CN109602702A - 一种具有脑靶向作用的白藜芦醇纳米粒及其制备工艺 - Google Patents
一种具有脑靶向作用的白藜芦醇纳米粒及其制备工艺 Download PDFInfo
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Abstract
一种具有脑靶向功能的白藜芦醇纳米粒制剂，包括组分：白藜芦醇、BCA、乳铁蛋白溶液、OH‑PEG‑MAL和注射用水。包括如下步骤：一、白藜芦醇纳米粒的制备；二、洗脱液A的配制；三、乳铁蛋白修饰白藜芦醇纳米粒的制备。本发明提供的白藜芦醇纳米粒制剂通过用乳铁蛋白修饰后，制成乳铁蛋白修饰的白藜芦醇纳米粒，具有脑靶向的纳米粒药物剂型，增强白藜芦醇的脑靶向和抗AD作用。
Description
技术领域
本发明涉及一种具有脑靶向作用的白藜芦醇纳米粒及其制备工艺，属于医药制剂技术领域。
背景技术
阿尔茨海默病(Alzheimer’s disease, AD)是老年人常见的一种神经系统退行性疾病，临床表现为记忆逐渐减退、认知和学习能力下降、精神萎靡和行为学障碍。其特征性病理学主要改变包括：脑神经元缺失、tau蛋白过度磷酸化导致的神经元纤维缠结(Neurofibrillary tangles, NFT)和β淀粉样蛋白(βeta amyloid peptides, Aβ)沉积所导致的神经细胞外的老年斑形成(Senile plaque, SP)。虽然AD的发病机制研究已百年有余，但至今仍没有定论，存在众多假说，包括胆碱能神经元假说、β淀粉样蛋白毒性假说、tau蛋白异常磷酸化假说和自由基损伤假说等。2016年由美国阿尔茨海默病协会调查分析报告中指出：在美国老年痴呆症的人年龄≥65年长期护理和临终关怀服务的估计是2360亿美元。可见，AD患者给家庭和社会都造成了极大的影响。尽管世界各国已投入大量资金用于阿尔茨海默病的研究，但临床上尚无根治AD的药物。目前，临床主要防治AD的药物有：抗胆碱能药物多奈哌齐、兴奋性氨基酸受体拮抗剂美金刚和抗氧化剂维生素E等一线药物。此外，还有针对Aβ疫苗、中药以及预防性保健药物等。AD的一线治疗药物及Aβ疫苗因疗效或毒副作用等原因在临床应用一直未取得突破性进展，故此人们开始关注中药及预防性保健药物对延缓AD的作用及机理。中医药因其特有的优势在治疗AD方面具有广阔的前景，引起广大学者的兴趣。现已研究发现银杏叶及其提取物、当归、白藜芦醇(Resveratrol, Res)等有改善AD模型大鼠的认知功能。尤其是Res良好的神经元保护效果已吸引了大量科研工作者去进一步的探索。
Res化学名为3,5,4-三羟基-1,2-二苯乙烯，是广泛存在于葡萄、虎杖、花生等植物性食物或药物中的多酚类化合物。研究发现，Res是一种植物抗毒素，大约有70多种植物可以产生Res用以抵御真菌和细菌等病原体攻击。动物实验证实，Res有广泛的药理作用，主要有以下几方面：抗癌、抗心血管疾病、抗细胞膜脂质过氧化、抗炎、神经保护以及类雌激素作用等。其中，Res抗β-淀粉样蛋白毒性作用、减少自由基的生成及改善AD海马组织中锥体细胞损伤的神经保护作用已成为研究的热点。本实验室前期研究发现，Res可改善AD小鼠模型学习和认知能力，有效抑制AD小鼠海马Aβ的表达，并呈一定的剂量相关性。但因Res为脂溶性化合物，其普通制剂通过口服或者注射给药在体内的生物利用度低，并且如何保证药物能顺利进入脑组织内并保持有效的治疗浓度尚未解决。因此，提高Res生物利用度和穿过血脑屏障(blood brain barrier, BBB)达到脑组织的浓度是有利于Res发挥治疗作用的必要条件。
目前，药物脑内递送系统的研究中，非侵袭性给药途径已成为国内外脑内靶向给药的研究重点。非侵袭性给药途径主要包括经鼻腔给药系统、单克隆抗体导向给药系统、纳米粒递送系统、载体或受体介导给药系统等。这些脑靶向给药系统能够“挑战”BBB的屏障作用，并取得一定的治疗效果。其中，尤以通过受体介导的微粒给药脑内递送系统的研究最为成功。其通过对网状内皮系统的功能抑制或对微粒给药系统的表面进行修饰以延长微粒给药系统在血液循环系统的保留时间，使药物有充足的时间穿透BBB进入脑内而起到治疗作用。在此类研究中，通常选择将纳米粒(Nanoparticles, NPs)作为给药载体，并在NPs上连接配体以配体-受体的形式将药物递送入脑。
NPs用于递送药物透过BBB的方法近年来已引起人们极大关注。NPs是药剂学领域研究中较为活跃的一系列新型超微小给药系统的统称，粒径在10-1000nm。最早研究的以NPs为载体传递入脑的药物是六肽化合物dalargin，其具有中枢镇痛作用，但外周给药无疗效，其药效学实验证明dalargin-PBCA-NPs经吐温-80表面修饰后静注给药可以将dalargin顺利输送入脑发挥镇痛作用。随后，对二肽化合物kytorpH in以及筒箭毒碱等的研究也获得了相似的结果。此外，采用优化的超顺磁性氧化铁纳米粒子作为药物载体也能透过BBB产生生物学效应。还有研究表明，单克隆抗体靶向治疗纳米载体系统药物ACNPs-DOX-DSPE-PEG2000-anti-CD20是一个单克隆抗体与化疗剂的有效制剂，可应用于靶向化疗特异性细胞。Koziara等采用在体灌注模型证明了紫杉醇-NPs能显著提高脑摄取量，且白蛋白结合紫杉醇的NPs注射混悬液(Abraxane®)在2005年初获得FDA的批准。在NPs的研究中，以聚氰基丙烯酸烷酯(Polyalkylcyanoacrylate，ACA)为代表的载体材料，是一种较为常见的生物降解型人工合成的高分子材料。ACA包括氰基丙烯酸甲酯、乙酯、正丁酯、异丁酯及异己酯等衍生物，在体内降解方式主要有两条途径：一是生成甲醛；二是在酯酶作用下降解为异丁醇通过肾脏代谢后尿液排出，在碱性环境下ACA主要通过酯酶降解，降解速度随烷基链的增长而降低。因氰基丙烯酸正丁酯(Butylcyanoacrylate, BCA)烷基链较长、降解慢、体内耐受性好，深受广大学者的青睐。由于ACA单体结构上含有氰基和羧基两个吸电子基团，使得β-C原子显示出很强的正电性。因此，只要有阴离子存在，即便在极少量的水、醇及弱碱作用下均会迅速使单体发生阴离子聚合。正是ACA这种特有化学活性，因此可以通过控制聚合速度来制备具有一定大小和形状的纳米微粒，调节聚合环境下液相pH值来引发或停止聚合反应。此制备工艺较为简单，适合于纳米粒药物递送系统的研究。
有研究发现，在AD和帕金森病等病理状态下脑微血管和神经元上的乳铁蛋白(lactoferrin, Lf)受体表达均会增加。Lf是一种存在于哺乳动物体内的阳离子糖蛋白，属于转铁蛋白家族。它由约690个氨基酸组成，这些氨基酸组成多肽链环形成了两个球形叶片，各包含一个铁结合位点。除了参与铁代谢，Lf还具有抗菌，抗病毒及免疫调节等多种生理功能。己有研究证实，Lf可以通过受体介导的胞吞转运入脑，且该转运过程为单向转运，无明显的跨细胞降解[27]。此外，有研究发现，在加入低密度脂蛋白受体相关蛋白拮抗剂后，该转运过程可以被抑制70%以上，说明Lf还可能通过低密度脂蛋白受体相关蛋白转运入脑。经Lf修饰的生物可降解NPs的脑内递药研究也证实了Lf-NPs比空白NPs的脑靶向指数增加2.49倍，且在Lf-NPs浓度为3mg/mL时细胞活力均保持在80以上，展示了Lf-NPs良好的安全性。
综上所述，本实验选择BCA为载体材料制备Res-NPs，借助羟基聚乙二醇马来酰亚胺(Hydroxyl-PEG-Maleimide, OH-PEG-MAL)作为中间“桥梁”链接Res-NPs与Lf，得到Lf修饰的Res-NPs(Lactoferrin modified Resveratrol NanoParticles, Lf-Res-NPs)，期望其借助Lf以受体介导胞吞转运方式增加Res的脑内转运，提高Res对AD的治疗作用。
发明内容
本发明的目的在于克服白藜芦醇体内生物利用度低以及脑部分布较少，提供一种生物利用度较高并具有脑靶向功能的白藜芦醇纳米粒制剂。
为了实现上述发明目的，本发明提供了以下技术方案：
一种具有脑靶向功能的白藜芦醇纳米粒制剂，包括组分：白藜芦醇、BCA、乳铁蛋白溶液、OH-PEG-MAL和注射用水。
一种具有脑靶向功能的白藜芦醇纳米粒制剂制备工艺，包括如下步骤：
步骤一、白藜芦醇纳米粒的制备：精密称量右旋糖酐70 50.0 mg溶解于8 mL注射用水中，调节pH至2.0，作为水相。再精密称量48 mg白藜芦醇，并精密量取BCA 52 μL，溶解于4mL丙酮中，作为油相。在搅拌的条件下，将油相缓慢滴加入水相中，搅拌速度为800 rpm，搅拌3.7 h后，调节pH至7.0，再继续搅拌0.5 h，最后将此混悬液于40 ℃旋转蒸发除去丙酮，制得白藜芦醇纳米粒混悬液；
步骤二、洗脱液A的配制：精密称量N-2-羟乙基哌嗪-Nˊ-2-乙磺酸2.3840g， NaCl8.7652g，用适量蒸馏水溶解，最后用1 mol/L NaOH调节pH 至7.0，蒸馏水定容至1000mL，备用；
步骤三、乳铁蛋白修饰白藜芦醇纳米粒的制备：按照上述步骤一操作，其中制备水相时还需加入精密称量的OH-PEG-MAL 10.0 mg，由此制备得到PEG-Res-NPs混悬液；取PEG-Res-NPs混悬液于3500 rpm离心10 min，弃去上清液，沉淀物质用0.5 mL洗脱液A重新分散，再加入活化巯基的浓度为1g/L乳铁蛋白溶液1mL，室温搅拌反应9 h，即得乳铁蛋白修饰的白藜芦醇纳米粒混悬液。
本发明提供的白藜芦醇纳米粒制剂通过用乳铁蛋白修饰后，制成乳铁蛋白修饰的白藜芦醇纳米粒，具有脑靶向的纳米粒药物剂型，增强白藜芦醇的脑靶向和抗AD作用。
附图说明
图1为白藜芦醇纳米粒的透射电镜图。
图2为未加入一抗的乳铁蛋白修饰白藜芦醇纳米粒的透射电镜图。
图3为乳铁蛋白修饰白藜芦醇纳米粒的透射电镜图。
图4为乳铁蛋白修饰白藜芦醇纳米粒粒径分布图。
图5为各组小鼠血浆药物浓度-时间曲线(n=10)图。
具体实施方式
实施例1 Res分析方法的建立：
精密称量Res 0.4mg，用甲醇溶解并定容至10mL得到Res溶液。分别在200-400nm范围内扫描Res溶液和空白NPs混悬液。
精密称量Res标准品4.0mg，甲醇溶解并定容至100mL，得到浓度为40μg/mL的Res储备液。从储备液中分别量取1mL、1.5mL、2mL、2.5mL、3mL，分别加入甲醇稀释定容至10mL，得到浓度为4、6、8、10、12μg/mL的Res标准溶液。紫外分光光度计于309nm下测定其吸光度，以吸光度A对浓度C进行线性回归。
精密称量Res标准品4.0mg，甲醇溶解并定容至100mL，得到浓度为40μg/mL的Res储备液。从储备液中分别量取5mL、10mL、15mL，分别加入甲醇稀释定容至50mL，得到浓度为4、8、12μg/mL的Res低、中、高标准溶液。紫外分光光度计于309nm下测其吸光度，代入标准曲线方程计算浓度。测定方法为日内精密度要求一天内测定5次；日间精密度要求每天测1次，连续测定5天。
实施例2 Res-NPs包封率及载药量的测定：
Res回收率试验：精密量取0.1mL浓度为4、8、12μg/mL Res标准溶液到2mL超滤离心管中，加入0.5mL乙醇混匀，4℃，15000rpm超速离心2h。精密量取滤液0.3mL，加入5mL DMSO溶解，最后用甲醇定容至10mL。紫外分光光度计于309nm下测其吸光度，代入标准曲线方程计算浓度。标准曲线方程A=0.1439C-0.0002，R2=0.9993。表明Res浓度在4~12 μg/mL内呈良好的线性关系。
Res-NPs中Res的含量测定：精密量取空白NPs混悬液1mL，加入5mL DMSO超声破乳后，用甲醇定容至10mL，于3500rpm离心10min，取上清液作为空白对照。精密量取Res-NPs混悬液1mL，加入5mL DMSO超声破乳，用甲醇定容至10mL，于3500rpm离心10min，紫外分光光度计于309nm下测其吸光度，并计算药物含量。
Res-NPs包封率及载药量的测定：精密量取0.1mL Res-NPs混悬液到2mL超滤离心管中，加入0.5mL乙醇混匀，4℃，15000rpm超速离心2h。精密量取滤液0.3mL，加入5mL DMSO超声破乳后，用甲醇定容至10mL。紫外分光光度计于309nm波长下测定吸光度，代入标准曲线方程计算其浓度并计算游离药物质量，按式EE%=[(投药量-游离药量)÷投药量] ×100%计算Res-NPs的包封率(Encapsulation efficiency, EE%)。
取已恒重的1.5mL EP管，编号为A，称量其重量记为W0。精密量取1mL Res-NPs混悬液于A管中，80℃干燥至恒重，称量其重量记为W总，1mL Res-NPs混悬液质量W=W总-W0。按式DL%=[(投药量-游离药量) ÷W] ×100%计算Res-NPs混悬液的载药量。
实施例3 Res-NPs处方工艺的研究：
Res-NPs的制备：右旋糖酐-70与泊洛沙姆188溶于纯化水中，调节pH至酸性，形成水相，备用。将Res与BCA溶于丙酮中，形成油相。搅拌条件下将油相缓慢滴加入水相中，搅拌一定时间后用0.1mol/L NaOH调节pH至7.0，继续搅拌一定时间，将此混悬液于40℃旋转蒸发除去丙酮，制得Res-NPs混悬液。
空白NPs的制备：与上述Res-NPs的制备方法相同，不加Res，即得空白NPs。
单因素考察：固定其它制备条件不变，只改变单个因素，以粒径、包封率和载药量为指标，来考察Res-NPs的处方和工艺。
（1）油相与水相比例的考察：
分别选择油水体积比为8mL:8mL、4mL:8mL、2.7mL:8mL、2mL:8mL。Res 32mg，BCA用量40μL，右旋糖酐-70与泊洛沙姆188各50mg，水相pH 2.0，搅拌速度800rpm，搅拌时间3h，继续搅拌时间0.5h，制备Res-NPs。
（2）水相pH值的考察：
水相pH值分别调至2.0，3.0，4.0。油水体积比为4mL:8mL，Res 32mg，BCA用量40μL，右旋糖酐-70与泊洛沙姆188各50mg，搅拌速度800rpm，搅拌时间3h，继续搅拌时间0.5h，制备Res-NPs。
（3）BCA用量的考察：
分别选择BCA单体投入量为20μL，40μL，64μL，96μL。油水体积比为4mL:8mL，Res 32mg，右旋糖酐-70与泊洛沙姆188各50mg，搅拌速度800rpm，搅拌时间3h，继续搅拌时间0.5h，制备Res-NPs。
（4）搅拌时间的考察
油相滴入水相后，搅拌时间设置为2h，3h，4h，5h，6h。油水体积比为4mL:8mL，Res 32mg，BCA用量40μL，右旋糖酐-70与泊洛沙姆188各50mg，搅拌速度800rpm，继续搅拌时间0.5h，制备Res-NPs。
（5）继续搅拌时间的考察
在调节pH至7.0后继续搅拌时间设置为0.5h，1h，2h。油水体积比为4mL:8mL，Res 32mg，BCA用量40μL，右旋糖酐-70与泊洛沙姆188各50mg，搅拌速度800rpm，搅拌时间3h，制备Res-NPs。
（6）搅拌速度的考察
搅拌速度分别设置为400、600、800和1000rpm。油水体积比为4mL:8mL，Res 32mg，BCA用量40μL，右旋糖酐-70与泊洛沙姆188各50mg，搅拌时间3h，继续搅拌时间0.5h，制备Res-NPs。
（7）表面活性剂种类及用量的考察
右旋糖酐-70 50mg，泊洛沙姆188 50mg，右旋糖酐-70 与泊洛沙姆188 各40mg，右旋糖酐-70 与泊洛沙姆188各80mg，右旋糖酐-70 与泊洛沙姆188各120mg。油水体积比为4mL:8mL，Res 32mg， BCA用量40μL，搅拌速度800rpm，搅拌时间3h，继续搅拌时间0.5h，制备Res-NPs。
（8）投药量的考察
分别投入Res的质量为6、16、32、48、64、80mg。油水体积比为4mL:8mL，BCA用量40μL，右旋糖酐-70 50mg，搅拌速度800rpm，搅拌时间3h，继续搅拌时间0.5h，制备Res-NPs。
Res-NPs的表征：将Res-NPs混悬液经0.5%磷钨酸负染后在透射电镜下观察纳米粒形态和大小。量取Res-NPs 0.5mL并用蒸馏水稀释到1.8mL，用激光粒度分析仪检测其粒径。
（9）Res-NPs的表征
透射电镜观察Res-NPs的形态，结果见图1。粒径测定结果见图4。
实施例4 Lf-Res-NPs的制备
相关溶液配制：（1）二抗缓冲液的配制：精密称量吐温20 0.050g，NaCL 2.922g，牛血清白蛋白0.100g，精密量取小牛血清5mL，蒸馏水溶解并定容至100mL。
（2）0.15mol/L硼酸钠溶液的配制：精密称量5.720g四硼酸钠，加90mL蒸馏水溶解，用1mol/L NaOH 调pH至8.5，加蒸馏水定容至100mL。
（3）2-亚氨基硫烷盐酸盐(2-Iminothiolane hydrochloride, Traut's)溶液的配制：精密称量Traut's试剂0.010g，加0.15 mol/L硼酸钠溶液溶解并定容至10mL，以200μL体积分装后于-20℃保存。
（4）pH 8.0，pH 7.0的0.1 mol/L磷酸盐缓冲液(PBS)的配制：溶液A的配制：精密称量NaH2PO42•H2O 15.601g溶解于500mL蒸馏水得到0.2mol/L NaH2PO4溶液。溶液B的配制：精密称量Na2HPO4•12H2O 35.524g溶解于500mL蒸馏水得到0.2mol/L Na2HPO4溶液。pH 8.0的0.1mol/L PBS的配制：将5.3mL溶液A与94.7mL溶液B混匀，加蒸馏水至200mL即得。pH 7.0的0.1mol/L PBS的配制：将39mL溶液A与61mL溶液B混匀，加蒸馏水至200mL即得。
（5）洗脱液A的配制：精密称量N-2-羟乙基哌嗪-Nˊ-2-乙磺酸2.383g， NaCl8.7750g，蒸馏水溶解，用1 mol/L NaOH调pH 至7.0，加蒸馏水定容至1000mL。
（6）Ellman’s 试剂的配制：精密称量5,5ˊ-二硫代双（2-硝基苯甲酸）[5,5'-Dithio bis-(2-nitrobenzoic acid), DTNB] 0.0396g，加pH7.0 0.1mol/L PBS溶解并定容至10mL即得，4℃冰箱避光保存。
PEG-Res-NPs的制备：精密称量OH-PEG-MAL 0.01g与右旋糖酐-70 0.050g于8mL纯化水中溶解，用0.1mol/L HCL调节pH至2.0，室温下800rpm磁力搅拌，形成水相，备用。精密称量适量Res溶于丙酮中，加入BCA形成油相。再将此油相缓慢滴加入水相中，持续搅拌3.7h后用0.1mol/L NaOH调节pH至7.0，继续搅拌30min后将此混悬液于40℃旋转蒸发除去丙酮，制得PEG-Res-NPs混悬液。
Lf-Res-NPs的制备：将PEG-Res-NPs于3500rpm离心10min，收集沉淀，用0.5mL洗脱液A分散后加入活化巯基的Lf溶液，室温搅拌反应9h后，得到的纳米粒混悬液以0.01mol/LPBS洗脱过Sepharose CL-4B凝胶柱(1.0×60cm, 0.2mL/min流速)，除去未连接蛋白，即得Lf-Res-NPs。
Lf-Res-NPs的表征：量取Res-NPs 0.5mL用蒸馏水稀释到1.8mL，检测其粒径。透射电镜鉴定Lf-Res-NPs表面连接的Lf ：取适量Lf-Res-NPs，分散在500μL 0.01mol/L PBS中，加入l:1000稀释的山羊抗Lf 抗体(一抗)20μL，37℃摇床(100rpm)孵育lh，用SepharoseCL-4B凝胶柱分离除去未结合的一抗，用PBS洗脱，收集含有纳米粒的PBS，于10000g、4℃离心lh，弃上清液。沉淀加入1mL二抗缓冲液分散，加入100μL胶体金标记兔抗山羊IgG抗体(二抗)，继续37℃摇床(100rpm)孵育l.5h，于10000g、4℃离心lh，弃上清液。沉淀加300μL PBS分散，用Sepharose CL-4B凝胶柱分离除去未结合的胶体金标记二抗，收集含有纳米粒的PBS，磷钨酸染色后进行透射电镜观察，结果见图3。另取Lf-Res-NPs不加入一抗重复上述试验步骤，考察是否有假阳性现象。取Res-NPs重复上述试验作为阴性对照，结果见图2。
实施例5 乳铁蛋白修饰白藜芦醇纳米粒在小鼠体内药代动力学及组织分布研究
给药方案：饲养环境温度维持在(24~28)℃，相对湿度50％~60％，专室分笼饲养，隔日更换垫料，自由摄水，摄食(标准饲料喂养)，饲养1周后腹腔注射给药，给药前小鼠禁食12h，根据小鼠体重给药，分3组：Res组、Res-NPs组和Lf-Res-NPs组(临用前制备)，Res给药剂量为44mg/kg；3%水合氯醛(0.1mL/10g)麻醉小鼠，给药后于0.17、0.5、0.75、1、2、6、10、12、24h采血，每个时间点10只。采血后立即用生理盐水心脏灌流至各组织呈白色，取出心、肝、脾、肺、肾、脑，生理盐水冲洗干净表面血渍，滤纸吸干水分，-80℃保存。
HPLC法分析血浆中的药物浓度
（1）Res储备液及标准溶液的配制
精密称量Res 10.0mg，甲醇定容至10mL得到Res浓度为1mg/mL的储备液。精密量取Res储备液0.0l、0.25、0.5、1、2、3、4mL，用甲醇定容至10mL得到浓度1、2.5、5、10、20、30、40μg/mL的Res标准溶液。
（2）HPLC色谱检测条件
流动相：甲醇:水=5:5；流速：1.0 mL/min；
检测波长：306nm；保留时间：14min；进样量：60μL。
（3）血浆样品的处理
摘眼球取血后3500rpm离心10min，分离血浆于4℃保存。精密量取0.2mL血浆，加入0.5mL的乙酸乙酯，涡旋振荡1min，充分提取药物。涡旋后静置可见有油水两层液体，将上层油相液体取出，加入1.5mL EP管中，40℃水浴氮气吹干，加入1mL甲醇，涡旋1min，4000rpm离心10min，取上清液按方法“2.2.2”进样检测。
（4）专属性考察
取小鼠空白血浆样品按“2.2.3”方法处理后按方法“2.2.2”进样检测作为对照；空白血浆中加入Res储备液按“2.2.3”方法处理后按方法“2.2.2”进样检测，对比其结果考察检测方法的专属性。
（5）血浆中Res标准曲线的建立
精密量取小鼠空白血浆0.2mL，加入Res系列标准溶液1mL，甲醇定容至10mL得到浓度为0.1、0.25、0.5、1、2、3、4μg/mL的Res血浆标准溶液。精密吸取Res血浆标准溶液1mL，HPLC测定Res浓度。
（6）精密度及回收率试验
精密量取配制的低、中、高Res血浆标准溶液1mL，样品经处理后，用HPLC测定Res浓度。日内精密度要求1天内测定5次；日间精密度要求每天测1次，连续测定5天。精密量取配制的低、中、高Res血浆标准溶液1mL，样品经处理后，用HPLC测定药物浓度，计算Res回收率。
（7）Res组、Res-NPs组、Lf-Res-NPs组小鼠体内药代动力学结果，结果见图5。主要的药动学参数见表1。
表1 各组药代动力学统计距参数(n=10)
备注:* P<0.05 vs Res 组. # P<0.05 vs Res-NPs 组.
实施例6 Res、Res-NPs及Lf-Res-NPs小鼠靶向性研究
根据各组织Res浓度与各组织脏器重量计算各组织中药物含量，通过公式DTI(%) = 靶向组织药物含量 ÷ 非靶向组织药物含量 × 100%计算各组织中各时间点的药物靶向指数(Drug targeting index，DTI)，评价Res组、Res-NPs组及Lf-Res-NPs组的组织靶向性。结果见表2~表7。
表2 Res溶液组在小鼠各组织中Res的含量(n=10)
"-": represents below the minimum detection line
表3 Res组在小鼠各组织中的靶向指数
表4 Res-NPs组在小鼠各组织中Res的含量(n=10)
表5 Res-NPs组在小鼠各组织中的靶向指数
表6 Lf-Res-NPs组在小鼠各组织中Res的含量(n=10)
表7 Lf-Res-NPs组在小鼠各组织中的靶向指数
Claims (2)
1.一种具有脑靶向功能的白藜芦醇纳米粒制剂，其特征在于：它包括组分：白藜芦醇、BCA、乳铁蛋白溶液、OH-PEG-MAL和注射用水。
2.一种具有脑靶向功能的白藜芦醇纳米粒制剂制备工艺，其特征在于：它包括如下步骤：
步骤一、白藜芦醇纳米粒的制备：精密称量右旋糖酐70 50.0 mg溶解于8 mL注射用水中，调节pH至2.0，作为水相；
再精密称量48 mg白藜芦醇，并精密量取BCA 52 μL，溶解于4 mL丙酮中，作为油相；
在搅拌的条件下，将油相缓慢滴加入水相中，搅拌速度为800 rpm，搅拌3.7 h后，调节pH至7.0，再继续搅拌0.5 h，最后将此混悬液于40 ℃旋转蒸发除去丙酮，制得白藜芦醇纳米粒混悬液；
步骤二、洗脱液A的配制：精密称量N-2-羟乙基哌嗪-Nˊ-2-乙磺酸2.3840g， NaCl8.7652g，用适量蒸馏水溶解，最后用1 mol/L NaOH调节pH 至7.0，蒸馏水定容至1000mL，备用；
步骤三、乳铁蛋白修饰白藜芦醇纳米粒的制备：按照上述步骤一操作，其中制备水相时还需加入精密称量的OH-PEG-MAL 10.0 mg，由此制备得到PEG-Res-NPs混悬液；取PEG-Res-NPs混悬液于3500 rpm离心10 min，弃去上清液，沉淀物质用0.5 mL洗脱液A重新分散，再加入活化巯基的浓度为1g/L乳铁蛋白溶液1mL，室温搅拌反应9 h，即得乳铁蛋白修饰的白藜芦醇纳米粒混悬液。
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| EP1239849A2 (en) * | 1999-10-29 | 2002-09-18 | Pharmascience Inc. | Pharmaceutical formulations comprising resveratrol and use thereof |
| CN104382888A (zh) * | 2014-11-06 | 2015-03-04 | 江苏隆力奇生物科技股份有限公司 | 一种白藜芦醇磷脂复合物自微乳颗粒的制备方法 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111166765A (zh) * | 2020-01-16 | 2020-05-19 | 华南理工大学 | 一种铁乳蛋白修饰的金-硒化铋量子点材料及其制备方法和应用 |
| CN116392601A (zh) * | 2023-04-26 | 2023-07-07 | 陕西微泌生物科技有限公司 | 一种复合修饰外泌体包裹白藜芦醇的制剂及其制备方法 |
| CN118178621A (zh) * | 2024-03-28 | 2024-06-14 | 浙江大学 | 乳铁蛋白在结合血红素上的应用 |
| CN118892197A (zh) * | 2024-07-10 | 2024-11-05 | 吉林大学 | 一种负载白藜芦醇的乳铁蛋白基纳米颗粒制备方法及应用 |
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