阅读说明：本技术 功能化多能干细胞纳米囊泡制剂的制备及其在防治肺炎中的用途 (Preparation of functionalized pluripotent stem cell nano vesicle preparation and application thereof in preventing and treating pneumonia ) 是由 汪洪 何建氢 于 2020-03-16 设计创作，主要内容包括：本发明涉及功能化多能干细胞纳米囊泡制剂的制备及其在防治肺炎中的用途,属于生物医药技术领域。所述多能干细胞来源的纳米囊泡制剂是将多能干细胞的培养上清去除死细胞、细胞碎片,富集浓缩后获得的。本发明还提供了载有药物的功能化纳米囊泡制剂。并进一步提供了所述多能干细胞来源的纳米囊泡制剂或载有药物的功能化纳米囊泡制剂在制备治疗肺炎或肺损伤药物上的应用。与现有技术相比,本发明利用EVs负载法匹拉韦等抗病毒药物,构建既具有抗炎调节免疫又具有直接抑制RNA病毒复制的EVs,很有可能为新型冠状病毒等烈性RNA病毒引起的难治性病毒性肺炎提供有效治疗手段。(The invention relates to preparation of a functionalized pluripotent stem cell nano vesicle preparation and application thereof in preventing and treating pneumonia, and belongs to the technical field of biological medicines. The nano vesicle preparation derived from the pluripotent stem cells is obtained by removing dead cells and cell fragments from the culture supernatant of the pluripotent stem cells, enriching and concentrating. The invention also provides a functionalized nano vesicle preparation carrying the medicine. And further provides application of the nano-vesicle preparation derived from the pluripotent stem cells or the functionalized nano-vesicle preparation carrying the medicine in preparation of medicines for treating pneumonia or lung injury. Compared with the prior art, the invention utilizes the EVs loading method Pilatvir and other antiviral drugs to construct the EVs which not only has anti-inflammatory and immune regulation function, but also has the function of directly inhibiting RNA virus replication, and is likely to provide an effective treatment means for intractable viral pneumonia caused by novel coronavirus and other virulent RNA viruses.)

1. A nano vesicle preparation derived from pluripotent stem cells is obtained by removing dead cells and cell debris from culture supernatant of pluripotent stem cells, enriching and concentrating, wherein nano vesicles of the nano vesicle preparation derived from pluripotent stem cells are in a cup-shaped structure; the pluripotent stem cell-derived nanovesicle preparation expresses CD63, CD9, Tsg101, and does not express GM 130; the vesicle particle size of the nano vesicle preparation derived from the pluripotent stem cells is mainly distributed in the range of 50-150 nm.

2. The pluripotent stem cell-derived nanovesicle preparation according to claim 1, wherein the pluripotent stem cell-derived nanovesicle preparation is an induced pluripotent stem cell-or embryonic stem cell-derived nanovesicle preparation.

3. A drug-loaded functionalized nanovesicle formulation, comprising the pluripotent stem cell-derived nanovesicle formulation of claim 1 or 2, wherein the nanovesicle formulation is loaded with a drug.

4. The functionalized nanovesicle formulation with a drug according to claim 3, wherein the drug comprises a drug that inhibits viral RNA polymerase or protein kinase or reverse transcriptase;

preferably, the medicament is one or more selected from the group consisting of Favipiravir, nelfinavir, lopinavir, ritonavir and emtricitabine propofol tenofovir.

5. The drug-loaded functionalized nanovesicle preparation according to claim 3, wherein the functionalized nanovesicle preparation is derived from induced pluripotent stem cells or embryonic stem cells loaded with Favipiravir.

6. The method for preparing the drug-loaded functionalized nanovesicle preparation of claim 3, wherein a drug solution containing a saturated concentration is mixed with the pluripotent stem cells, the mixture is passed through a liposome extruder and sequentially passes through micron-sized and nano-sized filtration membranes with different diameters from large to small to obtain a drug-loaded vesicle mixed solution, and the drug-loaded functionalized nanovesicle preparation is further obtained through ultrafiltration or ultracentrifugation.

7. Use of the pluripotent stem cell-derived nanovesicle formulation of claim 1 or the drug-loaded functionalized nanovesicle formulation of claim 4 in the preparation of a medicament for treating pneumonia or lung injury or pulmonary fibrosis.

8. The use of claim 7, wherein the pluripotent stem cell-derived nanovesicle preparation or the drug-loaded functionalized nanovesicle preparation is used for preparing a medicament for treating viral pneumonia or acute lung injury.

9. The use of claim 8, wherein the virus comprises influenza virus H1N1 or coronavirus, including novel coronavirus 2019-nCoV or SARS virus or MERS virus.

10. The use of claim 7, wherein the dosage form of the drug for treating pneumonia or lung injury prepared from the pluripotent stem cell-derived nano-vesicle preparation or the drug-loaded functionalized nano-vesicle preparation is a nebulized dosage form, an injection dosage form, a powder dosage form or a tablet dosage form.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to preparation of a functionalized pluripotent stem cell nano vesicle preparation and application of the preparation in preventing and treating pneumonia.

Background

Studies have shown that inflammatory monocyte-macrophages and neutrophils accumulate in The lung following human Coronavirus infection [ L. E.Gralinski, R.S. Baric, Molecular pathalogy of observing coronavir infections, J Pathol 235(2) (2015) 185-95; R.Channapaparvavar, S.Perlman, Pathological human coronavir infections: consumers and responses of pulmonary disorders, clinical study of pulmonary disorders of epithelial invasion, Seminnopathol 39, (5) 529. and 539. The rapid replication of viruses and vigorous pro-inflammatory cytokine/chemokine responses of lung epithelial cells and endothelial cells, destroy The microvascular and cellular barriers, cause vascular permeability and endothelial permeability of epithelial cells and cause acute lung edema in patients [ T.2017 ] 20135 ] acute lung edema of pulmonary hypertension, coronary heart lung tissue, lung epithelial edema, coronary heart tissue, lung tissue, lung tissue, lung tissue, lung tissue, lung tissue.

Pluripotent stem Cells including Embryonic Stem Cells (ESC) and Induced Pluripotent Stem Cells (iPSC), which are the most primitive and naive totipotent stem Cells, such as ESC, have immune privileges similar to those possessed by gestational embryos, evade allogeneic immunological responses, and have unique advantages in regulating immune responses, since ESC has very low levels of MHC I expression and MHC II, CD80, CD86, and CD 40L expression deletion studies have found that ESC has strong immune regulatory properties, and embryonic stem Cells of Human, mouse, and rat all have the ability to inhibit T lymphocyte proliferation, even in the presence of a fraction of antigen-presenting Cells [ L I, L, Baroja, m. L, jumdar, a., et al (2004), Human embryonic stem Cells of mouse, and rat, and thus cannot be used for the treatment of embryonic stem-viral diseases, stelms, 448, 22, 35, 22, 9, 27, 9, g.

Research shows that Stem cells mainly exert biological functions thereof through a paracrine mechanism [ C.B, &lTtTtranslation = L "&gTtL &lTt/T &gTt.Q, Z.B, W.Y, Stem Cell-Derived Extracellular Vesicles as a Novel therapeutic toolol for Tissue Repair, Stem Cell translation medicine 6(9) (2017)1753 and 1758 ] whether Extracellular Vesicles (PSC-EVs) secreted by pluripotent Stem cells have immune regulation functions similar to those of the pluripotent Stem cells is not reported so far.

Disclosure of Invention

Aiming at the problem of refractory viral pneumonia caused by virulent RNA viruses such as novel coronavirus and the like, the invention provides the preparation of a functionalized pluripotent stem cell nano vesicle preparation and the application of the preparation in preventing and treating pneumonia.

The purpose of the invention can be realized by the following technical scheme:

in a first aspect, the present invention provides a pluripotent stem cell-derived nanovesicle formulation.

Further, the pluripotent stem cell-derived nanovesicle preparation is an induced pluripotent stem cell-or embryonic stem cell-derived nanovesicle preparation.

In a second aspect, the present invention provides a method for preparing a pluripotent stem cell-derived nanovesicle preparation.

The preparation method of the pluripotent stem cell-derived nanovesicle preparations (PSC-EVs) can be obtained by adopting the following modes:

1. removing substances such as dead cells, cell debris and the like from the culture supernatant of the pluripotent stem cells, and enriching and concentrating to obtain the nano vesicle preparation derived from the pluripotent stem cells.

2. The physical extrusion method comprises the following steps: digesting and collecting a certain amount of iPSC single cells, re-suspending the cells by PBS, uniformly mixing the cells, sequentially passing through micron-scale and nano-scale filter membranes with different diameters from large to small by using a liposome extruder to obtain a vesicle mixed solution, and further obtaining a nano-vesicle preparation (PSC-EVs) by ultrafiltration or ultracentrifugation.

In one embodiment of the physical compression method, the concentration of the resuspended cells in PBS is 1-9 × 10⁸And (4) cells.

In one embodiment of the physical extrusion method, the micron-sized and nano-sized filtration membranes sequentially passing through different diameters from large to small are filtration membranes sequentially passing through 5 μm, 2 μm, 800nm, 500nm, 200nm, 100nm, and 50 nm.

Cells secrete limited number of EVs in a natural state, and the cells are long in period and difficult to directly carry out engineering production. The nano vesicle preparation (PSC-EVs) obtained by adopting the physical extrusion method has similar structure, particle size and membrane protein with natural secreted EVs, but the yield of the vesicles can be improved by hundreds of times, the preparation cost of the vesicles is effectively reduced, and the nano vesicle preparation has important practical application potential.

Further, the invention further provides a preparation method of a clinical-grade induced pluripotent stem cell line (iPSC) -derived nanovesicle preparation (PSC-EVs), which comprises the following steps:

the construction and production of the clinical induced pluripotent stem cell line (iPSC) use a serum-free culture system with clear chemical components. Construction of the cells can be carried out by conventional techniques known to those skilled in the art. During operation, the quality control is strictly carried out according to the quality control standard. And (3) amplifying a large amount of collected clinical-grade iPSC culture supernatant, removing substances such as dead cells and cell fragments through various modes such as centrifugation and filtration or a combined mode, enriching and concentrating to obtain a nano vesicle preparation (PSC-EVs), and subpackaging and freezing the PSC-EVs.

Characterization of induced pluripotent stem cell line (iPSC) -derived nanovesicle preparations (PSC-EVs):

the appearance of the prepared nano vesicles is observed through a transmission electron microscope, and the nano vesicles are in a cup-shaped structure; the markers of the nano vesicles are identified by an immunoblotting technology, and the results show that the nano vesicles express CD63, CD9 and Tsg101, but do not express GM 130; the grain diameter of the prepared vesicle is mainly distributed at 50-150nm by the detection of the nano-flow technology. It was demonstrated that PSC-EVs belong to vesicles with diameters between 50 and 150 nm.

In a third aspect, the invention provides a functionalized nanovesicle formulation loaded with a drug.

The functionalized nanovesicle preparation comprises a nanovesicle preparation, wherein the nanovesicle preparation is loaded with a drug, and the drug comprises one or more of Favipiravir, nelfinavir, lopinavir, ritonavir or emtricitabine propiophenol tenofovir.

The novel coronavirus is a single-stranded RNA positive-strand enveloped B coronavirus, the replication of which is dependent on RNA polymerase. Favipiravir is a broad-spectrum RNA polymerase inhibitor, which is converted into a bioactive Favipiravir nucleoside triphosphate after oral absorption, has a structure similar to purine, and can compete with purine for viral RNA polymerase. And the research finds that nucleoside triphosphates of the Favipiravir can be inserted into a virus RNA chain to induce fatal mutation of the virus. Therefore, Favipiravir has potential antiviral effects on various RNA viruses. In addition, nelfinavir, lopinavir/ritonavir inhibit HIV viral replication by inhibiting proteases; emtricitabine propofol tenofovir tablets inhibit viral replication by inhibiting reverse transcriptase.

Further, the functionalized nanovesicle preparation is an induced pluripotent stem cell or embryonic stem cell-derived nanovesicle preparation loaded with a drug such as favipiravir.

Further, functionalized nanovesicle preparations (F-PSC-EVs) derived from induced pluripotent stem cell lines (ipscs) are provided. The functionalized nanovesicle formulations (F-PSC-EVs) are loaded with drugs comprising one or more of Favipiravir, nelfinavir, lopinavir, ritonavir or emtricitabine Propofovir.

In a fourth aspect, the invention provides a preparation method of a functionalized nanovesicle preparation carrying a drug.

The functional nano vesicle preparation carrying the medicine is obtained by wrapping a certain dose of medicine into the nano vesicle preparation through one or a combination of incubation, electric conversion, extrusion, ultrasound, freeze thawing or saponin treatment.

In one embodiment of the invention, the invention provides a physical extrusion method for obtaining a functionalized nano vesicle preparation carrying a drug, which comprises the following steps: digesting and collecting a certain amount of iPSC single cells, resuspending the cells by PBS, adding a drug solution (such as Favipiravir) with saturated concentration, resuspending the iPSC, uniformly mixing the cells, sequentially passing through micron-scale and nano-scale filter membranes with different diameters from large to small by using a liposome squeezer to obtain a drug-loaded vesicle mixed solution, and further performing ultrafiltration or ultracentrifugation to obtain the drug-loaded functionalized nanocapsule preparation.

In one embodiment of the invention, the concentration of the PBS resuspended cells is 1-9 × 10⁸And (4) cells.

In one embodiment of the present invention, the micron-sized and nano-sized filtration membranes sequentially passing through different diameters from large to small are filtration membranes sequentially passing through 5 μm, 2 μm, 800nm, 500nm, 200nm, 100nm, and 50 nm.

In one embodiment of the present invention, the drug solution containing a saturation concentration is added in an amount of 20 to 100m L.

The invention directly mixes the cells and the medicine and then directly obtains the medicine-loaded functionalized nano vesicle preparation through physical extrusion, and the method has the advantages of high speed, effective reduction of the preparation cost of the vesicle preparation and important practical application potential.

In one embodiment of the present invention, the drug-loaded functionalized nanovesicle preparation is separated from the free drug by means of conventional ultrafiltration, ultracentrifugation or desalting column. And detecting the concentration of the medicine by an ultraviolet spectrophotometer.

In a fifth aspect, an application of a pluripotent stem cell-derived nanovesicle preparation or a drug-loaded functionalized nanovesicle preparation is provided.

The application of the nano-vesicle preparation derived from the pluripotent stem cells or the functionalized nano-vesicle preparation carrying the medicine in the preparation of the medicine for treating pneumonia or lung injury or pulmonary fibrosis.

Further, the pluripotent stem cell-derived nano-vesicle preparation or the drug-loaded functionalized nano-vesicle preparation is applied to preparation of a drug for treating viral pneumonia or acute lung injury.

The virus comprises influenza virus H1N1 or novel coronavirus 2019-nCoV or SARS virus or MERS virus and the like.

The dosage form of the medicine for treating pneumonia or lung injury prepared from the pluripotent stem cell-derived nano vesicle preparation or the medicine-loaded functionalized nano vesicle preparation is an atomized dosage form, an injection dosage form, a powder dosage form or a tablet dosage form. The route of administration may be: oral or nasal spray, aerosol inhalation, administration through bronchoscope or intubation trachea, oral administration, intravenous injection, etc.

The invention provides an application of a nano vesicle preparation derived from pluripotent stem cells or a functionalized nano vesicle preparation carrying a medicine in preparing a medicine for treating pneumonia or lung injury or pulmonary fibrosis, wherein the prepared medicine for treating pneumonia or lung injury is used for preventing and treating human diseases through oral or nasal cavity spraying, atomizing inhalation, intratracheal administration, oral administration, intravenous injection and other ways, the risks of tumorigenicity, immune rejection, embolism, possible chromosome mutation and the like faced by directly transplanted cells are avoided, and the biological safety of the medicine is obviously superior to that of the directly transplanted cells. Aiming at severe pneumonia, the nano vesicles directly enter the lung through atomization inhalation or intratracheal administration, and compared with systemic administration, the nano vesicles are safer and more reliable, and the drug utilization rate is higher.

In the production process of the invention, the nano-scale extracellular vesicles naturally secreted by pluripotent stem cells are found to repair damaged lung tissues possibly through regulating immunity, inhibiting inflammatory reaction, promoting interferon secretion and the like, but the nano-scale extracellular vesicles are still difficult to directly inhibit intracellular virus replication with strong efficiency, so that related antiviral drugs are delivered into cells by EVs to directly kill viruses, and further, the functional nano-vesicle preparation capable of regulating immunity, resisting inflammation and directly inhibiting virus replication with strong efficiency is prepared. In addition, most antiviral drugs have strong toxic and side effects on human bodies, and the efficiency of the antiviral drugs can be greatly improved by delivering the antiviral drugs to directly enter cells by using EVs, so that the dosage of the drugs is greatly reduced, namely the toxic and side effects are reduced.

The research of the invention finds that the functionalized nanovesicle preparation (F-PSC-EVs) carrying the medicine has an obvious protective effect on lung injury caused by H1N1 virus or 2019-nCoV and other viruses, meanwhile, the nanovesicle preparation (PSC-EVs) also has an obvious protective effect on lung injury caused by H1N1 virus or 2019-nCoV and other viruses, the F-PSC-EVs has the best protective effect, and the effect of a simple medicine group is not as good as that of the functionalized nanovesicle preparation (F-PSC-EVs) or the nanovesicle preparation (PSC-EVs) of the medicine.

The invention utilizes the pluripotent stem cells to generate the antiviral drugs such as the EVs loading Pilatvir, constructs the EVs with the functions of resisting inflammation, regulating immunity and directly inhibiting the RNA virus replication, and is likely to provide an effective treatment means for the intractable viral pneumonia caused by the virulent RNA viruses such as the novel coronavirus.

Drawings

FIG. 1: the results of identification of PSC-EVs;

fig. 1 includes the A, B, C panels, a. transmission electron micrograph of vesicles; B. the particle size distribution of the vesicles; C. immunoblot identification of surface proteins of vesicles.

FIG. 2.1 Effect of Nanobubular formulations on pathological structures of H1N1 and 2019-nCoV Virus infected lesions in Lung tissue (HE staining, 200 ×)

FIG. 2.2 histological scoring of lung tissue for each experimental group;

FIG. 2.3 Effect of nanovesicle preparations on pulmonary fibrosis caused by 2019-nCoV Virus infection (Masson staining, 200 ×)

FIG. 2.4 the results of quantitative analysis of collagen deposition in lung tissue of each experimental group;

FIG. 3: the inhibition effect of the nano vesicles on the virus replication in lung tissues (real-time fluorescence quantitative PCR method);

FIG. 4: the effect of the nano vesicles on the level of the inflammatory factors in the plasma of mice;

FIG. 5: the consequences of the influence of PSC-EVs on Treg proliferation;

FIG. 6.1 phenotypic modulation of L PS-stimulated macrophages by PSC-EVs;

FIG. 6.2 Regulation of L PS-stimulated macrophages release of inflammatory and anti-inflammatory factors by PSC-EVs.

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments.