CN108686208B

CN108686208B - Non-invasive near-infrared light-controlled nano material for repairing damaged nerve

Info

Publication number: CN108686208B
Application number: CN201810709649.9A
Authority: CN
Inventors: 刘坚; 刘耀波; 李晨曦; 黄振晖; 严俊; 吉喆
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2018-07-02
Filing date: 2018-07-02
Publication date: 2021-05-18
Anticipated expiration: 2038-07-02
Also published as: CN108686208A

Abstract

The invention relates to a non-invasive near-infrared light-controlled nano material for repairing damaged nerves, which can convert near-infrared light into visible light and/or ultraviolet light. The non-invasive near infrared light-controlled nano material is an up-conversion fluorescent nano material. The invention discloses a new application of an upconversion fluorescence nano material, in the process of neuron repair, electrical stimulation is not needed to be applied to neurons, invasive optical fibers are not needed to be implanted into animals in an operation mode, near infrared light with high tissue penetrability is used for exciting the upconversion nano material in organisms, the converted upconversion fluorescence can activate photosensitive ion channel protein on neuron cell membranes, the neuron cells are stimulated to generate membrane potential change, stimulation to neural circuits is enhanced, and damaged neurons are repaired.

Description

Non-invasive near-infrared light-controlled nano material for repairing damaged nerve

Technical Field

The invention relates to the field of nerve regeneration medicine, in particular to a non-invasive near-infrared light-controlled nano material for repairing damaged nerves.

Background

The optogenetic technology is a brand new technology generated by combining the genetic technology and the light control technology. The optogenetic technology transfers the receptor cells into related genes through genetic engineering, and selects and opens the receptor cells by using a light control method to realize the optical control of the cells. The function of specific cells is determined by activating or inhibiting the specific cells through light, and the activity of individual nerve cells is operated to regulate and control the function of related nerve cells, so that scientific researchers can be helped to perform in-vivo research on specific tissue cells, further analyze the change of the biological functions of the cells under the condition of pathophysiology, and realize the regulation on the function of the specific neuron cells through the influence of light-controlled stimulation. Meanwhile, by using similar optical and genetic means, protein expression in brain nerve cells, spinal cord nerve cells or other cells can be controlled, so that the aims of photoinduced protein expression, starting biological processes in different cells and further controlling biological behaviors are fulfilled.

Stimulation of neural circuits is a common treatment for neural trauma and disease in neurosurgery, and the aim of adjusting target circulation can be achieved in part by applying excitation caused by pulse current stimulation of fixed frequency, intensity and duration to a particular neural circuit.

The conventional approach of applying electrical stimulation to the neural circuit, although the parameters of the current are adjustable and reversible, works very inefficiently with this unexplained mechanism, accompanied by many side effects, thus limiting the development of this treatment. Therefore, the light sensitive ion channels of microorganisms, i.e., ion channel proteins, are widely accepted today as new therapeutic approaches. By stimulation with specific wavelengths, it can regulate in vivo the nerve activity of highly selective neuronal types, bringing its cell membrane to hyperpolarization or depolarization with high time resolution. Although ion channel proteins have an important role in therapy, conventional therapeutic approaches rely on the use of clinically used metal catheters and optical fibers, respectively, for delivery of drugs and light of specific wavelengths, which presents a number of technical problems to overcome. The excitation light for light-sensitive ion channel proteins (e.g., ChR1, ChR2, NpHR, etc.) is typically visible light, which presents great difficulties for modulating neural activity in vivo, producing specific stimuli, because visible light has very low tissue penetration depth and can cause photodamage to biological tissues. Therefore, the UCNPs (up-conversion fluorescent nano materials) which can convert near infrared light with strong tissue penetrability into visible light, are stable and have low signal-to-noise ratio and are easy to modify become very suitable carriers of the optogenetic regulation system. However, the UCNPs are mostly applied to the fields of medical imaging, biosensing, photothermal therapy, photodynamic therapy and the like, and are not widely applied to the field of nerve regeneration medicine by targeting tumor cells.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a non-invasive near-infrared light-controlled nano material for repairing damaged nerves, and discloses a new application of the non-invasive near-infrared light-controlled nano material.

The first purpose of the invention is to disclose the application of the non-invasive near infrared light controlled nano material in the preparation of a tool for repairing damaged nerves, wherein the non-invasive near infrared light controlled nano material can convert near infrared light into visible light and/or ultraviolet light.

The non-invasive means that in the nerve repair process, an animal does not need to be implanted with invasive optical fibers by operation, and due to the good tissue penetrability of near infrared light, the laser fixed-point irradiation is applied in vitro, so that the in-vivo near infrared light-controlled nano material can be excited to emit light with the wavelength required by repair.

Further, there are various types of tools for repairing damaged nerves: if the material can be taken up or adsorbed by appointed cells of a damaged part through molecular targeting after surface modification and phase inversion, the material can also be compounded with a biological material, the material is directly placed on the damaged part through an operation, and the repair is completed by applying light stimulation to excite the up-conversion material in vitro.

The non-invasive near infrared light-controlled nano material plays a role in repair: after targeting to a specific site or inside a cell, near infrared light is used to provide light of a desired wavelength in vivo.

Further, the non-invasive near infrared light controlled nano material is an up-conversion fluorescent nano material.

Furthermore, the upconversion fluorescence nanomaterial comprises a rare earth element doped inorganic nanomaterial and a water-soluble biomolecule, wherein the water-soluble biomolecule is connected to the surface of the rare earth element doped inorganic nanomaterial, and the water-soluble biomolecule selectively targets the neuron cell.

The upconversion fluorescent nano material is a composite system of a series of rare earth element doped inorganic nano materials and water-soluble biomolecules, wherein the inorganic nano materials in the composite system have an accurately adjustable energy band gap structure and can convert near infrared light (exciting light) into emitted light with specific wavelength combination, including visible light and ultraviolet light. Further, the rare earth element doped inorganic nano material comprises a matrix material and rare earth elements, wherein the matrix material is NaYF₄、Y₂O₃、YCl₃And Y (CH)₃COO)₃One or more of Yb as rare earth element³⁺、Er³⁺、Tm³⁺、Ce³⁺、Nd³⁺、Gd³⁺And Ho³⁺One or two of them.

Preferably, the rare earth element doped inorganic nano material is NaYF₄:Yb³⁺,Er³⁺、NaYF₄:Yb³⁺,Tm³⁺Or NaYF₄:Yb³⁺,Tm³⁺@NaYF₄(core-shell structure) wherein the material before @ is the core and the material after @ is the shell.

Further, the water-soluble biomolecule is one or more of glutamic acid (Glu), polyglutamic acid (pGlu), acetylcholine, dopamine, glycine, epinephrine and NO precursor. NO precursors are a class of substances that can release NO (nitric oxide) under light excitation or chemical excitation.

Further, the wavelength range of the near infrared light is 750nm to 1400 nm. Preferably, the wavelength is 980 nm. Near-infrared light has a significantly increased tissue penetration depth as excitation light compared to typical visible wavelengths.

When the upconversion fluorescent nano material is used as a medicine, various forms can be selected, such as injection, and the upconversion fluorescent nano material is injected to a specific damaged part. Or the upconversion fluorescence nanometer material is compounded with other matrixes, such as dispersed in a solid material, and then placed on the surface of the damaged part, or transplanted into the specific damaged part in a body, so as to play the functions of repairing and regenerating after the nerve is damaged.

According to the application disclosed by the invention, people in the field can develop an efficient functional nerve repair and regeneration technology based on the upconversion fluorescent nanomaterial after nerve damage, the upconversion fluorescent nanomaterial is selectively enriched on the surface of a neuron cell membrane of a specific neurotransmitter receptor and in cells, the regulation and control of physiological activities such as cell axon growth of superior neurons and the like are promoted in a non-invasive mode in a damaged area through light wavelength conversion (near infrared light is converted into visible light and/or ultraviolet light), and the selective connection between the superior neurons and inferior neurons is guided and attracted.

The second purpose of the invention is to provide a non-invasive near-infrared light-controlled nano material for repairing damaged nerves, which can convert near-infrared light into visible light and/or ultraviolet light, and the wavelength range of the near-infrared light is 750nm to 1400 nm.

Further, the upconversion fluorescent nanomaterial comprises a rare earth element doped inorganic nanomaterial and a water-soluble biomolecule, wherein the water-soluble biomolecule is connected to the surface of the rare earth element doped inorganic nanomaterial, and the water-soluble biomolecule selectively targets the neuronal cell.

Further, the rare earth element doped inorganic nano material comprises a matrix material and rare earth elements, wherein the matrix material is NaYF₄、Y₂O₃、YCl₃And Y (CH)₃COO)₃One or more of Yb as rare earth element³⁺、Er³⁺、Tm³⁺、Ce³⁺、Nd³⁺、Gd³⁺And Ho³⁺One or two of them.

Further, the water-soluble biomolecule is one or more of glutamic acid, polyglutamic acid, acetylcholine, dopamine, glycine, epinephrine and NO precursor.

Further, the wavelength range of the near infrared light is 750nm to 1400 nm.

By the scheme, the invention at least has the following advantages:

1. the application of a non-invasive near infrared light controlled nano material in the preparation of a tool for repairing damaged nerves is disclosed, and based on the technology, the treatment method which has low efficiency and many side effects and can avoid the stimulation of current in the process of repairing neurons can be realized. Compared with the traditional optogenetics, the method does not need to apply operation invasive implanted optical fibers, avoids a series of side reactions caused by trauma, gets rid of the constraint of the optical fibers, and improves the flexibility.

2. The invention uses the non-invasive near infrared light controlled nano material with high tissue penetrability, such as the up-conversion fluorescent nano material in the near infrared light excited organism, compared with visible light, the tissue penetrability is greatly improved. The non-invasive near-infrared light-controlled nano material is selectively enriched on the surface of a cell membrane of a specific subordinate neuron and in a cell, protein molecules in the optogenetic component are activated through light wavelength conversion, the damaged axon directional regeneration of the superior neuron cell is promoted, the connection with the subordinate neuron cell is repaired, and the neural loop reconstruction is completed. The laser can be used for accurately treating the damaged part, and the influence on other tissues and organs is avoided.

3. The non-invasive near-infrared light-controlled nano material can repair the damage of the central nervous system and repair and regenerate the damage of the peripheral nervous system.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.

Drawings

FIG. 1 is a TEM representation of UCNPs prepared in example 1 of the present invention;

FIG. 2 shows the results of dynamic light scattering measurements on the products prepared in the different examples;

FIG. 3 is a fluorescence spectrum and a potential diagram of a product prepared in different examples;

FIG. 4 shows the cytotoxicity and difference in material intake of HUVEC cells and Neuron cells for modified Glu-UCNP, pGlu-UCNP and PEG-UCNP;

FIG. 5 is a confocal view of fluorescence after staining HUVEC cells and Neuron cells with PEG-UCNP, pGlu-UCNP, and Glu-UCNP;

FIG. 6 is a fluorescence imaging diagram of the wound and a statistic diagram of the distribution fluorescence values of UCNPs of the small animal at different time points in example 5 of the present invention;

FIG. 7 shows the specific adsorption of Glu-UCNP to PSD 95-positive post-synaptic neurons of glutamic acid in vitro in example 5 of the present invention;

FIG. 8 shows the specific adsorption of Glu-UCNP to PSD 95-positive post-synaptic glutamate neurons in example 5 of the present invention.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1NaYF₄:Yb³⁺,Er³⁺Synthesis of nanoparticles

Get Y₂O₃And CF₃COOH was placed in a round bottom flask at a molar ratio of 1:6, 50mL of pure water was added, stirred to dissolve and distribute uniformly, heated to 120 ℃ and then refluxed for 2 hours with condensed water. After the reflux is finished, the reaction solution is cooled to room temperature, insoluble impurities are filtered out by using a Buchner funnel, the filtered clear solution is collected and placed on a heating plate to dry most of water, and then the clear solution is dried into powder in an oven at the temperature of 140 ℃.

Further, according to the above method, Yb is used₂O₃And Er₂O₃Replacing Y in the above step₂O₃Separately synthesizing Yb (CF)₃COO)₃And Er (CF)₃COO)₃。

Taking Y (CF)₃COO)₃(0.695mmol),Yb(CF₃COO)₃(0.030mmol), and Er (CF)₃COO)₃(0.010mmol),(CF₃COO) Na (4mmol) was placed in a 100mL three-necked flask, and 20mL oleic acid and 20mL octadecene were added. The reaction was warmed to 160 ℃ under nitrogen blanket and magnetically stirred for 0.5h to remove excess water. Then the temperature of the reaction mixture is raised to 320 ℃, and the reaction mixture is stirred for 1 hour to fully react. Cooling to room temperature after the reaction is finished, fully centrifuging and washing the product at 10000 revolutions by using ethanol and cyclohexane, and then re-dispersing by using cyclohexane to obtain NaYF₄:Yb³⁺,Er³⁺Upconversion nanoparticles (UCNPs). The morphology of the synthesized material was observed by a Transmission Electron Microscope (TEM). The results are shown in FIGS. 1A and 1B. The results of the images a and B and the detection by a Transmission Electron Microscope (TEM) show that the UCNPs prepared by the embodiment have uniform particle size, uniform regular hexagon shape and about 113nm particle size, respectively, in a large field and a small field.

EXAMPLE 2 preparation of Glu-UCNP

Glutamic acid (Glu) modifier was modified to UCNPs prepared in example 1 using carboxyl substitution. 60mg of Glu was dissolved in 15mL of deionized water and stirred for 30min to disperse uniformly. Then, 40mg of UCNP prepared in example 1 were each added slowly dropwise at 45 ℃ and the solution turned from a clear solution to a milky white suspension and stirred for 8 h. And cooling to room temperature after the reaction is finished, standing the reaction solution for 1h, separating the reaction solution by using a separating funnel, taking the lower-layer water phase, centrifugally washing the lower-layer water phase by using ethanol and water at 14800 rpm, washing the lower-layer water phase for three times, and re-dispersing the washed lower-layer water phase by using pure water to obtain the glutamic acid modified UCNPs, which are hereinafter referred to as Glu-UCNP.

Example 3

Polyglutamic acid-modified UCNPs (pGlu-UCNP) were prepared according to the method of example 2, except that 60mg of Glu of example 2 was replaced with 60mg of pGlu.

Comparative example

Polyethylene glycol-modified UCNPs (PEG-UCNPs) were prepared according to the procedure of example 2, except that 60mg of Glu of example 2 was replaced with 50mg of PEG. The properties of the product prepared in the comparative example and the product prepared in example 2 will be studied hereinafter.

The particle size distribution test was performed on the products prepared in examples 1 to 3 and comparative example, respectively, and the results are shown in fig. 2. Glu, pGlu and PEG are modified by a carboxyl substitution method to convert UCNPs into water-solubility, the product particle size is uniform, and the particle size after ligand modification is not changed greatly and is about 117nm because Glu, pGlu and PEG are all small molecular ligands.

The fluorescence values after photoconversion of the products prepared in examples 1 to 3 and comparative example were measured by fluorescence spectroscopy. As shown in FIG. 3A, under the excitation of 980nm laser, the main emission peaks of UCNPs converted light are at 520nm, 540nm and 650nm, and the light intensity of 540nm is 3-4 times of that of 650nm, so that the light of 540nm can be more effectively applied to a light control system. The unmodified UCNPs and modified UCNPs were then potential characterized by a Zeta-potentiostat. As can be seen from FIG. 3B, the potential values of the unmodified UCNPs and the PEG-modified UCNPs are almost 0mV and are neutral; the UCNPs potential values of Glu and pGlu which are modified are +27mV and +33mV respectively, and the modified UCNPs have positive electricity and have positive correlation effects on target cells and entering cells.

Example 4 cytotoxicity assay and Material uptake assay

In the present invention, the reagents for cell culture are all purchased from Byobo Co., Ltd. Neuronal cells (Neuron) and vascular endothelial cells (HUVEC) were cultured in a medium containing 10% fetal bovine serum at 37 ℃ and 5% CO₂Culturing is carried out in the environment.

The cytotoxicity test was carried out by using a cell viability assay kit (CTG) using a luminescence method. Neuron cells and HUVEC cells were plated in 96-well plates at 8000 cell densities per well. After 24 hours, uniformly mixing the modified water-soluble nano materials Glu-UCNP, pGlu-UCNP and PEG-UCNP with culture media according to different concentrations (10 concentrations are selected according to a 0-200 mu g/mL medium-fold ratio), standing for 2 hours, absorbing the culture media in a 96-well plate, adding a new culture medium mixed with the materials into the 96-well plate to perform incubation culture with cells, taking out the cells after 24 hours or 48 hours of culture, adding 20 mu L of CTG solution into each well, shaking the cells on a shaking table for 5 minutes, and measuring the fluorescence value by using a multifunctional microplate reader.

As shown in fig. 4A-B, the corresponding columns at each concentration were Glu-UCNP, pGlu-UCNP and PEG-UCNP in order from left to right, and the survival rate of Neuron cells incubated with the material for 24h was above 85%, while the survival rate of HUVEC cells was above 90%; after the material is incubated for 48 hours, the survival rates of the Neuron cells and the HUVEC cells are respectively more than 80% and 83%, which shows that the three modified water-soluble UCNPs do not show obvious cytotoxicity within 48 hours of incubation with the cells, are materials with very good cell compatibility, and can be used for the next experiment.

To observe the targeting differences of Glu, pGlu and PEG molecules to neuronal cells, experiments were performed on the difference in material uptake for Neuron cells and HUVEC cells, respectively. The Neuron cells and HUVEC cells were taken at 1X 10⁵Is spread in a medium dish. After 24h, Glu-UCNP, pGlu-UCNP and PEG-UCNP were mixed with the medium, left to stand for 2h, and then added to a petri dish to incubate the two cells for 1h, 4h, 8h, 12h and 24h, respectively. At the corresponding time point, the mixed material medium was aspirated from the petri dish and the material not taken up by the cells was washed away by three gentle washes with PBS. Then, the cells were sufficiently digested with pancreatin, and after counting, the cell sap was digested and diluted with aqua regia at 300 ℃ and the concentration of UCNPs in the cell sap was measured by ICP-MS.

As shown in FIGS. 4C-D, there was no significant difference in the uptake rate of the three materials by HUVEC cells within 24h, since there was no selectivity in the uptake of the three materials because there were no glutamate-related receptors on the vascular endothelial cell surface; meanwhile, the ingestion amount of Glu-UCNP by Neuron cells is twice that of pGlu-UCNP and PEG-UCNP, which indicates that the glutamic acid has specific targeting property on Neuron cells, and the modified Glu can greatly increase the ingestion amount of the Neuron cells on UCNPs.

After the low toxicity of the material was verified by the CTG method, the concentration of the material was selected to be 40. mu.g/mL, at which PEG-UCNP, pGlu-UCNP, and Glu-UCNP were incubated with HUVEC cells and Neuron cells. After 12h, HUVEC were subjected to dead-live double staining (Calcein-AM/PI staining), as shown in FIGS. 5A-C, the vast majority of green cytograms showed that after incubation with the three materials, the cells were almost not dead, and the vast majority of cells remained very viable; meanwhile, the Neuron cells under the same conditions are stained with tubulin on the cell nucleus and the axon, as shown in fig. 5D-F, the fluorescence diagram well shows the form of the nerve cells, the cells are completely spread, and the axon extension and the peripheral nerve cells form good connection, which also shows that the cell form and survival meet the experimental requirements, and the in vivo experiment can be carried out under the concentration. In FIG. 5, A, D corresponds to PEG-UCNP, B and E correspond to pGlu-UCNP, and C and F correspond to Glu-UCNP test results.

Example 5 nerve terminal repair experiment

In order to further verify whether the light-operated ion channel protein stimulates a specific neural circuit system to repair the neuron which is mechanically damaged, the following experiment is carried out in the invention. The photosensitive channel protein Channelrhodosis 1(ChR1) is a light-controlled ion channel protein found in algae, is excited by 540nm light and has important influence on the activity of neurons, so that the photosensitive channel protein has an important position in the field of neuroscience. Since the light with the wavelength of 540nm is weak in penetrability in vivo, and optogenetic related drugs such as light-sensitive channel protein and the like are excited by the light with the wavelength of 540nm and the like, UCNPs are introduced, and the UCNPs absorbed by the mice are deeply excited in vivo to convert the UCNPs into the light with the wavelength of 540nm to activate ChR1 through the high penetrability and the low absorptivity of tissues of 980nm long-wavelength laser, so that the purpose of activating the neuron activity is achieved.

First, a plasmid containing the ChR1 gene was coated with a virus (VChR1), and injected into the mouse brain together with the RCAMP virus containing the calcium excitation-associated protein gene from the sensory and motor cortex region of the brain, four weeks later, the dorsal spinal cord part was surgically cut through dorsal hemisection, and PEG-UCNP or Glu-UCNP was injected on both sides of the wound. In order to verify whether UCNPs can be gathered at a wound site to carry out light conversion to activate ChR1 after being injected into the wound, but not diffuse into intercellular matrix near the wound, so that the UCNPs amount per unit area is too small to cause too low light intensity of 540nm after conversion, the UCNPs distribution at the wound site at different time points is characterized by a small animal fluorescence imaging system. Glu-UCNP and PEG-UCNP were dissolved at a concentration of 200. mu.g/mL in PBS, pH 7.4. Mice were anesthetized and 5-10 μ L of the material solution was injected 0.5mm across the spinal cord wound of mice after dorsal hemisection. After the relevant mice are anesthetized 1, 14, 28 and 84 days after the operation and the injection of UCNPs, the mice are fixed on a pure black thin plate in a prone position, the skin and the muscle at the wound position are sequentially separated (the experiment uses the black mice, the black fur can absorb light so that the light in the wavelength range required by the experiment cannot be specifically observed), the 540nm fluorescence signals converted by the UCNPs near the wound on the back of the mice are observed under the excitation of 980nm laser by using a living body fluorescence imaging system of the mice to detect the distribution of the UCNPs at the wound position, and the average signal intensity of each area at the wound position is subjected to standardized analysis by using relevant software to further quantitatively compare. Each group of mice had 5 replicates. The results are shown in fig. 6A, six time points of day 1 (1D), day 14 (14D), day 28 (28D) and day 84 (84D) are selected, the mice are fixed on a pure black thin plate in a prone position after anesthesia at the corresponding time points, and in order to avoid the influence of black fur on the imaging result, the skin and the muscle at the wound are separated in advance and then placed in the system for observation. From the experimental results, it was found that the water-soluble UCNPs were well distributed around the wound with little diffusion phenomenon as time passed. From the imaging images, the fluorescence at the wound site was statistically analyzed, i.e., the relative content of UCNPs at the wound site, as seen in fig. 6B, there was a slight diffusion of UCNPs after the second week (14D), but most of the material was still distributed at and near the wound site.

Subsequently, in vitro and in vivo experiments were performed on whether Glu-UCNP was specifically adsorbed by PSD 95-positive glutamate postsynaptic neurons. FIG. 7 is an in vitro cell culture experiment showing the specific adsorption of Glu-UCNP to PSD95 positive post-synaptic glutamate neurons. (A) The representative graph shows the absorption of Glu-UCNP by PSD 95-positive glutamatergic post-synaptic neurons in culture; (B-D) displaying a neuron-labeled antibody (Tuj1), a glutamatergic postsynaptic antibody (PSD95), and Glu-UCNP (D), respectively. And (E-H) is an enlarged view of the square area of the A picture in the (A-D) pictures respectively. The experimental result proves that the neuron containing the glutamate receptor has high specific adsorption capacity to Glu-UCNP.

FIG. 8 is a graph showing the specific adsorption of Glu-UCNP to PSD95 positive post-synaptic glutamate neurons in vivo. (A) The representative figures show the absorption of Glu-UCNP by PSD 95-positive glutamatergic post-synaptic neurons under in vivo conditions; (B) is an enlarged view of the boxed area of panel a. (C-F) shows signals (D) of virus-labeled corticospinal tracts (VchR1-GFP), neuron-labeled antibodies (Tuj1), glutamatergic postsynaptic antibodies (PSD95) and Glu-UCNP single channels, respectively. (G-K) are the reconstructed images of the corresponding XY and YZ in the (B-F) diagrams, respectively. The experimental results show that the damaged neuron axons grow towards the postsynaptic neurons of glutamate under the induction of light.

Example 6NaYF₄:Yb³⁺,Tm³⁺Synthesis of nanoparticles

Separately taking YCl₃(0.695mmol),YbCl₃(0.30mmol), and TmCl₃(0.005mmol) was placed in a 50mL three-necked flask, and 12mL of oleic acid and 15mL of octadecene were added. After introducing nitrogen for 5min, the reaction was heated to 160 ℃ and magnetically stirred for 0.5h to remove oxygen and moisture. The reaction was then cooled to room temperature. 4mmol of ammonium fluoride (148mg) and 2.5mmol of sodium hydroxide (100mg) were dissolved in 10mL of methanol. It was dissolved completely by sonication for 10min and then added dropwise to a three-necked flask cooled to room temperature. After keeping magnetic stirring for 2h, the reaction system was heated to 100 ℃ and incubated for 15min to remove methanol, then warmed to 300 ℃ and kept for 1 h. Cooling to room temperature after the reaction is finished, fully centrifuging and washing the product at 10000 revolutions by using ethanol, and then re-dispersing by using cyclohexane to obtain NaYF₄:Yb³⁺,Tm³⁺Up-converting the nanoparticles.

Example 7

Mixing YCl₃(0.695mmol) was added to a 50mL three-necked flask, 12mL oleic acid and 15mL octadecene were added, the temperature was raised to 160 ℃ and maintained for 30min, and then the reaction system was cooled to 80 ℃. 6mL of NaYF-containing solution was added₄:Yb³⁺,Tm³⁺And incubated for 30min to remove the solvent cyclohexane, and then cooled to room temperature. 4mmol of ammonium fluoride (148mg) and 2.5mmol of sodium hydroxide (100mg) were dissolved in 10mL of methanol. Dissolving completely by ultrasonic treatment for 10min, adding dropwise into a three-neck flask cooled to room temperature, stirring at room temperature for 2h, and heating to 75 deg.C for 10min to remove solvent methanol. After removing methanol, the reaction system was warmed to 300 ℃ at 30 DEGStirring at 0 deg.C for 1h, and cooling to room temperature after reaction. The product is fully centrifuged and washed by ethanol under 10000 revolutions, and then is re-dispersed by cyclohexane to obtain NaYF₄:Yb³⁺,Tm³⁺@NaYF₄Nanoparticles of structure which are core-shell structures, wherein NaYF₄Is a shell.

Following the procedure of example 2, in NaYF₄:Yb³⁺,Tm³⁺@NaYF₄The surface of the nanoparticle with the structure is modified with a targeting molecule to prepare another kind of UCNPs modified by glutamic acid, the product can also be used as a nerve cell repair preparation, light converted under excitation light of 980nm is at 480nm, and ChR2 is excited, and the product is also applied to nerve repair. In addition, the targeting molecule can also be selected from water-soluble biomolecules such as acetylcholine, dopamine, glycine and epinephrine.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Claims

1. The application of the non-invasive near-infrared light-controlled nano material in the preparation of a tool for repairing damaged nerves is characterized in that: the non-invasive near infrared light controlled nano material can convert near infrared light into visible light and/or ultraviolet light; the non-invasive near-infrared light-controlled nano material is used for activating photosensitive ion channel protein on a neuron cell membrane; the non-invasive near-infrared light-controlled nano material is an up-conversion fluorescent nano material; the up-conversion fluorescent nano material comprises a rare earth element doped inorganic nano material and water-soluble biomolecules, wherein the water-soluble biomolecules are connected to the surface of the rare earth element doped inorganic nano material and selectively target neuronal cells.

2. Use according to claim 1, characterized in that: the rare earth element doped inorganic nanoThe rice material comprises a matrix material and rare earth element ions, wherein the matrix material is NaYF₄、Y₂O₃、YCl₃And Y (CH)₃COO)₃One or more of the rare earth element ions are Yb³⁺、Er³⁺、Tm³⁺、Ce³⁺、Nd³⁺、Gd³⁺And Ho³⁺One or two of them.

3. Use according to claim 1, characterized in that: the water-soluble biological molecules are one or more of glutamic acid, acetylcholine, dopamine, glycine, epinephrine and NO precursors.

4. Use according to claim 1, characterized in that: the wavelength range of the near infrared light is 750nm-1400 nm.

5. A non-invasive near-infrared light controlled nanomaterial for repairing damaged nerves, comprising: the non-invasive near-infrared light-controlled nano material can convert near-infrared light into visible light and/or ultraviolet light, and the wavelength range of the near-infrared light is 750nm-1400 nm; the non-invasive near-infrared light-controlled nano material is used for activating photosensitive ion channel protein on a neuron cell membrane; the non-invasive near-infrared light-controlled nano material is an up-conversion fluorescent nano material; the up-conversion fluorescent nano material comprises a rare earth element doped inorganic nano material and water-soluble biomolecules, wherein the water-soluble biomolecules are connected to the surface of the rare earth element doped inorganic nano material and selectively target neuronal cells.

6. The non-invasive near-infrared light controlled nanomaterial for repairing a damaged nerve according to claim 5, characterized in that: the rare earth element doped inorganic nano material comprises a matrix material and rare earth element ions, wherein the matrix material is NaYF₄、Y₂O₃、YCl₃And Y (CH)₃COO)₃One or more ofThe rare earth element ion is YB³⁺、Er³⁺、Tm³⁺、Ce³⁺、Nd³⁺、Gd³⁺And Ho³⁺One or two of them, the water-soluble biological molecule is one or several of glutamic acid, acetylcholine, dopamine, glycine, adrenalin and NO precursor.