Marcelo Ganzarolli de Oliveira

Thermal and photochemical nitric oxide release from S-nitrosothiols incorporated in Pluronic F127 gel: potential uses for local and controlled nitric oxide release

The local delivery of nitric oxide (nitrogen monoxide, NO) by thermal or photochemical means to target cells or organs has a great potential in several biomedical applications, especially if the NO donors are incorporated into non-toxic viscous matrices. In this work, we have shown that the NO donors S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylcysteine (SNAC) can be incorporated into F127 hydrogels, from where NO can be released thermally or photochemically (with lambda(irr)>480nm). High sensitivity differential scanning calorimetry (HSDSC) and a new spectrophotometric method, were used to characterize the micellization and the reversal thermal gelation processes of the F127 hydrogels containing NO donors, and to modulate the gelation temperatures to the range 29-32 degrees C. Spectral monitoring of the S-NO bond cleavage showed that the initial rates of thermal and photochemical NO release (ranging from 2 to 45 micromoll(-1)min(-1)) are decreased in the hydrogel matrices, relative to those obtained in aqueous solutions. This stabilization effect was assigned to a cage recombination mechanism and offers an additional advantage for the storage and handling of S-nitrosothiols. These results indicate that F127 hydrogels might be used for the thermal and photochemical delivery of NO from S-nitrosothiols to target areas in biomedical applications.

Antibacterial Nitric Oxide‐Releasing Polyester for the Coating of Blood‐Contacting Artificial Materials

The emergence of multidrug-resistant bacteria associated with blood-contacting artificial materials is a growing health problem, which demands new approaches in the field of biomaterials research. In this study, a poly(sulfhydrylated polyester) (PSPE) was synthesized by the polyesterification reaction of mercaptosuccinic acid with 3-mercapto-1,2-propanediol and blended with poly(methyl methacrylate) (PMMA) from solution, leading to solid PSPE/PMMA films, with three different PSPE : PMMMA mass ratios. These films were subsequently S-nitrosated through the immersion in acidified nitrite solution, yielding poly(nitrosated)polyester/PMMA (PNPE/PMMA) films. A polyurethane intravascular catheter coated with PNPE/PMMA was shown to release nitric oxide (NO) in phosphate buffered saline solution (pH 7.4) at 37 degrees C at rates of 4.6 nmol/cm(2)/h in the first 6 h and 0.8 nmol/cm(2)/h in the next 12 h. When used to coat the bottom of culture plates, NO released from these films exerted a potent dose- and time-dependent antimicrobial activity against Staphylococcus aureus and a multidrug-resistant Pseudomonas aeruginosa strains. This antibacterial effect of PSPE/PMMA films opens a new perspective for the coating of blood-contacting artificial materials, for avoiding their colonization with highly resistant bacteria.

Nitric oxide donor improves healing if applied on inflammatory and proliferative phase

BACKGROUND Nitric oxide (NO) is an important molecule synthesized during wound repair. Studies have reported the use of NO donors on cutaneous wound repair, but their effects in different phases of healing are still not elucidated. The aim of this work was to investigate the effects of topical application of a NO donor (S-nitrosoglutathione, GSNO)-containing hydrogel on excisional wounds in the inflammatory ((inf)), proliferative ((prol)), and inflammatory and proliferative phases ((inf+prol)) of rat cutaneous wound repair. MATERIAL AND METHODS In each group (control, GSNO(inf), GSNO(prol), and GSNO(inf+prol)), excisional wounds on the dorsal surface were made and wound contraction and re-epithelialization were evaluated. Fourteen days after wounding, wounds and adjacent skin were formalin-fixed and paraffin-embedded. Collagen fibers organization, mast cells, myofibroblasts and vessels were evaluated. RESULTS Wound contraction of the GSNO(inf+prol) group was faster than control, GSNO(inf), and GSNO(prol) groups, 5 and 7 d after wounding. Topical application of GSNO accelerated re-epithelialization 14 d after wounding, mainly in GSNO(inf+prol) group. In addition, the GSNO(inf+prol) group showed improved collagen fibers maturation and tissue organization, and lower amount of inflammatory cells in the superficial and deep areas of the granulation tissue, compared with the other groups. CONCLUSIONS NO is important in all phases of rat cutaneous wound repair, but if applied on inflammatory and proliferative phases, the improvement in wound healing (accelerating wound closure, wound re-epithelialization, and granulation tissue organization) is more impressive.

Characterization of the hypotensive effect of S-nitroso-N-acetylcysteine in normotensive and hypertensive conscious rats.

S-Nitrosothiols (RSNOs) are potent vasodilators found naturally in vivo. A variety of synthetic RSNOs have been considered as potential nitric oxide (NO) donors for biomedical applications. We have characterized the hypotensive effect of the RSNO S-nitroso-N-acetylcysteine (SNAC) in normotensive and hypertensive conscious rats. SNAC reduced the medium arterial pressure in a dose-response manner in both normotensive and hypertensive animals. At the same doses (EC(50) of SNAC), SNAC showed a vasodilator effect in normotensive rats more potent and more prolonged than that of sodium nitroprusside (SNP). The hypotensive effect of SNAC was also more potent in methylene blue-treated rats, where the cGMP-dependent pathway had been blockaded. These data indicate that SNAC acts by both cGMP-dependent and cGMP-independent pathways. It was also shown that the thiol N-acetylcysteine (NAC) potentiates the action of SNP in hypertensive rats, pointing to the mediation of thiols in the vasodilator action of SNP in this condition. Such mediation may involve the formation of a more potent thiol complex with the nitroprusside anion or the transfer of NO to NAC, generating SNAC as a primary vasoactive species. The kinetic monitoring of the decomposition reactions of SNAC and SNP showed that both compounds are quite stable under the infusion conditions used. Therefore, their vasodilator action cannot be assigned to their breakdown with release of free NO in solution. As the two compounds are unlikely to cross the plasmalemma of smooth muscle cells, their actions are probably associated with the mediation of endogenous thiols in transnitrosation reactions.

Polyethylene Glycol Matrix Reduces the Rates of Photochemical and Thermal Release of Nitric Oxide from S-nitroso-N-acetylcysteine

Abstract S-nitrosothiols have many biological activities and may act as nitric oxide (NO) carriers and donors, prolonging NO half-life in vivo. In spite of their great potential as therapeutic agents, most S-nitrosothiols are too unstable to isolate. We have shown that the S-nitroso adduct of N-acetylcysteine (SNAC) can be synthesized directly in aqueous and polyethylene glycol (PEG) 400 matrix by using a reactive gaseous (NO/O2) mixture. Spectral monitoring of the S–N bond cleavage showed that SNAC, synthesized by this method, is relatively stable in nonbuf-fered aqueous solution at 25°C in the dark and that its stability is greatly increased in PEG matrix, resulting in a 28-fold decrease in its initial rate of thermal decomposition. Irradiation with UV light (λ = 333 nm) accelerated the rate of decomposition of SNAC to NO in both matrices, indicating that SNAC may find use for the photogeneration of NO. The quantum yield for SNAC decomposition decreased from 0.65 ± 0.15 in aqueous solution to 0.047 ± 0.005 in PEG 400 matrix. This increased stability in PEG matrix was assigned to a cage effect promoted by the PEG microenvironment that increases the rate of geminated radical pair recombination in the homolytic S–N bond cleavage process. This effect allowed for the storage of SNAC in PEG at −20°C in the dark for more than 10 weeks with negligible decomposition. Such stabilization may represent a viable option for the synthesis, storage and handling of S-nitrosothiol solutions for biomedical applications.

Antithrombogenic Polynitrosated Polyester/Poly(methyl methacrylate) Blend for the Coating of Blood‐Contacting Surfaces

A nitric oxide (NO) donor polyester containing multiple S-nitrosothiol (S-NO) groups covalently attached to the polymer backbone was synthesized through the esterification of poly(ethylene glycol) with mercaptosuccinic acid, followed by the nitrosation of the -SH moieties. The polynitrosated polyester (PNPE) obtained was blended with poly(methyl methacrylate) (PMMA), yielding solid films capable of releasing NO. Scanning electron microscopy analysis showed that acrylic plates and stainless steel intracoronary stents can be coated with continuous and adherent PNPE/PMMA films. After an initial NO burst, these films release NO spontaneously in dry condition or immersed in aqueous solution at constant rates of 1.8 and 180 nmol/g/h, respectively, for more than 24 h at physiological temperature. PNPE/PMMA coated surfaces were shown to inhibit platelet adhesion when in contact with whole blood. These results show that PNPE/PMMA blend can be used for the coating of blood-contacting surfaces, with potential to inhibit thrombosis and restenosis after stenting.

Prevention and Reversion of Nonalcoholic Steatohepatitis in OB/OB Mice by S-Nitroso-N-Acetylcysteine Treatment

Objective: To evaluate the role oral administration of S-nitroso-N-acetylcysteine (SNAC), a NO donor drug, in the prevention and reversion of NASH in two different animal models. Methods: NASH was induced in male ob/ob mice by methionine-choline deficient (MCD) and high-fat (H) diets. Two animal groups received or not SNAC orally for four weeks since the beginning of the treatment. Two other groups were submitted to MCD and H diets for 60 days receiving SNAC only from the 31st to the 60th day. Results: SNAC administration inhibited the development of NASH in all groups, leading to a marked decrease in macro and microvacuolar steatosis and in hepatic lipid peroxidation in the MCD group. SNAC treatment reversed the development of NASH in animals treated for 60 days with MCD or H diets, which received SNAC only from the 31st to the 60th day. Conclusions: Oral administration of SNAC markedly inhibited and reversed NASH induced by MCD and H diets in ob/ob mice.

Nitric oxide donor superparamagnetic iron oxide nanoparticles

This work reports a new strategy for delivering nitric oxide (NO), based on magnetic nanoparticles (MNPs), with great potential for biomedical applications. Water-soluble magnetic nanoparticles were prepared through a co-precipitation method by using ferrous and ferric chlorides in acidic solution, followed by a mercaptosuccinic acid (MSA) coating. The thiolated nanoparticles (SH-NPs) were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), and vibrating sample magnetometry (VSM). The results showed that the SH-NPs have a mean diameter of 10nm and display superparamagnetic behavior at room temperature. Free thiol groups on the magnetite surface were nitrosated through the addition of an acidified nitrite solution, yielding nitrosated magnetic nanoparticles (SNO-NPs). The amount of NO covalently bound to the nanoparticles surface was evaluated by chemiluminescense. The SNO-NPs spontaneously released NO in aqueous solution at levels required for biomedical applications. This new magnetic NO-delivery vehicle has a great potential to generate desired amounts of NO directed to the target location.

Combined nitric oxide-releasing poly(vinyl alcohol) film/F127 hydrogel for accelerating wound healing.

Nitric oxide (NO) releasing biomaterials represent a potential strategy for use as active wound dressings capable of accelerating wound healing. Topical NO-releasing poly(vinyl alcohol) (PVA) films and Pluronic F127 hydrogels (F127) have already exhibited effective skin vasodilation and wound healing actions. In this study, we functionalized PVA films with SNO groups via esterification with a mixture of mercaptosucinic acid (MSA) and thiolactic acid (TLA) followed by S-nitrosation of the SH moieties. These films were combined with an underlying layer of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), i.e., PEO-PPO-PEO (Pluronic F127) hydrogel and used for the topical treatment of skin lesions in an animal model. The mixed esterification of PVA with MSA and TLA led to chemically crosslinked PVA-SNO films with a high swelling capacity capable of spontaneously releasing NO. Real time NO-release measurements revealed that the hydrogel layer reduces the initial NO burst from the PVA-SNO films. We demonstrate that the combination of PVA-SNO films with F127 hydrogel accelerates wound contraction, decreases wound gap and cellular density and accelerates the inflammatory phase of the lesion. These results were reflected in an increase in myofibroblastic differentiation and collagen type III expression in the cicatricial tissue. Therefore, PVA-SNO films combined with F127 hydrogel may represent a new approach for active wound dressings capable of accelerating wound healing.

Poly(vinyl alcohol) films for topical delivery of S-nitrosoglutathione: Effect of freezing–thawing on the diffusion properties

Poly(vinyl alcohol) (PVA) is a biocompatible polymer already used in several pharmaceutical products. The purpose of this work was to investigate the influence of freezing-thawing cycles (F/T) on the in vitro diffusion and skin vasodilator properties of S-nitrosoglutathione (GSNO)-releasing PVA films. Films subjected to 1-, 3-, and 5-F/T showed an increase in crystallinity, which is associated with an increase in the radius of gyration of macropores from 155 to 180 nm. Diffusion coefficients (D) of GSNO decreased from 5.7 x 10(-7) to 2.0 x 10(-7) cm(2) s(-1) in 1 and 3 F/T films, respectively, and were inversely correlated with the increase in crystallinity, whereas 5-F/T films showed an anomalous increase in D (5.0 x 10(-7) cm(2) s(-1)). Topical release of GSNO from PVA films on the skin of healthy volunteers led to local vasodilation measured by laser Doppler flowmetry. A higher increase in local blood flow was observed for 5-F/T films reaching maximum tissue perfusion at 45 min with return toward basal level after 45 min, whereas 1-F/T films led to a lower increase in blood flow up to 98 min. These results show that F/T treatment can be used to modulate the diffusion properties and the topical vasodilator profile of GSNO-containing PVA films, what might allow the use of these materials as dermal wound dressings or for promoting local vasodilation in ischemic tissues.