Dexamethasone liposome targeting synovial fibroblasts and preparation method of dexamethasone liposome

Abstract

The invention relates to the technical field of dexamethasone pharmaceutical preparations, and discloses a dexamethasone liposome targeting synovial fibroblasts and a preparation method of the dexamethasone liposome, the liposome comprises dexamethasone and a lipid bilayer used for wrapping the dexamethasone, and the lipid bilayer comprises DSPE-PEG2000-GPA-KLVFF. The preparation method comprises the following steps: step S1, preparation of DSPE-PEG2000-GPA-KLVFF (Distearoyl Pyrrolidone-Polyethylene Glycol 2000); s2, the soybean lecithin, the cholesterol, the DSPE-PEG2000-GPA-KLVFF and the dexamethasone are weighed according to the proportion, and the soybean lecithin, the cholesterol, the DSPE-PEG2000-GPA- And evaporating the solvent, and preparing the dexamethasone liposome by adopting a film dispersion method. The dexamethasone liposome prepared by the invention has definite in-vivo synovial fibroblasts and RA inflammation joint targeting characteristics, is good in dispersity and stability and high in FLS targeting efficiency, reduces MHC-II expression and inhibits RA inflammation development.

Description

Technical Field

[0001] The invention relates to the technical field of dexamethasone pharmaceutical preparations, and in particular to a dexamethasone liposome targeting synovial fibroblasts and a preparation method thereof. Background Art

[0002] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0003] Rheumatoid arthritis (RA) is a chronic autoimmune joint disease. The exact cause is still unclear, but studies have shown that genetics and environment are the two main factors. There is no cure for RA in clinical practice. The current means are mainly drug therapy, and the treatment goal is often to alleviate or reduce disease activity. In addition, long-term systemic use of dexamethasone (Dex), a steroid hormone drug used as a first-line drug, can cause extensive and serious side effects. Synovial fibroblasts (FLS) are a key factor driving the development of RA and are almost involved in the entire pathological process of RA. Their destructive effects in RA are mainly manifested in: 1) Recruiting and activating a variety of immune cells to promote the formation of ectopic lymphoid structures, secretion of inflammatory cytokines, and activation of innate immune responses; 2) Activated FLS upregulate the expression of major histocompatibility complex-II (MHC-II), promoting MHC-II-dependent self-antigen presentation and adaptive immune responses. 3) Through the combined effects of 1) and 2), FLS invasion, synovial hyperplasia and pannus formation are further accelerated; and by attaching to cartilage, bone degradation and bone erosion are accelerated, leading to joint deformities. In summary, FLS stimulates and amplifies inflammation by continuously producing inflammatory cytokines, inflammatory small molecule mediators, proteases that degrade the extracellular matrix and cartilage, and upregulating the expression of MHC-II, thereby promoting the degradation of the extracellular matrix and exacerbating joint damage.

[0004] Therefore, influencing the action of FLS through pharmaceutical techniques may be expected to alleviate the effects of RA.

[0005] Nano drug delivery technology is beneficial to enhance drug targeting and improve bioavailability, thereby achieving toxicity reduction and efficacy enhancement. In addition, when nano drugs pass through the proliferating blood vessels in the inflammatory site of RA, they can reside in the inflammatory site through the "extravasation through leaky vasculature and subsequent inflammatory cell-mediated sequestration" (ELVIS) effect, achieving passive joint targeting.

[0006] Since 1971, liposomes (Lip) have been used to transport bioactive substances and have become a new type of drug delivery system. Liposomes are composed of phospholipid bilayers and have both hydrophilic and lipophilic properties, which enable them to encapsulate both hydrophilic and hydrophobic drugs. Hydrophilic drugs are usually encapsulated in the central aqueous phase, while hydrophobic drugs are encapsulated inside the phospholipid bilayer. Liposomes can fuse with cell membranes and release drugs into cells, making them an efficient targeted delivery system. In addition, liposomes have good biocompatibility and biodegradability, are non-immunogenic, and therefore have low toxicity to normal tissues.

[0007] Fibroblast activation protein α (FAP-α) is a type II transmembrane glycoprotein highly expressed on the cell membrane of activated FLS and belongs to the serine proline oligopeptidase family. The oligopeptide glycine-proline-alanine (Gly-Pro-Ala, GPA) is the substrate peptide of FAP-α and can be specifically cleaved by FAP-α from GP-"X", thereby achieving targeting of FLS.

[0008] The polypeptide Lys-Leu-Val-Phe-Phe (KLVFF) is a peptide motif that can form a β-folded structure from β-amyloid protein (Aβ), and can self-assemble into a stable fiber structure through intermolecular hydrogen bonds. Based on the property that KLVFF can be highly fibrillated into a β-folded structure through hydrogen bonds and inhibit Aβ aggregation, KLVFF can self-assemble into mesh nanofibers around target cells and wrap around their surface, so that surface molecules (MHC-II, etc.) expressed on the cell membrane are blocked. Therefore, KLVFF has the potential to curb the development of RA by inhibiting the expression of MHC-II by FLS and the subsequent adaptive immune response, but no relevant research has been seen so far. Summary of the invention

[0009] The purpose of the present invention is to address the problems of insufficient efficacy and widespread side effects of current clinical treatment drugs for RA, provide a dexamethasone liposome targeting synovial fibroblasts and a preparation method thereof, design a nano drug delivery system that can accurately target FLS, combine passive targeting and active targeting to accurately deliver drugs to FLS cells, and thus curb the development of RA.

[0010] The technical solution of the present invention is as follows:

[0011] A dexamethasone liposome targeting synovial fibroblasts, comprising dexamethasone and a lipid bilayer for encapsulating dexamethasone, wherein the lipid bilayer comprises DSPE-PEG 2000 -GPA-KLVFF, the DSPE-PEG 2000The structural formula of -GPA-KLVFF is shown in formula (I).

[0012]

[0013] According to a preferred embodiment, the lipid bilayer is further made of soybean lecithin and / or cholesterol.

[0014] According to a preferred embodiment, the hydrated particle size of the dexamethasone liposome is 111.5 nm to 130.1 nm.

[0015] Another aspect of the present invention provides use of the above-mentioned dexamethasone liposome targeting synovial fibroblasts in the preparation of a drug for preventing / improving / treating rheumatoid arthritis.

[0016] Another aspect of the present invention provides a pharmaceutical composition for preventing / improving / treating rheumatoid arthritis, wherein the active ingredient of the pharmaceutical composition comprises the above-mentioned dexamethasone liposome targeting synovial fibroblasts.

[0017] Another aspect of the present invention provides a method for preparing the above-mentioned dexamethasone liposomes targeting synovial fibroblasts, comprising the following steps:

[0018] Step S1: DSPE-PEG 2000 - Preparation of GPA-KLVFF;

[0019] Weigh an appropriate amount of DSPE-PEG 2000 -NHS was dissolved in N,N-dimethylformamide (DMF), GPA-KLVFF peptide and triethylamine were added and dissolved completely, and the reaction was carried out at room temperature for 12 hours; the reaction solution was transferred to a dialysis bag, the dialysis medium was purified water, the dialysate was collected and freeze-dried, and the product was obtained by purification through molecular sieves.

[0020] Step S2: Preparation of dexamethasone liposomes targeting synovial fibroblasts;

[0021] Weigh appropriate amount of soybean lecithin, cholesterol, DSPE-PEG 2000 -GPA-KLVFF and dexamethasone were added, and Kolliphor HS15 was completely dissolved in anhydrous ethanol solution. The organic solvent was removed by reduced pressure distillation using a rotary evaporator in a 37°C water bath to form a lipid film.

[0022] 0.9% sodium chloride solution was added for hydration, and the mixture was incubated in a 37°C water bath and then ultrasonically treated to obtain dexamethasone liposomes Dex@GF-Lipos targeting synovial fibroblasts, which were stored at 4°C for future use.

[0023] Preferably, the DSPE-PEG in step S1 2000The molar ratio of -NHS, DMF, GPA-KLVFF peptide and triethylamine is 1:8-9:1:2-3.

[0024] Preferably, the molar mass ratio of soybean lecithin, cholesterol, DSPE-PEG2000-GPA-KLVFF, dexamethasone and Kolliphor HS15 in step S2 is (50-100):(9-18):1:(9-14):(50-110).

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present invention provides a dexamethasone liposome targeting synovial fibroblasts, which uses the GPA-KLVFF peptide sequence on the liposome membrane to target and inhibit FLS overexpression of MHC-II, and simultaneously delivers Dex to FLS in inflamed joints based on passive targeting and active targeting; it exhibits high uptake efficiency on in vitro synovial fibroblast cell lines, and has clear in vivo synovial fibroblast and RA inflamed joint targeting characteristics;

[0027] 2. The dexamethasone liposome Dex@GF-Lipos provided by the present invention, which is targeted to synovial fibroblasts, has good stability, an average particle size of 111.5nm to 130.1nm, an encapsulation rate of 87.6% to 95.3%, good dispersibility and stability, high FLS targeting efficiency, and good effect of downregulating MHC-II expression; in addition, it can significantly improve the clinical drug safety of dexamethasone, improve the anti-inflammatory efficacy of Dex, and reduce the toxic and side effects of Dex, and has the value of clinical transformation;

[0028] 3. The present invention provides a method for preparing dexamethasone liposomes targeting synovial fibroblasts, which utilizes a solubilizing agent to increase the solubility of dexamethasone in a lipid material, and prepares dexamethasone into a nano-preparation with good dispersion, good biocompatibility, and both passive targeting and active targeting. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a DSPE-PEG in dexamethasone liposomes targeting synovial fibroblasts. 2000 -Structural formula of GPA-KLVFF;

[0030] Figure 2 DSPE-PEG 2000 -Schematic diagram of the synthesis process of GPA-KLVFF;

[0031] Figure 3 DSPE-PEG prepared in Example 1 2000 -H NMR spectrum of GPA-KLVFF;

[0032] Figure 4 It is the hydrated particle size spectrum of Dex@GF-Lipos and Dex@Lipos prepared in Examples 1 to 3 and Comparative Example 1;

[0033] Figure 5 1 is a transmission electron micrograph of Dex@GF-Lipos prepared in Example 1 and Dex@Lipos prepared in Comparative Example 1;

[0034] Figure 6 This is a stability change trend diagram of Dex@GF-Lipos prepared in Example 1 at 4°C;

[0035] Figure 7 is a concentration-dependent graph of the inhibition of the growth of synovial fibroblast cell lines in vitro by Dex@GF-Lipos of Example 1;

[0036] Figure 8 This is a graph showing the uptake experiment of Dex@GF-Lipos in Example 1 on a synovial fibroblast cell line in vitro;

[0037] Fig. 9 is the TNF-α concentration in the solution after FLS were treated by different groups in Example 6;

[0038] Fig.10 This is an experimental diagram of Dex@GF-Lipos in Example 7 in inhibiting the expression of MHC-II by synovial fibroblasts;

[0039] Fig.11 is a trend chart showing the distribution of Dex@GF-Lipos and Dex@Lipos loaded with fluorescent molecules in rheumatoid arthritis rats over time in Example 8;

[0040] Fig.12 is the fluorescence intensity graph of the heart, liver, spleen, lung, kidney, and sole taken at 48h in Example 8;

[0041] Fig.13 This is a flow cytometry result of liposome targeting synovial fibroblasts in inflammatory joints in Example 8. DETAILED DESCRIPTION

[0042] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, the equal transformations and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturers are not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In other embodiments, the methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the purport of the present invention.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0044] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0045] Example 1 Preparation of dexamethasone liposomes targeting synovial fibroblasts

[0046] Prepare according to the following steps:

[0047] Step S1: Figure 1 DSPE-PEG 2000 - Preparation of GPA-KLVFF;

[0048] Weigh DSPE-PEG 2000 -NHS 500 mg was dissolved in 15 mL N,N-dimethylformamide (DMF), GPA-KLVFF peptide (200 mg) and triethylamine (50 μL, 36 mg) were added and dissolved completely, and the following was carried out at room temperature: Figure 2 The reaction was performed for 12 h; the reaction solution was transferred to a dialysis bag (molecular weight cutoff 1000 Da), dialyzed in pure water for 48 h, the dialyzate was collected and freeze-dried, and the product DSPE-PEG was obtained by purification with molecular sieves. 2000 -GPA-KLVFF, the structure was confirmed by H NMR. Figure 3 shown.

[0049] Step S2: Preparation of dexamethasone liposomes targeting synovial fibroblasts;

[0050] Components Weight(mg) Molar mass ratio Soy lecithin 120 58.3 cholesterol 10 9.5 <![CDATA[DSPE-PEG 2000 -GPA-KLVFF]]> 10 1 Dexamethasone 10 9.4 Kolliphor HS15 50 53.5

[0051] According to the weight in the table above, weigh appropriate amounts of soybean lecithin, cholesterol, and DSPE-PEG prepared in step S1. 2000 -GPA-KLVFF and dexamethasone were added, and Kolliphor HS15 was completely dissolved in anhydrous ethanol solution. The organic solvent was removed by reduced pressure distillation using a rotary evaporator in a 37°C water bath to form a lipid film.

[0052] 10 mL of 0.9% sodium chloride solution (physiological saline, pH = 7.4) preheated to 37°C was added for hydration. After shaking and incubating in a 37°C water bath for 30 min, probe ultrasonic dispersion (power 10%, 5s on, 3s off) was used for 20 min to obtain FAP-α enzyme-responsive dexamethasone liposomes Dex@GF-Lipos, which were stored at 4°C for future use.

[0053] Example 2 Preparation of dexamethasone liposomes targeting synovial fibroblasts

[0054] Example 2 is a further improvement of Example 1. The preparation steps are the same. The only difference from Example 1 is that soybean lecithin, cholesterol, DSPE-PEG prepared in step S1 2000 - The dosage of GPA-KLVFF and dexamethasone is different. The dosage of each component in Example 2 is as follows:

[0055] Components Weight(mg) Molar mass ratio Soy lecithin 100 60.8 cholesterol 15 17.9 <![CDATA[DSPE-PEG 2000 -GPA-KLVFF]]> 8 1 Dexamethasone 12 14.0 Kolliphor HS15 80 106.9

[0056] Example 3 Preparation of dexamethasone liposomes targeting synovial fibroblasts

[0057] Example 3 is a further improvement of Example 1. The preparation steps are the same. The only difference from Example 1 is that soybean lecithin, cholesterol, DSPE-PEG prepared in step S1 2000 - The dosage of GPA-KLVFF and dexamethasone is different. The dosage of each component in Example 3 is as follows:

[0058] Components Weight(mg) Molar mass ratio Soy lecithin 200 97.2 cholesterol 10 9.5 <![CDATA[DSPE-PEG 2000 -GPA-KLVFF]]> 10 1 Dexamethasone 12 11.3 Kolliphor HS15 80 85.6

[0059] Comparative Example 1 Preparation of Dexamethasone Liposomes

[0060] This comparative example 1 is a further improvement of Example 1. The difference from Example 1 is that DSPE-PEG is not added in this application. 2000 -GPA-KLVFF and DSPE-PEG 2000 , and the addition amounts of the remaining substances are also different.

[0061] Weigh soybean lecithin, cholesterol, DSPE-PEG according to the table below 2000 and dexamethasone:

[0062] Components Weight(mg) Soy lecithin 120 cholesterol 10 <![CDATA[DSPE-PEG 2000 ]]> 10 Dexamethasone 10 Kolliphor HS15 50

[0063] Soy lecithin, cholesterol, DSPE-PEG 2000 , dexamethasone, and Kolliphor HS15 were completely dissolved in anhydrous ethanol solution, and the organic solvent was removed by vacuum distillation using a rotary evaporator in a 37°C water bath to form a lipid film. 10 mL of 0.9% sodium chloride solution (normal saline, pH = 7.4) preheated to 37°C was added for hydration, and after shaking and incubating in a 37°C water bath for 30 minutes, a probe ultrasonic dispersion (power 10%, 5s on, 3s off) was used for 20 minutes to obtain dexamethasone liposomes Dex@Lipos, which were stored at 4°C for later use.

[0064] The hydrated particle size and zeta potential of Dex@GF-Lipos prepared in Examples 1-3 and Dex@Lipos prepared in Comparative Example 1 were measured, the appearance morphology was observed by transmission electron microscopy, the encapsulation efficiency was measured, and the stability test methods and results were as follows:

[0065] 1. Determination of hydrated particle size and zeta potential

[0066] Three samples of each of Examples 1-3 and Comparative Example 1 were prepared, and pure water was used as the test medium. The test temperature was set at 25°C. The hydrated particle size distribution and Zeta potential of the prepared Dex@GF-Lipos and Dex@Lipos were measured using a Malvern Panalytical Zetasizer Lab laser particle size analyzer. Figure 4 As shown in A, 4B, 4C, and 4D, the Dex@GF-Lipos prepared in Examples 1-3 and the Dex@Lipos prepared in Comparative Example 1 are 126.0nm±3.33nm, 121.1nm±6.42nm, 130.0nm±2.74nm, and 119.7nm±8.04nm, respectively.

[0067] 2. Observe the appearance using transmission electron microscopy

[0068] Take 10 μl of the 50-fold diluted liposome solution and drop it onto the copper grid. Use 2% phosphotungstic acid to stain for 3 minutes. After drying at room temperature, use transmission electron microscopy (TEM) to observe the appearance of the liposome. Figure 5 As shown in A and 5B, the Dex@GF-Lipos prepared in Example 1 and the Dex@Lipos prepared in Comparative Example 1 both present a relatively uniform spherical structure.

[0069] 3. Encapsulation Efficiency Determination

[0070] Take 200 μL of the prepared liposomes and add them to a Sephadex G-75 gel column. Use pure water as the eluent to separate the free drug and the drug-loaded liposomes by column chromatography. Collect the liposome components and break the emulsion with 5 times the volume of 1% hydrochloric acid ethanol solution. Determine the content of dexamethasone by high performance liquid chromatography (HPLC). Chromatographic conditions: reverse phase C 18 Chromatographic column 250mm×4.6mm×5μm, mobile phase: water-acetonitrile (70-30), detection wavelength: 242nm. Take another liposome sample 200μL and dilute it to the same volume with pure water, then demulsify it with hydrochloric acid ethanol and dilute it to the same multiple. Under the same conditions, its concentration is measured as C'. The encapsulation efficiency (i.e. the ratio of the amount of drug encapsulated in the liposome to the total amount of drug in the preparation) is calculated as follows: EE (Encapsulation Efficiency) = C / C'×100%. The dexamethasone encapsulation efficiencies of the liposomes of Examples 1-3 and Comparative Example 1 are 88.1%, 91.3%, 92.9%, and 87.6%, respectively.

[0071] 4. Stability test

[0072] The liposome solution of Example 1 was placed at 4°C, and the particle size changes were detected at 0d, 1d, 2d, 5d, 10d, 15d, and 30d. Figure 6 As shown in the figure, the particle size of Dex@GF-Lipos did not change significantly and had good stability.

[0073] Example 4 In vitro cytotoxicity experiment of dexamethasone liposomes targeting synovial fibroblasts

[0074] 1) Cell culture

[0075] Rheumatoid arthritis synovial fibroblast cell line (RA-FLS) was purchased from ATCC and cultured and preserved in this laboratory. The culture conditions of RA-FLS are: RPMI-1640 incomplete medium, which is prepared into complete medium containing 10% fetal bovine serum, 1% penicillin and streptomycin before use, and stored at 4°C for future use. The cells are cultured in a constant temperature incubator at 5% CO2 and 37°C, and passaged when they grow to 80-90%.

[0076] 2) CCK-8 detection of cytotoxicity

[0077] RA-FLS cells in the logarithmic phase were taken and plated in a 96-well plate, with about 1×10 4After the cells adhered to the wall, the set grouping solutions (300μM, 200μM, 100μM, 50μM, and 10μM, respectively, according to dexamethasone) were added, and blank and negative control wells were set up. After incubation for 24 hours, 10L of CCK-8 solution was added to each well, incubated in the dark for 2 hours, the absorbance (A) was measured at 450nm, and the cell survival rate was calculated according to the following formula:

[0078]

[0079] As: absorbance of experimental wells (containing cells, culture medium, CCK-8 solution and drug solution);

[0080] Ac: absorbance of control well (containing cells, culture medium, CCK-8 solution, but no drug);

[0081] Ab: absorbance of blank wells (containing culture medium and CCK-8 solution, but not cells or drugs).

[0082] The results are as follows Figure 7 As shown, compared with the free group, the Dex@GF-Lipos group of Example 1 did not show obvious cytotoxicity.

[0083] Example 5 Experiment on the efficient targeting of synovial fibroblasts by dexamethasone liposomes to synovial fibroblasts in vitro

[0084] 1) Preparation of fluorescent liposomes

[0085] According to the liposome preparation method of Example 1 and Comparative Example 1, the fluorescent substance coumarin-6 was added while the lipid material was dissolved in ethanol to prepare fluorescently labeled Dex@GF-Lipos and Dex@Lipos, respectively.

[0086] 2) Intake experiment

[0087] RA-FLS cells in the logarithmic growth phase were seeded in 12-well plates and incubated overnight at 37°C and 5% CO2 incubator. The next day, the culture medium in the well plate was aspirated, the cells were washed twice with PBS, 1 ml of 1640 culture medium containing each preparation (the concentration of coumarin-6 was 50 ng / mL) was added, and the cells were placed in an incubator for 1 hour, the culture medium was discarded, PBS was washed once, 300 μl of trypsin was added to digest the cells for 2 minutes, the trypsin was discarded, 200 μl of complete culture medium and 800 μl of PBS were added to collect the cells by blowing, centrifuged at 400 g for 4 minutes, washed three times with PBS, and finally resuspended with 500 μl of PBS and detected by flow cytometry.

[0088] The results are as follows Figure 8As shown, compared with the free group and the Dex@Lipos group, the uptake efficiency of Dex@GF-Lipos on RA-FLS was the highest, indicating that Dex@GF-Lipos has the ability to actively target RA-FLS.

[0089] Example 6: Experiment on the anti-inflammatory effect of dexamethasone liposomes targeting synovial fibroblasts in vitro

[0090] RA-FLS cells in the logarithmic growth phase were seeded into 96-well plates and incubated overnight in a 37°C, 5% CO2 incubator. The next day, the culture medium in the well plate was aspirated, and fresh culture medium containing IL-1β was added and incubated for 4 hours. The culture medium in the well plate was aspirated again, and fresh culture medium solutions containing blank liposome carriers, free Dex, Comparative Example 1 Dex@Lipos or Example 1 Dex@GF-Lipos were added according to the grouping, and incubated together for 24 hours. Take out and collect the culture supernatant solution in the well plate. The concentration of TNF-α in the solution was detected by enzyme-linked immunosorbent assay.

[0091] result:

[0092] like Fig. 9 As shown, after the activated FLS was treated with blank liposome carrier (blank liposome carrier without Dex in Example 1), free Dex, Dex@Lipos in Comparative Example 1 or Dex@GF-Lipos in Example 1, the concentration of TNF-α in the solution decreased successively, and there was a significant difference between Dex@GF-Lipos and each group. This indicates that Dex@GF-Lipos has a good anti-inflammatory effect on cells in vitro.

[0093] Example 7: An experiment on down-regulating the expression of MHC-II by synovial fibroblasts using dexamethasone liposomes targeting synovial fibroblasts

[0094] RA-FLS cells in the logarithmic growth phase were inoculated into 12-well plates and incubated overnight at 37°C and 5% CO2 incubator. The next day, the culture medium in the well plate was aspirated, and fresh culture medium solutions containing Dex@Lipos in Comparative Example 1 or Dex@GF-Lipos in Example 1 were added according to the grouping, and incubated together for 6 hours. Take out, discard the culture medium, wash once with PBS, add 300μl of trypsin to digest the cells for 2min, discard the trypsin, add 200μl of complete culture medium and 800μl of PBS to collect the cells, and label the MHC-II on the surface of FLS with MHC-II monoclonal antibodies carrying fluorescent molecules. After incubation for 40min, centrifuge at 400g for 4min, wash three times with PBS, and finally resuspend the cells with 500μl of PBS and detect with flow cytometry.

[0095] result:

[0096] like Fig.10 As shown, compared with the Dex@Lipos group, the MHC-II expression level of FLS was significantly decreased after Dex@GF-Lipos pretreatment.

[0097] Example 8: Experiment on the efficient targeting of liposomes to synovial fibroblasts and inflamed joints in vivo

[0098] 1) Preparation of fluorescent liposomes

[0099] According to the liposome preparation method of Example 3 and Comparative Example 1, the fluorescent substance DID was added during the ethanol dissolution to prepare fluorescently labeled Dex@GF-Lipos and Dex@Lipos, respectively.

[0100] 2) Establishment of adjuvant-induced arthritis rat model

[0101] Complete Freund's adjuvant (CFA) was used to induce the onset of AIA model rats. 180±20g healthy male SD rats were subcutaneously injected with 50μL CFA (10mg / mL) in the hind paws on the left and right sides of the 0th day. The CFA suspension needed to be shaken before injection. After modeling, the disease development was monitored every day to ensure that the animals had enough food and water. Generally, the onset of the disease reached its peak 2 weeks after modeling.

[0102] 3) In vivo experiments targeting inflamed joints

[0103] On the 5th day after AIA modeling, AIA rats were randomly divided into 3 groups (3 rats / group), and 100 μL of free Did solution (5% 1,2-propylene glycol, Did was 10 μg / 100 μL), Did-labeled Dex@Lipos (Did was 10 μg / 100 μL), and Did-labeled Dex@GF-Lipos (Did was 10 μg / 100 μL) were injected into the tail vein, respectively, and recorded as hour 0. Subsequently, at different set time points (1h, 6h, 48h), the in vivo fluorescence distribution was collected using the small animal in vivo imaging system (IVIS), and the rats were killed at hour 48, and the main organs (heart, liver, spleen, lung, kidney) and soles were removed, and the fluorescence distribution of the ex vivo tissues was collected using the IVIS system.

[0104] result:

[0105] Fig.11 The results showed that after administration, the average fluorescence intensity of the free Did group at the inflamed sole was significantly weaker than that of the Dex@Lipos and Dex@GF-Lipos groups, and the gap widened rapidly; the average fluorescence intensity of the Dex@GF-Lipos group was also significantly stronger than that of the Dex@Lipos group. The difference between the three continued until 48 hours after administration, and the fluorescence intensity of the organs removed at 48 hours further confirmed the result ( Fig.12 A, 12B).

[0106] 4) In vivo experiments targeting synovial fibroblasts in inflammatory joints

[0107] After the rats were killed at 48h, the inflamed paws of the rats in each group were removed, the paws were depilated and cut into pieces, 1.5mL of type IV collagenase digestion solution (1mg / mL DPBS solution) was added, and the mixture was evenly mixed with a pipette, and incubated on a shaker at 37℃ for 1.5-2h. The paws were taken out, centrifuged at 3000g, the supernatant was discarded, and 1.5mL of PBS was added to wash again, and the supernatant was discarded after centrifugation.

[0108] Next, according to the cell surface molecule staining procedure, the cell pellet was incubated with the fluorescently labeled monoclonal antibody PE-Vimentin at 4°C for 40 min. The supernatant was removed by centrifugation at 600 g, and the cells were washed with PBS and resuspended in 400 μL PBS. The content of Did+Vimentin+ positive cells was detected by flow cytometry.

[0109] result:

[0110] Fig.13 The results showed that the positive rate of Did+Vimentin+ cells in the single cell suspension of the inflamed sole of the foot in the Dex@GF-Lipos group was 2.5 times that of the free group; the positive rate of Did+Vimentin+ cells in the Dex@Lipos group was 2 times that of the free group. The positive rate of Did+Vimentin+ cells in the Dex@GF-Lipos group was higher than that in the Dex@Lipos group, and the difference was significant. This shows that Dex@GF-Lipos can effectively target synovial fibroblasts in vivo.

[0111] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A dexamethasone liposome targeting synovial fibroblasts, characterized in that: The invention comprises dexamethasone and a lipid bilayer for encapsulating dexamethasone, wherein the lipid bilayer comprises DSPE-PEG 2000 -GPA-KLVFF, the DSPE-PEG 2000 The structural formula of -GPA-KLVFF is shown in formula (I).

2. The dexamethasone liposome targeting synovial fibroblasts according to claim 1, characterized in that: The lipid bilayer is also made of soybean lecithin and / or cholesterol.

3. The dexamethasone liposome targeting synovial fibroblasts according to claim 1, characterized in that: The hydrated particle size of the dexamethasone liposome is 111.5 nm to 130.1 nm.

4. Use of the dexamethasone liposome targeting synovial fibroblasts as claimed in any one of claims 1 to 3 in the preparation of a drug for preventing / improving / treating rheumatoid arthritis.

5. A pharmaceutical composition for preventing / improving / treating rheumatoid arthritis, characterized in that: The effective component thereof comprises the dexamethasone liposome targeted to synovial fibroblasts as claimed in any one of claims 1 to 3.

6. The method for preparing the dexamethasone liposome targeting synovial fibroblasts according to any one of claims 1 to 3, comprising the following steps: Step S1: DSPE-PEG 2000 - Preparation of GPA-KLVFF; Weigh an appropriate amount of DSPE-PEG 2000 -NHS was dissolved in N,N-dimethylformamide, GPA-KLVFF peptide and triethylamine were added to dissolve completely, and the reaction was carried out at room temperature; the reaction solution was transferred to a dialysis bag, the dialysis medium was purified water, the dialysate was collected and freeze-dried, and DSPE-PEG was obtained after purification by molecular sieve. 2000 -GPA-KLVFF; Step S2: Preparation of dexamethasone liposomes targeting synovial fibroblasts; Weigh soybean lecithin, cholesterol, DSPE-PEG in proportion 2000 -GPA-KLVFF and dexamethasone, and add Kolliphor HS15 to completely dissolve in anhydrous ethanol solution, and remove the organic solvent by reduced pressure distillation using a rotary evaporator in a 37°C water bath to form a lipid film; Sodium chloride solution was added for hydration, and then the mixture was incubated in a water bath and then subjected to ultrasonic treatment to obtain dexamethasone liposomes targeting synovial fibroblasts.

7. The method for preparing a dexamethasone liposome targeting synovial fibroblasts according to claim 6, characterized in that: The DSPE-PEG in step S1 2000 The molar ratio of -NHS, N,N-dimethylformamide, GPA-KLVFF peptide and triethylamine is 1:8-9:1:2-3.

8. The method for preparing a dexamethasone liposome targeting synovial fibroblasts according to claim 6, characterized in that: The molar mass ratio of soybean lecithin, cholesterol, DSPE-PEG2000-GPA-KLVFF, dexamethasone and Kolliphor HS15 in step S2 is 50-100:9-18:1:9-14:50-110.

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