Panaxanthin Nano-targeting Preparation And Anti-tumor Synergy Study

Annonaceous acetogenins(ACGs)are a series of natural substances with a long aliphatic lactones chain isolated species of the Annonaceae family.ACGs have attracted much attention due to their strong antitumor activity,unique pharmacological mechanism and the ability to reverse multiple drug resistance in many tumor cell lines.However,their clinical apply has been restricted by their poor solubility,complex compositions,severe toxicity together with the narrow therapeutic window.Nanosuspensions(NSps)can effectively tackle the poor solubility problems of many hydrophobic drugs,and can also display passive tumor targetability due to the EPR effect after i.v.administration.Moreover,it can also realize active tumor targetability through modifying the ligand on the surface of nanosuspensions,which is hopeful to break the bottleneck of putting ACGs into use.The first part was to preparing ACGs nanosuspensions(ACGs-NSps)using hydroxypropyl-?-cyclodextrin(HP-P-CD)and soya lecithin(SPC)as supramolecular self-assembly stabilizer and then examined their in vitro and in vivo characteristics.ACGs-NSps were successfully prepared through a precipitation-stirring method with a mean particle size of 144.4 nm,a PDI value of 0.08 and a zeta potential of-22.9mV.ACGs-NSps could in vitro release encapsulated drug in a nearly first-order kinetics manner and the accumulative release reached 86.38%within 72 h.ACGs-NSps also demonstrated good stability in simulated gastrointestinal fluid as well as plasma,anhemolytic below 2mg/mL and met the demand of both intravenous injection(iv)and oral administration.The best chosen cryoprotectant was 0.5%(w/v)lactose.The MTT assay indicated that the ACGs-NSps was more potent against tumor cell lines and demonstrated significantly increased cytotoxicity against HeLa ? HepG2 compared to ACGs solution—the IC50 of ACGs solution was 13.39 and 1.31 times of ACGs-NSps respectively.An in vivo anti-tumor activity in H22-tumor bearing mice displayed that when orally administered,ACGs-NSps achieved a similar tumor inhibition rate at only 1/10th the dose of ACGs in an oil solution(P>0.05).Improved therapeutic efficacy was further achieved when ACGs-NSps were intravenously injected compared to ACGs oil solution(70.31%vs 49.74%,P<0.05).The second part was to conjugate folic acid(FA)molecules with ?-cyclodextrin in order to prepare a FA mediated active tumor-targeting ACGs nanosuspensions.In this study,FA-?-CD was successfully synthesized and then combined with SPC to prepare a FA modified ACGs nanosuspensions(FA-ACGs-NSps).The resultant FA-ACGs-NSps were spherical with the mean particle size of 199.5 nm and the PDI value of 0.07.The in vitro release of FA-ACGs-NSps displayed a two-phase release behavior in PBS(0.1 mol/L,pH=7.4)which followed a nearly Higuchi kinetics manner and exhibited a sustained release until 146 h(93.05%).The cellular uptake of FA-ACGs/Cy5.5-NSps by 4T1 cells(FA receptor positive)was significantly higher than that of ACGs/Cy5.5-NSps(P<0.00).In MTT assay,FA-ACGs-NSps showed significantly enhanced cytotoxicity against 4T1 cells compared to ACGs-NSps,but no significant difference against A549 cells(FA receptor negative).The in vivo real-time imaging displayed that FA-ACGs-NSps could enhance tumor accumulation(P<0.05)and tumor targeting efficiency(P<0.001).An in vivo anti-tumor activity and toxicity assessment in H22-tumor bearing mice displayed that FA-ACGs-NSps showed a superior antitumor therapeutic efficacy over ACGs-NSps(68.76%vs 41.82%,P<0.05)at the same dose of 0.4 mg/kg with no abnormalities in spleen index and serum biochemical indices.The third part was to prepare FA mediated active tumor targeting and long-circulated ACGs nanosuspensions(FA-PEG-ACGs)using FA-PEG-DSPE as stabilizer.The finally determined ratio of ACGs/FA-PEG-DSPE/SPC was 2:1:1(w/w/w).The resultant FA-PEG-ACGs had a mean particle size of 119.7 nm,a PDI value of 0.20 and a zeta potential of-23.0mV.FA-PEG-ACGs was quite stable in 0.9%NaCI,5%Glu,PBS as well as plasma.The in vivo pharmacodynamics against HeLa-tumor bearing mice demonstrated that FA-PEG-ACGs had an outstanding tumor suppression effect,with a tumor inhibition rate over 70%during drug withdrawal period after 7 times of intravenous injection and no mice was died.The acute toxicity experiment demonstrated the LD50 of FA-PEG-ACGs was 1.742mg/kg,and the toxic symptom together with death rate of mice was positive correlated with the dose of FA-PEG-ACGs.In conclusionn,ACGs nanosuspension based on cyclodextrin and SPC self-assembly stabilizer could solve the problem of the poor solubility and difficuty in drug administration of ACGs.Moreover,ACGs nanosuspensions mofidied with FA and PEG could further enhance the tumor targetability of ACGs and meanwhile decrease the distribution to the non-target organs like liver and spleen to finally achieve the purpose of toxicity reduction and efficacy enhancement.The above research lays solid foundation for ACGs' further investigation and future clinical application.