羟基化多壁碳纳米管对RAW264.7细胞增殖及功能影响研究

目的研究羟基化多壁碳纳米管对巨噬细胞RAW264.7的活性、吞噬功能及氧化应激的影响。方法将质量浓度分别为1、10、100、200μg/mL的羟基化多壁碳纳米管与原始多壁碳纳米管分别与小鼠巨噬细胞系RAW264.7细胞共育24、48、72 h,采用CellTiter-GloR发光法进行细胞活性测定,用2’,7’-二氯荧光黄双乙酸盐法(DCFH-DA)检测细胞内活性氧自由基(ROS)的生成。选24只小鼠,雌雄不限,鼠龄5～6周,体质量18～25 g,随机分为4组,同时通过鸡红细胞吞噬实验检测细胞吞噬能力的变化。结果CellTiter-GloR发光法检测显示碳纳米管的细胞毒性作用呈现浓度依赖性,只有在质量浓度为10μg/mL时,羟基化多壁碳纳米管比原始碳纳米管细胞毒性小,其他浓度两者之间差异无统计学意义(P>0.05)。鸡红细胞吞噬实验证实两种碳纳米管具有促进小鼠腹腔巨噬细胞吞噬功能的作用。结果还显示,在碳纳米管质量浓度为1μg/mL和10μg/mL时,羟基化多壁碳纳米管诱导细胞内ROS含量升高程度高于原始多壁碳纳米管,而在高浓度组(100μg/mL和200μg/mL),随着孵育时间延长,原始多壁碳纳米管诱导细胞内ROS含量不断增加,明显高于羟基化多壁碳纳米管对细胞的作用。结论两种碳纳米管可显著抑制巨噬细胞增殖并提高细胞吞噬活性;不同浓度的多壁碳纳米管与细胞相互作用时,羟基化多壁碳纳米管与原始多壁碳纳米管诱导细胞内ROS升高机制不同。     

低分子质量壳聚糖修饰多壁碳纳米管的制备及细胞毒性研究

目的 利用低分子质量壳聚糖(CS)制备可在水溶液中稳定分散的壳聚糖/多壁碳纳米管(CS/MWCNTs)材料,并观察其与人脐静脉内皮细胞(HUVEC)的相互作用.方法 以物理吸附法对MWCNTs进行CS修饰,利用透射电镜、纳米粒度及Zeta电位分析仪对其进行表征.将CS/MWCNT进行荧光标记,以不同浓度与细胞作用24 h,通过激光共聚焦显微镜观察细胞摄入情况,并检测细胞毒性及细胞内活性氧自由基含量.结果 当低分子质量CS与MWCNTs的质量比大于10∶1时,可很好地将MWCNTs进行分散,CS/MWCNTs可在水相中稳定存在.细胞摄入实验显示,进入细胞内的碳纳米管主要位于胞浆内.毒性检测结果显示,在较高质量浓度(10、20 μg/ml)时,CS分散后的MWCNTs毒性较小.而与2种碳纳米管(MWCNTs与CS/MWCNTs)作用的细胞内的活性氧含量均随着浓度升高而显著提高,差别无统计学意义(P＞0.05).结论 水溶性的CS/MWCNTs材料拥有极好的分散性,性状稳定,细胞毒性低,这对后期将其应用于以MWCNTs为载体的治疗研究具有重要意义.     

功能化修饰多壁碳纳米管对人外周血单个核细胞的毒性研究

目的 探讨不同功能化修饰的多壁碳纳米管(F-MWCNTs)对人外周血单个核细胞(PBMC)的细胞毒性的影响.方法 利用透射电镜表征5种直径和长度均相同的MWCNTs(羟基、羧基、氨基、镀镍修饰和未修饰的MWCNTs(P-MWCNTs))在生理盐水溶液中的分散性.体外实验中先通过Ficoll密度梯度离心从人外周血中分离出PBMC,再将5种MWCNTs分别超声分散于含血清的培养基中,与PBMC共培养12、24、48、72 h,通过CCK-8试剂盒检测5种MWCNTs对PBMC的细胞毒性.结果 5种MWCNTs的分散性相对良好,尤其是各F-MWCNTs.细胞毒性实验结果 表明,MWCNTs的细胞毒性呈剂量-效应关系和一定的时间-效应关系.F-MWCNTs与P-MWCNTs相比,细胞毒性发生显著变化,其中羟基、羧基和氨基修饰的MWCNTs的细胞毒性减小,尤以氨基修饰的细胞毒性减小最为显著(P＜0.05);而镀镍修饰的MWCNTs的细胞毒性反而明显增大,其处理细胞24 h和48 h时的细胞存活率较同剂量(25 μg/ml)的P-MWCNTs均有所降低,差异均有统计学意义(P＜O.01,P＜0.05).结论 功能化修饰不仅影响MWCNTs在水溶液中的分散性,还影响MWCNTs对人外周血淋巴细胞的细胞毒性.     

TRAIL对多种肿瘤细胞的杀伤作用

目的： 研究肿瘤坏死因子相关凋亡诱导配体（TRAIL）作用下多种肿瘤细胞的生长抑制效应及凋亡诱导情况。方法： 利用大肠杆菌基因工程菌表达非融合rhsTRAIL，进行目的蛋白的分离纯化，得到rhsTRAIL样品，纯度为97%。通过倒置显微镜下观察、MTT法、流式细胞仪法检测其对细胞的生长抑制和诱导凋亡情况。结果： 一定浓度的TRAIL 可有效抑制LS174-T细胞、MCF-7细胞、GLC细胞、7402细胞、Jurkut T细胞生长，其生长抑制率具剂量依赖性，且各细胞对TRAIL敏感性不同，其中Jurkat T细胞最为敏感。用2 mg/L TRAIL作用Jurkat T细胞0-72 h，6 h后细胞即发生明显凋亡，其细胞凋亡率具时间依赖性。结论： 所制备的TRAIL可抑制多种肿瘤细胞生长，并诱导Jurkat T细胞凋亡。     

LXR-ABCA1/ABCG1通路对BCG感染的巨噬细胞凋亡和炎性反应的调控作用

目的 揭示LXR-ABCA1/ABCG1通路对BCG感染后巨噬细胞凋亡和炎性反应的调控作用。方法 采用T0901317预处理RAW264.7巨噬细胞2 h和BCG感染24 h,设置4个实验组：对照组、T0901317组、BCG感染组和T0901317+BCG感染组。采用Western blot方法检测凋亡相关蛋白cleaved-Caspase3、cleaved-Caspase8、cleaved-Caspase9和TLR信号通路相关蛋白TLR2、TLR4、MyD88、TRAF6、p-NF-κB p65、IRF3、TBK1的表达,采用ELISA方法检测细胞培养上清中促炎细胞因子TNF-α、IL-6、IL-1β的含量。结果 与对照组比较,BCG感染组凋亡相关蛋白cleaved-Caspase3、cleaved-Caspase8、cleaved-Caspase9及依赖MyD88途径蛋白TLR2、TLR4、MyD88、TRAF6、p-NF-κBp65的表达均上调（P<0.01）,促炎细胞因子TNF-α、IL-6、IL-1β表达水平上升（P<0.01）;与BCG感染组相比,T09013...     

2型登革病毒诱导RAW264.7细胞凋亡的机制及凋亡对病毒复制的影响

 目的：探讨2型登革病毒（DENV2）感染能否诱导RAW264.7细胞凋亡,并初步探讨凋亡对病毒复制的影响。方法：用DENV2感染RAW264.7细胞,MTT检测细胞活性,Hoechst 33342染色检测细胞核变化,Annexin V-FITC/PI双染流式细胞术检测细胞凋亡,Western blotting检测caspase-3和caspase-8活化片段的变化,比色法检测caspase-9活性变化,JC-1染色检测线粒体膜电位变化,Z-VAD-FMK抑制细胞凋亡后以TCID50检测感染细胞上清病毒滴度。结果：DENV2感染RAW264.7细胞24 h、36 h及48 h后细胞活性受到抑制,免疫荧光检测有核固缩现象,流式细胞术检测发现病毒感染诱导了细胞凋亡,Western blotting检测发现活化caspase-3和caspase-8的表达增加,caspase-9活性也增加,JC-1染色发现病毒感染诱导RAW264.7细胞线粒体膜电位降低,用Z-VAD-FMK抑制凋亡后感染细胞上清病毒滴度增加。结论：登革病毒感染可以通过内、外源性途径诱导RAW264.7细胞发生凋亡;凋亡发生抑制了病毒的产生。     

胞壁酰二肽激活大鼠巨噬细胞抗肿瘤免疫效应的研究

目的:探讨胞壁酰二肽(MDP)激活大鼠巨噬细胞抗肿瘤效应的途径和机制。方法:以SD大鼠为骨肉瘤动物模型进行肿瘤抑制试验;采用中性红吞饮实验、间接MTT法及硝酸还原酶法,测定大鼠腹腔巨噬细胞的功能。结果:MDP(50μg～100μg/鼠)皮下注射可明显抑制UMR106骨肉瘤细胞在SD大鼠体内的生长,并可显著提高巨噬细胞的吞噬功能、杀伤活性及TNF和NO的分泌水平,且呈现一定的量效关系。结论:MDP激活的巨噬细胞可能是机体非特异性免疫抗瘤效应的主要基础。     

多壁碳纳米管改善激素性股骨头坏死模型兔的股骨头形态

背景：碳纳米管可以促进成骨细胞的增殖和分化,可能对激素性股骨头坏死发挥治疗作用。 目的：观察多壁碳纳米管在激素性股骨头坏死模型建立过程中的作用。方法：36只新西兰大耳白兔随机选出32只等分为治疗组和模型组,剩余4只作为空白组。治疗组兔每天臀肌注射地塞米松2.5 mg/kg,每周向双侧股骨的骨髓腔内各注射0.1 g/L多壁碳纳米管悬浊液1 mL;模型组每天臀肌注射地塞米松2.5 mg/kg,每周向双侧股骨的骨髓腔内各注射生理盐水1 mL,空白组每天臀肌注射生理盐水2 mL,每周向双侧股骨的骨髓腔内各注射生理盐水1 mL。结果与结论：模型组兔股骨头组织中骨小梁开始少量出现变细断裂,骨髓脂肪化十分明显,脂肪细胞肥大,微血管开始出现血栓;而经多壁碳纳米管悬浊液治疗的兔股骨头病理损伤出现明显改善。提示多壁碳纳米管能在一定程度上改善激素性股骨头坏死的病理损伤。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

碳纳米管对肺组织病理损伤

目的:探讨碳纳米管对肺组织的病理损伤.方法:雄性SD大鼠鼻腔滴注0.5 mg/ml的单壁碳纳米管(SWNTS)和多壁碳纳米管(MWNTS)颗粒悬液25 d,双蒸水滴注作为对照,取大鼠肺组织.采用光学显微镜和透射电子显微镜观察2种碳纳米管材料对肺组织的病理损伤.结果:SWNTS染毒组,观察到肺泡壁增厚,部分肺泡隔断裂,肺泡融合成肺大泡,肺间质有炎症细胞浸润,较高倍数下可以观察到肺组织中沉积有疑似SWNTS颗粒;在肺泡Ⅱ型上皮细胞中,板层体溶解,出现空泡化;在肺泡巨噬细胞中,次级溶酶体增多,细胞核染色质异常;MWNTS染毒组肺组织损伤情况与SWNTS染毒组相似,但较SWNTS染毒组要轻.结论:鼻腔滴注SWNTS和MWNTS均引起肺组织的病理学损伤和超微结构变化.相同的质量浓度下,SWNTS毒理效应强于MWNTS.     

α-硫辛酸降低脂多糖诱导的Raw264.7细胞TNF-α的分泌

目的观察α-硫辛酸(ALA)对脂多糖(LPS)在体外诱导的Raw264.7细胞TNF-α释放及机制。方法用ALA或者ALA联合ERK或NF-κB的抑制剂或者激活剂预处理2 h,再用LPS(1 mg/L)体外刺激巨噬细胞Raw264.7;采用MTT法检测ALA对Raw264.7细胞活力影响,利用ELISA方法对Raw264.7释放在培养上清中的细胞因子肿瘤坏死因子(TNF-α)的浓度,及用Western blot方法检测T-p ERK、p ERK和NF-κB的表达。结果 ALA可抑制LPS诱导的Raw264.7细胞TNF-α表达(P0.05),用ALA联合ERK或NF-κB的激活剂预处理后可减弱ALA产生的抑制效果(P0.05)。同时ALA抑制LPS诱导的Raw264.7细胞p ERK和NF-κB的表达(P0.05),用ALA联合ERK或NF-κB的抑制剂预处理后可加强ALA产生的抑制效果(P0.05)。结论 ALA可能通过抑制LPS诱导的Raw264.7细胞的ERK和NF-κB信号通路来抑制TNF-α释放。     

Self-assembly of nano-hydroxyapatite on multi-walled carbon nanotubes

Inspired by self-assembly of nano-hydroxyapatite (nHA) on collagen associated with the 67nm periodic microstructure of collagen, we used multi-walled carbon nanotubes (MWCNTs) with approximately 40nm bamboo periodic microstructure as a template for nHA deposition to form a nHA-MWCNT composite. The assembled apatite was analyzed by transmission electron microscopy and scanning electron microscopy. Defects that were analogous to edge dislocations along the carbon nanotubes' multi-walled surfaces were the nucleation sites for nHA after these defects had been functionalized principally into carboxylic groups. Spindle-shaped units consisting of an assembly of near parallel, fibril-like nHA polycrystals were formed and oriented at a certain angle to the long axis of the carbon nanotubes, unlike nHA-collagen in which the nHA is oriented along the longitudinal axis of the collagen molecule. One possible explanation for this difference is that there are more bonds for calcium chelation (-COOH, >CO) on the collagen fibril surface than on the surface of MWCNTs. Spindle-shaped units that are detached from the MWCNT template are able to maintain the ordered parallel structure of the nHA polycrystal fibril. We have thus created a self-assembled hydroxyapatite on MWCNTs.     

Toxicity and imaging of multi-walled carbon nanotubes in human macrophage cells

Multi-walled carbon nanotubes (MWNTs) have been proposed for use in many applications and concerns about their potential effect on human health have led to the interest in understanding the interactions between MWNTs and human cells. One important technique is the visualisation of the intracellular distribution of MWNTs. We exposed human macrophage cells to unpurified MWNTs and found that a decrease in cell viability was correlated with uptake of MWNTs due to mainly necrosis. Cells treated with purified MWNTs and the main contaminant Fe₂O₃ itself yielded toxicity only from the nanotubes and not from the Fe₂O₃. We used 3-D dark-field scanning transmission electron microscopy (DF-STEM) tomography of freeze-dried whole cells as well as confocal and scanning electron microscopy (SEM) to image the cellular uptake and distribution of unpurified MWNTs. We observed that unpurified MWNTs entered the cell both actively and passively frequently inserting through the plasma membrane into the cytoplasm and the nucleus. These suggest that MWNTs may cause incomplete phagocytosis or mechanically pierce through the plasma membrane and result in oxidative stress and cell death.     

The response effect of pheochromocytoma (PC12) cell lines to oxidized multi-walled carbon nanotubes (o-MWCMTs)

Background

The applications of oxidized carbon nanotubes (o-CNTs) have shown potentials in novel drug delivery including the brain which is usually a challenge. This underscores the importance to study its potential toxic effect in animals. Despite being a promising tool for biomedical applications little is known about the safety of drugs in treating brain diseases. The toxicity of oxidized multi-walled carbon nanotubes (o-MWCNTs) are of utmost concern and in most in-vitro studies conducted so far are on dendritic cell (DC) lines with limited data on PC12 cell lines.

Objectives

We focused on the effect of o-MWCNTs in PC12 cells in vitro: a common model cell for neurotoxicity.

Methods

The pristine multi-walled carbon nanotubes (p-MWCNTs) were produced by the swirled floating catalytic chemical vapour deposition method (SFCCVD). The p-MWCNTs were then oxidized using purified H₂SO₄/HNO₃ (3:1v/v) and 30% HNO₃ acids to produce o-MWCNTs. The Brunauer-Emmett-Teller (BET), transmission electron microscopy (TEM), Scanning electron microscopy (SEM), thermogravimetric analyser (TGA) and Raman spectroscopy techniques were used to characterize the MWCNTs. The PC12 cells were cultured in RPMI medium containing concentrations of o-MWCNTs ranging from 50 to 200 µg/ml.

Results

The o-MWCNTs demonstrated slight cytotoxicity at short time period to PC12 neuronal cells whilst at longer time period, no significant (p > 0.05) toxicity was observed due to cell recovery.

Conclusion

In conclusion, the o-MWCNTs did not affect the growth rate and viability of the PC12 cells due to lack of considerable toxicity in the cells during the observed time period but further investigations are required to determine cell recovery mechanism.     

Synthesis and characterization of nano-hydroxyapatite/polyamide 66 biocomposites reinforced with multi-walled carbon nanotubes

In this work, we investigate the enhanced mechanical properties of nano-hydroxyapatite/polyamide 66 (nHA/PA66) composites reinforced with multi-walled carbon nanotubes (MWCNTs) by means of the blending method. The MWCNTs–nHA/PA66 composites were characterized by various techniques, and the obtained results indicated that the MWCNTs were evenly distributed in the composite and that good interfacial bonding was formed between MWCNTs and PA66. The addition of MWCNTs improved the crystallinity of PA66, while it had little or no effect either on the composition or on the crystal structure of the composites. Moreover, the addition of MWCNTs in nHA/PA66 significantly improved the mechanical strength, and the tensile and compressive strengths attained maximum values of 90.3 and 126.8 MPa, respectively, with the addition of 0.1 wt% MWCNTs, whereas the bending strength attained a maximum value of 105.5 MPa with the addition of 0.05 wt% MWCNTs. Finally, L929 cells co-cultured with the MWCNTs-nHA/PA66 composite exhibited comparatively uninhibited cell growth, indicating that the addition of MWCNTs had negligible effect on the cytocompatibility of the original nHA/PA66 composite.     

Fatigue and biocompatibility properties of a poly(methyl methacrylate) bone cement with multi-walled carbon nanotubes

Composites of multi-walled carbon nanotubes (MWCNT) of varied functionality (unfunctionalised and carboxyl and amine functionalised) with polymethyl methacrylate (PMMA) were prepared for use as a bone cement. The MWCNT loadings ranged from 0.1 to 1.0 wt.%. The fatigue properties of these MWCNT-PMMA bone cements were characterised at MWCNT loading levels of 0.1 and 0.25 wt.% with the type and wt.% loading of MWCNT used having a strong influence on the number of cycles to failure. The morphology and degree of dispersion of the MWCNT in the PMMA matrix at different length scales were examined using field emission scanning electron microscopy. Improvements in the fatigue properties were attributed to the MWCNT arresting/retarding crack propagation through the cement through a bridging effect and hindering crack propagation. MWCNT agglomerates were evident within the cement microstructure and the degree of agglomeration was dependent on the level of loading and functionality of the MWCNT. The biocompatibility of the MWCNT-PMMA cements at MWCNT loading levels upto 1.0 wt.% was determined by means of established biological cell culture assays using MG-63 cells. Cell attachment after 4h was determined using the crystal violet staining assay. Cell viability was determined over 7 days in vitro using the standard colorimetric MTT assay. Confocal scanning laser microscopy and SEM analysis was also used to assess cell morphology on the various substrates.     

Preparation,mechanical properties and in vitro cytocompatibility of multi-walled carbon nanotubes/poly(etheretherketone) nanocomposites

Desired bone repair material must have excellent biocompatibility and high bioactivity. Moreover, mechanical properties of biomaterial should be equivalent to those of human bones. For developing an alternative biocomposite for load-bearing orthopedic application, combination of bioactive fillers with polymer matrix is a feasible approach. In this study, a series of multi-walled carbon nanotubes (MWCNTs)/poly(etheretherketone) (PEEK) bioactive nanocomposites were prepared by a novel coprecipitation-compounding and injection-molding process. Scanning electron microscope (SEM) images revealed that MWCNTs were adsorbed on the surface of PEEK particles during the coprecipitation-compounding process and dispersed homogeneously in the nanocomposite because the conjugated PEEK polymers stabilized MWCNTs by forming strong π–π stack interactions. The mechanical testing revealed that mechanical performance of PEEK was significantly improved by adding MWCNTs (2–8 wt%) and the experimental values obtained were close to or higher than that of human cortical bone. In addition, incorporation of MWCNTs into PEEK matrix also enhanced the roughness and hydrophilicity of the nanocomposite surface. In vitro cytocompatibility tests demonstrated that the MWCNTs/PEEK nanocomposite was in favor of cell adhesion and proliferation of MC3T3-E1 osteoblast cells, exhibiting excellent cytocompatibility and biocompatibility. Thus, this MWCNTs/PEEK nanocomposite may be used as a promising bone repair material in orthopedic implants application.     

Reinforcement of calcium phosphate cement with multi-walled carbon nanotubes and bovine serum albumin for injectable bone substitute applications

This paper presents the development of novel alternative injectable calcium phosphate cement (CPC) composites for orthopaedic applications. The new CPC composites comprise β-tri-calcium phosphate (β-TCP) and di-calcium phosphate anhydrous (DCPA) mixed with bovine serum albumin (BSA) and incorporated with multi-walled carbon nanotubes (MWCNTs) or functionalized MWCNTs (MWCNTs-OH and MWCNTs-COOH). Scanning electron microscopy (SEM), compressive strength tests, injectability tests, Fourier transform infrared spectroscopy and X-ray diffraction were used to evaluate the properties of the final products. Compressive strength tests and SEM observations demonstrated particularly that the concomitant admixture of BSA and MWCNT improved the mechanical properties, resulting in stronger CPC composites. The presence of MWCNTs and BSA influenced the morphology of the hydroxyapatite (HA) crystals in the CPC matrix. BSA was found to act as a promoter of HA growth when bounded to the surface of CPC grains. MWCNT-OH-containing composites exhibited the highest compressive strengths (16.3?MPa), being in the range of values for trabecular bone (2-12?MPa).     

The targeted delivery of anticancer drugs to brain glioma by PEGylated oxidized multi-walled carbon nanotubes modified with angiopep-2

In this study, a dual-targeting drug delivery system based on PEGylated oxidized multi-walled carbon nanotubes (O-MWNTs) modified with angiopep-2 (O-MWNTs-PEG-ANG) was successfully developed for treatment of brain glioma. O-MWNTs can not only distribute in brains but also accumulate in tumors, and have ultrahigh surface area with remarkably high loading anticancer drug of doxorubicin (DOX), which was selected as drug carrier. Angiopep-2 can specifically combine to the low-density lipoprotein receptor-related protein (LRP) receptor overexpressed on the blood-brain barrier (BBB) and glioma cells, which was selected as targeting ligand. The cooperative dual-targeting to brain glioma by O-MWNTs-PEG-ANG was evaluated by intracellular tracking in vitro and fluorescence imaging in vivo, which demonstrated that the combination of O-MWNTs-PEG and angiopep-2 constituted an ideal dual-targeting drug delivery system. The anti-glioma effect of DOX-loaded O-MWNTs-PEG-ANG (DOX-O-MWNTs-PEG-ANG) was assessed by C6 cytotoxicity and median survival time of glioma bearing mice, which showed a better anti-glioma effect than DOX. The biological safety of O-MWNTs-PEG-ANG was evaluated by BCEC and C6 cytotoxicity, hematology analysis and CD68 immunohistochemical analysis, which proved O-MWNTs-PEG-ANG was good biocompatibility and low toxicity. The biological safety of DOX-O-MWNTs-PEG-ANG was evaluated by histopathological analysis, which suggested a lower cardiac toxicity than DOX. In conclusion, O-MWNTs-PEG-ANG is a promising dual-targeting carrier to deliver DOX for the treatment of brain tumor.