CN109876147A - A kind of preparation method and applications of fibroin albumen microsphere drug slow-released carrier - Google Patents
A kind of preparation method and applications of fibroin albumen microsphere drug slow-released carrier Download PDFInfo
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- CN109876147A CN109876147A CN201910153624.XA CN201910153624A CN109876147A CN 109876147 A CN109876147 A CN 109876147A CN 201910153624 A CN201910153624 A CN 201910153624A CN 109876147 A CN109876147 A CN 109876147A
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- preparation
- slow
- fibroin albumen
- released carrier
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- 239000003814 drug Substances 0.000 title claims abstract description 79
- 108010022355 Fibroins Proteins 0.000 title claims abstract description 78
- 229940079593 drug Drugs 0.000 title claims abstract description 69
- 239000004005 microsphere Substances 0.000 title claims abstract description 41
- 238000002360 preparation method Methods 0.000 title claims abstract description 41
- 210000000988 bone and bone Anatomy 0.000 claims abstract description 56
- 241001494479 Pecora Species 0.000 claims abstract description 50
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims abstract description 48
- 239000002245 particle Substances 0.000 claims abstract description 38
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 30
- 239000012460 protein solution Substances 0.000 claims abstract description 26
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 25
- 239000004408 titanium dioxide Substances 0.000 claims abstract description 24
- 229920001872 Spider silk Polymers 0.000 claims abstract description 23
- 239000002105 nanoparticle Substances 0.000 claims abstract description 23
- 239000002086 nanomaterial Substances 0.000 claims abstract description 19
- 239000000725 suspension Substances 0.000 claims abstract description 19
- 238000000502 dialysis Methods 0.000 claims abstract description 10
- 238000001914 filtration Methods 0.000 claims abstract description 9
- 238000004108 freeze drying Methods 0.000 claims abstract description 8
- 230000020764 fibrinolysis Effects 0.000 claims abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 235000019687 Lamb Nutrition 0.000 claims description 11
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- 239000002374 bone meal Substances 0.000 claims description 11
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- UGVQELHRNUDMAA-BYPYZUCNSA-N Gly-Ala-Gly Chemical compound [NH3+]CC(=O)N[C@@H](C)C(=O)NCC([O-])=O UGVQELHRNUDMAA-BYPYZUCNSA-N 0.000 description 8
- JURQXQBJKUHGJS-UHFFFAOYSA-N Ser-Ser-Ser-Ser Chemical compound OCC(N)C(=O)NC(CO)C(=O)NC(CO)C(=O)NC(CO)C(O)=O JURQXQBJKUHGJS-UHFFFAOYSA-N 0.000 description 8
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- 238000012360 testing method Methods 0.000 description 6
- INLIXXRWNUKVCF-JTQLQIEISA-N Gly-Gly-Tyr Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 INLIXXRWNUKVCF-JTQLQIEISA-N 0.000 description 5
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- CSMYMGFCEJWALV-WDSKDSINSA-N Gly-Ser-Gln Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(N)=O CSMYMGFCEJWALV-WDSKDSINSA-N 0.000 description 4
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- RJIVPOXLQFJRTG-LURJTMIESA-N Gly-Arg-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N RJIVPOXLQFJRTG-LURJTMIESA-N 0.000 description 2
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- CCBIBMKQNXHNIN-ZETCQYMHSA-N Gly-Leu-Gly Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O CCBIBMKQNXHNIN-ZETCQYMHSA-N 0.000 description 2
- HQSKKSLNLSTONK-JTQLQIEISA-N Gly-Tyr-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CC1=CC=C(O)C=C1 HQSKKSLNLSTONK-JTQLQIEISA-N 0.000 description 2
- AFCARXCZXQIEQB-UHFFFAOYSA-N N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CCNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 AFCARXCZXQIEQB-UHFFFAOYSA-N 0.000 description 2
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- SRSPTFBENMJHMR-WHFBIAKZSA-N Ser-Ser-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O SRSPTFBENMJHMR-WHFBIAKZSA-N 0.000 description 2
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- GYDFRTRSSXOZCR-ACZMJKKPSA-N Ser-Ser-Glu Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O GYDFRTRSSXOZCR-ACZMJKKPSA-N 0.000 description 1
- 229910003077 Ti−O Inorganic materials 0.000 description 1
- HIINQLBHPIQYHN-JTQLQIEISA-N Tyr-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H](N)CC1=CC=C(O)C=C1 HIINQLBHPIQYHN-JTQLQIEISA-N 0.000 description 1
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Abstract
The present invention relates to slow releasing carrier of medication preparation fields, and in particular to a kind of preparation method and applications of fibroin albumen microsphere drug slow-released carrier.The preparation method of fibroin albumen microsphere drug slow-released carrier provided by the invention, including by recombinant spider silk protein Fibrinolysis, silk fibroin protein solution is obtained after filtering, dialysis, concentration；Silk fibroin protein solution is placed in bag filter, nano material is added in Xiang Suoshu bag filter, then bag filter is placed in 22-25 DEG C of water-bath, is handled at the uniform velocity shaking table, obtain microballoon suspension；Microballoon suspension is washed with deionized, is centrifugated, freeze-drying obtains fibroin albumen microsphere drug slow-released carrier；The nano material is titanium dioxide nanoparticle and nanometer sheep bone particle.The slow releasing carrier of medication that the preparation method provided through the invention obtains can take into account faster drug starting rate of release and excellent slow release effect, have a good application prospect.
Description
Technical field
The present invention relates to slow releasing carrier of medication preparation fields, and in particular to a kind of fibroin albumen microsphere drug slow-released carrier
Preparation method and applications.
Background technique
Since some drugs are irritant to esophagus and gastric mucosa, be easy to volatilize or easily sucking tracheae etc. is former in daily life
Cause is often loaded into capsule, avoids the influence of moisture, air, light, both medicine property of medicine was prevented not to be destroyed, and also protects digestion
Organ and respiratory tract.In addition, capsule can delay release and the positioning release medicine of drug, reach sustained release prolongation of effect and positioning intelligent administration
Effect.Currently, the common carrier material of Loaded Microspheres Drug Delivery System has sodium alginate, poly lactide-glycolide acid (PLGA), shell
Glycan (CS) and its biodegradation materials such as derivative and fibroin.Wherein from the fibroin albumen of silk because of its biofacies
Capacitive is good, biodegradable, physical and chemical and biological stability, extremely low toxicity and the advantages that higher load pharmacological property, uses extensively
Make the carrier of drug, enzyme and vaccine.But the fibroin microsphere preparation method for the nanometer and submicron-scale reported at present is complicated, delays
Release the slow release effect that carrier is more focused on drug, be allowed to drug starting rate of release it is slow, drug slow effect cannot be considered in terms of drug and see
The fast double requirements with sustained release of effect.
Prior art CN105832682A discloses a kind of system of silk fibroin porous microsphere drug slow-released carrier of honeycomb
Preparation Method in turn includes the following steps: (1) dissolving the fibrous fibroin formed after degumming silkworm cocoons, by filtering, dialysis, dense
Silk fibroin water solution is obtained after contracting；(2) the silk fibroin protein solution concentration in step (1) is adjusted to 0.1-50mg/mL and surpassed
Sound is dispersed, and is fitted into the human simulation body fluid for being placed in different times concentration in bag filter and is impregnated, and solution temperature is 37 DEG C, even
It is handled in fast shaking table；(3) the shaking table processing time is 1-14 days, and the every 24 hours human simulation body fluid replaced in step (2) obtains micro-
Ball suspension；(4) by step (3) microballoon suspension deionized water and dehydrated alcohol wash for several times, be centrifugated, freezing
It is dry or 60 DEG C at it is 5-48 hour dry, obtain partial size for 0.2-5 μm of apatite/fibroin albumen microsphere powder, as drug
Slow-released carrier.Its drug of the slow releasing carrier of medication obtained through the above scheme starting rate of release is slow, drug slow effect, Bu Nengjian
Care for the double requirements that drug is quick and is sustained.
Summary of the invention
It is an object of the invention to overcome in the prior art microsphere sustained-release carrier cannot be considered in terms of drug starting rate of release it is fast
The problem of with sustained release, and then a kind of preparation method and applications of fibroin albumen microsphere drug slow-released carrier are provided.
In order to achieve the above objectives, the present invention adopts the following technical scheme:
A kind of preparation method of fibroin albumen microsphere drug slow-released carrier, comprising the following steps:
1) by recombinant spider silk protein Fibrinolysis, silk fibroin protein solution is obtained after filtering, dialysis, concentration；
2) silk fibroin protein solution for obtaining step 1) is placed in bag filter, and nano material is added in Xiang Suoshu bag filter,
Then bag filter is placed in 22-25 DEG C of water-bath, is handled at the uniform velocity shaking table, obtain microballoon suspension；
3) the microballoon suspension that step 2) obtains is washed with deionized, is centrifugated, freeze-drying obtains fibroin egg
White microsphere drug slow-released carrier；
Nano material described in step 2) is titanium dioxide nanoparticle and nanometer sheep bone particle.
Preferably, the amino acid sequence of recombinant spider silk protein such as SEQ ID NO.1 institute in the recombinant spider silk protein fiber
Show.
High efficient expression process of the above-mentioned recombinant spider silk protein in Escherichia coli is as follows:
(1) use of the recombinant spider silk protein sequence according to shown in SEQ ID NO.1 and Escherichia coli preference codon is former
Then, the nucleotide sequence of design coding and the recombinant spider silk protein of recombinant spider silk protein consensus amino acid sequence, is shown in sequence table
The nucleotide sequence of SEQ ID NO.2.Above-mentioned nucleotide sequence is named as MaSp1 gene, company synthesizes by gene chemical synthesis
MaSp1 gene is cloned into the prokaryotic expression carrier pET-28a+ containing T7 strong promoter, is built into containing artificial
The recombinant plasmid pET-28a+-MaSp1 of the MaSp1 gene of synthesis；
(2) e. coli bl21 (DE3) competent cell is prepared, with heat shock (42 DEG C heat shock 45 seconds) by recombinant plasmid
PET-28a+-MaSp1 is converted into host cell BL21 (DE3), obtains the engineered strain containing recombinant plasmid；
(3) engineered strain is inoculated into LB culture medium solution, 37 DEG C, 220rpm shaking table culture, when recombinant liquid
When concentration OD600 reaches 0.6-0.8, be added 0.5mmol/L IPTG, 37 DEG C inducing expression 6 hours, then cracked, by institute
The cell pyrolysis liquid obtained carries out SDS-PAGE identification, and the result is shown in Figure 1 takes the cell pyrolysis liquid to carry out recombinant spider silk protein
Extraction, the purifying of MaSp1, carries out Western blotting identification for recombinant spider silk protein MaSp1 after purification, as a result sees figure
2.Expression vector pET-28a of the present invention, e. coli bl21, LB culture medium, TEV enzyme enzyme solution are commercial product.
Recombination spider's thread egg is prepared by above-mentioned recombinant spider silk protein using conventional technical means in the art in the present invention
White fiber, process are as follows: above-mentioned recombinant spider silk protein being dissolved in the formic acid that mass fraction is 98%, obtain 25% (w/
V) electrostatic spinning solution, then obtain as-spun fibre through conventional spinning processes, then by as-spun fibre drawn, drying, crimp, cut
The aftertreatment technologies such as disconnected, packing obtain recombinant spider silk protein fiber.
Preferably, the concentration of the silk fibroin protein solution is 0.8-1.0wt%；Bag filter described in step 2) retains molecule
Amount is 8-10kD.
Preferably, the mass ratio of the nano material and the silk fibroin protein solution is 0.1-0.3:100.
Preferably, the mass ratio of the titanium dioxide nanoparticle and nanometer sheep bone particle is 0.3-0.5:1.
Preferably, the nanometer sheep bone particle the preparation method is as follows: by black bone sheep sheep bone degreasing, pulverize and sieve, obtain
Lamb bone meal；Lamb bone meal is dissolved in water, is digested with pepsin, a nanometer sheep bone particle is obtained.
Preferably, the partial size of the titanium dioxide nanoparticle is 30-50nm；The partial size of the nanometer sheep bone particle is
30-50nm。
The present invention provides a kind of drug of the preparation method preparation of fibroin albumen microsphere drug slow-released carrier described above
Slow-released carrier.
The present invention provides a kind of slow releasing carrier of medication described above and is preparing the application in slow releasing pharmaceutical.
The invention has the advantages that:
1, the present invention provides a kind of preparation method of fibroin albumen microsphere drug slow-released carrier, recombination used in the present invention
Spider's thread protein has excellent biocompatibility, while titanium dioxide nanoparticle and nanometer sheep being added into silk fibroin protein solution
Osseous granules, by the mating reaction of titanium dioxide nanoparticle and nanometer sheep bone particle, so that the slow releasing carrier of medication of preparation can
To take into account faster drug starting rate of release and excellent slow release effect, and there is degradability, before there is good application
Scape.
Ti-O bond polarity is larger in nano-titanium dioxide, easy adsorbed water molecule, and the hydrone of absorption is solved because polarizing
From, easily formation hydroxyl, reticular structure is formed in the form of hydrogen bond with carboxyl in spider's thread protein or amino groups.Nanometer sheep bone
Grain has preferably porosity and biggish average pore size, and fibroin is added in nanometer sheep bone particle and titanium dioxide nanoparticle cooperation
The slow release effect of slow releasing carrier of medication is greatly strengthened in protein solution, while improving drug starting rate of release, is allowed to medicine
Object takes effect faster.
2, the present invention provides a kind of preparation method of fibroin albumen microsphere drug slow-released carrier, further limits the nanometer
The mass ratio of titanium dioxide granule and nanometer sheep bone particle is that 0.3-0.5:1 further enhances medicament slow release under this ratio
The slow release effect of carrier.
3, the present invention provides a kind of preparation method of fibroin albumen microsphere drug slow-released carrier, further limits the nanometer
The preparation method of sheep bone particle, the nanometer sheep bone particle that the preparation method of nanometer sheep bone particle obtains through the invention have more excellent
Porosity, be allowed to the more preferable cooperation with titanium dioxide nanoparticle, be conducive to enhance slow releasing carrier of medication slow release effect, together
Shi Tigao drug originates rate of release.Wherein sheep bone used in the present invention is black bone sheep sheep bone, black bone sheep since the speed of growth is slower,
Skeleton density is closeer, and the porosity for the nanometer sheep bone particle being allowed to is more excellent, and aperture more evenly, more conducively enhances slow-released carrier
Slow release effect and discharge drug stability.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the SDS-PAGE qualification figure of the recombinant spider silk protein MaSp1 in the present invention, and 1 is M1 in figure, and 2 be recombination spider
Silk-fibroin MaSp1；
Fig. 2 is the Western blotting qualification figure of the recombinant spider silk protein MaSp1 in the present invention, and 1 is M1 in figure, 2
For recombinant spider silk protein MaSp1.
Specific embodiment
There is provided following embodiments is to preferably further understand the present invention, it is not limited to the best embodiment party
Formula is not construed as limiting the contents of the present invention and protection scope, anyone under the inspiration of the present invention or by the present invention and its
The feature of his prior art is combined and any and identical or similar product of the present invention for obtaining, all falls within of the invention
Within protection scope.
Specific experiment step or condition person are not specified in embodiment, according to the literature in the art described routine experiment
The operation of step or condition can carry out.Reagents or instruments used without specified manufacturer, being can be by commercially available acquisition
Conventional reagent product.
Embodiment 1
The present embodiment provides a kind of preparation methods of fibroin albumen microsphere drug slow-released carrier, comprising the following steps:
1) recombinant spider silk protein fiber deionized water and ethyl alcohol are dissolved, is placed in retention molecule through filtered through gauze after dissolution
Amount be 8-10kD bag filter in dialyse 4 days, filtering and concentrating obtain the silk fibroin protein solution that concentration is 0.8wt%；
2) silk fibroin protein solution for obtaining step 1) is placed in bag filter (dialysis bag retention molecular weight 8-10kD), to
Nano material is added in bag filter, and (nano material is titanium dioxide nanoparticle and nanometer sheep bone particle, the nanometer material
The mass ratio of material and the silk fibroin protein solution is 0.1:100, the matter of the titanium dioxide nanoparticle and nanometer sheep bone particle
Amount is than being 0.3:1, and the partial size of the titanium dioxide nanoparticle is 30-50nm, and the partial size of the nanometer sheep bone particle is 30-
50nm), then bag filter is placed in 22 DEG C of water-bath, handles 15 days (100 revs/min of revolving speed), obtains at the uniform velocity shaking table
Microballoon suspension；
3) the microballoon suspension that step 2) obtains is washed with deionized 3 times, is centrifugated, freeze-drying obtains silk
Fibroin microsphere drug slow-released carrier.
The nanometer sheep bone particle the preparation method is as follows: by black bone sheep sheep bone degreasing, pulverizing and sieving, obtaining partial size is
The lamb bone meal of 30-50nm；Lamb bone meal is dissolved in water, be added pepsin digested (enzymatic hydrolysis condition is that pH value is 8.2,
Temperature 50 C), it is during which stirred continuously, makes its reaction uniformly, obtain a nanometer sheep bone particle.
Embodiment 2
The present embodiment provides a kind of preparation methods of fibroin albumen microsphere drug slow-released carrier, comprising the following steps:
1) recombinant spider silk protein fiber deionized water and ethyl alcohol are dissolved, is placed in retention molecule through filtered through gauze after dissolution
Amount be 8-10kD bag filter in dialyse 4 days, filtering and concentrating obtain the silk fibroin protein solution that concentration is 1.0wt%；
2) silk fibroin protein solution for obtaining step 1) is placed in bag filter (dialysis bag retention molecular weight 8-10kD), to
Nano material is added in bag filter, and (nano material is titanium dioxide nanoparticle and nanometer sheep bone particle, the nanometer material
The mass ratio of material and the silk fibroin protein solution is 0.3:100, the matter of the titanium dioxide nanoparticle and nanometer sheep bone particle
Amount is than being 0.5:1, and the partial size of the titanium dioxide nanoparticle is 30-50nm, and the partial size of the nanometer sheep bone particle is 30-
50nm), then bag filter is placed in 25 DEG C of water-bath, handles 15 days (100 revs/min of revolving speed), obtains at the uniform velocity shaking table
Microballoon suspension；
3) the microballoon suspension that step 2) obtains is washed with deionized 3 times, is centrifugated, freeze-drying obtains silk
Fibroin microsphere drug slow-released carrier.
The nanometer sheep bone particle the preparation method is as follows: by black bone sheep sheep bone degreasing, pulverizing and sieving, obtaining partial size is
The lamb bone meal of 30-50nm；Lamb bone meal is dissolved in water, be added pepsin digested (enzymatic hydrolysis condition is that pH value is 8.2,
Temperature 50 C), it is during which stirred continuously, makes its reaction uniformly, obtain a nanometer sheep bone particle.
Embodiment 3
The present embodiment provides a kind of preparation methods of fibroin albumen microsphere drug slow-released carrier, comprising the following steps:
1) recombinant spider silk protein fiber deionized water and ethyl alcohol are dissolved, is placed in retention molecule through filtered through gauze after dissolution
Amount be 8-10kD bag filter in dialyse 3 days, filtering and concentrating obtain the silk fibroin protein solution that concentration is 0.9wt%；
2) silk fibroin protein solution for obtaining step 1) is placed in bag filter (dialysis bag retention molecular weight 8-10kD), to
Nano material is added in bag filter, and (nano material is titanium dioxide nanoparticle and nanometer sheep bone particle, the nanometer material
The mass ratio of material and the silk fibroin protein solution is 0.2:100, the matter of the titanium dioxide nanoparticle and nanometer sheep bone particle
Amount is than being 0.4:1, and the partial size of the titanium dioxide nanoparticle is 30-50nm, and the partial size of the nanometer sheep bone particle is 30-
50nm), then bag filter is placed in 25 DEG C of water-bath, handles 15 days (100 revs/min of revolving speed), obtains at the uniform velocity shaking table
Microballoon suspension；
3) the microballoon suspension that step 2) obtains is washed with deionized 3 times, is centrifugated, freeze-drying obtains silk
Fibroin microsphere drug slow-released carrier.
The nanometer sheep bone particle the preparation method is as follows: by black bone sheep sheep bone degreasing, pulverizing and sieving, obtaining partial size is
The lamb bone meal of 30-50nm；Lamb bone meal is dissolved in water, be added pepsin digested (enzymatic hydrolysis condition is that pH value is 8.2,
Temperature 50 C), it is during which stirred continuously, makes its reaction uniformly, obtain a nanometer sheep bone particle.
Comparative example 1
This comparative example provides a kind of preparation method of fibroin albumen microsphere drug slow-released carrier, comprising the following steps:
1) recombinant spider silk protein fiber deionized water and ethyl alcohol are dissolved, is placed in retention molecule through filtered through gauze after dissolution
Amount be 8-10kD bag filter in dialyse 3 days, filtering and concentrating obtain the silk fibroin protein solution that concentration is 0.9wt%；
2) silk fibroin protein solution for obtaining step 1) is placed in bag filter (dialysis bag retention molecular weight 8-10kD), to
Nano material is added in bag filter, and (nano material is titanium dioxide nanoparticle, the nano material and the fibroin egg
The mass ratio of white solution is 0.2:100, and the partial size of the titanium dioxide nanoparticle is 30-50nm), then bag filter is placed in
In 25 DEG C of water-bath, is handled at the uniform velocity shaking table 15 days (100 revs/min of revolving speed), obtain microballoon suspension；
3) the microballoon suspension that step 2) obtains is washed with deionized 3 times, is centrifugated, freeze-drying obtains silk
Fibroin microsphere drug slow-released carrier.
Comparative example 2
This comparative example provides a kind of preparation method of fibroin albumen microsphere drug slow-released carrier, comprising the following steps:
1) recombinant spider silk protein fiber deionized water and ethyl alcohol are dissolved, is placed in retention molecule through filtered through gauze after dissolution
Amount be 8-10kD bag filter in dialyse 3 days, filtering and concentrating obtain the silk fibroin protein solution that concentration is 0.9wt%；
2) silk fibroin protein solution for obtaining step 1) is placed in bag filter (dialysis bag retention molecular weight 8-10kD), to
Nano material is added in bag filter, and (nano material is nanometer sheep bone particle, and the nano material and the fibroin albumen are molten
The mass ratio of liquid is 0.2:100, and the partial size of the nanometer sheep bone particle is 30-50nm), then bag filter is placed in 25 DEG C of water
In bath, is handled at the uniform velocity shaking table 15 days (100 revs/min of revolving speed), obtain microballoon suspension；
3) the microballoon suspension that step 2) obtains is washed with deionized 3 times, is centrifugated, freeze-drying obtains silk
Fibroin microsphere drug slow-released carrier.
The nanometer sheep bone particle the preparation method is as follows: by black bone sheep sheep bone degreasing, pulverizing and sieving, obtaining partial size is
The lamb bone meal of 30-50nm；Lamb bone meal is dissolved in water, be added pepsin digested (enzymatic hydrolysis condition is that pH value is 8.2,
Temperature 50 C), it is during which stirred continuously, makes its reaction uniformly, obtain a nanometer sheep bone particle.
Test example 1
Using 5-Fluorouracil (5-FU) as model drug, embodiment 3 is evaluated, the fibroin egg that comparative example 1-2 is prepared
The slow-release capability of white microsphere drug slow-released carrier.The application test process of embodiment 3 is as follows:
(1) weigh fibroin albumen microballoon that 5g is prepared by embodiment 34 DEG C, frequency of oscillation be 150-200 turn/
Under conditions of point, decentralized processing is carried out using constant temperature oscillator in ultrapure water, the fibroin albumen microballoon that configuration obtains 5g/L is molten
Concentration is being added in fibroin albumen microsphere suspension liquid without fluorouracil (5-Fu) 500mL for 1mg/mL, by mixed solution by liquid
Decentralized processing 10min at room temperature, then (4 DEG C) centrifugations of high speed freezing, obtains carrying medicine fibroin albumen microballoon；
(2) it weighs measurement 3g load medicine fibroin albumen microballoon to be placed in bag filter, the phosphate buffer solution of pH=7.4 is added
5mL, concussion make it be uniformly dispersed, are then placed in bag filter in the flask of the phosphate buffer solution of 1000mLpH=7.4, in 37
DEG C isothermal vibration carries out drug release.Absorbance using ultraviolet specrophotometer come periodic detection maceration extract at 264nm
(taking 10mL maceration extract, supplement 10mL phosphate buffer), calculates according to langbobier law and carries target in medicine fibroin albumen microballoon
The burst size of drug.
Pass through the same embodiment of application test process of the comparative example 1-2 fibroin albumen microsphere drug slow-released carrier being prepared
3。
Test result see the table below.
As can be seen from the above table, the release amount of medicine in 30min of embodiment 3 reaches 38%, then 2 hours, 6 hours,
Slow release at 12 hours.It can be seen that the slow-released carrier that the embodiment of the present invention 3 obtains has rapid-action and sustained release two-fold advantage.And
Release amount of medicine of the comparative example 1 and 2 in 30min is well below embodiment 3, in subsequent 2 hours, 6 hours, 12 hours
Drug releasing rate is significantly larger than embodiment 3, it is seen that both titanium dioxide nanoparticle and nanometer sheep bone particle phase in the present invention
Mutually cooperation, has obvious synergistic function.
Test example 2
The slow-released carrier 10mg being prepared by embodiment 1 is taken, is added in the blood of people, wherein the blood is (by curing
Institute provide) dosage be 50ml, in 37 DEG C degrade 6 hours, observation degradation situation.Test result passes through embodiment after showing 5 hours
The 1 slow-released carrier degradation being prepared can reach 30%.Show that slow-released carrier provided by the invention has certain degradability
Energy.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
SEQUENCE LISTING
<110>Suzhou Bai Yuan gene technology Co., Ltd
<120>a kind of preparation method and applications of fibroin albumen microsphere drug slow-released carrier
<130> SHA201900049
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 432
<212> PRT
<213>artificial synthesized (artificial synthesis)
<400> 1
Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Ser Gln Gly Ala Gly
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Ala Ala Lys Lys Lys Lys Ser Ser Asp Ser Ser Glu Ser Ser Arg Ser
35 40 45
Ser Ser Ser Ser Ser Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu
50 55 60
Gly Ser Gln Gly Ala Gly Arg Gly Gly Leu Gly Gly Gln Gly Ala Gly
65 70 75 80
Ala Ala Ala Ala Ala Ala Ala Ala Ala Lys Lys Lys Lys Ser Ser Asp
85 90 95
Ser Ser Glu Ser Ser Arg Ser Ser Ser Ser Ser Ser Gly Ala Gly Gln
100 105 110
Gly Gly Tyr Gly Gly Leu Gly Ser Gln Gly Ala Gly Arg Gly Gly Leu
115 120 125
Gly Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Lys Lys
130 135 140
Lys Lys Ser Ser Asp Ser Ser Glu Ser Ser Arg Ser Ser Ser Ser Ser
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Ser Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Ser Gln Gly
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Ala Gly Arg Gly Gly Leu Gly Gly Gln Gly Ala Gly Ala Ala Ala Ala
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Ala Ala Ala Ala Ala Lys Lys Lys Lys Ser Ser Asp Ser Ser Glu Ser
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Ser Arg Ser Ser Ser Ser Ser Ser Gly Ala Gly Gln Gly Gly Tyr Gly
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Gly Leu Gly Ser Gln Gly Ala Gly Arg Gly Gly Leu Gly Gly Gln Gly
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Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Lys Lys Lys Lys Ser Ser
245 250 255
Asp Ser Ser Glu Ser Ser Arg Ser Ser Ser Ser Ser Ser Gly Gly Ala
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Gly Gln Gly Gly Tyr Gly Gly Leu Gly Ser Gln Gly Ala Gly Arg Gly
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Gly Leu Gly Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala
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Ser Ser Arg Ser Ser Ser Ser Ser Ser Gly Gly Ala Gly Gln Gly Gly
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Claims (10)
1. a kind of preparation method of fibroin albumen microsphere drug slow-released carrier, which comprises the following steps:
1) by recombinant spider silk protein Fibrinolysis, silk fibroin protein solution is obtained after filtering, dialysis, concentration；
2) silk fibroin protein solution for obtaining step 1) is placed in bag filter, nano material is added in Xiang Suoshu bag filter, then
Bag filter is placed in 22-25 DEG C of water-bath, is handled at the uniform velocity shaking table, obtains microballoon suspension；
3) the microballoon suspension that step 2) obtains is washed with deionized, is centrifugated, it is micro- to obtain fibroin albumen for freeze-drying
Ball slow releasing carrier of medication；
Nano material described in step 2) is titanium dioxide nanoparticle and nanometer sheep bone particle.
2. the preparation method of fibroin albumen microsphere drug slow-released carrier according to claim 1, which is characterized in that described heavy
The amino acid sequence of recombinant spider silk protein is as shown in SEQ ID NO.1 in group spider's thread protein fiber.
3. the preparation method of fibroin albumen microsphere drug slow-released carrier according to claim 1 or 2, which is characterized in that institute
The concentration for stating silk fibroin protein solution is 0.8-1.0wt%.
4. the preparation method of fibroin albumen microsphere drug slow-released carrier according to claim 1-3, feature exist
In dialysis bag retention molecular weight described in step 2) is 8-10kD.
5. the preparation method of fibroin albumen microsphere drug slow-released carrier according to claim 1, which is characterized in that described to receive
The mass ratio of rice material and the silk fibroin protein solution is 0.1-0.3:100.
6. the preparation method of fibroin albumen microsphere drug slow-released carrier according to claim 1-5, feature exist
In the mass ratio of the titanium dioxide nanoparticle and nanometer sheep bone particle is 0.3-0.5:1.
7. the preparation method of fibroin albumen microsphere drug slow-released carrier according to claim 1-6, feature exist
In, the nanometer sheep bone particle the preparation method is as follows: by black bone sheep sheep bone degreasing, pulverize and sieve, lamb bone meal is obtained；By sheep bone
Powder is dissolved in water, and is digested with pepsin, and a nanometer sheep bone particle is obtained.
8. the preparation method of fibroin albumen microsphere drug slow-released carrier according to claim 1-7, feature exist
In the partial size of the titanium dioxide nanoparticle is 30-50nm；The partial size of the nanometer sheep bone particle is 30-50nm.
9. drug prepared by a kind of preparation method of described in any item fibroin albumen microsphere drug slow-released carriers of claim 1-8
Slow-released carrier.
10. slow releasing carrier of medication described in a kind of claim 9 is preparing the application in slow releasing pharmaceutical.
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