PH response bionic nano system for enhancing photodynamic therapy, preparation and application

Abstract

The invention provides a pH response bionic nano system for enhancing photodynamic therapy, preparation and application, and belongs to the field of pharmaceutical preparations, hyaluronic acid is used as an initial carrier to load an endogenous hydrogen sulfide donor SPRC and modify an acid response connexon, and then the hyaluronic acid is crosslinked with a pegylated photosensitizer Ce6 to form acid response nano particles. And finally, coating nano particles with the macrophage membrane through a nano-membrane co-extrusion technology to prepare the pH response type bionic nano system (RAW-HSC) coated with the macrophage membrane. In-vivo and in-vitro experiments show that the pH response type bionic nano system can effectively target an inflammation joint part, a small amount of hydrogen sulfide generated by SPRC release can effectively enhance the photodynamic therapy effect, meanwhile, the pH response type bionic nano system has the advantages of biological safety, pH response release and the like, a new thought is provided for research, development and design of a novel medicament, and the pH response type bionic nano system has wide application prospects. The important significance is realized on the treatment of rheumatoid arthritis.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical preparations, and in particular to a pH-responsive bionic nanosystem for enhancing photodynamic therapy, and its preparation and application. Background Art

[0002] Rheumatoid arthritis (RA) is a highly heterogeneous autoimmune disease characterized by localized synovial inflammatory cell infiltration and bone erosion within the joint cavity. RA has become a major health problem worldwide, as approximately 1% of the global population and 0.28% of the Chinese population are affected by it. Similar to the pathological environment in which tumor cells grow, abnormal cell proliferation, metabolism, and invasiveness of RA synovial tissue play an important role in the pathogenesis of RA. In addition, autoantibodies and immune cells activated during the RA process promote the infiltration of inflammatory cells into the joint synovium. Inflammatory cells, such as plasma cells, neutrophils, and macrophages, release inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), chemokines, and matrix metalloproteinases (MMPs), which activate osteoclasts and subsequently lead to damage to articular cartilage and bone.

[0003] Photodynamic therapy (PDT) is a non-invasive, highly selective and cell-killing treatment. It is a clinically approved treatment for various cancers and proliferative diseases and shows great potential for the treatment of RA. Recently, scientists have used photodynamic therapy to produce reactive oxygen species (ROS) to kill inflammatory cells such as macrophages, synovial fibroblasts and other inflammatory cells in RA lesions, thereby reducing the levels of related inflammatory factors (TNF-α, IL-1β). The hypoxic microenvironment in the RA inflammation site will lead to insufficient oxygen supplementation, thereby reducing the efficacy of PDT in the treatment of RA. Therefore, it is necessary to propose and design clever and effective strategies to alleviate RA inflammatory hypoxia and improve the efficacy of PDT for RA treatment. Hydrogen sulfide (H 2 S) is an important endogenous gas signal molecule in medical care and has effective therapeutic effects on various diseases. 2 S can modify oxygen metabolism by inhibiting the activity of cytochrome c oxidase to execute mitochondrial dysfunction, thereby inhibiting cellular respiration and oxygen consumption. 2 High levels of H in S therapy 2 S damages mitochondria and eventually neutralizes oxygen, hindering oxygenation, but producing reasonably low levels of H 2 S can lead to the desired inhibition of oxygen consumption and thus enhance the efficacy of PDT for RA treatment. 2 S-propargyl-cysteine ​​(SPRC) is a S donor, which has anti-inflammatory effects. 2Inhibition of mitochondrial function of S to improve the oxygen environment may be used as an original strategy to promote PDT for RA treatment. Summary of the invention

[0004] The purpose of the present invention is to construct a macrophage membrane-coated pH-responsive bionic nanosystem for targeted enhanced photodynamic therapy, and its preparation and application.

[0005] A pH-responsive bionic nanosystem for enhanced photodynamic therapy consists of pH-responsive nanoparticles loaded with endogenous hydrogen sulfide donor SPRC and photosensitizer and macrophage membranes. The pH-responsive nanoparticles are obtained by chemical covalent cross-linking, and the nanoparticles and macrophage membranes are co-extruded by ultrasound and a membrane extruder to achieve outer membrane coating.

[0006] Preferably, the macrophages are RAW264.7 cell lines, and the cell membrane surface of the macrophages overexpresses relevant adhesion molecules.

[0007] Preferably, the macrophage membrane is obtained by mechanical grinding and differential centrifugation extraction, and the cell membrane vesicles are obtained by ultrasonic treatment.

[0008] Preferably, the relevant adhesion molecules include Mac-1 and LFA-1 membrane proteins.

[0009] Preferably, the hydrogen sulfide donor loaded on the pH-responsive nanoparticles is SPRC, and the photosensitizer is Ce6.

[0010] A method for preparing a pH-responsive bionic nanosystem for enhancing photodynamic therapy, wherein pH-responsive nanoparticles are synthesized by chemical covalent crosslinking: a prescribed amount of hyaluronic acid, SPRC and an acid-responsive linker BRM are weighed, dissolved in an appropriate amount of an alkaline aqueous solvent, stirred and reacted for 12-24 hours under the action of a catalyst, free small molecules are removed by dialysis, an intermediate product is obtained by freeze-drying, and then a pH-responsive nanoparticle HSC is obtained by chemical crosslinking reaction with PEGylated Ce6 in an alkaline aqueous solvent.

[0011] Preferably, the nanoparticles and the macrophage membrane are coated with an outer layer of membrane by ultrasound and membrane extrusion, and the mass ratio thereof is 1:1 to 100:1.

[0012] Preferably, the average particle size of the nanoparticles is 90 to 150 nm, and the particle size of the bionic nanoparticles coating the macrophage membrane is 120 to 180 nm.

[0013] A pH-responsive bionic nanosystem for enhanced photodynamic therapy is used in the preparation of targeted photodynamic therapy drugs for rheumatoid arthritis. The prepared drug produces a small amount of hydrogen sulfide at the site of inflammation by loading the hydrogen sulfide donor SPRC, inhibiting mitochondrial function and oxygen consumption, thereby enhancing ROS generation and strengthening the photodynamic therapy effect of rheumatoid arthritis.

[0014] The present invention uses hyaluronic acid as a carrier to load hydrogen sulfide donor SPRC and acid response linker BRM, and prepares pH-responsive bionic nanoparticles through chemical covalent cross-linking reaction with pegylated photosensitizer. Cell membrane-coated nanoparticles extracted from RAW264.7 cells are used to construct a pH-responsive bionic nanosystem coated with macrophage membranes. The pH-responsive bionic nanosystem can not only increase the biocompatibility of the system by using macrophage membrane surface molecules, but also actively tend to the inflammatory site by using the macrophage membrane, target rheumatoid arthritis lesions, and effectively deliver hydrogen sulfide donor SPRC and photosensitizer to the inflammatory lesions. SPRC and photosensitizer are released in an inflammatory slightly acidic environment, and the increase of a small amount of hydrogen sulfide is beneficial to improving the photodynamic therapy effect of rheumatoid arthritis.

[0015] In the present invention, the photosensitizer used is Ce6. Studies have shown that Ce6 can be used to treat inflammatory diseases such as rheumatoid arthritis through photodynamic therapy. The loaded hydrogen sulfide donor is SPRC. Studies have shown that SPRC, as an endogenous hydrogen sulfide donor, can effectively increase the hydrogen sulfide content and has a certain anti-inflammatory effect. Studies have shown that the generation of a small amount of hydrogen sulfide can inhibit mitochondrial function and oxygen consumption, thereby increasing reactive oxygen species (ROS) and enhancing the photodynamic therapy effect. The outer layer of the system is coated with a macrophage membrane to improve drug enrichment in rheumatoid arthritis inflammatory lesions and enhance the photodynamic therapy effect of rheumatoid arthritis through a small amount of hydrogen sulfide provided by SPRC.

[0016] In the present invention, the macrophage membrane is extracted from RAW264.7 cells, the cell membrane surface-related adhesion molecules are overexpressed, and the macrophage membrane is extracted by differential centrifugation after mechanical grinding.

[0017] In the present invention, among the macrophage membrane-related adhesion molecules, Mac-1 and LFA-1 membrane proteins are overexpressed on the RAW264.7 cell membrane.

[0018] The present invention constructs a macrophage membrane-coated pH-type bionic nanoparticle delivery system by the following method:

[0019] RAW264.7 cells were mechanically ground and subjected to differential centrifugation to extract macrophage membranes and dispersed in double-distilled water by ultrasonication;

[0020] Hyaluronic acid was used as a carrier to load the endogenous hydrogen sulfide donor SPRC, which was chemically cross-linked with the PEGylated photosensitizer Ce6 through an acid-responsive linker to form pH-sensitive nanoparticles.

[0021] The pre-prepared pH-type nanoparticles were co-incubated with macrophage membranes and repeatedly extruded through the membrane using a membrane extruder.

[0022] In the present invention, the inner core of the pH-type bionic nanosystem is nanoparticles, which interact with the outer cell membrane through electrostatic force or hydrophobic force to form a pH-type bionic nanoparticle drug delivery system coated with macrophage membrane.

[0023] In the present invention, the average particle size of the nanoparticles is 90-150 nm, and the final average particle size after loading SPRC and Ce6 and coating the macrophage membrane is 120-180 nm. The cell membrane is dispersed on the surface of the bionic nanoparticles, which is beneficial to the role of membrane surface-related protein molecules.

[0024] The present invention uses human rheumatoid arthritis fibroblasts (MH7A) to construct an in vitro rheumatoid arthritis inflammation model under lipopolysaccharide (LPS) stimulation to evaluate the photodynamic effect of the macrophage membrane-coated pH-type bionic nanosystem in vitro. This method is common and recognized in the art.

[0025] The present invention uses Mycobacterium tuberculosis (Mtb) to construct an in vivo rheumatoid arthritis model, namely an adjuvant-induced arthritis (AIA) rat model, to evaluate the photodynamic in vivo efficacy of the macrophage membrane-coated pH-type bionic nanosystem, which is common and recognized in the art.

[0026] The administration mode of the macrophage membrane-coated pH-type bionic nanosystem prepared by the present invention is intravenous injection.

[0027] The advantages of the present invention are:

[0028] The prepared macrophage membrane-coated pH-type bionic nanosystem is different from other targeted nanocarriers modified with a single molecule or multiple molecules. This bionic nanosystem has unique advantages in the treatment of rheumatoid arthritis:

[0029] Using macrophage membrane surface molecules to actively target inflammatory lesions in rheumatoid arthritis;

[0030] The nanoparticles are loaded with SPRC and Ce6 through hyaluronic acid, have a good grafting rate, and have pH responsive properties. The small amount of hydrogen sulfide provided by SPRC can effectively inhibit mitochondrial function, inhibit oxygen consumption, and enhance the photodynamic therapy effect of rheumatoid arthritis.

[0031] The pH-type bionic nanosystem increases the biocompatibility of the system and reduces phagocytosis of the endothelial system through coating of the macrophage membrane, thus having high biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a characterization diagram of the particle size and potential of the pH-responsive bionic nanosystem in the present invention.

[0033] Figure 2 It is a transmission electron microscope image of the nanoparticles and bionic nanoparticles in the present invention.

[0034] Figure 3 This is the qualitative detection result of the surface protein of the nano drug delivery system of the present invention.

[0035] Figure 4 This is the in vitro release diagram of SPRC in the pH-responsive bionic nanosystem of the present invention.

[0036] Figure 5 It is a fluorescence image of the cell uptake ability of the pH-responsive bionic nanosystem of the present invention in an in vitro cell model and a corresponding ImageJ quantitative analysis image.

[0037] Figure 6 The present invention is a photodynamic therapy evaluation of inflammatory cells in an in vitro cell model using the pH-responsive bionic nanosystem.

[0038] Figure 7 The flow cytometry results of cell apoptosis of the pH-responsive bionic nanosystem in the present invention in an in vitro cell model.

[0039] Figure 8 It is a fluorescence image of the pH-responsive bionic nanosystem of the present invention detecting the hydrogen sulfide level using WSP-1 as a probe in an in vitro cell model and the corresponding ImageJ quantitative analysis image.

[0040] Fig. 9 It is a fluorescence image and a corresponding ImageJ quantitative analysis image of the pH-responsive bionic nanosystem of the present invention detecting the change of mitochondrial membrane potential by using JC-1 test in an in vitro cell model.

[0041] Fig.10 It is the determination of the cell oxygen consumption rate (OCR) level of the pH-responsive bionic nanosystem in the in vitro cell model.

[0042] Fig.11 It is a fluorescence image of the pH-responsive bionic nanosystem of the present invention detecting the change of reactive oxygen species (ROS) using DCFH-DA as a probe in an in vitro cell model and the corresponding ImageJ quantitative analysis image.

[0043] Fig.12The flow cytometric results of the pH-responsive bionic nanosystem of the present invention detecting the changes in reactive oxygen species (ROS) using DCFH-DA as a probe in an in vitro cell model.

[0044] Fig.13 The diagrams are in vivo fluorescence imaging and quantitative analysis of the targeted experiment of the pH-responsive bionic nanosystem in the in vivo animal model.

[0045] Fig.14 The paw volume change and inflammation score of the in vivo treatment experiment of the pH-responsive bionic nanosystem in the present invention in an in vivo animal model. DETAILED DESCRIPTION

[0046] In order to make the technical solution of the present invention easier to understand, the technical solution of the present invention is now clearly and completely described in the form of specific embodiments in conjunction with the accompanying drawings.

[0047] Example 1

[0048] Extraction and preparation of macrophage membranes:

[0049] RAW264.7 cells were passaged and expanded in a cell culture incubator at 37°C and 5% CO2 using DMDM ​​medium + 10% FBS + 1% PS. The cells were centrifuged at 1000 rpm / min for 3 min to remove the supernatant and washed three times with PBS (4°C, 1000 rpm / min, 3 min). RAW264.7 cells were resuspended in prepared membrane extraction TM buffer (pH 7.4; 10 mM Tris + 1 mM MgCl2) and 1% protease inhibitor was added to control the concentration to 2.0 × 10 7 / mL. Place on ice and grind mechanically for 300 times, grind twice. Then mix 1M sucrose with the cell homogenate and adjust to 0.25M sucrose concentration, centrifuge (4℃, 2000g, 15min), remove the cell nucleus and other structures, collect the supernatant and further centrifuge (4℃, 3000g, 30min), collect the precipitate to obtain the cell membrane. Wash twice with ice-cold TM buffer in the presence of 0.25M sucrose to purify the cell membrane. After centrifugation, resuspend the cell membrane in distilled water, freeze-dry and store at -80℃ for later use.

[0050] Example 2

[0051] Preparation of pH-responsive nanoparticles:

[0052] 1) Using hyaluronic acid as a carrier to load SPRC and modified acid-responsive linker: Synthesis is performed according to a mass ratio of hyaluronic acid to SPRC of 5:1 to 5:8, with a preferred ratio of 5:3. Taking the preferred dosage as an example, 20 mg of hyaluronic acid and 12 mg of SPRC are added to a 0.1M sodium bicarbonate solution, and stirred for reaction at room temperature for 24 hours under the action of an appropriate amount of catalytic reagent (N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride, EDC, 24 mg). Free small molecules are then removed by dialysis, and the primary product is obtained by freeze-drying. It is then redissolved in HEPE buffer (pH 8.5), 4 mg of acid-responsive linker BRM is added, reacted at room temperature for 24 hours, and then dialyzed to remove free small molecules, and freeze-dried to obtain an intermediate product. The SPRC grafting rate is calculated according to the formula: SPRC grafting rate = (total SPRC dosage - free SPRC) / total mass × 100%. According to the above preferred formulation, the SPRC grafting rate is about 10%. In addition, the above operation synthesis is carried out according to two formulations with a mass ratio of hyaluronic acid to SPRC of 5:1 and 5:8, and the SPRC grafting rates are about 4% and 10%, respectively. The grafting rate mainly depends on the SPRC dosage and the total number of carboxyl groups on the carrier.

[0053] 2) PEGylated photosensitizer Ce6: First, 10 mg of Ce6 was dissolved in a small amount of DMSO solvent and activated under the catalytic action of 12.3 mg of EDC and 8.1 mg of NHS (stirring reaction for 30 minutes). Then it was slowly added to a buffer solution containing 100 mg of eight-arm PEG-NH2 (10 KDa) and stirred for 24 hours. After the reaction was completed, the free small molecules and a small amount of DMSO were removed by dialysis, and the intermediate product was lyophilized.

[0054] 3) Preparation of pH-responsive nanoparticles: Nanoparticles are prepared according to the mass ratio of the intermediate products synthesized in 1 and 2 above in the range of 5:1 to 1:5, with a preferred ratio of 2:1. Taking the preferred dosage as an example, the synthetic products of 1 and 2 above are added to HEPE buffer (pH 8.5) and stirred for reaction for 24 hours. After the reaction is completed, unreacted molecules are removed by ultrafiltration to obtain the final product. Taking the preferred dosage as an example, the average particle size of the pH-responsive nanoparticles measured by the particle size analyzer is about 100nm, and the zeta potential results are shown in Figure 1 .

[0055] 4) Determination of SPRC content: The SPRC content is determined indirectly by measuring the free SPRC collected by ultrafiltration centrifugation in the above 3 steps by high performance liquid chromatography, and the SPRC content is calculated according to the formula: drug loading rate = (total SPRC amount - free SPRC) / total mass × 100%. The nanoparticles are synthesized according to the preferred prescription ratios in the above 3, and the SPRC drug loading rate is about 5%. The drug loading rate is mainly affected by the grafting rate in the above 1 and the input ratio in the above 3. In addition, the nanoparticles are prepared by the above 3 operations according to the two prescriptions with the mass ratio of the intermediate products synthesized in the above 1 and 2 being 5:1 and 1:5, and the SPRC drug loading rates are about 7% and 1%, respectively. Considering the drug loading rate, the dispersibility and uniformity of the nanoparticles, the preferred ratio is 2:1.

[0056] Example 3

[0057] Preparation of pH-responsive biomimetic nanosystem coated with macrophage membrane:

[0058] The cell membrane and the pH-responsive nanoparticles (as described above) are mixed and coated in a mass ratio of 50:1 to 1:50, and the preferred ratio is 1:1. Taking the preferred prescription as an example, the cell membrane and the nanoparticles are mixed in a ratio of 1:1, ultrasonically treated for 5 minutes, and extruded through 400nm and 200nm polycarbonate membranes in a film extruder to obtain a regular macrophage membrane-coated pH-responsive bionic nanosystem. The average particle size of the pH-responsive bionic nanoparticles is about 100nm as measured by a particle size analyzer, and the particle size of the nanoparticles increases after being coated with macrophage membranes of different ratios. In addition, the cell membrane and the nanoparticles are mixed and coated in the above steps according to two prescriptions of a mass ratio of 50:1 and 1:50, wherein the nanoparticles coated in a ratio of 50:1 have a Zeta potential that is almost the same as that of a simple cell membrane due to an excessive amount of cell membrane, while the nanoparticles coated in a ratio of 1:50 have an insignificant change in particle size before and after the coating due to an excessive amount of cell membrane. Taking into account the stability, uniformity and cellular uptake of the nanoparticles, the preferred ratio is 1:1.

[0059] For example, in a 1:1 ratio Figure 1 After the macrophages were coated with pH-responsive nanoparticles, the particle size was about 120-180nm. The original potential of the nanoparticles, about -11mV, changed significantly after being coated with the negatively charged macrophage membrane, and the Zeta potential became more negative at -21mV. The morphology was characterized by transmission electron microscopy. Figure 2 It can be observed that the pH-responsive biomimetic nanoparticles have a relatively regular spherical structure with a particle size of about 140 nm. The nanoparticles, macrophage membranes and biomimetic nanoparticles were subjected to gel electrophoresis and stained with Coomassie brilliant blue. Figure 3 It can be observed that the biomimetic nanoparticles have associated proteins consistent with macrophage membranes.

[0060] Example 4

[0061] SPRC release of pH-responsive biomimetic nanoparticles coated with macrophage membrane in vitro:

[0062] The preparation method of pH-responsive biomimetic nanoparticles encapsulating SPRC was the same as above. The in vitro release behavior of SPRC from free SPRC and biomimetic nanoparticles under different pH conditions was investigated by dialysis. PBS at pH 7.4 or pH 5.0 was used as the release solution. Free SPRC and biomimetic nanoparticles were respectively encapsulated into 1 ml dialysis bags (MWCO = 8000-14000Da), immersed in the release solution in a 50 ml centrifuge tube, and the centrifuge tube was placed in a 37°C water bath shaker. 1 ml of the release solution was taken out at a fixed point to determine the released motherwort alkaloid, and 1 ml of fresh release solution was added at the same time. The SPRC content was determined under high performance liquid chromatography HPLC, and the SPRC release percentage was determined according to the standard curve. Figure 4 The release of free SPRC was close to 95% in 2 hours, while the bionic nanoparticles continued to release until 12 hours, and the release curve became flat. In addition, SPRC was released faster and more under pH 5.0 conditions, indicating that the pH-responsive bionic nanoparticles have a certain sustained release ability and can specifically release more SPRC under acidic conditions, providing a basis for the generation of hydrogen sulfide.

[0063] Example 5

[0064] In vitro cellular uptake and photodynamic effect evaluation of pH-responsive biomimetic nanoparticles coated with macrophage membranes

[0065] 1) In vitro cell uptake: MH7A cells were seeded into 12-well plates, the culture medium was DMDM ​​medium + 10% FBS + 1% PS, and 1 μg / mL LPS was given to stimulate the cells to form an inflammatory cell model. The pH-responsive biomimetic nanoparticles before and after macrophage membrane coating were added to each well plate and incubated for 1.5 hours, and then placed under a laser confocal fluorescence microscope for observation. Figure 5 The inflammatory cell uptake rate of pH-responsive bionic nanoparticles coated with macrophage membranes was significantly higher than that of uncoated bionic nanoparticles, indicating that macrophage membranes have important functions and significance in targeting inflammatory cells.

[0066] 2) In vitro evaluation of photodynamic effect: MH7A cells were seeded into 96-well plates. The culture medium was DMDM ​​medium + 10% FBS + 1% PS, and 1 μg / mL LPS was given to stimulate the cells to form an inflammatory cell model. Free Ce6 and macrophage membrane-coated pH-responsive biomimetic nanoparticles were added to each well plate and incubated for 24 hours, and the irradiation group was irradiated at 660nm for 10 minutes 2 hours after addition. The cell survival rate was determined by the Alamar Blue method, that is, the cell survival was calculated by measuring the absorption values ​​at 570nm and 600nm with an enzyme reader. Figure 6 The pH-responsive bionic nanoparticles coated with macrophage membranes can effectively kill inflammatory cells under light irradiation, and their killing ability is stronger than that of the free Ce6 group, indicating that the SPRC in the pH-responsive bionic nanosystem coated with macrophage membranes provides a small amount of hydrogen sulfide to enhance the photodynamic effect. In addition, the apoptosis of cells after irradiation is shown in Figure 2. Figure 7 Compared with the normal group and the free Ce6 group, the apoptosis in the macrophage membrane-coated pH-responsive bionic nanosystem group was more significant, indicating that the bionic nanosystem had a significant in vitro photodynamic effect and the small amount of hydrogen sulfide provided by SPRC could further enhance the photodynamic effect in vitro.

[0067] Example 6

[0068] Study on the mechanism of enhanced photodynamic effect of pH-responsive biomimetic nanoparticles coated with macrophage membrane in vitro

[0069] MH7A cells were seeded into 24-well plates in a medium of DMDM ​​medium + 10% FBS + 1% PS, and 1 μg / mL LPS was given to stimulate the cells to form an inflammatory cell model. Nanoparticles containing or not containing SPRC or biomimetic nanoparticles coated with macrophage membranes were added to each well plate and incubated for 2 hours. WSP-1 was added to each well plate as a fluorescent probe to detect the generation of hydrogen sulfide at the cellular level. Figure 8 , both the nanoparticles containing SPRC or the biomimetic nanoparticles can observe obvious fluorescence signals, indicating the generation of hydrogen sulfide. At the same time, the biomimetic nanoparticles coated with macrophage membranes can produce more hydrogen sulfide, because the targeting of macrophage membranes makes them interact more strongly with cells.

[0070] The inflammatory cell model was established in the above manner, and nanoparticles containing or not containing SPRC or biomimetic nanoparticles coated with macrophage membranes were added to each well plate and incubated for 2 hours. JC-1 working solution was added to each well plate and the staining operation was performed according to the instructions of the kit, and finally the fluorescence signal in each well plate was observed under a fluorescence microscope. Fig. 9The proportion of JC-1 monomers in the positive CCCP group and the nanogroup or biomimetic nanogroup containing SPRC increased significantly, and mitochondrial dysfunction became more serious, indicating that hydrogen sulfide produced by SPRC can inhibit mitochondrial function. Fig.10 , by using the OCR kit to detect the oxygen consumption of each group of cells, the results showed that the nanogroup or bionic nanogroup containing SPRC could reduce its oxygen consumption by inhibiting mitochondrial function.

[0071] In addition, if Fig.11 and Fig.12 , using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) as a ROS probe, the in vitro ROS level was observed and detected by fluorescence microscopy and flow cytometry. The pH-responsive bionic nanosystem coated with macrophage membrane containing SPRC can produce more ROS at the cellular level, ultimately enhancing the effect of in vitro photodynamic therapy, providing a basis and foundation for its enhanced photodynamic effect.

[0072] Example 7

[0073] In vivo targeting of pH-responsive biomimetic nanosystems coated with macrophage membranes

[0074] The AIA rat rheumatoid arthritis animal model was established: Complete Freund's adjuvant emulsion containing inactivated Mycobacterium tuberculosis (Mtb) was subcutaneously injected into the rats at the base of the tail (100 μL per rat). The symptoms of arthritis were monitored by three indicators: rat body weight, arthritis score and hind paw volume.

[0075] The AIA rat rheumatoid arthritis animal model was established as described above. On the 12th day, free Ce6 and pH-responsive biomimetic nanoparticles (Ce6: 1 mg / kg) before and after macrophage membrane coating were intravenously injected into AIA rats and normal rats. The living imaging fluorescence images of the ankle joints of each group of rats were observed and photographed at 1 hour, 2 hours, 4 hours, 6 hours, 12 hours and 24 hours using the Living Imaging System (Perkin Elmer, USA). Fig.13 The pH-responsive biomimetic nanoparticles coated with macrophage membranes were significantly enriched in the ankles of AIA rat models, which was significantly higher than that in other groups, indicating the key role and significance of macrophage membranes in targeting the inflammatory lesions of rheumatoid arthritis.

[0076] Example 8

[0077] Photodynamic therapy of rheumatoid arthritis by pH-responsive biomimetic nanosystem coated with macrophage membrane

[0078] The AIA rat rheumatoid arthritis animal model was established as described above. On day 12, the AIA rats were randomly divided into 7 groups: 1) model group; 2) SPRC; 3) Ce6+ irradiation; 4) RAW@HSC NP; 5) HSC NP+ irradiation; 6) RAW@HC NP+ irradiation; 7) RAW@HSC NP+ irradiation. AIA rats were intravenously injected with nanoparticles of each group every other day, each time at a dose of 1 mg / kg Ce6 and 1 mg / kg SPRC by tail vein injection for one week. In the irradiation group, rats were irradiated with 660nm light for 30 minutes 6h after injection. The body weight, hind paw volume and arthritis score of these rats were recorded and evaluated every three days. In the arthritis score assessment, each paw and tail were scored from 0 to 4. The scoring criteria are as follows: 0 = no abnormality; 1 = mild erythema and swelling; 2 = mild to moderate erythema and swelling; 3 = obvious erythema and swelling; 4 = severe erythema and swelling. On the 30th day, the rats in the experimental group were euthanized and their hind paws were photographed. In addition, their blood, organs (heart, liver, spleen, lung, kidney), synovial tissue and hind paws were collected for further study. During the treatment, the symptoms of arthritis were monitored by three indicators: the body weight of the rats, the arthritis score and the hind paw volume. Fig.14 (AC), the irradiated pH-responsive bionic nanosystem group was significantly better than the other groups, which was the result of the targeting effect of the macrophage membrane and the release of a small amount of hydrogen sulfide by SPRC to enhance the photodynamic therapy effect. Fig.14 (D) During the treatment period, the body weight of rats in the normal group and irradiation treatment group increased steadily, while the body weight of rats in the model group was relatively light.

[0079] It should be noted that the embodiments described herein are only partial embodiments of the present invention, rather than all implementations of the present invention, and the embodiments are only exemplary, and their role is only to provide a more intuitive and clear way to understand the content of the present invention, rather than to limit the technical solutions described in the present invention. Without departing from the concept of the present invention, all other implementation methods that can be thought of by ordinary technicians in this field without creative work, and other simple replacements and various changes to the technical solutions of the present invention, all belong to the protection scope of the present invention.

Claims

1. A pH-responsive bionic nanosystem for enhanced photodynamic therapy, characterized in that: The pH-responsive bionic nanosystem for enhanced photodynamic therapy consists of pH-responsive nanoparticles loaded with endogenous hydrogen sulfide donor SPRC and photosensitizer and macrophage membranes. The nanoparticles are obtained by chemical covalent cross-linking, and the nanoparticles and macrophage membranes are co-extruded by ultrasound and a membrane extruder to achieve outer membrane coating.

2. A pH-responsive bionic nanosystem for enhancing photodynamic therapy according to claim 1, characterized in that: The macrophage cells are RAW264.7 cell lines, and the cell membrane surface of the macrophages overexpresses related adhesion molecules.

3. A pH-responsive bionic nanosystem for enhancing photodynamic therapy according to claim 2, characterized in that: Macrophage membranes were obtained by mechanical grinding and differential centrifugation extraction, and cell membrane vesicles were obtained by ultrasonic treatment.

4. The pH-responsive bionic nanosystem for enhancing photodynamic therapy according to claim 2, characterized in that: Relevant adhesion molecules include Mac-1 and LFA-1 membrane proteins.

5. The pH-responsive bionic nanosystem for enhancing photodynamic therapy according to claim 2, characterized in that: The photosensitizer is Ce6.

6. The method for preparing a pH-responsive bionic nanosystem for enhancing photodynamic therapy according to any one of claims 1 to 5, characterized in that: pH-responsive nanoparticles are synthesized and prepared by chemical covalent cross-linking: the prescribed amount of hyaluronic acid, hydrogen sulfide donor and acid-responsive linker BRM are weighed, dissolved in an appropriate amount of alkaline aqueous solvent, stirred and reacted for 12 to 24 hours under the action of a catalyst, free small molecules are removed by dialysis, and the intermediate product is obtained by freeze-drying, which is then chemically cross-linked with a pegylated photosensitizer in an alkaline aqueous solvent to obtain pH-responsive nanoparticles HSC.

7. The method for preparing a pH-responsive bionic nanosystem for enhancing photodynamic therapy according to claim 6, characterized in that: During the preparation process, 1) the mass ratio of the carrier hyaluronic acid to the hydrogen sulfide donor is 5:1 to 5:8, and the optimal ratio is 5:3; 2) when synthesizing nanoparticles, the mass ratio of the hyaluronic acid loaded with the hydrogen sulfide donor to the pegylated photosensitizer is 5:1 to 1:5, and the optimal ratio is 2:

1.

8. The method for preparing a pH-responsive bionic nanosystem for enhancing photodynamic therapy according to claim 6, characterized in that: The nanoparticles and the macrophage membrane are coated with an outer layer membrane by ultrasound for 3-10 minutes and a membrane extrusion method, and the mass ratio thereof is 50:1-1:50, and the preferred ratio is 1:

1.

9. The method for preparing a pH-responsive bionic nanosystem for enhancing photodynamic therapy according to claim 8, characterized in that: The average particle size of the nanoparticles is 90-150 nm, and the particle size of the bionic nanoparticles covering the macrophage membrane is 120-180 nm.

10. The use of a pH-responsive bionic nanosystem for enhancing photodynamic therapy according to any one of claims 1 to 5, characterized in that: A pH-responsive bionic nanosystem for enhancing photodynamic therapy is used in the preparation of drugs for targeted treatment of rheumatoid arthritis; the prepared drug produces a small amount of hydrogen sulfide at the inflammatory site by loading the hydrogen sulfide donor SPRC, inhibiting mitochondrial function and oxygen consumption, thereby enhancing ROS generation and strengthening the photodynamic therapy effect of rheumatoid arthritis.

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