【作者】 谢芳；

【导师】 蔡杰；

【作者基本信息】 武汉大学 ， 高分子化学与物理， 2022， 博士

【摘要】 随着健康中国发展战略和可持续发展战略提出,以及人们对生命健康意识的不断增强,迫切需要研发新型生物医用材料以提高生物医用材料的自主研发能力。甲壳素作为自然界储量第二的天然聚多糖,是一种来源广泛的可再生资源,其有效开发和利用具有非常重要的意义。甲壳素脱乙酰化可转变成壳聚糖,甲壳素和壳聚糖都具有优异的生物相容性、生物可降解性、低免疫原性和化学反应活性,是开发生物医用材料的理想原料。然而,甲壳素和壳聚糖分子链间存在大量的分子内和分子间氢键相互作用,化学性质稳定,难以溶解于水和大部分有机溶剂中,而且由于缺乏能够同时实现甲壳素和壳聚糖溶解的溶剂体系,限制了甲壳素和壳聚糖在同一均相衍生化平台中的改性与应用。此外,甲壳素和壳聚糖在生物功能和活性上有很大的不同,系统研究甲壳素和壳聚糖衍生物的合成及其生物医学功能具有非常重要的研究意义和应用前景。本工作的主要创新点如下:(1)实现了季铵化甲壳素和季铵化壳聚糖在KOH/尿素水溶液中的均相合成,揭示了季铵化甲壳素和季铵化壳聚糖的脱乙酰度和阳离子取代度对抗菌选择性的影响,阐明其抗菌机制和促伤口愈合性能;(2)构建了负载纳米酶和胚胎干细胞外泌体的多功能季铵化壳聚糖基水凝胶,揭示其自凝胶行为、自愈合性能和自适应性,阐明了促MRSA感染慢性创面愈合机制;(3)利用季铵化甲壳素/壳聚糖表面改性医用聚氨酯,揭示其表面抗菌活性、生物相容性,阐明体内预防感染性能和机制;(4)合成了苯硼酸改性的季铵化甲壳素/壳聚糖,制备包裹红景天的季铵化甲壳素/壳聚糖水凝胶,揭示其物理交联机制和自凝胶行为,发现了季铵化甲壳素的抗炎效果优于季铵化壳聚糖,阐明其抗炎机制;(5)创建了仿细胞外基质的季铵化甲壳素-纤维蛋白水凝胶,阐明了水凝胶的黏弹性和细胞行为与功能的构效关系,初步构建出功能性心肌组织。本论文的主要研究内容和结论包括以下几个部分。在KOH/尿素体系中均相制备一系列不同脱乙酰度(DD’)和不同阳离子取代度(DQ)的季铵化甲壳素(QC)和季铵化壳聚糖衍生物(QCS),研究其化学结构与生物活性的构效关系。核磁共振氢谱(1H NMR)、核磁共振碳谱(13C NMR)和傅立叶红外光谱(FT-IR)表征其化学结构,揭示其脱乙酰度和阳离子取代度对抗菌性、细胞相容性和血液相容性的影响:脱乙酰度和阳离子取代度与抗菌性呈正相关,阳离子取代度与细胞相容性呈负相关,而在阳离子取代度相同时,脱乙酰度不影响细胞相容性,所有的材料都显示出优异的血液相容性。其中QCS-2被选为最优的抗菌剂,其DQ为0.46,DD’为82%,QCS-2对革兰氏阳性和革兰氏阴性细菌均表现优异的抗菌性,同时具有良好的生物相容性。由于QCS-2通过物理破坏细菌细胞膜的抗菌机制,它不会产生抗菌耐药性,还表现出对细菌生物膜形成的有效抑制和对成熟细菌生物膜的有效清除。此外,QCS-2表现出优异的体内抗感染效果,并通过杀死细菌、减轻局部炎症反应、促进血管生成和胶原蛋白的沉积,从而有效地促进MRSA感染的伤口愈合。为探索季铵化壳聚糖的促慢性创面愈合效果,利用季铵化壳聚糖(QCS)和3-羧基苯硼酸的酰胺化反应制备苯硼酸化壳聚糖季铵盐(QCS-PBA),并封装二氧化铈纳米颗粒和外泌体得到QCSP@Ceria@Exo水凝胶用于MRSA感染糖尿病创面修复。QCS-PBA分子链在水溶液中通过氢键、离子键、疏水相互作用和链缠结形成QCSP水凝胶,该水凝胶具有良好自凝胶行为、可注射性和自愈合性质,并表现出优异的抗菌性,可抑制MRSA生物膜生成和清除成熟的细菌生物膜,同时还具有ROS清除性能。糖尿病小鼠合并MRSA感染的伤口愈合实验表明,与临床上用于治疗糖尿病足的Tegaderm3M水凝胶相比,该多功能纳米递送水凝胶系统具有优异的细菌生物膜清除效果,可有效清除伤口细菌感染、降低伤口组织的氧化应激和伤口炎症反应,促进血管再生和胶原沉积,从而有效加快MRSA感染的糖尿病小鼠伤口愈合。QCSP@Ceria@Exo水凝胶作为一款高效、安全的促愈合水凝胶敷料,有望解决糖尿病患者伤口难愈合的难题。为探索季铵化甲壳素/壳聚糖的表面抗菌活性,利用聚多巴胺(PDA)表面修饰的方法,选择相同季铵化取代度和不同脱乙酰度的季铵化甲壳素(QC)和季铵化壳聚糖(QCS)对医用聚氨酯(TPU)进行表面改性,探索QC和QCS表面物理化学结构以及抗感染性能。研究发现,QC和QCS表面改性后,TPU表面亲水性增强。QC和QCS修饰的TPU均表现出良好的细胞相容性。抗菌活性最佳的TPU-QCS2对革兰氏阳性和阴性细菌表现出高效的接触杀菌性能和抗细菌粘附性,而且具有稳定的抗菌活性(>2个月)。小鼠皮下植入抗感染实验证实,TPU-QCS2对降低周围组织感染和炎症有明显效果,在预防临床导管相关感染方面具有潜在的应用前景。为探究季铵化甲壳素/壳聚糖的抗炎活性,利用季铵化甲壳素和季铵化壳聚糖与3-羧基苯硼酸(3-PBA)进行酰胺化反应,合成出苯硼酸改性季铵化甲壳素(QC-PBA)或苯硼酸改性季铵化壳聚糖(QCH-PBA),并负载红景天苷制备出QCP@Sal和QCHP@Sal水凝胶探究其抗炎活性。实验结果证明,QC-PBA和QCH-PBA通过氢键、离子键、疏水相互作用形成物理交联水凝胶,QCP和QCHP水凝胶具有良好的可注射自愈合性能。小鼠特应性皮炎治疗实验表明,季铵化甲壳素水凝胶比季铵化壳聚糖水凝胶表现出更优异的抗炎效果,而且包裹红景天苷的QC-PBA水凝胶组展现出最佳的抗炎效果。该系列水凝胶可以有效降低血液中白细胞、淋巴细胞和中心粒细胞的数量,通过抑制TNF-α和IL-6的表达来降低炎症反应,在特应性皮炎治疗领域有潜在应用前景。基于季铵化甲壳素良好的生物活性,利用季铵化甲壳素(QC)和纤维蛋白(FM)构建了一系列强度可调的人工细胞外基质仿生水凝胶(QC-FM),并研究该水凝胶性质和细胞行为与功能的关系。实验结果证明,QC-FM仿生水凝胶呈现三维微纤维网络结构,水凝胶的强度随着FM组分的增加而降低,应力松弛行为随着FM组分的增加而加快。通过对水凝胶性质和封装培养细胞行为的分析,内皮细胞(HUVECs)倾向于在凝胶范围强度为222-1380 Pa的QC-FM水凝胶中扩散和形成内皮细胞网络。细胞扩散归因于水凝胶特性的改变,包括纤维蛋白的细胞粘附配体密度,以及基质水凝胶的强度和应力松弛。这项工作可以获得强度可调控的QC-FM仿生水凝胶,以调节细胞行为,适应不同的要求和应用。最重要的是,优选QC2-1.0-FM 1-1可用于封装培养HUVECs,并在体内诱导血管化。此外,本工作中实现了悬浮凝胶中该水凝胶的体外3D打印,QC2-1.0-FM 1-1水凝胶可以快速制造和封装人类多功能干细胞分化的心肌细胞,随后培养形成功能性心脏组织,并且心肌的微观结构和电生理功能可以在体外的仿生水凝胶中重现,表明这项工作构建的QC-FM水凝胶在构建功能性心脏组织上具有广阔的应用前景。综上所述,本论文基于绿色高效的KOH/尿素水溶液体系均相合成了一系列不同脱乙酰度和阳离子取代度的季铵化甲壳素和季铵化壳聚糖,并分别构建出多功能纳米酶封装的季铵化壳聚糖水凝胶系统、季铵化甲壳素/壳聚糖抗菌表面、红景天苷封装的季铵化甲壳素/壳聚糖水凝胶递送系统和聚多糖-蛋白仿生水凝胶,阐明构效关系和生物医学功能,揭示它们在缓解抗生素耐药性、MRSA感染型创面和糖尿病小鼠MRSA感染型创面修复、炎症调节、以及构建心肌组织片等方面展现出重要应用价值。这些研究为构建季铵化甲壳素/壳聚糖基生物医用材料提供新的研究方法和思路,具有重要的研究价值和广阔应用前景。更多还原

【Abstract】 With the concept of "Healthy China" and "Sustainable Development Strategy.",people’s awareness of life and health have been enhanced.There is an urgent need to create innovative biomedical materials and increase the independent research and development capabilities of biomedical materials in China.Chitin,the second most prevalent polysaccharide in Nature,is a renewable resource with a broad variety of sources,and its optimal development and usage is of considerable significance.The deacetylation product of chitin is chitosan,and both chitin and chitosan have good biocompatibility,biodegradability,low immunogenicity,and chemical reactivity,making them attractive raw materials for the production of biomedical products.However,the presence of many intra-and inter-molecular hydrogen bonding interactions between chitin and chitosan molecular chains results in their chemical stability,which makes them difficult to dissolve in water and most organic solvents,and the lack of solvent systems that can achieve simultaneous dissolution of chitin and chitosan limits the modification and application of chitin and chitosan on the same homogeneous derivatization platform.In addition,chitin and chitosan are quite distinct in biological functions and activities,and the systematic investigation of the synthesis of chitin and chitosan derivatives and their structures and biomedical functions has very significant scientific relevance and practical potential.The main innovations in this work are as follows:(1)In aqueous KOH/urea solution,homogeneous synthesis of quaternized chitin and chitosan was achieved,revealing the effects of deacetylation and cation substitution on antibacterial selectivity and elucidating their antibacterial mechanisms and wound healing abilities.(2)Multifunctional quaternized chitosan-based hydrogels loaded with nanoenzymes and embryonic stem cell exosomes were created to demonstrate their self-gelling,self-healing,and self-adaptability,as well as to understand the mechanism by which hydrogels promote the healing of chronic MRSA wounds.(3)Quaternized chitin/chitosan was utilized to modify the surface of medical polyurethane in order to demonstrate its antibacterial activity and biocompatibility,as well as to elucidate its in vivo infection prevention capabilities and mechanism.(4)We synthesized quaternized chitin/chitosan modified with phenylboronic acid to create quaternized chitin/chitosan hydrogels wrapped with salidroside,elucidating the physical cross-linking mechanism and self-gel behavior,and discovered that quaternized chitin had a superior anti-inflammatory effect to quaternized chitosan.(5)We constructed quaternized chitosan-fibrin hydrogels that imitate extracellular matrix,established the structureproperties relationships between the hydrogels’ viscoelasticity and cellular behaviors and functions,and initially generated in vitro cardiac tissue slices.The following sections summarize the thesis’s main contents and conclusions.A series of quaternized chitin(QC)and quaternized chitosan derivatives(QCS)with different degrees of deacetylation(DD’)and degrees of quaternization(DQ)were homogeneously prepared in the KOH/urea system.The structure-activity relationship between chemical structure and biological activity was studied.The chemical structures were characterized by 1H NMR,13C NMR and FT-IR spectrum.It was found that the amphiphilic quaternized chitin/chitosan derivatives could self-assemble to form cationic micelles in aqueous solution,and the formation and size reduction of micelles increased the local positive charge density,thus enhancing the antibacterial activity.The effects of their deacetylation and cation substitution on antimicrobial properties,cytocompatibility and hemocompatibility were revealed,that was deacetylation and cation substitution were positively correlated with antimicrobial properties,cation substitution was negatively correlated with cytocompatibility,while deacetylation did not affect cytocompatibility when cation substitution was the same,and all materials showed excellent hemocompatibility.Among them,QCS-2 was selected as the optimal antimicrobial agent with DQ of 0.46 and DD’of 82%.QCS-2 showed excellent antimicrobial activity against both Gram-positive and Gram-negative bacteria and had good biocompatibility.Since QCS-2 had an antibacterial mechanism through physical disruption of bacterial cell membranes,it did not develop antibacterial resistance and exhibited effective inhibition of bacterial biofilm formation and eradication of mature bacterial biofilms.In addition,QCS-2 exhibited excellent antiinfective effects,effectively killing bacteria,reducing local inflammatory response,promoting angiogenesis and collagen deposition,thus effectively promoting MRS A infected wound healing.To explore the healing-promoting effect of quaternized chitosan on chronic wounds,phenylborated chitosan quaternary ammonium salt(QCS-PBA)was prepared by the amidation reaction of 3-carb oxy pheny lb oroni c acid and quaternized chitosan.QCSP@Ceria@Exo hydrogel was prepared by encapsulated cerium dioxide nanoparticles and exosomes in QCSP hydrogel for MRSA-infected diabetic wound repair.Physically cross-linked QCSP hydrogel was obtained by hydrogen bonding,ionic bonding,and hydrophobic interactions of QCS-PBA chains in aqueous solution.The self-gelling behavior,injectability and self-healing properties of the hydrogel were investigated by rheometry.It was found that the hydrogels exhibited excellent antibacterial properties,inhibiting MRSA biofilm,and eradicating mature bacterial biofilms;in addition,the hydrogels exhibited good ROS scavenging properties due to the formation of borate ester bonds between QCSP hydrogels and glucose,and the encapsulated cerium dioxide nanoparticles.Results of MRSA-infected wound healing in diabetic mice showed that this multifunctional nanodelivery hydrogel system has excellent bacterial biofilm eradication ability compared with Tegaderm3M hydrogel,a commercialized hydrogel dressing for the treatment of diabetic foot,which can effectively reduce bacterial infection,oxidative stress and wound inflammatory response in wound tissues,promote vascular regeneration and collagen deposition,and thus effectively accelerate MRSA-infected diabetic mice wound healing.As a result,QCSP@Ceria@Exo hydrogel is projected to enter the clinic as a commercial wound dressing in the future to address diabetes patients’ wound healing difficulties.To explore the surface antibacterial activity of quaternized chitin/chitosan,quaternized chitin(QC)and quaternized chitosan(QCS)with the same quaternized substitution degree and different deacetylation degrees were selected for surface modification of medical polyurethane(TPU)by the method of surface modification of polydopamine(PDA),and the surface physicochemical structures and antimicrobial activities of QC and QCS were investigated.The surface modification of QC and QCS resulted in enhanced hydrophilicity of TPU surface.Both QC and QCS modified TPUs exhibited good cytocompatibility.Both QC and QCS-modified TPU exhibited good cytocompatibility.TPU-QCS2 with the best antibacterial activity showed high contact bactericidal performance and anti-bacterial adhesion against Gram-positive and gram-negative bacteria,and had stable antibacterial activity(>2 months).In addition,anti-infection experiments in mice subcutaneously confired that TPU-QCS2 was significantly effective in reducing peripheral tissue infection and inflammation.All of the aforementioned findings indicate that quaternized chitin(QC)and quaternized chitosan(QCS)may have a role in the prevention of clinical catheter-related infections.To investigate the anti-inflammatory activity of quaternized chitin/chitosan,by amidation of 3-carboxyphenylboronic acid(3-PBA)with quaternized chitin(QC)or quaternized chitosan(QCH-PBA),a series of benzeneboronic acid modified quaternized chitin(QCPBA)or quaternized chitosan(QCH-PBA)was created(QCH).The hydrogels QCP@Sal and QCHP@Sal were produced by loading salidroside.Through hydrogen bonding,ionic bonding,and hydrophobic interactions,the QC-PBA and QCH-PBA chains may produce physically cross-linked hydrogels(QCP and QCHP hydrogels)in aqueous solution.The hydrogels composed of quaternized chitin(QCP)and quaternized chitosan(QCHP)displayed excellent injectability and self-healing characteristics.In a mouse model of atopic dermatitis,QCHP hydrogels shown better anti-inflammatory properties over QCP hydrogels,whereas the chitosan hydrogel group encapsulated with salidroside demonstrated the greatest anti-inflammatory properties.This set of hydrogels successfully decreased the amount of leukocytes,lymphocytes,and centrophils in the blood and inhibited the inflammatory response by decreasing TNF-and IL-6 production,indicating a possible use in the treatment of atopic dermatitis.Based on the excellent bioactivity of quaternized chitin,we constructed a series of artificial extracellular matrix bionic hydrogels(QC-FM)with adjustable strength using quaternized chitin(QC)and fibronectin(FM)for bionic simulation,and investigated the viscoelasticity of hydrogels as well as the structure-activity relationship between cell behavior and function.The findings indicated that the QC-FM hydrogel exhibited a threedimensional microfibre network structure,that the hydrogel’s strength reduced as the FM concentration increased,and that the stress relaxation behavior accelerated as the FM concentration increased.We discovered that human endothelial cells(HUVECs)prefer to disperse and vascularize in QC-FM hydrogels with a viscoelasticity of 222-1380 Pa.The hydrogel QC2-1.0-FM 1-1 was utilized to encapsulate and stimulate vascularization in HUVECs in vivo.In addition,we achieved in vitro 3D printing of this hydrogel in suspension gel,and QC2-1.0-FM 1-1 hydrogel can rapidly fabricate and encapsulate human multifunctional stem cell differentiated cardiomyocytes,which are subsequently cultured to form functional cardiac tissues,and the microstructure and electrophysiological functions of cardiac muscle can be reproduced in vitro in the bionic hydrogel,indicating that the QC-FM hydrogel constructed in this work is useful in building functional cardiac tissues,indicating that the QC-FM hydrogels constructed in this work have promising applications for the construction of functional cardiac tissues.In summary,this thesis developed a range of quaternized chitin and quaternized chitosan with varying degrees of deacetylation using the green and efficient KOH/urea homogeneous modification technique.Then,a multifunctional nanoenzyme-encapsulated quaternized chitosan hydrogel system was built,as well as antibacterial quaternized chi tin/chitosan surfaces,a salidroside-encapsulated quaternized chitin/chitosan hydrogel delivery system,and polysaccharide-protein bionic hydrogels.Further investigation of the structure-activity relationships and biological applications of these quaternized chitin/chitosan-based materials was performed.It was shown that they had critical biological functions in a variety of biomedical fields,including the prevention of antibiotic resistance,the healing of MRSAinfected wounds in diabetic mice,inflammatory control,and the building of cardiac tissue patches.These works provide novel research methodologies and concepts for the development of biomedical materials based on chitin/chitosan,which have significant research value and vast application potential.更多还原