In-Bath 3D Printing of Anisotropic Shape-Memory Cryogels Functionalized with Bone-Bioactive Nanoparticles.

Cryogels exhibit unique shape memory with full recovery and structural stability features after multiple injections. These constructs also possess enhanced cell permeability and nutrient diffusion when compared to typical bulk hydrogels. Volumetric processing of cryogels functionalized with nanosized units has potential to widen their biomedical applications, however this has remained challenging and relatively underexplored. In this study, we report a novel methodology that combines suspension 3D printing with directional freezing for the fabrication of nanocomposite cryogels with configurable anisotropy. When compared to conventional bulk or freeze-dried hydrogels, nanocomposite cryogel formulations exhibit excellent shape recovery (>95%) and higher pore connectivity. Suspension printing, assisted with a prechilled metal grid, was optimized to induce anisotropy. The addition of calcium- and phosphate-doped mesoporous silica nanoparticles into the cryogel matrix enhanced bioactivity toward orthopedic applications without hindering the printing process. Notably, the nanocomposite 3D printed cryogels exhibit injectable shape memory while also featuring a lamellar topography. The fabrication of these constructs was highly reproducible and exhibited potential for a cell-delivery injectable cryogel with no cytotoxicity to human-derived adipose stem cells. Hence, in this work, it was possible to combine a gravity defying 3D printed methodology with injectable and controlled anisotropic macroporous structures containing bioactive nanoparticles. This methodology ameliorates highly tunable injectable 3D printed anisotropic nanocomposite cryogels with a user-programmable degree of structural complexity.

Natural Polymer-Polyphenol Bioadhesive Coacervate with Stable Wet Adhesion, Antibacterial Activity, and On-Demand Detachment.

Medical adhesives are emerging as an important clinical tool as adjuvants for sutures and staples in wound closure and healing and in the achievement of hemostasis. However, clinical adhesives combining cytocompatibility, as well as strong and stable adhesion in physiological conditions, are still in demand. Herein, a mussel-inspired strategy is explored to produce adhesive coacervates using tannic acid (TA) and methacrylate pullulan (PUL-MA). TA|PUL-MA coacervates mainly comprise van der Waals forces and hydrophobic interactions. The methacrylic groups in the PUL backbone increase the number of interactions in the adhesives matrix, resulting in enhanced cohesion and adhesion strength (72.7 Jm-2), compared to the non-methacrylated coacervate. The adhesive properties are kept in physiologic-mimetic solutions (72.8 Jm-2) for 72 h. The photopolymerization of TA|PUL-MA enables the on-demand detachment of the adhesive. The poor cytocompatibility associated with the use of phenolic groups is here circumvented by mixing reactive oxygen species-degrading enzyme in the adhesive coacervate. This addition does not hamper the adhesive character of the materials, nor their anti-microbial or hemostatic properties. This affordable and straightforward methodology, together with the tailorable adhesivity even in wet environments, high cytocompatibility, and anti-bacterial activity, enables foresee TA|PUL-MA as a promising ready-to-use bioadhesive for biomedical applications.

In situ generated hemostatic adhesives: From mechanisms of action to recent advances and applications.

Conventional surgical closure techniques, such as sutures, clips, or skin closure strips, may not always provide optimal wound closure and may require invasive procedures, which can result in potential post-surgical complications. As result, there is a growing demand for innovative solutions to achieve superior wound closure and improve patient outcomes. To overcome the abovementioned issues, in situ generated hemostatic adhesives/sealants have emerged as a promising alternative, offering a targeted, controllable, and minimally invasive procedure for a wide variety of medical applications. The aim of this review is to provide a comprehensive overview of the mechanisms of action and recent advances of in situ generated hemostatic adhesives, particularly protein-based, thermoresponsive, bioinspired, and photocrosslinkable formulations, as well as the design challenges that must be addressed. Overall, this review aims to enhance a comprehensive understanding of the latest advancements of in situ generated hemostatic adhesives and their mechanisms of action, with the objective of promoting further research in this field.

Biomimetic Adhesive Micropatterned Hydrogel Patches for Drug Release.

The optimized physical adhesion between bees' leg hairs and pollen grains-whereby the latter's diameter aligns with the spacing between the hairs-has previously inspired the development of a biomimetic drug dressing. Combining this optimized process with the improved natural mussels' adhesion in wet environments in a dual biomimetic process, it is herein proposed the fabrication of a natural-derived micropatterned hydrogel patch of methacrylated laminarin (LAM-MET), with enriched drug content and improved adhesiveness, suitable for applications like wound healing. Enhanced adhesion is accomplished by modifying LAM-MET with hydroxypyridinone groups, following the patch microfabrication by soft lithography and UV/vis-irradiation, resulting in a membrane with micropillars with a high aspect ratio. Following the biomimetics rational, a drug patch is engineered by combining the microfabricated dressing with drug particles milled to fit the spaces between pillars. Controlled drug release is achieved, together with inherent antibacterial activity against Escherichia coli and Pseudomonas aeruginosa, and enhanced biocompatibility using the bare micropatterned patches. This new class of biomimetic dressings overcomes the challenges of current patches, like poor mechanical properties and biocompatibility, limited adhesiveness and drug dosage, and lack of prolonged antimicrobial activity, opening new insights for the development of high drug-loaded dressings with improved patient compliance.

Self-Supporting Hyaluronic Acid-Functionalized G-Quadruplex-Based Perfusable Multicomponent Hydrogels Embedded in Photo-Cross-Linkable Matrices for Bioapplications.

Dynamic G-quadruplex supramolecular hydrogels have aroused great interest in a broad range of bioapplications. However, neither the development of native extracellular matrix (ECM)-derived natural biopolymer-functionalized G-quadruplex hydrogels nor their use to create perfusable self-supporting hydrogels has been explored to date, despite their intrinsic potential as carrier vehicles of therapeutic agents, or even living cells in advanced regenerative therapies, or as platforms to enable the diffusion of nutrients and oxygen to sustain long-term cell survival. Herein, we developed a dynamic co-assembling multicomponent system that integrates guanosine (G), 3-aminophenylboronic acid functionalized hyaluronic acid (HA-PBA), and potassium chloride to bioengineer strong, homogeneous, and transparent HA-functionalized G-quadruplex hydrogels with injectable, thermo-reversible, conductive, and self-healing properties. The supramolecular polymeric hydrogels were developed by hydrogen bonding and π-π stacking interactions between G coupled via dynamic covalent boronate ester bonds to HA-PBA and stabilized by K+ ions, as demonstrated by a combination of experiments and molecular dynamics simulations. The intrinsic instability of the self-assembled G-quadruplex structures was used to bioengineer self-supporting perfusable multicomponent hydrogels with interconnected size and shape-tunable hollow microchannels when embedded in 3D methacrylated gelatin supporting matrices. The microchannel-embedded 3D constructs have shown enhanced cell viability when compared to the bulk hydrogels, holding great promise for being use as artificial vessels for enabling the diffusion of nutrients and oxygen essential for cell survival. The proposed approach opens new avenues on the use of ECM-derived natural biopolymer-functionalized dynamic G-quadruplex hydrogels to design next-generation smart systems for being used in tissue regeneration, drug screening, or organ-on-a-chip.

Marine-origin polysaccharides-based free-standing multilayered membranes as sustainable nanoreservoirs for controlled drug delivery.

The layer-by-layer (LbL) assembly technology has been widely used to functionalise surfaces and precisely engineer robust multilayered bioarchitectures with tunable structures, compositions, properties, and functions at the nanoscale by resorting to a myriad of building blocks exhibiting complementary interactions. Among them, marine-origin polysaccharides are a sustainable renewable resource for the fabrication of nanostructured biomaterials for biomedical applications owing to their wide bioavailability, biocompatibility, biodegradability, non-cytotoxicity, and non-immunogenic properties. Chitosan (CHT) and alginate (ALG) have been widely employed as LbL ingredients to shape a wide repertoire of size- and shape-tunable electrostatic-driven multilayered assemblies by exploring their opposite charge nature. However, the insolubility of CHT in physiological conditions intrinsically limits the range of bioapplications of the as-developed CHT-based LbL structures. Herein, we report the preparation of free-standing (FS) multilayered membranes made of water-soluble quaternised CHT and ALG biopolymers for controlled release of model drug molecules. The influence of the film structure in the drug release rate is studied by assembling two distinct set-ups of FS membranes, having the model hydrophilic drug fluorescein isothiocyanate-labelled bovine serum albumin (FITC-BSA) either as an intrinsic building block or added as an outer layer after the LbL assembly process. Both FS membranes are characterised for their thickness, morphology, in vitro cytocompatibility, and release profile, with those having FITC-BSA as an intrinsic LbL ingredient denoting a more sustained release rate. This work opens up new avenues for the design and development of a wide array of CHT-based devices for biomedical applications, overcoming the limitations associated with the insolubility of native CHT under physiological conditions.

Engineering natural based nanocomposite inks via interface interaction for extrusion 3D printing.

Nanocomposites and low-viscous materials lack translation in additive manufacturing technologies due to deficiency in rheological requirements and heterogeneity of their preparation. This work proposes the chemical crosslinking between composing phases as a universal approach for mitigating such issues. The model system is composed of amine-functionalized bioactive glass nanoparticles (BGNP) and light-responsive methacrylated bovine serum albumin (BSAMA) which further allows post-print photocrosslinking. The interfacial interaction was conducted by 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide crosslinking agent and N-Hydroxysuccinimide between BGNP-grafted amines and BSAMA's carboxylic groups. Different chemical crosslinking amounts and percentages of BGNP in the nanocomposites were tested. The improved interface interactions increased the elastic and viscous modulus of all formulations. More pronounced increases were found with the highest crosslinking agent amounts (4 % w/v) and BGNP concentrations (10 % w/w). This formulation also displayed the highest Young's modulus of the double-crosslinked construct. All composite formulations could effectively immobilize the BGNP and turn an extremely low viscous material into an appropriate inks for 3d printing technologies, attesting for the systems' tunability. Thus, we describe a versatile methodology which can successfully render tunable and light-responsive nanocomposite inks with homogeneously distributed bioactive fillers. This system can further reproducibly recapitulate phases of other natures, broadening applicability.

Bioinspired Oxidation-Resistant Catechol-like Sliding Ring Polyrotaxane Hydrogels.

Adaptable hydrogels have been used in the biomedical field to address several pathologies, especially those regarding tissue defects. Here, we describe unprecedented catechol-like functionalized polyrotaxane (PR) polymers able to form hydrogels. PR were functionalized with the incorporation of hydroxypyridinone (HOPO) moieties into the polymer backbone, with a degree of substitution from 4 to 22%, depending on the PR type. The hydrogels form through the functionalized supramolecular systems when in contact with a Fe(III) solution. Despite the hydrogel formation being at physiological pH (7.4), the HOPO derivatives are extremely resistant to oxidation, unlike common catechols; consequently, they prevent the formation of quinones, which can lead to irreversible bounds within the matrix. The resulting hydrogels demonstrated properties lead to unique hydrogels with improved mechanical behavior obtained by metallic coordination crosslinking, due to the synergies of the sliding-ring PR and the non-covalent (reversible) catechol analogues. Following this strategy, we successfully developed innovative, cytocompatible, oxidative-resistant, and reversible crosslinked hydrogels, with the potential of being used as structural self-materials for a variety of applications, including in the biomedical field.

Unveiling the Assembly of Neutral Marine Polysaccharides into Electrostatic-Driven Layer-by-Layer Bioassemblies by Chemical Functionalization.

Marine-origin polysaccharides, in particular cationic and anionic ones, have been widely explored as building blocks in fully natural or hybrid electrostatic-driven Layer-by-Layer (LbL) assemblies for bioapplications. However, the low chemical versatility imparted by neutral polysaccharides has been limiting their assembly into LbL biodevices, despite their wide availability in sources such as the marine environment, easy functionality, and very appealing features for addressing multiple biomedical and biotechnological applications. In this work, we report the chemical functionalization of laminarin (LAM) and pullulan (PUL) marine polysaccharides with peptides bearing either six lysine (K6) or aspartic acid (D6) amino acids via Cu(I)-catalyzed azide-alkyne cycloaddition to synthesize positively and negatively charged polysaccharide-peptide conjugates. The successful conjugation of the peptides into the polysaccharide's backbone was confirmed by proton nuclear magnetic resonance and attenuated total reflectance Fourier-transform infrared spectroscopy, and the positive and negative charges of the LAM-K6/PUL-K6 and LAM-D6/PUL-D6 conjugates, respectively, were assessed by zeta-potential measurements. The electrostatic-driven LbL build-up of either the LAM-D6/LAM-K6 or PUL-D6/PUL-K6 multilayered thin film was monitored in situ by quartz crystal microbalance with dissipation monitoring, revealing the successful multilayered film growth and the enhanced stability of the PUL-based film. The construction of the PUL-peptide multilayered thin film was also assessed by scanning electron microscopy and its biocompatibility was demonstrated in vitro towards L929 mouse fibroblasts. The herein proposed approach could enable the inclusion of virtually any kind of small molecules in the multilayered assemblies, including bioactive moieties, and be translated into more convoluted structures of any size and geometry, thus extending the usefulness of neutral polysaccharides and opening new avenues in the biomedical field, including in controlled drug/therapeutics delivery, tissue engineering, and regenerative medicine strategies.

Green approaches for extraction, chemical modification and processing of marine polysaccharides for biomedical applications.

Over the past few decades, natural-origin polysaccharides have received increasing attention across different fields of application, including biomedicine and biotechnology, because of their specific physicochemical and biological properties that have afforded the fabrication of a plethora of multifunctional devices for healthcare applications. More recently, marine raw materials from fisheries and aquaculture have emerged as a highly sustainable approach to convert marine biomass into added-value polysaccharides for human benefit. Nowadays, significant efforts have been made to combine such circular bio-based approach with cost-effective and environmentally-friendly technologies that enable the isolation of marine-origin polysaccharides up to the final construction of a biomedical device, thus developing an entirely sustainable pipeline. In this regard, the present review intends to provide an up-to-date outlook on the current green extraction methodologies of marine-origin polysaccharides and their molecular engineering toolbox for designing a multitude of biomaterial platforms for healthcare. Furthermore, we discuss how to foster circular bio-based approaches to pursue the further development of added-value biomedical devices, while preserving the marine ecosystem.

Phthalocyanine-Functionalized Magnetic Silica Nanoparticles as Anion Chemosensors.

Anionic species are one of the most common pollutants in residual and freshwaters. The presence of anthropogenic anions in water drastically increases the toxicity to living beings. Here, we report the preparation of a new optical active material based on tri(tosylamino)phthalocyanines grafted to ferromagnetic silica nanoparticles for anion detection and removal. The new unsymmetrical phthalocyanines (Pcs) proved to be excellent chemosensors for several anions (AcO-, Br-, Cl-, CN-, F-, H2PO4-, HSO4-, NO2-, NO3-, and OH-) in dimethyl sulfoxide (DMSO). Furthermore, the Pcs were grafted onto magnetic nanoparticles. The resulting novel hybrid material showed selectivity and sensitivity towards CN-, F-, and OH- anions in DMSO with limit of detection (LoD) of ≈4.0 µM. In water, the new hybrid chemosensor demonstrated selectivity and sensitivity for CN- and OH- anions with LoD of ≈0.2 µM. The new hybrids are easily recovered using a magnet, allowing recyclability and reusability, after acidic treatment, without losing the sensing proprieties.

Modular Functionalization of Laminarin to Create Value-Added Naturally Derived Macromolecules.

With society's growing awareness of climate change, novel renewable and naturally sourced materials have received increasing attention as substitutes for petroleum-based products. Laminarin (LAM-OH) is a highly abundant, nontoxic, degradable polysaccharide found in marine organisms and hence is a promising sustainable polymeric candidate. This work reports on a simple, environmentally friendly, and customizable functionalization strategy for producing a toolbox of LAM-OH derivatives under mild conditions. Herein, natural-origin macromolecules exhibiting specific chemical moieties, namely, allyl, amine, carboxylic acid, thiol, aldehyde, and catechol, were prepared and chemically characterized. Furthermore, the obtained polymers were processed into cytocompatible hydrogels, obtained by employing distinct cross-linking mechanisms, to assess their potential for biomedical purposes. The application scope of such polymers could be extended to fields such as catalysis, cosmetics, life sciences, and food packaging, which can also benefit from having sustainable, nontoxic, and degradable materials. Moreover, it is anticipated that the methodology employed to create this library of new natural-based products could be adapted to modify other polysaccharides and biopolymers in general.

Assembly of High-Potency Photosensitizer-Antibody Conjugates through Application of Dendron Multiplier Technology.

Exploitation of photosensitizers as payloads for antibody-based anticancer therapeutics offers a novel alternative to the small pool of commonly utilized cytotoxins. However, existing bioconjugation methodologies are incompatible with the requirement of increased antibody loading without compromising antibody function, stability, or homogeneity. Herein, we describe the first application of dendritic multiplier groups to allow the loading of more than 4 porphyrins to a full IgG antibody in a site-specific and highly homogeneous manner. Photophysical evaluation of UV-visible absorbance and singlet oxygen quantum yields highlighted porphyrin-dendron 14 as the best candidate for bioconjugation; with subsequent bioconjugation producing a HER2-targeted therapeutic with average loading ratios of 15.4:1. In vitro evaluation of conjugate 18 demonstrated a nanomolar photocytotoxic effect in a target cell line, which overexpresses HER2, with no observed photocytotoxicity at the same concentration in a control cell line which expresses native HER2 levels, or in the absence of irradiation with visible light.

Porphyrin modified trastuzumab improves efficacy of HER2 targeted photodynamic therapy of gastric cancer.

Gastric cancer (GC) is the 3rd deadliest cancer worldwide, due to limited treatment options and late diagnosis. Human epidermal growth factor receptor-2 (HER2) is overexpressed in ∼20% of GC cases and anti-HER2 antibody trastuzumab in combination with conventional chemotherapy, is recognized as standard therapy for HER2-positive metastatic GC. This strategy improves GC patients' survival by 2-3 months, however its optimal results in breast cancer indicate that GC survival may be improved. A new photoimmunoconjugate was developed by conjugating a porphyrin with trastuzumab (Trast:Porph) for targeted photodynamic therapy in HER2-positive GC. Using mass spectrometry analysis, the lysine residues in the trastuzumab structure most prone for porphyrin conjugation were mapped. The in vitro data demonstrates that Trast:Porph specifically binds to HER2-positive cells, accumulates intracellularly, co-localizes with lysosomal marker LAMP1, and induces massive HER2-positive cell death upon cellular irradiation. The high selectivity and cytotoxicity of Trast:Porph based photoimmunotherapy is confirmed in vivo in comparison with trastuzumab alone, using nude mice xenografted with a HER2-positive GC cell line. In the setting of human disease, these data suggest that repetitive cycles of Trast:Porph photoimmunotherapy may be used as an improved treatment strategy in HER2-positive GC patients.

Porphyrin-based photosensitizers and their DNA conjugates for singlet oxygen induced nucleic acid interstrand crosslinking.

The development of methods for the generation of site-selective interstrand crosslinks (ICLs) in synthetic oligonucleotides provides a platform for the study of ICL repair mechanisms and the stabilisation of DNA-based materials. Our group has previously reported on the use of a furan moiety as a masked reactive functionality for ICL generation and recently introduced the use of 1O2 as an efficient light-induced oxidant. Here, the use of porphyrin-based photosensitizers (PSs) has been explored for ICL generation. The efficiency of the ICL reaction has been investigated using PSs added into solution as well as attached to oligonucleotide probes. Interestingly, even a highly hydrophobic phthalocyanine was able to produce ICLs. Either in solution or conjugated to an ON, chlorin e6 was the most efficient ICL generator for the current purpose.

New porphyrin derivatives for phosphate anion sensing in both organic and aqueous media.

New porphyrin-based anion receptors have been prepared from meso-tetrakis(pentafluorophenyl)porphyrin () and diamine derivatives. These receptors (compounds ) interact preferentially with phosphate anions both in organic solutions and in basic aqueous media when coated on gold piezoelectric quartz crystals. A single crystal X-ray structure of the bis-HF complex of a imine derivative of was obtained; it serves to highlight the main interactions between the anion and cation receptors in the solid state.

5-[4-(diethoxyphosphoryl)-2,3,5,6-tetrafluorophenyl]-10,15,20-tris(pentafluorophenyl)porphyrin.

The title compound, C(48)H(20)F(19)N(4)O(3)P, prepared by the nucleophilic attack of triethyl phosphite on one of the 4-fluoro atoms of 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin, contains a single molecule in the asymmetric unit. The porphyrin unit is almost planar [largest non-H atom deviation = 0.174 (6) Å], and has the planes of the neighbouring benzene rings oriented at angles ranging from 64.3 (2) to 89.6 (3)° relative to the porphyrin core. The P=O group is almost coplanar with the attached benzene ring, subtending an angle of 4.0 (3)°. Several weak supramolecular interactions, namely C-H...π, C-F...π, P=O...π, C-H...(O,F) and F...F contacts, contribute to the crystal packing.