WO2021043231A1 - 一种双靶向材料及其在药物传递中的应用 - Google Patents

一种双靶向材料及其在药物传递中的应用 Download PDF

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WO2021043231A1
WO2021043231A1 PCT/CN2020/113360 CN2020113360W WO2021043231A1 WO 2021043231 A1 WO2021043231 A1 WO 2021043231A1 CN 2020113360 W CN2020113360 W CN 2020113360W WO 2021043231 A1 WO2021043231 A1 WO 2021043231A1
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tyrosine
dual
targeting
targeting material
liposomes
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王永军
王振杰
何仲贵
刘洪卓
孙进
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沈阳药科大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/331—Polymers modified by chemical after-treatment with organic compounds containing oxygen
    • C08G65/332—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing carboxyl groups, or halides, or esters thereof
    • C08G65/3322—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing carboxyl groups, or halides, or esters thereof acyclic
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33303—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing amino group
    • C08G65/3331—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing amino group cyclic
    • C08G65/33313—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing amino group cyclic aromatic

Definitions

  • the invention belongs to the field of new excipients and new dosage forms for pharmaceutical preparations, and specifically relates to a novel amphiphilic dual-targeting functional material and its application as a targeting material in an active targeting drug delivery system.
  • Tumors need high-nutrient support to maintain rapid growth and metastasis, so tumor cells generally highly express nutrient transporters, such as glucose transporters and amino acid transporters.
  • the amino acid transporters are divided into many types, such as glutamine transporters, large and medium amino acid transporters (LAT1), ATB 0,+ amino acid transporters.
  • the LAT1 transporter is encoded by the SLC7A5 gene on human chromosome 16.
  • Human LAT1 is a membrane protein composed of 507 amino acids with a relative molecular weight of 55kD, composed of 12 transmembrane units.
  • LAT1 is a non-sodium ion-dependent transporter that mainly transports neutral amino acids with large molecular weight.
  • the ATB 0,+ transporter is encoded by the human SLC6A14 gene and contains 642 amino acids with a molecular weight of 72kD amino acid transporter.
  • the ATB 0,+ transporter is sodium ion and chloride ion dependent, and it mainly mediates the transmembrane transport of basic and neutral amino acids and some amino acid derivatives such as nitric oxide synthase inhibitors and carnitine.
  • Human tumors are heterogeneous. For example, they are all breast cancer cells.
  • MCF-7 has high expression of LAT1 and ATB 0,+
  • MDA-MB-231 has high expression of LAT1 and ATB 0,+ low expression
  • T47D has low LAT1. Expression and ATB 0,+ high expression. Therefore, it is difficult for a single targeted agent to completely kill the tumor, and it is necessary to improve the targeting efficiency to offset the tumor heterogeneity, so materials and agents with dual targeting functions have attracted much attention.
  • the purpose of the present invention is to provide a method that can target both the LAT1 transporter and the ATB 0,+ transporter, with active tumor targeting, which can not only assemble to form micelles by itself, but also be modified in liposomes and nanometers.
  • the second purpose of the present invention is to provide the amphiphilic targeting functional carrier material modified nano-formulation, which simultaneously targets the LAT1 transporter and the ATB 0,+ transporter to achieve targeted delivery of active drugs.
  • the present invention provides an amphipathic tumor targeting functional carrier material, which can target both the LAT1 transporter highly expressed by tumor cells and the ATB 0,+ transporter.
  • the targeted functional carrier material uses A as the hydrophobic end, polyethylene glycol (PEG) and Linker in the middle, and L-tyrosine as the biological target head.
  • PEG polyethylene glycol
  • Linker in the middle
  • L-tyrosine L-tyrosine
  • A is C8-C22 fatty acid (such as stearic acid, palmitic acid, palmitic acid), cholesterol, various phosphatidylethanolamines, such as distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE) , Di-erucylphosphatidylethanolamine (DEPE), dimyristoylphosphatidylethanolamine (DMPE), dioleoylphosphatidylethanolamine (DOPE), etc.
  • DSPE distearoylphosphatidylethanolamine
  • DPPE dipalmitoylphosphatidylethanolamine
  • DEPE Di-erucylphosphatidylethanolamine
  • DMPE dimyristoylphosphatidylethanolamine
  • DOPE dioleoylphosphatidylethanolamine
  • the molecular weight of PEG is 100-10000.
  • Linker includes n Wherein R can be any group, preferably C1-C4 alkyl, C1-C4 alkoxy.
  • the present invention preferably has a targeted functional carrier material with the following general structure:
  • A is stearic acid or DSPE, the molecular weight of PEG is 500-5000, and the Linker is 0-10 CH 2 , preferably 2-10 CH 2 , more preferably 2-4 CH 2 .
  • the present invention also provides a method for preparing the targeted functional carrier material, which includes the following steps:
  • reaction formula is as follows:
  • the tyrosine-modified amphiphilic carrier material of the present invention has the function of simultaneously targeting LAT1 and ATB 0,+ and can be used to prepare nano-formulations and encapsulate anti-tumor drugs. It has good stability and slowness. Release characteristics and active tumor targeting characteristics. Experiments have proved that the transporter-targeted nano-formulations of the present invention have tumor targeting properties, and loading anti-tumor drugs can significantly improve the efficacy of chemotherapy.
  • the tyrosine-modified amphiphilic carrier material of the present invention can adopt active or passive drug loading mode to encapsulate antineoplastic drugs, and the drugs can be taxanes, camptothecins, and anthraquinones.
  • Antitumor drugs, dihydropyridines, non-steroidal anti-inflammatory drugs, genetic drugs, any substance or derivatives thereof; genetic drugs are DNA or siRNA.
  • Said nano preparations are emulsions, liposomes, polymer nanoparticles, inorganic nanoparticles, polymer micelles, nano lipid carriers and the like.
  • the present invention further provides the application of the tumor-targeting nano preparation in the preparation of anti-tumor drug preparations.
  • the present invention synthesizes tyrosine-modified amphiphilic carrier material, and uses it for the preparation of nano-formulations, which can make the nano-formulations target LAT1 and ATB 0,+ transporters at the same time, so it can effectively improve the drug in tumor tissues.
  • the distribution of medium while improving the efficacy of the drug, overcomes tumor heterogeneity, and achieves the effect of completely killing tumors in a large range, which has great application prospects.
  • Figure 1 is the 1 H-NMR spectrum of tyrosine polyethylene glycol monostearate in Example 1 of the present invention
  • Figure 2 is a DSPE-PEG-tyrosine 1 H-NMR spectrum in Example 1 of the present invention
  • Figure 3 is a transmission electron microscope image and a particle size image measured by dynamic light scattering of tyrosine dual-targeted liposomes in Example 2 of the present invention
  • FIG. 4 shows the expression of LAT1 and ATB 0,+ two amino acid transporters in different cells measured by Western-blot method in Example 3 of the present invention
  • Figure 5 shows the uptake of different target liposomes in the BxPC-3 cell line measured by flow cytometry in Example 5 of the present invention
  • Figure 6 is the accumulation of 6h and 24h drugs in tumors of nude mice in Example 6 of the present invention
  • Fig. 7 is a graph of tumor growth curve, body weight change, tumor-bearing rate, and tumor inhibition rate (TIR) graph of nude mice in Example 7 of the present invention
  • Figure 8 shows the values of alanine aminotransferase, aspartate aminotransferase, blood creatinine, and blood urea nitrogen of nude mice in different preparation groups in Example 7 of the present invention
  • Figure 9 is the pathological section results of heart, liver, spleen, lung and kidney tumors of nude mice in different preparation groups in Example 7 of the present invention
  • Figure 10 is a schematic diagram of liposomes in Example 2 with targetless liposomes and single-targeted liposomes as controls.
  • DSPC distearoyl phosphatidyl choline
  • the inner water phase is 0.25M triethylamine-sucrose octasulfate (pH5.0-6.0)
  • the outer water phase is 4.05mg/ml 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES)+8.42mg/ml chlorine Sodium.
  • HEPES 4-hydroxyethylpiperazine ethanesulfonic acid
  • single-targeted liposomes are liposomes modified with glutamate target head (only targeted to LAT1) and liposomes modified with lysine target head (only targeted to ATB 0,+ ), double Targeting agents are liposomes modified with glutamate and lysine target heads and liposomes modified with tyrosine target heads (both target LAT1 and ATB 0,+ )
  • the particle size of liposomes modified with tyrosine target head is about 130nm, PDI is 0.050, and Zeta potential is negative.
  • the liposome encapsulation efficiency was measured by Sephadex G-50 column chromatography. The liposome encapsulation efficiency was all above 90%, and the drug-to-lipid ratio was 0.33.
  • a Hitachi HT7700 transmission electron microscope was used to characterize the appearance of the liposomes, with uniform particle size and a round surface.
  • the logarithmic growth phase of human pancreatic cancer cells BxPC-3, human breast cancer cells MCF-7 and mouse embryonic fibroblasts NIH/3T3 were buried in a 96-well plate at 3000 cells/well/0.1ml of DMEM medium. After culturing for 12 hours in the cell incubator, add the drug-loaded liposomes prepared in Example 2 to each well at different dilution concentrations, add 0.2ml of liposome-containing solution to each hole, 6 parallel holes for each concentration, and place in the cell incubator Incubate.
  • Table 1 The IC50 values of different preparations in BXPC-3, MCF-7, NIH/3T3 cells at different times
  • O non-target liposome
  • G glutamate target liposome
  • L lysine target liposome
  • GL glutlysine target liposome
  • T tyrosine target liposome Liposomes
  • the human pancreatic cancer cell BxPC-3 in the logarithmic growth phase was buried in a 12-well plate with 300,000 cells/well/1ml of DMEM medium, and placed in a cell incubator for 24 hours.
  • the drug-loaded liposome prepared in Example 2 Dilute with culture medium and add 50 ⁇ g/ml drug loading concentration to each well, add 1ml liposome-containing cell culture medium to each well, 3 parallel wells for each group of preparations, incubate in a cell culture incubator for 12h, 24h, then discard The culture solution was washed 3 times with cold PBS to stop the uptake, then the cells were digested with trypsin, centrifuged at 1000 rpm for 5 minutes to discard the supernatant, and 300 ⁇ l PBS was added to redistribute the cell pellet, and then put it into a flow tube after passing through a 200-mesh cell sieve. Use flow cytometry to detect the amount of drug uptake in the cells.
  • BxPC-3 cells were connected to the armpits of Balb/c-nu male nude mice, and when the tumors of the nude mice grew to about 500 mm 3 , they were administered in groups, the dosage was 20 mg/kg, and the commercially available preparation Onivyde was injected into the tail vein. Glutamic acid target, lysine target, glutlysine target, and tyrosine target liposomes. Nude mice were sacrificed after 6h and 24h respectively. Heart, liver, spleen, lung and kidney tumors were dissected and weighed 200 mg tissue scissors.
  • BxPC-3 cells were connected to the armpits of Balb/c-nu male nude mice, and when the tumors of the nude mice grew to about 200mm 3 , they were administered in groups.
  • the dosage was 10 mg/kg, and the commercially available preparation Onivyde was injected into the tail vein.
  • Glutamic acid target, lysine target, glutlysine target, tyrosine target liposome, tumor volume and nude mouse body weight were measured every two days, and the tail vein was administered every five days.
  • nude mice were sacrificed on the 18th day, the heart, liver, spleen, lung and kidney tumor vesicles were cut out and fixed in 4% paraformaldehyde, followed by pathological section studies.
  • mice Before the nude mice were sacrificed, the eyes were removed and blood was taken to determine the difference in liver and kidney function of the mice in different preparation groups, and the concentrations of alanine aminotransferase, aspartate aminotransferase, creatinine, and urea nitrogen in serum were determined.
  • the dual-targeted formulation has a significantly better tumor-inhibiting effect than the single-targeted formulation than the commercially available formulation, and the tyrosine target formulation has the best efficacy.
  • Figure 8 shows that there is no difference in liver and kidney function between different preparation groups, which proves that the preparation does not cause liver and kidney damage in nude mice.
  • Figure 9 shows the results of pathological slices of the heart, liver, spleen, lung and kidney tumors of nude mice in different preparation groups. There is no significant difference between the heart, liver, spleen, lung and kidney of the dual-targeted preparation group and the control group.
  • the tumor slices show that the tyrosine target liposome group has the fewest nuclei and tumors The tissue is loose and the gap is large, and it also shows the excellent anti-tumor effect of the dual-targeting preparation group.

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Abstract

一种属于药物制剂新辅料和新剂型领域的新型两亲性双靶向功能材料，及其作为靶向材料在主动靶向药物传递系统中的应用。所述的两亲性靶向材料的结构通式如下：其中，A，Linker如权利要求和说明书所述。所述的两亲性靶向材料以酪氨酸为靶头，经化学修饰后，该靶向材料可以自组装形成胶束也可以修饰到脂质体、纳米粒表面，作为抗肿瘤药物靶向传递的载体。该材料通过表面修饰的酪氨酸能同时与肿瘤细胞膜上高表达的大中型氨基酸转运体1(LAT1)和氨基酸转运体ATB 0,+相互作用，有效提高纳米制剂的细胞摄取和抗肿瘤活性。

Description

一种双靶向材料及其在药物传递中的应用 技术领域
本发明属于药物制剂新辅料和新剂型领域，具体涉及一种新型两亲性双靶向功能材料，及其作为靶向材料在主动靶向药物传递系统中的应用。
背景技术
肿瘤维持快速增长和转移需要高营养物质的支持，所以肿瘤细胞一般高表达营养性转运体，如葡萄糖转运体，氨基酸转运体。而氨基酸转运体又分为很多种，如谷氨酰胺转运体，大中型氨基酸转运体(LAT1)，ATB 0,+氨基酸转运体。其中LAT1转运体是由人类第16号染色体上的SLC7A5基因所编码。人类LAT1是由507个氨基酸组成相对分子量为55kD的膜蛋白，由12个跨膜单元构成。LAT1是一种非钠离子依赖的，以转运分子质量较大的中性氨基酸为主的转运蛋白。ATB 0,+转运体是由人类SLC6A14基因编码，含有642个氨基酸分子量为72kD的氨基酸转运蛋白。ATB 0,+转运体是钠离子和氯离子依赖的，主要介导碱性和中性氨基酸及一些氨基酸衍生物如一氧化氮合酶抑制剂和肉毒碱的跨膜转运。而人类肿瘤具有异质性，比如都是乳腺癌细胞，MCF-7中LAT1和ATB 0,+均高表达，MDA-MB-231中LAT1高表达和ATB 0,+低表达，T47D中LAT1低表达和ATB 0,+高表达。所以单一的靶向制剂很难完全杀死肿瘤，需要提高靶向效率来抵消肿瘤异质性，从而具有双靶向功能的材料及制剂备受关注。
发明内容
本发明的目的在于提供一种既能靶向到LAT1转运体又能靶向到ATB 0,+转运体，具有肿瘤主动靶向，既可以自身组装形成胶束又可以修饰在脂质体、纳米粒表面的两亲性靶向功能载体材料。
本发明的第二个目的在于提供该两亲性靶向功能载体材料修饰的纳米制剂，同时靶向LAT1转运体和ATB 0,+转运体而实现活性药物的靶向传递。
本发明的技术方案如下：
本发明提供了一种两亲性肿瘤靶向功能载体材料，既可以靶向肿瘤细胞高表达的LAT1转运体又可以靶向ATB 0,+转运体。
所述的靶向功能载体材料以A为疏水端，中间侨联聚乙二醇(PEG)和Linker，以L-酪氨酸作为生物靶头。结构通式如下：
Figure PCTCN2020113360-appb-000001
其中A为C8-C22脂肪酸(如硬脂酸、软脂酸、棕榈酸)，胆固醇，各种磷脂酰乙醇胺，如二硬脂酰磷脂酰乙醇胺(DSPE)、二棕榈酰磷脂酰乙醇胺(DPPE)、二芥酰基磷脂酰乙醇胺(DEPE)、二肉豆蔻酰基磷脂酰乙醇胺(DMPE)、二油酰磷脂酰乙醇胺(DOPE)等。
PEG的分子量为100-10000。
Linker包括n个
Figure PCTCN2020113360-appb-000002
其中R可以为任意基团，优选为C1-C4烷基、C1-C4烷氧基。
本发明优选具有如下通式结构的靶向功能载体材料：
Figure PCTCN2020113360-appb-000003
A为硬脂酸或DSPE，PEG分子量为500-5000，Linker为0-10个CH 2，优选为2-10个CH 2，更优选为2-4个CH 2。
本发明还提供了所述靶向功能载体材料的制备方法，包括如下步骤：
(1)以聚乙二醇单硬脂酸酯为原料，先连上丁二酸，再用丁二酸另一端的羧基与L-酪氨酸的酚羟基进行连接，再脱去酪氨酸的保护基得终产物。反应式如下：
Figure PCTCN2020113360-appb-000004
(2)以DSPE-PEG2000-COOH为原料，直接与酪氨酸的酚羟基进行反应，再脱去酪氨酸的保护基得终产物。反应式如下：
Figure PCTCN2020113360-appb-000005
本发明的酪氨酸修饰的两亲性载体材料，具有同时靶向LAT1和ATB 0,+双靶向的功能，可以用于制备纳米制剂，包载抗肿瘤药物，其稳定性好，具有缓释特性和肿瘤主动靶向特性。实验证明本发明的转运体靶向纳米制剂均具备肿瘤靶向性，装载抗肿瘤药物能显著地提高化疗疗效。
本发明所述的酪氨酸修饰的两亲性载体材料可以采用主动或被动载药方式的包载抗肿瘤药物，所述的药物可以为：紫杉烷类、喜树碱类、蒽醌类抗肿瘤药 或二氢吡啶类、非甾体抗炎药、基因类药物中的任一物质或其衍生物；基因类药物为DNA或siRNA。
所述的纳米制剂为乳剂、脂质体、聚合物纳米粒、无机纳米粒、聚合物胶束、纳米脂质载体等。
本发明进一步提供了所述肿瘤靶向纳米制剂在制备抗肿瘤药物制剂中的应用。
本发明具有以下有益效果：
本发明合成了酪氨酸修饰的两亲性载体材料，将其用于纳米制剂的制备，可使得纳米制剂能同时靶向到LAT1和ATB 0,+转运体，因此可有效提高药物在肿瘤组织中的分布，在提高药效的同时克服肿瘤异质性，达到大范围彻底杀灭肿瘤的效果，具有很大的应用前景。
附图说明
图1为本发明实施例1中酪氨酸聚乙二醇单硬脂酸酯 1H-NMR谱图
图2为本发明实施例1中DSPE-PEG-酪氨酸 1H-NMR谱图
图3为本发明实施例2中酪氨酸双靶向脂质体的透射电镜图和动态光散射测得的粒径图
图4为本发明实施例3中Western-blot法测得的不同细胞LAT1和ATB 0,+两种氨基酸转运体的表达情况
图5为本发明实施例5中用流式细胞仪测定的不同靶头脂质体在BxPC-3细胞株中的摄取情况
图6为本发明实施例6中6h和24h药物在裸鼠肿瘤中的蓄积量
图7为本发明实施例7中裸鼠肿瘤生长曲线图、裸鼠体重变化图、荷瘤率、肿瘤抑制率(TIR)图
图8为本发明实施例7中不同制剂组裸鼠谷丙转氨酶、谷草转氨酶、血肌酐、血尿素氮值
图9为本发明实施例7中不同制剂组裸鼠心肝脾肺肾肿瘤病理切片结果
图10为实施例2中以无靶头脂质体和单靶向脂质体作为对照的脂质体示意图。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将发明限制在所述的实施例范围之中。
实施例1
制备靶向功能材料
(1)酪氨酸聚乙二醇单硬脂酸酯靶向材料合成
称量聚乙二醇单硬脂酸酯(PEG分子量2000)10.2g，丁二酸1.2g，1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDCI)1.05g，4-二甲基吡啶(DMAP)0.65g，N,N-二异丙基乙胺(DIEA)915μl，溶于30ml无水N,N-二甲基甲酰胺(DMF)中，30℃搅拌过夜，之后旋蒸除去DMF，用二氯甲烷复溶，分别加蒸馏水，5％柠檬酸，饱和碳酸氢钠溶液，饱和氯化钠溶液洗去多余的丁二酸和催化剂，用无水硫酸钠干燥后旋干得产物1进行下一步反应。
称量8.64g产物1，3.24g苄酯保护的酪氨酸，0.84g EDCI，0.52g DMAP，732μl DIEA，溶于20ml二氯甲烷中，30℃搅拌反应24h，分别用5％柠檬酸，饱和碳酸氢钠溶液，饱和氯化钠溶液洗去催化剂，加无水硫酸钠除水，过硅胶柱分离纯化产物2。流动相先用二氯甲烷：甲醇为100:1比例除去小极性杂质，再用二氯甲烷：甲醇为50:1比例分离产物2。
称量3.3g产物2，溶于15ml四氢呋喃中，加入10％钯碳加氢催化剂0.8g，在氢气存在的情况下30℃搅拌过夜，过滤除去钯碳，过硅胶柱分离纯化产物3。流动相先用二氯甲烷：甲醇为50:1比例除去小极性杂质，再用二氯甲烷：甲醇为10:1比例分离产物3。合成路线如下所示：
Figure PCTCN2020113360-appb-000006
采用核磁共振 1H-NMR氢谱来确定实施例1中的酪氨酸聚乙二醇单硬脂酸酯结构，选用溶剂为氘代氯仿，结果如图1。8.1ppm为酪氨酸上氨基的峰，3.6ppm为PEG的特征峰，1.25ppm为硬脂酸-CH 2特征峰，证明酪氨酸聚乙二醇单硬脂酸酯成功合成。
(2)DSPE-PEG-酪氨酸靶向材料的合成
称量100mg羧基封端的DSPE-PEG2000，40mg苄酯保护的酪氨酸，10mg EDCI，6mg DMAP，90μl DIEA，溶于10ml二氯甲烷中，30℃搅拌反应12h，用5％柠檬酸洗去催化剂，然后过硅胶柱进行分离纯化。先用二氯甲烷：甲醇＝100:1的比例除去小极性物质，再用二氯甲烷：甲醇＝50:1分离产物。将得到的产物纯品和20mg 10％钯碳加氢催化剂溶于10ml四氢呋喃中，在氢气存在的情况下30℃搅拌过夜，过滤除去钯碳，过硅胶柱分离纯化产物。先用二氯甲烷：甲醇＝50:1的比例除去小极性物质，再用二氯甲烷：甲醇＝10:1分离最终产物。合成路线如下所示：
Figure PCTCN2020113360-appb-000007
采用核磁共振 1H-NMR氢谱来确定实施例1中的DSPE-PEG-Tyr结构，选用溶剂为氘代氯仿，结果如图2。8.1ppm为酪氨酸上氨基的峰，3.6ppm为PEG的特征峰，1.25ppm为DSPE中-CH 2特征峰，证明DSPE-PEG-Tyr成功合成。
实施例2
制备双靶向脂质体
称量68.1mg二硬脂酰磷脂酰胆碱(DSPC)、22.2mg胆固醇，18mg酪氨酸聚乙二醇单硬脂酸酯，利用薄膜分散法制备脂质体。其内水相为0.25M三乙胺-蔗糖八硫酸酯(pH5.0-6.0)，外水相为4.05mg/ml 4-羟乙基哌嗪乙磺酸(HEPES)+8.42mg/ml氯化钠。再称量4mg伊立替康粉末与1ml脂质体于70℃孵育1小时，冷却15分钟，即得载药脂质体。为了能更好的体现酪氨酸修饰的双靶向脂质体的优势，我们又以同样的方法做了无靶头脂质体和单靶向脂质体作为对照。脂质体如图10所示。
其中单靶向脂质体为谷氨酸靶头修饰的脂质体(只能靶向到LAT1)和赖氨酸靶头修饰的脂质体(只能靶向到ATB 0,+)，双靶向制剂为谷氨酸和赖氨酸靶头混合修饰的脂质体和酪氨酸靶头修饰的脂质体(既能靶向LAT1又能靶向ATB 0,+)
如图3所示，使用马尔文粒径电位仪测量脂质体粒径，酪氨酸靶头修饰的脂质体粒径在130nm左右，PDI为0.050，Zeta电位为负值。用葡聚糖凝胶G-50柱层析测脂质体包封率，脂质体包封率均在90％以上，药脂比为0.33。用Hitachi  HT7700透射电子显微镜表征了脂质体外貌，粒径均一，表面圆整。
实施例3
Western blot测转运体表达量
将对数生长期的人胰腺癌细胞BxPC-3，人乳腺癌细胞MCF-7，小鼠胚胎成纤维细胞NIH/3T3用细胞刮刀刮下，冷PBS洗一次收集细胞沉淀，加入200μl RIPA强裂解液(含1mM PMSF)冰浴条件下用移液枪吹打20次，100W探头超声1min，冰上静置30min，4℃，12000rpm，离心10min，取上清，采用BCA蛋白定量试剂盒进行蛋白浓度测定后，加入电泳上样缓冲液，稀释至蛋白浓度1μg/μl，煮沸5min使蛋白充分变性，每个孔道吸取20μl上述蛋白溶液，加入10％PAGE凝胶中，150V恒压电泳60min，裁胶，覆盖上提前用甲醇泡过的0.45μm PVDF膜，250mA恒流转膜60min。之后用5％脱脂奶粉室温封闭1h，TBST洗三次后加入内参蛋白和目标蛋白一抗，室温孵育2h，TBST洗三次后加入HRP羊抗兔二抗室温孵育1h，TBST洗三次后加入ECL发光液，用BIO-RAD ChemiDoc TM XRS+进行仪器显影。
结果如图4所示，MCF-7与BxPC-3细胞中LAT1与ATB 0,+转运体均高表达作为阳性细胞进行后续的实验，NIH/3T3两个转运体均低表达，作为阴性对照细胞进行后续的实验。
实施例4
细胞毒性实验
将对数生长期的人胰腺癌细胞BxPC-3，人乳腺癌细胞MCF-7和小鼠胚胎成纤维细胞NIH/3T3以3000个/孔/0.1ml的DMEM培养液埋于96孔板中，放细胞培养箱培养12h后将实施例2制备的载药脂质体以不同稀释浓度加入各孔，每孔加入0.2ml含脂质体溶液，每个浓度6个平行孔，置于细胞培养箱中孵育。培养48h、72h、96h后，取出96孔板，每孔加入20μl的5mg/ml噻唑蓝，培养箱中继续孵育4h，然后倒出板中溶液，每孔加入200μl二甲基亚砜，置于振荡器上震摇10min后用酶标仪测定各孔在490nm处的吸光度，计算IC 50值。
表1 不同制剂在BXPC-3,MCF-7,NIH/3T3细胞中不同时间的IC50值
Figure PCTCN2020113360-appb-000008
O:无靶头脂质体,G:谷氨酸靶头脂质体,L:赖氨酸靶头脂质体,GL:谷赖氨酸靶头脂质体,T:酪氨酸靶头脂质体
MTT法测定载伊立替康脂质体细胞毒性结果如表1所示，不同浓度载药纳米粒作用于BxPC-3和MCF-7细胞株48h、72h、96h后，细胞抑制率随药物浓度和孵化时间增加而增大，并且对细胞的抑制作用双靶向脂质体强于单靶向脂质体强于无靶头脂质体，酪氨酸靶头脂质体的细胞毒性最强。而在NIC/3T3阴性对照细胞中，单靶向、双靶向与市售脂质体并没有表现出明显的差异，证明细胞毒性的增强依赖LAT1和ATB 0,+转运体的高表达。
实施例5
细胞摄取实验
将对数生长期的人胰腺癌细胞BxPC-3以30万个/孔/1ml的DMEM培养液埋于12孔板中，放细胞培养箱培养24h后将实施例2制备的载药脂质体用培养液稀释后以50μg/ml的载药浓度加入各孔，每孔加入1ml含脂质体细胞培养液，每组制剂3个平行孔，置于细胞培养箱中孵育12h、24h后弃去培养液并用冷PBS洗3次终止摄取，然后用胰酶将细胞消化下来，1000rpm离心5min弃去上清，加300μl PBS重新分散细胞沉淀，过200目细胞筛网后装入流式管中，用流式细胞仪检测细胞中摄取药量。
结果如图5所示，细胞对脂质体的摄取呈时间依赖性，24h摄取量明显大于12h摄取量，而且不同制剂摄取量为双靶向制剂大于单靶向制剂大于市售制剂。证明双靶向脂质体确实增加了细胞摄取。
实施例6
组织分布实验
将BxPC-3细胞接于Balb/c-nu雄性裸鼠腋下，待裸鼠肿瘤长到约500mm 3时进行分组给药，给药量为20mg/kg，分别尾静脉注射市售制剂Onivyde，谷氨酸靶头，赖氨酸靶头，谷赖氨酸靶头，酪氨酸靶头脂质体，分别于6h，24h后处死裸鼠，剖出心肝脾肺肾肿瘤，称200mg组织剪碎放入EP管中加1ml生理盐水，10000rpm进行组织匀浆，之后3500rpm离心10min，取上清100μl加入400μl甲醇涡旋2min充分提取，再13000rpm离心10min，取200μl上清加入黑色96孔板中，用酶标仪测量荧光，激发波长368nm，发射波长426nm。
结果如图6所示，在肿瘤部位24h的蓄积量明显大于6h，证明脂质体有很好的缓释效果，双靶头脂质体大于单靶头脂质体大于市售脂质体，证明经过酪氨酸配体修饰明显增加了肿瘤部位伊立替康药物的蓄积量。
实施例7
药效实验
将BxPC-3细胞接于Balb/c-nu雄性裸鼠腋下，待裸鼠肿瘤长到约200mm 3时进行分组给药，给药量为10mg/kg，分别尾静脉注射市售制剂Onivyde，谷氨酸靶头，赖氨酸靶头，谷赖氨酸靶头，酪氨酸靶头脂质体，每两天量一次肿瘤体积和裸鼠体重，每五天尾静脉给药一次。给四次药后，于第18天处死裸鼠，剖出心肝脾肺肾肿瘤泡在4％多聚甲醛中进行组织固定，继而进行后续的病理切片研究。处死裸鼠前进行摘眼球取血以测定不同制剂组老鼠的肝肾功能差异，分别测定血清中谷丙转氨酶、谷草转氨酶、肌酐、尿素氮的浓度。
药效结果如图7所示，在没有明显系统毒性的前提下，双靶向制剂抑制肿瘤效果明显优于单靶向制剂优于市售制剂且酪氨酸靶头制剂药效最好。图8显示不同制剂组肝肾功能没有差异，证明制剂并不造成裸鼠肝肾损伤。图9为不同制剂组裸鼠心肝脾肺肾肿瘤病理切片结果，双靶向制剂组心肝脾肺肾与对照组并没有明显差异，肿瘤切片显示酪氨酸靶头脂质体组细胞核最少而且肿瘤组织疏松空隙大，也显示出双靶向制剂组优秀的抗肿瘤效果。

Claims (10)

  1. 一种双靶向材料，分子结构中以酪氨酸为靶头，结构通式如下：
    Figure PCTCN2020113360-appb-100001
    A是C8-C22脂肪酸，胆固醇，各种磷脂酰乙醇胺，
    Linker为n个
    Figure PCTCN2020113360-appb-100002
    其中，n＝0-10，R为任意基团，优选为C1-C4烷基、C1-C4烷氧基。
  2. 如权利要求1所述的双靶向材料，其特征在于，所述的各种磷脂酰乙醇胺为二硬脂酰磷脂酰乙醇胺、二棕榈酰磷脂酰乙醇胺、二芥酰基磷脂酰乙醇胺、二肉豆蔻酰基磷脂酰乙醇胺、二油酰磷脂酰乙醇胺。
  3. 如权利要求1所述的双靶向材料，其特征在于，PEG分子量为100-10000。
  4. 如权利要求1所述的双靶向材料，其特征在于，A为硬脂酸或DSPE，PEG分子量为500-5000。
  5. 如权利要求1所述的双靶向材料，其特征在于，n＝2-10，优选为2-4。
  6. 如权利要求1所述的双靶向材料，其特征在于，其结构式为：
    Figure PCTCN2020113360-appb-100003
    A为硬脂酸或DSPE，PEG分子量为500-5000，Linker为2-10个CH 2，更优选为2-4个CH 2。
  7. 如权利要求1所述的双靶向材料的制备方法，其特征在于：
    以聚乙二醇单硬脂酸酯为原料，先连上丁二酸，再用丁二酸另一端的羧基与L-酪氨酸的酚羟基进行连接，再脱去酪氨酸的保护基即得；
    或以DSPE-PEG2000-COOH为原料，直接与酪氨酸的酚羟基进行反应，再脱去酪氨酸的保护基即得。
  8. 权利要求1-6任何一项所述的双靶向材料在制备抗肿瘤药物中的应用。
  9. 权利要求1-6任何一项所述的双靶向材料在制备靶向纳米制剂中的应用。
  10. 如权利要求9所述的应用，其特征在于，所述的纳米制剂为脂质体、聚合物纳米粒、无机纳米粒、聚合物胶束、纳米脂质载体。
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WANG ZHENJIE; CHI DONGXU; WU XINGCHEN; WANG YINGLI; LIN XINXIN; XU ZHAOCHU; LIU HONGZHUO; SUN JIN; HE ZHONGGUI; WANG YONGJUN: "Tyrosine modified irinotecan-loaded liposomes capable of simultaneously targeting LAT1 and ATB0,+ for efficient tumor therapy", JOURNAL OF CONTROLLED RELEASE, ELSEVIER, AMSTERDAM, NL, vol. 316, 30 October 2019 (2019-10-30), AMSTERDAM, NL, pages 22 - 33, XP085944496, ISSN: 0168-3659, DOI: 10.1016/j.jconrel.2019.10.037 *

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