CN109276557A - A kind of immune suppressant drug and preparation method thereof using PBAE as pharmaceutical carrier - Google Patents
A kind of immune suppressant drug and preparation method thereof using PBAE as pharmaceutical carrier Download PDFInfo
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- CN109276557A CN109276557A CN201711129962.7A CN201711129962A CN109276557A CN 109276557 A CN109276557 A CN 109276557A CN 201711129962 A CN201711129962 A CN 201711129962A CN 109276557 A CN109276557 A CN 109276557A
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- 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
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
- A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
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- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
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- 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
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Abstract
The invention belongs to field of biotechnology, a kind of immune suppressant drug using PBAE as pharmaceutical carrier is disclosed, the immune suppressant drug is followed successively by immunosuppressor, lipid layers, PBAE layers, the PBAE layer of modification polyethylene glycol 2000, polyglutamic acid layer from inside to outside.The purpose of the present invention is to provide a kind of targeting height, the immunocompetent immune suppressant drug using PBAE as pharmaceutical carrier that can be effectively reduced T cell and B cell and preparation method thereof.
Description
Technical field
The present invention relates to field of biotechnology, especially a kind of immune suppressant drug using PBAE as pharmaceutical carrier and
Preparation method.
Background technique
Immunosuppressor is the inhibited drug of the immune response to body, can inhibit and be immunoreacted related thin
The proliferation and function of born of the same parents' (macrophages such as T cell and B cell), can reduce organism immune response.Immunosuppressor is mainly used for
The anti-rejection of organ transplant and autoimmunity disease such as rheumatoid arthritis, lupus erythematosus, dermatomycosis, film kidney ball kidney
Scorching, inflammatory bowel disease and autoimmune hemolytic anemia anaemia etc..Mainstream therapeutic agent has glucocorticoid, cyclosporine at present, replaces wheat
Examine the immunosuppressants drug such as phenolic ester, imuran.The main function of these immunosuppressor, which has, lowers T, bone-marrow-derived lymphocyte function
Can, it reduces specific autoantibody and generates, inhibit cell factor generation etc..But the long-time service of systemic immune inhibitor is not
It is evitable to bring some serious toxic side effects, such as inhibit reproductive system function, increase Tumor incidence, destroys hematopoiesis function
Deng causing normal body's immunity impaired, so as to cause other a series of side effects.The drug of existing immunosuppressor
Carrier targeting is low.
Summary of the invention
In order to solve the above shortcomings and deficiencies, that the present invention provides a kind of targetings is high, can be effectively reduced T cell and
The immunocompetent immune suppressant drug and preparation method thereof using PBAE as pharmaceutical carrier of B cell.
It is the specific scheme is that a kind of immune suppressant drug using PBAE as pharmaceutical carrier, the immunosuppressor
PBAE layer, the polyglutamic that drug is followed successively by immunosuppressor, lipid layers, PBAE layers from inside to outside, modifies polyethylene glycol 2000
Acid layer；
The polyglutamic acid layer, which is dispatched workers to do on-site repairs, is decorated with CD19 or CD3.
In the above-mentioned immune suppressant drug using PBAE as pharmaceutical carrier, the immunosuppressor is sugared cortical hormone
Plain class, calcineurin inhibitor, cyclosporine, tacrolimus, antimetabolite, imuran, methotrexate (MTX), mercaptopurine or mould phenol
Acid esters.
In the above-mentioned immune suppressant drug using PBAE as pharmaceutical carrier, the immune suppressant drug partial size
For 20-300nm.
Meanwhile the preparation method for the immune suppressant drug that the invention also discloses a kind of using PBAE as pharmaceutical carrier,
The following steps are included:
Step 1: immunosuppressive drug is coated with by liposome；
Step 2: preparation PBAE polymer；
Step 3: the PBAE polymer of preparation surface modification PEG2000；
Step 4: preparation modification CD19 or CD3 polyglutamic acid；
Step 5: substance prepared by step 1-4 is assembled into immune suppressant drug.
In the above-mentioned preparation method using PBAE as the immune suppressant drug of pharmaceutical carrier, step 2 tool
Body are as follows: 1,4-butanediol diacrylate is mixed with diacrylate with 1.1:1 molar ratio, 90 DEG C is heated to and stirs 24 hours
To generate poly- (the 4- amino-n-butyl alcohol -co- 1,4-butanediol diacrylate) of acrylate ended, by the 2.3g polymer
2ml tetrahydrofuran (THF) is dissolved in mix with the 1- of 60.5mg/ml (3- aminopropyl) -4- methyl piperazine tetrahydrofuran solution afterwards,
The PBAE polymer for obtaining piperazine sealing end for 2 hours is stirred at 37 DEG C, the ether of 5 times of volumes of polymer of sealing end is precipitated, collected
Vacuum freeze drying is precipitated, will finally be collected into polymer and be dissolved in dimethyl sulfoxide (DMSO) to concentration is 100mg/ml,
It is stored in -20 DEG C.
In the above-mentioned preparation method using PBAE as the immune suppressant drug of pharmaceutical carrier, step 3 tool
Body are as follows: 1,4-butanediol diacrylate is mixed with diacrylate with 1.1:1 molar ratio, 90 DEG C is heated to and stirs 24 hours
To generate poly- (the 4- amino-n-butyl alcohol -co- 1,4-butanediol diacrylate) of acrylate ended, obtained polymer is in second
Vacuum freeze drying after purification in alcohol.By the polymer and methoxyl group PEG- sulfydryl and 1- (3- aminopropyl) -4- methyl piperazine
Molecule is mixed into the DMSO solution of 100mg/mL with 1:2.5:0.2M ratio, and stirs 24 hours at 1000rpm at room temperature.
Final block copolymer is freeze-dried with after precipitating in ether at room temperature, will be finally collected into polymer and is dissolved in dimethyl
In sulfoxide (DMSO) to concentration be 100mg/ml, be stored in -20 DEG C it is spare.
In the above-mentioned preparation method using PBAE as the immune suppressant drug of pharmaceutical carrier, step 4 tool
Body are as follows: polyglutamic acid is soluble in water to 20mg/ml, and then ultrasound 10 minutes in bathing ultrasonoscope, are added isometric N'-
(3- dimethylaminopropyl) carbodiimide HCL aqueous solution (4mg/ml), and the solution is mixed 5 minutes at room temperature, it will
The PGA of obtained activation is added in the antibody-solutions in phosphate buffered saline (PBS) with the molar ratio of 4:1, and mixed at room temperature
6 hours, excessive reagent is removed by dialysing 24 hours to above-mentioned solution, is then filtered by 40kZeba column spinner, it is spare.
In the above-mentioned preparation method using PBAE as the immune suppressant drug of pharmaceutical carrier, step 5 tool
Body are as follows: all components are diluted to following concentration in sodium acetate buffer: being coated with the liposome nanosphere of immunosuppressive drug,
0.1mg/ml；It is modified with the PBAE polymer of PEG2000,3.14mg/ml；PGA antibody, 0.45mg/ml；
The liposome nanosphere of PBAE polymer and coating immunosuppressive drug is mixed into gently whirlpool with the mass ratio of 15:1
10s is revolved, is incubated for 2h at room temperature；
Again by the PBAE for being modified with PEG2000 with the lipid body mass ratio with coating immunosuppressive drug in the mixture system
It is added in mixture above-mentioned for 15:1, is gently incubated at room temperature 10min after vortex 10.Finally by antibody to exempt from coating in system
The mass ratio of the liposome nanosphere mass ratio 2.5:1 of epidemic disease depressant is added, and is gently incubated at room temperature 10min after vortex 10；
Sucrose is added as cryoprotector, final concentration of 30mg/ml.Mixture is freezed in liquid nitrogen mesoscale eddies, then
Use freeze drier lyophilization system；
Finally freeze-drying particle is resuspended in physiological saline.
The beneficial effects of the present invention are:
Immune suppressant drug of the invention has targeting height, extends drug effect, improve curative effect, avoid tolerance, reduce
The advantages that drug toxic side effect, changeable administration route.
PBAE contains a large amount of amino cations, has electropositive, can effectively carry with electronegative cell membrane interaction
Drug is mediated by endocytosis enters cell, simultaneously because " the proton sponge effect " of amino cation is (or interior, it can be achieved that endocytosis body
Contain body) escape and drug controlled release intracellular.However a large amount of positive charge is but also PBAE is steady in the circulatory system in vivo
Qualitative difference, easily causes erythrocyte hemolysis, has certain toxicity in vivo, cannot be directly used to using it as the medicine-carried system of material quiet
Arteries and veins injection.Given this, grafting polyethylene glycol 2000 (PEG-2000) can make liposome hide catching for reticuloendothelial system (RES)
It catches, extends the time that carrier recycles in vivo, and Targeting Effect will not be reduced, so that enhancing it is used as antineoplastic drug carrier
Feasibility.PGA polymer is a kind of water-soluble, biodegradable, energy specific recognition T/B cell after connection CD19 or CD3.From
And drug is accurately loaded into cell, reduce the adverse reaction of whole body.
Detailed description of the invention
Fig. 1 is a schematic structural view of Embodiment 1 of the present invention.
Specific embodiment
With reference to embodiment, technical solution of the present invention is described in further detail, but do not constituted pair
Any restrictions of the invention.
In order to which more clearly the present invention will be described, embodiment is listed below to illustrate superiority of the invention.
Such as Fig. 1, a kind of immune suppressant drug using PBAE as pharmaceutical carrier, the immune suppressant drug is by interior
And it is followed successively by immunosuppressor 1, lipid layers 2, PBAE layer 3, the PBAE layer 4 for modifying polyethylene glycol 2000, polyglutamic acid layer outside
5；
The PBAE layer is outer to be modified with polyethylene glycol 2000；
The polyglutamic acid layer, which is dispatched workers to do on-site repairs, is decorated with CD19 or CD3.
Specifically, included the following steps using PBAE as the preparation method of the immune suppressant drug of pharmaceutical carrier.
The preparation of liposome coating immunosuppressive drug: the lecithin of precise 30mg is dissolved in a small amount of anhydrous second respectively
In alcohol, 10mg tacrolimus, 40mg stearic acid is separately taken to be dissolved in methylene chloride, ultrasound dissolves it sufficiently in 30 minutes, by gained
Ethanol solution is mixed with dichloromethane baked solution constitutes organic phase.Precision weighs PLURONICS F87 and Brij78 (the two 1；1)
150mg is added in distilled water, constitutes water phase.By mutually synthermal oil mutually with syringe be slowly injected into 1200r/min stir it is auspicious
It in (65 ± 2) C ° of constant temperature water phase, persistently stirs 3 hours, make organic solvent evaporating completely and system is made to be concentrated into about formation nanometer
Cream.Resulting lotion is quickly mixed in another 0 DEG C of ice water, and at ice-water bath ultrasound 30 minutes to get nanoparticle suspension.
2, the preparation of PBAE polymer:
1,4-butanediol diacrylate is mixed with diacrylate with 1.1:1 molar ratio, 90 DEG C of stirrings 24 are heated to
Hour to generate poly- (the 4- amino-n-butyl alcohol -co- 1,4-butanediol diacrylate) of acrylate ended, 2.3g should be gathered
Close object be dissolved in 2ml tetrahydrofuran (THF) afterwards with the 1- of 60.5mg/ml (3- aminopropyl) -4- methyl piperazine tetrahydrofuran solution
The PBAE polymer for obtaining piperazine sealing end for 2 hours is stirred in mixing at 37 DEG C, and the ether of 5 times of volumes of polymer of sealing end precipitates,
Collect precipitating vacuum freeze drying.It is 100mg/ that polymer, which will be finally collected into, and be dissolved in dimethyl sulfoxide (DMSO) to concentration
Ml is stored in -20 DEG C.
3, it is modified with the preparation of the PBAE of PEG2000:
1,4-butanediol diacrylate is mixed with diacrylate with 1.1:1 molar ratio, 90 DEG C of stirrings 24 are heated to
Hour to generate poly- (the 4- amino-n-butyl alcohol -co- 1,4-butanediol diacrylate) of acrylate ended, obtained polymer
Vacuum freeze drying after purification in ethanol.By the polymer and methoxyl group PEG- sulfydryl and 1- (3- aminopropyl) -4- methyl
Piperazine moieties are mixed into the DMSO solution of 100mg/mL with 1:2.5:0.2M ratio, and stir 24 at 1000rpm at room temperature
Hour.Final block copolymer is freeze-dried with after precipitating in ether at room temperature.Polymer will be finally collected into be dissolved in
In dimethyl sulfoxide (DMSO) to concentration be 100mg/ml, be stored in -20 DEG C it is spare.
4, it is modified with the PGA preparation of CD19 or CD3:
Polyglutamic acid (PGA) is soluble in water to 20mg/ml, then ultrasound 10 minutes in bath ultrasonoscope.Be added etc.
N'- (3- dimethylaminopropyl) carbodiimide HCL aqueous solution (4mg/ml) of volume, and the solution is mixed at room temperature
5 minutes.The PGA of obtained activation is added in the antibody-solutions in phosphate buffered saline (PBS) (PBS) with the molar ratio of 4:1,
And it mixes 6 hours at room temperature.Excessive reagent is removed by dialysing 24 hours to above-mentioned solution, is then revolved by 40kZeba
Rotary column filtering.It is spare.
5, the assembling of drug-carrying nanometer particle:
The all components of aforementioned preparation are diluted to following concentration in sodium acetate buffer (25mM, pH5.2): being coated with him
The liposome nanosphere of Ke Mosi, 0.1mg/ml；It is modified with the PBAE polymer of PEG2000,3.14mg/ml；PGA antibody,
0.45mg/ml.We are mixed the liposome nanosphere of PBAE polymer and coating tacrolimus with the mass ratio of 15:1 first
The 10s that is gently vortexed is closed, is incubated for 2h at room temperature.Again by the PBAE for being modified with PEG2000 with immune with coating in the mixture system
The lipid body mass ratio of depressant is that 15:1 is added in mixture above-mentioned, is gently incubated at room temperature 10min after vortex 10.Finally will
Antibody is added with the mass ratio with the liposome nanosphere mass ratio 2.5:1 for being coated with tacrolimus in system, is gently vortexed 10
After be incubated at room temperature 10min.Sucrose is added as cryoprotector, final concentration of 30mg/ml.Mixture is cold in liquid nitrogen mesoscale eddies
Freeze, then uses freeze drier lyophilization system.Finally freeze-drying particle is resuspended in physiological saline.
Above-described is only presently preferred embodiments of the present invention, all made within the scope of the spirit and principles in the present invention
What modifications, equivalent substitutions and improvements etc., should all be included in the protection scope of the present invention.
Claims (8)
1. a kind of immune suppressant drug using PBAE as pharmaceutical carrier, which is characterized in that the immune suppressant drug
It is followed successively by immunosuppressor, lipid layers, PBAE layers, the PBAE layer of modification polyethylene glycol 2000, polyglutamic acid layer from inside to outside；
The polyglutamic acid layer, which is dispatched workers to do on-site repairs, is decorated with CD19 or CD3.
2. the immune suppressant drug according to claim 1 using PBAE as pharmaceutical carrier, which is characterized in that described to exempt from
Epidemic disease inhibitor is glucocorticoids, calcineurin inhibitor, cyclosporine, tacrolimus, antimetabolite, imuran, first
Aminopterin, mercaptopurine or mycophenolate.
3. the immune suppressant drug according to claim 1 using PBAE as pharmaceutical carrier, which is characterized in that described
Immune suppressant drug partial size is 20-300nm.
4. a kind of preparation side of the immune suppressant drug a method according to any one of claims 1-3 using PBAE as pharmaceutical carrier
Method, which comprises the following steps:
Step 1: immunosuppressive drug is coated with by liposome；
Step 2: preparation PBAE polymer；
Step 3: the PBAE polymer of preparation surface modification PEG2000；
Step 4: preparation modification CD19 or CD3 polyglutamic acid；
Step 5: substance prepared by step 1-4 is assembled into immune suppressant drug.
5. the preparation method of the immune suppressant drug according to claim 4 using PBAE as pharmaceutical carrier, feature
It is, the step 2 specifically: 1,4-butanediol diacrylate is mixed with diacrylate with 1.1:1 molar ratio, is added
Heat stirs 24 hours to generate poly- (the 4- amino-n-butyl alcohol -co- 1,4- butanediol diacrylate of acrylate ended to 90 DEG C
Ester), by the 2.3g polymer be dissolved in 2ml tetrahydrofuran (THF) afterwards with the 1- of 60.5mg/ml (3- aminopropyl) -4- methyl piperazine
The PBAE polymer for obtaining piperazine sealing end for 2 hours is stirred in the mixing of piperazine tetrahydrofuran solution at 37 DEG C, and the polymer of sealing end is with 5 times
The ether of volume precipitates, and collects precipitating vacuum freeze drying, will finally be collected into polymer and be dissolved in dimethyl sulfoxide (DMSO)
In to concentration be 100mg/ml, be stored in -20 DEG C.
6. the preparation method of the immune suppressant drug according to claim 4 using PBAE as pharmaceutical carrier, feature
It is, the step 3 specifically: 1,4-butanediol diacrylate is mixed with diacrylate with 1.1:1 molar ratio, is added
Heat stirs 24 hours to generate poly- (the 4- amino-two propylene of n-butyl alcohol -co- 1,4- butanediol of acrylate ended to 90 DEG C
Acid), obtained polymer vacuum freeze drying after purification in ethanol.By the polymer and methoxyl group PEG- sulfydryl and 1- (3-
Aminopropyl) -4- methyl piperazine molecule is mixed into the DMSO solution of 100mg/mL with 1:2.5:0.2M ratio, and exists at room temperature
It is stirred 24 hours under 1000rpm.Final block copolymer is freeze-dried with after precipitating in ether at room temperature, will finally be collected
To polymer be dissolved in dimethyl sulfoxide (DMSO) to concentration be 100mg/ml, be stored in -20 DEG C it is spare.
7. the preparation method of the immune suppressant drug according to claim 4 using PBAE as pharmaceutical carrier, feature
It is, the step 4 specifically: polyglutamic acid is soluble in water to 20mg/ml, it is then 10 points ultrasonic in bath ultrasonoscope
Clock is added isometric N'- (3- dimethylaminopropyl) carbodiimide HCL aqueous solution (4mg/ml), and the solution is existed
It mixes 5 minutes at room temperature, it is molten that the PGA of obtained activation with the molar ratio of 4:1 is added to the antibody in phosphate buffered saline (PBS)
It in liquid, and mixes 6 hours at room temperature, removes excessive reagent by dialysing 24 hours to above-mentioned solution, then pass through
The filtering of 40kZeba column spinner, it is spare.
8. the preparation method of the immune suppressant drug according to claim 4 using PBAE as pharmaceutical carrier, feature
It is, the step 5 specifically: all components are diluted to following concentration in sodium acetate buffer: coating immunosupress
The liposome nanosphere of medicine, 0.1mg/ml；It is modified with the PBAE polymer of PEG2000,3.14mg/ml；PGA antibody,
0.45mg/ml；
The liposome nanosphere of PBAE polymer and coating immunosuppressive drug is mixed with the mass ratio of 15:1 and is gently vortexed
10s is incubated for 2h at room temperature；
Again by the PBAE for being modified with PEG2000 to be with the lipid body mass ratio for being coated with immunosuppressive drug in the mixture system
15:1 is added in mixture above-mentioned, is gently incubated at room temperature 10min after vortex 10.Finally by antibody with immune with coating in system
The mass ratio of the liposome nanosphere mass ratio 2.5:1 of depressant is added, and is gently incubated at room temperature 10min after vortex 10；
Sucrose is added as cryoprotector, final concentration of 30mg/ml.Mixture is freezed in liquid nitrogen mesoscale eddies, is then used
Freeze drier lyophilization system；
Finally freeze-drying particle is resuspended in physiological saline.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101953797A (en) * | 2010-09-06 | 2011-01-26 | 中华人民共和国卫生部肝胆肠外科研究中心 | Method for preparing medicament carrying controlled-release nanometer material and application |
| US20140050671A1 (en) * | 2012-08-16 | 2014-02-20 | University Of Washington Through Its Center For Commercialization | Theranostic nanoparticle and methods for making and using the nanoparticle |
| WO2015108912A1 (en) * | 2014-01-16 | 2015-07-23 | MUSC Foundation for Research and Development | Targeted nanocarriers for the administration of immunosuppressive agents |
| CN105232462A (en) * | 2015-11-06 | 2016-01-13 | 中南大学湘雅医院 | Tacrolimus liposome and gel and preparation method thereof |
-
2017
- 2017-11-15 CN CN201711129962.7A patent/CN109276557A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101953797A (en) * | 2010-09-06 | 2011-01-26 | 中华人民共和国卫生部肝胆肠外科研究中心 | Method for preparing medicament carrying controlled-release nanometer material and application |
| US20140050671A1 (en) * | 2012-08-16 | 2014-02-20 | University Of Washington Through Its Center For Commercialization | Theranostic nanoparticle and methods for making and using the nanoparticle |
| WO2015108912A1 (en) * | 2014-01-16 | 2015-07-23 | MUSC Foundation for Research and Development | Targeted nanocarriers for the administration of immunosuppressive agents |
| CN105232462A (en) * | 2015-11-06 | 2016-01-13 | 中南大学湘雅医院 | Tacrolimus liposome and gel and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| 李伟男等: "聚(β-氨基酯)肿瘤靶向给药系统的研究进展", 《药学学报》 * |
| 肖忠革等主编: "《口腔药理学与药物治疗学》", 31 March 2009, 上海世界图书出版公司 * |
| 谢瑾等: "他克莫司长效脂质体的药物动力学研究", 《中国药业》 * |
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