CN110279672A - A kind of pair carries medicine loaded into erythrocyte, preparation method and application - Google Patents
A kind of pair carries medicine loaded into erythrocyte, preparation method and application Download PDFInfo
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- CN110279672A CN110279672A CN201910619067.6A CN201910619067A CN110279672A CN 110279672 A CN110279672 A CN 110279672A CN 201910619067 A CN201910619067 A CN 201910619067A CN 110279672 A CN110279672 A CN 110279672A
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- erythrocyte
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
- A61K9/5068—Cell membranes or bacterial membranes enclosing drugs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention discloses a kind of double load medicine loaded into erythrocyte and the preparation method and application thereof.Double medicine loaded into erythrocyte that carry include red blood cell, the second drug molecule for containing in endoerythrocytic first drug molecule and being coupled at erythrocyte surface.First drug molecule can be taxol etc., and the second drug molecule can be Cetuximab etc..Double load medicine loaded into erythrocyte of the invention utilize red blood cell while two kinds of therapeutic molecules of one drug molecule of load regulation and the second drug molecule, on the one hand containing by Carrier erythrocytes, reduce drug bring drug resistance risk and toxic side effect, increase the safety of administration, on the other hand it is also supported by the drug of two kinds of different function, better therapeutic effect can be played for different problems.Meanwhile double load medicine loaded into erythrocyte preparation processes of the invention are simple, raw material economics is easy to get, and is not need to rely on complex device, easy to industrialized production.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to a kind of pair carries medicine loaded into erythrocyte and preparation method thereof.
Background technique
Red blood cell (Red blood cell, RBC) is a kind of unique natural drug carrier.From 1970s,
Since red blood cell shows many advantageous properties in terms of biodegrade, release profiles, carried out as drug delivery vehicle
The research in nearly halfth century, and various molecules have successfully been contained, including antibody/enzyme, polypeptide, nucleic acid, nanoparticle and chemistry
Drug etc., some of them are successfully converted into human clinical trial, such as L-ASP, dexamethasone etc..But mesh
It is preceding with red blood cell to make carrier and be largely limited in by contain in red blood cell or be coupled at the mode of Surface of Erythrocytes and support
Single molecule.
Taxol (Paclitaxel, PTX) is a kind of diterpenoids taxanes, is clinically usually used in treating kinds of tumors
Disease.But in recent years, research finds its differentiation ratio that can significantly improve neuron in the treatment of central nervous system disease
Rate.Cetuximab (Cetuximab, Cet) is the monoclonal antibody of mosaic type immunoglobulin 1, and molecular target is raw for epidermis
Growth factor receptor body (Epidermal Growth Factor Receptor, EGFR).To neuron studies have shown that EGFR antibody
Myelin protein etc., which can significantly be inverted, inhibits molecule to the detrimental effect of neural axon growth, and Cetuximab is in conjunction with EGFR
Affinity is about 5 to 10 times of its endogenic ligand, therefore Cetuximab blocks the combination of EGFR and its endogenic ligand, thus
Inhibit its function of receptors.
Currently, the limited efficacy that single drug can play, two or more are medication combined when many clinical disease treatments
Treatment has become effective administrated method.And red blood cell supports drug can reduce drug bring drug resistance risk and toxic side effect,
Increase the safety of administration；It is supported simultaneously by the drug of two kinds of different function, for different problems, plays preferably treatment effect
Fruit.It therefore, is the demand for adapting to drug combination in various treatments, research red blood cell combines that support be to need to solve to two kinds of drugs
Certainly the problem of.
Summary of the invention
The main purpose of the present invention is to provide a kind of double load medicine loaded into erythrocyte and preparation method thereof, to overcome existing skill
The deficiency of art.
For realization aforementioned invention purpose, the technical solution adopted by the present invention includes:
The embodiment of the invention provides a kind of double load medicine loaded into erythrocyte comprising red blood cell is contained in endoerythrocytic
First drug molecule and the second drug molecule for being coupled at erythrocyte surface.
Further, first drug molecule includes taxol, but not limited to this, wherein the first drug passes through hypotonic
Enter in red blood cell with the mode of expansion.
Further, second drug molecule includes Cetuximab, but not limited to this, wherein the second drug molecule
Erythrocyte surface can be coupled to by amphipathy macromolecule.
Further, amphipathy macromolecule is embedded in the cell membrane of the red blood cell, the amphipathy macromolecule can
It is reacted by Streptavidin with biotinylated second drug molecule, so that the second drug molecule be made to be coupled at red blood cell table
Face.
Further, the amphipathy macromolecule includes distearoylphosphatidylethanolamine-polyethylene glycol-biotin
(DSPE-PEG-biotin), but not limited to this.
The embodiment of the invention also provides a kind of aforementioned double preparation methods for carrying medicine loaded into erythrocyte comprising:
S1. it will be mixed from the red blood cell of health adult's blood with the first drug molecule solution, and high sepage weight be added
Envelope, then it is sonicated, obtain the red blood cell for containing the first drug molecule；
S2. by Streptavidin and DSPE-PEG-biotin hybrid reaction, the DSPE-PEG of Avidin is obtained, then
With the second drug molecule hybrid reaction, the conjugate of the second drug molecule of DSPE-PEG- is obtained；
S3. the second medicine of DSPE-PEG- in the solution and step S2 of the red blood cell of the first drug molecule will be contained in step S1
Object molecular mixing, and it is continuously extruded by polycarbonate perforated membrane, obtain double load medicine loaded into erythrocyte.
The embodiment of the invention also provides a kind of aforementioned double load medicine loaded into erythrocyte application in preparations of anti-tumor drugs.
The embodiment of the invention also provides a kind of aforementioned double medicine loaded into erythrocyte that carry in the repair medicine of preparation spinal cord injury
In application.
The embodiment of the invention also provides a kind of pharmaceutical compositions comprising aforementioned double load medicine loaded into erythrocyte and pharmacy
Upper acceptable auxiliary material.
Compared with prior art, double load medicine loaded into erythrocyte of the invention support the first drug molecule using red blood cell simultaneously
With two kinds of therapeutic molecules of the second drug molecule, on the one hand containing by Carrier erythrocytes, reduces drug bring drug resistance wind
Danger and toxic side effect increase the safety of administration, are on the other hand also supported by the drug of two kinds of different function, can be for difference
Problem plays better therapeutic effect.Meanwhile double load medicine loaded into erythrocyte preparation processes of the invention are simple, raw material economics is easy
, it is not need to rely on complex device, it is easy to industrialized production.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
The some embodiments recorded in invention, for those of ordinary skill in the art, without creative efforts,
It is also possible to obtain other drawings based on these drawings.
Fig. 1 is a kind of double preparation process schematic diagrams for carrying medicine loaded into erythrocyte in a typical embodiments of the invention；
Fig. 2A-Fig. 2 B is 1 pair of photo for supporting taxol-Cetuximab loaded into erythrocyte of the embodiment of the present invention respectively
And transmission electron microscope picture；
Fig. 3 is the embodiment of the present invention 1 pair and supports taxol-Cetuximab loaded into erythrocyte high performance liquid chromatography；
Fig. 4 is the embodiment of the present invention 1 pair and supports taxol-Cetuximab loaded into erythrocyte protein electrophoresis Western
Blot figure；
Fig. 5 is the embodiment of the present invention 1 pair and supports taxol-Cetuximab loaded into erythrocyte dynamic particle size figure；
Fig. 6 is the embodiment of the present invention 1 pair and supports releasing in vitro for taxol in taxol-Cetuximab loaded into erythrocyte
Put curve.
Fig. 7 is the embodiment of the present invention 1 pair cell differentiation effect for supporting taxol-Cetuximab loaded into erythrocyte
Immunostaining figure.
Specific embodiment
In view of the defect of the prior art, inventor is studied for a long period of time and is largely practiced, and is able to propose skill of the invention
Art scheme mainly being contained the first drug molecule into a manner of hypotonic and expansion, while utilizing one kind two
Parent's property macromolecule is embedded into erythrocyte membrane, and then by Streptavidin, biotinylated second drug molecule is coupled to
Erythrocyte surface (its principle can be refering to fig. 1).
One kind that the one aspect of the embodiment of the present invention provides is double to carry medicine loaded into erythrocyte comprising red blood cell, contain in
Endoerythrocytic first drug molecule and the second drug molecule for being coupled at erythrocyte surface.
Further, first drug molecule includes taxol, but not limited to this, wherein the first drug passes through hypotonic
Enter in red blood cell with the mode of expansion.
Further, second drug molecule includes Cetuximab, but not limited to this, wherein the second drug molecule
Erythrocyte surface can be coupled to by amphipathy macromolecule.
Further, amphipathy macromolecule is embedded in the cell membrane of the red blood cell, the amphipathy macromolecule can
It is reacted by Streptavidin with biotinylated second drug molecule, so that the second drug molecule be made to be coupled at red blood cell table
Face.
Further, the amphipathy macromolecule includes distearoylphosphatidylethanolamine-polyethylene glycol-biotin
(DSPE-PEG-biotin), but not limited to this.
The other side of the embodiment of the present invention additionally provides double preparation methods for carrying medicine loaded into erythrocyte, packet
It includes:
S1. it will be mixed from the red blood cell of health adult's blood with the first drug molecule solution, and high sepage weight be added
Envelope, then it is sonicated, obtain the red blood cell for containing the first drug molecule；
S2. by Streptavidin and DSPE-PEG-biotin hybrid reaction, the DSPE-PEG of Avidin is obtained, then
With the second drug molecule hybrid reaction, the conjugate of the second drug molecule of DSPE-PEG- is obtained；
S3. the solution that the red blood cell of the first drug molecule is contained in step S1 is mixed with DSPE-PEG-Cet in step S2
It closes, and continuously extruded by polycarbonate perforated membrane, obtains double load medicine loaded into erythrocyte.
In some embodiments, as follows including preparing the method for the red blood cell in step S1: will to be derived from health adult's
After blood is with anticoagulant heparin, it is centrifuged off top blood plasma and buffy coat, the 1X for being 7.2~7.4 by remaining object pH value
PBS buffer solution washes repeatedly 3~5 times, at the hypotonic medium that pre-cooling then is added into once purged remaining object at 0~4 DEG C
15~45min is managed, hemoglobin is removed later, then washed, obtains the red blood cell.
Further, the hypotonic medium is 0.25X PBS buffer solution.
Further, the volume ratio of the once purged remaining object and hypotonic medium is 1:3~6.
Further, step S1 is specifically included: the red blood cell being mixed with the first drug molecule solution, later in 25-
High sepage is added under the conditions of 37 DEG C, seals 30~60min again, is then ultrasonically treated 3~5min, is centrifuged off free first drug point
Son obtains the red blood cell for containing the first drug molecule.
Further, the volume ratio of the red blood cell and the first drug molecule solution is 1:1~3.
Further, the high sepage is 10X PBS buffer solution.
Further, step S2 is specifically included: Streptavidin being mixed with DSPE-PEG-biotin, in 0~4 DEG C of item
20~30min is reacted under part, the 1X PBS buffer solution dialysis for being later 7.2~7.4 with pH value removes free Streptavidin,
Obtain the DSPE-PEG of Avidin, then mixed with biotinylated second drug molecule, under the conditions of 0~4 DEG C be incubated for 20~
30min obtains the conjugate of the second drug molecule of DSPE-PEG-.
Further, the pore size of polycarbonate perforated membrane described in step S3 is 0.2 μm, continuously extruded time therein
Number is 11~21 times.
In some more specifically embodiments, double load taxol-Cetuximab loaded into erythrocyte preparation sides
Method includes:
S1. it will be mixed from the red blood cell of health adult's blood with paclitaxel solution, and high sepage be added and seals again, then passes through
Ultrasonic treatment, obtains the red blood cell for containing taxol；
S2. by Streptavidin and DSPE-PEG-biotin hybrid reaction, the DSPE-PEG of Avidin is obtained, then
With Cetuximab hybrid reaction, the conjugate of DSPE-PEG-Cet is obtained；
S3. the solution for containing the red blood cell of taxol in step S1 is mixed with DSPE-PEG-Cet in step S2, and led to
It is continuously extruded to cross polycarbonate perforated membrane, obtains double load medicine loaded into erythrocyte.
Further, taxol in every milliliter of red blood cell, Cetuximab loading range be respectively 0.2~400ug
With 0.5~5ug.
The other side of the embodiment of the present invention additionally provide it is a kind of it is aforementioned it is double carry medicine loaded into erythrocyte prepare it is antitumor
Application in drug.
The embodiment of the invention also provides a kind of aforementioned double medicine loaded into erythrocyte that carry in the repair medicine of preparation spinal cord injury
In application.
Further, the drug is while supporting the loaded into erythrocyte drug of taxol and Cetuximab.
The other side of the embodiment of the present invention additionally provides a kind of pharmaceutical composition comprising aforementioned double load medicine red blood cells
Carrier and pharmaceutically acceptable auxiliary material.
The technical scheme of the invention is further explained by means of specific implementation.Those skilled in the art are easy to
Understand, the described embodiments are merely helpful in understanding the present invention, should not be regarded as a specific limitation of the invention.
Embodiment 1: it is double support taxol-Cetuximab loaded into erythrocyte (also known as " double load medicine loaded into erythrocyte " or
" carry medicine red blood cell ") preparation
1. top blood plasma and buffy coat are centrifuged off after the blood of health adult will be derived from anticoagulant heparin, use
1XPBS buffer (pH value 7.3) washes repeatedly 4 times and obtains red blood cell, and the hypotonic medium (0.25X of pre-cooling is then added at 2 DEG C
PBS buffer solution) processing 30min, it is centrifuged off hemoglobin later, and wash until supernatant is colourless, collection bottom red blood cell is heavy
It forms sediment, wherein the volume ratio of red blood cell and hypotonic medium is 1:4.
2. gained erythrocyte membrane in step 1 is mixed with paclitaxel solution with volume ratio for 1:2, later under the conditions of 30 DEG C
High sepage (10XPBS buffer) is added, seals 40min again, is then ultrasonically treated 4min, be centrifuged off free paclitaxel, wrapped
The red blood cell of taxol is carried, 4 DEG C save backup；
3. Streptavidin is mixed with DSPE-PEG-biotin, 25min is reacted under the conditions of 4 DEG C, uses 1X PBS later
Buffer (pH value 7.3) dialysis removes free Streptavidin, obtains the DSPE-PEG of Avidin, then single with western appropriate former times
Anti- mixing, 2 DEG C of incubation 25min remove free molecule, obtain DSPE-PEG- Cetuximab；
4. the red blood cell for containing taxol in step 2 is mixed with DSPE-PEG- Cetuximab in step 3, pass through 0.2
μm polycarbonate perforated membrane, continuously extruded 15 times, obtain and double support taxol -- the loaded into erythrocyte of Cetuximab.
The photo of the load medicine red blood cell of the embodiment 1 preparation is as shown in Figure 2 A, and the red blood cell suspension after carrying medicine is in yellowish
Color.
The load medicine red blood cell of the embodiment 1 is characterized, transmission electron microscope picture is as shown in Fig. 2, by load after extruding
Medicine erythrocyte size is uniform, and partial size is in 200nm or so；By high performance liquid chromatography (see Fig. 3) and protein electrophoresis Western
Blot (see Fig. 4) detection carries medicine red blood cell and successfully realizes supporting for taxol and Cetuximab.
Extracorporeal biology evaluation
1, the vitro stability of medicine red blood cell is carried
Embodiment 1 is obtained and carries medicine red blood cell using the fixed negative staining of phosphotungstic acid, after drying at room temperature, medicine red blood cell suspension will be carried
In PBS, 4 DEG C of standings save 10d.The size and PDI index that carry medicine red blood cell are measured daily.As shown in fig.5, carrying
Partial size of 0-7 days erythrocyte membranes under the conditions of 25 DEG C increases to 233.9 ± 7.2nm from 189 ± 3.4nm after medicine, after 7 days
PDI index is 0.169 ± 0.03, shows that the load medicine red blood cell of preparation has preferable stability.Therefore, the load of the embodiment 1
Medicine red blood cell has the function of stabilization in vitro storage.
2, release in vitro
Embodiment 1 is obtained to the solution progress dialysis treatment for carrying medicine red blood cell, uses bag filter (molecular cut off 8-
10kDa), the additional PBS containing 0.5%Tween80, the dissolution medium of pH value 7.4, above-mentioned delivery system, which is placed in revolving speed, is
In 37 DEG C of constant-temperature tables of 200rpm.1mL volume was taken out at 0.5,1,2,4,8,12,24,36,48 and 72 hour each time point
Dissolution medium, supplement equal fresh dissolution medium immediately, utilize the content of taxol in high performance liquid chromatography detection external solution.
The In-vitro release curves of taxol are as shown in Figure 6.The result shows that the naked medicine group not supported releases 42 in initial 4 hours
± 1.3%, in 24 hours, all completely, and loaded into erythrocyte has apparent slow release effect to naked medicine group for release, at 24 hours and
48.61 ± 4.22% and 65.11 ± 2.62% are released respectively in 48 hours.Therefore, the load medicine red blood cell of the embodiment 1 has
Extend the function of drug half-life.
3, cell differentiation
It will be originated from the neural stem cell NSCs in ICR hippocampus of mice area, is seeded to 96 holes according to the cell density in every hole 3.5 ten thousand
In plate, the pretreated differential medium containing myelin protein, 37 DEG C, 5%CO are added2It is cultivated in CMC model case.It second day will training
Feeding base removal changes into and the different differential mediums for carrying medicine red blood cells that example 1 is obtained is added, and contains naked medicine (PTX) respectively, supports
The red blood cell (RBC-PTX) of taxol, the red blood cell (RBC-Cet) for supporting Cetuximab and double load medicine red blood cell (RBC-
PTX-Cet).It carries out cell after dosing 7 days to fix and immunofluorescence dyeing, detection each group is neural stem cell differentiating at neuron
Ratio.The coloration result of early stage neuronal cell as shown in fig. 7, two kinds of molecules of taxol and western appropriate former times myelin protein presence
The neuron differentiation ratio 10.2 ± 1.05% of naked medicine taxol group is compared in the differentiation that can promote neuronal cell down, red thin
The containing of born of the same parents has the effect of promoting neuron differentiation, and neuron ratio is improved to 14.7 ± 2.9%, and compares a kind of molecule
Single therapy, double systems that support can promote the differentiation ratio of neuron, be 23.1 ± 2.72%.Realize two kinds for the treatment of molecules
Effect improve.
Embodiment 2: double preparations for supporting taxol-Cetuximab loaded into erythrocyte
1. being centrifuged off top blood plasma and buffy coat after being derived from the blood anticoagulant heparin of health adult, using 1X
PBS buffer solution (pH value 7.2) washes repeatedly 3 times and obtains red blood cell, and hypotonic medium (the 0.25X PBS of pre-cooling is then added at 0 DEG C
Buffer) processing 15min, it is centrifuged off hemoglobin later, and wash until supernatant is colourless, collects bottom erythroprecipitin,
Wherein, the volume ratio of red blood cell and hypotonic medium is 1:2.
2. with volume ratio be that 1:1 is mixed by gained erythrocyte membrane and paclitaxel solution in step 1, later again under the conditions of 25 DEG C
High sepage (10X PBS buffer solution) is added, seals 30min again, is then ultrasonically treated 3min, be centrifuged off free paclitaxel, obtain
The red blood cell of taxol is contained, 4 DEG C save backup；
3. Streptavidin is mixed with DSPE-PEG-biotin, 20min is reacted under the conditions of 0 DEG C, it is slow with PBS later
Fliud flushing (pH value 7.2) dialysis removes free Streptavidin, obtains the DSPE-PEG of Avidin, then and Cetuximab
Mixing, 0 DEG C of incubation 20min remove free molecule, obtain DSPE-PEG- Cetuximab；
4. the red blood cell for containing taxol in step 2 is mixed with DSPE-PEG- Cetuximab in step 3, pass through 0.2
μm polycarbonate perforated membrane, continuously extruded 11 times, obtain and double support taxol -- the loaded into erythrocyte of Cetuximab.
Embodiment 3: double preparations for supporting taxol-Cetuximab loaded into erythrocyte
1. being centrifuged off top blood plasma and buffy coat after being derived from the blood anticoagulant heparin of health adult, use
1XPBS buffer (pH value 7.4) washes repeatedly 5 times and obtains red blood cell, and the hypotonic medium (0.25X of pre-cooling is then added at 4 DEG C
PBS buffer solution) processing 45min, it is centrifuged off hemoglobin later, and wash until supernatant is colourless, collection bottom red blood cell is heavy
It forms sediment, wherein the volume ratio of red blood cell and hypotonic medium is 1:6.
2. with volume ratio be that 1:3 is mixed by gained erythrocyte membrane and paclitaxel solution in step 1, later again under the conditions of 37 DEG C
High sepage (10XPBS buffer) is added, seals 60min again, is then ultrasonically treated 5min, be centrifuged off free paclitaxel, wrapped
The red blood cell of taxol is carried, 4 DEG C save backup；
3. Streptavidin is mixed with DSPE-PEG-biotin, 30min is reacted under the conditions of 4 DEG C, it is slow with PBS later
Fliud flushing (pH value 7.4) dialysis removes free Streptavidin, obtains the DSPE-PEG of Avidin, then and Cetuximab
Mixing, 4 DEG C of incubation 30min remove free molecule, obtain DSPE-PEG- Cetuximab；
4. the red blood cell for containing taxol in step 2 is mixed with DSPE-PEG- Cetuximab in step 3, pass through 0.2
μm polycarbonate perforated membrane, continuously extruded 21 times, obtain and double support taxol -- the loaded into erythrocyte of Cetuximab.
The mode of reference implementation example 1, which obtains embodiment 2,3, carries the progress extracorporeal biology evaluation of medicine red blood cell, also can be obtained
Evaluation result similar to Example 1.
It should be appreciated that the technical concepts and features of above-described embodiment only to illustrate the invention, its object is to allow be familiar with this
The personage of item technology cans understand the content of the present invention and implement it accordingly, and it is not intended to limit the scope of the present invention, example
Such as, aforementioned taxol is also substitutable for other chemicals, and it is raw that aforementioned Cetuximab also can be replaced other monoclonal antibodies, how anti-etc.
Object molecule.Any equivalent change or modification in accordance with the spirit of the invention, should all cover protection scope of the present invention it
It is interior.
Claims (14)
1. a kind of double load medicine loaded into erythrocyte, it is characterised in that including red blood cell, contain in endoerythrocytic first drug molecule
With the second drug molecule for being coupled at erythrocyte surface.
2. double load medicine loaded into erythrocyte according to claim 1, it is characterised in that: first drug molecule includes Japanese yew
Alcohol, wherein the first drug molecule is entered in red blood cell by way of hypotonic and expansion；
And/or second drug molecule includes Cetuximab, wherein the second drug molecule passes through amphipathy macromolecule idol
It is coupled to erythrocyte surface.
3. double load medicine loaded into erythrocyte according to claim 1, it is characterised in that: be embedded in the cell membrane of the red blood cell
There is amphipathy macromolecule, the amphipathy macromolecule can be anti-by Streptavidin and biotinylated second drug molecule
It answers, so that the second drug molecule be made to be coupled at erythrocyte surface.
4. double load medicine loaded into erythrocyte according to claim 3, it is characterised in that: the amphipathy macromolecule includes two hard
Fatty acyl group phosphatidyl-ethanolamine-polyethylene glycol-biotin.
5. double preparation methods for carrying medicine loaded into erythrocyte described in any one of claim 1-4, characterized by comprising:
S1. it will be mixed from the red blood cell of health adult's blood with the first drug molecule solution, and high sepage be added and seals again, then
It is sonicated, obtain the red blood cell for containing the first drug molecule；
S2. it by Streptavidin and distearoylphosphatidylethanolamine-polyethylene glycol-biotin hybrid reaction, obtains affine
The distearoylphosphatidylethanolamine-polyethylene glycol of elementization obtains distearyl then with the second drug molecule hybrid reaction
Base phosphatidyl-ethanolamine-polyethylene glycol and the second drug molecule conjugate；
S3. distearyl acyl group phosphatidyl ethanol in the solution and step S2 of the red blood cell of the first drug molecule will be contained in step S1
Amine-polyethylene glycol is mixed with the conjugate of the second drug molecule, and continuously extruded by polycarbonate perforated membrane, obtains double load medicines
Loaded into erythrocyte.
6. preparation method according to claim 5, it is characterised in that include the method for preparing the red blood cell in step S1:
After the blood of health adult will be derived from anticoagulant heparin, it is centrifuged off top blood plasma and buffy coat, by remaining object pH value
It washes repeatedly 3~5 times for 7.2~7.4 1X PBS buffer solution, is then added at 0~4 DEG C into once purged remaining object
The hypotonic medium of pre-cooling handles 15~45min, removes hemoglobin later, then washed, obtains the red blood cell.
7. preparation method according to claim 6, it is characterised in that: the hypotonic medium is 0.25X PBS buffer solution；
And/or the volume ratio of the once purged remaining object and hypotonic medium is 1:3~6.
8. preparation method according to claim 5, it is characterised in that step S1 is specifically included: by the red blood cell and first
Drug molecule solution mixing, high sepage is added under the conditions of 25~37 DEG C later, again seal 30~60min, then be ultrasonically treated 3~
5min is centrifuged off free first drug molecule, obtains the red blood cell for containing the first drug molecule.
9. the preparation method according to claim 5 or 8, it is characterised in that: the red blood cell and the first drug molecule solution
Volume ratio be 1:1~3；
And/or the high sepage is 10X PBS buffer solution.
10. preparation method according to claim 5, it is characterised in that step S2 is specifically included: by Streptavidin and two
The mixing of stearoyl phosphatidyl ethanol amine-polyethylene glycol-biotin, 20~30min is reacted under the conditions of 0~4 DEG C, uses pH later
Value removes free Streptavidin for 7.2~7.4 1X PBS buffer solution dialysis, obtains the distearyl acyl group phosphorus of Avidin
Acyl ethanol amine-polyethylene glycol is then mixed with biotinylated second drug molecule, under the conditions of 0~4 DEG C be incubated for 20~
30min obtains the conjugate of distearoylphosphatidylethanolamine-polyethylene glycol and the second drug molecule.
11. preparation method according to claim 5, it is characterised in that: the hole of polycarbonate perforated membrane described in step S3
Diameter size is 0.2 μm, and continuously extruded number therein is 11~21 times.
12. double described in any one of claim 1-4 carry medicine loaded into erythrocyte application in preparation of anti-tumor drugs.
13. double described in any one of claim 1-4 carry medicine loaded into erythrocyte answering in the repair medicine of preparation spinal cord injury
With, it is preferred that the drug is while supporting the loaded into erythrocyte drug of taxol and Cetuximab.
14. a kind of pharmaceutical composition, it is characterised in that including described in any one of claim 1-4 it is double carry medicine loaded into erythrocyte with
And pharmaceutically acceptable auxiliary material.
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