Tat-TOS,磷脂酶D抑制剂和阿霉素的多组分脂质体递送系统的制备及体内外评价（英文）
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摘要
本研究的目的是将α-维生素E琥珀酸酯的偶联物Tat-TOS与磷脂酶D抑制剂FIPI以及抗肿瘤药阿霉素共同包载于脂质体递送系统中,用于抗肿瘤转移。首先采用固相合成法合成了Tat-TOS,并对其进行结构确证,用诱导凋亡法考察了游离Tat-TOS和Tat-脂质体的体外诱导细胞凋亡的能力,并评价了表面修饰了Tat-TOS脂质体的肺部靶向特性,采用薄膜分散法结合pH梯度法与后插入法制备了修饰有Tat-TOS的载FIPI和阿霉素的多组分脂质体,并进行理化性质测定,最后评价了细胞对该制剂的体外摄取能力。研究结果表明,所制备脂质体具备粒径分布均一,粒径小的特点,FIPI和DOX的包封率均超过85%,无论是游离还是脂质体包载的Tat-TOS均能显著提高诱导肿瘤细胞凋亡的能力,表面修饰了Tat-TOS的脂质体具有明显聚集于健康肺组织和肿瘤转移肺组织,多组分脂质体具有最强的细胞摄取能力,预示该脂质体制剂具有更强的体外抗转移功效。
In the present study, we aimed to co-load the α-TOS conjugate Tat-TOS with the phospholipase D inhibitor FIPI and the antitumor drug doxorubicin(DOX) in a liposome delivery system for antitumor metastasis. Firstly, Tat-TOS was synthesized by solid-phase synthesis, and its structure was confirmed. The ability of free and liposomal Tat-TOS to induce apoptosis in vitro was evaluated by flow cytometry. Biodistribution of Tat-TOS-loaded liposomes was investigated by a molecular imaging system. Multi-component-loaded liposomes modified with Tat-TOS containing FIPI and DOX was prepared by thin film dispersion method in combination with p H gradient method and post-insertion method. Physicochemical properties were determined, and the in vitro uptake ability of the formulations was evaluated. The results showed that the prepared liposomes were characterized by a uniform particle size distribution and small particle size. The encapsulation efficiency of FIPI and DOX exceeded 85%. Both free and liposomal Tat-TOS significantly improved the activity of inducing apoptosis of tumor cells. The liposomes modified with Tat-TOS were apparently accumulated in normal lung tissue and tumor metastasized lung. Multi-component-loaded liposomes exhibited the strongest cell uptake capacity, suggesting a stronger anti-metastatic effect and anti-tumor activity in vivo.
In the present study, we aimed to co-load the α-TOS conjugate Tat-TOS with the phospholipase D inhibitor FIPI and the antitumor drug doxorubicin(DOX) in a liposome delivery system for antitumor metastasis. Firstly, Tat-TOS was synthesized by solid-phase synthesis, and its structure was confirmed. The ability of free and liposomal Tat-TOS to induce apoptosis in vitro was evaluated by flow cytometry. Biodistribution of Tat-TOS-loaded liposomes was investigated by a molecular imaging system. Multi-component-loaded liposomes modified with Tat-TOS containing FIPI and DOX was prepared by thin film dispersion method in combination with p H gradient method and post-insertion method. Physicochemical properties were determined, and the in vitro uptake ability of the formulations was evaluated. The results showed that the prepared liposomes were characterized by a uniform particle size distribution and small particle size. The encapsulation efficiency of FIPI and DOX exceeded 85%. Both free and liposomal Tat-TOS significantly improved the activity of inducing apoptosis of tumor cells. The liposomes modified with Tat-TOS were apparently accumulated in normal lung tissue and tumor metastasized lung. Multi-component-loaded liposomes exhibited the strongest cell uptake capacity, suggesting a stronger anti-metastatic effect and anti-tumor activity in vivo.
引文
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[26]Yu,Y.;Wang,Z.H.;Zhang,L.;Yao,H.J.;Zhang,Y.;Li,R.J.;Ju,R.J.;Wang,X.X.;Zhou,J.;Li,N.;Lu,W.L.Mitochondrial targeting topotecan-loaded liposomes for treating drug-resistant breast cancer and inhibiting invasive metastases of melanoma.Biomaterials.2012,33,1808–1820.
[27]Guo,Z.;Li,W.;Yuan,Y.;Zheng,K.;Tang,Y.;Ma,K.;Cui,C.;Wang,L.;He,B.;Zhang,Q.Improvement of chemosensitivity and inhibition of migration via targeting tumor epithelial-to-mesenchymal transition cells by ADH-1-modified liposomes.Drug Deliv.2018,25,112–121.
[28]Santos-Cuevas,C.L.;Ferro-Flores,G.;Arteaga de Murphy,C.;Ramirez Fde,M.;Luna-Gutierrez,M.A.;Pedraza-Lopez,M.;Garcia-Becerra,R.;Ordaz-Rosado,D.Design,preparation,in vitro and in vivo evaluation of(99m)Tc-N2S2-Tat(49-57)-bombesin:a target-specific hybrid radiopharmaceutical.Int.J.Pharm.2009,375,75–83.
[2]Maishi,N.;Hida,K.Tumor endothelial cells accelerate tumor metastasis.Cancer Sci.2017,108,1921–1926.
[3]Luo,X.;Qiu,Y.;Jiang,Y.;Chen,F.;Jiang,L.;Zhou,Y.;Dan,H.;Zeng,X.;Lei,Y.L.;Chen,Q.Long non-coding RNA implicated in the invasion and metastasis of head and neck cancer:possible function and mechanisms.Mol.Cancer.2018,17,14.
[4]Zhang,Y.;Frohman,M.A.Cellular and Physiological Roles for Phospholipase D1 in Cancer.J.Biol.Chem.2014,289,22567–22574.
[5]Frohman,M.A.The phospholipase D superfamily as therapeutic targets.Trends Pharmacol.Sci.2015,36,137–144.
[6]Chen,Y.;Zheng,Y.;Foster,D.A.Phospholipase D confers rapamycin resistance in human breast cancer cells.Oncogene.2003,22,3937–3942.
[7]Saito,M.;Iwadate,M.;Higashimoto,M.;Ono,K.;Takebayashi,Y.;Takenoshita,S.Expression of phospholipase D2 in human colorectal carcinoma.Oncol.Rep.2007,18,1329–1334.
[8]Uchida,N.;Okamura,S.;Kuwano,H.Phospholipase D activity in human gastric carcinoma.Anticancer Res.1999,19,671–675.
[9]Zhao,Y.;Ehara,H.;Akao,Y.;Shamoto,M.;Nakagawa,Y.;Banno,Y.;Deguchi,T.;Ohishi,N.;Yagi,K.;Nozawa,Y.Increased activity and intranuclear expression of phospholipase D2 in human renal cancer.Biochem.Biophys.Res.Commun.2000,278,140–143.
[10]Noh,D.Y.;Ahn,S.J.;Lee,R.A.;Park,I.A.;Kim,J.H.;Suh,P.G.;Ryu,S.H.;Lee,K.H.;Han,J.S.Overexpression of phospholipase D1 in human breast cancer tissues.Cancer Lett.2000,161,207–214.
[11]Brown,H.A.;Thomas,P.G.;Lindsley,C.W.Targeting phospholipase D in cancer,infection and neurodegenerative disorders.Nat.Rev.Drug Discov.2017,16,351–367.
[12]Qu,Q.;Ma,X.;Zhao,Y.Anticancer Effect of alphaTocopheryl Succinate Delivered by Mitochondria-Targeted Mesoporous Silica Nanoparticles.ACS Appl.Mater.Interfaces.2016,8,34261–34269.
[13]Barnett,K.T.;Fokum,F.D.;Malafa,M.P.Vitamin E succinate inhibits colon cancer liver metastases.J.Surg.Res.2002,106,292–298.
[14]Zhang,M.;Altuwaijri,S.;Yeh,S.RRR-alpha-tocopheryl succinate inhibits human prostate cancer cell invasiveness.Oncogene.2004,23,3080–3088.
[15]Crispen,P.L.;Uzzo,R.G.;Golovine,K.;Makhov,P.;Pollack,A.;Horwitz,E.M.;Greenberg,R.E.;Kolenko,V.M.Vitamin E succinate inhibits NF-kappa B and prevents the development of a metastatic phenotype in prostate cancer cells:implications for chemoprevention.Prostate.2007,67,582–590.
[16]Kang,D.W.;Min do,S.Platelet derived growth factor increases phospholipase D1 but not phospholipase D2expression via NFkappa B signaling pathway and enhances invasion of breast cancer cells.Cancer Lett.2010,294,125–133.
[17]Kang,D.W.;Park,M.H.;Lee,Y.J.;Kim,H.S.;Kwon,T.K.;Park,W.S.;Min,D.S.Phorbol ester up-regulates phospholipase D1 but not phospholipase D2 expression through a PKC/Ras/ERK/NF kappa B-dependent pathway and enhances matrix metalloproteinase-9 secretion in colon cancer cells.J.Biol.Chem.2008,283,4094–4104.
[18]Abramkin,S.;Valiahdi,S.M.;Jakupec,M.A.;Galanski,M.;Metzler-Nolte,N.;Keppler,B.K.Solid-phase synthesis of oxaliplatin-TAT peptide bioconjugates.Dalton Trans.2012,41,3001–3005.
[19]Sylvester,B.;Porfire,A.;Muntean,D.M.;Vlase,L.;Luput,L.;Licarete,E.;Sesarman,A.;Alupei,M.C.;Banciu,M.;Achim,M.;Tomuta,I.Optimization of prednisolone-loaded long-circulating liposomes via application of Quality by Design(Qb D)approach.J.Liposome Res.2018,28,49–61.
[20]Zhen,S.;Takahashi,Y.;Narita,S.;Yang,Y.C.;Li,X.Targeted delivery of CRISPR/Cas9 to prostate cancer by modified g RNA using a flexible aptamer-cationic liposome.Oncotarget.2017,8,9375–9387.
[21]Liu,Y.;Yoo,S.D.;Li,L.;Fang,L.;Wen,Z.;Li,T.Formulation and characterization of boanmycin-loaded liposomes prepared by p H gradient experimental design.Drug Deliv.2012,19,90–101.
[22]Lei,J.;Cong,S.;Song,M.;Zhang,W.;Peng,G.;Li,X.;Liu,Y.Combination of doxorubicin with harmine-loaded liposomes exerting synergistic antitumor efficacy.Drug Dev.Ind.Pharm.2018,44,570–581.
[23]Song,Y.;Tang,C.;Yin,C.Enhanced antitumor efficacy of arginine modified amphiphilic nanoparticles co-delivering doxorubicin and i Sur-p DNA via the multiple synergistic effect.Biomaterials.2018,150,1–13.
[24]Wang,Z.;Yu,Y.;Dai,W.;Cui,J.;Wu,H.;Yuan,L.;Zhang,H.;Wang,X.;Wang,J.;Zhang,X.;Zhang,Q.A specific peptide ligand-modified lipid nanoparticle carrier for the inhibition of tumor metastasis growth.Biomaterials.2013,34,756–764.
[25]Minn,A.J.;Gupta,G.P.;Siegel,P.M.;Bos,P.D.;Shu,W.;Giri,D.D.;Viale,A.;Olshen,A.B.;Gerald,W.L.;Massague,J.Genes that mediate breast cancer metastasis to lung.Nature.2005,436,518–524.
[26]Yu,Y.;Wang,Z.H.;Zhang,L.;Yao,H.J.;Zhang,Y.;Li,R.J.;Ju,R.J.;Wang,X.X.;Zhou,J.;Li,N.;Lu,W.L.Mitochondrial targeting topotecan-loaded liposomes for treating drug-resistant breast cancer and inhibiting invasive metastases of melanoma.Biomaterials.2012,33,1808–1820.
[27]Guo,Z.;Li,W.;Yuan,Y.;Zheng,K.;Tang,Y.;Ma,K.;Cui,C.;Wang,L.;He,B.;Zhang,Q.Improvement of chemosensitivity and inhibition of migration via targeting tumor epithelial-to-mesenchymal transition cells by ADH-1-modified liposomes.Drug Deliv.2018,25,112–121.
[28]Santos-Cuevas,C.L.;Ferro-Flores,G.;Arteaga de Murphy,C.;Ramirez Fde,M.;Luna-Gutierrez,M.A.;Pedraza-Lopez,M.;Garcia-Becerra,R.;Ordaz-Rosado,D.Design,preparation,in vitro and in vivo evaluation of(99m)Tc-N2S2-Tat(49-57)-bombesin:a target-specific hybrid radiopharmaceutical.Int.J.Pharm.2009,375,75–83.