| Objective RGD cyclopeptide and R8 peptide-modified Ergosterol and Cisplatin liposome with lung cancer active targeting:Preparation,Antitumor effect research in vitro and in vivo and Targeted delivery in transplanted tumor in vivo evaluation,which based on the research of A549 lung cancer cell resistance mechanism and Lewis tumor-bearing mice in vivo pharmacodynamics of Ergosterol combined Cisplatin.Methods First,in order to investigate whether it can exert synergy of anti-lung cancer and study its mechanism and inspect whether Ergosterol can reverse the drug resistance of Cisplatin,when Ergosterol and Cisplatin are combine applied.In the process of experiment,set ergosterol,cisplatin alone,and two drugs combination groups,proceeding A549 cell proliferation inhibition test(determined by MTT test),comparing each group of inhibition rate and IC50 value.Employing flow cytometry to determine the A549 lung cancer cell apoptosis rate under different concentration of each group at 48 h.According to the results determined by MTT test inspects A549 cell cycle after the each group treatment.After the experiment,through the protein immunoblot analysis(Western-blots)test detect the expression of cell apoptosis related proteins activated cysteine protease-3(cleved Caspase-3)and Cisplatin resistance related proteins poly adenosine diphosphate ribose transferase-1(PARP-1).Second,in order to prove the in vitro research in the body level and discuss tumor inhibition of Lewis lung cancer on ICR mice when given Ergosterol and Cisplatin,established body weight,tumor weight,inhibitory rate as indexs of tumor inhibitory effect,white blood cells and lymphocyte number,thymus and spleen index in blood routine examination as indexs of immunity,simultaneously proceeded the thymus of HE staining,observing the histopathologic characteristics,and determined the content of vascular endothelial growth factor(VEGF),vascular inhibition(AS)and endothelial inhibition(ES)in serum.Second,to carry out experiments on animals and to discuss tumor inhibition of Lewis lung cancer on ICR mice when given Ergosterol and Cisplatin,establishing body weight,tumor weight,inhibitory rate as indexs of tumor inhibitory effect,white blood cells and lymphocyte number,thymus and spleen index in blood routine examination as indexs of immunity,simultaneously proceeded the thymus of HE staining,observing the histopathologic characteristics,and determined the content of vascular endothelial growth factor(VEGF),vascular inhibition(AS)and endothelial inhibition(ES)in serum.Third,determined the optimum preparation technology of RGD and R8 peptides-modified Ergosterol combined Cisplatin targeted liposome.The first step is to establish the content determination methodology of Ergosterol and Cisplatin;The second step is to determined the liposome optimum film dispersion method preparation technology of ergosterol,which selected lecithin and ergosterol ratio(molar ratio),probe ultrasonic time,ergosterol drug loading for the three factors in the single factor test and the response surface design with ergosterol coating rate as the index.The third step is to determine the dialysis end point(The formation of the pH active drug-loading gradient)and methodology with electrical conductivity method,after confirmed ammonium chloride gradient method for cisplatin active drug-loading method.Ergosterol and Cisplatin concentration ratio,incubation time,incubation temperature as three factors to carry out the single factor and orthogonal experiment design,with cisplatin coating rate as the index,so as to determine the ergosterol and cisplatin liposome optimum preparation technology;then research on its quality for shape,encapsulation efficiency and drug loading,particle size and its distribution,Zeta potential,pH value and releasing rate.The fourth step is modifying the RGD cyclic peptide and R8 peptide through chemical synthesis method and purificating with semi-preparative high performance liquid chromatography,which characterized by MADLI-TOF-MS,then obtained the DSPE-PEG3400-c(RGDfk)and DSPE-PEG1000-R8,investigated the cytotoxicity by MTT method;The fifth step is preparation of RGD cyclo peptide and R8 peptide-modified Ergosterol combined Cisplatin active load medicine liposome drug delivery system;The sixth step is mixing RGD cyclo peptide and R8 peptide-modified,single modified,no modified liposome with fetal bovine serum in the ratio of 1:1(v:v)and determination its serum stability at different time point;The seventh step is to determine the cell uptake rate of each liposome by flow cytometry and to qualitative observe the A549 cell intakes through laser scanning confocal microscope and the penetration of A549 tumor ball;The eighth step is to determine the inhibition rate of A549 cells after given different liposome by MTT test.Finally,the first step,injected RGD cyclo peptide and R8 peptides modified,single modified or no modified Ergosterol combined Cisplatin liposome in the caudal vein of nude mouse bearing the tumor,observing the body distribution and targeting of each group under the different time points through small animals living imager.The second step,continuously,dose every other day for 14 days,observating the weight of mice and the tumor growth situation.The animals were drawed blood and then were put to death,removing the tumor,the spleen and the lung tissue of all the mice.As the indexs of the tumor weight,the tumor suppression effect,the level of TGF-β1、TIMPs、TNF-α in serum,the spleen index and changes of the tumor and lung tissue,investigate the tumor suppression effect in mice of the liposomes preliminary.Results 1.When compared with equal concentration of Ergosterol and Cisplatin single used groups(10 μg·mL-1),the inhibition rate to A549 cell of combination group had a certain degree of increasing,which shows that Ergosterol and Cisplatin combination group had significantly enhanced sensitivity to cisplatin of A549.The result of Hoechst 33258 dyeing shows that the control group of A549 cells emit blue fluorescence uniformly with no obvious morphological change of the nucleus;the apoptotic cells in experimental group,however,appeared hyperchromatic nuclei,fracture,emiting strong fluorescence.The number of apoptosis cells appeared in Ergosterol(12.5 μg·mL-1)and Cisplatin(10 μg·mL-1)combination group significantly more than two single given groups.2.According to in vivo animal experiment data,it can be obtained that Ergosterol has inhibitory effect on Lewis lung tumor-bearing mice(P<0.01),and has synergistic inhibitory with Cisplatin on tumor(P<0.05).Therefore,it can comes to that Ergosterol can enhance the sensitivity of Cisplatin chemotherapy,and play a role of synergy on Lewis lung cancer.3.The result of Ergosterol liposome optimum preparation technique revealed that the mole ratio of lecithin and cholesterol is 5:1,the probe ultrasound time is 20 min and the drug loading of Ergosterol is 10%.The result of Cisplatin liposome optimum preparation technique revealed that concentration of Cisplatin is 0.150 mg·mL-1,the incubation temperature is 50℃ and the incubation time is 10 min.After explorating high performance liquid chromatography(HPLC)conditions for modified peptides and purificating it,analysing the purified samples through MALDI-TOF-MS,which identified that the molecular weight of DSPE-PEG1000-R8 and DSPE-PEG3400c(RGDfk)peptides was 3015.6 and 4751.8,respectively.Preparing RGD and R8 peptides-modified liposome based on the mole ratio of SPC,Chole,DSPE-PEG1000-R8,DSPE PEG3400-c(RGDfk)is 5:1:0.07:0.07.The liposome quality evaluation results show that the shape of it is round with double-layer structure and the particle size distribution is uniform.The average particle size of liposome without modified is 153.4 nm,the dispersion coefficient of PDI is 0.156,and the Zeta potential is-10.9 mV;the average particle size of liposome with RGD peptide modified is 156.7 nm,the dispersion coefficient of PDI is 0.164,and the Zeta potential is-1.29 mV;the average particle size of liposome with R8 peptide modified is 154.3 nm,the dispersion coefficient of PDI is 0.178,and the Zeta potential is 0.661 mV;the average particle size of liposome with RGD and R8 peptides modified is 155.2 nm,the dispersion coefficient of PDI is 0.102,and the Zeta potential is 4.74 mV,which shows that the particle size distribution of each group of liposome is relatively concentrated.The drug loading and the encapsulation efficiency of Ergosterol in Ergosterol liposome prepared by the second step is 10%and 90.49%,respectively;the drug loading and the encapsulation efficiency of Cisplatin in Cisplatin liposome prepared by the third step is 2.44%and 52.24%,respectively;the pH value and peroxide value of liposome solution is 6.64 and 0.1095,respectively,the accumulated release rate of Cisplatin is more than 80%at 24 h.The liposomes are stable in the serum.The liposomes have obvious inhibition function to A549 lung cancer cells,and liposome modified with RGD and R8 peptides shows the strongest inhibitory effect,RGD modified liposome is second,and no modified liposome is third.Since A549 was given liposomes,the cell uptake rate is highest at 4 h and RGD and R8 peptides-modified liposome has the highest cell uptake rate combined with the others,which also presenting the concentration dependence.The experiment results show that the intake by A549 cells of liposomes is mainly by the endocytosis pathway mediated by clathrin.4.Finally,the targeting result of tumor-bearing nude mice displays that the fluorescence intensity of RGD and R8 peptides modified liposome is the highest and the targeting is most obvious under high concentration and other group of liposome are weaker.Preliminary pharmacodynamics results show that each dosage group of mice have no obvious change in body weight and the high and middle dose group of RGD and R8 peptides-modified liposome has tumor suppression effect obviously.The high dose group of RGD and R8 peptides modified liposome is the most significant,up to 46.76%.It has high expression of cytokines(TNF-α)in serum.The spleen index of middle and low dose group of RGD and R8 peptides-modified liposome significantly increased compared with positive medicine group.Conclusion This paper completed the resistance mechanism to A549 lung cancer cells and in vivo pharmacodynamics experiment of Lewis tumor-bearing mice which confirmed that two drug combination have synergy,and Ergosterol can partly reverse the Cisplatin resistance.Meanwhile,this paper successfully developed the RGD cyclo peptide and R8 peptide modified Ergosterol combined Cisplatin targeting liposome drug delivery system,further improved the anti-lung cancer effect and tumor targeting in vitro and vivo. |
