CN110437667A - A kind of biometric print ink composition and its method for preparing ingredients thereof - Google Patents
A kind of biometric print ink composition and its method for preparing ingredients thereof Download PDFInfo
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- CN110437667A CN110437667A CN201910790233.9A CN201910790233A CN110437667A CN 110437667 A CN110437667 A CN 110437667A CN 201910790233 A CN201910790233 A CN 201910790233A CN 110437667 A CN110437667 A CN 110437667A
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- norbornene
- sulfydryl
- gelatin
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- 239000000203 mixture Substances 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 title description 14
- 239000004615 ingredient Substances 0.000 title description 2
- 230000004048 modification Effects 0.000 claims abstract description 69
- 238000012986 modification Methods 0.000 claims abstract description 69
- 108010010803 Gelatin Proteins 0.000 claims abstract description 44
- 239000008273 gelatin Substances 0.000 claims abstract description 44
- 229920000159 gelatin Polymers 0.000 claims abstract description 44
- 235000019322 gelatine Nutrition 0.000 claims abstract description 44
- 235000011852 gelatine desserts Nutrition 0.000 claims abstract description 44
- JFNLZVQOOSMTJK-KNVOCYPGSA-N norbornene Chemical compound C1[C@@H]2CC[C@H]1C=C2 JFNLZVQOOSMTJK-KNVOCYPGSA-N 0.000 claims abstract description 33
- 238000006243 chemical reaction Methods 0.000 claims abstract description 31
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims abstract description 26
- 229920002683 Glycosaminoglycan Polymers 0.000 claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000007853 buffer solution Substances 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 78
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 33
- 229920000669 heparin Polymers 0.000 claims description 33
- 229960002897 heparin Drugs 0.000 claims description 33
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 16
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 14
- 238000010189 synthetic method Methods 0.000 claims description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 238000003756 stirring Methods 0.000 claims description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
- 239000000126 substance Substances 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 6
- KNDQHSIWLOJIGP-UMRXKNAASA-N (3ar,4s,7r,7as)-rel-3a,4,7,7a-tetrahydro-4,7-methanoisobenzofuran-1,3-dione Chemical compound O=C1OC(=O)[C@@H]2[C@H]1[C@]1([H])C=C[C@@]2([H])C1 KNDQHSIWLOJIGP-UMRXKNAASA-N 0.000 claims description 5
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 4
- 239000005864 Sulphur Substances 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- 125000002091 cationic group Chemical group 0.000 claims description 4
- 238000000502 dialysis Methods 0.000 claims description 4
- 229920005989 resin Polymers 0.000 claims description 4
- 239000011347 resin Substances 0.000 claims description 4
- 238000004090 dissolution Methods 0.000 claims description 3
- 239000003292 glue Substances 0.000 claims description 2
- 235000016127 added sugars Nutrition 0.000 claims 1
- 150000005846 sugar alcohols Chemical class 0.000 claims 1
- 229910052760 oxygen Inorganic materials 0.000 abstract description 7
- 239000001301 oxygen Substances 0.000 abstract description 7
- 238000006467 substitution reaction Methods 0.000 abstract description 5
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical compound CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 abstract description 4
- 230000004071 biological effect Effects 0.000 abstract description 3
- 238000006116 polymerization reaction Methods 0.000 abstract description 3
- 239000003102 growth factor Substances 0.000 abstract description 2
- JUYQFRXNMVWASF-UHFFFAOYSA-M lithium;phenyl-(2,4,6-trimethylbenzoyl)phosphinate Chemical compound [Li+].CC1=CC(C)=CC(C)=C1C(=O)P([O-])(=O)C1=CC=CC=C1 JUYQFRXNMVWASF-UHFFFAOYSA-M 0.000 description 13
- 108010041308 Endothelial Growth Factors Proteins 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 9
- NHJVRSWLHSJWIN-UHFFFAOYSA-N 2,4,6-trinitrobenzenesulfonic acid Chemical compound OS(=O)(=O)C1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O NHJVRSWLHSJWIN-UHFFFAOYSA-N 0.000 description 6
- 239000003642 reactive oxygen metabolite Substances 0.000 description 6
- 238000004132 cross linking Methods 0.000 description 5
- 239000003999 initiator Substances 0.000 description 5
- 238000007711 solidification Methods 0.000 description 5
- 230000008023 solidification Effects 0.000 description 5
- 239000012224 working solution Substances 0.000 description 5
- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 3
- UNXHWFMMPAWVPI-QWWZWVQMSA-N D-threitol Chemical compound OC[C@@H](O)[C@H](O)CO UNXHWFMMPAWVPI-QWWZWVQMSA-N 0.000 description 3
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- UFULAYFCSOUIOV-UHFFFAOYSA-N cysteamine Chemical compound NCCS UFULAYFCSOUIOV-UHFFFAOYSA-N 0.000 description 3
- 210000002889 endothelial cell Anatomy 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 229960003151 mercaptamine Drugs 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 125000003396 thiol group Chemical group [H]S* 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- 210000003606 umbilical vein Anatomy 0.000 description 3
- QUSNBJAOOMFDIB-UHFFFAOYSA-N Ethylamine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 238000007259 addition reaction Methods 0.000 description 2
- 150000001336 alkenes Chemical class 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- ZQTYQMYDIHMKQB-UHFFFAOYSA-N exo-norborneol Chemical compound C1CC2C(O)CC1C2 ZQTYQMYDIHMKQB-UHFFFAOYSA-N 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000005311 nuclear magnetism Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 208000011580 syndromic disease Diseases 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- SLAMLWHELXOEJZ-UHFFFAOYSA-N 2-nitrobenzoic acid Chemical compound OC(=O)C1=CC=CC=C1[N+]([O-])=O SLAMLWHELXOEJZ-UHFFFAOYSA-N 0.000 description 1
- KWOLFJPFCHCOCG-UHFFFAOYSA-N Acetophenone Natural products CC(=O)C1=CC=CC=C1 KWOLFJPFCHCOCG-UHFFFAOYSA-N 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 238000011481 absorbance measurement Methods 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 235000021050 feed intake Nutrition 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000006384 oligomerization reaction Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000036279 refractory period Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
- C09D11/00—Inks
- C09D11/02—Printing inks
- C09D11/04—Printing inks based on proteins
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
- C09D11/00—Inks
- C09D11/02—Printing inks
- C09D11/10—Printing inks based on artificial resins
- C09D11/101—Inks specially adapted for printing processes involving curing by wave energy or particle radiation, e.g. with UV-curing following the printing
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Materials For Medical Uses (AREA)
Abstract
The present invention provides a kind of biometric print ink composition, sulfydryl modification mucopolysaccharide component is introduced into gelatin, assigns ink additional biological activity, while using sulfydryl-norbornene step-reaction polymerization pair, substitution tradition is methacrylate modified, and the oxygen radical reduced in reaction generates.Biometric print ink composition is mainly water or buffer solution, photoinitiator, norbornene modification gelatin, sulfydryl modification mucopolysaccharide (or dithiothreitol (DTT) of inanimate object function), can additionally contain growth factor and cell according to actual needs.In the component of above-mentioned biometric print ink, the content that norbornene modifies gelatin is 5~40%, and the content of sulfydryl modification mucopolysaccharide is 0~5%.In addition, the molar ratio of sulfydryl and norbornene is not limited to 1:1, but utilization rate and efficiency are all higher when 1:1.
Description
Technical field
The present invention relates to biometric print technical fields.More specifically to a kind of biometric print ink composition and its group
Divide preparation method.
Background technique
The features such as biometric print technology is personalized, without mold, higher precision because of its height, can meet multiclass tissue
Engineering process requirements.But fault is in the rigors of print performance and biocompatibility, and biometric print (Bioprint) can be at present
The material used is extremely limited.In limited selection, methacrylate gelatin (GelatinMethacrylamide,
GelMA) because of its is temperature sensitive, double bond is crosslinked dual cure feature and outstanding biocompatibility, degradation property, it is most wide to become use
One of biometric print ink.
But GelMA has two, first is that being usually associated with the generation of a large amount of oxygen radicals in solidification process, wraps to it
There are oxidation toxicities for the cell wrapped up in, or reduce active constituent effect；Second is that GelMA itself is functional insufficient, it is difficult to which satisfaction makes
With needs.
Summary of the invention
The present invention provides a kind of biometric print ink composition, and sulfydryl modification mucopolysaccharide component is introduced into gelatin, assigns ink
The additional biological activity of water, while sulfydryl-norbornene step-reaction polymerization pair is used, substitution is methacrylate modified,
The oxygen radical reduced in reaction generates.
Biometric print ink composition in the present invention include water or buffer solution, photoinitiator, norbornene modification gelatin,
Sulfydryl modification mucopolysaccharide or dithiothreitol (DTT).
In above-mentioned biometric print ink composition, the content of the norbornene modification gelatin is 5~40%, the sulfydryl
The content for modifying mucopolysaccharide or dithiothreitol (DTT) is 0~5%.
In above-mentioned biometric print ink composition, the sulfydryl modification mucopolysaccharide is sulfydryl modification heparin.
In above-mentioned biometric print ink composition norbornene modification gelatin synthetic method the following steps are included:
Step 1 is heated to 50 DEG C by 10g Gelatin in the 0.1M PBS solution of 100mlpH=8, stirs to molten
Solution；
Step 2, then 5~30g nadic anhydride is added thereto, it stirs evenly；
Step 3, then by several times, be slowly added to NaOH solution, adjust pH value to 8, react 48 hours；
Step 4 stops reaction to which the 0.1M PBS solution of 200ml after reaction, is added, and reaction solution is packed into and is dialysed
Bag, and dialyse three days in 40 DEG C of pure water, during which replace dialyzate twice.
Acquired solution is lyophilized after dialysis for step 5, obtains norbornene modification gelatin.
The synthetic method of sulfydryl modification heparin in above-mentioned biometric print ink composition is main are as follows:
Heparin is dissolved in the 0.1M PBS solution of pH=6.8 by step 1, to be configured to the heparin solution of 10mg/mL；
Step 2 calculates the concentration of carboxyl-COOH in heparin solution, is put in proportion into several EDC, HOBt, Cys, compares
Example is-COOH:EDC:HOBt:Cys=1:0.75~7.5:0.75~7.5:1~15；
Step 3, after feeding intake adjust pH to 6.8, at room temperature react 3~12 hours, end of reaction, by reaction solution in
It dialyses 2 days in 0.1MNaCl solution, dialyses 1 day in pure water, be lyophilized, obtain White Flocculus；
White Flocculus is redissolved in pure water by step 4, and two of the amount of carboxyl substance in 10 times of heparin are added
Sulphur threitol adjusts pH to 7.5, reacts 3~5 hours；
Enough strong acid type cationic resins are added in step 5 after reaction, and stirring is filtered after 15 minutes, by acquired solution
It dialyses 3 days in the hydrochloric acid solution of pH 3.5, is then lyophilized, obtains sulfydryl modification heparin
Compared with the bio-ink of traditional methacrylic acid modification gelatin, ink of the invention uses sulfydryl-norbornene
Gradually addition reaction system replaces the reaction system of methacrylic acid addition oligomerization；It will not thus be produced in light-initiated solidification process
Raw a large amount of active oxygen, can largely reduce adverse effect of the ink to cell during biometric print；Improve cell
Survival rate, and then improve cell utilization rate；Gradually addition reaction can resist sulfydryl-norbornene to a certain extent simultaneously
Oxygen inhibition phenomenon, thus have faster crosslinking rate, higher printing precision, faster print speed may be brought.
Compared with the bio-ink of traditional methacrylic acid modification gelatin, ink of the invention possesses richer biology
Performance can protect VEGF (endothelial growth factors) from degrading, inactivating by taking the component of the heparin containing sulfydryl as an example.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
The embodiment of invention for those of ordinary skill in the art without creative efforts, can also basis
The attached drawing of offer obtains other attached drawings；
Fig. 1 is the schematic diagram of biological marking ink component；
Fig. 2 is to mark ROS in HUVEC horizontal using dihydro second ingot；
Fig. 3 is using 2', and 7'- dichlorofluorescein diacetate esters mark ROS in HUVEC horizontal；
Fig. 4 is the dead situation living of HUVCE cell in gel after ultraviolet light cross-linking；
Fig. 5 is the elastic modulus change of ink after ultraviolet light；
Fig. 6 is after ultraviolet light/LAP initiator system solidifies, and the VEGF's of 100ng/mL retains situation in gel.
Specific embodiment
The present invention provides a kind of biometric print ink composition, and sulfydryl modification mucopolysaccharide component is introduced into gelatin, assigns ink
The additional biological activity of water, while sulfydryl-norbornene step-reaction polymerization pair is used, substitution is methacrylate modified,
The oxygen radical reduced in reaction generates.
One, the proportion of biometric print ink
Biometric print ink composition is mainly water or buffer solution, photoinitiator, norbornene modification gelatin, sulfydryl modification
Mucopolysaccharide (or dithiothreitol (DTT) of inanimate object function) can be rapid after special wavelength light corresponding with photoinitiator irradiation
Crosslinking.
In addition, biometric print ink composition can also contain growth factor and cell according to actual needs.
In the component of above-mentioned biometric print ink, buffer solution can be PBS solution (phosphate buffer solution), norborneol
The content that alkene modifies gelatin is 5~40%, and the content of sulfydryl modification mucopolysaccharide or dithiothreitol (DTT) is 0~5%.In addition, sulfydryl
It is not limited to 1:1 with the molar ratio of norbornene, but utilization rate and efficiency are all higher when 1:1.
Photoinitiator is different because of type, as LAP (Lithium phenyl-2,4,6-
Trimethylbenzoylphosphinate content) is 0.03%~6%.
It should be noted that the print performance of biometric print ink is directly related with the norbornene modification content of gelatin, and
The content of both intensity and sulfydryl modification mucopolysaccharide, norbornene modification gelatin after photo-crosslinking is all related, and the speed of crosslinking is then
It is related with the former two and photoinitiator type, content.
Two, the preparation of norbornene modification gelatin
It is modified gelatin that norbornene, which modifies gelatin, and wherein the carbic anhydride degree of modification of modified gelatin is (with TNBS
Method measures) it is 0~0.35mmol/g.
The synthetic method that norbornene modifies gelatin is main are as follows:
10g gelatin (intensity about 300bloom) is dissolved in the 0.1M PBS solution of 100ml pH=8, adds by step 1
Heat is to 50 DEG C, stirring to dissolution.It should be noted that M in English=mol/L, so in 0.1MPBS solution PBS concentration
For 0.1mol/L.
Step 2, then 5~30g nadic anhydride is added thereto, it stirs evenly.
Step 3, then by several times, be slowly added to NaOH solution, adjust pH value to 8, react 48 hours.
Step 4 stops reaction, will react to after reaction, the 0.1M PBS solution of two volumes (200ml) be added
Liquid is packed into bag filter, and dialyses three days in 40 DEG C of pure water, during which replaces dialyzate twice.
Acquired solution is lyophilized after dialysis for step 5, obtains white sponge solid, i.e. norbornene modifies gelatin.
The norbornene modification gelatin of this method synthesis uses TNBS (2,4,6-Trinitrobenzenesulfonic
Acid) method measurement gelatin amino is substituted degree, and is proved using nuclear-magnetism.
The concrete operations of TNBS method are as follows；
Solution allocation to be measured: the norbornene modification gelatin and gelatin material for taking the above method to prepare are dissolved in pH=8's
In 0.1M PBS solution, it is configured to the solution to be measured of 5mg/mL.
Working solution configuration: precise 60mg glycine, and be dissolved in the 0.1M PBS solution of pH=8, it is fixed
Hold to 100mL.It takes 0,0.5mL, 1mL, 1.5mL, 2mL solution from the solution after constant volume respectively again, dilutes, is settled to
10mL is configured to the glycine solution of the amount concentration of predetermined substance.
Absorbance measurement: 1mL solution to be measured or working solution and 1mL1%TNBS are separately added into each band plug centrifuge tube
Solution reacts 2 hours in 37 DEG C.0.5mL 1M hydrochloric acid solution is added into each centrifuge tube after reaction and stops reaction, then
The sodium dodecyl sulfate solution that 1mL 10% is added prevents solution from precipitating.Take prepare liquid or working solution in each centrifuge tube
It is several, use absorbance at microplate reader measurement 345nm.Working solution amino content and absorbance working curve are drawn, and is passed through
Solution absorbance to be measured calculates the amino content in sample to be tested, to calculate amino group substitution degree indirectly.
Amino group substitution degree and the relationship that feeds intake:
Three, the preparation of sulfydryl modification mucopolysaccharide
Sulfydryl modification mucopolysaccharide can be sulfydryl modification heparin.
The synthetic method of sulfydryl modification heparin is main are as follows:
Heparin is dissolved in the 0.1M PBS solution of pH=6.8 by step 1, to be configured to the heparin solution of 10mg/mL.
Step 2 calculates the substance withdrawl syndrome of carboxyl in solution.With carboxyl (- COOH) for 1 equivalent, if being put in proportion into
Dry EDC (1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride), HOBt (I-hydroxybenzotriazole), Cys (sulfydryl
Ethamine or cysteamine), its ratio be-COOH:EDC:HOBt:Cys=1:0.75~7.5:0.75~7.5:1~15.
Step 3 adjusts pH to 6.8 after feeding intake, react 3~12 hours at room temperature.End of reaction, by reaction solution in
It dialyses 2 days in 0.1MNaCl solution, is lyophilized, obtains White Flocculus.
Products therefrom is redissolved in pure water by step 4, and two sulphur of the amount of carboxyl substance in 10 times of heparin are added
Threitol (DTT) adjusts pH to 7.5, reacts 3~5 hours.
Step 5, is added enough strong acid type cationic resins after reaction, and stirring is filtered after 15 minutes, acquired solution in
It dialyses 3 days in the hydrochloric acid solution of pH 3.5, White Flocculus is then lyophilized to obtain.
The sulfydryl modification heparin of this method synthesis measures sulfhydryl content using Ellman method, and using nuclear-magnetism result as assistant
Card.
Ellman method concrete operations are as follows:
Solution allocation to be measured: above-mentioned sulfydryl modification mucopolysaccharide is dissolved in the Tris solution of pH=8,1mg/mL is configured to
Solution to be measured.
Working solution configuration: precise 121.2mg cysteine, and be dissolved in the Tris solution of pH=8, it is fixed
Hold to 100mL.It takes 0,0.25mL, 0.5mL, 0.75mL, 1mL solution from the solution after constant volume respectively again, dilutes, is settled to
10mL is configured to the cysteine solution that thiol concentration is 0,0.25mM, 0.5mM, 0.75mM and 1mM.
Ellman reagent: 30mg DTNB (bis- thiobis of 5,5'- (2- nitrobenzoic acid)) is dissolved in the Tris of pH=8
In solution, it is settled to 100mL.
Test: 1:1 mixing Ellman reagent and standard solution or solution to be measured measure extinction at 405nm by microplate reader
Degree, calculates sulfhydryl content in solution to be measured.
Sulfhydryl content and the relationship (by taking heparin as an example) to feed intake:
| The ratio between the amount of-COOH:EDC:HOBt:Cys substance | Content/mmolg-1 of sulfydryl in heparin |
| 1:0.75:0.75:1 | 0.20 |
| 1:1:1:1.5 | 0.35 |
| 1:2:2:3 | 0.67 |
| 1:7.5:7.5:15 | 1.05 |
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Embodiment 1
Meaning GelNB/HepSH group in attached drawing, consisting of the norbornene that 8% degree of modification is 0.17mmol/g is modified
Gelatin, 1.4% degree of modification are the sulfydryl modification heparin of 0.97mmol/g, 0.03% photoinitiator LAP (Lithiumphenyl-
2,4,6-trimethylbenzoylphosphinate), 100ng/mL VEGF (endothelial growth factors) and 1 × 106/mL
HUVEC (Human umbilical vein endothelial cells).
Wherein the norbornene modification gelatin synthetic method of 0.17mmol/g is as follows:
10g gelatin (intensity about 300bloom) is dissolved in the 0.1M PBS solution of 100ml pH=8, adds by step 1
Heat is to 50 DEG C, stirring to dissolution.It should be noted that M in English=mol/L, so in 0.1MPBS solution PBS concentration
For 0.1mol/L.
Step 2, then 20g nadic anhydride is added thereto, it stirs evenly.
Step 3, then by several times, be slowly added to NaOH solution, adjust pH value to 8, react 48 hours.
Step 4 stops reaction, will react to after reaction, the 0.1M PBS solution of two volumes (200ml) be added
Liquid is packed into bag filter, and dialyses three days in 40 DEG C of pure water, during which replaces dialyzate twice.
Step 5, after dialysis by acquired solution be lyophilized to get.It is measured by TNBS method, norborneol obtained by the example
The degree of modification that alkene modifies gelatin is about 0.17mmol/g.
The sulfydryl modification heparin of degree of modification 0.97mmol/g, synthetic method are as follows:
Heparin is dissolved in the 0.1M PBS solution of pH=6.8 by step 1, to be configured to the heparin solution of 10mg/mL.
Step 2 calculates the substance withdrawl syndrome of carboxyl in solution.With carboxyl (- COOH) for 1 equivalent, if being put in proportion into
Dry EDC (1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride), HOBt (I-hydroxybenzotriazole), Cys (sulfydryl
Ethamine or cysteamine), its ratio be-COOH:EDC:HOBt:Cys=1:7.5:7.5:15.
Step 3 adjusts pH to 6.8 after feeding intake, react 3~12 hours at room temperature.End of reaction, by reaction solution in
It dialyses 2 days in 0.1MNaCl solution, is lyophilized, obtains White Flocculus.
Products therefrom is redissolved in pure water by step 4, and two sulphur of the amount of carboxyl substance in 10 times of heparin are added
Threitol (DTT) adjusts pH to 7.5, reacts 3~5 hours.
Step 5, is added enough strong acid type cationic resins after reaction, and stirring is filtered after 15 minutes, acquired solution in
It dialyses 3 days, is then lyophilized in the hydrochloric acid solution of pH 3.5 to obtain the final product.It is measured using Ellman reagent, degree of modification is about
0.97mmol/g。
Embodiment 2
Meaning GelNB/DTT group i.e. in attached drawing, the norbornene modification that the degree of modification of component 8% is 0.17mmol/g are bright
Glue, and 6.8mM DTT (dithiothreitol (DTT)), 0.03% photoinitiator LAP (Lithium phenyl-2,4,6-
Trimethylbenzoylphosphinate), 100ng/mL VEGF (endothelial growth factors) and 1 × 106/mL HUVEC
(Human umbilical vein endothelial cells).Wherein the norbornene modification gelatin synthetic method of 0.17mmol/g is identical as example 1.
Embodiment 3
Bio-ink in the present invention, the norbornene modification that composition can be 0.08mmol/g for 5% degree of modification
Gelatin, 0.65% degree of modification are the sulfydryl modification heparin of 0.60mmol/g, 0.03% photoinitiator LAP
(Lithiumphenyl-2,4,6-trimethylbenzoylphosphinate), 100ng/mL VEGF (endothelial growth factor
Son) and 1 × 106/ mL HUVEC (Human umbilical vein endothelial cells).
Wherein, compared with example 1, difference is in step 2 the synthetic method of 0.08mmol/g norbornene modification gelatin
The inventory of norbornene is 5g.
Wherein, compared with example 1, difference is in step 2 the sulfydryl modification heparin synthetic method of 0.60mmol/g, with
Carboxyl (- COOH) is 1 equivalent, is put in proportion into several EDC (1- (3- dimethylamino-propyl) -3- ethyl carbodiimide hydrochloride
Salt), HOBt (I-hydroxybenzotriazole), Cys (mercaptoethylmaine or cysteamine), its ratio be-COOH:EDC:HOBt:Cys
=1:2:2:3.
Embodiment 4
Bio-ink in the present invention, the norbornene modification that composition can be 0.13mmol/g for 40% degree of modification
Gelatin, 5% degree of modification are the sulfydryl modification heparin of 0.97mmol/g, 0.5% photoinitiator I2959 (2- hydroxyl -4- (2- hydroxyl
Ethyoxyl) -2- methyl phenyl ketone).
Wherein compared with example 1, difference is in step 2 the synthetic method of 0.13mmol/g norbornene modification gelatin
The inventory of norbornene is 7.5g.
Wherein the synthetic method of the sulfydryl modification heparin of 0.97mmol/g is identical as example 1.
GelMA group in all figures is the biometric print ink that traditional methacrylic acid modifies gelatin, the sheet of GelNB/DTT
Biometric print ink of one of the invention containing norbornene modification gelatin and dithiothreitol (DTT), GelNB/HepSH is this hair
A kind of bright middle biometric print ink containing norbornene modification gelatin and sulfydryl modification heparin.
1, ROS (reactive oxygen species) level difference: either being characterized using which kind of fluorescent dye it can be seen from Fig. 1, Fig. 2,
After ultraviolet light and LAP initiator cause, GelMA group all has the ROS of highest level, compares GelMA group, GelNB/DTT with
The reduction of the equal conspicuousness of the ROS of GelNB/HepSH group, and there is no significant difference between two groups.
2, cell survival rate difference: as seen from Figure 3, GelMA group, GelNB/DTT group and GelNB/HepSH group are compared
HUVEC possess higher cell survival rate after ultraviolet light solidification, and its survival rate is similar to non-treated control group cell.
3, ultraviolet lighting/LAP initiator system curing rate difference: as seen from Figure 4, comparing GelMA group,
In the presence of LAP initiator, modulus immediately rises GelNB/HepSH group after ultraviolet light is opened；And GelMA group is at one section
For time memory in refractory period, this may be since there are oxygen inhibition phenomenons by GelMA.
4, after the solidification of ultraviolet light/LAP initiator system, the VEGF of 100ng/mL retains situation in gel: In
After ultraviolet light solidification, GelNB/HepSH can significantly guarantee VEGF content therein；And GelMA group and GelNB/DTT
Group can only retain about 50%.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although with reference to the foregoing embodiments
Invention is explained in detail, those skilled in the art should understand that: it still can be to aforementioned each implementation
Technical solution documented by example is modified or equivalent replacement of some of the technical features；And these modification or
Replacement, the spirit and scope for technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution.
Claims (5)
1. a kind of biometric print ink composition, which is characterized in that including water or buffer solution, photoinitiator, norbornene modification
Gelatin, sulfydryl modification mucopolysaccharide or dithiothreitol (DTT).
2. biometric print ink composition according to claim 1, which is characterized in that the norbornene modification gelatin contains
Amount is 5~40%, and the content of the sulfydryl modification mucopolysaccharide or dithiothreitol (DTT) is 0~5%.
3. biometric print ink composition according to claim 2, which is characterized in that the sulfydryl modification mucopolysaccharide is sulfydryl
Modify heparin.
4. the preparation method of biometric print ink composition as claimed in claim 3, which is characterized in that the norbornene modification is bright
The synthetic method of glue the following steps are included:
Step 1 is heated to 50 DEG C, stirring to dissolution by 10g Gelatin in the 0.1M PBS solution of 100mlpH=8；
Step 2, then 5~30g nadic anhydride is added thereto, it stirs evenly；
Step 3, then by several times, be slowly added to NaOH solution, adjust pH value to 8, react 48 hours；
Step 4 stops reaction, reaction solution is packed into bag filter to which the 0.1M PBS solution of 200ml after reaction, is added,
And dialyse three days in 40 DEG C of pure water, during which replace dialyzate twice.
Acquired solution is lyophilized after dialysis for step 5, obtains norbornene modification gelatin.
5. the preparation method of biometric print ink composition as claimed in claim 3, which is characterized in that the sulfydryl modification heparin
Synthetic method is main are as follows:
Heparin is dissolved in the 0.1M PBS solution of pH=6.8 by step 1, to be configured to the heparin solution of 10mg/mL；
Step 2 calculates the concentration of carboxyl-COOH in heparin solution, is put in proportion into several EDC, HOBt, Cys, its ratio be-
COOH:EDC:HOBt:Cys=1:0.75~7.5:0.75~7.5:1~15；
Step 3, after feeding intake adjust pH to 6.8, at room temperature react 3~12 hours, end of reaction, by reaction solution in
It dialyses 2 days in 0.1MNaCl solution, dialyses 1 day in pure water, be lyophilized, obtain White Flocculus；
White Flocculus is redissolved in pure water by step 4, and the two sulphur Soviet Union of the amount of carboxyl substance in 10 times of heparin is added
Sugar alcohol adjusts pH to 7.5, reacts 3~5 hours；
Enough strong acid type cationic resins are added in step 5 after reaction, and stirring is filtered after 15 minutes, by acquired solution in pH
It dialyses 3 days in 3.5 hydrochloric acid solution, is then lyophilized, obtains sulfydryl modification heparin.
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|---|---|---|---|---|
| CN106478841A (en) * | 2016-09-20 | 2017-03-08 | 海南大学 | The cysteine conjugates of hyaluronic acid prepared with mercaptan alkene clicking chemistry by lyophilization and its synthetic method and application |
| CN108367100A (en) * | 2015-12-02 | 2018-08-03 | 奥塔哥创新有限公司 | It is prepared by the photoactivation of hydrogel |
| US20190106673A1 (en) * | 2017-10-11 | 2019-04-11 | Wake Forest University Health Sciences | Bioink compositions and methods of preparing and using the same |
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2019
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| CN108367100A (en) * | 2015-12-02 | 2018-08-03 | 奥塔哥创新有限公司 | It is prepared by the photoactivation of hydrogel |
| CN106478841A (en) * | 2016-09-20 | 2017-03-08 | 海南大学 | The cysteine conjugates of hyaluronic acid prepared with mercaptan alkene clicking chemistry by lyophilization and its synthetic method and application |
| US20190106673A1 (en) * | 2017-10-11 | 2019-04-11 | Wake Forest University Health Sciences | Bioink compositions and methods of preparing and using the same |
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Application publication date: 20191112 |