PH-responsive Multifunctional Nanoparticles For Imaging And Treatment Of Acute Ischemic Stroke

Stroke is the second leading cause of death globally and the most common cause of severe disability.Ischemic stroke predominates in stroke cases,accounting for approximately 80%of all strokes.Patients with ischemic stroke do not benefit very well from revascularisation due to the limited time window for stroke and the low recanalization rate.Neuroprotective drug therapy,another essential treatment for ischemic stroke,is not ideal for stroke due to the non-specific accumulation of drugs,BBB permeability,solubility,and rapid metabolism.With the development of nanomedicine,drug delivery has been greatly improved.These nanocarriers demonstrate exciting benefits in targeted delivery,controlled release,and even great potential for diagnostics and tracking disease progression.However,the need for more precise drug release into pathological areas and more accurate real-time drug tracing and monitoring of lesion area changes is still critical to be overcome.Therefore,innovative techniques and approaches are still necessary to improve the diagnosis and treatment of ischemic stroke.At present,some studies have designed a series of environmentally responsive nano-materials based on the pathophysiological characteristics of cerebral ischemia,such as p H and ROS responsiveness.Developments in this field have led to more accurate treatment for ischemic stroke.Therefore,based on the microenvironment characteristics of the disease,this study aimed to develop a bioresponsive multifunctional theranostic nanoplatform with“on-demand”drug release and signal amplification,improving the shortcomings of traditional neuroprotective drugs and overcoming the limitations of single imaging.The platform enables drugs to be released at therapeutically relevant concentrations and real-time,accurate monitoring of nano-drug distribution in the body to be implemented by using magnetic resonance（MR）and near-infrared fluorescence（NIRF）imaging.The development of this system provides a scientific basis for assessing drug efficacy and the development of individualized treatment protocols in the future.At the same time,they may also become a more accurate method for in vivo imaging and generalized treatment of other diseases with similar microenvironmental characteristics.ChapterⅠPreparation and functional evaluation of p H-responsive rapamycin-loaded multifunctional nanoparticlesObjective:Achieving more precise drug release into pathological regions and reducing the side effects of off-target areas,more accurate real-time drug tracing and monitoring changes in the pathological areas remain critical to be overcome in disease treatment.The present study aimed to develop a multifunctional theranostic nanoplatform with microenvironment response to realize targeted drug release and precision imaging.Materials and Methods:Considering that specific disease tissues（e.g.,brain ischemia,tumors,etc.）present a low p H acidic microenvironment compared with normal tissues,a p H-sensitive theranostic nanoparticle system was designed to diagnose and treat diseases with a similar pathological microenvironment.The system was characterized by effective RAPA loading,“on-demand”acid-triggered drug release,and drug distribution monitoring.First,p H-sensitive amphiphilic block copolymer（m PEG-b-P（DPA-co-HEMA）-Ce6）was prepared by RAFT polymerization and post-modification.p H-sensitive PDPA segment was used to achieve acid-triggered drug release,and chlorin e6（Ce6）was chosen as a near-infrared fluorescence imaging agent and gadolinium（III）(Gd³⁺)chelator for dual-modal imaging（MRI and NIRF）.The RAPA/Gd³⁺-loaded nanoparticles(denoted as RAPA/Gd³⁺@NPs)were prepared by self-assembly of m PEG-b-P（DPA-co-HEMA）-Ce6 in the presence of RAPA and Gd³⁺.Then,the nanoparticles were characterized by transmission electron microscopy（TEM）and dynamic light scattering（DLS）.The acid-triggered drug release profile and dual-modal imaging properties of nanoparticles were validated in vitro,and their biocompatibility and cell uptake were assessed.Results:The nanoparticles demonstrated good stability and biocompatibility and could efficiently load rapamycin,followed by its rapid release in acidic environments.The result showed more than 80%of RAPA was rapidly released at p H 6.0 within four hours.The system also exhibited acid-enhanced MRI and NIRF signal properties.we observed a longitudinal correlation coefficient（r₁）of 11.01 m M^-1·s^-1 at p H 6.0,which was 9-fold higher than that at p H7.4.The fluorescence intensity measured by the near-infrared imaging system gradually increased with the decrease in p H,demonstrating acid-enhanced NIRF imaging capability.The nanoparticles were effectively taken up by cells in cellular uptake experiments.Conclusion:We have successfully prepared a p H-responsive multifunctional theranostic system(RAPA/Gd³⁺@NPs).They could be a universal solution to achieve accurate in vivo imaging and treatment of other diseases.Chapter II p H-responsive theranostic nanoparticles for imaging andtreatment of acute ischemic strokeObjective:Stroke is the second leading cause of death globally and the most common cause of severe disability.Several barriers need to be addressed more effectively to treat stroke,including efficient delivery of therapeutic agents,rapid release at the infarct site,precise imaging of the infarct site,and drug distribution monitoring.The present study aimed to develop a bio-responsive theranostic nanoplatform with the signal-amplifying capability to deliver RAPA to ischemic brain tissues and visually monitor drug distribution,to make it more conducive to the management and treatment of ischemic stroke.Materials and Methods:Considering that ischemic tissues present a low p H compared with normal tissues,we applied the p H-sensitive RAPA-loaded multifunctional nanoparticle system to transient middle cerebral artery obstruction（t MCAO）rat models to validate its imaging and therapeutic capabilities.First,focal cerebral ischemia models were produced by intraluminal occlusion of the right middle cerebral artery and reperfused after two hours.RAPA/Gd³⁺@NPs were given in the caudal vein along with reperfusion.MRI and NIRF imaging were performed at defined times to examine the dual-modal imaging capability and observe their cellular localization under pathological sections.Secondly,t MCAO rats included in the treatment groups were randomly divided into sham-operated,PBS-treated,RAPA-treated,and RAPA@NPs-treated groups.At 24 hours after treatment,neurological function was evaluated by Zea Longa,tentacle-induced forelimb placement test,and foot-failure test.Apoptosis in the ischemic penumbra area was detected by TUNEL staining.The T₂-mapping,wet and dry weight methods were used to assess the brain edema.Evans blue leakage was used to determine the extent of damage to the blood-brain barrier.ELISA was used to determine the expression levels of inflammatory factors in ischemic brain tissues.Results:The multimodal imaging and pathological results confirmed that the nanoparticles significantly accumulated in the ischemic area and were internalized by cerebral cells.The T₁contrast of the nanoparticles in the cerebral ischemic hemisphere increased with injection time,reflecting the gradual accumulation of nanoparticles.The fluorescence intensity measured by the NIR system in the ischemic hemisphere was 4.10 times higher than that in the normal hemisphere,further demonstrating the effective accumulation of nanoparticles.Finally,in vivo experiments confirmed that the nanoparticles exerted a neuroprotective effect in t MCAO rats.The sensorimotor function significantly improved in the RAPA@NPs-treated group compared to the PBS and RAPA groups.In the RAPA@NPs-treated group,there were scattered TUNEL positive cells,while the PBS group appeared with numerous positive cells in the peri-ischemia area.The degree of brain edema,blood-brain barrier damage,and inflammatory factor expression levels significantly reduced in the RAPA@NPs-treated rats compared to the other treatment groups.Conclusion:We have successfully applied the theranostic system to the ischemic stroke models.MRI,NIRF imaging,and pathological results demonstrate that the nano-delivery system effectively reaches the ischemic area and is internalized by neuronal cells,indicating that the nanoparticles exert a cerebral protective effect by effectively delivering RAPA to the ischemic area.We have also used the dual-modal imaging of the nanoparticles to monitor their dynamic changes in the brain in real-time and non-invasively and respond more precisely to the location of the ischemic area,which is an essential guide to achieving individualized and precise treatment.