CN115245521B - Nose drops containing stem cell extracellular vesicles and application thereof in treating cerebral neurovascular diseases - Google Patents
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Abstract
The invention provides a nose drop preparation of stem cell vesicles modified by genetic engineering and application thereof in treating cerebral nerve vascular diseases. Specifically, the invention provides a nose drop preparation taking active substances in stem cell vesicle contents with nerve cell targeting capability as main active ingredients, a preparation method thereof and application of the preparation in treating Alzheimer disease. After the preparation is instilled and administrated through nasal cavity, the main components of the preparation can effectively penetrate through blood brain barrier and be absorbed by microglial cells and nerve cells to release the main effective components in stem cell vesicles, so that obvious inflammation inhibition effect is generated; meanwhile, the expression level of the amyloid precursor protein hydrolase BACE1 is reduced, the expression level of ADAM10 is maintained, and the generation of amyloid Abeta is inhibited, so that neurodegenerative diseases such as Alzheimer disease and the like are effectively prevented or treated.
Description
Technical Field
The invention relates to the field of stem cell therapeutic drugs, in particular to a nose drop preparation of extracellular vesicles secreted by stem cells and application thereof in treating cerebral nerve vascular diseases.
Background
Alzheimer's Disease (AD), commonly known as Alzheimer's disease, is an age-related severe neurodegenerative disease characterized by amyloid beta (Abeta) aggregation to form plaques, tau protein hyperphosphorylation to induce formation and accumulation of neurofibrillary tangles, loss of synapses and neurons, and decline of cognitive function (Zhang et al, 2016). Studies have shown that Amyloid Precursor Protein (APP) forms aβ under the action of BACE1 hydrolase, whereas aβ is able to induce intracellular iκb phosphorylation and subsequent degradation, allowing NF- κb to activate into the nucleus, regulating inflammation-related gene transcription (Li et al, 2018). Thus, AD occurs during the course of development with chronic inflammation.
With the growth of the aging population and the extension of life expectancy, alzheimer's disease will become the most common neurological disease in the world. In 2019, it was estimated that 4400 thousands of AD cases have been reported worldwide (Chopra et al 2020). AD prevalence is expected to double every 20 years due to the increase in life expectancy of the global population, meaning that 1.31 million people will have such diseases by 2050 (Shaik et al, 2018).
Currently approved therapeutic agents for AD mainly include cholinesterase inhibitors and N-methyl-D-aspartic acid (NMDA) receptor antagonists. The use of these drugs can cause patients to develop varying degrees of side effects or to have a lower efficacy (Briggs et al, 2016; shik et al, 2018). Furthermore, preclinical studies have shown that reduced expression or activity of BACE1 can reverse pathology in AD mice (Hu et al, 2018), but clinical trials have remained unsuccessful. In fact, most anti-AD drug candidates are BACE1 inhibitors, but have been withdrawn due to their toxicity or lack of efficacy (Chopra et al 2020). Because these drugs systemically reduce the expression level or activity of BACE1, thereby impairing the important role played by the protein in liver metabolism, liver dysfunction is prone to occur (Kitazume et al, 2005;Lahiri et al, 2014; shaik et al, 2018). Therefore, in order to avoid the side effects caused by the conventional AD drugs, reduce the uncontrollable liver tissue toxicity caused by systemic administration therapy, control the disease progression of patients, and improve the survival quality of patients, it is highly desirable to find a new therapeutic strategy and therapeutic method for AD.
Numerous studies have shown that many miRNAs play an important role in regulation of BACE1 expression and inhibition of inflammation-related NF- κb signaling pathways during the development of AD (Shaik et al., 2018). Thus, these miRNAs can be potential molecular targets for the treatment of AD. Extracellular vesicles or exosomes produced by mesenchymal stem cells are able to penetrate the blood-brain barrier (Morales-primeo et al 2009), and are rich in miRNAs related to tissue repair and anti-inflammatory effectsEt al 2020). Therefore, stem cell extracellular vesicles or exosomes have great potential for use in the prevention and treatment of neurological related diseases.
Disclosure of Invention
The invention aims to provide a nose drop preparation taking an active ingredient of a target point related to the formation of inflammation and amyloid beta in stem cell extracellular vesicles after genetic engineering modification as an active ingredient, a preparation method thereof and application of the preparation in treatment of neurodegenerative diseases such as Alzheimer disease.
In a first aspect, the present invention provides a formulation comprising stem cell extracellular vesicles, the stem cell extracellular vesicles of the formulation comprising an active ingredient that modulates a target associated with inflammation and amyloid β formation; and, the formulation is for:
(a) Inhibiting inflammatory response of brain nerve tissue;
(b) Inhibiting the production of amyloid aβ; and
(C) Preventing and/or treating neurodegenerative diseases.
In another preferred embodiment, the formulation contains 1X 10 5～9×108 extracellular vesicles/ml of stem cells.
In another preferred embodiment, the stem cell extracellular vesicles are derived from supernatants collected during in vitro culture of genetically engineered human stem cells.
In another preferred embodiment, the human stem cells are selected from the group consisting of: human umbilical cord blood-derived stem cells, human peripheral blood-derived stem cells, human umbilical cord mesenchymal stem cells, human placental mesenchymal stem cells, human adipose mesenchymal stem cells, human bone marrow-derived stem cells, or a combination thereof.
In another preferred example, the human stem cells are human adipose mesenchymal stem cells.
In another preferred embodiment, the stem cell extracellular vesicles comprise an intact lipid bilayer structure, wherein the membrane surface comprises specifically expressed CD9, CD63, CD81, and the membrane comprises TSG101, HSP70.
In another preferred embodiment, the stem cell extracellular vesicles further comprise within the membrane certain non-coding functional DNA and RNA.
In another preferred embodiment, the non-coding functional DNA and RNA include non-coding functional DNA and RNA molecules produced by stem cells cultured in a natural state, non-coding functional DNA and RNA molecules produced by expression of genetically engineered stem cells, non-coding functional DNA and RNA molecules transduced into stem cells after chemical synthesis, or a combination thereof.
In another preferred embodiment, the RNA comprises miRNA, tRNA, rRNA, snoRNA and snRNA.
In another preferred embodiment, the RNA comprises a miRNA or a precursor miRNA thereof having a length of 17 to 100 nucleotides.
In another preferred example, the cells used to produce the stem cell extracellular vesicles include cells of the following sources: cells obtained by specific gene modification, specific gene editing, specific gene transduction, and specific microribonucleic acid miRNA introduction in GMP laboratories.
In another preferred embodiment, the stem cell extracellular vesicles have the following characteristics: the extracellular vesicles are obtained by adopting a polyethylene glycol (PEG) precipitation method, the particle size of the extracellular vesicles is concentrated and distributed between 80 nm and 200nm, and the particle size is uniform.
In another preferred embodiment, the polyethylene glycol (PEG) precipitation comprises the steps of: PEG 3000-9000 PEG is used, PEG mother liquor which is prepared into 8% -20% by PBS is added into a treated conditioned medium (the conditioned medium is obtained by subjecting a culture supernatant of human adipose-derived mesenchymal stem cells to differential centrifugation and filtration) according to a certain proportion (such as about 1:1 volume ratio) after filtering and sterilizing (such as filtering by a 0.22 mu m filter), and the mixture is placed at 4 ℃ for overnight incubation; centrifuging at 4deg.C for 30-60 min at 3,000-5000g, discarding supernatant, adding pre-cooled PBS, and re-suspending to precipitate; and (3) ultra-high speed centrifugation is carried out at 100000-120000g for 60-120 minutes at the temperature of 4 ℃, and the supernatant is discarded, so that the stem cell extracellular vesicles are obtained.
In another preferred embodiment, the stem cell extracellular vesicles have the following characteristics: (a) The particle size is small, and the blood brain barrier can be penetrated into the brain nerve tissue; (b) The miRNA small molecules in the cell can be released after the stem cell extracellular vesicles enter the machine body, so that the cell reaches the brain.
In another preferred embodiment, the formulation has the following characteristics: the stem cell extracellular vesicles in the preparation are genetically engineered to have the brain nerve tissue specific targeting capability, and the active ingredients in the stem cell extracellular vesicles can be targeted and delivered into the brain nerve tissue to be taken up by nerve cells and microglia cells.
In another preferred embodiment, the active ingredient within the extracellular vesicles of stem cells is capable of simultaneously exerting an inflammatory response inhibiting effect and reducing the expression of the amyloid precursor protein hydrolase BACE1 and maintaining the expression level of ADAM 10.
In another preferred embodiment, the active ingredient within the stem cell extracellular vesicles is selected from the group consisting of: antisense oligonucleotides of hsa-miR-1290, hsa-miR-126-5p, hsa-miR-130a-3p, hsa-miR-24-3p, hsa-let-7b-3p and precursor forms thereof, hsa-let-7b-5p, hsa-let-7a-5p, hsa-miR-92a-3p, hsa-miR-151a-3p, hsa-miR-1246 and precursor forms thereof, and any combination of the foregoing.
In another preferred embodiment, the active ingredients comprise hsa-miR-1290 and hsa-miR-126-5p antisense oligonucleotides.
In another preferred embodiment, the formulation comprises a pharmaceutically acceptable carrier or adjuvant.
In another preferred embodiment, the pharmaceutically acceptable carrier or adjuvant is selected from the group consisting of: sodium chloride, sodium phosphate, polyethylene glycol, chitosan, sodium hyaluronate, trehalose, chlorophosphonate, heparin, or combinations thereof.
In another preferred embodiment, the formulation is cell-free and cell-debris-free.
In another preferred embodiment, the term "cell-free" means that the formulation does not contain living cells or dead cells.
In another preferred embodiment, the neurodegenerative disease is selected from the group consisting of: alzheimer's disease, parkinson's disease, cerebral ischemia, cerebral apoplexy, or combinations thereof.
In a second aspect of the present invention, there is provided a pharmaceutical composition comprising: (a) A formulation comprising stem cell extracellular vesicles according to the first aspect of the invention, and (b) a pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition is a cell-free and cell-free fragment pharmaceutical composition.
In another preferred embodiment, the term "cell-free" means that the pharmaceutical composition does not contain living cells or dead cells.
In another preferred embodiment, the pharmaceutically acceptable carrier is selected from the group consisting of: sodium chloride, sodium phosphate, polyethylene glycol, chitosan, sodium hyaluronate, trehalose, chlorophosphonate, heparin, or combinations thereof.
In another preferred embodiment, the pharmaceutical composition is in a dosage form selected from the group consisting of: liquid dosage forms, solid dosage forms (e.g., lyophilized dosage forms).
In another preferred embodiment, the pharmaceutical composition is in a dosage form selected from the group consisting of: nose drops, aerosol inhalants, eye drops and injections.
In another preferred embodiment, the pharmaceutical composition is in the form of nasal drops.
In another preferred embodiment, the pharmaceutical composition has the following characteristics: the stem cell extracellular vesicles in the pharmaceutical composition are genetically engineered to have a brain nerve tissue specific targeting capability, and can target and deliver active ingredients in the stem cell extracellular vesicles into brain nerve tissue to be taken up by nerve cells and microglia.
In another preferred embodiment, the pharmaceutical composition has the following characteristics: the active ingredient in the extracellular vesicles of stem cells in the pharmaceutical composition can reach the brain nasally.
In another preferred embodiment, the active ingredient is selected from the group consisting of: antisense oligonucleotides of hsa-miR-1290, hsa-miR-126-5p, hsa-miR-130a-3p, hsa-miR-24-3p, hsa-let-7b-3p and precursor forms thereof, hsa-let-7b-5p, hsa-let-7a-5p, hsa-miR-92a-3p, hsa-miR-151a-3p, hsa-miR-1246 and precursor forms thereof, and any combination of the foregoing.
In another preferred embodiment, the active ingredients comprise hsa-miR-1290 and hsa-miR-126-5p antisense oligonucleotides.
In another preferred embodiment, the pharmaceutical composition is used for preventing and/or treating neurodegenerative diseases.
In another preferred embodiment, the neurodegenerative disease is selected from the group consisting of: alzheimer's disease, parkinson's disease, cerebral ischemia, cerebral apoplexy, or combinations thereof.
In another preferred embodiment, the neurodegenerative disease is Alzheimer's disease.
In a third aspect of the present invention, there is provided a method of preparing a pharmaceutical composition comprising stem cell extracellular vesicles, the method comprising the steps of:
(S1) culturing the genetically engineered human stem cells to a predetermined confluence (e.g., 75-90%);
(S2) continuing to culture said cells for a period of time T1 under conditions suitable for the production of extracellular vesicles; wherein said T1 is generally 24-72 hours, preferably 30-60 hours;
(S3) removing cells from the culture system, thereby separating and obtaining a culture solution containing stem cell extracellular vesicles, namely a conditional culture solution (conditioned medium);
(S4) mixing said conditioned medium (conditioned medium) with polyethylene glycol (PEG) to form a first mixture, and allowing to stand for a period of time T2, thereby forming PEG-modified extracellular vesicles of stem cells; wherein said T2 is usually 6-60 hours, preferably 12-48 hours;
(S5) centrifuging the first mixture of the previous step to precipitate the PEG-modified stem cell extracellular vesicles, and discarding the supernatant to obtain PEG-modified stem cell extracellular vesicle precipitates;
(S6) resuspending the PEG-modified stem cell extracellular vesicle pellet obtained in the previous step, thereby obtaining a first suspension mixture;
(S7) centrifuging the first resuspended mixture to pellet the PEG-modified stem cell extracellular vesicles, and discarding the supernatant to obtain PEG-modified stem cell extracellular vesicle pellets;
(S8) resuspending the PEG-modified stem cell extracellular vesicle pellet obtained in the previous step, thereby obtaining a stem cell extracellular vesicle preparation for medical use.
In another preferred embodiment, the method further comprises:
(S9) mixing a pharmaceutically acceptable stem cell extracellular vesicle preparation (or active substance) with a pharmaceutically acceptable carrier, thereby preparing a pharmaceutical composition.
In another preferred embodiment, the method further comprises preparing the pharmaceutical composition into a nasal drop, an aerosol inhalation, an eye drop, an injection, or a lyophilized formulation.
In another preferred embodiment, the genetically engineered human stem cells are cells obtained by transducing an active ingredient into human stem cells.
In another preferred embodiment, the human stem cells are human adipose mesenchymal stem cells.
In another preferred embodiment, the active ingredient is selected from the group consisting of: antisense oligonucleotides of hsa-miR-1290, hsa-miR-126-5p, hsa-miR-130a-3p, hsa-miR-24-3p, hsa-let-7b-3p and precursor forms thereof, hsa-let-7b-5p, hsa-let-7a-5p, hsa-miR-92a-3p, hsa-miR-151a-3p, hsa-miR-1246 and precursor forms thereof, and any combination of the foregoing.
In another preferred embodiment, the active ingredients comprise hsa-miR-1290 and hsa-miR-126-5p antisense oligonucleotides.
In another preferred embodiment, the active ingredients hsa-miR-1290 and hsa-miR-126-5p antisense oligonucleotides are introduced into (i.e., overexpressed in) human adipose mesenchymal stem cells in a molar (pmol) combination ratio of 1 (1-6) or (1-6): 1, including but not limited to 1:1, 1:2, 1:3, 1:4, 1:5 and 1:6, and 2:1, 3:1, 4:1, 5:1 and 6:1.
In a fourth aspect, the present invention provides a use of a formulation comprising stem cell extracellular vesicles according to the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention for the preparation of a medicament or formulation for the prevention and/or treatment of neurodegenerative diseases.
In another preferred embodiment, the neurodegenerative disease is selected from the group consisting of: alzheimer's disease, parkinson's disease, cerebral ischemia, cerebral apoplexy, or combinations thereof.
In another preferred embodiment, the neurodegenerative disease is Alzheimer's disease.
In a fifth aspect of the invention, there is provided a method of treating a neurodegenerative disease comprising the steps of: administering to a subject in need thereof a formulation according to the first aspect of the invention, or a pharmaceutical composition according to the second aspect.
In another preferred embodiment, the subject in need thereof is a human or non-human mammal.
In another preferred embodiment, the desired subject is a human.
In another preferred embodiment, the subject in need thereof suffers from a neurodegenerative disease.
In another preferred embodiment, the neurodegenerative disease is selected from the group consisting of: alzheimer's disease, parkinson's disease, cerebral ischemia, cerebral apoplexy, or combinations thereof.
In another preferred embodiment, the neurodegenerative disease is Alzheimer's disease.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows extracellular vesicles of human adipose mesenchymal stem cells prepared by the invention and identification of biological characteristics thereof.
FIG. 2 shows the detection of the content of the antisense oligonucleotides of the major active ingredients hsa-miR-1290 and hsa-let-7b-5p in the modified pharmaceutical nasal drop preparation of human adipose mesenchymal stem cell extracellular vesicles.
FIG. 3 shows that a drug nasal drop formulation of extracellular vesicles of human adipose mesenchymal stem cells overexpressing hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides is specifically taken up by nerve cells.
FIG. 4 shows the detection of hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotide levels after specific uptake of drug nasal drops of genetically engineered human adipose mesenchymal stem cell extracellular vesicles by nerve cells and microglia.
FIG. 5 shows a schematic representation of drug nasal drip formulations of human adipose mesenchymal stem cell extracellular vesicles overexpressing hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides for treatment of Abeta 42 induced AD mice.
Detailed Description
The present inventors have unexpectedly developed a preparation containing genetically engineered stem cell extracellular vesicles for the first time through extensive and intensive studies. In the invention, the human adipose mesenchymal stem cells produced under the condition of GMP (good manufacturing practice) are preferably used as parent cells for producing stem cell extracellular vesicles, and are subjected to a series of genetic engineering transformation to obtain the drug nasal drop preparation of the stem cell vesicles with the brain nerve tissue specific targeting delivery function, and the drug nasal drop preparation is used for treating neurodegenerative diseases such as Alzheimer disease and the like through an administration route in a nasal cavity so as to provide a new effective treatment means for the neurodegenerative diseases. On this basis, the present invention has been completed.
Terminology
Neurodegenerative diseases
As used herein, the term "cerebral neurovascular disease" includes primarily neurodegenerative diseases.
Neurodegenerative diseases are caused by the loss of neurons and/or their myelin sheath, worsening over time, and presenting with dysfunction. It can be classified into acute neurodegenerative diseases and chronic neurodegenerative diseases, the former mainly including Cerebral Ischemia (CI), brain Injury (BI), epilepsy; the latter include Alzheimer's Disease (AD), parkinson's Disease (PD), huntington's Disease (HD), amyotrophic Lateral Sclerosis (ALS), different types of spinocerebellar ataxia (SCA), pick's disease, and the like.
Stem cell extracellular vesicles of the invention
There are three main types of stem cell extracellular vesicles (extracellular vesicles, EVs), exosomes (Exosomes), microvesicles (Microvesicles) and apoptotic bodies (Apoptotic bodies), respectively. All three major types of EVs are encapsulated by lipid bilayers, with diameters ranging from 30-2000nm.
The term exosomes refers to the subset of EVs derived from endosomes with diameters between 50-100nm, which are the major components of paracrine secretions of a variety of cell types, including mesenchymal stem cells (MESENCHYMAL STEM CELLS, MSCs).
MSCs Exosomes (Exosomes) are a class of EVs with a diameter in the range of 50-100nm, with a complete lipid bilayer membrane structure, among MSCs-derived EVs.
The invention provides an extracellular vesicle derived from human stem cells, which comprises but is not limited to stem cells derived from human umbilical cord blood, stem cells derived from human peripheral blood, human umbilical cord mesenchymal stem cells, human placenta mesenchymal stem cells, human adipose mesenchymal stem cells, stem cells derived from human bone marrow and the like. In a preferred embodiment of the invention, the extracellular vesicles are derived from supernatants of genetically engineered human stem cells collected during in vitro culture.
The stem cell extracellular vesicles contain active components for regulating and controlling targets related to inflammation and amyloid beta formation, and can simultaneously play a role in inhibiting inflammatory reaction and reducing the expression of amyloid precursor protein hydrolase BACE1 and maintain the expression level of ADAM 10. In a preferred embodiment of the present invention, the active ingredients mainly include: antisense oligonucleotides of miR-1290, miR-126-5p, miR-130a-3p, miR-24-3p, let-7b-3p and its precursor form, and also can include let-7b-5p, let-7a-5p, miR-92a-3p, miR-151a-3p, miR-1246 and its precursor form, and any combination of the foregoing.
In another preferred embodiment, the active ingredients comprise hsa-miR-1290 and hsa-miR-126-5p antisense oligonucleotides.
In addition, the stem cell extracellular vesicles of the present invention have the following characteristics: the extracellular vesicles are obtained by adopting a polyethylene glycol (PEG) precipitation method, the particle size of the extracellular vesicles is concentrated and distributed between 80 nm and 200nm, and the particle size is uniform.
Preparation method of preparation containing stem cell extracellular vesicles
The invention provides a preparation method of a nose drop preparation taking an active ingredient of a target spot related to inflammation and amyloid beta formation in stem cell extracellular vesicles after genetic engineering improvement as an active ingredient, which comprises the following specific technical scheme:
in a preferred embodiment, GMP-grade human adipose mesenchymal stem cells are mainly used as parent cells for the production of stem cell extracellular vesicles.
The preparation operation of the nose drops preparation of the stem cell vesicle mainly comprises the following steps:
1) Constructing a skeleton carrier containing fusion of RVG polypeptide sequence and Lamp2b sequence, and constructing target sequences hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotide sequences on the skeleton plasmid;
2) Transfecting the preferred plasmid which overexpresses hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotide into human adipose-derived mesenchymal stem cells through liposome 3000 (Lipofectamine 3000), selecting cell clones which stably express a target sequence, and preparing the human adipose-derived mesenchymal stem cells after genetic engineering; in another preferred embodiment, the active ingredients hsa-miR-1290 and hsa-miR-126-5p antisense oligonucleotides are introduced into (i.e., overexpressed in) human adipose mesenchymal stem cells in a molar (pmol) combination ratio of 1 (1-6) or (1-6): 1, including but not limited to 1:1, 1:2, 1:3, 1:4, 1:5 and 1:6, and 2:1, 3:1, 4:1, 5:1 and 6:1.
3) Collecting culture supernatant of the modified human adipose-derived mesenchymal stem cells produced in a GMP scale by effective condition treatment, performing differential centrifugation and filtration treatment to obtain a conditioned medium, and then performing vesicle separation by combining a polymer precipitation method; wherein the polymer includes but is not limited to polyethylene glycol (PEG 3000-9000) of different molecular weights and their compositions in different proportions (1:1-1:8);
4) Preparing PEG mother liquor with proper concentration (8% -20% g/ml) by PBS, filtering, mixing with the treated conditioned medium according to the volume ratio of 1:1, and incubating overnight at low temperature (4-8 ℃);
5) Centrifuging (4 ℃, 3000-5000 g, 45-60 min), discarding supernatant, adding precooled PBS, re-suspending for precipitation, and centrifuging (4 ℃, 100000-120 g, 1-2 h) at ultrahigh speed, discarding supernatant;
6) Adding a proper volume (3-10 ml) of medical normal saline, re-suspending and precipitating, and identifying the biological characteristics of the obtained modified extracellular vesicles of the human adipose-derived mesenchymal stem cells by the technologies of particle size analysis, scanning electron microscope observation, immunoblotting and the like;
7) Detecting the expression quantity of miRNAs which are main active ingredients in the extracellular vesicles of the stem cells by using real-time fluorescence quantitative PCR (qRT-PCR);
8) And diluting the obtained stem cell extracellular vesicles to proper concentration by adopting 3-10 ml of pharmaceutically acceptable carriers or auxiliary materials.
In a preferred embodiment of the invention, the preparation of the nasal drop preparation of the stem cell extracellular vesicles preferably uses allogenic human adipose mesenchymal stem cell cells as parent cells for producing extracellular vesicles after genetic engineering.
In a preferred embodiment of the invention, the nasal drop preparation of the stem cell extracellular vesicles is preferably a pharmaceutically acceptable carrier or adjuvant, including but not limited to sodium chloride, sodium phosphate, polyethylene glycol, chitosan, sodium hyaluronate, trehalose, chlorophosphonate, heparin, and the like, and any combination thereof.
In addition, the expression levels of hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides in the pharmaceutical nasal drop preparation of the extracellular vesicles of the preferably produced human adipose mesenchymal stem cells are subjected to real-time fluorescent quantitative PCR detection, and the result shows that the sequences are expressed at high levels. Meanwhile, the vesicles are also used as uptake assays for in vitro neural cells. The lipophilic fluorescent dye PKH26 marks the extracellular vesicles (1× 5～9×108 extracellular vesicles) of the modified human adipose mesenchymal stem cells, and the vesicles in the pharmaceutical preparation can be specifically ingested by the cells after being added into the cells for culturing for 24-48 hours and then observed under an inverted fluorescent microscope.
Process scale for producing stem cell extracellular vesicles under GMP conditions
A HYPERFLASK cell factory can produce 500ml of stem cell extracellular vesicles of conditioned medium, and it is expected that the total amount of stem cell extracellular vesicles is isolated 5X 10 10-10×1010.
At present, the laboratory can produce capacity that one production operation unit can separate stem cell extracellular vesicles of 800-1200ml of condition culture medium in total of two cell factories, and the separation of the stem cell extracellular vesicles of 2-5X 10 11 is expected, and according to the use amount of 2-5X 10 9 of one patient, the batch production amount of one production operation unit can meet the use amount of 100-250 patients.
Use of the preparation containing stem cell extracellular vesicles of the invention
On the other hand, the invention also provides application of the nasal drop preparation taking the active ingredient of the stem cell extracellular vesicle internal regulation and control related target spot formed by inflammation and amyloid beta after genetic engineering as the active ingredient in treating neurodegenerative diseases such as Alzheimer disease and the like. Such neurodegenerative diseases include, but are not limited to, alzheimer's disease, parkinson's disease, cerebral ischemia, or cerebral stroke.
In a preferred embodiment, 4 sets of experiments (Table 1) were set up, one PBS-induced control (PBS), A.beta.42-molded and physiological saline-treated (A.beta.42+saline), A.beta.42-molded and dimethyladamantane-treated (A.beta.42+memantine) and A.beta.42-molded and stem cell extracellular vesicular nose drops administered (A.beta. 42+modified haMSC-Exos). Firstly, in the first 2-5 days (D-2-D-5) of preparing an AD animal model, physiological saline, memantine (1 mg/kg of the weight of the mice) and modified stem cell vesicles are respectively given to the 4 groups of mice for treatment by a medicinal nasal drop preparation, and the treatment is carried out 1 time a day; wherein, the dosage of the nasal drop preparation is 1X 10 5～1×108 extracellular vesicles/mouse each time, and the nasal drop preparation is continuously treated for 2 to 5 days; then, AD mice model was induced by Aβ42 (5-20. Mu.g), and the mice of the test group were subjected to physiological saline drop nasal treatment and nasal drug administration treatment of the above-mentioned stem cell extracellular vesicle (1X 10 5～1×108 extracellular vesicle/mouse) respectively, once daily for 5-10 days. The cognitive function of the brain of the mouse is detected, then the mouse is euthanized, the brain tissue of the mouse is taken out to prepare paraffin sections, and then the Abeta 42 level and the microglial activation level of the sea horse area and the cortex area of the mouse are detected. The results showed a significant improvement in AD symptoms in mice.
TABLE 1 pharmaceutical nasal drop preparation of extracellular vesicles of human adipose tissue-derived stem cells for treatment of Abeta 42AD mice group
Pharmaceutical composition and application
The invention provides a pharmaceutical composition, which comprises: (a) A formulation comprising stem cell extracellular vesicles according to the first aspect of the invention, and (b) a pharmaceutically acceptable carrier.
The pharmaceutical composition of the invention is a cell-free and cell-free fragment pharmaceutical composition comprising a therapeutically effective amount of the stem cell extracellular vesicles of the invention. The term "therapeutically effective amount" refers to an amount of a therapeutic agent that treats, alleviates, or prevents a disease or condition of interest, or that exhibits a detectable therapeutic or prophylactic effect. The precise effective amount for a subject will depend on the size and health of the subject, the nature and extent of the disorder, and the therapeutic agent and/or combination of therapeutic agents selected for administration.
The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent (e.g., a stem cell extracellular vesicle of the present invention). The term refers to such agent carriers: they do not themselves induce the production of antibodies harmful to the individual receiving the composition and do not have excessive toxicity after administration. Suitable carriers may be large, slowly metabolizing macromolecules such as proteins, polysaccharides, polylactic acid (polylactic acid), polyglycolic acid and the like. Such vectors are well known to those of ordinary skill in the art. A sufficient discussion of pharmaceutically acceptable carriers or excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub.Co., N.J.1991).
Pharmaceutically acceptable carriers in the compositions can include liquids such as water, saline, glycerol, and ethanol. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers. In general, the compositions may be formulated as injectables, either as liquid solutions or suspensions; it can also be made into solid form suitable for formulation into solution or suspension, and liquid excipient prior to injection. Liposomes are also included in the definition of pharmaceutically acceptable carrier.
In a preferred embodiment of the invention, the pharmaceutically acceptable carrier is selected from the group consisting of: sodium chloride, sodium phosphate, polyethylene glycol, chitosan, sodium hyaluronate, trehalose, chlorophosphonate, heparin, or combinations thereof.
The pharmaceutical composition of the present invention can be prepared in various conventional dosage forms, such as liquid dosage forms, solid dosage forms (e.g., lyophilized dosage forms), the dosage forms of the pharmaceutical composition being selected from the group consisting of: nose drops, aerosol inhalants, eye drops and injections, preferably nose drops.
Once the composition of the invention is formulated, it may be administered directly to a subject. The subject to be treated may be a mammal, in particular a human. The subject to be treated suffers from neurodegenerative diseases, representative diseases include, but are not limited to: alzheimer's disease, parkinson's disease, cerebral ischemia, or cerebral apoplexy.
Furthermore, the pharmaceutical composition of the present invention may be administered in combination with or with other therapeutic agents for neurodegenerative diseases.
The main advantages of the present invention include:
(1) The stem cell extracellular vesicles modified by genetic engineering are rich in specific miRNAs which simultaneously play the roles of resisting inflammation, inhibiting amyloid precursor protein hydrolase BACE1 and maintaining ADAM10 expression.
(2) The stem cell extracellular vesicles are prepared by adopting a polyethylene glycol (PEG) precipitation method, the extracellular vesicles have the particle size which is concentrated and distributed between 80 and 200nm, and the particle size is uniform; the particle size is small, and the miRNA small molecules in the micro-RNA can penetrate through the blood brain barrier to enter the brain nerve tissue, and can be released after the stem cell extracellular vesicles enter the machine body, so that the micro-RNA reaches the brain.
(3) Compared with the conventional drug treatment, the nasal drop preparation of the stem cell extracellular vesicles can effectively prevent or treat neurodegenerative diseases such as Alzheimer disease.
The invention is further described below in conjunction with the specific embodiments. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.
Test material and instrument
Human adipose mesenchymal stem cells, DMEM medium, 5% serum replacement, lipofectamine 3000 (L3000015, invitrogen), wild-type BALB/c mice (7 weeks old, female, SPF grade), anesthetic (50 mg/kg ketamine and 30mg/kg cetlazine), neuro2A, microglial BV-2, aβ42 (AG 968, sigma-Aldrich), memantine (# 187836, sigma-Aldrich), medical saline, PEG of different molecular weights, sterile Phosphate Buffer (PBS), 1ml sterile syringe and 20G needle, PKH26 dye, paraffin block, H & E dye, PAS dye, trizol kit (# 12183555, invitrogen),III RT Supermix (R323-01, norweizan), chloroform, 10% neutral formaldehyde fixation, OCT embedding medium, liquid nitrogen, ELISA detection kit of mouse IL-1 beta (PI 301, biyundian) and Abeta (#KMB3441, thermo Scientific), centrifuge tube, 12-well cell culture plate, centrifuge, paraffin embedding machine, paraffin microtome, PCR instrument, real-time fluorescence quantitative PCR instrument, nanodrop2000 instrument, common bright field microscope (40X objective), normal fluorescence microscope (40X objective), inverted fluorescence microscope (20X objective), cell incubator.
Data statistical analysis method
All experimental data were statistically analyzed using GRAPHPAD PRISM 6.0.0 software and presented as Mean ± standard error (Mean ± SEM). When comparing the two groups of data, adopting Mann-Whitney Utest to carry out data comparison analysis; for comparison between the sets of data, one-way ANOVA and Tukey were used for data comparison analysis. P values less than 0.05 are considered significant differences.
Example 1
Preparation of pharmaceutical nasal drops preparation of human adipose mesenchymal stem cell extracellular vesicles over-expressing hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides
In all embodiments of the present invention, human adipose mesenchymal stem cells that overexpress the sequence of interest, preferably produced under GMP conditions, are used as parent cells for further production of extracellular vesicles of stem cells, and it should be emphasized that the parent cells for production of vesicles may also be derived from stem cells derived from tissues such as cord blood, umbilical cord, placenta, bone marrow, etc. The main process for preparing the medicinal nasal drop preparation of the human adipose mesenchymal stem cell extracellular vesicles which preferably overexpress hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides is as follows:
(1) Constructing a framework vector containing RVG polypeptide sequence and Lamp2b sequence, expressing the fusion protein and GFP label, and constructing hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotide sequences on the plasmid framework;
(2) Transfecting the preferred plasmid which overexpresses hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotide into human adipose-derived mesenchymal stem cells through liposome 3000 (Lipofectamine 3000), selecting cell clone which stably expresses a target sequence, and preparing the human adipose-derived mesenchymal stem cells modified by genetic engineering means;
(3) Preparing and culturing human adipose-derived mesenchymal stem cells over-expressing hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides under GMP (good manufacturing practice) conditions;
(4) Collecting culture supernatant of the human adipose-derived mesenchymal stem cells which are treated under effective conditions and produced on a large scale, performing differential centrifugation and filtration to remove cell debris to obtain a conditioned medium, and separating by combining a polymer precipitation method, wherein the polymer is preferably PEG9000;
(5) Preparing 20% g/ml PEG9000 mother liquor with PBS, filtering with 0.22 μm filter, adding into the treated conditioned medium according to a volume ratio of 1:1, standing at 4deg.C overnight for incubation, centrifuging at 4deg.C for 50min with 4000g, discarding supernatant, and retaining precipitate;
(6) Adding precooled PBS to resuspend sediment, carrying out ultra-high speed centrifugation for 1h at 4 ℃ and 120000g, removing supernatant, and adding 5ml of medical carrier solution to resuspend sediment;
(7) The stem cell extracellular vesicles obtained above were diluted to an appropriate concentration using 3ml of a medical carrier solution.
The results show that a pharmaceutical nasal drop formulation of extracellular vesicles of human adipose mesenchymal stem cells with uniformly dispersed vesicle particles can be obtained (fig. 1).
Example 2
The content of the main active ingredients hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides in the pharmaceutical nasal drop preparation of the genetically engineered human adipose tissue stem cell extracellular vesicles is increased
In order to determine the expression level of the target sequence in the extracellular vesicles of the modified human adipose-derived mesenchymal stem cells, the relative expression level of hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides in the vesicles is detected by adopting real-time fluorescence quantitative PCR (qRT-PCR), and the main operation steps are as follows:
(1) In a fume hood, 0.5ml Trizol reagent was added, and the mixture was allowed to stand at room temperature for 5min, followed by centrifugation (4 ℃ C., 12000g,5 min);
(2) Sucking the supernatant into an EP tube of a new RNase-free, adding 100. Mu.l of chloroform, manually shaking and mixing, standing at room temperature for 5min, and centrifuging (12000 g,15min at 4 ℃);
(3) Slowly sucking the colorless supernatant of the uppermost layer into an EP tube of a new RNase-free;
(4) Adding isopropanol with equal volume, mixing gently, standing at room temperature for 10min, centrifuging (12000 g,10min at 4deg.C);
(5) Gently pouring the supernatant, draining the residual liquid by inverting on a piece of absorbent paper, adding 500. Mu.l of DEPC-water prepared 75% ethanol to resuspend the precipitate, and centrifuging (4 ℃,7500g,5 min);
(6) Gently pouring the supernatant, inversely sucking the residual liquid on a piece of absorbent paper at room temperature, and adding 10 mu lDEPC-TREATED DDH 2 O to dissolve RNA;
(7) Mu.l of RNA concentration was determined in a Nanodrop2000 instrument by pipetting and diluting it to 1. Mu.g/. Mu.l;
(8) Kit for synthesizing cDNA III RT Supermix instructions for cDNA reverse transcription;
(9) Real-time fluorescent quantitative PCR is adopted to detect the expression level of hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotide sequences.
The results show that the content of hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides in the genetically engineered human adipose mesenchymal stem cell extracellular vesicles is significantly increased compared to the non-engineered human adipose mesenchymal stem cell extracellular vesicles (FIG. 2).
Example 3
A nasal drop preparation of human adipose mesenchymal stem cell extracellular vesicles over-expressing hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides is specifically ingested by nerve cells
In order to demonstrate that the above-mentioned modified human adipose mesenchymal stem cell extracellular vesicles expressing RVG-Lamp2b fusion protein can be taken up by nerve cells, an in vitro experiment was designed, and the vesicles collected by using a lipophilic fluorescent dye PKH26 marker were incubated with nerve cell Neuro2A, and then the taking-up condition was detected by using a fluorescence microscope, and the following specific steps were carried out:
(1) Incubating the stem cell extracellular vesicles with PKH26 dye for 12h with the stem cell extracellular vesicles which are modified or not modified, so that the stem cell extracellular vesicles are marked on the membranes of the vesicles; the vesicles were then pelleted using a 120000g ultracentrifugation for 1h, and after resuspension, the free dye and vesicles remaining in the solution were separated using size exclusion chromatography.
(2) Neuro2A cells were seeded at a density of 5X 10 4 cells per well in 12-well plates and after 24h incubation in a cell incubator (37 ℃, 5%), 5. Mu.g/ml of the pre-and post-remodelled vesicles described above were added.
(3) After 3h of co-incubation, the uptake of vesicles by the cells was observed under a fluorescence microscope.
The results showed a significant increase in cells that exhibited a red fluorescent signal in Neuro2A cells incubated with the human adipose mesenchymal stem cell extracellular vesicles transfected with RVG-Lamp2b fusion protein plasmid described above (fig. 3), indicating that vesicles transfected with the fusion protein are more readily taken up by neural cells.
Example 4
Increased levels of hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides following ingestion of genetically engineered human adipose mesenchymal stem cell extracellular vesicle drug nasal drops by neural cells and microglia
To determine whether the level of hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides was increased following uptake of the vesicles by Neuro2A and microglial BV-2, real-time fluorescent quantitative PCR assays were performed, and were essentially as follows:
(1) According to the procedure of example 2, neuro2A cells and microglial cells BV-2 were seeded into 12-well plates at a density of 5X 10 4 cells per well, respectively, and cultured in a cell incubator (37 ℃, 5%) for 24 hours;
(2) Adding or not adding 5 mug/ml of the PKH 26-labeled vesicle into the cell culture dish, placing the cells into a cell culture box (37 ℃ C., 5%) for further culture for 3 hours;
(3) Observing under a fluorescence microscope that green fluorescence in cells after taking vesicles increases, taking out the cells, and placing the cells on ice;
(4) RNA extraction, purification and cDNA synthesis, as well as real-time fluorescent quantitative PCR detection of hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotide levels, were carried out according to the procedure of example 2.
The results showed that the content of hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides was significantly increased after the Neuro2A cells and microglial cells BV-2 ingested the genetically engineered human adipose mesenchymal stem cell extracellular vesicles compared with the control (FIG. 4).
Example 5
Pharmaceutical nasal drop preparation for preventing or treating Abeta 42 induced AD mice by over-expressing hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotide
In order to explore the cognitive function of the mice with the AD model through nasal administration of the drug nasal drop preparation of the modified human adipose-derived mesenchymal stem cell extracellular vesicles, an AD mice disease model induced by Abeta 42 is firstly established, then the mice disease model is treated (figure 5), and the relevant indexes of the cognitive function of the brain are detected, wherein the method mainly comprises the following steps:
(1) Firstly, setting 4 groups of experiments, namely a PBS-induced control group (PBS), wherein A beta 42 is molded and treated by normal saline (A beta 42+saline), A beta 42 is molded and treated by memantine (A beta 42+memantine) and A beta 42, and a treatment group (A beta 42+modified haMSC-Exos) of extracellular vesicle nose drops of stem cells is given;
(2) On the first 2 days (D-2) of preparation of the AD animal model, the above 4 groups of mice were respectively given physiological saline, memantine (1 mg/kg of mouse body weight) and a drug nasal drop preparation treatment of the stem cell vesicles after modification, 1 time per day; wherein, the dosage of the nasal drop preparation is 1X 10 6 parts of drugs/mouse each time, and the nasal drop preparation is continuously treated for 2 days;
(3) Inducing AD mice model by using 10 mu g A β42, and respectively administering physiological saline drop nasal treatment and drug nasal preparation (1×10 6 parts/mouse) of the stem cell extracellular vesicles to the mice of the test group except the control group, wherein the administration is carried out once a day for 5 days;
(4) The cognitive function of the mice is tested by adopting a Barns maze; thereafter, the mice were euthanized, brain tissues of the mice were removed to prepare paraffin sections, and then the levels of aβ42 and microglial activation in their hippocampal and cortical areas were examined.
After the modified human adipose mesenchymal stem cell extracellular vesicle drug nasal drop preparation is used for treating AD mice by nasal administration, the generation level of Abeta 42 in the hippocampal region and the cortical region is also obviously reduced, the activation level of microglial cells is reduced, and the cognitive level of the mice is obviously improved.
Reference to the literature
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All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Claims (6)
1. A nasal drop formulation comprising stem cell extracellular vesicles, wherein the stem cell extracellular vesicles of the nasal drop formulation comprise an active ingredient that modulates a target associated with inflammation and amyloid β formation; and, the formulation is for:
(a) Inhibiting inflammatory response of brain nerve tissue;
(b) Inhibiting the production of amyloid aβ; and
(C) Preventing and/or treating the neurodegenerative disease,
Wherein, the stem cell extracellular vesicle is a genetically engineered human adipose mesenchymal stem cell extracellular vesicle which overexpresses hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides.
2. The nasal drip formulation of claim 1, wherein the stem cell extracellular vesicles have the following characteristics: the extracellular vesicles are obtained by adopting a polyethylene glycol (PEG) precipitation method, the particle size of the extracellular vesicles is concentrated and distributed between 80 nm and 200nm, and the particle size is uniform.
3. The nasal drip formulation of claim 1, wherein the stem cell extracellular vesicles have the following characteristics: (a) The particle size is small, and the blood brain barrier can be penetrated into the brain nerve tissue; (b) The miRNA small molecules in the cell can be released after the stem cell extracellular vesicles enter the machine body, so that the cell reaches the brain.
4. A nasal drip formulation according to any one of claims 1 to 3, characterized in that it has the following characteristics: the stem cell extracellular vesicles in the nasal drop preparation are genetically engineered to have the brain nerve tissue specific targeting capability, and can target and deliver the active ingredients in the stem cell extracellular vesicles into the brain nerve tissue to be taken up by nerve cells and microglia cells.
5. The nasal drip formulation of claim 4, wherein the stem cell extracellular vesicles further comprise an active ingredient selected from the group consisting of: antisense oligonucleotides of hsa-miR-126-5p, hsa-miR-130a-3p, hsa-miR-24-3p, hsa-let-7b-3p and precursor forms thereof, hsa-let-7a-5p, hsa-miR-92a-3p, hsa-miR-151a-3p, hsa-miR-1246 and precursor forms thereof.
6. Use of a nasal drop formulation containing stem cell extracellular vesicles as claimed in any one of claims 1 to 5 in the manufacture of a medicament for the treatment of alzheimer's disease.
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| WO2021003403A1 (en) * | 2019-07-02 | 2021-01-07 | Ohio State Innovation Foundation | Neurodegenerative disease therapies utilizing the skin-brain axis |
| CN112402456A (en) * | 2020-07-06 | 2021-02-26 | 医微细胞生物技术(广州)有限公司 | Application of an induced extracellular vesicle derived from mesenchymal stem cells in the treatment of Alzheimer's disease |
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