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The epithelial-mesenchymal transition (EMT), a normal biological process by which epithelial cells acquire a mesenchymal phenotype, is associated with migration, metastasis, and chemoresistance in cancer cells, and with poor prognosis in patients with esophageal cancer. However, therapeutic strategies to inhibit EMT in tumor environments remain elusive. Here, we show the therapeutic potential of telomerase-specific replication- competent oncolytic adenovirus OBP-301 in human esophageal cancer TE4 and TE6 cells with an EMT phenotype. Transforming growth factor-β (TGF-β) administration induced the EMT phenotype with spindleshaped morphology, upregulation of mesenchymal markers and EMT transcription factors, migration, and chemoresistance in TE4 and TE6 cells. OBP-301 significantly inhibited the EMT phenotype via E1 accumulation. EMT cancer cells were susceptible to OBP-301 via massive autophagy induction. OBP-301 suppressed tumor growth and lymph node metastasis of TE4 cells co-inoculated with TGF-β-secreting fibroblasts. Our results suggest that OBP-301 inhibits the TGF-β-induced EMT phenotype in human esophageal cancer cells. OBP-301-mediated E1A overexpression is a promising antitumor strategy to inhibit EMT-mediated esophageal cancer progression.

Age hardening of Ti-2.3 mass pct Cu (Ti-2.3Cu) at 673 K to 873 K (400 °C to 600 °C) after solution treatment at 1063 K (790 °C) was observed. The relationship between precipitates formed during aging and changes in hardness was investigated. During aging at 673 K (400 °C), the hardness increased rapidly up to 200 hours, and subsequently increased more slowly up to 1000 hours. At 873 K (600 °C), the hardness began to decrease immediately. Transmission electron microscopy showed that fine disk-shaped precipitates of 20 to 40 nm in diameter grew in the α phase. It is concluded that these precipitates interacted with dislocations and increased the hardness. At 873 K (600 °C), precipitates of 1 µm in length and Ti2Cu particles of 200 nm in length were observed. The decrease in hardness may have resulted from the precipitate formation decreasing the concentration of Cu in the α phase. Bright/dark contrast of the three atomic layers and small atomic shift of the hcp structure were observed in the atomic resolution imaging of the precipitates. This suggests that the precipitates are not just Cu-enriched zones and have structures with similar periodicity to the Ti2Cu phase, which is thermally stable at those aging temperatures.

We previously reported on a stearylated INF7 peptide (str-INF7), which enhances the endosomal escape of an octaarginine (R8)-modified liposomal particle encapsulating plasmid DNA (pDNA) in a fusion-independent manner. This study examined whether this peptide derivative enhanced the endosomal escape and gene expression of PEGylated liposomes encapsulating pDNA. We used a PEGylated, R8-modified multifunctional envelope-type nanodevice (R8–MEND) as a model for PEGylated liposomes. Polyethylene glycol 2000 (PEG2000) attached to two different anchors, distearoylphosphatidylethanolamine (DSPE–PEG) or dimyristoylphosphatidylethanolamine (DMPE–PEG), was used to modify the R8–MEND in the presence or absence of two different concentrations of str-INF7. Modification of the PEGylated R8–MEND with str-INF7 resulted in luciferase gene expression levels in HeLa cells that were 73-fold and 24-fold higher than the corresponding value for an unmodified MEND in the case of DSPE–PEG and DMPE–PEG, respectively. The endosomal escape of the PEGylated R8–MEND was improved by str-INF7, as confirmed by confocal laser scanning microscopy. Furthermore, modification with str-INF7 enhanced the hepatic gene expression of the R8–MEND modified with DSPE–PEG and DMPE–PEG by 95-fold and 1885-fold, respectively, after intravenous injection in mice. Collectively, these data demonstrate that str-INF7 can be a useful device for enhancing the endosomal escape even for PEGylated liposomes encapsulating pDNA. © 2011 Wiley Periodicals, Inc. and the American Pharmacists Association.

Pre-applied process followed by mounting chip and underfill at the same time is considered as promising underfill process for coming finer pitch and narrower gap flip chip package with Cu pillar and also TSV package with micro bumps. On the other hand, conventional mass reflow could introduce too much stress on low-k chip with advanced node. Pre-applied underfill process can reduce stress on low-k chip by dispersing stress into underfill material at the connection with thermal compression bonding process. One of the challenges of thermal compression bonding process with NCP is trapping material and poor connection at the bonding area. Creeping and voiding are other challenges for NCP. This presentation reports material design of a new NCP, features, and reliability results. Our newly developed NCP material was dispensed smoothly, and didn't cause creeping up on the backside of the chip. While low thixotropic NCP tends to create voids because of easier NCP spreading out when chip is mounted, this NCP material showed good performance in terms of voiding because of high thixotropic index, less spreading out even under heating condition (

The successful control of intracellular trafficking (i.e., endosomal escape and nuclear delivery) is prerequisite for the development of a gene delivery system. In the present study, we developed an in vivo hepatic gene delivery system using a plasmid DNA (pDNA)-encapsulating lipid envelope-type nanoparticle, to which we refer as a multifunctional envelope-type nanodevice (MEND). The critical structural elements of the MEND are a DNA/protamine condensed core coated with lipid bilayers including serum-resistant cationic lipids. Intravenous administration of bare MEND represents minimal transfection activity. For the surface modification of functional devices, hydrophobic moieties were chemically attached, which are shed in the spontaneous orientation outward from the MEND surface by anchoring to the lipid bilayers. Modification of the pH-dependent fusogenic peptide GALA as an endosome escape induced transfection activity by 1 and 2 orders of magnitude. In an attempt to induce the nuclear delivery of pDNA, maltotriose, a recently characterized nuclear localization signal, was additionally modified. As a result, transfection activity further enhanced by 1 order of magnitude, and it reached to the higher level obtained for a conventional lipoplex and an in vivo jetPEI-Gal, with less hepatic toxicity. The data show that the combination of GALA and maltotriose results in a highly potent functional device that shows an enhanced endosomal escape and nuclear delivery in vivo.

We previously reported that modification of GALA peptide on the surface of liposomes enhanced fusion with endosomal membrane, and cytoplasmic release of encapsulated macromolecules. We report herein that an additional coating of GALA-modified liposomes with 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer resulted in a two order of magnitude enhancement in the transfection activity of encapsulating plasmid DNA (pDNA). Quantification of the delivered gene copies in whole cells and isolated nuclei revealed that the increase of transfection activity can be attributed to improved efficiencies in cellular uptake and post-nuclear delivery processes. Imaging studies revealed that the intracellular dissociation of pDNA from the lipid envelope is enhanced by GALA modification and further coating with MPC polymer in a stepwise manner. The MPC polymer-coating decreased the zeta-potential of GALA-modified liposomes, suggesting that it assisted in the functional display of negatively charged GALA on the cationic liposomes by providing shielding from mutual electrostatic interactions. Collectively, these data indicate that MPC polymer-coating induced the fusogenic activity of the GALA-modified envelope with endosomes, leading to a more effective cytoplasmic release pDNA. The extensive fusion of the lipid envelope may also reduce electrostatic interactions between mRNA and cationic lipid components, thereby resulting in an enhancement in the translation process.

Background: Epithelial-mesenchymal transition (EMT) is known to be one of the poor prognostic factors. EMT is a biological process, by which epithelial cancer cells lose their epithelial characteristics and gain mesenchymal properties, including invasion, metastasis and drug resistance, resulting in tumor recurrence and poor prognosis. Esophageal cancer is one of the most common cause of cancer-related death worldwide, especially in East Asia. Despite the recent advances in the treatment of esophageal cancer, antitumor therapy to inhibit EMT program remains to be developed. We developed a telomerase-specific replication-selective oncolytic adenovirus (OBP-301; Telomelysin), in which the human telomerase reverse transcriptase promoter drives the expression of E1A and E1B for virus replication. In this study, we explored the biological effect of OBP-301 on EMT in human esophageal cancer cells. Methods: Transforming growth factor-β (TGF-β) was used to induce EMT in human esophageal cancer TE-4 cells. To investigate whether OBP-301 infection affects TGF-β-induced EMT, we performed western blot and real-time PCR analysis for EMT-related markers. We also examined the migration capability using transwell migration assay. In vitro antitumor effect of OBP-301 and chemotherapeutic agents on TGF-β-treated TE-4 cells were assessed by a XTT assay. Results: Administration of TGF-β induced mesenchymal characteristics, including spindle-shaped morphology, increased migration capability, and upregulation of mesenchymal markers N-cadherin and vimentin, in TE-4 cells. When TGF-β-treated TE-4 cells were infected with OBP-301, upregulation of N-cadherin and vimentin expression was attenuated. Upregulation of EMT-transcription factors, Snail, Slug, and ZEB1 by TGF-β was reduced by OBP-301 infection. OBP-301 also inhibited TGF-β-induced enhancement of migration capability. Moreover, interestingly, EMT-induced TE-4 cells acquired chemoresistance, whereas they were sensitive to OBP-301-mediated lytic effect. Conclusion: Our data suggest that telomerase-specific oncolytic virotherapy is a promising antitumor strategy to inhibit TGF-β-induced EMT and warrants clinical trials in human esophageal cancer. Currently, we are conducting a phase I/II clinical study of OBP-301 in combination with radiotherapy for esophageal cancer. Citation Format: Tomoya Masuda, Hiroshi Tazawa, Takeshi Ieda, Yuuri Hashimoto, Shunsuke Tanabe, Kazuhiro Noma, Yasuo Urata, Shunsuke Kagawa, Yasuhiro Shirakawa, Toshiyoshi Fujiwara. Transforming growth factor-b-induced epithelial-mesenchymal transition is attenuated by telomerase-specific oncolytic virotherapy in human esophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2033.

It is previously reported that octaarginine (R8)-modified liposome (R8-Lip) was taken up via macropinocytosis, and subsequently delivered to the nuclear periphery. In the present study, we investigated the mechanism for the cytoplasmic transport of R8-Lips, comparing with that for adenovirus. Treatment with microtubule-disruption reagent (nocodazole) inhibited the transfection activity of plasmid DNA (pDNA)-encapsulating R8-Lip more extensively than that of adenovirus. The directional transport of R8-Lips along green fluorescent protein (GFP)–tagged microtubules was observed; however, the velocity was slower than those for adenovirus or endosomes that were devoid of R8-Lips. These directional motions were abrogated in R8-Lips by nocodazole treatment, whereas adenovirus continued to undergo random motion. This finding suggests that the nuclear access of R8-Lip predominantly involves microtubule-dependent transport, whereas an apparent diffusive motion is also operative in nuclear access of adenovirus. Furthermore, quantum dot-labeled pDNA underwent directional motion concomitantly with rhodamine-labeled lipid envelopes, indicating that the R8-Lips were subject to microtubule-dependent transport in the intact form. Dual particle tracking of carriers and endosomes revealed that R8-Lip was directionally transported, associated with endosomes, whereas this occurs after endosomal escape in adenovirus. Collectively, the findings reported herein indicate that vesicular transport is a key factor in the cytoplasmic transport of R8-Lips.

Lipid envelope-type nanoparticles are promising carriers for gene delivery. The modification of liposomes with polyethyleneglycol (PEG) can often be useful in liposomal formation and pharmacokinetics. However, there is a dilemma concerning the use of PEG because of its poor intracellular trafficking properties. To overcome this problem, in the present study, we report on a strategy for improving the intracellular trafficking of PEG-modified lipid particles by incorporating a short PEG lipid. The findings presented here show that the incorporation of tetra(ethylene)glycol (TEG)-conjugated cholesterol into a liposome composition is useful in controlling the number of lipid envelopes, resulting in an improvement in particle uniformity with a reduced particle size. The TEG-modified lipid particles were found to enhance transfection activity by more than 100-fold. This increase is attributed to an enhancement of cellular uptake, and nuclear transcription by improving intracellular decoating. Moreover, the use of a various short PEG lipids in lipid particle formation showed a clear threshold polymerization degree (less or equal 25: PEG1100), for achieving stimulated transfection activity. Collectively, the use of short PEG lipid promises to be useful in developing an efficient non-viral gene vector.

The nuclear delivery process is a crucial barrier to successful gene delivery, especially in non-dividing cells. We previously proposed a novel strategy for the nuclear delivery of plasmid DNA (pDNA), in which the pDNA is encapsulated in lipid bilayers that had been modified with nucleus-targeting signals, including nuclear localizing signals derived from SV40 (NLS) or sugar units. In the present study, we report on an investigation of the effect of the topology of the liposome-modified NLS on its ability to bind to the isolated nucleus. NLS was directly attached to a liposome (NLS-Lip) by incorporating stearylated NLS (STR-NSL), or by modification with a polyethyleneglycol (PEG) spacer (NLS-PEG-Lip). NLS-unmodified liposomes (PEG-Lip) were used as a control. The liposomes, after labeling with 7-nitrobenz-2-oxa-1,3-diazole (NBD), were incubated with a cell homogenate derived from JAWS II cells, followed by isolation of the nuclear fraction by centrifugation. The PEG-Lip preparation showed negligible binding to the nucleus. In contrast, the binding of NLS-Lips to the nucleus gradually increased in a STR-NLS density-dependent manner. Interestingly, the binding of NLS-PEG-Lips to the nucleus is highly effective even at low density, suggesting that the presence of the PEG spacer is an important factor in improving the binding activity of NLS-modified liposomes to the nucleus. This information will be useful for the design of nucleus-targeting carriers.

Background The efficient nuclear delivery of plasmid DNA (pDNA) is essential for the development of a promising non-viral gene vector. In an attempt to achieve nuclear delivery, NLS-mu, a novel pDNA condenser, was prepared. This consists of mu, a highly potent polypeptide for condensing the pDNA, and a SV40 T antigen-derived nuclear localization signal (NLSSV40). Methods The utility of NLS-mu was assessed in terms of green fluorescent protein (GFP) expression after cytoplasmic and nuclear microinjection of GFP-encoding pDNA along with the transfection, and compared with mu and poly-L-lysine (PLL). Trans-gene expression after cytoplasmic microinjection was affected by the efficiencies of nuclear transfer and following intra-nuclear transcription. To evaluate the nuclear transfer process separately, we introduced a parameter, a nuclear transfer score (NT score), which was calculated as the trans-gene expression after cytoplasmic microinjection divided by that after nuclear microinjection. Results As expected, the rank order of trans-gene expression after the transfection and cytoplasmic microinjection was NLS-mu > mu > PLL. However, the calculated NT scores were unexpectedly ranked as mu = NLS-mu > PLL, suggesting that mu, and not NLSSV40, is responsible for the nuclear delivery of pDNA. In addition, confocal images of rhodamine-labeled pDNA indicated that pDNA condensed with mu and NLS-mu was delivered as a condensed form. In comparing the nuclear transcription, the rank order of trans-gene expression after nuclear microinjection was PLL = NLS-mu > mu, suggesting that intra-nuclear transcription is inhibited by efficient condensation by mu, and is avoided by the attachment of NLSSV40. Conclusions Collectively, NLS-mu, which consists of chimeric functions, is an excellent DNA condenser, and the process is based on mu-derived nuclear transfer and NLSSV40-derived efficient intra-nuclear transcription. Copyright © 2005 John Wiley & Sons, Ltd.

In this study, we developed novel double-membranous non-viral gene delivery system modified with SV-40 T antigen-derived nuclear localization signal (NLS-DMEND) for delivery of luciferase plasmid DNA to nucleus of non-dividing mouse bone marrow-derived dendritic cells (BMDC). Intracellular trafficking and gene expression of NLS-DMEND in the BMDC were evaluated. Condensed DNA was observed in the nucleus by confocal laser scanning microscopy, and the NLS-DMEND induced significant luciferase activity in the BMDC. It was suggested that the condensed DNA particle transferred into nucleus via energy dependent manner, since the nuclear transfer was inhibited by metabolic inhibitors. In conclusion, condensed plasmid DNA was delivered into the nucleus of non-dividing BMDC by NLS-DMEND.

The reuse system proposed by the authors is an overall business system for realizing a cyclic reuse flow through the processes of design, fabrication, construction, maintenance, demolition and storage. The reuse system is one of the methods to reduce the environmental burden in the field of building steel structures. These buildings are assumed to be demolished within approximately 30 years or more for physical, architectural, economic and social reasons in Japan. In this paper, focusing on building steel structures used for plants, warehouses and offices without fire protection, the performance of steel structural members for reuse is evaluated by a non-destructive test. First, performance evaluation procedures for a non-destructive test, such as mechanical properties, chemical compositions, dimension and degradation, are shown. Tensile strengths are estimated using Vickers hardness measured by a portable ultrasonic hardness tester, and chemical compositions are measured by a portable optical emission spectrometer. The weldability of steel structural members is estimated by carbon equivalent and weld crack sensitivity composition using chemical compositions. Finally, the material grade of structural members of the building steel structure for reuse is estimated based on the proposed procedures.

A man in his twenties had intermittent abdominal pain in the right lower quadrant for more than 4 years. The abdominal pain persisted after a meal, and he visited our hospital emergency department. We performed an emergency colonoscopy and found a 3-cm mobile polypoid lesion located on the antimesenteric side of the ileum 40 cm from the ileocecal valve and was 85×26×23 mm in size. On the basis of characteristic ultrasound and contrast-enhanced CT findings, our preoperative diagnosis was intussusception due to Meckel's diverticulum translation and performed a laparoscopic ileocecal resection. The pathological diagnosis was Meckel's diverticulum translation with ectopic pancreatic and gastric tissue. Furthermore, we aggregated the cases of adult intussusception due to Meckel's diverticulum translation reported in Japan, and investigated preoperative diagnoses and treatment plans.

An octaarginine-modified multifunctional envelope-type nano device (R8-MEND) was previously reported to be an efficient nonviral vector for the delivery of plasmid DNA, in vitro and after topical administration. We report herein on a novel stearylated derivative of the INF7 peptide, a derivative of the N-terminal domain of the HA2 protein of the influenza virus envelope, which enhances the endosomal escape of R8-MEND through a mechanism independent of fusion between the MEND coat and the endosomal membrane. The use of the novel peptide derivative would permit the gene expression of the R8-MEND to be improved, both in vitro and in vivo. R8-MEND modified with stearylated INF7 resulted in gene expression levels that were 77-fold higher than unmodified and 20-fold higher than the free INF7 peptide-modified R8-MEND with no cellular toxicity. Spectral imaging in live cells confirmed that the stearylated INF7 modification did not mediate fusion between liposomes and the endosomal membrane. The inclusion of DOPE to the R8-MEND coat was synergistic with the peptide in improving gene transfection. The intravenous injection of an R8-MEND modified with stearylated INF7 to ICR mice resulted in luciferase expression levels 240-fold higher in liver and 115-fold higher in spleen than that of the R8-MEND.

Efficient targeting of DNA to the nucleus is a prerequisite for effective gene therapy. The gene-delivery vehicle must penetrate through the plasma membrane, and the DNA-impermeable double-membraned nuclear envelope, and deposit its DNA cargo in a form ready for transcription. Here we introduce a concept for overcoming intracellular membrane barriers that involves step-wise membrane fusion. To achieve this, a nanotechnology was developed that creates a multi-layered nanoparticle, which we refer to as a Tetra-lamellar Multi-functional Envelope-type Nano Device (T-MEND). The critical structural elements of the T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes and two endosome-fusogenic outer envelopes, which are shed in stepwise fashion. A double-lamellar membrane structure is required for nuclear delivery via the stepwise fusion of double layered nuclear membrane structure. Intracellular membrane fusions to endosomes and nuclear membranes were verified by spectral imaging of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores that had been dually labeled on the liposome surface. Coating the core with the minimum number of nucleus-fusogenic lipid envelopes (i.e., 2) is essential to facilitate transcription. As a result, the T-MEND achieves dramatic levels of transgene expression in non-dividing cells.

Efficient nuclear gene delivery is essential for successful gene therapy. This study developed a novel system that mimics the mechanism of nuclear entry of adenovirus (Ad) by means of a Multifunctional Envelope-type Nano Device (MEND). In this system, plasmid DNA (pDNA) was condensed with polycation, followed by encapsulation in a lipid membrane. To target MEND to the nuclear pore complex (NPC), sugar served as a NPC-mediated nuclear targeting device was modified on the surface of the lipid envelope. This was accomplished via synthesis of a sugar-cholesterol conjugate. After binding of the MEND to the NPC, the pDNA core was transferred into the nucleus in conjunction with a breakdown of the lipid envelope. Sugar-modified MEND showed higher transfection efficiency compared with unmodified MEND, in non-dividing and dividing cells. Confocal microscopy confirmed that nuclear transfer of pDNA was improved by sugar modification of MEND. Furthermore, destabilization of the lipid envelope significantly enhanced transfection activity: therefore, nuclear-delivery efficiency was closely related to lipid envelope stability. Moreover, quantitative evaluation of cellular uptake and nuclear transfer processes by real-time PCR confirmed that the surface sugars affected nuclear transfer, but not cellular uptake. In summary, a novel system for the nuclear delivery of pDNA was successfully developed by using a sugar-modified MEND and by optimizing the lipid envelope stability.

Samarium-mediated 7-endo-trig radical cyclization afforded excellent stereocontrol of the four contiguous asymmetric centers present in the 6-7-6 tricyclic cores of the (sugar-fused) erinacines E, F, and G.

In the present study, the nuclear delivery of a green fluorescence protein (GFP)-encoding pDNA condensed by protamine was investigated in terms of trans-gene expression after cytoplasmic (E(cyt)) and nuclear (E(nuc)) microinjection. To compare the nuclear transfer process, a novel parameter; the nuclear transfer (NT) score was introduced. The E(cyt) value for protamine/pDNA particles increased in a charge ratio-dependent manner. The calculated NT score showed that this increase results from an enhancement in nuclear transfer efficiency, which was also quantitatively confirmed by a recently developed confocal image-assisted three-dimensionally integrated quantification (CIDIQ) method. Moreover, E(nuc) for protamine/pDNA particles was significantly higher than that for poly-l-lysine/pDNA particles, suggesting that pDNA, when condensed with protamine, is more accessible to intra-nuclear transcription. Collectively, protamine is an excellent DNA condenser, with bi-functional advantages: improvement in nuclear delivery and efficient intra-nuclear transcription.

Purpose: For the efficient trans-gene expression by non-viral gene vector, improvement of nuclear transfer of the exogenous plasmid DNA (pDNA) is essential. Since protamine consists of four possible NLS-like regions rich in basic amino acids, it is considered that condensation with this peptide ensure the nuclear delivery of pDNA.

Top of pageAbstract [Objective] To develop a non-viral gene vector successfully, highly efficient nuclear delivery system of plasmid DNA (pDNA) is essential. In the present study, we developed a nuclear delivery system based on the novel concept of |[ldquo]|degradable particle on the nuclear membrane|[rdquo]|, which mimic the mechanism of nuclear entry of adenovirus by means of multifunctional envelope-type nano device (MEND). In this system, pDNA was condensed with polycation, followed by encapsulation with lipid membrane. Devices for the nuclear targeting were modified on the surface of lipid envelope. After the particle binds to the nuclear pore complex (NPC), lipid envelope was drawn into the nucleus with condensed pDNA particle. As a device for nuclear targeting, various sugars were modified. [Methods] To modify the sugars on the surface of the lipid envelope, sugars were conjugated with cholesterol, which enables it to insert in the lipid layer. To enhance a cellular uptake via macropinocytosis, which is advantageous to avoid a lysosomal degradation, stearylated octaarginine (STR-R8) was further modified on the envelope. We evaluated transfection activities of sugar chain modified MEND in HeLa cells which is synchronized in G1 phase by hydroxyurea. As a control, sugar-conjugated cholesterol was replaced with unconjugated cholesterol. [Results] Sugar modified MEND showed drastically higher transfection activity compared with sugar-unmodified MEND in dividing cells. Moreover, in synchronized cells, transfection activity of sugar- unmodified MEND was prominently decreased. Even this condition, high transfection activity was also observed with sugar-modified MEND. These results suggested that sugar chain improved the nuclear transfer of pDNA. Optimum density of sugar on the MEND was depended on the type of sugar. Furthermore it was demonstrated that trans-gene expression was decreased when lipid envelope was stabilized by increasing the cholesterol content. Therefore, destabilization of lipid envelope on the nuclear membrane was closely related to the transfection activity. [Conclusion] Collectively, we succeeded in developing the novel system for the nuclear delivery of pDNA with MEND by optimizing the density of sugar and the stability of lipid envelope.