CN118059037A - Preparation method of super-soft growth factor-loaded photocrosslinked hydrogel and application of super-soft growth factor-loaded photocrosslinked hydrogel in brain trauma treatment - Google Patents
Preparation method of super-soft growth factor-loaded photocrosslinked hydrogel and application of super-soft growth factor-loaded photocrosslinked hydrogel in brain trauma treatment Download PDFInfo
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Abstract
The invention discloses a preparation method and application of super-soft growth factor-loaded photocrosslinked hydrogel. The preparation method comprises the following steps: firstly, reacting heparin salt with dopamine to obtain heparin-dopamine copolymer, and then reacting with growth factors to obtain the heparin-dopamine copolymer grafted with the growth factors; then mixing with the precursor solution of the methacryloylated gelatin hydrogel, and irradiating with an ultraviolet curing lamp to obtain the super soft photo-crosslinked hydrogel carrying the growth factors. The preparation method is simple, and the prepared hydrogel has high gel forming speed, is close to the modulus of brain tissue, and has good injectability, biocompatibility, wound adhesion and hemostatic performance; and the growth factor is rapidly released in the acute stage of injury, the inflammatory reaction is effectively inhibited, the residence time of FGF2 in the brain injury area is prolonged, the FGF2-FGFR1 signal path is activated for a long time, and pharmacological actions such as FGF2 nerve function repair, blood brain barrier reconstruction, brain tissue repair and the like are enhanced.
Description
Technical Field
The invention belongs to the technical field of preparation of brain trauma drugs, and particularly relates to a preparation method of a super-soft growth factor-loaded photocrosslinked hydrogel and application of the super-soft growth factor-loaded photocrosslinked hydrogel in brain trauma treatment.
Background
In daily life, traumatic brain tissue injury caused by external causes (car accidents, collisions) is one of the brain diseases with high mortality. After a patient has suffered a traumatic brain injury, a large number of neurons may necrose, resulting in temporary or permanent cognitive dysfunction or other related neurological injury, and thus timely treatment after a brain injury is necessary.
The current common treatment means in clinic are operation treatment and administration of neurotrophic drugs, the operation only aims at saving the life of patients, and the improvement effects of the common neurotrophic drugs on the aspects of recovery of nerve functions after brain trauma, reconstruction of blood brain barrier, regeneration of neuron axons and the like are not clear. In order to improve the life quality of patients, it is urgently required to find a candidate drug capable of alleviating the generation of acute phase inflammation, promoting the recovery of blood brain barrier function and promoting nerve repair after traumatic brain injury. The research team of the present inventors has been devoted to develop a therapeutic effect of a fibroblast growth factor (Fibroblastgrowth factor, FGF) family in brain nerve injury diseases such as traumatic brain injury, ischemic cerebral apoplexy, alzheimer's disease, etc., and found that basic fibroblast growth factor (Basicfibroblastgrowthfactor, bFGF or FGF 2) and its downstream signal pathway play an important role in the recovery of nerve function and the reconstruction of blood brain barrier after traumatic brain injury according to the previous research results. One of the difficulties faced in the clinical use of FGF2 is its unstable protein activity, susceptibility to degradation and inactivation during storage and transport, susceptibility to off-target effects, etc. As the hydrogel is used as a clinically common wound dressing, the hydrogel has high water content, so that the growth factors can be better stored in matrix materials, and the inventor hopes to develop a gel preparation which has certain visibility, similar hardness of brain tissues and can be used for treating open brain injury, the activity and stability of the growth factors are protected, the retention time of the growth factors in brain injury areas is prolonged, and the nerve repair promoting effect of the growth factors is better exerted.
Disclosure of Invention
The invention aims to provide a preparation method of a super-soft growth factor-loaded photocrosslinked hydrogel and application of the super-soft growth factor-loaded photocrosslinked hydrogel in brain trauma treatment. The preparation method is simple, and the prepared hydrogel has high gel forming speed, is close to the modulus of brain tissue, and has good injectability, biocompatibility, wound adhesion and hemostatic performance; and the growth factor is rapidly released in the acute stage of injury, the inflammatory reaction is effectively inhibited, the residence time of FGF2 in the brain injury area is prolonged, the FGF2-FGFR1 signal path is activated for a long time, and pharmacological actions such as FGF2 nerve function repair, blood brain barrier reconstruction, brain tissue repair and the like are enhanced.
Specifically, the invention is realized through the following technical schemes:
in a first aspect, the present invention provides a method for preparing a super soft growth factor loaded photocrosslinked hydrogel, the method comprising the steps of:
(1) Mixing heparin salt buffer solution with dopamine, performing a condensation reaction, and performing dialysis and freeze-drying to obtain heparin-dopamine copolymer; the molar ratio of heparin to dopamine in the heparin salt is 1-10:10-1;
(2) Mixing the growth factor with the heparin-dopamine copolymer obtained in the step (1) for reaction to obtain a heparin-dopamine copolymer grafted with the growth factor;
(3) Dissolving a freeze-dried methacrylic acylated gelatin (GelMA) matrix material in a sterile PBS buffer solution to prepare a 5% GelMA solution, adding 0.25% photoinitiator LAP, and filtering with a microporous filter membrane of 0.22 mu m to obtain a hydrogel precursor solution;
(4) Mixing the heparin-dopamine copolymer grafted with the growth factors obtained in the step (2) with the hydrogel precursor solution obtained in the step (3), and irradiating for 5-10 seconds by using an ultraviolet curing lamp (365 nm,100mW cm –2) to obtain the super-soft growth factor-loaded photocrosslinked hydrogel.
In a preferred embodiment, the heparin salt in step (1) is a carboxy-activated heparin salt, and the step of activating the carboxy group of the heparin salt comprises: mixing a heparin salt buffer solution with a carboxyl activating agent under the protection of inert gas, and reacting for 20-50 min; the molar ratio of heparin in the heparin salt to the carboxyl activating agent is 1-10:1, a step of; the carboxyl activating agent comprises 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), and the molar ratio of the 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) to the N-hydroxysuccinimide (NHS) is 1:1.
In a preferred embodiment, the heparin salt in step (1) is selected from at least one of heparin sodium, heparin calcium, heparin potassium, heparin lithium, preferably heparin sodium.
In a preferred embodiment, the growth factor in step (2) is selected from at least one of basic fibroblast growth factor (FGF 2), nerve Growth Factor (NGF), vascular Endothelial Growth Factor (VEGF), fibroblast growth factor 9 (FGF 9), preferably basic fibroblast growth factor (FGF 2).
In a preferred embodiment, the growth factor in step (4) is present in the ultra-soft photo-crosslinked hydrogel in an amount of 0.4-0.7mg/mL, preferably 0.5mg/mL.
In a second aspect, the present invention also provides a super soft growth factor loaded photocrosslinked hydrogel, which is prepared by the above preparation method.
In a third aspect, the invention also provides an application of the super-soft growth factor-loaded photocrosslinked hydrogel in treating brain wounds.
Compared with the prior art, the invention has the following beneficial effects:
The preparation method of the super-soft growth factor-loaded photocrosslinked hydrogel provided by the invention is simple, and the prepared hydrogel has high gel forming speed, is close to the modulus of brain tissues, and has good injectability, biocompatibility, wound adhesion and hemostatic performance. The hydrogel can regulate the release speed and release behavior of growth factors by responding to the rise of local tissue Matrix Metalloproteinases (MMPs) in an acute trauma stage in a brain trauma model, and compared with a wild type growth factor, the growth factors combined with heparin-dopamine copolymer have enhanced combination degree of the growth factors with receptors and initiation effect of downstream signal paths, so that the rapid release of the growth factors in an acute stage of injury is realized, inflammatory reaction is effectively inhibited, the retention time of FGF2 in a brain injury region is prolonged, the FGF2-FGFR1 signal paths are activated for a long time, and pharmacological actions such as FGF2 nerve function repair, blood brain barrier reconstruction and brain tissue repair are enhanced.
Drawings
FIG. 1 is a nuclear magnetic characterization spectrum of a heparin-dopamine copolymer prepared by the invention.
FIG. 2 shows the mechanical properties of hydrogels prepared with different concentrations of GelMA solution over time.
FIG. 3 is an external view of the ultra-soft FGF 2-loaded photocrosslinked hydrogel prepared by the present invention, wherein the left figure is an external view without photocrosslinking, and the right figure is an external view after photocrosslinking into a gel.
FIG. 4 is a scanning electron microscope image of the ultra soft FGF 2-loaded photocrosslinked hydrogel prepared by the present invention.
FIG. 5 is a graph showing the injectability test of the ultra-soft FGF 2-loaded photocrosslinked hydrogel prepared by the present invention.
Fig. 6 is a graph of in vitro hemostatic effect of the hydrogel prepared according to the present invention, wherein fig. a is a schematic view of the hydrogel applied, and fig. b, fig. c, fig. d, and fig. e are graphs of in vitro hemostatic effect of the first, second, third, and fourth groups, respectively.
Fig. 7 is a schematic illustration of a brain trauma model versus hydrogel.
FIG. 8 is a graph showing the detection of the downstream activation level of FGF2-FGFR1 in brain injury regions of each group.
FIG. 9 shows the degradation levels of the FGF 2-grafted heparin-dopamine copolymer prepared according to the present invention in three environments.
Detailed Description
The following detailed description of the embodiments of the present invention is provided for better illustration of the present invention, but is not to be construed as limiting the invention.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase through regular channels, with no manufacturer noted.
Example 1
Preparation of heparin-dopamine copolymer grafted with basic fibroblast growth factor (FGF 2)
(1) Preparation of heparin-dopamine copolymers
100Mg of heparin sodium is dissolved in 50mLMES buffer solution, 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) are added according to the mol ratio of 2:1:1 with heparin sodium, after stirring for 40min under the protection of nitrogen, 66mg of dopamine is added, stirring is carried out for 3h, then the reaction solution is put into a dialysis bag for dialysis for 72h, water is replaced every 12h, and finally the dialysis solution is freeze-dried, so that the heparin-dopamine copolymer is obtained, wherein the grafting rate of the dopamine is 5-18%. The nuclear magnetic characterization spectrogram of the heparin-dopamine copolymer is shown in figure 1.
(2) Preparation of heparin-dopamine copolymer grafted with basic fibroblast growth factor (FGF 2)
Mixing and reacting basic fibroblast growth factor (FGF 2) with the heparin-dopamine copolymer obtained in the step (1) to obtain the heparin-dopamine copolymer grafted with the basic fibroblast growth factor (FGF 2).
Example 2
Experiment of the Effect of methacryloylated gelatin (GelMA) solution concentration on hydrogel Properties
(1) Preparation of hydrogels
The matrix material of methacryloylated gelatin (GelMA) in lyophilized form was dissolved in sterile PBS buffer to prepare GelMA solutions with concentrations of 2.5%,5%,10% and 15%, respectively, 0.25% photoinitiator LAP was added and filtered through a 0.22 μm microporous filter membrane, and then irradiated with an ultraviolet curing lamp (365 nm,100mW cm –2) for 8 seconds to form a hydrogel.
(2) Rheological property detection
And (3) detecting the rheological property of the hydrogel prepared in the step (1) by adopting a rheometer, and examining the mechanical strength of the hydrogel. The concentration of the super soft hydrogel suitable for the modulus of brain tissue is selected by selecting 8mm diameter cone plates, 1000 mu mgap, the temperature is 25+/-0.1 ℃, the oscillation frequency is set to be 1Hz, the time is 0-300 seconds, and the strength change of the mechanical properties of the hydrogels prepared by GelMA solutions with the concentration of 2.5%,5%,10% and 15% respectively along with the time change is sequentially detected. The storage modulus of hydrogels prepared with different concentrations of GelMA solution are shown in table 1. The mechanical properties of hydrogels prepared with different concentrations of GelMA solution are shown in fig. 2 as a function of time.
Table 1: storage modulus of hydrogels prepared with GelMA solutions of different concentrations
As can be seen from Table 1 and FIG. 2, the GelMA solution having a concentration of 2.5% failed to form a gel state, and the GelMA solutions having a concentration of 5%,10% and 15% formed hydrogels having stable mechanical properties and gel forming properties, but the GelMA solutions having a concentration of 10% and 15% formed hydrogels having a larger mechanical strength. Whereas a GelMA solution with a concentration of 5% can form a very soft and mechanically stable hydrogel.
Example 3
Preparation of the ultra-soft FGF 2-loaded photo-crosslinked hydrogel
Dissolving a freeze-dried methacrylic acylated gelatin (GelMA) matrix material in a sterile PBS buffer solution to prepare a 5% GelMA solution, adding 0.25% photoinitiator LAP, adjusting the pH to 7.4, and filtering with a microporous filter membrane of 0.22 mu m to obtain a hydrogel precursor solution; then, the heparin-dopamine copolymer grafted with basic fibroblast growth factor (FGF 2) prepared in example 1 was added, and after mixing, the mixture was irradiated with ultraviolet curing light (365 nm,100mW cm –2) for 8 seconds, to prepare the ultra-soft FGF 2-loaded photocrosslinked hydrogel of the present invention. The appearance of the ultra-soft FGF 2-loaded photo-crosslinked hydrogel is shown in FIG. 3, and a scanning electron microscope image is shown in FIG. 4. As can be seen from fig. 4, the hydrogel has a better three-dimensional space network structure. The ultra-soft FGF 2-loaded photocrosslinked hydrogel can be detected by needle injection of 27G, and as shown in FIG. 5, the hydrogel has good injectability as can be seen from FIG. 5.
Comparative example 1
Preparation of FGF 2-loaded hydrogel 1:
Dissolving a freeze-dried methacrylic acylated gelatin (GelMA) matrix material in a sterile PBS buffer solution to prepare a 5% GelMA solution, adding 0.25% photoinitiator LAP, adjusting the pH to 7.4, and filtering with a microporous filter membrane of 0.22 mu m to obtain a hydrogel precursor solution; then, basic fibroblast growth factor (FGF 2) was added, and after mixing, irradiation was performed with an ultraviolet curing lamp (365 nm,100mW cm –2) for 8 seconds, to prepare FGF 2-loaded hydrogel 1.
Comparative example 2
Preparation of FGF 2-loaded hydrogel 2:
Dissolving a freeze-dried methacrylic acylated gelatin (GelMA) matrix material in a sterile PBS buffer solution to prepare a 5% GelMA solution, adding 0.25% photoinitiator LAP, adjusting the pH to 7.4, and filtering with a microporous filter membrane of 0.22 mu m to obtain a hydrogel precursor solution; then adding dopamine solution grafted with basic fibroblast growth factor (FGF 2), mixing, and irradiating with ultraviolet curing lamp (365 nm,100mW cm –2) for 8 seconds to obtain FGF 2-loaded hydrogel 2.
Example 4
Performance test experiments on hydrogels prepared in example 3, comparative example 1 and comparative example 2
Experiment 1: in vitro hemostatic Performance test
The gel time of the hydrogels was first measured as shown in table 2:
table 2: gel time test results of the prepared hydrogels
| Sequence number | Gel time/s |
| Example 3 hydrogel | 10s |
| Hydrogel of comparative example 1 | 12s |
| Hydrogel of comparative example 2 | 8s |
As can be seen from Table 2, the hydrogels prepared in example 3, comparative example 1 and comparative example 2 all had very fast gel formation times, and a hemostatic effect could be achieved rapidly.
Practical application experiment of hydrogel: SD rats were randomly divided into 4 groups, the abdominal liver position was used as the experimental site, the bleeding wound surface of about 2cmx1cm was cut with scissors, three prepared hydrogel precursor solutions were rapidly injected into the wound of each group of rats with a syringe, and simultaneously ultraviolet irradiation was given, so that a solid gel was rapidly formed at the wound. Meanwhile, filter paper is attached below the wound, and the weight of the filter paper is weighed to calculate the blood loss. The first group of rats was given physiological saline as a Control group, the second group of rats was given the hydrogel prepared in comparative example 2, the third group of rats was given the hydrogel prepared in comparative example 1, and the fourth group of rats was given the hydrogel prepared in example 3. The in vitro hemostatic effect is shown in figure 6. As can be seen from fig. 6, the hydrogel prepared in example 3 has better adhesiveness in situ on the wound surface and better hemostatic effect on the wound surface.
Experiment 2: constructing and administering a controllable cortical impact brain trauma animal model:
A controlled cortical impact (controlled cortical impact, CCI) molding machine, manufactured by German leica company, was used to prepare a repeatable traumatic brain injury model. Healthy C56BL/6 mice (23-27 g) are selected, and after anesthesia, the head hair of the mice is shaved, and the mice are fixed on a brain stereotactic instrument, wherein the fixing standard is to keep the top fixing left-right level and the front-back tangential level. After being smeared with iodine for disinfection, the head skin is cut according to the median sagittal trend, and the bregma, the lambdoidal point and the left brain part on the skull are exposed. 0.6mm behind bregma, 1mm beside midline, drilling with dental drill, grinding to obtain circular bone window with diameter of 4mm, removing bone flap, and exposing dura mater. The striking tube of the striker is adjusted to be perpendicular to the brain contact portion, thereby ensuring a perpendicular striking. The striking coefficient is: the striking speed was 4m/s, the striking diameter was 3mm, the striking depth was 1mm, and the striking time was 100ms. After sucking the bleeding, the incision was sutured, the mice were placed in a 37 ℃ incubator, and after waking up, were transferred to a squirrel cage for feeding. And whether the modeling is successful or not is confirmed by observing the behavior change and the related indexes.
Mice were randomly divided into six groups of 5 mice each.
The first group of mice was a sham-operated group, the second group of mice was given normal saline as a control group, the third group of mice was given a pure hydrogel group, the fourth group of mice was given FGF2, the fifth group of mice was given the hydrogel prepared in comparative example 2, and the sixth group of mice was given the hydrogel prepared in example 3. To ensure dosing consistency, groups four to six each had a FGF2 content of 1.6 μg/10 μl, a second group was given 10 μl of PBS buffer, and a third group was given 10 μl of hydrogel without FGF 2. Fig. 7 is a schematic illustration of a brain trauma model versus hydrogel. Brain wound repair was observed for each group of mice on day 28 post-dosing, and the statistical mNSS scores were shown in table 3. Each group of mice was then sacrificed and heart perfused, brain tissue was removed and the level of activation of FGFR1 signaling pathway after administration was detected by WesternBlot, and the results are shown in fig. 8.
Table 3: repair results of mice with brain injury of each group
As can be seen from table 3, the ultra-soft FGF 2-loaded photocrosslinked hydrogel of the present invention prepared in the sixth group, i.e., example 3, has a very good repairing effect on traumatic brain injury of mice, so that the wound surface is almost completely recovered.
As can be seen from table 8, the sixth group of the ultra-soft FGF 2-loaded photocrosslinked hydrogels prepared in example 3 can activate FGF2-FGFR1 signaling pathway for a long time, thereby playing pharmacological roles of nerve repair, blood brain barrier repair and traumatic brain injury repair.
Experiment 3
FGF2 stability experiment
100 Mu LFGF of the heparin-dopamine copolymer solution grafted with FGF2 and the heparin-dopamine copolymer solution grafted with FGF2 were placed in a water bath at 37 ℃ and a refrigerator at 65 ℃ respectively, samples were taken from the water bath at 65 ℃ after 1 hour, samples were taken from the environment at 37 ℃ and 4 ℃ after 12 hours, the concentration of FGF2 was detected by ELISA kit, and the degradation degree of FGF2 under three conditions was calculated, and the result is shown in FIG. 9.
As can be seen from fig. 9, the heparin-dopamine copolymer grafted with FGF2 can significantly improve the stability of the growth factor FGF2 under different conditions.
Fgf2 Release assay
200. Mu.L of the ultra soft FGF 2-loaded photocrosslinked hydrogel prepared in example 3 was placed in a 37℃incubator, 100. Mu.L of the centrifugate was taken at 0h,12h,24h,48h,96h,144h,192h,240h, respectively, and then 100. Mu. LPBS buffer (pH 7.4) was added thereto, and the concentration of the obtained liquid was measured by ELISA kit to calculate the percent release. The results are shown in Table 4.
Table 4: the average release rate of FGF2 in the super soft FGF 2-loaded photocrosslinked hydrogel prepared by the invention
| Time (day) (%) | FGF2 release |
| 0 | 0 |
| 1/2 | 9.3 |
| 1 | 10.5 |
| 2 | 15.8 |
| 4 | 27.1 |
| 6 | 35.8 |
| 8 | 50.4 |
| 10 | 60.2 |
From Table 4, it can be seen that the ultra-soft FGF 2-loaded photocrosslinked hydrogel prepared by the invention has a good slow release effect, and can effectively improve the stability of the growth factors, so that the growth factors can be released for a long time to repair brain tissues.
It is apparent that the above examples are only illustrative of the present invention and are not limiting of the embodiments of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (9)
1. A method for preparing a super-soft growth factor-loaded photo-crosslinked hydrogel, which is characterized by comprising the following steps:
(1) Mixing heparin salt buffer solution with dopamine, performing a condensation reaction, and performing dialysis and freeze-drying to obtain heparin-dopamine copolymer; the molar ratio of heparin to dopamine in the heparin salt is 1-10:10-1;
(2) Mixing the growth factor with the heparin-dopamine copolymer obtained in the step (1) for reaction to obtain a heparin-dopamine copolymer grafted with the growth factor;
(3) Dissolving a freeze-dried methacrylic acylated gelatin (GelMA) matrix material in a sterile PBS buffer solution to prepare a 5% GelMA solution, adding 0.25% photoinitiator LAP, and filtering with a microporous filter membrane of 0.22 mu m to obtain a hydrogel precursor solution;
(4) Mixing the heparin-dopamine copolymer grafted with the growth factors obtained in the step (2) with the hydrogel precursor solution obtained in the step (3), and irradiating for 5-10 seconds by using an ultraviolet curing lamp (365 nm,100mW cm –2) to obtain the super-soft growth factor-loaded photocrosslinked hydrogel.
2. The method according to claim 1, wherein the heparin salt in the step (1) is a carboxyl-activated heparin salt, and the step of activating the carboxyl group of the heparin salt comprises: mixing a heparin salt buffer solution with a carboxyl activating agent under the protection of inert gas, and reacting for 20-50 min; the molar ratio of heparin in the heparin salt to the carboxyl activating agent is 1-10:1, a step of; the carboxyl activating agent comprises 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), and the molar ratio of the 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) to the N-hydroxysuccinimide (NHS) is 1:1.
3. The method according to claim 1, wherein the heparin salt in step (1) is at least one selected from the group consisting of heparin sodium, heparin calcium, heparin potassium and heparin lithium.
4. The method according to claim 1, wherein the growth factor in step (2) is at least one selected from the group consisting of basic fibroblast growth factor (FGF 2), nerve Growth Factor (NGF), vascular Endothelial Growth Factor (VEGF), and fibroblast growth factor 9 (FGF 9).
5. The method of claim 1, wherein the growth factor in step (2) is basic fibroblast growth factor (FGF 2).
6. The method according to claim 1, wherein the content of the growth factor in the super soft growth factor-loaded photocrosslinked hydrogel in the step (4) is 0.4-0.7mg/mL.
7. The method according to claim 1, wherein the content of the growth factor in the super soft growth factor-loaded photocrosslinked hydrogel in the step (4) is 0.5mg/mL.
8. The super soft growth factor-loaded photocrosslinked hydrogel prepared by the preparation method as claimed in any one of claims 1 to 7.
9. Use of the super soft growth factor loaded photocrosslinked hydrogel of claim 8 in the treatment of brain trauma.
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