CN117180443B - Application of cell membrane of synovial myofibroblast in preparation of osteoarthritis medicine - Google Patents
Application of cell membrane of synovial myofibroblast in preparation of osteoarthritis medicine Download PDFInfo
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- CN117180443B CN117180443B CN202311376131.5A CN202311376131A CN117180443B CN 117180443 B CN117180443 B CN 117180443B CN 202311376131 A CN202311376131 A CN 202311376131A CN 117180443 B CN117180443 B CN 117180443B
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Abstract
The invention discloses an application of cell membranes of synovial myofibroblasts in preparing an osteoarthritis drug; the invention also discloses a medicine for treating osteoarthritis and a preparation method thereof. The medicine prepared from the cell membrane of the synovial myofibroblasts has the targeting effect of the synovial myofibroblasts, can maintain the long-time effect in the joint cavity, and has a better treatment effect on relieving OA inflammation. The method is suitable for accurate targeted therapy of medicines for relieving OA inflammation, and is believed to have wide application prospects in the aspects of knee osteoarthritis, wound injury repair and the like.
Description
Technical Field
The invention relates to the field of biological medicine; in particular to the application of cell membranes of synovial myofibroblasts in preparing osteoarthritis drugs.
Background
Osteoarthritis (OA) is the most common form of arthritis affecting more than 5 million people worldwide (about 7% of the global population), with a particularly high prevalence in the elderly (> 65 years). As a complex disease, it is characterized by pathological changes of all joint tissues, including cartilage, subchondral bone, ligaments, meniscus, joint capsule and synovial membrane OA, are the most common joint diseases associated with pain and disability. Its main clinical symptoms are chronic pain, joint instability, stiffness and radiological joint gap stenosis. Osteoarthritis is mainly associated with aging, and its prevalence will steadily rise, being predicted to be the largest single cause of disability in the general population by 2030. This not only affects the quality of life of the individual, but also places a heavy burden on the global health care system. However, current analgesic therapies are limited in efficacy and apparent toxicity, and there are no licensed disease modifying drugs.
Synovium is a special connective tissue that lines around the knee joint, surrounding tendons, forming lining of bursa and fat pads. In the synovial joint, the synovium seals the synovial cavity and the fluid of the surrounding tissue. Synovial membranes are responsible for maintaining the volume and composition of synovial fluid, mainly by producing lubricin and hyaluronic acid. Since articular cartilage has no intrinsic vascular or lymphatic supply, synovial membrane also contributes to the nutrition of chondrocytes by synovial fluid. Synovitis, which is one of the most common pathological changes in the development of osteoarthritis, can occur at an early stage of osteoarthritis, and when no significant cartilage degeneration has occurred, it can be detected by ultrasound, magnetic resonance, etc., and it is considered to be closely related to the severity of osteoarthritis. A variety of inflammatory mediators are found in synovial fluid of osteoarthritis patients, all of which induce chondrocytes and synoviocytes to secrete matrix metalloproteinases, resulting in degradation of the cartilage matrix, loss of stable living environment of cartilage, and eventually damage of cartilage. Thus, targeting early OA synovial cells is an effective way to delay OA progression.
Early single cell transcriptome sequencing results showed that a synovial myofibroblast population derived from fibroblasts in synovial tissue was greatly increased during the progression of OA, especially in the early stages of progression, and that α -SMA was specifically highly expressed. Therefore, the accurate delivery of the drug to synovial myofibroblasts and the regulation of the inflammatory response thereof are expected to become a new therapeutic approach for alleviating OA synovial inflammation and further regulating OA progression.
The medicine with clinical value is usually cleared quickly by the joint and cannot reach the deep part of the tissue, so that the clinical management of the osteoarthritis becomes more complex. The nanometer medicine carrying system constructed based on nanometer materials can alleviate the metabolism and intake of pure medicine in vivo to some extent, but inevitably encounters the problems: 1) Drug stability problem: some drugs may have stability problems in nanocarriers, such as leakage of the drug from the carrier, drug inactivation by drug-carrier interactions, etc.; 2) Biocompatibility problems: although many nanomaterials exhibit good biocompatibility in vivo, some nanomaterials may cause immune reactions or toxicity, thereby causing damage to the body; 3) In vivo distribution and targeting problems: the distribution and targeting effects of nanodrug delivery systems in vivo can be difficult to predict and control. Although targeting can be improved by various surface modifications and targeting ligands, there are several complications that may occur, such as drug non-specific aggregation or clearance by macrophages in the body; 4) Is easy to be absorbed by non-targeted tissues of joints, and causes off-target effect of medicines.
Disclosure of Invention
Synovitis is closely related to the severity of OA. The synovial fluid secreted by the cartilage matrix contains various inflammatory mediators, exacerbates inflammatory immune cell recruitment, induces degradation and injury of the cartilage matrix, and exacerbates OA progression. Thus, targeting early OA synovial cells is an effective way to delay OA progression. The invention discovers a myofibroblast subtype differentiated from a fibroblast-like synoviocyte based on single cell sequencing of an arthritic synoviocyte. How to regulate the inflammation level by delivering drugs to myofibroblasts with obvious early proliferation of OA and finally relieving synovial inflammation is an important point and difficulty to be solved at present.
Specifically, the invention constructs a bionic drug delivery system by extracting deactivated autologous synovial myofibroblasts of intact membrane receptors. On the one hand, endogenous receptors act as decoys to regulate the action of cytokines, thereby recognizing, sequestering and eliminating myofibroblast activating factors, while avoiding triggering myofibroblast internal signal activation, preventing the induction of aggravated synovial inflammatory responses; secondly, the membrane bait is supported by polylactic acid-glycolic acid (PLGA) based nanoparticle cores to enhance stability and facilitate the delivery of anti-inflammatory drugs into the inside of homologous myofibroblasts, achieving interaction of the homologous biomimetic membrane system with the myofibroblasts and becoming a powerful means of drug delivery by its high-efficiency uptake technology.
Before describing the technical scheme of the present invention, the terms used herein are defined as follows:
the term "PLGA" refers to: polylactic acid-glycolic acid copolymer.
The term "PVA" refers to: polyvinyl alcohol.
The term "TGF- β" refers to: transforming growth factor beta.
The term "PBS" refers to: phosphate buffered saline.
The term "FLS" refers to: fibroblast-like synoviocytes.
The term "Myo" refers to: myofibroblasts.
The term "M Myo "means: myofibroblast membranes.
The term "M Myo The term/PT "means: myofibroblast membranes encapsulate PLGA nuclei with a nanoparticle comprising Trip.
The term "PT" refers to: PLGA core contains nanoparticles of trip.
The term "Trip" refers to: tripterine (Tripterin, trip).
The term "DMM" refers to: medial meniscal destabilization surgery.
To achieve the above object, the first aspect of the present invention extracts human primary fibroblast-like synoviocytes, induces them to differentiate into myofibroblasts using TGF- β, and extracts cell membranes derived from synovial myofibroblasts. Wherein the TGF-beta inducing cells are selected from one or more of the following: primary human synovial tissue fibroblast-like synoviocytes (FLS), mouse embryonic fibroblasts (NIH/3T 3 cell line), mouse fibroblasts (3T 3-L1 cell line), human lung fibroblasts (WI-38 cell line), human lung fibroblasts (MRC-5 cell line); preferably human primary FLS cells. In a second aspect, the invention provides a homogeneous engineered myofibroblast biomimetic membrane drug carrier nano-delivery system capable of targeting synovial tissue myofibroblasts, namely osteoarthritis drugs, wherein the nano-system is the engineered biofilm as nanocapsules according to the first aspect. The nano-delivery system according to the second aspect of the present invention, wherein the material of the nanocapsule is selected from one or more of the following: PLGA, liposomes, organometallic frameworks, inorganic silica, chitosan, and polylactic acid; PLGA is preferred. The nano-delivery system according to the second aspect of the present invention, wherein the mass ratio of PLGA to exemplary anti-inflammatory agent triperin in the nanocapsule is 5:1 to 100:1, preferably 10:1 to 50:1, most preferably 30:1. According to the nano delivery system of the second aspect of the invention, the mass parts of the engineering biological film, the nanocapsule and the Trip are as follows: 1.5 to 100 parts of biological film, 5 to 100 parts of nanocapsules and 0.5 to 5 parts of Trip drugs; preferably 20-60 parts of biological film, 5-40 parts of nanocapsules and 0.5-1 part of Trip; most preferably 31 parts of biological film, 30 parts of nanocapsules and 1 part of Trip drug; preferably, when the nanocapsule material is selected from PLGA, the mass ratio of the biofilm to the drug loaded nanocapsules is 1:1. In a third aspect the invention provides a natural anti-inflammatory agent, tripterine, the example anti-inflammatory agent being selected from one or more of the following: tripterine (Trip), glycyrrhizin, curcumin, baicalin, triamcinolone acetonide; preferably, it is Trip. A third aspect of the present invention provides a method for preparing the engineered biomimetic membrane targeting nano targeted delivery system according to the second aspect, the method may comprise the steps of: (1) preparing an anti-inflammatory drug solution; (2) Dripping the drug solution prepared in the step (1) into the solution of the nanocapsule material; (3) Dripping the mixed solution obtained in the step (2) into a solvent, ultrasonically stirring and centrifuging to remove supernatant, and re-suspending the obtained precipitate; (4) Mixing the heavy suspension obtained in the step (3) with a biological membrane, and performing ultrasonic treatment to obtain the nano delivery system. The preparation method according to the third aspect of the present invention, wherein, in the step (3), the solvent is selected from one or more of the following: PVA aqueous solution, tween 20 aqueous solution, tween 80 aqueous solution; preferably an aqueous PVA solution, more preferably 1% to 10% aqueous PVA solution, most preferably 3% aqueous PVA solution. In a fourth aspect the invention provides the use of an engineered synovial fibroblast membrane according to the first aspect and a nano-delivery system according to the second aspect for the preparation of a medicament for the treatment of osteoarthritis for delivery to synovial myofibroblasts. The invention aims to find a high-efficiency drug delivery system based on targeting synovial myofibroblast engineering bionic membrane camouflage, so that an anti-inflammatory drug is delivered to myofibroblasts with high efficiency, and the inflammatory properties of synovial cells are inhibited, and the treatment effect of osteoarthritis is relieved. In order to achieve the purpose, the invention adopts the following technical scheme:
according to the invention, firstly, single cell sequencing of OA model making mice by a row DMM method is used for verifying a large number of proliferation myofibroblast subgroups of OA acute phase joint synovial tissue. Next, human primary fibroblast-like synoviocytes were extracted based on single cell sequencing results, induced to differentiate into myofibroblasts by TGF-beta, and synoviocyte-derived cell membranes (M myo ) As a bionic membrane tool, the highly differentiated and proliferated myofibroblasts in the inflammatory synovial tissue are identified in a highly homologous and targeted manner, and the drugs are delivered into the inflammatory myofibroblasts in a highly efficient manner without any other transfection reagent, so that the cell uptake rate is improved; finally, M is myo The target medicine is efficiently delivered into target cells based on the polylactic acid-polyglycolic acid copolymer (PLGA) coated small-molecule anti-inflammatory medicine with biocompatibility and degradability, so that the therapeutic effects of inhibiting synovial cell inflammation and relieving OA progression are achieved, and the application value is important. The specific flow is shown in fig. 3. The invention provides a system which can identify a myofibroblast group which is highly differentiated and proliferated in OA acute synovitis tissues in a homologous targeting way and accurately deliver relevant anti-inflammatory drugs into cells of the myofibroblast group: m in the invention myo PLGA and the proportion of the drug are fixed. The nanometer capsule core component is PLGA, and the proportion is fixed. The method of the invention adopts myofibroblasts based on TGF-beta induction source, and extracts the cell membrane (M myo ) As biomimetic membrane to modify PLGA nuclei. This structure confers the ability of drug-loaded nanoparticles to target synovial myofibroblasts with high efficiency. On the one hand, endogenous receptors act as decoy to regulate cytokines, thereby recognizing, sequestering and eliminating myofibroblast activation factors while avoiding triggering myofibroblast internal signaling activation, preventingInduce exacerbating synovial inflammatory response; secondly, the membrane bait is supported by the PLGA-based nanoparticle core to enhance stability and facilitate the delivery of anti-inflammatory drugs into the inside of the homologous myofibroblasts, achieving interaction of the homologous biomimetic membrane system with the myofibroblasts and thus becoming a powerful means of drug delivery by its high-efficiency uptake technology. This structure can finally achieve safe and efficient inhibition of synovial inflammation in vivo, thereby alleviating OA progression. The delivery system of the present invention may have, but is not limited to, the following benefits:
1): one abnormally proliferative synovial myofibroblast subtype was found in early OA based on osteoarthritis joint synovial single cell sequencing.
2): the following disadvantages of simple drugs and traditional nano drug-loaded particles are pertinently overcome: the pure medicine is easy to be metabolized by the fast flowing joint liquid in the joint cavity; the pure drugs and/or nano drug-loaded particles lack high efficiency when ingested by tissues; the applied carrier is a foreign object and has poor biocompatibility; the internal medicine is not easy to release due to the easy sealing surfaces of serum or plasma proteins and the like in the circulatory system; is easily cleared by mononuclear cells, macrophages and the like of reticuloendothelial systems, and the efficacy is reduced.
3) Compared with the traditional cell membrane, the targeting of synovial myofibroblasts is greatly improved, and theoretical guidance and subsequent technical support are provided for subsequent treatment of synovitis. The method has higher synovial myofibroblast targeting effect, can maintain long-time action in the joint cavity, and has better treatment effect on relieving OA inflammation. The method is suitable for accurate targeted therapy of medicines for relieving OA inflammation, and is believed to have wide application prospects in the aspects of knee osteoarthritis, wound injury repair and the like.
Drawings
In order to clearly demonstrate the specific embodiments of the present invention and certain detection techniques employed in the experiments, the embodiments and techniques employed will be described below, mainly by way of introduction in the accompanying drawings.
FIG. 1 shows single cell sequencing of normal (FIG. 1A) and row DMM (FIG. 1B) OA model proliferation synovial tissue by 10 Xgenomics and analysis of the results. The 2 batches of samples were subjected to integration analysis, pretreatment and quality control, and macrophages and mesenchymal stem cells were analyzed according to the expression of the marker gene, to obtain late-period mesenchymal stem cells (Late mesenchymal stem cell, cluster 0), synovial cells (synoviduct, cluster 1, 5), myofibroblasts (myofbrobust, cluster 2), early-period mesenchymal stem cells (Early mesenchymal stem cell, cluster 3) and adipogenic precursor cells (Pre-adipocell, cluster 4). The mouse synovial tissue of the OA group significantly increased myofibroblast number at the early OA stage (day 7 post-surgery) compared to the normal group.
FIG. 2 shows the induction of OA models by constructing Dpp4-Creer, tdtomato mice parallel ACLT modeling. On day 7 knee tissue sections of Dpp4-Creer, tdtomato line OA molding mice were stained, a significantly high enrichment of the alpha-SMA positive cell population (red), i.e., the high myofibroblast cell population, with F4/80 labeled macrophage cell population (green) was observed in the Dpp4 labeled (white) synovial tissue.
FIG. 3 shows targeting of synovial myofibroblasts M myo A preparation process of a coated nanocapsule drug-loaded delivery system. Wherein FIG. 3A shows the differentiation into myofibroblasts induced by TGF-beta by extraction of primary fibroblast-like synoviocytes, and extraction of M myo A process; fig. 3B shows the construction of drug-loaded nanoparticles of polylactic-co-glycolic acid (PLGA) and encapsulated anti-inflammatory drug Tripterin (Trip). Fig. 3 shows in its entirety the process of preparing a drug-loaded delivery system for targeting synovial myofibroblasts using myofibroblasts as the nanocapsules for engineering biomimetic membrane encapsulation of anti-inflammatory drugs.
FIG. 4 shows PT and M in example 3 myo PT transmission electron microscopy and hydrated particle size analysis. FIG. 4A shows M myo Before wrapping the PT core; FIG. 4B shows M myo Wrapping the PT core. Wherein M is myo The mass ratio of PT to Trip is 31/30/1 in turn.
Fig. 5 shows the drug loading efficiency of the nanocapsules of example 3 on Trip. FIG. 5A shows drug loading rates of 5/1, 10/1, 20/1, 30/1, 40/1, 50/1, 60/1, 70/1, 80/1, 90/1 and 100/1 for PLGA to Trip mass ratios, respectively, in PT nanocapsules; FIG. 5B shows the variation of the particle size of the membrane protein in terms of the mass ratio of Trip-loaded PLGA (PT) to 0/1,0.05/1,0.1/1,0.25/1,0.5/1,0.75/1,1/1,1.25/1,1.5/1 and 2/1, respectively.
FIG. 6 shows cytotoxicity of the different group systems of test example 1.
FIG. 7 shows the fluorescent drug-loaded nanocapsules (PLGA-DID) and M in test example 2 myo Nanometer capsule of fluorescence medicine (M) myo -PLGA-DID) myofibroblast uptake after stimulation with TGF- β.
FIG. 8 shows qRT-PCR detection of synovial cell inflammation levels after administration of different sets of material systems in test example 3, the detection indicators including IL1, IL6, IL8.
FIG. 9 shows the results of safranin O/fast green staining of joint tissue sections of different treatment groups of the joint cavity after OA molding in a DMM mouse of test example 4.
Detailed description of the preferred embodiments
The specific embodiments of the present invention are explained by way of example, and the described embodiments are intended to be part of the invention, and are intended to be within the scope of the present invention as defined by the appended claims, unless the technology used for the detection is not limited in any way.
This section generally describes the materials used in the test of the present invention and the test method. Although many materials and methods of operation are known in the art for accomplishing the objectives of the present invention, the present invention will be described in as much detail herein. It will be apparent to those skilled in the art that in this context, the materials and methods of operation used in the present invention are well known in the art, if not specifically described.
The reagents and instrumentation used in the following examples were as follows:
recombinant human TGF- β1, available from PeproTech U.S.A.;
tripterin, available from MedChem Express united states;
collagenase type 2, available from Thermo Fisher united states;
PLGA, available from guangzhou sijia limited;
methylene chloride, purchased from guangzhou chemical reagent plant, china;
ethyl acetate, purchased from guangzhou chemical reagent plant, china;
PVA, available from Sigma-Aldrich, USA;
PBS, available from Life technology, USA;
microfilament Green fluorescent probe (action-Tracker Green-488), purchased from Biyun, china;
immunostaining permeation solution (saponine), purchased from bi yun tian, china;
BSA, purchased from Sigma-Aldrich, USA;
c57BL/6 mice, purchased from Guangzhou City Sea Bai Nuo Biotech Co., ltd;
transmission electron microscopy, available from Japan under the model JEM 1400PLUS.
Confocal laser microscopy was purchased from Zeiss germany.
Flow cytometry, available from U.S. under the model BD, san Jose, CA.
Example 1
This example is used to demonstrate the discovery of myofibroblasts in synovial tissue following DMM surgery
1) All animal experiments and related work performed in this study were approved by the animal ethics committee of experiments at university of south China. To investigate the characteristics of macrophages in varying degrees of synovitis, applicant constructed a model of mouse traumatic osteoarthritis using DMM surgery.
2) Normal mice and 7 days after surgery knee synovial tissue samples were collected and stained with hematoxylin/eosin, and it was found that DMM surgery caused an acute inflammatory response in synovial tissue with a significant increase in synovial score.
3) Normal mice and mouse knee synovial tissue samples 7 days after DMM surgery were collected, and cells were extracted for single cell transcriptome sequencing.
4) Single cell sequencing results were analyzed. The 2 batches of samples were subjected to integration analysis, pretreatment and quality control, and macrophages and mesenchymal stem cells were analyzed according to the expression of the marker gene, to obtain late-period mesenchymal stem cells (Late mesenchymal stem cell, cluster 0), synovial cells (synoviduct, cluster 1, 5), myofibroblasts (myofbrobust, cluster 2), early-period mesenchymal stem cells (Early mesenchymal stem cell, cluster 3) and adipogenic precursor cells (Pre-adipocell, cluster 4).
5) Analysis of the 2 lot single cell sequencing results, separated, found a significant increase in the early OA group synovial myofibroblast number compared to the normal group (fig. 1A-B, cluster 2). Immunofluorescent staining showed that the number of dpp4+α -sma+ synovial cells was also significantly increased in OA mouse synovial tissue (fig. 2).
Among them, for the extraction of the mouse synovial tissue of the OA group, the present invention first selected 8-week-old male C57BL/6J mice and performed DMM surgery on their right knees to construct an OA model. The method comprises the following specific steps:
1) Mice were anesthetized with tribromoethanol in combination with isoflurane, shaved and disinfected near the knee joint.
2) An incision approximately 5a mm a long was made over the patellar ligament using sterile scissors, blunt to separate the skin from the subcutaneous tissue. Further, the knee joint was opened along the medial border of the patellar ligament with fibrous surgical scissors, and the infrapture was separated from the infrapatellar adipose tissue.
3) After the inner meniscus transverse ligament is found, the special scissors for microsurgery are used for disconnecting, and whether the operation is successful is judged by observing whether free meniscus broken ends exist or not.
4) Finally, the joint capsule and skin were sutured and the surgical incision was sterilized, and after the mice were awakened, the mice were returned to the animal center and post-operative observations were made. And collecting mouse synovial membrane tissues of normal group and row DMM modeling group for subsequent single cell sequencing, tissue embedding, slice staining and the like.
FIG. 1 shows the analysis of single cell sequencing of normal and row DMM OA-model proliferation synovial tissue of mice by 10 Xgenomics. The 2 batches of samples were subjected to integration analysis, pretreatment and quality control, and macrophages and mesenchymal stem cells were analyzed according to the expression of the marker gene, to obtain late-period mesenchymal stem cells (Late mesenchymal stem cell, cluster 0), synovial cells (synoviduct, cluster 1, 5), myofibroblasts (myofbrobust, cluster 2), early-period mesenchymal stem cells (Early mesenchymal stem cell, cluster 3) and adipogenic precursor cells (Pre-adipocell, cluster 4). The mouse synovial tissue of the OA group significantly increased myofibroblast number at the early OA stage (day 7 post-surgery) compared to the normal group.
FIG. 2 shows the induction of OA models by constructing Dpp4-Creer, tdtomato mice parallel ACLT modeling. On day 7 knee tissue sections of Dpp4-Creer, tdtomato line OA molding mice were stained, a significantly high enrichment of the alpha-SMA positive cell population (red), i.e., the high myofibroblast cell population, with F4/80 labeled macrophage cell population (green) was observed in the Dpp4 labeled (white) synovial tissue.
Example 2
This example is for illustrating the induction of artificial myofibroblasts and the extraction of biomimetic cell membranes
Clinical samples and animal experiments used in this study were approved by the first affiliated hospital ethics committee of south university and received full written consent prior to surgery. Synovial tissue, which is the source of primary synovial cell extraction, is obtained from young patients receiving arthroscopic anterior cruciate ligament reconstruction or meniscus repair.
Primary synovial cell extraction:
1) Sample collection: the surgically excised synovial tissue was placed on an operating table with a nurse placing the specimen in low temperature saline, and at the completion of the surgery the specimen was placed in PBS ice water and brought back to the laboratory in a sterile container.
2) Reagent preparation: 2mg/mL type II collagenase (DMEM formulated), complete medium (containing 10% fetal bovine serum, 100 units/mL penicillin and 100 g/mL streptomycin).
3) Instrument preparation: surgical instruments, including scissors and forceps.
4) The excised tissue was placed on an operating table with the nurse placing the specimen in cold saline (stationary), and at the completion of the procedure the specimen was placed in PBS ice water and brought back to the laboratory in a sterile container. The specimens were placed in a petri dish in an ultra clean bench, and washed with DMEM. After carefully removing the adipose tissue with scissors, the tissue was placed in a 5 mL centrifuge tube and minced to paste with dissection shears. A centrifuge tube of 15 mL was placed in DMEM high-sugar medium containing 2mg/mL type II collagenase at 5 times the synovial tissue volume, and incubated at a constant temperature shaker at 37℃for 120 minutes at 110 rpm.
5) Taking out the centrifuge tube in the shaking table, centrifuging at 2000rpm for 5 min, discarding the supernatant, re-suspending the precipitate with EDTA-containing pancreatin in a 37-degree incubator for 30 min, standing at normal temperature in the centrifuge, centrifuging at 2000rpm for 5 min, discarding the supernatant, re-suspending the supernatant with DMEM culture medium, filtering with a 70 μm cell filter screen, and centrifuging at 2000rpm for 5 min.
6) The cell pellet is resuspended and then inoculated into a culture flask for culture, and the liquid is changed every 2-3 days.
2 induction of myofibroblasts and extraction of myofibroblast membranes:
when the cells grow to the 2 nd generation density and mature, the cells are plated in a 10 cm cell culture dish, and TGF-beta is added to induce synovial cells to differentiate into myofibroblasts.
Extraction of myofibroblast membranes. Cell membrane (M) obtained by repeated freeze thawing myo ). First, synovial myofibroblasts (about 2000 ten thousand to 5000 ten thousand cells) which were successfully induced to differentiate were collected. The cells were washed 3 times with a PBS solution containing protease inhibitors. The cells were scraped with a cell scraper, collected by centrifugation, and the supernatant was aspirated, leaving a cell pellet ready.
Cell membrane and cytoplasmic protein extraction kit a reagent and protease inhibitor without 1×edta were added to the cell pellet, and cells were lysed at 4 ℃ for 30 minutes and sonicated for 5 minutes.
And (3) sequentially and repeatedly freezing and thawing the sample twice in liquid nitrogen and at room temperature, and then taking a small amount of sample to detect the degree of cell disruption under a microscope until the degree of cell disruption is more than 70%.
Centrifuging at 4 ℃ for 10 minutes at 700g, carefully collecting the supernatant into a new centrifuge tube, and ensuring that the extracted supernatant has higher purity.
Centrifuging at 4 ℃ for 30 minutes at 14000 and g, precipitating cell membrane fragments by PBS, and storing the extracted cell membrane fragments at-80 ℃ for later use.
Example 3
This example is intended to illustrate the invention's drug carrier (M) myo -PT).
1) Will M myo Mixing with PLGA-Tripterin (PT) in a specified weight ratio, continuously swirling for 5 min, and performing ultrasonic treatment for 5 min;
2) Using Miniextruder (Avanti), the mixture was coextruded 15 times back and forth with 400 nm, 200 nm, 100 nm polycarbonate film to give M myo Coated PT (M) myo -PT)。
Wherein, the synthesis of PT is as follows:
1) 10mg of PLGA particles were dissolved in 700 uL dichloromethane and 300 uL ethyl acetate mixed organic solvent.
2) Trip drug dissolved in DMSO solvent was added to the organic solvent mixed with PLGA.
3) After the mixture was sonicated in a water bath for 5 minutes, the sonic probe was placed in a 3% polyvinyl alcohol (PVA) solution with a power setting of 50w, on for 3s, off for 2s. Under ultrasonic stimulation, the PLGA-Trip organic solvent mixture is fully emulsified in PVA solution drop by drop for 5 minutes to form an oil-in-water mixed system.
4) The fully emulsified organic system was placed in a 0.3% PVA solution and magnetically stirred for 4 hours to fully volatilize the organic solvent.
5) After stirring thoroughly, the solution in the conical flask was poured into a 50 mL centrifuge tube, balanced centrifuged at 12000 rpm for 40 minutes, after the first centrifugation was completed, the solution was poured out, the PBS was resuspended and precipitated, centrifuged at 12000 rpm for 40 minutes again, this step was repeated 2 times, after the second PBS was resuspended and centrifuged, the liquid in the tube was poured out, and after the precipitation was resuspended with 1mL PBS, the solution was stored in a 4℃refrigerator.
FIG. 3 showsM targeting synovial myofibroblasts myo A preparation process of a coated nanocapsule drug-loaded delivery system. Wherein FIG. 3A shows the differentiation into myofibroblasts induced by TGF-beta by extraction of primary fibroblast-like synoviocytes, and extraction of M myo Fig. 3B shows the process of constructing drug-loaded nanoparticles with polylactic-co-glycolic acid (PLGA) and encapsulated anti-inflammatory drug Tripterin (Trip). Fig. 3 shows in its entirety the process of preparing a drug-loaded delivery system for targeting synovial myofibroblasts using myofibroblasts as the nanocapsules for engineering biomimetic membrane encapsulation of anti-inflammatory drugs.
FIG. 4 shows PT and M in example 3 myo PT transmission electron microscopy and hydrated particle size analysis. FIG. 4A shows M myo Before wrapping the PT core, FIG. 4 shows M myo Wrapping the PT core.
Fig. 5A shows the drug loading efficiency of the nanocapsules of example 3 on Trip. The mass ratio of PLGA to Trip was 5/1, 10/1, 20/1, 30/1, 40/1, 50/1, 60/1, 70/1, 80/1, 90/1 and 100/1, respectively. Wherein, the mass ratio of PLGA to Trip is 30/1; FIG. 5B shows the variation of the particle size of the membrane protein in terms of the mass ratio of Trip-loaded PLGA (PT) to 0/1,0.05/1,0.1/1,0.25/1,0.5/1,0.75/1,1/1,1.25/1,1.5/1 and 2/1, respectively. Wherein M is myo The mass ratio to PT was 1/1.
Test example 1
The test example is used for explaining the experiment of the influence of the targeting nano drug-loaded delivery system on the bioactivity of inflammatory synovial cells
1) Myofibroblasts were induced as in example 2, i.e., M was synthesized as in example 3 myo PT and PT groups. PT group is a pure material drug-loaded group without targeting myofibroblasts, trip is a pure drug treatment group.
2) Synovial cells were seeded in six well plates with 1X 10 cells per well 5 After the cells are attached, the IL-1 beta drug treatment is performed to induce an inflammatory cell model, and the different drugs are added at the same time.
3) After culturing for 24 hours, the original culture medium is discarded, cells are digested by using EDTA-free pancreatin digestive juice, the supernatant is discarded, and Annexin V-FITC and propidium iodide staining solution are respectively added and mixed gently.
4) After incubation for 10-20 minutes at room temperature and in dark place, the apoptosis effect is detected by an upper machine. The apoptosis rate is detected and quantified by adopting an Annexin V-FITC/PI apoptosis detection kit.
As shown in FIG. 6, M synthesized according to the present invention myo PT, PT and Trip have no obvious toxic and side effects on cells in vitro.
Test example 2
The test example is used for explaining the experiment of the uptake effect of synovial tissue myofibroblasts on the targeting nano drug delivery system.
As shown in FIG. 7, in order to study cell uptake, fluorochrome DID was loaded into PLGA core (PLGA-DID) instead of Trip, M myo Coated on PLGA-DID to form M myo PLGA-DID Complex nanoparticle M carrying a fluorescent drug DID was synthesized as described in example 3 myo PLGA-DID, a simple material-loaded fluorescent drug group without targeting myofibroblasts. Synovial cells induced by TGF-beta were treated with fluorescent nanoparticles for in vitro uptake assessment.
Cell uptake was observed by laser confocal microscopy.
1) To study cellular uptake, fluorochrome DID was loaded into PLGA core (PLGA-DID) instead of Trip, M myo Coated on PLGA-DID to form M myo PLGA-DID complex, as previously described.
2) Cells were first seeded in confocal dishes at a density of 1X 10 5 Individual cells/wells. After the cells grow to a confluency of 70%, PLGA-DID and M are mixed myo After incubation of PLGA-DID with myofibroblasts, respectively, for the indicated time, the medium was removed, the cells were gently washed 3 times with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature, twice with PBS.
3) The membrane was broken with 0.1% triton for 10 min and washed twice with PBS.
4) The cytoskeleton was stained with action-Tracker Green-488, incubated in a 37℃incubator for 50 minutes in the absence of light, and then the nuclei were counterstained with DAPI dye for 15 minutes before repeated washing with PBS.
5) Cell imaging was performed on CLSM (LSM 880, zeiss).
Test example 3
The test example is used for explaining the experiment of the invention for evaluating the inflammatory synovial cell inflammation inhibition effect of the targeting nano drug delivery system
1) Nanoparticle M was synthesized as described in example 3 myo PT, trip, PT and M were synthesized separately according to the method of test example 1 myo PT group.
2) Synovial cells were seeded in six well plates and cultured as in test example 1, each well containing 1X 10 cells 5 And (3) after the cells are attached to the wall, carrying out inflammatory induction treatment on the synovial cells by IL-1 beta, and adding the medicines of different groups after 24 hours of induction treatment.
3) After culturing for 24 hours, the original medium was discarded, and cell RNA was extracted with Trizol. qRT-PCR for detecting inflammatory factor change, as shown in FIG. 8, M synthesized by the present invention myo PT has similar anti-inflammatory effects in vitro as the Trip group, which is compared to Trip and M myo PT group has a relatively weak anti-inflammatory effect.
Test example 4
This test example is used to illustrate the treatment of knee Osteoarthritis (OA) with the nanoparticles of the present invention.
Synthesis of M as in example 2 myo And M was synthesized as in example 3 myo PT group and PT group, treatment of OA. M is M myo PT is the experimental group, PT is the pure material drug loaded group without targeted myofibroblasts, trip is the pure drug treated group, sham is the surgery free group, PBS is the blank control group.
Preparation of model therapeutic strategy for knee osteoarthritis in mice
1) 40 male C57BL/6 mice (8 weeks old, weight about 20 g) were randomly divided into the five groups, and after one injection of the joint cavity medicine for two weeks after OA molding, the mice were sacrificed after 12 weeks, and the knee joints of the mice on the treatment side were stained with safranin O/fast green.
As shown in fig. 9. The invention synthesized targeted synovial muscle fiberCompared with a Trip simple drug group and a PT non-targeting drug-carrying group, the nano-carrier of the vitamin cells has higher effect of relieving cartilage matrix degradation caused by OA in a mouse body, and the relieving effect of the PT group on cartilage matrix degradation in a joint cavity is compared with M with a synovial myofibroblast targeting effect myo PT group was significantly attenuated and statistically significant. The Trip group only has weak influence on the alleviation effect of cartilage matrix degradation, thus showing that the treatment effect of the pure drug in the joint cavity is poor, and simultaneously showing the great superiority of the targeted drug-carrying system invention on disease alleviation in vivo.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Claims (10)
1. Use of cell membranes of synovial myofibroblasts in the preparation of a pharmaceutical carrier for osteoarthritis.
2. The use according to claim 1, characterized in that: the synovial myofibroblasts are formed by TGF-beta induced cell differentiation.
3. Use according to claim 2, characterized in that: TGF-beta induced cells include any of the following: primary human synovial tissue fibroblast-like synoviocytes, mouse embryonic fibroblasts, mouse fibroblasts, human lung fibroblasts.
4. An osteoarthritis drug characterized by comprising: cell membranes of synovial myofibroblasts, nanocapsules, and anti-inflammatory agents.
5. The osteoarthritis drug of claim 4, wherein: the nanocapsule is made of one or more of the following materials: PLGA, liposomes, organometallic frameworks, inorganic silica, chitosan, polylactic acid.
6. The osteoarthritis drug of claim 4, wherein: the anti-inflammatory agent is at least one of tripterine, glycyrrhizin, curcumin, baicalin and triamcinolone acetonide.
7. The osteoarthritis drug of any one of claims 4-6, comprising the following components in parts by weight: 1.5 to 100 parts of cell membrane of synovial myofibroblast, 5 to 100 parts of nanocapsule and 0.5 to 5 parts of drug.
8. The osteoarthritis drug of claim 7, wherein: the nanocapsule is made of PLGA; the anti-inflammatory is tripterine; the mass ratio of the cell membrane of the synovial myofibroblast to the nanocapsule loaded with the anti-inflammatory agent is 1:1.
9. The preparation method of the osteoarthritis medicine is characterized by comprising the following steps:
(1) Preparing an anti-inflammatory drug solution;
(2) Dripping the drug solution prepared in the step (1) into the solution of the nanocapsule material;
(3) Dripping the mixed solution obtained in the step (2) into a solvent, ultrasonically stirring and centrifuging to remove supernatant, and re-suspending the obtained precipitate;
(4) Mixing the heavy suspension obtained in the step (3) with a biomembrane, and performing ultrasonic treatment to obtain the osteoarthritis medicine; the biological membrane is a cell membrane of synovial myofibroblast.
10. The method for preparing an osteoarthritis drug as claimed in claim 9, wherein:
the anti-inflammatory agent in step (1) is: the preparation method comprises mixing radix Tripterygii Wilfordii with red,
the solvent in step (3) is selected from one or more of the following: PVA aqueous solution, tween 20 aqueous solution, tween 80 aqueous solution.
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