European Journal of Pharmaceutics and Biopharmaceutics 89 (2015) 1–8

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Research paper

Time lasting S-nitrosoglutathione polymeric nanoparticles delay cellular protein S-nitrosation Wen Wu a, Caroline Gaucher a,⇑, Roudayna Diab a, Isabelle Fries a, Yu-Ling Xiao b, Xian-Ming Hu b, Philippe Maincent a, Anne Sapin-Minet a a

Université de Lorraine, CITHEFOR EA3452, Faculté de Pharmacie, Nancy, France State Key Laboratory of Virology, Ministry of Education Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Wuhan University School of Pharmaceutical Sciences, Wuhan, China b

a r t i c l e

i n f o

Article history: Received 5 September 2014 Accepted in revised form 9 November 2014 Available online 18 November 2014 Keywords: Nitric oxide donors S-nitrosoglutathione Polymeric nanoparticles Drug delivery Protein S-nitrosation Vascular diseases treatment

a b s t r a c t Physiological S-nitrosothiols (RSNO), such as S-nitrosoglutathione (GSNO), can be used as nitric oxide (NO) donor for the treatment of vascular diseases. However, despite a half-life measured in hours, the stability of RSNO, limited by enzymatic and non-enzymatic degradations, is too low for clinical application. So, to provide a long-lasting effect and to deliver appropriate NO concentrations to target tissues, RSNO have to be protected. RSNO encapsulation is an interesting response to overcome degradation and provide protection. However, RSNO such as GSNO raise difficulties for encapsulation due to its hydrophilic nature and the instability of the S-NO bound during the formulation process. To our knowledge, the present study is the first description of the direct encapsulation of GSNO within polymeric nanoparticles (NP). The GSNO-loaded NP (GSNO-NP) formulated by a double emulsion process, presented a mean diameter of 289 ± 7 nm. They were positively charged (+40 mV) due to the methacrylic acid and ethylacrylate polymer (EudragitÒ RL) used and encapsulated GSNO with a satisfactory efficiency (i.e. 54% or 40 mM GSNO loaded in the NP). In phosphate buffer (37 °C; pH 7.4), GSNO-NP released 100% of encapsulated GSNO within 3 h and remained stable still 6 h. However, in contact with smooth muscle cells, maximum protein nitrosation (a marker of NO bioavailability) was delayed from 1 h for free GSNO to 18 h for GSNONP. Therefore, protection and sustained release of NO were achieved by the association of a NO donor with a drug delivery system (such as polymeric NP), providing opportunities for vascular diseases treatment. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Since the discovery of the pivotal role of nitric oxide (NO) in several physiological processes in 1999 by Ignarro [1], NO has rapidly emerged as a promising candidate for the treatment of numerous disorders, mainly in cardiovascular function but also in stroke [2], asthma [3] and erectile dysfunction [4]. Nitric oxide is a second messenger in vivo, produced endogenously by three distinct nitric oxide synthases via L-arginine conversion [5–7] and it corresponds to the major endothelial relaxing factor that relaxes smooth muscle not only in the vasculature, but also in the gastrointestinal tract [8–10]. The biological activity of NO can be explained by its high chemical reactivity. It is a free radical species, carrying a single unpaired electron in its outer shell. The substances that are known ⇑ Corresponding author. Université de Lorraine, CITHEFOR EA 3452, Faculté de Pharmacie, BP 80403, F-54001 Nancy Cedex, France. Tel.: +33 3 83 68 23 76. E-mail address: [email protected] (C. Gaucher). http://dx.doi.org/10.1016/j.ejpb.2014.11.005 0939-6411/Ó 2014 Elsevier B.V. All rights reserved.

to react with NO include other radicals, transition metal ions and nucleophiles such as thiols (RSH) and amines [11]. NO acts by two main signaling pathways to regulate vascular function in vivo. The first is nitrosylation, corresponding to reversible bounding of NO to transition metal ions, such as ferrous (FeII) heme prosthetic groups within proteins (such as soluble guanylyl cyclase); this leads to enzyme activation and increased conversion of guanosine-3,50 -triphosphate to cyclic guanosine monophosphate (cGMP). The elevated cGMP activates specific kinases and finally vasorelaxation [12–14]. The second is S-nitrosation, targeting sulfhydryl-containing proteins and resulting in NO being covalently bound to cysteine. The formation of a mixed disulfide between a thiol group on an effector protein or peptide and a low molecular mass thiol, is able to modulate the function of the former. This posttranslational modification of proteins is as important as phosphorylation [15–17]. The imbalance of NO production and bioavailability is at the center of many cardiovascular diseases such as atherosclerosis,

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pulmonary hypertension, thrombosis, ischemia and cardiac arrhythmia [18]. The direct application of gaseous NO (such as in the treatment of pulmonary hypertension) is limited by its high reactivity. Expense, complex operational conditions and potential toxicity are also reported [19]. In this context, over the past few decades, several NO-related therapeutics based on more complex chemical system have emerged, such as nitrosamines [20], organic nitrates [21], metal–NO complexes [22], N-diazeniumdiolates [23], and S-nitrosothiols (RSNO) [24]. Organic nitrates and nitrate esters have been used in therapy of cardiovascular diseases [25–29], for example in the treatment of angina pectoris. However, these compounds induce undesirable effects such as oxidative stress, tolerance, thunderclap headache, and hypotension [30]. Without any recorded side effects in preclinical studies, RSNO (such as S-nitrosoglutathione (GSNO), S-nitroso-N-acetylcysteine, NACNO) therefore represent an especially promising class of NO donors for in vivo applications [31]. Endogenous RSNO, such as GSNO, Snitrosoalbumin and S-nitrosocysteine, are formed by the nitrosation of free thiols by reactive nitrogen species (e.g., N2O3) [32] and constitute a physiological pool of NO. Many investigations relating to the therapeutic potential of RSNO in the cardiovascular system have focused on GSNO, which is a powerful antiplatelet agent [33] with arterioselective vasodilator effects and also with well-documented antimicrobial [34] and antithrombotic effects [35,36]. With half-lives measured in hours [37], the stability of RSNO is actually too low for clinical applications. Therefore, the combination of RSNO with a delivery system represents a very promising strategy for the pharmaceutical and medical applications of NO [38]. Three different strategies to achieve this have been described in the literature. The first is the development of new macromolecular RSNO (thiomers), assembled as nanostructures [39,40]. For example, NO has been covalently bound to PEG-conjugated bovine serum albumin (PEG-poly SNO-BSA) via a S-nitrosothiol linkage (by nitrosation of cysteine) in the study of Katsumi et al. [41], which increased in vivo stability and prolonged NO release. A possible second strategy is the nitrosation of encapsulated free thiols, thereby constructing a S-nitrosothiol-loaded carrier. Marcato et al. [42] have developed polymeric NP based on alginate/chitosan to encapsulate GSH. After nitrosation of GSH, they obtained the GSNO-loaded NP. As a rarer third strategy, the direct encapsulation of S-nitrosothiol in liposomes [43], inorganic NP delivery system [44] and polymeric films [45,46] has also been described. This third option is often hampered by the sensitivity of RSNO to many factors (such as light, temperature, and oxygen) and to the biological environment, making it difficult to maintain a therapeutic concentration. Taking into account the fragility of RSNO, mild pharmaceutical processes would be preferred. In the current study, polymeric nanocarriers based on poly (methyl)methacrylate were elaborated as potential delivery systems of GSNO, able to preserve S-NO bound throughout the formulation process. Drastic handling conditions were conducted to efficiently encapsulate this small hydrophilic and labile molecule. The platform showed its ability to protect GSNO from physicochemical and enzymatic degradations in vitro. Protein S-nitrosation in cell culture gave proof of concept of NO donor activity from encapsulated GSNO. To our knowledge, this report represents the first description of polymeric NP for efficient encapsulation and prolonged release of effective GSNO, as a pharmaceutical drug.

generous gift from Evonik industries (Germany). The BCA Protein Assay Kit was purchased from Pierce. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin, 2,3-diaminonaphthalene (DAN), sulfanilamide, N-(1-naphthyl)ethylenediamine and all other reagents were obtained from Sigma–Aldrich (France). All manipulations and assays involving GSNO were conducted under conditions of subdued light and at 4 °C, in order to minimize light-induced GSNO degradation. 2.1. Preparation of GSNO-loaded NP GSNO-loaded NP (GSNO-NP) were prepared by a double emulsion (water–oil–water) and solvent evaporation method. Briefly, an aqueous solution of GSNO (5 mg) in 500 lL of 0.1% (w/w) PluronicÒ F-68 solution was emulsified by sonication for 60 s (11 W, 80% amplitude, Vibra cell™ 72434, France) over an ice bath in 5 mL of methylene chloride containing 500 mg of EudragitÒ RL PO. This primary emulsion was further emulsified in 20 mL of 0.1% (w/w) PluronicÒ F-68 solution by sonication (30 W, Vibra cell™ 75022, France) for 30 s, over an ice bath to form a water– oil–water emulsion. An opalescent emulsion was obtained. The NP were hardened by solvent evaporation. Finally the GSNO-NP were collected by ultracentrifugation at 287,000g, 30 min, 4 °C (Optima™ TLX ultracentrifuge, USA) before use. 2.2. Physicochemical characterization of NP The hydrodynamic diameter, size distribution and polydispersity index (PDI) of the NP were measured in 1 mM NaCl by dynamic light scattering (DLS) (ZetasizerÒ Nano ZS, MalvernÒ Instrument, France). All DLS measurements were performed at 25 °C with an angle detection of 173° backscatter (NIBS default). The samples were measured after 30 s autocorrelation and 15 runs were performed on each sample. For zeta potential measurements by electrophoretic migration (ZetasizerÒ Nano ZS, MalvernÒ Instrument, France), samples were diluted with 1 mM NaCl. All measurements were performed in triplicate. 2.3. Determination of GSNO encapsulation efficiency and core loading Encapsulation efficiency (EE) describes the quantity of the drug entrapped within NP compared with the total amount of initial drug. It was determined according to the following equation: EE = me/mi  100, where EE is encapsulation efficiency (%), me is the mass of drug entrapped in NP, and mi is the mass of initial drug. The mass of encapsulated GSNO was determined by liquid–liquid extraction. The mass of initial GSNO was calculated by the addition of the quantity of encapsulated GSNO and that remaining in the supernatant. The concentrations of GSNO and nitrite ions (a product of GSNO decomposition) were quantified by Griess-Saville and Griess reaction, respectively [20]. The concentration of loaded GSNO describes the capacity of NP matrix to carry GSNO. It was determined with the following equation: C = ne/vf, where C is the concentration of loaded GSNO (mM), ne is the quantity (mol) of entrapped GSNO in the NP, and vf is the volume of opalescent nanosuspension (mL). Both the encapsulation efficiency and the concentration of loaded GSNO were detected immediately after the GSNO-NP preparation. 2.4. In vitro release of GSNO from NP

2. Materials and methods All reagents were of analytical grade and all solutions prepared with ultrapure deionized water (>18.2 mX cm). Sodium nitrite was purchased from Merck (Germany). GSNO was synthesized according to a previously described method [42]. EudragitÒ RL PO was a

GSNO-NP were suspended in 1 mL of 0.148 M phosphate buffered saline (PBS, pH 7.4) and were laid in dialysis tubing cellulose membrane (average flat width 10 mm (0.4 in), cut-off is 14,000 Da). Release kinetics were studied in 200 mL of PBS at 37 °C, protected from light. Released GSNO and nitrite ions were

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monitored at different time intervals (every 30 min during two hours and every hour from two to six hours) and immediately quantified by a spectrofluorometric method, the DAN-Hg2+ [47]. 2.5. Vascular smooth muscle cell culture The cytocompatibility and function of NP were evaluated on rat smooth muscle cells (SMC) (A-10) ATCCÒ CRL-1476™. SMC were grown in complete medium made of Dulbecco’s Modified Eagle’s Medium DMEM supplemented with 10% (v/v) FBS, 2% (v/v) glutamine, 100 U/mL penicillin and 100 lg/mL streptomycin mixture, and 4 mM sodium pyruvate. Cells were cultivated at 37 °C under 10% CO2 (v/v) of CO2 in a humidified incubator. 2.6. Cytocompatibility of GSNO-NP SMC were seeded in 12-wells plates at 90,000 cells/well 48 h before exposure to free GSNO (50 lM), blank-NP (3 mg/mL) or GSNO-NP (50 lM GSNO and 3 mg/mL of polymer) for 18 h, and complete medium was used as control. After incubation, cell morphology was observed by phase contrast microscopy (10 objective; NA 0.25) coupled to a digital sight camera using the NIS-Elements F 3.0 software (Nikon, Europe). Cytocompatibility through metabolic activity was checked with MTT assay. Briefly, 0.5 mg/mL MTT was incubated with the cells for 4 h. Then, 250 lL DMSO was added under stirring for 10 min to extract the formazan crystals. The absorbance was read at 570 nm with a reference at 630 nm using EL 800 microplate reader (Bio-TEK Instrument, INCÒ, France). Metabolic activity in the presence of treatments was compared to the control condition. In addition, the size of NP and agglomerates obtained after incubation with SMC in the cell culture medium, was measured with the help of a MastersizerÒ 2000 (MalvernÒ Instrument, France). 2.7. Protein S-nitrosation of vascular SMC by GSNO-NP Intracellular protein S-nitrosation was used to determine the biological activity of the GSNO released from the polymeric delivery system. The SMC were seeded in 6-wells-plates at 180,000 cells/well and incubated with 50 lM free GSNO or 50 lM GSNO encapsulated in 3 mg/mL of polymeric NP. Complete medium was used as the control condition. Blank-NP were used as a negative control. All the cells were incubated at 37 °C under 10% CO2 for 1, 4 and 18 h. After incubation the medium was collected to quantify nitrite ions, free GSNO and GSNO remaining in NP (when applicable) by the Griess and Griess-Saville reaction, respectively. The cells were washed with PBS and lysed with 0.4% (m/v) Triton X-100 in 0.1 M HCl. The intracellular nitrosated proteins were quantified by the DAN, DAN-Hg2+ fluorometric method [47]. 2.8. Confocal laser scanning microscopy of NP-cell interaction Fluorescently-labeled NP were prepared by replacing 5 mg GSNO with 5 mg rhodamine-B as reported in Section 2.1. The SMC seeded on 8-well Lab-tekÒ chambered coverglass were incubated with free rhodamine-B or rhodamine-B loaded NP for 1, 4 and 18 h. Cells were fixed with paraformaldehyde 4% (w/v) during

Fig. 1. Release kinetics study of GSNO-NP in phosphate buffer (pH 7.4) at 37 °C. GSNO released from free GSNO (A) or from GSNO-NP (B); Nitrite ions released from free GSNO (C) or from GSNO-NP (D). The GSNO and nitrite ions concentrations were measured by the DAN, Hg2+ method. Data are shown as mean ± SD, n = 3.

15 min and nuclei were stained with Hoechst 33258 at 1 lg/mL during 10 min. Confocal observations of samples were carried out with a LEICA TCS SPX AOBS CLSM. Images at a 0.756 lm side length square pixel size were obtained for each case in 512  512 matrices at 40 magnification (numerical aperture = 0.8) of the CLSM. Fluorescence emissions were recorded within an Airy disk confocal pinhole setting (Airy 1). The two channels were acquired sequentially. First channel detection was set from 569 to 616 nm with a 555 nm excitation laser line. Second channel detection was set from 650 to 665 nm with a 630 nm excitation laser line. 2.9. Statistical analysis Results are shown as either mean ± standard deviation (SD) or mean ± standard error of mean (sem). For the evaluation of protein nitrosation, significant differences between groups were determined with a two-way ANOVA (variables: treatments and incubation time) followed by a posthoc Bonferroni test. Differences were considered significant when p < 0.05. For the other biological experiment, significant differences between groups were determined by a one-way ANOVA. All statistical analyses were done using GraphPad PrismÒ version 5.0.

3. Results 3.1. Preparation and characterization of GSNO-NP EudragitÒ RL NP formulation was successfully accomplished by the water–oil–water double emulsion and solvent evaporation method. The average size represented by hydrodynamic diameter of NP (Table 1) showed to be 272 ± 19 nm for blank-NP and 289 ± 14 nm for GSNO-NP. The mean zeta potential was +35 ± 2 mV for blank-NP and +40 ± 6 mV for GSNO-NP. GSNO was entrapped within the NP with a satisfactory 54% encapsulation efficiency. Consequently, the NP presented sufficient concentration of GSNO in their core loading of around 40 mM (17 nmol/mg polymer).

Table 1 Particle size, polydispersity index (PDI), surface charge (zeta potential), and encapsulation efficiency of blank and GSNO-NP. Data are shown as mean ± SD, n = 3.

Blank-NP GSNO-NP

Mean diameter (nm)

PDI

Zeta potential (mV)

Encapsulation efficiency (%)

GSNO (mM)

272 ± 19 289 ± 14

0.168 ± 0.02 0.174 ± 0.01

35 ± 2 40 ± 6

– 54 ± 2

– 40 ± 6

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Fig. 2. Morphology of SMC before (A) and after (B) exposure to Blank-NP or GSNO-NP. The cell surface showed a rough edge after incubation with NPs. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.2. Release kinetics of encapsulated GSNO and free GSNO The release profile of GSNO-NP was measured in 0.148 M phosphate buffer at pH 7.4. An initial burst release phase and a plateau were observed (Fig. 1). In the first 3 h, about 100% of GSNO was released from NP, and keep stable still 6 h. After 6 h, the released GSNO was degraded till 24 h (data not shown). Nitrite ions (NO 2 ), which are degradation products of GSNO, were present in a small amount (5% of initial GSNO) throughout the release period with GSNO-NP. In contrast, in the case of free GSNO, 20% of the initial GSNO was found as NO 2 under the same conditions.

In culture medium without serum (i.e. DMEM), NP maintained their initial size around 0.35 lm. However, the size increased from nanoscale to microscale when FBS was present regardless of the incubation duration (Table 2). Indeed, after 18 h, blank-NP increased in size 26 times from an initial 0.341 ± 0.01 lm to a final 9.08 ± 3.50 lm. Similarly, GSNO-NP increased in size 24 times from 0.323 ± 0.012 lm to 8.0 ± 0.2 lm. To further study this increase in size and to complete for the drug release experiment in contact with cells in the next paragraph, NP size was also checked after 1, 4 h and 18 h of incubation. The size and PDI or span of GSNONP and blank NP increased all along the incubation time in complete medium containing FBS (Table 3).

3.3. Cytocompatibility of GSNO-NP GSNO-NP were incubated 18 h in contact with smooth muscle cells. Phase microscopy observations showed that cells retained their spindle shape after 18 h of exposure to free GSNO (50 lM) and appeared similar to controls. However, the cell surface showed a rough edge after incubation with blank-NP (3 mg/mL polymer) and GSNO-NP (50 lM GSNO, 3 mg/mL polymer) (Fig. 2). To check whether the change in cell morphology after incubation with blank loaded NP was accompanied by cytotoxicity, cell viability was studied by metabolic activity monitoring with MTT. In the results shown in Fig. 3, the values obtained with untreated control cells were taken as 100% of metabolic activity. Whatever the conditions, cells exhibited almost 100% metabolic activity after 18 h of treatment. However to find an explanation to the cell surface changing, the sizes of NP were checked after various times of incubation in different media including complete culture medium with 10% FBS (Table 2).

Table 2 Influence of the incubation medium on the size of NP. The size of NP was measured after incubation with DMEM with and without 10% FBS or SMC cultivated in complete medium for 1, 4 and 18 h. Size (lm)

1h

4h

18 h

DMEM Blank-NP GSNO-NP

0.346 ± 0.020 0.352 ± 0.021

0.338 ± 0.008 0.320 ± 0.070

0.341 ± 0.005 0.323 ± 0.012

8.5 ± 2.7* 4.3 ± 0.4#

8.3 ± 1.6* 5.9 ± 0.6#

9.08 ± 3.5* 8.0 ± 0.2#

SMC + complete medium Blank-NP 6.5 ± 0.7* GSNO-NP 3.9 ± 0.3#

9.7 ± 0.6* 7.4 ± 0.4#

12.1 ± 0.4* 8.8 ± 0.8#

DMEM + 10% FBS Blank-NP GSNO-NP

* #

p < 0.05 vs blank-NP in DMEM. p < 0.05 vs GSNO-NP in DMEM. Data are shown as mean (SD), n = 3.

Table 3 Influence of the incubation medium on the PDI of NP. The PDI of NP was measured after incubation with DMEM with and without 10% FBS or SMC cultivated in complete medium for 1, 4 and 18 h.

Fig. 3. Cytocompatibility of NP. SMC were treated with free GSNO (50 lM), blank NP (3 mg/mL) or GSNO-NP (50 lM GSNO and 3 mg/mL of polymer). Data are shown as mean ± sem, n = 3. No result was statically different from the control.

* #

PDI/span

1h

4h

18 h

DMEM Blank-NP GSNO-NP

0.247 ± 0.012 0.264 ± 0.010

0.247 ± 0.010 0.252 ± 0.007

0.254 ± 0.006 0.247 ± 0.003

DMEM + 10% FBS Blank-NP GSNO-NP

1.230 ± 0.10* 1.136 ± 0.02#

1.269 ± 0.11* 1.337 ± 0.08#

1.498 ± 0.02* 1.306 ± 0.02#

SMC + complete medium Blank-NP 1.451 ± 0.06* GSNO-NP 1.639 ± 0.45#

1.371 ± 0.01* 1.449 ± 0.03#

1.516 ± 0.01* 1.371 ± 0.01#

p < 0.05 vs blank-NP in DMEM. p < 0.05 vs GSNO-NP in DMEM. Data are shown as mean (SD), n = 3.

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3.4. GSNO release and degradation after contact with cells

nitrosation (0.323 ± 0.03 nmol/mg protein) was reached after 1 h incubation. From 1 h to 18 h, protein nitrosation decreased. Similarly, GSNO released from GSNO-NP delivered NO into the cells and produced intracellular protein S-nitrosation that was higher than control levels at all incubation times but with different kinetics from free GSNO. In the case of GSNO-NP, intracellular protein Snitrosation increased with time. The maximum level observed at 18 h (0.336 ± 0.02 nmol/mg protein) was the same that achieved at 1 h with free GSNO. This difference in kinetics was confirmed by studies of the interaction of SMC with rhodamine-B-loaded NP or free rhodamine-B (Fig. 6). Free rhodamine-B started to enter the cells after 1 h of incubation and carried on loading the cells up to 18 h, whereas uptake from rhodamine-B-loaded NP was not observed before 18 h. These results corroborate the delay of release of the encapsulated compound observed for GSNO-NP.

GSNO-NP and free GSNO were incubated with cells for 1, 4 and 18 h. After the incubation, nitrite ions, nitrosospecies (the GSNO in the medium may transfer NO to protein with free cysteine residues by transnitrosation referred to nitrosospecies RSNO) and the GSNO remaining in NP were quantified in the culture medium. Extracellular concentrations of all these species decreased with time whatever the conditions and of the incubation time (Fig. 4). In the free GSNO group, 11.5 lM (23% of initial GSNO), 12.5 lM (25% of initial GSNO), 16.86 lM (34% of initial GSNO) GSNO decomposed into nitrite ions after 1, 4 and 18 h of incubation, respectively. However, for GSNO-NP, a smaller proportion of GSNO was decomposed. Indeed, the nitrite ion concentration was maintained at around 10% of initial GSNO from 1 h to 18 h (Fig. 4). The total amount of all GSNO species was higher for GSNO-NP compared with free GSNO after 1 h and 4 h (Fig. 4.). The sum of the concentration of nitrite ion and of RSNO was higher for free GSNO than for GSNO-NP after 18 h of incubation. However, after 18 h of incubation, free GSNO led to 0.22 lM as RSNO species whereas there was 0.9 lM of RSNO and 2.58 lM GSNO were still present in the extracellular compartment.

4. Discussion Endothelium derived nitric oxide (NO) regulates vasodilation via relaxation of the SMC and inhibits platelet aggregation among a number of other biological processes. NO donors that release NO within the body, such as nitroglycerine, sodium nitroprusside, diazeniumdiolates (NONOates) [48] and RSNO have been developed and used for the delivery of NO in order to compensate for the very short in vivo half-life of NO. Among these NO donors, RSNO have several advantages: (i) they represent endogenous NO reservoirs circulating in the blood, and (ii) they do not induce oxidative stress or vascular tolerance that is sometimes observed with other NO

3.5. Smooth muscle cell protein nitrosation by GSNO-NP The basal level of protein nitrosation (0.08 ± 0.02 nmol/mg of protein) was not affected by the presence of blank NP (0.09 ± 0.02 nmol/mg of protein) whatever the incubation time (Fig. 5). Free GSNO increased the level of S-nitrosated the intracellular proteins at all incubation times, but the maximum level of

0.4 0.3

Ψ

*

1h 4h 18 h

*

0.2

Ψ

*

*

0.1

*

Ψ

*

*

-N

P

O N

G

S

N

O

S G

-N nk B la

on

tr o

P

l

0.0

C

Quantity of RSNO (nmol/mg protein)

Fig. 4. Concentration of extracellular nitrite ions and GSNO. SMC were incubated with free GSNO (50 lM) or GSNO (50 lM)-NP for 1 h, 4 h and 18 h. The concentrations of nitrite ions and RSNO in the medium were determined by the Griess or Griess-Saville reaction, respectively. The concentration of GSNO remaining in the GSNO-NP was assayed by DAN, Hg2+ method. Data are shown as mean ± sem, n = 4.

Fig. 5. Concentration of intracellular RSNO. SMC were incubated with free GSNO (50 lM) or GSNO (50 lM)-NP for 1 h, 4 h and 18 h. Data are shown as mean ± sem, n = 4. p values (two ways ANOVA test): ⁄ptreatment < 0.05; ptime < 0.05 vs control. Wp < 0.05 vs blank-NP.

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Fig. 6. Representative confocal microscopy images (magnification 40; numerical aperture = 0.8) of SMC after incubation with free rhodamine-B (A, C, E) or rhodamine-Bloaded NP (B, D, F) for 1 h (A, B), for 4 h (C, D), 18 h (E, F). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

donors, such as nitroglycerine [49,50]. It has been reported that RSNO, such as GSNO, improved recovery from female sexual dysfunction [51]. However, a high dose of RSNO is required in order to obtain therapeutic benefits in the treatment. This is mainly because these RSNO release NO very quickly, not only in the vascular compartment, or because they are rapidly cleared from the systemic circulation. Therefore, both to obtain a better understanding of the pharmacological and pharmacokinetic properties of NO donors and to improve therapeutic activity, novel RSNO delivery systems are urgently required. In the present study, EudragitÒ RL NP were developed to alleviate these problems by directly encapsulating and protecting GSNO, and providing a time lasting release. Hydrophobic EudragitÒ polymers are biocompatible polymers classically used for coating conventional oral formulations, and have also already been used for the encapsulation of hydrophilic drugs such as heparin [52] and insulin [53] and also for the encapsulation of the tripeptide glutathione, the ‘‘precursor’’ of GSNO [54]. The method of double emulsion and solvent evaporation was optimized and adapted for the properties of GSNO. The obtained NP presented a positive charge due to the methacrylic acid and ethylacrylate polymer used to form them. Fifty-four percent of the added GSNO was encapsulated, leading to 40 mM GSNO in the dosage form. Compared with other reported NP delivery scaffolds, prepared by sol–gel technique [55] or a coacervation process [33], the new developed EudragitÒ RL NP showed better encapsulation efficiency and a higher level of GSNO concentration loading than other hydrophilic or hydrophobic nanocarriers. For example, the liposomes developed by Rotta et al. [43] contained 2 mM GSNO, the hydrogel NP platform described in the work of Nacharaju et al. [56], carried less than 5 mM GSNO, and the polymeric alginate/chitosan NP indicated by Marcato et al. [42] were able to carry 0.4 mM GSNO. The high encapsulation efficiency

obtained in the present study is partly facilitated by mild electrostatic interaction, due to the fact that GSNO (pKa (COOH) = 2.21) is negatively charged during fabrication of NP (pH = 5.78). The present GSNO formulation based on polymeric NP increased GSNO loading by a large margin, which is a considerable achievement for a hydrophilic drug. Furthermore, the loaded GSNO can be delivered by the NP. In contrast to other NO-releasing formulations, the present NP released the payload as GSNO, rather than as the very labile NO. This property indicates the improved stability of NO in the application. We hypothesized that part of the GSNO was absorbed onto the surface of the NP, which is supported by the ‘‘burst’’ release in the first 2 h. Due to its hydrophilic nature, the surface-bound GSNO can diffuse out of the NP rapidly in contact with the release medium. The stability of GSNO in PBS over 6 h was also in accord with the study of Parent et al. [47]. Subsequent experiments evaluated the biological activity of GSNO released from encapsulated donor. A study of the cytocompatibility of NP for SMC is a prerequisite for the evaluation of the ability of GSNO-NP to S-nitrosated proteins. The well established MTT test revealed that free GSNO, blank-NP and GSNO-NP had no impact on SMC metabolic activity. The size of NP was increased by a factor of thousands after incubation with SMC in complete medium containing 10% FBS in a timedependent manner. The probable reason is that the negatively charged proteins in FBS like albumin aggregate positively charged NP via electrostatic interactions. After incubation, the rough-edged cell morphology observed may be attributed to particles aggregates on the remaining surface of the cell membrane. Indeed, the cell membrane is also negatively charged, which would attract the polymeric NP. This phenomenon could favor the direct transfer of GSNO to the immediate environment of membrane proteins,

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which include enzymes such as protein disulfide isomerase and gamma-glutamyl transpeptidase at their exofacial face. These enzymes are known to metabolize GSNO and to permit the delivery of NO [57,58]. S-nitrosation of various proteins has been implicated in physiological processes such as vasodilation, antimicrobial activity and other NO mediated cellular functions. To investigate whether our GSNO formulation improved NO delivery to cells compared with free GSNO, we selected protein S-nitrosation as a biomarker of the penetration of NO derived from GSNO into cells and thereby proof of GSNO activity. Indeed, as a NO donor, GSNO can nitrosate the free cysteine residues of proteins. Thus, S-nitrosated proteins can be considered as an experimental proof of NO release [38]. In vitro, GSNO is susceptible to decomposition by a number of factors (temperature, oxygen, metal. . .). Based on the extracellular concentration of nitrite and GSNO released from GSNO-NP after incubation with cells, it appears that encapsulated GSNO is stabilized and protected from degradation by the polymeric nanocarrier compared with free GSNO. In addition, measurements of the intracellular concentration of S-nitrosated proteins gave some even more interesting information. They indicated that both free GSNO and GSNO released from the NP were active as shown by their ability to elevate the intracellular nitrosated proteins concentration. Furthermore, the maximum level of nitrosated proteins was found for free GSNO at 1 h whereas it was at 18 h for GSNO-NP. This indicates that our GSNO-NP platform allowed a slower protein S-nitrosation as a result of delayed GSNO release. This slower NO release would be beneficial for its therapeutic activity. One hypothesis to explain the sustained release is that the proteins contained in the FBS (mostly albumin) surround the NP and formed a ‘‘bio-wall’’, which could slow down the diffusion speed of GSNO throughout the NP. In order to confirm our hypothesis, the release kinetics of GSNO from GSNO-NP were evaluated in the presence of BSA (0.45 g/dL similar to protein concentration in culture medium). The time for 50% GSNO release was delayed from 15 min (without BSA) to 1 h in the presence of BSA. However, in the presence of BSA only 60–70% of GSNO was released from the NP compared with that observed in the absence of BSA (data not shown). So the GSNO-NP covered by protein could adhere to the cell membrane surface, leading to the protection of GSNO and time-lasting delivery as a direct consequence. Further evidence for the delay of active compound release from the NP was provided by fluorescence microscopy monitoring of rhodamine-B-loaded NP, which showed accumulation by SMC of rhodamine-B released from NP after 18 h of incubation in contrast to1 h for free rhodamine-B.

5. Conclusion As a representative S-nitrosothiol for NO supplementation, the usefulness of GSNO is limited by poor stability and high hydrophily. In the present study, we developed an effective new polymeric NP NO donor platform. This GSNO-NP exhibited high encapsulation efficiency, high drug protection and controlled release. The delivered GSNO retained its biological activity as evidenced by protein S-nitrosation and GSNO release delayed by 17 h compared with free GSNO. Therefore, protection and sustained presence of NO are achieved by the association of a NO donor and a drug delivery system (hydrophobic polymeric NP), providing potential opportunities for vascular treatment optimization. As previously described [59], Eudragit RL is non-degradable and permeable in gastrointestinal tract. This NO donor delivery system based on Eudragit RL with highly improved loading capacity and suitable size range [5], provides an approach to increase bioavailability and shows promise for oral administration and chronic treatment applications.

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Conflict of interest There is no conflict of interest. Acknowledgments The authors are grateful to Pr Pierre Leroy (Université de Lorraine, CITHEFOR EA3452) and Pr Raphael Schneider (Université de Lorraine UPR 3349 CNRS, LRGP,) for their helpful discussion, advice and contribution to experimental studies, Pr Gillian Barratt (UMR CNRS 861, Paris-Sud University) for manuscript spelling corrections and Simone Filiaggi for his technical support. We thank Sébastien Hupont (from the imaging core facility (PTIBC IBISA Nancy) from the Federation de Recherche (FR3209 CNRS – BMCT) based at the Biopôle on the biology-health campus at Université de Lorraine for the formation and use of confocal imaging systems. The authors acknowledge Université de Lorraine and Région Lorraine (UHP_2011_EA3452_BMS_0062, CPER 2007-13 PRST «Ingénierie Moléculaire et Thérapeutique – Santé »), the National Agency for Research (France) (NANOSNO, 2010) and the program of Chinese Scholarships Council for their financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. References [1] L.J. Ignarro, Nitric oxide: a unique endogenous signaling molecule in vascular biology, Biosci. Rep. 19 (1999) 51–71. [2] B.N. King, S.M. Haque, M.C. Stoner, Z.M. Ellis, J.M. Kellum, Inhibition of neural nitric oxide synthase attenuates the chloride secretory response to stroking in human jejunum, Surgery 134 (2003) 255–259. [3] S.A. Kharitonov, D. Yates, R.A. Robbins, R. Logan-Sinclair, E.A. Shinebourne, P.J. Barnes, Increased nitric-oxide in exhaled air of asthmatic-patients, Lancet 343 (1994) 133–135. [4] M.E. Sullivan, C.S. Thompson, M.R. Dashwood, M.A. Khan, J.Y. Jeremy, R.J. Morgan, D.P. Mikhailidis, Nitric oxide and penile erection: is erectile dysfunction another manifestation of vascular disease?, Cardiovasc Res. 43 (1999) 658–665. [5] D.E. Heck, D.L. Laskin, C.R. Gardner, J.D. Laskin, Epidermal growth factor suppresses nitric oxide and hydrogen peroxide production by keratinocytes. Potential role for nitric oxide in the regulation of wound healing, J. Biol. Chem. 267 (1992) 21277–21280. [6] K. Ivanova, I.C. Le Poole, R. Gerzer, W. Westerhof, P.K. Das, Effects of nitric oxide on the adhesion of human melanocytes to extracellular matrix components, J. Pathol. 183 (1997) 469–476. [7] J. MacMicking, Q.W. Xie, C. Nathan, Nitric oxide and macrophage function, Annu. Rev. Immunol. 15 (1997) 323–350. [8] A.R. Butler, F.W. Flitney, D.L. Williams, NO, nitrosonium ions, nitroxide ions, nitrosothiols and iron-nitrosyls in biology: a chemist’s perspective, Trends Pharmacol. Sci. 16 (1995) 18–22. [9] K.A. Hanafy, J.S. Krumenacker, F. Murad, NO, nitrotyrosine, and cyclic GMP in signal transduction, Med. Sci. Monit. 7 (2001) 801–819. [10] L.J. McDonald, F. Murad, Nitric oxide and cyclic GMP signaling, Proc. Soc. Exp. Biol. Med. 211 (1996) 1–6. [11] V.G. Kharitonov, A.R. Sundquist, V.S. Sharma, Kinetics of nitrosation of thiols by nitric oxide in the presence of oxygen, J. Biol. Chem. 270 (1995) 28158– 28164. [12] R.G. Knowles, S. Moncada, Nitric oxide synthases in mammals, Biochem. J. 298 (1994) 249–258. [13] D. Yao, A.G. Vlessidis, N.P. Evmiridis, Determination of nitric oxide in biological samples, Microchim. Acta 147 (2004) 1–20. [14] C. Lindermayr, J. Durner, S-Nitrosylation in plants: pattern and function, J. Proteom. 73 (1994) 1–9. [15] A. Martinez-Ruiz, S. Lamas, Signalling by NO-induced protein s-nitrosylation and s-glutathionylation: convergences and divergences, Cardiovasc. Res. 75 (2007) 220–228. [16] L. Heikal, P.I. Aaronson, A. Ferro, M. Nandi, G.P. Martin, L.A. Dailey, Snitrosophytochelatins: investigation of the bioactivity of an oligopeptide nitric oxide delivery system, Biomacromolecules 12 (2011) 2103–2113. [17] M.W. Foster, D.T. Hess, J.S. Stamler, Protein S-nitrosylation in health and disease: a current perspective, Trends Mol. Med. 15 (2009) 391–404. [18] B.A. Maron, S.S. Tang, J. Loscalzo, S-nitrosothiols and the S-nitrosoproteome of the cardiovascular system, Antioxid. Redox Signal. 18 (2013) 270–287. [19] G. Hagan, J. Pepke-Zaba, Pulmonary hypertension, nitric oxide and nitric oxide-releasing compounds, Expert Rev. Respir. Med. 5 (2011) 163–171. [20] R. Messin, G. Boxho, J. De Smedt, I.M. Buntinx, Acute and chronic effect of molsidomine extended release on exercise capacity in patients with stable angina, a double-blind crossover clinical trial versus placebo, J. Cardiovasc. Pharmacol. 25 (1995) 558–563.

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