Second Near‐Infrared Activatable Nitric Oxide Releasing Nanoactuators for Photothermal Combinational Modulation of Epileptogenic Focus

Laser interstitial thermal therapy (LITT), which ablates diseased brain tissues via hyperthermia, has offered novel treatment approach for epilepsy; however, the current LITT protocols still lack ablation selectivity and disease‐modifying efficacy. A second near‐infrared (NIR‐II) activatable nitric oxide (NO) releasing nanoactuator is reported here for photothermal combinational modulation of epileptogenic focus. The nanoactuator is prepared by conjugating NO donor S‐nitrosoglutathione (GSNO) onto polydopamine‐coated gold (Au) nanoparticles (NPs), which shows excellent photothermal conversion capability and controlled NO‐releasing property upon illumination in the NIR‐II window. Both in vitro and in vivo study demonstrate that the nanoactuator could exert stronger cell killing effects as compared to its counterpart. Furthermore, the NIR‐II‐activated NO release could inhibit P‐gp expression by regulating NF‐κb pathway, resulting in reprogramming of epileptogenic microenvironment. This study thus offers a novel regulating strategy for the treatment of drug‐resistant epilepsy.


Introduction
[3] Laser interstitial thermal therapy (LITT), a clinically approved photothermal therapy (PTT) protocol, is an efficient treatment modality that has been introduced into DRE. [4][7][8] Nevertheless, this treatment modality still suffers from low ablation selectivity and specificity, [5,7,8] largely due to poor photothermal conversion efficiency of biological tissues. [9]lthough the photothermal transduction agent (PTA)-assisted PTT modality provides a novel approach to address these issues, [10] the applications of PTA-based PTT in epilepsy treatment are still very limited.Moreover, the treatment modality based on PTA-based PTT has not yet been clinically available, possibly due to the limited tissue penetration depth.Our preliminary study showed that polydopamine (PDA) nanoparticles (NPs) could significantly enhance the ability of the first near-infrared (NIR-I, 808 nm) to ablate deep brain tissue wirelessly. [11]ecently, we have demonstrated that epileptogenic focus in epileptic rats could be effectively ablated by the second nearinfrared (NIR-II, 1064 nm) laser with the assistance of ferrous Laser interstitial thermal therapy (LITT), which ablates diseased brain tissues via hyperthermia, has offered novel treatment approach for epilepsy; however, the current LITT protocols still lack ablation selectivity and disease-modifying efficacy.A second near-infrared (NIR-II) activatable nitric oxide (NO) releasing nanoactuator is reported here for photothermal combinational modulation of epileptogenic focus.The nanoactuator is prepared by conjugating NO donor S-nitrosoglutathione (GSNO) onto polydopamine-coated gold (Au) nanoparticles (NPs), which shows excellent photothermal conversion capability and controlled NO-releasing property upon illumination in the NIR-II window.Both in vitro and in vivo study demonstrate that the nanoactuator could exert stronger cell killing effects as compared to its counterpart.Furthermore, the NIR-II-activated NO release could inhibit P-gp expression by regulating NF-κb pathway, resulting in reprogramming of epileptogenic microenvironment.This study thus offers a novel regulating strategy for the treatment of drug-resistant epilepsy.
disulfide-based photothermal nanocatalysts, leading to significant reduction of epileptic attacks after ablation of epileptogenic focus. [12]Nonetheless, the therapeutic efficacy may be compromised due to inadequate peripheral temperature caused by heat dissipation when extrapolating to the clinical setting.Paradoxically, increase of laser power intensity will induce side damages to surrounding normal tissues. [13]Therefore, refinement of current PTA-based PTT strategy is still urgent to facilitate its clinical translation in epilepsy.
[16][17] Overexpression of multidrug efflux transporters in epileptogenic focus is an important contributor to multidrug resistance (MDR) in epilepsy. [1,2]-glycoprotein (P-gp), a major multidrug efflux transporter in the blood-brain barrier, severely restricts the penetration of anti-seizure drugs to the brain parenchyma, thus resulting in attenuated drug efficacy.[18,19] Despite the discovery of novel P-gp inhibitors, there is still a lack of small-molecule inhibitors that can safely and effectively modulate P-gp in the brain.[20] Previous studies have demonstrated that nanodrug delivery systems can bypass P-gp to overcome multidrug resistance.[21] For example, the introduction of Pluronic materials with biological response modifying activity could significantly increase lamotrigine penetration into epileptogenic focus.[22] In addition, nanodelivery systems can enhance the local accumulation of phenytoin in target locations.[23][24][25] However, the P-gp regulation capability of these nanocarriers is still low.
28][29] Unfortunately, therapeutic NO gas has a very short half-life and diffuses aimlessly, making it difficult to accumulate effectively in desired sites.NO donors such as N-diazeniumdiolates (NONOates) and S-nitrosothiols (SNOs) present relatively high stability and bioavailability, showing promise for controlled NO delivery. [30]However, these small-molecule NO donors are prone to rapid clearance and degradation.Therefore, the amount of low molecular weight NO donors required for a biological response is often toxic to mammalian cells and tissues. [31]By utilizing nanomaterials, the half-life of NO has been significantly prolonged, and the capability of targeted delivery and controlled release can be achieved. [26,31]As an example, polynitrosated polyesters-based NO nanogenerators can induce immunogenic cell death and potentiate the effect of cancer immunotherapy. [32]urthermore, activatable NO-releasing nanomaterials showed unique merits of extreme spatial and temporal controllability upon stimuli. [33]For example, near infrared (NIR) light-activated NO release can enhance the efficacy of chemotherapeutic drugs in tumors by modulating the expression and activity of P-gp. [34,35]owever, these studies still focus on NIR-I light to control NO release in P-gp modulation, which was greatly limited by the moderate tissue penetration depth (less than 1 cm) and the relatively low maximum permissible exposure (MPE) of NIR-I (0.33 W cm À2 for 808 nm).With much stronger tissue penetration capability (3-5 cm), NIR-II light also has an increased MPE (1 W cm À2 for 1064 nm). [36]However, the NIR-II light activatable nanomaterials for NO delivery to modulate epileptic microenvironment have never been explored.
In this work, a NIR-II activatable NO releasing nanoactuator was designed by conjugating NO donor S-nitrosoglutathione (GSNO) onto PDA-coated gold (Au) NPs, which was used for modulating epileptogenic focus microenvironment (Figure 1a).The nanoactuator exhibited strong photothermal conversion effect and photoinduced NO release upon NIR-II (1064 nm) laser irradiation.Moreover, the nanoactuator can produce heat to kill abnormally discharging nerve cells in epileptogenic focus.Meanwhile, NO release was triggered after laser irradiation, which not only enhanced the sensitivity of neuronal cells to thermicidal effect, but also downregulated the P-gp expression in the target region via NF-κb signaling pathway (Figure 1b).A combination of brain ablation and P-gp modulation in epileptogenic focus could be beneficial for the reversal of DRE.This study should present the first NIR-II activatable nanoactuator that can achieve ablation of epileptogenic focus and P-gp regulation simultaneously, which may provide a novel insight into the treatment of DRE.

Synthesis and Characterization of Nanoactuators
To prepare NO donor-conjugated nanoactuators, NO donor (GSNO) was reacted with heterobifunctional polyethylene glycol (SH-PEG-NHS) linker to obtain SH-PEG-GSNO.The amino (-NH 2 ) group on GSNO could be covalently bond to the NHS end of SH-PEG-NHS via amide bond (Figure 2a).The UV-Vis spectrum showed characteristic absorption bands with peaks at 330 and 545 nm (Figure 2b), which were assigned to the π !π* and n N !π* electronic transitions of SNO groups, respectively. [32]The GSNO conjugation was confirmed by Fourier transform infrared (FTIR) spectroscopy (Figure 2c).The successful grafting of SNO groups onto polyethylene glycol was verified by the disappearance of the peaks attributed to the N-hydroxy succinimide (NHS) group (1700-1800 cm À1 ), as well as the appearance of C═O and N═O stretching vibration bands at 1700-1750 cm À1 .Moreover, 1 H nuclear magnetic resonance (NMR) was applied to validate the conjugation of GSNO onto SH-PEG-NHS.As shown in Figure S1a, Supporting Information, resonance peaks characteristic for NHS group appeared at ≈2.5 ppm in SH-PEG-NHS.After reacting with GSNO, these characteristic resonance peaks disappeared together with the appearance of resonance peaks (2.0 and 8.0-8.5 ppm) corresponding to GSNO (Figure S1b, Supporting Information), indicating the coupling reaction of -NH 2 and -NHS group.Thermogravimetric analysis revealed that the mass ratio of SNO in the SH-PEG-GSNO was determined to be 8.03% (Figure S2, Supporting Information).
The nanoactuators (AuGSNO) were then constructed by conjugating SH-PEG-GSNO onto the surface of PDA-coated AuNPs.PDA-coated AuNPs were prepared using a simple one-pot method based on mussel-inspired dopamine. [37,38]The thiolated PEG could effectively bind to the surface of PDA-coated AuNPs through Michael addition and/or Schiff base reactions. [39]herefore, it is plausible to postulate that the GSNO can be conjugated onto AuNPs by the use of the reaction between SH-PEG-GSNO and PDA-coated AuNPs.The UV-Vis spectra of AuGSNO demonstrated characteristic absorption peak at 330 nm and 545 nm, indicating the successful grafting of SNO group onto AuNPs (Figure 2d).Dynamic light scattering (DLS) measurements showed that the hydrodynamic size of AuGSNO was comparable to that of the counterpart AuPEG NPs without GSNO (≈200 nm, Figure 2e).The zeta potential of AuGSNO (À28.6 mV) was also similar to that of AuPEG (À31.9 mV) (Figure 2f ).Transmission electron microscope (TEM) showed highly uniform nanoparticles with hyperbranched Au nanorods for both AuPEG and AuGSNO (Figure 2g).The particle sizes of both AuNPs measured from TEM were around 170 nm, slightly smaller than the measurements obtained from DLS.This difference can be attributed to the strong adhesion of AuNPs to the surface of hydron.The selected area electron diffraction (SAED) pattern of AuPEG and AuGNSO exhibited similar crystalline structure, indicating the core-shell structure of AuNPs.

Photothermal and NO-Releasing Performance Evaluation
To assess the photothermal property of the synthesized AuPEG and AuGSNO, the temperature fluctuations of the AuNPs suspensions exposed to NIR-II laser (1064 nm) irradiation were continually monitored.The temperatures of AuPEG and AuGSNO solutions increased rapidly in the initial 2 min of laser irradiation, followed by a plateau phase after 4 min (Figure 3a).The temperature curves of AuPEG and AuGSNO nearly overlapped, as well as the unremarkable temperature increase of GSNO solution upon NIR-II irradiation, indicating that the grafting of GSNO did not significantly affect the photothermal performance of AuNPs.Based on our previous calculation, the photothermal conversion efficiency of this kind of AuNPs could reach ≈80.0%. [37]The photothermal conversion capability of PDA-coated AuNPs can be ascribed to the localized surface plasmon resonance effects of hyperbranched Au nanostructures. [37,38]As shown in Figure 3b, increasing the laser power intensity could increase the temperature of AuGSNO solutions under the same irradiation duration.For example, after 6 min of NIR-II laser irradiation at a power of 1.5 W cm À2 , the temperature of AuNPs solution reached 76.8 °C.This represented a temperature increase of 10.3 and 24.4 °C compared to the solution irradiated at 1.0 and 0.5 W cm À2 , respectively (Figure 3c).The photothermal effects of AuGSNO also exhibited irradiation timeand nanoparticle concentration-dependent manners.Increasing the concentration of AuNPs notably amplified the heating effect (Figure 3d).After 6 min of NIR-II laser irradiation at the same power intensity, the solution containing AuNPs at a concentration of 200 μg mL À1 achieved a temperature of 71.7 °C.In contrast, the solution containing AuNPs at a concentration of 25 μg mL À1 only attained a temperature of 44.9 °C, marking a mere 13.4 °C increase compared to water (Figure 3e).During 5 cycles of on/off NIR-II irradiation, the heating and cooling curve was almost invariable (Figure 3f ), indicating the excellent photothermal stability of AuGSNO.
The NO release behaviors of AuGSNO with or without laser irradiation were investigated by Griess method.In the absence of NIR-II illumination, very minimal NO was released in the first 60 min, and only 20 μM NO was released after 180 min (Figure 3g).In contrast, 5 min of NIR-II laser irradiation could induce remarkable NO release.The NO released immediately in response to laser irradiation, with released NO increased by more than 3-fold after 180 min, suggesting the NIR-II activatable NO release property of AuGSNO.The SÀNO bonds in NO donors could be broken down by UV/vis light, X-rays, ultrasound, and heat to release NO. [26,33]Considering excellent photothermal conversion capability of AuGSNO, the activatable property of this AuNP could be mainly ascribed to the cleavage of the S─NO bond by heat due to the conversion of NIR light into thermal energy via AuGSNO. [34]As the Griess assay only detects nitrite ions instead of NO radicals, [40] another detection method was applied to confirm the activatable release of NO from GSNO.4,5-Diamino-N, N, N 0 , N 0 -tetraethylrhodamine (DAR-1) was a NO-specific probe which could emit remarkably enhanced fluorescence after reacting with NO (Figure S3, Supporting Information). [41]In accordance with the NO release, AuGSNO alone only induced slight fluorescence (Figure 3h).After NIR irradiation, however, AuGSNO induced markedly enhanced fluorescent signal (Figure S4, Supporting Information), indicating the massive release of NO.NO could react with 2-(4carboxyphenyl)-4,4 0 ,5,5 0 -tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) to form PTI and nitric dioxide (NO 2 ) (Figure S5, Supporting Information).The PTIO to PTI transition can be demonstrated by the decay of characteristic five-line electron paramagnetic resonance (EPR) signal pattern of PTIO. [42]onsistent with previous report, [32] the EPR signal of PTIO was almost completely suppressed after treated with GSNO and white light illumination (Figure S6, Supporting Information).However, white light has poor tissue penetration and is not suitable for the treatment of brain diseases.AuGSNO itself resulted in slight attenuation of PTIO signal, and the signal attenuation effect was greatly enhanced after NIR laser irradiation (Figure 3i).These results indicated that AuGSNO could be used as a NIR-II activatable nanomaterials to achieve controlled release of NO.

Assessment of Intracellular NO Generation and Cell Killing Effect on Neurons
The intracellular production of NO by AuGSNO was assessed using a NO fluorescent probe.As illustrated in Figure 4a, only neurons treated with AuGSNO þ NIR-II laser (1.0 W cm À2 , 5 min) could induce obvious intracellular NO generation, while cells treated with other procedures did not show visible intracellular NO.Quantitative analysis revealed that the intracellular NO level in AuGSNO þ laser group was approximately 8-fold higher than those in other groups (Figure 4b).Considering the size, shape, surface functionalization, and topography, the as-prepared AuNPs may be internalized via clathrin-and caveolae-mediated endocytosis, although the negative surface charge may decrease the efficiency of endocytosis. [43]The biocompatibility of AuNPs were assessed by cell counting kit-8 (CCK-8) assay.The cell viability remained above 80% after incubating with various PDA-coated AuNPs at the concentration of 125 μg mL À1 (Figure 4c), suggesting minimal AuNPs-induced nanotoxicity. [44]Similar results were found in C6 and GL261 glioma cells (Figure S7, Supporting Information), further confirming the excellent cytocompatibility of the as-prepared nanoactuator.NO per se can directly induce cell apoptosis or sensitize cells to noxious insults. [33]Although AuGSNO could release NO spontaneously, it was found that the cell viabilities after incubating with AuPEG and AuGSNO were comparable, suggesting that GSNO grafting onto AuNPs did not increase the cytotoxicity of nanoparticles.Our result confirmed that slowly released NO did not exert significant cytotoxicity, thus highlighting the importance of NO nanocarriers. [26]When NIR-II laser was applied, the survival rate of AuGSNO-treated cells was almost 4-fold and 8-fold lower than that of AuPEG-pretreated cells for the power intensity of 1.0 and 1.5 W cm À2 , respectively (Figure 4d).The enhanced cell killing efficacy by AuGSNO and NIR laser irradiation was also illustrated by calcein-acetylmethoxylate (calcein-AM)/propidium iodide (PI) double staining (Figure 4e).Compared with AuPEG, laser irradiation could induce 4-fold more cell death in AuGSNO-treated neurons (Figure 4f ).This enhanced cell killing efficacy could be attributed to the pro-apoptotic effects of the instant production of large quantities of NO upon NIR-II irradiation. [45]These results indicated that AuGSNO were highly biocompatible but exhibited enhanced cell killing efficacy under NIR-II irradiation.

In vitro Modulation of P-gp Expression via NF-κb Signaling in BCECs
Previous studies have demonstrated that NO can reverse MDR of cancer via inhibiting the expression of P-gp. [34,46,47]We envisioned that this NO donor-based strategy could be extrapolated to P-gp-dependent drug resistant brain disorders.Overexpression of P-gp in brain capillary endothelial cells (BCECs) was proposed to be a major contributing factor to the drug resistance in epilepsy. [1]Herein, the effect of AuGSNO on P-gp expression was investigated in BCECs.Consistent with the results in HT-22 neurons, combination of AuGSNO with NIR-II laser irradiation induced approximately 5-fold more intracellular NO generation in BCECs compared with other treatment groups (Figure 5a,b).Appropriate concentration of glutamate has been reported to upregulate P-gp on BCECs. [48]In line with previous studies, [48,49] glutamate could increase the expression of P-gp on BCECs (Figure 5c).Western blotting assay demonstrated a 2.5fold increase of P-gp expression by incubating BCECs with 50 μM glutamate (Figure 5d and S8, Supporting Information).Both immunofluorescent staining and western blotting assay showed that AuGSNO with NIR-II laser illumination could reverse P-gp overexpression to the basal level.Such reversal was not found in AuGSNO-or AuPEG þ NIR-treated BCECs.It should be noted that the P-gp expression in glutamate-activated BCECs was inversely correlated with the amount of NO released, suggesting the vital role of NO in modulating P-gp in BCECs.Given that NF-κb-mediated signaling was paramount in seizure-induced P-gp overexpression, [50] the effect of AuGSNO and NIR-II laser irradiation on protein level of NF-κb was investigated.As expected, glutamate significantly increased NF-κb protein expression level in BCECs, which could be completely reversed by AuGSNO in the presence of NIR-II irradiation (Figure 5e and S9, Supporting Information).In contrast, neither AuPEG with or without NIR-II laser irradiation nor AuGNSO alone exerted similar effects.Overall, these data indicated that AuGNSO could modulate P-gp expression of BCECs in a NIR light-dependent fashion via a NF-κb-mediated signaling pathway.

In vivo Photothermal Ablation and P-gp Modulation of Epileptogenic Focus
Encouraged by the excellent photothermal killing and P-gp modulation effects in vitro, the in vivo effects of AuNPs and NIR-II irradiation on epileptogenic focus were then investigated.Upon NIR-II laser illumination (1.0 W cm À2 ) for 10 min, intrahippocampally administered AuNPs (depth, 5 mm) could increase the temperature of targeted brain regions to around 50 °C, as compared with 41.2 °C in phosphate buffer saline (PBS)-treated rats (Figure 6a and S10, Supporting Information).Consistent with in vitro results, the in vivo photothermal performance of AuGSNO was comparable with that of AuPEG.Hematoxylin and eosin (H&E) staining revealed that AuPEG and AuGSNO could induce necrosis upon NIR-II irradiation (Figure 6b).The necrosis was more pronounced in AuGSNO þ laser than in AuPEG þ laser, which could be attributed to the generation of NO by AuGSNO under laser irradiation (Figure 6c).Subsequently, the P-gp expression level in hippocampus was evaluated by immunofluorescence staining and western blotting.In accordance with previously published data, [19,22,51] LiCl-pilocarpine-induced epileptic rats exhibited increased P-gp expression in epileptogenic focus, i.e., hippocampus (Figure 6d).Accordingly, AuGSNO in the presence of NIR-II could reverse the P-gp overexpression in the hippocampus of LiCl-pilocarpine-induced epileptic rats (Figure 6d and S11, Supporting Information).By contrast, neither AuGSNO nor AuPEG þ laser exert modulating effect on P-gp expression in epileptogenic focus.NF-κb expression in hippocampus was also evaluated using western blot.In line with the results in BCECs, the expression of NF-κb was increased significantly after induction of epilepsy, which could be reverted to its basal level by AuGSNO and NIR-II irradiation (Figure 6f and S12, Supporting Information).Overall, our findings provide compelling evidence that upon NIR-II irradiation, the nanoactuator exhibited a robust PTT-induced cell killing effect on epileptogenic focus, which was potentiated by the NIR-II-triggered release of NO.Notably, the released NO within the local microenvironment also downregulated the expression of P-gp in a manner dependent on NF-κb signaling.Consequently, the novel AuGSNO-based approach presented in this study holds great promise for reprogramming epileptogenic microenvironment by combining photothermal ablation with P-gp modulation.

Conclusion
We have developed a NIR-II activatable NO releasing nanoactuators for ablating epileptogenic focus and modulating P-gp expression in the lesion.Pegylated GSNO was covalently bound to the surface of PDA-coated AuNPs to construct AuGSNO.The prepared AuGSNO demonstrated excellent biocompatibility and efficient photothermal conversion capacity under NIR-II laser irradiation.NO release from the AuGSNO could be triggered by NIR-II irradiation in a controllable manner.The AuGSNO showed potent cell-killing efficacy under the NIR-II irradiation and could reverse the P-gp overexpression in epileptogenic focus via NF-κb signaling pathway.To the best of our knowledge, this study provides the first evidence of the application of NIR-II activatable NO-releasing nanoplatform for modulating epileptogenic focus.The therapeutic efficacy of this NPs-based laser irradiation in various chronic P-gp-mediated pharmacoresistant epilepsy models will be evaluated in the future.Hopefully, this nanoplatform may offer a novel strategy for the treatment of DRE, potentially leading to new therapeutic approaches to brain disorders.
Synthesis of SH-PEG-GSNO Conjugate: SH-PEG-NHS and GSNO at a 1:3 molar ratio were dissolved in anhydrous DMSO and stirred for 3 d at room temperature under light-proof condition.Afterwards, the reaction mixture was dialyzed against distilled water for 3 d by dialysis membrane (Mw cutoff = 1000 Da).The final product SH-PEG-GSNO conjugate was obtained by freeze-drying and stored at À20 °C for further use.
Preparation of Au Nanoparticles Conjugated with SH-PEG-GSNO: Gold (Au) nanoparticles (AuNPs) were prepared according to our previously reported method using mussel-inspired polydopamine (PDA). [37]riefly, 3.6 mL of dopamine hydrochloride (4 mg mL À1 ) was added into 72 mL TRIS buffer (10 mM, PH = 8.5) and stirred vigorously for 3 min.Then, HAuCl 4 (1 mg mL À1 in 7.2 mL solution) was added into the above solution.The mixed solution was further stirred for 8 h.The obtained AuNPs were washed and purified with ultrapure water for 3 times.To conjugate NO prodrug, PDA-coated AuNPs (1 mg) and SH-PEG-GSNO (10 mg) were dispersed in 10 mL of bicine buffer (10 mM, pH = 8.5) and stirred for 36 h at room temperature in the dark.After centrifugation and three washes with distilled water, AuNPs conjugated with SH-PEG-GSNO (AuGSNO) were obtained.The purified samples were freeze-dried and stored from the light at À20 °C.The AuNPs conjugated with SH-PEG-NHS (AuPEG) were also prepared following the same procedure except that SH-PEG-GSNO was substituted by SH-PEG-NHS.
In vitro and In vivo Photothermal Performance: To evaluate the photothermal performance of AuNPs, 100 μL of AuGSNO or AuPEG in phosphate buffer saline (PBS, 10 mM, pH = 7.4) solutions at different concentrations (25, 50, 100, 200 μg mL À1 ) were illuminated by second near-infrared (NIR-II) laser (1064 nm) with variable power densities for 6 min.The temperature was measured at every 30 s using infrared thermal imaging camera (FOTRIC, Shanghai, China).The photothermal stability of AuNPs was assessed through 5 cycles of alternating NIR-II laser irradiation and natural cooling.In each cycle, 200 μL of a 100 μg mL À1 AuNP solution was exposed to NIR-II laser (1.0 W cm À2 ) for 6 min and then allowed to cool to room temperature.
Measurement of NO Release from AuGSNO: The Griess kit was used to measure the release of NO from AuGSNO in an aqueous solution. [32]odium nitrite (NaNO 2 ) was employed to establish a standard calibration curve.The as-prepared AuGSNO (50 μg mL À1 ) was mixed with Griess solution and then the mixture was exposed to NIR-II irradiation (1.0 W cm À2 , 5 min).Dynamic changes of the absorbance at 540 nm were monitored before or after laser irradiation at predetermined times.
EPR spectroscopy was also conducted to detect the generation of NO.Carboxy-PTIO (20 μM) was used as NO trapping agent.EPR experiments were performed on X-band EPR (9.8 GHz) spectrometer.AuGSNO (50 μg mL À1 ) with or without NIR-II irradiation was subjected to EPR measurement.The EPR spectra of GSNO (100 μM) with or without white light exposure was also measured.
Detection of NO Generation In Vitro and In Vivo: HT-22 or bEnd.3 cells were seeded in 24-well cell plates at a density of 5 Â 10 4 cells per well and incubated for 24 h in a humidified atmosphere with 5% CO 2 to allow for adherence.The cells were pretreated with AuPEG or AuGSNO (50 μg mL À1 ) for 12 h.Then the cells were incubated with medium containing 5 μM of NO fluorescent marker DAF-FM DA for 20 min before NIR-II irradiation.After being irradiated for 5 min and washed three times with PBS, the intracellular NO was observed by fluorescence microscope (Leica DMi8, Germany).To confirm NO generation in vivo, DAF-FM DA (500 μM, 4 μL) was intrahippocampally injected into rat brain.Afterwards, AuNPs (4 μL, 200 μg mL À1 ) were topically injected into the same brain region.Half an hour later, NIR-II light (1.0 W cm À2 , 10 min) was applied on the rat head.Rat brains were obtained and sectioned into 10 μm-thick slices for fluorescent microscopy imaging.
In Vitro Cytotoxicity Test: In vitro cytotoxicity of as-prepared AuPEG and AuGSNO NPs on HT-22, C6, and GL261 cells were examined with CCK-8 assay.Cells were cultured in 96-well plates (10 4 per well).Following adherence, the cells were exposed to varying concentrations (25, 50, 75, 100, and 125 μg mL À1 ) of AuPEG or AuGSNO for 24 h.Subsequently, CCK-8 solution was introduced into the DMEM medium at a ratio of 1:10 v/v to assess cell viability.The impact of NIR-II irradiation on cell viability was investigated using the same approach.After 24 h of attachment, cells were treated with AuPEG or AuGSNO at a concentration of 50 μg mL À1 for 12 h, followed by irradiation with a 1064 nm laser (at 1.0 or 1.5 W cm À2 ) for 5 min.Following a further 12-hour incubation, cell viability was assessed using the CCK-8 assay.
The combination of calcein-AM/PI double staining was utilized to assess the cell-killing effects of the AuNPs and NIR-II irradiation both qualitatively and semi-quantitatively.HT-22 cells were planted in 24-well culture plates (10 5 cells well À1 ) and incubated for 24 h.Then cells were treated with or without AuNPs (50 μg mL À1 ).After 6 h of treatments, cells were irradiated by NIR-II laser (1.0 W cm À2 , 5 min) and then incubated for another 6 h.Afterwards, calcein-AM (2 μM) and PI (8 μM) were applied to stain the live and dead cells, respectively.After 20 min of staining, the cells were observed and recorded by a fluorescence microscope.
Detection of P-gp and NF-κb in Vitro: P-gp expression of microvascular endothelial cells was evaluated by immunofluorescence and western blot assay.For immunofluorescence assay, cells were seeded in 6-well culture plates plated with cell climbing slices for 48 h.Then, cells were treated with AuNPs (50 μg mL À1 in PBS) for 4 h.Afterwards, the cells were washed with PBS and then co-incubate with culture medium containing glutamate (50 μM) for 30 min.NIR-II irradiation (1.0 W cm À2 , 5 min) was applied if needed.After another 24 h incubation in normal growth medium, cells were fixed with 4% paraformaldehyde for 15 min, and P-gp primary antibody (1:50) and fluorescein isothiocyanate (FITC)-labeled secondary antibody (1:400) were sequentially used in a standard procedure.The cells were then stained with DAPI solution (5 μg mL À1 ) for 5 min at room temperature.Confocal images were acquired using a Nikon confocal laser scanning microscope (CLSM, Eclipse C1, Nikon, Melville, NY).For western blotting, the treated cells were lysed and the total protein was collected after centrifugation.Following the determination of total protein concentration using the BCA assay, the proteins from each sample were subsequently separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).The separated proteins were then electrotransferred onto polyvinylidene-difluoride (PVDF) membranes and blocked in Tris-buffered saline and 0.5% Tween-20 (TBST) with 5% skim milk for 1 h at room temperature.Afterwards, the membranes were incubated with primary antibodies (1:1000 and 1:3000 for P-gp and β-actin, respectively) overnight at 4 °C, followed by HRP-conjugated secondary antibodies for 30 min at room temperature.Subsequently, the membranes were stained using an electrochemiluminescence (ECL) detection kit and visualized using a chemiluminescent imaging system (Tanon, Shanghai, China).NF-κb was also determined using similar protocol.Optical densities of immunoreactive bands related to proteins of interest were quantified using ImageJ software (ImageJ, Maryland, USA) and normalized by the expression of β-actin.
Animals and Establishment of Epileptic Model: Male Sprague-Dawley (SD) rats weighing 220-300 g were obtained from Shanghai Jie Si Jie Laboratory Animal Co., Ltd (China).All animal procedures were performed in compliance with the guidelines approved by the Animal Care and Use Committee (IACUC) of Donghua University.The LiCl-pilocarpine-induced epileptic animal model was constructed as described in our previous study. [22]In brief, rats were intraperitoneally (i.p.) administered with LiCl (127 mg kg À1 ) and methylscopolamine (1 mg kg À1 ) 15-19 h and 30 min, respectively, before pilocarpine treatment.Pilocarpine (10 mg kg À1 , i.p.) was administered at every 30 min until the onset of status epilepticus (SE).Each rat received a maximum of 5 administrations of pilocarpine in total.After 90 min of SE, diazepam (10-20 mg kg À1 , i.p.) was used to terminate the SE.
Histological Evaluation of Epileptogenic Focus: Pure PBS or AuNPs dissolved in PBS solution (200 μg mL À1 , 4 μL) were intrahippocampally injected (anterior-posterior, À5.0 mm; lateral, þ/À5.0 mm; ventral, À5.0 mm) into epileptic rats after termination of SE.NIR-II irradiation (1.0 W cm À2 , 10 min) was applied through cranial hole.48 h after SE, rats were euthanized to extract brain for histological evaluation.Hematoxylin and eosin (H&E) staining of brain slices was conducted to assess ablation efficacy.Additionally, brain slices stained with TUNEL and DAPI were examined and imaged using a fluorescence microscope to evaluate cell apoptosis in the hippocampus. [11,12]ssessment of P-gp and NF-κb Expression in Epileptogenic Focus: Epileptic rats were treated in the same way as described above.The frozen brain slices were prepared according to standard procedures. [22]Afterwards, slices were stained with anti-P-gp primary antibody (1:50) and FITC-labelled secondary antibody (1:400).Nuclei were stained with DAPI.Fluorescence microscope was used to observe the stained slices.Semi-quantitative evaluation of P-gp and NF-κb expression was performed using western blotting.Hippocampal tissues were homogenized in RIPA buffer supplemented with protease inhibitor and centrifuged to harvest protein supernatants.After determining the protein concentration, the protein samples were subjected to SDS-PAGE and transferred onto a PVDF membrane.The membrane was then blocked with TBST containing 5% skim milk at room temperature.After blocking for 1 h, the membrane was incubated with primary antibodies (1:1000 and 1:2000 for P-gp and β-actin, respectively) for 12 h at 4 °C.Then, the membrane was subjected to TBST washout and secondary antibodies (1:5000) incubation for 30 min.Finally, the protein band was detected using an ECL kit.The determination of NF-κb followed a similar protocol.
Statistical Analysis: The data were presented as the mean AE standard deviation (SD).The sample sizes (n) were provided in the data.Statistical analysis between groups was performed using GraphPad Prism 8.The difference between two groups was analyzed using unpaired two-tailed Student's t-tests, while one-way analysis of variance (ANOVA) with Tukey's multiple comparisons test was employed for data involving multiple groups.A P-value < 0.05 was employed to establish the statistical significance for all tests.

Figure 1 .
Figure 1.NIR-II activatable NO releasing nanoactuator for modulating epileptogenic focus microenvironment.a) Preparation of NO donor-conjugated nanoactuator.b) Schematic illustration of mechanisms of the nanoactuator for modulation of epileptogenic focus via inducing PTT and NO-mediated P-gp inhibition.NO: nitric oxide; P-gp: P-glycoprotein; BCECs: brain capillary endothelial cells.

Figure 4 .
Figure 4. Intracellular NO generation and cell killing effects of AuGSNO on HT-22 cells.a) NO staining of HT-22 cells after different treatments.b) Analysis of the NO fluorescence in treated cells.The results were plotted as mean AE SD (n = 6).Statistical differences are represented as ***P < 0.001; statistical comparisons were performed using one-way analysis of variance (ANOVA) with Tukey's post-hoc test.c) Cell viability of HT-22 treated with different concentrations of AuPEG and AuGSNO for 24 h.The results were plotted as mean AE SD (n = 6).d) Cell viability of HT-22 cells treated with AuPEG and AuGSNO (50 μg mL À1 ) and NIR-II irradiation of different power intensities (1.0 and 1.5 W cm À2 ).The results were shown as mean AE SD (n = 3).Statistical differences are represented as **P < 0.01, ***P < 0.001 for ANOVA with Tukey's post-hoc test (vs.PBS); # P < 0.05, ### P < 0.001 for ANOVA with Tukey's post-hoc test (vs.AuPEG).e) Calcein-AM/PI double staining of live (green) and dead (red) cells after different treatments.f ) Analysis of live and dead cells in (e).Values shown: mean AE SD (n = 4).

Figure 5 .
Figure 5. Intracellular NO generation and P-gp modulation in BCECs.a) Fluorescent images of BCECs after NO probe DAF-FM DA staining.b) Analysis of NO fluorescence in differently treated conditions.The results were shown as mean AE SD (n = 6).Statistical differences are represented as ***P < 0.001; statistical comparisons were performed using ANOVA with Tukey's post-hoc test.c) Immunofluorescence staining of P-gp in BCECs after different treatments.Western blotting analysis for P-gp d) and NF-κb e) expression levels in BCECs.Laser indicates NIR-II irradiation (1.0 W cm À2 , 5 min); Glu denotes glutamate at a concentration of 50 μM.

Figure 6 .
Figure 6.In vivo photothermal ablation and P-gp modulation of epileptogenic focus.a) Infrared thermal images of PBS, AuPEG, or AuGSNO-treated rats after NIR-II laser irradiation (1.0 W cm À2 ) for 10 min.The images were captured from rat brains focusing on hippocampus.b) Histological H&E staining of hippocampal slices from epileptic rats after different treatments.c) Fluorescent images of NO signal in hippocampal slices determined by NO probe.d) Immunofluorescent images of P-gp in hippocampal slices from sham or epileptic rats after different treatments.Western blotting for P-gp e) and NF-κb f ) expression levels of hippocampus from sham control or differently treated epileptic rats.PILO: LiCl-pilocarpine model.