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Abstract
Acquired brain injury (ABI), which is the umbrella term for all brain injuries, is one of the most dangerous diseases resulting in high morbidity and mortality, making it extremely significant to early diagnosis of ABI. Current methods, which are mainly composed of X-ray computed tomography and magnetic resonance angiography, remain limited in diagnosis of ABI with respect to limited spatial resolution and long scanning times. Here, we reported through-skull fluorescence imaging of mouse cerebral vasculature without craniotomy, utilizing the fluorescence of down-conversion nanoparticles (DCNPs) in the 1.3 - 1.7 μm near-infrared window (NIR-II window). Due to its high spatial resolution of 22.79 μm, the NIR-II fluorescence imaging method could quickly distinguish the brain injury region of mice after performing the stab wound injury (traumatic brain injury) and ischemic stroke (non-traumatic brain injury), enabling it a powerful tool in the noninvasive and early diagnosis of ABI.
© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Acquired brain injury (ABI), which is mainly composed of traumatic brain injury (TBI) and non-traumatic brain injury (non-TBI), is brain damage caused by events after birth. TBI is a major global health issue with millions of people suffering each year from traumatic events in accidents, military conflicts, etc [1]. While non-TBI is resulted from either an internal or external source, such as stroke, brain tumor, etc. Since ABI could cause the impairments in emotion, recognition, behavior, etc., it is extremely important to early diagnose of ABI for the subsequent and successful treatment [2]. In the past four decades, various imaging technologies including computed tomography (CT) and magnetic resonance imaging (MRI), etc., have been developed for ABI diagnosis by detecting anatomic and functional changes in organs (cerebral edema and hemorrhage). It has been reported that cerebral blood vessels were sensitive and vulnerable to ABI, making vascular change the marker for ABI diagnosis [3]. Meanwhile, blood-brain barrier biomarkers (GFAP and UCH-L1) were also used for diagnosis and assessment of ABI [4,5]. Due to the limited resolution, these common imaging technologies can’t early diagnose the vessel- and molecular-level changes underlie ABI [6–8].
Noninvasive optical imaging with photons, featured with high resolution, paved a new avenue to extract the interested biological information [9,10], making it a power tool in early diagnosis of ABI. Fluorescence imaging, utilizing fluorescent agents as molecular processes or structures labels, could provide location, morphology and dynamic information [11,12]. However, most of the existing fluorescent agents exhibited the absorption spectra in the visible band, the imaging depth is limited due to the poor penetration capability of visible light in biological tissue [13]. Considering that the NIR (near-infrared) light could provide a strategy to increase the bioimaging depth, NIR light based multiphoton microscopy attracted much interests in the past decades [14,15]. It has been demonstrated that the imaging depth of two-photon microscopy could reach several hundreds of micrometers, and three-photon microscopy could image the mouse brain structure and function through the intact skull at > 500 μm [16]. The NIR light excited multi-photon microscopy achieved great improvement in imaging depth, but the improved imaging depth still suffered from the limitation from the emitted fluorescence, which was normally located in the visible band. Thus, it is urgent to explore new optical imaging method with high sensitivity and large imaging depth for early diagnosis of ABI.
With the rapid development of fluorescent agents with emission band extended to the second NIR window (NIR-II, 1000 - 1700 nm), including organic molecules [17–19], AIEgens [20–23], down-conversion nanoparticles (NPs) [24–26], carbon nanotubes [27], etc., fluorescence imaging at the NIR-II range achieved great progress. Due to the diminished autofluorescence and reduced photon scattering in both excitation and emission processes, deeper imaging could be achieved as compared with traditional fluorescence imaging at visible band (400 - 750 nm) and NIR-I band (750 - 900 nm) [28,29], e.g. the fluorescence imaging in the NIR-IIa window (1300 - 1400 nm) and NIR-IIb window (1500 - 1700 nm) could provide the imaging depth of 2-3 mm with sub-10 microns and sub-5 microns imaging resolutions, respectively [30,31]. Meanwhile, wide-field and confocal fluorescence imaging in the NIR-II range afforded the field of view from micrometer to centimeter [32]. Moreover, video rate dynamic imaging was achievable, enabling vascular hemodynamics monitoring [33]. All these features made fluorescence imaging in the NIR-II range a promising and powerful tool for diagnosis of ABI.
Herein, we reported the noninvasive and early diagnosis of acquired brain injury by fluorescence imaging in the NIR-II window (1.3-1.7 μm), enabling penetrate through intact skull and image cerebral vasculatures with a high spatial resolution of 22.79 μm. The NIR-II-emissive fluorescent agents of down-conversion nanoparticles (DCNPs) with a core-shell structure of NaYF4:50%Yb3+,2%Er3+,2%Ce3+@NaYF4 were synthesized by co-precipitation strategy. NIR-II fluorescence imaging method could efficiently and quickly distinguish the brain injury region of mice suffering from traumatic brain injury and non-traumatic brain injury once DCNPs were intravenously injected.
2. Materials and methods
2.1 Materials
Erbium (III) acetate hydrate (99.9%), yttrium (III) acetate hydrate (99.9%), ytterbium (III) acetate hydrate (99.9%), cerium (III) acetate hydrate (99.9%) and tetrahydrofuran (THF, ≥99.9%) were purchased from Sigma-Aldrich. Oleic acid (OA, AR), 1-octadecene (ODE, ≥90%(GC)), sodium hydroxide (NaOH, 99%), ammonium fluoride (NH4F, ≥99.99%) and cyclohexane (AR, 99.5%) were purchased from Aladdin Biochemical Technology Co. Ltd., Shanghai, China. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG) was purchased from Laysan-Bio. Methanol (AR) and ethanol (AR) were purchased from Sinopharm Chemical Reagent Co., Ltd., Beijing, China.
2.2 Synthesis of down-conversion nanoparticles (DCNPs)
The co-doped NaYF4:50%Yb3+,2%Er3+,2%Ce3+@NaYF4 core-shell nanoparticles were synthesized using a two-step co-precipitation strategy with modifications. In a typical synthesis, 5 mL Ln(CH3CO2)3 (Ln = 2%Er/46%Y/50%Yb/2%Ce) stock solution (0.2 M), 7.5 mL OA and 17.5 mL ODE were added into a 100-mL round-bottom flask, the mixture was heated to 150 ℃ under stirring for 40 min, and then cooled down to room temperature. Then, 2.5 mL NaOH-methanol stock solution (1 M) and 10 mL NH4F-methanol stock solution (0.4 M) were quickly added and then mixed with lanthanide-oleate precursor solution uniformly. Next the reaction system was heated to 100 ℃ and kept at this temperature for 30 min under an argon atmosphere, followed by incubated at 300 ℃for 1.5 h. After the reaction, the solution was cooled down to room temperature naturally, precipitated by adding 15 mL ethanol, and then centrifugated at 7,500 rpm for 5 min. The core NaYF4:50%Yb3+,2%Er3+,2%Ce3+ were washed for three times using ethanol and cyclohexane, and then re-dispersed in 8 mL fresh cyclohexane.
To epitaxially grow an inert NaYF4 shell, 5 mL Y(CH3CO2)3 stock solution (0.2 M) were added into a 100-mL round-bottom flask, together with 7.5 mL oleic acid and 17.5 mL ODE. The mixture was heated to 150 ℃ under stirring for 40 min, and then cooled down to room temperature. Then, 4 mL core nanoparticles suspension (NaYF4: 50%Yb3+,2%Er3+,2%Ce3+) was injected into the reaction flask as a growth template, followed by 2.5 mL NaOH-methanol stock solution (1 M) and 10 mL NH4F-methanol stock solution (0.4 M). The subsequent steps are the same as the synthesis of the core nanoparticles. Finally, the sample was re-dispersed in 8 mL fresh cyclohexane for later use (the mass concentration was 40 mg/mL).
2.3 Synthesis of DCNP@DSPE-PEG nanoparticles
100 μL of DCNPs solution was mixed with 600 μL of ethanol, which was followed by centrifugation at 10,000 rpm for 10 min. After resuspended in 2 mL of tetrahydrofuran (THF), the precipitate was mixed with DSPE-PEG (8 mg). And then, the THF mixture solution was injected into 20 mL deionized water, followed by sonication for 2 min. Finally, DCNP@DSPE-PEG NPs were obtained after THF was volatilized by stirring at room temperature for 12 h. The obtained samples were purified with centrifugal ultrafiltration for three times to remove residual THF.
2.4 Characterizations of synthesized nanoparticles
Transmission electron microscopy (TEM) images were recorded using the TALOS F200X microscope operating at 200 kV (FEI, United States). Dynamic light scattering (DLS) and zeta potentials were measured by Zetasizer Nano ZSP (Malvern, United Kingdom). The emission spectra were measured by a FLS1000 spectrofluorometer (Edinburgh Instruments, UK).
2.5 Animal model
All animal experiments were approved by the Ethical Committee of Animal Experiments in the School of Biomedical Engineering, Shanghai Jiao Tong University, and were consistent with regulations for the care and use of experimental animals in China.
6-8 weeks old male Institute of Cancer Research (ICR) mice (JSJ, Shanghai, China) were anaesthetized with 2% isoflurane and 30/70% oxygen/nitrous oxide. The body temperature was maintained at 37 ± 0.5 °C using a heating pad. Stroke injury was induced by transiently occluding middle cerebral artery (tMCAO). Briefly, the common carotid artery, the internal carotid artery (ICA), and the external carotid artery (ECA) were carefully isolated. A 6-0 monofilament suture (Covidien, Mansfield, MA, USA) coated with silicon was gently inserted from the external carotid artery to the internal carotid artery. The success of occlusion was determined by measuring the decrease of cerebral blood flow (CBF) to 10% of baseline CBF using laser Doppler flowmetry (Moor Instruments, Devon, UK). Reperfusion was performed by withdrawing the suture 90 min after MCAO.
The mouse model of TBI was based on brain damage caused by hitting the head with a sharp object. The mild brain damage was induced in the left side of the head of mouse by a syringe fixed on a stereotaxic instrument. The scalp of the mouse was cut open to expose the skull, and the tip of a syringe was inserted into the striatum with the following coordinates: AP -0.5 mm; L -2.0 mm; V -5.0 mm. Then, the needle was slowly pulled out and the scalp was sutured. After the hair on the head was removed and scalp was clipped, the mice were prepared for the in vivo imaging of cerebral vasculatures.
2.6 NIR-II wide-field fluorescence imaging system
The NIR-II wide-field fluorescence imaging system was schematically shown in Fig. 1. In brief, a continuous-wave 980 nm laser beam (Changchun New Industries Optoelectronics Technology Co., Ltd.) was employed for the excitation. The emitted NIR-II fluorescence from the sample was filtered with one dichroic mirror and one 1290 nm long-pass filter (Daheng Optics and Fine Mechanics Co., Ltd, China), then focused using a near infrared lens pair (SWIR-35, Navitar, US), and finally recorded using a 640 × 512-pixel 2D InGaAs/SWIR camera (Photonic Science, UK), achieving the final imaging area of ∼14.4 × 11.52 mm2.
3. Results and discussion
3.1 Characterization of DCNP@DSPE-PEG nanoparticles
NIR-II-emissive fluorescent agents could provide excellent in vivo fluorescence imaging quality, which could be further improved by detecting the fluorescence signal in the NIR-IIb window (1500 - 1700 nm). As compared with previously reported NIR-II fluorescence agents, erbium doped rare-earth nanoparticles (NPs) show more promising in biological imaging applications [34]. Organic nanomaterials have relatively low NIR-II fluorescence quantum yield (QY) and photostability; quantum dots (QDs) such as PbS QDs, InAs QDs, contained Class I toxic metals, defined by United States Pharmacopeia, limiting its biomedical applications; while carbon nanotubes were even un-excretable. The NIR-II fluorescence QY of erbium doped rare-earth NPs could reach about 5% [35]. Except for some ultra-small NPs, the final deposition site of erbium doped rare-earth NPs was mainly the liver and spleen, leading to the biliary excretion of the administrated NPs, while ultra-small NPs, normally less than 10 nm, could be quickly cleared out via renal excretion [36]. All these features make the erbium doped rare-earth NPs a good fluorescent agent for biomedical applications. As shown in Fig. 2(a), in the typical erbium-activated nanoparticles, the Yb3+ ions harvested 980-nm photon efficiently and transferred the energy to excite Er3+ ions to the 4I11/2 state, which could non-radiatively relax to the 4I13/2 state and subsequently radiatively to the 4I15/2 state to generate the NIR-II 1565 nm down-conversion (DC) luminescence. However, the inevitable upconversion (UC) processes (4I11/2 → 4F7/2, 4I13/2 → 4F9/2) and quenching of the excited 4I13/2 state caused by the OH- group limited the brightness of DC emission. Here, Ce3+ ions were introduced into the Er3+-activated system for enhancing the DC emission. Since the energy spacing between 2F5/2 and 2F7/2 levels of Ce3+ only slightly mismatched with the 4I11/2 to 4I13/2 energy difference of Er3+, an efficient phonon-assisted cross relaxation process between Er3+ and Ce3+ can occur (4I11/2(Er3+) + 2F5/2(Ce3+) → 4I13/2(Er3+) + 2F7/2(Ce3+) + phonon), accelerating the nonradiative relaxation from 4I11/2 to 4I13/2 and boosting the NIR-II emission from 4I13/2 state of Er3+. In addition, the quenching issue was efficiently alleviated by coating the NaYF4 inert shell (Fig. 2(b)). Thus, the synthesized co-doped NaYF4:50%Yb3+,2%Er3+,2%Ce3+@NaYF4 core-shell DCNP emitted strong NIR-II fluorescence signal centered at 1565 nm as shown in Fig. 2(c).
Fig. 2. Characterization of DCNP@DSPE-PEG NPs. (a) The down-conversion mechanism and (b) schematic illustration of the proposed NIR-II-emissive fluorescent agents. (c) Normalized absorption (blue) and emission spectra (red) of DCNP@DSPE-PEG NPs under 980 nm laser irradiation. (d) Size distribution of DCNP@DSPE-PEG NPs measured by DLS. Inset showed the corresponding TEM image. (e) The size distribution and NIR-II fluorescence (inset) of DCNP@DSPE-PEG NPs in PBS solution at different time. (f) NIR-II fluorescence intensity decrement of DCNP@DSPE-PEG NPs in PBS solution with increasing irradiation time (980 nm, 30 mW/cm2).
In order to further improve the biocompatibility, DCNPs were encapsulated with amphiphilic polymer DSPE-mPEG to form DCNP@DSPE-PEG NPs. The zeta potential of the NPs changed from -1 mV to -28.1 mV after the DCNPs were encapsulated with DSPE-PEG, indicating that DCNPs was successful modified with DSPE-PEG. The size and morphology of the synthesized DCNP@DSPE-PEG NPs were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurement as shown in Fig. 2(d). The TEM images shown that the DCNP@DSPE-PEG NPs had dispersive and uniform morphology. The DLS results shown that the average hydrodynamic diameter of DCNP@DSPE-PEG NPs was about 69 nm, ensuring the efficient blood circulation time. Before performing the bioimaging study, the stability of DCNP@DSPE-PEG NPs was evaluated by measuring hydrodynamic size and emission intensity. Both the average hydrodynamic diameter and fluorescence of DCNP@DSPE-PEG NPs exhibited negligible fluctuations when incubated in 1x PBS for up to 48 h as shown in Fig. 2(e), confirming the excellent physiological stability. The ability to resist photobleaching is an important requirement for fluorescent contrast agents, since this optical property determines their performance in bioimaging. The photostability of DCNP@DSPE-PEG NPs was investigated under continuous irradiation with 980 nm light (30 mW/cm2). Even after 60 min irradiation, the fluorescence intensity still strong (fluorescence loss < 5%) as shown in Fig. 2(f), indicating the superior photostability of the DCNP@DSPE-PEG NPs.
3.2 In vivo cerebrovascular imaging in the NIR-II window through intact skull
With the bright NIR-II emissive fluorescent agent i.e. DCNP@DSPE-PEG NPs, noninvasive in vivo cerebrovascular imaging was performed through the intact mouse skull by exciting the DCNP@DSPE-PEG NPs with a 980 nm laser (15 mW/cm2) and detecting the emitted fluorescence in NIR-II window (1290–1700 nm). Immediately after tail vein injection of DCNP@DSPE-PEG NPs solution (40 mg/mL, 200 μL), fluorescence imaging of mouse brain was performed and various venous vessels, including the inferior cerebral veins (ICVs), the superior sagittal sinus (SSS), and the transverse sinus (TS), etc., showed up quickly and clearly as shown in Fig. 3(a). By plotting the cross-sectional intensity profiles, the signal-to-background ratios (SBRs) of the vessel were measured to be 1.18 and 1.38 (Fig. 3(b)), which were comparable with previously reported SBRs obtained with dimmer QDs [37]. Furthermore, DCNP@DSPE-PEG NPs can be employed for in vivo imaging under a lower and safer power density as compared with some previously reported NIR-II fluorescent agents [38,39]. Moreover, the tiny capillary vessels (diameter = 22.79 μm) could be distinguished easily (Fig. 3(c)), the high sensitivity could be attributed to the high spatial resolution feature of NIR-II fluorescence imaging method. The high SBR and sensitivity of fluorescence imaging in NIR-II window made it a powerful tool in noninvasive and early diagnosis of acquired brain injury.
Fig. 3. (a) In vivo NIR-II fluorescence imaging of cerebral vessels in a mouse with intact skull (Ex: 980 nm, Em: 1290 - 1700 nm, power density: 15 mW/cm2, exposure time: 500 ms). The cross-sectional fluorescence intensity profiles (gray) and the corresponding Gaussian fitting curve (red) along (b) the white-dashed line and (c) the blue-dashed line in (a).
3.3 Noninvasive and early diagnosis of acquired brain injury
Since ABI could cause the various impairments, it is extremely important to early diagnose of ABI for the subsequent and successful treatment. Considering its distinguished features, fluorescence imaging in the NIR-II window is a powerful and promising tool for noninvasive and early diagnosis of ABI, and thus both TBI model and non-TBI model were established to investigate its diagnosis capability.
TBI is a critical health problem, and millions of people suffer from TBI every year [40]. Here the TBI was induced in the striatum on the left side of the head as shown in Fig. 4(a). In order to demonstrate the superior diagnosis performance of NIR-II fluorescence imaging technology, TBI region induced in this work was much smaller than that reported in previous works. Three hours after TBI, DCNP@DSPE-PEG NPs (40 mg/mL, 200 μL) were intravenously injected for fluorescence imaging of the mouse brain without craniotomy. As shown in Fig. 4(b), obvious NIR-II fluorescence signals in the contralateral hemisphere showed up immediately. Meanwhile, the left hemisphere except the TBI region, was also stained by the NIR-II fluorescence signal. Only one blood vessel was impaired by TBI and measured as 123.18 μm. It means that tiny injury by TBI could be noninvasively and quickly detected by NIR-II fluorescence imaging method at its early stage.
Fig. 4. In vivo NIR-II fluorescence imaging in a TBI mouse model. (a) Schematic illustration of the TBI procedure. (b) NIR-II fluorescence imaging of cerebral vasculature in mouse suffering from TBI (Ex: 980 nm, Em: 1290 - 1700 nm, power density: 30 mW/cm2, exposure time: 500 ms). Area marked in blue indicated the blood vessels impaired by TBI. (c) The cross-sectional fluorescence intensity profiles (gray) and the corresponding Gaussian fitting curve (red) along the white-dashed line in (b).
Ischemic stroke, one typical non-TBI and one of the most common cerebrovascular diseases, is featured with high morbidity, disability rate and mortality, making its early diagnosis extremely meaningful. The ischemic stroke model was induced by middle cerebral artery occlusion (MCAO), which is a standard model with great significance for clinical transformation study. MCAO was performed in the left cerebral hemisphere of the female mouse to induce cerebral ischemia as shown in Fig. 5(a), while the right cerebral hemisphere was used as the control. Three hours after MCAO, DCNP@DSPE-PEG NPs (40 mg/mL, 200 μL) were intravenously injected for non-TBI analysis without craniotomy. As shown in Fig. 5(b), strong NIR-II fluorescence signal lighted up the whole right hemisphere including ICVs, SSS, TS. While little signal was observed in the left hemisphere and was only partially visible, indicating the significant impairments induced by MACO. Quantitative analysis confirmed that the blood perfusion in the left hemisphere was significantly less than that in the right hemisphere as shown in Fig. 5(c). These results demonstrated that fluorescence imaging in NIR-II window could quickly and efficiently diagnose the ischemic stroke with high sensitivity.
Fig. 5. In vivo NIR-II fluorescence imaging in a stroke mouse model. (a) Schematic illustration of the MCAO procedure. (b) NIR-II fluorescence imaging of cerebrovascular in mouse suffering from MCAO (Ex: 980 nm, Em: 1290 - 1700 nm, power density: 30 mW/cm2, exposure time: 500 ms). (c) Fluorescence intensity of areas outlined in red and blue in (b).
4. Conclusion
In summary, we developed the NIR-II emissive fluorescence imaging agent of down-conversion nanoparticles (DCNPs) and performed the non-invasive brain imaging in the NIR-II window by penetrating through the intact skull with a high spatial resolution of 22.79 μm. The brain cerebral vasculatures of mice suffering from stab wound injury (traumatic brain injury) and ischemic stroke (non-traumatic brain injury) were resolved in an epifluorescence imaging mode. The truly non-invasive nature and dynamic imaging capability of NIR-II cerebrovascular imaging endow high spatial and temporal resolution to investigate biological processes in the brain at the molecular scale. Future work will focus on the development of NIR-II fluorescence imaging agents with high fluorescence quantum-yield and biocompatibility, making NIR-II fluorescence imaging methods a powerful clinical tool.
Funding
Ministry of Science and Technology of the People's Republic of China (2019YFC1604604); National Natural Science Foundation of China (11974123, 61805135, 6212202); Shanghai Jiao Tong University (ZH2018QNA43); Science and Technology Commission of Shanghai Municipality (19DZ2280300); Shanghai Municipal Education Commission (ZXWF082101); Wuhan National Laboratory for Optoelectronics (2019WNLOKF019); Guangdong Provincial Science Fund (2018B030306015); Guangdong Provincial Science and Technology Project (2019A050510037).
Acknowledgments
This work was supported by the National Key Research and Development Program of China (2019YFC1604604), the Natural Science Foundation of China (61805135, 11974123, 62122028), Shanghai Jiao Tong University (ZH2018QNA43), the Science and Technology Commission of Shanghai Municipality (19DZ2280300), the Innovation Research Plan supported by Shanghai Municipal Education Commission (ZXWF082101), Open Project Program of Wuhan National Laboratory for Optoelectronics (2019WNLOKF019) and the Guangdong Provincial Science Fund for Distinguished Young Scholars (2018B030306015), the Guangdong Provincial Science and Technology Project (2019A050510037).
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this paper may be obtained from the authors upon request.
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