CN112402625A - 负载光敏剂的peg化肝素纳米胶束及其制备方法 - Google Patents
负载光敏剂的peg化肝素纳米胶束及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种负载光敏剂的PEG化肝素纳米胶束,该胶束为负载卟啉类光敏剂的PEG化肝素分子。将生物可降解、相容性好、利用度高的天然多糖肝素作为药物载体,通过结合PEG改性和卟啉类光敏剂,实现药物的靶向和长效。
Description
技术领域
本发明涉及医药技术领域,具体涉及一种负载光敏剂的PEG化肝素纳米胶束及其制备方法。
背景技术
光敏剂(Photosensitizer PS)是一种本身稳定性良好且没有暗毒性的有机或无机物,在特定波长光线的照射下会产生单线态氧等强氧化性分子,通常采用其纳米尺度的颗粒以增强治疗效果。光动力治疗(Photodynamic Therapy,PDT)是非常重要的现代肿瘤治疗手段,它具有微创、靶向性好、不良反应小等优点。由于肿瘤组织细胞的高吸收、低代谢特性,一定时间后,纳米尺度的光敏剂粒子会特异性地聚集在肿瘤组织中;继而以特定波长的光照射肿瘤部位,光敏剂激发产生可以杀死肿瘤细胞的单线态氧和氧自由基等活性氧物种,最终达到消除肿瘤的目的。
上世纪70年代末,卟啉类(Ppa)就在世界各地被广泛用于肿瘤光动力治疗和荧光诊断。直到1993年,Ppa提纯后的产物Photofrin才正式被批准用于临床。此后,与Photofrin组成相近的Photogem、Photosan和喜泊分分别在俄罗斯、德国和我国上市。卟啉结构特殊的光敏性,一直以来的都是研究的热点。然而由于多数的光敏剂都是水难溶性和非特异性的,聚合物胶束作为给药载体正受到越来越多的关注。
发明内容
基于光敏剂的水难溶性和非特异性,本发明提供了一种负载光敏剂的PEG化肝素纳米胶束,以肝素骨架为载体,引入两亲性的亲生物高分子链PEG结构,改变药物在生物体中的存在形式,接着将Ppa光敏剂通过物理-化学的方法接在修饰的肝素上,实现药物的靶向性和长效性。
为了实现本发明的目的,本发明采用的技术方案是:
负载光敏剂的PEG化肝素纳米胶束,该胶束为负载卟啉类光敏剂的PEG化肝素分子;具体结构如下:
其中,X和Y为肝素分子上的固有基团,X为H或者SO3 -,Y为COCH3、H或者SO3 -;
R为PEG基团和D基团;
该基团是酰基与聚乙二醇末端的羟基通过酯键连接;
该基团与Ppa分子通过酰胺键连接。
本发明所述Ppa分子的结构为:
肝素钠(Heparin sodium,HP)是一种含有硫酸基的酸性粘多糖类天然抗凝血物质,可以从动物来源中广泛提取,在体内通过主动与抗凝血酶Ⅲ(AT-Ⅲ)结合发挥作用,即自发具有血液系统的靶向性,利用该性质,可以实现药物结构的特异性治疗,发挥其他结构不具有的优势。
肝素结构中本身有大量的氨基、羧基和磺酸基,水溶性极好最适于注射给药。但是考虑到保证药物的成药性,需要继续对其结构修饰,改变logP、蛋白结合率,从而实现改变药物的PKPD。本发明以肝素骨架为载体,引入两亲性的亲生物高分子链PEG结构,转变成水油两亲性结构;再引入柔性的乙基巯基链做为连接键,既可以降低肝素糖环位阻效应对后续连接化合物的位阻,又有利规范Ppa分子调节在胶束中的分布状态;以巯基为结合位点,将卟啉类光敏剂负载在修饰的肝素上,实现普通药物的靶向性和长效性。
本发明所述的聚乙二醇为mPEG2000(MeO-PEG2000-OH)。
本发明还提供了负载光敏剂的PEG化肝素纳米胶束的制备方法,其特征在于,包括以下合成路线:
(1)制备PEG衍生物
(2)制备中间体HP-SH
(3)制备Ppa衍生物mal-Ppa
(4)将中间体HP-SH与制得的Ppa衍生物mal-Ppa反应,得到负载有Ppa分子的肝素HP-Ppa;
(5)将负载Ppa分子的肝素与PEG衍生物mPEG2000-mal或者mPEG2000-SS-Py反应,得到载有Ppa的PEG化肝素纳米胶束。
在步骤(1)的反应中加入催化剂DIEA,中和反应中生成的HCl,提供碱性环境促进反应。
在步骤(1)的反应中加入催化剂DIEA和DMAP,中和反应中生成的HCl,提供碱性环境促进反应。
优选地,所述反应中,将依诺肝素钠溶于吗啉乙磺酸(MeS)缓冲液中,加入4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐(DMTMM)活化,另取S-(2-胺基乙硫基)-2-硫代吡啶(Py-SS-NH2·HCl)溶于MeS缓冲液,滴加入体系中反应,得中间体HP-SH。
进一步优选地,所述MeS缓冲液的制备方法:称取吗啉乙磺酸溶于纯化水中,滴加氢氧化钠溶液调节pH至5.5,定容,得到MeS缓冲溶液。
在步骤(3)的反应中,加入HOTu(O-[(乙氧基羰基)氰基甲胺]-N,N,N',N'-四甲基硫脲六氟磷酸盐)参与反应。HOTu作为缩合剂,用于催化羧基和胺基(羟基)缩合反应,形成酰胺(酯)。
优选地,在步骤(3)的反应完成后,置换氮气保护,避光,加入DMF和DIEA,在室温下搅拌过夜。DMF是反应溶剂,DIEA催化缩合反应进行。因为Ppa对光敏感,易被氧化变质,所以反应需要充氮避光。
在步骤(4)的反应中,中间体HP-SH与Ppa衍生物mal-Ppa反应,加入催化剂三乙胺。巯基显示弱酸性,加入三乙胺增加巯基的电离程度,提高亲核加成的活性。
本发明的有益效果在于:
1、本发明的纳米载药系统为肝素纳米胶束,肝素是天然生物内源性多糖结构,本身就可以注射药用,回避了合成/半合成材料的代谢毒性;对载体进行PEG修饰,降低载体的刚性,提高了载体的两亲性以及成胶束能力,克服了现有大分子载药系统生物相容性差的问题。
2、载药系统利用柔性脂肪链连接Ppa分子,更有利于体系在水中自组装成合适的胶束,使Ppa分子稳定地包裹于胶束内部,可以防止外界因素的降解或是非必要性代谢失活,克服了现有大分子载药系统自身稳定性差的问题;通过控制胶束的粒径分布,达到被动靶向的作用。高分子胶束在肿瘤部位富集,在体外光的辐照下,可以杀死肿瘤细胞。
3、PEG为高分子化合物,端头OH活性不高,不利于后续反应。本发明采用合成反应中用高活性的酰氯,可以高产率的得到中间体mPEG2000-OCOO-Ph-NO2。对硝基酚是一个好的离去基团,可以在后续反应中方便的发生亲核取代,连接目标分子。
4、由于肝素的糖环上羧基受糖环位阻影响,合成的活性偏低,影响收率,本发明引入柔性巯基脂肪链,不仅增加反应活性,还有助于与Ppa的结合,得到的中间体HP-SH带2-巯基吡啶,是一个极好的离去基,碰到更有亲和性的脂肪巯基时会因亲核取代被置换下来,方便在PEG衍生物反应中,2-巯基吡啶被取代得到PEG化的肝素。
5、Ppa分子结构为一个大平面,位阻较大且刚性,无法直接连接在肝素主链上,本发明引入马来酰亚胺的柔性脂肪链进行过渡,且马来酰亚胺的α,β-共轭不饱和结构,可以高效的和肝素的-SH发生加成反应,连接两个分子片段。
附图说明
图1为负载有Ppa分子的肝素HP-Ppa的HNMR谱图。
图2为负载有Ppa的PEG化肝素纳米胶束HP-Ppa-mPEG的HNMR谱图。
图3为负载有Ppa的PEG化肝素纳米胶束HP-Ppa-SS-mPEG的HNMR谱图。
图4为DLS测量HP-Ppa,HP-Ppa-mPEG和HP-Ppa-SS-mPEG在水中的粒径。
图5为HP-Ppa,HP-Ppa-mPEG和HP-Ppa-SS-mPEG在水中的粒径分布图。
图6为流式细胞仪分析仪检测细胞内的Ppa荧光值。
图7为流式细胞仪检测4T1细胞凋亡比例。
图8为CCK-8试剂盒测定各组细胞增殖活力。
图9为荧光成像系统检测各组织中Ppa的荧光信号定量统计。
图10为CD31单克隆抗体和TUNEL试剂盒对肿瘤组织切片的结果。
图11为肝素纳米药物处理后小鼠体重变化。
图12为肝素纳米药物处理后对小鼠主要组织的损伤作用。
具体实施方式
为了更加清楚、详细地说明本发明的目的技术方案,下面通过相关实施例对本发明进行进一步描述。以下实施例仅为具体说明本发明的实施方法,并不限定本发明的保护范围。
实施例1
一种负载光敏剂的PEG化肝素纳米胶束,其特征在于,该载药系统为胺基抗肿瘤药物加载在PEG化肝素分子上的偶联物;具体结构如下:
其中,X和Y为肝素分子上的固有基团;
R为PEG基团和D基团;
R为PEG基团和D基团;
该基团是酰基与聚乙二醇末端的羟基通过酯键连接;
该基团与Ppa分子通过酰胺键连接。
实施例2
本实施例在实施例1的基础上:
所述的聚乙二醇为mPEG2000,
实施例3
负载光敏剂的PEG化肝素纳米胶束的制备方法,包括以下合成路线:
(1)制备PEG衍生物mPEG2000-mal或者mPEG2000-SS-Py
(2)制备中间体HP-SH
肝素聚合物中部分单元的酰基与4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐(DMTMM)作用,得到中间体HP-SS-Py,进一步脱硫,得到中间体HP-SH。
(3)制备Ppa衍生物mal-Ppa
(4)将中间体HP-SH与制得的Ppa衍生物mal-Ppa反应,得到负载有Ppa分子的肝素HP-Ppa;
(5)将负载Ppa分子的肝素与PEG衍生物mPEG2000-mal或者mPEG2000-SS-Py反应,中间体HP-SH的聚合物单元中未被Ppa分子取代的-SH与PEG衍生物发生反应,得到载有Ppa的PEG化肝素纳米胶束HP-Ppa-mPEG或者HP-Ppa-SS-mPEG。
实施例4
本实施例在实施例3的基础上:
在步骤(1)的反应中加入催化剂DIEA,中和反应中生成的HCl,提供碱性环境促进反应。
所述反应中,将依诺肝素钠(HP·Na)溶于吗啉乙磺酸(MeS)缓冲液中,加入4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐(DMTMM)活化,另取S-(2-胺基乙硫基)-2-硫代吡啶(Py-SS-NH2·HCl)溶于MeS缓冲液,滴加入体系中反应,得中间体HP-SH。
在步骤(4)的反应中,中间体HP-SH与Ppa衍生物反应,加入催化剂三乙胺。巯基显示弱酸性,加入三乙胺增加巯基的电离程度,提高亲核加成的活性。
实施例5
本实施例在实施例3的基础上:
在步骤(1)的反应中加入催化剂DIEA,中和反应中生成的HCl,提供碱性环境促进反应。
所述反应中,将依诺肝素钠(HP·Na)溶于吗啉乙磺酸(MeS)缓冲液中,加入4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐(DMTMM)活化,另取S-(2-胺基乙硫基)-2-硫代吡啶(Py-SS-NH2·HCl)溶于MeS缓冲液,滴加入体系中反应,得中间体HP-SH。
所述MeS缓冲液的制备方法:称取吗啉乙磺酸溶于纯化水中,滴加氢氧化钠溶液调节pH至5.5,定容,得到MeS缓冲溶液。
在步骤(3)的反应中,加入HOTu参与反应,反应完成后,置换氮气保护,避光,加入DMF和DIEA,在室温下搅拌过夜。
在步骤(4)的反应中,中间体HP-SH与Ppa衍生物反应,加入催化剂三乙胺。巯基显示弱酸性,加入三乙胺增加巯基的电离程度,提高亲核加成的活性。
实施例6
PEG衍生物mPEG2000-mal的合成
将mPEG2000 20g溶于100mL DCM中,冰浴下滴入二异丙基乙基胺(DIEA)8mL,体系呈无色透明状。将氯甲酸对硝基苯酯8g溶于50mL DCM中,冰浴下滴入前述溶液中,滴毕,体系缓慢升至室温。体系此时未见明显变化,反应过夜。
分离纯化:体系为亮黄色微混状,过滤后旋干,得到黄色粘稠液体。向体系中先加入200mL醋酸乙酯(EA),室温下剧烈搅拌,黄色液体逐渐变为白色固体分散于体系中,液体变为亮黄色,再于搅拌下滴加100mL Et2O,保持室温下打浆0.5h,搅拌,得白色固体。白色固体转移至烧杯中,搅拌下先加入200mL EA,15min后滴入100mL Et2O,打浆0.5h,抽滤得白色固体,固体减压干燥得白色固体mPEG2000-OCOO-Ph-NO2 16.1g。
将DIEA 0.65g称入圆底烧瓶中,冰浴下加入DCM溶液,再加入Maleimide-NH2·TFA(N-(2-氨基乙基)马来酰亚胺三氟乙酸盐)0.47g搅拌0.5h,最后滴加mPEG2000-OCOO-Ph-NO24.3g于DCM溶液,滴毕自然升温过夜。第二天,抽滤,滤液旋干后得黄色油状物。用EA/叔甲醚=2/1的液体300mL打浆两次,产品减压干燥,得白色固体mPEG2000-mal 3.9g。
实施例7
PEG衍生物mPEG2000-SS-Py的合成
mPEG2000-OCOO-Ph-NO2的合成同实施例6。
将DIEA 0.65g和DMAP(4-二甲胺基吡啶)0.12g称入圆底烧瓶中,冰浴下加入DCM溶液,再加入S-(2-胺基乙硫基)-2-硫代吡啶(Py-SS-NH2)0.45g搅拌0.5h,最后滴加mPEG2000-OCOO-Ph-NO2 4.3g的DCM溶液,滴加完毕自然升温过夜。第二天,抽滤后旋干液体得到黄色油状物,用EA/叔甲醚=2/1的液体300mL打浆两次,产品减压干燥,得mPEG2000-SS-Py 4.2g。
实施例8
中间体HP-SH的合成
将依诺肝素钠(HP·Na)2.88g溶于MeS 10mL缓冲液中,加入4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐(DMTMM)4.14g活化10min,另取Py-SS-NH2·HCl 3.34g溶于10mL MeS缓冲液,滴加入体系中反应24h之后透析三天,冻干得白色固体中间体HP-SS-Py1.5g。
MeS缓冲液的制备:称取2g氢氧化钠溶于20mL纯化水中冷却待用。称取吗啉乙磺酸9.8g溶于250mL纯化水中,滴加氢氧化钠溶液调节pH至5.5,定容至500mL。
将中间体HP-SS-Py 1.5g溶于水中,室温下加入二硫苏糖醇(DTT)1.5g,反应过夜,第二天用1KDa的半透膜透析三天,冻干后得白色固体中间体HP-SH 1.1g。
实施例9
Ppa衍生物mal-Ppa的合成
将Ppa(2mmol,1.07g)、Maleimide-NH2·TFA(N-(2-氨基乙基)马来酰亚胺三氟乙酸盐)(2.2mmol,0.52g)、HOTu(O-[(乙氧基羰基)氰基甲胺]-N,N,N',N'-四甲基硫脲六氟磷酸盐)(2.6mmol,0.99g)称入圆底烧瓶中,置换氮气保护后,用锡箔纸包好避光。加入DMF(15mL)和DIEA(二异丙基乙基胺)(4mmol,0.51g),在室温下搅拌过夜。次日加入25mL二氯甲烷后,用饱和氯化钠溶液清洗-分液处理3次。得到的黑色液体旋干至粘性液体状态,用柱色谱(300-400硅胶,石油醚/乙酸乙酯=1.5/1)纯化,滤液旋干得到深墨绿色固体mal-Ppa1.23g。
实施例10
HP-Ppa的合成
用4mL水将中间体HP-SH 0.2g溶解澄清,再加入12mL DMSO,体系保持澄清状并大量放热,用锡箔包好。用4mL DMSO将30mg mal-Ppa溶清,得到墨绿色溶液,滴入体系后,加入催化量的NEt3,搅拌过夜,生成HP-Ppa。取少量冻干得到墨绿色固体产品HP-Ppa0.08g;1HNMR(D2O/d6-DMSO:1/5)(图1):2.2-2.4(=C-CH3),2.8-3.0(SCH2CH2N),3.0-3.7(肝素钠糖环碳氢),3.8(CH3O-),5.4-5.8(-HC=CH2),Ppa吡咯环上的氢(δ=9.56,9.29,8.84,8.11,7.38,7.27,6.35,6.17ppm)。
采用UV(SP-1920UV,上海光谱仪器有限公司),测得载药量为3.7%。
实施例11
PEG化HP-Ppa偶联物的合成
(1)HP-Ppa-mPEG的合成
(2)HP-Ppa-SS-mPEG的合成
将实施例10得到的溶液等分成两份,分别加入对应的mPEG2000-mal和mPEG2000-SS-Py各0.4g,保持避光下室温反应过夜。体系用3.5KDa的半透膜透析三天,产品冻干得到墨绿色固体。得到HP-Ppa-mPEG 0.287g;1HNMR(D2O/d6-DMSO:1/5)(图2):2.2-2.4(=C-CH3),2.8-3.0(SCH2CH2N),3.0-3.3(肝素钠糖环碳氢),3.4-3.7(PEG中亚甲基氢),3.8(CH3O-),5.4-5.8(-HC=CH2),Ppa吡咯环上的氢(δ=9.56,9.29,8.84,8.11,7.38,7.27,6.35,6.17ppm)。
采用UV(SP-1920UV,上海光谱仪器有限公司),测得载药量为1.6%。
得到HP-Ppa-SS-mPEG 0.325g;1HNMR(D2O/d6-DMSO:1/5)(图3):2.2-2.4(=C-CH3),2.7(CO-CH2),2.9(与NH相连的CH2)3.0-3.3(肝素钠糖环碳氢),3.4-3.7(PEG中亚甲基氢),3.8(CH3O-),3.9(S-CH2-CO),5.4-5.8(-HC=CH2),Ppa吡咯环上的氢(δ=9.56,9.29,8.84,8.11,7.38,7.27,6.35,6.17ppm)。
采用UV(SP-1920UV,上海光谱仪器有限公司),测得载药量为2.4%。
反应原料来源:
4-二甲氨基吡啶(DMAP) 成都科龙化工试剂厂
4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐(DMTMM) 百灵威科技有限公司
氯甲酸对硝基苯酯 天津希恩思生化科技有限公司
DL-二硫苏糖醇(DTT) 阿拉丁生化科技有限公司
吗啉乙磺酸(MeS) 阿拉丁生化科技有限公司
S-(2-氨基乙硫基)-2-硫代吡啶 上海瀚香生物科技有限公司
N-(2-氨基乙基)马来酰亚胺三氟乙酸盐 阿拉丁生化科技有限公司
Ppa 上海先辉医药科技公司
依诺肝素 南京健友生化制药股份有限公司
MeO-PEG2000-OH 阿拉丁生化科技有限公司
形态学分析和表面电荷
利用DLS测量HP-Ppa,HP-Ppa-mPEG和HP-Ppa-SS-mPEG在水中的粒径。结果表明(图4),HP-Ppa的粒径为41.18±3.41nm,PDI为0.266;HP-Ppa-mPEG的粒径为75.10±17.93nm,PDI为0.309;HP-Ppa-SS-mPEG的粒径为97.31±1.05nm,PDI为0.266。TEM照片进一步证实了3种纳米粒子的粒径分布(图5)。在10mM DTT条件下,HP-Ppa-SS-mPEG分子中HP与mPEG之间的二硫键被打断清除,使其粒径减小至80.52±11.29nm。HP-Ppa,HP-Ppa-mPEG和HP-Ppa-SS-mPEG的Zeta电位分别为-15.18±1.72mv,-12.34±2.32mv和-14.63±2.75mv。与带正电的聚合物相比,带负电荷的纳米粒子在体内具有较好的稳定性,较长的血液循环时间,其可通过EPR效应实现肿瘤富集。
细胞摄取研究
影响光动力治疗效率的一个重要因素是光敏剂在肿瘤细胞内的积累量。利用CLSM观察HP-Ppa,HP-Ppa-mPEG,HP-Ppa-SS-mPEG和Ppa在4T1细胞中的分布及荧光强度。4T1细胞系购自中国科学院典型培养物保藏中心(上海),并在RPMI-1640培养基(含有10%胎牛血清,100U mL-1penicillin G,100μg mL-1streptomycin)中培养(培养条件:5%CO2,37℃)。将2×104数量4T1细胞铺于35mm玻璃底皿中,细胞贴壁培养24h后,HP-Ppa,HP-Ppa-mPEG,HP-Ppa-SS-mPEG和Ppa(1μg/mL Ppa当量)孵育细胞12h。使用Hoechst 33342探针染色细胞核30min,利用4℃预冷PBS洗细胞两次。利用激光共聚焦扫描显微镜(CLSM,激发波长405nm和630nm)观察细胞核和Ppa的荧光。结果表明,3种基于肝素的纳米粒子和游离Ppa都可以有效地进入4T1细胞。细胞中HP-Ppa-mPEG和HP-Ppa-SS-mPEG的荧光信号比HP-Ppa的荧光信号更强,表明PEG功能化有利于肝素纳米颗粒被4T1细胞摄取。
同时,利用流式细胞仪测定基于肝素的纳米粒子和游离Ppa的细胞摄取量。将4T1细胞培养在6孔板中(2×105细胞/孔)。贴壁培养24h后,弃去培养基,各孔分别加入HP-Ppa,HP-Ppa-mPEG,HP-Ppa-SS-mPEG和Ppa(1μg/mL Ppa当量)孵育细胞12h,利用PBS洗细胞两次。通过流式细胞仪分析仪(APC-A通道,Beckman Coulter Cytomics FC-500,美国)检测细胞内Ppa荧光值。结果表明(图6),HP-Ppa-SS-mPEG组的细胞摄取量是HP-Ppa组的5.4倍,而HP-Ppa-SS-mPEG组的MFI值与HP-Ppa-mPEG组相近。由于游离Ppa进入4T1细胞的途径是自由扩散,因此4T1细胞中游离Ppa的荧光信号强于3组NPs。
ROS测定
当肿瘤细胞内积累大量光敏剂时,在激光照射下会产生细胞毒性ROS。将4T1细胞培养在6孔板中(2×105细胞/孔)。贴壁培养24h后,弃去培养基,各孔分别加入HP-Ppa,HP-Ppa-mPEG,HP-Ppa-SS-mPEG和Ppa(1μg/mL Ppa当量),孵育细胞12h后,PBS洗细胞两次,加入新鲜RPMI-1640培养基,利用660nm激光照射细胞(1J cm-2)。DCFH-DA荧光探针孵育细胞40min,PBS洗细胞两次;Hoechst 33342探针染色细胞核30min,PBS洗细胞两次,通过倒置荧光显微镜观察细胞核及ROS荧光并拍照。结果表明,HP-Ppa-mPEG和HP-Ppa-SS-mPEG组的DCF荧光信号均比HP-Ppa组的荧光信号强,而PBS组中未观察到荧光信号。这表明ROS产生的量与材料入胞量呈正相关。
细胞凋亡测定
细胞内ROS可诱导细胞质中大分子的氧化并破坏线粒体内膜系统,从而促进肿瘤细胞凋亡。将4T1细胞培养在6孔板中(2×105细胞/孔),贴壁培养24h后,弃去培养基,各孔分别加入HP-Ppa,HP-Ppa-mPEG,HP-Ppa-SS-mPEG和Ppa(1μg/mL Ppa当量)孵育细胞12h,PBS洗细胞两次,加入新鲜RPMI-1640培养基。使用660nm激光照射细胞(1J cm-2),将细胞收集于1.5mL EP管中。利用Annexin V-FITC/PI细胞凋亡检测探针(BD Pharmingen)孵育细胞15min,通过流式细胞仪检测4T1细胞凋亡比例。实验结果表明(图7),HP-Ppa-mPEG和HP-Ppa-SS-mPEG处理组细胞凋亡比例高于HP-Ppa处理组,而4T1细胞凋亡比例与细胞内ROS产生量正相关。
体外抗肿瘤效果评价
5×103数量4T1细胞铺于96孔板中,培养12h后,各孔分别加入梯度浓度HP-Ppa,HP-Ppa-mPEG,HP-Ppa-SS-mPEG和Ppa孵育细胞12h,PBS洗细胞两次,加入新鲜RPMI-1640培养基。使用660nm激光照射细胞(1J cm-2),然后置于培养箱中孵育24h,利用CCK-8试剂盒测定各组细胞增殖活力。结果表明(图8),HP-Ppa,HP-Ppa-mPEG和HP-Ppa-SS-mPEG组都具有抑制4T1细胞增殖的作用。三组的IC50值分别为1.89μg/mL、0.54μg/mL和0.67μg/mL。HP-Ppa组光动力抗肿瘤效果弱于HP-Ppa-mPEG和HP-Ppa-SS-mPEG组,而HP-Ppa-mPEG和HP-Ppa-SS-mPEG两组之间没有显著差异。基于肝素的纳米粒子细胞毒性差异取决于其在细胞内的积累量,ROS产生量及凋亡诱导能力。
肿瘤组织聚集
光敏剂在肿瘤组织中的聚集和渗透是影响体内PDT效率的关键因素。将5×105数量4T1细胞(50μL)皮下注射于BALB/c小鼠的右后肢。当肿瘤体积达到约100mm3时,通过尾静脉注射5mg/kg小鼠体重的HP-Ppa,HP-Ppa-mPEG,HP-Ppa-SS-mPEG和Ppa 4组药物(n=3)。在给药1h,3h,6h,24h,48h和72h检测Ppa的荧光信号,同时在给药72h时,处死荷瘤小鼠并取心、肝、脾、肺、肾和肿瘤组织,利用荧光成像系统检测各组织中Ppa的荧光信号。HP-Ppa,HP-Ppa-mPEG和HP-Ppa-SS-mPEG三种纳米粒子通过EPR效应和肝素的肿瘤靶向作用聚集在肿瘤组织中,在72h时,基于肝素的纳米粒子在肿瘤组织的荧光强度仍然很强。离体荧光图片表明,HP-Ppa-SS-mPEG组肿瘤组织中荧光信号最强,而Ppa组中的肿瘤组织荧光信号较弱。定量统计荧光信号结果表明(图9),HP-Ppa-SS-mPEG组肿瘤组织的平均信号强度是HP-Ppa组的1.5倍,是HP-Ppa-mPEG组的3.2倍,是游离Ppa组的39.2倍,表明PEG化的肝素纳米粒子促进药物在肿瘤组织的富集。HP-Ppa-SS-mPEG组在肿瘤部位的荧光信号比HP-Ppa-mPEG强,可能是由于PEG脱除促进了肝素纳米颗粒向肿瘤组织内部的渗透。BALB/c小鼠(雌性,6-8周)购自成都达硕生物技术有限公司,所有的动物研究和实验程序均遵循国家规定,并经四川大学华西医院动物伦理委员会批准。
肿瘤组织渗透
肿瘤球渗透:将1%的无菌琼脂铺于6孔板中(每孔1mL),加入4T1细胞悬液培养7天,当细胞球的直径达到约150μm时,将球体转移至35mm规格玻璃底皿中。各皿分别加入HP-Ppa,HP-Ppa-mPEG,HP-Ppa-SS-mPEG和Ppa(1μg/mL Ppa当量)孵育细胞24h。利用CLSM观察并拍照细胞球各层面荧光(每层5μm),并利用ImageJ软件制作图像的拓扑3D视图。结果表明,在肿瘤球的内部仅有少量的HP-Ppa-mPEG,原因可能是HP-Ppa-mPEG纳米粒子表面PEG层阻碍了HP-Ppa-mPEG向肿瘤组织内部渗透。由于HP-Ppa入胞能力较弱,因此HP-Ppa组大部分位于肿瘤球的外周边缘。而HP-Ppa-SS-mPEG在肿瘤球外周边缘及内部都有大量分布,表明HP-Ppa-SS-mPEG在肿瘤微环境中脱除PEG促进药物向肿瘤组织内部渗透。
实体瘤渗透:将5×105数量4T1细胞(50μL)皮下注射于BALB/c小鼠的右后肢。当肿瘤体积达到约100mm3时,通过尾静脉注射5mg/kg小鼠体重的HP-Ppa,HP-Ppa-mPEG,HP-Ppa-SS-mPEG和Ppa4组药物。在给药72h时,处死荷瘤小鼠并取肿瘤组织制备冷冻切片。DAPI探针染色细胞核10min,CLSM观察并拍照各组药物在实体瘤中的分布(激发波长:405nm和630nm)。4T1肿瘤组织冷冻切片CLSM结果进一步证实,与HP-Ppa-mPEG组和HP-Ppa组相比,HP-Ppa-SS-mPEG组具有更强的实体瘤渗透作用。
体内抗肿瘤效果评价
将5×105数量4T1细胞(50μL)皮下注射于BALB/c小鼠的右后肢。当肿瘤体积达到约50mm3时,通过尾静脉注射5mg/kg小鼠体重的HP-Ppa,HP-Ppa-mPEG,HP-Ppa-SS-mPEG和Ppa 4组药物,以及生理盐水,每组6只小鼠。每3天给一次药,共计3次。在给药48h后,使用660nm激光器照射肿瘤(200mW cm-2,8min)。用游标卡尺测量肿瘤的长度(L)和宽度(W),并计算肿瘤的体积(V)(V=L×W2/2)。在第18天或当肿瘤直径达到10mm时处死小鼠。收集肿瘤,拍照并称重,计算肿瘤生长抑制率(TGI)。利用Ki67单克隆抗体,CD31单克隆抗体和TUNEL试剂盒对肿瘤组织切片进行染色并拍照。结果表明(图10),Ppa组,HP-Ppa-mPEG组,HP-Ppa组和HP-Ppa-SS-mPEG组的抗肿瘤效果依次增强。三组肝素NPs组对肿瘤生长的抑制作用强于Ppa组。HP-Ppa-SS-mPEG组的TGI值高达94.3%,而Ppa组为51.9%,HP-Ppa组为86.2%,HP-Ppa-mPEG组为79.2%。HP-Ppa-SS-mPEG组较高的PDT效率与其肿瘤富集量高和渗透能力强有关。另外,通过分析组织切片,进一步研究各组药物的抗肿瘤效果。与HP-Ppa,HP-Ppa-mPEG和Ppa组相比,HP-Ppa-SS-mPEG组具有较低的Ki67阳性细胞百分比和较低的CD31阳性细胞百分比,以及较高的TUNEL阳性细胞比。上述结果表明,PEG可脱除的肝素纳米药物具有较强的抑制肿瘤细胞增殖和抑制肿瘤血管新生的作用,同时可高效地诱导肿瘤细胞凋亡。与生理盐水组和游离Ppa组相比,基于肝素的纳米药物具有抑制肿瘤细胞向肺组织和肝组织转移的作用。
体内毒性评价
治疗过程中测定小鼠体重,治疗结束后,取心、肝、脾、肺和肾组织制作切片,并用利用苏木精-伊红(H&E)染色。结果显示3组肝素纳米药物处理组小鼠体重没有降低(图11),并且未观察到对主要组织(心、肝、脾、肺和肾)的损伤作用(图12),表明PEG和肝素(HP-Ppa-SS-mPEG的主要组成单位)具有良好的生物相容性。
统计分析
对实验数据进行t检验(两尾)分析,所有数据均以mean±SD表示。显着性水平设置如下:“*”表示P<0.05,“**”表示P<0.01,“NS”表示P>0.05。
以上所述实施例仅表达了本发明的具体实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。
Claims (10)
2.根据权利要求1所述负载光敏剂的PEG化肝素纳米胶束,其特征在于,所述的聚乙二醇为mPEG2000。
5.根据权利要求4所述负载光敏剂的PEG化肝素纳米胶束的制备方法,其特征在于,在步骤(1)的反应中均加入催化剂DIEA。
6.根据权利要求4所述负载光敏剂的PEG化肝素纳米胶束的制备方法,其特征在于,所述反应中,将依诺肝素钠溶于MeS缓冲液中,加入DMTMM活化,另取S-(2-胺基乙硫基)-2-硫代吡啶溶于MeS缓冲液,滴加入体系中反应,继续加入DTT,得中间体HP-SH。
7.根据权利要求6所述负载光敏剂的PEG化肝素纳米胶束的制备方法,其特征在于,所述MeS缓冲液的制备方法:称取吗啉乙磺酸溶于纯化水中,滴加氢氧化钠溶液调节pH至5.5,定容,得到MeS缓冲溶液。
8.根据权利要求4所述负载光敏剂的PEG化肝素纳米胶束的制备方法,其特征在于,在步骤(3)的反应中,加入HOTu参与反应。
9.根据权利要求8所述负载光敏剂的PEG化肝素纳米胶束的制备方法,其特征在于,在步骤(3)的反应完成后,置换氮气保护,避光,加入DMF和DIEA,在室温下搅拌过夜。
10.根据权利要求4所述负载光敏剂的PEG化肝素纳米胶束的制备方法,其特征在于,在步骤(4)的反应中,中间体HP-SH与Ppa衍生物反应,加入催化剂三乙胺。
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