新型纳米DNA转运体系的构建及其在软骨损伤治疗等方面的应用研究
摘要
目的通过对高分子量壳聚糖进行控制性降解,使其成为低分子量壳聚糖,构建一种新型的纳米基因传送系统,能够在体内外安全高效地进行基因传递,表征其理化性质,为关节软骨损伤提供简便有效的治疗措施。研究纳米局部转运系统(Nanometer Local Delivery System, NLDS)在体内外的转运机制,评估局部的基因表达量及基因转染的持续性、安全性、有效性、可控性。检测其生物学特性。分离、培养、冻存、复苏和传代兔关节软骨细胞。评估该系统体内外转染软骨细胞的效果,以及对软骨缺损的动物模型的转染效果。探索体内外应用纳米局部转运系统传递生长因子基因来促进软骨损伤修复的条件和方法,并根据体外的实验数据进行必要的条件优化,进而利用其携带生长因子基因对软骨缺损的动物模型进行转染,促进软骨修复。同时,将其与抗TNF的反义核酸技术结合,对其在假体无菌性松动和类风湿关节炎的应用进行探索性研究。
方法采用NaNO2氧化法降解,将HMWC降解为合适分子量的LMWC,以FITC标记壳聚糖后,用凝胶渗透层析(GPC)的方法测定壳聚糖的分子量。以Xylidine Tonceau 2R (C.I. Acid Red 26)为指示剂的比色滴定法测定氨基百分含量。以增强型绿色荧光蛋白(EGFP)质粒、β-半乳糖酐酶(β-Galactosidase)、TGF-β1质粒等作为目的基因,通过分子自组装的方法制备载基因纳米壳聚糖微粒。以透射电镜(TEM)和原子力显微镜(AFM)观察微粒的形态,以激光粒径分析仪测定其粒径和表面的Zeta电位,行DNaseⅠ酶切保护实验研究壳聚糖对质粒DNA的保护作用,行MTT试验研究纳米微粒对细胞的毒性作用。对体外培养的关节软骨细胞进行转染,评估该系统体外转染软骨细胞的效果;切取软骨组织薄片体外行组织培养,评价该系统对软骨组织体外转染的效果;在兔膝关节上复制关节软骨缺损的模型,用该系统携带生长因子基因对软骨缺损的动物模型进行转染,观察其促进软骨修复的效果,同时观察该目的基因表达的时空及丰度特征等指标。在体内转染的同时,对其安全性进行评估。另外,采用ICR小鼠建立磨损微粒诱导的骨溶解的动物模型,采用该系统给药,观察其在假体无菌性松动应用方面的效果。采用DBA1小鼠建立II型胶原诱导的类风湿关节炎模型,同样采用该系统给药,观察其抗类风湿方面的效果。
结果随着与NaNO2反应时间增加,从GPC测定中得到的壳聚糖的分子量从145kD降到了18kD。相应地,通过比色滴定得到的各种壳聚糖的-NH2百分含量也从71%降低到了51%。壳聚糖可以和GFP质粒相互作用形成带有表面正电荷的纳米微粒,TEM和AFM观察到的纳米微粒的形状是近似球形的,大小较均一,表面光滑,粒径在70nm左右。该系统能在很大程度上保护质粒DNA不受DNaseⅠ的降解,对细胞的毒性小。体外对培养的软骨细胞的定性和定量转染均显示该系统有良好的转染特性;对体外培养的软骨组织转染结果与细胞转染结果相近;在体内时应用,LMWC/ TGF-β1基因纳米复合物可以提高目的基因的转移效率和延长相应蛋白的表达时程,目的基因表达的时程和分布特征良好,在软骨缺损动物模型的应用中取得了良好的治疗效果。该系统携带靶向TNF-α的ASO进行基因传递用于干预微粒诱导的骨溶解模型时,可抑制小鼠颅骨局部植入Co-Cr-Mo合金后TNF-α的mRNA的转录上调,与此同时TNF-α的蛋白定量测定也相应的下降,对应的破骨细胞数量,TRAP定量结果,以及骨吸收陷窝的数量,均有所下降。而且这种改变在再次给予了TNF-α后可以逆转。该系统携带靶向TNF-α的ASO进行基因传递用于干预II型胶原诱导的类风湿关节炎模型时,可以抑制造模小鼠的体重下降,明显缓解小鼠后足垫和前足腕部的肿胀,X线结果和功能学观察也表明该系统对类风湿关节炎具有明显的抑制作用。
结论高分子量(HMWC)降解而成的低分子量壳聚糖(LMWC)具有很好的溶解性,对细胞基本无毒,在生理环境下可以有效结合质粒DNA和反义核酸药物,形成稳定存在的纳米复合物颗粒。基于LMWC的纳米核酸药物传输系统对软骨细胞在体内外均有良好的转染效率。在体外能够保护目的DNA并延长其作用时间,在体内则对软骨缺损区域表现出一定的被动靶向性,更易于在缺损附近进行转染。使用该系统携带TGF-β1质粒应用于兔关节软骨缺损模型,能够有效地表达治疗因子,促进软骨修复。使用该系统携带抗TNF-α的反义核酸(ASO),应用于假体无菌性松动的动物模型中,可以有效地抑制小鼠模型中由微粒诱导的骨溶解。使用该系统携带抗TNF-α的反义核酸(ASO),应用于鸡胶原诱导的类风湿关节炎(RA)模型,可以有效地缓解RA的病情,抑制病情的发展。载基因低分子量壳聚糖纳米微粒能够有效地输送目的DNA,是一个很有潜力的纳米局部传输系统(Nanometer Local Delivery System, NLDS)。
Objective To study the preparation of low molecular weight chitosan (LMWC) by controllable degradation of high molecular weight chitosan (HMWC). To construct a novel nanometer DNA transferring system to deliver gene in vitro and in vivo safely and efficiently with DNA-loaded low molecular weight chitosan nanoparticles. Physico-chemical property and biological characteristics were observed for the further application in the articular cartilage injury. To investigate the transferring mechanism of this nanometer local delivery system (NLDS). To evaluate the quantity and location of target gene expression with the premise of safe, controllable, persistent and validity transfection. And to study the separation, cultivation, freezing and preservation, resuscitation and passage of chondrocytes from rabbit’s cartilage articular. To evaluate the activities of transfection to chondrocytes in vitro and in vivo. To explore the necessary conditions and methods of developing this system into a clinically applicable approach to enhance natural repair mechanisms by in vivo transfection using a non-viral gene delivery system targeting at chondrocytes. And according to the experiment results, some modification should be made. At the same time, to investigated the effect of this system in other fields of joint surgery by combination of ASO: prosthesis failure and rheumatoid arthritis.
Methods High molecular weight chitosan was chemically treated with NaNO2 in 0.1mol/L acetic acid at 25°C for 10h to produce low molecular weight chitosan. After marking LMWC with FITC, the average molecular weight of HMWC and LMWC were determined by HPLC. The average amino group content of chitosan was determined by metachromatic titration using Xylidine Tonceau 2R (C.I. Acid Red 26). The plasmids of EGFP,β-Galactosidase and TGF-β1 were used as target gene to generate DNA-loaded low molecular weight chitosan nanoparticles by molecular self-assembly. Transmission electron microscope (TEM) and atomic force microscope (AFM) were employed to observe these particles and a laser particle analyzer were used to survey the particle sizes and Zeta potentials. And DNase I assay was performed to test the gene protential of the nanopaticles. The cytotoxity of the nanopaticles was examined by the MTT assay. The transfection activities to the chondrocytes of the nanopaticles were valued by in vitro and in vivo gene transfection test. Then these nano-complexes then were employed to animal models with full-thick cartilage defect to demonstrate the feasibility of delivering the growth factor gene in vivo, with the aim to promoting cartilage healing by intra-articular injections. At the same time the quality and location of target gene expression also been observed, and their fluctuation changed with time as well. Before a in vivo transfection was performed, a safety assessment was done. Besides, the murine calvaria osteolysis model in ICR mice and type II collagen induced RA model in DBA1 mice also were employed to evaluate this system in wide rang.
Results LMWC had far lower viscosity than HMWC, and could dissolve in the 0.1mol/L acetic acid quite easily. After being treated with NaNO2, the average molecular weight of HMWC decreased from 145 KDa to 18 KDa. Although the average amino group content of chitosan also decreased from 71% to 51% as one of the results of oxidation reaction. From the ultrastructure observation, we can find that LMWC can form stable complexes with DNA effectively, and the LMWC/ DNA complexes are found to be near spherical of 70 nm in diameter with homogeneous structure. Preliminary studies were also performed to identify the experimental conditions for the formation of LMWC/DNA complexes. Results show that LMWC can condense plasmid DNA effectively. When the N/P ratio of LMWC/DNA complex increased from 1 to 6, the zeta potential increased from -7 mV to 13 mV, and the effective diameter of complexes decreased from 340nm to 250nm. At the condition of N/P ratio of 5 , the zeta potential was 7.35mV±0.22mV and the average effective diameter of complexes was 258nm±2.5nm. Nanopaticles with positive charges could be formed by the interaction of chitosan and plasmid, that can protect the DNA from degradation by DNase I in certain degree. There is almost no toxicity to chondrocytes. Gene could be effectively expressed in the chondrocytes, when the latter were transfected by the gene-loaded low molecular weight chitosan nanoparticles. When used in vivo, LMWC/ TGF-β1 gene nano-complexes could enhance the transfection efficiency and prolong the expression of TGF-β1 gene. In the animal models of articular osteochondral defect of rabbits, much better healing and much gentler degeneration could be observed comparing with the controls. And when carrying ASO, this system also can suppress particle-induced osteolysis after the implantation of Co-Cr-Mo alloy particles. The expression of TNF-αmRNA and protein descented dramatically, and at the same time, the quantity of osteoclast, the quality of TRAP, and the area of resorption pit decented too. And this suppression can reestablish by the addition of TNF-α. When applied in the treatment of rheumatoid arthritis, this system with anti-TNF-αASO could suppress the body weight loosing in the type II collagen induced rheumatoid arthritis model mice. And arthrocele could be also suppressed by this system. X ray and functional test verified this suppression.
Conclusion LMWC from HMWC has very good dissolubility and very low toxicity, and can carry DNA to form a nanometer local delivery system. Gene-Loaded Low molecular weight chitosan nanoparticles can delivery DNA effectively as a vectors to chondrocytes in vitro and in vivo. During this course, some superiority such as DNA protection, delayed release and passive targeting were observed. When carrying TGF-β1 plasmids, this system can delivery the therapeutic factor gene efficiently, and harvest very good result in rabbit’s articular cartilage defect model. When carrying anti- TNF-αASO, this system also can delivery oligonucleotide to cells, suppress the TNF-αdramatically, and provide a new treatment strategy for particle induced osteolysis and rheumatoid arthritis. Gene-Loaded Low molecular weight chitosan nanoparticles is a novel nanometer DNA transferring system and has a very nice prospect.
方法采用NaNO2氧化法降解,将HMWC降解为合适分子量的LMWC,以FITC标记壳聚糖后,用凝胶渗透层析(GPC)的方法测定壳聚糖的分子量。以Xylidine Tonceau 2R (C.I. Acid Red 26)为指示剂的比色滴定法测定氨基百分含量。以增强型绿色荧光蛋白(EGFP)质粒、β-半乳糖酐酶(β-Galactosidase)、TGF-β1质粒等作为目的基因,通过分子自组装的方法制备载基因纳米壳聚糖微粒。以透射电镜(TEM)和原子力显微镜(AFM)观察微粒的形态,以激光粒径分析仪测定其粒径和表面的Zeta电位,行DNaseⅠ酶切保护实验研究壳聚糖对质粒DNA的保护作用,行MTT试验研究纳米微粒对细胞的毒性作用。对体外培养的关节软骨细胞进行转染,评估该系统体外转染软骨细胞的效果;切取软骨组织薄片体外行组织培养,评价该系统对软骨组织体外转染的效果;在兔膝关节上复制关节软骨缺损的模型,用该系统携带生长因子基因对软骨缺损的动物模型进行转染,观察其促进软骨修复的效果,同时观察该目的基因表达的时空及丰度特征等指标。在体内转染的同时,对其安全性进行评估。另外,采用ICR小鼠建立磨损微粒诱导的骨溶解的动物模型,采用该系统给药,观察其在假体无菌性松动应用方面的效果。采用DBA1小鼠建立II型胶原诱导的类风湿关节炎模型,同样采用该系统给药,观察其抗类风湿方面的效果。
结果随着与NaNO2反应时间增加,从GPC测定中得到的壳聚糖的分子量从145kD降到了18kD。相应地,通过比色滴定得到的各种壳聚糖的-NH2百分含量也从71%降低到了51%。壳聚糖可以和GFP质粒相互作用形成带有表面正电荷的纳米微粒,TEM和AFM观察到的纳米微粒的形状是近似球形的,大小较均一,表面光滑,粒径在70nm左右。该系统能在很大程度上保护质粒DNA不受DNaseⅠ的降解,对细胞的毒性小。体外对培养的软骨细胞的定性和定量转染均显示该系统有良好的转染特性;对体外培养的软骨组织转染结果与细胞转染结果相近;在体内时应用,LMWC/ TGF-β1基因纳米复合物可以提高目的基因的转移效率和延长相应蛋白的表达时程,目的基因表达的时程和分布特征良好,在软骨缺损动物模型的应用中取得了良好的治疗效果。该系统携带靶向TNF-α的ASO进行基因传递用于干预微粒诱导的骨溶解模型时,可抑制小鼠颅骨局部植入Co-Cr-Mo合金后TNF-α的mRNA的转录上调,与此同时TNF-α的蛋白定量测定也相应的下降,对应的破骨细胞数量,TRAP定量结果,以及骨吸收陷窝的数量,均有所下降。而且这种改变在再次给予了TNF-α后可以逆转。该系统携带靶向TNF-α的ASO进行基因传递用于干预II型胶原诱导的类风湿关节炎模型时,可以抑制造模小鼠的体重下降,明显缓解小鼠后足垫和前足腕部的肿胀,X线结果和功能学观察也表明该系统对类风湿关节炎具有明显的抑制作用。
结论高分子量(HMWC)降解而成的低分子量壳聚糖(LMWC)具有很好的溶解性,对细胞基本无毒,在生理环境下可以有效结合质粒DNA和反义核酸药物,形成稳定存在的纳米复合物颗粒。基于LMWC的纳米核酸药物传输系统对软骨细胞在体内外均有良好的转染效率。在体外能够保护目的DNA并延长其作用时间,在体内则对软骨缺损区域表现出一定的被动靶向性,更易于在缺损附近进行转染。使用该系统携带TGF-β1质粒应用于兔关节软骨缺损模型,能够有效地表达治疗因子,促进软骨修复。使用该系统携带抗TNF-α的反义核酸(ASO),应用于假体无菌性松动的动物模型中,可以有效地抑制小鼠模型中由微粒诱导的骨溶解。使用该系统携带抗TNF-α的反义核酸(ASO),应用于鸡胶原诱导的类风湿关节炎(RA)模型,可以有效地缓解RA的病情,抑制病情的发展。载基因低分子量壳聚糖纳米微粒能够有效地输送目的DNA,是一个很有潜力的纳米局部传输系统(Nanometer Local Delivery System, NLDS)。
Objective To study the preparation of low molecular weight chitosan (LMWC) by controllable degradation of high molecular weight chitosan (HMWC). To construct a novel nanometer DNA transferring system to deliver gene in vitro and in vivo safely and efficiently with DNA-loaded low molecular weight chitosan nanoparticles. Physico-chemical property and biological characteristics were observed for the further application in the articular cartilage injury. To investigate the transferring mechanism of this nanometer local delivery system (NLDS). To evaluate the quantity and location of target gene expression with the premise of safe, controllable, persistent and validity transfection. And to study the separation, cultivation, freezing and preservation, resuscitation and passage of chondrocytes from rabbit’s cartilage articular. To evaluate the activities of transfection to chondrocytes in vitro and in vivo. To explore the necessary conditions and methods of developing this system into a clinically applicable approach to enhance natural repair mechanisms by in vivo transfection using a non-viral gene delivery system targeting at chondrocytes. And according to the experiment results, some modification should be made. At the same time, to investigated the effect of this system in other fields of joint surgery by combination of ASO: prosthesis failure and rheumatoid arthritis.
Methods High molecular weight chitosan was chemically treated with NaNO2 in 0.1mol/L acetic acid at 25°C for 10h to produce low molecular weight chitosan. After marking LMWC with FITC, the average molecular weight of HMWC and LMWC were determined by HPLC. The average amino group content of chitosan was determined by metachromatic titration using Xylidine Tonceau 2R (C.I. Acid Red 26). The plasmids of EGFP,β-Galactosidase and TGF-β1 were used as target gene to generate DNA-loaded low molecular weight chitosan nanoparticles by molecular self-assembly. Transmission electron microscope (TEM) and atomic force microscope (AFM) were employed to observe these particles and a laser particle analyzer were used to survey the particle sizes and Zeta potentials. And DNase I assay was performed to test the gene protential of the nanopaticles. The cytotoxity of the nanopaticles was examined by the MTT assay. The transfection activities to the chondrocytes of the nanopaticles were valued by in vitro and in vivo gene transfection test. Then these nano-complexes then were employed to animal models with full-thick cartilage defect to demonstrate the feasibility of delivering the growth factor gene in vivo, with the aim to promoting cartilage healing by intra-articular injections. At the same time the quality and location of target gene expression also been observed, and their fluctuation changed with time as well. Before a in vivo transfection was performed, a safety assessment was done. Besides, the murine calvaria osteolysis model in ICR mice and type II collagen induced RA model in DBA1 mice also were employed to evaluate this system in wide rang.
Results LMWC had far lower viscosity than HMWC, and could dissolve in the 0.1mol/L acetic acid quite easily. After being treated with NaNO2, the average molecular weight of HMWC decreased from 145 KDa to 18 KDa. Although the average amino group content of chitosan also decreased from 71% to 51% as one of the results of oxidation reaction. From the ultrastructure observation, we can find that LMWC can form stable complexes with DNA effectively, and the LMWC/ DNA complexes are found to be near spherical of 70 nm in diameter with homogeneous structure. Preliminary studies were also performed to identify the experimental conditions for the formation of LMWC/DNA complexes. Results show that LMWC can condense plasmid DNA effectively. When the N/P ratio of LMWC/DNA complex increased from 1 to 6, the zeta potential increased from -7 mV to 13 mV, and the effective diameter of complexes decreased from 340nm to 250nm. At the condition of N/P ratio of 5 , the zeta potential was 7.35mV±0.22mV and the average effective diameter of complexes was 258nm±2.5nm. Nanopaticles with positive charges could be formed by the interaction of chitosan and plasmid, that can protect the DNA from degradation by DNase I in certain degree. There is almost no toxicity to chondrocytes. Gene could be effectively expressed in the chondrocytes, when the latter were transfected by the gene-loaded low molecular weight chitosan nanoparticles. When used in vivo, LMWC/ TGF-β1 gene nano-complexes could enhance the transfection efficiency and prolong the expression of TGF-β1 gene. In the animal models of articular osteochondral defect of rabbits, much better healing and much gentler degeneration could be observed comparing with the controls. And when carrying ASO, this system also can suppress particle-induced osteolysis after the implantation of Co-Cr-Mo alloy particles. The expression of TNF-αmRNA and protein descented dramatically, and at the same time, the quantity of osteoclast, the quality of TRAP, and the area of resorption pit decented too. And this suppression can reestablish by the addition of TNF-α. When applied in the treatment of rheumatoid arthritis, this system with anti-TNF-αASO could suppress the body weight loosing in the type II collagen induced rheumatoid arthritis model mice. And arthrocele could be also suppressed by this system. X ray and functional test verified this suppression.
Conclusion LMWC from HMWC has very good dissolubility and very low toxicity, and can carry DNA to form a nanometer local delivery system. Gene-Loaded Low molecular weight chitosan nanoparticles can delivery DNA effectively as a vectors to chondrocytes in vitro and in vivo. During this course, some superiority such as DNA protection, delayed release and passive targeting were observed. When carrying TGF-β1 plasmids, this system can delivery the therapeutic factor gene efficiently, and harvest very good result in rabbit’s articular cartilage defect model. When carrying anti- TNF-αASO, this system also can delivery oligonucleotide to cells, suppress the TNF-αdramatically, and provide a new treatment strategy for particle induced osteolysis and rheumatoid arthritis. Gene-Loaded Low molecular weight chitosan nanoparticles is a novel nanometer DNA transferring system and has a very nice prospect.
引文
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