Selectively Positioned Catechol Moiety Supports Ultrashort Self-Assembling Peptide Hydrogel Adhesion for Coral Restoration.

Coral reef survival is threatened globally. One way to restore this delicate ecosystem is to enhance coral growth by the controlled propagation of coral fragments. To be sustainable, this technique requires the use of biocompatible underwater adhesives. Hydrogels based on rationally designed ultrashort self-assembling peptides (USP) are of great interest for various biological and environmental applications, due to their biocompatibility and tunable mechanical properties. Implementing superior adhesion properties to the USP hydrogel compounds is crucial in both water and high ionic strength solutions and is relevant in medical and marine environmental applications such as coral regeneration. Some marine animals secrete large quantities of the aminoacids dopa and lysine to enhance their adhesion to wet surfaces. Therefore, the addition of catechol moieties to the USP sequence containing lysine (IIZK) should improve the adhesive properties of USP hydrogels. However, it is challenging to place the catechol moiety (Do) within the USP sequence at an optimal position without compromising the hydrogel self-assembly process and mechanical properties. Here, we demonstrate that, among three USP hydrogels, DoIIZK is the least adhesive and that the adhesiveness of the IIZDoK hydrogel is compromised by its poor mechanical properties. The best adhesion outcome was achieved using the IIZKDo hydrogel, the only one to show equally sound adhesive and mechanical properties. A mechanistic understanding of this outcome is presented here. This property was confirmed by the successful gluing of coral fragments by means of IIZKDo hydrogel that are still thriving after more than three years since the deployment. The validated biocompatibility of this underwater hydrogel glue suggests that it could be advantageously implemented for other applications, such as surgical interventions.

Marcromolecular Architecture and Encapsulation of the Anticancer Drug Everolimus Control the Self-Assembly of Amphiphilic Polypeptide-Containing Hybrids.

Macromolecular architecture plays an important role in the self-assembly process of block copolymer amphiphiles. Herein, two series of stimuli-responsive amphiphilic 3-miktoarm star hybrid terpolypeptides and their corresponding linear analogues were synthesized exhibiting the same overall composition and molecular weight but different macromolecular architecture. The macromolecular architecture was found to be a key parameter in defining the morphology of the nanostructures formed in aqueous solutions as well as to alter the self-assembly behavior of the polymers independently of their composition. In addition, it was found that the assemblies prepared from the star-shaped polymers showed superior tolerance against enzymatic degradation due to the increased corona block density on the outer surface of the nanoparticles. Encapsulation of the hydrophobic anticancer drug Everolimus resulted in the formation of intriguing non-spherical and non-symmetric pH-responsive nanostructures, such as "stomatocytes" and "multi-compartmentalized suprapolymersomes", while the pH-triggered release of the drug was also investigated. Owing to the similarities of the developed "stomatocytes" with red blood cells, in combination with their pH-responsiveness and superior stability over enzymatic degradation, they are expected to present advanced drug delivery properties and have the ability to bypass several extra- and intracellular barriers to reach and effectively treat cancer cells.

A Novel Poly(vinylidene fluoride)-Based 4-Miktoarm Star Terpolymer: Synthesis and Self-Assembly.

A well-defined amphiphilic miktoarm polymer incorporating poly(vinylidene fluoride) (PVDF), polystyrene (PS), and poly(ethylene glycol) (PEG) blocks was synthesized via a combination of atom-transfer radical polymerization (ATRP), iodine transfer radical polymerization (ITP), and copper-catalyzed azide-alkyne cycloaddition (CuAAC). Morphology and self-assembly of this star polymer were examined in organic solvents and in water. The aggregates formed in water were found to possess unusual frustrated topology due to immiscibility of PS and PVDF. The polymer was evaluated for transport of small hydrophobic molecules in water.

Poly(sarcosine)-Based Nano-Objects with Multi-Protease Resistance by Aqueous Photoinitiated Polymerization-Induced Self-Assembly (Photo-PISA).

Poly(sarcosine) (PSar) is a non-ionic hydrophilic polypeptoid with numerous biologically relevant properties, making it an appealing candidate for the development of amphiphilic block copolymer nanostructures. In this work, the fabrication of poly(sarcosine)-based diblock copolymer nano-objects with various morphologies via aqueous reversible addition-fragmentation chain-transfer (RAFT)-mediated photoinitiated polymerization-induced self-assembly (photo-PISA) is reported. Poly(sarcosine) was first synthesized via ring-opening polymerization (ROP) of sarcosine N-carboxyanhydride, using high-vacuum techniques. A small molecule chain transfer agent (CTA) was then coupled to the active ω-amino chain end of the telechelic polymer for the synthesis of a poly(sarcosine)-based macro-CTA. Controlled chain-extensions of a commercially available water-miscible methacrylate monomer (2-hydroxypropyl methacrylate) were achieved via photo-PISA under mild reaction conditions, using PSar macro-CTA. Upon varying the degree of polymerization and concentration of the core-forming monomer, morphologies evolving from spherical micelles to worm-like micelles and vesicles were accessed, as determined by dynamic light scattering and transmission electron microscopy, resulting in the construction of a detailed phase diagram. The resistance of both colloidally stable empty vesicles and enzyme-loaded nanoreactors against degradation by a series of proteases was finally assessed. Overall, our findings underline the potential of poly(sarcosine) as an alternative corona-forming polymer to poly(ethylene glycol)-based analogues of PISA assemblies for use in various pharmaceutical and biomedical applications.

Self-Healing pH- and Enzyme Stimuli-Responsive Hydrogels for Targeted Delivery of Gemcitabine To Treat Pancreatic Cancer.

A novel, multifunctional hydrogel that exhibits a unique set of properties for the effective treatment of pancreatic cancer (PC) is presented. The material is composed of a pentablock terpolypeptide of the type PLys- b-(PHIS- co-PBLG)-PLys- b-(PHIS- co-PBLG)- b-PLys, which is a noncytotoxic polypeptide. It can be implanted via the least invasive route and selectively delivers gemcitabine to efficiently treat PC. Simply mixing the novel terpolypeptide with an aqueous solution of gemcitabine within a syringe results in the facile formation of a hydrogel that has the ability to become liquid under the shear rate of the plunger. Upon injection in the vicinity of cancer tissue, it immediately reforms into a hydrogel due to the unique combination of its macromolecular architecture and secondary structure. Because of its pH responsiveness, the hydrogel only melts close to PC; thus, the drug can be delivered directionally toward the cancerous rather than healthy tissues in a targeted, controlled, and sustained manner. The efficacy of the hydrogel was tested in vivo on human to mouse xenografts using the drug gemcitabine. It was found that the efficacy of the hydrogel loaded with only 40% of the drug delivered in one dose was equal to or slightly better than the peritumoral injection of 100% of the free drug delivered in two doses, the typical chemotherapy used in clinics so far. This result suggests that the hydrogel can direct the delivery of the encapsulated drug effectively in the tumor tissue. Enzymes lead to its biodegradation, avoiding removal by resection of the polypeptidic carrier after cargo delivery. The unique properties of the hydrogel formed can be predetermined through its molecular characteristics, rendering it a promising modular material for many biological applications.

Self-Assembly of Telechelic Tyrosine End-Capped PEO and Poly(alanine) Polymers in Aqueous Solution.

The self-assembly in aqueous solution of three novel telechelic conjugates comprising a central hydrophilic polymer and short (trimeric or pentameric) tyrosine end-caps has been investigated. Two of the conjugates have a central poly(oxyethylene) (polyethylene oxide, PEO) central block with different molar masses. The other conjugate has a central poly(L-alanine) (PAla) sequence in a purely amino-acid based conjugate. All three conjugates self-assemble into ß-sheet based fibrillar structures, although the fibrillar morphology revealed by cryogenic-TEM is distinct for the three polymers--in particular the Tyr5-PEO6k-Tyr5 forms a population of short straight fibrils in contrast to the more diffuse fibril aggregates observed for Tyr5-PEO2k-Tyr5 and Tyr3-PAla-Tyr3. Hydrogel formation was not observed for these samples (in contrast to prior work on related systems) up to quite high concentrations, showing that it is possible to prepare solutions of peptide-polymer-peptide conjugates with hydrophobic end-caps without conformational constraints associated with hydrogelation. The Tyr5-PEO6k-Tyr5 shows significant PEO crystallization upon drying in contrast to the Tyr5-PEO2k-Tyr5 conjugate. Our findings point to the remarkable ability of short hydrophobic peptide end groups to modulate the self-assembly properties of polymers in solution in model peptide-capped "associative polymers". Retention of fluidity at high conjugate concentration may be valuable in potential future applications of these conjugates as bioresponsive or biocompatible materials, for example exploiting the enzyme-responsiveness of the tyrosine end-groups.

Self-assembly of a model peptide incorporating a hexa-histidine sequence attached to an oligo-alanine sequence, and binding to gold NTA/nickel nanoparticles.

Amyloid fibrils are formed by a model surfactant-like peptide (Ala)10-(His)6 containing a hexa-histidine tag. This peptide undergoes a remarkable two-step self-assembly process with two distinct critical aggregation concentrations (cac's), probed by fluorescence techniques. A micromolar range cac is ascribed to the formation of prefibrillar structures, whereas a millimolar range cac is associated with the formation of well-defined but more compact fibrils. We examine the labeling of these model tagged amyloid fibrils using Ni-NTA functionalized gold nanoparticles (Nanogold). Successful labeling is demonstrated via electron microscopy imaging. The specificity of tagging does not disrupt the ß-sheet structure of the peptide fibrils. Binding of fibrils and Nanogold is found to influence the circular dichroism associated with the gold nanoparticle plasmon absorption band. These results highlight a new approach to the fabrication of functionalized amyloid fibrils and the creation of peptide/nanoparticle hybrid materials.

Fusing Peptide Epitopes for Advanced Multiplex Serological Testing for SARS-CoV-2 Antibody Detection.

The tragic COVID-19 pandemic, which has seen a total of 655 million cases worldwide and a death toll of over 6.6 million seems finally tailing off. Even so, new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise, the severity of which cannot be predicted in advance. This is concerning for the maintenance and stability of public health, since immune evasion and increased transmissibility may arise. Therefore, it is crucial to continue monitoring antibody responses to SARS-CoV-2 in the general population. As a complement to polymerase chain reaction tests, multiplex immunoassays are elegant tools that use individual protein or peptide antigens simultaneously to provide a high level of sensitivity and specificity. To further improve these aspects of SARS-CoV-2 antibody detection, as well as accuracy, we have developed an advanced serological peptide-based multiplex assay using antigen-fused peptide epitopes derived from both the spike and the nucleocapsid proteins. The significance of the epitopes selected for antibody detection has been verified by in silico molecular docking simulations between the peptide epitopes and reported SARS-CoV-2 antibodies. Peptides can be more easily and quickly modified and synthesized than full length proteins and can, therefore, be used in a more cost-effective manner. Three different fusion-epitope peptides (FEPs) were synthesized and tested by enzyme-linked immunosorbent assay (ELISA). A total of 145 blood serum samples were used, compromising 110 COVID-19 serum samples from COVID-19 patients and 35 negative control serum samples taken from COVID-19-free individuals before the outbreak. Interestingly, our data demonstrate that the sensitivity, specificity, and accuracy of the results for the FEP antigens are higher than for single peptide epitopes or mixtures of single peptide epitopes. Our FEP concept can be applied to different multiplex immunoassays testing not only for SARS-CoV-2 but also for various other pathogens. A significantly improved peptide-based serological assay may support the development of commercial point-of-care tests, such as lateral-flow-assays.

Post-Polymerization Modification of Fluoropolymers via UV Irradiation in the Presence of a Photoacid Generator.

Fluorinated polymers have unique wettability and protein adsorption properties. The site-specific alteration of these properties could expand their application to different research areas. In this work, a fluorinated homopolymer and two of its copolymers with 4-vinylbenzyl glycidyl ether (VBGE) are synthesized by free radical polymerization. The produced polymers are then used to develop resist formulations by the addition of a photoacid generator. Films of these formulations are exposed to ultraviolet radiation through a binary mask and heated to create the pattern. It is found that the water contact angle values of the exposed films areas are reduced compared to those of the unexposed ones, with the exception of pentafluorophenyl methacrylate (PFMA) homopolymer film. This is attributed to the reaction of the epoxy groups creating x-links and producing hydroxyl groups and the cleavage of the pentafluorophenyl group from the ester group leading to carboxylic acid groups. Both modifications on the exposed areas are verified by FTIR spectroscopy and ToF-SIMS analysis. In addition, the biomolecules adsorption ability of the exposed area is increasing 10-15 times compared to the unexposed one for the PFMA homopolymer and the PFMA/VBGE 1:1 copolymer. Thus, the proposed polymers and patterning procedure could find application to spatially directed immobilization of biomolecules and/or cells onto a surface for both biosensing and tissue engineering purposes.

Dipeptide-Based Photoreactive Instant Glue for Environmental and Biomedical Applications.

Nature-inspired smart materials offer numerous advantages over environmental friendliness and efficiency. Emulating the excellent adhesive properties of mussels foot proteins, where the lysine is in close proximity with the 3,4-dihydroxy-l-phenylalanine (DOPA), we report the synthesis of a novel photocurable peptide-based adhesive consisting exclusively of these two amino acids. Our adhesive is a highly concentrated aqueous solution of a monomer, a cross-linker, and a photoinitiator. Lap-shear adhesion measurements on plastic and glass surfaces and comparison with different types of commercial adhesives showed that the adhesive strength of our glue is comparable when applied in air and superior when used underwater. No toxicity of our adhesive was observed when the cytocompatibility on human dermal fibroblast cells was assessed. Preliminary experiments with various tissues and coral fragments showed that our adhesive could be applied to wound healing and coral reef restoration. Given the convenience of the facile synthesis, biocompatibility, ease of application underwater, and high adhesive strength, we expect that our adhesive may find application, but not limited, to the biomedical and environmental field.

Evaluation of Potential Peptide-Based Inhibitors against SARS-CoV-2 and Variants of Concern.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has greatly affected all aspect of life. Although several vaccines and pharmaceuticals have been developed against SARS-CoV-2, the emergence of mutated variants has raised several concerns. The angiotensin-converting enzyme (ACE2) receptor cell entry mechanism of this virus has not changed despite the vast mutation in emerging variants. Inhibiting the spike protein by which the virus identifies the host ACE2 receptor is a promising therapeutic countermeasure to keep pace with rapidly emerging variants. Here, we synthesized two ACE2-derived peptides, P1 and P25, to target and potentially inhibit SARS-CoV-2 cell entry. These peptides were evaluated in vitro using pseudoviruses that contained the SARS-CoV-2 original spike protein, the Delta-mutated spike protein, or the Omicron spike protein. An in silico investigation was also done for these peptides to evaluate the interaction of the synthesized peptides and the SARS-CoV-2 variants. The P25 peptide showed a promising inhibition potency against the tested pseudoviruses and an even higher inhibition against the Omicron variant. The IC50 of the Omicron variant was 60.8 µM, while the IC50s of the SARS-CoV-2 original strain and the Delta variant were 455.2 µM and 546.4 µM, respectively. The in silico experiments also showed that the amino acid composition design and structure of P25 boosted the interaction with the spike protein. These findings suggest that ACE2-derived peptides, such as P25, have the potential to inhibit SARS-CoV-2 cell entry in vitro. However, further in vivo studies are needed to confirm their therapeutic efficacy against emerging variants.

Fabrication of a Lateral Flow Assay for Rapid In-Field Detection of COVID-19 Antibodies Using Additive Manufacturing Printing Technologies.

The development of lateral flow immunoassay (LFIA) using three-dimensional (3D) printing and bioprinting technologies can enhance and accelerate the optimization process of the fabrication. Therefore, the main goal of this study is to investigate methods to speed up the developing process of a LFIA as a tool for community screening. To achieve this goal, an in-house developed robotic arm and microfluidic pumps were used to print the proteins during the development of the test. 3D printing technologies were used to design and print the housing unit for the testing strip. The proposed design was made by taking into consideration the environmental impact of this disposable medical device.

Ecologically Friendly Biofunctional Ink for Reconstruction of Rigid Living Systems Under Wet Conditions.

The development of three-dimensional (3D)-printable inks is essential for several applications, from industrial manufacturing to novel applications for biomedical engineering. Remarkably, biomaterials for tissue engineering applications can be expanded to other new horizons; for instance, restoration of rigid living systems as coral reefs is an emergent need derived from recent issues from climate change. The coral reefs have been endangered, which can be observed in the increasing bleaching around the world. Very few studies report eco-friendly inks for matter since most conventional approaches require synthetic polymer, which at some point could be a pollutant depending on the material. Therefore, there is an unmet need for cost-effective formulations from eco-friendly materials for 3D manufacturing to develop carbonate-based inks for coral reef restoration. Our value proposition derives from technologies developed for regenerative medicine, commonly applied for human tissues like bone and cartilage. In our case, we created a novel biomaterial formulation from biopolymers such as gelatin methacrylate, poly (ethylene glycol diacrylate), alginate, and gelatin as scaffold and binder for the calcium carbonate and hydroxyapatite bioceramics needed to mimic the structure of rigid structures. This project presents evidence from 2D/3D manufacturing, chemical, mechanical, and biological characterization, which supports the hypothesis of its utility to aid in the fight to counteract the coral bleaching that affects all the marine ecosystem, primarily when this is supported by solid research in biomaterials science used for living systems, it can extend tissue engineering into new approaches in different domains such as environmental or marine sciences.

Anionic Polymerization of Styrene and 1,3-Butadiene in the Presence of Phosphazene Superbases.

The anionic polymerization of styrene and 1,3-butadiene in the presence of phosphazene bases (t-BuP4, t-BuP2 and t-BuP1), in benzene at room temperature, was studied. When t-BuP1 was used, the polymerization proceeded in a controlled manner, whereas the obtained homopolymers exhibited the desired molecular weights and narrow polydispersity (Р< 1.05). In the case of t-BuP2, homopolymers with higher than the theoretical molecular weights and relatively low polydispersity were obtained. On the other hand, in the presence of t-BuP4, the polymerization of styrene was uncontrolled due to the high reactivity of the formed carbanion. The kinetic studies from the polymerization of both monomers showed that the reaction rate follows the order of [t-BuP4]/[sec-BuLi] >>> [t-BuP2]/[sec-BuLi] >> [t-BuP1]/[sec-BuLi] > sec-BuLi. Furthermore, the addition of t-BuP2 and t-BuP1 prior the polymerization of 1,3-butadiene allowed the synthesis of polybutadiene with a high 1,2-microstructure (~45 wt %), due to the delocalization of the negative charge. Finally, the one pot synthesis of well-defined polyester-based copolymers [PS-b-PCL and PS-b-PLLA, PS: Polystyrene, PCL: Poly(ε-caprolactone) and PLLA: Poly(L-lactide)], with predictable molecular weights and a narrow molecular weight distribution (Р< 1.2), was achieved by sequential copolymerization in the presence of t-BuP2 and t-BuP1.

Well-Defined Cyclic Triblock Terpolymers: A Missing Piece of the Morphology Puzzle.

Two well-defined cyclic triblock terpolymers, missing pieces of the terpolymer morphology puzzle, consisting of poly(isoprene), polystyrene, and poly(2-vinylpyridine), were synthesized by combining the Glaser coupling reaction with anionic polymerization. An α,ω-dihydroxy linear triblock terpolymer (OH-PI1,4-b-PS-b-P2VP-OH) was first synthesized followed by transformation of the OH to alkyne groups by esterification with pentynoic acid and cyclization by Glaser coupling. The size exclusion chromatography (SEC) trace of the linear terpolymer precursor was shifted to lower elution time after cyclization, indicating the successful synthesis of the cyclic terpolymer. Additionally, the SEC trace of the cyclic terpolymer produced, after cleavage of the ester groups, shifted again practically to the position corresponding to the linear precursor. The first exploratory results on morphology showed the tremendous influence of the cyclic structure on the morphology of terpolymers.

Development of multiple stimuli responsive magnetic polymer nanocontainers as efficient drug delivery systems.

Magnetic nanodevices based on poly[(methacrylic acid)-co-(N-isopropylacrylamide)] [P(MAA-co-NIPAAm)] are prepared and used as drug delivery systems employing daunorubicin (DNR) as a model drug. The magnetic nanocontainers exploit the pH, temperature, and magnetic response of the polymeric shell constituents and magnetic nanoparticles, respectively, for controlled pH, temperature and alternating magnetic field triggered drug release. The in vitro cytotoxicity of both DNR-loaded and empty nanocontainers is examined on MCF-7 breast cancer cells along with the intracellular distribution of DNR. The results show that the DNR-loaded nanocontainers have an anti-tumor effect comparable to the free drug. The current observations provide important information for potent drug delivery and release systems.

Nanoscale rings fabricated using self-assembled triblock terpolymer templates.

Although there has been extensive work on the use of self-assembled diblock copolymers for nanolithography, there are few reports of the use of multiblock copolymers, which can form a more diverse range of nanoscale pattern geometries. Pattern transfer from a self-assembled poly(butadiene-b-styrene-b-methyl methacrylate) (PB-b-PS-b-PMMA) triblock terpolymer thin film has been investigated. Polymers of different total molecular weight were synthesized with a predicted morphology consisting of PMMA-core/PS-shell cylinders in a PB matrix. By adjusting the solvent-annealing conditions and the film thickness, thin films with vertically oriented cylinders were formed. The PMMA cylinder cores and the PB matrix were then removed using selective etching to leave an array of PS rings, and the ring pattern was transferred into a silica film by reactive ion etching to form 19 nm high silica rings. This result illustrates the design and use of triblock terpolymers for self-assembled lithography.