治疗原发性肺癌的美乐托宁脂质体粉雾剂研究
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摘要
美乐托宁(melatonin, MLT)又称褪黑素,是人体内源性物质,大剂量给药时有抗肿瘤作用,但口服生物利用度低,体内半衰期短,成药性差。本文制备了美乐托宁脂质体粉雾剂(liposomal melatonin dry powder inhalers,LMD),经肺部给药治疗大鼠原发性肺癌。用乙醇注入法制备美乐托宁脂质体(liposomal melatonin, LM),包封率为98.9%,加入甘露醇冻干得到LMD,扫描电镜显示为类球形颗粒,加水复溶后粒径为65.15 nm, zeta电位为-14.2 mV。LMD的空气动力学粒径为6.73μm,微细粒子比例(fine particle fraction, FPF<8.06μm)为22.2%,适合肺部给药。在相同药物浓度时, LMD对A549肺癌细胞抑制作用显著高于MLT原料和吉西他滨,因此脂质体增强了MLT药效。大剂量LMD不影响人正常支气管细胞(BEAS-2B)生长,安全性好。用3-甲基胆蒽及N,N-二甲基亚硝胺经气管喷入大鼠肺中, 45天后得到原发性肺癌模型(动物实验经单位伦理委员会批准且实验均按照相关指导原则和规定进行)。将MLT、吉西他滨、LMD经气管喷入肺癌大鼠肺中,与模型组比较,治疗组均呈现明显少的肿瘤结节和炎性细胞数量,其中LMD的药效最强。治疗组NF-κB p65减少,以及Tunel检测表明细胞凋亡;丙二醛水平降低,其中LMD组的效果最强,与其他组有显著性差异。美乐托宁脂质体粉雾剂直接将药物递送至肺部肿瘤,是一种有前景的治疗肺癌的肺吸入给药剂型。
Melatonin(MLT) is an endogenous chemical that has antitumor effects at high doses. However, it shows low oral bioavailability and short in vivo half-life, leading to drug resistance. Here, liposomal melatonin dry powder inhalers(LMD) were prepared, and were used for treatment of primary rat lung cancer by pulmonary delivery. Liposomal melatonin(LM) was prepared by the ethanol injection method to achieve an entrapment efficiency of 98.89%. LMD was obtained by freeze-drying after LM was mixed with mannitol. LMD appeared as spherical particles under a scanning electron microscope. The rehydrated liposomes had a small size of 65.15 nm and the zeta potential of-14.2 mV without change inentrapment efficiency. LMD had an aerodynamic particle size of 6.73 ± 0.012 μm and a fine particle fraction(FPF<8.06 μm) of 22.2%, suitable for pulmonary delivery. When administered with the same dose, LMD showed much higher inhibition on A549 lung cancer cells than MLT and gemcitabine. LMD of a large dose had no effect on the growth of normal lung epithelial cells(BEAS-2 B). Rat lung cancer models were established after 45 days by instilling 3-methylcholanthrene(MCA) and N,N-dimethylni‐trosamine(DEN) into the rat lungs once(the experiments had been approved by the ethics committee and carried out in accordance with relevant guidelines and regulations). Decreases of tumor nodules and inflammatory cells in the tumor-bearing rat lungs were observed after treatment of MLT, gemcitabine and LMD by pulmonary delivery compared with the models, wherein LMD was most effective. The efficiencies of inhibition of NF-κB p65, increase of Tunel detection(indicating enhancement of apoptosis), and decrease of malondialdehyde corresponded to LMD being most effective. Therefore, given the fact that LMD can deliver the drug into the tumor tissues of lungs, and it presents as a promising pulmonary inhalable regiment for treatment of lung cancer.
Melatonin(MLT) is an endogenous chemical that has antitumor effects at high doses. However, it shows low oral bioavailability and short in vivo half-life, leading to drug resistance. Here, liposomal melatonin dry powder inhalers(LMD) were prepared, and were used for treatment of primary rat lung cancer by pulmonary delivery. Liposomal melatonin(LM) was prepared by the ethanol injection method to achieve an entrapment efficiency of 98.89%. LMD was obtained by freeze-drying after LM was mixed with mannitol. LMD appeared as spherical particles under a scanning electron microscope. The rehydrated liposomes had a small size of 65.15 nm and the zeta potential of-14.2 mV without change inentrapment efficiency. LMD had an aerodynamic particle size of 6.73 ± 0.012 μm and a fine particle fraction(FPF<8.06 μm) of 22.2%, suitable for pulmonary delivery. When administered with the same dose, LMD showed much higher inhibition on A549 lung cancer cells than MLT and gemcitabine. LMD of a large dose had no effect on the growth of normal lung epithelial cells(BEAS-2 B). Rat lung cancer models were established after 45 days by instilling 3-methylcholanthrene(MCA) and N,N-dimethylni‐trosamine(DEN) into the rat lungs once(the experiments had been approved by the ethics committee and carried out in accordance with relevant guidelines and regulations). Decreases of tumor nodules and inflammatory cells in the tumor-bearing rat lungs were observed after treatment of MLT, gemcitabine and LMD by pulmonary delivery compared with the models, wherein LMD was most effective. The efficiencies of inhibition of NF-κB p65, increase of Tunel detection(indicating enhancement of apoptosis), and decrease of malondialdehyde corresponded to LMD being most effective. Therefore, given the fact that LMD can deliver the drug into the tumor tissues of lungs, and it presents as a promising pulmonary inhalable regiment for treatment of lung cancer.
引文
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[25]Kim KJ,Choi JS,Kang I,et al.Melatonin suppresses tumor progression by reducing angiogenesis stimulated by HIF-1 in a mouse tumor model[J].J Pineal Res,2013,54:264-270.
[26]Goradel NH,Asghari MH,Moloudizargari M,et al.Melatonin as an angiogenesis inhibitor to combat cancer:mechanistic evidence[J].Toxicol Appl Pharmacol,2017,335:56-63.
[27]Anisimov VN,Popovich IG,Zabezhinski MA,et al.Melatonin as antioxidant,geroprotector and anticarcinogen[J].Biochim Biophys Acta,2006,1757:573-589.
[28]Szewczyk-Golec K,Rajewski P,Gackowski M,et al.Melatonin supplementation lowers oxidative stress and regulates adipokines in obese patients on a calorie-restricted diet[J].Oxidat Med Cell Longev,2017,2017:8494107.
[29]Habtemariam S,Daglia M,Sureda A,et al.Melatonin and respiratory diseases:a review[J].Curr Top Med Chem,2017,17:467-488.
[30]Lu JJ,Fu LY,Tang ZP,et al.Melatonin inhibits AP-2β/hTERT,NF-κB/COX-2 and Akt/ERK and activates caspase/Cyto Csignaling to enhance the antitumor activity of berberine in lung cancer cells[J].Oncotarget,2016,7:2985-3001.
[31]Tsai M,Kuo Y,Chiu Y,et al.Down-regulation of rad51 expres‐sion overcomes drug resistance to gemcitabine in human nonsmall-cell lung cancer cells[J].J Pharmacol Exp Ther,2010,335:830-840.
[32]Borin TF,Arbab AS,Gelaleti GB,et al.Melatonin decreases breast cancer metastasis by modulating Rho-associated kinase protein-1 expression[J].J Pineal Res,2016,60:3-15.
[2]Jin J,Hu KJ,Zhou YZ,et al.Clinical utility of the modified glasgow prognostic score in lung cancer:a meta analysis[J].PLoS One,2017,12:e0184412.
[3]Song N,Kim AJ,Kim HJ,et al.Melatonin suppresses doxorubi‐cin-induced premature senescence of A549 lung cancer cells by ameliorating mitochondrial dysfunction[J].J Pineal Res,2012,53:335-343.
[4]Ma ZQ,Yang Y,Fan CX,et al.Melatonin as a potential anticar‐cinogen for non-small-cell lung cancer[J].Oncotarget,2016,7:46768-46784.
[5]Majka J,Wierdak M,Brzozowska I,et al.Melatonin in prevention of the sequence from reflux esophagitis to barrett's esophagus and esophageal adenocarcinoma:experimental and clinical perspectives[J].Int J Mol Sci,2018,19:2033.
[6]Kuzmov A,Minko T.Nanotechnology approaches for inhalation treatment of lung diseases[J].J Control Release,2015,219:500-518.
[7]S?rli JB,Sivars KB,Da Silva E,et al.Bile salt enhancers for inhalation:correlation between in vitro and in vivo lung effects[J].Int J Pharm,2018,550:114-122.
[8]Zhu L,Li M,Liu X,et al.Inhalable oridonin-loaded poly(lacticco-glycolic)acid large porous microparticles for in situ treatment of primary non-small cell lung cancer[J].Acta Pharm Sin B,2017,7:80-90.
[9]Zhang T,Chen Y,Ge Y,et al.Inhalation treatment of primary lung cancer using liposomal curcumin dry powder inhalers[J].Acta Pharm Sin B,2018,8:440-448.
[10]Zhang J,Li Y,Xiang ML,et al.Preparation and pharmacody‐namic study of curcumin liposome dry powder inhalation[J].Mil Med Sci(军事医学),2013,37:692-695.
[11]Shen H,Wang LF,Chen WQ,et al.Tissue distribution and tumor uptake of folate receptor-targete depothilone folate conjugate,BMS-753493,in CD2F1 mice after systemic administration[J].Acta Pharm Sin B,2016,6:460-467.
[12]Pilcer G,Amighi K.Formulation strategy and use of excipients in pulmonary drug delivery[J].Int J Pharm,2010,392:1-19.
[13]Hu YZ,Li M,Zhang TT,et al.Preparation of liposomal artesunate dry powder inhalers and the effect on the acute lung injury of rats[J].Acta Pharm Sin(药学学报),2016,51:1906-1912.
[14]Vanbever R,Mintzes JD,Wang J,et al.Formulation and physical characterization of large porous particles for inhalation[J].Pharm Res,1999,16:1735-1742.
[15]Depreter F,Pilcer G,Amighi K.Inhaled proteins:challenges and perspectives[J].Int J Pharm,2013,447:251-280.
[16]Elversson J,Millqvist-Fureby A,Alderborn G,et al.Droplet and particle size relationship and shell thickness of inhalable lactose particles during spray drying[J].J Pharm Sci,2003,92:900-910.
[17]Sdraulig S,Franich R,Tinker RA,et al.In vitro dissolution studies of uranium bearing material in simulated lung fluid[J].JEnviron Radioact,2008,99:527-538.
[18]Zhang L,Chen F,Zheng JT,et al.Chitosan-based liposomal thermogels for the controlled delivery of pingyangmycin:design,optimization and in vitro and in vivo studies[J].Drug Deliv,2018,25:690-702.
[19]Choi Y,Attwood SJ,Hoopes MI,et al.Melatonin directly interacts with cholesterol and alleviates cholesterol effects in dipalmitoylphosphatidylcholine monolayers[J].Soft Matter,2014,10:206-213.
[20]Choo WH,Park CH,Jung SE,et al.Long-term exposures to low doses of silver nanoparticles enhanced in vitro malignant cell transformation in non-tumorigenic BEAS-2B cells[J].Toxicol In Vitro,2016,37:41-49.
[21]Dai T,Jiang K,Lu W.Liposomes and lipid disks traverse the BBB and BBTB as intact forms as revealed by two-step F?rster resonance energy transfer imaging[J].Acta Pharma Sin B,2018,8:261-271.
[22]Yang S.A pooled study on combination of gemcitabine and nedaplatin for treating patients with non-small cell lung cancer[J].Asian Pac J Cancer Prev,2015,16:5963-5966.
[23]Zhou QY,Gui SY,Zhou Q,et al.Melatonin inhibits the migra‐tion of human lung adenocarcinoma A549 cell lines involving JNK/MAPK pathway[J].PLoS One,2014,9:e101132.
[24]Roursgaard M,Knudsen KB,Northeved H,et al.In vitro toxicity of cationic micelles and liposomes in cultured human hepatocyte(HepG2)and lung epithelial(A549)cell lines[J].Toxicol In Vitro,2016,36:164-171.
[25]Kim KJ,Choi JS,Kang I,et al.Melatonin suppresses tumor progression by reducing angiogenesis stimulated by HIF-1 in a mouse tumor model[J].J Pineal Res,2013,54:264-270.
[26]Goradel NH,Asghari MH,Moloudizargari M,et al.Melatonin as an angiogenesis inhibitor to combat cancer:mechanistic evidence[J].Toxicol Appl Pharmacol,2017,335:56-63.
[27]Anisimov VN,Popovich IG,Zabezhinski MA,et al.Melatonin as antioxidant,geroprotector and anticarcinogen[J].Biochim Biophys Acta,2006,1757:573-589.
[28]Szewczyk-Golec K,Rajewski P,Gackowski M,et al.Melatonin supplementation lowers oxidative stress and regulates adipokines in obese patients on a calorie-restricted diet[J].Oxidat Med Cell Longev,2017,2017:8494107.
[29]Habtemariam S,Daglia M,Sureda A,et al.Melatonin and respiratory diseases:a review[J].Curr Top Med Chem,2017,17:467-488.
[30]Lu JJ,Fu LY,Tang ZP,et al.Melatonin inhibits AP-2β/hTERT,NF-κB/COX-2 and Akt/ERK and activates caspase/Cyto Csignaling to enhance the antitumor activity of berberine in lung cancer cells[J].Oncotarget,2016,7:2985-3001.
[31]Tsai M,Kuo Y,Chiu Y,et al.Down-regulation of rad51 expres‐sion overcomes drug resistance to gemcitabine in human nonsmall-cell lung cancer cells[J].J Pharmacol Exp Ther,2010,335:830-840.
[32]Borin TF,Arbab AS,Gelaleti GB,et al.Melatonin decreases breast cancer metastasis by modulating Rho-associated kinase protein-1 expression[J].J Pineal Res,2016,60:3-15.