Co-delivery Of Two Pin1 Inhibitors Using Albumin Nanoparticles To Inhibit Hepatocellular Carcinoma Metastasis

Cancer is a serious threat to human health.How to effectively block the occurrence and development of cancer is one of the major issues in the field of medicine.Prolyl isomerase Pin1 has been found to be the operator of the central "proline phosphorylation event" in several carcinogenic signaling pathways.Related experiments have shown that all-trans-retinoic acid(ATRA)and Arsenic trioxide(ATO)can cooperatively and effectively inhibit and degrade the active Pin1,block several Pin1-regulated Cancer-driving pathways,and produce significant tumor inhibition effect.However,ATRA has some defects such as short half-life in vivo,poor water solubility and sensitivity to light and heat,and ATO has some defects such as short half-life and high toxicity and side effects,which limit the clinical application of ATRA and ATO.Therefore,this topic uses human serum albumin(HSA)nanoparticles with high biological safety and good application prospects as a means to improve the defects of ATRA and ATO.By constructing HSA nanoparticles coated with ATRA and ATO and taking HCC as a model,this project studied and verified the pharmacokinetics,acute biotoxicity,in vivo and in vitro inhibition of lung metastasis and its mechanism.The aim is to provide a new,safe and effective medication method of ATRA combined with ATO for clinical use,and promote its application progress in solid tumors.Methods(1)The Human Serum Albumin-All-trans retinoic acid-arsenic trioxide(HSA-ATRA-ATO)nanoparticles were constructed by reducing and desolventizing.The ATRA drug load was tested by ultraviolet absorption wavelength,the ATO drug load was tested by ICP-MS.Dynamic light Scatterometer and scanning electron microscope were used to investigate its particle size,electric potential and morphology.Its release in vitro was measured by direct dispersion method.(2)In the pharmacokinetics and tissue distribution experiments,HPLC-MS was used to determine the ATRA concentration in plasma and tissue samples,ICP-MS was used to determine the ATO concentration in plasma and tissue samples.To observe the cellular uptake of HSA nanoparticles used the method of modifying HSA nanoparticles with a fluorescent group FITC.(3)Under the conditions of a single dose of 150 mg/kg and 600 mg/kg,the histological and hematological changes of the mice were observed for 48 hours,the behavioral changes of the mice were observed within 14 days.(4)Using MTT colorimetry investigate the inhibitory effect of HSA-ATRA-ATO on the proliferation of HCC and determine the concentration of ATRA and ATO to inhabit metastasis.Using metastasis experiments,lung metastasis models and other methods determine the synergy between ATRA and ATO and the effect of nanoparticles in inhibiting HCC metastasis.And,exploring its mechanism of inhibiting HCC used immunoblotting technology.Results(1)The HSA-ATRA-ATO nanoparticles solution is a fine,uniform and creamy yellow.After freeze-drying,they are dry,loose and uniform powders.The drug loading coefficient of ATRA is 3-5%,and the ATO is about 0.3%.The drug loading coefficient of ATRA is about 75%,and the ATO is about 60%.The shape is spherical with an average particle size of about 170 nm.Zeta potential is about +20m V.The ATRA releases effective of HSA-ATRA-ATO in DMEM + 5% Serum in vitro environment is for 20 days.(2)The pharmacokinetic results showed that HSA-ATRA-ATO has a high sustained-release efficiency.In the blood,the release of ATRA is prolonged for 18 hours.In the tissues,the distribution of ATO is flat,the peak of ATRA drug concentration is delayed by 3 hours.The pictures of Cell uptake show that HSA-ATRA-ATO is absorbed rapidly,and the absorbed amount within 24 h gradually increases with time.(3)In the acute toxicity animal experiment,there was no change in histology and hematology within 48 hours,and the mice behaved normally within 14 days.(4)MTT experiments show that HSA-ATRA-ATO nanoparticles have a better inhibition effect of metastasis,and it is determined that ATRA 10 μM and ATO 1μM are used for subsequent inhibition experiments.Metastasis experiments and lung metastasis models proved that the synergy of ATRA and ATO can exert a better inhibitory effect on HCC metastasis,and the performance of nanoparticles is more prominent than simple synergy.The changes of Pin1 and metastasis-related proteins are consistent with the trend of cell experiments.Conclusion In this project,HSA-ATRA-ATO nanoparticles prepared by the reduction and solvent removal method are stable,safe and non-toxic,effectively extend the half-life of ATRA in vivo,balance the toxicity of ATO tissue,and improve the bioavailability of ATRA and ATO.In HCC models,HSA-ATRA-ATO effectively enhances the synergistic inhibition of metastasis by ATRA and ATO,and affects the expression of Pin1 and EMT-related proteins in HCC.