A long afterglow / nuclear magnetic nanoprobes based on ape1 amplification for miRNA-21 signal amplification, preparation method
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HUNAN UNIV
- Filing Date
- 2026-04-07
- Publication Date
- 2026-08-07
AI Technical Summary
上述方法普遍存在以下不足：（1）多依赖体外检测，难以实现活体、实时和动态监测；（2）检测过程复杂，样品前处理繁琐，难以用于肿瘤体内成像；（3）在复杂生物环境中易受背景信号干扰，灵敏度和特异性受限
[0044]（1）本发明提供了一种长余辉/核磁纳米探针，长余辉/核磁纳米探针通过DNA碱基互补配对作用组装，该探针仅在肿瘤处特异性激活，最大限度地减少了自发荧光背景，提供持续发光，并为miRNA21成像提供解剖信息，有效地改善了传统技术的固有局限性。
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Figure CN122516397A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical imaging and nucleic acid molecular probe technology, specifically relating to a long-persistence / NMR nanoprobe based on APE1 amplification for miRNA-21 signal amplification and its preparation method. Background Technology
[0002] MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs approximately 18–25 nucleotides in length that regulate gene expression at the posttranscriptional level and play a crucial role in tumorigenesis, development, invasion, and metastasis. Among them, miRNA-21 is one of the most widely studied oncogenic miRNAs, exhibiting significantly high expression in various solid tumors (such as breast cancer, lung cancer, and colorectal cancer), and is considered an important molecular marker for tumor diagnosis, prognostic assessment, and efficacy monitoring. Therefore, achieving highly sensitive, specific, and real-time visualized detection of miRNA-21 in the in vivo tumor microenvironment is of great significance for precision tumor diagnosis and treatment.
[0003] Currently, the main methods for detecting miRNA-21 include qRT-PCR, Northern blot, in situ hybridization, and in vitro detection techniques based on fluorescence or electrochemistry. These methods generally have the following shortcomings: (1) They rely heavily on in vitro detection, making it difficult to achieve in vivo, real-time, and dynamic monitoring; (2) The detection process is complex, and sample pretreatment is cumbersome, making it difficult to use for in vivo tumor imaging; (3) They are easily affected by background signals in complex biological environments, limiting their sensitivity and specificity.
[0004] In recent years, nanoprobe technology based on molecular imaging has provided a new research direction for the in vivo detection of miRNAs. Among them, magnetic resonance imaging (MRI) is widely used in tumor imaging research due to its good tissue penetration and spatial resolution. However, traditional MRI probes have low sensitivity and are difficult to respond effectively to low-abundance miRNAs. Although fluorescence imaging has high sensitivity, it generally suffers from strong autofluorescence interference and limited penetration depth, which seriously restricts its application in imaging deep tumor tissues.
[0005] Long-persistence imaging technology, due to its ability to continue emitting light after the excitation light is removed, can effectively eliminate tissue autofluorescence interference, showing significant advantages in the field of bioimaging. However, most existing long-persistence probes are single-modal imaging systems, lacking specific response mechanisms for specific miRNAs, and have limited signal amplification capabilities, making it difficult to meet the high-sensitivity detection requirements of low-abundance miRNAs.
[0006] On the other hand, enzyme-mediated signal amplification strategies, as a highly efficient molecular amplification mechanism, have been extensively studied in the field of nucleic acid detection. However, existing technologies are mostly limited to in vitro systems and are difficult to effectively integrate with in vivo imaging probes. There is still a lack of a nanoprobe system that can achieve signal cascade amplification through enzyme-mediated mechanisms in the tumor microenvironment, while also possessing multimodal imaging capabilities. Summary of the Invention
[0007] To address the shortcomings of existing technologies, the present invention aims to provide a long-persistence / NMR nanoprobe based on APE1 amplification for miRNA-21 signal amplification and its preparation method. This probe can be specifically triggered by miRNA-21 in the tumor microenvironment and achieve signal amplification through the APE1 enzyme-mediated mechanism. It combines the high signal-to-noise ratio advantage of long-persistence imaging with the deep tissue imaging capability of NMR imaging to achieve highly sensitive, specific, and real-time dynamic visualization monitoring of miRNA-21, thereby providing a reliable molecular imaging tool for the precise diagnosis and treatment assessment of tumors.
[0008] To achieve the above objectives, the present invention provides the following technical solution:
[0009] This invention provides a long-persistence / NMR nanoprobe based on APE1 amplification for miRNA-21 signal amplification, with long-persistence nanoparticles as the core and magnetic nanoparticles as satellite units;
[0010] The surface of the long-afterglow nanoparticles is linked to amino-modified DNA1 via an amide coupling reaction to obtain long-afterglow nanoparticles-DNA1.
[0011] The magnetic nanoparticles are modified with amino-substituted hydroquinone (HQ-N3) on their surface, and then reacted with alkynyl-modified DNA2 via a click chemical reaction to obtain magnetic nanoparticles@HQ-N3-DNA2.
[0012] The long-afterglow nanoparticle-DNA1 and the magnetic nanoparticle@HQ-N3-DNA2 were assembled by hybridization of the two DNAs to obtain the long-afterglow / NMR nanoprobe.
[0013] The nucleotide sequence of DNA1 is: CGCCAGTTGT (SEQ ID NO.1);
[0014] The nucleotide sequence of the DNA2 is as follows:
[0015] GCGGTCAACATCA / dSpacer / GTCTGATA.
[0016] The nucleotide sequence of the DNA2 includes: a complementary sequence that undergoes a strand substitution (SDR) reaction with miRNA21 and an active site that is recognized and cleaved by the APE1 enzyme.
[0017] In the preferred embodiment, a covalent CO-NH bond is formed between the long-afterglow nanoparticles -COOH and DNA1-NH2.
[0018] In a preferred embodiment, the 3' modified amino group of the DNA1;
[0019] The 5' modified alkyne group of the DNA2 has a nucleotide sequence that specifically binds to miRNA21 in more than 60% of cases, and the terminal is modified with an APE1 active site.
[0020] In a preferred embodiment, the long afterglow / NMR nanoprobe has a nucleus-satellite structure with a particle size of 65~225 nm.
[0021] More preferably, the particle size of the nanoprobe is 85~150 nm.
[0022] In a preferred embodiment, the long afterglow nanoparticles include, but are not limited to, TA NPs, MEH NPs, and PFO NPs.
[0023] In a preferred embodiment, the long afterglow nanoparticles have a uniform spherical structure with a particle size of 15~85 nm.
[0024] More preferably, the particle size of the long afterglow nanoparticles is 20~55 nm.
[0025] In a preferred embodiment, the magnetic nanoparticles include, but are not limited to, FeOx, FeZnOx, and FeMnOx.
[0026] In a preferred embodiment, the magnetic nanoparticles have a uniform spherical structure with a particle size of 2~15 nm.
[0027] More preferably, the magnetic nanoparticles have a particle size of 3 to 7 nm.
[0028] As a whole inventive concept, this invention also provides a method for preparing the long-persistence / NMR nanoprobe based on APE1 amplification for miRNA-21 signal amplification, comprising:
[0029] (1) Synthesis of long afterglow nanoparticles;
[0030] (2) Synthesis of long afterglow nanoparticles-DNA1: The long afterglow nanoparticles obtained in step (1) were mixed with amino-modified DNA1 in PBS buffer, and then EDC was added. The reaction was carried out under stirring and purified to obtain the DNA1.
[0031] (3) Synthesis of magnetic nanoparticles;
[0032] (4) Magnetic nanoparticles @HQ-N3: The magnetic nanoparticles obtained in step (3) are mixed with N,N-dimethylformamide, and p-PEG-N3, BMPA, ascorbic acid (AA), and amino-substituted hydroquinone (HQ-N3) are added to the reaction system. The mixture is shaken at a set temperature. After the reaction is completed, the product is precipitated and then purified by centrifugation and filtration to obtain magnetic nanoparticles @HQ-N3.
[0033] (5) Magnetic nanoparticles @HQ-N3-DNA2: CuSO4, tri-hydroxypropyltriazolamide (THPTA), ascorbic acid (AA), alkynyl-modified DNA2 and magnetic nanoparticles @HQ-N3 obtained in step (4) were mixed, purified by ultrafiltration, and then dispersed in ultrapure water;
[0034] (6) Assembly: The long afterglow nanoparticles-DNA1 obtained in step (2) and the magnetic nanoparticles @HQ-N3-DNA2 obtained in step (5) are mixed in DPBS containing NaCl and MgCl2 to obtain the long afterglow / NMR nanoprobe.
[0035] Furthermore, in step (2), the molar ratio of long afterglow nanoparticles to DNA1 is (6~8):1, the pH value of PBS buffer is 7.4, the final concentration of EDC is 250 mM, the reaction time is 4~12 h, the ultrafiltration speed is 6000 rpm, the ultrafiltration time is 5 min, and the ultrapure water is used for purification and cycled three times.
[0036] Furthermore, in step (4), the mass ratio of magnetic nanoparticles, p-PEG-N3, BMPA, AA and HQ-N3 is (6~8):5:(160~170):(25~32):(0.05~0.09); the incubation temperature is 30 ℃, the shaking time is 12~15 h, the ultrafiltration speed is 6000 rpm, the ultrafiltration time is 5 min, and the ultrapure water is purified and circulated three times.
[0037] Furthermore, in step (5), the molar ratio of CuSO4, tri-hydroxypropyltriazolamide (THPTA) and ascorbic acid (AA) is (1~1.5):4:10; the incubation temperature is 37 ℃, the shaking time is 4~12 h, the ultrafiltration speed is 6000 rpm, the ultrafiltration time is 5 min, and the ultrapure water is used for purification and circulation three times.
[0038] Furthermore, in step (6), the assembly mass ratio of long afterglow nanoparticles-DNA1 and magnetic nanoparticles@HQ-N3-DNA2 is (32~40):1; the concentrations of NaCl and MgCl2 are 50 mM and 2.5 mM, respectively; the incubation temperature is 37 ℃; the shaking time is 12~24 h; the ultrafiltration speed is 6000 rpm; the ultrafiltration time is 5 min; and the ultrapure water is used for purification and circulation three times.
[0039] The present invention also provides an application based on the long-persistence / NMR nanoprobe, which is used to prepare contrast agents for tumor imaging; the probe based on the long-persistence / NMR nanoprobe can specifically react with miRNA21 in the tumor microenvironment and release the "binding" of the long-persistence nanoparticles to accurately perform tumor afterglow imaging.
[0040] This invention also provides an application based on the long-persistence / NMR nanoprobe, which is used to prepare contrast agents for tumor imaging; by using changes in T1 magnetic resonance images to precisely "activate" the long-persistence / NMR nanoprobe in the tumor microenvironment for imaging, T1 magnetic resonance imaging of miRNA21 activation can be achieved.
[0041] This invention provides a dual-mode probe that combines anatomical images from MRI with the contrast of tumor and normal tissue obtained through afterglow imaging. The probe is assembled via DNA hybridization between afterglow and MRI units. DNA2-miRNA21 interaction triggers probe degradation, generating amplified long-persistence and T1-weighted MRI signals. Simultaneously, the metabolic inhibitor anti-miRNA21 in cells and tumors reduces miRNA21 levels, enabling real-time dynamic monitoring of miRNA21 levels in tumors using a long-persistence / MRI nanoprobe.
[0042] The principle of this invention: This invention utilizes a long-persistence / NMR nanoprobe to achieve tumor-specific triggering of its decomposition, enabling real-time dynamic monitoring of miRNA21 levels guided by long-persistence signals and magnetic resonance imaging. The long-persistence / NMR nanoprobe is assembled from functional DNA nucleic acid molecules between long-persistence nanoparticles (acting as luminescent donors) and magnetic nanoparticles (MRI contrast agents). In the tumor microenvironment, the base structure of miRNA21 can capture the DNA2 strand assembled on satellites (magnetic nanoparticles) surrounding the nucleus (long-persistence nanoparticles). In the presence of APE1, the active site at the DNA2 terminus of the formed double-stranded miRNA21-DNA2 is cleaved, causing miRNA21-DNA2 to unwind. The released miRNA21 further binds to the next DNA2 strand on the nucleus, undergoing the same process until all magnetic nanoparticles are stripped from the long-persistence nanoparticles. The released long-persistence nanoparticles and magnetic nanoparticles can then be used for long-persistence imaging and magnetic resonance imaging, respectively. Because miRNA21 and APE1 can repeatedly dissociate and cleave DNA2, the sensitivity of dual-mode probes in afterglow imaging and T1 NMR imaging is significantly improved. Due to these effects, long-persistence / NMR nanoprobes can greatly reduce the detection limit of miRNA21 in tumors, allowing for real-time visualization of miRNA21 and thus providing insights into the metabolic state of tumors. Therefore, the long-persistence / NMR nanoprobes of this invention can significantly improve the accuracy and specificity of cancer imaging and have great application potential.
[0043] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0044] (1) The present invention provides a long-persistence / NMR nanoprobe, which is assembled through DNA base complementary pairing. The probe is specifically activated only at the tumor site, minimizing the autofluorescence background, providing continuous luminescence, and providing anatomical information for miRNA21 imaging, effectively improving the inherent limitations of traditional techniques.
[0045] (2) The long-afterglow / NMR nanoprobe provided by this invention uses long-afterglow nanoparticles labeled with quencher molecules as quenchers of long-afterglow nanoparticles. Under tumor environment stimulation, miRNA21 can strip the long-afterglow nanoparticles. In this process, miRNA21 does not act as a reaction fuel. After miRNA21 is assembled with magnetic nanoparticles @HQ-N3-DNA2, DNA2 can be cleaved by APE1, and miRNA21 can be repeatedly stripped, further breaking the afterglow seal of TA NPs of long-afterglow nanoparticles. This achieves signal amplification of miRNA21 in tumors, which can effectively assess the malignancy of tumors and optimize treatment plans (the process is as follows). Figure 1 (As shown).
[0046] (3) The present invention prepares long afterglow nanoparticles TA NPs by a simple nano-coprecipitation method, which has a short preparation process, simple operation and low cost.
[0047] (4) This invention also provides the application of the long-persistence / NMR nanoprobe in tumor detection. Utilizing the superior T1 signal of the magnetic nanoparticles, magnetic resonance imaging was used to image tumors and accurately assess their malignancy. Therefore, this nanoprobe has clinical guiding significance for the diagnosis and treatment of cancer.
[0048] Instruction manual illustrations
[0049] Figure 1 A schematic diagram of the response process of the long afterglow / NMR nanoprobe of this invention to miRNA-21;
[0050] Figure 2 A diagram illustrating the fabrication process of the long afterglow / NMR nanoprobe of this invention;
[0051] Figure 3 TEM image of the long afterglow / NMR nanoprobe prepared in Example 1;
[0052] Figure 4 This refers to the afterglow quenching of long afterglow nanoparticles by magnetic nanoparticles in Example 1;
[0053] Figure 5 The particle size of the long afterglow / NMR nanoprobe in Example 2 before and after its response to miRNA-21;
[0054] Figure 6 The afterglow response of the long afterglow / NMR nanoprobe to miRNA-21 in Example 2;
[0055] Figure 7 The NMR response of the long afterglow / NMR nanoprobe to miRNA-21 in Example 2 is shown. Detailed Implementation
[0056] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.
[0057] The assembly process of long afterglow / NMR nanoprobes is as follows: Figure 2 As shown, it includes the following steps:
[0058] (1) Synthesis of long-afterglow nanoparticles: PSMA, F127, quinoxaline-based polymers (PQx) and long-afterglow polymers (such as the previously reported TA, MEH-PPV, or PFODBT polymers) were dissolved in THF, rapidly introduced into ultrapure water, sonicated, and then rotary evaporated. The process was carried out in the dark. After ultrafiltration, the nanoparticles were stored at 4 °C.
[0059] (2) Synthesis of long-afterglow nanoparticles-DNA1: The long-afterglow nanoparticles prepared in step (1) were mixed with DNA1 in PBS buffer, briefly vortexed, and EDC was added. The mixture was stirred and then purified three times with ultrapure water.
[0060] (3) Synthesis of magnetic nanoparticles: FeCl3 and erucic acid were dissolved in methanol, and NaOH pre-dissolved in methanol was added dropwise under constant stirring to form ferric erucic acid; MnCl2 and oleic acid were mixed in methanol, and NaOH pre-dissolved in methanol was added dropwise under continuous stirring to synthesize manganese oleate. Manganese oleate, ferric erucic acid, oleyl alcohol and oleic acid were added to dibenzyl ether, heated under argon protection for a set time, and then acetone was added to induce the precipitation of nanoparticles. The solid was separated by centrifugation, and the obtained FeMnOx was dispersed in chloroform for storage;
[0061] (4) Magnetic nanoparticles @HQ-N3: The magnetic nanoparticles prepared in step (3) are mixed with N,N-dimethylformamide, and p-PEG-N3, BMPA, AA and amino-substituted hydroquinone (HQ-N3) are added to the reaction system. The mixture is shaken at the set temperature, and finally, ice-cold diethyl ether is added to precipitate the FeMnOx@HQ-N3 product. The product is purified by centrifugation and filtration, and then dispersed in H2O for storage.
[0062] (5) Magnetic nanoparticles @HQ-N3-DNA2: Freshly prepared CuSO4, tri-hydroxypropyltriazolamide (THPTA), ascorbic acid (AA), alkyne-modified DNA2 and magnetic nanoparticles @HQ-N3 prepared in step (4) were mixed, purified by ultrafiltration, and dispersed in ultrapure water;
[0063] (6) Assembly of long-afterglow nanoparticles-DNA1 and magnetic nanoparticles @HQ-N3-DNA2: The long-afterglow nanoparticles-DNA1 prepared in step (2) and the magnetic nanoparticles @HQ-N3-DNA2 prepared in step (5) were mixed in DPBS containing NaCl and MgCl2, and the sample was purified by ultrafiltration of DPBS.
[0064] When synthesizing control probes that do not respond to miRNA-21, DNA2 is modified to a sequence that does not respond to miRNA-21, and the other steps are similar to the preparation method of long afterglow / NMR nanoprobes.
[0065] The following description, through specific embodiments and accompanying drawings, provides further details.
[0066] Example 1
[0067] The assembly process of long afterglow / NMR nanoprobes includes the following steps:
[0068] (1) Synthesis of long afterglow nanoparticles: Na2CO3 solution (0.40 mL) was rapidly injected into ultrapure water (6 mL) into a solution containing TA (1 mg / mL), a quinoxaline-based polymer (PQx) (1 mg / mL), PSMA (10 mg / mL), and F127 (10 mg / mL). The mixture was sonicated for 4 min, and excess THF was rotary evaporated at 40 °C.
[0069] (2) Synthesis of long-afterglow nanoparticles-DNA1: 80 μg mL -1 TA NP was mixed with 100 μM DNA1 in 2 mL of 1×PBS buffer. The mixture was briefly vortexed, and EDC (pH 7.4) was added. The reaction was carried out with stirring for 4 hours. Subsequently, the mixture was purified three times with ultrapure water using ultrafiltration (MWCO 100 kDa, 6000 rpm, 5 min per cycle).
[0070] (3) Synthesis of magnetic nanoparticles: 2.7 g FeCl3 and 10.2 g erucic acid were dissolved in 50 mL methanol, and then 1.2 g NaOH, which was pre-dissolved in 100 mL methanol, was added dropwise at 40 °C with constant stirring to form ferric erucic acid. Similarly, 1.44 g MnCl2 and 5.65 g oleic acid were mixed in 50 mL methanol, and 0.8 g NaOH was added to 100 mL methanol at 40 °C with continuous stirring to form manganese oleate. 1.0 g manganese oleate, 2.14 g ferric erucic acid, 1.61 g oleyl alcohol, and 0.57 g oleic acid were completely added to 10 g dibenzyl ether, and then stirred at 5 °C for 1 minute. -1 The mixture was heated to 265 °C and maintained under argon protection for 30 min. 20 mL of acetone was added to induce precipitation of the nanoparticles, followed by centrifugation to separate the solid. The resulting magnetic nanoparticles were redispersed in chloroform and stored at 4 °C.
[0071] (4) Synthesis of magnetic nanoparticles @HQ-N3: 7.5 mg of magnetic nanoparticles were dispersed in chloroform (4 mL) and N,N-dimethylformamide (4 mL) containing 5 mg of p-PEG-N3, 150 mg of BMPA, 15 mg of AA, and 80 μg of amino-substituted hydroquinone (NH2-HQ). The reaction was shaken at 30 °C for 12 hours. After completion, the modified FeMnOx@HQ-N3 product was precipitated by adding ice-cold diethyl ether, and then the final product was purified by centrifugation and filtration and redispersed in H2O and stored at 4 °C.
[0072] (5) Synthesis of magnetic nanoparticles @HQ-N3-DNA2: 10 nmol of DNA2 was mixed with magnetic nanoparticles @HQ-N3 and a small volume of 200 μM CuSO4, 2 mM ascorbic acid (AA) and 800 μM THPTA mixture was added. The mixture was purified by ultrafiltration (MWCO 10kDa, 6000 rpm, 5 min), redispersed in ultrapure water, and stored at 4 °C.
[0073] (6) Assembly of long-afterglow nanoparticles-DNA1 and magnetic nanoparticles-DNA2: Long-afterglow nanoparticles-DNA1 (50 μg mL) -1 ) and magnetic nanoparticles-DNA2 (1 mg mL) -1 The sample was supplemented with phosphate-buffered saline (1×PBS) containing 50 mM NaCl and 2.5 mM MgCl2 and purified at 37 °C for 6 h, 6000 rpm, MWCO 100 kDa, 5 min. The purified long-afterglow / NMR nanoprobe was then redispersed in 1×PBS buffer for further use.
[0074] TEM testing was performed on the long afterglow / NMR nanoprobe prepared in this embodiment, and the results are as follows: Figure 3 As shown, from Figure 3 It can be seen that the average size of the long afterglow / NMR nanoprobe prepared in this embodiment is 95.3 nm.
[0075] The long-persistent nanoparticles-DNA1 and long-persistent / NMR nanoprobes prepared in this embodiment were subjected to 12 mW cm⁻¹ spectroscopy. -2 Under constant light intensity, the subjects were exposed to light for varying durations of 10 seconds. Afterglow images and intensities were collected using IVIS Spectrum (Lumina XR) in bioluminescence mode over a 60-second acquisition time. The results are as follows: Figure 4 As shown, from Figure 4 It can be seen that the magnetic nanoparticles @HQ-N3 have an afterglow quenching effect on the long afterglow nanoparticles-DNA1.
[0076] Example 2
[0077] 1. Performance testing of miRNA-21 response based on long-persistence / NMR nanoprobes:
[0078] Long afterglow / NMR nanoprobe (5 μg mL) prepared using Example 1 -1 The long-persistent light / NMR nanoprobes were incubated with miRNA-21 (10 µM) at 37°C in 1×PBS buffer (total volume: 100 μL). The particle size of the long-persistent light / NMR nanoprobes before and after incubation was determined using a Malvern Dynamic Light Scattering (DLS) analyzer, and the results are shown below. Figure 5 As shown, from Figure 5 It can be seen that the particle size of the long-persistent / NMR nanoprobes changed from 95.3 nm to 25 nm after reacting with miRNA-21. This is because miRNA-21 disassembled the long-persistent / NMR nanoprobes.
[0079] 2. Response afterglow assay based on long-persistence / NMR nanoprobes and different concentrations of miRNA-21:
[0080] Long afterglow / NMR nanoprobe (5 μg mL) prepared using Example 1 -1 Samples were incubated at 37 °C with miRNA-21 concentration gradients (0, 0.01, 0.05, 0.1, 0.2, 0.4, 0.8, 1, and 5 nM) in 1×PBS buffer (total volume: 100 μL), and each sample was exposed to white light (12 mW cm⁻¹). -2 ) 10 s, then use the IVIS Spectrum system (Lumina XR) to collect data for 60 s in bioluminescent mode.
[0081] Figure 6 The image shows the afterglow effect of long-persistence / NMR nanoprobes on different concentrations of miRNA-21. After incubation with the long-persistence / NMR nanoprobes, miRNA-21 produces afterglow, with a detection limit (LOD) of 0.2 nM. In the presence of APE1, the LOD is further reduced to 4.2 pM, representing a 46.5-fold increase in sensitivity. This is because, in the presence of APE1, the long-persistence / NMR nanoprobes not only decompose, relieving the quenching of long-persistence nanoparticles-DNA1, but also, after the magnetic nanoparticles-DNA2 bind to miRNA-21, APE1 can release miRNA-21 again to relieve the quenching of long-persistence nanoparticles-DNA1. This cycle continues until the magnetic nanoparticles-DNA2 on the surface of long-persistence nanoparticles-DNA1 are largely detached, thus achieving afterglow amplification of miRNA-21.
[0082] 3. NMR assay based on long-persistence / NMR nanoprobes and different concentrations of miRNA-21 response:
[0083] Will contain 5 μg mL -1 Long-persistent nanoparticles—long-persistent / NMR nanoprobes of DNA1—were exposed to different concentrations of miRNA-21 (range 0 to 1 nM: 0, 0.01, 0.05, 0.1, 0.2, 0.4, 0.8, and 1 nM). T1 MRI images were obtained using a 0.5 TBruker Minispec system and on a 7 Tesla Bruker Biospec system.
[0084] Figure 7 The image shows the afterglow effect of long-persistence / NMR nanoprobes on different concentrations of miRNA-21. After incubation with the long-persistence / NMR nanoprobes, miRNA-21 produces afterglow, with a detection limit (LOD) of 0.4 nM. In the presence of APE1, the LOD is increased to 13.8 pM, representing a 29-fold increase in sensitivity. This is because, in the presence of APE1, the long-persistence / NMR nanoprobes not only decompose, releasing magnetic nanoparticles-DNA2, which then pair with miRNA-21, but APE1 further cleaves the magnetic nanoparticles-DNA2 to release miRNA-21. This cycle continues until the magnetic nanoparticles-DNA2 are largely dispersed, thereby amplifying the NMR signal of miRNA-21.
[0085] This invention's long-persistence / NMR nanoprobe can be specifically activated by highly expressed miRNA-21 in the tumor microenvironment: miRNA-21 undergoes a strand displacement reaction with DNA2 on the surface of magnetic nanoparticles, and under the action of APE1 enzyme, DNA2 is cleaved and cyclically released, thereby triggering the stepwise stripping of magnetic nanoparticles from the surface of long-persistence nanoparticles, thus relieving the quenching of the long-persistence signal. This process not only significantly enhances the long-persistence luminescence signal but also simultaneously releases and triggers the magnetic nanoparticles, improving the contrast of T1-weighted magnetic resonance imaging and achieving synergistic amplification of long-persistence imaging and MRI. Since miRNA-21 is not consumed during the reaction, cascade amplification of the signal can be achieved, thereby realizing highly sensitive, specific, and real-time dynamic monitoring of miRNA-21 levels in tumors. This nanoprobe effectively reduces background interference, combining the advantages of high signal-to-noise ratio long-persistence imaging with the deep tissue penetration and anatomical resolution of MRI, and can be used for precise tumor imaging, malignancy assessment, and treatment guidance, showing promising biomedical application prospects.
[0086] The above content is only a specific implementation example of the present invention, and not all application examples of the present invention. All schemes that follow the technical concept of the present invention or make modifications based on the technical concept of the present invention are within the protection scope of the claims of the present invention.
Claims
1. A long-persistence / NMR nanoprobe based on APE1 amplification for miRNA-21 signal amplification, comprising long-persistence nanoparticles as the core and magnetic nanoparticles as satellite units, characterized in that: The surface of the long-afterglow nanoparticles is linked to amino-modified DNA1 via an amide coupling reaction to obtain long-afterglow nanoparticles-DNA1. The magnetic nanoparticles are modified with amino-substituted hydroquinone (HQ-N3) on their surface, and then reacted with alkynyl-modified DNA2 via a click chemical reaction to obtain magnetic nanoparticles@HQ-N3-DNA2. The long-afterglow nanoparticle-DNA1 and the magnetic nanoparticle@HQ-N3-DNA2 were assembled by hybridization of the two DNAs to obtain the long-afterglow / NMR nanoprobe. The nucleotide sequence of DNA1 is: CGCCAGTTGT (SEQ ID NO.1); The nucleotide sequence of the DNA2 is as follows: GCGGTCAACATCA / dSpacer / GTCTGATA.
2. The long-persistence / NMR nanoprobe based on APE1 amplification for miRNA-21 signal amplification according to claim 1, characterized in that: The 3' modified amino group of the DNA1; The 5' modified alkyne group of the DNA2 has a nucleotide sequence that specifically binds to miRNA21 in more than 60% of cases, and the terminal is modified with an APE1 active site.
3. The long-persistence / NMR nanoprobe for miRNA-21 signal amplification according to claim 1, characterized in that: The long afterglow / NMR nanoprobe has a nucleus-satellite structure and a particle size of 65~225 nm.
4. The long-persistence / NMR nanoprobe based on APE1 amplification for miRNA-21 signal amplification according to claim 1, characterized in that: The long afterglow nanoparticles include, but are not limited to, TA NPs, MEH NPs, and PFO NPs.
5. The long-persistence / NMR nanoprobe for miRNA-21 signal amplification according to claim 1 or 4, characterized in that: The long afterglow nanoparticles have a uniform spherical structure with a particle size of 15~85 nm.
6. The long-persistence / NMR nanoprobe based on APE1 amplification for miRNA-21 signal amplification according to claim 1, characterized in that: The magnetic nanoparticles include, but are not limited to, FeOx, FeZnOx, and FeMnOx.
7. The long-persistence / NMR nanoprobe based on APE1 amplification for miRNA-21 signal amplification according to claim 1 or 6, characterized in that: The magnetic nanoparticles have a uniform spherical structure with a particle size of 2~15 nm.
8. The method for preparing the long afterglow / NMR nanoprobe according to any one of claims 1 to 7, characterized in that, include: (1) Synthesis of long afterglow nanoparticles; (2) Synthesis of long afterglow nanoparticles-DNA1: The long afterglow nanoparticles obtained in step (1) were mixed with amino-modified DNA1 in PBS buffer, and then EDC was added. The reaction was carried out under stirring and purified to obtain the DNA1. (3) Synthesis of magnetic nanoparticles; (4) Magnetic nanoparticles @HQ-N3: The magnetic nanoparticles obtained in step (3) are mixed with N,N-dimethylformamide, and p-PEG-N3, BMPA, ascorbic acid (AA), and amino-substituted hydroquinone (HQ-N3) are added to the reaction system. The mixture is shaken at a set temperature. After the reaction is completed, the product is precipitated and then purified by centrifugation and filtration to obtain magnetic nanoparticles @HQ-N3. (5) Magnetic nanoparticles @HQ-N3-DNA2: CuSO4, tri-hydroxypropyltriazolamide (THPTA), ascorbic acid (AA), alkynyl-modified DNA2 and magnetic nanoparticles @HQ-N3 obtained in step (4) were mixed, purified by ultrafiltration, and then dispersed in ultrapure water; (6) Assembly: The long afterglow nanoparticles-DNA1 obtained in step (2) and the magnetic nanoparticles @HQ-N3-DNA2 obtained in step (5) are mixed in DPBS containing NaCl and MgCl2 to obtain the long afterglow / NMR nanoprobe.
9. The method for preparing the long afterglow / NMR nanoprobe according to claim 8, characterized in that, In step (2), the molar ratio of long afterglow nanoparticles to DNA1 is (6~8):1, the pH value of PBS buffer is 7.4, the final concentration of EDC is 250 mM, the reaction time is 4~12 h, the ultrafiltration speed is 6000 rpm, the ultrafiltration time is 5 min, and the ultrapure water is used for purification and cycled three times. In step (4), the mass ratio of magnetic nanoparticles, p-PEG-N3, BMPA, AA and HQ-N3 is (6~8):5:(160~170):(25~32):(0.05~0.09); the incubation temperature is 30 ℃, the shaking time is 12~15 h, the ultrafiltration speed is 6000 rpm, the ultrafiltration time is 5 min, and the ultrapure water is purified and circulated three times. In step (5), the molar ratio of CuSO4, tri-hydroxypropyltriazolamide (THPTA) and ascorbic acid (AA) is (1~1.5):4:10; the incubation temperature is 37 ℃, the shaking time is 4~12h, the ultrafiltration speed is 6000 rpm, the ultrafiltration time is 5 min, and the ultrapure water is purified and circulated three times. In step (6), the assembly mass ratio of long afterglow nanoparticles-DNA1 and magnetic nanoparticles @HQ-N3-DNA2 is (32~40):1; the concentrations of NaCl and MgCl2 are 50 mM and 2.5 mM, respectively; the incubation temperature is 37 ℃; the shaking time is 12~24 h; the ultrafiltration speed is 6000 rpm; the ultrafiltration time is 5 min; and the product is purified with ultrapure water and cycled three times.
10. The application of the long afterglow / NMR nanoprobe according to any one of claims 1 to 7, characterized in that, It is used to prepare contrast agents for tumor imaging.