槲皮素纳米混悬剂的制备、表征及抗乳腺癌研究
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摘要
目的为解决槲皮素水溶性差的问题,制备一种高载药量、适合静脉给药的纳米混悬剂,并探究其体内外抗肿瘤作用。方法采用反溶剂沉淀联合高压均质法,以聚乙二醇1000维生素E琥珀酸酯(TPGS)为稳定剂制备了槲皮素纳米混悬剂(quercetinnanosuspensions,Q-NSps)。动态光散射法测定粒径,扫描电镜观察其形态,HPLC法测定其载药量和体外药物释放情况,并考察了其冻干保护、放置稳定性、溶血和静脉注射的适宜性;MTT法检测槲皮素及其纳米混悬剂对4T1、HeLa、Hep G2细胞的生长抑制作用;以4T1荷瘤小鼠模型对比考察其体内抗肿瘤效果。结果制备的Q-NSps大小均匀,呈球型,平均粒径143.9 nm,PDI为0.231,表面电位-22.6 mV;载药量(45.82±1.73)%,用1%麦芽糖为保护剂冻干复溶后粒径变化不大;Q-NSps 30 d放置稳定,不溶血,可静脉注射;体外有良好的缓释作用,144 h累积释放82.86%;对4T1、HeLa、Hep G2的生长抑制均显著高于游离药物,在体内研究中,45 mg/kg的Q-NSps与阳性药紫杉醇注射液(8 mg/kg)表现出相同的抑瘤效果(56.78%vs55.08%,P>0.05)。结论制备的Q-NSps粒径小,稳定性好,显著提高了槲皮素体内外抗肿瘤效果,有望成为一种抗肿瘤药物用于临床。
Objective In order to solve the problem of poor water solubility of quercetin, quercetin nanosuspensions(Q-NSps) with high drug loading and suitable for intravenous administration was prepared, and study its anti-tumor effect in vitro and in vivo. Methods Quercetin was made into nanoparticles(Q-NSps) via the method of anti-solvent precipitation combined with high-pressure homogenization using polyethylene glycol 1 000 vitamin E succinate(TPGS) as stabilizer. The particle size of the resultant nanoparticles was measured by dynamic light scattering and the morphology was observed by scanning electron microscopy. The drug loading and the in vitro drug release were measured using HPLC analysis. In the meantime, the lyoprotectants were screened, the storage stability, hemolysis, and the suitability for intravenous injection were also studied; The in vitro anti-tumor activity of quercetin and its nanoparticles were assessed in contrast using MTT assay and the in vivo anti-tumor therapeutic efficacy was investigated using 4 T1 tumor bearing mice. Results Q-NSps had uniform size and spherical shape, the average particle size was 143.9 nm, the polydispersity index(PDI) was 0.231, and the Zeta potential was-22.6 mV. The drug loading content was(45.82 ± 1.73) %. Using 1% maltose as lyoprotectant, Q-NSps could be lyophilized and then reconstituted into nanoparticles of the similar size. Q-NSps were stable for 30 d at storage, showed no hemolysis, and were suitable to intravenous administration. The resultant nanosuspensions displayed a good sustained in vitro release, and the cumulative release reaching 82.86% at 144 h. Q-NSps showed significantly higher growth inhibition against 4 T1, HeLa, and Hep G2 cell lines. The in vivo study demonstrated that Q-NSps(45 mg/kg, iv) had the similar antitumor therapeutic efficacy as paclitaxel injections(56.78% vs 55.08%, P > 0.05). Conclusion The obtained Q-NSps had small particle size, good stability, and significantly improved anti-tumor effect of quercetin in vitro and in vivo, so Q-NSps are promising to be a antitumor drug for application in clinic.
Objective In order to solve the problem of poor water solubility of quercetin, quercetin nanosuspensions(Q-NSps) with high drug loading and suitable for intravenous administration was prepared, and study its anti-tumor effect in vitro and in vivo. Methods Quercetin was made into nanoparticles(Q-NSps) via the method of anti-solvent precipitation combined with high-pressure homogenization using polyethylene glycol 1 000 vitamin E succinate(TPGS) as stabilizer. The particle size of the resultant nanoparticles was measured by dynamic light scattering and the morphology was observed by scanning electron microscopy. The drug loading and the in vitro drug release were measured using HPLC analysis. In the meantime, the lyoprotectants were screened, the storage stability, hemolysis, and the suitability for intravenous injection were also studied; The in vitro anti-tumor activity of quercetin and its nanoparticles were assessed in contrast using MTT assay and the in vivo anti-tumor therapeutic efficacy was investigated using 4 T1 tumor bearing mice. Results Q-NSps had uniform size and spherical shape, the average particle size was 143.9 nm, the polydispersity index(PDI) was 0.231, and the Zeta potential was-22.6 mV. The drug loading content was(45.82 ± 1.73) %. Using 1% maltose as lyoprotectant, Q-NSps could be lyophilized and then reconstituted into nanoparticles of the similar size. Q-NSps were stable for 30 d at storage, showed no hemolysis, and were suitable to intravenous administration. The resultant nanosuspensions displayed a good sustained in vitro release, and the cumulative release reaching 82.86% at 144 h. Q-NSps showed significantly higher growth inhibition against 4 T1, HeLa, and Hep G2 cell lines. The in vivo study demonstrated that Q-NSps(45 mg/kg, iv) had the similar antitumor therapeutic efficacy as paclitaxel injections(56.78% vs 55.08%, P > 0.05). Conclusion The obtained Q-NSps had small particle size, good stability, and significantly improved anti-tumor effect of quercetin in vitro and in vivo, so Q-NSps are promising to be a antitumor drug for application in clinic.
引文
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[22]袁志平,陈俐娟,魏于全,等.纳米脂质体槲皮素对肝癌腹水抑制效应实验研究[J].癌症,2006,24(8):941-945.
[23]宋逍,段玺,赵鹏,等.槲皮素亚微乳在小鼠体内药代动力学及组织分布研究[J].中药材,2016,39(1):160-163.
[24]伍贤学.生物活性黄酮槲皮素的前药设计与合成研究[D].成都:四川大学,2005.
[25]Hong J,Li Y,Xiao Y,et al.Annonaceous acetogenins(ACGs)nanosuspensions based on a self-assembly stabilizer and the significantly improved anti-tumor efficacy[J].Colloids Surf B Biointerfaces,2016,145:319-327.
[26]苏文晶,肖瑶,张明珠,等.番荔素纳米混悬剂的制备及其抗肿瘤作用研究[J].药物评价研究,2016,39(6):966-972.
[27]申宝德,连王权,沈成英,等.微型化介质研磨法制备难溶性黄酮类化合物纳米混悬剂[J].中草药,2017,48(21):4413-4418.
[28]王义,王文倩,尹萌,等.1,3-二羟基异丙氧基-琥珀酸-8-莪术醇酯纳米混悬剂的制备及其性能研究[J].现代药物与临床,2017,32(4):579-583.
[29]童学飞,吴雪,杨智海.高效液相色谱法同时测定滋肝益气丸中槲皮素和丹皮酚含量[J].中国药业,2016,25(16):67-69.
[30]阮婧华,杨付梅,张金洁,等.槲皮素纳米结构脂质载体增加口服吸收机制研究[J].中国药学杂志,2013,48(5):368-373.
[31]杜宁.高槲皮素黄酮银杏叶注射液对荷瘤鼠抑瘤作用研究[D].保定:河北大学,2015.
[32]Lee E S,Na K,Bae Y H.Doxorubicin loaded pH-sensitive polymeric micelles for reversal of resistant MCF-7 tumor[J].J Control Release,2005,103(2):405-418.
[33]郏亚静,陈红丽,唐红波,等.普鲁兰基肿瘤靶向纳米粒的体外抑瘤效应和细胞摄取途径研究[J].医学研究生学报,2015,28(2):127-130.
[2]Andres S,Tejido M L,Bodas R,et al.Quercetin dietary supplementation of fattening lambs at 0.2%rate reduces discolouration and microbial growth in meat during refrigerated storage[J].Meat Sci,2013,93(2):207-212.
[3]Johari J,Kianmehr A,Mustafa M R,et al.Antiviral activity of baicalein and quercetin against the Japanese encephalitis virus[J].Int J Mol Sci,2012,13(12):16785-16795.
[4]Boots A W,Wilms L C,Swennen E L,et al.In vitro and ex vivo anti-inflammatory activity of quercetin in healthy volunteers[J].Nutrition,2008,24(7/8):703-710.
[5]Du G,Zhao Z,Chen Y,et al.Quercetin protects rat cortical neurons against traumatic brain injury[J].Mol Med Rep,2018,17(6):7859-7865.
[6]王秋红,匡海学,吴伦,等.二氢槲皮素对异丙肾上腺素致大鼠心肌缺血的保护作用[J].中国实验方剂学杂志,2011,17(17):177-180.
[7]Yang D K,Kang H S.Anti-diabetic effect of cotreatment with quercetin and resveratrol in streptozotocin-induced diabetic rats[J].Biomol Ther Seoul,2018,26(2):130-138.
[8]Yang Q L,Chen Y F.The effect of quercetin on the long-term memory and PARP-1/AIF signal path in neonatal rats with hypoxic-ischemic brain damage[J].JClin Pediatr,2016,12(34):936-941.
[9]杨扬,郭举.具有抗肿瘤活性的槲皮素衍生物研究进展[J].中草药,2018,49(6):1468-1475.
[10]付丽娜,刘维红,徐爱军,等.槲皮素对人胃癌MKN45细胞的抑制作用研究[J].现代药物与临床,2017,32(6):983-986.
[11]牟丽秋,杜俊,胡旖耘,等.杜仲中槲皮素、京尼平苷及桃叶珊瑚苷对小鼠成骨样细胞系MC3T3-E1增殖和分化的影响[J].药物评价研究,2015,38(2):165-169.
[12]Chan S T,Chuang C H,Lin Y C,et al.Quercetin enhances the antitumor effect of trichostatin A and suppresses muscle wasting in tumor-bearing mice[J].Food Funct,2018,9(2):871-879.
[13]Li S,Yuan S,Zhao Q,et al.Quercetin enhances chemotherapeutic effect of doxorubicin against human breast cancer cells while reducing toxic side effects of it[J].Biomed Pharmacother,2018,100(2):441-447.
[14]陈丽娜,任晓亮,刘亚男,等.槲皮素平衡溶解度的测定及热力学计算[J].药物分析杂志,2015,35(6):1006-1009.
[15]van Zanden J J,Wortelboer H M,Bijlsma S,et al.Quantitative structure activity relationship studies on the flavonoid mediated inhibition of multidrug resistance proteins 1 and 2[J].Biochem Pharmacol,2005,69(4):699-708.
[16]Li H,Zhao X,Ma Y,et al.Enhancement of gastrointestinal absorption of quercetin by solid lipid nanoparticles[J].J Control Release,2009,133(3):238-244.
[17]邵伟,谢清春,王春香,等.槲皮素-羟丙基-β-环糊精包合物的研究[J].中药材,2002,25(2):121-123.
[18]宋全道,丁易,李新民,等.羧甲基-β-环糊精的溶血作用及对槲皮素的增溶作用[J].山东大学学报:医学版,2005,43(2):166-169.
[19]李韶静,廖应芬,杨慧慧,等.槲皮素固体分散体的制备及大鼠体内生物利用度研究[J].中草药,2017,48(20):4229-4234.
[20]庞鑫.透明质酸--槲皮素结合物自组装胶束的研究[D].济南:山东大学,2014.
[21]吴金花,段金虹,许海燕,等.载有槲皮素的PEG-PE胶束对乳腺癌细胞耐药性的逆转效应[J].基础医学与临床,2015,35(2):174-177.
[22]袁志平,陈俐娟,魏于全,等.纳米脂质体槲皮素对肝癌腹水抑制效应实验研究[J].癌症,2006,24(8):941-945.
[23]宋逍,段玺,赵鹏,等.槲皮素亚微乳在小鼠体内药代动力学及组织分布研究[J].中药材,2016,39(1):160-163.
[24]伍贤学.生物活性黄酮槲皮素的前药设计与合成研究[D].成都:四川大学,2005.
[25]Hong J,Li Y,Xiao Y,et al.Annonaceous acetogenins(ACGs)nanosuspensions based on a self-assembly stabilizer and the significantly improved anti-tumor efficacy[J].Colloids Surf B Biointerfaces,2016,145:319-327.
[26]苏文晶,肖瑶,张明珠,等.番荔素纳米混悬剂的制备及其抗肿瘤作用研究[J].药物评价研究,2016,39(6):966-972.
[27]申宝德,连王权,沈成英,等.微型化介质研磨法制备难溶性黄酮类化合物纳米混悬剂[J].中草药,2017,48(21):4413-4418.
[28]王义,王文倩,尹萌,等.1,3-二羟基异丙氧基-琥珀酸-8-莪术醇酯纳米混悬剂的制备及其性能研究[J].现代药物与临床,2017,32(4):579-583.
[29]童学飞,吴雪,杨智海.高效液相色谱法同时测定滋肝益气丸中槲皮素和丹皮酚含量[J].中国药业,2016,25(16):67-69.
[30]阮婧华,杨付梅,张金洁,等.槲皮素纳米结构脂质载体增加口服吸收机制研究[J].中国药学杂志,2013,48(5):368-373.
[31]杜宁.高槲皮素黄酮银杏叶注射液对荷瘤鼠抑瘤作用研究[D].保定:河北大学,2015.
[32]Lee E S,Na K,Bae Y H.Doxorubicin loaded pH-sensitive polymeric micelles for reversal of resistant MCF-7 tumor[J].J Control Release,2005,103(2):405-418.
[33]郏亚静,陈红丽,唐红波,等.普鲁兰基肿瘤靶向纳米粒的体外抑瘤效应和细胞摄取途径研究[J].医学研究生学报,2015,28(2):127-130.