The Construction Of Clot-specific Nanoparticles And Their Theranostics On Arterial Thrombosis

Atherothrombosis（AS）is an ischemic vascular disease triggered by the sudden rupture of atherosclerotic plaque,resulting in platelet activation and thrombosis.AS is one of the most serious public health problems.The resulting fatal vascular diseases caused by local occlusion or distal embolism are named atherosclerotic thrombotic diseases,such as myocardial infarction,stroke,acute ischemia in lower limbs.Thrombus refers to the formation of clots made from blood components such as activated platelets,fibrin,coagulation factors and red blood cells in the heart or vessels.Usually,the coagulation system and antagonistic fibrinolytic system in the blood maintain a dynamic balance.When the endothelia are damaged,the blood flow slows down or the blood coagulability increases,the balance is broken and the coagulation process occurs.The coagulation of blood in the vascular lumen results in numerous fatal thrombotic diseases.Cardiovascular diseases,with higher fatality than other diseases such as tumor or AIDS,have become the“No.1 killer”threatening human health.Therefore,the exploitation of theranostic agents for thrombosis has huge market potential.Generally,high doses are needed for traditional fibrinolytic or antiplatelet drugs to exert their antithrombotic effect,and the hemorrhagic side effect of high doses limits their clinical application.Therefore,we intend to administer pharmaceutical approaches and design reasonable nanoscale drug delivery system based on the pathological characteristics of thrombosis to solve this problem.The designed system should have thrombus-targeting function and intelligent imaging performance,realizing improved antithrombotic effect and the clot-specific imaging.It is a potential strategy to design a specific nanotheranostic system,but the establishment of such a system is one major difficulty at present.It is reported that one of the main components within thrombus is fibrin.The activated factor XIII（FXIIIa）,a biomarker of thrombosis,can cross-linkαandγchains of fibrin and play a vital role in stabilizing newly formed thrombus.Therefore,in our first part,we focused on the construction of a functionalized polymeric hybrid micelle（FPHM）system based on fibrin stabilizing factor and its theranostics on thrombosis.The FPHM system,self-assembled from the amphiphilic polycaprolactone-polyethyleneimine（PCL-PEI）and polycaprolactone-polyethylene glycol-carboxyl（PCL-PEG-COOH）,was modified with FXIII peptide via amido bond.IR780molecules were entrapped in the hydrophobic core via hydrophobic interaction,while acidic protein drugs were adsorbed on the cationic hydrophilic shell via electrostatic interaction.The co-delivery of lumbrokinase（LK）and near-infrared IR780 can achieve the aim of specific imaging and effective thrombolysis within embolized vessels.LK is the most easily available and cheapest fibrinolytic agent.It can digest fibrin directly or catalyze transformation of plasminogen to plasmin,decomposing insoluble fibrin into soluble fibrin degradation products such as D-dimer.However,the clinical use of lumbrokinase is limited by the narrow therapeutic window and severe bleeding consequences.To overcome this defect,we encapsulate LK into cationic hydrophilic shell by electrostatic interactions,and the FXIII peptide on the surface of the system can actively bind to clots by ligand-receptor interaction,improving the local LK concentration and fibrin-dissolving effect within damaged vessels.In addition,photoacoustic imaging technique detects ultrasonic signals and reflects the difference of energy absorption.Deep vessels can be imaged with high-resolution and high-contrast under the excitation of laser through intravenous injection of NIR probes,but their imaging specificity needs to be improved.Therefore,IR780was physically embedded in the hydrophobic core of the functionalized polymeric hybrid micelles.In a ferric chloride（Fe Cl₃）-induced mouse carotid thrombosis model,IR780/FPHM/LK nanoparticles can target early thrombus actively and specifically,accompanied by the amplified photoacoustic signal,enhanced thrombolytic effect within damaged vessels,elevated D-dimer level in plasma and reduced nonspecific bleeding risk.Due to its clot-specific imaging and therapeutic capability,this multifunctional nanoagent has certain potential for the theranostics on thrombosis.In our first part,the designed nanotheranostic system has obvious drawbacks.（1）We found that PEI segment within the carrier has certain hemolytic risk,hepatotoxity and pulmonary toxicity.（2）The electrostatic interactions between acidic proteins and carriers are unstable in circulation.（3）The hemoglobin signal overlapped with the IR780 signal to some extent,suggesting the poor imaging specificity based on peptide modification and IR780.Therefore,in our second part,we focused on how to further improve the clot-specific imaging and antithrombotic capability of nanoparticles.Besides,carriers with better biocompatibility and stimuli-sensitive bonds are better choices.Inspired by the large number of reactive oxygen species（ROS）produced during the thrombosis process,a ROS-triggered nanoscale system can be developed.ROS play a vital role in platelet recruitment and activation.ROS level correlated positively with platelet activity.When ROS level increases,platelets can benefit from ROS and maintain an active state.When ROS level is reduced by drug administration,the thrombosis process is inhibited.Researchers found previously that fucoidan extracted from brown algea had a high affinity for P-selectin expressed on activated platelets by mimicking the structure of P-selectin glycoprotein ligand 1（PSGL-1）.Therefore,we introduced the hydrophobic phenylboronic ester side chain into hydrophilic fucoidan/maltodextrin mixture to synthetic amphiphilic ROS-sensitive PHB-Fuco/Malto material.Besides,a ROS-sensitive signal“off-on”near-infrared probe CyBA was synthesized.CyBA can be physically embedded into hydrophobic core of the nanoparticle via hydrophobic effect to build a ROS-responsive theranostic CyBA/PFM NPs based on P-selectin.CyBA/PFM NPs can identify thrombus with high-expressed P-selectin.After targeting to the thrombus actively through ligand-receptor interaction,the nanoparticles depolymerized due to high ROS level at the thrombosis site,scavenged ROS and inhibited thrombosis.At the same time,the ROS-sensitive probe was transformed from the non-fluorescent“CyBA”form to the fluorescent“Cy OH”form,enhancing photoacoustic signal under laser excitation and achieving more accurate imaging of thrombi.In a Fe Cl₃-induced mouse carotid thrombosis model,oxidizable CyBA/PFM NPs could exert antioxidant,antiplatelet and antithrombotic effects within embolized vessels,accompanied by the amplification of photoacoustic signal.These results suggest that CyBA/PFM NPs have great translational potential as a nanotheranostic agent for H₂O₂-related cardiovascular diseases.In summary,in our first part,from the perspective of dissolving fibrin skeleton of the clot,a functional polymeric hybrid micelle system based on fibrin stabilizing factor was constructed and its thrombolytic effect was preliminarily studied.The photoacoustic temography of damaged vessels was achieved by embedded IR780 in hydrophobic core,and the fibrin degradation effect was achieved by lumbrokinase adsorbed on cationic shell.The theranostics of the nanoparticles was initially verified in a murine model of carotid thrombosis.In our second part,from the perspective of inhibiting the platelet activation process,a H₂O₂-triggered“off-on”theranostic nanoparticles based on P-selectin was constructed to achieve accurate diagnosis and treatment of arterial thrombosis.The natural macromolecule fucoidan can actively target to P-selectin highly expressed on activated platelets,and phenylboronic ester bond can be oxidized to eliminate ROS,inhibiting platelet activation and aggregation.These results suggest that functionalized nanoscale theranostic systems are potential tools for arterial thrombosis.