CN115627261A

CN115627261A - Double-drug-loading erythrocyte carrier and preparation method and application thereof

Info

Publication number: CN115627261A
Application number: CN202210785677.5A
Authority: CN
Inventors: 赵押金; 梁鲁彪; 喻永林; 徐高强; 王苑茹
Original assignee: Oral Hospital Affiliated To Zunyi Medical University
Current assignee: Oral Hospital Affiliated To Zunyi Medical University
Priority date: 2022-07-04
Filing date: 2022-07-04
Publication date: 2023-01-20

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a double-drug-loading erythrocyte carrier and a preparation method and application thereof. The invention uses the erythrocyte lift technology to improve the encapsulation rate of the drug-loaded erythrocytes and the recovery rate of the erythrocytes, and solves the problems of serious liver and spleen ingestion, poor in-vivo targeting property and the like of nano-carrier drugs.

Description

Double-drug-loading erythrocyte carrier and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a double-drug-loading erythrocyte carrier, and a preparation method and application thereof.

Background

Red Blood Cells (RBCs) are a unique natural drug carrier. Since the last 70 th century, erythrocytes showed many advantageous properties in terms of biodegradation, release profile, were studied as drug delivery vehicles for nearly half a century and have successfully entrapped various molecules including antibodies/enzymes, polypeptides, nucleic acids, nanoparticles and chemical drugs, some of which have been successfully converted to human clinical trials, such as L-asparaginase, dexamethasone, and the like. However, the current use of erythrocytes as carriers is mostly limited to carrying a single molecule by entrapment in erythrocytes or by coupling to the surface of the erythrocyte membrane.

Paclitaxel (PTX), a diterpenoid taxane, is commonly used clinically in the treatment of various neoplastic diseases. In recent years, however, studies have found that it can significantly increase the rate of neuronal differentiation in the treatment of central nervous system diseases. Cetuximab (Cetuximab, cet) is a monoclonal antibody of chimeric immunoglobulin 1, and the molecular target is Epidermal Growth Factor Receptor (EGFR). The study on neurons shows that the EGFR antibody can remarkably reverse the adverse effect of inhibitory molecules such as myelin protein and the like on the growth of nerve axons, and the binding affinity of the cetuximab and the EGFR is about 5 to 10 times that of an endogenous ligand, so that the cetuximab blocks the binding of the EGFR and the endogenous ligand, thereby inhibiting the receptor function of the EGFR.

Chinese patent with publication number CN110279672A discloses a double-drug-loading erythrocyte carrier, a preparation method and application thereof, wherein the double-drug-loading erythrocyte carrier comprises erythrocytes, first drug molecules loaded in the erythrocytes and second drug molecules coupled on the surfaces of the erythrocytes. The first drug molecule may be paclitaxel or the like and the second drug molecule may be cetuximab or the like. The double-drug-loading erythrocyte carrier simultaneously loads two therapeutic molecules, namely the first drug molecule and the second drug molecule, by utilizing the erythrocytes, on one hand, the drug resistance risk and toxic and side effects brought by the drugs are reduced and the drug administration safety is increased by the loading of the erythrocytes, and on the other hand, better therapeutic effects can be exerted aiming at different problems by the loading of two drugs with different functions. Meanwhile, the double-drug-loading erythrocyte carrier disclosed by the invention is simple in preparation process, economic and easily available in raw materials, free from dependence on complex equipment and easy for industrial production.

However, the following problems still exist:

the encapsulation efficiency and the erythrocyte recovery rate of the existing drug-loaded erythrocyte carrier are low, and the targeting property of the nanometer carrier drug in vivo is poor;

disclosure of Invention

Aiming at the defects in the prior art, the invention provides a double-drug-loading erythrocyte carrier and a preparation method and application thereof, which are used for solving the problems of low encapsulation efficiency and erythrocyte recovery rate, poor targeting property of a nano-carrier drug in vivo and the like of the traditional drug-loading erythrocyte carrier in the prior art; the operation is simple, and each step can work independently, so that the operability of the method is improved.

In order to solve the technical problems, the invention adopts the following technical scheme:

a double-drug-loading erythrocyte carrier and a preparation method and application thereof comprise erythrocyte extraction and separation and purification of erythrocytes, wherein the erythrocyte extraction is preposed and hematopoietic stem cells are collected, and the hematopoietic stem cells are collected by screening, detecting and collecting donors.

Preferably, the donor screening relative donors is not age-restricted, regardless of donor designation from 18 to 40 years. HLA-A, B, DR, mixed lymphocyte culture meeting clinical requirements, blood types of ABO and Rh, serological examination of infectious diseases and the like are required to be tested before the collection. Before collection, 1300-1600 ml of peripheral blood should be collected by the corresponding donor for use in the operation. The collection amount is preferably controlled to be 20-30 ml/kg body weight, the minimum leukocyte amount is 2xl58/kg, and 3x158/kgMl is generally collected.

Preferably, the collection process should be kept sterile, with only 9-12 puncture points; when the whole posterior iliac eminence is selected, the puncture reaches 350-450, but the suction amount at each puncture point is less than 8ml, so as to prevent the blood dilution to the maximum extent. The collected blood needs to be filtered by a filter screen of 200-300 mm. The volume collected varies depending on the weight of the recipient, but is generally 10 to 15ml/kg, based on the weight of the donor. The lowest dose of nucleated cell number was 2xl8/kg calculated as the recipient weight. Collected blood contains small particles and fat and is dangerous to cause embolism in direct infusion, after the small particles are removed by filtration in the collection process, bone marrow is centrifuged or placed at 4Y to lead the fat to be coagulated and extrude out of the fat.

Preferably, the collecting step is:

the four-limb large blood vessel with good elasticity and small injury is selected before the acquisition, and the large vein puncture is performed under the general condition, and the central vein catheterization is needed when the peripheral vein puncture is difficult;

operating a CS-3000 Plus blood cell separator in advance, wherein the in vitro blood volume is about 200-400 ml, a donor possibly with hypovolemic syndrome can be generated, and filling a separation pipeline with 300-300 ml of erythrocyte suspension before collection;

the blood volume is closely related to the collection efficiency, and the collection process needs to process at least 3 times of the circulating blood volume;

the acquisition efficiency, since the relative percentage amount of CD34+ is affected by the total number of peripheral blood leukocytes, is often difficult to accurately estimate the exact number of HSCs in the periphery. The volumetric leukocyte harvesting is a body circulation volume with the circulation blood volume being more than 3 times or not less than 15L during the collection, and is an important means for improving the yield of stem cells and reducing the collection times in recent years. For most donors, 4x106/kgo (4) can be obtained by only taking twice, and for adults, the blood flow rate is 50-70 ml/min, and the blood flow rate of children is reduced; ACD/whole blood ratio, adult ratio was controlled at 1: (9-11), the child remains at 1:13 to prevent sodium citrate morinda citrifolia from poisoning; the CD34+ cell absolute counting method by using the Pro-COUNT method can reliably predict the collection effect of the autologous peripheral stem cells.

Preferably, the separation and purification steps of the red blood cells are as follows:

blood was mixed with PBS at 1: diluting according to a proportion of 1, adding 15ml of Ficoll-Hypague into a 50ml centrifuge tube, and slowly adding 30m1 of PBS diluted blood onto the surface of the Ficoll-Hypague liquid to obviously see a layer of cells, namely a mononuclear cell layer. The mononuclear cell layer was aspirated by a pipette and transferred to another centrifuge tube, PBS was added, the supernatant was decanted by centrifugation at 1000 rpm at room temperature for 10min, the cells were resuspended in PBSA, and lOmin was centrifuged at 1000 rpm at room temperature.

And (3) a separation process: adding PBS for washing, centrifuging at room temperature of 200g for 15min, adding an appropriate amount of buffer solution for resuspending cells, selecting an appropriate separation column type according to the total number of MNCs (MNCs), putting the column into a magnetic field of a MACs (adsorption monoclonal antibody-magnetic bead separation system) separator, adding the buffer solution for rinsing the column, filtering the cells by a 40 Xm nylon filter screen, removing cell clusters, adding cell suspension into the column, washing unbound cells by the buffer solution, removing the column from the separator, putting the column into an appropriate tube, and filling the column with the buffer solution. The bound cells were washed out by applying pressure to the inner plug of the column, the CD34+ cells were washed once with PBS, centrifuged at 200g for 15min at room temperature, and the cells were resuspended in IMDM for hematopoietic stem cell culture.

Amplification culture: the cells were resuspended in cytokine-supplemented medium and CD34+ cells were seeded at a cell concentration of 2X154 cells/ml into T25 flasks. Half the liquid change twice a week. Every 108 MNCs are resuspended in buffer with a final volume of 300 awkward, and 100,11FcR blocker is added to every 108 MNCs suspension to inhibit nonspecific binding of CD34 microbeads or to non-target cells mediated by Fc receptors. Adding 100 dishes of CD34 microbeads to each 108 MNCs suspensions for cell marking, fully and uniformly mixing, and placing in a refrigerator of 6-12Y for 30min.

Preferably, the red blood cells are separated and purified and then stored in a storage step, wherein the storage step comprises two storage modes, namely liquid nitrogen freezing and-80 dragon refrigerator deep low temperature freezing, and the two storage modes are respectively as follows:

one is storing by liquid fluorine vapor at-194-100T; the other is stored in liquid nitrogen-196 dragon, DMSO is combined with HES, lecchi and the like, 10% dimethyl sulfoxide and 4% HES are used as protective agents, and a two-step cooling method is adopted for programmed cooling to store the erythrocyte carrier: cooling from 4 Dragon to-45Y at the speed of 1 Dragon/min, then cooling from-45Y to-110T at the speed of 5Y/min, transferring into liquid nitrogen for preservation, and recovering the cells with the survival rate of 81.4-94.8% and the recovery rate of CFU-GM of 66.1%.

And (4) freezing and storing at a deep low temperature by using an-80Y or-70Y refrigerator as a refrigerating source and a storage place for non-program-control cooling and freezing storage, wherein the cooling rate of the sample is controlled to be l-3T/min, and the cooling platform period is controlled to be within 4 min. The program-controlled cooling and the cooling rate of liquid nitrogen preservation are precisely controlled by a computer, so that the cells are not unstable due to the fluctuation of the cooling rate in the freezing preservation process, and the damage of the cooling to the cells is reduced; the method has the advantages that a program temperature reduction instrument is not needed in deep low-temperature freezing storage of an 80-dragon refrigerator, time and labor are saved, the operation is easy, the red blood cell carrier concentration can be used for 4X108/mlo thawing which is the reverse process of the freezing process, rapid thawing is adopted for multiple main cells, the cell thawing swelling process is shortened, and meanwhile, recrystallization and ice cube reformation can be avoided. The cryopreservation effect of the red blood cell carrier is usually determined by trypan blue dye exclusion detection, in-vitro semi-synsomal colony culture, monoclonal antibody detection and the like.

Preferably, the application of the double-drug-loaded erythrocyte carrier comprises the drug loading method that drugs are encapsulated into active erythrocytes, and the erythrocytes loaded with drug particles are injected into a patient body to crack the erythrocytes and release the drug particles, so that the disease condition control is completed. Infusion into a patient by intravenous injection or arterial cannulation; after entering blood circulation, the nanoparticles are desorbed due to shear stress or direct contact with endothelial cells, so that the problems of serious liver and spleen uptake, poor in-vivo targeting property and the like of a nano-carrier medicament are effectively solved, the organ targeting capability of the nano-carrier medicament can be greatly improved by matching with a clinically used blood vessel intubation treatment mode, the treatment medicament is targeted to circulating red blood cells to prepare a medicament-antibody/polypeptide compound in vitro, then the compound is injected into the body and is connected to the circulating red blood cells in a targeted manner, the problems caused by in-vitro operation and re-infusion can be avoided to a certain extent, and the nano-carrier medicament can be used for clearing pathogens in the body. Conjugated binding of antibody-C3 b of erythrocyte surface antigen CR1 and antibody of pathogen to obtain compound-bispecific monoclonal antibody compound, which can specifically bind erythrocyte surface antigen CR1 and pathogen simultaneously; the injection of model pathogens followed by injection of the complex into experimental animals was found to be highly effective in achieving red blood cell mediated pathogen clearance.

Compared with the prior art, the invention has the following beneficial effects:

the double-drug-loading erythrocyte carrier has the advantages of long circulation period, easiness in obtaining, large specific surface area and volume, high biocompatibility and safe elimination mechanism, the encapsulation rate reaches 58.36%, the recovery rate of the erythrocyte reaches 84.94%, the preparation method is suitable for targeting drugs to the 1 st capillary vessel enrichment organ at the downstream of injection, the method can effectively improve the problems of serious liver and spleen uptake, poor in-vivo targeting property and the like of nano-carrier drugs, and the organ targeting capability of the nano-carrier drugs can be greatly improved by matching with a clinically used blood vessel intubation treatment mode.

Drawings

FIG. 1 is a line drawing of stability of a nanocarrier in an embodiment of a method for preparing a dual drug-loaded erythrocyte carrier and a use thereof of the invention;

FIG. 2 is a histogram of erythrocyte membranes and volume in an embodiment of a dual drug-loaded erythrocyte carrier of the invention, a method for preparing the same, and an application thereof;

FIG. 3 is a diagram of drug-loaded red blood cells in a method for preparing the dual drug-loaded red blood cell carrier and an application example thereof;

Detailed Description

In order that those skilled in the art can better understand the present invention, the following technical solutions are further described with reference to the accompanying drawings and examples. It should be noted that all directional indicators (such as upper, lower, left, right, front, and rear … …) in the embodiment of the present invention are only used to explain the relative position relationship between the components, the movement situation, and the like in a specific posture (as shown in the drawing), and if the specific posture is changed, the directional indicator is changed accordingly.

In addition, the descriptions related to "first", "second", etc. in the present invention are for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention.

Examples

As shown in figures 1-3, a double-drug loading erythrocyte carrier and its preparation method and application include erythrocyte extraction and erythrocyte separation and purification, erythrocyte extraction pre-stage also includes hematopoietic stem cell collection, which includes donor screening detection and collection.

Donor screening the parental donors is not age-restricted, and no donor is specified for 18-40 years. To explain further, HLA-A, B, DR, mixed lymphocyte culture meeting clinical requirements, blood types of ABO and Rh, serological tests of infectious diseases and the like are necessary for fully understanding the history of donors before collection. Before collection, 1300-1600 ml of peripheral blood should be collected by the corresponding donor for use in the operation. The collection amount is preferably controlled to be 20-30 ml/kg body weight, the minimum leukocyte amount is 2xl58/kg, and 3x158/kgMl is generally collected.

The collection process should be kept sterile, and only 9-12 puncture points are needed; when the whole posterior iliac eminence is selected, the puncture reaches 350-450, but the suction amount at each puncture point is less than 8ml, so as to prevent the blood dilution to the maximum extent. Further, the collected blood needs to be filtered by a 200-300 mm filter screen. The volume collected varies depending on the weight of the recipient, but is generally 10 to 15ml/kg, based on the weight of the donor. The lowest dose of nucleated cell count was 2xl8/kg calculated on the weight of the recipient. Collected blood contains small particles and fat and is dangerous to cause embolism in direct infusion, after the small particles are removed by filtration in the collection process, bone marrow is centrifuged or placed at 4Y to lead the fat to be coagulated and extrude out of the fat.

The collection steps are as follows:

the blood volume to be processed is closely related to the collection efficiency, and the collection process needs to process at least 3 times of the circulating blood volume;

the acquisition efficiency, since the relative percentage amount of CD34+ is affected by the total number of peripheral blood leukocytes, is often difficult to accurately estimate the exact number of HSCs in the periphery. Further, the volume leukocyte sampling is a body circulation volume of which the circulation blood volume is more than 3 times or not less than 15L during collection, and is an important means for improving the yield of stem cells and reducing the collection times in recent years. For most donors, 4x106/kgo (4) can be obtained by adopting two times, and for adults, the blood flow rate is 50-70 ml/min, and the blood flow rate of children is reduced; ACD/whole blood ratio, adult ratio was controlled at 1: (9-11), the child remains at 1:13 to prevent sodium citrate Morinda citrifolia from poisoning; the CD34+ cells are absolutely counted by using a Pro-COUNT method, so that the collection effect of autologous peripheral stem cells can be reliably predicted.

The separation and purification steps of the red blood cells are as follows:

Amplification culture: the cells were resuspended in cytokine-supplemented medium and CD34+ cells were seeded at a cell concentration of 2X154 cells/ml into T25 flasks. Half the liquid change twice a week. Further, every 108 MNCs were resuspended in a final volume of 300 awkward buffer and 100,11fcr blocker was added to each 108 MNCs suspension to inhibit nonspecific binding of CD34 microbeads or non-target cells mediated by Fc receptors. Adding 100 dishes of CD34 microbeads to each 108 MNCs suspensions for cell marking, fully and uniformly mixing, and placing in a refrigerator of 6-12Y for 30min.

The method comprises a storage step after separation and purification of the erythrocytes, wherein the storage step comprises liquid nitrogen freezing and deep low-temperature freezing in a-80-dragon refrigerator, and the two storage modes respectively comprise:

And (4) freezing and storing at a deep low temperature by using an-80Y or-70Y refrigerator as a refrigerating source and a storage place for non-program-control cooling and freezing storage, wherein the cooling rate of the sample is controlled to be l-3T/min, and the cooling platform period is controlled to be within 4 min. Further, the programmed cooling and the cooling rate of liquid nitrogen preservation are precisely controlled by a computer, so that the cells are not unstable due to fluctuation of the cooling rate in the freezing and storing process, and the damage of the cooling to the cells is reduced; the freezing storage of the deep low temperature in the 80-dragon refrigerator does not need a program cooling instrument, time and labor are saved, the operation is easy, the red blood cell carrier concentration can be used for 4X108/mlo thawing, the reverse process of the freezing process is realized, the rapid thawing is adopted for a plurality of main sheets, the cell thawing swelling process is shortened, and meanwhile, the recrystallization and the ice cube reformation can be avoided. The cryopreservation effect of the red blood cell carrier is usually determined by trypan blue dye exclusion detection, in-vitro semi-synsomal colony culture, monoclonal antibody detection and the like.

The application of the double-drug-loading erythrocyte carrier comprises the steps of encapsulating the drug into the active erythrocyte, injecting the erythrocyte loaded with drug particles into a patient body, cracking the erythrocyte to release the drug particles, and completing the disease control. Further, the infusion is performed to the patient by intravenous injection or arterial vessel intubation; after entering blood circulation, the nanoparticles are desorbed due to shear stress or direct contact with endothelial cells, so that the problems of serious liver and spleen uptake, poor in-vivo targeting property and the like of a nano-carrier medicament are effectively solved, the organ targeting capability of the nano-carrier medicament can be greatly improved by matching with a clinically used blood vessel intubation treatment mode, the treatment medicament is targeted to circulating red blood cells to prepare a medicament-antibody/polypeptide compound in vitro, then the compound is injected into the body and is connected to the circulating red blood cells in a targeted manner, the problems caused by in-vitro operation and re-infusion can be avoided to a certain extent, and the nano-carrier medicament can be used for clearing pathogens in the body. Conjugated binding of antibody-C3 b of erythrocyte surface antigen CR1 and antibody of pathogen to obtain compound-bispecific monoclonal antibody compound, which can specifically bind erythrocyte surface antigen CR1 and pathogen simultaneously; the injection of model pathogens followed by injection of the complex into experimental animals was found to be highly effective in achieving red blood cell mediated pathogen clearance.

The advantages of this embodiment are: the red blood cells have the advantages of long circulation period, easy obtainment, larger specific surface area and volume, high biocompatibility and safe elimination mechanism, the encapsulation rate reaches 58.36 percent, the recovery rate of the red blood cells reaches 84.94 percent, and the preparation method is suitable for targeting the medicament to the capillary enrichment organ at the 1 st position downstream of injection. The method can effectively improve the problems of serious liver and spleen ingestion, poor in-vivo targeting property and the like of the nano-carrier medicament, and can greatly improve the organ targeting capability of the nano-carrier medicament by matching with a clinically used blood vessel intubation treatment mode.

The foregoing is merely an example of the present invention, and common general knowledge in the field of known specific structures and characteristics is not described herein in any greater extent than that known in the art at the filing date or prior to the priority date of the application, so that those skilled in the art can now appreciate that all of the above-described techniques in this field and have the ability to apply routine experimentation before this date can be combined with one or more of the present teachings to complete and implement the present invention, and that certain typical known structures or known methods do not pose any impediments to the implementation of the present invention by those skilled in the art. It should be noted that, for those skilled in the art, without departing from the structure of the present invention, several variations and modifications can be made, which should also be considered as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the utility of the patent.

Claims

1. A double-drug-loading erythrocyte carrier and a preparation method and application thereof are characterized in that: the method comprises the steps of erythrocyte extraction and separation and purification of erythrocytes, wherein the erythrocyte extraction is preceded by the collection of hematopoietic stem cells, and the collection of the hematopoietic stem cells comprises the screening detection and the collection of donors.

2. The double-drug-loaded erythrocyte carrier of claim 1, which is prepared by the method and the application thereof, and is characterized in that: the donor screening relatives donors is not age limited and is specified to be 18-40 years of age regardless of the donor.

3. The double-drug-loaded erythrocyte carrier of claim 1, which is prepared by the method and the application thereof, and is characterized in that: the collection process is kept sterile, and only 9-12 puncture points are needed; when the whole posterior iliac eminence is selected, the puncture reaches 350-450, but the suction amount at each puncture point is less than 8ml, so as to prevent the blood dilution to the maximum extent.

4. The double-drug-loaded erythrocyte carrier of claim 1, which is prepared by the method and the application thereof, and is characterized in that: the acquisition steps are as follows:

the four-limb large blood vessel with good vessel elasticity and small injury is selected before the acquisition set, and generally, large descending vein puncture is performed, and a central vein is required to be placed when peripheral vein puncture is difficult;

the acquisition efficiency, since the relative percentage amount of CD34+ is affected by the total number of peripheral blood leukocytes, is often difficult to accurately estimate the exact number of HSCs in the periphery.

5. The double-drug-loaded erythrocyte carrier of claim 1, which is prepared by the method and the application thereof, and is characterized in that: the separation and purification steps of the red blood cells are as follows:

blood was mixed with PBS at 1:1 proportion dilution, 15ml of Ficoll-Hypague is added into a 50ml centrifuge tube, and 30m1 of PBS diluted blood is slowly added on the surface of the Ficoll-Hypague to obviously see a layer of cells, namely a mononuclear cell layer. The mononuclear cell layer was aspirated by a pipette and transferred to another centrifuge tube, PBS was added, the supernatant was decanted by centrifugation at 1000r/min at room temperature for 10min, the cells were resuspended in PBSA, and lOmin was centrifuged at 1000r/min at room temperature.

And (3) a separation process: adding PBS for washing, centrifuging for 15min at room temperature of 200g, adding an appropriate amount of buffer solution for resuspending cells, selecting an appropriate separation column type according to the total cell number of MNCs, putting the column into a magnetic field of a MACs (absorption monoclonal antibody-magnetic bead separation system) separator, adding the buffer solution for rinsing the column, filtering the cells by a nylon filter screen of 40 mm, removing cell clusters, adding cell suspension into the column, washing away unbound cells by the buffer solution, removing the column from the separator, putting the column into an appropriate tube, and filling the column with the buffer solution. The bound cells were washed out by applying pressure to the inner plug of the column, the CD34+ cells were washed once with PBS, centrifuged at 200g for 15min at room temperature, and the cells were resuspended in IMDM for hematopoietic stem cell culture.

Amplification culture: the cells were resuspended in cytokine-supplemented medium and CD34+ cells were seeded at a cell concentration of 2X154 cells/ml into T25 flasks. Half the liquid change twice a week.

6. The double-drug-loaded erythrocyte carrier of claim 1, and the preparation method and the application thereof are characterized in that: the method comprises the following steps of separating and purifying red blood cells, and then storing the red blood cells, wherein the storing step comprises two storage modes of liquid nitrogen freezing and deep low temperature freezing in a-80 dragon refrigerator, and the two storage modes respectively comprise:

one is storing by liquid fluorine vapor at-194-100T; the other is stored in liquid nitrogen-196 dragon, using DMSO together with HES, lecchi, etc., as protective agent, using 10% dimethyl sulfoxide and 4% HES, and programmed cooling by two-step cooling method to preserve erythrocyte carriers: cooling from 4 to-45Y at a rate of 1 Dragon/min, and then from-45Y to 5Y/min

And (5) transferring the cells to liquid nitrogen for storage at-110T, wherein the survival rate of the recovered cells is 81.4-94.8%, and the recovery rate of CFU-GM is 66.1%.

And (4) freezing and storing at a deep low temperature by using an-80Y or-70Y refrigerator as a refrigerating source and a storage place for non-program-control cooling and freezing storage, wherein the cooling rate of the sample is controlled to be l-3T/min, and the cooling platform period is controlled to be within 4 min.

7. The double-drug-loaded erythrocyte carrier of claim 1, which is prepared by the method and the application thereof, and is characterized in that: the application of the double-drug-loading erythrocyte carrier comprises the steps of encapsulating the drug into the active erythrocyte, injecting the erythrocyte loaded with drug particles into a patient body, cracking the erythrocyte to release the drug particles, and completing the disease control.