WO2008011812A1 - Biomimetic cell membrane of phospholipid polymer, preparation method and use thereof - Google Patents
Biomimetic cell membrane of phospholipid polymer, preparation method and use thereof Download PDFInfo
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- WO2008011812A1 WO2008011812A1 PCT/CN2007/002197 CN2007002197W WO2008011812A1 WO 2008011812 A1 WO2008011812 A1 WO 2008011812A1 CN 2007002197 W CN2007002197 W CN 2007002197W WO 2008011812 A1 WO2008011812 A1 WO 2008011812A1
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- C—CHEMISTRY; METALLURGY
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- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F230/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal
- C08F230/02—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal containing phosphorus
Definitions
- the invention belongs to the technical field of polymer material membranes, and relates to a novel method for preparing tissue cell membranes and the application thereof in drug permeation.
- Bioavailability is one of the important indicators for measuring whether a drug can be developed into an effective clinical drug.
- the absorption of the drug in the human gut depends on three related properties: permeability, solubility, and i:. .
- permeability permeability
- solubility i:. .
- absorbable data can be used for in vitro activity analysis in early drug development. .
- tissue cell models have been widely used as tools for drug absorption research at home and abroad, including Caco-2 cell monolayer model, MDCK cell model and BBMEC model.
- the Caco 2 cell model is derived from human colon cancer cells and is a well-known model of intestinal absorption. The results of this model have a good correlation with drug disposal in vivo.
- the Caco-2 cell model has now been approved as an in vitro screening model for the oral absorption of small fractions of sub-drugs, which has been widely adopted by drug research institutions or development companies in developed countries. .
- the MDCK cell model is derived from the kidney cells of canines and can form a tight cell monolayer.
- the BBMEC model which became an important tool for blood-brain barrier research at the beginning of its establishment, is still being used today. It is the most commonly used in vitro model for simulating the blood-brain barrier.
- ⁇ is a solution film formation, It causes errors in some test tests; (3) ⁇ is a bimolecular membrane made of small molecule phospholipids, the stability of the membrane is not good, affecting the results of drug detection; (4) At present, lecithin membrane is mainly used to simulate intestinal tract Absorption performance, simulated cell membrane
- the invention aims to predict candidate drugs in the early stage of rational drug design, and eliminates some unsatisfactory drugs in time, thereby greatly reducing the cost of drug screening, and providing a novel in vitro simulated tissue cell membrane drug penetration model.
- the invention provides several synthetic phospholipid polymers of cell-like membrane structure, constructs a simulated tissue membrane model by polymer self-assembly, and is applied to the analysis of drug permeability, and provides an early evaluation and screening method for drug permeability in vitro. .
- the present invention relates a hydrophilic phospholipid group or a derivative thereof, such as lecithin, cephalin, inositol phospholipid, serine phospholipid or dipalmitoylphosphatidylcholine, to a terminal group containing an aliphatic or fluorocarbon hydrophobic chain having a double bond.
- a pH-sensitive micro-homogeneous structure capable of self-assembly is formed, which is similar to the biofilm.
- the phospholipid polymer introduces different polar phospholipid groups or their derivatives on the hydrophobic chain to establish a simulated cell membrane model of different target targets.
- Lecithin-based biomimetic membrane to establish a small intestine simulated cell membrane model cephalin-based biomimetic membrane to establish brain tissue mimic cell membrane model, inositol phospholipid-based biomimetic membrane to establish gastric tissue mimic cell membrane model or the regulation and combination of various phospholipid groups to simulate various human tissues and organs simulation Models such as skin, oral mucosa and other models.
- the phospholipid membrane fluidity is changed, and the screening of the drug prescription is optimized by infiltration analysis of the drug excipients in various tissue cells.
- the self-assembled biomimetic membrane of the invention is stable, low in cost, simple and rapid to prepare, and the constructed model has a similar drug permeation and absorption function as the classical drug screening cell model such as Caco-2, and the related mathematical model can be established for the early stage of drug development. Screening studies.
- the object of the present invention is to overcome the deficiencies of the prior art and to provide a self-assembling biomimetic phospholipid polymer tissue cell membrane.
- the object of the present invention is also to provide a method for preparing the self-assembled biomimetic phospholipid polymer tissue cell membrane.
- Another object of the present invention is to provide the use of the above-described biomimetic phospholipid polymer tissue cell membrane for drug absorption.
- the self-assembling biomimetic phospholipid polymer tissue cell membrane of the present invention comprises a hydrophobic aliphatic hydrocarbon terminal and a hydrophilic phospholipid group oligomer, wherein the biomimetic membrane body comprises a hydrophilic phospholipid polar head end group and a hydrophobic aliphatic hydrocarbon tail
- the segment, its oligomer is used as a raw material for polymer film formation.
- the oligomer consists of three parts (see Figure 1, three types of oligomers Rl, 2, and R3 are different), and each part can be adjusted as needed. For example:
- the weight of the phospholipid is 0. 1wt%.
- the molecular weight is 0 ⁇ 1%; the molecular weight is 1000 ⁇ 9 ⁇ 10 6 ; the number of carbon atoms or fluorine atoms or silicon atoms in the hydrophobic segment is 8 ⁇ '20 ; 99wt%.
- the present invention employs a hydrophilic phospholipid group or a derivative thereof to bond a hydrophobic aliphatic hydrocarbon having a double bond at a terminal group, and is capable of self-assembly to form a structure very similar to a biofilm.
- the linking end group contains a hydrophobic aliphatic hydrocarbon of a double bond (including an aliphatic chain or a siloxane chain or a fluorocarbon chain, etc.).
- hydrophilic phospholipid group or derivative thereof is selected from the group consisting of lecithin, cephalin, inositol phospholipid, serine phospholipid or dipalmitoylphosphatidylcholine (Fig. 2, Table 1). ' '
- the hydrophilic phospholipid group or derivative thereof has a charged phospholipid structure, and an example is shown in FIG.
- the preparation method of the self-assembled biomimetic phospholipid polymer tissue cell membrane of the present invention is based on the following principles:
- the cell membrane structure has a hydrophilic head, a hydrophobic tail, and the phospholipid in the membrane is a liquid crystal structure, which allows ion transport and molecular motion.
- a polar phosphorylcholine or a derivative thereof is introduced into a hydrophobic fatty chain, and the material can self-assemble into a hydrophilic head or a hydrophobic tail structure in an aqueous solution or under physiological conditions. After self-assembly of the material, the double bond at the end of the hydrophobic chain is opened to condense the hydrophobic end of the molecule, thereby ensuring that the hydrophobic chain is always inside and the hydrophilic end is always on the surface.
- This phospholipid polymer ensures that the material will better mimic the structure and properties of the tissue cell membrane after film formation.
- the present invention prepares a biomimetic film by controlling the molecular weight of the polymer, the length of the hydrophobic aliphatic hydrocarbon end, and the phospholipid content to adjust the stability and elasticity of the synthetic phospholipid polymer.
- Synthetic phospholipid polymers contain both cation and amphoteric end groups and alkane non-polar molecular chains, which makes the phospholipid polymer readily self-assembled to form microscopic heterogeneous structures very similar to biofilms. Due to the presence of amphoteric end groups, the molecular morphology of the phospholipid polymer changes with the pH of the surrounding environment, and thus exhibits environmental sensitivity. When the pH of the surrounding environment is between 2 and 6.5, it is expressed as a lipid bimolecular. The layered structure, the surrounding environment pH value of 7. 3 ⁇ 10. 5, the performance of the monolayer, the ambient pH value of > 10. 5, the performance of the bilayer microtubules ( Figure 4). Adjustable osmotic liquid The pH of the pH, while simulating the pH changes of different organ tissues. This is a remarkable feature of the present invention.
- the present invention uses a phospholipid polymer to introduce different polar phospholipid groups or derivatives thereof such as lecithin, cephalin, inositol phospholipid, serine phospholipid and dipalmitoylphosphatidylcholine on the hydrophobic chain, according to polymers containing different phospholipids.
- a phospholipid-containing rock fat polymer can be used as a main film-forming material to construct an intestinal absorption and permeation model; a phosphatidylamine-containing phospholipid polymer as a main film-forming material can construct a blood-brain barrier BBB permeation absorption simulation model; containing lecithin, cephalin
- a mixture of phospholipid polymers such as serine phospholipids and phosphatidylinositol as the main film-forming materials can be used to construct skin penetration, oral absorption and corneal drug penetration absorption simulation models.
- the model diagram is shown in Figure 5.
- the invention adopts different phospholipid polymers to simulate the cell membrane drug permeation model, and by adjusting the anionic phospholipid content in the phospholipid mixture and the unsaturated phospholipid content to change the fluidity of the phospholipid membrane, the permeation characteristics of the drug excipients in the intestinal epithelial cells can be studied, and the screening of the drug prescription can be optimized.
- Vdn (ml) volume of the donor tank (0.2 ml)
- Vac (ml) volume of the receiving tank (0.30 or 0.35 ml)
- Cac concentration of the receiving tank after incubation with the added drug (m)
- Cdo initial concentration of the donor tank (mM)
- S (cm 2 ) membrane area (0.3 cm 2 )
- s) hatching time.
- Pm is the permeability of the membrane, is the inherent permeability of the undissociated sample, and pKa is the dissociation constant
- the self-assembled biomimetic membrane of the invention is stable, low in cost, simple in preparation, and has a similar model of drug permeation and absorption with a classical drug screening cell model such as Caco-2, and a related mathematical model can be established for the drug.
- a classical drug screening cell model such as Caco-2
- FIG. 1 Chemical structure of various oligomers (R1 represents various unsaturated alkanes, R2 represents hydrophobic chains of different chain lengths, and R3 represents different phospholipid polar groups);
- Figure 2 shows the general structural formula of phospholipids
- Figure 4 Possible forms of phospholipid polymers in aqueous solution
- FIG. 5 Schematic diagram of the operation of PAMPA-CN
- the synthetic biomimetic membrane monomer generally has the structure of formula (II):
- Ethyl/n-hexane 20/80 and ethyl acetate/n-hexane 50/50 Accurately weigh 32.96g (0.154mol) of octyl methacrylate, 15.6g of triethylamine (0.154 mol) dissolved in 200mL of dry tetrahydrofuran, cooled to -20 ° C, 21.9g (0.154mol) COP dissolved In 100 mL of dry tetrahydrofuran, slowly add the solution dropwise while stirring, keep the temperature at -20 ⁇ - 30 e C3h, filter, wash and dry to obtain 2 - (2-oxo-l, 3, 2-dioxaphospholoyloxy) ethyl methacrylate colorless liquid.
- the PAMPA-CN consists of two upper and lower chambers.
- the upper layer is the donor fluid (buffer solution and drug), and the lower layer is the receiving fluid (buffer solution).
- the buffer solution consists of 50ra sodium phosphate (pH 6. 0-7.5), 50mM sodium citrate (pH 3. 0-5. 5), 50mM sodium borate (pH 8. 0-10. 0), culture at 30 °C 2 ⁇ 15 hours to form a lecithin polymer bionic cell hire.
- the concentration of the solution in the receiver is measured by the UV spectrum.
- the lecithin polymer was dissolved in dodecane to prepare a solution of 0.2 mg/ml, and 4 ul of the phospholipid polymer solution was accurately placed on the micropores of PAMPA-CN, and the PAMPA-CN was composed of two upper and lower chambers.
- the upper layer is the donor liquid (buffer solution and drug)
- the lower layer is the receiving liquid (buffer solution)
- the buffer solution is 50raM sodium phosphate (pH 6. 0-7. 5), 50 mM sodium citrate (pH 3. 0-5. 5), Composition of 50 mM sodium borate (pH 8. 0-10. 0), cultured at 30 ° C for 2 to 15 hours to form a lecithin polymer biomimetic cell membrane.
- the concentration of the solution in the receiving liquid is measured by the UV spectrum.
- a small amount of test for the permeability of norfloxacin, famotidine, atenolol, cimetidine, ibuprofen and other drugs was used.
- the results showed that the drug penetration properties of several synthetic phospholipids were similar to those of the commercial PAMPA BLM-0 phospholipid model.
- the parameters obtained from the model were compared with those of in vivo drugs, especially those passively absorbed. Research The results have a greater correlation (Table 2).
- the lecithin polymer was dissolved in dodecane to prepare a solution of 0.2 mg/ml, and 4 ul of the phospholipid polymer solution was accurately placed on the micropores of PAMPA-CN.
- PAMPA-CN consisted of two upper and lower chambers. The upper layer is the donor fluid (buffer solution and drug), and the lower layer is the receiving solution (buffer solution). The pH of the drug solution is adjusted according to the buffer solution to 4. 6. 4. 92, 6. 5, 7. 4, 8. 2, 9 57, The buffer solution consists of 50 mM sodium phosphate (pH 6. 0 - 7.5), 50 raM sodium citrate (pH 3. 0-5. 5), 50 mM sodium borate (pH 8. 0-10.
- the lecithin polymer biomimetic cell membrane was formed in 16 hours.
- the concentration of the solution in the receiving liquid is measured by the UV spectrum.
- a small amount of test for the permeability of norfloxacin, famotidine, atenolol, cimetidine, ibuprofen and other drugs is measured. The results of the study indicate that the permeability of several synthetic phospholipids varies according to pH and varies.
Abstract
A mimetic cell membrane of phospholipid polymer comprising a hydrophobic terminal of aliphatic hydrocarbon and a hydrophilic group of phospholipid, which can construct a mimetic biomembrane by self-assembly of polymer, and be used in the analysis of drug permeability for drug screening.
Description
仿生磷脂聚合物细胞膜、 及其制备方法和应用 技术领域 Biomimetic phospholipid polymer cell membrane, preparation method and application thereof
本发明属高分子材料膜技术领域, 涉及一种新型模拟组织细胞膜制备方法及其在 药物渗透中的应用。 The invention belongs to the technical field of polymer material membranes, and relates to a novel method for preparing tissue cell membranes and the application thereof in drug permeation.
背景技术 Background technique
生物利用度是衡量一种药物能否开发成有效的临床药物的重要指标之一。药物在人 体肠道中的吸收同时取决于三个相关的特性: 渗透性, 溶解性和 ; i:。。 具有关报道, 80% 的药物通过肠上皮细胞的被动扩散渗透进入血液。有研究表明, 吸收性的数据能够用于 早期药物开发中的体外活性分析。 . Bioavailability is one of the important indicators for measuring whether a drug can be developed into an effective clinical drug. The absorption of the drug in the human gut depends on three related properties: permeability, solubility, and i:. . With the report, 80% of the drugs penetrate into the bloodstream through the passive diffusion of intestinal epithelial cells. Studies have shown that absorbable data can be used for in vitro activity analysis in early drug development. .
近十余年来, 国内外已普遍采用组织细胞模型作为药物吸收性研究的工具, 主要有 Caco-2细胞单层模型、 MDCK细胞模型和 BBMEC模型等。 For more than ten years, tissue cell models have been widely used as tools for drug absorption research at home and abroad, including Caco-2 cell monolayer model, MDCK cell model and BBMEC model.
Caco 2细胞模型来源于人的结肠癌细胞,是著名的小肠吸收模型, 该模型的研究结 果与体内药物的处置有较好的相关性。 Caco-2细胞模型现已被认可为小分.子药物口服吸 收的体外筛选模型, 已为发达国家的药物研究机构或开发公司广泛采用。. The Caco 2 cell model is derived from human colon cancer cells and is a well-known model of intestinal absorption. The results of this model have a good correlation with drug disposal in vivo. The Caco-2 cell model has now been approved as an in vitro screening model for the oral absorption of small fractions of sub-drugs, which has been widely adopted by drug research institutions or development companies in developed countries. .
' MDCK细胞模型来源于犬科动物的肾细胞, 可以形成紧密结^ ^的单细胞层。 The MDCK cell model is derived from the kidney cells of canines and can form a tight cell monolayer.
BBMEC模型, 在建立初期即成为血脑屏障研究的重要工具, 至今仍被应用。 是目 前模拟血脑屏障的最常用的体外模型。 The BBMEC model, which became an important tool for blood-brain barrier research at the beginning of its establishment, is still being used today. It is the most commonly used in vitro model for simulating the blood-brain barrier.
上述细胞模型在使用时均需要实验室培养, 传统的培养方法需要培养至少 21天才 具有模拟细胞膜的性质, 不同研究者建立细胞模型的方法存在一些差异, 不同实验室对 同一模型的评价结果也可能不尽相同, 除此之外, 还存在储存时间短, 操作麻烦, 成本 高等缺陷。 The above cell models require laboratory culture when used. Traditional culture methods require at least 21 days of culture to simulate the properties of cell membranes. There are some differences in the methods used by different researchers to establish cell models. The evaluation results of the same model may be different from different laboratories. In addition, there are also shortcomings such as short storage time, troublesome operation, and high cost.
建立一种用于分析药物渗透和吸收性的体外仿生膜模型已成为世界药物学家关注 的前沿。 2002年 7月在旧金山召开了第一届人工膜渗透性研究的国际会议,会上介绍了 一种新的人工膜 PAMPA模型, 但该模型尚存在不足之处, 主要在于: (l ) PAMPA价格 虽然比 Caco-2细胞模型低, 但此模型自动化程度高, 对少量样品的试验来说, 其成本 较大, 于普通实验室的少样测试不方便; (2 ) ΡΑΜΡΑ 是溶液成膜, 会对某些试验测 试造成误差; (3) ΡΑΜΡΑ 是小分子磷脂制成的双分子膜, 膜的稳定性不好, 影响药物 检测的结果; (4) 目前 ΡΑΜΡΑ主要采用卵磷脂膜以模拟肠道的吸收性能, 模拟的细胞膜 The establishment of an in vitro biomimetic membrane model for the analysis of drug penetration and absorption has become a frontier of concern to the world's pharmacologists. The first international conference on artificial membrane permeability research was held in San Francisco in July 2002. A new artificial membrane PAMPA model was introduced, but the model still has some shortcomings, mainly: (l) PAMPA price Although it is lower than the Caco-2 cell model, the model is highly automated. For a small number of samples, the cost is large, and it is inconvenient to test in a small laboratory. (2) ΡΑΜΡΑ is a solution film formation, It causes errors in some test tests; (3) ΡΑΜΡΑ is a bimolecular membrane made of small molecule phospholipids, the stability of the membrane is not good, affecting the results of drug detection; (4) At present, lecithin membrane is mainly used to simulate intestinal tract Absorption performance, simulated cell membrane
确认本
部位尚嫌单一。 Confirmation The location is still a single.
发明目的 Purpose of the invention
本发明为在合理药物设计过程中, 早期对候选药物进行预测, 及时淘汰一些性质 不理想的药物, 从而大大降低药物筛选的费用, 提供一种体外新型模拟组织细胞膜药物 渗透模型。 The invention aims to predict candidate drugs in the early stage of rational drug design, and eliminates some unsatisfactory drugs in time, thereby greatly reducing the cost of drug screening, and providing a novel in vitro simulated tissue cell membrane drug penetration model.
发明概要 Summary of invention
本发明提供几种人工合成的仿细胞膜结构的磷脂聚合物,通过聚合物自组装构建模 拟组织膜模型, 并应用于药物渗透性的分析中, 提供一种体外药物渗透性的早期评价和 筛选方法。 The invention provides several synthetic phospholipid polymers of cell-like membrane structure, constructs a simulated tissue membrane model by polymer self-assembly, and is applied to the analysis of drug permeability, and provides an early evaluation and screening method for drug permeability in vitro. .
本发明将亲水性的磷脂基团或其衍生物, 如卵磷脂, 脑磷脂, 肌醇磷脂, 丝氨酸磷 脂或二棕榈酰磷脂酰胆碱连接端基含有双键的脂肪族或碳氟疏水链上,合成出能自组装 形成与生物膜相似的 pH值敏感微观非均相结构, 磷脂聚合物在疏水链上引入不同极性 磷脂基团或其衍生物分别建立不同靶目标的模拟细胞膜模型, 卵磷脂基仿生膜建立小肠 模拟细胞膜模型、脑磷脂基仿生膜建立脑组织模拟细胞膜模型、肌醇磷脂基仿生膜建立 胃组织模拟细胞膜模型或各磷脂基的调控与组合模拟人体各种组织器官模拟模型,如皮 肤、口腔粘膜等模型。改变磷脂膜流动性,通过对药物辅料在各种组织细胞的渗透分析, 从而优化药物处方的筛选。 本发明的自组装仿生膜稳定, 成本低, 制备简单快速, 构建 的模型与 Caco-2等经典药物筛选细胞模型有相似的药物渗透吸收功能, 建立的相关数 学模型, 可以用于药物开发的早期筛选研究中。 The present invention relates a hydrophilic phospholipid group or a derivative thereof, such as lecithin, cephalin, inositol phospholipid, serine phospholipid or dipalmitoylphosphatidylcholine, to a terminal group containing an aliphatic or fluorocarbon hydrophobic chain having a double bond. On the other hand, a pH-sensitive micro-homogeneous structure capable of self-assembly is formed, which is similar to the biofilm. The phospholipid polymer introduces different polar phospholipid groups or their derivatives on the hydrophobic chain to establish a simulated cell membrane model of different target targets. Lecithin-based biomimetic membrane to establish a small intestine simulated cell membrane model, cephalin-based biomimetic membrane to establish brain tissue mimic cell membrane model, inositol phospholipid-based biomimetic membrane to establish gastric tissue mimic cell membrane model or the regulation and combination of various phospholipid groups to simulate various human tissues and organs simulation Models such as skin, oral mucosa and other models. The phospholipid membrane fluidity is changed, and the screening of the drug prescription is optimized by infiltration analysis of the drug excipients in various tissue cells. The self-assembled biomimetic membrane of the invention is stable, low in cost, simple and rapid to prepare, and the constructed model has a similar drug permeation and absorption function as the classical drug screening cell model such as Caco-2, and the related mathematical model can be established for the early stage of drug development. Screening studies.
发明内容 Summary of the invention
本发明的目的是克服现有技术的缺陷,提供一种自组装仿生磷脂聚合物组织细胞膜 本发明的目的还提供一种上述自组装仿生磷脂聚合物组织细胞膜的制备方法。 本发明的另一目的是提供上述仿生磷脂聚合物组织细胞膜在药物吸收中的用途。 本发明自组装仿生磷脂聚合物组织细胞膜含有疏水性脂肪烃端和亲水性磷脂基团 齐聚物, 所述仿生膜其体包含亲水性磷脂极性头部端基和疏水性脂肪烃尾部链段, 其齐 聚物作为聚合物成膜的原料。 齐聚物由三部分组成 (见图 1, 三类齐聚物 Rl , 2, R3 均不相同), 每部分都可以根据需要进行调节。 比如: SUMMARY OF THE INVENTION The object of the present invention is to overcome the deficiencies of the prior art and to provide a self-assembling biomimetic phospholipid polymer tissue cell membrane. The object of the present invention is also to provide a method for preparing the self-assembled biomimetic phospholipid polymer tissue cell membrane. Another object of the present invention is to provide the use of the above-described biomimetic phospholipid polymer tissue cell membrane for drug absorption. The self-assembling biomimetic phospholipid polymer tissue cell membrane of the present invention comprises a hydrophobic aliphatic hydrocarbon terminal and a hydrophilic phospholipid group oligomer, wherein the biomimetic membrane body comprises a hydrophilic phospholipid polar head end group and a hydrophobic aliphatic hydrocarbon tail The segment, its oligomer is used as a raw material for polymer film formation. The oligomer consists of three parts (see Figure 1, three types of oligomers Rl, 2, and R3 are different), and each part can be adjusted as needed. For example:
所述齐聚物特征在于: 原料中水份 0〜1 %; 分子量 1000〜9χ106; 疏水链段碳原子 或氟原子或硅原子的数目 8〜'20; 极性磷脂含量 O. lwt %〜99wt%。
本发明采用亲水性的磷脂基团或其衍生物连接端基含有双键的疏水性脂肪烃上,合:) 成出能自组装形成与生物膜非常相似的结构。所述的连接端基含有双键的疏水性脂肪烃 - (包括脂肪链或硅氧烷链或氟碳链等)。 The weight of the phospholipid is 0. 1wt%. The molecular weight is 0~1%; the molecular weight is 1000~9χ10 6 ; the number of carbon atoms or fluorine atoms or silicon atoms in the hydrophobic segment is 8~'20 ; 99wt%. The present invention employs a hydrophilic phospholipid group or a derivative thereof to bond a hydrophobic aliphatic hydrocarbon having a double bond at a terminal group, and is capable of self-assembly to form a structure very similar to a biofilm. The linking end group contains a hydrophobic aliphatic hydrocarbon of a double bond (including an aliphatic chain or a siloxane chain or a fluorocarbon chain, etc.).
所述的亲水性的磷脂基团或其衍生物选自卵磷脂, 脑磷脂, 肌醇磷脂, 丝氨酸磷脂 或二棕榈酰磷脂酰胆碱 (图 2, 表 1 ) 。 ' ' The hydrophilic phospholipid group or derivative thereof is selected from the group consisting of lecithin, cephalin, inositol phospholipid, serine phospholipid or dipalmitoylphosphatidylcholine (Fig. 2, Table 1). ' '
表 1 X基团和磷脂名称 Table 1 X group and phospholipid name
X基团 中 文 名 称 英 文 名 称 X group Chinese name English name
-H 磷脂酸 Phosphatidie acid (PA) -H phosphatidic acid Phosphatidie acid (PA)
-CH2CH2NH3 磷脂酰胆胺(脑磷脂) .. Phosphatidylethanolamine (PE) -CH 2 CH 2 NH 3 phosphatidylcholine (cephalin) : Phosphatidylethanolamine (PE)
-CH2CH2N(CH3)3 磷脂酰胆碱 (卵磷脂) Phosphatidylcholine (PC) -CH 2 CH 2 N(CH 3 ) 3 Phosphatidylcholine (lecithin) Phosphatidylcholine (PC)
-CH2CH(NH2) COOH 磷脂酰丝氨酸 Phosphatidylserine (PS) -CH 2 CH(NH 2 ) COOH Phosphatidylserine Phoshapidylserine (PS)
-CH2CH(OH)CH2OH 磷脂酰甘油 Phosphatidylglycerol (PG) -CH 2 CH(OH)CH 2 OH Phosphatidylglycerol Phosphatidylglycerol (PG)
-C6H6(0H)5 磷脂酷肌醇 Phosphatidylinoitide (PI) -C 6 H 6 (0H) 5 phospholipid coolositol Phosphatidylinoitide (PI)
所述的亲水性的磷脂基团或其衍生物其带电荷的磷脂结构, 实例见图 3。 The hydrophilic phospholipid group or derivative thereof has a charged phospholipid structure, and an example is shown in FIG.
本发明的自组装仿生磷脂聚合物组织细胞膜的制备方法基于下列原则: The preparation method of the self-assembled biomimetic phospholipid polymer tissue cell membrane of the present invention is based on the following principles:
分子生物学理论表明,细胞膜结构具有亲水的头、疏水的尾,膜中磷脂呈液晶结构, 这种结构容许离子传递和分子运动。 将极性磷酸胆碱基或其衍生物引入疏水脂肪链上, 这种材料在水溶液中或在生理条件下可以自组装成亲水头、 疏水尾的结构。材料自组装 以后,将疏水链末端的双键打开,使分子间疏水端缩合,从而保证了疏水链始终在内部, 亲水端始终在表面。 这种磷脂聚合物保证了材料在成膜以后, 更好模拟组织细胞膜的结 构和性能。 Molecular biology theory shows that the cell membrane structure has a hydrophilic head, a hydrophobic tail, and the phospholipid in the membrane is a liquid crystal structure, which allows ion transport and molecular motion. A polar phosphorylcholine or a derivative thereof is introduced into a hydrophobic fatty chain, and the material can self-assemble into a hydrophilic head or a hydrophobic tail structure in an aqueous solution or under physiological conditions. After self-assembly of the material, the double bond at the end of the hydrophobic chain is opened to condense the hydrophobic end of the molecule, thereby ensuring that the hydrophobic chain is always inside and the hydrophilic end is always on the surface. This phospholipid polymer ensures that the material will better mimic the structure and properties of the tissue cell membrane after film formation.
本发明通过控制聚合物的分子量大小、疏水性脂肪烃端的长度和磷脂含量, 调节合 成磷脂聚合物的稳定性和弹性, 制备仿生膜。 The present invention prepares a biomimetic film by controlling the molecular weight of the polymer, the length of the hydrophobic aliphatic hydrocarbon end, and the phospholipid content to adjust the stability and elasticity of the synthetic phospholipid polymer.
合成的磷脂聚合物含有阴阳两性端基和烷烃非极性分子链,'这使得磷脂聚合物容易 自组装形成与生物膜非常相似的微观非均相结构。 由于两性端基的存在, 磷脂聚合物的 分子形态随着周围环境 pH值的变化而变化, 因而表现出环境敏感性, 当周围环境 pH值 在 2〜6. 5时, 表现为脂质双分子层样结构, 周围环境 pH值在 7. 3〜10. 5时, 表现为单 分子层, 周围环境 pH值在〉 10. 5时, 表现为双分子层微管 (图 4)。 可以调节渗透液体
的 pH值的大小, 而模拟不同器官组织的 pH值变化。 这是本发明的显著特色。 Synthetic phospholipid polymers contain both cation and amphoteric end groups and alkane non-polar molecular chains, which makes the phospholipid polymer readily self-assembled to form microscopic heterogeneous structures very similar to biofilms. Due to the presence of amphoteric end groups, the molecular morphology of the phospholipid polymer changes with the pH of the surrounding environment, and thus exhibits environmental sensitivity. When the pH of the surrounding environment is between 2 and 6.5, it is expressed as a lipid bimolecular. The layered structure, the surrounding environment pH value of 7. 3~10. 5, the performance of the monolayer, the ambient pH value of > 10. 5, the performance of the bilayer microtubules (Figure 4). Adjustable osmotic liquid The pH of the pH, while simulating the pH changes of different organ tissues. This is a remarkable feature of the present invention.
本发明用磷脂聚合物在疏水链上引入不同极性磷脂基或其衍生物如卵磷脂、 脑磷 脂、 肌醇磷脂、 丝氨酸磷脂和二棕榈酰磷脂酰胆碱等, 根据含不同磷脂的聚合物为主 要成膜材料建立不同靶目标的模拟细胞膜药物渗透模型。如含卵磷脂的磯脂聚合物为主 要成膜材料可以构建肠吸收渗透模型;含脑磷脂的磷脂聚合物为主要成膜材料可以构建 血脑屏障 BBB渗透吸收模拟模型; 含卵磷脂, 脑磷脂, 丝氨酸磷脂, 磷脂酰肌醇等磷脂 聚合物混合物为主要成膜材料可以构建皮肤穿透性, 口腔吸收和角膜药物渗透吸收模拟 模型。 其模型图见图 5。 The present invention uses a phospholipid polymer to introduce different polar phospholipid groups or derivatives thereof such as lecithin, cephalin, inositol phospholipid, serine phospholipid and dipalmitoylphosphatidylcholine on the hydrophobic chain, according to polymers containing different phospholipids. A simulated cell membrane drug penetration model for different target targets for major film-forming materials. For example, a phospholipid-containing rock fat polymer can be used as a main film-forming material to construct an intestinal absorption and permeation model; a phosphatidylamine-containing phospholipid polymer as a main film-forming material can construct a blood-brain barrier BBB permeation absorption simulation model; containing lecithin, cephalin A mixture of phospholipid polymers such as serine phospholipids and phosphatidylinositol as the main film-forming materials can be used to construct skin penetration, oral absorption and corneal drug penetration absorption simulation models. The model diagram is shown in Figure 5.
本发明采用不同磷脂聚合物模拟细胞膜药物渗透模型,通过调节磷脂混合物中阴离 子磷脂含量, 不饱和磷脂含量改变磷脂膜流动性, 可以研究药物辅料在小肠上皮细胞间 的渗透特性, 优化药物处方的筛选。 The invention adopts different phospholipid polymers to simulate the cell membrane drug permeation model, and by adjusting the anionic phospholipid content in the phospholipid mixture and the unsaturated phospholipid content to change the fluidity of the phospholipid membrane, the permeation characteristics of the drug excipients in the intestinal epithelial cells can be studied, and the screening of the drug prescription can be optimized. .
所述的建立不同靶目标的模拟细胞膜模型是与体内实验数据或药物参数比较, 建立 模拟模型: P=VdC/ACodT ;其中 P—渗透率, V—接受液体积, A—膜表面积, Co—供体液 浓度, dC/dT-浓度变化速率; The simulated cell membrane model for establishing different target targets is compared with experimental data or drug parameters in vivo to establish a simulation model: P=VdC/ACodT; wherein P-permeability, V-receiving fluid volume, A-membrane surface area, Co- Feed fluid concentration, dC/dT-concentration rate of change;
通过人工膜的表观渗透率由方程计算;.其计算公式如下: The apparent permeability through the artificial membrane is calculated by the equation; its calculation formula is as follows:
, , Vdn Vac 1 , „ F7ux%、 , , Vdn Vac 1 , „ F7ux%,
Pam = -2.303 x― ~― x— x log(l—— - ~ ) ( 1 ) ' Pam = -2.303 x― ~― x— x log(l—— - ~ ) ( 1 ) '
Vdn + Vac St 100 Vdn + Vac St 100
Flux% = vdn + Vac.,io ( 2 ) Flux% = vdn + Vac ., io ( 2 )
Cdn Vdn Cdn Vdn
Vdn (ml) =供体槽的体积 (0.2 ml), Vac (ml) =接受槽的体积 (0.30 or 0.35 ml), Vdn (ml) = volume of the donor tank (0.2 ml), Vac (ml) = volume of the receiving tank (0.30 or 0.35 ml),
Cac =接收槽在加入药物孵化后的浓度 (m ), Cdo =供体槽最初的浓度 (mM), S (cm2) =膜的面积 (0.3 cm2), s) =孵化时间. Cac = concentration of the receiving tank after incubation with the added drug (m), Cdo = initial concentration of the donor tank (mM), S (cm 2 ) = membrane area (0.3 cm 2 ), s) = hatching time.
Po Po
Pm = Pm =
\+\opKa-pH (base) ( 3 ) \+\o pKa - pH (base) ( 3 )
Po Po
Pm =■ „ „ (acid) (4 ) Pm =■ „ „ (acid) (4 )
Pm是膜的渗透性 , 是未解离样品固有的渗透性, pKa是解离常数 Pm is the permeability of the membrane, is the inherent permeability of the undissociated sample, and pKa is the dissociation constant
1 1 1 1 1 1
+ · (5) + · (5)
Pam Pm Puwl Pam Pm Puwl
Pam是表观渗透性, UWL表示不搅拌水层。 联合 (3 ) - ( 5 ) 得到:
l ι+ιορΚα- Η 1 Pam is apparently permeable, and UWL means no agitating aqueous layer. Union (3) - (5) get: l ι+ιο ρΚα - Η 1
Pam Po P wl (base) (6) Pam Po P wl (base) (6)
1 i + io'"'-^ 1 , .j 1 i + io'"'-^ 1 , .j
+ ~ 7 (acid、) (,,7、) + ~ 7 (acid,) (,, 7,)
Pam Po Puwl 本发明的自组装仿生膜稳定, 成本低, 制备简单快速, 构建的模型与 Caco-2等经 典药物筛选细胞模型有相似的药物渗透吸收功能, 建立的相关数学模型, 可以用于药物 开发的早期筛选研究中。 附图说明 Pam Po Puwl The self-assembled biomimetic membrane of the invention is stable, low in cost, simple in preparation, and has a similar model of drug permeation and absorption with a classical drug screening cell model such as Caco-2, and a related mathematical model can be established for the drug. Developed in early screening studies. DRAWINGS
图 1 各类齐聚物的化学结构 (R1代表各种不饱和烷烃, R2代表不同链长的疏水链, R3代 表不同磷脂极性基团); Figure 1 Chemical structure of various oligomers (R1 represents various unsaturated alkanes, R2 represents hydrophobic chains of different chain lengths, and R3 represents different phospholipid polar groups);
图 2 磷脂的一般结构式; Figure 2 shows the general structural formula of phospholipids;
图 3 带电荷的磷脂结构 (R代表酰基链) ; Figure 3 Charged phospholipid structure (R stands for acyl chain);
图 4 磷脂聚合物在水溶液中可能的形态; Figure 4 Possible forms of phospholipid polymers in aqueous solution;
图 5 PAMPA-CN的工作示意图; Figure 5 Schematic diagram of the operation of PAMPA-CN;
图 6 PAMPA-CN模型与 Caco-2细胞/ DS-PAMPA渗透性值比较。 Figure 6. Comparison of PAMPA-CN model with Caco-2 cell/DS-PAMPA permeability values.
具体实施方式 detailed description
下面以实施例对本发明加以进一步的说明, 但是不限制本发明的内容。 The invention is further illustrated by the following examples without restricting the invention.
实施例 1: 磷脂聚合物的制备方法 Example 1: Preparation method of phospholipid polymer
( 1 ) 按式 ( I ) 的路线合成单体: (1) Synthesize monomers according to the route of formula (I):
—? H —? H
H2°— L H2°— L
(R=C8~C2o)(R=C 8 ~C 2 o)
( I ) (I)
称取一定量脂肪族二元醇, 三乙胺溶于干燥的四氢呋喃中, 冷却至 o'c , 搅拌缓慢 滴加一定量甲基丙稀酰氯, 保持温度在 0〜3'C, 混合物升温至 50'C反应 2h, 经过滤, 洗漆, 层析柱分离得无色液体甲基丙稀酸脂肪族二元醇酯, 展开剂为乙酸乙酯 /正己垸: 20Z80和乙酸乙酯 /正己垸: 50/50。称取一定量甲基丙稀酸脂肪族二元醇酯,三乙胺溶于 干燥的四氢呋喃中, 冷却至 -20'C , —定量 COP溶于干燥的四氢呋喃中, 搅拌缓慢滴加 溶液, 保持温度在- 20〜- 30'C 3h, 经过滤, 洗涤, 干燥得单体中间体无色液体。 称取 一定量单体中间体和干燥乙腈, 冷却至 - 2(TC, 快速向溶液中加入无水三甲胺, 混合物 升温至 60Ό反应 16h, 冷却至 - 2(TC, 氮气下过滤沉淀, 干燥的乙腈洗涤, 减压干燥得 磷脂聚合物单体。 Weigh a certain amount of aliphatic diol, triethylamine dissolved in dry tetrahydrofuran, cooled to o'c, slowly add a certain amount of methacrylic acid chloride with stirring, keep the temperature at 0~3'C, and warm the mixture to 50'C reaction for 2h, filtered, washed, and separated by chromatography to obtain a colorless liquid methacrylic acid aliphatic diol ester. The developing solvent is ethyl acetate/n-hexane: 20Z80 and ethyl acetate/n-hexane : 50/50. Weigh a certain amount of methyl acrylate aliphatic diol ester, dissolve triethylamine in dry tetrahydrofuran, cool to -20 'C, - Quantitative COP dissolved in dry tetrahydrofuran, slowly add the solution while stirring, keep The temperature is between - 20 and 30 'C for 3 h, filtered, washed and dried to give a monomer intermediate as a colorless liquid. Weigh a certain amount of monomer intermediate and dry acetonitrile, cool to - 2 (TC, quickly add anhydrous trimethylamine to the solution, heat the mixture to 60 ° reaction for 16 h, cool to - 2 (TC, filter the precipitate under nitrogen, dry The mixture was washed with acetonitrile and dried under reduced pressure to give a phospholipid polymer monomer.
所述合成仿生膜单体的一般具有式(II ) 的结构:
The synthetic biomimetic membrane monomer generally has the structure of formula (II):
( II ) 其中: n=8〜20, X基团和磷脂名称如表 1 , (II) where: n=8~20, X group and phospholipid name are shown in Table 1.
(2)按式(III) 的路线制备齐聚物:
(2) Prepare the oligomer according to the route of formula (III):
(m) 一定量的单体在引发剂偶氮二异丁腈(AIBN)存在下 50〜70Γ反应 10〜24h, 得到 齐聚物产物。 (m) A certain amount of monomer is reacted in the presence of the initiator azobisisobutyronitrile (AIBN) at 50 to 70 Torr for 10 to 24 hours to obtain an oligomer product.
实施例 2 Example 2
精确称取 1, 8-辛二醇 28.76g (0.197mol), 三乙胺 33. lg (0.327mol)溶于 200mL 干燥的四氢呋喃中, 冷却至 0'C, 搅拌缓慢滴加甲基丙稀酰氯 20.6g (0.197mol), 保持 温度在 0〜; TC, 混合物升温至 50'C反应 2h, 经过滤, 洗涤, 层析柱分离得无色液体甲 基丙稀酸辛二醇酯,展开剂为乙酸乙酯 /正己垸 20/80和 酸乙酯 /正己烷 50/50。精确 称取甲基丙稀酸辛二醇酯 32.96g (0.154mol), 15.6g三乙胺 (0.154 mol ) 溶于 200mL 干燥的四氢呋喃中, 冷却至 -20°C, 21.9g (0.154mol) COP溶于 lOOmL干燥的四氢呋喃 中, 搅拌缓慢滴加溶液, 保持温度在 -20〜- 30eC3h, 经过滤, 洗涤, 干燥得 2 - (2-oxo-l, 3, 2-dioxaphospholoyloxy) ethyl methacrylate 无色液体。 精确称取 2- (2-oxo-l, 3, 2-dioxaphospholoyloxy ) ethyl methacrylate5.0g |P 30ml干燥乙腈, 冷 却至- 20°C, 快速向溶液中加入 2ml无水三甲胺, 混合物升温至 60°C反应 16h, 冷却至 - 20'C, 氮气下过滤沉淀, 干燥的乙腈洗涤, 减压干燥得磷脂聚合物单体。 称量适当的 磷脂聚合物单体和 AIBN.溶于甲醇一四氢呋喃混合液中置入聚合试管中, 氮气保护下封 闭试管, 试管 60°C振荡反应 16h, 冷却停止反应, 减压收集得到磷脂聚合物。 实施例 3 Accurately weighed 8.76 g (0.197 mol) of 1,8-octanediol, 33. lg (0.327 mol) of triethylamine dissolved in 200 mL of dry tetrahydrofuran, cooled to 0 ° C, and slowly added dropwise methacrylic acid chloride with stirring. 20.6g (0.197mol), keep the temperature at 0~; TC, the mixture is heated to 50'C for 2h, filtered, washed, and separated by chromatography column to obtain the colorless liquid octyl methacrylate, the developing solvent is acetic acid. Ethyl/n-hexane 20/80 and ethyl acetate/n-hexane 50/50. Accurately weigh 32.96g (0.154mol) of octyl methacrylate, 15.6g of triethylamine (0.154 mol) dissolved in 200mL of dry tetrahydrofuran, cooled to -20 ° C, 21.9g (0.154mol) COP dissolved In 100 mL of dry tetrahydrofuran, slowly add the solution dropwise while stirring, keep the temperature at -20~- 30 e C3h, filter, wash and dry to obtain 2 - (2-oxo-l, 3, 2-dioxaphospholoyloxy) ethyl methacrylate colorless liquid. Accurately weigh 2-(2-oxo-l, 3, 2-dioxaphospholoyloxy) ethyl methacrylate 5.0g |P 30ml dry acetonitrile, cool to - 20 ° C, quickly add 2 ml of anhydrous trimethylamine to the solution, and warm the mixture to 60 The mixture was reacted for 16 h at ° C, cooled to - 20 ° C, and the precipitate was filtered under nitrogen, washed with dry acetonitrile, and dried under reduced pressure to give a phospholipid polymer monomer. Weigh the appropriate phospholipid polymer monomer and AIBN. Dissolve in a mixture of methanol and tetrahydrofuran and place it in a polymerization tube. The tube is sealed under nitrogen. The tube is shaken at 60 ° C for 16 h. The reaction is stopped by cooling. Things. Example 3
精确称取 1, 9-壬二醇 31.52g (0.197mol), 三乙胺 33. lg (0.327mol)溶于 200mL 干燥的四氢呋喃中, 冷却至 0Ό, 搅拌缓慢滴加甲基丙稀酰氯 20.6g (0.197mol), 保持 温度在 0〜3'C, 混合物升温至 50'C反应 2h, 经过滤, 洗涤, 层析柱分离得无色液体甲
棊丙稀酸辛二醇酯,展开剂为乙酸乙酯 /正己烷 20/80和乙酸乙酯 /正己烷 50/50。精确 称取甲基丙稀酸辛二醇酯 34. 25g (0. 154mol ), 15. 6g三乙胺(0. 154 mol )溶于 200mL 干燥的四氢呋喃中, 冷却至 - 20'C, 21. 9g (0. 154mol ) COP溶于 lOOmL干燥的四氢呋喃 中, 搅拌缓慢滴加溶液, 保持温度在 -20〜- 30 'C 3h, 经过滤, 洗涤, 干燥得 2- (2-OXO-1, 3, 2-dioxaphospholoyloxy ) ethyl methacrylate无色液体。 精确称取 2_ (2-OXO-1, 3, 2-dioxaphospholoyloxy ) ethyl methacrylate5. 0g和 30ml干燥乙腈, 冷 却至- 20'C, 快速向溶液中加入 2ml无水三甲胺, 混合物升温至 60°C反应 16h, 冷却至 -20Ό , 氮气下过滤沉淀, 干燥的乙腈洗涤, 减压干燥得磷脂聚合物单体。 称量适当的 磷脂聚合物单体和 AIBN溶于甲醇一四氢呋喃混合液中置入聚合试管中, 氮气保护下封 闭试管, 试管 60Ό摒荡反应 16h, 冷却停止反应, 减压收集得到磷脂聚合物。 Accurately weighed 11.52 g (0.197 mol) of 1,9-nonanediol, 33. lg (0.327 mol) of triethylamine dissolved in 200 mL of dry tetrahydrofuran, cooled to 0 Torr, and slowly added dropwise 20.6 g of methacrylic acid chloride with stirring. (0.197mol), keep the temperature at 0~3'C, warm the mixture to 50'C for 2h, filter, wash, and separate the column to obtain colorless liquid A The octyl propylene glycol acrylate was developed with ethyl acetate/n-hexane 20/80 and ethyl acetate/n-hexane 50/50. Accurately weighed 34. 25g (0. 154mol ) of methyl octyl methacrylate, 15. 6g of triethylamine (0.154 mol) dissolved in 200mL of dry tetrahydrofuran, cooled to - 20'C, 21. 9g (0. 154mol) COP was dissolved in 100 mL of dry tetrahydrofuran, and the solution was slowly added dropwise while stirring, keeping the temperature at -20~- 30 'C for 3 h, filtered, washed and dried to give 2-(2-OXO-1, 3, 2-dioxaphospholoyloxy ) ethyl methacrylate colorless liquid. Accurately weigh 2_(2-OXO-1, 3, 2-dioxaphospholoyloxy) ethyl methacrylate 5. 0g and 30ml of dry acetonitrile, cool to - 20'C, quickly add 2ml of anhydrous trimethylamine to the solution, and warm the mixture to 60 °C After reacting for 16 h, it was cooled to -20 Torr, and the precipitate was filtered under nitrogen, washed with dry acetonitrile, and dried under reduced pressure to give a phospholipid polymer monomer. The appropriate phospholipid polymer monomer and AIBN were dissolved in a mixture of methanol and tetrahydrofuran, and placed in a polymerization tube. The tube was sealed under a nitrogen atmosphere, and the tube 60 was shaken for 16 hours. The reaction was stopped by cooling, and the phospholipid polymer was collected under reduced pressure.
实施例 4 Example 4
将少量的合成的含卵磷脂聚合物放置在 PAMPA- CN的微孔上, PAMPA- CN由上下两个 腔室组成, 上层为供体液(缓冲溶液和药物), 下层为接收液(缓冲溶液), 缓冲溶液由 50ra 磷酸钠 (pH6. 0- 7. 5), 50mM柠檬酸钠(pH3. 0-5. 5), 50mM硼酸钠 (pH8. 0-10. 0) 组成, 30°C培养 2〜15小时形成卵磷脂聚合物仿生细胞聘。 接收液中溶液的浓度由 UV 光谱来测量。 少量测试磺胺撒克西啶, 诺福克, 双氢克尿塞, 立痛定, 心得安, 戊脉安 等几种药物渗透率分别为 16. 0, 45. 0, 16. 0, 128. 0, 133. 0, 148. 0ntn/s。 研究结果表 明, 构建的模型与 Caco-2细胞模型有相似的药物渗透吸收功能, 通过比较发现, 采用 该模型得出的参数与体内药物特别是那些被动吸收的药物的研究结 有较大的相关性 (图 6)。 A small amount of synthetic lecithin-containing polymer is placed on the micropores of PAMPA-CN. The PAMPA-CN consists of two upper and lower chambers. The upper layer is the donor fluid (buffer solution and drug), and the lower layer is the receiving fluid (buffer solution). The buffer solution consists of 50ra sodium phosphate (pH 6. 0-7.5), 50mM sodium citrate (pH 3. 0-5. 5), 50mM sodium borate (pH 8. 0-10. 0), culture at 30 °C 2 ~15 hours to form a lecithin polymer bionic cell hire. The concentration of the solution in the receiver is measured by the UV spectrum. A small amount of test sulfamethoxine, Norfolk, hydrochlorothiazide, Litongding, propranolol, valproate and other drugs have a permeability of 16. 0, 45. 0, 16. 0, 128. 0, 133. 0, 148. 0ntn/s. The results showed that the constructed model had similar drug permeation and absorption functions as the Caco-2 cell model. By comparison, the parameters obtained by the model were significantly correlated with the in vivo drugs, especially those of passively absorbed drugs. Sex (Figure 6).
实施例 5 Example 5
将几种卵磷脂聚合物溶解于十二烷中配制成 0. 2mg/ml溶液, 准确取 4ul磷脂聚合 物溶液放置在 PAMPA- CN的微孔上, PAMPA- CN由上下两个腔室组成, 上层为供体液(缓 冲溶液和药物), 下层为接收液(缓冲溶液), 缓冲溶液由 50raM磷酸钠 (pH6. 0-7. 5), 50mM柠檬酸钠 (pH3. 0- 5. 5), 50mM硼酸钠(pH8. 0-10. 0)组成, 30°C培养 2〜15小时 形成卵磷脂聚合物仿生细胞膜。 接收液中溶液的浓度由 UV光谱来测量。 少量测试诺氟 沙星, 法莫替丁, 阿替洛尔, 西米替丁, 布洛芬等几种药物的渗透率。 研究结果表明, 几种合成磷脂的药物渗透性能与商用 PAMPA BLM-0磷脂模型有相似的药物渗透吸收功 能, 通过比较发现, 采用该模型得出的参数与体内药物特别是那些被动吸收的药物的研
究结果有较大的相关性 (表 2)。 The lecithin polymer was dissolved in dodecane to prepare a solution of 0.2 mg/ml, and 4 ul of the phospholipid polymer solution was accurately placed on the micropores of PAMPA-CN, and the PAMPA-CN was composed of two upper and lower chambers. The upper layer is the donor liquid (buffer solution and drug), the lower layer is the receiving liquid (buffer solution), the buffer solution is 50raM sodium phosphate (pH 6. 0-7. 5), 50 mM sodium citrate (pH 3. 0-5. 5), Composition of 50 mM sodium borate (pH 8. 0-10. 0), cultured at 30 ° C for 2 to 15 hours to form a lecithin polymer biomimetic cell membrane. The concentration of the solution in the receiving liquid is measured by the UV spectrum. A small amount of test for the permeability of norfloxacin, famotidine, atenolol, cimetidine, ibuprofen and other drugs. The results showed that the drug penetration properties of several synthetic phospholipids were similar to those of the commercial PAMPA BLM-0 phospholipid model. By comparison, the parameters obtained from the model were compared with those of in vivo drugs, especially those passively absorbed. Research The results have a greater correlation (Table 2).
表 2. 四种不同磷脂在 pH7.4 下的渗透率 Table 2. Permeability of four different phospholipids at pH 7.4
药物 渗透率 X lO^ ctn/s Drug permeability X lO^ ctn/s
C8卵磷脂聚合物 C1()卵磷脂聚合物 C12卵磷脂聚合物 BLM-0 西米替丁 0 C 8 lecithin polymer C 1 () lecithin polymer C 12 lecithin polymer BLM-0 cimetidine 0
诺氟沙星 0.03 法莫替丁 0.06 阿替洛尔 0.06 布洛芬
2.3 Norfloxacin 0.03 famotidine 0.06 atenolol 0.06 ibuprofen 2.3
实施例 6 Example 6
将几种卵磷脂聚合物溶解于十二烷中配制成 0. 2mg/ml溶液, 准确取 4ul磷脂聚合 物溶液放置在 PAMPA- CN的微孔上, PAMPA-CN由上下两个腔室组成, 上层为供体液(缓 冲溶液和药物), 下层为接收液 (缓冲溶液), 药物溶液 pH值根据缓冲溶液调节为 4. 6, 4. 92, 6. 5, 7. 4, 8. 2, 9. 57, 缓冲溶液由 50mM磷酸钠 (pH6. 0—7. 5), 50raM柠檬酸钠 (pH3. 0-5. 5), 50mM硼酸钠 (pH8. 0-10. 0) 组成, ' 30Ό培养 16小时形成卵磷脂聚合物 仿生细胞膜。 接收液中溶液的浓度由 UV光谱来测量。 少量测试诺氟沙星, 法莫替丁, 阿替洛尔, 西米替丁, 布洛芬等几种药物的渗透率。 研究结果表明, 几种合成磷脂的药 物渗透性能根据 pH值变化, 有不同的变化。
The lecithin polymer was dissolved in dodecane to prepare a solution of 0.2 mg/ml, and 4 ul of the phospholipid polymer solution was accurately placed on the micropores of PAMPA-CN. PAMPA-CN consisted of two upper and lower chambers. The upper layer is the donor fluid (buffer solution and drug), and the lower layer is the receiving solution (buffer solution). The pH of the drug solution is adjusted according to the buffer solution to 4. 6. 4. 92, 6. 5, 7. 4, 8. 2, 9 57, The buffer solution consists of 50 mM sodium phosphate (pH 6. 0 - 7.5), 50 raM sodium citrate (pH 3. 0-5. 5), 50 mM sodium borate (pH 8. 0-10. 0), '30 Ό culture The lecithin polymer biomimetic cell membrane was formed in 16 hours. The concentration of the solution in the receiving liquid is measured by the UV spectrum. A small amount of test for the permeability of norfloxacin, famotidine, atenolol, cimetidine, ibuprofen and other drugs. The results of the study indicate that the permeability of several synthetic phospholipids varies according to pH and varies.
Claims
1、 一种仿生瞵脂聚合物细胞膜, 其特征在于含有疏水性脂肪烃端和亲水性磷脂基 团齐 物, 其单体包括含亲水性磷脂极性头部端基和疏水性脂肪烃尾部链段, 其齐聚物 作为成膜聚合物的原料。 A biomimetic rouge polymer cell membrane characterized by comprising a hydrophobic aliphatic hydrocarbon end and a hydrophilic phospholipid group, the monomer comprising a hydrophilic phospholipid polar head end group and a hydrophobic aliphatic hydrocarbon The tail segment, its oligomer is used as a raw material for the film-forming polymer.
2、 按权利要求 1所述的仿生磷脂聚合物细胞膜, 其特征在于所述的亲水性磷脂选 自磷脂酰胆碱、 磷脂酰胆胺、 肌醇磷脂、 丝氨酸磷脂、 二棕榈酰磷脂酰胆碱、 磷脂酸、 或磷脂酰甘油; 所述的疏水性包括脂肪链、 硅氧垸链或氟碳链。 The biomimetic phospholipid polymer cell membrane according to claim 1, wherein the hydrophilic phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylcholine, inositol phospholipid, serine phospholipid, dipalmitoylphosphatidylcholine a base, phosphatidic acid, or phosphatidylglycerol; said hydrophobicity comprising an aliphatic chain, a siloxane chain or a fluorocarbon chain.
3、 按权利要求 1所述的仿生磷脂聚合物细胞膜, 其特征在于所述的疏水性链段是 C8-20的不饱和烃链。 A biomimetic phospholipid polymer cell membrane according to claim 1, wherein said hydrophobic segment is a C8-20 unsaturated hydrocarbon chain.
4、 按权利要求 1所述的磷脂聚合物, 其特征在于含有带电荷的磷脂。 4. A phospholipid polymer according to claim 1 which comprises a charged phospholipid.
5、 按权利要求 1所述的仿生磷脂聚合物细胞膜, 其特征在于其分子形态随环境 pH 值变化, 当 pH值为 2〜6. 5时, 表现为脂质双分子层样结构; pH值为 7. 3〜10. 5时, 表 现为单分子层; 11值〉10. 5时, 表现为双分子层微管。 5. The biomimetic phospholipid polymer cell membrane according to claim 1, wherein the molecular morphology varies with the pH of the environment, and when the pH is 2 to 6.5, it is expressed as a lipid bilayer-like structure; When it is 7. 3~10. 5, it is expressed as a monolayer; when the value is >10. 5, it is expressed as a bilayer microtubule.
6、 按权利要求 5所述的仿生磷脂聚合物细胞膜, 其特征在于所述分子形态为单分 子层其厚度〈0. 75mm, 分子形态为双分子层其厚度〉0. 75画。 And a thickness of the molecular layer is a thickness of the bilayer, and the thickness is <0.75 mm.
7、 一种按权利要求 1的仿生磷脂聚合物细胞膜的制备方法, 其特征在于通过控制 聚合物的分子量大小、疏水性脂肪烃端的长度和磷脂含量, 调节合成磷脂聚合物的稳定 性和弹性。 A method for producing a biomimetic phospholipid polymer cell membrane according to claim 1, which is characterized in that the stability and elasticity of the synthetic phospholipid polymer are adjusted by controlling the molecular weight of the polymer, the length of the hydrophobic aliphatic hydrocarbon end, and the phospholipid content.
8、 按权利要求 7的方法, 其特征在于该方法包括下列 (1 ) 〜 (3 ) 的步骤: 8. A method according to claim 7, characterized in that the method comprises the following steps (1) to (3):
( 1 ) 单体制备: (1) Monomer preparation:
取脂肪族二元醇, 三乙胺溶于干燥四氢呋喃, 冷却至 0'C, 搅拌滴加甲基丙稀酰 氯, 温度为 0〜3°C , 混合物升温至 50'C反应 2h, 过滤, 洗涤, 层析柱分离得甲基丙 稀酸脂肪族二元醇酯液体, 展开剂为乙酸乙酯 /正己垸 20/80 和乙酸乙酯 /正己垸 50/50; Take the aliphatic diol, dissolve the triethylamine in dry tetrahydrofuran, cool to 0 ° C, add dropwise methyl methacrylate chloride, the temperature is 0~3 ° C, the mixture is heated to 50 ° C for 2 h, filtered, washed The chromatographic column is separated to obtain a methyl acrylate aliphatic diol ester liquid, and the developing solvent is ethyl acetate/n-hexane 20/80 and ethyl acetate/n-hexane 50/50;
取甲基丙稀酸脂肪族二元醇酯,三乙胺溶于干燥四氢呋喃中, 冷却至 - 20Ό , —定 量 COP溶于干燥四氢呋喃中, 搅拌滴加溶液, 温度为 - 20〜- 30°C , 3h, 过滤, 洗涤, 干燥得单体中间体无色液体; Take methacrylic acid aliphatic diol ester, triethylamine dissolved in dry tetrahydrofuran, cooled to - 20 Ό, - quantitative COP dissolved in dry tetrahydrofuran, stirred dropwise solution, temperature - 20 ~ - 30 ° C , 3h, filtration, washing, drying to obtain a monomer intermediate colorless liquid;
取单体中间体和干燥乙腈,冷却至 -20'C, 向溶液中加无水三甲胺,混合物升温至
60Ό反应 16h, 冷却至 -20Ό, 氮气下过滤沉淀, 干燥的乙腈洗搽, 减压干燥得磷脂聚 合物单体; Take the monomer intermediate and dry acetonitrile, cool to -20 'C, add anhydrous trimethylamine to the solution, and warm the mixture to 60 Ό reaction for 16 h, cooled to -20 Torr, filtered under nitrogen, precipitated, dried acetonitrile, dried under reduced pressure to give a phospholipid polymer monomer;
(2) 齐聚物制备: (2) Preparation of oligomers:
上述单体在引发剂偶氮二异丁腈存在下 50〜70°C反应 10~24h, 得到齐聚物产物; The above monomer is reacted in the presence of the initiator azobisisobutyronitrile at 50 to 70 ° C for 10 to 24 hours to obtain an oligomer product;
(3) 聚合物制备: (3) Polymer preparation:
上述齐聚物加入弓 I发剂或直接用紫外光照射, 弓 I发双键聚合的聚合物得仿生物膜。 The above oligomer is added to the hair or directly irradiated with ultraviolet light, and the polymer which is double-bonded is obtained as a biofilm.
9、 按权利要求 8的方法, 其特征在于所述齐聚物原料中水份为 0〜1%; 分子量为 1000〜9χ106; 疏水链段碳或氟或硅原子的数目为 8〜20; 极性磷脂含量 0. l~90wt%; 玻璃转化温度为 37〜73.2'C; 接触角为 70。 〜120。 。 The method according to claim 8, wherein the oligomer raw material has a water content of 0 to 1%; a molecular weight of 1000 to 9 χ 10 6 ; and a hydrophobic segment carbon or fluorine or silicon atom number of 8 to 20; The polar phospholipid content is from 0.1 to 90% by weight; the glass transition temperature is from 37 to 73.2 'C ; and the contact angle is 70. ~120. .
10、 一种按权利要求 1 的仿生磷脂聚合物细胞膜的应用, 其特征是在用于制备建立 不同靶目标的模拟细胞膜模型。 10. Use of a biomimetic phospholipid polymer cell membrane according to claim 1, characterized in that it is used in the preparation of a simulated cell membrane model for establishing different targets.
11、按权利要求 10的用途, 其特征是所述的建立不同靶目标的模拟细胞膜模型是卵 磷脂基仿生膜建立小肠模拟细胞膜模型、 脑磷脂基仿生膜建立脑组织模拟细胞膜模型、 肌醇磷脂基仿生膜建立胃组织模拟细胞膜模型或磷脂基的调控与组合模拟人体各种组 织器官模拟模型。 11. The use according to claim 10, characterized in that the simulated cell membrane model for establishing different target targets is a lecithin-based biomimetic membrane to establish a small intestinal simulated cell membrane model, a cephalin-based biomimetic membrane to establish a brain tissue mimic cell membrane model, and an inositol phospholipid. The base-like biofilm establishes a gastric tissue mimicking cell membrane model or a phospholipid-based regulation and combination simulation of various human tissue and organ simulation models.
12、按权利要求 10的用途, 其特征是所述的建立不同的模拟细胞膜模型是调节磷脂 混合物中丝氨酸磷脂的阴离子磷脂含量、不饱和磷脂含量改变磷脂膜流动性分析药物辅 料在小肠上皮细胞间的渗透特性或优化药物处方的筛选。 12. The use according to claim 10, characterized in that said establishing a different simulated cell membrane model is to modulate the anionic phospholipid content of the serine phospholipid in the phospholipid mixture, and the unsaturated phospholipid content changes the phospholipid membrane fluidity analysis drug adjuvant in the intestinal epithelial cells. Permeation characteristics or screening for optimized drug prescriptions.
13、 按权利要求 10的用途, 其特征是所述的建立不同靶目标的模拟细胞膜模型是与 体内实验数据或药物参数比较, 建立模拟模型: P=VdC/ACodT:其中 P—渗透率, V—接受 液体积, A—膜表面积, Co—供体液浓度, dC/dT—浓度变化速率; 13. The use according to claim 10, characterized in that said simulated cell membrane model for establishing different targets is compared with in vivo experimental data or drug parameters to establish a simulation model: P = VdC / ACodT: wherein P - permeability, V - receiving liquid volume, A - membrane surface area, Co - donor fluid concentration, dC / dT - concentration change rate;
通过人工膜的表观渗透率由方程计算; 其计算公式如下: The apparent permeability through the artificial membrane is calculated by the equation; its calculation formula is as follows:
„ , Vdn-Vac 1 , /t Flux%、 „ , Vdn-Vac 1 , /t Flux%,
Pam―-— - -2.303 χ x— Iog(I ) (1) Pam―-— - -2.303 χ x— Iog(I ) (1)
Vdn + Vac St 100 Vdn + Vac St 100
„, 0/ Cac Vdn + Vac „, 0/ Cac Vdn + Vac
Fl x% = x χ 100 (2) Fl x% = x χ 100 (2)
Cdn Vdn Cdn Vdn
Vdn(ml)=供体槽的体积 (0.2ml), Vac (ml) =接受槽的体积 (0.30 or Vdn (ml) = volume of the donor tank (0.2 ml), Vac (ml) = volume of the receiving tank (0.30 or
0.35 ml), 0.35 ml),
Cac=接收槽在加入药物孵化后的浓度 (mM), Cdo=供体槽最初的浓度 (mM), S (cm2) =膜的面积 (0.3 cm2), t(s) =孵化时间. Cac = concentration of the receiving tank after incubation with the drug (mM), Cdo = initial concentration of the donor tank (mM), S (cm 2 ) = membrane area (0.3 cm 2 ), t(s) = hatching time.
rm二 Po r m two Po
m~ l+\0pKa'pH (base) (3)
m~ l+\0 pKa ' pH (base) (3)
Pm是膜的渗透性, Po 是未解离样品固有的渗透性, p a是解离常数 1 1 1 Pm is the permeability of the membrane, Po is the permeability inherent to the undissociated sample, and p a is the dissociation constant 1 1 1
(5) (5)
Pam Pm Puwl 户^是表观渗透性, UWL表示不搅拌水层。 联合 (3) ― (5) 得到:
Pam Pm Puwl household ^ is apparent permeability, UWL means no water layer is stirred. Union (3) - (5) get:
+ (acid) (7)+ (acid) (7)
Pam Po Puwl
Pam Po Puwl
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Cited By (4)
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WO2017147215A1 (en) * | 2016-02-22 | 2017-08-31 | The Methodist Hospital | Biomimetic proteolipid vesicle compositions and uses thereof |
WO2021020275A1 (en) * | 2019-07-26 | 2021-02-04 | 国立大学法人東京大学 | Surface modifying material for lipid membrane structure |
WO2022193759A1 (en) * | 2021-03-19 | 2022-09-22 | 广东丁沃生医疗器械有限公司 | Phospholipid polymer, preparation method therefor and application thereof |
CN115486539A (en) * | 2022-09-14 | 2022-12-20 | 厦门遇见今生生物科技有限公司 | Herbal extract biomimetic membrane with anti-aging and telomere lengthening effects and preparation method thereof |
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US5274101A (en) * | 1993-01-07 | 1993-12-28 | Siltech Inc. | Polymeric phospholipid polymers |
JP2005239988A (en) * | 2004-02-24 | 2005-09-08 | Kazuhiko Ishihara | Water soluble reactive polymer, method for producing the same and biological sample modifier |
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US5237035A (en) * | 1992-12-28 | 1993-08-17 | Siltech Corp. | Silicone phospholipid polymers |
US5274101A (en) * | 1993-01-07 | 1993-12-28 | Siltech Inc. | Polymeric phospholipid polymers |
JP2005239988A (en) * | 2004-02-24 | 2005-09-08 | Kazuhiko Ishihara | Water soluble reactive polymer, method for producing the same and biological sample modifier |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2017147215A1 (en) * | 2016-02-22 | 2017-08-31 | The Methodist Hospital | Biomimetic proteolipid vesicle compositions and uses thereof |
WO2021020275A1 (en) * | 2019-07-26 | 2021-02-04 | 国立大学法人東京大学 | Surface modifying material for lipid membrane structure |
WO2022193759A1 (en) * | 2021-03-19 | 2022-09-22 | 广东丁沃生医疗器械有限公司 | Phospholipid polymer, preparation method therefor and application thereof |
CN115486539A (en) * | 2022-09-14 | 2022-12-20 | 厦门遇见今生生物科技有限公司 | Herbal extract biomimetic membrane with anti-aging and telomere lengthening effects and preparation method thereof |
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