Boosted Cross-Linking and Characterization of High-Performing Self-Assembling Peptides

Abstract

Tissue engineering (TE) strategies require the design and characterization of novel biomaterials capable of mimicking the physiological microenvironments of the tissues to be regenerated. As such, implantable materials should be biomimetic, nanostructured and with mechanical properties approximating those of the target organ/tissue. Self-assembling peptides (SAPs) are biomimetic nanomaterials that can be readily synthesized and customized to match the requirements of some TE applications, but the weak interactions involved in the self-assembling phenomenon make them soft hydrogels unsuited for the regeneration of medium-to-hard tissues. In this work, we moved significant steps forward in the field of chemical cross-linked SAPs towards the goal of stiff peptidic materials suited for the regeneration of several tissues. Novel SAPs were designed and characterized to boost the 4-(N-Maleimidomethyl) cyclohexane-1-carboxylic acid 3-sulpho-N-hydroxysuccinimide ester (Sulfo-SMCC) mediated cross-linking reaction, where they reached G′ values of ~500 kPa. An additional orthogonal cross-linking was also effective and allowed to top remarkable G′ values of 840 kPa. We demonstrated that cross-linking fastened the pre-existing self-aggregated nanostructures, and at the same time, a strong presence of ß-structures is necessary for an effective cross-linking of (LKLK)3-based SAPs. Combining strong SAP design and orthogonal cross-linking reactions, we brought SAP stiffness closer to the MPa threshold, and as such, we opened the door of the regeneration of skin, muscle and lung to biomimetic SAP technology.

Low-power microwaves: a cell-compatible physical treatment to enhance the mechanical properties of self-assembling peptides

“…79 It is well known that substrate stiffness can influence the fate decisions of various stem cells. 80,81 In our previous works, we demonstrated the negligible cytotoxicity of GP 17 and Sulfo-SMCC 26,27 cross-linkings of SAPs, as well as their limited effect on hNSC proliferation and differentiation. hNSCs are a well-standardized biological source for the in vitro screening of biomaterials in nervous tissue engineering and are also very sensitive to toxic compounds.…”

“…For this purpose, the tested SAPs are reported in Table 1 with their respective net charge. LDLK12 is a non-functionalized SAP widely used in regenerative medicine applications; 58 the functional motif of FAQ-LDLK12 was panned from a phage display against murine neural stem cells; 58 BM3 is a BMHP1-derived SAP, 60 linked with biotin at the N-terminal group; HYDROSAP was developed to obtain a 3D in vitro model of densely cultured hNSCs; 17 CK 1 belongs to co-assembling SAPs used for hetero-bifunctional cross-linking, 26,27 and a representative example of cationic peptides. As previously reported, 17 the purified linear sequences Ac-(Leu-Asp-Leu-Lys) 3 -CONH 2 , Ac-Lys-Leu-Pro-Gly-Trp-Gly-Gly-Gly-Gly-(Leu-Asp-Leu-Lys) 3 -CONH 2 , and Ac-Ser-Ser-Leu-Ser-Val-Asn-Asp-Gly-Gly-Gly(Leu-Asp-Leu-Lys) 3 -CONH 2 and branched tris(Leu-Asp-Leu-Lys) 3 -CONH 2 were combined and dissolved in distilled H 2 O to obtain the so-called pure HYDROSAP.…”

“…We found that small energy and prolonged doses of microwaves significantly improved the mechanical properties of a variety of peptide hydrogels (Table 1), representative of different classes of SAPs. LDLK12 is a non-functionalized SAP widely used in regenerative medicine applications; 58,59 the functional motif of FAQ-LDLK12 was panned from a phage display against murine-NSC; 58 BM3 is a BMHP1-derived SAP; 60 HYDROSAP, comprising branched and linear SAPs, was developed to obtain an optimal 3D in vitro model of densely cultured hNSCs; 17 CK 1 belongs to co-assembling SAPs used for hetero-bifunctional cross-linking; 26,27 Pal1 is an amphiphilic SAP conjugated with a palmitoyl moiety as hydrophobic tail.…”

Long-term cultures of human pancreatic islets in self-assembling peptides hydrogels

“…Purified peptides powder was subsequently dissolved in 0.1 M HCl to remove the presence of possible TFA salts. Three different SAPs were used for this study: pureHYDROSAP (Marchini et al, 2019;Marchini et al, 2020), FAQ (NH 2 -FAQRVPP-GGG-LDLKLDLKLDLK-CONH 2 ) (Gelain et al, 2012) and CK 1 (Ac-CGGLKLKLKLKLKLKGGC-CONH 2 ) (Pugliese et al, 2018c;Ciulla et al, 2022). As previously described, pureHYDROSAP is composed by linear SAPs Ac-(LDLK) 3 -CONH 2 , Ac-KLPGWSGGGG-(LDLK) 3 -CONH 2 (Caprini et al, 2013) and Ac-SSLSVNDGGG-(LDLK) 3 -CONH 2 (Gelain et al, 2012) and branched SAP tris(LDLK) 3 -CONH 2 (Pugliese et al, 2018a).…”

“…SAPs used in this work were widely applied in the field of neural stem cells (Gelain et al, 2012;Pugliese et al, 2018c;Marchini et al, 2019;Marchini et al, 2020). It is well-known that brain tissue is very soft, with a stiffness ranging from 0.1kPa to 1 kPa (Ciulla et al, 2022): pancreas stiffness lays in the similar range (Guimarães et al, 2020), with healthy pancreas tissue ~900 Pa and with increased ECM stiffness in pancreatic cancer (~2,900 Pa) (LeSavage et al, 2022). Three different SAPs (HYDROSAP, FAQ and CK 1 ) were evaluated in terms of rheological analysis to investigate their viscoelastic and mechanical properties, as well as to compare their characteristics to that of pancreatic tissue.…”