Cell Membrane Perforation Research Articles

1P1-P04 大規模光化学細胞膜穿孔法の穿孔条件高速スクリーニングシステムの構築

In recent years, the importance of intracellular material introduction technology is increasing. Our large scale cell membrane perforation method is a new cell modification technique. The feature of this perforation method is efficient in order to make the perforation to the group of cells, with high cell survival ratio. The problem on the current technique is, only limited part of the sequential process is automated. The target of this research is to automate sequences of operations associated with the cell modification. As the result, we could successfully construct the system for the automation and shorten the whole process time from 3-4 hours to 45min.

Intracellular Microelectrode by Photochemical Cell Membrane Perforation

Intracellular Microelectrode by Photochemical Cell Membrane Perforation

Localized high pressure near an aspheric encapsulated microbubble

Based on hydrodynamics, the pressure of the liquid outside an aspheric encapsulated bubble driven by ultrasound is studied, and its analytical expression is derived. Numerical simulation shows that 1) the aspheric shape of an encapsulated bubble makes little influence on the pressure of the liquid far away from the bubble; 2) the pressure is extremely high at some local places of the liquid near an aspheric encapsulated bubble, and the pressure values at these places are apparently larger than those for a spherical encapsulated bubble at the same conditions. This phenomenon is of significance in the applications such as high intensity ultrasound therapy, drug delivery, cell membrane perforation, etc. As the ultrasound frequency shifts to the resonance frequency of an encapsulated bubble, or bubble shape deviates from sphericity, the localized high pressure becomes even greater.

Intracellular Material Injection to Cell Population by Photochemical Local Cell Membrane Perforation

Intracellular Material Injection to Cell Population by Photochemical Local Cell Membrane Perforation

Study of the mechanisms underlying the reversal of multidrug resistance of human neuroblastoma multidrug-resistant cell line SK-N-SH/MDR1 by low-intensity pulsed ultrasound

The aim of the present study was to investigate the underlying mechanism(s) involved in reversing multidrug resistance (MDR) of SK-N-SH/MDR1 by low-intensity pulsed ultrasound (LIPUS). Membrane alteration of SK-N-SH/MDR1 cells exposed to LIPUS was analyzed by scanning electron microscopy (SEM). Immunofluorescence and western blotting were used\ to detect changes in the expression of the MDR-related proteins P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1) and glutathione-s-transferase-π (GST-π) after the optimum ultrasonic. The optimum ultrasonic conditions were 0.3 MHz, 1.0 W/cm², 40 sec and the chemosensitivity of SK-N-SH/MDR1 cells was significantly increased (P<0.05). The optimum ultrasonic-induced perforation of the irradiated cell membranes was observed by SEM. The expression of P-gp was significantly decreased in the group treated by optimum ultrasonic (P<0.05), but not the expressions of MRP1 or GST-π (P>0.05). We demonstrated that LIPUS effectively reverses the MDR of SK-N-SH/MDR1, presumably via augmenting membrane permeability and decreasing the P-gp expression of SK-N-SH/MDR1.

Visible and near infrared resonance plasmonic enhanced nanosecond laser optoporation of cancer cells

In this paper, we report a light driven, non-invasive cell membrane perforation technique based on the localized field amplification by a nanosecond pulsed laser near gold nanoparticles (AuNPs). The optoporation phenomena is investigated with pulses generated by a Nd:YAG laser for two wavelengths that are either in the visible (532 nm) or near infrared (NIR) (1064 nm). Here, the main objective is to compare on and off localized surface plasmonic resonance (LSPR) to introduce foreign material through the cell membrane using nanosecond laser pulses. The membrane permeability of human melanoma cells (MW278) has been successfully increased as shown by the intake of a fluorescent dye upon irradiation. The viability of this laser driven perforation method is evaluated by propidium iodide exclusion as well as MTT assay. Our results show that up to 25% of the cells are perforated with 532 nm pulses at 50 mJ/cm2 and around 30% of the cells are perforated with 1064 nm pulses at 1 J/cm2. With 532 nm pulses, the viability 2 h after treatment is 64% but it increases to 88% 72 h later. On the other hand, the irradiation with 1064 nm pulses leads to an improved 2 h viability of 81% and reaches 98% after 72 h. Scanning electron microscopy images show that the 5 pulses delivered during treatment induce changes in the AuNPs size distribution when irradiated by a 532 nm beam, while this distribution is barely affected when 1064 nm is used.

A Lectin from the Mussel Mytilus galloprovincialis Has a Highly Novel Primary Structure and Induces Glycan-mediated Cytotoxicity of Globotriaosylceramide-expressing Lymphoma Cells

A novel lectin structure was found for a 17-kDa α-D-galactose-binding lectin (termed "MytiLec") isolated from the Mediterranean mussel, Mytilus galloprovincialis. The complete primary structure of the lectin was determined by Edman degradation and mass spectrometric analysis. MytiLec was found to consist of 149 amino acids with a total molecular mass of 16,812.59 Da by Fourier transform-ion cyclotron resonance mass spectrometry, in good agreement with the calculated value of 16,823.22 Da. MytiLec had an N terminus of acetylthreonine and a primary structure that was highly novel in comparison with those of all known lectins in the structure database. The polypeptide structure consisted of three tandem-repeat domains of ∼50 amino acids each having 45-52% homology with each other. Frontal affinity chromatography technology indicated that MytiLec bound specifically to globotriose (Gb3; Galα1-4Galβ1-4Glc), the epitope of globotriaosylceramide. MytiLec showed a dose-dependent cytotoxic effect on human Burkitt lymphoma Raji cells (which have high surface expression of Gb3) but had no such effect on erythroleukemia K562 cells (which do not express Gb3). The cytotoxic effect of MytiLec was specifically blocked by the co-presence of an α-galactoside. MytiLec treatment of Raji cells caused increased binding of anti-annexin V antibody and incorporation of propidium iodide, which are indicators of cell membrane inversion and perforation. MytiLec is the first reported lectin having a primary structure with the highly novel triple tandem-repeat domain and showing transduction of apoptotic signaling against Burkitt lymphoma cells by interaction with a glycosphingolipid-enriched microdomain containing Gb3.

Saponins can perturb biologic membranes and reduce the surface tension of aqueous solutions: A correlation?

Saponins are secondary plant compounds. They have a triterpenoid or steroidal backbone. Sugars are attached to one or more points of this structure, forming chains that can be branched. This appearance leads to amphiphilic properties giving saponins the ability to interact with both lipophilic and hydrophilic structures. The surfactant behavior lets them lower the surface tension in aqueous solutions and form micelles when reaching the critical micelle concentration (cmc). It also lets them interact with biologic membrane layers that usually consist of phospholipids and cholesterol. This action may perturb the membrane and its function leading to membrane perforation or complete lysis. Thus saponins are also known for their cytotoxicity and membranolytic, respectively hemolytic features. In our studies we wanted to answer the question if there is a correlation between the unspecific detergent behavior when lowering the surface tension and the ability to perforate cell membranes and to act cytotoxic. Do saponins showing a considerable reduction in the surface tension also reveal an evident cytotoxicity or/and a marked cell membrane perforation?We tested a variety of saponins with distinct structures. The reduction in the surface tension and the cmc were analyzed on a tensiometer using the Wilhelmy plate method. The general cytotoxicity was determined in a cell model by DNA quantification. The cell membrane toxicity or membrane perforation was explored in a cell model by quantification of the leakage of the intracellular enzyme lactate dehydrogenase (LDH).The experiments revealed a correlation between the membrane toxicity and the reduction in surface tension.

In vitro perforation of human epithelial carcinoma cell with antibody-conjugated biodegradable microspheres illuminated by a single 80 femtosecond near-infrared laser pulse

Pulsed laser interaction with small metallic and dielectric particles has been receiving attention as a method of drug delivery to many cells. However, most of the particles are attended by many risks, which are mainly dependent upon particle size. Unlike other widely used particles, biodegradable particles have advantages of being broken down and eliminated by innate metabolic processes. In this paper, the perforation of cell membrane by a focused spot with transparent biodegradable microspheres excited by a single 800 nm, 80 fs laser pulse is demonstrated. A polylactic acid (PLA) sphere, a biodegradable polymer, was used. Fluorescein isothiocyanate (FITC)-dextran and short interfering RNA were delivered into many human epithelial carcinoma cells (A431 cells) by applying a single 80 fs laser pulse in the presence of antibody-conjugated PLA microspheres. The focused intensity was also simulated by the three-dimensional finite-difference time-domain method. Perforation by biodegradable spheres compared with other particles has the potential to be a much safer phototherapy and drug delivery method for patients. The present method can open a new avenue, which is considered an efficient adherent for the selective perforation of cells which express the specific antigen on the cell membrane.

Femtosecond laser-induced fusion of nonadherent cells and two-cell porcine embryos

Cell fusion is a fundamental biological process that can be artificially induced by different methods. Although femtosecond (fs) lasers have been successfully employed for cell fusion over the past few years, the underlying mechanisms are still unknown. In our experimental study, we investigated the correlation between fs laser-induced cell fusion and membrane perforation, and the influence of laser parameters on the fusion efficiency of nonadherent HL-60 cells. We found that shorter exposure times resulted in higher fusion efficiencies with a maximum of 21% at 10 ms and 100 mJ/cm(2) (190 mW). Successful cell fusion was indicated by the formation of a long-lasting vapor bubble in the irradiated area with an average diameter much larger than in cell perforation experiments. With this knowledge, we demonstrated, for the first time, the fusion of very large parthenogenetic two-cell porcine embryos with high efficiencies of 55% at 20 ms and 360 mJ/cm(2) (670 mW). Long-term viability of fused embryos was proven by successful development up to the blastocyst stage in 70% of cases with no significant difference to controls. In contrast to previous studies, our results indicate that fs laser-induced cell fusion occurs when the membrane pore size exceeds a critical value, preventing immediate membrane resealing.

Chemically modified MEMS probe driven by self-alignment mechanism for photooxidative cell membrane perforation

This paper describes a photooxidative cell membrane perforator that can improve intracellular manipulations. The cell membrane perforator was developed as a MEMS interface employing an ultraviolet excitation of a photosensitizer. The perforator consists of a movable planar probe and a suction hole having a self-alignment mechanism to aid manipulation. The tip of the probe modified with α-terthienyl is introduced to the cell clamped at the pre-aligned suction hole (Φ 5μm in diameter). The α-terthienyl is immobilized using the Au–S bond at the tip of the probe, which is 5μm wide and 400nm thick. The cell membrane is perforated by ultraviolet excitation of the α-terthienyl as a photosensitizer. Photooxidative perforation of the cell membrane was successfully performed using PC12 cells with a diameter as small as tens of micrometers.

Self-organized ZnO nanorod with photooxidative cell membrane perforation enables large-scale cell manipulation

Various devices have been developed for verification and application of cellular functions in recent years. In our previous study, we found that local oxidation reactions in the cell membrane could produce submicron sizes of reversible membrane perforations in cells, while more than 80% of treated cells were viable even after perforations; therefore, to date, we have attempted some applications of this mechanism and analyzed their feasibility. In the present study, we developed a rod-shaped device in which the function of membrane perforation is added by utilizing a photosensitizer and, using the device, we have attempted to produce membrane perforations in a large number of cells. Zinc oxide nanorods were synthesized on the basis of the vapor-liquid-solid mechanism and alpha-terthienyl (photosensitizer) was adsorbed onto gold at the top of the rods to add a membrane perforation function. We studied the effect of the oxidation catalytic ability of the rods on rat PC12 cells after pressing and making the rods' growth side come into contact with the base plate pressed onto the cells in a culture plate followed by photoexcitation of the photosensitizer for a certain period of time. It was revealed that water-soluble fluorescent marker molecules added extracellularly were taken up by the cells when the rods were applied at a pressure of 70 g/cm(2), with a light intensity of 0.82 W/cm(2), and with light irradiation for 30 s, as found in the case of the conventional photochemical cell membrane perforation method targeted at a single cell. These results suggest that cell membrane perforation can be successfully achieved in a large number of cells at a time.

Endothelial cell membrane perforation of aorta and pulmonary artery in the electrocution victims

Some electrocution deaths occur without detectable current marks on the skin, making forensic examination to determine the true cause of death more difficult. Because arterial thrombosis was a frequent finding in victims of electrocution, we investigated injury to the endothelium of the aorta and pulmonary artery with a scanning electron microscope in five cases of death known to be caused by electrocution. We found large pores on the surface of endothelial cells of the aorta and pulmonary artery in those who died of electrocution, but no endothelial membrane perforation was found in those who died of cardiac diseases. These findings were present within 12 h after death. Therefore, scanning electron microscopic evidence of endothelial perforation in the aorta and pulmonary artery could be a useful marker to identify electrocution for those victims without detectable current marks on the skin.

Characterization of the branched antimicrobial peptide M6 by analyzing its mechanism of action and in vivo toxicity

We analyzed functional activity of the antimicrobial peptide M6 in vitro and in vivo. The peptide was identified by our group by phage library selection, rational modification and synthesis in a tetrabranched form (Pini et al., Antimicrob. Agents Chemother. 2005; 49: 2665-72). We found that it binds lipopolysaccharide, causes perforation of cell membranes without destroying external cell morphology and strongly binds DNA. The latter feature suggests that it could inhibit metabolic pathways, blocking DNA replication and/or transcription. We also observed that M6 does not stimulate humoral immune response when repeatedly administered to animals. We also analyzed M6 toxicity when administered to animals by intraperitoneal or by intravenous injection, determining a preliminary LD50 (125 and 37.5 mg/kg, respectively), which suggested that M6 could be used in vivo. These features make the antimicrobial branched peptide M6 a promising candidate for the development of a new antibacterial drug.

Biological effects of two successive shock-waves focused on liver tissue and melanoma cells

A new generator of two successive shock waves focused to a common focal point has been developed. Cylindrical pressure waves created by multichannel electrical discharges on two cylindrical composite anodes are focused by a metallic parabolic reflector - cathode, and near the focus they are transformed to strong shock waves. Schlieren photos of the focal region have demonstrated that mutual interaction of the two waves results in generation of a large number of secondary short-wavelength shocks. Interaction of the focused shockwaves with liver tissues and cancer cell suspensions was investigated. Localized injury of rabbit liver induced by the shock waves was demonstrated by magnetic resonance imaging. Histological analysis of liver samples taken from the injured region revealed that the transition between the injured and the healthy tissues is sharp. Suspension of melanoma B16 cells was exposed and the number of the surviving cells rapidly decreased with increasing number of shocks and only 8 % of cells survived 350 shocks. Photographs of cells demonstrate that even small number of shocks results in perforation of cell membranes.

Sonoporation: Mechanisms of cell membrane perforation and rapid resealing

Sonoporation is a technique for making membrane perforation by exposure of cells to ultrasound, and it is an attractive method for realizing nonvirus gene transfection. A continuous or quasicontinuous wave is frequently used for this technique because a higher duty ratio gives higher efficiency of sonoporation. Addition of microbubbles during insonification greatly improves the efficiency of sonoporation, and, especially when microbubbles exist in the vicinity of the cells, ultrasound pulses from diagnostic ultrasound equipment can cause sonoporation. In this study, we examined sonoporation induced by single-shot pulsed ultrasound and the role of microbubbles in induction of cell membrane perforation. Bubble behavior and cell membrane damage were observed using a high-speed camera and light and scanning electron microscopes. Results of observation showed that mechanical stress induced by bubble motion could cause cell membrane perforation. We also studied repair of the perforation using a fluorescence microscope and found that the membrane of mammalian cells has the ability to reseal the perforation within several seconds. [Research partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan.]

Oiling the key hole

Many bacteria have been found to interact with specialized domains, rich in cholesterol and sphingolipids, of the host plasma membrane, termed lipid rafts. The mechanisms that underlie this interaction are starting to be unravelled. In this issue, Hayward et al. show that early effector proteins secreted by type III secretion harbouring Gram-negative bacteria are in fact cholesterol-binding proteins. Combined with other recent findings, this work shows that multiple steps leading to infection by these bacteria depend on raft components: activation of secretion, binding, perforation of the host cell membrane and signalling to trigger bacterial engulfment.

Selective detection of viable bacteria using dielectrophoretic impedance measurement method

This paper describes a selective detection technique of viable bacteria based on dielectrophoresis and electrical impedance measurements. The authors have previously proposed a detection technique of biological particles called dielectrophoretic impedance measurement (DEPIM) method using positive dielectrophoretic force to capture biological cells in suspension onto an interdigitated microelectrode array. By combining antigen-antibody reaction with the DEPIM, selective detection of a particular species of bacteria was demonstrated. In this present work, the authors demonstrated another selective DEPIM method utilizing cell viability dependency of dielectrophoretic force without introducing the antigen-antibody reaction. It was found that dielectrophoresis of heat-treated Escherichia coli showed strong dependency on viability when applied field frequency was as high as 1 MHz. As a result, viable bacteria could be exclusively collected by positive dielectrophoresis and selectively detected by the DEPIM technique from a suspension also containing heat-treated nonviable cells. On the other hand, nonviable bacteria obtained by UV irradiation showed little dielectrophoresis dependency on viability. According to a theoretical analysis of the dielectrophoretic force, it is suggested that heat treatment alters the dielectric properties of treated cells. In particular, a decrease in cytoplasmic conductivity, which might be caused by heat-induced perforation of cell membrane, was expected to considerably affect dielectrophoresis characteristics. Proposed selective DEPIM method was also applied to evaluation of heat sterilization effect on a real time basis. It was experimentally proved that DEPIM could evaluate viable cell number variation with heat treatment time in a considerably shorter time than conventional microbiological method based on cell incubation.

Keratoconus morphology and cell dystrophy: a SEM study.

The SEM-pictures represent a keratoconus in a 59-year-old patient with a Descemet rupture, which had led to a so-called Kammerwassereinbruch, where the failure of the endothelial structure has become manifest in the epithelium. In the Descemet-rupture the onset of stromal pathology is observable, while outside the rupture severe degradation of endothelial cells is seen. This is manifested as cell-membrane perforation, loss of cell contents and oedema formation in general. On the epithelial side the pathology is expressed as cell-membrane degradation and the presence of very irregularly shaped, sometimes swollen and in other cases shrunken, epithelial cells, in particular in the areas of the surface depressions. The topographical correspondence of these pathological phenomena on the endothelial and the epithelial sides is stressed.

Evidence of voltage-induced channel opening in Na/K ATPase of human erythrocyte membrane.

Previous studies have shown that human erythrocytes when subjected to a high voltage pulsation, in the microsecond time range, lysed in an isotonic medium. The hemolysis was the result of the colloid osmotic swelling, which, in turn, was caused by the voltage perforation of the red cell membranes. In this work we demonstrate that in a low ionic medium at least 35% of the pores was related to the opening of Na+/K4 ATPase channels. The membrane conductance generated by the externally applied electric field could be partially blocked by a specific inhibitor, ouabain, or by a specific cross-linkin g reagent, Cu++-phenanthroline, of the ATPase. The effect of ouabain was saturable and had a mid-point of saturation at 0.15 microM. This value agrees with the physiological inhibition constant of the drug. K+ ion in the external medium suppressed the effect of ouabain, as has also been demonstrated n physiological studies. Experiment presented in this communicaton also suggests that the Na+/K+ ATPase was not perforable in a high ionic medium, and that a large fraction of the voltage-induced pores occurred at as yet unidentified sites.