CN116672365A - Preparation method and application of fat source biological nano particles - Google Patents
Preparation method and application of fat source biological nano particles Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention relates to the technical field of biological nano-particle preparation, in particular to a preparation method and application of fat-source biological nano-particles. The invention provides a method for preparing subcutaneous fat-derived Lipo-NPs by using tangential flow filtration, wherein the Lipo-NPs prepared by the method have good activity of protecting ARDS early pulmonary fibrosis and can be used for treating or preventing acute respiratory distress syndrome. Experiments show that after the Lipo-NPs prepared by the invention are administered, the pulmonary fibrosis degree is lower, the TGF-beta content in BALF is obviously reduced, the expression of E-cadherein and alpha-SMA is reversed, the EMT process is inhibited, and finally the LPS-induced early pulmonary fibrosis of mice is improved.
Description
Technical Field
The invention relates to the technical field of biological nano-particle preparation, in particular to a preparation method and application of fat-source biological nano-particles.
Background
Acute respiratory distress syndrome (acute respiratory distress syndrome, ARDS) is a life threatening clinical syndrome of the respiratory system. Is an acute diffuse lung injury caused by various pathogenic factors in and out of the lung, and further develops into acute respiratory failure. An important cause of poor ARDS prognosis is the occurrence of pulmonary fibrosis, a typical pathological change of which is sustained and irreversible epithelial cell injury, proliferation and aggregation of fibroblasts and myofibroblasts, accompanied by a large amount of collagen fiber deposition.
Treatment of acute respiratory distress syndrome includes both mechanical and non-mechanical ventilation therapy. Mechanical ventilation is the primary treatment for patients with acute respiratory distress syndrome. According to the different mechanical ventilation modes, the method can be divided into noninvasive ventilation and invasive ventilation, wherein the noninvasive ventilation is performed by a mask, the invasive ventilation is performed by an endotracheal tube or an tracheostomy tube, and the two modes are selected according to specific conditions to determine the time. The non-mechanical ventilation treatment means comprises: pulmonary water clearance and fluid management, alveolar surfactant supplementation therapy, beta receptor agonist application, statin application, glucocorticoid application, anticoagulant application, antioxidant and enzyme inhibitor application, blood purification treatment, nutritional intervention, and the like; effective treatments for these diseases are still continually being explored.
Exosomes are a special extracellular vesicle (extracellular vesicles, EVs) that, with extensive research on its biological origin, substance constitution and transport, intercellular signaling and distribution in body fluids, has been found to have a wide variety of functions. Studies have shown that exosomes function depends on the cell type from which they are derived, and can be involved in a variety of aspects such as immune responses in the body, antigen presentation, cell migration, cell differentiation, tumor invasion, etc. However, large-scale cell reproduction is required for obtaining the cell-derived exosomes, and the exosomes are high in cost, low in yield and difficult to store. Thus, it is necessary to obtain a greater amount of protective substances that are convenient and effective. Whereas non-cell derived EVs and other biological nanoparticles (biogenic nanoparticles, biNPs) are of little research as therapeutic agents. Further optimizing the preparation process of the biological nano particles is beneficial to further developing the application of the biological nano particles in medicine.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a method for preparing fat-derived biological nanoparticles (lipoaspirate nanoparticles, lipo-NPs) and application thereof in protecting pulmonary fibrosis.
The invention provides a preparation method of fat source biological nano particles, which comprises the following steps:
the subcutaneous fat is crushed and thinned, and then centrifuged, and the supernatant is filtered by tangential flow, and the fat source biological nano particles with the particle size of 50-nnm-650 nm are intercepted.
In some embodiments, the parameters of the centrifugation include: centrifuge at 800g for 30min at normal temperature.
In some embodiments, the tangential flow filtration is performed 2 times, wherein:
the pore diameter of the filter membrane for the first filtration is 0.65 μm, the filtrate is collected,
the second filtration has a membrane pore size of 500kd and the retentate is collected.
According to the invention, subcutaneous adipose tissue with better biological effect compared with visceral adipose tissue is selected, adipose tissue is thinned through nano converters with different apertures, subcutaneous adipose Lipo-NPs are extracted through a tangential flow filtration method (TFF), and identification of transmission electron microscopy, refrigeration electron microscopy, nanoparticle tracking analysis (Nanoparticle Tracking Analysis, NTA) and western blot biomarkers is performed. The result shows that the method provided by the invention is simple and easy to implement, the prepared Lipo-NPs are in a typical cup shape, the particle size is 152.6+/-67.6 nm, and markers CD63, CD81 and cytoplasmic protein Annexin V are enriched, but intracellular protein Calnexin is not present. In addition, the Lipo-NPs prepared by the method have better physiological activity, and compared with other processes (such as other processes except tangential flow filtration) or preparation parameters (such as other molecular weight cut-off and other centrifugal parameters), the Lipo-NPs prepared by the method can effectively play a role in protecting early stage fibrosis of ARDS lung.
Furthermore, the invention also provides the fat source biological nano-particles prepared by the preparation method.
Furthermore, the invention also provides application of the fat source biological nano-particles prepared by the preparation method in preparing medicines for preventing and/or improving acute respiratory distress syndrome.
The research of the invention finds that Lipo-NPs injected by tail vein has protective effect on early pulmonary fibrosis of ARDS mice, and mainly shows that:
1) Less lung fibrosis, reduced collagen fiber deposition was seen following Masson staining of early stage lung fibrosis mice lung tissue with Lipo-NPs intervention.
2) Transforming growth factor beta (transforming growth factor-beta, TGF-beta) plays an important role in the pulmonary fibrosis process, and it can transform normally differentiated lung tissue cells into mesenchymal cells, thereby promoting the formation of pulmonary fibrosis. The invention measures the TGF-beta content in bronchoalveolar lavage fluid (bronchoalveolar lavage fluid, BALF) by enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA), and the result shows that the TGF-beta content in BALF is obviously reduced after Lipo-NPs are dried along with the gradual increase of the TGF-beta level in BALF in the early stage of pulmonary fibrosis.
3) After repeated injury to lung tissue, normal proliferation is not possible, and Epithelial-to-mesenchymal transition (EMT) of repaired Epithelial cells is a fibroblast, which is an important mechanism for the development of pulmonary fibrosis after inflammatory injury. According to the research of the invention, ARDS early stage fibrosis mouse lung tissue section immunofluorescence staining shows that the epithelial cell marker E-cadherin is obviously reduced, the expression of the mesenchymal cell representative alpha-SMA is increased, and the intervention of Lipo-NPs can obviously weaken the effect, reverse the expression of E-cadherin and alpha-SMA, inhibit the EMT process and finally improve LPS-induced mouse lung fibrosis.
In the present invention, the control and/or improvement comprises: protecting early pulmonary fibrosis in patients with acute respiratory distress syndrome.
The protecting early pulmonary fibrosis in patients with acute respiratory distress syndrome includes: inhibit lung fibrotic hyperplasia, reduce lung fibrosis score, reduce pro-fibrotic factor levels, or inhibit EMT progression.
In some embodiments, the pro-fibrotic factor is TGF- β.
In some embodiments, inhibiting the EMT process comprises increasing the level of the epithelial cell marker E-cadherein and/or decreasing the level of the mesenchymal representative α -SMA.
Further, the invention also provides a medicament for preventing and/or improving acute respiratory distress syndrome, which comprises the fat-source biological nano-particles prepared by the preparation method.
The preparation form of the medicine is injection, wherein the density of the fat-source biological nano-particles is 1 multiplied by 10 6 And each 100 mu L. The injections were resuspended in PBS for Lipo-NPs.
The invention also provides a method of preventing and/or ameliorating acute respiratory distress syndrome comprising administering a medicament according to the invention.
In the present invention, the administration is injection, in particular intravenous injection.
In the present invention, the control includes treatment and prevention. The prevention comprises administering the agents of the invention prior to the occurrence of ARDS. The treatment comprises administration of the agents of the invention after the occurrence of ARDS.
The invention provides a method for preparing Lipo-NPs from subcutaneous fat by tangential flow filtration, which has good activity of protecting pulmonary fibrosis and can treat or prevent acute respiratory distress syndrome. Experiments show that after the Lipo-NPs prepared by the invention are administered, the pulmonary fibrosis degree is lower, the TGF-beta content in BALF is obviously reduced, the expression of E-cadherein and alpha-SMA is reversed, the EMT process is inhibited, and finally the LPS-induced early pulmonary fibrosis of mice is improved.
Drawings
FIG. 1 shows a method of extracting Lipo-NPs;
FIG. 2 shows a transmission electron microscope detection map of Lipo-NPs;
FIG. 3 shows a cryo-electron microscopy image of Lipo-NPs;
FIG. 4 shows NTA detection patterns of Lipo-NPs;
FIG. 5 shows the Western blot analysis results of Lipo-NPs;
FIG. 6 shows the results of Masson staining of mouse lung tissue;
FIG. 7 shows mouse lung fibrosis scores;
FIG. 8 shows the amount of TGF-beta secretion by each group of mice;
FIG. 9 shows an immunofluorescence of E-cadherin tissue from each group of mice;
FIG. 10 shows the immunofluorescence intensity statistics of E-cadherin tissue for each group of mice;
FIG. 11 shows immunofluorescence of mouse α -SMA tissue for each group;
figure 12 shows immunofluorescence intensity statistics for individual groups of mice α -SMA tissue.
Detailed Description
The invention provides a preparation method and application of fat source biological nano particles, and a person skilled in the art can properly improve the technological parameters by referring to the content of the fat source biological nano particles. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
example 1 extraction and characterization of fat-derived biological nanoparticles (Lipo-NPs)
1. Method for extracting Lipo-NPs (figure 1)
Step 1 taking subcutaneous fat of obese mice,
step 2 mechanical refinement of fat: after cutting the fat, the fat is thinned by a five-knife fat refiner, and then is thinned by a seven-hole fat refiner (the fat is thinned into milky white)
Step 3, centrifuging and collecting supernatant: centrifugation (800 g;30 minutes) to remove large debris
Step 4 tangential flow filtration (0.65 μm filter): tangential flow filtration using a 0.65 μm filter membrane, collecting filtrate (shown in FIG. 1 as red frame, clarified after filtration) to obtain <0.65 μm material,
step 5 tangential flow filtration (500 kd filter): and (3) tangential flow filtration is carried out on the filtered liquid by using a 500kD filter membrane, and the trapped liquid is collected to obtain substances with the particle size of more than 50nm, and finally the fat source biological nano particles with the particle size of 50 nnm-650 nm are obtained.
2. Identification of Lipo-NPs
Transmission electron microscope
The sample was dropped onto a copper mesh of a carbon support film and left for about 1min, and the filter paper was blotted off the excess liquid. Dropping uranium acetate on a carbon support film copper net, standing for about 1min, sucking excessive liquid by filter paper, and drying at room temperature. And (5) observing by a transmission electron microscope and collecting images.
(II) freezing electron microscope
Spreading the sample in the solution state on a copper mesh, sucking the redundant sample by filter paper, putting the copper mesh into liquid ethane for quick freezing, observing the sample at a low temperature by using a transmission electron microscope, and collecting an image. Nanoparticle tracking analysis (nanoparticle tracking analysis, NTA)
The extracted samples were resuspended in approximately 800 μl cold PBS and the size and distribution of the samples to be tested were measured using the Malvern NTA system.
(IV) Western blot experiments (western blot, WB)
Extracting total protein of lung tissue, measuring protein concentration, fully denaturing protein, preparing SDS-PAGE gel, electrophoresis, transferring film, sealing, incubating primary antibody, incubating secondary antibody, developing and analyzing.
(V) animal experiment
SPF-grade C57BL/6 mice, male, 8-9 weeks old, were adaptively fed for 1 week and then divided into a normal control group, an ARDS early stage fibrosis group and an ARDS early stage fibrosis+lipo-NPs group according to a random number table method, each group comprising 5 animals. The three times of compound striking of LPS can successfully establish an early-stage pulmonary fibrosis animal model after ARDS, and the first time of LPS (3 mg/kg) tracheal injection, the intraperitoneal injection (1.5 mg/kg) after 48 hours and the intratracheal injection (3 mg/kg) after 72 hours. Pulmonary fibrosis occurred early (day 3). 100 μl of Lipo-NPs (1×10≡6 particles) were injected tail vein-wise daily the day before the first LPS administration, and the ARDS early stage pulmonary fibrosis group mice were injected tail vein-wise with an equal amount of sterile PBS; normal control mice were injected with an equivalent amount of sterile PBS by tail vein.
(six) Masson staining
Paraformaldehyde fixes the lung tissue of the mice and is embedded in paraffin; dewaxing paraffin sections to water; sequentially washing with tap water and distilled water; the nuclei are stained with Regaud hematoxylin dye for 5-10min; washing with distilled water; masson ponceau acid reddening solution for 5-10min; immersing and washing with 2% glacial acetic acid aqueous solution; differentiating the 1% phosphomolybdic acid aqueous solution for 3-5min; aniline blue dyeing for 5min; immersing and washing with 0.2% glacial acetic acid aqueous solution; 95% alcohol, absolute alcohol, xylene transparent, neutral gum sealing. Microscopic examination and image acquisition.
(seventh) ELISA determination of TGF-beta content in BALF
Mouse BALF supernatants were collected and TGF- β levels in BALF supernatants were determined using ELISA kits. A blank hole, a standard hole and a sample hole to be tested are arranged on the enzyme-labeled coated plate; after membrane sealing by a sealing plate, incubating for 30min at 37 ℃; removing the sealing plate film, discarding the liquid, spin-drying, filling each hole with the washing liquid, discarding after 30s, repeating for 5 times, and beating to dry; adding 50 mu L of enzyme-labeled reagent into each hole except the blank hole; after membrane sealing by a sealing plate, incubating for 30min at 37 ℃; removing the sealing plate film, discarding the liquid, spin-drying, filling each hole with the washing liquid, discarding after 30s, repeating for 5 times, and beating to dry; adding 50 mu L of a color developing agent A and 50 mu L of a color developing agent B into each hole, slightly vibrating and uniformly mixing, and carrying out light-shielding reaction for 15 minutes at 37 ℃; adding 50 mu L of stop solution into each hole to stop the reaction; OD values for each well were measured with a blank Kong Diaoling, 450nm wavelength.
Eight E-cadherin and alpha-SMA tissue immunofluorescence staining
After the animal experiment is finished, the lung tissue is taken and fixed, paraffin embedding and slicing are carried out, paraffin dissolution, dewaxing, gradient hydration, endogenous peroxidase blocking, antigen retrieval, blocking, primary E-cadherin and alpha-SMA antibody incubation overnight, PBS (phosphate buffered saline) cleaning for 3 times, secondary antibody incubation, DAPI (DAPI) nuclear staining, sealing, microscopic observation and image acquisition are carried out.
3. Experimental results
3.1 The identification of Lipo-NPs is shown in figure 2, which shows typical cuplike results, similar to extracellular vesicles, by transmission electron microscopy.
3.2 the detection result of the freeze electron microscope is shown in figure 3,
3.3NTA detection results show that the particle size of Lipo-NPs is 152.6+ -67.6 nm (FIG. 4).
3.4 Western Blot results are shown in FIG. 5, with extracellular vesicle enrichment markers CD63, CD81, cytoplasmic protein Annexin V present in Lipo-NPs, but no intracellular protein Calnexin.
3.5, FIGS. 6-7 show the protective effect of Lipo-NPs on early pulmonary fibrosis in ARDS mice, wherein Masson staining results show (FIG. 6) that the lung tissue of mice in the control group remained essentially normal, with no fibrous tissue proliferation; the lung tissue of the ARDS group mice was seen as significant fibrous tissue hyperplasia; and fibrous tissue proliferation was reduced compared to the ARDS group following Lipo-NPs intervention. The pulmonary fibrosis scores showed (fig. 7) that the ARDS group score was significantly higher than the Control group, whereas the scores were significantly lower after Lipo-NPs intervention, and the differences were all statistically significant. * Representing P < 0.05.
3.6, after Lipo-NPs intervention, the secretion of TGF-beta in the BALF of ARDS mice is obviously inhibited, and the result is shown in figure 8, compared with the Control group, the secretion of the TGF-beta of the pro-fibrosis factor in the BALF of the ARDS group mice is obviously increased (P is less than 0.05); the Lipo-NPs intervention group significantly reduced the secretion of TGF-beta, a pro-fibrotic factor (P < 0.05), compared to the ARDS group.
3.7, tissue immunofluorescence results showed (FIGS. 9-12), that the intervention of Lipo-NPs reversed EMT. The ARDS group immunofluorescence staining is obviously reduced compared with the Control group epithelial cell marker E-cadherin, the expression of the mesenchymal cell representative alpha-SMA is obviously increased, and the intervention of Lipo-NPs can obviously weaken the effect, reverse the expression of the E-cadherin and the alpha-SMA, inhibit the EMT process and finally improve the pulmonary fibrosis of the ARDS mice. * Representing P < 0.05.
4. Conclusion(s)
Lipo-NPs extracted by tangential flow filtration have protective effects on mice with early ARDS pulmonary fibrosis, and are mainly characterized by lower pulmonary fibrosis degree, remarkably reduced TGF-beta content in BALF, reversed E-cadherin and alpha-SMA expression, inhibited EMT process, and finally improved LPS-induced pulmonary fibrosis of mice.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. The preparation method of the fat source biological nano-particles is characterized by comprising the following steps:
the subcutaneous fat is crushed and thinned, and then centrifuged, and the supernatant is filtered by tangential flow, and the fat source biological nano particles with the particle size of 50-nnm-650 nm are intercepted.
2. The method of claim 1, wherein the parameters of centrifugation comprise: centrifuge at 800g for 30min at normal temperature.
3. The method of claim 1, wherein the tangential flow filtration is performed 2 times, wherein:
the pore diameter of the filter membrane for the first filtration is 0.65 μm, the filtrate is collected,
the second filtration has a membrane pore size of 500kd and the retentate is collected.
4. A fat-derived biological nanoparticle produced by the production method according to any one of claims 1 to 3.
5. Use of the fat-derived biological nanoparticle prepared by the method of any one of claims 1 to 3 in the preparation of a medicament for the prevention and/or amelioration of acute respiratory distress syndrome.
6. The use according to claim 5, wherein the controlling and/or improving comprises: protecting early pulmonary fibrosis in patients with acute respiratory distress syndrome.
7. The use of claim 6, wherein the protecting the lung fibrosis in the patient with acute respiratory distress syndrome comprises: inhibit lung fibrous tissue proliferation, reduce lung fibrosis score, reduce levels of pro-fibrotic factors, and/or inhibit EMT progression.
8. The use according to claim 7, wherein,
the pro-fibrotic factor is TGF-beta;
the inhibiting of the EMT process comprises increasing the level of the epithelial cell marker E-cadherein and/or decreasing the level of the mesenchymal representative α -SMA.
9. A medicament for the prevention and/or amelioration of acute respiratory distress syndrome comprising the fat-derived biological nanoparticle prepared by the method of any one of claims 1 to 3.
10. The medicament according to claim 9, wherein the dosage form is an injection, wherein the fat-derived bio-nanoparticles have a density of 1 x 10 6 And each 100 mu L.
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