Liver-targeting ghost preparation as well as preparation method and application thereof

Abstract

The invention relates to the technical field of ghost preparations. The invention provides a liver-targeting ghost preparation as well as a preparation method and application thereof. The preparation method comprises the following steps: centrifuging a red blood cell suspension, mixing the centrifuged red blood cell suspension with a NaCl solution, mixing the centrifuged red blood cell suspension with a hypertonic solution, incubating, centrifuging, and resuspending to obtain a ghost cell suspension; and mixing the ghost cell suspension and the MOG35-55 solution to obtain the ghost preparation. According to the invention, the use of chemical reagents is abandoned, the possible harm of the preparation to human bodies is effectively reduced, and a milder solution is provided for safe drug delivery. The prepared product is high in drug loading capacity, simple and convenient in preparation process, free of various chemical reagents and good in safety, and the problems that the drug loading capacity is low, the preparation process is complex due to chemical modification, the safety is not clear after long-term use and the like can be effectively solved.

Description

Technical Field

[0001] The present invention relates to the technical field of blood ghost preparations, and in particular to a blood ghost preparation with liver targeting, and a preparation method and application thereof. Background Art

[0002] The liver is a key organ for controlling immune tolerance. Antigens targeted to antigen-presenting cells in the liver can induce immune tolerance responses to autoantigens in the liver itself or in tissues outside the liver. A certain amount of research has been accumulated on liver-targeted drug delivery. Liver-targeted nano-drug delivery systems can be divided into active targeting (mainly based on the principles of "receptor-ligand binding" and "antigen-antibody binding", such as galactose receptor-mediated liposomes), passive targeting (mainly based on particle size effects, such as PLGA nanoparticles) and physicochemical targeting (mainly based on magnetic fields, ultrasound and other technologies, such as Fe3O4 magnetic nanoparticles) according to the targeting mechanism. While the above-mentioned liver-targeted nano-drug delivery system based on nanomaterials has achieved a certain liver-targeted delivery effect, it also has shortcomings such as low drug loading, complex preparation process due to chemical modification, and unclear long-term safety. It is still in the conceptual research stage.

[0003] In recent years, in order to solve these problems, researchers have begun to shift their attention to bionic drug delivery systems based on natural materials. Cellular drugs are a disruptive technology in the field of drug research. Red blood cells account for 1 / 4 of all human cells, have no nucleus and mitochondria, and are very suitable as drug carriers. Current research in the field of red blood cell preparations focuses on the development of long-acting red blood cell drug-loaded preparations based on the physiological law that red blood cells have a lifespan of 120 days (two varieties have entered Phase III clinical trials internationally), while research on targeted red blood cell delivery of drugs is still in its infancy.

[0004] The maintenance of a normal number of red blood cells is the result of a dynamic balance between continuous production and destruction. Red blood cells gradually age during the circulation process, and the final destination of aged red blood cells is to be specifically identified, retained, destroyed, and decomposed in the liver and spleen. Therefore, based on the physiological law that aged red blood cells are specifically cleared by the liver, aged red blood cells can be used as drug delivery carriers to deliver drugs to the liver.

[0005] Under certain physiological and pathological conditions, a specific hypotonic environment will be formed locally in the body. The cell membrane of the red blood cells in this environment will rupture, releasing hemoglobin and other cytoplasmic contents to the extracellular space. After returning to the isotonic environment, the rupture of the red blood cell membrane will close again, and the hollow red blood cells formed are called erythrocyte ghosts (Erythrocyte Ghost, EGs), also known as ghosts. Ghosts are a special type of aged red blood cells. Because ghosts have completely lost their oxygen-carrying capacity, and the phosphatidylserine (PS) eversion on the surface of the cell membrane is particularly significant, they are aged red blood cells that are preferentially recognized and captured by the liver and spleen. Accordingly, the present technical invention intends to process normal red blood cells through artificial intervention to make them into ghosts, thereby obtaining quality-controlled aged red blood cells on a large scale.

[0006] The cell membrane of normal red blood cells has strong fluidity and softness, and can resist the influence of high shear force by deformation and other means. Compared with normal red blood cells, the cell membrane of blood ghosts becomes thinner and more brittle. When encountering high shear force, the cell membrane of blood ghosts will be forced to open temporarily. Microfluidics (MF) is a new technology that can accurately control and manipulate liquid flow within the micron size. Based on microfluidic technology, the microparticle controller can squeeze the blood ghost in a high-speed physical way through the microfluidic chip to temporarily open the cell membrane, and diffuse the drug into the blood ghost before the cell membrane of the blood ghost closes again, thereby completing the drug loading process. Compared with the osmotic pressure regulation drug loading method, the microparticle control drug loading method is simple to operate, less time-consuming, and more efficient, and is more suitable for drug loading of aging red blood cells such as blood ghosts.

[0007] In previous studies, the applicant conducted in-depth exploration on the topic of using aged erythrocyte membranes as carriers to carry dithiothreitol to treat liver chromium poisoning ("Aging erythrocyte membranes as biomimeticnanometer carriers of liver-targeting chromium poisoning treatment. Drug Delivery. 2021, 28 (1): 1455-1465. IF6.8"). This study successfully confirmed the potential and feasibility of using aged erythrocytes as a liver and spleen targeted delivery platform. However, some urgent problems were exposed during the design and implementation process: First, the naturally aged erythrocytes separated from the blood have inconsistent aging degrees and limited numbers, which makes it difficult to meet the needs of drug delivery platforms. Therefore, it is urgent to solve the problem of the source of aged erythrocytes and the unification of their quality standards. Secondly, compared with normal erythrocytes, aged erythrocytes have undergone significant changes in morphology, specific gravity, and cell membranes. This results in the osmotic pressure gradient drug loading method originally suitable for normal erythrocytes no longer being suitable for aged erythrocytes. Therefore, it is necessary to develop new drug loading technologies to adapt to the characteristics of aged erythrocytes. The implementation scheme of the existing technology is as follows: Figure 1 shown.

[0008] Liver-targeted drug delivery is a field that has accumulated a considerable research foundation. According to the different targeting mechanisms, liver-targeted nano-drug delivery systems can be divided into three categories: active targeting, passive targeting, and physicochemical targeting. Active targeting systems mainly rely on the principles of "receptor-ligand binding" and "antigen-antibody binding", such as targeting through galactose receptor-mediated liposomes; passive targeting is mainly based on the size effect of nanoparticles, such as polylactic-co-glycolic acid (PLGA) nanoparticles; physicochemical targeting uses magnetic fields, ultrasound and other technologies, such as Fe3O4 magnetic nanoparticles.

[0009] Although these nanomaterial-based liver-targeted nanodrug delivery systems have made some progress in achieving liver-targeted delivery, they still have some limitations, including low drug loading, complex preparation processes due to chemical modification, and the safety of long-term use needs to be further verified. Due to these challenges, most of these systems are still in the conceptual research stage and have not yet been widely used in clinical practice. Future research needs to address these key issues to promote the development and application of liver-targeted nanodrug delivery systems.

[0010] Currently, encapsulating drugs in aged red blood cell membranes to achieve targeted liver treatment usually relies on chemical reagents such as hydrogen peroxide to accelerate the aging process of red blood cells. However, long-term exposure to hydrogen peroxide may increase an individual's risk of cancer, cardiovascular disease, and neurodegenerative diseases, and adversely affect the function of the immune system. The present invention proposes an innovative method that abandons the use of chemical reagents, effectively reduces the harm that the preparation may cause to the human body, and provides a gentler solution for safe drug delivery.

[0011] The traditional hypotonic method is to load drugs into red blood cells, which has the problems of limited drug loading and low targeting. However, loading drugs into blood ghosts through microfluidic technology can not only achieve a better red blood cell aging effect, but also significantly enhance the targeting to the liver and greatly increase the drug loading. Summary of the invention

[0012] The purpose of the present invention is to provide a liver-targeted blood ghost preparation and its preparation method and application. Aiming at the actual demand for treating the rare disease - multiple sclerosis, a universal platform for liver-directed drug delivery using artificial blood ghosts is designed by utilizing the physiological law of liver clearance of blood ghosts.

[0013] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0014] The present invention provides a method for preparing a liver-targeted blood ghost preparation, comprising the following steps:

[0015] (1) Centrifuging the red blood cell suspension to obtain a red blood cell pellet;

[0016] (2) mixing the red blood cell precipitate and the NaCl solution, allowing the mixture to stand and then centrifuging to obtain a precipitate;

[0017] (3) mixing the precipitate with a hypertonic solution, incubating and centrifuging to obtain a ghost precipitate, and resuspending to obtain a ghost cell suspension;

[0018] (4) MOG 35-55 Mix with saline to obtain MOG 35-55 Solution;

[0019] (5) Mix the ghost cell suspension and MOG 35-55 The solutions were mixed to obtain the ghost preparation GHOST-MOG. 35-55 .

[0020] Preferably, the red blood cell suspension in step (1) is a mouse red blood cell suspension, which is obtained by mixing a 0.8-1% NaCl solution with mouse red blood cells;

[0021] The centrifugation conditions in step (1) are 1200-1400×g, 2-6° C., and the centrifugation time is 4-6 min.

[0022] Preferably, in step (2), the volume ratio of the red blood cell precipitate to the NaCl solution is 1:2-10, and the concentration of the NaCl solution is 0.10-0.50%;

[0023] Preferably, the standing time in step (2) is 2 to 8 minutes, the centrifugation conditions are 4000 to 8000×g, 2 to 6° C., and the centrifugation time is 8 to 12 minutes.

[0024] Preferably, the hypertonic solution in step (3) is an 8-10% NaCl solution, the amount of the hypertonic solution is 8-12 times the volume of the red blood cell sediment, the incubation time is 10-60 min, the centrifugation conditions are 4000-8000×g, 2-6°C, and the centrifugation time is 8-12 min;

[0025] During the resuspending in step (3), physiological saline or red blood cell preservation solution is used to blow and evenly distribute the blood ghost precipitate.

[0026] Preferably, the MOG in step (4) 35-55 MOG in solution 35-55 The concentration is 20-200 μg / mL.

[0027] Preferably, in step (5), the ghost cell suspension and MOG are mixed 35-55 The solutions are transported and mixed by pumps respectively, and the flow rate during transport is 0.2-0.8 mL / min. The blood ghost cell suspension and MOG 35-55 The volume ratio of the solution is 1:0.5~1.5.

[0028] The present invention also provides a blood ghost preparation prepared by the preparation method.

[0029] The present invention also provides the use of the blood ghost preparation in preparing a carrier for liver-targeted drug delivery.

[0030] The present invention provides a liver-targeted ghost preparation and a preparation method and application thereof, comprising the following steps: centrifuging a red blood cell suspension and mixing it with a NaCl solution, centrifuging it and mixing it with a hypertonic solution, incubating it and centrifuging it, and resuspending it to obtain a ghost cell suspension; mixing the ghost cell suspension with MOG 35-55The solutions are mixed to obtain a blood ghost preparation. The present invention abandons the use of chemical reagents, effectively reduces the harm that the preparation may cause to the human body, and provides a more gentle solution for safe drug delivery. The prepared product has a high drug loading, a simple preparation process, and does not require a variety of chemical reagents, and has good safety. This can effectively solve the problems of low drug loading, complex preparation process due to chemical modification, and unclear long-term use safety.

[0031] Beneficial effects:

[0032] The ghost preparation prepared by the present invention, as a carrier for liver-targeted drug delivery, can achieve rapid liver-targeted drug delivery and increase drug loading. This strategy utilizes the natural characteristics of ghost cells and, through specific processing and optimization, enhances the ability to deliver drugs directly to the liver, thereby improving the therapeutic effect while reducing potential effects on other organs. In addition, the ability to load high drug loads means that therapeutic effects can be achieved at lower doses, which may help reduce the number of dosing times, improve patient compliance, and provide a new strategy for targeting the liver. Therefore, EGs containing autoantigens represent a potential effective strategy that not only has good biocompatibility, but is also independent of drugs and has no toxic side effects. Our research results provide proof of concept for EGs-derived bionanosystems, confirming their use as a potential clinical approach to reduce the burden of central nervous system diseases by regulating peripheral immune cells and implementing antigen-specific targeted delivery strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 A flow chart for the preparation of existing hypotonic drug-loaded ghost cells;

[0034] Figure 2 It is the overall technical solution process and hardware connection schematic diagram of the present invention;

[0035] Figure 3 A flow chart of the microfluidic method for preparing blood ghost cells loaded with drugs according to the present invention;

[0036] Figure 4 This is the PS eversion degree diagram of each group;

[0037] Figure 5 It is a bar graph of the percentage of PS eversion in each group;

[0038] Figure 6 The fluorescence distribution of different drugs in animals;

[0039] Figure 7 The fluorescence distribution of different drugs in various organs;

[0040] Figure 8 For RBC and GHOST-MOG 35-55Pharmacokinetics. DETAILED DESCRIPTION

[0041] The present invention provides a method for preparing a liver-targeted blood ghost preparation, comprising the following steps:

[0042] (1) Centrifuging the red blood cell suspension to obtain a red blood cell pellet;

[0043] (2) mixing the red blood cell precipitate and the NaCl solution, allowing the mixture to stand and then centrifuging to obtain a precipitate;

[0044] (3) mixing the precipitate with a hypertonic solution, incubating and centrifuging to obtain a ghost precipitate, and resuspending to obtain a ghost cell suspension;

[0045] (4) MOG 35-55 Mix with saline to obtain MOG 35-55 Solution;

[0046] (5) Mix the ghost cell suspension and MOG 35-55 The solutions were mixed to obtain the ghost preparation GHOST-MOG. 35-55 .

[0047] In the present invention, the red blood cell suspension in step (1) is a mouse red blood cell suspension, and the mouse red blood cell suspension is preferably obtained by mixing a 0.8-1% NaCl solution with mouse red blood cells, and more preferably a 0.9% NaCl solution;

[0048] The centrifugation conditions in step (1) are preferably 1200-1400×g, 2-6°C, more preferably 1300×g, 4°C, and the centrifugation time is preferably 4-6 min, more preferably 5 min.

[0049] In the present invention, the volume ratio of the red blood cell precipitate to the NaCl solution in step (2) is preferably 1:2-10, more preferably 1:5-6, and the concentration of the NaCl solution is preferably 0.10-0.50%, more preferably 0.20-0.30%;

[0050] The standing time in step (2) is preferably 4 to 8 minutes, more preferably 6 minutes. The centrifugation conditions are preferably 4000 to 8000×g, 2 to 6°C, more preferably 6000×g, 4°C. The centrifugation time is preferably 8 to 12 minutes, more preferably 10 minutes.

[0051] In the present invention, the hypertonic solution in step (3) is an 8-10% NaCl solution, the amount of the hypertonic solution is preferably 8-12 times the volume of the red blood cell sediment, more preferably 10 times, the incubation time is preferably 10-60 min, more preferably 30-40 min, the centrifugation conditions are preferably 4000-8000×g, 2-6°C, more preferably 6000×g, 4°C, the centrifugation time is preferably 8-12 min, more preferably 10 min;

[0052] During the resuspending in step (3), physiological saline or red blood cell preservation solution is used to blow and evenly distribute the blood ghost precipitate.

[0053] In the present invention, the MOG in step (4) 35-55 MOG in solution 35-55 The concentration is preferably 20 to 200 μg / mL, more preferably 50 μg / mL.

[0054] In the present invention, the ghost cell suspension and MOG are mixed in step (5). 35-55 The solutions are transported and mixed by pumps respectively, and the flow rate during the transport is preferably 0.5-0.7 mL / min, and more preferably 0.6 mL / min. The ghost cell suspension and MOG 35-55 The volume ratio of the solution is preferably 1:0.5 to 1.5, more preferably 1:1.

[0055] In the present invention, the mixing in step (5) is preferably performed using a microfluidic device, wherein a pump is used in the microfluidic device to provide thrust to mix the liquids evenly.

[0056] Principle of drug loading of ghost cells by microfluidics: Microfluidic devices have parallel channels, which are composed of constrictions smaller than the cell diameter. Ghost cells pass through narrow constrictions, causing temporary damage to the cell membrane, allowing drugs to passively diffuse from the surrounding buffer into the cell cytoplasm, and then the physiological properties of red blood cells reseal the cell membrane, and the drug is loaded into the ghost cells.

[0057] The present invention also provides a blood ghost preparation prepared by the preparation method.

[0058] The present invention also provides the use of the blood ghost preparation in preparing a carrier for liver-targeted drug delivery.

[0059] In the present invention, the abbreviations and key terms involved are defined as follows:

[0060] Erythrosomes Ghosts (EGs) are hollow cell membrane structures formed after artificial treatment of red blood cells to rupture the plasma membrane and remove hemoglobin and other intracellular soluble proteins in the red blood cells. They retain the morphology of red blood cells and some surface markers and are relatively pure cell membrane structures.

[0061] MOG 35-55 :It is a specific peptide segment of myelin oligodendrocyte glycoprotein (MOG), consisting of amino acid residues 35 to 55.

[0062] Multiple Sclerosis (MS) is a chronic disease that affects the central nervous system. It is characterized by the immune system mistakenly attacking nerve cells in the brain and spinal cord, causing damage to the myelin sheath of nerve fibers and the formation of scattered plaques or sclerosis.

[0063] Experimental Allergic Encephalomyelitis (EAE) model: An animal model used to simulate human multiple sclerosis (MS) by stimulating an immune response to myelin components of the central nervous system (CNS) in animals.

[0064] Kupffer cells: also known as Kupffer cells (KC), are a special type of macrophage located in the liver and are part of the mononuclear phagocyte system. They are mainly distributed in the hepatic sinusoidal cavity, have irregular shapes, and have strong phagocytic functions, which can remove foreign matter, microorganisms, antigen-antibody complexes, and aging blood cells in the blood.

[0065] Liver targeting: refers to technologies or methods specifically designed to deliver drugs, therapeutic molecules or diagnostic agents to the liver.

[0066] Immune tolerance: refers to a state in which the body does not produce an immune response to a specific antigen.

[0067] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0068] Example 1

[0069] 1. Preparation method of blood ghost cells: Take 1.67mL of 6% mouse red blood cell suspension, centrifuge at 1300×g and 4°C for 5min, and collect the obtained 100μL red blood cell precipitate. Add 500μL of 0.27% NaCl solution (equivalent to 5 times the volume of the red blood cell precipitate) to the precipitate, and allow the red blood cells to swell in the solution for 6min. Subsequently, centrifuge at 6000×g and 4°C for 10min, remove the supernatant, and incubate the precipitate with 1mL of hypertonic solution for 30 minutes. Centrifuge again at 6000×g and 4°C for 10min, and discard the supernatant. Finally, centrifuge the blood ghost precipitate at 6000×g and 4°C for 10min, and wash three times with 400μL of normal saline to obtain pure blood ghost cells.

[0070] 2. MOG 35-55 Solution preparation: 5 mg MOG 35-55 Dissolve in 1 mL of pre-cooled saline to prepare a stock solution. Take 100 μL of the stock solution and dilute it to 10 mL with pre-cooled saline to prepare a MOG solution with a concentration of 50 μg / mL. 35-55 Solution.

[0071] 3. GHOST-MOG 35-55 Preparation of the group: Build a microfluidic device, adjust the flow rate of the two pumps to 0.6 mL / min, and add 200 μL of blood ghost cell suspension and 200 mL of MOG 35-55 The solution was delivered by a pump. Through this precise microfluidic operation, GHOST-MOG was successfully prepared. 35-55 Group.

[0072] Experimental Example 1: Phosphatidylserine (PS) Exposure Rate Experiment on the Surface of Drug-loaded Ghost Cell Membrane

[0073] Experimental methods: This study set up several experimental groups, namely RBC group, RBC-MOG 35-55 Group, GHOST Group, GHOST-WATER Group and GHOST-MOG 35-55 group. Count the cells using a hematology analyzer and accurately obtain 5×10^5 cells. Subsequently, centrifuge at 6000×g for 5 minutes at 4°C and remove the supernatant. Next, add 500μL of binding solution and gently resuspend the cells. Add 5μL of AnnexinV-FITC reagent to each sample, mix thoroughly, and incubate at room temperature in the dark for 25 minutes. Finally, use a flow cytometer to detect the samples.

[0074] Analysis of test results:

[0075] Phosphatidylserine (PS) is located on the inner side of the red blood cell membrane under normal conditions. In the early stages of apoptosis, PS can flip from the inner side of the cell membrane to the surface of the cell membrane. Annexin V, a Ca^2+-dependent phospholipid binding protein with a molecular weight of 35-36 kDa, can bind to PS with high affinity. In the experiment, we combined Annexin V with fluorescein isothiocyanate (FITC) and observed the externalization of PS by flow cytometry as an indicator of the state of apoptosis. Experimental results Figure 4 and Figure 5 Shows that the order of PS eversion is GHOST-MOG 35-55 The GHOST-WATER group and the GHOST-WATER group were comparable, and both were higher than the GHOST group, while the GHOST group was significantly higher than the RBC-MOG 35-55 This result shows that the red blood cells treated by hypotonic method and microfluidic method have the highest degree of senescence, among which GHOST-MOG 35-55 The erythrocytes in the GHOST-WATER group and the GHOST-WATER group showed more significant PS eversion, which may be related to their processing during the preparation process, thus affecting the integrity and function of the erythrocyte membrane. More accurately evaluate the effects of different processing methods on the aging state of erythrocytes.

[0076] Test Example 2 Liver targeting assay

[0077] Thirty ICR mice were randomly divided into five groups, with six mice in each group, namely, saline group, DID-RBC group, DID-RBC-MOG group, and 35-55 GROUP AND DID-GHOST GROUP AND DID-GHOST-MOG 35-55 group. 200 μl of each drug was injected into the tail vein. 4 h after administration, the mice were anesthetized with 3% isoflurane, and the whole body of each group of mice was scanned with a living imaging instrument to analyze the fluorescent parts. Three mice in each group were killed at 4 h, and the heart, spleen, kidney and brain tissue samples were taken after the mice were killed by dislocating the neck. The blood on the surface of the tissues and organs was washed with saline and the surface moisture was absorbed with filter paper, and the same method was used for in vitro imaging analysis. Using living image software, the bioluminescence and fluorescence signals were quantified.

[0078] Analysis of test results:

[0079] The experimental results are as follows Figure 6 and Figure 7 As shown in the figure, the results of the in vivo imaging experiment showed that in the PBS control group, no fluorescent signals were detected in the mouse organs, which confirmed that the fluorescence emitted by the mouse organs themselves did not cause significant interference in the experiment. 35-55The group showed a very strong fluorescence signal in the liver. This result clearly showed that GHOST-MOG 35-55 It has significant liver targeting.

[0080] Test Example 3 Carrier Pharmacokinetics Determination

[0081] Evaluation of RBC and GHOST-MOG by fluorescence labeling 35-55 The model mice were randomly divided into 2 groups, 6 in each group, and DiD-labeled RBC and GHOST-MOG were injected into the tail vein respectively. 35-55 30 μL of blood was collected from the orbit at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h after injection. The collected blood samples were placed in a 96-well plate and diluted with 70 μL PBS for fluorescence measurement using an enzyme-labeled instrument.

[0082] Analysis of test results:

[0083] Experimental Results Figure 8 and Table 1 shows the pharmacokinetic evaluation of RBC and GHOST-MOG 35-55 Dynamic changes in animals. Through these data, we calculated the half-life of the drug and the average residence time of the drug in the body. 35-55 The elimination half-life of GHOST-MOG was 9.3 hours, which was significantly shorter than that of erythrocytes (40.65 hours). 35-55 After entering the blood circulation, it can achieve targeted delivery quickly. The rapid targeting ability means that GHOST-MOG 35-55 It can reach the site of action more effectively in a shorter time, which is GHOST-MOG 35-55 Provides strong evidence as an effective liver-targeted drug delivery system.

[0084] Table 1 RBC and GHOST-MOG 35-55 Vector half-life and average residence time of vector in the body

[0085] RBC Ghost <![CDATA[T 1 / 2 b / h]]> 40.653±4.935 9.359±1.139 <![CDATA[MRT (0-∞) / h]]> 41.332±9.768 21.622±7.75

[0086] As can be seen from the above embodiments, the present invention provides a liver-targeted ghost preparation and its preparation method and application, comprising the following steps: centrifuging a red blood cell suspension and mixing it with a NaCl solution, centrifuging it and mixing it with a hypertonic solution, incubating it and centrifuging it, and resuspending it to obtain a ghost cell suspension; mixing the ghost cell suspension with MOG 35-55The solutions are mixed to obtain a blood ghost preparation. The present invention abandons the use of chemical reagents, effectively reduces the harm that the preparation may cause to the human body, and provides a more gentle solution for safe drug delivery. The prepared product has a high drug loading, a simple preparation process, and does not require a variety of chemical reagents, and has good safety. This can effectively solve the problems of low drug loading, complex preparation process due to chemical modification, and unclear long-term use safety.

[0087] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a liver-targeted blood ghost preparation, characterized in that: The steps include: (1) Centrifuging the red blood cell suspension to obtain a red blood cell pellet; (2) mixing the red blood cell precipitate and the NaCl solution, allowing the mixture to stand and then centrifuging to obtain a precipitate; (3) mixing the precipitate with a hypertonic solution, incubating and centrifuging to obtain a ghost precipitate, and resuspending to obtain a ghost cell suspension; (4) MOG 35-55 Mix with saline to obtain MOG 35-55 Solution; (5) Mix the ghost cell suspension and MOG 35-55 The solutions were mixed to obtain the ghost preparation GHOST-MOG. 35-55 .

2. The preparation method according to claim 1, characterized in that: The red blood cell suspension described in step (1) is a mouse red blood cell suspension, which is obtained by mixing a 0.8-1% NaCl solution with mouse red blood cells; The centrifugation conditions in step (1) are 1200-1400×g, 2-6° C., and the centrifugation time is 4-6 min.

3. The preparation method according to claim 2, characterized in that: In step (2), the volume ratio of the red blood cell precipitate to the NaCl solution is 1:2-10, and the concentration of the NaCl solution is 0.10-0.50%.

4. The preparation method according to claim 2, characterized in that: The standing time in step (2) is 2 to 8 minutes, the centrifugal conditions are 4000 to 8000×g, 2 to 6° C., and the centrifugal time is 8 to 12 minutes.

5. The preparation method according to claim 3, characterized in that: The hypertonic solution in step (3) is an 8-10% NaCl solution, the amount of the hypertonic solution is 8-12 times the volume of the red blood cell sediment, the incubation time is 10-60 min, the centrifugation conditions are 4000-8000×g, 2-6°C, and the centrifugation time is 8-12 min; During the resuspending in step (3), physiological saline or red blood cell preservation solution is used to blow and evenly distribute the blood ghost precipitate.

6. The preparation method according to claim 4, characterized in that: MOG described in step (4) 35-55 MOG in solution 35-55 The concentration is 20-200 μg / mL.

7. The preparation method according to any one of claims 1 to 6, characterized in that: When mixing in step (5), the ghost cell suspension and MOG 35-55 The solutions are transported and mixed by pumps respectively, and the flow rate during transport is 0.2-0.8 mL / min. The blood ghost cell suspension and MOG 35-55 The volume ratio of the solution is 1:0.5~1.

5.

8. The blood ghost preparation obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the blood ghost preparation according to claim 8 in preparing a carrier for liver-targeted drug delivery.

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