CN115120772A - Multifunctional DNA hydrogel integration dressing for maintaining dryness of stem cells and preparation method thereof - Google Patents
Multifunctional DNA hydrogel integration dressing for maintaining dryness of stem cells and preparation method thereof Download PDFInfo
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- CN115120772A CN115120772A CN202210700066.6A CN202210700066A CN115120772A CN 115120772 A CN115120772 A CN 115120772A CN 202210700066 A CN202210700066 A CN 202210700066A CN 115120772 A CN115120772 A CN 115120772A
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Abstract
The invention belongs to the technical field of medical dressings, and particularly relates to a multifunctional DNA hydrogel integration dressing for maintaining the dryness of stem cells and a preparation method thereof L The ascorbic acid 2-phosphate dynamic cross-linking forms compact hydrogen bonds, borneol and adipose mesenchymal stem cells are doped, the DNA hydrogel similar to the extracellular matrix is obtained to maintain the dryness of the stem cells and enhance the activity of the stem cells, and the obtained DNA hydrogel provides a 'hotbed' similar to the structure of the extracellular matrix in vivo for the growth and development of the stem cells, so that the cell proliferation can be regulated, and the cell activity can be maintained. The dressing has reasonable structural design and mature and different manufacturing process, and can be widely used for the ulcerative pain lastingAnd the treatment of complicated and chronic wounds such as burn, diabetes and the like, and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of medical dressings, and particularly relates to a multifunctional DNA hydrogel integration dressing for maintaining the dryness of stem cells and a preparation method thereof.
Background
Chronic complex wounds, such as ulcerative wounds, burn wounds, and diabetic wounds, have become a significant global public health problem and burden. Chronic complex wounds further aggravate the wound by the activation of inflammatory mediators to produce oxygen radicals due to the patient's hypoimmunity. The main clinical features of chronic complex wound patients are severe pain and itching, large amounts of exudate, activation of pro-inflammatory factors, free radical production, peripheral nerve and vascular lesions and even ulceration. However, most wound dressings do not completely address the clinical features described above. To effectively promote healing of chronic complex wounds, advanced biofunctional materials should be combined with the wound microenvironment. The urgent need is to develop a micro-environment triggering regeneration that has both antibacterial and immune system activation, and drives the 'signal factor' to regulate chronic complex wounds.
Cell therapy is a promising and safe alternative to natural skin, especially stem cell therapy, which can rapidly cover and accelerate wound healing and skin regeneration by inducing secretion of beneficial cytokines. Pluripotent stem cells with the ability to self-replicate and differentiate in multiple directions have attracted considerable attention in the treatment of many injuries and diseases. However, during long-term in vitro culture, stem cells gradually lose the capacity for directed and spontaneous differentiation, resulting in a diminished stem cell sternness. To maintain and promote dryness, encapsulation of stem cells in 3D spheres is the most common method, as stem cells produce hypoxic and paracrine effects in the 3D space. In addition, it can be prepared by adding drugs or growth factors, such as melatonin, fibroblast growth factor and L ascorbic acid 2-phosphate to maintain dryness. As the culture generation increases, the volume of stem cells also changes. The extracellular matrix is a 3D scaffold that directs "regeneration and signaling". The network structure and size thereof vary according to each particular organization. Therefore, how to efficiently load stem cells into "suitable space" has become a delicate and challenging problem.
The intelligent support is just like a 'hotbed' for the growth and development of stem cells, can participate in the regulation of cell proliferation, maintains the dryness of the cells and provides a microenvironment which is the same as or similar to that of extracellular matrix for the cells. In addition, the loaded stem cells can achieve "perfect in vivo release" in a suitable environment. Currently, scaffolds made of biological materials, such as fibrin and collagen, have been widely used as matrices for transporting or supporting cells. In contrast to other materials, DNA is called an active regenerative scaffold and can direct biological development and vital functions. The hydrogel constructed by the DNA polymer has unique characteristics including designable sequence, rigid structure, biocompatibility and biodegradability, so that the DNA hydrogel can promote cell proliferation and provide a 'hotbed' for the survival of stem cells;
however, the DNA hydrogel used in the prior art does not have the ability to maintain dryness, and the DNA hydrogel used in the prior art as a wound dressing has no analgesic effect.
Disclosure of Invention
In order to solve the technical problems, the invention provides a multifunctional DNA hydrogel integrated dressing for maintaining the dryness of stem cells and a preparation method thereof.
The invention is realized by the following technical scheme:
a method of preparing a multifunctional DNA hydrogel, the method comprising:
(1) three DNA chain segments are adopted to prepare a DNA chain segment copolymer, and the addition amount of the three DNA chain segments can be adjusted;
(2) subjecting the DNA segment copolymer obtained in step (1) to L Carrying out chemical crosslinking on ascorbic acid 2-phosphoric acid to form a compact hydrogen bond, so as to obtain multifunctional DNA hydrogel; and (3) adjusting the pore diameter of the multifunctional DNA hydrogel by controlling the addition amount of the three DNA strands in the step (1).
Further, the step (1) is specifically as follows:
dissolving 3-9mM of an acrylic anhydride-modified first DNA strand, 3-9mM of an acrylic anhydride-modified second DNA strand, 3-9mM of an acrylic anhydride-modified third DNA strand and acrylamide in 150. mu.L of a HEPES buffer solution (150. mu.L, 10mM HEPES, 1) under a vacuum environment00mM MgCl 2 pH 7.0) and uniformly mixing, wherein the volume concentration of acrylamide in the mixture is 2-10%, so as to obtain a DNA chain segment mixture; the first DNA strand sequence is:
5’-acrydite-GGAGGGGAGGGGAGGTTTACCTCCCCTCCCCTCCCTTTGCCTCCCCTCCCCTCCGTACTC-3’；
the second DNA strand sequence is: 5 '-acrydite-GAGTACGGAGGG-3';
the third DNA strand sequence is: 5 '-acrydite-TTCTTTTCTTTTCTTTTCTT-3'.
The base sequence of the first DNA chain is shown as SEQ ID NO 1 in the sequence table; the base sequence of the second DNA chain is shown as SEQ ID NO. 2 in the sequence table; the base sequence of the third DNA chain is shown as SEQ ID NO. 3 in the sequence table;
adding 10-40mg of ammonium persulfate and 5-20 mu L N of N, N ', N' -tetramethylethylenediamine aqueous solution into the DNA chain segment mixture, reacting for 5-15min at room temperature, transferring to 4-10 ℃, continuing to polymerize for 12-16h, centrifugally purifying by using a microporous spin-filtration device, drying by placing under nitrogen gas flow to obtain a DNA chain segment copolymer, and storing at 2-10 ℃ for later use.
Further, the step (2) is specifically as follows: under the protection of nitrogen, the 3-9mM DNA segment copolymer obtained in the step (1) and 200- L Ascorbic acid 2-phosphate to 10 μ L HEPES buffer solution (10-12mM HEPES,100-120mM MgCl) 2 pH 7.0-7.4) to form a multifunctional DNA hydrogel;
further, the pore diameter of the multifunctional DNA hydrogel is adjusted by controlling the addition amount of the three DNA strands in the step (1), and the pore diameter of the prepared multifunctional DNA hydrogel is 40-240 μm.
A method of making a multifunctional DNA hydrogel integration dressing that maintains stem cell dryness, the method comprising:
(1) three DNA chain segments are adopted to prepare a DNA chain segment copolymer, and the addition amount of the three DNA chain segments can be adjusted;
(2) subjecting the DNA segment copolymer obtained in step (1) to L When the-ascorbic acid 2-phosphoric acid is mixed, a certain amount of adipose mesenchymal stem cells are added through in-situ doping to obtain loaded adipose cellsMesenchymal stem cells, multifunctional DNA hydrogel integration dressing capable of maintaining dryness.
Further, the step (1) is specifically as follows:
dissolving 3-9mM of acrylic anhydride modified first DNA chain, 3-9mM of acrylic anhydride modified second DNA chain, and 3-9mM of acrylic anhydride modified third DNA chain in 150 μ L of HEPES buffer solution (10-12mM HEPES,100- 2 pH 7.0-7.4) and uniformly mixing, wherein the volume concentration of acrylamide in the mixture is 2-10%, so as to obtain a DNA chain segment mixture; the first DNA strand sequence is:
5’-acrydite-GGAGGGGAGGGGAGGTTTACCTCCCCTCCCCTCCCTTTGCCTCCCCTCCCCTCCGTACTC-3’；
the second DNA strand sequence is: 5 '-acrydite-GAGTACGGAGGG-3';
the third DNA strand sequence is: 5 '-acrydite-TTCTTTTCTTTTCTTTTCTT-3'. The base sequence of the first DNA chain is shown as SEQ ID NO 1 in the sequence table; the base sequence of the second DNA chain is shown as SEQ ID NO 2 in the sequence table; the base sequence of the third DNA chain is shown as SEQ ID NO. 3 in the sequence table;
adding 10-40mg of ammonium persulfate and 5-20 mu L N of N, N ', N' -tetramethylethylenediamine aqueous solution into the DNA chain segment mixture, reacting for 5-15min at room temperature, transferring to 4-10 ℃, continuing to polymerize for 12-16h, centrifugally purifying by using a microporous spin-filtration device, drying by placing under nitrogen gas flow to obtain a DNA chain segment copolymer, and storing at 2-10 ℃ for later use.
Further, the step (2) is specifically as follows: under the protection of nitrogen, 3-9mM polymer and 200-260. mu.M L Ascorbic acid 2-phosphate was added to HEPES buffer solution, while adding a certain amount of adipose-derived mesenchymal stem cells (2000-3000 cells) to form a multifunctional DNA hydrogel integration dressing capable of maintaining dryness.
Further, the step (2) is specifically as follows: under the protection of nitrogen, 3-9mM polymer and 200-260. mu.M L Ascorbic acid 2-phosphate was added to HEPES buffer solution with (0.4-3.2W) Borneol /W gels+borneol %) Chinese herbal medicine borneol and adipose tissue-derived mesenchymal stem cells, and can relieve pain and maintain drynessA multifunctional DNA hydrogel integration dressing.
Further, the pore size of the DNA hydrogel in the multifunctional DNA hydrogel integrated dressing is controlled to be 40-80 μm by controlling the addition amount of the three DNA strands in the step (1).
The multifunctional DNA hydrogel dressing with the effects of relieving pain and maintaining dryness is prepared by the method and comprises a DNA hydrogel with the pore size of 40-80 mu m, borneol doped in the multifunctional DNA hydrogel in situ and adipose-derived mesenchymal stem cells.
For most wounds, pain and itching are unpleasant and persistent symptoms both early and late in the healing process, especially in patients with chronic complex wounds. Clinically, the main method for relieving pain is systemic medication, such as non-steroidal anti-inflammatory drugs, but it causes adverse reactions in the intestinal tract, liver and kidney diseases and central nervous system diseases. Over thousands of years ago, traditional Chinese medicine was widely used in Asia to treat various diseases, especially pathological pain. The signaling mechanisms for pain and itch are diverse. Traditional Chinese medicine research shows that local analgesics can treat or relieve specific pain and itching. The external treatment method of the traditional Chinese medicine is a wonderful aspect of the traditional Chinese medicine for treating pain and pruritus. After the drug is administered directly through the body surface and absorbed through the skin mucosa, the drug can reach the disease directly. Has obvious curative effect, small dosage and no side effect. Therefore, the Chinese medicinal dressing with the functions of locally relieving pain and itching opens up a new way for treating burn wounds.
The invention has the beneficial technical effects that:
(1) according to the preparation method of the multifunctional DNA hydrogel dressing with the effects of relieving pain and maintaining dryness, the pore size of the pores of the multifunctional DNA hydrogel prepared in the dressing is 40-80 mu m by adjusting the addition amount of three DNA chains. When the number of the adipose-derived stem cells is increased from 3 generations to 5 generations, the number is about 20-40 μm, and the stem cells can mutually permeate and mutually promote to generate paracrine effect under the pore size of 40-80 μm in an anoxic environment or a micro-aerobic environment; if too large a pore size is used, the stem cells will travel equivalently in the 2D plane, and too small a pore size will not penetrate as well.
(2) In the preparation method of the multifunctional DNA hydrogel provided by the invention, the DNA chain segment copolymer and the DNA chain segment copolymer are mixed L Ascorbic acid 2-phosphate produces DNA hydrogels with stem cell dryness maintenance through dense hydrogen bonding and chemical crosslinking, through physical and chemical crosslinking; wherein, L ascorbic acid 2-phosphate has the characteristic of maintaining the dryness of stem cells, therefore, the invention is to L The DNA hydrogel prepared after mixing ascorbic acid 2-phosphate into the DNA segment by physicochemical crosslinking can maintain the dry property by itself.
(3) According to the multifunctional DNA hydrogel dressing method with the functions of relieving pain and maintaining dryness, borneol and adipose-derived mesenchymal stem cells are doped in the prepared DNA hydrogel precursor, so that the DNA hydrogel similar to extracellular matrix is obtained, the dryness of the stem cells is maintained, the activity of the stem cells is enhanced, the pain characteristic of a patient can be reduced, and the dressing is more humanized, reasonable and effective. The prepared 'hotbed' type multifunctional DNA hydrogel integrated dressing with a similar extracellular matrix structure can maintain the proliferation and the activity of stem cells, and can perfectly release the stem cells and the pain relieving Chinese herbal medicine borneol by fully combining the wound microenvironment, so that the clinical problem of integrally treating the chronic complex ulcerative wound is realized.
Drawings
FIGS. 1a-d are a topographical view and corresponding statistical view of a multifunctional DNA hydrogel with different pore sizes in accordance with an exemplary embodiment of the present invention;
FIG. 2 is a graph showing the dry-state maintaining ability of a multifunctional DNA hydrogel in an embodiment of the present invention relative to that of a single-function DNA hydrogel.
Fig. 3a-c are schematic diagrams illustrating the proliferation effect of the multifunctional DNA hydrogel integrated system on adipose-derived mesenchymal stem cells according to the embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
On the contrary, the invention is intended to cover alternatives, modifications, equivalents and alternatives which may be included within the spirit and scope of the invention as defined by the appended claims. Furthermore, in the following detailed description of the present invention, certain specific details are set forth in order to provide a better understanding of the present invention. It will be apparent to one skilled in the art that the present invention may be practiced without these specific details.
Example 1
A method for preparing a multifunctional DNA hydrogel, which is characterized by comprising the following steps:
(1) three DNA chain segments are adopted to prepare a DNA chain segment copolymer, and the addition amount of the three DNA chain segments can be adjusted;
(2) subjecting the DNA segment copolymer obtained in step (1) to L Carrying out chemical crosslinking on ascorbic acid 2-phosphoric acid to form a compact hydrogen bond, so as to obtain multifunctional DNA hydrogel; and (3) adjusting the pore diameter of the multifunctional DNA hydrogel by controlling the addition amount of the three DNA strands in the step (1).
And (2) adjusting the pore diameter of the multifunctional DNA hydrogel by controlling the addition amount of the three DNA strands in the step (1), wherein the pore diameter of the prepared multifunctional DNA hydrogel is 40-240 mu m.
The step (1) is specifically as follows:
in a vacuum environment, a first acrylic anhydride-modified DNA strand (sequentially added in four tests: 3, 5, 7, 9mM), a second acrylic anhydride-modified DNA strand (sequentially added in four tests: 3, 5, 7, 9mM), a third acrylic anhydride-modified DNA segment (sequentially added in four tests: 3, 5, 7, 9mM), and acrylamide were dissolved in 150. mu.L of a HEPES buffer solution (10-12mM HEPES, 100-phase 120mM MgCl. MgCl) 2 pH 7.0-7.4) and uniformly mixing, wherein the volume concentration of acrylamide in the mixture is 2-10%, so as to obtain a DNA chain segment mixture;
adding 10-40mg of ammonium persulfate and 5-20 mu L N of N, N ', N' -tetramethylethylenediamine aqueous solution into the DNA chain segment mixture, reacting for 5-15min at room temperature, transferring to 4-10 ℃, continuing to polymerize for 12-16h, centrifugally purifying by using a microporous spin-filtration device, drying by placing under nitrogen gas flow to obtain a DNA chain segment copolymer, and storing at 2-10 ℃ for later use.
Wherein, the base sequence of the first DNA chain is shown as SEQ ID NO 1 in the sequence table; the base sequence of the second DNA chain is shown as SEQ ID NO 2 in the sequence table; the base sequence of the third DNA chain is shown as SEQ ID NO. 3 in the sequence table;
specifically, the first DNA strand sequence is:
5’-acrydite-GGAGGGGAGGGGAGGTTTACCTCCCCTCCCCTCCCTTTGCCTCCCCTCCCCTCCGTACTC-3’；
the second DNA strand sequence is: 5 '-acrydite-GAGTACGGAGGG-3';
the third DNA strand sequence is: 5 '-acrydite-TTCTTTTCTTTTCTTTTCTT-3'.
The step (2) is specifically as follows: under the protection of nitrogen, the 3-9mM DNA segment copolymer obtained in the step (1) and 200- L Ascorbic acid 2-phosphate was added to 10. mu.L of HEPES buffer solution (10-12mM HEPES,100-120mM MgCl) 2 pH 7.0-7.4) to form multifunctional DNA hydrogel; the porosity of the hydrogel obtained at this time is about 40-80 μm, the microscopic morphology is shown in fig. 1, fig. 1 is a topographic map of multifunctional DNA hydrogel with pores of different pore sizes, and it can be seen that DNA hydrogel without pore sizes can be prepared by using DNA strands of different molar concentrations, and the pore sizes gradually decrease with increasing concentration.
Example 2
A method of making a multifunctional DNA hydrogel integration dressing that maintains stem cell dryness, the method comprising:
(1) three DNA chain segments are adopted to prepare a DNA chain segment copolymer, and the addition amount of the three DNA chain segments can be adjusted; the method specifically comprises the following steps:
in a vacuum environment, 7mM of the first DNA strand modified with acrylic anhydride, 7mM of the second DNA strand modified with acrylic anhydride, 7mM of the third DNA strand modified with acrylic anhydride and acrylamide were dissolved in 150. mu.L of a HEPES buffer solution (10-12mM HEPES,100-120mM MgCl. sub.C) 2 pH 7.0-7.4) and uniformly mixing, wherein the volume concentration of acrylamide in the mixture is 2-10%, so as to obtain a DNA chain segment mixture; the base sequence of the first DNA chain is shown as SEQ ID NO. 1 in the sequence table; the base sequence of the second DNA strand is as shown in SEQ ID NO 2 of the sequence listShown in the specification; the base sequence of the third DNA chain is shown as SEQ ID NO. 3 in the sequence table;
adding 20mg of ammonium persulfate and 10 mu L N of aqueous solution of N, N ', N' -tetramethylethylenediamine into the DNA chain segment mixture, reacting for 6min at room temperature, transferring to 4 ℃, continuing to polymerize for 12h, centrifugally purifying by using a microporous spin-filtration device (MWCO 10KD), drying by placing under nitrogen flow to obtain a DNA chain segment copolymer, and storing at 2-10 ℃ for later use.
(2) Subjecting the DNA segment copolymer obtained in step (1) to L And when the ascorbic acid 2-phosphate is mixed, a certain amount of adipose mesenchymal stem cells are added through in-situ doping to obtain the multifunctional DNA hydrogel integration dressing which is loaded with the adipose mesenchymal stem cells and can maintain the dryness. The method comprises the following specific steps:
reacting the DNA segment copolymer obtained in step (1) with L Ascorbic acid 2-phosphate (added in six sets of experiments at 200mM, 220mM, 230mM, 240mM, 250mM, 260mM, respectively) was added to HEPES buffer solution (10. mu.L), followed by 2.8W doping Borneol /W gels+borneol % of borneol and a certain quantity of fat mesenchymal stem cells to form multifunctional DNA hydrogel with the functions of relieving pain and maintaining dryness.
In addition, the embodiment of the invention provides a preparation method of single-function DNA hydrogel, which comprises the following steps: and (2) drying the DNA chain segment copolymer obtained in the step (1) under nitrogen airflow, and storing at 4 ℃ for later use. The DNA segment copolymer was added to HEPES buffer solution (10. mu.L) to form a single-functional DNA hydrogel.
After serum-free culture, the ability of the multifunctional DNA hydrogel integrated dressing and the single-function DNA hydrogel to maintain dryness was examined, and the relative mRNA expression is shown in fig. 2: comprises L DNA hydrogel of ascorbic acid 2-phosphate (c) L DNA hydrogel) was 2.4-2.8 times higher than the control group, and further did not contain mRNA L The significant increase in mRNA expression compared to the DNA hydrogel of ascorbic acid 2-phosphate (monofunctional DNA hydrogel) indicates that L DNA hydrogels are able to maintain the dryness of stem cells.
Example 3
A method of making a multifunctional DNA hydrogel integration dressing that maintains stem cell dryness, the method comprising:
(1) three DNA chain segments are adopted to prepare a DNA chain segment copolymer, and the addition amount of the three DNA chain segments can be adjusted; the method comprises the following specific steps:
in a vacuum environment, 7mM of the first DNA strand modified with acrylic anhydride, 7mM of the second DNA strand modified with acrylic anhydride, 7mM of the third DNA strand modified with acrylic anhydride and acrylamide were dissolved in 150. mu.L of a HEPES buffer solution (10-12mM HEPES,100-120mM MgCl. sub.C) 2 pH 7.0-7.4) and uniformly mixing, wherein the volume concentration of acrylamide in the mixture is 2-10%, so as to obtain a DNA chain segment mixture; the base sequence of the first DNA chain is shown as SEQ ID NO 1 in the sequence table; the base sequence of the second DNA chain is shown as SEQ ID NO 2 in the sequence table; the base sequence of the third DNA chain is shown as SEQ ID NO. 3 in the sequence table;
adding 20mg of ammonium persulfate and 10 mu L N of aqueous solution of N, N ', N' -tetramethylethylenediamine into the DNA chain segment mixture, reacting for 6min at room temperature, transferring to 4 ℃, continuing to polymerize for 12h, centrifugally purifying by using a microporous spin-filtration device (MWCO 10KD), drying by placing under nitrogen flow to obtain a DNA chain segment copolymer, and storing at 2-10 ℃ for later use.
(2) Subjecting the DNA segment copolymer obtained in step (1) to L And when the ascorbic acid 2-phosphate is mixed, a certain amount of adipose mesenchymal stem cells are added through in-situ doping to obtain the multifunctional DNA hydrogel integration dressing which is loaded with the adipose mesenchymal stem cells and can maintain the dryness. The method specifically comprises the following steps:
reacting the DNA segment copolymer obtained in step (1) with L Ascorbic acid 2-phosphate (in six experiments, the amount of addition was 250mM to HEPES buffer solution (10. mu.L), followed by 2.8W doping Borneol /W gels+borneol % of borneol and a certain amount of adipose tissue-derived mesenchymal stem cells to form the multifunctional DNA hydrogel with the functions of relieving pain and maintaining dryness.
In order to study the proliferation effect of the multifunctional DNA hydrogel integrated system on adipose-derived mesenchymal stem cells, as shown in fig. 3. The cell proliferation experimental study was divided into 3 groups, which were: control without material, no L Mono of-ascorbic acid 2-phosphateFunctional DNA hydrogel group (DNA hydrogel/borneol) containing L A multifunctional DNA hydrogel group of ascorbic acid 2-phosphate (see below) L -DNA hydrogel/borneol), co-culturing with adipose-derived mesenchymal stem cells under a serum-free condition, and observing the proliferation condition of the cells; it is seen that comprises L A group of multifunctional DNA hydrogels of ascorbic acid 2-phosphoric acid: ( L DNA hydrogel/borneol) adipose mesenchymal stem cells were at most densest in cell proliferation number compared to the other two groups.
The multifunctional dressing provided by the invention can be combined with a multifunctional bioengineering high molecular DNA material by utilizing the characteristics of a chronic wound microenvironment, and by controlling the molar concentration of the added DNA chain segment and adding the DNA chain segment with a certain concentration L Ascorbic acid 2-phosphate, a multifunctional DNA hydrogel with properties similar to those of the extracellular matrix is prepared. The 'hotbed' type multifunctional DNA hydrogel integrated system not only has the function of being suitable for the survival of the adipose-derived mesenchymal stem cells, but also can realize the maintenance of the dryness and the activity of the stem cells, thereby providing a clinical transformation basis for the in vitro survival of the stem cells. Most importantly, the released borneol can realize the effect of local pain relief, and good news is brought to patients who comprehensively treat chronic complicated wounds.
The DNA hydrogel is pH responsive, and the microenvironment ulceration of the wound is at about pH 4-6. In addition, pain from skin ulcers or burns can cause mental harm to the patient. In the burn ulcer environment, the DNA hydrogel controls the release of the traditional Chinese medicine, the medicine can directly reach the wound of a patient, the local pain of the patient is reduced, the curative effect is obvious, and no side effect is caused.
In addition, the integrated system can promote macrophage transformation, angiogenesis and regulate pathological pain and pruritus through neuroimmune interaction, and is beneficial to promoting tissue regeneration characteristics. The dressing has reasonable structural design and mature and different manufacturing process, can be widely used for treating complex chronic wounds such as ulcerous wounds, burn wounds, diabetes and the like with lasting pain, and has good application prospect.
Sequence listing
<110> Beijing university of science and technology
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<141> 2022-06-20
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Claims (10)
1. A method for preparing a multifunctional DNA hydrogel, which is characterized by comprising the following steps:
(1) three DNA chain segments are adopted to prepare a DNA chain segment copolymer, and the addition amount of the three DNA chain segments can be adjusted;
(2) subjecting the DNA segment copolymer obtained in step (1) to L Carrying out chemical crosslinking on ascorbic acid 2-phosphoric acid to form a compact hydrogen bond, so as to obtain multifunctional DNA hydrogel; and (3) adjusting the pore diameter of the multifunctional DNA hydrogel by controlling the addition amount of the three DNA strands in the step (1).
2. The method for preparing the multifunctional DNA hydrogel according to claim 1, wherein the step (1) is specifically as follows:
dissolving 3-9mM of a first DNA chain modified by acrylic anhydride, 3-9mM of a second DNA chain modified by acrylic anhydride, 3-9mM of a third DNA chain modified by acrylic anhydride and acrylamide in 150 mu L of HEPES buffer solution under a vacuum environment, and uniformly mixing, wherein the volume concentration of the acrylamide in the mixture is 2-10%, so as to obtain a DNA chain mixture; the base sequence of the first DNA chain is shown as SEQ ID NO 1 in the sequence table; the base sequence of the second DNA chain is shown as SEQ ID NO 2 in the sequence table; the base sequence of the third DNA chain is shown as SEQ ID NO. 3 in the sequence table;
adding 10-40mg of ammonium persulfate and 5-20 mu L N of N, N ', N' -tetramethylethylenediamine aqueous solution into the DNA chain segment mixture, reacting for 5-15min at room temperature, transferring to 4-10 ℃, continuing to polymerize for 12-16h, centrifugally purifying by using a microporous spin-filtration device, drying by placing under nitrogen gas flow to obtain a DNA chain segment copolymer, and storing at 2-10 ℃ for later use.
3. The method for preparing the multifunctional DNA hydrogel according to claim 1, wherein the step (2) is specifically as follows: under the protection of nitrogen, the 3-9mM DNA segment copolymer prepared in the step (1) and 200- L Ascorbic acid 2-phosphate was added to 10 μ L HEPES buffer solution to form multifunctional DNA hydrogel.
4. The method for preparing a multifunctional DNA hydrogel according to claim 1, wherein the pore size of the multifunctional DNA hydrogel is adjusted by controlling the addition amount of the three DNA strands in step (1), and the pore size of the multifunctional DNA hydrogel prepared is 40-240 μm.
5. A method for preparing a multifunctional DNA hydrogel integration dressing for maintaining the dryness of stem cells, the method comprising:
(1) three DNA chain segments are adopted to prepare a DNA chain segment copolymer, and the addition amount of the three DNA chain segments can be adjusted;
(2) subjecting the DNA segment copolymer obtained in step (1) to L When ascorbic acid 2-phosphoric acid is mixed, a certain amount of adipose-derived mesenchymal stem cells are added through in-situ doping to obtain adipose-derived mesenchymal stem cells loaded with vitamin CA dry-holding multifunctional DNA hydrogel integration dressing.
6. The preparation method of the multifunctional DNA hydrogel integration dressing for maintaining the dryness of the stem cells according to claim 5, wherein the step (1) is specifically as follows:
dissolving 3-9mM of a first DNA chain modified by acrylic anhydride, 3-9mM of a second DNA chain modified by acrylic anhydride, 3-9mM of a third DNA chain modified by acrylic anhydride and acrylamide in 150 mu L of HEPES buffer solution under a vacuum environment, and uniformly mixing, wherein the volume concentration of the acrylamide in the mixture is 2-10%, so as to obtain a DNA chain mixture; the base sequence of the first DNA chain is shown as SEQ ID NO 1 in the sequence table; the base sequence of the second DNA chain is shown as SEQ ID NO 2 in the sequence table; the base sequence of the third DNA chain is shown as SEQ ID NO. 3 in the sequence table;
adding 10-40mg of ammonium persulfate and 5-20 mu L N of N, N ', N' -tetramethylethylenediamine aqueous solution into the DNA chain segment mixture, reacting for 5-15min at room temperature, transferring to 4-10 ℃, continuing to polymerize for 12-16h, centrifugally purifying by using a microporous spin-filtration device, drying by placing under nitrogen gas flow to obtain a DNA chain segment copolymer, and storing at 2-10 ℃ for later use.
7. The method for preparing the multifunctional DNA hydrogel integration dressing for maintaining the dryness of the stem cells according to claim 5, wherein the step (2) is specifically: under the protection of nitrogen, 3-9mM polymer and 200-260. mu.M L Ascorbic acid 2-phosphate was added to HEPES buffer solution, while adding a certain amount of adipose-derived mesenchymal stem cells, to form a multifunctional DNA hydrogel integration dressing capable of maintaining dryness.
8. The method for preparing the multifunctional DNA hydrogel integration dressing for maintaining the dryness of the stem cells according to claim 5, wherein the step (2) is specifically as follows: under the protection of nitrogen, 3-9mM polymer and 200-260. mu.M L Adding ascorbic acid 2-phosphoric acid into HEPES buffer solution, and adding Chinese medicinal materials including Borneolum Syntheticum and adipose mesenchymal stem cells to form multifunctional composition with analgesic and dryness keeping effectsThe dressing can be integrated with the DNA hydrogel.
9. The method for preparing the multifunctional DNA hydrogel integration dressing for maintaining the dryness of the stem cells as claimed in claim 5, wherein the pore size of the DNA hydrogel in the multifunctional DNA hydrogel integration dressing is controlled to be 40-80 μm by controlling the addition amount of the three DNA strands in the step (1).
10. A multifunctional DNA hydrogel dressing having analgesic and dryness-maintaining properties, prepared by the method according to claim 8, wherein the multifunctional DNA hydrogel dressing comprises a DNA hydrogel having a pore size of 40-80 μm, borneol doped in situ in the multifunctional DNA hydrogel, and adipose-derived mesenchymal stem cells.
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