Material & Methods
Reagents
Catalase from bovine liver was purchased from Calbiochem (CA, USA). Bis-(sulfosuccinimidyl)suberate sodium salt (BS3), 3,3'-dithiobis-(sulfosuccinimidyl propionate) (DTSSP), 6-hydroxydopamine (6-OHDA), lipopolysaccharides (LPS), PEI (2K, branched, 50% aqueous solution), rhodamine isothiocyanate, and Triton™ X-100 were from Sigma-Aldrich (MO, USA). Methoxypolyethylene glycol epoxy was from Shearwater Polymer Inc. (AL, USA). PEI (2K)–PEG (10K) was synthesized as described previously by conjugation of PEI and methoxypolyethylene glycol epoxy.[11] 3H-labeled catalase was custom synthesized by PerkinElmer Life (MA, USA). IFN-γ was purchased from Peprotech Inc. (NJ, USA). A XenoLight™ RediJect D-Luciferin Ultra inflammation probe was purchased from Caliper LifeSciences (MA, USA). A description of the different antibodies (Abs) for catalase intracellular localization studies can be found in the Supplementary Material https://www.futuremedicine.com/doi/suppl/10.2217/nnm.13.115/suppl_file/suppl_material.doc.
Assembling & Cross-linking Catalase Nanozymes
A description of nanozymes used in this study is presented in Table 1. The BICs were produced by mixing catalase and a block copolymer (PEI–PEG) that bind electrostatically to each other and form nanoparticles with an enzyme–polyion complex core and PEG corona. Catalase and the block copolymer were separately dissolved in phosphate-buffered saline (PBS) at room temperature. Precalculated volumes of the block copolymer solution (0.25 mg/ml) were added drop-by-drop to the enzyme solution (0.5 mg/ml) to achieve the desired charge ratio (charge ratio [Z] = 2–9). The Z was calculated by dividing the number of amino groups in PEI–PEG protonated at pH 7.4[22] by the total amount of glutamate and aspartate in the catalase. The mixture was supplemented with a six-times excess of either a biodegradable linker (DTSSP) or nonbiodegradable linker (BS3; with respect to catalase charges), incubated overnight at 4°C, and unreacted cross-linker was removed by desalting using NAP-10 Columns (GE Healthcare Bio-Sciences Corp., NJ, USA). Obtained nanozymes were characterized by electrophoretic retention, dynamic light scattering (DLS), nanoparticle tracking analysis and atomic force microscopy (AFM). A description of these methods can be found in the Supplementary Material https://www.futuremedicine.com/doi/suppl/10.2217/nnm.13.115/suppl_file/suppl_material.doc.
Macrophage Isolation, Differentiation & Cultivation
Bone marrow-derived macrophages (BMMs) were extracted from the femurs of 6–7-week-old C57Bl/6 male mice according to previously published protocols[23] and cultured for 12–14 days in DMEM medium (Invitrogen, CA, USA) supplemented with 1000 U/ml macrophage colony-stimulating factor (MCSF), a generous gift from Wyeth Pharmaceutical, MA, USA. RAW 264.7, a mouse macrophage cell line, was purchased from the American Type Culture Collection (VA, USA; catalog # TIB-71), and cultured in the DMEM supplemented with 10% fetal bovine serum. Human monocyte-derived macrophages (HMDMs) were obtained from the leukopaks of healthy donors, purified by countercurrent centrifugal elutriation[24] and cultured with MCSF.[23]
To promote specific differentiation of RAW 264.7 macrophages, the cells were cultured in the presence of: IL-4 (to promote the M2 anti-inflammatory subtype), or IFN-γ and LPS (to obtain the M1 proinflammatory subtype). For M2 subset differentiation, macrophages were supplemented with IL-4 (40 ng/ml) for 48 h. For M1 subset differentiation, the cells were cultured in a mixture of IFN-γ (20 ng/ml) and LPS (100 ng/ml) for 48 h. Following incubation, the media was replaced with a mixture of: Abs to the mannose receptor (M2 type marker, anti-CD 206; 1 µg/ml; BD Biosciences, CA, USA), and Abs to CD 86 (M1 type marker, anti-CD 86; 2 µg/ml; BD Biosciences). The cells were incubated with the Abs for 1 h, washed, fixed and examined by confocal microscopy as described below (CD 86: λ = 405 nm; and CD 206: λ = 647 nm). An overexpression of specific markers related to the M1 or M2 subset of macrophages upon cell differentiation was confirmed by FACS.
Measures of Nanozyme Activity
The catalase activity of the obtained nanozymes in a cell-free system was measured using hydrogen peroxide decomposition.[13] The initial catalase activity was 38,000 U/mg protein. Next, the effect of cross-linking on the stability of the enzyme in BIC was examined upon incubation of different nanozymes (0.5 mg/ml catalase) with trypsin (10–5 M) or pronase (2 × 10−1 mg/ml) for 3 h at 37°C. Following incubation, the aliquots were subjected to catalytic activity assessment as described before. The stability of catalase was expressed in residual activity versus initial activity of nanozyme.
For evaluation of the activity of catalase released from the cell carriers, RAW 264.7 macrophages were loaded with different nanozyme formulations for 2 h, then the loading solutions were washed with PBS, and fresh complete media with serum was added to the cells. Following different periods of time, the media was collected and the antioxidant activity of the enzymes released from macrophages was assayed as described before. The concomitant media collected from nonloaded macrophages was examined in the control experiments, and the activity of released endogenous catalase was subtracted from the samples with nanozyme-preloaded macrophages.
Nanozyme Accumulation & Release
First, cytotoxicity of different nanozyme formulations in BMMs or RAW 267.4 macrophages were evaluated using a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay,[25] as described in.[13] A tracer dose of 3H-labeled catalase (4 µCi, 2 µl) was mixed with nonlabeled catalase solution (0.03 mg/ml), supplemented with PEI–PEG block copolymer and cross-linked (Cl) to obtain nanozyme, as described before. For accumulation studies, RAW 264.7 macrophages grown on 24-well plates (2.5 × 106 cells/plate) were treated with the 3H-labeled catalase alone or various nanozyme formulations in the assay buffer for different periods of time in the absence of serum.[26,27] The concentration of catalase was kept the same in all samples (4 µCi/ml). After incubation, the cells were washed with PBS and solubilized in Triton X-100 (1%). For release studies, the cells were loaded with nanozymes for 2 h, washed with PBS and then incubated in fresh media for various times. A total of 100 µl of the cell concomitant media was then placed into 4 ml of a liquid scintillation cocktail, and the radioactivity levels were determined using a Tri-Carb® 4000 (Packard Bioscience; now PerkinElmer) scintillation counter. Assuming that radioactivity levels corresponded to the amount of 3H-labeled nanozyme, the obtained values were normalized for total cell protein content and expressed in ng of catalase per mg of total protein for loading experiments, and ng of catalase per ml of media.
Confocal Microscopy
To study intracellular localization, the catalase was labeled with the Alexa Fluor® 680 or Alexa Fluor 488 Protein Labeling Kit (Invitrogen); the PEI or PEI–PEG were labeled with rhodamine isothiocyanate.[11] The intracellular localization studies were performed in HMDMs due to the specificity of the Abs used for different intracellular compartments staining. HMDMs grown in the chamber were treated with labeled Cl-6-BS3 (0.2 mg/ml) in assay buffer for various periods of time (5–60 min) at 37°C.[28] A detailed description of these evaluations can be found in the Supplementary Material https://www.futuremedicine.com/doi/suppl/10.2217/nnm.13.115/suppl_file/suppl_material.doc.
To evaluate the transport of the macrophages into the brain with inflammation, RAW 264.7 macrophages were transduced with green fluorescent protein (GFP) firefly luciferase viral vector, as described in.[29] To eliminate nontransduced cells, macrophages were selected against puromycin (0.5 µg/ml). GFP-overexpressing macrophages were injected into 6-OHDA-intoxicated BALB/C mice on day 21 after intracranial (ic.) injection of the toxin. A total of 24 h later, mice were sacrificed and perfused with PBS and 4% paraformaldehyde. Brains were frozen, sectioned with a cryostate (10 µm thick) and examined by confocal microscopy (60× magnification). Healthy mice without brain inflammation (with PBS ic. injections) were used as a control group. The glass slides (five slides per brain) were sequentially treated with primary monoclonal mouse anti-CD11b Abs (BD Pharmingen, NJ, USA), the Abs defining macrophages; and secondary fluorescently labeled anti-Mouse-IgG-atto 647N (red; Sigma-Aldrich; 1:200 dilution), and then analyzed by confocal fluorescence microscopy.
For evaluation of catalase activity in the mouse brain, tissues from control animals were ic. injected with PBS or 6-OHDA, as described later, or PD mice were injected with 6-OHDA following intrajugular vein (intravenous; iv.) injection of BMM loaded with nanozyme (5 × 106 cells/mouse in 100 µl PBS) 48 h later. A total of 7 days after BMM treatment, animals were anesthetized with a ketamine/xylazine anesthetic cocktail and perfused transcardially with ice-cold PBS for 5 min. Ventral midbrain areas were dissected and placed in an ice-cold assay buffer (50 mM potassium phosphate buffer; pH 7.0), homogenized and centrifuged at 4000 rpm. Collected supernatant samples were analyzed for catalase activity using the Catalase Assay Kit (Cayman Chemical Company, MI, USA). Enzyme activity was expressed in nmol/min/ml.
Amplex® Red Dye Fluorescence Assay
The decomposition of ROS in Cath.a neurons stimulated with 6-OHDA by different nanozymes released from preloaded macrophages was measured by fluorescence, as described before.[11] The effect of the supernatants collected from nonloaded macrophage ROS decomposition was evaluated with the control experiments. Specific details of this experiment can be found in the Supplementary Material https://www.futuremedicine.com/doi/suppl/10.2217/nnm.13.115/suppl_file/suppl_material.doc.
Bioimaging & Infrared Spectroscopy of Neuroinflammation
C57Bl/6 male mice were used in the in vivo image visualization and infrared spectroscopy (IVIS) experiments (five mice per group). The animals were treated in accordance with the Principles of Animal Care outlined by the US NIH and approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center (USA). For induction of brain inflammation, mice were ic. injected with LPS solution, as described in.[14] Specific details of these procedures can be found in the Supplementary Material https://www.futuremedicine.com/doi/suppl/10.2217/nnm.13.115/suppl_file/suppl_material.doc. A total of 24 h after LPS intoxication, animals were injected iv. with macrophages (5 × 106 cells/mouse in 100 µl PBS) preloaded with Cl nanozyme (Cl-6-BS3). Saline-treated animals, which were intoxicated with LPS 24 h before the treatment, were used in the control group. A total of 10 min before luminescent imaging (exposure time: 10 min), each animal received an intraperitoneal injection of XenoLight RediJect Inflammation probe. Animals were imaged at 1–30 days following the treatment using an IVIS® 200 Series imaging system (Xenogen Co., now Caliper LifeSciences). The chemoluminescent signal was quantified by Living Image® 2.50 software (Caliper LifeSciences) and presented as a radiance ratio of treated animal versus 24 h after LPS injection.
Immunohistochemical & Stereological Analyses
6-OHDA-intoxicated mice (six animals per group) were iv. injected either with PBS, nanozyme-preloaded macrophages (5 × 106 cells/mouse/100 µl), macrophages preloaded with naked catalase, macrophages alone, or Cl nanozyme alone (Cl-6-BS3; 0.5 mg/ml catalase) 48 h after intoxication. Healthy, nonintoxicated animals were ic. injected with PBS instead of 6-OHDA and used in control groups. A total of 5 weeks later, animals were sacrificed, perfused according to standard perfusion protocol, brains were removed, and immunohistochemical analysis was performed, as described in.[29] A detailed description can be found in the Supplementary Material https://www.futuremedicine.com/doi/suppl/10.2217/nnm.13.115/suppl_file/suppl_material.doc.
Statistical Analysis
For all experiments, data are presented as the mean ± the standard error of the mean. Tests for significant differences between the groups were performed using a one-way analysis of variance with multiple comparisons (Fisher's pairwise comparisons) using GraphPad Prism® 6.0 (GraphPad software, CA, USA). A minimum p-value of 0.05 was chosen as the significance level for all tests.
Nanomedicine. 2014;9(9):1403-1422. © 2014 Future Medicine Ltd.
