Liposomal Reagent Research Articles

Effect of FLOT2 Gene Expression on Invasion and Metastasis of Colorectal Cancer and Its Molecular Mechanism under Nanotechnology and RNA Interference.

The study is aimed at investigating the effect of the FLOT2 gene on invasion and metastasis of colorectal cancer (CRC) cells and the corresponding molecular mechanism by preparing polylysine-silicon nanoparticles. Specifically, polylysine was used to modify the silica nanoparticles prepared by the emulsification method to obtain polylysine-silicon nanoparticles. The characterization of polylysine-silicon nanoparticles was completed by nanoparticle size analyzer, laser particle size potentiometer, and transmission microscope. The influence of polylysine-silicon nanoparticles on the survival rate of CRC cell line HT-29 was detected using the method of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). The FLOT2-siRNA expression vector was constructed and transfected with HT-29. The HT-29 transfected with empty plasmid was used as the negative control (NC). Western Blot (WB) and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect expression levels of FLOT2 gene and epithelial-mesenchymal transition- (EMT-) related genes. Transwell invasion assay, Transwell migration assay, and CCK8 assay were used to detect the cell invasion, migration, and proliferation. The results showed that the average particle size of polylysine-silicon nanoparticles was 30 nm, the potential was 19.65 mV, the particle size was 65.8 nm, and the dispersion coefficient was 0.103. At the same concentration, the toxicity of silicon nanoparticles to HT-29 was significantly lower than that of liposome reagent, and the transfection efficiency was 60%, higher than that of liposome reagent (40%). The mRNA level and protein expression of the FLOT2 gene in the FLOT2-siRNA group were significantly lower than those in the NC group (P < 0.01). The optical density (OD) value of the NC group and the blank control (CK) group were significantly higher than that of FLOT2-siRNA cells (P < 0.01). The OD value of FLOT2-siRNA cells was lower than that of NC cells at 48 h, 72 h, and 96 h (P < 0.01). The mRNA levels and protein expressions of MMP2 and vimentin in the FLOT2-siRNA group were significantly lower than those in the NC group and CK group (P < 0.01). The mRNA level and protein expression of the E-cadherin gene in the FLOT2-siRNA group were significantly higher than those in the NC group and CK group (P < 0.01). In conclusion, an RNA interference plasmid with high transfection efficiency and low cytotoxicity was established based on nanotechnology. siRNA-mediated FLOT2 protein inhibits the invasion, migration, and proliferation of CRC cells by regulating the expression changes of EMT-related genes, which provides a scientific basis for clinical treatment of CRC.

MRNA Transfection-Induced Activation of Primary Human Monocytes and Macrophages: Dependence on Carrier System and Nucleotide Modification

Monocytes and macrophages are key players in maintaining immune homeostasis. Identifying strategies to manipulate their functions via gene delivery is thus of great interest for immunological research and biomedical applications. We set out to establish conditions for mRNA transfection in hard-to-transfect primary human monocytes and monocyte-derived macrophages due to the great potential of gene expression from in vitro transcribed mRNA for modulating cell phenotypes. mRNA doses, nucleotide modifications, and different carriers were systematically explored in order to optimize high mRNA transfer rates while minimizing cell stress and immune activation. We selected three commercially available mRNA transfection reagents including liposome and polymer-based formulations, covering different application spectra. Our results demonstrate that liposomal reagents can particularly combine high gene transfer rates with only moderate immune cell activation. For the latter, use of specific nucleotide modifications proved essential. In addition to improving efficacy of gene transfer, our findings address discrete aspects of innate immune activation using cytokine and surface marker expression, as well as cell viability as key readouts to judge overall transfection efficiency. The impact of this study goes beyond optimizing transfection conditions for immune cells, by providing a framework for assessing new gene carrier systems for monocyte and macrophage, tailored to specific applications.

Suicide Gene Therapy of Oral Squamous Cell Carcinoma and Cervical Carcinoma In Vitro.

Suicide gene therapy induced by the Herpes Simplex Virus thymidine kinase/ganciclovir (HSV-tk/GCV) system has been utilized to successfully treat various cancers. We describe TransfeX-mediated transfection of pCMV.Luc into HeLa cervical carcinoma and HSC-3, FaDu, and H357 oral squamous cell carcinoma (OSCC) cell lines in the presence of 10% serum. This method has proved to be highly efficient, with low nonspecific cytotoxicity. The plasmid pNGVL1-tk encoding HSV-tk under the control of the CMV promoter was delivered to the cells in vitro via TransfeX, a cationic liposomal reagent, followed by treatment with ganciclovir. The Alamar Blue cell viability assay was used to determine levels of thesuicide effect.

Impact of PRDM1 gene inactivation on C-MYC regulation in diffuse large B-cell lymphoma

Objective: To investigate the role of PRDM1 gene inactivaion in the regulation of C-MYC in diffuse large B-cell lymphoma (DLBCL), and to explore the correlation of its immunophenotype and prognosis. Methods: 100 cases paraffin-embedded DLBCL tissues were collected from January 2009 to December 2015 at the First Affiliated Hospital of Xinjiang Medical University along with 20 cases of reactive proliferative lymph nodes as control. Immunohistochemical methods were used to detect the expression of CD20, CD10, MUM1, Ki-67, bcl-6, PRDM1/Blimp1, C-MYC and PAX5 protein. The tumors were classified into two subtypes according to Hans classification.The expression of PRDM1 and C-MYC gene in tumor group and control group was detected by reverse transcription PCR (RT-PCR) and the relationship between PRDM1 and C-MYC gene was analyzed.OCI-LY1 (GCB subtype) and OCI-LY3 (non-GCB subtype) cell lines were transfected with small interfering RNA by cationic liposome reagent transfection, and the expression of C-MYC in the transfected cell lines was detected by RT-PCR and Western blot. The Kaplan-Meier method was used to analyze the prognostic significance of PRDM1/Blimp1 and C-MYC at protein and mRNA levels. Results: There were 27 cases of GCB subtype and 73 cases of non-GCB subtype according to Hans classification. The positive expression of Blimp1 in DLBCL group and proliferative lymph nodes in control group was seen in 26(26.0%) and 20 cases(100%), respectively. There were 58 cases with high expression of PRDM1 at mRNA level, including 22 cases of GCB subtype and 36 cases non-GCB subtype, and the difference was statistically significant (P=0.004). There were differences in PRDM1 gene expression between the two immunological subtypes, serum lactate dehydrogenase (serum LDH) level, presence of B symptoms, tumor primary sites and other clinical pathological parameters, while C-MYC expression was different in gender, IPI score, and serum LDH levels. Upon PRDM1/Blimp1 gene silencing in the two cell lines, C-MYC protein and gene expression were up-regulated in the transfection group, compared with the blank control group and negative control group by reverse transcription PCR and Western blot analyses. Moreover, PRDM1 expression was significantly associated with C-MYC(χ(2)=7.648, P=0.006) at mRNA level. Conclusion: The up-regulation of C-MYC gene expression induced by PRDM1 inactivation in DLBCL may play an important role for the development of DLBCL.PRDM1 protein and mRNA are associated with immunophenotyping and PRDM1 mRNA is a marker of poor prognosis.

Establishment of protocol for preparation of gene-edited bovine ear-derived fibroblasts for somatic cell nuclear transplantation.

Recently, gene-editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) technique has attempted to utilize fibroblasts of livestock animals for somatic cell nuclear transfer. In this study, we establish the procedure for preparing skin fibroblast clones whose genes were edited by the CRISPR/Cas9 technique. After isolating fibroblasts from earlobes of Japanese Black cattle, subsequent collagenase-digestion and extensive wash procedures enabled us to avoid contamination of fungi. Electroporation using NEPA21, rather than lipofection using commercially available liposome reagents, allowed us to perform more efficient transfection of plasmid constructs. Although bovine ear-derived fibroblasts were not able to proliferate in single cell cultures in Dulbecco's modified Eagle medium containing 10% fetal calf serum, supplementation with insulin-transferrin-selenium mixture, human recombinant epidermal growth factor, or human recombinant basic fibroblast growth factor promoted proliferation of the cells, even in a single cell culture. Taking advantage of our established protocol, we eventually obtained eight ear-derived fibroblast clones with a recessive mutation in the isoleucyl-tRNA synthetase gene corrected by the CRISPR/Cas9 technique.

A review of genetic engineering biotechnologies for enhanced chronic wound healing.

Traditional methods for addressing chronic wounds focus on correcting dysfunction by controlling extracellular elements. This review highlights technologies that take a different approach - enhancing chronic wound healing by genetic modification to wound beds. Featured cutaneous transduction/transfection methods include viral modalities (ie adenoviruses, adeno-associated viruses, retroviruses and lentiviruses) and conventional non-viral modalities (ie naked DNA injections, microseeding, liposomal reagents, particle bombardment and electroporation). Also explored are emerging technologies, focusing on the exciting capabilities of wound diagnostics such as pyrosequencing as well as site-specific nuclease editing tools such as CRISPR-Cas9 used to both transiently and permanently genetically modify resident wound bed cells. Additionally, new non-viral transfection methods (ie conjugated nanoparticles, multi-electrode arrays, and microfabricated needles and nanowires) are discussed that can potentially facilitate more efficient and safe transgene delivery to skin but also represent significant advances broadly to tissue regeneration research.

FABP4-mediated homocysteine-induced cholesterol accumulation in THP-1 monocyte-derived macrophages and the potential epigenetic mechanism

Hyperhomocysteinemia (HHcy) is an independent risk factor for the development of atherosclerosis (AS), according to overwhelming number of clinical and epidemiological studies. However, the underlying pathogenic molecular mechanisms by which HHcy promotes AS remain to be fully elucidated. Fatty acid binding protein 4 (FABP4) has been shown to be important in macrophage cholesterol trafficking. The objective of the present study was to determine whether homocysteine (Hcy) accelerates AS through regulating FABP4, and then mediates cholesterol accumulation in macrophages. Hcy concentrations of 0, 50, 100, 200 and 500µM, and 100µM Hcy+30µMvitamin B12 (VB12)+30µM folic acid (FA) were respectively added to cultured THP‑1 monocyte‑derived macrophages for 24h. The levels of FABP4, which acts as a key factor connecting cellular lipid accumulation to inflammation, were determined using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analyses in the macrophages. The present study used a nested touchdown methylation‑specific PCR assay to detect the DNA methylation status of the FABP4 promoter region. In addition, the FABP4 gene fragment was inserted into the cloning vector, pcDNA3.1‑EGFP, to construct the recombinant plasmid, pcDNA3.1‑EGFP/FABP4, which was identified using restriction endonuclease digestion analysis and DNA sequencing. The pcDNA3.1‑EGFP/FABP4 expression plasmid was transfected into THP‑1 monocyte‑derived macrophages, mediated by liposome reagent, following which the expression levels of FABP4 were detected using RT‑qPCR and western blot analyses. The present study also determined the intracellular accumulation of total cholesterol in the macrophages. The results indicated that Hcy decreased the levels of FABP4 promoter methylation, but increased the mRNA and protein expression levels of FABP4 in the macrophages, compared with the control group (0µMHcy). However, no dose‑dependent changes were observed with increasing concentrations of Hcy. The recombinant fluorescent eukaryotic expression vector, pcDNA3.1‑EGFP/FABP4, was successfully constructed and effectively expressed in the THP‑1 macrophages. The results also showed that FABP4 accelerated the accumulation of cholesterol in the macrophages. Taken together, the results of the present study suggested that FABP4 DNA hypomethylation induced by Hcy may be involved in the overexpression of FABP4, thereby inducing cholesterol accumulation in macrophages.

680. TransfeX-Mediated HSV-tk/Ganciclovir Suicide Gene Therapy in HeLa Cervical Carcinoma and HSC-3, FaDu, and H357 Oral Cancer Cells

INTRODUCTION: Herpes Simplex Virus thymidine kinase/ganciclovir (HSV-tk/GCV)-induced suicide gene therapy has been used to successfully treat various cancers. TransfeX-mediated transfection of pCMV.Luc into HeLa cervical carcinoma and HSC-3, FaDu, and H357 oral squamous cell carcinoma (OSCC) cell lines in the presence of 10% serum has proved to be highly efficient, with low non-specific cytotoxicity. The plasmid pNGVL1-tk encoding HSV-tk under the control of the CMV promoter was delivered to the cells in vitro via the novel cationic liposomal reagent, TransfeX, followed by treatment with ganciclovir. METHODS: HeLa, HSC-3, FaDu, and H357 cells were seeded in 48-well culture plates one day prior to transfection, at a density of 1.5 × 105 cells per well in 1 ml of complete DMEM medium containing 10% serum (DME/10). Lipoplexes for transfection were prepared by mixing 1 µg pCMV.Luc or pNGVL1-tk per 100 µl of serum-free DMEM followed by the addition of 2 µl of TransfeX. Lipoplexes were added directly to cells grown in the presence of 10% serum. Twenty-four hours after transfection, 20 µg of ganciclovir was added per well and cells were incubated an additional 24 h. The Alamar blue assay was used to determine cell viability after 24, 48, and 120 h. Fresh medium and ganciclovir were added after each of these readings. RESULTS: After 24 h, 65% and 50% cytotoxicity were seen in HeLa cells and HSC-3 cells, respectively. FaDu and H357 cells were more resistant to treatment, showing 15% and 5% cytotoxicity, respectively, after 24 h. After 120 h, cytotoxicity remained the same in HeLa, and increased to 90% in HSC-3. In FaDu and H357, 65% and 30% cytotoxicity, respectively, were observed after 120 h. CONCLUSIONS: Suicide gene therapy is most effective against HSC-3 OSCC among the cells examined. Longer periods of incubation with ganciclovir or prolonged gene expression via enhanced episome vectors may be necessary to increase cytotoxicity in FaDu and H357 cells.View Large Image | Download PowerPoint SlideView Large Image | Download PowerPoint Slide

Abstract 479: A novel dynamic delivery system enabling high efficiency transfection of cells

Abstract Transfection is an enabling technique for cellular and molecular biologists to study expression and knockdown of gene expression. Over the past decade, many researchers have utilized liposomal systems to deliver nucleic acids; however, this method has many caveats including low transfection efficiency and high cellular toxicity in some cell types. To address these shortcomings, Mirus Bio has developed a novel non-liposomal technology called the TransIT-X2™ Dynamic Delivery System. This unique polymeric delivery system is able to effectively condense negatively charged nucleic acids (plasmid DNA and/or siRNA), enter cells and break out of the endosomal compartment, a critical barrier for transfection. The plasmid and siRNA can then be delivered to their respective functional locations, the nucleus and the cytoplasm. In this study, we demonstrate functional transfection of labeled plasmid DNA co-delivered with labeled siRNA illustrating the dual abilities of the TransIT-X2 Dynamic Delivery System. Additionally, extensive head-to-head comparisons with a popular liposomal reagent, Lipofectamine® 2000 were performed using a luciferase expression assay in 30 cell types; TransIT-X2 demonstrated superior performance in 20 cell types with 11 showing greater than 2-fold higher expression compared to Lipofectamine 2000. Transfection efficiency of green fluorescent protein (GFP) was measured using flow cytometry and visualized by microscopy; most cell types had greater than 50% GFP efficiency. Lastly, gene knockdown after delivery of siRNA or miRNA was assessed. Two endogenous genes, GAPDH and AHA1, were targeted for siRNA knockdown and mRNA levels were assessed by qRT-PCR. miRNA function was assessed by the decrease in the miR-1 target mRNA PTK9. The TransIT-X2 Dynamic Delivery System shows effective knockdown via siRNA or miRNA. The TransIT-X2 Dynamic Delivery System offers researchers exceptional broad-spectrum transfection of plasmid DNA and siRNA. This cutting edge, non-liposomal polymeric delivery method enhances endocytic release of the transfection complexes allowing researchers to obtain very high transfection efficiencies. Citation Format: James Ludtke, Anthony Lauer, Austin Storck, Nicholas Rossi, Karen Neder, Anatoly Pinchuk, Laura Juckem. A novel dynamic delivery system enabling high efficiency transfection of cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 479. doi:10.1158/1538-7445.AM2014-479

A simple and efficient method for transfecting mouse embryonic stem cells using polyethylenimine

Mouse embryonic stem cells (ESCs) can be transfected by electroporation, liposomal reagents, and viral transduction methods. The cationic polymer polyethylenimine (PEI) has been shown to transfect a variety of differentiated mammalian cell types, including mouse ESCs, but existing methods require the use of additional equipment that is not readily accessible to most labs. Here we describe conditions that permit for the efficient transfection of mouse ESCs with low cytotoxicity and without the need for specialized equipment. Our goal was to devise a protocol for the PEI-mediated transfection of mouse ESCs that was comparable in ease to commercial transfection reagents. For these studies, we compared PEI transfection efficiency and cytotoxicity to a well-known liposomal transfection reagent, Lipofectamine2000™ (LF2K), using fluorescence microscopy, flow cytometry, cell viability assays, and Western blotting. We provide evidence that PEI transfection of mouse ESCs compares favorably to LF2K. Our optimized protocol for efficient transfection of mouse ESCs with PEI is detailed in this report.

Behavior of calf Sertoli cells and fibroblast cells transfected with the human HNP-1 gene.

Human neutrophil peptide-1 (HNP-1) is an important defense molecule in neutrophils and Sertoli cells and plays an important role in the blood-testis barrier. In this study, we investigated the behavior of Sertoli cells transfected with the HNP-1 gene and compared the ability of Sertoli cells and fibroblast cells to resist transfection. Total RNA was isolated from human blood. The DNA coding sequence of HNP-1 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and the eukaryotic expression vector pEGFP-N3-HNP-1 was identified by PCR, endonuclease digestion, and sequencing. Bovine Sertoli cells and fibroblast cells were transfected with pEGFP-N3-HNP-1 using Liposome reagent. The transfection efficiency and the behavior of the transfected cells were evaluated at 24, 48 and 72 h as well as at other times after transfection. The plasmid pEGFP-N3-HNP-1 was successfully constructed. The cells achieved maximum transfection efficiency at 48 h. Two weeks after transfection, the cells began to stop dividing. The ability of Sertoli cells to resist transfection was higher compared to fibroblast cells. The ability of the 2 cell types to resist transfection was higher with plasmid pEGFP-N3-HNP-1 than with the plasmid pEGFP-N3. The injury to Sertoli cells caused by transfection with the HNP-1 gene was less pronounced than in fibroblast cells, which may be closely correlated with the physiological function of Sertoli cells.

CXCR4 transfection of cord blood mesenchymal stromal cells with the use of cationic liposome enhances their migration toward stromal cell–derived factor-1

Background aimsThe interaction between stromal cell–derived factor (SDF)-1 and its receptor CXCR4 is one of the mechanisms by which mesenchymal stromal cells (MSCs) are recruited to sites of injury. SDF-1 is upregulated in damaged tissues, but because the surface expression of CXCR4 on cultured MSCs is low, we investigated whether the delivery of CXCR4 into MSCs with the use of the cationic liposomal reagent IBAfect would increase their migration toward SDF-1. MethodsWe examined (i) the effect of MSC confluency, passage number, duration of transfection and amount of IBAfect and plasmid on transfection efficiency as determined by flow cytometric analysis of CXCR4 and (ii) whether IBAfect-mediated CXCR4 transfection affected the viability, proliferation and differentiation of MSCs as well as their response toward an SDF-1 gradient in a trans-Matrigel migration assay. ResultsWe found that transfection efficiency of up to 40% was achieved after 24-h transfection of 50% confluent MSCs (at passage 4) with an IBAfect:plasmid ratio of 3.6 μL:0.6 μg, and CXCR4 transcript expression in transfected MSCs was 105-fold higher than in non-transfected cells. Transfected MSCs retained their ability to differentiate to osteocytes and chondrocytes but had lower proliferation. Importantly, overexpression of surface CXCR4 with the use of IBAfect significantly increased (>3-fold) the number of cells migrating toward an SDF-1 gradient relative to cells migrating to media alone, compared with non-transfected cells (1.3-fold). ConclusionsOur results suggest that IBAfect-mediated delivery of CXCR4 into MSCs is a highly efficient technique that may be useful for enhancing the recruitment of systemically infused MSCs for tissue repair.

Enhancing the Migration Ability of Mesenchymal Stromal Cells by Targeting the SDF-1/CXCR4 Axis

Mesenchymal stromal cells (MSCs) are currently being investigated in numerous clinical trials of tissue repair and various immunological disorders based on their ability to secrete trophic factors and to modulate inflammatory responses. MSCs have been shown to migrate to sites of injury and inflammation in response to soluble mediators including the chemokine stromal cell-derived factor-(SDF-)1, but during in vitro culture expansion MSCs lose surface expression of key homing receptors particularly of the SDF-1 receptor, CXCR4. Here we review studies on enhancement of SDF-1-directed migration of MSCs with the premise that their improved recruitment could translate to therapeutic benefits. We describe our studies on approaches to increase the CXCR4 expression in in vitro-expanded cord blood-derived MSCs, namely, transfection, using the commercial liposomal reagent IBAfect, chemical treatment with the histone deacetylase inhibitor valproic acid, and exposure to recombinant complement component C1q. These methodologies will be presented in the context of other cell targeting and delivery strategies that exploit pathways involved in MSC migration. Taken together, these findings indicate that MSCs can be manipulated in vitro to enhance their in vivo recruitment and efficacy for tissue repair.

The influence of the structural orientation of amide linkers on the serum compatibility and lung transfection properties of cationic amphiphiles

Understanding the structural parameters of cationic amphiphiles which can influence gene transfer efficiencies of cationic amphiphiles continues to remain important for designing efficient liposomal gene delivery reagents. Previously we demonstrated the influence of structural orientation of the ester linker (widely used in covalently tethering the polar head and the non-polar tails) in modulating in vitro gene transfer efficiencies of cationic amphiphiles. However, our previously described cationic amphiphiles with ester linkers failed to deliver genes under in vivo conditions. Herein we report on the development of a highly serum compatible cationic amphiphile with circulation stable amide linker which shows remarkable selectivity in transfecting mouse lung. We also demonstrate that reversing structural orientation of the amide linker adversely affects both serum compatibility and the lung selective gene transfer property. Dynamic laser light scattering and atomic force microscopic studies revealed smaller average hydrodynamic sizes of the liposomes of transfection efficient lipid than those for the liposomes of transfection incompetent analog (148 ± 1 nm vs 214 ± 4 nm). Average surface potential of the liposomes of transfection competent amphiphiles were found to be significantly higher than that for the liposomes of transfection incompetent analog (10.7 ± 5.4 mV vs 2.8 ± 1.3 mV, respectively). Findings in fluorescence resonance energy transfer and dye entrapment experiments support lower rigidity and higher biomembrane fusogenicity of the liposomes of the transfection efficient amphiphiles. Importantly, cationic lipoplexes of the novel amide-linker based amphiphile exhibited higher mouse lung selective gene transfer properties than DOTAP, one of the widely used commercially available liposomal lung transfection kits. In summary, the present findings demonstrate for the first time that amide linker structural orientation profoundly influences the serum compatibility and lung transfection efficiencies of cationic amphiphiles.

Dendrimer-Mediated Transfection

INTRODUCTIONThe efficient delivery of exogenous DNA to cells for expression and function studies is an essential technique of modern cell biology. Polymer-based systems can be used to transfect cells, some of which rely on one type of molecule, whereas others contain a mixture of polymers and lipids. Reagents such as SuperFect or PolyFect use activated dendrimers, which are positively charged and resemble snowflakes in structure. One advantage of using dendrimer-based reagents is that they appear to be less sensitive than liposomal reagents to the presence of serum during transfection. Thus, they could be a better choice for cells that show greater dependence on the continual presence of serum (to avoid initiation of stress responses or induction of differentiation). This protocol describes a procedure for dendrimer-mediated transfection.

Effects of antisense oligodeoxynucleotides targeting hypoxia inducible factor 1α gene on huinan glioma cell line U87 in vitro

Objective To investigate the effects of antisense oligodeoxynucleotides （ODNs） targe- ting hypoxia inducible factor-1α（HIF-1α） on the apoptosis, proliferation and invasion of U87 glioma cell line. Methods Antisense ODNs were constructed and transfected into U87 cells by Dosper liposomal reagent. The HIF-1α gene expression was detected by Western blotting, the cell proliferative index was determined by methyl thiazol tetrazolium （MTT） assay, the cells cycle and apoptosis of the cells were examined by flow cytometry and the changes of the U87 cells invasive ability were measured by Transwell chamber. Results The protein expression of HIF-1α in U87 ceils was down-regulated by HIF-1α ASONDN. The cell proliferative index in transfected group, empty vector group and control group was 5.46%, 21.25% and 22. 32% respectively. Transwell chamber assay showed that the cell number in transfected group, empty vector group and control group was （22 ±4）, （124±3） and （122 ±6） respectively; and the apoptosis rate was （53.35 ± 2.80） %, （ 12.02 ± 1.60） %, （ 10. 19 ± 3. 15 ） % respectively, and there was significant difference between transfected group and other groups （ P 〈 0. 05）. Conclusion Silencing the expression of HIF-1α protein can inhibit proliferation and invasion, and promote apoptosis of human glioma U87 cells. HIF-1α is expected to become a target for cancer therapy. Key words: Glioma; HIF-1α; Invasion; Proliferation

Optimalized transient transfection of chondrogenic primary cell cultures

Abstract We aimed to find a transfection method which provides high efficiency with minimal cytotoxic and/or apoptotic effects for gene transfer into multilayer primary chondrogenic cell cultures. The pEGFP-C1 plasmid was introduced into the cell culture and the efficiency of transformation quantified by GFP fluorescence; the resulting nucleofection was effective but resulted in severe apoptosis. Two liposomal reagents designed to allow transfection into adherent cells did not deliver the plasmids sufficiently and cartilage formation did not occur. In addition, a third liposomal compound, recommended for transfection into either adherent or suspension cell cultures, lead to acceptable transfection efficiency but no cartilage formation. When an amphiphilic reagent was used however, there was acceptable transfection efficiency as well as cartilage formation. The viability of the cells which were transfected using the amphiphilic reagent remained unaffected but proliferation was severely diminished, particularly in the presence of GFP. In addition, the amount of cartilage decreased when GFP was expressed, despite unchanged levels of mRNAs of sox9 and aggrecan core protein, factors reflecting on the efficiency of chondrogenesis. Overexpression of both the constitutively active delta and gamma isoforms of catalytic subunit of calcineurin, a protein phosphatase described as a positive regulator of chondrogenesis, decreased protein level of Sox9 and subsequent cartilage formation. In conclusion, we found that amphiphilic reagent applied prior to the adhesion of cells provides a useful means to transfer plasmids to primary differentiating chondrogenic cells.

Tissue inhibitor of metalloproteinase-2 inhibits ameloblastoma growth in a new mouse xenograft disease model

Ameloblastomas are odontogenic neoplasms characterized by local invasiveness. This study was conducted to develop a new animal model of ameloblastoma and to address the role of tissue inhibitor of metalloproteinase-2 (TIMP-2) and matrix metalloproteinase-2 (MMP-2) in the growth and invasiveness of ameloblastomas. Donated fresh human ameloblastoma tissue was finely minced, screened, and subcutaneously implanted in three locations on each of 10 BALB/c-nu/nu nude mice. Newly established tumors on each mouse were injected with: (i) transfection reagent; (ii) liposome and transfection reagent; or (iii) liposome, transfection reagent, and the expression plasmid pcDNA3.1(+)/green fluorescent protein (GFP)-TIMP-2. Tumors were monitored for 5 weeks and excised for histopathology, RNA, and protein analyses. The ameloblastoma xenografts were established with high frequency and contained a variety of typical features, validating this new model system. Xenografts injected with the TIMP-2 expression plasmid showed reduced growth, increased TIMP-2 mRNA and protein, and decreased MMP-2 protein compared with the control groups. We successfully established a new experimental model of ameloblastoma consisting of subcutaneous human xenografts in nude mice. In addition, we demonstrated the successful introduction of the TIMP-2 gene in tumor xenograft cells in vivo, resulting in xenograft growth inhibition. This growth inhibition may have resulted from TIMP-2 overexpression specifically inhibiting MMP-2 protein expression and activity.

135 Dramatic enhancement of uptake and trafficking of HIV-1 Tat protein via modulation of endocytosis pathways

HIV-1 Tat protein can be secreted from infected cells and taken up by other target cells. Tat uptake and trafficking has been widely investigated. This property is also exploited for drug delivery. We initially confirmed that Tat mainly entered Hela cells by clathrin-dependent endocytosis and was degraded in lysosomes, whereas chloroquine could inhibit the degradation and enhanced its trafficking to the nucleus. Interestingly, a similar effect was noted in presence of chlorpromazine (CPZ). Most likely, the uptake of Tat was shifted to caveolar endocytosis after the clathrin pathway was blocked by CPZ. However, the LTR transactivation of Tat could be enhanced by at least 50 fold when a liposome reagent was introduced into the Tat delivery system. Further evidence showed that liposome reagents could switch the Tat uptake from clathrin-dependent endocytosis to lipid raft pathway and bypass the degradation in lysosomes. This was supported by both pharmacological assays using inhibitors of caveolae-mediated endocytosis (MβCD, etc.) or microtubules (cytochalasin D & lutrunculin A) and co-localization using Tat101venus and immunstaining with anti-clathrin1, anti-caveolin1, anti-EEA1, anti-Lamp1 or anti-Rab7. Subsequently, chemical molecules carrying “+” or “−” charges were tested for blocking the Tat uptake facilitated with the liposome reagent, including macromolecules (e.g., heparin, polybrene) and small molecules (e.g., spermine). Both “+” and “-” charged molecules could inhibit the uptake of Tat, but the “+” molecules actually attached to the membrane carrying “-” charges and the “-” molecules bound to the Tat-liposome complex with “+” charges. However, the Tat-liposome complex was probably formed by hydrophobic bonds linking liposome's lipid to Tat hydrophobic domain because both Tat and the liposome reagent carried “+”charges. A peptide containing partial hydrophobic domain of Tat (CFITKGLGISYGRKK) could competitively inhibit the uptake of Tat-liposome complex. In conclusion, the liposome reagent could facilitate Tat protein to traverse the membrane by changing endocytosis pathways and significantly enhance its delivery to the nucleus in a lysosome-independent manner. These observations have important implications for elucidating the mechanism of Tat uptake and trafficking and exploring drug delivery.

Supramolecular assemblies of zwitterionic nanoliposome-polynucleotide complexes as gene transfer vectors: Nanolipoplex formulation and in vitro characterisation

Synthetic gene transfer vectors based on zwitterionic nanoliposome-DNA assemblies (nanolipoplexes), formed by the mediation of magnesium ions, were prepared by a scalable method without employing volatile solvents, high-shear force treatments or extrusion. The zwitterionic nanolipoplexes (NLP) were formulated with PC (phosphatidylcholine) and DPPC (a natural lung surfactant) incorporating different amounts of cholesterol (CHOL). The resulting structures were characterised in terms of their morphology, size and DNA content. In addition, the toxicity and transfection efficiency of the nanolipoplexes were evaluated in cultured Chinese hamster ovary-K1 (CHO-K1) cells. The effects of the multivalent cation Mg2+ on nanoliposome-DNA transfection potency were evaluated. Formulations containing 10% CHOL showed maximum transfection efficiency and the optimum amount of Mg2+ ions for transfection with minimum cytotoxicity was ca. 20 mM. The zwitterionic formulations showed significantly less cytotoxicity compared to a commercially available cationic liposome reagent or polyethylenimine (PEI) while they were superior in terms of gene transfer potency. The zwitterionic vectors formulated in this study avoid the use of toxic cationic lipids as well as toxic solvents and may have potential application in gene therapy. The new method will enable scale-up and manufacture of safe and efficacious transfection vehicles required for preclinical and clinical studies. Based on the advantages and superiority of the formulated nanolipoplexes, this method allows for the acceleration of nanolipoplex formulation, enabling the rapid development and evaluation of novel carrier systems for genes and other drugs.