Multifunctional nanoformulation with thrombin targeting and method of preparation thereof

Abstract

This invention provides a multifunctional nanoparticle formulation with thrombin targeting capability, comprising a gold nanoparticle matrix, a paramagnetic small molecule, and a thrombin-targeting nucleic acid aptamer. The thrombin-targeting nucleic acid aptamer endows the nanoparticle formulation with thrombin-targeting binding ability; the paramagnetic small molecule endows the nanoparticle formulation with T1-weighted magnetic resonance imaging capability and reactive oxygen species scavenging capability; the gold nanoparticle matrix endows the nanoparticle formulation with CT imaging capability and near-infrared photothermal conversion capability; the nanoparticle formulation can achieve thrombin-dependent enrichment within the thrombus.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a multifunctional nano-formulation with thrombin targeting, its preparation method, and its application in the diagnosis and treatment of thrombosis, especially suitable for the identification of thrombus active centers, bimodal imaging diagnosis, photothermal thrombolysis, and synergistic antithrombotic therapy. Background Technology

[0002] Thrombotic diseases (including ischemic stroke, myocardial infarction, pulmonary embolism, etc.) are the leading causes of disability and death among cardiovascular diseases worldwide. Their core pathological mechanism is the formation of abnormal thrombi in blood vessels, which obstructs blood flow and causes ischemia and hypoxia in downstream tissues, seriously threatening the life and health of patients.

[0003] Currently, the clinical diagnosis of thrombosis mainly relies on imaging techniques such as computed tomography angiography (CTA), magnetic resonance angiography (MRA), and digital subtraction angiography (DSA). These techniques can only determine the anatomical location of the thrombus by detecting vascular filling defects or blood flow obstruction, but cannot identify the active state inside the thrombus. Thrombus activity can help in formulating treatment plans and assessing prognosis. Old thrombi with high thrombin activity are thrombolytic resistant and have poor efficacy with conventional treatment.

[0004] In terms of treatment, mainstream thrombolytic drugs in clinical practice have drawbacks such as poor thrombus targeting, short circulation time in the body, and high risk of bleeding. They are also difficult to overcome the antifibrinolytic mechanism of hyperthrombin thrombi, which can easily lead to re-occlusion. At the same time, existing treatment methods cannot monitor the amount of drug accumulation at the thrombus site in real time, making it difficult to achieve precise thrombolysis.

[0005] In recent years, researchers have developed a variety of thrombus-targeting imaging probes. Among them, thrombin-responsive probes can assess thrombus activity, but near-infrared fluorescent probes have the problem of limited tissue penetration depth and are only applicable to superficial thrombi, which cannot meet the clinical diagnostic needs of deep vascular thrombi. At the same time, existing probes are difficult to achieve in situ accurate quantification of drug concentration within thrombi, and cannot provide effective guidance for thrombolytic therapy.

[0006] Thrombin is a core biomarker for assessing thrombus activity, directly reflecting the potential for thrombus formation and growth; however, its accurate in-situ detection remains a challenge. Current technologies cannot simultaneously achieve integrated diagnosis and treatment of thrombus activity centers, precise dual-modal imaging (CT and MR), intrathrombus drug quantification, and photothermal thrombolysis. Therefore, developing a novel nano-therapeutic agent that combines thrombin targeting, dual-modal imaging, precise quantification, and synergistic thrombolysis is urgently needed for the precise diagnosis and treatment of thrombotic diseases. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a multifunctional nanoparticle formulation with thrombin-targeting properties. The nanoparticle formulation comprises a gold nanoparticle matrix, paramagnetic small molecules, and a thrombin-targeting nucleic acid aptamer. The thrombin-targeting nucleic acid aptamer endows the nanoparticle formulation with thrombin-targeting binding ability; the paramagnetic small molecules endow the nanoparticle formulation with T1-weighted magnetic resonance imaging (MRI) capability and reactive oxygen species (ROS) scavenging ability; the gold nanoparticle matrix endows the nanoparticle formulation with CT imaging capability and near-infrared photothermal conversion capability; the nanoparticle formulation can achieve thrombin-dependent enrichment within thrombi.

[0008] In some embodiments, the gold nanoparticle matrix is ​​rod-shaped or spherical.

[0009] In some embodiments, the paramagnetic small molecule is any one of TEMPO, gadobutrol, and gadotonic acid.

[0010] In some embodiments, the nanoformulation includes a poly-T oligonucleotide sequence, which is covalently modified on the surface of the gold nanoparticle matrix via gold-sulfur bonds to form poly-T-AuNR. The paramagnetic small molecule is TEMPO, which forms a TEM-Apt conjugate with the thrombin-targeting nucleic acid aptamer via a covalent grafting reaction mediated by phosphate thioester groups. The TEM-Apt conjugate has a poly-A tail complementary to the poly-T oligonucleotide sequence and hybridizes with the poly-T-AuNR through base pairing to form TEM-Apt-AuNR.

[0011] In some embodiments, the thrombin-targeting nucleic acid aptamer has six thiophosphate groups and retains its secondary structure, and its target binding sites are the Arg-89, Lys-174, Arg-245, Lys-247, Lys-248 and Lys-252 sites of α-thrombin.

[0012] In some embodiments, the gold nanorod matrix has an average length of 50-60 nm, an average width of 10-12 nm, and an aspect ratio of 4.5-5.5.

[0013] In some embodiments, the poly-T oligonucleotide sequence is poly-T 15 sequence.

[0014] This invention also provides a method for preparing a multifunctional nanoparticle formulation with thrombin targeting, comprising: preparing a TEM-Apt conjugate: covalently grafting a thrombin-targeting nucleic acid aptamer targeting α-thrombin with a paramagnetic small molecule via a thiophosphate group to obtain a TEM-Apt conjugate; preparing poly-T-AuNR: covalently modifying a poly-T oligonucleotide sequence onto the surface of a gold nanorod matrix via gold-sulfur bonds to obtain poly-T-AuNR; and hybridization assembly: hybridizing the TEM-Apt conjugate with poly-T-AuNR via base complementary pairing to obtain the multifunctional nanoparticle formulation TEM-Apt-AuNR with thrombin targeting.

[0015] In some embodiments, the covalent grafting reaction is carried out at a temperature of 55°C for 1 hour.

[0016] In some embodiments, a salt aging method is used to achieve gold-sulfur covalent modification of the poly-T oligonucleotide sequence with the gold nanorod matrix. The salt aging method employs a NaCl gradient addition, with the final NaCl concentration in the system being 0.5 mol / L.

[0017] In some embodiments, the gold nanorod matrix is ​​synthesized using a seed growth method.

[0018] In some embodiments, the incubation time for the hybridization assembly is 24 hours.

[0019] This invention also provides the application of a multifunctional nano-formulation with thrombin targeting properties in the preparation of thrombosis diagnostic and therapeutic products. The nano-formulation includes a gold nanorod matrix, a poly-T oligonucleotide sequence, and a TEM-Apt conjugate. The thrombosis diagnostic and therapeutic products include one or more of the following: CT contrast agents, MR contrast agents, thrombus active site recognition reagents, photothermal thrombolytic reagents, reactive oxygen species scavenging reagents, and anti-inflammatory and synergistic antithrombotic reagents.

[0020] In some embodiments, the thrombosis diagnosis and treatment product is used for the diagnosis and treatment of at least one thrombotic disease, including ischemic stroke, myocardial infarction, and pulmonary embolism.

[0021] In some embodiments, the photothermal thrombolytic agent achieves photothermal ablation of the thrombus by irradiation with an 808nm near-infrared laser.

[0022] In some embodiments, the CT contrast agent is used in conjunction with a photon counting computed tomography (PCCT) device to achieve quantitative detection of the concentration of gold nanorods within the thrombus; the quantitative concentration results of the gold nanorods are used to guide the implementation of photothermal thrombolysis.

[0023] Compared with the prior art, the present invention has the following significant advantages: Excellent targeting: By targeting the nucleic acid aptamer of the α-thrombin anion binding site II, the nano-formulation achieves thrombin-dependent enrichment inside the thrombus, accurately identifies the active site of the thrombus, and solves the problem of poor targeting of existing probes.

[0024] Superior imaging performance: It can simultaneously achieve computed tomography (CT) and magnetic resonance (MR) contrast enhancement. Specifically, CT contrast enhancement is achieved through gold nanorod matrix, and T1-weighted magnetic resonance contrast enhancement is achieved through TEMPO. It solves the defect of limited tissue penetration depth of near-infrared fluorescent probes, and can clearly show the anatomical location and activity status of deep vascular thrombosis, providing accurate basis for thrombosis diagnosis. Integrated diagnosis and treatment: It integrates dual-modal imaging diagnosis, thrombus activity identification, photothermal thrombolysis, reactive oxygen species removal, and anti-inflammatory synergistic antithrombotic therapy to solve the problems of separation of diagnosis and treatment, poor thrombolysis effect, and easy recurrence in existing technologies, and realizes precise intervention in the entire process of thrombosis from diagnosis to treatment; Quantitative controllability: The concentration of gold nanorods in thrombi can be precisely quantified by using CT contrast agents in conjunction with PCCT (photonic CT) equipment, providing real-time guidance for the implementation of photothermal thrombolysis. Attached Figure Description

[0025] Other features, objects, and advantages of this disclosure will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is a schematic diagram illustrating the synthesis of TEM-Apt-AuNR nanoparticles and their PCCT and MR dual-modal imaging and thrombolysis function in the diagnosis and treatment of thrombosis. Figure 2 The figure shows the characterization results of the TEMPO aptamer conjugate and the thrombin-targeted TEM-Apt-AuNR nanoformulation. Figure 3 This is a graph showing the results of photon counting computed tomography (PCCT) imaging and the decomposition of gold components in a gold nanorod solution; Figure 4 The in vitro characterization of the thrombin binding ability of TEM-Apt-AuNR nanoparticles to fibrin was performed using PCCT. Figure 5 It is in vivo MR and quantitative PCCT imaging of thrombi; Figure 6 This is an in vitro characteristic analysis of light-triggered thrombolysis. Figure 7 Analysis of the in vivo light-triggered thrombolysis characteristics; Figure 8 This is an in vivo biosafety assessment of the TEM-Apt-AuNR nanoformulation. Detailed Implementation

[0026] The present disclosure will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present disclosure, but do not limit the present disclosure in any way. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present disclosure. These all fall within the protection scope of the present disclosure.

[0027] This invention develops a multifunctional nano-formulation based on gold nanomaterial AuNR, which has dual-modal contrast enhancement capabilities for computed tomography (CT) and magnetic resonance imaging (MR). It can specifically bind to active thrombin in thrombi to achieve accurate diagnosis of active thrombi, and can also trigger photo-controlled synergistic thrombolytic therapy.

[0028] This multifunctional nanoparticle formulation comprises a gold nanoparticle matrix, paramagnetic small molecules, and a thrombin-targeting nucleic acid aptamer. The thrombin-targeting nucleic acid aptamer endows the nanoparticle formulation with thrombin-targeting binding capability; the paramagnetic small molecules endow the nanoparticle formulation with T1-weighted magnetic resonance imaging capability and reactive oxygen species scavenging capability; the gold nanoparticle matrix endows the nanoparticle formulation with CT imaging capability and near-infrared photothermal conversion capability; the nanoparticle formulation can achieve thrombin-dependent enrichment within the thrombus.

[0029] The gold nanomaterial matrix can be rod-shaped (gold nanorods), spherical, or other shaped nanomaterials. The paramagnetic small molecule can be any one of TEMPO (2,2,6,6-tetramethylpiperidine nitroxide radical), gadobutrol, or gadotonic acid.

[0030] In some embodiments, the preparation process of the multifunctional nanoformulation TEM-Apt-AuNR is as follows: First, polyT sequences are modified onto the surface of gold nanorods via gold-sulfur bonds (Au-S bonds). Then, a base-complementary hybridization is performed with a thrombin-targeting aptamer grafted with a paramagnetic reactive oxygen species (ROS) scavenger (2,2,6,6-tetramethylpiperidine nitroxide radical, TEMPO) to obtain the therapeutic gold nanorod formulation (TEM-Apt-AuNR). Due to their high atomic number and localized surface plasmon resonance effect, gold nanorods naturally possess excellent properties as CT contrast agents and photothermal agents, and can be used for CT contrast enhancement and visualized treatment of thrombosis. Using photon-counting computed tomography (PCCT) imaging technology, we successfully separated gold from other materials in complex mixed environments, achieving precise quantification of the concentration of gold nanorods within thrombi, thus providing guidance for photocontrolled thrombolysis therapy. Furthermore, the combination of TEMPO and thrombin-targeting aptamers endows gold nanorods with magnetic resonance imaging (MRI), anti-inflammatory, and thrombin-inhibiting functions, which can effectively inhibit the formation and progression of thrombi.

[0031] Experimental results show that TEM-Apt-AuNR can achieve precise in vivo visualization of thrombus anatomical location, diagnosis of thrombus length, and quantitative detection of gold nanorods accumulated within the thrombus through magnetic resonance angiography (MRA), T1-weighted magnetic resonance (T1-weighted MR), and PCCT imaging. This provides guidance for photocontrolled thrombolysis and synergistic antithrombotic therapy, and offers a promising new approach for direct imaging, activity assessment, and treatment of thrombi.

[0032] like Figure 1 The diagram shows the synthesis of the multifunctional TEM-Apt-AuNR nanoparticles and their PCCT and MR dual-modal imaging and thrombolysis functions in thrombosis diagnosis and treatment. Figure 1 A shows the chemical synthesis route of the TEMPO-aptamer conjugate and its G-tetramer (G4) structural diagram; Figure 1 B represents the synthesis process of TEM-Apt-AuNR nanoparticles that can target and bind to thrombin. Figure 1 C represents the in vivo functional validation of the TEM-Apt-AuNR nanoformulation in a ferric chloride (FeCl3)-induced carotid artery thrombosis model.

[0033] The following describes the completed experiments: Experimental materials 4-(2-Iodoacetamido)-tetramethylpiperidine nitroxide radical (TEMPO-I), dimethyl sulfoxide (DMSO), acetic acid, and ethyl acetate (EA) were purchased from Shanghai Macklin Biochemical Co., Ltd. Sodium dodecyl sulfate (SDS), sodium chloride (NaCl), ferric chloride (FeCl3) solution, and nucleic acid aptamers were purchased from Sangon Biotech (Shanghai) Co., Ltd. Bovine fibrinogen (Fibrinogen) and Luminex assay kits were purchased from Shanghai Universal Biotech Co., Ltd. Type I collagen from rat tail and thrombin were purchased from Sigma-Aldrich Corporation.

[0034] Synthesis of TEMPO-coupled aptamer (TEM-Apt) First, the phosphate-thioester-modified nucleic acid aptamers (10 OD) were dried by vacuum rotary evaporation and then reconstituted in 5 μL of 10× phosphate-buffered saline (PBS). TEMPO-I was dissolved in dimethyl sulfoxide (DMSO) to prepare a 100 mM stock solution. Next, 55 μL of the TEMPO-I solution was added to the aptamer solution, and the mixture was reacted at 55 °C for 1 h. Subsequently, a certain volume of water was added to the mixture, and the mixture was extracted and washed six times with ethyl acetate (EA). The aqueous phase was collected and concentrated by vacuum rotary evaporation. To further purify the TEM-Apt conjugate, the final product was dissolved in water and purified by ultrafiltration using a Millipore ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa. The purified TEM-Apt conjugate was characterized by denaturing polyacrylamide gel electrophoresis (PAGE) and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS).

[0035] Synthesis and characterization of poly(T-AuNR) and poly(TEM-Apt-AuNR) First, gold nanorods (AuNRs) were synthesized using a seed growth method. Then, polyT sequences were coupled to the surface of the gold nanorods using a salt aging method: thiol-modified polyT sequences were... 15 Sequence (polyT) 15 Poly(T-S) modified gold nanorods (polyT-AuNR) were mixed with the prepared gold nanorods at a molar ratio of 1:3000 and sonicated for 5 minutes. The mixture was then placed in a metal bath and incubated with shaking at 45°C. After overnight incubation with shaking, 10% sodium dodecyl sulfate (SDS) solution was added to the mixture to bring the final concentration of SDS in the system to 0.01%. After sonication for 30 seconds, 3 mol / L sodium chloride (NaCl) solution was added to the polyT-modified gold nanorod (polyT-AuNR) mixture to bring the final concentration of NaCl in the system to 0.01 mol / L. The mixture was sonicated again for 30 seconds and incubated with shaking at 45°C for 1 hour. Subsequently, 3 mol / L NaCl solution was added dropwise every hour to sequentially increase the NaCl concentration to 0.05 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, and 0.5 mol / L. Once the NaCl concentration reached 0.5 mol / L, the mixture was incubated overnight at 45°C with shaking to complete the covalent modification reaction. After the reaction, the supernatant was removed by centrifugation to remove uncoupled polyTT. 15-SH; The precipitate was washed three times with phosphate-buffered saline (PBS) containing 0.3 mol / L NaCl to obtain the final polyT-modified gold nanorod (polyT-AuNR) intermediate. Excess TEMPO-aptamer conjugate (TEM-Apt) was added to polyT-AuNR and incubated for 24 hours. After incubation, unbound free TEM-Apt was removed by centrifugation, and the precipitate was resuspended in PBS solution containing 0.3 mol / L NaCl (PBS-0.3M-NaCl) to obtain the final TEM-Apt-AuNR nanoformulation. The structure and properties of the successfully prepared polyT-AuNR and TEM-Apt-AuNR were characterized by agarose gel electrophoresis, zeta potential, UV-Vis-NIR spectroscopy, and element mapping.

[0036] Quantitative Isolation of Gold Components in Gold Nanorods Based on Photon Counting Computed Tomography (PCCT) The concentration of synthesized gold nanorods was determined using inductively coupled plasma atomic emission spectrometry (ICP). Different concentrations of gold nanorod solutions (0.59, 1.18, 2.88, 4.32, and 6.92 mg / mL) were prepared, and PCCT imaging was performed using United Imaging Healthcare's uCT microPCCT system. This system is equipped with a photon counting detector and has two energy windows with thresholds of 20 keV and 30 keV. The Feldkamp-Davis-Kress (FDK) algorithm was used to reconstruct the acquired data, obtaining pixel values ​​(i.e., CT values) under the low-energy window (20-30 keV), high-energy window (30-70 keV), and total energy window (20-70 keV). CT values ​​of pixels in different regions of interest (ROIs) were extracted, and the linear relationship of signals under the low-energy and high-energy windows was analyzed to achieve precise quantification of the concentration of gold nanorods within the thrombus.

[0037] The specific scanning parameters are as follows: voltage 70 kV peak (70 kVp); current 200 μA (200 μA); exposure time 125 ms (125 ms); scanning pitch 0.5; pixel size 52.9 μm (52.9 μm). The contrast-to-noise ratio (CNR) is calculated according to the following formula: Basic Substance Decomposition (Water / Gold Decomposition): Water and gold were selected as the basic substances for the basic substance decomposition experiment. The decomposition process was carried out according to the following formula: Among them, HU HighWith HU Low These represent the CT values ​​acquired in the high-energy window and the low-energy window, respectively; μ w,H With μ w,L μ represents the linear decay coefficient of water under high and low energy windows, respectively. Au,H With μ Au,L ρ represents the linear decay coefficient of gold nanorods (AuNR) under high and low energy windows, respectively; w With ρ Au The values ​​represent the densities of water and gold nanorods, respectively.

[0038] Photothermal properties characterization of gold nanorods This study evaluated the photothermal properties of TEM-Apt-AuNR nanoparticles under different sample concentrations and 808 nm laser power densities. Phosphate-buffered saline (PBS) samples and TEM-Apt-AuNR samples at concentrations of 20, 40, and 80 μg / mL were irradiated with an 808 nm laser at a power density of 2 W / cm² for 120 seconds. Subsequently, a TEM-Apt-AuNR sample at a concentration of 40 μg / mL was irradiated with an 808 nm laser at different power densities of 0.5, 1, and 2 W / cm² for 120 seconds. Infrared thermal imaging was used to record the temperature changes of the samples throughout the process. Furthermore, to evaluate the thermal stability of TEM-Apt-AuNR, a sample at a concentration of 40 μg / mL was irradiated with an 808 nm laser at a power density of 2 W / cm² for 150 seconds. The laser was then turned off, and the samples were allowed to cool naturally to room temperature. This heating-cooling cycle was repeated twice to obtain the heating-cooling curves of the samples.

[0039] Preparation of fibrin clots and in vitro PCCT imaging Fibrinogen was dissolved in PBS at a concentration of 3 mg / mL. Thrombin was then added to the fibrinogen solution to achieve final thrombin concentrations of 1 U / mL and 5 U / mL, respectively. After overnight incubation, fibrin clots containing different concentrations of thrombin were obtained. TEM-Apt-AuNR with a gold concentration of 0.1 mg / mL and polyT-modified gold nanorods (polyT-AuNR) were added to the fibrin network and incubated at 37°C for 4 hours. The incubated fibrin clots were collected and PCCT imaging was performed using the parameters described above, with simultaneous water-gold decomposition and reconstruction.

[0040] Establishment of an in vitro model of endothelial injury and thrombosis First, the expanded polytetrafluoroethylene (ePTFE) artificial blood vessel was rinsed with phosphate-buffered saline (PBS). Type I collagen (1 mg / mL) dissolved in 0.2N acetic acid was injected into the ePTFE artificial blood vessel and incubated overnight at 4°C to complete the collagen coating. Subsequently, the collagen-coated ePTFE artificial blood vessel was connected to a peristaltic pump via a silicone rubber tube. Fresh rat blood was collected and injected into a sodium citrate anticoagulant blood collection tube, followed by the addition of thrombin and calcium ions (Ca²⁺). + A coagulation mixture was used to activate coagulation. The treated blood was then pumped into collagen-coated ePTFE artificial blood vessels using a peristaltic pump, creating a circulating blood flow at a rate of 40 mL / min. After incubation for at least 2 hours, thrombi formed in the collagen-coated area (simulating vascular endothelial injury sites). Subsequently, poly(T-AuNR) intermediates and TEM-Apt-AuNR nanoparticles were introduced into the thrombus model for further incubation. PCCT imaging was then performed on the thrombus model to verify the targeted enrichment and imaging effects of the nanoparticles.

[0041] Blood clot preparation and in vitro thrombolysis efficacy evaluation Take 15 μL of collected blood and mix it with 5 μL of coagulation solution

[34] , and incubate at 37°C for 1 hour to form a blood clot. After the blood clot is formed, add 100 μL of different test preparations: phosphate buffer (PBS, blank control group), poly-T-AuNR (negative control group), and TEM-Apt-AuNR (experimental group), and incubate with the blood clot for 1 hour; then irradiate with 808 nm near-infrared laser for 20 minutes for photo-controlled thrombolysis. The absorbance at 560 nm is detected by an enzyme-linked immunosorbent assay (ELISA) reader. This absorbance reflects the amount of contents released from the blood clot after photo-controlled thrombolysis and is used to quantitatively evaluate the thrombolytic effect. The positive control group is a mixture of 15 μL of blood and 85 μL of PBS (complete thrombolysis control). The thrombolysis rate is calculated according to the following formula: animal experiments All animal experiments were conducted in strict accordance with the principles of laboratory animal care and use, and all procedures were approved by the Animal Ethics Committee of Shanghai Jiao Tong University (Ethics Approval No.: A2023179). All experiments involving animals were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiao Tong University (China). Six- to seven-week-old male C57BL / 6 mice were used in this study.

[0042] Construction of an in vivo ferric chloride (FeCl3) induced thrombosis model After anesthetizing male C57BL / 6 mice, a minor surgical procedure was performed to expose and dissect the left common carotid artery (LCCA), and syringe packaging paper was placed beneath the artery. Subsequently, filter paper soaked in 5% ferric chloride (FeCl3) solution was applied to the arterial surface for 3 minutes. The FeCl3 filter paper and syringe packaging paper were removed, and the wound was sutured. Magnetic resonance angiography (PC-MRA) and T1-weighted magnetic resonance imaging (T1-MRI) were used to characterize the formation of vascular stenosis.

[0043] In vivo MRI and quantitative PCCT dual-modal imaging of thrombi One day prior to the procedure, mice were used to create a thrombosis model. TEM-Apt-AuNR nanoparticles were injected via the tail vein at a dose of 20 mg / kg. Subsequently, dual-modal imaging using photon-counting computed tomography (PCCT) and magnetic resonance imaging (MR) was performed to observe the location of the thrombus. Specific scanning parameters are as follows: PCCT scan parameters: Voltage 70 kV peak (70 kVp); Current 200 μA (200 μA); Exposure time 125 ms (125 ms); Scan pitch 0.5; Pixel size 23 μm (23 μm) MRI scan parameters: uMR 9.4T magnetic resonance imaging system, sequence fse_mx_3d; repetition time (TR) 800 ms; echo time (TE) 6.5 ms; slice thickness 0.1 mm. In vivo photodynamic thrombolysis One day prior to administration, thrombosis-inducing mice were prepared. Different test formulations (including poly(T-AuNR) intermediates and TEM-Apt-AuNR nanoformulation) were then administered via tail vein injection into the mice at a dose of 15 mg / kg. One and a half hours after administration, the thrombosis-inducing mice were anesthetized, and the left common carotid artery (LCCA) with thrombus formation was exposed. This vessel was then subjected to 20 minutes of laser irradiation (photodissolving treatment). Phosphate-buffered saline (PBS) was used to keep the vessel moist throughout the irradiation process. Photon-counted computed tomography (PCCT) and magnetic resonance imaging (MRI) were performed before and after irradiation to assess the thrombolytic effect. The following day, whole blood and serum were collected from the mice for subsequent biosafety assessment; serum inflammatory factor levels were measured using a Luminex assay kit. In addition, the thrombus-containing segment of the left common carotid artery was isolated, fixed with paraformaldehyde, and embedded in paraffin to prepare tissue sections of the segment. At the same time, tissues from major organs such as the heart, liver, spleen, lungs, and kidneys were collected and stained with hematoxylin and eosin (H&E) to comprehensively evaluate the in vivo safety of the TEM-Apt-AuNR nano-formulation.

[0044] Statistical analysis Experimental data are expressed as mean ± standard deviation. Statistical significance was analyzed using analysis of variance (ANOVA) and two-tailed Student's t-test. The significance criteria are as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0045] Results and Discussion Preparation and characterization of thrombin-targeted gold nanorods (AuNR) First, gold nanorods (AuNR) were synthesized using a seed growth method. The morphology of the gold nanorods was characterized by transmission electron microscopy (TEM), revealing a regular rod-like structure. ImageJ software was used to analyze the length and width distribution frequencies of the gold nanorods in the TEM images. The results showed that the average length of the prepared gold nanorods was 53.7 nm, the average width was 10.6 nm, and the high-frequency aspect ratio (L / W) was 4.5–5.5, consistent with near-infrared light absorption characteristics. Subsequently, to achieve thrombin-dependent targeting and imaging of thrombi, this study selected a thrombin aptamer (Apt) that specifically binds to the α-thrombin anion-binding external site II to prepare thrombin-targeting gold nanorods. The aptamer designed in this study carries polyA... 10 The protruding end is modified with 6 phosphate thioester (PS) groups; such as Figure 2 A. The paramagnetic free radical scavenger TEMPO (tetramethylpiperidine nitroxide radical) is grafted onto the aptamer via the reaction of the thiophosphate (PS) group with 4-(2-iodoacetamido)-TEMPO.

[0046] The conjugated products were characterized by denaturing polyacrylamide gel electrophoresis and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). As shown in Figure 2B, the TEMPO-aptamer conjugate (TEM-Apt) exhibited a single, clear band with a slower migration rate than the free aptamer (Apt), confirming successful TEMPO grafting. Furthermore, the molecular weight of the conjugated product TEM-Apt increased to 13705.4, an increase of 1428 compared to the free aptamer (Figure 2C), demonstrating successful covalent grafting of TEMPO molecules to all six PS groups. Since the function of the aptamer depends on its secondary structure, this study further analyzed the secondary structures of the aptamer and TEM-Apt using circular dichroism (CD) chromatography. TEM-Apt maintained the same G-tetramer (G4) conformation as the unmodified aptamer, proving that TEMPO grafting did not disrupt the G-tetramer structure of the aptamer, thus preserving its targeting activity. Furthermore, to investigate the binding ability of TEM-Apt to thrombin, this study conducted molecular docking experiments with free aptamers, TEM-Apt conjugates, and thrombin using a Haddock 2.4 server, with anion-binding external sites II (Arg-89, Lys-174, Arg-245, Lys-247, Lys-248, Lys-252) as anchoring sites. As shown in Figures 2D-2E, the binding modes of TEM-Apt and the unmodified aptamers were highly consistent, further validating the thrombin-targeting ability of TEM-Apt and laying a core foundation for the subsequent thrombus-targeted enrichment of nano-formulations.

[0047] Figure 2 shows the characterization results of the TEMPO-aptamer conjugate and the thrombin-targeted TEM-Apt-AuNR nanoformation. Figure 2A is a schematic diagram of the structure of the TEMPO-aptamer conjugate; Figure 2B is the denaturing polyacrylamide gel electrophoresis (PAGE) characterization of the free thrombin-targeting aptamer (Apt) and the TEMPO-aptamer conjugate (TEM-Apt); Figure 2C is the matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) characterization of the free aptamer (Apt) and the TEM-Apt conjugate; Figures 2D-2E are the binding energy analysis diagram (D) and molecular docking simulation diagram (E) of TEM-Apt and thrombin, respectively; Figure 2F is a schematic diagram of the preparation process of polyT-modified gold nanorods (polyT-AuNR) and the TEM-Apt-AuNR nanoformation; Figures 2G-2I are the gold nanorods (AuNR) and polyT-AuNR, respectively. Figure 2J shows the zeta potential characterization (G), 0.5% agarose gel electrophoresis characterization (H), and UV-Vis absorption spectroscopy characterization (I) of polyT-AuNR; the elemental mapping characterization of polyT-AuNR is shown in Figure 2J, with a scale bar of 20 nm.

[0048] Subsequently, the prepared gold nanorods (AuNRs) were covalently coupled with thiol-modified poly(T) nanorods via gold-sulfur bonds (Au-S bonds). 15Oligonucleotide binding was employed, and a salt aging method was used to generate polyT-modified gold nanorods (polyT-AuNR) nanoparticles. The polyT-AuNR and polyA-tailed modified TEM-Apt conjugates were hybridized and assembled through base complementarity pairing to obtain gold nanorods (TEM-Apt-AuNR) capable of targeting thrombin binding (see Figure 2F). The successful synthesis of polyT-AuNR and TEM-Apt-AuNR was characterized by 1% agarose gel electrophoresis, zeta potential measurement, and UV-Vis-NIR absorption spectroscopy. Due to the hexadecyltrimethylammonium bromide (CTAB) coating on the surface of the gold nanorods during synthesis, the unmodified AuNRs exhibited a weak positive charge in the zeta potential detection (Figure 2G), theoretically migrating from the positive to the negative electrode in agarose gel electrophoresis. However, unmodified AuNRs exhibit poor stability and are prone to aggregation at high salt concentrations, leading to aggregates at the loading wells (Figure 2H). After salt aging and base pairing modification, both polyT-AuNR and TEM-Apt-AuNR showed negative zeta potentials, proving that the polyT sequence and aptamer (Apt) were successfully modified onto the surface of gold nanorods. Therefore, both polyT-AuNR and TEM-Apt-AuNR migrated towards the positive electrode in agarose gel electrophoresis, presenting a single, clear band; this also indicates that nucleic acid modification can improve the dispersibility and uniformity of gold nanorods. Furthermore, the migration rate of TEM-Apt-AuNR was slower than that of polyT-AuNR, due to its increased molecular weight, further demonstrating that TEM-Apt and polyT-AuNR successfully achieved base complementary hybridization. In UV-Vis-NIR spectroscopy, the synthesized AuNRs exhibited a characteristic absorption peak at 937 nm and a lateral localized surface plasmon resonance (LSPR) absorption peak at 505 nm. As shown in Figure 2I, after nucleic acid modification onto the surface of gold nanorods, the longitudinal LSPR absorption peak position significantly blue-shifted from 937 nm to 889 nm, while the lateral LSPR absorption peak position remained unchanged at 505 nm. This result further confirms that the nucleic acid was successfully coupled to the surface of the gold nanorods. Furthermore, the elemental distribution of the prepared TEM-Apt-Au nanoformulation was observed under field emission transmission electron microscopy (TEM): As shown in Figure 2J, the characteristic elements (Au and S) of AuNRs and the characteristic elements (P) of nucleic acid showed a highly consistent distribution in the elemental mapping diagram, confirming at the microscopic level that the gold nanorods and nucleic acid were successfully coupled.

[0049] Photon-counting computed tomography (PCCT) imaging and gold component decomposition in solution Subsequently, PCCT imaging of gold nanorods (AuNR) at different concentrations in pure water was performed using the United Imaging uCT micro-photon counting CT system equipped with a photon counting detector. This system has two energy windows with thresholds of 20 keV and 30 keV, enabling precise differentiation of different substances. The Feldkamp-Davis-Kress (FDK) algorithm was used to reconstruct the acquired data, obtaining pixel values ​​under the low energy window (20-30 keV), high energy window (30-70 keV), and total energy window (20-70 keV) (Figure 3A). As shown in Figures 3B-3E, under different regions of interest (ROIs), the HU value reconstructed by the total energy window showed an excellent linear correlation with gold concentration, with the HU value increasing linearly with increasing gold concentration. The contrast-to-noise ratio (CNR) was calculated using the following formula: The results showed that the CNR of all samples was higher than 4.4. Furthermore, the experimental results indicated that the HU values ​​reconstructed from the gold nanorod solution under both low and high energy windows exhibited good linearity, providing performance assurance for the accurate differentiation of gold. Subsequently, water and gold were selected as the basic substances for material decomposition experiments; as shown in Figure 3B, the gold mapping obtained after decomposition could be perfectly distinguished from water. Altman-Bland consistency analysis (Figures 3F and 3G) verified that the gold concentration measured by PCCT gold energy dispersive spectroscopy was highly consistent with the gold concentration measured by inductively coupled plasma atomic emission spectrometry (ICP), proving that the water / gold decomposition method established in this study has extremely high accuracy. Meanwhile, as... Figure 3 H. The CNR was analyzed under different ROIs. The results showed that the CNR of gold energy spectroscopy imaging was still higher than 2 under an ROI of 0.005 cm², which provides a reliable guarantee for the in vivo application of this technology.

[0050] like Figure 3 The image shown is a graph illustrating the decomposition of gold components in a gold nanorod (AuNR) solution using photon-counted computed tomography (PCCT). Figure 3 Figure A is a schematic diagram of the principle of PCCT imaging and gold component decomposition; Figure 3B shows the reconstructed images of AuNR at different concentrations after PCCT imaging, including high-energy window, low-energy window, and total energy window images, as well as the gold mapping map after gold component decomposition. Figure 3Figure C shows the linear analysis results of the CT values ​​(HU values) obtained from high-energy and low-energy window reconstructions; Figures 3D-3E show the HU values ​​(D) and contrast-to-noise ratios (CNR) (E) of AuNR in different regions of interest (ROIs) in the total energy window reconstructed images, respectively; Figure 3F shows the linear analysis results of the gold concentration measured by PCCT and the gold concentration measured by inductively coupled plasma atomic emission spectrometry (ICP) in different ROIs; Figure 3G shows the Bland-Altman consistency analysis results of the gold concentration measured by PCCT and the gold concentration measured by ICP; Figure 3H shows the contrast-to-noise ratio (CNR) results of the gold concentration measured by PCCT in different ROIs.

[0051] TEM-Apt-AuNR nano-formulations exhibit thrombin-dependent binding to fibrin. Thrombin plays a central role in thrombus formation: it catalyzes the conversion of fibrinogen into fibrin monomers, activates various coagulation factors, promotes fibrin cross-linking, and activates platelets. Studies have shown that elevated thrombin levels not only indicate thrombus core formation but also promote the development of stable thrombi, thereby reducing the thrombolytic effect of plasmin. Therefore, designing nanotherapeutic agents targeting thrombin is crucial for the diagnosis and treatment of stable thrombi. Previous studies have reported that thrombin-binding aptamers can inhibit the activity of thrombin by anchoring to thrombin's external site II, thereby inhibiting the formation of fibrin clots. In this study, an enzyme-linked immunosorbent assay (ELISA) reader was used to monitor the inhibitory effect of TEM-Apt-AuNR nanoparticles on fibrin network formation: after adding thrombin to the fibrinogen solution, thrombin cleaved fibrinogen to generate fibrin, resulting in an increase in absorbance at 350 nm. The absorbance at 350 nm in the PBS control group increased rapidly, indicating rapid fibrin network formation; the non-targeted poly-T-AuNR intermediate had no significant effect on fibrin network formation. Conversely, both uncoupled TEMPO-modified aptamer gold nanorods (Apt-AuNR) and TEM-Apt conjugate-modified gold nanorods (TEM-Apt-AuNR) significantly delayed fibrin network formation. This is because the nanostructures, upon binding to thrombin, created steric hindrance, inhibiting thrombin activity. These results indicate that TEMPO coupling does not affect the binding affinity of the aptamer, consistent with molecular docking experiments.

[0052] Furthermore, to verify the thrombin-dependent binding of TEM-Apt-AuNR to pre-formed fibrin, this study constructed fibrin clot models with different thrombin concentrations in vitro using fibrinogen and thrombin (Figure 4A). Fibrin networks were constructed using different concentrations of thrombin and fibrinogen. Subsequently, fibrin clots containing different thrombin concentrations were co-incubated with TEM-Apt-AuNR and poly(T-AuNR) at a gold concentration of 0.1 mg / mL. After incubation, photon-counting computed tomography (PCCT) imaging was performed on the fibrin clots to quantitatively detect the concentration of gold nanorods within the clots, and a water-gold decomposition mapping was reconstructed based on the PCCT data. As shown in Figure 4B, for fibrin clots containing a low concentration of thrombin (1 U / mL), the gold concentrations measured in the gold mapping images after incubation with TEM-Apt-AuNR and poly-T-AuNR were 2.9 mg / mL and 2.3 mg / mL, respectively, with no significant difference between the two. However, for fibrin clots containing a high concentration of thrombin (5 U / mL), the gold concentration significantly increased to 8.7 mg / mL after incubation with TEM-Apt-AuNR, confirming that TEM-Apt-AuNR can achieve specific enrichment in highly active thrombus regions through thrombin-dependent binding. The experimental results indicate that TEM-Apt-AuNR nanoparticles can be enriched to 87-fold in fibrin clots via a thrombin-dependent mechanism (based on an initial solution concentration of 0.1 mg / mL), demonstrating the excellent in vivo bioavailability of TEM-Apt-Au nanoparticles. In contrast, the poly-T-AuNR intermediate did not exhibit thrombin-dependent enrichment behavior. Furthermore, at a gold concentration of 3.3 mg / mL, this study further validated the enrichment and diffusion potential of AuNR within thrombi: human fibrin plates were prepared using agarose, fibrinogen, and thrombin to assess the diffusion capacity of poly-T-AuNR and TEM-Apt-AuNR. The targeted modified gold nanorods (TEM-Apt-AuNR) showed significantly better diffusion range than the untargeted poly-T-AuNR, confirming the thrombin-mediated enrichment and diffusion characteristics of AuNR within thrombi, providing significant potential for the diagnosis and treatment of thrombosis.

[0053] To further investigate the thrombus targeting ability of TEM-Apt-AuNR under blood flow conditions, this study established an in vitro thrombosis model to simulate the in vivo thrombus formation process: First, expanded polytetrafluoroethylene (ePTFE) grafts (low-hemorrhagic) were coated with 1 mg / mL collagen and rinsed with PBS; this collagen-coated graft triggered platelet-dependent thrombus formation. Subsequently, the collagen-coated graft was connected to a peristaltic pump through a silicone rubber tube; blood was collected and pumped in through the peristaltic pump to establish blood flow. To promote thrombus formation within the collagen-coated graft, low concentrations of thrombin (1 U / mL) and calcium ions (25 mM) were added. After 2 hours of blood flow circulation, thrombi formed in the collagen-coated area (Figure 4D). The graft containing the thrombus was collected and imaged under a microscope and photon-counting computed tomography (PCCT), revealing a thrombus structure of a certain thickness forming on the inner wall of the ePTFE tube. Subsequently, poly-T-AuNR and TEM-Apt-AuNR were introduced into the established thrombus models and circulated at a low concentration of 0.1 mg / mL for 2 hours for PCCT imaging. As shown in Figures 4E-4F, the gold concentration in the TEM-Apt-AuNR group within the thrombus was measured to be 2.8 mg / mL, significantly higher than the 1.7 mg / mL in the poly-T-AuNR group. These results demonstrate that TEM-Apt-AuNR exhibits excellent targeting and enrichment capabilities in thrombin-rich thrombi, providing a promising diagnostic and therapeutic strategy for the direct imaging of thrombin-rich thrombi.

[0054] Figure 4 shows the in vitro experimental results of the TEM-Apt-AuNR nanoparticles' thrombin binding ability on fibrin, characterized by photon-counted computed tomography (PCCT). Figure 4A is a schematic diagram of the principle of thrombin-induced fibrin clot formation. Figures 4B-4C are the water / gold component decomposition mapping (B) and the measured gold concentration results (C) of fibrin clots formed under different thrombin activities after incubation with poly(T-AuNR) and TEM-Apt-AuNR, respectively. Figure 4D is a schematic diagram of the device used to prepare a thrombosis model in expanded polytetrafluoroethylene (ePTFE) grafts, where the yellow arrow indicates the ePTFE graft and the blue arrow indicates the formed thrombus. Figure 4E is the gold component mapping / total energy window reconstruction image of the ePTFE-thrombosis model after perfusion of 0.1 mg / mL gold nanorod (AuNR) solution for 2 hours using a peristaltic pump. The red layer is the gold concentration mapping reconstructed within the thrombus, and the gray layer is the total energy window CT. Image; Figure 4F shows the gold concentration results measured from the gold component mapping map. Statistical significance is indicated by: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0055] In vivo MR / PCCT dual-modal imaging of thrombi After confirming that thrombin-targeted TEM-Apt-AuNR can accumulate in thrombi, we next investigated the in vivo thrombus imaging behavior of TEM-Apt-AuNR. First, the in vitro MRI characteristics of the tempo-coupled aptamer were evaluated on a 9.4 T MRI scanner. (For example...) Figure 5 As shown in A-5B, the T1 relaxation time decreases with increasing tempo concentration, while the T1 gray value increases, indicating excellent T1 magnetic resonance imaging capabilities. Furthermore, to evaluate the reactive oxygen species scavenging ability of tempo, superoxide anions (•O2) were prepared using xanthine and xanthine oxidase. - ), and residual •O2 was determined by DCFH. - This ion can be activated by •O2 - Oxidation produces a fluorescent signal, which is related to •O2. - The fluorescence intensity decreased significantly after incubation, confirming the reactive oxygen species scavenging ability of TEM-Apt. Next, we evaluated the PCCT / MR dual-modal imaging capability of TEM-AptAuNR using a ferric chloride (in vivo)-induced mouse carotid artery thrombosis model. First, the left common carotid artery (LCCA) of the mouse was exposed and isolated, followed by placing a piece of syringe packaging paper underneath it. Then, filter paper soaked in 5% FeCl3 was applied to the artery for 3 minutes. Both phase-contrast magnetic resonance angiography (PC-MRA) and laser speckle contrast imaging showed obstruction of blood flow in the left common carotid artery, indicating the presence of a thrombus. Subsequently, using thrombotic mice, we investigated the targeting and retention effects of TEM-Apt-AuNR on the thrombus using MR / PCCT dual-modal imaging of the neck region. Figure 5 As shown in Figure C, isointense signals were observed in the left common carotid artery lumen of thrombotic mice in T1-MR images before TEM-Apt-AuNR injection, indicating thrombosis. Subsequently, TEM-Apt-AuNR was intravenously injected into the thrombotic mice at a gold dose of 20 mg / kg, and magnetic resonance (MR) and PCCT imaging of the thrombus was performed at different time points. After injection of TEM-Apt-AuNR, the thrombus site showed high signal intensity, confirming the T1-enhanced MRI function of gold nanorods in vivo. Specifically, the thrombus length measurement and contrast-to-noise ratio (CNR) in the MR images showed that the retention rate of TEM-Apt-AuNR continuously increased in the first 4 hours, reaching a peak at 3-4 hours. Figure 5 E). Simultaneously, the accumulation concentration of TEM-Apt-AuNR was quantitatively analyzed using PCCT imaging. For example... Figure 5As shown in D and 5F, TEM-Apt-AuNR showed a gradually increasing accumulation trend within the thrombus over 4 hours, eventually reaching a maximum concentration of 2.4 mg / mL, occurring at 3-4 hours. The thrombus length measured in PCCTAu images was consistent with the quantitative data, with the longest length occurring at 3-4 hours, providing precise guidance for subsequent photo-triggered thrombolysis. In contrast, the measured gold concentration in mouse thrombi treated with non-targeted polyT-AuNR decreased 3 hours after injection, with the highest concentration occurring in the first 1.5 hours post-injection (2.1 mg / mL). Figure 5 G). Consistently, the measured length decreased after 1.5 hours. Of particular note, thrombotic mice treated with thrombin-bound TEM-Apt-AuNR showed significantly higher gold concentrations (G) 5 hours after injection compared to mice treated with untargeted polyT-AuNR. Figure 5 H). The measured thrombus length and gold distribution map showed that TEMApt-AuNR had a longer retention time and higher enrichment in the thrombus, which can be attributed to the thrombin binding capacity of TEMApt-AuNR.

[0056] like Figure 5 The images show in vivo MR and quantitative PCCT imaging of thrombi. (A) T1 mapping and T1-weighted MR images of TEM-Apt solutions at different concentrations. (B) T1 relaxation time of TEM-Apt solution. (C) MR images of thrombotic mice before and after injection of TEM-Apt-AuNR. (D) Gold concentration mapping and CT images of thrombotic mice after injection of TEM-Apt-AuNR. The red layer represents the reconstructed gold concentration mapping image within the thrombus; the gray layer represents the total tomographic CT image. (E) Calculated CNR value and measured thrombus length in MR images of thrombotic mice after injection of TEM-Apt-AuNR. (FG) Measured gold concentration and thrombus length in the thrombus after injection of TEM-Apt-AuNR (F) and polyTAuNR (G). (H) Comparison of measured gold concentration in the thrombus after treatment with polyT-AuNR and TEM-Apt-AuNR at different time points. Statistical significance: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0057] In vitro light-triggered thrombolysis After verifying the thrombin-targeting effect of TEM-Apt-AuNR, we investigated its in vitro photothermal thrombolytic effect. Gold nanorods possess excellent optical properties by converting laser energy into heat, demonstrating broad potential for thrombolytic therapy. Therefore, we characterized the photothermal conversion properties of the prepared TEM-Apt-AuNR in vitro. The heating curves of TEM-Apt-AuNR were measured using an 808 nm laser at different gold concentrations and laser intensities. Figure 6 As shown in A-6B, the temperature of the gold nanorod solution increases with increasing gold nanorod concentration, laser intensity, and laser irradiation time. At laser intensities of 0.5, 1.0, and 2.0 W·cm⁻¹, the temperature of the solution also increases. -2 At the same concentration, the TEM-Apt-AuNR solution (40 μg / mL) showed temperature increases of 15.1, 29.1, and 68.9 °C, respectively, indicating that at the same TEM-Apt-AuNR solution concentration, higher laser intensity resulted in a greater temperature increase. Simultaneously, the temperature changes of different concentrations of TEM-Apt-AuNR solution (10, 20, and 40 μg / mL) after irradiation with an 808 nm laser at an intensity of 2 W / cm² for 2 minutes were measured. The temperatures increased by 38.9 °C, 63.0 °C, and 68.9 °C, respectively, while phosphate-buffered saline (PBS) only increased by 6.8 °C after laser irradiation. Furthermore, the photothermal stability of TEM-Apt-AuNR was also verified. Figure 6 As shown in Figure C, TEM-Apt-AuNR exhibits excellent photothermal conversion stability during three heating / cooling cycles, demonstrating significant application potential in photo-triggered thrombolytic therapy.

[0058] Subsequently, the photo-triggered thrombolysis was characterized in vitro. Equal weights of freshly prepared thrombi were incubated for 1 hour with different formulations (including PBS solution, polyT-AuNR, and TEM-Apt-AuNR (0.1 mg / mL)), followed by incubation with an 808 nm laser at 1 W / cm². 2 The thrombus mixture was irradiated with appropriate power. The temperature of the thrombus mixture rose to 39.1, 51.8, and 55.6 °C after incubation with PBS, polyT-AuNR, and TEM-AptAuNR, respectively, followed by irradiation. Twenty minutes after irradiation, the absorbance at 560 nm due to thrombolysis caused by erythrocyte release was assessed using a microplate reader. Figure 6 As shown in Figures D and 6E, the 808 nm laser did not induce thrombolysis, showing no significant visual difference compared to the unirradiated PBS group, with a thrombolysis rate of 3.0%. In contrast, the thrombolysis rates of polyT AuNR and TEM-Apt-AuNR were 65.3% and 72.6%, respectively. To further analyze the effect of photothermal activity on the fibrin network, TEM-Apt-AuNR was co-incubated with the fibrin network overnight, followed by laser irradiation for 30 minutes. The morphological characteristics of the fibrin network before and after irradiation were characterized by scanning electron microscopy (SEM). Figure 6 As shown in Figure F, after TEM-Apt-AuNR incubation and subsequent laser irradiation, the originally non-flowable fibrin network underwent structural disruption and transformed into a flowable solution. Scanning electron microscopy (SEM) images further confirmed that the photothermal effect effectively disrupted the structural integrity of the fibrin network, providing direct evidence for the photo-triggered thrombolytic effect.

[0059] Figure 6 This study analyzes the in vitro characteristics of photo-triggered thrombolysis. (A) Measured temperatures of PBS and AuNR solutions at different concentrations after irradiation with an 808 nm laser at 2 W / cm² intensity for different durations. (B) Measured temperatures of AuNR solution (40 μg / mL) after irradiation with an 808 nm laser at different energies for different durations. (C) Photothermal stability of AuNR during three cycles of laser switching. (D) Digital images of blood clots after different treatments. (E) Thrombolysis rates after different treatments. (F) Digital images of formed fibrin clots, comparing fibrin clots incubated with TEM-Apt-AuNR before and after irradiation. Statistical significance: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0060] In vivo photo-triggered thrombolysis therapy PCCT and MR imaging revealed the aggregation behavior of TEM-Apt-AuNR, providing important guidance for photo-triggered thrombolysis. Next, we investigated the potential of TEM-Apt-AuNR as a thrombosis management strategy. Increasing evidence suggests that local hyperthermia accelerates thrombus ablation by disrupting non-covalent fibrin interactions, providing a safe thrombolytic method. The synthesized AuNR exhibits excellent photothermal efficiency, converting laser light into heat and disrupting the solid structure of collagen fibers, leading to erythrocyte release and effective vascular recanalization. To characterize the efficiency of photo-triggered thrombolysis, a mouse model of thrombosis was established by pre-applying 5% FeCl3 to the left common carotid artery (LCCA) for 33 minutes. First, PC-MRA imaging characterized embolism formation and thrombus location. Compared to the right common carotid artery RCCA, the embolism in the left carotid artery LCCA was observably significantly narrowed. Subsequently, targeted and non-targeted gold nanorods (AuNR) were injected via the tail vein, supplemented by PC-MRA, T1 MRI, and quantitative PCCT imaging techniques. This allowed for visualization of vascular embolism, thrombus length, and drug concentration measurements, guiding the photo-triggered thrombolysis process. Figure 7As shown in Figure A, PCMRA images revealed stenosis rates of 69.8%, 72.7%, 65%, and 69.6% in the control group, laser-only group, polythiophene gold nanorod group, and TEM-Apt-AuNR treatment group, respectively. One and a half hours after drug administration, thrombus length was analyzed and in-situ gold nanorod concentration was calculated using PCCT / MR dual-modal imaging. Subsequently, the embolized LCCA was irradiated with an 808 nm laser for 20 minutes to observe the thrombolysis rate. As expected, the stenosis degree of the LCCA in the laser-only group remained essentially the same as before drug administration. Irradiation experiments demonstrated that ordinary lasers do not produce a thermal effect in vivo, confirming the excellent biocompatibility of lasers. Consistent with in vitro results, the highest temperature in the lesion area of ​​the control group mice was 37.2℃. In contrast, the highest temperatures in the lesion areas of the polyT-AuNR and TEM-Apt-AuNR treatment groups reached 47.5℃ and 50.6℃, respectively. Therefore, after irradiation, PC-MRA images showed that mice treated with polyT-AuNR and TEM-Apt-AuNR exhibited significant thrombolytic efficiency (stenosis rates of 29.5% and 14.2%, respectively). Notably, the thrombin-targeting TEM-Apt-AuNR thrombolytic rate (79.6%) was higher than that of polyT-AuNR, possibly due to less accumulation of polyT-AuNR in the thrombus. Furthermore, T1-weighted magnetic resonance imaging was used to observe the formation and distribution of thrombi within the vascular lumen after photo-triggered thrombolysis. Consistent with the PC-MRA results, no significant morphological changes were observed in thrombi after conventional laser irradiation compared to before irradiation. Moreover, a significant reduction in thrombus length was observed in irradiated mice treated with polyT-AuNR and TEM-Apt-AuNR, providing further visual evidence for photothermal mediated thrombolysis. PCCT imaging was also performed before and after irradiation to obtain images of gold distribution. Figure 7 C As shown in Figure 7E, the thrombus length was significantly reduced after irradiation. PCCT analysis revealed that the thrombus length in the polyT-AuNR-treated group was significantly reduced from 1.48 mm to 0.45 mm, with an antithrombolytic rate of 69.5%. In contrast, the TEM-Apt-AuNR-treated group demonstrated a superior antithrombolytic effect of 79.3%, dissolving the thrombus from 1.63 mm to 0.34 mm, a result consistent with MRI data.

[0061] Figure 7Analysis of in vivo photo-triggered thrombolysis characteristics. (A) PC-MRA and T1-MR images of mice with thrombosis after 20 minutes of 808 nm laser irradiation. White arrows indicate the lumen of blood vessels. (B) Stenosis rate of thrombotic LCCAs before and after laser irradiation. (C) Thrombus length measured in PCCT images of mice treated with TEM-Apt-AuNR and polyT-AuNR. (DE) Gold spectrum CT images before and after laser irradiation following intravenous injection of TEM-Apt-AuNR (D) and polyT-AuNR (E). (F) H&E staining images of thrombotic LCCAs after different treatments. (G) Concentration of inflammatory cytokines in the serum of mice with thrombosis after different treatments. (H) Schematic diagram of thrombotic inflammation-related pathways. Statistical significance: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0062] Furthermore, we investigated the pathological morphological characteristics of the thrombus using hematoxylin and eosin (H&E) staining. Figure 7 As shown in Figure F, significant embolism was observed in the vascular lumen of both the control and laser treatment groups. H&E staining images of the control group showed prominent platelet bundles, fibrin network structures, and embedded erythrocytes, exhibiting typical characteristics of mixed thrombi. Neutrophil infiltration was also observed, suggesting an inflammatory response within the thrombus. The pathological features of the laser treatment group were similar to those of the control group, but after treatment with polythiophene gold nanorods (polyT-AuNR) combined with laser irradiation, the cellular components within the vascular lumen were significantly reduced. Furthermore, the fibrous tissue formation structure was not prominent, resulting in relatively patent lumens, indicating good thrombolysis and successful recanalization. Notably, the thrombin-conjugated TEM-Apt-AuNR group showed the most significant thrombolytic effect, with only a small amount of residual thrombus observed, demonstrating superior recanalization. Literature studies suggest that due to endothelial damage and platelet activation, an inflammatory microenvironment exists within the thrombus, and reactive oxygen species (ROS) levels are elevated, which may further exacerbate the coagulation process. Therefore, the anti-inflammatory effect of the tempo ROS scavenger contained in TEM-Apt-AuNR was evaluated by detecting serum inflammatory cytokine levels using a Luminex assay kit. Figure 7As shown in G, compared with the control group, laser treatment group and polyT-AuNR treatment group, the expression levels of multiple cytokines in mice treated with TEM-Apt-AuNR were reduced, including interleukin-2 (IL-2), interleukin-5 (IL-5), interleukin-12p70 (IL-12p70), IL-10 and TNFα

[49] . This confirms that TEM-Apt-AuNR has potential anti-inflammatory effects in photothermal-triggered thrombolysis. Since these cytokines are involved in the activation of platelets and tissue factor (TF), they enhance the coagulation response through multiple signaling pathways. Figure 7 The downregulation of TEM-Apt-AuNR (H) expression indicates a significant preventive effect against the formation of new thrombus centers, revealing the substance's remarkable multifaceted antithrombotic function.

[0063] Biosafety assessment The biosafety of TEM-Apt-AuNR was studied both in vitro and in vivo. First, a hemolysis assay was performed by incubating erythrocytes with different concentrations of TEM-Apt-AuNR (15, 30, 60, and 120 μg / mL) at 37°C for 4 hours. The mixture was then centrifuged, and the supernatant was collected. The absorbance of the dissolved hemoglobin at 540 nm was measured. TEM-Apt-AuNR showed a slight hemolysis rate with no significant visual difference compared to the PBS-treated group, indicating that TEM-Apt-AuNR has excellent biosafety. Furthermore, in vivo biosafety was assessed by detecting liver and kidney function, routine blood tests, and histopathological analysis after antithrombolytic therapy.

[0064] Figure 8 The in vivo biosafety assessment of TEM-Apt-AuNR is shown. (AD) Plasma concentrations of ALT, AST, BUN, and creatinine in mice after different treatments. (E) Blood cell concentrations in mice after different treatments. (F) H&E staining images of heart, liver, spleen, lung, and kidney tissues in mice after different treatments.

[0065] like Figure 8 As shown in A-8D, mice treated with polyT-AuNR and TEM-Apt-AuNR maintained stable levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CERA) compared to untreated thrombotic mice, indicating that these two drugs did not cause damage to the liver and kidneys. Hematological tests further confirmed that TEM-Apt-AuNR has good biocompatibility and safety, with all blood cell indicators remaining within the normal range. Figure 8 E). To assess tissue toxicity, heart, liver, spleen, lungs, and kidneys were collected for pathological analysis after antithrombotic therapy. Figure 8The H&E staining images in F showed no obvious pathological changes, which provides strong evidence for the excellent biosafety of TEM-Apt-AuNR.

[0066] in conclusion This study presents a novel therapeutic nanoformulation, TEM-Apt-AuNR, integrating thrombus active site targeting technology, precise quantitative analysis of intrathrombus drugs, and PCCT / MRI dual-modal imaging-guided photothermal therapy into a single system. Due to the high atomic number of gold nanomaterials, they can serve as excellent CT contrast agents for disease imaging. This study selected gold nanorods (AuNRs) with photothermal properties and established a gold element decomposition method based on PCCT technology to achieve precise quantification of gold content. The gold concentration measured by this method is consistent with the results of inductively coupled plasma (ICP) analysis, providing a new approach for the quantitative analysis of AuNRs. Furthermore, to achieve the identification and imaging of thrombus active sites, this study selected thrombin (a direct indicator of thrombus formation activity) as the target. Nucleotide aptamers were linked to thrombin extra-site II via thiophosphate modification and grafted with aspartic methionine peptide (tempo) molecules, enabling the thrombin-binding aptamer to possess both MRI imaging and reactive oxygen species scavenging functions. Importantly, by optimizing the coupling site, the secondary structure and thrombin binding affinity of the aptamer remained unchanged after conjugation with temozolomide. We constructed a multifunctional TEM-Apt-AuNR platform by combining the synthesized multifunctional aptamer with gold nanorods (AuNR). This TEM-Apt-AuNR system can achieve thrombin-dependent aggregation in fibrin clots and thrombi. By integrating PCCT imaging, gold decomposition, phosphorylcholine MRA, and T1 magnetic resonance imaging, we can not only directly visualize the thrombus location and active site but also achieve in-situ precise quantification of drug concentration within the lesion. Compared with traditional fluorescence methods or ex vivo drug concentration analysis methods, this in-situ quantitative method provides real-time feedback on drug concentration. Through the phenomenon of drug accumulation within the lesion, we established a novel and convenient method for in vivo drug quantification. Based on the inherent photothermal effect of gold nanorods (AuNR), we further implemented laser irradiation therapy, achieving effective thrombolysis 24 hours after thrombosis in mice. This localized photothermal thrombolysis technology not only overcomes the inefficiency caused by plasmin resistance but also demonstrates advantages in high efficiency and controllability. Furthermore, the reactive oxygen species scavenger (tempo) integrated into AuNR synergistically reduces the levels of inflammatory factors related to platelet activation and coagulation in the serum of thrombotic mice, thereby producing a synergistic antithrombotic effect. This comprehensive strategy successfully solves several long-standing challenges in the treatment of highly active plasmin-resistant thrombosis, providing new ideas and methodological support for thrombosis imaging, activity analysis, dosing, and thrombosis treatment. We anticipate that this innovative paradigm combining quantitative imaging and targeted nanomedicine will open up entirely new pathways for the precision treatment of cardiovascular diseases.

[0067] The specific embodiments of this disclosure have been described above. It should be understood that this disclosure is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the substantive content of this disclosure. The above-described preferred features can be used in any combination without conflict.

Claims

1. A multifunctional nano-formulation with thrombin-targeting properties, characterized in that, The nanoformulation includes gold nanoparticles, paramagnetic small molecules, and thrombin-targeting nucleic acid aptamers.

2. The nano-formulation according to claim 1, characterized in that, The gold nanoparticles are rod-shaped or spherical.

3. The nano-formulation according to claim 1, characterized in that, The paramagnetic small molecule is any one of TEMPO, gadobutrol, and gadotonic acid.

4. The nano-formulation according to claim 1, characterized in that, It also includes a poly-T oligonucleotide sequence, which is covalently modified on the surface of the gold nanoparticle matrix by gold-sulfur bonds to form poly-T-AuNR.

5. The nano-formulation according to claim 1, characterized in that, The paramagnetic small molecule is TEMPO, and the TEMPO and the thrombin-targeting nucleic acid aptamer form a TEM-Apt conjugate through a covalent grafting reaction mediated by thiophosphate groups.

6. The nano-formulation according to claim 5, characterized in that, The TEM-Apt conjugate has a poly-A tail that is complementary to the poly-T oligonucleotide sequence, and hybridizes with the poly-T-AuNR through base complementary pairing to form TEM-Apt-AuNR.

7. The nano-formulation according to claim 1, characterized in that, The thrombin-targeting nucleic acid aptamer endows the nano-formulation with thrombin-targeting binding ability; the paramagnetic small molecule endows the nano-formulation with T1-weighted magnetic resonance imaging ability and reactive oxygen species scavenging ability; the gold nanoparticle matrix endows the nano-formulation with CT imaging ability and near-infrared photothermal conversion ability; the nano-formulation can achieve thrombin-dependent enrichment inside the thrombus.

8. The nano-formulation according to claim 7, characterized in that, The thrombin-targeting nucleic acid aptamer has six thiophosphate groups and retains its secondary structure. Its target binding sites are Arg-89, Lys-174, Arg-245, Lys-247, Lys-248 and Lys-252 sites of α-thrombin.

9. A method for preparing a multifunctional nano-formulation with thrombin targeting properties, characterized in that, include: Preparation of TEM-Apt conjugates: Thrombin-targeting nucleic acid aptamers targeting α-thrombin are covalently grafted with paramagnetic small molecules via thiophosphate groups to obtain TEM-Apt conjugates. Preparation of poly-T-AuNR: Poly-T oligonucleotide sequences were covalently modified onto the surface of gold nanorod matrix via gold-sulfur bonds to obtain poly-T-AuNR; Hybridization assembly: The TEM-Apt conjugate is hybridized with poly-T-AuNR through base complementary pairing to obtain the multifunctional nano-formulation TEM-Apt-AuNR with thrombin targeting.

10. The application of a multifunctional nano-formulation with thrombin targeting properties in the preparation of thrombosis diagnostic and therapeutic products, wherein the nano-formulation comprises a gold nanorod matrix, a poly-T oligonucleotide sequence, and a TEM-Apt conjugate; and the thrombosis diagnostic and therapeutic products comprise one or more of the following: CT contrast agents, MR contrast agents, thrombus active site recognition reagents, photothermal thrombolytic reagents, reactive oxygen species scavenging reagents, and anti-inflammatory and synergistic antithrombotic reagents.

11. The application according to claim 10, characterized in that, The thrombosis diagnosis and treatment product is used for the diagnosis and treatment of at least one of the following thrombotic diseases: ischemic stroke, myocardial infarction, and pulmonary embolism.

12. The application according to claim 10, characterized in that, The photothermal thrombolytic reagent achieves photothermal ablation of thrombi through irradiation with an 808nm near-infrared laser.

13. The application according to claim 10, characterized in that, The CT contrast agent, used in conjunction with a photon counting computed tomography (CT) scanner, enables quantitative detection of the concentration of gold nanorods within the thrombus; the quantitative results of the gold nanorod concentration are used to guide the implementation of photothermal thrombolysis.

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