Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Ribonuclease S (RNase S) is an enzyme that exhibits anticancer activity by degrading RNAs within cancer cells; however, the cellular uptake efficiency is low due to its small molecular size. Here we generated RNase S-decorated artificial viral capsids with a size of 70-170 nm by self-assembly of the β-annulus-S-peptide followed by reconstitution with S-protein at neutral pH. The RNase S-decorated artificial viral capsids are efficiently taken up by HepG2 cells and exhibit higher RNA degradation activity in cells compared with RNase S alone. Cell viability assays revealed that RNase S-decorated capsids have high anticancer activity comparable to that of standard anticancer drugs.

DOI： 10.1002/cbic.202200220

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

We have developed a series of attenuated cationic amphiphilic lytic (ACAL) peptides that can efficiently bring immunoglobulin G (IgG) and other functional proteins into cells. Delivery is generally achieved through the coadministration of ACAL peptides with cargo proteins. However, conjugation of ACAL peptides with cargos may be a promising approach for in vivo application to link in vivo outcomes of ACAL peptides and cargos. This study describes the creation of a new cell-permeable ACAL peptide, L17ER4. L17E is an optimized prototype of ACAL peptides previously developed in our laboratory for efficient delivery of IgGs into cells. Delivery was improved by functionalizing L17E with a tetra-arginine (R4) tag. Compared to the use of R8, a representative cell-penetrating peptide with high intracellular delivery efficacy, conjugation with L17ER4 afforded approximately four-fold higher cellular uptake of model small-molecule cargos (fluorescein isothiocyanate and HiBiT peptide). L17ER4 was also able to deliver proteins to cells. Fused with L17ER4, Cre recombinase was delivered into cells. Intracerebroventricular injection of Cre-L17ER4 into green red reporter mice, R26GRR, led to significant in vivo gene recombination in ependymal cells, suggesting that L17ER4 may be used as a cell-penetrating peptide for delivering protein therapeutics into cells in vivo.

DOI： 10.1016/j.bmc.2022.116728

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Nanocarriers that deliver functional proteins to cell interiors are an attractive platform for the intracellular delivery of intact proteins without further modification, with in vivo compatibility. Development of efficient methods for cargo protein encapsulation and release in recipient cell cytosol is needed. Herein, we assess the feasibility of the abovementioned requirements using a protein nanocage (artificial nanocage) without compromising the structure and functions of the original protein and allowing for design flexibility of the surfaces and interiors. The protein nanocage formed via the self-assembly of the β-annulus peptide (24-amino acid peptide) in water was used as a model framework. The nitrilotriacetic acid moiety was displayed on the nanocage lumen for effective encapsulation of hexahistidine-tagged proteins in the presence of Ni2+, and the amphiphilic cationic lytic peptide HAad was displayed on a nanocage surface to attain cell permeability. Successful intracellular delivery of cargo proteins and targeting of cytosolic proteins by a nanobody were achieved, indicating the validity of the approach employed in this study.

DOI： 10.1021/acs.bioconjchem.1c00534

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Fc region binding peptide conjugated with attenuated cationic amphiphilic lytic peptide L17E trimer [FcB(L17E)3 ] was designed for immunoglobulin G (IgG) delivery into cells. Particle-like liquid droplets were generated by mixing Alexa Fluor 488 labeled IgG (Alexa488-IgG) with FcB(L17E)3 . Droplet contact with the cellular membrane led to spontaneous influx and distribution of Alexa488-IgG throughout cells in serum containing medium. Involvement of cellular machinery accompanied by actin polymerization and membrane ruffling was suggested for the translocation. Alexa488-IgG negative charges were crucial in liquid droplet formation with positively charged FcB(L17E)3 . Binding of IgG to FcB(L17E)3 may not be necessary. Successful intracellular delivery of Alexa Fluor 594-labeled anti-nuclear pore complex antibody and anti-mCherry-nanobody tagged with supernegatively charged green fluorescence protein allowed binding to cellular targets in the presence of FcB(L17E)3 .

DOI： 10.1002/anie.202105527

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

We previously reported an approach for intracellular protein delivery by attenuating membrane-lytic activity of cationic amphiphilic peptides on cell surfaces. HAad is one such peptides that cytosolically delivers proteins of interest, including antibodies, by stimulating their endosomal escape. Additionally, HAad elicits ruffling of cell membrane, accompanied by transient membrane permeabilization, allowing for the efficient cytosolic translocation of proteins. In this study, we prepared a conjugate of HAad with pyrenebutyric acid as a membrane-anchoring unit (pBu-HAad). pBu-HAad demonstrated protein delivery into cells with only 1/20 concentration of HAad. However, the conjugates with cholesteryl hemisuccinate and aliphatic fatty acids (C = 3, 6, and 10) did not yield such marked effects. The results of time-course and inhibitor studies suggest that the membrane anchoring of HAad by a pyrene moiety leads to enhanced peptide-membrane interaction and to loosen lipid packing, thus facilitating cytosolic translocation through membranes.

DOI： 10.1021/acs.bioconjchem.1c00101

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Our research group has been studying the design of intracellular delivery peptides based on cationic lytic peptides. By placing negatively charged amino acids on potentially hydrophobic faces of the peptides, membrane lytic activity is attenuated on the cell surface, whereas it recovers in endosomes, enabling cytosolic delivery of proteins including antibodies. These lytic peptides generally contain multiple lysines, facilitating cell surface interaction and membrane perturbation. This study evaluated the effect of lysine-to-homoarginine substitution using HAad as a model delivery peptide. The resulting peptide had a comparable or better delivery efficacy for Cre recombinase, antibodies, and the Cas9/sgRNA complex with one-quarter of the concentration of HAad, implying that a subtle structural difference can affect delivery activity.

DOI： 10.1016/j.bmcl.2021.127925

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Endocytic pathways are practical routes for the intracellular delivery of biomacromolecules. Along with this, effective strategies for endosomal cargo release into the cytosol are desired to achieve successful delivery. Focusing on compositional differences between the cell and endosomal membranes and the pH decrease within endosomes, we designed the lipid-sensitive and pH-responsive endosome-lytic peptide HAad. This peptide contains aminoadipic acid (Aad) residues, which serve as a safety catch for preferential permeabilization of endosomal membranes over cell membranes, and His-to-Ala substitutions enhance the endosomolytic activity. The ability of HAad to destabilize endosomal membranes was supported by model studies using large unilamellar vesicles (LUVs) and by increased intracellular delivery of biomacromolecules (including antibodies) into live cells. Cerebral ventricle injection of Cre recombinase with HAad led to Cre/loxP recombination in a mouse model, thus demonstrating potential applicability of HAad in vivo.

DOI： 10.1002/anie.202005887

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Intracellular delivery of biomacromolecules is a challenging research field in chemical biology and drug delivery. We previously reported a peptide named L17E, which successfully delivered functional proteins, including antibodies, into cells. However, relatively high concentrations of L17E and proteins are needed. In this study, we prepared dimers of L17E and its analog L17E/Q21E. Dimerization of L17E increased cytotoxicity leading to reduced intracellular delivery compared with L17E. On the other hand, the dimers of the L17E analog, L17E/Q21E, especially when tethered at the N-termini, yielded a comparable level of intracellular delivery with L17E at decreased amounts of delivery peptides and cargoes.

DOI： 10.1016/j.bmcl.2020.127362

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Intracellular delivery of bioactive macromolecules via endocytic pathways has utility in biotechnological and medicinal applications. Various endosomolytic peptides bearing glutamic acid (Glu) residues have been developed with the aim to achieve selective lysis of endosomal membranes without damaging cell membranes (plasma membranes) to release endosome-entrapped macromolecules and obtain their bioactivity. Glu residues on peptides are negatively charged in the extracellular medium, and substitution of this residue onto membrane-lytic peptides prevents its peptide-membrane interaction and its lytic activity. On the other hand, within endosomes, which have a reduced pH of ∼5, Glu is protonated, resulting in the reduction of the hydrophilicity of the peptide, unmasking its lytic activity. Despite this, a limited number of studies have elucidated the optimum positions for Glu substitution. This report investigated the positioning of Glu and the endosomolytic activities of cationic lytic peptides, ponericin-W3, and melittin. By cell-based assays, biophysical analyses, and molecular dynamics simulations, we found that analogues with Glu positioned on the borders between the hydrophobic and hydrophilic faces of the helical structures showed better performance than placing Glu within said faces.

DOI： 10.1021/acs.molpharmaceut.0c00312

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1002/pep2.24144

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

One of the major obstacles in intracellular targeting using antibodies is their limited release from endosomes into the cytosol. Here we report an approach to deliver proteins, which include antibodies, into cells by using endosomolytic peptides derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin. The delivery peptides were developed by introducing one or two glutamic acid residues into the hydrophobic face. One peptide with the substitution of leucine by glutamic acid (L17E) was shown to enable a marked cytosolic liberation of antibodies (immunoglobulins G (IgGs)) from endosomes. The predominant membrane-perturbation mechanism of this peptide is the preferential disruption of negatively charged membranes (endosomal membranes) over neutral membranes (plasma membranes), and the endosomolytic peptide promotes the uptake by inducing macropinocytosis. The fidelity of this approach was confirmed through the intracellular delivery of a ribosome-inactivation protein (saporin), Cre recombinase and IgG delivery, which resulted in a specific labelling of the cytosolic proteins and subsequent suppression of the glucocorticoid receptor-mediated transcription. We also demonstrate the L17E-mediated cytosolic delivery of exosome-encapsulated proteins.

DOI： 10.1038/nchem.2779

PubMed