Protein Nanoparticles Modified with PDGF-B as a Novel Therapy After Acute Cerebral Infarction

Expression and purification of HSPNPs

The pET21a(+) vector-encoded HSPG41C mutant, in which the Gly 41 residue of wild-type HSP was substituted with a Cys residue, was prepared by polymerase chain reaction-mediated mutagenesis using appropriate primers (Toita et al., 2012, 2013). Successful mutagenesis was confirmed by DNA sequencing. The HSPG41C mutant protein was obtained from E. coli and was purified by anion-exchange chromatography and size-exclusion chromatography (SEC). The BL21 (DE3) strain of E. coli (Merck KGaA) was used for HSPG41C expression. E. coli containing the pET21a (+) plasmid vector was grown in 2 × YT medium containing 100 μg/ml of ampicillin at 37°C. When the culture medium’s optical density at 600 nm reached 0.5−0.6, the expression of the recombinant protein was induced by exposure to 1 mm isopropyl-β-D-thiogalactopyranoside (Wako Pure Chemical Industries) for 4 h at 37°C. After cell harvesting by centrifugation, cells were suspended in 25 mm monopotassium phosphate solution containing 1 mm EDTA and 2 mm dithiothreitol, pH 7.0, and stored at −80°C pending purification. The cell suspensions were then sonicated to disrupt the cell membrane. The resulting cell lysates were centrifuged at 15,000 × g for 30 min at 4°C, and the supernatant was collected. Proteins were purified using a HiPrep Q HP 16/10 anion exchange column (GE Healthcare) and a SEC column (TSKgel G4000SW; Tosoh Corporation). Successful purification was confirmed by electrophoresis with 15% SDS-PAGE.

Synthesis of PDGF-B modified HSPNPs (PDGF-B HSPNPs)

To introduce a chelating function to the surface of HSPG41C, isothiocyanobenzyl-NTA (N-[5-(4-isothiocyanatobenzyl)amido-1-carboxypentyl]iminodiacetic acid; Dojindo Laboratories) was used. Through the NTA moiety attached to the surface, a genetically edited protein bearing a hexahistidine extension (His-tag) at its terminus can be immobilized to the surface via chelation bridging through nickel (II). HSPNPs were modified through amide bond formation with isothiocyanobenzyl-NTA using a 20 × excess of the protein monomer in HEPES (0.1 m, pH 8.5) for 24 h at room temperature, followed by removal of unreacted isothiocyanobenzyl-NTA using ultrafiltration. Successful modification of the HSPNPs was confirmed with matrix assisted laser desorption ionization-time of flight MS. For modification of PDGF-B, recombinant human PDGF-B protein bearing a His-tag (SPEED BioSystems) was prepared. HSPNP modified-NTA and PDGF-B protein (2 equiv. to HSPG41C) were dissolved in HEPES (0.1 m, pH 8.0) with 1 mm nickel (II) and stirred for 24 h at 4°C. Unreacted protein was removed by ultrafiltration with a molecular weight cutoff of 100,000.

NP characterization

The hydrodynamic diameter of the NPs (10 μmol/l) was measured using dynamic light scattering at a detection angle of 173° and a temperature of 25°C using a Zetasizer Nano ZS Analyzer (Malvern Instruments). The 633 nm line of a helium-neon laser was used as the incident beam. All samples and buffer solutions were filtered using Ultrafree-MC centrifugal filter units with 0.22-μm pores (Millipore) before analysis. NPs were applied to carbon-coated copper grids to prepare samples for analysis using TEM. After adsorption of the NPs, the grids were rinsed with droplets of deionized water and stained with 2% uranyl acetate.

In vitro experiments

Human brain pericytes isolated from normal adult human cortices were purchased from ScienCell Research Laboratories. The cells were plated on poly-D-lysine plates and cultured in pericyte medium supplemented with 10% fetal bovine serum and pericyte growth supplement at 37°C in 5% CO₂ in a humidified incubator. Cells at passages 4–8 were used for experiments. To elucidate the role of PDGF-BB, we incubated cultured human brain pericytes with PBS (n = 5, 20 μl), HSPNPs (n = 5, 10 ng/ml, 20 μl), PDGF-BB (n = 5, 10 ng/ml, 20 μl), or PDGF-B HSPNPs (n = 5, 10 ng/ml, 20 μl) at 37°C for 10 min. We extracted the proteins from the cultured cells for immunoblot analysis.

Immunoblot analyses

Cultured cells and brain tissue were homogenized in RIPA lysis buffer (Wako), phosphatase inhibitor cocktail (Thermo Fisher Scientific), and protease inhibitor cocktail (Cell Signaling Technology Japan). Protein concentration was determined using a microplate BCA protein assay kit (Thermo Fisher Scientific). The samples were subjected to Super Sep Ace (20 μg/lane, Fujifilm) and then transferred onto PVDF membranes. The membranes were incubated at room temperature with ECL prime blocking agent (GE Healthcare) for 1 h and probed with primary antibodies to Akt, phospho-Akt (Ser 473; all from Cell Signaling Technology Japan), and β-actin (Sigma) at 4°C overnight. The membranes were then washed and incubated for 1 h at room temperature with the secondary antibody (1:2000 dilution, Cell Signaling Technology Japan). Blots were developed using the ECL prime Western blot detection reagent (GE Healthcare) according to the manufacturer’s instructions.

Measurement of neurotrophic factors

The concentrations of neurotrophic factors in protein samples from infarcted brain tissue were measured with a multi-neurotrophin (NT) rapid screening ELISA kit for NGF, BDNF, NT-3, and NT-4/5 (Biosensis Pty Ltd.), according to the manufacturer’s instructions. Absorbance was measured at 450 nm with a microplate reader (Bio-Rad).

Mouse stroke model

All animal procedures were performed in accordance with the Kyushu University animal care committee’s regulations. All animal experiments complied with the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines. CB-17 (CB-17/lcr-+/+Jcl) wild-type mice were purchased from CLEA Japan. We used both male and female mice aged 7–19 weeks and weighing 18–30 g. A transient middle cerebral artery occlusion (t-MCAO) model mouse was created using the following method (Kasahara et al., 2013). We used this mouse stroke model because individual differences of ischemic volume might be small because of less collateral circulation in CB-17 strain compared with other mouse strains, and we could focus on the effect of NPs. The mice were randomly assigned to surgery and administered intraperitoneal anesthesia with ketamine (100 mg/kg) and xylazine (10 mg/kg). We maintained rectal temperatures at 37°C with a heating pad. Following a midline skin incision between the left orbit and the external auditory canal, we dissected the temporal muscle to expose the temporal bone. A burr hole was made using a microdrill to expose the MCA. A 6–0 nylon suture was then threaded under the MCA, distal to the olfactory tract. The suture was rotated 180° clockwise, horizontally with the artery, for complete interruption of blood flow. The mice were returned to cages controlled at 35–37°C without anesthesia until reperfusion. After occlusion for 2 h, anesthesia was again induced and cerebral blood flow was restored by rotating the suture counterclockwise, back to its original position. The number of mice used in each experiment and related information is shown in Table 1. The following conditions excluded mice from the analyses: (1) premature death (n = 6); (2) hemorrhagic infarction on MRI (n = 2); or (3) failure of surgery and incomplete reperfusion (n = 2).

In vivo MRI

MRI was performed using a 9.4 T Biospec MRI scanner (Bruker). Before the experiment, mice were anesthetized with 1.6% isoflurane in 70% N₂O and 30% O₂, fixed in an MR holder and placed in a 3.8 cm-diameter birdcage coil. t-MCAO model mice were imaged with MRI 1, 4, and 8 d after surgery. We captured T2-weighted images of mice and measured the ischemic volume (Wild et al., 2000) using an image analysis system (NIH ImageJ; repetition time = 3000 ms, echo time = 33 ms, number of signals averaged = 8, matrix resolution = 128 × 80, slice thickness = 1 mm, interslice distance = 1 mm, number of slices = 15–20). The infarct volume was calculated with the following formula: infarct volume ratio = (ipsilateral T2 high intensity area)/(contralateral hemisphere × 2). Because of individual differences in the cerebral infarct volume, we evaluated the infarct volume before injection of NPs as a control.

Cylinder test

Because the degree of paresis was small, we used the cylinder test for behavioral evaluation (Li et al., 2004). Cylinder tests were performed on the day of surgery and on days 1, 4, and 8 after surgery. The mouse was placed in an acrylic cylinder, 9 cm in diameter and 20 cm in height, and recorded with a video camera. Two mirrors were placed behind the cylinder and movements of the forelimbs were recorded. The contacts of the forelimbs with the wall were recorded, and when both forelimbs were used, it was considered as “both.” However, if the right forelimb slipped after the simultaneous use of both forelimbs, it was recorded as “both” and “left.” A total of 20 sets were recorded to complete the test. Score = (left forelimb exercise – right forelimb exercise)/(left forelimb exercise + right forelimb exercise + both exercises). Under normal conditions, the score was 0 on average.

Accumulation of HSPNPs in infarct area

PDGF-B HSPNPs were fluorescently labeled with Alexa Fluor 488-maleimide (Alexa Fluor- maleimide; 1 equiv to protein monomer; Invitrogen) in 0.1 m phosphate buffer (pH 7.2) for 24 h at 4°C. Unreacted Alexa Fluor was removed by ultrafiltration using Amicon Ultra Centrifugal Filters with a molecular weight cutoff of 100,000 (Merck KGaA) and a Zeba spin column (Thermo Fisher Scientific). Fluorescently labeled HSPNPs were prepared using the Michael addition reaction, which involves the formation of a stable bond between maleimide and a thiol group under physiological conditions. To investigate the effects of the dose of PDGF-B HSPNPs, concentrations of 0.104 and 1.04 μmol/l were used, based on previous reports. One day after surgery, PDGF-B HSPNPs (concentration of 0.104 μmol/l: n = 5, 0.2 ml; concentration of 1.04 μmol/l: n = 13, 0.2 ml), PBS (n = 5, 0.2 ml), HSPNPs (concentration of 1.04 μmol/l: n = 12, 0.2 ml), and PDGF-BB protein (concentration of 1.04 μmol/l: n = 5, 0.2 ml) were intravenously injected into the tail vein of t-MCAO mice. We administered PDGF-BB protein at the maximum amount, assuming that PDGF-B was attached to all HSPNPs. We determined the dose and administration time based on preliminary experiments. The mice were exsanguinated on days 3, 7, and 15 after drug administration and transcardially perfused with 20 ml of ice-cold, non-heparinized saline and 20 ml of 4% paraformaldehyde. Mouse brains were sliced into 2-mm-thick coronal sections using a mouse brain slicer (Muromachi). Thereafter, fluorescence was measured using an in vivo imaging system (IVIS; PerkinElmer). Some slices were stained with 2,3,5-triphenyltetrazolium chloride (TTC; Wako). In addition, the infarct area, which was unstained after TTC staining, was isolated, homogenized, and used for immunoblotting analyses.

Brains of t-MCAO model mice on days 3 and 7 after the injection of PDGF-B HSPNPs were embedded in optimal cutting temperature compound (Sakura Finetek), and then frozen in dry ice/ethanol. Frozen sections (10 μm) were prepared using a cryostat microtome (HM 505E, Microm). The slides were observed with a BIOREVO BZ-9000 fluorescence microscope (Keyence Corporation). Green fluorescence emitted from PDGF-B HSPNPs was detected at a 488-nm excitation wavelength and a 507-nm emission wavelength.

Immunostaining was performed using the VECTASTAIN ABC kit (Funakoshi); 2-mm-thick coronal sections were fixed with 4% paraformaldehyde in PBS for immunohistochemistry. Paraformaldehyde-fixed coronal sections were embedded in paraffin and cut into 4-μm-thick slices. The sections were then deparaffinized and activated with microwaves in 10 mmol/l Na citrate or 1 mmol/l EDTA for 10–15 min. The sections were immersed in 0.3% hydrogen peroxide-methanol solution and peroxidase-blocked for 30 min. The sections were then blocked for 1 h at room temperature with TBS-T containing 5% goat serum albumin. The sections were incubated with the following primary antibodies: anti-phospho-Akt (CST), anti-microtubule-associated protein 2 (MAP-2; Abcam) and overnight at 4°C. The mixture was incubated with biotinylated affinity-purified anti-rabbit IgG as a secondary antibody for 30 min at room temperature. The enzyme reagent reaction was conducted at room temperature for 30 min with ABC reagent. The slides were incubated with DAB until an appropriate staining intensity was obtained. The slides were observed with a BIOREVO BZ-9000 fluorescence microscope (Keyence Corporation). The sum of positive cells was measured in five randomly selected sections (60×) in the peri-infarct area (inside the MCA area of the bregma slice) and the ischemic core. The infarct volume ratio was evaluated based on the following formula: infarct volume ratio = (ipsilateral MAP-2-negative area or T2 high intensity area)/(contralateral hemisphere × 2). MAP-2-negative areas and infarct volumes were quantified using ImageJ in a blinded manner.

To examine the distribution of phosphorylated Akt in the cerebral infarction, immunofluorescent double-labeling was performed. Staining was performed using VECTASTAIN ABC kit (Funakoshi). Sections were incubated with the primary antibody anti-phospho-Akt (1:100, CST) followed by incubation with the fluorescence-labeled secondary antibody anti-PDGFRβ (1:50, CST). Then, the slides were washed and mounted with Vectashield mounting medium with DAPI (Vector Laboratories) for nuclear staining. The slides were imaged with a BZ-9000 fluorescence microscope (Keyence Corporation).

To examine the fibrotic response in peri-infarct area, immunofluorescent staining of GFAP was performed. Staining was performed using the VECTASTAIN ABC kit (Funakoshi). Sections were incubated with primary antibody GFAP (1:50, CST) and then with fluorescently labeled secondary antibody Alexa Fluor 594 goat anti-rabbit IgG (1:250, Thermo Fisher Scientific). The slides were imaged with a BZ-9000 fluorescence microscope (Keyence Corporation). Peri-infarct GFAP-positive areas were calculated using three sections. The sum of positive cells was measured in five randomly selected sections (60×) in the peri-infarct area (inside the MCA area of the bregma slice). Results are presented as cells/mm².

Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) staining

To assess apoptosis in cerebral infarctions, a TUNEL assay was conducted with a direct MEBSTAIN Apoptosis TUNEL kit (MBL) based on the manufacturer’s instructions. Then, the slides were washed and mounted with Vectashield mounting medium with DAPI (Vector laboratories) for nuclear staining. The percentage of TUNEL-positive cells in the cerebral infarct was measured by counting cells using the BIOREVO BZ-9000 microscope. In addition, fluorescent/TUNEL double staining was performed to examine cells leading to apoptosis. For the primary antibody, we used the same mouse monoclonal antibody against NeuN (1:50) that we had used for immunohistochemistry at different concentrations. The secondary antibody was Alexa Fluor 594 goat anti-rabbit IgG (1:250, Thermo Fisher Scientific). The images were acquired using a BIOREVO BZ-9000 fluorescence microscope (Keyence Corporation). Green fluorescence emitted from TUNEL-positive cells was detected at a 488 nm excitation wavelength and a 507 nm emission wavelength. The sum of TUNEL-positive, TUNEL/NeuN double-positive, and NeuN-positive cells was counted in five randomly selected sections (60×) in the peri-infarct area and the ischemic core. Results are presented as cells/mm².

Statistical analysis

Data are expressed as mean ± standard deviation. Statistical analyses were performed using the unpaired t test with the JMP software (version 14; SAS Institute); p < 0.05 was considered statistically significant.