Self-enhanced DNA hydrogel as well as preparation method and application thereof

Abstract

According to the self-enhanced DNA hydrogel as well as the preparation method and the application thereof, DNA molecules with modified groups are subjected to oxidation treatment, and a supramolecular non-covalent network is slowly converted into a covalent network, so that the mechanical strength and the stability are improved. The obtained DNA hydrogel can maintain dynamic characteristics in an initial state, such as self-repairing and shear thinning characteristics. Along with the subsequent slow covalent cross-linking, the stability and mechanical strength of the DNA hydrogel are gradually improved. The method provided by the invention has universality in the aspects of sequence programmability and oxidation strategy, and provides a new platform for researching cell behaviors in the process of dynamically increasing the mechanical strength of the extracellular matrix.

Description

Technical Field

[0001] The present invention relates to the technical field of DNA hydrogels, and in particular to a self-strengthening DNA hydrogel, a preparation method thereof, and an application thereof. Background Art

[0002] Hydrogels are water-retaining materials with a three-dimensional polymer network, and are well-known for their potential to mimic the natural extracellular matrix. It has been demonstrated that hydrogels can provide strong physiological and 3D structural support for cells with well-designed biochemical and physical properties. In the past few decades, it has been found that the mechanical properties of the cell matrix have a significant impact on cell functions, including adhesion, migration, proliferation, and differentiation. Therefore, various synthetic and natural polymer hydrogels with appropriate mechanical strength have been developed and applied to drug delivery, immunotherapy, and tissue engineering. However, the natural cell environment is dynamic, and increasing evidence shows that the process of gradual hardening of the matrix is particularly important during tissue development and wound healing, which has not been achieved in artificial mimics of the extracellular matrix, and the precise design of network molecules also poses challenges for achieving this goal.

[0003] Recently, due to good permeability, thixotropy, and biocompatibility, DNA supramolecular hydrogels have shown great potential in cell culture and tissue engineering. Using its excellent programmability, DNA hydrogels with adjustable mechanical strength can be constructed. It should be noted that the good biodegradability of DNA usually leads to a decrease in mechanical strength, which will affect the fate of cells. Therefore, in order to regulate the mechanical properties of DNA hydrogels, many efforts have been made by predecessors, including strategies such as constructing L-DNA hydrogels, double-network hydrogels, and introducing kinetic interlocking units. Although the cultivation of cells in DNA hydrogels with a slowly decreasing or relatively constant mechanical strength has been successfully achieved, the cultivation of cells in gradually hardening DNA hydrogels has never been reported.

[0004] Therefore, there is an urgent need to provide a slowly self-strengthening DNA hydrogel and apply it to cell culture. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems in the related art to some extent. For this purpose, the present invention provides a self-strengthening DNA hydrogel, a preparation method thereof, and an application thereof. By oxidizing DNA molecules with modifying groups, the supramolecular non-covalent network is slowly transformed into a covalent network, thereby improving mechanical strength and stability. The obtained self-strengthening DNA hydrogel can be used in cell culture to provide a powerful platform for understanding the mechanism of the complex dynamic environment in the extracellular matrix.

[0006] To this end, a first aspect of the present invention provides a method for preparing a self-reinforcing DNA hydrogel, the preparation method comprising:

[0007] Oxidizing multiple single-stranded DNA molecules having a modifying group to obtain the self-reinforcing DNA hydrogel;

[0008] Wherein the modifying group includes at least one selected from the group consisting of a mercapto group, an aldehyde group, and an alkynyl group.

[0009] The dynamic mechanical strength of the extracellular matrix has been shown to play an important role in determining cell behavior. Increasing evidence indicates that the gradual hardening process of the extracellular matrix plays a particularly decisive role in tissue development and wound healing. To address the deficiencies in the prior art, the present invention slowly transforms the supramolecular non-covalent network into a covalent network by oxidizing DNA molecules with modifying groups, thereby improving the mechanical strength and stability of the DNA hydrogel. At the initial stage of preparation, the DNA hydrogel still maintains dynamic properties such as self-healing and shear-thinning properties. With subsequent slow covalent crosslinking, the stability and mechanical strength of the DNA hydrogel will gradually increase. This method is versatile in terms of sequence programmability and oxidation strategy, providing a new platform for studying cell behavior during the dynamic increase in the mechanical strength of the extracellular matrix.

[0010] According to an embodiment of the present invention, the preparation method further comprises:

[0011] Mixing multiple single-stranded DNA molecules having a modifying group with an oxidant for oxidation treatment.

[0012] According to an embodiment of the present invention, the molar ratio of the single-stranded DNA molecule to the modifying group is (1:1)-(1:3).

[0013] According to an embodiment of the present invention, the molar ratio of the single-stranded DNA molecule to the oxidant is (1:2)-(1:150).

[0014] According to an embodiment of the present invention, the oxidant includes at least one selected from the group consisting of hydrogen peroxide, (NH2C2NHCOC3Se)2, I2, and ammonium persulfate.

[0015] According to an embodiment of the present invention, the preparation method further comprises:

[0016] Mixing a single-stranded DNA molecule having a modifying group with a polymer monomer having a functional group for oxidation treatment. The polymer monomer having a functional group and the DNA molecule having a modifying group can undergo a crosslinking reaction to further improve the stability and mechanical strength of the DNA hydrogel.

[0017] According to an embodiment of the present invention, the molar ratio of the single-stranded DNA molecule to the polymer monomer is (1:2)-(1:8).

[0018] According to an embodiment of the present invention, the functional group includes at least one selected from the group consisting of a mercapto group, an aldehyde group, and an alkynyl group.

[0019] According to an embodiment of the present invention, the polymer monomer includes at least one selected from the group consisting of a polyethylene glycol monomer, a polyglycolic acid monomer, and a polylactic acid monomer.

[0020] According to an embodiment of the present invention, the polymer monomer is at least one of a four-arm polymer monomer, an eight-arm polymer monomer, and a linear polymer monomer.

[0021] According to an embodiment of the present invention, there is at least one single-stranded DNA molecule, and the single-stranded DNA molecule forms a double-stranded DNA molecule through complementary base pairing. This is more conducive to the construction of the hydrogel and subsequent cell culture.

[0022] According to an embodiment of the present invention, the single-stranded DNA molecule further includes n bases starting from the 5'-end, and the n bases are consecutive identical bases;

[0023] where n is an integer from 2 to 6. This reduces the influence of subsequent oxidation reactions on DNA base complementary pairing.

[0024] According to an embodiment of the present invention, the single-stranded DNA molecule further includes m bases at the end of the 3'-end, and the m bases are consecutive identical bases;

[0025] where m is an integer from 2 to 6. This reduces the influence of subsequent oxidation reactions on DNA base complementary pairing.

[0026] According to an embodiment of the present invention, the n bases starting from the 5'-end and the m bases at the end of the 3'-end are non-complementary bases.

[0027] According to an embodiment of the present invention, the double-stranded DNA molecule is obtained by complementary base pairing of two single-stranded DNA molecules having modification groups;

[0028] wherein, the first single-stranded DNA molecule includes at least sequence 1, sequence 2, and sequence 3 from the 5'-end to the 3'-end;

[0029] The second single-stranded DNA molecule includes at least sequence 4, sequence 5, and sequence 6 from the 5'-end to the 3'-end;

[0030] Sequence 2 and sequence 5 include palindromic sequences, sequence 1 and sequence 4 are at least partially reverse complementary, and sequence 3 and sequence 6 are at least partially reverse complementary. This is more conducive to the construction of the hydrogel and subsequent cell culture.

[0031] According to an embodiment of the present invention, the sequence 1 and the sequence 3 are not reverse complementary sequences, and the sequence 4 and the sequence 6 are not reverse complementary sequences.

[0032] According to an embodiment of the present invention, the length of the sequence 1 is 10–30 bp, the length of the sequence 2 is 10–30 bp, the length of the sequence 3 is 10–30 bp, the length of the sequence 4 is 10–30 bp, the length of the sequence 5 is 10–30 bp, and the length of the sequence 6 is 10–30 bp.

[0033] According to an embodiment of the present invention, the double-stranded DNA molecule is obtained by complementary pairing of a single-stranded DNA molecule having a modifying group;

[0034] wherein, the single-stranded DNA molecule includes at least the sequence 7, the sequence 8, and the sequence 9 from the 5'-end to the 3'-end;

[0035] The sequence 7, the sequence 8, and the sequence 9 all include palindromic sequences. This is more conducive to the construction of the hydrogel and subsequent cell culture.

[0036] According to an embodiment of the present invention, the sequence 7 and the sequence 9 are not reverse complementary sequences.

[0037] According to an embodiment of the present invention, the length of the sequence 7 is 10–30 bp, the length of the sequence 8 is 10–30 bp, and the length of the sequence 9 is 10–30 bp.

[0038] The second aspect of the present invention provides a self-reinforcing DNA hydrogel, which is obtained according to the preparation method described in the first aspect.

[0039] The method described in the first aspect slowly transforms the supramolecular non-covalent network into a covalent network in the presence of an oxidant, and the obtained DNA hydrogel has better stability and mechanical strength.

[0040] The third aspect of the present invention provides an application of the self-reinforcing DNA hydrogel described in the second aspect in cell culture.

[0041] The self-reinforcing DNA hydrogel described in the second aspect has better stability and mechanical strength, can be used in cell culture, and can alleviate cell precipitation, providing a powerful platform for understanding the mechanism of the complex dynamic environment in the extracellular matrix.

[0042] The fourth aspect of the present invention provides a cell culture method, which includes:

[0043] Mix a single-stranded DNA molecule solution a, a cell suspension, an oxidant, and a culture medium to form a hydrogel coated with cells. The hydrogel undergoes an oxidation reaction, and as the oxidation reaction proceeds, the hydrogel gradually hardens.

[0044] Or

[0045] Mix a single-stranded DNA molecule solution b and a culture medium to form solution 1. Mix a single-stranded DNA molecule solution c, a cell suspension, an oxidant, and a culture medium to form solution 2. Mix solution 1 and solution 2 to form a hydrogel coated with cells. The hydrogel undergoes an oxidation reaction, and as the oxidation reaction proceeds, the hydrogel gradually hardens.

[0046] Among them, the single-stranded DNA molecules in the single-stranded DNA molecule solution a, the single-stranded DNA molecule solution b, and the single-stranded DNA molecule solution c are DNA molecules with modifying groups, and the modifying groups include at least one selected from the group consisting of a mercapto group, an aldehyde group, and an alkynyl group.

[0047] The oxidant includes at least one selected from the group consisting of hydrogen peroxide, (NH2C2NHCOC3Se)2, I2, and ammonium persulfate.

[0048] The self-strengthening DNA hydrogel provided by the present invention has better stability and mechanical strength. When it is used in cell culture, uniformly distributed cells can be observed, and cell precipitation is alleviated, broadening the application field of the DNA hydrogel.

[0049] According to an embodiment of the present invention, the molar ratio of the single-stranded DNA molecule to the modifying group is (1:1)-(1:3).

[0050] According to an embodiment of the present invention, the molar ratio of the single-stranded DNA molecule to the oxidant is (1:2)-(1:150).

[0051] According to an embodiment of the present invention, the cell culture method further includes:

[0052] Mix a single-stranded DNA molecule solution a, a cell suspension, an oxidant, a culture medium, and a polymer monomer with a functional group to form a hydrogel coated with cells. The hydrogel undergoes an oxidation reaction, and as the oxidation reaction proceeds, the hydrogel gradually hardens.

[0053] Or

[0054] Mix a single-stranded DNA molecule solution b and a culture medium to form solution 1. Mix a single-stranded DNA molecule solution c, a cell suspension, an oxidant, a culture medium, and a polymer monomer with a functional group to form solution 2. Mix solution 1 and solution 2 to form a hydrogel coated with cells. The hydrogel undergoes an oxidation reaction, and as the oxidation reaction proceeds, the hydrogel gradually hardens.

[0055] According to an embodiment of the present invention, the molar ratio of the single-stranded DNA molecule to the polymer monomer is (1:2)-(1:8).

[0056] According to an embodiment of the present invention, the functional group includes at least one selected from the group consisting of a thiol group, an aldehyde group, and an alkynyl group.

[0057] According to an embodiment of the present invention, the polymer monomer includes at least one selected from the group consisting of a polyethylene glycol monomer, a polyglycolic acid monomer, and a polylactic acid monomer.

[0058] According to an embodiment of the present invention, the single-stranded DNA molecule in the single-stranded DNA molecule solution b includes at least sequence 1, sequence 2, and sequence 3 from the 5' end to the 3' end;

[0059] The single-stranded DNA molecule in the single-stranded DNA molecule solution c includes at least sequence 4, sequence 5, and sequence 6 from the 5' end to the 3' end;

[0060] Sequence 2 and sequence 5 include palindromic sequences, sequence 1 and sequence 4 are at least partially reverse complementary, and sequence 3 and sequence 6 are at least partially reverse complementary.

[0061] According to an embodiment of the present invention, sequence 1 and sequence 3 are not reverse complementary sequences, and sequence 4 and sequence 6 are not reverse complementary sequences.

[0062] According to an embodiment of the present invention, the length of sequence 1 is 10–30 bp, the length of sequence 2 is 10–30 bp, the length of sequence 3 is 10–30 bp, the length of sequence 4 is 10–30 bp, the length of sequence 5 is 10–30 bp, and the length of sequence 6 is 10–30 bp.

[0063] According to an embodiment of the present invention, the single-stranded DNA molecule in the single-stranded DNA molecule solution a includes at least sequence 7, sequence 8, and sequence 9 from the 5' end to the 3' end;

[0064] Sequence 7, sequence 8, and sequence 9 all include palindromic sequences.

[0065] According to an embodiment of the present invention, sequence 7 and sequence 9 are not reverse complementary sequences.

[0066] According to an embodiment of the present invention, the length of sequence 7 is 10–30 bp, the length of sequence 8 is 10–30 bp, and the length of sequence 9 is 10–30 bp.

[0067] Advantages of the present invention over the prior art:

[0068] The present invention provides a new strategy for preparing DNA hydrogels with gradually increasing mechanical strength. Such DNA hydrogels can maintain dynamic properties in their initial state, such as self-healing and shear-thinning properties. With subsequent slow covalent cross-linking, the stability and mechanical strength of the DNA hydrogels will gradually increase. This method is versatile in terms of sequence programmability and oxidation strategy, providing a new platform for studying cell behavior during the process of dynamically increasing the mechanical strength of the extracellular matrix.

[0069] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. Brief Description of the Drawings

[0070] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, wherein:

[0071] Figure 1-2 respectively show the design mechanism diagram of the D1SS-DNA hydrogel based on the thiol-modified SH-D1 strand in Example 1 of the present invention;

[0072] Figure 3 shows the electron micrograph of the D1SS-DNA hydrogel based on the thiol-modified SH-D1 strand in Example 1 of the present invention;

[0073] Figure 4 shows the rheological test comparison diagram of the slowly enhanced D1SS-DNA hydrogel formed by the thiol-modified SH-D1 strand in Example 1 of the present invention. Among them, the black solid curve is the storage modulus (G’) of the DNA hydrogel after adding hydrogen peroxide; the red solid curve represents the storage modulus (G’) of the DNA hydrogel after adding DTT; the black hollow curve represents the loss modulus (G”) of the DNA hydrogel after adding hydrogen peroxide; the red hollow curve represents the storage modulus (G”) of the DNA hydrogel after adding DTT. Each curve takes time as the abscissa, showing the trend of G’ and G” changing with time;

[0074] Figure 5 shows the design mechanism diagram of the D1SS-DNA / PEG hybrid hydrogel based on the thiol-modified SH-D1 strand in Example 2 of the present invention;

[0075] Figure 6Shows the rheological test comparison chart of D1SS-DNA / PEG hybrid hydrogel based on mercapto-modified SH-D1 chain in Example 2 of the present invention. Among them, the black solid curve represents the storage modulus (G’) of D1SS-DNA / hybrid hydrogel after adding hydrogen peroxide; the red solid curve represents the storage modulus (G’) of D1SS-DNA / hybrid hydrogel after adding DTT; the black hollow curve represents the loss modulus (G”) of D1SS-DNA / hybrid hydrogel after adding hydrogen peroxide; the red hollow curve represents the storage modulus (G”) of D1SS-DNA / hybrid hydrogel after adding DTT. Each curve takes time as the abscissa, indicating the trend of G’ and G” changing with time;

[0076] Figure 7 Shows the design mechanism diagram of D 23 SS-DNA hydrogel based on mercapto-modified SH-D2 chain and SH-D3 chain in Example 3 of the present invention;

[0077] Figure 8 Shows the D 23 SS-DNA hydrogel and D 23 SS-DNA / PEG hybrid hydrogel based on mercapto-modified SH-D2 chain and SH-D3 chain in Example 3 of the present invention under the oxidation of hydrogen peroxide. Among them, the black solid curve represents the storage modulus (G’) of D 23 SS-DNA / PEG hybrid hydrogel after adding hydrogen peroxide; the red solid curve represents the storage modulus (G’) of D 23 SS-DNA hydrogel after adding hydrogen peroxide; the black hollow curve represents the loss modulus (G”) of D 23 SS-DNA / PEG hybrid hydrogel after adding hydrogen peroxide; the red hollow curve represents the storage modulus (G”) of D 23 SS-DNA hydrogel after adding hydrogen peroxide. Each curve takes time as the abscissa, indicating the trend of G’ and G” changing with time;

[0078] Figure 9 Shows the D 23 SS-DNA hydrogel and D 23 SS-DNA / PEG hybrid hydrogel based on mercapto-modified SH-D2 chain and SH-D3 chain in Example 3 of the present invention under the oxidation of (NH2C2NHCOC3Se)2. Among them, the red solid curve represents the storage modulus (G’) of the hybrid hydrogel after adding (NH2C2NHCOC3Se)2; the black solid curve represents D 23Storage modulus (G’) of the SS-DNA hydrogel; the red hollow curve represents the loss modulus (G”) of the hybrid hydrogel after adding (NH2C2NHCOC3Se)2; the black hollow curve represents D after adding (NH2C2NHCOC3Se)2 23 Storage modulus (G”) of the SS-DNA hydrogel. Each curve uses time as the abscissa, showing the trend of G’ and G” changing with time;

[0079] Figure 10 Shows D in Example 4 of the present invention 23 -DNA hydrogel, D 23 SS-DNA hydrogel and D 23 SS-DNA / PEG hybrid hydrogel in the mechanical strength change diagram during cell culture;

[0080] Figure 11 Shows D in Example 4 of the present invention 23 -DNA hydrogel, D 23 SS-DNA hydrogel and D 23 Confocal microscope images of 293T cells wrapped with SS-DNA / PEG hybrid hydrogel for 24 hours. Detailed implementation manners

[0081] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as a limitation to the present invention.

[0082] It should be noted that the terms “first” and “second” are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with “first” and “second” may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, the meaning of “plurality” is two or more.

[0083] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0084] To make it easier to understand the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0085] In this text, the term "comprising" or "including" is an open-ended expression, that is, it includes the content specified in the present invention, but does not exclude other aspects of the content.

[0086] In this text, the terms "optionally", "optional" or "option" generally mean that the subsequent events or conditions may or may not occur, and this description includes the cases where the events or conditions occur, as well as the cases where the events or conditions do not occur.

[0087] According to an embodiment of the present invention, the present invention provides a self-reinforcing DNA hydrogel and a preparation method thereof, and the preparation method includes:

[0088] Performing an oxidation treatment on multiple single-stranded DNA molecules having a modification group to obtain the self-reinforcing DNA hydrogel;

[0089] Wherein the modification group includes at least one selected from the group consisting of a mercapto group, an aldehyde group, and an alkynyl group.

[0090] According to a specific embodiment of the present invention, the preparation method further includes: mixing multiple single-stranded DNA molecules having a modification group with an oxidant for oxidation treatment. The modification group includes but is not limited to a mercapto group, an aldehyde group, and an alkynyl group, and can be used to modify the 5'-end and / or 3'-end of the DNA molecule, and preferably the 5'-end and 3'-end of the DNA molecule are modified with a mercapto group. The oxidant includes but is not limited to hydrogen peroxide, (NH2C2NHCOC3Se)2, I2, and ammonium persulfate. Among them, hydrogen peroxide has a relatively fast oxidation rate, and the oxidation process does not require light and oxygen; (NH2C2NHCOC3Se)2 has less toxicity to cells, and the oxidation process requires light and oxygen.

[0091] According to a specific embodiment of the present invention, the molar ratio of the single-stranded DNA molecule to the modification group is (1:1)-(1:3). The molar ratio of the single-stranded DNA molecule to the oxidant is (1:2)-(1:150).

[0092] According to a specific embodiment of the present invention, the preparation method further includes: mixing a single-stranded DNA molecule having a modification group with a polymer monomer having a functional group for oxidation treatment. The molar ratio of the single-stranded DNA molecule to the polymer monomer is (1:2)-(1:8). The functional group includes but is not limited to a mercapto group, an aldehyde group, and an alkynyl group. The polymer monomer includes but is not limited to at least one of a polyethylene glycol monomer, a polyglycolic acid monomer, and a polylactic acid monomer. The polymer monomer can be a four-arm polymer monomer, an eight-arm polymer monomer, or a linear polymer monomer, and preferably a four-arm polymer monomer, such as a four-arm polyethylene glycol monomer, etc.

[0093] According to a specific embodiment of the present invention, the single-stranded DNA molecule is at least one kind, and the single-stranded DNA molecule forms a double-stranded DNA molecule through complementary pairing.

[0094] According to a specific embodiment of the present invention, the single-stranded DNA molecule further includes n bases starting from the 5'-end, and the n bases are consecutive identical bases; where n is an integer from 2 to 6. The single-stranded DNA molecule further includes m bases at the end of the 3'-end, and the m bases are consecutive identical bases; where m is an integer from 2 to 6. Thus, the influence of subsequent oxidation reactions on DNA base complementary pairing is reduced. The n bases starting from the 5'-end and the m bases at the end of the 3'-end are non-complementary bases. For example, the base sequences starting from the 5'-end and ending at the 3'-end of the single-stranded DNA molecule are both "TT", "AAA", "CCCC", "GGGGGG"; or, the base sequence starting from the 5'-end of the single-stranded DNA molecule is "TT", and the base sequence ending at the 3'-end is "CCCC", etc.

[0095] According to a specific embodiment of the present invention, the double-stranded DNA molecule is formed by complementary pairing of two single-stranded DNA molecules with modification groups;

[0096] wherein, the first single-stranded DNA molecule includes at least sequence 1, sequence 2, and sequence 3 from the 5'-end to the 3'-end;

[0097] The second single-stranded DNA molecule includes at least sequence 4, sequence 5, and sequence 6 from the 5'-end to the 3'-end;

[0098] The sequence 2 and sequence 5 are palindromic sequences, the sequence 1 and sequence 4 are at least partially reverse complementary, and the sequence 3 and sequence 6 are at least partially reverse complementary.

[0099] According to a specific embodiment of the present invention, the sequence 1 and sequence 3 are not reverse complementary sequences, and the sequence 4 and sequence 6 are not reverse complementary sequences. The length of sequence 1 is 10–30bp, the length of sequence 2 is 10–30bp, the length of sequence 3 is 10–30bp, the length of sequence 4 is 10–30bp, the length of sequence 5 is 10–30bp, and the length of sequence 6 is 10–30bp.

[0100] According to a specific embodiment of the present invention, the double-stranded DNA molecule is formed by complementary pairing of a single-stranded DNA molecule with a modification group;

[0101] wherein, the single-stranded DNA molecule includes at least sequence 7, sequence 8, and sequence 9 from the 5'-end to the 3'-end;

[0102] The sequence 7, sequence 8, and sequence 9 are all palindromic sequences. The sequence 7 and sequence 9 are not reverse complementary sequences.

[0103] According to specific embodiments of the present invention, the length of sequence 7 is 10 - 30 bp, the length of sequence 8 is 10 - 30 bp, and the length of sequence 9 is 10 - 30 bp.

[0104] The solution of the present invention will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specifying specific techniques or conditions in the embodiments, the techniques or conditions described in the literature in the art or according to the product specifications are followed. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0105] Table 1 shows the DNA sequence information used in the following embodiments of the present invention. Among them, sequence SH-D1 contains three palindromic sequences, sequence SH-D2 and SH-D3 each contain one palindromic sequence, and the sequences at both ends are complementary sequences, which can be paired according to the base complementary pairing principle. The two bases at the 5' end and 3' end of the three single-stranded DNA molecules are both "TT" to reduce the influence of the subsequent oxidation of sulfhydryl groups into disulfide bonds on DNA base complementary pairing.

[0106] Table 1 DNA single-stranded sequences used

[0107]

[0108] Example 1 Preparation of D1SS-DNA hydrogel

[0109] The design mechanism of the D1SS-DNA hydrogel based on the SH-D1 chain modified with sulfhydryl groups is as Figure 1-2 shown:

[0110] Mix the mother liquor of the SH-D1 chain containing three palindromic sequences with the buffer solution containing the oxidant solution, and form a hydrogel through the base complementary pairing principle under the action of the buffer solution. After the hydrogel is formed, the sulfhydryl groups are oxidized by the oxidant to form disulfide bonds in the hydrogel, thereby improving the strength of the hydrogel.

[0111] The specific preparation method is as follows:

[0112] (1) Synthesize a DNA single-stranded SH-D1 chain containing three palindromic sequences and with sulfhydryl modifications at both the 3' and 5' ends;

[0113] (2) Obtain a mother liquor of the SH-D1 chain with a concentration of 4 mM through concentration, and add distilled water and a concentrated PBS buffer solution containing 0.4% hydrogen peroxide by mass fraction to obtain a D1SS-DNA hydrogel with a concentration of 2 mM of the SH-D1 chain. Its electron micrograph is as Figure 3 shown.

[0114] As a control, the mother liquor of the SH-D1 strand was mixed with a buffer solution containing dithiothreitol (DTT, a reducing agent that prevents thiols from being oxidized to disulfide bonds) to form an SS-DNA hydrogel. The rheological test results of it and the D1SS-DNA hydrogel in the time mode are as Figure 4 shown. The results show that compared with the SS-DNA hydrogel, the D1SS-DNA hydrogel exhibits obvious slow enhancement properties.

[0115] Example 2 Preparation of D1SS-DNA / PEG Hybrid Hydrogel

[0116] The design mechanism of the D1SS-DNA / PEG hybrid hydrogel based on the thiol-modified SH-D1 strand is as Figure 5 shown:

[0117] The mother liquor of the SH-D1 strand containing three palindromic sequences was mixed with hydrogen peroxide solution and tetra-arm polyethylene glycol monomer, and PBS buffer solution was added to form a hydrogel through the base complementary pairing principle. After the hydrogel was formed, the thiols were oxidized by hydrogen peroxide to form disulfide bonds in the hydrogel, and the addition of the tetra-arm polyethylene glycol monomer could further improve the strength of the hydrogel.

[0118] The specific preparation method is as follows:

[0119] (1) Synthesize a DNA single strand SH-D1 strand containing three palindromic sequences and thiol modifications at both the 3' and 5' ends;

[0120] (2) Obtain a high-concentration mother liquor of the SH-D1 strand by concentration, add a thiol-modified tetra-arm polyethylene glycol monomer, supplement distilled water and a concentrated PBS buffer solution containing hydrogen peroxide to obtain a D1SS-DNA / PEG hybrid hydrogel with a SH-D1 strand concentration of 2 mM and a PEG content of 3%.

[0121] As a control, hydrogen peroxide in the above preparation method was replaced with DTT to obtain a hybrid hydrogel after adding DTT. The rheological test results of it and the D1SS-DNA / PEG hybrid hydrogel formed after adding hydrogen peroxide in the time mode are as Figure 6 shown. The results show that compared with the hybrid hydrogel after adding DTT, the D1SS-DNA / PEG hybrid hydrogel containing hydrogen peroxide exhibits more obvious slow enhancement properties.

[0122] Example 3 D 23 SS-DNA Hydrogel and D 23 SS-DNA / PEG Hybrid Hydrogel Preparation

[0123] 1. When the oxidant is hydrogen peroxide, D 23 SS-DNA Hydrogel and D23 The SS-DNA / PEG hybrid hydrogel can be obtained by the following method.

[0124] 1. Based on the thiol-modified SH-D2 and SH-D3 chains of D 23 The design mechanism of the SS-DNA hydrogel is as Figure 7 shown:

[0125] Mix the PBS buffer solution containing the SH-D3 chain with the hydrogen peroxide solution, and add the mother liquor of the SH-D2 chain to form a hydrogel. After the hydrogel is formed, the thiol groups are oxidized by hydrogen peroxide to form disulfide bonds in the hydrogel, thereby increasing the strength of the hydrogel.

[0126] The specific preparation method is as follows:

[0127] (1) Synthesize DNA single strands SH-D2 and SH-D3 containing a palindromic sequence and thiol modifications at both the 3' and 5' ends respectively;

[0128] (2) Dissolve the SH-D2 chain in the PBS buffer solution to form a mother liquor with a concentration of 2 mM. Dissolve the SH-D3 chain in the PBS buffer solution and mix it with the hydrogen peroxide solution to form a mother liquor with a concentration of 2 mM of the SH-D3 chain and a mass fraction of hydrogen peroxide of 0.4%. Mix the mother liquor of the SH-D2 chain and the mother liquor of the SH-D3 chain containing hydrogen peroxide in equal volumes to form a hydrogel. D 23 The DNA content in the SS-DNA hydrogel is 2 mM, and the mass fraction of hydrogen peroxide is 0.2%.

[0129] 2. Based on the thiol-modified SH-D2 and SH-D3 chains of D 23 SS-DNA / PEG hybrid hydrogel:

[0130] Mix the PBS buffer solution containing the SH-D3 chain with the hydrogen peroxide solution and the tetra-arm polyethylene glycol monomer, and add the mother liquor of the SH-D2 chain to form a hydrogel. After the hydrogel is formed, the thiol groups are oxidized by hydrogen peroxide to form disulfide bonds in the hydrogel, thereby increasing the strength of the hydrogel.

[0131] The specific preparation method is as follows:

[0132] (1) Synthesize DNA single strands SH-D2 and SH-D3 containing a palindromic sequence and thiol modifications at both the 3' and 5' ends respectively;

[0133] (2) Dissolve the SH-D3 chain in PBS buffer and mix it with hydrogen peroxide solution and tetra-arm polyethylene glycol monomer to form a mother liquor with a SH-D3 chain concentration of 2 mM, a hydrogen peroxide mass fraction of 0.4%, and a tetra-arm polyethylene glycol monomer mass fraction of 6%. Mix the SH-D2 chain mother liquor with the SH-D3 chain mother liquor containing hydrogen peroxide and tetra-arm polyethylene glycol monomer in equal volume to form a hydrogel. D 23 The total DNA content in the SS-DNA / PEG hybrid hydrogel is 2 mM, the hydrogen peroxide mass fraction is 0.2%, and the tetra-arm polyethylene glycol mass fraction is 3%.

[0134] D 23 SS-DNA hydrogel and D 23 The rheological test results of the SS-DNA / PEG hybrid hydrogel in the time mode are as Figure 8 shown, and both show obvious slow enhancement properties.

[0135] Second, when the oxidant is (NH2C2NHCOC3Se)2, D 23 SS-DNA hydrogel and D 23 The preparation of the SS-DNA / PEG hybrid hydrogel is similar to that when the oxidant is hydrogen peroxide as described above.

[0136] 1, where D 23 The SS-DNA hydrogel can be obtained by the following method:

[0137] Obtain high-concentration mother liquors of SH-D2 chain and SH-D3 chain by concentration respectively. (NH2C2NHCOC3Se)2 is dissolved in DMSO to form a solution. The (NH2C2NHCOC3Se)2 solution is mixed with the SH-D3 solution, and distilled water and concentrated PBS buffer are added to obtain mother liquors of SH-D2 chain and SH-D3 chain with a concentration of 2 mM, where the SH-D3 mother liquor contains 12 mg / ml (NH2C2NHCOC3Se)2 solution. Finally, the SH-D2 chain and SH-D3 chain mother liquors are mixed in equal volume to obtain a SS-DNA hydrogel with a total DNA chain concentration of 2 mM and a (NH2C2NHCOC3Se)2 concentration of 6 mg / ml. D 23 SS-DNA hydrogel.

[0138] 2, D 23 The SS-DNA / PEG hybrid hydrogel can be obtained by the following method:

[0139] High-concentration mother liquors of SH-D2 chain and SH-D3 chain were obtained by concentration respectively. (NH2C2NHCOC3Se)2 was dissolved in DMSO to form a solution. The (NH2C2NHCOC3Se)2 solution was mixed with the SH-D3 solution, and distilled water and concentrated PBS buffer were added to obtain a SH-D2 chain solution with a concentration of 2 mM. Distilled water, concentrated PBS buffer and thiol-modified tetra-arm polyethylene glycol monomer were added to obtain a solution containing SH-D3 chain with a concentration of 2 mM, 6% thiol-modified tetra-arm polyethylene glycol monomer and (NH2C2NHCOC3Se)2 with a concentration of 12 mg / ml. Finally, the SH-D2 chain and the SH-D3 chain mother liquor containing thiol-modified tetra-arm polyethylene glycol monomer were mixed in equal volume to obtain a DNA chain with a total chain concentration of 2 mM, a content of thiol-modified tetra-arm polyethylene glycol monomer of 3% and a concentration of (NH2C2NHCOC3Se)2 of 6 mg / ml. 23 SS-DNA / PEG hybrid hydrogel.

[0140] Among them, the synthesis method of (NH2C2NHCOC3Se)2 (Dise) is as follows:

[0141] (1) Synthesis of γ-butyrolactone

[0142] Se powder (1.00 g, 12.66 mmol) and sodium borohydride (0.96 g, 25.32 mmol) were dissolved in water (15 mL), and the reaction was carried out for 30 minutes under a nitrogen atmosphere and an ice bath condition. 4-bromobutyryl chloride (2.35 g, 12.67 mmol) and tetrabutylammonium bromide (0.1 g, 0.31 mmol) were dissolved in toluene (20 ml) and added to the flask containing Se powder and sodium borohydride at room temperature. After reacting overnight, the mixture was further extracted with CH2Cl2 (3×20 mL) and washed three times with saturated aqueous sodium bicarbonate solution. After drying with anhydrous sodium sulfate, the crude product was further purified by column chromatography using a mixture of petroleum ether and ethyl acetate (volume ratio 20:1) as the eluent. After evaporating the solvent, a golden-yellow γ-butyrolactone liquid was finally obtained;

[0143] (2) Synthesis of Dise

[0144] Ethylenediamine (403.06 mg, 6.71 mmol) was dissolved in tetrahydrofuran (5 mL) and added to a flask at room temperature. The γ-butyrolactone (200 mg, 1.34 mmol) obtained in step (1) was dissolved in tetrahydrofuran (20 mL) and added dropwise to the ethylenediamine in the flask. After reacting overnight, the tetrahydrofuran was evaporated. The crude product was further dried in a vacuum oven at 70 °C for 4 hours to obtain a golden-yellow solid.

[0145] D 23 SS-DNA hydrogel and D23 The rheological test results of the SS-DNA / PEG hybrid hydrogel in the time mode are as follows Figure 9 shown, and both show obvious slow enhancement properties.

[0146] The hydrogel of Example 4 is used for three-dimensional cell culture

[0147] Respectively, D 23 -DNA hydrogel, the slowly enhanced D formed by the oxidation of (NH2C2NHCOC3Se)2 23 SS-DNA hydrogel and D 23 SS-DNA / PEG hybrid hydrogel are used for the culture of three-dimensional cells.

[0148] (1) Preparation of D 23 -DNA hydrogel and its use for the culture of three-dimensional cells

[0149] High-concentration mother liquors of SH-D2 chain and SH-D3 chain are obtained by concentration respectively. Distilled water and concentrated PBS buffer are added to obtain mother liquors of SH-D2 chain and SH-D3 chain with a concentration of 2 mM. Finally, the mother liquors of SH-D2 chain and SH-D3 chain are mixed in equal volume to obtain D 23 -DNA hydrogel with a total DNA strand concentration of 2 mM.

[0150] Using 293T cells as model cells, the concentrated mother liquors of SH-D2 chain and SH-D3 chain are mixed with DMEM medium. The final concentrations of SH-D2 chain and SH-D3 chain solutions are 2 mM and 4 mM respectively. Then the 293T cell suspension is added to the mother liquor of SH-D3 chain to obtain a mixed suspension with a cell concentration of 1×10 6 cells / mL and a SH-D3 chain concentration of 2 mM. The mixed suspension containing 293T cells and SH-D3 chain is mixed with the SH-D2 chain solution in equal volume and added to a 96-well plate and quickly mixed to encapsulate 293T cells in D 23 -DNA hydrogel with a cell density of 5×10 5 cells / mL. Finally, the D 23 -DNA hydrogel is irradiated under white light for 0.5 hour and then placed in a cell culture incubator for culture.

[0151] To monitor the change in the strength of the hydrogel during cell culture, the D 23 -DNA hydrogel after culturing cells for 0 hour, 8 hours, 16 hours, and 24 hours is subjected to rheological test, and the results are as follows Figure 10 shown. After 24 hours, the 293T cells stained with calcein AM and propidium iodide are observed to be distributed in D 23 -DNA hydrogel by confocal microscopy, and the results are as shown in Figure 11 Figure a in it.

[0152] (2)D 23 SS-DNA hydrogel for three-dimensional cell culture

[0153] Using 293T cells as model cells, the concentrated mother liquors of SH-D2 chain and SH-D3 chain were mixed with DMEM medium, and the final concentrations of SH-D2 chain and SH-D3 chain solutions were 2 mM and 4 mM respectively. Then, the 293T cell suspension and (NH2C2NHCOC3Se)2 were added to the mother liquor of SH-D3 chain to obtain a mixed suspension with a cell concentration of 1×10 6 cells / mL, an SH-D3 chain concentration of 2 mM, and an (NH2C2NHCOC3Se)2 concentration of 12 mg / mL. The mixed suspension containing 293T cells, (NH2C2NHCOC3Se)2 and SH-D3 chain was mixed with an equal volume of the SH-D2 chain solution and added to a 96-well plate and quickly mixed to encapsulate the 293T cells in D 23 SS-DNA hydrogel with a cell density of 5×10 5 cells / mL. Finally, the D 23 SS-DNA hydrogel was irradiated under white light for 0.5 h and then placed in a cell culture incubator for culture.

[0154] To monitor the change in the strength of the hydrogel during cell culture, the D 23 SS-DNA hydrogels after culturing cells for 0 h, 8 h, 16 h, and 24 h were subjected to rheological tests, and the results are as Figure 10 shown. After 24 h, the distribution of 293T cells stained with calcein AM and propidium iodide (PI) was observed by confocal microscopy in the D 23 SS-DNA hydrogel, and the results are as shown in Figure 11 Figure b. During the 0-24 h cell culture period, the strength of the D 23 SS-DNA hydrogel first increased due to the formation of disulfide bonds, and then decreased due to the hydrolysis and destruction of the D23SS-DNA hydrogel network by cells.

[0155] (3)D 23 SS-DNA / PEG hybrid hydrogel for three-dimensional cell culture

[0156] Using 293T cells as model cells, the concentrated mother liquors of SH-D2 chain and SH-D3 chain were mixed with DMEM medium, and the final concentrations of SH-D2 chain and SH-D3 chain solutions were 2 mM and 4 mM respectively. Then, the 293T cell suspension, (NH2C2NHCOC3Se)2 and the mother liquor of thiol-modified four-arm polyethylene glycol monomer were added to the mother liquor of SH-D3 chain to obtain a cell concentration of 1×10 6A mixed suspension with a density of 2×10⁶ cells / mL, a concentration of SH-D3 chain of 2 mM, a concentration of (NH₂C₂NHCOC₃Se)₂ of 12 mg / mL, and 3% of thiol-modified tetra-arm polyethylene glycol monomer. A mixed suspension containing 293T cells, thiol-modified tetra-arm polyethylene glycol monomer, (NH₂C₂NHCOC₃Se)₂, and SH-D3 chain was mixed with an equal volume of SH-D2 chain solution and quickly mixed in a 96-well plate to encapsulate 293T cells in D 23 SS-DNA / PEG hybrid hydrogel at a cell density of 5×10 5 cells / mL. Finally, the D 23 SS-DNA / PEG hybrid hydrogel was irradiated under white light for 0.5 h and then placed in a cell culture incubator for 24 h.

[0157] To monitor the change in the strength of the hydrogel during cell culture, rheological tests were performed on the D 23 SS-DNA / PEG hybrid hydrogel after culturing cells for 0 h, 8 h, 16 h, and 24 h. The results are as Figure 10 shown. After 24 h, 293T cells stained with calcein AM and propidium iodide were observed to be more evenly distributed in the D 23 SS-DNA / PEG hybrid hydrogel by confocal microscopy. The results are as Figure 11 shown in Figure c.

[0158] By comparison, it can be seen that the D 23 SS-DNA hydrogel and D 23 SS-DNA / PEG hybrid hydrogel provided by the present invention both have stronger mechanical strength than D 23 -DNA hydrogel, and at the same time can promote cell adhesion and slow down cell sedimentation in the hydrogel.

[0159] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0160] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A preparation method of a self-reinforcing DNA hydrogel, characterized in that, The preparation method includes: Performing oxidation treatment on multiple single-stranded DNA molecules with modification groups to obtain the self-enhancing DNA hydrogel; Wherein the modification group includes at least one selected from the group consisting of a mercapto group, an aldehyde group, and an alkynyl group.

2. The preparation method according to claim 1, wherein The preparation method further includes: Mixing multiple single-stranded DNA molecules with modification groups with an oxidant for oxidation treatment.

3. The preparation method according to claim 2, wherein, The molar ratio of the single-stranded DNA molecule to the modification group is (1:1)-(1:3); Optionally, the molar ratio of the single-stranded DNA molecule to the oxidant is (1:2)-(1:150); Optionally, the oxidant includes at least one selected from the group consisting of hydrogen peroxide, (NH2C2NHCOC3Se)2, I2, and ammonium persulfate.

4. The preparation method according to claim 1, characterized in that, The preparation method further includes: Mixing a single-stranded DNA molecule with a modification group with a polymer monomer with a functional group for oxidation treatment; Optionally, the molar ratio of the single-stranded DNA molecule to the polymer monomer is (1:2)-(1:8); Optionally, the functional group includes at least one selected from the group consisting of a mercapto group, an aldehyde group, and an alkynyl group; Optionally, the polymer monomer includes at least one selected from the group consisting of a polyethylene glycol monomer, a polyglycolic acid monomer, and a polylactic acid monomer; Optionally, the polymer monomer is at least one of a four-arm polymer monomer, an eight-arm polymer monomer, and a linear polymer monomer.

5. The preparation method according to claim 1, wherein The single-stranded DNA molecule is at least one kind, and the single-stranded DNA molecule forms a double-stranded DNA molecule through complementary pairing; Optionally, the single-stranded DNA molecule further includes n bases starting from the 5' end, and the n bases are consecutive identical bases; Where n is an integer from 2 to 6; Optionally, the single-stranded DNA molecule further includes m bases at the end of the 3' end, and the m bases are consecutive identical bases; Where m is an integer from 2 to 6; Optionally, the n bases starting from the 5' end and the m bases at the end of the 3' end are non-complementary bases.

6. The preparation method according to claim 5, wherein, The double-stranded DNA molecule is obtained by complementary pairing of two single-stranded DNA molecules with modification groups; Wherein, the first single-stranded DNA molecule includes at least sequence 1, sequence 2, and sequence 3 from the 5' end to the 3' end; The second single-stranded DNA molecule includes at least sequence 4, sequence 5, and sequence 6 from the 5' end to the 3' end; The sequence 2 and sequence 5 include palindromic sequences, the sequence 1 and sequence 4 are at least partially reverse complementary, and the sequence 3 and sequence 6 are at least partially reverse complementary; Optionally, the sequence 1 and sequence 3 are not reverse complementary sequences, and the sequence 4 and sequence 6 are not reverse complementary sequences; Optionally, the length of sequence 1 is 10–30 bp, the length of sequence 2 is 10–30 bp, the length of sequence 3 is 10–30 bp, the length of sequence 4 is 10–30 bp, the length of sequence 5 is 10–30 bp, and the length of sequence 6 is 10–30 bp.

7. The preparation method according to claim 5, wherein The double-stranded DNA molecule is obtained by complementary pairing of a single-stranded DNA molecule with a modification group; Wherein, the single-stranded DNA molecule includes at least sequence 7, sequence 8, and sequence 9 from the 5' end to the 3' end; Sequences 7, 8, and 9 all include palindromic sequences; Optionally, sequences 7 and 9 are not reverse complementary sequences; Optionally, sequence 7 has a length of 10–30 bp, sequence 8 has a length of 10–30 bp, and sequence 9 has a length of 10–30 bp.

8. A self-reinforced DNA hydrogel, characterized in that, The self-strengthening DNA hydrogel is obtained according to the preparation method described in any one of claims 1-7.

9. Use of the self-strengthening DNA hydrogel according to claim 8 in cell culture.

10. A cell culture method, characterized in that, The cell culture method includes: Mixing single-stranded DNA molecule solution a, cell suspension, oxidant, and culture medium to form a hydrogel coated with cells, and the hydrogel undergoes an oxidation reaction. As the oxidation reaction proceeds, the hydrogel gradually hardens; Or Mixing single-stranded DNA molecule solution b and culture medium to form solution 1, mixing single-stranded DNA molecule solution c, cell suspension, oxidant, and culture medium to form solution 2, and mixing solution 1 and solution 2 to form a hydrogel coated with cells. The hydrogel undergoes an oxidation reaction. As the oxidation reaction proceeds, the hydrogel gradually hardens; Wherein, the single-stranded DNA molecules in single-stranded DNA molecule solution a, single-stranded DNA molecule solution b, and single-stranded DNA molecule solution c are single-stranded DNA molecules with modifying groups, and the modifying groups include at least one selected from the group consisting of mercapto group, aldehyde group, and alkynyl group; The oxidant includes at least one selected from the group consisting of hydrogen peroxide, (NH2C2NHCOC3Se)2, I2, and ammonium persulfate.

11. The cell culture method according to claim 10, wherein The molar ratio of the single-stranded DNA molecule to the modifying group is (1:1)-(1:3); Optionally, the molar ratio of the single-stranded DNA molecule to the oxidant is (1:2)-(1:150); Optionally, the cell culture method further includes: Mixing single-stranded DNA molecule solution a, cell suspension, oxidant, culture medium, and polymer monomer with functional groups to form a hydrogel coated with cells, and the hydrogel undergoes an oxidation reaction. As the oxidation reaction proceeds, the hydrogel gradually hardens; Or Mixing single-stranded DNA molecule solution b and culture medium to form solution 1, mixing single-stranded DNA molecule solution c, cell suspension, oxidant, culture medium, and polymer monomer with functional groups to form solution 2, and mixing solution 1 and solution 2 to form a hydrogel coated with cells. The hydrogel undergoes an oxidation reaction. As the oxidation reaction proceeds, the hydrogel gradually hardens; Optionally, the molar ratio of the single-stranded DNA molecule to the polymer monomer is (1:2)-(1:8); Optionally, the functional groups include at least one selected from the group consisting of mercapto group, aldehyde group, and alkynyl group; Optionally, the polymer monomers include at least one selected from the group consisting of polyethylene glycol monomers, polyglycolic acid monomers, and polylactic acid monomers.

12. The cell culture method according to claim 10, wherein The single-stranded DNA molecule in single-stranded DNA molecule solution b includes at least sequences 1, 2, and 3 from the 5' end to the 3' end; The single-stranded DNA molecule in single-stranded DNA molecule solution c includes at least sequences 4, 5, and 6 from the 5' end to the 3' end; The sequence 2 and sequence 5 include palindromic sequences, the sequence 1 and sequence 4 are at least partially reverse complementary, and the sequence 3 and sequence 6 are at least partially reverse complementary; Optionally, the sequence 1 and sequence 3 are not reverse complementary sequences, and the sequence 4 and sequence 6 are not reverse complementary sequences; Optionally, the length of the sequence 1 is 10–30 bp, the length of the sequence 2 is 10–30 bp, the length of the sequence 3 is 10–30 bp, the length of the sequence 4 is 10–30 bp, the length of the sequence 5 is 10–30 bp, and the length of the sequence 6 is 10–30 bp; Optionally, the single-stranded DNA molecule in the single-stranded DNA molecule solution a includes at least the sequence 7, sequence 8, and sequence 9 from the 5'-end to the 3'-end; The sequence 7, sequence 8, and sequence 9 all include palindromic sequences; Optionally, the sequence 7 and sequence 9 are not reverse complementary sequences; Optionally, the length of the sequence 7 is 10–30 bp, the length of the sequence 8 is 10–30 bp, and the length of the sequence 9 is 10–30 bp.

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