CN115554409A - 一种具有线粒体靶向性的高分子载体材料及其制备方法和用途 - Google Patents
一种具有线粒体靶向性的高分子载体材料及其制备方法和用途 Download PDFInfo
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Abstract
本发明公开一种具有线粒体靶向性的高分子载体材料及其制备方法和用途,以线粒体靶向性的高分子共聚物为载体材料,采用乳化‑溶剂挥发法制备熊果酸靶向纳米粒,增强其在肿瘤细胞的浓集能力,延长作用时间。采用MTT法和细胞划痕实验,研究熊果酸纳米粒对肿瘤细胞生长的抑制作用,激光共聚焦成像实验验证线粒体的靶向效果,结果显示,熊果酸纳米粒具有线粒体靶向作用,相对于熊果酸原料药,熊果酸纳米粒具有更强的抑制肿瘤细胞增殖的作用。
Description
技术领域
本发明内容属于医药技术领域。具体涉及一种线粒体靶向高分子载体材料TPP-PEG-PLLA-PAEEP的合成、载体材料包载熊果酸纳米粒的制备方法和用途。
背景技术
熊果酸可以从多种天然植物中提取出来的五环三萜类化合物,具有镇静、抗炎、抗菌、抗糖尿病、抗溃疡、抗氧化、抗肿瘤等多种生物学效应。相关研究结果显示,熊果酸能诱导多种肿瘤细胞死亡和抑制增殖。但熊果酸不溶于水,导致其在体内生物利用度很差,且药动学数据显示其在体内代谢较快,半衰期短,进一步限制了其在临床上的使用。纳米粒属于纳米释药系统,能通过载体材料包载药物增加生物利用度和稳定性,还可以对纳米粒进行修饰达到缓控释等目的。
采用线粒体靶向高分子材料制备熊果酸纳米粒,能改善熊果酸的溶解度、延长代谢时间、增加药物浓集浓度、提高抗肿瘤活性。
发明内容
为了解决上述技术问题,本发明提供一种线粒体靶向高分子载体材料TPP-PEG-PLLA-PAEEP。
本发明的另一个发明目的是载体材料包载熊果酸纳米粒的制备方法及在靶向性高分子载体材料上的用途。
本发明提供的技术方案如下:
一种具有线粒体靶向性的高分子载体材料TPP-PEG-PLLA-PAEEP,其结构如下:
进一步地,上述线粒体靶向基团选自三苯基膦。
一种线粒体靶向性高分子载体材料TPP-PEG-PLLA-PAEEP的制备方法,包
含步骤如下:
(1) 中间体N-叔丁氧羰基乙醇胺(EABoc)的合成
称取碳酸氢钠适量溶于纯化水中,加入溶有乙醇胺的四氢呋喃溶液;
称取一定量二碳酸二叔丁酯溶于四氢呋喃溶液,在0℃、磁力搅拌的情况下用恒压滴液漏斗逐滴加入到前者混合溶液中,反应30 min后升至室温,连续反应12 h;反应液用乙醚萃取,收集上层有机相,然后加入适量的无水硫酸钠,搅拌2 h后过滤,滤液经旋蒸去除有机溶剂,并用三氯甲烷洗涤得到N-叔丁氧羰基乙醇胺(EABoc);
(2) 中间体2-(N-叔丁氧羰基胺基)乙氧基-2-氧-1,3,2-二氧磷杂环戊烷(PEEABoc)的合成
反应装置保持密闭状态,先抽真空充氮气;称取除水三乙胺、EABoc,加入到除水四氢呋喃中,通过恒压滴液漏斗加入到双口烧瓶中,体系温度恒温在-5℃,磁力搅拌,氮气保护;于手套箱中称取COP,与适量四氢呋喃混合后加入到恒压漏斗中,在30 min内将COP的四氢呋喃溶液逐渐滴入双口烧瓶内,体系在恒温下反应8 h;将生成的沉淀过滤,滤液旋蒸去除溶剂,将产物用三氯甲烷洗涤,旋蒸温度为40℃;
(3) 中间体聚乙二醇-聚乳酸(PEG-PLLA)的合成
重结晶左旋丙交酯:称取一定量的左旋丙交酯于蒸馏水中,水浴60℃溶解,维持2~3 min后自然冷却,然后放置于4℃环境,使之析出大量晶体;然后弃去溶剂,晶体40℃真空干燥;重复以上操作两次,以达到纯化目的;
称取重结晶左旋丙交酯、PEG6000、辛酸亚锡置于圆底烧瓶中,放入搅拌子,装置体系抽真空充氮气后油浴120℃反应2 h;
将产物用一定量三氯甲烷溶解后静置4 h,然后将之滴入一定量乙醚中,封存于-20℃环境,待产品沉淀;
反复用乙醚沉淀几次,弃去溶剂,沉淀于40℃真空干燥,得白色干燥粉末状固体;
(4) 中间体聚乙二醇-聚乳酸-聚磷酸酯(PEG-PLLA-PAEEP)的合成
称取一定量的PEG-PLLA、PEEABoc、辛酸亚锡,加入到除水四氢呋喃中,用注射器将混合溶液通过恒压漏斗加入到干燥的、真空氮气保护环境的圆底烧瓶中;体系在30℃条件下反应2 h;将产物沉淀于乙醚中,过滤,沉淀于40℃真空干燥,即得;
(5) PEG-PLLA-PAEEP脱Boc保护基团
在0℃下,将干燥后的PEG-PLLA-PAEEP加入到 4 mol/L 氯化氢的无水四氢呋喃溶液中,搅拌1 h,反应液沉淀于冷的无水乙醚溶液中,过滤,沉淀于室温下真空干燥,得到产物;
(6) 中间体三苯基膦溴丙酸(TPP-COOH)的合成
将TPP加入到3-溴丙酸的乙腈溶液中;抽真空充N2,将混合物在80℃下搅拌24 h,旋干溶剂,用三氯甲烷溶解浓缩物后,用乙醚沉淀,沉淀物真空干燥,即得;
(7) PEG-PLLA-PAEEP接三苯基膦溴丙酸盐
取适量三苯基膦溴丙酸、NHS、EDCI溶于适量乙腈中,室温搅拌4 h活化羧基;PEG-PLLA-PAEEP溶于适量二氯甲烷中,加入适量三乙胺;
将二氯甲烷混合溶液缓缓加入乙腈溶液体系中,室温继续搅拌反应24 h,接着用超纯水洗涤反应物,将有机层用无水乙醚进行沉淀,沉淀物真空干燥,即得;
(8) 熊果酸纳米粒的制备
采用乳化-溶剂挥发法制备纳米粒:
称取材料TPP-PEG-PLLA-PAEEP,溶解于适量二氯甲烷中;称取熊果酸溶解于无水甲醇中;将二氯甲烷溶液与甲醇溶液混匀,在超声条件下加入 SDS溶液,继续超声;然后常温缓慢搅拌,待挥干溶剂,即得纳米粒溶液;
(9) 纳米粒的平均粒径与表面电位测定
采用动态光散射法测定纳米粒平均粒径和表面电位;取一定量纳米粒溶液,用超纯水稀释后,利用马尔文激光粒度仪测定粒径与表面电位。
进一步地,上述步骤(4)中PEG-PLLA与PEEABoc的摩尔比为1:50~1:100,辛酸亚锡与PEEABoc的摩尔比为1:50~1:100。
进一步地,上述步骤(7)中三苯基膦溴丙酸、NHS、EDCI的比例为质量比43:25:40。
进一步地,上述步骤(7)中PEG-PLLA-PAEEP与三苯基膦溴丙酸盐的比例分别为质量比1:1或1:3或1:5。
进一步地,上述TPP-PEG-PLLA-PAEEP选用接合摩尔比例为TPP:PEG-PLLA-PAEEP=3:1的载体材料,载体材料与药物熊果酸质量之比为1:1,溶剂二氯甲烷、无水甲醇和2.5%SDS溶液体积之比为3:1:5。
进一步地,上述步骤(8)固化时间为4小时,超声由超声细胞破碎仪提供,频率为195 W,时间为15 min。
一种如上述中任意所述的的制备方法制备的高分子载体材料TPP-PEG-PLLA-PAEEP作为线粒体靶向性高分子载体材料的应用。
本发明的有益效果:
本发明的技术方案以线粒体靶向性的高分子共聚物为载体材料,采用乳化-溶剂挥发法制备熊果酸靶向纳米粒,增强其在肿瘤细胞的浓集能力,延长作用时间。
采用MTT法和细胞划痕实验,研究熊果酸纳米粒对肿瘤细胞生长的抑制作用,激光共聚焦成像实验验证线粒体的靶向效果,结果显示,熊果酸纳米粒具有线粒体靶向作用,相对于熊果酸原料药,熊果酸纳米粒具有更强的抑制肿瘤细胞增殖的作用。
附图说明
图1是N-叔丁氧羰基乙醇胺的核磁共振氢谱图;
图2是N-叔丁氧羰基乙醇胺的红外光谱图;
图3是PEEABoc的核磁共振氢谱图;
图4是PEG-PLLA的核磁共振氢谱图;
图5是PEG-PLLA-PAEEP的核磁共振氢谱图;
图6是TPP-PEG-PLLA-PAEEP的核磁共振氢谱图;
图7:A是纳米粒溶液;B是不同材料制备的纳米粒平均粒径分布图;C是不同材料制备的纳米粒表面电位分布图;
图8是不同比例的TPP与PEG-PLLA-PAEEP接合合成的载体材料制备的纳米粒包载荧光素与C6细胞共孵育的荧光成像图;
图9是不同浓度的空白纳米粒、熊果酸原料药、熊果酸纳米粒给药C6细胞后的细胞存活率统计图;
图10:A是空白组、熊果酸原料药组、熊果酸纳米粒组的细胞划痕图片;
图11:B是空白组、熊果酸原料药组、熊果酸纳米粒组的细胞迁移率统计图。
具体实施方案
下面通过借助实施例更加详细地说明本发明,但以下实施例仅是说明性的,本发明的保护范围并不受这些实施例的限制。
实施例一
(1) 中间体N-叔丁氧羰基乙醇胺(EABoc)的合成
称取碳酸氢钠0.8400 g溶于10 mL纯化水中,加入溶有0.6100 g乙醇胺的10 mL四氢呋喃溶液。称取2.1800 g二碳酸二叔丁酯溶于四氢呋喃溶液,在0℃、磁力搅拌的情况下用恒压滴液漏斗逐滴加入到前者混合溶液中,反应30 min后升至室温,反应过夜。反应液用乙醚萃取三次,收集上层有机相,然后加入适量的无水硫酸钠除水,搅拌2 h过滤,滤液经过旋蒸去除有机溶剂,并用三氯甲烷洗涤三次得到N-叔丁氧羰基乙醇胺(EABoc)。
采用核磁共振氢谱法和红外光谱法验证结构,如图1、图2。核磁氢谱中δ=1.49×10-6处为叔丁基9个氢的特征峰;氨基氢的化学位移因为羰基的存在,从高场向低场移动,出现在δ=5.15×10-6处。红外谱图中,仲胺的伸缩振动峰出现在3065 cm-1处,弯曲振动峰位于1520 cm-1;羰基吸收峰位于1697 cm-1;叔丁基的裂分峰出现在1367、1393 cm-1;羟基吸收峰出现在3386 cm-1。
(2) 中间体2-(N-叔丁氧羰基胺基)乙氧基-2-氧-1,3,2-二氧磷杂环戊烷(PEEABoc)的合成
反应装置保持密闭状态,先抽真空充氮气。称取除水三乙胺0.7030 g、EABoc1.9787 g,加入到5 mL除水四氢呋喃中,用注射器通过恒压滴液漏斗加入到双口烧瓶中,体系温度恒温在-5℃,磁力搅拌,氮气保护。于手套箱中用1 mL注射器称取COP 0.9600 g打入到恒压漏斗中,再注射入5 mL四氢呋喃溶液,在30 min内将COP的四氢呋喃溶液逐渐滴入双口烧瓶内,体系在恒温下反应8 h。将生成的沉淀过滤,滤液旋蒸去除溶剂,将产物用三氯甲烷洗涤多次,旋蒸温度40℃。
采用核磁共振氢谱法验证结构,如图3。五元磷环上4个亚甲基氢因为氧的吸电子效应,化学位移位于低场,于δ=4.1×10-6出现。
(3) 中间体聚乙二醇-聚乳酸(PEG-PLLA)的合成
重结晶左旋丙交酯:称取一定量左旋丙交酯于蒸馏水中,水浴60℃溶解,维持2~3min后,自然冷却,然后放置于4℃环境使之析出晶体。然后取出弃去溶剂,真空40℃干燥。重复以上操作两次,以达到纯化目的。称取重结晶左旋丙交酯5.3126 g、PEG6000 2.2136 g、辛酸亚锡0.1494 g置于圆底烧瓶中,放入搅拌子,装置体系抽真空充氮气后油浴120℃反应2 h。将产物用一定量三氯甲烷溶解后静置4 h,然后将之滴入一定量乙醚中,封存于-20℃环境,待产品沉淀,反复用乙醚沉淀几次,挥干溶剂, 40℃真空干燥,得白色干燥粉末状固体。
采用核磁共振氢谱法验证结构,如图4。聚合链段中的甲基裂分二重峰出现在δ=1.51×10-6处;次甲基氢因为羰基与氧的吸电子作用存在,化学位移向低场移动,于δ=5.2×10-6出现;亚甲基氢同样受到氧的吸电子效应影响,化学位移移动到δ=3.7×10-6处。
(4) 中间体聚乙二醇-聚乳酸-聚磷酸酯(PEG-PLLA-PAEEP)的合成
称取PEG-PLLA 1.0020 g 、PEEABoc 1.3350 g、辛酸亚锡0.0203 g加入到5 mL除水四氢呋喃中,用注射器将混合溶液通过恒压漏斗加入到干燥的、真空氮气保护环境的圆底烧瓶中。体系在30℃条件下反应2 h。将产物沉淀于乙醚中,过滤,沉淀于真空40℃干燥,即得。
采用核磁共振氢谱法验证结构,如图5。
(5) PEG-PLLA-PAEEP脱Boc保护基团
在0℃下,将干燥后的PEG-PLLA-PAEEP加入到 4 mol/L 氯化氢的无水四氢呋喃溶液中,搅拌1 h,反应液沉淀于冷的无水乙醚溶液中,过滤,沉淀于室温下真空干燥,得到产物。
(6) 中间体三苯基膦溴丙酸(TPP-COOH)的合成
将TPP(8.5 g,32.5 mmol)加入到3-溴丙酸(5 g,32.5 mmol)的乙腈溶液中。抽真空充N2,将混合物在80℃下搅拌24 h,旋干溶剂,用三氯甲烷溶解浓缩物后,用乙醚沉淀,沉淀物真空干燥,即得。
(7) PEG-PLLA-PAEEP接三苯基膦溴丙酸盐
称取三苯基膦溴丙酸0.1000 g、NHS 0.0581 g、EDCI 0.0930 g溶于适量乙腈中,室温搅拌4 h活化羧基。PEG-PLLA-PAEEP 0.1000 g溶于适量二氯甲烷中,加入适量三乙胺。将二氯甲烷混合溶液缓缓加入乙腈溶液体系中,室温继续搅拌反应24 h,接着用超纯水洗涤反应物,将有机层用无水乙醚进行沉淀,沉淀物真空干燥,即得。
采用核磁共振氢谱法验证结构,如图6。叔丁基的9个氢峰已经消失;δ=7.75×10-6处出现了苯环氢峰;由于酰胺键的形成,支链中的亚甲基氢峰化学位移聚集于δ=3.15~3.5×10-6;其他氢峰化学位移变化不明显,可能因为反应前后各自的化学环境变化不明显。
(8) 熊果酸纳米粒的制备
采用乳化-溶剂挥发法制备纳米粒。称取材料TPP-PEG-PLLA-PAEEP 0.0300 g左右,溶解于3 mL二氯甲烷中;称取熊果酸0.0030 g左右,溶解于1 mL无水甲醇中。将二氯甲烷溶液与甲醇溶液混匀,在功率195 W的超声条件下加入5 mL 2.5%SDS溶液,继续超声5min。缓慢搅拌,待挥发溶剂,即得纳米粒溶液。根据各指标筛选出的适宜材料制备的纳米粒溶液呈淡蓝色半透明状,如图7A。
(9) 纳米粒的平均粒径与表面电位测定
采用动态光散射法测定纳米粒平均粒径和表面电位。取一定量纳米粒溶液,用超纯水稀释后,利用马尔文激光粒度仪测定粒径与表面电位。各接合比例材料制备的纳米粒的平均粒径与表面电位对比如图7B、C。
(10) 荧光成像
采用激光共聚焦仪器拍摄。调整细胞浓度为每毫升5×104个左右,以每皿1mL细胞悬液接种于共聚焦小皿,置于细胞培养箱培养24小时。每皿加入含包载荧光素的TPP-PEG-PLLA-PAEEP纳米粒的培养基1 mL。作用12小时后弃去旧培养基,用PBS清洗两次,加入MitoTracker Red染线粒体20 min;除去染料,用PBS清洗两次。清洗完毕加入1 mL培养基保持细胞活力,于激光共聚焦显微镜下观察。
结果如图8,荧光强度统计结果显示TPP/PEG-PLLA-PAEEP=3:1接合比例的材料靶向线粒体效果较好且不破坏细胞结构;TPP/PEG-PLLA-PAEEP=1:1的材料靶向效果较差;细胞内的荧光强度较高且均匀,提示TPP/PEG-PLLA-PAEEP=5:1的材料存在破环细胞膜与线粒体膜结构的可能性。
(11) 细胞增殖
采用MTT法。调整细胞浓度为每毫升1×104个左右,以每孔100 µL细胞悬液的量均匀接种到96孔板中,边缘孔补入相同量的PBS。置于细胞培养箱中培养24小时,每孔加入100µL含药培养基,每个样品重复5个孔。作用12小时后,每孔加入20 µL MTT溶液,孵育4小时后弃去孔中所有液体,每孔加入150 µL DMSO,振荡15分钟左右。于酶标仪450 nm下检测吸光度。计算细胞存活率。
结果如图9,给药12 h后,在规定浓度范围内,Drug-free nanoparticles具有一定的抑制细胞增殖能力,原因是三苯基膦具有一定的细胞毒性;熊果酸和熊果酸纳米粒对细胞增殖存在明显的浓度抑制,且熊果酸纳米粒的抑制效果更明显,存在显著统计学差异。
(12) 细胞迁移
调整细胞浓度为每毫升5×104个左右,以每孔1 mL细胞悬液接种于六孔板,置于细胞培养箱培养24小时,待细胞长至90%左右密度进行划痕操作。采用200 µL移液枪枪头进行垂直划线,划完后吸走旧培养基,PBS清洗一次,加入2 mL新培养基,于培养箱中缓和20min后拿出拍照,记为0 h。弃去培养基,分为Control组、UA组、 UA nanoparticles组分别加入2 mL含药培养基,孵育12 h后显微镜下拍照。运用软件进行图片处理,计算细胞迁移率。
结果如图10,给药12 h后熊果酸纳米粒的划痕面积最大,与空白组、熊果酸组存在显著性差异,其结果与细胞增殖结果一致。
实施例2
方法同实施例1,区别在于:
步骤(7)中各物质用量变为三苯基膦溴丙酸0.3000 g、NHS 0.1743 g、EDCI0.2790 g。
实施例3
方法同实施例1,区别在于:
步骤(7)中各物质用量变为三苯基膦溴丙酸0.5000 g、NHS 0.2905 g、EDCI0.4650 g。
实施例4
方法同实施例1,区别在于:
步骤(8)中载体材料TPP-PEG-PLLA-PAEEP 0.0200 g,熊果酸0.0040 g,甲醇2 mL,SDS浓度为1%。
实施例5
方法同实施例1,区别在于:
步骤(8)中载体材料TPP-PEG-PLLA-PAEEP 0.0200 g,熊果酸0.0020 g,甲醇1.5mL,SDS浓度为1.5%。
上述步骤(1)中无水硫酸钠的量应适量,不能过多也不能过少,过多会吸附产品造成产量减少,过少会除水不完全,一般加入量为溶液中含有少量未凝聚结块的无水硫酸钠即可。
步骤(2)中反应物于手套箱中进行称取;反应装置多次抽真空充氮气保证无水无氧;四氢呋喃和三乙胺需要进行除水处理。
步骤(2)中的三乙胺能与产物之一的氯化氢反应,适量加入三乙胺有利于反应的进行。
步骤(2)中整体反应属于放热反应,低温条件有利于反应的进行。
步骤(3)中反复溶解结晶析出有利于除去左旋丙交酯在储存过程中产生的杂质。
以上,仅为本发明较佳的具体实施方式,但本发明保护的范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内所做的任何修改,等同替换和改进等,均应包含在发明的保护范围之内。
Claims (9)
2.根据权利要求1所述的线粒体靶向性高分子载体材料TPP-PEG-PLLA-PAEEP,其特征在于:所述线粒体靶向基团选自三苯基膦。
3.一种如权利要求1所述的线粒体靶向性高分子载体材料TPP-PEG-PLLA-PAEEP的制备方法,其特征包含步骤如下:
(1)中间体N-叔丁氧羰基乙醇胺(EABoc)的合成
称取碳酸氢钠适量溶于纯化水中,加入溶有乙醇胺的四氢呋喃溶液;
称取一定量二碳酸二叔丁酯溶于四氢呋喃溶液,在0℃、磁力搅拌的情况下用恒压滴液漏斗逐滴加入到前者混合溶液中,反应30 min后升至室温,连续反应12 h;反应液用乙醚萃取,收集上层有机相,然后加入适量的无水硫酸钠,搅拌2 h后过滤,滤液经旋蒸去除有机溶剂,并用三氯甲烷洗涤得到N-叔丁氧羰基乙醇胺(EABoc);
(2)中间体2-(N-叔丁氧羰基胺基)乙氧基-2-氧-1,3,2-二氧磷杂环戊烷(PEEABoc)的合成
反应装置保持密闭状态,先抽真空充氮气;称取除水三乙胺、EABoc ,加入到除水四氢呋喃中,通过恒压滴液漏斗加入到双口烧瓶中,体系温度恒温在-5℃,磁力搅拌,氮气保护;于手套箱中称取COP,与适量四氢呋喃混合后加入到恒压漏斗中,在30 min内将COP的四氢呋喃溶液逐渐滴入双口烧瓶内,体系在恒温下反应8 h;将生成的沉淀过滤,滤液旋蒸去除溶剂,将产物用三氯甲烷洗涤,旋蒸温度为40℃;
(3)中间体聚乙二醇-聚乳酸(PEG-PLLA)的合成
重结晶左旋丙交酯:称取一定量的左旋丙交酯于蒸馏水中,水浴60℃溶解,维持2~3min后自然冷却,然后放置于4℃环境,使之析出大量晶体,然后弃去溶剂,晶体40℃真空干燥;重复以上操作两次,以达到纯化目的;
称取重结晶左旋丙交酯、PEG6000、辛酸亚锡置于圆底烧瓶中,放入搅拌子,装置体系抽真空充氮气后油浴120℃反应2 h;
将产物用一定量三氯甲烷溶解后静置4 h,然后将之滴入一定量乙醚中,封存于-20℃环境,待产品沉淀;
反复用乙醚沉淀几次,弃去溶剂,沉淀于40℃真空干燥,得白色干燥粉末状固体;
(4)中间体聚乙二醇-聚乳酸-聚磷酸酯(PEG-PLA-PAEEP)的合成
称取一定量的PEG-PLA 、PEEABoc、辛酸亚锡,加入到除水四氢呋喃中,用注射器将混合溶液通过恒压漏斗加入到干燥的、真空氮气保护环境的圆底烧瓶中;体系在30℃条件下反应2 h;将产物沉淀于乙醚中,过滤,沉淀于40℃真空干燥,即得;
(5)PEG-PLLA-PAEEP脱Boc保护基团
在0℃下,将干燥后的PEG-PLLA-PAEEP加入到 4 mol/L 氯化氢的无水四氢呋喃溶液中,搅拌1 h,反应液沉淀于冷的无水乙醚溶液中,过滤,沉淀于室温下真空干燥,得到产物;
(6)中间体三苯基膦溴丙酸(TPP-COOH)的合成
将TPP加入到3-溴丙酸的乙腈溶液中;抽真空充N2,将混合物在80℃下搅拌24 h,旋干溶剂,用三氯甲烷溶解浓缩物后,用乙醚沉淀,沉淀物真空干燥,即得;
(7)PEG-PLLA-PAEEP接三苯基膦溴丙酸盐
取适量三苯基膦溴丙酸、NHS、EDCI溶于适量乙腈中,室温搅拌4 h活化羧基;PEG-PLLA-PAEEP溶于适量二氯甲烷中,加入适量三乙胺;
将二氯甲烷混合溶液缓缓加入乙腈溶液体系中,室温继续搅拌反应24 h,接着用超纯水洗涤反应物,将有机层用无水乙醚进行沉淀,沉淀物真空干燥,即得;
(8)熊果酸纳米粒的制备
采用乳化-溶剂挥发法制备纳米粒:
称取材料TPP-PEG-PLLA-PAEEP ,溶解于适量二氯甲烷中;称取熊果酸溶解于无水甲醇中;将二氯甲烷溶液与甲醇溶液混匀,在超声条件下加入 SDS溶液,继续超声;然后常温缓慢搅拌,待挥干溶剂,即得纳米粒溶液;
(9)纳米粒的平均粒径与表面电位测定
采用动态光散射法测定纳米粒平均粒径和表面电位;取一定量纳米粒溶液,用超纯水稀释后,利用马尔文激光粒度仪测定粒径与表面电位。
4.根据权利要求3所述的制备方法,其特征在于:步骤(4)中PEG-PLLA与PEEABoc的摩尔比为1:50~1:100,辛酸亚锡与PEEABoc的摩尔比为1:50~1:100。
5.根据权利要求3所述的制备方法,其特征在于:步骤(7)中三苯基膦溴丙酸、NHS、EDCI的比例为质量比43:25:40。
6.根据权利要求3所述的制备方法,其特征在于:步骤(7)中PEG-PLLA-PAEEP与三苯基膦溴丙酸盐的比例分别为质量比1:1或1:3或1:5。
7.根据权利要求3所述的筛选方法,其特征在于:TPP-PEG-PLLA-PAEEP选用接合摩尔比例为TPP:PEG-PLLA-PAEEP=3:1的载体材料,载体材料与药物熊果酸质量之比为1:1,溶剂二氯甲烷、无水甲醇和2.5%SDS溶液体积之比为3:1:5。
8.根据权利要求3所述的制备方法,其特征在于:所述步骤(8)固化时间为4小时,超声由超声细胞破碎仪提供,频率为195 W,时间为15 min。
9.一种如权利要求3-8中任意所述的的制备方法制备的高分子载体材料TPP-PEG-PLLA-PAEEP作为线粒体靶向性高分子载体材料的应用。
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