Red blood cell microreactor as well as preparation method and application thereof

Abstract

The invention provides an erythrocyte microreactor as well as a preparation method and application thereof, and belongs to the technical field of biomedical engineering. Glucose oxidase (GOx) is entrapped in erythrocytes under a hypotonic condition, endogenous hemoglobin (Hb) is retained, and palmitate modified catalase (CAT) is fixed on the surface of an erythrocyte membrane, so that the erythrocyte microreactor is prepared. According to the invention, spatial separation and function synergy of enzyme distribution and synergistic effect of GOx / Hb intracellular catalysis and membrane surface CAT removal of by-product H2O2 are realized, cascade reaction efficiency and stability are effectively improved, and accurate regulation and control of NO generation can be realized. Experiments show that the microreactor has remarkable effects in the aspects of regulating NO steady state, improving glucose tolerance, relieving insulin resistance, stabilizing blood glucose and the like, has a lasting curative effect, low medication frequency and high biological safety, and has a wide application prospect in the aspects of biological catalysis and prevention and treatment of metabolic diseases such as diabetes mellitus.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical engineering, and particularly relates to a red blood cell micro-reactor and a preparation method and application thereof. BACKGROUND

[0002] Nitric oxide (NO) is a gaseous signal molecule, which participates in the regulation of numerous physiological processes in the human body, especially in metabolic regulation. Its main functions include improving insulin sensitivity, protecting vascular endothelial function, promoting glucose metabolism, regulating fat metabolism, and inhibiting oxidative stress. At present, the main NO donors include organic nitrate compounds (nitroglycerin), organic nitrite compounds (butyl nitrite), metal-NO complexes (sodium nitroprusside), hydroxylamine compounds, hydroxyl guanidine compounds, and hydroxyl urea compounds (literatures: Wilkinson I B, Franklin S S, Cockcroft J R. Nitric oxide and the regulation of large artery stiffness: from physiology to pharmacology. Hypertension, 2004, 44(2): 112-116. and Walford G, Loscalzo J. Nitric oxide in vascular biology. Journal of Thrombosis and Haemostasis, 2010, 1(10): 2112-2118.). By introducing exogenous NO donors, the physiological functions of diseased tissues can be intervened, which can achieve the purpose of preventing or treating certain diseases such as diabetes. However, the pure NO donor can cause adverse reactions while playing a certain role, and the bioavailability of NO is low, making it difficult to achieve controllable release.

[0003] Cascade enzyme reaction has great potential in the fields of biosensing, medical diagnosis and green synthesis by simulating the multi-enzyme synergistic pathway in the body. However, there are still problems in constructing an efficient cascade enzyme system in vitro: simple blending of different enzymes often causes incompatibility between enzymes; the by-products produced during the reaction process may inhibit enzyme activity, limiting its long-term application. Red blood cells, as a natural biological carrier, are an ideal platform for constructing biomimetic micro-reactors. Some studies have attempted to load exogenous enzymes into red blood cells, but most of them are limited to single-function development, and there are problems such as low enzyme loading efficiency, uncontrollable spatial distribution of enzymes, easy damage to cell integrity, uncontrollable by-products, etc., making it difficult to achieve the functional synergy of multi-enzyme system and efficient management of by-products. Especially, the uncontrollable hazardous by-products pose great limitations to the application of enzyme-loaded red blood cells.

[0004] Therefore, in view of the low bioavailability, poor controllability and adverse side effects of existing NO donors, and the poor compatibility between enzymes and the inhibition of by-products in cascade enzyme systems, the application innovatively proposes a cascade enzyme micro-reactor using natural red blood cells as carriers to solve the above problems, and provides a preparation method of the red blood cell micro-reactor and further verifies the feasibility of its practical application. SUMMARY

[0005] The application aims to provide a red blood cell micro-reactor, a preparation method and application thereof. The micro-reactor introduces glucose oxidase (GOx) into red blood cells by low-osmotic encapsulation, retains endogenous hemoglobin (Hb), and then fixes hydrophobic modified catalase (CAT) on the cell membrane surface to form a red blood cell micro-reactor (Re-RBC) with spatially cooperative catalytic function. Without damaging the integrity of the red blood cells, the application realizes spatial separation and functional cooperation of enzyme distribution: GOx catalyzes the generation of H2O2 in the presence of glucose, and further catalyzes the oxidation of L-arginine (L-Arg) by H2O2 using the naturally existing Hb inside the red blood cells to achieve controllable NO generation; at the same time, in order to avoid the excessive accumulation of H2O2, the hydrophobic modified CAT is further fixed on the red blood cell membrane surface to degrade excess H2O2 and avoid the oxidative damage caused by it.

[0006] In a first aspect, the application provides an application of a red blood cell micro-reactor in the preparation of a drug for preventing and / or treating diabetes and related metabolic diseases, wherein the red blood cell micro-reactor uses red blood cells as carriers, the red blood cells contain an encapsulated concentration of 0.5-3 mg / mL of exogenous glucose oxidase and retain endogenous hemoglobin, and the red blood cell membrane surface is fixed with an attached concentration of 0.05-0.6 mg / mL of catalase modified by palmitate; the drug exerts its effect by administering the red blood cell micro-reactor to a subject, so that it generates NO in the body by using glucose and L-arginine as substrates through a cascade catalytic reaction, and the generated NO is used to inhibit the production of pro-inflammatory cytokines and / or promote the uptake of glucose by peripheral tissues.

[0007] Optionally, the diabetes and related metabolic diseases include insulin resistance, impaired glucose tolerance or obesity.

[0008] Optionally, the red blood cell micro-reactor generates NO through a cascade catalytic reaction, and the generation rate and / or total amount of the NO can be regulated by adjusting one or more of the glucose concentration, the L-arginine concentration, the glucose oxidase concentration and the catalase concentration.

[0009] In a second aspect, the application provides a preparation method of the above-mentioned red blood cell micro-reactor, comprising:

[0010] S1, using low-osmotic pressure-resealing method to encapsulate glucose oxidase in red blood cells, while retaining endogenous hemoglobin in red blood cells, and centrifuging to obtain red blood cells encapsulating glucose oxidase;

[0011] S2, modifying catalase by palmitate, the molar ratio of palmitate to catalase being 56:1, configuring the modified catalase solution, and using low-osmotic pressure-resealing method to fix catalase on the surface of the red blood cell membrane prepared in step S1, and centrifuging to obtain red blood cell microreactor.

[0012] Optionally, the low-osmotic pressure-resealing method in S1 includes mixing red blood cells, glucose oxidase solution and low-osmotic buffer to form a first mixed system, incubating, isotonically sealing after the incubation, and centrifuging for standby; the osmotic pressure of the first mixed system is 100-120 mOsm / kg.

[0013] Optionally, the volume ratio of red blood cells to glucose oxidase solution in the above-mentioned first mixed system is 1:(0.4-0.6).

[0014] Optionally, the concentration of the above-mentioned glucose oxidase solution is 0.5-5 mg / mL.

[0015] Optionally, the temperature for incubating the above-mentioned red blood cells in the first mixed system is 2-6℃, and the time is 10-20 min.

[0016] Optionally, the low-osmotic pressure-resealing method in S2 includes mixing the red blood cells obtained in S1, the modified catalase solution and low-osmotic buffer to form a second mixed system, incubating, isotonically sealing after the incubation, and centrifuging to obtain red blood cell microreactor; the osmotic pressure of the second mixed system is 190-210 mOsm / kg.

[0017] Optionally, the volume ratio of the red blood cells obtained in S1 to the modified catalase solution in the above-mentioned second mixed system is 1:(1.5-1.7).

[0018] Optionally, the concentration of the above-mentioned modified catalase solution is 0.1-2 mg / mL.

[0019] Optionally, the temperature for incubating the red blood cells obtained in S1 in the second mixed system is 2-6℃, and the time is 30-50 min.

[0020] The beneficial effects of the present application are:

[0021] (1) The red blood cell microreactor Re-RBC provided by the application contains exogenous glucose oxidase and endogenous hemoglobin inside the cell, and the cell membrane surface is fixed with catalase. The structure realizes spatial separation of enzyme distribution and functional synergy: GOx catalyzes the generation of H2O2 in the presence of glucose, the naturally existing Hb in the cell is further used to catalyze the oxidation of L-Arg by H2O2, the CAT on the cell membrane surface removes excess byproduct H2O2, avoids causing oxidative damage, and maintains the integrity and biocompatibility of the red blood cell. As shown in the hemolysis rate experiment, it does not cause obvious hemolysis in a wide dose range; and the Re-RBC can be used to generate NO, and precise regulation and release can be realized.

[0022] (2) Animal experiments show that in a diabetes model, Re-RBC can effectively improve a plurality of metabolic indicators: significantly reduce fasting blood glucose, alleviate weight loss, enhance glucose clearance capacity, and significantly improve insulin resistance; in addition, Re-RBC can be enriched in key metabolic organs such as liver and kidney through the red blood cell metabolic pathway, which is beneficial to its delivery and functional release in metabolic related diseases. The application has good application prospect in the fields of biological catalysis and metabolic disease treatment due to its biological safety, high catalytic efficiency and controllable treatment.

[0023] (3) When the red blood cell microreactor provided by the application is used in the preparation of a drug for preventing and / or treating diabetes and related metabolic diseases, the following effects can be realized: first, the slow-release effect can ensure more effective and sustained release of NO. As shown in the controllable NO generation experiment of Re-RBC in Example 1 of the application, 10 μM NO can be released within 24-48 h, which is durable and beneficial to maintaining long-term effect; second, the in-vivo long-circulation effect can effectively reduce the frequency of drug administration. As shown in the in-vivo circulation animal experiment of Re-RBC in Example 1 of the application, the in-vivo circulation time of Re-RBC injected into C57 mice intravenously can be up to 4 days, which provides guarantee for sustained therapeutic effect; third, the use of endogenous substrate improves biological safety. The use of L-arginine as an NO donor in the body not only has better biocompatibility and reduces self-rejection, but also can reduce the potential cytotoxicity or side effect risk caused by exogenous substances. The application for drug preparation can realize long-lasting effect, low drug frequency, high biological safety, and is more in line with the clinical needs of long-term intervention for chronic metabolic diseases. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 It is a schematic diagram of the construction of Re-RBC and its reaction mechanism;

[0025] Figure 2 It is a verification diagram of the GOx loading effect in Example 1, wherein A is the distribution diagram of Cy5-GOx; B is the fluorescence spectrum of Cy5-GOx; C is the ultraviolet-visible absorption spectrum of Cy5-GOx;

[0026] Figure 3 Validation chart of CAT loading effect in Example 1, wherein A is the distribution chart of FITC-CAT; B is the fluorescence spectrum of FITC-CAT; C is the UV-visible absorption spectrum of FITC-CAT;

[0027] Figure 4 Controllability detection result chart of Re-RBC generating NO in Example 1, wherein A, B, C and D respectively correspond to the experimental results with Glu, L-Arg, GOx and CAT as variables;

[0028] Figure 5 Re-RBC concentration gradient hemolysis test chart in Example 1, wherein A is the actual photograph of samples in tubes 1-11; B is the hemolysis rate chart of sample groups in tubes 3-11;

[0029] Figure 6 Fluorescence detection chart of Re-RBC in vivo circulation in Example 1, wherein A is the fluorescence distribution of Re-RBC in organs and blood of mice killed on day 2; B is the fluorescence distribution of Re-RBC in organs and blood of mice killed on day 4;

[0030] Figure 7 Functional characterization chart of Re-RBC on maintaining mouse FBG and body weight in Example 1, wherein A is the influence chart on mouse FBG; B and C are respectively the influence chart and its change rate chart on mouse body weight;

[0031] Figure 8 Functional characterization chart of Re-RBC on improving mouse glucose tolerance in Example 1, wherein A is the influence chart on mouse glucose tolerance, and B is the corresponding AUC value change chart thereof;

[0032] Figure 9 Functional characterization chart of Re-RBC on improving mouse insulin tolerance in Example 1, wherein A is the influence chart on mouse insulin tolerance, and B is the corresponding AUC value change chart thereof;

[0033] Figure 10The results of the anti-inflammatory and antioxidant indicators for different Re-RBC concentrations in Example 1 are shown in the following figures. Figure A shows the effect of different concentrations of Re-RBC on the content of serum SOD, Figure B shows the effect of different concentrations of Re-RBC on the content of serum MDA, Figure C shows the effect of different concentrations of Re-RBC on the content of serum IL-1β, Figure D shows the effect of different concentrations of Re-RBC on the content of serum TNF-α, Figure E shows the effect of different concentrations of Re-RBC on the content of kidney SOD, Figure F shows the effect of different concentrations of Re-RBC on the content of kidney MDA, Figure G shows the effect of different concentrations of Re-RBC on the content of kidney IL-1β, and Figure H shows the effect of different concentrations of Re-RBC on the content of kidney TNF-α. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. Unless otherwise defined, the technical terms or scientific terms used herein should have the usual meanings understood by those skilled in the art.

[0035] In the following examples, the operations not described in detail are conventional technical operations, which can be referred to existing published journal literature.

[0036] The present application provides a use of a red blood cell microreactor (Re-RBC) in the preparation of a medicament for preventing and / or treating diabetes and its related metabolic diseases, wherein the red blood cell microreactor (Re-RBC) takes red blood cells as carriers, the inside of the red blood cells contains exogenous glucose oxidase (GOx) with a loading concentration of 0.5-3 mg / mL, and retains endogenous hemoglobin (Hb), and the membrane surface of the red blood cells is modified by palmitate (PA) to fix hydrogen peroxidase (CAT) with a loading concentration of 0.05-0.6 mg / mL; the medicament exerts its effect by the following way: the red blood cell microreactor is administered to a subject, so that it generates NO in the body by taking glucose (Glu) and L-arginine (L-Arg) as substrates through a cascade catalytic reaction, and the generated NO is used to inhibit the production of pro-inflammatory cytokines and / or promote the uptake of glucose by peripheral tissues.

[0037] In fact, in the total reaction system, Glu generates gluconic acid and hydrogen peroxide under the catalysis of GOx, hydrogen peroxide diffuses to the vicinity of the red blood cell membrane to generate NO and water under the action of Hb and L-Arg, and the excess hydrogen peroxide will overflow into the red blood cells, and the Re-RBC provided in the application is loaded and fixed with CAT on the surface of the red blood cell membrane, which is used to regulate the concentration of hydrogen peroxide, which not only helps to improve the generation rate of NO, but also can effectively avoid the harm of hydrogen peroxide: high concentration of hydrogen peroxide can cause harm to the body, and when the concentration is too high, it can rapidly decompose and release a large amount of oxygen, causing corrosive gastrointestinal injury, acute gastric perforation, and more seriously, causing gas embolism - oxygen entering the portal vein, inferior vena cava or heart, which can cause cerebral infarction, myocardial infarction, pulmonary embolism-like symptoms, and even sudden death; at the same time, its strong oxidizing property triggers lipid peroxidation chain reaction, damages cell membrane, DNA and mitochondria, and causes acute renal tubular necrosis, liver necrosis, nervous system dysfunction and multiple organ failure.

[0038] In some embodiments, the rate and / or total amount of NO generated by the red blood cell microreactor can be precisely regulated by adjusting one or more of the concentrations of Glu, L-Arg, GOx, and CAT.

[0039] The application provides a preparation method of the above-mentioned red blood cell microreactor, comprising: S1, using a low-osmotic pressure-resealing method to encapsulate GOx in red blood cells while retaining endogenous Hb in the red blood cells, and centrifuging to obtain red blood cells encapsulating GOx; S2, modifying CAT with palmitate, the molar ratio of PA to CAT being 56:1, configuring a solution of the modified catalase (PA-CAT), and using the low-osmotic pressure-resealing method again to fix CAT on the surface of the red blood cell membrane prepared in step S1, and centrifuging to obtain the red blood cell microreactor.

[0040] Specifically, the construction of Re-RBC and the schematic diagram of its reaction mechanism are shown in Figure 1 .

[0041] In fact, the key to realizing efficient insertion of membrane anchoring is to modify CAT with hydrophobic chain palmitate (PA-NHS). Red blood cells have no transfection, no endocytosis, no receptor, and only a highly ordered phospholipid bilayer membrane; in order to "insert" a hydrophilic 240kDa tetramer protein into it, two major bottlenecks must be solved: one is the energy barrier, the membrane surface is a hydrophobic core + a hydrophilic head, and when the hydrophilic protein approaches, it faces "dehydration energy"> 100 kT, and the spontaneous insertion probability is approximately 0; the second is the space barrier, the red blood cell membrane protein / skeleton is dense, and a 10nm diameter foreign particle will be "bounced" back by the Band3-Ankyrin-Spectrin network. The modification of palmitate can change the "hydrophilic large protein" into a "membrane-like lipid molecule", which solves the four problems of thermodynamic insertion, topological orientation, immune compatibility and activity retention at one time.

[0042] In some embodiments, the low-osmolarity-high-osmolarity sealing method in S1 comprises mixing red blood cells, GOx solution and low-osmolarity buffer to form a first mixed system, incubating, and then isotonic sealing and centrifugation after the incubation; the osmotic pressure of the first mixed system in this process is 100-120 mOsm / kg.

[0043] In some embodiments, the red blood cells and the GOx solution in the first mixed system are mixed at a volume ratio of 1:(0.4-0.6).

[0044] In some embodiments, the concentration of the GOx solution added in the first mixed system is 0.5-5 mg / mL.

[0045] In some embodiments, the incubation temperature of the red blood cells in the first mixed system is 2-6°C, and the incubation time is 10-20 min.

[0046] In some embodiments, the low-osmolarity-high-osmolarity sealing method in S2 comprises mixing the red blood cells obtained in S1, the modified hydrogen peroxidase solution and the low-osmolarity buffer to form a second mixed system, incubating, and then isotonic sealing and centrifugation after the incubation; the osmotic pressure of the second mixed system in this process is 190-210 mOsm / kg.

[0047] In some embodiments, the red blood cells obtained in S1 and the modified hydrogen peroxidase solution in the second mixed system are mixed at a volume ratio of 1:(1.5-1.7).

[0048] In some embodiments, the concentration of the modified hydrogen peroxidase solution in the second mixed system is 0.1-2 mg / mL.

[0049] In some embodiments, the incubation temperature of the red blood cells obtained in S1 in the second mixed system is 2-6°C, and the incubation time is 30-50 min.

[0050] Embodiment 1

[0051] This embodiment 1 provides a red blood cell microreactor and a preparation method thereof, and evaluates and determines the controllability of NO generation, biocompatibility and functionality of injection into the body of the red blood cell microreactor prepared in this embodiment 1.

[0052] (1) Preparation of red blood cell microreactor (Re-RBC)

[0053] GOx loading procedure: Take 200 μL of erythrocyte suspension, add 100 μL of 1.8 mg / ml Cy5-GOx solution, and add 200 μL of 0.1×PBS. The final reaction system should be in a 0.4×PBS environment (osmolarity 115 mOsm / kg), and react at 4°C in the dark for 15 min. After the reaction, add 2 mL of 1×PBS solution to restore the erythrocytes to 1×PBS solution (osmolarity 290 mOsm / kg), react for 5 min, and then perform isotonic blocking. Centrifuge at 2000 rpm for 5 min, discard the supernatant, and repeat this step 6-8 times until the supernatant is clear. Collect the centrifuged erythrocytes, resulting in a total 60 μL precipitate. This erythrocyte is the GOx-loaded erythrocyte.

[0054] Calculation of Cy5-GOx partition coefficient: During the preparation of Re-RBCs, the total fluorescence intensity of 100 μL of 1.8 mg / mL Cy5-GOx added during the preparation process was detected using an ELISA reader and recorded as fluorescence intensity 1. The total fluorescence intensity in the erythrocyte precipitate loaded with Cy5-GOx was detected using an ELISA reader and recorded as fluorescence intensity 2. The partition coefficient of Cy5-GOx is equal to: fluorescence intensity 2 / fluorescence intensity 1, and the partition coefficient is calculated to be 49.15%.

[0055] Encapsulation concentration / attached concentration: The amount of enzyme encapsulated / attached per unit volume of red blood cells.

[0056] Calculate the GOx concentration in the final 60 μL red blood cell volume: Total volume of added enzyme (100 μL) × Concentration of added enzyme (1.8 mg / ml) × Partition coefficient / Red blood cell pellet volume (60 μL), and the GOx concentration is calculated to be 1.5 mg / ml, that is, the loading concentration is 1.5 mg / ml.

[0057] Volume normalization: The initial reaction system was 500 μL, and the GOx concentration in the initial reaction system was 0.36 mg / ml; the GOx loading concentration was 1.5 mg / ml, and the volume normalized final concentration was 0.18 mg / ml.

[0058] Calculation of GOx loading rate: After volume normalization, the final GOx concentration in the collected red blood cell pellet (0.18 mg / ml) is divided by the initial GOx concentration in the 500 μL reaction system (0.36 mg / ml), which gives the loading rate of 50%.

[0059] CAT loading procedure: Take 60 μL of erythrocyte pellet loaded with GOx, add 100 μL of 0.2 mg / mL PA-CAT solution prepared with 1×PBS (molar ratio of palmitate to CAT is 56:1), add 173 μL of 1×PBS solution and 167 μL of 0.1×PBS solution to make the total reaction volume 500 μL. The final reaction system is kept in a 0.7×PBS environment (osmolarity of 203 mOsm / kg) and reacted at 4°C in the dark for 40 min. After the reaction, add 2 mL of 1×PBS solution to restore the erythrocytes to 1×PBS solution (osmolarity of 290 mOsm / kg), react for 5 min, and then perform isotonic blocking. Centrifuge at 2000 rpm for 5 min, discard the supernatant, and repeat this step 3-5 times until the supernatant is clear. Collect the centrifuged erythrocyte pellet, which is the erythrocyte loaded with GOx (0.18 mg / mL) and PA-CAT (0.012 mg / mL).

[0060] The partition coefficient of PA-CAT-FITC was calculated as follows: During the preparation of Re-RBC, the total fluorescence intensity of 100 μL of 0.2 mg / mL PA-CAT-FITC added during the preparation process was detected using an ELISA reader and recorded as fluorescence intensity 1. The total fluorescence intensity in the final collected Re-RBC precipitate was detected using an ELISA reader and recorded as fluorescence intensity 2. The partition coefficient of PA-CAT-FITC is equal to: fluorescence intensity 2 / fluorescence intensity 1. The calculated partition coefficient is 29.89%.

[0061] Calculation of CAT loading rate: During the preparation process, the reaction system is in 0.7 times the amount of PBS solution, and the red blood cells will not rupture, so the final collected red blood cell precipitate is also 60 μL.

[0062] Calculate the CAT concentration in the final 60 μL red blood cell volume: Total volume of added enzyme (100 μL) × Concentration of added enzyme (0.02 mg / ml) × Partition coefficient (29.89%) / Red blood cell volume (60 μL), and the CAT concentration is calculated to be 0.1 mg / ml, that is, the loading concentration is 0.1 mg / ml.

[0063] Volume normalization: The initial reaction system was 500 μL, and the initial CAT concentration in the reaction system was 0.04 mg / ml; the CAT loading concentration was 0.1 mg / ml, and the volume normalized final concentration was 0.012 mg / ml.

[0064] Calculation of CAT loading rate: After volume normalization, the final CAT concentration in the collected red blood cell pellet (0.012 mg / ml) is divided by the initial CAT concentration in the 500 μL reaction system (0.04 mg / ml) to obtain the loading rate of 30%.

[0065] To verify the spatial distribution and anchoring effect of enzymes in the constructed microreactor, GOx and CAT were fluorescently labeled with Cy5 and FITC fluorescent dyes, respectively. The localization and distribution of GOx and CAT were observed by confocal laser scanning microscopy, and the successful loading of the biological enzymes was confirmed by combining fluorescence spectroscopy and UV-Vis absorption spectroscopy.

[0066] Results: The distribution map, fluorescence spectrum, and UV-Vis absorption spectrum of Cy5-GOx are shown in the figure. Figure 2 The distribution map, fluorescence spectrum, and UV-Vis absorption spectrum of FITC-CAT are shown in [reference needed]. Figure 3 As shown in the figure, the Cy5-GOx fluorescence signal is uniformly distributed inside the erythrocytes, while Cy5-GOx is distributed in a continuous ring around the cell membrane periphery, indicating that GOx was successfully internalized and CAT was stably anchored on the membrane surface. Fluorescence spectroscopy analysis showed that Cy5-GOx had a significant emission peak at the excitation wavelength of 643 nm, and FITC-CAT also showed a strong fluorescence response at 520 nm. The corresponding UV-Vis absorption spectra showed characteristic absorption peaks at 667 nm and 492 nm, respectively. These results further verified the specificity of the enzyme label and its successful loading in Re-RBCs.

[0067] (2) Controllability of NO generation by Re-RBC

[0068] To evaluate the effects of different substrate and enzyme concentrations on NO production, experimental systems were set up with glucose (Glu), L-Arg, GOx, and CAT as variables. The detection procedure was as follows: 10 μL of Re-RBC was added to a 1.5 mL EP tube, the substrate was added, and PBS was added to bring the total volume to 1 mL. After initiating the reaction, it was incubated at 4°C. At 0, 2, 4, 8, 12, 24, 36, and 48 h, 50 μL of the reaction solution was taken, and equal volumes of 50 μL of Griess reagent I and 50 μL of Griess reagent II were added. After mixing, the reaction was carried out in the dark for 5 min. After the reaction, the absorbance was measured at 540 nm, and the NO concentration was calculated using a standard curve to analyze the NO release efficiency.

[0069] By changing the GOx loading concentration, CAT loading concentration, glucose (Glu) substrate concentration, and L-Arg substrate concentration of Re-RBC, four experimental groups were set up for each variable to detect and analyze the controllability of NO generation by Re-RBC.

[0070] The specific experimental group settings are as follows:

[0071] The substrate glucose (Glu) was set as a variable: four experimental groups were set with Glu concentrations of 0 mM, 1 mM, 5 mM and 25 mM, respectively; all other conditions were kept the same, with L-Arg concentration of 0.2 mM and Re-RBC (GOx loading concentration of 1.5 mg / ml and CAT loading concentration of 0.1 mg / ml) prepared in Example 1.

[0072] The substrate L-Arg was set as a variable: four experimental groups were formed with L-Arg concentrations of 0 mM, 0.2 mM, 1.6 mM, and 10 mM, respectively; all other conditions were kept the same, with Glu concentration at 5 mM and Re-RBC (GOx loading concentration of 1.5 mg / ml and CAT loading concentration of 0.1 mg / ml) prepared in Example 1.

[0073] GOx was set as a variable: the experiment was divided into 4 groups, with GOx loading concentrations of 0 mg / ml, 0.5 mg / ml, 1.5 mg / ml and 3 mg / ml for Re-RBC; other conditions were kept the same, Glu concentration was 5 mM, L-Arg concentration was 0.2 mM and CAT loading concentration of Re-RBC was 0.1 mg / ml.

[0074] CAT was set as a variable: the experiment was divided into 4 groups, with CAT loading concentrations of 0 mg / ml, 0.05 mg / ml, 0.1 mg / ml and 0.5 mg / ml of Re-RBC, respectively; other conditions were kept the same, with Glu concentration of 5 mM, L-Arg concentration of 0.2 mM and GOx loading concentration of Re-RBC of 1.5 mg / ml.

[0075] The difference between the preparation of Re-RBCs with GOx loading concentrations of 0 mg / ml, 0.5 mg / ml, and 3 mg / ml and the preparation of Re-RBCs in Example 1 is that the added GOx concentrations are 0 mg / ml, 1 mg / ml, and 3 mg / ml, respectively, with partition coefficients of 0, 30%, and 59.8%, and loading rates of 0, 30%, and 60%, respectively. The difference between the preparation of Re-RBCs with CAT loading concentrations of 0 mg / ml, 0.05 mg / ml, and 0.5 mg / ml and the preparation of Re-RBCs in Example 1 is that the added PA-CAT concentrations are 0 mg / ml, 0.125 mg / ml, and 0.75 mg / ml, respectively, with partition coefficients of 0, 24.1%, and 40.21%, and loading rates of 0, 24%, and 40%, respectively.

[0076] Results: See below for detailed experimental results. Figure 4As shown in the figure, different concentrations of substrate or enzyme have different effects on NO production. Therefore, the amount of NO released can be effectively controlled by adjusting the concentrations of enzyme and substrate in the cascade enzyme reaction.

[0077] (3) Biocompatibility of Re-RBC

[0078] Re-RBC concentration gradient hemolysis test: Sheep red blood cells were precipitated and diluted with 1×PBS at a volume ratio of 1:9 to prepare red blood cell storage solution. 100 μL of the red blood cell storage solution was placed into 11 1.5 mL EP tubes. Tube 1 was added with 300 μL of 1×PBS (negative sample group), tube 2 was added with 300 μL of ultrapure water (positive sample group), and tubes 3-11 were designated as experimental sample groups. Re-RBC precipitate was added at concentrations of 1, 20, 40, 60, 80, 100, 120, 140, and 160 μL respectively, and the total volume was brought up to 400 μL with 1×PBS. The experiment was repeated three times. Centrifuge at 2,000 rpm for 3 min after 90 min, photograph and record the color of the supernatant of each group, and take 300 μL of supernatant to measure the absorbance at 540 nm using a UV-Vis spectrophotometer to evaluate the effect of different Re-RBC concentrations on erythrocyte membrane integrity and calculate the hemolysis rate.

[0079] Re-RBC in vivo circulation experiment: SPF-grade male C57BL / 6J mice were used as experimental animals. 12 mg / kg of Cy5-labeled Re-RBC precipitate was injected via the tail vein, with PBS as the suspension medium. On days 2 and 4 post-injection, mice were randomly sacrificed, and their major organs (heart, liver, lung, kidney, and spleen) and blood were collected for fluorescence detection. The surfaces of each organ were thoroughly washed with PBS to remove residual blood, and then placed in pre-cooled sterile culture dishes. Fluorescence imaging was performed using the IVIS Lumina II in vitro imaging system to record the fluorescence distribution of Cy5 signals in each tissue and organ, assessing the enrichment and homing characteristics of Re-RBCs in vivo.

[0080] Results: The results of the Re-RBC concentration gradient hemolysis test are shown in […]. Figure 5 , Figure 5 In the image, A represents actual photographs of samples from tubes 1-11. Figure 5 Figure B shows the hemolysis rate of experimental samples in tubes 3-11, specifically: tube 1 (PBS, positive control), tube 2 (water, negative control), tube 3 (volume ratio: 0), tube 4 (volume ratio: 0.125), tube 5 (volume ratio: 0.25), tube 6 (volume ratio: 0.5), tube 7 (volume ratio: 0.75), tube 8 (volume ratio: 1), tube 9 (volume ratio: 1.25), tube 10 (volume ratio: 1.5); tube 11 (volume ratio: 1.75). The fluorescence detection results of Re-RBC in vivo circulation are shown in [the figure].Figure 6 , Figure 6 In the figure, A represents the fluorescence distribution of Re-RBCs in organs and blood of mice sacrificed on day 2. Figure 6 In the figure, B represents the fluorescence distribution of Re-RBCs in organs and blood of mice sacrificed on day 4. Figure 5 It can be seen that Re-RBCs in different dose ranges (1-160 μL) did not induce significant erythrocyte lysis, demonstrating good blood compatibility and erythrocyte membrane stability; Figure 6 In vivo fluorescence imaging showed that the fluorescence in the blood was still relatively strong on day 2, but no fluorescence was observed in the blood on day 4, indicating that Re-RBCs had been metabolized and were no longer circulating in the blood, suggesting that the half-life of Re-RBCs is 4 days. In vivo fluorescence detection results of mice sacrificed on day 2 showed that Re-RBCs were mainly enriched in the liver and kidneys, with moderate signal in the lungs, and weak distribution in the heart and spleen, exhibiting tissue distribution characteristics consistent with the metabolic homing pathway of erythrocytes, which is conducive to their delivery and functional release in metabolic-related diseases.

[0081] (4) Functional effects of Re-RBC injection into animals

[0082] Based on the multiple metabolic regulatory roles of NO in improving insulin sensitivity, promoting glucose transport, and alleviating inflammatory oxidative stress, this study further evaluated the functional characterization of Re-RBCs in an STZ-induced type 2 diabetes mouse model.

[0083] A type 2 diabetes model was established in male C57BL / 6J mice using streptozotocin (STZ). After successful model establishment, mice were randomly divided into a normal control group (NC group), a model control group (MC group), and intervention groups with low (Re-RBC-L group; dose of 0.4 ml / kg Re-RBC precipitate), medium (Re-RBC-M group; dose of 0.8 ml / kg Re-RBC precipitate), and high (Re-RBC-H group; dose of 1.2 ml / kg Re-RBC precipitate) doses in the erythrocyte microreactors. Each of the NC, MC, and low / medium / high dose groups consisted of 6 mice. The intervention groups received the corresponding dose of Re-RBC via tail vein injection once daily for 4 weeks. The normal control and model control groups received an equal volume of PBS as controls. During the intervention period, fasting blood glucose (FBG) levels were monitored regularly, and weight changes were recorded to assess overall metabolic status.

[0084] In week 5, the intraperitoneal glucose tolerance test (IPGTT) was conducted. Mice were injected intraperitoneally with glucose solution (2 g / kg) after fasting for 12 h. Blood glucose concentrations were measured at 0, 15, 30, 60 and 120 min, and glucose-time curves were plotted. The area under the curve (AUC) was calculated to evaluate glucose clearance efficiency.

[0085] Three days after the IPGTT, an insulin tolerance test (ITT) was conducted. Mice were injected intraperitoneally with insulin (0.5 U / kg) after fasting for 6 hours. Blood glucose concentrations were measured at 0, 15, 45, 60, 90, and 120 minutes, and glucose-time curves were plotted. The intervention effect of Re-RBC on glucose metabolism in a diabetic model was comprehensively evaluated by combining fasting blood glucose (FBG), weight change, IPGTT, and ITT results.

[0086] Results: The functional characterization diagram of Re-RBC is shown in... Figures 7 to 9 The results showed that during four consecutive weeks of monitoring, the fasting blood glucose level in the model group mice remained consistently >23 mmol / L, while the Re-RBC treatment group showed a significant trend of blood glucose reduction, with the intervention effect increasing with increasing dosage. The low, medium, and high dose groups were all significantly lower than the model group. Figure 7 In addition, the model group mice continued to lose weight, with a body weight loss of approximately 20.3% within 4 weeks, while Re-RBC intervention significantly delayed weight loss, and the body weight of the high-dose group mice remained basically stable, close to the normal control level. Figure 7 B in Figure 7 The presence of C in the figure suggests its ability to alleviate diabetes-related metabolic failure. IPGTT was used to examine its regulatory effect on glucose metabolism. After glucose injection, blood glucose in the model group rapidly increased and remained at a high level, with AUC significantly higher than in the normal group. Re-RBC treatment effectively promoted blood glucose reduction, and the AUC value gradually decreased with increasing dose, reflecting its dose-dependent effect in enhancing glucose clearance efficiency and improving glucose tolerance. Figure 8 A in Figure 8 In type 2 diabetes, insulin resistance occurs, affecting the biological activity of insulin. In the diabetic model group mice, blood glucose decreased slowly after insulin injection. In the three microreactor treatment groups, blood glucose decreased sharply after insulin injection, and the rate of change in blood glucose was significantly greater than that in the model group. This indicates that Re-RBC treatment can effectively improve insulin resistance. Figure 9 A in Figure 9 (B)

[0087] (5) Validation of the anti-inflammatory and antioxidant indices of Re-RBC

[0088] After establishing a type 2 diabetes model in male C57BL / 6J mice induced by streptozotocin, the mice were randomly divided into a normal control group (NC group), a model control group (MC group), and intervention groups with low (Re-RBC-L group; dose of 0.4 ml / kg Re-RBC precipitate), medium (Re-RBC-M group; dose of 0.8 ml / kg Re-RBC precipitate), and high (Re-RBC-H group; dose of 1.2 ml / kg Re-RBC precipitate) doses of erythrocyte microreactors. The intervention groups were injected with the corresponding dose of Re-RBC via tail vein once a day for 4 consecutive weeks. The normal control group and the model control group were injected with the same volume of PBS as controls.

[0089] After the last administration, samples were collected from mouse serum and kidney tissue homogenate. Enzyme-linked immunosorbent assay (ELISA) was performed using an ELISA kit (Nanjing Jiancheng Company), strictly following the instructions, to detect changes in superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) content in mouse serum and kidney tissue homogenate under different concentrations of Re-RBC intervention.

[0090] For details, see Figure 10 : Figure 10 In the figure, A represents the effect of different concentrations of Re-RBC on serum SOD levels. Figure 10 In the figure, B represents the effect of different concentrations of Re-RBC on serum MDA levels. Figure 10 In the figure, C represents the effect of different concentrations of Re-RBC on serum IL-1β levels. Figure 10 In the figure, D represents the effect of different concentrations of Re-RBC on serum TNF-α levels. Figure 10 E in the figure represents the effect of different concentrations of Re-RBC on the content of SOD in the kidney. Figure 10 F in the figure represents the effect of different concentrations of Re-RBC on the renal MDA content. Figure 10 In the figure, G represents the effect of different concentrations of Re-RBC on the renal IL-1β content. Figure 10 H in the figure represents the effect of different concentrations of Re-RBC on the renal TNF-α content.

[0091] Experimental data show that the erythrocyte microreactor has systemic antioxidant and anti-inflammatory effects and can target and improve the inflammatory microenvironment of the kidney (a common target organ of diabetes).

[0092] In summary, Re-RBCs, by regulating NO homeostasis, demonstrated significant intervention effects on multiple metabolic indicators, including stabilizing blood glucose, maintaining body weight, improving glucose tolerance, and reducing insulin resistance. Furthermore, the anti-inflammatory and antioxidant effects of Re-RBCs were verified. The erythrocyte microreactors prepared in this invention have application potential and translational value in the treatment of chronic metabolic diseases such as diabetes.

[0093] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. Use of a red blood cell microreactor for the preparation of a medicament for the prevention and / or treatment of diabetes mellitus and its associated metabolic disorders, characterized in that, The red blood cell microreactor takes red blood cells as carriers, the inside of the red blood cells contains exogenous glucose oxidase with a loading concentration of 0.5-3 mg / mL, and retains endogenous hemoglobin, and the membrane surface of the red blood cells is fixed with immobilized catalase with a loading concentration of 0.05-0.6 mg / mL through palmitate modification; the drug exerts effects in the following manner: the red blood cell microreactor is administered to a subject, so that it generates NO in the body by taking glucose and L-arginine as substrates through cascade catalytic reactions, and the generated NO is used to inhibit the production of pro-inflammatory cytokines and / or promote the uptake of glucose by peripheral tissues.

2. Use according to claim 1, characterized in that, The diabetes and related metabolic diseases include insulin resistance, impaired glucose tolerance or obesity.

3. Use according to claim 1, characterized in that, The generation rate and / or total amount of NO can be regulated by adjusting one or more of the glucose concentration, the L-arginine concentration, the glucose oxidase concentration and the catalase concentration.

4. A method for the preparation of a red blood cell microreactor for use in any one of the uses of claims 1 to 3, characterized in that, Comprise: S1, using a low-osmotic pressure-resealing method to encapsulate glucose oxidase in red blood cells while retaining endogenous hemoglobin in the red blood cells, and centrifuging to obtain red blood cells encapsulating glucose oxidase; S2, modifying catalase through palmitate, configuring a solution of the modified catalase with a molar ratio of palmitate to catalase of 56:1, and again using a low-osmotic pressure-resealing method to fix the catalase on the membrane surface of the red blood cells prepared in step S1, and centrifuging to obtain a red blood cell microreactor.

5. The preparation method according to claim 4, characterized in that, The low-osmotic pressure-resealing method in S1 comprises mixing red blood cells, a glucose oxidase solution and a low-osmotic buffer to form a first mixed system, incubating, isotonically sealing after the incubation is completed, and centrifuging for standby use; the osmotic pressure of the first mixed system is 100-120 mOsm / kg.

6. The production method according to claim 5, wherein The volume ratio of red blood cells to the glucose oxidase solution in the first mixed system is 1:(0.4-0.6); and / or, the concentration of the glucose oxidase solution is 0.5-5 mg / mL; and / or, the incubation temperature is 2-6°C, and the incubation time is 10-20 min.

7. The preparation method according to claim 4, characterized in that, The low-osmotic pressure-resealing method in S2 comprises mixing the red blood cells obtained in S1, the solution of the modified catalase and a low-osmotic buffer to form a second mixed system, incubating, isotonically sealing after the incubation is completed, and centrifuging to obtain a red blood cell microreactor; the osmotic pressure of the second mixed system is 190-210 mOsm / kg.

8. The production method according to claim 7, characterized by, The volume ratio of the red blood cells obtained in S1 to the solution of the modified catalase in the second mixed system is 1:(1.5-1.7); and / or, the concentration of the solution of the modified catalase is 0.1-2 mg / mL; and / or, the incubation temperature is 2-6°C, and the incubation time is 30-50 min.

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