Stem cell preparation for treating pulmonary fibrosis as well as preparation method and application thereof

Abstract

The invention discloses a stem cell preparation for treating pulmonary fibrosis. The stem cell preparation is mesenchymal stem cell surface modified nanoparticles. The nanoparticles are ROS (reactive oxygen species) response type lipid polymer hybrid nanoparticles loaded with an anti-fibrosis drug. According to the invention, a hydrophobic drug and a hydrophilic anti-fibrosis drug can be simultaneously loaded through the reductive hybrid nanoparticles, and can be delivered to the lung by utilizing the inflammation targeting capability of stem cells. The polymer in the nanoparticles reacts with active oxygen in the microenvironment to promote drug release, and then the effect of protecting stem cells is achieved. The released medicine can induce dedifferentiation of myofibroblasts and inhibit apoptosis of vascular endothelial cells and endothelial mesenchymal transformation, and the mesenchymal stem cells release growth factors at the same time to promote angiogenesis, reconstruct capillary structures deleted in fibrosis areas, promote lung tissue regeneration and recover lung functions to achieve the effect of treating pulmonary fibrosis.

Description

Technical Field

[0001] The present invention specifically relates to a stem cell preparation for treating pulmonary fibrosis and a preparation method and application thereof, belonging to the technical field of pharmaceutical preparations. Background Art

[0002] Pulmonary fibrosis is a progressive disease characterized by damage to the respiratory system and tissue function. The pathogenesis of pulmonary fibrosis has not been fully elucidated, and vascular involvement is an important pathological feature in the development of pulmonary fibrosis. As pulmonary fibrosis worsens, pulmonary capillaries are damaged and permeability increases, until the number of vascular endothelial cells in the late fibrotic area decreases and vascular density decreases. The pulmonary vascular system is an important component of the lung parenchyma, mostly composed of capillaries, wrapped around the alveoli, not only providing nutritional support and gas exchange, but also playing a vital role in lung tissue repair, development and regeneration. Pulmonary capillary endothelial cells construct a specific vascular niche. When lung tissue is damaged, they activate alveolar epithelial cells by secreting angiogenic factors including epidermal growth factor and hepatocyte growth factor, promote the differentiation of type II alveolar epithelial cells, and regenerate alveolar structures. Therefore, targeted angiogenesis has a positive effect on the treatment of pulmonary fibrosis.

[0003] Stem cell therapy is an effective way to promote vascular regeneration and can promote angiogenesis by secreting vascular endothelial growth factor. In addition, stem cells have good immune immunity and can effectively target the lungs through chemokine receptors and implant into diseased tissues. However, mesenchymal stem cells are easily affected by the pathological environment of oxidative stress, resulting in reduced activity and a shorter retention time in the body, which limits their application in the treatment of pulmonary fibrosis.

[0004] Therefore, there is an urgent need to design a stem cell preparation that can target the lungs and effectively repair damaged pulmonary blood vessels while having good biological activity. Summary of the invention

[0005] Purpose of the Invention

[0006] One of the purposes of the present invention is to provide a stem cell preparation for treating pulmonary fibrosis by targeting angiogenesis. The stem cell preparation of the present invention comprises mesenchymal stem cells and ROS-responsive nanoparticles, wherein the ROS-responsive nanoparticles are modified on the cell membrane surface of the mesenchymal stem cells.

[0007] Technical Solution

[0008] A stem cell preparation, characterized in that it comprises mesenchymal stem cells and ROS-responsive lipid polymer hybrid nanoparticles; wherein the ROS-responsive lipid polymer hybrid nanoparticles are modified on the surface of the stem cells.

[0009] The stem cell preparation is characterized in that the ROS lipid polymer hybrid nanoparticles are nanocarriers carrying drugs, and the nanocarriers include phospholipids, cholesterol, reducing polymers, DSPE-PEG2000-MAL, and A6 peptide; the amino acid sequence of the A6 polypeptide is KPSSPPEECGPLGIAGQC, see SEQ ID NO.1.

[0010] The stem cell preparation is characterized in that the mesenchymal stem cells are selected from adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells or skin mesenchymal stem cells.

[0011] The stem cell preparation is characterized in that the phospholipid is one or more of soybean lecithin, egg yolk lecithin, HSPC, DPPC, DOPC and DPPE.

[0012] The stem cell preparation is characterized in that the reducing polymer includes one or more of PPS-PEG, PEG-b-PBA, and PTE-PEG.

[0013] The stem cell preparation is characterized in that the drugs include drugs for protecting vascular endothelial cells and anti-fibrosis drugs.

[0014] The stem cell preparation is characterized in that the drug for protecting vascular endothelial cells is: CYM-5442 or macitentan;

[0015] Antifibrotic drugs are dihydrotestidine, prostaglandin E2, metformin, or bosentan.

[0016] The stem cell preparation is characterized in that the mass ratio of phospholipids to cholesterol is 5:1-10:1, the mass ratio of phospholipids to DSPE-mPEG2000-MAL is 8:1-10:1, the mass ratio of phospholipids to PPS-PEG is 2:1-5:1, the mass ratio of phospholipids to metformin is 5:1-10:1, and the mass ratio of phospholipids to macitentan is 10:1-15:1.

[0017] The application of the stem cell preparation in the preparation of drugs for treating pulmonary fibrosis.

[0018] The specific preparation method of the stem cell preparation is as follows;

[0019] 1. Preparation of ROS-responsive nanoparticles

[0020] 2. A6 peptide is modified on the surface of nanoparticles to obtain nanoparticles

[0021] 3. Incubate mesenchymal stem cells with nanoparticles to obtain stem cell preparations

[0022] Furthermore, the mesenchymal stem cells include adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells or skin-derived mesenchymal stem cells. Mesenchymal stem cells have low immunogenicity and can target diseased tissues through a homing effect, effectively delivering drugs to the lungs. In the embodiments of the present invention, adipose-derived mesenchymal stem cells are used.

[0023] Furthermore, the connection modes of mesenchymal stem cells and ROS-responsive nanoparticles include: ligand binding, electrostatic adsorption, and covalent binding. In the present invention, A6 peptide is used to connect with CD44 receptor on mesenchymal stem cells by biological coupling reaction.

[0024] Furthermore, the ROS-responsive nanoparticles carry the anti-fibrosis drug in the inner aqueous phase and the hydrophobic layer, respectively. The nanoparticles are composed of phospholipids, cholesterol, ROS-responsive polymers, and DSPE-PEG-MAL, and are surface-modified with A6 connecting peptides. The ROS-responsive polymer PPS-PEG has ROS responsiveness, and the A6 peptide binds to the CD44 receptor on the cell membrane of mesenchymal stem cells.

[0025] Beneficial Effects

[0026] The present invention specifically targets pulmonary fibrosis tissue with the homing effect of mesenchymal stem cells. Nanoparticles on the surface of the mesenchymal stem cell membrane are responsively separated, and the nanoparticles react with reactive oxygen in the pathological environment, releasing anti-fibrotic drugs while also protecting mesenchymal stem cells from oxidative stress damage. Anti-fibrotic drugs induce myofibroblast dedifferentiation and inhibit endothelial cell apoptosis and endothelial-mesenchymal transition. Combined with mesenchymal stem cells to mediate the formation of new blood vessels, the vascular structure of the fibrotic area is rebuilt, thereby restoring the integrity of the alveoli and achieving recovery of lung function.

[0027] 1. Nanoparticles on the surface of mesenchymal stem cell membranes can not only carry hydrophobic and hydrophilic drugs at the same time, but also responsively release anti-fibrotic drugs while also protecting mesenchymal stem cells from reactive oxygen damage in pathological environments.

[0028] 2. Through the combined therapeutic effect of anti-fibrotic drugs and mesenchymal stem cells, it is possible to regenerate the missing blood vessels in the fibrotic area, restore the alveolar capillary barrier, and rebuild lung function. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the morphological characterization of stem cells in Example 1.

[0030] Figure 2 These are the stem cell surface markers detected by flow cytometry in Example 1; A is CD90, B is CD29, and C is CD35.

[0031] Figure 3This is the preparation screening process in Example 1.

[0032] Figure 4 The particle size and polydispersity coefficient of the ROS-responsive nanoparticles in Example 1 were investigated (A) and their stability was characterized (B). Figure 5 1 is a fluorescence diagram of the active oxygen response release and ROS scavenging effect of the ROS responsive nanoparticles in Example 1; A is the drug release curve, and B is the intracellular ROS fluorescent probe detection result.

[0033] Figure 6 This is the immunofluorescence image of the stem cell preparation in Example 1.

[0034] Figure 7 These are fluorescence images of the drug-loaded nanoparticles regulating myofibroblast dedifferentiation and inhibiting endothelial-mesenchymal transition in Example 2; A is the α-SMA labeling result of myofibroblasts, and B is the morphology of microvascular endothelial cells labeled with phalloidin.

[0035] Figure 8 Figure 3 shows the promotion of angiogenesis by stem cells; A is a bright field image of HUVEC tube formation, and B is a quantitative image

[0036] Fig. 9 This is the in vivo distribution diagram of the stem cell preparation in Example 4; A is an in vitro organ fluorescence photo, and B is a quantitative diagram.

[0037] Fig.10 The figure shows the therapeutic effect of the stem cell preparation in Example 5 on mice with pulmonary fibrosis; A is double staining of lung tissue blood vessels (CD31) and myofibroblasts (α-SMA), B is HE staining and Masson staining of lung tissue; C is the mouse survival rate curve; D is quantification of hydroxyproline. DETAILED DESCRIPTION

[0038] Example 1 Preparation and characterization of stem cell preparations

[0039] 1. Extraction of Adipose-Derived Mesenchymal Stem Cells

[0040] Adipose tissue comes from the fat around the organs of rats. The specific operation is as follows: the rats are anesthetized and killed, and then soaked in 75% ethanol for disinfection for 3-5 minutes. The abdomen is cut open under sterile conditions to separate the fat around the epididymis. After cutting, transfer to a 15ml centrifuge tube, add 3-4 times the volume of 0.1% type I collagenase solution, and digest in a 37℃ water bath for 15 minutes until it becomes a milky and thick liquid. After centrifugation at 1600rpm for 5 minutes at 4℃, the upper layer of fat is aspirated, 2ml PBS is added and blown evenly, and then passed through a cell sieve, and centrifuged again at 1600rpm for 5 minutes at 4℃ to collect the bottom cells. Add 1ml of red blood cell lysis solution, let it stand at room temperature for 3-5 minutes, add an equal amount of PBS to stop the lysis, and centrifuge at 1600rpm for 5 minutes. Collect the bottom cells, add 2ml PBS to wash again, aspirate the supernatant, add complete culture medium and transfer to the culture dish for continued culture. After the cells grow for 48 hours, change the medium and remove the cells that are not attached to the wall.

[0041] result:

[0042] like Figure 1 The cells shown exhibited a single layer, fusiform, or multi-branched cell morphology; Figure 2 Flow cytometry results showed that the cells highly expressed CD29 and CD90 and lowly expressed CD45 on their surface, and were identified as mesenchymal stem cells.

[0043] 2. Preparation and characterization of ROS-responsive nanoparticles

[0044] In the following abbreviations of the names of nanoformulations: LPHN is lipid polymer hybrid nanoparticles, including phospholipids, cholesterol, DSPE-PEG2000-MAL, and PPS-PEG; Lip is nanoparticles; MM is metformin and macitentan; MAC is macitentan; MET is metformin.

[0045] 1. Screening of MM@LPHN:

[0046] The phospholipids, cholesterol, PPS-PEG (purchased from Xi'an Qiyue) and macitentan required for different groups were weighed and dissolved in 1 ml of dichloromethane. 200 μl of water was added under the condition of ice bath by probe ultrasound to form an emulsion. The emulsion was dispersed in 1% PVA solution while stirring, and a double emulsion was formed by probe ultrasound for 5 min under the condition of ice bath. The organic reagent was removed by vacuum rotary evaporation to form nanoparticles. Free macitentan was removed by low-speed centrifugation at 3500 rpm for 15 min. The particle size and encapsulation efficiency of the nanoparticles were detected by DLS and HPLC, respectively.

[0047] Table 1. Formulation screening of ROS-responsive nanoparticles

[0048]

[0049] Results: As shown in Table 1 and Figure 3 As shown, when the ratio of phospholipid: cholesterol: DSPE-PEG in group 9 was 5:1:2, nanoparticles with an average particle size of 220.3 nm and a macitentan encapsulation efficiency of 97.03% were formed. Compared with other groups, the particle size was uniform and the encapsulation efficiency was the highest.

[0050] 2. Preparation of MM@LPHN:

[0051] Weigh 10 mg of phospholipids, 2 mg of cholesterol, 5 mg of PPS-PEG, 4 mg of DSPE-PEG-MAL, and 2 mg of macitentan and dissolve them in 1 ml of dichloromethane. Then weigh the water-soluble drug metformin and dissolve it in 200 μl of water. After mixing the two, ultrasonic probe was used for 5 minutes under ice bath to form an emulsion. The emulsion was dispersed in 1% PVA solution while stirring, and ultrasonic probe was used for 5 minutes under ice bath to form a double emulsion. The organic reagent was removed by vacuum rotary evaporation to form nanoparticles. The unencapsulated drug was removed by G50 dextran gel column to obtain drug-loaded nanoparticles.

[0052] 3. Preparation of A6-MM@LPHN:

[0053] Weigh 2 mg of A6 connecting peptide, dissolve it in a small amount of PBS, add it to the nanoparticles in step 1, and stir at 3°C ​​for 6-8 hours to obtain A6 modified nanoparticles. The particle size and polydispersity index of the nanoparticles were detected by DLS.

[0054] result:

[0055] Figure 4 A The results show that the dynamic light scattering particle size of A6-MM@LPHN is 259.20±1.92nm, and the transmission electron microscopy results show that the particles are spherical with uniform size. The PDI is 0.21, with uniform size and good dispersion. Figure 4 B is the particle size change of the nanoparticles in PBS containing 10% serum for 7 days. The results show that it can exist stably in the serum solution for 7 days, indicating that MM@LPHN still has good stability under physiological conditions.

[0056] 4. Evaluation of the reactive oxygen species reaction performance of ROS-responsive nanoparticles

[0057] The dialysis method was used to investigate the in vitro responsive release performance of drug-loaded nanoparticles in the presence of hydrogen peroxide. 1 ml of drug-loaded nanoparticles MM@LPHN was placed in a dialysis bag with a molecular weight cutoff of 3500, and then the dialysis bag was placed in a blank release medium and a release medium containing 100 mM hydrogen peroxide. The release medium was PBS containing 0.5% Tween 80, and then incubated in a constant temperature oscillating box (37°C, 60 rpm). 1 ml was sampled from the release medium at a pre-set time point (0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 24h), and an equal volume of blank release medium was added at the same time. The established HPLC method was used to detect the contents of macitentan and metformin, respectively, and the cumulative release rate was calculated.

[0058] result:

[0059] Figure 5 A release curve shows that the drug-loaded nanoparticles can quickly and completely release drugs in a hydrogen peroxide environment. Compared with the blank release medium, when hydrogen peroxide exists in the release medium, the release rate of MET and MAC in 0-10h is much higher than that of the control group, and the drug is almost completely released at 24h, while the release of the blank group does not exceed 50%, indicating that MM@LPHN has a strong reaction ability with reactive oxygen species. In the pathological microenvironment enriched with ROS, the nanoparticles can effectively remove reactive oxygen species and promote drug release.

[0060] 5. Evaluation of ROS scavenging ability of polymeric nanoparticles

[0061] Fluorescent probe DCFH-DA was used to detect the ability of drug-loaded nanoparticles to scavenge reactive oxygen species in stem cells. Nanoparticles Lip without PPS-PEG and nanoparticles LPHN containing PPS-PEG were prepared respectively. Mesenchymal stem cells were inoculated in cell culture plates, induced by adding 400μM hydrogen peroxide (H2O2), and PBS, Lip, and LPHN were added for co-incubation for 24h, and then the fluorescent probe (10μM) was added, incubated at 37℃ for 20min, and the cells were washed three times with serum-free cell culture medium to remove DCFH-DA that did not enter the cells. The content of reactive oxygen species in the cells was observed under a fluorescence microscope at a wavelength of 488nm.

[0062] result:

[0063] Figure 5 B ROS fluorescence probe diagram shows that LPHN containing PPS-PEG has good ROS scavenging effect and MSC protection. Compared with the control group, the green fluorescence of the LPHN group was significantly reduced, indicating that LPHN nanoparticles containing PPS-PEG can effectively reduce the level of reactive oxygen species and have the effect of scavenging ROS and protecting mesenchymal stem cells.

[0064] 3. Preparation and Characterization of Stem Cell Preparations

[0065] 1. Preparation of DiO@LPHN

[0066] Preparation of nanoparticles loaded with DiO (DiO@LPHN): 10 mg of phospholipids, 2 mg of cholesterol, 5 mg of PPS-PEG, 4 mg of DSPE-PEG-MAL, and 500 μg of DIO were weighed and dissolved in 1 ml of dichloromethane, added to 200 ml of saline, and ultrasonicated for 5 min under ice bath conditions to form an emulsion, which was dispersed in 1% PVA solution while stirring, and ultrasonicated for 5 min under ice bath conditions to form a double emulsion. The organic reagent was removed by vacuum rotary evaporation to form nanoparticles. Unencapsulated DiO was removed by a dextran gel column.

[0067] 2. Preparation and characterization of MSC-DiD@LPHN

[0068] Weigh 2 mg of A6 connecting peptide, dissolve it in a small amount of PBS, and stir at 4°C for 6-8 h to obtain the modified nanoparticles A6-DiO@LPHN.

[0069] Mesenchymal stem cells were cultured in a T25 flask, digested with a small amount of trypsin, and centrifuged for 5 minutes (800 rpm) to collect the cells. The cells were dispersed in a culture medium containing DiI (5 μM) and stained for 30 minutes to stain the cell membrane of the mesenchymal stem cells with DiI. The free DiI was removed by washing with PBS. Nanoparticles connected with A6 peptide were added and incubated at 37°C for 2 hours to obtain the stem cell preparation MSC-DiD@LPHN.

[0070] result:

[0071] Figure 6 The fluorescence image shows that the DiO@LPHN group without A6 peptide only shows red fluorescence of stem cells, and the green fluorescence preparation fails to co-localize with the red stem cell fluorescence, indicating that the preparation without A6 peptide cannot connect to the stem cell surface. The A6-modified nanoparticles clearly co-localize with the cell membrane of mesenchymal stem cells, and the red fluorescence overlaps with the green fluorescence to form orange fluorescence, indicating that the nanoparticles are effectively connected to the surface of the mesenchymal cell membrane through the A6 peptide.

[0072] Example 2 Drug-loaded nanoparticles regulate myofibroblast dedifferentiation and inhibit endothelial-mesenchymal transition

[0073] 1. MM@LPHN regulates myofibroblast dedifferentiation

[0074] Mouse fibroblasts were cultured in a cell culture dish. When the confluence reached 70-80%, trypsin was added for digestion. The cells were collected by centrifugation and inoculated in a confocal dish (5×10 5cells / well), TGF (10 ng / ml) was added to stimulate for 48 hours to transform them into myofibroblasts, and the nanoformulations MM@LPHN, MAC and MET prepared according to Example 1 were added. The activation degree of myofibroblasts was characterized by immunofluorescence staining of α-SMA;

[0075] result:

[0076] Figure 7 A shows that MM@LPHN can effectively induce myofibroblast dedifferentiation. Compared with the free drug PFD, MAC, and MET groups, the green fluorescence of the MM@LPHN group was significantly reduced, indicating that the number of activated myofibroblasts was significantly reduced, dedifferentiation occurred and returned to a normal state, effectively inhibiting the aggravation of fibrosis.

[0077] 2. Inhibit endothelial-mesenchymal transition

[0078] Mouse pulmonary microvascular endothelial cells (PMVECs) were cultured in a cell culture dish. When the confluence reached 70-80%, trypsin was added for digestion. The cells were collected by centrifugation and inoculated in a confocal dish (5×10 5 cells / well), TGF (10 ng / ml) was added to stimulate for 72 hours to induce endothelial-mesenchymal transformation, and the nanoformulations MM@LPHN, MAC and MET prepared according to Example 1 were added. The endothelial-mesenchymal transformation process was characterized by immunofluorescence staining of α-SMA and cytoskeletal proteins;

[0079] result:

[0080] Figure 7 B shows that MM@LPHN can effectively inhibit the endothelial-mesenchymal transition of PMVEC. Compared with the free drug MAC and MET groups, the MM@LPHN group can significantly improve the morphological changes of endothelial cells induced by TGF-β. The endothelial cells in the induced group are long spindle-shaped, similar to the morphology of mesenchymal cells, while the MM@LPHN group presents a cobblestone-like cell morphology similar to that of the normal group. This shows that the MM@LPHN group can effectively inhibit the endothelial-mesenchymal transition induced by TGF-β.

[0081] Example 3 Stem cells promote angiogenesis

[0082] After freezing and thawing, centrifuge at 4°C for several minutes and operate on ice. Precool the 24-well plate and 1ml pipette tip for 30 minutes, take 90μL of matrix gel and add it to the 96-well plate, and place it in the cell culture incubator for 60 minutes to solidify. Culture human umbilical vein endothelial cells (HUVEC) in a cell culture dish. After trypsin digestion of cells, add serum-containing culture medium to terminate digestion. After counting, resuspend the cells in culture medium and adjust the cell density to 2×10 4After the gel solidifies, take out the 96-well plate, add 200 μl of cell suspension to each well, add stem cell supernatant, take pictures at the set time, and observe the tube-forming morphology of endothelial cells.

[0083] result:

[0084] Figure 8 It shows that stem cell secretions can effectively promote angiogenesis. Figure 8 A shows that the tube length and branch number of the stem cell supernatant group at 6 hours after adding the stem cell supernatant were significantly higher than those of the control group. When the tube was completely formed at 12 hours, the tube length and branch number of the stem cell supernatant group were still significantly higher than those of the control group, indicating that the stem cell supernatant has the effect of promoting HUVEC tube formation.

[0085] Example 4 Distribution of stem cell preparations in vivo

[0086] 1. Preparation of DiR@LPHN

[0087] Weigh 10 mg of phospholipids, 2 mg of cholesterol, 4.5 mg of PPS-PEG, 2 mg of DSPE-PEG-MAL, and 1 ml of DIR, dissolve in 1 ml of dichloromethane, add to 200 μL of physiological saline, and form an emulsion by probe ultrasonication for 5 min under ice bath conditions. Disperse it in 1% PVA solution while stirring, and form a double emulsion by probe ultrasonication for 5 min under ice bath conditions. Remove the organic reagent by vacuum rotary evaporation to form nanoparticles. Remove the unencapsulated DiR by passing through a dextran gel column.

[0088] 2. Preparation of MSC-DiR@LPHN

[0089] Mesenchymal stem cells were cultured in a T25 flask, digested with a small amount of trypsin, and centrifuged for 5 min (800 rpm) to collect the cells. The cells were dispersed in 1 ml of PBS solution, A6-DiR@LPHN was added, and incubated at 37°C for 2 h to obtain MSC-DiR@LPHN.

[0090] 3. Distribution in vivo detection

[0091] First, 6-8 week old male C57BL / 6 mice were used to model pulmonary fibrosis. Bleomycin hydrochloride (2 USP / Kg) was injected into the mouse lungs by tracheal instillation, and a pulmonary fibrosis model was created 10 days later. MSC-DIR@LHPN, DiR@LPHN and DiR were injected through the tail vein at a dose of 50 μg / kg, with three mice in each group. After 24 hours, the mice were euthanized, and the heart, liver, spleen, lung, and kidney were dissected and arranged on black cardboard in sequence. The fluorescence images of each organ were collected using a small animal in vivo imager under the conditions of an excitation wavelength of 752 nm and an emission wavelength of 779 nm.

[0092] result:

[0093] like Fig. 9 A and B show that stem cell preparations can be effectively enriched in lung tissue. 24 hours after administration, DiR, DiR@LPHN, and MSC-DiR@LPHN were mainly enriched in the liver. In addition to the liver, they were mainly concentrated in the lungs. In addition, the final preparation group MSC-DiR@LPHN had the most lung accumulation at 24 hours, indicating that the final preparation group had good lung tropism.

[0094] Example 5 In vivo therapeutic effect of stem cell preparation

[0095] MSC-MM@LPHN and MM@LPHN were prepared by the method described in Example 1;

[0096] Male C57BL / 6 mice aged 6 to 8 weeks were used to model pulmonary fibrosis, and a pulmonary fibrosis model with vascular loss was induced by multiple stimulation of the bleomycin model. Bleomycin hydrochloride (1 USP / Kg) was injected into the mouse lungs by tracheal instillation once a week for three weeks. There were 6 groups of mice, with 7 mice in each group. After 15 days, treatment was performed by tail vein injection of MSC-MM@LHPN, mesenchymal stem cells, MM@LPHN and drugs. The non-modeled group served as a negative control, and the PBS group served as a positive group. After three weeks of treatment, lung tissues were stained and analyzed by HE and Masson staining to evaluate the therapeutic effect. At the same time, blood vessels were immunofluorescently stained to investigate the effect of vascular regeneration. At the same time, blood vessels and myofibroblasts were immunofluorescently stained to investigate the effect of vascular regeneration;

[0097] result:

[0098] The results are as follows Fig.10 As shown in A, the final preparation MSC-MM@LPHN group can effectively promote capillary formation, the expression of endothelial cell marker CD31 is significantly increased, and the expression of myofibroblast marker α-SMA is significantly reduced, promoting the formation and repair of normal vascular structure. Fig.10 B HE and Masson staining results showed that compared with the BLM group, the degree of pulmonary fibrosis in the treatment group was significantly weakened. Among them, the final preparation MSC-MM@LPHN group had the best effect, with clear alveolar structure and reduced collagen deposition. Fig.10 C The survival rate curve of mice in different groups showed that the survival rate of mice in the final preparation group was significantly increased; Fig.10 D-hydroxyproline quantification showed that collagen deposition in the final preparation group was significantly reduced compared with the BLM group, reflecting a good therapeutic effect.

Claims

1. A stem cell preparation, characterized in that: The invention comprises mesenchymal stem cells and ROS-responsive lipid polymer hybrid nanoparticles; wherein the ROS-responsive lipid polymer hybrid nanoparticles are modified on the surface of the stem cells.

2. The stem cell preparation according to claim 1, characterized in that The ROS lipid polymer hybrid nanoparticles are nanocarriers carrying drugs, and the nanocarriers include phospholipids, cholesterol, reducing polymers, DSPE-PEG2000-MAL, and A6 peptide; the amino acid sequence of A6 peptide is KPSSPPEECGPLGIAGQC, see SEQ ID NO.

1.

3. The stem cell preparation according to claim 1, characterized in that The mesenchymal stem cells are selected from adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells or skin mesenchymal stem cells.

4. The stem cell preparation according to claim 2, characterized in that The phospholipids are one or more of soybean lecithin, egg yolk lecithin, HSPC, DPPC, DOPC and DPPE.

5. The stem cell preparation according to claim 2, characterized in that The reducing polymer includes one or more of PPS-PEG, PEG-b-PBA, and PTE-PEG.

6. The stem cell preparation according to claim 2, characterized in that The drugs include drugs for protecting vascular endothelial cells and anti-fibrosis drugs.

7. The stem cell preparation according to claim 2, characterized in that The drug for protecting vascular endothelial cells: CYM-5442 or macitentan; Antifibrotic drugs are dihydrotestidine, prostaglandin E2, metformin, or bosentan.

8. The stem cell preparation according to claim 2, characterized in that The mass ratio of phospholipids to cholesterol is 5:1-10:1, the mass ratio of phospholipids to DSPE-mPEG2000-MAL is 8:1-10:1, the mass ratio of phospholipids to PPS-PEG is 2:1-5:1, the mass ratio of phospholipids to metformin is 5:1-10:1, and the mass ratio of phospholipids to macitentan is 10:1-15:

1.

9. The method for preparing a stem cell preparation according to claim 8, characterized in that: Follow the steps below to obtain: A. Preparation of ROS-responsive nanoparticles; B, A6 peptide was modified on the surface of nanoparticles to obtain nanoformulation; C. Incubate mesenchymal stem cells and nanoparticles together to obtain a stem cell preparation.

10. Use of the stem cell preparation according to any one of claims 1 to 8 in the preparation of a drug for treating pulmonary fibrosis.

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