| Background: Stroke is one of the leading causes of death and disability in the world.Stroke is characterized by high morbidity,high recurrence rate,high disability rate and high mortality rate.There are two main types of stroke,ischemic and hemorrhagic.Acute ischaemic stroke（AIS）is the main category of stroke.Cytotoxic oedema develops,which although reversible,releasing oxygen-derived free radicals within minutes to hours.However,the irreversible vasogenic oedema peaks can precipitate raised intracranial pressure（ICP）.At present,the clinical treatment of ischemic stroke is mainly surgical and drug treatment.Surgical treatment is directly effective,but its cost and risk are high;drug treatment is mainly symptomatic treatment,including thrombolytics,anticoagulants,and neuroprotective drugs.However,existing drugs have limitations such as short therapeutic time window,central nervous system toxicity,bleeding,short in vivo circulation time,and low targeting ability.In recent years,nanoparticle（NP）-based drug delivery systems have been investigated as promising paradigms for targeted treatment of different central nervous system disorders,due to their multiple advantages such as high capacity of loading therapeutics with diverse properties,desirable permeability to the BBB,tailorable capabilities of targeting diseased sites at multiple scales,and spatiotemporally controlled on-demand therapeutic release.Taking advantage of the increased permeability of the BBB after stroke,various types of NPs without targeting modifications were used to deliver molecular payloads across the BBB via the transcellular or paracellular transport.In addition to utilizing passive targeting effects,ligand-decorated NPs have been examined to further enhance targeting efficiency.On the other hand,the components of nanovehicle-forming materials can be tailored to release loaded drug molecules in response to pathologically relevant molecular signals.Therefore,NPs based on regulatable responsive materials are still necessary to enable site-specific drug release performance under the pathological microenvironment of ischemic stroke.The above NPs is expected to provide a new therapeutic strategy for ischemic stroke.Based on the above analysis and in view of the crucial role of inflammation and oxidative stress at the acute stage of ischemic stroke,herein we designed and developed multiple-targeting NPs based on a cyclodextrin-derived ROS-responsive material.Herein,ROS responsive group,phenylboronic acid pinacol ester（PBAP）,was used as functional unit and was respectively bonded to β-cyclodextrin（β-CD）to obtained OCD.Then we combined the stroke homing peptide（SHp）with OCD,and then developed an NBP-loaded brain multiple-targeted NBP nanotherapy for site-specific delivery to the in ischemic site in AIS.Both in vitro and in vivo studies were executed to explore the targeted therapeutic effects and the underlying mechanisms of this NBP-derived multiple-targeting nanoplatform.Methods: 1.Synthesis of a ROS-responsive material OCD based on β-cyclodextrin Specifically,PBAP was activated by CDI.Then CDI-activated PBAP and β-CD were dissolved in anhydrous DMF,the obtained mixture was precipitated by anhydrous ether and finally to give a ROS-responsive material OCD.1H NMR spectroscopy,Matrix-assisted laser desorption/ionization time-of-flight（MALDI-TOF）mass spectra,and Fourier transform-infrared（FT-IR）spectroscopy was recorded to characterize OCD.2.Fabrication of nanoparticles by a nanoprecipitation/self-assembly method Briefly,OCD was dissolved in methanol,the obtained solution was added dropwise into deionized water containing DSPE-PEG and lecithin.After vortexing,the blank OCD nanoparticles（OCD NP,defined as OCN）were harvested by lyophilization.A similar method was employed to prepare the NBP-loaded PLGA or OCD nanoparticles（NBP/PLGA NP or NBP/OCD NP,defined as NPN or NON）.First,PLGA or OCD and NBP were co-dissolved in methanol,the subsequent method is the same as the preparation of OCN.Using the stroke targeting peptide containing free sulfhydryl and maleimide unit contained in DSPE-PEG-MAL,the targeting unit DSPE-PEG-SHp was constructed by Michael addition reaction;brain targeted OCD nanoparticles（SOCD NP,defined as SON）and NBP-containing brain targeted OCD nanoparticles（NBP/SOCD NP,defined as NSN）based on DSPE-PEG-SHp were produced as the synthesis of NON.3.Characterization of nanoparticles The particle size,size distribution profiles,and δ-potential values of different NPs were measured on a Malvern Zetasizer Nano ZS instrument at 25℃.The morphology of NPs was observed by transmission electron microscopy（TEM）and scanning electron microscopy（SEM）.4.Hydrolysis of nanoparticles under different solutions In brief,newly prepared OCN were dispersed in PBS containing various concentrations of H2O2 and incubated for varied time periods.The degree of hydrolysis was calculated based on the transmittance values.In a separate experiment,PLN and OCN were separately dissolved in PBS containing H2O2 for varied time periods.The size distribution profiles,particle size,and δ-potential values changes were measured on a Malvern Zetasizer Nano ZS instrument.5.Quantification of drug loading contents and entrapment efficiency of the NBP-loaded NPs（NPN,NON,NSN）To quantify the NBP loading content,the NBP-loaded NPs（NPN,NON,NSN）was separately dissolved in methanol.The concentration of NBP dissolved in methanol was quantified by high performance liquid chromatography（HPLC）.6.In vitro drug release profiles Aqueous solution containing NBP-loaded NPs was placed into dialysis tubing,which was immersed in PBS with or without H2O2.The NBP concentration was quantified by HPLC.7.Cellular uptake profiles of NPs in PC12,BV2,BEND3,and primary astroglial cells BEND3 cells were seed plates and incubated with PLN or OCN,followed by incubation at 37℃ for different time.Fluorescence images were acquired by confocal laser scanning microscopy.In addition,the fluorescence intensity of Cy5 in BEND3 cells was also analyzed by flow cytometry.By similar methods,we also investigated the time-dependent and dose-dependent experiments of OCN or SON uptake by PC12,BV2 cells and primary astrocytes.8.In vitro anti-proliferative activity of various nanotherapies in cells PC12,BV2 cells,and primary astroglia were separately seeded in plates and treated with different doses of NON or NSN.After incubation for various time periods,cell viability was determined by CCK-8 assay.9.In vitro targeting capability of NPs in cells BEND3 cells were separately seeded into plates and were induced with H2O2.PC12 and BV2 cells were separately seeded plates and were induced with（the model group）or without（the normal group）CoCl2.Then cells were incubated with PLN or OCN.Fluorescence images were acquired by confocal microscopy,and the fluorescence intensities were determined by flow cytometry.BEND3,PC12 and BV2 cells were seeded into plates and were induced with CoCl2.Subsequently,the cells were stained with DCFH-DA for.CLSM was performed to acquire fluorescence images to determine intracellular ROS Level.In a separate experiment,BEND3 cells were induced with CoCl2 as aforementioned.Subsequently,cells were incubated with PLN or OCN,followed by staining with DiO.CLSM was performed to observe the interaction of BEND3 cells membrain and NPs.In a separate study,PC12 and BEND3 cells were induced with CoCl2.Then cells were incubated with OCN or SON.Fluorescence images were acquired by confocal microscopy,while fluorescence intensities were determined by flow cytometry.Furthermore,BEND3,PC12 and BV2 cells were induced with CoCl2.After stimulation,cells were treated with SON with or without free SHp.Fluorescence intensities were determined by flow cytometry.Similarly,cellular internalization of SON with or without transferrin in three cells were examined by the similar methods.10.In vitro antioxidant and anti-inflammatory activities of NBP-loading nanoparticles PC12 cells were induced with glutamic acid and treated with different concentrations of NBP nanotherapies or free NBP and stained with DCFH-DA.Fluorescence images and quantification flow cytometry profiles were acquired.In separate experiments,the level of malondialdehyde（MDA）was determined using the corresponding kit,and the cell viability was measured by CCK-8 assay after the treatment.On the other hand,PC12 cells treated with CoCl2 to establish a model of oxygen-glucose deprivation（OGD）.After stimulation,cells were treated with different concentrations of NBP nanotherapies or NBP.The levels of tumor necrosis factor（TNF）-α,interleukin（IL）-1β,and IL-6 were separately measured by ELISA,and the cell viability was measured by CCK-8 assay.11.In vitro hemolysis assay Fresh blood was collected from male C57BL/6J mice,and suspensions of 2% red blood cells（RBCs）were prepared,which were incubated with different concentrations of OCN,NON,or NSN.The obtained supernatant was separated and the absorbance at 570 nm was measured by UV-Visible spectrophotometry.12.In vivo targeting of the ischemic infarct and distribution of NPs in MCAO mice Establishment of a mouse model of middle cerebral artery occlusion（MCAO）.First,mice were anesthetized using isoflurane.A midline neck incision was made to isolate the soft tissues.The left common carotid artery（LCCA）,left external carotid artery（LECA）,and left internal carotid artery（LICA）were isolated.Through a small incision in the middle of LECA,Nylon 6 filament was inserted into the LICA.The Nylon filament was further inserted to block the blood supply from the left middle cerebral artery（LMCA）.Mice neck wound was sutured.At 120 min post-occlusion,the Nylon filament was withdrawn to initiate reperfusion.The distribution of i.v.delivered OCN or SON in mice.The distribution of NPs in normal mice was also examined after i.v.injection of OCN or SON.At specific time points after administration of NPs,mice were euthanized.Blood samples and main organs were collected from mice.Fluorescence intensities were determined by ex vivo imaging.Evaluation of the BBB permeability by Evans blue（EB）extravasation assay.Mice were i.v.administered EB after ischemic injury.The EB levels in the brain samples were quantified using a fluorescence spectrophotometer.MCAO mice were i.v.administered with PLN or OCN.Whole brain tissues were harvested and ex vivo imaging was conducted using an IVIS Spectrum system.Fluorescence intensities were then analyzed.Furthermore,immunofluorescence analysis was performed to observe cellular adhering of NPs to the BEND3 cells.Similarly,OCN or SON was i.v.administered in MCAO mice.Fluorescence intensities of whole brain tissues were then analyzed.In a separate study,immunofluorescence analysis was performed to interrogate cellular distribution of NPs.OCN or SON was i.v.administered in MCAO mice.The brain slice was obtained as s previously mentioned.Subsequently,antibodies to CD31,Iba-1,Neu N,and transferrin receptor were separately added,followed by incubation with fluorescent secondary antibodies.After the sections were sealed with an anti-fluorescence quenching agent,they were observed by confocal microscopy.13.In vivo therapeutic effects of different formulations in mice with ischemic stroke After different treatments,mice were euthanized,and then brain slices were stained with TTC.The TTC-stained sections were photographed,and the percentage area of brain infarct was evaluated using Image J.Similarly,the brain water content（BWC）was estimated.In addition,the levels of H2O2 and MDA in the collected brains were determined using the corresponding assay kits.Also,the levels of TNF-α,IL-1β,and IL-6 were separately measured by ELISA.14.Evaluation of neurological deficits Mice were treated by the same procedures as described above.Neurological deficits were scored.The behavior analysis of a limb placement test was carried out at specific time points during of treatment.15.Therapeutic effects of the active targeting nanotherapy in MCAO mice Mice were treated by the same procedures as described above.Then the brain infarct area and brain water content as well as the levels of H2O2,TNF-α,IL-1β,and IL-6 were measured as aforementioned.Evaluation of neurological deficits: Mice were treated as described above.Neurological deficits were scored.16.Preliminary study on potential therapeutic mechanisms Mice were treated with the above methods.The paraffin sections of brain tissues were prepared.Morphologic observation of neurons by Nissl staining,which was performed on coronal sections 2 mm behind the optic chiasm,followed by morphological observation of brain sections by optical microscopy.In addition,brain sections of 100-μm thickness were prepared.Golgi staining was performed to analyze dendritic spines.Panoramic images of brain sections were acquired by a digital slice scanner.Mice were treated with the above methods,followed by received i.v.injection of FITC-dextran.Mice were sacrificed,and the isolated brain tissues were fixed in 4% paraformaldehyde and processed for vibratome traverse sectioning.The cryosections was observed by confocal microscopy to determine the cortical cerebrovascular permeability.17.Toxicity evaluation of nanoparticles in mice After different treatments,mice were euthanized.Blood samples were collected for hematological and biochemical analyses.The organ index was calculated as the ratio of organ weight to the body weight of each mouse.Histopathological sections were prepared and stained with H&E.In addition,Male C57BL/6J mice were treated with different doses of NON by i.v.injection via the tail vein.After administration,the body weight of mice and their behaviors were monitored for 2 weeks.Blood samples were collected for hematological and biochemical analyses.The organ index was calculated as the ratio of organ weight to the body weight of each mouse.Histopathological sections were prepared and stained with H&E.Results: 1.Preparation of ROS-responsive nanoparticles A ROS-responsive material OCD was synthesized by covalently conjugating PBAP onto a cyclic oligosaccharide β-CD,which was characterized by FTIR,1H NMR,and MALDI-TOF mass spectrometry.In addition,PLGA was used as a control material.OCN were prepared by a modified nanoprecipitation/self-assembly method.Similarly,PLN were fabricated.Regardless of different materials,spherical NPs were obtained,as implicated by TEM and SEM observation.The mean diameter was 143 ± 4 and 120 ± 2 nm for PLN and OCN,respectively.Both PLN and OCN displayed negative Zeta-potential.2.Cellular uptake of ROS-responsive nanoparticles Both PLN and OCN showed rapid and time-dependent cellular internalization in BEND3 cells.At examined time points,OCN exhibited significantly higher internalization than PLN,in the presence of H2O2.In neurons and microglial cells,OCN showed effective cellular internalization in both time and dose dependent manners.Further,we also observed significantly enhanced cellular internalization of OCN in PC12 neuron-like cells and BV2 murine microglial cells under oxygen-glucose deprivation（OGD）.3.In vitro targeting of brain-targeted nanoparticles We investigated the cellular uptake ability of PLN and OCN in BEND3 cells.Fluorescence intensity of the OCN group was higher than that of the PLN group in the model BEND3 cells.While there was no difference between PLN and OCN groups of the normal BEND3 cells.Subsequently,we compared the cellular uptake of PLN and OCN in the normal and the OGD PC12 cells.Fluorescence intensity of the OCN group was higher than that of the PLN group in the OGD PC12 cells.Similarly,OCN can also be uptaken more effectively in the OGD BV2 cells than the PLN.While there was no difference between PLN and OCN groups of the normal PC12 and BV2 cells.In view of the experimental results,compared with the PLN,the ROS-responsive nanoparticles OCN can accumulate more effectively on BEND3 cells under oxidative stress and the ischemic injury nervous cells.We then explored mechanisms responsible for enhanced cellular uptake of OCD by stroke-relevant cells under pathological conditions.First,we obtained OCD and the hydrolysates of OCD incubated in PBS with H2O2.According to the NMR spectrum revealed hydrogen peroxide-triggered removal of pinacol from PBAP units of OCD.The FT-IR spectrometry shows that PBAP units was removed from β-CD.The OCN displayed inhomogenous size distribution and notably decreased negative zeta-potential value,which was upon exposure to hydrogen peroxide.While the PLN had no obviously changes.To evaluate the targeting efficiency binding to BEND3 cells of OCN,we further assessed the interaction of BEND3 cells membrain and OCN.We found that BEND3 cells membrain and OCN co-localize.However,there was no significant cellular interaction of membrain and PLN.4.Effective in vivo targeting capability of OCN in a MCAO mice First,we evaluated the BBB permeability by EB extravasation assay.At 2 h after ischemic injury,EB-treated MCAO mice showed notably higher accumulation of EB in the brain,compared to the sham mice.At 24 h after MCAO,we still observed considerable accumulation of EB.This result implied that MCAO-induced opening of the BBB can maintain for at least 24 h.Indeed,after i.v.injection of PLN or OCN in MCAO mice,ex vivo imaging of brain tissues revealed that OCN displayed significantly higher fluorescence intensities relative to PLN.Accordingly,these results suggested that the OCD-derived ROS-responsive nanoparticles OCN can more effectively accumulate in the brain tissue of MCAO,compared to PLN.To further confirm the transportation way on BEND3 cells of nanoparticles,we performed immunofluorescence analysis by staining with brain sections with CD31 in MCAO mice after injection of PLN or OCN.The co-localization of endothelial cells and nanoparticles could be evidently observed in the brain tissue of OCN group,compared to those of the PLN group.5.Engineering of NBP-derived nanotherapies for targeted treatment of ischemic strokeWe subsequently developed a control nanotherapy of NBP-loaded PLN（NPN）and NBP-loaded OCN（NON）.Both NON and NPN showed well-defined spherical morphology.The mean diameter was 156 ± 3 and 128 ± 3 nm for PLN and OCN,respectively.The loadings of NBP in NPN and NON measured by HPLC were separately 7.0 ± 0.2% and 8.1 ± 0.3%.In vitro release tests indicated that NON displayed a rapid hydrolysis profile in PBS containing H2O2.As for NPN,relative slow release profiles were observed.Accordingly,NBP release from NON could be selectively triggered by ROS,which was associated with ROS-responsive hydrolysis of OCN.6.In vitro bioactive effects of NBP nanotherapies in PC12 cells We evaluated the neuroprotective effect of NBP nanotherapies in neurons induced with glutamate.NON treatment most effectively reduced intracellular ROS levels in PC12 cells.Consistently,NON can significantly attenuate glutamate-induced the level of MDA and cell death of neurons.In addition,NON inhibited the expression of typical inflammatory cytokines,i.e.,TNF)-α,IL-1β,and IL-6 in OGD PC12 cells.7.In vivo neuroprotective effects of NBP nanotherapies in MCAO mice We examined therapeutic effects of different NBP nanotherapies in mice with MCAO-induced AIS.NON can significantly reduced the infarct size,water content,typical oxidative mediators（H2O2 and MDA）and pro-inflammatory cytokines（TNF-α,IL-1β,and IL-6）were also significantly decreased after therapy with different NBP formulations.NON notably decreased neurologic deficits,promoted better functional recovery of the mice.8.Engineering of an OCN-derived multiple-targeting nanoplatform for stroke SON was prepared by nanoprecipitation/self-assembly.The mean diameter of SON was 127 ± 4 nm.Subsequently,in vivo targeting capability of SON was examined in MCAO mice,using OCN as a control.After injection of OCN or SON,ex vivo imaging of the brain tissues showed significantly higher fluorescent signals in the SON group compared with the OCN group.Furthermore,fluorescence intensities of the ipsilateral brain tissue of SON-treated MCAO mice were significantly stronger than that of the corresponding contralateral brain tissue.Then,we compared the cellular uptake of OCN and SON in the nervous system cells.Fluorescence intensity of the SON group was higher than that of the OCN group in model PC12 and BEND3 cells.We further examined in vitro targeting capability of brain-targeted nanoparticles in cells subjected to hypoxic/ischemic injury.For injured PC12 and BEND3 cells,fluorescence intensities of incubated with SON mixed with free SHp or transferrin significantly reduced compared to those incubated with SON alone.Moreover,we examined cellular distribution of SON by immunofluorescence analysis.For MCAO mice treated with OCN or SON,co-localization of Cy5 fluorescence with neuron,microglia,or brain microvessel endothelial cells could be observed in the ipsilateral brain.In particular,the SON group showed notably higher ipsilateral accumulation and co-localization,compared with the OCN group.Consequently,further studies were performed to examine possible mechanisms underlying this endothelial cell targeting effect.We found that SON showed notable co-localization with the Tf R in the brain tissue of MCAO mice,in comparison to that of the OCN group.We also found that the brain tissue of MCAO mice exhibited more notable co-localization between endothelial cells with Tf R,compared to that of sham mice,indicating that the expression of Tf R on endothelial cells was notably increased under ischemic conditions.9.Development of a multiple-targeting NBP nanotherapy for the treatment of stroke NBP-loaded SON（NSN）was also prepared and displayed spherical shape,with mean diameter of 141 ± 3 nm.The NBP content was 7.5 ± 0.5% in NSN.In addition,NSN showed good stability in different media and after incubation in different media for various time periods.Also,NSN exhibited a notable ROS-responsive release profile.We then conducted in vivo therapeutic effects in MCAO mice.NSN significantly reduced infarct area,inhibited brain edema,as well as more significantly attenuated oxidative stress and inflammatory responses in the brain tissue.NSN could also effectively reduce morphological alterations of neurons in the damaged brain region and improved the dendritic spine density and the structure of dendritic trunks.And treatment with NSN more significantly reduced fluorescence intensities of FITC-dextran in brain tissues.NSN can significantly decreased deficit scores and promoted better functional recovery of the mice.10.Safety studies First,cytotoxicity of NON and NSN was tested in PC12,BV2,and primary astroglial cells.After incubation,relatively high cell viability was observed regardless of various doses of NON or NSN.Also,there was no significant in vitro hemolysis was observed in either NON or NSN.Secondly,in vivo toxicity test shouwed that there was no significant difference in body weight,organ index,Typical hematological parameters and biomarker between all treatment groups and normal mice.Examination on H&E-stained sections of major organs from administered mice indicated that there were no pathological patterns.Conclusions: 1.we found that NPs（i.e.,OCN）based on a cyclodextrin-derived ROS-responsive material showed significantly increased targeting capability to the ischemic brain tissue,largely resulting from enhanced endocytosis by brain microvascular endothelial cells.2.By loading a candidate drug NBP into OCN,the engineered nanotherapy NON with surface chemistry transformable capability showed notably enhanced neuroprotective effects in a mouse model of acute ischemic stroke.3.Targeting efficiency of OCN for the cerebral ischemia/reperfusion injury site can be further improved by decorating with a stroke-homing peptide SHp.Besides previously documented neuron-targeting capability of SHp,we discovered that SHp-functionalized OCN（i.e.,SON）can be transported by transferrin-mediated endothelial endocytosis,thereby leading to an additional active targeting effect in MCAO mice.4.The NBP nanotherapy based on the triple-targeting nanovehicle SON exhibited a more potent neuroprotective activity than the non-functionalized nanotherapy NON at a 5-fold higher dose,likely due to considerably enhanced site-specific NBP delivery in the injured brain tissue.5.Preliminary experiments also indicated that NBP nanotherapies displayed good safety for i.v.treatment,and therefore they can be further developed as efficacious and safety medications for targeted treatment of ischemic stroke. |
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