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Background Aims: With the objective of improving the ex vivo production of therapeutic chimeric antigen receptor (CAR) T cells, we explored the addition of three-dimensional (3D) polystyrene scaffolds to standard suspension cell cultures., Methods: We aimed to mimic the structural support given by the lymph nodes during in vivo lymphocyte expansion., Results: We observed an increase in cell proliferation compared with standard suspension systems as well as an enhanced cytotoxicity toward cancer cells. Moreover, we directly obtained the CAR T cells from peripheral blood mononuclear cells, thus minimizing the ex vivo manipulation of the therapeutic cells and opening the way to synergies among different cell populations., Conclusions: We propose the use of commercially available 3D polystyrene systems to improve the current immune cell cultures and resulting cell products for emerging cellular (immuno)therapies., Competing Interests: Declaration of Competing Interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article., (Copyright © 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Cardiac medical devices (CMDs) are required when the patient's cardiac capacity or activity is compromised. To guarantee its correct functionality, the building materials in the development of CMDs must focus on several fundamental properties such as strength, stiffness, rigidity, corrosion resistance, etc. The challenge is more significant because CMDs are generally built with at least one metallic and one polymeric part. However, not only the properties of the materials need to be taken into consideration. The biocompatibility of the materials represents one of the major causes of the success of CMDs in the short and long term. Otherwise, the material will lead to several problems of hemocompatibility (e.g., protein adsorption, platelet aggregation, thrombus formation, bacterial infection, and finally, the rejection of the CMDs). To enhance the hemocompatibility of selected materials, surface modification represents a suitable solution. The surface modification involves the attachment of chemical compounds or bioactive compounds to the surface of the material. These coatings interact with the blood and avoid hemocompatibility and infection issues. This work reviews two main topics: 1) the materials employed in developing CMDs and their key characteristics, and 2) the surface modifications reported in the literature, clinical trials, and those that have reached the market. With the aim of providing to the research community, considerations regarding the choice of materials for CMDs, together with the advantages and disadvantages of the surface modifications and the limitations of the studies performed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Coronel-Meneses, Sánchez-Trasviña, Ratera and Mayolo-Deloisa.)

Antibiotic resistance has exponentially increased during the last years. It is necessary to develop new antimicrobial drugs to prevent and treat infectious diseases caused by multidrug- or extensively-drug resistant (MDR/XDR)-bacteria. Host Defense Peptides (HDPs) have a versatile role, acting as antimicrobial peptides and regulators of several innate immunity functions. The results shown by previous studies using synthetic HDPs are only the tip of the iceberg, since the synergistic potential of HDPs and their production as recombinant proteins are fields practically unexplored. The present study aims to move a step forward through the development of a new generation of tailored antimicrobials, using a rational design of recombinant multidomain proteins based on HDPs. This strategy is based on a two-phase process, starting with the construction of the first generation molecules using single HDPs and further selecting those HDPs with higher bactericidal efficiencies to be combined in the second generation of broad-spectrum antimicrobials. As a proof of concept, we have designed three new antimicrobials, named D5L37βD3, D5L37D5L37 and D5LAL37βD3. After an in-depth exploration, we found D5L37D5L37 to be the most promising one, since it was equally effective against four relevant pathogens in healthcare-associated infections, such as methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus , methicillin-resistant Staphylococcus epidermidis (MRSE) and MDR Pseudomonas aeruginosa , being MRSA, MRSE and P. aeruginosa MDR strains. The low MIC values and versatile activity against planktonic and biofilm forms reinforce the use of this platform to isolate and produce unlimited HDP combinations as new antimicrobial drugs by effective means.

Hypothesis: Quatsome nanovesicles, formed through the self-assembly of cholesterol (CHOL) and cetyltrimethylammonium bromide (CTAB) in water, have shown long-term stability in terms of size and morphology, while at the same time exhibiting high CHOL-CTAB intermolecular binding energies. We hypothesize that CHOL/CTAB quatsomes are indeed thermodynamically stable nanovesicles, and investigate the mechanism underlying their formation., Experiments: A systematic study was performed to determine whether CHOL/CTAB quatsomes satisfy the experimental requisites of thermodynamically stable vesicles. Coarse-grain molecular dynamics simulations were used to investigate the molecular organization in the vesicle membrane, and the characteristics of the simulated vesicle were corroborated with experimental data obtained by cryo-electron microscopy, small- and wide-angle X-ray scattering, and multi-angle static light scattering., Findings: CHOL/CTAB quatsomes fulfill the requisites of thermodynamically stable nanovesicles, but they do not exhibit the classical membrane curvature induced by a composition asymmetry between the bilayer leaflets, like catanionic nanovesicles. Instead, CHOL/CTAB quatsomes are formed through the association of intrinsically planar bilayers in a faceted vesicle with defects, indicating that distortions in the organization and orientation of molecules can play a major role in the formation of thermodynamically stable nanovesicles., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Inc.)

Fluorescent organic nanoparticles (FONs) are a large family of nanostructures constituted by organic components that emit light in different spectral regions upon excitation, due to the presence of organic fluorophores. FONs are of great interest for numerous biological and medical applications, due to their high tunability in terms of composition, morphology, surface functionalization, and optical properties. Multifunctional FONs combine several functionalities in a single nanostructure (emission of light, carriers for drug-delivery, functionalization with targeting ligands, etc.), opening the possibility of using the same nanoparticle for diagnosis and therapy. The preparation, characterization, and application of these multifunctional FONs require a multidisciplinary approach. In this review, we present FONs following a tutorial approach, with the aim of providing a general overview of the different aspects of the design, preparation, and characterization of FONs. The review encompasses the most common FONs developed to date, the description of the most important features of fluorophores that determine the optical properties of FONs, an overview of the preparation methods and of the optical characterization techniques, and the description of the theoretical approaches that are currently adopted for modeling FONs. The last part of the review is devoted to a non-exhaustive selection of some recent biomedical applications of FONs.

The growing emergence of microorganisms resistant to antibiotics has prompted the development of alternative antimicrobial therapies. Among them, the antimicrobial peptides produced by innate immunity, which are also known as host defense peptides (HDPs), hold great potential. They have been shown to exert activity against both Gram-positive and Gram-negative bacteria, including those resistant to antibiotics. These HDPs are classified into three categories: defensins, cathelicidins, and histatins. Traditionally, HDPs have been chemically synthesized, but this strategy often limits their application due to the high associated production costs. Alternatively, some HDPs have been recombinantly produced, but little is known about the impact of the bacterial strain in the recombinant product. This work aimed to assess the influence of the Escherichia coli strain used as cell factory to determine the activity and stability of recombinant defensins, which have 3 disulfide bonds. For that, an α-defensin [human α-defensin 5 (HD5)] and a β-defensin [bovine lingual antimicrobial peptide (LAP)] were produced in two recombinant backgrounds. The first one was an E. coli BL21 strain, which has a reducing cytoplasm, whereas the second was an E. coli Origami B, that is a strain with a more oxidizing cytoplasm. The results showed that both HD5 and LAP, fused to Green Fluorescent Protein (GFP), were successfully produced in both BL21 and Origami B strains. However, differences were observed in the HDP production yield and bactericidal activity, especially for the HD5-based protein. The HD5 protein fused to GFP was not only produced at higher yields in the E. coli BL21 strain, but it also showed a higher quality and stability than that produced in the Origami B strain. Hence, this data showed that the strain had a clear impact on both HDPs quantity and quality., (© 2022. The Author(s).)

Fabricating polymeric scaffolds using cost-effective manufacturing processes is still challenging. Gas foaming techniques using supercritical carbon dioxide (scCO 2 ) have attracted attention for producing synthetic polymer matrices; however, the high-pressure requirements are often a technological barrier for its widespread use. Compressed 1,1,1,2-tetrafluoroethane, known as Freon R134a, offers advantages over CO 2 in manufacturing processes in terms of lower pressure and temperature conditions and the use of low-cost equipment. Here, we report for the first time the use of Freon R134a for generating porous polymer matrices, specifically polylactide (PLA). PLA scaffolds processed with Freon R134a exhibited larger pore sizes, and total porosity, and appropriate mechanical properties compared with those achieved by scCO 2 processing. PLGA scaffolds processed with Freon R134a were highly porous and showed a relatively fragile structure. Human mesenchymal stem cells (MSCs) attached to PLA scaffolds processed with Freon R134a, and their metabolic activity increased during culturing. In addition, MSCs displayed spread morphology on the PLA scaffolds processed with Freon R134a, with a well-organized actin cytoskeleton and a dense matrix of fibronectin fibrils. Functionalization of Freon R134a-processed PLA scaffolds with protein nanoparticles, used as bioactive factors, enhanced the scaffolds' cytocompatibility. These findings indicate that gas foaming using compressed Freon R134a could represent a cost-effective and environmentally friendly fabrication technology to produce polymeric scaffolds for tissue engineering approaches.

Recent achievements in the field of immunotherapy, such as the development of engineered T cells used in adoptive cell therapy, are introducing more efficient strategies to combat cancer. Nevertheless, there are still many limitations. For example, these T cells are challenging to manufacture, manipulate, and control. Specifically, there are limitations in producing the large amounts of therapeutic T cells needed for these therapies in a short period of time and in an economically viable manner. In this study, three-dimensional (3D) poly(ethylene) glycol (PEG) hydrogels covalently combined with low molecular weight heparin are engineered to resemble the lymph nodes, where T cells reproduce. In these hydrogels, PEG provides the needed structural and mechanical properties, whereas heparin is used as an anchor for the cytokine CCL21, which is present in the lymph nodes, and can affect cell migration and proliferation. The 3D structure of the hydrogel in combination with its loading capacity result in an increased primary human CD4 + T cell proliferation compared to the state-of-the-art expansion systems consisting of artificial antigen presenting cells. Thus, we present a new tool for adoptive cell therapy to help achieving the large numbers of cells required for therapy of selected phenotypes targeted against cancer cells, by mimicking the lymph nodes., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Engineering new materials which are capable of trapping biomolecules in nanoscale quantities, is crucial in order to achieve earlier diagnostics in different diseases. This article demonstrates that using free radical copolymerization, polyacrylamide can be successfully functionalized with specific synthons for nanotrapping positively charged molecules, such as numerous proteins, through electrostatic interactions due to their negative charge. Specifically, two functional random copolymers, acrylamide/acrylic acid (1) and acrylamide/acrylic acid/ N -(pyridin-4-yl-methyl)acrylamide (2), whose negative net charges differ in their water solutions, were synthetized and their ability to trap positively charged proteins was studied using myoglobin as a proof-of-concept example. In aqueous solutions, copolymer 1, whose net charge for a 100 chain fragment ( Q pH 6 / M ) is -1.323 × 10 -3 , interacted with myoglobin forming a stable monodisperse nanosuspension. In contrast, copolymer 2, whose value of Q pH 6 / M equals -0.361 × 10 -3 , was not able to form stable particles with myoglobin. Nevertheless, thin films of both copolymers were grown using a dewetting process, which exhibited nanoscale cavities capable of trapping different amounts of myoglobin, as demonstrated by bimodal AFM imaging. The simple procedures used to build protein traps make this engineering approach promising for the development of new materials for biomedical applications where trapping biomolecules is required., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Adoptive cell therapy, i.e., the extraction, manipulation, and administration of ex vivo generated autologous T cells to patients, is an emerging alternative to regular procedures in cancer treatment. Nevertheless, these personalized treatments require laborious and expensive laboratory procedures that should be alleviated to enable their incorporation into the clinics. With the objective to improve the ex vivo expansion of large amount of specific T cells, we propose the use of three-dimensional (3D) structures during their activation with artificial antigen-presenting cells, thus resembling the natural environment of the secondary lymphoid organs. Thus, the activation, proliferation, and differentiation of T cells have been analyzed when cultured in the presence of two 3D systems, Matrigel and a 3D polystyrene scaffold, showing an increase in cell proliferation compared to standard suspension systems., Competing Interests: The authors declare no competing financial interest.

Stimuli-responsive self-assembled monolayers (SAMs) are used to confer switchable physical, chemical, or biological properties to surfaces through the application of external stimuli. To obtain spatially and temporally tunable surfaces, we present microcontact printed SAMs of a hydroquinone molecule that are used as a dynamic interface to immobilize different functional molecules either via Diels-Alder or Michael thiol addition reactions upon the application of a low potential. In spite of the use of such reactions and the potential applicability of the resulting surfaces in different fields ranging from sensing to biomedicine through data storage or cleanup, a direct comparison of the two functionalization strategies on a surface has not yet been performed. Although the Michael thiol addition requires molecules that are commercial or easy to synthesize in comparison with the cyclopentadiene derivatives needed for the Diels-Alder reaction, the latter reaction produces more homogeneous coverages under similar experimental conditions.

The understanding of the crystal structure of organic compounds, and its relationship to their physical properties, have become essential to design new advanced molecular materials. In this context, we present a computational study devoted to rationalize the different crystal packing displayed by two closely related organic systems based on the TTF-PTM dyad (TTF = tetrathiafulvalene, PTM = polychlorotriphenylmethane) with almost the same molecular structure but a different electronic one. The radical species (1), with an enhanced electronic donor-acceptor character, exhibits a herringbone packing, whereas the nonradical protonated analogue (2) is organized forming dimers. The stability of the possible polymorphs is analyzed in terms of the cohesion energy of the unit cell, intermolecular interactions between pairs, and molecular flexibility of the dyad molecules. It is observed that the higher electron delocalization in radical compound 1 has a direct influence on the geometry of the molecule, which seems to dictate its preferential crystal structure.

Unlabelled: Inclusion bodies (IBs) are protein-based nanoparticles formed in Escherichia coli through stereospecific aggregation processes during the overexpression of recombinant proteins. In the last years, it has been shown that IBs can be used as nanostructured biomaterials to stimulate mammalian cell attachment, proliferation, and differentiation. In addition, these nanoparticles have also been explored as natural delivery systems for protein replacement therapies. Although the production of these protein-based nanomaterials in E. coli is economically viable, important safety concerns related to the presence of endotoxins in the products derived from this microorganism need to be addressed. Lactic acid bacteria (LAB) are a group of food-grade microorganisms that have been classified as safe by biologically regulatory agencies. In this context, we have demonstrated herein, for the first time, the production of fully functional, IB-like protein nanoparticles in LAB. These nanoparticles have been fully characterized using a wide range of techniques, including field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared (FTIR) spectroscopy, zymography, cytometry, confocal microscopy, and wettability and cell coverage measurements. Our results allow us to conclude that these materials share the main physico-chemical characteristics with IBs from E. coli and moreover are devoid of any harmful endotoxin contaminant. These findings reveal a new platform for the production of protein-based safe products with high pharmaceutical interest., Statement of Significance: The development of both natural and synthetic biomaterials for biomedical applications is a field in constant development. In this context, E. coli is a bacteria that has been widely studied for its ability to naturally produce functional biomaterials with broad biomedical uses. Despite being effective, products derived from this species contain membrane residues able to trigger a non-desired immunogenic responses. Accordingly, exploring alternative bacteria able to synthesize such biomaterials in a safe molecular environment is becoming a challenge. Thus, the present study describes a new type of functional protein-based nanomaterial free of toxic contaminants with a wide range of applications in both human and veterinary medicine., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

In nature, cells respond to complex mechanical and biological stimuli whose understanding is required for tissue construction in regenerative medicine. However, the full replication of such bimodal effector networks is far to be reached. Engineering substrate roughness and architecture allows regulating cell adhesion, positioning, proliferation, differentiation and survival, and the external supply of soluble protein factors (mainly growth factors and hormones) has been long applied to promote growth and differentiation. Further, bioinspired scaffolds are progressively engineered as reservoirs for the in situ sustained release of soluble protein factors from functional topographies. We review here how research progresses toward the design of integrative, holistic scaffold platforms based on the exploration of individual mechanical and biological effectors and their further combination.

Slow protein release from amyloidal materials is a molecular platform used by nature to control protein hormone secretion in the endocrine system. The molecular mechanics of the sustained protein release from amyloids remains essentially unexplored. Inclusion bodies (IBs) are natural amyloids that occur as discrete protein nanoparticles in recombinant bacteria. These protein clusters have been recently explored as protein-based functional biomaterials with diverse biomedical applications, and adapted as nanopills to deliver recombinant protein drugs into mammalian cells. Interestingly, the slow protein release from IBs does not significantly affect the particulate organization and morphology of the material, suggesting the occurrence of a tight scaffold. Here, we have determined, by using a combined set of analytical approaches, a sponge-like supramolecular organization of IBs combining differently folded protein versions (amyloid and native-like), which supports both mechanical stability and sustained protein delivery. Apart from offering structural clues about how amyloid materials release their monomeric protein components, these findings open exciting possibilities for the tailored development of smart biofunctional materials, adapted to mimic the functions of amyloid-based secretory glands of higher organisms., (Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

In spite of the considerable understanding and development of perchlorotriphenylmethyl (PTM) radical derivatives, the preparation of crystals of the pure unsubstituted PTM radical, C19Cl15, suitable for single-crystal X-ray diffraction has remained a challenge since its discovery, and only two studies dealing with the crystal structure of the unsubstituted PTM radical have been published. In one study, the radical forms clathrates with aromatic solvents [Veciana, Carilla, Miravitlles & Molins (1987). J. Chem. Soc. Chem. Commun. pp. 812-814], and in the other the structure was determined ab initio from powder X-ray diffraction data [Rius, Miravitlles, Molins, Crespo & Veciana (1990). Mol. Cryst. Liq. Cryst. 187, 155-163]. We report here the preparation of PTM crystals for single-crystal X-ray diffraction and their resolution. The structure, which shows monoclinic symmetry (C2/c), revealed a nonsymmetric molecular propeller conformation (D3 symmetry) caused by the steric strain between the ortho-Cl atoms, which protect the central C atom (sp(2)-hybridization and major spin density) and give high chemical and thermal persistence to the PTM. The supramolecular structure of PTM shows short Cl...Cl intermolecular interactions and can be described in terms of layers formed by rows of molecules positioned in a head-to-tail manner along the c axis.

The electronic properties of a new set of cytotoxic 2-amino-naphtho[2,3-b]furan-4,9-dione derivatives (1-8) are evaluated. The electron delocalization of these compounds is described by means of their redox potentials and solvatochromic properties. The large solvatochromism of their intramolecular electron transfer band is analyzed using the linear solvation energy relationship method. In addition, this method determined the importance of the molecular environment, quantifying the interactions that compounds (1-8) establish with their surrounding media, with the capacity of acting as hydrogen-bond acceptors (HBA) and hydrogen-bond donors (HBD) and the dipolarity/polarizability being the most significant ones. As a result, a relationship between the electronic and the cytotoxic properties of these compounds is proposed.

The chemical and mechanical properties of bacterial inclusion bodies, produced in different Escherichia coli genetic backgrounds, have been characterized at the nanoscale level. In regard to wild type, DnaK(-) and ClpA(-) strains produce inclusion bodies with distinguishable wettability, stiffness and stiffness distribution within the proteinaceous particle. Furthermore it was possible to observe how cultured mammalian cells respond differentially to inclusion body variants when used as particulate materials to engineer the nanoscale topography, proving that the actual range of referred mechanical properties is sensed and discriminated by biological systems. The data provide evidence of the mechanistic activity of the cellular quality control network and the regulation of the stereospecific packaging of partially folded protein species in bacteria. This inclusion body nanoscale profiling offers possibilities for their fine genetic tuning and the resulting macroscopic effects when applied in biological interfaces., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Essential state models are presented to discuss absorption spectra of two related donor-acceptor (DA) chromophores that show two solvatochromic bands in the near-infrared spectral region. The two-state model only accounts for the lowest energy band and results in a very small value of mu(0), the dipole moment associated with the D(+)A(-) state. The model is then extended to account for the active role of the bridge: the resulting three-state model satisfactorily reproduces the double solvatochromism, leading at the same time to a roughly doubled estimate of mu(0). This result, supported by a detailed analysis of an N-state model that explicitly accounts for bridge states, rationalizes the well-known discrepancy between the geometrical DA distance and the dipole length extracted from the analysis of optical spectra of DA chromophores as reflecting the active role of bridge states, not explicitly accounted for in essential state models.

A test paper for high-selectivity detecting Hg 2+ ions in mixed acetonitrile-watersolutions has been achieved using a bis(ferrocenyl) azine, as chromogenic chemosensormolecule, and a solid cellulose fibre, as a substrate. Depending on the amount of mercuryions in contact with the detecting molecule a spectacular color change in the celluloseindicator is produced, being possible to determine the concentration of Hg 2+ ions either bynaked eye or spectroscopically.