CN102718991A - High strength injectable hydrogel and preparation method thereof - Google Patents
High strength injectable hydrogel and preparation method thereof Download PDFInfo
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- CN102718991A CN102718991A CN2012100975229A CN201210097522A CN102718991A CN 102718991 A CN102718991 A CN 102718991A CN 2012100975229 A CN2012100975229 A CN 2012100975229A CN 201210097522 A CN201210097522 A CN 201210097522A CN 102718991 A CN102718991 A CN 102718991A
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Abstract
The invention relates to a high strength injectable hydrogel and a preparation method thereof, and specifically relates to the hydrogel formed by using the double bond of polyethyleneglycol diacrylate (PEGDA) and the mercapto group of a sulfhydrylation natural polymer to undergo a Michael addition reaction and at the same time taking the nanoparticles of the triblock copolymer of polyethylene glycol and polycaprolactone (PEG-PCL-PEG) as a reinforcing agent. This kind of hydrogel has relatively high mechanical strength, is injectable hydrogel and degradable hydrogel with good biocompatibility, and has a high gelating speed. The hydrogel has the characteristics of cheap and easily available raw materials and simple preparation method, and therefore has a good application prospect in the biomedical field.
Description
Technical field
The present invention relates to a kind of HS injection aquagel and preparation method thereof; More particularly; Relate to that the Michael addition reaction takes place the link coupled sulfydryl on a kind of two keys that utilize polyethyleneglycol diacrylate (PEGDA) and the natural polymer; Triblock copolymer nanoparticle with polyoxyethylene glycol and polycaprolactone (PEG-PCL-PEG) is the hydrogel that toughener forms simultaneously, intensity that tool is higher and syringeability.
Background technology
Hydrogel be a kind of can be in water swelling, absorb and keep large quantity of moisture and can not be dissolved in the three-dimensional polymer swelling body of water.Hydrogel is first kind and develops by the biomaterial of human body use that when contacting with blood, body fluid and tissue, show excellent biological compatibility, it neither influences the metabolic process of life entity, and meta-bolites can be discharged through hydrogel again.All near biological tissue, it is similar to the extracellular matrix part to hydrogel in nature, can reduce friction and mechanical effect to surrounding tissue after the suction, significantly improves the biology performance of material than other any synthesising biological material.Therefore, hydrogel is widely used in fields such as biological medicine, organizational project.Hydrogel is the good timbering material of organizational project, and is mostly nontoxic or toxicity is little, machine-shaping easily, and character is similar to many tissues and extracellular matrix.A lot of natural water gels and synthetic water gel all can be applicable to organizational project and regenerative medicine [Kopecek J.Hydrogel biomaterials:A smart future Biomaterials, 2007,28:5185-5192; Jagur-Grodzinski J.Polymeric gels and hydrogels for biomedical and pharmaceutical applications.Polymers for Advanced Technologies, 2010,21:27-47].
The formation of hydrogel mainly contains physical crosslinking and two kinds of main paties of chemically crosslinked.The formation of physical cross-linking hydrogel can realize through the variation of temperature, ionic strength, also can rely on approach such as charge effect, hydrogen bond action to realize, chemically crosslinked aquagel then forms through covalent linkage.The sorting technique of hydrogel has multiple: according to the difference of hydrogel network bonding, can be divided into physical cross-linking hydrogel and chemically crosslinked aquagel; Can be divided into natural water gel and synthetic hydrogel according to its source; The response condition that stimulates to external world according to hydrogel can be divided into ortho-water gel and intelligent aqueous gel capable; Also can whether degrade in addition and be divided into degradability hydrogel and non-degradable property hydrogel etc. according to hydrogel.
The injectable biomaterial obtains broad research and will have great effect to drug release and field of tissue engineering technology in recent years.Injection aquagel is the functional aquagel that one type of original position forms, and its rapid shaping characteristics make this type biomaterial have broad application prospects at clinicing aspect.Injection aquagel is a gel state in vivo the time, is solution state when external, can embedding under this state such as biologically active substance such as polypeptide, protein, cell; After being expelled to subcutis or muscle tissue; Because the generation of variation of temperature or chemical crosslink reaction, the solution that includes active substance is converted into gel rapidly, and then active substance is under the dual promotion of diffusion and gel self Degradation; In gel, discharge reposefully, reach the effect of long-term release.In the embedding process,, can not make the activity of embedding substance lose easily owing to can not relate to organic solvent.Another advantage of injection aquagel is that it can implant through injecting method; Make mobile biomaterial solution can be full of the whole erose damaged part that has; Little and easy operation [the Hoffman A S.Hydrogels for biomedical applications.Advanced Drug Delivery Reviews of operation wound; 2002,54:3-12].On the other hand, because DNA recombinant technology and other development of technologies have promoted the commercialization of polypeptide and pharmaceutical grade protein.But polypeptide and pharmaceutical grade protein are prone to be degraded in stomach, and the transformation period is short, and these shortcomings have all limited the application of polypeptide and pharmaceutical grade protein.And the syringeability hydrogel material is as carrier embedding and these polypeptide of load and pharmaceutical grade protein equably; Can directly inject subcutaneous and avoided being degraded by stomach; Can also reach the purpose of curing the sickness to save the patient, so injection aquagel obtain further development [Sakiyama-Elbert S M W by diffusion intravasation, arrival damaged part; Faxel TE; David I G.et al.The effects of soluble growth factors on embryonic stem cell differentiation inside of fibrin scaffolds.Stem Cells, 2007,25:2235-2244].
As the high water-keeping material of a kind of high suction, hydrogel is widely used in multiple field, but the overwhelming majority utilizes the mechanical property of natural or the hydrogel that synthetic materials prepares all very poor.The hydrogel of ordinary construction often has certain mechanical property than low water content the time, and (as＞90%) mechanical strength significantly descends when higher moisture, even just chipping under utmost point low pressure.Hydrogel has limited its application at medical field because of its mechanical property is relatively poor.Their cross-linking set distributes and has different length between irregular and the cross-linking set, and pressure can not be evenly distributed between the polymer chain, thereby crackle more is prone to, and has caused its relatively poor mechanical property.Therefore many researchs make great efforts all to concentrate on the intensity that improves hydrogel, but still can not satisfy request for utilization.
In recent years, synthetic research work with high intensity hydrogel has attracted numerous investigators' interest, and obtains certain progress.Prepare the DN hydrogel like people such as Nagayama; They adopt fine and close crosslinked polymkeric substance is that first network, loose crosslinked polymkeric substance are that second network is prepared double-network hydrogel with two kinds of network interpenetratings, and the mechanical property of this type of hydrogel is compared have significant improvement [Nakayama A, Kakugo A with the single network hydrogel; Gong J P; Osada Y, Takai M, Erata T; Kawano S.High mechanical strength double-network hydrogel with bacterial cellulose.Advanced Functional Materials 2004,14:1124-1128].And people such as Fleury prepare " 8 shape " the crosslinked ring that can on polymer chain, the be free to slide topological shape hydrogel as the may command cross-linking set; The tensile elongation of this type of hydrogel can reach about 20 times of original length; And the equilibrium swelling degree can reach about 500 [Fleury G; Schlatter G, Brochon C, Travelet C; Lapp A; Lindner P, Hadziioannou G.Topological polymer networks with sliding cross-link points:the " sliding gels " .relationship between their molecular structure and the viscoelastic as well as the swelling properties.Macromolecules 2007,40:535-543].The inorganic dispersant that Haraguchi etc. will have a nanoscale is scattered in and has prepared Nanometer composite hydrogel in the polymeric matrix; Homodisperse inorganic component can make the mechanical property of hydrogel that tens times raising [Haraguchi K is arranged in the hydrogel; Farnworth R; Ohbayashi A; Takehisa T.Compositional effects on mechanical properties of nanocomposite hydrogels composed of poly (N, N-dimethylacrylamide) and clay.Macromolecules 2003,36:5732-5741].Through irradiation initiation grafting water-soluble monomer, and with this microballoon to be arranged be the hydrogel that regular crosslinking structure is provided in the dispersion-s preparation to people such as Huang on macromolecular microspheres surface.Because macromolecular microspheres serves as the node of graft copolymerization; Homodisperse and obtain uniform crosslinking structure in compound system simultaneously; Therefore this type of composite aquogel has significantly on mechanical property and improves [Huang T, Xu H G, Jiao K X; Zhu L P; Brown H R, Wang H L.A novel hydrogel with high mechanical strength:a macromolecular microsphere composite hydrogel.Advanced Materials 2007,19:1622-1626].People such as Tan are raw material is prepared controllable particle size distribution through self-assembling method nano-starch particle with the mixing starch ester; And be that linking agent has synthesized SEPIGEL 305 and poly N-isopropyl acrylamide hydrogel with this nanoparticle; Result of study confirms that the swelling rate that goes of this type of novel hydrogels sharply accelerates, and the mechanical strength of hydrogel also be improved largely [Tan Y, Xu K; Wang P; Li W, Sun S, Dong L S; High mechanical strength and rapid response rate of poly (N-isopropyl acrylamide) hydrogel crosslinked by starch-based nanospheres.Soft Matter 2010,6:1467-1471].
Can find out that adopt multiple strategy and method can obtain having the hydrogel of special construction, these hydrogels all are greatly increased or improve on mechanical strength and other performances.The present invention adopts the material of good biocompatibilities such as natural copolymer chitosan, gelatin, is the crucial part of the above-mentioned novel hydrogels of difference as toughener with nanoparticle.
Summary of the invention
The objective of the invention is to improve the mechanical property of natural polymer hydrogel; A kind of injection-type high-strength natural polyalcohol hydrogel and preparation method thereof is provided; With the nanoparticle is that toughener is an innovative point of the present invention; Under the prerequisite that does not influence the raw materials mix performance, significantly improve the compressive strength of natural polymer hydrogel, thereby can overcome the low defective of natural polymer hydrogel mechanical strength.This hydrogel is the injectable type hydrogel at 37 ℃ of following gels and rapid shaping.Natural polymer and synthetic polymer good biocompatibilities such as polycaprolactone, polyoxyethylene glycol such as the chitosan of being selected for use, gelatin can be degraded by organism.
A kind of HS injection aquagel of the present invention, structural formula is following:
Wherein: a is the agent structure of hydrogel, and b is the toughener structure of hydrogel;
Wherein R is the structure of natural copolymer chitosan or gelatin
A kind of HS injection aquagel of the present invention is a toughener with the nanoparticle, and hydrogel is 37 ℃ of following PhastGel moulding, injectable, and intensity reaches more than the 10kPa.
The method of hydrogel of the present invention may further comprise the steps:
(1) polyoxyethylene glycol is dissolved in methylene dichloride, its concentration is 2.5 * 10
-5～5.0 * 10
-5Mol/mL, frozen water cooling, nitrogen protection are constantly stirred down and are added triethylamine and acrylate chloride; The triethylamine that wherein adds, the mole number of acrylate chloride are respectively 2～4 times, 2～5 times of polyoxyethylene glycol mole number, and following reaction is after 12～24 hours, with reacting liquid filtering; Deposition, decompress filter, the vacuum-drying of final product normal temperature; Collect subsequent usely, this step obtains polyethyleneglycol diacrylate;
(2) taking by weighing natural polymer, to be made into concentration be 1～10% solution, and wherein natural polymer is chitosan or gelatin, stirs to form suspension-s; Add the N-hydroxy benzo triazole, be stirred to the solution clarification, add N-acetyl-L-cysteine then; Add 1-ethyl-(3-dimethylaminopropyl) carbodiimide subsequently, regulating pH is 3～5, stirring at room 3～5 hours; Lucifuge was dialysed freeze-drying, stored refrigerated three days down for 4 ℃; The mole number of the N-hydroxy benzo triazole that adds, N-acetyl-L-cysteine, carbodiimide is respectively 1～3 times, 2～4 times and 4～8 times of amino mole number on the natural polymer, and this step obtains the sulfhydrylation natural polymer;
(3) polycaprolactone and purified acetic acid ethyl ester are joined reactor drum, after the heating for dissolving, be cooled to 20～60 ℃; Add hexamethylene diisocyanate and 5～10mL purified acetic acid ethyl ester; Be warming up to 70～90 ℃, add polyoxyethylene glycol and 5～10mL purified acetic acid ethyl ester then, continue reaction; Hexamethylene diisocyanate that adds and polyoxyethylene glycol and polycaprolactone mol ratio are respectively 2～4 times and 2～5 times; Precipitating, suction filtration obtain the triblock copolymer of polyethylene glycol-caprolactone-polyoxyethylene glycol then;
(4) triblock copolymer is dissolved in methylene dichloride, the frozen water cooling, nitrogen protection adds triethylamine and acrylate chloride down, constantly stirs, and triblock copolymer: triethylamine: the mol ratio of acrylate chloride is 1:2～4:2～4; Down reaction 12～24 hours of nitrogen protection, temperature of reaction is 20～25 ℃, with reacting liquid filtering, deposition, decompress filter, the vacuum-drying of final product normal temperature obtains the triblock copolymer of acrylated;
(5) triblock copolymer that takes by weighing 100～200mg acrylated is dissolved in the 100mL methylene dichloride in the ampere ware, under the room temperature magnetic agitation, solution is splashed in the distilled water; Along with the volatilization of methylene dichloride, kernel solidifies balling-up, and the triblock copolymer of propylene acidylate is self-assembled into nano-micelle, and big aggregate particles is removed in spinning, and freeze-drying obtains the nanoparticle toughener;
(6) mol ratio of lip-deep pair of key of two keys of the sulfydryl on the natural polymer, polyethyleneglycol diacrylate, nanoparticle is 1:1～3:0.5～2.5; Reaction mixture is carried out ultrasonic abundant stirring; Method through being inverted bottle is measured gelation time; Per 30～60 seconds handstand bottles once, all samples all reacted 30～60 minutes in 25～37 ℃ of water-baths, made high intensity hydrogel after this step.
The present invention utilizes link coupled sulfydryl generation Michael addition reaction on two keys of polyethyleneglycol diacrylate (PEGDA) and the natural polymer, and the triblock copolymer nanoparticle with polyoxyethylene glycol and polycaprolactone (PEG-PCL-PEG) is a kind of novel high intensity hydrogel that toughener forms simultaneously.
Introduce method of the present invention below in detail:
(1) polyoxyethylene glycol (PEG) is dissolved in methylene dichloride, its concentration is 2.5 * 10
-5～5.0 * 10
-5Mol/mL, frozen water cooling, nitrogen protection add triethylamine and acrylate chloride down, and the triethylamine that wherein adds, the mole number of acrylate chloride are respectively 2～4 times, 2～5 times of PEG mole number, and constantly stir.Nitrogen protection is reaction down.End reaction liquid is filtered, deposition, decompress filter, the vacuum-drying of final product normal temperature is collected subsequent usely, and this step obtains PEGDA.PEG wherein: its reaction principle is as follows:
The reaction principle chemical formula of step (1)
(2) temperature in the step (1) is 20～25 ℃, and nitrogen protection was reacted 12～24 hours down.
(3) taking by weighing natural polymer (gelatin or chitosan), to be made into concentration be 1～10% solution, stirs to form suspension-s, adds N-hydroxy benzo triazole (HOBt), is stirred to the solution clarification.Add N-acetyl-L-cysteine (NAC) then, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDAC.HCL) subsequently.Regulating pH is 3～5.Stirring at room 3～5 hours, lucifuge was dialysed freeze-drying, stored refrigerated three days down for 4 ℃.The mole number of the HOBt that adds, NAC, EDAC.HCI is respectively 1～3 times, 2～4 times and 4～8 times of amino mole number on the natural polymer, and this step obtains the sulfhydrylation natural polymer.Its reaction principle is as follows:
The reaction principle chemical formula of step (3) (wherein R is natural copolymer chitosan or gelatin)
(4) polycaprolactone (PCL) and purified acetic acid ethyl ester (EAC) are joined reactor drum, after the heating for dissolving, be cooled to 20～60 ℃; The EAC that adds hexamethylene diisocyanate (HDI) and 5～10mL; Be warming up to 70～90 ℃, add the EAC of PEG and 5～10mL then, continue reaction.HDI that adds and PEG and PCL mol ratio are respectively 2～4 times and 2～5 times.Precipitating, suction filtration obtain triblock copolymer PECL then, and its reaction principle is as follows:
The reaction principle chemical formula of step (4)
(5) PECL is dissolved in methylene dichloride, the frozen water cooling, nitrogen protection adds triethylamine and acrylate chloride down, constantly stirs, and PECL: triethylamine: the mol ratio of acrylate chloride is 1: (2～4): (2～4).Nitrogen protection reaction was down carried out in 12～24 hours, and temperature of reaction is 20～25 ℃.。End reaction liquid is filtered, deposition, decompress filter, the vacuum-drying of final product normal temperature obtains the PECL of acrylated.This step reaction principle is similar with the chemical formula of step 3, and PECL is write a Chinese character in simplified form into following form, and its reaction principle is as follows:
The reaction principle chemical formula of step (5)
(6) take by weighing the above-mentioned sample of 100～200mg in the ampere ware, be dissolved in the 100mL methylene dichloride, under the room temperature magnetic agitation, solution is slowly splashed in the distilled water.Along with the volatilization of methylene dichloride, kernel solidifies balling-up, and the PECL of propylene acidylate is self-assembled into nano-micelle, and big aggregate particles, freeze-drying are removed in spinning.This step obtains the nanoparticle toughener.
(7) mol ratio of lip-deep pair of key of two keys of the sulfydryl on the natural polymer, PEGDA, nanoparticle is 1: (1～3): ultrasonic abundant stirring is carried out with reaction mixture in (0.5～2.5).Method through being inverted bottle is measured gelation time, and per 30～60 seconds handstand bottles once.All samples all reacted 30～60 minutes in 25～37 ℃ of water-baths.Make high intensity hydrogel after this step.Wherein the reaction principle of natural polymer and PEGDA is as follows, and its enhancement principle is as follows:
The reactive chemistry formula of the sulfydryl of natural polymer and PEGDA
The enhancing chemical formula of hydrogel (PECL of acrylated being write a Chinese character in simplified form into the form of reactant 1)
The present invention has following advantage: the novel hydrogels that the present invention makes has favorable biological degradability and biocompatibility.Nanoparticle plays the effect of toughener, thereby improves the intensity of hydrogel.The hydrogel intensity of preparation can reach the kPa level, can be described as high intensity hydrogel.In addition, this hydrogel gel time is shorter, has syringeability.
With the chitosan is example, and the compressive strength of test water gel can find out that through data the adding of nanoparticle has improved the intensity of hydrogel greatly shown in table-1, and nanoparticle has played the effect of toughener, but the cytotoxicity of hydrogel material is not influenced.
The compressive strength of hydrogel changes behind table-1 adding nanoparticle
With the chitosan is example, and the gel time of test is shown in table-2, and the time of the gel of hydrogel, all below 30min, wherein the fastest can be within 5min.
Table-1 adds the influence of nanoparticle to the gel time of hydrogel
Embodiment
Below in conjunction with specific embodiment the present invention is further described, but should limit protection scope of the present invention with this.
Embodiment 1
(1) with 5.0 * 10
-5Mol PEG (Mw=2000) is dissolved in the 100mL methylene dichloride, the frozen water cooling, and nitrogen protection adds 0.01mol triethylamine and 0.025mol acrylate chloride down, constantly stirs.Be warming up to room temperature, nitrogen protection was reacted 12 hours down.End reaction liquid is filtered, deposition, decompress filter, the vacuum-drying of final product normal temperature is collected subsequent usely, and this step obtains PEGDA2000.
(2) 1.00g chitosan (Mn=30,0000) is dissolved in the pure water of 100ml, adds the HOBt of 697.2mg (5.16mmol) behind the stirring formation suspension-s, be stirred to the solution clarification.The NAC that adds 3.368g (20.64mmol) then adds EDACHCL 7.914g (41.28mmol) subsequently, and logical nitrogen, lucifuge, adjusting pH are 5.The stirring at room reaction was dialysed freeze-drying, stored refrigerated three days down for 4 ℃ in lucifuge after 3 hours.
(3) with 10g (5.0 * 10
-5Mol) EAC of PCL (Mn=2000) and 10mL joins whisking appliance is housed, temperature is taken into account in the 100mL four-hole bottle of prolong, after the heating for dissolving, is cooled to 40 ℃, adds 0.017g (1.0 * 10
-4Mol) EAC of HDI and 5mL is warming up to 80 ℃ of reactions 3 hours, adds 20g (1.0 * 10 then
-4Mol) EAC of PEG (Mn=2000) and 10mL continues reaction about 3 hours.Crude product is dissolved in chloroform, joins precipitating in 20 times the cold diethyl ether then, and decompress filter obtains the PECL amphipathic three block copolymer.
(4) get synthetic sample 0.015mol in the step (3), be dissolved in the 300ml methylene dichloride, frozen water is cooled to 0 ℃, and nitrogen protection adds 0.03mol triethylamine and 0.075mol acrylate chloride down, constantly stirs.Be warming up to room temperature, nitrogen protection is 12 hours (in good time adding methylene dichloride therebetween) of reaction down.End reaction liquid is filtered, with cold diethyl ether deposition, decompress filter.Products therefrom is dissolved in trichloromethane once more, with the cold diethyl ether deposition, and decompress filter, triplicate like this, final product normal temperature vacuum-drying 24 hours is collected subsequent use.
(5) learn from else's experience the sample 100mg of step (4) after handling in the ampere ware, be dissolved in 3mL methylene dichloride (DCM), under the room temperature magnetic agitation, splash into solution in the 10mL distilled water.Along with the volatilization of DCM, kernel solidifies balling-up, and PECL is self-assembled into nano-micelle, and big aggregate particles is removed in spinning, and clear liquid gets the PECL nano freeze-dried powder through lyophilize.
(6) get the sulfydryl on the chitosan in (2): two keys of PEGDA2000 in (1): the mol ratio of the lip-deep pair of key of (5) nanoparticle is 1:2:1, and reaction mixture is carried out ultrasonic abundant stirring.The gel time of measuring through the method for being inverted bottle under 37 ℃ of conditions is 4 minutes, and the compressive strength of mensuration is 17.8kPa, and this embodiment is gel under 37 ℃ of conditions, and gel time is shorter, is fit to injection.
Embodiment 2
(1) with 5.0 * 10
-5Mol PEG (Mw=2000) is dissolved in the 100mL methylene dichloride, the frozen water cooling, and nitrogen protection adds 0.01mol triethylamine and 0.02mol acrylate chloride down, constantly stirs.Be warming up to room temperature, nitrogen protection was reacted 12 hours down.End reaction liquid is filtered, deposition, decompress filter, the vacuum-drying of final product normal temperature is collected subsequent usely, and this step obtains PEGDA2000.
(2) 1.00g chitosan (Mn=30,0000) is dissolved in the pure water of 100ml, adds the HOBt of 2091.6mg (15.48mmol) behind the stirring formation suspension-s, be stirred to the solution clarification.The NAC that adds 1.684g (10.32mmol) then adds EDACHCL 3.957g (20.64mmol) subsequently, and logical nitrogen, lucifuge, adjusting pH are 4.The stirring at room reaction was dialysed freeze-drying, stored refrigerated three days down for 4 ℃ in lucifuge after 5 hours.
(3) with 10g (5.0 * 10
-5Mol) EAC of PCL (Mn=2000) and 10mL joins whisking appliance is housed, temperature is taken into account in the 100mL four-hole bottle of prolong, after the heating for dissolving, is cooled to 20 ℃, adds 0.017g (1.0 * 10
-4Mol) EAC of HDI and 5mL is warming up to 70 ℃ of reactions 3 hours, adds 20g (1.0 * 10 then
-4Mol) EAC of PEG (Mn=2000) and 5mL continues reaction about 3 hours.Crude product is dissolved in chloroform, joins precipitating in 20 times the cold diethyl ether then, and decompress filter obtains the PECL amphipathic three block copolymer.
(4) get synthetic sample 0.015mol in the step (3), be dissolved in the 300ml methylene dichloride, frozen water is cooled to 0 ℃, and nitrogen protection adds 0.015mol triethylamine and 0.045mol acrylate chloride down, constantly stirs.Be warming up to room temperature, nitrogen protection is 12 hours (in good time adding methylene dichloride therebetween) of reaction down.End reaction liquid is filtered, with cold diethyl ether deposition, decompress filter.Products therefrom is dissolved in trichloromethane once more, with the cold diethyl ether deposition, and decompress filter, triplicate like this, final product normal temperature vacuum-drying 24 hours is collected subsequent use.
(5) learn from else's experience the sample 100mg of step (4) after handling in the ampere ware, be dissolved in 3mL methylene dichloride (DCM), under the room temperature magnetic agitation, splash into solution in the 10mL distilled water.Along with the volatilization of DCM, kernel solidifies balling-up, and PECL is self-assembled into nano-micelle, and big aggregate particles is removed in spinning, and clear liquid gets the PECL nano freeze-dried powder through lyophilize.
(6) get the sulfydryl on the chitosan in (2): two keys of PEGDA2000 in (1): the mol ratio of the lip-deep pair of key of (5) nanoparticle is 1:2:1, and reaction mixture is carried out ultrasonic abundant stirring.The gel time of measuring through the method for being inverted bottle under 37 ℃ of conditions is 4 minutes, and the compressive strength of mensuration is 17.4kPa, and this embodiment is gel under 37 ℃ of conditions, and gel time is shorter, is fit to injection.
Embodiment 3
(1) with 5.0 * 10
-5Mol PEG (Mw=2000) is dissolved in the 100mL methylene dichloride, the frozen water cooling, and nitrogen protection adds 0.02mol triethylamine and 0.025mol acrylate chloride down, constantly stirs.Be warming up to room temperature, nitrogen protection was reacted 18 hours down.End reaction liquid is filtered, deposition, decompress filter, the vacuum-drying of final product normal temperature is collected subsequent usely, and this step obtains PEGDA2000.
(2) 1.00g chitosan (Mn=30,0000) is dissolved in the pure water of 100ml, adds the HOBt of 1045.8mg (7.74mmol) behind the stirring formation suspension-s, be stirred to the solution clarification.The NAC that adds 3.368g (22.64mmol) then adds EDACHCL 7.914g (41.28mmol) subsequently, and logical nitrogen, lucifuge, adjusting pH are 5.The stirring at room reaction was dialysed freeze-drying, stored refrigerated three days down for 4 ℃ in lucifuge after 5 hours.
(3) with 10g (5.0 * 10
-5Mol) EAC of PCL (Mn=2000) and 10mL joins whisking appliance is housed, temperature is taken into account in the 100mL four-hole bottle of prolong, after the heating for dissolving, is cooled to 60 ℃, adds 0.034g (2.0 * 10
-4Mol) EAC of HDI and 10mL is warming up to 90 ℃ of reactions 3 hours, adds 40g (2.0 * 10 then
-4Mol) EAC of PEG (Mn=2000) and 10mL continues reaction about 3 hours.Crude product is dissolved in chloroform, joins precipitating in 20 times the cold diethyl ether then, and decompress filter obtains the PECL amphipathic three block copolymer.
(4) get synthetic sample 0.015mol in the step (3), be dissolved in the 300ml methylene dichloride, frozen water is cooled to 0 ℃, and nitrogen protection adds 0.060mol triethylamine and 0.060mol acrylate chloride down, constantly stirs.Be warming up to room temperature, nitrogen protection is 18 hours (in good time adding methylene dichloride therebetween) of reaction down.End reaction liquid is filtered, with cold diethyl ether deposition, decompress filter.Products therefrom is dissolved in trichloromethane once more, with the cold diethyl ether deposition, and decompress filter, triplicate like this, final product normal temperature vacuum-drying 24 hours is collected subsequent use.
(5) learn from else's experience the sample 100mg of step (4) after handling in the ampere ware, be dissolved in 3mL methylene dichloride (DCM), under the room temperature magnetic agitation, splash into solution in the 10mL distilled water.Along with the volatilization of DCM, kernel solidifies balling-up, and PECL is self-assembled into nano-micelle, and big aggregate particles is removed in spinning, and clear liquid gets the PECL nano freeze-dried powder through lyophilize.
(6) get the sulfydryl on the chitosan in (2): two keys of PEGDA2000 in (1): the mol ratio of the lip-deep pair of key of (5) nanoparticle is 1:2:1, and reaction mixture is carried out ultrasonic abundant stirring.The gel time of measuring through the method for being inverted bottle under 37 ℃ of conditions is 3 minutes, and the compressive strength of mensuration is 18.0kPa, and this embodiment is gel under 37 ℃ of conditions, and gel time is shorter, is fit to injection.
Embodiment 4
Raw material and embodiment 1 are basic identical, and (1) and (6) of embodiment 1 is adjusted into:
(1) with 5.0 * 10
-5The PEG of mol (Mw=575) is dissolved in the 100mL methylene dichloride, the frozen water cooling, and nitrogen protection adds the triethylamine of 0.01mol and the acrylate chloride of 0.025mol down, constantly stirs.Be warming up to room temperature, nitrogen protection was reacted 12 hours down.With the filtration of end reaction liquid, deposition, decompress filter, the vacuum-drying of final product normal temperature.This step obtains PEGDA575.
(6) get the sulfydryl on the chitosan in (2): two keys of PEGDA575 in (1): the mol ratio of the lip-deep pair of key of (5) nanoparticle is 1:2:1, and reaction mixture is carried out ultrasonic abundant stirring.The gel time of measuring through the method for being inverted bottle under 37 ℃ of conditions is for being about 10 minutes, and the compressive strength of mensuration is 10.1kPa.This embodiment is gel under 37 ℃ of conditions, and gel time is not long, can inject.
Embodiment 5
Raw material and embodiment 1 are basic identical, and (2) and (6) of embodiment 1 are adjusted into:
(2) the A type gelatin with 2.00g is dissolved in the pure water of 100ml, after stirring forms suspension-s, adds HOBt0.5067g (3.75mol), is stirred to the solution clarification.The NAC that adds 2.448g (15.02mol) then adds EDACHCL5.751g (29.97mol) subsequently, lucifuge, logical nitrogen, and regulating pH is 5.Stirring at room 3 hours, 4 ℃ of dialysis freeze-drying after three days down of lucifuge, stored refrigerated.
(6) get the sulfydryl on the gelatin in (2): two keys of PEGDA2000 in (1): the mol ratio of the lip-deep pair of key of (5) nanoparticle is 1:2:1, and reaction mixture is carried out ultrasonic abundant stirring.The gel time of measuring through the method for being inverted bottle under 37 ℃ of conditions is for being about 10 minutes, and the compressive strength of mensuration is 11.4kPa.This embodiment is gel under 37 ℃ of conditions, and gel time is not long, can inject.
Embodiment 6
Raw material and embodiment 3 are basic identical, and (2) and (6) of embodiment 3 are adjusted into:
(2) the A type gelatin with 2.00g is dissolved in the pure water of 100ml, after stirring forms suspension-s, adds HOBt0.6135g (4.54mol), is stirred to the solution clarification.The NAC that adds 2.7236g (16.71mol) then adds EDACHCL4.8353g (25.18mol) subsequently, lucifuge, logical nitrogen, and regulating pH is 5.Stirring at room 4 hours, 4 ℃ of dialysis freeze-drying after three days down of lucifuge, stored refrigerated.
(6) get the sulfydryl on the gelatin in (2): two keys of PEGDA2000 in (1): the mol ratio of the lip-deep pair of key of (5) nanoparticle is 1:2:1, and reaction mixture is carried out ultrasonic abundant stirring.The gel time of measuring through the method for being inverted bottle under 37 ℃ of conditions is for being about 9 minutes, and the compressive strength of mensuration is 11.7kPa.This embodiment is gel under 37 ℃ of conditions, and gel time is not long, can inject.
Claims (3)
1. HS injection aquagel is characterized in that structural formula is following:
Wherein: a is the agent structure of hydrogel, and b is the toughener structure of hydrogel;
Wherein R is the structure of natural copolymer chitosan or gelatin.
2. a kind of HS injection aquagel as claimed in claim 1 is characterized in that with the nanoparticle being toughener, and hydrogel is 37 ℃ of following PhastGel moulding, injectable, and intensity reaches more than the 10kPa.
3. prepare the method for claim 1 or 2 described hydrogels, it is characterized in that may further comprise the steps:
(1) polyoxyethylene glycol is dissolved in methylene dichloride, its concentration is 2.5 * 10
-5～5.0 * 10
-5Mol/mL, frozen water cooling, nitrogen protection are constantly stirred down and are added triethylamine and acrylate chloride; The triethylamine that wherein adds, the mole number of acrylate chloride are respectively 2～4 times, 2～5 times of polyoxyethylene glycol mole number, and following reaction is after 12～24 hours, with reacting liquid filtering; Deposition, decompress filter, the vacuum-drying of final product normal temperature; Collect subsequent usely, this step obtains polyethyleneglycol diacrylate;
(2) taking by weighing natural polymer, to be made into concentration be 1～10% solution, and wherein natural polymer is chitosan or gelatin, stirs to form suspension-s; Add the N-hydroxy benzo triazole, be stirred to the solution clarification, add N-acetyl-L-cysteine then; Add 1-ethyl-(3-dimethylaminopropyl) carbodiimide subsequently, regulating pH is 3～5, stirring at room 3～5 hours; Lucifuge was dialysed freeze-drying, stored refrigerated three days down for 4 ℃; The mole number of the N-hydroxy benzo triazole that adds, N-acetyl-L-cysteine, carbodiimide is respectively 1～3 times, 2～4 times and 4～8 times of amino mole number on the natural polymer, and this step obtains the sulfhydrylation natural polymer;
(3) polycaprolactone and purified acetic acid ethyl ester are joined reactor drum, after the heating for dissolving, be cooled to 20～60 ℃; Add hexamethylene diisocyanate and 5～10mL purified acetic acid ethyl ester; Be warming up to 70～90 ℃, add polyoxyethylene glycol and 5～10mL purified acetic acid ethyl ester then, continue reaction; Hexamethylene diisocyanate that adds and polyoxyethylene glycol and polycaprolactone mol ratio are respectively 2～4 times and 2～5 times; Precipitating, suction filtration obtain the triblock copolymer of polyethylene glycol-caprolactone-polyoxyethylene glycol then;
(4) triblock copolymer is dissolved in methylene dichloride, the frozen water cooling, nitrogen protection adds triethylamine and acrylate chloride down, constantly stirs, and triblock copolymer: triethylamine: the mol ratio of acrylate chloride is 1: 2～4: 2～4; Down reaction 12～24 hours of nitrogen protection, temperature of reaction is 20～25 ℃, with reacting liquid filtering, deposition, decompress filter, the vacuum-drying of final product normal temperature obtains the triblock copolymer of acrylated;
(5) triblock copolymer that takes by weighing 100～200mg acrylated is dissolved in the 100mL methylene dichloride in the ampere ware, under the room temperature magnetic agitation, solution is splashed in the distilled water; Along with the volatilization of methylene dichloride, kernel solidifies balling-up, and the triblock copolymer of propylene acidylate is self-assembled into nano-micelle, and big aggregate particles is removed in spinning, and freeze-drying obtains the nanoparticle toughener;
(6) mol ratio of lip-deep pair of key of two keys of the sulfydryl on the natural polymer, polyethyleneglycol diacrylate, nanoparticle is 1: 1～3: 0.5～2.5; Reaction mixture is carried out ultrasonic abundant stirring; Through being inverted the method for bottle, measure gelation time, per 30～60 seconds handstand bottles are once; All samples all reacted 30～60 minutes in 25～37 ℃ of water-baths, made high intensity hydrogel after this step.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1425706A (en) * | 2003-01-14 | 2003-06-25 | 天津大学 | Polyglycol block modified polyhexanolactone and its preparing method |
| US20100158979A1 (en) * | 2007-07-18 | 2010-06-24 | The Board Of Trustees Of The University Of Illinois | Temporal release of growth factors from 3d micro rod scaffolds for tissue regeneration |
| WO2011002249A2 (en) * | 2009-07-02 | 2011-01-06 | Ajou University Industry-Academic Cooperation Foundation | In situ forming hydrogel and biomedical use thereof |
| CN102241837A (en) * | 2011-06-08 | 2011-11-16 | 天津大学 | Thiolated-chitosan-based temperature-sensitive in-situ hydrogel as well as preparation method and uses thereof |
-
2012
- 2012-04-05 CN CN2012100975229A patent/CN102718991A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1425706A (en) * | 2003-01-14 | 2003-06-25 | 天津大学 | Polyglycol block modified polyhexanolactone and its preparing method |
| US20100158979A1 (en) * | 2007-07-18 | 2010-06-24 | The Board Of Trustees Of The University Of Illinois | Temporal release of growth factors from 3d micro rod scaffolds for tissue regeneration |
| WO2011002249A2 (en) * | 2009-07-02 | 2011-01-06 | Ajou University Industry-Academic Cooperation Foundation | In situ forming hydrogel and biomedical use thereof |
| CN102241837A (en) * | 2011-06-08 | 2011-11-16 | 天津大学 | Thiolated-chitosan-based temperature-sensitive in-situ hydrogel as well as preparation method and uses thereof |
Non-Patent Citations (6)
| Title |
|---|
| 《Macromolecules》 20030701 Kazutoshi Haraguchi et al "Compositional Effects on Mechanical Properties of Nanocomposite Hydrogels Composed of Poly(N,N-dimethylacrylamide) and Clay" 第5732-5741页，实验部分，结论部分 1-3 , 第36期 * |
| 《Soft Matterial》 20100209 Ying Tan et al "High mechanical strength and rapid response rate of poly(N-isopropyl acrylamide) hydrogel crosslinked by starch-based nanospheres" 第1467-1471页，图示1，结论部分 1-3 , 第6期 * |
| 《化学通报》 20050618 娄翔等 "巯基化壳聚糖/明胶的制备研究" 第452-457页，第1.2部分 3 , 第6期 * |
| KAZUTOSHI HARAGUCHI ET AL: ""Compositional Effects on Mechanical Properties of Nanocomposite Hydrogels Composed of Poly(N,N-dimethylacrylamide) and Clay"", 《MACROMOLECULES》 * |
| YING TAN ET AL: ""High mechanical strength and rapid response rate of poly(N-isopropyl acrylamide) hydrogel crosslinked by starch-based nanospheres"", 《SOFT MATTERIAL》 * |
| 娄翔等: ""巯基化壳聚糖/明胶的制备研究"", 《化学通报》 * |
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