CN113336977A - 一种壳聚糖纳米纤维微球及其制备方法 - Google Patents
一种壳聚糖纳米纤维微球及其制备方法 Download PDFInfo
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Abstract
本发明涉及生物医学工程领域,为一种壳聚糖纳米纤维微球及其制备方法,包括以下步骤:(1)得到澄清透亮的壳聚糖溶液;(2)将表面活性剂加入溶剂中,再加入所述壳聚糖溶液,继续搅拌;(3)使壳聚糖完全凝胶化;(4)将反应体系进行破乳,沉淀物洗涤、干燥得到微球;(5)将得到的微球先用乙醇进行梯度置换,再用叔丁醇进行溶剂置换并冷冻干燥,得到壳聚糖纳米纤维微球。本发明的制备方法制备的壳聚糖纳米纤维微球结构稳定、粒径均匀、分散性好、易于细胞粘附,生物安全性高,可以有效地支撑细胞的生长,并促进细胞进行三维粘附和增殖,具有良好的生物相容性,可以作为优良的细胞微载体作为组织工程支架材料,具有良好的生物医用前景。
Description
技术领域
本发明涉及生物医学工程领域,具体为一种壳聚糖纳米纤维微球及其制备方法。
背景技术
骨缺损会对人类的生活质量和活动能力造成严重损害([1]Harris,J.S.;Bemenderfer,T.B.;Wessel,A.R.;Kacena,M.A.A review of mouse critical sizedefect models in weight bearing bones.Bone 2013,55,(1),241-247.[2]El-Rashidy,A.A.;Roether,J.A.;Harhaus,L.;Kneser,U.;Boccaccini,A.R.Regenerating bone withbioactive glass scaffolds:A review of in vivo studies in bone defectmodels.Acta Biomater.2017,62,1-28)。由于骨组织再生能力有限,一旦发生严重损伤,会导致骨不连以及骨功能丧失(Vellucci,R.;Mediati,R.D.;Ballerini,G.Use of opioidsfor treatment of osteoporotic pain.Clinical Cases in Mineral and BoneMetabolism 2014,11,(3),173-176)。尽管自体骨移植目前仍是修复大型骨缺损的主要手段,但是移植手术供体部位发病率高、感染风险高,存在一定的隐患(Pina,S.;Oliveira,J.M.;Reis,R.L.Natural-based nanocomposites for bone tissue engineering andregenerative medicine:A review.Adv.Mater.2015,27,(7),1143-1169)。近年来,利用组织工程支架促进细胞增殖并引导骨再生已经成为骨组织再生领域的研究热点(Li,J.J.;Ebied,M.;Xu,J.;Zreiqat,H.Current approaches to bone tissue engineering:Theinterface between biology and engineering.Adv.Healthcare Mater.2018,7,(6),1701061.)。在组织工程中,通常需要支架具有良好的生物相容性和生物可降解性,并且可以模拟细胞外基质(ECM)从而为细胞的粘附、迁移以及组织的形成提供良好的基础(Alice,C.;Zvi,S.;Adrian,K.;Xiyu,L.;Zhenxing,S.;Muyang,S.;Yingfang,A.;D.,B.B.;Haifeng,C.Advances in porous scaffold design for bone and cartilage tissueengineering and regeneration.Tissue Engineering Part B:Reviews 2019,25,(1),14-29.Ma,H.;Feng,C.;Chang,J.;Wu,C.3D-printed bioceramic scaffolds:From bonetissue engineering to tumor therapy.Acta Biomater.2018,79,37-59.)。
壳聚糖是自然界中含量仅次于纤维素的甲壳素脱乙酰之后得到的衍生物,是唯一带正电荷的碱性天然多糖(Ahsan,S.M.;Thomas,M.;Reddy,K.K.;Sooraparaju,S.G.;Asthana,A.;Bhatnagar,I.Chitosan as biomaterial in drug delivery and tissue engineering.Int.J.Biol.Macromol.2018,110,97-109.M.Ways,T.M.;Lau,W.M.;Khutoryanskiy,V.V.Chitosan and its derivatives for application inmucoadhesive drug delivery systems.Polymers 2018,10,(3),267.)。壳聚糖不仅安全无毒,还具有良好的生物相容性、生物可降解性和抗菌性等,在生物医用领域具有良好的应用前景(LogithKumar,R.;KeshavNarayan,A.;Dhivya,S.;Chawla,A.;Saravanan,S.;Selvamurugan,N.A review of chitosan and its derivatives in bone tissue engineering.Carbohydr.Polym.2016,151,172-188)。众所周知,壳聚糖一般溶解在低浓度的酸性水溶液中(Geng,X.;Kwon,O.-H.;Jang,J.Electrospinning of chitosan dissolved inconcentrated acetic acid solution.Biomaterials 2005,26,(27),5427-5432.),在这种溶剂条件下制备得到的壳聚糖材料往往力学性能较差,不能很好的用于骨组织工程支架。因此,构建高强度壳聚糖支架材料应用于骨修复领域具有重要的意义。本实验室已报道,壳聚糖在低温条件下可以直接溶解在碱/尿素水溶液中(Duan,J.;Liang,X.;Cao,Y.;Wang,S.;Zhang,L.High strength chitosan hydrogels with biocompatibility vianew avenue based on constructing nanofibrous architecture.Macromolecules2015,48,(8),2706-2714.,)并且通过化学交联成功构建出了一系列高强度的水凝胶和微球材料(Duan,J.;Liang,X.;Guo,J.;Zhu,K.;Zhang,L.Ultra-stretchable andforcesensitive hydrogels reinforced with chitosan microspheres embedded inpolymer networks.Adv.Mater.2016,28,(36),8037-8044.)。然而,化学交联剂的引入会对壳聚糖本身的生物相容性造成一定的影响,甚至可能存在一定的毒性危害(Mirzaei B,E.;Ramazani S.A,A.;Shafiee,M.;Danaei,M.Studies on glutaraldehyde crosslinkedchitosan hydrogel properties for drug delivery systems.International Journalof Polymeric Materials and Polymeric Biomaterials 2013,62,(11),605-611.。)此外,大块的水凝胶材料由于形状固定,并不能很好的适用于骨组织缺损部位。据报道,具有纳米纤维结构的微/纳米材料更有利于细胞的粘附和生长,并能促进组织再生(Liu,X.;Jin,X.;Ma,P.X.Nanofibrous hollow microspheres self-assembled from star-shapedpolymers as injectable cellcarriers for knee repair.Nat.Mater.2011,10,398-406.Duan,B.;Zheng,X.;Xia,Z.;Fan,X.;Guo,L.;Liu,J.;Wang,Y.;Ye,Q.;Zhang,L.Highlybiocompatible nanofibrous microspheres self-assembled from chitin in NaOH/Urea aqueous solution as cell carriers.Angew.Chem.Int.Ed.2015,54,(17),5152-5156.)。因此,构建一种具有纳米纤维结构的壳聚糖微球材料用作骨组织工程支架具有重要的意义。
发明内容
本发明的目的之一在于提供一种壳聚糖纳米纤维微球的制备方法,制备工艺简便,易于调节。
本发明的目的之二在于提供一种壳聚糖纳米纤维微球,结构稳定、粒径均匀、分散性好、易于细胞粘附。
本发明实现目的之一所采用的方案是:一种壳聚糖纳米纤维微球的制备方法,包括以下步骤:
(1)将一定量的壳聚糖粉末分散在碱、尿素和去离子水组成的混合液中,经冷冻-解冻多个循环后,离心脱泡并去除未溶解的杂质,得到澄清透亮的壳聚糖溶液;
(2)将表面活性剂加入溶剂中,在0-5℃下搅拌预冷,再加入所述壳聚糖溶液,继续搅拌;
(3)将反应体系在一定温度保温,使壳聚糖完全凝胶化;
(4)将步骤(3)的反应体系转移至乙醇/水混合溶剂中搅拌进行破乳;静置后将下层沉淀,洗涤,得到微球;
(5)将得到的微球先用乙醇进行梯度置换,再用叔丁醇进行溶剂置换并冷冻干燥,得到壳聚糖纳米纤维微球。
优选地,所述步骤(1)中,壳聚糖溶液中壳聚糖粉末与混合液的质量比为2%~10%。
优选地,所述步骤(1)中,碱为氢氧化钠、氢氧化钾、氢氧化锂中的至少一种;碱的加入量为混合液总质量的2%~30%,尿素的加入量为混合液总质量的1%~10%。
优选地,所述步骤(2)中,表面活性剂为Span85或Span80,在溶剂中的质量浓度为1%~10%。
优选地,所述步骤(2)中,溶剂为异辛烷。
优选地,所述步骤(2)中,壳聚糖溶液和溶剂的体积比为1-2:1。
优选地,所述步骤(3)中,保温温度范围为20~80℃。
优选地,所述步骤(4)中,乙醇/水混合溶剂中乙醇和水的体积比为9~3:1,破乳的搅拌速度为600-1200rpm。
本发明实现目的之二所采用的方案是:一种壳聚糖纳米纤维微球,由所述的制备方法制得。
优选地,所述壳聚糖纳米纤维微球呈现出规则的球形,尺寸分布为30~90μm;微球表面及内壁均呈现出均匀的纳米纤维结构,纳米纤维的平均直径约为22±5nm。
本发明具有以下优点和有益效果:
本发明的制备方法基于壳聚糖在碱/尿素水溶剂体系中的低温溶解机理,通过热诱导壳聚糖分子链平行自组装排列形成纳米纤维,采用乳液法构建得到了物理再生的壳聚糖纳米纤维微球。通过对纳米纤维的形成过程进行探究,揭示了壳聚糖纳米纤维的形成机理。同时,本发明壳聚糖纳米纤维微球制备工艺较简便,工艺参数较易控制,微球的尺寸和纳米纤维结构可以通过改变制备条件、温度和溶液浓度来调控。微球的尺寸可以通过表面活性剂的用量、乙醇/水溶剂的比例、搅拌速度来进行调节,而纳米纤维结构则可以通过改变初始溶液的浓度和热诱导温度来进行有效调控。
本发明的制备方法制备的壳聚糖纳米纤维微球结构稳定、粒径均匀、分散性好、易于细胞粘附,生物安全性高,可以有效地支撑细胞的生长,并促进细胞进行三维粘附和增殖,具有优良的生物相容性,可以作为细胞微载体及组织工程支架材料,生物医用前景良好。
附图说明
图1是本发明实施例得到的壳聚糖纳米纤维微球的形貌图:(a)光镜;(b、d、e、f)扫描电镜;(c)微球直径分布;
图2是本发明实施例1制备过程中步骤(3)的反应体系在80℃时的AFM图像;
图3是本发明实施例1得到的壳聚糖纳米纤维微球的压缩应力应变曲线图;
图4是本发明实施例1得到的壳聚糖纳米纤维微球的细胞活性(a)和MTT(b)结果数据图;
图5是为本发明实施例1得到的壳聚糖纳米纤维微球微球与成骨细胞MC3T3-E1共培养的生长情况图;
图6本发明实施例1得到的壳聚糖纳米纤维微球微球对成骨细胞MC3T3-E1粘附行为的电镜表征图;
图7为本发明实施例2得到的壳聚糖纳米纤维微球的扫描电镜形貌图和纤维直径分布;
图8为本发明实施例3在不同温度条件下(20℃、30℃、40℃、50℃、60℃)制备得到的壳聚糖纳米纤维微球微球表面纳米纤维的形貌和尺寸分布。
具体实施方式
为更好的理解本发明,下面的实施例是对本发明的进一步说明,但本发明的内容不仅仅局限于下面的实施例。
实施例1
(1)将4.5g LiOH、7g KOH和8g尿毒依次溶解于80.5g去离子水中,形成混合液,然后将4.5g壳聚糖粉末分散于所述混合液中,得到悬浮液。将悬浮液在-40℃下冷冻若干小时之后,常温下搅拌解冻。
(2)冷冻-解冻重复三次后得到澄清透亮的壳聚糖溶液,以7000rpm的转速离心10min除去溶液中的气泡和杂质。将175g异辛烷和10g Span 85加入三口烧瓶中,在冰水浴下搅拌预冷30min,然后再加入壳聚糖溶液,以1000rpm的转速继续搅拌1h。
(3)将冰水浴换为80℃的热水浴并恒温加热30min,使壳聚糖完全凝胶化。
(4)将步骤(3)的反应体系转移至乙醇/水混合溶剂中(乙醇和水的体积比为9:1),以1000rpm速度搅拌进行破乳。静置后倒去上层油相并保留下层沉淀,依次用无水乙醇和去离子水反复洗涤,去除残留的表面活性剂和溶剂,得到微球。
(5)将得到的微球先用乙醇进行梯度置换,再用叔丁醇进行溶剂置换并冷冻干燥,得到壳聚糖纳米纤维微球,保存于干燥器中备用,标记为NCSM。
图1为本发明实施例1得到的壳聚糖纳米纤维微球的形貌图:(a)光镜;(b、d、e、f)扫描电镜;(c)壳聚糖纳米纤维微球直径分布。如图所示,制备得到的壳聚糖纳米纤维微球呈现出规则的球形,并且具有相对较窄的尺寸分布(30~90μm)。壳聚糖纳米纤维微球表面呈现出均匀的纳米纤维结构,纳米纤维的平均直径约为22±5nm,长度约数微米。同时,壳聚糖纳米纤维微球的内部也呈现出均匀的纳米纤维结构,说明整个壳聚糖纳米纤维微球从内到外均由纳米纤维构成,展现出均一稳定的结构。
图2为本发明实施例1的制备过程中步骤(3)的反应体系在80℃时的AFM图像。如图所示,壳聚糖溶液在受热时,分子链发生了平行聚集形成了长纤维状的聚集体,并逐渐缠绕形成网络状结构。结果表明,通过加热的方式可以有效的诱导壳聚糖分子链发生自组装并平行聚集形成纳米纤维,从而进一步构建由纳米纤维编织而成的微球。
图3为本发明实施例1制备的壳聚糖纳米纤维微球的压缩应力应变曲线,显示得到的壳聚糖纳米纤维微球具有良好的力学性能。
图4为本发明实施例1制备的壳聚糖纳米纤维微球的细胞活性(a)和MTT(b)结果。结果显示,随着壳聚糖纳米纤维微球浓度的增加,细胞的存活率先增加后降低,但是均维持在100%以上,说明壳聚糖纳米纤维微球对成骨细胞MC3T3-E1的生长具有很好的促进作用。随着培养天数的增加,细胞数量也明显增加,表明微球具有很好的细胞相容性,能够促进MC3T3-E1细胞的增殖。
图5为本发明实施例1得到的壳聚糖纳米纤维微球与成骨细胞MC3T3-E1共培养的生长情况。如图所示,MC3T3-E1细胞与NCSM共培养3天(图5a和b)和5天(图5c和d)后在明场和荧光场下的显微镜照片。可以看出,在细胞培养过程中,壳聚糖纳米纤维微球具有良好的细胞相容性,可以与MC3T3-E1细胞很好的共存,几乎没有死细胞出现。培养3天后,MC3T3-E1细胞出现了明显的增殖行为,开始逐渐连接在一起。培养到5天时,MC3T3-E1细胞已经形成了网络状的细胞群,表现出了良好的增殖情况。
图6本发明实施例1制备的壳聚糖纳米纤维微球对成骨细胞MC3T3-E1粘附行为的电镜表征。可以看出,MC3T3-E1细胞与壳聚糖纳米纤维微球共培养三天后,通过丝状伪足紧密的粘附在壳聚糖纳米纤维微球的表面,从而呈现出3D粘附和增殖,展现出良好的粘附能力。
实施例2
(1)将4.5g LiOH、7g KOH和8g尿毒依次溶解于80.5g去离子水中,形成混合液,然后将2g壳聚糖粉末分散于所述混合液中,得到悬浮液。将悬浮液在-40℃下冷冻若干小时之后,常温下搅拌解冻。
(2)冷冻-解冻重复三次后得到澄清透亮的壳聚糖溶液,以7000rpm的转速离心10min除去溶液中的气泡和杂质。将175g异辛烷和10g Span 85加入三口烧瓶中,在冰水浴下搅拌预冷30min,然后再加入壳聚糖溶液,以1000rpm的转速继续搅拌1h。
(3)将冰水浴换为80℃的热水浴并恒温加热30min,使壳聚糖完全凝胶化。
(4)将乳液转移至乙醇/水混合溶剂中(乙醇和水的体积比为9:1),以1000rpm速度搅拌进行破乳。静置后倒去上层油相并保留下层沉淀,依次用无水乙醇和去离子水反复洗涤,去除残留的表面活性剂和溶剂。
(5)将得到的微球先用乙醇进行梯度置换,再用叔丁醇进行溶剂置换并冷冻干燥,得到壳聚糖纳米纤维微球,保存于干燥器中备用。
图7为本发明实施例2得到的壳聚糖纳米纤维微球的扫描电镜形貌图和纤维直径分布。如图所示,制备得到的NCSM仍为规则的球形,但大小分布相对不均匀,表面伴随着缺陷和松散的结构,纳米纤维结构依然存在,直径约19±3nm。
实施例3
(1)将4.5g LiOH、7g KOH和8g尿毒依次溶解于80.5g去离子水中,形成混合液,然后将4.5g壳聚糖粉末分散于所述混合液中,得到悬浮液。将悬浮液在-40℃下冷冻若干小时之后,常温下搅拌解冻。
(2)冷冻-解冻重复三次后得到澄清透亮的壳聚糖溶液,以7000rpm的转速离心10min除去溶液中的气泡和杂质。将175g异辛烷和10g Span 85加入三口烧瓶中,在冰水浴下搅拌预冷30min,然后再加入壳聚糖溶液,以1000rpm的转速继续搅拌1h。
(3)将冰水浴换分别换为20℃、30℃、40℃、50℃、60℃的水浴并恒温保持30min,使壳聚糖完全凝胶化。
(4)将乳液转移至乙醇/水混合溶剂中(乙醇和水的体积比为9:1),以1000rpm速度搅拌进行破乳。静置后倒去上层油相并保留下层沉淀,依次用无水乙醇和去离子水反复洗涤,去除残留的表面活性剂和溶剂。
(5)将得到的微球先用乙醇进行梯度置换,再用叔丁醇进行溶剂置换并冷冻干燥,得到壳聚糖纳米纤维微球,保存于干燥器中备用。
图8为本发明实施例在不同温度条件下制备得到的NCSM表面纳米纤维的形貌和尺寸分布。如图所示,当温度从20℃升高到60℃时,微球表面纳米纤维的平均直径从39nm减小到21nm,尺寸分布也随之变窄,说明温度越高,形成的纳米纤维越均匀。
实施例4
(1)将4.5g LiOH、7g KOH和8g尿毒依次溶解于80.5g去离子水中,形成混合液,然后将10g壳聚糖粉末分散于所述混合液中,得到悬浮液。将悬浮液在-40℃下冷冻若干小时之后,常温下搅拌解冻。
(2)冷冻-解冻重复三次后得到澄清透亮的壳聚糖溶液,以7000rpm的转速离心10min除去溶液中的气泡和杂质。将175g异辛烷和19.5g Span 85加入三口烧瓶中,在冰水浴下搅拌预冷30min,然后再加入壳聚糖溶液,以1000rpm的转速继续搅拌1h。
(3)将冰水浴换为80℃的热水浴并恒温加热30min,使壳聚糖完全凝胶化。
(4)将乳液转移至乙醇/水混合溶剂中(乙醇和水的体积比为6:1),以1000rpm速度搅拌进行破乳。静置后倒去上层油相并保留下层沉淀,依次用无水乙醇和去离子水反复洗涤,去除残留的表面活性剂和溶剂。
(5)将得到的微球先用乙醇进行梯度置换,再用叔丁醇进行溶剂置换并冷冻干燥,得到壳聚糖纳米纤维微球,保存于干燥器中备用。
本发明实施例4制备壳聚糖纳米纤维微球的与实施例1得到的壳聚糖纳米纤维微球形貌类似。
实施例5
(1)将4.5g LiOH、7g KOH和8g尿毒依次溶解于80.5g去离子水中,形成混合液,然后将2g壳聚糖粉末分散于所述混合液中,得到悬浮液。将悬浮液在-40℃下冷冻若干小时之后,常温下搅拌解冻。
(2)冷冻-解冻重复三次后得到澄清透亮的壳聚糖溶液,以7000rpm的转速离心10min除去溶液中的气泡和杂质。将175g异辛烷和1.77g Span 85加入三口烧瓶中,在冰水浴下搅拌预冷30min,然后再加入壳聚糖溶液,以1000rpm的转速继续搅拌1h。
(3)将冰水浴换为80℃的热水浴并恒温加热30min,使壳聚糖完全凝胶化。
(4)将乳液转移至乙醇/水混合溶剂中(乙醇和水的体积比为3:1),以1000rpm速度搅拌进行破乳。静置后倒去上层油相并保留下层沉淀,依次用无水乙醇和去离子水反复洗涤,去除残留的表面活性剂和溶剂。
(5)将得到的微球先用乙醇进行梯度置换,再用叔丁醇进行溶剂置换并冷冻干燥,得到壳聚糖纳米纤维微球,保存于干燥器中备用。
本发明实施例5制备壳聚糖纳米纤维微球的与实施例1得到的壳聚糖纳米纤维微球形貌类似。
以上所述是本发明的优选实施方式而已,当然不能以此来限定本发明之权利范围,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和变动,这些改进和变动也视为本发明的保护范围。
Claims (10)
1.一种壳聚糖纳米纤维微球的制备方法,其特征在于,包括以下步骤:
(1)将一定量的壳聚糖粉末分散在碱、尿素和去离子水组成的混合液中,经冷冻-解冻多个循环后,离心脱泡并去除未溶解的杂质,得到澄清透亮的壳聚糖溶液;
(2)将表面活性剂加入溶剂中,在0-5℃下搅拌预冷,再加入所述壳聚糖溶液,继续搅拌;
(3)将反应体系在一定温度保温,使壳聚糖完全凝胶化;
(4)将步骤(3)的反应体系转移至乙醇/水混合溶剂中搅拌进行破乳;静置后将下层沉淀,洗涤,得到微球;
(5)将得到的微球先用乙醇进行梯度置换,再用叔丁醇进行溶剂置换并冷冻干燥,得到壳聚糖纳米纤维微球。
2.根据权利要求1所述的壳聚糖纳米纤维微球的制备方法,其特征在于:所述步骤(1)中,壳聚糖溶液中壳聚糖粉末与混合液的质量比为2%~10%。
3.根据权利要求1所述的壳聚糖纳米纤维微球的制备方法,其特征在于:所述步骤(1)中,碱为氢氧化钠、氢氧化钾、氢氧化锂中的至少一种;碱的加入量为混合液总质量的2%~30%,尿素的加入量为混合液总质量的1%~10%。
4.根据权利要求1所述的壳聚糖纳米纤维微球的制备方法,其特征在于:所述步骤(2)中,表面活性剂为Span85或Span80,在溶剂中的质量浓度为1%~10%。
5.根据权利要求1所述的壳聚糖纳米纤维微球的制备方法,其特征在于:所述步骤(2)中,溶剂为异辛烷。
6.根据权利要求1所述的壳聚糖纳米纤维微球的制备方法,其特征在于:所述步骤(2)中,壳聚糖溶液和溶剂的体积比为1-2:1。
7.根据权利要求1所述的壳聚糖纳米纤维微球的制备方法,其特征在于:所述步骤(3)中,保温温度范围为20~80℃。
8.根据权利要求1所述的壳聚糖纳米纤维微球的制备方法,其特征在于:所述步骤(4)中,乙醇/水混合溶剂中乙醇和水的体积比为9~3:1,破乳的搅拌速度为600-1200rpm。
9.一种壳聚糖纳米纤维微球,其特征在于:由所述权利要求1-8中任一项所述的制备方法制得。
10.根据权利要求9所述的壳聚糖纳米纤维微球,其特征在于:所述壳聚糖纳米纤维微球呈现出规则的球形,尺寸分布为30~90μm;微球表面及内壁均呈现出均匀的纳米纤维结构,纳米纤维的平均直径约为22±5nm。
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