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1.
RNA干扰技术已被广泛应用于心血管领域,壳聚糖纳米粒以其良好的生物特性而作为基因递送载体成为现在研究的热点.就BNA干扰技术与纳米技术在心血管领域的应用及目前常采用的制备壳聚糖纳米粒的方法、影响质粒与壳聚糖纳米粒结合效率的因素、质粒壳聚糖复合物纳米粒转染的影响因素及体外释药行为作一简单的回顾.  相似文献   

2.
背景:作为非病毒基因转染载体,由可降解的高聚物形成的纳米载体目前被广泛由于基因转染,因为他们具有良好的缓释性,靶向性和生物相容性。 目的:制备mPEG-CS纳米粒,探讨mPEG-CS作为Livin shRNA基因转染载体的可行性。 方法:通过离子交联法制备mPEG-CS纳米粒,利用聚乙二醇对壳聚糖进行改性,通过静电吸附法制备载livin shRNA的基因纳米复合物。Zeta-size分析仪和透射电镜检测空白纳米粒和载livin shRNA的基因纳米复合物的形态、粒径和zeta电位,测定基因纳米复合物的包封率,凝胶电泳阻滞实验和DNase I酶消化实验验证纳米粒对基因的保护作用。利用最佳条件下制备的基因纳米复合物,转染大肠癌HT-29细胞,考察转染效率。 结果及结论:成功制备出约60 nm的mPEG-CS纳米粒,当纳米粒与基因体积比为3∶1时,得到的基因纳米复合物形较规则,粒径100 nm左右;其包封率为(94.32±0.35)%。凝胶电泳阻滞实验表明纳米粒能够紧密结合DNA,对基因具有良好的基因保护作用。该基因纳米复合物转染大肠癌细胞的转染效率高,持续作用时间长。mPEG-CS纳米粒作为基因转染载体,对基因具有保护作用,能够将livin shRNA重组质粒高效转染入大肠癌细胞,能够在大肠癌细胞内长时间表达,克服了RNA干扰在基因治疗肿瘤中基因作用时间较短的缺点。  相似文献   

3.
制备负载重组质粒 PGenesil-TGF-β1的壳聚糖纳米粒,并研究其结构特征和性能特点。采用复凝聚法制备壳聚糖-PGenesil-TGF-β1(报告基因)纳米微粒体,通过投射电镜测定其形态、粒径;采用全光谱分光光度计测定该复合物的包封率;凝胶电泳阻滞实验分析纳米粒载体与质粒的结合能力;DNase I 消化实验观察壳聚糖纳米粒保护质粒抵抗核酸酶的能力。制备的壳聚糖-PGenesil -TGF -β1纳米粒均呈球形,微粒直径在100~200 nm 之间,平均约(127.37±19.75)nm;其包封率为(94.38±0.45)%;凝胶电泳阻滞实验结果表明壳聚糖纳米粒能通过静电作用有效结合质粒,将其包裹在内;DNase I 消化实验显示壳聚糖纳米粒能有效地保护质粒免受核酸酶的降解。用复凝聚法成功制得壳聚糖-PGenesil -TGF -β1纳米粒,为后续研究基因药物奠定了实验基础。  相似文献   

4.
聚乙交酯丙交酯牙周片的体内降解和组织相容性研究   总被引:1,自引:0,他引:1  
引言随着现代医学科学和材料科学的迅猛发展 ,生物降解类材料在临床医疗实践中发挥着重要的作用 ,如医用可吸收缝线、骨折内固定、组织工程支架材料、组织充填、创面保护和药物缓释载体等医用产品[1- 4] ,由于这类材料具有在生物体内逐渐解体变成小分子物质而参与机体代谢的特性 ,因而它们在人体应用的安全性问题越来越引起人们的关注 ,如何对这类材料进行临床前评价 ?如何了解材料的降解时段能否与实际应用的要求相匹配 ?这是目前在应用和发展生物降解类材料中亟待解决的问题。迄今为止 ,涉及该领域的研究工作还相对比较缺乏 ,国内外文献检…  相似文献   

5.
背景:壳聚糖对软骨细胞具有良好的生物相容性和可降解性,但存在基因转染效率偏低的缺陷。 目的:构建负载增强型绿色荧光蛋白基因的聚乙烯亚胺-壳聚糖/DNA纳米粒,检测其理化性能,以及体外对关节软骨细胞的基因转染效率。 方法:将聚乙烯亚胺共价连接于壳聚糖骨架上构建聚乙烯亚胺-壳聚糖复合物,再将聚乙烯亚胺-壳聚糖与负载增强型绿色荧光蛋白基因的质粒DNA以复凝聚法制成纳米粒,以扫描电镜检测纳米粒形态,Zeta电位粒度分析仪测定其粒径、表面电位;凝胶电泳阻滞实验观察聚乙烯亚胺-壳聚糖和质粒DNA的结合力。以聚乙烯亚胺-壳聚糖/DNA纳米粒、裸质粒DNA、脂质体2000及壳聚糖/DNA纳米粒转染体外培养的兔关节软骨细胞,流式细胞仪及荧光显微镜检测基因转染率;激光共聚焦显微镜检测DNA的入核情况。 结果与结论:聚乙烯亚胺-壳聚糖/DNA纳米粒多呈球形,粒径为(154.6±18.6) nm,表面Zeta电位为(24.68± 6.82) mV,可有效保护质粒DNA免受 DNaseⅠ的降解。体外转染实验证明聚乙烯亚胺-壳聚糖/DNA纳米粒能介导增强型绿色荧光蛋白基因转染关节软骨细胞并在细胞内表达绿色荧光蛋白,转染率达(23.80±1.74)%,转染率高于裸质粒DNA组及壳聚糖/DNA纳米粒组(P < 0.05),与脂质体2000组无显著差别(P=0.522)。表明聚乙烯亚胺-壳聚糖/DNA纳米粒能有效保护质粒DNA免受核酸酶降解,对关节软骨细胞有良好的基因转染能力。  相似文献   

6.
PEG化壳聚糖质粒纳米粒的制备及其转染的研究   总被引:1,自引:1,他引:0  
目的制备壳聚糖纳米粒并构建聚乙二醇(PEG)化壳聚糖质粒纳米粒,研究其对大鼠主动脉内皮细胞的转染能力及细胞毒性。方法采用离子交联法制备壳聚糖纳米粒,应用喷金扫描电子显微镜检测壳聚糖纳米粒粒径的分布与形态;通过静电吸附作用连接上pGenesil-1质粒(报告基因);对壳聚糖质粒纳米粒进行PEG化的修饰;应用PEG化壳聚糖质粒纳米粒转染大鼠主动脉内皮细胞;采用噻唑蓝(MTT)法测定壳聚糖纳米粒对细胞的毒性作用。结果喷金扫描电镜检测显示壳聚糖纳米粒呈均匀分散的球形颗粒,平均直径为5nm;PEG化壳聚糖质粒纳米粒能转染大鼠主动脉内皮细胞;MTT结果显示壳聚糖纳米粒对细胞无毒性作用;壳聚糖质粒纳米粒对内皮细胞的转染效率为26%,PEG修饰壳聚糖质粒纳米粒转染细胞,转染率为63.4%。结论对壳聚糖质粒纳米粒进行化学修饰不仅能提高其转染效率,且对细胞无毒性作用。  相似文献   

7.
目的:制备壳聚糖纳米粒并构建聚乙二醇(PEG)化壳聚糖质粒纳米粒,研究其对大鼠主动脉内皮细胞的转染能力及细胞毒性.方法:采用离子交联法制备壳聚糖纳米粒,应用喷金扫描电子显微镜检测壳聚糖纳米粒粒径的分布与形态;通过静电吸附作用连接上pGenesil-1质粒(报告基岗);对壳聚糖质粒纳米粒进行PEG化的修饰;应用PEG化壳聚糖质粒纳米粒转染大鼠主动脉内皮细胞;采用噻唑蓝(MTT)法测定壳聚糖纳米粒对细胞的毒性作用.结果:喷金扫描电镜检测显示壳聚糖纳米粒呈均匀分散的球形颗粒,平均直径为5 nm;PEG化壳聚糖质粒纳米粒能转染大鼠主动脉内皮细胞;MTT结果显示壳聚糖纳米粒对细胞无毒性作用;壳聚糖质粒纳米粒对内皮细胞的转染效率为26%,PEG修饰壳聚糖质粒纳米粒转染细胞,转染率为63.4%.结论:对壳聚糖质粒纳米粒进行化学修饰不仅能提高其转染效率,且对细胞无毒性作用.  相似文献   

8.
目的:制备壳聚糖纳米粒并构建聚乙二醇(PEG)化壳聚糖质粒纳米粒,研究其对大鼠主动脉内皮细胞的转染能力及细胞毒性.方法:采用离子交联法制备壳聚糖纳米粒,应用喷金扫描电子显微镜检测壳聚糖纳米粒粒径的分布与形态;通过静电吸附作用连接上pGenesil-1质粒(报告基岗);对壳聚糖质粒纳米粒进行PEG化的修饰;应用PEG化壳聚糖质粒纳米粒转染大鼠主动脉内皮细胞;采用噻唑蓝(MTT)法测定壳聚糖纳米粒对细胞的毒性作用.结果:喷金扫描电镜检测显示壳聚糖纳米粒呈均匀分散的球形颗粒,平均直径为5 nm;PEG化壳聚糖质粒纳米粒能转染大鼠主动脉内皮细胞;MTT结果显示壳聚糖纳米粒对细胞无毒性作用;壳聚糖质粒纳米粒对内皮细胞的转染效率为26%,PEG修饰壳聚糖质粒纳米粒转染细胞,转染率为63.4%.结论:对壳聚糖质粒纳米粒进行化学修饰不仅能提高其转染效率,且对细胞无毒性作用.  相似文献   

9.
目的:制备壳聚糖纳米粒并构建聚乙二醇(PEG)化壳聚糖质粒纳米粒,研究其对大鼠主动脉内皮细胞的转染能力及细胞毒性.方法:采用离子交联法制备壳聚糖纳米粒,应用喷金扫描电子显微镜检测壳聚糖纳米粒粒径的分布与形态;通过静电吸附作用连接上pGenesil-1质粒(报告基岗);对壳聚糖质粒纳米粒进行PEG化的修饰;应用PEG化壳聚糖质粒纳米粒转染大鼠主动脉内皮细胞;采用噻唑蓝(MTT)法测定壳聚糖纳米粒对细胞的毒性作用.结果:喷金扫描电镜检测显示壳聚糖纳米粒呈均匀分散的球形颗粒,平均直径为5 nm;PEG化壳聚糖质粒纳米粒能转染大鼠主动脉内皮细胞;MTT结果显示壳聚糖纳米粒对细胞无毒性作用;壳聚糖质粒纳米粒对内皮细胞的转染效率为26%,PEG修饰壳聚糖质粒纳米粒转染细胞,转染率为63.4%.结论:对壳聚糖质粒纳米粒进行化学修饰不仅能提高其转染效率,且对细胞无毒性作用.  相似文献   

10.
目的:制备壳聚糖纳米粒并构建聚乙二醇(PEG)化壳聚糖质粒纳米粒,研究其对大鼠主动脉内皮细胞的转染能力及细胞毒性.方法:采用离子交联法制备壳聚糖纳米粒,应用喷金扫描电子显微镜检测壳聚糖纳米粒粒径的分布与形态;通过静电吸附作用连接上pGenesil-1质粒(报告基岗);对壳聚糖质粒纳米粒进行PEG化的修饰;应用PEG化壳聚糖质粒纳米粒转染大鼠主动脉内皮细胞;采用噻唑蓝(MTT)法测定壳聚糖纳米粒对细胞的毒性作用.结果:喷金扫描电镜检测显示壳聚糖纳米粒呈均匀分散的球形颗粒,平均直径为5 nm;PEG化壳聚糖质粒纳米粒能转染大鼠主动脉内皮细胞;MTT结果显示壳聚糖纳米粒对细胞无毒性作用;壳聚糖质粒纳米粒对内皮细胞的转染效率为26%,PEG修饰壳聚糖质粒纳米粒转染细胞,转染率为63.4%.结论:对壳聚糖质粒纳米粒进行化学修饰不仅能提高其转染效率,且对细胞无毒性作用.  相似文献   

11.
We studied three different types of scaffolds, encapsulating bone morphogenetic protein-2 (BMP-2) plasmid, in terms of their performances in bone regeneration in nude mice. The plasmid was loaded into fibrous matrices in three different ways: coating of naked DNA (Group A) or DNA/chitosan nanoparticles (Group B) onto scaffolds after fiber fabrication by dripping, and encapsulation of DNA/chitosan nanoparticles into scaffold by mixing them with PLGA/DCM solution before fiber fabrication (Group C). Their individual performances were examined by soft X-ray observation, histological analysis and immunostaining of bone tissue. In addition, the BMP-2 protein concentration and alkaline phosphatase (ALP) activity in serum were monitored. The results revealed that the bioactivity of BMP-2 plasmid released from all three kinds of scaffolds was well maintained; this eventually helped improve the healing of segmental defects in vivo. Interestingly, the three kinds of scaffolds released DNA or DNA nanoparticles in different modes and their performances in bone healing were diverse. These observations demonstrate that the in vivo performance of these newly developed DNA delivery devices correlates well with their in vitro release profiles.  相似文献   

12.
目的 探讨心肌细胞吞噬聚乳酸—乙醇酸 ( polylactic polyglycolicacid ,PLGA)纳米粒子的形态学机理 ,制备组织细胞吞噬纳米粒子的相关模型 ,为纳米粒子作为包载各类药物或基因载体提供实验形态学依据。方法 :将制备的PLGA纳米粒子加入生理盐水中 ,超声振荡成注射用纳米粒子悬浮液后 ,注射至活体家兔心肌组织内 ,4 8小时后取材切片、电镜下观察。结果 :心肌细胞胞质内及胞核内可见大量被吞噬的纳米粒子 ,并且出现了PLGA纳米粒子被吸收、降解的迹象。结论 :心肌细胞完全可以通过吞噬方式大量摄取 ;PLGA纳米粒子 ,将PLGA纳米粒子作为向心肌细胞内转运某种药物或基因的载体是完全可行的。  相似文献   

13.
Polyethylenimine (PEI) was combined with plasmid DNA and freeze dried following the addition of sucrose as a lyoprotectant and pore-forming agent. Freeze-dried PEI DNA condensates were dry mixed with granular polylactideglycolic acid (PLGA) then compression molded and sponged to encapsulated PEI DNA. A measurement of the elastic modulus indicated that 91 wt% sucrose substituted for 95 wt% sodium chloride as a porogen, resulting in PLGA sponges with a mechanical modulus of 100 kPa. The PEI DNA was retained (80%) within PLGA sponges prepared with sucrose during the leaching and subsequent 2-week release studies, whereas sodium chloride PLGA sponges caused the premature release (100%) of PEI DNA within 2 days. In vitro gene transfer studies with PEI DNA PLGA sponges established that adherent and infiltrating fibroblasts expressed reporter gene for 15 days compared with the short, 3-day expression mediated by direct gene of PEI DNA on cells in culture. The results demonstrate an approach to encapsulate condensed DNA in a PLGA sponge for the purpose of retaining DNA within the matrices and creating efficient gene transfer during tissue engineering.  相似文献   

14.
《Acta biomaterialia》2014,10(6):2643-2652
Polymeric nanoparticles (NPs) are promising carriers of biological agents to the lung due to advantages including biocompatibility, ease of surface modification, localized action and reduced systemic toxicity. However, there have been no studies extensively characterizing and comparing the behavior of polymeric NPs for pulmonary protein/DNA delivery both in vitro and in vitro. We screened six polymeric NPs: gelatin, chitosan, alginate, poly(lactic-co-glycolic) acid (PLGA), PLGA–chitosan and PLGA–poly(ethylene glycol) (PEG), for inhalational protein/DNA delivery. All NPs except PLGA–PEG and alginate were <300 nm in size with a bi-phasic core compound release profile. Gelatin, PLGA NPs and PLGA–PEG NPs remained stable in deionized water, serum, saline and simulated lung fluid (Gamble’s solution) over 5 days. PLGA-based NPs and natural polymer NPs exhibited the highest cytocompatibility and dose-dependent in vitro uptake, respectively, by human alveolar type-1 epithelial cells. Based on these profiles, gelatin and PLGA NPs were used to encapsulate plasmid DNA encoding yellow fluorescent protein (YFP) or rhodamine-conjugated erythropoietin (EPO) for inhalational delivery to rats. Following a single inhalation, widespread pulmonary EPO distribution persisted for up to 10 days while increasing YFP expression was observed for at least 7 days for both NPs. The overall results support both PLGA and gelatin NPs as promising carriers for pulmonary protein/DNA delivery.  相似文献   

15.
Highly porous poly(D,L-lactic-co-glycolic acid) (PLGA) scaffolds were fabricated by a thermally-induced phase-separation (TIPS) method to deliver plasmid DNA in a controlled manner. A variety of TIPS parameters directly affecting pore structures and their interconnectivities of the scaffold, such as polymer concentration, solvent/non-solvent ratio, quenching methods and annealing time, were systematically examined to explore their effects on sustained release behaviors of plasmid DNA. Plasmid DNA was directly loaded into the inner pore region of the scaffold during the TIPS process. By optimizing the parameters, PLGA scaffolds releasing plasmid DNA over 21 days were successfully fabricated. DNA release profiles were mainly affected by the pore structures and their interconnectivities of the scaffolds. Plasmid DNA released from the scaffolds fully maintained its structural integrity and showed comparable transfection efficiency to native plasmid DNA. These biodegradable polymeric scaffolds capable of sustained DNA release can be potentially applied for various tissue engineering purposes requiring a combined gene delivery strategy.  相似文献   

16.
Preparation and characterization of cationic PLGA nanospheres as DNA carriers   总被引:21,自引:0,他引:21  
Ravi Kumar MN  Bakowsky U  Lehr CM 《Biomaterials》2004,25(10):1771-1777
Nanoparticles formulated from biodegradable polymers such as poly(lactic acid) (PLA) and poly(lactide-co-glycolide) (PLGA) are being extensively investigated as non-viral gene delivery systems due to their controlled release characteristics and biocompatibility. PLGA nanoparticles for DNA delivery are mainly formulated by an emulsion-solvent evaporation technique using PVA as a stabilizer generating negatively charged particles and heterogeneous size distribution. The objective of the present study was to formulate cationically modified PLGA nanoparticles with defined size and shape that can efficiently bind DNA. An Emulsion-diffusion-evaporation technique to make cationic nanospheres composed of biodegradable and biocompatible co-polyester PLGA has been developed. PVA-chitosan blend was used to stabilize the PLGA nanospheres. The nanospheres were characterized by atomic force microscopy (AFM), photon-correlation spectroscopy (PCS), and Fourier transform infrared spectroscopy (FTIR). Zeta potential and gel electrophoresis studies were also performed to understand the surface properties of nanospheres and their ability to condense negatively charged DNA. The designed nanospheres have a zeta potential of 10mV at pH 7.4 and size under 200nm. From the gel electrophoresis studies we found that the charge on the nanospheres is sufficient to efficiently bind the negatively charged DNA electrostatically. These cationic PLGA nanospheres could serve as potential alternatives of the existing negatively charged nanoparticles.  相似文献   

17.
Jeon SY  Park JS  Yang HN  Woo DG  Park KH 《Biomaterials》2012,33(17):4413-4423
Some genes expressed in stem cells interrupt and/or enhance differentiation. Therefore, the aim of this study was to inhibit the expression of unnecessary genes and enhance the expression of specific genes involved in stem cell differentiation by using small interfering RNA (siRNA) and plasmid DNA (pDNA) incorporated into cationic polymers as co-delivery factors. To achieve co-delivery of siRNA and pDNA to human mesenchymal stem cells (hMSCs), two different genes were complexed with poly(ethyleneimine) (PEI) and then coated onto poly(lactide-co-glycolic acid) (PLGA) nanoparticles (NP). To evaluate co-delivery of siRNA and pDNA into hMSCs, cells were transfected with green fluorescence protein (GFP) pDNA (GFP pDNA) and GFP siRNA (GFP siRNA). The percentage of GFP-expressing hMSCs decreased from 25.35 to 3.7% after transfection with GFP-DNA/PLGA NP (NPs) or GFP siRNA/PLGA NPs, whereas GFP-DNA/PLGA NPs and scramble siRNA (MOCK)/PLGA NPs had no effect on GFP expression. hMSCs cotransfected with coSOX9-pDNA/NPs and Cbfa-1-siRNA/NPs were tested both in vitro and in vivo using gel retardation, dynamic light scattering (DLS), and scanning electron microscope (SEM). The expression of genes and proteins associated with chondrogenesis was evaluated by FACS, RT-PCR, real time-qPCR, Western blotting, immunohistochemistry, and immunofluorescence imaging.  相似文献   

18.
以可生物降解材料聚乳酸聚乙醇酸共聚物(Poly—dl—lactic—cp—glycolic,PLGA)为原料,采用多相乳化技术制备载VEGP纳米粒子。并对纳米粒子的粒径,VEGF含量,体外释放等进行了测定。VEGF纳米粒子和VEGF裸质粒被注射到兔下肢缺血模型的缺血部位,通过RT—PCR,免疫组化和血管造影等技术来验证基因治疗的效果,评价VEGF纳米粒子作为基因载体在动物模型基因治疗中的效率。制备的VEGF纳米粒子的平均粒径约为300nm,包埋效率在96%以上,纳米粒子中VEGF含量约4%。可在体外维持恒定释放约两周。两周基因注射结果表明VEGF-NP治疗组与裸质粒VEGF治疗组的毛细血管密度明显高于对照组,VEGF纳米粒子组(81.22per mm^2),对照组(29.54mm^2),两者有显著性差异(P〈0.05)。RT—PCR结果显示VEGF纳米粒子组表达(31.79au*mm)明显高于VEGF裸质粒组(9.15au*mm)。在动物模型中VEGF纳米粒子是比裸质粒DNA更好的基因载体系统,结果显示了纳米粒子可望在人类基因治疗中得到很好的应用。  相似文献   

19.
以可生物降解材料聚乳酸聚乙醇酸共聚物(Poly-dl-lactic-co-glycolic,PLGA)为原料,采用多相乳化技术制备载VEGF纳米粒子。并对纳米粒子的粒径,VEGF含量,体外释放等进行了测定。VEGF纳米粒子和VEGF裸质粒被注射到兔下肢缺血模型的缺血部位,通过RT-PCR,免疫组化和血管造影等技术来验证基因治疗的效果,评价VEGF纳米粒子作为基因载体在动物模型基因治疗中的效率。制备的VEGF纳米粒子的平均粒径约为300nm,包埋效率在96%以上,纳米粒子中VEGF含量约4%。可在体外维持恒定释放约两周。两周基因注射结果表明VEGF-NP治疗组与裸质粒VEGF治疗组的毛细血管密度明显高于对照组,VEGF纳米粒子组(81.22permm2),对照组(29.54mm2),两者有显著性差异(P<0.05)。RT-PCR结果显示VEGF纳米粒子组表达(31.79au*mm)明显高于VEGF裸质粒组(9.15au*mm)。在动物模型中VEGF纳米粒子是比裸质粒DNA更好的基因载体系统,结果显示了纳米粒子可望在人类基因治疗中得到很好的应用。  相似文献   

20.
目的载紫杉醇聚乳酸聚羟基乙酸共聚物(PLGA)/F68纳米粒逆转耐紫杉醇人乳腺癌细胞MCF-7/Taxol细胞多药耐药的可行性研究。方法使用超声乳化溶剂挥发法分别制备载紫杉醇PLGA和载紫杉醇PLGA/F68纳米粒(10%),并对载紫杉醇纳米粒进行表征。载紫杉醇纳米粒的体外释放研究使用高效液相色谱进行分析。最后研究载紫杉醇纳米粒在耐紫杉醇人乳腺癌细胞MCF-7/Taxol细胞的细胞摄取和细胞毒性(PLGA/F68组、PLGA组和泰素组)。结果纳米粒呈球形,表面粗糙多孔,平均粒径250 nm左右,粒径分布比较窄,体外药物释放呈双相释放模型。载紫杉醇PLGA/F68纳米粒能够被耐紫杉醇人乳腺癌细胞MCF-7/Taxol细胞摄取。载紫杉醇PLGA/F68纳米粒比载紫杉醇PLGA纳米粒(P〈0.05)和泰素(TaxolR)(P〈0.05)有更高的细胞毒性。结论载紫杉醇PLGA/F68纳米粒能够逆转耐紫杉醇人乳腺癌细胞MCF-7/Taxol细胞的多药耐药,药用辅料Pluronic F68在乳腺癌治疗中具有潜在的应用前景。  相似文献   

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