Titanium Boston keratoprosthesis with corneal cell adhesive and bactericidal dual coating.

The Boston keratoprosthesis (BKPro) is a medical device used to restore vision in complicated cases of corneal blindness. This device is composed by a front plate of polymethylmethacrylate (PMMA) and a backplate usually made of titanium (Ti). Ti is an excellent biomaterial with numerous applications, although there are not many studies that address its interaction with ocular cells. In this regard, despite the good retention rates of the BKPro, two main complications compromise patients' vision and the viability of the prosthesis: imperfect adhesion of the corneal tissue to the upside of the backplate and infections. Thus, in this work, two topographies (smooth and rough) were generated on Ti samples and tested with or without functionalization with a dual peptide platform. This molecule consists of a branched structure that links two peptide moieties to address the main complications associated with BKPro: the well-known RGD peptide in its cyclic version (cRGD) as cell pro-adherent motif and the first 11 residues of lactoferrin (LF1-11) as antibacterial motif. Samples were physicochemically characterized, and their biological response was evaluated in vitro with human corneal keratocytes (HCKs) and against the gram-negative bacterial strain Pseudomonas aeruginosa. The physicochemical characterization allowed to verify the functionalization in a qualitative and quantitative manner. A higher amount of peptide was anchored to the rough surfaces. The studies performed using HCKs showed increased long-term proliferation on the functionalized samples. Gene expression was affected by topography and peptide functionalization. Roughness promoted α-smooth muscle actin (α-SMA) overexpression, and the coating notably increased the expression of extracellular matrix components (ECM). Such changes may favour the development of unwanted fibrosis, and thus, corneal haze. In contrast, the combination of the coating with a rough topography decreased the expression of α-SMA and ECM components, which would be desirable for the long-term success of the prosthesis. Regarding the antibacterial activity, the functionalized smooth and rough surfaces promoted the death of bacteria, as well as a perturbation in their wall definition and cellular morphology. Bacterial killing values were 58 % for smooth functionalised and 68 % for rough functionalised samples. In summary, this study suggests that the use of the dual peptide platform with cRGD and LF1-11 could be a good strategy to improve the in vitro and in vivo performance of the rough topography used in the commercial BKPro.

Protease-degradable hydrogels with multifunctional biomimetic peptides for bone tissue engineering.

Mimicking bone extracellular matrix (ECM) is paramount to develop novel biomaterials for bone tissue engineering. In this regard, the combination of integrin-binding ligands together with osteogenic peptides represents a powerful approach to recapitulate the healing microenvironment of bone. In the present work, we designed polyethylene glycol (PEG)-based hydrogels functionalized with cell instructive multifunctional biomimetic peptides (either with cyclic RGD-DWIVA or cyclic RGD-cyclic DWIVA) and cross-linked with matrix metalloproteinases (MMPs)-degradable sequences to enable dynamic enzymatic biodegradation and cell spreading and differentiation. The analysis of the intrinsic properties of the hydrogel revealed relevant mechanical properties, porosity, swelling and degradability to engineer hydrogels for bone tissue engineering. Moreover, the engineered hydrogels were able to promote human mesenchymal stem cells (MSCs) spreading and significantly improve their osteogenic differentiation. Thus, these novel hydrogels could be a promising candidate for applications in bone tissue engineering, such as acellular systems to be implanted and regenerate bone or in stem cells therapy.

Comparison of the Antibacterial Effect of Silver Nanoparticles and a Multifunctional Antimicrobial Peptide on Titanium Surface.

Titanium implantation success may be compromised by Staphylococcus aureus surface colonization and posterior infection. To avoid this issue, different strategies have been investigated to promote an antibacterial character to titanium. In this work, two antibacterial agents (silver nanoparticles and a multifunctional antimicrobial peptide) were used to coat titanium surfaces. The modulation of the nanoparticle (≈32.1 ± 9.4 nm) density on titanium could be optimized, and a sequential functionalization with both agents was achieved through a two-step functionalization method by means of surface silanization. The antibacterial character of the coating agents was assessed individually as well as combined. The results have shown that a reduction in bacteria after 4 h of incubation can be achieved on all the coated surfaces. After 24 h of incubation, however, the individual antimicrobial peptide coating was more effective than the silver nanoparticles or their combination against Staphylococcus aureus. All tested coatings were non-cytotoxic for eukaryotic cells.

Functionalization of 3D printed polymeric bioresorbable stents with a dual cell-adhesive peptidic platform combining RGDS and YIGSR sequences.

Biomimetic surface modification with cell-adhesive peptides is a promising approach to improve endothelialization of current bioresorbable stents (BRS). Among them, RGDS and YIGSR sequences have been reported to mediate adhesion and migration of endothelial cells (ECs) while preventing platelet activation. This work presents the functionalization of novel 3D-printed poly-L-lactic acid (PLLA) and poly(L-lactic-co-ε-caprolactone) (PLCL) BRS with linear RGDS and YIGSR sequences, as well as a dual platform (PF) containing both motifs within a single biomolecule. Functionalized surfaces were characterized in terms of static contact angle, biomolecule distribution under confocal fluorescence microscopy and peptide quantification via detachment from the surface, showing a biomolecule density in the range of 0.5 to 3.5 nmol cm-2. Biological evaluation comprised a cell adhesion test on functionalized films with ECs and a blood perfusion assay on functionalized stents to assess ECs response and device hemocompatibility, respectively. Cell adhesion assays evidenced significantly increased cell number and spreading onto functionalized films with respect to control samples. Regarding stents' hemocompatibility, platelet adhesion onto PLCL stents was severely decreased with respect to PLLA. In addition, functionalization with RGDS, YIGSR and the PF rendered BRS stents displaying even further reduced platelet adhesion. In conclusion, the combination of intrinsically less prothrombogenic materials such as PLCL and its functionalization with EC-discriminating adhesive biomolecules paves the way for a new generation of BRS based on accelerated re-endothelialization approaches.

Hydroxyapatite nanoparticles-cell interaction: New approaches to disclose the fate of membrane-bound and internalised nanoparticles.

Hydroxyapatite nanoparticles are popular tools in bone regeneration, but they have also been used for gene delivery and as anticancer drugs. Understanding their mechanism of action, particularly for the latter application, is crucial to predict their toxicity. To this end, we aimed to elucidate the importance of nanoparticle membrane interactions in the cytotoxicity of MG-63 cells using two different types of nanoparticles. In addition, conventional techniques for studying nanoparticle internalisation were evaluated and compared with newer and less exploited approaches. Hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles were used as suspensions or compacted as specular discs. Comparison between cells seeded on the discs and those supplemented with the nanoparticles allowed direct interaction of the cell membrane with the material to be ruled out as the main mechanism of toxicity. In addition, standard techniques such as flow cytometry were inconclusive when used to assess nanoparticles toxicity. Interestingly, the use of intracellular calcium fluorescent probes revealed the presence of a high number of calcium-rich vesicles after nanoparticle supplementation in cell culture. These structures could not be detected by transmission electron microscopy due to their liquid content. However, by using cryo-soft X-ray imaging, which was used to visualise the cellular ultrastructure without further treatment other than vitrification and to quantify the linear absorption coefficient of each organelle, it was possible to identify them as multivesicular bodies, potentially acting as calcium stores. In the study, an advanced state of degradation of the hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles within MG-63 cells was observed. Overall, we demonstrate that the combination of fluorescent calcium probes together with cryo-SXT is an excellent approach to investigate intracellular calcium, especially when found in its soluble form.

Mimicking Bone Extracellular Matrix: From BMP-2-Derived Sequences to Osteogenic-Multifunctional Coatings.

Cell-material interactions are regulated by mimicking bone extracellular matrix on the surface of biomaterials. In this regard, reproducing the extracellular conditions that promote integrin and growth factor (GF) signaling is a major goal to trigger bone regeneration. Thus, the use of synthetic osteogenic domains derived from bone morphogenetic protein 2 (BMP-2) is gaining increasing attention, as this strategy is devoid of the clinical risks associated with this molecule. In this work, the wrist and knuckle epitopes of BMP-2 are screened to identify peptides with potential osteogenic properties. The most active sequences (the DWIVA motif and its cyclic version) are combined with the cell adhesive RGD peptide (linear and cyclic variants), to produce tailor-made biomimetic peptides presenting the bioactive cues in a chemically and geometrically defined manner. Such multifunctional peptides are next used to functionalize titanium surfaces. Biological characterization with mesenchymal stem cells demonstrates the ability of the biointerfaces to synergistically enhance cell adhesion and osteogenic differentiation. Furthermore, in vivo studies in rat calvarial defects prove the capacity of the biomimetic coatings to improve new bone formation and reduce fibrous tissue thickness. These results highlight the potential of mimicking integrin-GF signaling with synthetic peptides, without the need for exogenous GFs.

New trends in the development of multifunctional peptides to functionalize biomaterials.

Improving cell-material interactions is a major goal in tissue engineering. In this regard, functionalization of biomaterials with cell instructive molecules from the extracellular matrix stands out as a powerful strategy to enhance their bioactivity and achieve optimal tissue integration. However, current functionalization strategies, like the use of native full-length proteins, are associated with drawbacks, thus urging the need of developing new methodologies. In this regard, the use of synthetic peptides encompassing specific bioactive regions of proteins represents a promising alternative. In particular, the combination of peptide sequences with complementary or synergistic effects makes it possible to address more than one biological target at the biomaterial surface. In this review, an overview of the main strategies using peptides to install multifunctionality on biomaterials is presented, mostly focusing on the combination of the RGD motif with other peptides sequences. The evolution of these approaches, starting from simple methods, like using peptide mixtures, to more advanced systems of peptide presentation, with very well defined chemical properties, are explained. For each system of peptide's presentation, three main aspects of multifunctionality-improving receptor selectivity, mimicking the extracellular matrix and preventing bacterial colonization while improving cell adhesion-are highlighted.

A versatile click chemistry-based approach for functionalizing biomaterials of diverse nature with bioactive peptides.

A novel and versatile toolkit approach for the functionalization of biomaterials of different nature is described. This methodology is based on the solid-phase conjugation of specific anchoring units onto a resin-bound azido-functionalized peptide by using click chemistry. A synergistic multifunctional peptidic scaffold with cell adhesive properties was used as a model compound to showcase the versatility of this new approach. Titanium, gold and polylactic acid surfaces were biofunctionalized by this method, as validated by physicochemical surface characterization with XPS. In vitro assays using mesenchymal stem cells showed enhanced cell adhesion on the functionalized samples, proving the capacity of this strategy to efficiently bioactivate different types of biomaterials.

An Engineered Biomimetic Peptide Regulates Cell Behavior by Synergistic Integrin and Growth Factor Signaling.

Recreating the healing microenvironment is essential to regulate cell-material interactions and ensure the integration of biomaterials. To repair bone, such bioactivity can be achieved by mimicking its extracellular matrix (ECM) and by stimulating integrin and growth factor (GF) signaling. However, current approaches relying on the use of GFs, such as bone morphogenetic protein 2 (BMP-2), entail clinical risks. Here, a biomimetic peptide integrating the RGD cell adhesive sequence and the osteogenic DWIVA motif derived from the wrist epitope of BMP-2 is presented. The approach offers the advantage of having a spatial control over the single binding of integrins and BMP receptors. Such multifunctional platform is designed to incorporate 3,4-dihydroxyphenylalanine to bind metallic oxides with high affinity in a one step process. Functionalization of glass substrates with the engineered peptide is characterized by physicochemical methods, proving a successful surface modification. The biomimetic interfaces significantly improve the adhesion of C2C12 cells, inhibit myotube formation, and activate the BMP-dependent signaling via p38. These effects are not observed on surfaces displaying only one bioactive motif, a mixture of both motifs or soluble DWIVA. These data prove the biological potential of recreating the ECM and engaging in integrin and GF crosstalk via molecular-based mimics.

Chemically Diverse Multifunctional Peptide Platforms with Antimicrobial and Cell Adhesive Properties.

Bacterial infections and incomplete biomaterial integration are major problems that can lead to the failure of medical implants. However, simultaneously addressing these two issues remains a challenge. Here, we present a chemical peptide library based on a multifunctional platform containing the antimicrobial peptide LF1-11 and the cell-adhesive motif RGD. The scaffolds were customized with catechol groups to ensure straightforward functionalization of the implant surface, and linkers of different length to assess the effect of peptide accessibility on the biological response. The peptidic platforms significantly improved the adhesion of mesenchymal stem cells and showed antimicrobial effects against Staphylococcus aureus. Of note is that peptides bearing spacers that were too long displayed the lowest efficiency. Subsequently, we designed a platform replacing linear RGD by cyclic RGD; this further enhanced eukaryotic cell adhesion while retaining excellent antimicrobial properties, thus being a suitable candidate for tissue engineering applications.

Chemical Modification of Microcin J25 Reveals New Insights on the Stereospecific Requirements for Antimicrobial Activity.

In this study, microcin J25, a potent antimicrobial lasso peptide that acts on Gram-negative bacteria, was subjected to a harsh treatment with a base in order to interrogate its stability and mechanism of action and explore its structure-activity relationship. Despite the high stability reported for this lasso peptide, the chemical treatment led to the detection of a new product. Structural studies revealed that this product retained the lasso topology, but showed no antimicrobial activity due to the epimerization of a key residue for the activity. Further microbiological assays also demonstrated that it showed a high synergistic effect with colistin.

A Lasso-Inspired Bicyclic Peptide: Synthesis, Structure and Properties.

The chemical synthesis of a bicycle inspired by the natural lasso peptide sungsanpin using a combination of solid-phase and in-solution chemistries is described. The bicyclic-derived topoisomer was designed by introducing a covalent linkage between the ring and the loop, which allowed the tying of these two parts of the peptide, rendering the bicyclic structure. Several structural techniques, such as MS fragmentation, ion-mobility and NMR spectroscopic analysis were used to characterize the bicycle. Ion-mobility spectroscopy studies revealed that it showed lasso-like behavior. Its 3D structure was predicted on the basis of the NMR restraints. In addition, the high proteolytic and thermal stability of the bicycle potentially make it a suitable scaffold for epitope grafting.

Investigation of the Biosynthesis of the Lasso Peptide Chaxapeptin Using an E. coli-Based Production System.

Lasso peptides are natural products belonging to the family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) and are defined by their unique topology. Even though lasso peptide biosynthetic gene clusters are found in many different kinds of bacteria, most of the hitherto studied lasso peptides were of proteobacterial or actinobacterial origin. Despite this, no E. coli-based production system has been reported for actinobacterial lasso peptides, while there are numerous examples of this for proteobacterial lasso peptides. Here, a heterologous production system of the lasso peptide chaxapeptin was established in E. coli. Chaxapeptin, originally isolated from Streptomyces leeuwenhoekii strain C58, is closely related to the lasso peptide sungsanpin (produced by a marine Streptomyces sp.) and shares its inhibitory activity against cell invasion by the human lung cancer cell line A549. Our production system not only allowed isolation of the mature lasso peptide outside of the native producer with a yield of 0.1 mg/L (compared to 0.7 mg/L from S. leeuwenhoekii) but also was used for a mutational study to identify residues in the precursor peptide that are important for biosynthesis. In addition to these experiments, the stability of chaxapeptin against thermal denaturation and proteases was assessed.

Lasso peptides: chemical approaches and structural elucidation.

The discovery and development of novel constrained peptides that combine the advantages of therapeutic proteins with those of small molecules has partially prompted the re-emergence of peptides as therapeutics. In this regard, lasso peptides are characterized by both the selectivity and potency of larger protein biologics but with no or low immunogenicity, and the stability and bioavailability of small molecules. Moreover, the diverse functionality of lasso peptides and their extraordinary stability against chemical, thermal and proteolytic degradation make them attractive candidates for drug discovery. However, the chemical synthesis of lasso peptides remains a challenge due to the difficulty in building and maintaining their threaded structure. From a therapeutic point of view, these small and constrained structures would provide a new paradigm in drug discovery.