CN101864178A - Injected chemical crosslinking protein/polypeptide hydrogel and preparation method thereof - Google Patents
Injected chemical crosslinking protein/polypeptide hydrogel and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical fields of biotechnology, biological medical materials and tissue engineering, in particular to injected chemical crosslinking protein/polypeptide hydrogel and a preparation method thereof. The injected chemical crosslinking protein/polypeptide hydrogel consists of natural or synthesized protein/polypeptide with a crosslinking phenolic hydroxyl group, horse radish peroxidase and hydrogen peroxide which quickly form the chemical crosslinking hydrogel under physiological condition. The protein/polypeptide hydrogel has good biocompatibility and easily regulated mechanical strength, and is degradable, and the degraded product is amino acids capable of being absorbed by a human body. In addition, the framework of the protein/polypeptide hydrogel very approaches a protein three-dimensional network which supports the cell growth in extracellular matrix, and the invention can be widely applied to multiple biological and medical fields such as drug controlled-release carriers, cell culture supports and the like.
Description
Technical field
The invention belongs to biotechnology, bio-medical material and tissue engineering technique field, be specifically related to a kind of injectable chemical crosslinking protein/polypeptide hydrogel and preparation method thereof.
Background technology
In recent years, new bio medical material and development of technologies make the diagnosis of numerous disease and treatment level that obvious improvement arranged.According to estimates, at present existingly surpass 8000 kinds of medicine equipments, used in 2500 kinds of diagnostics classes products and the 40000 kinds of pharmaceutical preparations various types of (comprising polymkeric substance, pottery and metal etc.) bio-medical material (Adv.Mater., 2006,18,1345-1360).Yet, more excellent and be more suitable for the bio-medical material that uses in the biological friendly environment and the demand of technology of preparing still grows to even greater heights for performance.
Hydrogel is the cross-linked network material that is rich in large quantity of moisture, the normally natural or synthetic polymkeric substance macromolecular chain of its network skeleton.Recently emerge many supramolecular hydrogels that form by non covalent bond self-assembly (self-assembly) by small molecules again.Because it is quite similar with the body tissue of full a large amount of aqueous solution; the medicine such as polypeptide, protein, oligonucleotide and the DNA etc. that more help protecting cell and easy inactivation compared to other types of material; so hydrogel is the material that important being used to of a class makes up tissue engineering cell scaffold, be again the controlled release carrier material of medicines such as a kind of polypeptide that has a prospect and protein.
In numerous hydrogels, the syringeability hydrogel of formed in situ is little and operate simple and easy tool clinical value because of wound.The syringeability hydrogel can be filled into complex-shaped defect easily in external maintenance flow state, simultaneously can load has the bioactive molecule of therapeutic efficiency, is injected in lesions position and realizes topical.Can respond and polymkeric substance that sol-gel transition takes place is the important syringeability hydrogel of a class physical stimulation.ReGel be the temperature sensitive property injection aquagel that constitutes by the PLGA-PEG-PLGA amphipathic three block copolymer (J.Control.Release, 2001,72,203-215).It keeps flowable solution state in room temperature or when being lower than room temperature, becomes gel state when temperature reaches human body temperature.ReGel can improve the solubility property of hydrophobic small-molecule drug, and himself belongs to degradable polymer, and degradation cycle in vivo reaches several weeks, and degraded product is the absorbable lactic acid of human body, oxyacetic acid and low-molecular-weight polyoxyethylene glycol.BTG company has developed the ReGel of load antitumor drug taxol, and commodity are called OncoGel
TM, the medicine as the treatment esophagus cancer is carrying out clinical second phase test at present.Although ReGel has a extensive future as the injection aquagel solid support material, itself be a kind of viscous solid as segmented copolymer ReGel, bring very big inconvenience for transportation and preparation production, and its synthetic requirement is harsh, the preparation cost is higher.
Utilize natural polymer also can prepare the syringeability hydrogel, its biocompatibility and biodegradability are all fine, and have avoided the loaded down with trivial details preparation process of synthetic polymer.Patent US 6129761 and patent WO 99015211 have reported and have utilized hyaluronic acid, alginates to form hydrogel in the presence of divalence or the above high-valence cationic of divalence (as calcium ion, aluminum ion etc.) that partly solidified hydrogel has syringeability.But the defective of this type of hydrogel is the high-valence cationic that plays crosslinked action and can be lost to gradually in the surrounding medium by ion-exchange, make gel dissolve, and this process is difficult to control.
Because the three-dimensional network of sustenticular cell growth is made of collagen protein and elastin in the extracellular matrix of body tissue, feasible hydrogel based on protein or polypeptide becomes another kind of noticeable biomaterial.Commercial polypeptide hydrogel has the PuraMatrix of BD Biosciences company
TMHydrogel, it is to constitute with upper amino acid by 6 or 6, these amino acid whose characteristics are to have comprised positive charge side group (as arginine R), negative charge side group (as aspartic acid D) and hydrophobicity side group (as L-Ala A), and form aminoacid sequence alternately, as (RARADADA)
n(relevant patent has WO 2002062969A2).PuraMatrix
TMThe microtexture of hydrogel is the three-dimensional network that nanofiber is formed, and is suitable for cultivating various types of cells.Yet, the intensity of this type of hydrogel a little less than, modulus G ' (during 1Hz) only have only 50Pa (Proc.Natl.Acad.Sci.USA, 2005,102,8414-8419), limited it in the application of repairing aspect the high-modulus tissue (as cartilage and bone).Patent WO 2007043048A2 discloses a kind of polypeptide hydrogel that is made of 6 following amino acid, characteristics are to have at least an amino acid to have aromatic ring side group (as the phenylalanine F of band phenyl ring) in the peptide sequence, and an end of sequence has fluorenylmethyloxycarbonyl (Fmoc) or carbobenzoxy-(Cbz) (Cbz) capping group.The modulus G ' of this polypeptide hydrogel (during 1Hz) reaches 1000-10,000Pa, but redilution is in water after needing when its defective is to prepare hydrogel earlier polypeptide is dissolved in organic solvents such as having toxicity and corrosive hexafluoroisopropanol.(Proc.Natl.Acad.Sci.USA such as patent WO 2009117497A1 and Haines-Butterick, 2007,104,7791-7796) reported the polypeptide hydrogel of a class syringeability, characteristics are to be made of 20 amino acid, wherein the 10th and the 11 two adjacent amino acid be respectively D type proline(Pro) (
DP) and L type proline(Pro) (
LP), single peptide molecule can be folded into the beta sheet conformation and further assemble becomes the three-dimensional manometer fibre network.This polypeptide hydrogel is under shearing action, and network structure is destroyed, and modulus and viscosity reduce greatly; After removing shearing, network structure can spontaneously be recovered, and therefore has syringeability.But, synthetic step and cost have been increased greatly because this type of amino acid sequence of polypeptide is long and accurately control.
Except above-mentioned physical gel (refer to not to form by chemical covalent linkage network), the method by chemically crosslinked also can prepare the syringeability hydrogel, and common cross-linking method has photo-crosslinking, Michael addition reaction and forms the reaction etc. of Schiff alkali.Wherein, the rate of crosslinking of Michael addition and Schiff alkali reaction relatively slow (generally need 30 minutes or more than), thus may limit its clinical application (Biomaterials, 2004,25,1339-1348 to a certain extent; Int.J.Pharm., 2006,322,44-51).The photo-crosslinking method is meant in the presence of light trigger, the pre-polymer solution that contains unsaturated double-bond by visible or UV-irradiation, make prepolymer form the network of chemically crosslinked, for example, U.S. Pat 20050238678A1 discloses a kind of photo-crosslinking polyglutamic acid hydrogel, and Chinese patent CN101502655A then discloses a kind of preparation method of photo-crosslinking carboxymethyl chitosan hydrogel.Though the gel time of photocrosslinkable hydrogel short (therefore can form gel fast), physical strength higher (depending on cross-linking density) by being injected at by the injection site, but the necessary light trigger of photo-crosslinking often has cytotoxicity, and the irradiation of UV-light also may cause the necrocytosis (Biomaterials at illuminated position, 2005,26,1211-1218; Biomaterials, 2009,30,344-353), limited the application of photocrosslinkable hydrogel to a great extent as the Injectable in-situ shaped hydrogel.
Summary of the invention
The objective of the invention is to propose injectable chemical crosslinking protein/polypeptide hydrogel that a kind of three-dimensional net structure is stable, rate of crosslinking is fast and preparation method thereof.
Injectable chemical crosslinking protein/polypeptide hydrogel provided by the invention, be by natural or synthetic (comprising that chemosynthesis and biotechnology the are synthetic) protein/polypeptide that has crosslinkable phenolic hydroxyl group group, formed chemically crosslinked network under the condition of horseradish peroxidase and hydrogen peroxide existence.
But the described protein/polypeptide generation of described enzyme quick catalysis chemically crosslinked forms hydrogel network.
Among the present invention, protein/polypeptide is partly or entirely for being rich in tyrosine (Tyr) or 3, the natural protein of 4-dopa (DOPA) is (as the animal silk-protein, Keratin sulfate, casein, mussel attachment proteins Mefp-1, mussel attachment proteins Mefp-2, mussel attachment proteins Mefp-3, mussel attachment proteins Mefp-4, mussel attachment proteins Mefp-5 etc.) in any or any several combination.
Among the present invention, protein/polypeptide partly or entirely for synthetic polypeptide or poly-peptide (polypeptide), contains 2 amino acid with phenolic hydroxyl group side chain at least in its aminoacid sequence, as tyrosine, D-pHPG, 2,3-dopa, 2,4-dopa, 2, the 5-dopa, 2, the 6-dopa, 3, the 4-dopa, 3, any or any several combination in the 5-dopa etc.
The present invention also provides the preparation method of above-mentioned chemical crosslinking protein/polypeptide hydrogel, and concrete steps are:
(1) obtains natural protein (solution) by pre-treatment (method determines according to protein source); Adopt chemistry or synthetic polypeptide of biological (transgenosis) technology or poly-peptide.
(2) mixing solutions of preparation protein/polypeptide and horseradish peroxidase, wherein the concentration of protein/polypeptide is 1～20% (w/v), the ultimate density of horseradish peroxidase is 0.01～10mg/mL, and the solvent of solution is that pure water, buffered soln (as phosphoric acid (PBS), Tutofusin tris (tris), boric acid, citric acid etc.), human body or animals and plants body fluid, tissue culture medium and other be not based on the solvent medium of organic solvent;
(3) the pH value of adjusting mixing solutions is 4～8;
(4) add hydrogen peroxide in this mixing solutions, the concentration of hydrogen peroxide is 0.001～1% (v/v), carries out under 20～40 ℃ crosslinked.
The present invention has following characteristics and beneficial effect:
(1) protein/polypeptide hydrogel that proposes of the present invention is the chemically crosslinked gel, and three-dimensional net structure is stable, is not vulnerable to the influence of the various factors (as the chemical substance that exists in the physiological environment, mechanicals efforts etc.) in the environment for use.In this, the chemically crosslinked gel is better than physical crosslinking gel (polypeptide hydrogel that passes through self-assembly formation as described in the background art).
(2) the chemically crosslinked gel of the present invention's proposition is under enzyme catalysis, realizes down that in eco-friendly condition (normal temperature and pressure, the aqueous solution) rate of crosslinking is fast, the fastest can finishing in 30s.By changing the concentration of component (enzyme, protein/polypeptide and hydrogen peroxide), can not change substantially under the prerequisite of network structure, the physical strength of rate of crosslinking, crosslinked formed gel, the performances such as degradation rate of gel are regulated.The enzyme that adopts is the horseradish peroxidase that contains (trace) in the human body, and this enzyme mainly extracts from the horseradish plant roots, has realized commercialization production.What the necessary superoxide of crosslinking reaction was selected for use is often to be used as the low concentration hydrogen peroxide (hydrogen peroxide) that sterilizing agent uses in surgical operation.
(3) protein/polypeptide hydrogel of the present invention's proposition is the aqueous solution of good fluidity before crosslinking reaction, and rate of crosslinking is fast, can form hydrogel at short notice in position after injecting, thereby can use as injectable materials.
(4) protein/polypeptide hydrogel water content height, good biocompatibility, the safety non-toxic and biodegradable of the present invention's proposition, and degraded product is the absorbable amino acid of human body.In addition, the gel network skeleton very near the protein three-dimensional network of sustenticular cell growth in the extracellular matrix, is with a wide range of applications at many biomedical sectors such as slow releasing carrier of medication, cell culturing brackets.
Embodiment
Further describe the present invention below by example, but be not limited to these examples.
Embodiment 1-3 provides the preparation method of silk fibroin (containing Tyr) hydrogel:
Embodiment 1
20g silkworm raw silk is put into 4L Na
2CO
3(0.5wt%) came unstuck 40 minutes at 100 ℃ in the aqueous solution, take out the back and use deionized water rinsing, dry.10g degumed silk solution in the LiBr of the 100mL 9.3mol/L aqueous solution, constantly is stirred to the silk dissolving under 40 ℃.Filter, remove undissolved silk.The silk fibroin salts solution that obtains is removed LiBr salt with the deionized water dialysis.Insoluble other impurity of the centrifugal removal of silk fibroin protein solution that dialysis is good stays clear liquid, obtains concentration after further concentrating and be 12% regenerated silk fibroin water solution.
(HRP 100unit/mg) is dissolved in the PBS buffered soln (pH=6.0) of 2mL 0.1mol/L, is made into the HRP solution of 2.5mg/mL, preserves in 4 ℃ of refrigerators with the 5mg horseradish peroxidase.0.3mLHRP solution is added in the 2.5mL regenerated silk fibroin water solution (12%), mix, place 37 ℃ of water-bath preheatings 5 minutes.Add 0.75% aqueous hydrogen peroxide solution that 0.2mL prepares then in mixing solutions, can form light yellow transparent hydrogel in the time at 60s, the modulus G ' of gel (during 1Hz, using rotational rheometer to measure) reaches 3000Pa.
Embodiment 2
20g silkworm raw silk is put into 4L NaHCO
3(0.5wt%) came unstuck 40 minutes at 100 ℃ in the aqueous solution, take out the back and use deionized water rinsing, dry.10g degumed silk solution in the LiBr of the 100mL 9.3mol/L aqueous solution, constantly is stirred to the silk dissolving under 40 ℃.Filter, remove undissolved silk.The silk fibroin salts solution that obtains is removed LiBr salt with the deionized water dialysis.Insoluble other impurity of the centrifugal removal of silk fibroin protein solution that dialysis is good stays clear liquid.With the clear liquid lyophilize, obtain powder or foaming shape regenerated silk protein solid.The regenerated silk protein solid is dissolved in the deionized water, and centrifugal removal insolubles stays clear liquid, and concentration is 15-20%, is diluted to desired concn (being 12% in this example), obtains regenerated silk fibroin water solution.
(HRP 100unit/mg) is dissolved in the PBS buffered soln (pH=7.4) of 2mL 0.1mol/L, is made into the HRP solution of 0.25mg/mL, preserves in 4 ℃ of refrigerators with the 0.5mg horseradish peroxidase.Under the room temperature (20 ℃), 0.3mL HRP solution is added in the 2.5mL regenerated silk fibroin water solution (12%), mix.Add 0.05% aqueous hydrogen peroxide solution that 0.2mL prepares then in mixing solutions, can form light yellow transparent hydrogel in the time at 600s, the modulus G ' of gel (during 1Hz, using rotational rheometer to measure) reaches 1000Pa.
Embodiment 3
20g silkworm raw silk is put into 4LNa
2CO
3(0.5wt%) came unstuck 60 minutes at 100 ℃ in the aqueous solution, take out the back and use deionized water rinsing, dry.10g degumed silk solution in the LiBr of the 100mL9.3mol/L aqueous solution, constantly is stirred to the silk dissolving under 40 ℃.Filter, remove undissolved silk.The silk fibroin salts solution that obtains is removed LiBr salt with the deionized water dialysis.Insoluble other impurity of the centrifugal removal of silk fibroin protein solution that dialysis is good stays clear liquid.With the clear liquid lyophilize, obtain powder or foaming shape regenerated silk protein solid.The regenerated silk protein solid is dissolved in the deionized water, and centrifugal removal insolubles stays clear liquid, and concentration is 15-20%, is diluted to desired concn (being 18% in this example), obtains regenerated silk fibroin water solution.
(HRP 100unit/mg) is dissolved in the PBS damping fluid (pH=7.4) of 2mL 0.1mol/L, is made into the HRP solution of 10mg/mL, preserves in 4 ℃ of refrigerators with the 20mg horseradish peroxidase.0.3mLHRP solution is added in the 2.5mL regenerated silk fibroin water solution (18%), mix, place 37 ℃ of water-bath preheatings 5 minutes.Add 1% aqueous hydrogen peroxide solution that 0.2mL prepares then in mixing solutions, can form light yellow transparent hydrogel in the time at 30s, the modulus G ' of gel (during 1Hz, using rotational rheometer to measure) reaches 5000Pa.
Embodiment 4
Present embodiment provides mussel attachment proteins Mefp-1 (containing DOPA) preparation method of hydrogel
At the Mefp-1 of 12mg/mL (Cell-Tak
TM, BD Biosciences, 5% acetum) and the middle Tris adjusting pH to 8 that adds.(HRP 100unit/mg) is dissolved in the PBS buffered soln (pH=7.4) of 2mL 0.1mol/L, is made into the HRP solution of 5mg/mL, preserves in 4 ℃ of refrigerators with the 10mg horseradish peroxidase in addition.0.3mL HRP solution is added in the 2.5mL Mefp-1 aqueous solution, mix, place 37 ℃ of water-bath preheatings 5 minutes.Add 0.5% aqueous hydrogen peroxide solution that 0.2mL prepares then in mixing solutions, can form hydrogel at 300s in the time, the modulus G ' of gel (during 1Hz, using rotational rheometer to measure) reaches 800Pa.
Embodiment 5-6 provides poly-(L-Methionin
m-L-tyrosine
n) preparation method of hydrogel:
Embodiment 5
(1) preparation of O-carbobenzoxy-(Cbz)-L-tyrosine-N-carboxyl inner-acid anhydride (O-Z-L-TyrNCA)
In the 500ml there-necked flask, add 12g O-carbobenzoxy-(Cbz)-L-tyrosine, add the 5.0g triphosgene then, at room temperature vacuumize 2 hours after, add the 300mL anhydrous tetrahydro furan, then under argon gas atmosphere, 40 ℃ of stirrings reaction 3 hours down.After reaction finishes (reaction system becomes settled solution by suspension), revolve to steam to remove and desolvate, obtain the solid head product, vacuum-drying is spent the night.Head product is dissolved in the anhydrous tetrahydro furan, adds normal hexane then and make its recrystallization, the ratio of anhydrous tetrahydro furan and normal hexane is 3: 1, and final product is a colourless acicular crystal, and productive rate is about 75%.
(2) N-ε-carbobenzoxy-(Cbz)-L-Methionin N-carboxyl inner-acid anhydride (preparation of N-ε-Z-L-LysNCA)
In the 500ml there-necked flask, add 10g N-ε-carbobenzoxy-(Cbz)-L-Methionin, add the 5.0g triphosgene then, at room temperature vacuumize 2 hours after, add the 300mL anhydrous tetrahydro furan, then under argon gas atmosphere, 50 ℃ of stirrings reaction 2 hours down.After reaction finishes (reaction system becomes settled solution by suspension), revolve to steam to remove and desolvate, obtain the solid head product, vacuum-drying is spent the night.Head product is dissolved in the ethyl acetate, adds normal hexane then and make its recrystallization obtain final product, the ratio of ethyl acetate and normal hexane is 1: 1, and productive rate is about 85%.
(3) poly-(L-Methionin
m-L-tyrosine
n) preparation (m=45, n=5)
6.5g N-ε-Z-L-LysNCA and 0.8g O-Z-L-TyrNCA are dissolved in the 75mL tetrahydrofuran (THF), add the sodium tert-butoxide 4.8ml of 0.1mol/L then, at room temperature stirred 1 day, stirred 2 days down at 40 ℃ then, stirred 4 hours down at 80 ℃ again.Reaction finishes, and adds ether sedimentation and obtains white product A, and vacuum-drying is spent the night, and productive rate is about 80%.
5g is gone up the step product A be dissolved in about 50ml trifluoracetic acid, under agitation add the acetum of 20mL 33wt% hydrogen bromide then.React after 1 hour, add ether sedimentation and gathered (L-Methionin
m-L-tyrosine
n) crude product.Crude product is dissolved in less water/methyl alcohol (1: 4) mixed solvent, and ether sedimentation is gathered (L-Methionin
m-L-tyrosine
n), vacuum-drying is spent the night, and productive rate is about 90%.
(4) poly-(L-Methionin
m-L-tyrosine
n) preparation of cross-linked hydrogel
With poly-(the L-Methionin of 0.75g
m-L-tyrosine
n) be dissolved in the 5mL deionized water, be made into 15% solution.(HRP 100unit/mg) is dissolved in the PBS buffered soln (pH=7.4) of 2mL 0.1mol/L, is made into the HRP solution of 5mg/mL, preserves in 4 ℃ of refrigerators with the 10mg horseradish peroxidase in addition.0.3mL HRP solution is added poly-(the L-Methionin of 2.5mL
m-L-tyrosine
n) in the aqueous solution (15%), mix, place 37 ℃ of water-bath preheatings 5 minutes.Add 0.5% aqueous hydrogen peroxide solution that 0.2mL prepares then in mixing solutions, can form light yellow transparent hydrogel in the time at 60s, the modulus G ' of gel (during 1Hz, using rotational rheometer to measure) reaches 2000Pa.
Embodiment 6
Take by weighing poly-(L-Methionin among the embodiment 5
m-L-tyrosine
n) 0.6g is dissolved in the 5mL deionized water, is made into 12% solution.(HRP 100unit/mg) is dissolved in the PBS buffered soln (pH=7.4) of 2mL 0.1mol/L, is made into the HRP solution of 5mg/mL, preserves in 4 ℃ of refrigerators with the 10mg horseradish peroxidase in addition.Under the room temperature (20 ℃), 0.3mL HRP solution is added poly-(the L-Methionin of 2.5mL
m-L-tyrosine
n) in the aqueous solution (12%), mix, in mixing solutions, add 0.5% aqueous hydrogen peroxide solution that 0.2mL prepares then, can form light yellow transparent hydrogel in the time at 100s, the modulus G ' of gel (during 1Hz, using rotational rheometer to measure) reaches 1200Pa.
Claims (5)
1. injectable chemical crosslinking protein/polypeptide hydrogel, it is characterized in that it being natural or synthetic protein/polypeptide, formed chemically crosslinked network under the condition that horseradish peroxidase and hydrogen peroxide exist by having crosslinkable phenolic hydroxyl group group.
2. chemical crosslinking protein/polypeptide hydrogel according to claim 1, it is characterized in that described protein/polypeptide is partly or entirely for being rich in tyrosine or 3, any in the natural protein of 4-dopa, or wherein any several combination.
3. chemical crosslinking protein/polypeptide hydrogel according to claim 1 is characterized in that described protein/polypeptide partly or entirely for synthetic polypeptide or poly-peptide, contains 2 amino acid with phenolic hydroxyl group side chain at least in its aminoacid sequence.
4. chemical crosslinking protein/polypeptide hydrogel according to claim 3 is characterized in that described amino acid is to have any in the amino acid of phenolic hydroxyl group side chain or wherein any several combination.
5. one kind as claim 1, and the preparation method of one of 2,3 described chemical crosslinking protein/polypeptide hydrogels is characterized in that concrete steps are as follows:
(1) obtains natural protein by pre-treatment; Adopt synthetic polypeptide of chemistry or biotechnology or poly-peptide;
(2) mixing solutions of preparation protein/polypeptide and horseradish peroxidase, wherein the ultimate density of protein/polypeptide is 1～20% (w/v), the ultimate density of horseradish peroxidase is 0.01～10mg/mL, and solvent is pure water, buffered soln, human body or animals and plants body fluid, tissue culture medium, other is based on the solvent medium of organic solvent;
(3) the pH value of adjusting mixing solutions is 4～8;
(4) add hydrogen peroxide in this mixing solutions, the concentration of hydrogen peroxide is 0.001～1% (v/v), carries out crosslinked under 20～40 ℃.
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