Nanothermometer Based on Polychlorinated Trityl Radicals Showing Two-Photon Excitation and Emission in the Biological Transparency Window: Temperature Monitoring of Biological Tissues.

Nanothermometers are emerging probes as biomedical diagnostic tools. Especially appealing are nanoprobes using NIR light in the range of biological transparency window (BTW) since they have the advantages of a deeper penetration into biological tissues, better contrast, reduced phototoxicity and photobleaching. This article reports the preparation and characterization of organic nanoparticles (ONPs) doped with two polychlorinated trityl radicals (TTM and PTM), as well as studies of their electronic and optical properties. Such ONPs having inside isolated radical molecules and dimeric excimers, can be two-photon excited showing optimal properties for temperature sensing. Remarkably, in TTM-based ONPs the emission intensity of the isolated radical species is unaltered increasing temperature, while the excimer emission intensity decreases strongly being thereby able to monitor temperature changes with an excellent thermal absolute sensitivity of 0.6-3.7% K-1 in the temperature range of 278-328 K. The temperature dependence of the excimeric bands of ONPs are theoretically simulated by using electronic structure calculations and a vibronic Hamiltonian model. Finally, TTM-doped ONPs as ratiometric NIR-nanothermometers are tested with two-photon excitationwith enucleated pig eye sclera, as a real tissue model, obtaining a similar temperature sensitivity as in aqueous suspensions, demonstrating their potential as NIR nanothermometers for bio applications.

Three-dimensional cell culture of chimeric antigen receptor T cells originated from peripheral blood mononuclear cells towards cellular therapies.

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.

Molecular One- and Two-Qubit Systems with Very Long Coherence Times.

General-purpose quantum computation and quantum simulation require multi-qubit architectures with precisely defined, robust interqubit interactions, coupled with local addressability. This is an unsolved challenge, primarily due to scalability issues. These issues often derive from poor control over interqubit interactions. Molecular systems are promising materials for the realization of large-scale quantum architectures, due to their high degree of positionability and the possibility to precisely tailor interqubit interactions. The simplest quantum architecture is the two-qubit system, with which quantum gate operations can be implemented. To be viable, a two-qubit system must possess long coherence times, the interqubit interaction must be well defined and the two qubits must also be addressable individually within the same quantum manipulation sequence. Here results are presented on the investigation of the spin dynamics of chlorinated triphenylmethyl organic radicals, in particular the perchlorotriphenylmethyl (PTM) radical, a mono-functionalized PTM, and a biradical PTM dimer. Extraordinarily long ensemble coherence times up to 148 µs are found at all temperatures below 100 K. Two-qubit and, importantly, individual qubit addressability in the biradical system are demonstrated. These results underline the potential of molecular materials for the development of quantum architectures.

Ratiometric Nanothermometer Based on a Radical Excimer for In Vivo Sensing.

Ratiometric fluorescent nanothermometers with near-infrared emission play an important role in in vivo sensing since they can be used as intracellular thermal sensing probes with high spatial resolution and high sensitivity, to investigate cellular functions of interest in diagnosis and therapy, where current approaches are not effective. Herein, the temperature-dependent fluorescence of organic nanoparticles is designed, synthesized, and studied based on the dual emission, generated by monomer and excimer species, of the tris(2,4,6-trichlorophenyl)methyl radical (TTM) doping organic nanoparticles (TTMd-ONPs), made of optically neutral tris(2,4,6-trichlorophenyl)methane (TTM-αH), acting as a matrix. The excimer emission intensity of TTMd-ONPs decreases with increasing temperatures whereas the monomer emission is almost independent and can be used as an internal reference. TTMd-ONPs show a great temperature sensitivity (3.4% K-1 at 328 K) and a wide temperature response at ambient conditions with excellent reversibility and high colloidal stability. In addition, TTMd-ONPs are not cytotoxic and their ratiometric outputs are unaffected by changes in the environment. Individual TTMd-ONPs are able to sense temperature changes at the nano-microscale. In vivo thermometry experiments in Caenorhabditis elegans (C. elegans) worms show that TTMd-ONPs can locally monitor internal body temperature changes with spatio-temporal resolution and high sensitivity, offering multiple applications in the biological nanothermometry field.

Stable Organic Radical for Enhancing Metal-Monolayer-Semiconductor Junction Performance.

The preparation of monolayers based on an organic radical and its diamagnetic counterpart has been pursued on hydrogen-terminated silicon surfaces. The functional monolayers have been investigated as solid-state metal/monolayer/semiconductor (MmS) junctions showing a characteristic diode behavior which is tuned by the electronic characteristics of the organic molecule. The eutectic gallium-indium liquid metal is used as a top electrode to perform the transport measurements and the results clearly indicate that the SOMO-SUMO molecular orbitals impact the device performance. The junction incorporating the radical shows an almost two orders of magnitude higher rectification ratio (R(|J1V/J-1V|) = 104.04) in comparison with the nonradical one (R(|J1V/J-1V|) = 102.30). The high stability of the fabricated MmS allows the system to be interrogated under irradiation, evidencing that at the wavelength where the photon energy is close to the band gap of the radical there is a clear enhancement of the photoresponse. This is translated into an increase of the photosensitivity (Sph) value from 68.7 to 269.0 mA/W for the nonradical and radical based systems, respectively.

Stable nanovesicles formed by intrinsically planar bilayers.

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.

Ultrabright Föster Resonance Energy Transfer Nanovesicles: The Role of Dye Diffusion.

The development of contrast agents based on fluorescent nanoparticles with high brightness and stability is a key factor to improve the resolution and signal-to-noise ratio of current fluorescence imaging techniques. However, the design of bright fluorescent nanoparticles remains challenging due to fluorescence self-quenching at high concentrations. Developing bright nanoparticles showing FRET emission adds several advantages to the system, including an amplified Stokes shift, the possibility of ratiometric measurements, and of verifying the nanoparticle stability. Herein, we have developed Förster resonance energy transfer (FRET)-based nanovesicles at different dye loadings and investigated them through complementary experimental techniques, including conventional fluorescence spectroscopy and super-resolution microscopy supported by molecular dynamics calculations. We show that the optical properties can be modulated by dye loading at the nanoscopic level due to the dye's molecular diffusion in fluid-like membranes. This work shows the first proof of a FRET pair dye's dynamism in liquid-like membranes, resulting in optimized nanoprobes that are 120-fold brighter than QDot 605 and exhibit >80% FRET efficiency with vesicle-to-vesicle variations that are mostly below 10%.

Hierarchical Quatsome-RGD Nanoarchitectonic Surfaces for Enhanced Integrin-Mediated Cell Adhesion.

The synthesis and study of the tripeptide Arg-Gly-Asp (RGD), the binding site of different extracellular matrix proteins, e.g., fibronectin and vitronectin, has allowed the production of a wide range of cell adhesive surfaces. Although the surface density and spacing of the RGD peptide at the nanoscale have already shown a significant influence on cell adhesion, the impact of its hierarchical nanostructure is still rather unexplored. Accordingly, a versatile colloidal system named quatsomes, based on fluid nanovesicles formed by the self-assembling of cholesterol and surfactant molecules, has been devised as a novel template to achieve hierarchical nanostructures of the RGD peptide. To this end, RGD was anchored on the vesicle's fluid membrane of quatsomes, and the RGD-functionalized nanovesicles were covalently anchored to planar gold surfaces, forming a state of quasi-suspension, through a long poly(ethylene glycol) (PEG) chain with a thiol termination. An underlying self-assembled monolayer (SAM) of a shorter PEG was introduced for vesicle stabilization and to avoid unspecific cell adhesion. In comparison with substrates featuring a homogeneous distribution of RGD peptides, the resulting hierarchical nanoarchitectonic dramatically enhanced cell adhesion, despite lower overall RGD molecules on the surface. The new versatile platform was thoroughly characterized using a multitechnique approach, proving its enhanced performance. These findings open new methods for the hierarchical immobilization of biomolecules on surfaces using quatsomes as a robust and novel tissue engineering strategy.

Liposomal formulations for treating lysosomal storage disorders.

Lysosomal storage disorders (LSD) are a group of rare life-threatening diseases caused by a lysosomal dysfunction, usually due to the lack of a single enzyme required for the metabolism of macromolecules, which leads to a lysosomal accumulation of specific substrates, resulting in severe disease manifestations and early death. There is currently no definitive cure for LSD, and despite the approval of certain therapies, their effectiveness is limited. Therefore, an appropriate nanocarrier could help improve the efficacy of some of these therapies. Liposomes show excellent properties as drug carriers, because they can entrap active therapeutic compounds offering protection, biocompatibility, and selectivity. Here, we discuss the potential of liposomes for LSD treatment and conduct a detailed analysis of promising liposomal formulations still in the preclinical development stage from various perspectives, including treatment strategy, manufacturing, characterization, and future directions for implementing liposomal formulations for LSD.

Emergent Insulator-Metal Transition with Tunable Optical and Electrical Gap in Thin Films of a Molecular Conducting Composite.

Composites exhibit unique synergistic properties emerging when components with different properties are combined. The tuning of the energy bandgap in the electronic structure of the material allows designing tailor-made systems with desirable mechanical, electrical, optical, and/or thermal properties. Here, we study an emergent insulator-metal transition at room temperature in bilayered (BL) thin-films comprised of polycarbonate/molecular-metal composites. Temperature-dependent resistance measurements allow monitoring of the electrical bandgap, which is in agreement with the optical bandgap extracted by optical absorption spectroscopy. The semiconductor-like properties of BL films, made with bis(ethylenedithio)-tetrathiafulvalene (BEDT-TTF or ET) α-ET2I3 (nano)microcrystals as two-dimensional molecular conductor on one side and insulator polycarbonate as a second ingredient, are attributed to an emergent phenomenon equivalent to the transition from an insulator to a metal. This made it possible to obtain semiconducting BL films with tunable electrical/optical bandgaps ranging from 0 to 2.9 eV. A remarkable aspect is the similarity close to room temperature of the thermal and mechanical properties of both composite components, making these materials ideal candidates to fabricate flexible and soft sensors for stress, pressure, and temperature aiming at applications in wearable human health care and bioelectronics.

Quatsomes Formulated with l-Prolinol-Derived Surfactants as Antibacterial Nanocarriers of (+)-Usnic Acid with Antioxidant Activity.

The efficacy of the treatment of bacterial infection is seriously reduced because of antibiotic resistance; thus, therapeutic solutions against drug-resistant microbes are necessary. Nanoparticle-based solutions are particularly promising for meeting this challenge because they can offer intrinsic antimicrobial activity and sustained drug release at the target site. Herein, we present a newly developed nanovesicle system of the quatsome family, composed of l-prolinol-derived surfactants and cholesterol, which has noticeable antibacterial activity even on Gram-negative strains, demonstrating great potential for the treatment of bacterial infections. We optimized the vesicle stability and antibacterial activity by tuning the surfactant chain length and headgroup charge (cationic or zwitterionic) and show that these quatsomes can furthermore serve as nanocarriers of pharmaceutical actives, demonstrated here by the encapsulation of (+)-usnic acid, a natural substance with many pharmacological properties.

Enhanced human T cell expansion with inverse opal hydrogels.

Advanced personalized immunotherapies still have to overcome several biomedical and technical limitations before they become a routine cancer treatment in spite of recent achievements. In adoptive cell therapy (ACT), the capacity to obtain adequate numbers of therapeutic T cells in the patients following ex vivo treatment should be improved. Moreover, the time and costs to produce these T cells should be reduced. In this work, inverse opal (IOPAL) 3D hydrogels consisting of poly(ethylene) glycol (PEG) covalently combined with heparin were engineered to resemble the environment of lymph nodes, where T cells get activated and proliferate. The introduction of an IOPAL strategy allowed a precise control on the porosity of the hydrogels, providing an increase in the proliferation of primary human CD4+ T cells, when compared with state-of-the-art expansion systems. Additionally, the IOPAL hydrogels also showed a superior expansion compared to hydrogels with the same composition, but without the predetermined pore structure. In summary, we have shown the beneficial effect of having an IOPAL architecture in our 3D hydrogels to help achieving large numbers of cells, while maintaining the desired selected phenotypes required for ACT.

Photoswitching activation of a ferrocenyl-stilbene analogue by its covalent grafting to gold.

Until now, surface-deposited stilbenes have been much less studied than other photochromic systems. Here, an asymmetrically substituted styrene incorporating a redox-active ferrocene moiety and a terminal alkyne group has been synthesised to investigate its photoisomerization in solution, and upon the formation of chemisorbed self-assembled monolayers through a carbon-gold bond formation. Charge transport measurements across the monolayers reveal that upon chemical linkage to the gold substrate there is an alteration of the isomerization pathway, which favours the trans to cis conversion, which is not observed in solution. The experimental observations are interpreted based on quantum chemistry calculations.

DELOS Nanovesicles-Based Hydrogels: An Advanced Formulation for Topical Use.

Topical delivery has received great attention due to its localized drug delivery, its patient compliance, and its low risk for side effects. Recent developments have focused on studying new drug delivery systems as a strategy for addressing the challenges of current topical treatments. Here we describe the advances on an innovative drug delivery platform called DELOS nanovesicles for topical drug delivery. Previously, the production of DELOS nanovesicles demonstrated potentiality for the topical treatment of complex wounds, achieving well-tolerated liquid dispersions by this route. Here, research efforts have been focused on designing these nanocarriers with the best skin tolerability to be applied even to damaged skin, and on exploring the feasibility of adapting the colloidal dispersions to a more suitable dosage form for topical application. Accordingly, these drug delivery systems have been efficiently evolved to a hydrogel using MethocelTM K4M, presenting proper stability and rheological properties. Further, the integrity of these nanocarriers when being gellified has been confirmed by cryo-transmission electron microscopy and by Förster resonance energy transfer analysis with fluorescent-labeled DELOS nanovesicles, which is a crucial characterization not widely reported in the literature. Additionally, in vitro experiments have shown that recombinant human Epidermal Growth Factor (rhEGF) protein integrated into gellified DELOS nanovesicles exhibits an enhanced bioactivity compared to the liquid form. Therefore, these studies suggest that such a drug delivery system is maintained unaltered when hydrogellified, becoming the DELOS nanovesicles-based hydrogels, an advanced formulation for topical use.

Methods for the Characterization of Protein Aggregates.

The physicochemical characterization of protein aggregates yields an important contribution to further our understanding on many diseases for which the formation of protein aggregates is one of the pathological hallmarks. On the other hand, bacterial inclusion bodies (IBs) have recently been shown to be highly pure proteinaceous aggregates of a few hundred nanometers, produced by recombinant bacteria supporting the biological activities of the embedded polypeptides. Despite the wide spectrum of uses of IBs as functional and biocompatible materials upon convenient engineering, very few is known about their physicochemical properties.In this chapter we present methods for the characterization of protein aggregates as particulate materials relevant to their physicochemical and nanoscale properties.Specifically, we describe the use of dynamic light scattering (DLS) for sizing, nanoparticle tracking analysis for sizing and counting, and zeta potential measurements for the determination of colloidal stability. To study the morphology of protein aggregates we present the use of atomic force microscopy (AFM) and scanning electron microscopy (SEM). Cryo-transmission electron microscopy (cryo-TEM) will be used for the determination of the internal structuration. Moreover, wettability and nanomechanical characterization can be performed using contact angle (CA) and force spectroscopic AFM (FS-AFM) measurements of the proteinaceous nanoparticles, respectively. Finally, the 4'4-dithiodipyridine (DTDP) method is presented as a way of relatively quantifying accessible sulfhydryl groups in the structure of the nanoparticle .The physical principles of the methods are briefly described and examples are given to help clarify capabilities of each technique.

Methods for Processing Protein Aggregates into Surfaces.

The processing of inclusion bodies (IBs) into surfaces is of great interest for cell culture applications due to the combined physical and biological cues these particles provide. The arrangement of these IBs into defined and tunable micropatterns can be useful for basic research purposes regarding the mechanical properties needed for cell adhesion and migration, among other responses. There are several approaches that can be used when functionalizing a substrate with IBs, regarding both the strategy used and also the kind of surface-particle interaction. The interaction between surface and IB can be mainly of three types: physisorption, electrostatic or covalent. This interaction can be controlled by depositing an appropriate self-assembled monolayer (SAM) on top of a substrate as an interface. Furthermore, several strategies can be used to immobilize IBs on surfaces in various configurations, like random deposition, micrometric printed geometries or gradient patterns.

Engineering pH-Sensitive Stable Nanovesicles for Delivery of MicroRNA Therapeutics.

MicroRNAs (miRNAs) are small non-coding endogenous RNAs, which are attracting a growing interest as therapeutic molecules due to their central role in major diseases. However, the transformation of these biomolecules into drugs is limited due to their unstability in the bloodstream, caused by nucleases abundantly present in the blood, and poor capacity to enter cells. The conjugation of miRNAs to nanoparticles (NPs) could be an effective strategy for their clinical delivery. Herein, the engineering of non-liposomal lipid nanovesicles, named quatsomes (QS), for the delivery of miRNAs and other small RNAs into the cytosol of tumor cells, triggering a tumor-suppressive response is reported. The engineered pH-sensitive nanovesicles have controlled structure (unilamellar), size (<150 nm) and composition. These nanovesicles are colloidal stable (>24 weeks), and are prepared by a green, GMP compliant, and scalable one-step procedure, which are all unavoidable requirements for the arrival to the clinical practice of NP based miRNA therapeutics. Furthermore, QS protect miRNAs from RNAses and when injected intravenously, deliver them into liver, lung, and neuroblastoma xenografts tumors. These stable nanovesicles with tunable pH sensitiveness constitute an attractive platform for the efficient delivery of miRNAs and other small RNAs with therapeutic activity and their exploitation in the clinics.

Polylactide, Processed by a Foaming Method Using Compressed Freon R134a, for Tissue Engineering.

Fabricating polymeric scaffolds using cost-effective manufacturing processes is still challenging. Gas foaming techniques using supercritical carbon dioxide (scCO2) 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 CO2 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 scCO2 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.

Application of Quality by Design to the robust preparation of a liposomal GLA formulation by DELOS-susp method.

Fabry disease is a lysosomal storage disease arising from a deficiency of the enzyme α-galactosidase A (GLA). The enzyme deficiency results in an accumulation of glycolipids, which over time, leads to cardiovascular, cerebrovascular, and renal disease, ultimately leading to death in the fourth or fifth decade of life. Currently, lysosomal storage disorders are treated by enzyme replacement therapy (ERT) through the direct administration of the missing enzyme to the patients. In view of their advantages as drug delivery systems, liposomes are increasingly being researched and utilized in the pharmaceutical, food and cosmetic industries, but one of the main barriers to market is their scalability. Depressurization of an Expanded Liquid Organic Solution into aqueous solution (DELOS-susp) is a compressed fluid-based method that allows the reproducible and scalable production of nanovesicular systems with remarkable physicochemical characteristics, in terms of homogeneity, morphology, and particle size. The objective of this work was to optimize and reach a suitable formulation for in vivo preclinical studies by implementing a Quality by Design (QbD) approach, a methodology recommended by the FDA and the EMA to develop robust drug manufacturing and control methods, to the preparation of α-galactosidase-loaded nanoliposomes (nanoGLA) for the treatment of Fabry disease. Through a risk analysis and a Design of Experiments (DoE), we obtained the Design Space in which GLA concentration and lipid concentration were found as critical parameters for achieving a stable nanoformulation. This Design Space allowed the optimization of the process to produce a nanoformulation suitable for in vivo preclinical testing.

A Trapezoidal Octacyanoquinoid Acceptor Forms Solution and Surface Products by Antiparallel Shape Fitting with Conformational Dipole Momentum Switch.

A new compound (1) formed by two antiparallelly disposed tetracyano thienoquinoidal units has been synthesized and studied by electrochemistry, UV/Vis-NIR, IR, EPR, and transient spectroscopy. Self-assembly of 1 on a Au(111) surface has been investigated by scanning tunneling microscopy. Experiments have been rationalized by quantum chemical calculations. 1 exhibits a unique charge distribution in its anionic form, with a gradient of charge yielding a neat molecular in-plane electric dipole momentum, which transforms out-of-plane after surface deposition due to twisted→folded conformational change and to partial charge transfer from Au(111). Intermolecular van der Waals interactions and antiparallel trapezoidal shape fitting lead to the formation of an optimal dense on Au(111) two-dimensional assembly of 1.