【作者】 姜宇；

【导师】 李妍妍；

【作者基本信息】 东北林业大学 ， 药学， 2023， 硕士

【摘要】 天然化合物小檗碱是一种常用的抗菌药物,在治疗胃肠炎、细菌性痢疾等方面发挥着重要作用。近年来,大量的体内外实验研究结果表明,小檗碱也可以用于肿瘤治疗,对胃癌、乳腺癌和肝癌等有明显的治疗效果。然而,天然化合物自身水溶性低、靶向性差和生物利用度低等一系列问题严重阻碍了其发挥药效。寻找方法或途径提高小檗碱的生物利用度,将会有效提高这种低毒、价廉的天然化合物的利用价值,造福病患。一氧化氮（Nitric oxide,NO）是一种气体内源性信使,在哺乳动物组织和细胞中广泛存在。近年来的研究表明,高浓度的NO具有较好的抗菌抗肿瘤效果。NO主要依靠与活性氧（Reactive oxygen species,ROS）之间相互作用产生的活性氮氧化物（Reactive Nitrogen Oxides,RNOS）发挥抗菌抗肿瘤疗效。然而NO的半衰期极短,对氧气、超氧化物、金属酶和硫醇等存在的高化学反应性和有限的水溶性,导致NO难以靶向并大量积累到目标位置。利用NO供体构建NO控释系统是提高NO在目标位置浓度较为有效的方法。稀土掺杂的上转换纳米材料（Upconversion nanoparticles,UCNP）可以吸收低能光（近红外光）并将其转换为高能光（可见光或紫外光）,可以满足多种化学键断键的能量要求;介孔二氧化硅（Si O₂）具有高比表面积、可调节孔径以及优异的生物相容性等优势,近年来已逐步成为一种高效的纳米载体材料。基于以上背景,本论文利用上转换纳米材料通过表面包覆介孔硅材料装载NO供体构建了一种NO控释体系,将其与小檗碱共装载于热敏水凝胶中制备一种多功能水凝胶体系（UCMS+BBR-gel）,可实现NO控释和小檗碱缓控释效果,实现NO和小檗碱用于抗肿瘤和抗菌的联合治疗。样品形貌表征结果显示,所制备的包覆有二氧化硅壳层的上转换纳米材料（UCM）呈椭圆球形态,UCM表面的二氧化硅壳层为介孔硅结构,平均粒径约为90 nm;氮气吸附脱附实验结果表明样品总孔体积为642.95 m²·g^-1,平均孔径为12.8 nm;X射线能谱分析结果显示,样品表面主要由Y、Yb、Tm、O、F五种元素组成。对样品的性能检测结果显示,NO释放可利用近红外激光实现NO的控制释放,且NO的释放具有浓度相关性和时间累积性。通过优化筛选不同装载制备方式的水凝胶,选择几组装载方式中NO释放量最多的NO控释体系与小檗碱共装载于水凝胶中,制备成多功能水凝胶。药物释放实验结果表明,水凝胶在水中具有自主溶胀降解的能力,能够缓慢并完全地释放药物。体外抗肿瘤实验结果表明,4T1细胞与经过优化筛选的UCMS+BBR-gel共孵育作用下,细胞存活率不足20%,通过与NO共作用一定程度上提高了BBR的抗肿瘤性能。体外抗菌实验结果表明,UCMS+BBR-gel对金黄色葡萄球菌和大肠杆菌的抑制率达到了97%和80%,减少了细菌存活量。通过构建小鼠金黄色葡萄球菌感染模型,利用激光辐照UCMS+BBR-gel,提高NO的释放量联合小檗碱发挥抗菌作用,在生物相容性良好的情况下,安全有效的实现细菌杀伤效果,大幅提高了感染伤口的愈合速度;材料发挥抑菌作用后,水凝胶对创口覆盖起到保护和修复的作用,也加速了伤口愈合。综上所述,本论文利用小檗碱和基于上转换材料的NO控释系统建立了一种多功能水凝胶体系,可持续缓慢释放药物和按需释放一氧化氮,用于抗肿瘤和抗菌治疗。所合成的UCM用于负载NO供体SNO,获得的具有NO控释功能的纳米颗粒和天然药物BBR都被包裹在Pluronic F127/F68制备的热敏水凝胶体系中。UCNP在980 nm近红外光照射下,低能量的红外光转化为高能量的蓝紫光,导致S-NO键断裂释放NO,同时热敏水凝胶缓慢释放BBR。在980nm近红外辐照后,抗肿瘤实验中,水凝胶处理组可实现80%的4T1癌细胞活性抑制作用;抗菌实验中,水凝胶可实现约97%的金黄色葡萄球菌和近80%的大肠杆菌的活性抑制作用,小鼠皮肤细菌感染模型中可观察到较为明显的细菌活性抑制作用和较快的伤口愈合速率。本论文为提高NO的递送效率并利用NO联合BBR用于抗菌抗肿瘤提供了新方法,为NO联合BBR有效应用于抗菌抗肿瘤治疗提供了参考数据。更多还原

【Abstract】 At present,both bacteria and cancer are serious diseases that threaten human health.Berberine,a natural compound,is a commonly used antibacterial agent,which plays an important role in treating gastroenteritis and bacillary dysentery.In recent years,a large number of experimental results show that berberine can be used for tumor treatment,and berberine has obvious therapeutic effects on gastric cancer,breast cancer and liver cancer.However,kinds of problems such as poor water-solubility,poor targeting and low bioavailability of natural compounds have hindered their efficacy seriously.It can be seen that finding ways to improve the bioavailability of berberine with low-toxicity will effectively improve the utilization value.Nitric oxide（NO）is a gas endogenous messenger that is almost ubiquitous in mammalian cells.Recent studies have shown that high concentrations of NO have good antibacterial and good anti-tumor effects.NO mainly relies on active nitrogen oxides（RNOS）produced by the interaction with reactive oxygen species（ROS）to exert antibacterial and anti-tumor effects.However,NO has disadvantages such as the extremely short half life,limited water solubility and high chemical reactivity to oxygen,superoxide,metalloenzymes,and mercaptans,make it difficult to target to the lesion site and accumulate a large amount of NO to the target location.Using NO donors to construct a controlled release system is an effective method to increase the concentration of NO at the target location.Rare earth doped upconversion nanomaterials（UCNP）can absorb low energy light（near infrared light）and convert it into high energy light（visible or ultraviolet light）,which can break chemical bonds.Mesoporous silicon dioxide（Si O₂）has advantages such as high specific surface area,adjustable pore size,and excellent biocompatibility.In recent years,Si O₂has gradually become an efficient carrier.Based on the above background,this paper uses up conversion nanomaterials with surface modified Si O₂to load NO donor,and establish a NO controlled release system.The system was co-loaded with berberine in a thermosensitive hydrogel to achieve NO controlled release and berberine sustained release,and achieve the joint action of NO and berberine in anti-tumor and antibacterial treatment.The sample morphology characterization results show that UCM exhibits an elliptical spherical shape with pores,the average particle size is about 90 nm;The results of nitrogen adsorption and desorption experiments show that the total pore volume of the sample is 642.95 m²·g^-1,and the average pore diameter is 12.8 nm;The X-ray energy spectrum analysis results show that the sample is mainly composed of five elements:Y,Yb,Tm,O,and F.The NO release performance test experiment shows that the controlled release of NO can be achieved using near-infrared laser,and the release of NO has concentration dependence and time accumulation;The results of drug release experiment showed that hydrogel had the ability of self swelling and degradation in water,and could release drug slowly and completely.Finally,the results of anti-tumor experiments in vitro showed that the survival rate of 4T1 cells co incubated with UCMS+BBR-gel was less than 20%,which indicated that the anti-tumor performance of BBR was improved to some extent by co-incubating with NO.In vitro and in vivo antibacterial experiments showed that the inhibition rates against Staphylococcus aureus and Escherichia coli reached 97%and 80%,reducing bacterial survival;After the antibacterial effect of the material,the hydrogel further protects and repairs the wound coverage,and accelerates the wound healing.In conclusion,this paper established a multifunctional hydrogel system consist of berberine and up conversion materials based NO controlled release system,which can realize sustained drug release and controlled nitric oxide release for anti-tumor and antibacterial treatment.The synthesized UCM is used to load the NO donor SNO,and the obtained nanoparticles with NO controlled release function and natural drug BBR are wrapped in the thermal sensitive hydrogel system prepared by Pluronic F127/F68.Under 980 nm near-infrared light irradiation of UCNP,low-energy infrared light converted into high-energy blue violet light,leading to the breaking of S-NO bond to release NO,and the thermosensitive hydrogel slowly releases BBR.After 980nm near-infrared radiation,the hydrogel treatment group could achieve 80%inhibition of 4T1 cancer cell activity;It can inhibit the activity of about 97%of S.aureus and nearly 80%of E.coli,and accelerate wound healing.更多还原