Membrane electroporation increases photodynamic effects

Electroporation of membranes is used widely for drug delivery. Photodynamic action consists of three main steps: (A) incorporation of the sensitizer through a membrane into cells; (B) photoxidation of cell constituents and (C) reoxidation of the reduced sensitizer by oxygen etc. The mechanisms of (B) and (C) have been studied widely in past decades. However, the mechanism of transport (A) of sensitizers to targets as the rate limiting step has not been studied to the same extent. Therefore we applied membrane and cell wall electroporation of human histiocytic lymphoma U937 and Saccharomyces cerevisiae cells in order to incorporate rapidly the reliable photodynamic agents thiopyronine, protoporphyrin, zinc phthalocyanine, copper phthalocyanine sulfonate, adriamycin and daunomysin, well-tried cytostatic agents. Depending on field strength and pulse width, 50–90% of cells become electroporated, then the dye diffuses rapidly into the cells, which reseal their membranes over a period of 6–10 min. Illumination for 10–15 min destroys all resealed cells faster than the same amount of unporated cells as in the case of the control (without pulse treatment) either by oxidation of cell components caused by excited dyes or singlet oxygen treatment. By this synergism of electroporation and photodynamic action at the same time, it is possible to kill all cells in a much shorter time than under usual conditions, e.g. in the control suspension. A combination of electrochemotherapeutic needle electrodes with a light conductor for a LASER connection will be effective for therapy.     

在体电穿孔辅助基因转染视网膜色素上皮细胞层和光感受器细胞层的实验研究

目的　探讨在体电穿孔辅助基因转染视网膜色素上皮（RPE）细胞层和光感受器（PR）细胞层的可行性。方法　147只健康雄性Spragne-Dawley(SD)大鼠根据电穿孔刺激模式电压值分为5、10、15、20、25、30、35 V组,右眼视网膜下腔注射增强型绿色荧光蛋白（EGFP）真核表达质粒pEGFP N1为实验眼,左眼注射TE Buffer 为对照眼。各组根据不同转染方向再分为RPE和PR两个亚组。各组除电压不同外,其余参数均为脉宽99 ms,脉冲间期0.5 s,连续5个脉冲。将带有负电荷的质粒在电场作用下,拟转染到RPE细胞层,反向置电极,拟转染到PR细胞层。转染后7 d 取各组视网膜铺片,荧光显微镜下观察EGFP表达强弱、范围及荧光细胞计数分析;采用蛋白质免疫印迹法（Western blot）、实时逆转录聚合酶链反应（RT-PCR）半定量观察EGFP蛋白和mRNA的表达。结果　转染后7 d,荧光显微镜观察发现,各组RPE亚组对照眼RPE细胞层内均未见特异性荧光表达,实验眼可见绿色荧光表达;各组RPE亚组对照眼视网膜铺片均未见荧光表达,实验眼视网膜铺片均可见转染了EGFP的RPE细胞。Western blot检测显示,随电压增加,EGFP蛋白与β-肌动蛋白条带灰度相对比值呈上升趋势。RT-PCR检测显示,各组均产生阳性扩增条带,随电压增高,EGFP mRNA与GADPH mRNA扩增条带灰度相对比值逐渐增高。结论　在体电穿孔方法可以有效辅助基因转染大鼠RPE细胞层。      

Processing of tetanus and botulinum A neurotoxins in isolated chromaffin cells

Tetanus and botulinum A neurotoxins were introduced into the cytosol of chromaffin cells by means of an electric field in which the plasma membrane is forced to form pores of approximately 1 m at the sites facing the electrodes. As demonstrated by electron microscopy, both [¹²⁵I] and gold-labelled tetanus toxin (TeTx) diffuse through these transient openings. Dichain TeTx, with its light chain linked to the heavy chain by means of a disulfide bond, causes the block of exocytosis to develop more slowly than does the purified light chain. The disulfide bonds, which in both toxins hold the subunits together, were cleaved by the intrinsic thioredoxin-reductase system. Single chain TeTx, in which the heavy and light chains are interconnected by an additional peptide bond, was far less effective than dichain TeTx at blocking exocytosis, which indicates that proteolysis is the rate-limiting step. The toxins were degraded further to low-molecular weight fragments which, together with intact toxins and subunits, were released by the cells. The intracellular half-life of [¹²⁵ I] dichain TeTx was approximately three days. The number of light-chain molecules required to maintain exocytosis block in a single cell, as calculated by two different methods, was less than 10. The long duration of tetanus poisoning may result from the persistence of intracellular toxin due to a scarcity of free cytosolic proteases. This may also hold for the slow recovery from botulism.     

Development of murine embryos following electroporation

Purpose: The objective was to establish the parameters for reversible electroporation of murine embryos. Methods: In Trial 1, murine presumptive zygotes received an electrical pulse of 5, 10, or 20-s duration, and one of five voltages (100, 200, 250, 300, or 400 V). In Trial 2, embryo orientation within the electroporation chamber was evaluated with 250 or 400 V at a pulse period of 10 s. Results: Presumptive zygotes that received 400 V at each pulse length and zygotes exposed to 20 s at each voltage had the lowest embryonic development (P < 0.05). Presumptive zygotes that received 250 V had higher development compared to 400 V, irrespective of orientation (P < 0.01), but development was lower than the controls (P < 0.01). Conclusions: Electrical stimulation of presumptive zygotes can have a detrimental impact on early embryo development, but low amounts of stimulation may allow for potential gene transfer in transgenic experimentation.     

Influence of biological parameters and gene transfer technique on transformation of Fusarium oxysporum

Summary An improved method for PEG-mediated transformation of the fungal pathogen Fusarium oxysporum was developed which led to at least a 10-fold increase in transformation frequency. This improvement was gained through the analysis of biological factors, viz., protoplast origin, plasmid modifications and protoplast/plasmid ratio. Elecroporation, a recently developed method for introducing DNA into many cell types, was successfully applied. It gave similar transformation efficiencies to those obtained with the chemical method, and thus appeared a valuable alternative. Qualitative features such as integration events were also analysed. Molecular analysis of transformants revealed that a single copy of plasmid DNA was preferentially integrated by electroporation. The respective advantages of the two DNA transfer methods are discussed.Abbreviations (CM) complete medium (Daboussi 1980) - (MM) minimal medium (Daboussi 1980) - (MS) 10 mM MOPS, pH 6.3, 1 M sorbitol - (MSC) MS with 10 mM CaCl₂ - (HS) 5 mM HEPES, pH 6.3, 20% sucrose - (PEG) polyethylene glycol - (FD) microfarad     

电穿孔介导的基因治疗对兔下颌骨牵引过程中细胞周期蛋白表达的影响

目的 探索电穿孔介导的基因治疗对兔下颌骨牵引成骨过程中细胞周期调节蛋白表达的影响。方法 45只新西兰大白兔,双侧下颌骨截骨后3d开始以0.8 mm/d速度行下颌骨牵引,连续牵引7d后,随机分为A、B、C、D、E5组,每组9只,分别在牵引区注射2μg（0.1 μg/μ1）重组质粒plRES-hVEGF165-hBMP2、pIRES-hBMP2、plRES-hVEGF165、空质粒pIRES和相同剂量的生理盐水后,均施加电穿孔刺激。各组分别于固定期第7、14、28天处死动物取材,行免疫组织化学检查细胞周期蛋白Cyclins A、D1、E的表达情况,并利用CMIAS-2001A病理图像分析系统分析,结果采用单因素方差分析和q检验。结果 Cyclin A、D1、E主要在肉芽组织中的炎性细胞如单核细胞、成纤维细胞及少量沿牵张方向排列的新生幼稚骨小梁表面的成骨细胞、骨细胞和骨周围结缔组织中表达;固定7d时表达最强烈,14 d下降,28 d时表达较弱。图像分析结果显示,固定7d时C组阳性表达蛋白的吸光度A值(0.59 +0.14)表达较强,与A(0.41±0.13)、B(0.38 +0.14)、D(0.34±0.12)、E(0.31 +0.10)组比较差异有统计学意义(P ＜0.05,P＜0.01);A、B组间比较差异无统计学意义(P＞0.05),但与D、E组比较差异有统计学意义(P＜0.05);固定14 d和28 d时,A(0.39±0.11)、B(0.34±0.10)、C(0.33 +0.09)组间比较差异无统计学意义(P＞0.05),但与D(0.19±0.12)、E(0.14 +0.04)组比较差异有统计学意义(P ＜0.05,P＜0.01)。各时相点基因治疗组明显强于对照组。结论 电穿孔介导的基因治疗能使细胞周期蛋白CyclinsA、D1、E在牵引区的表达增强、时限延长,可能促进细胞的分裂增殖与分化,促进牵引区细胞基质的形成和新骨生成。     

Placental growth factor-2 gene transfer by electroporation restores diabetic sensory neuropathy in mice

Placental growth factor-2 (PlGF-2) exhibits neurotrophic activity in dorsal root ganglion (DRG) neurons through the neuropilin-1 (NP-1) receptor in vitro. To examine the potential utility of PlGF-2 therapy for treating diabetic neuropathy, we performed intramuscular PlGF-2 gene transfer by electroporation, and examined its effects on sensory neuropathy in diabetic mice. PlGF-2 was overexpressed in the tibial anterior (TA) muscles of streptozotocin-induced diabetic mice with hypoalgesia using a PlGF-2 plasmid injection with electroporation. The nociceptive threshold was measured using a paw-pressure test. In addition, we overexpressed PlGF-1, an isoform of PlGF that does not bind NP-1. The sciatic nerve and skin were examined 3 weeks after PlGF-2 electro-gene transfer. The overexpression and secretion of PlGF-2 in TA muscles were confirmed by an increase in PlGF levels in TA muscles and plasma, and strongly PlGF positive myofibers in TA muscles. Two weeks after electro-gene transfer into the bilateral TA muscles, the previously elevated nociceptive threshold was found to be significantly decreased in all treated mice. PlGF-1 gene transfer by electroporation did not significantly decrease the nociceptive threshold in diabetic mice. No increase in the number of endoneurial vessels in the sciatic nerve was found in the PlGF-2 plasmid-electroporated mice. A reduction of area of immunoreactivity in epidermal nerves in diabetic mice was restored by PlGF-2 gene transfer. These findings suggest that PlGF-2 electro-gene therapy can significantly ameliorate sensory deficits (i.e. hypoalgesia) in diabetic mice through NP-1 in DRG and peripheral nerves.     

Effect of the electroporation in the field calculation in biological tissues

This article presents results from the field calculation in a model of biological tissue taking into account the electroporation process. The electroporation model used in this study was proposed and experimentally verified by Glaser et al. for planar membranes. The numerical method for field calculation is based on a two-step process: (1) a cell-scale analysis, used for obtaining the field and current in a small volume containing only one cell and its nearest neighbors; and (2) a tissue-scale analysis, based on averaged values of conductivity and permittivity (obtained from the cell-scale analysis) and used for field calculation in a large volume of the tissue. The results for a high voltage applied between two electrodes in a two-dimensional analysis show that the electric field in the porated tissue extends beyond the limits obtained when the electroporation process is not taken into account. Furthermore, due to the electroporation, the tissue conductivity increases in the space between the electrodes. These results show that the electroporation process cannot be ignored in the field calculation in biological tissues when high strength electric fields are present.