Application of carbon dots in preparation of photoacoustic tomography and two-photon fluorescence dual-mode imaging contrast agent

Abstract

The application discloses application of carbon dots in preparation of photoacoustic tomography and two-photon fluorescence bimodal imaging contrast agent, and belongs to the technical field of nanobiomedicine and diagnosis and treatment. The carbon dot contrast agent takes different graphite layers as core active components, and has photoacoustic tomography capability and two-photon fluorescence microscopic imaging capability in the near infrared two regions. The application breaks the bottleneck that traditional single mode contrast agent cannot simultaneously consider imaging depth and spatial resolution, and for the first time realizes seamless combination of single-component contrast agent in centimeter-level depth three-dimensional photoacoustic tomography and micron-level high-resolution two-photon fluorescence imaging in a living body. The carbon dot contrast agent has simple chemical composition, good biocompatibility, high target accumulation rate and can be metabolized and removed through the kidney, and has wide clinical application prospects in multi-scale accurate early diagnosis and intraoperative navigation of tumors.

Description

Technical Field

[0001] This invention belongs to the field of interdisciplinary technology of nanomaterials and biomedical imaging, specifically relating to a carbon quantum dot contrast agent for photoacoustic tomography and two-photon fluorescence dual-modal imaging, its preparation method, and its application in multi-scale biological diagnosis and treatment. Background Technology

[0002] Early and accurate diagnosis of major diseases such as cancer relies on highly sensitive and high-resolution bioimaging technologies. Among numerous imaging technologies, optical imaging and photoacoustic imaging have attracted much attention due to their advantages such as being radiation-free, having high spatiotemporal resolution, and enabling real-time visualization. Two-photon fluorescence (TPF) microscopy, utilizing near-infrared nonlinear optical effects, can provide ultra-high spatial resolution at the micrometer or even sub-micrometer level, demonstrating outstanding advantages in the fine imaging of microscopic structures such as cells and microvessels. However, limited by the strong scattering and absorption of light by tissues, the penetration depth of TPF imaging is usually shallow (<1 mm), making it difficult to obtain macroscopic three-dimensional structural information of deep tumors.

[0003] In contrast, three-dimensional photoacoustic tomography (3D PAT) combines the high contrast of optical imaging with the high penetration depth of ultrasound imaging, enabling deep tissue imaging at centimeter-level depths. This makes it highly suitable for acquiring the three-dimensional anatomical structure and blood supply distribution of macroscopic tumors. However, its spatial resolution is still insufficient compared to two-photon microscopy, failing to meet the demands for detailed observation of microscopic cellular pathological structures. Therefore, organically combining 3D photoacoustic tomography with two-photon fluorescence imaging to construct a complementary dual-modal imaging system with "centimeter-level depth to micrometer-level resolution" is an effective approach to achieving accurate multi-scale diagnosis of diseases.

[0004] However, the core bottleneck in achieving this dual-modal imaging lies in the lack of an ideal single-component dual-modal contrast agent. Traditional composite contrast agents often combine photoacoustic contrast agents with two-photon fluorescent molecules through physical mixing or chemical coupling. This not only limits the chemical purity and batch stability of the materials, but also easily leads to serious problems such as inconsistent in vivo metabolic kinetics and increased potential toxicity.

[0005] Carbon quantum dots (CQDs), as a novel zero-dimensional carbon-based nanomaterial, have shown great potential in the biomedical field due to their excellent biocompatibility, resistance to photobleaching, and ease of surface modification. However, carbon dots synthesized in existing technologies often suffer from an underlying physical contradiction: a competition between fluorescence quantum yield and photothermal conversion efficiency. Photoluminescence requires efficient radiative transitions, while the generation of photoacoustic signals depends on efficient non-radiative thermal relaxation processes. This inherent conflict often limits existing carbon dots to a single imaging function, making it extremely difficult to simultaneously achieve high-brightness near-infrared two-photon fluorescence and strong photoacoustic signals on a single carbon dot nanoplatform. This severely restricts the practical application of carbon dots in deep, high-resolution dual-modal imaging.

[0006] Here, the inventors cleverly utilized the differential distribution of electronic states on the surface and inside of carbon dots with concentric multilayer graphitized structures to achieve spatial decoupling of excited-state radiative and non-radiative transition pathways. On a single carbon dot nanoplatform, they simultaneously achieved a near-infrared II fluorescence quantum yield of up to 37.8% and a high photothermal conversion efficiency of 50.3%, breaking the bottleneck of the traditional inability to simultaneously achieve luminescence and photoacoustic properties of carbon dots. Furthermore, based on the endogenous protein corona regulation mechanism, they achieved excellent tumor-targeted enrichment and efficient renal metabolic clearance.

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] The primary objective of this invention is to provide a carbon dot contrast agent for photoacoustic tomography and two-photon fluorescence dual-modal imaging, as well as its preparation method and application. This aims to solve the technical problems in the prior art, such as the lack of single-component dual-modal contrast agents and the competition between fluorescence quantum yield and photothermal conversion efficiency in traditional carbon dots, which makes it impossible to achieve both deep penetration and high-resolution microscopic imaging.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides an application of a carbon dot contrast agent in the preparation of a multimodal bioimaging reagent, wherein the multimodal bioimaging is a combination of photoacoustic tomography and two-photon fluorescence imaging; the contrast agent uses carbon dots as a single core imaging component.

[0010] The present invention also provides a dual-modal bioimaging contrast agent comprising carbon dots; the carbon dots simultaneously possess photoacoustic signal generation capability and two-photon fluorescence emission capability in the near-infrared region, for synchronous or asynchronous implementation of three-dimensional photoacoustic tomography and two-photon fluorescence microscopy.

[0011] Preferably, the near-infrared II fluorescence quantum yield of the carbon dots is 30% to 45% (e.g., 37.8%), and the photothermal conversion efficiency is 40% to 60% (e.g., 50.3%).

[0012] Preferably, the carbon dots have a concentric multilayer graphitized structure; the multilayer graphitized structure effectively separates the surface fluorescent active state from the internal / edge nonradiative thermal relaxation channels in space, thereby achieving spatial decoupling between the radiative transition pathway and the nonradiative transition pathway of the excited state.

[0013] Preferably, the hydrodynamic particle size or the average particle size measured by transmission electron microscopy of the carbon dots is 1 nm to 10 nm.

[0014] Preferably, after intravenous injection into a living organism, the contrast agent can selectively adsorb endogenous plasma proteins to form a specific protein corona, thereby mediating the selective enrichment and long-term retention of the contrast agent in tumor tissue.

[0015] This invention also provides a method for preparing the above-mentioned dual-modal bioimaging contrast agent, comprising the following steps: (1) Dissolve the precursor containing a carbon source (such as citric acid or its salts) and a nitrogen source (such as formamide, urea, diethylenetriamine, ethylenediamine) in a solvent (formic acid or other carboxylic acid) to form a homogeneous reaction solution; (2) The reaction solution is subjected to a solvothermal reaction at a temperature of 160 ℃~240 ℃ for a time of 4 h~24 h; (3) The reaction product is purified by dialysis, centrifugation or ultrafiltration to obtain the carbon dot contrast agent.

[0016] The present invention also provides a dual-modal bioimaging method, comprising the following steps: (1) Apply the dual-modal bioimaging contrast agent to the organism or ex vivo tissue to be tested; (2) Irradiate the target area with an excitation light source (such as near-infrared II laser) and synchronously or asynchronously collect the photoacoustic signal and two-photon fluorescence signal emitted by the target area; (3) A three-dimensional photoacoustic tomographic image is reconstructed based on the photoacoustic signal, and a two-photon fluorescence microscopy image is reconstructed based on the two-photon fluorescence signal.

[0017] Preferably, the three-dimensional photoacoustic tomography is used to acquire macroscopic three-dimensional structural information of tissues with a depth greater than 5 cm, and the two-photon fluorescence microscopy is used to acquire cellular or microvascular structural information with a resolution of less than 10 micrometers.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) Breaking the physical competition mechanism and realizing high dual-modal performance of single component: The present invention, through a unique concentric multilayer graphitized structure design, has for the first time realized the decoupling of excited-state radiative transition and non-radiative transition on a single-component carbon dot nanoplatform. While maintaining a photothermal conversion efficiency of up to 50.3% (providing strong photoacoustic signal), it endows the material with a near-infrared II fluorescence quantum yield of up to 37.8%, breaking the bottleneck that traditional contrast agents cannot achieve both performance. (2) Combining deep penetration and microscopic high resolution to achieve multi-scale accurate diagnosis: The present invention combines three-dimensional photoacoustic tomography (3D PAT) and two-photon fluorescence imaging (TPF) in a single contrast agent for the first time. PAT is used to realize macroscopic three-dimensional panoramic imaging of tumors and their vascular networks at a depth of centimeters, and TPF is used to realize micron-level high-resolution cell-level fine observation, providing complementary information of "macroscopic + microscopic" for early diagnosis and precise delineation of tumor boundaries. (3) Targeting of endogenous protein corona and excellent in vivo biocompatibility: The carbon dots of this invention can induce the formation of specific endogenous protein corona after intravenous injection, achieving highly efficient active / passive accumulation of tumors without ligand modification, and reaching an extremely high tumor-to-normal tissue signal ratio (I0.05) in about 16 hours. T / I N = 3.2). In addition, the carbon dots have uniform particle size, a clear metabolic pathway, and are mainly excreted through the kidneys. They have no obvious toxic side effects on major organs such as the heart, liver, spleen, lungs, and kidneys, and have extremely high potential for clinical translation. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 shows the preparation and structural characterization of carbon dots (CQDs) and their protein crowns in the embodiments of the present invention; wherein, (a) is a schematic diagram of the solvothermal synthesis process of carbon dots; (b) is an electron paramagnetic resonance (EPR) spectrum of carbon dots in water and dimethyl sulfoxide (DMSO); (c) is a representative transmission electron microscope (TEM) image of carbon dots, with the inset being a high-resolution transmission electron microscope (HRTEM) image (showing lattice fringes); (d) is a three-dimensional morphology image of carbon dots obtained by atomic force microscopy (AFM), and the elevation profile in the inset confirms that it is a uniform spherical morphology with an average height of about 3 nm; (e) is a Raman spectrum of carbon dots; (f) is a representative TEM image of carbon dot protein crowns; (g) is a dynamic light scattering (DLS) hydrodynamic particle size distribution diagram of carbon dots in water and serum; and (h) is a schematic diagram of the mechanism of carbon dot protein crown formation and structure.

[0021] Figure 2 shows the optical and photothermal properties characterization of carbon dots (CQDs) in the embodiments of the present invention; wherein, (a) is the UV-Vis-NIR absorption spectrum of carbon dots dispersed in water, serum and DMSO solution respectively; (b) and (c) are the two-dimensional fluorescence excitation-emission matrix (EEMs) spectra of carbon dots in serum and water respectively; (d) is the two-photon fluorescence (TPF) emission spectrum of carbon dots under 1080 nm femtosecond laser excitation; (e) is the emission spectrum under 1064 nm laser irradiation (power density of 1 W cm⁻¹). -2 (f) Comparison curves of photothermal heating of carbon dot aqueous solutions of different concentrations and pure water; -1 Photothermal heating curves of carbon dot aqueous solution under different laser power densities; (g) is the carbon dot aqueous solution (500 μg mL) -1 (h) The photothermal heating and natural cooling curves of the light source after 600 s of 1064 nm laser irradiation were obtained by cutting off the light source; (h) The system heat transfer time constant (τ) was calculated based on the linear regression line of the negative natural logarithm of the temperature driving force during the cooling stage and time. s =671.22s); (i) is a schematic diagram of the functional mechanism model of decoupling of radiative transition (outer layer) and non-radiative transition (equatorial layer) of carbon dots under near-infrared II (NIR-II) excitation.

[0022] Figure 3 shows the near-infrared II photoacoustic tomography (NIR-II PAT) performance evaluation of carbon dots (CQDs) in this embodiment of the invention; where (a) represents different concentrations (0, 100, 150, 200, 300 and 600 μg mL). -1(a) Photoacoustic imaging of carbon dot aqueous solution; (b) Linear correlation fitting curve of photoacoustic signal intensity and carbon dot concentration (data expressed as mean ± standard deviation); (c) Carbon dot (200 μL, concentration 1000 μg / mL) injected into the tail vein of tumor-bearing mice. -1 (d) Quantitative analysis of photoacoustic signal intensity in major organs at different time points after 1064 nm laser excitation; (d) Representative whole-body photoacoustic images of mice at different time points after intravenous injection of carbon dots under 1064 nm laser excitation (scale bar is 1 cm).

[0023] Figure 4 shows multiscale in vivo fluorescence imaging and near-infrared two-photon microscopy (NIR-II TPF) images of carbon dots (CQDs) in the embodiments of the present invention; where (a) are representative in vivo fluorescence images at different time points (0-32 h) after tail vein injection of carbon dots in tumor-bearing mice, with dashed white arrows and accompanying I T / I N Numerical values ​​show that the contrast between tumor and normal tissue gradually increases over time, reaching a peak at 16 h (scale bar 1 cm); (b) shows a carbon dot aqueous solution (500 μg mL). -1 (a) Wavelength-dependent NIR-II TPF images under 1080 nm femtosecond laser excitation, showing broad-spectrum and high-intensity fluorescence emission in the 555-645 nm spectral range (scale bar 200 μm); (b) In vivo high-resolution two-photon imaging of tumor microvessels. The composite image shows the superposition and reconstruction of the NIR-II TPF signal emitted by carbon dots (red) and the intrinsic second harmonic generation (SHG, cyan) generated by the collagen matrix surrounding the tumor. Through time-series observation (control group, 1 h and 2 h after injection), the fine structure of the tumor vascular system is clearly revealed at subcellular resolution (white arrow, scale bar 50 μm). Detailed Implementation

[0024] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the chemical reagents used in the embodiments of the present invention are commercially available analytical grade reagents. Example 1

[0025] Preparation and structural characterization of near-infrared II carbon dots (CQDs) and their protein corona (1) Solvent-thermal synthesis of near-infrared II carbon dots: 1 g of citric acid (carbon source) was completely dissolved in a mixed solvent of 4 mL formamide and 6 mL formic acid, and the solution was magnetically stirred until a homogeneous and clear solution was formed. The reaction liquid was then transferred to a 20 mL polytetrafluoroethylene-lined high-pressure reactor for a solvothermal reaction at a temperature of 180 °C for 4 h.

[0026] (2) Purification and drying: After the reaction was completed and allowed to cool naturally to room temperature, 60 mL of anhydrous ethanol was added to the resulting reaction solution to induce precipitation. The mixture was centrifuged at 8000 rpm, the supernatant was discarded, and the lower precipitate was retained. The ethanol washing and centrifugation steps were repeated three times to thoroughly remove unreacted precursors and byproducts. The lower precipitate was collected and freeze-dried to obtain a black solid powder, which is carbon dots (CQDs), with a synthesis yield of approximately 5%.

[0027] (3) Characterization of microstructure and crystal structure: As shown in Figure 1c, transmission electron microscopy (TEM) shows that CQDs exhibit a monodisperse spherical structure; high-resolution TEM (HRTEM, inset of Figure 1c) clearly observes highly ordered concentric multilayer lattice fringes.

[0028] As shown in Figure 1d, atomic force microscopy (AFM) and elevation profile (inset) confirm that CQDs have a uniform spherical morphology with an average height of about 3 nm.

[0029] As shown in Figure 1e, the Raman spectrum of the material at 1350 cm⁻¹ is [missing value]. -1 (D belt) and 1580cm -1 The (G band) exhibits characteristic absorption peaks, confirming that it has a highly graphitized carbon skeleton.

[0030] As shown in Figure 1b, electron paramagnetic resonance (EPR) spectroscopy compared the free radical and unpaired electron signals of CQDs in water and DMSO, confirming the existence of highly localized magnetic states in the equatorial plane and edge regions.

[0031] (4) Induction and structural characterization of protein crowns in vitro: CQDs (500 μg mL) -1The CQDs were incubated with mouse serum (FBS) at 37 °C for 1 h to simulate the in vivo circulatory environment. As shown in Figure 1f, TEM revealed a uniform protein coating (protein crown) on the surface of the CQDs. Dynamic light scattering (DLS) hydrodynamic particle size analysis, as shown in Figure 1g, showed that the CQDs had a particle size of approximately 3.5 nm in water, which significantly increased in size after binding to the protein crown in serum. Figure 1h illustrates the structural model of spontaneously induced endogenous protein crown formation by CQDs in blood circulation. Example 2

[0032] Verification of the photophysical / photothermal properties and "transition decoupling" mechanism of carbon QDs (1) Ultraviolet-Vis-NIR absorption spectroscopy and solvation colorimetric effect: The optical characteristics of CQDs were characterized using steady-state absorption spectroscopy, as shown in Figure 2a. The main absorption transition peak at 570 nm of CQDs remained consistent in different solvents; however, in aqueous media, its absorption cross-section in the near-infrared II (NIR-II) window was significantly amplified, laying a solid absorption spectral foundation for NIR-II biophotonics applications.

[0033] Further analysis revealed that the absorption spectra of CQDs exhibited a significant solvatochromic dependence: they displayed highly resolved discrete transition peaks in DMSO, while transforming into broadband continuous spectra in aqueous solutions. This spectral evolution is attributed to the high dielectric constant and proton properties of water, which shield long-range dipole interactions and promote relaxation of radical species, ultimately inducing significant inhomogeneous broadening.

[0034] (2) Serum environmental fluorescence quantum yield (QY) and two-photon fluorescence (TPF) performance: Enhanced fluorescence quantum yield: As shown in Figures 2b and 2c, the two-dimensional fluorescence excitation-emission matrices (EEMs) reveal that in a physiological serum environment, the luminescent centers of CQDs are mainly concentrated in the 590-650 nm region, achieving a fluorescence quantum yield (QY) as high as 37.8%, a significant enhancement of nearly 6 times compared to 6.7% in pure water. This remarkable enhancement of radiative transitions in the serum environment provides a solid foundation for its subsequent efficient nonlinear optical imaging.

[0035] Two-photon fluorescence emission: As shown in Figure 2d, under 1080 nm femtosecond laser excitation, CQDs exhibit strong two-photon fluorescence (TPF) emission with the emission center located at approximately 640 nm, confirming their strong application potential in micron-scale high-resolution two-photon microscopy imaging.

[0036] (3) Near-infrared 1064 nm photothermal conversion kinetics and photothermal conversion efficiency (PCE) calculation: The photothermal conversion performance of CQDs was evaluated within a clinical NIR-II bio-transparent window. All tests were conducted within the maximum permissible dose (MPE) limit for humans (laser power density controlled at 1 W cm⁻¹). -2 ).

[0037] Concentration and power dependence: As shown in Figure 2e, after 10 minutes of 1064 nm laser irradiation, the concentrations were 50, 100, 250, and 500 μg mL, respectively. -1 The aqueous solutions of CQDs exhibited significant temperature increases, reaching 25.4 °C, 28.6 °C, 36.4 °C, and 46.0 °C, respectively. As shown in Figure 2f, the photothermal response exhibited a strong dose- and power-density dependence.

[0038] Photothermal conversion efficiency calculation: As shown in Figure 2 (g), take 500 μg mL -1 The aqueous solution of CQDs was irradiated with a 1064 nm laser for 600 s, and the light source was cut off. The natural cooling trajectory was recorded. As shown in h in Figure 2, the characteristic cooling curve was analyzed by performing linear regression fitting on the cooling time and the negative natural logarithm of the normalized temperature difference. The photothermal conversion efficiency (PCE) of CQDs was found to be as high as 50.3%.

[0039] (4) Physical mechanism model of "spatial decoupling" of radiative / nonradiative transitions: To elucidate the underlying photophysical mechanism of the aforementioned dual-modal imaging, this invention proposes a unified structure-function model (as shown in i in Figure 2): Outer graphite layer (radiation center): The outer graphitized structure of carbon dots serves as the main radiative transition center. Its surface engineered state promotes strong red light emission and efficient two-photon absorption, dominating two-photon fluorescence microscopy (TPF). Equatorial graphite layer (non-radiative oscillator): The equatorial graphitized layer of carbon dots acts as a highly efficient non-radiative oscillator, which can efficiently absorb NIR-II photons and generate a significant thermal gradient, dominating photoacoustic tomography (PAT).

[0040] Technical effect: The inherent structural duality strategically decouples the two imaging contrasts in space, successfully breaking the bottleneck of the traditional contrast agent's "incompatibility between luminescence and photothermal effect", and providing a rational material design principle for subsequent dual-modal precision diagnosis and treatment. Example 3

[0041] Near-infrared II 3D photoacoustic tomography (NIR-II 3D PAT) performance and in vivo whole-body metabolic kinetic monitoring (1) In vitro 1064 nm near-infrared II photoacoustic signal and quantitative linear response: The in vitro performance of engineered carbon dots (CQDs) as a contrast agent for high-contrast photoacoustic tomography (PAT) in near-infrared II (NIR-II) region was evaluated.

[0042] Under 1064 nm laser pulse excitation, CQDs exhibit extremely strong and stable photoacoustic (PA) signal energy output.

[0043] As shown in Figures 3a and 3b, different concentrations (0, 100, 150, 200, 300, 600 μg mL) were studied. -1 The photoacoustic signal intensity of the CQDs solution was measured. Results showed that at concentrations up to 600 μg / mL... -1 Within a wide concentration range, the photoacoustic signal intensity exhibits a strong linear positive correlation with the mass concentration of CQDs, with a linear correlation coefficient as high as R0. 2 =0.99. This highly predictable linear response characteristic provides a solid and reliable physical basis for subsequent precise quantitative diagnosis and treatment of deep tissues in vivo.

[0044] (2) 360° panoramic in vivo 3D photoacoustic tomography and high-resolution reconstruction of the whole-body vascular network: To overcome the limitations of depth and angular blind spots in traditional unidirectional photoacoustic imaging, this embodiment employs a 360° rotating scanning configuration, simultaneously combining four orthogonal laser beams for high-sensitivity acquisition of integrated ultrasound signals, thereby achieving high-fidelity three-dimensional visualization of the entire mouse body. This system was used to perform live mouse injections (200 μL via tail vein, 1 mg / mL) -1 Real-time longitudinal monitoring of the systemic biodistribution and excretion dynamics of CQDs in solution (in vivo): Before drug administration: Only weak endogenous hemoglobin photoacoustic background signals from background tissues and major blood vessels could be detected throughout the mouse body (as shown in Figure 3c). Post-injection: CQDs rapidly diffuse throughout the body via the bloodstream, significantly enhancing the photoacoustic contrast of the entire systemic circulation. Thanks to their high photothermal-photoacoustic conversion efficiency and deep penetration capability at the NIR-II window, the 3D reconstructed photoacoustic images clearly delineate an unprecedented microvascular network distribution map with extremely high spatial resolution; Timeliness and imaging depth: The fine branching structures of superficial subcutaneous microvessels and deep main blood vessels are visualized with high contrast, and the high-resolution vascular imaging effect remains clearly visible throughout the continuous observation period of up to 180 minutes, demonstrating excellent blood circulation stability.

[0045] (3) Organ clearance dynamics and targeted metabolic mechanisms mediated by endogenous protein corona: Longitudinal 3D PAT whole-body imaging further dynamically elucidated the metabolic clearance pathways and organ distribution characteristics of CQDs in vivo: Spatiotemporal distribution evolution: Between 30 and 180 minutes after injection, PA signals in the systemic blood vessels gradually shifted to excretory and immune organs, and strong photoacoustic signals began to selectively accumulate in the kidney and spleen regions (Figure 3c and Figure 3d). Targeted substructure analysis: Although a very small number of nanoparticles were taken up and retained by the liver endothelial reticulum system, the amplification of photoacoustic signals was significantly concentrated in the renal cortex and splenic red pulp regions.

[0046] Protein Corona-Mediated Mechanism: This specific deep organ enrichment behavior is closely related to the endogenous protein corona spontaneously induced in vivo by CQDs in Example 1, confirming that the protein corona effectively alters the biocompatibility and interfacial physicochemical properties of nanoparticles, mediating efficient systemic circulatory transport and specific renal / splenic metabolic clearance.

[0047] (4) Summary of Implementation Examples and Technical Effects: This embodiment demonstrates that CQDs engineered with equatorial graphite layers possess the dual capabilities of high-resolution whole-body vascular mapping and dynamic imaging of deep tissues and organs, truly showcasing their enormous application potential as a next-generation precision multimodal bio-optical imaging contrast agent. Example 4

[0048] Multiscale in vivo near-infrared II (NIR-II) fluorescence imaging and high-resolution two-photon microscopy (TPF) (1) Macroscopic in vivo near-infrared II region tumor targeting dynamics and endogenous protein corona-mediated mechanism: By fully utilizing the high fluorescence quantum yield and large two-photon absorption cross-section characteristics of engineered carbon dots (CQDs) in graphite layers, multi-scale in vivo fluorescence imaging studies were conducted under the NIR-II window.

[0049] Spatiotemporal distribution evolution: At the macroscopic level, longitudinal in vivo fluorescence imaging was used to monitor the tail vein injection of CQDs (200 μL, 1 mg / mL). -1 The tumor-targeting enrichment capacity and systemic biodistribution evolution of CQDs in tumor-bearing mice were shown in Figure 4a. After intravenous administration, the fluorescence signal in the tumor region showed a significant increasing accumulation trend over time.

[0050] Signal ratio quantitative analysis: The ratio of fluorescence intensity between tumor and normal tissue (I0.05) T / I N It gradually climbed from 0.8 at 0 h and reached a very high peak of 3.2 at 16 h.

[0051] Protein crown-mediated mechanism: This significant tumor enrichment achieved without any exogenous targeting ligand modification confirms that the endogenous protein crown spontaneously induced by CQDs in vivo can greatly mediate and promote the efficient passive / active targeted accumulation and high-contrast imaging of contrast agents in tumor lesions through enhanced permeation and retention effects (EPR effect) and endogenous homing mechanisms.

[0052] (2) Wavelength-dependent nonlinear spectral characteristics of near-infrared II two-photon fluorescence (NIR-II TPF): The nonlinear optical properties of the material were thoroughly evaluated using wavelength-dependent NIR-II TPF testing.

[0053] As shown in Figure 4b, under 1080 nm femtosecond laser pulse excitation, CQDs aqueous solution (500 μg mL) -1 It exhibits a robust emission pattern that is tunable over a wide spectral range of 555-645 nm.

[0054] The fluorescence emission intensity reaches its maximum peak at approximately 645 nm. This broadband, high-intensity two-photon fluorescence emission characteristic provides an ideal optical spectral window for high-resolution microscopic imaging of deep tissues with extremely low background interference.

[0055] (3) Applications of subcellular high-resolution in vivo tumor microvascular two-photon microscopy and angiography: To demonstrate the superior spatial resolution of the nanoplatform of this invention, two-photon microscopy was performed on microvessels of living tumors (as shown in Figure 4c).

[0056] Co-reconstruction imaging: By co-localizing and fusing the NIR-II two-photon fluorescence signal (TPF, displayed in red in pseudocolor) from CQDs with the intrinsic second harmonic signal (SHG, displayed in cyan in pseudocolor) from collagen fibers surrounding the tumor, high-contrast, dual-channel real-time visualization of the tumor microenvironment was achieved.

[0057] High-contrast imaging over time: Before injection of CQDs (Control), only cyan SHG signals from the extracellular matrix could be observed; after injection of CQDs (1 h and 2 h), the TPF red channel revealed the fine three-dimensional architecture of the tumor vascular system with extremely clear subcellular resolution.

[0058] Microcirculation and capillary characterization: In particular, CQDs enable the extremely fine capillary network and internal hemodynamics to be clearly characterized with high fidelity within 1 h and 2 h after injection, highlighting the ultra-high luminescence brightness and excellent photostability of the material due to its concentric multilayer graphitized structure.

[0059] (4) Summary of Implementation Examples and Technical Effects: This embodiment demonstrates that CQDs have the ability to analyze deep blood vessels and microscopic tumor lesions with high fidelity, and confirms their great clinical application potential in fields such as precision angiography and guided surgical interventions.

[0060] Comparative Example 1: A comparative experiment on ordinary carbon dots without decoupled structures was conducted using a conventional hydrothermal method to synthesize ordinary carbon dots (C-Dots) without concentric multilayer graphitized structures. Tests showed that the photothermal conversion efficiency (1064 nm excitation) of these ordinary carbon dots was only 10.2%, the fluorescence quantum yield in serum was only 0.5%, and there was no high-intensity two-photon fluorescence signal. When injected into tumor-bearing mice according to the method in Example 4, the tumor was essentially indistinguishable from normal tissue at 16 h, failing to achieve high-contrast imaging of microvessels and centimeter-level photoacoustic reconstruction. This fully demonstrates the unexpected technical effects of the concentric multilayer graphitized structure of this invention in achieving spatial decoupling of radiative / non-radiative transitions and providing "photoacoustic tomography / two-photon" dual-modal multi-scale imaging.

[0061] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. The application of a carbon dot contrast agent in the preparation of a dual-modal bioimaging reagent, characterized in that, The dual-modal bioimaging is a combination of photoacoustic tomography and two-photon fluorescence imaging; the contrast agent uses carbon dots as a single core imaging component.

2. A dual-modal bioimaging contrast agent, characterized in that, It includes carbon dots; the carbon dots simultaneously possess photoacoustic signal generation capability and two-photon fluorescence emission capability in the near-infrared region, and are used to simultaneously perform three-dimensional photoacoustic tomography and two-photon fluorescence microscopy.

3. The dual-modal bioimaging contrast agent according to claim 2, characterized in that: The near-infrared II fluorescence quantum yield of the carbon dots is 30%–45%, and the photothermal conversion efficiency is 40%–60%.

4. The dual-modal bioimaging contrast agent according to claim 2, characterized in that: The carbon dots have a concentric multilayer graphitized structure; the multilayer graphitized structure enables spatial decoupling between the radiative and non-radiative transition pathways of their excited states.

5. The dual-modal bioimaging contrast agent according to claim 2, characterized in that: The average particle size of the carbon dots is 1 nm to 10 nm.

6. A method for preparing a dual-modal bioimaging contrast agent as described in any one of claims 2-5, characterized in that, Includes the following steps: (1) Dissolve the precursor containing carbon and nitrogen sources in a solvent to form a homogeneous reaction solution; (2) The reaction solution is subjected to a solvothermal reaction at a temperature of 160 ℃~240 ℃ for a time of 4 h~24 h; (3) The reaction product is purified to obtain the contrast agent.

7. The preparation method according to claim 6, characterized in that: The carbon source in step (1) includes citric acid or its salts; the nitrogen source includes at least one of formamide, urea, diethylenetriamine, and ethylenediamine; and the solvent includes formic acid or other carboxylic acids.

8. A dual-modal bioimaging method, characterized in that, Includes the following steps: (1) Applying the dual-modal bioimaging contrast agent of any one of claims 2-5 to an isolated tissue; (2) Irradiate the target area with an excitation light source and synchronously or asynchronously collect the photoacoustic signal and two-photon fluorescence signal of the target area; (3) A three-dimensional photoacoustic tomographic image is reconstructed based on the photoacoustic signal, and a two-photon fluorescence microscopy image is reconstructed based on the two-photon fluorescence signal.

9. The dual-modal bioimaging method according to claim 8, characterized in that: The three-dimensional photoacoustic tomography image is used to obtain macroscopic three-dimensional structural information of tissue at the centimeter level, and the two-photon fluorescence microscopy image is used to obtain microscopic structural information of cells or blood vessels at the micrometer level.

Images