The Study Of Disulfiram-loaded PH-triggered PEG-shedding TAT-modified Lipid Nanocapsules

Disulfiram(DSF),which exhibits marked tumor inhibition mediated by copper in tumor cells,has been assessed as a potential antitumor drug.However its water insolubility and instability in stomach and blood both become the main limited factors in the clinical treatment of DSF.In this study,DSF was encapsulated in lipid nanocapsules modified with TAT peptide(TATp)and pH-triggered sheddable PEG to PEG sheddable DSF lipid nanocapsules(DSF-S-LNCs)for improving the stability in vivo and targeting cancer cells based on tumor environmental specificity.DSF-LNCs with particle size at 20-100 nm was prepared through phase-inversion method.The proportion of water phase(20-80%),oil phase(10-30%)and surfactant(10-50%)was determined to form DSF-LNCs.DSF-LNCs showed the characteristics of sustained release,the rate of release accelerated with the decreasing of particle size.DSF-LNCs,whose drug loading was 4%,exhibited good stability when stored at 4℃.HS-PEG1000-TATp and PGA-g-PEG2000,which contained TAT peptide and pH sensibility,were synthesized and verified by 1H-NMR and MS.Through post-insertion method and electrostatic adsorption method,PEG sheddable lipid nanocapsules(S-LNCs)were fabricated from LNCs by decorating short PEG chains with TATp(HS-PEG1000-TATp)to form TATp-LNCs,and then covered by pH-sensitive graft copolymers of long PEG chains(PGA-g-PEG2000).DSF-S-LNCs was negatively charged(-2.17～-40.13 mV)with particle size ranging from c.a.60 to 90 nm,exhibited sustained release and good stability in plasma.DSF-S-LNCs was pH sensitive and shed PEG layer and completely exposed TAT peptide with a positive potential(about+5 mV)under the condition of pH 6.5.Copper-enriched Hep G2 cells was cultivated to verify whether the cytotoxicity of DSF was dependent on the accumulation of copper ions within tumor cells.DSF-S-LNCs showed better uptake ability of tumor cells under pH 6.5 condition than pH 7.4,so as the enhanced cell apoptosis and cytotoxicity(pH 6.5 IC50=1.291 μM,pH 7.4 IC50=6.144 μM).Pharmacokinetic studies showed the markedly increased bioavailability of DSF-S-LNCs(AUC=3921.391 mg/L·h,t1/2z=1.294 h)compared with free DSF(AUC=907.724 mg/L·h,t1/2z=0.252 h).The in vivo distribution of S-LNCs was investigated using Cy5.5 as a fluorescent probe.In tumor-bearing mice,the delivery efficiency of S-LNCs was found to be 496.5%higher than free Cy5.5 and 74.5%higher than LNCs in tumors.In vivo antitumor efficiency test in H22 tumor bearing mice,combinations of copper gluconate p.o.and saline,DSF-sol,DSF-LNCs and DSF-S-LNCs i.v.were evaluated.The TIRs of DSF-sol,DSF-LNCs and DSF-S-LNCs were 5.34%,43.22%and 80.68%%respectively,showed that DSF-S-LNCs owned better tumor suppressing effect.The preliminary experiments indicated that DSF-S-LNCs could enhance DSF antitumor effect while reduce its toxicity to liver.