Photoinduced elimination of senescent microglia cells in vivo by chiral gold nanoparticles

Parkinson's disease (PD) is an age-related neurodegenerative disease, and the removal of senescent cells has been proved to be beneficial for improving age-associated pathologies in neurodegeneration disease. In this study, chiral gold nanoparticles (NPs) with different helical directions were synthesized to selectively induce the apoptosis of senescent cells under light illumination. By modifying anti-B2MG and anti-DCR2 antibodies, senescent microglia cells could be cleared by chiral NPs without damaging the activities of normal cells under illumination. Notably, l-P+ NPs exhibited about a 2-fold higher elimination efficiency than d-P− NPs for senescent microglia cells. Mechanistic studies revealed that the clearance of senescent cells was mediated by the activation of the Fas signaling pathway. The in vivo injection of chiral NPs successfully confirmed that the elimination of senescent microglia cells in the brain could further alleviate the symptoms of PD mice in which the alpha-synuclein (α-syn) in cerebrospinal fluid (CFS) decreased from 83.83 ± 4.76 ng mL−1 to 8.66 ± 1.79 ng mL−1 after two months of treatment. Our findings suggest a potential strategy to selectively eliminate senescent cells using chiral nanomaterials and offer a promising strategy for alleviating PD.

Next, a growth solution was formed by adding 0.08 mL of 100 mM CTAB to 3.95 mL of deionized water. Then, 0.2 mL of 10 mM HAuCl 4 was added to form an AuBr 4complex. After incubation for 5 min, 0.475 mL of 50 mM VC and 5μL of 4mM CF dipeptide were injected rapidly to the growth solution. Finally, 100μL of seeds was added with vigorously stringing. Polarized light (light wavelength: 594 nm; intensity: 84 mW/cm 2 ) was applied during the growth process. The reaction solution was immediately illuminated with various types of polarized light for 30 min immediately after the injection of seeds. Right circularly polarized light (RCP) was applied to form D-P -NPs and left circularly polarized light (LCP) was applied to form L-P + NP. As for the synthesis of DL-NP, DL-CF and 594 nm laser were used following the same steps.
Finally, the chiral NPs were modified with 4 mM mPEG-SH (MW: 2000) for 4 h for further biological application.

Preparation of Au@anti-B2MG and anti-DCR2
We prepared different concentration of anti-DCR2 (0, 0.4, 0.6, 0.8, and 1.0 nM) and anti-B2MG in 200 μL DMF and mixed these with 1.89 mg of EDC and 0.57 mg of NHS. The solution was stirred for 4h at 37 ℃ in the dark. Then the mixture was added dropwise into the NPs and the solution was stirred for 6 h at 37 ℃ in the dark. Finally, unconjugated antibodies were removed by centrifugation at 1700×g for 3 min. The successful conjugation of NPs and antibodies was characterized by CD and the UV spectrum.

Cell lines and incubation conditions
All cells were cultured at 37 °C in a humidified atmosphere of 5% CO 2 and 95% air.
MA-C and MN9D cells were grown in DMEM medium with 1.0 mg/mL d-glucose supplemented with 10% FBS and 1% penicillin streptomycin. BV-2 cells were grown in DMEM medium with 4.5 mg/mL d-glucose supplemented with 10% FBS and 1% penicillin streptomycin.

Cellular survival assays for nanoparticles
BV-2, MA-C and MN9D cells were placed in 96-well plates and incubated for 24 h.
Then, new culture medium, containing different concentrations of nanomaterials, was added to replace the old medium. After 24 h of incubation, cell viability was assessed using the standard Cell Counting Kit-8 (CCK-8) in accordance with the manufacturer's instructions (Beyotime).

Clearance of senescent cell in vitro
The clearance of senescent cells in vitro was determined by the fluorescence intensity of caspases-3, a known marker of apoptosis. Generally, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 2 min, and then blocked with 1% bovine serum albumin (BSA) for 2 h at 20 ℃. Then, the cells were washed three times in PBS before being incubated with anti-caspase-3 for another 2 h at 26 ℃. Subsequently, the cells were washed with PBS and then incubated with Alex488-conjugated (goat-anti-rabbit) secondary antibody (1:500) for 2 h at 26 ℃.
After washing, the cells were imaged using a Leica LSM880 confocal fluorescence microscope.

Transcriptomic genes analysis
RNA was extracted from BV-2 cells with Total RNA Extractor (Sangon Biotechnology, Shanghai) in accordance with the manufacturer's protocol. In total, 10 6 cells were collected and washed three times in PBS. Then, 1mL of Total RNA Extractor was added immediately. Each sample was stored at -80 °C for further treatment.

Rotarod test
All animal experiments were approved by the Committee on Animal Welfare of Jiangnan University. The rotarod test was carried out to determine the motor ability of PD mice undergoing different treatments. For the rotarod test, mice were placed on a rotarod cylinder and the speed was slowly increased speed from 4 to 40 rpm over a 5 min period. We then measured the amount of time each animal remained on the rotarod at least three times after training for 5 days.

Morris water maze test
The Morris water maze test was used to investigate the memory of PD mice undergoing different treatments. The training phase was carried out four times per day for 5 continuous days, in which we tested the ability of the animals to find the position of the platform. We recorded the latency for escape to the platform was recorded. After training for 5 days, the spatial memory of the mice was characterized by the proportion of time spent in the target, the time spent in the target quadrant, and the number of platform crossings in a probe test.

Running wheel test
The running wheel test was used to monitor the improvement of the movement disorder of PD mice undergoing different treatments. The training phase was carried out for 5 continuous days to allow the mice to become familiar with the running wheel. Then, mice were given 60-min periods of wheel-running exercise. For each mouse, we recoded the maximum speed and calculative distance in 60 min.

Immunofluorescence and immunohistochemistry staining
The brains of each mice were stored in 10% formaldehyde and then sectioned into thin slices. Each piece was fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min and blocked overnight with 1% BSA at 4°C. Then, brain sections were incubated with IBA-1 and TH antibodies (1:500) at 20℃. Subsequently, sections were washed with PBS and then incubated with Alex488-conjugated (goatanti-rabbit) and Alex555-conjugated (goat-anti-mouse) secondary antibodies (1:500) for 2 h at room temperature. After washing with PBS, the cells were imaged using a Leica LSM880 confocal fluorescence microscope. The same antibodies were used for IHC.

Histological examination
An H&E staining kit was used to investigate the biocompatibility of gold nanoparticles in major organs

Statistical analyzes
All data are presented as mean ± standard deviation. For each experimental series, at least five replicates (n = 5) were performed unless otherwise stated. For many experimental series at least five independent experiments were carried out; n values given in the figure legends indicate the number of independent experiments performed or the number of mice used. Group differences were considered statistically significant when p < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001). Analysis of variance test was used for multicomponent comparison.