Materials and methods
Cholesterol, DOPA (1,2-dioleoyl-sn-glycero-3-phosphate), DOTAP (1,2-dioleoyl-3-trimethylammonium-propane (chloride salt)), DSPE-PEG-2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt)), LissRd B-DSPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt)) and 1,2-distearoyl-sn-glycero-3-phosphocholine were purchased from Avanti Polar Lipids Inc. All other chemicals were purchased from Sigma-Aldrich and used as received unless otherwise stated. Rabbit polyclonal antibody to SOD1 was purchased from abcam (ab13498) and goat anti-rabbit IgG (H + L) secondary antibody HRP conjugate was purchased from Invitrogen to be used in western blot experiments. Cy5-SOD1 ASO adapted from McCampbell et al. (2018)13 was synthesised by Integrated DNA Technologies and was used as received (target sequence 5’ Cy5-C*AG*GA*TA*C*A*T*T*T*C*T*A*CA*GC*T 3’ where * indicates phosphorothioate bonds and italic bases are 2′-O-methoxyethylribose).
In vivo biodistribution of Cy5-SOD1 ASO CaP lipid NPs
All animal experiments including biodistribution and FUS were approved by the University of Queensland Animal Ethics Committee and conformed to the guidelines of the Australian Code of Practice for the care and use of animals for scientific purposes (AEC approval number: AIBN/CAI/SCMB/223/20). For all animal studies, 6–8 week-old male and female C57BL/6 mice were acquired from the Animal Resource Centre and were allowed access to food and water ad libitum throughout the course of the stay and experiments.
For the biodistribution study, mice were randomly divided into 3 sub-groups (n = 4, 2 male and 2 female). The first group was administered with free Cy5-SOD1 ASO (0.25 mg/kg ASO dose) intravenously and the second group was administered with Cy5-SOD1 ASO CaP lipid NPs (0.25 mg/kg ASO dose/ 5 µg SOD1 ASO per mouse) and were imaged at 0 min, 30 min, 3 h and 6 h time points. The third group of mice were administered with Cy5-SOD1 ASO CaP lipid NPs (0.25 mg/kg ASO dose) and were imaged at 0 min, 8 h, 24 h and 48 h time points. After the final imaging time point, the mice were sacrificed and the major organs (kidney, liver, gut, spleen, lungs, heart, brain and spinal cord) of all animals were harvested and imaged immediately for ex vivo fluorescence using an IVIS Lumina X5 imaging system (PerkinElmer Inc., Waltham, MA, USA) and analyzed using the Living Image software (PerkinElmer Inc.). The 620Ex/670Em wavelengths were used to analyse the Cy5 fluorescence from SOD1 ASO.
Generation of microbubbles
Phospholipid-shelled microbubbles with octafluoropropane gas core were prepared in-house, following a previously described chemical synthesis protocol.51,52 In brief, a 9:1 molar ratio of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] (DSPE-PEG2000 Amine) were mixed and dissolved in the sterile chloroform. The chloroform was evaporated in the vacuum desiccator overnight. The dried lipid film was then rehydrated with sterile filtered phosphate-buffered saline (PBS) with 10% glycerol and 10% propylene glycol to a concentration of 1 mg/mL and sonicated in a water bath sonicator (Branson, M1800) for 10 min at 55 °C. Resulting solution was placed in the glass 1.5 mL HPLC vials, air was removed and octafluoropropane gas (C3F8; Arcadophta) was introduced into the empty headspace of the vial. At the day of experiment, the vials were equilibrated to the room temperature and lipid solution was diluted with 0.5 mL of sterile 0.9% NaCl. Microbubbles were generated by agitation in a dental amalgamator at 4000 rpm for 45 s. Following activation, vials were sealed with parafilm to minimise the gas exchange between the vial and the external environment.
Determining the optimal focused ultrasound conditions required to safely open the BBB
For determining the optimal parameters to safely open the BBB using FUS, female C57BL/6 mice (6-8 weeks old) were intravenously administered with two different doses of MBs (high dose – 0.4 mL/kg, low dose – 0.15 mL/kg) diluted up to 100 µL with sterile saline through lateral tail vein using a catheter. The mice were then subjected to FUS at different exposure levels (0.33 MI, 0.325 MI, 0.319 MI, 0.22 MI and 0.14 MI) for different durations (120 sec and 30 sec). The centre frequency was 250 kHz with a burst length of 10 ms and a burst repetition frequency of 1 Hz. During all injections, FUS experiments and imaging time points, mice were anesthetized with 2% isoflurane in oxygen at a flow rate of 2.5 L min-1. Following FUS exposure, the mice were shifted to the MR machine. The MR images were acquired on a 7T Bruker ClinScan using a 23 mm ID volume mouse head coil. Dual-echo dynamic gradient echo (GRE) images were acquired with the following parameters: GRE_6slice: flip angle = 45°, 6 X 1 mm slices, FOV = 20 X 20 mm, in-plane resolution = 313 x 313 µm, TR = 75 ms, TE = 3 and 6 ms, acquisition time = 3.6 sec. Following localiser images, a T1 weighted fl3d-vibe and T2-weighted images were acquired with the same slice positioning as the dynamic GRE_6slice images. Dynamic GRE_6slices images with 200 measurements were acquired (acquisition time = 12 min). After a 2 min baseline period the mice were administered with 50 µL of Gadovist (Gadolinium contrast agent) and 100 µL of 1% (w/v) Evans blue dye in sterile saline mixed in 50 µL of sterile saline through lateral tail vein using a catheter. Following the dynamic GRE images, high resolution T2_TSE images were acquired in coronal, sagittal and transverse planes then the GRE_6slices were repeated with 20 measurements. High resolution T1_tse images in the three planes, fl3d_swi, fl3d_dixon_T1mapit were acquired followed by another GRE_6slices with 20 measurements. After MRI the mice were allowed to recover to observe any behavioural changes. After a specific period, they were euthanized through cervical dislocation and the brains were removed to observe Evans blue extravasation. The optimal parameters, including the FUS exposure level, time and the MB dose that can be used to safely and transiently open the BBB in mice without causing adverse effects were selected to be used in the following nanoparticle delivery experiments.
FUS-mediated delivery of Cy5-SOD1 ASO CaP lipid NPs into the brain of mice
To deliver Cy5-SOD1 ASO CaP lipid NPs into the brain, female C57BL/6 mice (6-8 weeks old) were intravenously administered with 0.15 mL/kg dose of MBs diluted with sterile saline through lateral tail vein using a catheter. The mice were then subjected to FUS at 0.33 MI exposure levels for 30 sec. The centre frequency was 250 kHz with a burst length of 10 ms and a burst repetition frequency of 1 Hz. During all injections, FUS experiments and imaging time points, mice were anesthetized with 2% isoflurane in oxygen at a flow rate of 2.5 L min-1. Following FUS exposure, the mice were shifted to the MR machine. The MR images were acquired on a 7T Bruker ClinScan using a 23 mm ID volume mouse head coil. Dual-echo dynamic gradient echo (GRE) images were acquired with the following parameters: GRE_6slice: flip angle = 45°, 6 X 1 mm slices, FOV = 20 X 20 mm, in-plane resolution = 313 x 313 µm, TR = 75 ms, TE = 3 and 6 ms, acquisition time = 3.6 sec. Following localiser images, a T1 weighted fl3d-vibe and T2-weighted images were acquired with the same slice positioning as the dynamic GRE_6slice images. Dynamic GRE_6slices images with 200 measurements were acquired (acquisition time = 12 min). After a 2 min baseline period the mice were administered with 50 µL of Gadovist (Gadolinium contrast agent) and Cy5-SOD1 ASO CaP lipid NPs (equivalent to 0.25 mg/kg ASO dose) through lateral tail vein using a catheter. Following the dynamic GRE images, high resolution T2_TSE images were acquired in coronal, sagittal and transverse planes then the GRE_6slices were repeated with 20 measurements. High resolution T1_tse images in the three planes, fl3d_swi, fl3d_dixon_T1mapit were acquired followed by another GRE_6slices with 20 measurements. After MRI the mice were allowed to recover to observe any behavioural changes. After 6 h from NP administration, the mice were euthanized through cervical dislocation and the organs were harvested and imaged for ex vivo fluorescence using an IVIS Lumina X5 imaging system (PerkinElmer Inc., Waltham, MA, USA) and analyzed using the Living Image software (PerkinElmer Inc.). The 620Ex/670Em wavelengths were used to analyse the Cy5 fluorescence from SOD1 ASO. The same experiment was repeated for the control study without the administration of MBs and instead administering saline only.
Histological examination of the brain tissue
To assess for potential injury associated with FUS, the extracted brains of were fixed in 4% PFA for 48 h at 4 oC. Following fixation, brains were subsequently washed with PBS and cryoprotected in 30% sucrose for 48 h at 4 oC, before snap frozen and stored at -80 oC. Mouse brains were coronally sectioned (18 µM) using a cryostat (Jung CM 3000, Leica Instruments GmbH, Nussloch, Germany).
Hematoxylin and eosin (H&E) stain
Sections were stained with freshly filtered Lillie-Mayer’s hematoxylin (POCD Healthcare, Australia) for 3 min, rinsed in running tap water for 1 min, differentiated by quickly dipping 10 times in 1% acid alcohol, washed in running tap water until clear, blued in saturated aqueous carbonate solution for 1 min, washed in running tap water for 30 seconds, counterstained in filtered 1% alcoholic eosin for 1 minute, dehydrated in 3 × 3-minute changes of absolute ethanol, cleared in 2 × 3-minute changes of xylene, mounted in DePex mounting medium and cover slipped.
GFAP and IBA1 immunohistochemistry
The primary antibodies used were; rabbit anti-glial fibrillary acidic protein ([GFAP], #Z0334, Dako, Glostrup, Denmark) diluted 1/13 000 and rabbit anti-ionized calcium binding adaptor molecule 1 (Iba1, #016-20001, Wako, Richmond, VA) diluted 1/500 in 2% normal donkey serum (NDS). The secondary antibody used was biotinylated donkey antirabbit (#715-065-152, Jackson ImmunoResearch, West Grove, PA) diluted 1/1000 in phosphate buffered saline (PBS). Sections were mounted on charged glass microscope slides (Superfrost Plus microscope slides, Lomb Menzel-Glaser, Thermo Scientific, Waltham, MA) and heated at 60°C for 20 minutes. The sections were then dewaxed in 2 changes of xylene (2 min each change), followed by 3 changes of absolute alcohol (2 min each change), and then gently washed in running tap water (3 min). After a brief rinse in distilled water, antigen retrieval was performed by microwave heating the sections to a gentle boil in 10 mM sodium citrate buffer, pH 6 for 10 (GFAP) or 20 (Iba1) min. Slides were removed from the microwave and once the buffer cooled to 50°C, they were gently washed in running tap water (3 min). All subsequent steps were performed at room temperature in a humidified chamber. Sections were circled with a peroxidase-antiperoxidase pen (Dako), rinsed in 2 changes of PBS (5 min each) and then incubated in 10% NDS (Jackson ImmunoResearch) in PBS for 60 min. The NDS was drained from the slides and the sections were incubated with the primary antibody overnight. The following day, the sections were rinsed in 3 changes of PBS (5 min each) prior to blocking endogenous peroxidase by incubating sections in 0.3% hydrogen peroxide in PBS for 30 min. Slides were rinsed in 3 changes of PBS (5 min each), and then incubated in the secondary antibody for 60 min. Slides were rinsed in 3 changes of PBS (5 min each), incubated in Vector Avidin-Biotin Complex (ABC) reagent (Vector Laboratories, Burlingame, CA) for 60 min and rinsed again in 3 changes of PBS (5 min each). The antibody reaction was visualized using diaminobenzidine. Sections were then rinsed in running tap water for 5 min, dehydrated in 3 changes of absolute ethanol (3 min each), cleared in 2 changes of xylene (3 minutes each), mounted in DePex mounting medium (Sigma Life Science, Madrid, Spain) and cover slipped.
Images of GFAP and IBA1-stained tissue sections were acquired using a Leica Confocal SP8 FALCON (Leica, Germany) with an attached colour camera. A 10× air objective was used with white balancing performed to equalise colour intensity for each image. Images were processed with LASX software (Leica, Germany) and FIJI software (version 1.53q)53.
Statistics
Statistical comparison by analysis of variance was performed using student’s t-test or one-way analysis of variance (ANOVA) using a Tukey’s multiple comparisons post-test (GraphPad Prism 8.3.1.). P < 0.05 was considered statistically significant.