• International Journal of Advanced Culture Technology
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• v.8 no.1
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• pp.226-235
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• 2020

In this paper, we present to evaluate physiological activity of Robinia pseudo-acacia leaf and its skin penetration using liposome and cell penetrating peptide. After extraction with Robinia pseudo-acacia leaf using the distilled water and supercritical, various physiological activities were examined. In antioxidants experiments, the total concentration of polyphenol compounds was determined to be 56.88 mg/g in hydrothermal extract, 45.07 mg/g in supercritical extract. The DPPH radical scavenging ability at 1,000 ㎍/mL was 33.97% in supercritical extract. The scavenging effect on SOD experiment at 500 ㎍/mL was 76.41% in supercritical extract. In the antimicrobial experiments, the hydrothermal extract had no effect, but supercritical extract represented maximum clear zone of 14.00 mm in Staphylococcus aureus strain. Liposome containing the RSE (Robinia pseudo-acacia leaf supercritical extract) reduced particle size and stabilized zeta potential. In the epidermal permeability experiment, it was confirmed that the permeation of liposome containing the RSE and cell penetrating peptides was remarkable.

• BMB Reports
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• v.52 no.5
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• pp.324-329
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• 2019

Recent progress in cellular reprogramming technology and lineage-specific cell differentiation has provided great opportunities for translational research. Because virus-based gene delivery is not a practical reprogramming protocol, protein-based reprogramming has been receiving attention as a safe way to generate reprogrammed cells. However, the poor efficiency of the cellular uptake of reprogramming proteins is still a major obstacle. Here, we reported key factors which improve the cellular uptake of these proteins. Purified red fluorescent proteins fused with 9xLysine (dsRED-9K) as a cell penetrating peptide were efficiently delivered into the diverse primary cells. Protein delivery was improved by the addition of amodiaquine. Furthermore, purified dsRED-9K was able to penetrate all cell lineages derived from mouse embryonic stem cells efficiently. Our data may provide important insights into the design of protein-based reprogramming or differentiation protocols.

• Prospectives of Industrial Chemistry
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• v.24 no.6
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• pp.32-40
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• 2021

세포 투과성 펩타이드(cell penetrating peptide; CPP)는 강력한 세포막 투과성을 보유하고 있어 난투과성 중거대분자 약물의 세포 내 전달체 개발에 있어 중요한 요소 기술로 부각되고 있다. 하지만 대부분의 세포 투과성 펩타이드는 타겟 세포에 대한 선택성 없이 투과하므로, 전신 투여시 심각한 부작용이 발생할 수 있다. 이 글에서는 선택적 세포 투과성 펩타이드를 개발하는 최근 연구 전략 중, 타겟 세포 표면에 존재하는 수용체에 결합하는 리간드를 이용한 전략과, 타겟 세포 주변의 물리, 화학, 생물학적 신호 변화를 이용하는 전략에 대해 소개한다. 특히, 최근 논문에 발표된, 어피버디(affibody)와 세포 투과성 펩타이드 결합체를 이용하여 HER2 수용체를 지닌 유방암 세포에 선택적 투과성을 부여하는 방법과, 암세포 주변의 작은 pH 변화를 감지하여 양전하성을 조절함으로써 수용체가 없는 유방암 세포에도 선택적으로 투과성을 보이는 방법에 대해 자세하게 소개한다.

• Korean Chemical Engineering Research
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• v.58 no.1
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• pp.21-28
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• 2020

The stem extract from Pyrus serotina var has natural antioxidant ability, but the extraction method does not result in a soluble compound in cosmetic formulations. This study investigated the cosmetic efficacy of the Pyrus serotina var stem extract and its epidermis permeation ability when combined with polymer micelles and a cell penetrating peptide. The total concentration of polyphenol compounds was determined to be 103.1644 ± 1.38 mg/g in the ethanol extract and 78.97 ± 1.45 mg/g in the hydrothermal extract. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging effects were 55.94 ± 0.22% in the ethanol extract at 1,000 mg/L. Superoxide dismutase (SOD) activity rates were 104.05 ± 3.28% in the ethanol extract at 62.5 mg/L. The elastase inhibition rate was 67.21 ± 2.72% in the ethanol extract at 1,000 mg/L. An antimicrobial effect was observed in the Propionibacterium acnes strain. In the epidermal permeability experiment, it was confirmed that formulation of the polymer micelle containing the Pyrus serotina var stem extract and cell penetrating peptide (R6, hexa-D-arginine) showed small particle size and much better skin permeability. The cumulative amount of total Pyrus serotina var stem extract that penetrated to the skin over time increased over 24 hours in three formulations. The three formulations showed 51.61 ㎍/㎠ (Formulation 0), 75.97 ㎍/㎠ (Formulation 1) and 95.23 ㎍/㎠ (Formulation 2) skin penetration, respectively. Therefore, it was confirmed that the ethanol extracts of Pyrus serotina var stem showed good cosmetic efficacy and excellent epidermis permeation ability when combined with a polymer micelle and cell penetrating peptide. Thus, this extract has the potential to be used as a safe and natural cosmetic material in the future.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.4
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• pp.239-245
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• 2022

Protein and peptide candidates are screened to apply therapeutic application as a drug. Ensuring that these candidates are delivered and maximized effectiveness is still challenging and a variety of studies are ongoing. As drug delivery system vehicles, cell-penetrating peptide (CPP) can deliver various kinds of cargo into the cell cytosol. In a previous study, we developed Ara27 CPP, which are a zinc knuckle family protein of Arabidopsis, and confirmed internalization in human dermal fibroblasts and human dental pulp stem cells at low concentration with short time treatment condition without any toxicity. Ara27, an amphipathic CPP, could be modified and utilized in the biomedical field excluding the risk of toxicity. Therefore, we would like to confirm the non-toxic induced penetrating ability of Ara27 in various cell lines. The purpose of this study was to screen the cell internalization ability of Ara27 in various cell lines and to confirm Ara27 as a promising core CPP structure. First, Ara27 was screened to confirm non-toxicity concentration. Then, fluorescence-labeled Ara27 was treated on human normal cell lines, cancer cell lines and animal cell lines to identify the cellular internalization of Ara27. Ara27 was well intracellular localized in all cell lines and the intensity of fluorescence was remarkably increased in time pass manner. These results indicate that Ara27 has the potential as a core structure for applications in various drug delivery systems.

• Clinical and Experimental Reproductive Medicine
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• v.41 no.1
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• pp.1-8
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• 2014

Objective: Estrogen related receptor ${\beta}$ (Esrrb) is a member of the orphan nuclear receptors and may regulate the expression of pluripotencyrelated genes, such as Oct4 and Nanog. Therefore, in the present study, we have developed a method for delivering exogenous ESRRB recombinant protein into embryos by using cell-penetrating peptide (CPP) conjugation and have analyzed their effect on embryonic development. Methods: Mouse oocytes and embryos were obtained from superovulated mice. The expression of Oct4 mRNA and the cell number of inner cell mass (ICM) in the in vitro-derived and in vivo-derived blastocysts were first analyzed by real time-reverse transcription-polymerase chain reaction and differential staining. Then 8-cell embryos were cultured in KSOM media with or without $2{\mu}g/mL$ CPP-ESRRB protein for 24 to 48 hours, followed by checking their integration into embryos during in vitro culture by Western blot and immunocytochemistry. Results: Expression of Oct4 and the cell number of ICM were lower in the in vitro-derived blastocysts than in the in vivo-derived ones (p<0.05). In the blastocysts derived from the CPP-ESRRB-treated group, expression of Oct4 was greater than in the non-treated groups (p<0.05). Although no difference in embryonic development was observed between the treated and non-treated groups, the cell number of ICM was greater in the CPP-ESRRB-treated group. Conclusion: Treatment of CPP-ESRRB during cultivation could increase embryos' expression of Oct4 and the formation rate of the ICM in the blastocyst. Additionally, an exogenous delivery system of CPP-conjugated protein would be a useful tool for improving embryo culture systems.

• Molecules and Cells
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• v.28 no.4
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• pp.341-345
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• 2009

MiRNAs are non-coding RNAs that play a role in the regulation of major processes. The inhibition of miRNAs using antisense oligonucleotides (ASOs) is a unique and effective technique for the characterization and subsequent therapeutic targeting of miRNA function. Recent advances in ASO chemistry have been used to increase both the resistance to nucleases and the target affinity and specificity of these ASOs. Peptide nucleic acids (PNAs) are artificial oligonucleotides constructed on a peptide-like backbone. PNAs have a stronger affinity and greater specificity to DNA or RNA than natural nucleic acids and are resistant to nucleases, which is an essential characteristic for a miRNA inhibitor that will be exposed to serum and cellular nucleases. For increasing cell penetration, PNAs were conjugated with cell penetrating peptides (CPPs) at N-terminal. Among the tested CPPs, Tat-modified peptide-conjugated PNAs have most effective function for miRNA inhibition. PNA-based ASO was more effective miRNA inhibitor than other DNA-based ASOs and did not show cytotoxicity at concentration up to 1,000 nM. The effects of PNA-based ASOs were shown to persist for 9 days. Also, PNA-based ASOs showed considerable stability at storage temperature. These results suggest that PNA-based ASOs are more effective ASOs of miRNA than DNA-based ASOs and PNA-based ASO technology, compared with other technologies used to inhibit miRNA activity can be an effective tool for investigating miRNA functions.

• Journal of Microbiology and Biotechnology
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• v.28 no.3
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• pp.367-374
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• 2018

RNA interference provides an effective tool for developing antitumor therapies. Cell-penetrating peptides (CPPs) are delivery vectors widely used to efficiently transport small-interfering RNA (siRNA) to intracellular targets. In this study, we investigated the efficacy of the cancer-specific CPP carrier BR2 to specifically transport siRNA to cancer-target cells. Our results showed that BR2 formed a complex with anti-vascular endothelial growth factor siRNA (siVEGF) that exhibited the appropriate size and surface charge for in vivo treatment. Additionally, the BR2-VEGF siRNA complex exhibited significant serum stability and high levels of gene-silencing effects in vitro. Moreover, the transfection efficiency of the complex into a cancer cell line was higher than that observed in non-cancer cell lines, resulting in downregulated intracellular VEGF levels in HeLa cells and comprehensively improved antitumor efficacy in the absence of significant toxicity. These results indicated that BR2 has significant potential for the safe, efficient, and specific delivery of siRNA for diverse applications.

• Development and Reproduction
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• v.17 no.1
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• pp.9-16
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• 2013

Coactivator-associated arginine methyltransferase 1 (CARM1) is included in the protein arginine methyltransferase (PRMT) family, which methylates histone arginine residues through posttranslational modification. It has been proposed that CARM1 may up-regulate the expression of pluripotency-related genes through the alteration of the chromatin structure. Mouse embryonic stem cells (mESCs) are pluripotent and have the ability to self-renew. The cells are mainly used to study the genetic function of novel genes, because the cells facilitate the transmission of the manipulated genes into target mice. Since the up-regulated methylation levels of histone arginine residue lead to the maintenance of pluripotency in embryos and stem cells, it may be suggested that CARM1 overexpressing mESCs elevate the expression of pluripotency-related genes in reconstituted embryos for transgenic mice and may resist the differentiation into trophectoderm (TE). We constructed a fusion protein by connecting CARM1 and 7X-arginine (R7). As a cell-penetrating peptide (CPP), can translocate CARM1 protein into mESCs. CPP-CARM1 protein was detected in the nuclei of the mESCs after a treatment of 24 hours. Accordingly, the expression of pluripotency-related genes was up-regulated in CPP-CARM1-treated mESCs. In addition, CPP-CARM1-treated mESC-derived embryoid bodies (EBs) showed an elevated expression of pluripotency-related genes and delayed spontaneous differentiation. This result suggests that the treatment of recombinant CPP-CARM1 protein elevates the expression of pluripotency-related genes of mESCs by epigenetic modification, and this protein-delivery system could be used to modify embryonic fate in reconstituted embryos with mESCs.

• Proceedings of the Korean Vacuum Society Conference
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• 2013.02a
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• pp.110-111
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• 2013

FcBP consisting of 13 amino acids specifically binds to Immunoglobulin G Fc domain. Initially, we utilized this peptide for preparation of antibody chip as a PEG composite for enhanced solubility. After then, the peptide conjugate was immobilized on agarose resin, resulting in highly efficient affinity column for antibody purification. The efficiency was comparable to commercial Protein A column. Recently, this peptide was conjugated with cell penetrating peptide (CPP) on a backbone of GFP, affording antibody transducer, which carries antibody into live cells by simple mixing of antibody and the transducer in cell culture media. Antibody transduction into cells was monitored by live cell imaging. More recently, the FcBP was fused to ferritin cage, which consists of 24 ferritin protein molecules. The FcBP-ferritin cage showed greatly increased binding affinity to human IgG. Its binding was analyzed by QCM and SPR analysis. Finally, it was selectively delivered by Herceptin to SKBR3, a breast cancer cell, over MCF10A, non-tumorigenic cells (Fig. 1). Fig. 1. Fluorescent microscopic images of SKBR3 breast cancer cells (A~C) and MCF10A breast cells (D~F) treated with Cy3-trastuzumab/fFcBP-Pf_Fn complexes. Trastuzumab and FcBP-Pf_Fn, which were labeled with Cy3 (Cy3-trastuzumab) and fluorescein (fFcBP-Pf_Fn), respectively, selectively targeted SKBR3 over MCF10A.