Sparsely PEGylated poly(beta-amino ester) polyplexes enhance antigen specific T-cell response of a bivalent SARS-CoV-2 DNA vaccine.

DNA technology has emerged as a promising route to accelerated manufacture of sequence agnostic vaccines. For activity, DNA vaccines must be protected and delivered to the correct antigen presenting cells. However, the physicochemical properties of the vector must be carefully tuned to enhance interaction with immune cells and generate sufficient immune response for disease protection. In this study, we have engineered a range of polymer-based nanocarriers based on the poly(beta-amino ester) (PBAE) polycation platform to investigate the role that surface poly(ethylene glycol) (PEG) density has on pDNA encapsulation, formulation properties and gene transfectability both in vitro and in vivo. We achieved this by synthesising a non-PEGylated and PEGylated PBAE and produced formulations containing these PBAEs, and mixed polyplexes to tune surface PEG density. All polymers and co-formulations produced small polyplex nanoparticles with almost complete encapsulation of the cargo in all cases. Despite high gene transfection in HEK293T cells, only the fully PEGylated and mixed formulations displayed significantly higher expression of the reporter gene than the negative control in dendritic cells. Further in vivo studies with a bivalent SARS-CoV-2 pDNA vaccine revealed that only the mixed formulation led to strong antigen specific T-cell responses, however this did not translate into the presence of serum antibodies indicating the need for further studies into improving immunisation with polymer delivery systems.

A Self-Assembled 3D Model Demonstrates How Stiffness Educates Tumor Cell Phenotypes and Therapy Resistance in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense and stiff extracellular matrix (ECM) associated with tumor progression and therapy resistance. To further the understanding of how stiffening of the tumor microenvironment (TME) contributes to aggressiveness, a three-dimensional (3D) self-assembling hydrogel disease model is developed based on peptide amphiphiles (PAs, PA-E3Y) designed to tailor stiffness. The model displays nanofibrous architectures reminiscent of native TME and enables the study of the invasive behavior of PDAC cells. Enhanced tuneability of stiffness is demonstrated by interacting thermally annealed aqueous solutions of PA-E3Y (PA-E3Yh) with divalent cations to create hydrogels with mechanical properties and ultrastructure similar to native tumor ECM. It is shown that stiffening of PA-E3Yh hydrogels to levels found in PDAC induces ECM deposition, promotes epithelial-to-mesenchymal transition (EMT), enriches CD133+/CXCR4+ cancer stem cells (CSCs), and subsequently enhances drug resistance. The findings reveal how a stiff 3D environment renders PDAC cells more aggressive and therefore more faithfully recapitulates in vivo tumors.

Bioactive and chemically defined hydrogels with tunable stiffness guide cerebral organoid formation and modulate multi-omics plasticity in cerebral organoids.

Organoids are an emerging technology with great potential in human disease modelling, drug development, diagnosis, tissue engineering, and regenerative medicine. Organoids as 3D-tissue culture systems have gained special attention in the past decades due to their ability to faithfully recapitulate the complexity of organ-specific tissues. Despite considerable successes in culturing physiologically relevant organoids, their real-life applications are currently limited by challenges such as scarcity of an appropriate biomimetic matrix. Peptide amphiphiles (PAs) due to their well-defined chemistry, tunable bioactivity, and extracellular matrix (ECM)-like nanofibrous architecture represent an attractive material scaffold for organoids development. Using cerebral organoids (COs) as exemplar, we demonstrate the possibility to create bio-instructive hydrogels with tunable stiffness ranging from 0.69 kPa to 2.24 kPa to culture and induce COs growth. We used orthogonal chemistry involving oxidative coupling and supramolecular interactions to create two-component hydrogels integrating the bio-instructive activity and ECM-like nanofibrous architecture of a laminin-mimetic PAs (IKVAV-PA) and tunable crosslinking density of hyaluronic acid functionalized with tyramine (HA-Try). Multi-omics technology including transcriptomics, proteomics, and metabolomics reveals the induction and growth of COs in soft HA-Tyr hydrogels containing PA-IKVAV such that the COs display morphology and biomolecular signatures similar to those grown in Matrigel scaffolds. Our materials hold great promise as a safe synthetic ECM for COs induction and growth. Our approach represents a well-defined alternative to animal-derived matrices for the culture of COs and might expand the applicability of organoids in basic and clinical research. STATEMENT OF SIGNIFICANCE: Synthetic bio-instructive materials which display tissue-specific functionality and nanoscale architecture of the native extracellular matrix are attractive matrices for organoids development. These synthetic matrices are chemically defined and animal-free compared to current gold standard matrices such as Matrigel. Here, we developed hydrogel matrices with tunable stiffness, which incorporate laminin-mimetic peptide amphiphiles to grow and expand cerebral organoids. Using multi-omics tools, the present study provides exciting data on the effects of neuro-inductive cues on the biomolecular profiles of brain organoids.

Intestinal Cell Differentiation and Phenotype in 2D and 3D Cell Culture Models.

Three-dimensional (3D) culture models are more physiologically relevant than two-dimensional (2D) cell culture models. 2D approaches cannot reproduce the complexity of the tumor microenvironment and are less able to translate biological insights; and drug response studies have many limitations to be extrapolated to the clinics. Here, we use the Caco-2 colon cancer cell line, which is an immortalized human epithelial cell line that under specific conditions can polarize and differentiate into a villus-like phenotype. We describe cell differentiation and cell growth in both 2D and 3D culture conditions, concluding that cell morphology, polarity, proliferation and differentiation are highly dependent on the type of cell culture system.

Predictors and efficacy of surgical treatment for consecutive exotropia.

PURPOSE: To study the surgical outcomes of consecutive exotropia and predictive factors, and to compare the medial rectus (MR) advancement, lateral rectus (LR) recession, or a combination of both procedures. METHODS: A retrospective study of patients diagnosed with consecutive exotropia that were operated on (2000-2020) were included. The convergence was classified from 0 to +++, with good: ++/+++ and poor: 0/+. A good outcome was considered when the final horizontal deviation was < 10 prism diopters (pd). Follow-up since the surgery and the number of reoperations were registered. RESULTS: A total of 88 cases were analyzed, mean age: 33.98 ± 17.68 years (57.95%: women). The near and distance horizontal deviation mean (±SD) was 34.3 pd (±16.45) and 34.36 pd (±16.33), respectively. MR advancement was performed in 36.36%, LR recession in 27.27%, and a combination of both in 36.36%. Surgery was unilateral in 65.91% (bilateral in 34.09%). A good outcome was obtained in 69.32% and reoperations in 11.36%. The insufficiency convergence was associated with a bad outcome. The near horizontal deviation (P = .006), the vertical deviation (VD) association (P = .036) and the combination of both MR advancement and LR recession (P = .017) were predictors of a bad result. The mean follow-up was 56.5 months ± 57.65. CONCLUSION: A long-term good surgical result was obtained in most patients. The greatest near deviation, the VD association, and the combination of MR advancement and the LR recession were predictive factors for bad results.

Synthetic extracellular matrices with function-encoding peptides.

The communication of cells with their surroundings is mostly encoded in the epitopes of structural and signalling proteins present in the extracellular matrix (ECM). These peptide epitopes can be incorporated in biomaterials to serve as function-encoding molecules to modulate cell-cell and cell-ECM interactions. In this Review, we discuss natural and synthetic peptide epitopes as molecular tools to bioengineer bioactive hydrogel materials. We present a library of functional peptide sequences that selectively communicate with cells and the ECM to coordinate biological processes, including epitopes that directly signal to cells, that bind ECM components that subsequently signal to cells, and that regulate ECM turnover. We highlight how these epitopes can be incorporated in different biomaterials as individual or multiple signals, working synergistically or additively. This molecular toolbox can be applied in the design of biomaterials aimed at regulating or controlling cellular and tissue function, repair and regeneration.

Elastin-like recombinamer-mediated hierarchical mineralization coatings on Zr-16Nb-xTi (x = 4,16 wt%) alloy surfaces improve biocompatibility.

The biocompatibility of biomedical materials is vital to their applicability and functionality. However, modifying surfaces for enhanced biocompatibility using traditional surface treatment techniques is challenging. We employed a mineralizing elastin-like recombinamer (ELR) self-assembling platform to mediate mineralization on Zr-16Nb-xTi (x = 4,16 wt%) alloy surfaces, resulting in the modification of surface morphology and bioactivity while improving the biocompatibility of the material. We modulated the level of nanocrystal organization by adjusting the cross-linker ratio. Nanoindentation tests revealed that the mineralized configuration had nonuniformity with respect to Young's modulus and hardness, with the center areas having higher values (5.626 ± 0.109 GPa and 0.264 ± 0.022 GPa) compared to the edges (4.282 ± 0.327 GPa and 0.143 ± 0.023 GPa). The Scratch test results indicated high bonding strength (2.668 ± 0.117 N) between the mineralized coating and the substrate. Mineralized Zr-16Nb-xTi (x = 4,16 wt%) alloys had higher viability compared to untreated alloys, which exhibited high cell viability (>100 %) after 5 days and high alkaline phosphatase activity after 7 days. Cell proliferation assays indicated that MG 63 cells grew faster on mineralized surfaces than on untreated surfaces. Scanning electron microscopy imaging confirmed that the cells adhered and spread well on mineralized surfaces. Furthermore, hemocompatibility test results revealed that all mineralized samples were non-hemolytic. Our results demonstrate the viability of employing the ELR mineralizing platform to improve alloy biocompatibility.

Peptide-Protein Coassemblies into Hierarchical and Bioactive Tubular Membranes.

Multicomponent self-assembly offers opportunities for the design of complex and functional biomaterials with tunable properties. Here, we demonstrate how minor modifications in the molecular structures of peptide amphiphiles (PAs) and elastin-like recombinamers (ELs) can be used to generate coassembling tubular membranes with distinct structures, properties, and bioactivity. First, by introducing minor modifications in the charge density of PA molecules (PAK2, PAK3, PAK4), different diffusion-reaction processes can be triggered, resulting in distinct membrane microstructures. Second, by combining different types of these PAs prior to their coassembly with ELs, further modifications can be achieved, tuning the structures and properties of the tubular membranes. Finally, by introducing the cell adhesive peptide RGDS in either the PA or EL molecules, it is possible to harness the different diffusion-reaction processes to generate tubular membranes with distinct bioactivities. The study demonstrates the possibility to trigger and achieve minor but crucial differences in coassembling processes and tune material structure and bioactivity. The study demonstrates the possibility to use minor, yet crucial, differences in coassembling processes to tune material structure and bioactivity.

Single-molecule magnet behaviour and catalytic properties of tetrahedral Co(II) complexes bearing chloride and 1,2-disubstituted benzimidazole as ligands.

Five cobalt(II) complexes of formula [CoCl2(Ln)2] [1 with L1 = 1-benzyl-2-phenyl-1H-benzimidazole, 2 with L2 = 2-(furan-2-yl)-1-(furan-2-ylmethyl)-1H-benzimidazole, 3 with L3 = 1-(4-chlorobenzyl)-2-(4-chlorophenyl)-1H-benzimidazole, 4 with L4 = 1-(2-methoxybenzyl)-2-(2-methoxyphenyl)-1H-benzimidazole and 5 with L5 = 2-(thiophen-2-yl)-1-(thiophen-2-ylmethyl)-1H-benzimidazole] have been synthesised, spectroscopically characterised and cryomagnetically investigated. The crystal structures of 1, 3, 4 and 5 have been determined by X-ray diffraction on single crystals. Each cobalt(II) ion is four-coordinate in a distorted tetrahedral environment built by two chloride anions and two benzimidazole ligands. The neutral molecules are well separated from each other, shortest intermolecular cobalt⋯cobalt distances being greater than 9.0 Å. Static (dc) magnetic susceptibility measurements in the temperature range 2.0-300 K of 1-5 reveal the occurrence of a Curie law behaviour of magnetically non-interacting spin quadruplets in the high-temperature domain with a downturn at low temperatures due to magnetic anisotropy. The values of the D and E/D parameters for these compounds vary in the ranges -8.75 to +8.96 cm-1 and 0.00140 to 0.23, respectively. Dynamic (ac) magnetic susceptibility measurements of 1-5 show slow magnetic relaxation in the lack (1) or under the presence (1-5) of applied dc magnetic fields, a feature which is typical of single-molecule magnet behaviour (SMM). The analysis of the ac data shows that a thermally activated Orbach relaxation mechanism dominates this behaviour. Complexes 1-5 also act as efficient and highly selective eco-friendly catalysts in the coupling reaction between CO2 and epoxides to produce cyclic carbonates under solvent-free conditions. Under optimized reaction conditions, different epoxides were converted to the respective cyclic carbonate, with excellent conversions, using catalyst 4.

Mineralizing Coating on 3D Printed Scaffolds for the Promotion of Osseointegration.

Design and fabrication of implants that can perform better than autologous bone grafts remain an unmet challenge for the hard tissue regeneration in craniomaxillofacial applications. Here, we report an integrated approach combining additive manufacturing with supramolecular chemistry to develop acellular mineralizing 3D printed scaffolds for hard tissue regeneration. Our approach relies on an elastin-like recombinamer (ELR) coating designed to trigger and guide the growth of ordered apatite on the surface of 3D printed nylon scaffolds. Three test samples including a) uncoated nylon scaffolds (referred to as "Uncoated"), b) ELR coated scaffolds (referred to as "ELR only"), and c) ELR coated and in vitro mineralized scaffolds (referred to as "Pre-mineralized") were prepared and tested for in vitro and in vivo performance. All test samples supported normal human immortalized mesenchymal stem cell adhesion, growth, and differentiation with enhanced cell proliferation observed in the "Pre-mineralized" samples. Using a rabbit calvarial in vivo model, 'Pre-mineralized' scaffolds also exhibited higher bone ingrowth into scaffold pores and cavities with higher tissue-implant integration. However, the coated scaffolds ("ELR only" and "Pre-mineralized") did not exhibit significantly more new bone formation compared to "Uncoated" scaffolds. Overall, the mineralizing coating offers an opportunity to enhance integration of 3D printed bone implants. However, there is a need to further decipher and tune their immunologic response to develop truly osteoinductive/conductive surfaces.

Rational design of hydrogels for immunomodulation.

The immune system protects organisms against endogenous and exogenous harm and plays a key role in tissue development, repair and regeneration. Traditional immunomodulatory biologics exhibit limitations including degradation by enzymes, short half-life and lack of targeting ability. Encapsulating or binding these biologics within biomaterials is an effective way to address these problems. Hydrogels are promising immunomodulatory materials because of their prominent biocompatibility, tuneability and versatility. However, to take advantage of these opportunities and optimize material performance, it is important to more specifically elucidate, and leverage on, how hydrogels affect and control the immune response. Here, we summarize how key physical and chemical properties of hydrogels affect the immune response. We first provide an overview of underlying steps of the host immune response upon exposure to biomaterials. Then, we discuss recent advances in immunomodulatory strategies where hydrogels play a key role through (i) physical properties including dimensionality, stiffness, porosity and topography; (ii) chemical properties including wettability, electric property and molecular presentation;and (iii) the delivery of bioactive molecules via chemical or physical cues. Thus, this review aims to build a conceptual and practical toolkit for the design of immune-instructive hydrogels capable of modulating the host immune response.

Embracing complexity in biomaterials design.

Animate materials, man-made materials behaving like living systems, are attracting enormous interest across a range of sectors, from construction and transport industry to medicine. In this leading opinion article, we propose that embracing complexity in biomaterials design offers untapped opportunities to create biomaterials with innovative life-like properties that extend their capabilities and unleash new paradigms in medical treatment.

Peptide Amphiphile Hydrogels Based on Homoternary Cucurbit[8]uril Host-Guest Complexes.

Supramolecular hydrogels based on peptide amphiphiles (PAs) are promising materials for tissue engineering and model extracellular matrixes for biological studies. While PA hydrogels are conventionally formed via electrostatic screening, new hydrogelation mechanisms might help to improve the design and functionality of these materials. Here, we present a host-guest-mediated PA hydrogelation method that relies on the formation of a host-guest homoternary complex with cucurbit[8]uril (CB[8]) and aromatic amino-acid-bearing PA nanofibers. As a result of the host-guest cross-linking between PA nanofibers, hierarchical morphologies and increased stiffness were found when host-guest-mediated PA hydrogels were compared to their ion-based equivalents. Additionally, both families of hydrogels exhibited similar biocompatibilities. These results demonstrate that CB[8]-mediated hydrogelation can be used as an alternative cross-linking method to upgrade the design of PA materials and extend their biomedical applications.

Disordered Protein Stabilization by Co-Assembly of Short Peptides Enables Formation of Robust Membranes.

Molecular self-assembly is a spontaneous natural process resulting in highly ordered nano to microarchitectures. We report temperature-independent formation of robust stable membranes obtained by the spontaneous interaction of intrinsically disordered elastin-like polypeptides (ELPs) with short aromatic peptides at temperatures both below and above the conformational transition temperature of the ELPs. The membranes are stable over time and display durability over a wide range of parameters including temperature, pH, and ultrasound energy. The morphology and composition of the membranes were analyzed using microscopy. These robust structures support preosteoblast cell adhesion and proliferation as well as pH-dependent cargo release. Simple noncovalent interactions with short aromatic peptides can overcome conformational restrictions due to the phase transition to facilitate the formation of complex bioactive scaffolds that are stable over a wide range of environmental parameters. This approach offers novel possibilities for controlling the conformational restriction of intrinsically disordered proteins and using them in the design of new materials.

Exploiting the fundamentals of biological organization for the advancement of biofabrication.

The field of biofabrication continues to progress, offering higher levels of spatial control, reproducibility, and functionality. However, we remain far from recapitulating what nature has achieved. Biological systems such as tissues and organs are assembled from the bottom-up through coordinated supramolecular and cellular processes that result in their remarkable structures and functionalities. In this perspective, we propose that incorporating such biological assembling mechanisms within fabrication techniques, offers an opportunity to push the boundaries of biofabrication. We dissect these mechanisms into distinct biological organization principles (BOPs) including self-assembly, compartmentalization, diffusion-reaction, disorder-to-order transitions, and out-of-equilibrium processes. We highlight recent work demonstrating the viability and potential of these approaches to enhance scalability, reproducibility, vascularization, and biomimicry; as well as current challenges to overcome.

Modeling Experimental Parameters for the Fabrication of Multifunctional Surfaces Composed of Electrospun PCL/ZnO-NPs Nanofibers.

In this work, a one-step electrospinning technique has been implemented for the design and development of functional surfaces with a desired morphology in terms of wettability and corrosion resistance by using polycaprolactone (PCL) and zinc oxide nanoparticles (ZnO NPs). The surface morphology has been characterized by confocal microscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM) and water contact angle (WCA), whereas the corrosion resistance has been evaluated by Tafel polarization curves. Strict control over the input operational parameters (applied voltage, feeding rate, distance tip to collector), PCL solution concentration and amount of ZnO NPs have been analyzed in depth by showing their key role in the final surface properties. With this goal in mind, a design of experiment (DoE) has been performed in order to evaluate the optimal coating morphology in terms of fiber diameter, surface roughness (Ra), water contact angle (WCA) and corrosion rate. It has been demonstrated that the solution concentration has a significant effect on the resultant electrospun structure obtained on the collector with the formation of beaded fibers with a higher WCA value in comparison with uniform bead-free fibers (dry polymer deposition or fiber-merging aspect). In addition, the presence of ZnO NPs distributed within the electrospun fibers also plays a key role in corrosion resistance, although it also leads to a decrease in the WCA. Finally, this is the first time that an exhaustive analysis by using DoE has been evaluated for PCL/ZnO electrospun fibers with the aim to optimize the surface morphology with the better performance in terms of corrosion resistance and wettability.

Modeling the Tumor Microenvironment of Ovarian Cancer: The Application of Self-Assembling Biomaterials.

Ovarian cancer (OvCa) is one of the leading causes of gynecologic malignancies. Despite treatment with surgery and chemotherapy, OvCa disseminates and recurs frequently, reducing the survival rate for patients. There is an urgent need to develop more effective treatment options for women diagnosed with OvCa. The tumor microenvironment (TME) is a key driver of disease progression, metastasis and resistance to treatment. For this reason, 3D models have been designed to represent this specific niche and allow more realistic cell behaviors compared to conventional 2D approaches. In particular, self-assembling peptides represent a promising biomaterial platform to study tumor biology. They form nanofiber networks that resemble the architecture of the extracellular matrix and can be designed to display mechanical properties and biochemical motifs representative of the TME. In this review, we highlight the properties and benefits of emerging 3D platforms used to model the ovarian TME. We also outline the challenges associated with using these 3D systems and provide suggestions for future studies and developments. We conclude that our understanding of OvCa and advances in materials science will progress the engineering of novel 3D approaches, which will enable the development of more effective therapies.

Bioengineered 3D models of human pancreatic cancer recapitulate in vivo tumour biology.

Patient-derived in vivo models of human cancer have become a reality, yet their turnaround time is inadequate for clinical applications. Therefore, tailored ex vivo models that faithfully recapitulate in vivo tumour biology are urgently needed. These may especially benefit the management of pancreatic ductal adenocarcinoma (PDAC), where therapy failure has been ascribed to its high cancer stem cell (CSC) content and high density of stromal cells and extracellular matrix (ECM). To date, these features are only partially reproduced ex vivo using organoid and sphere cultures. We have now developed a more comprehensive and highly tuneable ex vivo model of PDAC based on the 3D co-assembly of peptide amphiphiles (PAs) with custom ECM components (PA-ECM). These cultures maintain patient-specific transcriptional profiles and exhibit CSC functionality, including strong in vivo tumourigenicity. User-defined modification of the system enables control over niche-dependent phenotypes such as epithelial-to-mesenchymal transition and matrix deposition. Indeed, proteomic analysis of these cultures reveals improved matrisome recapitulation compared to organoids. Most importantly, patient-specific in vivo drug responses are better reproduced in self-assembled cultures than in other models. These findings support the use of tuneable self-assembling platforms in cancer research and pave the way for future precision medicine approaches.

Polymyxin B-Triggered Assembly of Peptide Hydrogels for Localized and Sustained Release of Combined Antimicrobial Therapy.

Repurposing old antibiotics into more effective and safer formulations is an emergent approach to tackle the growing threat of antimicrobial resistance. Herein, a peptide hydrogel is reported for the localized and sustained release of polymyxin B (PMB), a decade-old antibiotic with increasing clinical utility for treating multidrug-resistant Gram-negative bacterial infections. The hydrogel is assembled by additing PMB solution into a rationally designed peptide amphiphile (PA) solution and its mechanical properties can be adjusted through the addition of counterions, envisioning its application in diverse infection scenarios. Sustained release of PMB from the hydrogel over a 5-day period and prolonged antimicrobial activities against Gram-negative bacteria are observed. The localized release of active PMB from the hydrogel is shown to be effective in vivo for treating Pseudomonas aeruginosa infection in the Galleria mellonella burn wound infection model, dramatically reducing the mortality from 93% to 13%. Complementary antimicrobial activity against Gram-positive Staphylococcus aureus and enhanced antimicrobial effect against the Gram-negative Acinetobacter baumannii are observed when an additional antibiotic fusidic acid is incorporated into the hydrogen network. These results demonstrate the potential of the PMB-triggered PA hydrogel as a versatile platform for the localized and sustained delivery of combined antimicrobial therapies.

Carboxylated-xyloglucan and peptide amphiphile co-assembly in wound healing.

Hydrogel wound dressings can play critical roles in wound healing protecting the wound from trauma or contamination and providing an ideal environment to support the growth of endogenous cells and promote wound closure. This work presents a self-assembling hydrogel dressing that can assist the wound repair process mimicking the hierarchical structure of skin extracellular matrix. To this aim, the co-assembly behaviour of a carboxylated variant of xyloglucan (CXG) with a peptide amphiphile (PA-H3) has been investigated to generate hierarchical constructs with tuneable molecular composition, structure, and properties. Transmission electron microscopy and circular dichroism at a low concentration shows that CXG and PA-H3 co-assemble into nanofibres by hydrophobic and electrostatic interactions and further aggregate into nanofibre bundles and networks. At a higher concentration, CXG and PA-H3 yield hydrogels that have been characterized for their morphology by scanning electron microscopy and for the mechanical properties by small-amplitude oscillatory shear rheological measurements and compression tests at different CXG/PA-H3 ratios. A preliminary biological evaluation has been carried out both in vitro with HaCat cells and in vivo in a mouse model.