| Objective:Researches nowadays have reported many ligands modified liposomes which used galactose,folic acid,transferrin or lactoferrin as a target head.Compared to conventional liposomes,these ligands modified liposomes had significantly targeting effect on cancer cells.The galactosylated modified liposome was studied the most of all the ligands modified liposomes.These galactosylated modified liposomes can be specifically recognized by the asialoglycoprotein receptor(ASGPR),and then they will be endocytosed by cells.ASGPR is a high-efficient and specific receptors and it is highly and only expressed on the surface of hepatoma cells.Therefore,the liposomes which have the terminal of galactose residues can be an ideal carrier for targeting treatment of liver cancer.It can enhance the drug uptake by hepatoma cells.However,the galactosylated modified liposomes reported by the researches now were not stable in plasma and the loading drugs were easy to leak from the liposomes,resulting in burst release of drug and reducing drug effect.Fortunately,it has been proved that a hydrophilic protective layer will form on the surface of liposomes after liposomes are modified with long-circulating material-polyethylene glycol(PEG).This protective layer can prevent liposomes interaction with serum opsonins and fast phagocytosis of liposomes by the reticuloendothelial system(RES),thus increasing the stability of liposomes in plasma and prolonging the retention time of liposomes.In addition,PEG modified liposomes can reach the cancer cells nearby by the enhanced permeability and retention effect(EPR effect),result in enhancing the probability of drug uptake by cancer cellsDocetaxel(DOC)is a semisynthetic anti-cancer drug derivative from 10-deacetyl baccatin III,which is extracted from the needles or barks of Taxus baccata.It has been demonstrated that DOC has strong antitumor activity for hepatoma cells in vitro.It can increase the apoptosis of hepatoma cells and cause the cell cycle stay in G2/M phase.But it has no effect on patients with hepatocellular carcinoma.The II period clinical trials showed that DOC did not appear to be safe and effective enough in patients with advanced hepatocellular carcinoma.The main cause is that DOC is lack of selectivity for hepatoma cells in vivo.From above,our study chose DOC as a model drug and constructs a liposome(CG-SSL-DOC)which was anchored a long-circulating material of DSPE-PEG2000 and a liver targeting ligand of cholesterol gatactosyl derivate(Chol-Gal)simultaneously.There are two purposes of this study.First,CG-SSL-DOC can increase the stability of liposomes in plasma,resulting in avoiding the burst release of loading drugs and prolonging the retention time of liposomes.Second,CG-SSL-DOC can enhance the DOC uptake by hepatoma cells and achieve targeting delivery of DOC by the ASGPR-mediated endocytosis,thus enhancing its antitumor efficacies for hepatocellular carcinoma and reducing the dosage of DOC in patients.Methods:1.The pharmaceutical study of sterically stabilized docetaxel liposomes(SSL-DOC)(1)The preformulation of SSL-DOC was studied,including the establishing determination method of DOC content and determining the oil-water partition coefficient of DOC.HPLC method was used to determine the DOC content and the feasibility of HPLC method was investigated.Shake-flask method and water-octanol system were used to measure the partition coefficient of DOC.(2)The preparation and preparation technology parameters optimizing study on SSL-DOC were carried out.First,the entrapment efficiency of liposomes was investigated to select the preparation methods of liposomes.Then,the effect of various factors on entrapment efficiency of liposomes was investigated through the single factor experiment.Finally,the preparation of liposomes was further optimized by the orthogonal design.(3)The lyophilization study on the SSL-DOC was carried out.Freeze drying was used to prepare lyophilized SSL-DOC.The effect of various critical factors of lyophilization on entrapment efficiency and particle size of liposomes was investigated through the single factor experiment.(4)The thin-film dispersion method was used to prepare SSL-DOC,and their pharmaceutical properties were evaluated.The appearance of liposomes was observed by naked eyes and the transmission electron microscope was used to observe the morphology of liposomes.Then,Sephadex gel method was used to measure the entrapment efficiency of liposomes and the particle size analyser was used to determine the particle size and zeta potential of liposomes.Finally,the dialysis method was used to detect the in vitro release characteristic of liposomes and the oxidation product value of liposomes was detected by TBA-reaction method.(5)According to the stability experiment guiding principles of preparations,the study on the stability of SSL-DOC was carried out,including influence factor experiment,accelerated experiment,long-term experiment and compatibility experiment.Influence factor experiment:SSL-DOC was put into high temperature(40?),high humidity(RSH 75%±5%)and strong light(45001x±5001x)conditions for 10 days to investigate the change of pharmaceutical properties of liposomes.Accelerate experiment:SSL-DOC was put into the conditions of 25 ?±2 ?,RH60%± 10%for 6 months to investigate the change of pharmaceutical properties of liposomes.Long-term experiment:SSL-DOC was put into 4? for 12 months to investigate the change of pharmaceutical properties of liposomes.Compatibility experiment:SSL-DOC was diluted by 21 kinds of infusion solutions and then put in the room temperature(25 ?)for 24h to investigate the change of pharmaceutical properties of liposomes.2.The enzymatic synthesis and structural identification of Chol-Gal,and the preparation and pharmaceutical properties evaluation of CG-SSL-DOC(1)Chol-Gal was synthesis by enzymatic catalyze method.MS and NMR were used to identify the structure of Chol-Gal.(2)The thin-film dispersion method was used to prepare CG-SSL-DOC,and its pharmaceutical properties were evaluated.The appearance of liposomes was observed by naked eyes and the transmission electron microscope was used to observe the morphology of liposomes.Then,Sephadex gel method was used to measure the entrapment efficiency of liposomes and the particle size analyser was used to determine the particle size and zeta potential of liposomes.Finally,the dialysis method was used to detect the in vitro release characteristic of liposomes and the oxidation product value of liposomes was detected by TBA-reaction method.3.The safety study,in vitro anti-hepatoma cells study and pharmacokinetic study of liposomes(1)According to the Chinese pharmacopoeia method,we did the pyrogen and hemolysis tests of SSL-DOC and CG-SSL-DOC,and the DOC-I was used as reference preparation.Subsequently,the acute toxicity test of SSL-DOC and CG-SSL-DOC in KM mice was determined by median lethal dose method and the LD50 value was calculated by SPSS software.The toxicity of different DOC preparations in the mice was evaluated through comparison their LD50 value.(2)The MTT method was used to determine the proliferation inhibition effect of different DOC preparations on HepG2 cells,A549 cells and Hela cells.The in vitro anti-tumor effect of different DOC preparations was evaluated by comparison their IC50 value.Subsequently,Coumarin-6 was used as a fluorescent indicator and the coumarin-6 long-circulating liposomes(SSL-Coumarin-6)and Chol-Gal modified SSL-Coumarin-6(CG-SSL-Coumarin-6)were prepared.Fluorescent microplate reader was used to quantitative analysis of the binding rate between the cells and liposomes,and the fluorescent microscope was used to qualitative analysis of cellular uptake of liposomes.(3)The pharmacokinetic rule of SSL-DOC and CG-SSL-DOC in newzeland rabbits was investigated,and the DOC-I was used as reference preparation.The paclitaxel(PTX)was used as internal standard,methyl tert-butyl ether was used as extraction solvent and the LC-MS/MS method was used to detect the DOC concentration in plasma.The pharmacokinetic characteristics of different DOC preparations were evaluated through comparison their pharmacokinetic parameters which were analyzed by DAS 2.0 software.Results:1.The pharmaceutical study of SSL-DOC(1)First,the HPLC method for determining DOC content was established.The result showed that the specificity,linearity precision,recovery and stability of this method were conformed to the requirement,and also this method was stable and feasible.Second,the result showed that the oil-water partition coefficient of DOC was 3.17±0.32,which implied that DOC was a strong lipophilic drug.(2)According to the result,thin-film dispersion method was chose to prepare liposomes.The optimal incubation temperature and probe ultrasonic time were 50? and 10 min,respectively.Through single factor experiment,we found that factors which had significant effects on entrapment efficiency of liposomes were the mass ratio of EPCS and CHO-HP,the mass ratio of drug and lipids and the amount of DSPE-PEG2000.Besides,the results of orthogonal design showed that the optimal preparation of SSL-DOC was EPCS to CHO-HP mass ratio of 3:1,drug to lipids mass ratio of 1:15 and the DSPE-PEG2000 of 2%(mol%).(3)The results of lyophilization study of SSL-DOC showed that sucrose and mannitol together were chosen as cryoprotectant.The liposomes exhibited the most satisfactory results when adding mannitol to sucrose mass ratio of 1:1 and cryoprotectant to lipid mass ratio of 8:1.Besides,the optimal freeze drying technology parameters were freezed in-80? for 2h in a fast freezing method.The appearance and surface of lyophilized SSL-DOC were full and smooth,respectively.There was no significant difference between lyophilization and non-lyophilization in entrapment efficiency and particle size of liposomes.(4)The pharmaceutical properties result showed that the appearance and surface of SSL-DOC were full and smooth.The morphology of SSL-DOC was spheroids with regular shape and a size of 100-200nm.Besides,Sephadex gel method was stable and feasible for measuring the entrapment efficiency of liposomes.The entrapment efficiency of SSL-DOC was 92.70±2.05%,particle size was 156.70±2.26 nm,PDI was 0.14±0.01 and Zeta potential was-21.70±3.57 mV.The oxidation product value and pH value of both liposomes were conformed to the requirement.(5)The results of influence factor experiment showed that the particle size,oxidation product value and leakage rate of SSL-DOC increased after it was put into high temperature and strong light conditions for 10 days.While the reconstituted time and leakage rate of SSL-DOC increased after it was put into high humidity conditions for 10 days.Besides,the results of accelerated experiment showed that the external conditions had no influence on the appearance,reconstituted time,zeta potential,pH value and oxidation product value of SSL-DOC,but increased the leakage rate and particle size of SSL-DOC.In additions,the results of long-term experiment showed that SSL-DOC was stable in 4? for 9 months.But it was abnormal after 12 months.Finally,the results of compatibility experiment showed that all the properties of SSL-DOC was normal when it diluted by 14 kinds of infusion solutions,except salinger lactate injection,salinger lactate and glucose injection,potassium chloride injection,potassium chloride and glucose injection,fat enmulsion injection,dextran 40 glucose injection and dextran 40 sodium chloride injection.2.The enzymatic synthesis and structural identification of Chol-Gal,and the preparation and pharmaceutical properties evaluation,of CG-SSL-DOC(1)The yield and purity of Chol-Gal were above 95%,respectively.The structure of Chol-Gal was confirmed through the MS,1H-NMR and 13C-NMR method.(2)The pharmaceutical properties result showed that the appearance and surface of CG-SSL-DOC were full and smooth.The morphology of CG-SSL-DOC was spheroids with regular shape and a size of 100-200nm.The entrapment efficiency of CG-SSL-DOC was 92.54±2.74%,particle size was 157.47±1.37 nm,PDI was 0.18±0.01 and Zeta potential was-23.90±4.93 mV.The oxidation product value and pH value of both liposomes were conformed to the requirement.What's more,the dialysis method can be used to detect the in vitro release rule of liposomes and PBS 7.4 containing 0.5%Tween-80 was chosen as the release medium.The release characteristic of SSL-DOC and CG-SSL-DOC was similar to Higuchi release model,which indicated that they showed the sustain release effect.3.The safety study,in vitro anti-hepatoma cells study and pharmacokinetic study of liposomes(1)The results of hemolysis tests showed that SSL-DOC and CG-SSL-DOC did not cause hemolysis,while DOC-I can cause hemolysis.Besides,three DOC preparations did not cause pyrogen phenomenon.The results of acute toxicity test showed that the LD50 value of SSL-DOC and CG-SSL-DOC were 1.63-fold and 1.66-fold compared with that of DOC-I,respectively,which indicated that the in vivo safety of two liposomal preparations was higher than that of DOC-I.(2)The results of MTT experiment showed that the proliferation inhibition effect of CG-SSL-DOC on HepG2 cells improved 3.80-fold and 9.06-fold compared with that of SSL-DOC and DOC-I,respectively.However,there was no significant difference between the proliferation inhibition effect of three DOC preparations on A549 cells and Hela cells.Besides,the results of cellular uptake experiment showed that the amount of CG-SSL-Coumarin-6 uptook by HepG2 cells was 1.77-fold compared with that of SSL-Coumarin-6.It was suggested that Chol-Gal can improve the uptake amount of liposomes by HepG2 cells.This was dependent on the endocytosis mediated by ASGPR which expresses on the surface of HepG2 cells.(3)In the pharmacokinetic study,the LC-MS/MS method for determining DOC concentration in plasma was established,and this method was stable and feasible.The analysis results of pharmacokinetic parameters showed that the AUC and half-time of two liposomal preparations were significantly higher than that of DOC-?,but their clearance rate were significantly lower than that of DOC-?.Besides,CG-SSL-DOC was eliminated fastly with a shorter half-time,compared with SSL-DOC.This phenomenon may be related to the targeting of CG-SSL-DOC to hepatocyte.Conclusion:In conclusion,our CG-SSL-DOC showed high entrapment efficiency and uniform distribution of particle size.Also,it had sustain release effects and good stability in vitro.Through the pharmacokinetic study,we found that CG-SSL-DOC released slowly and had long-circulating effect in vivo.Meanwhile,the anti-hepatoma cells study in vitro proved that CG-SSL-DOC can significantly enhance the DOC uptake by hepatoma cells and significantly improve the proliferation inhibition effect of DOC for hepatoma cells through the ASGPR-mediated endocytosis.We have reason to believe that CG-SSL-DOC could be a novel drug delivery system for targeting treatment of liver cancer in the future. |
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