• Title/Summary/Keyword: DNA delivery

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Polyethyleneimine based Delivery System Coated with Hyaluronate Amine for Improved pDNA Transfection Efficiency (개선된 플라스미드 DNA 전달 효율을 위한 히알루론 아민 코팅 폴리에틸렌이민 기반 전달 시스템)

  • Oh, Kyoung-yeon;Jang, Yongho;Lee, Eunbi;Kim, Tae-ho;Kim, Hyuncheol
    • Applied Chemistry for Engineering
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    • v.33 no.1
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    • pp.83-89
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    • 2022
  • Since the pandemic of COVID-19, active investigation to develop immunity to infectious disease by delivering nucleic acids has been proceeded. Particularly, many studies have been conducted on non-viral vector as several vital side-effects which were found on nucleic acid delivery system using viral vectors. In this study, we have developed plasmid DNA (pDNA) loaded-hyaluronic acid derivative (HA) coated-polyethyleneimine (PEI) based polyplex for enhanced nucleic acid delivery efficiency. We have optimized the ratio of pDNA : PEI : HA by measuring size and protein transcription efficiency. The final product, polyplex-HA, was characterized through measuring size, zeta-potential and TEM image. Intracellular uptake and protein transcription efficiency were compared to commercially available transfection reagent, lipofectamine, through fluorescence image and flow cytometry. In conclusion, polyplex-HA presents a novel gene delivery system for efficient and stable protein transcription since it is available for delivering various genetic materials and has less immunoreactivity.

New Cationic Liposome with Enhanced Stability and Transfection Efficiency for Gene Delivery (안정성 및 Transfection 효율이 우수한 양이온성 리포좀 유전자 전달시스템의 개발)

  • Kim, Kyoung-Mi;Nam, Bang-Hyun;Sohn, Dong-Hwan
    • Journal of Pharmaceutical Investigation
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    • v.28 no.2
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    • pp.93-98
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    • 1998
  • We have developed liposomes which can be easily prepared with inexpensive lipid, have enhanced stability, and can efficiently deliver DNA into the COS-l cells, Liposome formulations were prepared using cationic materials such as dimethyldioctadecyl ammonium bromide (DDAB), cetyltrimethyl ammonium bromide(CTAB), We investigated the effect of cationic liposome formulations on in vitro DNA transfection, DDAB-containing liposomes showed increased transfection efficiency which was 3.2-fold as much as that by $Lipofectin^{\circledR}$, but CTAB-containing liposomes were inactive in gene transfection. The effect of colipid of DDAB-containing liposome was also investegated. As a colipid, dioleylphosphatidylethanolamine(DOPE) and cholesterol did altered the transfection efficiency of DDAB-containing liposomes. And increased DDAB concentration lowered the transfection efficiency. The optimum amount of liposomal formulation was $10\;{\mu}M$ for $1\;{\mu}g$ of DNA. In the experiment of stability, DOPE-containing liposomes formulation showed a broad size distribution and separation of two major peaks on a 5th day of preparation, but liposomes containing cholesterol was stable for 10 days. DDAB-containing liposomal DNA delivery system was prepared easily and was stable.

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Chitosan and Its Derivatives for Gene Delivery

  • Lee, Knen-Yong
    • Macromolecular Research
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    • v.15 no.3
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    • pp.195-201
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    • 2007
  • Non-viral vectors, including lipid- or polymer-based systems, have attracted much attention to date as a gene delivery vehicle, due to safety issues with viral vectors. Chitosan, a naturally existing cationic polymer, has shown great potential as a gene delivery carrier, as it has low immunogenicity and toxicity, excellent transcellular transport ability, and is relatively easy to chemically modify. This review summarizes and discusses the general features of chitosan and its applications as a delivery carrier of DNA and RNA.

Immunogenicity of a DNA and Recombinant Protein Vaccine Combining LipL32 and Loa22 for Leptospirosis Using Chitosan as a Delivery System

  • Umthong, Supawadee;Buaklin, Arun;Jacquet, Alain;Sangjun, Noppadol;Kerdkaew, Ruthairat;Patarakul, Kanitha;Palaga, Tanapat
    • Journal of Microbiology and Biotechnology
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    • v.25 no.4
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    • pp.526-536
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    • 2015
  • Leptospirosis is a worldwide zoonotic disease caused by pathogenic Leptospira, a genus of which more than 250 serovars have been identified. Commercial bacterin vaccines are limited in that they lack both cross-protection against heterologous serovars and long-term protection. This study investigated in mice the immunogenicity of an anti-leptospirosis vaccine, using the outer membrane proteins LipL32 and Loa22 as antigens. The immunogenicity of this vaccine formulation was compared with those induced by vaccines based on LipL32 or Loa22 alone. A DNA-encapsulated chitosan nanoparticle was used for in vivo DNA delivery. Using a unique DNA plasmid expressing both lipL32 and loa22 for vaccination, higher antibody responses were induced than when combining plasmids harboring each gene separately. Therefore, this formulation was used to test the immunogenicity when administered by a heterologous prime (DNA)-boost (protein) immunization regimen. The specific antibody responses against LipL32 (total IgG and IgG1) and Loa22 (IgG1) were higher in mice receiving two antigens in combination than in those vaccinated with a single antigen alone. Although no significant difference in splenic CD4+ T cell proliferation was observed among all groups of vaccinated mice, splenocytes from mice vaccinated with two antigens exhibited higher interferon-γ and IL-2 production than when using single antigens alone upon in vitro restimulation. Taken together, the immunogenicity induced by LipL32 and Loa22 antigens in a heterologous primeboost immunization regimen using chitosan as a DNA delivery system induces higher immune response, and may be useful for developing a better vaccine for leptospirosis.

The outcome of perinatal prophylaxis for HBeAg positive mothers according to the maternal HBV-DNA levels at the delivery time (HBeAg 양성 산모의 분만 직후 HBV-DNA 수치에 따른 주산기 예방조치의 결과)

  • Jeong, On;Kim, Jong-Hyun
    • Clinical and Experimental Pediatrics
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    • v.50 no.4
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    • pp.348-354
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    • 2007
  • Purpose : Perinatal hepatitis B viral infection is decreasing; however, 10% of babies to HBeAg positive mothers still become chronic carriers despite perinatal prophylaxis. Although, the cause of prophylaxis failure is still unclear, an importance of maternal HBV-DNA level at the delivery time has been suggested. This study was established to certify if it would be a useful predictable factor for the outcome of perinatal prophylaxis. Methods : Twenty-nine HBeAg positive mothers whose babies had known outcomes of prophylaxis were selected. To determine the amount of maternal HBV-DNA, a quantitative PCR was performed with the WHO International Standard for HBV DNA NAT assays. Results : The mean logarithm HBV-DNA level of mothers with failed outcomes was significantly higher than that of mothers with succeessful outcomes (7.99 vs. 6.72, P=0.015). The predictable maternal HBV-DNA cut-off level to prophylaxis outcome was $2.83{\times}10^7copies/mL$ (100 pg/mL). None out of the case 16 (0%) who had below this level, and 5 out of 13 (38.5%) who had above this level of maternal HBV-DNA failed in perinatal prophylaxis. Conclusion : Mothers with higher levels of HBV-DNA at delivery time would be prone to a worse outcome of prophylaxis using the conventional approach. Perinatal prophylaxis failure rate can be reduced, if we try to introduce more potent prophylactic treatment into the cases with this risk factor.

Antiviral Efficacy of a Short PNA Targeting microRNA-122 Using Galactosylated Cationic Liposome as a Carrier for the Delivery of the PNA-DNA Hybrid to Hepatocytes

  • Kim, Hyoseon;Lee, Kwang Hyun;Kim, Kyung Bo;Park, Yong Serk;Kim, Keun-Sik;Kim, Dong-Eun
    • Bulletin of the Korean Chemical Society
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    • v.34 no.3
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    • pp.735-742
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    • 2013
  • Peptide nucleic acids (PNAs) that bind to complementary nucleic acid sequences with extraordinarily high affinity and sequence specificity can be used as antisense oligonucleotides against microRNAs, namely antagomir PNAs. However, methods for efficient cellular delivery must be developed for effective use of PNAs as therapeutic agents. Here, we demonstrate that antagomir PNAs can be delivered to hepatic cells by complementary DNA oligonucleotide and cationic liposomes containing galactosylated ceramide and a novel cationic lipid, DMKE (O,O'-dimyristyl-N-lysyl glutamate), through glycoprotein-mediated endocytosis. An antagomir PNA was designed to target miR-122, which is required for translation of the hepatitis C virus (HCV) genome in hepatocytes, and was hybridized to a DNA oligonucleotide for complexation with cationic liposome. The PNA-DNA hybrid molecules were efficiently internalized into hepatic cells by complexing with the galactosylated cationic liposome in vitro. Galactosylation of liposome significantly enhanced both lipoplex cell binding and PNA delivery to the hepatic cells. After 4-h incubation with galactosylated lipoplexes, PNAs were efficiently delivered into hepatic cells and HCV genome translation was suppressed more than 70% through sequestration of miR-122 in cytoplasm. PNAs were readily released from the PNA-DNA hybrid in the low pH environment of the endosome. The present study indicates that transfection of PNA-DNA hybrid molecules using galactosylated cationic liposomes can be used as an efficient non-viral carrier for antagomir PNAs targeted to hepatocytes.

Magnetofection is an efficient tool for ectopic gene expression into oral cells

  • Ji, Jae-Hoon;Ko, Seon-Yle;Jang, Young-Joo
    • International Journal of Oral Biology
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    • v.32 no.1
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    • pp.7-11
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    • 2007
  • It is difficult to introduce DNA in non-invasive manner into oral cancer cells as well as primary cells for gene manipulation and expression in vivo. So far, several methods for a gene delivery have been performed to solve this problem. Magnetofection is one of the recent methods for gene transfer, and nanoparticles are applied under a magnetic field for DNA delivery. We investigated whether the magnetofection increases the efficiency of a gene delivery into several oral cell lines. By using a plasmid coding the green fluorescent protein (GFP), the efficiency of gene transfer by magnetofection was compared with those by using the calcium phosphate and the commercial transfection agent. Indeed, the magnetofection increased the green fluorescent signal in cells, suggested that this method apparently enhance the efficiency of gene delivery without any defects in various oral cancer cell lines. Finally, we have shown that magnetofection can be a useful technique for gene delivery to difficult-to-transfect cells to perform a functional study of genes in vivo.

Enhancement of Gene Delivery Using Novel Homodimeric Tat Peptide Formed by Disulfide Bond

  • Lee, Soo-Jin;Yoon, Sung-Hwa;Doh, Kyung-Oh
    • Journal of Microbiology and Biotechnology
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    • v.21 no.8
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    • pp.802-807
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    • 2011
  • Cationic liposomes have been actively used as gene delivery vehicle because of their minimal toxicity, but their relatively low efficiency of gene delivery is the major disadvantage of these vectors. Recently, cysteine residue incorporation to HIV-1 Tat peptide increased liposomemediated transfection compared with unmodified Tat peptide. Therefore, we designed a novel modified Tat peptide having a homodimeric (Tat-CTHD, Tat-NTHD) and closed structure (cyclic Tat) simply by using the disulfide bond between cysteines to develop a more efficient and safe nonviral gene delivery system. The mixing of Tat-CTHD and Tat-NTHD with DNA before mixing with lipofectamine increased the transfection efficiency compared with unmodified Tat peptide and lipofectamine only in MCF-7 breast cancer cells and rat vascular smooth muscle cells. However, cyclic Tat did not show any improvement in the transfection efficiency. In the gel retardation assay, Tat-CTHD and Tat-NTHD showed more strong binding with DNA than unmodified Tat and cyclic Tat peptide. This enhancement was only shown when Tat-CTHD and Tat-NTHD were mixed with DNA before mixing with lipofectamine. The effects of Tat- CTHD and Tat-NTHD were also valid in the experiment using DOTAP and DMRIE instead of lipofectamine. We could not find any significant cytotoxicity in the working concentration and more usage of these peptides. In conclusion, we have designed a novel transfection-enhancing peptide by easy homodimerization of Tat peptide, and the simple mix of these novel peptides with DNA increased the gene transfer of cationic lipids more efficiently with no additional cytotoxicity.

Biodistribution and Genotoxicity of Transferrin-Conjugated Liposomes/DNA Complexes in Mice

  • Lee Sang Mi;Kim Jin-Seok;Oh Yu-Kyoung;Lee Yong-Bok;Sah Hongkee
    • Macromolecular Research
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    • v.13 no.3
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    • pp.218-222
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    • 2005
  • Transferrin-conjugated liposomes ($T_f$-liposomes) were made and formulated with pCMVluc DNA to form a lipoplex. Among the various formulations studied, the $T_f$-liposome: pCMVluc DNA complex at a ratio of 5: 1 (wt/wt) showed the highest transfection efficiency, which was twice that of $Lipofectin^{TM}$ on HeLa cells. The maxi-mum tolerated dose (MTD) of this lipoplex formulation from a single intravenous injection was over 10 mg/kg in healthy ICR mice. The RT-PCR results showed that the highest level of luciferase mRNA was detected in the lungs, followed by the liver, spleen, heart and kidneys, after an intravenous injection into mice. Two weeks after the injection, the levels of luciferase mRNA decreased gradually in the liver, spleen, heart, and kidney, but not in the lungs. The micro-array study showed that the cancer-related genes, including the bcl 6 gene, were highly up-regulated by the treatment with $T_f$-liposome/ pCMVluc DNA complex on HeLa cells, indicating that there were possible interactions between the host chromosomal DNA and the $T_f$-liposome within the cells. The results obtained from this study are expected to be useful for designing a safe and efficient gene delivery system using transferrin-conjugated liposomes.

Production of virus-like particles of nervous necrosis virus displaying partial VHSV's glycoprotein at surface and encapsulating DNA vaccine plasmids

  • Yang, Jeong In;Bessaid, Mariem;Kim, Ki Hong
    • Journal of fish pathology
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    • v.33 no.2
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    • pp.103-109
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    • 2020
  • In order to use nervous necrosis virus (NNV) virus-like particles (VLPs) as a delivery tool for heterologous antigens or plasmids, we attempted to produce red-spotted grouper nervous necrosis virus (RGNNV) VLPs displaying a partial region of viral hemorrhagic septicemia virus (VHSV) glycoprotein at the surface and VLPs that are harboring DNA vaccine plasmids within the VLP. A peptide encoding 105 amino acids of VHSV glycoprotein was genetically inserted in the loop region of NNV capsid gene, and VLPs expressing the partial part of VHSV glycoprotein were successfully produced. However, in the transmission electron microscope analysis, the shape and size of the partial VHSV glycoprotein-expressing NNV VLPs were irregular and variable, respectively, indicating that the normal assembly of capsid proteins was inhibited by the relatively long foreign peptide (105 aa) on the loop region. To encapsulate by simultaneous transformation with both NNV capsid gene expressing plasmids and DNA vaccine plasmids (having an eGFP expressing cassette under the CMV promoter), NNV VLPs containing plasmids were produced. The encapsulation of plasmids in the NNV VLPs was demonstrated by PCR and cells exposed to the VLPs encapsulating DNA vaccine plasmids showed fluorescence. These results suggest that the encapsulation of plasmids in NNV VLPs can be done with a simple one-step process, excluding the process of disassembly-reassembly of VLPs, and NNV VLPs can be used as a delivery tool for DNA vaccine vectors.