CN114767850B - Cell membrane targeting nano probe and preparation thereof and application thereof in regulating and controlling neuron calcium ion flow through light response - Google Patents
Cell membrane targeting nano probe and preparation thereof and application thereof in regulating and controlling neuron calcium ion flow through light response Download PDFInfo
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- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 title claims abstract description 41
- 229910001424 calcium ion Inorganic materials 0.000 title claims abstract description 41
- 239000000523 sample Substances 0.000 title claims abstract description 41
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- 230000004298 light response Effects 0.000 title description 7
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 59
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- AXURXPXGTBQYGB-UHFFFAOYSA-N C1(=CC=CC=C1)S(=O)(=O)O.C(CCCCCCCCCCCCCCC)[Na] Chemical compound C1(=CC=CC=C1)S(=O)(=O)O.C(CCCCCCCCCCCCCCC)[Na] AXURXPXGTBQYGB-UHFFFAOYSA-N 0.000 claims description 4
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- HEBRGEBJCIKEKX-UHFFFAOYSA-M sodium;2-hexadecylbenzenesulfonate Chemical compound [Na+].CCCCCCCCCCCCCCCCC1=CC=CC=C1S([O-])(=O)=O HEBRGEBJCIKEKX-UHFFFAOYSA-M 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
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Abstract
The invention discloses a cell membrane targeting nano probe and preparation thereof and application thereof in regulating and controlling neuron calcium ion flow in a photoresponse manner. The invention adopts gold nanorods with small size and high length-diameter ratio as a main structure, and HS-PEG-NHS and Anti-TRPV-1 antibodies as target modification materials, and the assembled probe can realize a non-invasive and non-destructive long-range safety probe. The gold nanorods with small size and high length-diameter ratio are used as a main structure, and the modification of the Anti-TRPV-1 antibody is introduced to improve the cell membrane targeting characteristic of the nano probe, so that the nano probe can be combined to the TRPV-1 antibody on the surface of the neuron membrane. The characteristic that TRPV-1 is sensitive to thermal stimulation is combined with the photo-thermal conversion of the gold nanorod to near infrared light stimulation, so that a synergistic effect is achieved, and under the thermal conversion of the gold nanorod, the switch of a TRPV-1 channel can be rapidly stimulated, so that the movement of neuron calcium ions can be reversibly regulated and controlled.
Description
Technical Field
The invention belongs to the technical field of medical materials, and particularly relates to a cell membrane targeting nano probe and preparation thereof and application thereof in regulating and controlling neuron calcium ion flow through light response.
Background
With the rapid development of nanotechnology, the appearance of nanoprobes provides a more accurate intervention strategy for regulating and controlling cell activities. Wherein, because of the characteristics of noninvasive, nondestructive, non-invasive and the like, the nano-probe is combined with the optical stimulus to regulate and control the life activities of the cells, and the nano-probe is focused by researchers in the field of life medicine. By adjusting parameters such as the wavelength of light, the stimulation time length, the stimulation position and the like, researchers can realize the time-space specific intervention on the vital activities of cells, thereby further regulating and controlling the biochemical changes in vivo and assisting in the treatment of diseases.
Calcium ions are important constituent elements of the human body, and play a special role in various physiological activities, especially in brain activities. When calcium ions flow or overflow through the cell membrane, neurons produce nerve impulses, i.e., nerve electrical signals. The generation of an electrical signal causes the neuron to secrete neurotransmitters and pass on to the next neuron. After the next neuron receives the transmitter, calcium ion flow is further generated, and then the electric signal is transmitted step by step. By repeating the ion flow and the signal transmission, people can develop consciousness, emotion and learn advanced skills such as cognition, learning and memory. When abnormal calcium ion flow occurs in neurons, cognitive difficulties and reaction retardation may occur, and even senile dementia, epilepsy and other diseases may occur. Therefore, after abnormal calcium ion activity, human intervention on the flow is necessary to achieve the purpose of preventing or treating diseases.
At present, two main schemes for regulating and controlling the flow of neuron calcium ions are respectively drug treatment and electrode stimulation. The drug treatment is to take or inject a calcium ion receptor antagonist or an agonist to inhibit the inflow or outflow of calcium ions, which is only applicable to a wide area of abnormal calcium ion flow, but cannot be interfered in a specific space (brain area). Another approach to electrode stimulation, while accurate, requires destructive implantation into the brain for stimulation, and this invasive intervention strategy is necessarily not suitable for long-term regulation. Based on the defects, an intervention strategy for safely and accurately regulating and controlling the flow of calcium ions is needed, and has important significance for basic research and clinical application.
Disclosure of Invention
The first aim of the invention is to provide a preparation method of a neuron cell membrane targeting nano probe, aiming at the defects that the existing calcium ion flow regulation technology cannot realize nondestructive regulation, reversible regulation and insufficient response time. The nanometer probe has the advantages of high tissue response depth, good sensitivity, good monodispersity, clear synthesis path, strong repeatability, simple and convenient operation when regulating and controlling calcium ions, and is suitable for regulating and controlling the calcium ion flow of different types of neurons and preventing and curing related diseases.
The nano probe related to the invention is prepared step by adopting a step method.
Step (1): preparing gold nanorods with the length of 30-70nm, the diameter of 5-10nm and the length-diameter ratio of not less than 5;
preferably, in the step (1), sodium hexadecyl benzene sulfonate and gold chlorate are mixed, silver nitrate is added and stirred for a period of time, then a pH value is regulated to 1-2, and then a reducing agent hydroquinone aqueous solution is added to prepare gold nano seeds (goldenoseeds); and (3) placing the gold nano seeds in a water bath with a certain temperature (4-60 ℃) to react for more than 15 minutes, and rapidly adding sodium borohydride solution as an aging agent to promote the gold nano seeds to longitudinally grow to form gold nano rods.
More preferably, the step (1) is specifically to prepare 10mL of 0.01-0.2M hexadecyl sodium benzenesulfonate solution as a stabilizer, add 500 mu L of 1-20mM gold chlorate solution, stir rapidly, then add 20 mu L of 0.1-2M silver nitrate solution, stir, finally add 10-30 mu L of 1M hydrochloric acid to adjust pH, then add 500 mu L of 0.1-1M hydroquinone aqueous solution as a reducing agent, and prepare gold nano seeds; the gold nano-seeds are reacted in water bath with a certain temperature (4-60 ℃) for more than 15 minutes, and sodium borohydride solution is rapidly added as an aging agent to promote the gold nano-seeds to longitudinally grow to form gold nano-rods.
Step (2): preparation of thiol polyethylene glycol-Anti-capsaicin receptor antibody (HS-PEG-Anti-TRPV-1) dimer.
In a weak alkaline environment, adding Anti-TRPV-1 into a sulfhydryl polyethylene glycol succinamate (HS-PEG-NHS) solution, reacting for more than 2 hours at a low temperature (< 25 ℃), removing unreacted substances by a dialysis bag, and freeze-drying the rest solution to obtain the HS-PEG-Anti-TRPV-1 dimer.
Preferably, the pH is in the range of 5 to 9;
preferably, the molar ratio of Anti-TRPV-1 to HS-PEG-NHS is 1:10-10:1;
step (3): modification of HS-PEG-Anti-TRPV-1 dimer.
In an aqueous phase environment system, uniformly mixing the gold nanorods and the HS-PEG-Anti-TRPV-1 dimer, and obtaining the gold nanorods modified by the HS-PEG-Anti-TRPV-1 dimer, namely a crude product of the nano probe by utilizing an Au-S bond with high affinity. After centrifugal water washing, unreacted substances can be removed, and the purified nano probe is obtained.
Preferably, the molar ratio of the gold nanorods to the HS-PEG-Anti-TRPV-1 dimer is 1:10-10:1.
The second object of the invention is to provide a neuron cell membrane targeting nano probe which is prepared by the method.
The third object of the invention is to provide an application of the neuron cell membrane targeting nano probe in regulating and controlling the flow of neuron calcium ions in a light response manner.
The fourth object of the invention is the use of a neuronal cell membrane targeting nanoprobe for the intervention or treatment of a disease kit associated with an imbalance in calcium ion flow.
A fifth object of the present invention is a kit for intervention or treatment of a disease associated with an imbalance in calcium ion flow, comprising a neuronal cell membrane targeting nanoprobe as described above.
The beneficial effects of the invention are as follows:
1) The invention adopts gold nanorods with small size and high length-diameter ratio as a main structure, and HS-PEG-NHS and Anti-TRPV-1 antibodies as target modification materials, and the assembled probe can realize a non-invasive and non-destructive long-range safety probe.
2) The invention provides a gold nanorod with small size and high length-diameter ratio as a main structure, and modification of an Anti-TRPV-1 antibody is introduced to improve the cell membrane targeting characteristic of the nano probe, so that the nano probe can be combined to the TRPV-1 antibody on the surface of a neuron membrane. The characteristic that TRPV-1 is sensitive to thermal stimulation is combined with the photo-thermal conversion of the gold nanorod to near infrared light stimulation, so that a synergistic effect is achieved, and under the thermal conversion of the gold nanorod, the switch of a TRPV-1 channel can be rapidly stimulated, so that the movement of neuron calcium ions can be reversibly regulated and controlled.
3) The invention adopts the Anti-TRPV-1 to precisely target the nano material to the vicinity of the TRPV-1, realizes in-situ close-range neuron regulation, and has space-time specificity, sensitive response and quick response.
4) The invention adopts gold nanorods with proper size and length-diameter ratio, thereby realizing proper absorption spectrum of the nanoprobe, and directly influencing the tissue depth of the nanoprobe which can respond in the biological brain.
5) The introduction of PEG groups in the HS-PEG-NHS polymer adopted by the invention further improves the monodispersity of the nano probe, and prevents aggregation of the nano probe, thereby causing calcium ion regulation failure.
In summary, the invention adopts the special nano probe to realize the light-regulated neuron calcium ion flow, the operation is simple, the thought is clear, the structural components of the obtained nano probe are clear, the functional properties are clear, and the synergistic effect is fully exerted among the three materials. Further research on neuron cells and in-vitro brain slices proves that the invention provides a high-sensitivity, space-time specificity and repeatability nano probe for remotely controlling calcium ion flow in a light response mode.
Drawings
Fig. 1. A) photographs of small-sized gold nanorod solutions with different aspect ratios and corresponding maximum absorption wavelengths. b) Visible-near infrared absorption spectra of small-sized gold nanorods with different length-diameter ratios.
FIG. 2 is a transmission electron micrograph of a nanoprobe solution of different aspect ratios.
FIG. 3 surface charge analysis of gold nanorods (sAR), PEG-modified gold nanorods (sAR-PEG), nanoprobes (sAR-PEG-AA).
FIG. 4. Research on cell membrane targeting of nanoprobes (nanoprobes were previously red fluorescence-labeled). Scale bar: 20 μm.
Fig. 5. Influence of near infrared light stimulation on calcium ion flow of nerve cells (same region) incubated with gold nanorods (without targeting modification) (green fluorescence is calcium ions). Scale bar: 150 μm.
Fig. 6. Influence of near infrared light stimulation on calcium ion flow conditions of neural cells (same region) incubated with nanoprobes (green fluorescence is calcium ions). Scale bar: 150 μm.
Figure 7. Influence of near infrared light stimulation on ex vivo brain slice discharge conditions of nanoprobe incubation. The red line area is the light-administration stimulation period.
Detailed Description
As described above, in view of the shortcomings of the prior art, the present inventors have long studied and practiced in a large number of ways, and have proposed the technical solution of the present invention, which is based on at least: 1) The invention designs a nano probe for regulating and controlling the flow of neuron calcium ions by taking gold nano rods with small size and high length-diameter ratio as light response materials, combining an anti-capsaicin receptor antibody and utilizing sulfhydryl polyethylene glycol succinamide ester for biological coupling. Gold nanorods with proper size and length-diameter ratio are selected to realize the required brain delivery requirement and deeper photo-thermal conversion, which are important for brain regulation. 2) Anti-TRPV-1 can be precisely combined with a capsaicin receptor (TRPV-1) on the surface of a neuron, and the nanomaterial is precisely targeted to the vicinity of the TRPV-1. When the nanomaterial generates specific stimulus, such as local temperature rise and mechanical intervention, TRVP-1 can be opened as channel protein to guide rapid inflow of calcium ions, and the inflow speed is directly related to the stimulus intensity. 3) HS-PEG-NHS is used as a functional polymer, which not only plays a role in connection, but also can form hydrogen bonds with water molecules in living environment to prevent aggregation of nano materials so as to prolong a regulation time window. 4) The gold nanorod, the Anti-TRPV-1 and the HS-PEG-NHS are subjected to gradual reaction, so that the obtained nano probe has high responsiveness to near infrared light, has the effect of accurately interfering neuron calcium ions, and fully plays the synergistic effect among different material units. The nano probe prepared by the invention has extremely strong absorption near a near infrared two-region, and can realize the regulation and control under deep tissues; the size is small, which is beneficial to penetrating the blood brain barrier; the monodispersity is good, and the rapid and accurate targeting is facilitated. The nano probe prepared by the invention can be used for safely and accurately regulating and controlling the flow of the calcium ions of neurons, thereby realizing the intervention of a specific biochemical reaction process and the treatment of diseases related to the imbalance of the flow of the calcium ions.
Example 1:
1) Preparing gold nanorods with the length of 70nm, the diameter of 9nm, the length-diameter ratio of 8.3 and the maximum absorption wavelength of 1129 nm.
Preparing 10mL of 0.1M hexadecyl sodium benzenesulfonate solution as a stabilizer, adding the newly prepared 500 mu L of 10mM gold chlorate solution as a raw material, rapidly stirring, adding 20 mu L of 0.1M silver nitrate solution, stirring, then adding 20 mu L of 1M hydrochloric acid to adjust pH, and finally adding 500 mu L of 0.1M hydroquinone aqueous solution as a reducing agent to prepare gold nano seeds (goldenna seeds). In a water bath at 30 ℃ for 15 minutes, 10 mu L of sodium borohydride solution with the concentration of 10mM is rapidly added as an aging agent to promote the longitudinal growth of gold nano seeds to form gold nano rods, and the obtained gold nano rods have a length of 70nm, a width of 9nm, an aspect ratio of 8.3 and a near infrared two-region with the maximum absorption wavelength of 1129 nm.
2) Preparation of HS-PEG-Anti-TRPV-1 dimer.
In a weakly alkaline environment, 5mg/mL of HS-PEG-NHS solution is prepared, wherein the molecular weight of the HS-PEG-NHS is about 5000Da, anti-TRPV-1 with the same molar weight as the HS-PEG-NHS is added for reaction for 12 hours at the temperature of 4 ℃, unreacted substances are removed by a dialysis bag, and the residual solution is freeze-dried to obtain the HS-PEG-Anti-TRPV-1 dimer.
3) Modification of HS-PEG-Anti-TRPV-1 dimer.
In a water phase environment system, uniformly mixing a gold nanorod and an HS-PEG-Anti-TRPV-1 dimer in a certain molar ratio (1:1), stirring and reacting for 12 hours, and obtaining the gold nanorod modified by the HS-PEG-Anti-TRPV-1 dimer, namely a crude product of the nano probe by utilizing an Au-S bond with high affinity. After ultra-high speed centrifugation at 12000rpm and washing with ultra-pure water 2 times, unreacted materials can be removed to obtain purified nanoprobe.
4) The nano probe regulates and controls the calcium ion inflow of nerve cells.
The nano probe is incubated with ND7/23 nerve cells at the concentration of 0.1mg/mL, and the target enrichment can be carried out rapidly on the surface of the nerve cell membrane within 2 hours. The nerve cells are subjected to near infrared two-region optical stimulation, the stimulation time is 5 minutes, and the stimulation intensity is 0.3W/cm 2 The confocal microscope is used for observation to confirm the rapid inflow of calcium ions in nerve cells, so as to realize the regulation and control of the calcium ion flow by light response.
As shown in figure 1, the scheme provided by the invention can be used for preparing gold nanorods absorbed in a near infrared two-region of 900-1200nm, and the color of the obtained gold nanorod solution is directly related to the absorption spectrum.
As shown in fig. 2, the scheme proposed by the present invention can prepare nanoprobes with different aspect ratios.
As shown in FIG. 3, the gold nanorods (sAR), the PEG modified gold nanorods (sAR-PEG) and the Anti-TRPV-1-PEG modified gold nanorods (sAR-PEG-AA) prepared by the method have different indicated charges, and the preparation of the nano probe is proved.
As shown in FIG. 4, the nano-probe prepared by the invention can be rapidly enriched on the surface of nerve cells, and the gold nano-rod without targeted modification has no cell membrane targeting effect.
As shown in fig. 5 and fig. 6, under the same light stimulation, the gold nanorods (sAR) which are not subjected to targeted modification cannot regulate and control the flow of calcium ions, while the nano-probes (sAR-PEG-AA) can promote the calcium ion inflow of nerve cells, and the higher the stimulation intensity, the more obvious the inflow.
Example 2:
1) Preparing gold nanorods with the length of 35nm, the width of 7nm, the length-diameter ratio of 5 and the maximum absorption wavelength of 953nm.
Preparing 10mL of 0.1M hexadecyl sodium benzenesulfonate solution as a stabilizer, adding the newly prepared 500 mu L of 10mM gold chlorate solution as a raw material, rapidly stirring, adding 20 mu L of 0.1M silver nitrate solution, stirring, then adding 10 mu L of 1M hydrochloric acid to adjust pH, and finally adding 500 mu L of 0.1M hydroquinone aqueous solution as a reducing agent to prepare the gold nano seeds. In a water bath at 30 ℃ for 15 minutes, 10 mu L of sodium borohydride solution with the concentration of 10mM is rapidly added as an aging agent to promote the longitudinal growth of gold nano seeds to form gold nano rods, and the obtained gold nano rods have the length of 35nm, the width of 7nm, the length-diameter ratio of 5 and the maximum absorption wavelength of 953nm.
2) Preparation of HS-PEG-Anti-TRPV-1 dimer.
In a weak alkaline environment, preparing 0.1-10mg/mL of HS-PEG-NHS solution, wherein the molecular weight of the HS-PEG-NHS is about 2000Da, adding a certain mass of Anti-TRPV-1 (the molar ratio of the Anti-TRPV-1 to the HS-PEG-NHS is 1:2), reacting for 12 hours at 4 ℃, removing unreacted substances by a dialysis bag, and freeze-drying the residual solution to obtain the HS-PEG-Anti-TRPV-1 dimer.
3) Modification of HS-PEG-Anti-TRPV-1 dimer.
In a water phase environment system, uniformly mixing a gold nanorod and an HS-PEG-Anti-TRPV-1 dimer in a certain molar ratio (1:1), stirring and reacting for 12 hours, and obtaining the gold nanorod modified by the HS-PEG-Anti-TRPV-1 dimer, namely a crude product of the nano probe by utilizing an Au-S bond with high affinity. After ultra-high speed centrifugation at 12000rpm and washing with ultra-pure water 2 times, unreacted materials can be removed to obtain purified nanoprobe.
4) The nanoprobe regulates the release of electrical signals by neurons.
Incubating the nanoprobe with ICR young mouse brain slice at concentration of 0.1mg/mL, and applying near infrared light stimulation to Hippocampus area of brain slice with stimulation intensity of 0.4W/cm 2 The discharge of neurons is recorded in real time by using an electrophysiological technology. The study confirms that the nano probe can respond to the activation of brain slices in real time under the light stimulation to promote the discharge of neurons.
As shown in fig. 7, the neurons treated with the nanoprobe can respond rapidly (within 20 seconds) after the near infrared light stimulus is given, releasing the electrical signal, while the untreated neurons do not respond to the light stimulus.
Claims (7)
1. The preparation method of the neuron cell membrane targeting nano probe is characterized by comprising the following steps of:
step (1): preparation of gold nanorods:
preparing 10mL of 0.01-0.2M hexadecyl sodium benzenesulfonate solution as a stabilizer, adding 500 mu L of 1-20mM gold chloroauric acid solution, stirring rapidly, then adding 20 mu L of 0.1-2M silver nitrate solution, stirring, finally adding 10-30 mu L of 1M hydrochloric acid, regulating pH, and then adding 500 mu L of 0.1-1M hydroquinone aqueous solution as a reducing agent to prepare gold nano seeds; reacting the gold nano seeds in a water bath at the temperature of 4-60 ℃ for more than 15 minutes, and rapidly adding sodium borohydride solution as an aging agent to promote the gold nano seeds to longitudinally grow to form gold nano rods with the length of 30-70nm, the diameter of 5-10nm and the length-diameter ratio of not less than 5;
step (2): preparation of thiol polyethylene glycol-Anti-capsaicin receptor antibody HS-PEG-Anti-TRPV-1 dimer;
adding Anti-TRPV-1 into a sulfhydryl polyethylene glycol succinamate HS-PEG-NHS solution in a weak alkaline environment, reacting for more than 2 hours at the temperature of <25 ℃, removing unreacted substances by a dialysis bag, and freeze-drying the rest solution to obtain an HS-PEG-Anti-TRPV-1 dimer;
step (3): modification of HS-PEG-Anti-TRPV-1 dimer;
in an aqueous phase environment system, uniformly mixing a gold nanorod and an HS-PEG-Anti-TRPV-1 dimer, and obtaining a gold nanorod modified by the HS-PEG-Anti-TRPV-1 dimer, namely a crude product of the nano probe by utilizing an Au-S bond with high affinity; after centrifugal water washing, unreacted substances can be removed, and the purified nano probe is obtained.
2. The process of claim 1, wherein the reaction system of step (2) has a pH of from 5 to 9.
3. The method of claim 1, wherein the molar ratio of Anti-TRPV-1 to HS-PEG-NHS in step (2) is from 1:10 to 10:1.
4. The method of claim 1, wherein the molar ratio of gold nanorods to HS-PEG-Anti-TRPV-1 dimer of step (3) is 1:10 to 10:1.
5. A neuronal cell membrane targeting nanoprobe prepared by the method of any of claims 1-4.
6. Use of a neuronal cell membrane targeting nanoprobe according to claim 5 for the preparation of a kit for the intervention or treatment of a disease associated with an imbalance in calcium ion flow.
7. A kit for the intervention or treatment of a disease associated with an imbalance in calcium ion flow comprising a neuronal cell membrane targeting nanoprobe according to claim 5.
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| WO2013029025A1 (en) * | 2011-08-24 | 2013-02-28 | The Rockefeller University | Compositions and methods to modulate cell activity |
| CN112620646A (en) * | 2020-12-17 | 2021-04-09 | 上海纳米技术及应用国家工程研究中心有限公司 | Preparation method of large length-diameter ratio gold nanorod with platinum particles growing at two ends and product thereof |
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