Brain-targeted neuronal ion signal specificity bimodal nanoprobe as well as preparation method and application thereof

Abstract

The invention provides a brain-targeted neuron ion signal specificity bimodal nanoprobe and a preparation method and application thereof.The nanoprobe comprises mesoporous silicon nanoparticles, and a micromolecular magnetic resonance T1 contrast agent and a potassium ion indicator are loaded in pore channels of the mesoporous silicon nanoparticles; the surfaces of the meso-porous silicon nanoparticles are modified with potassium ion selective filter membranes and brain-targeted ligands. The neuron ion signal specificity fluorescence-magnetic resonance bimodal nanoprobe provided by the invention can effectively combine high-resolution structural magnetic resonance imaging with high-sensitivity functional fluorescence imaging, so that real-time dynamic high-resolution imaging of a neuron potassium ion signal is realized; the method has good research and application prospects in the aspects of precise positioning and operation guidance of the structure-hidden epilepsy focus.

Description

Technical Field

[0001] The present invention relates to the technical field of functional nanoprobes, and in particular to a brain-targeted neuron ion signal-specific dual-modal nanoprobe and a preparation method and application thereof. Background Art

[0002] Epilepsy is a common and complex neurological disease characterized by repeated spontaneous seizures, unpredictability and high disability rates, which seriously affect the patient's physical and mental health and quality of life. According to statistics, it has become the third largest factor in the global burden of neurological diseases. In China, there are about 10 million epilepsy patients, and the number of new cases continues to rise each year, which not only brings heavy economic and mental burdens to patients' families, but also constitutes a major public health problem.

[0003] Although anti-epileptic drugs play a certain role in reducing the frequency of epileptic seizures, they are still unable to completely cure epilepsy, especially about 30% of patients with drug-resistant epilepsy still cannot effectively control seizures after drug treatment. Therefore, surgical resection of the epileptogenic focus has become an important way to treat refractory epilepsy because of its long-lasting efficacy, high cost-effectiveness and ability to significantly prolong the patient's survival. However, the key to surgical treatment lies in the accurate positioning of the epileptogenic focus before surgery. Currently, there is a lack of effective and accurate positioning technology in clinical practice, which makes the surgery difficult to carry out or the treatment is ineffective. Only less than 1% of patients can successfully undergo surgery. This problem has become a bottleneck restricting the application of epilepsy surgery.

[0004] From a pathophysiological perspective, epileptic seizures are caused by abnormal neuronal discharge activity, a process that is closely related to the regulation of neuronal ion signals. Potassium ions (K+) play an important role in this process as a key factor in activating and inhibiting voltage-gated channels and maintaining neuronal membrane potential. Normally, the extracellular potassium ion concentration ([K + ] ex ) fluctuates around a stable baseline level (about 3 mM), but its brief rise can trigger an epileptic seizure, and further K+ outflow intensifies neuronal depolarization, forming a positive feedback process that increases action potentials. When the seizure ends, ([K + ] ex ) will gradually return to the baseline. Based on this phenomenon, the development of new intelligent imaging probe technology to accurately identify the spatiotemporal changes of neuronal potassium ion signals in the epileptic brain is expected to promote the development of epilepsy pathology research and diagnosis. Summary of the invention

[0005] In view of the defects in the prior art, the purpose of the present invention is to provide a brain-targeted neuronal ion signal-specific dual-modal nanoprobe and its preparation method and application, which solves the problem in the prior art that it is difficult to accurately locate structurally hidden epileptic foci, making surgical treatment difficult.

[0006] The first aspect of the present invention provides a brain-targeted neuronal ion signal-specific dual-modal nanoprobe, which includes mesoporous silicon nanoparticles, the pores of which are loaded with small molecule magnetic resonance T1 contrast agents and potassium ion indicators, and the surfaces of the mesoporous silicon nanoparticles are modified with a potassium ion selective filter membrane and a brain-targeting ligand.

[0007] In one embodiment of the present invention, the particle size of the mesoporous silicon nanoparticles is 10 to 120 nm.

[0008] Preferably, the particle size of the mesoporous silicon nanoparticles is 30 to 100 nm.

[0009] More preferably, the particle size of the mesoporous silicon nanoparticles is 40 to 80 nm.

[0010] In one embodiment of the present invention, the mesoporous silicon nanoparticles are surface-carboxyl-modified mesoporous silicon nanoparticles.

[0011] In one embodiment of the present invention, the small molecule magnetic resonance T1 contrast agent includes at least one of a small molecule gadolinium chelate and a small molecule manganese chelate.

[0012] In one embodiment of the present invention, the potassium ion indicator is a small molecule potassium ion fluorescent probe.

[0013] In one embodiment of the present invention, the potassium ion indicator includes at least one of PBFI, APG-1, APG-2, and APG-4.

[0014] In one embodiment of the present invention, the brain targeting ligand includes one or more ligands that promote the probe to cross the blood-brain barrier and target the epileptic lesion.

[0015] Preferably, the brain targeting ligand comprises at least one of Angiopep-2 and TAT polypeptide.

[0016] In one embodiment of the present invention, the potassium ion selective filter membrane is assembled from molecules of structural formula (I), and the potassium ion selective filter membrane can specifically capture potassium ions, and then diffuse the ions into the pores of the mesoporous nanoparticles to bind to the potassium ion indicator;

[0017]

[0018] The second aspect of the present invention provides a method for preparing the above-mentioned brain-targeted neuron ion signal-specific dual-modal nanoprobe, comprising the following steps:

[0019] S1. Preparation of carboxyl-modified mesoporous silica nanoparticles;

[0020] S2, loading a small molecule magnetic resonance T1 contrast agent and a potassium ion indicator into the pores of the carboxyl-modified mesoporous silicon nanoparticles;

[0021] S3, modifying the surface of the mesoporous silicon nanoparticles obtained in step S2 with a potassium ion selective filter membrane precursor by an in-situ deposition method;

[0022] S4. Connect the brain-targeting ligand to the surface of the mesoporous silica nanoparticles obtained in step S3 through an amidation reaction to obtain a brain-targeting neuron ion signal-specific dual-modal nanoprobe.

[0023] The third aspect of the present invention provides the use of the above-mentioned brain-targeted neuron ion signal-specific dual-modal nanoprobe in the preparation of surgical navigation drugs.

[0024] Compared with the prior art, the embodiments of the present invention have the following beneficial effects:

[0025] (1) The neuronal ion signal-specific fluorescence-magnetic resonance dual-modal nanoprobe provided by the present invention can cross the blood-brain barrier and selectively target the epileptic lesion area.

[0026] (2) The neuronal ion signal-specific fluorescence-magnetic resonance dual-modal nanoprobe provided by the present invention can effectively combine high-resolution structural magnetic resonance imaging with high-sensitivity functional fluorescence imaging to achieve real-time dynamic high-resolution imaging of neuronal potassium ion signals, and has good research and application prospects in the precise positioning of structurally latent epileptic lesions and surgical guidance.

[0027] (3) The reaction system of the preparation method of the present invention is mild and the conditions are controllable. The prepared materials have good biocompatibility, wide applicability, strong promotion potential, and good clinical transformation potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0029] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of Si-DTTA in Example 2;

[0030] Figure 2 is the hydrogen nuclear magnetic resonance spectrum of the potassium ion selective filter membrane precursor in Example 3;

[0031] Figure 3 This is a transmission electron microscopy image of the neuron ion signal-specific fluorescence-magnetic resonance dual-modal nanoprobe in Example 4;

[0032] Figure 4This is the EDS mapping diagram of the neuron ion signal-specific fluorescence-magnetic resonance dual-modal nanoprobe in Example 4;

[0033] Figure 5 This is the EDS element linear scan diagram of the neuron ion signal specific fluorescence-magnetic resonance dual-modal nanoprobe in Example 4;

[0034] Figure 6 is a T1 magnetic resonance weighted image of the neuron ion signal-specific fluorescence-magnetic resonance dual-modality nanoprobe in Example 4;

[0035] Figure 7 is the fluorescence signal intensity of the neuron ion signal-specific fluorescence-magnetic resonance dual-modal nanoprobe in Example 4 under the conditions of potassium ion and other different types of physiological ion concentrations;

[0036] Figure 8 This is the neuronal potassium ion signal imaging result in Application Example 1;

[0037] Fig. 9 This is the EEG after epilepsy treatment in Application Example 2;

[0038] Fig.10 This is a graph showing the results of the learning and memory function evaluation after epilepsy treatment in Application Example 2. DETAILED DESCRIPTION

[0039] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several changes and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0040] The development of new intelligent imaging probe technology to accurately identify the spatiotemporal changes of neuronal potassium ion signals in the epileptic brain is expected to promote the development of epilepsy pathology research and diagnosis.

[0041] Among many research techniques, fluorescence imaging is widely used in the study of brain potassium signals due to its high detection sensitivity. However, its penetration depth is limited and it is only suitable for local imaging. Its spatial resolution is insufficient, making it difficult to accurately locate lesions in vivo. In contrast, magnetic resonance imaging (MRI) has the advantages of being non-invasive, radiation-free, with high soft tissue resolution and high spatial resolution, but it is insufficient in terms of sensitivity in epilepsy diagnosis. By combining high-sensitivity fluorescence imaging technology with high-resolution MRI, the shortcomings of a single imaging technology can be made up, while providing anatomical information on the location of the lesion and functional information on the characteristics of the disease microenvironment, thereby improving the efficiency of disease diagnosis. Therefore, the development of imaging technology that combines these advantages is of great significance for improving the level of epilepsy diagnosis.

[0042] Based on this, the present invention provides a brain-targeted neuron ion signal-specific dual-modal nanoprobe and a preparation method and application thereof.

[0043] The brain-targeted neuron ion signal-specific dual-modal nanoprobe comprises mesoporous silicon nanoparticles, the pores of which are loaded with a small molecule magnetic resonance T1 contrast agent and a potassium ion indicator, and the surface of the mesoporous silicon nanoparticles is modified with a potassium ion selective filter membrane and a brain-targeted ligand.

[0044] The brain-targeted neuron ion signal-specific dual-modal nanoprobe is prepared by the following steps:

[0045] S1. Preparation of carboxyl-modified mesoporous silica nanoparticles;

[0046] S2, loading a small molecule magnetic resonance T1 contrast agent and a potassium ion indicator into the pores of the carboxyl-modified mesoporous silicon nanoparticles;

[0047] S3, modifying the surface of the mesoporous silicon nanoparticles obtained in step S2 with a potassium ion selective filter membrane precursor by an in-situ deposition method;

[0048] S4. Connect the brain-targeting ligand to the surface of the mesoporous silica nanoparticles obtained in step S3 through an amidation reaction to obtain a brain-targeting neuron ion signal-specific dual-modal nanoprobe.

[0049] Preferably, the preparation of carboxyl-modified mesoporous silicon nanoparticles specifically includes the following steps: using tetraethyl silicate as a silicon source and hexadecyltrimethylammonium chloride as a template, dissolving them in deionized water, and reacting at 85-95° C. for 2-6 hours; washing the product with a sodium chloride methanol solution; and carboxyl-modifying the mesoporous silicon nanoparticles with carboxyethylsilanetriol sodium salt to obtain carboxyl-modified mesoporous silicon nanoparticles.

[0050] Preferably, the loading of the small molecule magnetic resonance T1 contrast agent and the potassium ion indicator specifically comprises the following steps: dispersing the carboxyl-modified mesoporous silicon nanoparticles and the silicon-oxygen-containing small molecule magnetic resonance T1 contrast agent in toluene, and reacting them at 90-110° C. overnight; mixing them with the potassium ion indicator after washing, stirring them in the dark at room temperature for one day, and washing them by centrifugation to obtain mesoporous silicon nanoparticles loaded with the small molecule magnetic resonance T1 contrast agent and the potassium ion indicator.

[0051] Preferably, the preparation of the potassium ion selective filter membrane precursor comprises the following steps: adding N-benzyl salicylamide and anhydrous potassium carbonate to N,N-dimethylformamide, heating to 85-95° C., and continuously adding 1,1,1-tri(p-toluenesulfonyloxy-methyl)ethane and 2-aminoterephthalic acid to react to obtain the potassium ion selective filter membrane precursor.

[0052] Preferably, the modification of the potassium ion selective filter membrane and the brain targeting ligand specifically comprises the following steps: dispersing mesoporous nanoparticles loaded with a small molecule magnetic resonance T1 contrast agent and a potassium ion indicator in an acetonitrile solution; adding an acetonitrile solution containing a filter membrane precursor to the above solution under vigorous stirring for reaction, annealing at room temperature for 10 to 15 hours, and modifying the filter membrane precursor on the surface of the mesoporous nanoparticles by an in situ deposition method; further connecting the brain targeting ligand to the surface of the nanoparticles by an amidation reaction, and finally obtaining a neuronal ion signal-specific fluorescence-magnetic resonance dual-modal nanoprobe.

[0053] The neuron ion signal-specific fluorescence-magnetic resonance dual-modal nanoprobe provided by the present invention can be used in epileptic focus visualization and surgical guidance. The nanoprobe can cross the blood-brain barrier to target epileptic lesions, enhance the MRI signal at the lesion site, and further dynamically monitor the fluctuation of neuronal potassium ion signals in the MRI signal enhancement area through fluorescence imaging to confirm the epileptic focus. After surgical treatment guided by the imaging results, the frequency of epileptic seizures is significantly reduced, and learning and memory functions are restored.

[0054] The present invention is further described in detail below with reference to specific embodiments.

[0055] Example 1: Synthesis of surface carboxyl-modified mesoporous silica nanoparticles (MSN-COOH)

[0056] 2g hexadecyltrimethylammonium chloride and 0.07g triethanolamine were dispersed in deionized water, stirred at 95°C for 1 hour, and then 3mL tetraethyl silicate was added dropwise, and the reaction was continued for 1 hour. After the reaction was completed, the supernatant was discarded by centrifugation, and the product was washed 4 times with sodium chloride methanol solution under heating and stirring overnight to obtain mesoporous silicon nanoparticles (MSN), which were dispersed in deionized water for use. 10mg MSN and 30μL 25wt.% carboxyethylsilanetriol sodium salt solution were added to the HEPES solution in sequence, and reacted for 3 hours under stirring at room temperature. The product was centrifuged and washed with ethanol and water in sequence to obtain MSN-COOH, which was dispersed in deionized water for use.

[0057] Example 2: Synthesis of small molecule gadolinium contrast agent

[0058] (1) Dissolve 0.5558 g of bromoacetic acid and 0.2654 g of 3-(trimethoxysilylpropyl)diethylenetriamine in 1.0 mL of deionized water and 2.0 mL of 2 M sodium hydroxide solution. Heat the reaction solution to 50°C, and add 3.0 mL of 2 M sodium hydroxide solution within 30 min using a constant flow pump. After stirring at 50°C for 2 hours, remove the solvent by rotary evaporation, separate the off-white powder, and vacuum dry to obtain Si-DTTA. The hydrogen nuclear magnetic resonance spectrum verifies the structure of the small molecule, as shown in FIG. Figure 1 shown.

[0059] (2) 108.6 mg Si-DTTA was dissolved in 4 mL deionized water, and 380 μL 0.50 M gadolinium chloride solution was slowly added until the formed precipitate no longer dissolved, and stirred at room temperature for 2 h. After removing excess metal ions, the solution volume was concentrated to 1 mL to obtain a Gd-DTTA-Si aqueous solution with a concentration of about 0.20 M.

[0060] Example 3: Synthesis of Potassium Ion Selective Filter Membrane Precursor

[0061] Add 2.5g of anhydrous potassium carbonate and 3.4g of N-benzyl-2-hydroxybenzamide to 25mL of anhydrous dimethylformamide, heat to 90°C under argon protection, then add 2.9g of 1,1,1-tris(p-toluenesulfonyloxymethyl)ethane and 0.3mL of 2-aminoterephthalic acid, and continue stirring the reaction at 90°C for 24 hours. After cooling to room temperature, add the reaction mixture to 200mL of deionized water, centrifuge and collect the precipitate. Using petroleum ether-ethyl acetate (2:1) as the eluent, the obtained precipitate is treated with silica gel column chromatography to obtain a potassium ion selective filter membrane precursor. The hydrogen nuclear magnetic resonance spectrum verifies the structure of the filter membrane precursor, as shown in Figure 2 shown.

[0062] Example 4: Synthesis of neuronal ion signal-specific fluorescence-magnetic resonance dual-modal nanoprobe

[0063] (1) Disperse 50 mg of MSN-COOH prepared in Example 1 and 400 μL of Gd-DTTA-Si prepared in Example 2 in 4 mL of toluene, and condense and reflux under stirring at 105° C. overnight to obtain MSN-Gd after washing. -1 ) and 1 mL potassium ion indicator APG aqueous solution (0.5 mg mL -1 ) and stirred at room temperature in the dark for 24 hours. After centrifugation, the unadsorbed APG was washed twice with methanol to remove the unadsorbed APG and dispersed in acetonitrile for later use.

[0064] (2) Under vigorous stirring, the acetonitrile solution (2 mL, 15 mg) of the potassium ion selective filter membrane precursor synthesized in Example 3 was slowly added to 5 mL of the acetonitrile solution of the nanoparticles synthesized in step (1) (2 mg mL -1 ), react at 50°C for 30 min, anneal at room temperature for 24 h, wash by centrifugation, and disperse in deionized water for later use.

[0065] (3) 10 mg of the nanoparticles synthesized in step (2) were dispersed in 1 mL of morpholineethanesulfonic acid buffer (pH = 5.6), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added, and after stirring at room temperature for 1 hour, the precipitate was collected by centrifugation and mixed with 2 mL of Angiopep-2 aqueous solution (1 mg mL -1 ) were mixed and stirred at room temperature for 12 hours, and the product was dialyzed to purify the product to obtain a neuronal ion signal-specific fluorescence-magnetic resonance dual-modal nanoprobe.

[0066] The probe transmission electron microscope prepared in this example is as follows Figure 3 As shown, it proves that the obtained nanoparticles have uniform size and morphology; the EDS mapping of the probe is as follows Figure 4 As shown, it proves that Gd-DTTA-Si and APG are evenly distributed in the mesoporous silicon nanochannels; the EDS linear element scanning diagram of the probe is shown in Figure 5 As shown, it proves the successful modification of potassium ion selective filter membrane; the T1-weighted image of the probe is shown Figure 6 As shown; the potassium ion selective fluorescence imaging effect of the probe is shown Figure 7 shown.

[0067] Application example 1: Neuronal ion signal-specific fluorescence-magnetic resonance dual-modality nanoprobe for visualization of epileptic foci

[0068] (1) Animal model construction: C57BL / 6 male mice were used to construct epilepsy model, inflammation model and sham operation model using kainic acid, neocuproine and normal saline, respectively.

[0069] (2) The neuronal ion signal-specific fluorescence-magnetic resonance dual-modal nanoprobe was injected into epilepsy model mice, inflammation model mice, and Sham model mice via tail vein administration (the dosage was: [Gd] 0.1 mmol / kg -1 ). The control material without brain targeting ligand was injected into the epilepsy model mice by tail vein injection. Before administration and 1.5 hours after administration, MRI scans were performed on each group of mice using a 9T high-field MRI apparatus.

[0070] (3) Using a brain stereotaxic instrument, optical fibers were placed in the brain regions of epilepsy model mice and inflammation model mice where T1 signals were enhanced as shown by MRI scans, and the optical fiber recording system was used to continuously monitor the fluorescence signal for 1 hour in a dual-channel continuous excitation mode of 410nm and 470nm. At the same time, electrodes were implanted on the surface of the mouse skull and connected to the Pinnacle-Small Animal EEG Signal Recording System to record the mouse EEG during the fluorescence signal recording period.

[0071] The imaging results obtained in this experiment are as follows Figure 8 As shown, the probe can cross the blood-brain barrier and specifically target the inflammatory microenvironment in vivo for MRI. The fluorescence signal increase in the epilepsy model group was significantly higher than that in the inflammatory model group, and was consistent with the electroencephalogram results, indicating that epileptic lesions can be accurately diagnosed through high-temporal and spatial resolution imaging of neuronal potassium ion signals.

[0072] Application Example 2: Neuronal Ion Signal-Specific Fluorescence-Magnetic Resonance Dual-Mode Nanoprobe Guiding Epilepsy Surgery

[0073] A drug-resistant epilepsy model was constructed by intraperitoneal injection of kainic acid. The epileptic lesions were located under the guidance of neuronal ion signal-specific fluorescence-magnetic resonance dual-modal nanoprobe imaging, and surgical treatment was performed by electrode thermal ablation. A sham surgery group and a drug treatment group were set up as controls. The epileptic seizures were monitored after a period of time after treatment, and the learning and memory functions of the animals were evaluated by the Morris water maze experiment.

[0074] The treatment results obtained in this application example are as follows Fig. 9 As shown in the figure, it is proved that the surgical treatment guided by the neuronal ion signal-specific fluorescence-magnetic resonance dual-modality nanoprobe has a significantly improved therapeutic effect compared with the drug treatment group and the sham operation group; the results of learning and memory function evaluation are shown in the figure. Fig.10 As shown, since probe-guided surgical treatment can effectively control epileptic seizures, learning and memory functions are significantly restored compared with the drug treatment group and the sham operation group.

[0075] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. A brain-targeted neuron ion signal-specific dual-modal nanoprobe, characterized in that: The nanoprobe comprises mesoporous silicon nanoparticles, the pores of which are loaded with small molecule magnetic resonance T1 contrast agent and potassium ion indicator, and the surface of the mesoporous silicon nanoparticles is modified with potassium ion selective filter membrane and brain targeting ligand.

2. The brain-targeted neuron ion signal-specific dual-modal nanoprobe according to claim 1, characterized in that: The particle size of the mesoporous silicon nanoparticles is 10 to 120 nm.

3. The brain-targeted neuron ion signal-specific dual-modal nanoprobe according to claim 2, characterized in that: The mesoporous silicon nanoparticles are mesoporous silicon nanoparticles with surface carboxylation modification.

4. The brain-targeted neuron ion signal-specific dual-modal nanoprobe according to claim 1, characterized in that: The small molecule magnetic resonance T1 contrast agent includes at least one of a small molecule gadolinium chelate and a small molecule manganese chelate.

5. The brain-targeted neuron ion signal-specific dual-modal nanoprobe according to claim 1, characterized in that: The potassium ion indicator is a small molecule potassium ion fluorescent probe.

6. The brain-targeted neuron ion signal-specific dual-modal nanoprobe according to claim 5, characterized in that: The potassium ion indicator includes at least one of PBFI, APG-1, APG-2, and APG-4.

7. The brain-targeted neuron ion signal-specific dual-modal nanoprobe according to claim 1, characterized in that: The brain targeting ligand includes at least one of Angiopep-2 and TAT polypeptide.

8. The brain-targeted neuron ion signal-specific dual-modal nanoprobe according to claim 1, characterized in that: The potassium ion selective filter membrane is assembled from molecules of structural formula (I) 9. The method for preparing the brain-targeting neuron ion signal-specific dual-modal nanoprobe according to any one of claims 1 to 8, characterized in that: The following steps are involved: S1. Preparation of carboxyl-modified mesoporous silica nanoparticles; S2, loading a small molecule magnetic resonance T1 contrast agent and a potassium ion indicator into the pores of the carboxyl-modified mesoporous silicon nanoparticles; S3, modifying the surface of the mesoporous silicon nanoparticles obtained in step S2 with a potassium ion selective filter membrane precursor by an in-situ deposition method; S4. Connect the brain-targeting ligand to the surface of the mesoporous silica nanoparticles obtained in step S3 through an amidation reaction to obtain a brain-targeting neuron ion signal-specific dual-modal nanoprobe.

10. Use of the brain-targeted neuron ion signal-specific dual-modal nanoprobe according to any one of claims 1 to 8 in the preparation of surgical navigation drugs.

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