s2fieldsofstudy: Biology Medicine

text:
Evolutional Characterization of Photochemically Induced Stroke in Rats: a Multimodality Imaging and Molecular Biological Study

Photochemically induced cerebral ischemia is an easy-manipulated, reproducible, relatively noninvasive, and lesion controllable model for translational study of ischemic stroke. In order to longitudinally investigate the characterization of the model, magnetic resonance imaging, 18F-2-deoxy-glucose positron emission tomography, fluorescence, and bioluminescence imaging system were performed in correlation with triphenyl tetrazolium chloride (TTC), hematoxylin-eosin staining, and immunohistochemistry examinations of glial fibrillary acidic protein, CD68, NeuN, von willebrand factor, and α-smooth muscle actin in the infarct zone. The results suggested that the number of inflammatory cells, astrocytes, and neovascularization significantly elevated in peri-infarct region from day 7 and a belt of macrophage/microglial and astrocytes was formed surrounding infarct lesion at day 14. Both vasogenic and cytotoxic edema, as well as blood brain-barrier leakage, occurred since day 1 after stroke induction and gradually attenuated with time. Numerous cells other than neuronal cells infiltrated into infarct lesion, which resulted in no visible TTC negative regional existence at day 14. Furthermore, recovery of cerebral blood flow and glucose utilization in peri-infarct zone were noted and more remarkably than that in infarct core following the stroke progression. In conclusion, these characterizations may be highly beneficial to the development of therapeutic strategies for ischemic stroke.

Introduction
Cerebral ischemia accounts for 80 % of all human strokes and has a major impact on the public health [1]. It causes primary neuronal death in the ischemic region and leads to delayed neuronal degeneration in the penumbra [2]. Stroke disables the patients more than it kills. This fact has led a recent effort to develop strategies for neural repair after stroke. Several small animal models of stroke have been developed to identify mechanisms of cerebral ischemia for developing novel recanalyzing, neuroprotective, neuroregenerative, or antiinflammatory drugs at a preclinical level [3,4]. Among them, middle cerebral artery occlusion (MCAo) by an intraluminal filament technique is the most widely used method [5]. Another approach that is technically simpler involves induction of cerebral ischemia and infarction in the cortical vasculature of rats by photochemical reaction triggered by systemic administration of rose bengal (disodium tetraiodo-tetrachlorofluorescein) and focal illumination of the brain [6]. Illumination leads to production of singlet oxygen via dye triplet energy transfer, which in turn induces peroxidative damage to the endothelium and a vasoconstriction. Consequently, platelet aggregation is produced, with the development of a thrombus and vascular occlusion, and a distal territory ischemia is formed [6][7][8]. Rats with small infarct volume induced by this method have low mortality, and even performed on aged rats, the mortality of rats with photothrombotic stroke remained much lower than those with global, permanent stroke [9,10]. An animal model of cerebral ischemia, providing both reproducibility and precise control of lesion size, is critical for translational studies. MCAo produces large ischemic lesions of varying size, which may be considered as a burden to research conducting. Further, MACo lacks the participation of platelet aggregation which is primary initiator of clinical ischemic events [11]. The photothrombotic model not only overcomes the drawbacks of MCAo but can also be used to characterize inflammatory response and apoptosis following thrombosis [12][13][14] and to monitor structural and functional plasticity of neurons [15].
Current managements of acute stroke include restoring cerebral blood flow (CBF) to ischemic penumbral area by thrombolytic therapy, interventional procedures, surgery, and/or cell-based therapy to enhance tissue repair and functional recovery after ischemic stroke [16][17][18]. Noninvasive imaging modalities, such as magnetic resonance imaging (MRI) and positron emission tomography (PET), are promising for investigating the evolution of stroke, confirming the validity of models, monitoring the effect of revascularization interventions, and evaluating the efficacy of novel therapeutic drugs [19][20][21][22][23]. MRI has been used in several preclinical studies of stroke using MCAo model [8,20,21]. When ischemic brain injury occurs, glucose metabolism changes in the infarct and peri-infarct regions. This was demonstrated in earlier reports using 14 C-2-deoxy-D-glucose ( 14 C-2-DG) autoradiography for the study of photochemically induced ischemic stroke [24] and MCAo model [25]. However, method of 14 C-2-DG with autoradiography is limited for in vivo longitudinal evaluation of glucose metabolism on the same animal. Until recently, 18 F-2-deoxy-glucose (FDG) PET imaging was used to evaluate glucose metabolism in transient and permanent MCAo models of rats [26,27]. The powerful imaging tools of 18 F-FDG/PET and MRI offer the information of metabolic changes, lesion structure, edematous and CBF status, respectively. In order to characterize the temporal evolution of stroke lesion induced by photothrombotic method in 14 days, 18 F-FDG/PET, T2-weighted image (T 2 WI), perfusion-weighted image (PWI), and diffusion-weighted image (DWI) were performed to measure cerebral glucose metabolism, edematous lesion, tissue perfusion, and water diffusion, respectively. This image information was further correlated with the cellular and molecular analysis including tissue viability, morphological changes, inflammatory response, astrocyte scar formation, neovascularization, and blood-brain barrier (BBB) permeability, performed by 2, 3, 5-triphenyl tetrazolium chloride (TTC), hematoxylin-eosin (H&E) staining, immunohistochemistry (IHC) staining, and ex vivo Evans blue (EB) imaging in the photochemically induced stroke model of rat.

Animals and Study Design
Seven-week-old male Sprague-Dawley rats were kept under standardized condition (12-12-h light-dark cycle, with free access to food and water). The animals received serial MRI and 18 F-FDG PET/computed tomography (CT) imaging at day 1, 3, 7, and 14 after photothrombotic stroke induction. For TTC, H&E, IHC, and EB staining, rats were sacrificed at day 1, 3, 7, and 14 after stroke induction, and intact brain was carefully removed for subsequent manipulation. All animal experiments in this study were conducted according to the guidelines set by the National Laboratory Animal Center and approved by the Institutional Animal Care and Use Committee of National Yang-Ming University and Macau University of Science and Technology. Reporting of this work complies with Animal Research: Reporting in Vivo Experiments (ARRIVE) guidelines.

Photochemically Induced Stroke
Rats were temporarily anesthetized with isoflurane (induction 3.0 % in air) followed by intramuscular injection of Dexdomitor and Zoletil 50 mixture (1:1) (Orion, Finland and Virbac, France, respectively) with a dose of 100 μl per 300 g body weight and placed in a stereotaxic frame (Narishige Instruments, Tokyo, Japan). After a small incision was made on the scalp, a craniotomic window (3 mm × 6 mm) was made over the somatosensory cortex with the center at the coordinate of 1 mm rostrally from the bregma and 3.5 mm lateral to the midline. A laser beam of 1.5 mm diameter and 532 nm wavelength (GPD105-M-12, Onset Electro-Optics, Taiwan) was stereotactically positioned at the middle of the craniotomic window and illuminated for 20 min. During the first 2 min of illumination, rose bengal (2 ml/kg body weight, concentration: 10 mg/ml saline) was slowly injected through the tail vein. Two control groups were performed following full procedure except for laser illumination or rose bengal injection. All rats after stroke induction were able to survive until they were sacrificed at the end point in this study.

Regional Cerebral Vasculature Examination with Laser Speckle Contrast Imaging
Laser speckle contrast imaging (LSCI) is a technique in which coherent light incidence on a surface produces a reflected speckle pattern that is related to the underlying movement of optical scatters, such as red blood cells, indicating blood flow [28]. Before and after thrombosis was induced by photochemical method, regional cerebral vasculature was examined by LSCI. Briefly, a moorFLPI-2 Full-Field Laser Perfusion Imager (Moor Instruments, Axminster, UK) was placed at the center of cranial window on the somatosensory cortex where the laser beam illuminated. Before and after laser illumination, the laser speckle imaging was acquired with 25-Hz sampling frequency, 1 frame/s, 580 × 752 pixels resolution, and zoom size of 5.6 mm × 7.5 mm.

TTC Assay and H&E Staining
At day 1, 3, 7, and 14 after initiation of photothrombotic stroke, selected rats (n = 3 at each day) were sacrificed by overdose injection of pentobarbital for tissue viability and histopathological examinations. Following sacrifice, fresh brain was removed from the skull, washed in iced phosphate buffer saline (PBS), and placed in a brain mold. Coronal sections of 1 mm in thickness were cut through the cerebrum and placed in 2 % TTC (Sigma) for 15 min in a 37°C incubator for the macroscopic determination of tissue viability. All sections were photographed for delineation of infarct as revealed with TTC. The brain sections were then fixed with 10 % formalin and processed with H&E staining for microscopic examination.

T 2 WI, DWI, and PWI Examinations by Magnetic Resonance Imaging
BioSpec-70/30 7T system (Bruker, Ettlingen, Germany) using a birdcage head-coil of 75 mm inner diameter for radio frequency (RF) transmission and a 20 mm diameter surface coil for reception was used for MRI experiment. The same rat at day 1, 3, 7, and 14 after thrombotic stroke was anesthetized with initial inhalation of 4 % isoflurane for 3 min and maintained with 2 % isoflurane in a mixture of 20 % oxygen and 80 % room air. Prior to imaging, rats under anesthesia were placed in the stereotaxic holder of MRI machine equipped with a heating system to maintain body temperature and a pressure detector to monitor respiration. MRI data sets consisting TWI, DWI, and PWI were acquired at corresponding time points. T 2 WIs were acquired with a multislice multiecho Carr, Purcell Eight coronal and eight horizontal slices were acquired covering a volume extending 10 mm in the rostrocaudal direction. Coronal slices were centered around the infarction lesion, whereas horizontal slices were aligned with the skullcap. DWIs were recorded with a multislice Stejskal-Tanner spin-echo sequence: TR = 4000 msec, echo time = 22 msec, field of view = 25 × 25 mm 2 , slice thickness = 1 mm, matrix = 128 × 128. Data were recorded in the same coronal and horizontal slices as chosen for T 2 WI. Two sets of images were acquired with two different diffusionencoding gradient strengths (b = 30, 1000 s/mm 2 ) in the rostrocaudal direction. PWIs were performed with pulsed arterial spin labeling (PASL) technique using a flow-sensitive alternating inversion-recovery echo planar imaging (FAIR-EPI) sequence with matrix = 96 × 96, FOV = 25 × 25 mm 2 , inversion recovery time (TIR) = 30 to 2300 msec, number of TIR values = 22, recovery time = 10,000 msec, TE/TR > = 10/ 18,000 msec. The T 1 changes between slice selective inversion sequence and nonselective inversion sequence were used for CBF quantification [29]. The infarct volume and CBF ratio were measured by using PMOD software (version 3.0; PMOD Technologies). The CBF difference indexes (CBFDI) of the infarct lesion, peri-infarct region, ipsilateral remote cortex, a n d h i p p o c a m p u s w e r e c a l c u l a t e d a s f o l l o w s : CBFDI = (CBF of the mirror site of contralateral hemisphere-CBF of the selected region in the affected hemisphere)/CBF of the mirror site of contralateral hemisphere.
Evaluation of Cerebral Glucose Metabolism by MicroPET/CT Imaging 18 F-FDG-PET/CT was performed to assess the evolutional change of cerebral glucose utilization. After a 12-h fasting, thrombotic stroke rats (n = 5) at 1, 3, 7, and 14 days were anesthetized with isoflurane and intravenously administered approximately 37 MBq (~1 mCi) of 18 F-FDG. Sixty minutes later, microPET/CT images were acquired for 30 min using a FLEX X-PET and X-O small animal imaging system (GE Healthcare) with the spatial resolution of 1.6 mm and the voxel size of 0.4 mm × 0.4 mm × 1.2 mm. CT images were acquired with 256 projections over 2 min for attenuation correction and anatomy landmarks. PET data were reconstructed using 3D ordered subset expectation maximization (OSEM) method. CT images were reconstructed using a cone-beam reconstruction algorithm. PET and CT images were coregistered using commercial software (Visage Imaging) with 72-μm isotropic CT spatial resolution and 2 mm for PET imaging. For quantitative analysis, volume of interests (VOIs) were drawn on the infarct lesion, one-voxel width surrounding the lesion, and remote region on the ipsilateral cortex. VOIs were also selected at the mirror sites of the contralateral hemispheres. Percent-injected dose of 18 F-FDG per c.c. of brain tissue (%ID/cm 3 ) was obtained from each VOI. The metabolic difference index (MDI) of each VOI was calculated with the following formula: MDI = (%ID/c.c. of contralateral side VOI − %ID/c.c. of lesion VOI)/%ID/c.c. of contralateral side VOI.

Assessment of Blood-Brain Barrier by EB Dye Staining
Disruption of BBB was evaluated at day 1, 3, 7, and 14 after photothrombotic stroke induction. Briefly, 2 h after EB dye (Sigma, 10 mg/ml in saline, 2.5 ml/kg rat weight) was injected via tail vein, animals were sacrificed with an overdose of pentobarbital injection (RMB, Animal Health Ltd., UK). The brains were then removed from the skulls immediately after death. To detect the presence of EB, the intact brain was imaged using in vivo fluorescence and bioluminescence imaging system (IVIS 50, Perkin Elma, UK) with following steps and image acquisition settings: the brains were placed at the center of imaging field, and images were acquired for 2 s using Cy5.5 band pass filter channel (excitation/emission wavelength: 615~665 nm/695~770 nm). Region of interest (ROI) selection and quantification were performed using living image software 3.2 (IVIS Imaging System, Perkin Elma, UK).

Statistical Analysis
The numerical data were reported as means ± standard deviation (SD). Statistical analysis was carried out with the SPSS for windows software package (release 13.0, SPSS Inc., Chicago IL). One-way ANOVA and Tukey post hoc test were used to compare the MDIs and fluorescent signal intensity of EB staining between different time points after stroke. A significant difference was considered if the p value was less than 0.05.

Cerebral Vascular Occlusion by Photothrombotic Induction
Photochemical induction of embolic stroke was completed by 532-nm LASER illumination at somatosensory cortex through the cranial window upon rose bengal infusion. Vasoconstriction and blockade of blood flow were examined by LSCI before and after stroke induction. As shown in Fig. 1, the surface vessel network was clearly appeared before induction in each group. Thirty minutes after stroke induction, remarkably vanishing of blood vessel network was observed in rats injected with rose bengal through the cranial window where the LASER beam illuminated at, while hyperemic blood vessel network was presented in the group of only Fig. 1 Laser speckle contrast imaging (LSCI) before and after photochemical induction of stroke. Cerebral vasculature occlusion was presented after photothrombotic stroke. Either illumination of laser beam or sole injection of rose bengal did not block cerebral blood flow as shown on LSCI illuminated by laser beam and there was no change of blood flow in the group of sole injection of rose bengal. The rapid and massive coagulation of vessels within the illumination area features the pathomechanism of this method which is not similar to that in clinical ischemic stroke usually caused by an embolus or two.

Tissue Viability and Morphological Characteristics of Infarct Lesion
Tissue viability and morphologic change of the photochemically induced stroke were evaluated by TTC and H&E stains at day 1, 3, 7, and 14 post-induction. Results of TTC and H&E staining at each time point were shown in Fig. 2. TTCnegative region which represents the necrotic and non-viable tissue was well demonstrated on the first day after stroke induction, and gradually decreased in size afterward. At day 3, in addition to the shrinkage of ischemic lesion, the boundary between TTC-positive and TTC-negative regions became blurred, as compared to a sharp, clear dividing line at day 1, indicating an increasing number of cells which were migrating and infiltrating to the ischemic margin from outside the region. The volume of the infarct lesions at day 3 to day 14 reduced gradually and remarkably compared to that of the initial infarct volume at day 1. TTC-negative region became hardly observed at day 14 (Fig. 2a). Microscopic examination of the H&E-stained tissues showed a typical stroke-induced liquefactive necrosis at day 1, dilated vessels at day 3, infiltration of cells into the surrounding area of ischemia at days 3 and 7, and neovascular formation at day 14 (Fig. 2b, c).

The Change of Inflammatory Cells and Astrocytes in Infarct Region
As a marker of macrophage and microglia, CD68 expression represents the evolution of inflammatory response post-stroke [30]. Figure 3a shows the presence of CD68-positive cells in the border zone of infarction lesion at day 7, and the cell number increased in the lesion at day 14. IHC staining using antibody against GFAP showed the infiltration of astrocytes into infarct boundary at day 7, and interdigitating compact astrocyte scar surrounding the lesion core was observed at day 14 (Fig. 3b). In addition, NeuN-expressing mature neuronal cells were absent in stroke region throughout 14 days after induction (Fig. 3c). These results indicated that the cellular evolutional changes in the lesion were mainly the inflammatory response and astrocyte scar formation, instead of neurogenesis.

Angiogenesis and Vasculogenesis in Infarct Lesion
As shown in Fig. 4, there were scattered vWF (a marker of endothelial cell) positive vessels in infarct boundary at day 3, and the number of capillaries and αSMA (a marker of vascular smooth muscle cells) positive vessels significantly increased along the infarct margin at day 7. Further, neovascularization was detected in the core of infarct lesion at day 14. These results suggested that angiogenesis and vasculogenesis occurred in the regions of infarct and peri-infarct lesion within 2 weeks after photochemically induced stroke.

The Dynamic Evaluation of Brain Edema and CBF
To monitor the evolutional brain edema and hemodynamic change of photothrombotic stroke, T 2 WI, DWI, and PWI were performed on rats at day 1, 3, 7, and 14 after stroke induction. The ischemic lesion showed a hyperintensity in T 2 WI with sharp margin at day 1, and the signals gradually declined afterward (Fig. 5a). Quantitative analysis indicated that the lesion volume declined from 11.4 mm 3 at day 1 to 0.7 mm 3 at day 14 (Fig. 5b). In DWI, restriction of water diffusion was observed at the margin of stroke shown as an intense halo at Morphological change in H&E-stained tissue sections. It showed a typical stroke-induced liquefactive necrosis at day 1, dilated vessels at day 3, infiltration of cells into the surrounding area of ischemia at days 3 and 7, and neovascular formation at day 14 day 1 and day 3, but not in the core of the lesion which showed a hypointense signal instead. At day 7, the halo was getting smaller. The hypointense lesion almost completely disappeared at day 14 (Fig. 5a). As T 2 WI and DWI have been the reliable methods for investigation of vasogenic edema and cytotoxic edema, respectively [20,21,23], gradual reduction of hypointense signal of T 2 WI and DWI suggested the improvement of brain edema, which contributed to decrease of infarct volume. PWI at day 1 after photochemical induction demonstrated a region of compromised CBF on arterial spin labeling (ASL) corresponding to the photothrombotic infarct lesion (6 ± 51 ml/100 g/min vs 200 ± 95 ml/100 g/min of contralateral cortex), and slightly decreased CBF in the remote cortex of ipsilateral hemisphere. The CBF compromised zone was getting smaller with time. At day 14, a tiny infarct core was still noted, suggesting a persisted damage of brain tissue. CBF of the ischemic region recovered to 195 ± 55 ml/100 g/ min (Fig. 5c). CBFDI of the infarct region (zone A) and the peri-infarct region (zone B) declined rapidly at day 7. CBFDIs of the infarct region and the peri-infarct region showed no significant difference with the contralateral cortex at day 7 and day 14, respectively. CBFDI of ipsilateral remote cortex (zone C) and hippocampus (zone D) closed to zero means CBF almost identical to that of the contralateral hemisphere throughout the whole study of 14 days (Fig. 5d). In summary, the MRI results revealed progressive shrinkage of stroke volume at the periphery of the infarct lesion early after stroke and almost complete recovery of CBF within 2 weeks after stroke induction. Moreover, the remarkable improvement of CBF in infarct lesion and peri-infarct region at day 7 might be related to the synchronous enhancement of angiogenesis and vasculogenesis.

Glucose Metabolism of Stroke Lesion by 18 F-FDG PET Imaging
To investigate the metabolic change of the photochemically induced brain stroke with time, 18 F-FDG PET imaging was performed at day 1, 3, 7, and 14 after induction of stroke. As shown in Fig. 6a, a remarkably reduced 18 F-FDG uptake was noted at the site of infarct lesion and moderately reduced uptake in the remote region of ipsilateral hemisphere at day 1 following induction of stroke. At day 3, the cortical metabolic defect partially recovered in the outer region of the ischemic Fig. 3 Immunohistochemical staining of GFAP, CD68, and NeuN at day 1, 3, 7, and 14 after stroke induction. At day 7 poststroke, the result of staining indicated that GFAP-and CD68positive cells accumulated at the peri-infarct zone and formed a belt surrounding the infarct lesion at day 14. No NeuN expression was observed in ischemic zone throughout 14 days. Scale bar 100 μm Fig. 4 Immunohistochemical staining of vWF and αSMA at day 1, 3, 7, and 14 after stroke induction. Scattered vWF-vessels were observed in the margin of infarct zone at day 3, and the number of vWF-and αSMApositive vasculature began to increase at day 7. At day 14, neovascularization notably occurred in the core of infarct zone zone and almost completely recovered in the remote cortex. At day 14, the 18 F-FDG uptake at the infarct site almost completely recovered. MDI was calculated to monitor the change of glucose utilization in the infarct lesion (zone A), in the region at one-pixel width surrounding the lesion (zone B), in the remote region of the ipsilateral cortex (zone C), as well as in the hippocampus (zone D). MDIs of zones A and B were decreasing with time, indicating that the glucose consumption was gradually restored at both the infarct region and the margin (Fig. 6b). There was significant decrease in MDIs with time in zone A and zone B in 14 days (ANOVA, p < 0.05). From day 7 to day 14, MDI of zone A decreased by 10 % compared to 20 % decrease in zone B, indicating that the recovery of glucose metabolism at the margin of the infarct is better than that in the infarct core. There was no significant statistical difference of MDI observed for 14 days in zones C and D (ANOVA, p > 0.05), suggesting no obvious compromise of glucose metabolism observed in the ipsilateral remote cortex and hippocampus. Using photothrombotic method, infarct induction at somatosensory cortex did not interfere the glucose utilization in the remote ipsilateral cortex and hippocampus and contralateral cortex as well.

Evolution of BBB Permeability in Photothromboic Stroke
Evidence indicates that BBB disruption occurs after ischemic stroke and leads to the leakage of normally excluded substances into interstitial fluid of the brain. To evaluate the extent of BBB disruption and its restoration, we performed the EB staining and ex vivo fluorescence imaging at day 1, 3, 7, and 14 post-stroke. Figure 7a demonstrated an infarction core surrounded by an EB-stained zone in the somatosensory cortex at day 1 after photochemical induction. Subsequently, the leakage of BBB gradually increased at day 3 and began to restore at day 7; a small EB-stained lesion still remained at day 14. The ex vivo fluorescence imaging showed a high intensity fluorescence corresponding to the EB-stained ischemic lesion. The fluorescence signals were much less intense and less extensive at day 14. Quantitative analysis of EB fluorescence at the ROIs of ischemic region showed a markedly reduced signal intensity at day 14 compared to the highest intensity at day 3 (Fig. 7b, p < 0.05). Fluorescent signal at peri-infarct region was much lower than that at infarct core throughout 7 days and greatly reduced to a background level while the infarct core still showed a high signal at day 14. These results suggest a favorable self-repair of BBB in 14 days after photothrombotic stroke.

Discussion
In the present study, we have successfully assessed the evolutional changes of cellular viability in the lesion, infarct volume, brain edema, infiltration of inflammatory cells and astrocytes, neovascularization, CBF, glucose metabolism, and BBB permeability (Fig. 8). To the best of our knowledge, this is the first study conducted using 18 F-FDG/PET and MRI to longitudinally evaluate the metabolic and hemodynamic Evolutional changes and CBFDI, which was calculated from the regional CBF obtained from the PWI of the infarct lesion (zone A), peri-lesional region (zone B, one-pixel width surrounding the infarct lesion), ipsilateral remote cortex (zone C), and hippocampus (zone D). It suggests that CBF of zone B improved more significantly than that of zone A from day 7 after induction changes of the brain in the rat model of photochemically induced cerebral infarction.
The method applied in current study was categorized as Bend-artery occlusion in the cortex^described by Chen et al. [23]. It is the classical and simplest method of photochemically induced stroke model. Although this method can be achieved with intact skull, however, to avoid the scatter and reflection of light when illuminating through skull, a cranial window was made before illumination. In this way, the infarction is anticipated to be more sharply demarcated and more consistent in lesion size than using intact skull. Also, the volume of the ischemic lesion can be controlled by manipulating the intensity of irradiating light and the size of irradiated zone in the cranial window [11]. In the current study, a laser beam of 532 nm wavelength was applied instead of arc beam irradiated system with 560 nm wavelength. Less intensity of the irradiating light causing less severity of the induced stroke may account for the short period of ipsilateral hypometabolism and lack of compromised glucose utilization in contralateral cortex.
To confirm the success of photochemical induction, we applied LSCI through the cranial window of the rat before and after thrombosis was induced to assess the occlusion of cerebral vasculature caused by photochemically induced thrombosis. Laser speckle imaging is useful to assess cerebral vasculature and CBF noninvasively with high temporal and spatial resolution [28]. It has been used to study changes of CBF after distal middle cerebral artery ligation in mice [31]. Upon photochemical induction, LSCI demonstrated blockade of the majority of blood vessels in the illuminated region, Fig. 6 Sequential 18 F-FDG PET imaging of rats with photochemical thrombosis on the right somatosensory cortex. a Transaxial tomographic images at day 1, 3, 7, and 14 after stroke induction. b Metabolic difference index (MDI) at ROIs of infarct zone (zone A), one-pixel width surrounding the lesion (zone B, one-pixel width surrounding the infarct lesion), remote region of the ipsilateral cortex (zone C), and hippocampus (zone D) showed at each time point. There was significant decrease in MDIs with time in zone A and zone B in 14 days (p < 0.05). Stroke rats, n = 5; shame rats, n = 3 indicating successful induction of cerebral ischemia and/or infarction.
In the evaluation of tissue viability and morphologic change in infarct lesion, a wedge-shaped TTC-negative area appeared at day 1 after stroke induction and the size gradually reduced through day 3 to day 7 and was eventually not detectable at day 14. The result of H&E stain showed increased number of infiltrated cells in infarct region since day 3, and some cells were detected in the infarct core at day 14. The identity of these infiltrated cells was further confirmed to be macrophages, glial, endothelial, and smooth muscle cells, but not neuronal cells by IHC staining. Infiltration of cells into the infarct region caused the gradual shrinkage of TTC-negative area.
Inflammation is strongly linked to the development of stroke. Macrophages and microglia, two important types of inflammatory cells in response to stroke, have significant effect on removing debris and activating inflammatory cascades involved in repairing tissue damage [32,33]. In the present study, IHC staining showed that CD68 positive macrophages/ microglia and GFAP-expressing astrocytes notably accumulated along the boundary of infarct lesion at day 14, forming a belt consisting of inflammatory cells and astrocytes. Simultaneously, some microglia/macrophages and astrocytes were detected in the core of infarct lesion. Previous reports suggested that astrocytes produce pro-inflammatory cytokines and chemokines which subsequently recruit microglia/ macrophages for dead cell clearance [34,35]. However, excessive inflammation may have adverse effect, i.e., inducing free radicals which are harmful to neuronal cells [36]. Also, excessive thickness of astrocyte-associated scar blocks neuronal cells from migrating into the injured site. This evidence further supports our result in which neuronal cells were not detectable in the ischemic lesion throughout 14 days after stroke induction.
Vasogenic edema examined by T 2 WI is strongly correlated with BBB disruption after stroke [37,38]. This was also observed in present study. Hyperintensity in T 2 WI at day 1 and day 3 post-stroke was paralleled with the intensity of EB fluorescence, indicating the linkage between vasogenic edema and BBB permeability in photothrombotic stroke. Serial DWI in the current study revealed an intense halo located at the periphery of the stroke lesion seen on T 2 WI. This hyperintense halo seen on DWI indicated the zone with cytotoxic edema and began to gradually reduce since day 3 post-stroke. MR images of photochemical stroke in a previous report also showed early increase in T 2 signal and decreased diffusion of water, indicating the simultaneous development of substantial vasogenic edema and ischemic infarction [39]. This pattern is different from that seen in human stroke, where infarcts develop with cytotoxic edema, followed by a vasogenic edema which is delayed by several hours [3].
The PWI demonstrated a region of absent CBF at the infarct site as well as the edematous region. CBFDI revealed progressive restoration of CBF in the infarct zone and returned to normal at day 14. CBF in the peri-infarct zone was less compromised than that in the infarct zone and returned to normal at day 7. The status of CBF was further confirmed and well correlated with IHC results which showed dilated vessels close to the infarct core at day 3 and neovascularization at day 7 to 14.
In current study, progressive recovery of glucose utilization was noted at the infarct lesion, peri-infarct zone, and ipsilateral remote cortex. CBF recovery and progressive cell infiltration subsequently causing enhancement of glucose utilization may partially account for the rapid recovery of 18 F-FDG uptake in Fig. 7 Detection of blood-brain barrier disruption by EB staining. a Representative images of EB staining and fluorescence imaging of rat brain at day 1, 3, 7, and 14 after ischemia induction. b Quantitative analysis of EB fluorescence at the ROIs of ischemic region (n = 3 at each time point). It suggested that the leakage of BBB was most remarkable at day 3 and was subsequently decreasing with time the infarct zone and its vicinity. Analysis of the MDI is more sensitive than visual inspection of hypometabolic lesion. MDI at day 14 revealed relatively higher restoration of glucose consumption at the peri-infarct zone than that in the infarct core (10 vs 20 %, respectively). Several preclinical studies of small animal ischemic stroke models have consistently revealed an increased 18 F-FDG uptake in the peri-infarct regions due to the effects of activation of glucose transporters, hexokinase, and neuroinflammation [40]. Previous study which used the model of photothrombotic MCAo in rats showed that 18 F-FDG uptake in the peri-ischemic areas was comparable to the normal brain regions at day 1 and 3 and notably elevated at days 7 and 14 [41]. In the present study, glucose utilization in the peri-infarct region was decreased compared to that in the normal region throughout 14 days after stroke induction. However, progressive recovery of glucose utilization in this region was noted. These different results of glucose metabolism in peri-infarct area might attribute to different animal stroke models, time of evaluation, and imaging techniques.
It is commonly agreed that no salvageable penumbral tissue exists in the model of photochemically induced stroke. This disadvantageous phenomenon of this model is different to that of other embolic models where penumbra is much more like those in human stroke and much larger. Hilger and colleagues named peri-infarct zone of photothrombotic stroke as Bregion-at-risk,^where a little viable cells, low energy metabolism, and vasogenic edema existed [42]. In present study, the number of inflammatory cells and astrocytes, as well as neovascularization, gradually increased in peri-infarct region. At day 14, a belt consisting of inflammatory cells and astrocytes was observed surrounding the infarct lesion. In addition, vasogenic edema and cytotoxic edema progressively reduced, and CBF and glucose metabolism gradually improved. The leakage of EB dye at the peri-infarct zone significantly reduced than that in the infarct core. These data suggests the recoverable BBB leakage, neovascularization, CBF, and glucose metabolism at peri-infarct region rather than at infarct core.
Although we have longitudinally characterized the pathophysiological changes of photothrombotic brain ischemia by multiple imaging modalities and cellular immunostaining, some limitations should be addressed in the current study. First, photochemically induced occlusion occurred in vessels within the irradiation area, where the mechanism is different from that of clinical ischemic stroke which was usually caused by an embolus or two. Only a little or no local collateral flow/ reperfusion and ischemic penumbra occur in this model. Despite that the cellular changes in this model are still evolving and follow a similar trend to those of other occlusive models, the pathomechanism is different from those seen in human stroke. Second, evaluation was not performed at earlier time points (3-12 h post-stroke) due to poor animal condition early after stroke induction for 18 F-FDG/PET and MRI examination. Sequential assessment of the evolutional change of Fig. 8 Evolutional characterization of photochemically induced stroke. The changes of tissue viability, infarct lesion volume, brain edema, inflammatory cells and astrocyte infiltration, neovascularization, CBF, glucose metabolism, and BBB permeability at each time point were summarized photochemically induced rat stroke model during the first 24 h following stroke will be carried out in the future study. Third, we did not perform quantitation of blood flow of regional cerebral vasculature by LSCI because the measurement of CBF is limited to the superficial cortex. Fourth, the experiments of multi-modality imaging were performed on the same animal as possible as we could for serial imaging protocol. However, to avoid being anesthetized for a long period of time on the same animal, PET and MRI imaging were not performed on the same day. Despite these drawbacks and limitations of this model, the advantages of easy manipulation, highly reproducible, lesion controllable, and low mortality have made this method comprehensively applied. Further, the consistent lesion made in the cortex where cellular changes evolve and follow a similar trend to those of the occlusive models makes it a suitable model for the study of neurorestorative therapy of pharmaceuticals or stem cells. In the scope of translational purpose, this method had been applied on animals other than rodents, such as rabbits and pigs [43][44][45][46].

Conclusion
The evolution of photochemically induced stroke model in rats has been longitudinally characterized for 14 days by 18 F-FDG/PET, MRI, IVIS, histopathology, and immunohistochemistry examination. Within 14 days after stroke induction, we found the occurrence of early brain edema, infiltration of inflammatory cells and astrocytes, neovascularization in the infarct core and peri-infarct zone, improvement of CBF, glucose metabolism, and BBB permeability. These serial changes characterized in this study provide better understanding of cerebral ischemia and are highly beneficial to the development of therapeutic strategies for ischemic stroke.


extfieldsofstudy:
Materials Science
Medicine

s2fieldsofstudy:
Biology
Materials Science

text:
Mesoporous silica nanoparticles for stimuli-responsive controlled drug delivery: advances, challenges, and outlook

With the development of nanotechnology, the application of nanomaterials in the field of drug delivery has attracted much attention in the past decades. Mesoporous silica nanoparticles as promising drug nanocarriers have become a new area of interest in recent years due to their unique properties and capabilities to efficiently entrap cargo molecules. This review describes the latest advances on the application of mesoporous silica nanoparticles in drug delivery. In particular, we focus on the stimuli-responsive controlled release systems that are able to respond to intracellular environmental changes, such as pH, ATP, GSH, enzyme, glucose, and H2O2. Moreover, drug delivery induced by exogenous stimuli including temperature, light, magnetic field, ultrasound, and electricity is also summarized. These advanced technologies demonstrate current challenges, and provide a bright future for precision diagnosis and treatment.

Introduction
Chemotherapy is a common cancer treatment approach that uses chemotherapeutic agents to kill cancer cells. 1 High-dose conventional chemotherapeutic agents could not only rapidly kill growing cancer cells, but also non-specifically distribute in the whole body where they affect both cancerous and normal cells, thereby limiting the accumulated dose within the tumor cells and resulting in undesirable treatment due to excessive toxicities. 2 Whereas an inadequate dose will limit its effectiveness and result in incomplete treatment and stingy recovery period. Thus, delivery of drugs at an optimal dosage and appropriate duration will make them more effective and powerful in cancer therapy. To overcome this hurdle, a widely pursuit strategy is to design a target-specific and controlled drug-delivery system (DDS) that can transport an effective dosage of drug molecules to the targeted tissues and release the drugs in a sustainable manner. 3 The main challenge of such DDS is its ability to carry an effective amount of drugs with no effects or significantly decreased side effects than those of current therapy approaches.
Recently, nanomaterials have attracted increasing attentions in the fields of drug delivery. Over the past decades, several nano-sized DDSs have been developed and applied by targeted drug delivery to cancer cells, such as liposomes, 4-7 polymeric micelles, [8][9][10][11] dendrimers, [12][13][14][15][16] carbon nanotubes, [17][18][19][20][21][22] inorganic nanoparticles, [23][24][25][26] and silica-based materials. [27][28][29][30][31] Among them, mesoporous silica nanoparticles (MSNs) have attracted much attention due to their unique physiochemical properties, such as large specific surface area and pore volume, controllable particle size, remarkable stability and biocompatibility, and high drug-loading capacity. In 1992, scientists at the Mobil Corporation synthesized ordered mesoporous silica nanomaterials, and this discovery was recognized as a critical breakthrough in material science that could lead to a variety of applications ranging from food manufacturing to pharmaceutical technology. 32 In 2001, Vallet-Regí et al 33 published their first report on the application of MSN as a DDS for controlled drug release. Ibuprofen (IB), an extensively employed analgesic and antiinflammatory drug, was loaded into the MSN and the release process was recorded. Results proved that up to 30 wt% of IB could be loaded into the MSN, and MSN showed a sustained drug release feature. With a gradually increase in studies, a series of MSN-based stimuli-responsive systems have been reported. 34,35 The drug release is subsequently to be triggered by environmental stimuli including physical signals (eg, temperature, 36,37 electricity, 38 magnetic field, 39 and photons [40][41][42] ) and chemical signals (eg, pH values, 43 redox potential, [44][45][46] and enzymatic activities 47,48 ).
In this review, we intend to discuss about the advanced progress related to MSN for drug delivery associated with special focus on environmental responsive mechanisms and highlight the effect of endogenous stimuli in drug delivery such as pH, redox, enzyme, ATP, glucose, and H 2 O 2 , as well as exogenous stimuli including thermo, light, ultrasound, and magnetic field. We have also summarized current challenges that need to be addressed in order to bring this highly promising MSN to practical uses as drug-delivery vehicles in the bright upcoming future.

Endogenous stimuli-responsive drug delivery
Endogenous stimuli are intrinsic conditions of cancerous tissues such as a tough redox condition, an acidic environment, and presence of certain types of enzymes. The design of nanocarriers sensitive to endogenous stimuli may represent an attractive alternative for targeted and controlled drug delivery. In this section, we will discuss DDS that takes advantage of variations in pH value, redox potential, active enzymes, ATP, glucose, and H 2 O 2 .

pH-responsive drug delivery
Among the various stimuli-responsive DDS, pH-triggered drug release attracted much attention since it is well documented that the pH in tumor and inflammatory tissues is more acidic (pH 6.0-7.0) than that in blood and normal tissue (pH 7.4), with even lower pH values in some organelles, such as endosomes (pH 5.5) and lysosomes (pH ,5.5). 49 Thus, the abnormal pH gradients combined with the advantages of MSN provide opportunities to design nanocarriers that are sensitive to physiopathological pH signals to trigger selective drug release in cancer cells. A number of studies have reported the developments of pH-responsive MSN nanocarriers through surface functionalization of MSN with various materials as gatekeepers. The triggered release of anti-cancer drugs from MSN nanopores was achieved mainly by using polyelectrolytes, supramolecular nanovalves, pH-sensitive linkers, and acid-decomposable inorganic materials.
Polyelectrolyte is a commonly used blocking material in pH-responsive drug delivery. Feng et al 50 constructed an MSN-based pH-responsive DDS with polyelectrolyte multilayers. Polyallylamine hydrochloride and polystyrene sulfonate were coated onto the surface of MSN via a layerby-layer technique, and doxorubicin hydrochloride (DOX) was loaded into the nanopores of the as-prepared polyelectrolyte multilayer (PEM)-MSN. The biocompatibility and the influence of the layer numbers on the release profiles were evaluated. A schematic illustration of the construction and release mechanism of PEM-MSN is shown in Figure 1. Results showed that there was a tendency of layer thicknessdependent drug release, and MSN with 20 layers exhibited the highest DOX release rate. Moreover, MSN at acidic condition (pH 5.2) showed a significant higher drug release rate than those at neutral condition (pH 7.4). Popat et al 51 reported a core-shell pH-responsive nanocarrier based on surface coating onto phosphonate-functionalized MSN. The coating was realized by phosphoramidate covalent bonding between phosphonate groups on the MSN surface and amino groups on chitosan. IB was loaded as a model drug. Only approximately 20% of IB was released in pH 7.4 due to chitosan's low degradability and solubility. Whereas ~90% of IB was released in the first 8 hours when the pH value decreased to 5, below the isoelectric point (pI =6.3) of chitosan. Chitosan as a polycation led to the fast dissolution and burst drug release. Hu et al 52 also investigated the chitosan-capped MSN as pH-responsive nanocarriers for controlled drug release. Recently many studies reported efficient pH-responsive delivery systems using other polyelectrolytes as gatekeepers, such as poly-4-vinyl pyridine, 53 poly [2-(diethylamino)ethyl methacrylate], 54 polyacrylic acid, 55,56 poly(methyl acrylic acid), 57 and poly-l-glutamic acid. 58 Thus, the tumor tissues with weak acidity make pH-responsive release systems suitable for controlled release of anti-cancer drugs.
In addition, the application of supramolecule was also reported as a gatekeeper to develop pH-responsive DDS for controlled cargo release such as pseudorotaxanes, rotaxanes, and analogues. [59][60][61][62][63] Herein, the aromatic amines/ammonium stalks were immobilized on the MSN surface, and mobile cyclic molecular gates were introduced to encircle the stalks via non-covalent interactions for controlling the transport of the model drugs loaded in the nanopores. 64 Under certain conditions (especially acidic conditions), the weak binding constant between cyclic caps and stalks resulted in largeamplitude sliding motions of the caps and burst drug release. Li et al 64 have developed several pH-responsive mesoporous silica DDS utilizing the pH-dependent pseudorotaxanes and rotaxanes. In 2009, they constructed a β-cyclodextrin (β-CD) and N-methylbenzimidazole-based pH-responsive MSN DDS, as shown in Figure 2. N-methylbenzimidazole was immobilized onto the MSN surface to serve as stalks and β-CD was introduced to encircle the stalks and form a polypseudorotaxane complex. 59 The nanovalves remain closed at pH 7 with no cargo leakage; however, by decreasing the pH to 5, a rapid release of the guest molecules was detected. Upon the drug-loaded MSN was internalized by THP-1 and KB-31 cells, the acidic pH in lysosome triggered the decomposition of the nanovalves and resulted in an observable drug release. Park et al 63 also reported a cyclodextrin (CD) rotaxane-based pH-responsive DDS, in which the guest molecules were entrapped in the pores of MSN and then polyetherimide (PEI)/CD poly pseudorotaxane was grafted onto the surface to block the nanopores. PEI was used as the guest polymer for CD hosts. At a pH of 11, a stable poly pseudorotaxane complex formed by PEI and α/γ-CD tightly covered the MSN and blocked the pore of the MSN to make the guest molecules reside in the pores. On decreasing the pH values to 5.5, a burst release of guest molecule was observed, which was attributed to the dissociation of CD rings from the PEI stalks due to the weak interaction of the protonated PEI chain with the hydrophobic interior of CDs.
The pH-sensitive linkers, such as acetal bond, [65][66][67] hydrazine bond, [68][69][70][71] hydrazone bond, 72 and ester bond, 73,74 can be cleaved under acidic condition, thus providing opportunities for designing pH-responsive DDS applied in cancer treatment. Liu et al 65 reported a new pH-responsive nanocarrier by capping gold nanoparticles onto the surface of mesoporous silica through acid-labile acetal linkers ( Figure 3). [Ru(bipy) 3 ]Cl 2 dye was loaded as a model drug to investigate the pH-responsive release behavior, and the dye-loaded MSN was dispersed in water at different pH values to test the release profiles. At pH 7.0, no free dye was observed as the intact acetal linker and gold nanoparticles blocked the nanopores to inhibit cargo release. However, the solution at pH 4.0 induced a quick release of dye molecules and almost 100% dye molecules were totally released in 13 hours. On decreasing the pH to 2.0, an even faster molecular transport was observed with 90% release within 30 minutes and reached equilibrium in 2 hours. Similarly, a pH-responsive nanocarrier was designed by Chen et al 66 via capping graphene quantum dot (GQD) onto the nanopores of mesoporous silica through an acid-cleavable acetal bond. The amount of drug leaked from GQD@MSN remained negligible (nearly 3.5%) after 24 hours of incubation, indicating that GQD caps can efficiently block the nanopores. However, ~48% and 86% of the drug molecule were released when the pH value decreased to 5.0 and 4.0, respectively. The accelerated DOX release was ascribed to the cleavage of the acetal bond under the acidic conditions and the continuous separation of GQDs from MSN. Besides that, hydrazine bond is another widely studies acid-labile linkers. Li et al 68 reported a novel intelligent DDS by conjugation of DOX to MSN through acid-labile hydrazone bonds. Results showed that ~15% of DOX was released after 100 hours of incubation at pH 7.4. However, a much faster release rate was observed

91
MSNs for stimuli-responsive controlled drug delivery when the pH value decreased to 6.5, and approximately 55% of DOX was released after 100 hours. To further decrease the pH value to 5.0 in order to simulate the pH condition in endosomes and lysosomes, ~60% of DOX was released in the first 10 hours, and 90% of DOX was released after 100 hours of incubation, which was attributed to the acidic conditions induced cleavage of hydrazone bond uncapped the sealing materials from the MSN.
Acidic-decomposable inorganic materials have been reported as gatekeepers to control drug release, offering opportunities to design promising pH-responsive DDS for therapeutic applications. Muhammad et al 75 reported a pH-responsive DDS based on acid-decomposable, luminescent ZnO quantum dots (QDs)-capped MSN to inhibit premature drug release ( Figure 4). Following cellular uptake by HeLa cells, the ZnO QDs were fast dissolved in the acidic intracellular condition that resulted in a burst drug release into the cytosol. In this research, the ZnO QDs behaved not only as a cap but also as a synergistic antitumor drug for efficient cancer therapy, as the dissolved Zn 2+ would induce the production of reactive oxygen species (ROS), lipid peroxidation, and DNA damage. Rim et al 76 developed absorbable calcium phosphate (CaP)-capped MSN-based pH-responsive DDS for controlled drug release. Under acidic conditions, the entrapped drugs within the nanopores would be released via the dissolution of the capped CaP. To record the release profiles, the drug loaded nanocarriers were immersed at different pH conditions, and the results showed a fast DOX release rate under low pH conditions (pH 4.5) after 24 hours compared to physiological pH (pH 7.4). Furthermore, the pH-dependent dissolution kinetics of hydroxyapatite-like coating from the DOX-Si-MP-CaP complex was also investigated to support the DOX release profiles. The increased Ca 2+ concentration further confirmed

Redox-responsive drug delivery
The development of redox-responsive vehicles for targeted intracellular drug/gene delivery is a very efficient cancer therapeutic strategy. The basic principle of redox-responsive DDS is based on the significant differences in redox concentrations between tumors and normal tissues. It is well documented that concentration of reducing agents such as glutathione (GSH) existing in tumor cells is approximately three times higher than that in normal cells. 77 As a redoxsensitive group, the disulfide bond (S-S) could be easily cleaved in the presence of GSH, which makes it an attracting receptor site in the design of redox-responsive DDS. Since Lai et al 44 reported the first redox-responsive DDS using CdS nanoparticles to block the pore entrances of mesoporous silica through a disulfide bond, several capped systems driven by GSH and other reducing agents (such as dithiothreitol, DTT) have been described. To establish redox-responsive DDS, disulfide bonds were absolutely necessary, and the gatekeepers may vary, but mainly based on nanoparticles, 44,45,78,79 supramolecule or biomacromolecule, [80][81][82][83][84][85][86][87][88] and polymers. [89][90][91][92][93][94] Many inorganic nanoparticles have been used as nanovalves to seal the drug molecule into the channels of MSN through covalently functionalizing MSN with disulfide containing linkers, such as CdS, 44 Fe 3 O 4 , 45 gold, 78 and ZnO. 79 Giri et al 45 synthesized a controlled-release delivery system that was based on MCM-41-type MSN capped with superparamagnetic Fe 3 O 4 nanoparticles through disulfide bonds and was stimuli-responsive and chemically inert to guest molecules entrapped in the matrix. 45 As shown in Figure 5, the disulfide bonds between the MSN and the Fe 3 O 4 nanoparticles are labile

93
MSNs for stimuli-responsive controlled drug delivery and could be cleaved with disulfide reducing agents (DTT) to release the trapped guest molecules from the mesopores.
Moreover, some supramolecule can also be introduced as gatekeeper onto the surface of MSN. As illustrated in Figure 6, Zhang et al 80 reported redox-responsive nanogated MSN by grafting β-CD or adamantane onto the nanopores of MSN through disulfide. After the drug-loaded nanoparticles internalized and then escaped from the endosome to diffuse into the cytoplasm of cancer cells, the high concentration of GSH in the cytoplasm leads to the removal of the β-CD/adamantane caps by cleaving the pre-installed disulfide bonds, further promoting the release of drugs from the nanocarriers. 80,81 Similarly, some biomacromolecules including collagen, 84 cytochrome c, 85 peptides, 86 hyaluronic acid, 87 and heparin 88 can also be covalently immobilized onto the surface of MSN through disulfide-containing linker and act as gatekeeper for controlled drug release.
Polymers are the most popular organic materials used for stimuli-responsive drug delivery. Gong et al 89 successfully synthesized redox-sensitive MSN functionalized with polyethylene glycol (PEG) through a disulfide bond linker. Results showed that drug release was markedly accelerated with the increasing concentration of GSH, while PEG-functionalized system maintained close at low GSH concentrations. Other reports using PEG as gatekeepers also showed similar results. 90-92 Liu et al 95 reported a redox-responsive DDS by using crosslinked poly N-acryloxysuccinimide as a gatekeeper. Poly N-acryloxysuccinimide was anchored to the outlet of silica mesopore through reversible addition-fragmentation chain transfer polymerization. The loaded molecules were released from the hybrid materials by the cleavage of the disulfide linker of the polymeric network with the addition of disulfide reducing agents DTT. It is believed that the developments of such a biocompatible system with non-covalent polymergatekeepers in MSN-based nanocarriers provide a versatile method for hydrophilic drug delivery and enhance the effectiveness of cancer therapy.

Enzyme-responsive drug delivery
The upregulated expression profile of specific enzymes in pathological conditions such as cancer or inflammation makes it an interesting stimulus to achieve enzyme-mediated drug release. 96 Recently, the developments of enzymetriggered DDS based on functionalized MSN have attracted much attention.
Patel et al 97 designed an enzyme-responsive snap-top covered SiO 2 nanocarriers. MSN with an azide-terminated surface was achieved by alkylating amine-modified MSN with a tri(ethylene glycol) monoazide monotosylate motif. The α-CD tori was then threaded onto the tri(ethylene glycol) chains at low temperature (at 5°C) to effectively block the nanopores, while the azide served as a handle to attach a stoppering group that was chemically attached to the snap-top precursors using the Cu(I)-catalyzed azidealkyne cycloaddition. Porcine liver esterase was designed to test the viability of an enzyme-responsive snap-top motif by catalyzing the hydrolysis of an adamantyl ester stopper, which resulted in a dethreading of the α-CD and quick release of the cargo molecules from the pores. Similarly, Sun et al 98   onto the surface of mesostructured silica. These versatile systems are capable of entrapment and controlled release of cargo molecules in response to three different types of exogenous stimuli, such as enzyme, pH variation, and addition of competitive binding agent. In the presence of esterase and urease, SC [4]A-capped MSN showed a rapid cargo release.
As one of the important physiological changes in the tumor microenvironment, matrix metalloproteinases (MMPs), especially MMP2 and MMP9, are overexpressed in almost all the types of cancer cells and associated with tumor invasiveness, metastasis, and angiogenesis, whereas they are minimally expressed in healthy tissues. [99][100][101][102][103][104][105][106] Recently, specific protease-sensitive peptide sequences have been designed as linkers that allow the controlled  Cathepsin B is another kind of protease that is widely used for enzyme-responsive drug release. As a late endosomal and lysosomal protease that can specifically hydrolyze Gly-Phe-Leu-Gly (GFLG) sequence, cathepsin B is overexpressed in various types of tumors. 107,108 Cheng et al 109 reported an MSN-based cathepsin-B responsive DDS. As described in Figure 8, the classic rotaxane structure formed with alkoxysilane tether and α-CD was employed to anchor onto the orifices of MSN to serve as gatekeeper and further modified with multi-functional peptide azido-GFLGR7RGDS for drug loading, tumor targeting, and cathepsin B responsive functions. The functional nanoparticles could specifically target and internalize to HeLa cells, and the overexpressed cathepsin B in endosomes and lysosomes could specifically hydrolyze the GFLG sequences to switch on the nanovalves and resulted in ~80% release of the loaded DOX within 24 hours. Furthermore, in vitro cellular experiments indicated that DOX-loaded MSN resulted in a high rate of cell apoptosis. Li et al 110 also obtained similar results by using enzyme-responsive cell-penetrating peptides to functionalize silica nanoparticles.
Also, other enzyme-responsive DDSs have been reported. Bernardos et al 48 synthesized lactose-capped silica nanoparticles that could be selectively uncapped using β-d-galactosidase by the cleavage of a glycosidic bond. Thornton and Heise 111 described enzyme-mediated release

97
MSNs for stimuli-responsive controlled drug delivery of guest molecules from silica nanoparticles coated with a bioactive peptide shell. In the presence of elastase, specific enzymatic hydrolysis of the peptide shell removes the bulky peptide-terminated Fmoc groups, permitting the selective release of the entrapped guest molecules. Schlossbauer et al 47 described the use of biotin-avidin complex for biomoleculebased, enzyme-responsive drug release and demonstrated the mechanism of action of this system with controlled release of guest molecules as a result of enzymatic hydrolysis of trypsin. These representative examples highlight the potential of enzyme-triggered drug delivery.

Glucose-responsive drug delivery
Glucose-responsive materials have attracted much attention in recent years due to their capability of competitive combination with glucose, which shows great potential application in drug delivery. 112 Zhao et al 113 reported the synthesis of a glucose-responsive MSN-based double delivery system for both insulin and cyclic AMP with precise control over the sequence of release. As depicted in Figure 9, gluconic acid-modified insulin (G-Ins)8 proteins were immobilized on the exterior surface of boronic acid-functionalized MSN through reversible covalent bonding between phenyl boronic acid and vicinal diols of FITC-G-Ins, giving rise to the desired FITC-G-Ins-MSN material and also serving as caps to encapsulate cyclic AMP molecules inside the mesopores of MSN. It is well known that phenyl boronic acid can form much more stable cyclic esters with the adjacent diols of saccharides than with acyclic diols. The release of both G-Ins and cyclic AMP from MSN could be triggered by introducing glucose to cleave the linkage between FITC-G-Ins and boronic acid-functionalized MSN. This glucose-responsive drug release system was expected to be a novel and promising therapeutic strategy to cure diabetes in the future, instead of the frequently used insulin injection approach.
Chen et al 114 reported a glucose-responsive controlledrelease system based on the competitive combination between glucose oxidase, glucosamine, and glucose. MSN was first Zhao et al 115 introduced a novel glucose-responsive controlled release of insulin system by coating glucose oxidase and catalase multilayers onto the surface of MSN. The MSN serves as a drug reservoir and the glutaraldehyde cross-linked enzymatic multilayers act as a valve to control the release of insulin in response to the exogenous glucose level.

H 2 O 2 -responsive drug delivery
The main culprit in the pathogenesis of ischemia/reperfusion injury is the overproduction of ROS. Hydrogen peroxide (H 2 O 2 ) is the most abundant form of ROS produced during ischemia/reperfusion, which usually induces the release of pro-inflammatory cytokines and triggers apoptosis, leading to the oxidative damage of tissues. [116][117][118] Therefore, targeting H 2 O 2 as a diagnostic marker and therapeutic agent shows great potentials.
Geng et al 119 reported a biocompatible and H 2 O 2 -responsive controlled-release system to realize target delivery of Alzheimer's disease therapeutic metal chelator. As illustrated in Figure 10, the system was consisted of a mesoporous nanoparticle functionalized with a derivative of aryl boronic acids by forming stable cyclic esters with the adjacent diols of saccharides. Human immunoglobulin G (IgG) was chosen as a nanoscopic cap. The delivery of the entrapped guest molecules depended on the oxidization of the aryl boronic esters to phenols in the presence of H 2 O 2 . The complete breakage of aryl boronic esters resulted in the release of IgG and guest molecules, and the release rate was sensitive to H 2 O 2 concentration. Negligible guest molecules were released in the absence of H 2 O 2 at room temperature, indicating that IgG acted as an efficient cap for guest molecules retention. Whereas with increasing H 2 O 2 concentrations of 5 mM, approximately 91% of the guest molecules were released after 12 hours of incubation, which was attributed to the rupture of boronate ester bonds that link the MSN and IgG to the opening of the nanovalves. This novel kind of delivery system shows a potential application in controlled drug release.

ATP-responsive drug delivery
As one of the important biogenic molecules, adenosine triphosphate (ATP) is a multifunctional nucleotide that provides ubiquitous energy for all biological processes, including muscle contraction, cells function, synthesis and degradation of essential cellular compounds, and membrane transport, by breaking the phosphoanhydride bond. There is growing evidence that the upregulated ATP levels are correlated with many pathological processes, such as chemoresistance, uncontrolled tumor growth, and synaptic transmission in neurons, 120,121 which make it a significant marker in distinguishing cancerous cells from normal cells. Owning to the upregulated ATP level, many researchers have tried to design ATP-responsive drug-release systems that can specifically recognize ATP as well as the competitive binding with ATP aptamer. [122][123][124][125][126] Lai et al 122 developed an ATP-responsive DDS for realtime monitoring of drug release. As shown in Figure 11, the mesoporous-silica-coated up-conversion nanoparticle (UCNP@MSN) was wrapped with a compact branched polypeptide, poly(Asp-Lys)-b-Asp, and then functionalized with zinc-dipicolylamine analogue (TDPA-Zn 2+ ) on its exterior surface. In the absence of ATP, the loaded drugs remained entrapped within the UCNP-MSN due to the multivalent interactions between Asp moieties in the polypeptide and the TDPA-Zn 2+ complex present on the surface of UCNP-MSN. Whereas in the presence of ATP, a competitive displacement of the surface-bound polypeptide by ATP due to its higher affinity to TDPA-Zn 2+ was observed, which led to the opening of the channel and sustained drug release. He et al designed a facile ATP-responsive controlled release system consisting of MSN functionalized with aptamers as a cap. 123 In this system, the ATP aptamer was first hybridized with arm single-stranded DNA1 (arm ssDNA1) and arm singlestranded DNA2 (arm ssDNA2) to form a sandwich-type DNA structure and then grafted onto the MSN surface through click chemistry approach, which resulted in blocked guest molecules in the nanopores. Seven hours after the addition of ATP (20 mM), ~83.2% of the total load guest molecule

100
Song et al was released due to the aptamer-containing sandwich-type DNA structure was dissociated through a competitive binding with ATP, leaving the flexible arm ssDNA1 and ssDNA2 on the surface of MSN. Zhu et al 124 reported an ATP-responsive MSN-dependent drug-release system based on aptamer target interactions. The pores of MSN were capped with ATP aptamer-modified Au nanoparticles. In the presence of ATP molecule, a competitive displacement reaction occurred, the Au nanoparticles were uncapped from the MSN, and the cargo was released.

Exogenous stimuli-responsive drug delivery
Unlike endogenous stimuli, exogenous stimuli are carried out via an external physical treatment. Although this approach seems unappealing, exogenous stimuli-responsive DDSs might be more encouraging and favorable due to the heterogeneous physiological conditions of human population. In this section, drug delivery triggered by externally applied stimuli, including temperature changes, magnetic fields, ultrasounds, and light and electric fields, are discussed.

Thermo-responsive drug delivery
Thermo-responsive drug delivery is one the most investigated stimuli-responsive strategies and has been widely explored in tumor therapy. 127 Thermo-responsive MSN DDSs are usually composed of MSN and surface-coated thermo-responsive materials. The drug release was closely dependent on the variation of the surrounding temperature to control the switch of the nanovalves. Poly N-isopropyl acrylamide (PNIPAM) has been well known as a thermoresponsive polymer. As depicted in Figure 12, Chen et al 128 reported a thermo-responsive DDS synthesized by covalent functionalization of silica nanoparticles with PNIPAM by a combination of reversible addition-fragmentation chain transfer and click chemistry. The thermal-induced conformational changes of the NIPAM polymer layer on the exterior surface of the MSN could be used as a switch to control the cargo release. When the temperature increases above 30°C, PNIPAM acquires hydrophobic character and the chains of polymers aggregated on the SiO 2 surface and the drug molecule are blocked in the nanopores of MSN. At a temperature between 25°C and 30°C, the outer zone of PNIPAM brushes still remains hydrophilic and the chains of polymers are extended and swollen in an aqueous solution. Thus, the entrapped drug could be quickly released. Singh et al 129 developed a temperature-sensitive MSN for triggered drug release by a relatively simple technique. The anionic surface of MSNPs was functionalized with bifunctional N-(3-aminopropyl) methacrylamide hydrochloride, and the acrylamide group was subsequently covalently cross-linked to NIPAM and poly(ethylene glycol) diacrylate by radical copolymerization at room temperature. DOX was used as a model drug molecule and the polymer-coated MSN showed a high drug-loading capability (~50% of total DOX added). At temperatures (37°C) greater than the lower critical solution temperature (LCST, 31°C), approximately 50% DOX was released within the first 2 hours, which is relatively higher than those maintained at room temperature. Other researchers also investigated the PNIPAM-based thermo-responsive DDS using MSN as the drug container. [130][131][132][133][134][135][136] This new core-shell thermo-responsive nanocarrier showed promising potential in controlled drug/gene delivery.
Moreover, researchers have tried to employ nuclear acid as gatekeeper to develop thermo-responsive DDS. The double-stranded DNA has been recognized as attractive capping materials due to their unique self-recognition properties of duplex DNA, as well as temperature-dependent assembly. As reported by Schlossbauer et al, 37 Chen et al, 137 and Chang et al, 138 duplex DNA strand or oligonucleotide was anchored onto the openings of the nanovalves and was utilized as a cap for blocking the guest molecules within the porous channels. The duplex DNA cap could be denatured by DNA strand melting at a specific melting temperature of the oligonucleotide, thus opening the valves and releasing the cargo.
In general, the biggest challenge in designing thermoresponsive nanocarriers lies in the development of novel materials that are safe and sensitive enough to respond to slight temperature changes around the physiological temperature of 37°C.

Light-responsive drug delivery
Due to their non-invasiveness property and the possibility of remote spatiotemporal control, a variety of light-responsive systems have been developed in recent years to achieve on-demand drug release in response to light irradiation at a specific wavelength (in the ultraviolet [UV], visible, or near-infrared regions). The mechanism relies on the photo-sensitiveness-induced conformational transition of the nano-carriers.
Azobenzene (AB) is a type of light-sensitive molecule. When irradiated with UV light at a wavelength of 351 nm, AB is able to isomerize from the more-stable trans to a less-stable cis configuration. Previous studies have demonstrated that there was a high binding affinity

101
MSNs for stimuli-responsive controlled drug delivery between β-CD and trans-AB derivatives and a low binding affinity between β-CD and cis-AB derivatives in aqueous solutions. 139,140 According to this principle, a lot of AB-liable light-responsive DDSs were designed. 41,42,[141][142][143] Ferris et al 42 developed AB-derivative-dependent DDS and MSN-based light-responsive DDS. As shown in Figure 13, two different AB derivatives, prepared from 4-(3-triethoxysilylpropylureido) azobenzene and (E)-4-((4-(benzylcarbamoyl)phenyl) diazenyl) benzoic acid, were used to modify MSN and act as stalks. Then, pyrene-modified β-CD was threaded onto the stalks that bind to trans-AB units to cap the nanopores and seal the preloaded model drug molecule. Upon irradiation with UV light of 351 nm, isomerization of AB units from trans form to cis form occurs, which leads to the dissociation of β-CD rings from the stalks, uncapping the container and releasing the cargo outside.
Coumarin is also an attractive photo-responsive molecule. Mal et al 144 described an UV light-induced reversible drug-release system for the first time. 7-[(3-Trihydroxysilyl) ropoxy]coumarin was attached to the silanol groups of cholestane-loaded MSN that acted as "hinged double doors" to block the drug molecules in the nanopores. When the system was irradiated at the UV light wavelength greater than 310 nm, coumarin underwent a photodimerization reaction and cyclobutane dimer rings were formed that spanned the pores to hinder drug diffusion. Whereas when the system was irradiated with UV light of wavelength of ~250 nm, cyclobutane rings were photocleaved, yielding new coumarin

103
MSNs for stimuli-responsive controlled drug delivery monomers to release the entrapped drug molecules. Guardado-Alvarez et al 145 introduced a light-responsive DDS based on photolabile coumarin-based molecules. Coumarinbased molecules were bound to the surface of MSN, and then bulky β-CD molecules were non-covalently associated with the substituted coumarin molecules, blocking the pores and preventing the cargo diffusion. Upon one-photon excitation at 376 nm or two-photon excitation at 800 nm, the bond that bound coumarin to the nanopore was cleaved, which resulted in the fast release of both the CD cap and cargo molecules. The major drawback of light-triggered drug delivery is its low penetration depth (~10 mm) in UV-visible region (wavelength ,700 nm). However, it is possible to replace UV-visible light with near infrared (NIR) laser (range 700-1,000 nm), which showed deeper tissue penetration effect, lower scattering properties, and minimal harm to tissues.
Gold nanoparticles presented plasmonic properties and showed high efficiency to transform NIR radiation into thermal energy. Chang et al 138 have reported an NIR light-responsive oligonucleotide-gated ensembles for intracellular drug delivery. As depicted in Figure 14, the system is composed of gold nanorods-encapsulated MSN and surface-decorated DNA double strands as gatekeepers. When this device was irradiated with NIR laser (808 nm, 1.5 W/cm 2 ), the generated heat enables denaturing of the duplex oligonucleotides of the DNA strands opening the pores and allowing the drugs to diffuse out of the carrier. Yang et al 146 developed a novel multifunctional NIR-stimuluscontrolled drug-release system based on gold nanocages as photothermal cores, mesoporous silica shells as supporters to increase the anticancer drug loading, and thermo-responsive PNIPAM as NIR-stimulus gatekeepers (Au-nanocage@ mSiO 2 @PNIPAM). The Au nanocage cores can effectively absorb and convert light into heat upon irradiation with an NIR laser, thereby resulting in the collapse of the PNIPAM shell covering the surface of mesoporous silica and exposing the nanochannels outside, realizing the triggered release of entrapped DOX. Vivero-Escoto et al 147 synthesized a gold There are also other strategies to construct light-responsive DDS, such as grafting pore blockers using light-sensitive linkers or polymers, which suffer physicochemical changes or induced rupture under light irradiation. [148][149][150] Magnetic-responsive drug delivery Magnetic-responsive DDS relies on the delivery of magnetic and drug-loaded nanoparticles to the tumor site under the influence of external magnetic field. The external magnetic field can not only drive the magnetic nanoparticles to the desired location precisely, but also can act as an exogenous stimulus to induce the controlled drug release. Superparamagnetic iron oxide nanoparticle is one of the most widely employed magnetic particles.
As illustrated in Figure 15, Chen et al 151 constructed a novel nanocarrier (MSN@Fe 3 O 4 ) using a facile technology by capping amine-modified MSN with 2,3-dimercaptosuccinic acid-functionalized Fe 3 O 4 nanoparticles through chemical amidation. In the absence of magnetic field, a negligible amount of the drug was released from the MSN@Fe 3 O 4 . However, some nanocaps can be removed by breaking the chemical bonds when subjected to an external controllable magnetic field, which subsequently leads to a fast drug release. Also, the results showed that the release profiles were dependent on the strength and time duration. Moreover, MSN@Fe 3 O 4 nanocarriers could perform well as T 2 -weighted magnetic resonance contrast enhancement agents for molecular imaging.
Magnetic field-triggered drug release is usually dependent on the temperature, which is based on magnetic nanoparticleembedded MSN that is capable of generating thermal energy under an external magnetic field so as to induce the conformational changes of the thermo-sensitive materials capped on the surface of MSN. Baeza et al 152

105
MSNs for stimuli-responsive controlled drug delivery magnetic-responsive nanodevice that is able to control the release of small molecules and proteins in response to an alternating magnetic field. This device was based on iron oxide-encapsulated MSN and surface decorated with a temperature-sensitive NIPAM-based polymer. The copolymer was designed to act as thermo-responsive gatekeeper for the drug-loaded MSN and the release of the encapsulated drug could be modulated by the duration of the alternating magnetic field on-off states, which affects the shrinkage of the mesh size and the recovery of polymer. Cargos could be entrapped into nanocarriers at temperatures below LCST and released at temperatures above LCST. Under alternating magnetic field, the quickly enhanced temperature induced the conformational change of the surface-coated polymer to attain a more hydrophobic property thereby leading to a rapid release of the entrapped cargos. Ruiz-Hernández et al 154 also reported a magneticresponsive DDS through double-helix DNA strand selfassembly to cap the nanochannels. Single-stranded DNA was immobilized onto the surface of thiol-/disulfide-functionalized MSN in which iron oxide superparamagnetic nanoparticles were encapsulated. The magnetic MSN was loaded with fluorescein as model drug molecule and subsequently capped with the complementary DNA strand. When exposed to an alternating magnetic field of 24 kA⋅m -1 and 100 kHz, the fluorescein-loaded particles could quickly heat the environment to reach hyperthermia in a few minutes, so as to induce progressive double-stranded DNA melting, giving rise to uncapping and subsequent drug release. Therefore, this novel strategy could play an important role in the development of thermo-and magnetic-responsive DDS against cancer.
Many other temperature-sensitive materials are also used as gatekeepers, such as lipid bilayer 155 and pseudorotaxanes, 39 and the heat generated from alternating magnetic field induced the disassembly of the blockers and sustained drug release.

Ultrasound responsive drug delivery
Ultrasound is one of the most promising exogenous stimuli for drug delivery with the advantages of noninvasiveness, ability of deep tissue penetration, and controllable frequency. 156 Moreover, ultrasonic irradiation could enhance drug release rate from both biodegradable and non-biodegradable polymer matrices. 157 Paris et al developed a new ultrasound-responsive system based on MSN for controlled drug release. 158 As illustrated in Figure 16, the temperature-ultrasound dual responsive random copolymer p(2-(2-methoxyethoxy) ethyl methacrylate-co-tetrahydropyranyl methacrylate) was grafted on the MSN surface to act as gatekeeper, where 2-(2methoxyethoxy)ethyl methacrylate is a kind of temperatureresponsive monomer and tetrahydropyranyl methacrylate is a kind of ultrasound-responsive monomer. The temperature conversation is an on-off switch for model drug loading. At 4°C, 2-(2-methoxyethoxy)ethyl methacrylate showed a hydrophilic coil-like conformation and the model drug molecules could be diffused into the open nanopores, whereas at a temperature of 37°C, the hydrophobic polymer tightly collapsed onto the MSN to block the drug molecules in the nanopores. Upon ultrasound irradiation, the sensitive polymer changes its hydrophobicity and conformation toward coil-like gate-opening and cargo-releasing. Upon

Cargo loading
Cargo transport

106
Song et al ultrasound irradiation with a frequency of and 1.3 MHz and power of 100 W at 37°, the hydrophobic tetrahydropyranyl methacrylate groups were hydrolyzed into hydrophilic methacrylic acid and methacrylic acid groups. This polarity change provokes the opening of the gates of the mesoporous channels resulting in a release of drug molecules. Kim et al 159 reported a silica nanoparticle-based DDS that could be triggered by ultrasound. The system was composed of mesoporous silica as the drug reservoir and poly dimethylsiloxane (PDMS) as gatekeeper to suppress the initial fast release. When the system was exposed to ultrasound for 10 minutes, sustained drug release was observed. These data confirmed that these hybrid MSNs can act as effective systems for ultrasound-responsive drug delivery.

Electro-responsive drug delivery
Weak electric fields can be used to achieve pulsed or sustained drug release through a variety of actuation mechanisms with the advantages of simplicity, accurate dosage control, and easy coupling to bioelectronics. For instance, nanoparticles based on conductive polypyrrole exhibited tailored drug-release profiles as a result of a synergistic process of electrochemical reduction-oxidation and electric-field-driven movement of charged molecules. 160 Zhao et al 161 developed a tunable pH and electro-responsive drug release system based on chitosan-capped MSN. IB-loaded MSNs (IB-MSNs) were dispersed in chitosan solution and co-deposited with chitosan hydrogel on a titanium plate. The co-deposition was performed by immersing a titanium plate (as a cathode) and a platinum wire to the chitosan solution (pH ~5) containing IB-MSN. After removal of the titanium plate from the chitosan solution, an opaque chitosan hydrogel with IB-MSN could be observed on the titanium plate and the stimuli-triggered IB release from chitosan/MSN complex hydrogel was studied. Results showed that the release of ibuprofen could be activated by applying a cathodic voltage (~ -5.0 V) to the titanium plate, resulting in a burst drug release and ~95% of the drug molecule was released in the first 3 hours. Also, this system showed a non-acid-dependent release manner, and a pH value of 7.4 and 10.0 accelerated the drug release, respectively. Li et al 162 established a dual thermo-and electro-responsive DDSs by coating functional macromolecules onto the outlets of MSN. The thermo-responsive moiety N-isopropylacrylamide was copolymerized with the electro-responsive moiety 4-nitrophenyl methacrylate (NPMA) to form a macromolecular coating on the surface of MSN. When exposed to an external electric field, the electro-sensitive NPMA could rotate and reorient its conformation, resulting in an increased drug release no matter whether the surrounding temperature was below or above the LCST.
Electrostimuli provide a new option in the design of the stimuli-triggered drug release and shows a promising potential in cancer therapy.

Conclusion
In this review, we have highlighted the advanced progress on mesoporous silica-based materials as stimuli-responsive controlled-release systems. The application of SiO 2 particles in the preparation of stimuli-responsive DDS is mainly based on their biocompatibility, large surface area, high drug-loading capacity, nontoxicity, easy functionalization, and pore volume modulation. Moreover, it is simple to functionalize the surface of MSN with polymer/molecular ensembles to develop gated DDS. These smart DDS can be delivered into targeted cells and released drugs in some controlled manner by the exogenous stimuli including temperature, light, magnetic field, ultrasound, and electricity, as well as internal stimuli such as pH, ATP, GSH, enzyme, glucose, and H 2 O 2 . The application of MSN-based DDS shows great promise in biomedical applications such as bioimaging, disease diagnosis, and treatment which serves the precision and personalized therapy with high efficiency.