季铵盐壳聚糖及其纳米粒子作为基因载体的研究

评价N,N,N-三甲基壳聚糖盐酸盐(TMC)及其纳米粒子对质粒DNA(pDNA)的负载及保护能力,考察了其纳米复合物对人类肝癌细胞株(HepG 2细胞)的转染能力。通过复凝聚法制备TMC/pDNA纳米粒子,并采用透射电镜及原子力显微镜表征粒子形态和粒径;采用凝胶阻滞分析观察其对pDNA的保护情况;采用四噻氮唑盐(MTT)比色法测定细胞毒性;以Lipofectamine 2000转染试剂作为阳性对照,检测其对HepG 2细胞的转染活性;采用荧光倒置显微镜观察转染情况。结果表明负载pDNA的TMC纳米粒子多呈球形,粒径为100～300nm,能有效地包裹和保护基因不被DNaseⅠ酶消化;当TMC与pDNA的质量比为10∶1时,在48h达到最高的转染效率。     

负载过氧化铜的介孔二氧化硅纳米粒子协同化学动力学疗法和化疗联合治疗肿瘤

化学动力学治疗(CDT)是通过使用芬顿催化剂将细胞内过氧化氢(H₂O₂)催化成羟基自由基(·OH)以杀死癌细胞的一种新型治疗方法。然而，内源性H₂O₂含量不足以及单一治疗的局限性限制了CDT的治疗效率。本工作报道了一种负载过氧化铜(CuO₂)的介孔二氧化硅纳米颗粒(MSN)。其中，MSN内部负载化疗药物阿霉素(DOX),其表面负载催化剂CuO₂封堵孔道，避免药物提前早释。在肿瘤微环境(TME)刺激下，CuO₂分解产生外源性H₂O₂和类芬顿离子Cu²⁺,Cu²⁺消耗细胞内谷胱甘肽(GSH)生成Cu⁺,Cu⁺催化外源性H₂O₂生成高细胞毒性的·OH,协同化疗药物DOX实现CDT和化疗的联合治疗，提高抗肿瘤疗效。此外，体外细胞实验研究表明，该芬顿催化剂表现出良好的细胞...     

基于主客体识别的自组装金粒子基因载体的构建与性能评价

利用金纳米粒子及环糊精-二茂铁主客体识别作用,制备了2种不同形状的H2O2响应性基因载体GP2(球形)、GR2(棒状),并与传统转染试剂Lipo2000及PEI25k进行了转染及毒性的系统比较。结果表明,2种形状的粒子在同等条件下的转染效率超过了Lipo2000及PEI25k,且棒状粒子的转染效率更高。同时,在可用于转染的范围内,2种粒子表现出与PEI10k相当的较低细胞毒性。     

生物活性玻璃纤维的基因转染研究

生物活性玻璃由于含有钙、磷成分, 因此在基因转染方面具有应用潜能。本研究通过溶胶-凝胶结合静电纺丝技术制备了具有连续介孔或分级纳米孔的微纳米生物活性玻璃纤维(BGF), 并将其用于基因转染。实验结果显示, BGF在无血清培养基中可大量释放Ca²⁺和PO₄^3-, 这些释放的离子在沉积的同时可装载质粒DNA, 起到基因转染载体的作用, 转染效率与BGF之间具有剂量依赖性。当质粒浓度为1 μg/mL、BGF浓度为1000 μg/mL时, 细胞的转染效率可达对照组(脂质体转染试剂)的50%以上。其转染机理与传统的磷酸钙基因转染类似, 而其较好的离子溶出保障了其使用的稳定性, 有望替代纳米磷酸钙转染体系用于基因传输。     

pH响应铜掺杂介孔硅纳米催化剂增强肿瘤化疗–化学动力学联合治疗的研究

化学动力学疗法(CDT)利用肿瘤细胞内源性H₂O₂与芬顿催化剂反应生成高毒性的羟基自由基(·OH),从而杀死肿瘤细胞,但内源性H₂O₂不足和纳米粒子转运效率较低导致抗癌效果不理想。本研究制备了一种分散性良好、尺寸较小的铜掺杂介孔二氧化硅(Cu-MSN),负载化疗药物阿霉素(DOX)和抗坏血酸盐(AA)后,表面经叶酸(FA)和二甲基马来酸酐(DMMA)改性的壳聚糖(FA-CS-DMMA)以及羧甲基壳聚糖(CMC)包裹,得到pH响应型靶向纳米催化剂FA-CS-DMMA/CMC@Cu-MSN@DOX/AA(缩写为FCDC@Cu-MSN@DA)。扫描电镜显示纳米粒子FCDC@Cu-MSN@DA粒径约为100nm。体外48h内Cu²⁺释放量可达80%,药物DOX释放达到57.3%。释放的AA经自氧化后产生H₂O₂,诱导Cu²⁺发生类芬顿反应,从而增强CDT。细胞实验证明, FCDC@Cu-MSN@DA联合化疗药物表现出优异...     

球形活性炭担载MnOx-CeO2和三聚氰胺的低温脱硝行为研究

以MnO_x-CeO₂为活性组分, 以三聚氰胺为还原剂, 制备了球形活性炭(SAC)担载MnO_x-CeO₂和三聚氰胺的复合催化剂, 并考察了该催化剂在低温下(120~180℃)对NO的选择性催化还原(SCR)反应行为。实验还利用扫描电子显微镜-能谱仪(SEM-EDS)、X射线衍射(XRD)和低温N₂吸附法等技术对催化剂进行了表征。结果表明, 当反应温度为180℃、空速为6000 h^-1、NO和O₂浓度分别为0.1%和8%时, 担载8% Mn(摩尔比Mn:Ce固定为1:1)和10%三聚氰胺的催化剂可在8.8 h内实现99%的NO_x转化率。煅烧温度高于400℃将促使MnO_x-CeO₂形成更大的晶体颗粒和更加规整的晶型结构, 从而降低其melamine-SCR活性。三聚氰胺担载量高于15%将造成催化剂比表面积和孔容的急剧减小, 最终导致其稳态NO_x转化率的下降。而金属氧化物担载量的增加和反应温度的升高都有利于Melamine-SCR反应, 且在较宽的NO和O₂浓度范围内, 催化剂的稳态NO_x转化率都能维持在99%左右。     

电纺纳米纤维诱导LDH生长促进CO2分离

先进的功能膜材料是实现高效膜分离的关键，要求兼顾选择性和渗透通量.层状双金属氢氧化物(LDH)表面存在丰富的-OH基团，对CO₂具有较高吸附选择性.利用晶种外延生长策略(SES),通过溶剂热合成在电纺纤维载体上诱导生长LDH,并用聚乙二醇二丙烯酸酯(PEGDA)在纤维间隙间原位光聚合得到致密的PEO/HPAN-LDH MMM,用于CO₂的高效分离.研究结果表明，沿纤维连续的低结晶LDH具有丰富的亲CO₂基团，提供连续亲和CO₂的传递通路.通过增加LDH生长次数，提高LDH的担载量.性能最佳的PEO/HPAN-LDH-2 MMM的CO₂渗透性能为132.1 Barrer, CO₂/N₂选择性高达99.4,相较于PEO/HPAN MMM,CO₂渗透性能提升46.8%,CO₂/N₂选择性提升25.8%.     

AgNO3真空浸渍制备杀菌银缓释椰壳活性炭研究

对椰壳活性炭在真空条件下浸渍硝酸银(AgNO₃),制备具有杀菌作用的银缓释椰壳活性炭(Ag/AC)。以大肠埃希杆菌(E.coli)为实验菌种考察Ag/AC的抑菌杀菌作用,采用扫描电子显微镜(SEM)、X射线衍射(XRD)、比表面积分析仪分别对Ag/AC表面形态、晶体结构、比表面积和孔结构进行分析。结果表明：银离子在AC表面被还原成粒径为200～800 nm的单质银;随着AgNO₃浸渍浓度的增加,银担载量上升、粒径增大,Ag/AC的比表面积、总孔容、平均孔径减小,杀菌性能与抗银流失性能提高。当AgNO₃溶液浸渍浓度为8.0 g·L^-1时,载银量最大(0.44%),杀菌性能最佳(99.26%);在水中振荡15 d,银流失率为30%,对比相同条件下常压浸渍得到Ag/AC银流失率为94.23%,真空浸渍制备的载银椰壳活性炭在保持高杀菌率的同时实现了银缓释。     

pH响应/H2O2自供型金属过氧化物在肿瘤化学动力学治疗中的应用

基于活性氧自由基的抗肿瘤疗法近些年来得到了人们的广泛关注,主要包含光动力疗法、声动力疗法以及化学动力学疗法。其中,化学动力学疗法无需借助外部能量(光能或超声)和氧气,主要依赖金属催化剂(Fe²⁺、Cu⁺等)与H₂O₂分子发生芬顿或类芬顿反应,即可产生高细胞毒性的羟基自由基(·OH)等强氧化性活性物种,该活性物种可破坏细胞脂质、蛋白质和DNA等生物大分子,引发细胞凋亡,从而达到肿瘤治疗的目的。相比应用于传统化学动力学疗法的纳米材料(Fe₃O₄、Cu₂O等),金属过氧化物材料具有在低pH下响应降解、自供H₂O₂等特点,在应用于肿瘤化学动力学疗法时展现出巨大的优势,逐渐得到了人们的重视。金属过氧化物材料可以在肿瘤病灶区弱酸微环境下生成H₂O₂与金属离子,依靠肿瘤病灶区H₂O₂水平的提高和金属离子过载,或者通过产...     

多粒径TiB2/Cu复合材料耐电弧侵蚀行为

采用粉末冶金工艺制备了不同配比的多粒径(2 μm+10 μm+50 μm) TiB₂/Cu复合材料。通过JF04C触点材料测试系统对多粒径TiB₂/Cu复合材料进行耐电弧侵蚀性能试验,研究(2 μm+10 μm+50 μm) TiB₂颗粒质量比分别为1∶1∶1、1∶1∶3、1∶3∶1、3∶1∶1时,TiB₂/Cu复合材料的耐电弧侵蚀性能及电弧侵蚀形貌变化规律,探究多粒径配比对TiB₂/Cu复合材料表层耐电弧侵蚀行为的影响。结果表明:当(2 μm+10 μm+50 μm) TiB₂颗粒质量比为1∶1∶1时,TiB₂/Cu复合材料相对密度和导电率最高,分别为99.1%和87.1%IACS。当(2 μm+10 μm+50 μm) TiB₂颗粒质量比为1∶1∶1和1∶3∶1时,TiB₂/Cu复合材料的组织均匀性较好,电弧侵蚀后材料损失相同,材料转移量最少。其中,质量比为1∶3∶1时,TiB₂/Cu复合材料平均燃弧能量最低,且燃弧时间和燃弧能量最稳定。研究表明,这与复合材料的综合物理性能密切相关。在颗粒增强Cu基复合材料设计过程中,引入合适配比的多粒径TiB₂颗粒有助于提高TiB₂/Cu复合材料的密度、导电率等综合物理性能。电弧侵蚀过程中,不同粒径的TiB₂颗粒相互协同作用,有助于提高TiB₂/Cu复合材料的耐电弧侵蚀性能和服役稳定性。      

Encapsulation of Plasmid DNA by Nanoscale Metal–Organic Frameworks for Efficient Gene Transportation and Expression

The intracellular delivery and functionalization of genetic molecules play critical roles in gene‐based theranostics. In particular, the delivery of plasmid DNA (pDNA) with safe nonviral vectors for efficient intracellular gene expression has received increasing attention; however, it still has some limitations. A facile one‐pot method is employed to encapsulate pDNA into zeolitic imidazole framework‐8 (ZIF‐8) and ZIF‐8‐polymer vectors via biomimetic mineralization and coprecipitation. The pDNA molecules are found to be well distributed inside both nanostructures and benefit from their protection against enzymatic degradation. Moreover, through the use of a polyethyleneimine (PEI) 25 kD capping agent, the nanostructures exhibit enhanced loading capacity, better pH responsive release, and stronger binding affinity to pDNA. From in vitro experiments, the cellular uptake and endosomal escape of the protected pDNA are greatly improved with the superior ZIF‐8‐PEI 25 kD vector, leading to successful gene expression with high transfection efficacy, comparable to expensive commercial agents. New cost‐effective avenues to develop metal–organic‐framework‐based nonviral vectors for efficient gene delivery and expression are provided.     

Quaternized chitosan/rectorite intercalative materials for a gene delivery system

Non-viral vectors have gained increasing attention in gene therapy because of their safety, but with the shortcoming of low transfection efficiency. We have developed a hybrid material as a novel non-viral vector, which combines the advantages of both biopolymer and clay in a gene delivery system. Quaternized chitosan was intercalated into the interlayers of rectorite to obtain a new polymer/layered silicate nanocomposite. In vitro and in vivo toxicity studies revealed that the nanocomposites were biocompatible and non-toxic. At the nanocomposite:pDNA mass ratio of 8:1, they achieved 100% pDNA adsorption capacity. In vitro cell transfection revealed a transfection efficiency of 32.1% at 96?h as shown by a flow-cytometric study, and the intensive green fluorescence protein (GFP) expression could last for up to 120?h. Furthermore, an in vivo transfection study showed that the most prominent GFP expression was observed in the gastric and duodenum mucosa, and good transfection efficiency was also obtained when injected into the muscle. All the results suggest that quaternized chitosan/rectorite nanocomposite is a novel and potential non-viral gene carrier.     

Insight into the role of N,N-dimethylaminoethyl methacrylate (DMAEMA) conjugation onto poly(ethylenimine): cell viability and gene transfection studies

In the present study, the effect of N,N-dimethylaminoethyl methacrylate (DMAEMA) conjugation onto branched poly(ethylenimine) (PEI) with different grafting degree was examined for gene delivery applications. The DMAEMA-grafted-PEI conjugates were characterized and complexed with plasmid DNA (pDNA) at various concentrations, and the physicochemical properties, cell viability, and in vitro transfection efficiency of the complexes were evaluated in HEK 293T cells. Computational techniques were used to analyze the interaction energies and possible binding modes between DNA and conjugates at different grafting degrees. The cytotoxicity analysis and in vitro transfection efficiency of the conjugate/pDNA complexes exhibited a beneficial effect of DMAEMA conjugation when compared to PEI alone. The computational results revealed that the DNA/vector interaction energy decreases with increasing grafting degree, which can be associated to an enhanced release of the pDNA from the carrier once inside cells. The results indicate the significance of DMAEMA conjugation onto PEI as a promising approach for gene delivery applications.     

Novel redox nanomedicine improves gene expression of polyion complex vector

Abstract

Gene therapy has generated worldwide attention as a new medical technology. While non-viral gene vectors are promising candidates as gene carriers, they have several issues such as toxicity and low transfection efficiency. We have hypothesized that the generation of reactive oxygen species (ROS) affects gene expression in polyplex supported gene delivery systems. The effect of ROS on the gene expression of polyplex was evaluated using a nitroxide radical-containing nanoparticle (RNP) as an ROS scavenger. When polyethyleneimine (PEI)/pGL3 or PEI alone was added to the HeLa cells, ROS levels increased significantly. In contrast, when (PEI)/pGL3 or PEI was added with RNP, the ROS levels were suppressed. The luciferase expression was increased by the treatment with RNP in a dose-dependent manner and the cellular uptake of pDNA was also increased. Inflammatory cytokines play an important role in ROS generation in vivo. In particular, tumor necrosis factor (TNF)-α caused intracellular ROS generation in HeLa cells and decreased gene expression. RNP treatment suppressed ROS production even in the presence of TNF-α and increased gene expression. This anti-inflammatory property of RNP suggests that it may be used as an effective adjuvant for non-viral gene delivery systems.     

A Highly Emissive Conjugated Polyelectrolyte Vector for Gene Delivery and Transfection

An intrinsically fluorescent cationic polyfluorene ( CCP ) has been designed, synthesized, characterized, and examined as a plasmid DNA (pDNA) delivery vector. This material facilitates nucleic acid binding, encapsulation and efficient cellular uptake. CCP can effectively protect pDNA against nuclease degradation, which is necessary for gene carriers. Green fluorescent protein (GFP) expression experiments reveal that CCP can achieve efficient delivery and transfection of pDNA encoding GFP gene with 92% efficiency, which surpasses that of commercial transfection agents, lipofectamine 2000 (Lipo) and polyethylenimine (PEI). CCP is also highly fluorescent, with 43% quantum yield in water, and exhibits excellent photostability, which allows for real‐time tracking the location of gene delivery and transfection. These features and capabilities represent a major step toward designing and applying conjugated polymers that function in both imaging and therapeutic applications.     

Silicalite nanoparticles that promote transgene expression

Here, we report on a new zeolite-based silicalite nanoparticle that can enhance the transfection efficiencies generated by poly ethylene imine-plasmid DNA (PEI-pDNA) complexes via a sedimentation mechanism and can enhance the transfection efficiencies of pDNA alone when surface functionalized with amine groups. The silicalite nanoparticles have a mean size of 55?nm. Functionalizing the silicalite nanoparticles with amine groups results in a clear transition in zeta potential from -25.9 ± 2.3?mV (pH 7.4) for unfunctionalized silicalite nanoparticles to 4.9 ± 0.7?mV (pH 7.4) for amine functionalized silicalite nanoparticles. We identify that silicalite nanoparticles used to promote non-viral vector acceleration to the cell surface are found in acidic vesicles or the cytoplasm but not the nucleus. An MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay showed that the silicalite nanoparticles were non-toxic at the concentrations tested for transfection. We show that surface functionalization of silicalite nanoparticles with amine groups results in a significant (230%) increase in transfection efficiency of pDNA when compared to unfunctionalized silicalite nanoparticles. Silicalite nanoparticles enhanced pDNA-PEI induced transfection of human embryonic kidney (HEK-293) cells by over 150%.     

Polymeric micelles comprising stearic acid-grafted polyethyleneimine as nonviral gene carriers

Non-viral vectors composed of biodegradable polymers or lipids have been considered as a safer alternative for gene carriers over viral vectors. Among some of the cationic polymers, polyethyleneimine (PEI) possess high pH-buffering capacity that can provide protection to nucleotides from acidic degradation and promotes endosomal and lysosomal release. However, it has been reported that cytotoxicity of PEI depends on the molecular weight of the polymer. Hence modifications of PEI structure for clinical application have been developed in order to reduce the cytotoxicity, or improve the insufficient transfection efficiency of lower molecular weight PEI. In this study, 10 k PEI was modified by grafting stearic acid (SA) and formulated to polymer micelles with positive surface charge and evaluated for pDNA delivery. The amine group on PEI was crosslinked with the carboxylic group of stearic acid by 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC) as linker. PEI-SA micelles were then prepared using oil in water (o/w) solvent evaporation method. The success of PEI-SA conjugation structure was confirmed with 1H NMR. The average diameter and zeta potential determined by photon correlation spectroscopy was 149.6 +/- 1.2 nm and 64.1 +/- 1.5 mV, respectively. These self-assemble positive charge micelles showed effective binding to pDNA for transfection. PEI-SA micelles exhibited lower cytotoxicity compared to that of PEI only, while flow cytometry analysis revealed PEI-SA/pEGFP complex provided 62% high EGFP expression. Luciferase activity also showed high transfection efficiency of PEI-SA micelles for weight ratio above 4.5 that was comparable to PEI only. These results demonstrated that stearic acid grafted PEI micelles can provide high transfection efficiency comparable to unmodified PEI, and exhibit low cytotoxicity. Stearic acid grafted PEI micelles can be promising polymer carriers in genetic therapy.     

Transferrin-mediated PEGylated nanoparticles for delivery of DNA/PLL

The purpose of this work was to determine the stability of pDNA/poly(L-lysine) complex (DNA/PLL) during microencapsulation, prepare transferrin (TF) conjugated PEGylated nanoparticles (TF-PEG-NP) loading DNA/PLL, and assess its physicochemical characteristics and in vitro transfection efficiency. The DNA/PLL was prepared by mixing plasmid DNA (pDNA) in deionized water with various amounts of PLL. PEGylated nanoparticles (PEG-NP) loading DNA/PLL were prepared by a water-oil-water double emulsion solvent evaporation technique. TF-PEG-NP was prepared by coupling TF with PEG-NP. The physicochemical characteristics of TF-PEG-NP and in vitro transfection efficiency on K562 cells were measured. The results showed that free pDNA reserved its double supercoiled form (dsDNA) for only on average 25.7% after sonification, but over 70% of dsDNA was reserved after pDNA was contracted with PLL. The particle size range of TF-PEG-NP loading DNA/PLL was 150-450?nm with entrapment efficiency over 70%. TF-PEG-NP exhibited the low burst effect (<10%) within 1 day. After the first phase, DNA/PLL displayed a sustained release. The amount of cumulated DNA/PLL release from TF-PEG-NP with 2% polymer over 7 days was 85.4% for DNA/PLL (1:0.3 mass ratio), 59.8% and 43.1% for DNA/PLL (1:0.6) and DNA/PLL (1:1.0), respectively. To TF-PEG-NP loading DNA/PLL without chloroquine, the percentage of EGFP expressing cells was 28.9% for complexes consisting of DNA/PLL (1:0.3), 38.5% and 39.7% for DNA/PLL (1:0.6) and DNA/PLL (1:1.0), respectively. In TF-PEG-NP loading DNA/PLL with chloroquine, more cells were transfected, the percentage of positive cells was 37.6% (DNA/PLL, 1:0.3), 47.1% (DNA/PLL, 1:0.6) and 45.8% (DNA/PLL, 1:1.0), which meant that the transfection efficiency of pDNA was increased by over 50 times when PLL and TF-PEG-NP were jointly used as a plasmid DNA carrier, in particular, the maximal percentage of positive cells (47.1%) from TF-PEG-NP loading DNA/PLL (1:0.6) was about 70 times the transfection efficiency of free plasmid DNA. The average cell viability of TF-PEG-NP loading DNA/PLL was about 90%, which meant that TF-PEG-NP appeared to be safer than PLL alone. As a result, TF-PEG-NP loading DNA/PLL could be a more effective non-viral vector for the delivery of pDNA.     

Polyethylenimine functionalized magnetic nanoparticles as a potential non-viral vector for gene delivery

Polyethylenimine (PEI) functionalized magnetic nanoparticles were synthesized as a potential non-viral vector for gene delivery. The nanoparticles could provide the magnetic-targeting, and the cationic polymer PEI could condense DNA and avoid in vitro barriers. The magnetic nanoparticles were characterized by Fourier transform infrared spectroscopy, X-ray powder diffraction, dynamic light scattering measurements, transmission electron microscopy, vibrating sample magnetometer and atomic force microscopy. Agarose gel electrophoresis was used to asses DNA binding and perform a DNase I protection assay. The Alamar blue assay was used to evaluate negative effects on the metabolic activity of cells incubated with PEI modified magnetic nanoparticles and their complexes with DNA both in the presence or absence of an external magnetic field. Flow cytometry and fluorescent microscopy were also performed to investigate the transfection efficiency of the DNA-loaded magnetic nanoparticles in A549 and B16-F10 tumor cells with (+M) or without (?M) the magnetic field. The in vitro transfection efficiency of magnetic nanoparticles was improved obviously in a permanent magnetic field. Therefore, the magnetic nanoparticles show considerable potential as nanocarriers for gene delivery.     

Cholesterol tethered bioresponsive polycation as a candidate for gene delivery

The efficient unpacking of viral protein shell gave the inspiration for the synthesized vectors. In this research, novel cholesterol tethered bioresponsive polyethylenimine (PEI) was specially designed via disulfide-containing cross-linker. The cholesterol lipid had proved to increase the permeability of gene vector through cell membrane. The acid–base titration indicated that the synthesized polycation possessed efficient proton sponge effect, which was suggested to increase endosomal release of pDNA complexes into the cytoplasm. The cholesterol tethered polycation could effectively induce DNA condensation and form spherical particles with diameter about 200 nm at N/P ratio of 10. At glutathione concentration of 3 mM, the polyplexes were unpacked due to the bioresponsive cleavage of the disulfide bonds. The in-vitro experiment indicated that the polyplexes showed efficient transfection efficiency to HEK293T cells. All the results indicated that the bioresponsive polycation could be served as an effective trigger to control the release of DNA at the intracellular environment. The novel bioresponsive polycation might have great potential in non-viral gene delivery research and application.