反义寡核苷酸-聚乙烯亚胺纳米微粒逆转耐甲氧西林金黄色葡萄球菌耐药性的研究

目的：以聚乙烯亚胺（polyethyleneimine,PEI）为载体,将硫代寡聚脱氧核苷酸（PS-ODNs）转染到耐甲氧西林金黄色葡萄球菌（methicillin resistant staphylococcus aureus,MRSA）体内,通过药效学观察PS-ODNs逆转MRSA对β-内酰胺类抗生素的耐药性。方法：制备PEI与PS-ODNs结合的纳米微粒（PEI-ODNs纳米微粒）;PEI-ODNs纳米微粒的粒径分析及结合率的测定;平板克隆形成实验计数菌落数（CFU）;微量法测定细菌生长曲线;液体稀释法测定细菌的最小抑菌浓度（MIC）。结果：PEI-ODNs纳米微粒的粒径为（85±22）nm,PEI与PS-ODNs的结合率最高为（97.3±1.1）%。含苯唑西林（6mg/L）的M-H琼脂板上,30μg/mL的PEI-ODNs纳米微粒组MRSA的菌落数为6.4×10^8/mL,而空白对照组的菌落数为3.3×10^9/mL,二者相比给药组菌落数明显减少,差异具有统计学意义（P〈0.01）,而其他对照组与空白对照组比较差异无统计学意义。实验结果显示PEI-ODNs纳米微粒组的苯唑西林对MRSA生长有抑制作用,30μg/mL的PEI-ODNs纳米微粒可将苯唑西林对MRSA最小抑菌浓度由1024μg/mL降低至16μg/mL。结论：PEI与PS-ODNs的结合率很高且粒径较小,PS-ODNs可以部分逆转MRSA对β-内酰胺类抗生素的耐药性。药效学结果显示PEI可以有效地将反义寡核苷酸转入MRSA体内,可考虑将其作为反义寡核苷酸进入细菌的载体。     

A simplified approach for efficient isolation of functional microglial cells: Application for modeling neuroinflammatory responses in vitro

Purified microglial cells in culture are frequently used to model brain inflammatory responses but obtaining large yields of these cells on a routine basis can be quite challenging. Here, we demonstrate that it is possible to achieve high‐yield isolation of pure microglial (MAC‐1⁺/Fcrls⁺/Ccr2^‐) cells from postnatal brain tissue through a simple culture procedure that mainly relies on the adhesion preference of these cells to the polycation polyethyleneimine (PEI) in serum‐supplemented DMEM medium. Accordingly, other synthetic or biological substrates failed to mimic PEI effects under the same culture conditions. Replacement of DMEM by DMEM/F12 nutrient mixture did not permit microglial cell isolation on PEI coating, indicating that PEI effects were context‐dependent. Remarkably, the lack of culture feeding during progression of microglial cell isolation strongly improved cell yield, suggesting that nutritional deprivation was required to optimize this process. When generated in large culture flasks coated with PEI, cultures of microglial cells were easily recovered by trypsin proteolysis to produce subcultures for functional studies. These cultures responded to lipopolysaccharide (LPS, 1–10 ng/ml) treatment by secreting pro‐inflammatory cytokines such as TNF‐α, IL‐6, IL‐1β and by generating nitric oxide and reactive oxygen species. Most interestingly, this response was curtailed by appropriate reference drugs. Microglial cells were also strongly responsive to the mitogenic cytokine GM‐CSF, which confirms that the functional repertoire of these cells was well preserved. Because of its high yield and simplicity, we believe that the present method will prove to be especially convenient for mechanistic studies or screening assays. GLIA 2016;64:1912–1924     

Poly(ester amine) constructed from polyethylenimine and pluronic for gene delivery in vitro and in vivo

A series of poly (ester amines) (PEAs) constructed from low molecular weight polyethyleneimine (LPEI, Mw: 0.8k, 1.2k Da) and Pluronic (different molecular weight (Mw) and hydrophilic-lipophilic-balance (HLB)) components were synthesized, and evaluated in vitro and in vivo as gene delivery carriers. Most PEA polymers were able to bind and condense plasmid DNA effectively into particles of approximately 150?nm in solution at the polymer/DNA ratio of 5 and above. Transfection efficiency of the PEA polymers depends on particle size of the polymer/DNA complex, molecular weight and HLB of the Pluronics and the size of PEI within PEA composition, as well as the cell type. Significant improvement in gene delivery efficacy was achieved with PEA01/04/05 composed of Pluronic size (Mw: 3000–5000?Da), and HLB (12–18) in CHO, C2C12 and HSkM cell lines; and the effective transfection was reflected with PEA 01/04/07 composed of Pluronics with size (2000–5000?Da) and HLB (12–23) in mdx mice. The best formulation for pDNA delivery was obtained with PEA 01 producing transgene expression efficiency 5, 19-folds of that of PEI 25k in vitro and in vivo, respectively. These results potent some of these PEA polymers as attractive vehicles for gene or oligonucleotide delivery.     

叶酸-聚乙烯亚胺复合碳点的跨膜机制和胞内分布

目的：探讨以叶酸和聚乙烯亚胺（PEI）为原料合成的荧光碳点跨MC3T3-E1细胞膜转运途径、胞内分布其对细胞的影响,并阐明其机制。方法：以叶酸和PEI为原料,通过水热法合成具有细胞成像功能的荧光碳点,采用MTT法筛选碳点的最佳使用浓度。将MC3T3-E1细胞分为空白对照组、叶酸组和碳点组,通过细胞周期、细胞凋亡和细胞活性氧（ROS）水平检测评估碳点的生物相容性;通过胞膜窖途径抑制剂制霉菌素、巨胞饮途径抑制剂诺考达唑的使用,探讨细胞摄取碳点的途径;利用碳点在紫外光激发下发射蓝色荧光的特点,通过各种细胞器探针进行荧光共定位以观察碳点在细胞内的分布。结果：与空白对照组比较,24 h时100~450mg·L^-1碳点组细胞增殖率明显升高（P<0.05）;在48h时,当碳点浓度达到350 mg·L^-1时,细胞增殖率仅有空白对照组的68.4%（P<0.05）。与空白对照组比较,碳点组G₀期和G₁期细胞比例明显下降（P<0.05）,S期细胞比例明显升高（P<0.05）;G₂期和M期细胞比例亦升高,但差异无统计学意义（P>0.05）;与空白对照组比较,叶酸组和碳点组细胞凋亡率无明显改变（P>0.05）,但细胞内ROS水平明显降低（P<0.05）。与空白对照组比较,制霉菌素组细胞摄取碳点量减少（P<0.05）。倒置荧光显微镜观察,碳点的蓝色荧光与线粒体的红色荧光较好重叠,与溶酶体的红色荧光较好重叠,与内质网的红色荧光不完全重叠,与高尔基体的红色荧光重叠得较差。结论：碳点生物相容性较好,被细胞摄取后可以分布至细胞质的主要细胞器上,可作为一种非病毒载体应用到转基因治疗中。     

Development and evaluation of polyethyleneimine-treated calcium alginate beads for sustained release of diltiazem

The objective of this investigation is to develop a multi-unit sustained release dosage form of a water soluble drug from a completely aqueous environment avoiding the use of any organic solvent. The drug was complexed with resin and calcium alginate or polyethyleneimine-treated calcium alginate beads loaded with the resinate were prepared by a ionic/polyelectrolyte complexation method. The effect of different formulation variables on the characteristics of the beads was investigated. Although the drug release from spherical and smooth-surfaced calcium alginate beads in both acidic and alkaline dissolution media were slower than those obtained from plain resinate, none of the variables were found to prolong the drug release considerably due to rapid swelling and disintegration of calcium alginate beads in alkaline medium. On the other hand, drug release from polyethyleneimine-treated calcium alginate beads in acidic medium did not increase appreciably following a burst release. However, in alkaline medium, the drug release was found to increase gradually and extend over a different period of time depending on the intensity of polyethyleneimine treatment. Scanning electron micrographs revealed the formation of a dense membrane around the resinate-loaded calcium alginate matrix. The membrane appeared to be responsible for reduced swelling and protracted disintegration of the beads resulting in slow release of the drug. The results indicate that sustained release of a water soluble drug from polyethyleneimine-treated calcium alginate beads could be achieved by adjusting the formulation variables.     

An update on oral drug delivery via intestinal lymphatic transport

Orally administered drug entities have to survive the harsh gastrointestinal environment, penetrate the enteric epithelia and circumvent hepatic metabolism before reaching the systemic circulation. Whereas the gastrointestinal stability can be well maintained by taking proper measures, hepatic metabolism presents as a formidable barrier to drugs suffering from first-pass metabolism. The pharmaceutical academia and industries are seeking alternative pathways for drug transport to circumvent problems associated with the portal pathway. Intestinal lymphatic transport is emerging as a promising pathway to this end. In this review, we intend to provide an updated overview on the rationale, strategies, factors and applications involved in intestinal lymphatic transport. There are mainly two pathways for peroral lymphatic transport—the chylomicron and the microfold cell pathways. The underlying mechanisms are being unraveled gradually and nowadays witness increasing research input and applications.     

Enhanced gene delivery by polyethyleneimine coated mesoporous silica nanoparticles

Due to large surface area, tunable pore size, easy surface manipulation, and low-toxicity mesoporous silica nanoparticles (MSNs) may act as a suitable vector for gene delivery. In order to make MSNs as a suitable gene delivery system, we modified the surface of phosphonated MSNs (PMSN) with polyethyleneimine (PEI) 10 and 25?KDa. Then nanoparticles were loaded with chloroquine (CQ) (a lysosomotropic agent) and complexed with plasmid DNA. The transfection efficiency and cytotoxicity of these nanoparticles was examined using green fluorescent protein plasmid (pGFP) and cytotoxicity assay. All PEI coated nanoparticles showed positive zeta potential and mean size was ranged between 170 and 215?nm with polydispersity index bellow 0.35. PEI-coated MSNs significiantly enhanced GFP gene expression in Neuro-2?A cells compared to PEI 10 and 25?KDa. The results of the cytotoxicity assays showed that these nanoparticles have an acceptable level of viability but CQ loaded nanoparticles showed higher cytotoxicity and lower transfection activity than CQ free nanoparticles.     

Modulation of quantum dots and clearance of Helicobacter pylori with synergy of cell autophagy

Helicobacter pylori (Hp) is one type of Gram-negative pathogenic bacterium that colonizes and causes a wide range of gastric diseases. Once Hp penetrates into cells, the currently recognized triple or quadruple therapy often loses effectiveness. Recent evidence suggests that autophagy is closely associated with Hp infection, and can play an important role in the eradication of Hp. More importantly, certain types of quantum dots (QDs) can induce and modulate cellular autophagy, and can be developed into conjugates making QDs potential candidates as new anti-Hp agents.     

A method for optical imaging and monitoring of the excretion of fluorescent nanocomposites from the body using artificial neural networks

In this study, a new approach to the implementation of optical imaging of fluorescent nanoparticles in a biological medium using artificial neural networks is proposed. The studies were carried out using new synthesized nanocomposites — nanometer graphene oxides, covered by the poly(ethylene imine)-poly(ethylene glycol) copolymer and by the folic acid. We present an example of a successful solution of the problem of monitoring the removal of nanocomposites based on nGO and their components with urine using fluorescent spectroscopy and artificial neural networks. However, the proposed method is applicable for optical imaging of any fluorescent nanoparticles used as theranostic agents in biological tissue.     

聚合物强化超滤法分离镧和铕离子稀土废水

采用经过预处理的聚乙烯基亚胺(PEI)为聚合物络合剂,探讨了pH、装载量比(L)、离子强度、PEI浓度等对聚合物强化超滤过程的影响。结果表明:采用PEI为聚合物络合剂,pH=2.5时,每毫克PEI对Eu3 络合容量为0.5 mg;pH、L对稀土离子的截留率影响趋势相同;溶液中的含盐量对稀土离子的截留率有一定影响;在体积稀释因子(VDF)为3时,恒容超滤过程可以回收92%以上的PEI,将回收后的PEI重新用于聚合物强化超滤过程处理稀土离子废水,效果与新鲜的PEI没有明显差别。在大量的La3 存在下,络合剂PEI对La3 /Eu3 混合溶液中Eu3 的截留率影响甚微。因此,可用聚合物强化超滤法选择性分离出La3 /Eu3 混合溶液中的镧和铕离子。