【作者】 徐晓玲；

【导师】 杜永忠；

【作者基本信息】 浙江大学 ， 药剂学， 2019， 博士

【摘要】 类风湿关节炎(Rheumatoid Arthritis,RA)是一种以多关节滑膜炎症为主要特征的系统性自身免疫疾病,常伴关节外器官受累及血清类风湿因子阳性,最终导致关节畸形及功能丧失,高发于30-50岁的女性,发病率呈逐年上升的趋势。目前临床上RA的治疗以药物治疗和手术治疗为主,但手术治疗易造成关节腔二次损伤,滑膜的切除使关节失去了合成滑液的基本功能,进一步加剧了关节功能的受损。甲氨蝶呤(Methotrexate,MTX)是临床一线的抗风湿药,具有免疫抑制、抗炎作用,能够减轻疼痛、缓解症状、控制病情发展。但因其在体内的非特异性分布,使得进入炎症关节部位的药物分子数量有限,而在正常组织中大量分布,服用高剂量甲氨蝶呤常产生溃疡性口腔炎、腹泻甚至致命的肠穿孔等毒副作用,严重影响了患者的生活质量,而低剂量的治疗又严重制约RA疗效,且长期小剂量使用仅能延缓骨质侵蚀的进展,而不能阻止长期炎症反应导致的骨质疏松的发生,更不能促进已受损骨质的修复与再生。新型纳米给药系统是改善药物体内分布,降低药物毒副作用的有效手段,且可通过载体材料的功能化实现RA的多靶点治疗。唾液酸(SialicAcid,SA)是一类位于细胞膜最外层的九碳糖,可与E选择素特异性结合,且唾液酸化的糖复合物可通过抑制破骨细胞成熟,促进成骨细胞分化,进而促进骨的修复和再生。本研究采用唾液酸、葡聚糖和硬脂酸为原料合成了唾液酸-葡聚糖-硬脂酸(SA-Dex-OA,SDO)嫁接物;SDO可在水性介质中自组装形成胶束,临界胶束浓度(Critical micelle concentration,CMC)为 55.06 μg/mL,胶束粒径为75.20 ± 11.91 nm,胶束能有效负载抗类风湿药物MTX,包封率为89.39 ±0.26%。唾液酸-葡聚糖-硬脂酸载药胶束(SA-Dex-OA/MTX,SDM)粒径为117.33±5.77 nm,载药胶束对药物有明显的缓释效应,药物可持续释放48 h以上。以人脐静脉内皮细胞(HUVEC)为模型细胞,对嫁接物的细胞毒性进行考察,结果显示其在1-1000 μg/mL浓度范围内对细胞的生长及增殖均无影响,细胞毒性较低。采用脂多糖刺激HUVEC构建E-选择素受体高表达的体外炎症细胞模型用于考察SDO嫁接物胶束的主动靶向能力,以葡聚糖-硬脂酸(Dex-OA,DO)嫁接物胶束为对照,结果表明炎症HUVEC对SDO嫁接物胶束具有更好的摄取行为;采用游离唾液酸阻断细胞表面E-选择素受体后,炎症细胞对SDO嫁接物胶束的摄取量降低;E-选择素荧光与SDO胶束荧光信号呈现共定位现象,证实SDO嫁接物胶束的炎症血管内皮细胞主动转运是由SA与E-选择素的特异性结合所介导的。以小鼠胚胎成骨细胞前体细胞(MC3T3-E1)为模型细胞,对嫁接物的骨修复与再生作用进行考察,结果显示SDO嫁接物胶束可通过唾液酸介导的促胞内碱性磷酸酶活力和胞外矿化结节形成,促进成骨细胞分化和矿化。采用胶原诱导关节炎法构建大鼠RA模型,尾静脉给药考察包载荧光探针的嫁接物胶束在模型动物体内的分布情况,结果显示SDO嫁接物胶束可通过唾液酸与炎症关节周围血管内皮细胞表面高表达E选择素的特异性结合,实现在炎症关节部位的大量蓄积。经尾静脉注射给药14天后,SDM组足爪肿胀程度显著减轻,关节炎评分指数显著下降;滑膜组织增生和胞内线粒体肿胀情况得到有效抑制,显著改善了细胞缺血缺氧状态;同时炎性细胞浸润程度明显减轻,TNF-a和IL-6的合成和释放能力显著降低,炎症反应得到有效抑制。另外,SDM载药胶束还可通过唾液酸介导的骨修复调控作用,上调OPG表达,下调RANKL表达,使骨代谢向骨形成倾斜,提高骨密度,发挥骨修复与再生作用;MTX给药14天后,血清中谷草转氨酶、谷丙转氨酶、尿素氮和血肌酐的浓度显著升高,对肝肾均有一定毒性,而SDM组生化指标与正常组接近,显示SDM载药胶束的安全性。纳米羟基磷灰石(Hydroxyapatite,HAP)作为骨骼的天然组分之一,具有生物粘附性强、可吸附药量大、酸敏感、骨诱导性能良好等特性,是优良的人体骨仿生再生材料。本研究在前期研究的基础上,以具有修复软骨作用的壳寡糖为胶束主要材料,通过酰胺反应合成唾液酸-壳寡糖-脱氧胆酸嫁接物胶束(SA-CSO-DA,SD);SD可在水性介质中自组装形成胶束,CMC为38.45 μg/mL;以具有较好表面活性剂特性的该嫁接物胶束作为稳定剂,通过水热法制得棒状的唾液酸修饰壳寡糖/羟基磷灰石(SH)纳米粒,大小均一,粒径约为30.03±6.54nm;该纳米粒可有效吸附MTX,制得的载药纳米粒(SHM)外观仍呈棒状,分布均一,粒径大小增加至46.08 ± 7.88 nm,包封率为68.67±2.80%。体外释放结果显示,SHM呈现明显的酸敏感释放特性,在pH 7.4中性介质中释放缓慢,而在pH 5.5酸性条件下释放加快,48h内的累积释放百分率可达87.95%左右。以原代大鼠软骨细胞为模型细胞,考察SH纳米粒的促软骨修复作用,结果表明,SH纳米粒具有较低的细胞毒性;IL-1β刺激的原代大鼠软骨细胞经SH处理后,凋亡细胞的比例显著降低,糖胺多糖含量显著增加,Ⅱ型胶原表达明显上调,而HAP预处理基本不影响凋亡细胞比例、糖胺多糖含量和Ⅱ型胶原的表达。以原代成骨细胞为模型细胞,考察SH纳米粒的促成骨修复与再生作用,结果显示,与SH共孵育后,胞内碱性磷酸酶活力明显增强,矿化结节数目显著增加,Ⅰ型胶原和OPG表达明显上调,显著促进了成骨细胞的分化及矿化进程。采用胶原诱导关节炎法建立RA模型,SH纳米粒经静脉给药后,其可有效分布至靶部位,实现在炎症关节部位的大量蓄积;经尾静脉注射给药28天后,SHM组足爪肿胀程度显著减轻,关节炎评分指数显著下降;脾脏指数和胸腺指数有效降低,免疫功能亢进情况得到显著改善,同时滑膜组织中炎性细胞合成和释放TNF-α和IL-6的量显著降低,炎症反应得到有效抑制;还可通过补充软骨组织中糖胺多糖含量、上调Ⅱ型胶原表达及抑制基质金属蛋白酶的活性,发挥促软骨修复作用;SHM纳米给药系统可通过上调骨形成阶段关键蛋白OPG、下调破骨细胞激活因子RANKL,使新骨生成速度显著加快,显著增加骨质及骨量,实现骨修复与再生作用;MTX给药28天后,血清中尿素氮的浓度和谷草转氨酶/谷丙转氨酶比值显著升高,对肝肾均有一定毒性,而SHM组生化指标与正常组接近,肝脏和肾脏无明显病变区域,显示SHM纳米给药系统安全性较高。本研究构建的功能性甲氨蝶呤纳米给药系统均能通过药物向炎症关节部位的靶向递送,实现类风湿关节炎炎症反应的有效抑制,同时,残留的壳寡糖可促进软骨修复;纳米羟基磷灰石协同唾液酸,发挥成骨修复与再生功能,最终实现类风湿性关节炎的安全高效治疗,为RA治疗方法提供新策略和新思路。更多还原

【Abstract】 Rheumatoid arthritis(RA)is a systemic autoimmune disease characterized by multi-joint synovial inflammation.It is often accompanied by involvement of extraarticular organs and positive serum rheumatoid factor,which eventually leads to joint deformity and functional loss.The incidence of RA is increasing year by year in women aged 30-50.At present,the treatment of RA is mainly based on drug therapy and surgical treatment,but surgical treatment is liable to cause secondary injury of joint cavity.The removal of synovium makes the joint lose the basic function of synthesizing synovial fluid,which further aggravates the damage of joint function.Methotrexate is a clinical first-line anti-rheumatic drug with immunosuppressive and anti-inflammatory effects.It can relieve pain,relieve symptoms and control the development of the disease.However,due to its non-specific distribution in the body,the number of drug molecules entering the inflammatory joints is limited.High-dose methotrexate often produces side effects such as ulcerative stomatitis,diarrhea and even fatal intestinal perforation,which seriously affects the life quality of patients,while low-dose methotrexate often restricts the efficacy of treating RA.And long-term application of low-dose methotrexate can only delay the progress of bone erosion rather than prevent the occurrence of osteoporosis caused by long-term inflammatory response,or promote the repair and regeneration of damaged bone.Adopting new nano-drug delivery system is an effective means to improve drug distribution in vivo and reduce side effects.It can realize multi-target therapy of RA by functionalization of carrier materials.Sialic acid(S A)is a kind of nine-carbohydrate located in the outermost layer of cell membrane,which can specifically bind to E-selectin.Sialic acid glycoconjugates can inhibit osteoclast maturation,promote osteoblast differentiation,and then improve bone repair and regeneration.In this study,sialic acid-dextran-octadecanoic acid(SA-Dex-OA,SDO)were synthesized.SDO can self-assemble into micelles in aqueous media with a critical micelle concentration of 55.06 ug/mL and a particle size of 75.20 ± 11.91nm.The micelles can effectively encapsulate anti-rheumatoid drug MTX.The encapsulation efficiency was 89.39 ± 0.26%.The particle size of drag-loaded micelles(SDM)was 117.33 ± 5.77 nm.SDM showed a significant sustained release effect on the drug,and the drug release lasted for more than 48 hours.Human umbilical vein endothelial cells(HUVEC)were used as model cells to investigate the cytotoxicity.The results showed that the grafts had no effect on cell proliferation in the concentration range of 1 and 1000μg/mL.An in vitro inflammatory cell model with high expression of E-selectin receptor stimulated by lipopolysaccharide was constructed to investigate the active targeting ability of SDO.Dex-OA(DO)were used as control.The results showed that inflammatory HUVEC had better uptake behavior on SDO.After blocking the E-selectin receptor on the cell surface,the uptake of SDO by inflammatory cells decreased.The co-localization of E-selectin and SDO micelle confirmed that the active transport of SDO to inflammatory vascular endothelial cells was mediated by the specific binding of SA and E-selectin.Using mouse embryonic osteoblast precursor cells(MC3T3-E1)as model cells,the bone repair and regeneration effect of micelles were investigated.The results showed that SDO could promote the differentiation and mineralization of osteoblasts by promoting the activity of intracellular alkaline phosphatase and the formation of extracellular mineralized nodules.The targeting efficiency of SDO in vivo was evaluated in a rat model of collagen induced arthritis(CIA)The results showed that more SDO/ICG micelles were accumulated in the arthritic paws by the specific combination of sialic acid and E-selectin overexpressed on the surface of vascular endothelial cells around inflammatory joints.After treatment for 14 days,the paw thickness and arthritis score index of SDM group were significantly reduced.Synovial tissue proliferation and intracellular mitochondrial swelling were effectively inhibited.And cell ischemia and hypoxia were significantly improved.At the same time,inflammatory cell infiltration was significantly reduced,and secretion of TNF-alpha and IL-6 decreased.In addition,SDM also up-regulate expression of OPG,down-regulate expression of RANKL,increasing bone mineral density,so as to regulate bone repair effect mediated by sialic acid.After treatment for 14 days,the serum concentrations of alanine transaminase(ALT),aspartate aminotransferase(AST),blood urea nitrogen(BUN)and serum creatininealanine aminotransferase(Scr)were significantly increased.However,the indexes of SDM group were similar to those of normal group,indicating the safety of SDM.Hydroxyapatite(HAP),as one of the natural components of bone,has the characteristics of strong biological adhesion,excellent capacity for large amount of drugs adsorbtion,acid sensitivity,bone induction and so on.It is an excellent bionic regeneration material for human bone.On the basis of previous studies,sialic acid-chitooligosaccharide-deoxycholic acid(SA-CSO-DA,SD)were synthesized.SD could self-assemble into micelles in aqueous media,CMC was 38.45μg/mL.The sialic acid modified chitosan oligosaccharide/hydroxyapatite(SH)nanoparticles were prepared by hydrothermal method using micelles as the stabilizer.The size of the rod-like SH nanoparticles was uniform,with the average size of 30.03±6.54 nm.The nanoparticles could effectively adsorb MTX,and the drug-loaded nanoparticles(SHM)were rod-like in appearance,uniform in distribution.And the size of SHM was 46.08±7.88 nm,the encapsulation efficiency was 68.67 ± 2.80%.The results of in vitro release showed that SHM exhibited obvious acid-sensitive release characteristics.The release was slow in neutral medium at pH 7.4,but accelerated in acidic medium at pH 5.5.The cumulative release was about 87.95%within 48 hours.Using primary rat chondrocytes as model cells,the effect of SH nanoparticles on cartilage repair was investigated.The results showed that SH nanoparticles had low cytotoxicity.After SH treatment of chondrocytes stimulated by IL-1β,the proportion of apoptotic cells decreased significantly,the content of glycosaminoglycan increased significantly,and the expression of type II collagen increased.However,HAP pretreatment did not affect the percentage of apoptotic cells,the content of glycosaminoglycan and the expression of type II collagen.Using primary osteoblasts as model cells,the effect of SH nanoparticles on bone repair and regeneration was investigated.The results showed that after co-incubation with SH,the activity of alkaline phosphatase in cells increased significantly,the number of mineralized nodules increased,the expression of type Ⅰcollagen and OPG enhanced,which promoted the differentiation and mineralization process.RA model was established by collagen-induced arthritis.After intravenous administration,SH nanoparticles could effectively distribute to the target site and accumulate in inflammatory joints.After treatment of SHM nanoparticles for 28 days,the paw thickness,the arthritis score index,the spleen index,thymus index,inflammatory cytokines(TNF-alpha and IL-6)were significantly reduced.It could also exert cartilage repair effect by supplementing the content of glycosaminoglycan,increasing the expression of type Ⅱ collagen and inhibiting the activity of matrix metalloproteinase.SHM can significantly accelerate the formation of new bone by up-regulating expression of OPG,down-regulating expression of RANKL.After treatment of MTX for 28 days,the ratio of ALT and AST and the concentration of BUN increased significantly.However,the biochemical indexes of SHM were close to those of normal group,and there were no obvious lesion areas in liver and kidney,indicating the safety of SDM.In conclusion,the two functional drug delivery systems designed in the study could effectively inhibit the inflammatory response through the targeted delivery of MTX to the arthritic paws of CIA rats.Simultaneously,the residual chitosan oligosaccharides could promote cartilage repair and the residual nano-hydroxyapatite cooperating with sialic acid could exert bone repair and regeneration effect,thus safe and effective therapy of RA was achieved through the synergistic therapy between drug and the delivery systems.Our findings might provide new insights and strategies for RA treatment.更多还原