Publications by authors named "Kosuke Noguchi"

7 Publications

  • Page 1 of 1

Environmental pH stress influences cellular secretion and uptake of extracellular vesicles.

FEBS Open Bio 2021 03 18;11(3):753-767. Epub 2021 Feb 18.

Keio University School of Medicine, Tsukuba, Japan.

Exosomes (extracellular vesicles/EVs) participate in cell-cell communication and contain bioactive molecules, such as microRNAs. However, the detailed characteristics of secreted EVs produced by cells grown under low pH conditions are still unknown. Here, we report that low pH in the cell culture medium significantly affected the secretion of EVs with increased protein content and zeta potential. The intracellular expression level and location of stably expressed GFP-fused CD63 (an EV tetraspanin) in HeLa cells were also significantly affected by environmental pH. In addition, increased cellular uptake of EVs was observed. Moreover, the uptake rate was influenced by the presence of serum in the cell culture medium. Our findings contribute to our understanding of the effect of environmental conditions on EV-based cell-cell communication.
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http://dx.doi.org/10.1002/2211-5463.13107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7931216PMC
March 2021

Effects of Lyophilization of Arginine-rich Cell-penetrating Peptide-modified Extracellular Vesicles on Intracellular Delivery.

Anticancer Res 2019 Dec;39(12):6701-6709

Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Osaka, Japan

Background/aim: Extracellular vesicles (exosomes, EVs) (30-200 nm in diameter) are secreted by various cells in the body. Owing to the pharmaceutical advantages of EVs, an EV-based drug delivery system (DDS) for cancer therapy is expected to be the next-generation therapeutic system. However, preservation methods for functional and therapeutic EVs should be developed. Here, we developed the method of lyophilization of arginine-rich cell penetrating peptide (CPP)-modified EVs and investigated the effects of lyophilization on the characteristics of EVs.

Materials And Methods: Particle size, structure, zeta-potential, and cellular uptake efficacy of the arginine-rich CPP-modified EVs were analyzed. The model protein saporin (SAP), having anti-cancer effects, was encapsulated inside the EVs to assess the cytosolic release of EV content after cellular uptake.

Results: Lyophilization of the EVs did not affect their particle size, structure, zeta-potential, and cellular uptake efficacy; however, the biological activity of the encapsulated SAP was inhibited by lyophilization.

Conclusion: Lyophilization of EVs may affect SAP structures and/or reduce the cytosolic release efficacy of EV's content after cellular uptake and needs attention in EV-based DDSs.
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http://dx.doi.org/10.21873/anticanres.13885DOI Listing
December 2019

Effects of gefitinib treatment on cellular uptake of extracellular vesicles in EGFR-mutant non-small cell lung cancer cells.

Int J Pharm 2019 Dec 11;572:118762. Epub 2019 Oct 11.

NanoSquare Research Institute, Research Center for the 21st Century, Organization for Research Promotion, Osaka Prefecture University, Sakai, Osaka 599-8570, Japan; Graduate School of Science, Osaka Prefecture University, Sakai, Osaka 599-8570, Japan. Electronic address:

Extracellular vesicles (exosomes, EVs) are cell membrane particles (30-200 nm) secreted by virtually all cells. During intercellular communication in the body, secreted EVs play crucial roles by carrying functional biomolecules (e.g., microRNAs and enzymes) into other cells to affect cellular function, including disease progression. We previously reported that the macropinocytosis pathway contributes greatly to the efficient cellular uptake of EVs. The activation of growth factor receptors, such as epidermal growth factor receptor (EGFR), induces macropinocytosis. In this study, we demonstrated the effects of gefitinib, a tyrosine kinase inhibitor of EGFR, on the cellular uptake of EVs. In EGFR-mutant HCC827 non-small cell lung cancer (NSCLC) cells, which are sensitive to gefitinib, macropinocytosis was suppressed by gefitinib treatment. However, the cellular uptake of EVs was increased by gefitinib treatment, whereas that of liposomes was reduced. In accordance with the results of the cellular uptake studies, the anti-cancer activity of doxorubicin (DOX)-loaded EVs in HCC827 cells was significantly increased in the presence of gefitinib, whereas the activity of DOX-loaded liposomes was reduced. The digestion of EV proteins by trypsin did not affect uptake, suggesting that the cellular uptake of EVs might not be mediated by EV proteins. These results suggest that gefitinib can enhance cell-to-cell communication via EVs within the tumor microenvironment. In addition, EVs show potential as drug delivery vehicles in combination with gefitinib for the treatment of patients harboring EGFR-mutant NSCLC tumors.
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http://dx.doi.org/10.1016/j.ijpharm.2019.118762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899172PMC
December 2019

Acid-Tolerant Reversibly Switchable Green Fluorescent Protein for Super-resolution Imaging under Acidic Conditions.

Cell Chem Biol 2019 10 15;26(10):1469-1479.e6. Epub 2019 Aug 15.

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Japan; The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki 567-0047, Japan. Electronic address:

Reversibly switchable fluorescent proteins (RSFPs) are crucial tags for super-resolution observation of protein localization and dynamics inside living cells. However, due to the high fluorescence pK (∼5-6) of most RSFPs, their usage in acidic conditions (pH 4.5-6.0) has been limited. Here, we investigated a new photochromic mechanism in Gamillus, a recently developed green fluorescent protein with acid tolerance. Gamillus exhibits negative switching with especially high contrast in acidic conditions, and its off switching is caused by trans-to-cis isomerization of the chromophore hydroxyphenyl ring that accompanies protonation. Through a combination of rational design and saturation mutagenesis, we developed two variants with enhanced switching contrasts and off-switching speeds, designated rsGamillus-S and rsGamillus-F, respectively. The fluorescence intensity, off-switching speed, and switching contrast of the rsGamillus variants are only slightly affected by changes in pH between 4.5 and 7.5. Exploiting these properties, we succeeded in high-contrast super-resolution imaging of cellular architectures in acidic conditions.
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http://dx.doi.org/10.1016/j.chembiol.2019.07.012DOI Listing
October 2019

Arginine-rich cell-penetrating peptide-modified extracellular vesicles for active macropinocytosis induction and efficient intracellular delivery.

Sci Rep 2017 05 16;7(1):1991. Epub 2017 May 16.

Institute for Chemical Research, Kyoto University, Uji, Kyoto, 611-0011, Japan.

Extracellular vesicles (EVs) including exosomes have been shown to play crucial roles in cell-to-cell communication because of their ability to carry biofunctional molecules (e.g., microRNAs and enzymes). EVs also have pharmaceutical advantages and are highly anticipated to be a next-generation intracellular delivery tool. Here, we demonstrate an experimental technique that uses arginine-rich cell-penetrating peptide (CPP)-modified EVs to induce active macropinocytosis for effective cellular EV uptake. Modification of arginine-rich CPPs on the EV membrane resulted in the activation of the macropinocytosis pathway, and the number of arginine residues in the peptide sequences affected the cellular EV uptake efficiency. Consequently, the ribosome-inactivating protein saporin-encapsulated EVs modified with hexadeca-arginine (R16) peptide effectively attained anti-cancer activity.
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http://dx.doi.org/10.1038/s41598-017-02014-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5434003PMC
May 2017

Vectorization of biomacromolecules into cells using extracellular vesicles with enhanced internalization induced by macropinocytosis.

Sci Rep 2016 10 17;6:34937. Epub 2016 Oct 17.

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.

Extracellular vesicles (EVs, exosomes) are approximately 30- to 200-nm-long vesicles that have received increased attention due to their role in cell-to-cell communication. Although EVs are highly anticipated to be a next-generation intracellular delivery tool because of their pharmaceutical advantages, including non-immunogenicity, their cellular uptake efficacy is low because of the repulsion of EVs and negatively charged cell membranes and size limitations in endocytosis. Here, we demonstrate a methodology for achieving enhanced cellular EV uptake using arginine-rich cell-penetrating peptides (CPPs) to induce active macropinocytosis. The induction of macropinocytosis via a simple modification to the exosomal membrane using stearylated octaarginine, which is a representative CPP, significantly enhanced the cellular EV uptake efficacy. Consequently, effective EV-based intracellular delivery of an artificially encapsulated ribosome-inactivating protein, saporin, in EVs was attained.
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http://dx.doi.org/10.1038/srep34937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5066177PMC
October 2016

Mitochondrial protein-derived cryptides: Are endogenous N-formylated peptides including mitocryptide-2 components of mitochondrial damage-associated molecular patterns?

Biopolymers 2016 Nov;106(4):580-7

Laboratory of Peptide Science, Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, 526-0829, Japan.

Recently, much attention has been paid to "nonclassical" bioactive peptides, which are fragmented peptides simultaneously produced during maturation and degradation of various functional proteins. We identified many fragmented peptides derived from various mitochondrial proteins including mitocryptide-1 and mitocryptide-2 that efficiently activate neutrophils. These endogenous, functionally active, fragmented peptides are referred to as "cryptides." Among them, mitocryptide-2 is an N-formylated cryptide cleaved from mitochondrial cytochrome b that is encoded in mitochondrial DNA (mtDNA). It is known that 13 proteins encoded in mtDNA are translated in mitochondria as N-formylated forms, suggesting the existence of endogenous N-formylated peptides other than mitocryptide-2. Here, we investigated the effects of N-formylated peptides presumably cleaved from mtDNA-encoded proteins other than cytochrome b on the functions of neutrophilic cells to elucidate possible regulation by endogenous N-formylated cryptides. Four N-formylated cryptides derived from cytochrome c oxidase subunit I and NADH dehydrogenase subunits 4, 5, and 6 among 12 peptides from mtDNA-encoded proteins efficiently induced not only migration but also β-hexosaminidase release, which is an indicator of neutrophilic phagocytosis, in HL-60 cells differentiated into neutrophilic cells. These activities were comparable to or higher than those induced by mitocryptide-2. Although endogenous N-formylated peptides that are contained in mitochondrial damage-associated molecular patterns (DAMPs) have yet to be molecularly identified, they have been implicated in innate immunity. Thus, N-formylated cryptides including mitocryptide-2 are first-line candidates for the contents of mitochondrial DAMPs to promote innate immune responses. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 580-587, 2016.
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http://dx.doi.org/10.1002/bip.22788DOI Listing
November 2016
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