| Objective: Preparation of single-methoxy polyethylene glycol-polylactic acid contained curcumin micelles(m PEG- PLA/CUR). By observing the drug loadings, the encapsulation efficiency, particle size and in vitro drug release of m PEG- PLA/CUR nano micelles is to understand its physical and chemical properties; To further investigate the m PEG- PLA/CUR in vitro of cervical cancer Hela proliferation inhibition, cell uptake and in vivo antitumor effect, and discussed its possible mechanism against cervical cancer. Methods: The study is divided into two stages. The first stage(in vitro) :The three different concentrations of single-methoxy polyethylene glycol-polylactic acid micelles contained curcumin(m PEG- PLA/CUR) were prepared by solid dispersion. Using high-performance liquid chromatograph determined of three groups of different concentrations of m PEG- PLA/CUR nano micelles encapsulation efficiency and drug loadings, the selected which has high encapsulation efficiency and drug loadings concentrations of a set of nano drug used for subsequent experiment. Observed under AFM selectedconcentration of single-methoxy polyethylene glycol- polylactic acid micelles contained curcumin(m PEG- PLA/CUR) nano micelles morphology, Using laser particle size analyzer to determine the m PEG- PLA/CUR nano micelles particle size. Choose the selected concentration of m PEG- PLA/CUR nano micelles drug release in vitro experiments, by high performance liquid chromatograph to determine its in vitro drug release. By determined by MTT method, determination of physiological saline, blank carrier m PEG- PLA polymers, free of curcumin, the m PEG- PLA/CUR nano micelles cervical cancer Hela inhibitory effect. Through inversion fluorescence microscope observation of cervical cancer Hela m PEG of blank carrier- PLA polymers, m PEG- PLA/CUR nano micelles, free of curcumin. The second phase(in vivo): Animal model was constructed, The first 36 nude mice with stud earrings Numbers, subcutaneous inoculation of human cervical cancer cells into nude mice were randomly divided into 4 groups(tumor after each group were 9 rats), to extract only 3 of them to do immunohistochemical, each group with the rest of the 6 only nude mice to observe the survival period, the grouping situation as follows:(1) : physiological saline group(negative group);(2) : blank carrier m PEG- PLA polymer group(carrier);(3) : free of curcumin groups(25 mg/Kg)(free);(4) : the m PEG- PLA/CUR nano micelle group(in terms of curcumin dose of 25 mg/Kg) group(compound). Treat tumors grow to about 0.1 cm3 drugs through the nude mouse tail vein injection of four groups respectively(according to 0.005 ml/g) 10, injection once every one day, a total of two weeks.Every 2 days use vernier caliper to measure a plaque, long and short diameter calculation of tumor volume, plot of tumor growth; Growth state, general situation and at the same time observe nude mice survival, drug in each group separately executed after two weeks, three nude mice stripping plaque, measurement of tumor weight, draw the tumor weight curve, and then by immunohistochemical method to determine the Ki- 67, MVD(CD31), VEGF preliminarily the m PEG- PLA/CUR, possible mechanisms of nano micelles of cervical cancer. Results: 1, The three groups of different concentration of m PEG-PLA/CUR nano micelles were successful prepared. 2, The three different concentrations of m PEG- PLA/CUR nano micelles were determined, Drug loadings were 3.22±0.51%, 7.22 ±0.42% and 8.06 ± 1.5%, and the encapsulation efficiencys were80.58 ± 0.13%, 90.42 ± 0.05% and 80.6 ±0.15%, Confirmed the m PEG- PLA(92 mg) of curcumin(8 mg) nano micelles has good drug loading and encapsulation efficiency. 3,The average particle size of the nano micelles of m PEG- PLA(92 mg) /CUR(8 mg) is 119.6nm, uniform particle size of dispersion, unimodal distribution. 4, free of curcumin in curcumin within 12 hours about 66% released from dialysis bag, about 80% of curcumin in 24 hours released from dialysis bag; However the m PEG-PLA/CUR nano micelles curcumin in 14 days only 59% released from nano micelles, Confirmed the m PEG- PLA/CUR nano micelle has good slow release properties. 5, Through the inverted fluorescence microscope after dealing with the blank carrier m PEG- PLA of Hela never see green fluorescence, after usingfree curcumin treated Hela cell,Faint green fluorescence was observed after 2 hours, after4 hours a strong green fluorescence was observed, but after the m PEG- PLA/CUR nano micelles treated Hela cell a strong green fluorescence was observed after 2 hours, the bright green fluorescence was observed after 4 hours. Confirm the m PEG- PLA/CUR nano micelles has good cell permeability characteristics. 6, 6.25 ug/ml, 12.5 ug/ml, 25 ug/ml and 50 ug/ml, 100 ug/ml m PEG- PLA/CUR nano micelles can significantly inhibit the growth of human cervical cancer Hela cell, inhibitory effect with a concentration in accordance with the patient. And free of the same concentration of curcumin m PEG- PLA/CUR nano micelle of cervical cancer Hela inhibition was stronger than the free of curcumin(P < 0.05). MPEG- PLA polymer is a kind of ideal, safety and low toxicity of nanometer carrier. 7, determination of four groups of nude mice tumor after growth of composite group(1.21 ± 1.16cm3), negative(2.82 ±1.34cm3), carrier group(2.35 ± 1.31cm3), the free group(1.67±1.16cm3), compared with other three groups of composite group was obviously inhibit the growth of cervical cancer cells(P < 0.05), statistically significant differences, while carrier group did not show anticancer activity. The median survival time of four groups of nude mice respectively for the composite group of 160 days, free 135 days, carrier group 115 days, negative group 87 days, The composite group compared with the remaining three groups significantly prolong survival time and the difference is statistically significant(P < 0.05). 8, Four groups were determined byimmunohistochemical tumor weight were composite tumor weight was(0.08 + 0.14g), free group(0.10 +0.03g), vector group(0.39 + 0.17g), negative group(0.37 + 0.14g), composite group and free group and vector group, negative group compared with tumor weight was significantly reduced(P < 0.05), the difference was statistically significant. Ki- 67 positive cells was observed in compound group number is(53.33 ±8.82%), the ratio of the number of positive cells significantly free(80 ± 5%, P < 0.01), the vehicle group(90±5%, P < 0.01), and negative groups(93.33±2.89%, P < 0.01) is low, and the difference is statistically significant; VEGF positive cells number ratio in the compound group(25±13.23%) and free group(28.33 ± 12.58%), and the carrier(56.67± 15.28%), and the negative group(65 ± 18.03%) compared to the compound group and free group were lower(P < 0.05), shows obvious difference; And negative groups(3 ± 1, P < 0.01), the vehicle group(2.67 ±0.58, P < 0.01) compared with compound group(MPEG- PLA/CUR nano micelle) block tumor microvascular article number(0.67± 0.58) less obvious, and has significant statistical significance, but the number of composite group compared with the free set of CD31 no statistical difference(P > 0.05).Conclusion: 1, the solid dispersion method successfully prepared m PEG- three groups of different concentration of MPEG- PLA/CUR, Drug Loading also increased with the increase of the concentration. 8% concentration group of MPEG- PLA(92 mg) of curcumin(8 mg) nano micelle has good drug loadings and the coating ratio, uniform particle size of dispersion, showunimodal distribution, has a good slow-release character; 2, At the same time, In vitro cytotoxicityof m PEG-PLA/CUR has obvious concentration dependence; 3, the m PEG- PLA polymers can obviously improve the bioavailability of curcumin, is a safe, ideal and low toxicity of drug carrier; 4, compared with the negative group, the carrier, free group, composite group have significantly inhibited tumor growth and prolong the median survival period of the nude mice; The m PEG-PLA/CUR nano micelles antitumor lies advantage of the m PEG- PLA delivery system which improves the water solubility and slow release of curcumin. |
PDF Full Text Request