WO2023011287A1 - Vesicle nanomedicine carrying chloroquine compound, and preparation method and application thereof - Google Patents

Abstract

A vesicle nanomedicine carrying a chloroquine compound, and a preparation method and application thereof. Using a polymer and a chloroquine compound as raw materials to prepare a vesicle nanomedicine carrying a chloroquine compound; the polymer comprises a hydrophilic segment and a hydrophobic segment, wherein, a side chain of the hydrophobic segment is dithiolane containing a disulfide bond. A macrophage-targeting nanomedicine is used for treating rheumatoid arthritis, efficient polymer vesicle loading, targeted delivery and controlled release of medicinal hydroxychloroquine or chloroquine, and increased enrichment of drugs in the cytoplasm; repolarizing M1M to M2M, thereby reducing secretion of pro-inflammatory cytokines, increasing secretion of anti-inflammatory cytokines, inhibiting DC activation, removing ROS, and enrichment in inflamed joints.

Description

A kind of vesicular nano drug loaded with chloroquine compound and its preparation method and application technical field

The invention belongs to nano-medicine technology, in particular to vesicle nano-medicine loaded with chloroquine compound and its preparation method and application.

Background technique

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint pain, cartilage damage, and bone loss (D. L. Scott, F. Wolfe, T. W. J. Huizinga, Rheumatoid arthritis, The Lancet, 2010, 376, 1094-1108). At present, the etiology of RA is still unclear, and there is no radical cure, and it is necessary to take medicine for life. The existing clinical therapies mostly use the combination of non-steroidal drugs, glucocorticoids, antirheumatic drugs (DMARDs) and biological agents. For example, low-dose MTX has been the most commonly used clinical therapy to control the progress of RA since 1980. Often used as the first choice, but the effective concentration of MTX at the site of inflammation is low, and the systemic toxicity is high (M. TR, O. D. J, The changing face of rheumatoid arthritis therapy: results of serial surveys, Arthritis & rheumatology, 2000, 43, 464-465). Hydroxychloroquine (HCQ) is a clinically common drug used for autoimmune diseases. It has curative effect on RA and systemic lupus erythematosus (SLE), and has little systemic toxicity. However, its daily oral dose is 200-400 mg, which is a large amount , slow onset. Biological drugs developed in recent years, including trastuzumab, etanercept, and infliximab, can antagonize IL-6 receptors or TNF-α receptors to alleviate the process of RA. However, antibody drugs have the disadvantages of low bioavailability, cumbersome production process, and high price (P. R. Stocco Romanelli, Biologics for rheumatoid arthritis: an overview of Cochrane reviews, Sao Paulo Medical Journal, 2010, 128, 309-357).

The prior art uses nanocarriers such as liposomes, lipid nanoparticles, PLGA, and silver nanoparticles to load anti-inflammatory drugs such as dexamethasone, prednisolone, p-coumaric acid, MTX, or siTNF-α for treatment RA has achieved certain results; however, there are still the following problems: poor stability, early release of the drug leads to systemic toxicity; the effective concentration of the drug at the inflammatory site is low, and effective treatment cannot be achieved. Therefore, it is urgent to develop a nano-delivery system that can specifically target the inflammatory site of RA to achieve efficient targeted therapy of RA.

technical problem

The invention discloses vesicle nanomedicine loaded with chloroquine compound and its preparation method and application. The delivery system based on nanocarrier can change the biodistribution of medicine, can be enriched in joints, reduce toxic and side effects, and be applied to the treatment of RA to overcome Limitations of anti-RA drugs.

technical solution

The present invention adopts the following technical scheme: vesicle nanomedicine loaded with chloroquine compound, including polymer vesicles and chloroquine compound; the polymer includes a hydrophilic segment and a hydrophobic segment, and the side chain of the hydrophobic segment is a disulfide bond-containing of dithiolane. Preferably, the polymer is a non-targeting polymer, or the polymer is a mixture of a non-targeting polymer and a targeting polymer; the non-targeting polymer includes PEG-P (TMC-DTC) , PEG-P (CL-DTC) or PEG-P (LA-DTC); targeting polymers include B-PEG-P (TMC-DTC), B-PEG-P (CL-DTC), or B-PEG -P(LA-DTC), B is targeting molecules, such as folic acid, mannose, dextran, hyaluronic acid, galactose, etc.

In the present invention, polymers are self-assembled to form polymer vesicles; polymers and chloroquine compounds are used as raw materials to prepare vesicle nanomedicines loaded with chloroquine compounds. Preferably, the chloroquine compound is loaded into the vesicle by a pH gradient method to obtain a vesicle nanomedicine loaded with the chloroquine compound. Specifically, the polymer is self-assembled in an acid buffer to form a polymer vesicle solution, and then a chloroquine compound solution is added to the polymer vesicle solution under alkaline conditions to obtain a vesicle nanomedicine loaded with a chloroquine compound.

In the present invention, the molecular weight of the polymer is 10-50 kg/mol, preferably, the molecular weight of the hydrophilic segment is 2-10 kg/mol.

In the present invention, when the polymer is a mixture of a non-targeting polymer and a targeting polymer, the molar ratio of the non-targeting polymer to the targeting polymer is 1: (0-0.8), excluding 0.

In the present invention, the chloroquine compound is an anti-autoimmune disease drug, which is a small molecule drug, such as hydroxychloroquine, chloroquine and the like. As a specific example, the present invention discloses mannose-modified polymer vesicles for efficient loading, targeted delivery and controlled release of hydroxychloroquine (Man-PS-HCQ), in order to realize the effect on zymosan-induced RA mouse model ( Efficient and safe treatment of ZIA), the vesicles are made of block polymer polyethylene glycol- b -poly(trimethylene carbonate- co -dithiolane trimethylene carbonate) (PEG-P(TMC- DTC)) and mannose-coupled Man-PEG-P (TMC-DTC) self-assembled and loaded with HCQ by the pH gradient method. Targeted delivery of hydroxychloroquine to macrophages at the site of inflammation through vesicles can increase the accumulation of the drug in diseased joints, increase the pH in cell lysosomes, reduce enzyme activity, and reduce the expression of MHC-II on the surface of APCs. Reduce the dosage of hydroxychloroquine and reduce its toxic and side effects. The experiment found that in the arthritis mouse model, the tail vein injection of Man-PS-HCQ showed better curative effect than the free HCQ and PS-HCQ groups, significantly reduced the joint swelling of the mice, and increased the M2M concentration in the joints. enrichment, while M1M, DC (CD11c ⁺ CD80 ⁺ CD86 ⁺ ), CD8 ⁺ T cells and CD4 ⁺ T cells were all decreased, indicating that Man-PS-HCQ can effectively improve the immune microenvironment in diseased joints and help relieve Inflammation, cartilage damage and bone loss.

The invention discloses a freeze-dried powder of a vesicular nano-medicine loaded with a chloroquine compound. The preparation method is as follows: mixing the vesicular nano-medicine loaded with a chloroquine compound with a freeze-drying protective agent, and then freeze-drying to obtain a freeze-dried powder of a vesicular nano-medicine loaded with a chloroquine compound. dry powder. Preferably, the lyoprotectant is sucrose and/or mannitol; more preferably, the amount of the lyoprotectant is 2-6 wt%.

The invention discloses the application of vesicle nano-medicine loaded with chloroquine compound in the preparation of medicine for treating rheumatoid arthritis. The invention discloses the application of a vesicle nano-medicine loaded with a chloroquine compound in preparing a medicine for repolarizing M1M macrophages into M2M macrophages. The invention discloses the application of a vesicle nano-medicine loaded with a chloroquine compound in the preparation of a drug for eliminating ROS. The invention discloses the application of a vesicle nano-medicine loaded with a chloroquine compound in the preparation of a drug for inhibiting BMDC activation. The invention discloses the application of vesicle nano-medicine loaded with chloroquine compound in the preparation of medicine for protecting articular cartilage and bone tissue. The invention discloses the application of a vesicle nano-medicine loaded with a chloroquine compound in the preparation of an anti-inflammatory drug or an anti-autoimmune disease drug.

Beneficial effect

In recent years, studies have found that antigen-presenting cells (APCs) such as macrophages and dendritic cells play an important role in the occurrence and development of rheumatoid arthritis (RA). The content of macrophages in the joints of RA patients Many, activated macrophages can release a large number of pro-inflammatory cytokines, which can exacerbate inflammation, cause cartilage damage and bone loss. The present invention develops safe and efficient macrophage-targeted nano-medicines for the treatment of RA, and designs polymer vesicles for efficient loading, targeted delivery and controlled release of anti-RA drugs. Taking hydroxychloroquine as an example, the results of in vitro cell experiments show that Man-PS-HCQ can target macrophages to deliver HCQ, increase the enrichment of HCQ in the cytoplasm, repolarize M1M to M2M, reduce the secretion of pro-inflammatory cytokines, Increased secretion of anti-inflammatory cytokines can inhibit DC activation and clear ROS; it can also be enriched in inflammatory joints of ZIA mice. The study found that the targeting effect of Man-PS-HCQ on M2M was slightly stronger than that of M1M, while the endocytic ability of M1M was much higher than that of M2M, and M1M was the main source of pro-inflammatory cytokines in the RA site. Strong endocytosis is more conducive to its repolarization into M2M, reducing the secretion of pro-inflammatory cytokines and increasing the secretion of anti-inflammatory cytokines. Animal experiments in ZIA mice found that the content of M1M in RA joints increased significantly, and Man-PS-HCQ could gather more in the inflammatory sites of mice, and repolarize M1M to M2M. Both in vitro and in vivo experimental results confirmed that Man-PS-HCQ can target the treatment of RA.

Description of drawings

Figure 1 is the ¹ H NMR charts (400 MHz, DMSO- d ₆ ) of NHS-PEG-P(TMC-DTC) (a) and Man-PEG-P(TMC-DTC) (b).

Figure 2 shows the physicochemical properties of Man-PS-HCQ and PS-HCQ (a) Size and size distribution determined by DLS (inset: TEM image). (b) Changes in size and size distribution of PS-HCQ and Man PS-HCQ at different concentrations (1.0 mg/mL, 50-fold dilution (0.02 mg/mL), (c) in PB containing 10% FBS solution (pH 7 .4) for 24 hours, or (d) PB (pH 7.4, 10 mM GSH) at 37°C for 24 hours, (e) at 37°C with or without 10 mM GSH (vesicle concentration: 1.3 mg/mL) In vitro release profile of Man-PS-HCQ, (f) PS-HCQ and Man-PS-HCQ in PB (pH 6.0, 3% H2O2) after 24 hours at 37°C (n=3) Size and size distribution changes in (g) pH 6.0, 3% H2O2, 37 ℃ (vesicle concentration: 1.3mg/mL), the in vitro release curves of PS-HCQ and Man-PS-HCQ, in (h ) 4.4 wt.% and (i) 2.2 wt.% concentration, the particle size distribution of lyophilized PS-HCQ powder in deionized water.

Figure 3 shows the effect of Man-PS-HCQ and PS-HCQ on the expression of cytokine LPS/IFN-γ in LPS-treated RAW 264.7 cells and LPS/IFN-γ co-treated BMDM (10g HCQ/mL). RAW 264.7 cells stimulated by LPS (100ng/mL) were incubated with PBS, free HCQ, PS-HCQ or Man-PS-HCQ for 24h (a, b), and the protein levels of IL-6 and IL-10 were determined by ELISA kit (c , d), qRT-PCR detection of mRNA expression of IL-6 and IL-10. Data were normalized by GAPDH expression level. BMDM cells treated with LPS (100 ng/mL) and IFN-γ (20 ng/mL) were incubated with PBS, human PS, free HCQ, PS-HCQ or Man-PS-HCQ for 24 hours (e, f) IL-6 and Protein level of IL-10, (g) ratio of IL-10 to IL-6. (h) Effect of HCQ concentration on IL-10 production by BMDMs (n=6).

Figure 4 shows the stimulation of BMDMs (CD11b ⁺ F4/80 ⁺ CD206 ⁺ Cells) incubated for 24 h to analyze the repolarization effect by flow cytometry.

Figure 5 shows (a) flow cytometry analysis of BMDM with different phenotypes after incubation with PS-Cy5 and Man-PS-Cy5 (0.2 μg Cy5/mL) for 4 hours, (b) CD206 expression in M0M, M1M and M2M.

Figure 6 shows (CD80+CD86+) (a), MHC-II ⁺ expression (b) and The effect of quantification (c) was compared with PBS, free HCQ or PS-HCQ (n=3).

Fig. 7 CLSM images of ROS in (a) RAW 264.7 cells and (b) BMDM cells stained with DCFH-DA (green). LPS or LPS/IFN-γ pretreated cells were incubated with HCQ preparations for 24 hours. Scale bars: (a) 500 μm and (b) 100 μm (n=3).

Figure 8 shows the cytotoxicity of PS-HCQ and Man-PS-HCQ (HCQ concentration is 5, 10 μg/mL); (a) RAW 264.7 cells and (b) BMDMs cells were cultured for 24 hours (n=6).

Figure 9 shows the intravenous injection of Cy5-labeled Man-PS-HCQ and PS-HCQ (0.3 μg Cy5 equv./mice, 1.2 mg HCQ/kg, n = 3) Fluorescence images of ZIA mouse model after (a) intravital imaging and (b) left leg semiquantification (n = 3).

Figure 10 is a preliminary study of the effect of Man-PS-HCQ on ZIA mice (n=5) (a) Treatment scheme. Drugs administered at 0.6, 1.2, or 2.4 mg HCQ/kg on days 0 and 3, respectively (b) leg circumference and (c) knee diameter (d) relative body weight, (e, f, g, h, i, j ) Serum transforming growth factor TGF-β concentrations under different treatments on day 1, day 0, day 3 or day 7 (n=5).

Figure 11 is the anti-RA treatment of Man-PS-HCQ on ZIA mouse model (a) treatment plan, (b) mouse leg circumference, (c) knee diameter or (d) body weight changes; on day 0 and On day 3, HCQ preparations were injected intravenously at 1.2 mg HCQ/kg; serum IL-6 (e) and TGF-β concentrations (f) were detected on days 0, 3, and 7; serum (g ) and IL-6, TNF-α, Expression of IL-1β, IL-10 and TGF-β (n=6).

Figure 12 is the section image of the mouse joints stained by H&E, safranin fast green, and Trap, and the mice were treated with the scheme shown in Figure 11.

Figure 13 is the H&E staining analysis of the major organ sections of mice treated with the scheme shown in Figure 11.

Fig. 14 is the staining analysis of CD206 antibody in bone and joint sections of mice treated with the scheme shown in Fig. 11 .

Figure 15 shows the establishment of the ZIA model (PBS group) and the cell characterization data in the mouse joints.

Embodiments of the present invention

Hydroxychloroquine sulfate (HCQ, > 99%, Beijing Yinuokai Technology Co., Ltd.), chloroquine phosphate (CQ, > 99%, Aladdin Reagent (Shanghai) Co., Ltd.), D-mannosamine hydrochloride (> 98%, Bailingwei), glutathione (GSH, > 99%, Roche), CpG (Shanghai Gemma Pharmaceutical Technology Co., Ltd.), lipopolysaccharide (LPS, sigma-aldrich) were used directly after purchase. IL-4, IFN-γ, M-CSF, and GM-CSF were all > 99%, purchased from PeproTech and used directly. Mouse Interleukin-6, 10, 1β (IL-6, 10, 1β), Transforming Growth Factor-β (TGF-β) and Necrosis Factor-α (TNF-α) ELISA Kit (Invivogen) , and mouse fluorescently labeled various antibodies (Biolegend) CD80-APC, CD86-PE, CD11c-FITC, CD11b-FITC, CD206-APC, F4/80-PE, CD3-APC, CD4-PE, and CD8-FITC After the kits and antibodies were purchased, they were used according to the instructions.

Proton nuclear magnetic resonance spectrum ( ¹ H NMR) was tested with a nuclear magnetic resonance spectrometer model Unity Inova 400, the deuterated reagent was DMSO- d ₆ , and the chemical shift was based on the residual DMSO signal peak. The particle size, PDI and surface zeta potential of polymersomes were determined by Zetasizer Nano-ZS (Malven Instruments, UK), using a He-Ne laser light source with a wavelength of 633 nm and a backscattering detector. The microscopic morphology of the samples was determined by a Tecnai G220 transmission electron microscope (TEM) at an accelerating voltage of 120 kV, and the samples were stained with 1% phosphotungstic acid solution. The uptake of vesicles by cells was determined by using a BD FACSVerse flow cytometer (Becton Dickinsion, FACSVerse, USA). The drug loading of HCQ in vesicles was determined by UV-Vis spectrometer (HITACHI) at 343 nm. Nanoparticle freeze-dried powder preparations were obtained by freeze-drying with a freeze dryer (CHIRIST, ALPHA1-2). In the in vitro drug release experiment, the concentration of HCQ was measured by a high-performance liquid chromatography (HPLC) equipped with a reversed-phase C18 chromatographic column (4.6×150 mm, 5 μm). Phase A was acetonitrile, and phase B was an aqueous phase (50 mM phosphoric acid Potassium dihydrogen, 6.5 mM sodium ethanesulfonate, 7 mM triethylamine, pH adjusted to 3.0 with phosphoric acid). The mobile phase was A:B = 22:78 (v/v), the flow rate was 1 mL/min, and the detection wavelength was 330 nm. A microplate reader (Thermo Multiskan FC) was used to measure the absorbance at 570 nm of purple formazan formed by living cells and MTT. A multifunctional microplate reader (Varioskan LUX, Thermo Scientific) was used for ELISA detection of cytokines. In vivo and ex vivo images of mice were taken by infrared fluorescence imager (IVIS, Lumina II; Caliper, MA).

Cell culture and experimental animals: The mouse macrophage cell line RAW 264.7 was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The extraction steps of bone marrow-derived macrophages (BMDM) and bone marrow-derived dendritic cells (BMDC) were as follows: the mice were killed by cervical dislocation, sprayed with alcohol all over the body, and transferred to an ultra-clean table. The bones of the limbs of the mice were taken out, soaked in PBS, transferred to the ultra-clean bench in the cell room, and the cells in the bone marrow of the limbs of the mice were washed by PBS perfusion into a centrifuge tube. Filter the impurities in the cells with a membrane filter, and then centrifuge (1500 rpm) for 5 min. The supernatant was discarded, and 3 mL of cell lysis red solution was added to the lower layer of cells to lyse the red blood cells for 5-8 min. Add 10 mL of PBS to neutralize and then centrifuge, discard the supernatant, and spread the lower layer of cells into a 6-well plate at a density of 3×10 ⁶ /well. After culturing with 1640 medium containing 25 ng/mL M-CSF for 3 days, the medium was changed completely, after 5 days, the medium was half changed, and BMDM was obtained after 7 days; after culturing with 1640 medium containing 20 ng/mL GM-CSF for 2 days, the medium was completely changed After 4 days, 6 days and 8 days, the medium was half changed, and BMDC was obtained after 9 days.

The mice used for cell extraction were female C57BL/6 at the age of 5-6 weeks, and the mice for establishing the ZIA model were female C57BL/6 at the age of 12. The mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. All animal experiments and manipulations were approved by the Experimental Animal Center of Soochow University and the Animal Care and Use Committee of Soochow University.

Preparation example: The polymer disclosed in the present invention is a prior art, and its preparation and characterization can refer to the applicant’s published literature or patent application, such as CN2016105581166, Y. Fang, W. Yang, L. Cheng, F. Meng, J . Zhang, Z. Zhong, EGFR-targeted multifunctional polymersomal doxorubicin induces selective and potent suppression of orthotopic human liver cancer in vivo, Acta Biomaterialia, 2017, 64, 323-333. Take the preparation of PEG-P(TMC-DTC) (5k-15k-2k) and NHS-PEG-P(TMC-DTC) (6.5k-15k-2k) used in the examples as an example to briefly explain.

Under nitrogen atmosphere, weigh MeO-PEG-OH ( M _n = 5.0 kg/mol, 0.009 mmol), TMC (1.93 mmol) and DTC (0.21 mmol) successively and dissolve in dichloromethane (DCM, 6.8 mL) , stirring and adding the catalyst diphenyl phosphate (DPP, DPP/OH molar ratio is 10/1). The airtight reactor was sealed and placed under magnetic stirring in an oil bath at 40 °C for 24 hours. After terminating the reaction with glacial acetic acid, precipitate twice in glacial ether, filter with suction, and dry under vacuum at room temperature to obtain the product PEG-P(TMC-DTC) (5k-15k-2k). Yield: 91.8%.

The reaction is shown below.

.

.

The above-mentioned initiator MeO-PEG-OH is replaced by NHS-PEG6.5k-OH functionalized by N-hydroxysuccinimide, and ring-opening polymerization TMC and DTC obtain NHS-PEG-P (TMC-DTC) (6.5k- 15k-2k).

Man-PEG-P (TMC-DTC) can be prepared by amidation reaction of NHS on PEG of D-mannosamine and NHS-PEG-P (TMC-DTC), and the preparation route is as follows.

.

The specific method is as follows: firstly dissolve NHS-PEG-P(TMC-DTC) (523 mg, 22.2 nmol) in anhydrous DMF (100 mg/mL) under nitrogen atmosphere. Then D-mannosamine hydrochloride (24 mg, 111 nmol) was dissolved and desalted with triethylamine (22.5 mg, 222 nmol) to obtain D-mannosamine solution, which was then slowly added dropwise to 37 ^o C NHS- In the DMF solution of PEG-P(TMC-DTC), the reaction was continued for 48 h after the dropwise addition was completed. The reaction solution was dialyzed in DMF for two days (MWCO 7000 Da), DCM for one day, and finally precipitated twice in 30 times the volume of glacial ether, filtered by suction, and dried in vacuum for 24 hours to obtain a white solid Man-PEG-P(TMC -DTC). Yield: 92%. The proton NMR spectrum (Figure 1, DMSO- d ₆ , 400 MHz, ppm) shows the characteristic peaks of the polymer: δ1.91(-OCOCH2CH2CH2CO-), 3.04 (-C(CH2SSCH2)C-), 3.48 (- CH2CH2O-) _, 4.11 (-OCOCH2CH2CH2O-), 4.22 (-OCOCH2( _{CH2SSCH2 ) CH2O-} ). The characteristic peak of Man appeared around 4.9, and the functionalization degree of Man was calculated as 95% through the ratio of the reduction of NHS characteristic peak at 2.51 to the PEG peak at 3.48.

Polymers with different molecular weights can be obtained by adjusting the proportion of raw materials used or replacing PEG with different end-capping groups, as shown in Table 1.

.

Similarly, block polymers with reactive end groups were reacted with D-mannosamine hydrochloride (Man) to obtain mannose-targeted amphiphilic polymers.

Example 1 Preparation and characterization of vesicles of mannose-modified chloroquine compounds.

Preparation and characterization of mannose-modified hydroxychloroquine-loaded vesicles (Man-PS-HCQ): Hydroxychloroquine was loaded into vesicles by a pH gradient method. The two polymers Man-PEG-P (TMC-DTC) and PEG-P (TMC-DTC) were dissolved in DMF respectively, then mixed at a molar ratio of 10/90, and added to 150 nM citric acid-sodium citrate (pH=3.0) buffer solution, stirred for 3 minutes to form vesicles, and after standing at room temperature for 1 hour, NaOH aqueous solution was added to adjust the pH to 8.5 to establish a pH gradient inside and outside the vesicles. Then add a certain amount of HCQ aqueous solution, place in a shaker (37oC, 100 rpm) overnight, and then dialyze with secondary water for 8 hours, changing the medium every hour to obtain drug-loaded vesicles 10Man-PS-HCQ.

According to the above method, only use PEG-P (TMC-DTC) as the polymer, without adding Man-PEG-P (TMC-DTC), to obtain the drug-loaded vesicle PS-HCQ; according to the above method, according to the molar ratio of 20/80 Mix Man-PEG-P (TMC-DTC) and PEG-P (TMC-DTC) to obtain drug-loaded vesicle 20Man-PS-HCQ. According to the above method, without adding hydroxychloroquine, empty vesicles are obtained, that is, according to the molar content of Man-PEG-P (TMC-DTC) is 0, 10%, or 20% ratio and PEG-P (TMC-DTC) The empty polymersome Man-PS can be obtained by mixing the DMF solution.

The particle size and particle size distribution of freshly prepared vesicles, 50-fold dilution, 10% FBS, and in vitro simulated reducing conditions were measured by dynamic laser light scattering (DLS). HCQ is quantified based on the UV absorbance at 343 nm and based on a standard curve, and the drug loading capacity (DLC) and drug loading efficiency (DLE) can be routinely calculated:

.

The calculation results of drug loading of HCQ showed that the increase in the density of targeting molecules did not affect the loading efficiency of HCQ in vesicles (Table 2). When the drug loading increased from 20 wt.% to 33.3 wt.%, the final drug loadings were 13 wt.% and 20 wt.%. The increase of Man density and drug loading will not have a significant impact on the surface potential of the vesicle drug, which is electrically neutral. DLS results showed that the vesicles with drug loading at 20 wt.% and 33.3 wt.% had smaller sizes (46-49 nm), and the particle size distribution PDI was 0.12-0.15 (Figure 2 a). It is shown that Man-PS-HCQ has an obvious vesicle structure, and the particle size is also similar to the results measured by DLS. The hydroxychloroquine liposome particle size disclosed in the prior art is between 90-100 nm, and the PDI reaches 0.18-0.2. Unless otherwise stated below, the vesicle drugs used are Man with a targeting density of 10%, abbreviated as Man-PS-HCQ, and the corresponding empty vesicles are Man-PS.

Preparation and characterization of mannose-modified chloroquine-loaded vesicles (Man-PS-CQ): Chloroquine was loaded into vesicles by a pH gradient method. The two polymers Man-PEG-P (TMC-DTC) and PEG-P (TMC-DTC) were dissolved in DMF respectively, then mixed at a molar ratio of 10/90, and added to 150 nM citric acid-sodium citrate (pH=3.0) buffer solution, stirred for 3 minutes to form vesicles, and after standing at room temperature for 1 hour, NaOH aqueous solution was added to adjust the pH to 8.5 to establish a pH gradient inside and outside the vesicles. Then add a certain amount of CQ aqueous solution, place in a shaker (37°C, 100 rpm) overnight, and then dialyze with secondary water for 8 hours, changing the medium every hour to obtain drug-loaded vesicles 10Man-PS-CQ. In the same way, the ratio of the two polymers Man-PEG-P (TMC-DTC) and PEG-P (TMC-DTC) was changed to obtain PS-CQ and 20Man-PS-CQ. The characterization results are shown in Table 2.

.

Replace Man-PEG-P (TMC-DTC) and PEG-P (TMC-DTC) with the polymers in Table 1 or other amphiphilic polymers containing DTC units previously disclosed by the applicant, and the above method can also be used to obtain Polymer vesicles loaded with hydroxychloroquine and targeted polymer vesicles loaded with hydroxychloroquine can be used as nano-medicines. Or replace hydroxychloroquine with chloroquine to obtain polymer vesicles loaded with chloroquine and targeted polymer vesicles loaded with chloroquine, which can be used as nano-medicines.

Hydroxychloroquine was loaded into vesicles by the pH gradient method. Dissolve PEG-P(TMC-DTC) polymer in DMF, add to 150 nM citric acid-sodium citrate (pH=3.0) buffer solution, stir for 3 minutes to form vesicles, leave at room temperature for 1 hour, add NaOH Adjust the pH of the aqueous solution to 7.4 to establish a pH gradient inside and outside the vesicle. Then add a certain amount of HCQ aqueous solution, put it in a shaker (37oC, 100 rpm) overnight, and then dialyze with secondary water for 8 hours, and change the medium every hour to obtain drug-loaded vesicles PS-HCQ, with a theoretical drug-loading capacity of 20%. , the actual drug loading was 6.3±0.2%. In the same way, hydroxychloroquine was replaced by chloroquine to obtain drug-loaded vesicle PS-CQ. When the theoretical drug-loaded amount was 20%, the actual drug-loaded amount was 8.1±0.2%.

Example 2 In vitro drug release of Man-PS-HCQ.

Add 1 mL of Man-PS-HCQ (concentration: 1.3 mg/mL, HCQ drug loading: 12.5wt.%) into the dialysis bag (MWCO 12000 Da), submerge in 20 mL of secondary water with pH 7.4 and simulate In the reducing environment (pH 7.4, 10 mM GSH) aqueous solution in the cytoplasm, placed in a 37-degree air shaker (200 rpm), at the set time points (0.25, 0.5, 1, 2, 4, 6, 8, 12 , 24 hours), take out 5 mL of dialysate, and then add 5 mL of corresponding fresh dialysate. The concentration of HCQ in the medium taken out was tested by HPLC and the cumulative release amount was calculated (n=3, the experimental results were averaged). The cumulative release amount can be calculated by the following formula:

.

In the formula: E _r is the cumulative release of HCQ, %; Ve: the replacement volume of the medium, 5 mL; V ₀ : the total volume of the medium, 20 mL; Ci: the concentration of HCQ in the medium at the i-th sampling, μg/ mL; m: the total mass of HCQ in Man-PS-HCQ, μg; n: the number of medium replacements.

The prepared vesicle nanomedicine has very good reproducibility. In addition, the particle size of Man-PS-HCQ not only remained unchanged when stored for 1-2 months, but also after dilution of simulated intravenous injection (Figure 2 b), in the presence of 10% In the solution of FBS (Figure 2 c), the particle size can still be kept stable, showing better colloidal stability. However, in simulated intracellular reducing conditions (10 mM GSH), 100-1000 vesicles appear within 8 hours nm peak (Fig. 2d), Man-PS-HCQ released 70-80% of HCQ in 4 hours under the same reducing conditions, while under physiological conditions, the drug release was < 15% in 12 hours (Fig. 2e ), showing the possibility of its rapid release of HCQ inside macrophages.

In order to study the degradation and drug release of nanomedicine at the _site of inflammation, 1 mL of PS-HCQ or Man-PS _- HCQ (capsule Bubble concentration: 1.3 mg/mL, HCQ drug loading: 12.5 wt.%) into the dialysis bag, respectively submerged in 20 mL of the above simulated inflammation solution, placed in a 37-degree air shaker (200 rpm), at the set At time points (2, 4, 12, and 24 hours), 4 mL of dialysate was withdrawn, and 4 mL of corresponding fresh dialysate was added. The dialysate was used for HPLC determination, and an appropriate amount of nanomedicine was taken out at the corresponding time point to test its particle size change by DLS.

In the in vitro simulated inflammatory microenvironment (pH 6.0, PB containing 3% H ₂ O ₂ ), the vesicles swelled significantly after 12 hours (Fig. 2 f), and 60% of the vesicles could be released in 2 hours under these conditions About HCQ, the release amount can reach more than 90% after 24 h (Fig. 2 g). Combined with the previous drug release of vesicles under reducing conditions in simulated cells, these results confirm that Man-PS-HCQ may achieve circulation stability in vivo, rapidly release drugs in inflammatory sites and cells, and achieve the purpose of regulating the inflammatory microenvironment.

In order to achieve long-term storage and long-distance transportation of nano-medicines, lyophilized powders of vesicles were prepared using lyoprotectants. influence of the diameter. A typical example is: add 4.4 wt.% sucrose/mannitol (50:50, w/w) to the prepared vesicle solution (4 mg/mL, 200 μL), mix well, and place in the solution Nitrogen quick-frozen for 2 min, and then freeze-dried with a freeze dryer for 24 h (pressure 0.37 mbar, temperature -30°C) to obtain a freeze-dried powder preparation and store it in a -20 ^° C refrigerator. Take it out before use and return to room temperature, then add 800 μL of secondary water and pipette evenly to obtain 1 mg/mL PS-HCQ or Man-PS-HCQ. The morphology observation and particle size of Man-PS-HCQ before and after lyophilization were determined by DLS. The study found that the best lyoprotectant was sucrose:mannitol=50:50 (w/w), the concentration in the aqueous phase was 4.4 wt.%. 20-30 nm increase compared to , but the PDI remained less than 0.2 (Fig. 2 h, i). The preparation of freeze-dried powder is not only convenient to use, but also provides a basis for long-term storage and transportation.

Example 3 In vitro anti-inflammatory experiment of Man-PS-HCQ in macrophages.

The mechanism of the anti-inflammatory effect of HCQ is still unclear. The present invention first studies the anti-inflammatory effect of Man-PS-HCQ in macrophages that simulate inflammation in vitro. Lipopolysaccharide (LPS) and γ-interferon (IFN-γ) are the most commonly used reagents to induce oxidative stress in cells to produce a large number of inflammation-related cytokines, and they are used to stimulate two macrophage RAW 264.7 and BMDM to induce M1M, which plays a major role in inflammation, and then study the effect of HCQ preparations therein.

Add LPS (100 ng/mL) to RAW 264.7 cells plated in 12-well plates, then add PBS, free HCQ, PS-HCQ or Man-PS-HCQ (10 μg HCQ/mL) and incubate for 24 h without LPS stimulation Cells are negative controls (control). Subsequently, the cell culture medium was taken to test the concentration of pro-inflammatory cytokine IL-6 and anti-inflammatory cytokine IL-10 with the corresponding Elisa kit, and at the same time extract the RNA of the adherent cells in the lower layer, and test the IL in the cells by qRT-PCR -6 and IL-10 mRNA expression. Add LPS (100 ng/mL) and IFN-γ (10 ng/mL) to BMDM inoculated in a 12-well plate (1× ¹⁰⁶ /well), then add PBS, Man-PS, free HCQ, PS -HCQ, or Man-PS-HCQ (10 μg HCQ/mL) were incubated for 24 h, and unstimulated cells were used as negative control (control). The concentration of IL-6 and IL-10 in the cell culture medium was tested. At the same time, the cells were scraped and suspended with a scraper, and anti-CD11b, anti-F4/80, and anti-CD206 antibodies were added to label macrophages, and the ratio of M1M (CD206 ^- ) and M2M (CD206 ⁺ ) in macrophages was tested by FACS , to characterize the anti-inflammatory effect of Man-PS-HCQ.

The results are shown in Figure 3, the control group is unstimulated cells, while the PBS group is the corresponding induced M1M, and the pro-inflammatory cytokine IL-6 can be seen to be significantly increased. It can be seen from Figure 3 a, c that compared with the free HCQ and PBS groups, Man-PS-HCQ can significantly inhibit the secretion of IL-6 by RAW 264.7, and reduce the expression of IL-6 mRNA in cells. In addition, Man-PS-HCQ can also greatly stimulate RAW 264.7 to produce anti-inflammatory cytokine IL-10, and significantly increase the expression of IL-10 mRNA in cells (Figure 3 b, d). Free HCQ had no significant effect on the secretion of IL-6 and IL-10 by RAW 264.7. Both PS-HCQ and Man-PS-HCQ showed good effects on inhibiting the production of IL-6 by RAW 264.7, but Man-PS Compared with -HCQ, it can stimulate cells to secrete significantly more IL-10 (**** p ), and RAW 264.7 has a higher expression of IL-10 (* p ), which is crucial for alleviating inflammation.

In addition, the amount of secreted IL-6 and IL-10 increased significantly in primary mouse cell BMDM stimulated by LPS (100 ng/mL) and IFN-γ (10 ng/mL). Notably, Man-PS-HCQ also significantly inhibited the secretion of IL-6 from BMDM (** p , **** p ), and promoted the secretion of IL-10 (Fig. 3 e, f). The ratio of IL-10/IL-6 in the Man-PS-HCQ group was much higher than that in both the free HCQ and PS-HCQ groups (**** p , Fig. 3 g), which indicated that Man-PS-HCQ could provide a very favorable The anti-inflammatory environment has a stronger effect on alleviating inflammation. It can be seen from Figure 3 e, f that empty vesicles (Man-PS) have basically no effect on the secretion of IL-6 and IL-10 in BMDM, and free HCQ has little effect on promoting the secretion of IL-10, which shows that The anti-inflammatory effect of Man-PS-HCQ is mainly caused by the synergistic effect of Man-PS and HCQ. Within the concentration range of HCQ, the greater the concentration, the stronger the anti-inflammatory ability (Fig. 3 h); at 2.5 μg/mL, there was basically no effect on the secretion of IL-10, and when HCQ was at 5 μg/mL, the concentration of IL-10 increased significantly (* p ), and when the concentration of HCQ was 10 μg/mL, Man-PS-HCQ induced BMDM to produce significantly increased IL-10 (*** p ).

Repolarizing effect of Man-PS-HCQ on macrophages. It is well known that macrophages can be divided into two phenotypes, pro-inflammatory M1 macrophages (M1M, CD206-) and anti-inflammatory M2 macrophages (M2M, CD206+), and these two types of cells are also It is the main source of pro-inflammatory cytokines such as IL-6 and anti-inflammatory cytokines such as IL-10. From the results of flow cytometry (Figure 4), it can be concluded that compared with the extracted BMDM control group (mostly CD206+ M2M), the M2M of the stimulated PBS group decreased and the M1M increased. Compared with the PBS group, PS-HCQ and Man-PS-HCQ could reduce the proportion of proinflammatory M1M (CD206-), while the proportion of M2M (CD206+) increased from 61.8% to 68.0% and 72.5%, respectively. Empty vesicles (Man-PS) had little effect on cell phenotype changes. Therefore, Man-PS-HCQ can repolarize part of M1M into M2M, which will increase the secretion of anti-inflammatory IL-10 and inhibit the secretion of pro-inflammatory cytokines, which is also consistent with the above results.

Example 4 Study on the uptake of Man-PS-HCQ in BMDM.

Three subtypes of BMDM cells (M0M, M1M and M2M) were seeded in 6-well plates (5×10 ⁵ cells/well), and 200 μL of Cy5-labeled vesicle PS-Cy5 or Man-PS- Cy5 (Cy5 concentration was 0.2 μg/mL), and the PBS group was the control. After incubation for 4 h, scrape the cells with a cell scraper, centrifuge (1000 rpm, 3 min), wash twice with PBS, and finally disperse with 500 μL of PBS and add to the flow tube, and use a flow cytometer (FACS) within one hour Determination. M1M and M2M were stimulated by LPS and IL-4, respectively. Analysis of flow cytometry results showed that the uptake of Man-PS-Cy5 by M1M and M2M was 1.20 and 1.36 times that of PS-Cy5, respectively (Fig. 5 a). It seems that Man-PS-Cy5 has a stronger targeting effect on M2M. The expression of CD206 on M0M, M1M, and M2M was tested, and the results showed that the expression of CD206 on M2M was indeed significantly higher than that of M0M and M1M ( *** p , Fig. 5b). It is worth noting that the uptake of Man-PS-Cy5 and PS-Cy5 by M1M is 3-4 times higher than that of M2M, which shows that M1M does have a stronger endocytic ability, which is also beneficial to the development of inflammation-related diseases. treat.

Example 5 Inhibition of BMDC activation by Man-PS-HCQ.

BMDC cells were inoculated in a 12-well plate (1×10 ⁶ cells/well) and cultured overnight, replaced with fresh medium containing CpG (0.4 μg/mL), and then added PBS, free HCQ, PS-HCQ, or Man- PS-HCQ (10 μg HCQ/mL) was incubated for 24 h, and cells without CpG were used as negative control group (Control). Subsequently, anti-CD11c, anti-CD80, anti-CD86, and anti-MHC-II antibodies were added to label DC surface markers, and the inhibitory ability of Man-PS-HCQ to DC activation was characterized by FACS test.

Using CpG (0.4 μg/mL) to activate BMDC, it was found that two typical markers of DC activation have been greatly improved (Figure 6): the proportion of CD80 ⁺ CD86 ⁺ BMDC increased from 22.6% (control group) to 46.5% % (PBS group), the proportion of MHC-II ⁺ BMDCs increased from 25.9% (control group) to 49.6% (PBS group), indicating that BMDCs were successfully activated. From the analysis of flow cytometry results (Fig. 6c), it can be seen that HCQ and PS-HCQ significantly inhibited the maturation of BMDCs (** p ), while Man-PS-HCQ more significantly reduced the proportion of mature BMDCs, CD80 ⁺ CD86 ⁺ BMDC decreased from 46.5% to 22.8%. In addition, MHC-II molecules expressed on APCs can present antigenic peptides to CD4 ⁺ T cells for recognition, thereby activating the immune system. The results showed that Man-PS-HCQ could greatly down-regulate the MHC-II expression of activated BMDCs from 49.6% (PBS group) to 29.2%. It can be seen that the higher cellular uptake capacity of Man-PS-HCQ may inhibit DC activation by inhibiting the TLR9 pathway, and inhibit the immune response of T cells by inhibiting the expression of MHC-II.

Example 6 Research on ROS scavenging ability of Man-PS-HCQ.

LPS (100 ng/mL) was added to RAW 264.7 cells, and then PBS, free HCQ, PS-HCQ, or Man-PS-HCQ were added and incubated for 24 h. Unstimulated cells were used as negative control (Control). Afterwards, the medium was replaced with serum-free medium, incubated in the incubator for 30 minutes, and then the ROS dye 2ʹ,7ʹ-dichlorofluorescein diacetate DCFH-DA (1 mL, 20 μM) was added, and stained at room temperature for 10 minutes After washing with PBS for 3 times, observe and take pictures with an inverted microscope. Add LPS (100 ng/mL) and IFN-γ (10 ng/mL) to BMDM, then add PBS, free HCQ, PS-HCQ, or Man-PS-HCQ and incubate for 24 h, unstimulated cells were used as negative control ( Control). The rest of the operations are the same as the above RAW 264.7 cells are the same.

As shown in Figure 7a, compared with the unstimulated group (control), the green fluorescence of DCF in the cells of the LPS group was significantly enhanced, confirming that LPS can not only promote the differentiation of M1M and the secretion of related pro-inflammatory cytokines, but also stimulate RAW 264.7 Cells produce large amounts of ROS. After the cells were incubated with free HCQ, PS-HCQ or Man-PS-HCQ, the content of ROS was greatly reduced (**** p ), and the ROS scavenging ability of Man-PS-HCQ was higher than that of free HCQ and PS-HCQ Even stronger ( ** p ). In BMDM cells activated by LPS/IFN-γ co-stimulation, free HCQ and PS-HCQ can also reduce the concentration of ROS, but Man-PS-HCQ also exhibits stronger ROS scavenging ability than free HCQ and PS-HCQ (Fig. 7b).

These results indicate that targeting macrophages to deliver HCQ can increase the accumulation of HCQ in the cytoplasm, which in turn helps to clear ROS and play an anti-inflammatory role. The local production of a large amount of ROS in RA lesions is the main cause of cartilage damage in RA patients. Excessive ROS can inhibit the proliferation of chondrocytes and induce their apoptosis. Both ROS and NO can increase the content of matrix metalloproteinase (MMP) in joint tissues, Reduce the ability of cartilage to repair itself. Therefore, the ability of Man-PS-HCQ to significantly reduce ROS at inflammatory sites may protect mouse cartilage from damage in an animal model.

Example 7 Cytotoxicity experiment.

The toxicity of PS-HCQ and Man-PS-HCQ was evaluated using two mouse macrophage cell lines (RAW 264.7 and BMDM). BMDM extracted from the bone marrow of healthy C57BL/6 mice (5× ¹⁰⁴ cells/well) were plated in a 96-well plate, cultured in 1640 medium for 24 h, and then cultured under three conditions: cultured at 1640 M0 type macrophages (M0M) were cultured in medium for 24 h; M2 type macrophages (M2M) were obtained after cultured in 1640 medium containing IL-4 (40 ng/mL) for 24 h; (100 ng/mL) and IFN-γ (10 ng/mL) in 1640 medium for 24 h to obtain M1 macrophages (M1M). Add 20 μL of different concentrations of PS-HCQ or Man-PS-HCQ to the 96-well plate and incubate for 24 hours. The final concentration of HCQ in the well is 5 and 10 μg/mL (PBS is used as the control). Then add 10 μL of MTT solution (5 mg/mL) and incubate for 4 hours, aspirate the medium, add 150 μL of DMSO solution, and incubate for 30 min in a shaker at 37°C and 100 rpm, so that the living cells and the MTT produced The blue-purple crystalline formazan was completely dissolved, and finally the ultraviolet absorbance at 570 nm was measured with a microplate reader. The cell survival rate was equal to the ratio of the absorbance of the experimental group to the absorbance of the PBS group (n = 6).

RAW 264.7 cells were seeded in 96-well plates (1×10 ⁴ cells/well), and cultured under the same three conditions as above, except that the cells were stimulated to M1M with LPS (100 ng/mL), and the rest of the operations were the same as above.

The effect of PS-HCQ and Man-PS-HCQ on RAW was investigated by MTT method Cytotoxicity of 264.7 cells and BMDM. As shown in Figure 8, PS-HCQ and Man-PS-HCQ have no obvious toxicity when incubated with two macrophages for 24 hours at 5-10 μg HCQ/mL or below. The same method was used to investigate the toxicity of PS-CQ and Man-PS-CQ to RAW 264.7 cells, and it was found that it was greater than PS-HCQ and Man-PS-HCQ.

Example 8 Establishment of zymosan-induced rheumatoid arthritis (ZIA) mouse model and study on the biodistribution of Man-PS-HCQ.

To establish the ZIA mouse model, add secondary water to the zymosan at a concentration of 10 mg/mL, heat to boil and continue to cook for 5-10 minutes to form an emulsion, and then sonicate for 20 minutes before use. Inject 50 μL of zymosan emulsion into the joint cavity of the left leg of C57BL/6 mice to induce acute arthritis. After 24 hours, obvious swelling and inflammation can be seen in the knee joint of the mouse, and the leg circumference reaches the maximum value, and ZIA is established. Mouse models are available for experiments. In order to study the targeting effect of mannose-modified vesicles on the inflammatory site of ZIA mice, 24 hours after the establishment of the model, the mice were divided into two groups (3 mice in each group), and 200 μL of Cy5-labeled vesicles were injected through the tail vein. Drug-loaded vesicles PS-HCQ-Cy5 or Man-PS-HCQ-Cy5 (0.3 μg Cy5/mouse) were used for live imaging of mice at predetermined time points. The enrichment of nanomedicine in the inflammatory site of ZIA mice has a significant impact on the therapeutic effect, and a large amount of accumulation in normal tissues can cause severe toxic side effects. The ZIA model was established in the left leg joint of the mouse. After 24 hours, the swelling degree of the mouse leg reached the peak, and the Cy5-labeled vesicle PS-HCQ or Man-PS-HCQ was injected into the mouse body through the tail vein (1.2 mg HCQ/kg), the distribution of vesicles in major organs and inflammatory sites of mice was observed over time by in vivo fluorescence imaging. The results showed that Man-PS-HCQ was rapidly enriched in the RA joint of the left leg of the mice, and the average enrichment of the three mice reached the peak at 8 h, and then decreased slightly (Fig. 9 a, b). Within 48 hours of observation, the fluorescence at the RA joints of the left leg of PS-HCQ in the no-target group was significantly lower, and the enrichment of Man-PS-HCQ in RA joints was 2.4-5.0 times that of PS-HCQ group (** p ), And the residence time is longer, and the fluorescence intensity is still high after 48 h.

Example 9 The curative effect of Man-PS-HCQ on ZIA mice.

To study the effect of HCQ dose and surface density of mannose, mice were divided into 8 groups (5 mice in each group) 24 hours after zymosan induction. ZIA mice were injected with 200 μL of free HCQ (1.2 mg HCQ/kg), PS-HCQ (1.2 mg HCQ/kg), 10Man-PS-HCQ (0.6 mg HCQ/kg), 10Man-PS-HCQ ( 1.2 mg HCQ/kg), 10Man-PS-HCQ (2.4 mg HCQ/kg) or 20Man-PS-HCQ (1.2 mg HCQ/kg), given once every three days, twice in total, PBS group and healthy Group of mice served as controls (Fig. 10a). The start of treatment was recorded as day 0. Observe the joint swelling every day, measure the leg circumference and body weight of the left leg, take blood on day -1, 0, 1, 3 and 7, and test the content of TGF-β in the plasma. The calculation formula of mouse leg circumference (leg circumference.) is as follows:

.

In the formula: T is the thickness of the mouse leg, W is the width of the mouse leg.

From the swelling of the left leg of the mice (Fig. 10 b, c), it can be seen that compared with free HCQ, all groups of HCQ exhibited excellent joint swelling relief (* p , ** p ), three 10% Man The diameters of the diseased joints in the Man-PS-HCQ group showed a downward trend all the time, and the 1.2 mg HCQ/kg group had the best treatment effect, and there was no statistical difference from the healthy group. The content of anti-inflammatory TGF-β produced in the serum of mice was monitored, and the results showed that the content of TGF-β in healthy mice was relatively low. Both have risen. After 24 hours (i.e. day 1), the TGF-β content in the 10Man-PS-HCQ (1.2 mg HCQ/kg) group was significantly higher than that in the free HCQ group (* p ). Four hours after administration of the second injection (that is, day 3), the secretion of TGF-β increased more than that after the first injection, which indicates that the nanomedicine of the present invention can further increase the secretion of TGF-β after multiple administrations , while at day 7, TGF-β secretion in all groups decreased greatly. Body weight monitoring found that the body weight of mice decreased significantly due to acute inflammation 24 hours after ZIA modeling (Fig. 10 d), and returned to the normal range as time went by. Therefore, it can be preliminarily judged that Man-PS-HCQ with a Man density of 10% and an HCQ dose of 1.2 mg/kg has the best ZIA efficacy (Figure 10 ei), and this formula will be used in the next systematic ZIA mouse treatment study and immunoassays.

In order to study the excellent effect of Man-PS-HCQ on the elimination of inflammation in ZIA mice, the protection of joint cartilage and bone, and the regulation of the immune environment in diseased joints, we then increased each group of ZIA mice To 12 rats (n = 12), tail vein injection of free HCQ, PS-HCQ, Man-PS-HCQ (1.2 mg HCQ/kg), administered twice every three days (Fig. 11 a), mice in PBS group and healthy group served as controls. The start of treatment was recorded as day 0. Observe the joint swelling every day, measure the leg circumference and weight of the left leg, and take blood on the 0th, 3rd, and 7th days to test the IL-6 and TGF-β levels in order to evaluate the curative effect. Then, on the seventh day, six mice were randomly selected from each group for dissection, and the cartilage and synovium of the diseased part were taken, homogenized, and micro BCA was used to measure the protein content in the grinding solution, and the corresponding Elisa kit was used to determine the content of cytokines (IL-6, TNF-α, IL-1β, IL-10 and TGF-β). In addition, three other mice were randomly sacrificed in each group, and the diseased articular cartilage was removed, and sections were used for CD206 antibody labeling, H&E, safranin fast green and Trap staining to evaluate the damage of cartilage and synovium. The remaining three mice in each group continued to be observed until the third week, and the joints and leg bones were dissected out, and micro CT was scanned to analyze the bone loss in the joints of the mice.

It was also found that on the 0th day, the legs and knees of ZIA mice showed obvious redness and swelling, the IL-6 content in the serum was significantly increased compared with the healthy group, the TGF-β was similar to the healthy group, and the body weight decreased slightly. The body weight of the mice did not decrease significantly (Fig. 11 ac). After administration of HCQ preparations, the leg circumference and knee diameter of the mice were continuously and significantly decreased. Free HCQ showed a certain ability to inhibit pro-inflammatory cytokines, but it did not alleviate the symptoms of redness and swelling in the joints of mice. In contrast, on the 6th day, Man-PS-HCQ significantly relieved the redness and swelling of the joints, which was significantly smaller than that of the other groups, and there was no redness and swelling visible to the naked eye, and there was no significant difference from the healthy group (Figure 11 ac). Serum tests found that HCQ preparations significantly down-regulated the secretion of IL-6 and up-regulated the secretion of TGF-β on the 3rd and 7th days, but the Man-PS-HCQ and PS-HCQ groups were significantly better than free HCQ. Overall, since this model is an acute inflammation model, the concentration of IL-6 decreased sharply on the 7th day, while the decrease of TGF-β was not much (Fig. 11 e, f). The mice were sacrificed on the 7th day, and the Elisa test found that Man-PS-HCQ significantly inhibited the three pro-inflammatory cytokines IL-6, TNF-α and IL-1β in the serum of the study, which was no different from the healthy group; while Compared with PS-HCQ, it had a stronger promoting effect on the secretion of anti-inflammatory cytokines IL-10 and TGF-β (* p ) (Fig. 11 g).

Next, the regulation of pro-inflammatory and anti-inflammatory cytokine concentrations in the joint cavity of mice after treatment (day 7), immune microenvironment, and cartilage and bone joint damage were studied, which will be discussed separately. Firstly, the ratio of representative pro-inflammatory and anti-inflammatory cytokines in the mouse joint grinding fluid to the total protein extracted was determined, and the trend and conclusion were found to be the same as those in the serum, that is, the mice in the Man-PS-HCQ group The treatment effect was the best: it had a more obvious effect on relieving the symptoms of joint redness and swelling, down-regulating the content of pro-inflammatory cytokines and up-regulating the content of anti-inflammatory cytokines, reaching the same level as the healthy group mice (Figure 11 h). These results confirm that Man-PS-HCQ regulates the balance of pro-inflammatory and anti-inflammatory cytokines, achieving excellent anti-inflammatory effects.

Example 8 Establishment of zymosan-induced rheumatoid arthritis (ZIA) mouse model and study on the biodistribution of Man-PS-HCQ.

To establish the ZIA mouse model, add secondary water to the zymosan at a concentration of 10 mg/mL, heat to boil and continue to cook for 5-10 minutes to form an emulsion, and then sonicate for 20 minutes before use. Inject 50 μL of zymosan emulsion into the joint cavity of the left leg of C57BL/6 mice to induce acute arthritis. After 24 hours, obvious swelling and inflammation can be seen in the knee joint of the mouse, and the leg circumference reaches the maximum value, and ZIA is established. Mouse models are available for experiments. In order to study the targeting effect of mannose-modified vesicles on the inflammatory site of ZIA mice, 24 hours after the establishment of the model, the mice were divided into two groups (3 mice in each group), and 200 μL of Cy5-labeled vesicles were injected through the tail vein. Drug-loaded vesicles PS-HCQ-Cy5 or Man-PS-HCQ-Cy5 (0.3 μg Cy5/mouse) were used for live imaging of mice at predetermined time points. The enrichment of nanomedicine in the inflammatory site of ZIA mice has a significant impact on the therapeutic effect, and a large amount of accumulation in normal tissues can cause severe toxic side effects. The ZIA model was established in the left leg joint of the mouse. After 24 hours, the swelling degree of the mouse leg reached the peak, and the Cy5-labeled vesicle PS-HCQ or Man-PS-HCQ was injected into the mouse body through the tail vein (1.2 mg HCQ/kg), the distribution of vesicles in major organs and inflammatory sites of mice was observed over time by in vivo fluorescence imaging. The results showed that Man-PS-HCQ was rapidly enriched in the RA joint of the left leg of the mice, and the average enrichment of the three mice reached the peak at 8 h, and then decreased slightly (Fig. 9 a, b). Within 48 hours of observation, the fluorescence at the RA joints of the left leg of PS-HCQ in the no-target group was significantly lower, and the enrichment of Man-PS-HCQ in RA joints was 2.4-5.0 times that of PS-HCQ group (** p ), And the residence time is longer, and the fluorescence intensity is still high after 48 h.

Example 9 The curative effect of Man-PS-HCQ on ZIA mice.

To study the effect of HCQ dose and surface density of mannose, mice were divided into 8 groups (5 mice in each group) 24 hours after zymosan induction. ZIA mice were injected with 200 μL of free HCQ (1.2 mg HCQ/kg), PS-HCQ (1.2 mg HCQ/kg), 10Man-PS-HCQ (0.6 mg HCQ/kg), 10Man-PS-HCQ ( 1.2 mg HCQ/kg), 10Man-PS-HCQ (2.4 mg HCQ/kg) or 20Man-PS-HCQ (1.2 mg HCQ/kg), given once every three days, twice in total, PBS group and healthy Group of mice served as controls (Fig. 10a). The start of treatment was recorded as day 0. Observe the joint swelling every day, measure the leg circumference and body weight of the left leg, take blood on day -1, 0, 1, 3 and 7, and test the content of TGF-β in the plasma. The calculation formula of mouse leg circumference (leg circumference.) is as follows:

.

In the formula: T is the thickness of the mouse leg, W is the width of the mouse leg.

From the swelling of the left leg of the mice (Fig. 10 b, c), it can be seen that compared with free HCQ, all groups of HCQ exhibited excellent joint swelling relief (* p , ** p ), three 10% Man The diameter of the diseased joints of the mice in the Man-PS-HCQ group has always shown a downward trend, and the treatment effect of the 1.2 mg HCQ/kg group is the best, and there is no statistical difference from the healthy group. The content of anti-inflammatory TGF-β produced in the serum of mice was monitored, and the results showed that the content of TGF-β in healthy mice was relatively low. Both have risen. After 24 hours (i.e. day 1), the TGF-β content in the 10Man-PS-HCQ (1.2 mg HCQ/kg) group was significantly higher than that in the free HCQ group (* p ). Four hours after administration of the second injection (that is, day 3), the secretion of TGF-β increased more than that after the first injection, which indicates that the nanomedicine of the present invention can promote the secretion of TGF-β to further increase after multiple administrations , while at day 7, TGF-β secretion in all groups decreased greatly. Body weight monitoring found that the body weight of mice decreased significantly due to acute inflammation 24 hours after ZIA modeling (Fig. 10 d), and returned to the normal range as time went by. Therefore, it can be preliminarily judged that Man-PS-HCQ with a Man density of 10% and an HCQ dose of 1.2 mg/kg has the best ZIA efficacy (Figure 10 ei), and this formula will be used in the next systematic ZIA mouse treatment study and immunoassays.

In order to study the excellent effect of Man-PS-HCQ on the elimination of inflammation in ZIA mice, the protection of joint cartilage and bone, and the regulation of the immune environment in diseased joints, we then increased each group of ZIA mice To 12 rats (n = 12), tail vein injection of free HCQ, PS-HCQ, Man-PS-HCQ (1.2 mg HCQ/kg), administered twice every three days (Fig. 11 a), mice in PBS group and healthy group served as controls. The start of treatment was recorded as day 0. Observe the joint swelling every day, measure the leg circumference and weight of the left leg, and take blood on the 0th, 3rd, and 7th days to test the IL-6 and TGF-β levels in order to evaluate the curative effect. Then, on the seventh day, six mice were randomly selected from each group for dissection, and the cartilage and synovium of the diseased part were taken, homogenized, and micro BCA was used to measure the protein content in the grinding solution, and the corresponding Elisa kit was used to determine the content of cytokines (IL-6, TNF-α, IL-1β, IL-10 and TGF-β). In addition, three other mice were randomly sacrificed in each group, and the diseased articular cartilage was removed, and sections were used for CD206 antibody labeling, H&E, safranin fast green and Trap staining to evaluate the damage of cartilage and synovium. The remaining three mice in each group continued to be observed until the third week, and the joints and leg bones were dissected out, and micro CT was scanned to analyze the bone loss in the joints of the mice.

It was also found that on the 0th day, the legs and knees of ZIA mice showed obvious redness and swelling, the IL-6 content in the serum was significantly increased compared with the healthy group, the TGF-β was similar to the healthy group, and the body weight decreased slightly. The body weight of the mice did not decrease significantly (Fig. 11 ac). After administration of HCQ preparations, the leg circumference and knee diameter of the mice were continuously and significantly decreased. Free HCQ showed a certain ability to inhibit pro-inflammatory cytokines, but it did not alleviate the symptoms of redness and swelling in the joints of mice. In contrast, on the 6th day, Man-PS-HCQ significantly relieved the redness and swelling of the joints, which was significantly smaller than that of the other groups, and there was no redness and swelling visible to the naked eye, and there was no significant difference from the healthy group (Figure 11 ac). Serum tests found that HCQ preparations significantly down-regulated the secretion of IL-6 and up-regulated the secretion of TGF-β on the 3rd and 7th days, but the Man-PS-HCQ and PS-HCQ groups were significantly better than free HCQ. Overall, since this model is an acute inflammation model, the concentration of IL-6 decreased sharply on the 7th day, while the decrease of TGF-β was not much (Fig. 11 e, f). The mice were sacrificed on the 7th day, and the Elisa test found that Man-PS-HCQ significantly inhibited the three pro-inflammatory cytokines IL-6, TNF-α and IL-1β in the serum of the study, which was no different from the healthy group; while Compared with PS-HCQ, it had a stronger promoting effect on the secretion of anti-inflammatory cytokines IL-10 and TGF-β (* p ) (Fig. 11 g).

Next, the regulation of pro-inflammatory and anti-inflammatory cytokine concentrations in the joint cavity of mice after treatment (day 7), immune microenvironment, and cartilage and bone joint damage were studied, which will be discussed separately. Firstly, the ratio of representative pro-inflammatory and anti-inflammatory cytokines in the mouse joint grinding fluid to the total protein extracted was determined, and the trend and conclusion were found to be the same as those in the serum, that is, the mice in the Man-PS-HCQ group The treatment effect was the best: it had a more obvious effect on relieving the symptoms of joint redness and swelling, down-regulating the content of pro-inflammatory cytokines and up-regulating the content of anti-inflammatory cytokines, reaching the same level as the healthy group mice (Figure 11 h). These results confirm that Man-PS-HCQ regulates the balance of pro-inflammatory and anti-inflammatory cytokines, achieving excellent anti-inflammatory effects.

Example 10 Man-PS-HCQ protects the articular cartilage and bone tissue of ZIA mice.

In addition to joint swelling and pain, the main symptoms of RA include severe cartilage damage and bone loss. According to the treatment scheme in Figure 11, slices of mouse joints were prepared, and H&E, safranin fast green, and Trap staining were used to study and analyze cartilage damage, bone loss, immune cell infiltration, and destruction of mouse joints treated with different HCQ preparations. bone cell content, etc. The results of H&E and safranin fast green (SO-FG) staining pictures showed that the joints of mice in PBS, free HCQ and PS-HCQ groups had obvious synovial inflammation and cartilage defects (black arrows), and the H&E pictures showed that the PBS group was small There are a large number of immune cells and new blood vessels in the mouse synovium (red arrow), which can be alleviated by the treatment of free HCQ and PS-HCQ groups, while the infiltration of immune cells and new blood vessels in the joints of the mice in the Man-PS-HCQ group The formation and cartilage damage were significantly reduced, and the shape of cartilage in the joints was similar to that of the healthy mice. According to literature reports, the formation of new blood vessels plays an important role in the maintenance of pannus in arthritis, and new blood vessels are conducive to the recruitment of immune cells, which can cause continuous damage to joint tissues. The results of Trap staining showed that compared with the mice in the healthy group, there were a large number of osteoclasts (triangles) in the joints of the mice in the PBS, free HCQ and PS-HCQ groups, while the number of osteoclasts in the joints of the mice treated with Man-PS-HCQ Less, close to the healthy mice (Figure 12). In addition, after two administrations as shown in Figure 11, the mice were sacrificed on the 7th day, and the main organ sections were taken and analyzed by H&E staining to evaluate the toxic and side effects. The results showed that there was no significant difference between Man-PS-HCQ and healthy mice in heart, liver, spleen, lung, and kidney, and there was no serious side effect on mice (Figure 13).

Immunofluorescent staining of the M2M marker CD206 in the joint sections of the mice showed that compared with healthy mice, the green fluorescence in the joints of the mice in the PBS group and the free HCQ group was weaker, indicating that the content of anti-inflammatory M2M was significantly less. This may be caused by the release of a large number of cytokines and chemokines in the diseased joints of ZIA mice and the recruitment of a large number of M1M infiltration. Compared with the former two groups, the M2M of the joints in the PS-HCQ group increased significantly (**** p ), while the Man-PS-HCQ group further significantly increased the M2M content in the joints (**** p ), reaching the highest, Embodies its significant in vivo targeting macrophage effect. More M2M was recruited to the diseased joints of mice, which was conducive to the release of more anti-inflammatory cytokines (such as IL-10 and TGF-β), and modulated the inflammatory microenvironment in the diseased joints.

In order to further reveal the anti-inflammatory mechanism of Man-PS-HCQ in ZIA mice, the joint mixture was ground to obtain leukocytes, the immune microenvironment in the mouse joints was tested, and the regulation of Man-PS-HCQ on the immune microenvironment was analyzed. Cells in mouse joint grinding fluid were stained with the following antibodies: anti-CD11b, anti-F4/80, anti-CD206, anti-CD11c, anti-CD80, anti-CD86, anti-CD3, anti-CD4 or anti- CD8. The results showed that after the establishment of the ZIA model (PBS group), the proportions of CD11b ⁺ F4/80 ⁺ macrophages, CD11c ⁺ DCs and CD3 ⁺ T cells in the joints of the mice were significantly increased compared with those in the healthy group ( Figure 15). It is worth noting that the proportion of macrophages in the mouse joints can reach about 30.4% of all cells, which is much higher than the content of DC (about 7.2%) and T cells (about 4.5%), which also confirms that Macrophages are indeed the dominant immune cells in RA. Compared with the healthy group mice, the total content of macrophages in the joints treated with Man-PS-HCQ was significantly increased (* p ), and the proportion of CD206 ⁺ M2M was also significantly further increased (** p ), which is Man-PS- HCQ repolarizes the pro-inflammatory M1M to the anti-inflammatory M2M, so the secretion of anti-inflammatory IL-10 and TGF-β in mice is significantly increased, and the reduction of M1M also leads to a corresponding decrease in pro-inflammatory cytokines. In addition, the infiltration of CD11c ⁺ DC and CD3 ⁺ T cells in the RA joints of ZIA mice was increased, and Man-PS-HCQ treatment can greatly reduce their infiltration in the joints (* p ), especially mature DC (CD11c ⁺ CD80 ⁺ CD86 ⁺ ) and CD3 ⁺ CD4 ⁺ T cells were significantly reduced, and Man-PS-HCQ greatly down-regulated the infiltration of mature DC and T cells, which could reduce the expression of MHC-II and the presentation of related antigens, thereby reducing pro-inflammatory The secretion of cytokines helps to relieve the symptoms of RA. The test found that the content of CD3 ⁺ CD8 ⁺ T cells in RA joints was very low before and after treatment, and remained basically unchanged, indicating that these cells played little role in RA.

For rheumatoid arthritis RA, the present invention uses HCQ as an example to design a reduction-responsive polymer vesicle loaded with HCQ for targeted therapy of mouse RA. The representative Man-PS-HCQ has the advantages of simple preparation, adjustable surface Man density, small and uniform size (~46 nm), high stability and strong reduction response. Man-PS-HCQ has a small size and is easy to accumulate at the site of inflammation. It has a targeted uptake in macrophages, and its reduction-responsive drug release can increase the effective drug concentration at the site of inflammation, enhance the anti-inflammatory effect, reduce toxic side effect. Cell experiments show that Man-PS-HCQ can regulate the secretion of cytokines in macrophages, remove ROS, and exhibit excellent anti-inflammatory effects. Experiments in ZIA mice showed that Man-PS-HCQ was rapidly enriched in RA joints, which could reduce the secretion of pro-inflammatory cytokines and increase the secretion of anti-inflammatory cytokines in mouse serum and synovial fluid; it could increase the secretion of anti-inflammatory cytokines in the joints. Repolarization of M1M to anti-inflammatory M2M reduces the number of activated DC and T cells. Therefore, Man-PS-HCQ can significantly eliminate the swelling of diseased joints, reduce the infiltration of inflammatory cells, reduce the number of osteoclasts, protect the joint synovium, cartilage and bone tissue, and show excellent anti-inflammatory and immune micro-regulatory effects. environmental effects. Therefore, this biodegradable nanomedicine provides an avenue for safe and efficient treatment of RA.

All data in the present invention are mean ± standard deviation (SD). Differences between groups were evaluated using ANOVA one-way analysis of variance, * p < 0.05 indicated a significant difference, ** p < 0.01 and *** p < 0.001 indicated a highly significant difference.

Claims (10)

1. A vesicular nano-medicine loaded with a chloroquine compound, characterized in that it comprises a polymer vesicle and a chloroquine compound; the polymer includes a hydrophilic segment and a hydrophobic segment, and the side chain of the hydrophobic segment is a disulfide bond-containing Dithiolane.
2. According to claim 1, the vesicle nano-medicine loaded with chloroquine compound is characterized in that polymer self-assembly forms polymer vesicles; the polymer is a non-targeting polymer, or the polymer is a non-targeting polymer and a targeting polymer A mixture of polymers; the chloroquine compound is an anti-autoimmune disease drug.
3. According to claim 2, the vesicular nano-medicine of loaded chloroquine compound is characterized in that the non-targeting polymer comprises PEG-P (TMC-DTC), PEG-P (CL-DTC) or PEG-P (LA-DTC ); the targeting polymer includes B-PEG-P (TMC-DTC), B-PEG-P (CL-DTC) or B-PEG-P (LA-DTC), and B is a targeting molecule.
4. The vesicle nanomedicine loaded with chloroquine compound according to claim 1, characterized in that the molecular weight of the polymer is 10-50 kg/mol.
5. The vesicle nanomedicine loaded with chloroquine compound according to claim 4, characterized in that the molecular weight of the hydrophilic segment is 2-10 kg/mol.
6. The preparation method of the vesicle nano-medicine loaded with chloroquine compound according to claim 1 is characterized in that, the vesicle nano-medicine loaded with chloroquine compound is prepared with polymer and chloroquine compound as raw materials.
7. according to the preparation method of the described vesicle nano medicine of chloroquine compound loaded in claim 6, it is characterized in that, polymer is non-target polymer, or polymer is the mixture of non-target polymer and targeting polymer; Adopt pH The chloroquine compound is loaded into the vesicle by the gradient method to obtain the vesicle nanomedicine loaded with the chloroquine compound.
8. According to the preparation method of the described vesicle nano-medicine of chloroquine compound loaded in claim 7, it is characterized in that, when polymer is the mixture of non-targeting polymer and targeting polymer, the difference between non-targeting polymer and targeting polymer The molar ratio is 1: (0 to 0.8), excluding 0.
9. A vesicle nano-medicine freeze-dried powder of chloroquine compound, its preparation method is, freeze-dry after mixing the vesicle nano-medicine of chloroquine compound described in claim 1 with a freeze-drying protective agent, obtain the vesicle of chloroquine compound Nano drug freeze-dried powder.
10. The application of the vesicle nanomedicine loaded with chloroquine compound described in claim 1 in the preparation of a drug for the treatment of rheumatoid arthritis or in the preparation of a drug for repolarizing M1M macrophages into M2M macrophages or in the preparation of scavenging ROS The application in the medicine or the application in the preparation of the medicine for inhibiting the activation of BMDC or the application in the preparation of the medicine for protecting articular cartilage and bone tissue or the application in the preparation of the anti-inflammatory medicine or the application in the preparation of the anti-autoimmune disease medicine application.