【作者】 陈翔；

【导师】 俞豪杰； 王立；

【作者基本信息】 浙江大学 ， 化学工程与技术， 2022， 博士

【摘要】 糖敏微针贴可根据血糖浓度自动调节胰岛素释放速度,并且具有微创无痛等优点,有助于糖尿病人更好地管理血糖,提升生活质量。现有文献报道的糖敏微针贴主要有组装式、一体式和核壳结构式微针贴等三类,还存在着胰岛素负载量低、组分溶解和制备复杂等问题。本论文采用苯硼酸基为糖敏基团,以简单温和的方法制备了一系列一体式和核壳结构式糖敏凝胶微针贴,研究了其溶胀行为、机械强度、胰岛素分布和胰岛素控释性能,探讨了其糖敏机理,探索了其在大鼠降血糖和预防低血糖方面的应用。(1)以N-异丙基丙烯酰胺、N-乙烯基-2-吡咯烷酮和3-丙烯酰胺苯硼酸为单体,在微针模具中共聚制备了微针贴,并利用滴/干法在微针贴上负载胰岛素。研究了微针贴的溶胀度、胰岛素分布以及胰岛素控释性能。结果表明:微针贴在pH 9.0、27℃时表现出良好的糖敏溶胀性能。滴/干法负载方式使胰岛素同时分布在微针贴的表面和内部,处于微针贴内部的胰岛素约占43.2%。处于微针贴表面的胰岛素可以在～10 min内释放,以快速降低血糖,而微针贴内胰岛素根据葡萄糖浓度释放,4g/L葡萄糖浓度下12h的胰岛素释放量是1g/L葡萄糖浓度下的1.6倍。(2)合成了4-((2-氨基乙基)氨基甲酰)-3-氟基苯硼酸改性透明质酸钠(HA-FPBA),将其与聚乙烯醇(PVA)混合通过硼酸酯键制备凝胶。微针贴可以由这种可流动凝胶直接制备,并通过反复冻融法使内部的PVA链结晶形成物理交联增加强度。探索了纤维素纳米纤维(CNF)的引入对微针贴机械强度和形貌的影响,研究了微针贴的胰岛素负载量、糖敏释放性能以及对糖尿病大鼠的降血糖作用。研究发现:添加CNF可以显著提高微针贴的机械强度。微针贴最多可以负载53.1 wt%胰岛素而不影响其机械强度,具有高负载量的优势。由于HA-FPBA的苯硼酸基团接枝率较低,微针贴主要起到缓释胰岛素的效果。糖尿病大鼠的降血糖实验证明微针贴可以有效地刺穿皮肤并在较长时间内维持正常血糖浓度,而且处于针体和基底内的胰岛素均可以扩散进入皮肤。简单的制备工艺和高负载能力使这种微针贴有很好的应用前景。(3)合成了4-羧基-3-氟基苯硼酸改性聚烯丙基胺(PAH-FPBA),并将其与PVA混合通过硼酸酯键形成凝胶。交联密度可调糖敏微针贴可以由这种可流动凝胶直接制备,并通过反复冻融法使内部的PVA链结晶形成物理交联提高强度。聚烯丙基胺上的氨基不仅可以作为反应基团简化糖敏材料制备过程,而且还有助于苯硼酸基团和PVA的结合。通过研究凝胶在不同糖浓度下的流变和溶胀行为,根据橡胶弹性理论计算了有效交联密度,证实了葡萄糖可以破坏硼酸酯键,使得凝胶的交联密度降低。微针贴的胰岛素释放实验表明了微针贴可以有效控制胰岛素的释放,并且通过引入额外的离子交联点证实了微针贴的控释性能与微针贴的网络尺寸是否匹配胰岛素的分子尺寸相关。探索了微针贴在糖尿病大鼠降血糖方面的应用,表明微针贴可以有效递送胰岛素。(4)合成了 4-羧基-3-氟基苯硼酸改性ε-聚赖氨酸(PLL-FPBA)。以PLL-FPBA和PVA为材料,通过温和的方法制备了交联密度可调的核壳结构凝胶微针贴。微针贴最外层的糖敏壳由PLL-FPBA和PVA构成,内部的针体由纯PVA负载胰岛素组成。研究了不同组成糖敏壳的结晶性能、玻璃化转变温度、溶胀行为和胰岛素控释性能。结果表明:糖敏壳内主要是通过硼酸酯键形成交联,其控释机理可能是在葡萄糖和水的协同作用下,硼酸酯键的分解引起溶胀度的差异,从而控制胰岛素的释放速率。探究了不同糖敏壳厚度微针贴的机械强度、胰岛素分布和胰岛素控释性能。发现糖敏壳层厚度的增加有助于核壳结构微针贴整体机械强度的提高以及对胰岛素的控释,但会引起处于针体内的胰岛素占比逐步减少。可溶胀PVA针体的存在会提高糖敏壳层在初始阶段的溶胀度,进而提高糖敏壳的胰岛素释放速度。在大鼠实验中,该种核壳结构微针贴具有很高的刺穿效率,在皮肤内只溶胀并保持结构完整,可以有效递送胰岛素,其达到完全溶胀后降血糖速率与直接注射相似,而且可以有效预防低血糖,具有很好的应用前景。(5)以PLL-FPBA、PVA和PVP为原料制备了核壳结构糖敏微针贴。研究了含不同分子量PVP的糖敏壳的表面形貌、结晶性能、玻璃化转变温度和溶胀行为。PVP的分子量对糖敏壳的形貌和硼酸酯键形成的影响不明显,但会显著影响壳的溶胀性能。在糖敏壳的胰岛素控释实验中,随着糖敏壳添加的PVP分子量增大,胰岛素的释放速度越来越快,但是不同糖浓度下的释放差异不断变小。研究了添加PVP的核壳结构微针贴的形貌、机械强度和胰岛素控释行为。发现添加PVP后的完整核壳结构微针贴具有很好的形貌,并且添加不同分子量的PVP对微针贴的机械强度影响不明显,但随着添加的PVP分子量变大,微针贴对胰岛素的释放速度变快,控释性能变弱。通过大鼠实验发现该种核壳结构微针贴有较高的刺穿率,并且溶胀的微针贴可以紧密地贴在皮肤表面。该核壳结构微针贴能够较长时间地维持血糖处在正常范围内,并可以通过提高添加的PVP分子量缩短起效时间。更多还原

【Abstract】 Glucose-responsive microneedle patches can automatically adjust the insulin release rate according to the blood glucose level and has the advantages of minimally invasive and painless,which help diabetic patients better manage blood glucose and improve their quality of life.There are three main types of glucose-responsive microneedle patches reported in the existing literature,such as integrated,all-in-one or core-shell microneedle patch,which still have problems such as low insulin loading capacity,component dissolution and complex preparation.In this thesis,a series of allin-one and core-shell glucose-responsive gel microneedle patches using phenylboronic acid group as glucose-responsive group were prepared by simple and mild methods.Their swelling behavior,mechanical strength,insulin distribution and controlled insulin release properties were studied,the glucose-responsive mechanism was discussed,and their applications on reducing blood glucose and preventing hypoglycemia in rats were studied.(1)The microneedle patch was prepared by copolymerization of N-isopropyl acrylamide,N-vinyl-2-pyrrolidone,and 3-acrylamidoephenylboronic acid in the microneedle mold,and insulin was loaded by drop/dry method.The swelling degree,insulin distribution and insulin-controlled release performance of microneedle patch were studied.It was found that the microneedle patch showed good glucose-responsive swelling property at pH 9.0 and 27℃.The drop/dry loading method made insulin distributed both on the surface and inside of the microneedle patch,and the insulin inside the microneedle patch accounted for about 43.2%.Insulin on the surface of the microneedle patch was released in～10 min to rapidly reduce blood glucose.The release of insulin inside the microneedle patch depended on the glucose concentration.The release of insulin at 4 g/L glucose concentration was 1.6 times higher than that at 1 g/L glucose concentration at 12 h.(2)Sodium hyaluronate modified by 4-((2-aminoethyl)carbamoyl)-3fluorophenylboronic acid(HA-FPBA)was synthesized and mixed with polyvinyl alcohol(PVA)to prepare gels through the borate ester bond.Microneedle patches were prepared directly through the fluxible hydrogel,and the internal PVA chain could be crystallized to form physical crosslinking to enhance mechanical strength by repeated freezing and thawing.The effect of the introduction of cellulose nanofiber(CNF)on mechanical strength and morphology of microneedle patch was explored.The insulin loading capacity,glucose-responsive release property and hypoglycemic effect of the microneedle patch on diabetic rats were studied.It was found that adding CNF could significantly improve the mechanical strength of microneedle patch.The microneedle patch could load 53.1 wt%of insulin at most without affecting its mechanical strength,which showed the advantage of high loading capacity.Due to the low grafting rate of phenylboric acid groups on HA-FPBA,microneedle patch mainly played the effect of slow-release insulin.The hypoglycemic test in diabetic rats showed that the microneedle patch could effectively pierce the skin and maintain normal blood glucose level for a long time,and insulin in the needle and base could both diffuse into the skin.The simple preparation process and high loading capacity make this microneedle patch have great application prospects.(3)Polyallylamine modified by 3-carboxy-4-fluorophenylboronicacid(PAHFPBA)was synthesized and mixed with PVA to form gels through the borate ester bond.Crosslinking density changeable microneedle patches were prepared directly through the fluxible hydrogel,and the internal PVA chain could be crystallized to form physical crosslinking to enhance mechanical strength by repeated freezing and thawing.The amino groups on polyallylamine not only could be used as a reaction group to simplify the preparation process of glucose-responsive materials,but also help the combination of phenylboronic acid groups and PVA.By studying the rheological and swelling behavior of the hydrogel at different glucose concentrations,and calculating the effective crosslinking density according to the theory of rubber elasticity,it was confirmed that glucose could break borate ester bond,resulting in decrease of crosslinking density of hydrogel.The insulin release experiment of the microneedle patch showed that microneedle could effectively control the release of insulin,and it was confirmed by introducing additional ionic crosslinking points that the controlled release performance of the microneedle patch was related to whether the network size of microneedle patch matched the molecular size of insulin.The application of the microneedle patch in reducing blood glucose level in diabetic rats was explored,indicating that microneedle patch could effectively transfer insulin.(4)ε-Polylysine modified by 3-carboxy-4-fluorophenylboronicacid(PLL-FPBA)was synthesized.Using PLL-FPBA and PVA as materials,the crosslinking density changeable core-shell structure gel microneedle patches were prepared by a mild method.The outer layer of glucose-responsive shell was formed by PLL-FPBA and PVA,and the inner needle was composed of pure PVA loaded insulin.The crystallinity,glass transition temperature,swelling behavior and controlled insulin release performance of glucose-responsive shells with different composition were studied.It was found that the glucose-responsive shell was mainly crosslinked by borate ester bond.The controlled release mechanism may be that under the synergistic action of glucose and water,the decomposition of borate ester bonds leads to the difference of swelling degree,so as to control the release rate of insulin.The mechanical strength,insulin distribution and controlled insulin release property of the microneedle patch with different glucose-responsive shell thickness were investigated.It was found that the increase of shell thickness was helpful to the improvement of the overall mechanical strength and the controlled release of insulin,but it would lead to the gradual reduction of the proportion of insulin in the needles.The existence of swellable inner PVA needle increase the swelling degree of glucose-responsive shell in the initial stage,and then improve the insulin release speed of shell.In rat experiments,the core-shell structure microneedle patch showed high puncture efficiency,only swelled,kept intact in the skin,and could effectively transfer insulin.Its hypoglycemic rate after complete swelling was similar to that of direct injection,and effectively prevented hypoglycemia,which showed a good application prospect.(5)The core-shell structure gel microneedle patches were prepared using PLLFPBA,PVA and PVP as materials.The surface morphology,crystallinity,glass transition temperature and swelling behavior of the shell containing different molecular weight PVP were studied.The molecular weight of PVP had no obvious effect on the morphology of the glucose-responsive shell and the formation of borate ester bonds,but it would significantly affect the swelling property of the shell.In the in vitro controlled release experiment of glucose-responsive shell,the release rate of insulin became faster with the increase of PVP molecular weight added to shell,but the release difference under different glucose concentrations decreased.The morphology,mechanical strength and controlled insulin release behavior of core-shell microneedle patches added with PVP were studied.It was found that the intact core-shell structure microneedle patch added with PVP showed a good morphology,and the addition of PVP with different molecular weight had no obvious effect on the mechanical strength of microneedle patch.However,with the increase of PVP molecular weight,the insulin release speed was improved,but the controlled insulin release ability of microneedle patch became weaker.Through the rat experiment,the core-shell structure microneedle patch had a high puncture rate,and the swollen microneedle patch could be closely attached to the skin surface.The core-shell structure microneedle patch could maintain the blood glucose in the normal range for a long time and its onset time could be shortened by increasing the molecular weight of introduced PVP.更多还原