WO2019214219A1 - 一种pH/还原响应性聚氨基酸两性离子纳米颗粒的制备方法 - Google Patents

一种pH/还原响应性聚氨基酸两性离子纳米颗粒的制备方法 Download PDF

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WO2019214219A1
WO2019214219A1 PCT/CN2018/118862 CN2018118862W WO2019214219A1 WO 2019214219 A1 WO2019214219 A1 WO 2019214219A1 CN 2018118862 W CN2018118862 W CN 2018118862W WO 2019214219 A1 WO2019214219 A1 WO 2019214219A1
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zwitterionic
nanoparticles
benzyl
acid
polyamino acid
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张丽萍
倪才华
刘仁
王大伟
石刚
桑欣欣
夏晓峰
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Jiangnan University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/351—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom not condensed with another ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to a preparation method of pH/reduction responsive polyamino acid zwitterionic nanoparticles, belonging to the technical field of polymer synthesis and biomedical materials.
  • Cancer is considered to be the number one killer of death.
  • an important method for treating cancer is chemotherapy, but the method not only has a killing effect on cancer cells, but also has a large toxic side effect on normal cells, and is easily removed by the reticuloendothelial system, resulting in poor cell absorption.
  • the nano drug carrier system can enhance the permeability and retention (EPR) effect, and can escape the recognition and capture of the reticular cortex system of the human body, prolong the circulation time of the drug-loading system in the blood, and improve the bioavailability of the drug. .
  • EPR permeability and retention
  • the zwitterionic polymer has high hydration ability and anti-bacterial adhesion ability, can resist non-specific protein adsorption, effectively avoid macrophage uptake, realize "invisible" function in the blood circulation system, and can also charge and chemically modify Therefore, there is hope for an excellent substitute for polyethylene glycol.
  • Most of the zwitterionic polymers prepared in the literature are currently non-biodegradable. This will create a series of problems, such as residuals in polymer degradation leading to incomplete release of the drug and undesirable side reactions. Another problem is that the zwitterionic polymers presently have no pH/reduction responsiveness and it is difficult to control the release of the drug at the tumor site.
  • the pH and reducing environment in tumor cells are special, the tumor cells are slightly acidic, and the concentration of glutathione is 100 to 1000 times higher than that in body fluids or other tissues in the body, which has a strong reducing effect on disulfide bonds. . Therefore, the design of environmentally responsive drug carriers, using the pH of the tumor site and the reducing environment for drug controlled release, has become an effective method.
  • Polyglutamic acid has good biocompatibility and is a biodegradable polymer. Poly L-glutamic acid and its derivatives are slowly degraded in vivo, and as a drug carrier, the circulation time of the drug in the body can be prolonged. Helps with sustained release and targeted drug delivery.
  • the polyglutamic acid side chain contains a ⁇ -carboxy group and is capable of undergoing an amidation reaction with an amino group.
  • Lysine is one of the essential amino acids in the human body and is often used in the preparation of pharmaceutical carriers.
  • the polylysine side chain has a free amino group and a carboxyl group, which have positive and negative charges, respectively, and can form zwitterions.
  • the method for preparing nanoparticles by using L-lysine as a matrix is complicated and takes a long time. Most of them have acid sensitivity and no pH/reduction responsiveness, resulting in less drug release.
  • an object of the present invention is to provide a method for preparing a pH/reduction responsive polyamino acid zwitterionic nanoparticle, which firstly synthesizes a polyglutamic acid main chain by ring-opening polymerization, and then introduces a leaching reaction by a side chain group reaction. The is a zwitterion and the final cross is combined into a nanoparticle.
  • the nanoparticles have anti-non-specific protein adsorption and pH responsiveness due to the presence of zwitterions. Since cystamine contains disulfide bonds, the nanoparticles have sensitive reduction responsiveness. Nanoparticles can be loaded with anticancer drugs and controlled release at tumor targeting sites.
  • a first object of the present invention is to provide a method for preparing a pH/reduction responsive polyamino acid zwitterionic nanoparticle, the preparation method comprising the steps of:
  • Step 1 using an initiator to initiate ring-opening polymerization of ⁇ -benzyl-L-glutamic acid-N-carboxy anhydride to obtain a polybenzyl benzyl ester intermediate;
  • Step 2 reacting the benzyl polyglutamic acid intermediate obtained above with lysine to form an amphoteric ion polymer;
  • Step 3 The zwitterionic polymer obtained above is crosslinked with cystamine, and purified to obtain a pH/reduction responsive polyamino acid zwitterionic nanoparticle of a crosslinked structure.
  • the initiator in step 1, is a fatty amine.
  • the fatty amine is n-pentylamine, n-hexylamine, n-heptylamine, n-octylamine, cyclopentylamine, cyclohexylamine or benzylamine.
  • step 1 dichloromethane, chloroform or dichloroethane is used as the solvent.
  • step 1 in step 1, the molar ratio of initiator to gamma-benzyl-L-glutamic acid-N-carboxy anhydride is from 1:25 to 30.
  • step 2 the molar ratio of lysine to gamma-benzyl-L-glutamic acid-N-carboxyanhydride is from 0.5 to 0.8:1.
  • the reaction conditions are such that the reaction is stirred at 20 to 30 ° C for 15 to 24 hours under a nitrogen atmosphere.
  • step 2 the reaction conditions are such that the reaction is stirred at 20-30 ° C for 10-15 hours.
  • the cross-linking of the zwitterionic polymer with cystamine specifically comprises the steps of: adding a cystamine hydrochloride solution containing an acid-binding agent to the zwitterionic polymer, at 20 The reaction was stirred at -40 ° C for 10-15 hours; water was added dropwise to the obtained reaction solution until the micelles appeared, and the micelle solution was dialyzed for 40-60 hours to obtain pH/reduction responsive polyamino acid zwitterionic nanoparticles.
  • the molar ratio of cystamine hydrochloride to ⁇ -benzyl-L-glutamic acid-N-carboxy anhydride is from 0.25 to 0.1:1.
  • the molar ratio is 1 to 3 times that of the cystamine hydrochloride.
  • Step 1 Dissolve the initiator, add ⁇ -benzyl-L-glutamic acid-N-carboxy anhydride, and stir the reaction at 20-30 ° C for 15-24 hours under nitrogen to make the fatty amine initiate ⁇ -benzyl group. Ring-opening polymerization of -L-glutamic acid-N-carboxy anhydride to obtain a polybenzyl benzyl ester intermediate;
  • Step 2 The intermediate product of the above step 1 is dissolved, L-lysine is added, and the reaction is stirred at 20-30 ° C for 10-15 hours to obtain a zwitterionic polymer;
  • Step 3 Dissolve the zwitterion in step 2 above, add the cystamine hydrochloride solution containing the acid-binding agent, and continue stirring the reaction at 20-40 ° C for 10-15 hours; add water to the reaction solution while stirring; The micelles appeared, and the micelle solution was dialyzed for 40-60 hours, and lyophilized to obtain pH/reduction responsive polyamino acid zwitterionic nanoparticles.
  • step 1 the fatty amine is dissolved in dichloromethane, and a solution of ⁇ -benzyl-L-glutamic acid-N-carboxy anhydride in dichloromethane is added to protect the nitrogen.
  • the reaction is stirred at 20-30 ° C for 15-24 hours to initiate ring-opening polymerization of ⁇ -benzyl-L-glutamic acid-N-carboxy anhydride, and the polymerization product is precipitated with anhydrous petroleum ether, and the product is washed with water.
  • the vacuum was dried at a constant temperature of 20-30 ° C for 40-50 h to obtain a polybenzyl glutamate intermediate product.
  • the concentration of gamma-benzyl-L-glutamic acid-N-carboxy anhydride in dichloromethane is 16-20 w%.
  • the intermediate product obtained in the step 1 is dissolved in tetrahydrofuran, L-lysine is added, and the reaction is stirred at 20-30 ° C for 10-15 hours, using anhydrous petroleum.
  • the ether precipitated the polymer and dried under vacuum at 20-30 ° C to obtain a zwitterionic polymer.
  • step 3 the zwitterion obtained in step 2 is dissolved in N,N-dimethylformamide, and a solution of cystamine hydrochloride and triethylamine is added at 20-40. Stir the reaction for 10-15 hours at °C, add ultrapure water to the solution under magnetic stirring until the micelles appear. Transfer the micelle solution to the dialysis bag for dialysis for 40-60 hours, freeze-dry to obtain pH/reduction responsiveness. Polyamino acid zwitterionic nanoparticles.
  • a second object of the present invention is to provide a pH/reduction responsive polyamino acid zwitterionic nanoparticle prepared by the method.
  • a third object of the present invention is to provide the use of the pH/reduction responsive polyamino acid zwitterionic nanoparticles in the preparation of a pharmaceutical carrier for dissolving a drug, the pH/reduction responsive polyamino acid
  • the ionic nanoparticles are dissolved in the above drug solution, stirred and dialyzed, and then freeze-dried to obtain drug-loaded nanoparticles.
  • the drug is doxorubicin or paclitaxel.
  • a hydrophobic small molecule fatty amine is used to initiate the ring-opening polymerization of ⁇ -benzyl-L-glutamic acid-N-carboxy anhydride compound to prepare polyglutamic acid-benzyl ester, and the reaction is stable and the product structure is regular.
  • the molecular weight and its nanoparticle size are controllable and stable, which can improve the performance of the nanoparticles.
  • a small molecule lysine is reacted with a side chain group of benzyl polyglutamic acid, and the reaction is irreversible, and the conversion ratio of the group can be controlled by the feed ratio to make the product performance more controllable; Moreover, the reaction of the present invention is simple and easy to handle.
  • the disulfide bond in cystamine is introduced into the crosslinked structure to make the nanoparticles sensitive to reduction responsiveness; the zwitterionic structure of lysine makes the nanoparticles have pH responsiveness. Conducive to loading anti-cancer drugs, controlled release at the tumor targeting site.
  • 2 is a transmission electron micrograph of the pH/reduction responsive polyamino acid zwitterionic nanoparticles of the present invention, a: CPLG-1; b: CPLG-2; c: CPLG-3; d: CPLG-4; e: CPLG- 5;f:CPLG-6;
  • 3 is a fluorescence emission spectrum of the cerium solubilized nanoparticle CPLG-1 of the present invention which was incubated at 10 ° C for various times after being treated with 10 mM GSH.
  • Monomer preparation 80 g of L-glutamic acid and 65 g of benzyl alcohol were added to a 500 mL flask, and the temperature was slowly raised to 70 ° C. The mixture was uniformly stirred by magnetic stirring, and 86 g of 50% sulfuric acid was added dropwise thereto, and distilled under reduced pressure, and the reaction liquid was poured into NaHCO. 3 The solution was neutralized to neutrality, cooled to a standstill at 4 ° C, filtered under reduced pressure, and washed with ethanol and distilled water for 3 times, then recrystallized at 70 ° C and 5% ethanol solution, and cooled at 4 ° C overnight. Finally, it was vacuum dried at 35 ° C for 48 h to obtain a white flake solid as L-glutamic acid- ⁇ -benzyl ester.
  • Ring-opening polymerization 40 g of anhydrous dichloromethane was placed in a three-necked flask, and 0.383 g of heptylamine was added to dissolve in dichloromethane, and 26.3 g of ⁇ -benzyl-L-glutamic acid-N-carboxy anhydride was previously dissolved. 80 g of dichloromethane solvent was slowly added to the above solution, and the reaction was stirred under a nitrogen atmosphere at 25 ° C for 20 hours to cause ring-opening polymerization of ⁇ -benzyl-L-glutamic acid-N-carboxy anhydride by aliphatic amine.
  • the polymerization product is precipitated with anhydrous petroleum ether, and the product is washed several times with deionized water, and finally dried at a constant temperature of 25 ° C for 48 hours to obtain a polybenzyl benzyl ester intermediate product;
  • the polymer PLG obtained above was dissolved in N,N-dimethylformamide, and 5.63 g of cystamine hydrochloride and 5.1 g of triethylamine were added, and the reaction was further stirred at 30 ° C for 12 hours, and the mixture was stirred under magnetic stirring. Ultrapure water was added dropwise until the micelles appeared. The micelle solution was transferred to a dialysis bag for dialysis for 48 hours, and lyophilized to obtain pH/reduction responsive polyamino acid zwitterionic nanoparticles, which were labeled as CPLG-1 in Table 1.
  • Example 1 As in Example 1, except that the weight of the lysine was changed to 8.76 g, the remaining steps were unchanged, and the synthesis was carried out as CPLG-2 in Table 1.
  • Example 1 As in Example 1, except that the weight of the lysine was changed to 10.22 g, the remaining steps were unchanged, and the synthesis was carried out as CPLG-3 in Table 1.
  • Example 1 As in Example 1, except that the weight of the lysine was changed to 11.68 g, the remaining steps were unchanged, and the synthesis was carried out as CPLG-4 in Table 1.
  • Example 1 As in Example 1, the amount of ⁇ -benzyl-L-glutamic acid-N-carboxy anhydride was changed to 21.9 g, lysine was 8.03 g, cystamine hydrochloride 5.60 g, and triethylamine 5.07 g.
  • the synthesis was obtained as CPLG-5 in Table 1.
  • Hydration particle size and zeta potential of the nanoparticles the obtained dry nanoparticles were formulated into an aqueous solution having a concentration of 0.15 mg/mL, and the pH of the drug-loaded nanoparticle solution was adjusted using 0.1 M NaOH and 0.1 M HCl solution, and the difference was determined. The hydrated particle size and zeta potential of the drug nanoparticles were downloaded under pH conditions, and the test temperature was 25 ° C. The results are shown in Table 2.
  • the drug loading of the nanoparticles firstly, the anticancer drug doxorubicin or paclitaxel was dissolved in N,N-dimethylformamide to form a solution having a concentration of 2 mg/mL, and the nanoparticles of Examples 1 to 6 were prepared. 50 mg of the nanoparticles was dissolved in 5 mL of the above anticancer solution, stirred for 24 hours, transferred to a dialysis bag for dialysis for 12 hours, and lyophilized to obtain drug-loaded nanoparticles.
  • Reduction sensitivity of nanoparticles 5 mL of acetone solution with a concentration of 0.5 mg/L hydrazine was placed, 10 mL of 0.1 mg/mL of the nanoparticle solution prepared in Example 4 was added to the sample vial, and 100 L of 0.5 mg/capture was pipetted with a pipette. The acetone solution of L ⁇ was added to the sample bottle, and the mixture was shaken evenly. The solution was placed under normal temperature and normal pressure for 24 hours. After the acetone was completely evaporated, glutathione was added to make the final concentration to 10 mM, and then placed in a constant temperature oscillator at 37 ° C.
  • the polybenzyl benzyl ester intermediate was prepared according to the method of Example 1, the polybenzyl benzyl ester intermediate was dissolved in tetrahydrofuran, 7.3 g of L-lysine was added, and evenly mixed, cystamine hydrochloride was added. 5.63g and 5.1g of triethylamine were stirred at 30 ° C for 12 hours. Ultrapure water was added dropwise to the solution under magnetic stirring until the micelles appeared. The micelle solution was transferred to a dialysis bag for dialysis for 48 hours, freeze-dried. Nanoparticles are obtained. However, the zeta potential of the obtained nanoparticles was measured by the method of Example 7, and it was found that the obtained nanoparticles did not have pH responsiveness.

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Abstract

本发明提供了一种pH/还原响应性聚氨基酸两性离子纳米颗粒的制备方法，用脂肪胺引发γ-苄基-L-谷氨酸-N-羧酸酐的开环聚合，将所得到的聚谷氨酸苄酯与赖氨酸反应，形成两性离子聚合物，最后将上述得到的产物用胱胺交联，纯化后得到交联结构的pH/还原响应性聚氨基酸两性离子纳米颗粒。

Description

一种pH/还原响应性聚氨基酸两性离子纳米颗粒的制备方法 技术领域
本发明涉及一种pH/还原响应性聚氨基酸两性离子纳米颗粒的制备方法，属于高分子合成及生物医用材料技术领域。
背景技术
癌症被认为是致使人死亡的第一大杀手。目前治疗癌症的重要方法是化疗，但该法不仅对癌细胞有杀灭作用，对正常细胞的也有较大的毒副作用，且易被网状内皮系统进行清除，造成细胞吸收差。纳米药物载体体系能够增强渗透性和停留(EPR)效应，并能躲过人体的网状内皮质系统的识别和捕获，延长了载药系统在血液中的循环时间，提高了药物的生物利用度。
两性离子聚合物具有高度的水合能力和抗细菌黏附能力，可抵抗非特异性蛋白质吸附，有效规避巨噬细胞的摄取，实现在血液循环系统内的“隐形”功能，同时还可电荷翻转及化学修饰，因此有希望成为聚乙二醇的优秀替代品。目前文献报道制备的两性离子聚合物多数是非生物降解性的。这将产生一系列的问题，如聚合物降解的残余部分会导致药物的不完全释放以及产生不良的副反应。另一个问题是目前所存在的两性离子聚合物没有pH/还原响应性，难以控制药物在肿瘤部位释放。
肿瘤细胞内的pH和还原性环境特殊，肿瘤细胞内呈微酸性，所含谷胱甘肽的浓度比在体液或身体其他组织内高100到1000倍，对于双硫键具有较强的还原作用。因此设计环境响应性的药物载体，利用肿瘤部位的pH和还原性环境进行药物控释，成为一种有效方法。
聚谷氨酸具有较好的生物相容性，是可生物降解的聚合物，聚L-谷氨酸及 其衍生物在体内的降解缓慢，作为药物载体可延长药物在体内的循环时间，有助于药物的缓释和靶向给药。聚谷氨酸侧链含有γ-羧基，能够与氨基发生酰胺化反应。
赖氨酸是人体必需氨基酸之一，在制备药物载体时也经常用到。聚赖氨酸侧链有游离氨基和羧基，分别带正、负电荷，可以形成两性离子。文献报道的以L-赖氨酸为基体制备纳米颗粒的方法是复杂的且所需时间较长，大多只具有酸敏感性，没有pH/还原响应性，导致药物释放量较少。
曾经有文件报道：直接将天然产物聚谷氨酸用胱胺交联，合成纳米微凝胶，但是天然产物聚谷氨酸分子量分布宽，杂质多，纳米颗粒粒径不易控制，而且通过羧基与氨基的直接酰胺化反应速率慢，反应程度难控制。还有报道聚(N-异丙基丙烯酰胺-co-聚乙二醇)-聚(L-谷氨酸-γ-苄酯)嵌段共聚物形成的纳米颗粒，虽然有温度响应性，但是没有pH/还原响应性，纳米颗粒的稳定性受温度影响较大。另外有报道聚赖氨酸与聚谷氨酸的嵌段共聚物纳米颗粒，但是所用迈克尔反应合成时间长，产率低。
发明内容
为解决这些问题，本发明的目的是提供一种pH/还原响应性聚氨基酸两性离子纳米颗粒的制备方法，首先通过开环聚合合成聚谷氨酸主链，然后通过侧链基团反应引入赖氨酸为两性离子、最后交联合成纳米颗粒。该纳米颗粒由于两性离子的存在，所以具有抗非特异性蛋白质吸附性和pH响应性，由于胱胺含有二硫键，因此纳米颗粒有敏感的还原响应性。纳米颗粒可以负载抗癌药物，在肿瘤靶向部位控制释放。
本发明的第一个目的是提供一种pH/还原响应性聚氨基酸两性离子纳米颗粒的制备方法，该制备方法包括如下步骤：
步骤1：采用引发剂引发γ-苄基-L-谷氨酸-N-羧酸酐的开环聚合反应，得到聚谷氨酸苄酯中间产物；
步骤2：将上述得到的聚谷氨酸苄酯中间产物与赖氨酸反应，形成两性离 子聚合物；
步骤3：将上述得到的两性离子聚合物用胱胺交联，纯化后得到交联结构的pH/还原响应性聚氨基酸两性离子纳米颗粒。
在本发明的一种实施方式中，在步骤1中，引发剂为脂肪胺。
在本发明的一种实施方式中，所述脂肪胺为n-戊胺、n-己胺、n-庚胺、n-辛胺、环戊胺、环己胺或苄基胺。
在本发明的一种实施方式中，在步骤1中，以二氯甲烷、氯仿或二氯乙烷作为溶剂。
在本发明的一种实施方式中，在步骤1中，引发剂与γ-苄基-L-谷氨酸-N-羧酸酐的摩尔比为1:25～30。
在本发明的一种实施方式中，在步骤2中，赖氨酸与γ-苄基-L-谷氨酸-N-羧酸酐的摩尔比为0.5～0.8:1。
在本发明的一种实施方式中，在步骤1中，反应条件为，在氮气保护下，在20-30℃搅拌反应15-24小时。
在本发明的一种实施方式中，在步骤2中，反应条件为，在20-30℃下搅拌反应10-15小时。
在本发明的一种实施方式中，在步骤3中，两性离子聚合物用胱胺交联具体包括以下步骤：向两性离子聚合物中添加含有缚酸剂的胱胺盐酸盐溶液，在20-40℃下搅拌反应10-15小时；在搅拌下，向所得反应溶液中滴加水直至胶束出现，将胶束溶液透析40-60小时，得到pH/还原响应性聚氨基酸两性离子纳米颗粒。
在本发明的一种实施方式中，胱胺盐酸盐与γ-苄基-L-谷氨酸-N-羧酸酐的摩尔比为0.25～0.1:1。
在本发明的一种实施方式中，按摩尔数计，缚酸剂是胱胺盐酸盐的1～3倍。
在本发明的一种实施方式中，所述方法具体步骤为：
步骤1：将引发剂溶解，添加γ-苄基-L-谷氨酸-N-羧酸酐，在氮气保护下，在20-30℃搅拌反应15-24小时，使脂肪胺引发γ-苄基-L-谷氨酸-N-羧酸酐的开 环聚合反应，得到聚谷氨酸苄酯中间产物；
步骤2：将上述步骤1的中间产物溶解，加入L-赖氨酸，在20-30℃下搅拌反应10-15小时，得到两性离子聚合物；
步骤3：将上述步骤2得到两性离子溶解，添加含有缚酸剂的胱胺盐酸盐溶液，在20-40℃下继续搅拌反应10-15小时；在搅拌下，向反应溶液中滴加水直至胶束出现，将胶束溶液透析40-60小时，冷冻干燥得到pH/还原响应性聚氨基酸两性离子纳米颗粒。
在本发明的一种实施方式中，在步骤1中，将脂肪胺溶解在二氯甲烷中，添加γ-苄基-L-谷氨酸-N-羧酸酐的二氯甲烷溶液，在氮气保护下在20-30℃搅拌反应15-24小时，使脂肪胺引发γ-苄基-L-谷氨酸-N-羧酸酐的开环聚合，用无水石油醚沉淀聚合反应产物，用水洗涤产物数遍后真空20-30℃恒温干燥40-50h，得到聚谷氨酸苄酯中间产物。
在本发明的一种实施方式中，γ-苄基-L-谷氨酸-N-羧酸酐的二氯甲烷溶液浓度为16-20w％。
在本发明的一种实施方式中，在步骤2中，将步骤1得到的中间产物溶解于四氢呋喃，加入L-赖氨酸，在20-30℃下搅拌反应10-15小时，用无水石油醚沉淀聚合物，真空20-30℃恒温干燥，得到两性离子聚合物。
在本发明的一种实施方式中，在步骤3中，将步骤2得到的两性离子溶于N,N-二甲基甲酰胺，添加胱胺盐酸盐和三乙胺溶液，在20-40℃下继续搅拌反应10-15小时，在磁力搅拌下向溶液中滴加超纯水直至胶束出现，将胶束溶液转入透析袋中透析40-60小时，冷冻干燥得到pH/还原响应性聚氨基酸两性离子纳米颗粒。
本发明的第二个目的是提供所述方法制备得到的pH/还原响应性聚氨基酸两性离子纳米颗粒。
本发明的第三个目的是提供所述的pH/还原响应性聚氨基酸两性离子纳米颗粒在制备药物载体中的应用，所述应用是将药物溶解，将所述pH/还原响应性聚氨基酸两性离子纳米颗粒溶于上述药物溶液中，搅拌透析后冷冻干燥，得 到负载药物的纳米颗粒。
在本发明的一种实施方式中，所述药物为阿霉素或紫杉醇。
本发明的有益效果：
1.第一步中选用疏水性小分子脂肪胺引发γ-苄基-L-谷氨酸-N-羧酸酐化合物的开环聚合制备聚谷氨酸-苄酯，反应平稳、产物结构规整、分子量及其纳米颗粒尺寸可控、性能稳定，可提升纳米颗粒的性能。
2.本发明第二步中采用小分子赖氨酸与聚谷氨酸苄酯的侧链基团反应，此反应不可逆，通过投料比就可以控制基团转化率，使产品性能更加可控；并且本发明的反应简单，操作容易。
3.选用疏水性脂肪胺引发γ-苄基-L-谷氨酸-N-羧酸酐的开环聚合，增加了聚合物在水溶液中自组装的趋势，更易形成纳米颗粒。
4.所有原料生物相容性好，无细胞毒性，对人体安全无害，产物具有应用前景。
5.将胱胺中的二硫键引入交联结构，使纳米颗粒有敏感的还原响应性；赖氨酸的两性离子结构使纳米颗粒具有pH响应性。有利于负载抗癌药物，在肿瘤靶向部位控制释放。
附图说明
图1是本发明的pH/还原响应性聚氨基酸两性离子纳米颗粒的制备原理反应式；
图2是本发明的pH/还原响应性聚氨基酸两性离子纳米颗粒的透射电镜图，a:CPLG-1；b:CPLG-2；c:CPLG-3；d:CPLG-4；e:CPLG-5；f:CPLG-6；
图3是本发明的芘增溶纳米颗粒CPLG-1经10mM GSH处理后在37℃下孵育不同时间的荧光发射光谱图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人 员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
实施例1
单体制备：在500mL烧瓶中加入80g L-谷氨酸和65g苯甲醇，慢慢升温至70℃，磁力搅拌混合均匀，逐滴加入50％硫酸86g，减压蒸馏，将反应液倒入NaHCO 3溶液中和至中性，在4℃温度下冷却静止，减压过滤，用乙醇和蒸馏水洗涤产物3遍，然后在70℃下和5％的乙醇溶液中重结晶，4℃冷却静止过夜，最后在35℃下真空干燥48h得白色片状固体，为L-谷氨酸-γ-苄酯。
在盛有120mL四氢呋喃的三口烧瓶中加入L-谷氨酸-苄酯12.2g，水浴下逐渐升温至40℃，使苄酯溶解，将三聚光气17.0g添加到反应溶液中，在氮气保护下反应5h得澄清透明溶液，继续通氮气驱赶反应产生的HCl气体和剩余光气，将反应液缓慢滴加到过量石油醚中，冰浴冷却，减压抽滤分离白色针状晶体，用四氢呋喃/无水石油醚提纯产物，在35℃下真空干燥后得到γ-苄基-L-谷氨酸-N-羧酸酐；
开环聚合：在三口烧瓶中置入无水二氯甲烷40g，加入庚胺0.383g溶解在二氯甲烷中，预先将γ-苄基-L-谷氨酸-N-羧酸酐26.3g溶于80g二氯甲烷溶剂中，缓慢加入到上述溶液中，在氮气保护下、25℃下搅拌反应20小时，使脂肪胺引发γ-苄基-L-谷氨酸-N-羧酸酐的开环聚合，用无水石油醚沉淀聚合反应产物，用去离子水洗涤产物数遍，最后真空25℃恒温干燥48h，得到聚谷氨酸苄酯中间产物；
聚谷氨酸苄酯的侧链取代反应：将上述得到的聚谷氨酸苄酯溶解于四氢呋喃，加入7.3g L-赖氨酸，在25℃下搅拌反应12小时，用无水石油醚沉淀聚合物，真空25℃恒温干燥，产物标记为PLG；
将上述得到聚合物PLG溶于N,N-二甲基甲酰胺，添加胱胺盐酸盐5.63g和三乙胺5.1g，在30℃下继续搅拌反应12小时，在磁力搅拌下向溶液中滴加超纯水直至胶束出现，将胶束溶液转入透析袋中透析48小时，冷冻干燥得到pH/还原响应性聚氨基酸两性离子纳米颗粒，标记为表1中的CPLG-1。
实施例2
如同实施例1，但是改变赖氨酸的重量为8.76g，其余步骤不变，合成得到标记为表1中的CPLG-2。
实施例3
如同实施例1，但是改变赖氨酸的重量为10.22g,其余步骤不变，合成得到标记为表1中的CPLG-3。
实施例4
如同实施例1，但是改变赖氨酸的重量为11.68g,其余步骤不变，合成得到标记为表1中的CPLG-4。
实施例5
如同实施例1，但是改变γ-苄基-L-谷氨酸-N-羧酸酐的量为21.9g，赖氨酸为8.03g，胱胺盐酸盐5.60g,三乙胺5.07g。合成得到标记为表1中的CPLG-5。
实施例6
如同实施例1，但是改变胱胺盐酸盐2.25g,三乙胺5.10g。合成得到标记为表1中的CPLG-6。
表1 pH/还原响应性聚氨基酸两性离子纳米颗粒的制备配方
Figure PCTCN2018118862-appb-000001
实施例7
纳米颗粒的水合粒径和Zeta电位：将所得干燥纳米颗粒配制成浓度为0.15mg/mL的水溶液，采用0.1M的NaOH和0.1M的HCl溶液对载药纳米颗粒溶液的pH进行调节，测定不同pH条件下载药纳米颗粒的水合粒径和Zeta电位，测试温度为25℃，结果见表2。
表2纳米颗粒的表征及其性能
Figure PCTCN2018118862-appb-000002
实施例8
纳米颗粒的载药性：首先将抗癌药物阿霉素或紫杉醇用N,N-二甲基甲酰胺溶解，形成浓度为2mg/mL的溶液，再制备实施例1～6的纳米颗粒，取该纳米颗粒50mg溶于5mL上述抗癌溶液中，搅拌24小时后转入透析袋透析12小时，冷冻干燥得到负载药物的纳米颗粒。将干燥后的纳米颗粒10mg用DMSO溶解，超声0.5h，后将其定容至10mL，用紫外分光光度计在483nm测定阿霉素的吸光度。利用标准曲线，测定载药纳米颗粒载药率，计算公式如下：
LC(％)＝(W L/W N)×100
式中W L—纳米颗粒中负载药物的质量，mg；W N—载药纳米颗粒样品的质量，mg；测试结果见表2。
实施例9
纳米颗粒的透射电镜图：将纳米颗粒溶液20μL滴到铜网上，然后滴加一滴1.0wt％磷钨酸溶液进行染色，铜网在室温下自然干燥，用透射电子显微镜在高真空状态下观察不同配方制备的纳米颗粒的形貌，透射电镜加速电压为120kV，结果见图2。
实施例10
纳米颗粒的还原敏感性：配置5mL浓度为0.5mg/L芘的丙酮溶液，向样品瓶中加入10mL 0.1mg/mL的实施例4制备的纳米颗粒溶液，用移液枪移取100L 0.5mg/L芘的丙酮溶液加进样品瓶，振荡均匀，常温常压下敞口放置24h， 待丙酮全部挥发完加入谷胱甘肽，使其最终浓度为10mM，随后放在恒温振荡器中37℃下孵育，用荧光分光光度计测定溶液在不同时间点的荧光光谱图，发射波长设定在395nm，狭缝宽度设为5cm。测试结果见图3，芘是一种具有荧光性质的疏水性分子，可以很好地被增溶到纳米颗粒内，一旦疏水性环境遭到破坏，芘会泄露到水中，引起其荧光强度的变化。
对比例1
按照实施例1的方法制备得到聚谷氨酸苄酯中间产物，将所述聚谷氨酸苄酯中间产物溶解于四氢呋喃，加入7.3g L-赖氨酸，混合均匀后添加胱胺盐酸盐5.63g和三乙胺5.1g，在30℃下搅拌反应12小时，在磁力搅拌下向溶液中滴加超纯水直至胶束出现，将胶束溶液转入透析袋中透析48小时，冷冻干燥得到纳米颗粒。但是按照实施例7的方法测定得到的纳米颗粒的Zeta电位，发现得到的纳米颗粒不具有pH响应性。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。

Claims (10)

  1. 一种pH/还原响应性聚氨基酸两性离子纳米颗粒的制备方法，其特征在于，包括如下步骤：
    步骤1：采用引发剂引发γ-苄基-L-谷氨酸-N-羧酸酐的开环聚合反应，得到聚谷氨酸苄酯中间产物；
    步骤2：将上述得到的聚谷氨酸苄酯中间产物与赖氨酸反应，形成两性离子聚合物；
    步骤3：将上述得到的两性离子聚合物用胱胺交联，得到交联结构的pH/还原响应性聚氨基酸两性离子纳米颗粒。
  2. 根据权利要求1所述的方法，其特征在于，在步骤1中，所述引发剂为脂肪胺，所述脂肪胺为n-戊胺、n-己胺、n-庚胺、n-辛胺、环戊胺、环己胺或苄基胺。
  3. 根据权利要求1所述的方法，其特征在于，在步骤2中，按摩尔数计,所述赖氨酸的添加量为γ-苄基-L-谷氨酸-N-羧酸酐的50％～80％。
  4. 根据权利要求1所述的方法，其特征在于，在步骤1中，聚合反应在氮气保护下进行，反应温度为20-30℃，反应时间为15-24小时。
  5. 根据权利要求1所述的方法，其特征在于，在步骤2中，反应温度为20-30℃，反应时间为10-15小时。
  6. 根据权利要求1所述的方法，其特征在于，在步骤3中，两性离子聚合物用胱胺交联包括:
    向两性离子聚合物中添加含有缚酸剂的胱胺盐酸盐溶液，在20-40℃下反应10-15小时；及
    在搅拌下，向所得反应液中滴加水直至胶束出现，将胶束溶液透析40-60小时，得到pH/还原响应性聚氨基酸两性离子纳米颗粒。
  7. 根据权利要求6所述的方法，其特征在于，按摩尔数计，所述胱胺盐酸盐的添加量为γ-苄基-L-谷氨酸-N-羧酸酐的10％～25％。
  8. 根据权利要求6所述的方法，其特征在于，按摩尔数计，所述缚酸剂是胱胺盐酸盐的1～3倍。
  9. 一种根据权利要求1～8中任一项所述方法制备得到的pH/还原响应性聚氨基酸两性离子纳米颗粒。
  10. 权利要求9所述的pH/还原响应性聚氨基酸两性离子纳米颗粒在制备药物载体中的应用，其特征在于，所述应用是先将药物溶解，然后将pH/还原响应性聚氨基酸两性离子纳米颗粒溶于上述药物溶液中，搅拌透析后冷冻干燥，得到负载药物的纳米颗粒。
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