Application Of Oxidation-Responsive Liposomes Co-Loaded With Cisplatin And Glutaminase Inhibitor In Ovarian Cancer Treatment

Ovarian cancer is the gynecological tumor with the highest mortality rate,and its first-line treatment is surgery combined with platinum（Pt）-based combination chemotherapy.However,in the course of repeated treatment,the sensitivity of most patients to platinum drugs would gradually decline.Cisplatin（CDDP）is the most classic platinum drug,and its resistance mechanisms involves decreased drug uptake and increased drug excretion,drug detoxification of sulfhydryl substances,enhancement of DNA repair,and abnormality of specific cell signaling pathways.Glutamine metabolic reprogramming is an important feature of tumor metabolic reprogramming.It not only replenishes the tricarboxylic acid（TCA）cycle to ensure energy supply as well as provide raw materials for the synthesis of glutathione,nucleic acids and so on,but also regulates specific intracellular signal pathways.Studies have shown that inhibiting glutamine metabolism can suppress tumor growth and sensitize tumor cell to CDDP,which is achieved by disturbing the intracellular redox homeostasis.However,a more comprehensive and in-depth mechanism of it has not been explored.The pattern of administration of the combination therapy plays an important role in the optimal efficacy of the drug.Dual drug co-delivery under nanomedicine technology is currently an ideal combined drug delivery strategy.Tumor environmentally responsive liposomes have high biocompatibility and biodegradability.They can simultaneously carry a variety of drugs with different solubility and release drugs in response to the tumor microenvironment,thus to increase drug efficacy without accumulating toxicity and delay drug resistance,which have a very broad application prospect.Objective and significanceBased on the existing problems of CDDP therapy and the advantages of glutaminase inhibitors in anti-tumor and CDDP sensitization,this research intended to co-load CDDP and BPTES,a glutaminase inhibitor,into intelligent oxidationresponsive liposomes,which were expected to passively target tumor sites due to high permeability and retention（EPR）effect.In response to the oxidative environment in cells,drugs were released from liposomes to achieve the effect of chemo/starvation therapy as well as CDDP sensitization.This study has important theoretical significance and practical value for enhancing the therapeutic effect of ovarian cancer and improving the low survival rate of ovarian cancer patients.Methods（1）DOPC,cholesterol and DSPE-mPEG₂₀₀₀ were used as raw materials for liposomes preparation.The liposomes loaded CDDP（CDDP LPs）and the liposomes co-loaded with CDDP and BPTES（C@B LPs）were prepared by thin-film dispersion method.Inductive coupled plasma emission spectrometer（ICP）and High-performance liquid chromatography（HPLC）were used to determine the content of CDDP and ray photoelectron spectroscopy（XPS）was used to determine the element component of liposomes.The particle size,poly disperse index（PDI）and zeta potential of liposomes were determined by dynamic laser light scattering（DLS）.The morphology of the liposomes was observed by transmission electron microscopy（TEM）.The stability of the liposomes was investigated by measuring the changes of the particle size and PDI of the liposomes in the serum-containing environment.The responsive ability and drugs release of the liposomes in the oxidative environment was evaluated by DLS detection,TEM observation and dialysis.（2）The cytotoxicity of CDDP LPs and C@B LPs in vitro was investigated by MTT and flow cytometry.The synergistic index of CDDP and BPTES was then calculated based on the MTT results.Endocytosis experiment,endocytosis mechanism study,platinum uptake experiment,metabolites measurement,GLS activity,GSH and ATP detection and Western blot were used to explore the synergistic mechanism of the two drugs in C@B LPs at the aspects of drug accumulation and detoxification,DNA repair and abnormality of specific signaling pathway.（3）The tumor bearing mouse model was established,and the drug was administered by tail vein.The biological distribution of liposomes was studied by in vivo fluorescence imaging and ICP detection.The efficacy and toxicity of C@B LPs were evaluated by the curve of tumor growth and weight change of mice,serum biochemical detection,HE and TUNEL staining.The in vivo antitumor mechanism of C@B LPs was investigated by GSH detection,immunohistochemistry and Liquid Chromatography Mass Spectrometry（LC-MS）untargeted metabolomics.Results（1）CDDP LPs and C@B LPs were successfully prepared by thin-film dispersion method.XPS and HPLC results indicated CDDP and BPTES were successfully encapsulated in C@B LPs.The Pt encapsulation rate of CDDP LPs was 80.3±2.6%.The Pt and BPTES encapsulation rates of C@B LPs were 18.7±0.9%and 17.8±1.2%,respectively.The molar ratio of CDDP and BPTES in C@B LPs was 1:1.4.TEM observation showed that CDDP LPs and C@B LPs were spherical and evenly distributed.The particle sizes of CDDP LPs and C@B LPs were 138.70± 0.92 nm and 156.33± 17.40 nm,respectively.The PDI of both liposomes were less than 0.3 and the zeta potentials were negative whose absolute values were more than 30.The stability of CDDP LPs and C@B LPs in serum-containing solution was tested for 7 consecutive days,whose result was that the particle sizes of liposomes were stable within 7 days,and the average PDI was below 0.3.TEM,DLS and dialysis revealed cleavage of liposomes incubated in H2O2 solution,with cumulative Pt and BPTES release of more than 80%and 65%,respectively within 24 hours,while the cumulative Pt and BPTES release of liposomes incubated in PBS solution was less than 40%and 30%,respectively within 24 hours.（2）MTT results showed that for SKOV3 cells,the IC50 of CDDP,CDDP LPs and C@B LPs were 11.84 ± 1.51 μM,9.00 ± 0.94 μM and 3.34±0.15 μM,respectively,while for SKOV3DDP cells,the IC50 of which were 84.34±4.27 μM,66.95± 6.64 μM,9.26 ± 1.07 μM.At the molar ratio of CDDP to BPTES of 1:1.4,the two drugs showed synergistic effect.Studies on intracellular endocytosis and its mechanism showed that liposomes entered SKOV3DDP cells through endocytosis in a time-dependent manner.Compared with CDDP,CDDP LPs promoted Pt absorption in SKOV3DDP cells.However,C@B LPs could further increase cellular Pt accumulation.In addition,BPTES and C@B LPs significantly reduced GLS activity,caused glutamine accumulation,and decreased glutamate,α-ketoglutarate,oxaloacetate,aspartate,GSH and ATP production in SKOV3DDP cells.Western blot experiment showed that C@B LPs could significantly increase the expression of γ-H2AX,Bax and Caspase-3 in SKOV3DDP cells,while significantly decrease the expression of mTOR and Bcl-2.（3）In vivo distribution experiments showed that the circulation time of the liposomes was more than 24 hours.C@B LPs could be enriched and retained in the tumor site of SKOV3 cell-bearing mice,which could significantly improve the Pt accumulation of tumor.In vivo efficacy and toxicity studies showed that C@B LPs had excellent in vivo antitumor effect,and significantly lower liver and kidney toxicity compared with CDDP,while the toxicity could not be completely eliminated.In addition,compared with CDDP LPs,C@B LPs did not aggravate the side effects in vivo.Immunohistochemical analysis showed that CDDP,CDDP LPs,and C@B LPs increased the expression of γ-H2AX and Cleaved caspase-3 in tumor tissue.C@B LPs can also significantly reduce the level of GSH in tumor area.The results of LC-MS untargeted metabolomics suggested that C@B LPs leaded to significant changes in the levels of multiple metabolites in tumors,which woulf further disturbed a series of metabolic pathways,such as glycine,aspartate and glutamine metabolism,TCA cycle,ABC transporters,and protein digestion and absorption,etc.Conclusion（1）CDDP LPs and C@B LPs were successfully prepared in this study,with appropriate particle size,uniform dispersion,good stability in physiological media,and also with the ability to cleave and release drugs in response to an oxidative environment.（2）For SKOV3 and SKOV3DDP cells,CDDP LPs and C@B LPs significantly enhanced the sensitivity of cells to CDDP,and C@B LPs had a stronger sensitization effect with significant difference.C@B LPs increased the sensitivity of SKOV3DDP to cisplatin due to a combination of the following mechanisms:1)Delivery by liposome to bypass the uptake barrier of CDDP;2)Reduce the detoxification of CDDP by inhibiting GSH synthesis;3)Reduce ATP production to inhibit the efflux of CDDP by ATP-dependent transports;4)affecting nucleotides synthesis by reducing aspartate thus to inhibit DNA damage repair;5)Inhibiting mTOR pathway by reducing αketoglutarate thus to promote caspase-3 mediated apoptosis signal.（3）C@B LPs showed long circulation in vivo and can be passively enriched and retained in tumor sites of SKOV3 tumor cell-bearing mice through EPR effect,thus to significantly increasing the accumulation of CDDP in tumor tissues.C@B LPs have excellent antitumor effects in vivo,and can significantly reduce the systemic toxicity of CDDP,which but cannot completely eliminate.C@B LPs play a tumor-killing role in vivo by disrupting tumor tissue metabolism,reducing local GSH,damaging cell DNA,and inducing programmed cell death.