Construction Of Estrogen Receptor Targeting Liposomes Co-Loaded With Paclitaxel And Carboplatin And Its Anti-Ovarian Cancer Study

Ovarian cancer（OC）is the deadliest gynecological malignancies and about 70percent of patients are diagnosed with advanced tumors.Despite advances in cancer therapy,OC remains a challenging disease.At present,the main therapeutic methods of OC include surgery,chemotherapy,targeted therapy and immunotherapy,among which surgical therapy combined with chemotherapy is still the main therapeutic regimen.Compared with single drug chemotherapy,combination chemotherapy can improve efficacy and reduce drug resistance,which has been widely used in the treatment of OC.Combined chemotherapy with paclitaxel（PTX）and carboplatin（CBP）is the first-line chemotherapy regimen for the clinical treatment of OC.These two drugs play different mechanisms in inhibiting the growth of OC,thus producing synergistic anti-tumor effects by triggering different apoptosis signaling pathways.Like all chemotherapies,PTX and CBP combination chemotherapy also has severe adverse reactions,such as myelosuppression,neuropathy,and renal toxicity.Therefore,a drug delivery system with better therapeutic efficacy and lower adverse reactions is urgently needed in clinic.Nanocarriers,such as liposomes,micelles,dendrimers,nanocapsules and artificial cells,can passively target tumor sites by enhancing permeability and retention effect,which has become a research hotspot for many years.Among nanocarriers for drug delivery,liposomes achieve a safe and effective therapeutic efficacy because of their non-toxic,non-immunogenic and high loading capacity for drugs with different physical and chemical properties.They also could be surface modified for prolonging the half-life of drugs and targeting delivery of drugs.In OC cells,the amount of estrogen receptor（ER）expression is much higher than that in normal tissue cells,therefore ER can be served as a potential target for nanoparticles delivering chemo-drugs to ovarian tumors.Estrone（ES）,as an estrogen,can bind specifically to ER with a high affinity.Therefore,in this study PTX and CBP co-loading long-acting targeted liposomes（ES-PEG-Lip-PTX/CBP）was designed and prepared by using ES as the targeted fragment and polyethylene glycol（PEG）as the long-acting fragment.Its physical and chemical indicators,in vitro and in vivo targeting,pharmacokinetics,anti-OC effect and preliminary safety were systematically investigated.Firstly,a high performance liquid chromatography（HPLC）method for detection of PTX and CBP was established which showed a good linear relationship.The HPLC method was validated for specificity,precision and recovery rate.The results were accurate and reliable.The CCK-8 method was used to determine the optimal synergistic effect of PTX and CBP at a molar ratio of 1:1.ES-PEG-Lip-PTX/CBP was prepared by thin film hydration method,and the optimal prescription and preparation conditions was determined by single factor experiment combined with orthogonal experiment as follows:egg yolk lecithin-cholesterol ratio 3:1,drug-lipids ratio 1:20,CBP concentration 2.0 mg/m L,hydration time 1 h.When PEG ratio was5%,the encapsulation efficiency of liposome was slightly increased,and the proportion of targeted fragments was 0.5%,liposome had high targeting ability.The characterization results of ES-PEG-Lip-PTX/CBP was determined by transmission electron microscopy,Malvern particle size analyzer and HPLC.The ES-PEG-Lip-PTX/CBP presented a uniform and dispersed spherical shape with a particle size of 105.4±0.2 nm,a PDI of 0.156±0.010,and a Zeta potential of-29.6±0.5 m V.The PTX and CBP encapsulation efficiency was 79.61±3.82%and 48.86±4.25%,respectively.The stability results demonstrated that ES-PEG-Lip-PTX/CBP was stable stored at 4℃for 28 days and had a low leakage within 48 h.ES-PEG-Lip-PTX/CBP could remain stable in PBS medium.After PEG modification,ES-PEG-Lip-PTX/CBP showed continuous and slow release behavior without burst release phenomenon.In vitro cellular targeting and uptake mechanism was studied by fluorescence microscopy.The results showed that the uptake of ES-PEG-Lip-PTX/CBP by SKOV-3 cells was significantly higher than that of PTX/CBP co-loaded long-acting liposomes（PEG-Lip-PTX/CBP）,indicating that the modification of ES targeting fragments improved the targeting ability of liposomes.Its uptake mechanism was mainly ER receptor-mediated endocytosis.Meanwhile,ES-PEG-Lip-PTX/CBP could also enter the cell through macropinocytosis and caveolin-mediated endocytosis.The results of blood compatibility experiment showed that ES-PEG-Lip-PTX/CBP had good blood compatibility.In vitro cytotoxicity study showed that the blank liposome had no cytotoxicity on SKOV-3 cells,indicating that blank preparation had high safety.ES-PEG-Lip-PTX/CBP showed the highest cytotoxicity after loading PTX and CBP.The colony formation ability of SKOV-3 cells was also significantly inhibited after incubation with ES-PEG-Lip-PTX/CBP.Meanwhile,the migration ability of SKOV-3 cells was significantly decreased after ES-PEG-Lip-PTX/CBP treatment,and the wound closure rate was significantly lower than that of PEG-Lip-PTX/CBP.In addition,flow cytometry showed that ES-PEG-Lip-PTX/CBP induced more cell apoptosis,and its killing ability on SKOV-3 cells was significantly higher than that of PEG-Lip-PTX/CBP.Cell cycle analysis showed that ES-PEG-Lip-PTX/CBP could induce more cells to arrest in G₂/M phase so as to result in the cell apoptosis.Compared with the control group,ES-PEG-Lip-PTX/CBP also increased the expression of pro-apoptotic protein Bax and down-regulated the expression of anti-apoptotic protein Bcl-2 in SKOV-3 cells.It was proved that ES-PEG-Lip-PTX/CBP showed an excellent tumor targeting ability in the SKOV-3-xenografted nude mouse models by in vivo imaging system of small animals.Compared with PEG-Lip-PTX/CBP,the fluorescence intensity of ES-PEG-Lip-PTX/CBP in the tumor site significantly increased,and more enrichment of the drugs in the tumor site was achieved by the specific targeting of ER.The pharmacokinetic results in mice showed that the blood drug concentration of ES-PEG-Lip-PTX/CBP increased at all the time points due to PEG modification,and the half-lives of PTX and CBP were 2.7 times and 2.2 times that of conventional liposomes,respectively.The bioavailability was improved.In the in vivo anti-tumor studies,ES-PEG-Lip-PTX/CBP showed significantly better therapeutic effect than free PTX/CBP on SKOV-3 tumor bearing nude mice,and the tumor inhibition rate reached 81.83%,with the strongest anti-tumor effect for SKOV-3 tumor.The tumor histopathology also showed also the same result.The number of tumor cells was significantly reduced and the structure of tumor tissue was destroyed.The preliminary safety study showed that after ES-PEG-Lip-PTX/CBP treatment,nude mice did not lose weight during the whole anti-tumor process.H&E staining results of major organs showed no pathological changes.The whole blood indexes and biochemical indexes for liver and kidney functions showed no statistical difference with those of the normal control group,indicating that ES-PEG-Lip-PTX/CBP had good safety profiles.In addition,acute toxicity was investigated in ICR mice with a single intravenous injection of different formulations.The results showed that,compared with free PTX/CBP,ES-PEG-Lip-PTX/CBP significantly increased median lethal dose,and minimized the acute toxicity of free PTX/CBP.A preliminary long-term toxicity was carried out by multiple intravenous administration of ES-PEG-Lip-PTX/CBP in SD rats.The results proved that ES-PEG-Lip-PTX/CBP did not cause toxic reactions with a good safety profile.In summary,ES-PEG-Lip-PTX/CBP was successfully designed and prepared in this study.The characterization study confirmed that the ES-PEG-Lip-PTX/CBP presented good preparation characteristics,and particle size,encapsulation efficiency,stability and other indicators all met the requirements.Both in vivo and in vitro experiments have proved that ES-PEG-Lip-PTX/CBP with a strong tumor targeting ability could prolong the drugs circulation time and enhance the therapeutic effect of PTX/CBP on OC,and had a good safety profile,which might be a potential strategy for the treatment of OC.