Nerve-targeted imaging nano fluorescent probe constructed by near-infrared two-region aggregation-induced emission material and NP41 polypeptide as well as preparation method and application of nerve-targeted imaging nano fluorescent probe

Abstract

The invention relates to a nerve-targeted imaging nano fluorescent probe constructed by a near-infrared two-region aggregation-induced emission material and NP41 polypeptide as well as a preparation method and application of the nerve-targeted imaging nano fluorescent probe, belongs to the technical field of medical image contrast agents, and particularly relates to a nerve-targeted imaging nano fluorescent probe constructed by combining a near-infrared two-region fluorescent material TTB with AIE characteristics and a nerve-targeted peptide NP41. The invention also relates to a preparation method of the fluorescent probe and application of the fluorescent probe in long-term continuous imaging of central and peripheral nerve living bodies.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical imaging contrast agents, and specifically relates to a nerve-targeted near-infrared zone II nano fluorescent probe constructed of near-infrared zone II aggregation-induced luminescence material and NP41 polypeptide, and a preparation method and application thereof. Background Art

[0002] Molecular imaging of nerves is the basis for exploring the function of the nervous system and precision medicine. At present, there are many methods for observing neural molecular imaging in vitro, such as conventional immunofluorescence staining and microscopic imaging. However, in vitro experiments cannot immediately reflect the activity changes of neurons and synapses, as well as glial cells, and their value for clinical diagnosis is far lower than that of in vivo experiments. At present, the main methods for studying nerves in vivo include CT, MRI, calcium imaging, two-photon imaging, near-infrared zone II imaging, etc. However, the resolution of CT and MRI cannot reach the micron level to observe neural structure, and calcium imaging only indirectly reflects neural activity by changes in calcium signals. Two-photon microscopy can observe neuronal morphology, but its observation field is small, it is highly destructive to tissues, and the imaging depth is limited. The advantage of near-infrared zone II imaging is that it has stronger penetration, lower tissue absorption and scattering, higher spatial resolution and wider imaging range, and has the potential for minimally invasive imaging and intraoperative navigation. It is a more suitable method for studying neural morphology in vivo.

[0003] Unlike blood vessels that run throughout the body, due to the particularity of nerve binding targets and the limitations of applied materials, the difficulty in observing nerves using near-infrared zone II lies in the need to innovatively design a molecular probe that can specifically label nerves while having the advantages of in vivo imaging (high brightness, strong penetration, and non-toxicity).

[0004] PCT / US2015 / 065816 describes a cyclic peptide, a nanoparticle bound to the cyclic peptide, and a method for intraoperative neural tissue imaging using the cyclic peptide and / or the nanoparticle bound to the cyclic peptide. The highly selective and significantly enhanced fluorescence signal of neural tissue is achieved, which can be displayed early or delayed during surgery, thereby reducing the risk of neural damage and improving the accuracy of surgical operations.

[0005] The webpage http: / / www.nanoer.net / showinfo-4-13892.html suggests a nanoparticle (RGD 4R MPD / TTB NPS) prepared by encapsulating a fluorescein (TTB) containing an electron-accepting benzo[1,2-b:4, 5-b']dithiophene 1,1,5,5-tetraoxide core and an electron-donating 4,4'-(2,2-diphenylethylene-1,1-diyl)bis(N,N-diphenylaniline) group with obvious aggregation-induced emission (AIE) characteristics in a polymer matrix and then modifying it with RGD 4R peptide. The nanoparticle RGD 4R MPD / TTB NPS has near-infrared emission (about 730 nm), high photostability and low dark cytotoxicity for real-time, long-term image-guided photodynamic therapy (PDT). This probe has not yet been shown to be combined with a neural targeting peptide.

[0006] The webpage https: / / zhuanlan.zhihu.com / p / 271241951 suggests a neuropeptide NP41 that can bind to the surface of peripheral nerve cells. NP41 is a neuropeptide screened using phage display technology, and its sequence is NTQTLAKAPEHT. Studies have shown that laminin-421 and -211 are target proteins bound by NP41. It is only necessary to combine the corresponding luminescent material with NP41, and the luminescent material will bind to the nerves along with NP41, thereby staining the nerves. It has not yet been combined with a probe with near-infrared 2 emission properties.

[0007] The webpage https: / / ir.pku.edu.cn / handle / 20.500.11897 / 699641 shows the imaging effect of a near-infrared fluorescent targeted probe ICG-NP41 on the neurovascular bundles (NVB) around the prostate in rats in vivo experiments. It has not yet been applied to the central nervous system, and the emission of ICG in the near-infrared 2 region is only its tailing effect, and the intensity is significantly lower than its own emission peak.

[0008] The website https: / / pmc.ncbi.nlm.nih.gov / articles / PMC3364105 / suggests the imaging effect of a near-infrared fluorescent targeted probe NP41-FAM on the brachial plexus and sciatic nerve in experiments in mice. It has not yet been applied to the central nervous system. The optimal imaging time window is 4 to 5 hours, and there is a lot of non-specific binding with surrounding tissues. Summary of the invention

[0009] The purpose of the present invention is to provide a nano fluorescent probe with high specificity and good photostability targeting the second near-infrared region, peripheral nerves and central nerves.

[0010] Another object of the present invention is to provide a method for preparing the nerve-targeted nano fluorescent probe.

[0011] Another object of the present invention is to provide applications of the nano fluorescent probe.

[0012] The object of the present invention is achieved by the following scheme: a nerve-targeted nano fluorescent probe, comprising a nerve-targeted nano fluorescent probe NP41-TTB NPs constructed by an aggregation-induced luminescent material TTB emitting in the second near-infrared region and an NP41 nerve-targeting peptide, wherein the aggregation-induced luminescent material TTB compound emitting in the second near-infrared region (1000-1700 nm) has a structural formula as shown in Formula 1, and the NP41 [AcHN-SHSNTQTLAKAPEHTG-C (SH) -NH2] polypeptide compound has a structural formula as shown in Formula 2: .

[0013] The preparation method of the near-infrared second-zone aggregation-induced luminescence fluorescent material and NP41 to form a nerve-targeted nano fluorescent probe, the reaction mechanism is as follows Figure 4 As shown, the following steps are included: The near-infrared second-zone aggregation-induced emission material TTB, the amphiphilic polymer carrier DSPE-mPEG2000 and maleimide (Mal)-modified DSPE-PEG2000 (DSPE-PEG2000-Mal) were co-assembled into nanoparticles (TTB NPs); The NP41 neurotargeting peptide undergoes a click chemical reaction with the Mal on the periphery of TTB NPs through the thiol (SH) in its structure, and is modified onto the surface of TTB NPs to prepare NP41-TTB NPs. By adjusting the ratio of NP41 on the nano-surface modification of TTB NPs, the nerve-targeted nano-fluorescent probe NP41-TTB NPs was obtained.

[0014] Furthermore, the near-infrared second-zone aggregation-induced emission fluorescent material TTB was physically encapsulated by a mixed carrier of DSPE-mPEG2000 and DSPE-PEG2000-Mal to prepare TTB NPs, and the ratio between the two carriers was adjusted so that the molar ratio between the two was 9:1 ~ 7:3.

[0015] To facilitate subsequent use, the prepared nanomaterial NP41-TTB NPs can be placed at 4°C and stored in a sealed container away from light for a storage period of one week.

[0016] The present invention also provides an application of the nano fluorescent probe NP41-TTB NPs in long-term continuous imaging of peripheral and central nerves.

[0017] The nano fluorescent probe NP41-TTB NPs obtained by the present invention can be used for peripheral nerve imaging. The obtained nano fluorescent probe NP41-TTB NPs are formulated into an intravenous injection preparation with an imaging window of 6 to 48 hours. The imaging equipment parameters need to meet the requirements of excitation light of 808 nm and emission light greater than 1000 nm. For peripheral thick nerves, such as sciatic nerves, common cervical nerves, etc., and peripheral micro nerves, such as cutaneous nerves with a diameter of about 10 μm, the morphological characteristics of nerve fiber running, distribution, bifurcation, etc. can be clearly distinguished in situ; or, The nano fluorescent probe NP41-TTB NPs obtained in the present invention can be used for central nervous system imaging. The obtained nano fluorescent probe NP41-TTB NPs are formulated into an injectable preparation and injected into the lateral ventricle, the third ventricle, or the intrathecally. The imaging window is 2 to 24 hours. The imaging equipment parameters must meet the requirements of excitation light of 808 nm and emission light greater than 1000 nm. For shallow brain regions, such as the cortex, and deep brain regions such as the hypothalamus, the morphological characteristics of neuron distribution density, fibers, cell bodies, etc. can be distinguished.

[0018] The present invention is aimed at the development of fluorescent contrast agents for peripheral and central nerves, and provides a preparation and application method of a nanoprobe NP41-TTB NPs based on near-infrared zone II aggregation-induced emission materials and their derivatives. As a fluorescent imaging probe in the near-infrared zone II window, the probe has a deeper penetration depth and higher spatial resolution, and can achieve specific targeted labeling of central and peripheral nerves. The probe preparation method is safe and simple, and has good stability and repeatability.

[0019] The present invention combines for the first time a near-infrared II region fluorescent probe with AIE properties and a nerve targeting peptide to achieve long-term continuous imaging of central and peripheral nerves in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the complex of particle size and potential changes after TTB NPs are connected to NP41; Figure 2 Representative images of mouse peripheral nerves at 6 and 24 h are shown for NP41-TTB NPs; Figure 3 Representative images of mouse CNS at 2 and 24 h are shown for NP41-TTB NPs; Figure 4 This is the reaction mechanism of the present invention. DETAILED DESCRIPTION

[0021] The specific steps of the present invention are described below by way of examples, but are not limited by the examples. The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. In the following examples, various processes and methods not described in detail are conventional methods known in the art. The reagents used in the following examples are purchased from common commercial sources, and the unspecified experimental operations and experimental conditions refer to the conventional operations and conventional conditions in the art. The specific embodiments of the present invention are described below in conjunction with the accompanying drawings.

[0022] Example 1 A nerve-targeted nano fluorescent probe includes a nerve-targeted nano fluorescent probe NP41-TTB NPs constructed by combining an aggregation-induced luminescent material TTB emitting in the second region of the near-infrared and a NP41 nerve-targeting peptide. The NP41 nerve-targeting peptide undergoes a click chemical reaction with Mal on the periphery of TTB NPs through the thiol (SH) in its structure, and is modified onto the surface of TTB NPs, according to the following steps: The TTB molecule was mixed with carriers DSPE-mPEG2000 and DSPE-PEG2000-Mal at a mass ratio of 1:5, and dissolved in tetrahydrofuran to prepare a mixed solution with an AIE material concentration of 1 mg / mL; the mixed solution was vortexed and dropped into ultrapure water, and the vortex was continued for 2 minutes to prepare a TTB NPs material with Mal on the periphery, and then the nanomaterial was transferred to an ultrafiltration tube for centrifugal concentration to obtain a concentrated nanomaterial TTB NPs material, the particle size of the nanomaterial TTB NPs was 100 ~120 nm, and the potential was -50 mV; NP41 was added to the concentrated nanomaterials and slowly shaken for 6 hours, so that the Mal on the periphery of the TTB NPs material and the SH on the NP41 polypeptide would undergo click chemical bonding; the reacted nanomaterials were added to a dialysis bag and dialyzed for 24 hours; the above nanomaterials were concentrated and quantified to obtain the nanomaterial NP41-TTB NPs after binding with NP41.

[0023] The particle size of the nanomaterial NP41-TTB NPs decreases and the potential increases. The particle size is 90 ~ 100 nm and the potential is about -20mV.

[0024] The prepared nanomaterial NP41-TTB NPs was dissolved in water to prepare 10 μM, and UV absorption and fluorescence emission tests were performed. The absorption peak of the nanomaterial is 700 nm, and the emission spectrum peak is 1000 nm, with significant emission properties in the near-infrared region II. The particle size and potential changes of TTB NPs before and after binding with peptide NP41 are shown in Figure 2. Figure 1After the neurotargeting peptide NP41 was connected with the fluorescent probe TTB NPs, the particle size was about 106.7 nm, which was consistent with the nanoparticles. The decrease in the site after connection proved that the connection was successful.

[0025] Example 2 The steps of this embodiment are similar to those of embodiment 1, except that the ratio of NP41 modified on the nano-surface of TTB NPs is adjusted, wherein: The molar ratio of DSPE-mPEG2000 and DSPE-PEG2000-Mal can also be 9:1, 4:1, and 7:3; The molar ratio of NP41 to DSPE-PEG2000-Mal was 1.2:1.

[0026] Example 3 In vivo imaging of nanomaterial NP41-TTB NPs in the peripheral sciatic nerve of mice: For anesthetized mice, 200 μl of 1.5 mM NP41-TTB NPs probe was injected into the tail vein, and good imaging effect was achieved after 6 to 48 hours of circulation in vivo. The near-infrared zone II in vivo imaging device meets the requirements of 808 nm external laser irradiation, and the laser power density is 100 mW / cm 2 , the 1000 nm filter can collect and observe emission light with a wavelength greater than 1000 nm, such as Figure 2 As shown, the dotted box shows the mouse sciatic nerve in situ, and the bright and sharp signals show the fine filamentous signals of the sciatic nerve, which can be clearly distinguished from the surrounding muscles and connective tissues in situ.

[0027] Example 4 In vivo imaging of the central hypothalamus of mice. Anesthetized mice were given 2 μl of 1.5 mM NP41-TTB NPs probe by intracerebroventricular injection. Two hours after intracerebroventricular injection, the mice were subjected to mandibular traction and the hypothalamic area was fully exposed. The near-infrared zone II in vivo imaging equipment meets the requirements of 808 nm external laser irradiation, the laser power density is 100 mW / cm2, and the 1000 nm filter can collect and observe the emission light with a wavelength greater than 1000 nm, such as Figure 3 As shown, the hypothalamic region is viewed from the perspective of the mouse skull base. The bright and sharp signals show the morphological characteristics of the dot-like cell bodies and filamentous axons of hypothalamic neurons.

[0028] The nerve-targeted nanofluorescent probe constructed with near-infrared zone II aggregation-induced emission fluorescent material TTB and NP41 is used for peripheral / central nervous system intraoperative navigation. The application scenarios include but are not limited to tumor resection, nerve injury, nerve repair, nerve biopsy, nerve activation, real-time monitoring of nerve activity, etc.

Claims

1. A nerve-targeted nano fluorescent probe, comprising a nerve-targeted nano fluorescent probe NP41-TTB NPs constructed by combining an aggregation-induced luminescent material TTB emitting in the second near-infrared region and a NP41 nerve-targeting peptide, wherein: The aggregation-induced luminescence material TTB compound emitting in the second near-infrared region (1000-1700 nm) has a structural formula as shown in Formula 1, and the NP41 [AcHN-SHSNTQTLAKAPEHTG-C (SH) -NH2] polypeptide compound has a structural formula as shown in Formula 2: 。 2. A method for preparing the nerve-targeted nano fluorescent probe according to claim 1, characterized in that: The steps include: The near-infrared second-zone aggregation-induced emission material TTB, the amphiphilic polymer carrier DSPE-mPEG2000 and maleimide (Mal)-modified DSPE-PEG2000 (DSPE-PEG2000-Mal) were co-assembled into nanoparticles (TTB NPs); The NP41 neurotargeting peptide undergoes a click chemical reaction with Mal on the periphery of TTB NPs through the thiol (SH) group in its structure and is modified onto the surface of TTB NPs; By adjusting the ratio of NP41 on the nano-surface modification of TTB NPs, the nerve-targeted nano-fluorescent probe NP41-TTBNPs was obtained.

3. The method for preparing a neural-targeted nano fluorescent probe according to claim 2, characterized in that: The steps include: When the near-infrared second-zone aggregation-induced emission fluorescent material TTB was physically encapsulated and prepared by using DSPE-mPEG2000 and DSPE-PEG2000-Mal mixed carriers to prepare TTB NPs, the molar ratio between the two carriers was 9:1 ~ 7:

3.

4. The method for preparing a nerve-targeted nano fluorescent probe according to claim 2 or 3, characterized in that: Follow these steps: The TTB molecule was mixed with carriers DSPE-mPEG2000 and DSPE-PEG2000-Mal at a mass ratio of 1:5 and dissolved in tetrahydrofuran to prepare a mixed solution with an AIE material concentration of 1 mg / mL; the mixed solution was vortexed and added dropwise to ultrapure water and vortexed for 2 minutes to prepare TTB NPs material with Mal on the periphery. Subsequently, the nanomaterial was transferred to an ultrafiltration tube for centrifugal concentration to obtain a nanomaterial TTB NPs material with a particle size of 100 to 120 nm after concentration; NP41 was added to the concentrated nanomaterials and slowly shaken for 6 hours, so that the Mal on the periphery of the TTB NPs material and the SH on the NP41 polypeptide underwent click chemical bonding. The reacted nanomaterials were added to a dialysis bag and dialyzed for 24 hours. The above nanomaterials were concentrated and quantified to obtain the nanomaterial NP41-TTB NPs with a particle size of 90 to 100 nm after bonding with NP41.

5. The method for preparing a nerve-targeted nano fluorescent probe according to claim 4, characterized in that: The molar ratio of DSPE-mPEG2000 and DSPE-PEG2000-Mal was 9:1, 4:1 or 7:

3.

6. The method for preparing a nerve-targeted nano fluorescent probe according to claim 4, characterized in that: The molar ratio of NP41 to DSPE-PEG2000-Mal was 1.2:

1.

7. Use of the nano fluorescent probe as claimed in claim 1 in long-term continuous imaging of peripheral and central nervous systems.

8. A fluorescent contrast agent comprising a nerve-targeted nanofluorescent probe, characterized in that: Prepared into intravenous preparations or injections.

9. A method for using the nano fluorescent probe according to claim 1 in peripheral nerve imaging, characterized in that: The steps include: Take the nano fluorescent probe and dilute it to 1.5 mM with sodium chloride solution for injection, and inject the probe by intravenous injection. The imaging time window is 6 to 48 hours; or, Take the nano fluorescent probe, dilute it to 1.5 mM with sodium chloride solution for injection, and inject the probe into the lateral ventricle, third ventricle, or intrathecally. The imaging time window is 2 to 24 hours.

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