An engineered nanovesicle for promoting hair regeneration and its preparation method and application

Abstract

This invention relates to an engineered nanovesicle for promoting hair regeneration, its preparation method, and its application. The engineered nanovesicle for promoting hair regeneration uses extruded vesicles derived from human induced pluripotent stem cells as a carrier, and the extruded vesicles are loaded with siGroups, including siTGF-β1, siLats1, and siSFRP1. The nucleotide sequence of siTGF-β1 is 5'-CGACAAGTTGAACGCATATTGCACA-3'; the nucleotide sequence of siLats1 is 5'-CATGAACACTTTCGCTGTAAGTAAA-3'; and the nucleotide sequence of siSFRP1 is 5'-TCAAGAGACAAACACTTCGAGTGAA-3'. Compared with existing technologies, the engineered nanovesicles prepared by the method of this invention utilize extruded vesicles derived from hiPSCs as carriers to replace traditional liposome or nanoparticle treatment methods. Furthermore, the gene cargo of active molecules loaded in synergy has the characteristics of multifunctionality and good therapeutic effect, and can be used to treat the regeneration and damage repair of hair follicles in seborrheic alopecia and telogen effluvium.

Description

Technical Field

[0001] The present invention belongs to the field of cell biology, molecular biology and drug research and development technology, and in particular relates to an engineered nanovesicle for promoting hair regeneration, and a preparation method and application thereof. Background Art

[0002] Hair growth is a continuous cycle that includes four phases: growth (anagen), regression (catagen), rest (telogen), and shedding (exogen). Hair loss is caused by changes in the phases of the hair follicle, which undergoes rhythmic, spontaneous, and cyclical organ transformations throughout the life cycle of its host. The hair follicle cycles between relatively "quiescent" states (telogen). Many exogenous, systemic, neural, endocrine, and nutritional factors affect these cyclic transformations of the hair follicle, leading to the appearance of hair loss. Androgenic alopecia is the most common form of hair loss. The hair follicle cycle of patients with androgenic alopecia is prolonged in the resting phase (telogen) and shortened in the active phase (anagen). The main treatments currently used are minoxidil and finasteride, but the side effects of these drugs limit their long-term use. Minoxidil can cause irritant dermatitis and transient facial hirsutism, and finasteride can cause adverse reactions such as sexual dysfunction and infertility. Therefore, finding new alternatives that can promote hair growth remains a top priority.

[0003] Many studies have shown that hair follicle regeneration and damage repair are regulated by multiple signaling pathways, such as the classic Wnt / β-catenin signaling pathway, TGF-β / Smad signaling pathway, Hippo signaling pathway, and PI3K-Akt signaling pathway. Studies have confirmed that a significant decrease in β-catenin expression and upregulation of GSK-3β activity can be observed in the hair follicle cells of patients with androgenic alopecia. Recent studies have confirmed that GSK-3β inhibitors can inhibit the activity of GSK-3β in hair papilla stem cells, increase the expression and stability of β-catenin, and further upregulate the activity and expression of hair growth marker proteins in human hair papilla stem cells.

[0004] In the TGF-β signaling pathway, dihydrotestosterone (DHT) is at a high level in patients with androgenic alopecia. DHT reduces the expression level of β-catenin and promotes the expression of TGF-β1, a negative regulator of hair follicle stem cell growth. Studies have confirmed that miR-122-5p can play a positive role in hair regeneration and skeletal muscle injury repair and regeneration by downregulating the function of the TGF-β / Smad pathway. The latest research also shows that bone morphogenetic proteins (BMPs) in the TGF-β family control the initiation of the growth phase of hair follicles by preventing cell proliferation and differentiation during epidermal development and preventing the growth of hair follicles in the resting phase.

[0005] The Hippo signaling pathway is a pathway that inhibits cell growth. After being affected by extracellular growth inhibitory signals, a series of kinase cascade phosphorylation reactions are activated, and the downstream effectors Yes-associated protein 1 (YAP) and Tafazzin protein (TAZ) are finally phosphorylated, reducing the nuclear activity of the factors, thereby achieving the regulation of organ size and volume. A study pointed out that the release of YAP enables the body to show stronger wound repair ability and new hair follicle formation ability. YAP phosphorylation is mainly regulated by the large tumor suppressor gene 1 (Lats1). Studies have confirmed that high expression of Lats1 will lead to increased phosphorylation levels of YAP, which will lead to hair follicle stem cell exhaustion and hair loss. This suggests that small RNA interference technology can be used to inhibit targeted genes for hair follicle repair in hair loss diseases, thereby promoting hair regeneration.

[0006] Unlike small molecule and protein drugs, RNA molecules must be inside cells to work.

[0007] Currently, non-viral vectors used for RNA delivery can be classified into four categories: lipid-based nanoparticles, polymer nanoparticles, inorganic nanoparticles, and biomimetic nanoparticles. However, due to their toxicity and effectiveness in vivo, most research remains at the preclinical research stage. Summary of the invention

[0008] In order to overcome the defects of the technology for obtaining nanoparticles, the present invention provides an engineered nanovesicle for promoting hair regeneration, and a preparation method and application thereof.

[0009] The purpose of the present invention can be achieved by the following technical solutions:

[0010] The first aspect of the present invention:

[0011] Provided is an engineered nanovesicle for promoting hair regeneration, wherein the engineered nanovesicle for promoting hair regeneration uses extruded vesicles (human induced pluripotent stem cells-small nanovesicles, hiPSC-sNVs) derived from human induced pluripotent stem cells (human induced pluripotent stem cells, hiPSCs) as a carrier, and the extruded vesicle is loaded with siGroup, wherein the siGroup includes siTGF-β1, siLats1 and siSFRP1,

[0012] The nucleotide sequence of the siTGF-β1 is:

[0013] 5'-CGACAAGTTGAACGCATATTGCACA-3';

[0014] The nucleotide sequence of the siLats1 is:

[0015] 5'-CATGAACACTTTCGCTGTAAGTAAA-3';

[0016] The nucleotide sequence of the siSFRP1 is:

[0017] 5'-TCAAGAGACAAACACTTCGAGTGAA-3'.

[0018] In the present invention, the engineered nanovesicles for promoting hair regeneration are hiPSC-sNVs loaded with siTGF-β1, siLats1 and siSFRP1, referred to as hiPSC-sNVs / siGroup, which can promote hair regeneration.

[0019] In one embodiment of the present invention, the extruded vesicles are uniform vesicles formed by mechanically extruding human induced pluripotent stem cells (hiPSCs), also referred to as human induced pluripotent stem cell sNVs or hiPSC-sNVs.

[0020] In one embodiment of the present invention, the average particle size of the engineered nanovesicles for promoting hair regeneration is 30-150 nm, preferably 80-120 nm.

[0021] In one embodiment of the present invention, the extruded vesicles are uniform vesicles formed by mechanically extruding human induced pluripotent stem cells (hiPSCs), also referred to as human induced pluripotent stem cell sNVs or hiPSC-sNVs.

[0022] In one embodiment of the present invention, the engineered nanovesicles for promoting hair regeneration are in the form of a suspension, and the concentration of hiPSC-sNVs ranges from 1×10 8 / mL–1×10 12 / mL, preferably 1×10 10 / mL.

[0023] In one embodiment of the present invention, the engineered nanovesicles for promoting hair regeneration are in the form of a suspension, and the concentration of siGroup is 100 nM.

[0024] The second aspect of the present invention:

[0025] Provided is a method for preparing engineered nanovesicles for promoting hair regeneration, comprising the following steps:

[0026] S1. Using a feeder-free culture method, culturing hiPSCs in a serum-free culture system, and collecting hiPSCs;

[0027] S2. Using a liposome extruder, hiPSCs were sequentially passed through polycarbonate filters with pore sizes of 5 μm, 1 μm, 400 nm, 200 nm, and 100 nm to obtain a crude suspension;

[0028] S3, the crude suspension was purified by gradient centrifugation combined with tangential flow to collect extruded vesicles to obtain hiPSC-sNVs;

[0029] S4. Incubate the hiPSC-sNVs obtained in step S3 with siGroup to obtain hiPSC-sNVs / siGroup, i.e., engineered nanovesicles for promoting hair regeneration. The siGroup includes siTGF-β1, siLats1 and siSFRP1.

[0030] In one embodiment of the present invention, in step S1, commercial human induced pluripotent stem cells (RC01001-A) are used, a feeder-free culture method is adopted, and the cells are collected after hiPSCs are cultured in a serum-free culture system.

[0031] In one embodiment of the present invention, in step S1, the serum-free culture system selects a commercially available culture medium of model RP01020 or RP01001.

[0032] In one embodiment of the present invention, in step S1, after culturing hiPSCs, human induced pluripotent stem cells are centrifuged at 4°C 400g for 10 min to collect hiPSCs.

[0033] In step S2, the collected hiPSCs are extruded through liposomes to obtain a crude suspension, that is, the hiPSCs are sequentially extruded through polycarbonate filter membranes with pore sizes of 5 μm, 1 μm, 400 nm, 200 nm, and 100 nm to obtain a crude suspension.

[0034] In one embodiment of the present invention, gradient centrifugation combined with tangential flow is used to collect and purify extruded vesicles, and the main steps are:

[0035] The crude suspension was centrifuged at 1500 g for 20 min at 4 °C to remove cell debris and obtain supernatant 1;

[0036] The supernatant 1 was centrifuged at 10000 g for 1 h at 4° C. to remove large vesicles, thereby obtaining supernatant 2;

[0037] Supernatant 2 was further purified and concentrated using a tangential flow filtration (TFF) device to obtain purified hiPSC-sNVs.

[0038] In one embodiment of the present invention, the specific operation process of purifying and concentrating using a tangential flow filtration (TFF) device is as follows: the flow rate is set to 100 mL / min, and the transmembrane pressure is set to 1.0 psi.

[0039] In one embodiment of the present invention, in step S3, after sNVs are collected, they are identified by electron microscopy, particle size analysis and other methods, and the results are consistent with the characteristics of nanoscale vesicles: diameter range is 30-150nm, and a double-layer membrane vesicle structure under transmission electron microscopy.

[0040] In one embodiment of the present invention, in step S4, the incubation treatment is performed as follows:

[0041] A suspension containing hiPSC-sNVs at a certain concentration is mixed with a siGroup, wherein the siGroup includes siTGF-β1, siLats1 and siSFRP1, and incubated at a certain temperature for a certain period of time;

[0042] Subsequently, the solution is placed in an ultrafiltration tube with a certain molecular weight cutoff, and ultrafiltration and washing are performed three times using a biocompatible medium to obtain a suspension containing engineered nanovesicles that promote hair regeneration.

[0043] In one embodiment of the present invention, the concentration range of the suspension containing hiPSC-sNVs is 1×10 8 / mL–1×10 12 / mL, preferably 1×10 10 / mL; the incubation temperature ranges from 4°C to 50°C, preferably 37°C; the incubation time ranges from 1h to 48h, preferably 4h; the molecular weight cutoff range of the ultrafiltration tube is 10KDa to 5000KDa, preferably 100KDa; the biocompatible medium is physiological saline, physiological buffer, cell culture medium, etc.

[0044] In one embodiment of the present invention, in the suspension containing engineered nanovesicles for promoting hair regeneration, the concentration of siGroup is 100 nM, and the concentration of hiPSC-sNVs ranges from 1×10 8 / mL–1×10 12 / mL, preferably 1×10 10 / mL.

[0045] The third aspect of the present invention provides a preparation based on the engineered nanovesicles (i.e., hiPSC-sNVs / siGroup) for promoting hair regeneration, wherein the preparation is a suspension: the hiPSC-sNVs / siGroup is dissolved in a solvent and exists in the form of a suspension.

[0046] In one embodiment of the present invention, the solvent of the suspension is physiological saline or phosphate buffer or cell culture medium. The suspension can be used in the form of microneedle injection, smear introduction or spraying.

[0047] The fourth aspect of the present invention:

[0048] Provided is the use of the engineered nanovesicles (i.e., hiPSC-sNVs / siGroup) for promoting hair regeneration in the preparation of drugs for treating and / or preventing hair loss.

[0049] In one embodiment of the present invention, the engineered nanovesicles (i.e., hiPSC-sNVs / siGroup) that promote hair regeneration are used in the preparation of drugs for repairing hair follicles in hair loss diseases.

[0050] In one embodiment of the present invention, the engineered nanovesicles (i.e., hiPSC-sNVs / siGroup) that promote hair regeneration are used in the preparation of drugs for the regeneration and damage repair of hair follicles in the treatment of seborrheic alopecia and telogen effluvium.

[0051] In one embodiment of the present invention, the engineered nanovesicles for promoting hair regeneration (i.e., hiPSC-sNVs / siGroup) are used to treat hair loss. A suspension containing the engineered nanovesicles for promoting hair regeneration (i.e., hiPSC-sNVs / siGroup) is injected into the scalp by microneedle injection to observe the therapeutic effect of the engineered nanovesicles for promoting hair regeneration on hair loss. The preventive effect of the engineered nanovesicles for promoting hair regeneration on hair loss is also studied.

[0052] The method of the present invention for verifying the therapeutic and preventive effects of the engineered nanovesicles for promoting hair regeneration on hair loss is:

[0053] T01, hiPSC-sNVs / siGroup was co-incubated with 293T cells to verify the silencing efficiency;

[0054] T02, co-incubate hiPSC-sNVs / siGroup with human keratinocytes to detect toxicity levels;

[0055] T03, inject hiPSC-sNVs / siGroup suspension into the back skin of mice using microneedle injection for animal level verification;

[0056] T04. Microneedle injection of hiPSC-sNVs / siGroup suspension into the human scalp to detect the efficiency of skin hair follicle repair.

[0057] The present invention designs engineered nanovesicles (sNVs) derived from human pluripotent stem cells and containing siTGF-β1, siLats1 and siSFRP1 for targeted treatment according to the characteristics of hair loss diseases.

[0058] By controlling the biologically active substances such as functional siRNAs carried by microvesicles, the biological role of regulating the activity of hair follicle stem cells to participate in hair regeneration can be exerted. Based on the safety and high feasibility of clinical applications, hiPSCs are selected as the source cells of extruded vesicles. hiPSCs have easy access, low immunogenicity, low tumorigenicity, low cytotoxicity and inherent homing properties. HiPSCs are used as the source to make drug delivery media-extruded vesicles. Because the vesicles contain cellular phospholipids, surface proteins and other substances, they have good cell compatibility and realize the functions of specific delivery of loaded cargo, cross-biological barrier delivery and immune exemption. At the same time, this application study proves that the vesicle structure can be loaded with siRNA (siTGF-β1, siLats1 and siSFRP1). The engineered vesicles can promote the proliferation and differentiation of hair follicle stem cell groups from the Hippo, Wnt / β-catenin and TGF-β / Smad signaling pathways and promote hair growth.

[0059] Compared with the prior art, the engineered nanovesicles prepared by the method of the present invention utilize extruded vesicles derived from hiPSCs as carriers to replace traditional liposome or nanoparticle treatment methods, and the gene cargo of active molecules synergistically loaded has the characteristics of multi-function and good therapeutic effect, and can be used to treat the regeneration and damage repair of hair follicles in seborrheic alopecia and telogen effluvium. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 : Particle size distribution and electron microscopy images of sNVs formed by extrusion of human induced pluripotent stem cells.

[0061] Figure 2 :The RNA interference efficiency detection results of hiPSC-sNVs / siGroup.

[0062] Figure 3 :Detection results of hiPSC-sNVs / siGroup cytotoxicity levels.

[0063] Figure 4 :hiPSC-sNVs / siGroup accelerates hair regeneration in the telogen phase.

[0064] Figure 5 :HE staining of the hair loss areas in the three groups showed the number of complete hair follicles.

[0065] Figure 6 :hiPSC-sNVs / siGroup promotes human hair regeneration. DETAILED DESCRIPTION

[0066] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0067] Example 1

[0068] This example provides a specific method for preparing sNVs derived from hiPSCs, comprising the following steps:

[0069] 1. Take out the hiPSCs stored in liquid nitrogen, put them into a water bath preheated at 37°C, shake them while thawing, and take them out when the ice crystals in the cell suspension are almost completely gone.

[0070] 2. Immediately pipette the cell suspension into a 15 mL centrifuge tube and add 9 mL of preheated complete cell culture medium dropwise. ncEpic, mix well; at the same time, take 1 mL of preheated cell culture medium to wash the cryovial.

[0071] 3. Centrifuge at 200g × 5min to remove the supernatant, collect the cells, add 1-2mL of complete culture medium to resuspend the cells, inoculate the cells in a six-well plate, add an appropriate amount of preheated fresh complete culture medium, and place the plate in a cell culture incubator for culture.

[0072] 6. When hiPSCs grow to the desired cell number, centrifuge the digested hiPSCs at 400 g for 10 min and collect them in a 50 ml centrifuge tube.

[0073] 7. After the collected cells are resuspended in PBS, they are extruded through a liposome extruder, and passed through polycarbonate filter membranes with pore sizes of 5 μm, 1 μm, 400 nm, 200 nm, and 100 nm, and extruded 10 times to obtain a crude suspension. The present invention has no particular limitation on the source of the liposome extruder, and a conventional commercially available liposome extruder well known to those skilled in the art can be used.

[0074] 8. The crude suspension was centrifuged at 4°C and 15000 g for 20 min to remove cell debris and obtain supernatant 1. The supernatant 1 was centrifuged at 4°C and 10000 g for 1 h to obtain supernatant 2.

[0075] 9. Supernatant 2 was directly subjected to TFF filtration as described above. The TFF system used was a KR2i TFF system equipped with a 750 kD modified polyethersulfone hollow fiber filter (MidiKros membrane surface area 115 cm 2 ). The flow rate was set to 100 mL / min (not exceeding 120 mL / min), the transmembrane pressure was set to 1.0 psi (the transmembrane pressure should not exceed 3.0 psi during rinsing, sample running and rinsing), and purified hiPSC-sNVs were obtained by filtration and concentration.

[0076] Example 2

[0077] Taking the vesicles collected in Example 1 as an example, further preliminary detection of the vesicles includes the following steps:

[0078] 1. Take 10 μL of the sample, drop it on the copper grid, let it stand for 90 seconds, and absorb the excess liquid with absorbent paper.

[0079] 2. In a dark environment, add 10 μL of uranyl acetate onto the copper grid. After staining for 30 seconds, use absorbent paper to absorb the excess liquid.

[0080] 3. Place the prepared copper mesh on a light-tight disk, dry it for 2 hours, and then perform TAE transmission electron microscopy examination.

[0081] 4. TAE transmission electron microscopy images ( Figure 1 ) showed that the prepared vesicles were cup-shaped and oval.

[0082] On the other hand, the prepared vesicles are sampled and the nanoparticle size distribution is detected by nanoflow cytometry. The specific steps are as follows:

[0083] 1. First, initialize the flow and remove the bubbles from the nanoflow cytometer.

[0084] 2. Use the standard sample to perform quality control on the nanoflow cytometer according to the instructions to achieve the best detection state (the signals of the scattering and fluorescence channels are both the strongest and uniform, and the particle count value range of the quality control standard sample is usually 5000-7000).

[0085] 3. Detect the particle count of phosphate buffer under LabelledExo sample measurement parameters to determine the cleanliness of phosphate buffer. Only phosphate buffer with a particle count of less than 200 can be used as blank control data collection and used for subsequent sample dilution.

[0086] 4. Pre-dilute the extruded vesicle sample 100 times with clean phosphate buffer, and collect data on the extruded vesicle sample under the vesicle sample measurement parameters. The nanoflow cytometer particle count is between 4000-8000 for accurate measurement. If the sample particle count is too high or too low, adjust the sample dilution multiple and then measure again, and record the final dilution multiple of the sample.

[0087] 5. Such as Figure 1 It is shown that the main diameter of the vesicles prepared in this application is 100nm, and the vesicle range is between 30nm-150nm, which is a typical nanoscale vesicle.

[0088] Example 3

[0089] Using the vesicles collected in Example 2, the vesicles are further incubated and loaded with drugs, comprising the following steps:

[0090] 1. Design and construction of siTGF-β1, siLats1 and siSFRP1 through gene library

[0091] The nucleotide sequence of the siTGF-β1 is:

[0092] 5'-CGACAAGTTGAACGCATATTGCACA-3';

[0093] The nucleotide sequence of the siLats1 is:

[0094] 5'-CATGAACACTTTCGCTGTAAGTAAA-3';

[0095] The nucleotide sequence of the siSFRP1 is:

[0096] 5'-TCAAGAGACAAACACTTCGAGTGAA-3'.

[0097] 2. The siTGF-β1, siLats1 and siSFRP1 were loaded into the vesicles in Example 2 by a simple incubation method. The specific operation was as follows:

[0098] Take 100 μL of hiPSC-sNVs (>10 9 / mL) in an EP tube, add 30 μL of 2 μM stock solutions of siTGF-β1, siLats1 and siSFRP1, invert to mix, and incubate at 37°C for 24 h.

[0099] The mixture obtained in the above steps was subjected to ultracentrifugation to remove free siTGF-β1, siLats1 and siSFRP1, thereby obtaining hiPSC-sNVs / siGroup co-loaded with siTGF-β1, siLats1 and siSFRP1.

[0100] Example 4

[0101] Taking the vesicles prepared in Example 3 as an example, the silencing efficiency and toxicity experiments of keratinocytes were further conducted, and the specific steps are as follows:

[0102] (I) Silencing efficiency experiment of hiPSC-sNVs / siGroup

[0103] The specific experimental process is:

[0104] First, 293T cells were cultured at 2 × 10 5The cells were plated in a 3 mL cell culture dish and grown to a confluence of 50%. 3 mL of 1×10 9 The cells were placed in an incubator for 24 h, and then the complete culture medium was replaced and cultured for another 24 h. The cell culture medium was removed, the cells were lysed with trizol, and total cell RNA was extracted. The expression levels of TGF-β1, Lats1, and SFRP1 were detected by qRT-PCR. Figure 2 As shown, compared with the control group, the expression levels of TGF-β1, Lats1 and SFRP1 were significantly decreased after hiPSC-sNVs / siGroup was co-incubated with cells.

[0105] (II) hiPSC-sNVs / siGroup cytotoxicity experiment

[0106] Human keratinocytes (HACAT) were plated in a 96-well plate. After the cells adhered to the wall and grew, 200 μL of 1×10 9 The hiPSC-sNVs / siGroup with a particle number / mL was placed in an incubator for 24 hours, and the complete medium was replaced and cultured for another 24 hours. At the same time, as a control, a control experiment was performed with the nanovesicles without drug loading in Example 2 (prepared by referring to the above operation).

[0107] The CCK8 kit was used to detect the effect of nanovesicles (hiPSC-sNVs / siGroup) on the proliferation and activity of HACAT cells. Figure 3 As shown, compared with the control group, hiPSC-sNVs / siGroup did not affect cell proliferation.

[0108] Example 5

[0109] Promoting hair regeneration using hiPSC-sNVs / siGroup loaded with siGroup from human induced pluripotent stem cells sNVs

[0110] Seven-week-old C57BL / 6 mice were selected, and after shaving their backs, microneedle injections were performed on the back skin using normal saline (as the control group), hiPSC-sNVs / siGroup suspension (as the hiPSC-sNVs / siGroup treatment group), and minoxidil (as the minoxidil treatment group). After treatment for 21 days, the skin of different groups at 7, 14, and 21 days was fixed with paraformaldehyde, gradient dehydrated, and transparentized with xylene, then embedded in paraffin and sliced. After dewaxing and rehydration, the sections were stained with hematoxylin-eosin (HE), and the complete hair follicles were counted. The effect of hiPSC-sNVs / siGroup suspension on hair regeneration in C57BL / 6 mice was evaluated by skin pathological sections and histological statistics. The experimental results showed that hiPSC-sNVs / siGroup suspension can significantly promote hair follicle regeneration in C57BL / 6 mice, as shown in Figure 2. Figure 4 , 5.

[0111] Example 6

[0112] Application example of promoting hair regeneration using hiPSC-sNVs / siGroup loaded with siGroup using human induced pluripotent stem cell sNVs

[0113] The scalp of volunteers with hair loss was treated with hiPSC-sNVs / siGroup microneedle injections in the selected area with hair follicles that had not yet completely atrophied. Statistics were taken on the 30th, 60th, and 90th days after use. Figure 6 , showing the results of use on the 30th, 60th, and 90th days of hiPSC-sNVs / siGroup treatment. The experimental results showed that although hair loss occurred on the 30th day, more hair growth was shown on the 90th day.

[0114] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. Those familiar with the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without performing creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the contents disclosed by the present invention should belong to the protection scope of the present invention.

Claims

1. An engineered nanovesicle for promoting hair regeneration, characterized in that: The engineered nanovesicles for promoting hair regeneration use extruded vesicles derived from human induced pluripotent stem cells as carriers, and the extruded vesicles co-load siTGF-β1, siLats1 and siSFRP1. The nucleotide sequence of the siTGF-β1 is: 5'-CGACAAGTTGAACGCATATTGCACA-3'; The nucleotide sequence of the siLats1 is: 5'-CATGAACACTTTCGCTGTAAGTAAA-3'; The nucleotide sequence of the siSFRP1 is: 5'-TCAAGAGACAAACACTTCGAGTGAA-3'.

2. The engineered nanovesicle for promoting hair regeneration according to claim 1, characterized in that: The average particle size of the engineered nanovesicles for promoting hair regeneration is 30-150 nm.

3. The engineered nanovesicle for promoting hair regeneration according to claim 2, characterized in that: The average particle size of the engineered nanovesicles for promoting hair regeneration is 80-120 nm.

4. The method for preparing an engineered nanovesicle for promoting hair regeneration according to claim 1, 2 or 3, characterized in that: The following steps are involved: S1. Using a feeder-free culture method, culturing hiPSCs in a serum-free culture system, and collecting hiPSCs; S2. Using a liposome extruder, hiPSCs were sequentially passed through polycarbonate filters with pore sizes of 5 μm, 1 μm, 400 nm, 200 nm, and 100 nm to obtain a crude suspension; S3, the crude suspension was purified by gradient centrifugation combined with tangential flow to collect extruded vesicles to obtain hiPSC-sNVs; S4. Incubate the hiPSC-sNVs obtained in step S3 with siGroup to obtain hiPSC-sNVs / siGroup, i.e., engineered nanovesicles for promoting hair regeneration. The siGroup includes siTGF-β1, siLats1 and siSFRP1.

5. The method for preparing engineered nanovesicles for promoting hair regeneration according to claim 4, characterized in that: In step S1, the serum-free culture system selects a commercially available culture medium of model RP01020 or RP01001; In step S1, after culturing hiPSCs, human induced pluripotent stem cells are centrifuged at 400 g for 10 min at 4°C to collect hiPSCs.

6. The method for preparing engineered nanovesicles for promoting hair regeneration according to claim 4, characterized in that: In step S3, gradient centrifugation combined with tangential flow is used to collect and purify extruded vesicles. The main steps are: The crude suspension was centrifuged at 1500 g for 20 min at 4 °C to remove cell debris and obtain supernatant 1; The supernatant 1 was centrifuged at 10000 g for 1 h at 4° C. to remove large vesicles, thereby obtaining supernatant 2; Supernatant 2 was further purified and concentrated using a tangential flow filtration device to obtain purified hiPSC-sNVs.

7. The method for preparing engineered nanovesicles for promoting hair regeneration according to claim 6, characterized in that: The specific operation process of purification and concentration using tangential flow filtration equipment is as follows: the flow rate is set to 100 mL / min, and the transmembrane pressure is set to 1.0 psi.

8. The method for preparing engineered nanovesicles for promoting hair regeneration according to claim 4, characterized in that: In step S4, the incubation process is performed as follows: The suspension containing hiPSC-sNVs is mixed with a siGroup including siTGF-β1, siLats1, and siSFRP1, and incubated; The resulting solution is then placed in an ultrafiltration tube and washed three times by ultrafiltration using a biocompatible medium to obtain a suspension containing engineered nanovesicles that promote hair regeneration; The concentration of the suspension containing hiPSC-sNVs ranged from 1 × 10 8 / mL–1×10 12 / mL, the incubation temperature range is 4℃–50℃, the incubation time range is 1h–48h, the retention molecular weight range of the ultrafiltration tube is 10KDa–5000KDa, and the biocompatible media are normal saline, physiological buffer, and cell culture medium.

9. The preparation of engineered nanovesicles for promoting hair regeneration according to claim 1, characterized in that: The preparation is a suspension: the engineered nanovesicles for promoting hair regeneration are dissolved in a solvent and exist in the form of a suspension; The solvent of the suspension is physiological saline or phosphate buffer or cell culture medium.

10. The use of the engineered nanovesicles for promoting hair regeneration according to claim 1, characterized in that: The use of the engineered nanovesicles for promoting hair regeneration in the preparation of drugs for treating and / or preventing hair loss.

11. The use of engineered nanovesicles for promoting hair regeneration according to claim 10, characterized in that: Use of the engineered nanovesicles for promoting hair regeneration in the preparation of a drug for repairing hair follicles in hair loss diseases; or, The engineered nanovesicles for promoting hair regeneration are used in preparing drugs for treating seborrheic alopecia and telogen effluvium to regenerate hair follicles and repair damage.

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