CN107226839B - 一种rgd多肽偶联的酞菁硅光敏剂的合成和应用 - Google Patents
一种rgd多肽偶联的酞菁硅光敏剂的合成和应用 Download PDFInfo
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- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
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- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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Abstract
本发明涉及一种以RGD多肽为靶向基团的酞菁硅光敏剂及其制备方法和在肿瘤光动力治疗方面的应用。以酞菁硅(SiPc)作为光敏部分与RGD多肽配基连接,并在结构中引入聚乙二醇(PEG)和含羧酸基团的片段,能制备出一系列具有肿瘤靶向性的新型光敏剂。其中的一个偶联化合物RGD‑(Linker)2‑Glu‑SiPc具有很好的光物理和光动力性能,对于受体阳性肿瘤细胞的EC50值在10‑20nM之间,并可在一次给药的情况下,治愈小鼠身上的恶性胶质瘤,且连续观察35天没有出现复发,在肿瘤光动力治疗领域显示出了很好的应用前景。
Description
本发明属于肿瘤光动力治疗用新型光敏剂领域,涉及一种以RGD多肽为靶向基团的酞菁硅光敏剂及其制备方法和在肿瘤光动力治疗方面的应用。
背景技术
作为光动力治疗(Photodynamic therapy,PDT)用第二代光敏剂的典型代表—酞菁光敏剂由于具有优越的光物理性质,在红外到近红外区域(>650nm)具有较强的光吸收(消光系数ε>1×105升/摩尔·厘米)和较高的单线态氧量子产率等诸多优点,一直是光敏剂领域的研究热点,但是该类光敏剂结构中含有疏水性极强的酞菁环,在水中溶解度很小,而且容易聚集导致光学淬灭,使得大多数酞菁光敏剂很难在PDT应用中取得好的实际效果。
将酞菁环与多肽配基结合是一种非常有用的优化酞菁类化合物物理性质的方法,因为多肽类配基有很好的水溶性和受体靶向能力,酞菁类化合物与多肽配基结合后可以明显增加整个化合物的水溶性,同时也可大大降低酞菁类化合物的聚集趋势,赋予其肿瘤靶向特异性。目前,国内外研究大多集中在酞菁锌-多肽相结合的化合物方面,虽然该类化合物具有一定的水溶性和肿瘤靶向性,但是由于酞菁锌光活性较弱,EC50值仅在1-10μM之间,尚不能满足作为光敏剂在体内癌症光动力治疗中应用的性能要求。
相对于酞菁锌,酞菁硅(SiPc)具有更高的光动力活性,EC50值在纳摩(nM)的范围内。因此,用酞菁硅与多肽结合构建的光敏剂比酞菁锌类光敏剂会具有更好的光动力活性。本发明中,我们以酞菁硅作为光敏部分,以Arg-Gly-Asp-d-Phe-Lys(RGDfK多肽,简称RGD)多肽环状序列作为配基,合成出一系列新型光敏剂,并进行了光物理以及体内外生物活性评价。研究发现,其中的一个偶联化合物RGD-(Linker)2-Glu-SiPc具有很好的光物理和光动力学性能,对于受体阳性肿瘤细胞的EC50值在10-20nM之间,并可在一次给药的情况下,可治愈小鼠身上的恶性胶质瘤,且连续观察35天没有出现复发,在肿瘤光动力治疗领域显示出了很好的应用前景。
发明内容
本发明以酞菁硅作为光敏部分,以Arg-Gly-Asp-d-Phe-Lys(RGDfK多肽,简称RGD)多肽环状序列作为配基,合成出一系列新型光敏剂,并进行了光物理以及体内外生物活性表征。简称为.RGD-SiPcRGD-Linker-SiPc,RGD-(Linker)2-SiPc和RGD-(Linker)2-Glu-SiPc的一系列化合物的结构式为:
4,RGD-SiPc(此时R=R1)
5.RGD-Linker-SiPc(此时R=R2)
6.RGD-(Linker)2-SiPc(此时R=R3)
7.RGD-(Linker)2-Glu-SiPc(此时R=R4)
在本发明中,我们用RGD多肽轴向取代与SiPc偶联,设计并合成出了新型具有肿瘤靶向性的光敏剂。用SiPc作为光敏部分,其在近红外区域有较强的吸收(吸收波长为λ=681nm,logε=5.23),并且具有较强的单线态氧量子产率(0.32)。RGD多肽用于提高酞菁环的水溶性和靶向性。考虑到酞菁硅很强疏水性可能会影响RGD与受体的结合力,我们在结构中引入一个或两个PEG连接链以增加配基和SiPc之间的距离,同时引入了含有羧基的具有较强亲水性的谷氨酸片段。我们利用三苯基二氯树脂采用Fmoc固相合成多肽的方法合成出了带有和不带PEG和/或谷氨酸连接链的RGD配基(RGD,RGD-Linker,RGD-(Linker)2和RGD-(Linker)2-Glu)。利用商业上可以买到的SiPcCl2作为起始原料与1-(2-羟乙基)哌嗪进行反应合成出SiPc-PQ,再加入二甘醇酐合成出SiPc-COOH。然后,SiPc-COOH与RGD或PEG改性的RGD多肽配基通过典型的缩合反应得到产物RGD-SiPc,RGD-Linker-SiPc,RGD-(Linker)2-SiPc和RGD-(Linker)2-Glu-SiPc。产物经HPLC纯化后加入乙醚沉淀,再用DMSO溶解。产物利用HPLC进行纯度分析。与没有偶联配基的酞菁类化合物相比,所有偶联了配基的化合物都表现出了很好的水溶性,尤其是引入了谷氨酸片段的化合物RGD-(Linker)2-Glu-SiPc。
我们进一步研究了这些化合物在不同溶液中的光学特性,RGD-(Linker)2-Glu-SiPc的吸收光谱几乎完全没有聚集,结果表明,与亲水性多肽偶联,并引入PEG连接链和羧基官能团能够显著提高SiPcs的水溶性,并有助于解决SiPcs在水溶液中的聚集问题。
通过MTT实验对这些偶联物在肿瘤细胞水平上体外光动力学活性进行了评价。我们用人体胶质瘤U87-MG细胞系、人前列腺癌22RV1和PC3细胞系,以及人类表皮鳞癌A431细胞系进行了评估。RGD-(Linker)2-Glu-SiPc具有最强的光活性,相比其他3个偶联物而言在肿瘤光动力治疗中是一种比较好的光敏剂。这种光敏剂在细胞水平上有最强的细胞杀伤力,具有较好的水溶性,并且在水溶液中不易发生聚集。因此,我们选择这个化合物进行体内动物水平上的评价。我们采用已经建立的U87-MG异种移植肿瘤模型进行了体内光动力活性的研究。对于注射过化合物RGD-(Linker)2-Glu-SiPc治疗组经光照后肿瘤的体积显著缩小。所有的肿瘤在第14天被全部治愈,并且在观察到第35天没有复发。在整个治疗过程中,没有观察到小鼠体重下降,表明光照和化合物RGD-(Linker)2-Glu-SiPc都没有对小鼠引起严重的毒副作用。RGD-(Linker)2-Glu-SiPc在U87-MG肿瘤模型上的功效表现出其在肿瘤光动力治疗上具有的极大的临床应用潜力。
附图说明
图1是RGD多肽的合成过程示意图
图2是RGD-Linker,RGD-(Linker)2和RGD-(Linker)2-Glu的合成过程示意图
图3是RGD-SiPc,RGD-Linker-SiPc,RGD-(Linker)2-SiPc和RGD-(Linker)2-Glu-SiPc的合成过程示意图
图4是RGD,RGD-Linker,RGD-(Linker)2和RGD-(Linker)2-Glu的高效液相色谱(HPLC)分析(检测器波长为220nm)
图5是SiPc-PQ,SiPc-COOH,RGD-SiPc,RGD-Linker-SiPc,RGD-(Linker)2-SiPc和RGD-(Linker)2-Glu-SiPc的高效液相色谱HPLC分析(检测器波长为220nm)
图6是SiPc-PQ,SiPc-COOH,RGD-SiPc,RGD-Linker-SiPc,RGD-(Linker)2-SiPc和RGD-(Linker)2-Glu-SiPc紫外吸收光谱图(吸收峰678nm)
表1SiPc-PQ,SiPc-COOH,RGD-SiPc,RGD-Linker-SiPc,RGD-(Linker)2-SiPc和RGD-(Linker)2-Glu-SiPc的光谱数据
图7是SiPc-COOH,RGD-SiPc,RGD-Linker-SiPc,RGD-(Linker)2-SiPc和RGD-(Linker)2-Glu-SiPc在不同细胞系上对照实验结果
图8是SiPc-COOH,RGD-SiPc,RGD-Linker-SiPc,RGD-(Linker)2-SiPc和RGD-(Linker)2-Glu-SiPc在细胞水平上药物活性研究
表2SiPc-COOH,RGD-SiPc,RGD-Linker-SiPc,RGD-(Linker)2-SiPc和RGD-(Linker)2-Glu-SiPc在对应细胞系的EC50值
图9是RGD-(Linker)2-Glu-SiPc对U87-MG小鼠皮下异种移植瘤的体内治疗效果研究
具体实施方式
下述实施例用于进一步说明本发明但不意味着限制本发明。
实施例1 RGD多肽的合成(附图1)
我们首先利用固相的方法合成RGD多肽化合物。合成之前1h先用二氯甲烷对树脂进行溶胀处理,我们使用的是载量为0.5mmol/g三苯基氯树脂。将Fmoc-Asp(OAll)-OH(1.0g,1.0mmol)和N,N-二异丙基乙胺(DIEA)(680μL,4.0mmol)溶解于10mL N,N-二甲基甲酰胺(DMF)中加入到固相合成器中,室温反应5h。然后将配置封闭液(二氯甲烷CH2Cl2:甲醇MeOH:DIEA=17:1:2),加入到固相合成器中封闭未反应的氯。用含有20%哌啶的DMF溶液脱除Fmoc保护基30min。四个氨基酸(Fmoc-Gly-OH,Fmoc-Arg(Pbf)-OH,Fmoc-Lys(Dde)-OHand Fmoc-D-Phe-OH)的偶联反应每步都用1.5eq.的Fmoc保护的衍生物原位加入缩合剂O-苯并三氮唑-四甲基脲六氟磷酸酯(HBTU)(2.0eq),1-羟基苯并三唑(HOBt)(1.5eq),andDIEA(4eq),溶于DMF中,室温反应5h。每步都需要DMF洗脱3次。在最后一个Fmoc去保护之前,加入苯基硅烷(PhSiH3)(24eq),四三苯基膦钯(Pd(PPh3)4)(0.25eq)溶于二氯甲烷中反应1.5h。加入1H-苯并三唑-1-基氧三吡咯烷基六氟磷酸盐(PyBOP)(1.5eq),1-羟基苯并三唑(HOBt)(1.5eq)和N,N-二异丙基乙胺DIEA(2equiv)溶于DMF溶液中室温反应过夜进行环化反应。
取环化后RGD(1.0g,0.5mmol)用2%的水合肼去保护,用DMF和二氯甲烷洗涤后真空干燥得产物resin-RGD。干燥后的树脂加入500μL洗脱液(三氟乙酸TFA/三异丙基硅烷Tris/水=95:2.5:2.5)室温下洗脱1h。洗脱液加无水乙醚(1.5mL)进行沉淀,并洗涤三次后室温干燥得到产物RGD(0.16g,收率52%)。(HRMS-ESI:m/zcalculated for C27H41N9O7[M+H]+604.3202,found 604.3202)
实施例2 RGD-Linker,RGD-(Linker)2和RGD-(Linker)2-Glu的合成(附图2)
1、简称为RGD-Linker的化合物的合成
去保护的resin-RGD(1.0g,0.5mmol)与二甘醇酐(116.1mg,1.0mmol)溶于5mL DMF中震荡反应5h得到产物resin-COOH。然后用N,N'-羰基二咪唑(0.5M CDI)对resin-COOH进行活化,反应1h。活化后的resin-COOH,0.5M 1-羟基苯并三唑(HOBt),双(3-氨丙基)二乙二醇(1.1g,5mmol)混合震荡反应5h,然后用DMF和二氯甲烷洗涤后真空干燥得产物resin-RGD-Linker。加入500μL洗脱液(三氟乙酸TFA/三异丙基硅烷Tris/水=95:2.5:2.5)室温下洗脱1h。洗脱液加无水乙醚(1.5mL)进行沉淀,并洗涤三次后室温干燥得到产物RGD-Linker(0.18g,产率39%)。(HRMS-ESI:m/z calculated for C41H67N11O13[M+H]+922.4993,found922.4987)
2、简称为RGD-(Linker)2的化合物的合成
Resin-RGD-Linker(1.0g,0.5mmol)与二甘醇酐(116.1mg,1.0mmol)一起溶于5mLDMF中震荡反应5h得到产物resin-COOH。然后用N,N'-羰基二咪唑(0.5M CDI)对resin-COOH进行活化,反应1h。活化后的resin-COOH,0.5M HOBt,双(3-氨丙基)二乙二醇(1.1g,5mmol)混合震荡反应5h,然后用DMF和二氯甲烷洗涤后真空干燥得产物resin-RGD-(Linker)2。加入500μL洗脱液(三氟乙酸TFA/三异丙基硅烷Tris/水=95:2.5:2.5)室温下洗脱1h。洗脱液加无水乙醚(1.5mL)进行沉淀,并洗涤三次后室温干燥得到产物RGD-(Linker)2(0.18g,产率30%)。(HRMS-ESI:m/z calculated for C55H93N13O19[M+H]+1240.6783,found1240.6788,[M+Na]+1262.6603,found 1262.6603)
3、简称为RGD-(Linker)2-Glu的化合物的合成
resin-RGD-(Linker)2(1.0g,0.5mmol)与Fmoc-Glu(OtBu)-OH(0.32g,0.75mmol),HBTU(0.38g,0.1mmol),HOBt(0.1g,0.75mmol)DIEA(0.26g,2mmol)一起溶于5mL DMF中震荡反应5h,然后用DMF和二氯甲烷洗涤后真空干燥得到产物resin-RGD-(Linker)2-Glu-Fmoc。取Fmoc保护的resin-RGD-(Linker)2-Glu-Fmoc(1.0g,0.5mmol)加入含有20%哌啶的DMF溶液脱除Fmoc保护基得产物resin-RGD-(Linker)2-Glu再,加入500μL洗脱液(三氟乙酸TFA/三异丙基硅烷Tris/水=95:2.5:2.5)室温下洗脱1h。洗脱液加无水乙醚(1.5mL)进行沉淀,并洗涤三次后室温干燥得到产物RGD-(Linker)2-Glu(0.15g,产率22%)。(HRMS-ESI:m/zcalculated for C60H100N14O22[M+H]+1369.7209,found 1369.7180)
实施例3简称为RGD-SiPc,RGD-Linker-SiPc,RGD-(Linker)2-SiPc和RGD-(Linker)2-Glu-SiPc的合成(附图3)
1、简称为SiPc-PQ的化合物的合成
1-(2-羟乙基)哌嗪(20.8mg,0.016mmol)和SiPcCl2(10.0mg,0.016mmol)加入到甲苯和吡啶的混合溶液中(1mL,v/v=5:1)回流10h后,减压脱除溶剂,加入二氯甲烷(CH2Cl2)稀释,再用饱和食盐水洗涤三次,合并有机相,加入无水硫酸钠(Na2SO4)干燥,脱除溶剂,硅胶柱进行纯化,洗脱液为甲醇(MeOH),二氯甲烷(CH2Cl2),并加入少量三乙胺(TEA),得到产物SiPc-PQ(5.0mg,收率42%)。(HRMS(ESI):m/z calculated for C44H42N12O2Si[M+H]+799.3396,found 799.3392)
2、简称为SiPc-COOH的化合物的合成
SiPc-PQ(10.0mg,0.013mmol)和二甘醇酐(9.0mg,0.078mmol)加入到200μL无水DMF中,室温搅拌2h后,加入无水乙醚沉淀,洗涤得到产物SiPc-COOH 3(10.0mg,收率76%)。(HRMS(ESI):m/z calculated for C52H51N12O10Si[M+H]+1031.3615,found 1031.3613)
3、简称为RGD-SiPc的光敏化合物的合成
SiPc-COOH(10.0mg,0.010mmol),1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC)(1.8mg,0.009mmol)和N-羟基丁二酰亚胺(NHS)(1.1mg,0.010mmol)加入到1.0mL无水DMF中,室温搅拌4h后,再加入DIEA(2.5mg,0.019mmol)和RGD(1.7mg,0.003mmol)继续室温搅拌过夜。经无水乙醚沉淀洗涤,再用二氯甲烷洗涤三次,经HPLC纯化后得产物RGD-SiPc(3.8mg,收率78%)。(HRMS(ESI):m/z calculated for C79H90N21O16Si[M+H]+1616.6638,found 1616.6705)
4、简称为RGD-Linker-SiPc的光敏化合物的合成
SiPc-COOH(10.0mg,0.010mmol),1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC)(1.8mg,0.009mmol)和N-羟基丁二酰亚胺(NHS)(1.1mg,0.010mmol)加入到200μL无水DMF中,室温搅拌4h后,再加入DIEA(2.5mg,0.019mmol)和RGD-Linker(2.6mg,0.003mmol)继续室温搅拌过夜。经无水乙醚沉淀洗涤,再用二氯甲烷洗涤三次,经HPLC纯化后得产物RGD-Linker-SiPc(3.3mg,收率57%)。(HRMS(ESI):m/z calculated for C93H116N23O22Si[M+H]+1934.8429,found 1934.8512)
5、简称为RGD-(Linker)2-SiPc的光敏化合物的合成
SiPc-COOH(10.0mg,0.010mmol),1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC)(1.8mg,0.009mmol)和N-羟基丁二酰亚胺(NHS)(1.1mg,0.010mmol)加入到200μL无水DMF中,室温搅拌4h后,再加入DIEA(2.5mg,0.019mmol)和RGD-(Linker)2(3.6mg,0.003mmol)继续室温搅拌过夜。经无水乙醚沉淀洗涤,再用二氯甲烷洗涤三次,经HPLC纯化后得产物RGD-(Linker)2-SiPc(2.6mg,收率40%)。
6、简称为RGD-(Linker)2-Glu-SiPc的光敏化合物的合成
SiPc-COOH(10.0mg,0.010mmol),1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC)(1.8mg,0.009mmol)和N-羟基丁二酰亚胺(NHS)(1.1mg,0.010mmol)加入到200μL无水DMF中,室温搅拌4h后,再加入DIEA(2.5mg,0.019mmol)和RGD-(Linker)2-Glu(4.0mg,0.003mmol)继续室温搅拌过夜。经无水乙醚沉淀洗涤,再用二氯甲烷洗涤三次,经HPLC纯化后得产物RGD-(Linker)2-Glu-SiPc(2.5mg,收率32%)。
实施例4 RGD-SiPc,RGD-Linker-SiPc,RGD-(Linker)2-SiPc和RGD-(Linker)2-Glu-SiPc的光谱性质(附图6,附表1)
(1)UV-Vis光谱
我们利用美国Cary 5000型紫外光谱仪进行紫外吸收光谱的测定。这些化合物配置成浓度为10μM的溶液,溶剂分别为DMSO,含有10%聚乙二醇辛基苯基醚的水溶液,含有1%聚氧乙烯蓖麻油(CEL)的水溶液(VCEL/Vwater=1:99),和磷酸盐缓冲液(PBS)。室温条件下,扫描波长范围从500nm到900nm,分辨率为1nm,扫描速率为每分钟600nm。
通过紫外吸收光谱对偶联产物RGD-SiPc,RGD-Linker-SiPc,RGD-(Linker)2-SiPc和RGD-(Linker)2-Glu-SiPc进行了表征,在DMSO溶液中均有较强的吸收峰,吸收波长为λ=680nm,表现出了典型的非聚集形式,严格符合Beer–Lambert定律。众所周知,DMSO可以防止酞菁类化合物的聚集,然而,DMSO并不是进行生物评价时一种合适的溶剂。与没有偶联配基的酞菁类化合物相比,所有偶联了配基的化合物都表现出了很好的水溶性,尤其是引入了谷氨酸片段的化合物RGD-(Linker)2-Glu-SiPc。因此,我们进一步研究了这些化合物在不同溶液中的光学特性,包括:含有10%聚乙二醇辛基苯基醚的水溶液,含有1%聚氧乙烯蓖麻油(CEL)的水溶液(VCEL/Vwater=1:99),和磷酸盐缓冲液(PBS)。如图6所示,所有的化合物在PBS溶液中都发生了聚集,吸收峰变宽,吸收波长范围为λ=600-700nm。然而,在含有10%聚乙二醇辛基苯基醚的水溶液中的光谱图与DMSO溶液的光谱图相似,特别是RGD-(Linker)2-Glu-SiPc,其吸收光谱几乎完全没有聚集。使用相对较弱的脂环境的含有1%聚氧乙烯蓖麻油(CEL)的水溶液作为溶剂,其中一些偶联物仍会聚集,然而RGD-(Linker)2-Glu-SiPc的吸收光谱几乎完全没有聚集,与DMSO溶液的光谱图相同。以上结果表明,与亲水性多肽偶联,并引入PEG连接链和羧基官能团能够显著提高SiPcs的水溶性,并帮助解决了SiPcs在水溶液中的聚集问题。尽管这些SiPcs与多肽的偶联物在PBS溶液中仍然会聚集,但是含有10%聚乙二醇辛基苯基醚的水溶液,和含有1%聚氧乙烯蓖麻油(CEL)的水溶液可以显著消除其聚集问题,尤其是引入了PEG连接链和羧基官能团的RGD-(Linker)2-Glu-SiPc。引入一个谷氨酸片段不仅能够明显提高偶联物的水溶性,而且可以很容易消除聚集问题。与纯水溶液相比脂质水溶液能够更好地模拟细胞膜的环境,尤其是脂质浓度较低的含有1%聚氧乙烯蓖麻油(CEL)的水溶液。由于只有消除聚集的光敏剂才有光活性,因此,光敏剂在生理条件下消除聚集在生物应用中非常重要。
(2)荧光激发和发射光谱
荧光发射光谱的记录范围从波长600nm到900nm,激发波长为680nm。荧光激发光谱的记录扫描范围从波长500nm到900nm,发射波长为695nm。样品均为2.0μM的DMSO溶液。激发和发射光谱宽度分别为1nm和2nm。
如表1所示,RGD对酞菁环的光学性能没有明显的影响。与没有偶联RGD多肽的酞菁硅SiPc-PQ和and SiPc-COOH相比这些偶联产物的最大发射,激发波长和荧光量子产量非常接近,荧光衰减时间稍有减少。所有的偶联物都有相当好的单线态氧量子产率,但与多肽和连接链连接后似乎还增加了其单线态氧量子产率。RGD-(Linker)2-Glu-SiPc具有最高的单线态氧量子产率,为0.39。单线态氧量子产率是PDT中影响细胞杀伤力最主要的因素,因此提高单线态氧量子产率对PDT非常有帮助。
实施例5 RGD-SiPc,RGD-Linker-SiPc,RGD-(Linker)2-SiPc和RGD-(Linker)2-Glu-SiPc在细胞水平上药物活性的评价
我们用人体胶质瘤U87-MG细胞系、人前列腺癌22RV1和PC3细胞系,以及人类表皮鳞癌A431细胞系在细胞水平上体外光动力学活性进行了评估。首先以1×106个细胞/孔的细胞密度铺于96孔板上,培养24h后加入含有偶联化合物RGD-SiPc,RGD-Linker-SiPc,RGD-(Linker)2-SiPc和RGD-(Linker)2-Glu-SiPc不同浓度(溶解在1.0%CEL水溶液)的细胞培养基继续培养4h后,用波长670nm,40mW/cm2的功率密度给予光照15min。4h后,更换含0.5mg/mL MTT的新鲜培养基在37℃下继续孵育4h。形成的蓝紫色的甲臜结晶沉积在细胞中,结晶物被DMSO溶解,用多功能酶联免疫检测仪在490nm处测量各孔的吸光值。同时,我们在各细胞系上也进行了不给光照的暗毒性实验。每个实验我们都重复3次。根据测定的吸光度计算细胞的存活率,然后绘制细胞存活率和加药浓度之间关系的药物活力抑制曲线,并计算相应的半抑制浓度(EC 50值)。
U87-MG,22RV1和PC3都是ανβ3蛋白受体高表达的细胞系,而A431是对ανβ3蛋白受体低表达细胞系。图7为不光照只给药和只给光照不给药条件下细胞存活率与相应剂量的曲线;图8为光照给药条件下不同细胞系细胞存活率与相应剂量的曲线;表2为这些偶联物在对应细胞系的EC50值。四个偶联化合物对以上细胞系都没有表现出明显的暗毒性,然而,在有光照条件下,四个偶联化合物在浓度很低的情况下,细胞的生存能力被显著地降低。所有的偶联化合物EC50值均在nM范围,并且RGD-(Linker)2-Glu-SiPc具有最强的光活性,其EC50值对应U87-MG、22RV1、PC3以及A431细胞系分别为17.1nM,16.7nM,16.2nM和50.4nM。含有羧基官能团的谷氨酸片段似乎对提高活性起到了非常重要的作用。
这些偶联物对受体阳性和受体阴性的细胞系活性差异并不明显。在最近的报道中我们也发现了类似的结果,RGD多肽与酞菁锌进行偶联的化合物,对于ανβ3 +U87-MG受体阳性的细胞系和ανβ3 +A431或MCF-7受体阴性的细胞系在药物孵育后有相似的光毒性。这些偶联化合物被细胞摄取可能不仅与细胞膜表面受体的数目有关系,可能还有其他途径。
实施例6 RGD-(Linker)2-Glu-SiPc的体内治疗效果的评价
我们采用已经建立的U87-MG异种移植肿瘤模型对偶联物RGD-(Linker)2-Glu-SiPc进行了体内光动力活性的研究。肿瘤模型的建立是通过给8周龄的雌性BALB/c裸鼠右侧腿部接种3×106个U87-MG肿瘤细胞,当小鼠肿瘤体积达到100mm3时,这些小鼠被随机分成两组,每组5只。治疗组每只小鼠通过尾静脉注射RGD-(Linker)2-Glu-SiPc(50nmol),给药4h后给予波长为670nm,强度为200mW/cm2的光照16min。对照组注射相同剂量的PBS,给予相同光照。在观察的35天内,对所有小鼠的肿瘤体积和体重进行了监测。每隔一天用游标卡尺测量记录各组小鼠肿瘤的体积变化,肿瘤体积的计算公式:肿瘤体积=长×宽2×0.5。每隔两天对小鼠进行拍照,以更直观地观察肿瘤的变化。当肿瘤达到1500mm2以上时,老鼠被认为死亡。
RGD-(Linker)2-Glu-SiPc相比其他3个偶联物而言在肿瘤光动力治疗中是一种更好的光敏剂。这种光敏剂在细胞水平上有最强的细胞杀伤力,具有较好的水溶性,并且在水溶液中不易发生聚集。因此,我们选择这个化合物进行动物水平上的评价。由于ανβ3受体在U87-MG肿瘤细胞表面高表达,因此可以选择性的结合偶联物上的RGD配基。在这个模型中,我们在BALB/c裸鼠右侧皮下注射U87-MG细胞。当肿瘤体积达到100mm3时,将化合物RGD-(Linker)2-Glu-SiPc和生理盐水通过尾静脉分别注射,然后给予强度为200mW/cm2的光照16min。如图9所示,对照组经过光照后,肿瘤体积显著增加,在不到25天的时间里增大到1500mm2。相对应的,治疗组经光照后肿瘤的体积显著缩小。所有的肿瘤在第14天被全部治愈,并且在观察到第35天没有复发。在整个治疗过程中,没有观察到小鼠体重下降,表明光照和化合物RGD-(Linker)2-Glu-SiPc都没有对小鼠引起严重的毒副作用。RGD-(Linker)2-Glu-SiPc在U87-MG肿瘤模型上的功效表现出其在肿瘤光动力治疗上具有的极大的临床应用潜力。
本发明中,为了开发新型具有肿瘤靶向性的光动力治疗用光敏剂,我们设计并合成了一系列RGD多肽轴向取代与SiPc偶联的产物。与RGD多肽偶联极大地提高了SiPc的水溶性,又引入了两个PEG连接链和一个含有强亲水性的羧酸官能团进一步提高了其亲水性能,制备出的RGD-(Linker)2-Glu-SiPc产物显示出了很强的光活性,其对于各种受体阳性细胞系其EC50值在10-20nM。在U87-MG异种移植肿瘤模型治疗中,一次给药后进行光动力治疗即可完全治愈,并且在观察到第35天没有复发。研究结果表明,RGD-(Linker)2-Glu-SiPc在肿瘤光动力治疗上具有的极大的临床应用潜力。如何进行用PEG连接链和羧酸官能团对光敏剂进行修饰来提高其光活性仍是一个有待探索的问题。
Claims (8)
2.如权利要求1所述的酞菁硅光敏剂在制备治疗肿瘤的光动力药物中的用途。
3.一种如权利要求1所述的酞菁硅光敏剂的制备方法,其合成路线为:
1)合成RGD多肽;
2)合成RGD-(Linker)2-Glu;
3)合成RGD-(Linker)2-Glu-SiPc。
7.如权利要求1所述的酞菁硅光敏剂在制备治疗肿瘤的药物中的用途。
8.如权利要求1所述的酞菁硅光敏剂在制备肿瘤成像诊断试剂中的用途。
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