CN102028936B - Method for preparing oral protein medicament - Google Patents
Method for preparing oral protein medicament Download PDFInfo
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- CN102028936B CN102028936B CN 201010588005 CN201010588005A CN102028936B CN 102028936 B CN102028936 B CN 102028936B CN 201010588005 CN201010588005 CN 201010588005 CN 201010588005 A CN201010588005 A CN 201010588005A CN 102028936 B CN102028936 B CN 102028936B
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- 239000003814 drug Substances 0.000 title claims abstract description 29
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 24
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 23
- 238000000034 method Methods 0.000 title abstract description 7
- 229920001661 Chitosan Polymers 0.000 claims abstract description 67
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 48
- 239000000017 hydrogel Substances 0.000 claims abstract description 17
- 230000009977 dual effect Effects 0.000 claims abstract description 15
- 238000003756 stirring Methods 0.000 claims abstract description 15
- 238000002156 mixing Methods 0.000 claims abstract description 13
- 238000005406 washing Methods 0.000 claims abstract description 8
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 7
- 230000001105 regulatory effect Effects 0.000 claims abstract description 7
- 239000000203 mixture Substances 0.000 claims abstract description 5
- 238000001035 drying Methods 0.000 claims abstract description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 38
- 229940098773 bovine serum albumin Drugs 0.000 claims description 37
- 235000010413 sodium alginate Nutrition 0.000 claims description 27
- 239000000661 sodium alginate Substances 0.000 claims description 26
- 229940005550 sodium alginate Drugs 0.000 claims description 26
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 25
- 238000002360 preparation method Methods 0.000 claims description 24
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 claims description 14
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 10
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 8
- HWXBTNAVRSUOJR-UHFFFAOYSA-N alpha-hydroxyglutaric acid Natural products OC(=O)C(O)CCC(O)=O HWXBTNAVRSUOJR-UHFFFAOYSA-N 0.000 claims description 7
- 229940009533 alpha-ketoglutaric acid Drugs 0.000 claims description 7
- 230000015572 biosynthetic process Effects 0.000 claims description 6
- 238000003786 synthesis reaction Methods 0.000 claims description 6
- WBZKQQHYRPRKNJ-UHFFFAOYSA-L disulfite Chemical compound [O-]S(=O)S([O-])(=O)=O WBZKQQHYRPRKNJ-UHFFFAOYSA-L 0.000 claims description 4
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 4
- 239000012279 sodium borohydride Substances 0.000 claims description 4
- IXIBAKNTJSCKJM-BUBXBXGNSA-N bovine insulin Chemical group C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)C(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 IXIBAKNTJSCKJM-BUBXBXGNSA-N 0.000 claims description 2
- 239000002253 acid Substances 0.000 abstract description 5
- JFCQEDHGNNZCLN-UHFFFAOYSA-N glutaric acid Chemical compound OC(=O)CCCC(O)=O JFCQEDHGNNZCLN-UHFFFAOYSA-N 0.000 abstract description 4
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 abstract description 3
- 239000001110 calcium chloride Substances 0.000 abstract description 3
- 229910001628 calcium chloride Inorganic materials 0.000 abstract description 3
- 239000011734 sodium Substances 0.000 abstract description 3
- 229910052708 sodium Inorganic materials 0.000 abstract description 3
- 238000004132 cross linking Methods 0.000 abstract 3
- 238000001914 filtration Methods 0.000 abstract 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 abstract 1
- 238000002791 soaking Methods 0.000 abstract 1
- 229910052938 sodium sulfate Inorganic materials 0.000 abstract 1
- 235000011152 sodium sulphate Nutrition 0.000 abstract 1
- 230000002194 synthesizing effect Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 49
- 239000000499 gel Substances 0.000 description 37
- 239000011324 bead Substances 0.000 description 30
- 230000008961 swelling Effects 0.000 description 17
- 238000004088 simulation Methods 0.000 description 14
- 239000000872 buffer Substances 0.000 description 10
- 210000001072 colon Anatomy 0.000 description 7
- 210000002784 stomach Anatomy 0.000 description 7
- 230000000968 intestinal effect Effects 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 230000001186 cumulative effect Effects 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000002329 infrared spectrum Methods 0.000 description 4
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 210000004347 intestinal mucosa Anatomy 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
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- 229960001126 alginic acid Drugs 0.000 description 2
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- 229920000615 alginic acid Polymers 0.000 description 2
- 150000004781 alginic acids Chemical class 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 229910001424 calcium ion Inorganic materials 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 210000001842 enterocyte Anatomy 0.000 description 2
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- 238000011049 filling Methods 0.000 description 1
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- 210000004051 gastric juice Anatomy 0.000 description 1
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- 229940088597 hormone Drugs 0.000 description 1
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Abstract
The invention discloses a method for preparing an oral protein medicament. The method comprises the following steps of: (a) synthesizing N-alpha-chitosan glutarate; and (b) preparing dual crosslinking hydrogel, namely, regulating pH of solution of N-alpha-chitosan glutarate to be 0.5 to 6.5 by using solution of sodium hydroxide, adding solution of sodium alga acid and mixing uniformly, adding the oral protein medicament and dripping into solution of calcium chloride; after dripping is completed, stirring and crosslinking for 0.5 to 3 hours, filtering, washing, soaking in solution of sodium sulfate and stirring and crosslinking for 0.5 to 4 hours, and filtering, washing and drying at room temperature until the mixture has constant weight. Compared with the prior art, the method has the advantages that: the prepared medicament carrier is hardly disintegrated under an acid condition, and the medicament is easily absorbed.
Description
Technical field
The present invention relates to the preparation method of oral protein medicament, particularly modification of chitosan is as the oral protein medicament preparation method of pharmaceutical carrier.
Background technology
Along with the development of DNA recombinant technique, the bioactive molecules such as a lot of protein, enzyme, polypeptide become commercial medicine and drop into clinical practice.Although oral is protein drug most convenient, the easiest administering mode of being accepted by patient, but because protein drug very easily sour degeneration and enzymatic degradation in digestive tract, the protein macromolecule medicine is difficult to see through the intestinal mucosa absorbed into serum in addition, so protein and polypeptide drugs are still mainly by the injection system administration, even this administering mode brings misery to patient and causes easily infecting at present.Oral protein class pharmaceutical carrier becomes the study hotspot of pharmaceutics in recent years, and the natural polymer hydrogel material of pH sensitivity has received great concern.
Chitosan is a kind of natural polysaccharide of commercial large-scale production, because the biocompatibility of its height, nontoxic, biodegradable, intestinal mucosa adhesiveness and open the closely performance such as connection of enterocyte, chitosan is widely used in pharmaceutical field.Chitosan has been used as the carrier of hormone, protein, enzyme and gene.Because its apparent pKa=5.6, chitosan is only soluble in the sour environment, and this has limited it as the application of protein drug oral carrier.
Sodium alginate is a kind of soluble-salt of alginic acid, can or form gel under lower sour environment in polyvalent cation such as calcium ion existence.Because sodium alginate gel is easy to preparation, add its inherent attributes such as porous, biocompatibility and mucosa-adherent, the sodium alginate derivant is widely used in field of medicaments.Various sodium alginates, chitosan complexes also have been used as the carrier of targeted drug delivery and controlled release.
At present, both at home and abroad so long as chitosan and sodium alginate are directly prepared hydrogel with methods such as emulsification and cross linkeds, hydrogel easily disintegrate under sour environment that this method is prepared.
Summary of the invention
Technical problem to be solved by this invention provide a kind of under the gastric juice environment swelling and at the process for preparing medicine of small intestinal environment swelling not.
The technical scheme of technical solution problem of the present invention is: a kind of preparation method of oral protein medicament comprises the synthesis step of (a) N-α-1,3-propanedicarboxylic acid chitosan, the preparation process of (b) dual cross-linked hydrogel;
The preparation process of described (b) dual cross-linked hydrogel is:
Be the N-α-1,3-propanedicarboxylic acid chitosan solution of 0.5-3% with weight concentration, regulate the pH to 0.5-6.5 of N-α-1,3-propanedicarboxylic acid chitosan solution with the sodium hydroxide solution of 0.5-2M, adding weight concentration is the sodium alginate soln mixing of 0.5-3% again, add oral protein medicament, splash in the weight concentration 1.5-2% calcium chloride solution, after dropwising, stir crosslinked 0.5-3h, filter, washing, the % metabisulfite solution that is immersed in again weight concentration 1.5-2 stirs crosslinked 0.5-4h, filters, washing, be dried to constant weight under the room temperature, sodium alginate, N-α-1,3-propanedicarboxylic acid chitosan, the mass ratio of oral protein medicament is 1: 0.3-0.9: 0.5-1.5.
Described oral protein medicament is insulin, bovine serum albumin.
The synthesis step of described (a) N-α-1,3-propanedicarboxylic acid chitosan:
In 1-2% (v/v) acetic acid solution that chitosan is dissolved in, add again α-ketoglutaric acid, the pH value of regulating mentioned solution with the 0.5-2M sodium hydroxide solution is 3.5-5.5, room temperature reaction 2-6 hour, add again sodium borohydride, the pH value of regulating said mixture with the 0.1-1M hydrochloric acid solution is 4.5-7.5, reaction is not less than 24 hours, add 90-95% (v/v) ethanol cessation reaction, filter washing, drying, namely obtain N-α-1,3-propanedicarboxylic acid chitosan, the mass ratio of chitosan, α-ketoglutaric acid, sodium borohydride is 1: 1.5-3.5: 0.3-1.5.
As shown in Figure 1: use the α-ketoglutaric acid beautify chitosan, can improve its water solublity, and chitosan has the biocompatibility of height, the sticking tack of nontoxic, biodegradable, intestinal mucosa and open the closely performance such as connection of enterocyte, sodium alginate is a kind of soluble-salt of alginic acid, can or form gel under lower sour environment in polyvalent cation such as calcium ion existence.After both mixings, dually more crosslinkedly may access substantially swelling not of acid condition, and under alkali condition swelling behavior.
The present invention compared with prior art, prepared pharmaceutical carrier, easy disintegrating not under acid condition, and medicine is absorbed easily.
Description of drawings
Fig. 1 is the synthetic principles of chemistry figure of N-α-1,3-propanedicarboxylic acid chitosan.
Fig. 2 is the infrared spectrum of sodium alginate/N-α-1,3-propanedicarboxylic acid chitosan gel rubber pearl, sodium alginate and N-α-1,3-propanedicarboxylic acid chitosan.
Fig. 3 is the swelling character of gel beads (embodiment 1,2,3,4,5) under the pH environment of simulation stomach.
Fig. 4 is the swelling character of gel beads (embodiment 1,2,3,4,5) under the pH environment of simulation small intestinal.
Fig. 5 is the swelling character of gel beads (embodiment 1,2,3,4,5) under the pH environment of simulation colon.
Fig. 6 is that the bag of bovine serum albumin BSA carries rate.
Fig. 7 is in the gel beads, and BSA is cumulative release in the buffer of simulation stomach (pH1.2).
Fig. 8 is in the gel beads, and BSA is cumulative release in the buffer of simulation small intestinal (pH6.8).
Fig. 9 is in the gel beads, and BSA is cumulative release in the buffer of simulation colon (pH7.4).
Figure 10 is that gel beads is put into simulation stomach (SGF) buffer 3h, and then puts into simulation colon (SCF) buffer 4h, BSA preparation.
The specific embodiment
Be described further below in conjunction with the technical scheme of specific embodiment to invention.
Embodiment 1
(a) N-α-1,3-propanedicarboxylic acid chitosan is synthetic:
The synthesis step of N-α-1,3-propanedicarboxylic acid chitosan: the chitosan of 4.5 grams is dissolved in 1% (v/v) acetic acid solution of 100ml, the α-ketoglutaric acid that adds again 7.2 grams, pH value with 1M sodium hydroxide solution regulator solution is 5, stirring at room 4 hours, add 2 gram sodium borohydrides, after stirring, the pH value of regulating said mixture with the 1M hydrochloric acid solution is 6.5 again, stirring reaction 24 hours, adding volume ratio is 95% ethanol cessation reaction.Filter and obtain N-α-1,3-propanedicarboxylic acid chitosan polymer, wash 3 to 4 times with ethanol and ether respectively, in infrared drier, be dried to constant weight.
(b) preparation of dual cross-linked hydrogel
Be 2% N-α-1,3-propanedicarboxylic acid chitosan solution 30ml with weight concentration, regulate the pH to 5.0 of N-α-1,3-propanedicarboxylic acid chitosan solution with the sodium hydroxide solution of 1M, add again weight concentration and be 2% sodium alginate soln 70ml mixing, splash in excessive weight concentration 2% calcium chloride solution, after dropwising, stirred crosslinked 0.5 hour, filter, remove the unreacted calcium chloride in gel beads surface for 3 to 4 times with distilled water flushing, the metabisulfite solution that is immersed in again excessive weight concentration 2% stirs crosslinked 1h, filter, repeatedly wash 3 to 4 times with distilled water, be dried to constant weight under the room temperature.
The infrared spectrum of sodium alginate/N-α-1,3-propanedicarboxylic acid chitosan gel rubber pearl, sodium alginate and N-α-1,3-propanedicarboxylic acid chitosan as shown in Figure 2, three's infrared spectrum relatively, wave number 1609cm in the made gel beads infrared spectrum of embodiment 1
-1The asymmetric shock absorbing peak of place's sodium alginate carboxyl obviously weakens and moves (blue shift) to high wave number, in addition wave number 1568cm
-1The characteristic of place's amino of chitosan is sheared the shock absorbing peak and is obviously weakened.
Embodiment 2:
Except in the preparation of (b) dual cross-linked hydrogel,
" be 2% N-α-1,3-propanedicarboxylic acid chitosan solution 10ml with weight concentration, regulate the pH to 4.5 of N-α-1,3-propanedicarboxylic acid chitosan solution with the sodium hydroxide solution of 1M, add again weight concentration and be 2% sodium alginate soln 90ml mixing, "
Outward, all the other are identical with embodiment 1.
Embodiment 3:
Except in the preparation of (b) dual cross-linked hydrogel,
" be 2% N-α-1,3-propanedicarboxylic acid chitosan solution 20ml with weight concentration, regulate the pH to 4.8 of N-α-1,3-propanedicarboxylic acid chitosan solution with the sodium hydroxide solution of 1M, add again weight concentration and be 2% sodium alginate soln 80ml mixing, "
Outward, all the other are identical with embodiment 1.
Embodiment 4:
Except in the preparation of (b) dual cross-linked hydrogel,
" be 2% N-α-1,3-propanedicarboxylic acid chitosan solution 40ml with weight concentration, regulate the pH to 4.5 of N-α-1,3-propanedicarboxylic acid chitosan solution with the sodium hydroxide solution of 1M, add again weight concentration and be 2% sodium alginate soln 60ml mixing, "
Outward, all the other are identical with embodiment 1.
Embodiment 5:
Except in the preparation of (b) dual cross-linked hydrogel,
" be 2% N-α-1,3-propanedicarboxylic acid chitosan solution 50ml with weight concentration, regulate the pH to 4.5 of N-α-1,3-propanedicarboxylic acid chitosan solution with the sodium hydroxide solution of 1M, add again weight concentration and be 2% sodium alginate soln 50ml mixing, "
Outward, all the other are identical with embodiment 1.
Embodiment 6
1. the swelling of gel beads is measured
Take by weighing a certain amount of (W
0) the xerogel pearl place respectively the buffer solution of 37 ℃ of slow joltings, buffer solution is simulated respectively the pH environment of stomach, small intestinal and colon.Take out gel beads at specific interval, paper using sucks the unnecessary water in gel beads surface, at scales/electronic balance weighing (W
t).Experiment repeats 3 times.The swelling ratio of gel beads calculates with following formula:
S(t)＝(W
t-W
0)/W
0
Wt and W
0Be respectively is gel beads weight in wet base and dry weight.
2. the swelling character of gel beads
Embodiment 1,2,3,4,5 swelling character are shown in Fig. 3,4,5 under the pH environment of simulation stomach, small intestinal and colon, the swelling ratio of gel pearl increases along with the increase of the ratio of N-α-1,3-propanedicarboxylic acid chitosan in sodium alginate/N-α-1,3-propanedicarboxylic acid chitosan, and embodiment 5 made gel beads behind 45min with regard to disintegrate, this phenomenon is because N-α-1,3-propanedicarboxylic acid chitosan has more negative charge than sodium alginate, and chitosan side-chain radical α-1,3-propanedicarboxylic acid has weakened the active force between the polymer.The high swelling ratio of the gel beads that embodiment 5 is made and disintegration rate cause being difficult to the protection medicine under the low pH environment on gastrointestinal top, the medicine that bag carries can discharge fast in the gastrointestinal bottom.
The result shows embodiment 1,2,3, when 4 swelling ratios pH6.8 and are pH1.2 respectively 6 times and 11 times at 7.4 o'clock.This is because in sodium alginate/N-α-1,3-propanedicarboxylic acid chitosan, and carboxyl is main functional group, is in the nonionic state in pH1.2, electrostatic repulsion a little less than, so the expansion ratio is less.Along with pH value is increased to 6.8 and 7.4, be higher than its pKa=4.75, carboxyl (COOH) becomes ionic species (COO fully
-).At COO
-Between have powerful electrostatic repulsion to cause high-hydroscopicity and high swelling ratio.
Embodiment 7
Bag carries the preparation of the dual cross-linked gel pearl of bovine serum albumin
(a) N-α-1,3-propanedicarboxylic acid chitosan is synthetic:
The synthesis step of N-α-1,3-propanedicarboxylic acid chitosan: the chitosan of 4.5 grams is dissolved in 1% (v/v) acetic acid solution of 100ml, the α-ketoglutaric acid that adds again 7.2 grams, pH value with 1M sodium hydroxide solution regulator solution is 5, stirring at room 4 hours, add 2 gram sodium borohydrides, after stirring, the pH value of regulating said mixture with the 1M hydrochloric acid solution is 6.5 again, stirring reaction 24 hours, adding volume ratio is 95% ethanol cessation reaction.Filter and obtain N-α-1,3-propanedicarboxylic acid chitosan polymer, wash 3 to 4 times with ethanol and ether respectively, in infrared drier, be dried to constant weight.
(b) preparation of dual cross-linked hydrogel
Be 2% N-α-1,3-propanedicarboxylic acid chitosan solution 30ml with weight concentration, regulate the pH to 5.0 of N-α-1,3-propanedicarboxylic acid chitosan solution with the sodium hydroxide solution of 1M, add again weight concentration and be 2% sodium alginate soln 70ml mixing, then 0.4 bovine serum albumin (BSA) that restrains is added in the mixed solution and dissolve, splash in excessive weight concentration 2% calcium chloride solution, after dropwising, stirred crosslinked 0.5 hour, filter, remove the unreacted calcium chloride in gel beads surface for 3 to 4 times with distilled water flushing, the metabisulfite solution that is immersed in again excessive weight concentration 2% stirs crosslinked 1h, filter, repeatedly wash 3 to 4 times with distilled water, be dried to constant weight under the room temperature.
Embodiment 8
Except in the preparation of (b) dual cross-linked hydrogel,
" being 2% N-α-1,3-propanedicarboxylic acid chitosan solution 10ml with weight concentration; regulate the pH to 4.5 of N-α-1,3-propanedicarboxylic acid chitosan solution with the sodium hydroxide solution of 1M; add again weight concentration and be 2% sodium alginate soln 90ml mixing; then the bovine serum albumin (BSA) of 0.4 gram is added in the mixed solution and dissolve, "
Outward, all the other are identical with embodiment 7.
Embodiment 9
Except in the preparation of (b) dual cross-linked hydrogel,
" being 2% N-α-1,3-propanedicarboxylic acid chitosan solution 20ml with weight concentration; regulate the pH to 4.5 of N-α-1,3-propanedicarboxylic acid chitosan solution with the sodium hydroxide solution of 1M; add again weight concentration and be 2% sodium alginate soln 80ml mixing; then the bovine serum albumin (BSA) of 0.4 gram is added in the mixed solution and dissolve, "
Outward, all the other are identical with embodiment 7.
Except in the preparation of (b) dual cross-linked hydrogel,
" being 2% N-α-1,3-propanedicarboxylic acid chitosan solution 40ml with weight concentration; regulate the pH to 4.5 of N-α-1,3-propanedicarboxylic acid chitosan solution with the sodium hydroxide solution of 1M; add again weight concentration and be 2% sodium alginate soln 60ml mixing; then the bovine serum albumin (BSA) of 0.4 gram is added in the mixed solution and dissolve, "
Outward, all the other are identical with embodiment 7.
Embodiment 11:
The BSA bag carries the mensuration of rate (LE)
Bag carries rate (LE) and is calculated as follows:
LE (%)=(total amount of the BSA that uses in the total amount of the BSA that bag carries in the pearl/experiment) * 100%
The bag of BSA carries rate (LE)
The bag of BSA carries rate as shown in Figure 6.We can see from figure, when the weight of BSA account for the gel beads quality 20% the time, bag carries a rate and can reach more than 92%.Simultaneously, along with the N-α-increase of 1,3-propanedicarboxylic acid chitosan in the gel beads total amount, the bag of BSA carries rate and also can constantly rise, because the BSA bag acid condition (pH5.0) when carrying gel beads is higher than the isoelectric point, IP (4.7) of BSA, but is lower than pKa value (6.3) amino on the chitosan chain.In this case, electronegative BSA will finish bag by electrostatic attraction with positively charged amino on the chitosan chain
Embodiment 12:
The mensuration of BSA release in vitro rate
At the external release rate of measuring in the following method BSA: 100mg embodiment 7,8,9,10 made gel beads are added respectively in the buffer of 20ml simulation stomach (pH1.2), simulation small intestinal (pH6.8), simulation colon (pH7.4), continue slow jolting under 37 ℃ of conditions.Every 15 minutes, get 5ml supernatant (filling into simultaneously the fresh buffer of equal volume in the solution) with ultraviolet spectrophotometer at the 280nm place, mensuration is the amount of contained BSA wherein.The cumulative release amount of BSA is calculated by standard curve.The experiment triplicate.
Its result is shown in Fig. 7,8,9, in the pH1.2 buffer, the release rate of BSA is relatively low, the BSA of 14-18% of only having an appointment in the 75min discharges from gel beads, and its cumulative release amount reaches balance subsequently, and this is low relevant with gel beads expansion rate in the solution of pH1.2, when pH reaches 6.8 and 7.4, along with gel beads constantly expands, the release rate of BSA improves rapidly, reaches about 100% in three hours.
Embodiment 13:
Claims (2)
1. the preparation method of an oral protein medicament is characterized in that: comprise the synthesis step of (a) N-α-1,3-propanedicarboxylic acid chitosan, the preparation process of (b) dual cross-linked hydrogel;
The synthesis step of described (a) N-α-1,3-propanedicarboxylic acid chitosan:
In 1-2% (v/v) acetic acid solution that chitosan is dissolved in, add again α-ketoglutaric acid, the pH value of regulating mentioned solution with the 0.5-2M sodium hydroxide solution is 3.5-5.5, room temperature reaction 2-6 hour, add again sodium borohydride, the pH value of regulating said mixture with the 0.1-1M hydrochloric acid solution is 4.5-7.5, reaction is not less than 24 hours, add 90-95% (v/v) ethanol cessation reaction, filter washing, drying, namely obtain N-α-1,3-propanedicarboxylic acid chitosan, the mass ratio of chitosan, α-ketoglutaric acid, sodium borohydride is 1: 1.5-3.5: 0.3-1.5;
The preparation process of described (b) dual cross-linked hydrogel is:
Be the N-α-1,3-propanedicarboxylic acid chitosan solution of 0.5-3% with weight concentration, regulate the pH to 0.5-6.5 of N-α-1,3-propanedicarboxylic acid chitosan solution with the sodium hydroxide solution of 0.5-2M, adding weight concentration is the sodium alginate soln mixing of 0.5-3% again, add oral protein medicament, splash in the weight concentration 1.5-2% calcium chloride solution, after dropwising, stir crosslinked 0.5-3h, filter, washing, the metabisulfite solution that is immersed in again weight concentration 1.5-2% stirs crosslinked 0.5-4h, filters, washing, be dried to constant weight under the room temperature, sodium alginate, N-α-1,3-propanedicarboxylic acid chitosan, the mass ratio of oral protein medicament is 1: 0.3-0.9: 0.5-1.5.
2. the preparation method of a kind of oral protein medicament according to claim 1, it is characterized in that: described oral protein medicament is insulin, bovine serum albumin.
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| CN1810867A (en) * | 2006-02-16 | 2006-08-02 | 武汉理工大学 | Prepn of sodium alginate/chitosan mixture gel |
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| CN1810867A (en) * | 2006-02-16 | 2006-08-02 | 武汉理工大学 | Prepn of sodium alginate/chitosan mixture gel |
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| 干扰素壳聚糖/海藻酸钠微囊控释制剂载体的初步研究;石晓丽;《中国生物制品学杂志》;20070228;第20卷(第2期);117-118 * |
| 石晓丽.干扰素壳聚糖/海藻酸钠微囊控释制剂载体的初步研究.《中国生物制品学杂志》.2007,第20卷(第2期),117-118. |
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