Core Crosslinked Nanoparticles Reduce Neuroinflammation and

Core-Cross-Linked Nanoparticles Reduce Neuroinflammation and Improve Outcome in a Mouse Model of Traumatic Brain Injury Dasom Yoo,† Alexander W. Magsam,‡ Abby M. Kelly,† Patrick S. Stayton,† Forrest M. Kievit,*,‡ and Anthony J. Convertine*,† †

Department of BioEngineering, Molecular Engineering and Sciences Institute, Box 355061, Seattle, Washington 98195, United States Department of Biological Systems Engineering, University of Nebraska, Lincoln, Nebraska 68583, United States

‡

S Supporting Information *

ABSTRACT: Traumatic brain injury (TBI) is the leading cause of death and disability in children and young adults, yet there are currently no treatments available that prevent the secondary spread of damage beyond the initial insult. The chronic progression of this secondary injury is in part caused by the release of reactive oxygen species (ROS) into surrounding normal brain. Thus, treatments that can enter the brain and reduce the spread of ROS should improve outcome from TBI. Here a highly versatile, reproducible, and scalable method to synthesize core-cross-linked nanoparticles (NPs) from polysorbate 80 (PS80) using a combination of thiol−ene and thiol−Michael chemistry is described. The resultant NPs consist of a ROS-reactive thioether cross-linked core stabilized in aqueous solution by hydroxy-functional oligoethylene oxide segments. These NPs show narrow molecular weight distributions and have a high proportion of thioether units that reduce local levels of ROS. In a controlled cortical impact mouse model of TBI, the NPs are able to rapidly accumulate and be retained in damaged brain as visualized through fluorescence imaging, reduce neuroinflammation and the secondary spread of injury as determined through magnetic resonance imaging and histopathology, and improve functional outcome as determined through behavioral analyses. Our findings provide strong evidence that these NPs may, upon further development and testing, provide a useful strategy to help improve the outcome of patients following a TBI. KEYWORDS: polysorbate 80, antioxidant, controlled cortical impact, gliosis, hippocampus, startle habituation decrease neurocognitive impairments following a TBI.5 In order to suppress the damage caused by ROS following a TBI, antioxidant nanoparticles (NPs) have been developed.4,6−8 These agents contain functional groups that inactivate ROS into less toxic species. For example, hydrogen peroxide degrading enzymes (i.e., superoxide dismutase; SOD) bound to NPs were shown to significantly reduce neuronal damage as a result of oxidative stress.9 Clinical trials with the free-radicalscavenging agents poly(ethylene glycol)-conjugated SOD (PEG-SOD) and tirilazad have been conducted for use in TBI. Unfortunately, these studies with both antioxidant formulations failed to improve patient outcome, likely as a result of poor delivery into the brain.10 Indeed, there are currently no treatments that have shown efficacy in improving outcome following a TBI in a large, multi-institution phase III trial.

T

he chronic progression of a variety of neurodegenerative diseases is partially caused and exacerbated by the persistence of oxidative stress within the brain, as neurons are highly sensitive to reactive oxygen species (ROS).1,2 Following a traumatic brain injury (TBI), reperfusion injury, delayed cortical edema, blood−brain barrier breakdown, and local electrolyte imbalance can result in the release of ROS well beyond the initial insult and are thought to be a major cause of the progression of a TBI.3,4 Furthermore, neuroinflammation, which includes reactive astrocytes and activated microglia recruited to the damaged tissue, releases additional ROS, inhibits the integration of newly formed neurons, and prevents axon development. As a result, many of the lifelong problems that arise following a TBI can at least partially be attributed to this secondary injury mechanism following the primary insult. This signifies ROS as a critical therapeutic target for neuroprotection to (1) reduce ROS-mediated neurodegeneration in surrounding tissue and importantly (2) reduce the reactivity of astrocytes and activation of microglia to minimize further ROS release into the brain in order to (3) © 2017 American Chemical Society

Received: May 16, 2017 Accepted: August 7, 2017 Published: August 7, 2017 8600

DOI: 10.1021/acsnano.7b03426 ACS Nano 2017, 11, 8600−8611

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ACS Nano Scheme 1. Synthesis of PS80-Based Core-Cross-Linked NPs via Sequential Thiol−Ene and Thiol−Michael Reactions

Figure 1. (A) 1H NMR of PS803SH in CDCl3 following thiol−ene conjugation of tetra-SH to PS80. (B) Overlay of the olefin resonance of PS80 before (red trace) and after (black trace) thiol conjugation confirming quantitative reaction of the cis double bond. (C) 1H NMR spectrum in CDCl3 for core-cross-linked NP1 prepared with the addition of pentaerythritol tetraacrylate (TAc) to an aqueous solution of PS803SH and at an overall olefin to thiol ratio of 1. (D) GPC chromatograms for PS80 and core-cross-linked NPs prepared from the reaction of PS803SH (with (F, G) or without (E) addition of free thiol) and TAc at an overall thiol to olefin ratio of 1. Absolute molecular weights and Đ values were determined via GPC in dimethylformamide eluent at a flow rate of 1 mL/min.

Polysorbate 80 (PS80) is a nonionic surfactant consisting of oleic acid that has been esterified with polyethoxylated sorbitan. Because of its low toxicity and cost, PS80 has been used as an emulsifier/stabilizer in a wide range of pharmaceutical, cosmetic, and food applications with an average consumption of 100 mg per day in Europe and America.17 Thus, PS80 has been widely investigated in drug delivery applications. For example, Borchard et al. have shown that coating therapeutic NPs with PS80 resulted in enhanced delivery across the blood− brain barrier in bovine brain blood vessel endothelial cell cultures.18 Subsequent in vivo studies with PS80-coated poly(butyl cyanoacrylate) NPs loaded with the analgesic

NPs offer the potential to improve delivery and retention in a brain injury through an enhanced permeability and retentionlike effect that occurs as a result of the traumatic injury to the brain and blood−brain barrier.11−16 Therefore, NPs can accumulate and be retained in damaged brain to exert a protective effect and prevent the spread of ROS to surrounding normal brain. However, the translation of many nanotechnologies into clinical use has been hindered by low reproducibility, high cost, and poor scalability. Therefore, scalable synthesis strategies that can produce NPs with a high density of ROS-reactive materials are needed to improve translation into clinical trials. 8601


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appearance of additional resonances corresponding to the tetra thiol alkyl (2.5−2.7 ppm) and ester (4.2 ppm) groups. Also absent from the conjugate spectrum is the peak at 2.1 ppm, which is associated with the four allylic protons adjacent to the cis double bond. These resonances shift upfield following the thiol−ene reaction, which converts the alkene to a single bond. Taken together, these data suggest the formation of the desired thioether-linked adduct with an average of three additional thiol groups available for subsequent reactions. The resultant thiol-functionalized PS80 (PS803SH) is soluble in most organic solvents (e.g., chloroform, acetone, methanol) and assembles in water to form micelles with a hydrodynamic diameter of 16 nm. This size is similar to the parent PS80, which has a diameter of 10.7 nm, corresponding to an aggregation number of 60. Core-cross-linked NPs with hydrophilic coronas and a reactive hydroxyl functionality were then synthesized from PS803SH using thiol−Michael chemistry. Here the thiol-functional surfactant was first dispersed in deionized water at a concentration of 44 mg/mL. Pentaerythritol tetraacrylate (TAc) in acetone (to cut the viscosity) was then added dropwise to the aqueous solution, yielding a translucent blue solution. In order to enhance the probability that an equilibrium morphology had been reached, the aqueous solution was then sonicated in a low-power ultrasonic bath for 1 h. Hexyl amine was then employed to catalyze the thiol− Michael reaction because it is inexpensive, is easy to handle, and has been shown to be highly effective at promoting thiol− Michael reactions.27 The 1H NMR spectrum of the core-cross-linked NPs in CDCl3 (Figure 1C) shows resonances associated with residues from PS803SH as well as the tetra-acrylate. For example, key PS803SH resonances remain clear at 4.2, 3.6, and 1.3 ppm, corresponding to the oleate ester, ethylene oxide, and alkyl protons, respectively. Resonances associated with the TAc residues at 4.0−4.5 ppm (overlapping ester) and overlapping alkyl residues at 2.5−3.0 ppm are also apparent. These resonances are somewhat broadened relative to the low molecular weight precursors, suggesting a solvent-swollen covalently cross-linked core. The lack of any vinyl resonances associated with the TAc, in combination with the presence of the additional ester and aliphatic residues, suggests that the acrylate groups were successfully reacted via thiol−Michael chemistry to from the desired thioether core. Trace unreacted thiols were subsequently scavenged by adding an excess of monofunctional acrylate (e.g., TBA or HEA) to the NP solution prior to purification. Core-cross-linked NPs were also prepared with the addition of free tetra-SH to the aqueous solution of the PS803SH micelles. Here, additional TAc was added in order to maintain an acrylate:thiol ratio of 1:1 over a range of thiol concentrations (3 SH groups per PS803SH + 4 SH groups per tetrathiol). This strategy allows for the synthesis of core-crosslinked NPs with progressively larger hydrodynamic sizes as the amount of hydrophobic core forming residues is increased relative to the hydrophilic stabilizing corona segments. Next GPC analysis in DMF was employed to provide evidence supporting the formation of stable core-cross-linked NPs. Dimethylformamide (DMF), which is a good solvent for PS80, caused the surfactant to elute as unimers with a retention volume of 26.7 mL, which is consistent with the molecular weight of PS80 (i.e., 1310 g/mol) (Figure 1D). In comparison, cross-linked NPs prepared at an equimolar alkene-to-thiol ratio (Figure 1E) eluted at a significantly lower retention volume (18.4 mL) corresponding to a molecular weight of 877 000 g/

hexapeptide dalargin showed a significant pharmaceutical affect.19 In another study, Gulyaev et al. have shown that the brain concentration of systemically administered doxorubicin can be enhanced 60-fold by encapsulating the drug within PS80-coated NPs.20 PS80 contains three reactive hydroxyl residues terminating the hydrophilic oligoethylene oxide residues as well as a cis alkene functionality at the 9-position of the hydrophobic oleate chain. These orthogonal functional groups can be employed to introduce chemical functionality at spatially discrete locations along the surfactant using efficient click reactions such as thiol−ene chemistry. Thiol−ene and thiol−Michael reactions represent a complementary set of click chemistries that can be applied to wide range of readily available olefin substrates.21−24 These reactions proceed either by a free-radical mechanism or through a basecatalyzed addition process, respectively. Free-radical thiol−ene reactions can be initiated via either thermal or photochemical means to generate thiyl radicals that are reactive toward olefins. In general, the reactivity of thiyl radicals toward enes are highest for electron-rich double bonds such as vinyl ethers, vinyl esters, and aliphatic alkenes.22,25,26 In contrast to radical thiol−ene reactions, thiol−Michael reactions occur between thiols and electron-deficient double bonds such as acrylates and maleates. Thiol−Michael addition reactions proceed rapidly to quantitative conversion even under dilute conditions in the presence of a suitable catalyst.27 These reactions typically employ a base catalyst (usually an amine or phosphine) to facilitate the reaction between thiols and electron-deficient olefins. If the reaction is catalyzed by common bases (e.g., triethyl amine), it proceeds by an initial deprotonation step where the base extracts a proton from the thiol. The versatility of thiol−ene and thiol−Michael click reactions has led to their widespread use in drug delivery as well as diagnostic and other biotechnology applications.28−32 Herein we describe the sequential application of thiol−ene and thiol−Michael chemistry to prepare ROS-reactive core-cross-linked NPs based on PS80 for neuroprotective applications in TBI.

RESULTS AND DISCUSSION Synthesis of Core-Cross-Linked NPs from PS80 via Successive Thiol−Ene and Thiol−Michael Reactions. Scheme 1 shows the synthetic strategy used to prepare corecross-linked NPs from thiol-functionalized PS80. The resultant NPs contain thioether groups within their hydrophobic cores, allowing them to effectively scavenge ROS released as a result of traumatic brain injury or as part of neurodegeneration (vide inf ra). A thiol functionality was introduced to the hydrophobic portion of PS80 via a photochemically induced thiol−ene reaction with a tetrafunctional thiol. Here, pentaerythritol tetrakis(3-mercaptopropionate) (tetra-SH) was reacted at 5 °C under bulk conditions with the cis double bond present at position 9 on the oleic acid chain. Because of the relatively low reactivity of the internal double bond, an alkene to photoinitiator ratio of 10 was employed. Following 7 h of UV irradiation at 365 nm, a sample was taken and characterized via 1 H NMR. The unmodified PS80 (Figure 1B, red trace) shows characteristic peaks associated with the olefinic protons at ∼5.3 ppm as well as the oleate ester (4.2 ppm), ethylene oxide (3.6 ppm), and alkyl (1.3 ppm) resonances (see Supporting Information Figure 1 for full spectrum). Analysis of the 1H NMR spectrum for the resultant PS803SH conjugate (Figure 1A,B black trace) shows that the signal associated with unsaturation at 5.3 ppm is completely consumed with the 8602


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ACS Nano Table 1. Formulation Parameters Used to Prepare Core-Cross-Linked NPs via Thiol−Michael Addition Reactions sample

PS803SH (mmol)

TSH (mol)

total SH (mmol)

TAc (mmol)

total Ac (mmol)

V H2O final (mL)

Mna (g·mol−1 × 106)

Đa

Dhb (nm)

NP1 NP2 NP3

1.11 1.11 1.11

0 1.02 2.05

3.33 7.43 11.52

0.83 1.86 2.88

3.33 7.43 11.52

45 45 45

0.88 6.0 20

1.24 1.20 2.04

16.4 24.2

a As determined by size exclusion chromatography using Tosoh SEC TSK-GEL α-3000 and α-4000 columns (Tosoh Bioscience, Montgomeryville, PA, USA) connected in series to an Agilent 1200 Series liquid chromatography system (Santa Clara, CA, USA) and Wyatt Technology miniDAWN TREOS 3 angle MALS light scattering instrument and Optilab TrEX refractive index detector (Santa Barbara, CA, USA). HPLC-grade DMF containing 0.1 wt % LiBr at 60 °C was used as the mobile phase at a flow rate of 1 mL min−1. bAs determined by dynamic light scattering in phosphate-buffered saline (20 mM NaCl + 20 mM phosphate) at pH 7.4. Samples were sterile filtered with a 0.22 μm filter to remove dust immediately prior to analysis.

mol. Under these solvent conditions any un-cross-linked materials would be expected to elute at long retention volumes as the physically assembled NPs dissociated. The absence of any additional peaks at longer elution volumes supports the formation of NPs derived from PS803SH that are covalently linked to tetra-acrylate residues at their cores via thioether bonds. Figure 1 also shows the molecular weight chromatograms for NPs prepared with the addition of 1.2 and 2.4 equiv of free thiol (relative to PS803SH thiol groups). The molecular weight distribution for NPs prepared with 1.2 equiv of additional SH (Figure 1F) is narrow and unimodal, yielding a molecular weight and molar mass dispersity of 6.00 × 106 g/ mol and 1.20, respectively. In contrast, NPs synthesized with the addition of 2.4 (Figure 1G) equiv of free thiol become multimodal with large molar mass dispersities. Molar compositions as well as Mn, molar mass dispersity values, and hydrodynamic diameters in buffer (Dulbecco’s phosphatebuffered saline (DPBS) pH 7.4) for the NPs described in Figure 1 are shown in Table 1. Comparison of the molecular weight chromatograms and 1H NMR spectra for different batches of NP1 confirm the reproducibility of the synthetic approach outlined in Scheme 1 (Supporting Information Figure 2). Here molecular weights and Đ values of 928 700 g mol−1/ 1.12 and 933 200 g mol−1/1.16 were observed for two batches of the nanoparticles. The high atom efficiency of this approach combined with the use of commercially available reagents and aqueous conditions combined with the facile purification strategy suggest that the NP synthesis can be significantly scaled beyond the 5 g batch sizes produced in these studies. Evaluation of the ROS Sponge Capacity of NP1 via DCFH Fluorescence. The core-cross-linked NPs are designed to scavenge ROS through the oxidation of thioether residues (present in the NP core) to sulfoxides and sulfones (Figure 2A). The ROS sponge capacity of NP1 was assessed using the dichlorodihydrofluorescein diacetate (DCFH-DA) assay. In this assay 2,7-dichlorodihydrofluorescein diacetate is initially hydrolyzed by either an enzymatic or base-catalyzed process to yield DCFH, which becomes highly fluorescent upon oxidation by ROS such as peroxide. DCFH fluorescence was measured as a function of H2O2 concentration in the presence of various concentrations of NP1 following a 1.0, 1.5, 2.0, and 2.5 h incubation period (Figure 2B). For all incubation times a sharp increase in DCFH fluorescence was observed for NP-free control solutions (red traces). The maximum fluorescence intensity was somewhat lower for the untreated samples at 1 h relative to later time points but remained relatively constant for subsequent times, suggesting that 1.5 h was sufficient to achieve near-quantitative oxidation of the substrate under these conditions. In strong contrast, the presence of even the lowest concentration of NP1 (0.01 mg/mL) caused a dramatic

Figure 2. (A) Schematic representation of therapeutic NPs scavenging reactive oxygen species release following traumatic brain injury. (B) DCFH fluorescence as a function of H2O2 concentration with and without the addition of NP1. In the absence of ROS-scavenging NPs a significant increase in fluorescence is observed above an H2O2 concentration of 1 mM. In contrast, experiments conducted in the presence of NP1 show significant reduction in fluorescence even at low concentration.

reduction of DCFH fluorescence. For example, at 2.5 h the DCFH fluorescence intensity dropped from a value of 50 000 ± 1000 at 10 mM H2O2 to 14 000 ± 20 at a NP concentration of 0.01 mg/mL, an approximate 3.5-fold reduction in ROS levels. At higher NP concentrations (i.e., 0.1, 0.5, and 1.0 mg/mL) nearly complete suppression of DCFH fluorescence was observed at 10 mM H2O2, which was not caused by reduction 8603


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ACS Nano in fluorescence caused by the presence of a hydrophobic NP core or PS80, a major component of NP1 (Supporting Information Figure 3). Therefore, NP1 achieved upward of a 140-fold reduction in ROS levels. This translates to an astounding 9.93 μmol of ROS per mg of NP1. This level of ROS reduction compares favorably with reported antioxidant small molecules, polymers, and NPs, which have been reported to scavenge 0.88−3.25 μmol of ROS per mg of therapeutic agent.5,33−35 The ability of NP1 to reduce intracellular levels of ROS was also assessed in vitro using the DCFH assay. In this assay, human astrocytes were exposed to H2O2 in the presence or absence of NP1 with intracellular green fluorescence from DCFH, indicating the presence of ROS (see Supporting Information Figure 4). Here a sharp increase in green fluorescence from DCFH in human astrocytes was observed following exposure to H2O2, indicating high intracellular levels of ROS, whereas control astrocytes not exposed to H2O2 had low background levels of ROS. Astrocytes treated with NP1 had much lower levels of intracellular ROS, even when exposed to H2O2, showing the ability of these NPs to protect astrocytes from ROS exposure. These data suggest that the NP1-mediated reduction in intracellular ROS in astrocytes should lead to reduced reactivity. This finding is consistent with work by Hubbell et al., where the authors demonstrated that poly(propylene sulfide) could be oxidized to poly(propylene sulfoxide) and poly(propylene sulfone) in the presence of H2O2.36 Also evident is the lack of overt toxicity caused by NP1, which is further supported by fluorescence microscopy and an MTS viability assay (Supporting Information Figure 5). ROS-Mediated Astrocyte Reactivity in the Presence of NP1. To corroborate the DCFH assay results of NP1-mediated reduction in ROS and to better assess the potential of NP1 for neuroprotection through reduction in neuroinflammation, we tested the ability of NP1 to reduce ROS-mediated astrocyte reactivity. In these studies human astrocytes were incubated with H2O2 with and without the addition of NP1 and reactivity was assessed through glial fibrillary acidic protein immunostaining (GFAP) (Figure 3). The media-only control treatment showed baseline levels of GFAP positivity (Figure 3A), which is indicative of astrocyte reactivity. Treatment of cells with NP1 resulted in no increase in the reactivity of astrocytes (Figure 3B), further confirming the biocompatibility of these NPs. Importantly, in the presence of H2O2, NP1-treated cells showed a marked decrease in GFAP positivity (Figure 3C) as compared to H2O2 alone (Figure 3C), indicating its protective properties beyond preventing DCFH-DA conversion. Quantification in low-powered field views revealed a significant decrease in the numbers of GFAP+ astrocytes (Figure 3I), suggesting NP1 reduced ROS-mediated astrocyte reactivity. This was further shown through high-magnification images where astrocytic processes could be visualized (Figure 3D−I). Astrocyte exposure to H2O2 resulted in an increase in the number of cellular processes, which further suggests increased astrocyte reactivity, whereas treatment with NP1 resulted in astrocytes displaying normal cell structure. These data support the DCFH assay data to show their biological relevance and suggest they may function well for neuroprotection in the setting of TBI. NP1 Accumulates in Injured Brain in a Mouse Model of TBI and Is Cleared through the Liver and Kidneys. Brain delivery continues to plague potential treatments for TBI. NPs are well suited to overcome brain delivery issues, as it is well established that the blood−brain barrier is highly disrupted

Figure 3. NP1 reduced ROS-mediated activation of human astrocytes as determined by GFAP immunostaining. (A) Media (10×), (B) 1 mg/mL NP (10×), (C) 0.1 mg/mL NP + 100 μM H2O2 (10×), (D) 100 μM H2O2 (10×), (E) media (60×), (F) 1 mg/mL NP (60×), (G) 0.1 mg/mL NP + 100 μM H2O2 (60×), (H) 100 μM H2O2 (40×). (I) Quantification of numbers of GFAP+ cells in low-powered field images as compared to total cell number determined by DAPI nucleus staining. There was a significant difference in the percentage of GFAP+ cells in vitro between treatments (P = 0.0002), with NP treatments significantly reducing the percentage of GFAP+ cells back to baseline as compared to H2O2 treatment (adjusted P values are media-only vs NP-only adjusted, 0.8797; media-only vs H2O2, 0.0258; media-only vs 1 mg/ mL NP + H2O2, 0.6865; media-only vs 0.01 mg/mL NP + H2O2, 0.2424; NP-only vs H2O2, 0.0027; NP-only vs 1 mg/mL NP + H2O2, 0.9955; NP-only vs 0.01 mg/mL NP + H2O2, 0.7597; H2O2 vs 1 mg/mL NP + H2O2, 0.0011; H2O2 vs 0.01 mg/mL NP + H2O2, 0.0001; 1 mg/mL NP + H2O2 vs 0.01 mg/mL NP + H2O2, 0.9251).

in a TBI, which can be passively targeted with NPs through an enhanced permeability and retention effect similar to that observed in tumors.11−16 The ability of NP1 to accumulate in a TBI was tested using a controlled cortical impact (CCI) mouse model of TBI. Mice were injected with 200 μL of NP1 at 10 mg/mL in phosphate-buffered saline (PBS) immediately after impact to provide an initial circulating NP1 concentration of approximately 1 mg/mL (80 mg/kg). This dose was chosen since it was able to nearly completely suppress ROS levels based on our DCFH and in vitro data to ensure sufficient ROS sequestration within the brain injury. Translating to humans, however, this would require a dose of approximately 500 mL to be delivered to achieve 80 mg/kg, which is a common translational problem in small-animal studies that commonly use this injection volume. Nevertheless, comparing NP1 to clinically tested antioxidant therapies such as PEG-SOD (sequesters ∼1 μmol of H2O2 per mg of PEG-SOD) that are given at 10 000 U/kg (∼7 mg/kg), NP1 dose was ∼10-fold higher but has approximately 10-fold higher antioxidant reactivity per mg. Therefore, it would be possible to significantly reduce the administered dose of NP1 to make translation to humans feasible. Brains were removed after 1 or 2 h and imaged with an iBox small-animal imaging system. Fluorescence from NP1 was observed at the impacted site at both 1 and 2 h postinjection (Figure 4A), showing accumulation in damaged brain occurs quickly after injection and is retained, similar to previous findings.11 This strongly suggests NP1 enters brain parenchyma through an impaired 8604


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Figure 4. (A) NP1 accumulates in damaged brain after intravenous injection. Fluorescence images show high NP1 accumulation quickly after injection, which was retained in damaged brain for over 2 h postinjection. (B) Biodistribution and clearance of NP1. Fluorescence images and quantification show NP1 mainly in the liver and kidneys. Organ layout: 1. liver, 2. kidneys, 3. spleen, 4. heart, 5. lungs. (C) Time-dependent biodistribution of NP1 showing clearance through the liver and kidneys at 2 and 6 h with nearly complete clearance achieved at 24 h postinjection.

Figure 5. NP1 reduces the spread of neuroinflammation in a CCI mouse model of TBI. (A) T2-weighted fast spin−echo sequences were used to image the spread of secondary injury as visualized by hyperintense regions surrounding the primary damage (arrows). Slices from 1 mm anterior and 1 mm posterior to the impact site are also shown. (B) Quantification of volume of hyperintense secondary damage through all slices revealed faster reduction in NP-treated mice as compared to untreated mice. (C−J) NP1 improves histological outcome in a CCI mouse model of TBI. (C, D) Histological assessment of structure and reactive astrogliosis in (C) CCI and (D) CCI with NP1 treatment at 1 week postinjury. No structural changes in the ipsilateral CA1, CA2, CA3, and dentate gyrus (DG) regions of the hippocampus were observed with H&E staining. GFAP immunostaining revealed a decrease in reactive astrocyte staining in brains from mice treated with NP1 after TBI. Iba1 immunostaining revealed a decrease in activated microglia in brains from mice treated with NP1 after TBI. Scale bars correspond to 0.25 mm. (E) Quantification of GFAP+ cell number per 40× field (0.05 mm2). There was a statistical difference (P = 0.0019) between CCI and CCI + NP as determined by paired t test. (F) Quantification of Iba1+ cell number per 40× field (0.05 mm2). There was a statistical difference (P = 0.0638) between CCI and CCI + NP as determined by paired t test. (G, H) Histological assessment of structure and reactive astrogliosis in (G) CCI and (H) CCI with NP1 treatment at 1 month postinjury. GFAP and Iba1 immunostaining revealed a decrease in reactive astrogliosis staining in brains from mice treated with NP1 after TBI. Scale bars correspond to 0.25 mm. (I) Quantification of GFAP+ cell number per 40× field (0.05 mm2). There was a statistical difference (P = 0.0481) between CCI and CCI + NP as determined by paired t test. (J) Quantification of Iba1+ cell number per 40× field (0.05 mm2). There was a statistical difference (P = 0.0133) between CCI and CCI + NP as determined by paired t test.

blood−brain barrier rather than being carried in by neutrophils, the first circulating immune cells that infiltrate the CNS after a TBI by around 24−48 h after injury.3,37 This is important to ensure the NPs are available for ROS sequestration. The clearance route for NP1 was tested through biodistribution measurements at various time points after injection. NP1 was observed mainly in the liver and kidneys (Figure 4B,C), suggesting clearance by these organs, which is expected for NPs of this size. NP1 was nearly completely cleared after 24

h, indicating it is not retained, which reduces the potential for long-term exposure. Analyses of Secondary Spread of Injury and Neuroinflammation. The ability of NP1 to effectively reduce the spread of secondary damage and neuroinflammation following an initial TBI was evaluated in vivo using a combination of MRI and histological studies. Here mice were given a TBI through CCI and then imaged 1, 3, 6, 10, 15, and 22 days after the impact to observe the extent of the damage through T28605


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Figure 6. NP1 improves functional outcome in a CCI mouse model of TBI. (A) Animal weights over 3 weeks from before and after the day of CCI surgery (day 0). A significant drop in weight was observed for animals receiving the CCI surgery at days 1 and 4, but animal recovery was improved in animals that received NP1 treatment. (B) Rotor-Rod analysis of motor function. Mice were trained on the Rotor-Rod (0−50 rpm over 5 min) for 1 week prior to CCI surgery. Mice that received CCI surgery or CCI surgery plus NP treatment showed a significant reduction in latency to fall at 1 day following the surgery as compared to control animals (adjusted P = 0.0428 and 0.0543, respectively, as determined by one-way ANOVA with Tukey’s multiple comparisons test), which fully recovered by day 4, with CCI and CCI + NP mice showing similar performance. (C) Startle response habituation test 1 week after TBI. Both control and CCI + NP mice showed nonzero slopes through regression analyses (P values of 0.0143 and 0.0713, respectively), whereas CCI mice did not show a nonzero slope (P = 0.9851). (D) There were no significant changes in startle amplitude, indicating no change in the reflex circuitry in these mice (P = 0.2569 by one-way ANOVA). (E) Histological analyses confirm there were no histopathological markers of astrogliosis in the PnC region, which is involved in the startle response. (F) Startle response habituation test 1 month after TBI. CCI + NP mice showed a nonzero slope through regression analysis (P = 0.0704), whereas CCI mice did not show a nonzero slope (P = 0.9851). (G) There was a significant difference in startle amplitude at 1 month post-TBI (P = 0.0402 by one-way ANOVA) with CCI significantly lower than CCI + NP (adjusted P value = 0.0370 by Tukey’s multiple comparisons test). Additionally, CCI mice showed significantly lower startle amplitude at 1 month post-TBI as compared to 1 week post-TBI (P = 0.0012 by unpaired t test), whereas startle amplitudes for control and CCI + NP mice were similar between these two time points (P = 0.8699 and 0.9420, respectively, by unpaired t test). (H) Histological analyses showing histopathological markers of astrogliosis in the PnC at 1 month post-TBI.

important for spatial memory and with CA3 is involved in contextual learning such as fear conditioning, CA2 is involved in social memory, and CA3 with the dentate gyrus (DG) is involved in pattern separation.43−46 Cognitive deficits commonly experienced by TBI patients including attention, concentration, memory, and judgment can partially be attributed to decreased function of these hippocampal regions. To assess changes in the hippocampus in our CCI mice, we performed histological assessment of neuroinflammation through immunostaining of reactive astrocytes (GFAP) and activated microglia ionized calcium binding adapter protein 1 (Iba1) 7 days after injury (Figure 5C−F). Significant immunopositivity for GFAP and Iba1 were observed on both the ipsilateral and contralateral side of the brain for CCI mice that did not receive treatment with NP1, indicating the spread of astrogliosis to the contralateral hemisphere. GFAP and Iba1 immunostaining was also apparent in both hemispheres in CCI mice that did receive treatment with NP1, but not quite as widespread. This suggests NP1 was able to reduce the chronic neuroinflammation caused by astrogliosis, further supporting the accelerated reduction in edema observed in the MR images. Quantification of GFAP- and Iba1-positive cells in hippocampal subfields confirmed NP1 was able to significantly reduce reactive astrocytes (P = 0.0019 as determined by paired t test) and activated microglia (P = 0.0638 as determined by paired t test) as compared to mice that did not receive NP1. To determine the long-term neuroinflammation in these mice, we

weighted imaging sequences to observe edema surrounding the damage. At 1 day following the injury, damage was observed as a hyperintense region on the top right of the brain at the impact point (Figure 5A, black arrows). Minor hyperintensity was also observed anterior and posterior of the damage. Significant hyperintensity was still observed at days 3−22 in the CCI mice, but mice that received NP treatment showed minimal edema at these time points where the void of damage can be seen as a hypointense region. This suggests an NP1-mediated decrease in the spread of secondary damage through decreased edema and neuroinflammation. Neurocognitive problems in TBI patients include anxiety, reduced attention, and reduced memory. The hippocampus plays a central role in learning and memory and modulates sensorimotor processes.38 Much of the cognitive impairment observed both clinically and experimentally following a TBI can be attributed to alterations within the hippocampus.39 Indeed, significant neurodegeneration is observed within the hippocampus 48−72 h following a TBI,40,41 suggesting alterations within the hippocampus are caused by secondary effects rather than the initial insult. This is further supported by the fact that a significant increase in neuroinflammation including astrogliosis, a commonly utilized measure of secondary injury, is observed for 1−3 months following blast injury where little gross structural damage occurs.42 The various subfields of the hippocampus are involved in different learning and memory tasks. For example, cornu ammonis region 1 (CA1) is 8606


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memory as well as pattern separation. Therefore, a reduction in habituation would be an indicator of a poorer outcome after a TBI and further supports the effects of NP treatment on outcome with our histological findings. Habituation was tested on mice 1 week after receiving a TBI using an acoustic startle response test. Mice were placed in an SR-LAB startle response system, which is an enclosed ∼1 ft3 box with a mouse holder attached to an accelerometer that measures mouse movements. Mice were placed into the chamber and allowed to acclimate for 5 min prior to sounding a 120 dB broadband noise pulse lasting 30 ms and measuring the startle response through the accelerometer (trial 1). This pulse was repeated 19 more times with 20 s between each pulse (trials 2−20). A general decrease in startle response from early to late trials was observed for control animals, indicating habituation as expected. CCI mice showed minimal decrease in startle response, indicating a lack of habituation caused by the TBI. The CCI + NP mice showed a significant decrease in startle response, indicating habituation and suggesting an improved outcome. Regression analyses of startle response revealed habituation was present in control and CCI + NP mice as indicated by their nonzero slopes (Figure 6C, P = 0.0143 and P = 0.0713, respectively), whereas there was not a nonzero slope for CCI mice (P = 0.9851). The startle response itself is a reflex controlled by the pons and spinal cord and is relayed through the caudal pontine reticular nucleus (PnC).48,49 We observed no difference in startle amplitude between treatment conditions (Figure 6D, P = 0.2569 by one-way ANOVA), suggesting the habituation was not solely caused by a change in the startle reflex between animals. This is further supported by the lack of astrogliosis observed in the PnC (Figure 6E) and further supports the lack of difference in motor function after day 4 observed in the Rotor-Rod tests. At one month after receiving a TBI, a similar trend in startle habituation was observed (Figure 6F), where CCI mice showed no habituation (P = 0.9851) but mice receiving NP treatment showed habituation (P = 0.0704). Interestingly, startle amplitude was significantly lower for CCI mice as compared to CCI + NP mice at 1 month (Figure 6G, P = 0.0370), suggesting progression of secondary damage beyond startle habituation to startle response circuitry. Indeed, startle amplitude was significantly lower in CCI mice at 1 month as compared to 1 week post-TBI (P = 0.0012), whereas there was no significant difference for control and CCI + NP mice (P = 0.8699 and 0.9420, respectively), indicating better protection from significant spread of secondary damage in NP-treated mice. Histological analysis of the PnC revealed minimal GFAP immunostaining but larger amounts of Iba1-positive cells in the PnC of CCI mice that did not receive NP1 treatment (Figure 6H). This suggests minor secondary injury spread well beyond the primary impact site that affects the startle reflex was prevented by treatment with NP1. In toto, our findings show the potential of NPs to accumulate in damaged brain and reduce local levels of ROS to decrease neuroinflammation and improve neurobehavioral outcome. Nevertheless, translating this work into human studies will require significant additional effort, as TBI is an extremely difficult disease to treat owing to the vast differences in patient presentation. Significant differences in outcome have been observed based on sex, treatment time point, and severity of injury, among others.50 Therefore, in order to gain a more thorough understanding of NP1 translatability, future work will require the use of male mice as well as female, generation of

performed histological assessment of reactive astrocytes and activated microglia 1 month postinjury (Figure 5G−J). Higher immunostaining can be seen in images from CCI mice as compared to mice that received NP treatment. Lower GFAP immunostaining can be observed at 1 month postinjury as compared to 1 week, suggesting a decrease in reactive astrocytes over time, but Iba1 staining appeared to increase, suggesting an increase in activated microglia during secondary injury. Quantification confirmed decreased GFAP immunostaining was present throughout the hippocampus after 1 month. Animals that received NP1 treatment still had fewer GFAPpositive cells than untreated animals (P = 0.0481 by paired t test), suggesting accelerated recovery. Iba1 immunostaining was increased at 1 month postinjury in CCI mice, with significantly fewer Iba1-positive cells throughout the hippocampus in mice that received NP1 treatment (P = 0.0133 by paired t test). These data, combined with our previous findings,11 strongly support the use of antioxidant NPs to reduce the chronic secondary astrogliosis and neuroinflammation resulting from a TBI. Behavior Outcome. To observe differences in recovery from a TBI, mouse weights were monitored for 1 week prior to CCI surgery (days −7 to −3) and then 1, 4, 7, and 14 days following the surgery. There was a significant difference between the weights of animals after surgery (Figure 6A). Control mice showed a continued upward trend in weight, as expected for mice of this age. On the other hand, mice showed a significant decrease in weight 1 day following surgery, likely as a result from the surgical procedure and brain injury. CCI mice continued to lose weight at 4 days postinjury, whereas mice that received treatment with NP1 showed recovery back to control mice by this time point. This significant difference in weight at day 4 suggests an accelerated recovery from injury with NP1 treatment, further supporting our MRI and histological data. To observe differences in motor function following a TBI, motor function performance was tested with the Rotor-Rod system for 1 week prior to CCI surgery and days 1, 4, 7, and 14 postinjury. Mice were trained for 1 week prior to surgery to best separate motor function from learning performance. Daily training on the Rotor-Rod improved latency to fall over 1 week prior to surgery as expected (Figure 6B). At 1 day postimpact both CCI and CCI + NP mice showed a significant decrease in latency to fall as compared to control animals (adjusted P = 0.0428 and 0.0543, respectively). By 4 days postimpact all mice recovered back to baseline, suggesting NP1 treatment had no effect on Rotor-Rod performance in these animals. This rapid recovery in motor performance in CCI mice regardless of NP1 treatment indicates that this CCI mouse model of TBI is not affected by long-term secondary injury. Instead, the reduction in latency to fall was likely caused by an acute response to the surgery and injury. To test learning outcome in these animals, the startle habituation test was performed. Startle habituation is thought of as the simplest form of learning. This is a cross-species phenomenon that under repeated exposure to a startling stimulus the magnitude of response will be reduced over time. Deficits in habituation are an indication of a pathology, with most of the reported literature on schizophrenia patients. Habituation deficits have more recently been observed in animal models of TBI.47 Furthermore, the hippocampus plays a critical role in the habituation process and is thought to be involved in habituation to startle response,38 as habituation would require functional short-term spatial and contextual 8607


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ACS Nano

μL of hexalmine, 0.362 μmol) in 1 mL of acetone was added dropwise over 10 min. The solution was then sonicated for an additional 15 min and then allowed to stir overnight. The following morning 3 mL of tert-butyl acrylate or 3 mL of hydroxyethyl acrylate in 3 mL of acetone was added to the aqueous solution, which was then allowed to react until the following morning. The NPs were then purified via dialysis against acetone followed by deionized water and then lyophilized. Conjugation of Rhodamine B to Core-Cross-Linked NPs. To a 22 mL glass scintillation vial was added NPs (400 mg) that were dissolved in 8 mL of methylene chloride. To this solution was then added 4-(dimethylamino)pyridine (DMAP) (26 mg, 213 μmol) and rhodamine B (20 mg, 42 μmol), which were allowed to completely dissolve before diisopropyl carbodiimide (33 μL, 213 μmol) was added. The solution was then allowed to react overnight, after which time the solvent was removed via rotary evaporation. The NPs were then dissolved in 2.5 mL of deionized water and purified via filtration through two PD10 columns in series according to the manufacturer’s instructions. Dynamic Light Scattering. Dynamic light scattering (DLS) studies of the NPs were conducted using a Malvern Instruments Zetasizer Nano series instrument equipped with a 22 mW He−Ne laser operating at 632.8 nm. Solutions of the copolymer and NPs were prepared in DPBS (pH 7.4) at a concentration of 0.5 mg/mL. The resulting solutions were filtered with 0.22 μm filters prior to measurement, and mean diameter was defined as the ±half-peak width. Gel Permeation Chromatography (GPC). Absolute molecular weights and molar mass dispersities were determined using Tosoh SEC TSK-GEL α-3000 and two α-e4000 columns (Tosoh Bioscience, Montgomeryville, PA, USA) connected in series to an Agilent 1200 Series liquid chromatography system (Santa Clara, CA, USA), a Wyatt Technology miniDAWN TREOS 3 angle MALS light scattering instrument, and an Optilab TrEX refractive index detector (Santa Barbara, CA, USA). HPLC-grade DMF containing 0.1 wt % LiBr at 60 °C was used as the mobile phase at a flow rate of 1 mL/min. GFAP Immunostaining. Human astrocytes were maintained in human astrocyte growth medium (Cell Applications, San Diego, CA, USA) in a humidified incubator at 37 °C and 95%/5% air/CO2. Cells were plated on 22 × 22 μm coverslips in six-well plates the day before H2O2 exposure. Cells were then exposed to 100 μM H2O2 in human astrocyte growth medium in the presence or absence of NP1 for 2 h, prior to washing with PBS and fixing with 10% formalin for 20 min. Cells were then washed with PBS and permeabilized with 0.01% Triton X-100 in PBS. Blocking was performed with PBS containing 10% FBS and 1% sodium azide (PSA) for 2 h. Sections were stained with rabbit anti-GFAP polyclonal antibody (Dako, 1:5000 dilution) in PSA overnight at 4 °C. After three washes with PSA, incubation was resumed with PSA containing FITC-conjugated goat anti-rabbit secondary antibody (Abcam, 1:1000 dilution) for 1 h at room temperature. Washed coverslips were counterstained with DAPI and mounted onto slides using ProLong Gold antifade reagent (Life Technologies). Cells were visualized by fluorescence microscopy using a Nikon Ri1 color cooled camera system (Nikon Instruments, Melville, NY, USA). DCFH Assay. DCFH-DA was dissolved in methanol to make a 1 mM stock solution, which was aliquoted and stored at −80 °C. A working solution (50 μM) was prepared by diluting the stock solution in PBS. NP1 was dissolved at 100 mg/mL in PBS and diluted with various concentrations of H2O2 up to 1 mg/mL in 630 μL. After 1 h to allow for NP1 to react with ROS, DCFA-DA (70 μL) was added and allowed to react for 1 h at room temperature in the dark before measuring fluorescence (ex: 480, em: 530) on a SpectraMax microplate reader (Molecular Devices). Fluorescence measurements were taken every 30 min up to the 2.5 h time point. For intracellular DCFH staining, human astrocytes were seeded the day before treatment in 24-well plates at 25 000 cells per well. Cells were washed with serum-free DMEM without sodium pyruvate (ROS medium), after which fresh ROS medium or ROS medium containing H2O2 (1 mM) was added. NP1 was then added to the treatment wells at 0.01 or 0.1 mg/mL and incubated for 30 min. Cells were subsequently washed three times with PBS; then fresh ROS medium containing DCFH-DA

various injury severities beyond a single moderate injury, treatment at various time points after injury in addition to immediately after, and assessments of systemic toxicity and immunogenicity. We are encouraged by these results, but appreciate the vast efforts required that eventually lead to making a clinical impact.

CONCLUSIONS Well-defined core-cross-linked NPs were synthesized from polysorbate 80 using a combination of thiol−ene and thiol− Michael chemistry. The initial neat reaction of PS80 with tetrathiol (TSH) proceeds with quantitative addition of the thiol to the olefin. The resultant PS803SH can be used without the need for purification to prepare core-cross-linked NPs. Selfassembly of PS803SH under aqueous conditions prior to the addition of the TAc and hexyl amine catalyst yields NPs with low molar mass dispersities. Purified NPs can be resuspended in buffer following lyophilization. In vitro assays show that NP1 has promise as a neuroprotective agent in TBI that can be readily modified to achieve therapeutic benefit in vivo. NP1 sequesters significant amounts of ROS, reduces intracellular ROS concentration in human astrocytes, and completely abolishes human astrocyte reactivity after exposure to H2O2. Furthermore, NP1 is able to accumulate in damaged brain quickly after injection and reduce the spread of damage during this early phase of the disease. Importantly, this translated into improved long-term outcome in a CCI mouse model of TBI with decreased neuroinflammation and improved recovery and learning, whereas no difference in motor function was observed. NP1 was cleared from the body through the liver and kidneys after around 24 h, suggesting its safety for use. Combined, our observations strongly support the further development and testing of NP1 as an NP-based antioxidant therapy for TBI. METHODS Materials. All chemicals and all materials were supplied by SigmaAldrich unless otherwise specified. Spectra/Por regenerated cellulose dialysis membranes (6−8 kDaA cutoff) were obtained from Fisher Scientific. G-25 prepacked PD10 columns were obtained from GE Life Sciences. MTS cytotoxicity kits were obtained from Promega. All cell culture reagents were purchased from Life Technologies unless otherwise specified. Antibodies were purchased from Wako (Richmond, VA, USA). RAW 264.7 cells and murine-derived macrophages (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing L-glutamine (Gibco), 4.5 g/L glucose, 10% fetal bovine serum (FBS, Invitrogen), and 1% penicillin− steptomycin (Gibco) at 37 °C and 5% CO2. Synthesis of PS803SH via a Photochemical Thiol−Ene Reaction. Synthesis of PS803SH was done by adding pentaerythritol tetrakis(3-mercaptopropionate) (37.3 g, 76.3 mmol) to polysorbate 80 (100 g, 76.3 mmol), achieving the initial molar ratio [tetraSH]0: [Ps80]0 of 1:1. 2,2-Dimethoxy-2-phenylacetophenone (1.95 g, 7.63 mmol) was added to the mixture, achieving the initial molar ratio [Ps80]0:[PI]0 of 10:1. The thiol−ene reaction was conducted in an ice bath under UV irradiation at 365 nm for 7 h with replacement of fresh ice every 2 h. Synthesis of Core-Cross-Linked NPs via Thiol−Michael Chemistry. To a 22 mL glass scintillation vial was added PS803SH (4.0 g, 2.22 mmol), which was then diluted with 4.0 mL of acetone. The solution was then diluted into 90 mL of deionized water in a 100 mL glass bottle containing a magnetic stir bar. The aqueous solution was then sonicated in a low-power ultrasonic bath for 10 min. The reaction vessel was then placed on a magnetic stir plate. To the stirred solution was then added pentaerythritol tetraacrylate (0.587 g, 1.66 mmol) in acetone (4.0 mL) dropwise over 10 min. The solution was then sonicated for an additional 6 h, after which time hexylamine (47.8 8608


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ACS Nano reagent (10 μM) was added to each well. After a 30 min incubation, cells were washed three times with PBS, fresh ROS medium was added, and then live cells were imaged by fluorescence microscopy using a Nikon Ri1 color cooled camera system. For GFAP immunostaining, human astrocytes were plated onto 22 × 22 mm No. 1 glass coverslips the day before treatment in six-well plates. Cells were washed with ROS medium, after which fresh ROS medium or ROS medium containing H2O2 (100 μM) was added. NP1 was then added to the treatment wells at 1 or 0.1 mg/mL and incubated for 2 h. Cells were subsequently washed three times with normal growth medium and incubated overnight. Cells were then fixed with 4% formaldehyde for 15 min, permeabilized with PBS containing 0.3% Tween 20 (PBT), blocked with 10% serum in PBT (blocking buffer), then stained with anti-GFAP antibody (Abcam, Cambridge, MA, USA) overnight at 4 °C, washed three times with blocking buffer, then stained with Alexa Fluor 488 conjugated secondary antibody (Abcam) for 1 h, washed three times with blocking buffer, counterstained with DAPI, and then mounted on slides using ProLong Gold antifade reagent (Invitrogen, Carlsbad, CA, USA). Cells were then imaged by fluorescence microscopy using a Nikon Ri1 color cooled camera system. In Vitro Cytotoxicity Assay. The cytotoxicity of the NPs was evaluated in RAW 264.7 cells using the CellTiter 96AQueous One Solution cell proliferation assay (MTS) (Promega Corp., Madison, WI, USA). RAW 264.7 cells were seeded in DMEM (Gibco, Life Technologies, Grand Island, NY, USA) containing 1% pen/strep and 10% FBS at a density of 50 000 cells/well in 96-well plates and allowed to adhere for 18 h at 37 °C with 5% CO2. After incubation, NPs diluted in supplemented DMEM at a concentration of 40 mg/mL total polymer were added to cells in triplicate wells in a 1:1 dilution and then serially diluted down the plate (20 mg/mL−9.77 μg/mL), and cells were incubated for 24 h. After the allotted time, cells were evaluated using the CellTiter MTS assay according to the manufacturer’s instructions. The absorbance at 490 nm was evaluated using a Tecan Safire 2 microplate reader. MTS reagent alone was used as a negative control, and all treatments were compared to untreated cells as a positive control to acquire percentage viability. All experiments were carried out in triplicate wells on duplicate days. Controlled Cortical Impact Mouse Model of TBI. All animal procedures were performed in accordance with University of Nebraska−Lincoln IACUC approved protocols. Six-week-old C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and allowed to acclimate for 1 week prior to procedures. Mice were induced with 4% isoflurane gas via inhalation and maintained at ∼1.5% on a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). Hair on the top of the skull was removed with Nair (Church and Dwight Co., Inc., Princeton, NJ, USA), and scalp disinfected with a betadine scrub and cleaned with 2propanol wipes. Lidocaine (0.05 mL at a 5 mg/mL) and bupivacaine (0.05 mL at 0.3 mg/mL) were injected into the scalp. An approximately 1 cm midline incision was made in the scalp over bregma. A small, 2 mm cranial window made in the skull over the left frontoparietal cortex (3 mm anterior and 2 mm left of lambda) using a surgical drill. A controlled cortical impactor (Hatteras Instruments, Cary, NC, USA) attached to the stereotaxic frame with a 1 mm convex tip was used to impact the brain normal to the dura surface at a depth of 1.5 mm at 4 m/s and remained in the brain for 80 ms. Incisions were closed using skin glue, and mice given a subcutaneous injection of buprenorphine SR (0.1 mg/kg). Within 5 min postsurgery 200 μL of NP1 at 10 mg/mL in PBS was injected through the tail vein. Mice were monitored until awake. iBox Imaging. For imaging of NP1 accumulation in TBI, mice were euthanized at 1 and 2 h postinjection through anesthetized cervical dislocation. Brains were removed, placed on the bottom of a black 96-well plate, and imaged with the iBox small animal imaging system (UVP, Upland, CA, USA) with appropriate filters. For imaging of NP1 biodistribution, mice were euthanized at 2, 6, and 24 h postinjection, and organs dissected and placed on a black 96-well plate for iBox imaging. Mice receiving no NP1 injection were used for background controls. Quantification of fluorescence intensity was

performed using the region of interest tool in the iBox software. Values shown are background-subtracted averages of three replicates. Magnetic Resonance Imaging. In vivo mouse brain MRI was conducted on a 9.4 T MR scanner (Varian, Walnut Creek, CA, USA) to generate T2 weighted images. Images were acquired using a fast spin−echo pulse sequence: TR/effective TE = 3000/60 ms, echo spacing = 15 ms, echo train length = 8, center of k-space = echo 4, 6 averages, matrix = 256 × 256, FOV = 20 × 20 mm, 12 slices of 1 mm thickness. Mice were serially imaged on days 1, 3, 6, 10, 15, and 22 after CCI surgery under isoflurane anesthesia. Volume of secondary damage was estimated from hyperintense regions indicative of edema in T2 weighted images using the OsiriX Lite software package (Pixmeo SARL, Bernex, Switzerland). Behavior Analyses. Startle response was tested using an SR-LAB startle response system at 1 week and 1 month following CCI surgery with control animals receiving no surgery or NPs. A startle habituation protocol was used with a test session consisting of a background noise (70 dB) presented alone for 5 min for the acclimation period and continued throughout the session. After the acclimation period a startle-inducing 120 dB broadband noise pulse was sounded and startle measured through an accelerometer. The noise pulse was sounded 19 more times with interpulse intervals of 20 s for a total of 20 trials. Habituation was determined through regression analyses of maximum startle amplitudes over sets of 4 trials. Motor function and learning were assessed using the Rotor-Rod motor function system (San Diego Instruments, San Diego, CA, USA). Mice were trained on the Rotor-Rod daily for 1 week prior to CCI surgery (days −7 to −3) and then 1, 4, 7, and 14 days following CCI with control animals receiving no surgery or NPs. Mice were placed onto the cylinders, which then began to rotate linearly, increasing in speed from 0 to 50 rpm over 5 min. Latency to fall was averaged over 5 separate runs for each animal on each day. Histology. Brains from mice were collected 1 week and 1 month post-CCI, trimmed, and fixed in 10% formalin for 48 h prior to exchanging with 70% ethanol. Brains were then submitted to the Veterinary Diagnostic Clinic for histological processing including paraffin embedding, sectioning, and immunohistochemistry for GFAP and Iba1. Images were acquired on an Olympus IX73 microscope (Olympus, Center Valley, PA, USA) with a DP80 CCD camera (Olympus). GFAP- and Iba1-positive cells were manually counted through a 40× objective with a 0.05 mm3 field of view. Statistics. Data are reported as mean ± standard deviation. Statistical analyses were performed using GraphPad Prism software with statistical significance defined as P values lower than 0.1. Comparisons between two groups were performed using t tests, and comparisons between three groups were performed using one-way ANOVA with Tukey’s multiple comparisons test for comparing between groups. Linear regressions were used for startle response analyses.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b03426. 1

H NMR, microscopy, and cell viability assays (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail (F. M. Kievit): [email protected]. Tel: (402) 472-2175. *E-mail (A. J. Convertine): [email protected]. Tel: (206) 8176011. ORCID

Patrick S. Stayton: 0000-0001-6939-6371 Forrest M. Kievit: 0000-0002-9847-783X Anthony J. Convertine: 0000-0002-3263-1523 8609


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The authors declare no competing financial interest.

ACKNOWLEDGMENTS We acknowledge the Biomedical and Obesity Research Core facility supported by a grant (P20GM104320) from the National Institute of General Medical Sciences, National Institutes of Health, for use of the iBox small-animal imaging system, startle response system, and Rotor-Rod. We also thank D. Yates for use of his microscope. We thank A. Manske, B. Murphy, and C. Gee for assistance with animal procedures. F.K. acknowledges support from an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health (P20GM103480) and the Nebraska Settlement Biomedical Research Development Funds. REFERENCES (1) Impellizzeri, D.; Campolo, M.; Bruschetta, G.; Crupi, R.; Cordaro, M.; Paterniti, I.; Cuzzocrea, S.; Esposito, E. Traumatic Brain Injury Leads to Development of Parkinson’s Disease Related Pathology in Mice. Front. Neurosci. 2016, 10, 458. (2) Barnham, K. J.; Masters, C. L.; Bush, A. I. Neurodegenerative Diseases and Oxidative Stress. Nat. Rev. Drug Discovery 2004, 3, 205− 214. (3) Simon, D. W.; McGeachy, M. J.; Bayir, H.; Clark, R. S. B.; Loane, D. J.; Kochanek, P. M. The Far-Reaching Scope of Neuroinflammation After Traumatic Brain Injury. Nat. Rev. Neurol. 2017, 13, 171−191. (4) Hall, E. D.; Vaishnav, R. A.; Mustafa, A. G. Antioxidant Therapies for Traumatic Brain Injury. Neurotherapeutics 2010, 7, 51−61. (5) Astete, C. E.; Dolliver, D.; Whaley, M.; Khachatryan, L.; Sabliov, C. M. Antioxidant Poly(Lactic-Co-Glycolic) Acid Nanoparticles Made with A-Tocopherol-Ascorbic Acid Surfactant. ACS Nano 2011, 5, 9313−9325. (6) Bitner, B. R.; Marcano, D. C.; Berlin, J. M.; Fabian, R. H.; Cherian, L.; Culver, J. C.; Dickinson, M. E.; Robertson, C. S.; Pautler, R. G.; Kent, T. A.; Tour, J. A. Antioxidant Carbon Particles Improve Cerebrovascular Dysfunction Following Traumatic Brain Injury. ACS Nano 2012, 6, 8007−8014. (7) Singhal, A.; Morris, V. B.; Labhasetwar, V.; Ghorpade, A. Nanoparticle-Mediated Catalase Delivery Protects Human Neurons From Oxidative Stress. Cell Death Dis. 2013, 4, e903. (8) Reddy, M. K.; Wu, L.; Kou, W.; Ghorpade, A.; Labhasetwar, V. Superoxide Dismutase-Loaded PLGA Nanoparticles Protect Cultured Human Neurons Under Oxidative Stress. Appl. Biochem. Biotechnol. 2008, 151, 565−577. (9) Reddy, M. K.; Labhasetwar, V. Nanoparticle-Mediated Delivery of Superoxide Dismutase to the Brain: an Effective Strategy to Reduce Ischemia-Reperfusion Injury. FASEB J. 2009, 23, 1384−1395. (10) McConeghy, K. W.; Hatton, J.; Hughes, L.; Cook, D. A. M. A Review of Neuroprotection Pharmacology and Therapies in Patients with Acute Traumatic Brain Injury. CNS Drugs 2012, 26, 613−636. (11) Xu, J.; Ypma, M.; Chiarelli, P. A.; Park, J.; Ellenbogen, R. G.; Stayton, P. S.; Mourad, P. D.; Lee, D.; Convertine, A. J.; Kievit, F. M. Theranostic Oxygen Reactive Polymers for Treatment of Traumatic Brain Injury. Adv. Funct. Mater. 2016, 26, 4124−4133. (12) Boyd, B. J.; Galle, A.; Daglas, M.; Rosenfeld, J. V.; Medcalf, R. Traumatic Brain Injury Opens Blood-Brain Barrier to Stealth Liposomes Via an Enhanced Permeability and Retention (EPR)-Like Effect. J. Drug Targeting 2015, 23, 847−853. (13) Clond, M. A.; Lee, B.-S.; Yu, J. J.; Singer, M. B.; Amano, T.; Lamb, A. W.; Drazin, D.; Kateb, B.; Ley, E. J.; Yu, J. S. Reactive Oxygen Species-Activated Nanoprodrug of Ibuprofen for Targeting Traumatic Brain Injury in Mice. PLoS One 2013, 8, e61819. (14) Cruz, L. J.; Stammes, M. A.; Que, I.; van Beek, E. R.; KnolBlankevoort, V. T.; Snoeks, T. J. A.; Chan, A.; Kaijzel, E. L.; Löwik, C. 8610


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