A nanomaterial for optical stimulation of nerve cells, its preparation method and application

Abstract

A nanomaterial for optical stimulation of nerve cells, its preparation method and application. The present invention belongs to the field of nano-biological nerve medicine diagnosis and treatment, and mainly relates to the preparation of an organic / inorganic composite nanomaterial system with a capacitance effect. The present invention uses the supramolecular self-assembly of metal porphyrin to construct functionalized ZnTPyP-Au nanoparticles, which retain the physical and chemical properties of the original assembled molecule ZnTPyP while endowing the material with new optical properties. With ZnTPyP-Au as the core, a manganese oxide shell layer is grown and acts together with nerve cells to stimulate neuron activity using the photocapacitance property. The nanoparticles have good light response performance and electrical characteristics, and the present invention can be used for the treatment of neurological diseases and has important significance in clinical medicine.

Description

Technical Field

[0001] The present invention belongs to the field of nano - biological neuro - medical diagnosis and treatment, and relates to a nano - material for optical stimulation of nerve cells, a preparation method thereof, and an application thereof. Background Art

[0002] With the deepening of the global population aging and the continuous increase of life pressure, the incidence of nervous system diseases such as epilepsy, visual impairment, and nerve deafness has been rising continuously. The common method for treating nervous system diseases is to implant electrodes in the brain region and stimulate the target tissue under a specific pulsed current to stimulate nerve cells to generate specific action potentials for remodeling damaged neurons. However, the implanted electrodes are relatively large in volume and may cause mechanical damage to organisms. Therefore, the use of nano - materials with smaller volume and higher precision for treating nervous system diseases has more application prospects. By activating neurons and generating action potentials under the excitation of a specific stimulus source, nano - materials can stimulate nerve cells to make them excited, achieving the treatment purpose. In fact, the charge distribution state of nerve cells at the resting potential is positive outside and negative inside. Therefore, to activate nerve cells to generate action potentials, a negative electric potential can be generated on the material attached to the cell membrane, thereby affecting the distribution of extracellular cations of the nerve cell membrane and breaking the membrane charge balance state. With the development of research, the application of nano - materials in the field of nerve disease treatment is increasing. For example, gold nanoclusters are used for the treatment of Parkinson's disease, and Au - TiO 2 nanowire arrays are used to restore the vision of blind mice, indicating that the application potential of nano - materials for optical stimulation of nerve cells has attracted much attention.

[0003] The working principle of existing nano - materials for stimulating nerve cells under light excitation is all photoelectrochemical reactions, that is, the Faraday effect occurs at the material - cell / tissue interface, and irreversible redox reactions occur. Such irreversible redox reactions inevitably cause damage to cells or tissues. Therefore, designing a material that undergoes a reversible redox reaction under light stimulation for nerve cell stimulation will have important research significance. Summary of the Invention

[0004] The object of the present invention is to overcome the defect that conventional nano - materials cause damage to nerve cells due to the photoelectrochemical effect under light stimulation, and provides a preparation method of a nano - material for optical stimulation of nerve cells with an organic photosensitizer ZnTPyP - Au (abbreviated as ZSN - Au) as the matrix and MnO 2 as the shell.

[0005] The present invention is driven by the supramolecular self-assembly of metal porphyrin to construct functionalized ZSN-Au nanoparticles (with good light response performance and electrical properties). While retaining the physicochemical properties of the original assembled molecule ZnTPyP, new optical properties are imparted to the material. Therefore, the present invention can be used for the treatment of neurological diseases and has important significance in clinical medicine.

[0006] To achieve the above object, the technical solution provided by the present invention is as follows:

[0007] The present invention provides a novel nanomaterial (nanocapacitor material) for stimulating nerve cells. Based on ZSN-Au nanoparticles, it provides photo-generated electrons; the outer layer is wrapped with flaky MnO 2 , combined with the electrons generated by photoexcitation of the ZSN-Au core and Na outside the nerve cell membrane + act together, that is, a reaction similar to that of a pseudocapacitive supercapacitor electrode occurs, thereby changing the ion distribution outside the nerve cell membrane and being used to activate nerve cells to generate action potentials.

[0008] The novel nanomaterial of the present invention can be applied to the cell level and has a nerve regulation effect on nerve cells.

[0009] The present invention also provides a preparation method of the nanomaterial. (1) Adopt an acid-base neutralization method to assemble and prepare ZSN-Au nanoparticles in surfactant micelles; (2) Then utilize the redox reaction between KMnO 4 and MES to uniformly coat a layer of flaky amorphous MnO on the surface of ZSN-Au 2 , and finally obtain a nanomaterial ZnTPyP-Au@MnO with pseudocapacitive properties 2 , simply referred to as ZAM.

[0010] In step (1), the preparation method of ZSN-Au is as follows: Dissolve ZnTPyP in hydrochloric acid, protonate to obtain ZnTPyP-H 4 4+ , and then add it and an aqueous solution of HAuCl 4 to a mixed solution of a surfactant and an alkali, and then add an aqueous solution of NaBH 4 until the solution turns green, and centrifuge to collect the assembled product ZSN-Au nanoparticles.

[0011] Among them, the concentration of the hydrochloric acid is 0.1 - 0.3 M; preferably, it is 0.2 M.

[0012] Among them, the surfactant is one or more of cetyltrimethylammonium bromide (CTAB), CTAC, MTAB, etc.; preferably, it is CTAB.

[0013] Among them, the concentration of the surfactant is 10 - 20 mM; preferably, it is 10 mM.

[0014] Among them, the base is one or more of NaOH, KOH, etc.; preferably, it is NaOH.

[0015] Among them, the molar ratio of the hydrochloric acid, ZnTPyP, base, and surfactant is 20:1:2:1. When the concentration of hydrochloric acid changes, the concentrations of the base and surfactant need to be changed accordingly.

[0016] Among them, the volume ratio of the ZnTPyP and HAuCl 4 solution is 25:(4 - 5); preferably, it is 25:4.

[0017] Among them, the volume ratio of the surfactant alkaline solution and NaBH 4 solution is 40:(3 - 5); preferably, it is 40:3.

[0018] In the present invention, the mechanism of "adding an aqueous solution of NaBH 4 until the solution turns green" is as follows: Before adding the aqueous solution of NaBH 4 the system is acidic, and at this time, the ZnTPyP monomers have not started to assemble. With the addition of the aqueous solution of NaBH 4 the alkalinity of the system gradually increases. When the pH of the solution system is about 7.4 after adding, the ZnTPyP monomers start to assemble into particles, and the color presented is green. At this time, about 600 μL of the aqueous solution of NaBH 4 is added.

[0019] In the present invention, ZnTPyP will generate electron-hole pairs under light excitation; after Au is combined with ZnTPyP to form ZSN-Au, a contact potential difference is formed between ZnTPyP and Au, and the photo-generated electrons generated by ZnTPyP will flow to Au, effectively promoting the separation of electrons and holes, inhibiting their recombination, greatly enhancing the charge density on the inner core surface, and being beneficial to further 2 reacting with the outer layer of MnO

[0020] Furthermore, during the light stimulation of nerve cells, the ZAM inner core generates electrons under light stimulation and conducts them to the surface of MnO 2 and jointly acts with Na + outside the nerve cell membrane to form a capacitance effect, affecting the charge distribution of neurons, thereby causing nerve cell excitation.

[0021] In a specific embodiment, the preparation of the ZnTPyP-Au includes the following specific steps: Mix 50 mM HAuCl 4An aqueous solution was added to a mixed solution of NaOH (20 mM) and CTAB (10 mM) and stirred for about 1 minute; ZnTPyP was dissolved in a 0.2 M hydrochloric acid solution, and the ZnTPyP molecules were protonated to obtain a 0.01 M ZnTPyP-H 4 4+ solution. 0.5 mL of it was taken and added to the mixed solution of NaOH and CTAB and stirred continuously; finally, 10 mM NaBH 4 aqueous solution was added in small batches, and finally about 600 μL was added. With the addition of NaBH 4 , on the one hand, HAuCl 4 was reduced to Au particles, and at the same time, the alkalinity of the system increased. ZnTPyP-H 4 4+ deprotonated in the presence of base, and the hydrophobic ZnTPyP entered the CTAB micelles. Through Zn-N bonding, ZSN-Au nanoparticles were self-assembled. The assembled nanoparticle solution was green. Then the ZSN-Au nanoparticles were washed 3 times with ethanol to remove the CTAB on the surface of the material.

[0022] The NaBH 4 was added in portions until the solution turned green, and then the system was stirred for about 10 minutes. ZnTPyP-H 4 4+ and OH - had been fully neutralized, and the assembly process was completed. The product was collected by centrifugation.

[0023] The pH of the reaction system solution was 7.2 - 7.8; preferably, it was 7.4.

[0024] The concentration of CTAB was 10 - 20 mM; preferably, it was 10 mM.

[0025] The pH of the reaction system solution and the concentration of CTAB will significantly affect the morphology and size of the product and must be strictly controlled; only when the pH of the reaction system solution and the concentration of CTAB are within the above ranges, can ZnTPyP-Au nanoparticles be synthesized, and their morphology is the same as that in Figure 1 . However, when the pH of the reaction system solution or the concentration of CTAB is not within the above ranges, ZnTPyP will not assemble, and ZnTPyP-Au nanoparticles cannot be successfully obtained, and the experiment fails.

[0026] The assembly time was 5 - 30 minutes; preferably, it was 10 minutes.

[0027] The assembled product ZSN-Au was repeatedly washed with ethanol to remove the surfactant CTAB. After centrifugal washing, it was dispersed in ethanol for standby.

[0028] In step (2), the volume ratio of ZSN-Au, KMnO 4 to the MES solution is (80 - 100):(1 - 3):(1 - 3); preferably, it is 100:2:2.

[0029] In step (2), the concentration of the MES solution is 10 - 100 mM; preferably, it is 25 mM.

[0030] In step (2), the concentration of the KMnO 4 solution is 10 - 100 mM; preferably, it is 20 mM.

[0031] In step (2), by using the redox reaction between KMnO 4 and MES, an amorphous flaky MnO 2 is grown on the surface of ZSN-Au nanoparticles, including the following steps: dispersing the ZSN-Au nanoparticles prepared in step (1) in a mixed solution of 10 mL of ethanol and water, adding 20 mM KMnO 4 aqueous solution and 25 mM MES aqueous solution respectively, and stirring to reduce KMnO 4 to MnO 2 on the surface of ZSN-Au. After that, it is washed with ethanol three times to obtain ZSN-Au@MnO 2 (abbreviated as ZAM) nanoparticles.

[0032] The concentration of the MES solution is 10 - 100 mM; preferably, it is 25 mM.

[0033] The concentration of the KMnO 4 solution is 10 - 100 mM; preferably, it is 20 mM.

[0034] The concentrations of the KMnO 4 and MES solutions should neither be too low nor too high. If the concentrations of both are too low, it is not easy to form a MnO 2 layer on the surface of ZSN-Au; if the concentrations are too high, the MnO 2 layer will be too thick, and the prepared nanomaterial will have poor dispersibility and be prone to agglomeration.

[0035] The stirring time after adding KMnO 4 and MES is 30 - 60 minutes; preferably, it is 30 minutes, and the final product ZAM coated with a layer of flaky MnO 2 can be obtained.

[0036] The finally synthesized product ZAM is repeatedly washed with ethanol to remove the excess KMnO 4 and MES solutions, and after centrifugal washing, it is dispersed in ethanol for standby.

[0037] The beneficial effects of the present invention include:

[0038] In the present invention, the organic porphyrin molecular assembly core ZnTPyP can be excited in the visible light range to generate photo-generated electrons, which react with the outer layer of MnO 2 The Au particles doped in the ZnTPyP assembly body of the present invention can not only improve the electron-hole separation efficiency of ZnTPyP, but also relatively improve the conductivity of the outer layer of MnO 2 (Appendix Figure 4 ). After the surface of the present invention is coated with MnO 2 , it can cooperate with the electrons generated by the photo-excitation of ZSN-Au to react with Na + outside the nerve cell membrane to stimulate the action potential of nerve cells. The present invention can be used for the treatment of nervous system diseases and will be of great significance in clinical applications.

[0039] The preparation method of a nanomaterial for photo-stimulating nerve cells provided by the present invention has the following characteristics: mild reaction conditions, few synthesis steps, and cheap and easily available raw materials. The Au particles doped in the ZnTPyP assembly body improve the electron-hole separation efficiency compared with the single ZnTPyP assembly body, thereby enhancing the stimulating effect of the material on nerve cells. After coating a layer of MnO 2 on the surface of ZSN-Au, the nanomaterial prepared by the present invention has a pseudocapacitance effect, which is different from traditional nanomaterials, such as gold nanoclusters, Au-TiO 2 nanowire arrays, etc., and can have a reversible redox reaction with Na + outside the nerve cell membrane, reducing cell damage. Description of the Drawings

[0040] Figure 1 are the scanning (SEM) and transmission electron microscope (TEM) photos of the nanoparticles prepared in Example 1 of the present invention. Among them, Figures a and b are the TEM images of ZSN-Au, Figure c is the SEM image of ZSN-Au, Figures d and e are the TEM images of ZAM, and Figure f is the SEM image of ZAM.

[0041] Figure 2 are the ultraviolet-visible absorption spectra of the ZnTPyP monomer molecules, ZSN-Au, and ZAM nanoparticles prepared in Example 1 of the present invention.

[0042] Figure 3 are the fluorescence spectra of the ZnTPyP monomer molecules, ZSN-Au, and ZAM nanoparticles prepared in Example 1 of the present invention at 450 nm and 500 nm lasers.

[0043] Figure 4 is the photocurrent response diagram of the ZAM nanoparticles prepared in Example 1 of the present invention.

[0044] Figure 5 This is the cyclic voltammogram of the ZAM nanoparticles prepared in Example 1 of the present invention.

[0045] Figure 6 This shows the effects of the ZAM nanoparticles prepared in Example 1 of the present invention on the excitability of PC12 cells and fetal rat cortical neurons under 405 nm laser stimulation. Detailed implementation manners

[0046] In combination with the following specific examples and drawings, the present invention will be further described in detail. These examples are only used to explain the present invention and do not constitute a limitation to the protection scope of the present invention.

[0047] Example 1: Preparation of ZSN-Au and ZAM nanoparticles:

[0048] 1. Preparation of ZSN-Au nanoparticles

[0049] (1) Preparation of ZnTPyP-H 4 4+ 13.64 mg of ZnTPyP was dissolved in 2 mL of 0.2 M hydrochloric acid solution to obtain a 0.01 M ZnTPyP solution.

[0050] (2) Preparation of the CTAB and NaOH mixed solution: 364.45 mg of CTAB and 80 mg of NaOH were dissolved in 100 mL of water to obtain a CTAB (10 mM) / NaOH (20 mM) mixed solution.

[0051] (3) 80 μL of 0.05 M HAuCl 4 aqueous solution was quickly added to 8 mL of the CTAB / NaOH solution. After stirring for about one minute, 0.5 mL of the ZnTPyP solution obtained in step 1 was quickly added and stirring continued.

[0052] (4) After one minute, 200 μL of 10 mM NaBH 4 solution was added every 30 seconds. When the solution turned green, stirring was continued for 10 minutes and then the reaction was terminated. Ethanol was added and centrifuged at 13,000 revolutions per minute to quickly collect the product, which was then dispersed in 2 mL of absolute ethanol.

[0053] Figure 1 (a) and (b) are the TEM maps of the ZSN-Au nanoparticles prepared in Example 1 of the present invention.

[0054] Figure 1 (c) is the SEM image of the ZSN-Au nanoparticles prepared in Example 1 of the present invention. In the figure: the size of the ZSN-Au is about 250 nm.

[0055] 2. ZSN-Au@MnO 2 (Preparation of (ZAM) nanoparticles):

[0056] Add 1 mL of deionized water and 7 mL of absolute ethanol to the 2 mL ZSN-Au ethanol solution obtained in Step 1. While stirring, sequentially add 200 μL of MES solution and 200 μL of KMnO 4 solution, and continue the reaction for 0.5 hour. Centrifuge to collect the product.

[0057] Figure 1 (d) and (e) are TEM images of the ZAM nanoparticles prepared in Example 1 of the present invention. In the figures: the nanoparticles have good dispersibility and no homogeneous nucleation.

[0058] Figure 1 (f) is the SEM image of the ZAM nanoparticles prepared in Example 1 of the present invention. In the figure: the outer layer of the nanoparticles is uniformly coated with MnO 2 , and there is no homogeneous nucleation.

[0059] Example 2: Detection of solution ultraviolet-visible absorption spectrum

[0060] When the ZAM nanoparticles prepared in Example 1 of the present invention are excited by light in the wavelength range of 250 nm to 800 nm, compared with the ZnTPyP monomer molecules, there are obvious absorption peaks at 415 nm and 455 nm, indicating the formation of J-aggregates. Due to the strong p-electron coupling between the closely arranged ZnTPyP structural units, it is more conducive to photoelectron transfer.

[0061] Figure 2 is the ultraviolet-visible absorption spectrum diagram of the ZSN-Au and ZAM nanoparticles prepared in Example 1 of the present invention. In the figure: the absorption light intensity of the ZAM nanoparticles is higher than that of the ZSN-Au nanoparticles because the outer layer of the ZAM nanoparticles is coated with brown MnO 2 .

[0062] Example 3: Detection of fluorescence spectrum

[0063] When the ZAM nanoparticles prepared in Example 1 of the present invention are irradiated with excitation light at 450 nm and 500 nm, fluorescence with wavelengths around 600 nm and 650 nm is emitted.

[0064] Figure 3Respectively, they are the fluorescence spectrograms of ZnTPyP monomer molecules, ZSN-Au and ZAM nanoparticles in this example under the irradiation of 450nm and 500nm lasers. In the figure: at about 600nm and 650nm, the fluorescence intensities of ZSN-Au and ZAM are significantly lower than that of ZnTPyP monomer molecules, indicating that the electrons generated by the ZnTPyP monomer under photoexcitation are received by Au, which verifies that Au can improve the efficiency of electron-hole separation.

[0065] Example 4: Detection of Photoelectrochemical Properties in Aqueous Solution

[0066] Add the ZAM nanoparticles prepared in Example 1 of the present invention to the Nafion solution. After ultrasonic treatment for 30 minutes, drop it on the glassy carbon electrode and test it in the extracellular fluid using an electrochemical workstation in combination with a three-electrode system.

[0067] Figure 4 It is the photocurrent diagram of the ZAM nanoparticles in this example measured by giving light stimulation every 20 seconds under a three-electrode system. In the figure: when the light stimulation is applied, the current immediately rises, and immediately drops after the light is removed, while there is a certain delay for ZSN. It is verified that compared with the single ZSN, the Au particles doped in the ZnTPyP assembly improve the conductivity of ZAM, making the material have good light response performance.

[0068] Figure 5 It is the cyclic voltammogram of the ZAM nanoparticles in this example measured under a three-electrode system. In the figure: the CV curve of ZAM has no obvious oxidation-reduction peak, verifying that under the action of MnO 2 the ZAM nanoparticles have pseudocapacitance effect.

[0069] Example 5: Detection of Cell Excitability

[0070] The ZAM nanoparticles prepared in Example 1 of the present invention are co-cultured with PC12 cells and fetal rat cortical neurons for 12h at a certain concentration. After calcium ion probe staining, under the stimulation of 405nm laser, the excitability of the cells is observed using a laser confocal microscope.

[0071] Figure 6 It is the synchronous light stimulation experiment of ZAM in this example, and a total of 30 seconds of laser stimulation is carried out. In the figure: the light stimulation starts after 5 seconds, and the fluorescence intensity of the cells with ZAM nanoparticles attached outside the membrane is significantly increased, realizing the optical stimulation of nerve cells.

[0072] In summary, compared with the existing nanomaterials used for optical stimulation of nerve cells, the metal porphyrin supramolecular assembly provided by the present invention can give full play to the respective advantages of the inner core ZSN-Au and the outer shell MnO 2 and +Reversible redox reactions similar to the working mechanism of pseudocapacitors occur, avoiding cell damage, which will be of great significance for clinically using light field stimulation of the lesion area to treat nervous system diseases.

[0073] The above embodiments are only used to further illustrate the technical solutions of the present invention in detail, and cannot be understood as a limitation on the protection scope of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention fall within the protection scope of the present invention.

Claims

1. A nanomaterial for stimulating nerve cells, characterized in that, The matrix of the nanomaterial is ZnTPyP-Au and the shell is MnO 2 .

2. A method for preparing a nanomaterial for stimulating nerve cells, characterized in that, the method comprises the following steps: Step (1) Using the acid-base neutralization method, ZSN-Au nanoparticles are assembled and prepared within surfactant micelles; In step (1), the preparation method of ZSN-Au is as follows: ZnTPyP is dissolved in hydrochloric acid and protonated to obtain ZnTPyP-H 4 4+ , and then it is added to a mixed solution of surfactant and base together with an aqueous solution of HAuCl 4 . Then, an aqueous solution of NaBH 4 is added until the solution turns green, and the assembled product ZSN-Au nanoparticles are collected by centrifugation; The pH of the reaction system solution is 7.2 - 7.8; the surfactant is CTAB, and its concentration is 10 - 20 mM; Step (2) Then, by using the redox reaction between the KMnO 4 solution and the MES solution, a flaky amorphous MnO is uniformly coated on the surface of ZSN-Au, 2 and finally the nanomaterial ZnTPyP-Au@MnO 2 is obtained; In step (2), the concentration of the MES solution is 10 - 100 mM; and / or, the concentration of the KMnO 4 solution is 10 - 100 mM.

3. According to the preparation method described in claim 2, characterized in that, the concentration of the hydrochloric acid is 0.1 - 0.3 M.

4. According to the preparation method described in claim 2, characterized in that, the molar ratio of the hydrochloric acid, ZnTPyP, base, and surfactant is 20:1:2:

1.

5. According to the preparation method described in claim 2, characterized in that, The volume ratio of the ZnTPyP and HAuCl 4 is 25:(4 - 5).

6. According to the preparation method described in claim 2, characterized in that, Alkaline solution of surfactant, NaBH 4 Volume ratio of the solution is 40:(3 - 5).

7. According to the preparation method described in claim 2, characterized in that, the base is one or more of NaOH and KOH.

8. According to the preparation method described in claim 2, characterized in that, In step (2), the volume ratio of ZSN-Au, KMnO 4 to the MES solution is (80-100):(1-3):(1-3).

Images