WO2017201686A1 - Dmaps-aa-aas共聚物及其制备方法和应用 - Google Patents

Dmaps-aa-aas共聚物及其制备方法和应用 Download PDF

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WO2017201686A1
WO2017201686A1 PCT/CN2016/083280 CN2016083280W WO2017201686A1 WO 2017201686 A1 WO2017201686 A1 WO 2017201686A1 CN 2016083280 W CN2016083280 W CN 2016083280W WO 2017201686 A1 WO2017201686 A1 WO 2017201686A1
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dmaps
copolymer
aas
monomer
preparation
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PCT/CN2016/083280
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English (en)
French (fr)
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陈少军
卓海涛
任换换
梅占奎
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深圳大学
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Priority to PCT/CN2016/083280 priority Critical patent/WO2017201686A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/44—Preparation of metal salts or ammonium salts
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/04—Acids; Metal salts or ammonium salts thereof
    • C08F220/06—Acrylic acid; Methacrylic acid; Metal salts or ammonium salts thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/10—Esters
    • C08F220/38—Esters containing sulfur
    • C08F220/387—Esters containing sulfur and containing nitrogen and oxygen

Definitions

  • the invention belongs to the field of preparation and application of high molecular polymers, in particular to DMAPS-AA-AAS copolymers and preparation methods and applications thereof.
  • Immune cells play an important role in the prevention of diseases, tumors and cancer treatment, especially macrophages play an important role in the immune response of various functions, from wound healing to resistance to microorganisms.
  • antibodies or active immune biomaterials are widely used to promote macrophage phagocytosis of tumor cells or bacteria.
  • nanoparticles or other grafts are typically surface treated with passive immunobiology materials, such as phosphorylcholine zwitterionic copolymers or polysaccharide surface coatings.
  • passive immunobiology materials such as phosphorylcholine zwitterionic copolymers or polysaccharide surface coatings.
  • human neutrophil phagocytosis can be activated by ⁇ -1,6-glucan. Therefore, we believe that the phagocytosis of immune cells can be regulated by suitable biological materials.
  • the technical problem to be solved by the present invention is to provide DMAPS-AA-AAS (DMAPS) :N,N-dimethyl(methacryloyloxyethyl)ammonium propanesulfonic acid inner salt, AA: acrylic acid, AAS: sodium acrylate) copolymer, preparation method and application thereof To further improve the regulation of macrophage biological activity.
  • DMAPS-AA-AAS DMAPS-AA-AAS
  • the present invention is achieved by the method for preparing DMAPS-AA-AAS, comprising the following steps:
  • Step 2 Formulating an initiator solution, adding the initiator to the mixture, and heating the resulting solution to 65 ⁇ 85 ° C, the reaction 4 to 6 hours, cooled to room temperature, to obtain DMAPS-AA copolymer;
  • Step three adding 5 to 15 wt% to the DMAPS-AA copolymer
  • concentration of sodium hydroxide solution was adjusted to pH and stirred for 2 to 5 hours to prepare a DMAPS-AA-AAS copolymer.
  • the present invention also provides a DMAPS-AA-AAS copolymer prepared by the method described above.
  • DMAPS-AA-AAS copolymer is used to prepare an immunosuppressive agent for macrophages.
  • DMAPS-AA-AAS copolymer is used to prepare an immunopotentiator for macrophages.
  • the present invention has an advantageous effect in comparison with the prior art in that the present invention passes the DMAPS-AA which will be produced.
  • the copolymer is reacted with a sodium hydroxide solution, and the pH of the reaction system is adjusted so that the mass ratio of AAS to the DMAPS-AA-AAS copolymer in the obtained DMAPS-AA-AAS copolymer is different.
  • the pH of the DMAPS-AA-AAS copolymer was adjusted.
  • the DMAPS-AA-AAS copolymer thus obtained, It has good biocompatibility and biomimeticity, and when the pH value of the surface is different, there is a significant difference in biological activity.
  • the present invention thus provides a new application of the DMAPS-AA-AAS copolymer
  • the DMAPS-AA-AAS copolymer is used to prepare a medicament for anti-inflammatory, treating tumor/cancer or promoting wound healing; An immunosuppressant or immunopotentiator for the preparation of macrophages.
  • the DMAPS-AA-AAS copolymer provided by the present invention opens up a new way for the adjustment of macrophage immune activity.
  • the preparation method provided is simple in operation and convenient for mass production.
  • FIG. 1 is a schematic diagram showing the results of biological activity test of macrophages cultured by copolymers of different AAS contents according to an embodiment of the present invention
  • Figure 2 is provided by an embodiment of the present invention. Schematic diagram of the results of DCFH-DA fluorescence intensity values after incubation of mouse macrophages (RAW264.7) with copolymers of different AAS contents;
  • Figure 3 is a graph showing the results of the change in the number of bacteria after cultured macrophages and E. coli cultured for 24 hours according to an embodiment of the present invention
  • Figure 4 is provided by an embodiment of the present invention. Schematic diagram of the results of changes in the number of bacteria after cultured macrophages and Staphylococcus aureus for 24 hours.
  • DMAPS-AA N,N-dimethyl(methacryloyloxyethyl)ammonium propanesulfonic acid inner salt (DMAPS) and acrylic acid DMAPS-AA is a zwitterionic copolymer with high hydration ability And the electrically neutral surface makes it better resistant to non-specific protein adsorption.
  • the surface modified with the zwitterionic copolymer greatly reduces fibrinogen adsorption, platelet adhesion, bacterial adhesion, and plasma coagulation.
  • the study found that the DMAPS-AA copolymer has reversible responsiveness to pH changes.
  • This external response not only changes the physical and chemical properties of the zwitterionic copolymer, but also affects their biological properties, including biocompatibility, biology. Activity and antibacterial properties.
  • the zwitterionic copolymer is non-toxic and therefore has good biocompatibility and biomimeticity.
  • the DMAPS-AA-AAS copolymer is prepared according to the technical scheme of the present invention, and the process is as follows:
  • Step 1 adding monomer DMAPS and monomer AA to deionized water to obtain a mixed solution
  • Step 2 preparing an initiator solution, adding the initiator to the mixed solution, then heating the resulting solution to 65-85 ° C, reacting for 4-6 hours, cooling to room temperature, to obtain a DMAPS-AA copolymer;
  • Step 3 adding a sodium hydroxide solution having a concentration of 5 to 15% by weight to the DMAPS-AA copolymer, adjusting the pH value, and stirring for 2 to 5 hours to obtain a DMAPS-AA-AAS copolymer.
  • the mass ratio of the monomer DMAPS to the monomer AA is 1:4-6, preferably 1:4; in the mixed liquid, the sum of the mass of the monomer DMAPS and the monomer AA
  • the mass ratio of deionized water is 20% to 30%: 1.
  • the mass ratio of the monomeric DMAPS to the monomer AA is different, and the DMAPS-AA copolymer obtained may have a difference in molecular mechanism, and its properties may be different.
  • the initiator is ammonium persulfate or potassium persulfate, preferably ammonium persulfate; the concentration of the initiator solution is 0.5 to 1.5 wt%, preferably 0.5 wt%; the preparation of the initiator solution is It is carried out in a shielding gas which may be an inert gas or nitrogen.
  • the DMAPS-AA copolymer is reacted with the sodium hydroxide solution, and the pH of the reaction system is different, and the AAS in the prepared DMAPS-AA-AAS copolymer accounts for the DMAPS.
  • the mass ratio of the -AA-AAS copolymer will vary.
  • the pH of the reaction system is 2.0 to 4.0
  • the mass ratio of the AAS to the DMAPS-AA-AAS copolymer is 0 to 40% by weight, preferably 0 to 10% by weight
  • the pH of the reaction system is 6.0 to 8.0.
  • the mass ratio of the AAS to the DMAPS-AA-AAS copolymer is from 70 to 80% by weight, preferably 80% by weight.
  • a reaction between a sodium hydroxide solution and a prepared DMAPS-AA copolymer is carried out, and a part of acrylic acid is converted into sodium acrylate by sodium hydroxide, that is, a monomer AAS is introduced into the DMAPS-AA copolymer.
  • the pH of the copolymer was adjusted.
  • the present invention proves that the copolymer of DMAPS-AA has a significant change in its biological activity after pH adjustment, in particular, a copolymer having a DMAPS mass percentage of 20% and an AA mass percentage of 80%.
  • the pH-adjusted copolymer has a regulatory effect on the immune function of macrophages and can be used for the preparation of drugs for anti-inflammatory, tumor/cancer treatment or wound healing.
  • the copolymer when AAS accounts for 70-80% by weight of the total monomer, the copolymer can make the macrophage organisms in an immune hyperactive state; when AAS accounts for 80% by weight of the total monomer weight The effect is best; therefore, it can be used to prepare an immunostimulating agent.
  • AAS accounts for 0-40% by weight of total monomer, the copolymer can reduce or lose the immune function of macrophages.
  • AAS accounts for 0-10% by weight of total monomer, the effect is better; therefore, it can be used Preparation of immunosuppressive agents for macrophages.
  • the biological activity of mouse macrophages was tested by cell viability assay (eg CCK-8); the superoxide radical (ROS) test method was used with fluorescent probes.
  • DCFH-DA (Olympus, Japan) detected the accumulation of ROS in mouse macrophages (RAW264.7) and copolymer cultured for 20 minutes. The content and the fluorescence intensity of DCFH-DA can be analyzed to analyze its biological activity.
  • Figure 1 is a graph showing the bioactivity of macrophages cultured with different AAS contents of DMAPS-AA-AAS copolymer. It can be seen from Fig. 1 that the cell activity of the copolymer with AAS content of 30% is significantly higher than that of blank cells. The decrease indicates that the bioactivity of the copolymer decreases.
  • the cell activity of the copolymer with AAS content of 60% is basically the same as that of the blank cells, indicating that the copolymer has good biocompatibility; the copolymer with 80% AAS content is cultured. Cell viability is significantly higher than that of blank cells, and macrophages are in a hyperactive state.
  • Figure 2 is a comparison of the fluorescence intensity values of DCFH-DA after incubation of mouse macrophages (RAW264.7) with copolymers of different AAS contents. It can also be seen from Fig. 2 that copolymers with AAS content of 30% are cultured. The DCFH-DA fluorescence intensity value of the cells was significantly higher than that of the endotoxin sample LPS, indicating that the copolymer has obvious toxicity and decreased the biological activity of macrophages; therefore, the copolymer can be used to inhibit the immunological activity of macrophages. Conducive to the release of drugs or shifts.
  • the DCFH-DA fluorescence intensity of the cells cultured with the AAS content of 60% was significantly lower than that of the endotoxin sample LPS, and higher than the blank sample, indicating that the copolymer was less toxic and biocompatible. This indicates that this type of copolymer has little effect on the phagocytosis of macrophages.
  • the DCFH-DA fluorescence intensity of the cells cultured with the AAS content of 80% was significantly lower than that of the endotoxin sample LPS, slightly higher than the blank sample, indicating that the copolymer was less toxic and biocompatible.
  • Figure 3 and Figure 4 show the changes in bacterial numbers after co-culture of macrophages with E. coli and S. Aureus for 24 hours after culture of different AAS copolymers and macrophages. Numerical diagram. It can be seen from Fig. 3 and Fig. 4 that the copolymer having an AAS content of 80% greatly promoted the ability of macrophages to phagocytose bacteria, and the number of cells was significantly less than that in blank cells. This indicates that the copolymer containing 80% AAS can promote the phagocytosis of macrophages and can be applied to immune cell promoters.

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Abstract

本发明适用于高分子聚合物制备及应用领域，提供了DMAPS-AA-AAS共聚物的制备方法，包括以下步骤：将单体DMAPS与单体AA加入到去离子水中，得混合液；配制引发剂溶液，将所述引发剂加入到所述混合液中，然后将所得溶液加热到65～85℃，反应4～6小时，冷却至室温，获得DMAPS-AA共聚物；向所述DMAPS-AA共聚物中加入5～15wt%浓度的氢氧化钠溶液，调整pH值，搅拌2～5小时，制得DMAPS-AA-AAS共聚物。本发明还提供了DMAPS-AA-AAS共聚物，采用上述的制备方法制成。本发明还提供了DMAPS-AA-AAS共聚物的应用。本发明提供的DMAPS-AA-AAS共聚物为巨噬细胞免疫活性的调整，开辟了新的途径。所提供的制备方法，操作简单，便于大批量生产。

Description

DMAPS-AA-AAS共聚物及其制备方法和应用 技术领域
本发明属于高分子聚合物制备及应用领域，尤其涉及DMAPS-AA-AAS共聚物及其制备方法和应用。
背景技术
免疫细胞在预防疾病、肿瘤和癌症治疗等方面具有重要的作用，特别是巨噬细胞在各种功能的免疫响应中起着重要的作用，包括从伤口愈合到抵抗微生物。目前，抗体或主动免疫生物材料被广泛应用促进巨噬细胞吞噬肿瘤细胞或细菌。为了弱化非特异吞噬作用，纳米粒子或其他移植物通常采用一些被动免疫生物材料进行表面处理，比如采用磷酰胆碱两性离子共聚物，或采用多糖类表面包覆。另外，也有研究报道说人的中性粒细胞的吞噬作用可以被β-1、6-葡聚糖激活。因此，我们相信免疫细胞的吞噬作用是可以通过合适的生物材料进行调节。
在现有研究中，一些天然化合物已经被用来调节巨噬细胞的吞噬作用，包括MAP1S蛋白质，TRPV4，细胞外基质凝胶，synaptotagmin蛋白质，脂多糖等。这些天然化合物对巨噬细胞的调节也确实起到了一定效果，但作用较为单一，作用效果有限。
因此，现有技术还有待改进。
 技术问题
本发明所要解决的技术问题在于提供 DMAPS-AA-AAS （ DMAPS ：N，N-二甲基（甲基丙烯酰氧乙基）铵基丙磺酸内盐，AA：丙烯酸 ，AAS：丙烯酸钠 ）共聚物及其制备方法和应用 ，旨在进一步提高人们对巨噬细胞生物活性的调节作用。
技术解决方案
本发明是这样实现的， DMAPS-AA-AAS 的制备方法，包括以下步骤：
步骤一：将单体DMAPS与单体AA加入到去离子水中，得混合液；
步骤二： 配制引发剂溶液，将所述引发剂 加入到所述混合液中，然后将所得溶液 加热到65 ～85℃，反应 4 ～6小时，冷却至室温，获得DMAPS-AA共聚物；
步骤三： 向所述DMAPS-AA共聚物中加入5 ～15 wt% 浓度的氢氧化钠溶液，调整pH值，搅拌2～5小时，制得DMAPS-AA-AAS共聚物。
本发明还提供了 DMAPS-AA-AAS 共聚物，采用上述所述的方法制备而成。
本发明还提供了 DMAPS-AA-AAS 共聚物的应用，将所述DMAPS-AA-AAS共聚物用于制备 抗炎症、治疗肿瘤/癌症或促进伤口愈合的药物。
进一步地， 将所述DMAPS-AA-AAS共聚物用于制备巨噬细胞的免疫抑制剂。
进一步地，将所述DMAPS-AA-AAS共聚物用于制备巨噬细胞的免疫促进剂。
 有益效果
本发明与现有技术相比，有益效果在于：本发明通过 将制得的 DMAPS-AA 共聚物与氢氧化钠溶液进行反应，同时调整了反应体系的pH值，使所制得的DMAPS-AA-AAS共聚物中AAS占所述DMAPS-AA-AAS共聚物的质量比不同，由此调整DMAPS-AA-AAS共聚物的pH值。由此制得的DMAPS-AA-AAS共聚物， 具有较好的生物相容性和仿生性，而且 其表面的pH值不同时，其具有的生物活性存在显著差别。本发明由此提供了DMAPS-AA-AAS共聚物的新应用， 所述DMAPS-AA-AAS共聚物用于制备 抗炎症、治疗肿瘤/癌症或促进伤口愈合的药物； 用于制备巨噬细胞的免疫抑制剂或者免疫促进剂。本发明提供的DMAPS-AA-AAS共聚物为巨噬细胞免疫活性的调整，开辟了新的途径。所提供的制备方法，操作简单，便于大批量生产。
附图说明
图1是本发明实施例提供的 不同AAS含量的共聚物培养的巨噬细胞的生物活性测试结果示意图；
图2 是本发明实施例提供的 小鼠巨噬细胞（RAW264.7）与不同AAS含量的共聚物培养后的DCFH-DA荧光强度值的结果示意图；
图3 是本发明实施例提供的 经培养的巨噬细胞与大肠杆菌（E.coli）培养24小时后细菌数量变化数值的结果示意图；
图4 是本发明实施例提供的 经培养的巨噬细胞与金黄色葡萄球菌培养24小时后细菌数量变化数值的结果示意图。
 本发明的实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
N，N-二甲基（甲基丙烯酰氧乙基）铵基丙磺酸内盐（DMAPS）与丙烯酸的共聚物DMAPS-AA为一种两性离子共聚物，其较高的水化作用能力和电中性表面，使得其具有较好的抵抗非特异蛋白吸附。采用该两性离子共聚物表面修饰的表面大大减少了纤维蛋白原吸附、血小板粘附、细菌粘附和血浆凝固等。通过研究发现，DMAPS-AA共聚物对pH值变化具有可逆响应性，这一外界响应不仅改变了两性离子共聚物的物理与化学性质，还影响了它们的生物性质，包括生物相容性，生物活性及抗菌性能等。此外，所述两性离子共聚物是非毒性的，因此具有较好的生物相容性和仿生性。
按照本发明的技术方案制备DMAPS-AA-AAS共聚物，过程如下：
步骤一：将单体DMAPS与单体AA加入到去离子水中，得混合液；
步骤二：配制引发剂溶液，将所述引发剂加入到所述混合液中，然后将所得溶液加热到65～85℃，反应4～6小时，冷却至室温，获得DMAPS-AA共聚物；
步骤三：向所述DMAPS-AA共聚物中加入5～15wt%浓度的氢氧化钠溶液，调整pH值，搅拌2～5小时，制得DMAPS-AA-AAS共聚物。
具体地，步骤一中，所述单体DMAPS与单体AA的质量比为1:4～6，优选1:4；所述混合液中，单体DMAPS与单体AA的质量之和与所述去离子水的质量比为20%～30%:1。单体DMAPS与单体AA的质量比不同，制得的DMAPS-AA共聚物在分子机构上会存在差别，则其性质会有所不同。
具体地，步骤二中，所述引发剂为过硫酸铵或过硫酸钾，优选过硫酸铵；所述引发剂溶液的浓度为0.5～1.5wt%，优选0.5wt%；引发剂溶液的配制是在保护气体中进行，保护气体可以是惰性气体或氮气。
具体地，步骤三中，所述DMAPS-AA共聚物与所述氢氧化钠溶液进行反应，反应体系的pH值不同，所制得的DMAPS-AA-AAS共聚物中所述AAS占所述DMAPS-AA-AAS共聚物的质量比会有所不同。反应体系的pH值为2.0～4.0时，所述AAS占所述DMAPS-AA-AAS共聚物的质量比为0～40wt%，优选0～10wt%；反应体系的pH值为6.0～8.0时，优选7.0，所述AAS占所述DMAPS-AA-AAS共聚物的质量比为70～80wt%，优选80wt%。
在本发明的技术方案中，通过氢氧化钠溶液与制备的DMAPS-AA共聚物进行反应，通过氢氧化钠将部分丙烯酸转化为丙烯酸钠，即在DMAPS-AA共聚物中引入单体AAS，以对共聚物的pH值进行调节。本发明证明，DMAPS-AA的共聚物经pH值调节后，其生物活性具有显著的变化，特别是研究发现DMAPS质量百分含量20%，AA质量百分含量80%的共聚物。其中最显著的特征是，经pH值调节后共聚物对巨噬细胞的免疫功能具有调节作用，可用于抗炎症、肿瘤/癌症治疗或伤口愈合等方面的药物的制备。在制备的DMAPS-AA-AAS共聚物中，当AAS占总单体重量为70～80wt%时，该共聚物可使巨噬细胞生物处于免疫亢奋状态；当当AAS占总单体重量为80wt%时，效果最佳；因此可用来制备免疫促进剂。当AAS占总单体重量为0～40wt%时，该共聚物可使巨噬细胞免疫功能下降或散失，当当AAS占总单体重量为0～10wt%时，效果更佳明显；因此可用来制备巨噬细胞的免疫抑制剂。
实施例1
将20g单体DMAPS和80g单体AA加入到反应容器中，加入一定量去离子水搅拌均匀；使单体DMAPS与AA总量占水溶液总量的质量百分比20%；在氮气保护下，将过硫酸铵配成1wt%的水溶液，当水浴温度升至65℃时，用滴液漏斗分次滴加入反应容器中；保温反应5小时, 反应结束后，冷却到室温即可出料,制得DMAPS-AA共聚物。然后在10%浓度的DMAPS-AA 共聚物中加入10wt%浓度的氢氧化钠溶液，调节pH值3.0～3.5，搅拌3小时，使共聚物中部分丙烯酸转化为丙烯酸钠（AAS），使得丙烯酸钠含量占总单体含量的30%，制得AAS含量为30%的DMAPS-AA-AAS共聚物。
实施例2
将20g单体DMAPS和80g单体AA加入到反应容器中，加入一定量去离子水搅拌均匀；使单体DMAPS与AA总量占水溶液总量的质量百分比20%；在氮气保护下，过硫酸铵配成1wt%的水溶液，当水浴温度升至65℃时，用滴液漏斗分次滴加入反应容器中，保温反应5小时, 反应结束后，冷却到室温即可出料,制得DMAPS-AA共聚物。然后在10%浓度的DMAPS-AA 共聚物中加入10wt%浓度的氢氧化钠溶液，调节pH值5.5～6.0，搅拌3小时，使共聚物中部分丙烯酸转化为丙烯酸钠（AAS），使得丙烯酸钠含量占总单体含量的60%，制得AAS含量为60%的DMAPS-AA-AAS共聚物。
实施例3
将20g单体DMAPS和80g单体AA加入到反应容器中，加入一定量去离子水搅拌均匀;使单体DMAPS与AA总量占水溶液总量的质量百分比20%；在氮气保护下，引发剂过硫酸铵配成1wt%的水溶液，当水浴温度升至65℃时，用滴液漏斗分次滴加入反应容器中；保温反应5小时, 反应结束后，冷却到室温即可出料,制得DMAPS-AA共聚物。然后在10%浓度的DMAPS-AA 共聚物中加入10wt%浓度的氢氧化钠溶液，调节pH值7.0～7.5，搅拌3小时，使共聚物中全部丙烯酸转化为丙烯酸钠（AAS），使得丙烯酸钠含量占总单体含量的80%，制得80%AAS含量的DMAPS-AA-AAS共聚物。
采用细胞活力测试方法（如CCK-8）测试小鼠巨噬细胞（RAW264.7）生物活性；采用超氧自由基（ROS）测试方法，用荧光探针 DCFH-DA(Olympus, Japan)检测小鼠巨噬细胞（RAW264.7）与共聚物培养20分钟后细胞内积累的 ROS 含量，检测DCFH-DA荧光强度可以分析其生物活性。
图1是不同AAS含量的DMAPS-AA-AAS共聚物培养的巨噬细胞的生物活性测试图，从图1中可以看到：AAS含量为30%的共聚物培养后的细胞活性明显比空白细胞下降，表明该共聚物生物活性下降；AAS含量为60%的共聚物培养后的细胞活性基本与空白细胞相同，表明该共聚物生物相容性理想；AAS含量为80%的共聚物培养后的细胞活性明显比空白细胞提高,巨噬细胞处于亢奋状态。
图2是小鼠巨噬细胞（RAW264.7）与不同AAS含量的共聚物培养后的DCFH-DA荧光强度值比较结果，从图2中也可以看到：AAS含量为30%的共聚物培养后的细胞的DCFH-DA荧光强度值明显比内毒素样品LPS的高很多，表明该共聚物具有明显的毒性，使巨噬细胞生物活性下降；因此该共聚物可以用来抑制巨噬细胞的免疫活性，有利于药物或移值物的释放。AAS含量为60%的共聚物培养后的细胞的DCFH-DA荧光强度值明显比内毒素样品LPS的低，而高于空白样品，表明该共聚物毒性较小,生物相容性可以接受。这表明该类共聚物对巨噬细胞的吞噬作用影响较小。AAS含量为80%的共聚物培养后的细胞的DCFH-DA荧光强度值明显比内毒素样品LPS的低，略高于空白样品，表明该共聚物毒性很小,生物相容性理想。
图3、图4分别是不同AAS含量共聚物与巨噬细胞培养后，巨噬细胞再与大肠杆菌（E.coli）、金黄色葡萄球菌(S.Aureus)共同培养24小时后，细菌数量变化数值图。从图3与图4中可以看到AAS含量为80%的共聚物大大促进了巨噬细胞的吞噬细菌能力，细胞数量明显比空白细胞中少。由此说明含量80%AAS的共聚物能促进巨噬细胞的吞噬作用，可应用于免疫细胞促进剂。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. DMAPS-AA-AAS共聚物的制备方法，其特征在于，包括以下步骤：
    步骤一：将单体DMAPS与单体AA加入到去离子水中，得混合液；
    步骤二：配制引发剂溶液，将所述引发剂加入到所述混合液中，然后将所得溶液加热到65～85℃，反应4～6小时，冷却至室温，获得DMAPS-AA共聚物；
    步骤三：向所述DMAPS-AA共聚物中加入5～15wt%浓度的氢氧化钠溶液，调整pH值，搅拌2～5小时，制得DMAPS-AA-AAS共聚物。
  2. 如权利要求1所述的DMAPS-AA-AAS共聚物的制备方法，其特征在于，步骤一中所述单体DMAPS与单体AA的质量比为1:4～6。
  3. 如权利要求1所述的DMAPS-AA-AAS共聚物的制备方法，其特征在于，所述引发剂溶液的浓度为0.5～1.5wt%，所述引发剂为过硫酸铵或过硫酸钾。
  4. 如权利要求1所述的DMAPS-AA-AAS共聚物的制备方法，其特征在于，步骤三中所述pH值为2.0～4.0，所述AAS占所述DMAPS-AA-AAS共聚物的质量比为0～40wt%。
  5. 如权利要求1所述的DMAPS-AA-AAS共聚物的制备方法，其特征在于，步骤三中所述pH值为6.0～8.0，所述AAS占所述DMAPS-AA-AAS共聚物的质量比为70～80wt%。
  6. 一种DMAPS-AA-AAS共聚物，其特征在于，采用权利要求1～4任意一项所述的制备方法制成。
  7. 一种DMAPS-AA-AAS共聚物，其特征在于，采用权利要求5所述的制备方法制成。
  8. 如权利要求6或7所述的DMAPS-AA-AAS共聚物的应用，其特征在于，所述应用为将所述DMAPS-AA-AAS共聚物用于制备抗炎症、治疗肿瘤/癌症或促进伤口愈合的药物。
  9. 如权利要求6所述的DMAPS-AA-AAS共聚物的应用，其特征在于，所述应用为将所述DMAPS-AA-AAS共聚物用于制备巨噬细胞的免疫抑制剂。
  10. 如权利要求7所述的DMAPS-AA-AAS共聚物的应用，其特征在于，所述应用为将所述DMAPS-AA-AAS共聚物用于制备巨噬细胞的免疫促进剂。
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