CN110279672B - Double-drug-loading erythrocyte carrier, preparation method and application thereof - Google Patents

Double-drug-loading erythrocyte carrier, preparation method and application thereof Download PDF

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CN110279672B
CN110279672B CN201910619067.6A CN201910619067A CN110279672B CN 110279672 B CN110279672 B CN 110279672B CN 201910619067 A CN201910619067 A CN 201910619067A CN 110279672 B CN110279672 B CN 110279672B
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戴建武
胡玲玲
陈艳艳
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Suzhou Institute of Nano Tech and Nano Bionics of CAS
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Abstract

The invention discloses a double-drug-loading erythrocyte carrier and a preparation method and application thereof. 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.

Description

Double-drug-loading erythrocyte carrier, 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 thereof.
Background
Red Blood Cells (RBCs) are a unique natural drug carrier. Since the last 70 th century, erythrocytes have shown many advantageous properties in terms of biodegradation, release profile, have been 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 transformed into 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.
At present, in the treatment of many clinical diseases, a single drug can exert limited drug effect, and the combined treatment of two or more drugs becomes an effective drug application method. The erythrocyte carries the medicine, so that the drug resistance risk and toxic and side effects caused by the medicine can be reduced, and the administration safety is improved; meanwhile, the drug carriers with two different functions are used, so that a better treatment effect is exerted aiming at different problems. Therefore, in order to meet the requirements of combination drugs in various treatments, the research on the combined loading of the two drugs by the erythrocytes is an urgent problem to be solved.
Disclosure of Invention
The invention mainly aims to provide a double-drug-loading erythrocyte carrier and a preparation method thereof, so as to overcome the defects of the prior art.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
the embodiment of the invention provides a double-drug-loading erythrocyte carrier, which comprises erythrocytes, first drug molecules loaded in the erythrocytes and second drug molecules coupled on the surfaces of the erythrocytes.
Further, the first drug molecule includes, but is not limited to, paclitaxel, wherein the first drug enters the red blood cells by hypotonic expansion.
Further, the second drug molecule includes cetuximab, but is not limited thereto, wherein the second drug molecule can be coupled to the surface of the red blood cell via an amphiphilic polymer.
Furthermore, an amphiphilic polymer is embedded in the cell membrane of the red blood cell, and the amphiphilic polymer can react with a biotinylated second drug molecule through streptavidin, so that the second drug molecule is coupled to the surface of the red blood cell.
Further, the amphiphilic polymer includes distearoylphosphatidylethanolamine-polyethylene glycol-biotin (DSPE-PEG-biotin), but is not limited thereto.
The embodiment of the invention also provides a preparation method of the double-drug-loading erythrocyte carrier, which comprises the following steps:
s1, mixing red blood cells derived from healthy adult blood with a first drug molecule solution, adding hypertonic solution for resealing, and carrying out ultrasonic treatment to obtain red blood cells carrying first drug molecules;
s2, mixing streptavidin and DSPE-PEG-biotin for reaction to obtain avidin DSPE-PEG, and then mixing the avidin DSPE-PEG with second drug molecules for reaction to obtain a conjugate of DSPE-PEG-second drug molecules;
s3, mixing the solution of the red blood cells loaded with the first drug molecules in the step S1 with the DSPE-PEG-second drug molecules in the step S2, and continuously extruding through a polycarbonate porous membrane to obtain the double-drug-loaded red blood cell carrier.
The embodiment of the invention also provides application of the double-drug-loading erythrocyte carrier in preparing an anti-tumor drug.
The embodiment of the invention also provides application of the double-drug-loading erythrocyte carrier in preparing a repairing drug for spinal cord injury.
The embodiment of the invention also provides a pharmaceutical composition which comprises the double-drug-loaded erythrocyte carrier and pharmaceutically acceptable auxiliary materials.
Compared with the prior art, the double-drug-loading erythrocyte carrier simultaneously loads two therapeutic molecules, namely the first drug molecule and the second drug molecule, by utilizing the erythrocyte, on one hand, the drug resistance risk and the toxic and side effect brought by the drug are reduced and the drug administration safety is increased by the loading of the erythrocyte, 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.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram of a process for preparing a dual drug-loaded erythrocyte carrier according to an exemplary embodiment of the invention;
FIGS. 2A-2B are photographs and TEM images of the double paclitaxel-cetuximab-loaded erythrocyte carrier of example 1 of the present invention;
FIG. 3 is the high performance liquid chromatography of the erythrocyte carrier of the double-loading paclitaxel-cetuximab of example 1 of the present invention;
FIG. 4 is a Western Blot of double paclitaxel-cetuximab-loaded erythrocyte carrier protein electrophoresis in example 1 of the present invention;
FIG. 5 is a dynamic particle size chart of the erythrocyte carrier of the double-loading paclitaxel-cetuximab in example 1 of the present invention;
FIG. 6 is the in vitro release profile of paclitaxel in the red blood cell vehicle with double-loading paclitaxel-cetuximab of example 1 of the present invention.
FIG. 7 is an immunostaining pattern showing the cell differentiation effect of the paclitaxel-cetuximab-doubly loaded erythrocyte carrier of example 1 of the present invention.
Detailed Description
In view of the defects of the prior art, the present inventors have conducted long-term research and extensive practice to propose the technical solution of the present invention, which is mainly to encapsulate a first drug molecule in a hypotonic and swelling manner, embed an amphiphilic polymer into a red blood cell membrane, and then couple a biotinylated second drug molecule to the surface of the red blood cell via streptavidin (the principle can be seen in fig. 1).
One aspect of the embodiments of the present invention provides a dual drug-loaded erythrocyte carrier, which includes erythrocytes, a first drug molecule loaded in the erythrocytes, and a second drug molecule coupled on the surface of the erythrocytes.
Further, the first drug molecule includes, but is not limited to, paclitaxel, wherein the first drug enters the red blood cells by hypotonic expansion.
Further, the second drug molecule includes cetuximab, but is not limited thereto, wherein the second drug molecule can be coupled to the surface of the red blood cell via an amphiphilic polymer.
Furthermore, an amphiphilic polymer is embedded in the cell membrane of the red blood cell, and the amphiphilic polymer can react with a biotinylated second drug molecule through streptavidin, so that the second drug molecule is coupled to the surface of the red blood cell.
Further, the amphiphilic polymer includes distearoylphosphatidylethanolamine-polyethylene glycol-biotin (DSPE-PEG-biotin), but is not limited thereto.
In another aspect of the embodiments of the present invention, a preparation method of the double drug-loaded erythrocyte carrier is also provided, which includes:
s1, mixing red blood cells derived from healthy adult blood with a first drug molecule solution, adding hypertonic solution for resealing, and carrying out ultrasonic treatment to obtain red blood cells carrying first drug molecules;
s2, mixing streptavidin and DSPE-PEG-biotin for reaction to obtain avidin DSPE-PEG, and then mixing the avidin DSPE-PEG with second drug molecules for reaction to obtain a conjugate of DSPE-PEG-second drug molecules;
s3, mixing the solution of the red blood cells loaded with the first drug molecules in the step S1 with the DSPE-PEG-Cet in the step S2, and continuously extruding through a polycarbonate porous membrane to obtain the double-drug-loaded red blood cell carrier.
In some embodiments, the method for preparing the red blood cells in step S1 is as follows: the method comprises the steps of carrying out anticoagulation on blood taken from healthy adults by heparin, centrifuging to remove upper plasma and a buffy coat, repeatedly washing residues for 3-5 times by 1XPBS buffer solution with the pH value of 7.2-7.4, then adding pre-cooled hypotonic solution into the washed residues at 0-4 ℃ for treatment for 15-45 min, then removing hemoglobin, and washing to obtain the red blood cells.
Further, the hypotonic solution is 0.25X PBS buffer.
Further, the volume ratio of the cleaned residues to the hypotonic solution is 1: 3-6.
Further, step S1 specifically includes: mixing the red blood cells with the first drug molecule solution, then adding hypertonic solution at the temperature of 25-37 ℃, resealing for 30-60 min, then carrying out ultrasonic treatment for 3-5 min, and centrifuging to remove free first drug molecules, thereby obtaining the red blood cells coated with the first drug molecules.
Furthermore, the volume ratio of the red blood cells to the first drug molecule solution is 1: 1-3.
Further, the hypertonic solution is 10X PBS buffer.
Further, step S2 specifically includes: mixing streptavidin and DSPE-PEG-biotin, reacting for 20-30 min at 0-4 ℃, dialyzing with 1XPBS buffer solution with the pH value of 7.2-7.4 to remove free streptavidin to obtain the avidin-modified DSPE-PEG, mixing with biotinylated second drug molecules, and incubating for 20-30 min at 0-4 ℃ to obtain the conjugate of the DSPE-PEG-second drug molecules.
Further, the pore size of the polycarbonate porous membrane in the step S3 is 0.2 μm, wherein the number of continuous extrusion times is 11 to 21.
In some more specific embodiments, the method for preparing the paclitaxel-cetuximab-loaded erythrocyte carrier comprises the following steps:
s1, mixing red blood cells derived from healthy adult blood with a paclitaxel solution, adding hypertonic solution for resealing, and carrying out ultrasonic treatment to obtain red blood cells carrying paclitaxel;
s2, mixing streptavidin and DSPE-PEG-biotin for reaction to obtain avidin DSPE-PEG, and then mixing the avidin DSPE-PEG with cetuximab for reaction to obtain a conjugate of DSPE-PEG-Cet;
s3, mixing the solution of the red blood cells loaded with the paclitaxel in the step S1 with the DSPE-PEG-Cet in the step S2, and continuously extruding through a polycarbonate porous membrane to obtain the double-drug-loaded red blood cell carrier.
Furthermore, the carrying amount of paclitaxel and cetuximab in each milliliter of red blood cells is respectively 0.2-400 ug and 0.5-5 ug.
The embodiment of the invention also provides application of the double-drug-loaded erythrocyte carrier in preparing anti-tumor drugs.
The embodiment of the invention also provides application of the double-drug-loading erythrocyte carrier in preparing a repairing drug for spinal cord injury.
Further, the medicine is an erythrocyte carrier medicine which simultaneously carries paclitaxel and cetuximab.
In another aspect of the embodiment of the invention, a pharmaceutical composition is also provided, which includes the double drug-loaded erythrocyte carrier and pharmaceutically acceptable auxiliary materials.
The technical solution of the present invention is further explained by the following embodiments. It is easily understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1: preparation of double-supported taxol-cetuximab erythrocyte carrier (also called double-drug-loading erythrocyte carrier or drug-loading erythrocyte)
1. Anticoagulating blood taken from healthy adults with heparin, centrifuging to remove upper plasma and buffy coat, washing with 1XPBS buffer (pH 7.3) for 4 times to obtain erythrocytes, adding precooled hypotonic solution (0.25 XPBS buffer) at 2 deg.C for 30min, centrifuging to remove hemoglobin, washing until supernatant is colorless, and collecting bottom erythrocyte precipitate, wherein the volume ratio of erythrocytes to hypotonic solution is 1: 4.
2. Mixing the erythrocyte membrane obtained in the step 1 with a paclitaxel solution according to a volume ratio of 1:2, then adding a hypertonic solution (10XPBS buffer solution) at the temperature of 30 ℃, resealing for 40min, then carrying out ultrasonic treatment for 4min, centrifuging to remove free paclitaxel to obtain paclitaxel-encapsulated erythrocytes, and storing at 4 ℃ for later use;
3. mixing streptavidin with DSPE-PEG-biotin, reacting at 4 ℃ for 25min, dialyzing with 1XPBS buffer (pH value 7.3) to remove free streptavidin to obtain avidin-modified DSPE-PEG, mixing with cetuximab, incubating at 2 ℃ for 25min, and removing free molecules to obtain DSPE-PEG-cetuximab;
4. and (3) mixing the red blood cells loaded with the paclitaxel in the step (2) with the DSPE-PEG-cetuximab in the step (3), and continuously extruding for 15 times through a polycarbonate porous membrane with the diameter of 0.2 mu m to obtain the red blood cell carrier loaded with the paclitaxel-cetuximab.
The photograph of the drug-loaded red blood cells prepared in example 1 is shown in fig. 2A, and the red blood cell suspension after drug loading is pale yellow.
The drug-loaded red blood cells of the embodiment 1 are characterized, and the transmission electron microscope image of the drug-loaded red blood cells is shown in fig. 2, the extruded drug-loaded red blood cells are uniform in size, and the particle size is about 200 nm; through the detection of high performance liquid chromatography (shown in figure 3) and protein electrophoresis Western Blot (shown in figure 4), the loading of the paclitaxel and the cetuximab by the drug-loaded red blood cells is successfully realized.
In vitro biological evaluation
1. In vitro stability of drug-loaded erythrocytes
The drug-loaded red blood cells obtained in example 1 were fixed and negatively stained with phosphotungstic acid, dried at room temperature, suspended in PBS, and stored at 4 ℃ for 10 days. The size and PDI index of the drug-loaded red blood cells were measured daily. As shown in figure 5, the particle size of the erythrocyte membrane 0-7 days after drug loading is increased from 189 +/-3.4 nm to 233.9 +/-7.2 nm at 25 ℃, and the PDI index after 7 days is 0.169 +/-0.03, which shows that the prepared drug-loaded erythrocyte has better stability. Therefore, the drug-loaded red blood cells of this example 1 have a function of stable storage in vitro.
2. In vitro release
The solution loaded with erythrocytes obtained in example 1 was dialyzed using a dialysis bag (molecular weight cut-off of 8-10kDa) with the addition of PBS containing 0.5% Tween80, a release medium with pH 7.4, and the above drug delivery system was placed in a 37 ℃ constant temperature shaker at 200 rpm. At each time point of 0.5, 1, 2, 4, 8, 12, 24, 36, 48 and 72 hours, a volume of 1mL of release medium was removed, followed by supplementation with an equivalent amount of fresh release medium, and the paclitaxel content in the external liquid was determined by high performance liquid chromatography. The in vitro release profile of paclitaxel is shown in figure 6. The result shows that the unsupported naked drug group is released by 42 +/-1.3% within the initial 4 hours, the naked drug group is completely released within 24 hours, and the erythrocyte carrier has obvious slow release effect and releases 48.61 +/-4.22% and 65.11 +/-2.62% within 24 hours and 48 hours respectively. Therefore, the drug-loaded erythrocytes of example 1 have a function of extending the half-life of the drug.
3. Cell differentiation
Neural stem cells NSCs derived from the hippocampal region of ICR mice were inoculated into a 96-well plate at a cell density of 3.5 ten thousand per well, and a differentiation medium pretreated with myelin-containing protein was added thereto at 37 ℃ with 5% CO2Culturing in a condition incubator. The following day the medium was removed and replaced with a differentiation medium containing different drug-loaded erythrocytes obtained in example 1, containing naked drug (PTX), paclitaxel-loaded erythrocytes (RBC-PTX), cetuximab-loaded erythrocytes (RBC-Cet) and double drug-loaded erythrocytes (RBC-PTX-Cet), respectively. After adding the medicine for 7 days, cell fixation and immunofluorescence staining are carried out, and the proportion of each group of neural stem cells differentiated into neurons is detected. The early stage staining results of the neuronal cells are shown in fig. 7, the paclitaxel and cetuximab molecules can promote the differentiation of the neuronal cells in the presence of myelin proteins, and compared with the neuronal differentiation ratio of 10.2 ± 1.05% of the naked paclitaxel, the coated erythrocyte has the effect of promoting the differentiation of the neuronal cells, the neuronal ratio is increased to 14.7 ± 2.9%, and compared with the single treatment of one molecule, the double-loading system can promote the differentiation ratio of the neuronal cells to be 23.1 ± 2.72%. An increase in the effect of the two therapeutic molecules is achieved.
Example 2: preparation of double-supported taxol-cetuximab erythrocyte carrier
1. Anticoagulating blood heparin taken from healthy adults, centrifuging to remove upper plasma and a buffy coat, repeatedly washing with 1XPBS buffer (pH 7.2) for 3 times to obtain erythrocytes, then adding precooled hypotonic solution (0.25 XPBS buffer) at 0 ℃ for treatment for 15min, centrifuging to remove hemoglobin, washing until supernatant is colorless, and collecting bottom erythrocyte precipitate, wherein the volume ratio of erythrocytes to hypotonic solution is 1: 2.
2. Mixing the erythrocyte membrane obtained in the step 1 with a paclitaxel solution according to a volume ratio of 1:1, then adding hypertonic solution (10XPBS buffer solution) at 25 ℃, resealing for 30min, then carrying out ultrasonic treatment for 3min, centrifuging to remove free paclitaxel to obtain paclitaxel-encapsulated erythrocytes, and storing at 4 ℃ for later use;
3. mixing streptavidin with DSPE-PEG-biotin, reacting at 0 ℃ for 20min, dialyzing with PBS buffer (pH 7.2) to remove free streptavidin to obtain avidin-modified DSPE-PEG, mixing with cetuximab, incubating at 0 ℃ for 20min, and removing free molecules to obtain DSPE-PEG-cetuximab;
4. and (3) mixing the red blood cells loaded with the paclitaxel in the step (2) with the DSPE-PEG-cetuximab in the step (3), and continuously extruding for 11 times through a polycarbonate porous membrane with the diameter of 0.2 mu m to obtain the red blood cell carrier loaded with the paclitaxel-cetuximab.
Example 3: preparation of double-supported taxol-cetuximab erythrocyte carrier
1. Anticoagulating blood heparin obtained from healthy adults, centrifuging to remove upper plasma and buffy coat, washing with 1XPBS buffer (pH 7.4) for 5 times to obtain erythrocytes, adding precooled hypotonic solution (0.25 XPBS buffer) at 4 deg.C for 45min, centrifuging to remove hemoglobin, washing until supernatant is colorless, and collecting bottom erythrocyte precipitate, wherein the volume ratio of erythrocytes to hypotonic solution is 1: 6.
2. Mixing the erythrocyte membrane obtained in the step 1 with a paclitaxel solution according to a volume ratio of 1:3, then adding a hypertonic solution (10XPBS buffer solution) at 37 ℃, resealing for 60min, then carrying out ultrasonic treatment for 5min, centrifuging to remove free paclitaxel to obtain paclitaxel-encapsulated erythrocytes, and storing at 4 ℃ for later use;
3. mixing streptavidin with DSPE-PEG-biotin, reacting at 4 ℃ for 30min, dialyzing with PBS buffer (pH 7.4) to remove free streptavidin to obtain avidin-modified DSPE-PEG, mixing with cetuximab, incubating at 4 ℃ for 30min, and removing free molecules to obtain DSPE-PEG-cetuximab;
4. and (3) mixing the red blood cells loaded with the paclitaxel in the step (2) with the DSPE-PEG-cetuximab in the step (3), and continuously extruding for 21 times through a polycarbonate porous membrane with the diameter of 0.2 mu m to obtain the red blood cell carrier loaded with the paclitaxel-cetuximab.
The drug-loaded erythrocytes obtained in examples 2 and 3 were subjected to in vitro biological evaluation in the same manner as in example 1, and similar evaluation results to those in example 1 were also obtained.
It should be understood that the above-mentioned embodiments are only for illustrating the technical concept and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the content of the present invention and to implement the present invention, and not to limit the scope of the present invention, for example, the paclitaxel can be replaced by other chemical drugs, and the cetuximab can be replaced by other biomolecules such as mab and polyclonal antibody. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims (13)

1. A double-drug-loading erythrocyte carrier is characterized by comprising erythrocytes, first drug molecules encapsulated in the erythrocytes and second drug molecules coupled on the surfaces of the erythrocytes;
the first drug molecule is selected from paclitaxel and the second drug molecule is selected from cetuximab; wherein, the first drug molecule enters the erythrocyte through hypotonic and swelling mode, the second drug molecule is coupled on the surface of the erythrocyte through amphiphilic polymer;
amphiphilic macromolecules are embedded in cell membranes of the erythrocytes, and can react with biotinylated second drug molecules through streptavidin, so that the second drug molecules are coupled on the surfaces of the erythrocytes;
the amphiphilic polymer is selected from distearoyl phosphatidyl ethanolamine-polyethylene glycol-biotin.
2. The method for preparing a double drug-loaded erythrocyte carrier of claim 1, which is characterized by comprising the following steps:
s1, mixing red blood cells derived from healthy adult blood with the first drug molecule solution, adding hypertonic solution for resealing, and carrying out ultrasonic treatment to obtain red blood cells entrapping the first drug molecules;
s2, mixing streptavidin with distearoyl phosphatidyl ethanolamine-polyethylene glycol-biotin for reaction to obtain avidin distearoyl phosphatidyl ethanolamine-polyethylene glycol, and then mixing the avidin distearoyl phosphatidyl ethanolamine-polyethylene glycol with a second drug molecule for reaction to obtain a conjugate of distearoyl phosphatidyl ethanolamine-polyethylene glycol and the second drug molecule;
s3, mixing the solution of the red blood cells loaded with the first drug molecules in the step S1 and the conjugate of the distearoyl phosphatidyl ethanolamine-polyethylene glycol and the second drug molecules in the step S2, and continuously extruding through a polycarbonate porous membrane to obtain the double-drug-loaded red blood cell carrier.
3. The method according to claim 2, wherein step S1 includes a method for preparing the red blood cell: the method comprises the steps of carrying out anticoagulation on blood taken from healthy adults by heparin, centrifuging to remove upper plasma and a buffy coat, repeatedly washing residues for 3-5 times by 1XPBS buffer solution with the pH value of 7.2-7.4, then adding pre-cooled hypotonic solution into the washed residues at 0-4 ℃ for treatment for 15-45 min, then removing hemoglobin, and washing to obtain the red blood cells.
4. The production method according to claim 3, characterized in that: the hypotonic solution is 0.25 XPBS buffer solution.
5. The production method according to claim 3, characterized in that: the volume ratio of the cleaned residues to the hypotonic solution is 1: 3-6.
6. The method according to claim 2, wherein step S1 specifically includes: mixing the red blood cells with the first drug molecule solution, then adding hypertonic solution at the temperature of 25-37 ℃, resealing for 30-60 min, then carrying out ultrasonic treatment for 3-5 min, and centrifuging to remove free first drug molecules, thereby obtaining the red blood cells coated with the first drug molecules.
7. The production method according to claim 2 or 6, characterized in that: the volume ratio of the red blood cells to the first drug molecule solution is 1: 1-3.
8. The production method according to claim 2 or 6, characterized in that: the hypertonic solution is 10XPBS buffer solution.
9. The method according to claim 2, wherein step S2 specifically includes: mixing streptavidin and distearoyl phosphatidyl ethanolamine-polyethylene glycol-biotin, reacting for 20-30 min at 0-4 ℃, dialyzing with 1XPBS buffer solution with the pH value of 7.2-7.4 to remove free streptavidin to obtain the avidin distearoyl phosphatidyl ethanolamine-polyethylene glycol, mixing with biotinylated second drug molecules, and incubating for 20-30 min at 0-4 ℃ to obtain the conjugate of the distearoyl phosphatidyl ethanolamine-polyethylene glycol and the second drug molecules.
10. The method of claim 2, wherein: in the step S3, the pore size of the polycarbonate porous membrane is 0.2 μm, wherein the continuous extrusion times are 11-21 times.
11. The use of the double drug loaded erythrocyte vector of claim 1 in the preparation of antineoplastic drugs.
12. The use of the double-drug loaded erythrocyte vector of claim 1 in the preparation of a drug for repairing spinal cord injury.
13. Use according to claim 12, characterized in that: the repairing medicine is an erythrocyte carrier medicine which simultaneously carries paclitaxel and cetuximab.
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