Hydrogel containing ATP cross-linking nodes, and preparation method and application thereof

Abstract

This invention discloses a hydrogel containing ATP crosslinking nodes, its preparation method, and its applications, belonging to the technical field of polymer hydrogel materials and functional soft materials. This invention utilizes the multi-point interaction between guanidine-containing polymer units and ATP phosphate groups, and the reversible borate ester interaction between phenylboronic acid-containing polymer units and ATP ribose diol, to synergistically construct a hydrogel network. Through this structural design, ATP is retained as a network crosslinking node within the hydrogel, and the formed network possesses a structural basis for enzymatic decrosslinking or degradation. Furthermore, the hydrogel provided by this invention exhibits excellent mechanical properties, dynamic recovery properties, antibacterial properties, and anti-biofilm properties.

Description

Technical Field

[0001] This invention belongs to the technical field of polymer hydrogel materials and functional soft matter, specifically relating to a hydrogel containing ATP crosslinking nodes, its preparation method, and its application. Background Technology

[0002] Hydrogels are a class of three-dimensional network soft materials composed of hydrophilic polymer segments, capable of absorbing and retaining large amounts of water. They have wide applications in tissue engineering, drug delivery, wound dressing, soft tissue interface materials, and flexible devices. In recent years, functional hydrogels possessing network stability, dynamic reversibility, and controllable degradation capabilities have attracted widespread attention. Existing biodegradable hydrogels typically rely on natural components such as proteins, peptides, and polysaccharides, or on breakable chemical structures such as ester bonds, Schiff base bonds, and acetal bonds to achieve degradation. While these systems can achieve material degradation under specific conditions, they generally suffer from the following problems: (1) The degradation triggering conditions are relatively simple, often depending on specific pH values, redox environments or specific enzyme systems, and the scope of application is limited; (2) The cross-linking effect of some biodegradable networks is weak, resulting in insufficient gel formation and mechanical stability; (3) Although some systems have dynamic reversible characteristics, it is difficult to simultaneously take into account network strength and controllable de-crosslinking behavior.

[0003] Adenosine triphosphate (ATP) is a small molecule widely found in biological systems, containing adenine, ribose, and a triphosphate chain. The phosphate group of ATP can interact at multiple points with positively charged groups or groups capable of hydrogen bonding, while the vicinal diol structure of the ribose moiety can form reversible borate ester bonds with the borate group. Furthermore, the phosphate structure of ATP can be recognized and acted upon by enzyme systems such as ATPase, alkaline phosphatase, and acid phosphatase, which are widely present in any organ and tissue of living organisms. Therefore, from a molecular structure perspective, ATP has the potential to serve as a functional cross-linking unit for constructing enzyme-responsive network materials and can be widely applied to any part of living organisms without requiring specific applications.

[0004] However, in existing technologies, ATP is more commonly used in small molecule self-assembly systems, complex condensation systems, micelles, vesicles, or supramolecular fiber systems, with relatively few reports on its utilization as a crosslinking node in macroscopic bulk hydrogel networks. This is because relying solely on the single interaction between ATP and a single functional group is usually insufficient to construct a three-dimensional gel network that possesses both stability and mechanical support.

[0005] Therefore, it is still necessary to provide a new network construction strategy that enables ATP to be stably introduced into the polymer network as a crosslinking node, thereby obtaining a hydrogel material that combines network stability, dynamic properties and enzyme-responsive decrosslinking ability. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a hydrogel containing ATP crosslinking nodes, its preparation method, and its applications. This invention utilizes the multi-point interaction between guanidine-containing polymer units and ATP phosphate groups, as well as the reversible borate ester interaction between phenylboronic acid-containing polymer units and ATP ribose diol, to synergistically construct a hydrogel network. Through this structural design, ATP is retained as a network crosslinking node within the hydrogel, and the formed network possesses a structural basis for enzymatic decrosslinking or degradation. Furthermore, the hydrogel provided by this invention exhibits excellent mechanical properties, dynamic recovery properties, antibacterial properties, and anti-biofilm properties.

[0007] To achieve the above objectives, the present invention provides the following technical solution: One of the technical solutions of this invention is to provide a method for preparing a hydrogel containing ATP cross-linking nodes, comprising the following steps: A precursor solution is obtained by dissolving a hydrophilic polymerizable monomer, a polymerizable monomer containing a phenylboronic acid group, a polymerizable monomer containing a guanidine group, and adenosine triphosphate in a solvent. Then, an initiator is added, and after polymerization, the hydrogel containing ATP crosslinking nodes is obtained.

[0008] The hydrogel containing ATP crosslinking nodes provided by this invention has a three-dimensional crosslinked network structure. Hydrophilic polymerizable monomers polymerize to form the hydrogel framework, with ATP distributed as crosslinking nodes within the three-dimensional crosslinked network. The phosphate groups of ATP form multi-point interactions (including one or more of electrostatic interactions, salt bridging, and hydrogen bonding) with guanidine-containing polymeric units. The vicinal diol structure of the ATP ribose moiety forms borate ester bonds with polymeric units containing phenylboronic acid groups, thereby enabling ATP to connect different polymeric segments and construct a three-dimensional hydrogel network. Because the phosphate structure of ATP in the hydrogel containing ATP crosslinking nodes can serve as a structural site recognized and acted upon by enzymes within the hydrogel network, the hydrogel possesses a structural basis for enzyme-responsive decrosslinking or degradation.

[0009] The structural formula of adenosine triphosphate in this invention is as follows: .

[0010] Preferably, the hydrophilic polymerizable monomer is a hydrophilic vinyl monomer.

[0011] More preferably, the hydrophilic vinyl monomer is an acrylamide monomer.

[0012] Preferably, the polymerizable monomer containing phenylboronic acid groups is a monomer having a polymerizable double bond and a phenylboronic acid structure. The polymerizable double bond is preferably vinyl, acryloyl, or methacryloyl.

[0013] Preferably, the polymerizable monomer containing a guanidine group is a monomer that simultaneously has a polymerizable double bond and a guanidine group structure. The polymerizable double bond is preferably vinyl, acryloyl, or methacryloyl.

[0014] Preferably, the concentration of the hydrophilic polymerizable monomer in the precursor solution is 1~4M, the concentration of the polymerizable monomer containing phenylboronic acid group is 0.01~0.1M, the concentration of the polymerizable monomer containing guanidine group is 0.01~0.1M, and the concentration of adenosine triphosphate is 0.01~0.1M.

[0015] Preferably, the initiator is a free radical initiator, and more preferably a redox initiator.

[0016] Optionally, the oxidant in the redox initiator is a persulfate, and the reducing agent is a tertiary amine promoter.

[0017] Preferably, the total concentration of the initiator is 10–50 mM.

[0018] Preferably, the polymerization conditions are a temperature of 10~50℃ and a time of 1~24h; more preferably, the time is 6~24h.

[0019] The second technical solution of the present invention provides a hydrogel containing ATP cross-linking nodes prepared according to the above-mentioned method for preparing hydrogels containing ATP cross-linking nodes.

[0020] The third technical solution of the present invention provides an application of the above-mentioned hydrogel containing ATP crosslinking nodes in the preparation of enzyme-responsive degradable soft materials, antibacterial materials or anti-biofilm materials.

[0021] The enzymes applicable to the enzyme-responsive degradable soft materials include one or more of alkaline phosphatase, acid phosphatase, and ATPase; the bacterial species in the antimicrobial materials include Gram-positive bacteria and / or Gram-negative bacteria; and the biofilms in the antibiofilm materials include biofilms formed by Staphylococcus aureus.

[0022] The beneficial technical effects of the present invention are as follows: This invention provides a novel strategy for constructing macroscopic bulk hydrogel networks using ATP. Instead of using ATP merely as a small molecule ligand or assembly inducing factor, this invention introduces it into the polymer system as a network crosslinking node, thereby expanding the application of ATP in functional gel materials.

[0023] The hydrogel provided by this invention utilizes dual recognition to synergistically construct a network, which improves gel formation and network stability. Through multi-point interactions between guanidine-containing units and ATP phosphate groups, and the borate ester interaction between phenylboronic acid-containing units and ATP ribose diol, synergistic crosslinking is formed, which helps overcome the problem that single interactions are insufficient to stably construct a macroscopic gel network.

[0024] The hydrogel provided by this invention can simultaneously possess network stability and dynamic characteristics. Due to the reversible interactions within the network, the resulting hydrogel exhibits good tensile properties, toughness, and self-healing ability in some embodiments.

[0025] The hydrogel provided by this invention has a structural basis for enzyme-responsive decrosslinking or degradation. Because the ATP crosslinking nodes contain phosphate structures that can be recognized and acted upon by relevant enzymes, the hydrogel can undergo network disruption, decrosslinking, or degradation in the presence of enzymes, making it suitable for constructing soft materials with controllable service life.

[0026] The hydrogel provided by this invention has the potential for antibacterial and anti-biofilm applications. Due to the introduction of guanidine-containing functional units into the network, the hydrogel can exhibit an inhibitory effect on bacterial growth and biofilm formation in some embodiments. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a schematic diagram illustrating the network construction principle of the hydrogel containing ATP crosslinking nodes in this invention.

[0029] Figure 2 The tensile stress-strain curves are for each hydrogel prepared in Examples 1-3.

[0030] Figure 3 The curves show the change in the remaining mass of each hydrogel in Example 4 over time in the presence of alkaline phosphatase.

[0031] Figure 4 The results show the antibacterial properties of each hydrogel against MRSA and E. coli in Example 5. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0033] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0034] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] Unless otherwise specified, room temperature in this invention refers to a temperature of 20±10℃.

[0038] A schematic diagram illustrating the network construction principle of the hydrogel containing ATP crosslinking nodes in this invention is shown below. Figure 1 .

[0039] Example 1 This embodiment provides a method for preparing a hydrogel containing ATP crosslinking nodes, the specific steps of which are as follows: Acrylamide (AAm), 4-acrylamidophenylboronic acid (AAPBA), N-(3-guanidinopropyl)acrylamide (AAGu), and adenosine triphosphate (ATP) were added to deionized water to prepare a precursor solution with the following concentrations: AAM 2.5 mol / L, AAPBA 0.06 mol / L, AAGu 0.05 mol / L, and ATP 0.08 mol / L. After thorough mixing, potassium persulfate and N,N,N',N'-tetraethyldiamine (a tertiary amine promoter) were added to the precursor solution as the initiation system. The final concentrations of potassium persulfate and N,N,N',N'-tetraethyldiamine were both 20 mmol / L.

[0040] The obtained precursor solution was quickly transferred to a pre-set mold and allowed to stand at room temperature for 12 h to polymerize, resulting in a homogeneous hydrogel, which is a hydrogel containing ATP crosslinking nodes, denoted as ATPGel-0.05.

[0041] The hydrogel obtained in Example 1 was subjected to mechanical property testing. The hydrogel sample dimensions were 75 mm × 10 mm × 5 mm (length × width × height), and the tensile rate was 50 mm / min. The results showed that the tensile strength of ATPGel-0.05 was 28 kPa, the elongation at break was 1350%, and the toughness was 264 kJ·m. -3 The hydrogel sample obtained in Example 1 was cut open and reattached. After being placed at room temperature for 2 hours, it was able to restore its integrity, indicating that the hydrogel has self-healing ability.

[0042] Example 2 The preparation method in this embodiment is basically the same as that in Example 1, except that the concentration of AAGu is adjusted to 0.20 mol / L, while the concentrations of other components and operating conditions remain unchanged, namely AAm 2.5 mol / L, AAPBA 0.06 mol / L, ATP 0.08 mol / L, the final concentration of potassium persulfate is 20 mmol / L, and the final concentration of N,N,N',N'-tetraethyldiamine is 20 mmol / L.

[0043] The obtained precursor solution was transferred to a mold and allowed to stand at room temperature for 12 h to polymerize, resulting in a hydrogel containing ATP crosslinking nodes, denoted as ATPGel-0.20.

[0044] The mechanical properties of the hydrogel obtained in Example 2 were tested. The results showed that ATPGel-0.20 had a tensile strength of 45 kPa, an elongation at break of 2030%, and a toughness of 610 kJ·m. -3 The hydrogel sample obtained in Example 2 was cut open and reattached. After being placed at room temperature for 1 hour, it was able to restore its integrity, indicating that the hydrogel has a fast self-healing ability.

[0045] Example 3 The preparation method in this embodiment is basically the same as that in Example 1, except that the concentration of AAGu is adjusted to 0.50 mol / L, while the concentrations of other components and operating conditions remain unchanged, namely, AAm 2.5 mol / L, AAPBA 0.06 mol / L, ATP 0.08 mol / L, potassium persulfate final concentration of 20 mmol / L, and N,N,N',N'-tetraethyldiamine final concentration of 20 mmol / L.

[0046] The obtained precursor solution was transferred to a mold and allowed to stand at room temperature for 12 h to polymerize, resulting in a hydrogel containing ATP crosslinking nodes, denoted as ATPGel-0.50.

[0047] The mechanical properties of the hydrogel obtained in Example 3 were tested. The results showed that the tensile strength of ATPGel-0.50 was 56 kPa, the elongation at break was 1830%, and the toughness was 732 kJ·m. -3 The hydrogel sample obtained in Example 2 was cut open and reattached. After being placed at room temperature for 1 hour, it was able to restore its integrity, indicating that the hydrogel also has good self-healing ability.

[0048] The tensile stress-strain curves of the hydrogels prepared in Examples 1-3 are shown below. Figure 2 .

[0049] Comparative Example 1 This comparative example illustrates the importance of guanidine-containing units for the construction of stable networks.

[0050] Compared to Example 1, AAGu was removed from the formulation, retaining only AAM, AAPBA, ATP, and the initiation system, while the remaining conditions were the same as in Example 1. The results showed that the resulting system was difficult to form a target hydrogel with a complete shape and stable mechanical properties, exhibiting only a weak viscoelastic system or a fluid state, indicating that AAGu is necessary for stable network construction.

[0051] Comparative Example 2 This comparative example illustrates the importance of phenylboronic acid-containing units for network construction.

[0052] Compared to Example 1, AAPBA was removed from the formulation, retaining only AAM, AAGu, ATP, and the initiation system, while the remaining conditions were the same as in Example 1. The results showed that the resulting system was difficult to form the same stable and homogeneous gel as in this invention, indicating that the interaction between AAPBA and ATP-ribose diol plays a crucial role in network construction.

[0053] Comparative Example 3 This comparative example is used to illustrate the key cross-linking role of ATP in the system of this invention.

[0054] Compared to Example 1, ATP was removed from the formulation, retaining only AAm, AAPBA, AAGu, and the initiation system, while the remaining conditions were the same as in Example 1. The results showed that the resulting system could not form the target hydrogel with ATP as the crosslinking node described in this invention, indicating that ATP is the key crosslinking component in the system of this invention.

[0055] Example 4 This embodiment is used to illustrate the enzymatic degradation performance of the hydrogel containing ATP crosslinking nodes of the present invention.

[0056] The hydrogels obtained in Examples 1-3 were used to prepare cylindrical samples with a diameter of 10 mm and a thickness of 3 mm. These samples were then placed in 20 mL Tris-HCl buffer containing alkaline phosphatase (ALP) for degradation experiments. The ALP concentration in the degradation solution was 10 U, the Tris-HCl buffer concentration was 100 mmol / L, and the pH was 7.4. The samples were incubated at 37 °C, and the degradation solution was replaced every 2 days.

[0057] The curves showing the change in residual mass of each hydrogel over time in the presence of alkaline phosphatase are shown below. Figure 3 .

[0058] The degree of degradation was evaluated by the remaining mass of the samples. The results showed that the mass of each group of hydrogel samples gradually decreased with prolonged incubation time. After 21 days of incubation, the remaining mass of the hydrogels obtained in Examples 1, 2, and 3 were 7%, 8%, and 31%, respectively, with corresponding average daily mass loss rates of approximately 4.4%, 3.9%, and 3.3%. This indicates that the hydrogel containing ATP crosslinking nodes prepared in this invention can undergo significant degradation in the presence of alkaline phosphatase, demonstrating its enzyme-responsive degradation characteristics.

[0059] Comparative Example 4 This comparative example is used to illustrate the stability of hydrogels under enzyme-free conditions.

[0060] The hydrogels obtained in Examples 1-3 were used to prepare cylindrical samples with a diameter of 10 mm and a thickness of 3 mm. These samples were placed in a Tris-HCl buffer solution (100 mmol / L, pH 7.4) without alkaline phosphatase and cultured at 37 °C, with the buffer solution replaced every 2 days. After 21 days of culture, the remaining mass of the hydrogels obtained in Examples 1-3 was greater than 71%.

[0061] The above results show that the hydrogel prepared by the present invention has good structural stability under enzyme-free conditions, but exhibits significant degradation behavior in the presence of alkaline phosphatase, further demonstrating that the degradation of the hydrogel is enzyme-responsive.

[0062] Example 5 This embodiment is used to illustrate the antibacterial properties of the hydrogel containing ATP crosslinking nodes of the present invention.

[0063] Methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli) were inoculated into brain heart infusion (BHI) medium and Luria-Bertani (LB) medium, respectively. After reaching the logarithmic growth phase, the cultures were centrifuged, washed, and resuspended in sterile buffer to adjust the bacterial concentration to 6 log. 10CFU / mL. The hydrogels obtained in Examples 1-3 were prepared into circular samples of the same size (10 mm in diameter, 3 mm in thickness), and each was added to a culture system containing 500 µL of bacterial suspension, and incubated at 37 °C for 24 h. A bacterial suspension system without hydrogel was used as a control group. The antibacterial properties of the hydrogels were evaluated by measuring colony forming units (CFU) and the absorbance of the culture medium.

[0064] The results of the antibacterial performance tests of each hydrogel against MRSA and E. coli are shown in the table below. Figure 4 .

[0065] For MRSA, the viable count after co-incubation with the control group was 5.55 log. 10 CFU / mL; After treatment with hydrogels in Example 1, the viable bacterial count decreased to 3.87 log. 10 CFU / mL; after hydrogel treatment in Example 2, the viable bacterial count decreased to 2.55 log. 10 CFU / mL; after hydrogel treatment in Example 3, the viable bacterial count further decreased to 1.96 log. 10 CFU / mL.

[0066] For E. coli, the viable count after co-incubation with the control group was 7.55 log. 10 CFU / mL; After treatment with hydrogels in Example 1, the viable bacterial count decreased to 4.55 log. 10 CFU / mL; after hydrogel treatment in Example 2, the viable bacterial count decreased to 3.45 log. 10 CFU / mL; After hydrogel treatment in Example 3, the viable bacterial count decreased to 1.51 log. 10 CFU / mL.

[0067] Simultaneously, the absorbance of the culture medium was measured. Using OD600 as an indicator, the absorbance of the control group MRSA bacterial culture was 0.56, while the absorbance of the treatment groups in Examples 1, 2, and 3 decreased to 0.22, 0.10, and 0.09, respectively; the absorbance of the control group E. coli bacterial culture was 0.48, while the absorbance of the treatment groups in Examples 1, 2, and 3 decreased to 0.38, 0.09, and 0.01, respectively.

[0068] The above results indicate that the hydrogel containing ATP crosslinking nodes prepared in this invention has a significant inhibitory effect on both Gram-positive bacteria MRSA and Gram-negative bacteria E. coli, and the antibacterial ability of the hydrogel is further enhanced with the increase of guanidine content.

[0069] Example 6 This embodiment is used to illustrate the anti-biofilm properties of the hydrogel containing ATP crosslinking nodes of the present invention.

[0070] MRSA bacterial culture was inoculated into BHI medium to achieve an initial bacterial concentration of 6 log₂. 10 The samples were cultured at CFU / mL and co-cultured with the hydrogel samples obtained in Examples 1-3 for 72 h (hydrogel samples with a diameter of 10 mm and a thickness of 3 mm were prepared and added to a culture system containing 500 µL of bacterial solution). After culture, the supernatant was discarded, and the samples were gently washed with phosphate buffer to remove unattached bacteria. The formed biofilm was then quantitatively analyzed using crystal violet staining, and the absorbance was measured at 570 nm. The system without hydrogel treatment served as the control group.

[0071] The results showed that the absorbance of the biofilm after staining in the control group was 0.48; the absorbance of the ATPGel-0.05 treatment group decreased to 0.24; the ATPGel-0.20 treatment group decreased to 0.06; and the ATPGel-0.50 treatment group further decreased to 0.05. Calculated with the control group as 100%, the inhibition rates of ATPGel-0.05, ATPGel-0.20, and ATPGel-0.50 on MRSA biofilm formation were 50%, 88%, and 90%, respectively.

[0072] The above results indicate that the hydrogel containing ATP crosslinking nodes prepared in this invention can significantly inhibit the formation of MRSA biofilms, and its anti-biofilm properties are enhanced with the increase of guanidine compound content.

[0073] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a hydrogel containing ATP crosslinking nodes, characterized in that, Includes the following steps: A precursor solution is obtained by dissolving a hydrophilic polymerizable monomer, a polymerizable monomer containing a phenylboronic acid group, a polymerizable monomer containing a guanidine group, and adenosine triphosphate in a solvent. Then, an initiator is added, and after polymerization, the hydrogel containing ATP crosslinking nodes is obtained.

2. The method for preparing a hydrogel containing ATP crosslinking nodes according to claim 1, characterized in that, The hydrophilic polymerizable monomer is a hydrophilic vinyl monomer.

3. The method for preparing a hydrogel containing ATP crosslinking nodes according to claim 2, characterized in that, The hydrophilic vinyl monomer is an acrylamide monomer.

4. The method for preparing a hydrogel containing ATP crosslinking nodes according to claim 1, characterized in that, The polymerizable monomer containing phenylboronic acid groups is a monomer with polymerizable double bonds and a phenylboronic acid structure.

5. The method for preparing a hydrogel containing ATP crosslinking nodes according to claim 1, characterized in that, The polymerizable monomer containing a guanidine group is a monomer that simultaneously has a polymerizable double bond and a guanidine group structure.

6. The method for preparing a hydrogel containing ATP crosslinking nodes according to claim 1, characterized in that, The precursor solution contains 1-4 M of hydrophilic polymerizable monomers, 0.01-0.1 M of polymerizable monomers containing phenylboronic acid groups, 0.01-0.1 M of polymerizable monomers containing guanidine groups, and 0.01-0.1 M of adenosine triphosphate.

7. The method for preparing a hydrogel containing ATP crosslinking nodes according to claim 1, characterized in that, The initiator is a free radical initiator; the total concentration of the initiator is 10–50 mM.

8. The method for preparing a hydrogel containing ATP crosslinking nodes according to claim 1, characterized in that, The polymerization conditions are a temperature of 10~50℃ and a time of 1~24h.

9. A hydrogel containing ATP crosslinking nodes prepared by the method of preparing a hydrogel containing ATP crosslinking nodes according to any one of claims 1 to 8.

10. The application of the hydrogel containing ATP crosslinking nodes as described in claim 9 in the preparation of enzyme-responsive degradable soft materials, antibacterial materials or antibiofilm materials.

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