Abstract
Clear elucidation of the connection between chemical structure and biological action mechanisms is the key issue preventing the successful development of nanomedicines. Herein, employing essential trace element selenium (Se) as an example, we fabricate organic-inorganic covalent Se hybrid by anchoring Se atom to polyethylene glycol chain during carbonization to form organic Se-C and inorganic Se-Se bonds in one system to integrate the advantages of both species. The weak covalent Se-Se bond breaks down in response to redox stimuli, thus releases organic Se with stronger electron transfer ability to scavenge free radicals, and forms highly active inorganic Se, which further releases free Se atom to trigger selenoprotein synthesis and activation, ultimately reverses reperfusion injury in male-mice ischemic stroke, and improves neurological restoration. This work provides a unique Se atom reprogramming strategy to design highly bioactive hybrid Se species with clear chemical nature and action mechanisms.
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Introduction
In the past decade, development of nanotechnology has witnessed great application prospectives in fields of bioimaging, sensing and biomedical applications through optimization of composition, chemical species and morphology, etc1,2,3. However, elucidation of the connection between the chemical structure and biological action mechanisms of the nanomedicine is still the key issue preventing their successful clinical translation4,5. Therefore, how to optimize the structure and composition of nanomaterials is becoming the key bottleneck in designing nano drugs. Many efforts have been made to balance the toxicity and enhance the bioactivity of conventional nanomedicines6,7. For instance, the strategy of synthesizing organic-inorganic hybrids serves as an effective method to realize this objective. Such kinds of nanocomposites has attracted intensively attention for drug carrier8,9, bioimaging10,11, sensor12,13, photothermal and chemodynamic therapy14,15,16,17,18, benefit from the unexpected effects originating from inorganic and organic components, and their synergistic phenomena interaction19,20. For instance, dual covalent cross-linked organic-inorganic hybrid hydrogel exhibits excellent mechanical properties, protease response release and antibacterial activity21. In comparison with organic or inorganic nanoparticles, hybrid nanocomposites exhibit broader application potential22,23 due to their high drug loading capacity, high bioavailability and large surface area24,25,26. Such features enable them exploited in the application of transistors, self-healing and stimulus response27,28,29, promoting bone regeneration30,31. Therefore, optimizing the chemical structure and simplifying the composition of hybrid materials is an important prerequisite for clarifying the chemical nature of its biological action and promoting its clinical transformation.
Single-element-based nanomaterials have shown better potential in clinical application32,33,34. For example, selenium (Se), an essential trace element, exhibits physiological functions by regulating the expression of selenoproteins through gene coding35,36. Historically, low Se level in blood serum was involved in the progression of Kashin-Beck and Keshan diseases and health problems like cancer, immune function, Type 2 diabetes, cognitive decline, etc. In contrast, excessive Se intake was usually associated with hypercholesterolemia, hypertension and diabetes mellitus, which suggests the narrow safe window of Se37,38,39. According to the World Health Organization, the normative requirement dose and the safe dietary of Se intake dose are 40 μg/day to 400 μg/day40. Furthermore, different species of Se (including inorganic, organic and elemental substances) exhibit distinct biological functions41,42. The high toxicity of inorganic Se and the low stability of organic Se greatly limited their successful clinical application in disease treatment 43. Interestingly, the microstructure of elemental Se also greatly influenced its bioactivity44. Triclinic crystal Se (t-Se, gray Se) is known as inactive to be transformed spontaneously as the cubic closet packing structure, thermal stability under the lowest formation energy45. In contrast, we and others found that, amorphous elemental Se (α-Se) exhibited high activities in antivirus, antioxidation, anticancer and immune-regulation46,47. In addition, from the chemical nature of Se, the diverse bond strength (bond energy for Se-O, Se-C, Se-N and Se-Se at 233, 244, 193, and 172 kcal mol−1), valence electronic state (ranging from − 2 to + 6) and redox potentials (E° HSeO4− /H2Se and E° H2SeO3/H2Se are + 0.54 and + 0.360 V), determine the biodiversity of Se. Se-Se bond in inorganic Se species with lower bond energy is more likely to break down and transform into selenoproteins under physiological conditions, than organic Se (Se-C, Se-N, Se-O)43,48. On the other hand, the unbounded electrons in organic Se species, such as selenabenzene-structure compounds, could bind to the unoccupied orbital of the substrate, thus exhibiting the catalytic activity49,50. Therefore, designing highly bioactive Se species integrating the advantages of organic and inorganic Se and balancing their drawbacks can be an innovative way to develop potent Se-based therapeutics with clinical translation potential.
In this work, the Se atom reprogramming strategy is employed to develop organic-inorganic covalent Se nanoparticles (Or/In-Se NPs) through anchoring Se atoms to carbon dots to form Se-C and Se-Se bonds as templated by polyethylene glycol (PEG) chain. Under highly oxidative environments like ischemic stroke, the weak covalent Se-Se bond can break down in response to redox stimuli, releasing organic Se (SeOrg) with stronger electron transfer ability to scavenge free radicals. Meanwhile, the highly active inorganic Se (SeInorg) releases a single Se atom to trigger selenoprotein synthesis and activation, ultimately reverses reperfusion injury in ischemic stroke, and improves neurological restoration. Overall, this study provides a unique strategy to design highly bioactive Se species with a clear chemical nature and action mechanisms in suppressing reperfusion-induced injury of ischemic stroke.
Results
Se atom reprogramming strategy for synthesizing Or/In-Se NPs
In this study, we have rationally designed and synthesized a highly bioactive organic-inorganic covalent Se nanoparticles by Se atom reprogramming strategy to combine the advantages of organic and inorganic Se in biomedical effects, which achieves enhanced free radical scavenging ability and selenoproteins activation capability for inhibition of neuronal oxidative damage in ischemic reperfusion injury (Fig. 1a). Briefly, we used selenium powder as t-Se to disperse in polyethylene glycol 400 (PEG400) system, which was fused into free Se atoms at 220 °C and reprogrammed by PEG to realize high-efficiency conversion of monodisperse Or/In-Se NPs. Transmission electron microscope (TEM) results of the reaction system showed that monodispersed Or/In-Se NPs with a uniform size of about 5 nm, which was much different from the morphology of t-Se (Fig. 1b, c). Furthermore, the colocalization for signals of Se, C and O also be observed by elemental mapping, implying the presence and interaction of Se and C in the nanoparticles (Fig. 1d). Spherical aberration corrected transmission electron microscope (ACTEM) analysis was conducted to get more detailed structural information of Se atoms appearing in Or/In-Se NPs. Aberration-corrected high-angle annular dark-field scanning transmission electron microscopy (AC-HAADF-STEM) images showed that Se atoms appear as clear luminous points in dark field, suggesting the uniform monodispersity of Se atoms on the surface of Or/In-Se NPs (Fig. 1e). We have interestingly found that, as the reaction took place, the color of the reaction system of t-Se and PEG400 changed from gray to light yellow and transparent dark red in 60 min (Supplementary Fig. 1a). While, different color variation can be observed in the free PEG400 solution under 220 °C, which exhibited the solution color lighter than that of Or/In-Se NPs. Then, we monitored the morphology of the intermediate products at the reaction time points of 10, 30 and 60 min with TEM and found that the nanoparticles reached a maximum size of 50 nm at 10 min (Supplementary Fig. 1b). However, after 30 min of reaction, the intermediate products have changed apparently and become quantum dots with the particle size reduced to under 5 nm. Subsequently, the reaction solution became homogeneous, and then the nanoparticle size increased slightly at 60 min when the reaction system changed to a homogeneously dark, which suggests that the structure of t-Se was significantly changed.
a Illustrative assembly process of free Se atom into Or/In-Se NPs under the command of PEG400 template. TEM images of (b) t-Se and (c) Or/In-Se NPs with particle size at about 5 nm. The length of the double-headed white arrow for the inset images in (c) is 5 nm. d Elemental mapping analysis and (e) Aberration Corrected Scanning TEM (ACSTEM) images of Or/In-Se NPs. Red solid line cycle in (e): Se atoms. b–e The experiments are repeated 3 times independently with similar results. XPS fine spectra of (f) C 1 s and (g) Se 3 d of Or/In-Se NPs reaction system at different reaction time. Source data are provided as a Source Data file.
Based on these changes, we further monitored the dynamic process of the intermediate product in the formation of Or/In-Se NPs. In ultraviolet-visible (UV-Vis) spectra, we also found a gradual enhancement of characteristic absorption at 253 nm, which is attributed to the Π-Π excitation of C=C bond in carbon dots formed by carbonization of PEG400 (C-PEG400) and could be found with the intensity increased and red shifted in the heating process (Supplementary Fig. 2a). Notably, fluorescence emission at 477 nm, the characteristic absorption at 350 nm could be detected, which is also consistent with the special absorption of carbon dots in reports (Supplementary Fig. 2b)51,52. Subsequently, typical peak of 002 crystal planes in powder X-ray diffraction (XRD) spectra ascertained the existence of carbon dots for both the reaction system of PEG400 alone and Or/In-Se NPs (Supplementary Fig. 3). To further investigate the formation of carbon dots, we recorded the Fourier transform infrared (FT-IR) spectra of the reaction system at different time points and found that the presence of the typical formants at 1743 and 1640 cm−1 that are known to belong C=O and C=C, which are derived from the structure of carbon dots formed by PEG carbonization (Supplementary Fig. 4). It is worthy to note that the decrease of peak strength of C=C bond from 10 min to 60 min, while the peak strength of C=O bond gradually increased, which indicates that some of the C=C bonds were oxidized to -COOH. To further evaluate the interaction of Se and carbon dots in Or/In-Se NPs, we examined the chemical structure of the reaction system at different time points by X-ray photoelectron spectroscopy (XPS). As shown in the fine spectra of C 1 s, with the prolongment of the reaction time, a remarkable peak of C-Se bond at binding energy of 288.5 eV emerged and increased in intensity, which suggest the bonding of Se and C in the reaction (Fig. 1f). The appearance of C-Se was also verified by the fine spectra of Se 3 d, where clear C-Se-C bond confirmed at binding energy of Se 3d3/2 (55.63 eV) and Se 3d5/2 (54.68 eV) (Fig. 1g). All these results confirm that t-Se under the molten state gradually transforms to free Se atoms, and then anchors on the carbon dots carbonized from PEG400, and finally forms an organic-inorganic covalent Se species through this Se atom reprogramming strategy.
Elucidating chemical structure of Or/In-Se NPs
X-ray absorption near-edge structure (XANES) was employed to elucidate the coordination environment and chemical structure of Se in Or/In-Se NPs. As shown in Fig. 2a, the main peak of Se in XANES (12654.62 eV) is located on the normalized Se K-edge, consistent with the Se-foil. Fitted Fourier transform spectrum for XANES demonstrates the main bonds of Se-C and Se-Se in Or/In-Se NPs, and the bond length is 1.6 Å and 2.1 Å (Fig. 2b), implying the existence of hybrid SeOrg and SeInorg in Or/In-Se NPs. What is more, the results of wavelet transform image in Fig. 2c, Extended X-ray absorption fine structure (EXAFS) in k2 weighted k-space (Supplementary Fig. 5 and Supplementary Table 1) reflected the coordination number of Se in Or/In-Se NPs is 1.9, which conforms to the classical valence bond theory of C-Se-C formation. It is worth noting that the existence of Se-Se bonds from SeInorg in both the K-edge XANES spectra and wavelength transformation images of Se K-edge XANES, although the intensity is lower than that of Se-C bonds from SeOrg. In order to elucidate the chemical structure of SeOrg and SeInorg in Or/In-Se NPs, we firstly clarified the C-Se-C bonds on carbon dots with different coordination numbers in the SeOrg, including four typical theoretical structures with coordination numbers 2 and 3. Among the four binding models, the cohesive energy of the structure of 1 was proven to be the lowest and the most stable, as the monodispersed electric charges in the electron localization function (ELF) images (Supplementary Figs. 6 and 7). While the other structures exhibit the increase of binding energy and instability due to the ring tension and spatial hindrance, especially the type 4. These results suggested that the selenopyrrole of the 1-Se atom binding to carbon dots is the chemical structure of the SeOrg in Or/In-Se NPs, and the charge density of the Se atom on this structure of SeOrg(1) was much higher than other atoms (Supplementary Fig. 8).
a k2 weighted Se K-edge XANES spectra of Or/In-Se NPs fit in R space and (b) the corresponding EXAFS curve. c The wavelength transformation images of Se spectra. d Se K-edge EXAFS spectra and theoretical fitting curves of Or/In-Se NPs and its simulated SeOrg(1)-SeInorg(5) structure supported by carbon dot through selenopyrrole. e Temperature and energy changes of the simplified system with 3 Se atoms and a PEG chain. f Snapshot for the chemical structure of composites of 3 Se atoms and PEG chain. g Bond length of C-H, Se-C and Se-H in simplified system with 3 Se atoms and PEG chain. Source data are provided as a Source Data file.
Furthermore, in order to clarify the chemical structure of SeInorg in Or/In-Se NPs, we further explored the system energy for XANES fitting curves with different atomic numbers of Se. Previous studies have reported that the elemental Se is a mixture of molecules with 2–8 atoms in the forms of rings and short chains under high temperature or in the fusion state53,54,55. In addition, the atomization energy (the binding energy of per atom) in small Se molecules with less than 8 atoms is close, while the molecular energy would increase and structure stability would decrease when the Se atoms number continued to increase56. Hence, we set the Se atom number at 1–7 as representatives to calculate the chemical structure of Or/In-Se NPs. As shown in Supplementary Fig. 9, the chemical potential of −3.43 eV is attributed to the system of Or/In-Se NPs with 1-Se atom, which belongs to the Se-C of SeOrg(1) and exhibits the relative higher stability. While with the formation of Se-Se bonds in the SeInorg, the chemical potential of Or/In-Se NPs rises as the number of Se atoms increases. When the total Se atom number in the system increases to 6, the chemical potential of Or/In-Se NPs reaches the lowest, which means that the Se atomic number in SeInorg should most likely be 5 in this nanoparticle. Furthermore, as shown in Fig. 2d, XANES fitting curves of Or/In-Se NPs matches best with the simulation, when SeInorg is a 5-atom ring structure, SeInorg(5). Density functional theory (DFT) calculation further showed that SeOrg(1)-SeInorg(5) hybrid was composed of a weak Se-Se bond at a length of 3.1 Å. To further confirm this hypothesis, we changed the Se atom number at 4, 5 and 6 in SeInorg of Or/In-Se NPs for XANES fitting to ascertain the atomic structure. As shown in Supplementary Table 2, when the Se atomic number in SeInorg is 5, the Se-Se fitting parameter coordination number (CN) and Debye-Waller factor were found at about 2 and 0.003-0.02, respectively, indicating the high reliability for this fitting. In contrast, when Se atomic number changes to 4 or 6 of SeInorg, the CN value and Debye-Waller factor increase to 4 and > 0.02, and the coordination number and disorder also increase, indicating that the simulated curves of SeOrg(1)-SeInorg(4) and SeOrg(1)-SeInorg(6) could not match well with the real chemical structure. Furthermore, the R factor is much less than 0.02 for all simulations, thus affirming the rationality of this fitting model. Taken together, these results thus confirm that the 5-Se atom is the major structure of the SeInorg(5) in Or/In-Se NPs.
The system was simplified to further clarify the dynamic process of binding of the Se atom to the PEG400 by DFT-based ab initio molecular dynamics (AIMD) simulation. The energy of absorption showed that both the O atoms, C atoms and C-C σ bond on the PEG400 main chain exhibited obvious binding effects with Se atoms, especially the strongest attraction between C and Se atoms (Supplementary Fig. 10). In the simplified model between tri(ethylene glycol) monoethyl ether chain and 3 Se atoms, the binding energy of the reaction system decreased gradually, and tended to reach an equilibrium as Se atoms successively bonded to C atom on the main chains of PEG (Fig. 2e and Supplementary Fig. 11). The dynamic binding process of Se atoms and PEG chain by the formation of C-Se bond in the simplified reaction system was also shown in Supplementary Movie 1. The theoretical model diagram of Fig. 2f directly illustrated that the C-Se-H structure formed by PEG and Se atoms was distributed on the sides of the PEG main chain. At the same time, during the kinetic process in 3000 fs, the C-H bonds length on the PEG400 chain gradually increased and finally broke, while the Se-C bonds length decreased obviously and eventually stabilized at about 2.5 Å (Fig. 2g). The bond length and system energy variation analysis reveal that Se atom could bond with C atom on the main chains sequentially under the command of PEG400 template. After carbonization of PEG400, this Se species with organic-inorganic covalent hybrid structure, SeOrg(1)-SeInorg(5) structure supported by carbon dot through selenopyrrole, was finally formed through the Se atom reprogramming strategy.
ROS-scavenging activity of Or/In-Se NPs based on the organic part of Se species
Based on the charge density of the Se atom in the Or/In-Se NPs is significantly higher than other regions, which indicates its high potential in the catalytic reaction as an electronic donator. After understanding the clear chemical structure of Or/In-Se NP, we commenced to explore the free radical scavenging ability of Or/In-Se NPs motivated by the potential catalytic performance based on SeOrg species. Firstly, the free radical cleaning ability of different Se species Or/In-Se NPs was tested by 2,2’-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS). As shown in Fig. 3a, t-Se and α-Se have no obvious free radical scavenging effects, and Or/In-Se NPs could clean ABTS radical in concentration dependence. For instance, Or/In-Se NPs could catalyze the ABTS free radical in a 100% clearance ratio at 10 μg/mL for 120 min incubation, while the clearance ratio was only 1.67% and 5.14% for t-Se and α-Se at the same concentration and time incubation. We also compared the ABTS clearance of C-PEG400 and found it was about 52.37% at 10 μg/mL for 120 min incubation, which was much lower than that of the Or/In-Se NPs (Supplementary Fig. 12). These results may be due to the fact that the dense packing structure of t-Se is not conducive to contact with free radicals, while the homogeneous phase of α-Se is not conducive to electron transport. In contrast, Or/In-Se NP combines with the high charge density of organic Se species in SeOrg(1) and the presence of holes on the surface of carbon dots, thus resulting in higher catalytic performance.
a Clearance ratio of ABTS free radical in different Se species by ABTS assay (mean ± SD, n = 3 biological replicates). b Work function of Or/In-Se NPs and t-Se. Ef: Fermi level. c Relative energy changes in the scavenging procedures of ROS, including ·O2−, ·OH and H2O2. d Scheme illustration for the process of Or/In-Se NPs decomposition into organic-Se to exhibit free radical scavenging activity. e ESR signals of ·O2−, H2O2 and ·OH scavenging ability of t-Se (20 μg/mL), α-Se (20 μg/mL) and Or/In-Se NPs (20 μg/mL). f Intermediates process and structure of catalytic ROS decomposition by Or/In-Se NP. Source data are provided as a Source Data file.
To further confirm this hypothesis, we also compared the work function value of Or/In-Se NPs and t-Se, which aim to predict the effect of the electron-donating ability and reducibility of the Se atom as the active center. As is shown in Fig. 3b, the work function value of the Or/In-Se NPs (5.05 eV) is remarkably lower than that of t-Se (5.31 eV), indicating that the energy required for electron escape in Or/In-Se NPs system is lower, resulting in stronger electron-giving ability and reducibility, thus exhibiting higher reactive oxygen species (ROS)-catalytic activity. Furthermore, the catalytic processes of free radicals were simulated by the theoretical calculations of DFT, and found that the decomposition of ·O2−, H2O2 and ·OH radicals catalyzed by Or/In-Se NPs mainly involves electron transfer, protonation and dehydration. As shown in Fig. 3c, comparing with the t-Se, the relative energy in the process for Or/In-Se NPs to scavenging ·O2− and H2O2 is close to 0, implying the higher potential to catalyze the decomposition of free radicals. In consideration that the length of weak Se-Se bond (3.1 Å) between SeOrg(1)-SeInorg(5) is much longer than that in SeInorg(5) (2.1 Å) and the energy of the weak Se-Se bond (172 kcal mol−1) is much lower than Se-C bond (244 kcal mol−1) in SeOrg(1), it is proposed that the weak Se-Se bond is cleaved by ROS and releases the organic Se part to exert the efficient ROS-catalytic decomposition ability (Fig. 3d). Meanwhile, the Se-doped carbon dots of SeOrg(1) confers Se atoms with well-defined atomic configuration and larger polarization to promote electron transfer in catalysis process. Hence, we executed electron spin resonance (ESR) to verify the ROS scavenging ability of Or/In-Se NPs against ·OH, ·O2− and H2O2. As shown in Fig. 3e, we prepared ·O2− radicals by the reaction of KO2 and 18-Crown-6 simultaneously, and the ·O2− signal was found to be completely cleared in Or/In-Se NPs co-incubation system. More interestingly, the ·OH signal was detected in the system where t-Se and α-Se were present, indicating that these two Se species catalyzed the decomposition of ·O2− only to obtain intermediate products of ·OH. In addition, after incubated H2O2 with different Se species, the obvious ·OH signal was detected in the Or/In-Se NPs catalytic system, while this signal was not detected in the t-Se and α-Se systems under the same conditions. The results demonstrate that Or/In-Se NPs could effectively catalyze H2O2 into ·OH, following by further decomposition, while t-Se and α-Se could not catalyze the decomposition of H2O2, suggesting the catalytic ability of Or/In-Se NPs is much stronger than that of other Se species. Furthermore, compared with t-Se and α-Se, Or/In-Se NPs exhibit a stronger ·OH scavenging ability. The above results further verify that Or/In-Se NPs could catalyze ·O2− and H2O2 to ·OH, and finally decomposed into low-activity water and oxygen. In conclusion, the ·O2− catalyzed by Or/In-Se NPs is a four-electron process. The ·O2− attached to the Se atom is protonated to form the less stable ·OOH, then combined with electrons and hydrogen ions to form ·OH, and finally, protonation and dehydration reactions occur to promote the clearance of ·O2−. In contrast, the H2O2 adsorbed on Or/In-Se NPs is cleaved into two ·OH, which is accompanied by the reduction of relative energy. Subsequently, ·OH is combined with two protons under the action of electron reduction, and eventually escapes as H2O (Fig. 3f).
Or/In-Se NPs inhibits neuronal oxidative damage by triggering exogenous antioxidant activity and endogenous selenoprotein-activation
Oxidative stress is one of the most important pathologic mechanisms of ischemia-reperfusion injury in ischemic stroke. Based on the superior ROS-catalytic activity of Or/In-Se NPs, we constructed the oxygen and glucose deprivation (OGD) model by human neuroblastoma cell line (SH-SY5Y cells) to simulate the oxidative injury of neurons in ischemic stroke, and further investigated the neuroprotective effects of Or/In-Se NPs in vitro. As shown in Fig. 4a, SH-SY5Y cells under the OGD conditions effectively inhibited the proliferation to about 80%, while Or/In-Se NPs reversed the cell viability, returned to 92%, which was much higher than that of the C-PEG400 (returned to 86%). Furthermore, the different Se species of t-Se and α-Se at the same concentration (5 μg/mL) treatment exhibited lower neuroprotective effects, which did not significantly improve cell survival and maintain at about 81% and 82%, respectively. We further detected the cytotoxicity of different Se species against SH-SY5Y cells to evaluate the biosafety of Se to neurons. As shown in Supplementary Fig. 13, Or/In-Se NPs, t-Se and selenomethionine (SeMet) exhibited much higher IC50 values than other Se species, indicating the high efficiency and low toxicity of Or/In-Se NPs as Se-based nanomedicine to neurons. We then further investigated the oxidative stress in the OGD cell model and the ROS scavenging ability of Or/In-Se NPs by 2’, 7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) assay. As shown in the Fig. 4b, Or/In-Se NPs significantly inhibited the ROS overproduction in the SH-SY5Y cells under OGD conditions in a concentration-dependent manner, which showed a much higher ROS scavenging effect than the carbon dots by C-PEG400. As expected, the other two Se species of t-Se and α-Se exhibited little ROS scavenging activity, especially t-Se. These results demonstrate that Or/In-Se NPs can effectively inhibit the excess ROS generation in the OGD cell model to reduce oxidative damage of SH-SY5Y cells.
a Cell viability of SH-SY5Y in OGD model after treating with t-Se, α-Se, C-PEG400 and Or/In-Se NPs (mean ± SD, n = 3 biological replicates, One-Way ANOVA test, ****p < 0.0001, compared with OGD group). b The intercellular ROS level of SH-SY5Y in OGD model detected by DCFH-DA (mean ± SD, n = 3 biological replicates). c The quantitation of fluorescence images in SH-SY5Y cells labeled by Fluo-4 AM under the OGD model with different concentrations of Or/In-Se (mean ± SD, n = 3 biological replicates, One-Way ANOVA test, ****p < 0.0001, compared with OGD group). d Or/In-Se NPs inhibit OGD model-induced mitochondrial fragmentation with Mito-tracker (Red) in SH-SY5Y cells (Scale bars: 10 μm), n = 3 biological replicates. e Se-related metabolic products in SH-SY5Y cells cultured with Or/In-Se NPs for 0, 6 and 12 h (Se(IV): SeO32-; Se(VI): SeO42-; SeCys2: selenocystine; MetSeCys: methylselenocystine; SeMet: selenomethionine. f Quantification of different Se species, including SeCys2, MetSeCys, SeMet, Se(IV) and Se(VI), cultured with Or/In-Se NPs in SH-SY5Y cells (mean ± SD, n = 3 biological replicates, One-Way ANOVA test, **p < 0.01, ***p < 0.001, ****p < 0.0001. g mRNA level of selenoproteins in Or/In-Se NPs (10 μg/mL) treated SH-SY5Y cells for 12 and 24 h. h Expression level of main selenoproteins in SH-SY5Y cells cultured with Or/In-Se NPs (10 μg/mL) for 8, 12 and 24 h, n = 3 biological replicates. i Schematic illustration for selenoprotein activation by Or/In-Se. Intracellular ROS breaks the weak Se-Se bond, and then inorganic Se would be transformed to selenides and selenophosphate, and eventually incorporated into selenoproteins as selenocysteine. Source data are provided as a Source Data file.
Excessive ROS generation during ischemia-reperfusion will produce Ca2+ overload in mitochondria and lead to mitochondrial dysfunction, which in turn promotes neuronal apoptosis in ischemic stroke. As presented in this study, the level of intracellular calcium concentration in the OGD cell model was detected by Fluo-4 acetoxymethyl ester (Fluo-4 AM) staining. As shown in Fig. 4c, Ca2+ concentration in SH-SY5Y cells under OGD conditions was apparently enhanced to about 500%, and Or/In-Se NPs at 2.5 and 5 µg/mL can significantly decrease it to about 158% and 137%, respectively. A similar trend was also observed by fluorescent imaging of SH-SY5Y cells, as evidenced by a significant decrease in the green fluorescence (Supplementary Fig. 14). As illustrated in Fig. 4d, the filamentous mitochondrial network is normally presented in healthy SH-SY5Y cells, after tracking with Mito-tracker, whereas in the OGD model, the mitochondria were evidently fragmented severely. Moreover, Or/In-Se NPs effectively inhibited mitochondrial fragmentation in the OGD model in a dose-dependent manner. Furthermore, we also used 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolyl carbocyanine iodide (JC-1) probe to detect the inversion of mitochondrial membrane potential in the OGD cell model by Or/In-Se NPs. As shown in the Supplementary Fig. 15, compared with the control group, the ratio of cell membrane potential inversion in the OGD-treated cells increased to 49.67% from 4.15%. Then, after being treated by Or/In-Se NPs with 5 and 10 μg/mL, the inversion of mitochondrial membrane potential ratio of SH-SY5Y cells decreased to 21.12% and 5.72% respectively, which was closed to the healthy cell state. In addition, intracellular Ca2+ concentration increase and mitochondrial membrane potential decrease are considered as the important factors of cell apoptosis. Therefore, the Annexin V and propidium iodide (PI) staining was used to examine the reversal of OGD-induced SH-SY5Y cell apoptosis by Or/In-Se NPs. As shown in Supplementary Fig. 16, the OGD model induced SH-SY5Y cells apoptosis mainly at the last stage to 41.7%, while the apoptosis ratio was decreased to 19.6% and 6.5% after treating with Or/In-Se NPs at 5 and 10 µg/mL, respectively. These results indicate that Or/In-Se NPs effectively inhibit OGD-induced Ca2+ overload and mitochondrial membrane potential decrease, thus alleviating SH-SY5Y cells mitochondrial dysfunction and cell apoptosis.
As an essential trace element for the human body, Se, as the 21st amino acid, selenocysteine, insert into selenoproteins to maintain the neuronal function of brain tissue. In the process of Se metabolism, the maintenance of Se concentration in brain tissue takes priority over other tissues, and Se deficiency can lead to irreversible brain damage. Therefore, we evaluated the derivatives of Se in SH-SY5Y cells after incubating with Or/In-Se NPs for 0, 6 and 12 h by high-performance liquid chromatography coupled to inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) assay, including SeO32−(Se(IV)), SeO42− (Se(VI)), selenocystine (SeCys2), methylselenocystine (MetSeCys) and SeMet. As shown in Fig. 4e, f, after being incubated with Or/In-Se NPs for 6 and 12 h, the intracellular metabolic products are mainly seleno-amino acids, including SeCys2, MetSeCys and SeMet. The contents of SeCys2 were significantly increased in SH-SY5Y cells in a time-dependent manner. While MetSeCys and SeMet contents were highest at 6 h and subsequently decreased, which may be transformed into SeCys2 and participate in selenoprotein biosynthesis. To further confirm this hypothesis, the mRNA expression of selenoproteins in SH-SY5Y cells was determined by quantitative RT-PCR analysis, and found that Or/In-Se NPs can effectively upregulate most of the selenoproteins expression, especially the mRNA level of antioxidant selenoenzyme of glutathione peroxidase (GPX) and thioredoxin reductase (TXNRD) (Fig. 4g). Studies have found that the mRNA expression of selenoproteins can be regulated through Nrf2/Keap1 signal under oxidative stimulation conditions57,58. Our previous studies also found that Se can bind to Keap1 as an electrophilic reagent, promote Nrf2 dissociation and nuclear transfer, and thus activate this signaling pathway to up-regulate the expression of selenoprotein-related genes59. Herein, we found that Or/In-Se NPs can significantly up-regulate the expression of Nrf2, and the activation of this signaling pathway may be a key factor for the promotion of selenoprotein gene expression by Or/In-Se NPs (Supplementary Fig. 17). Correspondingly, we also examined the selenoproteins expression level in SH-SY5Y cells affected by Or/In-Se NPs using Western blot. As shown in Fig. 4h and Supplementary Fig. 18, after incubation with Or/In-Se NPs for 8–24 h, the expression level of two important antioxidant selenoproteins, glutathione peroxidase 2 and 4 (Gpx2, Gpx4), were significantly increased. Meanwhile, the expression of the antioxidant selenoproteins, such as selenoprotein O, P and W (SelO, SelP and SelW), were also slightly upregulated. Furthermore, selenophosphate synthetase 2 (SPS2), related to selenoprotein synthesis, also increased to a certain extent after being incubated with Or/In-Se NPs. The expression of other selenoproteins, such as thioredoxin reductase1 (TrxR1) and selenoproteins S, K, T (SelS, SelK and SelT), demonstrated no significant difference after incubation with Or/In-Se NPs. These results imply that Gpx2 and Gpx4 may play a more important role in reversing neuronal oxidative damage by Or/In-Se NPs. Based on the structure of Or/In-Se NPs, the energy of the Se-Se bond (172 kcal mol−1) in SeInorg(5) is much lower than that of the Se-C bond (244 kcal mol−1) form the SeOrg(1), which triggers the preferential biotransformation of SeInorg(5). After ROS cleavage, the SeInorg(5) is more likely to be reduced and transformed to seleno-amino acids to participate in selenoprotein synthesis under physiological conditions, thus achieving endogenous antioxidant and neuroprotection (Fig. 4i).
Or/In-Se NPs attenuates ischemic stroke-induced oxidative damage in vivo
The pharmacokinetic analysis and biosafety evaluation of nanomedicine are the basis and prerequisite for its application60,61. Therefore, we have systematically examined the pharmacokinetics, distribution and metabolism of Or/In-Se NPs in vivo. The fitted blood concentration curve of Or/In-Se NPs quantified by Se revealed that the half-life (t1/2) and the area under the curve (AUC0-t) of Or/In-Se NPs were 14.9 h and 152.4 mg·h/L, respectively (Supplementary Fig. 19a), which are much higher than that of the clinically used Edaravone (t1/2: 4.47 h, AUC0-t: 2.3 mg·h/L)62, suggesting the high blood circulation and adequate drug exposure of Or/In-Se NPs in vivo. Furthermore, the distribution of Or/In-Se NPs in the main organs showed that the nanoparticles mainly accumulated in the liver and kidney (Supplementary Fig. 19b), thereby being metabolized by the kidney and excreted mainly in the urine (Supplementary Fig. 19c). Subsequently, we further evaluated the biosafety of Or/In-Se NPs by histopathology and hematology. The results of hematoxylin and eosin (H&E) staining showed that Or/In-Se NPs did not cause the obvious pathological changes in the main organs (Supplementary Fig. 20). Importantly, the hematological data also showed that the blood biochemical indexes of the Or/In-Se NPs treated mice maintained in the normal range (Supplementary Table 3). These results indicate that Or/In-Se NPs exhibit high biocompatibility and biosafety in vivo.
Based on the highly neuroprotective ability of Or/In-Se NPs in the OGD cell model, we then further evaluated its ROS scavenging ability and biotransformation of selenium-containing antioxidant proteins in vivo. Accordingly, a middle cerebral artery occlusion (MCAO) reperfusion mice model was established to simulate the pathophysiology of ischemic stroke reperfusion, and further evaluate the neuroprotective effects of Or/In-Se NPs in vivo (Fig. 5a). The infarct area of brain tissue and behavioral changes in MCAO mice were analyzed after injecting with Or/In-Se NPs (0.4 and 0.8 mg/kg) from the tail vein every 24 h for 3 times. As shown in Fig. 5b, observing the mobility and neurological scores of the mice, we found that mice in the MCAO group exhibited a typical hemiplegic symptom of unidirectional steering pattern and only moved in a smaller range. However, Or/In-Se NPs-treated mice were observed the recovery of motor behaviors, such as uprightness and hair grooming, and their activity trajectory was closer to that of the control mice (sham group). To further evaluate the therapeutic efficacy of Or/In-Se NPs in ischemia-reperfusion mice during the treatment period, we performed the behavioral analysis every day using Right-biased swings in the Hanging Test and Neurological scores behavioral testing assays. The results showed that the treatment of Or/In-Se NPs significantly improved the elapsed time of the mice on the rotarod, remarkably decreased the proportion of MCAO mice veering to the right and ameliorated hemiplegia (Fig. 5c, d). Furthermore, based on behavioral scores, Or/In-Se NPs treatment effectively mitigates mobility impairment associated with brain injury in MCAO mice, especially those treated with Or/In-Se NPs at 0.8 mg/kg for 3 days (Fig. 5e). Overall, Or/In-Se NPs treatment significantly reversed the MCAO-induced dyskinesia, as well as the decrease in neurological scores. Then, 2,3,5-triphenyltetrazolium chloride (TTC) staining was used to detect the cerebral ischemic infarction, which reflects as pale in brain tissue by the inability to stain with ischemic tissue. Compared with the MCAO group, Or/In-Se NPs reduced the infarct area and significantly improved the treatment effect with increasing dose of Or/In-Se NPs (Fig. 5f and Supplementary Fig. 21). In addition, Magnetic resonance imaging (MRI) imaging was widely used for noninvasive monitoring in patients with clinical ischemic stroke. Therefore, we further monitored the progression of cerebral infarction by MRI T2 imaging with MCAO mice after treatment with Or/In-Se NPs at different time points. As shown in Supplementary Fig. 22, the MRI T2 images indicated that the infarct area of the MCAO group mice was slightly increased with the extension of time. After treatment with Or/In-Se NPs, the infarct area of brain tissue was significantly reduced with the increase of the concentration and treatment time. Furthermore, we quantified these MRI images and also demonstrated that Or/In-Se NPs administration effectively inhibited the progression of cerebral infarct area, which was reduced almost ~ 40% after treatment with 0.8 mg/kg of Or/In-Se NPs for 72 h (Supplementary Fig. 23). These results indicate that Or/In-Se NPs can effectively attenuate ischemia reperfusion-induced cerebral infarction and motor dysfunction in vivo.
a Illustrated protocol for Or/In-Se NPs to treat mice in ischemic stroke-reperfusion injury. The icons of the mouse and brain tissues are created with BioRender.com. b Digital images of ischemic stroke mice with different treatments for evaluating action capability. Neuronal function evaluation of the treated MCAO mice by behavioral tests, including (c) tail suspension test, (d) Rota-Rod test and (e) neurological scores at different time points (mean ± SD, n = 4 biologically independent samples, One-Way ANOVA test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. f Representative brain tissue images of TTC staining in MCAO mice with different treatments for 72 h (n = 3 biologically independent samples). g MRI T2 images of MCAO mice at different brain layers with 72 h-treatment (Red cycle: Infarct area, n = 3 biologically independent samples). Source data are provided as a Source Data file.
Based on the dual effects of exogenous antioxidant activity and endogenous selenoprotein activation of Or/In-Se NPs, we then further evaluate the neuroprotective mechanism of Or/In-Se NPs in vivo (Fig. 6a). We firstly quantified the accumulation of Se in brain tissue, and found that the treatment of Or/In-Se NPs obviously increased the Se content in the damaged brain sites of MCAO mice (Supplementary Fig. 24). In this regard, we verified the selenoprotein-related mRNA expression levels in brain tissue on the injured side of MCAO mice by RT-PCR. As shown in Fig. 6b, MCAO-reperfusion significantly decreased the expression of SELENOP, that plays an important role in maintaining Se levels and neuronal function in brain tissue63. More importantly, Or/In-Se NPs treatment can effectively enhance the expression of SELENOP up to 4.8 times. Furthermore, we also found that some antioxidant selenoproteins were slightly upregulated in the brain tissue of MCAO mice, which may be used to suppress the oxidative damage caused by ischemia-reperfusion. While after three times of Or/In-Se NPs treatment, a significant increase in the expression of antioxidant selenoenzyme was clearly observed, especially the glutathione peroxidase2, 3 (GPX2, GPX3) and thioredoxin reductase1, 2 (TXNRD1, TXNRD2). In addition, endogenous related antioxidant enzymes in brain tissue on the injured side of MCAO mice were examined by enzyme-linked immunosorbent assay (ELISA). Consistent with the results of RT-PCR, the Or/In-Se NPs remarkably enhanced the expression level of SelP related to storage and transport of Se, indicating that Or/In-Se NPs promote the Se level in the brain tissues (Fig. 6c). What is more, Gpx and TrxR, as two important antioxidant enzymes of selenoproteins, play important roles in neuroprotection and recovery of stroke, which can be significantly up-regulated by Or/In-Se NPs treatment in MCAO mice (Fig. 6d, e). Then, we detected the other two important antioxidant enzymes associated with ROS scavenging at the injury side, including superoxide dismutase (SOD) and catalase (CAT), and found that Or/In-Se NPs also can effectively enhance their expression (Supplementary Fig. 25). For instance, after 0.8 mg/kg of Or/In-Se NPs treatment, SOD expression was restored to about 2 times the level of the MCAO group, while CAT expression was also elevated. Moreover, we also detected the expression level of malondialdehyde (MDA), an important product of intracellular lipid peroxidation, and found that MCAO reperfusion can significantly increase the MDA secretion, which further verified the occurrence of oxidative stress response in ischemic stroke (Fig. 6f). Importantly, Or/In-Se NPs can effectively reduce the level of MDA in the injured side of MCAO mice, which indicates that Or/In-Se NPs can prominently alleviate lipid peroxidation to protect neurons from oxidative stress.
a Schematic illustration of dual action mechanisms of Or/In-Se NPs in the treatment of ischemic stroke. Organic part of Or/In-Se NPs scavenges ROS to H2O for direct exogenous antioxidation, while the inorganic Se transforms to selenoproteins to exhibit endogenous antioxidation. The icons of brain tissues are created with BioRender.com. b mRNA expression level of selenoproteins in brain tissue on the injured side of MCAO mice after treated by Or/In-Se NPs. Concentration for antioxidative enzyme of (c) SelP, (d) Gpx, (e) TrxR and (f) MDA in brain tissue homogenate of the injured side (mean ± SD, n = 3 biologically independent samples, One-Way ANOVA test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with saline group). Immunofluorescence images of (g) ALOX15, (h) Gpx4 and (i) TfR1 expression in the damaged brain of MCAO mice with different treatments. j H&E staining, (k) Nissl staining, (l) NeuN expression and (m) TUNEL staining images of damaged brain tissues in MCAO mice with different treatments, n = 3 biologically independent samples. The red arrow in (k) represents a normal neuron. Source data are provided as a Source Data file.
Meanwhile, as the executor for Se function, selenoproteins like Gpx4 have been reported to be a regulator of ferroptosis, and play a crucial role in stroke treatment64. Therefore, in order to examine whether the neuroprotection action of Or/In-Se NPs involves blocking ferroptosis, we have performed immunofluorescence to assess the expression of Gpx4, arachidonate 15-lipoxygenase (ALOX15, a marker for lipid peroxidation) and transferrin receptor (TfR1) in the brain tissue of the Or/In-Se NPs treated MCAO mice65. As shown in Fig. 6g–i and Supplementary Fig. 26a–c, the administration of Or/In-Se NPs obviously enhanced the expression of Gpx4 and down-regulated the expression of ALOX15 and TfR1, suggesting that supplementation of Or/In-Se NPs could effectively suppress lipid peroxidation through upregulating Gpx4. These results indicate that Or/In-Se NPs exhibit the potential to inhibit ferroptosis and promote neuroprotection in ischemic stroke. Inspired by this finding, we further explored the role of ferroptosis in neuroprotection of Or/In-Se NPs in both the OGD cell model and (1S,3 R)-RSL3 (RSL3)-induced ferroptosis model. As shown in Supplementary Figs. 27 and 28, treatment of Or/In-Se NPs could significantly inhibit OGD- and RSL3-induced cell death and lipid peroxidation, and these effects were comparable with typical ferroptosis inhibitor ferrostatin-1 (Fer-1), indicating that Or/In-Se NPs could protect neuronal cells from OGD-induced cell oxidative damage by inhibiting ferroptosis. Furthermore, the neuroprotective ability of Or/In-Se NPs was elucidated by pathological analysis of brain tissue in MCAO mice. H&E staining of brain sections from the MCAO group showed substantial neuronal necrosis in the infarcted areas, while Or/In-Se NPs significantly reduced the proportion of cellular necrosis with dose dependence (Fig. 6j). Nissl staining has been used to detect the neuronal status and evaluate the normal function of brain tissue. As shown in Fig. 6k, the number of intact neurons in the infarct area was obviously reduced in the MCAO group mice, and the neural progenitor cells showed morphological collapse and no intact Nissl vesicles were observed. In contrast, Nissl vesicles and neuronal cell shape were evidently restored in the Or/In-Se NPs treatment group, suggesting that Or/In-Se NPs can protect neuronal cells from oxidative damage by MCAO-reperfusion. Furthermore, the increased expression of Neuronal nuclei antigen (NeuN) on mature neurons of the damaged brain tissues treated by Or/In-Se NPs, which further indicate the effective neuroprotection of Or/In-Se NPs (Fig. 6l and Supplementary Fig. 29a). We also used TUNEL staining to further detect the neuronal apoptosis in the injured side of MCAO mice, which reflect as the strong green fluorescence intensity (Fig. 6m and Supplementary Fig. 29b). After treatment of MCAO mice with 0.4 and 0.8 mg/kg of Or/In-Se NPs, a decrease of fluorescence in brain tissue could be clearly observed, further indicating the inhibition of neuronal damage by Or/In-Se NPs. In conclusion, Or/In-Se NPs reduce the effect of ischemic stroke on neuronal oxidative damage by the dual effects of exogenous antioxidant activity and endogenous selenoprotein activation.
Discussion
Although great development has been achieved for nanomedicine in the past decades, its successful clinical translation is still being hindered by the difficulty in clear elucidation of the connection between the chemical structure and biological action mechanisms. Therefore, many efforts have been made to balance the toxicity and enhance the bioactivity of conventional nanomedicines. Among them, the strategy of synthesizing organic-inorganic hybrids serves as an effective method to realize this objective. In this study, herein, we employ essential trace element Se as an example, we fabricate organic-inorganic covalent Se hybrid nanoparticles to integrate the advantages of both species, and to realize the unexpected biological application in reversing ischemic reperfusion injury.
In this study, we construct a facile chemical approach for the synthesis of Se hybrid employing the Se atom reprogramming strategy. Under 220 °C, t-Se powder fuses to form free Se atoms, which bind with carbon atoms on the PEG400 chain sequentially with C-Se bond. After carbonization of PEG400, the structure of C-Se formed selenopyrrole-based SeOrg(1). This process was accompanied by the change that the C-Se structure on the PEG400 template bound to free Se atoms, forming SeInorg(5). Finally, we obtained the highly bioactive Or/In-Se NPs. Furthermore, a weak covalent Se-Se bond was formed between SeOrg(1) and SeInorg(5), which can be cleaved by ROS, and thus promotes the formation of different Se species to play their respective advantages under physiological conditions. Second, high charge density and large polarization of Se atoms in SeOrg(1) endows it the ability to promote electron transfer as an electronic donator in the ROS reaction process, thus resulting in higher ROS scavenging ability. In contrast, the bond strength of SeInorg(5) is much lower than that of Se-C in SeOrg(1), and thus preferentially releases free Se atoms to convert to seleno-amino acids to participate in selenoprotein synthesis, thus achieving endogenous antioxidant activity and neuroprotection. Third, in the view of biological application, we take ischemic reperfusion injury as an oxidative damage model. Based on the dual effects of exogenous ROS scavenging activity and endogenous antioxidant selenoprotein activation, Or/In-Se NPs effectively inhibit OGD-induced Ca2+ overload and mitochondrial membrane potential decrease, thus alleviating SH-SY5Y cells mitochondrial dysfunction and cell apoptosis. Importantly, Or/In-Se NPs demonstrate neuroprotective ability to improve neuron survival and neurological functions, followed by the behavioral amelioration in the MCAO mice model. Taken together, our results demonstrate the promise of Se-based nanomedicine in reversing reperfusion injury in ischemic stroke. However, from a drug development perspective, we need to validate this finding in more animal models in the future. At the same time, by improving the synthesis process to promote industrial large-scale production, we then should promote its clinical application in the treatment of ischemic stroke patients to verify its clinical efficacy.
Overall, this study demonstrates a single-atom engineering strategy to fabricate highly bioactive organic-inorganic covalent Se nanoparticles, and elucidates its formation process and structural evolution, and also sheds light on the chemical nature-driven action mechanisms of this Se species in suppressing reperfusion-induced injury of ischemic stroke.
Methods
Animal ethics statement
Animal experiments were carried out under the supervision of the Experimental Animal Ethics Committee of Jinan University (No.: IACUC-20221121-03).
Materials
PEG400, Se powder and hydrogen peroxide (H2O2) were purchased from Aladdin Company (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco®. Thiazolyl blue tetrazolium bromide (MTT), 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1), (1S,3 R)-RSL3 (RSL3) and DCFH-DA were purchased from Sigma-Aldrich. BODIPY C11 was purchased from Thermo Fisher Scientific Inc. 2, 3, 5-Triphenyte-trazoliumchloride (TTC) was purchased from Sangon Biotech (Shanghai) Co., Ltd. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) and Fluo4-AM were purchased from Dojindo Company. TdT-mediated dUTP Nick-End Labeling (TUNEL) assay kit, Proteinase K and Pancreatin were purchased from Roche Inc. Annexin V-FITC/PI apoptosis kit was purchased from Multi Sciences Inc. Selenoprotein P (SelP), Catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (Gpx), thioredoxin reductase (TrxR) and malondialdehyde (MDA) Elisa kits were purchased from Shanghai Jianglai Industrial Ltd.
Synthesis and characterization of Or/In-Se NPs
Se powder (20 mg) was dispersed in PEG400 liquid (10 mL), followed by ultrasonic mixing and heated to 220 °C with a magnetic stirring heater. After 1 h, a brown mixed solution was obtained, and the mixed solution was cooled naturally to finally obtain organic-inorganic covalent Se nanoparticles (Or/In-Se NPs). The samples at different time points of 0, 5, 10, 20, 30 and 60 min during the reaction process were obtained and characterized by spectral and morphological analysis. The topography and structure of Or/In-Se NPs were characterized by transmission electron microscopy (TEM, Hitachi H-7650, 100 kV) and high-resolution TEM (HR-TEM, JEOL 2010). Energy disperse X-ray was performed in HR-TEM to analyze the elemental mapping of the Or/In-Se NPs. High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) was used to get more insight into the detailed selenium atom structures of Or/In-Se NPs. UV-vis-NIR spectrophotometry (UH-4150 Spectrophotometer, Hitachi), fluorescence spectrophotometry (Thermo Scientific LUMINA Fluorescence Spectrometer), Powder X-ray Diffraction (Rigaku Miniflex 600), Fourier Transform infrared Spectrum (Thermo Scientific Nicolet iS10) and XPS spectra (Thermo Scientific K-Alpha + ) were applied to analyze the chemical and morphological changes of Se in the Or/In-Se NPs reaction system at different time points.
The fine structures and coordination environment were detected by X-ray absorption structure (XAS) at the BL14W1 station. The detection was operated on a monochromator of Si (311) double-crystal with 3.5 GeV energy of the electron storage ring. The signals were collected under ambient conditions, and the obtained data were analyzed through Athena and Artemis software. The global amplitude extended x-ray absorption fine structure (EXAFS) (CN, R, σ2 and ΔE0) was obtained by using Artemis software. The least-squares refinement was performed for nonlinear fitting with the EXAFS equation to the Fourier-transformed data in R-space. EXAFS of the Se foil is fitted to obtain the amplitude reduction factor S02 value (0.7156) to determine the coordination numbers (CNs) of the sample. The Debye-Waller factors and delta Rs are calculated based on the guessing parameters and constrained for Se-C and Se-Se. Wavelet transformation (WT) is also implemented with a software package (Funke and Chukalina) by setting Morlet wavelet to κ = 10, σ = 1.
DFT calculation
The DFT calculations are carried out with the projector augmented plane-wave method under the framework of the density functional theory, which is the same as operation with Vienna ab initio simulation package (VASP) version 6.3.266. The generalized gradient approximation proposed by Perdew et al. is used for the derivation of the exchange-correlation potential67. The long-range van der Waals interaction is presented by the DFT-D368. 400 eV was set as the cut-off energy of the plane wave, and 10−7 eV is set as the energy criterion of iterative solution for the Kohn-Sham equation. A perpendicular vacuum layer with a thickness of 15 Å is added to the sheet in order to avoid artificial interaction within periodic images. A 2 x 2 x 1 k-mesh is used to conduct Brillouin zone integration. All structures are considered to be relaxed when the residual forces on the atoms less than 0.03 eV/Å.
ROS scavenging activity assessment
The total ROS scavenging ability of different Se species was detected by the total antioxidant capacity assay kit with the ABTS method (Betotime.inc). The Fenton reaction of Fe2+ (5 mM) and H2O2 (5 mM) was used to generate hydroxyl radicals (·OH). The superoxide radical (·O2−) was obtained by the reaction system of KO2 (0.7 mg/mL) and 18-Crown-6 (6 mg/mL). Then, the hydroxyl radical and superoxide radical scavenging abilities of Or/In-Se NPs were detected by electronic spin resonance (ESR, Magnettech ESR5000).
SH-SY5Y cell culture
Human neuroblastoma cell line of SH-SY5Y was purchased from the American Type Culture Collection (ATCC, #CRL-2266) and was incubated at 37 °C in a 5 % CO2 incubator with DMEM (10 %), penicillin (100 μ/mL) and streptomycin (50 μ/mL). The oxygen glucose deprivation (OGD) cells model was established by incubating in glucose-free DMEM medium and a hypoxic gas environment (0.1 % O2, 5 % CO2 and 94.9 % N2). SH-SY5Y cells were incubated in OGD conditions for 6 h, followed by treatment with different Se species (5 μg/mL) for another 24 h and cell viability measurement with MTT. SH-SY5Y cells were treated with 10 μM of RSL3 to induce ferroptosis. In addition, we used Se powder as t-Se, and the Se nanoparticles as α-Se. The Se nanoparticles was obtained by reducing Na2SeO3 with cysteine (CySH). Specifically, 4 mL CySH (100 mmol L−1) was slowly dropped into 1 mL solution of Na2SeO3 (100 mmol L−1) under an ice bath. The reaction volume was fixed to 20 mL and stirred for 24 h at ambient temperature. After being dialyzed for 48 h, the α-Se was obtained69.
Intracellular ROS detection
SH-SY5Y cells (2 × 105/mL) were seeded in 96-well plates to incubated for 12 h for attachment, and were incubated under OGD conditions for 6 h. DCFH-DA probe was used to the label SH-SY5Y cells for 30 min, followed by gradient concentrations of Or/In-Se NPs treatment. Then, fluorescence intensity was measured in the cell fluorescence reader to reflect the ROS level (Ex = 488, Em = 528 nm), (Bio Tek Cytation 5, Bio Tek Gen5).
Cell apoptosis, cellular Ca2+ concentration and lipid peroxidation level detection
SH-SY5Y (5 × 104 cells/mL, 5 mL) cells were plated in 60 mm dishes and incubated for 12 h. After incubating under OGD conditions for 6 h, the cells were treated with Or/In-Se NPs (5, 10 μg/mL) for another 24 h under normal incubated conditions. Then, after discarding the medium and washing with precooled PBS, the cells were marked by Annexin-V and PI double stanning and analyzed with a flow cytometer (Beckman Colter CytoFLEX S, CytExpert 2.1) for detection of cell apoptosis. Furthermore, the cells with different treatments were stained by Fluo-4AM probe and the green fluorescence signal of Fluo-4AM in SH-SY5Y cells was further detected by fluorescence microscopy to analyze the cellular Ca2+ concentration. The lipid peroxidation level for cells with different treatments was detected by a flow cytometer (Beckman Coulter CytoFLEX S, CytExpert 2.1) with the BODIPY C11 probe. The gating strategies for the flow cytometry of cell apoptosis and lipid peroxidation level are provided in Supplementary Figs. 30 and 31, respectively.
Mitochondrial fragmentation and mitochondrial membrane potential polarization analysis
SH-SY5Y cells (12 × 104 cells/mL, 2 mL) were seeded in 35 mm confocal dishes with a cover glass bottom and incubated for 24 h to attach. After being treated under OGD conditions for 6 h, the cells were further incubated with Or/In-Se NPs (2.5 and 5 μg/mL) for another 24 h. Then, the cells were labeled with MitoTracker (red) for mitochondrial morphology visualization, and observed under a fluorescence microscope (Thermo Scientific EVOS FL, 100 × objective lens). In addition, OGD model and Or/In-Se NPs (5 and 10 μg/mL) co-treated cells were stained by JC-1 probe and then examined with a flow cytometer to evaluate the mitochondrial membrane potential polarization. The gating strategy for the flow cytometry is provided in Supplementary Fig. 32.
MCAO mice model establishment and behavioral evaluation
C57BL/6 mice (wild type, male, 6–8 weeks old, 20 ~ 24 g, n = 48) were purchased from the Gempharmatech Co., Ltd, and the animal experiments were carried out under the supervision of the Experimental Animal Ethics Committee of Jinan University. The mice were housed under the constant temperature of 24 ± 1 °C and humidity of 51 ± 5% in a SPF-grade animal facility with a 12 h light/12 h dark cycle. All animal tests were performed according to ARRIVE guidelines for laboratory animal care and use. The MCAO mice model was performed as described in a previous study. Briefly, the randomized mice were blocked from the bloodstream in the middle cerebral artery (MCA) using a silicon-coated nylon tip (Jialing Biotechnology Company, China). Obstruction of the bloodstream by blocking the middle cerebral artery in unilateral brain tissue to simulate an ischemic process in ischemic stroke, the silicone tip was removed after 60 min of bloodstream blockage for reperfusion. The sham group was treated with the MCAO model, the same as other groups, but was administered without embolization.
The established MCAO mice (n = 36) were assigned to three groups randomly: saline, 0.4 and 0.8 mg/kg of Or/In-Se NPs treatment groups (n = 12 per group). The reperfusion was initiated from the point that the silicone tip was removed, and Or/In-Se NPs was immediately injected into the tail vein and repeated every 24 h for three consecutive times. Another 12 mice without obstruction of the bloodstream were assigned to the sham group and injected with saline. Then the Bederson’s scoring system, the Rotarod test, and the Elevated Body Swing Test (EBST) were used to evaluate the behavioristics of motor cortical dysfunction in MCAO-reperfusion model mice with Or/In-Se NPs -treatment for 3 days. The MRI imaging of these MCAO mice was implemented in the Animal Magnetic Resonance Center of the First Affiliated Hospital of Jinan University. After that, brain tissues were harvested and divided equally to five slices, then incubated by 2 % TTC prepared with normal saline for 20 min at 37 °C. Digital cameras were used to photograph brain sections after staining (n = 3). As a statistical parameter, the infarct size of five tissue slices was calculated for each brain. Then the brain frozen sections (10 μm) were post-fixed with 4 % PFA for 15 min, washed with PBS, and incubated with H&E staining (n = 3), Nissl staining (n = 3), TUNEL-Hoechst co-staining (n = 3) and immunofluorescent staining (n = 3). The immunofluorescence antibodies were used included Anti-15 Lipoxygenase 1 antibody (abcam, ab244205, 1:200), Anti-NeuN antibody (abcam, ab177487, 1:200), Anti-Glutathione Peroxidase 4 (abcam, ab125066, 1:200), CD71/Transferrin Receptor Rabbit pAb (ABclonal, A5865, 1:200), Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (abcam, ab150077, 1:500), Goat Anti-Rabbit IgG H&L (Alexa Fluor® 555) (abcam, ab150078, 1:500), Goat Anti-Mouse IgG H&L Alexa Fluor® 555 (abcam, ab150114, 1:500), Goat Anti-Mouse IgG H&L Alexa Fluor® 488 (abcam, ab150113, 1:500). The homogenate of brain tissues (n = 3) on injured side were grinded with a homogenizer, and centrifuged at 14000 × g to obtain the supernatant, and the protein concentration of the homogenate was detected by the BCA assay. The contents of SOD, SelP, Gpx, CAT, MDA and TrxR in brain damage side tissues were detected by ELISA kits (Shanghai Jianglai Industrial Ltd.) according to the specification method. Besides, a pharmacokinetics experiment was implemented by administration of Or/In-Se NPs (2 mg/kg, once injection) with 5 mice. Then the blood serum, feces and urine of the treated mice were collected at different time points, nitrified with nitric acid and hydrochloric acid (V/V = 1:3) and quantified Se content by ICP-MS. Meanwhile, the main organs were harvested after 72 h, and detected the Se content for tissue distribution analysis.
mRNA expression of selenonoproteins by quantitative real-time PCR (qPCR) assay
The selenoproteins mRNA expression in SH-SY5Y cells after treating with Or/In-Se NPs was assayed by qPCR assay. SH-SY5Y cells (about 105 cells) with Or/In-Se NPs (10 μg/mL) treatments for 12 or 24 h were digested, washed and centrifuged to form cell pellets, followed by resuspended with 1 mL TRIzol (Takara Biotechnology, Japan). The mice brain tissues with different administrations were dissected and ground under an ice bath. After lysed the red blood cells with lysis buffer, washing with PBS, the single cells (about 105 cells) were collected, and 1 mL TRIzol was added. The total RNA was extracted, and the cDNA was prepared by the PrimeScript™ RT Master Mix (Takara Biotechnology, Japan) following the protocols provided by the manufacturers (Bio-Rad, MJ Mini). qPCR analysis was conducted by SYBR ® Premix Ex TaqTM II (Takara Biotechnology, Japan) under CFX ConnectTM Real-Time PCR Detection System (Bio-Rad CFX Connect™, Bio-Rad CFX Maestro). The primers sequences used in this study are shown in the Supplementary Table 4 and 5.
Western blot analysis
SH-SY5Y cells (5 × 104 cells/mL, 10 mL) were seeded in 10 cm dishes, incubated for 12 h and then co-incubated with 10 μg/mL of Or/In-Se NPs for another 8, 12 and 24 h. Then, the treated SH-SY5Y cells were washed with cold PBS and fully lysed by the RIPA lysis buffer under an ice bath to obtain the total protein. The protein concentrations in the cell lysates were quantified by bicinchoninic acid (BCA) assay. 40 μg protein was subjected to electrophoresis with a 12 % SDS-PAGE gel, which was then transferred to polyvinylidene fluoride (PVDF) membrane and blocked with 5% non-fat milk. The membranes were incubated with specific antibodies (selenoproteins, Nrf2 and Keap1) and corresponding secondary antibodies according to the manufacturer’s instructions. The protein bands were visualized on medical X-ray film (FUJI, Super RX-N) using an enhanced chemiluminescence system (Jiangsu Taixing Hong-Ri Medical Equipment Factory, HR-380A). The targeted proteins were confirmed by the molecular weight through comparing with the protein ladder (Epizyme Biotech, #WJ103). The selenoproteins antibodies used in this study were included Anti-Glutathione Peroxidase 2/Gpx2 antibody (abcam, ab137431, 1:1000), Anti-Glutathione Peroxidase 4/Gpx4 (abcam, ab125066, 1:1000), Anti-SelS antibody (abcam, ab190247, 1:1000), Anti-SelK antibody (abcam, ab121276, 1:1000), Anti-Selenoprotein W antibody (Bioss, bs-0495R, 1:1000), Anti-Selenophosphate synthetase 2 antibody (abcam, ab96541, 1:1000), TrxR1 (D1T3D) Rabbit mAb (CST, 15140S, 1:1000), Anti-SELO antibody (abcam, ab172957, 1:1000), Anti-SELT antibody (abcam, ab176192, 1:1000), Anti-SEPP1 antibody (abcam, ab155185, 1:1000) and Anti-beta Actin antibody (abcam, ab8226, 1:1000). The other antibodies used were included Nrf2 (D1Z9C) XP® Rabbit mAb (CST, #12721, 1:1000), Keap1 (D6B12) Rabbit mAb (CST, #8047S, 1:1000), Anti-rabbit IgG, HRP-linked Antibody (CST, #7074, 1:2000), Anti-mouse IgG and HRP-linked Antibody (CST, #7076, 1:2000). The relative expression level of all proteins is quantified correspondingly based on the bands of specific molecular weight. The uncropped Western blot images were provided in Supplementary Data and Supplementary Fig. 33.
Selenium metabolite assay and detection
About 5 × 107 SH-SY5Y cells and mouse brain tissue cells with different treatments were collected and digested with 4 mL digestion solution containing with 4 mg mL−1 trypsin and Proteinase K, respectively. After ultrasonication under ice-bath, the mixture was blended at 37 °C for 24 h, following centrifugation (12000 × g, 15 min). The supernatant was filtered with a 220 μm filtration membrane and submitted to HPLC-ICP-MS detection (Thermo Scientific UltiMate 3000, iCAP RQ, Chromeleon™, Qtegra™) with a pH = 4.5 ammonium citrate buffer (10 mmol L−1) at a flow rate of 1 mL min−1. Selenite (SeO32−, Se(IV)), Selenate (SeO42− Se(VI)), Selenomethionine (SeMet), Selenocystine (SeCys2) and Methylselenocystine (MetSeCys) were used as standards to quantify the content of metabolites of Or/In-Se NPs in SH-SY5Y cells and mouse brain tissues.
Statistics and reproducibility
All experiments in this research were implemented at least in triplicate and repeated three times in biological independence, and the collected data were expressed as mean ± S.D. Measurements were carried out with distinct samples, and no data were excluded from the analyses. The fluorescence intensity, infarct area and Western blot expression level were quantified by ImageJ 1.46r. The difference among multiple groups was analyzed by a one-way ANOVA with Dunnett’s multiple comparisons test.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
All relevant data supporting the findings of this study are available within the article, Supplementary Information and Source Data files. All data underlying this study are available from the corresponding author upon request. Source data are provided in this paper.
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Acknowledgements
This work was supported by National Science Fund for Distinguished Young Scholars (T.C., 82225025), National Natural Science Foundation of China (L.H., 32271351, Z.X., 22305096, T.C., 32171296), Guangdong Basic and Applied Basic Research Foundation for Distinguished Young Scholars (L.H., 2024B1515020057), China Postdoctoral Science Foundation (G.H., 2023TQ0134, G.H., GZC20230968).
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T.C. conceived this research. Z.X. and G.H. synthesized the materials, conducted the preparation reaction and metabolism analysis, performed structure characterization, and carried out the cell experiments. L.H. helped to compile the figures and prepare the manuscript. J.H. performed the theorical calculation. Y.L. helped to conducted animal related experiments. Z.X., G.H., L.H., and T.C. prepared the manuscript.
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Xiong, Z., Huang, G., Huang, JR. et al. Organic-inorganic covalent selenium reversing ischemic reperfusion injury. Nat Commun 16, 8030 (2025). https://doi.org/10.1038/s41467-025-62986-2
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DOI: https://doi.org/10.1038/s41467-025-62986-2
