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CN101926775B - Preparation and application methods of difunctional naonparticle preparation entrapping vincristine sulphate - Google Patents

️Wed Sep 28 2011

Preparation and application methods of difunctional naonparticle preparation entrapping vincristine sulphate Download PDF

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Publication number

CN101926775B

CN101926775B CN2010102747321A CN201010274732A CN101926775B CN 101926775 B CN101926775 B CN 101926775B CN 2010102747321 A CN2010102747321 A CN 2010102747321A CN 201010274732 A CN201010274732 A CN 201010274732A CN 101926775 B CN101926775 B CN 101926775B Authority

China

Prior art keywords

plga

peg

preparation

vincristine sulfate

modified

Prior art date

2010-09-07

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Expired - Fee Related

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CN2010102747321A

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Chinese (zh)

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CN101926775A (en

Inventor

沈琦

李绍顺

陈家念

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Shanghai Jiao Tong University

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Shanghai Jiao Tong University

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2010-09-07

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2010-09-07

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2011-09-28

2010-09-07 Application filed by Shanghai Jiao Tong University filed Critical Shanghai Jiao Tong University

2010-09-07 Priority to CN2010102747321A priority Critical patent/CN101926775B/en

2010-12-29 Publication of CN101926775A publication Critical patent/CN101926775A/en

2011-09-28 Application granted granted Critical

2011-09-28 Publication of CN101926775B publication Critical patent/CN101926775B/en

Status Expired - Fee Related legal-status Critical Current

2030-09-07 Anticipated expiration legal-status Critical

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AQTQHPDCURKLKT-JKDPCDLQSA-N vincristine sulfate Chemical compound OS(O)(=O)=O.C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C=O)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 AQTQHPDCURKLKT-JKDPCDLQSA-N 0.000 title claims abstract description 53
238000002360 preparation method Methods 0.000 title claims abstract description 31
238000000034 method Methods 0.000 title claims abstract description 15
239000002105 nanoparticle Substances 0.000 claims abstract description 54
OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 claims abstract description 45
229920006022 Poly(L-lactide-co-glycolide)-b-poly(ethylene glycol) Polymers 0.000 claims abstract description 34
229960002110 vincristine sulfate Drugs 0.000 claims abstract description 31
230000001588 bifunctional effect Effects 0.000 claims abstract description 30
235000019152 folic acid Nutrition 0.000 claims abstract description 23
239000011724 folic acid Substances 0.000 claims abstract description 23
OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 claims abstract description 22
229960000304 folic acid Drugs 0.000 claims abstract description 22
108010051109 Cell-Penetrating Peptides Proteins 0.000 claims abstract description 16
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238000004945 emulsification Methods 0.000 claims abstract description 6
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238000009472 formulation Methods 0.000 claims abstract description 3
HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 20
239000000243 solution Substances 0.000 claims description 15
QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 14
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NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 9
239000004372 Polyvinyl alcohol Substances 0.000 claims description 8
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125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 7
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QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 6
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229920001223 polyethylene glycol Polymers 0.000 claims description 4
FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 claims description 3
FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 3
238000003756 stirring Methods 0.000 claims description 3
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BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical group CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 claims description 2
LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 2
OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 claims description 2
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WZVXLJYENHHFPD-UHFFFAOYSA-N methylaminomethanol;hydrochloride Chemical compound Cl.CNCO WZVXLJYENHHFPD-UHFFFAOYSA-N 0.000 claims 1
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OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 22
210000004027 cell Anatomy 0.000 description 9
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YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical group ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
206010028980 Neoplasm Diseases 0.000 description 5
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210000000170 cell membrane Anatomy 0.000 description 5
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VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 4
230000000259 anti-tumor effect Effects 0.000 description 4
239000002502 liposome Substances 0.000 description 4
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210000004881 tumor cell Anatomy 0.000 description 4
WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
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KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 2
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CWGFSQJQIHRAAE-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol tetrahydrochloride Chemical compound Cl.Cl.Cl.Cl.OCC(N)(CO)CO CWGFSQJQIHRAAE-UHFFFAOYSA-N 0.000 description 1
208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 1
208000014697 Acute lymphocytic leukaemia Diseases 0.000 description 1
241000208327 Apocynaceae Species 0.000 description 1
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FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
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238000000692 Student's t-test Methods 0.000 description 1
229930006000 Sucrose Natural products 0.000 description 1
CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
102000004243 Tubulin Human genes 0.000 description 1
108090000704 Tubulin Proteins 0.000 description 1
241000863480 Vinca Species 0.000 description 1
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ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
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QAOWNCQODCNURD-UHFFFAOYSA-L sulfate group Chemical group S(=O)(=O)([O-])[O-] QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
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OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 1

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Abstract

一种医药技术领域的包载硫酸长春新碱的双功能纳米粒制剂的制备方法，通过复乳法将硫酸长春新碱包封于叶酸/细胞穿透肽修饰的PLGA-PEG聚合物载体中，制得双功能纳米粒制剂。本发明在体内外表现出良好的药代动力学行为，制备的叶酸/细胞穿透肽修饰的PLGA-PEG双功能纳米粒粒径287.2±0.8nm，具有较高载药量和包封率，以及良好的稳定性。

A method for preparing a bifunctional nanoparticle preparation loaded with vincristine sulfate in the field of medical technology, encapsulating vincristine sulfate in a PLGA-PEG polymer carrier modified by folic acid/cell penetrating peptide by double emulsion method, A bifunctional nanoparticle formulation was prepared. The present invention exhibits good pharmacokinetic behavior in vivo and in vitro, and the prepared folic acid/cell penetrating peptide modified PLGA-PEG bifunctional nanoparticle has a particle size of 287.2±0.8nm, has a relatively high drug loading capacity and encapsulation efficiency, and good stability.

Description

包载硫酸长春新碱的双功能纳米粒制剂的制备和应用方法Preparation and application method of bifunctional nanoparticle preparation loaded with vincristine sulfate

技术领域technical field

本发明涉及一种医药技术领域的制剂及其制备和应用方法，具体是一种包载硫酸长春新碱的双功能纳米粒制剂的制备和应用方法。The invention relates to a preparation in the technical field of medicine and a preparation and application method thereof, in particular to a preparation and application method of a bifunctional nanoparticle preparation carrying vincristine sulfate.

背景技术Background technique

长春新碱为夹竹桃科植物长春花中提取的有效成分。其游离形式极不稳定，因此常以其硫酸盐形式(vincristine sulfate，VCR)存在。抗肿瘤作用靶点是微管，在细胞周期S期，VCR与微管蛋白结合，从而使中期细胞分裂停止，导致有丝分裂中的细胞群体明显增加，使细胞分裂停止于M期，因此是M期细胞周期特异性药物。还可干扰蛋白质代谢及抑制RNA多聚酶的活力，并抑制细胞膜类脂质的合成和氨基酸在细胞膜上的转运。临床上主要用于治疗急性淋巴细胞白血病、何杰金及非何杰金淋巴瘤，也用于乳腺癌、支气管肺癌、软组织肉瘤及神经母细胞等，但VCR对神经系统及注射局部正常组织刺激性大，剂量限制性毒性是神经系统毒性，在临床上需严格控制给药剂量，因而大大限制了其在临床上的使用。Vincristine is an active ingredient extracted from the vinca flower of the Apocynaceae plant. Its free form is extremely unstable, so it often exists in its sulfate form (vincristine sulfate, VCR). The target of anti-tumor effect is microtubules. In the S phase of the cell cycle, VCR binds to tubulin, thereby stopping the cell division in the metaphase, resulting in a significant increase in the cell population in mitosis, and stopping the cell division in the M phase, so it is the M phase Cell Cycle Specific Drugs. It can also interfere with protein metabolism and inhibit the activity of RNA polymerase, and inhibit the synthesis of cell membrane lipids and the transport of amino acids on the cell membrane. Clinically, it is mainly used for the treatment of acute lymphoblastic leukemia, Hodgkin's and non-Hodgkin's lymphoma, as well as for breast cancer, bronchial lung cancer, soft tissue sarcoma and neuroblastoma, etc. The dose-limiting toxicity is nervous system toxicity, and the dosage needs to be strictly controlled clinically, thus greatly limiting its clinical use.

诸多研究发现，相对于目前的VCR注射液，载VCR的脂质体可明显减轻VCR的毒副作用，如通过提高VCR对肿瘤细胞的被动靶向作用，减小对正常组织的损伤，从而达到提高抗肿瘤疗效的目的。但是这些脂质体缺乏对肿瘤细胞的主动靶向作用，因而毒副作用依然明显。叶酸受体是一种在多种癌细胞中广泛过量表达且能和叶酸分子高度识别并结合的物质。利用叶酸和叶酸受体特有的相互作用，人们将叶酸作为常用的靶向分子修饰在纳米尺寸的抗肿瘤药物载体表面，以期达到对肿瘤细胞的高度靶向作用，从而提高药物的抗肿瘤效果。本发明的目的之一即以叶酸作为靶向基团，修饰在PLGA-PEG纳米粒的表面，达到主动靶向的目的。然而，细胞膜作为细胞与环境进行物质交换的选择通透性屏障，在保证细胞内环境相对稳定，使各种生化反应能够有序运行的同时，却阻止了细胞外物质自由进入细胞，比如将抗肿瘤药物排斥在细胞膜外，是导致这类药物抗肿瘤疗效不佳的一个重要原因。为了解决这一难题，人们在给药系统中引入了细胞穿透肽。实验表明，在纳米粒给药系统的聚合物载体表面修饰以细胞穿透肽，如寡聚精氨酸后，可显著增强药物的吸收。虽然人们对细胞穿透肽如何携带纳米粒穿过细胞膜，进入细胞的机制还没有统一的认识，但是细胞穿透肽这种特殊的功能越来越引起了人们的重视。Many studies have found that compared with the current VCR injection, VCR-loaded liposomes can significantly reduce the toxic and side effects of VCR, for example, by improving the passive targeting effect of VCR on tumor cells and reducing the damage to normal tissues, thereby improving The purpose of antitumor efficacy. However, these liposomes lack the active targeting effect on tumor cells, so the toxic and side effects are still obvious. Folate receptor is a substance that is widely overexpressed in a variety of cancer cells and can highly recognize and bind to folic acid molecules. Taking advantage of the unique interaction between folic acid and folic acid receptors, people use folic acid as a commonly used targeting molecule to modify the surface of nano-sized anti-tumor drug carriers in order to achieve a high degree of targeting of tumor cells, thereby improving the anti-tumor effect of drugs. One of the objectives of the present invention is to use folic acid as a targeting group to modify the surface of PLGA-PEG nanoparticles to achieve the purpose of active targeting. However, the cell membrane acts as a selectively permeable barrier for the exchange of substances between cells and the environment. While ensuring the relative stability of the intracellular environment and enabling various biochemical reactions to run in an orderly manner, it prevents extracellular substances from freely entering the cell. The exclusion of tumor drugs outside the cell membrane is an important reason for the poor anti-tumor efficacy of these drugs. To solve this problem, cell-penetrating peptides have been introduced into the drug delivery system. Experiments have shown that modifying the surface of the polymer carrier of the nanoparticle drug delivery system with cell-penetrating peptides, such as oligoarginine, can significantly enhance drug absorption. Although there is no unified understanding of how cell-penetrating peptides carry nanoparticles across the cell membrane and into cells, the special function of cell-penetrating peptides has attracted more and more attention.

经过对现有技术的检索发现，有几种PEG化的硫酸长春新碱脂质体(Rodriguez M.A.，Pytlik R.，Kozak T.，et al.Cancer 2009，115：3475-3482；Thomas D.A.，Sarris A.H.，Cortes J.，et al.Cancer 2006，106：120-127)已进入临床前实验阶段，虽然该类脂质体在体内能达到长循环的效果，但是粒径较大，一般在500nm以上，有的粒子粒径高达几十乃至上百微米，而且粒径分布很宽。如此大的颗粒进入体内后，由于缺乏血管通透性，很容易直接被网状内皮系统吞噬而难以到达肿瘤部位，导致其靶向性不高，因而相对于传统的硫酸长春新碱注射液，疗效并无显著提高(Tokudome Y.，Oku N.，Doi K.，et al.Biochim.Biophys.Acta.1996，1279：70-74)。After searching the prior art, it was found that there are several PEGylated vincristine sulfate liposomes (Rodriguez M.A., Pytlik R., Kozak T., et al. Cancer 2009, 115:3475-3482; Thomas D.A., Sarris A.H., Cortes J., et al.Cancer 2006, 106:120-127) have entered the stage of preclinical experiments. Although this type of liposome can achieve the effect of long circulation in vivo, the particle size is relatively large, generally above 500nm , some particles have a particle size as high as tens or even hundreds of microns, and the particle size distribution is very wide. After such large particles enter the body, due to the lack of vascular permeability, they are easily swallowed by the reticuloendothelial system and difficult to reach the tumor site, resulting in low targeting. Therefore, compared with the traditional vincristine sulfate injection, The curative effect was not significantly improved (Tokudome Y., Oku N., Doi K., et al. Biochim. Biophys. Acta. 1996, 1279: 70-74).

发明内容Contents of the invention

本发明针对现有技术存在的上述不足，提供一种包载硫酸长春新碱的双功能纳米粒制剂的制备和应用方法，采用复乳法制备包载硫酸长春新碱的PLGA-PEG纳米粒，制备的叶酸/细胞穿透肽修饰的PLGA-PEG双功能纳米粒粒径287.2±0.8nm，具有较高载药量和包封率，以及良好的稳定性。与硫酸长春新碱水溶液，非修饰的PLGA-PEG纳米粒及叶酸修饰的PLGA-PEG纳米粒相比，该双功能纳米粒在体内外表现出良好的性能。因而，叶酸/细胞穿透肽修饰的PLGA-PEG双功能纳米载体是一种较为理想的药物载体，具有广阔的应用前景。The present invention aims at the above-mentioned deficiencies existing in the prior art, and provides a kind of preparation and application method of the bifunctional nanoparticle preparation of entrapped vincristine sulfate, adopts double emulsion method to prepare the PLGA-PEG nanoparticle of entrapped vincristine sulfate, The prepared folic acid/cell penetrating peptide modified PLGA-PEG bifunctional nanoparticles have a particle size of 287.2±0.8nm, high drug loading capacity and encapsulation efficiency, and good stability. Compared with vincristine sulfate aqueous solution, non-modified PLGA-PEG nanoparticles and folic acid-modified PLGA-PEG nanoparticles, the bifunctional nanoparticles showed good performance in vitro and in vivo. Therefore, the folic acid/cell penetrating peptide modified PLGA-PEG bifunctional nanocarrier is an ideal drug carrier and has broad application prospects.

本发明是通过以下技术方案实现的：The present invention is achieved through the following technical solutions:

本发明涉及一种包载硫酸长春新碱的双功能纳米粒制剂的制备方法，通过复乳法将硫酸长春新碱包封于叶酸/细胞穿透肽修饰的PLGA-PEG聚合物载体中，制得双功能纳米粒制剂。The invention relates to a preparation method of bifunctional nanoparticle preparations loaded with vincristine sulfate. Vincristine sulfate is encapsulated in folic acid/cell penetrating peptide modified PLGA-PEG polymer carrier by double emulsion method to prepare A bifunctional nanoparticle formulation was obtained.

所述的叶酸/细胞穿透肽修饰的PLGA-PEG聚合物载体由PLGA-mPEG、叶酸修饰的PLGA-PEG(PLGA-PEG-folate)以及七聚精氨酸修饰的PLGA-PEG(PLGA-PEG-R₇)组成。The PLGA-PEG polymer carrier modified by folic acid/cell penetrating peptide is composed of PLGA-mPEG, PLGA-PEG (PLGA-PEG-folate) modified by folic acid and PLGA-PEG (PLGA-PEG) modified by hepta-arginine -R ₇ ) composition.

所述的七聚精氨酸的化学式为R₇-NH₂，即末端羧基以氨基封闭。The chemical formula of the hepta-arginine is R ₇ -NH ₂ , that is, the terminal carboxyl group is blocked with an amino group.

所述的叶酸修饰的PLGA-PEG以及七聚精氨酸修饰的PLGA-PEG中，PEG的重均分子量1000～5000Da，PLGA的重均分子量8000～50000Da。In the folic acid-modified PLGA-PEG and hepta-arginine-modified PLGA-PEG, the weight-average molecular weight of PEG is 1000-5000 Da, and the weight-average molecular weight of PLGA is 8000-50000 Da.

所述的叶酸修饰的PLGA-PEG以及七聚精氨酸修饰的PLGA-PEG分别通过以下方式制备得到：从HO-PEG-OH出发，合成聚合物PEG二氨基化物(NH₂-PEG-NH₂)；以三氯甲烷为溶剂，N-羟基琥珀酰亚胺(NHS)为活化试剂，加入缩合剂，使NH₂-PEG-NH₂与PLGA-COOH轭合，得到PLGA-PEG-NH₂；将丁二酸酐(succinic anhydride)与PLGA-PEG-NH₂缩合，所得产物丁二酸修饰的PLGA-PEG(PLGA-PEG-succinate)，再与R₇-NH₂缩合，得到细胞穿透肽修饰的载体PLGA-PEG-R₇。以三氯甲烷为溶剂，NHS为活化试剂，加入缩合剂，使PLGA-PEG-NH₂与叶酸轭合，得到PLGA-PEG-folate。The folic acid-modified PLGA-PEG and the hepta-arginine-modified PLGA-PEG are respectively prepared in the following manner: starting from HO-PEG-OH, synthesizing polymer PEG diamide (NH ₂ -PEG-NH ₂ ); using chloroform as a solvent, N-hydroxysuccinimide (NHS) as an activation reagent, adding a condensing agent to conjugate NH ₂ -PEG-NH ₂ with PLGA-COOH to obtain PLGA-PEG-NH ₂ ; Condensate succinic anhydride with PLGA-PEG-NH ₂ to obtain succinic acid-modified PLGA-PEG (PLGA-PEG-succinate), and then condense with R ₇ -NH ₂ to obtain cell-penetrating peptide modification The vector PLGA-PEG-R ₇ . Using chloroform as a solvent and NHS as an activation reagent, a condensing agent was added to conjugate PLGA-PEG- _NH2 with folic acid to obtain PLGA-PEG-folate.

所述的PLGA-COOH、NHS以及缩合剂的摩尔比为1/5/5～1/10/10；The molar ratio of the PLGA-COOH, NHS and condensing agent is 1/5/5～1/10/10;

所述的复乳法是指：将硫酸长春新碱溶于三(羟甲基)氨基甲烷盐酸盐(Tris-HCl)缓冲液后，在超声冰浴条件下，滴加到含有PLGA-mPEG、PLGA-PEG-folate和PLGA-PEG-R₇的有机溶剂中得到初乳，在超声冰浴条件下，将初乳滴加至含PVA的Tris-HCl缓冲液中得到复乳，经过搅拌旋蒸后离心低温冻干，得到包载硫酸长春新碱的双功能纳米粒。The double-emulsion method refers to: after dissolving vincristine sulfate in tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) buffer solution, under the condition of ultrasonic ice bath, add dropwise to the mixture containing PLGA-mPEG , PLGA-PEG-folate and PLGA-PEG-R ₇ organic solvents to obtain colostrum, under the condition of ultrasonic ice bath, the colostrum was added dropwise to Tris-HCl buffer solution containing PVA to obtain double emulsion, after stirring and spinning After steaming, centrifuge and freeze-dry at low temperature to obtain bifunctional nanoparticles loaded with vincristine sulfate.

所述的缩合剂为N，N′-二异丙基碳二亚胺(DIC)，N，N′-二环己基碳二亚胺(DCC)或1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC·HCl)；The condensing agent is N, N'-diisopropylcarbodiimide (DIC), N, N'-dicyclohexylcarbodiimide (DCC) or 1-ethyl-(3-dimethyl Aminopropyl) carbodiimide hydrochloride (EDC HCl);

所述的硫酸长春新碱的投药量为4～9％(w/w)；The dosage of the vincristine sulfate is 4-9% (w/w);

所述的PLGA-PEG-folate的质量为PLGA-PEG-R₇质量的2倍，PLGA-mPEG的质量为PLGA-PEG-R₇质量的6～15倍；The quality of the PLGA-PEG-folate is twice that of PLGA-PEG-R ₇ , and the quality of PLGA-mPEG is 6 to 15 times that of PLGA-PEG- _R 7;

所述的Tris-HCl缓冲液的pH为5～7.4；The pH of the Tris-HCl buffer solution is 5-7.4;

所述的初乳中有机溶剂为二氯甲烷、三氯甲烷或乙酸乙酯；The organic solvent in the described colostrum is dichloromethane, chloroform or ethyl acetate;

所述的初乳中的Tris-HCl缓冲液与有机溶剂的体积比为1/10～1/30。The volume ratio of the Tris-HCl buffer solution in the colostrum to the organic solvent is 1/10˜1/30.

所述的含聚乙烯醇(PVA)的Tris-HCl缓冲液是指：含有0.6～2.5％PVA(w/v)且pH为5～7.4的Tris-HCl缓冲液；The Tris-HCl buffer solution containing polyvinyl alcohol (PVA) refers to: a Tris-HCl buffer solution containing 0.6-2.5% PVA (w/v) and having a pH of 5-7.4;

所述的离心低温冻干是指：以15000～50000rpm的转速离心处理30分钟后低温冻干；The centrifugal low-temperature freeze-drying refers to: low-temperature freeze-drying after being centrifuged at a speed of 15,000 to 50,000 rpm for 30 minutes;

所述的低温冻干是指：添加蔗糖或山梨醇D作为冻干保护剂。The low-temperature freeze-drying refers to adding sucrose or sorbitol D as a freeze-drying protective agent.

本发明涉及上述纳米粒的应用方法，通过将所述纳米粒的溶液放置于透析袋后置于释放介质中，经恒温摇床震荡实现药物释放。The present invention relates to the application method of the above-mentioned nanoparticle. The solution of the nanoparticle is placed in a dialysis bag and then placed in a release medium, and the drug release is realized by shaking on a constant temperature shaker.

所述的透析袋截留分子量为5000～10000Da；The molecular weight cut-off of the dialysis bag is 5000～10000Da;

所述的释放介质为磷酸盐缓冲液或Tris-HCl缓冲液；The release medium is phosphate buffer or Tris-HCl buffer;

所述的恒温是指37±0.5℃。The constant temperature refers to 37±0.5°C.

附图说明Description of drawings

图1聚合物PLGA-PEG-folate和PLGA-PEG-R₇的合成路线。Fig. 1 Synthetic routes of polymers PLGA-PEG-folate and PLGA-PEG-R ₇ .

图2聚合物PLGA-mPEG、PLGA-PEG-folate和PLGA-PEG-R₇的¹HNMR图谱。Fig. 2 ¹ H NMR spectra of polymers PLGA-mPEG, PLGA-PEG-folate and PLGA-PEG-R ₇ .

图3双功能纳米粒的扫描电镜图。Fig. 3 SEM images of bifunctional nanoparticles.

图4四种硫酸长春新碱剂型的体外释放行为(pH 6.8)：(A)在磷酸缓冲液中的释放；(B)在含有EDTA的磷酸缓冲液中的释放；(C)在Tris-HCl缓冲液中的释放。Figure 4 The in vitro release behavior (pH 6.8) of four vincristine sulfate dosage forms: (A) release in phosphate buffer; (B) release in phosphate buffer containing EDTA; (C) release in Tris-HCl release in buffer.

图5四种硫酸长春新碱剂型在大鼠体内的血药浓度-时间曲线。The plasma concentration-time curves of four kinds of vincristine sulfate dosage forms in the rat body of Fig. 5.

具体实施方式Detailed ways

下面对本发明的实施例作详细说明，本实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。The embodiments of the present invention are described in detail below. This embodiment is implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operating procedures are provided, but the protection scope of the present invention is not limited to the following implementation example.

实施例1Example 1

聚合物PLGA-PEG-folate和PLGA-PEG-R₇的合成，如图1所示，具体包括以下步骤：The synthesis of polymer PLGA-PEG-folate and PLGA-PEG-R ₇ , as shown in Figure 1, specifically includes the following steps:

1)从HO-PEG-OH出发，经过三步，合成聚合物NH₂-PEG-NH₂；1) Starting from HO-PEG-OH, through three steps, the polymer NH ₂ -PEG-NH ₂ is synthesized;

2)以三氯甲烷为溶剂，NHS为活化试剂，DIC为缩合剂，使NH₂-PEG-NH₂与PLGA-COOH轭合，得到PLGA-PEG-NH₂，以上合成过程见图1A；2) Using chloroform as a solvent, NHS as an activation reagent, and DIC as a condensation agent, conjugate NH ₂ -PEG-NH ₂ and PLGA-COOH to obtain PLGA-PEG-NH ₂ , the above synthesis process is shown in Figure 1A;

3)以三氯甲烷为溶剂，DIC为缩合剂，4-二甲氨基吡啶(DMAP)为催化剂，使丁二酸酐与PLGA-PEG-NH₂缩合，得到PLGA-PEG-succinate；3) using chloroform as a solvent, DIC as a condensing agent, and 4-dimethylaminopyridine (DMAP) as a catalyst to condense succinic anhydride and PLGA-PEG- _NH to obtain PLGA-PEG-succinate;

4)以DMF为溶剂，使末端羧基以氨基封闭的七聚精氨酸(R₇-NH₂)与PLGA-PEG-succinate反应，得到细胞穿透肽修饰的PLGA-PEG(PLGA-PEG-R₇)；¹HNMR见图2C。在第2)步反应中，PLGA-COOH/NHS/DIC的摩尔比为：1/5/5～1/10/10。第3)和4)步合成过程见图1B。4) Using DMF as a solvent, react the heptameric arginine (R ₇ -NH ₂ ) whose terminal carboxyl group is blocked with an amino group, with PLGA-PEG-succinate to obtain cell-penetrating peptide-modified PLGA-PEG (PLGA-PEG-R ₇ ); ¹ HNMR is shown in Figure 2C. In the second step reaction, the molar ratio of PLGA-COOH/NHS/DIC is: 1/5/5～1/10/10. The synthesis process of steps 3) and 4) is shown in Figure 1B.

5)聚合物PLGA-PEG-folate的合成采用与上述相似的方法：在得到PLGA-PEG-NH₂后，以三氯甲烷为溶剂，NHS为活化试剂，DIC为缩合剂，使PLGA-PEG-NH₂与叶酸轭合，即得产物；¹HNMR见图2B。5) The synthesis of polymer PLGA-PEG-folate adopts a method similar to the above: after obtaining PLGA-PEG-NH ₂ , use chloroform as a solvent, NHS as an activation reagent, and DIC as a condensation agent to make PLGA-PEG- NH ₂ is conjugated with folic acid to obtain the product; ¹ HNMR is shown in Figure 2B.

实施例2Example 2

叶酸/细胞穿透肽修饰的PLGA-PEG双功能纳米粒的制备，包括以下步骤：The preparation of the PLGA-PEG bifunctional nanoparticles modified by folic acid/cell penetrating peptide comprises the following steps:

(1)将硫酸长春新碱溶于pH(5～7.4)的Tris-HCl缓冲液中，配成浓溶液(称为相I)。将三种聚合物载体PLGA-mPEG，PLGA-PEG-folate和PLGA-PEG-R₇(质量比7/2/1)溶解于有机溶剂(二氯甲烷，或三氯甲烷，或乙酸乙酯)中(称为相II)。缓冲液与有机溶剂的比例为1/10～1/30。理论上硫酸长春新碱的投药量为4～9％(w/w)；(1) Dissolve vincristine sulfate in Tris-HCl buffer solution with pH (5-7.4) to form a concentrated solution (referred to as phase I). Dissolve the three polymer carriers PLGA-mPEG, PLGA-PEG-folate and PLGA-PEG-R ₇ (mass ratio 7/2/1) in an organic solvent (dichloromethane, or chloroform, or ethyl acetate) Middle (referred to as phase II). The ratio of the buffer solution to the organic solvent is 1/10-1/30. Theoretically, the dosage of vincristine sulfate is 4-9% (w/w);

(2)超声，冰水浴条件下，将相I滴加到相II中，得到初乳；(2) Ultrasound, under the condition of ice-water bath, add phase I dropwise to phase II to obtain colostrum;

(3)超声，冰水浴条件下，将上述初乳滴加到含0.6～2.5％PVA(w/v)，pH 5～7.4的Tris-HCl缓冲液中，得到w/o/w的复乳；(3) Ultrasound, under the condition of ice-water bath, add the above-mentioned colostrum dropwise to the Tris-HCl buffer solution containing 0.6-2.5% PVA (w/v), pH 5-7.4, to obtain w/o/w double emulsion ;

(4)室温下搅拌，然后旋转蒸发，除去复乳中有机溶剂；(4) stirring at room temperature, then rotary evaporation to remove the organic solvent in the double emulsion;

(5)对上述(4)得到的溶液进行高速离心(15000～50000rpm)30分钟，除去游离的VCR，得到带有蓝色乳光的纳米粒溶液；(5) Perform high-speed centrifugation (15000-50000 rpm) on the solution obtained in the above (4) for 30 minutes to remove free VCR and obtain a nanoparticle solution with blue opalescence;

(6)将纳米粒溶液低温冻干，即得包载硫酸长春新碱的双功能纳米粒NP3。用SEM检测，形貌见图3；(6) Freeze-drying the nanoparticle solution at low temperature to obtain bifunctional nanoparticle NP3 loaded with vincristine sulfate. Detected by SEM, the morphology is shown in Figure 3;

(7)采用相似的方法，制备PLGA-mPEG纳米粒NP1，叶酸修饰的PLGA-PEG纳米粒NP2。并对以上纳米粒粒径，Zeta电位，药物支载量和包封率进行表征。表1为所得到的结果。可见，所制备的三种纳米粒粒径在200～300nm之间，Zeta电位低于-15mV，显示了纳米粒良好的稳定性。(7) Using a similar method, prepare PLGA-mPEG nanoparticles NP1 and folic acid-modified PLGA-PEG nanoparticles NP2. And characterize the particle size, Zeta potential, drug loading capacity and encapsulation efficiency of the above nanoparticles. Table 1 shows the obtained results. It can be seen that the particle size of the prepared three kinds of nanoparticles is between 200-300nm, and the Zeta potential is lower than -15mV, showing the good stability of the nanoparticles.

表1Table 1

实施例3Example 3

叶酸/细胞穿透肽修饰的PLGA-PEG双功能纳米粒的释放Release of folate/cell-penetrating peptide-modified PLGA-PEG bifunctional nanoparticles

取双功能纳米粒的溶液0.5mL，置于透析袋(5000～10000Da)，于体积为30mL释放介质中(磷酸盐缓冲液或Tris-HCl缓冲液)，37℃恒温摇床震荡，定时取样用高效液相测定释放介质中VCR的含量，计算累计释放百分率。释放曲线见图4。Take 0.5mL of the bifunctional nanoparticle solution, put it in a dialysis bag (5000-10000Da), put it in a release medium (phosphate buffer or Tris-HCl buffer) with a volume of 30mL, shake it on a constant temperature shaker at 37°C, and use it for regular sampling. The content of VCR in the release medium was determined by high performance liquid chromatography, and the cumulative release percentage was calculated. The release curve is shown in Figure 4.

通过对样品在两种释放介质的释放性质的研究，结果表明，前8h，VCR在磷酸盐缓冲液(pH 6.8)出现突释；在8～20h，VCR呈现平稳释放；但是超过20h后，由于VCR在磷酸盐缓冲液中稳定性欠佳，浓度反而缓慢下降，累计释放随之下降(图4A)。但是向磷酸盐缓冲液中加入EDTA后，可显著提高VCR的稳定性，VCR在8～60h内可平稳释放(图4B)。当缓冲液换为Tris-HCl时，VCR也表现出良好的稳定性，前期出现突释，在接下来的时间内呈良好的释放特性。累计释放率达80％以上(图4C)。这说明VCR在不同的释放介质中，显示出不同的释放过程。Through the study of the release properties of the samples in two release media, the results showed that, in the first 8 hours, VCR appeared burst release in phosphate buffer (pH 6.8); in 8-20 hours, VCR showed a steady release; but after more than 20 hours, due to The stability of VCR in phosphate buffer was not good, but the concentration decreased slowly, and the cumulative release decreased accordingly (Figure 4A). However, adding EDTA to the phosphate buffer can significantly improve the stability of VCR, and VCR can be released smoothly within 8-60 hours (Fig. 4B). When the buffer was changed to Tris-HCl, VCR also exhibited good stability, with burst release in the early stage and good release characteristics in the following period. The cumulative release rate was over 80% (Fig. 4C). This shows that VCR exhibits different release processes in different release media.

实施例4Example 4

体外细胞毒实验In vitro cytotoxicity test

将不同浓度的NP3，NP1，NP2，VCR水溶液(F-VCR)分别与三种细胞株人白血病细胞毒，人乳腺癌细胞和人胃癌细胞株共孵化24h，然后用MTT检测细胞成活率，ORIGIN v8.0(OriginLab Corp.)软件计算其IC50值。Different concentrations of NP3, NP1, NP2, and VCR aqueous solution (F-VCR) were incubated with three cell lines of human leukemia cytotoxicity, human breast cancer cells and human gastric cancer cell lines for 24 hours, and then the cell survival rate was detected by MTT, ORIGIN The IC50 value was calculated by v8.0 (OriginLab Corp.) software.

表2为NP3，NP1，NP2，F-VCR体外对三种肿瘤细胞株的IC50值。比较得到，相同浓度下，三种纳米粒形式的VCR的细胞毒性均强于VCR水溶液；而且，双功能纳米粒NP3具有最低的IC₅₀值，表现出最强的细胞毒作用。Table 2 shows the IC50 values of NP3, NP1, NP2, and F-VCR against three tumor cell lines in vitro. By comparison, at the same concentration, the cytotoxicity of the three forms of VCR nanoparticles is stronger than that of VCR aqueous solution; moreover, the bifunctional nanoparticle NP3 has the lowest IC ₅₀ value, showing the strongest cytotoxicity.

表2Table 2

^*p＜0.05，采用t检验纳米粒组与F-VCR比较进行统计学分析；^**p＜0.01，采用t检验纳米粒组与F-VCR比较进行统计学分析。 ^* p<0.05, t test was used to compare the nanoparticle group with F-VCR for statistical analysis; ^** p<0.01, the t test was used for statistical analysis to compare the nanoparticle group with F-VCR.

实施例5Example 5

体内药代动力学评价In vivo pharmacokinetic evaluation

将16只SD大鼠(200～250g)按雌雄各半随机分为四组，尾静脉注射VCR纳米溶液(NP1，NP2，NP3)或F-VCR溶液，给药剂量0.6mg/kg(以VCR计)。分别于以下时刻：给药前，5，15，30，60，120，240和480min，眼眶采血0.25ml，肝素抗凝。全血经肝素抗凝，3000rpm离心15min分离血浆。乙腈沉淀处理后，HPLC检测。血药浓度-时间曲线如图5，体内药代动力学参数如表4。16 SD rats (200-250g) were randomly divided into four groups according to male and female half and half, and VCR nano solution (NP1, NP2, NP3) or F-VCR solution was injected into the tail vein at a dosage of 0.6 mg/kg (based on VCR count). At the following times: before administration, 5, 15, 30, 60, 120, 240 and 480 minutes, 0.25ml of orbital blood was collected and anticoagulated with heparin. The whole blood was anticoagulated with heparin, and the plasma was separated by centrifugation at 3000rpm for 15min. After acetonitrile precipitation treatment, HPLC detection. The plasma concentration-time curve is shown in Figure 5, and the pharmacokinetic parameters in vivo are shown in Table 4.

表4Table 4

^*p＜0.05，采用t检验纳米粒组与F-VCR比较进行统计学分析。 ^* p<0.05, statistical analysis was performed by t-test comparing the nanoparticle group with F-VCR.

由表4可见，纳米粒组与F-VCR的主要动力学参数AUC、C_max、T_max、MRT和等均具有显著性差异(P＜0.05)。虽然NP3，与NP1和NP2纳米粒溶液的药代动力学参数之间并没有显著性差异，但是，相对于F-VCR，这三种纳米粒溶液却表现出良好的药代动力学行为。在同等剂量下，由于VCR被包载于纳米粒内核，呈缓慢释放，因而纳米粒溶液的AUC和Cl明显低于对照制剂F-VCR，而分布相半衰期和消除相半衰期均明显长于F-VCR。这说明叶酸和细胞穿透肽修饰的双功能纳米粒NP3有着良好的开发前景。It can be seen from Table 4 that there are significant differences (P<0.05) in the main kinetic parameters AUC, C _max , T _max , MRT and so on between the nanoparticle group and F-VCR. Although there was no significant difference between the pharmacokinetic parameters of NP3, NP1 and NP2 nanoparticle solutions, these three nanoparticle solutions showed good pharmacokinetic behavior relative to F-VCR. At the same dose, since the VCR is entrapped in the inner core of the nanoparticle and released slowly, the AUC and Cl of the nanoparticle solution are significantly lower than those of the control preparation F-VCR, while the half-life of the distribution phase and the half-life of the elimination phase are significantly longer than those of the F-VCR . This shows that folic acid and cell penetrating peptide modified bifunctional nanoparticles NP3 has a good development prospect.

Claims (10)

1.一种包载硫酸长春新碱的双功能纳米粒制剂的制备方法，其特征在于，通过复乳法将硫酸长春新碱包封于叶酸/细胞穿透肽修饰的PLGA-PEG聚合物载体中，制得双功能纳米粒制剂。1. A preparation method of a bifunctional nanoparticle preparation loaded with vincristine sulfate, characterized in that, vincristine sulfate is encapsulated in the PLGA-PEG polymer carrier modified by folic acid/cell penetrating peptide by double emulsion method , prepared bifunctional nanoparticle formulations. 2.根据权利要求1所述的包载硫酸长春新碱的双功能纳米粒制剂的制备方法，其特征是，所述的叶酸/细胞穿透肽修饰的PLGA-PEG聚合物载体由PLGA-mPEG、叶酸修饰的PLGA-PEG以及七聚精氨酸修饰的PLGA-PEG组成。2. the preparation method of the bifunctional nanoparticle preparation of encapsulating vincristine sulfate according to claim 1, is characterized in that, the PLGA-PEG polymer carrier of described folic acid/cell penetrating peptide modification is made of PLGA-mPEG , PLGA-PEG modified by folic acid and PLGA-PEG modified by hepta-arginine. 3.根据权利要求1所述的包载硫酸长春新碱的双功能纳米粒制剂的制备方法，其特征是，所述的叶酸修饰的PLGA-PEG以及七聚精氨酸修饰的PLGA-PEG分别通过以下方式制备得到：从HO-PEG-OH出发，合成聚合物PEG二氨基化物；以三氯甲烷为溶剂，N-羟基琥珀酰亚胺为活化试剂，加入缩合剂，使聚合物PEG二氨基化物与PLGA-COOH轭合，得到PLGA-PEG-NH₂；将丁二酸酐与PLGA-PEG-NH₂缩合，所得产物丁二酸修饰的PLGA-PEG，再与R₇-NH₂缩合，得到七聚精氨酸修饰的PLGA-PEG；最后以三氯甲烷为溶剂，N-羟基琥珀酰亚胺为活化试剂，加入缩合剂，使PLGA-PEG-NH₂与叶酸轭合，得到叶酸修饰的PLGA-PEG。3. the preparation method of the bifunctional nanoparticle preparation of encapsulating vincristine sulfate according to claim 1, is characterized in that, the PLGA-PEG of described folic acid modification and the PLGA-PEG of hepta-arginine modification respectively It is prepared by the following method: starting from HO-PEG-OH, synthesizing polymer PEG diamide; using chloroform as solvent, N-hydroxysuccinimide as activation reagent, adding condensing agent to make polymer PEG diamino The compound is conjugated with PLGA-COOH to obtain PLGA-PEG-NH ₂ ; the succinic anhydride is condensed with PLGA-PEG-NH ₂ , and the resulting product succinic acid-modified PLGA-PEG is condensed with R ₇ -NH ₂ to obtain PLGA-PEG modified with hepta-arginine; finally, chloroform was used as solvent, N-hydroxysuccinimide was used as activation reagent, and condensing agent was added to conjugate PLGA-PEG- _NH2 with folic acid to obtain folic acid-modified PLGA-PEG. 4.根据权利要求3所述的包载硫酸长春新碱的双功能纳米粒制剂的制备方法，其特征是，所述的三氯甲烷、N-羟基琥珀酰亚胺以及缩合剂的摩尔比为1/5/5～1/10/10。4. the preparation method of the bifunctional nanoparticle preparation carrying vincristine sulfate according to claim 3 is characterized in that, the mol ratio of described chloroform, N-hydroxysuccinimide and condensing agent is 1/5/5～1/10/10. 5.根据权利要求3所述的包载硫酸长春新碱的双功能纳米粒制剂的制备方法，其特征是，所述的复乳法是指：将硫酸长春新碱溶于三(羟甲基)氨基甲烷盐酸盐缓冲液后，在超声冰浴条件下，滴加到含有叶酸/细胞穿透肽修饰的PLGA-PEG聚合物载体的有机溶剂中得到初乳，在超声冰浴条件下，将初乳滴加至含聚乙烯醇的三(羟甲基)氨基甲烷盐酸盐缓冲液中得到复乳，经过搅拌旋蒸后离心低温冻干，得到包载硫酸长春新碱的双功能纳米粒。5. the preparation method of the bifunctional nanoparticle preparation carrying vincristine sulfate according to claim 3 is characterized in that, described double emulsion method refers to: vincristine sulfate is dissolved in three (hydroxymethyl ) aminomethane hydrochloride buffer solution, under the condition of ultrasonic ice bath, drop into the organic solvent containing folic acid/cell penetrating peptide modified PLGA-PEG polymer carrier to obtain colostrum, under the condition of ultrasonic ice bath, The colostrum was added dropwise into tris(hydroxymethyl)aminomethane hydrochloride buffer solution containing polyvinyl alcohol to obtain double emulsion, and after stirring and rotary evaporation, it was centrifuged and freeze-dried at low temperature to obtain the bifunctional nano grain. 6.根据权利要求3所述的包载硫酸长春新碱的双功能纳米粒制剂的制备方法，其特征是，所述的缩合剂为N，N′-二异丙基碳二亚胺，N，N′-二环己基碳二亚胺或1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐。6. the preparation method of the bifunctional nanoparticle preparation of encapsulating vincristine sulfate according to claim 3, is characterized in that, described condensing agent is N, N '-diisopropyl carbodiimide, N , N'-dicyclohexylcarbodiimide or 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride. 7.根据权利要求1或5所述的包载硫酸长春新碱的双功能纳米粒制剂的制备方法，其特征是，所述的硫酸长春新碱的投药量为4～9％(w/w)；所述的叶酸修饰的PLGA-PE的质量为七聚精氨酸修饰的PLGA-PEG质量的2倍，PLGA-mPEG的质量为七聚精氨酸修饰的PLGA-PEG质量的6～15倍。7. according to the preparation method of the bifunctional nanoparticle preparation of entrapment vincristine sulfate described in claim 1 or 5, it is characterized in that, the dosage of described vincristine sulfate is 4～9% (w/w ); the quality of the PLGA-PE modified by folic acid is 2 times of the quality of PLGA-PEG modified by hepta-arginine, and the quality of PLGA-mPEG is 6～15 times of the quality of PLGA-PEG modified by hepta-arginine times. 8.根据权利要求5所述的包载硫酸长春新碱的双功能纳米粒制剂的制备方法，其特征是，所述的含聚乙烯醇的三(羟甲基)氨基甲烷盐酸盐缓冲液是指：含有0.6～2.5％聚乙烯醇(w/v)且pH为5～7.4的三(羟甲基)氨基甲烷盐酸盐缓冲液。8. the preparation method of the bifunctional nanoparticle preparation of entrapment vincristine sulfate according to claim 5 is characterized in that, described tris (hydroxymethyl) aminomethane hydrochloride buffer solution containing polyvinyl alcohol Refers to: Tris(hydroxymethyl)aminomethane hydrochloride buffer containing 0.6-2.5% polyvinyl alcohol (w/v) and pH 5-7.4. 9.一种包载硫酸长春新碱的双功能纳米粒制剂，其特征在于，根据上述任一权利要求所述方法制备得到。9. A bifunctional nanoparticle preparation loaded with vincristine sulfate, characterized in that it is prepared according to the method of any one of the preceding claims. 10.一种根据权利要求1至8种任一所述方法制备得到的双功能纳米粒制剂的释放液，其特征在于，通过将所述纳米粒的溶液放置于透析袋后置于释放介质中，经恒温摇床震荡得到。10. A release solution of a bifunctional nanoparticle preparation prepared according to any one of claims 1 to 8, wherein the solution of the nanoparticles is placed in a release medium after being placed in a dialysis bag , obtained by shaking on a constant temperature shaker.

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