Application of tumor-associated macrophage targeting nanoprobe in ultrasensitive magnetic resonance molecular imaging of in-vivo in-situ lung cancer

Abstract

The invention discloses an application of a tumor-associated macrophage targeting nanoprobe in ultrasensitive magnetic resonance molecular imaging of in-vivo in-situ lung cancer. The tumor-associated macrophage targeting nanoprobe prepared by the method provided by the invention is loaded with water-insoluble supramolecular celopphane, so that Xe'molecular cage 'celopphane can be uniformly dispersed in water and a biological system, and is used for in-vivo hyperpolarized 129Xe magnetic resonance imaging. According to the nanoprobe, mannose modification is carried out on the surface of the nanoprobe, so that the nanoprobe can effectively target tumor-related macrophages in a tumor area, and targeted detection of in-situ lung cancer tumors is realized; the surface of the nanoprobe is subjected to near-infrared fluorescence molecular modification, so that fluorescence / magnetic resonance bimodal detection of solid tumors can be realized. In combination with the advantage that local drug concentration can be improved by pulmonary drug delivery, a strong 129Xe magnetic resonance signal of the developed probe can be directly detected in vivo, and in-vivo 129Xe magnetic resonance molecular imaging is realized on an in-situ lung cancer model mouse through a direct sampling imaging mode.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of magnetic resonance imaging, and particularly relates to application of a tumor-associated macrophage-targeted nano probe in in vivo in situ lung cancer super-sensitive magnetic resonance molecular imaging. BACKGROUND

[0002] Tumor-associated macrophages (TAM) are mainly derived from peripheral blood mononuclear cells and infiltrate tumors, and are one of the most abundant infiltrating leukocytes in tumor tissues, participating in the construction of tumor immune microenvironments and participating in the regulation of various complex immune responses of tumors. Tumor-associated macrophages have high plasticity, and under the stimulation of corresponding cytokines, tumor-associated macrophages will polarize to M1 phenotype and M2 phenotype. M1 macrophages have phagocytosis, foreign body clearance and anti-tumor effects, while M2 macrophages mainly play an anti-inflammatory role and promote the occurrence, development and infiltration of tumors. The number of M2 tumor-associated macrophages is significantly higher than that of M1 tumor-associated macrophages at the site of solid tumors, and by targeting the mannose receptor (CD206) highly expressed on the surface of M2 tumor-associated macrophages, precise and efficient targeting of tumors can be achieved.

[0003] Magnetic resonance imaging is one of the most commonly used methods for disease diagnosis in clinical practice. Compared with other medical imaging methods such as CT and PET, it has the advantages of non-invasiveness, no ionizing radiation, high tissue penetration and tissue contrast, and can provide rich diagnostic information. However, the traditional magnetic resonance imaging signal is derived from the protons of water in the body, and there is strong background signal interference in the body, and the sensitivity is low, which greatly limits the further application of magnetic resonance imaging in clinical diagnosis. Hyperpolarized 129 Xe magnetic resonance imaging 129 (Xe MRI) provides a new technical means to solve the above problems. 129 Xe MRI uses hyperpolarized 129 Xe gas as the signal source. By spin-exchange optical pumping technology, the 129 Xe is hyperpolarized, which can increase its sensitivity by more than 50,000 times compared to the thermal equilibrium state. 129 Xe MRI can successfully realize the MRI visualization of the lung by virtue of its ultra-high sensitivity advantage, which can offset the influence of low tissue density in the lung. 129 Xe has good liposolubility and chemical shift sensitivity, and can be used to quantitatively visualize and evaluate the changes in ventilation function, microstructure and gas-blood exchange function of patients with lung diseases. However, 129 Xe is an inert atom and is difficult to bind to specific small molecules, proteins or RNA, etc.

[0004] In order to solve this problem, researchers have developed a series of probes that can capture 129The "cage" of Xe, and the detection of specific target analytes is achieved by targeting group modification of the cage. Among them, cucurbituril is a cage-shaped organic molecule, and 129 Xe has high affinity, 129 Xe combined with cucurbituril produces a new chemical shift, which is 60ppm different from the gaseous 129 Xe chemical shift. At present, based on cucurbituril molecules, various functional hyperpolarized 129 Xe molecular probes have been designed and developed, and the detection of proteins, enzymes, nucleic acids, thiols and other life substances and metal ions has been achieved. However, the existing 129 Xe molecular probes use indirect magnetic resonance sampling signals, such as chemical exchange saturation transfer (Chemical exchange saturation transfer, CEST), and the sampling time is long. The magnetic resonance signal of hyperpolarized 129 Xe in vivo is constantly relaxing, and the stability of the signal intensity cannot be guaranteed, making it difficult to use Xe CEST MRI in vivo. 129 In addition, cucurbituril has poor water solubility and biocompatibility, and it is difficult to be used directly in vivo, and the detection of analytes is limited to the solution and cell level. For the above reasons, so far, there has been no report on 129 Xe molecular probes at the level of in vivo. SUMMARY

[0005] The purpose of the present application is to overcome the shortcomings of the prior art, and provide a tumor-associated macrophage-targeted nanoprobe (Tumor-Associated Macrophages-Targeted Nanoparticles, TAMT NP) for in vivo hyperpolarized 129 Xe magnetic resonance molecular imaging of lung cancer. The TAMT NP probe constructed in the present application loads drug molecules cucurbituril, which retains the 129 Xe magnetic resonance properties of cucurbituril, and improves its biocompatibility, which can be used for in vivo 129 Xe magnetic resonance imaging. The mannose modified on the surface of the nanoprobe can effectively target the tumor-associated macrophages in the tumor area, so as to realize the targeted detection of solid tumors. Further combined with pulmonary drug delivery, the advantage of increasing local drug concentration can be achieved, and the targeted 129 Xe magnetic resonance molecular imaging of lung cancer in situ lung cancer model mice is successfully realized. A new, non-radiation, non-invasive molecular imaging detection method for the diagnosis of lung cancer in the future is provided.

[0006] The technical scheme adopted by the present application to achieve the above-mentioned purposes is as follows:

[0007] A tumor-associated macrophage-targeting nanoprobe TAMT NP, comprising graphene quantum dot-linear polyethylene glycol-adamantane, cryptophane Cryp, eight-arm polyethylene glycol-cyclodextrin, mannose-polyethylene glycol-adamantane,

[0008] The graphene quantum dot of the graphene quantum dot-linear polyethylene glycol-adamantane is connected with the cryptophane Cryp through π-π conjugation, and a mesh structure is formed by using PEG self-assembly cross-linking to wrap the cryptophane therein; the mass ratio of the graphene quantum dot-linear polyethylene glycol-adamantane to the cryptophane Cryp is 1:1.

[0009] The graphene quantum dot-linear polyethylene glycol-adamantane, the eight-arm polyethylene glycol-cyclodextrin, and the mannose-polyethylene glycol-adamantane are self-assembled according to the mass ratio.

[0010] The tumor-associated macrophage-targeting nanoprobe TAMT NP further comprises Cy5.5-linear polyethylene glycol-adamantane, and the mass ratio of the Cy5.5-linear polyethylene glycol-adamantane to the graphene quantum dot-linear polyethylene glycol-adamantane is 2:5.

[0011] The molecular formula of the cryptophane (Cryp, full name cryptophane) includes: five compounds shown in formula (1), a compound shown in formula (2), and a compound shown in formula (3).

[0012]

[0013] In formula 2, Y is OZO; -CH2ArCH2-, -CH2CH=CHCH2-, -CH2=CCH2-, -OCH2=CC=CCH2O-, -(OCH2CH2)O(OCH2CH2)-; Z=(CH2)n;

[0014] M is a transition metal;

[0015] L is a ligand;

[0016] R1 and R2 are independently H, (C1-C3)alkyl or (C1-C3)alkoxy X is an anionic group; m is an integer from 1 to 6;

[0017] and each n is independently 1 or 2;

[0018]

[0019] The average hydrated particle size of the tumor-associated macrophage-targeting nanoprobe TAMT NP is 143±6.5 nm.

[0020] The average hydrated particle size of the tumor-associated macrophage-targeting nanoprobe TAMT NP is 147±5.2 nm.

[0021] The synthesis steps of the tumor-associated macrophage targeting nanoprobe include: mixing graphene quantum dots-linear polyethylene glycol-adamantane and Holeban Cryp in equal amounts in a PBS solution, then slowly adding an eight-arm polyethylene glycol-cyclodextrin solution until the mass of the eight-arm polyethylene glycol-cyclodextrin is 3 times that of the graphene quantum dots-linear polyethylene glycol-adamantane, stirring at room temperature for 12 hours, then adding a mannose-linear polyethylene glycol-adamantane solution, and stirring at room temperature for 12 hours.

[0022] The synthesis steps of the tumor-associated macrophage targeting nanoprobe include: mixing graphene quantum dots-linear polyethylene glycol-adamantane and Holeban Cryp in equal amounts in a PBS solution, then slowly adding an eight-arm polyethylene glycol-cyclodextrin solution until the mass of the eight-arm polyethylene glycol-cyclodextrin is 3 times that of the graphene quantum dots-linear polyethylene glycol-adamantane, stirring at room temperature for 12 hours, then adding a mannose-linear polyethylene glycol-adamantane solution, and stirring at room temperature for 12 hours.

[0023] Then, a Cy5.5-linear polyethylene glycol-adamantane solution is added, and stirring is continued for 12 hours, and the mass of the Cy5.5-linear polyethylene glycol-adamantane in the Cy5.5-linear polyethylene glycol-adamantane solution is 2 / 5 of that of the graphene quantum dots-linear polyethylene glycol-adamantane.

[0024] Then, a Cy5.5-linear polyethylene glycol-adamantane solution is added, and stirring is continued for 12 hours, and the mass of the Cy5.5-linear polyethylene glycol-adamantane in the Cy5.5-linear polyethylene glycol-adamantane solution is 2 / 5 of that of the graphene quantum dots-linear polyethylene glycol-adamantane.

[0025] Further, the tumor-associated macrophage targeting nanoprobe TAMT NP is combined with hyperpolarized 129Xe to produce a strong 129Xe magnetic resonance signal. 1 2 9 The 129Xe magnetic resonance signal can be directly detected in vivo, and can meet the requirements of in vivo 129Xe magnetic resonance direct imaging. 129 129 Further, the tumor-associated macrophage targeting nanoprobe TAMT NP is combined with hyperpolarized 129Xe to produce a strong 129Xe magnetic resonance signal.

[0026] Further, the tumor-associated macrophage targeting nanoprobe TAMT NP is combined with hyperpolarized 129Xe to produce a strong 129Xe magnetic resonance signal.

[0027] ​Further, the tumor-associated macrophage-targeting nanoprobe TAMT NP is specifically combined with the tumor-associated macrophage surface receptor through the modified mannose molecules on the surface of the TAMT NP.

[0028] Further, the lung cancer model is constructed by inoculating A549-Luc cells in situ on Balb / c nude mice, and the tumor inoculation is verified by two imaging methods of bioluminescence and CT.

[0029] Further, the tumor-associated macrophage-targeting nanoprobe TAMT NP is specifically combined with the tumor-associated macrophage surface receptor through the modified mannose molecules on the surface of the TAMT NP. 129 Xe magnetic resonance signal.

[0030] Further, the tumor-associated macrophage-targeting nanoprobe 129 Xe magnetic resonance signal, and the tumor-targeting effect is good, so that the in vivo lung cancer can be detected by the direct imaging method. 129 Xe magnetic resonance molecular imaging.

[0031] An in vivo method for magnetic resonance imaging, comprising injecting the tumor-associated macrophage-targeting nanoprobe TAMT NP into an individual, ventilating the individual with xenon gas and hyperpolarized 129 Xe, and selecting 129 Xe chemical shift imaging sequence for imaging, and the imaging sequence parameters comprise:

[0032] TR=250 ms, FA=90°, sampling matrix=16x16, interpolation is 64x64, and the resolution is 0.78 mm.

[0033] In the method, pure oxygen is used to maintain the breathing of the individual, 129 Xe magnetic resonance imaging, and hyperpolarized 129 Xe is introduced.

[0034] Compared with the prior art, the present application has the beneficial effects and advantages that:

[0035] 1. The TAMT NP probe prepared in the present application can effectively load drug molecules, and the biological compatibility is improved under the premise that the Xe magnetic resonance signal intensity is not affected, so that the TAMT NP probe can be used for in vivo detection application. 129 Xe magnetic resonance signal intensity is not affected, so that the TAMT NP probe can be used for in vivo detection application.

[0036] 2. The main material of the TAMT NP probe prepared in the present application is polyethylene glycol, and when the lung is administered, the nanoparticle can effectively penetrate the mucus barrier, improve the drug delivery efficiency, and make more drugs enter the lung tissue to fully play a role.

[0037] 3. The TAMT NP probe prepared in this invention targets tumor-associated macrophages in the tumor region by modifying its surface with mannose, rather than directly targeting tumor cells. This enhances the probe's penetration efficiency in lung tissue and enables highly efficient targeting of in situ lung cancer tumor sites.

[0038] 4. The TAMT NP probe prepared in this invention, combined with lung administration, increases the concentration of probe molecules in the target area while reducing systemic toxicity.

[0039] 5. The nanoprobe prepared in this invention and hyperpolarization 129 After Xe binds, it has a strong... 129 Xe magnetic resonance signals can be detected directly in vivo, meeting the requirements for in vivo detection. 129 The requirements for direct Xe magnetic resonance imaging. Compared to existing indirect sampling methods such as CEST, this necessitates... 129 The probe for Xe magnetic resonance signals is better able to meet the signal intensity requirements in live detection, breaking through the limitations of probe use and extending the probe to live detection applications.

[0040] 6. After the nanoprobe prepared by this invention is administered to the lungs, it undergoes... 129 Xe magnetic resonance imaging has successfully achieved targeted detection of lung tumors in a mouse model of orthotopic lung cancer, marking the first time this has been achieved at the in vivo level. 129 The application of Xe molecular probes provides a novel molecular imaging method for the diagnosis of lung tumors, which is of great milestone significance. Attached Figure Description

[0041] Figure 1 Transmission electron microscopy image of a tumor-associated macrophage-targeting nanoprobe;

[0042] Figure 2 Dynamic light scattering pattern of the tumor-associated macrophage-targeting nanoprobe;

[0043] Figure 3 HPLC analysis of drug loading of succinate molecules in tumor-associated macrophage-targeting nanoprobes;

[0044] Figure 4 Nanoprobes for targeting tumor-associated macrophages 129 Xe NMR spectrum;

[0045] Figure 5 Nanoprobes for targeting tumor-associated macrophages 129 Plot showing the change in Xe NMR signal intensity over time in bronchoalveolar lavage fluid;

[0046] Figure 6 This is a bioluminescence imaging image of a mouse model of orthotopic lung cancer.

[0047] Figure 7 MicroCT imaging of a mouse model of orthotopic lung cancer;

[0048] Figure 8 After intratracheal instillation of tumor-associated macrophage-targeting nanoprobe TAMT NP into an orthotopic lung cancer model mouse, the following procedures were performed. 129 Xe magnetic resonance imaging image;

[0049] Figure 9 HE staining of lung tissue sections from mice with orthotopic lung cancer model;

[0050] Figure 10 Immunostaining of lung tissue sections from mice with orthotopic lung cancer. Detailed Implementation

[0051] The present invention will be further illustrated below with specific embodiments. These embodiments are merely illustrative of the technical concept and features of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made based on the essence of the present invention should be covered within the scope of protection of the present invention.

[0052] The term "cage" refers to a class of organic supramolecular compounds primarily studied and synthesized for molecular encapsulation and recognition. A cage, or tryptophan cage, is formed by two cup-shaped [1.1.1] ortho-cyclic aryl units connected by three or more bridges (represented as Q in the following structural formula), Q1 and Q2, which are attached to the aromatic ring of the unit. Most codons exhibit two diastereomeric forms (syn and anti), distinguished by their symmetry type. This general scheme provides a variety of choices (Q, Q1, Q2, and symmetry type) that allow modification of the shape, volume, and chemical properties of the typically hydrophobic sac inside the cage, making tryptophan suitable for encapsulating many types of small molecules and even chemical reactions.

[0053]

[0054] The main reagents and instruments used in the following examples are as follows:

[0055] The preparation method of the aril was based on the literature (J Am. Chem. Soc. 2006, 128, 6239-6246).

[0056] Nuclear magnetic resonance spectrometer (400MHz Bruker AV400 wide bore spectrometer).

[0057] 7.0T animal magnetic resonance imaging system (Bruker Biospec 70 / 20USR).

[0058] A549-Luc cells are a derivative of the human lung cancer cell line A549, where "Luc" stands for "Luciferase." Luciferase is an enzyme that emits light in the presence of a specific substrate and is commonly used as a reporter gene in biological experiments. By integrating the luciferase gene into A549 cells, researchers can utilize this cell line for a variety of biological studies, particularly in cancer research and drug screening.

[0059] Example 1

[0060] Synthesis of tumor-associated macrophage-targeting nanoprobe TAMT NP.

[0061] Experimental methods:

[0062] The specific experimental steps are as follows:

[0063] Weigh 5 mg of graphene quantum dot-linear polyethylene glycol-adamantane (GQD-PEG-Ad) fragment and 5 mg of cytoplasm (Cryp) and dissolve them in 2 mL of PBS (pH 7.4). After thorough mixing, slowly add branched polyethylene glycol-cyclodextrin (MPEG-CD) fragment (10 mL, 1.5 mg / mL) dropwise while stirring. React at room temperature for 12 hours. Then add Cy5.5-linear polyethylene glycol-adamantane (Cy5.5-PEG-Ad) fragment (1 mL, 2 mg / mL) dropwise to the reaction system and continue stirring for 12 hours. Finally, add mannose-linear polyethylene glycol-adamantane (Man-PEG-Ad) fragment (1 mL, 5 mg / mL) dropwise to the reaction system and continue stirring for another 12 hours. After the reaction is complete, dialysis purification yields the tumor-associated macrophage-targeting nanoprobe, denoted as Cryp@TAMT NP.

[0064] The Cryp@TAMT NP prepared in Example 1 was subjected to transmission electron microscopy (TEM), and the obtained TEM image is shown below. Figure 1 As shown, the nanoparticles prepared in this embodiment are spherical nanoparticles.

[0065] The Cryp@TAMT NP prepared in Example 1 was subjected to dynamic light scattering detection, and the resulting dynamic light scattering pattern is shown below. Figure 2 As shown, the average hydrated particle size is 143±6.5nm.

[0066] In Example 1, when preparing Cryp@TAMT NP, if Cy5.5-linear polyethylene glycol-adamantane is not present, the average hydrated particle size is 147±5.2 nm.

[0067] Free Cryp in the dialysate was quantified using high-performance liquid chromatography (HPLC), and the encapsulation efficiency of Cryp in the nanoparticles was calculated using an indirect method. The HPLC results are shown below. Figure 3 As shown, the calculated encapsulation efficiency of Cryp is 19.6%.

[0068] The following is used in this embodiment:

[0069] The molecular formula of graphene quantum dots-linear polyethylene glycol-adamantane (GQD-PEG-Ad) is: Prepared using the method described in CN202110076014.1, adamantane and graphene quantum dots are covalently linked to both ends of linear polyethylene glycol, with an average molecular weight of 2 kDa.

[0070] Branched polyethylene glycol-cyclodextrin (MPEG-CD), also known as eight-arm polyethylene glycol-cyclodextrin, is prepared using the method described in CN202110076014.1. The cyclodextrin is 6-benzenesulfonic acid-β-cyclodextrin, and the average molecular weight of the multi-arm polyethylene glycol is 10 Kda. The multi-arm polyethylene glycol is covalently linked to the cyclodextrin.

[0071] Cy5.5-linear polyethylene glycol-adamantane (Cy5.5-PEG-Ad) and mannose-linear polyethylene glycol-adamantane (Man-PEG-Ad) were both purchased from Chongqing Yusi Pharmaceutical Technology Co., Ltd. The average molecular weight of Cy5.5-PEG-Ad was 3101 da; the molecular weight of Man-PEG-Ad was 2452 da; and the molecular weight of linear polyethylene glycol was 2 kDa for both.

[0072]

[0073] The molecular formula of Cryp is 1; R = CH2COOH; X = H; m = 2; n = 2.

[0074] The graphene quantum dots-linear polyethylene glycol-adamantane and branched polyethylene glycol-cyclodextrin are used for self-assembly to form nanomedicine carriers. Cy5.5-linear polyethylene glycol-adamantane is a fluorescent module. After fluorescent labeling of the nanomaterials, the distribution of the nanomaterials in tumors can be detected using fluorescence imaging mode.

[0075] Example 2

[0076] Cryp@TAMT NP, a tumor-associated macrophage-targeting nanoprobe. 129 Xe NMR spectroscopy.

[0077] Experimental methods:

[0078] The specific experimental steps are as follows:

[0079] The Cryp@TAMTNP probe synthesized in Example 1 was dispersed in PBS, and 2 mL of the solution was transferred to a 10 mm NMR sample tube for analysis on a 400 MHz NMR spectrometer. 129 Xe NMR spectra were acquired using a 10mm dual-resonance probe. 129 Xe and 1H, PA BBO 400W1 / S2 BB-HD-10Z), the RF pulse frequency of the Xe core is 110.7MHz, and the temperature is set to 300K.

[0080] In the experiment, Xe-containing gas (a mixture of 10 vol% N2, 88 vol% He, and 2 vol% Xe (natural abundance)) was hyperpolarized using a permanent magnet polarizer as described in CN102364333B, and then introduced into the NMR tube of the magnetic resonance spectrometer at a flow rate of 100 mL / min. The gas flow was maintained for 20 seconds, then stopped, and a 90° pulse was applied to... 129 Xe is used to excite the sample, which is then sampled and accumulated 16 times.

[0081] Experimental results are as follows Figure 4 As shown, the signal peak of Cryp@TAMT NP is located at 60 ppm, indicating a strong direct sampling signal, while the signal peak at 194 ppm represents the dissolved state. 129 The Xe signal, the integral area of ​​the Cryp@TAMT NP signal peak is approximately that of the dissolved state. 129 The Xe signal integration area is 5 times larger.

[0082] The process by which an atomic nucleus with a magnetic moment is effectively perturbed by a radio frequency (RF) signal of a specific frequency is similar to the process of energy level transition. The Xe nucleus selects an RF pulse signal with a frequency of 110.7 MHz, extracting energy from it. The energy extracted is equal to the energy difference between the two energy states. After the RF interference is removed, the nucleus gradually returns to a stable low-energy state under thermal equilibrium and releases the RF signal, which is the signal detected by the nuclear magnetic resonance (NMR) device. The 90° pulse is used to flip the magnetic moment of the nucleus from the longitudinal direction (aligned with the main magnetic field B0) to the transverse plane (perpendicular to the B0 direction). This provides the necessary conditions for signal detection and imaging.

[0083] Example 3

[0084] Cryp@TAMT NP, a tumor-associated macrophage-targeting nanoprobe. 129 Stability testing of Xe NMR signal in bronchoalveolar lavage fluid.

[0085] Experimental methods:

[0086] The specific experimental steps are as follows:

[0087] The Cryp@TAMT NP probe synthesized in Example 1 was dispersed in bronchoalveolar lavage fluid, mixed thoroughly, and then 2 mL of the solution was transferred to a 10 mm NMR sample tube and analyzed on a 400 MHz NMR spectrometer. 129 Xe NMR spectra were acquired using a 10mm dual-resonance probe. 129 Xe and 1H, PA BBO 400W1 / S2BB-HD-10Z), the RF pulse frequency of the Xe core is 110.7MHz, and the temperature is set to 300K.

[0088] In the experiment, Xe-containing gas (a mixture of 10 vo1% N2, 88 vo1% He, and 2 vo1% Xe) was hyperpolarized by a permanent magnet polarizer as described in CN102364333B, and then introduced into the NMR tube of the magnetic resonance spectrometer at a flow rate of 100 mL / min. The gas flow was maintained for 20 seconds, then stopped, and a 90° pulse was applied to... 129 Xe is used to excite the sample, which is then sampled and accumulated 16 times.

[0089] Samples were subjected to different time points at 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, and 12 hours. 129 XeNMR spectral acquisition was performed in triplicate at each time point, and the average was calculated. The average signal intensity at hour 0 was used as 1, and the average signal intensity at other time points was normalized. The results are as follows: Figure 5 As shown in the results, although the signal intensity at other time points fluctuated slightly compared to the signal intensity at 0 hours, there was no significant difference. This indicates that the synthesized Cryp@TAMT NP is stable in bronchoalveolar lavage fluid and can be used for biological imaging via lung administration.

[0090] Example 4

[0091] Lung cancer model mice were treated with Cryp@TAMT NP via lung administration. 129 Xe magnetic resonance imaging.

[0092] 1. Construction of an orthotopic lung cancer mouse model.

[0093] Experimental methods:

[0094] The specific experimental steps are as follows:

[0095] A549-Luc cells were cultured, and when the cells were in the logarithmic growth phase, the cells were digested and collected. The cells were resuspended in PBS and washed, counted using a cell counter, and the cell density was adjusted to 4 × 10⁶ cells / year. 7 / mL, then add an equal volume of matrix gel and mix well. Anesthetize and fix nude mice using isoflurane gas. Draw 50μL of cell mixture into an insulin syringe and insert the needle perpendicularly to the body surface at a position 1.0-1.5cm above the left costal arch of the nude mouse, about 3mm, and slowly inject the cells. After stopping the needle for a few seconds, withdraw the needle.

[0096] Three weeks after modeling, the model mice were injected intraperitoneally with 0.2 mL of 15 mg / mL luciferin potassium salt solution, and bioluminescence detection was performed 15 minutes later (imaging parameters: Exposure time = Auto, Excitation filter = block, Emission filter = open). Figure 6 The image shows the bioluminescence imaging results. The imaging results show that there is a strong bioluminescence signal in the left lung of the mouse, indicating that tumor formation in the left lung was successful.

[0097] Simultaneously, MicroCT imaging was performed on the model mice to further verify the presence of a tumor in the left lung (imaging parameters: Image Pixel Size = 35µm, Filter = A1 0.5mm, Source Voltage = 50kV, Use360Rotation, Rotation Step = 0.7, Exposure = 60ms). Three-dimensional reconstruction was performed on the scan results to obtain... Figure 7 The three-dimensional CT image of the lung shown clearly shows an abnormal tumor signal area in the upper left lung (the maximum diameter of the tumor is 2 mm), which well verifies the successful construction of the in situ lung cancer model.

[0098] 2. Lung cancer model mice were treated with Cryp@TAMT NP via intrapulmonary administration. 129 Xe magnetic resonance imaging.

[0099] Experimental methods:

[0100] The specific experimental steps are as follows:

[0101] Mice with orthotopic lung cancer were endotracheally intubated and 0.2 mL of the Cryp@TAMT NP probe synthesized in Example 1 was instilled into the endotracheal tube.

[0102] Two hours after endotracheal instillation, mice were anesthetized and fixed with isoflurane gas, and tracheal intubation was performed. The intubation port was connected to the ventilator interface, and the mice were mechanically ventilated to maintain respiration (inhalation 150ms, exhalation 350ms, tidal volume calibrated to 0.4mL, high-purity oxygen was used to maintain vital signs, and 200% isoflurane gas was used to maintain the anesthetized state of the mice).

[0103] During imaging, the mice were fixed in a supine position on the animal bed and transported to the magnet center of the 7T MRI scanner. They were then controlled to inhale hyperpolarized [instrument name missing] via ventilator. 129 Xe gas, 0.5 mL hyperpolarized per inhalation 129 Exhale while holding your breath with Xe gas for 5 seconds, then exhale. Perform the breath-holding phase. 129 Xe magnetic resonance imaging. (Select) 129 Imaging was performed using a Xe chemical shift imaging sequence (imaging parameters: FOV = 50 × 50 mm). 2 TR = 250ms, FA = 90°, sampling matrix = 16×16, interpolation = 64×64, resolution = 0.78mm. 129 Xe chemical shift imaging results are as follows Figure 8 As shown, Cryp@TAMT NP's 129 The Xe MRI signal region anastomoses with the left lung tumor region and is consistent with the tumor location shown in bioluminescence and CT imaging in Example 4. This indicates that the constructed Cryp@TAMT NP can be used for targeted therapy of in vivo orthotopic lung cancer. 129 Xe magnetic resonance imaging.

[0104] After imaging was completed, the mice were dissected, and their lung tissue was removed, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and subjected to hematoxylin and eosin (HE) staining and immunostaining. The HE staining results are as follows: Figure 9 As shown, compared to normal lung tissue, the tumor region had denser and darker cell nuclei, indicating a tumor in the left lung lobe of the mouse. Immunostaining was performed at the same level to stain the F4 / 80 receptors on the surface of tumor-associated macrophages, and co-scanned with the Cy5.5 fluorescence inherent in Cryp@TAMT NP. The results are as follows... Figure 10 As shown, the tumor-associated macrophage signaling region (green light) and the Cryp@TAMT NP signaling region (red light) are co-localized, indicating that the constructed Cryp@TAMT NP can effectively target tumor-associated macrophages.

[0105] Imaging parameters:

[0106] FOV (Field of View): The field of view is 50×50mm. 2 , which represents the physical size of the imaging area.

[0107] TR (Repetition Time): 250ms, representing the time interval between two consecutive pulses, affecting the signal-to-noise ratio and contrast of the image.

[0108] FA (Flip Angle): 90°, indicating the flip angle during pulse excitation. A 90° flip can provide a higher signal strength.

[0109] Sampling matrix: 16×16, representing the number of original sampling points, which affects the resolution and signal-to-noise ratio of the image.

[0110] Interpolation: Interpolating to 64×64 is typically used to improve image resolution or for image reconstruction.

[0111] Resolution: 0.78mm, which is the actual physical size represented by each pixel in the image.

[0112] Experimental results:

[0113] The orthotopic lung cancer model constructed in Example 4 can be detected by bioluminescence imaging by injecting a luciferase substrate because the A549 cells used for modeling are labeled with the Luciferase gene. Figure 6 The results showed strong bioluminescent signals in the left lung region in both prone and supine positions, indicating that tumor cells survived and formed tumors after injection into the left lung lobe. Further validation was performed using CT, a commonly used imaging method for lung cancer screening. Three-dimensional reconstruction of the CT scan results was conducted, such as... Figure 7 As shown, the location of the in situ lung cancer tumor is clearly revealed: the tumor is located in the left lung lobe, with a maximum diameter of 2 mm. The constructed Cryp@TAMT NP probe was instilled into the in situ lung cancer model constructed in Example 4 via tracheal instillation, and the tumor was examined 2 hours after administration. 129 Xe magnetic resonance imaging, results as follows Figure 8 As shown, the Cryp@TAMT NP probe 129 The Xe MRI signal area was also located in the left lung of the mouse, and corresponded to the tumor location in bioluminescence and CT imaging. 129 Mouse lung tissue obtained after Xe magnetic resonance imaging was removed, fixed, sectioned, and subjected to histopathological staining and immunofluorescence staining. Histopathological staining results ( Figure 9 This study confirmed the presence of a solitary tumor measuring 2 mm in diameter in the left lung lobe of mice, validating the results of bioluminescence imaging and CT scans. It also demonstrated the effectiveness of the constructed Cryp@TAMT NP probe in targeting lung cancer. 129 Xe magnetic resonance imaging is accurate and reliable for this new method. Immunofluorescence staining results ( Figure 10 In the study, the Cy5.5 fluorescence signal region labeled by the Cryp@TAMT NP probe overlapped with the signal region labeled by tumor-associated macrophages, verifying that the constructed Cryp@TAMT NP probe achieves targeted detection of tumors by targeting tumor-associated macrophages. The constructed Cryp@TAMT NP probe can accurately target solid tumor regions, further confirming the effectiveness of using this probe for lung cancer detection. 129 Xe magnetic resonance imaging screening has important clinical significance.

Claims

1. A tumor-associated macrophage-targeting nanoprobe TAMT NP, characterized in that, Including graphene quantum dots-linear polyethylene glycol-adamantane, cryptanalyte Cryp, octagonal polyethylene glycol-cyclodextrin, and mannose-polyethylene glycol-adamantane. The graphene quantum dots-linear polyethylene glycol-adamantane and eight-arm polyethylene glycol-cyclodextrin self-assemble and cross-link into a mesh structure to encapsulate the Cryp within it, and the mass ratio of graphene quantum dots-linear polyethylene glycol-adamantane to Cryp is 1:

1. Graphene quantum dots, linear polyethylene glycol, adamantane, eight-arm polyethylene glycol, cyclodextrin, and mannose, polyethylene glycol, and adamantane are self-assembled at a mass ratio of 1:3:

1.

2. The tumor-associated macrophage-targeting nanoprobe TAMT NP according to claim 1, characterized in that, It also includes Cy5.5-linear polyethylene glycol-adamantane, with a mass ratio of Cy5.5-linear polyethylene glycol-adamantane to graphene quantum dots-linear polyethylene glycol-adamantane of 2:

5.

3. The tumor-associated macrophage-targeting nanoprobe TAMT NP according to claim 2, characterized in that, The average hydrated particle size of the tumor-associated macrophage-targeting nanoprobe TAMT NP is 143±6.5 nm.

4. The tumor-associated macrophage-targeting nanoprobe TAMT NP according to claim 2, characterized in that, The average hydrated particle size of the tumor-associated macrophage-targeting nanoprobe TAMT NP is 147±5.2 nm.

5. The method for preparing the tumor-associated macrophage-targeting nanoprobe TAMT NP as described in claim 1, characterized in that, Includes the following steps: Equal masses of graphene quantum dots-linear polyethylene glycol-adamantane and cytoplasmic precipitate (Cryp) were mixed in PBS solution. Then, an octagonal polyethylene glycol-cyclodextrin solution was slowly added dropwise until the mass of the octagonal polyethylene glycol-cyclodextrin reached three times the mass of the graphene quantum dots-linear polyethylene glycol-adamantane. After stirring and reacting at room temperature for 12 hours, a mannose-linear polyethylene glycol-adamantane solution was added dropwise, and the reaction was stirred and reacted at room temperature for another 12 hours. The mass of mannose-linear polyethylene glycol-adamantane in the mannose-linear polyethylene glycol-adamantane solution was equal to that of the graphene quantum dots-linear polyethylene glycol-adamantane.

6. The method for preparing the tumor-associated macrophage-targeting nanoprobe TAMT NP as described in any one of claims 2 or 3, characterized in that, Includes the following steps: Equal masses of graphene quantum dots-linear polyethylene glycol-adamantane and cyclodextrin Cryp were mixed in PBS solution, and then an eight-arm polyethylene glycol-cyclodextrin solution was slowly added dropwise until the mass of the eight-arm polyethylene glycol-cyclodextrin reached 3 times the mass of graphene quantum dots-linear polyethylene glycol-adamantane. The mixture was stirred at room temperature for 12 hours. Then, Cy5.5-linear polyethylene glycol-adamantane solution was added dropwise, and the reaction was stirred for another 12 hours. The mass of Cy5.5-linear polyethylene glycol-adamantane in the Cy5.5-linear polyethylene glycol-adamantane solution was 2 / 5 of that of graphene quantum dot-linear polyethylene glycol-adamantane. Then, a mannose-linear polyethylene glycol-adamantane solution was added dropwise, and the mixture was stirred at room temperature for 12 hours. The mass of mannose-linear polyethylene glycol-adamantane in the mannose-linear polyethylene glycol-adamantane solution was equal to that in the graphene quantum dot-linear polyethylene glycol-adamantane solution.

7. The application of the tumor-associated macrophage-targeting nanoprobe TAMT NP as described in any one of claims 1 or 2 in the preparation of magnetic resonance molecular imaging agents.

8. An in vivo method for magnetic resonance imaging, comprising, An individual containing the tumor-associated macrophage-targeting nanoprobe TAMT NP as described in any one of claims 1 or 2 was injected with xenon gas and hyperpolarized... 129 While Xe was ventilating the individual, he selected 129 Imaging was performed using Xe chemical shift imaging sequences, and the imaging sequence parameters included: TR = 250ms, FA = 90°, sampling matrix = 16×16, interpolation = 64×64, resolution = 0.78mm.

9. The method according to claim 8, characterized in that, When ventilating the individuals, pure oxygen was used to maintain their respiration. 129 Hyperpolarization in Xe magnetic resonance imaging 129 Xe.

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