Preparation method of L929 cell source exosome entrapped recombinant human cytochalasin 2 hybrid liposome nanoparticles

Abstract

The invention discloses a preparation method and application of RLN2 heterozygous liposome nanoparticles wrapped by L929 cell-derived exosomes, the nanoparticles are formed by carrying out ultrasonic co-incubation on the L929 cell-derived exosomes and RLN2, and the exosomes are extracted by adopting an ultracentrifugation method; the functionalized liposome is mainly composed of phospholipid, cholesterol and a fat-soluble active drug, and the preparation method is a film dispersion method. The preparation process is simple, the condition is mild, the cost is low, and the prepared nanoparticles have the advantages of high drug loading capacity, various drug loading types, biological targeting regulation, biological safety and the like. The nano-drug delivery platform can realize drug delivery, combined drug delivery and collaborative precise treatment, can realize targeted treatment of complex progressive diseases such as pulmonary fibrosis, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to a recombinant human cytochalasin 2 (RLN2) hybrid liposome nanoparticle encapsulated by L929 cell-derived exosomes, belonging to the technical field of biomedicine. Background Art

[0002] Pulmonary fibrosis is a chronic, progressive and even fatal interstitial lung disease with a five-year survival rate of 20%, second only to lung cancer (4% to 17%). In the past decade, the incidence of pulmonary fibrosis has increased at an annual rate of 11%. Existing therapeutic drugs can only delay the progression of pulmonary fibrosis to a certain extent, but cannot curb the structural damage to the lungs and have limited effect on prolonging the patient's survival. Therefore, it is crucial to enhance the therapeutic effect of anti-pulmonary fibrosis drugs.

[0003] The complex pathogenesis of pulmonary fibrosis involves multiple cell types, including fibroblasts activated by inflammatory factors, abnormally accumulated myofibroblasts, and macrophages that participate in the entire inflammatory process of pulmonary fibrosis. Among them, a type of "inflammatory" macrophages that highly express lymphocyte antigen 6C (Ly6C) hi ) has received extensive attention from researchers in recent years. In the process of pulmonary fibrosis, Ly6C hi After macrophages are highly recruited to the inflammatory site, they release inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin (IL-1β) and IL-13, as well as reactive oxygen species (ROS), which on the one hand aggravate the inflammatory response and on the other hand cause secondary damage to the lung tissue, causing the damage and inflammatory response to continue to develop. To repair the damage, Ly6C hi Macrophages then secrete large amounts of transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF), activating alveolar epithelial cells, pulmonary interstitial fibroblasts, and recruited circulating fibroblasts into myofibroblasts, and intensifying the cross-linking of collagen secreted by myofibroblasts, promoting the formation of excessive deposition of extracellular matrix (ECM), i.e., excessive tissue repair. hi Macrophage-promoted inflammatory response and excessive ECM deposition are key factors in promoting the continued development of pulmonary fibrosis.

[0004] Recombinant Human Relaxin 2 (RLN2) is an endogenous polypeptide hormone secreted by the human body, which can promote ECM degradation in various tissues and organs, inhibit fibroblast activation and myofibroblast contraction. hiRelaxin family peptide receptor-1 (RXFP1) is highly expressed on the surface of macrophages, which converts to Ly6C after binding to RLN2. lo Phenotype, reducing inflammatory response and promoting ECM degradation. Therefore, RLN2 is a promising candidate for anti-pulmonary fibrosis biotherapy. However, due to its poor in vitro stability, short blood circulation half-life, and lack of lung targeting, direct administration is difficult to achieve effective therapeutic effects.

[0005] Exosomes are a type of extracellular vesicles that are naturally released from all cells. They have a diameter of 40 to 160 nm (average ~100 nm). Due to their natural role in intracellular communication and biocompatibility, they have been used as a favorable delivery vehicle. Exosomes can specifically home to their source cells and undergo membrane fusion, so using fibroblast-derived exosomes as drug carriers can target drugs to the site of pulmonary fibrosis lesions. Liposomes are a type of multifunctional carrier used to encapsulate hydrophilic and hydrophobic drugs. They are widely used as drug carrier materials because of their biocompatibility and biodegradability.

[0006] The present invention encapsulates RLN2 in exosomes derived from L929 cells through exosome hybrid liposome technology, and then hybridizes the drug-loaded liposomes to prepare nanoparticles, which effectively improves the in vitro stability and in vivo targeting of RLN2 and enhances the therapeutic effect on pulmonary fibrosis. Summary of the invention

[0007] In view of the defects of the existing technology, the exosome-liposome nano-targeted drug delivery system is constructed by co-constructing exosomes derived from lung fibroblasts and liposomes, which has the function of targeting to the lungs and increasing the amount of drugs entering the lungs. Hybridization of liposomes and exosomes can also improve the stability of the drug delivery system, prolong its blood circulation half-life, and increase its accumulation in the lungs, thereby improving the therapeutic effect on pulmonary fibrosis.

[0008] The purpose of the present invention is to utilize the homing effect of exosomes derived from lung fibroblasts to construct an exosome liposome nano-targeted drug delivery system to increase the accumulation of drugs in fibrotic lungs. The drug delivery system contains RLN2, a drug that regulates the phenotype of macrophages, and Nintedanib (NIN), an anti-fibrotic drug, which can transform the phenotype of macrophages to inhibit inflammation and inhibit the activation and proliferation of fibroblasts, while degrading ECM. The nano-targeted drug delivery system is implemented by the following technical solutions:

[0009] A method for preparing L929 cell-derived exosome-encapsulated RLN2 hybrid liposome nanoparticles, characterized in that the exosomes are extracted by ultracentrifugation, the exosome-encapsulated RLN2 is prepared by ultrasonic co-incubation, and the drug-loaded liposomes are mainly composed of phospholipids, cholesterol and fat-soluble active drugs. The method comprises the following steps:

[0010] 1) preparing an organic solution A containing phospholipid, cholesterol (Chol), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) and a fat-soluble active drug: when the mass of the phospholipid is 20.00 mg, the mass of the organic solvent is 5 mL, and the solid mass ratio is phospholipid: Chol: DSPE-PEG: fat-soluble active drug = (15-20): (1-2): (5-10): (1-5);

[0011] 2) removing the organic solvent from the organic solution A by rotary evaporation, hydrating with PBS, ultrasonically disrupting, and passing through a membrane to obtain drug-loaded liposomes;

[0012] 3) The exosomes from L929 cells were extracted by ultracentrifugation, and the extracted L929 cell exosomes were mixed with equal volumes of RLN2, and the mixture was subjected to ultrasonic treatment for 10 min, and then allowed to stand at room temperature for 30 min to obtain exosomes loaded with RLN2;

[0013] 4) mixing the drug-loaded liposomes with the RLN2-loaded exosomes, wherein the drug-loaded liposomes are calculated based on the total amount of phospholipids, and the RLN2-loaded exosomes are calculated based on the protein content, and the mass ratio of the drug-loaded liposomes to the RLN2-loaded exosomes is (5-10):(1-2), and the drug-loaded liposomes and the RLN2-loaded exosomes are extruded 10-20 times using an extruder, and the hybrid liposome nanoparticles are obtained by collecting them;

[0014] Wherein, the phospholipid in the step 1) is dioleoylphosphatidylcholine (DOPC), and the fat-soluble active drug is nintedanib (NIN).

[0015] The organic solvent in step 1) is chloroform.

[0016] In the step 2), the time for rotary evaporation, hydration and ultrasound is 30 minutes, the time for ultrasonic crushing is 5 to 10 minutes, the temperature for ultrasound and hydration is 50° C., and the rotation speed is 300 to 100,000 g.

[0017] The RLN2 in step 3) is recombinant human cytochalasin 2, which is synthesized by Shanghai Xinyu Biotechnology Co., Ltd., and the protein sequence is:

[0018]

[0019] Advantages of the present invention:

[0020] (1) Strong endogenous property: Using cell-derived exosome hybrid liposomes as drug carriers makes the drug delivery system highly endogenous.

[0021] (2) Targeted drug delivery: Nanoparticles retain the specific protein receptors of exosomes and can bind to cells in the lesion site.

[0022] (3) High biosafety: The main component of liposomes is phospholipids, which are similar to the structure and composition of cell membranes; exosomes are extracted from cells, so both have good biocompatibility, are easily degraded in the body, and have low immunogenicity, so they have high biosafety.

[0023] (4) Multiple therapeutic mechanisms: By loading different drugs, the drug delivery system can treat different diseases.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] (1) The preparation method is simple.

[0026] (2) Improve biological targeting.

[0027] (3) Diseases can be treated through multiple pathways. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The expression of characteristic proteins on the surface of exosomes;

[0029] Figure 2A This is a transmission electron microscopy image of exosome hybrid liposome nanoparticles. Figure 2B is the size of the nanoparticles;

[0030] Figure 3A Fluorescence image of cellular uptake of exosome hybrid liposome nanoparticles. Figure 3B For fluorescence quantification;

[0031] Figure 4 The toxic effects of exosome hybrid liposome nanoparticles on cells;

[0032] Figure 5 To investigate the ability of exosome hybrid liposome nanoparticles to reverse the phenotype of macrophages. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further illustrated by the following embodiments, but the protection scope of the present invention is not limited thereto.

[0034] Example 1: Preparation of exosome hybrid liposome nanoparticles encapsulating RLN2 and NIN

[0035] 1.1 Preparation of liposomes (L-NIN) loaded with the anti-fibrotic drug nintedanib (NIN)

[0036] Take 20 mg of phospholipids, 1 mg of Chol, 5 mg of DSPE-PEG, and 1 mg of NIN powder, dissolve in 5 mL of chloroform, ultrasonicate for 1 min, and rotary evaporate at 50 ° C for 30 min to evaporate the organic solvent and form a film. Add 5 mL of PBS to hydrate for 30 min. Ultrasonicate in a water bath for 30 min, and probe ultrasonicate for 5 min (ice bath), and filter through a 0.22 μm filter membrane to obtain drug-loaded liposomes (L-NIN).

[0037] 1.2 Extraction and separation of exosomes

[0038] Extraction of exosome-free culture medium: 10% FBS (v / v) was added to the DMEM culture medium, and the serum exosomes were removed by ultracentrifugation at 100,000g and 4°C for 1 h. The supernatant was collected, filtered through a 0.22 μm sterile filter membrane, and 1% penicillin-streptomycin (v / v) was added to obtain a conditioned medium without serum exosomes.

[0039] Exosomes from fibroblasts were extracted by ultracentrifugation, and all centrifugations were performed at 4°C. The collected L929 culture supernatant was centrifuged at 300g for 10min, 2,000g for 20min to remove cells and cell debris, and the supernatant was collected; microvesicles were removed by centrifugation at 10,000g for 30min, and the supernatant was collected; and then centrifuged at 100,000g for 1h to obtain the precipitate, which was the exosome. The exosome precipitate was washed with sterile PBS, resuspended, and centrifuged again at 100,000g for 1h to obtain the purified exosome precipitate. The finally extracted exosomes were resuspended in 50-200μL of sterile PBS, filtered through a 0.22μm filter membrane, and stored at -80°C for later use.

[0040] 1.3 Preparation of exosome hybrid liposome nanoparticles

[0041] Exosome hybrid liposome nanoparticles were prepared using a liposome extruder, and the drug-loaded exosomes (E-RLN2) and drug-loaded liposomes (L-NIN) were repeatedly extruded to cause membrane fusion (EL-NIN-RLN2). The specific operation was to take 100 μg of E-RLN2 with an exosome protein content and 500 μg of L-NIN with a phospholipid content, and extrude them through a 200 nm polycarbonate membrane 10 times to obtain EL-NIN-RLN2.

[0042] Example 2: Investigation of the properties of exosome hybrid liposome nanoparticles

[0043] 1.1 Expression of characteristic proteins on the surface of exosomes

[0044] Protein concentration in exosomes: The BCA method was used to determine the protein concentration in the sample. The exosome solution was mixed with an equal volume of RIPA lysis solution, lysed on ice for 15 minutes, 10 μL of the mixture was added to a 96-well plate, 10 μL of PBS solution was added, and 200 μL of BCA working solution was added. The sample was allowed to stand at 37°C for 30 minutes, and the absorbance was measured at a wavelength of 562 nm. The protein concentration in the sample was calculated based on the standard curve. The mixed solution of exosomes and RIPA was stored at -20°C before use.

[0045] Detection of exosome marker proteins CD9 and HSP70: Immunoblotting was used to detect marker proteins contained in exosomes. Figure 1 As shown, CD9, HSP70 and β-actin were all positively expressed, proving the expression of surface marker proteins of fibroblast-derived exosomes.

[0046] 1.2 Morphology of exosome hybrid liposome nanoparticles

[0047] The morphology of exosome hybrid liposome nanoparticles was observed using a transmission electron microscope (TEM). The diluted exosome and exosome hybrid liposome nanoparticle solutions (10 μL) were dropped onto a copper grid and allowed to stand at room temperature for 20 min. After the droplets were removed, phosphotungstic acid (1%) solution was added for staining. After 20 s, the liquid was quickly dried and set aside. The morphology was observed using a TEM at a voltage of 120 kV and photographed and recorded.

[0048] The particle size, potential and PDI (polydispersity index) of exosomes and nanoparticles were measured using a Malvern particle size analyzer.

[0049] The results are shown in Figure 2. Transmission electron microscopy analysis showed that the nanoparticles were round, had a distinct lipid layer, a particle size of ∼100 nm, a zeta potential of -3.8 mV, and a PDI of 0.22.

[0050] 1.3 Cellular uptake of exosome hybrid liposome nanoparticles

[0051] Fluorescent liposomes (C6-L) containing coumarin 6 (C6) were prepared, and then exosome hybrid liposome nanoparticles (C6-EL) were prepared by extrusion method.

[0052] Take logarithmically growing L929 cells and use 1×10 5 Cells were inoculated in a 24-well plate at a density of 100 cells and cultured in a 37°C, 5% CO2 incubator until the cells adhered to the wall. After 24 hours, the culture medium was discarded, and the cells were washed twice with pre-cooled PBS. Then, 500 μL of each group of preparations (C6: 1 μg / mL) diluted with FBS-free culture medium was added and cultured for 0.5 hours and 4 hours. After washing with pre-cooled PBS, 4% paraformaldehyde was added for fixation for 10 minutes, and then photographed for observation.

[0053] As shown in Figure 3, the green fluorescence signals of L and EL in L929 cells at 4 h were greater than those at 0.5 h in the same group, indicating that the cell uptake of nanoparticles was time-dependent. Compared with the L group, the green fluorescence signal in the EL group had a higher intensity in L929 cells, indicating that under the homing effect of exosomes, L929 cells were able to take in more exosome hybrid liposome nanoparticles.

[0054] 1.4 Cytotoxicity of exosome hybrid liposome nanoparticles

[0055] 1.4.1 Preparation of EL-NIN-RLN2

[0056] Take 20.0 mg of phospholipids, 1.0 mg of Chol, 5.0 mg of DSPE-PEG, and 1.0 mg of NIN powder and dissolve them in 5 mL of chloroform, ultrasonicate for 5 min, and rotary evaporate at 50 ° C for 30 min to remove the organic solvent and form a film. Add 5 mL of PBS to hydrate for 30 min. Ultrasonicate in a water bath for 30 min, ultrasonicate with a probe for 5 min (ice bath), and filter through a 0.22 μm filter to obtain drug-loaded liposomes (L-NIN). Mix the extracted exosome solution with the RLN2 solution (protein mass ratio is 1:1), ultrasonicate for 10 min (ice bath), and then stand at room temperature for 1.5 h to obtain drug-loaded exosomes (E-RLN2), which are extruded with L-NIN to obtain EL-NIN-RLN2.

[0057] 1.4.2 Cytotoxicity

[0058] The prepared E, EL, EL-NIN, EL-RLN2 and EL-NIN-RLN2 were taken for gradient dilution.

[0059] Take logarithmically growing L929 fibroblasts, 1×10 4 The cells were inoculated in a 96-well plate at a density of 100 μL and cultured at 37°C and 5% CO2 until the cells adhered to the wall. When the cells grew to about 80%, the supernatant was discarded, and 100 μL of a series of gradient concentrations of PBS, E, EL, EL-NIN, EL-RLN2 and EL-NIN-RLN2 prepared with culture medium were added to each well. Three replicate wells were set in parallel for each concentration, and the cells were incubated for 48 hours. The culture medium was discarded, and 10% (v / v) cck8 solution was added to each well. The cells were placed in a cell culture incubator for 4 hours to react and the culture was terminated. The absorbance (OD value) of each well was measured at a wavelength of 450 nm using a plate reader.

[0060] 1.5 Exosome hybrid liposome nanoparticles reverse macrophage phenotype

[0061] 1.5.1 Sample preparation

[0062] L929 cells were cultured in 12-well plates. When the cell growth reached 60% to 70%, the old culture medium was aspirated and 100 ng / mL lipopolysaccharide was used to induce macrophages into inflammatory Ly6C hi Macrophages were then added with 50 μg / mL of EL-NIN-RLN2, EL-NIN, and 100 ng / mL of EL-RLN2 and incubated for 24 h.

[0063] Aspirate the liquid into a 1.5 mL centrifuge tube. Wash the adherent cells once with PBS and add an appropriate amount of trypsin cell digestion solution to digest the cells.

[0064] Add the collected liquid, mix briefly, transfer to a centrifuge tube, centrifuge at 1,000 g for 5 min, discard the supernatant, collect the cells, gently resuspend the cells with less than 0.5 mL of PBS, count the cells, and dilute to 1×10 6 / mL.

[0065] 1.5.2 Antibody incubation

[0066] According to the experimental design, except for the blank control, 5 μL of the corresponding antibody was added to the corresponding single-stained tube and full-stained tube, mixed, and incubated at 4°C (ice bath) in the dark for 30 minutes. 2 mL of Cell Staining Buffer was added to each tube. Then centrifuged at 600g for 5 minutes and the supernatant was discarded.

[0067] 1.5.3 On-machine testing

[0068] Add 200 μL Cell Staining Buffer and resuspend the cells. Adjust the instrument parameters and perform the test. Test results: The cells treated with EL-RLN2 and EL-NIN-RLN2 groups significantly expressed Ly6C lo The characteristic antigen of macrophages, while the cells treated with EL-NIN group still highly expressed Ly6C hi characteristic antigen of macrophages, indicating that RLN2 plays a role in converting Ly6C hi Phenotypic conversion to Ly6C lo The role of phenotype.

Claims

1. A method for preparing RLN2 hybrid liposome nanoparticles encapsulated by L929 cell-derived exosomes, characterized in that: The exosomes are extracted by ultracentrifugation, the exosome-encapsulated RLN2 is prepared by ultrasonic co-incubation, and the drug-loaded liposomes are mainly composed of phospholipids, cholesterol and fat-soluble active drugs. The method includes the following steps: 1) preparing an organic solution A containing phospholipid, cholesterol Chol, distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) and a fat-soluble active drug: when the mass of the phospholipid is 20.00 mg, the mass of the organic solvent is 5 mL, and the solid mass ratio is phospholipid: Chol: DSPE-PEG: fat-soluble active drug = (15-20): (1-2): (5-10): (1-5); 2) removing the organic solvent from the organic solution A by rotary evaporation, hydrating with PBS, ultrasonically disrupting, and passing through a membrane to obtain drug-loaded liposomes; 3) The exosomes from L929 cells were extracted by ultracentrifugation, and the extracted L929 cell exosomes were mixed with equal volumes of RLN2, and the mixture was subjected to ultrasonic treatment for 10 min, and then allowed to stand at room temperature for 30 min to obtain exosomes loaded with RLN2; 4) mixing the drug-loaded liposomes with the RLN2-loaded exosomes, wherein the drug-loaded liposomes are calculated based on the total amount of phospholipids, and the RLN2-loaded exosomes are calculated based on the protein content, and the mass ratio of the drug-loaded liposomes to the RLN2-loaded exosomes is (5-10):(1-2), and using an extruder to extrude the drug-loaded liposomes and the RLN2-loaded exosomes 10-20 times, and collecting to obtain the hybrid liposome nanoparticles; Wherein, the phospholipid in the step 1) is dioleic acid phospholipid, and the fat-soluble active drug is nintedanib.

2. The RLN2-loaded hybrid liposome nanoparticles according to claim 1, characterized in that: The organic solvent in step 1) is chloroform.

3. The RLN2-loaded hybrid liposome nanoparticles according to claim 1, characterized in that: In the step 2), the time for rotary evaporation, hydration and ultrasound is 30 minutes, the time for ultrasonic crushing is 5 to 10 minutes, the temperature for ultrasound and hydration is 50° C., and the rotation speed is 300 to 100,000 g.

4. The RLN2-loaded hybrid liposome nanoparticles according to claim 1, characterized in that: The RLN2 in step 3) is recombinant human cytochalasin 2, which is synthesized by Shanghai Xinyu Biotechnology Co., Ltd., and the protein sequence is:

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