CN108794771A - Dual network cross-linked cellulose/fibroin albumen high intensity hydrogel and its preparation and application - Google Patents
Dual network cross-linked cellulose/fibroin albumen high intensity hydrogel and its preparation and application Download PDFInfo
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Abstract
The invention discloses a kind of dual network cross-linked cellulose/fibroin albumen high intensity hydrogels and its preparation and application, wherein preparation method to include the following steps：(1) cellulose solution, silk fibroin protein solution, epoxychloropropane are uniformly mixed, crosslinking is allowed under stirring condition and obtains crosslinker solution；(2) it is poured into mold after crosslinker solution being centrifuged de-bubbled, low-density chemical crosslinking cellulose/silk fibroin hydrogel is obtained after reaction solidification；(3) obtained cellulose/silk fibroin hydrogel is positioned over progress atmosphere physical crosslinking in carbon dioxide incubator；(4) it is rinsed with flowing water, you can obtain dual network cross-linked cellulose/fibroin albumen high intensity hydrogel.The present invention can get the hydrogel with different mechanical strengths and hydrophilic and hydrophobic by the ratio of adjusting cellulose and fibroin albumen.The experimental results showed that this kind of dual network crosslinking high intensity hydrogel has good cell compatibility and blood compatibility, there is larger application potential in field of tissue engineering technology.
Description
Technical field
The invention belongs to the crossing domains in Chemistry and Physics of Polymers field and organizational project, more particularly, to one kind
Dual network cross-linked cellulose/fibroin albumen high intensity hydrogel and the preparation method and application thereof.
Background technology
With the development of life science, advanced biomaterial such as hydrogel, composite sponge, nanoparticle etc. constantly comes out,
New scheme is provided to solve clinical problem.Hydrogel refers to one kind has three-dimensional by what physically or chemically crosslinking method was formed
The polymeric system (Adv.Mater., 2017,29,1700321) of network structure.It is presently used for preparing the raw material master of hydrogel
To include that the natural polymers and polyvinyl alcohol, polyethylene glycol, poly- second lactone etc. such as cellulose, chitosan, collagen synthesize macromolecule
(Biomacromolecules.,2018,4,1356-1364；Adv.Funct.Mater.,2017,27,1606497).It is natural high
Molecule hydrogel is because it is with higher water content and water holding capacity, and the specifically bioactivity of microstructure and raw material
(Biomaterials., 2017,122,34-37), in doctors such as skin tissue engineering, bone tissue engineer and drug release carriers
Medicine field shows wide application prospect.In practical applications, hydrogel should have the mechanics to match with tissue
Intensity.However, hydrogel generally have the shortcomings that matter it is soft, broken (ACS.Nano., 2017,11,2554-2560), promoted water
The mechanical property of gel receives the close attention of research staff.
Cellulose is to be distributed widest natural macromolecular material in the world, have rich reserves, can natural degradation, without thin
The advantages that cellular toxicity and good processing performance.The patent of invention of the patent No. 200810197646.8 is prepared for a kind of epoxychloropropane
Crosslinked cellulose aquagel, being excessively used for crosslinking agent cause hydrogel crosslink density excessive, and compressive strength is only tens of
KPa.Patent No. CN 107417934A disclose a kind of high-strength, high-ductility cellulose aquagel, it is first to use a small amount of epoxy chloropropionate
The cellulose aquagel of low chemical crosslinking density is obtained by the reaction in alkane, is then impregnated in acidic aqueous solution and carries out rapid physical friendship
Connection.The experimental results showed that the hydrogel compressive strength obtained has reached MPa grades, the disadvantage is that cellulose aquagel bioactivity
Difference, mechanical property not easy-regulating, and the use of acid reagent may aggravate environmental pollution.
Fibroin albumen is one of the main component of natural silk fiber, has good water solubility, cell compatibility, blood
Compatibility and low immunogenicity (Acta.Biomaterialia., 2016,31,1-16).Containing more in the polypeptide chain of fibroin albumen
Kind chemical active radical and cell active factor, such as cell adhesion molecules RGD, thus have certain bioactivity.Research
The result shows that the catabolite of fibroin albumen be conducive to skin, periodontal and liver organization regeneration (Prog.Polym.Sci.,
2014,39,251-267) while currently, fibroin albumen in bioreactor, organizational project organ and slow releasing carrier of medication
Equal fields achieve good application effect.
It yet there are no the report in relation to preparing cellulose and fibroin albumen high intensity hydrogel.
Invention content
For the disadvantages described above or Improvement requirement of the prior art, the purpose of the present invention is to provide a kind of crosslinking of dual network is fine
It ties up element/fibroin albumen high intensity hydrogel and its preparation and application, this method is molten using cellulose and fibroin albumen as primary raw material
Solution is blended in sodium hydroxide/urea liquid, low-density chemical crosslinking, CO2The techniques such as atmosphere crosslinking and bath prepare in pairs
Network cross-linked high intensity hydrogel.
It is primary raw material that the present invention, which selects cellulose and fibroin albumen, not only efficiently solves pure high strength fibre element water
The bad problem of bioactive hydrogel, and realize the easy regulation and control of mechanical strength.Content by changing fibroin albumen can
To adjust the mechanical strength of high intensity hydrogel, and then meets the needs of human body different tissues organ is to hydrogel mechanical strength.
The present invention uses CO2The crosslinked method of atmosphere carries out two level physical crosslinking to the hydrogel that low-density is chemically crosslinked, with existing skill
Art is compared as impregnated ethyl alcohol or acid solution, has the advantages such as mild reaction condition, good moldability, environmentally safe.It is obtained
Dual network cross-linked cellulose/fibroin albumen high intensity hydrogel there is adjustable compressive strength, hydrophilic and hydrophobic and excellent
Good cell compatibility and blood compatibility, it is very potential to be applied to field of tissue engineering technology.
To achieve the above object, according to one aspect of the present invention, a kind of dual network cross-linked cellulose/fibroin egg is provided
The preparation method of white high intensity hydrogel, which is characterized in that include the following steps：
(1) cellulose solution, silk fibroin protein solution, epoxychloropropane are uniformly mixed, are allowed to be crosslinked under stirring condition
To crosslinker solution；
(2) it pours into mold, is obtained after reaction solidification low after the crosslinker solution that the step (1) obtains being centrifuged de-bubbled
Density chemical cross-linked cellulose/silk fibroin hydrogel；
(3) cellulose/silk fibroin hydrogel that the step (2) obtains is positioned in carbon dioxide incubator and is carried out
Atmosphere is physical crosslinking；
(4) cellulose/silk fibroin hydrogel that the step (3) obtains is rinsed with flowing water, obtains dual network crosslinking
Cellulose/fibroin albumen high intensity hydrogel.
Preferably, in the step (1), the cellulose solution is prepared by the following method to obtain：By 7 parts by weight
Sodium hydroxide and 12 parts by weight urea co-dissolve in the deionized water of 81 parts by weight, it is molten to obtain sodium hydroxide/urea
Liquid, then cellulose and the sodium hydroxide/urea liquid are cooled to -12 DEG C in advance respectively and then mixed, it is vigorously stirred until dissolving i.e.
Cellulose solution can be obtained.
Preferably, the silk fibroin protein solution is prepared by the following method to obtain：By the sodium hydroxide of 7 parts by weight and
The urea co-dissolve of 12 parts by weight obtains sodium hydroxide/urea liquid in the deionized water of 81 parts by weight, then by fibroin egg
Mechanical agitation after being mixed in vain with the sodium hydroxide/urea liquid can be obtained silk fibroin protein solution until being completely dissolved.
Preferably, in the crosslinker solution that the step (1) obtains, the mass fraction of cellulose is 1.5~4.0%, fibroin
The mass fraction of albumen is 0~2.0%, and the mass fraction of epoxychloropropane is 0.5-10%.
Preferably, in the step (1), the crosslinking carries out at room temperature, crosslinking time 5-15min.
Preferably, in the step (2), the centrifugation de-bubbled condition is that 1500-3000rpm centrifuges 5-10min；
Preferably, in the step (4), described rinsed with flowing water is specifically to rinse 48h or more with deionized water.
Preferably, in the step (2), the reaction condition of cure is to stand 4-24h at room temperature.
Preferably, in the step (3), the physical crosslinking is at room temperature, in CO2Gas concentration is 3-30vol%
Contain CO22-12h is handled under atmosphere.
It is another aspect of this invention to provide that the present invention provides the dual network being prepared using above-mentioned preparation method crosslinkings
Cellulose/fibroin albumen high intensity hydrogel.
Another aspect according to the invention, the present invention provides above-mentioned dual network cross-linked cellulose/fibroin albumen high intensity
The application of hydrogel, which is characterized in that be applied to field of tissue engineering technology.
In dual network cross-linked cellulose/fibroin albumen high intensity hydrogel that the present invention is obtained, by adjusting cellulose
Different compressive strengths and hydrophilic and hydrophobic can be obtained with the mass ratio of fibroin albumen.
Vitro cytotoxicity is tested and hemolytic experiment shows that cellulose of the present invention/fibroin albumen high intensity hydrogel has
Good cell compatibility and blood compatibility.Therefore, the present invention has certain application potential in field of tissue engineering technology.
The invention has the advantages that and advantageous effect：(1) low-density chemical crosslinking and CO is used in combination2Atmosphere crosslinking is aobvious
The mechanical strength for improving hydrogel write；(2) bioactivity that hydrogel is improved by the way that fibroin albumen is added, realizes water
The easy regulation and control of gel mechanical strength and hydrophilic and hydrophobic；(3)CO2Item is reacted in atmosphere crosslinking compared with impregnating ethyl alcohol and acid flux material
Part milder, gel forming is more preferable, the cellulose that environmental pollution smaller (4) is obtained/fibroin albumen high intensity hydrogel tool
There are good cell compatibility and blood compatibility, it is very potential to be applied to field of tissue engineering technology.
Description of the drawings
Fig. 1 is the result for the dual network crosslinking high intensity hydrogel compression verification that comparative example 1 and embodiment 1 are obtained.
Fig. 2 is the dual network crosslinking high intensity hydrogel water contact angle measurement experiment that comparative example 1 and embodiment 1 are obtained
As a result.
Fig. 3 is the dual network crosslinking high intensity hydrogel vitro cytotoxicity experiment that comparative example 1 and embodiment 1 are obtained
As a result.
Fig. 4 be comparative example 1 and embodiment 1 obtained dual network crosslinking high intensity hydrogel hemolytic experiment as a result, its
In, DS represents distilled water (distilled water), and NS represents physiological saline (normal saline).
Fig. 5 is the schematic diagram of dual network cross-linked cellulose of the present invention/fibroin albumen high intensity hydrogel preparation flow, from a left side
To the right, it corresponds to be blended respectively from top to bottom, low-density chemical crosslinking, atmosphere crosslinking, wash by water.
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that described herein, specific examples are only used to explain the present invention, not
For limiting the present invention.As long as in addition, technical characteristic involved in the various embodiments of the present invention described below that
Conflict is not constituted between this to can be combined with each other.
As described in Figure 5, the preparation method of dual network cross-linked cellulose/fibroin albumen high intensity hydrogel is summarized in the present invention
For, it is that cellulose, fibroin albumen are individually dissolved separately in sodium hydroxide/urea liquid, it is blended, low-density is chemical
Crosslinking, CO2Atmosphere is crosslinked and dual network cross-linked cellulose/fibroin albumen high intensity hydrogel is prepared in the techniques such as bath.
Comparative example 1
7g sodium hydroxides and 12g urea are dissolved in 81g deionized waters, sodium hydroxide/urea liquid is obtained.By cellulose
It is cooled to -12 DEG C in advance respectively with sodium hydroxide/urea liquid, 4min is vigorously stirred after mixing, obtains the fibre that mass fraction is 2.5%
The plain solution of dimension.The epoxychloropropane for accounting for cellulose solute gross mass 100% is added dropwise into above-mentioned solution, continues stirring crosslinking
Crosslinker solution is poured into mold after centrifuging 10min under conditions of 2000rpm, is stood at room temperature by 15min, reaction solidification
12h is then transferred to 5vol%CO2Continue to be crosslinked 6h in incubator, it is fine that dual network crosslinking is obtained after rinsing 48h with deionized water
The plain high intensity hydrogel of dimension.
Embodiment 1
7g sodium hydroxides and 12g urea are dissolved in 81g deionized waters, sodium hydroxide/urea liquid is obtained.By cellulose
It is cooled to -12 DEG C in advance respectively with sodium hydroxide/urea liquid, 4min is vigorously stirred after mixing, obtains the fiber that mass fraction is 3%
Plain solution.Fibroin albumen is dissolved in and obtains the silk fibroin protein solution that mass fraction is 10% in sodium hydroxide/urea liquid.It will be fine
The plain solution of dimension and silk fibroin protein solution are uniformly mixed according to a certain percentage, and a small amount of sodium hydroxide/urea is then added thereto
The mass fraction that solution adjusts cellulose is 2.5%, and the mass fraction of fibroin albumen is followed successively by 0.5%, 1%, 1.5%.Upwards
The epoxychloropropane for accounting for cellulose and fibroin albumen solute gross mass 100% is added in the mixed solution stated, continues stirring crosslinking
Crosslinker solution is poured into mold after centrifuging 10min under conditions of 2000rpm, is stood at room temperature by 15min, reaction solidification
12h is then transferred to 5%CO2Continue to be crosslinked 6h in incubator, dual network cross filament is obtained after rinsing 48h with deionized water
Element/fibroin albumen high intensity hydrogel.CCS-0, CCS-0.5, CCS-1, CCS-1.5 respectively represent fibroin albumen in hydrogel
Final mass score is respectively 0%, 0.5%, 1% and 1.5%.CCS-0 is the product of corresponding comparative example 1.
Embodiment 2
The dual network crosslinking high intensity hydrogel that comparative example 1 and embodiment 1 are obtained is cut into a diameter of 10mm, height
For the cylinder of 8mm.The compressive strength of hydrogel, compression speed 2mm/min are detected using universal testing machine.
Fig. 1 is the dual network crosslinking high intensity hydrogel compression verification that comparative example 1 and embodiment 1 are obtained as a result, such as
Seen in figure, CCS-0, CCS-0.5, the compressive strength of CCS-1, CCS-1.5 is respectively 2.51MPa, 1.58MPa, 0.91MPa and
0.55MPa shows that hydrogel prepared by the present invention has good mechanical strength.With the increase of fibroin albumen content, water-setting
The mechanical strength of glue is decreased obviously, and significant difference (P is all had between each group<0.05).By the content for controlling fibroin albumen
The mechanical strength of regulation and control high intensity hydrogel that can be easy, to meet different tissues organ in practical applications to hydrogel
The demand of mechanical strength.
Embodiment 3
The dual network crosslinking high intensity hydrogel that comparative example 1 and embodiment 1 are obtained is positioned in 60 DEG C of baking ovens dry
For 24 hours, the sample for being then cut into 1cm × 1cm sizes is pasted on glass slide.Gel surface is measured using water contact angle measuring instrument
Hydrophilic and hydrophobic.
Fig. 2 is the dual network crosslinking high intensity hydrogel water contact angle measurement experiment that comparative example 1 and embodiment 1 are obtained
As a result.As shown in Fig. 2, the water contact angle of CCS-0, CCS-0.5, CCS-1, CCS-1.5 are respectively 53.8 °, and 64.7 °, 85.8 °,
115.8°.With the increase of fibroin albumen content, hydrogel hydrophilicity gradually weakens, and realizes by hydrophily to hydrophobic
Transformation.Cell material surface Adhering capacity and material hydrophilic and hydrophobic have significant correlation, for different tissues come
The cell in source should take the circumstances into consideration to increase and decrease the content of fibroin albumen.
Embodiment 4
It is cut into powder after the dual network crosslinking high intensity hydrogel that comparative example 1 and embodiment 1 are obtained is dried, high pressure is gone out
It is immersed in after bacterium in α-DMEM culture mediums.1mL culture mediums, 37 DEG C of incubation 72h is added to obtain hydrogel after filtering per 0.05g powder
Leaching liquor.The cell compatibility of hydrogel is detected using vitro cytotoxicity experiment (mtt assay).By l cell
(L929) it is inoculated in 96 orifice plates, inoculum density is 1 × 103Cells/well.Routine culture discards culture medium afterwards for 24 hours, and 200 μ are added
L material leaching liquors, continue culture 24,48 and 72h. uses α-DMEM to cultivate L929 as negative control, and blank culture plate is as empty
White control.20 μ L tetrazolium bromides (MTT) reagents are added into every hole.After continuing culture 4 hours, the liquid in culture plate is all abandoned
It goes, 150 μ L dimethyl sulfoxides (DMSO) is added per hole, after being protected from light concussion 10min, it is detected in 490nm using multi-function microplate reader
Absorbance at wavelength.Statistical analysis obtains the cell proliferation rate of L929.
Fig. 3 is the dual network crosslinking high intensity hydrogel vitro cytotoxicity experiment that comparative example 1 and embodiment 1 are obtained
As a result.As seen in figures, the cell proliferation rate of control group is set as 100%, compared with the control group, CCS-0, CCS-0.5, CCS-1,
The cell proliferation rate of CCS-1.5 was noticeably greater than 80% at the 1-3 days, showed good cell compatibility.
Embodiment 5
The sample that the dual network crosslinking high intensity hydrogel that comparative example 1 and embodiment 1 obtain is cut into 1cm × 1cm is put into
In 15mL centrifuge tubes, rinsed 3 times with physiological saline, each 30min.The addition 10mL physiological saline into every centrifuge tube, 37 DEG C
Water bath with thermostatic control 30min, it is 1 to be subsequently added into whole blood with physiological saline mass ratio:1.25 dilution whole blood 0.2mL mixings, 37 DEG C of perseverances
Tepidarium 60min centrifuges 10min under 1500rpm.It is positive control, 10mL physiology with 10mL distilled water dilution 0.2mL whole bloods
It is negative control that brine, which dilutes 0.2mL whole bloods,.The finally extinction with multi-function microplate reader detection supernatant at 545nm wavelength
Degree.Statistical analysis obtains hemolysis rate.
Fig. 4 is the result for the dual network crosslinking high intensity hydrogel hemolysis rate that comparative example 1 and embodiment 1 are obtained.Such as figure
Finding, the hemolysis rate of blood in deionized water are defined as 100%, and the hemolysis rate in physiological saline is defined as 0%.CCS-0,
The hemolysis rate of CCS-0.5, CCS-1, CCS-1.5 are respectively 0.12%, 2.66%, 0.11% and 1.20%, are substantially less than U.S.
5% hemolysis rate (P as defined in state's pharmacopeia<0.05).It is above-mentioned the experimental results showed that：Dual network of the present invention is crosslinked high intensity hydrogel
Meet the general requirement of medical equipment hemolysis.
The control of the mass ratio of cellulose and fibroin albumen is preferably 5 by being blended in the crosslinker solution formed by the present invention:
0 to 5:3.
As it will be easily appreciated by one skilled in the art that the foregoing is merely illustrative of the preferred embodiments of the present invention, not to
The limitation present invention, all within the spirits and principles of the present invention made by all any modification, equivalent and improvement etc., should all include
Within protection scope of the present invention.
Claims (10)
1. a kind of preparation method of dual network cross-linked cellulose/fibroin albumen high intensity hydrogel, which is characterized in that including following
Step：
(1) cellulose solution, silk fibroin protein solution, epoxychloropropane are uniformly mixed, are allowed to be handed under stirring condition
Join solution；
(2) it is poured into mold after the crosslinker solution that the step (1) obtains being centrifuged de-bubbled, low-density is obtained after reaction solidification
It is chemically crosslinked cellulose/silk fibroin hydrogel；
(3) cellulose/silk fibroin hydrogel that the step (2) obtains is positioned in carbon dioxide incubator and carries out atmosphere
Physical crosslinking；
(4) cellulose/silk fibroin hydrogel that the step (3) obtains is rinsed with flowing water, obtains dual network cross filament
Element/fibroin albumen high intensity hydrogel.
2. the preparation method of dual network cross-linked cellulose/fibroin albumen high intensity hydrogel as described in claim 1, feature exist
In in the step (1), the cellulose solution is prepared by the following method to obtain：By the sodium hydroxide of 7 parts by weight and
The urea co-dissolve of 12 parts by weight obtains sodium hydroxide/urea liquid in the deionized water of 81 parts by weight, then by cellulose
Be cooled to -12 DEG C in advance respectively with the sodium hydroxide/urea liquid then to mix, be vigorously stirred until dissolving to can be obtained cellulose molten
Liquid.
3. the preparation method of dual network cross-linked cellulose/fibroin albumen high intensity hydrogel as described in claim 1, feature exist
In the silk fibroin protein solution is prepared by the following method to obtain：By the urine of the sodium hydroxide of 7 parts by weight and 12 parts by weight
Plain co-dissolve obtains sodium hydroxide/urea liquid in the deionized water of 81 parts by weight, then by fibroin albumen and the hydroxide
Mechanical agitation can be obtained silk fibroin protein solution until being completely dissolved after sodium/urea liquid mixing.
4. the preparation method of dual network cross-linked cellulose/fibroin albumen high intensity hydrogel as described in claim 1, feature exist
In in the crosslinker solution that the step (1) obtains, the mass fraction of cellulose is 1.5~4.0%, the quality point of fibroin albumen
Number is 0~2.0%, and the mass fraction of epoxychloropropane is 0.5-10%.
5. the preparation method of dual network cross-linked cellulose/fibroin albumen high intensity hydrogel as described in claim 1, feature exist
In in the step (1), the crosslinking carries out at room temperature, crosslinking time 5-15min.
6. the preparation method of dual network cross-linked cellulose/fibroin albumen high intensity hydrogel as described in claim 1, feature exist
In in the step (2), the centrifugation de-bubbled condition is that 1500-3000rpm centrifuges 5-10min；
Preferably, in the step (4), described rinsed with flowing water is specifically to rinse 48h or more with deionized water.
7. the preparation method of dual network cross-linked cellulose/fibroin albumen high intensity hydrogel as described in claim 1, feature exist
In in the step (2), the reaction condition of cure is to stand 4-24h at room temperature.
8. the preparation method of dual network cross-linked cellulose/fibroin albumen high intensity hydrogel as described in claim 1, feature exist
In in the step (3), the physical crosslinking is at room temperature, in CO2Gas concentration is 3-30vol% containing CO2Under atmosphere
Handle 2-12h.
9. the dual network cross-linked cellulose/fibroin egg being prepared using the preparation method as described in claim 1-8 any one
White high intensity hydrogel.
10. the application of dual network cross-linked cellulose/fibroin albumen high intensity hydrogel as claimed in claim 9, which is characterized in that
Applied to field of tissue engineering technology.
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