Polydopamine(PDA)-Coated Diselenide-Bridged Mesoporous Silica-based Nanoplatform for Neuroprotection by Reducing Oxidative Stress and Targeting Neuroinflammation in Intracerebral Hemorrhage

Polydopamine(PDA)-Coated Diselenide-Bridged Mesoporous Silica-based Nanoplatform for Neuroprotection by Reducing Oxidative Stress and Targeting Neuroinflammation in Intracerebral Hemorrhage | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Polydopamine(PDA)-Coated Diselenide-Bridged Mesoporous Silica-based Nanoplatform for Neuroprotection by Reducing Oxidative Stress and Targeting Neuroinflammation in Intracerebral Hemorrhage Fangfang Zhou, Yongju He, Meiru Zhang, Xiyu Gong, Xiaoxuan Liu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4476509/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Nov, 2024 Read the published version in Journal of Nanobiotechnology → Version 1 posted 9 You are reading this latest preprint version Abstract Oxidative stress (OS) and neuroinflammation are critical pathological processes in secondary brain injury (SBI) after intracerebral hemorrhage(ICH), and their intimate interactions initiate and aggravate brain damage. Thus, targeting oxidative stress and neuroinflammation could be a promising therapeutic strategy for ICH treatment. Here, we report a high-performance platform using polydopamine (PDA)-coated diselenide bridged mesoporous silica nanoparticle (PDA-DSeMSN) as a smart ROS scavenger and ROS-responsive drug delivery system. Caffeic acid phenethyl ester (CAPE) was blocked in the pore of DSeMSN by covering the pore with PDA as a gatekeeper. PDA-DSeMSN @CAPE maintained high stability and underwent reactive oxygen species (ROS)-responsive degradation and drug release. The intelligent nanomaterial effectively eliminated ROS, promoted M1 to M2 microglial conversion and suppressed neuroinflammation in vitro and i n vivo . Importantly, intravenous administration of PDA-DSeMSN@CAPE specifically accumulated in perihematomal sites and demonstrated robust neuroprotection in an ICH mouse model with high biological safety. Taking together, the synergistic effect of ROS-responsive drug delivery ability and ROS scavenging ability of PDA-DSeMSN makes it a powerful drug delivery platform and provided new considerations into the therapeutic action to improve ICH-induce brain injury. Intracerebral hemorrhage polydopamine-coated diselenide bridged mesoporous silica nanoparticle ROS-responsive Microglia polarization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Intracerebral hemorrhage (ICH) is a devastating subtype of stroke, with severe morbidity and high mortality[ 1 ]. Classically, injury from ICH is broadly divided into primary injury, mass effect from the initial hematoma, and secondary injury, induced by a chain reaction of events responed to the hematoma [ 2 , 3 ]. Thus far, the seemingly feasible therapies for treating ICH-induced primary injury have not provided sufficient benefits in clinical trials[ 4 – 7 ], suggesting that secondary injury after ICH may represent an essential and attractive pathophysiological target. Secondary brain injury (SBI) is the main culprit of irreversible neurological deficits and contributes to the progressive neurological deterioration after ICH[ 8 – 10 ]. Various scenarios have elucidated that neuroinflammation and oxidative stress (OS) are funda-mental signatures of SBI after ICH, being inextricably interlinked and interdependent in their pathogenesis[ 11 , 12 ]. The main element of neuroinflammation is microglia activation[ 13 ]. Activated microglia release a myriad of inflammatory and cytotoxic agents, especially excessive production of reactive oxygen species (ROS) [ 14 , 15 ]. ROS are recognized as a crucial player in neuroinflammation-mediated OS. Moreover, ROS burst can trigger and exacerbate inflammation through redox-sensitive transcription factors activation and inflammatory cells recruitment [ 16 – 18 ]. An overactivated inflammatory response and an enhanced ROS generation act synergistically, forming a vicious cycle, and promoting the onset and development of SBI after ICH. Obviously, multi-target agents, which can dampen down neuroinflammation and counter oxidative stress, hold considerable promise as a therapeutic strategy for ICH. Caffeic acid phenethyl ester (CAPE), an efficient hydrophobic bioactive polyphenolic ester, shows a wide range of important biological activities[ 19 – 21 ]. It has been proved that CAPE protects neurons by reducing of ROS, attenuating neuroinflammation and suppressing apoptosis in various neurologic diseases [ 22 – 24 ]. However, its practical applications are seriously restricted by poor water solubility, low bioavailability and chemical instability[ 25 – 27 ]. Along with the rapid development of material science and nanotechnology, numerous fascinating nanoparticles used as carriage systems for drug delivery have evolved to achieve better efficacy and safety[ 28 – 30 ]. In particular, the large surface areas, pore tunability, great drug loading capacity facile functionalization, nontoxicity and excellent biocompatibility mesoporous silica nanoparticle (MSN) has sparked immense interest as drug carrier[ 31 – 34 ]. At present, MSN has been further tailored to respond to disease-specific stimuli and realize targeted and controlled drug delivery. Regarding the redox-imbalance and excessive ROS production in the perihematomal brain tissues, introducing ROS-cleavable linkers into the matrix of mesoporous silica is emerging as a promising “on-demand” smart drug delivery platform in the field of ICH treatment [ 35 , 36 ]. Among the cleavable species used for constructing MSN-based drug delivery systems, the diselenide is an ideal choice for ROS-responsive linker, which has high sensitivity towards oxidative milieu. Of particular importance, under ROS-rich circumstances, the diselenide bond was oxidized to selenenic acid, making it attractive ROS scavenger [ 37 – 39 ]. However, diselenide-bridged MSN(DSeMSN) still holds some disadvantages, including short-term circulation and premature drug leakage, which hamper ideal performance in vivo[ 40 ]. Fortunately, a simple surface modification method was invented based on oxidative self-polymerization of dopamine in an alkaline environment. Under this condition, dopamine can be oxidized to generate a water-insoluble polydopamine (PDA) film. As an excellent gatekeeper, PDA encapsulates prodrug nanoparticles internally, reducing the interplay among factors in complex physiological environ- ment and drug properties during blood circulation [ 41 – 43 ]. Additionally, PDA possesses excellent antioxidant activity, which effectively scavenge overproduced ROS[ 44 , 45 ]. In this study, we constructed a smart CAPE-loaded, ROS-responsive diselenide-bridged MSN by PDA coating (PDA-DSeMSN@CAPE)(Scheme 1 ). PDA coating fabricated on the surface of DSeMSN@CAPE acted as a protected capsule for long-term stability. As expected, the structure of PDA-DSeMSN@CAPE was effectively degraded in ROS condition. More importantly, PDA and diselenide can both significantly deplete ROS levels, enabling synergistic treatment of ICH with drug and materials. PDA-DSeMSN@CAPE displayed an extraordinary anti-oxidative and anti-inflammatory property, showing little cytotoxic effect in vitro . Furthermore, using a murine model of ICH, we demonstrated that PDA-DSeMSN@CAPE via intravenous administration accumulated in perihematomal regions and produced an efficient neuroprotective effect via concomitant antioxidative and anti-inflammatory effects. Besides, PDA-DSeMSN@CAPE can be metabolized out of the body more quickly, avoiding toxicity and side effects. Methods 1. Materials. Tetraethyl orthosilicate (TEOS, ≥ 99%), Bis[3-(triethoxysilyl) propyl] diselenide (BTESePD, ≥ 98%), triethanolamine (TEAH3, ≥ 99%), cetyltrimethylammonium tosylate (CTAT, ≥ 95%), and 3-aminopropyltriethoxysilane (APTES, ≥ 98%) were obtained from Titan Co., Ltd (Shanghai, China). Ethanol absolute (C 2 H 5 OH, ≥ 99.7%) and hydrochloride acid (HCl, 36–38%) were purchased from Hunan Huihong Reagent Co., Ltd (Hunan, China). Caffeic Acid Phenethyl Ester (CAPE, 97%) was purchased from Shaoyuan Chemical Technology (Shanghai, China). Dopamine hydrochloride (DA, 98%) was purchased from Shanghai Macklin Biochemical Co., Ltd (Shanghai, China). Near-infrared fluorescent dye (DiR, ≥ 99%) was bought from MedChemExpress Co., Ltd (Shanghai, China). 2. Characterizations Transmission electron microscopy (TEM, Talos F200X) and scanning electron microscopy (SEM, JEOL-4800) were used to detect the morphology and microstructures of nanoparticles. Fourier transform infrared (FTIR) spectrums were recorded on a Fourier transform infrared spectrometer (Nicolet 6700, Thermo Electron Scientific Instruments). UV-Vis absorption spectra were recorded on a UV-visible spectro-photometer (UV-6100, METASH). 3. Synthesis of CAPE-Loaded DSeMSN The diselenide-bridged mesoporous silica nanoparticle (DSeMSNs) was synthesized by a modified sol-gel method. Firstly, 75 mg of CTAT and 16.74 µL of TEAH 3 were dissolved in 5 mL of deionized water, and heated by continuous stirring until the temperature increased to 80 ℃. At this point, 400 µL of TEO and 200 µL of BTESePD were simultaneously added dropwise. The final mixture was stirred continuously at 80 ℃ for 4 h. Subsequently, the synthesized product was centrifuged, washed with pure water and ethanol and then dried in vacuum. To remove the surfactant template, the dried product was washed and refluxed at 70 ℃ in HCL and ethanol for 24 h. Finally, the precipitate after centrifugation was washed and vacuum dried to obtain DSeMSN. For drug loading, MSN-NH 2 and CAPE were dissolved in 5 mL of ethanol at a mass ratio of 5:4 under magnetic stirring at 30 ℃ for 24 h. The content of CAPE in the supernatant was measured by UV-Vis, and then the drug loading rate was calculated indirectly. 4. Synthesis of PDA-DSeMSN@CAPE For the successful encapsulation of PDA, 250 mg of DSeMSN was stirred and refluxed with 40 mL of toluene and 1 mL of APTES at 110 ℃ for 24 h to introduce amino groups on its surface. The product was centrifuged and washed three times with alcohol, followed by vacuum drying to obtain MSN-NH 2 . To synthesize PDA-DSeMSN @CAPE, 10 mg of DSeMSN@CAPE and 10 mg of dopamine hydrochloride were suspended in 2 mL of PBS (PH 8.5) containing 20 µL of Tris buffer (pH 8.5, 1 M). The mixed solution was magnetically stirred at 30 ℃ for 6 h. After centrifugation, washing and vacuum drying, black product was obtained and collected. 5. Drug Release Profile of PDA-DSeMSN@CAPE To test the release of CAPE, the CAPE-loaded, PDA-coated DSeMSN was dispersed in 1 mL of PBS (pH 7.4) in a dialysis bag and the sealed dialysis bag was immersed in into 7 mL of the corresponding PBS (pH 7.4). The release medium was slowly and constantly shaken in 37°C at 100 rpm. 3 mL of the dialysate was collected and replaced with an equivalent volume of fresh PBS at various time points. The released amount of CAPE in the buffer solution was monitored using the UV-vis spectrophotometry. 6. Catalase Activity of PDA-DSeMSN@CAPE To study the consumption of H 2 O 2 by CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE, respectively, potassium titanium oxalate (the H 2 O 2 indicator) was utilized to detect the changes of H 2 O 2 concentration. Specifically, 50 µL of H2O2 (5 mM) was added to 5 mL of PBS (pH 6.8), then 3 mg of DSeMSN, DSeMSN@CAPE and PDA-DSeMSN@CAPE was added in different groups, respectively. After that, each group was stirred at 37 ℃ for 2, 4, 8, 12, 24, 48, and 72 h. After incubation with potassium titanium oxalate, the absorbance at 540 nm was determined using UV-Vis, and then the specific value was obtained by the standard curve. Moreover, the standard curve was measured from different concentrations of H 2 O 2 after incubation with potassium titanium oxalate. 7. Cellular Uptake Murine microglia BV-2 cells, obtained from Wuhan University Cell Library (Wuhan, China), were incubated with RhB-labeled PDA-DSeMSN@CAPE (100 µg/mL). After co-incubation, the fluorescence intensity of PDA-DSeMSN@CAPE was performed at 2 h, 6 h and 8 h by confocal microscope. The nuclei were stained with DAPI. 8. MTT Assay Murine microglia BV-2 cells and human neuroblastoma SH-SY5Y cells were respectively seeded into 96-well plates at 1 × 10 5 /well and treated with different concentrations of PDA-DSeMSN@CAPE (DSeMSN dose of 2.5, 5.0, 10, 25, 50µg/mL). After 48 h of incubation, 20µL MTT solution (5 mg/mL) was added and continued incubation for another 4 h at 37°C. The MTT solution was then discarded and the metabolized MTT product was dissolved in 150 µL of DMSO. The value of optical density was measured at 562 nm using a microplate reader, and the cell survival of the different groups was calculated. 9. Antioxidant Effect in Vitro The protection capacity of CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE for BV-2 cells when incubated in the medium with high H 2 O 2 levels was explored. Firstly, BV-2 cells were seeded in a 96-well plate (5×10 3 cells/well) and stimulated by H 2 O 2 (100 µM). Subsequently, CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE (10 µg/mL) were added into the wells and incubated for 48 h. Furthermore, the experiment with PDA-DSeMSN@CAPE at different concentrations (0, 2.5, 5.0, 10, 25, 50 µg/mL) was also performed in the high level of H 2 O 2 medium. The production of intracellular ROS was evaluated using a DCFH-DA-ROS assay kit (Beyotime, Nanjing, China). The DCFH-DA fluorescence was monitored using flow cytometry. 10. Flow Cytometry Analysis After polarization of each group, cells were harvested and stained with FITC-A-conjugated monoclonal mouse iNOS antibody (Thermo Fisher Scientific, #14-0161-82) and PE-A conjugated monoclonal mouse CD206 antibody (eBioscience™, #12-2061-82) at room temperature in the dark. After 30 min incubation, cells were washed and then and then immediately assessed by flow cytometry. 11. Supernatant Cytokine ELISA Assays ELISA was used to measure the concentration of inflammatory cytokines in the supernatant of BV-2 cultures according to the manufacturer’s instructions. Interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and IL-10 ELISA kits were purchased from Abcam. 12. Co-culture of BV-2 with SH-SY5Y Cells A neuron-microglia coculture model was established on a 24-well transwell (pore size, 0.4 µm) to elucidate the role of PDA-DSeMSN@CAPE in the microglia-mediated protection of neurons. H 2 O 2 -stimulated BV-2 cells were seeded in transwell inserts while SH-SY5Y cells were seeded in the underwells. Subsequently, CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE (50 µg/mL) were added to transwell inserts and incubated for 48 h. The viability of SH-SY5Y cells was then measured by MTT assay. 13. Establishment of ICH Mice Models and Animal Groups All animal experiments complied with the ARRIVE guidelines and and should be carried out in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals. All animal experiments were approved by the Ethics Committee of the 2nd Xiangya Hospital of Central South University. After deeply anesthetized with 0.3% pentobarbital sodium (40 mg/kg), C57BL/6 mice were placed in a stereotactic head frame. A burr hole was drilled and the needle of a syringe (Hamilton) was inserted (coordinates: 2.4 mm (right), 0.2 mm (posterior), 3.7 mm (depth) relative to bregma). Collagen type IV (0.0375U in 0.5 µL) was slowly injected into the right basal ganglia through a micro-pump. C57BL/6 mice were randomly divided into the following four groups: Sham group, ICH group (normal saline + ICH), free CAPE + ICH (CAPE group), DSeMSN@CAPE group + ICH (DSeMSN@CAPE group) and PDA-DSeMSN-PDA@CAPE + ICH (PDA-DSeMSN @CAPE group). The mice were injected with the formulations (5 mg/kg) via tail vein every day until sacrifice. 14. Biodistribution Assay C57BL/6 mice suffered from ICH were intravenously administered with DiR-loaded PDA-DSeMSN@CAPE (10 mg/kg). The biodistribution of PDA-DSeMSN@CAPE was monitored by the in vivo imaging system (IVIS) spectral system at 1,4, 8,12 and 24 h. The mice were executed at 24 h after nanoparticle injection, and the major organs (heart, brain, liver, spleen, and kidney) were collected for ex vivo imaging to determine fluorescence intensity and assess biodistribution. 15. Behaviour Test The modified neurological severity score (mNSS) was used to evaluate the symptoms of brain injury sequelae in each group of mice at 1, 3, and 7 days after ICH. The sensory, motor, balance, and reflex abilities of mice were reflected by the comprehensive score of mNSS, with a total score of 18 points. The health level was divided into three levels: mild injuries (0–6 points), moderate injuries (7–12 points), and severe injuries (13–18 points). 16. H&E and TUNEL Staining Brain samples were collected and fixed with 4% PFA for 24 h, and then dehydrated and embedded in paraffin. Afterwards, the brain tissues were coronally sectioned into 10 µm-thick slices. After deparaffinization and dehydration, the sections were stained with hematoxylin (10 min) and eosin (3 min). Following neutral resin sealing, changes were observed under a light microscope. TUNEL assay was performed using a commercial kit, according to the manufacturer's instructions[ 46 ]. The nuclei were stained with DAPI (blue) and the fluorescent images were obtained using a microscope. 17. DHE Fluorescence Dihydroethidium (DHE) staining was performed to detect ROS production in brain tissues. Briefly, freshly prepared frozen brain sections were cut into 10-mm-thick coronal brain sections. The slices were incubated with 10µM of DHE at 37 ℃ for 30 min in the dark room. Thereafter, the sections were stained with DAPI for 5 min to detect nuclei. Finally, the sections were observed under a fluorescence microscope (Olympus). 18. Real-Time Quantitative PCR (RT-qPCR) TRIzol (Beyotime, Shanghai, China) was used to extract total RNA from the brain tissues of the perihematomal region, following the manufacturer's instructions. Then, reverse transcription was performed using a cDNA Synthesis Kit (Thermo Fisher Scientific). Standard RT-qPCR was performed using the SYBR Green Master Mix. All primers are listed in Table 1 . Table 1 Primers for qRT-PCR CD32 F-GCTCAAGGAAGACACGGTGA R-GTGTAGCTGGCTTGGACCTG iNOS F-ACGAGACGGATAGGCAGAGA R-CACATGCAAGGAAGGGAACT Arg1 F-GACCTGGCCTTTGTTGATGT R-CCATTCTTCTGGACCTCTGC CD206 F-GGGACTCTGGATTGGACTCA R-GCTCTTTCCAGGCTCTGATG 19. Statistical analysis All data were presented as the mean ± standard error of the mean (SEM). The comparison among multiple groups was analyzed using One-way ANOVA followed by Tukey’s post hoc test. P < 0.05 was considered statistically significant. Results 1. Preparation and characterization PDA-DSeMSN@CAPE The diselenide bond-bridged MSNs (DSeMSNs) were freshly synthesized by the modified sol-gel method. BTESePD was prepared as the precursor to ensure the successful introduction of the Se-Se bond into the MSN framework. Subsequently, BTESePD in conjunction with TEOS as silane sources, and TEAH3 as a catalyst, reacted with the pore-forming template of CTAT. In the present study, the mass ratio of TEOS to BTESePD was defined to be 4:1. As revealed by transmission electron microscopy (TEM), DSeMSN showed uniform and mono-disperse spherical structure, with average diameters (~ 100 nm) (Fig. 1 a). The elemental mappings revealed that the O, Si, and Se elements were evenly distributed within the DSeMSN matrices (Fig. 1 b). The results of Fourier transform infrared (FTIR) spectra showed a strong peak at 1092 cm -1 and 801 cm -1 of MSNs, corresponding to the Si-O-Si asymmetrical stretching vibration and symmetrical stretching. The characteristic absorptions at 570 cm-1 confirmed that Se-Se bond was formed in the silica framework (Fig. 1 e). The observation of CAPE characteristic peaks at 1483 cm -1 in the nanoparticles further demonstrated that CAPE was successfully loaded into DSeMSN (Fig. 1 f). The drug loading efficiency of CAPE was 14.34% with an encapsulation efficiency of 59.81%. Through the self-polymerization of dopamine under alkaline conditions, PDA was easily coated on the surface of DSeMSN@CAPE, yielding PDA-DSeMSN@CAPE. Compared with the bare DSeMSN@CAPE, the surface of PDA-DSeMSN@CAPE became rough and had no pores, and the particle size of PDA-DSeMSN@CAPE changed little (Fig. 1 c, d). The FT-IR spectra showed that after PDA coating, absorption peak around 1500 cm -1 for N-H stretching vibration after the oxidation and self-polymerization of dopamine (Fig. 1 g), indicating that the DSeMSN surface was effectively shielded by the PDA shell in coated systems. The zeta potential of nanoparticles was also determined. As displayed in Fig. 1 h, the zeta potential of DSeMSN was − 32.56 mV. After modification with positive amino groups, the zeta potential changed to 32.76 mV. When NH2-DSeMSN was loaded with the negatively charged CAPE, zeta potential of DSeMSN@CAPE has been changed to 20.23 mV. Because the Michael addition or Schiff base reaction between benzoquinone groups of PDA and amino groups of DSeMSN, the coating of PDA on the surface of DSeMSN@CAPE lead to a further decrease in zeta potential to 6.30 mV( Fig. 1 h). Then, the release profile of the CAPE in DSeMSN@CAPE and PDA-DSeMSN @CAPE was measured in pH 7.4 PBS with 10% FBS. The results showed that about 5% CAPE was released from DSeMSN@CAPE nanoparticle within 72h, while the cumulative release of CAPE was lower than 2% in 72 h in the physiological condition. These data illustrated that PDA-DSeMSN@CAPE maintained great colloidal stability during systemic circulation and inhibited premature drug leakage from nanocarriers. 2. ROS responsive degradation of PDA-DSeMSN@CAPE and H 2 O 2 catalytic property of PDA-DSeMSN@CAPE In order to investigate the ROS-responsiveness of PDA-DSeMSN@CAPE, the morphology change of the nanoclusters after H 2 O 2 treatment was studied by TEM. In solution containing 100µM H 2 O 2 simulating ROS conditions, the framework of DSeMSN@CAPE collapsed into irregular aggregates within 24 h, and dissembled into debris after 3 days (Fig. 2 a, upper panel). PDA coating on the DSeMSN surface didn’t affect the degradation profile of DSeMSN. As shown in Fig. 2 B, the PDA shell was responsive to ROS, and the structure of PDA-DSeMSN@CAPE went degradation in a time-dependent manner (Fig. 2 a, lower panel). The catalytic property of DSeMSN, DSeMSN@CAPE and PDA-SeMSN@CAPE for H 2 O 2 decomposition was evaluated by potassium titanium oxalate (PTO, K 2 TiO(C 2 O 4 ) 2 ·H 2 O) titration spectro-photometric method at a wavelength of 385 nm[ 47 ]. A plot of the mean intensities colour readings against the used H 2 O 2 concentration in PTO solutions under our reaction conditions gave a linear fit (Fig. 2 b), showing that the PTO image data enabled the determination of radical scavenging property of DSeMSN, DSeMSN @CAPE and PDA-DSeMSN@CAPE. The consumption of H 2 O 2 under our operating conditions was rapidly reached to approximately 1.0 mmol/g within the first 24h of reaction time by DSeMSN, DSeMSN@CAPE and PDA-DSeMSN@CAPE. Before 24h, catalytic activity for H 2 O 2 decomposition of DSeMSN, DSeMSN@CAPE and PDA-DSeMSN@CAPE is nearly the same. After initial burst, the capability to consume H 2 O 2 of DSeMSN and DSeMSN@CAPE reached a plateau, while PDA-SeMSN@CAPE induced a sustained H 2 O 2 consumption in a fairly wide range of time with accumulative H 2 O 2 concentration up to 1.3 mmol/g at 72h, higher than DSeMSN and DSeMSN@CAPE (Fig. 2 c). Quantitative analysis was carried out to obtain the absorption signal of different therapeutic agents at 72h of reaction time, which showed that PDA-DSeMSN@CAPE showed the best hydroxyl radical scavenging ability (Fig. 2 d). To evaluate the ROS scavenging effect of PDA-DSeMSN@CAPE under in vitro conditions, DCFH-DA staining was used to detect the ROS in BV-2 cells. Flow-cytometry analyses demonstrated that intracellular ROS production was significantly increased in BV-2 cells after exposure to H 2 O 2 compared with the control group. ROS content was decreased significantly in CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE treated cells, compared to the untreated cells (Fig. 2 e-f). There is a maximum decline in ROS level in PDA-DSeMSN@CAPE treatment group, indicating that PDA-DSeMSN@CAPE could emerge as a high-performance nanoparticulate ROS scavenger. We proposed that, under the ROS stimulus resulting from H 2 O 2 , PDA undergoes rapid degradation. With the gate unlocked, release of CAPE could be expedited through diselenide bond instantaneous cleavage in oxidative conditions. The synergistic ROS-scavenging activity of PDA, DSeMSNs and CAPE endowed the nanoparticle’s efficient H 2 O 2 -degrading capability. 3. Cellular uptake and cytotoxicity assessment The results revealed that PDA-DSeMSN@CAPE gradually gathered in the BV-2 cells after co-culturing the Rhodamine B (Rh B)-labeled nanoparticle with BV-2 cells for 2h, 6h, and 8h. The intracellular fluorescence became stronger along with prolonged incubation time, demonstrating that PDA-DSeMSN@CAPE was efficiently uptaken by the BV-2 cells in a time-dependent manner (Fig. 3 a). PDA-DSeMSN@CAPE did not affect microglial BV-2 cell viability over a wide concentration range (from 2.5–25µg/ml) even after 48h treatment (Fig. 3 b). In addition, no significant LDH leakage was observed even with high DSeMSN@CAPE concentration exposure (Fig. 3 c), indicating that PDA-DSeMSN@CAPE did not interfere with cell membrane. 4. PDA-DSeMSN@CAPE promoted the microglial phenotypic switch from M1 to M2 and inhibited proinflammatory responses in vitro Several lines of studies have indicated that microglia, as the immune-competent cells in the brain, respond immediately to brain injuries and serve as first line of defense[ 48 , 49 ]. After brain injury, microglia become activated and polarize towards classic M1-like or alternative M2-like phenotypes [ 50 ]. The classical M1 microglia exhibit pro-inflammatory activities, leading to neuronal damage. In contrast, the alternative M2 microglia play a role in inflammatory dampening[ 51 ]. After the onset of ICH, pro-inflammatory mediators produced by M1 microglia serve as a major contributor to SBI[ 52 , 53 ]. Conversely, switching the microglial shift to M2 phenotype could alleviate inflammatory response and exert neuroprotection. We measured the expression of iNOS (a marker of M1 polarization) and CD206 (a marker of M2 polarization) via immunofuorescence assay to determine the effect of PDA-DSeMSN@CAPE on phenotype switch in BV-2 cells. As expected, H 2 O 2 stimulation induced enhanced iNOS immunopositivity in BV-2 cells without dramatically affecting CD206 immuno-reactivity as compared to that in the control group. The increase in iNOS immunopositivity induced by H 2 O 2 treatment was inhibited by CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE. In addition, CD206 immunopositivity was increased in the CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE treatment group compared with the H 2 O 2 group (Fig. 4 a). These results were further confirmed by flow cytometry, which revealed a significant increase in iNOS in H 2 O 2 -induced BV-2 cells, with no significant difference in CD206 expression. The BV-2 cells treated with CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE expressed increased levels of CD206 and decreased levels of iNOS. PDA-DSeMSN@CAPE modulated microglial polarization more toward the M2 phenotype following H 2 O 2 stimulation more efficiently compared to CAPE, DSeMSN@CAPE (Fig. 4 b-d). Next, the secretion of pro-inflammatory and anti-inflammatory cytokines in BV-2 cell culture media were evaluated by ELISA. Pro-inflammatory cytokines, including TNF-α, IL-6 and IL-1β, were all increased significantly in H 2 O 2 group compared with the control group. Elevated production of TNF-α, IL-1β and IL-6 induced by H 2 O 2 treatment was dramatically counteracted by CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE. DSeMSN@CAPE showed more inhibitory effect on M1-associated pro-inflammatory cytokines than CAPE, and comparatively, PDA-DSeMSN@CAPE performed the best (Fig. 4 e). Regarding the anti-inflammatory cytokine level, the concentration of IL-10 was not significantly changed among the control, H 2 O 2 , and CAPE groups. However, DSeMSN@CAPE and PDA-DSeMSN@CAPE treatments significantly elevated the release of the typical M2-associated cytokine IL-10 in the supernatants compared with the H 2 O 2 group. As compared with DSeMSN@CAPE, PDA-DSeMSN@CAPE treatment group exhibited the higher level of IL-10 (Fig. 4 e). Taken together, our in vitro data demonstrated that PDA-DSeMSN@CAPE exhibited the best effect in promoting microglial M1 to M2 phenotype polarization shift and inhibiting inflammation. 5. PDA-DSeMSN@CAPE prevented H 2 O 2 -stimulated microglia-induced neurotoxicity in BV-2/SH-SY5Y coculture system We then investigated whether PDA-DSeMSN@CAPE could alleviate BV-2 microglial inflammation-mediated neuronal injury in a H 2 O 2 -activated BV-2/SH-SY5Y transwell coculture system (Fig. 5 A). Coculturing with H 2 O 2 -stimulated BV-2 microglia cells, rather than control cells, significantly decreased the viability of SH-SY5Y cells. Obviously, CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE treatment improved the SH-SY5Y viability in this coculture system, with PDA-DSeMSN@CAPE showing the most prominent effect (Fig. 5 B). Moreover, the efficiency of PDA-DSeMSN@CAPE showed a concentration-dependent manner(Fig. 5 C). As the membrane filter of the co-culture system allows for contact-independent intercellular communication through secreted factors, these data may indicate that BV-2 microglia exert harmful effects on SH-SY5Y cells via proinflammatory factors in the oxidative environment and PDA-DSeMSN@CAPE modulate microglial polarization and subsequent neuroinflammation, thus presenting a neuronal protective effect. 6. The biodistribution and biocompatibility of PDA-DSeMSN@CAPE in a mice model of ICH It is well established that the blood-brain barrier (BBB) is highly disrupted after ICH due to tight junction disruption facilitated by ICH-induced signaling milieu[ 54 ]. Similar to the phenomenon known as the enhanced permeability and retention (EPR) effect observed in tumor tissue, which offers the possibility for nano-sized drugs passively accumulation in tumors, the induced BBB hyperpermeability in IS bears great resemblance to EPR[ 55 ]. Nanoparticles have been demonstrated that accumulate across a disrupted BBB in the damaged brain passively through an enhanced permeability and retention (EPR)-like effect similar to that observed in tumors[ 56 , 57 ]. To evaluate the bio-distribution of PDA-DSeMSN@CAPE in ICH mice, the time-lapse fluorescence images of whole-body were analyzed after DiR-loaded PDA-DSeMSN@CAPE intravenously injection at different time points using the IVIS instrument. The signal in the brain was visible at 4 h post-injection and the fluorescence intensity continuously increased as time increased to the highest at 24 h (Fig. 6 a). The results were validated by the ex vivo image analysis of excised organs. PDA-DSeMSN@CAPE demonstrated great specificity to the perihematomal region (Fig. 6 b, d-f), indicating EPR-based nanoparticle passive targeting to access the injured area. The Liver, spleen and kidney also showed a relatively high amount of PDA-DSeMSN@CAPE (Fig. 6 b-c). This was the typical phenomenon of nanoparticles in vivo , which suggested the possible renal and hepatobiliary elimination pathway. The potential damage of the main organs of PDA-DSeMSN @CAPE was evaluated by histological analysis. H&E staining demonstrated that no noticeable pathological damage in main organs (heart, liver, spleen, lung, and kidney) after tail vein injection of PDA-DSeMSN@CAPE (Figure S1 ). The excellent biocompatibility makes it an excellent candidate for diverse biomedical applications and the clearance behavior of nanoparticles is a critical issue for nanosystems to minimize side effects. The excretion of the PDA-DSeMSN@CAPE from the body was also analyzed by detecting silicon in the feces and urine of mice using ICP-OES. The results indicated that at 24h post-administration about 13.51 and 2.95% of the supplied silicon dose was excreted via urine via urine and feces, respectively. The excreted Si content gradually increased over time. At 72h postinjection, 55.11% of the dose was excreted in urine and 6.36% was excreted in the feces, demonstrating that PDA-DSeMSN @CAPE was actively excreted mainly by the urinary tract, and to a lesser extent via biliary route (Figure s2 ). Taken together, these results indicated that PDA-DSeMSN @CAPE was rapidly cleared from the body, thus further demonstrating its good safety. 7. PDA-DSeMSN@CAPE improved neurological functions and reduced neuronal injury in mice ICH model To explore the role of PDA-DSeMSN@CAPE in ICH-induced brain injury in vivo, we compared neurobehavioral outcomes among groups. The mNSS test revealed significant neurological deficits in ICH groups at 1d, 3d, 7d compared with the sham group. CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE significantly promoted neurological function recovery of mice with ICH (Fig. 7 a). The overall behavioral performance showed that PDA-DSeMSN@CAPE held the best effect. To elucidate the mechanism underlying the therapeutic effect of PDA-DSeMSN@CAPE, H&E staining was performed to observe the histopathological changes of the peri-hematomal tissues at 24 h post-ICH. Compared with the sham group, the number of neurons decreased significantly and the neurons showed disorderly arrangement and interstitial edema in the ICH group. This pathological deterioration was significantly alleviated by treatment with CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE (Fig. 7 b). As shown in Fig. 7 c, a large number of TUNEL-positive cells were observed around the hematoma in ICH model, indicating massive neuronal cell death triggered by ICH. Administration of CAPE, DSeMSN@CAPE or PDA-DSeMSN @CAPE significantly reduced TUNEL-positive cells, and PDA-DSeMSN @CAPE showed the best anti-apoptotic ability. The inhibition of apoptosis by DSeMSN @CAPE was verified by double immunofluorescent staining of cleaved caspase-3 with the neuron marker NeuN. ICH resulted in obviously increased cleaved caspase-3 positive neurons in the perihematomal area compared with the sham group, which were significantly suppressed by CAPE, DSeMSN@CAPE and PDA-DSeMSN @CAPE administration. Importantly, the PDA-DSeMSN@CAPE demonstrated the best efficacy (Fig. 7 d). 8. PDA-DSeMSN@CAPE promoted a M1 to M2 polarization shift and alleviated neuroinflammation after ICH in vivo To specifically evaluate the effect of PDA-DSeMSN@CAPE on the M1/M2 polarization state of microglia after ICH, M1 marker(iNOS) or M2 marker(CD206) was labeled in the brain tissues surrounding the hematoma at 3 days post-ICH. ICH induced the perihematomal microglial activation with prominent M1 phenotype and a lesser extent M2 phenotype. CAPE, DSeMSN@CAPE or PDA-DSeMSN@CAPE treatment significantly decreased the number of iNOS + cells and increased CD206+, as compared to the ICH group. PDA-DSeMSN @CAPE exhibited a more significant effect on shifting microglia from M1 to M2 than CAPE and DSeMSN@CAPE (Fig. 8 a). Consistently, elevated level of IL-1β, TNF-α, and IL-6 mRNA in the perihematomal brain tissues after ICH was abolished by the administration of CAPE, DSeMSN@CAPE or PDA-DSeMSN@CAPE, and the reducing effect was most pronounced in PDA-DSeMSN@CAPE treatment group (Fig. 8 c). Additionally, treatment with PDA-DSeMSN@CAPE significantly upregulated the expression of IL-10 (Fig. 8 F). Taken together, these results demonstrate that modulated the microglia polarization shift from the M1 to M2 phenotype and suppressed microglia-induced inflammation. Conclusion In conclusion, the intelligent PDA-gated DSeMSN drug delivery system was successfully developed that featured on-demand drug release for synergistic ROS-scavenging and inflammation-suppressing effect for ICH treatment. PDA-DSeMSN@CAPE maintained great colloidal stability and underwent ROS-responsive degradation and controlled drug release. The oxidative reaction of the PDA shell and diselenide bond, together with CAPE, endowed the nanoparticle triple ROS degrading capability. In vitro experimental results demonstrated that PDA-DSeMSN@CAPE effectively reduced ROS accumulation, promoted microglial M1 to M2 polarization and suppressed neuroinflammation, thus improving SH-SY5Y viability. Importantly, intravenous injection of PDA-DSeMSN@CAPE preferentially accumulated in perihematomal area and effectively promote neurological recovery, which might be attributed to the synergistic effect of ROS elimination and inflammation inhibition. Last but not least, both in vitro and in vivo assessments demonstrated the low toxicity and excellent biocompatibility of PDA-DSeMSN@CAPE. Therefore, the nanomaterial presented here might stand out as a promising translatable solution for ICH treatment. Declarations CRediT authorship contribution statement F.Z. and B.Y. designed the research. Y.H., M.Z., F.Z., X.G., and X.L. performed the research. All authors analyzed and interpreted the data. B.Y., F.Z., R.T. and Y.H. wrote the paper. B.Y. supervised the project and revised the manuscript. All authors have given approval to the final version of the manuscript. Declaration of competing interest The authors declare no competing financial interest. Funding Natural Science Foundation of Hunan Province, China (Grant number 2022JJ40709) ; Natural Science Foundation of China (Grant number 82301668). Author Contribution F.Z. and B.Y. designed the research. Y.H., M.Z., F.Z., X.G., and X.L. performed the research. All authors analyzed and interpreted the data. B.Y., F.Z., R.T. and Y.H. wrote the paper. B.Y. supervised the project and revised the manuscript. All authors have given approval to the final version of the manuscript. 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Traumatic brain injury opens blood-brain barrier to stealth liposomes via an enhanced permeability and retention (EPR)-like effect. J Drug Target. 2015;23(9):847–53. Al-Ahmady ZS, Dickie BR, Aldred I, Jasim DA, Barrington J, Haley M, Lemarchand E, Coutts G, Kaur S, Bates J, Curran S, Goddard R, Walker M, Parry-Jones A, Kostarelos K, Allan SM. Selective brain entry of lipid nanoparticles in haemorrhagic stroke is linked to biphasic blood-brain barrier disruption. Theranostics. 2022;12(10):4477–97. Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Scheme1.tif Scheme 1 Schematic illustration of the fabrication process and the mechanism of PDA-DSeMSN@CAPE FigureS1.tif Figure S1 Representative H&E staining of heart, liver, spleen, lung, kidney FigureS2.tif Figure S2 Silicon in the feces and urine of mice using ICP-OES. Graphicalabstract.png Graphical abstract Cite Share Download PDF Status: Published Journal Publication published 23 Nov, 2024 Read the published version in Journal of Nanobiotechnology → Version 1 posted Editorial decision: Revision requested 09 Oct, 2024 Reviews received at journal 08 Oct, 2024 Reviewers agreed at journal 05 Oct, 2024 Reviews received at journal 04 Oct, 2024 Reviewers agreed at journal 24 Sep, 2024 Reviewers invited by journal 24 Sep, 2024 Submission checks completed at journal 02 Jun, 2024 Editor assigned by journal 02 Jun, 2024 First submitted to journal 25 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4476509","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":313318610,"identity":"7a0e8f8a-b937-45b3-b1bf-216d4aa05237","order_by":0,"name":"Fangfang Zhou","email":"","orcid":"","institution":"Second Xiangya Hospital of Central South University","correspondingAuthor":false,"prefix":"","firstName":"Fangfang","middleName":"","lastName":"Zhou","suffix":""},{"id":313318611,"identity":"08347588-6705-486f-b082-7c2b1a15a9c1","order_by":1,"name":"Yongju He","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Yongju","middleName":"","lastName":"He","suffix":""},{"id":313318612,"identity":"63c9a572-16b8-4251-b424-be4a0af89b19","order_by":2,"name":"Meiru Zhang","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Meiru","middleName":"","lastName":"Zhang","suffix":""},{"id":313318613,"identity":"504b5a4f-aa55-4e47-8023-bb0bcbf5340a","order_by":3,"name":"Xiyu Gong","email":"","orcid":"","institution":"Second Xiangya Hospital of Central South University","correspondingAuthor":false,"prefix":"","firstName":"Xiyu","middleName":"","lastName":"Gong","suffix":""},{"id":313318614,"identity":"279bf502-72fc-4787-b67a-ff054fd3e3a4","order_by":4,"name":"Xiaoxuan Liu","email":"","orcid":"","institution":"Second Xiangya Hospital of Central South University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoxuan","middleName":"","lastName":"Liu","suffix":""},{"id":313318615,"identity":"1b8df3b6-b94e-4aca-a1c7-aefd4692783c","order_by":5,"name":"Ranran Tu","email":"","orcid":"","institution":"Second Xiangya Hospital of Central South University","correspondingAuthor":false,"prefix":"","firstName":"Ranran","middleName":"","lastName":"Tu","suffix":""},{"id":313318616,"identity":"12a4a635-daaa-4857-8cba-9e3fa650422d","order_by":6,"name":"Binbin Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYJACZgYDhgQ2BuYDEO4B4rWwJZCihYEBqJzHgDgtBsfPHn5dUHAnj0+65+PHn20Mcnw3Ehg/F+DTciYvzXqGwbNiNpmzm6V52xiMJW8kMEvPwKflQI6ZMY/B4cQ2idxtzIxtDIkbbiSwMfPg03L+DUxLzjNGoMPqCWu5kWP8GKqFjQHosAQDQlokb7wxY55hcLiYTSLNWJrnnIThzDMPm6XxaeE7n2P8ueDP4Tz5GckPP/4os5HnO5588DM+LQoHGNgkkPggNmMDHg0MDPINDMwf8KoYBaNgFIyCUQAAx2hMHjagUQMAAAAASUVORK5CYII=","orcid":"","institution":"Second Xiangya Hospital of Central South University","correspondingAuthor":true,"prefix":"","firstName":"Binbin","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-05-25 11:08:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4476509/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4476509/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12951-024-03023-0","type":"published","date":"2024-11-23T15:56:52+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58354989,"identity":"b4260dc5-e633-4ea3-bb76-79ec42ea3ae3","added_by":"auto","created_at":"2024-06-14 09:47:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3920686,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhysicochemical characterization of DSeMSN@CAPE and PDA-DSeMSN@CAPE.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) TEM visualization of DSeMSN. (b) Representative elemental-mapping images of DSeMSN showing silicon (green), oxygen(red), and selenium (cyan). (c-d) TEM images of DSeMSN@CAPE and PDA-DSeMSN@CAPE. (e-g) Fourier transform infrared (FT-IR) spectrum of MSN, DSeMSN, DSeMSN@CAPE and PDA-DSeMSN@CAPE. (h) Zeta potential measurements of different nanoparticle. (i) Nitrogen adsorption-desorption isotherms and pore size distributions calculated for the DSeMSN, DSeMSN@CAPE and PDA-DSeMSN@CAPE. (j) The cumulative release profile of CAPE from DSeMSN@CAPE and PDA-DSeMSN@CAPE in PBS.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4476509/v1/3002f50153c36de8bc3a83a9.png"},{"id":58354203,"identity":"64b00899-e590-44cc-998f-eb18a1de2c0b","added_by":"auto","created_at":"2024-06-14 09:39:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2367808,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eROS responsiveness and H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e\u0026nbsp;scavenging efficiency of PDA-DSeMSN @CAPE \u003c/strong\u003e(a) TEM images of DSeMSN@CAPE (upper pannel) and PDA-DSeMSN@CAPE (lower pannel) showing degradation at 0, 1day and 3 days under 100 μM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. (b) \u003ca href=\"http://libdb.csu.edu.cn/topics/chemistry/analytical-calibration\" title=\"Learn more about Calibration curves from ScienceDirect's AI-generated Topic Pages\"\u003eCalibration curves\u003c/a\u003e\u0026nbsp;for the determination of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003eby the PTO spectro-photometric methods. (c) Quantitative scavenging activity toward H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. (d) UV-vis absorption spectra and photo (inset) of PTO after H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e degradation by different formulations. Decreased color intensity in association with increased catalase activity. (e-f)\u0026nbsp;Flow cytometric results of DCFH-DA fluorescence in the BV-2 cells treated with H2O2, CAPE, DSeMSN@CAPE, and PDA-DSeMSN@CAPE. ** p \u0026lt; 0.01 compared with indicated group.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4476509/v1/ff5f2e9103b6b106e0f67887.png"},{"id":58354202,"identity":"50884739-21b8-4bf3-bed7-5fe4ba8b6ed0","added_by":"auto","created_at":"2024-06-14 09:39:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4503460,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntracellular uptake and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e cytotoxicity.\u003c/strong\u003e (a) Confocal microscopy after 2, 4 and 6 h exposure in BV-2 cells with 100 μg/mL RhB-labeled PDA-DSeMSN@CAPE. The nuclei were stained with DAPI. Scale bar:20μm (b) \u003cem\u003eIn vitro\u003c/em\u003e cytotoxicity of different concentrations of PDA-DSeMSN@CAPEtoward cells incubated for 48 h measured by MTT assay. (c) LDH leakage assay ** p \u0026lt; 0.01 compared with indicated group.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4476509/v1/f81881eb72c1224e3eb795e8.png"},{"id":58354207,"identity":"01f33fd3-7227-4a7c-8914-c17a4344ad8b","added_by":"auto","created_at":"2024-06-14 09:39:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":856549,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePDA-DSeMSN@CAPE\u003c/strong\u003e \u003cstrong\u003eprimes microglial polarization toward M2 and decreased pro-inflammatory cytokines \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e(a) Representative photomicrographs of double-staining immunofluorescence of images of M1 (iNOS+ Iba-1+) and M2 (CD206+ Iba-1+) staining in the BV-2 cells. Scale bar: 20 μm. (b) Representative fluorescence-activated cell sorting plots showing the M1 (iNOS) and M2 (CD206) phenotypes by flow cytometry.(c-d) Quantitative analysis of iNOS-positive and CD206-positive microglia. (e) ELISA showing that the pro-inflammatory factors TNF-α, IL-6, IL-Iβand the anti-inflammatory factor IL-10. * p \u0026lt; 0.05 compared with indicated group, ** p \u0026lt; 0.01 compared with indicated group.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4476509/v1/c6b3e330c91c9ad8fb36a6a9.png"},{"id":58354992,"identity":"9bcf8c49-26c8-4dd9-b59e-b1950634feef","added_by":"auto","created_at":"2024-06-14 09:47:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":161051,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePDA-DSeMSN@CAPE promoted the viability of SH-SY5Y cells in H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-Activated BV-2 Coculture System. \u003c/strong\u003e(a) Schematic illustration of a Transwell® system. (b) The cell viability (MTT) of SH-SY5Y cells in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced BV-2 coculture system after 48h treatment of different formulations. (c) Cell viability (MTT) of SH-SY5Y cells in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced BV-2 coculture system after 48h treatment of different doses of PDA-DSeMSN@CAPE for 48h. ** p \u0026lt; 0.01 compared with indicated group.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4476509/v1/9426f40a491511a3ea0c0958.png"},{"id":58354209,"identity":"17785e02-706f-464d-a6cc-da0e612cdd82","added_by":"auto","created_at":"2024-06-14 09:39:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2033407,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of PDA-DSeMSN@CAPE in mice at different time points after tail vein injection. \u003c/strong\u003e(a) Representative \u003cem\u003ein vivo\u003c/em\u003e fluorescence images of ICH mice after intravenous injection of Dir-labeled PDA-DSeMSN@CAPE. (b) \u003cem\u003eEx vivo\u003c/em\u003e fluorescence images of brains after injection of Dir labeled for 24 h. (c) Semiquantification data of ex vivo fluorescence imaging results of major organs. (d) \u003cem\u003eEx vivo\u003c/em\u003e fluorescence images of peripheral organs after injection of Dir labeled for 24 h. (e) Semiquantification data of \u003cem\u003eex vivo \u003c/em\u003efluorescence imaging results of ipsilateral and contralateral hemisphere of hemorrhage. ** p \u0026lt; 0.01 compared with indicated group.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4476509/v1/01457c11c87270ec62a35474.png"},{"id":58354990,"identity":"c9afdd48-1c5b-4cb2-a4ed-b66aaf338102","added_by":"auto","created_at":"2024-06-14 09:47:24","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2670028,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e neuroprotective effect of PDA-DSeMSN@CAPE in ICH mice \u003c/strong\u003e\u0026nbsp;(a) Experimental design of animal study. (b) mNSS scores of each group at 24 h, 72 h and 7 days after ICH. (c) Representative images Hematoxylin and eosin (H\u0026amp;E) staining (upper pannel), TUNEL staining (middle pannel) and DHE staining of perihematomal region of ICH mice treated with CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE. Scale bar: 20μm. (d) Double immunofluorescent staining of cleaved caspase-3 with the neuron marker NeuN after different treatment. * p \u0026lt; 0.05, compared with indicated group, ** p \u0026lt; 0.01, compared with indicated group.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4476509/v1/65ffc941aa4ca64a3630b075.png"},{"id":58354214,"identity":"61b9362b-3078-453b-80fb-dfeb1c905c37","added_by":"auto","created_at":"2024-06-14 09:39:24","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1168042,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePDA-DSeMSN@CAPE\u003c/strong\u003e \u003cstrong\u003eskewed microglia toward M2 phenotypic polarization and suppressed neuroinflammation in mice ICH model \u003c/strong\u003e(a) Representative immunofluorescence of iNOS and CD206 on brain tissues surrounding the hematoma at 3 days post-ICH. Scale bar: 20μm. (b) qPCR analysis of mRNA expression levels of M1 markers (iNOS, CD32) and M2 markers (CD206, Arg1) in the peri-hematomal brain tissues. (c) The levels of proinflammatory cytokines(TNF-α, IL-6, IL-Iβ) and antiinflammatory cytokine (IL-10) were measured using ELISA. ** p \u0026lt; 0.01, compared with indicated group.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4476509/v1/07f1a25d042618526d3c80cb.png"},{"id":69834788,"identity":"639662d8-d113-46bf-82c1-0950e54cc592","added_by":"auto","created_at":"2024-11-25 16:08:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":22091117,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4476509/v1/da264abb-3683-4960-b9aa-221f120488d4.pdf"},{"id":58354205,"identity":"478516ff-68b1-451e-b656-d4876ac54a74","added_by":"auto","created_at":"2024-06-14 09:39:23","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1513626,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1\u003c/strong\u003e \u003cstrong\u003eSchematic illustration of the fabrication process and the mechanism of PDA-DSeMSN@CAPE\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Scheme1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4476509/v1/db55b26b6c9c63751cf21f65.tif"},{"id":58354216,"identity":"fab45c51-19ed-409f-bc8b-51d2af437a32","added_by":"auto","created_at":"2024-06-14 09:39:24","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":7496794,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S1 Representative H\u0026amp;E staining of heart, liver, spleen, lung, kidney\u003c/p\u003e","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4476509/v1/9011de3db2f9a27b1fb01128.tif"},{"id":58354206,"identity":"58a76e9b-7684-4150-b796-cab617b3baf7","added_by":"auto","created_at":"2024-06-14 09:39:23","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":175842,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S2 Silicon in the feces and urine of mice using ICP-OES.\u003c/p\u003e","description":"","filename":"FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-4476509/v1/a2d18083a15f0b0fa807742a.tif"},{"id":58354991,"identity":"2cd0c085-dadd-406f-b345-7e85e385f276","added_by":"auto","created_at":"2024-06-14 09:47:24","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":359894,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-4476509/v1/9f4377326859697148110068.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Polydopamine(PDA)-Coated Diselenide-Bridged Mesoporous Silica-based Nanoplatform for Neuroprotection by Reducing Oxidative Stress and Targeting Neuroinflammation in Intracerebral Hemorrhage","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIntracerebral hemorrhage (ICH) is a devastating subtype of stroke, with severe morbidity and high mortality[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Classically, injury from ICH is broadly divided into primary injury, mass effect from the initial hematoma, and secondary injury, induced by a chain reaction of events responed to the hematoma [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Thus far, the seemingly feasible therapies for treating ICH-induced primary injury have not provided sufficient benefits in clinical trials[\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e–\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], suggesting that secondary injury after ICH may represent an essential and attractive pathophysiological target. Secondary brain injury (SBI) is the main culprit of irreversible neurological deficits and contributes to the progressive neurological deterioration after ICH[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e–\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Various scenarios have elucidated that neuroinflammation and oxidative stress (OS) are funda-mental signatures of SBI after ICH, being inextricably interlinked and interdependent in their pathogenesis[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The main element of neuroinflammation is microglia activation[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Activated microglia release a myriad of inflammatory and cytotoxic agents, especially excessive production of reactive oxygen species (ROS) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. ROS are recognized as a crucial player in neuroinflammation-mediated OS. Moreover, ROS burst can trigger and exacerbate inflammation through redox-sensitive transcription factors activation and inflammatory cells recruitment [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e–\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. An overactivated inflammatory response and an enhanced ROS generation act synergistically, forming a vicious cycle, and promoting the onset and development of SBI after ICH. Obviously, multi-target agents, which can dampen down neuroinflammation and counter oxidative stress, hold considerable promise as a therapeutic strategy for ICH. Caffeic acid phenethyl ester (CAPE), an efficient hydrophobic bioactive polyphenolic ester, shows a wide range of important biological activities[\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e–\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It has been proved that CAPE protects neurons by reducing of ROS, attenuating neuroinflammation and suppressing apoptosis in various neurologic diseases [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e–\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, its practical applications are seriously restricted by poor water solubility, low bioavailability and chemical instability[\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e–\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlong with the rapid development of material science and nanotechnology, numerous fascinating nanoparticles used as carriage systems for drug delivery have evolved to achieve better efficacy and safety[\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e–\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In particular, the large surface areas, pore tunability, great drug loading capacity facile functionalization, nontoxicity and excellent biocompatibility mesoporous silica nanoparticle (MSN) has sparked immense interest as drug carrier[\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e–\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. At present, MSN has been further tailored to respond to disease-specific stimuli and realize targeted and controlled drug delivery. Regarding the redox-imbalance and excessive ROS production in the perihematomal brain tissues, introducing ROS-cleavable linkers into the matrix of mesoporous silica is emerging as a promising “on-demand” smart drug delivery platform in the field of ICH treatment [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Among the cleavable species used for constructing MSN-based drug delivery systems, the diselenide is an ideal choice for ROS-responsive linker, which has high sensitivity towards oxidative milieu. Of particular importance, under ROS-rich circumstances, the diselenide bond was oxidized to selenenic acid, making it attractive ROS scavenger [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e–\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, diselenide-bridged MSN(DSeMSN) still holds some disadvantages, including short-term circulation and premature drug leakage, which hamper ideal performance in vivo[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Fortunately, a simple surface modification method was invented based on oxidative self-polymerization of dopamine in an alkaline environment. Under this condition, dopamine can be oxidized to generate a water-insoluble polydopamine (PDA) film. As an excellent gatekeeper, PDA encapsulates prodrug nanoparticles internally, reducing the interplay among factors in complex physiological environ- ment and drug properties during blood circulation [\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e–\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Additionally, PDA possesses excellent antioxidant activity, which effectively scavenge overproduced ROS[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we constructed a smart CAPE-loaded, ROS-responsive diselenide-bridged MSN by PDA coating (PDA-DSeMSN@CAPE)(Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). PDA coating fabricated on the surface of DSeMSN@CAPE acted as a protected capsule for long-term stability. As expected, the structure of PDA-DSeMSN@CAPE was effectively degraded in ROS condition. More importantly, PDA and diselenide can both significantly deplete ROS levels, enabling synergistic treatment of ICH with drug and materials. PDA-DSeMSN@CAPE displayed an extraordinary anti-oxidative and anti-inflammatory property, showing little cytotoxic effect \u003cem\u003ein vitro\u003c/em\u003e. Furthermore, using a murine model of ICH, we demonstrated that PDA-DSeMSN@CAPE via intravenous administration accumulated in perihematomal regions and produced an efficient neuroprotective effect via concomitant antioxidative and anti-inflammatory effects. Besides, PDA-DSeMSN@CAPE can be metabolized out of the body more quickly, avoiding toxicity and side effects.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003e1. Materials.\u003c/b\u003e \u003c/p\u003e\u003cp\u003eTetraethyl orthosilicate (TEOS, ≥ 99%), Bis[3-(triethoxysilyl) propyl] diselenide (BTESePD, ≥ 98%), triethanolamine (TEAH3, ≥ 99%), cetyltrimethylammonium tosylate (CTAT, ≥ 95%), and 3-aminopropyltriethoxysilane (APTES, ≥ 98%) were obtained from Titan Co., Ltd (Shanghai, China). Ethanol absolute (C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eOH, ≥ 99.7%) and hydrochloride acid (HCl, 36–38%) were purchased from Hunan Huihong Reagent Co., Ltd (Hunan, China). Caffeic Acid Phenethyl Ester (CAPE, 97%) was purchased from Shaoyuan Chemical Technology (Shanghai, China). Dopamine hydrochloride (DA, 98%) was purchased from Shanghai Macklin Biochemical Co., Ltd (Shanghai, China). Near-infrared fluorescent dye (DiR, ≥ 99%) was bought from MedChemExpress Co., Ltd (Shanghai, China).\u003c/p\u003e\u003ch2\u003e2. Characterizations\u003c/h2\u003e\u003cp\u003eTransmission electron microscopy (TEM, Talos F200X) and scanning electron\u003c/p\u003e\u003cp\u003emicroscopy (SEM, JEOL-4800) were used to detect the morphology and microstructures of nanoparticles. Fourier transform infrared (FTIR) spectrums\u003c/p\u003e\u003cp\u003ewere recorded on a Fourier transform infrared spectrometer (Nicolet 6700, Thermo Electron Scientific Instruments). UV-Vis absorption spectra were recorded on a UV-visible spectro-photometer (UV-6100, METASH).\u003c/p\u003e\u003ch2\u003e3. Synthesis of CAPE-Loaded DSeMSN\u003c/h2\u003e\u003cp\u003eThe diselenide-bridged mesoporous silica nanoparticle (DSeMSNs) was synthesized by a modified sol-gel method. Firstly, 75 mg of CTAT and 16.74 µL of TEAH\u003csub\u003e3\u003c/sub\u003e were dissolved in 5 mL of deionized water, and heated by continuous stirring until the temperature increased to 80 ℃. At this point, 400 µL of TEO and 200 µL of BTESePD were simultaneously added dropwise. The final mixture was stirred continuously at 80 ℃ for 4 h. Subsequently, the synthesized product was centrifuged, washed with pure water and ethanol and then dried in vacuum. To remove the surfactant template, the dried product was washed and refluxed at 70 ℃ in HCL and ethanol for 24 h. Finally, the precipitate after centrifugation was washed and vacuum dried to obtain DSeMSN. For drug loading, MSN-NH\u003csub\u003e2\u003c/sub\u003e and CAPE were dissolved in 5 mL of ethanol at a mass ratio of 5:4 under magnetic stirring at 30 ℃ for 24 h. The content of CAPE in the supernatant was measured by UV-Vis, and then the drug loading rate was calculated indirectly.\u003c/p\u003e\u003ch3\u003e4. Synthesis of PDA-DSeMSN@CAPE\u003c/h3\u003e\u003cp\u003eFor the successful encapsulation of PDA, 250 mg of DSeMSN was stirred and refluxed with 40 mL of toluene and 1 mL of APTES at 110 ℃ for 24 h to introduce amino groups on its surface. The product was centrifuged and washed three times with alcohol, followed by vacuum drying to obtain MSN-NH\u003csub\u003e2\u003c/sub\u003e. To synthesize PDA-DSeMSN @CAPE, 10 mg of DSeMSN@CAPE and 10 mg of dopamine hydrochloride were suspended in 2 mL of PBS (PH 8.5) containing 20 µL of Tris buffer (pH 8.5, 1 M). The mixed solution was magnetically stirred at 30 ℃ for 6 h. After centrifugation, washing and vacuum drying, black product was obtained and collected.\u003c/p\u003e\u003ch2\u003e5. Drug Release Profile of PDA-DSeMSN@CAPE\u003c/h2\u003e\u003cp\u003eTo test the release of CAPE, the CAPE-loaded, PDA-coated DSeMSN was dispersed in 1 mL of PBS (pH 7.4) in a dialysis bag and the sealed dialysis bag was immersed in into 7 mL of the corresponding PBS (pH 7.4). The release medium was slowly and constantly shaken in 37°C at 100 rpm. 3 mL of the dialysate was collected and replaced with an equivalent volume of fresh PBS at various time points. The released amount of CAPE in the buffer solution was monitored using the UV-vis spectrophotometry.\u003c/p\u003e\u003ch2\u003e6. Catalase Activity of PDA-DSeMSN@CAPE\u003c/h2\u003e\u003cp\u003eTo study the consumption of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e by CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE, respectively, potassium titanium oxalate (the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e indicator) was utilized to detect the changes of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration. Specifically, 50 µL of H2O2 (5 mM) was added to 5 mL of PBS (pH 6.8), then 3 mg of DSeMSN, DSeMSN@CAPE and PDA-DSeMSN@CAPE was added in different groups, respectively. After that, each group was stirred at 37 ℃ for 2, 4, 8, 12, 24, 48, and 72 h. After incubation with potassium titanium oxalate, the absorbance at 540 nm was determined using UV-Vis, and then the specific value was obtained by the standard curve. Moreover, the standard curve was measured from different concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e after incubation with potassium titanium oxalate.\u003c/p\u003e\u003ch2\u003e7. Cellular Uptake\u003c/h2\u003e\u003cp\u003eMurine microglia BV-2 cells, obtained from Wuhan University Cell Library (Wuhan, China), were incubated with RhB-labeled PDA-DSeMSN@CAPE (100 µg/mL). After co-incubation, the fluorescence intensity of PDA-DSeMSN@CAPE was performed at 2 h, 6 h and 8 h by confocal microscope. The nuclei were stained with DAPI.\u003c/p\u003e\u003ch2\u003e8. MTT Assay\u003c/h2\u003e\u003cp\u003eMurine microglia BV-2 cells and human neuroblastoma SH-SY5Y cells were respectively seeded into 96-well plates at 1 × 10\u003csup\u003e5\u003c/sup\u003e/well and treated with different concentrations of PDA-DSeMSN@CAPE (DSeMSN dose of 2.5, 5.0, 10, 25, 50µg/mL). After 48 h of incubation, 20µL MTT solution (5 mg/mL) was added and continued incubation for another 4 h at 37°C. The MTT solution was then discarded and the metabolized MTT product was dissolved in 150 µL of DMSO. The value of optical density was measured at 562 nm using a microplate reader, and the cell survival of the different groups was calculated.\u003c/p\u003e\u003cp\u003e \u003cb\u003e9. Antioxidant Effect\u003c/b\u003e \u003cb\u003ein Vitro\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe protection capacity of CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE for BV-2 cells when incubated in the medium with high H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels was explored. Firstly, BV-2 cells were seeded in a 96-well plate (5×10\u003csup\u003e3\u003c/sup\u003e cells/well) and stimulated by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (100 µM). Subsequently, CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE (10 µg/mL) were added into the wells and incubated for 48 h. Furthermore, the experiment with PDA-DSeMSN@CAPE at different concentrations (0, 2.5, 5.0, 10, 25, 50 µg/mL) was also performed in the high level of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e medium. The production of intracellular ROS was evaluated using a DCFH-DA-ROS assay kit (Beyotime, Nanjing, China). The DCFH-DA fluorescence was monitored using flow cytometry.\u003c/p\u003e\u003ch2\u003e10. Flow Cytometry Analysis\u003c/h2\u003e\u003cp\u003eAfter polarization of each group, cells were harvested and stained with FITC-A-conjugated monoclonal mouse iNOS antibody (Thermo Fisher Scientific, #14-0161-82) and PE-A conjugated monoclonal mouse CD206 antibody (eBioscience™, #12-2061-82) at room temperature in the dark. After 30 min incubation, cells were washed and then and then immediately assessed by flow cytometry.\u003c/p\u003e\u003ch2\u003e11. Supernatant Cytokine ELISA Assays\u003c/h2\u003e\u003cp\u003eELISA was used to measure the concentration of inflammatory cytokines in the supernatant of BV-2 cultures according to the manufacturer’s instructions. Interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and IL-10 ELISA kits were purchased from Abcam.\u003c/p\u003e\u003ch2\u003e12. Co-culture of BV-2 with SH-SY5Y Cells\u003c/h2\u003e\u003cp\u003eA neuron-microglia coculture model was established on a 24-well transwell (pore size, 0.4 µm) to elucidate the role of PDA-DSeMSN@CAPE in the microglia-mediated protection of neurons. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-stimulated BV-2 cells were seeded in transwell inserts while SH-SY5Y cells were seeded in the underwells. Subsequently, CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE (50 µg/mL) were added to transwell inserts and incubated for 48 h. The viability of SH-SY5Y cells was then measured by MTT assay.\u003c/p\u003e\u003ch2\u003e13. Establishment of ICH Mice Models and Animal Groups\u003c/h2\u003e\u003cp\u003e All animal experiments complied with the ARRIVE guidelines and and should be carried out in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals. All animal experiments were approved by the Ethics Committee of the 2nd Xiangya Hospital of Central South University. After deeply anesthetized with 0.3% pentobarbital sodium (40 mg/kg), C57BL/6 mice were placed in a stereotactic head frame. A burr hole was drilled and the needle of a syringe (Hamilton) was inserted (coordinates: 2.4 mm (right), 0.2 mm (posterior), 3.7 mm (depth) relative to bregma). Collagen type IV (0.0375U in 0.5 µL) was slowly injected into the right basal ganglia through a micro-pump. C57BL/6 mice were randomly divided into the following four groups: Sham group, ICH group (normal saline + ICH), free CAPE + ICH (CAPE group), DSeMSN@CAPE group + ICH (DSeMSN@CAPE group) and PDA-DSeMSN-PDA@CAPE + ICH (PDA-DSeMSN @CAPE group). The mice were injected with the formulations (5 mg/kg) via tail vein every day until sacrifice.\u003c/p\u003e\u003ch2\u003e14. Biodistribution Assay\u003c/h2\u003e\u003cp\u003eC57BL/6 mice suffered from ICH were intravenously administered with DiR-loaded PDA-DSeMSN@CAPE (10 mg/kg). The biodistribution of PDA-DSeMSN@CAPE was monitored by the in vivo imaging system (IVIS) spectral system at 1,4, 8,12 and 24 h. The mice were executed at 24 h after nanoparticle injection, and the major organs (heart, brain, liver, spleen, and kidney) were collected for ex vivo imaging to determine fluorescence intensity and assess biodistribution.\u003c/p\u003e\u003ch2\u003e15. Behaviour Test\u003c/h2\u003e\u003cp\u003eThe modified neurological severity score (mNSS) was used to evaluate the symptoms of brain injury sequelae in each group of mice at 1, 3, and 7 days after ICH. The sensory, motor, balance, and reflex abilities of mice were reflected by the comprehensive score of mNSS, with a total score of 18 points. The health level was divided into three levels: mild injuries (0–6 points), moderate injuries (7–12 points), and severe injuries (13–18 points).\u003c/p\u003e\u003ch2\u003e16. H\u0026amp;E and TUNEL Staining\u003c/h2\u003e\u003cp\u003eBrain samples were collected and fixed with 4% PFA for 24 h, and then dehydrated and embedded in paraffin. Afterwards, the brain tissues were coronally sectioned into 10 µm-thick slices. After deparaffinization and dehydration, the sections were stained with hematoxylin (10 min) and eosin (3 min). Following neutral resin sealing, changes were observed under a light microscope. TUNEL assay was performed using a commercial kit, according to the manufacturer's instructions[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The nuclei were stained with DAPI (blue) and the fluorescent images were obtained using a microscope.\u003c/p\u003e\u003ch2\u003e17. DHE Fluorescence\u003c/h2\u003e\u003cp\u003eDihydroethidium (DHE) staining was performed to detect ROS production in brain tissues. Briefly, freshly prepared frozen brain sections were cut into 10-mm-thick coronal brain sections. The slices were incubated with 10µM of DHE at 37 ℃ for 30 min in the dark room. Thereafter, the sections were stained with DAPI for 5 min to detect nuclei. Finally, the sections were observed under a fluorescence microscope (Olympus).\u003c/p\u003e\u003ch2\u003e18. Real-Time Quantitative PCR (RT-qPCR)\u003c/h2\u003e\u003cp\u003eTRIzol (Beyotime, Shanghai, China) was used to extract total RNA from the brain tissues of the perihematomal region, following the manufacturer's instructions. Then, reverse transcription was performed using a cDNA Synthesis Kit (Thermo Fisher Scientific). Standard RT-qPCR was performed using the SYBR Green Master Mix. All primers are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimers for qRT-PCR\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD32\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-GCTCAAGGAAGACACGGTGA\u003c/p\u003e \u003cp\u003eR-GTGTAGCTGGCTTGGACCTG\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eiNOS\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-ACGAGACGGATAGGCAGAGA\u003c/p\u003e \u003cp\u003eR-CACATGCAAGGAAGGGAACT\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArg1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-GACCTGGCCTTTGTTGATGT\u003c/p\u003e \u003cp\u003eR-CCATTCTTCTGGACCTCTGC\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD206\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-GGGACTCTGGATTGGACTCA\u003c/p\u003e \u003cp\u003eR-GCTCTTTCCAGGCTCTGATG\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003ch2\u003e19. Statistical analysis\u003c/h2\u003e\u003cp\u003eAll data were presented as the mean ± standard error of the mean (SEM). The comparison among multiple groups was analyzed using One-way ANOVA followed by Tukey’s post hoc test. P \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e1. Preparation and characterization PDA-DSeMSN@CAPE\u003c/h2\u003e \u003cp\u003eThe diselenide bond-bridged MSNs (DSeMSNs) were freshly synthesized by the modified sol-gel method. BTESePD was prepared as the precursor to ensure the successful introduction of the Se-Se bond into the MSN framework. Subsequently, BTESePD in conjunction with TEOS as silane sources, and TEAH3 as a catalyst, reacted with the pore-forming template of CTAT. In the present study, the mass ratio of TEOS to BTESePD was defined to be 4:1. As revealed by transmission electron microscopy (TEM), DSeMSN showed uniform and mono-disperse spherical structure, with average diameters (~\u0026thinsp;100 nm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The elemental mappings revealed that the O, Si, and Se elements were evenly distributed within the DSeMSN matrices (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The results of Fourier transform infrared (FTIR) spectra showed a strong peak at 1092 cm\u003csup\u003e-1\u003c/sup\u003e and 801 cm\u003csup\u003e-1\u003c/sup\u003e of MSNs, corresponding to the Si-O-Si asymmetrical stretching vibration and symmetrical stretching. The characteristic absorptions at 570 cm-1 confirmed that Se-Se bond was formed in the silica framework (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). The observation of CAPE characteristic peaks at 1483 cm\u003csup\u003e-1\u003c/sup\u003e in the nanoparticles further demonstrated that CAPE was successfully loaded into DSeMSN (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). The drug loading efficiency of CAPE was 14.34% with an encapsulation efficiency of 59.81%. Through the self-polymerization of dopamine under alkaline conditions, PDA was easily coated on the surface of DSeMSN@CAPE, yielding PDA-DSeMSN@CAPE. Compared with the bare DSeMSN@CAPE, the surface of PDA-DSeMSN@CAPE became rough and had no pores, and the particle size of PDA-DSeMSN@CAPE changed little (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, d). The FT-IR spectra showed that after PDA coating, absorption peak around 1500 cm\u003csup\u003e-1\u003c/sup\u003e for N-H stretching vibration after the oxidation and self-polymerization of dopamine (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg), indicating that the DSeMSN surface was effectively shielded by the PDA shell in coated systems. The zeta potential of nanoparticles was also determined. As displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh, the zeta potential of DSeMSN was \u0026minus;\u0026thinsp;32.56 mV. After modification with positive amino groups, the zeta potential changed to 32.76 mV. When NH2-DSeMSN was loaded with the negatively charged CAPE, zeta potential of DSeMSN@CAPE has been changed to 20.23 mV. Because the Michael addition or Schiff base reaction between benzoquinone groups of PDA and amino groups of DSeMSN, the coating of PDA on the surface of DSeMSN@CAPE lead to a further decrease in zeta potential to 6.30 mV( Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). Then, the release profile of the CAPE in DSeMSN@CAPE and PDA-DSeMSN @CAPE was measured in pH 7.4 PBS with 10% FBS. The results showed that about 5% CAPE was released from DSeMSN@CAPE nanoparticle within 72h, while the cumulative release of CAPE was lower than 2% in 72 h in the physiological condition. These data illustrated that PDA-DSeMSN@CAPE maintained great colloidal stability during systemic circulation and inhibited premature drug leakage from nanocarriers.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e2. ROS responsive degradation of PDA-DSeMSN@CAPE and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e catalytic property of PDA-DSeMSN@CAPE\u003c/h2\u003e \u003cp\u003eIn order to investigate the ROS-responsiveness of PDA-DSeMSN@CAPE, the morphology change of the nanoclusters after H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment was studied by TEM. In solution containing 100\u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e simulating ROS conditions, the framework of DSeMSN@CAPE collapsed into irregular aggregates within 24 h, and dissembled into debris after 3 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, upper panel). PDA coating on the DSeMSN surface didn\u0026rsquo;t affect the degradation profile of DSeMSN. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, the PDA shell was responsive to ROS, and the structure of PDA-DSeMSN@CAPE went degradation in a time-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, lower panel). The catalytic property of DSeMSN, DSeMSN@CAPE and PDA-SeMSN@CAPE for H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decomposition was evaluated by potassium titanium oxalate (PTO, K\u003csub\u003e2\u003c/sub\u003eTiO(C\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO) titration spectro-photometric method at a wavelength of 385 nm[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. A plot of the mean intensities colour readings against the used H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration in PTO solutions under our reaction conditions gave a linear fit (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), showing that the PTO image data enabled the determination of radical scavenging property of DSeMSN, DSeMSN @CAPE and PDA-DSeMSN@CAPE. The consumption of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e under our operating conditions was rapidly reached to approximately 1.0 mmol/g within the first 24h of reaction time by DSeMSN, DSeMSN@CAPE and PDA-DSeMSN@CAPE. Before 24h, catalytic activity for H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decomposition of DSeMSN, DSeMSN@CAPE and PDA-DSeMSN@CAPE is nearly the same. After initial burst, the capability to consume H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e of DSeMSN and DSeMSN@CAPE reached a plateau, while PDA-SeMSN@CAPE induced a sustained H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e consumption in a fairly wide range of time with accumulative H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration up to 1.3 mmol/g at 72h, higher than DSeMSN and DSeMSN@CAPE (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Quantitative analysis was carried out to obtain the absorption signal of different therapeutic agents at 72h of reaction time, which showed that PDA-DSeMSN@CAPE showed the best hydroxyl radical scavenging ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). To evaluate the ROS scavenging effect of PDA-DSeMSN@CAPE under \u003cem\u003ein vitro\u003c/em\u003e conditions, DCFH-DA staining was used to detect the ROS in BV-2 cells. Flow-cytometry analyses demonstrated that intracellular ROS production was significantly increased in BV-2 cells after exposure to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e compared with the control group. ROS content was decreased significantly in CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE treated cells, compared to the untreated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-f). There is a maximum decline in ROS level in PDA-DSeMSN@CAPE treatment group, indicating that PDA-DSeMSN@CAPE could emerge as a high-performance nanoparticulate ROS scavenger. We proposed that, under the ROS stimulus resulting from H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, PDA undergoes rapid degradation. With the gate unlocked, release of CAPE could be expedited through diselenide bond instantaneous cleavage in oxidative conditions. The synergistic ROS-scavenging activity of PDA, DSeMSNs and CAPE endowed the nanoparticle\u0026rsquo;s efficient H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-degrading capability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3. Cellular uptake and cytotoxicity assessment\u003c/h2\u003e \u003cp\u003eThe results revealed that PDA-DSeMSN@CAPE gradually gathered in the BV-2 cells after co-culturing the Rhodamine B (Rh B)-labeled nanoparticle with BV-2 cells for 2h, 6h, and 8h. The intracellular fluorescence became stronger along with prolonged incubation time, demonstrating that PDA-DSeMSN@CAPE was efficiently uptaken by the BV-2 cells in a time-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). PDA-DSeMSN@CAPE did not affect microglial BV-2 cell viability over a wide concentration range (from 2.5\u0026ndash;25\u0026micro;g/ml) even after 48h treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). In addition, no significant LDH leakage was observed even with high DSeMSN@CAPE concentration exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), indicating that PDA-DSeMSN@CAPE did not interfere with cell membrane.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e4. PDA-DSeMSN@CAPE promoted the microglial phenotypic switch from M1 to M2 and inhibited proinflammatory responses\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSeveral lines of studies have indicated that microglia, as the immune-competent cells in the brain, respond immediately to brain injuries and serve as first line of defense[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. After brain injury, microglia become activated and polarize towards classic M1-like or alternative M2-like phenotypes [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The classical M1 microglia exhibit pro-inflammatory activities, leading to neuronal damage. In contrast, the alternative M2 microglia play a role in inflammatory dampening[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. After the onset of ICH, pro-inflammatory mediators produced by M1 microglia serve as a major contributor to SBI[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Conversely, switching the microglial shift to M2 phenotype could alleviate inflammatory response and exert neuroprotection. We measured the expression of iNOS (a marker of M1 polarization) and CD206 (a marker of M2 polarization) via immunofuorescence assay to determine the effect of PDA-DSeMSN@CAPE on phenotype switch in BV-2 cells. As expected, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e stimulation induced enhanced iNOS immunopositivity in BV-2 cells without dramatically affecting CD206 immuno-reactivity as compared to that in the control group. The increase in iNOS immunopositivity induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment was inhibited by CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE. In addition, CD206 immunopositivity was increased in the CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE treatment group compared with the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). These results were further confirmed by flow cytometry, which revealed a significant increase in iNOS in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced BV-2 cells, with no significant difference in CD206 expression. The BV-2 cells treated with CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE expressed increased levels of CD206 and decreased levels of iNOS. PDA-DSeMSN@CAPE modulated microglial polarization more toward the M2 phenotype following H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e stimulation more efficiently compared to CAPE, DSeMSN@CAPE (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-d). Next, the secretion of pro-inflammatory and anti-inflammatory cytokines in BV-2 cell culture media were evaluated by ELISA. Pro-inflammatory cytokines, including TNF-α, IL-6 and IL-1β, were all increased significantly in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group compared with the control group. Elevated production of TNF-α, IL-1β and IL-6 induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment was dramatically counteracted by CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE. DSeMSN@CAPE showed more inhibitory effect on M1-associated pro-inflammatory cytokines than CAPE, and comparatively, PDA-DSeMSN@CAPE performed the best (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Regarding the anti-inflammatory cytokine level, the concentration of IL-10 was not significantly changed among the control, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and CAPE groups. However, DSeMSN@CAPE and PDA-DSeMSN@CAPE treatments significantly elevated the release of the typical M2-associated cytokine IL-10 in the supernatants compared with the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group. As compared with DSeMSN@CAPE, PDA-DSeMSN@CAPE treatment group exhibited the higher level of IL-10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Taken together, our \u003cem\u003ein vitro\u003c/em\u003e data demonstrated that PDA-DSeMSN@CAPE exhibited the best effect in promoting microglial M1 to M2 phenotype polarization shift and inhibiting inflammation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e5. PDA-DSeMSN@CAPE prevented H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-stimulated microglia-induced neurotoxicity in BV-2/SH-SY5Y coculture system\u003c/h2\u003e \u003cp\u003eWe then investigated whether PDA-DSeMSN@CAPE could alleviate BV-2 microglial inflammation-mediated neuronal injury in a H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-activated BV-2/SH-SY5Y transwell coculture system (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Coculturing with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-stimulated BV-2 microglia cells, rather than control cells, significantly decreased the viability of SH-SY5Y cells. Obviously, CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE treatment improved the SH-SY5Y viability in this coculture system, with PDA-DSeMSN@CAPE showing the most prominent effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Moreover,\u003c/p\u003e \u003cp\u003ethe efficiency of PDA-DSeMSN@CAPE showed a concentration-dependent manner(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). As the membrane filter of the co-culture system allows for contact-independent intercellular communication through secreted factors, these data may indicate that BV-2 microglia exert harmful effects on SH-SY5Y cells via proinflammatory factors in the oxidative environment and PDA-DSeMSN@CAPE modulate microglial polarization and subsequent neuroinflammation, thus presenting a neuronal protective effect.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e6. The biodistribution and biocompatibility of PDA-DSeMSN@CAPE in a mice model of ICH\u003c/h2\u003e \u003cp\u003eIt is well established that the blood-brain barrier (BBB) is highly disrupted after ICH due to tight junction disruption facilitated by ICH-induced signaling milieu[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Similar to the phenomenon known as the enhanced permeability and retention (EPR) effect observed in tumor tissue, which offers the possibility for nano-sized drugs passively accumulation in tumors, the induced BBB hyperpermeability in IS bears great resemblance to EPR[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Nanoparticles have been demonstrated that accumulate across a disrupted BBB in the damaged brain passively through an enhanced permeability and retention (EPR)-like effect similar to that observed in tumors[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. To evaluate the bio-distribution of PDA-DSeMSN@CAPE in ICH mice, the time-lapse fluorescence images of whole-body were analyzed after DiR-loaded PDA-DSeMSN@CAPE intravenously injection at different time points using the IVIS instrument. The signal in the brain was visible at 4 h post-injection and the fluorescence intensity continuously increased as time increased to the highest at 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The results were validated by the \u003cem\u003eex vivo\u003c/em\u003e image analysis of excised organs. PDA-DSeMSN@CAPE demonstrated great specificity to the perihematomal region (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, d-f), indicating EPR-based nanoparticle passive targeting to access the injured area. The Liver, spleen and kidney also showed a relatively high amount of PDA-DSeMSN@CAPE (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb-c). This was the typical phenomenon of nanoparticles \u003cem\u003ein vivo\u003c/em\u003e, which suggested the possible renal and hepatobiliary elimination pathway. The potential damage of the main organs of PDA-DSeMSN @CAPE was evaluated by histological analysis. H\u0026amp;E staining demonstrated that no noticeable pathological damage in main organs (heart, liver, spleen, lung, and kidney) after tail vein injection of PDA-DSeMSN@CAPE (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The excellent biocompatibility makes it an excellent candidate for diverse biomedical applications and the clearance behavior of nanoparticles is a critical issue for nanosystems to minimize side effects. The excretion of the PDA-DSeMSN@CAPE from the body was also analyzed by detecting silicon in the feces and urine of mice using ICP-OES. The results indicated that at 24h post-administration about 13.51 and 2.95% of the supplied silicon dose was excreted via urine via urine and feces, respectively. The excreted Si content gradually increased over time. At 72h postinjection, 55.11% of the dose was excreted in urine and 6.36% was excreted in the feces, demonstrating that PDA-DSeMSN @CAPE was actively excreted mainly by the urinary tract, and to a lesser extent via biliary route (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003es2\u003c/span\u003e). Taken together, these results indicated that PDA-DSeMSN @CAPE was rapidly cleared from the body, thus further demonstrating its good safety.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e7. PDA-DSeMSN@CAPE improved neurological functions and reduced neuronal injury in mice ICH model\u003c/h2\u003e \u003cp\u003eTo explore the role of PDA-DSeMSN@CAPE in ICH-induced brain injury in vivo, we compared neurobehavioral outcomes among groups. The mNSS test revealed significant neurological deficits in ICH groups at 1d, 3d, 7d compared with the sham group. CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE significantly promoted neurological function recovery of mice with ICH (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). The overall behavioral performance showed that PDA-DSeMSN@CAPE held the best effect. To elucidate the mechanism underlying the therapeutic effect of PDA-DSeMSN@CAPE, H\u0026amp;E staining was performed to observe the histopathological changes of the peri-hematomal tissues at 24 h post-ICH. Compared with the sham group, the number of neurons decreased significantly and the neurons showed disorderly arrangement and interstitial edema in the ICH group. This pathological deterioration was significantly alleviated by treatment with CAPE, DSeMSN@CAPE and PDA-DSeMSN@CAPE (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec, a large number of TUNEL-positive cells were observed around the hematoma in ICH model, indicating massive neuronal cell death triggered by ICH. Administration of CAPE, DSeMSN@CAPE or PDA-DSeMSN @CAPE significantly reduced TUNEL-positive cells, and PDA-DSeMSN @CAPE showed the best anti-apoptotic ability. The inhibition of apoptosis by DSeMSN @CAPE was verified by double immunofluorescent staining of cleaved caspase-3 with the neuron marker NeuN. ICH resulted in obviously increased cleaved caspase-3 positive neurons in the perihematomal area compared with the sham group, which were significantly suppressed by CAPE, DSeMSN@CAPE and PDA-DSeMSN @CAPE administration. Importantly, the PDA-DSeMSN@CAPE demonstrated the best efficacy (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e8. PDA-DSeMSN@CAPE promoted a M1 to M2 polarization shift and alleviated neuroinflammation after ICH in vivo\u003c/h2\u003e \u003cp\u003eTo specifically evaluate the effect of PDA-DSeMSN@CAPE on the M1/M2 polarization state of microglia after ICH, M1 marker(iNOS) or M2 marker(CD206) was labeled in the brain tissues surrounding the hematoma at 3 days post-ICH. ICH induced the perihematomal microglial activation with prominent M1 phenotype and a lesser extent M2 phenotype. CAPE, DSeMSN@CAPE or PDA-DSeMSN@CAPE treatment significantly decreased the number of iNOS\u0026thinsp;+\u0026thinsp;cells and increased CD206+, as compared to the ICH group. PDA-DSeMSN @CAPE exhibited a more significant effect on shifting microglia from M1 to M2 than CAPE and DSeMSN@CAPE (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). Consistently, elevated level of IL-1β, TNF-α, and IL-6 mRNA in the perihematomal brain tissues after ICH was abolished by the administration of CAPE, DSeMSN@CAPE or PDA-DSeMSN@CAPE, and the reducing effect was most pronounced in PDA-DSeMSN@CAPE treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec). Additionally, treatment with PDA-DSeMSN@CAPE significantly upregulated the expression of IL-10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF). Taken together, these results demonstrate that modulated the microglia polarization shift from the M1 to M2 phenotype and suppressed microglia-induced inflammation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, the intelligent PDA-gated DSeMSN drug delivery system was successfully developed that featured on-demand drug release for synergistic ROS-scavenging and inflammation-suppressing effect for ICH treatment. PDA-DSeMSN@CAPE maintained great colloidal stability and underwent ROS-responsive degradation and controlled drug release. The oxidative reaction of the PDA shell and diselenide bond, together with CAPE, endowed the nanoparticle triple ROS degrading capability. \u003cem\u003eIn vitro\u003c/em\u003e experimental results demonstrated that PDA-DSeMSN@CAPE effectively reduced ROS accumulation, promoted microglial M1 to M2 polarization and suppressed neuroinflammation, thus improving SH-SY5Y viability. Importantly, intravenous injection of PDA-DSeMSN@CAPE preferentially accumulated in perihematomal area and effectively promote neurological recovery, which might be attributed to the synergistic effect of ROS elimination and inflammation inhibition. Last but not least, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e assessments demonstrated the low toxicity and excellent biocompatibility of PDA-DSeMSN@CAPE. Therefore, the nanomaterial presented here might stand out as a promising translatable solution for ICH treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCRediT authorship contribution statement\u003c/h2\u003e \u003cp\u003eF.Z. and B.Y. designed the research. Y.H., M.Z., F.Z., X.G., and X.L. performed the research. All authors analyzed and interpreted the data. B.Y., F.Z., R.T. and Y.H. wrote the paper. B.Y. supervised the project and revised the manuscript. All authors have given approval to the final version of the manuscript.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eNatural Science Foundation of Hunan Province, China (Grant number 2022JJ40709) ; Natural Science Foundation of China (Grant number 82301668).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eF.Z. and B.Y. designed the research. Y.H., M.Z., F.Z., X.G., and X.L. performed the research. All authors analyzed and interpreted the data. B.Y., F.Z., R.T. and Y.H. wrote the paper. B.Y. supervised the project and revised the manuscript. All authors have given approval to the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThanking for Natural Science Foundation of Hunan Province, China (Grant number 2022JJ40709) and Natural Science Foundation of China (Grant number 82301668).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMagid-Bernstein J, Girard R, Polster S, Srinath A, Romanos S, Awad IA, Sansing LH. Cerebral Hemorrhage: Pathophysiology, Treatment, and Future Directions. Circul Res. 2022;130(8):1204\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKeep RF, Hua Y, Xi G. Intracerebral haemorrhage: mechanisms of injury and therapeutic targets, The Lancet. Neurology. 2012;11(8):720\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBautista W, Adelson PD, Bicher N, Themistocleous M, Tsivgoulis G, Chang JJ. 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Theranostics. 2022;12(10):4477\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Intracerebral hemorrhage, polydopamine-coated diselenide bridged mesoporous silica nanoparticle, ROS-responsive, Microglia polarization","lastPublishedDoi":"10.21203/rs.3.rs-4476509/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4476509/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOxidative stress (OS) and neuroinflammation are critical pathological processes in secondary brain injury (SBI) after intracerebral hemorrhage(ICH), and their intimate interactions initiate and aggravate brain damage. Thus, targeting oxidative stress and neuroinflammation could be a promising therapeutic strategy for ICH treatment. Here, we report a high-performance platform using polydopamine (PDA)-coated diselenide bridged mesoporous silica nanoparticle (PDA-DSeMSN) as a smart ROS scavenger and ROS-responsive drug delivery system. Caffeic acid phenethyl ester (CAPE) was blocked in the pore of DSeMSN by covering the pore with PDA as a gatekeeper. PDA-DSeMSN @CAPE maintained high stability and underwent reactive oxygen species (ROS)-responsive degradation and drug release. The intelligent nanomaterial effectively eliminated ROS, promoted M1 to M2 microglial conversion and suppressed neuroinflammation \u003cem\u003ein vitro\u003c/em\u003e and i\u003cem\u003en vivo\u003c/em\u003e. Importantly, intravenous administration of PDA-DSeMSN@CAPE specifically accumulated in perihematomal sites and demonstrated robust neuroprotection in an ICH mouse model with high biological safety. Taking together, the synergistic effect of ROS-responsive drug delivery ability and ROS scavenging ability of PDA-DSeMSN makes it a powerful drug delivery platform and provided new considerations into the therapeutic action to improve ICH-induce brain injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Polydopamine(PDA)-Coated Diselenide-Bridged Mesoporous Silica-based Nanoplatform for Neuroprotection by Reducing Oxidative Stress and Targeting Neuroinflammation in Intracerebral Hemorrhage","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-14 09:39:18","doi":"10.21203/rs.3.rs-4476509/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-09T05:02:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-09T02:29:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"49324134074466514757891910896014544821","date":"2024-10-05T17:59:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-04T15:34:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"122150859239572493622814741921031308430","date":"2024-09-24T16:09:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-24T07:56:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-02T11:13:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-02T11:13:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2024-05-25T10:57:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1caf1cdf-698f-4ba8-a31e-85d465024706","owner":[],"postedDate":"June 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-25T15:59:45+00:00","versionOfRecord":{"articleIdentity":"rs-4476509","link":"https://doi.org/10.1186/s12951-024-03023-0","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2024-11-23 15:56:52","publishedOnDateReadable":"November 23rd, 2024"},"versionCreatedAt":"2024-06-14 09:39:18","video":"","vorDoi":"10.1186/s12951-024-03023-0","vorDoiUrl":"https://doi.org/10.1186/s12951-024-03023-0","workflowStages":[]},"version":"v1","identity":"rs-4476509","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4476509","identity":"rs-4476509","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}