Reperfusion Injury by the Targeted Delivery of

Article Cite This: ACS Appl. Bio Mater. 2019, 2, 2907−2919

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Reducing Ischemia/Reperfusion Injury by the Targeted Delivery of Nitric Oxide from Magnetic-Field-Induced Localization of S‑Nitrosothiol-Coated Paramagnetic Nanoparticles Mahantesh S. Navati,‡ Alfredo Lucas,† Celine Liong,† Marcelo Barros,† Jyothishree Tholalu Jayadeva,‡ Joel M. Friedman,‡,* and Pedro Cabrales*,† †

Department of Bioengineering, University of California, San Diego, La Jolla, California 92093, United States Department of Albert Einstein College of Medicine Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, United States

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ABSTRACT: Nitric oxide (NO) is an important factor during an ischemia/reperfusion (I/R) injury. Protective actions of NO during I/R are attributed to antioxidant and antiinflammatory effects, as well as cell-signaling-based inhibition of nuclear proteins. The therapeutic potential of supplemented NO during I/R is nonetheless uncertain, since peroxynitrite formed from NO near superoxide can be potentially harmful due to NF-κB up-regulation and direct cytotoxicity. This study investigates new technology to provide the magnet-assisted delivery of therapeutic levels of localized NO to targeted I/R tissues using biocompatible gadolinium-oxide-based paramagnetic nanoparticles coated with Snitrosothiols (SNO-PMNPs). Hamsters fitted with a window chamber were subjected to ischemia by application of a tourniquet at the periphery of the window chamber for 1 h. The SNO-PMNPs were intravenously infused (10 mg/kg) during the reperfusion phase, during which time a localized external magnetic field was either applied or not applied to the I/R area. The microvascular hemodynamics, functional capillary density (FCD), rolling and adherent leukocytes, reactive oxygen and nitrogen species, and tissue viability were assessed using intravital microscopy. Control animals did not receive SNO-PMNPs. Treatment with SNOPMNPs plus a magnet but not without a magnet increased reflow, decreased leukocytes rolling and sticking in postcapillary venules, limited cell death, and restored the FCD. The absence of the magnet resulted in systemic changes in heart rate and mean arterial blood pressure, consistent with the systemic delivery of NO by the SNO-PMNP. These results indicate that the localized delivery of NO during reperfusion counters the deleterious consequences of peroxynitrite and other reactive species generated upon reperfusion as reflected in localized increases in blood flow and tissue viability, all with minimal systemic effects. This technology can provide the basis for a timely treatment of a localized ischemia-associated disease to prevent injury in different tissues and organs. KEYWORDS: nitric oxide, peroxynitrite, ischemia/reperfusion, nanoparticles, S-nitrosothiols, paramagnetic, targeted delivery

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INTRODUCTION Ischemia/reperfusion is a complex and multifactorial phenomenon encountered in many clinical conditions such as a stroke, myocardial infarction, hemorrhage, and peripheral vascular disease and surgical interventions such as a cardiac bypass, organ transplantation, and aneurysm repair.1 Reestablishing the blood flow to ischemic tissue or organs (reperfusion) is a necessary step in many surgical procedures.2 However, reperfusion, especially after prolonged ischemia, leads to deleterious changes such as vasoconstriction, microcirculatory disturbances, and an increase of microvascular permeability causing tissue reperfusion edema.3 The vascular endothelium represents one of the most important elements of the inflammatory cascade by virtue of its ability to regulate the adhesion and subsequent tissue infiltration of potentially damaging leukocytes.4 An ischemia and reperfusion (I/R) injury is at the root of the pathophysiology of myocardial infarctions, cerebral ischemia, strokes, hemorrhagic shock, and organ transplantation (among others). An increased production of reactive oxygen species during an ischemia/ © 2019 American Chemical Society

reperfusion injury leads to consumption and depletion of endogenous scavenging antioxidants such as glutathione.5 The consequences of such injury, frequently observed in surgical patients, are both local and remote tissue destruction, as well as the occasional death of the patient. 6 Preischemic or postischemic administration of antioxidants, prostanoid-modulating drugs, adenosine-regulating agents, and antiendothelin antibodies, all of which act as vasodilators, has been shown to attenuate tissue damage post (IR) injury.7 Nitric oxide (NO), an endothelium-derived relaxing factor with various beneficial biologic actions, is another therapeutic candidate. Results show that NO may protect different organs and tissues against the noxious insult associated with IR injuries.8−15 The initial discoveries exposing the role of nitric oxide as a modulator of vascular tone have been followed by a flood of Received: April 2, 2019 Accepted: May 27, 2019 Published: May 27, 2019 2907

DOI: 10.1021/acsabm.9b00282 ACS Appl. Bio Mater. 2019, 2, 2907−2919

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ACS Applied Bio Materials

deactivate peroxynitrite, resulting in the formation of thiyl radicals, which can then rebind NO to reform the S-nitrosothiol moiety.19 This mechanism could result in the depletion of the IR-generated peroxynitrite and sustained delivery of NO bioactivity in the targeted tissues. This study presents a strategy for generating NO-releasing paramagnetic nanoparticles (SNO-PMNPs) and demonstrates their efficacy to mitigate an ischemia/reperfusion injury by increasing local NO levels during early reperfusion. The localization of the NO-releasing SNO-PMNPs at the ischemic site during reperfusion greatly limited the highly deleterious vascular consequences of the reperfusion injury. The results show both the efficacy of NO delivered within the circulation as a preventive of reperfusion injuries and the feasibility of the targeted delivery of NO.

follow up discoveries demonstrating the pivotal role of NO as a major player in almost every physiological function.16 These functionalities encompass not only those in the vasculature but also for such diverse functionalities as neurotransmission, inflammation, antimicrobial activity, management of both pain and wound repair, angiogenesis, and more.16 With respect to the circulation, NO has also emerged as a modulator of platelet activation, rolling, adhesion and activation of leucocytes, control of smooth muscle proliferation, and vascular inflammation. Because of these findings, it has become apparent that NO has significant promise as a therapeutic. A substantial number of cutting edge therapeutics are ultimately just triggers for an increased or decreased NO production. Given the properties of NO, such as it being an expensive, hard-to-administer gas and having a relatively short lifetime in many biological environments, it is not surprising that the major challenge in translating NO bioactivity into a therapeutic has been limited due to a lack of suitable delivery vehicles. Additionally, the challenge is further exacerbated because the physiological response to NO is dependent on the location of action, rate of release, and amount released with consequences ranging from therapeutic to toxic.17 In particular, the production of peroxynitrite from the reaction of NO and superoxide, two major products of the inflammatory response to IR, results in vascular dysfunction due in part to peroxynitrite-induced decoupling of eNOS and the ensuing loss of NO production in the vascular endothelium.18−20 The delivery of excess NO and thiols has the potential of scavenging and deactivating peroxynitrite as well as restoring the needed regulatory control of vascular tone.19 Our group has focused on developing nanoparticle-based NO delivery platforms.21 Several of these have been successfully tested in different animal models that include both topical22−26 and systemic applications.27−30 The topical applied NOreleasing nanoparticles were shown to be highly effective for accelerating wound closure, acting as a broad-spectrum antimicrobial agent in a variety of wound settings and as a topical treatment for erectile dysfunction using two different models.25−30 Systemic applications primarily via iv infusion resulted in demonstrated efficacy as a vasoactive agent and an inhibitor of inflammation in several models including hemorrhagic shock28 and acellular-Hb-induced toxicity.30 Similar results were also achieved using nanoparticles to deliver other bioactive forms of NO such as S-nitrosothiol-containing molecules.22−27 In all of these instances, there was no capability of specifically targeting a specific site other than in the case of topical applications to localized cutaneous lesions. There are instances where targeted delivery of NO to a specific site without complications arising from general systemic effects of NO would be of value. Such instances include local sites with poor tissue perfusion including those arising from crush injuries, pregangrenous tissues, local vascular occlusions, and tissues exposed to conditions, leading to either ischemia/reperfusion or ischemia/ reoxygenation injuries and tumors. To this end, we have developed a nanoparticle platform that is capable of targeted delivery of NO-utilizing S-nitrosothiol (SNO)-coated paramagnetic nanoparticles (PMNP) that allow for localization at the site of an externally applied magnetic field (U.S. Patent application no. 15/522,947). A similar strategy developed by this team was used to deliver L35, a potent red cell penetrating allosteric effector of hemoglobin, to a specific site with a resulting enhancement of tissue oxygenation at the site of the applied magnetic field.31 The use of S-nitrosothiols as a source of NO has the added potential advantage in that the thiols can

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METHODS

Preparation of NO-Releasing PMNPs. The basic strategy for this synthesis entails the use of a paramagnetic nanoparticle core derived from a nanocrystalline form of gadolinium oxide as describe previously. The paramagnetic core is coated with dimercaptosuccinic acid (DMSA) to introduce two thiols per coating molecule. DMSA has two thiols and two carboxylic acids side groups. Carboxylic acid binds very tightly to the surface of the trimethylamine-treated gadolinium oxide (GdO) nanoparticles. Two strategies were compared for introducing the NO to create S-nitroso groups on the surface of the GdO nanoparticles. In one case, DMSO is treated with NO prior to mixing with the trimethylamine-treated particles. The other attempted option was to treat the DMSO-coated particles with NO. The basic method of synthesizing the SNO-PMNP is as follows: GdO PMNP powder was purchased from Nanostructured & Amorphous Materials, Inc. (Houston, Texas; stock no. 2680ZQ). Briefly, GdO PMNP powder was prepared by means of evaporation condensation of Gd within an inert gas atmosphere to obtain a uniform size distribution function of GdO PMNP powder. Then, 100 mg of GdO PMNP was dispersed in 10 mL of chloroform followed by the addition of 50 μL of triethylamine, and 100 mg of DMSA was dissolved in 10 mL of dimethyl sulfoxide (DMSO) purged with gaseous NO (treated to remove higher oxides of NO) to form the SNO complex of DMSA. The SNO−DMSA solution is then mixed with the above solution/suspension containing the GdO PMNPs. The resulting solution was mixed on a lab rotor for 24 h at 4 °C. The pink turbid PMNP suspension was then centrifuged to collect the particles. The PMNPs were then washed with deionized water. This entire process can be repeated to further enhance coverage of the PMNP with DMSA. The particles were dried, stored, and used for the experiments. The strategy of first coating the PMNP with the DMSA and then exposing to NO proved to be not as effective as treating the PMNP with the SNO− DMSA. The pH changes occurring during the flushing of the coated PMNPs with NO resulted in the partial loss of the nanoparticle population likely due to an acid-induced breakdown of the GdO nanocrystals. As a result, the first protocol was used exclusively to test and evaluate the physical and biological properties. Preparation of PEG-Coated PMNPs. Rhodamine PEG (3K) and mPEG-DSPE (2k) were purchased from Nanocs Inc. A stock solution of 1 mg/mL in PBS (pH 7.5, 50 mM) was prepared and used for the experiments. Prior to any measurement, 10 mg of PMNP-SNO dried particles was suspended in 1 mL of PBS buffer (7.5 pH, 50 mM) treated with 20−50 μL of either rhodamine PEG or mPEG-DSPE stock solution for animal work or fluorescence studies immediately before testing or infusing. The reported results shown in the figures were obtained using the PEGylated SNO-PMNP. NonPEGylated particles aggregated and sedimented after 24 h; however, PEGylated particles do not aggregate or sediment after 24 h at room temperature in saline buffer. Additional studies with SNO-PMNP without PEG indicated decreased efficacy and evidence of red blood cell aggregation. Rhodamine-labeled PEGylated SNO-PMNP was used to establish the circulation time for the free circulating SNO-PMNP. 2908


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ACS Applied Bio Materials Size Distribution Characterization of the SNO-PMNP. The initial characterization of the untreated GdO nanocrystalline material was provided by the supplier. The GdO particles (99% purity) were described as nearly spherical with a size distribution ranging from 20 to 80 nm. Attempts at using dynamic light scattering (DLS) to establish the size distribution of the SNO-PMNPs proved difficult, as the NO release occurring during the measurement resulted in bubble formation. Instead, fluorescently labeled SNO-PMNPs were examined using a fluorescence microscope to provide a rough indication of the overall size distribution. NO Release Profiles: Characterization of the SNO-PMNP. Chemiluminescence was used to establish the NO release profiles using established techniques for calibration and analysis. Gaseous NO levels were measured using a NO analyzer (Sievers NO analyzer, Model 280i, Boulder, CO). NO donors/nitrite (for calibration) or SNO-PMNPs (5 mg) were added to 5 mL of phosphate buffer (pH 7.4). Suspensions of the PMNPs were continuously bubbled with pure nitrogen (0.2 L/ min). The flow rate of the gas into the purge vessel was adjusted to maintain the cell pressure between 6 and 7 Torr. The gas phase was collected into the NO analyzer every 1/4 of a second for a period of time (in this case approximately 4 h). The gaseous NO release was measured at 37 °C. The samples were kept in the dark throughout the measurement. Animal Preparation. Investigations were performed in 55−65 g male Syrian Golden hamsters (Charles River Laboratories) fitted with a dorsal chamber window. Animal handling and care followed the NIH Guide for the Care and Use of Laboratory Animals. All animal experimental protocols were approved by our institution local animal care committee. The hamster chamber window model is widely used for microvascular studies in nonanesthetized animals, and the complete surgical technique is described in detail elsewhere.32 Animals were first anesthetized with a 50 mg/kg intraperitoenal injection of sodium pentobarbital. Hair remover was subsequently applied to the animal’s back, after which sutures were used to lift the dorsal skin of the animal. The window chamber frame, consisting of two symmetric sides, was placed over the lifted skinfold one side at a time. At each side of the chamber frame, there is a circular region of 15 mm in diameter (12 mm in diameter of the field of view). On one side, following the circular outline, the dorsal skinfold is removed until only a thin layer of muscle and dermis is present in the field of view. A glass coverslip is then placed over this exposed region, held together by the chamber frame. The back side of the chamber is left unexposed. Animals are then allowed to recover for 2 days, during which the surgical preparation was observed for signs of bleeding or edema. After these 2 days, the animals were implanted with a carotid and a jugular catheter, which were exteriorized at the dorsal side of the neck. Prior to each experiment, the viewing region of the chamber was carefully studied under the microscope for signs of inflammation, low perfusion, or edema, all of which would lead to the exclusion of the animal from the study. Inclusion Criteria. Animals were considered suitable for inclusion in this study if the (1) mean arterial blood pressure (MAP) was above 80 mmHg, heart rate (HR) was above 340 bpm, arterial oxygen partial pressure (paO2) was above 60 mmHg, and systemic hematocrit was above 45% and if the (2) stereomicroscope examination of the chamber viewing region did not present signs of inflammation, bleeding, or edema. For the circulation time of the rhodamine-labeled SNO-PMNP, animals (n = 3, per group) fitted with the dorsal window chamber were injected intravenously with rhodamine-labeled SNO-PMNP, allowing for circulation before imaging.30 Low light fluorescent microscopy (ORCA 9247) was used to capture the fluorescence of the rhodaminelabeled SNO-PMNP. Circulation kinetics of fluorescent nanoparticles were generated over 60 min. Systemic Parameters. MAP and HR were recorded continuously (MP 150, Biopac System; Santa Barbara, CA) except during the actual blood exchange. Hematocrit (Hct) was measured from centrifuged arterial blood samples taken in heparinized capillary tubes (Readacrit; Becton Dickinson, Parsippany, NJ). Hemoglobin (Hb) content was determined spectrophotometrically from a single drop of blood (BHemoglobin, Hemocue, Stockholm, Sweden). Arterial blood was collected in heparinized glass capillaries (0.05 mL) and immediately

analyzed for PaO2, PaCO2, and pH (Blood Chemistry Analyzer 248, Bayer, Norwood, MA). The comparatively low PaO2 and high PaCO2 levels of these animals are a consequence of their adaptation to a fossorial or burrowing environment. Microvascular Measurements. As described,32 awake hamsters were placed in a hollow acrylic tube with a slit, through which the window chamber protruded. Prior to performing the microvascular measurements, the hamsters were allowed 30 min to adapt to the tube environment. The restrained animal was then imaged using a transillumination intravital microscope (BX51WI, Olympus, New Hyde Park, NY) equipped with a 40× water immersion objective (LUMPFL-WIR, numerical aperture 0.8, Olympus). The microscope projected to a charge-coupled device (CCD) camera (COHU 4815, San Diego, CA), which transmitted to a videocassette recorder (AG7355; JVC, Tokyo, Japan). The arteriolar and venular velocity was measured through the photodiode cross-correlation method (Photo Diode/Velocity Tracker Model 102B, Vista Electronics, San Diego, CA).33 To do so, the flow of RBCs was observed by increasing the RBCtissue contrast through a 420 nm bandpass filter. The vessel diameter was measured through a video image-shearing approach.34 With the availability of diameter and velocity, the blood flow (Q) was calculated as Q = π × V(D/2).35 The change in vascular tone was determined as the change in vessel diameter from the baseline. This approach has been proven to work for vessels ranging in diameter from 15 to 80 μm and for hematocrits ranging from 6 to 60%. Arteriole and venule locations were recorded prior to the baseline measurements to ensure that the same vessels were tracked throughout the entirety of the experiment. Local Nitric Oxide Measurement. The nanomolar NO concentration was measured using carbon fiber electrodes (Carbostar-1, Kation Scientific; Minneapolis, MN). The tip of the electrode was coated with three layers of 5% Nafion (Sigma; St. Louis, MO) in ethanol to reject active anions (e.g., nitrate, nitrite, and ascorbate). The microelectrodes were polarized at +0.8 V relative to a 0.8 mm diameter silver−silver chloride reference electrode (Cypress Systems; Lawrence, KS).36 NO measurements were obtained based on the current generated from the electrode system. Currents were measured with a potentiostat (Keithley model 610C; Cleveland, OH). The NO electrodes were calibrated at 37 °C with NO gas balanced with N2 at 2750 nM, 450 nM, and 0 nM (100% argon) (Airgas; Los Angeles, CA). Systemic, Microhemodynamics, and NO Levels Changes of the Target NO Delivery. Animals were randomly divided into three experimental groups: (1) received 10 mg/kg of SNO-PMNP (iv) and a magnetic field applied over the dorsal window post infusion (magnetic field ON); (2) received 10 mg/kg of SNO-PMNP (iv) and no magnetic field was applied over the dorsal window post infusion (magnetic field OFF); (3) received 10 mg/kg of control (without SNO) PMNP (iv) and no magnetic field was applied over the dorsal window post infusion (Control). Twelve animals (n = 4 per group) fitted with the dorsal window were included in the quantification of systemic, microvascular, and local NO levels. Briefly, after 30 min to adjust to the experimental conditions, baseline systemic parameters (MAP and HR) were measured. Treatments were given, and a magnetic field was applied to the dorsal window for 15 min post infusion of the SNO-PMNP. Systemic and microvascular parameters were quantified between 15 and 30 min after treatment. Perivascular NO measurements were obtained after removing the cover glass of the window chamber. Tissue was suffused with a physiological Krebs salt solution at 37 °C and equilibrated with 95% N2 and 5% CO2. NO measurements were initiated 10 min after the glass window removal, a period that was found to allow the tissue to stabilize. Perivascular measurements were made by placing the microelectrode on the perivascular tissue so that the tip was as close as possible to the microvessel without touching the wall. Animals were euthanized after measurements were completed. Functional Capillary Density (FCD). Functional capillaries are defined as those that allow for RBC transit of at least one RBC in a 60 s period.37 Following a procedure similar to that in ref 38, 10 successive microscopic fields of view were assessed, totaling a region of 0.46 mm2, with each field of view totaling between two and five capillaries with an RBC flow. The FCD was then quantified as the total length of RBC perfused capillaries divided by the area of the image field of view. 2909


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Figure 1. Schematic representation showing the general protocol for the IR experiments. An ischemic region is created by preventing blood flow to the window chamber model via a tourniquet applied on the edge of the window. Paramagnetic nanoparticles coated with S-nitrosothiols and PEG are infused iv at the start of the reperfusion phase. The particles are localized at the site of the ischemia using a Niobium-ased external magnet. Controls consist with experiments where either the nanoparticles are infused but there is no external magnet or there are no infused nanoparticles. Artwork created by Celine Liong. Measurement of NADH. NADH increases during ischemia because an oxygen shortage limits NADH oxidation at the electron transport chain. During reperfusion, NADH should decrease with the appropriate oxygen supply. Tissue fluorescence has been validated to measure NADH in vivo.30,39,40 Following a procedure similar to that in ref 39, fields of view were intravitally studied for the presence of NADH. The microscope was focused using the edge of the vessels through bright-field transillumination. Using fluorescence epi-illumination, the field of view was excited at 360 nm using a bandpass filter (Thorlabs), a dichroic mirror (XF2003 390DRLP, Omega Optical), and a 200 W mercury arc lamp as the light source. Each excitation lasted 2 s to prevent photobleaching of the window chamber. Emission signals were measured at 420 and 460 nm through a photomultiplier (model R928, Hamamatsu) connected to a Biopac acquisition system (MP 150, Biopac System). The fluorescence emission ratio at these two wavelengths provides an NADH-sensitive measurement.30 Tissue Viability. Following the procedure described,41 a mixture of equal parts Annexin V (Alexafluor 488 conjugate; Molecular Probes, Eugene, OR) and propidium iodide (PI, 0.2 mg/mL, Molecular Probes) was produced. Thirty minutes prior to the experimental procedure, 140 μL of the mixture was injected into the arterial line of the hamsters. Intravital microscopy imaging of the labeled cells was carried out 8 h after ischemia and reperfusion and SNO-PMNP treatment. Images were acquired with a low light video camera (ORCA 9247, Hamamatsu, Tokyo, Japan) and were recorded at 5 frames per second and 1344 × 1024 pixels per frame. Cells, single and doubly labeled, were counted in the window chamber, and the percentage of annexin V- and/or propidium iodide-labeled cells was calculated for each time point. Data is presented as the average number of fluorescent cells across 40 tissue and 40 endothelial visual fields (210 μm × 160 μm). As reported,41 sebaceous glands and hair follicles were identified and excluded from the cell counts due to their consistently high necrosis and apoptosis rate. Leukocyte−Endothelium Interaction. Microvascular leukocyte adherence and rolling in postcapillary venules (25−50 μm) was assessed through an intravenous (iv) injection of acridine orange (5 mg/kg solution in saline), as suggested.28 A low light fluorescent microscope (ORCA 9247, Hamamatsu) was used for arcidine orange bound leukocyte visualization. In this process, only straight venule portions were exposed to low intensity epi-illumination and recorded for 60 s at 10 frames per second. Venules, instead of arterioles, are were observed since they favor leukocyte adhesion and rolling due to an

increased presence of p-selectin. During the video playback, leukocytes were quantified and categorized according to their flow behavior following the convention proposed in ref 42, which included “passers”, “free-flowing”, “flowing with endothelial contact”, and “immobilized” leukocytes. ROS Detection. Dihydrorhodamine 123 (DHR, Calbiochem, La Jolla, CA) is an oxygen-sensitive probe and was used to quantify ROS generation in vivo.43 DHR freely permeates cellular membranes and, when oxidized, forms a fluorescent rhodamine. To determine relative ROS levels, fluorescent intensities were measured along the microvascular vessel selected for hemodynamic measures, before and after DHR. DHR was infused iv (5 mg/1 kg, and it was left to distribute for 10 min before measurements. Values of DHR fluorescence were averaged and were expressed in relative change in intensity versus initial signal. ROS = (I − I0)/I0, where I0 and I represent the fluorescence before ischemia and after for the same locations. Following the same location at the different time points in the same animal provided an internal control for the ROS levels.44 Microvascular Permeability of Labeled Dextran. Changes in vascular permeability were assessed using 100 μL of fluorescein isothiocyanate (FITC)-labeled 150 kDa dextran (100 mg/mL SigmaAldrich).45 Briefly, animals were injected intravenously, and the FITC− dextran tracer was allowed to circulate for 15 min before imaging. Low light fluorescent microscopy was used capture fluorescence (ORCA 9247, Hamamatsu, Tokyo, Japan). The straight portion of venules was exposed to low intensity epi-illumination for 5 s, and digital images were recorded. The area of extravasation was obtained by subtracting the area of bright fluorescence in the vessels from the total area fluorescence. Ischemia/Reperfusion Protocol. Ischemia was induced in the window chamber following a similar protocol.41 A thin, flat rubber ring, which served as a circular clamp, was used to compress the periphery of the window chamber (Figure 1). The flow was progressively occluded by pressing the rubber ring against the skin, toward the glass coverslip. Under the intravital transillumination microscope, complete cessation of flow from feeding and draining vessels was verified to ensure complete ischemia in the clamped area, in the absence of compression damage to the skin. Animals were randomly divided into three experimental groups: (1) received 10 mg/kg of SNO-PMNP (iv) and a magnetic field applied over the dorsal window post infusion (magnetic field ON); (2) received 10 mg/kg of SNO-PMNP (iv) and no magnetic field was applied over the dorsal window post infusion (magnetic field 2910


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Figure 2. Schematic representation of the SNO-PMNP (A). Electron micrograph (TEM) of the uncoated paramagnetic gadolinium oxide nanocrystal (b); image of rhodamine-labeled nanoparticles viewed through a fluorescence microscope (C); loss of fluorescence signal when comparing the signal from an initial stock solution containing rhodamine dye to the signal from the supernatant (SUP) obtained by the addition of paramagnetic nanoparticles to the stock solution, spinning down the nanoparticles and generating the fluorescence spectrum of the nanoparticle-free supernatant. The difference in intensity is attributed to the loss of rhodamine that became bound to the nanoparticles (D). Image of rhodamine-coated paramagnetic nanoparticles before and after being exposed to a powerful bar magnet. The images clearly demonstrate that the rhodamine is associated with paramagnetic nanoparticles that rapidly accumulate at the site of the magnet (E).

Figure 3. NO release profiles for 1 mg/mL of SNO-PMNP with no added PEG (black), with 50 μg PEG (red) and with 100 μg PEG (blue) (A). A photograph of SNO-PMNP as a compacted block of a fine powder after the lyophilization phase of preparation but prior to treatment to convert the material to a fine powder (B). Circulation time of iv infused fluorescently labeled PMNP (with added PEG) (C). The fluorescence of rhodaminelabeled SNO-PMNPs was measured, before infusion (BI) and after infusion for 240 min. 2911


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Figure 4. Impact of magnetic localization on both physiological parameters and local NO levels upon iv infusion of SNO-PMNPs (no ischemia and reperfusion). The presence of the magnet reduces systemic effects attributable to enhanced systemic NO levels. (A) Mean arterial pressure (MAP) and (B) heart rate (HR) seen in the absence of the magnet, whereas the presence of the magnet creates substantial physiological hemodynamic manifestations in the dorsal window, where (C) the arteriole diameter and (D) arteriole blood flow increased, as well as resulted in an increase in the (E) perivascular NO level in arterioles, venules. and tissues from the SNO-PMNPs localization with a magnet. †, P < 0.05 compared to the baseline. Artwork created by Celine Liong. OFF); (3) received 10 mg/kg of control (without SNO) PMNP (iv) and no magnetic field was applied over the dorsal window post infusion (control). Animals received SNO-PMNP (10 mg/kg) 10 min after the release of the ischemia, and right after, a strong conical magnet was aimed at the dorsal window tissue for 15 min in the SNO-PMNP magnetic field ON group. Thirty animals (n = 10 per group) fitted with the dorsal window were included in the quantification of systemic parameters (MAP and HR), microhemodynamics (diameter, blood flow, and FCD). Eighteen of the 30 animals (n = 6 per group) were included for the quantification of NADH levels and tissue viability. Twelve of the 30 animals (n = 4 per group) were included in the quantification of leukocyte interaction and ROS levels. Systemic and microvascular parameters were analyzed before ischemia, and 0.5, 2, and 24 h after the release of the ischemic clamp. Tissue viability was measured at 24 h after the release of the ischemia. Preliminary experiments using different doses of SNO-PMNPs (from 2 to 20 mg/ kg) during I/R were completed to optimize the dose that acutely (0.5 and 2 h) maximized the recovery of FCD. Final experiments competed at 10 mg/kg, as this dose restored FCD to near baseline during reperfusion. Data Analysis. Results are presented as mean ± standard deviation. Time course data within each group was analyzed using a repeated measures analysis of variance (ANOVA, Kruskal−Wallis test). Post hoc analyses were performed corrected for multiple comparisons using Dunn’s method. The intergroup analysis was done using a Bonferroni corrected two-way ANOVA. Statistical significance was achieved for P < 0.05. Microhemodynamic absolute values are shown in the corresponding figure legends. All statistics were calculated using GraphPad Prism 6 (GraphPad Software, Inc., San Diego, CA).

the supplier (Nanocs Inc.) of the uncoated PMNPs. Individual particles are considerably less than 100 nm. Figure 2C shows images of the rhodamine-labeled SNO-PMNP generated using a fluorescence microscope. The images indicate that the coated PMNPs are roughly similar in size to the original uncoated PMNPS. Figure 2D shows a loss of fluorescence intensity from a rhodamine stock solution after the addition of PMNP. The PMNPs are spun down, and the signal from the PMNP free solution is compared with that of the original stock solution. The loss of signal both shows evidence of the binding of the rhodamine to the PMNPs and allows for calculating the amount of rhodamine-PEG associated with the SNO-PMNPs (rhodamine without any PEG conjugation: 19 μg/mg of SNOPMNPS). Figure 2D shows the external magnet-induced localization of the rhodamine-labeled SNO-PMNPS on to the side of a cuvette, which further indicates that the rhodamine has associated with the SNO-PMNPs. Figure 3A shows the NO release profile in the dark of the SNO-PMNP as a function of added PEG. It can be seen that there are minor differences in the profiles due to PEGylation. The addition of PEG does, however, decrease the amount of SNO coverage. Figure 3B shows the compressed block of SNO-PMNP immediately after lyophilization. The pink color is attributed to the SNO moiety on the surface of the PMNP. Circulation Time. Intravascular fluorescence was used as an indicator SNO-PMNP concentration (Figure 3C). Infusion of the fluorescently labeled SNO-PMNP produced an increase in intravascular fluorescence. Circulating SNO-PMNP was observed up to 4 h after infusion. No evidence of the fluorescently labeled SNO-PMNP was observed the next day. Circulating

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RESULTS Physical Characterization. A schematic representation of the SNO-PMNP is presented in Figure 2A. Figure 2B shows the transmission electron microscopy (TEM) image provided by 2912


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Figure 5. Time-dependent changes in systemic parameters (MAP and HR) during the ischemia/reperfusion studies as a function of the time after reperfusion and the iv infused SNO-PMNP with and without a magnet compared with the control (no SNO-PMNP infused). †, P < 0.05 compared to the baseline.

Figure 6. Time-dependent changes in the local hemodynamic vascular parameters, (A) arteriole and (B) venular diameter and (C) arteriole and (D) venular blood flow at the site of an ischemic event during the reperfusion phase as a function of the time after reperfusion. The infusion SNO-PMNP with the magnet induced vasodilation and increased blood flow in arterioles and venules, and without a magnet, there were no differences from the control (no SNO-PMNP infused). †, P < 0.05, compared to the baseline.

field and control. Treatment with SNO-PMNP without a magnetic field induced minor vasodilation and an increase in blood flow. Figure 4E shows the perivascular NO levels in arterioles and venues and the NO levels in tissue. Treatment with SNO-PMNP with a magnetic field induced a statistically significant increase in perivascular and tissue NO levels compared to SNO-PMNP without a magnetic field and compared to the control. Treatment with SNO-PMNP without a magnetic field induced a minor increase in the perivascular NO concentration in arterioles, but not statistical compared to the control (p = 0.21). Systemic Responses to SNO-PMNP during Ischemia and Reperfusion. Figure 5 shows the changes with time of the MAP and HR after reperfusion. Treatment with SNO-PMNP caused a statistically significant reduction in MAP if a magnetic field was not applied, whereas the application of the magnetic field after treatment with SNO-PMNP prevented the decrease in MAP. All changes in MAP dissipated within 24 h. Similarly, treatment with SNO-PMNP caused a statistically significant increase in HR if a magnetic field was not applied, whereas the application of the magnetic field after treatment with SNO-

labeled SNO-PMNP without PEG was cleared from the circulation within minutes of infusion (data no shown). Systemic Physiological Responses to SNO-PMNP as a Function of Magnet-Induced Localization. Figure 4A,B shows changes in mean arterial blood pressure (MAP) and heart rate (HR) after treatment with SNO-PMNP with or without application of a magnetic field in the absence of ischemia and reperfusion. SNO-PMNPs cause a statistically significant reduction in MAP if a magnetic field was not applied, whereas the application of the magnetic field prevented the decrease in MAP. The magnet-treated animals show a substantially smaller decrease in MAP relative to the no magnet animals. Similarly, SNO-PMNP caused a statistically significant increase in HR without the magnetic field, and the magnet-treated animals showed a substantially smaller increase in HR, no different from the baseline. Figure 4C,D shows changes in the diameter and blood flow in arterioles after treatment with the SNO-PMNP with or without a magnetic field. Treatment with SNO-PMNP with a magnetic field induced statistically significant vasodilation and a statistically significant increase in blood flow compared to the baseline and compared to SNO-PMNP without a magnetic 2913


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Figure 7. Time-dependent changes in local physiological microvascular hemodynamic parameters. (A) Functional capillary density (FCD), (B) leucocyte immobilized, and (C) leucocyte rolling, during the reperfusion phase as a function treated with iv infusion of SNO-PMNP with and without a magnet compared with the control (no SNO-PMNP infused). †, P < 0.05, compared to the baseline. ‡, P < 0.05, compared to the control.

Figure 8. Time-dependent changes in local physiological parameters reflective of oxidative stress (ROS production (A), redox balance as reflected in the ratio of NADH to NAD+ (B), vascular leakiness (C) during the reperfusion phase as a function of iv infused SNO-PMNP with and without a magnet compared with the control (no SNO-PMNP infused). (D) A photograph of the vasculature at the site of the ischemic event, which illustrates a functional magnet treatment, the degree of vascular leakiness as reflected in the background green color derived from fluorescently labeled dextran that has passed out of the blood vessels. †, P < 0.05 compared to the baseline. ⧧, P < 0.05, compared to the control.

PMNP prevented the increase in HR. Changes in HR dissipated within 24 h. Microhemodynamic Responses to SNO-PMNP during Ischemia and Reperfusion. Figure 6 shows the diameter and blood flows for arterioles and venules during reperfusion.

Arteriolar diameters decreased in the SNO-PMNP without a magnet and the control relative to the baseline at 30 min during reperfusion. The enular diameter in SNO-PMNP with a magnet increased compared to the baseline and to both SNO-PMNP without a magnet and a control, at 2 h after reperfusion. The 2914


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Figure 9. Quantitative evaluation of both the number of cells undergoing necrosis and apoptosis (A) and the breakdown of the number of cells that are necrotic, early-stage apoptosis, and late-stage apoptosis (B) at the site of the ischemic event following reperfusion as a function of treatment: sham (no ischemia or SNO-PMNP), control, and treatment with and without a magnet). Reactions in vivo in excess of NO relative to superoxide and nitrogen dioxide (C). As NO and superoxide are both present, they react with nitrogen dioxide to form N2O3 and/or peroxynitrate. N2O3 can react with thiols to give nitrosothiols or with a hydroxide anion to give nitrite, and the peroxynitrate can decompose to nitrite and oxygen.

arteriolar blood flow decreased in SNO-PMNP without a magnet and a control, but it was preserved in SNO-PMNP with a magnet, at 30 min and 2 h during reperfusion. The venular blood flow increased in SNO-PMNP with a magnet compared to SNO-PMNP without a magnet and a control, at 30 min and 2 h during reperfusion. Functional Capillary Change (FCD) Responses to SNOPMNP during Ischemia and Reperfusion. Figure 7A shows that FCD was no different from the baseline in SNO-PMNP with a magnet, suggesting that it was preserved during reperfusion. Treatment with SNO-PMNP with a magnet preserved a statically significant higher FCD compared to the SNO-PMNP without a magnet and a control, at 30 min during reperfusion. Treatment with SNO-PMNP with a magnet had a statically significant higher FCD compared to that of the control, at 2 h during reperfusion. The localized accumulation of nanoparticles when the magnet is applied to the dorsal window does not have any negative hemodynamics effecting capillary plugging. The particles are very small, relative to the diameter of blood vessels (including capillaries), and the used dose is low, compared to the number of capillaries and blood vessels in the tissue. Thus, the blood flow or FCD was not affected by the accumulation of nanoparticles in the vasculature of the dorsal window. Leukocyte Adhesion and Rolling Responses to SNOPMNP during Ischemia and Reperfusion. Figure 7B,C shows the number of leukocytes immobilized and rolling per unit of area at the baseline, and during reperfusion. Treatment with SNO-PMNP with the magnetic field significantly reduced the number of leukocytes immobilized and rolling per unit of

area at all time points compared to SNO-PMNP without the magnetic field and control, respectively. Treatment with SNOPMNP without the magnetic field reduced the number of leukocytes immobilized and rolling per unit of area at 30 min and 2 h after reperfusion compared to the control. After 24 h, the number of leukocytes immobilized and rolling per unit of area decreased in all groups compared to 2 h after reperfusion. Previous studies using this I/R model show that sham-control animals (without I/R or treatment) have minimal changes in the hemodynamic, inflammatory, and tissue viability to the dorsal window over 24 and 48 h.41 ROS Levels, Anaerobic Metabolism, and Vascular Permeability: Responses to SNO-PMNP Treatment during Ischemia and Reperfusion. Figure 8A shows the relative amount of reactive oxygen species (ROS) in perivascular tissues. Treatment with SNO-PMNP with the magnetic field reduced ROS levels at a statistically significant level at all time points during reperfusion compared to SNO-PMNP without the magnetic field and control, respectively. Figure 8B shows the degree of anaerobic metabolism during reperfusion. Treatment with SNO-PMNP with the magnetic field reduced NADH/ NAD at statistically significant levels at 30 min, and 2 h during reperfusion compared to SNO-PMNP without the magnetic field and control, respectively. During reperfusion, NADH/ NAD levels were higher for all groups relative to the baseline. Figure 8C,D illustrates changes in vascular leakage of FITClabeled 150 kDa dextran with and without the magnet. Figure 8C shows the quantified changes in vascular permeability as the ratio of perivascular to intravascular fluorescence. Treatment with SNO-PMNP with the magnetic field significantly reduced 2915


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can undergo either homolytic cleavage to form a hydroxyl free radical and a nitrogen dioxide free radical or heterolytic cleavage to a nitronium cation and hydroxide anion. Three of these cleavage products (the hydroxyl free radical, nitrogen dioxide free radical, and nitronium cation) are among the most reactive and damaging species in biological systems.46 SNO-PMNP, when localized early on during reperfusion, limits the damage attributable to these reactive species, implying that the increase in delivered local NO either limits the formation and accumulation of peroxynitrous acid and its derived reactive species or limits the damage derived from peroxynitrous acid oxidation of biologically relevant molecules (Figure 9C). This hypothesis would not be unprecedented, since NO is known to inhibit and limit lipid peroxidation.47,48 In an indirect fashion, the NO-induced increase in blood flow can also function to dilute and wash away any local buildup of peroxynitrous acid. Finally, the presence of free thiols on the SNO-PMNPs due to either incomplete saturation with NO or loss of NO due to superoxide scavenging can contribute to the decrease in peroxinitrite-dependent IR damage. Low molecular weight thiols can deactivate peroxynitrite, and in the process, form thiyl radicals, which can then rebind any remaining excess NO, leading to the reformation of SNO.19 The existence of this cycle, in the presence of SNO-PMNPs, creates a tightly interconnected environment where reactive species can be neutralized by both the NO, through its action in superoxide, and the free thiols of SNO, through peroxinitrite neutralization and prevention of subsequent generation of peroxynitrous acidderived reactive species. Thus, the delivery of high-density Snitrosothiols may provide a unique combination of reactants that combat reperfusion injury in a 2-fold synergistic fashion. The same combination should prove effective for other peroxynitrite-mediated inflammatory tissue damage. Future studies will address the relative individual contributions of the thiols and the NO as well as the proposed synergy. The observed localized reduction in vascular inflammation and leukocyte infiltration as well as the preservation of tissue viability are most likely also the result of prolonged NO released by the localized SNO-PMNP treatment in the ischemic tissue. Aside from the reduction of ROS through the mechanisms already explained, other NO-dependent mechanisms are also likely to act in conjunction. NO plays an important role in enhancing the activity of various antioxidant enzymes through enhanced expression/induction of genes associated with the antioxidant defense system, contributing additional cytoprotective qualities.19 Furthermore, NO also participates in vasodilation (vascular smooth muscle relaxation, as observed in Figures 4C,D and 6A,B) and inhibition of platelet aggregation. A known mechanism for both, as suggested,49 involves the elevation of cyclic guanosine monophosphate following soluble guanylate cyclase activation by NO. NO is also known to have inhibitory effects over the endothelium−neutrophil interaction, partially through down-regulation of the expression of cell adhesion molecules such as p-selectins, both on endothelium and neutrophils. Furthermore, NO also downregulates the production of cytokines by inflammatory cells.49,50 These known mechanisms propose a potential pathway for the decrease in immobilized and rolling leukocytes for the magnetic field on the SNO-PMNP group (Figure 7B,C). The underlying hypothesis for the present study was based upon evidence that a decrease of NO production by endothelial cells, caused by eNOS decoupling due to multiple damaging pathways driven by enhanced ROS production, is one of the important mechanisms

the increase in vascular permeability at 2 h compared to SNOPMNP without the magnetic field and control. Figure 8D, taken at 2 h during reperfusion, shows an enhanced green coloration throughout the image of the no-magnet-treated vasculature, indicative of leakage of the labeled dextran. The magnet-treated vessels show much less evidence of leakiness. Tissue Viability Responses to SNO-PMNP during Ischemia and Reperfusion. Figure 9A,B shows the number of necrotic and apoptotic cells per field of view. The number of necrotic and apoptotic cells increased in all groups subjected to ischemia/reperfusion compared to animals without ischemia and reperfusion. Treatment with SNO-PMNP with the magnetic field resulted in a statistically significant reduction in the number of necrotic and apoptotic cells compared to SNOPMNP without the magnetic field and control, respectively. Treatment with SNO-PMNP without the magnetic field reduced the number of necrotic and apoptotic cells compared to the control. Treatment with SNO-PMNP with the magnetic field resulted in a statistically significant reduction in the number of early apoptotic, late apoptotic, and necrotic cells compared to SNO-PMNP without the magnetic field and control, respectively. Treatment with SNO-PMNP without the magnetic field reduced the number of early apoptotic, late apoptotic, and necrotic cells compared to the control.

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DISCUSSION Ischemia and reperfusion injuries remain a major unresolved clinical problem. Several mechanisms of a reperfusion injury have been previously postulated: (i) oxygen-derived free radicals, (ii) disturbed microcirculation, and (iii) local inflammation. NO has controversial effects on ischemia and reperfusion injuries; however, at low concentrations, NO can improve microcirculatory function. In earlier work, we showed that low levels of NO delivered into the circulation limits inflammation and leukocyte adhesion as well as increasing tissue perfusion via enhanced functional capillary density.28−30 Here we observed that targeted SNO-PMNP treatment maintains the integrity of the microcirculation post ischemia, as demonstrated by higher blood flows, reduced white blood cell vascular interaction, and preserved functional capillary density. These findings support the result that SNO-PMNP treatment during reperfusion can decrease ROS, restore oxygenation, and increase tissue viability post ischemia/reperfusion. In the model used in this study, ischemia and reperfusion likely initiates a high production of ROS, specifically superoxide, which leads to rapid scavenging of NO to form peroxynitrite. In fact, our results suggest significantly smaller ROS levels for the on magnetic field SMO-PMNP group (Figure 8A). During I/R, NO can be advantageous and disadvantageous depending on the local concentration. The formation of reactive nitrogen species is related to the local amount of NO, as NO is efficiently removed by reacting with hemoglobin to form nitrate in the intravascular compartment. However, during I/R, the simultaneous activation of superoxide synthesis along with limited production of NO completely transform the biological actions of NO to promote the formation of peroxynitrite. If the nonischemic NO increased levels (Figure 4E) were preserved during I/R, it is likely for the reaction of NO with superoxide, forming peroxynitrite, to be the cause of the decreased ROS. Peroxynitrite is relatively stable, but when protonated at a low pH, it forms peroxynitrous acid. Several research groups have suggested that peroxynitrous acid is responsible for the oxidation of biological molecules.18−20,46 Peroxynitrous acid 2916


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reperfusion injury. SNO-PMNP attenuated tissue injury rescued all tissues from the critical insult. An absence of localization through the use of the magnet resulted in systemic effects attributable to enhanced levels of NO in the circulation. These systemic effects were minimized through the magnet-based localization strategy. Although further studies are required to elucidate the exact mechanisms by which SNO-PMNP treatment prevents an ischemic reperfusion injury, the targeted supplementation of NO with SNO-PMNP treatment appears to be a possible strategy in clinical settings because it provides for delivery of therapeutic levels at the needed target site without systemic adverse hypotensive effects.

responsible for tissue injury associated with ischemia and reperfusion. To our knowledge, this is the first report to demonstrate not only that local NO enhancement reduces the ischemia and reperfusion injury but also that it is possible to localize the release of NO to a specific tissue via SNO-PMNP treatment. Although the NO levels were not determined directly during ischemia/reperfusion in this study, we believe that SNO-PMNP treatment augmented the local NO concentration in a similar fashion as in the absence of ischemia/reperfusion. It is this expected increase in local NO concentration, what we believe resulted in a decreased IR in the presence of magnetically targeted SNO-PMNP. This argument is consistent with the following evidence. First, NO is a known vasodilator. This effect can have positive consequences during reperfusion. Vasodilation allows for an increased flow that can flush dangerous byproducts that might have accumulated during ischemia. NO also has compensatory vasoconstrictive effects through the modulation of endothelin and prostanoids.49 It is then likely that this overall vasoactive functionality of NO, which includes both vasoconstriction and vasodilation, is one of the contributing factors for the SNO-PMNP’s benefits during ischemia/reperfusion. Second, in addition to its vasoactive properties, NO is also a superoxide scavenger,50 which can contribute to the decreased damage during a reperfusion injury. Our results also support this hypothesis as observed by the significant decrease in ROS in the presence of the SNO-PMNP. Third, SNO-PMNP demonstrated a decreased number of rolling and adhered leukocytes. It is known that leukocyte adherence to endothelial cells, specifically nutrophils, agravates tissue damage through the release of granules such as myeloperoxidase and elastase;51 therefore, the ability of SNO-PMNP to decrease leukocyte adherence to the endothelial wall, as suggested in the present study, might be another hypothesis for the decreased IR injury in the presence of SNO-PMNP. Finally, SNO-PMNP is also known to have effects in mitochondrial function during IR.52 In the mitochondria, NO has been shown to modulate aconitase as well as multiple electron transport cascades.49,52 This is important because, during ischemia, the lack of oxygen often leads to an electron backup in the electron transport chain. When oxygenation returns, some of the electrons will go toward the oxygen for the formation of water in complex IV of the electron transport chain. However, due to the electron excess, complex I and complex III will also contribute electrons to oxygen but will lead to the formation of ROS. It is then thought that reversible inhibition of an upstream electron transport chain enzyme (i.e., complexes I, II, and III) can lead to the slow reintroduction of electrons into the energetic cascade, mitigating the formation of excessive ROS.52−54 Furthermore, Krebs cycle intermediaries have also been shown to be influenced by NO, through interactions with iron−sulfur clusters in enzymes such as aconitase.55 These inhibitory interactions can also contribute to the slow reintroduction of electrons into the electron transport chain, contributing to the decreased formation of ROS, and the increased recovery in the presence of SNO-PMNP.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: (858) 822-4830. Telephone: (858) 534-5847. ORCID

Pedro Cabrales: 0000-0002-8794-2839 Funding

Funding was provided by NIH grants from the Heart Lung and Blood Institute, P01-HL110900, R01-HL126945, R01 HL138116, and a philanthropic foundation. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Cynthia Walser (UC San Diego) for surgical preparation of the animals. REFERENCES

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CONCLUSION In conclusion, we demonstrated through the magnetically induced localization of S-nitrosothiol-coated gadolinium-oxidebased paramagnetic nanoparticles (SNO-PMNP) that both NO can be delivered locally with minimal systemic consequences and that local delivery of NO via SNO-PMNP provides significant protective effects against severe ischemia and 2917


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