Near-Infrared Afterglow ONOO–-Triggered Nanoparticles for Real-Time Monitoring and Treatment of Early Ischemic Stroke

Early detection and drug intervention with the appropriate timing and dosage are the main clinical challenges for ischemic stroke (IS) treatment. The conventional therapeutic agents relay fluorescent signals, which require real-time external light excitation, thereby leading to inevitable autofluorescence and poor tissue penetration. Herein, we report endogenous peroxynitrite (ONOO–)-activated BDP-4/Cur-CL NPs that release NIR afterglow signals (λmax 697 nm) for real-time monitoring of the progression of ischemia reperfusion (I/R) brain injury while releasing curcumin for the safe treatment of IS. The BDP-4/Cur-CL NPs exhibited bright NIR afterglow luminescence (maximum 732-fold increase), superb sensitivity (LOD = 82.67 nM), high energy-transfer efficiency (94.6%), deep tissue penetration (20 mm), outstanding antiapoptosis, and anti-inflammatory effects. The activated NIR afterglow signal obtained in mice with middle cerebral artery occlusion (MCAO) showed three functions: (i) the BDP-4/Cur-CL NPs are rapidly activated by endogenous ONOO–, instantly illuminating the lesion area, distinguishing I/R damage from normal areas, which can be successfully used for endogenous ONOO– detection in the early stage of IS; (ii) real-time reporting of in situ generation and dynamic fluctuations of endogenous ONOO– levels in the lesion area, which is of great value in monitoring the evolutionary mechanisms of IS; and (iii) dynamic monitoring of the release of curcumin drug for safe treatment. Indeed, the released curcumin effectively decreased apoptosis, enhanced survival, alleviated neuroinflammation, reduced brain tissue loss, and improved the cognition of MCAO stroke mice. This work is the first example of afterglow luminescence for early diagnosis, real-time reporting, drug tracing, and treatment for IS.


■ INTRODUCTION
−3 Currently, thrombolysis within the effective time window has been considered as the gold standard for clinical IS treatment. 4,5−8 Notably, as a distinct component of oxidative stress, peroxynitrite (ONOO − ) shows ultrahigh toxicity compared to other RONS due to its destructive nitrification damage to lipid, mitochondria, and DNA. 9,10Also, the microenvironmental concentration of ONOO − in the lesion area correlates positively with neuroinflammation and neurotoxicity after the IS. 11,12It follows that the byproducts (notably ONOO − ) have a negative impact on the treatment of IS.Hence, how to fully utilize and consume ONOO − are crucial clinical factors.
−16 For example, aminophenol fluorescein (APF) and hydroxyphenyl fluorescein (HPF) are widely employed for the detection of ONOO − . 11A series of ONOO − -triggered NIR fluorescence (FL) probes constructed by Feng and co-workers have been exploited in biological applications. 17,18However, these reagents are far from ideal, and they possess significant drawbacks: (i) the required real-time light excitation results in inevitable autofluorescence and poor tissue penetration; 16,19−24 (ii) poor selectivity reduces the specific detection of ONOO − . 25−28 Notably, a series of ONOO − -triggered probes based on Schaap's adamantylidene-1,2-dioxetane show great potential for diagnosis because their afterglow signal can be "turned on" only in the presence of disease biomarkers/ microenvironments. 14,27,29,30Despite these exciting pioneer studies, near-infrared (NIR) afterglow luminescent (≥650 nm) materials are still scarce and are desirable for brain imaging with increased tissue penetration depth.Studies on the realtime visualization of ONOO − -targeted activatable NIR afterglow for I/R brain damage in IS are rare.
−34 Curcumin (Cur) is a naturally occurring substance that exhibits positive neuroprotective effects for IS because of its antioxidation, antiapoptosis, and anti-inflammatory properties. 35,36However, curcumin possesses poor water solubility, non-negligible cellular toxicity, and fast metabolism, causing suboptimal treatment efficacy for IS. 37−40 Therefore, exploring endogenous ONOO − as a trigger to obtain an NIR afterglow image-guided system that integrates real-time evaluation of ONOO − levels in the I/R brain injury area with dynamic monitoring of the release status of curcumin is of great significance for safe stroke treatment.
In this work, we turn an enemy into a friend and design ONOO − -activated NIR afterglow image-guided nanoparticles (NPs) (named BDP-4/Cur-CL NPs) consisting of BDP-4 and Cur-CL units (Figure 1A).The system can specifically and sensitively evaluate endogenous ONOO − levels to reveal the progression of I/R brain injury as well as dynamically monitor curcumin release for safe IS treatment.Upon the irradiation of BDP-4/Cur-CL NPs, Cur-CL was converted to CL-1 by generating 1 O 2 from BDP-4 (Figure 1B(I)).Subsequently, preirradiated BDP-4/Cur-CL NPs were cleaved by the endogenous ONOO − (at I/R brain injury areas) accompanying the simultaneous in situ-activated NIR afterglow luminescence (λ max 697 nm) and curcumin drug release (Figure 1B(II),C).The BDP-4/Cur-CL NPs exhibited bright NIR afterglow luminescence (maximum 732-fold increase), high sensitivity (limit of detection, LOD = 82.67 nM), high energy-transfer efficiency (94.6%), deep tissue penetration (20 mm), and outstanding antiapoptosis and anti-inflammatory effects.By using this intricate design, BDP-4/Cur-CL NPs activate NIR afterglow signals and offer triple functions during I/R brain injury onset and progression in vivo in mice.These functions are (i) accurately distinguishing between I/R injury and normal areas; (ii) real-time reporting of endogenous ONOO − levels in the lesion area; and (iii) dynamic monitoring of the release status of curcumin.In addition, the BDP-4/Cur-CL NPs effectively decreased apoptosis, enhanced survival rates, alleviated neuroinflammation, reduced infarct volume, and enhanced stroke outcomes.To the best of our knowledge, this is the first report of a theranostic prodrug with NIR afterglow turn-on and activation for synergistic IS therapy.

■ RESULTS AND DISCUSSION
Synthesis and Theoretical Calculations.In recent years, BODIPY-based theranostics have achieved significant success because their optical properties can be systematically tuned by chemical modifications. 41The introduction of strong electron donors (D) into the methylated BODIPY acceptor (A) core can significantly improve the photophysical properties and the 1 O 2 generation capacity of the compound, thereby optimizing its afterglow luminescence performance.The synthetic routes for Cur-CL and AIEgens (named BDP-1, BDP-3, and BDP-4) are shown in the Supporting Information (Schemes S1 and S2), and their chemical structures were validated by 1 H and 13 C NMR spectroscopy and mass spectrometry (Figures S2− S24).BDP-2 was synthesized based on the previously reported literature. 42The NPs were obtained by amphiphilic polymer (DSPE-PEG-MAL) encapsulation. 16Sulfhydrylated Lactoferrin (Lf-SH) is an iron-binding glycoprotein, which can effectively transport NPs into brain cells. 43Time-dependent density functional theory (TD-DFT) calculations gave insight into the electronic properties of BDP-1, BDP-2, BDP-3, and BDP-4 (Figure S25).The highest occupied molecular orbitals (HOMOs) are mainly localized on the triphenylamine (TPA) units, whereas the lowest unoccupied molecular orbitals (LUMOs) are located on the methylated BODIPY core, with significant HOMO−LUMO separation.The energy gap between the HOMO and the LUMO of BDP-1, BDP-2, BDP-3, and BDP-4 is 2.603, 2.416, 2.325, and 2.202 eV, respectively.The ΔE ST (the energy gap between the S1 and the T1 states) of BDP-1, BDP-2, BDP-3, and BDP-4 is 1.46, 1.37, 1.35, and 1.33 eV, respectively (Figure S25).The smaller ΔE ST value in BDP-4, compared with that in BDP-1, BDP-2, and BDP-3, promotes the generation of the triplet photoexcited state through intersystem crossing (ISC) and further enhances the generation of 1 O 2 . 15,44ptical Properties of AIEgens.To investigate the aggregation-induced emission (AIE) features of BDP-1, BDP-2, BDP-3, and BDP-4, the emission spectra were recorded in water−tetrahydrofuran (THF) mixtures with different water fractions (Figures 2B and S26).Upon gradual addition of water into THF (water fraction: fw ≤ 30%), all of the compounds show weakened fluorescence.However, photoluminescence (PL) intensities of BDP-1, BDP-2, BDP-3, and BDP-4 were then significantly enhanced (fw ≥ 30− 90%), indicating a typical AIE effect.However, the fluorescence intensity decreased when the water fraction was higher than 90%, which can be interpreted as the formation of amorphous particles. 45The dual emission peak of BDP-1 (Figure S26A) can be attributed to the combined effect of the locally excited (LE) state (569 nm) and twisted intramolecular charge-transfer (TICT) state (625 nm). 46,47Compared with the BODIPY complexes, the corresponding NPs exhibit similar emission peaks but with much brighter emission in water (Figures 2C and S27).The PL maxima of BDP-1 NPs, BDP-2 NPs, BDP-3 NPs, and BDP-4 NPs are 625, 645, 662, and 697 nm, respectively, demonstrating a beneficial red shift with the increasing electron-donating ability of the BODIPY core.The fluorescence quantum yields (QYs) of BDP-1 NPs (625 nm), BDP-2 NPs (645 nm), BDP-3 NPs (662 nm), and BDP-4 NPs (697 nm) in water are 39, 27, 23, and 22%, respectively.In Figure 2D, the shorter wavelength absorption bands at around 200−400 nm are attributed to the π−π* and n−π* transitions of the donors in the conjugated backbone, whereas the longer absorption bands at around 450−650 nm are ascribed to the π−π* transition of the BODIPY unit, which is beneficial for matching the chemiluminescence (CL) spectra of Cur-CL to facilitate efficient energy transfer within NPs.The UV−vis absorption and emission spectra of Cur-CL NPs were obtained.In Figure S28, the strong absorption of Cur-CL NPs in the 400−500 nm range is ascribed to the n−π* transition in the conjugated backbone.The Cur-CL NPs exhibited fluorescence around 510 nm, which is significantly different from their afterglow emission (550 nm).Furthermore, the BDP-1 NPs, BDP-2 NPs, BDP-3 NPs, BDP-4 NPs, and Cur-CL NPs exhibited emission and absorption spectra in PBS at different pH values (6.0−8.8)similar to those in water Furthermore, the 1 O 2 generation ability of the AIEgens NPs was monitored by using the standard dye indocyanine green (ICG) as an indicator. 48Upon irradiation of ICG solutions (5 μg mL −1 ) in the presence of BDP-1 NPs, BDP-2 NPs, BDP-3 NPs, and BDP-4 NPs (30 μg mL −1 ), the absorption peak of ICG at 790 nm gradually decreased in intensity (Figures 2E  and S35), confirming the efficient 1 O 2 generation by these NPs.As shown in Figure 2F, 1 O 2 generation follows first-order kinetics, with the slope in the order: BDP-1 NPs (1.39 × 10 −3 ) < BDP-2 NPs (1.74 × 10 −3 ) < BDP-3 NPs (3.17× 10 −3 ) < BDP-4 NPs (4.37 × 10 −3 ).A steeper slope represents a quicker decay rate of ICG and higher ability to generate 1 O 2 . 49o evaluate the type I ROS generation ability of the NPs, dihydrorhodamine 123 (DHR123) and HPF were used as an indicator for O 2 •− and • OH. 50As expected, upon irradiation of DHR123 (10 −5 M) solutions in the presence of BDP-1 NPs,  S40).The sizes observed by DLS are larger due to a hydrated surface layer on the NPs.The diameters of all of the NPs remain almost unchanged after 14 days in water (Figure 3A), indicating robust colloidal stability.The spherical morphology, appropriate size, and outstanding stability suggest that these NPs are suitable for transcellular transport to neurons.
The BDP-4/Cur-CL NPs were selected to construct an NIR afterglow image-guided drug delivery system because they have the most favorable NIR AIE emission and highest 1 O 2 generation ability.A series of reactive oxygen and nitrogen species (RONS), namely, NO 2 ClO − , and ONOO − , were used to study the selectivity of BDP-4/Cur-CL NPs (Figure 3B).Intriguingly, the bright afterglow signal was captured in the presence of ONOO − , verifying the very selective recognition with ONOO − .The signal intensity enhanced ≥732-fold compared to phosphate-buffered saline (PBS).Under physiological conditions (pH range of 6.0−7.4),only the afterglow signal of ONOO − activation was observed (Figure S42), while the H 2 O 2 -activated afterglow signal was observed only at pH 8.8 (outside the pathological range), proving the high specificity of the BDP-4/Cur-CL NPs toward ONOO − .The ONOO − -activated afterglow signal is pH-dependent (Figure S43), with a decreased intensity accompanying a decrease in the pH value.The BDP-4/Cur-CL NPs exhibited afterglow spectra similar to their fluorescence.The afterglow peaked at λ max 697 nm (Figure 3C), and no luminescence peak was observed from AGL (structure shown in Figure 1B).The energy-transfer efficiency between AGL and BDP-4 was calculated to be 94.6% by comparing the afterglow intensity in the presence and absence of BDP-4 (Figure S44).A negligible change in the fluorescence spectrum of the BDP-4/ Cur-CL NPs was observed in the presence of ONOO − , confirming the high tolerance of the NPs to ONOO − (Figure S45).As shown in Figure S46A, the activated afterglow signal of BDP-4/Cur-CL NPs is higher than those of BDP-1/Cur-CL NPs, BDP-2/Cur-CL NPs, and BDP-3/Cur-CL NPs, which can be attributed to the more enhanced NIR AIE emission and higher 1 O 2 generation ability of BDP-4.The optimized NPs with a BDP-4/Cur-CL molar ratio of 1:2 and a white-light (0.25 W cm −2 ) preirradiation time of 10 min produced the best afterglow signal (Figures S47 and S48).
The kinetics of chemiluminescence (CL) of the BDP-4/ Cur-CL NPs was further studied.After the addition of ONOO − (200 μM), the afterglow intensity initially increased, reaching a maximum within 35 min, and then gradually decreased (Figure S46B).The half-life of the afterglow luminescence was estimated to be 98.6 min, which provides sufficient time to monitor the drug release.Most importantly, the afterglow intensity of the BDP-4/Cur-CL NPs showed a linear response with increasing ONOO − concentration, and the limit of detection (LOD) was calculated to be 82.67 nM (Figure S46C).The low LOD predicts high sensitivity and excellent specificity for ONOO − , facilitating clear visualization of ONOO − levels at the infarct site in vivo.
It is well-known that deep tissue penetration is essential to ensuring the application of NPs for imaging in the brain.As shown in Figure 3D, the NIR luminescence intensity obviously decreased with increasing thickness of chicken tissue.It is noteworthy that the afterglow intensity is still clearly observable in tissues up to 20 mm thick, while the NIR fluorescence signal is barely detectable in tissues up to 10 mm thick, which is attributed to the increased tissue penetration of the afterglow imaging modality without external light excitation.The diameters of preirradiated BDP-4/Cur-CL NPs in the PBS solution remained almost unchanged after 7 days (Figure S49A), indicating excellent colloidal stability.There were no obvious changes of the fluorescence intensity and afterglow luminescence for these preirradiated BDP-4/ Cur-CL NPs during 7 days (Figures S49B and S50).These results indicate that the preirradiated BDP-4/Cur-CL NPs have high optical stability, which is beneficial for future commercial applications.
To analyze the drug release kinetics of BDP-4/Cur-CL NPs, liquid chromatograph−mass spectrometry (LC−MS) data were obtained.As shown in Figure S51, preirradiation caused a slight shift in the retention time due to the conversion of the BDP-4/Cur-CL NPs to CL-1 (Figure 1B). 26After ONOO − treatment, the peak at 7.5 min was replaced by a new peak at 4.9 min, signaling the release of curcumin.The BDP-4/Cur-CL NPs showed a progressive curcumin release after ONOO − treatment and reached a plateau at 29.5 h with a release rate of 86% (Figure 3E).As expected, there was negligible curcumin release in the absence of ONOO − , demonstrating that drug release is triggered at an infarct site only when ONOO − is abundant.
Real-Time Monitoring of Early Ischemic Stroke Process In Vivo.Motivated by the superior performance in vitro, BDP-4/Cur-CL NPs were further evaluated in vivo with an intraluminal MCAO mice model (an ischemic model). 52To monitor the cerebral blood flow for determining the stability of the model, laser speckle contrast imaging was performed (Figure S55).Subsequently, BDP-4/Cur-CL NPs were used to study the evolution process of IS and the underlying operational principle.The mice were first subjected to cerebral ischemia for 30 min followed by reperfusion for 30 min and then treated with preirradiated BDP-4/Cur-CL NPs (5 mg mL −1 , 10 μL).As a control, the sham group was treated with preirradiated BDP-4/Cur-CL NPs (Figure 5A).As shown in Figure 5B, the turn-on afterglow signal at the ischemia reperfusion injury site was significantly observed, and negligible afterglow was observed in the control sham groups, indicating that the BDP-4/Cur-CL NPs can successfully detect the signal for the ONOO − output in the mouse brain during I/ R injury.As expected, the real-time activated afterglow luminescence intensity increased to a peak at 8 h post injection, followed by a steady decrease from 8 to 24 h, reflecting the intense evolution of ONOO − during the process of IS (Figure 5C).However, with the "always-on" fluorescent nanoprobe technique, it was very difficult to distinguish the negligible increase in fluorescent signals for both the control sham and the I/R injury groups with the same injection of BDP-4/Cur-CL NPs (Figure S56).These results suggest that the targeted and activatable afterglow luminescence process has high sensitivity for detecting the IS, rather than the "always-on" fluorescent nanoprobe with an indistinguishable signal between the normal and injured regions.
Neutrophils are a type of white blood cell that plays a key role as a marker to judge the degree of inflammation for injury following IS. 53As shown in Figure 5D,E, the increase in the level of infiltrated neutrophils was in line with the afterglow luminescence intensity, which also reached a peak at 8 h, indicating that the ONOO − -activated NIR afterglow signals of BDP-4/Cur-CL NPs could serve to monitor the level of neutrophils.As anticipated, the increase in curcumin release was consistent with the time of the activated NIR afterglow signal.Also, the amount of curcumin for the MCAO model (318.3ng g −1 ) is much higher than for the sham model (29.1 ng g −1 ) at 8 h post light irradiation (Figure 5F).The data suggest that BDP-4/Cur-CL NPs can effectively probe the curcumin release status in IS in vivo.These results strongly validate that the BDP-4/Cur-CL NPs can be successfully used for ONOO − detection in the early phase of IS, showing great value for monitoring the progress of IS.
BDP-4/Cur-CL NPs Provide Cerebral Protective Effect In Vivo.Furthermore, the cerebral protective effect of BDP-4/ Cur-CL NPs was evaluated in vivo.The infarct volumes were observed at 72 h post stroke after treatment.As indicated in Figure 6A,B, compared with the group treated with PBS (90.49mm 3 ), the infarct volumes were significantly reduced for the group treated with BDP-4/Cur-CL NPs (12.97 mm 3 ), implying significant efficacy in decreasing brain injury.To evaluate the effect of BDP-4/Cur-CL NPs on neurological recovery at the indicated time points, the modified neurological severity score (mNSS) with a range of 0−18 is shown (Figure 6C).Low mNSS represents a milder neurological deficit and better effective neuroprotective function.Importantly, the score of the BDP-4/Cur-CL NP-treated group was significantly lower than that of the PBS-treated group at all time points, revealing that the BDP-4/Cur-CL NPs possess strong protection against ischemic injury in vivo.The behavioral changes were evaluated by the open field (Figures 6D and S58) and the elevated plus-maize tests (Figures 6E and  S59).Compared to the sham groups, the PBS-treated group exhibits lesser total distance and slower moving speed, indicating severely impaired motion due to the stroke. 8As expected, compared to the PBS-treated group, larger total distances and faster moving speeds were achieved by the BDP-4/Cur-CL NP group, showing a significant neuroprotective effect.The survival rate of the BDP-4/Cur-CL NP group was higher than that of the PBS-treated group (Figure S60A).Moreover, the BDP-4/Cur-CL NP treatment for 4 weeks had no effect on the mice body weight (Figure S60B).
Brain sections were collected after 72 h of MCAO.Notably, the BDP-4/Cur-CL NPs were found in neurons, indicating an active endocytosis of the BDP-4/Cur-CL NPs (Figure S61).The BDP-4/Cur-CL NP group had a significantly better functional outcome than the other groups, proving that the BDP-4/Cur-CL NPs protected against ischemic injury in vivo.As shown in Figure S62, the fluorescence intensity of BDP-4/ Cur-CL NPs from the brain in MCAO stroke mice gradually decreased within 14 days, indicating that NPs can be metabolized in the brain.
Safety Evaluations of BDP-4/Cur-CL NPs.To investigate the cytotoxicity and biosafety aspects, the BDP-4/Cur-CL NPs were further evaluated in vivo by serum biochemical detection and hematoxylin and eosin (H&E) staining of major organs at 3 and 28 days.No significant difference was observed between the untreated mice and the mice injected with BDP-4/Cur-CL NPs regarding the biochemical analysis of blood (Figures S63 and S64), demonstrating the favorable biocompatibility of the BDP-4/Cur-CL NPs.As presented in Figure S65, no pathological changes were found in the major organs of the six groups of mice (namely, with sham for 3 days; with MCAO for 3 days; with MCAO + BDP-4/Cur-CL NPs for 3 days; with sham for 28 days; with MCAO for 28 days; with MCAO + BDP-4/Cur-CL NPs for 28 days of sham).Also, negligible cell necrosis was observed in the brains treated with BDP-4/Cur-CL NPs, further indicating a positive therapeutic outcome.

■ CONCLUSIONS
In summary, we have successfully constructed ONOO −activated BDP-4/Cur-CL NPs with early diagnosis, real-time reporting, drug tracing, and treatment functions for IS.These outputs do not occur in the absence of ONOO − , whereas there is significant brightness (maximum 732-fold increase) and curcumin release rate (86%) enhancement with ONOO − stimulation and dependent upon the concentration of endogenous ONOO − .Notably, the BDP-4/Cur-CL NPs possess bright NIR emission at 697 nm, superb sensitivity (LOD = 82.67 nM), deep tissue penetration (20 mm), high energy-transfer efficiency (94.6%), and outstanding antiapoptosis and anti-inflammation effects.The activated NIR afterglow signal obtained in in vivo experiments showed three functions: (i) instantly and accurately distinguishing between I/R injury and normal areas for endogenous ONOO − detection in the early stage of IS; (ii) real-time reporting of endogenous ONOO − levels in the lesion area for monitoring the evolutionary mechanisms of IS; and (iii) dynamic monitoring of the release status of curcumin.The released curcumin effectively decreased apoptosis, enhanced survival rates, alleviated neuroinflammation, reduced brain tissue loss, and improved the cognition of MCAO stroke mice.
The special highlights of the BDP-4/Cur-CL NPs are as follows: (1) this work is the first example of NIR afterglow NPs applied to IS.The NPs successfully overcome the inherent limitation of conventional fluorescent materials that rely on real-time excitation; 16,19−24 they possess higher tissue penetration (20 mm), which ensures excellent imaging performance in the brain.(2) This work represents one of the very few examples of optical agents that distinguish I/R damage from normal areas, enabling ONOO − detection in the early stage of IS. 31−34 (3) More importantly, the ability to visualize the dynamic fluctuations of endogenous ONOO − levels in the lesion area is valuable for real-time monitoring of the development and evolution of the mechanisms of IS. (4) Compared to current activatable image-guided drug delivery systems, 11,17,18 the BDP-4/Cur-CL NPs enable dynamic monitoring of curcumin release for safe treatment.(5) Overall, the BDP-4/Cur-CL NPs were successfully applied to IS with the combined desirable features of early diagnosis, real-time reporting, drug tracking, therapeutic functions, and monitoring of drug release.Thus, this work provides new opportunities for the development of therapeutic agents for stroke treatment based on NIR afterglow luminescence for personalized IS therapy.
Statistical Analysis.All data are expressed as mean ± standard deviation (SD).GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA) was used for statistical analysis.The Brown−Forsythe test was used to test the homogeneity of the variance.P < 0.05 was considered statistically significant.The specific characterization and experimental methods are reported in the Supporting Information.

Figure 4 .
Figure 4. (A) Cell viability was measured after mice HT22 cells were exposed to the OGD at different times.(B) Injury in HT22 cells of different NPs in normal conditions.(C) Injury in HT22 cells of different NPs exposed to OGD/R conditions.(D) Afterglow and fluorescence cell imaging of BDP-4/Cur-CL NPs when present in culture with the corresponding OGD/R conditions.(E) Afterglow and (F) fluorescence intensities of the preirradiated BDP-4/Cur-CL NPs in different conditions.BDP-4/Cur-CL NPs decrease the level of expression of inflammatory factors induced by the OGD/R conditions.Concentrations of (G) IL-6, (H) IL-1β, and (I) TNF-α were determined by an enzyme-linked immunosorbent assay (ELISA).*p < 0.05, **p < 0.01, and ***p < 0.001 vs control group.# p < 0.05, ## p < 0.01, and ### p < 0.001 vs OGD/R-treated group.
and S52, in the presence of BDP-4/Cur-CL NPs, bright afterglow luminescence of the OGD/R-induced cell was observed, in contrast to negligible emission in healthy HT22 cells, confirming the outstanding sensitivity induced by the BDP-4/Cur-CL NPs.The cellular uptake of BDP-4 and BDP-4/Cur-CL NPs in HT22 cells was examined by confocal laser scanning microscopy (CLSM) (FigureS53).Bright fluorescence was observed, suggesting that BDP-4 and BDP-4/Cur-CL NPs can be internalized by living cells.Cerebral I/R injury progression is accompanied by a robust inflammatory response;44 therefore, treatment with the BDP-4/Cur-CL NPs was performed to verify the effect on the key inflammatory factors in vitro.First, the expression of inflammatory factors IL-1β, IL-6, and TNFα was studied after the injection of BDP-4/Cur-CL NPs (50 μg mL −1 ) into cells for 24 h.The results showed that a strong inflammatory response was induced by OGD/R.In addition, BDP-4/Cur-CL NPs exhibit an almost negligible effect on the expression of the levels of IL-1β, IL-6, and TNFα, while, after OGD/R stimulation, the levels of IL-1β, IL-6, and TNFα were obviously reduced with the preincubation of BDP-4/Cur-CL NPs (Figure 4G−I), indicating that the BDP-4/ Cur-CL NPs can significantly decrease the inflammatory responses of OGD/R-induced cells in vitro.In consideration of the RONS, • NO, ONOO − , and O 2 •− are key pathogenic species in IS.Therefore, the intracellular levels of O 2 •− were evaluated by dihydroethidium (DHE), • NO was measured by 4-amino-5-methylamino-2′,7′-fluorescein diacetate (DAF-FM DA) reagent, and ONOO − was detected by hydroxyphenyl fluorescin (HPF).As expected, compared to the control group, the BDP-4/Cur-CL NPs significantly decreased the intracellular levels of • NO, O 2 •−