CN114796515A - 一种双重响应的聚乙二醇前药及其制备方法和应用 - Google Patents
一种双重响应的聚乙二醇前药及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种双重响应的聚乙二醇前药,该聚乙二醇前药具有较高的稳定性,在病灶部位具有较高的药物释放性能。本发明还公开了双重响应的聚乙二醇前药的制备方法,包括将三羟甲基乙烷、肉桂醛和对甲苯磺酸加入四氢呋喃中,搅拌,蒸发,纯化得到中间产物1;将所述中间产物1和N,N’‑羰基二咪唑加入二氯甲烷,搅拌,蒸发,纯化得到中间产物2;将羧基化聚乙二醇单甲醚加入到氯化亚砜,加入甲苯共沸后加入四氢呋喃混合,将混合物与含有对羟基苯甲醇和三乙胺的四氢呋喃溶液反应,减压抽滤,纯化得到中间产物3;将中间产物2、中间产物3和4‑二甲基氨基吡啶混合后加入二氯甲烷反应,纯化得到双重响应的聚乙二醇前药。
Description
技术领域
本发明属于有机化合物领域,具体涉及一种双重响应的聚乙二醇前药及其制备方法和应用。
背景技术
近年来,恶性肿瘤的发病率和死亡率呈现明显上升趋势,已成为严重威胁人民健康的疾病,具有死亡率高、预后差的特点。目前在临床上,化疗仍然是肿瘤治疗中最常见的手段之一。然而,由于其较差的组织选择性和副作用过大等问题,化疗仍然无法达到令人满意的治疗效果。为了解决这些问题,近年来研究人员将纳米技术应用到了肿瘤的治疗中,构建了各种纳米递药系统,通过高通运性和滞留效应或主动靶向的方式,使药物在肿瘤部位聚集,增大药物浓度,同时提高抗肿瘤药物的戴药量、稳定性、生物利用度。
理想的纳米递药系统应该能使抗肿瘤药物在体内稳定运输,到达肿瘤之前无泄漏,达到肿瘤组织之后的药物释放是可控的,而刺激响应性纳米载体是解决这一问题的有效手段。公开号为CN113713121A的中国专利公开了一种开关型介孔二氧化硅纳米药物载体的制备方法及应用,其首先在含有氨基的介孔二氧化硅纳米颗粒表面及介孔内构建聚多巴胺涂层,使用含有侧链端羧基的仿细胞膜两性离子聚合物,与载药介孔二氧化硅纳米颗粒表面氨基的酰胺化反应进行多点锚定提高长循环性能,并通过含二硫键及两端羧基的分子将不同类型的靶向配体如含叶酸及RGD环肽偶联在介孔二氧化硅纳米药物表面提升对肿瘤细胞的靶向能力。但该药物载体抗癌药物毒性大、靶向性差。
聚合物前药是一种经生物体内转化才释放活性并发挥药效的前体药物。相比于传统小分子抗癌药物毒性大、靶向性差等缺点,聚合物前药的载药率确定,稳定性更高,因而被广泛应用于抗肿瘤药物递送领域。然而,药物和聚合物键合后在体外一般会失活或者减少活性,当聚合物前药到达病灶部位后,药物需要有效地脱离聚合物,才能达到释放药物,发挥药效的目的,因此,在聚合物与药物之间引入具有刺激响应型的化学键是提高药物释放,发挥药效的重要方式。
具有特定刺激响应型的前药,在体液循环过程中保持稳定,而在病灶部位独特的刺激条件下则能够特异性释放药物,既可以降低对正常组织的毒副作用,又可以提高疗效。
因此,亟需设计一种具有双重刺激和多重刺激响应性聚合物纳米粒子的策略,能够对两种或两种以上的信号组合作出响应,能更进一步提高药物释放性能,提高聚合物前药的药效。
发明内容
本发明提供了一种双重响应的聚乙二醇前药,该聚乙二醇前药再体内循环时具有较高的稳定性,在病灶部位具有较高的药物释放性能。
一种双重响应的聚乙二醇前药的结构通式如下:
其中,n为10-120。
将对羟基苯甲醇通过酯键键合至mPEG-COOH末端,通过酸敏感缩醛键接入肉桂醛,得到同时对酯酶和酸响应的聚乙二醇前药,该聚乙二醇前药能通过亲疏水作用自组装成纳米前药,其外层的聚乙二醇能增加药物的稳定性,延长药物在体内的循环时间,提高药物在病灶部位的富集,并且能在肿瘤细胞内的酯酶和酸性条件下特异性释放出能够引起活性氧(ROS)水平提高的肉桂醛,同时释放出能够和谷胱甘肽(GSH)结合的亚甲基醌,从而抑制GSH对ROS的抵抗作用,提高ROS引起的肿瘤细胞的杀伤,极具抗肿瘤应用潜力。
本发明还提供了一种双重响应的聚乙二醇前药的制备方法,包括:
合成中间产物1:将三羟甲基乙烷、肉桂醛和对甲苯磺酸加入四氢呋喃中,在50-90℃下搅拌6–10h得到第一混合溶液,冷却至室温,蒸发去溶剂得到第一混合物,对所述第一混合物纯化得到中间产物1;
合成中间产物2:将所述中间产物1和N,N’-羰基二咪唑加入二氯甲烷中,搅拌0.5–1h得到第二混合溶液,蒸发去溶剂得到第二混合物,对所述第二混合物纯化得到中间产物2;
合成中间产物3:将羧基化聚乙二醇单甲醚(mPEG-COOH)加入到氯化亚砜反应2–3h,减压除去剩余的氯化亚砜,然后加入甲苯共沸后得到第三混合物,将所述第三混合物加入四氢呋喃中得到第三混合溶液,将所述第三混合溶液加入至含有对羟基苯甲醇和三乙胺的四氢呋喃溶液中反应1-6h得到第四混合溶液,对第四混合溶液减压抽滤去除杂质,然后通过无水乙醚沉淀1-3次得到第四混合物,对所述第四混合物纯化得到中间产物3;
将所述中间产物2、所述中间产物3和4-二甲基氨基吡啶混合后加入二氯甲烷,在25-50℃下反应24–36h,纯化后得到双重响应的聚乙二醇前药。
一种双重响应的聚乙二醇前药的制备方法的反应路线如下:
本发明将中间产物2和中间产物3通过4-二甲基氨基吡啶进行催化反应得到了最终聚乙二醇前药。其中,中间产物3的羟基作为亲核试剂进攻中间产物2中羰基咪唑基的羰基发生取代反应,由于中间产物2和3的反应位阻较大及中间产物3的反应活性不高,在4-二甲基氨基吡啶催化下有利于咪唑基的脱去,发生亲核取代,显著提高了反应收率。
所述三羟甲基乙烷、肉桂醛和对甲苯磺酸的摩尔比为1:1–1.2:0.01–0.05。
所述mPEG-COOH的数均分子量为600–5000g/mol。进一步的,所述mPEG-COOH的数均分子量为600–5000g/mol。
所述中间产物1和N,N’-羰基二咪唑的摩尔比为1:1–2。
所述中间产物2、中间产物3和4-二甲基氨基吡啶的摩尔比为1–2:1:2。
所述双重响应的聚乙二醇前药通过自组装在制备纳米药物颗粒上的应用,包括:
将所述双重响应的聚乙二醇前药加入有机相中,然后加入纯水,搅拌0.5–2h,除去有机相溶剂得到所述双重响应的聚乙二醇前药纳米颗粒水溶液,浓缩、冻干得到纳米药物颗粒。
通过有机相溶解该聚合物前药,再加入至水相中,使其通过苯环疏水端的聚集作用发生自组装能有效形成纳米尺寸的颗粒。
所述纳米药物颗粒的粒径为60–500nm。
所述有机相溶剂为甲醇、乙醇、丙酮、乙腈、DMSO或DMF。进一步的,所述有机相溶剂为甲醇或DMSO。
所述双重响应的聚乙二醇前药溶解在有机相中的浓度为10–200mg/ml。
所述水相和有机相的体积比为3–10:1。
不同浓度的聚乙二醇前药有机相溶液以及不同的水相/有机相比例,会导致纳米药物颗粒的尺寸不同。有机相溶液浓度在一定范围内浓度越大,得到的颗粒尺寸越大,水相和有机相比例越高,颗粒的尺寸越小。以上选择的条件能保证纳米颗粒的尺寸在50–500nm内,且粒径分布较均一,超过这个范围不一定能保证分散度和尺寸大小。
与现有技术相比,本发明的有益效果为:
(1)本发明提供的双重响应的聚乙二醇前药,外层PEG能有效保持内部药物的稳定性,减少药物体内的非特异性排除,并增加药物病灶部位的富集。并且,该聚合物纳米前药能在酯酶和酸性条件下断裂,释放出肉桂醛和亚甲基醌两种有效药物,同时提高ROS水平和降低GSH水平,发挥协同增效的作用。
(2)本发明提供了该双重响应的聚乙二醇前药的高效合成方法,通过N,N’-羰基二咪唑将含羟基的肉桂醛缩醛化中间产物1和对聚乙二醇酯苯甲醇(中间产物3)键合,利用4-二甲基氨基吡啶催化得到了具有较高纯度,较高产率的双重响应的聚乙二醇前药。此外,该聚乙二醇前药由于其特殊的结构特点(适合长度的聚乙二醇作为亲水段,含两个苯环的疏水端能发生疏水聚集及π-π堆积作用)能自组装成纳米颗粒,能够有效制备纳米制剂。
附图说明
图1为实施例1制备得到的中间产物1的1H NMR谱图;
图2为实施例1制备得到的中间产物2的1H NMR谱图;
图3为实施例1制备得到的中间产物3的1H NMR谱图;
图4为实施例1制备得到的双重响应的聚乙二醇前药的1H NMR谱图;
图5为应用例制备得到的聚合物前药纳米制剂的粒径及分布图;
图6为应用例制备得到的聚合物前药纳米制剂的酸响应性图;
图7为应用例制备得到的聚合物前药纳米制剂的酯酶响应性图;
图8为应用例制备得到的聚合物前药纳米制剂的降低细胞内GSH图;
图9为应用例制备得到的聚合物前药纳米制剂的抑制肿瘤细胞活力情况图;
图10为应用例制备得到的聚合物前药纳米制剂的抑制肿瘤生长情况图。
具体实施方式
以下结合实施例对本发明进行技术方案进行详细说明,应当指出的是,具体实施方案只是对本发明的详细说明,不应视为对本发明的限定。对技术人员来说,本发明可以有多种更改和变化,凡是在本发明的保护权限范围内,所做的修改、替换或者改进,均应包含在本发明保护范围内。下列实施例中的实验方法,无特殊说明,均为常规方法;所采用的材料,无特殊说明,均为常规生化试剂厂商购买。
实施例1:
一种双重响应的聚乙二醇前药的合成
(1)合成及表征中间产物1:将三羟甲基乙烷(40mmol),肉桂醛(40mmol),对甲苯磺酸(1mmol)溶解在70ml的干燥的四氢呋喃(利用氢化钙除水,蒸馏纯化)中。在75℃下,搅拌反应6h。冷却至室温后,向反应瓶中添加100μl三乙胺,适当震荡后结束反应。利用旋转蒸发仪除去溶剂,油泵减压抽干,得到粗产物1。利用硅胶柱层析进行纯化,淋洗剂:正己烷/乙酸乙酯(7/3,v/v)。
利用1H NMR检测中间产物1的结构。结果如图1所示,中间产物1结构中各质子信号在谱图中均有显示,7.28ppm为氘代氯仿溶剂信号峰,7.33-7.59ppm之间对应苯环上各特征质子(a、b、c)信号,6.80ppm(d)、6.23ppm(e)为与苯环相连烯键CH信号峰,5.10ppm(i)、3.88ppm(m)、0.80ppm(h)、3.58ppm(j)和4.01ppm(j)分别对应三羟甲基乙烷上质子中亚甲基和甲基上质子的特征信号,且各信号峰积分面积比与质子数比一致,表明中间产物1结构的正确性。
(2)合成及表征中间产物2:将纯化后的产物1(10mmol)和N,N’-羰基二咪唑(20mmol)溶解于50ml二氯甲烷(利用氢化钙除水,蒸馏纯化)中,在室温下搅拌反应30min,将产物溶液用旋转蒸发仪浓缩,油泵减压抽干,得到粗产物2。利用硅胶柱层析进行纯化,淋洗剂为:乙酸乙酯。
利用1H NMR检测其结构,结果如图2所示,中间产物2中各质子峰均在谱图中出现,且化学位移与预期一致。谱图上出现咪唑环上质子信号峰8.17ppm(j)、7.44ppm(k)和7.99ppm(m),且信号峰积分面积比与质子数比一致,表明反应接上羰基咪唑,中间产物2结构正确。
(3)合成及表征中间产物3:取2g羧基化聚乙二醇单甲醚(mPEG45-COOH),加入10ml氯化亚砜进行反应,室温下反应2h,减压除去未反应的氯化亚砜,加入50ml甲苯进行减压共沸处理。将共沸后得到的产物用干燥的四氢呋喃溶解,加入至恒压滴液漏斗中,缓慢滴加至含对羟基苯甲醇(0.18g)及三乙胺(0.15g)的四氢呋喃溶液,室温搅拌反应约2h。反应溶液减压抽滤除少量沉淀,减压浓缩,加入至50ml无水乙醚中沉淀,重复沉淀3次,得到白色粉末粗产物3。粗产物通过硅胶柱层析法纯化,淋洗液:三氯甲烷/甲醇混合溶剂,三氯甲烷和甲醇体积比为10-15:1。
利用1H NMR检测中间产物3的结构,结果如图3所示,甲氧基聚乙二醇羧基中的质子峰在谱图中均显示:a(3.30ppm)、b和c(3.57ppm)、d(3.67-2.90ppm),此外,苯环质子e和f在谱图7.10ppm和7.42ppm均有信号峰,对位的亚甲基在4.55ppm(g)有信号峰,且积分面积比与质子数比相吻合,说明中间产物3的结构正确。
(4)终产物的合成:取中间产物2(0.2g)、中间产物3(0.6g)及4-二甲基氨基吡啶(0.075g)置于干燥的圆底烧瓶中,加20ml干燥的二氯甲烷溶解,40℃条件下进行搅拌反应30h。纯化:后将反应溶液用旋转蒸发仪进行浓缩,加二氯甲烷和无水乙醚(二氯甲烷/无水乙醚=1/10,v/v)沉淀3次。粗产物用5ml甲醇溶解,在去离子水中透析1-2天(透析袋截留分子量为3000),冻干得到白色絮状固体。
利用1H NMR检测其结构,结果如图4所示,在谱图中可以看出各个质子均有相应的谱峰,产物2中的氮杂环上的峰(8.17ppm)消失,说明产物2和产物3发生了反应,且二者其他峰均可以在谱图中找到相对应的峰,并且各信号峰积分面积比与预期结构中质子数比吻合,证明产物4结构的正确。
实施例2:
与实施例1不同的是,中间产物3选用的羧基化聚乙二醇单甲醚分子量是1000(mPEG22-COOH)。
(1)合成及表征中间产物1:与实施例1方法一致。
(2)合成及表征中间产物2:与实施例1方法一致。
(3)合成及表征中间产物3:取1g羧基化聚乙二醇单甲醚(mPEG22-COOH),加入10ml氯化亚砜进行反应,室温下反应2h,减压除去未反应的氯化亚砜,加入30ml甲苯进行减压共沸处理。将共沸后得到的产物用干燥的四氢呋喃溶解,加入至恒压滴液漏斗中,缓慢滴加至含对羟基苯甲醇(0.15g)及三乙胺(0.12g)的四氢呋喃溶液,室温搅拌反应约3h。反应溶液减压抽滤除少量沉淀,减压浓缩,加入至50ml无水乙醚中沉淀,重复沉淀3次,得到白色粉末粗产物3。粗产物通过硅胶柱层析法纯化,淋洗液:三氯甲烷/甲醇混合溶剂,三氯甲烷和甲醇体积比为10:1。1H NMR表征其结果,结果显示聚乙二醇单甲醚甲基质子信号(3.35ppm)、亚甲基信号(3.55ppm),此外,苯环质子信号分别在7.12ppm和7.45ppm均有信号峰,苯环对位的亚甲基在4.58ppm(g)有信号峰,且积分面积比与质子数比相吻合,说明中间产物3的结构正确。
(4)终产物的合成:取中间产物2(0.18g)、中间产物3(0.4g)及4-二甲基氨基吡啶(0.05g)置于干燥的圆底烧瓶中,加20ml干燥的二氯甲烷溶解,40℃条件下进行搅拌反应32h。纯化:后将反应溶液用旋转蒸发仪进行浓缩,-5℃下加无水乙醚下沉淀沉淀3次,得到的粗产物用3ml甲醇溶解,在去离子水中透析1天(透析袋截留分子量为1000),冻干得到白色絮状固体。利用1H NMR检测其结构,结果显示产物2中的氮杂环上的峰(8.17ppm)消失,中间产物2的其他质子信号和中间产物3的质子信号均可以在谱图中找到相对应的峰,并且各信号峰积分面积比与预期结构中质子数比吻合,证明中间产物4结构的正确。主要信号峰:两个苯环质子信号(8.0-7.0ppm)、苯环相连烯键信号(6.22ppm、6.75ppm),聚乙二醇单甲醚甲基质子信号(3.30ppm)、亚甲基信号(3.50ppm)。
应用例:
将实施例1中合成聚乙二醇前药溶于甲醇配制成10mg/ml溶液,逐滴加入至5ml超纯水中,搅拌0.5h,使用旋转蒸发仪除去甲醇,利用动态光散射仪检测水溶液中纳米颗粒的粒径及分布,其结果如图5所示,该聚乙二醇前药在水溶液中能自组装成尺寸为90nm左右的纳米颗粒,且尺寸较均一。将颗粒溶液在5000g离心5-10min,收集颗粒,冻干,得到聚合物前药纳米制剂PEG-Qcma。
聚合物前药纳米制剂PEG-Qcma的性能测试:
(1)双重响应的聚合物前药纳米制剂PEG-Qcma的酶响应性
将应用例中制备的聚合物前药纳米制剂PEG-Qcma加入至3组1ml谷胱甘肽(350μM)和1猪肝酯酶(100μg)的混合溶液中,使得聚合物前药纳米制剂终浓度分别为0μM、100μM、250μM,同时设置无酯酶对照组。在37℃下孵育3h,分别取50μl实验组和对照组溶液与50μl的艾尔曼试剂(0.5mM DTNB)进行混合。利用酶标仪测定在405nm下测定GSH含量变化。结果如图6所示,随着聚乙二醇前药含量的增加,溶液中GSH含量降低,并且未加酯酶组GSH含量未发生变化,表明了该聚乙二醇前药具有酯酶响应性断裂,并能释放出亚甲基醌中间体结合GSH,导致GSH水平下降。
(2)双重响应的聚合物前药纳米制剂PEG-Qcma的酸响应性
按应用例中制备的PEG-Qcma颗粒溶液,取0.9ml溶液,加入0.1ml10×PBS溶液(pH7.4和pH5.0),孵育48h,利用动态光散射仪检测聚乙二醇前药纳米颗粒在不同时间的粒径变化。结果如图7所示,pH7.4 PBS溶液中颗粒在2h内有所增加,可能是缓冲液中盐离子造成颗粒的部分聚集,但2-48h内粒径变化较小,说明该条件下稳定性较好。而pH5.0 PBS溶液中颗粒粒径快速变大,4h后粒径增加至1μm以上,48h粒径约3μm,且PDI均为1左右,这是结构中缩醛键发生了断裂,颗粒不稳定,发生了离散和重新团聚导致,进一步表明该聚合物前药具有酸敏感性。
(3)一种双重响应的聚合物前药纳米制剂PEG-Qcma降低肿瘤细胞内GSH:
在SW620细胞中,加入不同浓度(10-50μM)PEG-Qmca纳米前药(实施例2中制备)溶液孵育3h,收集细胞,用磷酸盐缓冲盐水(PBS)洗涤3次,加入40μL裂解液。将细胞裂解物离心(9800×g),并将上清液(10μL)与50μL艾尔曼试剂混合。用酶标仪测定在405nm下测定GSH含量。结果如图8所示,随着PEG-Qmca量的增加,SW620细胞内的GSH含量逐渐降低,50μM的PEG-Qmca可以是细胞中GSH含量下降约40%,结果表明PEG-Qmca可以进入细胞释放出亚甲基醌中间体,并且有效结合GSH。
(4)双重响应的聚合物前药纳米制剂PEG-Qcma有效抑制肿瘤细胞活力
在96孔板中培养SW620,加入50μM肉桂醛和50μM PEG-Qmca纳米前药(应用例中制备),培养24小时,利用你MTT溶液,孵育4h,加入二甲亚砜溶解,测量570nm处吸光度。结果如图9所示,加入肉桂醛的细胞抑制能力约15%,而PEG-Qmcap实验组,细胞活性抑制约65%,作为对照组加了过氧化氢酶的实验组细胞活力抑制约为25%,说明PEG-Qmca主要是通过提高ROS实现细胞活力的抑制。
(5)双重响应的聚合物前药纳米制剂PEG-Qcma有效抑制肿瘤生长
将SW620细胞通过皮下接种到裸鼠的下背部,将肉桂醛的DMSO溶液(10mg/mL)加入PBS溶液,使得浓度为1mg/ml,SW620细胞皮下接种到裸鼠的下背部,当小肿瘤约10cm3时,随机分组,每组5只,分别注射下列物质:PBS、肉桂醛(2mg/kg)、和PEG-Qmca纳米前药(肉桂醛含量2mg/kg)。每三天进行一次尾静脉给药,记录肿瘤尺寸。肿瘤的长度和宽度用游标卡尺测量,计算肿瘤体积使用以下公式V(mm3)=0.5×(a×b2),V为肿瘤体积,a为长度,b为宽度,单位mm。
结果如图10所示,2mg/kg游离肉桂醛对SW620肿瘤具有较弱的抑制能力,而含同剂量肉桂醛的PEG-Qmca纳米前药对肿瘤生长具有显著性抑制能力,肿瘤生长抑制效果约为肉桂醛的5倍,间接说明PEG-Qmca能有效富集至肿瘤部位,通过释放肉桂醛和亚甲基醌提高肿瘤抑制能力。
Claims (10)
2.一种根据权利要求1所述的双重响应的聚乙二醇前药的制备方法,其特征在于,包括:
合成中间产物1:将三羟甲基乙烷、肉桂醛和对甲苯磺酸加入四氢呋喃中,在50-90℃下搅拌6–10h得到第一混合溶液,冷却至室温,蒸发去溶剂得到第一混合物,对所述第一混合物纯化得到中间产物1;
合成中间产物2:将所述中间产物1和N,N’-羰基二咪唑加入二氯甲烷中,搅拌0.5–1h得到第二混合溶液,蒸发去溶剂得到第二混合物,对所述第二混合物纯化得到中间产物2;
合成中间产物3:将羧基化聚乙二醇单甲醚加入到氯化亚砜反应2–3h,减压除去剩余的氯化亚砜,然后加入甲苯共沸后得到第三混合物,将所述第三混合物加入四氢呋喃中得到第三混合溶液,将所述第三混合溶液加入含有对羟基苯甲醇和三乙胺的四氢呋喃溶液中反应1-6h得到第四混合溶液,对第四混合溶液减压抽滤去除杂质,然后通过无水乙醚沉淀1-3次得到第四混合物,对所述第四混合物纯化得到中间产物3;
将所述中间产物2、所述中间产物3和4-二甲基氨基吡啶混合后加入二氯甲烷,在25-50℃下反应24–36h,纯化后得到双重响应的聚乙二醇前药。
3.根据权利要求2所述的双重响应的聚乙二醇前药的制备方法,其特征在于,所述三羟甲基乙烷、肉桂醛和对甲苯磺酸的摩尔比为1:1–1.2:0.01–0.05。
4.根据权利要求2所述的双重响应的聚乙二醇前药的制备方法,其特征在于,所述羧基化聚乙二醇单甲醚的数均分子量为600–5000g/mol。
5.根据权利要求2所述的双重响应的聚乙二醇前药的制备方法,其特征在于,所述中间产物1和N,N’-羰基二咪唑的摩尔比为1:1–2。
6.根据权利要求2所述的双重响应的聚乙二醇前药的制备方法,其特征在于,所述中间产物2、中间产物3和4-二甲基氨基吡啶的摩尔比为1–2:1:2。
7.一种根据权利要求1所述的双重响应的聚乙二醇前药在制备纳米药物颗粒上的应用,其特征在于,包括:
将所述双重响应的聚乙二醇前药加入有机相中,然后加入纯水,搅拌0.5–2h,除去有机相溶剂得到所述双重响应的聚乙二醇前药纳米颗粒水溶液,浓缩、冻干得到纳米药物颗粒。
8.根据权利要求7所述的双重响应的聚乙二醇前药在制备纳米药物颗粒上的应用,其特征在于,所述纳米药物颗粒的粒径为100–500nm。
9.根据权利要求7所述的双重响应的聚乙二醇前药在制备纳米药物颗粒上的应用,其特征在于,所述有机相溶剂为甲醇、乙醇、丙酮、乙腈、DMSO或DMF。
10.根据权利要求7所述的双重响应的聚乙二醇前药在制备纳米药物颗粒上的应用,其特征在于,所述双重响应的聚乙二醇前药溶解在有机相中的浓度为10–200mg/ml。
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