CN115154651B - 一种生物矿化的牛血清白蛋白@钙硒纳米球、制备方法及应用 - Google Patents
一种生物矿化的牛血清白蛋白@钙硒纳米球、制备方法及应用 Download PDFInfo
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- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 claims abstract description 20
- 229940082569 selenite Drugs 0.000 claims abstract description 20
- 229910052711 selenium Inorganic materials 0.000 claims abstract description 20
- 239000011669 selenium Substances 0.000 claims abstract description 20
- MCAHWIHFGHIESP-UHFFFAOYSA-L selenite(2-) Chemical compound [O-][Se]([O-])=O MCAHWIHFGHIESP-UHFFFAOYSA-L 0.000 claims abstract description 19
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Abstract
本发明涉及一种生物矿化的牛血清白蛋白@钙硒纳米球、制备方法及其应用,属于生物材料技术领域。本发明提供的生物矿化的牛血清白蛋白@钙硒纳米球形貌、粒径均一,分散性良好,且硒元素含量高。牛血清白蛋白@钙硒纳米球作为墨水打印的3D生物支架在模拟体内环境下能有效地释放亚硒酸根,并诱导钙磷在白蛋白上矿化沉积。其作为一种高硒含量的钙磷生物活性材料,在3D打印、骨缺损修复以及肿瘤治疗等方面具有广阔的应用前景。
Description
技术领域
本发明涉及一种生物矿化的牛血清白蛋白@钙硒纳米球、制备方法及应用,属于生物材料技术领域。
背景技术
人体的天然骨是由钙和磷在胶原纤维中矿化形成的,钙磷元素是骨修复过程中的必须元素。硒元素是人体所必需的微量元素之一。研究发现,硒元素具有抗肿瘤,改善人体免疫力等功能,且硒在骨的形成方面具有重要作用。
目前已有很多含有硒钙磷生物活性材料的报道,如专利CN 102249206A公开了一种掺硒的羟基磷灰石及其制备方法;专利CN 106063947A公开了一种硒掺杂纳米羟基磷灰石的制备方法,;专利CN 107902636 A公开了一种微米级单晶含硒羟基磷灰石的制备方法。但是,这些发明中硒元素含量相对较低,限制了其在再生医学方面的应用。临床上,通过手术治疗骨肉瘤的过程,很难完全移除病灶,极容易导致肿瘤复发;并且移除病灶后的缺损部位需要使用生物材料进行填充,以促进缺损部位修复。发展一种既可以有效抗肿瘤,又可以促进骨再生的生物材料是有广阔应用前景的。
本发明旨在针对现有技术的不足提供一种牛血清白蛋白@钙硒纳米球生物活性材料。
发明内容
本发明针对现有技术的不足提出一种生物矿化的牛血清白蛋白@钙硒纳米球、制备方法及应用。该制备方法简单、易操作、反应条件温和,得到的牛血清白蛋白@亚硒酸钙纳米球中硒的质量分数高;能释放亚硒酸根,并诱导钙磷在蛋白模板上矿化。
为解决上述技术问题,本发明采用如下技术方案:
提供一种牛血清白蛋白@钙硒纳米球,其包括牛血清白蛋白和亚硒酸钙,为纳米球状结构,形貌均一。
进一步地,所述的牛血清白蛋白@钙硒纳米球的粒径为60nm-130nm。
进一步地,所述的牛血清白蛋白@钙硒纳米球中硒元素质量分数为10-32%。
提供一种上述牛血清白蛋白@钙硒的制备方法,将钙盐溶液和亚硒酸盐溶液依次逐滴加入到牛血清白蛋白的DMEM培养基溶液中,20-40℃控温搅拌10-15h,离心洗涤后得牛血清白蛋白@钙硒纳米球。
按上述方案,上述牛血清白蛋白@钙硒纳米球的制备方法,具体步骤为:
1)配制牛血清白蛋白的DMEM培养基溶液,在20-40℃控温搅拌的条件下,将钙盐溶液逐滴加入到牛血清白蛋白的DMEM溶液中;
2)搅拌10-20min后,将亚硒酸盐溶液逐滴加入到步骤1)的溶液体系中,搅拌10-15h;
3)离心,乙醇和去离子水交替洗涤,除去杂质,得牛血清白蛋白@钙硒纳米球。
按上述方案,所述步骤1)中配制牛血清白蛋白的DMEM培养基溶液为将牛血清白蛋白和DMEM培养基混合得到,牛血清白蛋白的浓度为1-2mg/mL;钙盐溶液浓度为25-50mg/mL。
按上述方案,所述步骤2)中亚硒酸盐溶液的浓度为12-80mg/mL;
按上述方案,所用的牛血清白蛋白与钙盐的质量比1∶(1.5-4.5);
按上述方案,所用的钙盐与亚硒酸盐的摩尔比为1∶(0.3-1.1);
按上述方案,优选的,所述的钙盐为氯化钙;优选的,所述亚硒酸盐为亚硒酸钠。
按上述方案,所述的生物矿化的牛血清白蛋白@钙硒纳米球进一步含有微量磷,磷含量0.5-1.5wt%。
按上述方案,DMEM培养基的主要组分为氨基酸、葡萄糖、维生素以及无机盐,无机盐中含有磷酸二氢钠。
提供上述生物矿化的牛血清白蛋白@钙硒纳米球在骨缺损修复中的应用。
提供上述生物矿化的牛血清白蛋白@钙硒纳米球在制备用于骨肉瘤治疗的生物材料中的应用。
按上述方案,所述的应用为:以生物矿化的牛血清白蛋白@钙硒纳米球配制打印墨水,3D打印得到生物支架;将3D打印生物支架填充到骨缺损修复部位或骨肉瘤手术切除病灶部位。
本发明的有益效果:
本发明提供的生物矿化的牛血清白蛋白@钙硒纳米球形貌、粒径均一,分散性良好,且硒元素含量高,作为打印墨水可以设计3D打印不同硒含量的生物支架;可以释放亚硒酸根,并诱导钙磷在白蛋白上矿化沉积,其中:亚硒酸根作为硒元素稳定存在的形式之一,不仅具有抗肿瘤活性,还兼顾促干细胞成骨分化和改善机体免疫等功能。其作为一种高硒含量的钙磷生物活性材料,在3D打印,骨缺损材料以及肿瘤治疗,骨缺损修复等方面具有广阔的应用前景。
本发明提供的生物矿化的牛血清白蛋白@钙硒纳米球的制备路线简单,反应条件温和,便于量化生产。
附图说明
图1为实施例1所制备的牛血清白蛋白@亚硒酸钙纳米球的透射电镜照片;
图2为实施例1所制备的牛血清白蛋白@亚硒酸钙纳米球的红外光谱图;
图3为实施例1所制备的牛血清白蛋白@亚硒酸钙纳米球的热重曲线图;
图4为实施例1所制备的牛血清白蛋白@亚硒酸钙纳米球中钙磷硒元素的含量;
图5为实施例2所制备的牛血清白蛋白@亚硒酸钙纳米球的透射电镜照片;
图6为实施例3所制备的牛血清白蛋白@亚硒酸钙纳米球的透射电镜照片;
图7为该牛血清白蛋白@亚硒酸钙纳在PBS溶液中重新矿化的透射电镜照片。
图8为牛血清白蛋白@亚硒酸钙纳米球3D打印生物支架的SEM。
图9为对比例1中牛血清白蛋白@亚硒酸钙纳米材料的透射电镜图。
图10为对比例2中牛血清白蛋白@亚硒酸钙纳米材料的透射电镜图。
具体实施方式
下面结合实施例对本发明的内容作详细说明,实施例在以本发明技术方案为前提下进行实施,给出了详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实施例。
实施例1
通过下述制备方法,合成含硒质量分数为30%的牛血清白蛋白@亚硒酸钙纳米球,具体步骤如下:
1)将25mg的牛血清白蛋白溶于25mL的DMEM培养液中,得到溶液Ⅰ;
2)将50mg的氯化钙溶于2mL的去离子水,得到溶液Ⅱ;
3)将80mg的亚硒酸钠溶于2mL的去离子水,得到溶液Ⅲ;
4)在37℃、搅拌条件下,将溶液Ⅱ逐滴加入到溶液Ⅰ中,得到溶液Ⅳ;10min搅拌后,将溶液Ⅲ逐滴加入到溶液Ⅳ中;
5)上述反应体系在37℃下,搅拌12h后5000rpm离心,乙醇和去离子水交替洗涤,除去杂质,得牛血清白蛋白@亚硒酸钙纳米球。
使用透射电子显微镜(TEM,TaLos F200X)对本实施例制备的牛血清白蛋白@亚硒酸钙纳米球进行表征,结果如图1所示,复合纳米球的粒径在60nm-130nm之间,分散性良好。使用红外光谱图仪(FT-IR,Nicolet iS50R)对本实施例制备得到的牛血清白蛋白@亚硒酸钙纳米球进行表征,结果如图2所示,存在肽键和亚硒酸根的特征吸收峰。使用热重仪(Diamond TG/DTA)对本实施例制备得到的牛血清白蛋白@亚硒酸钙纳米球进行分析,结果如图3所示,牛血清白蛋白@亚硒酸钙纳米球中白蛋白的质量分数为36%,亚硒酸钙的质量分数为58%。使用X射线荧光光谱仪(XRF)对本实施例制备得到的牛血清白蛋白@亚硒酸钙纳米球进行分析,结果如图4所示,样品中钙元素、硒元素和磷元素的质量百分数依次为44.4%、53.5%、2.1%,即质量比为1:1.2:0.047。结合牛血清白蛋白@亚硒酸钙纳米球的热重和X射线荧光光谱分析数据,计算得到牛血清白蛋白@亚硒酸钙纳米球中硒的质量分数为30%左右。
实施例2
通过下述制备方法,合成含硒质量分数为18%的牛血清白蛋白@亚硒酸钙纳米球,具体步骤如下:
1)将25mg的牛血清白蛋白溶于25mL的DMEM培养液中,得到溶液Ⅰ;
2)将50mg的氯化钙溶于2mL的去离子水,得到溶液Ⅱ;
3)将50mg的亚硒酸钠溶于2mL的去离子水,得到溶液Ⅲ;
4)在37℃、搅拌条件下,将溶液Ⅱ逐滴加入到溶液Ⅰ中,得到溶液Ⅳ;10min搅拌后,将溶液Ⅲ逐滴加入到溶液Ⅳ中;
5)上述反应体系在37℃下,搅拌12h后5000rpm离心,乙醇和去离子水交替洗涤,除去杂质,得牛血清白蛋白@亚硒酸钙纳米球。
图5为该牛血清白蛋白@亚硒酸钙纳米球的透射电镜照片.
实施例3
通过下述制备方法,合成含硒质量分数为13%的牛血清白蛋白@亚硒酸钙纳米球,具体步骤如下:
1)将25mg的牛血清白蛋白溶于25mL的DMEM培养液中,得到溶液Ⅰ;
2)将50mg的氯化钙溶于2mL的去离子水,得到溶液Ⅱ;
3)将25mg的亚硒酸钠溶于2mL的去离子水,得到溶液Ⅲ;
4)在37℃、搅拌条件下,将溶液Ⅱ逐滴加入到溶液Ⅰ中,得到溶液Ⅳ;10min搅拌后,将溶液Ⅲ逐滴加入到溶液Ⅳ中;
5)上述反应体系在37℃下,搅拌12h后5000rpm离心,乙醇和去离子水交替洗涤,除去杂质,得牛血清白蛋白@亚硒酸钙纳米球。
图6为该牛血清白蛋白@亚硒酸钙纳米球的透射电镜照片。
DMEM培养基的主要组分为氨基酸、葡萄糖、维生素以及无机盐,无机盐中含有磷酸二氢钠。具体可采用servicebio的DMEM培养基。
实施例4
牛血清白蛋白@亚硒酸钙纳米球在PBS中诱导钙磷矿化:将100μg/mL的牛血清白蛋白@亚硒酸钙分散在PBS溶液中,37℃下搅拌24h。
图7为该牛血清白蛋白@亚硒酸钙纳米球在PBS溶液中重新矿化的透射电镜照片。结果表明:本发明的牛血清白蛋白@亚硒酸钙纳米球在PBS(模拟体液环境)中能释放亚硒酸根,诱导钙和磷在蛋白模板上矿化沉积。
进一步地,以牛血清白蛋白@亚硒酸钙纳米球配制3D打印墨水,3D打印得到用于骨缺损修复的3D生物支架,该支架的SEM如图8所示。
打印墨水的制备方法:将明胶溶于0.5M Na2HPO4溶液中制备10wt%的明胶溶液,将5g制备好的热明胶溶液(温度60℃)与1.5g牛血清白蛋白@亚硒酸钙纳米球混合,使用混合消泡混合器在2000rpm下混合30分钟,得到打印墨水。
该支架材料可以释放具有抗肿瘤活性的亚硒酸根,同时诱导钙磷矿化,其基于牛血清白蛋白和亚硒酸钙的协同作用于骨肉瘤患者的治疗和骨缺损修复上具有优异的性能。具体地,可将上述打印的3D生物支架填充到骨肉瘤手术切除病灶部位,在体内微环境中能够有效释放亚硒酸根,并诱导钙磷在白蛋白上矿化沉积,基于各组分的协同作用进行治疗修复。
对比例1
制备方法具体步骤如下:
1)将25mg的牛血清白蛋白溶于25mL的DMEM培养液中,得到溶液Ⅰ;
2)将50mg的氯化钙溶于2mL的去离子水,得到溶液Ⅱ;
3)将10mg的亚硒酸钠溶于2mL的去离子水,得到溶液Ⅲ;
4)在37℃、搅拌条件下,将溶液Ⅱ逐滴加入到溶液Ⅰ中,得到溶液Ⅳ;10-30min搅拌后,将溶液Ⅲ逐滴加入到溶液Ⅳ中;
5)上述反应体系在37℃下,搅拌10-15h后5000rpm离心,乙醇和去离子水交替洗涤,除去杂质,得牛血清白蛋白@亚硒酸钙纳米球。
图9为该牛血清白蛋白@亚硒酸钙纳米材料的透射电镜图,因为牛血清白蛋白相对于钙盐和亚硒酸盐明显过量,反应体系中蛋白胶束过多,导致有很多小颗粒生成,很明显的没有有效地成球。
对比例2
制备方法具体步骤如下:
1)将10mg的牛血清白蛋白溶于50mL的DMEM培养液中,得到溶液Ⅰ;
2)将110mg的氯化钙溶于2mL的去离子水,得到溶液Ⅱ;
3)将170mg的亚硒酸钠溶于2mL的去离子水,得到溶液Ⅲ;
4)在37℃、搅拌条件下,将溶液Ⅱ逐滴加入到溶液Ⅰ中,得到溶液Ⅳ;10-30min搅拌后,将溶液Ⅲ逐滴加入到溶液Ⅳ中;
5)上述反应体系在37℃下,搅拌10-15h后5000rpm离心,乙醇和去离子水交替洗涤,除去杂质,得牛血清白蛋白@亚硒酸钙纳米球。
图10为该牛血清白蛋白@亚硒酸钙纳米材料的透射电镜图,因为钙盐和亚硒酸盐相对于牛血清白蛋白明显过量,反应体系中过量的钙盐和亚硒酸盐会在选择性在牛血清白蛋白@亚硒酸钙球表面生成小颗粒。
Claims (8)
1. 一种牛血清白蛋白@钙硒纳米球,其特征在于:所述牛血清白蛋白@钙硒纳米球由牛血清白蛋白和亚硒酸钙组成,为纳米球状结构,形貌均一,牛血清白蛋白@钙硒纳米球中硒元素质量分数为10-32%,牛血清白蛋白@钙硒纳米球的粒径为60 nm-130 nm,由氯化钙溶液和亚硒酸盐溶液依次逐滴加入到牛血清白蛋白的DMEM培养基溶液中,20-40℃控温搅拌10-15 h,离心洗涤后得到,所用的牛血清白蛋白与氯化钙的质量比1∶(1.5-4.5)。
2. 根据权利要求1所述的牛血清白蛋白@钙硒纳米球的制备方法,其特征在于:将氯化钙溶液和亚硒酸盐溶液依次逐滴加入到牛血清白蛋白的DMEM培养基溶液中,20-40℃控温搅拌10-15 h,离心洗涤后得牛血清白蛋白@钙硒纳米球,所用的牛血清白蛋白与氯化钙的质量比1∶(1.5-4.5)。
3.根据权利要求2所述的牛血清白蛋白@钙硒纳米球的制备方法,其特征在于:所用的氯化钙与亚硒酸盐的摩尔比为1∶(0.3-1.1)。
4.根据权利要求2所述的牛血清白蛋白@钙硒纳米球的制备方法,其特征在于:具体步骤为:
1)配制牛血清白蛋白的DMEM培养基溶液,在20-40℃控温搅拌的条件下,将氯化钙溶液逐滴加入到牛血清白蛋白的DMEM溶液中;
2)搅拌10-20 min后,将亚硒酸盐溶液逐滴加入到步骤1)的溶液体系中,搅拌10-15 h;
3)离心,乙醇和去离子水交替洗涤,除去杂质,得牛血清白蛋白@钙硒纳米球。
5. 根据权利要求4所述的牛血清白蛋白@钙硒纳米球的制备方法,其特征在于:所述步骤1)中配制牛血清白蛋白的DMEM培养基溶液为将牛血清白蛋白和DMEM培养基混合得到,牛血清白蛋白的浓度为1-2 mg/mL;所述的氯化钙溶液浓度为25-50 mg/mL;所述步骤2)中亚硒酸盐溶液的浓度为12-80 mg/mL;所述亚硒酸盐为亚硒酸钠。
6.权利要求1所述的牛血清白蛋白@钙硒纳米球在制备骨缺损修复材料中的应用。
7.权利要求1所述的牛血清白蛋白@钙硒纳米球在制备用于骨肉瘤治疗的生物材料中的应用。
8.根据权利要求6或7所述的应用,其特征在于:以权利要求1所述的牛血清白蛋白@钙硒纳米球作为打印墨水,通过3D打印得到生物支架。
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