Intracellular Delivery and Triggered Release of DNA Using Biodegradable Poly(2‐hydroxypropylene imine)s Containing Cystamine Units

Poly(2‐hydroxypropylene imine)s containing segments of cystamine (PHPI‐CA) are synthesized by polycondensation of 1,3‐dibromo‐2‐propanol with a mixture of 1,3‐diamino‐2‐propanol and cystamine. High molecular weight fractions of these polymers are collected by ultrafiltration and characterized by chemical analysis, ¹H and ¹³C‐NMR spectroscopy, size‐exclusion chromatography with triple detection, and potentiometric titration, and are tested for DNA delivery in vitro. It is shown that PHPI‐CA are highly branched polymers containing disulfide linkages. Transfection efficiency of PHPI‐CA for DNA gives similar results to that of PHPI with GFP⁺ cell percent reaching 80–90%. Cytotoxicity levels for PHPI‐CA are lower than that of PHPI. Novel polymers containing different amounts of disulfide linkages are able to disintegrate and release DNA following the treatment with reducing agent 1,4‐dithiothreitol. Downstream application of PHPI‐CA transfected cells for RNA purification shows that RNA yield is not affected even after the double transfection suggesting that these polymers could be great candidates for in vitro and in vivo transfection.

     

N,N,N-Trimethylchitosan chloride as a gene vector: synthesis and application

N,N,N-Trimethylchitosan chloride with different degrees of quaternization has been synthesized and characterized by (1)H NMR spectroscopy. The particle size ranges from 150 to 600 nm, which is dependent on the N/P ratio and is less influenced by the degree of quaternization. The majority of the particles have a spherical morphology. The zeta potential of the particles increases with the N/P ratio and the quaternization degree of TMC. Short-term contact experiments show good biocompatibility of TMC, but long-term contact experiments reveal its high toxicity. This study suggests that TMC is a promising gene carrier, but further modification is still required to improve its cytocompatibility.     

Intracellular triggered release of DNA-quaternary ammonium polyplex by ultrasound

2-Methacryloyloxy ethyl trimethyl ammonium chloride (TMA) is a potent polymeric plasma DNA (pDNA) carrier. The present study shows that TMA/pDNA polyplexes could be internalized into cells efficiently, but could not mediate gene transfection on its own. The transfection process of TMA/pDNA polyplexes is turned on only when ultrasound (US) was applied 4–8 h after incubating TMA/pDNA polyplexes with target cells (with a gene expression 1000 times that of the immediate US group). US is a widely used physical method for gene delivery; its transfection efficiency can be significantly enhanced when combined with cationic polymer vectors. Traditionally, US is given simultaneously with genetic materials, carriers and microbubbles to exert maximal efficacy. The unique on-off phenomenon of TMA/pDNA polyplexes, controlled by US exposure, was found to relate to the endosomal escape effect of US since the polyplexes colocalized well with the lysosome marker if no US was given or was given at inappropriate times. The proposed delivery system using US and TMA carriers has potential in many pharmaceutical applications requiring precise temporal and spatial release control.     

基于纳米材料的基因载体

基因疗法是治疗基因变异引起的先天性遗传疾病和后天获得性疾病以及癌症的新型有效方法。外源基因在细胞中安全、高效、稳定的表达是基因治疗成功的关键,这与基因治疗所使用的载体系统息息相关。基因载体主要分为病毒载体和非病毒载体两大类:病毒载体的转染效率较高,但副作用较大;非病毒载体作为一种新型的基因传递系统,可以弥补病毒载体的缺陷,尽管其转染效率稍逊于病毒载体,但在基因治疗领域具有不可替代的作用。随着纳米技术的出现和蓬勃发展,基于纳米材料的基因载体研究受到越来越多的关注。纳米基因载体具有如下潜在的优势:它制备相对简单,易于对其进行多功能的修饰;具有良好的生物相容性,一般不会引起强烈的机体免疫反应;粒径普遍很小,容易穿过人体的组织间隙而被细胞吸收,基因转运效率较高;可以较有效保护其所携带外源基因,利于基因更高效地表达。本文主要对基于金属、无机非金属、阳离子聚合物和脂质体纳米材料作为基因载体的研究进展进行综述和展望。     

重组质粒pIRES-EGFP-BCL 11B电转染幼稚T细胞的可视化研究

将BCL 11B基因插入pIRES-EGFP构建重组质粒真核表达载体pIRES-EGFP-BCL 11B,采用电转染法将重组质粒转入人幼稚T细胞,转染24 h后,用原子力显微镜(AFM)观察转染前后细胞的表面形态以及生物物理性质的变化.转染72 h后,用CCK-8试剂检测幼稚T细胞的增殖情况.分别对空转的幼稚T细胞组、空载体转染组(pIRES-EGFP naked plasmid)、重组载体转染组(pIRES-EGFP-BCL 11B recombinant vector)、无电转无质粒的幼稚T细胞组进行实验.结果表明,4组幼稚T细胞的体积、高度、半宽度、粗糙度、表面颗粒大小等参数发生了变化,细胞杨氏模量以及细胞硬度也呈现很大变化,CCK-8结果显示,重组质粒pIRES-EGFP-BCL 11B电转染人幼稚T细胞后影响细胞的增殖.     

Patterning of 293T fibroblasts on a mica surface

Controllable cell growth on the defined areas of surfaces is important for potential applications in biosensor fabrication and tissue engineering. In this study, controllable cell growth was achieved by culturing 293 T fibroblast cells on a mica surface which had been patterned with collagen strips by a microcontact printing (μCP) technique. The collagen area was designed to support cell adhesion and the native mica surface was designed to repel cell adhesion. Consequently, the resulting cell patterns should follow the micro-patterns of the collagen. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) measurement, atomic-force microscope (AFM) observation, and force-curve measurement were used to monitor property changes before and after the collagen adsorption process. Further data showed that the patterned cells were of good viability and able to perform a gene-transfection experiment in vitro. This technique should be of potential applications in the fields of biosensor fabrication and tissue engineering. Figure Controllable cells growth has been achieved by culturing 293T fibroblast cells on the mica surface which had been patterned with collagen strips by microcontact printing (μCP) technique     

壳聚糖辐射裂解制备基因载体及其体外转染性能研究

Chitosan (CS) is expected to be an ideal gene carrier for its high biosafety. In this work, CS with low molecular weight were prepared through the γ-ray radiation on the acetic acid solution of CS. The CS chains were scissioned under the γ-ray radiation, and the molecular weight (MW) of CS decreased with the absorbed dose. When the absorbed dose was above 30 kGy, the molecular weight of CS decreased about an order of magnitude. The γ-ray-radiation-scissioned CS can e ectively bind with plasmid (pEGFP) through complex coacervation method, forming pEGFP/ γ-ray-radiation-scissioned CS complex particles with a size of 200-300 nm. The complex particles have good stability and little cytotoxicity. The in uitro gene transfection efficiencies of the pEGFP/ γ-ray-radiation-scissioned CS complex particles were investigated by fluorescence microscope and flow cytometry. The results showed that the gene vectors using γ-ray-radiation-scissioned CS as the carrier will possess better gene transfection efficiency than those using natural high-MW CS as the carrier. The higher the absorbed dose, the smaller the MW of CS and the better transfection efficiency of the corresponding gene vector. This work provides a green and simple method on the preparation of CS-based gene vectors with high efficiency and biosafety.     

Stabilizing in vitro ultrasound-mediated gene transfection by regulating cavitation

It is well known that acoustic cavitation can facilitate the inward transport of genetic materials across cell membranes (sonoporation). However, partially due to the unstationary behavior of the initiation and leveling of cavitation, the sonoporation effect is usually unstable, especially in low intensity conditions. A system which is able to regulate the cavitation level during sonication by modulating the applied acoustic intensity with a feedback loop is implemented and its effect on in vitro gene transfection is tested. The regulated system provided better time stability and reproducibility of the cavitation levels than the unregulated conditions. Cultured hepatoma cells (BNL) mixed with 10 μg luciferase plasmids are exposed to 1-MHz pulsed ultrasound with or without cavitation regulation, and the gene transfection efficiency and cell viability are subsequently assessed. Experimental results show that for all exposure intensities (low, medium, and high), stable and intensity dependent, although not higher, gene expression could be achieved in the regulated cavitation system than the unregulated conditions. The cavitation regulation system provides a better control of cavitation and its bioeffect which are crucial important for clinical applications of ultrasound-mediated gene transfection.     

双子表面活性剂研究进展和应用

双子表面活性剂是一类新型的表面活性剂,它是由联结基团通过化学键将两个或多个单体表面活性剂连接在一起,由此产生优异的表面活性等一系列的性质,从而获得了广泛的应用.本文就它的合成进展及在生物技术、抗病毒、环境保护、新材料制备等方面的应用作一介绍。