Annonaceous Acetogenins Nanosuspensions:Preparation And Preliminary Evaluation In Vitro And In Vivo

Annonaceous acetogenins(ACGs)constitute a series of natural products isolated exclusively from Annonaceae species,which show significant cytotoxicity in 13 kinds of human cancer cell lines in vitro,such as the breast carcinoma,liver cancer,lung carcinoma and cervical carcinoma.Due to the excellent antitumor activities,annonaceous acetogenins have been attracting worldwide attention as another anticancer drug after taxol.It was reported that Bullatacin was identified to exhibit 100 times potency than DOX against the growth of breast carcinoma cells,10-100 times potency than 5-Fu against the growth of liver cancer cells.However,due to the poor water solubility and then difficult to be delivered in vivo,serious side effect and narrow therapeutic window,ACGs are limited in the further clinical application.Nanosuspensions are nearly pure drug nanoparticles stabilized by very small amount of surfactants.Nanosuspensions can effectively tackle the poor solubility of hydrophobic drugs and then difficult to be administrated,can also display tumor targetability due to the EPR effect after i.v.administration,thus is expected to be able to crack the bottleneck preventing ACGs from the further antitumor application.In the study,antisolvent precipitation method was used to prepare ACGs nanoparticles.And the effect of precipitation conditions,tempereture,stabilizer type and drug-stabilizer ratio were investigated separately in order to obtain stable ACGs nanosuspensions of small size.The optimized formulation and process conditions include a precipitation method at 25℃ ultrasonication,using PEG2000-PCL2000 as the stabilizing agent and the drug-stabilizer ratio of 3:1.The resultant ACGs nanosuspensions had high drug payload of 73.68 ± 0.12%and are only 123.2±3.54 nm in diameter with PDI value of 123.2±3.54 and Zeta potential of-25.0±0.21 mV.ACGs nanosuspensions were evaluated in vitro and in vivo.The obtained ACGs nanoparticles were spherical by TEM observation and could persistently release encapsulated drug within 96 h in vitro.In MTT assay,ACGs nanosuspensions showed stronger antitumor activity than free ACGs against MCF-7(0.273μg/mL vs.0.103μg/mL,IC50),HeLa(0.127μg/mL vs.0.060μg/mL,IC50),MDA-MB-231 and A549 cell lines.The near-infrared(NIR)fluorescence probe labelling and in vivo real-time imaging displayed thatACGs nanosuspensions were mainly distributed in the liver,spleen,and tumor in mice,suggesting nanosuspensions did realize tumor target ability to some extent.The in vivo antitumor tests using hepatocellular carcinoma H22 demonstrated that ACGs nanosuspensions(0.4mg/kg,i.g.)could reach the similar tumor inhibition effect to ACGs oil solution(4mg/kg,i.g.),indicating that nanosuspensions could decrease the oral dose to 1/10.When i.v.administrated,ACGs nanosuspensions at 0.4mg/kg showed significantly better tumor inhibition than high dose of oil solution(4mg/kg,traditional oral dosage form)in both H22-bearing(67.17%vs 49.74%)and 4T1-bearing(74.83%vs 45.53%)mice models.These results confirmed that nanosuspensions could effectively solve such problems as insolubility,difficult to be administrated that ACGs met,meanwhile can be expected to achieve enhanced efficacy and reduced side effect.The above research lays solid foundation for ACGs’ further investigation and future clinical application.