川芎嗪PEG-PE纳米胶束的体外评价、细胞摄取及抗心肌细胞凋亡研究
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摘要
目的制备川芎嗪PEG-PE纳米胶束,并评价该纳米胶束的细胞摄取和抗心肌细胞凋亡效果。方法采用薄膜水化法制备川芎嗪PEG-PE纳米胶束,并进行表征。采用体外释药、细胞摄取和细胞凋亡试验对该载药系统进行评价。结果川芎嗪PEG-PE纳米胶束粒径为(15.8±0.9)nm,Zeta电势为-(20.5±0.4)mV,载药量为(5.7±0.3)%,包封率为(87.2±5.4)%。电镜结果表明川芎嗪PEG-PE纳米胶束呈形态规则的圆球型结构;采用芘测定法测定PEG-PE纳米胶束的临界胶束浓度约为5.3μg·mL~(-1);细胞摄取试验结果表明,PEG-PE纳米胶束可以增强药物的细胞摄取量,细胞外残留量减少;川芎嗪PEG-PE纳米胶束在10%胎牛血清DMEM培养基稳定性良好,采用异丙肾上腺素诱导心肌细胞凋亡,Hoechst染色提示凋亡心肌细胞出现了大量形态学改变,而川芎嗪PEG-PE纳米胶束可以明显减少凋亡细胞和促凋亡Caspase-3活性、抑制促凋亡蛋白Bax表达,提高抗凋亡蛋白Bcl-2表达,均显著优于川芎嗪(P<0.01)。结论川芎嗪PEG-PE纳米胶束具有粒径小,载药量高,释药缓慢等优势,可很大程度上提高川芎嗪的心肌细胞摄取量,增强药物的抗心肌细胞凋亡作用。
OBJECTIVE To prepare tetramethylpyrazine PEG-PE(TMP-PEG-PE) micelles, and to evaluate the cellular uptake and anti-cardiomyocyte apoptosis of the nanomicelle. METHODS TMP-PEG-PE micelles were prepared by thin film hydration method and characterized. The drug delivery system was evaluated by in vitro release, cell uptake and apoptosis assays.RESULTS The particle size of TMP-PEG-PE micelles was(15.8±0.9)nm, the Zeta potential was -(20.5±0.4)mV, the drug loading was(5.7±0.3)% and the encapsulation efficiency was(87.2±5.4)%. The results of electron microscopy showed that the TMP-PEG-PE micelles were in the shape of a regular spherical structure. The critical micelle concentration of PEG-PE micelles was determined by Pyrene assay to be about 5.3 μg·mL~(-1). The results of cell uptake assay showed that PEG-PE nanomicelles could enhance the cellular uptake of the drug and reduce the extracellular residual amount. TMP-PEG-PE micelles had good stability in 10% fetal bovine serum DMEM medium. Isoproterenol was used to induce myocardial apoptosis, Hoechst staining indicated that there were a lot of morphological changes in apoptotic cardiomyocytes. TMP-PEG-PE micelles could significantly reduce the pro-apoptotic caspase-3 activity, inhibit the expression of the pro-apoptotic protein Bax, and increase the expression of anti-apoptotic protein Bcl-2, which were significantly better than the test results of TMP(P<0.01). CONCLUSION TMP-PEG-PE micelles have the advantages of small particle size, high drug loading and slow drug release, which can greatly improve the myocardial cell uptake of TMP and enhance its anti-cardiomyocyte apoptosis effect.
OBJECTIVE To prepare tetramethylpyrazine PEG-PE(TMP-PEG-PE) micelles, and to evaluate the cellular uptake and anti-cardiomyocyte apoptosis of the nanomicelle. METHODS TMP-PEG-PE micelles were prepared by thin film hydration method and characterized. The drug delivery system was evaluated by in vitro release, cell uptake and apoptosis assays.RESULTS The particle size of TMP-PEG-PE micelles was(15.8±0.9)nm, the Zeta potential was -(20.5±0.4)mV, the drug loading was(5.7±0.3)% and the encapsulation efficiency was(87.2±5.4)%. The results of electron microscopy showed that the TMP-PEG-PE micelles were in the shape of a regular spherical structure. The critical micelle concentration of PEG-PE micelles was determined by Pyrene assay to be about 5.3 μg·mL~(-1). The results of cell uptake assay showed that PEG-PE nanomicelles could enhance the cellular uptake of the drug and reduce the extracellular residual amount. TMP-PEG-PE micelles had good stability in 10% fetal bovine serum DMEM medium. Isoproterenol was used to induce myocardial apoptosis, Hoechst staining indicated that there were a lot of morphological changes in apoptotic cardiomyocytes. TMP-PEG-PE micelles could significantly reduce the pro-apoptotic caspase-3 activity, inhibit the expression of the pro-apoptotic protein Bax, and increase the expression of anti-apoptotic protein Bcl-2, which were significantly better than the test results of TMP(P<0.01). CONCLUSION TMP-PEG-PE micelles have the advantages of small particle size, high drug loading and slow drug release, which can greatly improve the myocardial cell uptake of TMP and enhance its anti-cardiomyocyte apoptosis effect.
引文
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[13]MUSACCHIO T,VAZE O,D’SOUZA G,et al.Effective stabilization and delivery of siRNA:reversible siRNA-phospholipid conjugate in nanosized mixed polymeric micelles[J].Bioconjug Chem,2010,21(8):1530-1536.
[2]GALAGUDZA M,KOROLEV D,POSTNOV V,et al.Passive targeting of ischemic-reperfused myocardium with adenosine-loaded silica nanoparticles[J].Int J Nanomedicine,2012(7):1671-1678.
[3]GU Y C,GUO W J,YU X L.Protective effect of tetramethylpyrazine postconditioning on ischemia-reperfusion injured myocardium in rats[J].Int J Cardiovasc Dis(国际心血管病杂志),2009,36(5):304-307.
[4]LV L,ZHANG J,YIN Y G,et al.An investigation on the effects of ligustrazine in attenuating apoptosis of myocardial ischemia reperfusion injury in rats and its mechanism[J].Mil Med J Southeast China(东南国防医药),2016,18(4):361-364,367.
[5]ZHENG Q,HUANG Y Y,ZHU P C,et al.Ligustrazine exerts cardioprotection in animal models of myocardial ischemia/reperfusion injury:preclinical evidence and possible mechanisms[J].Front Pharmacol,2018(9):729.
[6]LIU X Y,LIU H,ZENG Z W,et al.Pharmacokinetics of ligustrazine ethosome patch in rats and anti-myocardial ischemia and anti-ischemic reperfusion injury effect[J].Int JNanomed,2011(6):1391-1398.
[7]HUANG J G,LESHUK T,GU F X.Emerging nanomaterials for targeting subcellular organelles[J].Nano Today,2011,6(5):478-492.
[8]WANG J,WANG Y G,LIANG W.Delivery of drugs to cell membranes by encapsulation in PEG-PE micelles[J].JControl Release,2012,160(3):637-651.
[9]WU H,ZHU L,TORCHILIN V P.PH-sensitive poly(histidine)-PEG/DSPE-PEG co-polymer micelles for cytosolic drug delivery[J].Biomaterials,2013,34(4):1213-1222.
[10]QIAO H Z,LI J,WANG Y,et al.Synthesis and characterization of multi-functional linear-dendritic block copolymer for intracellular delivery of antitumor drugs[J].Int J Pharm,2013,452(1/2):363-373.
[11]KARINAGA R,KOUMOTO K,MIZU M,et al.PEG-appended beta-(1:>3)-D-glucan schizophyllan to deliver antisense-oligonucleotides with avoiding lysosomal degradation[J].Biomaterials,2005,26(23):4866-4873.
[12]WANG J,WANG Y G,LIANG W.Delivery of drugs to cell membranes by encapsulation in PEG-PE micelles[J].JControl Release,2012,160(3):637-651.
[13]MUSACCHIO T,VAZE O,D’SOUZA G,et al.Effective stabilization and delivery of siRNA:reversible siRNA-phospholipid conjugate in nanosized mixed polymeric micelles[J].Bioconjug Chem,2010,21(8):1530-1536.