• Asian Pacific Journal of Cancer Prevention
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• 제16권17호
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• pp.7749-7754
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• 2015

Background: Currently, cationic liposome has become the commonly used vehicles for gene transfection. Furthermore, one of the most significant steps in microRNAs expression studies is transferring microRNAs into cell cultures successfully. In this study we aim to approach the feasibility of transfection of cervical cancer cell lines mediated by liposome and to obtain the optimized transfection condition for cervical cancer cell lines. Materials and Methods: $Lipofectamine^{TM}2000$ as the carrier, miR-101 mimic was transfected into Hela cells and Siha cells. Using green fluorescent protein as reporter gene, to set different groups according to cell seeding density, the amount of miRNA, miRNA and the proportion of Liposomes, Whether to add serum into medium to study their impact on the liposomal transfection efficiency. Finally, MTT assay was used to analyze the relative minimal cell toxicity of liposome reagents. Results: The seeding density of Hela cell line and Siha are $1.5{\times}10^4$ (per well of 24 well plates), miRNA amount is 1ul of both, the ratio of miRNA and liposome is 1:0.5 of Hela cell line; 1:0.7 of Siha cell line respectively, after 24 hours we can get the highest transfection efficiency. Compared with serum medium, only Siha cells cultured with serum-free medium obtained higher transfection efficiency before transfection (P<0.01). MTT assay showed that according to the above conditions which has the lowest cytotoxicity. Conclusions: The method of Liposome to transfected is a suitable way and it can be an efficient reagent for miRNA delivery for Hela cells and Siha cells in vitro. It may serve as a reference for the further research or application.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.98-98
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• 2003

Direct gene transfer to mammalian tissues has significant potential for gene therapy and transgenesis. Liposome-mediated in vivo transfection has begun to gain attention as an alternative to viral vectors, and may also be a good mode of transfection in gene transfer. Interestingly, polymerized cationic liposomes are reported to be very stable in the bloods and efficient for in vivo gene transfer. To examine a possible gene delivery in vivo, we investigated the efficacy and safety of the liposome-mediated gene transfer using vein injection in chick or mouse as model animals. The number of injected pGFP-LacZ using either a commercial or home-made liposomes was 8 and 19 at 16 and 7 day of hatch, respectively. One of injected chick of each experiments was analyzed and the rest is being bred. In mouse, 4/22 showed expression of pGFP-LacZ but 8/22 showed no expression and the remaining animals are also being bred. After injection of liposome/pGFP-LacZ complex into wing vein of 7 or 16 day-old chick, pGFP-LacZ was detected in various tissues isolated from not only young chick but also old chick were turned out to possess. exogenous DNA. Transcripts and proteins of the transgene were also detected by RT-PCR or histochemical analysis, respectively. These results suggest that injected DNA were inserted to genome and produced mRNA and proteins in various tissues and may give an important tools for effective gene delivery in gene therapy or transgenesis.

• Journal of Microbiology and Biotechnology
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• 제21권8호
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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.

• 폴리머
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• 제37권1호
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• pp.94-99
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• 2013

폴리디아세틸렌(polydiacetylene, PDA)은 자기조립된 디아세틸렌(diacetylene) 단량체의 광중합에 의해 만들어진다. 디아세틸렌 단량체들이 조직적으로 배열되면 254 nm의 자외선 노광에 의해 1,4-첨가 중합이 일어나 고분자 주사슬에 이중결합과 삼중결합이 교대로 존재하는 폴리디아세틸렌이 만들어진다. 폴리디아세틸렌 수용액은 일반적으로 약 640 nm에서 최대흡수파장을 지니는 청색을 띠게 되며 여기에 온도나 pH의 변화, 다른 물질의 결합 등 외부 자극에 의해 약 550 nm의 최대 흡수 파장을 띠는 적색으로 색 전이가 일어나게 된다. 본 연구에서, 우리는 고체상 펩티드 합성을 이용하여 PCDA(10,12-pentacosadyinoic acid) 리포좀의 표면에 양이온성 올리고펩티드를 도입하였다. 또한 다양한 몰 비율로 리포좀 수용액을 제조하여 동물 세포에 트랜스펙션한 결과, 향상된 유전자 전달 효율과 낮은 독성을 보이는 것을 확인하였고, PCDA의 특성을 이용하여 세포에 처리 후 세포 관련 비표지 형광을 관찰하였다.

• Journal of Microbiology and Biotechnology
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• 제18권10호
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• pp.1729-1734
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• 2008

Chitosan, a cationic polysaccharide, has been widely used as a dietary supplement and in a variety of pharmacological and biomedical applications. The antifungal activity and mechanism of action of low molecular weight water-soluble chitosan (LMWS-chitosan) were studied in fungal cells and vesicles containing various compositions of fungal lipids. LMWS-chitosan showed strong antifungal activity against various pathogenic yeasts and hyphae-forming fungi but no hemolytic activity or cytotoxicity against mammalian cells. The degree of calcein leakage was assessed on the basis of lipid composition (PC/CH; 10:1, w/w). Our result showing that LMWS-chitosan interacts with liposomes demonstrated that chitosan induces leakage from zwitterionic lipid vesicles. Confocal microscopy revealed that LMWS-chitosan was located in the plasma membrane. Finally, scanning electron microscopy revealed that LMWS-chitosan causes significant morphological changes on fungal surfaces. Its potent antibiotic activity suggests that LMWS-chitosan is an excellent candidate as a lead compound for the development of novel anti-infective agents.

• Molecules and Cells
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• 제24권2호
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• pp.247-254
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• 2007

Improvement in the pharmacokinetic properties of short interfering RNAs (siRNAs) is a prerequisite for the therapeutic application of RNA interference technology. When injected into mice as unmodified siRNAs complexed to DOTAP/Chol-based cationic liposomes, all 12 tested siRNA duplexes caused a strong induction of cytokines including interferon ${\alpha}$, indicating that the immune activation by siRNA duplexes is independent of sequence context. When modified by various combinations of 2'-OMe, 2'-F, and phosphorothioate substitutions, introduction of as little as three 2'-OMe substitutions into the sense strand was sufficient to suppress immune activation by siRNA duplexes, whereas the same modifications were much less efficient at inhibiting the immune response of single stranded siRNAs. It is unlikely that Toll-like receptor 3 (TLR3) signaling is involved in immune stimulation by siRNA/liposome complexes since potent immune activation by ds siRNAs was induced in TLR3 knockout mice. Together, our results indicate that chemical modification of siRNA provides an effective means to avoid unwanted immune activation by therapeutic siRNAs. This improvement in the in vivo properties of siRNAs should greatly facilitate successful development of siRNA therapeutics.

• 대한의생명과학회지
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• 제9권2호
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• pp.67-74
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• 2003

Viral and non-viral vectors have been used in the delivery of genetic materials into animal cells and tissues, with each approach having pros and cons. Non-viral vectors have many useful merits such as easy preparation, low immunity and size tolerance of a transgene when compared to those of viral vectors. Delivery specificity may be achieved by complex formation between receptor ligands and a non-viral vector. In the present study, non-viral vector systems are investigated in an effort to find a practical delivery means for gene therapy, Receptor-ligand interaction between transferrin-receptor and transferrin was utilized for efficient gene transfer into cancer cells. A plasmid vector, pcDNA3 (LacZ) was ligated with a small duplexed oligo fragment in which a Biotin- VN$^{TM}$ phosphoramidite was placed in the middle of the oligo. The plasmid vector labeled by biotin was then conjugated with biotin-labeled transferrin via streptavidin. This trimeric conjugates were delivered to a hepatoma cell line, HepG2. The delivery efficiency of the trimeric conjugate was 2-fold higher than that of cationic liposomes used for transfection of a plasmid vector. These results demonstrate that a plasmid vector can be efficiently transferred into cells by forming a trimeric complex of plasmid vector-linker-ligand.

• 폴리머
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• 제30권4호
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• pp.279-285
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• 2006

유전자 치료에 있어서 안전성의 장점을 지니고 있는 비바이러스성 전달체에 대한 관심도가 높아져가고 있다. 비바이러스성 전달체 중, 양이온성 리포좀이나 합성 유전자 전달체는 in vitro계에서 효율적인 DNA 전달체이지만, 낮은 생체적합성으로 인하여 in vivo 계에서의 응용성은 크게 뒤떨어지고 있다. 한편, 천연 양이온성 다당류인 키토산은 낮은 독성과 강한 양전하를 띠고 있어 유전자 전달 시스템 (gene delivery system)에 있어 아주 기대되는 전달체이다. 본 연구에서는 저분자량 수용성 키토산 (low molecular water-soluble chitosan ; LMWSC)을 이용하여 동맥 벽 세포를 표적할 수 있는 표적성 유전자 전달체를 합성하였다. 상대 점도와 Kina 적정법을 이용하여 LMWSC의 점도 평균 분자량 $(M_W)$과 탈아세틸화도 (degree of de acetylation ; DDA)를 측정하였고 구조는 FTIR, $^1H-NMR$, 그리고 $^{13}C-NMR$을 통하여 분석하였다. 동맥 벽을 표적하기 위한 유전자 전달체로서 pegylated LMWSC 의 말단에 특이성 세포 표적 펩타이드인 artery wall binding peptide (AWBP)를 결합시킴으로써 AWBP-PEG-g-LMWSC을 합성하였고 FTIR, $^1H-NMR$. zeta potentiometer. 그리고 atomic force microscopy (AFM)을 이용하여 분석하였다.

• Journal of Microbiology and Biotechnology
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• 제28권1호
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• pp.136-144
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• 2018

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. Bacillus Calmette-$Gu\acute{e}rin$ (BCG) vaccine is the only TB vaccine currently available, but it is not sufficiently effective in preventing active pulmonary TB or adult infection. With the purpose of developing an improved vaccine against TB that can overcome the limitations of the current BCG vaccine, we investigated whether adjuvant formulations containing de-O-acylated lipooligosaccharide (dLOS) are capable of enhancing the immunogenicity and protective efficacy of TB subunit vaccines. The results revealed that the dLOS/dimethyl dioctadecyl ammonium bromide (DDA) adjuvant formulation significantly increased both humoral and Th1-type cellular responses to TB subunit vaccine that are composed of three antigens, Ag85A, ESAT-6, and HspX. The adjuvanted TB vaccine also effectively induced the Th1-type response in a BCG-primed mouse model, suggesting a potential as a booster vaccine. Finally, the dLOS/DDA-adjuvanted TB vaccine showed protective efficacy against M. tuberculosis infection in vitro and in vivo. These data indicate that the dLOS/DDA adjuvant enhances the Th1-type immunity and protective efficacy of the TB subunit vaccine, suggesting that it would be a promising adjuvant candidate for the development of a booster vaccine.

• 공업화학
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• 제35권3호
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• pp.230-238
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• 2024

본 연구에서는 인지질계 양쪽성 계면활성제 CDP-W 첨가가 지질 소포체 막과의 상호 작용에 미치는 영향에 관하여 살펴보았다. 이를 위하여 CDP-W 양쪽성 계면활성제와 레시틴 S100-3의 임계 마이셀 농도 및 표면장력 등의 계면 물성을 측정하였다. 또한 pH 변화에 따른 1 wt% 계면활성제 수용액의 제타 전위 측정을 통하여 양쪽성 계면활성제 CDP-W가 양이온 계면활성제에서 음이온 계면활성제로 작용이 전환되는 등전점을 결정하였으며, 이 결과를 바탕으로 pH 변화 및 CDP-W 첨가가 리포좀의 평균 입자 크기, 다분산 지수 및 제타 전위 등과 같은 안정성에 미치는 영향에 살펴보았다. 또한 가장 안정한 상태의 리포좀이 형성되는 pH 6의 조건에서 CDP-W 첨가에 따른 리포좀 막의 형광 이방성, 변형성, 녹는 점 측정 등을 통하여 리포좀 막의 유동성 특성을 측정하고 리포좀 막의 유동성이 포집효율 및 안정성에 미치는 영향에 관하여 살펴보았다.