CN111803449B - 一种用于免疫调控的高分子微球及其制备方法和应用 - Google Patents
一种用于免疫调控的高分子微球及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供一种用于免疫调控的高分子微球及其制备方法和应用，制备方法包括如下步骤：促巨噬细胞再生分型用药与纳米晶须分散于有机溶剂/水复合溶液中，得到溶液A；可降解高分子微球溶于易挥发溶剂中，得到溶液B；溶液A与溶液B进行乳化，得到乳化液；所述乳化液加入至含表面活性剂的水溶液中，搅拌，得微球；微球淬冷，得到用于免疫调控的高分子微球。利用有机溶剂和液氮淬冷处理协同调控纳米晶须和药物在微球中均匀分布的作用，从而制得可调控免疫反应的纳米增强可降解高分子微球。
Description
技术领域
本发明属于高分子微球技术领域，尤其涉及一种用于免疫调控的高分子微球及其制备方法和应用。
背景技术
用于组织修复与再生的材料必须对组织、细胞无毒等，且在植入后不应引起过度的炎症免疫反应。如此，才可有效保证组织的成功修复与再生。其中，巨噬细胞是关键的免疫细胞，它与植入体之间的相互作用将影响其促炎或再生分型，并将决定后续组织修复与再生是否成功。
高分子微球通常指直径在纳米至微米尺度，形状为球形的高分子聚集体。高分子微球以其可设计性和多功能性吸引了越来越多科学工作者的兴趣，并在组织修复与再生领域得到广泛的研究与应用。目前应用于微球的材料主要分为无机材料、天然高分子材料及合成的高分子材料。按降解性能又可分为可降解类材料及非可降解类材料。其中人工合成的可降解高分子材料可以通过改变其原材料化学组成、材料结构及表面性质等，来设计其生物应答特性。聚酯类是目前研究最多、应用最广泛的可生物降解的合成高分子材料，如聚乳酸、聚乙醇酸、聚ε-己内酯、聚β-羟基丁酸、聚β-羟基戊酸以及它们的共聚物。乳酸-乙醇酸的共聚物是被美国食品药品管理局批准应用于临床的高分子材料，具有良好的生物相容性。
但乳酸-乙醇酸的共聚物等人工合成高分子在体内降解形式为本体降解，期间会碎裂为块体颗粒，主要被促炎分型的巨噬细胞所吞噬。但同时，由于这过程中促进了巨噬细胞的炎症型分化，加重了局部炎症效应，不利于骨缺损的修复再生。而现有技术制得的低炎症反应的体系普遍存在1)需要外用装置来协同调控炎症反应或缺少有效地药物载体材料；2)免疫调控体系成份较为复杂，其潜在生物安全性需要多方评价的确认；3)制备工艺复杂、难以产业化的问题。
纳米晶须是一种由高纯度单晶生长而成的纳米级短纤维，晶须的高度取向结构使其具有高强度、高模量和高伸长率，可作为增强材料。
发明内容
本发明的目的在于提供一种用于免疫调控的高分子微球及其制备方法和应用，使用纳米晶须作为聚合物基微球的“钢筋”，调控纳米晶须在聚合物相中的分布及定向排列，并利用有机溶剂和液氮淬冷处理协同调控纳米晶须和药物在微球中均匀分布的作用，从而制得可调控免疫反应的纳米增强可降解高分子微球。制备的微球不仅具有良好的生物相容性及可控的降解动力学，可调控巨噬细胞的再生分型，可应用于创面修复材料、食品药品材料和药物载体材料中。
本发明所采取的技术方案是：
本发明的第一个方面，提供：
一种用于免疫调控的高分子微球的制备方法，包括如下步骤：
(1)促巨噬细胞再生分型用药与纳米晶须分散于有机溶剂/水复合溶液中，得到溶液A；
(2)可降解高分子微球溶于易挥发溶剂中，得到溶液B；
(3)溶液A与溶液B进行乳化，得到乳化液；所述乳化液加入至含表面活性剂的水溶液中，搅拌，得到微球；
(4)微球淬冷，得到用于免疫调控的高分子微球。
优选的，上述步骤(4)还包括在淬冷后，对用于免疫调控的高分子微球进行提纯，具体来说，提纯包括清洗和干燥，通过清洗和干燥的方法，可以进一步提高用于免疫调控的高分子微球的纯度。
优选的，上述步骤(1)中分散的速度为200～20000rpm，分散的时间为1min～24h。
优选的，上述步骤(3)中乳化的速度为6000～20000rpm，乳化的时间为0.5min～2min。
优选的，上述步骤(3)中搅拌的转速为200～1000rpm，搅拌的时间为30min～90min。
优选的，上述步骤(4)中淬冷为液氮淬冷，淬冷的温度为-80℃～-40℃，淬冷的时间为2s～10min。
优选的，上述干燥的方式选自室温风干干燥、热干燥、冷冻干燥中的至少一种，干燥的时间为24h～48h。
优选的，上述步骤(1)中促巨噬细胞再生分型用药选自干扰素4、干扰素17F、白藜芦醇、肿瘤坏死因子-α中的至少一种。
优选的，上述步骤(1)中纳米晶须选自碳化硅、氮化硼、氮化硅、纤维素、甲壳素、氧化锌、碳化钨、硫酸钙、氧化铝、钛酸钾、二氧化钛中的至少一种。
优选的，上述步骤(1)中有机溶剂选自甲醇、乙醇、乙醚、己烷、异丙醇、丙酮中的至少一种。
优选的，上述步骤(1)中有机溶剂与水的体积比为(0.4～1.2)：1。
优选的，上述步骤(1)中促巨噬细胞再生分型用药与所述有机溶剂/水复合溶液的质量体积比为(0.015～0.05)：1g/mL
优选的，上述步骤(1)中纳米晶须与有机溶剂/水复合溶液的质量体积比为(0.4～1.2)：1g/mL。
优选的，上述步骤(2)中可降解高分子选自聚乳酸、聚乳酸-羟基乙酸共聚物、聚己内酯、聚3-羟基烷酸酯、聚(3-羟基丁酸酯)、聚3-羟基丁酸酯-co-3-羟基戊酸酯、聚三亚甲基碳酸酯、聚丁二酸丁二酯中的至少一种。
优选的，上述步骤(2)中可降解高分子的分子量为3000～60000道尔顿。
优选的，上述步骤(2)中易挥发溶剂选自乙酸乙酯、二氯甲烷、三氯甲烷、四氢呋喃中的至少一种。
优选的，上述步骤(2)中可降解高分子与易挥发溶剂的质量体积比为(0.05～0.4)：1g/mL。
优选的，上述步骤(3)中乳化液中的纳米晶须与可降解高分子的质量比为(0.05～0.5)：1。
优选的，上述步骤(3)中表面活性剂选自明胶、甲基纤维素、聚乙烯醇中的至少一种。
优选的，上述步骤(3)中含表面活性剂的水溶液的表面活性剂质量浓度为0.5％～1.5％；
优选的，上述步骤(3)中乳化液与含表面活性剂的水溶液的体积比为(0.00125～0.08)：1。
本发明的第二个方面，提供：
一种用于免疫调控的高分子微球，由上述用于免疫调控的高分子微球的制备方法制得。
优选的，上述用于免疫调控的高分子微球的粒径为20～300μm。
本发明的第三个方面，提供：
一种用于免疫调控的高分子微球在创面修复材料、食品药品材料和药物载体材料中的应用，所述用于免疫调控的高分子微球为上述用于免疫调控的高分子微球。
本发明的有益效果为：
1.本发明的微球制备方法基于传统的乳化溶剂挥发法，针对不同种类的纳米晶须和促巨噬细胞再生分型用药，在内水相中加入有机溶剂，调控在易挥发性有机溶剂中纳米晶须与可降解高分子的相互作用力，从而达到调控纳米晶须与促巨噬细胞再生分型用药在微球中均匀分布的效果，调控用于免疫调控的高分子微球降解产物尺寸，在降低其引起的免疫炎症反应的同时，引入促巨噬细胞再生分型用药，能进一步促进巨噬细胞再生，能进一步促进巨噬细胞向能促进成骨矿化的再生型分化。
2.本发明针对含有不同纳米晶须的乳液体系，在微球成型过程中使用液氮淬冷的手段调控纳米晶须在微球中的分布及微球成型速率，与内水相中的有机溶剂达到协同调控纳米晶须和促巨噬细胞再生分型用药在微球中均匀分布的效果，并进一步达到调控巨噬细胞的再生分型的目的。
3.本发明乳化液中纳米晶须与可降解高分子聚合物的质量浓度比远小于1，使得本发明制得的高分子微球中纳米晶须可均匀分布在微球内部，与可降解高分子聚合物链段有良好的相互作用力，能作为聚合物材料的“钢筋”，从而在高分子微球降解时，调控降解产物的尺寸及其分布，使其不易激活巨噬细胞等免疫细胞或其炎症化。
4.本发明的的制备方法工艺简单，对设备的要求不高，原料均已产业化、来源易得，成本低廉，易于实现产业化。
附图说明
图1为实施例1～5和对比例1～4制得的微球的体外巨噬细胞共培养时CD206含量图。
具体实施方式
为了使本发明的发明目的、技术方案及其技术效果更加清晰，以下结合具体实施方式，对本发明进行进一步详细说明。应当理解的是，本说明书中描述的具体实施方式仅仅是为了解释本发明，并非为了限定本发明。实施例或对比例或测试例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施例1：一种用于免疫调控的高分子微球的制备方法：
(1)将400mg肿瘤坏死因子-α和400mg纳米氮化硅晶须分散于8mL乙醚/水(体积比为1.0：1)复合溶液中，15000rpm下搅拌1.5min，得到溶液A；。
(2)将1g聚乳酸溶于10mL乙酸乙酯，得到溶液B；
(3)将溶液A和溶液B进行乳化处理，300rpm下乳化处理18h，将该乳化液加入400mL质量浓度为1.0％的甲基纤维素的水溶液中，搅拌转速为500rpm，室温下搅拌30min后，得到微球；
(4)微球使用-80℃液氮进行淬冷处理1min，冷冻干燥36h后得到用于免疫调控的高分子微球。
实施例2：一种用于免疫调控的高分子微球的制备方法：
(1)将180mg干扰素4和200mg纳米硫酸钙晶须分散于4mL乙醇/水(体积比为0.5：1)复合溶液中，15000rpm下搅拌1.5min，得到溶液A；
(2)将2g聚乳酸-羟基乙酸共聚物溶于10mL二氯甲烷，得到溶液B；
(3)将溶液A和溶液B进行乳化处理，200rpm下乳化处理24h，将该乳化液加入200mL质量浓度为0.5％的1788型聚乙烯醇的水溶液中，搅拌转速为200rpm，室温下搅拌75min后，得到微球；
(4)微球使用-40℃液氮进行淬冷处理2min，冷冻干燥48h后得到用于免疫调控的高分子微球。
实施例3：一种用于免疫调控的高分子微球的制备方法：
(1)将200mg干扰素17F和300mg纳米硫酸钙晶须分散于6mL己烷/水(体积比为0.8：1)复合溶液中，6000rpm下搅拌2min，得到溶液A；
(2)将1.5g聚乳酸-羟基乙酸共聚物溶于10mL四氢呋喃，得到溶液B；
(3)将溶液A和溶液B进行乳化处理，300rpm下乳化处理18h，将该乳化液加入500mL质量浓度为0.8％的明胶的水溶液中，搅拌转速为300rpm，室温下搅拌45min后，得到微球；
(4)微球使用-60℃液氮进行淬冷处理0.5min，冷冻干燥24h后得到用于免疫调控的高分子微球。
实施例4：一种用于免疫调控的高分子微球的制备方法：
(1)将150mg干扰素4和600mg纳米纤维素晶须分散于10mL丙酮/水(体积比为0.4：1)复合溶液中，10000rpm下搅拌2min，得到溶液A；
(2)将0.5g聚己内酯溶于10mL二氯甲烷，得到溶液B；
(3)将溶液A和溶液B进行乳化处理，8000rpm下乳化处理5min，将该乳化液加入125mL质量浓度为0.8％的124型聚乙烯醇的水溶液中，搅拌转速为1000rpm，室温下搅拌90min后，得到微球；
(4)微球使用-50℃液氮进行淬冷处理1.5min，冷冻干燥24h后得到用于免疫调控的高分子微球。
实施例5：一种用于免疫调控的高分子微球的制备方法：
(1)将200mg白藜芦醇和200mg纳米甲壳素晶须分散于6.7mL乙醇/水(体积比为1.2：1)复合溶液中，20000rpm下搅拌0.5min，得到溶液A；
(2)将4g聚3-羟基丁酸酯-co-3-羟基戊酸酯溶于10mL三氯甲烷，形成溶液B；
(3)将溶液A和溶液B进行乳化处理，20000rpm下乳化处理1min，将该乳化液加入800mL质量浓度为的1.5％甲基纤维素的水溶液中，搅拌转速为600rpm，室温下搅拌60min后，得到微球；
(4)微球使用-70℃液氮进行淬冷处理10s，冷冻干燥48h后得到用于免疫调控的高分子微球。
对比例1：一种高分子微球的制备方法：
(1)将180mg干扰素4和200mg纳米硫酸钙晶须分散于4mL水溶液中，15000rpm下搅拌1.5min，得到溶液A；
(2)将2g聚乳酸-羟基乙酸共聚物溶于10mL二氯甲烷，得到溶液B；
(3)将溶液A和溶液B进行乳化处理，200rpm下乳化处理24h，将该乳化液加入200mL质量浓度为0.5％的1788型聚乙烯醇的水溶液中，搅拌转速为200rpm，室温下搅拌75min后，得到微球；
(4)微球使用-40℃液氮进行淬冷处理2min，冷冻干燥48h后得到高分子微球。
对比例2：一种高分子微球的制备方法：
(1)将180mg干扰素4和200mg纳米硫酸钙晶须分散于4mL乙醇/水(体积比为0.5：1)复合溶液中，15000rpm下搅拌1.5min，得到溶液A；
(2)将2g聚乳酸-羟基乙酸共聚物溶于10mL二氯甲烷，得到溶液B；
(3)将溶液A和溶液B进行乳化处理，200rpm下乳化处理24h，将该乳化液加入200mL质量浓度为0.5％的1788型聚乙烯醇的水溶液中，搅拌转速为200rpm，室温下搅拌10h后，停止搅拌，得到微球；
(4)对微球进行水洗并离心(1000rpm，10min，重复3次)，冷冻干燥48h后得到高分子微球。
对比例3：一种高分子微球的制备方法：
(1)将180mg干扰素4和200mg纳米硫酸钙晶须分散于4mL水溶液中，15000rpm下搅拌1.5min，得到溶液A；
(2)将2g聚乳酸-羟基乙酸共聚物溶于10mL二氯甲烷，得到溶液B；
(3)将溶液A和溶液B进行乳化处理，200rpm下乳化处理24h，将该乳化液加入200mL质量浓度为0.5％的1788型聚乙烯醇的水溶液中，搅拌转速为200rpm，室温下搅拌10h后，停止搅拌，得到微球；
(4)对微球进行水洗并离心(1000rpm，10min，重复3次)，冷冻干燥48h后得到高分子微球。
对比例4：一种高分子微球的制备方法：
(1)将2g聚乳酸-羟基乙酸共聚物溶于10mL二氯甲烷，得到溶液B；
(2)将溶液B加入200mL质量浓度为0.5％的1788型聚乙烯醇的水溶液中，搅拌转速为200rpm，室温下搅拌10h后，停止搅拌，得到微球；
(3)对微球进行水洗并离心(1000rpm，10min，重复3次)，冷冻干燥48h后得到高分子微球。
测试例：将实施例1～5制得的用于免疫调控的高分子微球和对比例1～4制得的高分子微球进行体外细胞毒性评价和体外干细胞成骨诱导分化性能检测。
1.体外细胞毒性评价
评价方法：本测试例选择MTT试剂来评价高分子微球的细胞毒性。种植有细胞的微球用无菌的PBS溶液洗涤2～3次；在每个孔中加入70μL的四甲基偶氮唑蓝和700μL无血清培养液，然后将孔板置于培养箱中；4h后，将孔板从培养箱取出。小心取出微球，将微球分别放入1.5mL的离心管中，每个离心管加入0.8mL的二甲基亚砜；待所有支架完全溶解以后，用酶标仪测定490nm波长下各孔的吸光值。
分别取实施例1～5制备得到的用于免疫调控的高分子微球和对比例1～4制备得到的高分子微球，按照上述方法进行体外细胞毒性评价和计分，结果如表1所示：
表1体外细胞毒性测试评价计分结果
| 实施例1 | 实施例2 | 实施例3 | 实施例4 | 实施例5 | 对比例1 | 对比例2 | 对比例3 | 对比例4 | |
| 计分 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
2.体外巨噬细胞再生分型诱导分化性能检测
体外实验采用小鼠外周巨噬细胞系RAW 264.7(RAW)细胞(中国科学院上海细胞库)。在细胞培养箱内采用两个细胞的培养基α-MEM(3T3)培养细胞，当细胞融合度达90％时采用胰酶消化，用于后续细胞实验。
实施例1～5制备得到的用于免疫调控的高分子微球和对比例1～4制备得到的高分子微球分别进行体外干细胞成骨诱导分化性能测试，每个实施例或对比例微球的测试方法为：24孔板每个孔的底部铺上250μL质量浓度为1％的无菌琼脂糖，在其完全凝固前加入微球，使微球固定于孔板。然后将微球浸渍于体积浓度70％的乙醇溶液2h，随后吸去乙醇，用PBS洗涤5遍，在紫外灯下照射20min。随后，每个孔加入500μL的培养液，将孔板放入培养箱中24h。将孔板中的培养液以及微球表面的培养液吸出，把250μL的细胞(小鼠外周巨噬细胞)悬液(1*106细胞数/mL)均匀地滴加在微球上。滴加完毕后，将孔板放入培养箱1h，待大部分细胞黏附于微球上以后，在每个孔中添加额外的750μL培养液。种植细胞的支架材料继续在温度为37℃且体积浓度为5％二氧化碳气氛下的培养箱中培养。
培养72h后，采用流式细胞仪检测巨噬细胞不同表现型的表面标记蛋白。步骤如下：实验开始前采用质量浓度为1％牛血清白蛋白的磷酸缓冲盐溶液稀释CD206抗体(M2marker，1:300dilution；Thermo Fisher)备用。将培养有小鼠外周巨噬细胞系RAW264.7细胞的孔板置于冰上。加磷酸缓冲盐溶液润洗两遍后用细胞刮刀将细胞刮下，1000rpm离心5min。用质量浓度为1％牛血清白蛋白的磷酸缓冲盐溶液重悬，在4℃离心机内1000rpm离心5min。倒掉上层液体后，加1mL质量浓度为4％的多聚甲醛冰上固定30min。离心去掉多聚甲醛后磷酸缓冲盐溶液洗1遍。加入1mL质量浓度为1％的TritonX-100(聚乙二醇辛基苯基醚)，置于冰上放置10min。磷酸缓冲盐溶液洗1遍后加入200μL抗体，孵育30min。孵育完成后加入96孔板内，放入流式细胞仪采用Guava Easycyte HT system(MerkMillipore)检测表面标记蛋白。结果如图1所示。
结果分析：由表1可知，本发明方法所制备的高分子微球均无细胞毒性。由图1可看出，实施例2与对比例1～4都是基于聚乳酸-羟基乙酸共聚物制备的高分子微球。其中，实施例2制得纳米晶须均匀分散的用于免疫调控的高分子微球，对比例1为无内水性有机溶剂辅助调控制备的纳米增强可降解高分子微球，对比例2为制备过程中不采用液氮淬冷处理纳米增强可降解高分子微球，对比例3为内水性不添加有机溶剂，制备过程中也不采用液氮淬冷处理的高分子微球，对比例4为非纳米增强可降解高分子微球。在这5组材料中，实施例2制备的用于免疫调控的高分子微球与巨噬细胞共培养后致其分泌的CD206最多，且相对于对比例1～4，实施例1、3～5制得用于免疫调控的高分子微球与巨噬细胞共培养后致其分泌的CD206也相对增多，表明这种纳米晶须分布均匀的用于免疫调控的高分子微球上的巨噬细胞的炎症反应趋势最低。相对于实施例1～5和对比例1～3，对比例4制得的高分子微球与巨噬细胞共培养后致其分泌的CD206最少，进一步表明对于本发明方法制得的微球中的药物和纳米纳米晶须的均匀分布对巨噬细胞的再生分型有更佳的促进作用。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (11)
1.一种用于免疫调控的高分子微球的制备方法，其特征在于：包括如下步骤：
（1）促巨噬细胞再生分型用药与纳米晶须分散于有机溶剂/水复合溶液中，得到溶液A；
（2）可降解高分子溶于易挥发溶剂中，得到溶液B；
（3）溶液A与溶液B进行乳化，得到乳化液；所述乳化液加入至含表面活性剂的水溶液中，搅拌，得微球；
（4）微球淬冷，得到用于免疫调控的高分子微球；
所述促巨噬细胞再生分型用药选自干扰素4、干扰素17F、白藜芦醇、肿瘤坏死因子-α中的至少一种。
2.根据权利要求1所述的用于免疫调控的高分子微球的制备方法，其特征在于：步骤（1）中所述有机溶剂与水的体积比为（0.4~1.2）：1。
3.根据权利要求1所述的用于免疫调控的高分子微球的制备方法，其特征在于：步骤（1）中所述促巨噬细胞再生分型用药与所述有机溶剂/水复合溶液的质量体积比为（0.015~0.05）：1g/mL。
4.根据权利要求1或2所述的用于免疫调控的高分子微球的制备方法，其特征在于：所述有机溶剂选自甲醇、乙醇、乙醚、己烷、异丙醇、丙酮中的至少一种。
5.根据权利要求1所述的用于免疫调控的高分子微球的制备方法，其特征在于：步骤（4）中所述淬冷为液氮淬冷，所述淬冷的温度为-80℃~-40℃，所述淬冷的时间为10s~2min。
6.根据权利要求1所述的用于免疫调控的高分子微球的制备方法，其特征在于：步骤（2）中所述可降解高分子与所述易挥发溶剂的质量体积比为（0.05~0.4）：1g/mL。
7.根据权利要求1或6所述的用于免疫调控的高分子微球的制备方法，其特征在于：所述易挥发溶剂选自乙酸乙酯、二氯甲烷、三氯甲烷、四氢呋喃中的至少一种。
8.根据权利要求1所述的用于免疫调控的高分子微球的制备方法，其特征在于：步骤（3）中所述乳化液中的纳米晶须与可降解高分子的质量比为（0.05~0.5）：1。
9.根据权利要求1或2或8任一项所述的用于免疫调控的高分子微球的制备方法，其特征在于：所述纳米晶须选自碳化硅、氮化硼、氮化硅、纤维素、甲壳素、氧化锌、碳化钨、硫酸钙、氧化铝、钛酸钾、二氧化钛中的至少一种。
10.一种用于免疫调控的高分子微球，其特征在于：由权利要求1~9任一项所述的用于免疫调控的高分子微球的制备方法制得。
11.一种用于免疫调控的高分子微球在制备创面修复材料和药物载体材料中的应用，其特征在于：所述用于免疫调控的高分子微球为权利要求10所述用于免疫调控的高分子微球。
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| CN103877029A (zh) * | 2014-03-10 | 2014-06-25 | 同济大学 | 一种新型磁性载5-氟尿嘧啶聚乳酸羟基乙酸共聚物材料的制备方法 |
| CN107714674A (zh) * | 2017-10-23 | 2018-02-23 | 天津双硕医药科技有限公司 | 一种plga微球的制备方法 |
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