WO2019095610A1 - 一种生理条件下蚕丝蛋白溶液快速凝胶化的方法 - Google Patents
一种生理条件下蚕丝蛋白溶液快速凝胶化的方法 Download PDFInfo
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- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
- C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
- C08J3/075—Macromolecular gels
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- C08J2389/00—Characterised by the use of proteins; Derivatives thereof
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- the invention relates to a method for rapidly gelling a silk protein solution under physiological conditions.
- Silk Fibroin also known as silk fibroin, is a natural high-molecular fibrin extracted from silk, containing about 70% to 80% of silk, and contains 18 kinds of amino acids, among which glycine (gly) and alanine ( Ala) and serine (ser) account for more than 80% of the total content.
- Silk fibroin has good mechanical properties and physical and chemical properties, such as good flexibility and tensile strength, breathable moisture permeability, sustained release, etc., and different treatments can be obtained in different forms, such as nanofibers, nanoparticles, silk Protein film, sponge-like porous scaffold material, silk fibroin hydrogel and various materials made by electrospinning.
- Time sonication allows the silk fibroin to change from a random coil structure at the time of solution to a ⁇ -fold structure at the gel state under physiological conditions.
- these additional conditions are greatly limited in the practical application of silk fibroin as a biological material.
- Scientific researchers have also adjusted by adding small organic molecules or high molecular polymers (such as ethanol, surfactant sodium dodecyl sulfate or hydrophilic polymer polyethylene glycol) to the silk protein solution. Its interaction with silk protein chains accelerates the gelation rate of silk fibroin solutions, but this series of gel processes may be incompatible with certain clinical use environments, such as potential cellular and tissue toxicity of organic molecules.
- the biological inertness of the polymer is difficult to degrade in the living body. Although these measures can shorten the gelation time of the silk protein to a certain extent, the obtained silk protein gel has poor biocompatibility and large size. Cytotoxicity, these problems have led to significant limitations in its use in biomedical materials.
- the object of the present invention is to provide a method for rapidly gelling a silk fibroin solution under physiological conditions, which can induce rapid gelation of a low concentration silk fibroin solution in a short time, and in vivo It can be degraded by hydrolysis by proteolytic enzymes in the human body, has no toxic side effects, has good biocompatibility, and can be used as a good biomaterial.
- the invention provides a method of rapid gelation of a silk fibroin solution under physiological conditions, comprising the steps of:
- step (2) heating is performed at 70 ° C using a microcomputer electric heating plate which can be precisely temperature controlled.
- the molar concentration of the HCl solution added is 1 mol/L.
- the concentration of the silk fibroin solution is 12.03 wt%.
- the volume is made up to 200 ⁇ L. .
- the solution has a pH of 7.4.
- the preparation step of the silk fibroin solution is:
- the first step silk degumming
- Step 2 Dissolve the silk
- the third step dialysis of silk protein solution
- the LiBr solution in which silk fibroin was dissolved was placed in a dialysis bag with an intercept molecular weight of 3,500, and dialysis was performed with ultrapure water, and the water was changed every 1 hour. After 10 hours, the interval was changed to 2-5 hours for ultrapure water, and dialysis for 4 days. ;
- the dialysis bag was taken out, and PEG20000 was evenly spread on the surface of the dialysis bag to concentrate the water. After the surface PEG20000 became viscous, the appropriate amount of PEG20000 was reapplied; the solution was concentrated for one day until the solution became yellowish;
- the concentrated solution was centrifuged at 4 ° C, 4000 r / min for 40 min, after centrifugation, the supernatant was taken, and a small amount of impurities were discarded;
- the formula is The concentration of each measurement should be measured in parallel with three glass evaporating dishes at the same time, that is, the silk protein solution (SF) having an average concentration of 12.03% was obtained. Further preferably, in the step (4), the volume of the silk fibroin solution is 34 ⁇ L. In another aspect, the invention also provides a silk fibroin gel prepared by the above method.
- the invention provides the use of the silk protein gel described above in a biomedical material.
- the method for rapidly gelling a silk protein solution under the physiological condition of the present invention can induce rapid gelation of a low concentration silk protein solution in a short time, and the biocompatibility of the small molecule NapFF of the polypeptide derivative is very high.
- it can be degraded by proteolytic enzymes in the human body and does not produce toxic and side effects. Therefore, the prepared polypeptide derivative small molecule and silk fibroin hybrid gel have good biocompatibility under physiological conditions.
- After the rapid induction of gelation of the silk protein solution it can still be used as a good biomaterial, and the traditional polymer, ethanol, surfactant and other series of inducers can not be used as a gel for the induction of silk protein solution.
- the dilemma of the use of biological materials can induce rapid gelation of a low concentration silk protein solution in a short time, and the biocompatibility of the small molecule NapFF of the polypeptide derivative is very high.
- it can be degraded by proteolytic enzymes in the human body and does not
- FIG. 4 is a dynamic mechanical rheological scan of Example 3;
- FIG. B is a gel point determination of the embodiment 3 in the dynamic rheological scan mode;
- FIG. C is an optical picture of the embodiment 3; Characterization of rheological properties of Example 3 under scanning.
- Figure 5 is a dynamic mechanical rheological scan of the embodiment 4;
- Figure B is the gel point determination of the embodiment 4 in the dynamic rheological scan mode;
- Figure C is the optical picture of the embodiment 4; Characterization of rheological properties of Example 4 under scanning.
- Figure 6 is a dynamic mechanical rheological scan of the embodiment 5;
- Figure B is the gel point determination of the embodiment 5 in the dynamic rheological scan mode;
- Figure C is the optical picture of the embodiment 5; Characterization of rheological properties of Example 5 under scanning.
- Figure 7 is a graph showing the results of the injectability test of Example 5;
- Figure B is a scanning electron micrograph of Example 5 which is formed by recovery;
- Figure C is a graph showing the storage modulus before and after being sheared and thinned by rheological mechanics monitoring Example 5. (G') and the change in energy consumption modulus (G").
- Figure 8 is a graph showing the gelation time of different concentrations of silk protein solution (0%, 0.1%, 0.5%, 1.0%, 2.0%) induced by 0.4wt% NapFF solution;
- Figure B is different for 0.4wt% NapFF solution induction. Comparison of gel storage modulus formed by concentration of silk protein solution (0%, 0.1%, 0.5%, 1.0%, 2.0%).
- Figure 9 is a cytotoxicity assay of the CCK-8 kit method for testing peptide small molecule gelatin and silk fibroin solution.
- Fig. 10 is a graph showing the results of the soaking stability test of the high sugar medium (DMEM) of Example 5.
- Fig. 11 is a graph showing the results of a 5-day culture experiment of human umbilical vein endothelial cells HUVEC on the surface of Example 5.
- the resin was washed five times in the order of anhydrous DMF-anhydrous DCM-anhydrous MeOH-n-hexane (n-hexane), each time for 30 s, and finally the resin was dried with high purity nitrogen;
- TFA trifluoroacetic acid
- a method for rapidly gelling a silk protein solution under physiological conditions comprising the steps of:
- a method for rapidly gelling a silk protein solution under physiological conditions comprising the steps of:
- a method for rapidly gelling a silk protein solution under physiological conditions comprising the steps of:
- a method for rapidly gelling a silk protein solution under physiological conditions comprising the steps of:
- a method for rapidly gelling a silk protein solution under physiological conditions comprising the steps of:
- a method for rapidly gelling a silk protein solution under physiological conditions comprising the steps of:
- a method for rapidly gelling a silk protein solution under physiological conditions comprising the steps of:
- a method for rapidly gelling a silk protein solution under physiological conditions comprising the steps of:
- the polypeptide derivative small molecule NapFF used in the invention has good biocompatibility, low toxicity, easy degradation and other excellent characteristics, and the synthetic technology route is mature, the purification is simple, and mass production and synthesis can be carried out.
- a 0.1% silk fibroin solution can be induced to form a stable hydrogel within 1.5 hours under physiological conditions of pH 7.4 using only 0.2 wt% of NapFF solution. After increasing the NapFF content to 0.4 wt%, the gel time of 0.1% silk protein solution was further shortened to 30 min.
- the CCK-8 assay was used to detect the activity of the cells.
- Human umbilical vein endothelial cells HUVEC were dispersed in 96-well plates (cell concentration: 4000 cells/well) and cultured in a 5% CO 2 atmosphere at 37 ° C for 24 hours.
- the medium was replaced with different concentrations of NapFF (10.0 ⁇ M, 20.0 ⁇ M, 50.0 ⁇ M, 100.0 ⁇ M, 200.0 ⁇ M) and silk protein solution (0.1%, 0.2%, 0.5%, 1.0%, 2.0%) of cell culture medium.
- the original medium was replaced with CCK-8 solution, and finally, the optical density value (OD value) of the solution at 450 nm was measured with a microplate reader.
- Cell viability is expressed as a percentage of the number of control (untreated) cells. The cell viability of the control group was set to 100% by default, and all experiments were performed in parallel five times.
- Example 5 was carried out on human umbilical vein endothelial cells HUVEC for 5 days, using cell counting method: cell counting using blood cell counting plate method, the specific steps are:
- the blood cell counting plate is placed on the stage of the microscope to be clamped, and the counting area is first found under the low power microscope, and then converted into a high power microscope to observe and count;
- the single counting area is composed of 16 squares, according to the diagonal orientation, the number of cells in the upper middle, lower left, upper right, and lower right four squares (ie, 16*4 small cells), in order to ensure The accuracy of the counting, avoiding repeated counting and missing recording, should be uniformly defined for the statistics of the cells on the grid line when counting. For example, if the cells are located on a double line of a large square, when counting, the number is not on the lower line, and the number on the left line is not on the right line to reduce the error. That is, the cells located on the upper line and the left line of the original grid are counted in the grid, and the cells on the lower line and the right line of the grid are counted into the corresponding grids according to regulations;
- the cell density in the original cell suspension is: n*10 4 /mL.
- the small molecule gel factor NapFF of the present application can induce gelation of a low concentration silk fibroin solution under physiological conditions, and the hydrogel obtained by mixing 2.0% silk fibroin solution with 0.4 wt% NapFF has an injectability, indicating that we
- the introduced small molecule gelatin factor has a very significant effect on the gelation of silk fibroin, which can rapidly gel the silk protein solution at low concentration and pH 7.4, and the gel time is only tens to hundreds of seconds, and at the same time
- the mechanical properties of the gel factor are more than ten times stronger than that of the individual silk protein hydrogel, and the resulting hybrid gel has good biocompatibility and can be used as a potential cell culture scaffold material.
- the small molecule of the polypeptide exhibits mild induction conditions, economical synthesis, and rapid induction time.
- the hybrid gel has good biocompatibility and can further extend and expand the ability of silk fibroin as a good biocompatible new biomaterial.
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Abstract
本发明提供了一种生理条件下蚕丝蛋白溶液快速凝胶化的方法,包括以下步骤:(1)称取NapFF固体粉末置入玻璃小瓶底部,加入超纯水;(2)加入NaOH溶液,超声辅助溶解,使用可以精确控温的微电脑电热板70℃加热小瓶底部1-2min,形成透明溶液;(3)慢慢加入HCl溶液,并用枪头搅匀,至溶液pH为7.2-7.5;(4)加入蚕丝蛋白溶液;(5)加入超纯水定容至200μL;(6)水平放置,通过倾斜和倒置玻璃小瓶法观察凝胶化进程。本发明的方法可以在短时间内诱导低浓度蚕丝蛋白溶液快速凝胶化,且在体内可以被人体中的蛋白水解酶水解而被降解,不产生毒副作用,具有良好的生物相容性,可以作为良好的生物材料使用。
Description
本发明涉及一种生理条件下蚕丝蛋白溶液快速凝胶化的方法。
蚕丝蛋白(Silk Fibroin)又名丝素蛋白,是从蚕丝中提取的天然高分子纤维蛋白,含量约占蚕丝的70%~80%,含有18种氨基酸,其中甘氨酸(gly)、丙氨酸(ala)和丝氨酸(ser)占总含量的80%以上。丝素本身具有良好的机械性能和理化性质,如良好的柔韧性和抗拉伸强度、透气透湿性、缓释性等,而且经过不同处理可以得到不同的形态,例如纳米纤维、纳米颗粒、丝素蛋白薄膜、海绵状多孔支架材料、丝素蛋白水凝胶和利用静电纺丝制成的各种材料等。蚕丝蛋白由于其生物降解较慢,生物相容性好,免疫原性较低等优良特性,使得其长久以来被科学研究者们广泛应用到生物医用材料方面。但是,在生理条件下,蚕丝蛋白溶液的凝胶化过程非常缓慢,通常需要在酸性条件下(pH4左右)经过相当长的时间(>5天)和高浓度(>4%)时才会发生凝胶化。种种这些因素极大地限制了其在生物医学领域的应用。最近的研究结果表明,为了改变蚕丝蛋白凝胶化pH低、浓度高、所需时间长的特点,科学家们做了很多尝试,例如通过对蚕丝蛋白溶液施加涡流、电场,或者是对其进行长时间超声处理,使得蚕丝蛋白在生理条件下从溶液时的无规线团结构向凝胶状态时的β折叠结构转变。但是这些外加条件在蚕丝蛋白作为生物材料实际应用时受到很大限制。科学研究者们还通过向蚕丝蛋白溶液中添加有机小分子物质或高分子聚合物(例如:乙醇、表面活性剂十二烷基磺酸钠或亲水性聚合物聚乙二醇等)来调节其与蚕丝蛋白链之间的相互作用,从而加速蚕丝蛋白溶液的凝胶化速率,但是这一系列的凝胶过程可能与某些临床使用环境不相容,例如有机分子的潜在细胞和组织毒性;高分子聚合物的生物惰性,在生物体内难以降解等特点,这些措施虽然可以在一定程度上可以缩短蚕丝蛋白凝胶化时间,但是得到的蚕丝蛋白凝胶生物相容性差,以及较大的细胞毒性,这些问题导致其在生物医用材料方面的应用受到了极大限制。
发明内容
为解决现有技术中的上述问题,本发明的目的是提供一种生理条件下蚕丝蛋白溶液快速凝胶化的方法,可以在短时间内诱导低浓度蚕丝蛋白溶液快速凝胶化,而且在体内可以被人体中的蛋白水解酶水解而被降解,不产生毒副作用,具有良好的生物相容性,可以作为良好的生物材料使用。
为实现上述目的,本发明采用了以下的技术方案:
在一方面,本发明提供了一种生理条件下蚕丝蛋白溶液快速凝胶化的方法,包括以下步 骤:
(1)称取0.4-0.8mg2-萘乙酸-苯丙氨酸-苯丙氨酸固体粉末置入玻璃小瓶底部,加入130-170μL超纯水;
(2)加入8-12μL用超纯水配制的1mol/L的NaOH溶液,超声辅助溶解,加热小瓶底部1-2min,形成透明溶液;
(3)慢慢加入用超纯水配制的HCl溶液,并用枪头搅匀,至溶液pH为7.2-7.5;
(4)加入1-34μL蚕丝蛋白溶液;
(5)加入超纯水;
(6)水平放置,每隔1min-1h通过倾斜和倒置玻璃小瓶法观察凝胶化进程,使得蚕丝蛋白溶液快速凝胶化
优选地,在步骤(2)中,使用可以精确控温的微电脑电热板70℃加热。
优选地,在步骤(3)中,加入的HCl溶液的摩尔浓度为1mol/L。
优选地,在步骤(4)中,所述蚕丝蛋白溶液的浓度为12.03wt%。
优选地,在步骤(4)中,定容至200μL。。
进一步优选地,在步骤(3)中,溶液pH为7.4。
进一步优选地,蚕丝蛋白溶液的制备步骤为:
第一步:蚕丝脱胶
称取6.36g无水碳酸钠溶解于3L超纯水中,加入7.5g蚕丝煮沸1小时,捞出蚕丝在室温下自然过夜风干,得脱胶后的蚕丝;
第二步:溶解蚕丝
配制40mL浓度为9.3M的LiBr溶液,在圆底烧瓶中加入脱胶后的蚕丝和30ml的LiBr溶液,在温度60℃,转速200rpm下搅拌4小时;
第三步:蚕丝蛋白溶液透析
将溶解有蚕丝蛋白的LiBr溶液放入拦截分子量为3500透析袋中,用超纯水进行透析,每隔1小时换水,10h后变为间隔2-5小时换一次超纯水,透析4天;
第四步:浓缩溶液
待LiBr透析完全后,取出透析袋,在透析袋表面均匀涂抹PEG20000进行吸水浓缩,至表面PEG20000变粘稠后重新涂抹适量的PEG20000;浓缩一天,至溶液变得微黄;
第五步:离心
将浓缩后的溶液在4℃,4000r/min的条件下离心40min,离心后取上层清液,弃除少量杂质;
第六步:测浓度
利用质量差减法测定蚕丝蛋白浓度,准备一个干净的玻璃蒸发皿,用万分之一分析天平准确称取其质量,记为m
0,用移液枪吸取100μL蚕丝蛋白溶液于蒸发皿上,用万分之一分析天平准确称取此时质量,记为m
1,将装有蚕丝蛋白溶液的玻璃蒸发皿放入60℃烘箱干燥5-6小时,取出冷却后准确称取此时质量,记为m
2,计算公式为
每次测定浓度均需同时平行三个玻璃蒸发皿测量取平均值,即得平均浓度为12.03%的蚕丝蛋白溶液(SF)。进一步优选地,在步骤(4)中,蚕丝蛋白溶液的体积为34μL。在另一方面,本发明还提供了上述方法所制备的蚕丝蛋白凝胶。
在又一方面,本发明提供了上述蚕丝蛋白凝胶在生物医用材料中的应用。
有益效果:本发明的一种生理条件下蚕丝蛋白溶液快速凝胶化的方法,可以在短时间内诱导低浓度蚕丝蛋白溶液快速凝胶化,而且由于多肽衍生物小分子NapFF生物相容性很好,在体内可以被人体中的蛋白水解酶水解而被降解,不产生毒副作用,因此在制备得到的多肽衍生物小分子和蚕丝蛋白混合凝胶具有良好的生物相容性,在生理条件下达到快速诱导蚕丝蛋白溶液凝胶化后,仍然可以作为良好的生物材料使用,改变了传统的高分子聚合物、乙醇、表面活性剂等一系列诱导剂对蚕丝蛋白溶液诱导凝胶后无法用作生物材料使用的窘境。
图1为对比例1的凝胶形成过程及性能测试,其中,图A为NapFF凝胶形成过程;图B为NapFF溶液的TEM(标尺=200nm),纤维宽度约为12nm;图C为应力扫描下的流变力学测试(G'表示储能模量,G”表示耗能模量);图D为频率扫描下的流变力学性能表征。
图2为对比例2的凝胶形成过程及性能测试,其中,图A为蚕丝蛋白溶液凝胶形成过程;图B为蚕丝蛋白溶液的TEM(标尺=100nm),纤维宽度约为7nm;图C为应力扫描下的流变力学测试;图D为频率扫描下的流变力学性能表征。
图3为实施例1的凝胶形成过程及性能测试,其中,图A为混合溶液的凝胶形成过程,凝胶时间为1.5h;图B为实施例1的TEM(标尺=100nm),两种组分的不同长度的纳米纤维均可以容易辨别出;图C为实施例1在频率扫描下的流变力学性能表征。
图4中图A为实施例3的动态力学流变扫描图;图B为实施例3在动态流变扫描模式下的凝胶点确定;图C为实施例3的光学图片;图D为频率扫描下的实施例3的流变力学性能表征。
图5中图A为实施例4的动态力学流变扫描图;图B为实施例4在动态流变扫描模式下 的凝胶点确定;图C为实施例4的光学图片;图D为频率扫描下的实施例4的流变力学性能表征。
图6中图A为实施例5的动态力学流变扫描图;图B为实施例5在动态流变扫描模式下的凝胶点确定;图C为实施例5的光学图片;图D为频率扫描下的实施例5的流变力学性能表征。
图7中图A为实施例5可注射性实验结果图;图B为恢复形成的实施例5扫描电子显微镜图;图C为流变力学监测实施例5被剪切变稀前后储能模量(G’)和耗能模量(G”)的变化。
图8中图A为0.4wt%NapFF溶液诱导不同浓度的蚕丝蛋白溶液(0%,0.1%,0.5%,1.0%,2.0%)凝胶化时间比较;图B为0.4wt%NapFF溶液诱导不同浓度的蚕丝蛋白溶液(0%,0.1%,0.5%,1.0%,2.0%)形成的凝胶储能模量的比较。
图9为CCK-8试剂盒方法测试多肽小分子凝胶因子和蚕丝蛋白溶液的细胞毒性实验。
图10为实施例5的高糖培养基(DMEM)浸泡稳定性实验结果图。
图11为人脐静脉血管内皮细胞HUVEC在实施例5表面进行5天培养实验结果图。
多肽衍生物小分子2-萘乙酸-苯丙氨酸-苯丙氨酸(NapFF)的固相合成:
a)溶胀树脂
将2g2-氯三苯甲基氯树脂(100~200目,0.3~0.8mmol/g)放入固相合成反应器后在通高纯(99.999%)氮气的保护下加入干燥除水后的二氯甲烷(DCM)并充分溶胀,所通氮气量使树脂恰好能在液体中充分翻滚但不过于剧烈,溶胀约1h,溶胀后用氮气将DCM挤出排净并用无水N,N-二甲基甲酰胺(DMF)冲洗树脂5遍,每次停留30s;
b)接Fmoc-Phe-OH
称取2712mg Fmoc-Phe-OH(平均每克树脂上有1.2-1.7mmol活性位点),用约20mL无水DMF溶解,加入3.04mL N,N-二异丙基乙胺(DIEA),超声溶解并充分混合后加入反应器中,在氮气流中震荡约2h,充分反应后用氮气将剩余废液排出,再用无水DMF冲洗树脂三遍,每次停留30s;
c)使树脂未反应活性点失活
取32mL无水DCM、6mL无水甲醇(MeOH)和2mL DIEA充分混匀,分两次冲洗树脂,每次停留10min,充分反应后用氮气将废液挤出,用无水DMF冲洗树脂三次,每次停留30s;
d)对Fmoc保护基的脱保护
配制20%的哌啶溶液(20mL哌啶:80mL无水DMF)50mL,分三次充分洗涤树脂,每 次洗涤10min,充分反应后将废液排出,并用无水DMF冲洗树脂三次,每次停留30秒;
e)接Fmoc-Phe-OH
称取2712mg Fmoc-Phe-OH,用20mL无水DMF溶解,加入3.04mL DIEA和2628mg O-苯并三氮唑-四甲基脲六氟磷酸盐(HBTU),超声溶解并充分混合后加入反应器中,在氮气流中震荡约2h,充分反应后用氮气将剩余废液挤出,再用无水DMF冲洗树脂三遍,每次停留30s;
f)对Fmoc保护基的脱保护
取20%的哌啶溶液50mL,分三次充分洗涤树脂,每次停留10min,充分反应后将废液挤出,并用无水DMF冲洗树脂三次,每次停留30s;
g)接2-萘乙酸
称取1304mg2-萘乙酸,用20mL无水DMF溶解,加入3.04mLDIEA和2628mg HBTU,充分混合后加入反应器,在氮气流中震荡1.5h,充分反应后用氮气将剩余废液排出;
h)冲洗树脂
以无水DMF-无水DCM-无水MeOH-无水正己烷(n-hexane)的顺序分别冲洗树脂五遍,每次洗涤30s,最后用高纯氮气吹干树脂;
i)从树脂上切下产物及产物处理
在持续通高纯(99.999%)氮气的保护下,用95%的三氟乙酸(TFA:水=95:5)溶液洗涤树脂3次,第一次洗涤2h,第二次和第三次各洗涤30min,充分反应后将切除液排出并收集,用空气泵吹干TFA,向切除液中加入适量冰乙醚,放入冰箱,-20℃下冷却过夜沉析,次日抽滤后向其中加少量超纯水放入冰箱冷冻,待冷冻干燥后称重,干燥好的粗产品经柱层析色谱纯化(淋洗剂为二氯甲烷:甲醇=30:1~15:1),纯化后产率为78%。
实施例1
一种生理条件下蚕丝蛋白溶液快速凝胶化的方法,包括以下步骤:
(1)称取0.4mg NapFF固体粉末置入玻璃小瓶底部,加入170μL的超纯水;
(2)加入8μL用超纯水配制的1mol/L的NaOH溶液,超声辅助溶解,使用可以精确控温的微电脑电热板70℃加热小瓶底部1min,形成透明溶液;
(3)慢慢加入用超纯水配制的1mol/L的HCl溶液,并用枪头搅匀,至溶液pH为7.4;
(4)加入1.7μL浓度为12.03wt%的蚕丝蛋白溶液;
(5)加入超纯水定容至200μL;
(6)水平放置,每隔10min通过倾斜和倒置玻璃小瓶法观察凝胶化进程。
在经过1.5h室温放置后,有凝胶化的迹象,可以形成透明度很高的混合水凝胶。
实施例2
一种生理条件下蚕丝蛋白溶液快速凝胶化的方法,包括以下步骤:
(1)称取0.8mg NapFF固体粉末置入玻璃小瓶底部,加入170μL的超纯水;
(2)加入10μL用超纯水配制的1mol/L的NaOH溶液,超声辅助溶解,使用可以精确控温的微电脑电热板70℃加热小瓶底部2min,形成透明溶液;
(3)慢慢加入用超纯水配制的1mol/L的HCl溶液,并用枪头搅匀,至溶液pH为7.4;
(4)加入1.7μL浓度为12.03wt%的蚕丝蛋白溶液;
(5)加入超纯水定容至200μL;
(6)水平放置,每隔5min通过倾斜和倒置玻璃小瓶法观察凝胶化进程。
发现在经过30min室温放置后,有凝胶化的迹象,可以形成透明度很高的混合水凝胶。
实施例3
一种生理条件下蚕丝蛋白溶液快速凝胶化的方法,包括以下步骤:
(1)称取0.8mg NapFF固体粉末置入玻璃小瓶底部,加入150μL的超纯水;
(2)加入12μL用超纯水配制的1mol/L的NaOH溶液,超声辅助溶解,使用可以精确控温的微电脑电热板70℃加热小瓶底部2min,形成透明溶液;
(3)慢慢加入用超纯水配制的1mol/L的HCl溶液,并用枪头搅匀,至溶液pH为7.4;
(4)加入8.3μL浓度为12.03wt%的蚕丝蛋白溶液;
(5)加入超纯水定容至200μL;
(6)水平放置,每隔5min通过倾斜和倒置玻璃小瓶法观察凝胶化进程。
发现在室温放置10min内可以快速凝胶化形成透明度一般的混合水凝胶,为了进一步确定凝胶点时间,我们通过动态流变力学监测凝胶化时间点为483s(图4)。
实施例4
一种生理条件下蚕丝蛋白溶液快速凝胶化的方法,包括以下步骤:
(1)称取0.8mg NapFF固体粉末置入玻璃小瓶底部,加入150μL的超纯水;
(2)加入12μL用超纯水配制的1mol/L的NaOH溶液,超声辅助溶解,使用可以精确控温的微电脑电热板70℃加热小瓶底部2min,形成透明溶液;
(3)慢慢加入用超纯水配制的1mol/L的HCl溶液,并用枪头搅匀,至溶液pH为7.4;
(4)加入16.6μL浓度为12.03wt%的蚕丝蛋白溶液;
(5)加入超纯水定容至200μL;
(6)水平放置,每隔2min通过倾斜和倒置玻璃小瓶法观察凝胶化进程。
发现在室温放置10min内可以快速凝胶化形成透明度较低的混合水凝胶。为了进一步确定凝胶点时间,我们通过动态流变力学监测凝胶化时间点为313.8s(图5)。
实施例5
一种生理条件下蚕丝蛋白溶液快速凝胶化的方法,包括以下步骤:
(1)称取0.8mg NapFF固体粉末置入玻璃小瓶底部,加入130μL的超纯水;
(2)加入15μL用超纯水配制的1mol/L的NaOH溶液,超声辅助溶解,使用可以精确控温的微电脑电热板70℃加热小瓶底部2min,形成透明溶液;
(3)慢慢加入用超纯水配制的1mol/L的HCl溶液,并用枪头搅匀,至溶液pH为7.4;
(4)加入33.2μL浓度为12.03wt%的蚕丝蛋白溶液;
(5)加入超纯水定容至200μL;
(6)水平放置,每隔1min通过倾斜和倒置玻璃小瓶法观察凝胶化进程。
发现在室温放置5min内可以快速凝胶化形成透明度较低的混合水凝胶。为了进一步确定凝胶点时间,我们通过动态流变力学监测凝胶化时间点为96.9s(图6)。
对比例1
一种生理条件下蚕丝蛋白溶液快速凝胶化的方法,包括以下步骤:
(1)称取0.8mg NapFF固体粉末置入玻璃小瓶底部,加入170μL的超纯水;
(2)加入10μL用超纯水配制的1mol/L的NaOH溶液,超声辅助溶解,使用可以精确控温的微电脑电热板70℃加热小瓶底部2min,形成透明溶液;
(3)慢慢加入用超纯水配制的1mol/L的HCl溶液,并用枪头搅匀,至溶液pH为7.4;
(4)加入超纯水定容至200μL;
(5)水平静置,通过倾斜和倒置玻璃小瓶法观察凝胶化进程。
发现12h后发现NapFF溶液已经形成了宏观上稳定透明的水凝胶。
对比例2
一种生理条件下蚕丝蛋白溶液快速凝胶化的方法,包括以下步骤:
(1)称取33.3μL浓度为12.03wt%的蚕丝蛋白溶液加入到玻璃小瓶底部;
(2)加入超纯水定容至200μL;
(3)水平放置,每隔1天通过倾斜和倒置玻璃小瓶法观察凝胶化进程。
(4)发现在经过14天室温放置下,蚕丝蛋白溶液可以形成宏观上不透明的水凝胶。
对比例3
一种生理条件下蚕丝蛋白溶液快速凝胶化的方法,包括以下步骤:
(1)称取0.2mg NapFF固体粉末置入玻璃小瓶底部,加入180μL的超纯水;
(2)加入5μL用超纯水配制的1mol/L的NaOH溶液,超声辅助溶解,使用可以精确控温的微电脑电热板70℃加热小瓶底部2min,形成透明溶液;
(3)慢慢加入用超纯水配制的1mol/L的HCl溶液,并用枪头搅匀,至溶液pH为7.4;
(4)加入1.7μL浓度为12.03%的蚕丝蛋白溶液;
(5)加入超纯水定容至200μL;
(6)水平放置,每隔1min通过倾斜和倒置玻璃小瓶法观察凝胶化进程。
发现在经过24h甚至7天室温放置后,仍然没有凝胶化的迹象。
表1 不同条件下NapFF和蚕丝蛋白溶液SF混合得到的凝胶性能一览表
[a]24h或更长时间没有凝胶化的迹象。
本发明所使用的多肽衍生物小分子NapFF具有良好生物相容性,细胞低毒性,易降解等优良特性,并且合成技术路线成熟,纯化简便,可以进行大量生产合成。在诱导蚕丝蛋白凝胶化时,可以在生理条件pH7.4下,只利用0.2wt%的NapFF溶液即可诱导0.1%的蚕丝蛋白溶液在1.5小时内形成稳定的水凝胶。在提高NapFF的含量到0.4wt%后,0.1%的蚕丝蛋白溶液凝胶时间进一步缩短为30min。在我们对二者组分比例作进一步筛选后,发现0.4wt%的NapFF和2.0%的蚕丝蛋白溶液混合室温下静置97s后即可形成非常稳固的水凝胶。通过动态流变力学测试我们确定了其凝胶点时间和形成的凝胶的力学性能。其储能模量较单独的2.0%蚕丝蛋白溶液形成的凝胶有了十多倍的增强。透射电镜(TEM)的结果也表明二者组分在混合前分别呈现不同宽度的纳米纤维,当混合后,在透射电镜图中我们可以清楚地分辨出二者组分别形成了相互穿插的物理交联纳米纤维网络,还有较粗的纳米纤维簇的形成。
采用CCK-8实验来检测细胞的活性,人脐静脉血管内皮细胞HUVEC被分散到96孔板(细胞浓度为4000个/孔)中在5%CO
2气氛下37℃培养24小时后,将原来的培养基替换为不同浓度的NapFF(10.0μM,20.0μM,50.0μM,100.0μM,200.0μM)和蚕丝蛋白溶液(0.1%,0.2%,0.5%,1.0%,2.0%)的细胞培养基,24小时后,用CCK-8溶液替代原来的培养基,最后,用酶标仪测试溶液在450nm的光密度值(OD值)。细胞活性表示为对照(未处理的)细胞数的百分比。对照组的细胞生存能力被默认设置为100%,所有实验平行五次进行的。
如图9所示的测试结果可以得出结论,我们合成的NapFF生物相容性很好,蚕丝蛋白溶液对细胞也无毒良好。
将实施例5对人脐静脉血管内皮细胞HUVEC进行5天培养实验,采用细胞计数方法:细胞计数采用血球计数板法,具体步骤为:
1.根据消化后的细胞悬液浓度,加DMEM适当稀释;
2.取洁净的血球计数板一块,在计数区上盖上一块盖玻片。
3.将细胞悬液摇匀,用10μL枪头吸取悬液并从计数板中间平台两侧的沟槽内沿盖玻片的边缘滴入,使之充满计数区,勿使气泡产生;
4.静置片刻,使细胞沉降到计数板上,不再随液体漂移。将血球计数板放置于显微镜的载物台上夹稳,先在低倍镜下找到计数区后,再转换高倍镜观察并计数;
5.单个计数区是由16个中方格组成,按对角线方位,数左上、左下、右上、右下的4个中方格(即16*4个小格)的细胞数,为了保证计数的准确性,避免重复计数和漏记,在计数时,对在格线上的细胞的统计应有统一的规定。如细胞位于大方格的双线上,计数时则数上线不数下线,数左线不数右线,以减少误差。即位于本格上线和左线上的细胞计入本格,本格的下线和右线上的细胞按规定计入相应的格中;
6.测数完毕,计算得到每个中方格中细胞的平均值n,取下盖玻片,用75%乙醇将血球计数板冲洗干净后晾干,放入盒内保存;
7.原细胞悬浮液中的细胞密度为:n*10
4个/mL。
8.染色后的细胞计数采用ImageJ软件计数,采用不同视野下的五个图来平均计数。
实验结果见图11。
本申请的小分子凝胶因子NapFF可以在生理条件下诱导低浓度蚕丝蛋白溶液凝胶化,当2.0%的蚕丝蛋白溶液与0.4wt%NapFF混合后得到的水凝胶具有可注射能力,表明我们引入的多肽小分子凝胶因子对蚕丝蛋白凝胶化具有非常显著的效果,可以使蚕丝蛋白溶液在低浓度和pH7.4时快速凝胶化,凝胶时间只有几十至上百秒,同时得到的凝胶因子的力学性能较单独蚕丝蛋白水凝胶增强十余倍,而且得到的混合凝胶生物相容性良好,可以作为潜在的细 胞培养支架材料。这是以往传统使用乙醇等有机小分子物质、表面活性剂和高分子聚合物等来诱导蚕丝蛋白凝胶来说,多肽小分子显示出其诱导条件温和化、合成方式经济化、诱导时间快速化、混合凝胶生物相容性好等特点,可以将蚕丝蛋白作为良好的生物相容性的新型生物材料的能力更进一步得到延伸和拓展。
Claims (10)
- 一种生理条件下蚕丝蛋白溶液快速凝胶化的方法,其特征在于:包括以下步骤:(1)称取0.4-0.8mg 2-萘乙酸-苯丙氨酸-苯丙氨酸固体粉末置入玻璃小瓶底部,加入130-170μL超纯水;(2)加入8-12μL用超纯水配制的1mol/L的NaOH溶液,超声辅助溶解,加热小瓶底部1-2min,形成透明溶液;(3)加入用超纯水配制的HCl溶液,并用枪头搅匀,至溶液pH为7.2-7.5;(4)加入1-34μL的蚕丝蛋白溶液;(5)加入超纯水定容;(6)水平放置,每隔1min-1h通过倾斜和倒置玻璃小瓶法使得蚕丝蛋白溶液快速凝胶化。
- 根据权利要求1所述的一种生理条件下蚕丝蛋白溶液快速凝胶化的方法,其特征在于:在步骤(2)中,使用可以精确控温的微电脑电热板70℃加热。
- 根据权利要求1所述的一种生理条件下蚕丝蛋白溶液快速凝胶化的方法,其特征在于:在步骤(3)中,加入的HCl溶液的摩尔浓度为1mol/L。
- 根据权利要求1所述的一种生理条件下蚕丝蛋白溶液快速凝胶化的方法,其特征在于:在步骤(4)中,所述蚕丝蛋白溶液的浓度为12.03wt%。
- 根据权利要求1所述的一种生理条件下蚕丝蛋白溶液快速凝胶化的方法,其特征在于:在步骤(4)中,定容至200μL。
- 根据权利要求1所述的一种生理条件下蚕丝蛋白溶液快速凝胶化的方法,其特征在于:在步骤(3)中,溶液pH为7.4。
- 根据权利要求4所述的一种生理条件下蚕丝蛋白溶液快速凝胶化的方法,其特征在于:所述蚕丝蛋白溶液的制备步骤为:第一步:蚕丝脱胶称取6.36g无水碳酸钠溶解于3L超纯水中,加入7.5g蚕丝煮沸1小时,捞出蚕丝在室温下自然过夜风干,得脱胶后的蚕丝;第二步:溶解蚕丝配制40mL浓度为9.3M的LiBr溶液,在圆底烧瓶中加入脱胶后的蚕丝和30ml LiBr溶液,在温度60℃,转速200rpm下搅拌4小时;第三步:蚕丝蛋白溶液透析将溶解有蚕丝蛋白的LiBr溶液放入拦截分子量为3500透析袋中,用超纯水进行透析,每隔1小时换水,10h后变为间隔2-5小时换一次超纯水,透析4天;第四步:浓缩溶液待LiBr透析完全后,取出透析袋,在透析袋表面均匀涂抹PEG20000进行吸水浓缩,至表面PEG20000变粘稠后重新涂抹适量的PEG20000;浓缩一天,至溶液变得微黄;第五步:离心将浓缩后的溶液在4℃,4000r/min的条件下离心40min,离心后取上层清液,弃除少量杂质;第六步:测浓度
- 根据权利要求1所述的一种生理条件下蚕丝蛋白溶液快速凝胶化的方法,其特征在于:在步骤(4),蚕丝蛋白溶液的体积为34μL。
- 一种根据权利要求1-8中任一项所述方法制备的蚕丝蛋白凝胶。
- 权利要求9所述的蚕丝蛋白凝胶在生物医用材料中的应用。
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