穿膜肽TAT修饰载丹酚酸B脂质体的制备及其抑制人皮肤成纤维细胞增殖与迁移初步研究
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摘要
目的制备具有防治增生性瘢痕(HS)作用的载丹酚酸B的穿膜肽TAT修饰脂质体(SAB-TAT-LIP),建立其质量评价方法,并初步考察其对体外人皮肤成纤维(HSF)细胞增殖和迁移的影响。方法采用pH梯度逆向蒸发法制备脂质体,超滤法测其包封率,以包封率为评价指标,采用Box-Behnken响应面法优化脂质体的处方工艺;考察其形态、粒径、Zeta电位、体外释放、体外透皮吸收和稳定性等理化性质;在此基础上采用MTT法考察其对HSF细胞增殖的作用,采用划痕法和Transwell小室法考察其对HSF细胞迁移和侵袭的作用。结果 SAB-TAT-LIP的药物包封率为(86.70±0.85)%,平均粒径为(219.90±5.09)nm,Zeta电位(-9.25±0.92)m V,体外24h累积释放率为62.49%,无突释效应,体外32h皮肤累积透过率为17.21%,透过速率为(28.33±4.9)μg/(cm2·h),真皮层滞留量为(44.39±6.87)μg/cm2,4℃放置10d稳定性良好。SAB-TAT-LIP能够显著地抑制HSF细胞的增殖、迁移和侵袭,与对照组相比,差异显著(P<0.01)。结论优化得到的SAB-TAT-LIP包封率较高、粒径较小,体外释放和透皮行为均满足局部透皮给药制剂的体外释放和透皮规律,对体外HSF细胞的增殖、迁移和侵袭具有抑制作用。
Objective To prepare the liposomes of salvianolic acid B modified with cell penetrating peptide TAT(SAB-TAT-LIP), of which has effects on preventing and treating hypertrophic scars(HS), and establish the method of quality evaluation, as well as preliminarily investigate the effect on the proliferation and migration of human skin fibroblasts(HSF). Methods Liposomes were prepared by pH gradient reverse-phase evaporation method, and the entrapment efficiency was measured by ultrafiltration. Box-Behnken design was performed to optimize the formulation of liposomes by using encapsulation rate as evaluating index. The physicochemical properties of liposomes including morphology, entrapment efficiency, particle size, zeta potential, in vitro release and transdermal absorption, and stability were studied. In addition, the effect of liposomes on proliferation of HSF was examined by MTT assay, and the effect of liposomes on migration of HSF was investigated by scratching method and Transwell assay. Results Based on the optimal formulation of SAB-TAT-LIP, the entrapment efficiency of salvianolic acid B was(86.70 ± 0.85)%, the average particle size was(219.90 ± 5.09) nm, and the zeta potential was(-9.25 ± 0.92). The in vitro 24 h cumulative release was 62.49% of the total drug with no burst effect. The in vitro 32 h cumulated skin penetration rate was 17.21%, the permeance rate was(28.33 ± 4.9) μg/(cm2·h), and the retention volume of dermis was(44.39 ± 6.87) μg/cm2. The stability was good when placed at 4 ℃ for 10 d. The in vitro cell studies showed that SAB-TAT-LIP can significantly inhibit the proliferation, migration and invasion of human skin fibroblasts, compared with the control group(P < 0.01). Conclusion The optimized SAB-TAT-LIP have higher encapsulation efficiency, smaller particle size, good sustained release effect, and good dermal retention effect which all satisfy the in vitro release and transdermal regulation of local transdermal preparation, and it can significantly inhibit the proliferation, migration and invasion of human skin fibroblasts in vitro.
Objective To prepare the liposomes of salvianolic acid B modified with cell penetrating peptide TAT(SAB-TAT-LIP), of which has effects on preventing and treating hypertrophic scars(HS), and establish the method of quality evaluation, as well as preliminarily investigate the effect on the proliferation and migration of human skin fibroblasts(HSF). Methods Liposomes were prepared by pH gradient reverse-phase evaporation method, and the entrapment efficiency was measured by ultrafiltration. Box-Behnken design was performed to optimize the formulation of liposomes by using encapsulation rate as evaluating index. The physicochemical properties of liposomes including morphology, entrapment efficiency, particle size, zeta potential, in vitro release and transdermal absorption, and stability were studied. In addition, the effect of liposomes on proliferation of HSF was examined by MTT assay, and the effect of liposomes on migration of HSF was investigated by scratching method and Transwell assay. Results Based on the optimal formulation of SAB-TAT-LIP, the entrapment efficiency of salvianolic acid B was(86.70 ± 0.85)%, the average particle size was(219.90 ± 5.09) nm, and the zeta potential was(-9.25 ± 0.92). The in vitro 24 h cumulative release was 62.49% of the total drug with no burst effect. The in vitro 32 h cumulated skin penetration rate was 17.21%, the permeance rate was(28.33 ± 4.9) μg/(cm2·h), and the retention volume of dermis was(44.39 ± 6.87) μg/cm2. The stability was good when placed at 4 ℃ for 10 d. The in vitro cell studies showed that SAB-TAT-LIP can significantly inhibit the proliferation, migration and invasion of human skin fibroblasts, compared with the control group(P < 0.01). Conclusion The optimized SAB-TAT-LIP have higher encapsulation efficiency, smaller particle size, good sustained release effect, and good dermal retention effect which all satisfy the in vitro release and transdermal regulation of local transdermal preparation, and it can significantly inhibit the proliferation, migration and invasion of human skin fibroblasts in vitro.
引文
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[2]Chawla S,Ghosh S.Regulation of fibrotic changes by the synergistic effects of cytokines,dimensionality and matrix:Towards the development of an in vitro human dermal hypertrophic scar model[J].Acta Biomater,2018,69(15):131-145.
[3]张慧迪,时军,陈桂添,等.丹皮酚VEGF抗体修饰脂质体的制备及其真皮滞留效应考察[J].中国实验方剂学杂志,2018,24(3):19-24.
[4]Curran T A,Ghahary A.Evidence of a role for fibrocyte and keratinocyte-like cells in the formation of hypertrophic scars[J].J Burn Care Res,2013,34(2):227-231.
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[6]Li G,Zhou R,Zhang Q,et al.Fibroproliferative effect of micro RNA-21 in hypertrophic scar derived fibroblasts[J].Exp Cell Res,2016,345(1):93-99.
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[8]鞠爱春,耿诗涵,杨欣鹏,等.丹酚酸B鼻腔给药对脑缺血损伤大鼠学习记忆能力及神经再生的影响[J].中草药,2017,48(12):2481-2485.
[9]夏杨,张惠军,聂亚莉.丹酚酸B预处理对心肌缺血/再灌注损伤能量代谢的影响[J].药物评价研究,2018,41(12):2210-2213.
[10]Liu Q,Chu H,Ma Y,et al.Salvianolic acid B attenuates experimental pulmonary fibrosis through inhibition of the TGF-βsignaling pathway[J].Sci Rep,2016,6(9):27610.
[11]Liu M,Xu H,Zhang L,et al.Salvianolic acid B inhibits myofibroblast transdifferentiation in experimental pulmonary fibrosis via the up-regulation of Nrf2[J].Biochem Biophys Res Commun,2018,495(1):325-331.
[12]Zhang J,An S J,Fu J Q,et al.Mixed aqueous extract of Salvia Miltiorrhiza reduces blood pressure through inhibition of vascular remodelling and oxidative stress in spontaneously hypertensive rats[J].Cell Physiol Biochem,2016,40(1/2):347-360.
[13]Wang M,Sun G B,Sun X,et al.Cardioprotective effect of salvianolic acid B against arsenic trioxide-induced injury in cardiac H9c2 cells via the PI3K/Akt signal pathway[J].Toxicol Lett,2013,216(2/3):100-107.
[14]Akhtar N,Khan R A.Liposomal systems as viable drug delivery technology for skin cancer sites with an outlook on lipid-based delivery vehicles and diagnostic imaging inputs for skin conditions[J].Prog Lipid Res,2016,64(10):192-230.
[15]马敏,瞿叶清,殳叶婷,等.应用复合磷脂脂质体人工皮肤膜表征香附四物汤活性部位生物药剂学性质[J].中草药,2018,49(5):1048-1055.
[16]高迪,王保军,裴玉,等.脂质体平衡透析与液质联用技术筛选脊痛宁片物质基础研究[J].中草药,2017,48(4):668-672.
[17]Fu H,Hu G L,He Q.Preparation of cell penetrating peptide TAT and cleavable PEG co-modified liposomes loaded with paclitaxel and its in vitro apoptosis assay[J].Acta Pharm Sin B,2014,49(7):1054-1061.
[18]Fu H,Shi K,Hu G,et al.Tumor-targeted paclitaxel delivery and enhanced penetration using TAT-decorated liposomes comprising redox-responsive poly(ethylene glycol)[J].J Pharm Sci,2015,104(3):1160-1173.
[19]Isacchi B,Fabbri V,Galeotti N,et al.Salvianolic acid Band its liposomal formulations:Anti-hyperalgesic activity in the treatment of neuropathic pain[J].Eur J Pharm Sci,2011,44(4):552-558.
[20]马金鹏,舒乐新,刘志东.丹酚酸B脂质体的制备及其体外释药的研究[J].中草药,2011,42(1):65-68.
[21]Zhang L,Han L,Sun X,et al.The use of PEGylated liposomes to prolong the circulation lifetime of salvianolic acid B[J].Fitoterapia,2012,83(4):678-679.
[22]Constable P D.Acid-base assessment:When and how to apply the Henderson-Hasselbalch equation and strong ion difference theory[J].Vet Clin North Am Food Anim Pract,2014,30(2):295-316.
[23]Yuan D F,Zong T L,Gao H L,et al.Cell penetrating peptide TAT and brain tumor targeting peptide T7 dual modified liposome preparation and in vitro targeting evaluation[J].Acta Pharm Sin B,2015,50(1):104-110.
[24]Fu H,Shi K,Hu G,et al.Tumor-targeted paclitaxel delivery and enhanced penetration using TAT-decorated liposomes comprising redox-responsive poly(ethylene glycol)[J].J Pharm Sci,2015,104(3):1160-1173.
[25]Wang R H,Cao H M,Tian Z J,et al.[Retracted]efficacy of dual-functional liposomes containing paclitaxel for treatment of lung cancer[J].Oncol Rep,2015,33(2):783-791.
[26]Qin Y,Zhang Q,Chen H,et al.Comparison of four different peptides to enhance accumulation of liposomes into the brain[J].J Drug Target,2012,20(3):235-245.
[27]Kwon S S,Kim S Y,Kong B J,et al.Cell penetrating peptide conjugated liposomes as transdermal delivery system of Polygonum aviculare L.extract[J].Int JPharm,2015,483(1/2):26-37.
[28]Wang Y,Su W,Li Q,et al.Preparation and evaluation of lidocaine hydrochloride-loaded TAT-conjugated polymeric liposomes for transdermal delivery[J].Int JPharm,2013,441(1/2):748-756.
[2]Chawla S,Ghosh S.Regulation of fibrotic changes by the synergistic effects of cytokines,dimensionality and matrix:Towards the development of an in vitro human dermal hypertrophic scar model[J].Acta Biomater,2018,69(15):131-145.
[3]张慧迪,时军,陈桂添,等.丹皮酚VEGF抗体修饰脂质体的制备及其真皮滞留效应考察[J].中国实验方剂学杂志,2018,24(3):19-24.
[4]Curran T A,Ghahary A.Evidence of a role for fibrocyte and keratinocyte-like cells in the formation of hypertrophic scars[J].J Burn Care Res,2013,34(2):227-231.
[5]Shu B,Xie J L,Xu Y B,et al.Directed differentiation of skin-derived precursors into fibroblast-like cells[J].Int JClin Exp Pathol,2014,7(4):1478-1486.
[6]Li G,Zhou R,Zhang Q,et al.Fibroproliferative effect of micro RNA-21 in hypertrophic scar derived fibroblasts[J].Exp Cell Res,2016,345(1):93-99.
[7]中国药典[S].一部.2015.
[8]鞠爱春,耿诗涵,杨欣鹏,等.丹酚酸B鼻腔给药对脑缺血损伤大鼠学习记忆能力及神经再生的影响[J].中草药,2017,48(12):2481-2485.
[9]夏杨,张惠军,聂亚莉.丹酚酸B预处理对心肌缺血/再灌注损伤能量代谢的影响[J].药物评价研究,2018,41(12):2210-2213.
[10]Liu Q,Chu H,Ma Y,et al.Salvianolic acid B attenuates experimental pulmonary fibrosis through inhibition of the TGF-βsignaling pathway[J].Sci Rep,2016,6(9):27610.
[11]Liu M,Xu H,Zhang L,et al.Salvianolic acid B inhibits myofibroblast transdifferentiation in experimental pulmonary fibrosis via the up-regulation of Nrf2[J].Biochem Biophys Res Commun,2018,495(1):325-331.
[12]Zhang J,An S J,Fu J Q,et al.Mixed aqueous extract of Salvia Miltiorrhiza reduces blood pressure through inhibition of vascular remodelling and oxidative stress in spontaneously hypertensive rats[J].Cell Physiol Biochem,2016,40(1/2):347-360.
[13]Wang M,Sun G B,Sun X,et al.Cardioprotective effect of salvianolic acid B against arsenic trioxide-induced injury in cardiac H9c2 cells via the PI3K/Akt signal pathway[J].Toxicol Lett,2013,216(2/3):100-107.
[14]Akhtar N,Khan R A.Liposomal systems as viable drug delivery technology for skin cancer sites with an outlook on lipid-based delivery vehicles and diagnostic imaging inputs for skin conditions[J].Prog Lipid Res,2016,64(10):192-230.
[15]马敏,瞿叶清,殳叶婷,等.应用复合磷脂脂质体人工皮肤膜表征香附四物汤活性部位生物药剂学性质[J].中草药,2018,49(5):1048-1055.
[16]高迪,王保军,裴玉,等.脂质体平衡透析与液质联用技术筛选脊痛宁片物质基础研究[J].中草药,2017,48(4):668-672.
[17]Fu H,Hu G L,He Q.Preparation of cell penetrating peptide TAT and cleavable PEG co-modified liposomes loaded with paclitaxel and its in vitro apoptosis assay[J].Acta Pharm Sin B,2014,49(7):1054-1061.
[18]Fu H,Shi K,Hu G,et al.Tumor-targeted paclitaxel delivery and enhanced penetration using TAT-decorated liposomes comprising redox-responsive poly(ethylene glycol)[J].J Pharm Sci,2015,104(3):1160-1173.
[19]Isacchi B,Fabbri V,Galeotti N,et al.Salvianolic acid Band its liposomal formulations:Anti-hyperalgesic activity in the treatment of neuropathic pain[J].Eur J Pharm Sci,2011,44(4):552-558.
[20]马金鹏,舒乐新,刘志东.丹酚酸B脂质体的制备及其体外释药的研究[J].中草药,2011,42(1):65-68.
[21]Zhang L,Han L,Sun X,et al.The use of PEGylated liposomes to prolong the circulation lifetime of salvianolic acid B[J].Fitoterapia,2012,83(4):678-679.
[22]Constable P D.Acid-base assessment:When and how to apply the Henderson-Hasselbalch equation and strong ion difference theory[J].Vet Clin North Am Food Anim Pract,2014,30(2):295-316.
[23]Yuan D F,Zong T L,Gao H L,et al.Cell penetrating peptide TAT and brain tumor targeting peptide T7 dual modified liposome preparation and in vitro targeting evaluation[J].Acta Pharm Sin B,2015,50(1):104-110.
[24]Fu H,Shi K,Hu G,et al.Tumor-targeted paclitaxel delivery and enhanced penetration using TAT-decorated liposomes comprising redox-responsive poly(ethylene glycol)[J].J Pharm Sci,2015,104(3):1160-1173.
[25]Wang R H,Cao H M,Tian Z J,et al.[Retracted]efficacy of dual-functional liposomes containing paclitaxel for treatment of lung cancer[J].Oncol Rep,2015,33(2):783-791.
[26]Qin Y,Zhang Q,Chen H,et al.Comparison of four different peptides to enhance accumulation of liposomes into the brain[J].J Drug Target,2012,20(3):235-245.
[27]Kwon S S,Kim S Y,Kong B J,et al.Cell penetrating peptide conjugated liposomes as transdermal delivery system of Polygonum aviculare L.extract[J].Int JPharm,2015,483(1/2):26-37.
[28]Wang Y,Su W,Li Q,et al.Preparation and evaluation of lidocaine hydrochloride-loaded TAT-conjugated polymeric liposomes for transdermal delivery[J].Int JPharm,2013,441(1/2):748-756.