In Situ Photo-crosslinkable Hyaluronic Acid/Gelatin Hydrogel for Local Nitric Oxide Delivery.

An increasing number of studies have shown that the local release of nitric oxide (NO) from hydrogels stimulates tissue regeneration by modulating cell proliferation, angiogenesis, and inflammation. The potential biomedical uses of NO-releasing hydrogels can be expanded by enabling their application in a fluid state, followed by controlled gelation triggered by an external factor. In this study, we engineered a hydrogel composed of methacrylated hyaluronic acid (HAGMA) and thiolated gelatin (GELSH) with the capacity for in situ photo-cross-linking, coupled with localized NO release. To ensure a gradual and sustained NO release, we charged the hydrogels with poly(l-lactic-co-glycolic acid) (PLGA) nanoparticles functionalized with S-nitrosoglutathione (GSNO), safeguarding SNO group integrity during photo-cross-linking. The formation of thiol-ene bonds via the reaction between GELSH's thiol groups and HAGMA's vinyl groups substantially accelerated gelation (by a factor of 6) and increased the elastic modulus of hydrated hydrogels (by 1.9-2.4 times). HAGMA/GELSH hydrogels consistently released NO over a 14 day duration, with the release of NO depending on the hydrogels' equilibrium swelling degree, determined by the GELSH-to-HAGMA ratio. Biocompatibility assessments confirmed the suitability of these hydrogels for biological applications as they display low cytotoxicity and stimulated fibroblast adhesion and proliferation. In conclusion, in situ photo-cross-linkable HAGMA/GELSH hydrogels, loaded with PLGA-GSNO nanoparticles, present a promising avenue for achieving localized and sustained NO delivery in tissue regeneration applications.

Evaluation of dried blood spots as an alternative sampling strategy for 5-fluorouracil monitoring: From method development to clinical application.

Therapeutic drug monitoring (TDM) of 5-Fluorouracil (5-FU) is strongly recommended because of its large inter-individual pharmacokinetic variability, narrow therapeutic window, and incidence of toxicity. However, there are several factors that limit the application of TDM in clinical settings. Considering the intrinsic advantages of dried microsamples, such as minimally invasive sampling, analyte stability, and cost-effective logistics, this study aimed to develop a method for the determination of 5-FU in dried blood spots (DBS) using ultra-high liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and to evaluate its clinical application. Sample preparation was based on an aqueous extraction followed by protein precipitation. Separation was performed in an Acquity UPLC® HSS C18 (150 ×2.1 mm, 1.8 µm), and the mobile phases were water and acetonitrile with 0.5% acetic acid. The total run time was 5.5 min. The method was linear from 100 to 2000 ng/mL, precise (maximum CV% of 7.5%), and accurate (98.3-115.4%). The average recovery was 70%. Blood hematocrit had a minimal impact on the assay. DBS samples were stable for 21 days at 4, 25, and 45 °C. A total of 40 paired samples of plasma, capillary DBS, and venous DBS were analyzed. Median 5-FU concentrations were 444.7, 637.0, and 499.7 ng/mL for plasma, capillary DBS, and venous DBS, respectively. Capillary and plasma concentrations were significantly correlated (r > 0.90), but there was a lack of agreement between the methods, as capillary DBS levels were on average 146% of plasma. Venous DBS corresponded to 110% of the measured plasma concentrations, with a strong correlation (r > 0.97) and agreement between the methods. Our study is the first to report the use of DBS samples to quantify 5-FU. Further studies are needed to establish whether capillary samples can replace plasma.

Photocurable Nitric Oxide-Releasing Copolyester for the 3D Printing of Bioresorbable Vascular Stents.

The design of bioresorbable vascular stents (BVS) capable of releasing nitric oxide (NO) at the implant site may enable BVS to mimic the antiplatelet, antiproliferative, and pro-endothelial actions of NO, overcoming complications of BVS such as late thrombosis and restenosis. In this study, the fabrication of BVS composed of methacrylated poly(dodecanediol citrate-co-dodecanediol S-nitroso-mercaptosuccinate) (mP(DC-co-DMSNO)), a novel elastomeric, bioabsorbable, and photocurable copolyester, containing covalently bound S-nitrosothiol groups in the carbon backbone of the polymer, is reported. The mP(DC-co-DMSNO) stents are manufactured via photoinduced 3D printing and allow deployment via a self-expansion process from a balloon catheter. After deployment, hydration of the stents triggers the release of NO, which is maintained during the slow hydrolysis of the polymer. Real-time NO release measurements show that by varying the copolyester composition and the strut geometry of the mP(DC-co-DMSNO) stents, it is possible to modulate their NO release rate in the range of 30-52 pmol min-1 cm-2 . Preliminary biological assays in cell culture show that endothelial cells adhere to the surface of the stents and that NO release favors their endothelization. Thus, mP(DC-co-DMSNO) may emerge as a new platform for the fabrication of advanced BVS.

Nitric Oxide-Releasing Supramolecular Cellulose Nanocrystal/Silsesquioxane Foams.

Cellulose nanocrystals (CNC)-based foams are promising tissue engineering materials that may facilitate implant-tissue integration and allow localized and controlled drug delivery. Herein, hybrid CNC-based foams, which are ultralightweight (30-100 mg cm-3 ), highly porous (>95%), ominiphilic and superabsorbent (1500-3000 wt% of water and/or toluene uptake) are obtained by the in situ condensation of poly(ethylene glycol) ditriethoxysilyl (TES-PEG-TES) into a 3D network, where silsesquioxane nanoparticles (SS-NP) are the crosslinking nodes, and CNC are entrapped forming ionic interactions, in a supramolecular structure. In a new approach, using 3-mercaptopropyltrimethoxysilane, sulfhydryl groups are inserted on the SS-NP periphery and S-nitrosated to enable the functionalization of SS-NP with S-nitrosothiol groups, which can nitric oxide (NO), in a process triggered by the hydration of the foams and modulated by their supramolecular structure. CNC-SS-PEG foams exhibit elevated thermal and structural stability, compressive strength compatible with various soft human tissues, and NO release rates (1-18 pmol mg-1 min-1 ) within the range of the beneficial NO actions. Thus, the CNC-SS-PEG foams herein described represent a new platform of supramolecular hybrid materials for localized delivery of NO, with potential uses in tissue engineering and other biomedical applications.

3D printed nitric oxide-releasing poly(acrylic acid)/F127/cellulose nanocrystal hydrogels.

Hydrogels have been used as matrices for the topical delivery of nitric oxide (NO) for achieving vasodilation, wound healing and analgesic actions. More recently, supramolecular hydrogels comprised of poly(acrylic acid) (PAA) and micellar Pluronic F127 (F127), prepared by thermal reaction, emerged as a suitable matrix for the incorporation of hydrophilic NO donors, such as S-nitrosoglutathione (GSNO). Herein, we describe an innovative method for the three-dimensional (3D) printing of cellulose nanocrystal (CNC)-containing and semi-interpenetrating PAA/F127 hydrogels by PAA photopolymerization via digital light processing (DLP), in the absence of organic solvents. Scanning electron microscopy showed that, differently from typical porous PAA-based hydrogels, the 3D printed PAA/F127/CNC hydrogels have dense morphology. By using transmission electron microscopy we confirmed for the first time the presence of F127 micelles in the printable resin, and their preservation after the photopolymerization process. The F127 micelles conferred compressive recoverability to the 3D printed PAA/F127/CNC hydrogels, widening their potential applications as soft biomaterials. PAA/F127/CNC hydrogels charged with GSNO are shown to release NO spontaneously upon hydration at initial rates that depend on the GSNO charge and are higher in the presence of CNC. As local NO release may exert cell proliferation action, 3D printed PAA/F127/CNC/GSNO hydrogels may serve as a versatile soft biomaterial for local NO delivery in regenerative medicine and other biomedical applications.

Visualization of supramolecular structure of Pluronic F127 micellar hydrogels using cryo-TEM.

Pluronic® F127 micellar hydrogels are of growing interest to the biomedical field due to their versatility as drug delivery systems. Pluronic® F127 is a symmetric and amphiphilic triblock copolymer which in aqueous medium self-assembles into micelles that pack togetherwith increasing temperature or concentration, leading to non-flowable hydrogels. The microstructure of these hydrogels is usually investigated by small-angle X-ray scattering, which is not a readily available technique. Conversely, cryo-TEM is a widespread technique used for investigating the morphology of aqueous systems. In the case of Pluronic® F127 micellar systems, the elevated viscosity poses a significant challenge for specimen preparation and, consequently, for cryo-TEM observation. Herein, we show a trustworthy, inexpensive and readily available methodology for preparing specimens of highly viscous micellar solutions and non-flowable hydrogels using an automated vitrification system. With this methodology we were able to visualize not only the morphology of individual Pluronic® F127 micelles -but also the supramolecular structure evolution as a function of concentration. This methodology opens up a wide range of opportunities for hydrogel characterization, although additional systematic studies might be required in order to optimize and replicate it for similar systems.

S-nitrosothiol-terminated Pluronic F127: Influence of microstructure on nitric oxide release.

HYPOTHESIS: Nitric oxide (NO)-releasing Pluronic F127 hydrogels (F127) containing dissolved S-nitrosothiols or pendant N-diazeniumdiolate (NONOate) groups have been described. The NO charging of these hydrogels is usually limited by their low stability or disruption of the micellar packing. S-nitrosothiol-terminated F127 may emerge as a new strategy for allowing NO delivery at different rates in biomedical applications. EXPERIMENTS: Terminal hydroxyl groups of F127 were esterified and reduced to produce F127-mercaptopropionate (HS-F127-SH), which was subsequently S-nitrosated to generate S-nitrosothiol-terminated F127 (ONS-F127-SNO). Micro-differential scanning calorimetry, 1H NMR spin-spin relaxation (T2), temperature-dependent small-angle X-ray scattering, and cryo-transmission electron microscopy, were used to determine the micellar packing structure, while real-time chemiluminescence NO detection and UV-Vis spectrophotometry were used to evaluate the kinetics of NO release. FINDINGS: HS-F127-SH micellization and gelation processes were analogous to native F127, however, with a decreased short-range ordering of the micelles. ONS-F127-SNO hydrogels released NO thorough a preferentially intramicellar SNO dimerization reaction. Increasing ONS-F127-SNO concentration reduces the rate of SNO dimerization and increases the overall rate of NO release to the gas phase, opening up new possibilities for tailoring NO delivery from F127-based hydrogels.

Wound healing action of nitric oxide-releasing self-expandable collagen sponge.

Mounting evidence showing that local nitric oxide (NO) delivery may significantly improve the wound healing process has stimulated the development of wound dressings capable of releasing NO topically. Herein, we describe the preparation of a self-expandable NO-releasing hydrolyzed collagen sponge (CS), charged with the endogenously found NO donor, S-nitrosoglutathione (GSNO). We show that cold pressed and GSNO-charged CS (CS/GSNO) undergo self-expansion to its original 3D shape upon water absorption to a swelling degree of 2,300 wt%, triggering the release of free NO. Topical application of compressed CS/GSNO on wounds in an animal model showed that exudate absorption by CS/GSNO leads to the release of higher NO doses during the inflammatory phase and progressively lower NO doses at later stages of the healing process. Moreover, treated animals showed significant increase in the mRNA expression levels of monocyte chemoattractant protein-1 (MCP-1), murine macrophage marker (F4/80), transforming growth factor beta (TGF-ß), stromal cell-derived factor 1 (SDF-1), insulin-like growth factor-1 (IGF-1), nitric oxide synthase(iNOS), and matrix metalloproteinase(MMP-9). Cluster differentiation 31 (CD31), vascular endothelial growth factor (VEGF), and F4/80 were measured on Days 7 and 12 by immunohistochemistry in the cicatricial tissue. These results indicate that the topical delivery of NO enhances the migration and infiltration of leucocytes, macrophages, and keratinocytes to the wounded tissue, as well as the neovascularization and collagen deposition, which are correlated with an accelerated wound closure. Thus, self-expandable CS/GSNO may represent a novel biocompatible and active wound dress for the topical delivery of NO on wounds.

Solvent-free and biocompatible multiphased organic-inorganic hybrid nanocomposites.

Biocompatible chemically cross-linked organic-inorganic (O-I) hybrid nanocomposites were developed using a new atoxic, simple and fast, solvent-free pathway. Poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG), which are both biocompatible, were used as the organic moieties (at different PCL/PEG ratios), while in situ synthesized polysilsesquioxanes made up the inorganic moiety. The O-I hybrid nanocomposites' molecular structures were characterized using solid-state 29Si NMR, TGA and ATR-IR. Results showed an unusually high condensation yield of approximately 90% and two distinct silsesquioxane structures. No traces of the remaining isocyanate groups were found. Advanced morphological characterization of the ternary O-I hybrids was performed using a combination of electron microscopy and X-ray scattering techniques such as SEM, TEM, ESI-TEM, WAXS and temperature-dependent SAXS. Results showed the occurrence of spherical nanoparticles, associated with polysilsesquioxane, and ordered network grains, associated with PCL and/or PEG chains cross-linked by silsesquioxane cages. As a consequence, a four-phased nanostructured morphology was proposed. In this model, PCL and PEG are undistinguishable, while polysilsesquioxane nanoparticles are uniformly distributed throughout a homogeneous cross-linked matrix, which shows gel-like behavior. Moreover, a mobile phase made up of unbound polymer chains occurs at the grain interface.

Amphiphilic Nucleating Agents to Enhance Calcium Phosphate Growth on Polymeric Surfaces.

Poly(ε-caprolactone) (PCL) is an aliphatic polyester widely explored in the preparation of guided bone regeneration (GBR) membranes because of its interesting mechanical properties and biodegradability. However, PCL high hydrophobicity often impairs cell adhesion and proliferation as well as calcium phosphate growth, all of which are crucial to achieving suitable bone-tissue integration. In this work, aimed at achieving less-hydrophobic surfaces, amphiphilic molecules were added at low concentrations to the polymeric dope solutions that generated the GBR membranes. During membrane formation, these molecules migrate to the solution/air interface in such a way that, upon liquid-solid phase transition, the negatively charged heads are exposed while the apolar tails are anchored to the polymer bulk. As a consequence, these molecules became nucleating agents for subsequent calcium phosphate growth using an alternating soaking process. Herein, PCL porous membranes containing different amphiphilic molecules, such as stearic acid and bis(2-ethylhexyl) phosphate, were investigated. This new, simple, and atoxic method to superficially treat polymeric membranes could be extended to a wide range of polymers and applications.