Nano / gas engineered erythrocyte medicinal preparation as well as preparation method and application thereof

Abstract

The invention discloses a nano / gas engineered red blood cell medicinal preparation as well as a preparation method and application thereof, belongs to the technical field of gas infusion treatment, and aims at accurately controlling delivery of carbon monoxide (CO) and nitric oxide (NO) into target cells and controlling the dosage and the dosage ratio of the two gases to prepare the nano / gas engineered red blood cell medicinal preparation. The compound has different synergistic effects of anti-inflammation, cell protection, tumor cell killing and the like, and plays an important role in treatment of different diseases (such as arthritis and tumor).

Description

Technical Field

[0001] The present invention relates to the technical field of gas infusion therapy, and in particular to a nano / gas engineered red blood cell pharmaceutical preparation and a preparation method and application thereof. Background Art

[0002] In recent years, gas therapy has attracted widespread attention in the treatment of various major diseases due to the unique therapeutic effects of physiological gas molecules such as O2, NO, CO and H2S.

[0003] In the treatment of arthritis, low-concentration CO inhibits proinflammatory factors (such as TNF-α and IL-1β) by activating the Nrf2 / HO-1 pathway, alleviating joint inflammation and possibly reducing chondrocyte apoptosis.

[0004] In tumor treatment, high concentrations of CO have direct adverse effects on the mitochondria of cancer cells by interfering with cytochrome c oxidase, leading to excessive production of the strong oxidant hydrogen peroxide (H2O2), ATP depletion, and pro-apoptotic effects. High concentrations of NO can react with superoxide anions (O2 - ) reacts to form peroxynitrite (ONOO - ), a potent oxidative and nitrating reactive nitrogen species (RNS), not only induces direct DNA damage but also mediates post-translational modification of proteins via S-nitrosylation of cysteine ​​residues, thereby disrupting the metabolic homeostasis of cancer cells.

[0005] However, gas therapy faces challenges in clinical applications, such as nonspecific gas release and biosafety issues. Red blood cells (RBCs), as natural gas carriers, have advantages such as high stability and strong affinity for gas molecules, but their micrometer-sized size limits their ability to reach lesions and enter target cells. Therefore, developing a new method to engineer RBCs to enable gas infusion therapy is of great clinical significance. Summary of the invention

[0006] The purpose of the present invention is to provide a nano / gas engineered red blood cell pharmaceutical preparation and its preparation method and application. The system can achieve safe and efficient delivery of CO and NO, and specifically release them in arthritis lesions or tumors, and utilize the regulatory effects of CO and NO on cells to reshape the immune microenvironment of arthritis lesions or tumor tissues, thereby effectively treating arthritis or tumors.

[0007] To achieve the above object, the present invention provides a method for preparing a nano / gas engineered red blood cell pharmaceutical preparation, comprising the following steps:

[0008] S1. Using a liposome extruder, the washed red blood cells are extruded through a polycarbonate porous membrane to obtain nano-engineered red blood cells nRBC;

[0009] S2. The nano-engineered red blood cells prepared in step S1 are loaded with CO or NO gas to prepare nano / gas engineered red blood cell preparation nRBCs.

[0010] Preferably, in step S1, the pore size of the polycarbonate porous membrane is 5-0.2 μm.

[0011] Preferably, in step S2, loading CO or NO gas comprises: exposing the nRBC obtained in step S1 to CO or NO gas under stirring, verifying the loading status of the gas in real time, and obtaining gas-saturated nRBCs.

[0012] In a second aspect, the present invention provides a nano / gas engineered red blood cell pharmaceutical preparation prepared by the above-mentioned preparation method, wherein the particle size of the nano / gas engineered red blood cell preparation nRBCs is 150 nm.

[0013] In a third aspect, the present invention provides the use of the above-mentioned nano / gas engineered red blood cell pharmaceutical preparation in gas infusion therapy for non-medical purposes.

[0014] In a fourth aspect, the present invention provides an application of the above-mentioned preparation method in preparing a gas infusion therapeutic drug preparation, wherein the gas infusion therapeutic drug preparation includes: an arthritis gas infusion therapeutic drug preparation and a tumor gas infusion therapeutic drug preparation;

[0015] The preparation method includes: co-extruding the target cell membrane and washed red blood cells to construct cell membrane / nano / gas engineered red blood cells, and the cell membrane / nano / gas engineered red blood cells can target homologous target cells.

[0016] Preferably, the method for preparing the arthritis gas infusion therapeutic pharmaceutical preparation comprises:

[0017] S71, mixing the washed red blood cells with the macrophage membrane and co-extruding the mixture through a polycarbonate porous membrane to obtain macrophage membrane / nano / gas engineered red blood cells M-nRBC;

[0018] S72. Exposing the macrophage membrane / nano / gas engineered red blood cells M-nRBC to CO gas to obtain M-nRBC CO .

[0019] Preferably, the method for preparing the drug preparation for tumor gas infusion therapy comprises:

[0020] S81, mixing the washed red blood cells with the tumor cell membrane and co-extruding the mixture through a polycarbonate porous membrane to obtain tumor cell membrane / nano / gas engineered red blood cells T-nRBC;

[0021] S82. Exposing tumor cell membrane / nano / gas engineered red blood cells T-nRBC to CO or NO gas to obtain T-nRBC CO and T-nRBC NO .

[0022] Preferably, in the drug preparation for tumor gas infusion therapy, T-nRBC CO :T-nRBC NO The molar ratio is 1~4:4~1.

[0023] Preferably, in the drug preparation for tumor gas infusion therapy, T-nRBC CO :T-nRBC NO The molar ratio is 1:3.

[0024] Therefore, the nano / gas engineered red blood cell pharmaceutical preparation and its preparation method and application of the present invention have the following beneficial effects:

[0025] (1) Nanoengineered red blood cells (nRBCs) are prepared by a polycarbonate porous membrane extruder. CO and NO gases bind to the sites on the hemoglobin molecule that originally bind to oxygen to form nRBCs. CO and nRBC NO ;

[0026] (2) The hemoglobin in the nano-erythrocytes contains hematoporphyrin, which can responsively release CO and NO under H2O2 conditions;

[0027] (3) Extract macrophage cell membrane and co-extrude with nRBC to construct macrophage cell membrane / nano / CO gas engineered red blood cells (M-nRBC) CO ), achieving targeted therapy of homologous macrophages; M-nRBC CO CO is specifically released in the high hydrogen peroxide (H2O2) microenvironment of macrophages in the joint cavity. CO can induce the expression of HO-1, coordinate the polarization homeostasis of macrophages through the Nrf2 / HO-1 pathway, promote the formation of anti-inflammatory macrophages (M2 type), and inhibit the activity of pro-inflammatory macrophages (M1 type), thereby exerting an anti-inflammatory effect;

[0028] (4) Extract tumor cell membrane and co-extrude with nRBC to construct tumor cell membrane / nano / gas engineered red blood cells (T-nRBC CO and T-nRB CNO ), achieving targeted therapy of homologous tumor cells; T-nRBC CO and T-nRBC NOIt specifically releases CO and NO in the high hydrogen peroxide (H2O2) microenvironment in tumor cells, and produces a large amount of ROS and RNS under the catalysis of peroxidase, which destroys the metabolic homeostasis of tumor cells and regulates the tumor immunosuppressive microenvironment, transforming "cold" tumors into "hot" tumors and enhancing immune-mediated tumor killing. It has been verified that the anti-tumor therapeutic effect is best when the molar ratio of CO to NO is 1:3.

[0029] (5) T-nRBC CO and T-nRB CNO In the high hydrogen peroxide (H2O2) microenvironment within tumor cells, CO specifically releases CO and NO, and CO promotes the release of OH and O2 ·- The production of NO and the increased O2 ·- The reaction produces more ONOO - , the dual-gas co-delivery and synergistic effect of CO and NO significantly enhances the therapeutic effect of dual gases in tumor treatment;

[0030] (6) The nano / gas engineered red blood cell pharmaceutical preparation of the present invention has excellent biosafety, does not cause acute or long-term toxicity, and has good biocompatibility.

[0031] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0033] Figure 1 The dynamic light scattering diagrams of different nano-red blood cell preparations, where A is nRBC, nRBC CO 、nRBC NO DLS diagram of M-nRBC, M-nRBC CO Dynamic light scattering diagram, C is T-nRBC, T-nRBC, T-nRBC NO Dynamic light scattering diagram of

[0034] Figure 2 TEM images of different nano-red blood cell preparations, where A is nRBC, nRBC CO 、nRBC NO TEM images of M-nRBC and M-nRBC. CO TEM images of T-nRBC and T-nRBC.CO 、T-nRBC NO TEM images of

[0035] Figure 3 For different nano red blood cell preparations (nRBC, nRBC CO 、nRBC NO 、M-nRBC、M-nRBC CO 、T-nRBC、T-nRBC CO 、T-nRBC NO )’s UV spectrum;

[0036] Figure 4 are the particle sizes and PDI indexes of different nano-red blood cell preparations stored at 4°C for 21 days, where A represents nRBC, nRBCCO, nRBC NO The particle size and PDI index of B are M-nRBC and M-nRBC. CO 、M-nRBC NO The particle size and PDI index of C are T-nRBC and T-nRBC CO 、T-nRBC NO Particle size and PDI index;

[0037] Figure 5 For free hemoglobin (Hb) and nano / gas engineered red blood cells (nRBC, nRBCCO, nRBC NO )’s circular dichroism spectrum;

[0038] Figure 6 nRBC CO 、nRBC NO Gas release test results, where A is nRBC CO Gas release under normal physiological conditions and simulated cellular oxidative microenvironment conditions, B is nRBC NO Gas release under normal physiological conditions and under conditions simulating cellular oxidative microenvironment;

[0039] Figure 7 Normal macrophages (Raw267.4), LPS-treated macrophages, LPS+dexamethasone (DMX)-treated macrophages, and LPS+M-nRBC CO The concentrations of inflammatory factors in the supernatant of treated macrophages, where A is the concentration of iNOS, B is the concentration of TNF-α, and C is the concentration of IL-1β;

[0040] Figure 8 Normal macrophages (Raw267.4), LPS-treated macrophages, LPS+DMX-treated macrophages, and LPS+M-nRBC CO CD86 immunofluorescence staining of treated macrophages;

[0041] Fig. 9 Western blot analysis of normal macrophages, LPS-treated macrophages, LPS+DMX-treated macrophages, and LPS+M-nRBC CO Processing of protein content in macrophages;

[0042] Fig.10 The articular cartilage was stained with hematoxylin-eosin (HE), safranin fast green (S / F) and toluidine blue (Tb);

[0043] Fig.11 This is the OARSI score diagram of osteoarthritis pathological tissue;

[0044] Fig.12 Immunohistochemical sections of joints for Nrf2 and HO-1, and immunohistochemistry sections for CD86;

[0045] Fig.13 HE images of major organs (heart, liver, spleen, lung, and kidney) stained with hematoxylin-eosin after drug administration;

[0046] Fig.14 To evaluate the nano / gas engineered red blood cells (nRBC) using MTT assay CO and nRBC NO ) against breast cancer 4T1, melanoma B16-F10 and colon cancer CT26 cell lines, wherein A is nRBC, nRBC CO 、nRBC NO and different molar ratios of nRBC CO With nRBC NO Survival rate of breast cancer 4T1 cells in the treatment group, B is nRBC, nRBC CO 、nRBC NO and different molar ratios of nRBC CO With nRBC NO The survival rate of melanoma B16-F10 cells in the treatment group, C is nRBC, nRBC CO 、nRBC NO and different molar ratios of nRBC CO With nRBC NO Survival rate of melanoma B16-F10 cells in the treatment group, D is different molar ratios of nRBC CO With nRBC NO The half-maximal inhibitory concentration (IC50) and combination index (CI) of the treatment group on breast cancer 4T1 cells, E is the nRBC at different molar ratios CO With nRBC NOThe half-maximal inhibitory concentration (IC50) and combination index (CI) of the treatment group on melanoma B16-F10 cells, F is the nRBC at different molar ratios CO With nRBC NO The half-maximal inhibitory concentration (IC50) and combination index (CI) of the treatment groups on colon cancer CT26 cells;

[0047] Fig.15 Nano / gas engineered red blood cells (nRBC CO and nRBC NO ) Flow cytometry results of in vitro ROS / RNS cascade generation after 6 h of co-incubation with 4T1 cells;

[0048] Fig.16 The results of the material targeting experiments of tumor cell membrane / nanoengineered red blood cells (nRBC and T-nRBC) in the 4T1 orthotopic breast tumor model in vivo at 3h, 12h, and 24h;

[0049] Fig.17 Tumor cell membrane / nano / gas engineered red blood cells (nRBC, nRBC CO 、nRBC NO 、nRBC CO / nRBC NO 、T-nRBC CO / T-nRBC NO ) Tumor inhibition curves after treatment in the 4T1 orthotopic breast tumor model;

[0050] Fig.18 Tumor cell membrane / nano / gas engineered red blood cells (nRBC, nRBC CO 、nRBC NO 、nRBC CO / nRBC NO 、T-nRBC CO / T-nRBC NO ) Tumor inhibition curves after treatment in the B16 melanoma lung metastasis model;

[0051] Fig.19 Tumor cell membrane / nano / gas engineered red blood cells (nRBC, nRBC CO 、nRBC NO 、nRBC CO / nRBC NO 、T-nRBC CO / T-nRBC NO ) Obvious bioluminescent signal patterns were observed in the lungs after treatment in the B16 melanoma lung metastasis model;

[0052] Fig. 20Evaluation of nano / gas engineered red blood cells (nRBC, nRBC) in SD rats CO 、nRBC NO 、nRBC CO / nRBC NO ) of acute toxicity and immunogenicity (IgG and IgM), wherein A is the IgG level of SD rats at 1 hour, B is the IgM level of SD rats at 1 hour, C is the IgG level of SD rats at 48 hours, and D is the IgM level of SD rats at 48 hours;

[0053] Fig.21 For transfusion of tumor cell membrane / nano / gas engineered red blood cells (nRBC, nRBC CO 、nRBC NO 、nRBC CO / nRBC NO 、T-nRBC CO / T-nRBC NO ) after the rats were treated with 4% paraformaldehyde, wherein A is blood pressure, B is blood oxygen saturation, and C is heart rate. DETAILED DESCRIPTION

[0054] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0055] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0056] The instruments, equipment, reagents and materials used in the examples were all obtained through commercial channels.

[0057] Embodiment 1

[0058] A nano / gas engineered red blood cell pharmaceutical preparation specifically comprises the following steps:

[0059] S1. Red blood cell (RBC) collection: Whole blood was collected from the heart of SD rats using a centrifuge tube containing EDTA-2K anticoagulant, and centrifuged at 3000 rap / min for 15 min to obtain an upper transparent yellow plasma layer and a lower dark red red red blood cell liquid layer.

[0060] Use a rubber-tipped dropper to aspirate the upper transparent yellow plasma, add PBS to the lower dark red red red blood cell liquid, mix gently, and centrifuge at 3000 rap / min for 15 minutes.

[0061] Repeat 3 times until the supernatant no longer appears yellow, indicating that the red blood cells are washed clean.

[0062] S2. Preparation of nano-engineered red blood cells (nRBC): Red blood cells are extruded through a polycarbonate porous membrane (pore size 5-0.2 μm) using a liposome extruder, retaining the natural membrane structure and internal hemoglobin (Hb) of red blood cells to obtain nRBC.

[0063] S3. Preparation of nano / gas engineered red blood cells (nRBCs)

[0064] The nRBCs obtained in step S2 were exposed to CO or NO gas under stirring. The gas was aerated for 20 minutes, and the gas loading was verified by UV spectrophotometer to obtain gas-saturated nRBCs (nRBC CO and nRBC NO ). Finally, nRBC CO and nRBC NO Store in sealed blood bags at 4°C.

[0065] Embodiment 2

[0066] A macrophage membrane / nano / gas engineered red blood cell pharmaceutical preparation specifically comprises the following steps:

[0067] S1. Red blood cell (RBC) collection: Whole blood was collected from the heart of SD rats using a centrifuge tube containing EDTA-2K anticoagulant, and centrifuged at 3000 rap / min for 15 min to obtain an upper transparent yellow plasma layer and a lower dark red red red blood cell liquid layer.

[0068] Use a rubber-tipped dropper to aspirate the upper transparent yellow plasma, add PBS to the lower dark red red red blood cell liquid, mix gently, and centrifuge at 3000 rap / min for 15 minutes.

[0069] Repeat 3 times until the supernatant no longer appears yellow, indicating that the red blood cells are washed clean.

[0070] S2. Extraction of macrophage membrane: Collect macrophages and suspend them in PBS, freeze and thaw repeatedly three times, centrifuge at 3200g / min for 10min, collect the supernatant, then centrifuge at 12000g / min for 30min, collect the precipitate which is the macrophage membrane.

[0071] S3. Preparation of macrophage membrane / nano / gas engineered red blood cells: Mix the macrophage membrane with the RBC obtained in step S1, and extrude the mixture of macrophage membrane and RBC through a polycarbonate porous membrane (pore size 5-0.2 μm) using a liposome extruder to obtain M-nRBC.

[0072] M-nRBCs were exposed to CO gas to obtain M-nRBCs. CO , M-nRBC CO Store in sealed blood bags at 4°C.

[0073] Embodiment 3

[0074] A tumor cell membrane / nano / gas engineered red blood cell pharmaceutical preparation specifically comprises the following steps:

[0075] S1. Red blood cell (RBC) collection: Whole blood was collected from the heart of SD rats using a centrifuge tube containing EDTA-2K anticoagulant, and centrifuged at 3000 rap / min for 15 min to obtain an upper transparent yellow plasma layer and a lower dark red red red blood cell liquid layer.

[0076] Use a rubber-tipped dropper to aspirate the upper transparent yellow plasma, add PBS to the lower dark red red red blood cell liquid, mix gently, and centrifuge at 3000 rap / min for 15 minutes.

[0077] Repeat 3 times until the supernatant no longer appears yellow, indicating that the red blood cells are washed clean.

[0078] S2. Extraction of tumor cell membrane: Collect tumor cells and suspend them in PBS, freeze and thaw them three times, centrifuge at 3200g / min for 10min, collect the supernatant, and then centrifuge at 12000g / min for 30min to collect the precipitate, which is the tumor cell membrane.

[0079] S3. Preparation of tumor cell membrane / nano / gas engineered red blood cells: Mix the tumor cell membrane with the RBC obtained in step S1, and extrude the mixture of tumor cell membrane and RBC through a polycarbonate porous membrane (pore size 5-0.2 μm) using a liposome extruder to obtain T-nRBC.

[0080] T-nRBCs were exposed to CO or NO gas to obtain T-nRBCs. CO and T-nRBC NO . T-nRBC CO and T-nRBC NO Store separately in sealed blood bags at 4°C.

[0081] Test Example 1

[0082] The engineered red blood cell preparations (nRBC, nRBC CO 、nRBC NO 、M-nRBC、M-nRBC CO 、T-nRBC、T-nRBC CO 、T-nRBC NO ) were tested for particle size, morphology and gas loading:

[0083] The particle size distribution of the engineered red blood cell preparation was measured by a dynamic light scattering (DLS) nanoparticle size analyzer (NanoBrook 90PlusZeta). Figure 1 As shown, where A is nRBC, nRBC CO 、nRBC NO DLS diagram of M-nRBC, M-nRBC CO Dynamic light scattering diagram, C is T-nRBC, T-nRBC, T-nRBC NO Dynamic light scattering diagram of

[0084] The results showed that the particle size distribution of cell membrane / nano-erythrocyte preparations carrying different gases was around 150nm.

[0085] Different cell membrane / nano red blood cell preparations (nRBC, nRBC CO 、nRBC NO 、M-nRBC、M-nRBC CO 、T-nRBC、T-nRBC CO 、T-nRBC NO ) were observed by transmission electron microscopy, and the results were as follows Figure 2 The results showed that different cell membrane / nano-erythrocyte preparations were distributed between 80-300 nm, with an average particle size of about 150 nm and were evenly dispersed.

[0086] UV spectrophotometry was used to measure the binding of CO and NO to hemoglobin (Hb). Figure 3 As shown, where A is nRBC, nRBC CO 、nRBC NO UV absorption diagram of M-nRBC, M-nRBC CO UV absorption diagram, C is T-nRBC, T-nRBC CO 、T-nRBC NO UV absorption diagram.

[0087] After binding, the characteristic absorption peak of hemoglobin at 415nm red-shifted to 419nm, proving that hemoglobin was converted into carbon monoxide state.

[0088] After binding, the characteristic absorption peak of 415nm blue-shifted to 413nm, and the characteristic absorption peak of hemoglobin at 340nm increased, proving that hemoglobin was converted into nitric oxide state.

[0089] Test Example 2

[0090] nRBC, nRBC CO 、nRBC NO、M-nRBC、M-nRBC CO 、T-nRBC、T-nRBC CO 、T-nRBC NO After being stored at 4°C for 21 days, the particle size and PDI index of different nano-erythrocyte preparations were measured by dynamic light scattering at 0, 7, 14 and 21 days to characterize their stability.

[0091] The results are as follows Figure 4 As shown, where A is nRBC, nRBCCO, nRBC NO The particle size and PDI index of B are M-nRBC and M-nRBC. CO 、M-nRBC NO The particle size and PDI index of C are T-nRBC and T-nRBC CO 、T-nRBC NO The particle size and PDI index of the engineered red blood cell preparation remained stable under storage conditions of 4°C.

[0092] After 21 days of storage, the engineered red blood cell preparations (nRBC, nRBC CO 、nRBC NO ) for circular dichroism detection, such as Figure 5 It is shown that the secondary structure of hemoglobin in engineered red blood cells remains stable after long-term storage.

[0093] Test Example 3

[0094] Nano / gas engineered red blood cells (nRBC CO 、nRBC NO ) Gas release

[0095] Example 1 nRBC prepared CO and nRBC NO After 21 days of storage, nRBCs were studied using UV-Vis spectroscopy CO and nRBC NO Gas release capacity under different conditions.

[0096] (1) The CO binding rate to hemoglobin (COHb%) was measured by dual-wavelength ultraviolet spectrophotometry to indirectly calculate the CO release. CO ), as the saturated group sample, the absorbance values ​​(A λ1 , A λ2 , A λ3 ), calculate ΔA1, ΔA2; where ΔA1 = A λ1 -Aλ2 , ΔA2=A λ3 -A λ2 .

[0097] Incubate in the dark at pH 7.4-0mM H2O2, pH 7.4-0.01mM, and pH 7.4-0.1mM H2O2 to measure saturated nRBC CO The UV absorption changes with time (0min, 3min, 5min, 10min, 20min, 30min, 40min, 50min, 60min, 90min, 120min), and the absorbance (A) at λ1'=530nm, λ2'=582nm, and λ3'=568nm at different times are recorded. λ1’ , A λ2’ , A λ3’ ), calculate ΔA1', ΔA2'; where ΔA1'=A λ1’ -A λ2’ , ΔA2'=A λ3’ -A λ2’ .

[0098] Finally, the COHb% content is calculated using the following formula:

[0099]

[0100] (2) The release behavior of NO was measured by Griess reagent method. The specific operation was as follows: Take the NO-saturated nano-red blood cells (nRBC) prepared in Example 1 NO ), incubated under the conditions of pH 7.4-0mM H2O2, pH7.4-0.01mM, and pH 7.4-0.1mM H2O2, respectively, in the dark, and took 50mL of the solution at the predetermined time points (0min, 3min, 5min, 10min, 20min, 30min, 40min, 50min, 60min, 90min, and 120min), and added 50mL of Griess reagent I and Griess reagent II in sequence. The absorption at 540nm was measured using an ELISA reader and an ultraviolet spectrophotometer, respectively.

[0101] The results are as follows Figure 6 As shown, A is the release measurement result of CO, and B is the release measurement result of NO.

[0102] The results showed that after incubation in a solution simulating physiological conditions (pH 7.4-0 mM H2O2 and PBS 7.4, 0.01 mM H2O2, 37°C), nRBC CO and nRBC NOAll showed excellent structural stability, with less than 7% of CO and NO released within 120 min. In contrast, in a solution simulating the highly oxidative microenvironment of inflammatory cells or cancer cells (PBS 5.5, 0.1 mM H2O2), nRBC CO and nRBC NO More than 90% of CO and NO were released within 20 min. This rapid release can be attributed to the oxidation of heme in Hb at high H2O2 concentrations (the UV absorption peak of the Hb Soret band blue-shifts to 405 nm), resulting in the loss of its gas-binding capacity.

[0103] Test Example 4

[0104] In vitro anti-inflammatory test

[0105] Example 3 In vitro macrophage anti-inflammatory experiment of macrophage membrane / nano / gas engineered red blood cells prepared: Raw267.4 cells were planted in 24-well plates, and after culturing for 24 hours, the cells were divided into 4 groups, namely control group, TNF-α group, TNF-α+dexamethasone (clinical first-line arthritis treatment drug, DXM) group, TNF-α+M-nRBC group CO Group.

[0106] After 24 h of culture, the cell supernatant was removed and the concentrations of inflammatory factors such as iNOS, TNF-α and IL-1β in the supernatant were detected using an Elisa kit.

[0107] The results are as follows Figure 7 As shown in the figure, TNF-α treatment can induce inflammatory response in macrophages. Compared with the control group, macrophages secrete a large amount of inflammatory factors. CO Afterwards, the concentration of inflammatory factors in the supernatant decreased, proving that engineered red blood cells can regulate inflammatory macrophages and reduce their secretion of inflammatory factors.

[0108] Example 3: In vitro macrophage phenotype verification of nano / gas engineered red blood cells prepared: Raw267.4 cells were planted in 24-well plates and cultured for 24 h before being divided into four groups, namely control group, TNF-α group, TNF-α+DXM group, TNF-α+M-nRBC group, and CO After culturing for 24 h, Raw267.4 cells were immunofluorescently stained with FITC anti-mouse CD86 and observed using laser confocal microscopy.

[0109] The results are as follows Figure 8As shown in the figure, compared with the control group, TNF-α treatment can induce macrophage polarization to pro-inflammatory type, and CD86 fluorescence is highly expressed, which is consistent with the results of anti-inflammatory experiments. After adding DXM and M-nRBCCO, the polarization degree of macrophages to pro-inflammatory type decreased, and CD86 fluorescence was basically the same as that of the control group, indicating that M-nRBC CO It has anti-inflammatory effects and can induce the transformation of pro-inflammatory macrophages into anti-inflammatory macrophages.

[0110] Example 3: Verification of the in vitro anti-inflammatory mechanism of the prepared nano / gas engineered red blood cells: Raw267.4 cells were planted in a 6-well plate and cultured for 24 hours before being divided into 4 groups, namely control group, TNF-α group, TNF-α+DXM group, TNF-α+M-nRBC group. CO After 24 h of culture, cells were collected and proteins were extracted, and Western blot technology was used to investigate the levels of Nrf2 / HO-1 / COX-2 proteins.

[0111] The results are as follows Fig. 9 As shown, compared with the TNF-α treated group, the TNF-α+DXM group and TNF-α+M-nRBC CO The levels of Nrf2 and HO-1 proteins in macrophages of the group were significantly increased, and the level of COX-2 protein was significantly decreased, indicating that M-nRBC CO It can regulate inflammatory proteins, exert anti-inflammatory effects, and inhibit the polarization of macrophages to M1 type.

[0112] Test Example 5

[0113] In vivo arthritis treatment experiment

[0114] The experimental group was divided into 4 groups, including 1 sham operation group (Sham), 3 CIOA groups, and 3 groups of saline administration group, DXM administration group and M-nRBC administration group. CO The drug-treated group was given the drug once a week for a total of three times. On the 35th day, the articular cartilage was collected for sectioning and the efficacy of osteoarthritis was evaluated by OARSI scoring. Cartilage degeneration is an important pathological manifestation of osteoarthritis.

[0115] like Fig.10 As shown, the articular cartilage is stained with hematoxylin-eosin (HE), safranin fast green (S / F), and toluidine blue (Tb). Fig.11 Shown is the OARSI score diagram of osteoarthritis pathological tissue.

[0116] The results showed that after CIOA modeling, the cartilage in the saline saline group showed wear and tear, the matrix was significantly reduced, and the OARSI score was significantly increased, while the DXM and M-nRBC prepared in Example 3 COThis phenomenon was restored to a certain extent, and M-nRBC CO The OARSI score was the lowest, proving that M-nRBC CO It can inhibit cartilage degeneration and is expected to be further used in the treatment of osteoarthritis.

[0117] Immunohistochemistry (Nrf2 and HO-1) and immunofluorescence staining (CD86) were performed on articular cartilage to examine inflammation-related proteins and macrophage phenotypes. Fig.12 As shown in Figure 2, after CIOA modeling, joint macrophages polarized into inflammatory macrophages (high expression of CD86), while the M-nRBC prepared in Example 3 CO In the treatment group, the expression of Nrf2 and HO-1 increased significantly, and the level of inflammatory macrophages in the joints decreased, indicating that M-nRBC CO It can exert anti-inflammatory effects and inhibit the polarization of macrophages to M1 type.

[0118] On the 35th day, different tissues were collected. First, the heart, liver, spleen, lung, and kidney tissues were sectioned and observed by hematoxylin-eosin staining. Fig.13 As shown, the M-nRBC prepared in Example 3 CO There is no obvious toxicity to major organs, indicating that it has good biocompatibility.

[0119] Test Example 6

[0120] In vitro antitumor assay

[0121] (1) In vitro tumor cell toxicity experiments of nano / gas engineered red blood cells

[0122] Evaluation of nRBC prepared in Example 2 by MTT assay CO and nRBC NO Cytotoxicity against breast cancer 4T1, melanoma B16-F10, and colon cancer CT26 cell lines.

[0123] Further research on nRBC CO With nRBC NO Potential synergistic effects of the combination. Cytotoxicity assays were performed in these cancer cell lines at different molar ratios of CO to NO (CO:NO = 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, using a fixed amount of Hb). Different molar ratios of nRBC CO and nRBC NO The half-maximal inhibitory concentration (IC50) and combination index (CI) were determined.

[0124] like Fig.14 As shown, no significant cytotoxicity of nRBC was observed in these cancer cell lines, whereas nRBCCO and nRBC NO The cytotoxicity of nRBC CO and nRBC NO Capable of releasing gases that are toxic to cancer cells.

[0125] Synergistic effects indicated by CI < 1 were observed at multiple molar ratios, with much lower IC50 values ​​after combination. CO With nRBC NO The strongest synergistic effect was achieved when the molar ratio of 1:3 was achieved in all three types of cancer cells.

[0126] (2) In vitro ROS / RNS cascade generation of nano / gas engineered erythrocytes (using the optimal ratio of CO:NO = 1:3)

[0127] Detection of nRBC by flow cytometry CO and nRBC NO The production of ROS and RNS in treated tumor cells. 4T1 cells were cultured at 1×10 5 The cells were seeded at a density of 10 cells / well in a six-well plate. After the cells adhered to the wall, the materials were added and incubated for 6 hours. The supernatant was aspirated and washed repeatedly, and then the cells were digested with trypsin. After that, the hydrogen peroxide fluorescent probe 37 was added, incubated in the dark for 30 minutes, centrifuged at 1000 rap / min for 5 minutes to remove the probe, and then washed three times with PBS, and finally 200 μL of PBS was added to resuspend, and then detected by flow cytometry.

[0128] The results are as follows Fig.15 As shown, nRBC CO Treatment further increased the H2O2 level in tumor cells, and the increased H2O2 level promoted the gas release of nano / gas-engineered red blood cells and promoted the release of ·OH and O2 ·- In contrast, nRBC and nRBC NO None of the treatments increased H2O2 levels.

[0129] nRBC NO NO release, NO and endogenous O2 in cancer cells ·- Reaction to form ONOO - , resulting in significant cytotoxicity. It is worth noting that compared with the use of nRBC alone CO or nRBC NO Compared with nRBC CO / nRBC NO In the combined group, CO promoted OH and O2 ·- The production of NO and the increased O2·- The reaction produces more ONOO - .

[0130] Test Example 7

[0131] In vivo antitumor assay

[0132] (1) Evaluation of the anti-tumor effect of tumor cell membrane / nano / gas engineered red blood cells in the 4T1 orthotopic breast tumor model

[0133] a. Establishment of 4T1 orthotopic breast tumor model

[0134] 4T1 mouse breast cancer cells were collected by digestion and 10 6 0.1 mL of cells were inoculated under the mammary pad of female Balb / c mice at a density of 100 / mL. After about 8 days, a 100 mm 3 Tumors of different sizes will form into 200-300mm in about 12 days 3 Large and small tumor masses.

[0135] b. Tumor cell membrane / nanoengineered erythrocyte targeting in vivo

[0136] nRBC and 4T1 tumor cell membrane modified T-nRBC were mixed with Cy5.5, stirred for 12 h at room temperature in the dark, and then placed in a dialysis bag with a molecular weight of 3500 and dialyzed for 12 h to obtain Cy5.5 loaded nRBC (nRBC Cy5.5 ) and MT-nRBC (T-nRBC Cy5.5 The tumor-bearing Balb / c mice were randomly divided into 2 groups, 3 mice in each group. When the tumor volume grew to 300 mm 3 When small molecule nRBC was injected into the tail vein Cy5.5 and T-nRBC Cy5.5 After 3h, 12h, and 24h of drug injection, the mice were anesthetized and near-infrared fluorescence imaging was performed to observe nRBC Cy5.5 and T-nRBC Cy5.5 Targeted accumulation in various tissues.

[0137] The results are as follows Fig.16 As shown, T-nRBC Cy5.5 Shows a higher ratio than nRBC Cy5.5 The more significant fluorescence demonstrated that T-nRBCs could enhance their accumulation at tumor sites by targeting homologous cancer cells.

[0138] c. Evaluation of the anti-tumor effect of tumor cell membrane / nano / gas engineered red blood cells

[0139] When the tumor size of the tumor-bearing Balb / c mice reached 100 mm3 The mice were randomly divided into 5 groups and intravenously injected with normal saline, nRBC CO 、nRBC NO 、nRBC CO / nRBC NO 、T-nRBC CO / T-nRBC NO Each mouse received a dose of 70 mg Hb / kg, a concentration of 30 mg Hb / mL, equivalent to 3.1% of its total blood volume. After monitoring tumor growth for 16 days, the tumor growth of each group was statistically analyzed. The results are as follows Fig.17 shown.

[0140] The results showed that after 16 days of tumor volume monitoring, nRBC CO and nRBC NO showed a modest antitumor effect, with tumor suppression rates (TCR) of 30.6% and 41.4%, respectively, due to nRBC CO CO release can increase ROS levels in tumors, nRBC NO The release of NO can increase RNS levels. CO / nRBC NO Treatment achieved a 68.6% tumor inhibition rate, while T-nRBC CO / T-nRBC NO Treatment resulted in the most significant tumor inhibition, with a tumor inhibition rate of 83.1%. This suggests that co-delivery of CO and NO by nRBCs can generate a large amount of ROS and RNS, thereby effectively inhibiting tumors. These findings indicate that engineered red blood cell-based gas delivery therapy effectively inhibits tumor growth in mice.

[0141] (2) Evaluation of the anti-tumor effect of tumor cell membrane / nano / gas engineered erythrocytes in the B16 melanoma lung metastasis model

[0142] a. Establishment of B16 melanoma lung metastasis model

[0143] In the B16 melanoma lung metastasis model, the same engineering technology was used to prepare melanoma cell membrane / nano / gas engineered red blood cells. Subsequently, the B16 melanoma lung metastasis model was established in female C57BL / 6 mice. In this model, 2×10 6 B16F10 cells were injected subcutaneously into the back of mice. On day 2, 1×10 5 Luciferase-labeled B16F10 (B16F10-LUC) cells.

[0144] b. Evaluation of the anti-tumor effect of tumor cell membrane / nano / gas engineered red blood cells:

[0145] The tumor volume on the back of C57 tumor-bearing mice grew to 60mm 3 The tumor-bearing mice were randomly divided into five groups: saline group, anti-PD-L1 group, nRBC group CO / nRBC NO Group, T-nRB CO / T-nRBC NO Group and T-nRBC CO / T-nRBC NO +anti-PD-L1 group. Engineered red blood cells were injected intravenously on days 7, 10, and 13, while anti-PD-L1 was injected intravenously on days 8, 11, and 14.

[0146] The results after treatment are as follows Fig.18 As shown, the subcutaneous tumor volume in the saline group exceeded 1450mm 3 . With nRB C CO / nRBC NO Compared with anti-PD-L1 therapy, T-nRB CO / T-nRBC NO The tumor suppression rate was 64.1%, while T-nRBC CO / T-nRBC NO The combination of + anti-PD-L1 further increased the tumor inhibition rate to 80.3%.

[0147] Thirteen days after intravenous injection of B16F10-LUC cells, obvious bioluminescent signals were observed in the lungs of mice treated with saline, indicating the presence of severe tumor lung metastasis. Fig.19 As shown for nRBC CO / nRBC NO Mice treated with either anti-PD-L1 or anti-PD-L1 showed a slight delay in metastasis. In contrast, T-nRB CO / T-nRBC NO Treatment significantly inhibited lung metastasis. CO / T-nRBC NO +anti-PD-L1 combined treatment almost eliminated lung metastasis in mice.

[0148] c. Acute toxicity and immunogenicity of nano / gas engineered erythrocytes were evaluated in SD rats.

[0149] The immunogenicity of nRBC was tested in healthy SD rats. CO 、nRBC NO or nRBCCO / nRBC NO Each rat received a dose of 70 mg Hb / kg, a concentration of 30 mg Hb / mL, equivalent to 3.7% of total blood volume. Blood samples were collected 1 hour and 48 hours after infusion for testing. The results are as follows Fig. 20 Serum immunoglobulin G (IgG) and immunoglobulin M (IgM) levels were similar between the control and treatment groups, indicating that the engineered nRBCs exhibited low immunogenicity.

[0150] Monitor the physiological indicators of rats. CO 、nRBC NO 、nRBC CO / nRBC NO or T-nRBC CO / T-nRBC NO After that, the vital signs of SD rats were monitored. The changes of mean arterial pressure (MAP), peripheral blood oxygen saturation (SpO2) and heart rate were recorded within 24 hours after administration. Fig.21 As shown, all physiological indicators of the drug-treated group were comparable to those of the control group, with no significant differences.

[0151] Perform routine blood tests. Intravenous infusion of nRBC, nRBC CO 、nRBC NO 、nRBC CO / nRBC NO or T-nRBC CO / T-nRBC NO . The results of routine blood tests 48 hours after administration were confirmed, and the results are shown in Table 1, showing that the hematological parameters of the rats remained within the normal range:

[0152] Table 1 Hematological parameters of rats

[0153]

[0154]

[0155] Therefore, the nano / gas engineered red blood cell pharmaceutical preparation of the present invention, its preparation method and application, can accurately control the delivery of carbon monoxide (CO) and nitric oxide (NO) into target cells, and by controlling the dosage and dosage ratio of the two gases, they can produce different synergistic effects such as anti-inflammatory, cell protection and tumor cell killing, playing an important role in the treatment of diseases such as arthritis and tumors.

[0156] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing a nano / gas engineered red blood cell pharmaceutical preparation, characterized in that: The steps include: S1. Using a liposome extruder, the washed red blood cells are extruded through a polycarbonate porous membrane to obtain nano-engineered red blood cells nRBC; S2. The nano-engineered red blood cells prepared in S1 are loaded with CO or NO gas to obtain nano / gas engineered red blood cell preparation nRBCs.

2. The method for preparing a nano / gas engineered red blood cell pharmaceutical preparation according to claim 1, characterized in that: In S1, the pore size of the polycarbonate porous membrane is 5 to 0.2 μm.

3. The method for preparing a nano / gas engineered red blood cell pharmaceutical preparation according to claim 1, characterized in that: In S2, the gas carrying CO or NO includes: The nRBCs obtained in S1 were exposed to CO or NO gas under stirring, and the gas loading was verified in real time to obtain gas-saturated nRBCs.

4. A nano / gas engineered red blood cell pharmaceutical preparation prepared by the preparation method according to any one of claims 1 to 3, characterized in that: The particle size of nano / gas engineered red blood cell preparation nRBCs is 150nm.

5. Use of the nano / gas engineered red blood cell pharmaceutical preparation as claimed in claim 4 in gas infusion therapy for non-medical purposes.

6. Use of the preparation method according to any one of claims 1 to 3 in the preparation of a gas infusion therapy pharmaceutical preparation, characterized in that: Gas infusion therapeutic drug preparations include arthritis gas infusion therapeutic drug preparations and tumor gas infusion therapeutic drug preparations; The preparation method includes: co-extruding the target cell membrane and washed red blood cells to construct cell membrane / nano / gas engineered red blood cells, and the cell membrane / nano / gas engineered red blood cells can target homologous target cells.

7. The use according to claim 6, characterized in that: The preparation method of the arthritis gas infusion therapeutic drug preparation comprises: S71, mixing the washed red blood cells with the macrophage membrane and co-extruding the mixture through a polycarbonate porous membrane to obtain macrophage membrane / nano / gas engineered red blood cells M-nRBC; S72. Exposing the macrophage membrane / nano / gas engineered red blood cells M-nRBC to CO gas to obtain M-nRBC CO .

8. The use according to claim 6, characterized in that: The method for preparing a drug preparation for tumor gas infusion therapy comprises: S81, mixing the washed red blood cells with the tumor cell membrane and co-extruding the mixture through a polycarbonate porous membrane to obtain tumor cell membrane / nano / gas engineered red blood cells T-nRBC; S82. Exposing tumor cell membrane / nano / gas engineered red blood cells T-nRBC to CO or NO gas to obtain T-nRBC CO and T-nRBC NO .

9. The use according to claim 8, characterized in that: T-nRBC in the drug preparation for tumor gas infusion therapy CO :T-nRBC NO The molar ratio is 1~4:4~1.

10. The use according to claim 9, characterized in that: T-nRBC in the drug preparation for tumor gas infusion therapy CO :T-nRBC NO The molar ratio is 1:3.

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