The Antibacterial Effect of PEGylated Carbosilane Dendrimers on P. aeruginosa Alone and in Combination with Phage-Derived Endolysin

The search for new microbicide compounds is of an urgent need, especially against difficult-to-eradicate biofilm-forming bacteria. One attractive option is the application of cationic multivalent dendrimers as antibacterials and also as carriers of active molecules. These compounds require an adequate hydrophilic/hydrophobic structural balance to maximize the effect. Herein, we evaluated the antimicrobial activity of cationic carbosilane (CBS) dendrimers unmodified or modified with polyethylene glycol (PEG) units, against planktonic and biofilm-forming P. aeruginosa culture. Our study revealed that the presence of PEG destabilized the hydrophilic/hydrophobic balance but reduced the antibacterial activity measured by microbiological cultivation methods, laser interferometry and fluorescence microscopy. On the other hand, the activity can be improved by the combination of the CBS dendrimers with endolysin, a bacteriophage-encoded peptidoglycan hydrolase. This enzyme applied in the absence of the cationic CBS dendrimers is ineffective against Gram-negative bacteria because of the protective outer membrane shield. However, the endolysin—CBS dendrimer mixture enables the penetration through the membrane and then deterioration of the peptidoglycan layer, providing a synergic antimicrobial effect.     

Immobilization of Alcalase on Silica Supports Modified with Carbosilane and PAMAM Dendrimers

Enzyme immobilization is a powerful strategy for enzyme stabilization and recyclability. Materials covered with multipoint molecules are very attractive for this goal, since the number of active moieties to attach the enzyme increases with respect to monofunctional linkers. This work evaluates different dendrimers supported on silica to immobilize a protease enzyme, Alcalase. Five different dendrimers were employed: two carbosilane (CBS) dendrimers of different generations (SiO₂-G₀Si-NH₂ and SiO₂-G₁Si-NH₂), a CBS dendrimer with a polyphenoxo core (SiO₂-G₁O₃-NH₂), and two commercial polyamidoamine (PAMAM) dendrimers of different generations (SiO₂-G₀PAMAM-NH₂ and SiO₂-G₁PAMAM-NH₂). The results were compared with a silica support modified with a monofunctional molecule (2-aminoethanethiol). The effect of the dendrimer generation, the immobilization conditions (immobilization time, Alcalase/SiO₂ ratio, and presence of Ca²⁺ ions), and the digestion conditions (temperature, time, amount of support, and stirring speed) on Alcalase activity has been evaluated. Enzyme immobilization and its activity were highly affected by the kind of dendrimer and its generation, observing the most favorable behavior with SiO₂-G₀PAMAM-NH₂. The enzyme immobilized on this support was used in two consecutive digestions and, unlike CBS supports, it did not retain peptides released in the digestion.     

Silver Nanoparticles Modified by Carbosilane Dendrons and PEG as Delivery Vectors of Small Interfering RNA

The fact that cancer is one of the leading causes of death requires researchers to create new systems of effective treatment for malignant tumors. One promising area is genetic therapy that uses small interfering RNA (siRNA). These molecules are capable of blocking mutant proteins in cells, but require specific systems that will deliver RNA to target cells and successfully release them into the cytoplasm. Dendronized and PEGylated silver nanoparticles as potential vectors for proapoptotic siRNA (siMCL-1) were used here. Using the methods of one-dimensional gel electrophoresis, the zeta potential, dynamic light scattering, and circular dichroism, stable siRNA and AgNP complexes were obtained. Data gathered using multicolor flow cytometry showed that AgNPs are able to deliver (up to 90%) siRNAs efficiently to some types of tumor cells, depending on the degree of PEGylation. Analysis of cell death showed that complexes of some AgNP variations with siMCL-1 lead to ~70% cell death in the populations that uptake these complexes due to apoptosis.     

Modeling the Interaction between Dendrimers and Nucleic Acids: a Molecular Perspective through Hierarchical Scales

Cationic dendrimers are promising nanocarriers for gene delivery thanks to their ability to establish strong interactions with oppositely charged strands of DNA and siRNA and to promote their aggregation. The binding between dendrimers and nucleic acids is typically a complex process that involves various types of interactions at different scales. To design efficient dendrimer candidates for DNA and siRNA binding it is necessary to have a detailed understanding of their interactions with oligonucleotides in the solvent. Molecular simulation can support experimental work, providing a privileged point of view on the aggregation process. This Minireview discusses recent computational efforts to unravel dendrimer–oligonucleotide binding, and proposes a perspective of the multiscale aggregation process based on hierarchy and on the transformations of the interacting “molecular units” following intermolecular interactions.     

PAMAM Dendrimers Mediate siRNA Delivery to Target Hsp27 and Produce Potent Antiproliferative Effects on Prostate Cancer Cells

RNA interference (RNAi) holds great promise for the treatment of inherited and acquired diseases, provided that safe and efficient delivery systems are available. Herein we report that structurally flexible triethanolamine (TEA) core PAMAM dendrimers are able to deliver an Hsp27 siRNA effectively into prostate cancer (PC‐3) cells by forming stable nanoparticles with siRNA, protecting the siRNA nanoparticles from enzymatic degradation, and enhancing cellular uptake of siRNA. The Hsp27 siRNA resulted in potent and specific gene silencing of heat‐shock protein 27, an attractive therapeutic target in castrate‐resistant prostate cancer. Silencing of the hsp27 gene led to induction of caspase‐3/7‐dependent apoptosis and inhibition of PC‐3 cell growth in vitro. In addition, the siRNA–dendrimer complexes are non‐cytotoxic under the conditions used for siRNA delivery. Altogether, TEA core PAMAM dendrimer‐mediated siRNA delivery, in combination with RNAi that specifically targets Hsp27, may constitute a promising approach for combating castrate‐resistant prostate cancer, for which there is no efficacious treatment.     

Polymeric siRNA delivery vectors: knocking down cancers with polymeric‐based gene delivery systems

One of the most promising routes for cancer therapy that has evolved over the previous decade is the use of small‐interfering RNA (siRNA) as a means of switching off genes that are responsible for tumour development. However, while siRNA and gene/antisense therapies provide alternatives to conventional chemotherapies, significant hurdles related to the delivery and efficacy of treatment must still be overcome before this technology can be used as an effective treatment for cancer and other diseases. This review highlights the issues associated with siRNA therapy in vivo, and describes the various approaches that are being explored using polymers as delivery vectors. In particular, the review focuses on targeted delivery as a means of improving efficacy of the gene therapy. © 2014 Society of Chemical Industry     

RNA干扰技术对MCF-7细胞Aurora-A基因表达的影响

目的构建针对Aurora-A基因的特异性小干扰RNA(siRNA)真核表达载体,并探讨其对人乳腺癌细胞株MCF-7中Aurora-A基因表达的抑制作用。方法将具有短发夹结构的2条DNA序列,经退火形成互补双链,再克隆至载体pGCsi-U6/Neo/GFP中,转化大肠杆菌DH5α,提取质粒进行序列测定。并通过脂质体法转染至MCF-7细胞中,48h后观察转染效率,并采用RT-PCR及Westernblot法检测siRNA对MCF-7细胞Aurora-A基因mRNA及蛋白表达的影响。结果测序鉴定证实目的寡核苷酸片段已被克隆至pGCsi-U6/Neo/GFP载体中,Aurora-A-siRNA质粒转染MCF-7细胞48h后转染效率约为60%,与对照组比较,Aurora-A-siRNA质粒转染的细胞Aurora-A基因mRNA和蛋白的表达均明显下降。结论已成功构建了Aurora-A-siRNA真核表达质粒,其能明显抑制MCF-7细胞Aurora-A基因的表达,为进一步研究Aurora-A基因的功能奠定了基础,并可能为肿瘤的生物学治疗提供新的方法。     

树形大分子的研究进展及现状

本文介绍了一类高度支化，带有很多末端官能团的新型特种结构大分子－树形大分子，主要介绍了树形大分子的研究进展及现状。     

Efficient Transfection of siRNA by Peptide Dendrimer–Lipid Conjugates

Efficient delivery of small interfering RNA (siRNA) into cells is the basis of target‐gene‐specific silencing and, ultimately, gene therapy. However, current transfection reagents are relatively inefficient, and very few studies provide the sort of systematic understanding based on structure–activity relationships that would provide rationales for their improvement. This work established peptide dendrimers (administered with cationic lipids) as siRNA transfection reagents and recorded structure–activity relationships that highlighted the importance of positive charge distribution in the two outer layers and a hydrophobic core as key features for efficient performance. These dendrimer‐based transfection reagents work as well as highly optimised commercial reagents, yet show less toxicity and fewer off‐target effects. Additionally, the degrees of freedom in the synthetic procedure will allow the placing of decisive recognition features to enhance and fine‐tune transfection and cell specificity in the future.     

TRIS封端树枝状大分子PAMAM的合成

采用发散法合成了以乙二胺为核的树枝状大分子聚酰胺-胺(PAMAM),以端基为酯基的半代PAMAM和三羟甲基氨基甲烷(TRIS)为原料,无水碳酸钾为催化剂,分别以二甲基亚砜和甲醇为溶剂,合成了端基被修饰的水溶性大分子PAMAM-OH和PAMAM-K,产率分别为80.3%和74.1%.采用红外光谱对产品结构进行了表征,同时对其水溶性和表面活性进行了初步的研究,结果表明:PAMAM被水溶性基团修饰后,失去了表面活性,却增强了水溶性.     

以聚酰胺-胺树形分子为模板制备AgI纳米簇

本文以聚酰胺-胺(PAMAM)树形分子为模板,原位制备AgI纳米簇.系统地研究了AgI纳米簇制备过程中各种反应条件如树形分子端基、反应时间、Ag 与PAMAM摩尔比等对AgI纳米簇粒径的影响,分别用紫外-可见光谱、荧光光谱、透射电镜等对所制备的纳米簇进行表征.在相同的条件下,以G4.5-COOH3为模板较以G5.0-NH2为模板制备的AgI纳米簇粒径小、分布均匀,这主要取决于G4.5-COOCH3PAMAM树形分子所起的“内模板”作用.G4·5-COOH3树形分子浓度为1×10-5mol/L,Ag 与树形分子摩尔比为30∶1时所制备的AgI纳米簇的粒径分布均匀、稳定性好,室温避光可稳定存在两个月以上.     

两性聚酰胺-胺树状聚合物的合成及絮凝性能

用Michael加成反应对G4.0聚酰胺-胺(PAMAM)树状聚合物分子进行了表面修饰改性。分别以丙烯酰氧基乙基三甲基氯化铵(DAC)、丙烯酸钠(SAA)、2-丙烯酰胺-2-甲基丙磺酸(AMPS)为端基改性剂,在N2气氛下50℃甲醇溶液中,反应投料比n(PAMAM)∶n(DAC)∶n(SAA或AMPS)=1∶16∶16时,反应96 h,合成出了两性聚酰胺-胺树状聚合物。絮凝性能实验结果表明,合成产物具有一定的絮凝性能,且与聚丙烯酰胺复配效果更好,最佳絮凝效果条件为:m(G4.0/DAC/AMPS)∶m(PAM)=1∶4;投药量16 mg/L;絮凝沉降时间20 min;pH=8~9。     

树形聚合物的合成和结构及应用研究进展

树形聚合物为纳米级单分散性大分子 ,它独特的核 -壳结构、表面端基的高密度及高反应活性 ,使其易于组装或修饰为功能化树形聚合物 ,用于催化体系、细胞识别及基因治疗、光学分子开关及光获体系等 ,而成为引人注目的 2 1世纪新材料     

A QCM‐D and SAXS Study of the Interaction of Functionalised Lyotropic Liquid Crystalline Lipid Nanoparticles with siRNA

Biophysical studies were undertaken to investigate the binding and release of short interfering ribonucleic acid (siRNA) from lyotropic liquid crystalline lipid nanoparticles (LNPs) by using a quartz crystal microbalance (QCM). These carriers are based on phytantriol (Phy) and the cationic lipid DOTAP (1,2‐dioleoyloxy‐3‐(trimethylammonium)propane). The nonlamellar phase LNPs were tethered to the surface of the QCM chip for analysis based on biotin–neutravidin binding, which enabled the controlled deposition of siRNA–LNP complexes with different lipid/siRNA charge ratios on a QCM‐D crystal sensor. The binding and release of biomolecules such as siRNA from LNPs was demonstrated to be reliably characterised by this technique. Essential physicochemical parameters of the cationic LNP/siRNA lipoplexes—such as particle size, lyotropic phase behaviour, cytotoxicity, gene silencing and uptake efficiency—were also assessed. The SAXS data show that when the pH was lowered to 5.5 the structure of the lipoplexes did not change, thus indicating that the acidic conditions of the endosome were not a significant factor in the release of siRNA from the cationic lipidic carriers.     

人CD147基因siRNA真核表达质粒的构建及其对HeLa细胞中CD147基因表达的影响

目的构建针对人CD147基因的siRNA真核表达质粒,并观察其对HeLa细胞中CD147基因表达的影响。方法根据GenBank中登录的人CD147基因序列,设计并合成针对CD147基因的特异性小干扰片段,并将其定向克隆至带有卡那霉素抗性和增强型绿色荧光蛋白的真核表达载体pGenesil-1中,对重组质粒进行酶切分析和DNA序列测定。用脂质体将重组质粒转染至HeLa细胞中,观察其对HeLa细胞CD147基因mRNA及蛋白表达水平的影响。结果酶切鉴定和测序结果表明,3个表达短发夹RNA的质粒及其阴性对照质粒构建正确。3个siRNA真核表达质粒对HeLa细胞CD147基因mRNA转录水平的抑制率分别为32.5%、66.8%和59.1%;对CD147蛋白表达水平的抑制率分别为28.3%、63.5%和56.2%。结论已成功构建针对人CD147基因的siRNA真核表达质粒,其对HeLa细胞CD147基因的表达具有明显的抑制作用,为进一步研究CD147基因的功能奠定了基础。     

Microfluidic-Based Cationic Cholesterol Lipid siRNA Delivery Nanosystem: Highly Efficient In Vitro Gene Silencing and the Intracellular Behavior

Safe and efficient delivery of small interfering RNA (siRNA) is essential to gene therapy towards intervention of genetic diseases. Herein, we developed a novel cationic cholesterol lipid derivative (CEL) in which cholesterol hydrophobic skeleton was connected to L-lysine cationic headgroup via a hexanediol linker as the non-viral siRNA delivery carrier. Well-organized CEL/siRNA nanocomplexes (100–200 nm) were prepared by microfluidic-assisted assembly of CEL and siRNA at various N/P ratios. The CEL and CEL/siRNA nanocomplexes have lower cytotoxicity compared with bPEI25k. Delightfully, we disclosed that, in Hela–Luc and H1299–Luc cell lines, the micro-fluidic-based CEL/siRNA nanocomplexes exhibited high siRNA transfection efficiency under both serum-free condition (74–98%) and low-serum circumstances (80–87%), higher than that of lipofectamine 2000. These nanocomplexes also showed high cellular uptake through the caveolae/lipid-raft mediated endocytosis pathway, which may greatly contribute to transfection efficiency. Moreover, the time-dependent (0–12 h) dynamic intracellular imaging demonstrated the efficient delivery to cytoplasm after lysosomal co-localization. The results indicated that the microfluidic-based CEL/siRNA nanosystems possessed good stability, low cytotoxicity, high siRNA delivery efficiency, rapid cellular uptake and caveolae/lipid raft-dependent internalization. Additionally, this study provides a simple approach for preparing and applying a “helper lipid-free” cationic lipid siRNA delivery system as potential nanotherapeutics towards gene silencing treatment of (tumor) diseases.     

端羧基PAMAM树枝状化合物的合成及其阻垢性能

以丙烯酸甲酯和乙二胺为原料,采用发散法合成了外围分别为4个、8个和16个—COOCH3的树枝状化合物(简记为MCMD4、MCMD8和MCMD16),在碱性条件下水解衍生为外围分别是4个、8个和16个的端羧基树枝状化合物(简记为MCD4、MCD8和MCD16)。水解衍生物用于模拟循环冷却水阻碳酸钙垢实验,结果表明,MCD4、MCD8和MCD16均具有良好的阻垢性能,MCD16、MCD8阻垢性能优于MCD4,在药剂用量为10 mg/L时,MCD4及MCD8的阻垢率分别为90%和96%。通过红外光谱及扫描电镜分析手段研究了该阻垢剂对碳酸钙垢晶形的影响,进一步探讨了该端羧基树枝状化合物的阻垢机理。     

Lactose‐Functionalized Dendrimers Arbitrate the Interaction of Galectin‐3/MUC1 Mediated Cancer Cellular Aggregation

By using lactose‐functionalized poly(amidoamine) dendrimers as a tunable multivalent platform, we studied cancer cell aggregation in three different cell lines (A549, DU‐145, and HT‐1080) with galectin‐3. We found that small lactose‐functionalized G(2)‐dendrimer 1 inhibited galectin‐3‐induced aggregation of the cancer cells. In contrast, dendrimer 4 (a larger, generation 6 dendrimer with 100 carbohydrate end groups) caused cancer cells to aggregate through a galectin‐3 pathway. This study indicates that inhibition of cellular aggregation occurred because 1 provided competitive binding sites for galectin‐3 (compared to its putative cancer cell ligand, TF‐antigen on MUC1). Dendrimer 4 , in contrast, provided an excess of ligands for galectin‐3 binding; this caused crosslinking and aggregation of cells to be increased.