（Ⅳ）NPs-c RGD were investigated on SKOV3 cells,A2780 cells andα_v integrin-downregulated SKOV3 cells in vitro.These results emphasized the significance of the c RGD ligand,which enhanced cytotoxicity through improving cellular uptake by integrin receptor-mediated endocytosis.Besides,the cellular uptake experiments based on confocal laser scanning microscopy and flow cytometry demonstrated that US further accelerated intracellular uptake of Pt（Ⅳ）NPs-c RGD.Therefore,Pt（Ⅳ）NPs-c RGD with US significantly inhibited SKOV3 cell proliferation and displayed excellent antitumor therapeutic efficiency in vitro.Antitumor mechanism of Pt（Ⅳ）NP-c RGD with US:The transformation of the Pt（Ⅳ）prodrug into Pt（II）complexes required a large consumption of GSH during drug release.The concentration of GSH decreased,the redox balance was destroyed,which resulted in an overproduction of ROS.In addition,US exposure induced the production of ROS.Thus,the combination of Pt（Ⅳ）NP-c RGD with US consumed GSH and overproduced ROS in tumor cells.Further researches confirmed that the Pt（Ⅳ）NP-c RGD+US induced the overproduction of different kinds of ROS instead of a specific one.The mechanistic studies demonstrated a route of ROS-induced mitochondria-mediated apoptosis.Pt（Ⅳ）NP-c RGD+US promoted tumor cell apoptosis,notably by consuming GSH and highly enhancing the levels of ROS,further inducing（35）ψ_m,decreasing and releasing cytochrome c into the cytosol,activating caspase 9 and caspase 3 and finally activating tumor cell apoptosis.Precise theranostics of Pt（Ⅳ）NP-c RGD with US in vivo:The prolonged circulation time properties of Pt（Ⅳ）NPs-c RGD may exhibit protection from hepatic phagocytosis and benefit for passive tumor targeting via EPR effect.The tumor-targeting ability of Pt（Ⅳ）NP-c RGD in vivo was investigated by using SKOV3tumor-xenografted nude mice as the model.A time-dependent biodistribution was observed with a in vivo imaging system.The results indicated that c RGD ligand modification improved the accumulation of the nanoparticles in the tumor tissue through active targeting.Pt（Ⅳ）NP-c RGD with US had the most effective inhibition on tumor growth compared with other drug formulations with an equivalent dose.And according to immunohisto-chemistry and TUNEL assay,the antitumor efficiency of Pt（Ⅳ）NP-c RGD with US was induced by mitochondrial apoptosis in vivo.In addition,Pt（Ⅳ）NP-c RGD exhibited strong echogenic signals and excellent echo persistence under an US field in vivo.ConclusionWe successfully constructed a GSH-sensitive and US-triggered drug delivery system using multifunctional nanosized UCAs that have prolonged blood circulation and tumor-targeting abilities for precise theranostics against ovarian cancer.Both the in vitro and in vivo experiments demonstrated that Pt（Ⅳ）NP-c RGD exhibited strong echogenic signals and excellent echo persistence under an US field.Furthermore,Pt（Ⅳ）NP-c RGD with US had an enhanced accumulation in the tumor tissue and displayed excellent antitumor therapeutic efficiency.The mechanistic studies demonstrated a route of ROS-induced mitochondria-mediated apoptosis.The nanosized UCAs in this work may provide valuable insight into the development of various multifunctional UCAs with improved US imaging for precise theranostics against various cancer.