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Enhancement of Gene Delivery Using Novel Homodimeric Tat Peptide Formed by Disulfide Bond
( Soo Jin Lee ),( Sung Hwa Yoon ),( Kyung Oh Doh ) 한국미생물 · 생명공학회 2011 Journal of microbiology and biotechnology Vol.21 No.8
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 liposome mediated 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.
Park, Ju-Young,Nam, Yoon-Sung,Kim, Jun-Oh,Han, Sang-Hoon,Chang, Ih-Seop The Korean Society of Pharmaceutical Sciences and 2004 Journal of Pharmaceutical Investigation Vol.34 No.2
This work aims at examining the cellular uptake behavior of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles derivatized with a protein transduction domain (PTD) using HeLa cells. For this purpose, $Tat_{49-57}$ peptide derived from transcriptional activation (Tat) protein of HIV type-1 was covalently conjugated to the terminal end of PLGA. Nanoparticles were ten prepared with the $Tat_{49-57}-PLGA$ conjugates by a spontaneous phase inversion method. The prepared particles had a mean diameter of ca. 84 nm, as measured by dynamic light scattering. The interaction of the Tat-PLGA nanoparticles with cells was examined by using confocal laser scanning microscopy. It was found tat Tat-PLGA nanoparticles incubated with HeLa cells could efficiently translocate into cytoplasm, while plain PLGA nanoparticles showed negligible cellular uptake. In addition, even at $4^{\circ}C$ or in the presence of sodium azide significant cellular internalization of Tat-PLGA nanoparticles was still observed. These results indicate that a non-endocytotic translocation mechanism might be involved in the cellular uptake of Tat-PLGA nanoparticles.
Tat Peptide에 의한 Cu-Peptide의 경피약물전달 증진 효과 연구
신문삼 한국공업화학회 2018 한국공업화학회 연구논문 초록집 Vol.2018 No.0
바이러스 세포감염 원리를 이용한 세포투과 펩티드에 대해 많은 연구가 진행되어 왔고, 세포막 구멍보다 큰 HIV 바이러스가 세포막을 급격하게 투과할 수 있는 것은 HIV 바이러스가 약물전달용 펩티드(Tat-Peptide)를 만들어내어 세포막을 투과하며 Tat-Peptide는 86개의 아미노산으로 구성되어 있고 약물전달을 나타내는 핵심 아미노산 배열은 47번~57번 아미노산이다. 또한 전통적인 주름개선 기능성 화장품 소재로는 Cu-Peptide 등의 펩티드 성분이 사용되고 있지만, 진피까지 약물전달이 되지 않아 효능효과에 많은 제약이 존재한다. 바이러스 세포감염 원리의 약물전달용 펩티드(Tat-Peptide)를 피부에 적용하여 약물전달효과를 측정하였고 그 대상은 Cu-Peptide의 주름개선 펩티드를 이용하였다. 약물전달용 펩티드의 자체 경피 흡수는 대조군에 비해 8배 이상의 효과를 확인하였고, 주름개선 펩티드에 약물전달용 펩티드를 함께 적용시 주름개선 펩티드의 약물전달이 Cu-Peptide의 경우는 3배의 경피 약물전달 증가효과를 확인하였고 이를 통해 주름개선 화장품으로의 적용가능성을 확인할 수 있었다.
이주연,설양조,조인호,이승진,김경화,Victor C. Yang,정창평,박정윤 한국생체재료학회 2006 생체재료학회지 Vol.10 No.4
Bone marrow stromal cells (BMSC) hold promise for osteogenic differentiation and can be augmented by the application of genes encoding bone morphogenetic proteins (BMPs). Several gene delivery approaches have been employed to these tissue engineering attempts. Despite some successes of the gene delivery protocol, however, both virus-based delivery and cationic lipid based delivery system have been beset by the immune response as well as inflammatory reaction to those applied delivery carriers. An alternative approach involving complexation of gene products with cell penetrating peptides seems promising. Herein, plasmid DNA coding BMP was condensed with cell penetrating peptide, TAT, and examined internalization, gene expression level as well as differentiation in BMSC. Synthetically prepared TAT peptides were able to condense and form complex with the BMP-coding pDNA, and efficiently transferred the pDNA into nucleus and cytoplasm in a short time period. The produced amount of BMP from the BMSC was higher when cells were treated with TAT-pDNA complex thereby inducing marked mineralization of the cultures. Taken together, the present study suggested that cell penetrating peptide TAT could be a useful and safe tool for enhancing delivery of BMP gene into stem cells, which opens wide applicability in the regenerative therapeutic strategy.
Park, Ju Young,Nam, Yoon Sung,Kim, Junoh,Han, Sang-Hoon,Chang, Ih-Seop 한국약제학회 2004 Journal of Pharmaceutical Investigation Vol.34 No.2
This work aims at examining the cellular uptake behavior of poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles derivatized with a protein transduction domain (PTD) using HeLa cells. For this purpose, Tat_(49-57) peptide derived from transcriptional activation (Tat) protein of HIV type-1 was covalently conjugated to the terminal end of PLGA. Nanoparticles were then prepared with the Tat_(49-57)-PLGA conjugates by a spontaneous phase inversion method. The prepared particles had a mean diameter of ca. 84 nm, as measured by dynamic light scattering. The interaction of the Tat-PLGA nanoparticles with cells was examined by using confocal laser scanning microscopy. It was found that Tat-PLGA nanoparticles incubated with HeLa cells could efficiently translocate into cytoplasm, while plain PLGA nanoparticles showed negligible cellular uptake. In addition, even at 4℃ or in the presence of sodium azide significant celluar internalization of Tat-PLGA nanoparticles was still observed. These results indicate that a non-endocytotic translocation mechanism might be involoved in the cellular uptake of Tat-PLGA nanoparticles.
최영숙,박윤정,이승진,이주연,권영민,이원규,정창평,Victor C. Yang 한국생체재료학회 2006 생체재료학회지 Vol.10 No.4
Targeted down-regulation of specific gene using antisense oligonucleotide (ODN) have gained considerable interests as therapeutic genes. The potential specificity for target gene binding and consequent inhibition of gene products makes antisense ODN attractive new class of drugs for broad clinical applications. The polyanionic charges carried by these antisense ODNs, however, present a limitation to efficient cellular uptake and consequent biological effects on gene regulation. To overcome this obstacle, a well-designed carrier system is desirable for antisense delivery. Herein the authors describe the synthesis, cellular translocation of carrier peptide and peptide-antisense ODN comprised of the cell-penetrating peptide (CPP) including TAT and protamine fragment termed as LMWP. The prepared LMWP and TAT peptide as well as their complexes with antisense ODN, for example, bcl-2 antisense ODN, were examined for cellular uptake behaviors and subcellular localization by using confocal microscopy and flow cytometry. Fluorescently tagged ODN were localized with the peptide in the cytoplasm shortly after incubation of peptide-ODN with MCF-7 breast tumor cells. The increased cell uptake achieved using the peptide compared to incubation of free ODN with cells resulted in significant down regulation of bcl-2 mRNA expression. Taken together, ODN can be delivered into the cells using cell penetrating peptides, thereby inducing marked suppression of target gene expression.
남윤성,Ju Young Park,Junoh Kim,Sang-Hoon Han,Ih-seop Chang 한국약제학회 2004 Journal of Pharmaceutical Investigation Vol.34 No.2
This work aims at examining the cellular uptake behavior of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles derivatized with a protein transduction domain (PTD) using HeLa cells. For this purpose, Tat49-57 peptide derived from transcriptional activation (Tat) protein of HIV type-1 was covalently conjugated to the terminal end of PLGA. Nanoparticles were then prepared with the Tat49-57 -PLGA conjugates by a spontaneous phase inversion method. The prepared particles had a mean diameter of ca. 84 nm, as measured by dynamic light scattering. The interaction of the Tat-PLGA nanoparticles with cells was examined by using confocal laser scanning microscopy. It was found that Tat-PLGA nanoparticles incubated with HeLa cells could efficiently translocate into cytoplasm, while plain PLGA nanoparticles showed negligible cellular uptake. In addition, even at 4oC or in the presence of sodium azide significant cellular internalization of Tat-PLGA nanoparticles was still observed. These results indicate that a non-endocytotic translocation mechanism might be involved in the cellular uptake of Tat-PLGA nanoparticles.
Jung, H.S.,Lim, K.S.,Kim, M.J.,Hwang, Y.H.,Yoo, C.,Lee, Y.k.,Kim, Y.H.,Lee, D.Y. Elsevier Science Publishers 2013 Journal of controlled release Vol.172 No.3
Subcutaneous site is ideal for clinical islet transplantation because it has the advantage of simple operation procedure under local anesthesia and can be biopsied when needed. However, the transplantation outcomes at subcutaneous site have been disappointing due to hypoxia-induced oxidative stress by poor vascularization. We hypothesized that subcutaneously transplanted islets would have hypoxia resistance by using internalization of metallothionein (MT), an antioxidant scavenging enzyme, which was mediated by fusion between MT and cell penetrating Tat peptide. The Tat-MT was dose-dependently transduced into islets without any damage. Tat-MT-treated islets could be protected from oxidative stress induced by intracellular nitric oxide donor, sodium nitroprusside (SNP). When Tat-MT-treated islets were subcutaneously transplanted into diabetic nude mice, they normally controlled the blood glucose levels without severe fluctuation (median survival time (MST): >30days), whereas most untreated islets were rejected (MST 17days). From the intraperitoneal glucose tolerance test 5days after posttransplantation, glucose responsiveness of Tat-MT-treated islets was similar to that of normal healthy mice, while untreated islets had delayed glucose responsiveness. From the results of immunohistochemical stain, Tat-MT-treated islets had strong anti-insulin positive cells and lower anti-HIF-1α positive cells. However, untreated islets had rare anti-insulin positive cells and strong anti-HIF-1α-positive cells. Collectively, these findings demonstrated that Tat-MT delivery into islet could offer a new strategy for successful islet transplantation under subcutaneous space.
HIV-1 Tat-mediated protein transduction of human brain creatine kinase into PC12 cells
( Min Seop Jeong ),( Dae Won Kim ),( Min Jung Lee ),( Yeom Pyo Lee ),( So Young Kim ),( Sun Hwa Lee ),( Sang Ho Jang ),( Kil Soo Lee ),( Jin Seu Park ),( Tae Cheon Kang ),( Sung Woo Cho ),( Oh Shin Kw 생화학분자생물학회 2008 BMB Reports Vol.41 No.7