A nano / gas-engineered red blood cell pharmaceutical preparation, and a preparation method and application thereof

Abstract

The application discloses a kind of nanometer / gas engineering red blood cell pharmaceutical preparation and its preparation method and application, belong to gas infusion treatment technical field, aim at by accurately controlling the delivery of carbon monoxide (CO) and nitric oxide (NO) into target cell, by controlling the dosing amount and dosing ratio of the two gases, make it produce different synergies, such as anti-inflammatory, cell protection and tumor cell killing, play an important role in different disease treatments (such as arthritis, tumor).

Description

Technical Field

[0001] This invention relates to the field of gas infusion therapy technology, and in particular to a nano / gas engineered red blood cell drug formulation, its preparation method, and its application. Background Technology

[0002] In recent years, gas therapy has received widespread attention for 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 concentrations of CO can reduce joint inflammation by activating the Nrf2 / HO-1 pathway to inhibit pro-inflammatory factors (such as TNF-α and IL-1β) and may also reduce chondrocyte apoptosis.

[0004] In cancer treatment, high concentrations of CO have a direct adverse effect 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 apoptosis-promoting effects. High concentrations of NO can react with superoxide anions (O2... - The reaction forms pernitrite (ONOO). - This is an effective oxidizing and nitrifying reactive nitrogen species (RNS) that not only induces direct DNA damage but also disrupts the metabolic homeostasis of cancer cells by mediating post-translational modifications of proteins through S-nitrosylation of cysteine ​​residues.

[0005] However, gas therapy faces challenges in clinical application due to issues such as non-specific gas release and biosafety. Red blood cells (RBCs), as natural gas carriers, possess advantages such as high stability and strong affinity for gas molecules, but their micrometer-sized dimensions limit their ability to reach lesion areas and enter target cells. Therefore, developing an engineered red blood cell method for gas infusion therapy has significant clinical implications. Summary of the Invention

[0006] The purpose of this invention is to provide a nano / gas-engineered erythrocyte drug formulation, its preparation method, and its application. This system can achieve safe and efficient delivery of CO and NO, and specific release at the site of arthritis lesions or within tumors. By utilizing the regulatory effects of CO and NO on cells, the immune microenvironment of the site of arthritis lesions or tumor tissues can be remodeled, thereby effectively treating arthritis or tumors.

[0007] To achieve the above objectives, the present invention provides a method for preparing a nano / gas-engineered erythrocyte drug formulation, comprising the following steps:

[0008] S1. Using a liposome extruder, washed red blood cells are extruded through a polycarbonate porous membrane to obtain nanoengineered red blood cells (nRBCs).

[0009] S2. Load the nano-engineered red blood cells prepared in step S1 with CO or NO gas to form nano / gas engineered red blood cell preparations 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 includes: exposing the nRBCs obtained in step S1 to CO or NO gas under stirring, verifying the gas loading status in real time, and obtaining gas-saturated nRBCs.

[0012] Secondly, the present invention provides a nano / gas engineered red blood cell drug formulation prepared by the above-mentioned preparation method, wherein the nano / gas engineered red blood cell formulation nRBCs has a particle size of 150 nm.

[0013] Thirdly, the present invention provides the application of the above-mentioned nano / gas-engineered erythrocyte drug formulation in gas infusion therapy for non-medical purposes.

[0014] Fourthly, the present invention provides an application of the above-mentioned preparation method in the preparation of gas infusion therapeutic drug formulations, the gas infusion therapeutic drug formulations including: arthritis gas infusion therapeutic drug formulations and tumor gas infusion therapeutic drug formulations;

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

[0016] Preferably, the preparation method of the arthritis gas infusion therapeutic drug formulation includes:

[0017] S71. After washing the red blood cells and mixing them with the macrophage membrane, the mixture is co-extruded through a polycarbonate porous membrane to obtain macrophage membrane / nano / gas engineered red blood cells M-nRBC.

[0018] S72. Macrophage membrane / nano / gas-engineered erythrocytes (M-nRBCs) were exposed to CO gas to obtain M-nRBCs. CO .

[0019] Preferably, the preparation method of the tumor gas infusion therapeutic drug formulation includes:

[0020] S81. After washing the red blood cells and mixing them with the tumor cell membrane, the mixture is co-extruded through a polycarbonate porous membrane to obtain tumor cell membrane / nano / gas engineered red blood cells T-nRBC;

[0021] S82. T-nRBCs, engineered from tumor cell membranes / nano / gas, are exposed to CO or NO gas to obtain T-nRBCs. CO and T-nRBC NO .

[0022] Preferably, in tumor gas infusion therapy formulations, T-nRBC CO :T-nRBC NO The molar ratio is 1-4:4-1.

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

[0024] Therefore, the nano / gas engineered erythrocyte drug formulation, its preparation method, and its application of the present invention have the following beneficial effects:

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

[0026] (2) The hemoglobin in nano-red blood cells contains hematoporphyrin, which can release CO and NO in response under H2O2 conditions;

[0027] (3) Macrophage cell membranes were extracted and co-extruded with nRBCs to construct macrophage cell membrane / nano / CO gas engineered red blood cells (M-nRBCs). CO This enables targeted therapy of homologous macrophages; M-nRBC CO CO is specifically released in the high hydrogen peroxide (H2O2) microenvironment within the intra-articular macrophages. CO can induce the expression of HO-1, which coordinates the polarization homeostasis of macrophages through the Nrf2 / HO-1 pathway, promotes the formation of anti-inflammatory macrophages (M2 type), and inhibits the activity of pro-inflammatory macrophages (M1 type), thereby exerting an anti-inflammatory effect.

[0028] (4) Extract tumor cell membranes and co-extrude them with nRBCs to construct tumor cell membrane / nano / gas engineered red blood cells (T-nRBCs). CO and T-nRB CNO This enables targeted therapy of homologous tumor cells; T-nRBC CO and T-nRBC NOThe tumor cells specifically release CO and NO in a high hydrogen peroxide (H2O2) microenvironment within the tumor cells. Under the catalysis of peroxidase, they generate a large amount of ROS and RNS, which disrupt the metabolic homeostasis of tumor cells. At the same time, they regulate the tumor immunosuppressive microenvironment, transforming "cold" tumors into "hot" tumors and enhancing immune-mediated tumor killing. It has been verified that the anti-tumor treatment 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 hyperhydrogen peroxide (H2O2) microenvironment within tumor cells, CO and NO are specifically released; CO promotes the release of ·OH and O2. ·- The production of NO and the increase in O2 ·- The reaction produces more ONOO - The co-delivery and synergistic effect of CO and NO significantly enhances the therapeutic efficacy of dual gases in tumor treatment;

[0030] (6) The nano / gas engineered red blood cell drug formulation 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 will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0032] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 The image shows dynamic light scattering patterns of different nano-red blood cell formulations, where A represents nRBC and nRBC. CO nRBC NO DLS diagram, B represents M-nRBC, M-nRBC CO Dynamic light scattering diagram, where C represents T-nRBC, T-nRBC, and T-nRBC. NO Dynamic light scattering diagram;

[0034] Figure 2 TEM images of different nano-red blood cell formulations, where A represents nRBC, nRBC CO nRBC NO TEM images, B represents M-nRBCs and M-nRBCs. CO TEM images, C represents T-nRBCs and T-nRBCs.CO T-nRBC NO TEM image;

[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 The ultraviolet spectrum of )

[0036] Figure 4 The particle size and PDI index of different nano-red blood cell formulations were measured after storage at 4°C for 21 days. Where A represents nRBC, nRBCCO, and nRBC... NO Particle size and PDI index, B represents M-nRBC, M-nRBC CO M-nRBC NO Particle size and PDI index, C for T-nRBC, 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 The circular dichroism chromatogram of )

[0038] Figure 6 nRBC CO nRBC NO Gas release test results, where A is nRBC CO Under normal physiological conditions and simulated cellular oxidation microenvironment conditions, gas release, B is nRBC. NO Gas release under normal physiological conditions and simulated cellular oxidation microenvironment conditions;

[0039] Figure 7 Normal macrophages (Raw 267.4), macrophages treated with LPS, macrophages treated with LPS + dexamethasone (DMX), and macrophages treated with LPS + M-nRBCs CO The concentrations of inflammatory factors in the supernatant of macrophages were determined, where α is the iNOS concentration, B is the TNF-α concentration, and C is the IL-1β concentration.

[0040] Figure 8 Normal macrophages (Raw 267.4), macrophages treated with LPS, macrophages treated with LPS+DMX, and macrophages treated with LPS+M-nRBCs. CO CD86 immunofluorescence staining of macrophages;

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

[0042] Figure 10 Images of articular cartilage stained with hematoxylin-eosin (HE), safranin-fast green (S / F), and toluidine blue (Tb).

[0043] Figure 11 OARSI score chart for osteoarthritis pathological tissue;

[0044] Figure 12 Immunohistochemical sections of joints containing Nrf2 and HO-1, and immunohistochemical sections containing CD86;

[0045] Figure 13 HE staining images of major organs (heart, liver, spleen, lung, and kidney) after drug administration;

[0046] Figure 14 To evaluate nano / gas-engineered erythrocytes (nRBCs) using the MTT assay CO and nRBC NO Cytotoxicity against breast cancer 4T1, melanoma B16-F10, and colon cancer CT26 cell lines, where A represents nRBCs and nRBCs. CO nRBC NO and nRBCs with different molar ratios CO With nRBC NO Survival rate of breast cancer 4T1 cells in the treatment group, B represents nRBC, nRBC CO nRBC NO and nRBCs with different molar ratios CO With nRBC NO Survival rate of melanoma B16-F10 cells in the treatment group, C represents nRBC, nRBC CO nRBC NO and nRBCs with different molar ratios CO With nRBC NO Survival rate of melanoma B16-F10 cells in the treatment group, where D represents different molar ratios of nRBCs. CO With nRBC NO The treatment group showed the half-maximal inhibitory concentration (IC50) and combination index (CI) of 4T1 breast cancer cells, where E represents different molar ratios of nRBCs. CO With nRBC NOThe treatment groups showed the half-maximal inhibitory concentration (IC50) and combination index (CI) of melanoma B16-F10 cells, where F represents different molar ratios of nRBCs. CO With nRBC NO The half-maximal inhibitory concentration (IC50) and combination index (CI) of the treatment group against CT26 colon cancer cells;

[0047] Figure 15 Nano / Gas Engineered Red Blood Cells (nRBCs) CO and nRBC NO Flow cytometry results of in vitro ROS / RNS cascade generation after co-incubation with 4T1 cells for 6 hours;

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

[0049] Figure 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 a 4T1 orthotopic breast tumor model;

[0050] Figure 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 suppression curves after treatment in a B16 melanoma lung metastasis model;

[0051] Figure 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 Significant bioluminescent signal was observed in the lungs after treatment in a B16 melanoma lung metastasis model;

[0052] Figure 20Evaluation of nano / gas engineered erythrocytes (nRBCs, nRBCs) in SD rats CO nRBC NO nRBC CO / nRBC NO Acute toxicity and immunogenicity (IgG and IgM) of SD rats, where A is the IgG level of SD rats at 1 h, B is the IgM level of SD rats at 1 h, C is the IgG level of SD rats at 48 h, and D is the IgM level of SD rats at 48 h.

[0053] Figure 21 For the infusion 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 Physiological indicators of rats after (A, B, and C are the blood pressure, blood oxygen saturation, and heart rate respectively). Detailed Implementation

[0054] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0055] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

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

[0057] Example 1

[0058] A nano / gas-engineered erythrocyte drug formulation specifically includes the following steps:

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

[0060] Use a dropper to aspirate the upper layer of clear yellow plasma and the lower layer of dark red red blood cell fluid. Add PBS to the mixture, mix gently, and centrifuge at 3000 rap / min for 15 min.

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

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

[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. After 20 minutes of aeration, the gas loading was verified by UV spectrophotometry to obtain gas-saturated nRBCs (nRBCs). CO and nRBC NO Finally, nRBC CO and nRBC NO Store in a sealed blood bag at 4°C.

[0065] Example 2

[0066] A macrophage membrane / nano / gas engineered erythrocyte drug formulation, specifically comprising the following steps:

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

[0068] Use a dropper to aspirate the upper layer of clear yellow plasma and the lower layer of dark red red blood cell fluid. Add PBS to the mixture, mix gently, and centrifuge at 3000 rap / min for 15 min.

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

[0070] S2. Extraction of macrophage membrane: Macrophages were collected and suspended in PBS, and subjected to three freeze-thaw cycles. The supernatant was collected by centrifugation at 3200g / min for 10 min, and then centrifuged at 12000g / min for 30 min. The precipitate was the macrophage membrane.

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

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

[0073] Example 3

[0074] A tumor cell membrane / nano / gas engineered erythrocyte drug formulation, specifically comprising the following steps:

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

[0076] Use a dropper to aspirate the upper layer of clear yellow plasma and the lower layer of dark red red blood cell fluid. Add PBS to the mixture, mix gently, and centrifuge at 3000 rap / min for 15 min.

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

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

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

[0080] T-nRBCs were obtained by exposing them to CO or NO gas. CO and T-nRBC NO T-nRBC CO and T-nRBC NO Store in sealed blood bags at 4°C.

[0081] Test Example 1

[0082] The engineered red blood cell preparations (nRBC, nRBC) in Examples 1 to 3 were respectively tested. CO nRBC NO M-nRBC, M-nRBC CO T-nRBC, T-nRBC CO T-nRBC NO The particle size morphology and gas loading were detected.

[0083] The particle size distribution of engineered red blood cell formulations was determined using a dynamic light scattering (DLS) nanoparticle size analyzer (NanoBrook 90PlusZeta). The results are as follows: Figure 1 As shown, where A represents nRBC and nRBC. CO nRBC NO DLS diagram, B represents M-nRBC, M-nRBC CO Dynamic light scattering diagram, where C represents T-nRBC, T-nRBC, and T-nRBC. NO Dynamic light scattering diagram;

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

[0085] Different cell membrane / nanoerythrocyte preparations (nRBC, nRBC) were used. CO nRBC NO M-nRBC, M-nRBC CO T-nRBC, T-nRBC CO T-nRBC NO Transmission electron microscopy was performed, and the results are as follows: Figure 2 As shown in the figure. The results show that different cell membrane / nano-red blood cell formulations are distributed between 80-300 nm, with an average particle size of about 150 nm, and are uniformly dispersed.

[0086] The binding of CO and NO to hemoglobin (Hb) was determined by ultraviolet spectrophotometry, and the results are as follows: Figure 3 As shown, where A represents nRBC and nRBC. CO nRBC NO The UV absorption spectrum, B represents M-nRBC and M-nRBC. CO The UV absorption spectrum, where C represents T-nRBC and T-nRBC. CO T-nRBC NO The ultraviolet absorption diagram.

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

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

[0089] Test Example 2

[0090] nRBC, nRBC CO nRBC NOM-nRBC, M-nRBC CO T-nRBC, T-nRBC CO T-nRBC NO The nano-red blood cell formulations were stored at 4°C for 21 days. The particle size and PDI index of different nano-red blood cell formulations were determined 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 represents nRBC, nRBCCO, and nRBC. NO Particle size and PDI index, B represents M-nRBC, M-nRBC CO M-nRBC NO Particle size and PDI index, C for T-nRBC, T-nRBC CO T-nRBC NO The particle size and PDI index showed that the engineered erythrocyte formulation remained stable under storage conditions at 4°C.

[0092] After 21 days of storage, the engineered red blood cell preparation (nRBC, nRBC) prepared in Example 1 was tested. CO nRBC NO Perform circular dichroism spectroscopy detection, such as Figure 5 As shown, this demonstrates that the secondary structure of hemoglobin in engineered red blood cells remains stable after prolonged storage.

[0093] Test Example 3

[0094] Nano / Gas Engineered Red Blood Cells (nRBCs) CO nRBC NO Gas release

[0095] nRBC prepared in Example 1 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 release was indirectly calculated by determining the CO-hemoglobin binding rate (COHb%) using a dual-wavelength ultraviolet spectrophotometric method. CO-saturated engineered nanored blood cells (nRBCs) were used. CO As a saturated group sample, its absorbance values ​​(A) were measured at wavelengths of λ1 = 530 nm, λ2 = 582 nm, and λ3 = 568 nm. λ1 A λ2 A λ3 ), calculate ΔA1 and ΔA2; where ΔA1 = A λ1 -Aλ2 ΔA2=A λ3 -A λ2 .

[0097] Incubation was performed in the dark under the conditions of pH 7.4-0 mM H2O2, pH 7.4-0.01 mM, and pH 7.4-0.1 mM H2O2, respectively, and the saturated nRBCs were measured. CO The ultraviolet absorption varied with time (0 min, 3 min, 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 90 min, 120 min), and the absorbance (A) at λ1' = 530 nm, λ2' = 582 nm, and λ3' = 568 nm was recorded at different times. λ1’ A λ2’ A λ3’ ), calculate ΔA1' and 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 determined using the Griess reagent method. The specific procedure was as follows: NO-saturated nanored blood cells (nRBCs) prepared in Example 1 were taken... NO The solution was incubated in the dark under the conditions of pH 7.4-0mM H2O2, pH 7.4-0.01mM, and pH 7.4-0.1mM H2O2. At predetermined time points (0 min, 3 min, 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 90 min, and 120 min), 50 mL of Griess reagent I and 50 mL of Griess reagent II were added sequentially. The absorbance at 540 nm was measured using an ELISA reader and a UV spectrophotometer.

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

[0102] The results showed that after incubation in solutions simulating physiological conditions (pH 7.4-0mM H2O2 and PBS 7.4, 0.01mM H2O2, 37℃), nRBCs... CO and nRBC NOBoth exhibited excellent structural stability, releasing less than 7% of CO and NO within 120 minutes. Conversely, in solutions simulating the highly oxidative microenvironment of inflammatory or cancer cells (PBS 5.5, 0.1 mM H2O2), nRBCs... CO and nRBC NO More than 90% of CO and NO are released within 20 minutes. 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 shifts to 405 nm), resulting in the loss of its ability to bind gases.

[0103] Test Example 4

[0104] In vitro anti-inflammatory test

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

[0106] After culturing for 24 hours, 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, TNF-α treatment can induce an inflammatory response in macrophages, with macrophages secreting a large number of inflammatory factors compared to the control group. Furthermore, the addition of DXM and M-nRBCs... CO Subsequently, 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 erythrocytes prepared: Raw267.4 cells were seeded in 24-well plates and cultured for 24 h. The cells were then divided into 4 groups: control group, TNF-α group, TNF-α+DXM group, and TNF-α+M-nRBC group. CO Group. After 24 hours of culture, 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, compared to the control group, TNF-α treatment induced macrophages to become pro-inflammatory, with high CD86 fluorescence expression, consistent with the anti-inflammatory results. However, the addition of DXM and M-nRBCCO reduced the degree of macrophage polarization towards pro-inflammatory, and the CD86 fluorescence was essentially the same as in the control group, indicating that M-nRBCCO... CO It has anti-inflammatory effects and can induce the transformation of pro-inflammatory macrophages into anti-inflammatory macrophages.

[0110] Example 3: Validation of the in vitro anti-inflammatory mechanism of nano / gas-engineered erythrocytes: Raw267.4 cells were seeded in 6-well plates and cultured for 24 h. The cells were then divided into 4 groups: control group, TNF-α group, TNF-α+DXM group, and TNF-α+M-nRBC group. CO Group. After 24 h of culture, cells were collected and proteins were extracted. The levels of Nrf2 / HO-1 / COX-2 proteins were examined using Western blot.

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

[0112] Test Example 5

[0113] In vivo arthritis treatment experiment

[0114] The experimental group was divided into 4 groups: one sham surgery group (Sham), three CIOA (induced arthritis model) groups (saline administration group, DXM administration group, and M-nRBC group), and three other groups. CO In the treatment group, the drug was administered once a week for a total of three times. On day 35, articular cartilage was collected for sectioning and the efficacy of the treatment for osteoarthritis was assessed using the OARSI score. Cartilage degeneration is an important pathological manifestation of osteoarthritis.

[0115] like Figure 10 The images shown are hematoxylin-eosin (HE), safranin-fast green (S / F), and toluidine blue (Tb) staining images of articular cartilage. Figure 11 The image shown is an OARSI score chart for pathological tissue of osteoarthritis.

[0116] The results showed that after CIOA modeling, the cartilage in the Saline saline group exhibited wear and significant matrix reduction, and the OARSI score was significantly increased, while the DXM and M-nRBCs prepared in Example 3 showed... COAll of these restored this phenomenon to some extent, and M-nRBC CO The lowest OARSI score indicates that M-nRBC CO It can inhibit cartilage degeneration and is expected to be further applied in the treatment of osteoarthritis.

[0117] Immunohistochemistry (Nrf2 and HO-1) and immunofluorescence staining (CD86) were performed on articular cartilage to examine its inflammation-related proteins and macrophage phenotype. Figure 12 As shown, after CIOA modeling, joint macrophages polarized into inflammatory macrophages (high expression of CD86), while the M-nRBCs prepared in Example 3... CO The treatment group showed a significant increase in Nrf2 and HO-1 expression, and a decrease in the level of joint inflammation-type macrophages, demonstrating the presence of M-nRBCs. CO It can exert anti-inflammatory effects and inhibit macrophage polarization towards the M1 type.

[0118] On day 35, different tissues were collected. First, sections of the heart, liver, spleen, lung, and kidney tissues were prepared and observed using hematoxylin-eosin staining. Figure 13 As shown, the M-nRBC prepared in Example 3 CO It showed no significant toxicity to major organs, indicating that it has good biocompatibility.

[0119] Test Example 6

[0120] In vitro antitumor test

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

[0122] The nRBCs prepared in Example 2 were evaluated using the MTT assay. CO and nRBC NO Cytotoxicity against breast cancer 4T1, melanoma B16-F10 and colon cancer CT26 cell lines.

[0123] Further research was conducted on nRBC CO With nRBC NO Potential synergistic effects of the combination were investigated. Cytotoxicity assays were performed in these cancer cell lines using 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). The cytotoxicity of nRBCs at different molar ratios was determined. CO and nRBC NO The half-maximum inhibitory concentration (IC50) and combination index (CI).

[0124] like Figure 14 As shown, no significant cytotoxicity of nRBCs was observed in these cancer cell lines, while nRBCs...CO and nRBC NO The cytotoxicity of nRBCs all showed a concentration-dependent increase, indicating that nRBCs... CO and nRBC NO It can release gases that are toxic to cancer cells.

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

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

[0127] nRBC detection by flow cytometry CO and nRBC NO Production of ROS and RNS in treated tumor cells. 4T1 cells were treated at a rate of 1×10⁻⁶. 5 Cells were seeded at a density of 1 / 2 well in six-well plates. After cell attachment, the plates were incubated for 6 hours. The supernatant was aspirated and the cells were washed repeatedly. The cells were then digested with trypsin. Hydrogen peroxide fluorescent probe 37 was added, and the plates were incubated in the dark for 30 minutes. The probe was removed by centrifugation at 1000 rpm for 5 minutes. The cells were washed three times with PBS and resuspended in 200 μL of PBS. Flow cytometry was then used for cell sorting.

[0128] The results are as follows Figure 15 As shown, nRBC CO The treatment further increased the H2O2 level within tumor cells. This elevated H2O2 level promoted gas release from nano / gas-engineered erythrocytes and facilitated the production of ·OH and O2. ·- The production of these substances causes significant cytotoxicity in cancer cells. In contrast, nRBCs and nRBCs... NO Neither treatment will increase H2O2 levels.

[0129] nRBC NO Release NO, NO interacts with endogenous O2 in cancer cells ·- The reaction forms ONOO - This resulted in significant cytotoxicity. Notably, compared to using nRBC alone... CO or nRBC NO In comparison, in nRBC CO / nRBC NO In the combination group, CO promotes the reaction of ·OH and O2. ·- The production of NO and the increase in O2·- The reaction produces more ONOO - .

[0130] Test Example 7

[0131] In vivo antitumor test

[0132] (1) Evaluation of the antitumor effect of tumor cell membrane / nano / gas engineered erythrocytes in the establishment of a 4T1 orthotopic breast tumor model

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

[0134] 4T1 mouse breast cancer cells were digested and collected, and then... 6 At a density of cells / mL, 0.1 mL of cells were seeded under the mammary pads of female Balb / c mice, and a 100 mm thick layer formed in about 8 days. 3 The tumor mass, ranging in size, formed in about 12 days, reaching 200-300 mm. 3 Tumor masses of varying sizes.

[0135] b. In vivo targeting of tumor cell membranes / nano-engineered erythrocytes

[0136] nRBCs and 4T1 tumor cell membrane-modified T-nRBCs were mixed with Cy5.5 and stirred at room temperature in the dark for 12 h. The mixtures were then placed in a dialysis bag with a molecular weight of 3500 and dialyzed for 12 h to obtain Cy5.5-loaded nRBCs (nRBCs). Cy5.5 ) and MT-nRBC (T-nRBC) Cy5.5 Quantification was performed using a fluorescence spectrophotometer. Tumor-bearing Balb / c mice were randomly divided into two groups of three. Tumors were quantified when they reached a volume of 300 mm². 3 At the same time, small molecule nRBC was injected via the tail vein. Cy5.5 and T-nRBC Cy5.5 The mice were given a saline solution. After 3, 12, and 24 hours following drug injection, they were anesthetized and near-infrared fluorescence imaging was performed to observe nRBCs. Cy5.5 and T-nRBC Cy5.5 Accumulated targeting status in various organizations.

[0137] The results are as follows Figure 16 As shown, T-nRBC Cy5.5 Shows more than nRBC Cy5.5 More pronounced fluorescence demonstrates that T-nRBCs can enhance accumulation at tumor sites by targeting homologous cancer cells.

[0138] c. Evaluation of the antitumor effects of tumor cell membrane / nano / gas engineered erythrocytes

[0139] Balb / c mice bearing tumors grew to 100 mm in size.3 Mice were randomly divided into 5 groups, and every 2 days, they were intravenously injected with saline and nRBC. CO nRBC NO nRBC CO / nRBC NO T-nRBC CO / T-nRBC NO Five doses were administered each time (CO:NO molar ratio 1:3). Each mouse received a dose of 70 mg Hb / kg, which is equivalent to 30 mg Hb / mL, or 3.1% of its total blood volume. Tumor growth was monitored for 16 days, and the results were recorded for each group. Figure 17 As shown.

[0140] The results showed that after 16 days of tumor volume monitoring, nRBC CO and nRBC NO It showed moderate antitumor activity, with tumor inhibition rates (TCR) of 30.6% and 41.4%, respectively, due to nRBC. CO CO release can increase ROS levels in tumors, nRBC NO Releasing NO can increase RNS levels. nRBC CO / nRBC NO The treatment achieved a tumor suppression rate of 68.6%, while T-nRBC... CO / T-nRBC NO The treatment resulted in the most significant tumor suppression, with a tumor inhibition rate of 83.1%. This indicates that the co-delivery of CO and NO by nRBCs can generate substantial amounts of ROS and RNS, thereby effectively inhibiting tumor growth. These findings demonstrate that gas delivery therapy based on engineered red blood cells effectively suppresses tumor growth in mice.

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

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

[0143] In the B16 melanoma lung metastasis model, melanoma cell membrane / nano / gas engineered erythrocytes were prepared using the same engineering techniques. Subsequently, a B16 melanoma lung metastasis model was established in female C57BL / 6 mice. In this model, 2 × 10⁻⁶ cells were injected on day 0. 6 One × 10 B16F10 cell was subcutaneously injected into the back of a mouse. On day 2, 1 × 10⁶ cells were injected intravenously. 5 Luciferase-labeled B16F10 (B16F10-LUC) cells.

[0144] b. Evaluation of the antitumor effect of tumor cell membrane / nano / gas engineered erythrocytes:

[0145] Tumor-bearing C57 mice showed a dorsal tumor volume of up to 60 mm. 3 At that time, tumor-bearing mice were randomly divided into five groups: saline group, anti-PD-L1 group, nRBC group, and anti-PD-L1 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] Post-treatment results Figure 18 As shown, the subcutaneous tumor volume in the saline group exceeded 1450 mm. 3 With nRB C CO / nRBC NO Compared with anti-PD-L1 treatment, T-nRB CO / T-nRBC NO The treatment showed a higher tumor suppression effect, with a tumor suppression rate of 64.1%, compared to T-nRBC. CO / T-nRBC NO The combination of +anti-PD-L1 further increased the tumor inhibition rate to 80.3%.

[0147] Mice treated with saline showed significant bioluminescent signals in their lungs 13 days after intravenous injection of B16F10-LUC cells, indicating severe lung metastasis of tumors. Figure 19 As shown, for nRBC CO / nRBC NO Mice treated with anti-PD-L1 showed a slight delay in metastasis. In contrast, T-nRB mice, due to their ability to target homologous tumor cells, showed... CO / T-nRBC NO Treatment significantly suppressed lung metastases. When T-nRBCs... CO / T-nRBC NO When combined with anti-PD-L1 therapy, lung metastases in mice were almost eliminated.

[0148] c. Evaluate the acute toxicity and immunogenicity of nano / gas-engineered erythrocytes in SD rats.

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

[0150] Monitor physiological indicators in rats. Intravenous infusion of nRBC and nRBC CO nRBC NO nRBC CO / nRBC NO Or T-nRBC CO / T-nRBC NO Subsequently, the vital signs of SD rats were monitored. Changes in mean arterial pressure (MAP), peripheral blood oxygen saturation (SpO2), and heart rate were recorded within 24 hours after drug administration. Results are as follows: Figure 21 As shown, all physiological indicators in the treatment group were comparable to those in the control group, with no significant differences.

[0151] Perform routine blood tests. Administer intravenous infusion of nRBC and nRBC. CO nRBC NO nRBC CO / nRBC NO Or T-nRBC CO / T-nRBC NO Blood routine examination 48 hours after drug administration confirmed the results, as shown in Table 1, indicating that the rats' hematological parameters remained within the normal range:

[0152] Table 1 Hematological parameters of rats

[0153]

[0154]

[0155] Therefore, the present invention provides a nano / gas-engineered erythrocyte drug formulation, its preparation method, and its application, which can precisely control the delivery of carbon monoxide (CO) and nitric oxide (NO) into target cells. By controlling the dosage and ratio of these 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 solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method of preparing a pharmaceutical preparation for gas infusion therapy, characterized by, Gas-infused therapeutic drug formulations are tumor gas-infused therapeutic drug formulations; Methods for preparing tumor gas infusion therapy drug formulations include: S81. After washing the red blood cells and mixing them with the tumor cell membrane, the mixture is co-extruded through a polycarbonate porous membrane with a pore size of 5~0.2μm to obtain tumor cell membrane / nano / gas engineered red blood cells T-nRBC; S82. Tumor cell membrane / nano / gas-engineered red blood cells (T-nRBCs) are exposed to CO or NO gas to obtain gas-saturated T-nRBCs. CO and T-nRBC NO ; T-nRBC in tumor gas infusion therapy formulations CO :T-nRBC NO The molar ratio is 1:3.

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