Architecture-Based Programming of Polymeric Micelles to Undergo Sequential Mesophase Transitions.

Di- and triblock amphiphiles can form different mesophases ranging from micelles to hydrogels depending on their chemical structures, hydrophilic to hydrophobic ratios, and their ratio in the mixture. In addition, their different architectures dictate their exchange rate between the assembled and unimer states and consequently affect their responsiveness toward enzymatic degradation. Here we report the utilization of the different reactivities of di- and triblock amphiphiles, having exactly the same hydrophilic to lipophilic balance, toward enzymatic degradation as a tool for programming formulations to undergo sequential enzymatically induced transitions from (i) micelles to (ii) hydrogel and finally to (iii) dissolved polymers. We show that the rate of transition between the mesophases can be programmed by changing the ratio of the amphiphiles in the formulation, and that the hydrogels can maintain encapsulated cargo, which was loaded into the micelles. The reported results demonstrate the ability of molecular architecture to serve as a tool for programming smart formulations to adopt different structures and functions.

Enhancement of Collagen-I Levels in Human Gingival Fibroblasts by Small Molecule Activation of HIF-1α.

Collagen is the most abundant protein in various mammalian tissues and has an essential role in various cellular processes. Collagen is necessary for food-related biotechnological applications such as cultivated meat, medical engineering, and cosmetics. High-yield expression of natural collagen from mammalian cells is challenging and not cost-effective. Thus, external collagen is obtained primarily from animal tissues. Under cellular hypoxia, overactivation of the transcription factor hypoxia-inducible factor (HIF) was shown to correlate with enhanced accumulation of collagen. Herein, we showed that the small molecule ML228, a known molecular activator of HIF, enhances the accumulation of collagen type-I in human fibroblast cells. We report an increase in collagen levels by 2.33 ± 0.33 when fibroblasts were incubated with 5 µM of ML228. Our experimental results demonstrated, for the first time, that external modulation of the hypoxia biological pathway can boost collagen levels in mammalian cells. Our findings pave the way for enhancing natural collagen production in mammals by altering cellular signaling pathways.

Ataxia Telangiectasia Mutated Signaling Delays Skin Pigmentation upon UV Exposure by Mediating MITF Function toward DNA Repair Mode.

Skin pigmentation is paused after sun exposure; however, the mechanism behind this pausing is unknown. In this study, we found that the UVB-induced DNA repair system, led by the ataxia telangiectasia mutated (ATM) protein kinase, represses MITF transcriptional activity of pigmentation genes while placing MITF in DNA repair mode, thus directly inhibiting pigment production. Phosphoproteomics analysis revealed ATM to be the most significantly enriched pathway among all UVB-induced DNA repair systems. ATM inhibition in mouse or human skin, either genetically or chemically, induces pigmentation. Upon UVB exposure, MITF transcriptional activation is blocked owing to ATM-dependent phosphorylation of MITF on S414, which modifies MITF activity and interactome toward DNA repair, including binding to TRIM28 and RBBP4. Accordingly, MITF genome occupancy is enriched in sites of high DNA damage that are likely repaired. This suggests that ATM harnesses the pigmentation key activator for the necessary rapid, efficient DNA repair, thus optimizing the chances of the cell surviving. Data are available from ProteomeXchange with the identifier PXD041121.

Molecular Engineering of Rigid Hydrogels Co-assembled from Collagenous Helical Peptides Based on a Single Triplet Motif.

The potential of ultra-short peptides to self-assemble into well-ordered functional nanostructures makes them promising minimal components for mimicking the basic ingredient of nature and diverse biomaterials. However, selection and modular design of perfect de novo sequences are extremely tricky due to their vast possible combinatorial space. Moreover, a single amino acid substitution can drastically alter the supramolecular packing structure of short peptide assemblies. Here, we report the design of rigid hybrid hydrogels produced by sequence engineering of a new series of ultra-short collagen-mimicking tripeptides. Connecting glycine with different combinations of proline and its post-translational product 4-hydroxyproline, the single triplet motif, displays the natural collagen-helix-like structure. Improved mechanical rigidity is obtained via co-assembly with the non-collagenous hydrogelator, fluorenylmethoxycarbonyl (Fmoc) diphenylalanine. Characterizations of the supramolecular interactions that promote the self-supporting and self-healing properties of the co-assemblies are performed by physicochemical experiments and atomistic models. Our results clearly demonstrate the significance of sequence engineering to design functional peptide motifs with desired physicochemical and electromechanical properties and reveal co-assembly as a promising strategy for the utilization of small, readily accessible biomimetic building blocks to generate hybrid biomolecular assemblies with structural heterogeneity and functionality of natural materials.

Exploiting Minimalistic Backbone Engineered γ-Phenylalanine for the Formation of Supramolecular Co-Polymer.

Ordered supramolecular hydrogels assembled by modified aromatic amino acids often exhibit low mechanical rigidity. Aiming to stabilize the hydrogel and understand the impact of conformational freedom and hydrophobicity on the self-assembly process, two building blocks based on 9-fluorenyl-methoxycarbonyl-phenylalanine (Fmoc-Phe) gelator which contain two extra methylene units in the backbone, generating Fmoc-γPhe and Fmoc-(3-hydroxy)-γPhe are designed. Fmoc-γPhe spontaneously assembled in aqueous media forming a hydrogel with exceptional mechanical and thermal stability. Moreover, Fmoc-(3-hydroxy)-γPhe, with an extra backbone hydroxyl group decreasing its hydrophobicity while maintaining some molecular flexibility, self-assembled into a transient fibrillar hydrogel, that later formed microcrystalline aggregates through a phase transition. Molecular dynamics simulations and single crystal X-ray analyses reveal the mechanism underlying the two residues' distinct self-assembly behaviors. Finally, Fmoc-γPhe and Fmoc-(3-OH)-γPhe co-assembly to form a supramolecular hydrogel with notable mechanical properties are demonstrated. It has been believed that the understanding of the structure-assembly relationship will enable the design of new functional amino acid-based hydrogels.

Stabilizing gelatin-based bioinks under physiological conditions by incorporation of ethylene-glycol-conjugated Fmoc-FF peptides.

Over the last decade, three-dimensional (3D) printing technologies have attracted the interest of researchers due to the possibility of fabricating tissue- and organ-like structures with similarities to the organ of interest. One of the most widely used materials for the fabrication of bioinks is gelatin (Gel) due to its excellent biocompatibility properties. However, in order to fabricate stable scaffolds under physiological conditions, the most common approach is to use gelatin methacrylate (GelMA) that allows the crosslinking and therefore the stabilization of the hydrogel through UV crosslinking. The crosslinking process can be harmful to cells thus decreasing total cell viability. To overcome the need for post-printing crosslinking, a new approach of bioink formulation was studied, incorporating the Fluorenylmethoxycarbonyl diphenylalanine (Fmoc-FF) peptide into the Gel bioink. However, although Fmoc-FF possesses excellent mechanical properties, the lack of elasticity and viscosity makes it unsuitable for 3D-printing. Here, we demonstrate that covalent conjugation of two different ethylene glycol (EG) motifs to the Fmoc-FF peptide increases the hydrophilicity and elasticity properties, which are essential for 3D-printing. This new approach for bioink formulation avoids the need for any post-printing manufacturing processes, such as chemical or UV crosslinking.

Thixotropic Red Microalgae Sulfated Polysaccharide-Peptide Composite Hydrogels as Scaffolds for Tissue Engineering.

Sulfated polysaccharides of red marine microalgae have recently gained much attention for biomedical applications due to their anti-inflammatory and antioxidant properties. However, their low mechanical properties limit their use in tissue engineering. Herein, to enhance the mechanical properties of the sulfated polysaccharide produced by the red marine microalga, Porphyridium sp. (PS), it was integrated with the fluorenylmethoxycarbonyl diphenylalanine (FmocFF) peptide hydrogelator. Transparent, stable hydrogels were formed when mixing the two components at a 1:1 ratio in three different concentrations. Electron microscopy showed that all hydrogels exhibited a nanofibrous structure, mimicking the extracellular matrix. Furthermore, the hydrogels were injectable, and tunable mechanical properties were obtained by changing the hydrogel concentration. The composite hydrogels allowed the sustained release of curcumin which was controlled by the change in the hydrogel concentration. Finally, the hydrogels supported MC3T3-E1 preosteoblasts viability and calcium deposition. The synergy between the sulfated polysaccharide, with its unique bioactivities, and FmocFF peptide, with its structural and mechanical properties, bears a promising potential for developing novel tunable scaffolds for tissue engineering that may allow cell differentiation into various lineages.

Molecular Co-Assembly of Two Building Blocks Harnesses Both their Attributes into a Functional Supramolecular Hydrogel.

Engineering ordered nanostructures through molecular self-assembly of simple building blocks constitutes the essence of modern nanotechnology to develop functional supramolecular biomaterials. However, the lack of adequate chemical and functional diversity often hinders the utilization of unimolecular self-assemblies for practical applications. Co-assembly of two different building blocks can essentially harness both of their attributes and produce nanostructured macro-scale objects with improved physical properties and desired functional complexity. Herein, the authors report the co-operative co-assembly of a modified amino acid, fluorenylmethoxycarbonyl-pentafluoro-phenylalanine (Fmoc-F5 -Phe), and a peptide, Fmoc-Lys(Fmoc)-Arg-Gly-Asp [Fmoc-K(Fmoc)-RGD] into a functional supramolecular hydrogel. A change in the morphology and fluorescence emission, as well as improvement of the mechanical properties in the mixed hydrogels compared to the pristine hydrogels, demonstrate the signature of co-operative co-assembly mechanism. Intriguingly, this approach harnesses the advantages of both components in a synergistic way, resulting in a single homogeneous biomaterial possessing the antimicrobial property of Fmoc-F5 -Phe and the biocompatibility and cell adhesive characteristics of Fmoc-K(Fmoc)-RGD. This work exemplifies the importance of the co-assembly process in nanotechnology and lays the foundation for future developments in supramolecular chemistry by harnessing the advantages of diverse functional building blocks into a mechanically stable functional biomaterial.

Bi-functional peptide-based 3D hydrogel-scaffolds.

Over the last few years, hydrogels have been proposed for many biomedical applications, including drug delivery systems and scaffolds for tissue engineering. In particular, peptides have been envisioned as excellent candidates for the development of hydrogel materials, due to their intrinsic biocompatibility, ease of handling, and intrinsic biodegradability. Recently, we developed a novel hybrid polymer-peptide conjugate, PEG8-(FY)3, which is able to self-assemble into a self-supporting soft hydrogel over dry and wet surfaces as demonstrated by molecular dynamics simulation. Here, we describe the synthesis and supramolecular organization of six novel hexapeptides rationally designed by punctual chemical modification of the primary peptide sequence of the ancestor peptide (FY)3. Non-coded amino acids were incorporated by replacing the phenylalanine residue with naphthylalanine (Nal) and tyrosine with dopamine (Dopa). We also studied the effect of the modification of the side chain and the corresponding PEGylated peptide analogues, on the structural and mechanical properties of the hydrogel. Secondary structure, morphology and rheological properties of all the peptide-based materials were assessed by various biophysical tools. The in vitro biocompatibility of the supramolecular nanostructures was also evaluated on fibroblast cell lines. We conclude that the PEG8-(Nal-Dopa)3 hydrogel possesses the right properties to serve as a scaffold and support cell growth.

UV-Protection Timer Controls Linkage between Stress and Pigmentation Skin Protection Systems.

Skin sun exposure induces two protection programs: stress responses and pigmentation, the former within minutes and the latter only hours afterward. Although serving the same physiological purpose, it is not known whether and how these programs are coordinated. Here, we report that UVB exposure every other day induces significantly more skin pigmentation than the higher frequency of daily exposure, without an associated increase in stress responses. Using mathematical modeling and empirical studies, we show that the melanocyte master regulator, MITF, serves to synchronize stress responses and pigmentation and, furthermore, functions as a UV-protection timer via damped oscillatory dynamics, thereby conferring a trade-off between the two programs. MITF oscillations are controlled by multiple negative regulatory loops, one at the transcriptional level involving HIF1α and another post-transcriptional loop involving microRNA-148a. These findings support trait linkage between the two skin protection programs, which, we speculate, arose during furless skin evolution to minimize skin damage.