| Objective:To reduce the toxicity of gambogic acid on normal cells and enhance its targeting performance,PVSG?Polyethylene glycol monomethyl ether-Valine-Citrulline-Cystine disulfide bond-Gambogic acid,mPEG-VC-SS-GA?was designed.As a dual sensitive polymer-drug conjugate,it can intelligently response to tumor micro-environment.With consideration that the bio-responsive materials may not effective enough,the single sensitive polymer-drug conjugate PSG?Polyethylene glycol monomethyl ether-Cystine disulfide bond-Gambogic acid,mPEG-SS-GA?was synthesized,and the gambogic acid solution?L-GA?was prepared at the same time as comparation.Method:PVSG were synthesized via disulfide bond and citrulline-valine fragments by a simple amide condensation method,then characterized by mass spectrometry,1H NMR,FT-IR and MALDI-TOF-MS.The melting point was determined by differential scanning calorimetry?DSC?.The particle size and potential were characterized by dynamic light scattering?DLS?.The morphology of particles was observed by transmission electron microscopy?TEM?.The saturated solubility and in vitro release profile were investigated by UV spectrophotometry.Cytotoxicity and mechanism were examined by MTT,AO-EB and flow cytometry.The protein adsorption was measured by fluorescence spectrophotometry.Results:The results demonstrated that PSG and PVSG were successfully synthesized.DSC showed that there was found no free gambogic acid in the polymer-drug conjugates.The average diameter of PSG-NPs was 117.80±9.27 nm with PDI in 0.315±0.049,when PVSG-NPs was 148.3±3.21 nm and with PDI in0.190±0.061.The potential of both particles was around 0.TEM showed the two kinds of particles were spherical with uniform morphology.In vitro drug release demonstrated that the cumulative release GA from PVSG-NPs at 7 h was81.95±3.93%while that of PSG-NPs was 39.92±0.84%.The saturated solubility of PVSG-NPs were 3.80 mg/mL,2.05 mg/mL,2.13 mg/mL,respectively in pure water,5%glucose and physiological saline,which were all more than 2000-folds higher than that of gambogic acid(7.25×10-4?4.44×10-4?4.58×10-4 mg/mL).MTT assay demonstrated that the IC50 of GA on HepG2 cell line were 2.930±0.166?M?24 h?,1.648±0.173?M?48 h?,0.676±0.038?M?72 h?,respectively.The IC50 of PSG-NPs on HepG2 cell line were 15.298±1.848?M?24 h?,13.573±3.229?M?48h?and 7.505±0.5?M?72 h?,respectively.While the IC50 of PVSG-NPs on HepG2cell line were 4.447±0.912?M?24 h?,1.074±0.143?M?48 h?and 0.651±0.086?M?72 h?,respectively.Flow cytometry showed that the apoptosis induced by PVSG-NPs was higher than that of GA.It mainly inhibited the growth of HepG2cells in G0/G1 phase.Protein adsorption test indicated that when the concentration was 32?g/ml?eq.GA?at 4h,the BSA adsorption rate of PVSG-NPs was 48.17±2.15%,while that of PSG-NPs and L-GA at the same concentration was 98.35±0.16%and 95.15±0.26%,respectively.Conclusions:Both PVSG-NPs and PSG-NPs had small particle size,high water solubility and good stealthy effect.Compared to PSG-NPs,the stability of PVSG-NPs was poorer at room temperature,but the in vitro release performance was much intelligent.The IC50 of PVSG-NPs on HepG2 cells was lower than that of GA at 48 h and 72 h.However,the toxicity of PVSG-NPs on normal cells was less than that of GA,especially at high concentration,showing excellent selectivity.As comparation,both the toxicity of PSG-NPs on three kinds of normal cells and HepG2 cells were not high.In addition,the biocompatibility of PVSG-NPs was better than that of GA and PSG-NPs,indicating that it was difficult to combine with BSA.These results showed that PVSG-NPs was better than PSG-NPs in response performance and biocompatibility.It may be a promising nano-drug carrier,providing thought for dual stimuli-responsive agents at the same time. |
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