Tumor-targeted Polymeric Micelle For The Delivery Of Poisonous Traditional Chinese Medicine CTD

Poisonous traditional Chinese medicine has been historically used in malignant tumor therapies in China following the principle of “fighting fire with fire”.Blister beetle(Mylabris phalerata)is a highly poisonous traditional chinese medicine.Cantharidin(CTD),the major bioactive compound in Mylabris,has been long acknowledged for its anti-tumor activity and it has potent and selective inhibitive ability on serine/threonine protein phosphatases 1 and 2A(PP1 and PP2A),but it has a narrow therapeutic window and would cause serious damage on gastrointestinal system and urinary system in case as overdose.Polymeric micelles system,as an efficient drug carrier,has received growing scientific attention in recent years owing to its solubilization,selective targeting,avoiding the recognition by macrophages and prolonging the circulation in the bloodstream.It is very meaningful to use polymeric micelle to deliver cantharidin and it was expected that the antitumor activity of CTD was improved and the side effect was reduced.Building polymeric micelle system to delivery CTD.Group contribution method was used to calculate the partial solubility parameters of the polymers and drug and the polymer-drug compatibility was predicted and compared,then the best polymer was chosed.The molecular weight of polymer and preparation method was evaluated to optimize the prescription of CTD micelle.CTD has a good compatibility with PLGA and then mPEG2000-PLGA2000 was selected as the carrier material.Using film dispersion method to prepare CTD micelle.The entrapping effiency and loading capacity were(89.88±1.01)% and(4.30±0.05)%,and the particle size and PDI was(25.32±1.25)nm and(0.08±0.02).Characterization of the CTD micelles.The morphology of micelles were observed by Transmission electron microscope.The CTD micelles were made of uniform spherical nano-sized particles and had a good dispersity.CTD was distributed in carrier with the amorphous form by DSC and XRD analysis.CTD micelles were stable at-4? for 20 days without the leakage of the drug.When CTD micelles were incubated with 0.9%Na Cl,200 U heparin and 0.1 mg/m L BSA at 37? for 24 h,the particle size of the micelles was not changed.Also,the micelle formulatios can improve the stability of CTD in plasma and the blank micelles and CTD micelles would not cause hemolysis.Drug release profile of CTD from micelles was studied with the dialysis method and the release manner was suited for first-order equation.The antitumor effect of CTD micelles.The anti-proliferation effects of blank micelles and CTD micelles on human hepatoma cells Hep G2 and human breast cancer celle MCF-7by MTT assays.The result showed that blank micelles has a good biocompatibility without significant inhibitory effect.When Hep G2 and MCF-7 were incubated with free CTD and CTD micelles for 48 h,the IC50 values of free CTD were(2.57±0.25)?g/m L,(2.50±0.18)?g/m L and the IC50 values of CTD micelles were(1.75±0.20)?g/m L,(2.10±0.13)?g/m L.Apoptotic nuclear morphology was assessed by Hoechst 33342 staining and AO/EB staining.The morphological changes of cell nuclei indicated that the apoptosis-inducing effects of CTD might be significantly enhanced by micellar formulation.Murine hepatocarcinoma tumor(H22)model was used to evaluate the therapeutic effect of CTD micelles in vivo.The mice in randomly divided groups were treated respectively with N.S(0.1m L/10 g).,cyclophosphamide(CTX)(20 mg/kg),free CTD and CTD micelles at an equivalent CTD dose of 400?g/kg i.v.on day 1,3,5,7,9and 11.Tumor size was monitored and calculated every day since the tumor can be touched..At the end of experiment,all the mice were sacrified and all the tissues except lung were weighted.tumor tissues were analyzed by hematoxylin-eosin(HE)staining assay.The tumor inhibiton rates of CTD and CTD micelles were 44.40%,28.05%,Tumor pathology showed that CTD micelles cause bigger tumor necrosis area than CTD,and nuclear condensation or fragments could been seen everywhere.Toxicity reducing study of CTD micelles.The anti-proliferation effects of blank micelles and CTD micelles on human normal hepatocyte L-02 by MTT assays.When L-02 was incubated for 24 h,the IC50 values of free CTD and CTD micelles were(2.12±0.22)?g/m L,(2.64±0.15)?g/m L.These results indicated that micelle formulations can improve the inhibitory effects of CTD on tumor cells and reduce the inhibitory effects on normal cells.Spleen index and kidney index were calculated.The collected kidney tissues were analyzed by hematoxylin-eosin(HE)staining assay.Serum biochemical indexes related to the renal function was measured.The result showed that compared to model group,Spleen index were reduced significantly in free CTD group,and CTD micelles group had no different with model group.33.3% of kidney indexs of mice in free CTD group were beyond the normal range.All the kidney indexs of mice in CTD micelles group were in the normal range.Kidney pathology showed that in free CTD group,some glomus was shrinked and Bowman's capsule cavity was largened and some glomerulis' volume was magnified and kidney tubules swelled.CTD micelles group had no difference with the model group.CTD micelles could lower the content of uric acid to normal level.Cell uptake of polymeric micelle.Using Coumarin 6 as a model drug to build C-6 micelle to study the cell uptake of the polymeric micelle.Qualitative study using fluorescence microscope and quantitative study using fluorescence spectrophotometer and high content screening were performed.All the results indicated that agents encapsulated in mPEG2000-PLGA2000 could enhance cellular internalization.In conclusion,a polymeric micelle system have been constructed for delivery of CTD.In vitro and in vivo studies all demonstrated that micellar formulation can improve the antitumor effect of CTD and reduce its toxicity as well.