CN116983269A - 一种载细胞多孔微球及其制备方法和应用 - Google Patents
一种载细胞多孔微球及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种PLGA多孔微球,具体涉及一种载细胞多孔微球及其制备方法和应用所述的载细胞多孔微球为PLGA/PDA/CGRP多孔微球,所述的PLGA/PDA/CGRP多孔微球为负载有CGRP的PLGA/PDA多孔微球,所述的PLGA/PDA多孔微球为通过PDA表面修饰的PLGA多孔微球。与现有技术相比,本发明解决炎症微环境中干细胞活性不足、功能性下降、组织再生效果差,而现有技术中多孔微球无法调控微环境的问题,本发明通过缓释多肽实现了形成有利于移植骨髓间充质干细胞(BMSCs)存活的微环境。
Description
技术领域
本发明涉及一种PLGA多孔微球,具体涉及一种载细胞多孔微球及其制备方法和应用。
背景技术
微球药物是指药物溶解或分散在成球材料中,形成的骨架型微小球形或类球形微粒,其粒径范围一般在1-250μm,可以供口服、注射、滴鼻或皮下埋植使用。
常用的合成材料,主要为已被美国FDA批准的可安全药用的聚酯类材料,包括聚乳酸(polylacticacid,PLA)、聚乙醇酸(Polyglycolic acid,PGA)、聚乳酸羟基乙酸共聚物(poly lactic-co-gly-colic acid,PLGA)和聚己内酯(Polycaprolactone,PCL)等。其中,PLA和PLGA以其良好的生物相容性和生物可降解性被广泛应用在缓控释注射给药系统。
PLGA具有良好的生物相容性和生物降解性,其材料降解产物与机体代谢产物相同,不会对机体产生不良反应,因此被广泛应用于医学工程材料和药物递送领域在所有已上市的微球产品中,PLGA是最常用的载体材料,Lurpon Depot、Zoladex、Sandotatin LAR、Risperdal Consta等均是以PLGA为载体制备的微球。此外,以PLGA微球为载体的研究,如:CN201410005888.8(一种载硫酸庆大霉素多孔羟基磷灰石/PLGA微球的制备方法)、CN201610881156.4(一种可控制备载紫杉醇PLGA多孔微球的方法)、胰高血糖素样肽-1缓释微球技术进展(刘波,阮思达,蔡挺.中国新药杂志,2021,30(13):1184-1191.)、Photothermal hydrogel platform for prevention of post-surgical tumorrecurrence and improving breast reconstruction(Yang X,Gao L,Wei Y,etal.Journal of Nanobiotechnology,2021,19(1):307.)等。
然而现有技术中公开的各种方案中均是针对于特定疾病所研制,而非调控炎症微环境,无法克服炎症微环境中干细胞活性不足、功能性下降、组织再生效果差的情况。
发明内容
本发明的目的就是为了解决上述问题至少其一而提供一种载细胞多孔微球及其制备方法和应用,以解决炎症微环境中干细胞活性不足、功能性下降、组织再生效果差,而现有技术中多孔微球无法调控微环境的问题,本发明通过缓释多肽实现了形成有利于移植骨髓间充质干细胞(BMSCs)存活的微环境。
本发明的目的通过以下技术方案实现:
本发明第一方面公开了一种载细胞多孔微球,
所述的载细胞多孔微球为PLGA/PDA/CGRP多孔微球,
所述的PLGA/PDA/CGRP多孔微球为负载有CGRP的PLGA/PDA多孔微球,
所述的PLGA/PDA多孔微球为通过PDA表面修饰的PLGA多孔微球。
优选地,所述的PLGA多孔微球的粒径为20-180μm。
优选地,所述的PLGA多孔微球的大孔孔径为5-40μm。
本发明第二方面公开了一种制备如上任一所述的载细胞多孔微球的方法,包括如下步骤:
S1:将PLGA多孔微球加入PDA溶液中,搅拌下反应,得到PLGA/PDA多孔微球;
S2:将步骤S1得到的PLGA/PDA多孔微球冷冻干燥;
S3:将步骤S2冻干的PLGA/PDA多孔微球浸泡于CGRP溶液中,再次冷冻干燥后得到PLGA/PDA/CGRP多孔微球。
优选地,步骤S1中,所述的PLGA多孔微球通过乳化法制得,包括如下步骤:
S1a:将PLGA溶解于二氯甲烷,得到PLGA溶液;
S1b:将造孔剂加入至步骤S1a得到的PLGA溶液中,均质使液体充分乳化;
S1c:将步骤S1b得到的乳化液加入PVA溶液中,搅拌得到PLGA多孔微球。
优选地,包括如下一项或几项:
i)步骤S1a中,PLGA与二氯甲烷的用量比为0.25g:10mL;
ii)步骤S1b中,造孔剂为碳酸氢铵溶液,碳酸氢铵溶液的浓度为5-25wt%;
iii)步骤S1b中,造孔剂与PLGA溶液中PLGA的用量比为5mL:0.25g;
iv)步骤S1b中,均质:采用高速均质机以10000rpm的转速均质2min;
v)步骤S1c中,PVA溶液的浓度为0.1wt%;
vi)步骤S1c中,PVA溶液与乳化液中PLGA的用量比为100mL:0.25g;
vii)步骤S1c中,搅拌:采用磁力搅拌器以600rpm的转速搅拌6h。
优选地,步骤S1中,反应的时间为6h。
优选地,步骤S1中,所述的PDA溶液由多巴胺、pH=8.0的tri-HCl和去离子水以0.02g:100μL:10mL的用量比制备得到。
优选地,步骤S2中,冷冻干燥的时间为24h。
优选地,步骤S3中,PLGA/PDA多孔微球与CGRP溶液的用量比为20mg:200μL,所述的CGRP溶液的浓度为10μM;冷冻干燥的时间为24h。
本发明第三方面公开了一种如上任一所述的载细胞多孔微球在制备炎症状态下用于促进骨形成的药物中的应用。
本发明的工作原理为:
该载细胞多孔微球为通过聚多巴胺(PDA)表面修饰构建的具有降钙素基因相关肽(CGRP)缓释功能的聚乳酸-羟基乙酸共聚物(PLGA)多孔微球;通过缓释多肽形成有利于移植骨髓间充质干细胞(BMSCs)存活的微环境。
与现有技术相比,本发明具有以下有益效果:
该载细胞多孔微球通过调控牙周炎微环境、保护干细胞的活性与功能,促进牙周炎性环境下骨组织再生。
不同浓度CGRP对BMSCs成骨相关基因表达影响不同,其中10-100nM下的多孔微球促成骨效果最强。
不同浓度CGRP与LPS(牙龈卟啉单胞菌来源的脂多糖Pg-LPS)刺激对BMSCs细胞活性的影响,10nM CGRP下的多孔微球可在一定程度上恢复细胞活性。
附图说明
图1为载细胞多孔微球的制备流程示意图;
图2为不同浓度的CGRP对BMSCs成骨相关基因表达的影响图,其中,图2a为ALP,图2b为Col1,图2c为Runx2,图2d为OPN,图2e为OCN,图2f为Osterix;
图3为不同浓度的CGRP与LPS刺激对BMSCs细胞活性的影响图;
图4为明场下PLGA多孔微球的光学显微镜图(200μm);
图5为PLGA多孔微球的电镜图,其中,图5a为多孔微球表面电镜图(20μm),图5b为图5a的放大图(8μm),图5c为多孔微球截面电镜图(20μm),图5d为图5c的放大图(8μm);
图6为不同大孔孔径的PLGA多孔微球表面接种BMSCs的活死细胞染色图(200μm);
图7为PLGA/PDA多孔微球与载细胞多孔微球表面接种BMSCs的OPN免疫荧光染色图(100μm)。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明。
如下示例中若未作特别说明,则所采用的实际可为市售产品,所使用的方法可为本领域常规手段。
制备基于PLGA的多孔微球:
使用水包油包气体(gas in oil in water,G/O/W)的乳化法制备不同孔径的PLGA多孔微球:
(1)将0.25g PLGA溶于10mL二氯甲烷中。
(2)分别将5mL碳酸氢铵溶液(5%、15%、25%)加入到PLGA溶液中,用高速均质机以10000rpm/min的转速均质2分钟使液体充分乳化。
(3)将(2)得到的乳化液滴加入100mL 0.1%PVA溶液中,以600rpm在磁力搅拌器上搅拌6小时,获得PLGA多孔微球。
(4)用去离子水清洗PLGA多孔微球三次,然后重新悬浮在去离子水中。
(5)显微镜下观察多孔微球并拍照。
(6)冷冻干燥PLGA多孔微球24小时,室温干燥条件下储存。
采用活死细胞染色观察不同孔径多孔微球上的细胞生长情况,具体为:
1)BMSCs与PLGA多孔微球共培养:
取生长状态良好的BMSCs细胞,消化、离心后用完全培养基重悬,进行细胞计数,以5×107个/mL的密度分别接种于不同孔径的PLGA多孔微球上,使用巴氏吸管反复吹打,使细胞与微球充分混匀。37℃、5%CO2培养箱中培养。
2)活死细胞染色:
(1)多孔微球上接种细胞后培养第3天时,使用Calcein-AM/PI活细胞/死细胞双染试剂盒检测PLGA多孔微球与PLGA/PDA多孔微球上负载的BMSCs细胞的存活情况。该试剂盒使用可渗透细胞的绿色荧光染料Calcein-AM对活细胞进行染色,死细胞则可以被非渗透性的红色荧光染料碘化丙啶(PI)染色。
(2)将Calcein-AM与PI染料以1:3的比例溶于检测缓冲液中,配制成工作液。
(3)将多孔微球浸泡于染色工作液中,避光、在37℃下孵育15-30分钟。
(4)激光共聚焦显微镜下观察并拍摄3D图像,将图像进行三维重建。
图6为不同大孔孔径的PLGA多孔微球接种BMSCs后的活死细胞染色,CLSM下观察,可见,微球表面大孔孔径越大,黏附的细胞越多,细胞形态更铺展。
根据细胞在多孔微球上的生长情况,优化制备条件后构建基于PLGA的载细胞多孔微球。
实施例1
制备载细胞多孔微球,制备步骤如图1所示:
(1)将5mL 10%碳酸氢铵溶液加入到1%PLGA溶液中,用高速均质机以10000rpm的转速均质2分钟使液体充分乳化。
(2)将(1)得到的乳化液滴加入100mL 0.1%PVA溶液中,以600rpm在磁力搅拌器上搅拌6小时,获得PLGA多孔微球。
(3)用去离子水清洗PLGA多孔微球三次,然后重新悬浮在去离子水中。
(4)将PLGA多孔微球加入聚多巴胺溶液(由0.02g多巴胺、100μL pH=8.0的tri-HCl和10mL去离子水制备得到)中,在轻柔的磁力搅拌下反应6小时,获得PLGA/PDA多孔微球。
(5)用去离子水清洗PLGA/PDA多孔微球三次,洗去多余的PDA溶液,然后重新悬浮在去离子水中。
(6)冷冻干燥PLGA多孔微球与PLGA/PDA多孔微球24小时,室温干燥条件下储存。
(7)称量冻干的PLGA/PDA多孔微球20mg,浸泡于200μL CGRP溶液(10μM)中,冷冻干燥24h后获得PLGA/PDA/CGRP多孔微球。
采用qRT-PCR检测CGRP对成骨相关基因在mRNA水平表达的影响,具体为:
(1)将BMSCs细胞接种于6孔细胞培养板中,培养箱中培养24小时。
(2)分别更换新鲜培养液,对照组(DMEM完全培养基)、10nM组(含10nM CGRP的DMEM完全培养基)、100nM组(含100nM CGRP的DMEM完全培养基)、1000nM组(含1000nM CGRP的DMEM完全培养基)。培养7天,每2天更换一次培养基。
(3)弃原有培养液,用PBS缓冲液清洗2遍。每孔加入1mL预冷的RNAiso Plus溶液,将培养板放置于冰上,用无RNA酶的枪头反复吹打裂解5分钟,收集裂解液至无RNA酶的EP管中。
(4)向裂解液中加入200μL氯仿,将EP管反复上下颠倒20秒,溶液充分乳化后于冰上静置10分钟。随后在4℃下,以12000rpm的转速离心15分钟。
(5)离心后EP管内液体分为三层,小心吸取上层液体约500μL至新的EP管中,随后向EP管中加入500μL预冷的异丙醇,将EP管上下颠倒混匀,冰上静置10分钟。在4℃下,以12000rpm的转速离心15分钟。
(6)小心倒掉EP管内的上清液,可见管底贴附的白色沉淀,加入1mL的75%乙醇(提前使用无水乙醇与DEPC水配制并置于冰上预冷),颠倒混匀后,在4℃下,以8000rpm的转速离心5分钟。
(7)小心倒去上清液,在4℃下,以8000rpm的转速离心5秒,小心吸去残留的上清液。室温下干燥10分钟。
(8)在EP管内加入50μL DEPC水,使用NanoDrop1000超微量分光光度计测定收集的RNA纯度和浓度。
(9)按照PrimeScriptTMRT reagent Kit试剂盒说明书配制逆转录反应,以20μL体系进行逆转录。将PrimeScriptTMRTase、5×PrimeScriptTMBuffer、引物oligo dT、引物random 6、模板RNA混合,加入总RNA的量为1000ng。37℃水浴15分钟;85℃水浴5秒终止反应。逆转录完成后,向每管加入灭菌去离子水180μL混匀,获得200μL cDNA溶液。
(10)按照Premix Ex TaqTMII说明书配制Real-time PCR反应,以体系20μL反应体系进行PCR扩增。将TB Green Premix Ex Taq(2×)、上下游引物(10μM)、cDNA模板与灭菌去离子水混合,以管家基因b-actin作为内参,目的基因为成骨相关基因ALP、OPN、Runx2和BMP-2,使用的引物序列如表3-1所示。将孔板离心后放入Real-time PCR仪进行PCR扩增反应。
(11)反应完成后,记录CT值,根据目的基因相对表达量=2-ΔΔCT的公式(其中ΔCT=CT目的基因-CT内参基因,ΔΔCT=ΔCT实验组-ΔCT对照组)计算ALP、Col1、Runx2、OPN、OCN、Osterix相对于b-actin的表达量。
图2示出了不同浓度CGRP对BMSCs成骨相关基因表达的影响,可见,10nM组与100nM组的促成骨效果最优,7d效果远超对照组和1000nM组。
采用CCK-8检测LPS与CGRP对BMSCs增殖-毒性的影响,具体为:
(1)将100μL BMSCs细胞悬液以5×103个细胞/孔的密度接种于96孔板中,在培养箱中培养24小时。
(2)分别更换新鲜培养液,每两天更换一次培养液。
研究LPS与不同浓度CGRP处理对BMSCs活性的影响时,更换为:对照组(DMEM完全培养基)、LPS组(含100ng/mL LPS的DMEM完全培养基)、LPS+10nM CGRP组(含10nM CGRP与100ng/mL LPS的DMEM完全培养基)、LPS+100nM CGRP组(含100nM CGRP与100ng/mL LPS的DMEM完全培养基)、LPS+1000nM CGRP组(含1mM CGRP与100ng/mL LPS的DMEM完全培养基)。
图3示出了不同浓度CGRP与LPS刺激对BMSCs细胞活性的影响,可见,LPS+10nMCGRP组可在一定程度上恢复细胞活性。
图4是PLGA微球的光镜下明场的照片(图中,左右两幅子图为不同位置下拍摄的照片),可见PLGA微球呈现良好的球状结构,且粒径在20-180μm之间。
如图5所示,使用扫描电镜观察基于PLGA多孔微球表面及截面的多孔结构(PLGA多孔微球与PLGA/PDA/CGRP多孔微球的SEM图一致),可见多孔微球表面具有大量的大孔,大孔孔径在5-40μm之间,多孔微球内部具有大量微通道。
采用免疫荧光染色观察多孔微球对BMSCs的OPN表达的影响:
1)BMSCs与PLGA多孔微球共培养
取生长状态良好的BMSCs细胞,消化、离心后用完全培养基重悬,进行细胞计数,以5×107/mL的密度分别接种于PLGA/PDA多孔微球和PLGA/PDA/CGRP多孔微球上,使用巴氏吸管反复吹打,使细胞与微球充分混匀。37℃、5%CO2培养箱中培养。
2)多孔微球上接种细胞的免疫荧光染色
(1)接种BMSCs细胞24小时后,将负载细胞的PLGA/PDA多孔微球与PLGA/PDA/CGRP多孔微球转移至6孔培养板中,分别加入DMEM完全培养基与含100ng/mL LPS的DMEM完全培养基。培养箱中培养7天。每2天更换一次培养基。
(2)4%多聚甲醛固定液固定细胞样品10分钟,用PBS缓冲液清洗3次,每次5分钟。
(3)用0.5%Triton X-100溶液通透5分钟,PBS缓冲液清洗3次,每次5分钟。
(4)加入驴血清,在室温下封闭1小时。
(5)加入OPN抗体(1:200)或ALP抗体(1:50),在4℃下孵育过夜。
(6)用PBS缓冲液在摇床上清洗3次,每次5分钟。
(7)加入Alexa Fluor 488标记驴抗山羊二抗(1:200),室温下孵育1小时。
(8)用PBS缓冲液在摇床上清洗3次,每次5分钟。
(9)加入DAPI染色液(1:1000),室温下孵育5分钟。
(10)用PBS缓冲液在摇床上清洗3次,每次5分钟。
(11)激光共聚焦显微镜下观察并拍摄3D图像,将图像进行三维重建。
图7为PLGA/PDA多孔微球与PLGA/PDA/CGRP多孔微球的OPN免疫荧光染色,CLSM下观察,负载了CGRP的微球表面的BMSCs的OPN阳性区域更多,多孔微球上的CGRP促进负载的BMSCs的OPN的表达。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改,并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此,本发明不限于上述实施例,本领域技术人员根据本发明的揭示,不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
Claims (10)
1.一种载细胞多孔微球,其特征在于,
所述的载细胞多孔微球为PLGA/PDA/CGRP多孔微球,
所述的PLGA/PDA/CGRP多孔微球为负载有CGRP的PLGA/PDA多孔微球,
所述的PLGA/PDA多孔微球为通过PDA表面修饰的PLGA多孔微球。
2.根据权利要求1所述的一种载细胞多孔微球,其特征在于,所述的PLGA多孔微球的粒径为20-180μm。
3.根据权利要求1所述的一种载细胞多孔微球,其特征在于,所述的PLGA多孔微球的大孔孔径为5-40μm。
4.一种制备如权利要求1-3任一所述的载细胞多孔微球的方法,其特征在于,包括如下步骤:
S1:将PLGA多孔微球加入PDA溶液中,搅拌下反应,得到PLGA/PDA多孔微球;
S2:将步骤S1得到的PLGA/PDA多孔微球冷冻干燥;
S3:将步骤S2冻干的PLGA/PDA多孔微球浸泡于CGRP溶液中,再次冷冻干燥后得到PLGA/PDA/CGRP多孔微球。
5.根据权利要求4所述的一种载细胞多孔微球的制备方法,其特征在于,步骤S1中,所述的PLGA多孔微球通过乳化法制得,包括如下步骤:
S1a:将PLGA溶解于二氯甲烷,得到PLGA溶液;
S1b:将造孔剂加入至步骤S1a得到的PLGA溶液中,均质使液体充分乳化;
S1c:将步骤S1b得到的乳化液加入PVA溶液中,搅拌得到PLGA多孔微球。
6.根据权利要求5所述的一种载细胞多孔微球的制备方法,其特征在于,包括如下一项或几项:
i)步骤S1a中,PLGA与二氯甲烷的用量比为0.25g:10mL;
ii)步骤S1b中,造孔剂为碳酸氢铵溶液,碳酸氢铵溶液的浓度为5-25wt%;
iii)步骤S1b中,造孔剂与PLGA溶液中PLGA的用量比为5mL:0.25g;
iv)步骤S1b中,均质:采用高速均质机以10000rpm的转速均质2min;
v)步骤S1c中,PVA溶液的浓度为0.1wt%;
vi)步骤S1c中,PVA溶液与乳化液中PLGA的用量比为100mL:0.25g;
vii)步骤S1c中,搅拌:采用磁力搅拌器以600rpm的转速搅拌6h。
7.根据权利要求4所述的一种载细胞多孔微球的制备方法,其特征在于,步骤S1中,反应的时间为6h。
8.根据权利要求4所述的一种载细胞多孔微球的制备方法,其特征在于,步骤S2中,冷冻干燥的时间为24h。
9.根据权利要求4所述的一种载细胞多孔微球的制备方法,其特征在于,步骤S3中,PLGA/PDA多孔微球与CGRP溶液的用量比为20mg:200μL,所述的CGRP溶液的浓度为10μM。
10.一种如权利要求1-3任一所述的载细胞多孔微球在制备炎症状态下用于促进骨形成的药物中的应用。
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