s2fieldsofstudy: Biology Chemistry Medicine

text:
Drug Synergy of Tenofovir and Nanoparticle-Based Antiretrovirals for HIV Prophylaxis

Background The use of drug combinations has revolutionized the treatment of HIV but there is no equivalent combination product that exists for prevention, particularly for topical HIV prevention. Strategies to combine chemically incompatible agents may facilitate the discovery of unique drug-drug activities, particularly unexplored combination drug synergy. We fabricated two types of nanoparticles, each loaded with a single antiretroviral (ARV) that acts on a specific step of the viral replication cycle. Here we show unique combination drug activities mediated by our polymeric delivery systems when combined with free tenofovir (TFV). Methodology/Principal Findings Biodegradable poly(lactide-co-glycolide) nanoparticles loaded with efavirenz (NP-EFV) or saquinavir (NP-SQV) were individually prepared by emulsion or nanoprecipitation techniques. Nanoparticles had reproducible size (d ∼200 nm) and zeta potential (-25 mV). The drug loading of the nanoparticles was approximately 7% (w/w). NP-EFV and NP-SQV were nontoxic to TZM-bl cells and ectocervical explants. Both NP-EFV and NP-SQV exhibited potent protection against HIV-1 BaL infection in vitro. The HIV inhibitory effect of nanoparticle formulated ARVs showed up to a 50-fold reduction in the 50% inhibitory concentration (IC50) compared to free drug. To quantify the activity arising from delivery of drug combinations, we calculated combination indices (CI) according to the median-effect principle. NP-EFV combined with free TFV demonstrated strong synergistic effects (CI50 = 0.07) at a 1∶50 ratio of IC50 values and additive effects (CI50 = 1.05) at a 1∶1 ratio of IC50 values. TFV combined with NP-SQV at a 1∶1 ratio of IC50 values also showed strong synergy (CI50 = 0.07). Conclusions ARVs with different physicochemical properties can be encapsulated individually into nanoparticles to potently inhibit HIV. Our findings demonstrate for the first time that combining TFV with either NP-EFV or NP-SQV results in pronounced combination drug effects, and emphasize the potential of nanoparticles for the realization of unique drug-drug activities.

Introduction
Sexual transmission is the primary cause of new HIV-1 infections worldwide, which today exceed 6,000 infections daily [1,2]. Sub-Saharan Africa bears the burden of the global HIV/ AIDS epidemic, and women in this region are disproportionately affected by the pandemic [3,4]. Women account for three-quarters of Africans between the ages of 15 and 24 who are HIV-positive, and HIV/AIDS is the leading cause of death among women of reproductive age [3]. In the absence of an effective vaccine, and as long as new infections continue to outpace advances made in treatment with antiretroviral (ARV) drugs [5], biomedical prevention strategies are critical for stemming the spread of HIV. Oral pre-exposure prophylaxis (PrEP) and topical microbicides are the lead strategies for preventing HIV infection, but there is still a critical need for methods with greater efficacy to protect women [6]. Long-acting ARV drug combinations have the potential to enhance the efficacy of current ARV-based prevention strategies by overcoming low user adherence, and harnessing drug combinations with synergistic activity and breadth of coverage against the global diversity of HIV variants. However, the physicochemical diversity of ARV drugs precludes their coformulation and limits access to all possible combinations of the .20 ARV drugs approved for clinical use [7,8,9]. Strategies that enable ARV drugs to be easily combined and provide sustained antiretroviral activity have the greatest potential to impact the efficacy of future biomedical prevention methods.
Nanocarrier systems provide an innovative approach for developing long-acting ARV drug combinations and have already been explored for use in HIV treatment and prevention [10,11,12,13,14]. The availability of different carrier systems, combined with the versatility of drugs that can be encapsulated for controlled release, motivate the use of nanocarrier systems for ARV drug delivery in different applications. In addition, nanocarriers have been shown in a number of examples to increase the activity and reduce cytotoxicity of several ARV drugs [15,16,17]. The selection of a nanocarrier system for drug delivery depends on the properties of the drug but also on the physical and chemical attributes of the final nanoformulation. The ability to control these attributes is important because the pharmacokinetics of the resulting nanoformulated drug can differ significantly from the parent compound. Properties of the delivery system such as carrier size, architecture and surface chemistry can also affect the activity of nanoformulated drugs. For example, Nowacek et al. demonstrated that physical characteristics of nanoparticles formed by wet-milling water-insoluble ARV drugs, i.e., particle size, surface charge, and shape, were correlated with macrophage uptake and resulted in greater antiretroviral efficacy [18]. The potential for rational design of drug carriers to enhance drug potency and efficacy may have important applications for prophylaxis.
Nanocarrier systems also have the capacity to address challenges associated with delivering drug combinations. The success of highly active antiretroviral therapy (HAART) provides a paradigm for developing the next generation ARV-based prevention strategies, giving rise to the possibility that a combination of potent and broadly active inhibitors will provide superior protection against HIV transmission and reduce the likelihood of emerging drug resistance. Drug combinations can also markedly expand the antiretroviral activity of single agents used alone by facilitating unique mechanisms of drug-drug activity when used in combination. For example, inhibition of drug metabolizing enzymes or efflux transporter systems has been implicated in the improved virological response to ARV drug combinations such as ritonavir in combination with saquinavir (SQV), and tenofovir (TFV) in combination with emtricitabine (FTC) or efavirenz (EFV) [19,20,21,22]. However, due to physicochemical incompatibilities, not all ARV drugs are amenable to combinations that may lead to beneficial combination drug activity. As such, nanocarrier mediated ARV delivery may allow for the exploration of unique drug-drug interactions of otherwise incompatible ARV compounds or modulate drug delivery profiles necessary to achieve drug synergy using specific drug combinations.
Among ARV-based prevention methods, tenofovir (TFV) has been the most extensively investigated. TFV is a nucleotide analog reverse transcriptase inhibitor that is effective against either CCR5 or CXCR4 HIV-1, and has been shown to be safe for both oral and vaginal use [23,24,25,26]. CAPRISA 004 was the first phase IIb double blind randomized controlled clinical trial to demonstrate protection against HIV-1 acquisition using TFV as a single ARV-based microbicide gel. In addition, oral TFV used either alone or in combination with emtricitabine (FTC) has been proven effective in three independent oral PrEP clinical trials (iPrEx, Partners PrEP, TDF2) [6]. Extensive safety and efficacy data exists for TFV, and it is furthest along in the development pipeline towards an ARV-based product that can protect against sexual HIV transmission. Improving the potency and long-acting efficacy of TFV by combining it with other ARV drugs is desirable for next generation biomedical prevention strategies. However, existing dosage forms may not be suitable for combining physicochemically diverse compounds and limits the realization of novel drug-drug interactions and synergies with TFV. Based on these observations, we sought to identify unique drug-drug activities mediated by our ARV loaded nanoparticles (NP-ARVs) when used in combination with free TFV.
Here we describe synergistic in vitro anti-HIV activity of novel combinations of NP-ARVs and free TFV. To overcome challenges associated with formulating multiple ARV compounds that are chemically incompatible, we fabricated polymeric nanoparticles encapsulating individual ARV drugs that were then delivered in combination. The non-nucleoside reverse transcriptase inhibitor (NNRTI) EFV and the protease inhibitor (PI) SQV were chosen based on their low aqueous solubility and different mechanisms of action. We show that EFV and SQV could be individually fabricated into biodegradable poly(lactide-co-glycolide) (PLGA) nanoparticles with high loading and encapsulation efficiency. NP-ARVs were nontoxic in cell culture and in mucosal tissue explants. In comparison to free ARVs, ARVs formulated in nanoparticles showed up to a 50-fold increase in antiviral activity. We also observed unique drug-drug activities when NP-ARVs were combined with free TFV, and observed in some cases drug synergy not seen with free drugs in combination. Collectively, our data show that PLGA-based nanoparticle formulations are a promising platform to deliver ARV combinations. The implications of our results may support a new paradigm for delivery of combination ARVs for HIV-1 prevention.

Fabrication of ARV loaded nanoparticles
Blank nanoparticles (vehicle control) and nanoparticles loaded with EFV or SQV were formulated individually. EFV loaded nanoparticles (NP-EFV) were formulated using a single emulsion technique as previously described [27,28]. All concentrations described below are expressed in % w/v unless noted otherwise. In each preparation, EFV was dissolved in dichloromethane (DCM) containing 1.5% PLGA (w/v, Lactel -B6010-2P). Mass percentage of drug initially dissolved in PLGA (theoretical drug loading) was 15% (w/w). This mixture was then added drop-wise to an aqueous phase containing an emulsifier (5% aqueous solution of polyvinyl alcohol, PVA) to form an oil-in-water emulsion (o/w). A probe sonicator (3 mm diameter, Sonicator XL, Misonix, Farmingdale, NY) was used to homogenize the emulsion for 60 sec at 65 W. After solvent evaporation in an aqueous solution of 0.25% PVA for 3 h, nanoparticles were washed with deionized water three times by centrifugation at 14,0006g for 10 min (Sorvall Ultra 80, Waltham, MA). To formulate SQV loaded nanoparticles (NP-SQV), we used a nanoprecipitation technique [29]. SQV were dissolved in acetone containing 0.33% PLGA (w/v, Resomer -502H) with 15% (w/w) theoretical drug loading. Then SQV-PLGA solution was added by syringe pump at a 1 mL/min flow rate to an aqueous solution containing 0.1% phosphate-buffered saline (0.01 M PBS, pH 7.4) and 0.1% dioctyl sulfosuccinate sodium (DSS) surfactant while it was stirring. Nanoparticles were formed instantly upon mixing due to the immiscibility of the polymer and non-solvent. After solvent evaporation, nanoparticles were washed as described above. The nanoparticles were suspended in deionized water and were lyophilized for 24 h under vacuum at 0.120 mbarr at 286uC (FreeZone 2.5 Plus, Labcono, Kansas City, MO). The dried nanoparticles were stored at 286uC until use.

Characterization of nanoparticles
Size and zeta potential of the fabricated nanoparticles were determined using a Zetasizer Nano ZS90 (Malvern Instruments, AR). Size and morphology of nanoparticles were confirmed by scanning electron microscopy (SEM) visualized with a JEOL-7000 (JEOL Ltd, Sheboygan, WI) scanning electron microscope. Samples of nanoparticles were dusted onto carbon tape, coated with gold, and imaged using a 10 kV electron beam.

Drug loading
Verification of drug-polymer association in nanoparticles was performed using Fourier transform infrared spectroscopy (FTIR). Briefly, 3-5 mg of nanoparticles were mixed with potassium bromide (KBr) using a mortar and pestle and analyzed in FTIR. Infrared spectra over a range of wavenumber 500 to 4000 cm 21 were monitored for the presence of the functional groups corresponding to the characteristic peaks of EFV or SQV. The amount of EFV and SQV actually loaded in nanoparticles was determined using a Shimadzu Prominence LC20AD high performance liquid chromatography (HPLC) system and LC Solutions software. A C18 column (Phenomenex, Torrance, CA), 5 mm, 25064.6 mm i.d., was used for analysis with isocratic mode at a flow rate of 1.0 mL/min. Methods used to analyze EFV and SQV were based on those described previously for detecting SQV [30]. The mobile phase consisted of a mixture of 10 mM ammonium acetate buffer in HPLC grade water and acetonitrile at a 35:65 ratio (v/v). SQV was detected at 238 nm and EFV was detected at 246 nm with retention times of 7.5 min and 8.6 min, respectively. Standard solutions of EFV and SQV were prepared at 1 mg/mL in dimethyl sulfoxide (DMSO) and diluted to generate the calibration curves. Analysis methods were validated with standard solutions and spiked samples. Linearity was established from 50 ng/mL to 10 mg/mL for EFV and 250 ng/ mL to 50 mg/mL for SQV using a 10 mL injection volume. Nanoparticles were dissolved in DMSO to assess drug loading and encapsulation efficiency. For all in vitro assays, the delivered dose of the formulated ARV drug is defined and calculated based on the total drug loaded in the polymeric nanoparticle. Therefore, we delivered a mass concentration of the drug-loaded polymer to achieve the desired molar concentration of the drug given the volume requirements for the particular assay.

In vitro release
To determine the release profiles of NP-ARVs in a physiological condition relevant to the vagina, an in vitro release study was conducted over 144 h using a vaginal fluid simulant (VFS) as the release medium [31]. Triplicate samples of approximately 2 mg of either NP-EFV or NP-SQV were suspended in 500 mL of VFS and added into individual dialysis tubes (1 kDa cut-off, GE Healthcare Bio-Sciences Corp., NJ). The dialysis tubes were placed in individual 50-mL tubes containing 5 or 15 mL of VFS for SQV and EFV, respectively, and incubated at 37uC on an orbital shaker at 100 rpm. At set time points (0.5, 1, 4, 8, 24, 48, 72, 144 h), 200 mL of samples were collected and replaced with fresh VFS. UV-HPLC methods were used to quantify the amount of drug in samples as described above.

Cellular viability assay
TZM-bl cells were seeded in a 96-well plate at 5,000 cells/well and incubated overnight to allow the cells to adhere to the well. Dilutions of drugs (free and nanoparticle forms) were added to triplicate wells of TZM-bl cells. Wells containing cells alone served as controls. Cell cultures in the absence and presence of drugs were incubated for 48 h. To determine cell culture viability, metabolic capacity of cells was measured using the Promega CellTiter-Blue TM assay according to the manufacturer's instructions. The ability of cells to reduce a resazurin indicator dye to fluorescent resorufin was measured using a plate reader at 560/ 590 nm (excitation/emission) and normalized to media onlytreated cells (100% viability). DMSO served as positive control.

Antiviral activity
The inhibitory activity of free and nanoparticle ARVs against HIV-1 BaL was determined in TZM-bl cells by luciferase quantification of cell lysates [37,38]. Cells were seeded at 5,000 cells/well and grown to approximately 50-60% confluence overnight prior to infection. Dilutions of each drug were added to triplicate wells. After 1 h, HIV-1 BaL at an approximate TCID 50 (50% tissue culture infectious dose) of 300 per well was added to each pre-treated well. Media controls (wells containing cells alone) were included in every run for luminescent background subtraction. Cells grown in the absence of virus served as the negative infectivity control (100% inhibition), while cells infected with virus in the absence of drug served as the positive infectivity control (0% inhibition). After 48 h, cells were lysed and luciferase expression was determined in relative light units (RLUs) using a luminometer. The percent inhibition was calculated for all test and control cultures to determine the 50% inhibition concentration (IC 50 ) value of each drug. The IC 50 values of NP-ARVs were calculated using drug concentrations that corresponded to the actual drug loading determined by HPLC.

Combination effect
Combined activity of dual drug combination was evaluated as described in Figure 1. First, median IC 50 values of each drug were obtained using the TZM-bl antiviral activity assay as described above. Next, each drug was added to free TFV at a 1:1 ratio based on their IC 50 values to create a mixture of combination drugs. For NP-ARVs, amounts of the individual agents used in combinations were determined using the measured drug loading. The drug mixtures were serially diluted and IC 50 values were determined with the TZM-bl assay. Finally, combination effects were evaluated by; 1) comparing IC 50 values to determine dose reduction; and 2) identifying combination indices (CI) to quantify drug synergy, with the median-effect analysis described by Chou and Talalay [39]. The CI of each drug combination was plotted as a function of the fractional inhibition (Fa) by computer simulation from Fa = 0.10 to 0.95. In this analysis, the combined effect at the 50% fractional inhibition (CI 50 ) was reported as synergistic, additive, or antagonistic when CI,1, = 1, or .1, respectively.

Ex vivo toxicity assay
Ectocervical tissues from two macaques (Tissue Banking and Distribution Program, the Washington National Primate Research Center) were processed for polarized explant cultures as previously described [40,41,42]. The explant cultures were set up on the day of surgery. Briefly, the macaque ectocervical explant cultures were established in duplicate by inserting a circular tissue punch through a hole in a transwell with the luminal epithelium side up. The edges around the explant were sealed with Matrigel TM (BD Biosciences, San Jose, CA). A 0.1 mg/mL suspension of either NP-EFV or NP-SQV in 200 mL of culture media (DMEM with 10% fetal bovine serum, 1% 100X penicillin/streptomycin, and 1% 200 mM L-glutamine) was added on the apical side of the tissue. Untreated explants (culture media) and explants treated with 0.4% nonoxynol-9 (N-9) gel served as controls. All explant cultures were maintained at 376C in a 5% CO 2 atmosphere. After 18-24 h, the explants were washed and one of each duplicate was incubated in Figure 1. Schematic diagram for combination effect analysis. Drug combinations were analyzed for their ability to affect dose reduction and synergy. First, individual drugs, either free or encapsulated, were used to create dose response curves using the TZM-bl assay. These curves were fit to a sigmoid curve using nonlinear least squares regression to estimate drug IC 50 and the Hill slope. Next, combinations of drugs at their equipotency ratio (1:1 ratios of IC 50 values) were used to create similar curves using the TZM-bl assay. These were also fit to a sigmoid curve using nonlinear least squares regression to estimate the IC 50 and Hill slope of the combination. Finally, comparison of IC 50 values combinations were used to estimate dose reduction. The median-effect analysis was performed to measure combination effects. doi:10.1371/journal.pone.0061416.g001 RPMI containing 250 mg/mL MTT [1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan] for 4 h. The explants were removed and placed in 1 mL of methanol overnight to extract the formazan dye produced by live tissue. The next day, the explants were removed from methanol and placed on a paper towel to dry and be weighed. The color extracted in methanol was read for optical density at 595 nm. Percent viability of the treated explants was determined by correcting the optical density (OD) with the weight of the corresponding explant. The other explant was frozen in an embedding medium (Tissue-Tek, Sakura Finetek USA Inc., CA) and processed for histology by cryosectioning and hematoxylineosin staining by Comparative Pathology Program/Histology and Imaging Core Research Laboratory, University of Washington School of Medicine at South Lake Union, Seattle, WA.

Statistical and mathematical analyses
The IC 50 values were calculated using a four-parameter sigmoid regression and bootstrapping (MATLAB R2010b software, Math-Works, Natick, Massachusetts) as previously described [43]. Briefly, confidence intervals were determined using a sampling procedure that created data sets by random sampling with replacement for curve fits 1000 times. The IC 50 values for each drug alone and in combination are presented as median IC 50 , 95% confidence interval (C.I.) based on percentiles from a histogram of IC 50 values, and the coefficient of variation (Cv). The combination effect was analyzed using MATLAB_R2010b software.

ARVs are effectively formulated into polymeric nanoparticles
We demonstrate that ARV compounds with low aqueous solubility can be formulated into PLGA nanoparticles with reproducible size, shape, and high drug loading content. We chose EFV and SQV as model drugs with low aqueous solubility (,0.1 mg/mL) that may be challenging to combine with TFV, especially for topical microbicide applications. The calculated value of the partition coefficient (logP) and aqueous solubility are useful parameters to determine the physicochemical properties of the ARVs [44]. The logP values of EFV and SQV are 3.8-4.5, and their aqueous solubility at 25uC are 8.55 mg/mL and 2.47 mg/mL, respectively [45]. Despite the similar logP and aqueous solubility, EFV and SQV required different methods for encapsulation into PLGA nanoparticles. For NP-EFV, a single emulsion-solvent evaporation technique was employed wherein the EFV and polymer were combined in DCM and aqueous PVA was used as a surfactant. NP-SQV formulated by the same technique resulted in low loading (,1% w/w) (data not shown). Therefore, NP-SQV were fabricated by a nanoprecipitation method in which two miscible solvents are employed to induce the precipitation of the drug and polymer mixture. Nanoprecipitation allows for instantaneous particle formation due to the miscibility of the polymer solvent and non-solvent, compared to the slower particle hardening process that occurs with the single emulsion process [27,29]. We also modified the formulation process by using acetone and adjusting the solvent/non-solvent ratio to prevent partition of SQV to the aqueous phase. Table 1 lists properties of NP-EFV and NP-SQV fabricated with emulsion and nanoprecipitation techniques, respectively. Both NP-EFV and NP-SQV showed a large negative zetapotential of approximately 225 mV, a value predictive of high colloidal stability due to large repulsive charges [46]. NP-EFV had a particle size of approximately 200 nm with low polydispersity (,0.08). However, in some cases, NP-SQV showed two distinctly sized populations, indicating bimodal distribution. One population had a mean diameter of ,100-200 nm, and we detected a second population with a mean diameter of ,600-2500 nm. We used scanning electron microscopy (SEM) to confirm the size and morphology of nanoparticles. SEM micrographs revealed that both NP-EFV and NP-SQV were spherical with an average particle diameter of ,200 nm ( Figure 2A). Owing to the SEM results, we expect the bimodal distribution observed with DLS can be attributed to a population of nanoparticle aggregates in aqueous suspension. Our findings are similar to those describing manufacturing of PLGA nanoparticles via emulsion and nanoprecipitation techniques [28,29,37,47,48]. Our results suggest that these two techniques are suitable for encapsulating hydrophobic drugs.
FTIR spectroscopy and UV-HPLC was used to confirm drug loading into polymer nanoparticles. FTIR absorption spectra for NP-ARVs detected characteristic bond vibrational frequencies for both the drug compound and the PLGA polymer. Infrared absorbance spectra of NP-SQV demonstrated characteristic frequencies of the phenyl (1500 cm 21 ) and amide carbonyl (1695 cm 21 ) present in the drug, as well as the ester stretching frequency (1750 cm 21 ) indicative of the polymer ( Figure 2B). FTIR spectra of NP-EFV showed absorption bands at 2300 cm 21 from the alkyne and at 600-800 cm 21 from the C-Cl alkyl halide stretching, in addition to the ester band from the PLGA polymer. These FTIR results strongly indicate drug loading within the polymer nanoparticles.
To quantify actual drug loading and encapsulation efficiency, we employed established methods for detection and separation of EFV and SQV from excipients of the nanoparticle formulation process using UV-HPLC. We demonstrate that nanoparticles prepared with a theoretical drug loading of 15% (weight of ARV to weight of polymer, w/w) achieved average actual drug loading of approximately 7% (w/w) and encapsulation efficiency of approximately 50% (Table 1). We validated our method for dissolution of the polymer matrix to release the drug for detection using vehicle control nanoparticles (no drug) spiked with known quantities of drug. These validation experiments indicated a high recovery (97-99%) and demonstrated the accuracy of our methods to quantify drug loading. As shown in Figure 2C, SQV and EFV were detected only in ARV loaded nanoparticles, whereas no compounds of similar retention time were detected in the vehicle control nanoparticles. NP-EFV had a comparatively high drug loading of 6.760.4% (w/w) sufficient for use in our in vitro efficacy studies [49,50,51]. Using the nanoprecipitation technique, we obtained NP-SQV that had 24 times higher drug loading and encapsulation efficiency of ,50% (Table 1) compared to NP-SQV formulated using a single emulsion technique.
To determine if we could achieve higher encapsulation efficiencies, we also prepared nanoparticles with a lower initial drug loading of 5-7.5% (w/w). For NP-EFV, we observed that decreasing the theoretical drug loading decreased the actual loading of EFV, but had no effect on the encapsulation efficiency. NP-EFV with 15% (w/w) theoretical drug loading improved the actual drug loading by 2-fold compared to preparing particles with 5% (w/w) theoretical drug loading (actual drug loading = 3.060.45% w/w). In contrast to NP-EFV, we observed that reducing the initial amount of SQV used in the nanoprecipitation process doubled the encapsulation efficiency without reducing the drug loading. The actual drug loading of NP-SQV was independent of the initial loading in the range tested. NP-SQV with a theoretical drug loading of 7.5% (w/w) or 15% (w/w) had similar actual drug loading of 6-7% (w/w).  Efavirenz (EFV) and Saquinavir (SQV) nanoparticles were synthesized using single emulsion and nanoprecipitation techniques, respectively. b The particle size, polydispersity indices (PDI) and zeta potential were determined using dynamic light scattering (DLS); data are the average of three batches. c Particles showed two peak sizes. This number represents the average peak intensity size for three batches of SQV nanoparticles. Two of the three batches displayed bimodal size distribution with one peak ,100-200 nm and another peak at ,600-2500 nm. Since the large peaks are likely indicative of aggregated clumps of particles and not individual particles, these large peaks were not included in the average size shown here but still affect the PDI value. doi:10.1371/journal.pone.0061416.t001

Nanoparticles modulate ARV release
We investigated the drug release profile of NP-ARVs using a vaginal fluid simulant (VFS) that mimics the composition and viscoelastic properties of cervico-vaginal secretions produced by healthy, non-pregnant, premenopausal women [31]. UV-HPLC was used to measure drug release from nanoparticles into VFS over 144 h. The in vitro release of both EFV and SQV from nanoparticles followed a biphasic release profile, where an initial burst release of 10-20% of drug was observed within 1 h followed by sustained drug release over 24 h. During the first 24 h, we observed a total cumulative release of 33.963.9% and 42.466.6% for EFV and SQV, respectively ( Figure 2D). Although we measured drug release out to 144 h, we did not detect a significant accumulation of drug release after 24 h. The percent cumulative release at 24 h corresponds to a mass ratio of 0.022 mg EFV/mg PLGA and 0.025 mg SQV/mg PLGA released at 24 h. Based on drug loading and release results, as well as the reported IC 50 values for free ARVs, we estimated that delivering approximately 10 23 mg/mL of NP-SQV or 10 26 mg/mL of NP-EFV would be sufficient to observe in vitro protection using the TZM-bl assay.

NP-ARVs are nontoxic to in vitro cell line and ex vivo ectocervical explants
PLGA nanoparticles loaded with EFV or SQV were neither cytotoxic to cells nor tissue explants over the range of concentrations evaluated. We evaluated cytotoxicity of our NP-ARVs in TZM-bl cell culture before testing their bioactivities to exclude effects of nanoparticles on the viability of TZM-bl cells. Cytotoxicity of our NP-ARVs was measured over a range of polymer concentrations from 1 to 10,000 mg/mL after 48 h of exposure. Compared with the negative control (media only), vehicle control nanoparticles at concentrations #5 mg polymer/ mL showed no reduction of viability (100%68%), suggesting that PLGA nanoparticles alone are not cytotoxic below these concentrations. We observed .80% viability of TZM-bl cells for NP-EFV and NP-SQV tested at #1,000 mg of polymer/mL (#48 mM EFV and #26 mM SQV) (Figure 3). Both NP-EFV and NP-SQV showed cytotoxicity at concentrations .5 mg polymer/ mL (.240 mM EFV and .130 mM SQV). Since anti-HIV bioactivity was measured at doses well below polymer concentrations that were cytotoxic, we did not expect toxicity to confound the outcome of the antiviral activity assays.
To confirm the results obtained with in vitro cytotoxicity in TZM-bl cells, we evaluated the safety of our NP-ARVs using polarized explant cultures. We chose to test nanoparticles (NP-ARVs or vehicle control) at a 0.1 mg/mL dose. This dose was shown to be nontoxic to TZM-bl cells and is several-fold higher than doses required for efficacy in vitro. We used two explant tissues per treatment -one for the MTT assay and the other for histology. For controls, explants were treated with either 0.4% nonoxynol-9 (N-9) or media (untreated). We evaluated explants for viability and tissue morphology at 18-24 h after application. The N-9-treated explants showed significant reduction in tissue viability, as measured by the MTT assay (n = 1), and destruction of the epithelial layer was observed by histology (n = 1) (Figure 4). The toxicity of N-9 found in our study was consistent with previous studies performed using human explant tissues [40,41]. Viability of explants determined by the MTT assay showed that neither NP-EFV (n = 1) nor NP-SQV (n = 1) altered tissue viability compared to the media control (untreated explants, n = 3) ( Figure 4B). Histological analysis of the ectocervical explants after 24 h of exposure to NP-ARV produced no visual changes to the integrity of epithelial layer ( Figure 4C). The results with ectocervical tissue explants confirmed the findings obtained using the TZM-bl cytotoxicity model, suggesting that our NP-ARV are nontoxic to the cells and are a biocompatible vehicle for drug delivery to the mucosal tissue, particularly the ectocervical tissue of the lower female reproductive tract.

NP-ARVs potently inhibit HIV-1 BaL infection
To ensure ARVs loaded into nanoparticles retained reproducible bioactivity against HIV-1, we tested three batches of NP-EFV using the TZM-bl assay. As described in the methods, a mass concentration of the drug-loaded polymer was delivered to achieve the desired molar drug concentration in the assay volume irrespective of the observed drug release kinetics. We observed consistent activity between three independent batches of NP-EFV to inhibit HIV-1 BaL at nanomolar levels, with an average IC 50 value of 0.5260.17 nM (mean 6 SD, n = 3). This value is lower than previously reported IC 50 values of unformulated (free) EFV, indicating no loss in drug activity due to the formulation processes [52].
We further evaluated antiviral activities of NP-ARVs in comparison with their free forms. After exposure of TZM-bl cells to NP-ARVs or free ARVs, we observed potent antiviral activity against HIV-1 BaL with estimated IC 50 values in the nanomolar and micromolar ranges for EFV and SQV, respectively ( Table 2). Compared with free EFV, NP-EFV showed higher HIV inhibitory activity, with a 50-fold reduction in IC 50 (Table 2 and Figure 5A). NP-SQV also showed higher HIV inhibitory activity when compared with free SQV, with a nearly 2-fold reduction of the IC 50 (Table 2 and Figure 5C). We observed that blank nanoparticles (vehicle control) tested at the same ranges of polymer concentrations showed no HIV inhibition and were comparable to the negative media control (,5%). This indicates that PLGA nanoparticles alone do not provide inhibition against HIV-1 infection. Together, our results suggest that ARVs loaded into nanoparticles possess potent bioactivity that is superior to that of unformulated ARVs. Since PLGA nanoparticles are known to enhance internalization and intracellular uptake [49,53,54,55], we hypothesize that the enhanced potency of our NP-ARVs formulated with PLGA may be due to higher intracellular ARV drug concentration.

NP-ARVs showed potent HIV inhibitory activity alone and in combination with TFV
We conducted drug combination studies to identify unique drug-drug activities facilitated by our nanoparticle delivery systems. The activity of drug combinations is typically assessed at their equipotency ratio (1:1 ratios of IC 50 values), which is the ratio at which the contribution of each drug to the combined efficacy is estimated to be equal [39]. However, there is no established precedence for conducting combination studies with nanoparticle-formulated drugs. Therefore, in addition to the equipotency ratio, we tested other ratios of the NP-ARVs in combination with free TFV.
The dose-response relationships showing the inhibitory activities of free TFV alone and in combination with either EFV or SQV (both free and nanoparticle formulations) against HIV-1 BaL are presented in Figure 5 and Table 2. Free drug combinations of EFV and TFV at their equipotency ratio (1:1 ratio of IC 50 , or 1:11 EFV:TFV molar ratio) had greater anti-HIV activity and showed a lower value of the IC 50 measured for either of the individual ARVs used alone. For example, the combination of free TFV and free EFV produced an IC 50 value of 10 nM, whereas the IC 50 values for free TFV and free EFV used alone were 1.8 mM and 0.2 mM, respectively. Therefore, the combination of the free drugs was 20-200 times more potent than either of the single ARVs.  However, NP-EFV combined with free TFV at their equipotency ratio (1:1 ratio of IC 50 , or 1:600 NP-EFV:TFV molar ratio) showed an IC 50 (,1.0 mM) that was intermediate in value between free TFV (1.8 mM) and NP-EFV (,3.0 nM) used alone. As such, in contrast to the enhanced potency obtained from combining the free drugs, and despite the 50-fold enhanced potency of NP-EFV compared to free EFV, the combination activity of NP-EFV and free TFV appeared to be dominated by the activity of free TFV alone. This result may be unsurprising because, at a molar ratio of 1:600 of NP-EFV:TFV near the IC 50 value, the number of nanoparticles per cell is ,10 whereas the number of molecules of TFV per cell is .10 14 . In the limit where the nanoparticle number is small relative to the number of cells, significant heterogeneity within the system may confound the true measure of combination activity of multiple drugs. Based on this observation, we combined NP-EFV and free TFV at the same molar ratio used to achieve equipotency of the free drugs (1:11 EFV:TFV molar ratio). At this ratio, we found that NP-EFV in combination with free TFV had a 3-fold reduction in the IC 50 value compared to the free drug combinations ( Figure 5B).   The combination activity of NP-SQV with free TFV also demonstrated favorable dose reduction that was superior to the combined activity of free SQV with free TFV ( Figure 5D). The antiviral activities of free TFV combined with either free SQV or NP-SQV were tested at their equipotency ratio, which corresponded to molar ratios of 1:5 TFV:SQV and 1:3 TFV:NP-SQV. We found that the combination of free TFV and free SQV did not significantly enhance antiviral potency as was observed when free TFV was combined with free EFV. The IC 50 measured for the combination of free TFV and free SQV was intermediate in value compared to the IC 50 measured for the individual ARVs (Table 2). Compared to free TFV alone, the IC 50 value for the combination of free TFV and free SQV increased by approximately 3-fold (5.95 mM vs. 1.81 mM). This indicates that the inhibitory activity of free TFV combined with free SQV was lower than that of free TFV alone. However, when compared to free SQV alone, the IC 50 value for the combination of free drugs decreased approximately 2-fold (9.79 mM vs. 5.95 mM). This result suggested that the inhibitory activity of combined drug was greater than that of free SQV alone. In contrast to the combination free TFV and free SQV, free TFV combined with NP-SQV showed greater inhibitory activity than that of free TFV and free SQV alone. We found that combination of free TFV and NP-SQV resulted in a 20-fold reduction in the IC 50 value (,3 nM) compared to the free drug combinations (,6 mM). Collectively, the dose reduction results indicated that NP-EFV and NP-SQV used alone or in combination with free TFV showed greater potency than the free drug equivalent.

NP-ARVs combined with free TFV exhibit strong synergistic activity
The CI was determined for ARV drugs combined at molar ratios to achieve equipotency (1:1 ratios of IC 50 values). The CI of each free drug or NP-drug combination was plotted as a function of the fractional inhibition (Fa) from 0.10 to 0.95 ( Figure 6). The plots show symmetric curves of fractional inhibition and combination indices (log CI), demonstrating that synergism and antagonism can vary depending on fractional inhibition values. For example, the CI plot of free SQV combined with free TFV demonstrated an additive effect at the 50% fractional inhibition (CI 50 = 1.04) and synergistic effects with CI 70 and CI 95 values of 0.8 and 0.2, respectively ( Figure 6C). We interpreted the combination effects at the CI 50 value, and defined drug synergy as a CI 50 value less than 1.
Free EFV and NP-EFV combined with free TFV at their respective equipotency ratio (1:1 ratio of IC 50 values) demonstrated a synergistic (CI 50 = 0.01) and additive (CI 50 = 1.05) effect, respectively ( Figure 6A and 6B). However, when combined at the equipotency ratio used for the free drugs (1:11 molar of EFV:TFV), NP-EFV and TFV demonstrated a strong synergistic effect with a measured CI 50 value of 0.07 ( Figure 6A). The free drug combinations of EFV and TFV were also measured at the equipotency ratio used for NP-EFV and free TFV (1:600 molar ratio, respectively), and we observed a CI 50 value of 0.58, which is indicative of moderate synergy ( Figure 6B). Thus, we observed that the combination activity depends on the dose ratio of the drugs and that unique drug-drug activities are observed when delivering drug combinations with polymeric nanocarriers.
Combination effects of TFV with either free or encapsulated SQV were tested at 1:1 ratios of their IC 50 values, which corresponded to a 1:5 TFV:SQV and 1:3 TFV:NP-SQV molar ratio ( Figure 6C). We found that free TFV combined with free SQV showed only an additive effect (CI 50 = 1.04). However, a synergistic effect (CI 50 = 0.07) was observed from combining of free TFV and nanoparticle-SQV. Collectively, drug synergy data demonstrate that combining free TFV with either NP-EFV or NP-SQV results in pronounced synergistic effects.

Discussion
Combination drug approaches are an emerging paradigm for the prevention of HIV-1 infection. However, chemical incompatibilities prevent the combination of many existing drugs in a manner that could enhance potency by realizing unique combination drug activities. We hypothesized that polymeric nanocarriers could facilitate the discovery of unexplored drug-drug activities by enabling the combination of chemically incompatible agents. We demonstrate that ARVs can be encapsulated within polymeric nanoparticles to provide synergistic prophylaxis in combination with TFV. We also report on novel combinations of NP-ARVs with TFV that show synergistic anti-HIV activity in vitro. Our findings reveal a relevant strategy for delivering multiple ARVs in combination to enhance drug potency, lower cytotoxicity and reduce the likelihood for developing drug resistance. We expect that the versatility of nanoparticle delivery platforms will result in broad applications for HIV chemoprophylaxis and treatment. ARV-encapsulating nanoparticles may overcome barriers in the delivery of agents with diverse physicochemical properties, particularly via administration routes with limited dosage forms for combination drug delivery such as topically to the genital and rectal mucosa.
Our NP-ARVs demonstrate enhanced antiviral activity against HIV-1 BaL compared to unformulated ARVs. Using an in vitro TZM-bl indicator cell model previously used to evaluate drug candidates for topical microbicides [37,38], we observed higher inhibitory activity of NP-EFV than free EFV with a 50-fold reduction in IC 50 . Similarly, NP-SQV showed greater anti-HIV activity when compared to the free drug. NP-ARVs formulated with PLGA have previously been demonstrated to facilitate drug uptake resulting in higher intracellular drug concentrations and greater inhibitory activity [49,55]. Destache et al. showed that monocyte-derived macrophage (MDM) cells incubated with ARV loaded PLGA nanoparticles exhibited higher intracellular drug concentrations than those incubated with free ARVs. Although we did not measure intracellular uptake of particles in this study, the 50-fold reduction in IC 50 values between NP-ARVs and free drugs suggests that the particulate nature of our delivery platform plays a major role in improving bioactivity. As Destache et al. observed, it is likely that the PLGA nanoparticles facilitate increased drug uptake and intracellular retention of our ARV drug candidates.
Based on the use of TFV in both oral and topical prophylactic prevention trials, we were motivated to explore the possible enhanced activity when combining free TFV with our NP-ARVs. These dose reduction results indicate that less of each drug was required to inhibit HIV-1 when compared with single-drug use. NP-ARVs alone and in combination mediated even greater dose reduction compared to the free drug equivalents. Therefore, NP-ARVs may be considered superior to free ARVs, since NP-ARVs combined with free TFV provided enhancement in dose reduction up to 600-fold. While dose reduction results can be used to predict the optimal therapeutic doses, they do not provide quantitative information to indicate the degree of synergism [32]. In other words, IC 50 values obtained from dose-response studies by themselves did not reflect whether the combination effect was additive, synergistic, or antagonistic. This is particularly evident when the IC 50 values of combined drugs are only slightly different from the single-drug treatments. As found in this study, the IC 50 values of free TFV combined with either free SQV or NP-SQV were of the same order of potency with an observed 2 to 7-fold dose reduction.
Using the median-effect analysis, we were able to quantify synergistic effects of combined drugs. We found that the combination of free TFV with free EFV at a 1:1 ratio of IC 50 values demonstrated a synergistic effect ( Figure 6A). Similar synergistic effects were observed when the combination of free TFV and free EFV was tested in MT-2 cells [52]. However, when we used a 1:1 ratio of IC 50 values to evaluate the combination effect of NP-EFV with free TFV, we observed only an additive effect ( Figure 6B). It may not be expected that free drugs used in combination would result in the same effect as the identical drug combinations delivered using NP-ARVs. Unlike many small molecule drugs whose cellular transport is dominated mostly by passive diffusion, the delivered drug dose from nanoparticles depends on the drug loading and release kinetics as well as the properties of the particles (number, size, shape, chemistry) and cells (density, internalization rate). At the equipotency ratio of NP-EFV and free TFV, the molar drug dose of TFV is in 600-fold excess to EFV delivered via nanoparticles. The molar excess of TFV may actually be higher given that only 10% of the loaded EFV was released from the nanoparticles within the first hour. The effective EFV dose delivered from nanoparticles also depends on the average number of NP-EFV per cell, which is at most ,6 at the IC 50 value for the combination dose. Therefore, at the equipotency ratio for NP-EFV and free TFV, these differences may bias the activity of the free drug and explain the observed IC 50 value and combination effect for NP-EFV and free TFV. When instead we tested the combination effect of NP-EFV and free TFV at the same molar ratio used for the free drug combinations, we observed synergistic activity ( Figure 6A). Our result supports observations that, while combination studies are recommended to be performed at the equipotency ratio, other combination ratios may be optimal to achieve maximum synergy. Experiments that evaluate combination effects at all possible combinations using different concentrations of NP-ARVs and free drug may be necessary to inform the use of nanocarriers for combination drug delivery [56].
The combination of NP-SQV and free TFV demonstrated superior action to that of free SQV/free TFV combination ( Figure 6C). SQV is the first HIV-protease inhibitor marketed for the treatment of HIV and is currently recommended in combination therapy regimen with nucleoside inhibitors of HIV reverse transcriptase [9,14]. Nevertheless, its poor absorption is a prominent pharmacologic characteristic of SQV [19,57,58,59]. We demonstrated a greater synergistic effect by combining NP-SQV with free TFV (CI 50 = 0.07) than that of combining free SQV with free TFV (CI 50 = 1.04). Previous studies have shown that administration of polymeric nanoparticles significantly increases the intracellular concentration and permeability of SQV [60,61]. We expect that the improved bioactivity of our NP-SQV is due to similarly increased bioavailability provided by the nanoparticle formulation.
The in vitro drug release profile of our NP-ARVs showed an initial burst release within 1 h followed by sustained release of drug over 24 h ( Figure 2D). We expected that this biphasic release profile would provide a sufficient amount of rapid release of ARVs to prevent HIV-1 infection soon after application and then sustain the release of drugs for prolonged protection. The in vitro release studies from EFV nanoparticles may have been limited by poor aqueous solubility of EFV and small release buffer volumes needed to maintain a concentration detectable by HPLC, resulting in sink conditions not being met. Future release experiments using a cosolvent such as polysorbate 80 or PEG 400 may prove useful in establishing sink conditions [62,63]. In vitro release profiles can be affected by the environmental conditions. As reported by Ham et al., the percent drug release from nanoparticles at pH 7.4 was lower compared to the release profiles obtained at pH 4.6 [37]. While we have not yet investigated in vitro release profiles at different pH conditions, further in vitro release studies are planned to determine the impact of pH on drug release and how the release patterns can help in determining the synergistic combination ratios.
Currently, nanoparticle encapsulation as a drug delivery system is being investigated in numerous therapeutic fields [17]; however, it is still emerging within the microbicide field [16]. In this study, we provide evidence for the efficacy of a PLGA nanocarrier system to achieve HIV-1 inhibition. Our results suggest that PLGA nanoparticles are a safe delivery platform for encapsulating and delivering ARVs. Furthermore, we show that our NP-ARVs act synergistically with TFV in preventing the infection of HIV-1 in vitro. Different models describe the classification of synergy, antagonism, and additive activity arising from the combination of multiple drugs [56]. We applied the approach of Chou and Talalay based on the median-effect principle for mutually exclusive drug combinations. This model is often chosen even though it assumes that the combinations involve multiple drugs competing for the same binding site. In general, the model provides a conservative estimate of inhibition and deviates from the alternative model for nonexclusive or independent drug action only at high drug concentrations [64]. Therefore, we expect that the synergy classification prescribed by our model for NP-ARV combinations to be supported by other methods used for combination studies, and deserves further study. To our knowledge, this work reports the first quantitative measure of synergy by combining ARV-nanoparticles and TFV. Our results also highlight new opportunities to design and quantify combination studies mediated by nanocarrier delivery systems.


Nitric Oxide Donors as Neuroprotective Agents after an Ischemic Stroke-Related Inflammatory Reaction

Cerebral ischemia initiates a cascade of detrimental events including glutamate-associated excitotoxicity, intracellular calcium accumulation, formation of Reactive oxygen species (ROS), membrane lipid degradation, and DNA damage, which lead to the disruption of cellular homeostasis and structural damage of ischemic brain tissue. Cerebral ischemia also triggers acute inflammation, which exacerbates primary brain damage. Therefore, reducing oxidative stress (OS) and downregulating the inflammatory response are options that merit consideration as potential therapeutic targets for ischemic stroke. Consequently, agents capable of modulating both elements will constitute promising therapeutic solutions because clinically effective neuroprotectants have not yet been discovered and no specific therapy for stroke is available to date. Because of their ability to modulate both oxidative stress and the inflammatory response, much attention has been focused on the role of nitric oxide donors (NOD) as neuroprotective agents in the pathophysiology of cerebral ischemia-reperfusion injury. Given their short therapeutic window, NOD appears to be appropriate for use during neurosurgical procedures involving transient arterial occlusions, or in very early treatment of acute ischemic stroke, and also possibly as complementary treatment for neurodegenerative diseases such as Parkinson or Alzheimer, where oxidative stress is an important promoter of damage. In the present paper, we focus on the role of NOD as possible neuroprotective therapeutic agents for ischemia/reperfusion treatment.

Introduction
When the brain blood flow is interrupted, it results in deprivation of oxygen and nutrients to the cells; this situation constitutes an ischemic stroke. Restoration of the flux, or reperfusion, can reduce the damage, but only when this is performed very early after the onset of ischemia, and its efficacy is restricted by secondary injuries, mainly by oxidative stress (OS) and an inflammatory reaction, which lead to cell death by apoptosis [1].
It is noteworthy that ischemic damage not only affects neurons. Thus, in recent years the concept of the neurovascular unit has been highlighted, emphasizing the need to protect not only the neurons, but also all cells in the brain [2][3][4][5].
In contrast to the known vulnerability of neurons and astrocytes, it is thought that endothelial cells tend to be more resistant to ischemic or oxidative injury [6]. Hence, to be successful, stroke therapies should be widely effective and must protect all neuronal, glial, and endothelial components in the brain [7].
After focal ischemia, primary neuronal death appears rapidly in the core area and is followed by secondary death in the ischemic penumbra, which evolves from the delayed activation of multiple cellular death pathways. At the core of the ischemic lesion, one of the first events is the rapid decline of adenosine triphosphate (ATP) reserves [8]. Consequently, all energy-dependent processes gradually cease their activity, leading to changes in transmembrane potential. The consequent depolarization (denominated anoxic depolarization) produces massive influx of Na + , Cl − , and Ca 2+ inside the cell with K + efflux [9].
Core anoxic ischemic depolarizations induce release of neurotransmitters such as glutamate. Once released, glutamate generates a phenomenon of peri-infarct depolarization, which increases energy consumption and promotes Ca 2+ influx into the cells [10].
The increase in intracellular Ca 2+ in neurons and glial cells initiates a set of nuclear and cytoplasmic events that produce deep brain tissue damage that includes the following: Ca 2+ mitochondrial overload (which compromises the already affected ATP production and promotes the opening of the mitochondrial transition pore); the increase in OS, and the activation of a number of Ca 2+ -dependent enzymes. Such enzymes include proteases, kinases, phospholipases, and endonucleases, which destroy biomolecules [10]. Additionally, increased intracellular Ca 2+ also promotes the production of NO from constitutive synthases that, together with acidosis and peri-infarct depolarization, contribute to the initiation of damage; later, inflammation and activation of apoptotic phenomena contribute to increased injury [2].
OS is a major mechanism implicated in stroke and in a variety of neurodegenerative diseases, mainly in Alzheimer and Parkinson (reviewed in [11,12]). The most accepted theory regarding neurodegeneration in Parkinson disease refers to OS as the main cause of damage to neurons in the substantia nigra. In addition, in Alzheimer disease, the OS generated by the action of -amyloid, which causes massive entry of Ca 2+ and caspase activation, leads to neuronal death [13,14].
During ischemia, reactive oxygen (ROS) and nitrogen species can be generated in the ischemic penumbra but can also be produced during reperfusion injury [15,16]. Indeed, it is now established that albeit maintenance of partial or complete blood flow is essential for preserving cerebral tissue, it is during reperfusion when it paradoxically induces excessive generation of ROS, such as superoxide anion radical (O •− 2 ), hydroxyl radical (OH • ), hydrogen peroxide (H 2 O 2 ), and nitric oxide (NO), which contribute to increased neuronal death by oxidizing proteins, damaging DNA, and inducing lipid peroxidation [17].
Reperfusion-induced ROS contribute to a decrease of the NO availability responsible for postischemic endothelial dysfunction [18,19]. During the ischemic period, reduction in O 2 availability reduces the activity of NO synthase, producing O 2− instead of NO; later, during reperfusion, the arrival of O 2 increases NO synthase activity. These can exert a deleterious effect by promoting nitrosative stress and diminishing the availability of NO for preserving endothelial integrity.
Over the past decade, remarkable advances have been made in understanding the basic molecular mechanisms underlying neuronal death. However, clinically effective neuroprotectants have not yet been discovered and no specific therapy for stroke is available at present. The body of experimental data supports the view that reducing OS should continue to be a potentially viable target for stroke therapy [20]. In addition, the inflammatory response requires consideration as a potential target of therapy for ischemic stroke [21]. Therefore, agents capable of modulating both elements will constitute promising therapeutic solutions [22][23][24][25].

Ischemic Lesion and Immune Response: Brain Inflammation
It has now been established that the Central nervous system (CNS) is able to raise an immune response to the majority of threatening stimuli, whereby resident cells generate inflammatory mediators including cytokines, prostaglandins, free radicals, complementary chemokines, and adhesion molecules that recruit immune cells and activate glia and microglia (reviewed in [21,[26][27][28]). The role of microglia and proinflammatory cytokines in the CNS has been characterized in models of brain insults, such as experimental stroke, the most common form of ischemic injury [26]. As mentioned previously, cerebral ischemia triggers acute inflammation, which exacerbates primary brain damage. Although inflammation should be adaptive, the release of proinflammatory cytokines has often been associated with harmful consequences to neurons and myelin [29]. The control of early CNS inflammation is a careful balancing act, as both too much and too little inflammation will lead to decreased or delayed recovery. Whether the inflammation is neurotoxic or protective may depend upon the context and the location of the inflammatory mediator in relation to an injury, and the timing of inflammatory response may determine the outcome (see Table 1 in [27]).
For example, tumor necrosis factor alpha (TNF-) upregulated in the proximity of an evolving lesion contributes to secondary infarct growth, whereas cytokine induction remote from the ischemic lesion confers neuroprotection [30]. TNF-could enhance apoptotic processes through its action on its tumor necrosis factor type 1 receptor (TNFR1) in models of acute (ischemia, excitotoxicity) and chronic (Alzheimer disease, multiple sclerosis) neurodegeneration [31]. TNF-and interleukin 1beta (IL-1 ) exert neurotoxicity in cerebral ischemia in the presence of elevated inducible nitric oxide synthase (iNOS), while in the absence of iNOS, both cytokines appear to contribute to neuroprotection and plasticity, highlighting the role of the context [32].
There is important recognition that protection of endothelial function and downregulation of vascular inflammation comprise part of neuroprotection phenomena and may possess added therapeutic benefit against stroke injury [33]. However, research on clinically effective neurovascular protective therapies for brain damage remains at an early phase [34]. Much attention has been focused on the role of NO in vessel protection from OS and inflammation [35]. Because OS coexists with inflammation and endothelial dysfunction, determining antioxidant status may be helpful in monitoring the progress of Nitric oxide donors (NOD) treatment. A variety of structurally different NOD, which release NO either as a free radical (NO • ) or as an NO ion (NO + /NO − ), have shown to reduce OS/inflammation and to increase cerebral blood flow [35][36][37][38]; thus, these can be considered attractive candidates for therapeutic agents in experimental models of stroke.

Nitric Oxide in Ischemic
Stroke. Nitric oxide (NO) plays a dual role, that is, neuroprotection and neurotoxicity, in the pathophysiology of cerebral ischemia-reperfusion injury [39]. NO is synthesized by NOS, of which there are three known isoforms: nNOS; eNOS, and inducible or immunological NOS (iNOS). The first two are constitutively expressed and their activity is dependent on changes at the intracellular Ca 2+ level, while iNOS acts in a Ca 2+ -independent manner [40]. Baseline concentration of NO in the brain is mainly due to nNOS activity, and secondarily to eNOS. iNOS is not expressed under physiological conditions [41,42].
In the brain, eNOS is mainly produced by the vascular endothelium and the choroid plexus [43]. Although eNOS-NO production is a minor part of total brain NOS activity, this enzyme is critical for the regulation of cerebrovascular hemodynamics and for the protection of endothelium integrity from inflammatory, oxidative, and procoagulant stimuli. It has been demonstrated that eNOS-derived NO scavenges ROS [44] and inhibits the expression of cellular adhesion molecules [45], platelet aggregation [46], and leukocyte adhesion [47].
During ischemia, NO concentration decreases because of oxygen deficiency [41]. However, immediately after reperfusion, biosynthesis of this molecule is triggered mainly by overactivation of nNOS, as evidenced in nNOS (−/−) mice [48] or with specific inhibitors such as 7-NI [41,49]. Glutamate-induced Ca 2+ overload in ischemic neurons is responsible for the rise of nNOS-derived NO [50]. Concentration of NO returns to physiological levels approximately 1 h after reperfusion [48,49,51] and increases again due to iNOS expression between 12 h and up to 8 days later [52,53]. iNOS sources at this stage comprise microglia [53], astrocytes [54], endothelial cells [55], and infiltrated leukocytes [56]. The amount of iNOS-NO derived is 100-1,000 times than that produced by nNOS [57]. Therefore, NO deriving from different sources (neurons, brain vessels, glia, and neutrophils) may exert an influence on the evolution of brain damage at different time-points after an ischemic insult [42]. Thus, the use of relatively selective inhibitors of NOS isoforms and genetically modified mice has contributed to clarifying the role of NO in cerebral ischemiareperfusion injury as follows.
iNOS expression is transcriptionally regulated by nuclear factor kappa B (NF-B) secondary to moderate inflammatory stimuli such as TNF- [69] and IL-1 [70], and also by oxidative radicals [71]. Due to the large amount of NO produced by iNOS, this enzyme is related with high peroxynitrite production and significant nitrosative damage of biological molecules [72].
Consequently, nNOS mediates early neuronal injury, while iNOS contributes to late neuronal injury, whereas eNOS activity might be protective [41,42,73]. Whether the effects of this molecule are beneficial or harmful depends on the cellular compartment in which NO is generated, on its concentration, on the environment's redox state, and on the evolutive stage of ischemic brain injury [42,48,73].
In addition to their involvement in ischemia, the expression of iNOS in astrocytes and microglia and the production of large amounts of NO contribute to dopaminergic neuronal death in the neuropathology of experimental Parkinson disease [74,75]. In the brains of patients with Alzheimer disease, nitrosylation of proteins is a hallmark of tissue damage [76] and is particularly high in hippocampus and cerebral cortex. The presence of beta amyloid is sufficient for triggering iNOS activation in astrocytes and microglia [77]. In addition, nNOS activity in reactive astrocytes surrounds beta-amyloid plaques in entorhinal cortex and is related with DNA fragmentation in CA1 and CA4 fields [78], while there is a correlation between neurofibrillary tangles and senile plaques with a reduction in eNOS capillary levels [79].
According to the dual role of NO in brain ischemia, there is a rationale for the use of NO for promoting treatments shortly after the occurrence of focal cerebral ischemia as neuroprotective strategies [42,53,80,81]. The neurovascular protective mechanism of eNOS-NO suggests that intervention with NO may be most effective when delivered at an optimal amount by a suitable source at the correct time in an appropriate environment [82].
Despite the fact that all of these are considered NOD, they have different pharmacokinetic and dynamic profiles that determine the type and extent of their biological effects. The main determinant of these effects is the manner in which NO is released, the amount of NO generated, and the time during which it is released. Moreover, some of these generate alternative products that may arise during their metabolism or   decomposition. These products may even be present in quantities exceeding NO with independent or side effects [92]. For example, organic nitrates, the most common NOD utilized in coronary artery disease, require enzymatic bioactivation in order to deliver NO [93]. Their principal effect is at the vascular level, by increasing venous capacitance and coronary vasodilation [84]. S-nitrosothiols are a heterogeneous group characterized by a nitroso group attached by a single chemical bond to the sulfur atom of a thiol [83]. S-nitrosoGlutathione (GSNO) is found in vivo and is an important intermediary in organic nitrate metabolism. The remaining nitrosothiols are synthetic. These compounds act as NO-carriers, NO-reservoirs, and intermediates in protein nitrosylation. They also possess the ability to transfer the different NO species through chains of thiols, without releasing the NO molecule itself. This feature decreases the possibility of NO reacting with O 2− to form ONOO − , or that of reacting with other molecules to nitrosylate these [94,95]. Sydnonimines release NO spontaneously, without enzymatic activity. Superoxide is generated concomitantly and may combine with NO to generate ONOO − . This process also releases significant quantities of hydroxyl radical, which increases its prooxidant potential [96]. Therefore, these compounds are considered peroxynitrite donors more than NOD and are utilized as nitrosative stress inducers in experimental models. NONOates decompose spontaneously in solution, at physiological pH and temperature, without interaction with biological molecules and in a concentrationdependent manner; thus, they usually are employed as NOrelease models [90].
Sodium nitroprusside (SNP) is a compound consisting of an iron core surrounded by five Cyanide ion (CN − ) molecules and one molecule of the Nitrosonium ion (NO + ). SNP does not liberate NO spontaneously in vitro, but does require partial reduction (one-electron transfer) by a variety of reducing agents present in membrane cells. It is also possible to release NO from SNP by photolysis. In addition to NO, SNP can release, in aqueous solution, a range of oxidant and free radical species, such as iron, cyanide, superoxide, H 2 O 2 , and hydroxyl radical in direct proportion to its concentration [97][98][99]. Because of the nitrosative and prooxidant potential inherent in the different NOD, these have been widely used as models of neuronal damage (for a more detailed review, see [83,84,94]).

Sodium Nitroprusside-Induced
Neurotoxicity. The potentially adverse effects of SNP on cells and tissues have been widely used in vivo and in vitro to study the mechanisms involved in nitrosative and OS injury. While many of the pharmacological effects elicited by SNP are attributed to the NO molecule, several in vitro studies [100,101] revealed other biological SNP properties that are independent of the NO moiety, due to the huge number of by-products released during its decomposition (e.g., cyanide, iron, and ROS).
In vitro, SNP is usually neurotoxic. This compound is able to cause cytotoxicity in the human neuroblastoma cell line SH-SY5Y by means of OS. In addition, SNP treatment activates the (ERK1/2) pathway and inactivates the Akt pathway, leading to cell death [102]. Additionally, inhibition of ERK activation or exogenous Superoxide dismutase (SOD) treatment protects human melanoma from SNP toxicity [103]. Furthermore, in hippocampal neurons, SNP and SIN-1 are capable of decreasing Bcl-2 and increasing Bax expression along with caspase-3 activation, leading to neuronal apoptosis [104]. Concentration-dependent neuronal death was induced in cerebellar granular cells after exposure to SNP by hydroxyl radical generation, as well as by increasing the level of iron and lipid peroxidation [105]. In cultured cholinergic cells, SNP impairs oxidative metabolism of Acetyl Co-A by suppression of choline acetyltransferase and pyruvate dehydrogenase activities in mitochondria and cytoplasm. This effect triggers OS and a reduction in neuronal viability [106].
In addition to SNP, other NOD are able to elicit cytotoxicity in vitro. In cortical neuronal cultures, SIN-1 induced neurotoxicity by ATP depletion and protein nitration, which was counteracted by the addition of hemoglobin (a NO scavenger), SOD, and an ONOO scavenger, demonstrating that the main mediator of damage in this case is ONOO − [107]. In the same cell culture type, neuronal viability was significantly reduced when compared with that of controls after DETANONOate exposure. This effect was associated with a decrease in catalase activity and expression [108].
Likewise, SNP has been used to induce neurotoxicity in vivo. While not mimicking a specific neuropathology, the rapid and localized neurodegeneration and demyelination caused by the SNP, when injected into the brain, provides a very practical tool for studying the role of the individual molecular players that can be involved in the immediate and consequent damage implicit in neurodegenerative processes. In animal models, SNP causes acute and localized excitotoxic cell death when infused within the brain parenchyma. This damage is also associated with a transient inflammatory response [109]. The neurodegeneration caused by SNP is accompanied by microglial activation and the induction of the proinflammatory cytokines TNF-and IL-1 . Injection of exogenous TNF-was shown to exacerbate the damage and inflammation caused by SNP through specific and transient activation of resident microglia [109]; in contrast, the abolition of the endogenous production of TNF-genetically is also detrimental, because it delays microglial activation, which is later expressed in an excessive manner. However, these effects do not extend to the IL-1B. Thus, this suggests that the source, timing, and dose of TNF-are preponderant in determining the fate of neurons and myelin during SNPinduced neurotoxicity [29].
Additionally, when infused into the substantia nigra, SNP induces an acute increase in lipid peroxidation, which is blocked by NO, oxyhemoglobin, and deferoxamine (an iron chelator), suggesting that OS is elicited, at least in part, by the iron moiety of SNP [110,111]. Thus, the addition of SNP and other NOD to neuronal cultures or into brain parenchyma causes damage through the establishment of OS, nitrosative stress, and the disturbance of cellular oxidative metabolism. The death pathway activated through these mechanisms is mainly apoptotic.
However, it should be considered that the cytotoxic effects of NOD are not necessarily due to the presence of NO, because its addition to culture media alone does not cause neurotoxicity [112]; therefore, other compounds that are also part of the NOD molecules can be delivered with different effects. Such is the case of SNP, in which its toxicity lies more in its content of iron [110,112,113] and cyanide ions [114] rather than in its NO content or, as the case of SIN-1, whose decomposition releases superoxide anion and hydroxyl radicals along with NO, leading to the production of large amounts of ONOO − [96]. 7 Finally, it should be taken into account that the cytoprotective and physiological effects of NO described (e.g., vasodilatation, neurotransmission, endothelial protection) require extremely small concentrations (pico-to nanomolar), while harmful effects take place at higher concentrations, particularly under OS [93]. In culture and in intracerebral application, the NOD concentrations usually administered fall within the micro-to millimolar range. Thus, direct contact of NOD with neurons in culture or intraparenchymally coupled with the high concentration of these could be mimicking overactivation of nNOS or iNOS during the postischemic reperfusion period or in other neurodegenerative disorders [83]. Figure 1 depicts the signaling pathways involved in the neurotoxic effects of NO.

Nitric Oxide as an Anti-Inflammatory and Neuroprotective Agent.
In contrast to the evidence presented in the previous section based on the reactivity of NO with iron and ROS, in 1994 Chiueh proposed that NO and related donor compounds may protect against the OS induced by small-molecular-weight iron complexes in the dopaminergic nigrostriatal system [115]. Since then, a growing number of reports have confirmed the potent neuroprotective and antioxidant actions of NO in the brain in experimental models of Parkinson disease [116][117][118][119][120]. In addition, NO has shown to inhibit lipid peroxidation of low-density lipoprotein oxidation [121,122] in order to protect against neurotoxininduced dopaminergic neurotoxicity [112,118,123,124] and to shield cells from OS [125][126][127], protecting these in vivo through both antioxidative and -apoptotic mechanisms [118].

NO Donors Exerted a Neuroprotective Effect against Cerebral Ischemia-Reperfusion Injury at Different Levels by
Influencing Cellular Oxidative Status. SNP and SPERMINE-NONOate are able to reduce infarct size after transient focal cerebral ischemia when administered early [87]. Likewise, S-nitrosothiols (GSNO and SNAP) and SIN-1 additionally reduced infarct volume and improved neurological performance [38,88,89]. Hemodynamically, SNP, GSNO, and SNAP increase CBF in the penumbral region when administered at the onset of reperfusion [38,80,89].
In addition, pre-and postischemic administration of SNP attenuates the ischemia-induced increase of caspase-3 at 6 h of reperfusion and downregulates neuronal apoptosis by inhibiting increased phosphorylation of JNK, c-Jun, and Bcl-2 [61,65]. This effect is achieved through nitrosylation of nNOS, which decreases its NO production. This means that SNP can regulate NO metabolism in the target cells. In focal ischemia, SNP and S-nitrosothiols decrease lipid peroxidation and nitrotyrosine formation in plasma, which is associated with less oxidative and nitrosative stress, neuroprotection, and fewer anti-inflammatory effects [38].
The effects of NOD on ischemia-reperfusion injury are also related with modulation of the inflammatory response, and these effects are probably the neuroprotective effects with the greatest impact after cerebral ischemia and reperfusion of this drug type. To shield nerve tissue from ischemiareperfusion injury is not sufficient to protect the brain parenchyma, but also must preserve the integrity of the Blood brain barrier (BBB). Thus, the cerebral vascular endothelium is essential in the control of vascular inflammatory and oxidative responses, leukocyte migration, and the production of inflammatory mediators capable of spreading to nerve tissue [26]. Under physiological conditions, eNOS-NO is responsible for maintaining the integrity of the vascular endothelium. But under ischemic conditions, endothelial dysfunction could be offset by mimicking eNOS-derived NO neuroprotective functions by intravascular administration of a NOD [137]. Control of endothelial inflammatory and oxidative responses in turn allows restriction of their impact on resident brain cells, particularly on those with an inflammatory phenotype, such as microglia and astrocytes. Therefore, effective neuroprotection should include protection of the BBB and of the elements within it [138,139].
In vivo, high expression of TNF-, IL-1 , and iNOS in microglia and astrocytes after focal cerebral ischemia is reduced by GSNO. Likewise, GSNO induces a reduction in microglial and macrophage cells in the penumbral region, which is associated with less expression of cellular adhesion molecules such as ICAM-1 in endothelial cells [56]. Decreased expression of adhesion molecules (ICAM-1 and Eselectin) was also demonstrated with SNP and SNAP in the same model [38].
Anti-inflammatory effects are also demonstrated in other neuronal-damaged models, such as experimental autoimmune encephalomyelitis [140] and traumatic brain injury [134]. Under both conditions, NOD inhibited the expression of cell adhesion molecules and infiltration of vascular immune cells into the CNS, which subsequently led to reduction in the expression of proinflammatory cytokines at the site of injury. This suggests less damage to BBB integrity, which is an indicator of neuroprotection.
Beyond the CNS, SNP protects other organs from inflammatory damage. In cardiac surgery, SNP decreases cardiac cytokine release [141][142][143] and improves postischemic cardiac function [143]. In experimental models of ischemiareperfusion injury, such as those in kidney [144] and lung [145], SNP attenuates the expression of proinflammatory cytokines and reduces leukocyte-endothelium adhesion, respectively.

Anti-Inflammatory Mechanisms of Nitric Oxide Donors.
In vitro studies have elucidated some of the mechanisms involved in the anti-inflammatory effect of NOD. It is well documented that cerebral ischemia, and particularly reperfusion, leads to nuclear translocation of NF-B into the core and ischemic penumbra [146,147], as well as into the microvessels of the affected region [148,149]. NF-B is a key regulator of innate immunity, inflammation, and of cell survival and proliferation [150]. This inducible transcription factor is comprised of two subunits. There are five subunits that can (TNF-and IL-1 ) ↑ Figure 1: In vitro and intracerebral effects of sodium nitroprusside and other nitric oxide donors (NOD) on neuronal survival. SNP is capable of releasing or producing diverse byproducts, such as nitric oxide (NO), iron, cyanide anions, hydroxyl radicals, and peroxynitrite. Collectively, these are all capable of inducing oxidative and nitrosative stress [1], with the possibility of modifying the structure and function of proteins, nucleic acids, and lipids by means of oxidation and nitrosylation. Iron, via the Fenton reaction, generates OH − that, together with ONOO − and other reactive species, damage membranes by lipid peroxidation [2] with decreased cellular viability. This effect is blocked by the addition of NO, oxyhemoglobin, and deferoxamine, which suggests the important role of iron and NO in this reaction. The oxidative stress (OS) produced by SNP increases the activation of MEK1/2 and its substrate ERK1/2 by phosphorylation [3]. Both effects are blocked by SOD, suggesting the participation of (O 2− ) in this reaction, probably in the form of ONOO − . Activation of ERK1/2 is associated with a reduction of Bcl2 and an increase in (Bax), and both conditions are associated with an activation of mitochondrial apoptotic pathways. Mitochondria are a target of SNP at different levels: SNP induces lipid peroxidation of its membrane with the subsequent activation of proapoptotic pathways via caspases. In addition, NO and CN − affect the functioning of the mitochondrial respiratory chain, thereby altering mitochondrial membrane potential, reducing ATP production and the generation of large amounts of reactive oxygen species [4]. The addition of ONOO − scavengers and SOD1 counteracts this effect. Also, SNP decreases Akt phosphorylation [5] and reduces the expression and function of SOD1 and catalase [6]. These actions decrease antioxidant responsiveness and the activation of neuronal survival pathways. OH − , hydroxyl radical; ONOO − , peroxynitrite; Akt, protein kinase B (PKB); Bax, Bcl-2-associated X protein; Bcl2, B-cell lymphoma 2; CN − , cyanide anion; ERK1/2, extracellular signalregulated kinase 1/2; IL-1 , interleukin 1 beta; MEK1/2, mitogen-activated protein kinase kinase 1/2; MMP, mitochondrial membrane potential; NO, nitric oxide; ROS, reactive oxygen species; SNP, sodium nitroprusside; SOD1, superoxide dismutase (Cu-Zn); TNF-, tumor necrosis factor alpha.
be combined to yield homo-or heterodimers of NF-B as follows: p50, p52, c-Rel, p65 (RelA), and RelB [151]. C-Relcontaining dimer activation increases neuron resistance to ischemia [152]. Moreover, the prevalent heterodimer during cerebral ischemia and reperfusion is formed by p50-and p65-inducible subunits, and its activation contributes to the pathogenesis of postischemic injury [146,152,153]. NF-B is maintained in latent form in the cytoplasm of cells bound to inhibitory I B proteins. Phosphorylation of I B releases NF-B by permitting its translocation into the nucleus, its binding with NF-B motifs, and the subsequent activation of its target genes. There is, in turn, an enzymatic complex responsible for I B phosphorylation in specific serine residues, the so-called I B kinases (IKK). Activation of IKK is essential to induce NF-B activity [154].
In the ischemic brain, a wide range of stimuli may trigger activation of NF-B including, among others, the following: hypoxia [155]; IL-1, and TNF- [156]; OS [157]; glutamate [158], and NOS activity, such as nNOS and iNOS [159]. Overactivation of NF-B after ischemia has been documented in neurons [147], astrocytes [53], microglia [160], and in endothelial cells [149]. Although in some hippocampal neurons NF-B have a constitutive action related with neuronal survival [150], overactivation of the p50/p65 heterodimer in neurons, glial, and endothelial cells due to ischemia, appears to contribute to acute neurodegeneration. In neurons, NF-B translocation has been associated with apoptosis [146,147], while in glia and in vascular endothelium, NF-B activates a proinflammatory phenotype [53,149,160].
Therefore, blocking inflammatory phenotype activation of NF-B could disrupt the cascade of events that culminate in proinflammatory brain tissue destruction. In human endothelial cells, the addition of exogenous NO through GSNO limits TNF-activation of NF-B in a time-and  Oxidative stress (OS) and inflammatory stimuli phosphorylate NIK. Subsequently, IKK phosphorylates NIK, which in turn phosphorylates IkB, resulting in I b degradation and NF-B translocation and activation. This action exerts different effects depending on the cell line. In endothelial cells, NF-B promotes a proinflammatory phenotype, with the expression of cellular adhesion molecules and proinflammatory cytokines that induce leukocyte migration to the ischemic territory and promote acute inflammation. In glial cells, NF-B leads to the expression of iNOS, COX-2, and proinflammatory cytokines. These effects contribute to nitrosative, oxidative, and inflammatory damage. Finally, in neurons, NF-B induces the expression of apoptosis pathways. NOD can act at different levels in this pathway: NOD-derived NO diffuses across target cell membranes, where it is able to nitrosylate kinases located upstream of NF-B, such as IKK, thereby suppressing their ability to phosphorylate [1]. This inhibition prevents I b phosphorylation and its degradation; thus the release of NF-B. NO can also increase I b gene transcription and stabilize the complex formed by I b and NF-B [2]. Furthermore, NOD-derived NO is capable of nitrosylating directly into the p50 and p65 subunits of NF-B, which blocks their ability to migrate to the nucleus [3]. All of these actions prevent the nuclear translocation of NF-B, therefore the expression of their target genes [4]. (B) Cerebral ischemia-reperfusion increases nNOS activity, which enhances its NO production. This NO can react with free radicals to produce ONOO − and also activates the JNK3 pathway. The result is c-Jun phosphorylation and mitochondrial dysfunction, with an increase in Bcl2 phosphorylation and cytochrome C release into the cytoplasm. In addition, this activates caspase-3 and leads to neuronal apoptosis. NOD-derived NO downregulates neuronal apoptosis by inhibiting increased phosphorylation of JNK, c-Jun, and Bcl-2 [5]. This is achieved by S-nitrosylation of nNOS, which interferes with its NO production [6]. NO is also capable of nitrosylating caspase-3 directly [7]. All of these effects, along with an increase in CBF, reduce brain damage after the ischemia-reperfusion event. Bcl2, B-cell lymphoma 2; CBF, cerebral blood flow; COX-2, cyclooxygenase 2; Cytoc, cytochrome; ICAM, intercellular adhesion molecule; IkB, inhibitors of kB; IKK, I b kinase; IL-1 , interleukin 1 beta; iNOS, inducible nitric oxide synthase; JNK3, c-Jun N-terminal kinases 3; NF-B, nuclear factor kappa B; NIK, NF-B-inducing kinase; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NOD, nitric oxide donors; ONOO − , peroxynitrite; TNF-, tumor necrosis factor alpha; [n] S-nitrosylation.
In astrocytes and microglial cells, NOD also exhibits an anti-inflammatory profile through downregulation of NF-B. In primary rat astrocytes and in a BV2 microglial cell line, GSNO mitigates iNOS production by inhibiting the ability of NF-B to bind to DNA [53]. Therefore, NOD are capable not only of regulating NF-B at the vascular level, but also they possess the capability of influencing glial cell reactivity and limiting their production of iNOS, proinflammatory cytokines, and other molecules and prooxidant enzymes that are potentially harmful to neuronal cells.
Inhibition of NF-B by exogenous NO has been documented at different levels. In the brain ischemic environment, activation of NF-B occurs, at least in part, via ROS [165]. Former researches have shown that one of the most significant sources of ROS in the ischemic brain is through the metabolism of arachidonic acid by Cyclooxygenase (COX) [166,167]. COX-2 expression is increased in brain tissue after global [168] and focal [167,169] cerebral ischemia. ROS are produced by the peroxidase step of the COX reaction in which prostaglandin G2 is converted into prostaglandin H2 [170,171]. Hence, reducing COX-2 activity reduces oxidative damage of the ischemic brain [167,169]. The NO donors GSNO and SNP are able to downregulate LPS-induced COX-2 protein expression via inhibition of NF-B DNA binding activity in murine monocytes [172]. Therefore, both drugs may be candidates for neuroprotective antioxidants in cerebral ischemia. In addition, NO is a superoxide scavenger; hence, NO may inhibit NF-B by scavenging superoxide anion [162].
Furthermore, SNP is capable of interfering directly with the ability of NF-B to translocate into the nucleus. Specifically, SNP inactivates NF-B by nitration of the p65 subunit at Tyr-66 and Tyr-152. This protein modification suppresses iNOS mRNA expression and prevents the activation of NF-B target genes by TNF-stimulation [164]. S-nitrosylation of the p50 subunit at Cys-62 has also been demonstrated as a major mechanism of NO regulation by inhibition of the p50 binding to its consensus DNA target sequence [173].
With respect to I B-, exogenous NO increases mRNA I B levels and stabilizes the complex formed with NF-B [161,172]. This stabilization is related with S-nitrosylation of the Cys-179 of IKKß, which decreases its ability to phosphorylate I B [174]. Additionally, NO interferes with the transient degradation of I B-induced by cytokines [70]. These three actions induce negative regulation of NF-B DNA-binding activity by NOD. Figure 2 sums up the neuroprotective actions of NOD.

Concluding Remarks
Understanding the interaction between the CNS and the immune system will provide greater insight into several different pathologies that involve CNS inflammation and the increase in the number of potential pharmacological targets.
The great variability in the observed effects elicited by NOD, from neuroprotection to toxicity, could be due to the great diversity in doses used in the experiments, which in fact are mainly distant from the existing physiological concentrations. Clarity about the NO concentrations that exists physiologically is essential for developing a quantitative understanding of NO signaling, for performing experiments with NO that emulate reality, and for knowing whether or not NO concentrations become abnormal in disease states [175]. Several independent lines of evidence suggest that NO operates physiologically at concentrations that are orders of magnitude lower than the near-micromolar order once considered correct.
Accordingly, physiological NO concentrations range from 100 pM (or below) up to 5 nM (reviewed in [175]). Therefore, the establishment of reliable methods for directing microelectrode measurement of NO concentrations and the (most foreseeable) progression of newly developed NO biosensors for quantitative imaging of NO signaling in subcellular dimensions and in real time in tissues in vivo will facilitate advances in this fundamental, but yet unsettled, area.
In addition to NOD concentration, it is relevant to consider the administration pathway (intravascular, intraperitoneal, or directly into the culture media in vitro), as well as the cell type in which the donor exerts its action, together with the cell redox state (reduction-oxidation), because these factors are determining ones in selecting the signaling pathway that will be affected or modified by the action of these.
Therefore, therapeutic use of these molecules must be performed carefully, because they can be beneficial for one tissue or cell type and harmful for others. Given their short therapeutic window, NOD appear appropriate for use during neurosurgical procedures involving transient arterial occlusions or in very early treatment of acute ischemic stroke [87].
At present, translation from in vitro to in vivo preclinical stroke models requires further research, as clearly as that required for the case for translation from in vivo animal models to the clinical condition of drugs for treatment of acute ischemic stroke, which requires overcoming phase III trials in patients.