CN112089886A - Hydrogel and preparation method thereof - Google Patents
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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Abstract
The invention relates to hydrogel and a preparation method thereof, relating to the technical field of medical materials. The main technical scheme adopted is as follows: a method for preparing a hydrogel, comprising the steps of: step 1) preparation of calcium carbonate nanoparticles CaCO loaded with DNA3@ DNA; step 2), mixing sodium alginate, chitosan and CaCO3@ DNA is prepared into a first reaction solution; adding gluconolactone into the first reaction solution and stirring to obtain a mixed solution; and standing the mixed solution to obtain the hydrogel. The invention is mainly used for preparing the hydrogel which can slowly release DNA and has good structural uniformity; when the hydrogel is applied to the sore surface of a wound, the hydrogel can absorb seepage, block bacteria and maintain a wet environment; simultaneously the salt in the body fluid and the Ca in the hydrogel2+Ion exchange occurs to slowly degrade the alginic acid network, releasing the DNA-loaded calcium carbonate nanoparticles. Compared with free DNA molecules, the calcium carbonate nanoparticles can transfer DNA into cells more efficiently, and promote the proliferation of the cells.
Description
Technical Field
The invention relates to the technical field of medical materials, in particular to hydrogel and a preparation method thereof.
Background
Acute and chronic wounds can cause serious physical injury to patients and carry a huge socio-economic burden. Wound dressings are a biomaterial used to treat skin lesions. The wound dressing can provide a favorable microenvironment for the wound surface healing of the skin, and has the effects of stopping bleeding, preventing wound infection, promoting wound healing and the like.
Hydrogels are considered to be effective wound dressings. Wherein sodium alginate is a natural polysaccharide, the molecule of which is connected by beta-D-mannuronic acid (M unit) and alpha-L-guluronic acid (G unit) according to (1 → 4), and the sodium alginate has pharmaceutical preparationStability, solubility, viscosity and safety required for adjuvants. Sodium alginate can be prepared by reacting with divalent cation (such as Ca) under mild conditions2+、Cu2+、Mn2+、Zn2+、Pb2+) Crosslinking rapidly forms a hydrogel. The sodium alginate hydrogel has excellent water absorption performance, can absorb wound exudates, and provides a moist environment for wound healing. However, conventionally by direct addition of Ca2+The gelling speed of the sodium alginate hydrogel prepared by the method is difficult to control, and the obtained hydrogel has the technical problem of nonuniform crosslinking.
In addition, the low molecular DNA compound (polydeoxyribonucleotide) can improve the internal environment of human skin, effectively eliminate the root of inflammation and promote the regeneration of skin cells, and has relatively low price. Wherein, the product containing salmon sperm low molecular DNA can be used for postoperative scars, laser treatment, skin repair problems after water light injection and daily skin emergency care. However, such products typically incorporate low molecular weight DNA directly into the emulsion; so that the free DNA molecules are easily hydrolyzed or degraded by DNase in vivo; meanwhile, because both the cell membrane and the DNA molecule are negatively charged, the DNA molecule is difficult to be taken up by the cell.
Disclosure of Invention
In view of the above, the present invention provides a hydrogel and a preparation method thereof, and the main objective of the present invention is to prepare a hydrogel capable of slowly releasing DNA, wherein the hydrogel has a good structural uniformity.
In order to achieve the purpose, the invention mainly provides the following technical scheme:
in one aspect, an embodiment of the present invention provides a method for preparing a hydrogel, including the following steps:
step 1): preparation of DNA-loaded calcium carbonate nanoparticles CaCO3@DNA;
Step 2): mixing sodium alginate, chitosan, and CaCO3@ DNA is prepared into a first reaction solution; adding gluconolactone into the first reaction solution and stirring to obtain a mixed solution; and standing the mixed solution to obtain the hydrogel.
Preferably, the step 1) includes:
step 11) dropwise adding a calcium salt solution into the DNA aqueous solution and stirring for a first set time, and then dropwise adding a carbonate solution into the DNA aqueous solution and stirring for a second set time to obtain a mixture containing a reaction product;
step 12) carrying out centrifugal separation treatment on the mixture containing the reaction product, and collecting the precipitate; freezing and drying the precipitate to obtain CaCO3@DNA。
Preferably, the DNA is low molecular weight DNA; preferably, the DNA is low molecular weight DNA of 25-700 bp; preferably, the DNA is salmon sperm low molecular weight DNA.
Preferably, in the step 11): the concentration of the DNA in the DNA water solution is 1-5 mg/mL; and/or the calcium salt solution is CaCl2Solution, preferably, the CaCl2The concentration of the solution is 0.1-0.5 mol/L; and/or the carbonate solution is Na2CO3Solution, preferably, the Na2CO3The concentration of the solution is 0.1-0.5 mol/L; and/or the CaCl2With Na2CO3In a molar ratio of 1: (1-5), preferably 1 (1-2); and/or the first set time is 0.5-2 h; and/or the second set time is 6-24 h.
Preferably, the CaCO3The particle size of the @ DNA particles was 100-400 nm.
Preferably, the CaCO3The loading of DNA in the @ DNA particles is 17-33% (i.e., CaCO)3The amount of DNA supported in @ DNA is 17-33%).
Preferably, in the step 2), the preparing step of the first reaction solution includes: mixing chitosan solution and sodium alginate solution, adding CaCO3@ DNA, stirring for a third set time to obtain the first reaction solution.
Preferably, the concentration of the chitosan solution is 20-100 mg/mL; and/or the concentration of the sodium alginate solution is 20-100 mg/mL; and/or the volume ratio of the chitosan solution to the sodium alginate solution is (1:1.1) - (1.1:1), preferably 1:1.
Preferably, the third set time is 10-30 min.
Preferably, in the step 2):
the mass ratio of the sodium alginate to the chitosan is 1: (1-5); and/or
The CaCO3The mass ratio of the @ DNA to the sodium alginate is 1: (2-10); and/or
The CaCO3The mass ratio of @ DNA to gluconolactone is 1: (1-7), preferably 1: (1-6), more preferably 1: (1-4).
In another aspect, the invention also provides a hydrogel, wherein the hydrogel has a three-dimensional porous structure and can slowly release calcium carbonate nanoparticle CaCO loaded with DNA3@DNA。
Preferably, the hydrogel comprises a first cross-linked network formed by calcium ions and sodium alginate and a second cross-linked network formed by electrostatic interaction between the alginate ions and chitosan.
Preferably, the DNA is a low molecular weight DNA; preferably, the DNA is salmon sperm low molecular weight DNA with molecular weight of 25-700 bp.
Preferably, the hydrogel is prepared by the method for preparing the hydrogel.
Compared with the prior art, the hydrogel and the preparation method and application thereof have at least the following beneficial effects:
in one aspect, the embodiment of the invention provides a method for preparing hydrogel, which comprises the steps of firstly preparing calcium carbonate nano-particles CaCO loaded with low-molecular-weight DNA3@ DNA, then with CaCO3The water gel is prepared by using @ DNA, chitosan, sodium alginate and gluconolactone as raw materials. In particular, calcium carbonate nanoparticles (CaCO)3@ DNA) can form a complex ionic crosslinking system with gluconolactone, and gluconolactone is hydrolyzed to slowly release H+Reacting CaCO3Part of Ca in @ DNA2+Slowly releases the calcium alginate, and forms hydrogel with uniform structure by crosslinking with sodium alginate molecules, thereby avoiding the nonuniform gel structure caused by the rapid generation of calcium alginate. The added chitosan with good biocompatibilityThe sugar molecules are electropositive and can generate electrostatic interaction with sodium alginate molecules to enhance the acting force among molecular chains, thereby increasing the strength of the hydrogel. Therefore, the preparation method of the hydrogel provided by the embodiment of the invention has the advantages of simple process, easy control, easily obtained raw materials and low cost, and all reactions are carried out at normal temperature; moreover, the prepared hydrogel can not only slowly release DNA, but also has uniform structure and good strength.
Further, according to the preparation method of the hydrogel provided by the embodiment of the invention, the calcium carbonate nanoparticles loaded with the low molecular weight DNA are prepared by adopting a coprecipitation method, and the loading capacity of the calcium carbonate nanoparticles can reach 33.38%. The low molecular weight DNA is preferably salmon sperm low molecular weight DNA having effects of improving cell activity and promoting cell growth.
Further, the preparation method of the hydrogel provided by the embodiment of the invention is to use CaCO3When the @ DNA, chitosan, sodium alginate and gluconolactone are made into hydrogel, CaCO is firstly added3The @ DNA, the chitosan and the sodium alginate are prepared into a first reaction solution, and then the gluconolactone is added into the first reaction solution, wherein the adding sequence can control the gelling speed, so that the hydrogel has a uniform structure (a uniform cross-linked structure). Further, CaCO3The @ DNA is added after the chitosan is dissolved, because CaCO3The @ DNA itself may slowly release calcium ions, thereby locally affecting the dispersion of chitosan; thus, CaCO3The @ DNA is added into the mixed solution of the chitosan and the sodium alginate, so that the uniformity of the hydrogel can be further improved.
On the other hand, the embodiment of the invention also provides a hydrogel, which is prepared by the preparation method of the hydrogel; therefore, the hydrogel has a uniform structure and good strength. When the hydrogel is applied to the sore surface of a wound, the hydrogel can absorb seepage, block bacteria and maintain a wet environment; simultaneous salt and Ca in body fluid2+Ion exchange occurs to slowly degrade the alginic acid network, thereby releasing the low molecular weight DNA loaded calcium carbonate nanoparticles. Compared with free DNA molecules, the calcium carbonate nanoparticles can transfer DNA into cells more efficiently, and promote the proliferation of the cells.The hydrogel can be used in the fields of wound closure and wound repair, and can also be used in the field of cosmetology to recover the regeneration capability of aged and atrophic skin and improve the function of skin.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.
Drawings
FIG. 1 is CaCO prepared according to example 1 of the present invention3Scanning electron microscope pictures of @ DNA nanoparticles;
FIG. 2 is CaCO prepared according to example 1 of the present invention3A schematic representation of the @ DNA nanoparticles promoting cell proliferation;
FIG. 3 is a photograph of the hydrogel prepared in example 1 of the present invention after being left for various periods of time;
FIG. 4 is a scanning electron microscope image of the hydrogel prepared in example 1 of the present invention;
FIG. 5 is a mechanical property test of the hydrogel prepared in example 1 of the present invention;
fig. 6 is a photograph of the hydrogel prepared in example 1 of the present invention used for wound repair on the back of a mouse (i.e., hydrogel dressing group), and a photograph of wound repair on the back of a mouse of a control group (dressing group).
Detailed Description
To further explain the technical means and effects of the present invention adopted to achieve the predetermined object, the following detailed description of the embodiments, structures, features and effects according to the present invention will be made with reference to the accompanying drawings and preferred embodiments. In the following description, different "one embodiment" or "an embodiment" refers to not necessarily the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
The embodiment of the invention provides a preparation method of hydrogel, which comprises the following steps:
step 1): preparation of DNA-loaded calcium carbonate nanoparticles CaCO3@DNA。
The method comprises the following steps: dissolving low molecular weight DNA (1-5mg/mL) in water, stirring, and slowly adding CaCl dropwise2The solution (0.1-0.5mol/L) is stirred for 0.5-2 h. Then Na was added dropwise2CO3Stirring the solution (0.1-0.5mol/L) for 12-24 h. After the reaction is finished, the precipitate is collected by centrifugal separation, and is frozen and dried to obtain CaCO3@DNA。
Wherein the molecular weight of the low molecular weight DNA is 25-700 bp. The low molecular weight DNA is salmon sperm low molecular weight DNA (preferably, the DNA used in the embodiment of the invention is 25-700bp salmon sperm DNA purchased by the inventor). CaCl2The concentration of the solution is 0.1-0.5 mol/L. Na (Na)2CO3The concentration of the solution is 0.1-0.5 mol/L. CaCl2With Na2CO3In a molar ratio of 1: (1-5). CaCO3The @ DNA particle size was 100-400 nm.
Step 2): mixing sodium alginate, chitosan, and CaCO3@ DNA is prepared into a first reaction solution; adding gluconolactone into the first reaction solution and stirring to obtain a mixed solution; and standing the mixed solution to obtain the hydrogel.
The method comprises the following steps: respectively dissolving sodium alginate (20-100mg/mL) and chitosan (20-100mg/mL) in water, and stirring. Mixing a sodium alginate solution and a chitosan solution according to a volume ratio of 1:1 after mixing uniformly, adding CaCO3@ DNA, stirring for 10-30 min. Gluconolactone was then added and stirred well. Pouring the mixed solution into a mould and standing to form the hydrogel.
Wherein the concentration of the sodium alginate aqueous solution is 20-100 mg/mL. The concentration of the chitosan water solution is 20-100 mg/mL. The mass ratio of the sodium alginate solution to the chitosan is 1: (1-5). CaCO added3The @ DNA is 0.01-0.1 g. CaCO3The mass ratio of @ DNA to gluconolactone is 1: (1-6).
In summary, the embodiment of the present invention provides a method for preparing hydrogel, which comprises preparing calcium carbonate nanoparticles CaCO loaded with low molecular weight DNA3@ DNA, then sodium alginate, chitosan, gluconolactone and low molecular weight DNA loaded calcium carbonate nanoparticles according to a certain ratioProportionally cross-linking to obtain hydrogel. Specifically, the calcium carbonate nanoparticles loaded with salmon sperm low molecular weight DNA are prepared by a coprecipitation method, and the loading capacity of the calcium carbonate nanoparticles can reach 33.38%. The low molecular weight DNA has the effects of improving cell activity and promoting cell growth. Calcium carbonate nanoparticles (CaCO)3@ DNA) can form a complex ionic crosslinking system with gluconolactone, and gluconolactone is hydrolyzed to slowly release H+Reacting CaCO3Part of Ca in @ DNA2+Slowly releases the calcium alginate, and forms hydrogel with uniform structure by crosslinking with sodium alginate molecules, thereby avoiding the nonuniform gel structure caused by the rapid generation of calcium alginate. The added chitosan molecule with good biocompatibility has electropositivity, can generate electrostatic interaction with sodium alginate molecules, and enhances the acting force among molecular chains, thereby increasing the strength of the hydrogel (a composite ion crosslinking system is formed by calcium carbonate nanoparticles loaded with DNA and gluconolactone, and calcium ions (Ca) are slowly released2+) Controllably cross-linking the sodium alginate to form a first cross-linked network; a second cross-linked network is formed by electrostatic interaction between sodium alginate and chitosan. Calcium carbonate nanoparticles loaded with low molecular weight DNA may be released during hydrogel degradation).
Therefore, the preparation method of the hydrogel provided by the embodiment of the invention has the advantages of simple process, easy control, easily obtained raw materials and low cost, and all reactions are carried out at normal temperature; moreover, the prepared hydrogel can not only slowly release DNA, but also has uniform structure and good strength.
When the hydrogel is applied to the sore surface of a wound, the hydrogel can absorb seepage, block bacteria and maintain a wet environment; simultaneous salt and Ca in body fluid2+Ion exchange occurs to slowly degrade the alginic acid network, thereby releasing the low molecular weight DNA loaded calcium carbonate nanoparticles. Compared with free DNA molecules, the calcium carbonate nanoparticles can transfer DNA into cells more efficiently, and promote the proliferation of the cells. The hydrogel prepared by the invention can be used in the fields of wound closure and wound repair, and can also be applied in the field of beauty to recover the regeneration capability of aged and atrophic skin and improve the function of the skin.
There is a need forThe description is as follows: the "hydrogel can slowly release DNA" of the invention refers to: the hydrogel mainly sustains CaCO in use3@ DNA, a small amount of DNA may be slowly released. And CaCO3@ DNA is released after entry into the cell.
The invention is further illustrated by the following specific experimental examples:
example 1
Example 1 preparation of a hydrogel, comprising the following steps:
step 1): 20mg of salmon sperm low molecular weight DNA (25-700bp) was dissolved in 10mL of deionized water and stirred uniformly to obtain an aqueous solution of DNA. 10mL of CaCl with the concentration of 0.1mol/L2The solution was slowly added dropwise to the DNA solution and stirred for 1 h. Then 10mL of Na with a concentration of 0.1mol/L2CO3The solution is slowly dripped into the solution, stirred for 12h, transferred into a centrifuge tube after the reaction is finished, and centrifuged for 20min at the rotating speed of 8000 r. Removing supernatant, collecting precipitate, and freeze drying to obtain CaCO3@ DNA nanoparticles.
Step 2): dissolving 0.5g of sodium alginate in 10mL of deionized water, and uniformly stirring to prepare a sodium alginate solution; 0.5g of chitosan is dissolved in 10mL of deionized water and stirred uniformly to prepare a chitosan solution. Mixing a sodium alginate solution and a chitosan solution according to a volume ratio of 1:1 after mixing well, 0.05g of CaCO was added thereto3@ DNA particles, stirring for 10min, adding 0.17g gluconolactone, stirring for 3min, pouring the mixture into a mold, and standing for 5min to form hydrogel.
Wherein CaCO prepared in step 1) of example 13Scanning electron microscope pictures of @ DNA nanoparticles are shown in FIG. 1; as can be seen from fig. 1: CaCO3The particle size of @ DNA is less than 400 nm.
Using Quant-iTTM CaCO calculation by dsDNA quantitative detection kit3@ DNA Loading of DNA particles. First, the fluorescence value (y) is plotted against the concentration (x) of the double-stranded DNA according to the kit instructionsQuasi-curve is y-138.37306 x +1.311 (R)20.999). Weighing 1mg of CaCO3@ DNA was added to 1mL of 10% hydrochloric acid solution and lysed for 20 min. Adding 100 mu L of Quant-iT into 100 mu L of lysate under the condition of keeping out of the sunTM And (3) incubating for 5 min. The excitation light and emission light of the microplate reader were 480nm and 520nm, respectively, and the fluorescence intensity of the sample at 520nm was measured. And calculating the content of the DNA in the sample according to the standard curve of the concentration of the DNA and the fluorescence value. Calculated as CaCO in this example3The loading of DNA in the @ DNA particles was 33.38%.
MTT method for testing CaCO prepared in step 1) of example 13Effect of @ DNA nanoparticles on cell proliferation. Specifically, 100. mu.L of a cell suspension (about 5000 cells/well) of mouse embryonic fibroblasts (NIH 3T3) was added to each well of a 96-well plate, and the plate was placed in a cell incubator to be cultured for 24 hours. After aspirating the culture medium from the well plate using a pipette in a clean bench, 100. mu.L of culture medium containing samples of different concentrations (100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.79, 0.39. mu.g/mL in each well of the experimental group, five wells in each group) was added to each well of the experimental group, and after further incubation for 24h, the culture medium was removed and 100. mu.L of PBS was added to each well and gently rinsed. PBS was then removed by pipette and 100. mu.L of MTT solution was added to each well in the dark (5 mg of MTT was weighed into 1mL of PBS and 9mL of culture solution was added). After 4h each well was tested for absorbance at 490nm using a microplate reader. The test was repeated three times. Wherein, CaCO is shown in FIG. 23Schematic representation of the enhancement of cell proliferation by @ DNA nanoparticles. As can be seen from fig. 2: at relatively low sample concentrations, the cells exhibit a pronounced tendency to proliferate as the sample concentration increases. To sample (CaCO)3@ DNA) at a concentration of 6.25. mu.g/mL, it is most suitable for promoting cell proliferation. Subsequently, as the concentration of the sample increases, there is a tendency for cell proliferation to decrease, which may be associated with high CaCO concentrations3Caused by the induced cytotoxicity.
FIG. 3 is a photograph of the hydrogel prepared in example 1. FIG. 4 shows a scanning electron micrograph of a fractured surface of a hydrogel after lyophilization; it was observed that the hydrogel exhibited a porous structure inside. By testing the rheological properties of the hydrogel, as shown in fig. 5, the storage modulus (G') is greater than the loss modulus (G ") over the range of angular frequencies studied, indicating that the hydrogel has elastomeric behavior.
FIG. 6 is a photograph showing the use of the hydrogel prepared in example 1 in wound repair on the back of a mouse. A wound model was constructed on the back of the mice, the experimental group was treated with the hydrogel prepared in example 1, and the control group was bandaged. The dressing was changed every two days. It was observed that, on day 4, the wounds on the backs of the mice developed scabs, regardless of the hydrogel dressing group or the control group. On day 8, the wound closure in the group treated with the hydrogel prepared in example 1 was 87.7%, which is higher than 68.1% of the control group. In the hydrogel-treated group prepared in example 1, the wound on the back of the mouse was substantially covered with newly formed skin, and the healing rate and quality thereof were significantly higher than those of the control group.
Example 2
Example 2 a hydrogel was prepared by the following specific steps:
step 1) 20mg of salmon sperm low molecular weight DNA (25-700bp) is dissolved in 10mL of deionized water and stirred uniformly to obtain an aqueous solution of DNA. 10mL of CaCl with the concentration of 0.1mol/L2The solution was slowly added dropwise to the DNA solution and stirred for 1 h. Then 10mL of Na with a concentration of 0.25mol/L2CO3Slowly dripping the solution into the solution, and stirring for 12 hours; after the reaction, the solution was transferred to a centrifuge tube and centrifuged at 8000r for 20 min. Discarding the supernatant, and collecting the precipitate; freezing and drying the precipitate to obtain CaCO3@ DNA nanoparticles.
Step 2), dissolving 0.5g of sodium alginate in 10mL of deionized water, and uniformly stirring to prepare a sodium alginate solution; 0.5g of chitosan is dissolved in 10mL of deionized water and stirred uniformly to prepare a chitosan solution. Mixing a sodium alginate solution and a chitosan solution according to a volume ratio of 1:1 after mixing well, 0.05g of CaCO was added thereto3@ DNA particles, stirred for 10 min. Subsequently, 0.17g of gluconolactone was added thereto and stirred for 3 min. Pouring the mixed solution into a mould for standingHydrogel formation took 5 min.
CaCO prepared in step 1) of example 2 by scanning Electron microscopy3@ DNA, the particle size was found to be less than 500 nm. In addition, CaCO was measured and calculated by the method described in example 13The loading of DNA in @ DNA was 22.53%. The above description: increase Na2CO3In such an amount that CaCO is used3The particle size of the @ DNA nanoparticles increased while the loading of DNA decreased.
Test of CaCO prepared in example 2 by MTT method3The effect of @ DNA nanoparticles on cell proliferation (see in particular the method described in example 1), the results of the test show that: the cell proliferation also shows a tendency of increasing and then decreasing with the increase of the sample concentration, and the optimum concentration is 12.5 mug/mL.
The morphology, rheological properties, and animal model wound repair of the hydrogel prepared in example 2 were characterized using the test methods in example 1. The characteristic structure is as follows: the hydrogel has a porous structure inside. The hydrogel has a storage modulus (G ') greater than a loss modulus (G'), and has elastomeric behavior.
Wound closure was 83.4% higher than 68.1% of the control group on day 8 in the group treated with the hydrogel prepared in example 2 during wound repair in animal models. The wound healing rate of the hydrogel dressing group prepared in example 2 was faster than that of the control group, but slightly lower than that of the experimental group in example 1. This is probably due to CaCO at larger particle size3The @ DNA nanoparticles are not conducive to cellular uptake, while CaCO3The decrease in the loading of DNA in @ DNA results in a decrease in the amount of DNA taken up by the cells, resulting in a slowing of the rate of wound healing.
Example 3
Example 3 a hydrogel was prepared by the following specific steps:
step 1): 20mg of salmon sperm low molecular weight DNA (25-700bp) was dissolved in 10mL of deionized water and stirred uniformly to obtain an aqueous solution of DNA. 10mL of CaCl with the concentration of 0.1mol/L2The solution was slowly added dropwise to the solution of DNA with stirring for 1h, and then 10mL of Na having a concentration of 0.1mol/L was added thereto2CO3The solution was slowly added dropwise to the above solution and stirred for 12 h. After the reaction, the solution was transferred to a centrifuge tube and centrifuged at 8000r for 20 min. Discarding the supernatant, and collecting the precipitate; freezing and drying the precipitate to obtain CaCO3@ DNA nanoparticles.
Step 2): dissolving 0.5g of sodium alginate in 10mL of deionized water, and uniformly stirring to prepare a sodium alginate solution; 0.5g of chitosan is dissolved in 10mL of deionized water and stirred uniformly to prepare a chitosan solution. Mixing a sodium alginate solution and a chitosan solution according to a volume ratio of 1:1 after mixing well, 0.05g of CaCO was added thereto3@ DNA particles were stirred for 10min, and then 0.34g of gluconolactone was further added thereto and stirred for 3 min. Pouring the mixed solution into a mold and standing for 5min to form hydrogel.
The CaCO prepared in step 1) of example 3 was observed by a scanning electron microscope3@ DNA nanoparticles, found to have a particle size of less than 400nm, and CaCO measured and calculated by the method described in example 13The loading of DNA in @ DNA was 33.38%.
MTT method for testing CaCO prepared in step 1) of example 33The effect of @ DNA nanoparticles on cell proliferation (see example 1 for test methods), test results indicate: with the sample (CaCO)3@ DNA), cell proliferation also tends to increase first and then decrease, with an optimum sample concentration of 6.25. mu.g/mL.
The morphology, rheological properties and animal model wound repair of the hydrogel prepared in example 3 were characterized using the test method in example 1. The characteristic structure is as follows: the hydrogel prepared in example 3 exhibited a porous structure inside, but the pores of the hydrogel prepared in example 3 were less uniform than those of the hydrogel prepared in example 1, compared to the internal structure of the hydrogel prepared in example 1. This is due to the action of CaCO at higher concentrations of gluconolactone3@ DNA Release of Ca2 +Is increased, thereby causing the crosslinking speed of the sodium alginate to be too high. The hydrogel prepared in example 3 had a storage modulus (G') greater than a loss modulus (G "), and exhibited elastomeric behavior. ByThe heterogeneity of the structure of the hydrogel resulted in that the wound closure was 84.0% in the group treated with the hydrogel prepared in example 3 on day 8 during the wound repair in the animal model, and the wound healing rate was faster than that of the control group but lower than that of the experimental group in example 1.
Comparative example 1
Comparative example 1 a hydrogel was prepared according to the conventional method, and the specific preparation procedure was as follows:
0.5g of sodium alginate is dissolved in 10mL of deionized water and stirred uniformly to prepare a sodium alginate solution. 10mL of CaCl with the concentration of 0.1mol/L2The solution was added to the sodium alginate solution and stirred rapidly. Pouring the mixed solution into a mould and standing for 3-5min to form hydrogel.
The morphology, rheological properties and animal model wound repair of the hydrogels prepared in the comparative examples were characterized using the test method in example 1. The characteristic structure is as follows: the hydrogel has a heterogeneous porous structure inside. This is due to the presence of sodium alginate in Ca2+Too fast and difficult to control. The hydrogel has a storage modulus (G ') greater than a loss modulus (G'), and has elastomeric behavior. Wound closure was 73.8% in the group treated with the hydrogel prepared in comparative example 1 on day 8 during wound repair in animal models. Although the wound healing rate of the hydrogel dressing group prepared using comparative example 1 was slightly faster than that of the control group, the healing rate of the wound of the mouse was significantly lower than that of the experimental groups of examples 1-3 due to the single function of the hydrogel in comparative example 1.
In summary, in the hydrogel and the preparation method thereof provided by the embodiment of the invention, in the preparation process, the DNA-loaded calcium carbonate nanoparticles and gluconolactone form a complex ionic crosslinking system, and calcium ions (Ca) are slowly released2+) Controllably cross-linking the sodium alginate to form a first cross-linked network; a second cross-linked network is formed by electrostatic interaction between sodium alginate and chitosan. Calcium carbonate nanoparticles loaded with low molecular weight DNA can be released during hydrogel degradation. When the prepared hydrogel is applied to wound surface, it can adsorb exudate, block bacteria, and maintainA wet environment; simultaneous salt and Ca in body fluid2+Ion exchange occurs to slowly degrade the alginic acid network, thereby releasing the low molecular weight DNA loaded calcium carbonate nanoparticles. Compared with free DNA molecules, the calcium carbonate nanoparticles can transfer DNA into cells more efficiently, and promote the proliferation of the cells. The hydrogel prepared by the invention can be used in the fields of wound closure and wound repair, and can also be applied in the field of beauty to recover the regeneration capability of aged and atrophic skin and improve the function of the skin.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent change and modification made to the above embodiment according to the technical spirit of the present invention are still within the scope of the technical solution of the present invention.
Claims (10)
1. A method for preparing a hydrogel, comprising the steps of:
step 1): preparation of DNA-loaded calcium carbonate nanoparticles CaCO3@DNA;
Step 2): mixing sodium alginate, chitosan, and CaCO3@ DNA is prepared into a first reaction solution; adding gluconolactone into the first reaction solution and stirring to obtain a mixed solution; and standing the mixed solution to obtain the hydrogel.
2. The method for preparing a hydrogel according to claim 1, wherein the step 1) comprises:
step 11) dropwise adding a calcium salt solution into the DNA aqueous solution and stirring for a first set time, and then dropwise adding a carbonate solution into the DNA aqueous solution and stirring for a second set time to obtain a mixture containing a reaction product;
step 12) carrying out centrifugal separation treatment on the mixture containing the reaction product, and collecting the precipitate; freezing and drying the precipitate to obtain CaCO3@DNA。
3. The method for preparing a hydrogel according to claim 1 or 2, wherein the DNA is a low molecular weight DNA;
preferably, the DNA is low molecular weight DNA of 25-700 bp;
preferably, the DNA is salmon sperm low molecular weight DNA.
4. The method for producing a hydrogel according to claim 2, wherein in the step 11):
the DNA concentration in the DNA water solution is 1-5 mg/mL; and/or
The calcium salt solution is CaCl2Solution, preferably, the CaCl2The concentration of the solution is 0.1-0.5 mol/L; and/or
The carbonate solution is Na2CO3Solution, preferably, the Na2CO3The concentration of the solution is 0.1-0.5 mol/L; and/or
The CaCl is2With Na2CO3In a molar ratio of 1: (1-5), preferably 1 (1-2); and/or
The DNA is reacted with CaCl2The mass ratio of (1): (3-6); and/or
The first set time is 0.5-2 h; and/or
The second set time is 6-24 h.
5. The method for producing a hydrogel according to any one of claims 1 to 4, wherein the CaCO is used as a raw material3The particle size of the @ DNA particle is 100-400 nm; and/or
The CaCO3The loading of DNA in the @ DNA particles was 17-33%.
6. The method for preparing a hydrogel according to claim 1, wherein in the step 2), the step of preparing the first reaction solution comprises:
mixing chitosan solution and sodium alginate solution, adding CaCO3@ DNA, stirring for a third set time to obtain the first reaction solution.
7. Method for producing the hydrogel according to claim 6
The concentration of the chitosan solution is 20-100 mg/mL; and/or
The concentration of the sodium alginate solution is 20-100 mg/mL; and/or
The volume ratio of the chitosan solution to the sodium alginate solution is (1:1.1) - (1.1:1), and preferably 1:1.
8. The method for preparing a hydrogel according to claim 6, wherein the third set time is 10 to 30 min.
9. The method for producing a hydrogel according to any one of claims 1 and 6 to 8, wherein in the step 2):
the mass ratio of the sodium alginate to the chitosan is 1: (1-5); and/or
The CaCO3The mass ratio of the @ DNA to the sodium alginate is 1: (2-10); and/or
The CaCO3The mass ratio of @ DNA to gluconolactone is 1: (1-7), preferably 1: (1-6), more preferably 1: (1-4).
10. The hydrogel is characterized in that the hydrogel is in a three-dimensional porous structure and can slowly release calcium carbonate nanoparticle CaCO loaded with DNA3@DNA;
Preferably, the hydrogel comprises a first cross-linked network formed by calcium ions and sodium alginate, and a second cross-linked network formed by electrostatic interaction between the alginate ions and chitosan;
preferably, the DNA is a low molecular weight DNA; preferably, the DNA is salmon sperm low molecular weight DNA of 25-700 bp;
preferably, the hydrogel is prepared by the method for preparing the hydrogel according to any one of claims 1 to 9.
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112843328A (en) * | 2021-02-25 | 2021-05-28 | 山东大学 | Preparation method of abalone shell powder/ZnO composite material-doped intelligent hydrogel wound dressing with antibacterial effect |
CN113181419A (en) * | 2021-03-23 | 2021-07-30 | 中山大学 | Novel composite hydrogel support prepared by 3D biological printing technology and application thereof |
WO2022101640A1 (en) * | 2020-11-16 | 2022-05-19 | Atelerix Limited | Storing and/or transporting extracellular nucleic acids |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101648023A (en) * | 2009-08-26 | 2010-02-17 | 天津大学 | DNA and nano lamellar hydroxylapatite compound as well as preparation method and application thereof |
CN108060176A (en) * | 2017-11-23 | 2018-05-22 | 华中农业大学 | A kind of functional form genophore and DNA/ carrier complexes |
CN108785738A (en) * | 2018-06-22 | 2018-11-13 | 中南大学 | A kind of preparation method and applications of hydrogel medical dressing |
CN109134885A (en) * | 2017-06-27 | 2019-01-04 | 华南理工大学 | A kind of sodium alginate-chitosan poly ion complexes hydrogel and preparation method thereof |
-
2020
- 2020-09-22 CN CN202011002679.XA patent/CN112089886A/en active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101648023A (en) * | 2009-08-26 | 2010-02-17 | 天津大学 | DNA and nano lamellar hydroxylapatite compound as well as preparation method and application thereof |
CN109134885A (en) * | 2017-06-27 | 2019-01-04 | 华南理工大学 | A kind of sodium alginate-chitosan poly ion complexes hydrogel and preparation method thereof |
CN108060176A (en) * | 2017-11-23 | 2018-05-22 | 华中农业大学 | A kind of functional form genophore and DNA/ carrier complexes |
CN108785738A (en) * | 2018-06-22 | 2018-11-13 | 中南大学 | A kind of preparation method and applications of hydrogel medical dressing |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2022101640A1 (en) * | 2020-11-16 | 2022-05-19 | Atelerix Limited | Storing and/or transporting extracellular nucleic acids |
CN112843328A (en) * | 2021-02-25 | 2021-05-28 | 山东大学 | Preparation method of abalone shell powder/ZnO composite material-doped intelligent hydrogel wound dressing with antibacterial effect |
CN112843328B (en) * | 2021-02-25 | 2022-05-13 | 山东大学 | Preparation method of abalone shell powder/ZnO composite material-doped intelligent hydrogel wound dressing with antibacterial effect |
CN113181419A (en) * | 2021-03-23 | 2021-07-30 | 中山大学 | Novel composite hydrogel support prepared by 3D biological printing technology and application thereof |
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