Polynitrosated Polyesters: Preparation, Characterization, and

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Polynitrosated Polyesters: Preparation, Characterization, and Potential Use for Topical Nitric Oxide Release Amedea B. Seabra, Regiane da Silva, and Marcelo G. de Oliveira* Instituto de Quı´mica, Universidade Estadual de Campinas, UNICAMP, CP 6154, CEP 13083-970 Campinas, SP, Brazil Received March 22, 2005; Revised Manuscript Received June 16, 2005

New nitric oxide (NO) donor macromolecules, containing multiple S-nitrosothiol (S-NO) groups covalently attached to the polymer backbone, were prepared through the polycondensation reaction of diols (ethylene glycol and poly(ethylene glycol)) with mercaptosuccinic acid, followed by the S-nitrosation of the SH groups by a gaseous NO/O2 mixture. The polynitrosated polyesters (PNPEs) obtained were characterized by IR spectroscopy and gel permeation chromatography and displayed biological activity as vasodilators, leading to local hyperaemia when applied topically on healthy skin. Kinetic measurements in either dry or aqueous conditions have shown that PNPEs can provide sustained NO release for more than 20 h at physiological temperature. Their increased viscosity at low temperatures greatly reduces the rate of NO release, allowing for their storage for more than 90 days at -20 °C without decomposition. These results indicate that PNPEs have potential for topical delivery of NO in biomedical applications. Introduction Nitric oxide (NO) is a key endogenous molecule, which mediates a wide range of physiological and pathophysiological processes.1-4 Some of the specific biological roles of NO, like the promotion of vasodilation through the activation of soluble guanylate cyclase, the inhibition of platelet aggregation and thrombosis, and its cytotoxic actions against invading bacteria and protozoans, have driven much attention to the development of NO-delivering biomaterials for topical applications and for the coating of biomedical devices.5-8 The role of NO in wound healing has led to the proposal of topical applications of NO donors for the treatment of chronic leg ulceration and wound infections in patients with diabetes and peripheral vascular disease.9-11 In such cases, the local release of NO can be beneficial to ischemic tissues by augmenting blood flow and by stimulating angiogenesis. In addition, NO can exert local cytotoxic effects against infective microorganisms. The antimicrobial action of NO has also led to topical applications of NO donors for treating infectious skin diseases such as cutaneous leishmaniosis with promising results.12-14 Several polymers, including poly(vinyl chloride), polyurethane, poly(dimethylsiloxane), and polymethacrylate containing NO-releasing N-diazeniumdiolates and other NO donors, have already been developed for the coating of medical devices and have proven to be effective in preventing bacterial adhesion and platelet aggregation and in promoting endothelial cell growth.15-21 In addition to N-diazeniumdiolates, a range of S-nitrosothiols (RSNOs) can also be used as NO-donor molecules. Some RSNOs, like S-nitrosoglutathione (GSNO) and Snitrosoalbumin, have already been identified as endogenous NO carriers in mammals.22,23 In such species, NO is covalently bound to a sulfur atom in a C-S-NO moiety * E-mail: [email protected].

and can be released through the homolytic or heterolytic S-N bond cleavage, allowing NO to be transferred to specific receptors such as iron-containing enzymes or thiolcontaining proteins.24-26 Similarly to N-diazeniumdiolates, RSNOs can also be incorporated into polymeric matrices or covalently bound to the backbone of some polymers. Bohl and West,27 for example, have prepared hydrogels of poly(ethylene glycol) with covalently bound S-nitrosocysteine and used such materials for a sustained and localized NO production, in the prevention of thrombosis. In a similar approach, Frost and Meyerhoff17,28 have attached S-nitrosoN-acetylpenicillamine (SNAP), S-nitrosocysteine (NO-Cys), and S-nitroso-N-acetylcysteine (SNAC) to fumed silica particles, obtaining a solid material capable of releasing NO. We have already reported that hydrogels and liquid and solid polymeric matrices can be used as vehicles for carrying dissolved hydrophilic RSNOs. In addition to allowing the controlled diffusion of both entire RSNO molecules and free NO to tissues, these matrices also exert stabilization effects on the RSNOs, increasing their half-lives.6,29-32 In this work, a new approach was used to obtain hydrophobic macromolecules with SNO groups covalently attached to the polymer backbone, which are capable of releasing only free NO in topical applications. The macromolecules obtained are polynitrosated polyesters, which were synthesized through the polycondensation reaction of diols (ethylene glycol and poly(ethylene glycol)) with the sulfhydryl (-SH)-containing mercaptosuccinic acid. Their NO releasing properties and potential uses in biomedical applications are presented and discussed. Materials and Methods Materials. Poly(ethylene glycol) (PEG) (average Mn ca. 300), ethylene glycol, mercaptosuccinic acid (Aldrich Chemical Co., Inc., U.S.A.), HCl (Synth, Brazil), tetrahydrofuran

10.1021/bm050216z CCC: $30.25 © 2005 American Chemical Society Published on Web 07/21/2005

Polynitrosated Polyesters for Topical NO Release

(THF HPLC Te´dica Company, Brazil), N-(1-naphythyl)ethylenediamine dihydrochloride (NEED), and sulfanilamide (SULF) (Merk, Germany) were used as received. Gaseous NO was obtained from a gas cylinder (White Martins, SP, Brazil). Synthetic air (N2/O2, 79/21 v/v, H2O < 2 ppm, THC, CO + CO2 < 0.3 ppm) was purchased from Air Liquide, SP, Brazil. All solvents were of analytical grade. Polysulfhydrylated Polyesters Synthesis. The polysulfhydrylated polyesters were synthesized through the general reaction of polycondensation of a diol with a dicarboxylic acid according to methods describe elsewere.33,34 The diols used in this work were either PEG or ethylene glycol, and the dicarboxylic acid employed was mercaptosuccinic acid. In brief, the diols (PEG or ethylene glycol), mercaptosuccinic acid and HCl, as a catalyst, were placed in a three-necked round-bottomed flask (100 mL) fitted with a Vigreaux fractionation condenser, a mechanical stirrer operated at approximately 300 rpm, and a nitrogen gas inlet for bubbling nitrogen during the reaction time course, to facilitate the removal of excess of water and oxygen from the system. The temperature of the reactants was maintained at 120 °C by a using a silicone oil bath, and the reactions were carried out under reflux for 22 h. For polyesterification using PEG as the diol, a 1:1 molar ratio of PEG/mercaptosuccinic acid was prepared by adding 3.6 mL of PEG and 2.0 g of mercaptosuccinic acid (corresponding to 13.3 mmol of each reactant) to the reaction flask, followed by the addition of 32 µL of 12 M HCl. For the polyesterification reaction with ethylene glycol as the diol, a 1:1 molar ratio of ethylene glycol/mercaptosuccinic acid was prepared by adding 745 µL of ethylene glycol and 2.0 g of mercaptosuccinic acid (corresponding to 13.3 mmol of each reactant) to the reaction flask, followed by the addition of 32 µL of 12 M HCl. The crude ethylene glycol mercaptosuccinate (polyester I) and PEG mercaptosuccinate (polyester II) were precipitated in cold water in an ice bath for purification. The precipitated polyesters I and II were transferred to open vials and dried in a freeze-drier for 24 h. Freeze-drying in this condition only removes the excess water from the precitation in cold water, but does not remove the water formed in the bulk of the materials in the polycondensation reactions (see below). The final products were obtained as viscous liquids at room temperature. Infrared Measurements. Infrared spectra of the polysulfhydrylated polyesters and of the reactant mixtures (without the addition of HCl and prior to the reflux at 120 °C for 22 h) were recorded with a Fourier transform (FT)IR (Bomem B-100, Hartmann & Braun, CA-USA) spectrophotometer at room temperature. Liquid samples were poured as a film onto the surface of calcium fluoride (CaF2) windows, freezedried for 24 h, and analyzed in the range 4000-1000 cm-1. Molar Mass Measurements. The molar masses of the two polyesters synthesized were calculated by gel permeation chromatography (GPC) using a GPC Waters 510 instrument equipped with a refractive index detector (Waters 410) and gel columns (AM 1.000 A; 5 µm) (American Polymer Standards Corporation, EUA). THF was used as the mobile phase at a flow rate of 1 mL min-1. Measurements were made at 40 °C, and commercial polystyrene standards with

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low polydispersity indices were employed for calibration. The number-average (Mn) and weight-average (Mw) molecular weights and the polydispersity indexes (PDI) were calculated using the calibration curve. S-nitrosation of Polysulfhydrylated Polyesters. The nitrosation of the -SH moieties of the synthesized polysulfhydrylated polyesters, yielding polynitrosated polyesters (PNPEs), was achieved by bubbling a mixture of NO/ synthetic air through the liquid polyester in a quartz spectrophotometer cuvette, under stirring. The gaseous mixture was prepared by mixing NO from a gas cylinder with O2 from a cylinder of synthetic air. The gas flows were controlled by flow meters (Aldrich) to give a mixture with known NO/O2 ratio. Preadjusted ratios of NO/O2 of 1:1 v/v (NO/synthetic air 5.6:26.7 mL min-1) were used. The use of synthetic air instead of pure oxygen offers the advantage of a more sensitive control of the reaction course and extent. Reactions were performed in a thermostatized quartz cuvette with a Teflon stopper crossed by four polypropylene tubes, which delivered the gas mixture to the bottom of the cuvette. A solenoid valve (Cole Palmer) controlled by an electronic circuit developed for this purpose was used to produce gas pulses with preadjusted frequency and duration. The reaction was followed spectrophotometrically using a diode array spectrophotometer (Hewlett-Packard, model 8453, Palo Alto, CA). The formation of -SNO groups in the polysulfhydrylated polyesters were monitored at λ ) 336 and λ ) 545 nm, which correspond to maximums of the UV and visible absorption bands of the S-NO bond, respectively. Effects of Temperature and Ambient Light on the Kinetics of NO Release from PNPEs in Dry Conditions. Samples of pure liquid PNPEs were poured into demountable quartz cell cuvettes with optical paths of 0.01 cm. Spectral changes of PNPEs were monitored in the range 220-1100 nm in the dark at 37 °C for 24 h. Kinetic curves of thermal S-NO bond cleavage were obtained from the absorption changes at 545 nm in time intervals of 20 min with the samples referenced against air. Liquid samples of PNPEs, poured in the demountable quartz cell cuvettes, were kept in a room with a controlled temperature maintained at 25 ( 2.0 °C for 24 h, in the dark and under ambient light. The cuvettes containing PNPEs were placed on the bench either protected from the ambient light or irradiated with an ambient light source composed by two sets of two fluorescent lamps (Sylvania Octron6500K, FO 32W/65K, Germany) positioned 2.5 m apart from each other, at a height of 2.0 m above the bench. The irradiated cuvett was placed between the two sets of lamps. The distance between the sample and each set of lamps was 2.1 m, measured at an angle of 45° relative to the normal axis of the sample. The emission spectrum of ambient light provided by the fluorescent lamps was measured with a radiometer (Newport 18030-C Optical Power Meter, Irvine, CA) in the wavelength range 250-1100 nm. The radiometer sensor was placed in the same position where the PNPEs were exposed to ambient light. Spectral changes at 545 nm were monitored at time zero and after 0.5, 2.0, 4.0, 6.0, 9.0, 12, and 24 h. Absorbance changes were not monitored

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continuously in order to minimize the irradiation of the samples by the spectrophotometer lamp. The amount of NO released over time was calculated from the amount of S-NO groups which undergo thermal homolytic cleavage. This calculation was based on the fact that the decay of the absorption band of the S-NO bond at 545 nm is associated solely to the homolytic S-N bond cleavage with release of NO,35 according to eq 1 RS-NO f RS• + •NO

Scheme 1. Esterification Reactions of Ethylene Glycol and Poly(ethylene glycol) with Mercaptosuccinic Acid Yielding Polyesters I and IIa

(1)

where R represents the polymer chain and the dots represent unpaired electrons at the sulfur and nitrogen atoms (used here to emphasize the homolytic bond cleavage). Thus, the increase in the concentration of NO released over time ([NO]t), was calculated from the changes in the concentration of S-NO groups present in the PNPEs ([S-NO]0 [S-NO]t), according to Beer’s law [NO]t ) [S-NO]0 - [S-NO]t ) (A0b/S-NO) - (Atb/S-NO) (2) where A0 and At are the S-NO bond absorbances at 545 nm at the beginning of the monitoring and at time t, respectively, [S-NO]0 and [S-NO]t are the concentrations of S-NO groups at the beginning of the reaction and at time t, respectively, S-NO is the molar absorption coefficient of the S-NO group at 545 nm, measured in a solution of Snitrosomercaptosuccinic acid in ethylene glycol as ) 16 mol-1 L cm-1 (which is the same value found for other RSNOs36), and b is the optical path length (0.01 cm). Therefore, by monitoring the disappearance of the absorption band of the S-NO groups of PNPEs, it was possible to calculate the amount of NO released from PNPEs over time. Initial rates (IR) of NO release of PNPEs were obtained by linear regression of the slopes of the initial sections (less than 10% of the reaction) of the NO concentration versus time plots, according to IR ) ∆[NO]/∆t

(3)

where ∆[NO] and ∆t are the changes in NO concentration and the corresponding time intervals, respectively. The kinetic curves of NO release versus time were fit to a firstorder exponential growth, according to [NO]t ) [NO]f - [NO]f e-kt

(4)

Where, [NO]t is the NO concentration at each time t, [NO]f is the final NO concentration, and k is the first-order rate constant. Each point in the kinetic curves represents the average of two experiments, with the error bars expressed by their standard error of the mean (SEM). Kinetics of NO Release from PNPEs Immersed in Aqueous Medium. The release of NO from PNPEs in aqueous medium was monitored as nitrite (NO2-) formation by using the Griess reaction.37 Typically, 3.0 mg of PNPE was immersed in a quartz cuvette containing Griess solution (SULF 0.1 mol L-1, NEED 0.1 mol L-1, HCl 4.0 mol L-1). The absorbance at 550 nm, which provides a quantitative measurement of NO released to the solution after its

a n refers to the number of monomeric units in the polyester, while m refers to the number of ethylene oxide units in the PEG chain.

conversion to NO2-, was kinetically monitored at 25 and 37 °C for 24 h. A calibration curve based on the absorption at 550 nm was obtained with acidic sodium nitrite solutions in the range 3-100 µmol L-1 (data not shown). Thermal Stability of PNPEs. The thermal stabilities of PNPEs regarding their S-NO bond cleavage with release of NO in the dark were evaluated by storing the PNPEs at temperatures of -20 °C for 90 days and 8 °C for 15 days, protected from light. During these periods of time, the intensity of the absorption band at 545 nm, which is assigned to the S-N bond, was monitored. Topical Application of PNPEs on Human Skin. Drops of liquid PNPEs and of a non-nitrosated polysulfhydrylated polyester (used as control), with ca. 1 cm2 of base area, were applied on the forearms of two of the authors of this work, by dropping the liquid polymers from a spatula. The drops were allowed to stay on the skin for 7 min under ambient light. After this time, the PNPEs were gently removed with tissue paper, and hyperaemia due to local vasodilation produced by the PNPEs was photographed with a digital camera. Further photographs were taken after 10 min to record the hyperaemia disappearance. The authors did not consume caffeine for at least 12 h before the experiments. Results and Discussion Polysulfhydrylated Polyester Synthesis. Scheme 1 shows the polycondensation reactions employed to synthesize polyesters I and II. It must be noted that the repeating units in these polymers have a free thiol group, which can be further S-nitrosated. Polyesterification reactions are known



Table 1. Number-Average Molecular Weight (Mn), Weight-Average Molecular Weight (Mw), and Polydispersity Index (PDI) Values Obtained by GPC for Polysulfhydrylated Polyesters Synthesized by Polycondensation Reactions of Ethylene Glycol (I) and Poly(ethylene glycol) (II) with Mercaptosuccinic Acid

Figure 1. FTIR spectrum of a mixture of poly(ethylene glycol) with mercaptosuccinic acid before the esterification reaction (i) and after the esterification reaction (ii), according to the conditions described in the experimental part.

to be affected by temperature, time, and the presence of acid catalysts. Higher temperatures or longer reaction times favor the yielding of polyesters with higher molar masses.33,38 The first step in the polyesterification reaction is the protolysis of the dicarboxylic acid by the acid catalyst, generating an oxonium ion (RCOOH2+). The bimolecular reaction between the hydroxyl groups (OH) of the glycols and the oxonium ion derived from the acid is considered to be the slow step in the esterification reaction. Thus, the rate-determining step in the esterification process is the nucleophilic attack of the OH group of the diol to the protonated carboxylic group of mercaptosuccinic acid (eq 5)39,40

where R′ is the organic radical of the corresponding diol. In this step, the oxonium ion can be present either as a free ion or as a component of an ion pair.40 Excess water formed in the reaction was removed from the system by the continuous bubbling of N2. Characterization of Polysulfhydrylated Polyesters. Polyesters I and II were characterized by FTIR and GPC, following procedures already described in the literature.41-43 Figure 1 shows the FTIR spectra of a mixture of PEG and mercaptosuccinic acid before the polyesterification reaction (i) and the final polyester II obtained after 22 h of reflux at 120 °C in the presence of the acid catalyst (ii). Figure 1i shows the presence of the characteristic carbonyl stretching vibration band at ν ) 1714 cm-1, assigned to the carboxylic group of mercaptosuccinic acid. Figure 1ii shows that, after the reaction, the carbonyl stretching vibration band was shifted to ν ) 1735 cm-1, which is the expected wavenumber for the carbonyl stretching vibration band of polyesters.38,40,41 This result confirms the polyesterification reaction between the hydroxyl groups of PEG and mercaptosuccinic acid. A similar spectral change (not shown) was observed in the polyesterification reaction using ethylene glycol. The formation of polyesters I and II was also confirmed by GPC analysis of the purified products. Table 1 shows

polyester

Mn/g mol-1

Mw/g mol-1

PDI

I II

66 063 74 404

74 612 89 159

1.1 1.2

the values of Mn, Mw, and PDI for polyesters I and II. These results show that relatively high molecular weight polyesters were obtained with low polydispersity. The Mn value for polyester II was found to be higher than that obtained for polyester I. The nominal Mn values found indicate that polyester II (Mn 74 404) has ca. 165 PEG 300mercaptosuccinate units per molecule, while polyester I (Mn 66 063) has ca. 311 ethylene glycol-mercaptosuccinate units per molecule. The higher Mn value obtained for polyester II can be assigned to the molar mass of PEG 300, which is 4.8-fold higher than the molar mass of ethylene glycol. On the other hand, the lower amount of glycol-mercaptosuccinate units formed per molecule with PEG 300 is probably a result of the higher viscosity of the reaction medium in this case. The increased viscosity of PEG 300, relative to ethylene glycol, lowers the diffusion of the reactants. In a bimolecular process such as the esterification reaction, a decrease in the diffusion rates implies a reduction in the reaction rate, leading to a lower extent of reaction and consequently to lower glycol-mercaptosuccinate units per molecule. Although polyester II has an ethylene oxide group (-OCH2-CH2-) in the place of the ethylene group (-CH2CH2-) of polyester I, both polymers have similar physical and chemical properties regarding their solubility (both are insoluble in water) viscosity, reactivity against NO/O2, and NO releasing properties. S-nitrosation of Polysulfhydrylated Polyesters. The nitrosation of the -SH groups of polyesters I and II lead to polynitrosated polyesters (PNPEs I and II, respectively) with -SNO groups capable of releasing free NO, like other S-nitrosothiols. In the gas mixture of NO/synthetic air bubbled through the liquid polyesters I and II, NO is oxidized by molecular oxygen to the brown gas nitrogen dioxide (•NO2).44 (eq 6). 2NO• + O2 f 2•NO2

(6)

•

NO2 can share the unpaired electron with another NO• molecule, yielding dinitrogen trioxide (N2O3) (eq 7). 2NO• + •NO2 f N2O3

(7)

N2O3 is capable of nitrosating thiol (SH) groups according to the reaction represented in Scheme 2. Nitrite (NO2-) formed in Scheme 2 hydrolyzes to nitrous acid (HONO) in the remaining water formed in the polycondensation reactions (eq 5). Nitrous acid is a very effective nitrosating agent, which exists in equilibrium with its chargeseparated structure (OH-- - -NO+) (eq 8). NO2- + H+ ) HO- -NO ) OH-- - -NO+

(8)

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Scheme 2. S-nitrosation Reaction of Polysulfhydrylated Polyester II, Yielding Polynitrosated Polyester II (PNPE II)a

a

n refers to the number of monomeric units in the polyester chain, while m refers to the number of ethylene oxide units in the PEG chain.

Figure 2. UV-vis spectra of polysulfhydrylated polyester I before its S-nitrosation reaction (i) and after the S-nitrosation reaction, showing the absorption bands assigned to the π f π* (336 nm) (ii) and nN f π* (545 nm) (iii) electronic transitions of the S-NO group.

S-nitrosation of the remaining -SH groups of the polysulfhydrylated proceeds through nucleophilic attack of the nitrosonium cation (NO+) to the sulfur atom of the thiol group, yielding a further PNPE molecule and water (eq 9) OH-- - -NO+ + RSH ) RSNO + H2O

(9)

where R represents the PNPE chain. This secondary Snitrosation process happens in the bulk of the liquid PNPE, where water is formed in a homogeneous medium. However, polysulfhydrylated polyesters cannot be efficiently nitrosated by the usual method, based on acidified sodium nitrite (NaNO2) solution, for they are insoluble in water, and in this case, S-nitrosation will occur only in the interface between the liquid polysulfhydrylated polyesters and the aqueous nitrous acid solution. Therefore, the method used in this work emerges as a very appropriate technique for the S-nitrosation of water-insoluble liquid substrates as well as for the S-nitrosation of thiols dissolved in water-free matrices. The homogeneity of the S-nitrosation reaction is assured by the rapid diffusion of the gas and by the continuous stirring of the liquid.45,32 This technique allowed accurate control of the nitrosation reaction through the monitoring of the two characteristic absorption bands of the S-NO group at 336 and 545 nm, assigned to π f π* and nN f π* electronic transitions, respectively.45 These bands are shown in Figure 2 (curves ii and iii, respectively) for PNPE II, selected as an example. Figure 2

Figure 3. Kinetic curves showing the total amount of NO released from PNPE II in dry condition over 24 h: (i) in the dark at 37 °C, (ii) under room light at 25 °C, (iii) in the dark at 25 °C.

also shows the UV-vis spectra of a polysulfhydrylated polyester (curve i) as a control. The absorption bands at 336 and 545 nm were also used to monitor the release of free NO from the PNPEs (see below), which are completely bleached after full decomposition. The S-nitrosation reaction can be conducted until its completion by monitoring the 336 and 545 nm bands until the achievement of a plateau. In the present case, the reaction was deliberately stopped before its completion in order to avoid a fast S-NO decomposition of full S-nitrosated polyesters, due to the autocatalytic effect present in Snitrosothiols.45 The extent of the S-nitrosation reaction after stopping the bubbling in this work was estimated as ca. 20%. PNPEs 100% S-nitrosated release NO at a much higher rate (data not shown) leading to high local concentrations and could not find medical applications. The maximal NO loading, obtained in 100% S-nitrosation, can be calculated on the basis of the molecular weights of the polyesters (Table 1) as 4.6 × 103 µmol/g (PNPE) for PNPE I and 2.2 × 103 µmol/g (PNPE) for PNPE II. Effects of Temperature and Ambient Light on the Kinetics of NO Release from PNPEs in Dry Conditions. Pure liquid PNPEs I and II were observed to release NO spontaneously when kept in an open cuvette in the dark at 37 °C, in dry conditions. In both cases, the NO release reaction was followed by monitoring the disappearance of the absorption band at 545 nm. The homolytic cleavage of the S-N bond associated with this band obeys first-order kinetics, according to eq 10. The fate of the thiyl radicals (RS•) in this reaction is dimerization, forming a sulfur bridge (eq 11) leading therefore to a cross-linked decomposition product. RSNO f RS• + NO• •

•

RS + RS f RSSR

(10) (11)

Figure 3i shows a representative kinetic curve of NO release, obtained for PNPE II in a 24 h period. The amount of NO released in the ordinate axis was calculated according to eq 2. The first-order rate constant (k) of NO release, calculated from eq 4, was found to be 9.0 × 10-2 ( 1.0 × 10 -3 h-1 corresponding to a half-life of 7.7 h, at 37 °C. A similar half-life was obtained from the kinetic curve of PNPE I (data not shown).


The corresponding initial rate of NO release is 5.2 mmol L-1 h-1 (or 5.2 µmol mL-1 h-1) at this temperature. The total amount of NO released after 24 h (ca. 45 µmol mL-1) allows one to estimate that a layer of PNPE 1 mm thick with a base area of 1 cm2 (i.e., of 0.1 mL) applied onto the skin would release a total amount of ca. 4.5 µmol of NO in 24 h. Although about one-half of the NO released in such application can be expected to escape through the PNPE/air interface, a total tissue concentration of ca. 2.3 µmol mL-1 (or 2.3 mmol L-1) could be achieved in the tissue below an application area of 1 cm2. Even if we consider that NO will be continuously scavenged after penetrating through the epidermis, this estimate represents a very high local NO concentration, compared to the minimal NO concentration required to stimulate the synthesis of cyclic guanosine monophosphate in vascular smooth muscle cells,46,47 which is ca. 10 nmol L-1. At the nanomolar level, NO usually has no cytotoxic effects;48 however, at concentrations 10-100 times normal levels (i.e., 0.1-1 µmol L-1), continuous administration of NO can lead to both acute and chronic cell damage through different mechanisms.49-52 Thus, the measured rates of NO released from the PNPEs imply that exposure of tissues to such materials for a few minutes could produce substantial toxicity. Of course, in applications aimed at increasing local vasodilation for recovering ischemic tissues or for enhancing the transdermal absorption of other drugs, the PNPEs would need to be diluted in an inert matrix to meet the desired doses. Such dilution could be easily achieved by mixing PNPEs with nonpolar vehicles already used in pharmaceutical formulations. Alternatively, the amount of available NO per volume of PNPE can be controlled by controlling the extent of the S-nitrosation reaction in the PNPE synthesis. On the other hand, in applications aimed at killing bacteria or leishmania protozoans, for instance, the high local NO concentrations provided by PNPEs can be a necessary condition. Curves ii and iii of Figure 3 show the kinetics of NO release from PNPEs kept in a room with controlled temperature maintained at 25 °C for 24 h, under irradiation with ambient light, and in the dark, respectively. By comparing curves i and iii, it can be seen that the rate of NO release increases 4 times with temperature, in accordance with a thermal decomposition process.31 The initial rates of NO release from PNPE II at 25 °C under exposure to ambient light and kept in the dark were found to be ca. 1.3 µmol mL-1 h-1 in the first 4 h. A close inspection of the error bars of curves ii and iii allows one to say that the effect of ambient light on the kinetics of NO release from PNPE II is nonsignificant in the first 6 h. Therefore, there must be no important effects of ambient light on the vasodilation action of PNPEs in topical applications, in this period. The intensities of ambient light, measured by the radiometer, were 95 and 38 nW/cm-2 at 336 and 545 nm, respectively. These low values of light intensity explain the negligible effect of ambient light on the photochemical S-N bond cleavage. It has already been shown31 that a light intensity of 33 nW/cm-2 is not capable of promoting an effective GSNO photodecomposition in either aqueous solution or poly(ethylene glycol) matrix. Significant photo-


Figure 4. Kinetic curves showing the increase in NO concentration, due to the release of NO from PNPE II immersed in a Griess solution at (i) 25 °C, (ii) 37 °C. The formation of the chromophoric azoderivative in the solution was monitored at λ ) 550 nm. Inset (A): detail of the sections of the kinetic curves in the first 2 h; (B) detail of the sections of the kinetic curves from 2 to 24 h.

chemical effects on the S-NO cleavage were only achieved by irradiation of GSNO with light intensities in the µW/ cm-2 range.31 NO Release from PNPEs Immersed in Aqueous Medium. Figure 4 shows the kinetic curves monitored at λ ) 550 nm, associated with the release of free NO from 3.0 mg of PNPEs immersed in aerated acidic aqueous solution containing Griess reactants, over a period of 24 h: (i) at 25 °C, (ii) at 37 °C. In this condition, NO released undergoes a very fast reaction with dissolved O2, forming NO2. The subsequent hydrolysis of NO2 in acidic conditions leads to the formation of nitrous acid, which, in turn, S-nitrosates sulfanilic acid of the Griess solution, forming a diazonium ion which couples to NEED, leading to the formation of a chromophoric azo-derivative detected through its absorbance band at 550 nm. The molar amount of the azo-derivative formed is equal to the molar amount of NO released from PNPEs to the solution. The Griess method is widely used to quantitatively measure nitrite (or NO after its conversion to nitrite).37 In the present case, this method was used to continually follow the kinetics of NO release from PNPEs in an aqueous medium. Figure 4 shows that there is an initial burst of NO in the first 2 h. After this time, an apparent plateau is reached in both temperatures. Inset A of Figure 4 shows the kinetic curves of NO release in Griess solution in the first 2 h at (i) 25 °C and (ii) 37 °C. The initial rate of NO release at 37 °C (475 µmol g-1 h-1) was 2.3-fold higher than at 25 °C (210 µmol g-1 h-1). It must be noted that the plateaus in Figure 4 are reached much more rapidly than in Figure 3. Consequently, the difference between the initial rates of NO release in this case is attenuated. The longer times for reaching the plateaus in Figure 3 can be assigned to a higher recombination rate between NO and thiyl radicals (reversal of eq 10) in dry condition, where the PNPE samples are enclosed between the quartz plates of the detachable cuvett. In aqueous solution, NO reaching the PNPE/water interface escapes to the solution, increasing the initial rates of NO release. In inset B of Figure 4, it can be seen that the conversion of the Griess reactants is still in progress in the period 2-24

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Figure 5. Topical application of non-nitrosated polyester II and of polynitrosated polyester II (PNPE II) on the forearm skin. (A) Non-nitrosated polyester II applied as a control. (B) Control (left drop) and PNPE II (right drop) immediately after application of the latter. (C) Hyperaemia detected after the removal of the PNPE II drop, which remained on the skin for 7 min. (D) Appearance of the application areas 3 min after the removal of the control and PNPE II drops, showing the disappearance of the hyperaemia detected in picture C. The black lines are reference marks drawn on the skin with a pen.

h after the immersion of the PNPEs in the solution, in both temperatures. This inset highlights the fact that the kinetics of NO release in aqueous solution has a bimodal behavior: in the initial section of the curve (ca. 2 h), there is a burst of NO release. The second section (from ca. 2.0 h to 24 h) corresponds to a slow release process (0.60 µmol g-1 h-1 for both 25 and 37 °C). Although the amount of NO released in the second section of the curve is very small, leading to an increase in the concentration of NO in the solution of ca. 12 µmol of NO released per gram of PNPEs, it may be enough to obtain biological effects. The Griess method also allowed the calculation that 3.0 mg of PNPEs immersed in aqueous solution release 0.8 µmol of NO in 24 h, corresponding to 0.27 mmol of NO released per gram of PNPE. In topical applications, where the PNPEs will be in contact with tissues (especially onto the skin), the rates of NO release can be expected to be lower that the rate measured in the Griess solution. Thermal Stability, Storage, and Formulation of PNPEs. The stabilities of PNPEs were monitored under storage at -20 °C. In this condition, PNPEs have shown negligible decomposition after three months of monitoring. This result shows that such materials are relatively stable under this condition and can be stored for at least several months. Freshly prepared PNPEs can also be stored at normal refrigerator temperature (ca. 8 °C) for at least seven days with negligible decomposition. Although their much lower

thermal stability at room temperature implies difficulty of handling, it must be remembered that this is an inescapable property of any material which releases NO spontaneously. Thus, like many other bioactive compounds, PNPEs must be stored under low temperature, and their shelf lives will not be long. As a possible alternative for a long storage, the precursor polysulfhydrylated polyesters could be S-nitrosated right before use, by bubbling NO/O2 from a gas ampule in an appropriate kit. In this case, the shelf life could be very long, once polysulfhydrylated polyesters are relatively stable. Characterization of the Local Vasodilation in Topical Application of PNPEs on Human Skin. Liquid PNPEs and their corresponding non-nitrosated polysulfhydrylated polyesters were applied topically onto the healthy skin of the forearms of two of the authors of this work. These preliminary tests were aimed at characterizing the local hyperaemia due to the diffusion of free NO across the skin. Figure 5 shows pictures taken in four different moments after the application of a sample of non-nitrosated polyester (left drop) and of PNPE II (right drop) on the forearm of one of the authors, as an example. It can be seen that both polymers are transparent viscous liquids. The colorless non-nitrosated polysulfhydrylated polyesters become deep red after Snitrosation. This color, which is typical of S-nitrosothiols, can be used as a visual confirmation of the S-nitrosation reaction and disappears after the complete release of NO. Figure 5A shows only the drop of the non-nitrosated


polysulfhydrylated polyester applied as a control. Figure 5B shows the drop of PNPE II immediately after its application. The PNPE II drop was allowed to stay on the skin under room light during 7 min. The effect obtained can be visualized in Figure 5C, after the gentle removal of the drop with tissue paper. It can be seen that a local hyperaemia, whose size matches exactly the perimeter of the drop, has developed after this short time. The hyperaemia was observed to disappear completely 3 min after the removal of the PNPEs drop (Figure 5D). At this time, the control drop was also removed with the same technique. It can be seen that the application of the control drop as well as that of the PNPE drop left no observable alterations in the skin after 10 min in contact with the skin. The fast disappearance of the hyperaemia after the removal of the PNPE drop and the absence of further hyperaemia or erythema formation after the removal of the control drop rules out the possibility that the hyperaemia was due to an allergic process. Such preliminary tests show that local hyperaemia develops very rapidly after the application of PNPEs on the skin and that the effect also vanishes rapidly after removing the PNPEs from contact with the skin. This result is in accordance with the expected fast diffusion of NO through tissues, due to its amphiphilic character. It must be emphasized here that non-nitrosated polysulfhydrylated polyesters and PNPEs are not water-soluble and also that, because of their polymeric nature, they are not able to penetrate the skin through either the appendageal or epidermal routes available to small molecules and ions. On the other hand, free NO that reaches the skin/PNPE interface can easily penetrate across the stratum corneum and diffuse across the viable epidermis and dermis reaching the cutaneous microvasculature. As a small amphiphilic molecule, NO can reach the smooth muscle cells through either transcellular or intercellular routes, cross their membranes, and activate guanylate cyclase, leading to local vasodilation and hyperaemia. The fast vasodilation obtained can be understood by considering that the blood supply reaches within 0.2 mm of the skin with a flow rate of 0.05 mL min-1 surface.53 At the same time, the fast disappearance of the local vasodilation effect after the removal of the PNPE drop can be attributed to this same generous blood volume, which also functions as a “sink” with respect to diffusing NO that reaches it during the percutaneous absorption. A similar local vasodilation effect has already been observed in a previous work where hydrogels containing S-nitrosothiols were applied topically on the skin of healthy human subjects. In this case, local vasodilation was characterized by a laser Doppler technique.6 Possible Advantages and Applications. We have recently reported that hydrogels and liquid and solid polymeric matrices can be appropriate vehicles for carrying RSNOs and can offer the advantages of stabilizing the RSNOs and providing their controlled release to tissues.29-32 However, in these cases, applications face two main limitations: The vehicles used can only be loaded with a limited amount of the NO donor, which is defined by its solubility in the matrix. If the NO donor is a water-soluble molecule and is not covalently bound to the polymer backbones of the matrices, diffusion to tissues is usually too fast.29 One approach to


overcome these limitations is to use a polymeric NO donor, which is not able to diffuse to tissues but is capable of releasing only free NO topically. PNPEs fulfill this requirement as they are insoluble in water. In adition, PNPEs can be S-nitrosated to different extents, allowing one to charge the material with potentially available NO in concentrations much higher those achieved by dissolving RSNOs in watersoluble polymers. This last possibility leads to an additional advantage: The non-nitrosated thiol groups remaining in the polymer can interact with cysteine-rich subdomains of mucus glycoproteins via disulfide exchange reactions, improving the adhesive properties of such polymer systems in mucous tissues.54 The ability of PNPEs to provide a sustained release of NO and to promote local vasodilation when applied on the skin indicates that such materials have several potential applications, including the improvement of wound healing, the treatment of chronic leg ulceration and wound infections associated with diabetic subjects or patients with peripheral vascular disease, the treatment of infectious skin diseases such as cutaneous leishmaniosis, and the enhancement of the transdermal absorption of other drugs.55 Evidently, further studies will be needed for evaluating the cytotoxicity of PNPEs and their decomposition products. Acknowledgment. A.B.S. and R.S. hold graduate fellowships from Fundaç aõ de Amparo a` Pesquisa do Estado de Saõ Paulo (FAPESP), projects 01/7869-9 and 03/120240, respectively. The authors thank FAPES for financial support. References and Notes (1) Moncada, S.; Radomski, M. W.; Palmer, R. M. J. Endotheliumderived relaxing factor: identification as nitric oxide and role in control of vascular tone and platelet function. Biochem. Pharmacol. 1988, 37, 2495. (2) Titheradge, M. A. Nitric oxide in septic shock. Biochim. Biophys. Acta 1999, 1411, 437. (3) Stuehr, D. J. Mammalian nitric oxide syntheses. Biochim. Biophys. Acta 1999, 1411, 217. (4) Ricardo, K. F. R.; Shishido, S. M.; de Oliveira, M. G.; Krieger, M. H. Characterization of the hypotensive effect of S-nitroso-Nacetylcysteine in normotensive and hypertensive conscious rats. Nitric Oxide 2002, 7, 57. (5) Ignarro, L. J.; Napoli, C.; Loscalzo, J. Nitric oxide and cardiovascular agents modulating the bioactivity of nitric oxide: An overview. Circ. Res 2002, 90, 21. (6) Seabra, A. B.; Fitzpatrick, A.; Paul, J.; de Oliveira, M. G.; Weller, R. Topically applied S-nitrosothiol-containing hydrogels as experimental and pharmacological NO donors in human skin. Br. J. Dermatol. 2004, 151, 977. (7) Mowery, K. A.; Schoenfisch, M. H.; Saavedra, J. E.; Keefer, L. K.; Meyerhoff, M. E. Preparation and characterization of hydrophobic polymeric films that are thromboresistant via nitric oxide release. Biomaterials 2000, 21, 9. (8) Frost, M. C.; Reynolds, M.; Meyerhoff, M. E. Polymers incorporating nitric oxide releasing/generating substances for improved biocompatibility of blood-contacting medical devices. Biomaterials 2005, 26, 1685. (9) Schewentker, A.; Vodovotz, Y.; Weller, R.; Billiar, T. R. Nitric oxide and wound repair: role of cytokines? Nitric Oxide 2002, 7, 1. (10) Masters, K. S. B.; Leibovich, S. J.; Belem, P.; West, J. L.; PooleWarren, L. A. Effects of nitric oxide releasing poly(vinyl alcohol) hydrogel dressing on dermal wound healing in diabetics mice. Wound Repair Regen. 2002, 10, 286. (11) Yuen, K.; Baker, C. J.; Rayman, N. R. Treatment of chronic painful diabetic neuropathy with isosorbide dinitrate spray. Diabetes Care 2002, 25, 1699.

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Polynitrosated Polyesters: Preparation, Characterization, and

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