Enveloped Viral Replica Equipped with Spike Protein Derived from SARS-CoV-2.

Synthetic viral nanostructures are useful as materials for analyzing the biological behavior of natural viruses and as vaccine materials. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped virus embedding a spike (S) protein involved in host cell infection. Although nanomaterials modified with an S protein without an envelope membrane have been developed, they are considered unsuitable for stability and functionality. We previously constructed an enveloped viral replica complexed with a cationic lipid bilayer and an anionic artificial viral capsid self-assembled from ß-annulus peptides. In this study, we report the first example of an enveloped viral replica equipped with an S protein derived from SARS-CoV-2. Interestingly, even the S protein equipped on the enveloped viral replica bound strongly to the free angiotensin-converting enzyme 2 (ACE2) receptor as well as ACE2 localized on the cell membrane.

Alkyl anchor-modified artificial viral capsid budding outside-to-inside and inside-to-outside giant vesicles.

The budding of human immunodeficiency virus from an infected host cell is induced by the modification of structural proteins bearing long-chain fatty acids, followed by their anchoring to the cell membrane. Although many model budding systems using giant unilamellar vesicles (GUVs) induced by various stimuli have been developed, constructing an artificial viral budding system of GUVs using only synthesized molecules remains challenging. Herein, we report the construction of an artificial viral capsid budding system from a lipid bilayer of GUV. The C-terminus of the ß-annulus peptide was modified using an octyl chain as an alkyl anchor via a disulfide bond. The self-assembly of the ß-annulus peptide with an octyl chain formed an artificial viral capsid aggregate. The fluorescence imaging and transmission electron microscopy observations revealed that the addition of the tetramethylrhodamine (TMR)-labeled octyl chain-bearing ß-annulus peptide to the outer aqueous phase of GUV induced the budding of the capsid-encapsulated daughter vesicle outside-to-inside the mother GUV. Conversely, the encapsulation of the TMR-labeled octyl chain-bearing ß-annulus peptide in the inner aqueous phase of GUV induced the budding of the capsid-encapsulated daughter vesicle inside-to-outside the mother GUV. Contrarily, the addition of the TMR-labeled ß-annulus peptide to GUV barely induced budding. It was demonstrated that the higher the membrane fluidity of GUV, the more likely budding would be induced by the addition of the alkyl anchor-modified artificial viral capsid. The simple virus-mimicking material developed in this study, which buds off through membrane anchoring, can provide physicochemical insights into the mechanisms of natural viral budding from cells.

Construction of an artificial viral budding system of GUVs using only synthesized molecules remains challenging. This study firstly demonstrates that budding outside-to-inside and inside-to-outside GUVs are induced by addition of alkyl anchor-modified artificial viral capsid.

Strategy toward In-Cell Self-Assembly of an Artificial Viral Capsid from a Fluorescent Protein-Modified ß-Annulus Peptide.

In-cell self-assembly of natural viral capsids is an event that can be visualized under transmission electron microscopy (TEM) observations. By mimicking the self-assembly of natural viral capsids, various artificial protein- and peptide-based nanocages were developed; however, few studies have reported the in-cell self-assembly of such nanocages. Our group developed a ß-Annulus peptide that can form a nanocage called artificial viral capsid in vitro, but in-cell self-assembly of the capsid has not been achieved. Here, we designed an artificial viral capsid decorated with a fluorescent protein, StayGold, to visualize in-cell self-assembly. Fluorescence anisotropy measurements and fluorescence resonance energy transfer imaging, in addition to TEM observations of the cells and super-resolution microscopy, revealed that StayGold-conjugated ß-Annulus peptides self-assembled into the StayGold-decorated artificial viral capsid in a cell. Using these techniques, we achieved the in-cell self-assembly of an artificial viral capsid.

An artificial viral capsid decorated with a DNA aptamer internalizing into lymphoma cells.

Tumor-specific drug-delivering nanocarriers could be a promising modality for next-generation tumor therapy. Here we developed a Burkitt lymphoma-specific DNA aptamer-labeled nanocarrier using the ß-Annulus peptide, which forms a spherical nanoassembly called artificial viral capsid. Dynamic light scattering and transmission electron microscopy of the DNA aptamer-decorated artificial viral capsid showed the formation of spherical assemblies with a diameter of approximately 50-150 nm. The artificial viral capsid was selectively internalized into the Burkitt lymphoma cell line, Daudi, and doxorubicin complexed with the capsid selectively killed Daudi cells.

Anticancer Activity of Reconstituted Ribonuclease S-Decorated Artificial Viral Capsid.

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 ß-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.

L17ER4: A cell-permeable attenuated cationic amphiphilic lytic peptide.

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.

Artificial Nanocage Formed via Self-Assembly of ß-Annulus Peptide for Delivering Biofunctional Proteins into Cell Interiors.

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 ß-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.

Liquid Droplet Formation and Facile Cytosolic Translocation of IgG in the Presence of Attenuated Cationic Amphiphilic Lytic Peptides.

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 .

Potentiating the Membrane Interaction of an Attenuated Cationic Amphiphilic Lytic Peptide for Intracellular Protein Delivery by Anchoring with Pyrene Moiety.

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.

Use of homoarginine to obtain attenuated cationic membrane lytic peptides.

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.

Improved cytosolic delivery of macromolecules through dimerization of attenuated lytic peptides.

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.

Optimizing Charge Switching in Membrane Lytic Peptides for Endosomal Release of Biomacromolecules.

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.

Rational Design Principles of Attenuated Cationic Lytic Peptides for Intracellular Delivery of Biomacromolecules.

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.

Tissue-specific bioaccumulation of long-chain perfluorinated carboxylic acids and halogenated methylbipyrroles in Dall's porpoises (Phocoenoides dalli) and harbor porpoises (Phocoena phocoena) stranded in northern Japan.

This study investigated accumulation of perfluoroalkyl substances (PFASs), persistent organochlorines (OCs), and naturally produced halogenated compounds (NHCs), including brominated methylbipyrroles and methoxylated bromodiphenyl ethers, in liver, blood, and blubber from Dall's porpoises (Phocoenoides dalli) and harbor porpoises (Phocoena phocoena) stranded in Hokkaido, northern Japan. Profiles of the PFASs were dominated by perfluoroundecanoic acid and perfluorotridecanoic acid, both of which accounted for 70% of the total measured PFAS concentrations in both porpoise species. The mean concentrations of the ∑PFCA were 573ng/g wet weight (ng/g-wet) in liver, 62ng/g-wet in whole blood, and 28ng/g-wet in blubber from the Dall's porpoises, and were significantly higher (p<0.05) than those in the harbor porpoises. The hepatic concentrations of perfluorooctane sulfonate (PFOS) were <14ng/g-wet, and accounted for only 3% of the total measured PFASs. The profiles of PFASs in the porpoises resembled those in fish species in this area, implying a common source of exposure to PFASs in East Asia. On the other hand, in the blubber of Dall's porpoises, NHCs were dominated by 2,3,3',4,4',5,5'-heptachloro-1'-methyl-1,2'-bipyrrole (867ng/g-wet), 5,5'-dichloro-1,1'-dimethyl-3,3',4,4'-tetrabromo-2,2'-bipyrrole (481ng/g-wet), and 6-methoxy-2,2',4,4'-tetrabromodiphenyl ether (30ng/g-wet), which were present at higher concentrations than in harbor porpoises. Factor analysis with varimax rotation revealed that factor 1 had higher eigenvectors (element in eigenvalues) for long-chain PFCAs and PFOS, which was found in the highest concentrations in the liver, whereas factor 2 was mainly associated with lipid soluble NHCs and OCs in both species. No correlations were observed between long-chain PFCAs and NHCs in the porpoises, probably because of the different sources and accumulation kinetics. Future research should assess the temporal trends and long-term effects of PFASs and NHCs in the tissues of mammals from the Asia-Pacific region.

Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide.

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.

[Two resected cases of pulmonary metastasis from post operative colorectal cancer after preoperative chemotherapy with FOLFOX].

UNLABELLED: We described two resected cases of pulmonary metastasis from postoperative colorectal cancer after preoperative FOLFOX chemotherapy. Pathologic histology inspection of the tumor was judged to be effective prior to resection. The first case is a 68-year-old male who underwent a sigmoidectomy as a stage III A sigmoid colon cancer in March 2003. Afterwards, distant metastases had occurred to the liver, the left lung and neck lymph nodes from 2003 to 2006. Three operations and systemic chemotherapy were performed. A new metastasis of the right lung occurred in November 2006. The FOLFOX chemotherapy was performed 7 times, the tumor was not changed in the image, and the effect judgment was SD. After the chemotherapy, a partial resection of the right lung was performed in November 2007. Half of the tumor resulted in necrosis on the specimen (Grade 1b). The second case is a 63-year-old female who underwent an anterior resection of the rectum as a stage II rectal cancer in January 2000, a partial resection of the right lung as a metachronous right pulmonary metastasis in March 2003, and post operative chemotherapy (IFL) were performed. A new metastasis of the right lung occurred in September 2005. The FOLFOX chemotherapy was performed 4 times, the tumor was not changed in the image, and the effect judgment was SD. After the chemotherapy, a partial resection of the right lung was performed in April 2006. One third of the tumor resulted in necrosis on the specimen (Grade 1a). COMMENT: It was thought that FOLFOX chemotherapy can be a promising candidate for neoadjuvant treatment of pulmonary metastasis from postoperative colorectal cancer.