CN101745114B - Method for preparing glucose-sensitive insulin controlled-release biological material - Google Patents
Method for preparing glucose-sensitive insulin controlled-release biological material Download PDFInfo
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- CN101745114B CN101745114B CN2009101557492A CN200910155749A CN101745114B CN 101745114 B CN101745114 B CN 101745114B CN 2009101557492 A CN2009101557492 A CN 2009101557492A CN 200910155749 A CN200910155749 A CN 200910155749A CN 101745114 B CN101745114 B CN 101745114B
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- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 title claims abstract description 60
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 title claims abstract description 39
- 239000008103 glucose Substances 0.000 title claims abstract description 39
- 238000013270 controlled release Methods 0.000 title claims abstract description 34
- 102000004877 Insulin Human genes 0.000 title claims abstract description 30
- 108090001061 Insulin Proteins 0.000 title claims abstract description 30
- 229940125396 insulin Drugs 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 24
- 239000012620 biological material Substances 0.000 title claims abstract description 18
- 239000000178 monomer Substances 0.000 claims abstract description 55
- 108010062580 Concanavalin A Proteins 0.000 claims abstract description 40
- 238000006243 chemical reaction Methods 0.000 claims abstract description 36
- 238000002360 preparation method Methods 0.000 claims abstract description 14
- 239000003054 catalyst Substances 0.000 claims abstract description 13
- 239000000376 reactant Substances 0.000 claims abstract description 8
- 238000000502 dialysis Methods 0.000 claims abstract description 7
- 229920001503 Glucan Polymers 0.000 claims description 50
- TWNIBLMWSKIRAT-VFUOTHLCSA-N levoglucosan Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@H]2CO[C@@H]1O2 TWNIBLMWSKIRAT-VFUOTHLCSA-N 0.000 claims description 30
- 125000005395 methacrylic acid group Chemical group 0.000 claims description 25
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 claims description 22
- 239000008367 deionised water Substances 0.000 claims description 21
- 229910021641 deionized water Inorganic materials 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 18
- 239000008363 phosphate buffer Substances 0.000 claims description 15
- DCUFMVPCXCSVNP-UHFFFAOYSA-N methacrylic anhydride Chemical compound CC(=C)C(=O)OC(=O)C(C)=C DCUFMVPCXCSVNP-UHFFFAOYSA-N 0.000 claims description 13
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 claims description 11
- 229910001870 ammonium persulfate Inorganic materials 0.000 claims description 11
- 238000006467 substitution reaction Methods 0.000 claims description 11
- ARJOQCYCJMAIFR-UHFFFAOYSA-N prop-2-enoyl prop-2-enoate Chemical compound C=CC(=O)OC(=O)C=C ARJOQCYCJMAIFR-UHFFFAOYSA-N 0.000 claims description 10
- 229960000549 4-dimethylaminophenol Drugs 0.000 claims description 9
- -1 dimethyl sulfoxine Chemical compound 0.000 claims description 9
- 239000000872 buffer Substances 0.000 claims description 8
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 4
- 238000005937 allylation reaction Methods 0.000 claims description 4
- 238000001212 derivatisation Methods 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- 239000000758 substrate Substances 0.000 claims description 4
- 241001597008 Nomeidae Species 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Natural products OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims 2
- 239000000243 solution Substances 0.000 abstract description 23
- 239000012876 carrier material Substances 0.000 abstract description 11
- 239000007853 buffer solution Substances 0.000 abstract 1
- 150000002303 glucose derivatives Chemical class 0.000 abstract 1
- 238000004519 manufacturing process Methods 0.000 abstract 1
- 238000010526 radical polymerization reaction Methods 0.000 abstract 1
- 239000008279 sol Substances 0.000 description 13
- 238000004108 freeze drying Methods 0.000 description 7
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 6
- 238000011056 performance test Methods 0.000 description 5
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- 235000013311 vegetables Nutrition 0.000 description 5
- HBOMLICNUCNMMY-KJFJCRTCSA-N 1-[(4s,5s)-4-azido-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1C1O[C@H](CO)[C@@H](N=[N+]=[N-])C1 HBOMLICNUCNMMY-KJFJCRTCSA-N 0.000 description 4
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 4
- 125000002769 thiazolinyl group Chemical group 0.000 description 4
- 241001000594 Tanna Species 0.000 description 3
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- 210000004369 blood Anatomy 0.000 description 3
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- 108090001090 Lectins Proteins 0.000 description 2
- 102000004856 Lectins Human genes 0.000 description 2
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- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 239000002523 lectin Substances 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 208000035408 type 1 diabetes mellitus 1 Diseases 0.000 description 2
- GHCZTIFQWKKGSB-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;phosphoric acid Chemical compound OP(O)(O)=O.OC(=O)CC(O)(C(O)=O)CC(O)=O GHCZTIFQWKKGSB-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
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- 238000001727 in vivo Methods 0.000 description 1
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- 230000002503 metabolic effect Effects 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 239000007981 phosphate-citrate buffer Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract
The invention discloses a new method for preparing an intelligent glucose-sensitive insulin controlled-release biological material. The method comprises the following steps of: firstly, dissolving glucose derivative monomers with C=C derivatives and concanavalin A derivative monomers with C=C double bonds in buffer solution to produce monomer solution; secondly, adding a catalyst in the monomer solution to perform free radical polymerization at a certain temperature; and finally, removing unreacted reactant through dialysis to obtain the glucose-sensitive insulin controlled-release biological material. The preparation method has the advantages of convenient implementation, fast reaction process, mild reaction condition and easy production preparation on a large scale. The obtained glucan-concanavalin A insulin controlled-release biological carrier material has the advantages of good glucose-sensitive property and excellent insulin controlled-release performance.
Description
(1) technical field
The present invention relates to a kind of preparation method of the biomaterial to glucose-sensitive, insulin controlled-release, particularly a kind of glucosan to glucose-sensitive-concanavalin A base insulin belongs to medicine controlled releasing and technical field of biological materials from controlled release bio-carrier material preparation new method.
(2) background technology
Insulin dependent diabetes mellitus (IDDM) (I type) is the chronic metabolic system disease of a kind of general, and is large to the harm of health.The insulin controlled release drug administration is the effective ways for the treatment of at present type i diabetes.In recent years for the intelligent insulin administration carrier system of self-interacting type vegetable lectin-carbohydrate derivative of type i diabetes research and development, for the treatment of diabetes provides a new way.This carrier system mainly utilizes the specific binding characteristic of vegetable lectin (such as concanavalin A) and oligosaccharide, polymer formation gel-sol change system with vegetable lectin and glucose or glucosan, by the competition combination of glucose molecule in plant external source collection element site that dissociate in the blood, so that carrier becomes dissolved colloidal state by gel state, uelralante; When blood sugar concentration was lower than certain value, colloidal sol became again gel network, restored to the original state, and insulin no longer discharges.This system is high to the specificity of glucose, can bring into play controlled-release function under the physiological environment in vivo, and the change between gel-sol is rapid, good reproducibility.Therefore, responsive to the variation of blood sugar concentration, response is good.
(Tanna etc. in the existing documents and materials, Biomaterials 2006,27:1586-1597) once utilize the ultraviolet induction polymerization, prepared glucosan to glucose-sensitive-concanavalin A base biological hydrogel material, can be used for the self-regulation controlled release of insulin.But this polymerization still is difficult for realization large-scale production, and resulting carrier system sensitive property reaches from the controlled release performance and still needs further to improve.
(3) summary of the invention
The purpose of this invention is to provide a kind of new method for preparing the intelligent insulin administration carrier system of self-interacting type vegetable lectin-carbohydrate derivative.
For reaching goal of the invention, the invention provides a kind of new method of utilizing free radical polymerisation process to prepare the intelligent insulin administration bio-carrier of self-interacting type vegetable lectin-carbohydrate derivative material, described method is as follows:
A kind of preparation method of the biomaterial to glucose-sensitive, insulin controlled-release, described method comprises: (1) is 1: 0.5～2.0 be dissolved in the buffer of pH5～9 with the glucan derivative monomer of the two keys of C=C with the concanavalin A derivatives monomer of the two keys of C=C with mass ratio, obtains the monomer solution that total monomer is 50～150mg/L; (2) in monomer solution, add catalyst, under 30～50 ℃, carry out Raolical polymerizable, react dialysis after finishing (dialyzer with deionized water and molecular cut off 8～15kDa was dialysed 3～7 days) and remove unreacted reactant, obtain described biomaterial; Described glucan derivative monomer with the two keys of C=C is the glucosan that C3～C6 thiazolinyl (preferred C3～C4 thiazolinyl) replaces, and is the Con A Concanavalin that C3～C6 thiazolinyl (preferred C3～C4 thiazolinyl) replaces with the concanavalin A derivant of the two keys of C=C; Described catalyst is the mixture of Ammonium persulfate. and tetramethylethylenediamine.
Preferably, described glucan derivative monomer with the two keys of C=C is pi-allyl glucosan or methacrylic glucosan, and molecular weight 10～100KDa, substitution value are 10～18%; Described concanavalin A derivant with the two keys of C=C is the pi-allyl Con A Concanavalin.
Described pH of buffer 5～9, concentration 10～200mM is preferably one of following: 1. phosphate buffer, 2. Tris-hydrochloride buffer, 3. phosphate-citric acid solution.
Glucan derivative monomer and concanavalin A derivatives monomer summation: Ammonium persulfate.: the mass ratio of tetramethylethylenediamine is 1: 0.02～0.1: 0.01～0.1.
Preferably, glucan derivative monomer and concanavalin A derivatives monomer summation: Ammonium persulfate.: the mass ratio of tetramethylethylenediamine is 1: 0.05: 0.02.
The preference temperature of Raolical polymerizable is at 30～60 ℃, response time 5～120min.
Described pi-allyl glucosan (or methacrylic glucosan) but and (Tanna etc. in the pi-allyl concanavalin A reference literature data, Biomaterials 2006, the response path that 27:1586-1597) provides prepares, but preparation condition is different.
Concrete, described methacrylic glucosan prepares by the following method: take the glucosan of molecular weight 10～100kDa and methacrylic anhydride as reaction substrate, take dimethyl sulfoxine as solvent, DMAP is catalyst, glucosan initial concentration 5.5～6.8mg/mL, methacrylic anhydride initial concentration 1.0～1.3mg/mL, the mass ratio of catalyst and methacrylic anhydride is 0.1～0.2: 1, under 48～55 ℃, carry out allylation derivatization reaction 12～36h, (8～15KDa) 1 weeks of dialyzer molecular cut off obtain the methacrylic glucosan of substitution value 10～18% in deionized water dialysis after reaction finishes.
Described pi-allyl glucosan prepares by the following method: take the glucosan of molecular weight 10～100kDa and acrylic anhydride as reaction substrate, take dimethyl sulfoxine as solvent, DMAP is catalyst, glucosan initial concentration 5.5～6.8mg/mL, acrylic anhydride initial concentration 1.0～1.3mg/mL, the mass ratio of catalyst and acrylic anhydride is 0.1～0.2: 1, under 48～55 ℃, carry out allylation derivatization reaction 12～36h, (8～15KDa) 1 weeks of dialyzer molecular cut off obtain the pi-allyl glucosan of substitution value 10～18% in deionized water dialysis after reaction finishes.
Described methacrylic Con A Concanavalin prepares by the following method: the phosphate buffer that concanavalin A and methacrylic anhydride is dissolved in mass ratio at 10: 1 20mM, pH7.4, be made into mass percent concentration and be 4.6% solution, at 51 ℃ of lower reaction 2h, (8～15KDa) 1 weeks of dialyzer molecular cut off obtained the methacrylic concanavalin A in the deionized water dialysis after reaction finished.
Described pi-allyl Con A Concanavalin prepares by the following method: the phosphate buffer that concanavalin A and methacrylic anhydride is dissolved in mass ratio at 10: 1 20mM, pH7.4, be made into mass percent concentration and be 4.6% solution, at 51 ℃ of lower reaction 2h, (8～15KDa) 1 weeks of dialyzer molecular cut off obtained the methacrylic concanavalin A in the deionized water dialysis after reaction finished.
Compare from controlled release bio-carrier material preparation technology with existing self-interacting type glucosan-concanavalin A base insulin, the difference of method provided by the invention is that preparation process adopts Raolical polymerizable.The beneficial effect of the inventive method is mainly reflected in: it is convenient to implement, course of reaction is rapid, mild condition, and the large-scale production preparation is very easy, gained glucosan-concanavalin A base insulin is good to the glucose-sensitive performance from controlled release bio-carrier material, from the controlled release function admirable.
(4) specific embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:
5.4g glucosan (molecular weight 10kDa) and 0.8g methacrylic anhydride are dissolved in the 80mL dimethyl sulfoxine, add the 0.08g DMAP, 48 ℃ of lower reaction 36h, dialyzer with deionized water and molecular cut off 8kDa was dialysed 7 days, lyophilization obtains the methacrylic glucosan monomer of substitution value 16%.
Getting concanavalin A 1g and methacrylic anhydride 0.1g is dissolved in the 24mL phosphate buffer (20mM, pH7.4) at 51 ℃ of lower reaction 2h, dialyzer with deionized water and molecular cut off 8kDa was dialysed 7 days, lyophilization obtains methacrylic concanavalin A monomer.
Get the above-mentioned methacrylic glucosan of 50mg monomer, the above-mentioned methacrylic concanavalin A of 25mg monomer is dissolved in 1.5mL phosphate-citrate buffer solution (50mM, pH 5), obtains monomer solution; In this monomer solution, add 7.5mg Ammonium persulfate. and 9.4mg tetramethylethylenediamine, in 30 ℃ of lower reaction 120min, dialyzer with deionized water and molecular cut off 8kDa was dialysed 3 days, removed unreacted reactant, obtained glucose-sensitive from the biomaterial of controlled release insulin.
Carry out with the phosphate buffer that contains 1mg/mL insulin and glucose that (method is seen document Tanna etc. from the controlled release performance test, Biomaterials 2006,27:1586-1597) show, the gel-sol change can occur according to the variation of environment glucose in solutions concentration in this carrier material, thereby well insulin is carried out from controlled release.This carrier material is to about 15～30min of sensitivity strain time of glucose, and concentration of glucose can be realized the gel-sol change in 1～15mg/mL scope, from the controlled release function admirable.
Embodiment 2:
5.5g glucosan (molecular weight 100kDa) and 1.3g methacrylic anhydride are dissolved in the 100mL dimethyl sulfoxine, add the 0.26g DMAP, 55 ℃ of lower reaction 12h, dialyzer with deionized water and molecular cut off 14kDa was dialysed 7 days, lyophilization obtains the methacrylic glucosan monomer of substitution value 12%; The preparation of methacrylic concanavalin A monomer is with embodiment 1.
Get the above-mentioned pi-allyl glucosan of 50mg monomer, the above-mentioned methacrylic concanavalin A of 50mg monomer is dissolved in 1mL Tris-hydrochloride buffer (10mM, pH 9), obtains monomer solution; In this monomer solution, add 2mg Ammonium persulfate. and 10mg tetramethylethylenediamine, in 60 ℃ of lower reaction 5min, dialysed 3 days with the dialyzer of deionized water and molecular cut off 10kDa, remove unreacted reactant, obtain glucose-sensitive from the biomaterial of controlled release insulin.
Carry out showing from the controlled release performance test that this carrier material can the gel-sol change occur according to the variation of environment glucose in solutions concentration with the phosphate buffer that contains 1mg/mL insulin and glucose, thereby well insulin is carried out from controlled release.This carrier material is to about 25～60min of sensitivity strain time of glucose, and concentration of glucose can be realized the gel-sol change in 3～15mg/mL scope, from the controlled release function admirable.
Embodiment 3:
6g glucosan (molecular weight 70kDa) and 1.2g acrylic anhydride are dissolved in the 100mL dimethyl sulfoxine, add the 0.18g DMAP, 50 ℃ of lower reaction 24h, dialyzer with deionized water and molecular cut off 10kDa was dialysed 7 days, lyophilization obtains the pi-allyl glucosan monomer of substitution value 18%.
Getting concanavalin A 1g and acrylic anhydride 0.1g is dissolved in the 24mL phosphate buffer (20mM, pH7.4) at 51 ℃ of lower reaction 2h, dialyzer with deionized water and molecular cut off 8kDa was dialysed 7 days, lyophilization obtains pi-allyl concanavalin A monomer.
Get the above-mentioned pi-allyl glucosan of 75mg monomer, the above-mentioned pi-allyl concanavalin A of 75mg monomer is dissolved in 1mL Tris-hydrochloride buffer (10mM, pH 8), obtains monomer solution; In this monomer solution, add 7.5mg Ammonium persulfate. and 3mg tetramethylethylenediamine, in 50 ℃ of lower reaction 20min, dialyzer with deionized water and molecular cut off 10kDa was dialysed 3 days, removed unreacted reactant, obtained glucose-sensitive from the biomaterial of controlled release insulin.
Carry out showing from the controlled release performance test with the phosphate buffer that contains 1mg/mL insulin and glucose, the gel-sol change can occur according to the variation of environment glucose in solutions concentration in this carrier material, about 10～20min of sensitivity strain time to glucose, concentration of glucose can be realized the gel-sol change in 0.5～20mg/mL scope, from the controlled release function admirable.
Embodiment 4:
6.2g glucosan (molecular weight 40kDa) and 1.1g methacrylic anhydride are dissolved in the 100mL dimethyl sulfoxine, add the 0.13g DMAP, 52 ℃ of lower reaction 24h, dialyzer with deionized water and molecular cut off 10kDa was dialysed 7 days, lyophilization obtains the methacrylic glucosan monomer of substitution value 15%; The preparation of pi-allyl concanavalin A monomer is with embodiment 3.
Get the above-mentioned methacrylic glucosan of 50mg monomer, the above-mentioned pi-allyl concanavalin A of 150mg monomer is dissolved in 2.4mL phosphate buffer (200mM, pH 7), obtains monomer solution; In this monomer solution, add 8mg Ammonium persulfate. and 5mg tetramethylethylenediamine, in 40 ℃ of lower reaction 60min, dialysed 3 days with the dialyzer of deionized water and molecular cut off 10kDa, remove unreacted reactant, obtain glucose-sensitive from the biomaterial of controlled release insulin.
Carry out showing from the controlled release performance test with the phosphate buffer that contains 1mg/mL insulin and glucose, the gel-sol change can occur according to the variation of environment glucose in solutions concentration in this carrier material, about 30～50min of sensitivity strain time to glucose, concentration of glucose can be realized the gel-sol change in 1.5～18mg/mL scope, from the controlled release function admirable.
Embodiment 5:
5.8g glucosan (molecular weight 20kDa) and 1g acrylic anhydride are dissolved in the 100mL dimethyl sulfoxine, add the 0.1g DMAP, 49 ℃ of lower reaction 12h, dialyzer with deionized water and molecular cut off 10kDa was dialysed 7 days, lyophilization obtains the pi-allyl glucosan monomer of substitution value 10%; The preparation of methacrylic concanavalin A monomer is with embodiment 1.
Get the above-mentioned methacrylic glucosan of 50mg monomer, the above-mentioned methacrylic concanavalin A of 75mg monomer is dissolved in 1.7mL phosphate buffer (150mM, pH 6), obtains monomer solution; In this monomer solution, add 4mg Ammonium persulfate. and 6.5mg tetramethylethylenediamine, in 45 ℃ of lower reaction 90min, dialyzer with deionized water and molecular cut off 10kDa was dialysed 3 days, removed unreacted reactant, obtained glucose-sensitive from the biomaterial of controlled release insulin.
Carry out showing from the controlled release performance test with the phosphate buffer that contains 1mg/mL insulin and glucose, the gel-sol change can occur according to the variation of environment glucose in solutions concentration in this carrier material, about 40～60min of sensitivity strain time to glucose, concentration of glucose can be realized the gel-sol change in 2.5～15mg/mL scope, from the controlled release function admirable.
Claims (4)
1. preparation method to the biomaterial of glucose-sensitive, insulin controlled-release, described method comprises: (1) is being dissolved in the buffer of pH5 ~ 9 with the glucan derivative monomer of the two keys of C=C with the concanavalin A derivatives monomer of the two keys of C=C of 1:0.5 ~ 2.0 with mass ratio, obtains the monomer solution that total monomer is 50 ~ 150mg/L; Described glucan derivative monomer with the two keys of C=C is pi-allyl glucosan or methacrylic glucosan, molecular weight 10 ~ 100KDa, substitution value 10 ~ 18%; Described concanavalin A derivant with the two keys of C=C is pi-allyl concanavalin A or methacrylic concanavalin A; (2) add catalyst in monomer solution, carry out Raolical polymerizable, unreacted reactant was removed in dialysis after reaction finished, and obtained described biomaterial; Described catalyst is the mixture of Ammonium persulfate. and tetramethylethylenediamine; Described glucan derivative monomer and concanavalin A derivatives monomer summation: Ammonium persulfate.: the mass ratio of tetramethylethylenediamine is 1:0.02 ~ 0.1:0.01 ~ 0.1;
Described methacrylic glucosan prepares by the following method: take the glucosan of molecular weight 10 ~ 100 kDa and methacrylic anhydride as reaction substrate, take dimethyl sulfoxine as solvent, DMAP is catalyst, glucosan initial concentration 5.5 ~ 6.8 mg/mL, methacrylic anhydride initial concentration 1.0 ~ 1.3 mg/mL, the mass ratio of catalyst and methacrylic anhydride is 0.1 ~ 0.2:1, under 48 ~ 55 ℃, carry out allylation derivatization reaction 12 ~ 36 h, deionized water was dialysed for 1 week after reaction finished, and obtained the methacrylic glucosan of substitution value 10 ~ 18%;
Described pi-allyl glucosan prepares by the following method: take the glucosan of molecular weight 10 ~ 100 kDa and acrylic anhydride as reaction substrate, take dimethyl sulfoxine as solvent, DMAP is catalyst, glucosan initial concentration 5.5 ~ 6.8 mg/mL, acrylic anhydride initial concentration 1.0 ~ 1.3 mg/mL, the mass ratio of catalyst and acrylic anhydride is 0.1 ~ 0.2:1, under 48 ~ 55 ℃, carry out allylation derivatization reaction 12 ~ 36 h, deionized water was dialysed for 1 week after reaction finished, and obtained the pi-allyl glucosan of substitution value 10 ~ 18%;
Described methacrylic concanavalin A prepares by the following method: with concanavalin A and methacrylic anhydride in mass ratio 10:1 be dissolved in the phosphate buffer of 20mM, pH7.4, be made into mass percent concentration and be 4.6% solution, at 51 ℃ of lower reaction 2 h, deionized water was dialysed for 1 week after reaction finished, and obtained the methacrylic concanavalin A;
Described pi-allyl concanavalin A prepares by the following method: with concanavalin A and acrylic anhydride in mass ratio 10:1 be dissolved in the phosphate buffer of 20mM, pH7.4, be made into mass percent concentration and be 4.6% solution, at 51 ℃ of lower reaction 2 h, deionized water was dialysed for 1 week after reaction finished, and obtained the pi-allyl concanavalin A.
2. the method for claim 1 is characterized in that described buffer is one of following: 1. phosphate buffer, 2. Tris-hydrochloride buffer, 3. Lin Suan Yan – citric acid solution.
3. the method for claim 1, it is characterized in that: glucan derivative monomer and concanavalin A derivatives monomer summation: Ammonium persulfate.: the mass ratio of tetramethylethylenediamine is 1:0.05:0.02.
4. the method for claim 1 is characterized in that described Raolical polymerizable carries out, response time 5 ~ 120min under 30 ~ 60 ℃.
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| CN103191437B (en) * | 2013-03-05 | 2015-03-11 | 常州大学 | Preparation method of controlled release carrier of glucose-responsive drug |
| CN106937948B (en) * | 2017-02-23 | 2019-07-05 | 青岛农业大学 | Concanavalin-starch nanometer granule preparation method and applications of glucose responding type |
| RU2652126C1 (en) * | 2017-10-17 | 2018-04-25 | Федеральное государственное бюджетное учреждение науки Ордена Трудового Красного Знамени Институт нефтехимического синтеза им. А.В. Топчиева Российской академии наук (ИНХС РАН) | Method of obtaining glucose-sensitive polymer hydrogels |
| CN109879977B (en) * | 2019-01-30 | 2022-03-04 | 中山大学 | Amphiphilic polysaccharide derivative containing cholesterol and phytohemagglutinin groups and preparation method and application thereof |
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| Jason D. Ehrick et al.,.Glucose Responsive Hydrogel Networks Based on Protein Recognition.《Macromolecular Bioscience》.2009,第9卷(第9期),864-868. * |
| Sangeeta Tanna et al.,.Glucose-responsive UV polymerised dextran–concanavalin A acrylic derivatised mixtures for closed-loop insulin delivery.《Biomaterials》.2005,第27卷(第8期),1586-1597. * |
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