【作者】 陈涛；

【导师】 汪琴；

【作者基本信息】 西南交通大学 ， 材料与化工（专业学位）， 2023， 硕士

【摘要】 类风湿性关节炎(Rheumatoid arthritis,RA)是一种以炎症浸润、骨侵蚀和软骨损伤为特征的全身性自身免疫疾病。参与RA炎症进展的细胞(如巨噬细胞、淋巴细胞、血管内皮细胞等)会发生明显的代谢重编程,它们往往对能量和生物合成前体有巨大的需求,以支持RA发展中细胞的快速激活、分化和增殖过程。这些炎症相关细胞的代谢转变的一个重要标志是葡萄糖的利用途径的转变。即使在有氧气存在的情况下,也可以观察到炎症细胞中糖酵解的急剧增加,这种现象被称为“Warburg”效应。虽然糖酵解不是一种高效的能量生产方式,但它能使细胞迅速获得足够的能量和物质,以促进其同化生长。有研究证实,糖酵解与最初的免疫激活和炎症反应的产生密切相关。因此,调节RA进展中升高的糖酵解水平有望实现炎症缓解。2-脱氧-D-葡萄糖(2-deoxy-D-glucose,2-DG)因其强大的糖酵解抑制能力,被广泛应用于各种异常代谢类疾病的治疗研究。2-DG与葡萄糖具有类似的结构,它可以竞争性地与葡萄糖争夺进入细胞的资格。然而2-DG进入细胞后不能像葡萄糖一样被正常代谢,因此它能抑制细胞糖酵解的过程,减少细胞能量的供应从而遏制RA病情的发展。当其应用于体内时,2-DG易被快速清除,并且它在体内的无差别分布阻碍了其进一步临床应用。因此,制备一种能够选择性地将2-DG输送到发炎关节的递送载体,有望特异性调节炎症代谢并最终实现慢性炎症的有效治疗。由于炎症部位血管通透性增加,纳米药物会渗漏出来并在病灶部位被炎症相关细胞摄取,最终停留在炎症关节处,该现象被称为“ELVIS”(Extravasation through leaky vasculature and subsequent inflammatory cell-mediated sequestration,ELVIS)效应。纳米载体虽然能提高药物在炎症部位的靶向分布能力,并具备提高药物的稳定性和生物利用度等优点,但是大多数纳米载体由于引入大量的载体材料存在着载药效率低、潜在的系统毒性等问题。相比于由纳米载体构成的药物制剂,由纯药物合成的无载体纳米药物因其生物安全性高、制备程序简单和载药率高等优点,已成为近几年科研人员探索的热点。论文设计了一种能自组装形成无载体纳米药物的制剂,通过糖酵解抑制剂2-DG和天然抗炎药物姜黄素(Curcumine,Cur)之间的分子间作用力,自组装形成了粒径为104.74nm的无载体纳米药物2-DCNP。2-DCNP会通过炎症部位的ELVIS效应进入到病灶部位,其中2-DG作为糖酵解抑制剂能通过调控炎症部位糖酵解水平,从而抑制炎症细胞的增生,降低炎症水平;Cur作为一种天然的抗炎药物,能抑制炎症因子如肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)、白细胞介素-6(Interleukin-6,IL-6)等的产生。两者间相辅相成,最终实现协同抗炎作用。体外实验表明,2-DCNP具有良好的细胞相容性,能够通过有效阻断该细胞的糖酵解过程从而缓解活化Raw264.7细胞产生的炎症反应。同时,相比于游离药物,2-DCNP具有更优异的细胞摄取能力,并且炎症状态下的细胞由于葡萄糖转运蛋白1(Glucose transporter 1,GLUT1)上调,会加剧其对2-DCNP的摄取吸收。通过建立大鼠佐剂性关节炎(Adjuvant-induced arthritis,AIA)模型,发现经静脉注射给药的2-DCNP,能下调组织中关键炎症因子白介素-17A(Interleukin-17A,IL-17A)和TNF-α的水平,并且改善患病大鼠关节组织处的软骨侵蚀和炎症浸润。经2-DCNP治疗的大鼠,其炎症关节组织显示下调的糖酵解水平。安全性评价结果表明,2-DCNP进入大鼠体内表现出良好的生物相容性,无明显毒性。综上所述,我们制备了一种无载体纳米药物2-DCNP,能通过靶向调控炎症关节组织的糖酵解水平实现关节炎大鼠的有效治疗。这些纯药物可能通过自组装方法的基本原理,开辟了另一条新的关节炎治疗途径,并为制造新的无载体纳米药物治疗RA提供了灵感。更多还原

【Abstract】 Rheumatoid arthritis(RA)is a systemic autoimmune disease characterized by inflammatory infiltration,bone erosion,and cartilage damage.Cells involved in the advancement of RA inflammation(such as macrophages,lymphocytes,vascular endothelial cells,etc.)undergo significant metabolic reprogramming.They often have a huge demand for energy and biosynthetic precursors to support the rapid activation,differentiation and proliferation of RA developing cells.An important sign of the metabolic transformation of these inflammation-related cells is the pathway of glucose utilization.Even in the presence of oxygen,a sharp increase in glycolysis in inflammatory cells can be observed.This phenomenon is called the “Warburg effect.” Although glycolysis is not an efficient way of energy production,it allows cells to quickly obtain enough energy and matter to promote their assimilation and growth.Studies have confirmed that glycolysis is closely related to the initial immune activation and the production of proinflammatory responses.Therefore,regulating the elevated glycolysis levels in the progress of RA is expected to achieve inflammation relief.2-deoxy-D-glucose(2-deoxy-D-glucose,2-DG)is widely used in the treatment and research of various abnormal metabolic diseases because of its strong glycolysis inhibition ability.2-DG has a similar structure to glucose,it can compete competitively with glucose for the right to enter the cell.However,2-DG cannot be metabolized normally like glucose after entering the cell.Therefore,it can inhibit the process of glycolysis of cells and reduce the supply of cellular energy to curb the development of RA.When applied in vivo,2-DG is easily cleared quickly,and its undifferentiated distribution in vivo hinders its further clinical application.Therefore,the preparation of a delivery vector capable of selectively delivering2-DG to inflamed joints is expected to specifically regulate inflammatory metabolism and ultimately achieve effective treatment of chronic inflammation.Due to the increased permeability of blood vessels at the site of inflammation,nano-drugs will leak out and be ingested by inflammation-related cells at the site of the lesion,and eventually stay at the inflammatory joint.This phenomenon is called “ELVIS”(Extravasation through leaky vasculature and subsequent inflammatory cell-mediated sequestration,ELVIS)effect.Although nanocarriers can improve the targeted distribution of inflammation and have the advantages of improving the stability and bioavailability of drugs,most nanocarriers have problems such as low drug carrying efficiency and potential systemic toxicity due to the introduction of a large number of carrier materials.Compared with pharmaceutical preparations composed of nanocarriers,carrier-free nano-drugs synthesized from pure drugs have become a hot topic for researchers to explore in recent years because of their high biosafety,simple preparation procedures and high drug carrying rate.In this paper,a preparation that can self-assemble to form a carrier-free nano-drug is designed.Through the intermolecular force between the glycolysis inhibitor 2-DG and the natural anti-inflammatory drug Curcumine(Cur),it self-assembles to form a carrier-free nanodrug 2-DCNP with a particle size of 104.74 nm.2-DCNP will enter the lesion site through the ELVIS effect of the inflammatory site.Among them,2-DG,as a glycolysis inhibitor,can regulate the glycolysis level of the inflammatory site,thereby inhibiting the proliferation of inflammatory cells and reducing the level of inflammation;Cur,as a natural anti-inflammatory drug,can inhibit the production of inflammatory factors such as Tumor necrosis factor-α(Tumor necrosis factor-α,TNF-α),Interleukin-6(Interleukin-6,IL-6),etc.).The two complement each other,and ultimately achieve a synergistic anti-inflammatory effect.In Vitro experiments have shown that 2-DCNP has good cytocompatibility and can effectively block the glycolysis process of the cell to alleviate the inflammatory response produced by activated Raw264.7 cells.At the same time,compared with free drugs,2-DCNP has a better cell uptake capacity,and cells in the inflammatory state due to the upregulation of Glucose transporter 1(GLUT1),it will promote the uptake and absorption of 2-DCNP by cells.By establishing a model of adjuvant-induced arthritis(AIA)in rats,it was found that 2-DCNP administered intravenously can downregulate the levels of key inflammatory factors Interleukin-17A(IL-17A)and TNF-α in tissues,and improve cartilage erosion and inflammatory infiltration in the joint tissues of diseased rats.Rats treated with 2-DCNP showed downregulated glycolysis levels in their inflammatory joint tissues.The safety evaluation results showed that 2-DCNP showed good biocompatibility and no obvious toxicity when entering rats.In summary,we have prepared a carrier-free nano-drug 2-DCNP,which can achieve effective treatment of arthritis rats by targeting and regulating the glycolysis level of inflammatory joint tissue.These pure drugs may open up another new way of arthritis treatment through the basic principles of the self-assembly method,and provide inspiration for the manufacture of new carrier-free nano-drugs to treat RA.更多还原