技术领域technical field
本发明公开一种曲妥株单抗修饰的载紫杉醇的靶向纳米粒传递系统，同时提供了该系统的制备方法，属于肿瘤靶向与缓释给药系统技术领域。The invention discloses a targeting nanoparticle delivery system loaded with paclitaxel modified by trastuzumab, and provides a preparation method of the system, belonging to the technical field of tumor targeting and sustained release drug delivery systems.
背景技术Background technique
紫杉醇是临床上广泛应用的抗肿瘤药物，可以诱导和促进微管蛋白聚合，防止解聚，使微管蛋白保持稳定，抑制有丝分裂，发挥抗肿瘤作用。紫杉醇具有高效、低毒及广谱抗癌活性，尤其对乳腺癌和卵巢癌有独特疗效，其注射液是治疗乳腺癌、卵巢癌、肺癌等实体肿瘤的一线用药。但由于紫杉醇水溶性差，制成注射制剂有一定困难。目前临床常用的紫杉醇注射液（Taxol）采用聚氧乙烯蓖麻油等作为溶媒，可促进组胺释放，引起严重的过敏反应。此外注射型紫杉醇靶向性差，易引起全身毒性，因此临床上迫切需要研发紫杉醇的新型给药系统，以克服缺陷。Paclitaxel is an anti-tumor drug widely used clinically. It can induce and promote tubulin polymerization, prevent depolymerization, stabilize tubulin, inhibit mitosis, and exert anti-tumor effects. Paclitaxel has high efficiency, low toxicity and broad-spectrum anticancer activity, especially has a unique curative effect on breast and ovarian cancer. Its injection is the first-line drug for the treatment of solid tumors such as breast cancer, ovarian cancer and lung cancer. However, due to the poor water solubility of paclitaxel, it is difficult to make injection preparations. Paclitaxel injection (Taxol), which is commonly used in clinical practice, uses polyoxyethylene castor oil as a solvent, which can promote the release of histamine and cause severe allergic reactions. In addition, injectable paclitaxel has poor targeting and is likely to cause systemic toxicity. Therefore, it is urgent to develop a new drug delivery system for paclitaxel in order to overcome the shortcomings.
PLGA，生物可降解性聚乳酸-羟基乙酸共聚物，作为药物载体具有良好的生物相容性和稳定性，能够包裹疏水性的紫杉醇，但PLGA纳米粒粒径普遍较大，不利于静脉注射。由磷脂材料构成的脂质体，类似生物膜结构，具有粒径小，生物相容性好，增加细胞摄取及内体逃逸，延缓药物释放等优势，但稳定性较差。聚合物脂质纳米粒是结合了聚合物和磷脂二者优点开发的一种具有核壳结构的新型给药系统。将紫杉醇载入聚合物脂质纳米粒，存在以下优点：粒径小，药物包封率高，生物相容性好，释放行为学可控，结合配体分子后靶向性增强。从而提高了主动靶向，降低紫杉醇毒副作用，减少紫杉醇用量，增加患者顺应性。PLGA, a biodegradable polylactic acid-glycolic acid copolymer, has good biocompatibility and stability as a drug carrier, and can wrap hydrophobic paclitaxel, but the particle size of PLGA nanoparticles is generally large, which is not conducive to intravenous injection. Liposomes composed of phospholipid materials, similar to biological membrane structures, have the advantages of small particle size, good biocompatibility, increased cell uptake and endosomal escape, and delayed drug release, but their stability is poor. Polymer lipid nanoparticles are a new type of drug delivery system with a core-shell structure developed by combining the advantages of both polymers and phospholipids. Loading paclitaxel into polymer lipid nanoparticles has the following advantages: small particle size, high drug encapsulation efficiency, good biocompatibility, controllable release behavior, and enhanced targeting after binding ligand molecules. Thereby, the active targeting is improved, the toxic and side effects of paclitaxel are reduced, the dosage of paclitaxel is reduced, and the compliance of patients is increased.
某些肿瘤细胞表面过量表达受体，可以结合特异性配体。利用这种配体导向作用，可以将化疗药物递送到特定肿瘤组织或细胞，从而实现靶向治疗。曲妥珠单抗（Trastuzumab）是人表皮生长因子受体-2（HER2）的单克隆抗体，可以识别并结合HER2过表达的肿瘤细胞，如乳腺癌，卵巢癌，肺癌细胞等。曲妥珠单抗具有蛋白类大分子分子量大及空间结构复杂等特点，在纳米粒制备过程中易受到操作环境的影响，从而失去活性。在载药纳米粒基本形成后，通过纳米粒表面引入的Mal基团，与抗体在温和的条件下反应，从而将抗体连接在纳米粒表面，可以保持抗体的天然活性，从而提高靶向效应。因此，通过HER2受体介导的纳米传递系统，为HER2过表达的肿瘤治疗提供了潜力的途径。Certain tumor cells overexpress receptors on the surface that can bind specific ligands. Using this ligand-directed effect, chemotherapy drugs can be delivered to specific tumor tissues or cells, thereby achieving targeted therapy. Trastuzumab is a monoclonal antibody against human epidermal growth factor receptor-2 (HER2), which can recognize and bind to tumor cells that overexpress HER2, such as breast cancer, ovarian cancer, and lung cancer cells. Trastuzumab has the characteristics of large molecular weight and complex spatial structure of protein macromolecules, and is easily affected by the operating environment during the preparation of nanoparticles, thus losing its activity. After the drug-loaded nanoparticles are basically formed, the Mal group introduced on the surface of the nanoparticles reacts with the antibody under mild conditions, thereby linking the antibody to the surface of the nanoparticle, which can maintain the natural activity of the antibody and improve the targeting effect. Therefore, the HER2 receptor-mediated nano-delivery system provides a potential way for the treatment of HER2 overexpressed tumors.
发明内容Contents of the invention
本发明提供一种曲妥株单抗修饰的载紫杉醇的靶向纳米粒传递系统，为特异性靶向HER2过表达肿瘤细胞的曲妥株单抗修饰的载紫杉醇的靶向纳米粒传递系统，解决了目前紫杉醇制剂存在的缺陷及纳米粒载体材料单一的缺陷。The present invention provides a paclitaxel-loaded targeting nanoparticle delivery system modified by trastuzumab, which is a paclitaxel-loaded targeting nanoparticle delivery system modified by trastuzumab specifically targeting HER2 overexpression tumor cells, The present invention solves the defects existing in the paclitaxel preparation and the single defect of the nano particle carrier material.
本发明进一步提供曲妥株单抗修饰的载紫杉醇的靶向纳米粒传递系统的制备方法。The present invention further provides a preparation method of the paclitaxel-loaded targeted nanoparticle delivery system modified by trastuzumab.
本发明所述的一种曲妥株单抗修饰的载紫杉醇的靶向纳米粒传递系统，通过以下技术方案实现：A targeted nanoparticle delivery system loaded with paclitaxel modified by trastuzumab according to the present invention is realized by the following technical scheme:
由聚合物PLGA和磷脂为载体材料制备的核壳结构的聚合物脂质纳米粒、包载于纳米粒中的紫杉醇及纳米粒表面的修饰配体曲妥株单抗构成。It consists of polymer lipid nanoparticles with core-shell structure prepared by polymer PLGA and phospholipids as carrier materials, paclitaxel entrapped in the nanoparticles, and trastuzumab, a modification ligand on the surface of the nanoparticles.
本发明所述的曲妥株单抗修饰的载紫杉醇的靶向纳米粒传递系统，其特征在于：The targeted nanoparticle delivery system loaded with paclitaxel modified by trastuzumab according to the present invention is characterized in that:
PLGA为羧基封端，型号5050 1A或5050 1.5A，黏度为0.16 ~ 0.20 dL/g。PLGA is carboxyl-terminated, model 5050 1A or 5050 1.5A, viscosity is 0.16 ~ 0.20 dL/g.
本发明所述的曲妥株单抗修饰的载紫杉醇的靶向纳米粒传递系统，其特征在于：The targeted nanoparticle delivery system loaded with paclitaxel modified by trastuzumab according to the present invention is characterized in that:
其中的磷脂为二棕榈酰磷脂酰胆碱与磷脂酰乙醇胺-聚乙二醇2000-马来酰亚胺。The phospholipids are dipalmitoylphosphatidylcholine and phosphatidylethanolamine-polyethylene glycol 2000-maleimide.
本发明所述的曲妥株单抗修饰的载紫杉醇的靶向纳米粒传递系统，其特征在于：The targeted nanoparticle delivery system loaded with paclitaxel modified by trastuzumab according to the present invention is characterized in that:
紫杉醇：PLGA：二棕榈酰磷脂酰胆碱：磷脂酰乙醇胺-聚乙二醇2000-马来酰亚胺的质量比为（10~15）：60：（15~20）：10。The mass ratio of paclitaxel: PLGA: dipalmitoylphosphatidylcholine: phosphatidylethanolamine-polyethylene glycol 2000-maleimide is (10~15):60:(15~20):10.
本发明所述的曲妥株单抗修饰的载紫杉醇的靶向纳米粒传递系统，其特征在于其中的曲妥株单抗与磷脂酰乙醇胺-聚乙二醇2000-马来酰亚胺的摩尔比为1：5~1。The targeted nanoparticle delivery system loaded with paclitaxel modified by trastuzumab according to the present invention is characterized in that the molar ratio of trastuzumab and phosphatidylethanolamine-polyethylene glycol 2000-maleimide wherein The ratio is 1:5~1.
本发明所述的曲妥株单抗修饰的载紫杉醇的靶向纳米粒传递系统的制备方法，包括以下步骤：The preparation method of the paclitaxel-loaded targeted nanoparticle delivery system modified by trastuzumab of the present invention comprises the following steps:
1）采用改良的乳化溶剂挥发法制备包裹并运载紫杉醇的PLGA /Lipid纳米粒；1) Prepare PLGA/Lipid nanoparticles that encapsulate and carry paclitaxel by the improved emulsified solvent evaporation method;
2）采用化学偶联的方法，使用活化试剂将曲妥株单抗的某个氨基巯基化，与纳米粒表面的Mal基团发生加成反应，形成硫醚键，进而将配基与纳米粒连接。2) Using the method of chemical coupling, an activating reagent is used to thiolate an amino group of trastuzumab, which undergoes an addition reaction with the Mal group on the surface of the nanoparticle to form a thioether bond, and then binds the ligand to the nanoparticle connect.
本发明所述的制备方法，其特征在于具体包括以下步骤：The preparation method of the present invention is characterized in that it specifically comprises the following steps:
1）称取10~15 mg的紫杉醇和60 mg 的PLGA溶解于2.4 mL的丙酮中，称取15~20 mg的DPPC和10 mg DSPE-PEG2000-Mal溶解于1.6 mL的乙醇中；混合丙酮乙醇溶液（积比为3：2）形成二元有机相（O相），加入适量1.0 ~ 2.0% 泊洛沙姆188水溶液（W相）, 0 ~ 4℃下间断超声分散制备O/W乳剂，再补充10~20 mL的1.0 ~ 2.0% 泊洛沙姆188水溶液，室温低速搅拌，过夜挥发除去二元有机相，过膜除去大粒径纳米粒，并离心收集纳米粒，冷冻干燥；1) Weigh 10-15 mg of paclitaxel and 60 mg of PLGA and dissolve in 2.4 mL of acetone, weigh 15-20 mg of DPPC and 10 mg of DSPE-PEG2000-Mal and dissolve in 1.6 mL of ethanol; mix acetone with ethanol The solution (volume ratio is 3:2) forms a binary organic phase (O phase), adding an appropriate amount of 1.0 ~ 2.0% poloxamer 188 aqueous solution (W phase), and intermittently ultrasonically disperses at 0 ~ 4°C to prepare an O/W emulsion. Add 10-20 mL of 1.0-2.0% poloxamer 188 aqueous solution, stir at room temperature at low speed, evaporate overnight to remove the binary organic phase, pass through the membrane to remove large-size nanoparticles, and collect the nanoparticles by centrifugation, freeze-drying;
2）在20℃、pH 8.0缓冲液孵育条件下，使用活化试剂将曲妥珠单抗的一部分氨基巯基化，与纳米粒表面的Mal基团发生加成反应，形成硫醚键，进而将配基与纳米粒连接，离心洗涤除去未反应的单抗，并收集纳米粒，即得曲妥珠单抗修饰的载紫杉醇的靶向纳米粒传递系统。2) Under the conditions of incubation at 20°C and pH 8.0 buffer solution, use an activation reagent to thiolate a part of the trastuzumab amino group, and undergo an addition reaction with the Mal group on the surface of the nanoparticle to form a thioether bond. The base is connected to the nanoparticle, and the unreacted monoclonal antibody is removed by centrifugation, and the nanoparticle is collected to obtain the trastuzumab-modified paclitaxel-loaded targeted nanoparticle delivery system.
本发明所述的制备方法，其特征在于：The preparation method of the present invention is characterized in that:
氨基活化试剂是2-亚氨氢氯化硫醇，使用浓度为6 ~ 7 mg/mL，用量范围为20 ~ 100 μL。The amino group activation reagent is 2-iminohydrothiol chloride, the concentration used is 6-7 mg/mL, and the dosage range is 20-100 μL.
本发明所介绍的二元溶剂乳化挥发法制备的PLGA/Lipid纳米粒传递系统，包裹的药物不仅限于紫杉醇，还可拓展为其它与PLGA具有良好生物相容性的疏水性化疗药物；偶联的配体也不仅限于曲妥株单抗，还可包括那些具有靶向性的小分子多肽及其它种类的单克隆抗体。The PLGA/Lipid nanoparticle delivery system prepared by the binary solvent emulsification and volatilization method introduced in the present invention is not limited to paclitaxel, but can also be expanded to other hydrophobic chemotherapeutic drugs with good biocompatibility with PLGA; coupled Ligands are not limited to trastuzumab, but may also include those targeting small molecule polypeptides and other types of monoclonal antibodies.
本发明制备的靶向纳米粒（T=PCNs）为冻干制剂，属于静脉给药系统。对于静脉给药系统，溶剂通常为无菌水溶液。适当的调整溶液pH值并使溶液等渗（如加入氯化钠或葡萄糖），即适合静脉给药，纳米粒采取过0.22 μm无菌滤膜的方式灭菌。The targeting nanoparticles (T=PCNs) prepared in the present invention are freeze-dried preparations and belong to an intravenous drug delivery system. For intravenous administration systems, the solvent is usually a sterile aqueous solution. Properly adjust the pH value of the solution and make the solution isotonic (such as adding sodium chloride or glucose), which is suitable for intravenous administration. The nanoparticles are sterilized by passing through a 0.22 μm sterile filter membrane.
本发明的积极效果在于：结合了PLGA和磷脂的优点并与单克隆抗体联合，制备了具有核壳结构的曲妥株单抗修饰的载紫杉醇的靶向纳米粒（T=PCNs），形成具有核壳结构的纳米级微粒；解决了现有紫杉醇制剂存在的缺陷及纳米粒载体材料单一的不足。具有粒径小（150 ~ 200 nm），更有利于细胞摄取（<200 nm），药物包封率高（75 ~ 83%），在溶液中稳定性好（电位0 mV左右），生物相容性好，增加内体逃逸，释放行为学可维持紫杉醇治疗水平，结合曲妥株单抗后更实现了药物的主动靶向性。提高了HER2过表达的肿瘤细胞的摄取效率，极大的提高了紫杉醇的安全性和靶向性。本发明构建的曲妥株单抗修饰的载紫杉醇的靶向纳米粒传递系统，是一种优良的递药系统，有望应用于HER2过表达的乳腺癌、卵巢癌及肺癌等实体肿瘤的治疗。The positive effects of the present invention are: combining the advantages of PLGA and phospholipids and combining with monoclonal antibodies to prepare trastuzumab-modified paclitaxel-loaded targeting nanoparticles (T=PCNs) with a core-shell structure, forming The nano-scale particle of the core-shell structure; solves the defect existing in the existing paclitaxel preparation and the single deficiency of the nano particle carrier material. Small particle size (150-200 nm), more conducive to cell uptake (<200 nm), high drug encapsulation efficiency (75-83%), good stability in solution (potential 0 mV or so), biocompatible Good sex, increase endosome escape, release behavior can maintain the therapeutic level of paclitaxel, combined with trastuzumab to achieve the active targeting of the drug. The uptake efficiency of tumor cells overexpressing HER2 is improved, and the safety and targeting of paclitaxel are greatly improved. The paclitaxel-loaded targeted nanoparticle delivery system modified by trastuzumab constructed in the present invention is an excellent drug delivery system and is expected to be applied to the treatment of solid tumors such as breast cancer, ovarian cancer and lung cancer with HER2 overexpression.
附图说明Description of drawings
图1为纳米粒的示意图；Fig. 1 is the schematic diagram of nanoparticle;
图2为纳米粒的粒径电位分布图：（A）PCNs粒径分布；（B）T=PCNs粒径分布；（C）PCNs电位分布；（D）T=PCNs电位分布；Figure 2 is the particle size and potential distribution diagram of nanoparticles: (A) PCNs particle size distribution; (B) T=PCNs particle size distribution; (C) PCNs potential distribution; (D) T=PCNs potential distribution;
图3为纳米粒的SEM图；（A）PCNs；（B）T=PCNs；Figure 3 is the SEM image of nanoparticles; (A) PCNs; (B) T=PCNs;
图4为纳米粒的体外释药曲线；Fig. 4 is the drug release curve in vitro of nanoparticles;
图5为PTX，HER，PCNs，T=PCNs对SKBR3（A）和MCF7细胞（B）的细胞毒性；Figure 5 shows the cytotoxicity of PTX, HER, PCNs, T=PCNs on SKBR3 (A) and MCF7 cells (B);
图6为SKBR3和MCF7细胞对T=PCNs摄取的激光共聚焦结果；Figure 6 is the laser confocal results of T=PCNs uptake by SKBR3 and MCF7 cells;
图7为SKBR3和MCF7细胞对T=PCNs摄取的流式细胞仪结果：（A）直方图（B）柱形图。Figure 7 shows the results of flow cytometry uptake of T=PCNs by SKBR3 and MCF7 cells: (A) histogram (B) bar graph.
具体实施方案specific implementation plan
下面结合具体实施例对本发明进行详细描述，但本发明的保护范围并不仅限于此：The present invention is described in detail below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:
实施例 1 ：Embodiment1:
1、称取紫杉醇10 mg和PLGA 1A 60mg溶解于2.4 mL丙酮中，称取DPPC 20 mg和DSPE-PEG2000-Mal 10 mg 溶解于1.6 mL的乙醇中。混合丙酮乙醇溶液形成二元有机相（O相），加入15 mL 1.0 ~ 2.0% 泊洛沙姆188水溶液（W相）, 0 ~ 4℃下间断超声（100 W，150 s）分散制备O/W乳剂，再补充15 mL的1.0 ~ 2.0% 泊洛沙姆188水溶液，室温低速搅拌（150 rpm），过夜挥发除去二元有机相，0.22 μm微孔滤膜过滤，并离心收集纳米粒（18000 rpm，15 min），冷冻干燥；1. Weigh 10 mg of paclitaxel and 60 mg of PLGA 1A and dissolve in 2.4 mL of acetone, weigh 20 mg of DPPC and 10 mg of DSPE-PEG2000-Mal and dissolve in 1.6 mL of ethanol. Mix the acetone-ethanol solution to form a binary organic phase (phase O), add 15 mL of 1.0-2.0% poloxamer 188 aqueous solution (phase W), and disperse with intermittent ultrasound (100 W, 150 s) at 0-4°C to prepare O/ W emulsion, supplemented with 15 mL of 1.0-2.0% poloxamer 188 aqueous solution, stirred at room temperature at low speed (150 rpm), evaporated overnight to remove the binary organic phase, filtered through a 0.22 μm microporous membrane, and centrifuged to collect nanoparticles (18000 rpm) rpm, 15 min), freeze-dried;
2、取20 μL、6 mg/mL Traut’s Reagent，加入1 mL、1 mg/mL曲妥株单抗溶液（pH 8.0 PBS），20℃条件下活化2 h，再加入5 mg 上述纳米粒，20℃条件下孵育18 h，离心洗涤除去未连接的单抗（18000 rpm，15 min），冷冻干燥，即获得曲妥珠单抗修饰的载紫杉醇的靶向纳米粒。2. Take 20 μL, 6 mg/mL Traut's Reagent, add 1 mL, 1 mg/mL Trastuzumab solution (pH 8.0 PBS), activate at 20°C for 2 h, then add 5 mg of the above nanoparticles, 20 Incubate at ℃ for 18 h, centrifuge and wash to remove unlinked monoclonal antibody (18000 rpm, 15 min), and freeze-dry to obtain trastuzumab-modified paclitaxel-loaded targeting nanoparticles.
实施例 2 ：Embodiment2:
1、称取紫杉醇15 mg和PLGA 1A 60mg溶解于2.4 mL丙酮中，称取DPPC 15 mg和DSPE-PEG2000-Mal 10 mg 溶解于1.6 mL的乙醇中。混合丙酮乙醇溶液形成二元有机相（O相），加入15 mL 1.0 ~ 2.0% 泊洛沙姆188水溶液（W相）, 0 ~ 4℃下间断超声（100 W，150 s）分散制备O/W乳剂，再补充15 mL的1.0 ~ 2.0% 泊洛沙姆188水溶液，室温低速搅拌（150 rpm），过夜挥发除去二元有机相，0.22 μm微孔滤膜过滤，并离心收集纳米粒（18000 rpm，15 min），冷冻干燥；1. Weigh 15 mg of paclitaxel and 60 mg of PLGA 1A and dissolve in 2.4 mL of acetone, weigh 15 mg of DPPC and 10 mg of DSPE-PEG2000-Mal and dissolve in 1.6 mL of ethanol. Mix the acetone-ethanol solution to form a binary organic phase (phase O), add 15 mL of 1.0-2.0% poloxamer 188 aqueous solution (phase W), and disperse with intermittent ultrasound (100 W, 150 s) at 0-4°C to prepare O/ W emulsion, supplemented with 15 mL of 1.0-2.0% poloxamer 188 aqueous solution, stirred at room temperature at low speed (150 rpm), evaporated overnight to remove the binary organic phase, filtered through a 0.22 μm microporous membrane, and centrifuged to collect nanoparticles (18000 rpm) rpm, 15 min), freeze-dried;
2、取20 μL、6 mg/mL Traut’s Reagent，加入1 mL、1 mg/mL曲妥株单抗溶液（pH 8.0 PBS），20℃条件下活化2 h，再加入5 mg 上述纳米粒，20℃条件下孵育18 h，离心洗涤除去未连接的单抗（18000 rpm，15 min），冷冻干燥，即获得曲妥珠单抗修饰的载紫杉醇的靶向纳米粒。2. Take 20 μL, 6 mg/mL Traut's Reagent, add 1 mL, 1 mg/mL Trastuzumab solution (pH 8.0 PBS), activate at 20°C for 2 h, then add 5 mg of the above nanoparticles, 20 Incubate at ℃ for 18 h, centrifuge and wash to remove unlinked monoclonal antibody (18000 rpm, 15 min), and freeze-dry to obtain trastuzumab-modified paclitaxel-loaded targeting nanoparticles.
实施例 3 ：Embodiment3:
1、称取紫杉醇10 mg和PLGA 1.5A 60mg溶解于2.4 mL丙酮中，称取DPPC 20 mg和DSPE-PEG2000-Mal 10 mg 溶解于1.6 mL的乙醇中。混合丙酮乙醇溶液形成二元有机相（O相），加入15 mL 1.0 ~ 2.0% 泊洛沙姆188水溶液（W相）, 0 ~ 4℃下间断超声（100 W，150 s）分散制备O/W乳剂，再补充15 mL的1.0 ~ 2.0% 泊洛沙姆188水溶液，室温低速搅拌（150 rpm），过夜挥发除去二元有机相，0.22 μm微孔滤膜过滤，并离心收集纳米粒（18000 rpm，15 min），冷冻干燥；1. Weigh 10 mg of paclitaxel and 60 mg of PLGA 1.5A and dissolve in 2.4 mL of acetone, weigh 20 mg of DPPC and 10 mg of DSPE-PEG2000-Mal and dissolve in 1.6 mL of ethanol. Mix the acetone-ethanol solution to form a binary organic phase (phase O), add 15 mL of 1.0-2.0% poloxamer 188 aqueous solution (phase W), and disperse with intermittent ultrasound (100 W, 150 s) at 0-4°C to prepare O/ W emulsion, supplemented with 15 mL of 1.0-2.0% poloxamer 188 aqueous solution, stirred at room temperature at low speed (150 rpm), evaporated overnight to remove the binary organic phase, filtered through a 0.22 μm microporous membrane, and centrifuged to collect nanoparticles (18000 rpm) rpm, 15 min), freeze-dried;
2、取20 μL、6 mg/mL Traut’s Reagent，加入1 mL、1 mg/mL曲妥株单抗溶液（pH 8.0 PBS），20℃条件下活化2 h，再加入5 mg 上述纳米粒，20℃条件下孵育18 h，离心洗涤除去未连接的单抗（18000 rpm，15 min），冷冻干燥，即获得曲妥珠单抗修饰的载紫杉醇的靶向纳米粒。2. Take 20 μL, 6 mg/mL Traut's Reagent, add 1 mL, 1 mg/mL Trastuzumab solution (pH 8.0 PBS), activate at 20°C for 2 h, then add 5 mg of the above nanoparticles, 20 Incubate at ℃ for 18 h, centrifuge and wash to remove unlinked monoclonal antibody (18000 rpm, 15 min), and freeze-dry to obtain trastuzumab-modified paclitaxel-loaded targeting nanoparticles.
实施例 4 ：Embodiment4:
1、称取紫杉醇15 mg和PLGA 1.5A 60mg溶解于2.4 mL丙酮中，称取DPPC 15 mg和DSPE-PEG2000-Mal 10 mg 溶解于1.6 mL的乙醇中。混合丙酮乙醇溶液形成二元有机相（O相），加入15 mL 1.0 ~ 2.0% 泊洛沙姆188水溶液（W相）, 0 ~ 4℃下间断超声（100 W，150 s）分散制备O/W乳剂，再补充15 mL的1.0 ~ 2.0% 泊洛沙姆188水溶液，室温低速搅拌（150 rpm），过夜挥发除去二元有机相，0.22 μm微孔滤膜过滤，并离心收集纳米粒（18000 rpm，15 min），冷冻干燥；1. Weigh 15 mg of paclitaxel and 60 mg of PLGA 1.5A and dissolve in 2.4 mL of acetone, weigh 15 mg of DPPC and 10 mg of DSPE-PEG2000-Mal and dissolve in 1.6 mL of ethanol. Mix the acetone-ethanol solution to form a binary organic phase (phase O), add 15 mL of 1.0-2.0% poloxamer 188 aqueous solution (phase W), and disperse with intermittent ultrasound (100 W, 150 s) at 0-4°C to prepare O/ W emulsion, supplemented with 15 mL of 1.0-2.0% poloxamer 188 aqueous solution, stirred at room temperature at low speed (150 rpm), evaporated overnight to remove the binary organic phase, filtered through a 0.22 μm microporous membrane, and centrifuged to collect nanoparticles (18000 rpm) rpm, 15 min), freeze-dried;
2、取20 μL、6 mg/mL Traut’s Reagent，加入1 mL、1 mg/mL曲妥株单抗溶液（pH 8.0 PBS），20℃条件下活化2 h，再加入5 mg 上述纳米粒，20℃条件下孵育18 h，离心洗涤除去未连接的单抗（18000 rpm，15 min），冷冻干燥，即获得曲妥珠单抗修饰的载紫杉醇的靶向纳米粒。2. Take 20 μL, 6 mg/mL Traut's Reagent, add 1 mL, 1 mg/mL Trastuzumab solution (pH 8.0 PBS), activate at 20°C for 2 h, then add 5 mg of the above nanoparticles, 20 Incubate at ℃ for 18 h, centrifuge and wash to remove unlinked monoclonal antibody (18000 rpm, 15 min), and freeze-dry to obtain trastuzumab-modified paclitaxel-loaded targeting nanoparticles.
实施例 5 ：Embodiment5:
1、称取紫杉醇10 mg和PLGA 1A 60mg溶解于2.4 mL丙酮中，称取DPPC 20 mg和DSPE-PEG2000-Mal 10 mg 溶解于1.6 mL的乙醇中。混合丙酮乙醇溶液形成二元有机相（O相），加入15 mL 1.0 ~ 2.0% 泊洛沙姆188水溶液（W相）, 0 ~ 4℃下间断超声（100 W，150 s）分散制备O/W乳剂，再补充15 mL的1.0 ~ 2.0% 泊洛沙姆188水溶液，室温低速搅拌（150 rpm），过夜挥发除去二元有机相，0.22 μm微孔滤膜过滤，并离心收集纳米粒（18000 rpm，15 min），冷冻干燥；1. Weigh 10 mg of paclitaxel and 60 mg of PLGA 1A and dissolve in 2.4 mL of acetone, weigh 20 mg of DPPC and 10 mg of DSPE-PEG2000-Mal and dissolve in 1.6 mL of ethanol. Mix the acetone-ethanol solution to form a binary organic phase (phase O), add 15 mL of 1.0-2.0% poloxamer 188 aqueous solution (phase W), and disperse with intermittent ultrasound (100 W, 150 s) at 0-4°C to prepare O/ W emulsion, supplemented with 15 mL of 1.0-2.0% poloxamer 188 aqueous solution, stirred at room temperature at low speed (150 rpm), evaporated overnight to remove the binary organic phase, filtered through a 0.22 μm microporous membrane, and centrifuged to collect nanoparticles (18000 rpm) rpm, 15 min), freeze-dried;
2、取100 μL、6 mg/mL Traut’s Reagent，加入1 mL、5 mg/mL曲妥株单抗溶液（pH 8.0 PBS），20℃条件下活化2 h，再加入5 mg 上述纳米粒，20℃条件下孵育18 h，离心洗涤除去未连接的单抗（18000 rpm，15 min），冷冻干燥，即获得曲妥珠单抗修饰的载紫杉醇的靶向纳米粒。2. Take 100 μL, 6 mg/mL Traut's Reagent, add 1 mL, 5 mg/mL Trastuzumab solution (pH 8.0 PBS), activate at 20°C for 2 h, then add 5 mg of the above nanoparticles, 20 Incubate at ℃ for 18 h, centrifuge and wash to remove unlinked monoclonal antibody (18000 rpm, 15 min), and freeze-dry to obtain trastuzumab-modified paclitaxel-loaded targeting nanoparticles.
实施例 6 ：Embodiment6:
1、称取紫杉醇15 mg和PLGA 1A 60mg溶解于2.4 mL丙酮中，称取DPPC 15 mg和DSPE-PEG2000-Mal 10 mg 溶解于1.6 mL的乙醇中。混合丙酮乙醇溶液形成二元有机相（O相），加入15 mL 1.0 ~ 2.0% 泊洛沙姆188水溶液（W相）, 0 ~ 4℃下间断超声（100 W，150 s）分散制备O/W乳剂，再补充15 mL的1.0 ~ 2.0% 泊洛沙姆188水溶液，室温低速搅拌（150 rpm），过夜挥发除去二元有机相，0.22 μm微孔滤膜过滤，并离心收集纳米粒（18000 rpm，15 min），冷冻干燥；1. Weigh 15 mg of paclitaxel and 60 mg of PLGA 1A and dissolve in 2.4 mL of acetone, weigh 15 mg of DPPC and 10 mg of DSPE-PEG2000-Mal and dissolve in 1.6 mL of ethanol. Mix the acetone-ethanol solution to form a binary organic phase (phase O), add 15 mL of 1.0-2.0% poloxamer 188 aqueous solution (phase W), and disperse with intermittent ultrasound (100 W, 150 s) at 0-4°C to prepare O/ W emulsion, supplemented with 15 mL of 1.0-2.0% poloxamer 188 aqueous solution, stirred at room temperature at low speed (150 rpm), evaporated overnight to remove the binary organic phase, filtered through a 0.22 μm microporous membrane, and centrifuged to collect nanoparticles (18000 rpm) rpm, 15 min), freeze-dried;
2、取100 μL、6 mg/mL Traut’s Reagent，加入1 mL、5 mg/mL曲妥株单抗溶液（pH 8.0 PBS），20℃条件下活化2 h，再加入5 mg 上述纳米粒，20℃条件下孵育18 h，离心洗涤除去未连接的单抗（18000 rpm，15 min），冷冻干燥，即获得曲妥珠单抗修饰的载紫杉醇的靶向纳米粒。2. Take 100 μL, 6 mg/mL Traut's Reagent, add 1 mL, 5 mg/mL Trastuzumab solution (pH 8.0 PBS), activate at 20°C for 2 h, then add 5 mg of the above nanoparticles, 20 Incubate at ℃ for 18 h, centrifuge and wash to remove unlinked monoclonal antibody (18000 rpm, 15 min), and freeze-dry to obtain trastuzumab-modified paclitaxel-loaded targeting nanoparticles.
实施例 7 ：Embodiment7:
1、称取紫杉醇10 mg和PLGA 1.5A 60mg溶解于2.4 mL丙酮中，称取DPPC 20 mg和DSPE-PEG2000-Mal 10 mg 溶解于1.6 mL的乙醇中。混合丙酮乙醇溶液形成二元有机相（O相），加入15 mL 1.0 ~ 2.0% 泊洛沙姆188水溶液（W相）, 0 ~ 4℃下间断超声（100 W，150 s）分散制备O/W乳剂，再补充15 mL的1.0 ~ 2.0% 泊洛沙姆188水溶液，室温低速搅拌（150 rpm），过夜挥发除去二元有机相，0.22 μm微孔滤膜过滤，并离心收集纳米粒（18000 rpm，15 min），冷冻干燥；1. Weigh 10 mg of paclitaxel and 60 mg of PLGA 1.5A and dissolve in 2.4 mL of acetone, weigh 20 mg of DPPC and 10 mg of DSPE-PEG2000-Mal and dissolve in 1.6 mL of ethanol. Mix the acetone-ethanol solution to form a binary organic phase (phase O), add 15 mL of 1.0-2.0% poloxamer 188 aqueous solution (phase W), and disperse with intermittent ultrasound (100 W, 150 s) at 0-4°C to prepare O/ W emulsion, supplemented with 15 mL of 1.0-2.0% poloxamer 188 aqueous solution, stirred at room temperature at low speed (150 rpm), evaporated overnight to remove the binary organic phase, filtered through a 0.22 μm microporous membrane, and centrifuged to collect nanoparticles (18000 rpm) rpm, 15 min), freeze-dried;
2、取100 μL、6 mg/mL Traut’s Reagent，加入1 mL、5 mg/mL曲妥株单抗溶液（pH 8.0 PBS），20℃条件下活化2 h，再加入5 mg 上述纳米粒，20℃条件下孵育18 h，离心洗涤除去未连接的单抗（18000 rpm，15 min），冷冻干燥，即获得曲妥珠单抗修饰的载紫杉醇的靶向纳米粒。2. Take 100 μL, 6 mg/mL Traut's Reagent, add 1 mL, 5 mg/mL Trastuzumab solution (pH 8.0 PBS), activate at 20°C for 2 h, then add 5 mg of the above nanoparticles, 20 Incubate at ℃ for 18 h, centrifuge and wash to remove unlinked monoclonal antibody (18000 rpm, 15 min), and freeze-dry to obtain trastuzumab-modified paclitaxel-loaded targeting nanoparticles.
实施例 8 ：Embodiment8:
1、称取紫杉醇15 mg和PLGA 1.5A 60mg溶解于2.4 mL丙酮中，称取DPPC 15 mg和DSPE-PEG2000-Mal 10 mg 溶解于1.6 mL的乙醇中。混合丙酮乙醇溶液形成二元有机相（O相），加入15 mL 1.0 ~ 2.0% 泊洛沙姆188水溶液（W相）, 0 ~ 4℃下间断超声（100 W，150 s）分散制备O/W乳剂，再补充15 mL的1.0 ~ 2.0% 泊洛沙姆188水溶液，室温低速搅拌（150 rpm），过夜挥发除去二元有机相，0.22 μm微孔滤膜过滤，并离心收集纳米粒（18000 rpm，15 min），冷冻干燥；1. Weigh 15 mg of paclitaxel and 60 mg of PLGA 1.5A and dissolve in 2.4 mL of acetone, weigh 15 mg of DPPC and 10 mg of DSPE-PEG2000-Mal and dissolve in 1.6 mL of ethanol. Mix the acetone-ethanol solution to form a binary organic phase (phase O), add 15 mL of 1.0-2.0% poloxamer 188 aqueous solution (phase W), and disperse with intermittent ultrasound (100 W, 150 s) at 0-4°C to prepare O/ W emulsion, supplemented with 15 mL of 1.0-2.0% poloxamer 188 aqueous solution, stirred at room temperature at low speed (150 rpm), evaporated overnight to remove the binary organic phase, filtered through a 0.22 μm microporous membrane, and centrifuged to collect nanoparticles (18000 rpm) rpm, 15 min), freeze-dried;
2、取100 μL、6 mg/mL Traut’s Reagent，加入1 mL、5 mg/mL曲妥株单抗溶液（pH 8.0 PBS），20℃条件下活化2 h，再加入5 mg 上述纳米粒，20℃条件下孵育18 h，离心洗涤除去未连接的单抗（18000 rpm，15 min），冷冻干燥，即获得曲妥珠单抗修饰的载紫杉醇的靶向纳米粒。2. Take 100 μL, 6 mg/mL Traut's Reagent, add 1 mL, 5 mg/mL Trastuzumab solution (pH 8.0 PBS), activate at 20°C for 2 h, then add 5 mg of the above nanoparticles, 20 Incubate at ℃ for 18 h, centrifuge and wash to remove unlinked monoclonal antibody (18000 rpm, 15 min), and freeze-dry to obtain trastuzumab-modified paclitaxel-loaded targeting nanoparticles.
实施例 9 ：Embodiment9:
纳米粒的表征Characterization of nanoparticles
采用激光粒度仪分析PCNs及T=PCNs的粒径及电位分布，其粒径及电位分布如图2所示。粒径检测结果显示，PCNs的平均粒径在130 ~ 160 nm之间，电位在 -10 mV左右。T=PCNs的平均粒径在 150 ~ 200 nm之间，电位在 0 mV左右（表1）。The particle size and potential distribution of PCNs and T=PCNs were analyzed by laser particle size analyzer, and the particle size and potential distribution are shown in Figure 2. The particle size detection results showed that the average particle size of PCNs was between 130 and 160 nm, and the potential was around -10 mV. The average particle size of T=PCNs is between 150 and 200 nm, and the potential is around 0 mV (Table 1).
采用扫描电子显微镜观测PCNs及T=PCNs的形貌，冻干纳米粒放置在硅片上喷金处理，结果如图3所示。扫面电镜结果显示，PCNs及T=PCNs均呈圆球形，大小均一，在100 ~ 200 nm之间，T=PCNs组微粒之间有少量聚集。Scanning electron microscopy was used to observe the morphology of PCNs and T=PCNs. Freeze-dried nanoparticles were placed on silicon wafers and sprayed with gold. The results are shown in Figure 3. The results of scanning electron microscopy showed that both PCNs and T=PCNs were spherical and uniform in size, between 100 and 200 nm, and there was a small amount of aggregation among the particles in the T=PCNs group.
表1.实施例1 ~ 8所制得的纳米粒粒径、电位分布及药物包封率Table 1. The prepared nanoparticles particle size, potential distribution and drug encapsulation efficiency of embodiment 1～8
*1 T：PCNs为曲妥株单抗与纳米粒的比例；*2 EE为紫杉醇包封率。*1 T: PCNs is the ratio of trastuzumab to nanoparticles; *2 EE is the encapsulation efficiency of paclitaxel.
实施例 10 ：Embodiment10:
纳米粒的载药量及体外释放行为考察Study on drug loading and in vitro release behavior of nanoparticles
采用高效液相色谱（HPLC）测定PCNs的包封率：称取10 mg冻干PCNs，溶解在1 mL 乙腈中，蜗旋溶解10 min，加入9 mL的75%甲醇溶液，0.22 μm微孔滤膜过滤，过滤液进行色谱分析。HPLC条件，流动相甲醇：水（75：25，v/v）溶液，流速1 mL/min，检测波长230 nm，进样量20 μL。根据以下公式计算包封率(EE, %) =测得紫杉醇的质量 / 投入紫杉醇的质量 × 100%。测得包封率为75% ~ 83%（表1）。The encapsulation efficiency of PCNs was determined by high performance liquid chromatography (HPLC): Weigh 10 mg of freeze-dried PCNs, dissolve in 1 mL of acetonitrile, vortex dissolve for 10 min, add 9 mL of 75% methanol solution, and filter through 0.22 μm microporous Membrane filtration, and the filtrate was subjected to chromatographic analysis. HPLC conditions, mobile phase methanol:water (75:25, v/v) solution, flow rate 1 mL/min, detection wavelength 230 nm, injection volume 20 μL. Encapsulation efficiency (EE, %) = measured paclitaxel mass / input paclitaxel mass × 100% was calculated according to the following formula. The encapsulation efficiency was measured to be 75%-83% (Table 1).
采用透析的方法分析PCNs及T=PCNs的体外释放行为：称取20 mg的冻干纳米粒，分散在1 mL pH 7.4 PBS（1% Tween-80，v/v）溶液中，装入透析袋（MWCO，10 kD）。将透析袋置入含40 mL pH 7.4 PBS（1% Tween-80，v/v）溶液中，置于37℃恒温摇床中，120 rpm持续震摇。分别于0.5 h，1 h，2 h，3 h，4 h，6 h，1 d，2 d，3 d，4 d取出透析液1 mL备用，同时补充1 mL新鲜的PBS溶液。透析液过μm微孔滤膜，过滤液HPLC检测紫杉醇含量，并计算紫杉醇的累积释放率。以市售紫杉醇注射液（Taxol）为对照，比较缓释纳米粒的释放能力，结果如图4所示。在起始的6 h内，三种制剂均呈现了不同程度的突释行为（61.2%，43.3%，34.4%），紫杉醇注射液组突释现象最明显，24 h内即释放完毕，无缓释行为。PCNs及T=PCNs组在突释后出现缓慢释放行为，T=PCNs组在96 h内累计释放率达78.2%。T=PCNs释放行为学先突释再缓释，且缓释效果明显。这归功于其PLGA与磷脂形成的核壳结构，PLGA表面的单分子磷脂层调节紫杉醇的释放，表面偶联单抗后，更进一步延缓了紫杉醇的释放，在一定时间内维持了紫杉醇的治疗浓度。The in vitro release behavior of PCNs and T=PCNs was analyzed by dialysis: 20 mg of freeze-dried nanoparticles were weighed, dispersed in 1 mL of pH 7.4 PBS (1% Tween-80, v/v) solution, and loaded into a dialysis bag (MWCO, 10 kD). The dialysis bag was placed in a solution containing 40 mL of PBS (1% Tween-80, v/v) at pH 7.4, placed in a constant temperature shaker at 37°C, and continuously shaken at 120 rpm. At 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 1 d, 2 d, 3 d, and 4 d, 1 mL of dialysate was taken out for use, and 1 mL of fresh PBS solution was added at the same time. The dialysate was passed through a μm microporous membrane, the paclitaxel content of the filtrate was detected by HPLC, and the cumulative release rate of paclitaxel was calculated. The commercially available paclitaxel injection (Taxol) was used as a control to compare the release ability of sustained-release nanoparticles, and the results are shown in Figure 4. In the first 6 hours, the three preparations all showed different degrees of burst release behavior (61.2%, 43.3%, 34.4%), and the burst release phenomenon was the most obvious in the paclitaxel injection group, and the release was completed within 24 hours without slowing down. release behavior. PCNs and T=PCNs groups showed slow release after burst release, and the cumulative release rate of T=PCNs group reached 78.2% within 96 hours. T=PCNs release behavior first burst release and then sustained release, and the sustained release effect is obvious. This is due to the core-shell structure formed by PLGA and phospholipids. The monomolecular phospholipid layer on the surface of PLGA regulates the release of paclitaxel. After the monoclonal antibody is coupled to the surface, the release of paclitaxel is further delayed and the therapeutic concentration of paclitaxel is maintained for a certain period of time. .
实施例 11 ：Embodiment11:
纳米粒的细胞毒实验Nanoparticle Cytotoxicity Experiment
人类乳腺癌细胞SKBR3及MCF7分别培养于含10% 胎牛血清、1%青霉素-链霉素的DMEM细胞培养液中，培养瓶置于37℃，含5% CO2培养箱中。取对数生长期的细胞进行以下实验。其中，SKBR3细胞过度表达HER2抗原，MCF7细胞低表达HER2抗原。Human breast cancer cells SKBR3 and MCF7 were respectively cultured in DMEM cell culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and the culture flasks were placed in a 37°C, 5% CO2 incubator. Cells in the logarithmic growth phase were taken for the following experiments. Among them, SKBR3 cells overexpressed HER2 antigen, and MCF7 cells underexpressed HER2 antigen.
以1×104个/孔的密度将处于对数期的癌细胞接种于在96微孔板中，37℃培养24 h使其贴壁，第二天去除培养液，分别加入100 μL含药培养基（PTX，HER，PCNs及T=PCNs），37℃孵育4 h后，去除含药培养基，更换新鲜培养基，分别继续培养24 h，48 h和72 h。去除培养基，每孔加入10 μL、5mg/mL MTT试剂，37℃孵育4 h，DMSO溶解生成的甲瓒结晶，酶标仪492 nm分析溶液吸光度，并计算细胞存活率。Inoculate cancer cells in the logarithmic phase in a 96-well plate at a density of 1×104 cells/well, culture at 37°C for 24 hours to allow them to adhere to the wall, remove the culture medium the next day, and add 100 μL drug-containing Medium (PTX, HER, PCNs and T=PCNs), after incubation at 37°C for 4 h, remove the drug-containing medium, replace with fresh medium, and continue to culture for 24 h, 48 h and 72 h, respectively. Remove the medium, add 10 μL, 5 mg/mL MTT reagent to each well, incubate at 37 °C for 4 h, dissolve the formed formazan crystals in DMSO, analyze the absorbance of the solution with a microplate reader at 492 nm, and calculate the cell survival rate.
实验结果如图5所示，T=PCNs对SKBR3细胞72 h后的杀伤作用（47.2%）明显高于PTX（76.3%）和PCNs（85.5%）。这归功于SKBR3过度表达的HER2抗原，说明其肿瘤靶向性良好。以MCF7细胞作为对照组实验，T=PCNs对MCF7细胞的杀伤作用（54.9%）与PCNs（56.6%）在作用72 h后并没有明显区别。细胞毒结果说明，T=PCNs体外抗肿瘤效果明显，离不开其载体材料良好的生物相容性及单抗的靶向作用。The experimental results are shown in Figure 5. The killing effect of T=PCNs on SKBR3 cells after 72 h (47.2%) was significantly higher than that of PTX (76.3%) and PCNs (85.5%). This is attributed to the HER2 antigen overexpressed by SKBR3, indicating its good tumor targeting. Using MCF7 cells as the control group, the killing effect of T=PCNs on MCF7 cells (54.9%) was not significantly different from that of PCNs (56.6%) after 72 hours of treatment. The results of cytotoxicity showed that T=PCNs had obvious anti-tumor effect in vitro, which could not be separated from the good biocompatibility of its carrier material and the targeting effect of monoclonal antibody.
实施例 12 ：Embodiment12:
纳米粒的细胞摄取实验Cellular uptake experiments of nanoparticles
细胞摄取实验中使用双荧光标记的T=PCNs，罗丹明B标记PCNs，FITC标记曲妥株单抗，再进行化学偶联，具体制备工艺如下：In the cell uptake experiment, double-fluorescence-labeled T=PCNs, rhodamine B-labeled PCNs, FITC-labeled trastuzumab, and then chemically coupled, the specific preparation process is as follows:
（A）载罗丹明B的PCNs的制备：称取罗丹明B 2 mg和PLGA 1A 60mg溶解于2.4 mL丙酮中，称取DPPC 20 mg和DSPE-PEG2000-Mal 10 mg 溶解于1.6 mL的乙醇中。混合丙酮乙醇溶液形成二元有机相（O相），加入15 mL 1.0 ~ 2.0% 泊洛沙姆188水溶液（W相）, 0 ~ 4℃下间断超声（100 W，150 s）分散制备O/W乳剂，再补充15 mL的1.0 ~ 2.0% 泊洛沙姆188水溶液，室温低速搅拌（150 rpm），过夜挥发除去二元有机相，0.22 μm微孔滤膜过滤，并离心洗涤并收集纳米粒（18000 rpm，15 min），冷冻干燥；(A) Preparation of rhodamine B-loaded PCNs: Weigh 2 mg rhodamine B and PLGA 1A 60 mg and dissolve in 2.4 mL acetone, weigh 20 mg DPPC and DSPE-PEG2000-Mal 10 mg and dissolve in 1.6 mL ethanol . Mix the acetone-ethanol solution to form a binary organic phase (phase O), add 15 mL of 1.0-2.0% poloxamer 188 aqueous solution (phase W), and disperse with intermittent ultrasound (100 W, 150 s) at 0-4°C to prepare O/ W emulsion, add 15 mL of 1.0 ~ 2.0% poloxamer 188 aqueous solution, stir at low speed at room temperature (150 rpm), evaporate overnight to remove the binary organic phase, filter through a 0.22 μm microporous membrane, and centrifuge to wash and collect nanoparticles (18000 rpm, 15 min), freeze-dried;
（B）FITC标记的曲妥株单抗的制备：（1）使用pH 9.0的交联液（NaHCO3，Na2CO3，NaCl）配制曲妥株单抗溶液，浓度为10 mg/mL。（2）FITC溶于DMSO中，浓度为1mg/mL。（3）将FITC溶液缓慢滴加到抗体溶液中，轻轻晃动使其与抗体混合均匀，暗处4℃反应6 h。（4）加入5 mol/L的NH4Cl至终浓度50 mmol/L，4℃终止反应1 h。（5）FITC-曲妥株单抗交联液pH 7.4 PBS中透析，除去未连接的FITC。(B) Preparation of FITC-labeled trastuzumab: (1) A trastuzumab solution was prepared at a concentration of 10 mg/mL using a pH 9.0 cross-linking solution (NaHCO3, Na2 CO3 , NaCl). (2) FITC was dissolved in DMSO at a concentration of 1 mg/mL. (3) Slowly add the FITC solution dropwise to the antibody solution, shake gently to mix it with the antibody evenly, and react for 6 h at 4°C in the dark. (4) Add 5 mol/L NH4Cl to a final concentration of 50 mmol/L, and stop the reaction at 4°C for 1 h. (5) Dialyze the FITC-trastuzumab cross-linking solution pH 7.4 in PBS to remove unlinked FITC.
（C）载罗丹明B的PCNs与FITC标记的曲妥株单抗的偶联：取20μL、6 mg/mL Traut’s Reagent，加入1 mL、1 mg/mL FITC-曲妥株单抗溶液（pH 8.0 PBS），20℃条件下活化2 h，再加入5 mg 上述纳米粒，20℃条件下孵育18 h，离心洗涤除去未连接的单抗（18000 rpm，15 min），冷冻干燥，即获得双荧光标记的曲妥珠单抗修饰的载紫杉醇的靶向纳米粒，避光保存。(C) Conjugation of rhodamine B-loaded PCNs to FITC-labeled trastuzumab: take 20 μL, 6 mg/mL Traut's Reagent, add 1 mL, 1 mg/mL FITC-trastuzumab solution (pH 8.0 PBS), activated at 20°C for 2 h, then added 5 mg of the above-mentioned nanoparticles, incubated at 20°C for 18 h, centrifuged and washed to remove unlinked monoclonal antibody (18000 rpm, 15 min), and freeze-dried to obtain the bismuth Fluorescently labeled trastuzumab-modified paclitaxel-loaded targeting nanoparticles were stored in the dark.
激光共聚焦显微成像laser confocal microscopy
以2×104个/孔的密度将处于对数期的癌细胞接种于玻底培养皿中，37℃培养24 h使其贴壁，第二天去除培养液，分别加入1 mL无血清含药培养基（双荧光标记T=PCNs），37℃孵育2 h后，去除含荧光纳米粒的培养基，pH 7.4 PBS清洗三次。4%甲醛固定细胞，DAPI溶液进行细胞核染色，激光共聚焦显微镜下成像。罗丹明B激发波长Ex=540 nm，发射波长Em=625 nm。FITC激发波长Ex=488 nm，Em=525 nm。Cancer cells in the logarithmic phase were inoculated on glass-bottom culture dishes at a density of2 ×104/well, cultured at 37°C for 24 h to allow them to adhere to the wall, and the culture medium was removed the next day, and 1 mL of serum-free containing Drug medium (double fluorescently labeled T=PCNs), incubated at 37°C for 2 h, removed the medium containing fluorescent nanoparticles, and washed three times with pH 7.4 PBS. Cells were fixed with 4% formaldehyde, nuclei were stained with DAPI solution, and imaged under a confocal laser microscope. The excitation wavelength of Rhodamine B is Ex=540 nm, and the emission wavelength is Em=625 nm. FITC excitation wavelength Ex=488 nm, Em=525 nm.
流式细胞仪定量分析Quantitative Analysis by Flow Cytometry
以1 ~ 2×105个/孔的密度将处于对数期的癌细胞接种于在24孔板中，37℃培养24 h使其贴壁，第二天去除培养液，分别加入1 mL无血清含药培养基（双荧光标记T=PCNs），37℃孵育3 h后，去除含荧光纳米粒的培养基，pH 7.4 PBS清洗三次。0. 25%胰酶（w/v）收集细胞，重悬于4%甲醛固定液中，进行流式细胞分析。分别采用激光通道FL1和FL3，检测FITC和罗丹明B的荧光。Cancer cells in the logarithmic phase were inoculated in 24-well plates at a density of 1-2×105 cells/well, cultured at 37°C for 24 h to allow them to adhere to the wall, and the culture medium was removed the next day, and 1 mL of no Serum drug-containing medium (double fluorescently labeled T=PCNs), incubated at 37°C for 3 h, removed the medium containing fluorescent nanoparticles, and washed three times with pH 7.4 PBS. Cells were harvested with 0.25% trypsin (w/v), resuspended in 4% formaldehyde fixative, and analyzed by flow cytometry. Laser channels FL1 and FL3 were used to detect the fluorescence of FITC and Rhodamine B, respectively.
细胞摄取实验结果如图6，7所示。罗丹明B标记的PCNs呈现红色荧光，FITC标记的曲妥株单抗呈现绿色荧光，当二者结合后，出现黄色的荧光（T=PCNs），其鲜艳程度取决于肿瘤细胞摄取的含量。这也间接的表明，曲妥株单抗成功的偶联在纳米粒的表面，并且在细胞摄取过程中，二者并没有分离。在SKBR3细胞中，亮黄色荧光出现在细胞质区域，说明肿瘤细胞对T=PCNs有较高的摄取率。对比MCF7细胞，黄色荧光极不明显，说明MCF7细胞对T=PCNs摄取率较低，T=PCNs不能发挥其靶向作用（图6）。在流式细胞分析中，肿瘤细胞对T=PCNs的摄取被定量分析。如图7 B所示，SKBR3细胞对罗丹明B-PCNs和FITC-HER的摄取均高于MCF7细胞。细胞实验结果说明了T=PCNs对HER2过表达的癌细胞具有良好的靶向性和细胞毒性。The results of cell uptake experiments are shown in Figures 6 and 7. Rhodamine B-labeled PCNs exhibits red fluorescence, FITC-labeled trastuzumab exhibits green fluorescence, and when the two are combined, yellow fluorescence (T=PCNs) appears, and the degree of brilliance depends on the amount of uptake by tumor cells. This also indirectly indicated that trastuzumab was successfully coupled to the surface of the nanoparticles, and the two were not separated during cellular uptake. In SKBR3 cells, bright yellow fluorescence appeared in the cytoplasmic region, indicating a high uptake rate of T=PCNs by tumor cells. Compared with MCF7 cells, the yellow fluorescence was very inconspicuous, indicating that the uptake rate of T=PCNs by MCF7 cells was low, and T=PCNs could not exert its targeting effect (Figure 6). In flow cytometric analysis, the uptake of T=PCNs by tumor cells was quantified. As shown in Figure 7B, the uptake of rhodamine B-PCNs and FITC-HER by SKBR3 cells was higher than that of MCF7 cells. The results of cell experiments showed that T=PCNs had good targeting and cytotoxicity to HER2 overexpressed cancer cells.
讨论discuss
本发明结合了PLGA和磷脂的优点并与单克隆抗体联合，制备了具有核壳结构的曲妥株单抗修饰的载紫杉醇的靶向纳米粒（T=PCNs）。磷脂包被在PLGA/紫杉醇的核心外形成单分子磷脂层，磷脂表面偶联单抗分子，从而形成具有核壳结构的纳米级微粒。T=PCNs具备了PLGA和磷脂的优点：具有粒径小（150 ~ 200 nm），更有利于细胞摄取（<200 nm），药物包封率高（75 ~ 83%），在溶液中稳定性好（电位0 mV左右），生物相容性好，增加内体逃逸，释放行为学可维持紫杉醇治疗水平，结合曲妥株单抗后更实现了药物的主动靶向性。对肿瘤细胞的杀伤作用明显高于PTX和PCNs，提高了HER2过表达的肿瘤细胞的摄取效率，极大的提高了紫杉醇的安全性和靶向性。尤其在治疗HER2高表达的乳腺癌十分见效。The present invention combines the advantages of PLGA and phospholipids and combines them with monoclonal antibodies to prepare trastuzumab-modified targeting nanoparticles (T=PCNs) loaded with paclitaxel with a core-shell structure. The phospholipid coating forms a monomolecular phospholipid layer outside the core of PLGA/paclitaxel, and the phospholipid surface is coupled with monoclonal antibody molecules to form nanoscale particles with a core-shell structure. T=PCNs has the advantages of PLGA and phospholipids: small particle size (150 ~ 200 nm), more conducive to cell uptake (<200 nm), high drug encapsulation efficiency (75 ~ 83%), and stability in solution Good (potential 0 mV or so), good biocompatibility, increased endosome escape, release behavior can maintain the therapeutic level of paclitaxel, combined with trastuzumab to achieve the active targeting of the drug. The killing effect on tumor cells is significantly higher than that of PTX and PCNs, improves the uptake efficiency of tumor cells with HER2 overexpression, and greatly improves the safety and targeting of paclitaxel. It is especially effective in the treatment of breast cancer with high expression of HER2.
本发明所介绍的二元溶剂乳化挥发法制备的PLGA/Lipid纳米粒传递系统，包裹的药物不仅限于紫杉醇，还可拓展为其它与PLGA具有良好生物相容性的疏水性化疗药物；偶联的配体也不仅限于曲妥株单抗，还可包括那些具有靶向性的小分子多肽及其它种类的单克隆抗体。The PLGA/Lipid nanoparticle delivery system prepared by the binary solvent emulsification and volatilization method introduced in the present invention is not limited to paclitaxel, but can also be expanded to other hydrophobic chemotherapeutic drugs with good biocompatibility with PLGA; coupled Ligands are not limited to trastuzumab, but may also include those targeting small molecule polypeptides and other types of monoclonal antibodies.
本发明制备的靶向纳米粒（T=PCNs）为冻干制剂，属于静脉给药系统。对于静脉给药系统，溶剂通常为无菌水溶液。适当的调整溶液pH值并使溶液等渗（如加入氯化钠或葡萄糖），即适合静脉给药，纳米粒采取过0.22 μm无菌滤膜的方式灭菌。The targeting nanoparticles (T=PCNs) prepared in the present invention are freeze-dried preparations and belong to an intravenous drug delivery system. For intravenous administration systems, the solvent is usually a sterile aqueous solution. Properly adjust the pH value of the solution and make the solution isotonic (such as adding sodium chloride or glucose), which is suitable for intravenous administration. The nanoparticles are sterilized by passing through a 0.22 μm sterile filter membrane.
本发明构建的曲妥株单抗修饰的载紫杉醇的靶向纳米粒传递系统，是一种优良的递药系统，有望应用于HER2过表达的乳腺癌、卵巢癌及肺癌等实体肿瘤的治疗。The paclitaxel-loaded targeted nanoparticle delivery system modified by trastuzumab constructed in the present invention is an excellent drug delivery system and is expected to be applied to the treatment of solid tumors such as breast cancer, ovarian cancer and lung cancer with HER2 overexpression.
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| CN201510205539.5ACN104814934A (en) | 2015-04-28 | 2015-04-28 | Herceptin modified paclitaxel-carried targeting nanoparticle transfer system |
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| CN201510205539.5ACN104814934A (en) | 2015-04-28 | 2015-04-28 | Herceptin modified paclitaxel-carried targeting nanoparticle transfer system |
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| CN201510205539.5APendingCN104814934A (en) | 2015-04-28 | 2015-04-28 | Herceptin modified paclitaxel-carried targeting nanoparticle transfer system |
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