Hydroxyapatite/alginate/gellan gum inks with osteoconduction and osteogenic potential for bioprinting bone tissue analogues.

There is a growing demand for engineered bone tissues custom-designed to match the patient-specific defect size and in vitro models for studying bone diseases and/or drug screening. Herein, we propose a bioprinted bone tissue construct using SaOs-2 cells within alginate/gellan gum/hydroxyapatite inks. Different ink formulations were developed with varying hydroxyapatite content and then evaluated for viscoelasticity, printability, biomineralization properties, post-printing viability, proliferation, metabolic activity, and osteogenic phenotype of SaOs-2-encapsulated cells. Results indicate that ink formulations exhibit non-Newtonian shear-thinning behaviour, maintaining shape integrity and structural stability post-printing. Ink mineralization rates increase with the hydroxyapatite content, rendering them suitable for bone defect strategies. Post-printed cells in the developed constructs remain live, spreading, and metabolically active but do not proliferate. Osteogenic gene and protein expression, both early and late, show upregulation at day 7 relative to day 1, followed by downregulation at day 14. Lower hydroxyapatite content inks demonstrate up to fourfold upregulation in genes and proteins at most time points. Additionally, these constructs release calcium and phosphate at levels conducive to mineralization. Overall, the tissue-engineered miniaturized constructs not only meet the criteria for early-stage bone defect/fracture regeneration but also serve as a promising platform for drug screening and evaluating potential therapeutic treatments.

Prevascularized spongy-like hydrogels maintain their angiogenic potential after prolonged hypothermic storage.

The chronic shortage of organs and tissues for transplantation represents a dramatic burden on healthcare systems worldwide. Tissue engineering offers a potential solution to address these shortages, but several challenges remain, with prevascularization being a critical factor for in vivo survival and integration of tissue engineering products. Concurrently, a different challenge hindering the clinical implementation of such products, regards their efficient preservation from the fabrication site to the bedside. Hypothermia has emerged as a potential solution for this issue due to its milder effects on biologic systems in comparison with other cold preservation methodologies. Its impact on prevascularization, however, has not been well studied. In this work, 3D prevascularized constructs were fabricated using adipose-derived stromal vascular fraction cells and preserved at 4 °C using Hypothermosol or basal culture media (α-MEM). Hypothermosol efficiently preserved the structural and cellular integrity of prevascular networks as compared to constructs before preservation. In contrast, the use of α-MEM led to a clear reduction in prevascular structures, with concurrent induction of high levels of apoptosis and autophagy at the cellular level. In vivo evaluation using a chorioallantoic membrane model demonstrated that, in opposition to α-MEM, Hypothermosol preservation retained the angiogenic potential of constructs before preservation by recruiting a similar number of blood vessels from the host and presenting similar integration with host tissue. These results emphasize the need of studying the impact of preservation techniques on key properties of tissue engineering constructs such as prevascularization, in order to validate and streamline their clinical application.

Extracellular matrix-derived materials for tissue engineering and regenerative medicine: A journey from isolation to characterization and application.

Biomaterial choice is an essential step during the development tissue engineering and regenerative medicine (TERM) applications. The selected biomaterial must present properties allowing the physiological-like recapitulation of several processes that lead to the reestablishment of homeostatic tissue or organ function. Biomaterials derived from the extracellular matrix (ECM) present many such properties and their use in the field has been steadily increasing. Considering this growing importance, it becomes imperative to provide a comprehensive overview of ECM biomaterials, encompassing their sourcing, processing, and integration into TERM applications. This review compiles the main strategies used to isolate and process ECM-derived biomaterials as well as different techniques used for its characterization, namely biochemical and chemical, physical, morphological, and biological. Lastly, some of their applications in the TERM field are explored and discussed.

Solvent-free synthesis of hydrophobic and amphiphilic esters using a chemically modified lipase from Thermomyces lanuginosus: a comparative study with native and immobilized forms.

Using lipases to catalyze the synthesis of the most differentiated type of compounds remains one of the major challenges among scientists. Seeking more economic and advantageous catalysts is a current goal of green chemistry. In this work, we demonstrate the potential of a chemically modified form of lipase from Thermomyces lanuginosus (cmLTL) for the synthesis of both hydrophobic (heptyl heptanoate, heptyl octanoate, heptyl decanoate, decyl heptanoate, decyl octanoate and decyl decanoate) and amphiphilic (2-(2-ethoxyethoxy)ethyl oleate and 2-(2-ethoxyethoxy)ethyl linoleate) esters, in bulk. The results were compared with its native (LTL) and immobilized (imLTL) forms. The data revealed that LTL showed poor activity for all reactions performed with n-heptane (η<20 %). ImLTL was able to synthesize all hydrophobic esters (η>60 %), with exception of the short ester, heptyl heptanoate. cmLTL was the only form of LTL capable of producing hydrophobic and amphiphilic esters, without compromising the yield when the reactions were performed under solvent-free conditions (>50 %). Molecular modeling showed that the active pocket of cmLTL is able to deeply internalize transcutol, with stronger interactions, justifying the outstanding results obtained. Furthermore, owing to the possibility of cmLTL filtration, the reusability of the catalyst is ensured for at least 6 cycles, without compromising the reaction yields.

Laccase-Catalyzed Synthesis of Added-Value Polymers from Cork and Grape Extracts.

The development of products from natural plant sources, including agriculture and food wastes, contributes significantly to the circular economy and global sustainability. Cork and grape wastes were employed as the primary sources in this study to obtain compounds of interest under mild extraction conditions. Laccase was applied to oxidize the cork and grape extracts, with the aim of producing value-added molecules with improved properties. Ultraviolet-visible (UV-vis) spectroscopy was assessed to monitor the oxidation process, and characterization of the end products was performed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) spectroscopy. The antioxidant and antiaging properties were evaluated by means of ABTS, DPPH, FRAP, and SPF testing. Overall, as compared to their monomeric counterparts, the polymeric compounds displayed remarkable antioxidant and antiaging characteristics after laccase oxidation, showing tremendous potential for applications in the food, pharmaceutical, cosmetic, and textile industries.

Deep Eutectic Solvents as Suitable Solvents for Lipase-Catalyzed Transesterification Reactions.

In this work, three deep eutectic mixtures (DES 1: choline chloride/urea; DES 2: choline chloride/glycerol; and DES 3: tetrabutylammonium bromide/imidazole) were investigated as mediums for the synthesis of glucose laurate and glucose acetate. Aiming to achieve a greener and more sustainable approach, the synthesis reactions were catalyzed by lipases from Aspergillus oryzae (LAO), Candida rugosa (LCR), and porcine pancreas (LPP). The hydrolytic activity of lipases against p-nitrophenyl hexanoate revealed no evidence of enzyme inactivation when DES were used as medium. Regarding the transesterification reactions, combining LAO or LCR with DES 3 resulted in the efficient production of glucose laurate (from glucose and vinyl laurate) (conversion >60 %). The best result for LPP was observed in DES 2, with 98 % of product production after 24 hours of reaction. When replacing vinyl laurate by a smaller hydrophilic substrate, vinyl acetate, a distinct behavior was observed. LCR and LPP performed better in DES 1, yielding more than 80 % of glucose acetate after 48 hours of reaction. The catalytic activity of LAO was less pronounced, reaching only nearly 40 % of product in DES 3. The results highlight the potential of combining biocatalysis with greener and environmentally-safer solvents, for the synthesis of differentiated chain-length sugar fatty acid esters (SFAE).

Addressing the Structural Organization of Silicone Alternatives in Formulations by Molecular Dynamics Simulations and a Novel Equilibration Protocol.

The world of cosmetics is an always-evolving field with constant updates on its formulation components. The current reality asks for an ever-increasing need for natural and sustainable replacements for synthetic compounds in all fields of modern consumer products. However, the research and development stages of finding these alternatives can be an expensive, time-consuming, and often wasteful process that turns this task into a laborious procedure. This study introduces the development of a computational methodology that will aid the research of silicone alternatives, disclosing their structural performance in a formulation. Additionally, an equilibration protocol was developed to measure the distribution and densities of these silicone alternatives to determine how they behave in relation to their counterparts, using molecular dynamics simulations. Two systems were tested, A and B, where the former is composed of one ester (Dipentaerythrityl Hexa C5 Acid Ester) and the latter by an ester combined with an alkane (Triheptanoin and C13-Isoalkane); all three molecules are commercially available and widely used. Both systems were subjected to a 3-step thermal regulation strategy. The systems went through an initial simulation at 25 °C and at 70 °C, then a temperature switch took place (25 °C « 70 °C), then a shock to 200 °C, and finally a Simulated Annealing protocol reaching 250 °C. In the end, all systems converged towards micelle-like structures. These results come to further ascertain the position of computational chemistry and Molecular Dynamics Simulations as an important part of R&D processes in modern sciences and investigation.

Lipase-Catalysed Polymerization of Eutectic Mixtures.

Aiming to reduce the toxicity and operational costs often associated to chemical processes, the enzymatic synthesis is applied herein as a sustainable route for producing polyesters. The use of NADES' (Natural Deep Eutectic Solvents) components as a source of monomers for the synthesis of polymers through lipase-catalyzed esterification in an anhydrous medium is detailed for the first time. Three NADES composed by glycerol and an organic base, or acid, were used to produce polyesters, through polymerization reactions catalyzed by Aspergillus oryzae lipase. High polyester conversion rates (above 70 %), containing at least 20 monomeric units (glycerol:organic acid/base (1 : 1)), were observed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) analysis. The NADES monomers' capacity for polymerization, along with their non-toxicity, cheap cost, and simplicity of production, sets up these solvents as a greener and cleaner approach for the synthesis of high value-added products.

Therapeutic Textiles Functionalized with Keratin-Based Particles Encapsulating Terbinafine for the Treatment of Onychomycosis.

Onychomycosis is the most common nail fungal infection worldwide. There are several therapy options available for onychomycosis, such as oral antifungals, topicals, and physical treatments. Terbinafine is in the frontline for the treatment of onychomycosis; however, several adverse effects are associated to its oral administration. In this work, innovative keratin-based carriers encapsulating terbinafine were designed to overcome the drawbacks related to the use this drug. Therapeutic textiles functionalized with keratin-based particles (100% keratin; 80% keratin/20% keratin-PEG) encapsulating terbinafine were developed. The controlled release of terbinafine from the functionalized textiles was evaluated against different mimetic biologic solutions (PBS buffer-pH = 7.4, micellar solution and acidic sweat solution-pH = 4.3). The modification of keratin with polyethylene glycol (PEG) moieties favored the release of terbinafine at the end of 48 h for all the solution conditions. When the activity of functionalized textiles was tested against Trichophyton rubrum, a differentiated inhibition was observed. Textiles functionalized with 80% keratin/20% keratin-PEG encapsulating terbinafine showed a 2-fold inhibition halo compared with the textiles containing 100% keratin-encapsulating terbinafine. No activity was observed for the textiles functionalized with keratin-based particles without terbinafine. The systems herein developed revealed therapeutic potential towards nail fungal infections, taking advantage of keratin-based particles affinity to keratin structures and of the keratinase activity of T. rubrum.

Chemical modification of lipases: A powerful tool for activity improvement.

The demand for adequate and ecologically acceptable procedures to produce the most differentiated products has been growing in recent decades, with enzymes being excellent examples of the advances achieved so far. Lipases are astonishing catalysts with a vast range of applications including the synthesis of esters, flavors, biodiesel, and polymers. The broad specificity of the substrates, as well as the regio-, stereo-, and enantioselectivity, are the differentiating factors of these enzymes. Structural modification is a current approach to enhance the activity of lipases. Chemical modification of lipases to improve catalytic performance is of great interest considering the increasingly broad fields of application. Together with the physical immobilization onto solid supports, different strategies have been developed to produce catalysts with higher activity and stability. In this review, practical insights into the different strategies developed in recent years regarding the modification of lipases are described. For the first time, the impact of the modifications on the activity and stability of lipases, as well as on the biotechnological applications, is fully compiled.

Changing the shape of wool yarns via laccase-mediated grafting of tyrosine.

The shape of wool yarns was changed by laccase-assisted grafting of tyrosine. Prior to tyrosine grafting a cysteine pre-treatment was optimized aiming to increase the amount of thiol reaction groups available. The best operational conditions for laccase-assisted tyrosine grafting were: i) pre-treatment with cysteine (2.2 mM) in a solution of 20 % ethanol, 15 % propylene glycol and 0.5 % benzyl alcohol, pH = 10, 40 °C; ii) tyrosine grafting with 3.0 mM tyrosine, 18 U/mL laccase, pH = 5, 40 °C. The shape modification was evaluated by number of curly twists determination on the grafted yarn samples. The thermal and mechanical properties of the grafted wool yarns was evaluated by TGA, DSC and breaking strength determination. The amount of free thiols and weight gain were assessed aiming to infer the role of the cysteine pre-treatment on the final tyrosine grafting and shape modification. The laccase-assisted grafting of tyrosine onto wool yarns have influenced the thermal and mechanical properties of the yarns however without compromising their structural integrity for the final application purposes. The developed methodology to impart new shape to wool yarns is presented herein as an environmentally friendly alternative to chemical methods. The new findings revealed great potentialities for application in similar fibers like hair.

Increased Encapsulation Efficiency of Methotrexate in Liposomes for Rheumatoid Arthritis Therapy.

Methotrexate (MTX) is a common drug used to treat rheumatoid arthritis. Due to the excessive side effects, encapsulation of MTX in liposomes is considered an effective delivery system, reducing drug toxicity, while maintaining its efficacy. The ethanol injection method is an interesting technique for liposome production, due to its simplicity, fast implementation, and reproducibility. However, this method occasionally requires the extrusion process, to obtain suitable size distribution, and achieve a low level of MTX encapsulation. Here, we develop a novel pre-concentration method, based on the principles of the ethanol injection, using an initial aqueous volume of 20% and 1:1 ratio of organic:aqueous phase (v/v). The liposomes obtained present small values of size and polydispersity index, without the extrusion process, and a higher MTX encapsulation (efficiency higher than 30%), suitable characteristics for in vivo application. The great potential of MTX to interact at the surface of the lipid bilayer was shown by nuclear magnetic resonance (NMR) studies, revealing mutual interactions between the drug and the main phospholipid via hydrogen bonding. In vivo experiments reveal that liposomes encapsulating MTX significantly increase the biological benefit in arthritic mice. This approach shows a significant advance in MTX therapeutic applications.

Carboxymethyl Cellulose (CMC) as a Template for Laccase-Assisted Oxidation of Aniline.

Polyaniline (PANi) is a conducting polymer which has been subject of intensive research on the exploitation of new products and applications. The main aim of the work is the development of a conductive bacterial cellulose (BC)-based material by enzymatic-assisted polymerization of aniline. For this, we study the role of carboxymethyl cellulose (CMC) as a template for the in situ polymerization of aniline. Bacterial cellulose was used as the supporting material for the entrapment of CMC and for the in situ oxidation reactions. The amount of CMC entrapped inside BC was optimized as well as the conditions for laccase-assisted oxidation of aniline. The new oligomers were evaluated by spectrometric techniques, namely 1H NMR and MALDI-TOF, and the functionalized BC surfaces were analyzed by thermogravimetric analysis (TGA), Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscope (SEM), and reflectance spectrophotometry. The conductivity of the developed materials was evaluated using the four-probe methodology. The oligomers obtained after reaction in the presence of CMC as template display a similar structure as when the reaction is conducted only in BC. Though, after oxidation in the presence of this template, the amount of oligomers entrapped inside BC/CMC is considerably higher conferring to the material greater electrical conductivity and coloration. The use of CMC as a template for aniline oxidation on BC seems to be a promising and cheap strategy to improve the yield of functionalization and increment the properties of the materials, namely electrical conductivity and coloration.

Poloxamer 407 based-nanoparticles for controlled release of methotrexate.

Poloxamer 407 (P407)-based nanoparticles were produced by the high pressure homogenization method for the encapsulation and delivery of methotrexate (MTX), aiming intravenous therapeutic applications. The surface of these nanoparticles was functionalized by conjugation of P407 with folic acid (FA) or with MTX, which served as targeting ligand agents. MTX-P407 conjugate was also developed to increase the final drug cargo. Two hydrophobic derivatives of MTX, MTX di-ethylated ester (MTX-OEt) and the ionic complex MTX-dimethyldioctadecylammonium bromide (MTX-DODAB) were produced and entrapped onto P407-based nanoparticles. All formulations developed revealed a monodisperse character comprising small and narrow nanoparticles (<100 nm). P407 nanoparticles (functionalized with FA) and MTX-P407 nanoparticles, both loaded with MTX-OEt, demonstrated a slow drug release profile. The effect of lipase from Aspergillus oryzae on the hydrolysis of the linkage between the P407 and MTX, and consequent MTX release profile, was also evaluated. We observed a controlled and slow release of MTX (<50% of release after 11 days) in the presence of enzyme. These MTX-P407 nanoparticles loaded with MTX-OEt induced a great effect against Caco-2 cancer cells (≈40% of cell death after 72 h of incubation), demonstrating higher efficiency than the free MTX at the same concentration.

Effect of Additives on the in situ Laccase-Catalyzed Polymerization of Aniline Onto Bacterial Cellulose.

Laccase-mediated systems are a green route to accelerate the oxidation of aniline and obtain polyaniline with conductive properties. The synthesis of green polyaniline (emeraldine salt) was herein improved by the inclusion of additives like sodium bis (2-ethyl hexyl) sulfosuccinate (AOT) and potassium hexacyanoferrate (II) (KHCF) in the medium. The aniline polymerization was confirmed by the detection of the absorption band typical of emeraldine salt at 420 nm, typical of the semiquinoid radical cation, and of the polaron absorption band at 700-800 nm, corresponding to the distinctive signal of doped or partial doped aniline. The oligomers and/or polymers obtained were characterized by spectrometry techniques, namely 1H NMR and MALDI-TOF, and the bacterial cellulose (BC) conductivity was assessed by means of a four-point probe electrical conductivity technique. The best polymerization results were obtained with 5 mM AOT, 10 mM KHCF, and 25 U/mL of laccase. The synergistic effect between both additives in the presence of a catalyst leads to obtaining BC samples coated with green polyaniline with promising electric conductive properties.

Ultrasound-Assisted Encapsulation of Sacha Inchi (Plukenetia volubilis Linneo.) Oil in Alginate-Chitosan Nanoparticles.

Sacha inchi oil is rich in essential and non-essential fatty acids and other types of bioactive agents like tocopherols and polyphenolic compounds, which are very well-known antioxidants. In this study, the encapsulation of sacha inchi oil in alginate (AL) and chitosan (CS) nanoparticles was achieved with the assistance of high-intensity ultrasound. Nanoemulsion is the most effective delivery and high stability system for lipophilic bioactive agents. Chitosan and surfactant concentrations were varied to study their effect on particle formulations. Size, zeta-potential, polydispersity, and stability of particles were determined in time to optimize the preparation conditions. Sacha inchi oil encapsulated in AL-CS nanoparticles showed a higher loading efficiency and stability for short and long periods compared with other vegetable oils such as olive and soybean. Also, because of the types of tocopherols present in sacha inchi oil (γ- and δ-tocopherols), a much higher antioxidant activity (95% of radical reduction in 15 min) was found in comparison with nanocapsules with olive oil, which contain α-tocopherols. The particles showed high efficiency of protein loading at high concentration of bovine serum albumin (BSA) and a low rate of leaching profiles in various testing media like simulated gastric and intestinal fluids with/without enzymes, that is, pepsin 0.1% (w/v) and pancreatin 0.1% (w/v), respectively.

PTS micelles for the delivery of hydrophobic methotrexate.

Polyoxyethanyl-α-tocopheryl sebacate (PTS) is an amphiphilic compound with self-emulsifying properties known to form micelles. In this work, we report the production of PTS micelles for the encapsulation and delivery of a hydrophobic derivative of methotrexate, MTX di-ethylated (MTX-OEt). We optimized the micelles production by testing two different techniques: auxiliary solvent and sonication. Small and homogeneous micelles (≈40 nm) were obtained through the auxiliary solvent method performed at 30 °C and using 15 mg/mL of PTS. The produced micelles with the most promising physicochemical properties did not induce cytotoxicity when tested in normal human cells (BJ5ta cells), being considered for the encapsulation of MTX-OEt. This prodrug was achieved by Fisher esterification using ethanol, being isolated in good yield (η = 68%). MTX-OEt was efficiently encapsulated onto the produced micelles which preserved their physicochemical properties. The PTS micelles loaded with MTX-OEt, free MTX-OEt and free unmodified MTX revealed similar biological effect against cancer cells (Caco-2 cells). These results demonstrated that the biological activity of MTX is not altered after modification. The developed PTS micelles revealed a promising intracellular delivery performance with potentiality for cancer therapy.

Quantification of drugs encapsulated in liposomes by 1H NMR.

Liposomes are one of the most important and extensively studied drug delivery system due to their ability to encapsulate different kinds of drugs. Exploiting the advantages of 1H Nuclear Magnetic Resonance (NMR) spectrometry, we established a rapid and easy method for quantification of drugs encapsulated in liposomes. An internal standard, pyridine, was used for quantitative determination of drug concentration. Two different drugs were involved in this work, one hydrophilic, methotrexate disodium salt, and another hydrophobic, tamoxifen. The specificity and selectivity of the suggested method were evaluated by the absence of overlapping of at least one signal of each drug with pyridine in the NMR spectrum. The accuracy and precision of the method were assessed by adding a known amount of each drug to unloaded liposomes. Results obtained by quantitative NMR (qNMR) were validated and confirmed by comparing with two other traditional techniques, Ultraviolet-Visible (UV-vis) spectrophotometry and High-Performance Liquid Chromatography (HPLC). It was found that the results were consistent with the ones obtained from our proposed qNMR method. Considering all the experiments conducted in this study, we deliberate that qNMR can be a suitable tool for the determination of drugs encapsulated in liposomes.

Conductive bacterial cellulose by in situ laccase polymerization of aniline.

Conductive and colored bacterial cellulose (BC) was developed by entrapment of polyaniline (PANi) onto dry BC membranes. The polyaniline was produced by in situ green polymerization of aniline by Myceliophthora thermophila laccase at pH = 4, 25°C, in the presence of a mediator, 1-hydroxybenzotriazol (HBT), using two different reactors, a water bath (WB) and an ultrasonic bath (US). MALDI-TOF and 1H NMR characterization of the experiment solutions confirmed the efficient polymerization of aniline by laccase. The dried BC membranes with entrapped polyaniline showed electrical conductive behavior and strong coloration, opening novel routes for the exploitation of functionalized bacterial cellulose as a green material for technical textiles, wearables, and other applications.

Strategies for the synthesis of fluorinated polyesters.

In this work we synthetized three fluorinated polyesters from dimethyl tetrafluorosuccinate (DMTFS), dimethyl hexafluoroglutarate (DMHFG), and dimethyl octafluoroadipate (DMOFA) and ethylene glycol. The influence of parameters like monomer's size, temperature, vacuum, ultrasound and catalyst, on the polyesters synthesis was evaluated. The conversion rates were assessed considering 1H NMR data and the results disclose the role of ultrasound (US) as crucial to attain high reaction conversion rates (≈20% of increase relatively to the reactions performed in absence of US). The effect of US was more relevant for the higher molecular weight monomers (DMHFG and DMOFA). The use of Candida antarctica lipase (immobilized CALB) marginally favors the synthesis reactions when fixing the other conditions. The size of the starting monomers influenced greatly the reaction conversion rates, as shorter monomers gave rise to high amount of product recovering. All the produced polyesters were isolated and fully characterized by NMR (1H and 19F), FTIR, TGA and MALDI-TOF.