Liposomal Reagent Research Articles

Unlimited Potential of Liposomes and Their Application to Medical Field

It was previously considered that the practical application of liposomal medicines was very difficult. The pharmaceutical technologies for mass production, long term stability during storage, encapsulation efficiency of the drug, etc. were seemed big problems to be solved, and it was well known that the liposomal particles are apt to be entrapped in vivo by the reticuloendothelial system (RES) such as liver, spleen, etc. Fortunately, owing to the progress of science, about 10 liposomal medicines containing the anticancer agents, the antifungal agents, etc. were launched out and are now contributed to medical treatment in the world. Recently liposomes are expected to be useful as the vectors for in vivo nucleic acid (plasmid DNA, siRNA, etc.) therapy and as the tools for target validation in the area of drug discovery. There are already many liposomal reagents for transfection, and some clinical trials are performed using liposomes. It is considered that liposomes, the membrane–structure particles, have unlimited potential in the medical field.

Analysis of Transgene Intergration Efficiencies into Porcine Fetal Fibroblast Using Different Transfection Methods and Twice Drug Selection.

Transgenic animals can be produced by nuclear transfer with genetically modified somatic cells in which the procedure of transgene transfection is required. The objective of this study was to compare transfection efficiencies of electroporation (Microporater [Seo Ryn Bio, Korea]) and liposome reagents (FuGENE 6, FuGENE HD [Roche, USA], Welfect-ex, Welfect-Q, Welfect-M [Welgene, Korea]) into pig fetal fibroblast cells using transgene vector containing human tPA cDNA fused to β-casein promoter sequence and neomycin-resistant gene (Neor) driven by PGK promoter and to determine the proportion of cells containing transgene in neomycin-resistant colonies. The linearized transgene vector DNA was transfected into pig fetal fibroblasts with electroporation or different transfection reagents according to manufacturer's procedures. Pig fetal fibroblast cells were selected following exposure to 400 μg/ml G418 for 14 days. Cell colonies surviving G418 selection were picked by cloning cylinder (Sigma, USA) and assayed by PCR amplification with tPA-specific primers. Also, single cell PCR analysis of growing colonies resistant to G418 selection was carried out to evaluate the percentage of cells containing transgene in G418-resistant colonies. Among transfection methods tested in this experiment, electroproation exhibited significantly higher transfection efficiency compared with liposome reagents (50% with microporater vs 20% with liposomes). When cells of G418-resistant colonies were performed by single-cell PCR, 60 to 100% of cells in each colony were identified to contain transgene regardless of transfection methods. The colonies carrying 60 to 80 % transgene-positive cells were cultured again with 400 μg/ml G418 for 14 days, and cell colonies surviving G418 selection were selected and assayed by PCR. Transfection frequencies in theses colonies for tPA gene were 95% to 100%. The present results suggested that electroproation may increase transfection efficiency of DNA into pig fetal fibroblast cells compared with liposome methods and twice drug-selection can improve transfection rate.

Antisense RNA of survivin gene enhances the taxol-induced apoptosis and sensitivity to chemotherapy drugs in bladder cancer cells: an in vitro experiment

To evaluate the effects of antisense oligodeoxynucleotide (ASODN) of survivin gene on taxol-induced apoptosis in bladder cancer cells. A survivin ASODN eukaryotic vector pcDNA3-SVVas was transfected into human bladder cancer cells of the line BIU87 mediated by liposomal reagent (BIU87 SVVas cells). The mRNA expression of survivin was measured by RT-PCR. Blank plasmid pcDNA3 was transfected as control (BIU87 neo cells). The cell growth curve was drawn using trypan blue dye exclusion assay. MTT assay was used to investigate the sensibility of transfected cells to taxol. The BIU87 SVVas cells, BIU87 neo cells, and BIU87 cells were cultured in the culture fluid with taxol and then DNA gel electrophoresis, Hoechst nuclear staining and fluorescent microscopy, and annexin-propidium iodide (PI) staining were used to examine the cell apoptosis. Compared to BIU87 and BIU87 neo cells, the mRNA expression of survivin in the BIU87-SVVas cells was obviously reduced. The growth of the BIU87-SVVas cells was significantly reduced in comparison with the BIU87 (P < 0.05). The sensibility of BIU87 SVVas cells to taxol increased significantly shown by cell proliferation and MTT assays. Agarose gel electrophoresis of genomic DNA showed typical DNA ladder only in the BIU87 SVVas cells, but not in other cells. Hoechst nuclear staining and fluorescent microscopy showed that the nuclei of the BIU87 SVVas cells become condense. Annexin-PI test showed that the cell apoptosis rate of the BIU87-SVVas cells treated with taxol was significantly higher than those of the other groups. survivin antisense RNA enhances the taxol-induced apoptosis in the bladder cancer cells. This may lay an experimental foundation for further research of biotherapy in bladder cancer.

CD98 Activation Increases the Invasion of Human Breast Carcinoma MCF-7 Cells

CD98, a disulfide-linked 125-kDa heterodimeric type II transmembrane glycoprotein, regulates the func-tions of β₁ integrin, suggesting that it may play a role in tumor cell invasion. In this study, the effects of CD98 signaling on the adhesion and invasion of tumor cells were investigated. The expression of CD98 on MCF-7 human breast carcinoma cells was confirmed by immunohistochemistry. The effects of CD98 activation on the adhesion to extracellular matrix (ECM) and invasion of MCF-7 cells were determined by adhesion assay and cell invasion assay. Dominant negative forms of focal adhesion kinase (FAK) were transiently transfected into MCF-7 cells using liposome reagents. CD98 stimulation increased the adhesion of MCF-7 cells to fibronectin, laminin and collagen IV. Activation of CD98 augmented the invasion rate of MCF-7 cells through ECM. EDTA or a function-blocking anti-β₁ integrin mAb suppressed the effect of CD98 on invasiveness. Inhibition of phosphorylation of FAK by PP2, an inhibitor of Src family kinase, reduced CD98-induced invasion of MCF-7 cells. This result was confirmed by over-expression of dominant negative forms of FAK. In addition, cytochalasin D or phalloidin inhibited CD98-mediated induction of tumor cell invasion. Inhibitory effects of PP2, cytochalasin D or phalloidin on CD98-stimulated invasion of MCF-7 cells were diminished by pretreatment of cells with Mn??, which is shown to induce conformational change of β₁ intgerin. These results provide the first evidence that CD98 activation increases tumor cell invasion by activating β₁ integrin affinity, and that FAK phosphorylation and subsequent cytoskeletal reorganization may be essential for CD98-mediated regulation of cell motility.

Liposome polymerase chain reaction assay for the sub-attomolar detection of cholera toxin and botulinum neurotoxin type A

We describe an ultrasensitive immunoassay for detecting biotoxins that uses a liposome with encapsulated DNA reporters, and ganglioside receptors embedded in the bilayer, as the detection reagent. After immobilization of the target biotoxin by a capture antibody and co-binding of the detection reagent, the liposomes are ruptured to release the reporters, which are quantified by real-time polymerase chain reaction. The new assays for cholera and botulinum toxins are several orders of magnitude more sensitive than current detection methods. A single 96-well microtiter plate can analyze approximately 20 specimens, including calibration standards and controls, with all measurements conducted in triplicate. Using pre-coated and blocked microtiter plates, and pre-prepared liposome reagents, a liposome polymerase chain reaction assay can be carried out in about 6 h.

Liposome-mediated transient transfection reduces cholesterol-dependent coxsackievirus infectivity

Liposome-mediated gene delivery provides a powerful strategy for the study of gene function and for gene therapy. Coxsackievirus B3 is an important human pathogen associated with various diseases. Here we reported that liposome-mediated transient transfection of plasmid cDNA inhibited coxsackieviral replication at the levels of RNA, protein and viral progeny release. These inhibitory effects were observed in various cell types and by using different liposome reagents. We further showed that the inhibition was likely due to the lack of virus attachment. Moreover, we showed that addition of cholesterol restored, at least in part, the viral infectivity. Interestingly, we found that membrane cholesterol levels were unchanged during transfection, indicating that disruption rather than depletion of membrane cholesterol contributes to the inhibitory effects of transfection. Our data suggest that liposome-mediated cDNA transient transfection inhibits coxsackievirus infectivity via inhibition of viral attachment, which is likely occurring through the changes of membrane cholesterol integrity.

Gene Therapy of Oral Cancer: Efficient Delivery of a ‘Suicide Gene’ to Murine Oral Cancer Cells in Physiological Milieu

Gene therapy is a new therapeutic modality in which defective genes are replaced with functional ones, or genes are delivered that can specifically kill cancer cells. Efficient gene delivery is an important component of gene therapy approaches. Potential safety problems with viral vectors necessitate the development of efficient nonviral vectors. DNA complexes with synthetic cationic liposomes or polymers constitute a simple means of transferring DNA into target cells. Gene delivery mediated by many nonviral vectors, however, is inhibited by serum components, and this is expected to limit the efficiency of gene delivery in vivo. In this study, the authors examined two novel gene transfection reagents, Metafectene and GeneJammer, for their ability to deliver a reporter gene to SCCVII murine squamous cell carcinoma cells in the presence of high concentrations of mouse serum. After establishing conditions that achieved significant gene delivery, the authors introduced the Herpes Simplex Virus Thymidine kinase (HSV-tk) gene into the cells using the cationic liposome reagent, Metafectene, followed by the administration of ganciclovir. After seven days of incubation, 90 percent and 82 percent cytotoxicity was observed in 0 percent and 60 percent mouse serum, respectively. The authors' observations suggest that Metafectene may be useful for the gene therapy of oral squamous cell carcinoma in a murine model involving the induction of oral tumors by SCCVII cells.

Constitutively Active Homo-oligomeric Angiotensin II Type 2 Receptor Induces Cell Signaling Independent of Receptor Conformation and Ligand Stimulation

Members of the G-protein-coupled receptor superfamily (GPCRs) undergo homo- and/or hetero-oligomerization to induce cell signaling. Although some of these show constitutive activation, it is not clear how such GPCRs undergo homo-oligomerization with transmembrane helix movement. We previously reported that angiotensin II (Ang II) type 2 (AT(2)) receptor, a GPCR, showed constitutive activation and induced apoptosis independent of its ligand, Ang II. In the present study, we analyzed the translocation and oligomerization of the AT(2) receptor with transmembrane movement when the receptor induces cell signaling. Constitutively active homo-oligomerization, which was due to disulfide bonding between Cys(35) in one AT(2) receptor and Cys(290) in another AT(2) receptor, was localized in the cell membrane without Ang II stimulation and induced apoptosis without changes in receptor conformation. These results provide the direct evidence that the constitutively active homo-oligomeric GPCRs by intermolecular interaction in two extracellular loops is translocated to the cell membrane and induces cell signaling independent of receptor conformation and ligand stimulation.

The use of liposomes for detection of the surface lipopolysaccharide antigen, Vibrio cholerae cells, and antibodies against them

A test system for determination of Vibrio cholerae cells, surface O-antigen, and antibodies against them was developed on the basis of complement-dependent lysis of liposomes sensitized by the lipopolysaccharide-dependent antigen from Vibrio cholerae 569B. The factors that affect the function of the liposomal reagent were studied, and the conditions for detecting antibodies and antigenic material were optimized. This system is highly specific and sensitive to be used for the determination of anticholeraic antibodies (30-50 times as effective as agglutination tests), lipopolysaccharide antigen (100 ng/ml, which corresponded to 3.0 ng of lipopolysaccharide in the sample studied), and Vibrio cholerae cells (3.3 x 10(7) m.b./ml, which corresponded to 10(6) m.b. in sample). It takes 30-40 min to detect the lipopolysaccharide antigen and 90 min to detect V. cholerae cells.

Evaluation of an mRNA Lipofection Procedure for Human Dendritic Cells and Induction of Cytotoxic T Lymphocytes against Enhanced Green Fluorescence Protein

We utilized an mRNA lipofection procedure in human dendritic cells (DCs) and attempted to induce cytotoxic T lymphocytes (CTLs) against enhanced green fluorescence protein (EGFP). EGFP mRNA was transfected into phytohemagglutinin (PHA)-stimulated lymphocytes or adherent peripheral blood mononuclear cell-derived DCs using a liposomal reagent. Lipofection efficiency was measured by flow cytometry. In PHA-stimulated lymphocytes, increasing concentrations of liposome or mRNA increased EGFP expression levels by up to 64.4%, but caused a decrease in cell viability. A similar trend was also observed in DCs. For 70% DC viability, the concentration of liposomes was 24 µl/ml, and the mRNA concentration was 6 µg/ml. Under these conditions, ELISPOT and ⁵¹Cr release assays were performed on CD8+ T cells stimulated twice with EGFP mRNA-transfected DCs. The number of interferon-γ-producing cells was increased when the CD8+ T cells were cocultured for 24 h with PHA-stimulated lymphocytes transfected with EGFP mRNA. The level of specific lysis of EGFP mRNA-transfected DCs also increased to approximately 80%, with an effector to target ratio of 40:1. These data suggest that EGFP is immunogenic for human T cells, confirming that our lipofection procedure may be of use for inducing specific CTLs.

CK2 Phosphorylation of the Armadillo Repeat Region of β-Catenin Potentiates Wnt Signaling

Protein kinase CK2 is a ubiquitous serine/threonine kinase involved in many biological processes. It is overexpressed in many malignancies including rodent and human breast cancer, and is up-regulated in Wnt-transfected mammary epithelial cells, where it can be found in a complex with dishevelled and beta-catenin. beta-Catenin is a substrate for CK2 and inhibition of CK2 reduces levels of beta-catenin and dishevelled. Here we report that inhibition of CK2 using pharmacologic agents or expression of kinase inactive subunits reduces beta-catenin-dependent transcription and protein levels in a proteasome-dependent fashion. The major region of phosphorylation of beta-catenin by CK2 is the central armadillo repeat domain, where carrier proteins like axin and the adenomatous polyposis coli gene product APC interact with beta-catenin. The major CK2 phosphorylation site in this domain is Thr393, a solvent-accessible residue in a key hinge region of the molecule. Mutation of this single amino acid reduces beta-catenin phosphorylation, cotranscriptional activity, and stability. Thus, CK2 is a positive regulator of Wnt signaling through phosphorylation of beta-catenin at Thr393, leading to proteasome resistance and increased protein and co-transcriptional activity.

Crossing the Membrane: Nonviral and Viral Delivery Methods for Use In Vitro and In Vivo

DNA and Cell BiologyVol. 21, No. 12 ArticlesCrossing the Membrane: Nonviral and Viral Delivery Methods for Use In Vitro and In VivoMarie A. BogoyevitchMarie A. BogoyevitchSearch for more papers by this authorPublished Online:6 Jul 2004https://doi.org/10.1089/104454902762053819AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail "Crossing the Membrane: Nonviral and Viral Delivery Methods for Use In Vitro and In Vivo." , 21(12), pp. 855–856FiguresReferencesRelatedDetailsCited byMicrofluidic platform for hepatitis B viral replication study29 December 2007 | Biomedical Microdevices, Vol. 10, No. 3 Volume 21Issue 12Dec 2002 To cite this article:Marie A. Bogoyevitch.Crossing the Membrane: Nonviral and Viral Delivery Methods for Use In Vitro and In Vivo.DNA and Cell Biology.Dec 2002.855-856.http://doi.org/10.1089/104454902762053819Published in Volume: 21 Issue 12: July 6, 2004PDF download

Deoxyribonuclease I-like III is an inducible macrophage barrier to liposomal transfection.

Extra- and intracellular nucleases are predicted to decrease the in vivo efficiency of liposomal transfection. DNASE1 (D1) has been proposed as the main nuclease barrier, yet liposome-complexed DNA and in vitro lipofection are generally immune to D1. In contrast, medium conditioned by the macrophage enzyme DNASE1-like 3 (DNASE1L3 or D3) erects a potent in vitro barrier to liposomal transfection. Although homologous to D1 over its amino-terminal sequence, D3 has a distinct, highly basic carboxy terminus, which resembles polylysine stretches often found in polycationic liposomal reagents. If this domain is truncated from D3, the resulting enzyme has more nuclease activity against naked DNA ("free DNA"-nuclease activity), yet does not block transfection. C-terminal fusion of this domain to D1 forms a chimeric protein able to block transfection. D3 can be immunodetected in both serum and macrophage lysates. Macrophage-conditioned medium contains both "free DNA"-nuclease activity and the ability to block transfection, and by zymogram only a 28-kDa DNASE, consistent by size with D3, is present. Thus, medium containing D3 confers to cells an in vivo shield to the nuclear acquisition of exogenous DNA. Modulation and further elucidation of this activity are likely to have importance for both gene therapy and autoimmune disorders.

Liposomes as signal amplification reagents for bioassays in microfluidic channels.

Liposomes with encapsulated carboxyfluorescein were used in an affinity-based assay to provide signal amplification for small-volume fluorescence measurements. Microfluidic channels were fabricated by imprinting in a plastic substrate material, poly(ethylene terephthalate glycol) (PETG), using a silicon template imprinting tool. Streptavidin was linked to the surface through biotinylated-protein for effective immobilization with minimal nonspecific adsorption of the liposome reagent. Lipids derivatized with biotin were incorporated into the liposome membrane to make the liposomes reactive for affinity assays. Specific binding of the liposomes to microchannel walls, dependence of binding on incubation time, and nonspecific adsorption of the liposome reagent were evaluated. The results of a competitive assay employing liposomes in the microchannels are presented.

Immunologic characterization of HIV-specific DNA vaccine.

We developed a method for applying HIV-1 DNA vaccine topically in mice. Topical application of DNA vaccine to the skin is useful against infections. To find a less expensive and less cumbersome vaccination method, we administered HIV-1 DNA vaccine to the skin of mice after elimination of keratinocytes using a fast-acting adhesive. HIV-1 DNA vaccine induced high levels of both humoral and cell-mediated immune activity against HIV-1 envelope antigen. A high level of HIV-1-specific cytotoxic T lymphocyte response was also observed, and a high level of IFN-gamma and IL-4 production was induced by the improved skin application of DNA vaccine. High levels of both HIV-specific cytotoxic T lymphocyte and delayed type hypersensitivity in topical application were induced by coadministration of the DNA vaccine with IL-12 expression plasmids and granulocyte-macrophage colony-stimulating factor expression plasmids. These immune responses were inhibited by intradermal injection of anti-CD11c or anti-I-A/I-E antibody. Therefore, topical administration of DNA vaccine is an effective route, and may be very useful for the prevention of infectious diseases.

Reduction of c‐myc expression by an antisense approach under Cre/loxp switching induces apoptosis in human liver cancer cells

c-Myc has been documented to be both a positive and a negative signal for the induction of apoptosis. It is well known that overexpression of the c-myc gene induces apoptosis of normal cells, but the result of a reduction in its expression is not fully understood. We examined whether a reduction in c-myc expression would induce apoptosis in human liver cancer cells. Specifically, antisense and sense oligodeoxynucleotides (oligos) against the human c-myc mRNA were synthesized, mixed with a liposome reagent at various ratios, and were applied to the liver cancer-derived cell lines, HCC-T, HepG2, and PLC/PRF/5. To exclude effects resulting from using oligos, plasmid vectors expressing the full-length c-myc cDNA in both sense and antisense orientations under the control of the Cre/loxP system were generated. Monoclonal cell lines including these plasmid vectors were produced and Cre was supplied by adenovirus infection. Apoptosis was determined morphologically and c-Myc and Bcl-2 expression was examined by Western blotting. The antisense myc significantly inhibited the proliferation of the cells within two days, while neither the liposome reagent alone nor sense myc did so. Most of the cells were rounded up by the antisense-treatment and nuclear fragmentation and DNA ladder formation were detected after two days in antisense c-myc-treated cells. Antisense c-myc largely reduced c-Myc and partially Bcl-2 expression; overexpression of Bcl-2 partially rescued from apoptosis in HCC-T and HepG2 cells. These results suggest that the massive reduction in c-myc mRNA induces apoptosis in liver cancer cell lines and consequent decrease in Bcl-2 enhances the cell death. c-Myc reduction under the Cre/loxP switching system may be a useful tool for the clarification of c-myc-related cellular mechanisms in differentiation and proliferation.

Activation of Interferon Regulatory Factor 3 in Response to DNA-damaging Agents

Genotoxic stress triggers signal transduction pathways that mediate either the protection or apoptosis of affected cells. The interferon regulatory factors (IRFs) are involved in a wide range of host defense mechanisms against environmental stresses. Treatment with DNA-damaging agents, including doxorubicin and UV radiation, caused phosphorylation of the IRF3 transcription factor. Phosphorylation of IRF3 induced its interaction with the transcriptional co-activator cAMP-response element binding protein-binding protein. Furthermore, genotoxic stress-induced phosphorylation of IRF3 resulted in its movement from the cytoplasm to the nucleus, where it activated transcription from its binding site. These observations suggest that IRF3 plays a role in the defensive responses induced by genotoxic stress.

Novel series of non-glycerol-based cationic transfection lipids for use in liposomal gene delivery.

A novel series of nontoxic and non-glycerol-based simple monocationic transfection lipids containing one or two hydroxyethyl groups directly linked to the positively charged nitrogen atom were synthesized. The in vitro transfection efficiencies of these new liposomal gene delivery reagents were better than that of lipofectamine, a widely used transfection agent in cationic lipid-mediated gene transfer. The most efficient transfection formulation was observed to be a 1:1:0.3 mol ratio of DHDEAB (N, N-di-n-hexadecyl-N,N-dihydroxyethylammonium bromide):cholesterol:HDEAB (N-n-hexadecyl-N,N-dihydroxyethylammonium bromide) using a DHDEAB-to-DNA charge ratio (+/-) of 0.3:1. Observation of good transfection at charge ratios lower than 1 suggests that the amphiphile-DNA complex may have net negative charge. Our results reemphasize the important point that in cationic lipid-mediated gene delivery, the overall charge of the lipid-DNA complex need not always be positive. In addition, our transfection results also imply that favorable hydrogen-bonding interactions between the lipid headgroups and the cell surface of biological membranes may have some role for improving the transfection efficiency in cationic lipid-mediated gene delivery.

Induction of interferon synthesis and activation of interferon-stimulated genes by liposomal transfection reagents.

Liposome-mediated transfection is a widely used technique for the introduction of exogenous DNA into mammalian cells. We observed a significant induction of endogenous interferon (IFN)-stimulated genes (ISGs) in cells treated with the liposomal reagents, lipofectamine and DOSPER, in the absence of DNA. Liposome treatment induced expression of reporter constructs driven by IFN-responsive promoter elements, demonstrating a generalized effect on ISG expression. The kinetics of ISG induction were markedly delayed in response to liposome as compared with IFN or double-stranded RNA. ISG induction could be transferred to naive cells with conditioned medium from liposome-treated cells, suggesting that a secreted factor was responsible for this activity. A cell line defective in IFN signaling was refractory to liposome-induced ISG expression, indicating a role for IFN in this induction. Indeed, liposome treatment directly induced IFN-beta gene expression and, thus, represents a novel IFN inducer. IFN induction by liposomal reagents and its potential effects on transgene expression should be considered in the choice of transfection reagent. The ability of liposomal gene delivery reagents to induce IFN synthesis in the host may prove useful in gene therapy approaches to viral and neoplastic diseases.

Highly Sensitive and Stable Phosphatidylserine Liposome Aggregation Assay for Annexins

Annexins are a gene family of Ca2+-dependent membrane binding proteins which interact specifically with acidic phospholipids. We describe here details of highly sensitive and precise assays for annexins I and V, utilizing turbidometric analysis of phosphatidylserine liposome aggregation. In the case of annexin I, the new assay is 7-fold more sensitive than the conventional chromaffin granule aggregation assay in terms of threshold for detection and the rate of increase of initial absorbance is 15-fold greater. Annexin V, which binds but does not aggregate liposomes, can be assayed on the basis of inhibition of Ca2+-dependent liposome aggregation. Further comparative advantages of the assay include lower expense and increased shelf life of the liposome reagent.