| Objective:To prepare liposomes loaded Paclitaxel-cholesterol complex(PTXL),explore the effect of PTXL on the proliferation,invasion,apoptosis and cycle of human keloid fibroblasts(HKFs),and investigated the effect of PTX on the expression of Akt/GSK3 β and fibrosis in keloids,establish nude mice subcutaneous keloid model and examine the inhibition in keloid growth with the treatment of PTXL.Method:(1)PTXL were prepared by film-ultrasound dispersion metho;(2)The PTXL prescription was optimized by single factor investigation and orthogonal experiment method;(3)the characterization of PTXL,such as particle size,zeta potential,morphology,drug loading,encapsulation efficiency,in vitro release,stability and leakage rate within 30 days were evaluated by high performance liquid chromatograph(HPLC),dynamic light scattering(DLS)and transmission electron microscope(TEM),respectively.(4)PTX was replaced by coumarin 6(C6)to prepare C6 liposomes(C6L),to investigate the uptake rate and mechanism of PTXL by flow cytometry and laser confocal experiment on HKFs;(5)CCK-8 experiment was conducted to detect the cytotoxicity of liposome materials,and evaluate the inhibition of PTXL on on the proliferation of HKFs;(6)Scratch healing assay was conducted to observe anti-migration effect of PTXL on HKFs;(7)Transwell assay was used to detect the effect of PTXL on the invasion ability of HKFs;(8)Flow cytometry was used to detect the apoptosis of HKFs induced by PTXL and its effect on cell cycle;(9)Enzyme-Linked ImmunoSorbent Assay(ELISA)was performed to detect the expression differences of TXF-α,IL-6 and TGF-β in normal human skin fibroblasts(NFs)and in HKFs,and evaluate the effect of PTX on the expression of above cytokines in HKFs;(10)Western Blot was used to detect the effects of PTX on the expression of Akt/GSK3 β signaling pathway and fibrosis marker including a-smooth muscle act in(a-SMA)and collagen I in HKFs;(11)A subcutaneous keloid model was established by subcutaneous inoculation of HKFs to investigate the effect of PTXL on the inhibition of keloid growth in vivo;(12)Hematoxylin-Eosin staining(HE)was used to observe the keloid structure and pathological changes in visceral tissues,TUXEL staining was carried on for observation of keloid tissue apoptosis;(13)Immunohistochemistry was used to detect the expression of Akt/GSK3β pathway and a-SMA and collagen I in keloid tissue after PTXL treatment.Results:(1)The preparation of PTXL:weigh 30 mg phosphatidylcholine,of which SPC:EPC =8:1,4 mg DSPE-mPEG2000,1.5 mg PTX-CHOL,CHOL 1 mg,and dissolve them with 3 mL chloroform.The mixture was transferred into eggplant bottlee and the chloroform eliminated by rotary evaporation at 100 rpm/min and 40℃ water bath to gain a uniform translucent thin film.Subsequently,the film was hydrated with 2 mL 5%glucose to obtain a liposomes solution,and then ultrasonicated the solution under a Ultrasonic cell pulverizer.The ultrasound probe parameter of prepared PTXL was 15 minutes(power;65W,time:ultrasonic 2 seconds,intermittent 1 second).Then filtered with 0.22 μ m water membrane to obtain a PTXL solution,which was a clear and transparent liquid.(2)The PTXL had a spherical shape with a uniform size,the average particle size was(101.43±0.54)nm in diameter with a PDI of(0.244 ± 0.006),zeta potential was(-41.63±0.83)mV,indicated that the liposome particles are moderate and uniform in size,and have a high negative charge on the surface,which is beneficial to the stability of the liposome solution;(3)The encapsulation efficiency of PTXL was(95.63±0.70)%,and the drug loading was(2.70±0.16)%.The in vitro drug release assay found that the released PTX could be detected after 6 hours and then gradually increased,the release rate reached at 51.21%after 72 hours,indicate that liposome could release drug at a slow and sustained pattern.There was no significant increase of the partical size within 30 days and the leakage rates at 1,7,15 and 30 days were(0.13± 0.05)%,(4.30±1.93)%,(8.74±0.06)%,and(18.23±0.98)%,respectively,indicated that PTXL has low leakage and good stability;(4)The cell uptake experiments showed that the fluorescence intensities of C6 and C6L groups in HKFs were(103.22+6.00)and(550.96±22.73)at 15 minutes,and were(861.33+47.37)and(3756.34±373.86)at 30 minutes,the uptake amount increased gradually with time(P<0.01),and the uptake of C6L by HKFs was significantly higher than that of C6(P<0.01).The uptake of C6L by HKFs could be inhibited 59.9%by caveolin endocytosis inhibitor methyl-beta-cyclodextrin(M-β-CD),not clathrin endocytic inhibitor chlorpromazine;(5)CCK8 experiments showed that when the drug concentration was lower than 1 μg/mL,the inhibition of liposome material(Blank-L)on HKFs proliferation was less than 5%and the inhibition of the commercially PTX solvent(anhydrous ethanol+polyoxyethylene castor oil,E+EL)was less than 15%within 48 hours.When the drug concentration was higher than 1 μg/mL,the cytotoxicity of Blank-L was lower than that of commercial PTX solvent at the same concentration(P<0.05 orP<0.01).Moreover,PTX inhibited the proliferation of HKFs in a time-and dose-dependent manner under the same concentration,the inhibition effect of PTXL on HKFs was significantly higher than that of PTX(P<0.01).(6)The scratch healing assay showed that PTX and PTXL could significantly delay the healing of scratches,inhibit the migration of cells to the scratches,and reduce the cell density;(7)Transwell experiments showed that HKFs have strong invasive ability in the control group.However,the invasive rate of the PTXL group was(5.77±4.10)%,which was significantly lower than that in the PTX group(28.21±7.95)%(P<0.01).(8)In cell apoptosis experiment,the apoptosis rates of the control group,PTX group and PTXL group were(4.59±0.94)%,(33.92±0.58)%and(44.42±1.31)%,respectively,after treated 24 hours.The apoptosis rates of PTX group and PTXL group were significantly higher than that of the control group(P<0.01),and the PTXL group was higher than the PTX group(P<0.01).(9)Cell cycle arrest assay showed that the ratio of G2/M phase cells increased from(14.60±3.48)%in the control group to(78.15 ± 5.37)%and(87.15±1.17)%af ter 24 hours of PTX and PTXL treatment,(there were significant differences in any two group,P<0.01);(10)The results of ELISA showed that the expression of TNF-α,IL-6 and TGF-β in HKFs was significantly higher than that of NFs,and the highly expressed cytokines could be inhibited by PTX.The PI3K/Akt inhibitor LY294002 also inhibited the above cytokines in HKFs.(11)Animal experiments showed that PTX and PTXL could significantly inhibit the growth of keloid.The tumor volume and tumor weight were significantly different from the control group after treatment(P<0.05 or P<0.01).The PTXL group had excellent inhibition effect on keloid than the PTX group(P<,0.05).HE staining showed that the cell density in the keloid was decreased in the treatment group,and no organic changes were observed in the visceral tissues.TUNEL staining revealed that PTX and PTXL could induce keloid tissue apoptosis.(12)Western Blot and immunohistochemistry were used to detect the expression of protein in HKFs and keloid tissues.It was found that PTX coud inhibit the expression of a-SMA and Collagen I in keloids to ameliorate keloid fibrosis,and this effect was related to the inhibition of the Akt/GSK3β pathway.Conclusions1.The prepared PTXL effectively improves the water solubility of PTX,with a moderate and uniform particle size,and a negative charge;2.PTXL released PTX in a slow manner,PTXL had good stability and low leakage of drugs;3.PTXL can inhibit the proliferation,migration and invasion of HKFs,and can arrest the cell cycle in G2/M phase and effectively induce cell apoptosis.4.The keloids model can be successfully established by subcutaneous inoculation with HKFs in nude nice.PTXL could induce apoptosis in keloid tissues and inhibit the growth of keloid in vivo.5.PTXL can significantly increase the effect of PTX on the treatment of keloids in vitro and in vivo,and with high safety;6.PTX can inhibit the expression of inflammatory factors TNF-a,IL-6 and TGF-β by inhibiting the expression of Akt/GSK3β pathway in HKFs,thereby restrain the fibrosis of keloids. |
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