Therapeutic system for realizing noninvasive photoinheritance by iPSCs (induced pluripotent stem cells) derived tentacle vector as well as preparation method and application of therapeutic system

Abstract

The invention discloses a treatment system for realizing noninvasive optogenetics by using an iPSCs (induced pluripotent stem cells) derived'tentacle 'carrier as well as a preparation method and application of the treatment system. The treatment system comprises a noninvasive optogenetics material and a targeted drug delivery carrier, the non-invasive optical genetic material is an adeno-associated virus containing ChR2 (H134R) photosensitive protein and RLuc renilla luciferase; the targeted drug delivery carrier is a'tentacle 'outer vesicle which is obtained by inducing and differentiating iPSCs into'tentacle' stem cells and carrying out gradient extrusion on the'tentacle 'stem cells. According to the constructed treatment system, an effective treatment means of degenerative neuron activation and tissue microenvironment remodeling can be achieved at the same time, the problem that an existing photogenetic technology is continuous and invasive is solved, and non-invasive treatment in the whole process is achieved. According to the treatment strategy generated by the invention, degenerative neurons are effectively activated, the pathological environment is improved, and a good treatment effect is shown on mice suffering from Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a treatment system for non-invasive optogenetics using iPSCs-derived "tentacle" vectors. Background Art

[0002] Neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, etc., are caused by severe neurodegeneration due to damaged pathological environments with deposited substances, resulting in slow reactions and loss of physical control in patients, seriously affecting the quality of life of patients. However, the pathogenesis of neurodegenerative diseases has not been fully clarified, and there is a lack of means to completely delay or terminate the disease process.

[0003] Optogenetics, a means of using light to cause specific ion currents to depolarize or hyperpolarize to manipulate individual neurons, achieving precise control of neural circuits with both spatial and temporal precision, has completely changed the ability to decode the operating mechanism of the nervous system and precisely sculpt neural behaviors. By transfecting the light-sensitive opsin ChR2 to promote the release of neurotransmitters and synaptic remodeling, etc., optogenetic technology can reactivate degenerated nerves and improve the memory of AD model mice. However, traditional optogenetic methods using adeno-associated virus (AAV) as an infection vector lack neural targeting, resulting in low transfection efficiency, uneven penetration and distribution of invasive light sources, unstable activation states, and continuous invasiveness during the treatment process, and there are still some challenges in its clinical application.

[0004] Bioluminescence refers to the means of generating light of a corresponding wavelength by the decomposition of a fluorescent substrate by luciferase. After expressing luciferase in the corresponding part of the body and injecting the substrate intravenously, a spontaneous light source can be generated in the body. Therefore, after fusing luciferase that generates excitation light of a specific wavelength with a photosensitive protein, the problems of light penetration and light distribution can be solved, and non-invasive in vivo light excitation operations can be achieved. Summary of the Invention

[0005] Object of the Invention: Aiming at the problems existing in the prior art, the present invention provides a treatment system for non-invasive optogenetics using iPSCs-derived "tentacle" vectors. The present invention uses "tentacle" extracellular vesicles derived from the directed differentiation of iPSCs to transport optimized bioluminescence-optogenetics fusion viruses, realizing non-invasive combined treatment of stem cells and optogenetics for neurodegenerative diseases, effectively solving the problem of invasiveness throughout the existing optogenetic treatment, and expanding the potential of optogenetic technology as a treatment means in neurodegenerative diseases.

[0006] The present invention also provides a preparation method and application of the treatment system for non-invasive optogenetics using the iPSCs-derived "tentacle" vector.

[0007] Technical solution: To achieve the above solution, the present invention provides a treatment system for non-invasive optogenetics using iPSCs-derived "tentacle" carriers, which includes a non-invasive optogenetic substance and a targeted drug delivery carrier; the non-invasive optogenetic substance is a bioluminescent-optogenetic adeno-associated virus; the bioluminescent-optogenetic adeno-associated virus is an adeno-associated virus containing the ChR2(H134R) photosensitive protein and the RLuc Renilla luciferase; the targeted drug delivery carrier is obtained by inducing iPSCs to differentiate into "tentacle" stem cells and obtaining "tentacle" extracellular vesicles through gradient extrusion; the non-invasive optogenetic substance is loaded into the targeted drug delivery carrier.

[0008] Preferably, the bioluminescent-optogenetic adeno-associated virus is optimized for partial virus uncoating and purified.

[0009] Preferably, the targeted drug delivery carrier is "tentacle" extracellular vesicles derived from the directed differentiation of iPSCs; the "tentacle" extracellular vesicles need to be pretreated for permeability before use.

[0010] Furthermore, the "tentacle" extracellular vesicles load the bioluminescent-optogenetic optimized adeno-associated virus system into the "tentacle" extracellular vesicles to form a treatment system for non-invasive optogenetics using "tentacle" carriers; the application of the treatment system for non-invasive optogenetics using iPSCs-derived "tentacle" carriers needs to be combined with corresponding optogenetic activation substances.

[0011] Among them, the non-invasive optogenetic substance is a bioluminescent-optogenetic adeno-associated virus, specifically, a fusion adeno-associated virus vector plasmid of the bioluminescent Renilla luciferase and the photosensitive channel protein based on ChR2(H134R) is constructed and packaged into an adeno-associated virus.

[0012] Among them, the iPSCs are commercial hiPSC cell lines.

[0013] The preparation method of the treatment system for non-invasive optogenetics using iPSCs-derived "tentacle" carriers according to the present invention includes the following steps:

[0014] (1) Construction of bioluminescent-optogenetic adeno-associated virus:

[0015] The gene sequences of the ChR2(H134R) photosensitive protein and the RLuc Renilla luciferase are constructed onto an adeno-associated virus vector, and the adeno-associated virus is packaged by the method of triple plasmid co-transfection.

[0016] (2) Construction of "tentacle" extracellular vesicles:

[0017] After culturing iPSCs stably, passage is carried out; the medium of the iPSCs with stable state after the above-mentioned passage culture is replaced with the pre-induction medium, and continuous culture is carried out. When the cells show a spindle shape and are slightly loosely arranged, neural preparatory cells are obtained; the medium of the above-mentioned neural preparatory cells is replaced with the maintenance medium until "neural rosette-like wreath" neural spheroid clusters with a higher degree of cell aggregation appear in the culture; the above-mentioned neural spheroid clusters are gently digested and passaged into a laminin-coated culture dish, and continuous culture is carried out with the "maintenance medium" until the cell state is stable, and induced preparatory cells are obtained; the medium of the above-mentioned induced preparatory cells is replaced with the post-induction medium, and culture is carried out until cell synapses appear, and differentiation is completed to obtain "tentacle" stem cells; the above-mentioned "tentacle" stem cells resuspended after digestion are purified by gradient extrusion to obtain "tentacle" extracellular vesicles;

[0018] (3) Construct a treatment system for transporting non-invasive optogenetics by "tentacle" extracellular vesicles:

[0019] The transfection reagent is co-incubated with the "tentacle" extracellular vesicles, and the uncoated virus in step (1) is mixed with the "tentacle" extracellular vesicles in a certain proportion, and after incubation, the mechanical co-extrusion method is used to obtain a complete treatment system.

[0020] Among them, the bioluminescence-optogenetic adeno-associated virus constructed in step (1) is optimized for partial virus uncoating by proton uncoating technology and purified by Nycodenz step density gradient centrifugation technology.

[0021] Preferably, the method for optimizing partial virus uncoating is specifically as follows: the packaged virus is dialyzed in an acetic acid buffer solution at pH 5.0 and 37°C for 10 minutes.

[0022] Among them, in step (3), the uncoated virus is mixed with the "tentacle" extracellular vesicles in a ratio of 10:1 and incubated in a water bath at 30-37°C for 1-2 hours.

[0023] Preferably, the "tentacle" extracellular vesicles in step (3) need to be pretreated for permeability before use, specifically by co-incubating with a transfection aid reagent and the "tentacle" extracellular vesicles in a water bath environment at 37°C for 15 minutes.

[0024] Among them, the "tentacle" extracellular vesicles load the bioluminescence-optogenetic optimized adeno-associated virus system onto the "tentacle" extracellular vesicles, and the loading technology is the mechanical co-extrusion method. The mechanical co-extrusion method is to extrude 5-10 times respectively under filter membranes with pore sizes of 400 nm, 200 nm, and 100 nm using a liposome extruder.

[0025] Among them, in the construction of the outer vesicles of "tentacles" in step (2): iPSCs are cultured in a culture environment coated with Matrigel and supported by ncTarget hPSC, cultured for 1-2 days until the cell state is stable for passage, and then passaged and cultured until the density convergence degree is more than 80%. Among them, the pre-induction medium includes N2, B27, Neurobasal DMEM / F12. After culturing for 5-6 days, some of the cells appear spindle-shaped and change from a tightly aggregated state to a slightly loose arrangement state, and are considered to have transformed into neural preparatory cells. The maintenance medium includes N2, B27, Neurobasal, DMEM / F12, bFGF. After culturing for 1-2 days, the cells maintain a proliferative state, and a large number of cells proliferate and form neural spherical clusters. The neural spherical clusters are digested and plated onto a culture dish pre-coated with Laminin at a cell plating density of 80%, and cultured with the maintenance medium for 10-12 hours to make the cells reach a stable state. Among them, the post-induction medium includes N2, B27, Neurobasal, DMEM / F12, bFGF, and purified neuron membranes. After culturing for 5-6 days, the cells gradually extend synapses, and finally obtain the stem cells of "tentacles". The gradient extrusion method is as follows: The "tentacle" stem cells resuspended in PBS are placed under ultrasonic treatment conditions in a water bath for ultrasonic treatment for minutes, and the suspension is sequentially passed through filter membranes with pore sizes of 10 μm, 5 μm, 1 μm, 400 nm, 200 nm, and 100 nm using a liposome extruder, and each particle size is extruded 3-4 times repeatedly to obtain a vesicle mixture.

[0026] Preferably, the method for generating multifunctional "tentacle" outer vesicles based on the directed differentiation of iPSCs includes the following steps:

[0027] S1, culture iPSCs in a culture environment coated with Matrigel and supported by ncTarget hPSC, passage after culturing until the convergence state reaches 80%, and culture for 1-2 days until the cell state is stable and the convergence degree is 80%;

[0028] S2, change the medium of the above-mentioned stable iPSCs to the "pre-induction medium", adjust the culture environment, and culture for 4 days. When the cells show a spindle shape and are arranged slightly loosely, neural preparatory cells are obtained;

[0029] S3, change the medium of the above-mentioned neural preparatory cells to the "maintenance medium", adjust the culture environment until neural spherical clusters with a relatively high cell convergence degree of "neural rose-like wreaths" appear;

[0030] S4, gently digest the above-mentioned neural spherical clusters, passage them into a culture dish coated with laminin, and continue to culture with the "maintenance medium" for 12 hours until the cell state is stable to obtain induced preparatory cells;

[0031] S5, replacing the culture medium of the above-mentioned induced preparatory cells with "post-induction medium", culturing for 5 days until cell synapses appear and differentiation is completed, thereby obtaining "tentacle" stem cells;

[0032] S6, the above-mentioned tentacle stem cells were digested and resuspended, and purified by gradient extrusion to obtain tentacle extracellular vesicles;

[0033] Further, in the above step S2, the composition of the "pre-induction medium" is: 49.5% DMEM / F12 medium + 49.5% Neurobasal medium + 0.5% N2 additive + 0.5% B27 additive;

[0034] Furthermore, in the above step S2, the culture environment is adjusted from 37°C, 5.0% carbon dioxide to: 37°C, 5.1% carbon dioxide;

[0035] Furthermore, in the above step S3, the composition of the "maintenance medium" is: 49.5% DMEM / F12 medium + 49.5% Neurobasal medium + 0.5% N2 additive + 0.5% B27 additive + 20 ng / mL bFGF;

[0036] Furthermore, in the above step S2, the culture environment is adjusted from 37° C. and 5.1% carbon dioxide to 37° C. and 5.0% carbon dioxide, and this culture environment is maintained during the subsequent induction process;

[0037] Furthermore, in the above step S4, the digestion method is as follows: digestion with EDTA digestion solution in a 37°C incubator for 5 minutes, gently shaking to obtain continuous digested cells, slowly blowing 3 times, and then slabbed into a culture dish at a cell slab density of 80%;

[0038] Further, in the above step S5, the composition of the "post-induction medium" is: 49.5% DMEM / F12 medium + 49.5% Neurobasal medium + 0.5% N2 additive + 0.5% B27 additive + 10 ng / mL bFGF + 60 μg / mL purified neuronal membrane;

[0039] Furthermore, in the above step S6, the gradient extrusion method is: the "tentacle" stem cells resuspended in PBS are placed in a 50W water bath ultrasonic condition for 10 minutes, and the suspension is sequentially passed through filter membranes with pore sizes of 10 μm, 5 μm, 1 μm, 400 nm, 200 nm, and 100 nm using a liposome extruder, and each particle size is repeatedly extruded 3 times to obtain a vesicle mixture;

[0040] Further, in the above step S6, the purification method is to centrifuge the above vesicle mixture at 2000 g for 10 minutes, remove the large particle precipitate, then centrifuge the supernatant at 10000 g for 30 minutes, remove the cell debris precipitate, and the obtained supernatant is the "tentacle" extracellular vesicle suspension.

[0041] Application of the iPSCs-derived "tentacle" vector-based non-invasive optogenetic therapy system of the present invention in the preparation of a drug or reagent capable of simultaneously achieving degenerative neuron activation and tissue microenvironment remodeling.

[0042] Application of the iPSCs-derived "tentacle" vector-based non-invasive optogenetic therapy system of the present invention in the preparation of drugs for treating Alzheimer's disease and Parkinson's disease.

[0043] Among them, the application of the therapy system needs to be combined with a corresponding optogenetic activation substance, and the optogenetic activation substance is coelenterazine.

[0044] In view of the problem that traditional optogenetic adeno-associated virus vectors lack neurotargeting, the present invention uses "tentacle" vesicles derived from the directed differentiation of iPSCs to target and deliver the optogenetic virus to neurons in damaged brain regions to achieve efficient transfection. At the same time, the rich stem cell therapeutic components it contains will synergistically improve the brain pathological state and reshape the microenvironment. Considering that the endosomal pathway after encapsulation of extracellular vesicles affects the infection pathway of the virus itself, the virus will be subjected to proton de-encapsulation treatment before uploading to accelerate the intracellular processing process and improve the stability of efficient expression of the target protein. The present invention uses "tentacle" extracellular vesicles derived from the directed differentiation of iPSCs to carry an optimized bioluminescence-optogenetic fusion virus to achieve non-invasive combined therapy of stem cells and optogenetics for neurodegenerative diseases.

[0045] In the present invention, iPSCs are induced to differentiate into "tentacle" stem cells with both neuron targeting and pathological environment repair functions through a specific method; the corresponding "tentacle" extracellular vesicles are obtained by the gradient extrusion method. The directed differentiation and extracellular vesicle production scheme provided by the present invention has good reproducibility, and the raw materials are easy to obtain and can be mass-produced. The produced multifunctional "tentacle" extracellular vesicles have rich biological functions and have broad application prospects in the treatment of various neurological diseases.

[0046] The present invention constructs an optimized virus for bioluminescence-activated optogenetics and uploads it into the "tentacle" extracellular vesicles generated from the directed differentiation of iPSCs, which have neuron-specific adhesion ability and the ability to repair damaged nerve tissues of stem cells, to produce an effective treatment method that can simultaneously achieve the activation of degenerative neurons and the remodeling of the tissue microenvironment, solve the problem of the continuous invasiveness of existing optogenetic technologies, and achieve non-invasive treatment throughout the process. The treatment strategy produced by the present invention effectively activates degenerative neurons, improves the pathological environment, and shows good therapeutic effects on Alzheimer's disease model mice.

[0047] The present invention provides a non-invasive optogenetic co-therapy system throughout the whole process. The treatment plan based on this treatment system can achieve non-invasive treatment throughout the whole process. Most of the existing optogenetic technologies require in-situ injection of viruses, and their activation also requires in-situ implantation of optical fibers, which has double invasiveness of transfection and activation. This re-injury to the already diseased brain area is obviously unrealistic. At the same time, single optogenetic therapy is not sufficient to improve the pathological environment. The treatment system of the present invention can target neurons in the pathological environment in vivo through the "tentacle" vesicles carrying the optogenetic system and non-invasively activate neurons through bioluminescence. At the same time, the therapeutic substances carried by stem cells will synergistically repair the pathological environment and create a better living environment for the activated neurons. Generally speaking, this invention first proposes the concept and technical method of using stem cells to cooperate with optogenetics for non-invasive treatment of neurodegenerative diseases throughout the whole process.

[0048] The present invention uses an independently designed induction differentiation protocol to regulate the differentiation of stem cells into stem cells with specific epigenetic states. The generated vesicles will have both nerve adhesion and pathological environment repair functions, can eliminate the invasiveness of optogenetic transfection while synergistically improving the pathological environment; further, Renilla luciferase is coupled with optogenetic elements, and bioluminescence is used to activate optogenetics to eliminate the invasiveness of optogenetic activation.

[0049] In addition, the unique design principle of the present invention lies in the utilization of the concept of "homologous attraction". During the differentiation of stem cells, "neuronal membranes" are used to stretch the stem cells, causing "tentacle" structures to appear on the cell membrane before the stem cells lose their unique content substances, thus generating "tentacle" stem cells. The vesicles of "tentacle" stem cells, namely "tentacle" vesicles, have the functions of both neuronal targeting and pathological environment repair, and are applicable to the drug targeting delivery and synergistic treatment of various neurodegenerative diseases. The present invention first proposes to generate functional carriers by directionally inducing the differentiation of stem cells. Compared with the existing stem cell differentiation technologies and the existing engineering vector modifications, it has extremely high value, which is reflected in: 1) creating a new culture method that can make stem cells differentiate into a specific epigenetic state different from somatic cells, enabling the vesicles produced to have ideal functions; 2) this method of generating engineering vectors through the entire process of stem cell differentiation naturally produces engineering vectors with more complete functional domains, making it easier for the vectors to achieve ideal biological functions. In contrast, the existing separate engineering vector modifications can only one-sidedly regulate the expression of certain proteins, etc., with incomplete functions and more complex operations. Therefore, the present invention has made great technological and conceptual progress in both the field of stem cell differentiation and the development of targeted drug delivery carriers.

[0050] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0051] 1. The method for generating functional extracellular vesicles provided by the present invention uses iPSCs that can be amplified in large quantities in vitro as raw materials, solving the problem of difficult access to human neural cells. At the same time, the induced differentiation protocol adopted is simple to operate, with a short operation duration, and a large number of products can be obtained in the same batch. Moreover, the protocol has good reproducibility, and the generated "tentacle" extracellular vesicles have been proven to have both neuronal specific adhesion and damaged neuron repair functions, and can be used as a direct nerve repair means or a nerve specific targeting functional carrier, realizing multi-level applications for the first time in the intervention and treatment of various neurological diseases.

[0052] 2. In the non-invasive optogenetic synergistic treatment system of the present invention, Renilla luciferase (RLuc) that produces 470 nm fluorescence after decomposing the substrate coelenterazine (CTZ) is fused and co-expressed with ChR2. The close spatial binding of the light source and the photosensitive protein avoids the limitation of insufficient tissue penetration ability of the light source, and precise quantitative and non-invasive control of deep brain neurons can be achieved by adjusting the dosage of the substrate.

[0053] 3. The present invention provides a non-invasive optogenetic co-therapy system that synergistically targets neural stem cell extracellular vesicles and non-invasive optogenetic technology to reshape degenerated neuronal synaptic networks, while cleaning the pathological environment to avoid the possibility of the repaired neurons being reversed. It synergistically improves the cognitive ability of AD mice, and the raw materials are easily available, biocompatible, and suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 It is the morphology and particle size distribution of "tentacle" vesicles.

[0055] Figure 2 It is the neuron-specific adhesion ability of "tentacle" vesicles. Green is NeuN (HT22), blue is CD133 (U87), red is DiR-vesicles, scale bar, 20 μm.

[0056] Figure 3 It is the aggregation degree of different nanoparticles in the mouse brain recorded by a small animal imaging system after administration.

[0057] Figure 4 It is the ability of "tentacle" vesicles to rescue the survival rate of damaged neurons.

[0058] Figure 5 It is the change in protein expression before and after the optimization treatment of adeno-associated virus.

[0059] Figure 6 It is the hydrodynamic diameter of adeno-associated virus before and after optimization and the constructed treatment system.

[0060] Figure 7 It is the treatment system to increase the number of dendritic spines of damaged neurons.

[0061] Figure 8 It is the treatment system to rescue the survival rate of damaged neurons.

[0062] Figure 9 It is the treatment system to clear amyloid deposited in the microenvironment of damaged neurons.

[0063] Figure 10 It is the treatment system to improve the cognitive state of Alzheimer's mice.

[0064] Figure 11 It is the treatment system to clear amyloid deposited in the pathological environment of the brain of Alzheimer's mice.

[0065] Figure 12 It is the treatment system to inhibit the inflammatory response in the pathological environment of the brain of Alzheimer's mice. DETAILED DESCRIPTION OF THE INVENTION

[0066] To make the present invention easier to understand, the following specific embodiments are provided to further illustrate the present invention without limiting it in any way. These embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Without departing from the technical solution of the present invention, any modification or change that can be easily implemented by those of ordinary skill in the art will fall within the scope of the claims of the present invention.

[0067] The materials, reagents, etc. used in the following embodiments are commercially available without special instructions. The experimental methods without specific conditions in the embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0068] The iPSCs were purchased from Nuwacell ZhongSheng Suyuan (product number RC01001-A) and were a hiPSC cell line - male.

[0069] Aβ25-35 was purchased from GenScript (product number RP10008);

[0070] HT22 hippocampal neurons were purchased from Cyagen Biosciences (Shanghai) Inc. (product number iCell-m020);

[0071] Neural stem cells NSC were provided by China Pharmaceutical University or purchased commercially;

[0072] U87 cells were purchased from Cyagen Biosciences (Shanghai) Inc. (product number iCell-h224).

[0073] Among them, the preparation of purified neuronal membranes: Collect HT22 hippocampal neuronal cells cultured in vitro, at a concentration of 10 7 / mL, and mix with 4 times the volume of hypotonic saline (prepared by mixing normal saline and distilled water at a volume ratio of 1:1). After standing at 4°C for 3 hours, centrifuge at 150 - 200g for 10 minutes to remove large particle precipitates. Centrifuge the supernatant at 10000g for 30 minutes to collect the neuronal membrane precipitate. Resuspend the precipitate with hypotonic saline and then centrifuge at 10000g for 30 minutes for washing. Wash repeatedly 3 times and resuspend with normal saline to obtain purified neuronal membranes.

[0074] The plasmids, viruses, and reagents used in each culture medium in the embodiments are all commercially available.

[0075] Example 1

[0076] 1. iPSCs (from the blood of a 36-year-old male) were purchased from Nuwacell and cultured in six-well plates coated with Matrigel (Corning, 354277) using the company's ncTarget hPSC culture medium (RP01020). The cells were placed at 37°C and 5% CO2, and mycoplasma testing was negative. After the cell culture state reached a stable state and the confluence state reached 80%, the cells were subcultured and cultured to a density of about 80%, and then prepared for differentiation induction. The process is as follows: Figure 2 shown.

[0077] 2. Replace the medium of the above-mentioned stable iPSCs after passage with "pre-induction medium" [0.5% N2 (Thermo Fisher, 17502001) + 0.5% B27 (Thermo Fisher, 17504044) + 49.5% Neurobasal (Thermo Fisher, 21103049)) + 49.5% DMEM / F12 (Thermo Fisher, 31331093)], the above ratio is a percentage volume ratio, and adjust the culture environment to 37°C, 5.1% CO2 culture environment. After 4 days of culture, some cells appeared spindle-shaped and changed from a tightly aggregated state to a slightly loosely arranged state, which was considered to be transformed into neural preparation cells.

[0078] 3. Replace the culture medium of the above neural preparation cells with "maintenance medium" [0.5% N2 (Thermo Fisher, 17502001) + 0.5% B27 (Thermo Fisher, 17504044) + 49.5% Neurobasal (Thermo Fisher, 21103049) + 49.5% DMEM / F12 (Thermo Fisher, 31331093), the above ratio is a percentage volume ratio, and then add a final concentration of 20ng / mL bFGF (iCellbioscience, cat no.c0468)], and adjust the culture environment to 37°C, 5.0% CO2 culture environment, culture for about 1 day, and the cells remain in a proliferative state. At this stage, the cells proliferate in large quantities and form neurosphere clusters. The neurosphere clusters were digested with EDTA digestion solution (Nuwacell Biotechnologies Co., Ltd, catno. RP01007), and the EDTA digestion solution was added to the incubator for digestion at 37°C for 5 minutes. The contiguous digested cells were obtained by gentle shaking. After slowly blowing 3 times, the cells were spread into a culture dish pre-coated with Laminin (10 μg / mL, Sigma-Aldrich, 11243217001) at a cell tiling density of 80%. The cells were continued to be cultured in a 37°C, 5.0% CO2 environment with "maintenance medium" for about 12 hours to allow the cells to reach a stable state and obtain induced ready cells.

[0079] 4. Replace the culture medium of the above-induced pre-state cells with the "post-induction culture medium" [0.5% N2 (Thermo Fisher, 17502001) + 0.5% B27 (Thermo Fisher, 17504044) + 49.5% Neurobasal (Thermo Fisher, 21103049) + 49.5% DMEM / F12 (Thermo Fisher, 31331093), the above ratios are volume percentages, and then add bFGF at a final concentration of 20 ng / mL (iCell bioscience, cat no. c0468) and 60 μg / mL purified neuron membrane]. Incubate at 37°C in a 5.0% CO2 culture environment for 5 days. The cells gradually extend synapses, and finally obtain stem cells with "tentacles".

[0080] 5. Digest the obtained stem cells with "tentacles" using EDTA digestive solution in a 37°C cell culture incubator for 15 minutes. After digestion, resuspend with PBS (pH = 7.0) as the solvent and place in a 50w water bath ultrasonic bath for 10 minutes.

[0081] Use a liposome extruder in combination with filter membranes of different pore sizes to complete the gradient extrusion method to generate "tentacle" vesicles. Pass the suspension through filter membranes of 10 μm, 5 μm, 1 μm, 400 nm, 200 nm, and 100 nm in sequence, and repeat the extrusion 3 times for each particle size. The supernatant obtained is the "tentacle" extracellular vesicle suspension. The generated "tentacle" vesicles conform to the vesicle standard in morphology, with a uniform particle size distribution, and a particle size of 143.51 nm ( Figure 1 ).

[0082] Example 2

[0083] Verification of the biological function of "tentacle" extracellular vesicles

[0084] For the verification of its neuron-specific adhesion function, a co-culture model of U87 cells and HT22 cells was constructed. Use coupled NeuN monoclonal antibody (ProteinTech, CL488-66836) and coupled CD133 polyclonal antibody (ProteinTech, CL594-18470) to co-incubate with the co-culture model at 37°C for 1 hour to label the two types of cells respectively. In addition, incubate the vesicles prepared in Example 1 with the DiR fluorescent probe (Yeasen Biotechnology Co., Ltd., Shanghai, cat no. 40757ES) for 1 hour to label the vesicles. Add the DiR-labeled vesicles to the culture environment (vesicle number: cell number = 5:1) and incubate at 37°C in a 5.0% CO2 culture environment for 1 hour. Use confocal imaging to record the difference in the vesicle uptake rate of the two types of cells. The results are as Figure 2 shown. More "tentacle" vesicles are taken up by neurons rather than glial cells, demonstrating its ability to target neurons.

[0085] At the same time, the pathological environment neuron targeting ability of this vesicle was also verified in APP / PS1 mice (purchased from Jiangsu Ai Lingfei Biotechnology Co., Ltd., male, 6 months old). AAV (purchased from Shanghai Kelei Biotechnology Co., Ltd., specifically AAV5) was incubated with 2 mg / mL of ICG fluorescent dye (purchased from Aladdin, item number 3599-32-4) in a 37°C water bath environment for 1 hour to obtain AAV labeled with ICG. After incubating it with the "tentacle" vesicles prepared in Example 4 at a number ratio of 10:1 at 37°C for 1 hour, it was extruded 5 times through a liposome extruder 200nm, and the AAV was uploaded into the "tentacle" vesicles. Two mice were injected with AAV and AAV+"tentacle" vesicles (10 in each group), respectively. 9 The fluorescence distribution in the mouse body was recorded using the IVIS imaging system. Figure 3 It was found that compared with only a small amount of AAV alone that could accumulate in the brain, the "tentacle" vesicles were able to carry more AAV and accumulate in brain tissue, demonstrating its potential to target neurons in pathological sites in vivo.

[0086] To verify its neural tissue repair function, 5 μM Aβ 25-35 The cells were incubated with HT22 neurons at 37°C for 48 hours to simulate the state of damaged neurons. The model was considered successful when the neurons shrank and the synapses retracted, but no more than half of the neurons died. The culture medium was removed and the "tentacle" vesicles prepared in Example 1 (10 8 Vesicles / 10 6 After 24 hours of treatment, the survival of damaged neurons was evaluated by MTT method. Figure 4 As shown, the survival rate of damaged neurons was significantly increased after treatment with "tentacle" vesicles, proving its neuronal rescue ability.

[0087] Example 3

[0088] Construction and optimization of bioluminescent-optogenetic adeno-associated virus

[0089] The gene sequences of ChR2(H134R) photosensitive protein (NCBI accession number NC_000068.8) and RLuc Renilla luciferase (NCBI accession number NC_000913.3) were constructed into the adeno-associated virus vector pAAV5.0 to generate the plasmid pAAV-hSyn-hChR2(H134R)-RLuc. The above plasmid was constructed by Shanghai CoreRay Biotechnology Co., Ltd. and can be directly purchased commercially, with the product number KZ28161. The adeno-associated virus was packaged by the method of triple plasmid co-transfection. The specific process is as follows: In the EZ Cel transfection reagent (Shanghai LFE-ILab Biotechnology Co., Ltd., AC04L098 / AC04L099), the plasmid pAAV-hSyn-ChR2(H134R)-RLuc was co-transfected with the helper plasmid pAAV-helper and pAAV-RC (Shanghai CoreRay Biotechnology Co., Ltd.) into HEK293T cells. After incubation at 37°C for 12 h, the cells were placed in high-glucose DMEM medium for further culture. After the CPE effect appeared in a large number of cells, the culture medium containing the cells was collected, frozen and thawed 4 times, and centrifuged at 5000 rpm for 10 min at 4°C to obtain the P0 generation suspension virus. Then, the P0 virus was used to infect HEK293T again. After isolation and purification, the P1, P2, and P3 generation viruses were obtained and combined into the virus stock solution. It was stored in liquid nitrogen at -80°C.

[0090] For the optimization of partial uncoating of the packaged virus, the virus stock solution was dialyzed in an acetic acid buffer at pH 5.0 and 37°C using a 15 kDa dialysis bag for 10 minutes, and then loaded onto a step density Nycodenz for purification to obtain the partially uncoated virus. SDS-PAGE combined with Coomassie blue staining was used to compare the protein expression of the virus before and after treatment. The results are as Figure 5 shown. The protein expression of the virus changed significantly before and after the optimization treatment, which was consistent with the location of the adeno-associated virus protein.

[0091] Example 4

[0092] Construct a "tentacle" extracellular vesicle-mediated non-invasive optogenetic therapy system

[0093] The virus is uploaded using the mechanical co - extrusion method. First, the EZ Cel transfection reagent is co - incubated with the "tentacle" outer vesicles prepared in Example 1 for 15 minutes (the dosage is in accordance with the instructions of the EZ Cel transfection reagent) to increase the permeability of the vesicles and create conditions for the entry of the virus. The partially uncoated virus constructed and optimized in Example 3 is mixed with the treated "tentacle" outer vesicles at a ratio of 10:1 by the number of particles, and incubated in a water bath at 37°C for 1 hour. Then, it is repeatedly extruded 10 times under 400nm, 200nm, and 100nm filter membranes using a liposome extruder to obtain a complete therapeutic system solution. The changes in the hydrated particle size of the nanoparticles before and after uploading are detected by dynamic light diffraction method, and the results are as follows Figure 6 shown, indicating that the particle size slightly decreases after virus treatment. After uploading to the "tentacle" outer vesicles, the average hydrated particle size of the resulting therapeutic system is about 150nm, which is relatively similar to the size of the individual "tentacle" outer vesicles (143.51nm).

[0094] Example 5

[0095] The therapeutic system repairs damaged neurons and improves the cellular pathological environment

[0096] First, 5μM Aβ 25-35 amyloid protein is co - incubated with HT22 neurons at 37°C for 48 hours to construct a neuron damage model to investigate the neuron activation and repair ability of the therapeutic system. After 48 hours, the culture medium is removed and replaced with fresh high - glucose DMEM medium. In the optogenetic group, "tentacle" vesicle group, and combination therapy group, optogenetic virus (the partially uncoated virus prepared in Example 3), "tentacle" vesicles (the treated "tentacle" outer vesicles prepared in Example 3), and the nano - therapeutic system (produced in Example 4) are added respectively (10 8 particles / 10 6 HT22 cells, or 10 8 vesicles / 10 6 HT22 cells, where the nano - therapeutic system is centrifuged and redispersed in the culture medium to measure the number of particles), and co - incubated at 37°C for 12 hours of treatment, and stimulated with 200μM final concentration of coelenterazine 2 times within 24 hours (with an interval of 12 hours). After treatment, the synaptic remodeling, cell survival, Aβ protein clearance, etc. are detected, and the results are respectively as follows Figure 7 , Figure 8 , Figure 9As shown, the number of dendritic spines of neurons increased significantly after treatment, and the survival rate increased substantially. In addition, optogenetic treatment-activated neurons can help extend dendritic spines, and this extension of dendritic spines is more obvious in the combination treatment group. This is because the "tentacle" vesicles endow optogenetic nerve targeting ability, prompting the optogenetic elements to more efficiently target and transform neurons. At the same time, compared with the single optogenetic treatment group that cannot maintain the activity of neurons for a long time, the combination treatment group can obviously better maintain the survival of neurons after the "vesicles" repair the pathological environment. The amyloid proteins deposited in the cell culture environments of the "tentacle" vesicle group and the combination treatment group that can improve the pathological environment are effectively degraded by the enkephalinase rich in the treatment system, which reflects the ability of the treatment system to remove the deposited substances in the AD pathological environment, making it possible to provide a more suitable pathological environment for the improved neurons to survive. This also proves that the pathological environment repair substances contained in the "tentacle" vesicles can cooperate with optogenetics to more effectively repair degenerative neurons and maintain the continuous survival of neurons.

[0097] Example 6

[0098] Treat Alzheimer's disease mice, improve cognitive ability, and reshape the pathological environment

[0099] For the treatment of Alzheimer's disease mice. The treatment plan is as follows: APP / PS1 mice (AD model, purchased from Jiangsu Ailingfei Biotechnology Co., Ltd.). Among them, the combination treatment group, the optogenetic group, and the "tentacle" vesicle group were respectively administered by gavage with 10 8 nanoparticle treatment system particles prepared in Example 4 (the nanoparticles prepared in Example 4 were centrifuged and resuspended in PBS to a concentration of 10 7 particles / μL, and the administration volume was 10 μL), 10 8 optogenetic viruses (the partially uncoated viruses prepared in Example 3 were centrifuged and the virus particles were resuspended in PBS to a concentration of 10 7 particles / μL, and the administration volume was 10 μL), 10 8 "tentacle" vesicles (the processed "tentacle" extracellular vesicles prepared in Example 3 were centrifuged and the vesicles were resuspended in PBS to a concentration of 10 7 particles / μL, and the administration volume was 10 μL). After 2 weeks, coelenterazine was administered nasally 5 times on the 14th, 16th, 18th, 20th, and 22nd days respectively to keep the coelenterazine concentration in the cerebrospinal fluid at 200 μM.

[0100] Analysis of mouse behavior. The new object test method was used to analyze the behavior state of mice. After the treatment, the mice were placed in an open field containing two identical objects A and B and allowed to freely explore for 10 minutes. One hour after the exploration ended, object B was replaced with a different object C and placed into the mice, and they were allowed to freely explore for 10 minutes. The differences in the number of times and time of the mice exploring the old and new objects were recorded. The results are as Figure 10As shown. The results showed that compared with the group treated with optogenetic virus alone and the group treated with "tentacle" vesicles, the mice in the treatment system group showed more exploratory tendencies towards new objects, demonstrating that the cognitive and memory functions of the mice were significantly restored after treatment. Consistent with the in vitro experimental results, this synergistic cognitive improvement ability was obviously significantly better than any single treatment method. This was because the "tentacle" vesicles helped repair the pathological environment and created a good survival environment for the neurons that regained their function after optogenetic activation, preventing them from returning to the "regressive" state again.

[0101] Therefore, the pathological environment after treatment was further analyzed. On the 28th day after the end of treatment, the mice were sacrificed and the cerebrospinal fluid of each group of mice was collected. An Aβ1-42 enzyme-linked immunosorbent assay kit (Thermo Fisher, KMB7011) was used to detect the clearance rate of deposited proteins, and the results were as Figure 11 shown; an IL-6 ELISA kit (Thermo Fisher, cat no. KMB0061) was used to detect the degree of inflammation, and the results were as Figure 12 shown. Both of these results showed that the treatment system could remove the deposited substances in the pathological sites of AD mice and relieve the degree of inflammation, demonstrating its good effect on repairing the pathological environment of AD mice. This also showed that compared with the group treated with optogenetic virus alone and the group treated with "tentacle" vesicles, the synergistic treatment group could more significantly and effectively create a more suitable pathological environment for the improved neurons to survive, which was more conducive to synaptic remodeling between neurons.

[0102] In summary, the above results showed that the present invention established a treatment system for non-invasive optogenetics using iPSCs-derived "tentacle" vectors, its preparation method and application. This treatment system was combined with a treatment plan of bioluminescence-activated optogenetics and synergized with the repair function of stem cell content substances to damaged tissues. It could target transfection and activate neurons in the lesion site in vivo and reshape the damaged pathological environment, and had good application potential for a variety of neurodegenerative diseases.

Claims

1. A therapeutic system for non-invasive optogenetics using iPSCs-derived "tentacle" carriers, characterized in that, The treatment system includes a non-invasive optogenetic substance and a targeted drug delivery vector; the non-invasive optogenetic substance is a bioluminescent-optogenetic adeno-associated virus; the bioluminescent-optogenetic adeno-associated virus is an adeno-associated virus containing the ChR2(H134R) photosensitive protein and the RLuc Renilla luciferase; the targeted drug delivery vector is a "tentacle" extracellular vesicle obtained by inducing the differentiation of iPSCs into "tentacle" stem cells and through the gradient extrusion method; the non-invasive optogenetic substance is loaded into the targeted drug delivery vector.

2. The iPSCs-derived "tentacle" vector-based non-invasive optogenetic treatment system according to claim 1, characterized in that The non-invasive optogenetic substance is a bioluminescent-optogenetic adeno-associated virus, and specifically preferably, a fusion adeno-associated virus vector plasmid of the bioluminescent Renilla luciferase and the photosensitive channel protein based on ChR2(H134R) is constructed and packaged into an adeno-associated virus.

3. The iPSCs-derived "tentacle" vector-based non-invasive optogenetic therapy system according to claim 1, characterized in that The iPSCs are a commercially available hiPSC cell line.

4. A method for preparing a non-invasive optogenetic therapy system using the iPSCs-derived "tentacle" vector described in claim 1, characterized in that, It includes the following steps: (1) Construct a bioluminescent-optogenetic adeno-associated virus: The gene sequences of the ChR2(H134R) photosensitive protein and the RLuc Renilla luciferase are constructed onto an adeno-associated virus vector, and the adeno-associated virus is packaged by the method of triple plasmid co-transfection. (2) Construction of "tentacle" extracellular vesicles: After the iPSCs are cultured stably, they are passaged; the medium of the above-mentioned stably passaged iPSCs is replaced with a pre-induction medium and cultured continuously. When the cells show a spindle shape and are arranged loosely, neural primed cells are obtained; the medium of the above-mentioned neural primed cells is replaced with a maintenance medium until a neural spherical cluster with a relatively high cell aggregation degree, namely a "neural rosette-like wreath", appears; the above-mentioned neural spherical cluster is gently digested, passaged, and cultured with the "maintenance medium" until the cell state is stable to obtain induced primed cells; the medium of the above-mentioned induced primed cells is replaced with a post-induction medium and cultured until cell synapses appear to complete differentiation and obtain "tentacle" stem cells; the above-mentioned "tentacle" stem cells after digestion and resuspension are purified by the gradient extrusion method to obtain "tentacle" extracellular vesicles; (3) Construction of a "tentacle" extracellular vesicle carrying non-invasive optogenetics treatment system: The transfection reagent is co-incubated with the "tentacle" extracellular vesicles. After partial uncoating treatment of the virus obtained in step (1), it is mixed with the "tentacle" extracellular vesicles in a certain proportion, and after incubation, a complete treatment system is obtained by the mechanical co-extrusion method.

5. The preparation method of the iPSCs-derived "tentacle" vector for realizing non-invasive optogenetic therapy system according to claim 4, characterized in that, The bioluminescent-optogenetic adeno-associated virus constructed in step (1) is optimized for partial virus uncoating through the proton uncoating technology and purified through the Nycodenz step density gradient centrifugation technology.

6. The preparation method of the iPSCs-derived "tentacle" vector-based non-invasive optogenetic therapy system according to claim 4, wherein In step (3), the uncoated virus and the "tentacle" extracellular vesicles are mixed at a number ratio of 100:8 - 12 and incubated in a water bath at 30 - 37 °C for 1 - 2 hours.

7. The preparation method of the iPSCs-derived "tentacle" vector for non-invasive optogenetic therapy system according to claim 4, characterized in that, The mechanical co-extrusion method is to extrude 5 - 10 times respectively under filter membranes with pore sizes of 400 nm, 200 nm, and 100 nm using a liposome extruder.

8. Use of the iPSCs-derived "tentacle" vector-based non-invasive optogenetic treatment system according to claim 1 in the preparation of a drug or reagent capable of simultaneously achieving degenerative neuron activation and tissue microenvironment remodeling.

9. Use of the iPSCs-derived "tentacle" vector-based non-invasive optogenetic therapy system according to claim 1 in the preparation of a medicament for treating Alzheimer's disease and Parkinson's disease.

10. The application according to claim 8 or 9, characterized in that The application of the said therapy system needs to be combined with a corresponding optogenetic activation substance, and the optogenetic activation substance is coelenterazine.

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