Characterization of Novel Cell-Adhesive Peptides for Biomaterial Development

Abstract

In previous studies, my group developed cell-adhesive peptide–polysaccharide complexes as biomaterials for tissue engineering. Having a wide variety of cell-adhesive peptides is important as the biological functions of peptide–polysaccharide complexes are highly dependent on the biological activity of peptides. This paper reviews the biological activities of two types of recently characterized cell-adhesive peptides. The first is peptides rich in basic amino acids originating from octaarginine. We analyzed the relationships between the amino acid composition of basic peptides and cell adhesion, elongation, and proliferation and identified the most suitable peptide for cell culture. The second was arginine-glycine-aspartic acid (RGD)-containing peptides that promote the adhesion of induced pluripotent stem cells (iPSCs). We identified the RGD-surrounding sequences necessary for iPSC adhesion, clarified the underlying mechanism, and improved cell adhesion by modifying the structure–activity relationships. The novel cell-adhesive peptides identified in our previous studies may aid in the development of novel peptide-based biomaterials.

1. INTRODUCTION

Cell-adhesive peptides are useful tools to confer cell adhesion ability to biomaterials in tissue engineering. My group previously identified more than 100 cell-adhesive peptides from the extracellular matrix protein, laminin.^1–16) These peptides interact with cell surface receptors, such as integrin and syndecan, a family of heparan sulfate proteoglycans, and promote various biological activities. Integrin-binding A99 (AGTFALRGDNPQG, integrin αvβ3),¹⁷⁾ EF1 (DYATLQLQEGRLHFMFDLG, integrin α2β1),¹⁸⁾ and A2G10 (SYWYRIEASRTG, integrin α6β1)¹²⁾ peptides, syndecan-binding AG73 (RKRLQVQLSIRT)²⁾ and A3G756 (KNSFMALYLSKGRLVFALG)¹⁵⁾ peptides, and both integrin- and syndecan-binding A13 (RQVFQVAYIIIKA)¹⁴⁾ and C16 (KAFDITYVRLKF)¹⁶⁾ peptides are representative laminin-derived cell-adhesive peptides. We previously combined peptides with polymers to facilitate their application as biomaterials.^{8,13,17,19–28)} For example, we developed peptide–polysaccharide complexes serving as two- and three-dimensional cell scaffolds using membranes and gels of chitosan, alginate, hyaluronate, and agarose and demonstrated their biological activities^{8,13,17,19,20,22–26,28)} (Fig. 1). These peptide–polysaccharide complexes control various cellular functions, such as cell adhesion, movement, differentiation, proliferation, and gene expression, in a receptor-specific manner. Therefore, cell-adhesive peptides are useful for the preparation of cell type-specific biomaterials. In addition to biological activities, physical properties, such as hydrophobicity and charge, are also important aspects of cell-adhesive peptides as they affect the biological functions of the peptide–polysaccharide complexes.^13,19,20,22) Therefore, having a wide selection of cell-adhesive peptides is desirable for the development of ideal biomaterials. In recent years, we have focused on cell-adhesive peptides other than those derived from laminin, analyzed their biological activities, and optimized them by modifying their structure–activity relationships.^29–32) This article describes our findings of two studies on novel cell-adhesive peptides with potential application for biomaterial development.

Fig. 1. Schematic Illustration of Application of Cell-Adhesive Peptides to Biomaterial Development by Preparation of Peptide–Polysacchride Complexes

2. OCTAARGININE (R8)-BASED CELL-ADHESIVE PEPTIDES

R8 is a cell-penetrating peptide commonly used in intracellular drug delivery studies.^33,34) R8 binds to various membrane proteins, including syndecan; binding to syndecan-4 is important for cellular uptake.³⁵⁾ Ruoslahti and colleagues have reported that R8 also binds to integrin β1.³⁶⁾ Although both syndecan and integrin are receptors mediating cell adhesion, no studies have evaluated the activity of R8 as a cell-adhesive peptide to date. Therefore, we analyzed the cell adhesion activities of R8 and octalysine (K8), a peptide with the same net charge as R8, and their receptors.²⁹⁾ To evaluate their activity, peptides must be immobilized on a substrate. However, peptides with strong positive charges, such as R8 and K8, interact electrostatically with the substrate, affecting their activity. Therefore, we used an agarose membrane, a polysaccharide that does not carry any charge, as a matrix. Agarose was modified to have aldehyde groups using the 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) oxidizing agent, and the aldehyde-functionalized agarose was then reacted with the N-terminal cysteine residue of the peptide to form a thiazolidine ring, allowing the peptide to covalently bond to agarose.²⁸⁾ We synthesized R8 and K8 with an N-terminal CGG sequence, prepared peptide–agarose matrices, and evaluated their cell adhesion to human dermal fibroblasts (HDFs). As a result, R8 and K8 showed cell adhesion activity as well as laminin-derived AG73 and A99 peptides (Fig. 2A). The activity of R8-matrices was stronger than that of the K8-matrices, as arginine forms strong hydrogen bonds with heparan sulfate. Adhesion inhibition experiments revealed that cell adhesion to R8- and K8-matrices was abolished by heparin, indicating that cell adhesion to R8- and K8-matrices was largely mediated by the heparan sulfate proteoglycan, such as syndecan (Fig. 2B). Cell adhesion to R8- and K8-matrices was also inhibited by ethylenediaminetetraacetic acid (EDTA) by approximately 20%, suggesting its partially divalent cation-dependent integrin-mediated adhesion. HDFs adhering to the R8- and K8-matrices showed a thin and elongated morphology with actin fiber formation, which is characteristic of integrin-mediated adhesion. Elongation of the cells on R8- and K8-matrices was inhibited by EDTA and anti-integrin β1 antibody, resulting in a round shape, confirming the contribution of integrin β1. These results were consistent with previous reports that R8 binds to syndecan and integrin β1.^35,36) Further evaluation of the cell proliferation activity under serum-rich conditions revealed that R8- and K8-matrices, but not AG73-matrices, promoted cell proliferation possibly due to integrin signaling by the R8- and K8-matrices (Fig. 2C). These results suggest R8 and K8 as useful cell-adhesive peptides for cell proliferation as they can bind to integrin β1 in addition to heparan sulfate proteoglycan (Fig. 2D).

Fig. 2. Cell Adhesion Activity of R8- and K8-Conjugated Agarose Matrices

(A) Morphology of HDFs attached to R8- and K8-matrices. Scale bar = 100 µm. (B) Inhibitory effect of heparin and EDTA on cell adhesion to R8 and K8-matrices. * p < 0.05, ** p < 0.0001 vs. None. (C) HDF proliferation on R8- and K8-matrices under serum-rich conditions. * p < 0.05 vs. AG73. (D) Schematic illustration of biological activities of R8- and K8-matrices. These data were cited by.²⁹⁾

AG73 (RKRLQVQLSIRT) is rich in basic amino acids but does not bind to integrin unlike R8. We hypothesized that the non-basic amino acids in AG73 affect its binding to integrins. Therefore, we examined the mechanism by which the biological activity of R8 is affected by replacing half of the arginine residues with non-basic amino acids.³⁰⁾ Thirteen XR8 peptides (CGG-XRXRXRXR, X = E, G, A, N, Q, S, T, V, I, L, Y, F, or W) were synthesized and evaluated for their cell adhesion activity. All XR8-matrices, except ER8-matrices, promoted cell adhesion, and the activity was relatively high when X was a hydrophobic or aromatic amino acid (Fig. 3A). Observation of cell morphology revealed that the YR8-, FR8-, and WR8-matrices promoted cell elongation with actin skeleton formation, similar to the R8-matrices (Fig. 3B), indicating that aromatic amino acids contribute to integrin-mediated cell spreading. Phosphorylation of focal adhesion kinase (FAK) was also observed in the YR8-matrices, which exhibited the highest cell elongation, confirming the activation of integrin signaling. In contrast, cells on the IR8- and LR8-matrices showed round morphology (Fig. 3B), and their shape was not changed by EDTA or anti-integrin β1 antibody, indicating that hydrophobic amino acids attenuate integrin signaling. Moreover, FAK phosphorylation was attenuated in the IR8-matrices. Next, cell proliferation on XR8-matrices was evaluated under serum-rich conditions. The IR8- and FR8-matrices did not show cell proliferation activity (Fig. 3C), and the cells on the IR8- and FR8-matrices did not elongate in the presence of serum. These results indicate that hydrophobic amino acids in basic-rich peptides reduced the contribution of integrin under serum-rich conditions, resulting in decreased cell proliferation. AG73 (RKRLQVQLSIRT) also contained four hydrophobic amino acid residues, suggesting that these residues may be responsible for the lack of integrin contribution to AG73. In contrast, cell proliferation on the YR8-matrices was significantly higher than that on the R8-matrices. Tyrosine is aromatic but less hydrophobic than phenylalanine and tryptophan. Therefore, the YR8-matrix most efficiently promoted cell adhesion, spreading, and proliferation among the XR8-matrices tested. Although the R8-matrices were cytotoxic under serum-free conditions owing to their strong positive charge, none of the XR8-matrices, including the YR8-matrices, showed cytotoxicity (Fig. 3D). Therefore, YR8 is superior to R8 in terms of cell proliferation and cytocompatibility and may be applied as a cell-adhesive peptide for biomaterial development (Fig. 3E).

Fig. 3. Effect of Amino Acid Substitution on Cell Adhesion Properties of R8

(A) HDF adhesion activity of XR8-matrices. (B) Morphology of HDFs attached to R8-, IR8-, and YR8-matrices. Scale bar = 100 µm. (C) HDF proliferation on XR8-matrices under serum-rich conditions. * p < 0.05, ** p < 0.0001 vs. R8. Dashed line indicates cell viability on a tissue culture plate on day 0. (D) Cytocompatibility of XR8-matrices under serum-free conditions. The percentages of live (white) and dead cells (gray) were determined using calcein-AM and propidium iodide, respectively. (E) Changes in biological activities of IR8 and YR8 compared to R8. These data were cited by.³⁰⁾

3. RGDX₁X₂-CONTAINING CELL-ADHESIVE PEPTIDES

Since its discovery in 1984 by Pierschbacher and Ruoslahti,³⁷⁾ RGD sequence is the most common integrin-binding motif. RGD-containing peptides are used to develop cell culture substrates, biomaterials, integrin inhibitors, and integrin-targeted drug delivery systems.^38–43) RGD-containing peptides are also used as substrates for the culture of PSCs, such as embryonic stem cells (ESCs) and induced PSCs (iPSCs). Synthemax is a commercially available stem cell culture substrate consisting of an acrylate polymer and a vitronectin-derived RGD-containing peptide. A report on Synthemax was published in 2010.⁴⁴⁾ In that study, RGD-containing peptides derived from vitronectin and bone sialoprotein promoted ESC adhesion, whereas those derived from fibronectin did not. These data suggest that the sequences surrounding RGD play an important role in PSC adhesion. To identify the RGD-surrounding sequences important for PSC adhesion, we synthesized RGD-containing peptides derived from eight extracellular matrix (ECM) proteins³¹⁾ (Fig. 4A). We also synthesized alaRGD, which contains only alanine residues around the RGD motif, as a control. All peptides were synthesized using a CGG sequence at the N terminus, covalently conjugated to maleimide-functionalized bovine serum albumin (BSA)-coated plates, and evaluated their adhesion activity using iPSCs. Only two peptides derived from vitronectin (vnRGD: CGG-PQVTRGDVFTnP, n = norleucine) and bone sialoprotein (bspRGD: CGG-NGEPRGDNYRAY) showed cell adhesion activity with iPSCs, whereas all the RGD-containing peptides promoted the adhesion of HDFs. These results indicate that the RGD-surrounding sequences of vnRGD and bspRGD contain important amino acids for iPSC adhesion. Alanine scanning of vnRGD and bspRGD peptides revealed RGDVF and RGDNY as the core sequences of the two peptides, respectively. We named them the RGDX₁X₂ motifs. As RGDVF was stronger than RGDNY, we focused on RGDVF for a more detailed analysis. The steric structure of X₁X₂ residues was found to be important for iPSC adhesion, as activity was completely lost when the VF residues were switched or when one of them was replaced by a D-body amino acid. The results of experiments using other cell lines showed that X₁X₂ residues were important for adhesion not only to iPSCs but also to HeLa and A549 cells. HDFs and human umbilical vein endothelial cells (HUVECs) attach to RGD peptides independent of the X₁X₂ sequence. To clarify the role of X₁X₂ residues for iPS, HeLa, and A549 cell adhesion, we focused on the integrin subtypes to which RGDVF binds. As vitronectin contains the RGDVF sequence and mediates cell adhesion primarily via integrin αvβ3 and αvβ5, inhibition experiments were performed using antibodies against these two subtypes, αvβ3 and αvβ5. Adhesion of iPS, HeLa, and A549 cells to RGDVF was found to be specifically mediated by integrin αvβ5 (Fig. 4C). In contrast, adhesion of HDFs and HUVECs to RGDVF was found to be mediated by both integrin αvβ3 and αvβ5, and their adhesion to RGDAA was integrin αvβ3-specific. Flow cytometry revealed that iPS, HeLa, and A549 cells expressed integrin αvβ5, but not αvβ3, whereas HDFs and HUVECs expressed both αvβ3 and αvβ5. These results indicate that RGD alone is not sufficient for binding to integrin αvβ5, which requires RGDX₁X₂ motif (Fig. 4D). iPS, HeLa, and A549 cells express integrin αvβ5, but not integrin αvβ3; therefore, they require the X₁X₂ sequence for cell adhesion. In addition to vitronectin, RGDVF motif is also present in ECM proteins, such as nephronectin,^45,46) milk fat globule–epidermal growth factor 8,⁴⁷⁾ and laminin α5,⁴⁸⁾ which can bind to integrin αvβ5. Experiments with mutant vitronectin confirmed that the VF sequence in the protein is also essential for binding to integrin αvβ5. To the best of our knowledge, the recognition mechanism of the RGD motif for integrin αvβ5 was first demonstrated in our study and is an important finding in the fields of matrix biology and cell biology.

Fig. 4. Identification of RGD-Containing Peptides Promoting iPSC Adhesion

(A) Sequences of synthetic peptides derived from RGD-containing proteins. (B) iPSC and HDF adhesion activity of RGD-containing peptides. (C) Effects of anti-integrin αvβ3 and αvβ5 antibodies on iPSC adhesion to RGDVF. (D) Schematic illustration of cell adhesion to RGDVF and RGDAA via integrin αvβ3 and αvβ5. These data were cited by.³¹⁾

Next, we performed structure–activity relationship study of RGDX₁X₂ motif using HeLa cells to identify more potent integrin αvβ5-binding peptides.³²⁾ First, V and F residues of CGG-RGDVF were individually substituted with other amino acids to optimize the X₁X₂ combination (Fig. 5A). The peptide was highly active when the X₁ residue was an amino acid with a relatively small side chain, such as V, A, S, T, or N; T was the most suitable for X₁ position. As the X₂ residue, F showed the strongest activity, and Y and W also showed high activity, indicating that the aromaticity of the X₂ position contributes to its binding to integrin αvβ5. Based on these results, TF was determined to be the optimal X₁X₂ combination. Then, we focused on the sequences surrounding the RGDX₁X₂ motif. As spacers between the RGD motifs and scaffolds affect the cell adhesion activity, we compared oligo-glycine, -alanine, and -proline as spacers. We found that rigid oligo-proline spacers were suitable for this activity, and tri-proline was the shortest spacer with an optimal effect. Furthermore, we found that addition of a hydrophobic amino acid, such as isoleucine, to the C-terminal X₃ position enhanced the cell adhesion activity. The best sequence obtained by combining the above-mentioned results was CPPP-RGDTFI. CPPP-RGDTFI showed higher integrin αvβ5-mediated cell adhesion activity than the original CGG-RGDVF sequence but almost the same activity as the CPPP-RGDTF sequence (Fig. 5B). In summary, we succeeded in improving the activity of CGG-RGDVF based on structure–activity relationships and obtained highly active CPPP-RGDTF and CPPP-RGDTFI (Fig. 5C). As potent ligands for integrin αvβ5, CPPP-RGDTF and CPPP-RGDTFI may aid in biomaterial development and other pharmaceutical applications.

Fig. 5. Structure−Activity Relationships of RGDX₁X₂-Containing Peptides

(A) Effect of the X₁X₂ combination on HeLa cell and HDF adhesion activity of RGDX₁X₂ peptides. * p < 0.05, ** p < 0.0001 vs. RGDVF. (B) Dose-dependent HeLa cell adhesion to CGG-vnRGD, CGG-RGDVF, CGG-RGDTF, CPPP-RGDTF, CPPP-RGDTFI, and CPPP-RGDAAI. (C) Summary of structure–activity relationships in integrin αvβ5-mediated cell adhesion activity of CGG-RGDVF. These data were cited by.³²⁾

4. CONCLUSION

We recently identified two types of peptides: YR8, which binds to heparan sulfate proteoglycan, such as syndecan, and integrin β1 (identified using R8), and RGDTF(I), which exhibits high affinity for integrin αvβ5 and has the most common RGD sequence. These peptides have better biological activities than previously reported laminin-derived cell adhesion peptides and may be used for biomaterial development in the future.

Acknowledgments

I sincerely thank Prof. Motoyoshi Nomizu, Department of Clinical Biochemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences for their support and guidance. I am also grateful to my colleagues, collaborators, and students for their contributions. This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI.

Conflict of Interest

The author declares no conflict of interest.

Notes

This review of the author’s work was written by the author upon receiving the 2023 Pharmaceutical Society of Japan Award for Young Scientists.

REFERENCES

• 1) Hayashi H, Horinokita I, Yamada Y, Hamada K, Takagi N, Nomizu M. Effects of laminin-111 peptide coatings on rat neural stem/progenitor cell culture. Exp. Cell Res., 400, 112440 (2021).
• 2) Hoffman MP, Nomizu M, Roque E, Lee S, Jung DW, Yamada Y, Kleinman HK. Laminin-1 and laminin-2 G-domain synthetic peptides bind syndecan-1 and are involved in acinar formation of a human submandibular gland cell line. J. Biol. Chem., 273, 28633–28641 (1998).
• 3) Hozumi K, Akizuki T, Yamada Y, Hara T, Urushibata S, Katagiri F, Kikkawa Y, Nomizu M. Cell adhesive peptide screening of the mouse laminin alpha1 chain G domain. Arch. Biochem. Biophys., 503, 213–222 (2010).
• 4) Hozumi K, Ishikawa M, Hayashi T, Yamada Y, Katagiri F, Kikkawa Y, Nomizu M. Identification of cell adhesive sequences in the N-terminal region of the laminin alpha2 chain. J. Biol. Chem., 287, 25111–25122 (2012).
• 5) Ishikawa M, Hamada K, Yamada Y, Kumai J, Katagiri F, Kikkawa Y, Nomizu M. Conformational dependence of integrin-binding peptides derived from homologous loop regions in the laminin alpha chains. J. Pept. Sci., 26, e3284 (2020).
• 6) Katagiri F, Hara T, Yamada Y, Urushibata S, Hozumi K, Kikkawa Y, Nomizu M. Biological activities of the homologous loop regions in the laminin alpha chain LG modules. Biochemistry, 53, 3699–3708 (2014).
• 7) Katagiri F, Ishikawa M, Yamada Y, Hozumi K, Kikkawa Y, Nomizu M. Screening of integrin-binding peptides from the laminin alpha4 and alpha5 chain G domain peptide library. Arch. Biochem. Biophys., 521, 32–42 (2012).
• 8) Katagiri F, Nomizu M. Peptide science for cell adhesive molecules. Seikagaku, 82, 463–473 (2010)
• 9) Kumai J, Yamada Y, Hamada K, Katagiri F, Hozumi K, Kikkawa Y, Nomizu M. Identification of active sequences in human laminin alpha5 G domain. J. Pept. Sci., 25, e3218 (2019).
• 10) Nomizu M, Kim WH, Yamamura K, Utani A, Song SY, Otaka A, Roller PP, Kleinman HK, Yamada Y. Identification of cell binding sites in the laminin alpha 1 chain carboxyl-terminal globular domain by systematic screening of synthetic peptides. J. Biol. Chem., 270, 20583–20590 (1995).
• 11) Sugawara Y, Hamada K, Yamada Y, Kumai J, Kanagawa M, Kobayashi K, Toda T, Negishi Y, Katagiri F, Hozumi K, Nomizu M, Kikkawa Y. Characterization of dystroglycan binding in adhesion of human induced pluripotent stem cells to laminin-511 E8 fragment. Sci. Rep., 9, 13037 (2019).
• 12) Urushibata S, Katagiri F, Takaki S, Yamada Y, Fujimori C, Hozumi K, Kikkawa Y, Kadoya Y, Nomizu M. Biologically active sequences in the mouse laminin alpha3 chain G domain. Biochemistry, 48, 10522–10532 (2009).
• 13) Yamada Y, Hozumi K, Nomizu M. Construction and activity of a synthetic basement membrane with active laminin peptides and polysaccharides. Chemistry, 17, 10500–10508 (2011).
• 14) Nomizu M, Kuratomi Y, Malinda KM, Song SY, Miyoshi K, Otaka A, Powell SK, Hoffman MP, Kleinman HK, Yamada Y. Cell binding sequences in mouse laminin alpha1 chain. J. Biol. Chem., 273, 32491–32499 (1998).
• 15) Araki E, Momota Y, Togo T, Tanioka M, Hozumi K, Nomizu M, Miyachi Y, Utani A. Clustering of syndecan-4 and integrin beta1 by laminin alpha 3 chain-derived peptide promotes keratinocyte migration. Mol. Biol. Cell, 20, 3012–3024 (2009).
• 16) Nomizu M, Kuratomi Y, Song SY, Ponce ML, Hoffman MP, Powell SK, Miyoshi K, Otaka A, Kleinman HK, Yamada Y. Identification of cell binding sequences in mouse laminin gamma1 chain by systematic peptide screening. J. Biol. Chem., 272, 32198–32205 (1997).
• 17) Mochizuki M, Kadoya Y, Wakabayashi Y, Kato K, Okazaki I, Yamada M, Sato T, Sakairi N, Nishi N, Nomizu M. Laminin-1 peptide-conjugated chitosan membranes as a novel approach for cell engineering. FASEB J., 17, 875–877 (2003).
• 18) Suzuki N, Nakatsuka H, Mochizuki M, Nishi N, Kadoya Y, Utani A, Oishi S, Fujii N, Kleinman HK, Nomizu M. Biological activities of homologous loop regions in the laminin alpha chain G domains. J. Biol. Chem., 278, 45697–45705 (2003).
• 19) Hozumi K, Otagiri D, Yamada Y, Sasaki A, Fujimori C, Wakai Y, Uchida T, Katagiri F, Kikkawa Y, Nomizu M. Cell surface receptor-specific scaffold requirements for adhesion to laminin-derived peptide-chitosan membranes. Biomaterials, 31, 3237–3243 (2010).
• 20) Yamada Y, Hozumi K, Katagiri F, Kikkawa Y, Nomizu M. Biological activity of laminin peptide-conjugated alginate and chitosan matrices. Biopolymers, 94, 711–720 (2010).
• 21) Yamada Y, Katagiri F, Hozumi K, Kikkawa Y, Nomizu M. Cell behavior on protein matrices containing laminin alpha1 peptide AG73. Biomaterials, 32, 4327–4335 (2011).
• 22) Yamada Y, Hozumi K, Aso A, Hotta A, Toma K, Katagiri F, Kikkawa Y, Nomizu M. Laminin active peptide/agarose matrices as multifunctional biomaterials for tissue engineering. Biomaterials, 33, 4118–4125 (2012).
• 23) Hozumi K, Sasaki A, Yamada Y, Otagiri D, Kobayashi K, Fujimori C, Katagiri F, Kikkawa Y, Nomizu M. Reconstitution of laminin-111 biological activity using multiple peptide coupled to chitosan scaffolds. Biomaterials, 33, 4241–4250 (2012).
• 24) Yamada Y, Hozumi K, Katagiri F, Kikkawa Y, Nomizu M. Laminin-111-derived peptide-hyaluronate hydrogels as a synthetic basement membrane. Biomaterials, 34, 6539–6547 (2013).
• 25) Otagiri D, Yamada Y, Hozumi K, Katagiri F, Kikkawa Y, Nomizu M. Cell attachment and spreading activity of mixed laminin peptide-chitosan membranes. Biopolymers, 100, 751–759 (2013).
• 26) Kumai J, Hozumi K, Yamada Y, Katagiri F, Kikkawa Y, Nomizu M. Effect of spacer length and type on the biological activity of peptide-polysaccharide matrices. Biopolymers, 106, 512–520 (2016).
• 27) Truong AT, Hamada K, Yamada Y, Guo H, Kikkawa Y, Okamoto CT, MacKay JA, Nomizu M. Evaluation of extracellular matrix mimetic laminin bioactive peptide and elastin-like polypeptide. FASEB J., 34, 6729–6740 (2020).
• 28) Yamada Y, Yoshida C, Hamada K, Kikkawa Y, Nomizu M. Development of three-dimensional cell culture scaffolds using laminin peptide-conjugated agarose microgels. Biomacromolecules, 21, 3765–3771 (2020).
• 29) Yamada Y, Onda T, Hamada K, Kikkawa Y, Nomizu M. Octa-arginine and octa-lysine promote cell adhesion through heparan sulfate proteoglycans and integrins. Biol. Pharm. Bull., 45, 207–212 (2022).
• 30) Yamada Y, Onda T, Hamada K, Kikkawa Y, Nomizu M. Effect of amino acid substitution on cell adhesion properties of octa-arginine. Biol. Pharm. Bull., 45, 1537–1543 (2022).
• 31) Yamada Y, Onda T, Hagiuda A, Kan R, Matsunuma M, Hamada K, Kikkawa Y, Nomizu M. RGDX₁ X₂ motif regulates integrin alphavbeta5 binding for pluripotent stem cell adhesion. FASEB J., 36, e22389 (2022).
• 32) Yamada Y, Onda T, Wada Y, Hamada K, Kikkawa Y, Nomizu M. Structure–activity relationships of RGD-containing peptides in integrin alphavbeta5-mediated cell adhesion. ACS Omega, 8, 4687–4693 (2023).
• 33) Kosuge M, Takeuchi T, Nakase I, Jones AT, Futaki S. Cellular internalization and distribution of arginine-rich peptides as a function of extracellular peptide concentration, serum, and plasma membrane associated proteoglycans. Bioconjug. Chem., 19, 656–664 (2008).
• 34) Khalil IA, Kogure K, Futaki S, Harashima H. Octaarginine-modified liposomes: enhanced cellular uptake and controlled intracellular trafficking. Int. J. Pharm., 354, 39–48 (2008).
• 35) Kawaguchi Y, Takeuchi T, Kuwata K, Chiba J, Hatanaka Y, Nakase I, Futaki S. Syndecan-4 is a receptor for clathrin-mediated endocytosis of arginine-rich cell-penetrating peptides. Bioconjug. Chem., 27, 1119–1130 (2016).
• 36) Vogel BE, Lee SJ, Hildebrand A, Craig W, Pierschbacher MD, Wong-Staal F, Ruoslahti E. A novel integrin specificity exemplified by binding of the alpha v beta 5 integrin to the basic domain of the HIV Tat protein and vitronectin. J. Cell Biol., 121, 461–468 (1993).
• 37) Pierschbacher MD, Ruoslahti E. Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature, 309, 30–33 (1984).
• 38) Hatley RJD, Macdonald SJF, Slack RJ, Le J, Ludbrook SB, Lukey PT. An alphav-RGD integrin inhibitor toolbox: drug discovery insight, challenges and opportunities. Angew. Chem. Int. Ed., 57, 3298–3321 (2018).
• 39) Slack RJ, Macdonald SJF, Roper JA, Jenkins RG, Hatley RJD. Emerging therapeutic opportunities for integrin inhibitors. Nat. Rev. Drug Discov., 21, 60–78 (2022).
• 40) Cheng Y, Ji Y. RGD-modified polymer and liposome nanovehicles: Recent research progress for drug delivery in cancer therapeutics. Eur. J. Pharm. Sci., 128, 8–17 (2019).
• 41) Park J, Singha K, Son S, Kim J, Namgung R, Yun CO, Kim WJ. A review of RGD-functionalized nonviral gene delivery vectors for cancer therapy. Cancer Gene Ther., 19, 741–748 (2012).
• 42) Alipour M, Baneshi M, Hosseinkhani S, Mahmoudi R, Jabari Arabzadeh A, Akrami M, Mehrzad J, Bardania H. Recent progress in biomedical applications of RGD-based ligand: from precise cancer theranostics to biomaterial engineering: a systematic review. J. Biomed. Mater. Res. A, 108, 839–850 (2020).
• 43) Hersel U, Dahmen C, Kessler H. RGD modified polymers: biomaterials for stimulated cell adhesion and beyond. Biomaterials, 24, 4385–4415 (2003).
• 44) Melkoumian Z, Weber JL, Weber DM, Fadeev AG, Zhou Y, Dolley-Sonneville P, Yang J, Qiu L, Priest CA, Shogbon C, Martin AW, Nelson J, West P, Beltzer JP, Pal S, Brandenberger R. Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells. Nat. Biotechnol., 28, 606–610 (2010).
• 45) Brandenberger R, Schmidt A, Linton J, Wang D, Backus C, Denda S, Muller U, Reichardt LF. Identification and characterization of a novel extracellular matrix protein nephronectin that is associated with integrin alpha8beta1 in the embryonic kidney. J. Cell Biol., 154, 447–458 (2001).
• 46) Magnussen SN, Toraskar J, Hadler-Olsen E, Steigedal TS, Svineng G. Nephronectin as a matrix effector in cancer. Cancers (Basel), 13, 959 (2021).
• 47) Andersen MH, Graversen H, Fedosov SN, Petersen TE, Rasmussen JT. Functional analyses of two cellular binding domains of bovine lactadherin. Biochemistry, 39, 6200–6206 (2000).
• 48) Sasaki T, Timpl R. Domain IVa of laminin alpha5 chain is cell-adhesive and binds beta1 and alphaVbeta3 integrins through Arg-Gly-Asp. FEBS Lett., 509, 181–185 (2001).