基于MEMS微电极芯片的电穿孔系统

报道了一种基于微加工金电极的细胞电穿孔芯片及一套完整的细胞电穿孔系统。该系统能够在多种细胞系中实现高效细胞电穿孔(对典型HEK-293A(人胚胎肾细胞)细胞的穿孔效率高于90%,3T3-L1(小鼠胚胎成纤维细胞)的电穿孔效率高达80%)。并且具有手动模式和自动模式。同时,由于基于独特的电极设计,其所需电压也远低于现有设备(典型工作电压为60 V),成本更低,操作更安全。     

One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis

Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting GGTA1, CMAH, and B4GALNT2 into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting GGTA1 and CMAH with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when GGTA1, CMAH, and B4GALNT2 were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.     

电致孔经皮给药：表面活性剂对孔道存在时间和药物传输的影响

Electroporation creates aqueous pathways by short high-voltage pulses resulting in a transient perme- abilization of stratum corneum and an increase in the transdermal delivery rate.However the aqueous pathways will reseal after pulsing,which leads to the rapid drop of transdermal flux.In the present study,the surfactants were added to the donor solution to hinder the shrinkage and resealing of the electropore,and to prolong the lifetime of the aqueous pathways with the consideration that the surfactants could reduce the surface energy of the electropore. These effects of surfactants were demonstrated by the dynamic electrical resistance of the skin and the fluorescent imaging of the local transport regions.Piroxicam（PIX）was transported percutaneously in the presence of surfactants in vitro.Owing to the longer lifetime of aqueous pathways,together with the promotion of PIX availability at the barrier exterior and the improvement in the partition of PIX into the aqueous pathways,the presence of surfactants led to a remarkable increase in the transdermal delivery rate during electroporation and a significant growth of the accumulative transdermal amount of PIX.     

不可逆电穿孔治疗肿瘤与冷热消融治疗的原理比较

目前,针对肿瘤的治疗,非血管介入技术已经成为了常规手段,但是,其治疗方法和原理各不相同。总结了各种治疗手段的原理,介绍和比较了最新的不可逆电穿孔技术的优点和缺点,以期为其今后的应用提供必要的参考。     

Curcumin Loaded Nanocarriers with Varying Charges Augmented with Electroporation Designed for Colon Cancer Therapy

(1) Background: The size and surface charge are the most significant parameters of nanocarriers that determine their efficiency and potential application. The poor cell uptake of encapsulated drugs is the main limitation in anticancer treatment. The well-defined properties of nanocarriers will enable to target specific tissue and deliver an active cargo. (2) Methods: In the current study, poly(D,L -lactide) (PLA) nanocarriers loaded with curcumin (CUR) and differing surface charge were evaluated for transport efficacy in combination with electroporation (EP) in dependence on the type of cells. The obtained CUR-loaded nanoparticles with diameters ranging from 195 to 334 nm (derived from dynamic light scattering (DLS)) were characterized by atomic force microscopy (AFM) (morphology and shape) and Doppler electrophoresis (ζ-potential) as well as UV-vis spectroscopy (CUR encapsulation efficiency (about 90%) and photobleaching rate). The drug delivery properties of the obtained PLA nanocarriers enhanced by electroporation were assessed in human colon cancer cells (LoVo), excitable normal rat muscle cells (L6), and free of voltage-gated ion channels cells (CHO-K1). CLSM studies, viability, and ROS release were performed to determine the biological effects of nanocarriers. (3) Results: The highest photodynamic activity indicated anionic nanocarriers (1a) stabilized by C₁₂(COONa)₂ surfactant. Nanocarriers were cytotoxic for LoVo cells and less cytotoxic for normal cells. ROS release increased in cancer cells with the increasing electric field intensity, irradiation, and time after EP. Muscle L6 cells were less sensitive to electric pulses. (4) Conclusions: EP stimulation for CUR-PLA nanocarriers transport was considered to improve the regulated and more effective delivery of nanosystems differing in surface charge.     

脉冲电场技术在食品工业上的应用进展

当前食品工业加工方法主要依赖于传统的热处理与化学方法,这些方法往往会对食品质量和安全性产生负面影响,且存在高成本、环境不友好等弊端,开发高效、绿色的食品加工方法已成为食品业界亟待解决的难题。脉冲电场(Pulsed Electric Field,PEF)技术是一种利用高电压振幅的电磁脉冲对物料进行处理的物理方法,作为一种新兴的非热加工技术,PEF技术因其独特的优势吸引了广大学者对其进行广泛而深刻的讨论,以期将其应用于食品工业。该研究综述了脉冲电场技术的原理、应用机制,包括电穿孔理论、电流体、等离子体以及电化学反应与自由基激活假说,并对其在食品工业上的应用进行总结与归纳,为食品加工领域提供了有效的解决方法,有利于推动脉冲电场技术在食品加工领域的发展与应用。     

不可逆电穿孔时组织电导率变化对电场和温度的影响

为了研究不可逆电穿孔过程中组织电导率对组织内电场分布及温度的影响,采用有限元分析软件COMSOL建立了小鼠肝脏的球形肿瘤及椭球形肿瘤的数值模型;通过改变组织的电导率,得到肿瘤及正常组织内不可逆电穿孔电场、温度的分布情况。结果表明:组织电导率随着穿孔过程的发展逐渐增大。组织电导率变化前后,2种肿瘤模型中的各项数据如下:1)不可逆电穿孔电场消融的肿瘤体积与肿瘤总体积之比分别从0.867 7升至0.927 0(球形肿瘤)以及从0.451 1升至0.772 1(椭球形肿瘤);2)不可逆电穿孔电场造成正常组织损伤的体积与肿瘤总体积之比分别从0.745 9降至0.678 2(球形肿瘤)以及从0.816 4降至0.603 6(椭球形肿瘤);3)温升1℃的正常组织的最大体积只有肿瘤体积的1.16%,说明正常组织的温升对组织整体温度变化的影响较小。因此,组织不可逆电穿孔过程中,增大电导率能在促进电脉冲对肿瘤的消融效果的同时减小其对正常组织的损伤,且脉冲作用造成的组织温升不会造成组织的热损伤。     

Design of a Microchannel‐Nanochannel‐Microchannel Array Based Nanoelectroporation System for Precise Gene Transfection

A micro/nano‐fabrication process of a nanochannel electroporation (NEP) array and its application for precise delivery of plasmid for non‐viral gene transfection is described. A dip‐combing device is optimized to produce DNA nanowires across a microridge array patterned on the polydimethylsiloxane (PDMS) surface with a yield up to 95%. Molecular imprinting based on a low viscosity resin, 1,4‐butanediol diacrylate (1,4‐BDDA), adopted to convert the microridge‐nanowire‐microridge array into a microchannel‐nanochannel‐microchannel (MNM) array. Secondary machining by femtosecond laser ablation is applied to shorten one side of microchannels from 3000 to 50 μm to facilitate cell loading and unloading. The biochip is then sealed in a packaging case with reservoirs and microfluidic channels to enable cell and plasmid loading, and to protect the biochip from leakage and contamination. The package case can be opened for cell unloading after NEP to allow for the follow‐up cell culture and analysis. These NEP cases can be placed in a spinning disc and up to ten discs can be piled together for spinning. The resulting centrifugal force can simultaneously manipulate hundreds or thousands of cells into microchannels of NEP arrays within 3 minutes. To demonstrate its application, a 13 kbp OSKM plasmid of induced pluripotent stem cell (iPSC) is injected into mouse embryonic fibroblasts cells (MEFCs). Fluorescence detection of transfected cells within the NEP biochips shows that the delivered dosage is high and much more uniform compared with similar gene transfection carried out by the conventional bulk electroporation (BEP) method.     

Peculiarities of Neurostimulation by Intense Nanosecond Pulsed Electric Fields: How to Avoid Firing in Peripheral Nerve Fibers

Intense pulsed electric fields (PEF) are a novel modality for the efficient and targeted ablation of tumors by electroporation. The major adverse side effects of PEF therapies are strong involuntary muscle contractions and pain. Nanosecond-range PEF (nsPEF) are less efficient at neurostimulation and can be employed to minimize such side effects. We quantified the impact of the electrode configuration, PEF strength (up to 20 kV/cm), repetition rate (up to 3 MHz), bi- and triphasic pulse shapes, and pulse duration (down to 10 ns) on eliciting compound action potentials (CAPs) in nerve fibers. The excitation thresholds for single unipolar but not bipolar stimuli followed the classic strength–duration dependence. The addition of the opposite polarity phase for nsPEF increased the excitation threshold, with symmetrical bipolar nsPEF being the least efficient. Stimulation by nsPEF bursts decreased the excitation threshold as a power function above a critical duty cycle of 0.1%. The threshold reduction was much weaker for symmetrical bipolar nsPEF. Supramaximal stimulation by high-rate nsPEF bursts elicited only a single CAP as long as the burst duration did not exceed the nerve refractory period. Such brief bursts of bipolar nsPEF could be the best choice to minimize neuromuscular stimulation in ablation therapies.     

The Effect of Electroporation of a Lyotroic Liquid Crystal Genistein-Based Formulation in the Recovery of Murine Melanoma Lesions

A lamellar lyotropic liquid crystal genistein-based formulation (LLC-Gen) was prepared in order to increase the aqueous solubility of the lipophilic phytocompound genistein. The formulation was applied locally, in a murine model of melanoma, with or without electroporation. The results demonstrated that, when the formulation was applied by electroporation, the tumors appeared later. During the 21 days of the experiment, the LLC-Gen formulation decreased the tumor volume, the amount of melanin and the degree of erythema, but when electroporation was applied, all these parameters indicated a better prognosis even (lower tumor volume, amount of melanin and degree of erythema). Although hematoxylin–eosin (HE) staining confirmed the above events, application of the LLC-Gen formulation by electroporation did not lead to a significant effect in terms of the serum concentrations of the protein S100B and serum neuron specific enolase (NSE), or the tissue expression of the platelet-derived growth factor receptor β (PDGFRβ) antibody.