Intelligent response nano-hydrogel capable of realizing charge overturning and preparation method and application of intelligent response nano-hydrogel
By preparing charge-flipped intelligent response nanohydrogels, the problem of neutrophil NETs generation and removal is solved, and precise targeting of neutrophils in an inflammatory environment is achieved, reducing NET generation, restoring tissue immune homeostasis, and providing efficient therapeutic effects.
Patent Information
- Application Number
- CN202510630765.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-16
- Publication Date
- 2025-08-12
AI Technical Summary
The prior art is difficult to effectively control the generation of neutrophil NETs and remove excessive NETs, resulting in inflammatory damage in periodontal tissues, and traditional treatment methods lack precise targeting and efficient drug delivery methods.
A smart responsive nanohydrogel that can achieve charge flip was prepared. The CMCS-PEI-SA@BA nanohydrogel was formed by combining the calcium ion chelating agent and sialic acid through carboxymethyl chitosan-polyetherimide nanohydrogel, which was able to responsively flip the charge in an inflammatory acidic environment, target neutrophils and bind and eliminate NETs.
It has achieved precise targeting of neutrophils in an inflammatory environment, reduced NETs generation, restored tissue immune homeostasis, and reduced inflammatory damage. It has high efficiency, low toxic side effects and high bioavailability.
Smart Images
Figure CN120459042A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biopolymer materials, and in particular to an intelligent responsive nano-hydrogel capable of realizing charge reversal, and a preparation method and application thereof. Background Art
[0002] Periodontitis is a microbiome-related, host-mediated, multifactorial inflammatory disease that leads to periodontal attachment loss. As the sixth most prevalent disease worldwide, affecting approximately 11% of the world's population, it is one of the leading causes of tooth loss in adults. The pathogenesis of periodontitis is complex, and the key role of immune regulation in its development and progression is increasingly recognized.
[0003] Neutrophils are the most abundant type of immune cell in humans. Neutrophil-mediated immunity is the host's first defense response to infection and plays a vital role in tissue remodeling. As part of this defense mechanism, neutrophils release depolymerized chromatin and granzymes, which fully bind to cytosolic proteins to form a network of neutrophil extracellular traps (NETs) that capture bacteria and eliminate pathogenic microorganisms. However, under pathological conditions, excessive production of NETs can cause tissue damage. Clinical studies have shown that there is a large amount of neutrophil infiltration and excessive accumulation of NET-related proteins in periodontal tissue lesions. Studies have shown that in the early stage after induction of animal models of periodontitis, neutrophils infiltrate gingival tissue and release NETs to trigger mucosal inflammation and bone destruction. Therefore, there is an urgent need to develop effective strategies to control the formation of neutrophil NETs and promptly remove excessive NETs, which is crucial for maintaining the health of periodontal tissues.
[0004] Many studies have confirmed that Ca 2+ Signaling plays an important role in the formation of NETs. In the NOX-dependent pathway, intracellular Ca 2+ The increase in NOX activates neutrophil elastase (NE) and myeloperoxidase (MPO), which are involved in chromatin depolymerization. In the NOX-independent pathway, mitochondria sense intracellular Ca 2+ The increase in Ca2+ also produces ROS, which directly activates PAD4 and promotes chromatin decondensation. 2+ Abnormally high concentrations of calcium lead to calcium overload, which can lead to excessive activation of neutrophils, activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and production of superoxide, thus inducing inflammatory responses and tissue damage. 2+ Concentration and inhibition of neutrophil calcium overload are potential therapeutic targets for inhibiting NETs release and improving inflammatory damage.
[0005] Research has shown that cationic nanomaterials can have therapeutic effects on many inflammatory diseases, such as cancer metastasis and inflammatory wound healing, by removing negatively charged free DNA. Researchers have also demonstrated that targeted removal of free DNA in periodontal tissues is an effective therapeutic strategy for reducing bone loss in periodontitis. Summary of the Invention
[0006] To solve the above technical problems, the first purpose of the present invention is to provide a method for preparing a smart responsive nanohydrogel that can achieve charge reversal, the second purpose is to provide such a hydrogel, and the third purpose is to provide its application. By responding to an inflammatory acidic environment, charge reversal is achieved, and the positive and negative charges are used to bind to nucleic acids and target neutrophils. The smart response expands and releases drugs to inhibit the generation of neutrophil NETs and absorb and remove excess NETs, thereby alleviating inflammatory damage to periodontal tissues.
[0007] To achieve the first objective, the present invention is implemented through the following technical solution: a method for preparing a smart responsive nanohydrogel capable of realizing charge reversal, characterized by being completed in accordance with the following steps:
[0008] (1) Preparation of carboxymethyl chitosan-polyetherimide nanohydrogel: carboxymethyl chitosan and polyetherimide were first dissolved in deionized water and then stirred at room temperature to obtain CMCS-PEI nanohydrogel;
[0009] (2) Preparation of CMCS-PEI@BA nanohydrogel: The calcium ion chelator BAPTA-AM was dissolved in acetonitrile and stirred with the CMCS-PEI nanohydrogel at room temperature in the dark to obtain the CMCS-PEI@BA nanohydrogel;
[0010] (3) Preparation of CMCS-PEI-SA@BA nanohydrogel: Sialic acid was dissolved in MES buffer containing N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to activate the carboxyl group of SA. CMCS-PEI@BA nanohydrogel was added and stirred at room temperature in the dark. The mixture solution was dialyzed and freeze-dried to obtain CMCS-PEI-SA@BA nanohydrogel.
[0011] Carboxymethyl chitosan (CMCS), polyetherimide (PEI), calcium ion chelator (BAPTA-AM), sialic acid (SA), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC).
[0012] In the above scheme, the mass ratio of carboxymethyl chitosan to polyetherimide is 5:1, the mass ratio of CMCS-PEI nanohydrogel to calcium ion chelator BAPTA-AM is 4:1, and the mass ratio of CMCS-PEI@BA to SA is 4:1. Within this ratio range, BAPTA-AM acts as a calcium ion chelating component, physically binding to the CMCS-PEI, while SA is linked to the CMCS-PEI@BA molecule via an amide bond, providing a neutrophil-targeting unit.
[0013] In the above scheme: in step (1), the stirring time is 1-2 hours, the molecular weight of the carboxylated chitosan is 100,000-200,000, and the degree of substitution is ≥80%. Polyethyleneimine is a water-soluble polymer produced by the polymerization of ethyleneimine. It is a partially branched polymer containing primary amines, secondary amines, and tertiary amines, and has a molecular weight of 1200 Da.
[0014] In the above scheme: in step (3), the carboxyl groups of SA were activated in an ice bath for 1-2 h; after adding CMCS-PEI@BA nanohydrogel, the mixture was stirred continuously at room temperature for 24-48 h, the reaction was maintained at pH 7-8, and dialyzed for 48-72 h, with water being changed every 2-3 h during the dialysis process.
[0015] In the above scheme: the molecular weight cut-off of the dialysis bag is 3500Da.
[0016] In the above scheme: in step (2), in the MES buffer of N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, the mass ratio of N-hydroxysuccinimide to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:2.
[0017] The method for preparing the charge-reversing smart-responsive nano-hydrogel prepared by the method has a particle size of about 100 nm.
[0018] A use of the intelligent responsive nanohydrogel capable of realizing charge reversal as an embedding material in the preparation of drugs for treating tissue inflammatory damage diseases caused by excessive NETs release.
[0019] The tissue inflammatory damage diseases caused by excessive NETs release include periodontitis, nephritis, and arthritis.
[0020] Carboxymethyl chitosan is a water-soluble derivative of chitosan, with amino groups (-NH3 +), carboxyl (-CH2COO-) active groups, similar to the molecular structure of natural extracellular matrix, have good biocompatibility and in vivo degradation and absorption. Polyethyleneimine is a partially branched polymer containing primary amines, secondary amines and tertiary amines. Neutrophil-targeted calcium ion chelating nanohydrogel main body, calcium ion chelating group position such as Figure 1 As shown, the main body is carboxymethyl chitosan and polyetherimide, the outer layer is modified with sialic acid small molecules, and the inner layer is wrapped with calcium chelator BAPTA-AM.
[0021] The smart responsive nanohydrogel material SA exhibits excellent neutrophil targeting and NETs binding capabilities. At the same time, BAPTA-AM chelates calcium ions, effectively regulating the release of neutrophil NETs and restoring the immune homeostasis of periodontal tissues.
[0022] Smart responsive nanohydrogels undergo charge reversal in inflammatory environments. By binding to released NETs through the cationic nanohydrogel, they can reduce excessive NET release from neutrophils caused by calcium overload, chelate excess calcium ions, and reduce the production of citH3 protein due to PAD4 activation. BAPTA-AM effectively reduces calcium overload in neutrophils during inflammation, effectively alleviating tissue inflammatory damage caused by excessive NET release.
[0023] There is no particular limitation on the drug administration of the prepared smart responsive nano-hydrogel material. Representative administration methods include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.
[0024] Beneficial effects:
[0025] (1) Compared with traditional drugs and existing treatment methods, the intelligent responsive nanohydrogel provided by the present invention has a simple preparation method, is easy to operate, and has low cost; it has many advantages in the process of treating neutrophil calcium ion overload diseases caused by inflammation, such as high efficiency, low toxicity and side effects, and long-lasting efficacy.
[0026] (2) The precise targeting of cells by smart responsive nanoparticles and the release of drugs by charge reversal under inflammatory acidic conditions can reduce tissue damage and improve bioavailability. The smart responsive nanohydrogel provided by the present invention can precisely target neutrophils and reduce tissue damage.
[0027] (3) The higher the loading amount of the chelating agent, the lower the dose of the conjugate, and the smaller the burden of elimination. The present invention provides a smart responsive nanohydrogel that chelates excess calcium ions inside and outside the cell by targeting neutrophils, with high calcium chelation efficiency. Charge reversal occurs in the inflammatory environment, and the cationic nanohydrogel binds to the released NETs, significantly reducing NET generation and effectively regulating tissue immune homeostasis. It is expected to overcome the difficulty of targeting inflammatory lesions and achieve long-term and safe drug delivery therapy. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 Schematic diagram of the synthesis route of CMCS-PEI-SA@BA.
[0029] Figure 2 The size of nano-hydrogel particles was detected by DLS and scanning electron microscopy.
[0030] Figure 3 The particle size of CMCS-PEI-SA nanohydrogel under different pH conditions is shown.
[0031] Figure 4 The zeta potential of CMCS-PEI-SA nanohydrogel under different pH conditions is shown.
[0032] Figure 5 demonstrated that nanohydrogels bind negatively charged ctDNA.
[0033] Figure 6 This figure shows the test results of CMCS-PEI-SA@BA's ability to target inflammatory neutrophils.
[0034] Figure 7 It was demonstrated that CMCS-PEI-SA@BA attenuated the expression of inflammatory factors. DETAILED DESCRIPTION
[0035] The present invention will be further described below with reference to the accompanying drawings and embodiments.
[0036] Example 1
[0037] 1) Preparation of CMCS-PEI nanohydrogel: Dissolve 100 mg of carboxymethyl chitosan and 20 mg of polyethyleneimine in 10 ml of deionized water. Stir magnetically at room temperature for 1 hour to obtain the CMCS-PEI nanohydrogel. The carboxymethyl chitosan has a molecular weight of 100,000-200,000 and a degree of substitution of ≥80%.
[0038] 2) Preparation of CMCS-PEI@BA nanohydrogel: 25 mg of BAPTA-AM was dissolved in 5 ml of acetonitrile and stirred with 100 mg of CMCS-PEI nanohydrogel at room temperature in the dark for 1 h to obtain CMCS-PEI@BA nanohydrogel;
[0039] 3) Dissolve 25 mg of SA in a MES buffer containing NHS and EDC (46 mg of N-hydroxysuccinimide, 96 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 10 mL of MES buffer) and place in an ice bath to activate the carboxyl groups of SA for 1 hour. Add 100 mg of CMCS-PEI@BA nanohydrogel, with a CMCS-PEI@BA to SA mass ratio of 4:1, and stir at room temperature in the dark for 24 hours. The reaction maintains a pH of 7-8. After dialyzing the mixture solution (dialysis for 72 hours (dialysis bag with a molecular weight cutoff of 3500 Da), changing the water every 2-3 hours), freeze-drying, the neutrophil-targeted calcium ion chelating smart responsive nanohydrogel CMCS-PEI-SA@BA nanohydrogel was obtained.
[0040] The prepared CMCS-PEI-SA@BA is used as a smart responsive nanohydrogel material to alleviate neutrophil calcium overload and NETs generation. Figure 2 (DLS and scanning electron microscopy) show that the selected CMCS-PEI-SA@BA has a regular morphology and uniform particle size, with an average particle size of about 100nm. Figure 3 (DLS) showed that the particle size of CMCS-PEI-SA nanohydrogel under different pH conditions increased under acidic conditions and recovered with the change of pH. The Zeta potential of the nanoparticles was measured by DLS. Figure 4 As shown in Figure 3, the zeta potential of CMCS-PEI-SA nanohydrogel is -15 mV at pH 7.4, and the zeta potential is reversed to 16 mV at pH 5.5.
[0041] Detection of CMCS-PEI-SA@BA's ability to bind DNA:
[0042] (1) ctDNA solution (4 μL, 1 mg / mL PBS storage solution) and EtBr (4 μL, 1 mg / mL PBS storage solution) were mixed in a 1.5 mL EP tube to form a ctDNA-EtBr complex.
[0043] (2) Add 100 μL of 1 mg / mL CMCS-PEI-SA@BA in dd water (enzyme-free water) to the ctDNA-EtBr complex in step (1), and then add 16 μL of PBS. Adjust the final volume to 160 μL with fresh PBS.
[0044] (3) The mixed solution was incubated at 37°C for 24 h, 100 μL of the supernatant was transferred to a 96-well plate, and the fluorescence intensity of the ctDNA-EtBr complex was detected by excitation at a wavelength of 485 nm.
[0045] (4) Binding efficiency of ctDNA and CMCS-PEI-SA@BA:
[0046] [(1-(X-X0) / (X1-X0))×100%]
[0047] X = fluorescence intensity of ctDNA-EtBr complex in the supernatant after addition of material;
[0048] X0 = fluorescence intensity of EtBr, X1 = fluorescence intensity of ctDNA-EtBr complex.
[0049] ⑤ Half maximal binding efficiency (BE50, ug / ml), which is 1 / 2 of the maximum binding efficiency of CMCS-PEI-SA@BA.
[0050] The research model was conducted by selecting primary neutrophils from mouse bone marrow. Figure 4 It can be seen that the half-maximal binding efficiency (BE50) shows that under acidic pH conditions, the nanohydrogel CMCS-PEI-SA@BA can bind more negatively charged ctDNA.
[0051] CMCS-PEI-SA@BA was labeled with RBITC and co-cultured with primary neutrophils stimulated with ionomycin. The results showed that after the addition of CMCS-PEI@BA, there was no fluorescence or sporadic fluorescence around neu, while CMCS-PEI-SA@BA appeared around neu or in the cells, and L-selectin expression increased ( Figure 6 ).
[0052] By detecting the changes in the mRNA levels of inflammatory factors in primary mouse neutrophils before and after CMCS-PEI-SA@BA treatment ( Figure 7 ), which proved that CMCS-PEI-SA@BA could effectively reduce the inflammatory factors IL-1β, TNFα and IL-6 that were elevated by ionomycin (iono) stimulation.
[0053] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.
Claims
1. A method for preparing a smart responsive nanohydrogel capable of realizing charge reversal, characterized in that: Follow these steps to complete: (1) Preparation of carboxymethyl chitosan-polyetherimide nanohydrogel: carboxymethyl chitosan and polyetherimide were first dissolved in deionized water and then stirred at room temperature to obtain CMCS-PEI nanohydrogel; (2) Preparation of CMCS-PEI@BA nanohydrogel: The calcium ion chelator BAPTA-AM was dissolved in acetonitrile and stirred with the CMCS-PEI nanohydrogel at room temperature in the dark to obtain the CMCS-PEI@BA nanohydrogel; (3) Preparation of CMCS-PEI-SA@BA nanohydrogel: Sialic acid was dissolved in MES buffer containing N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to activate the carboxyl group of SA. CMCS-PEI@BA nanohydrogel was added and stirred at room temperature in the dark. The mixture solution was dialyzed and freeze-dried to obtain CMCS-PEI-SA@BA nanohydrogel.
2. The method for preparing the charge-reversing smart responsive nanohydrogel according to claim 1, characterized in that: The mass ratio of carboxymethyl chitosan and polyetherimide was 5:1, the mass ratio of CMCS-PEI nanohydrogel and calcium ion chelator BAPTA-AM was 4:1, and the mass ratio of CMCS-PEI@BA and SA was 4:
1.
3. The method for preparing the charge-reversing smart responsive nanohydrogel according to claim 2, characterized in that: In step (1), the stirring time is 1 hour, the molecular weight of the carboxylated chitosan is 100,000-200,000, and the degree of substitution is ≥80%.
4. The method for preparing the charge-reversing smart responsive nanohydrogel according to claim 3, characterized in that: In step (3), the carboxyl groups of SA were activated in an ice bath for 1-2 h; after adding the CMCS-PEI@BA nanohydrogel, the mixture was stirred continuously at room temperature for 24-48 h, the reaction was maintained at pH 7-8, and dialyzed for 48-72 h, with the water being changed every 2-3 h during the dialysis process.
5. The method for preparing the charge-reversing smart responsive nanohydrogel according to claim 4, characterized in that: The molecular weight cut-off of the dialysis bag is 3500 Da.
6. The method for preparing the charge-reversing smart responsive nanohydrogel according to claim 5, characterized in that: In step (2), in the MES buffer of N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, the mass ratio of N-hydroxysuccinimide to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:
2.
7. A smart responsive nanohydrogel capable of realizing charge reversal prepared by the method for preparing a smart responsive nanohydrogel capable of realizing charge reversal according to any one of claims 1 to 6.
8. Use of the charge-reversing smart responsive nanohydrogel according to claim 7 as an embedding material in the preparation of a drug for treating tissue inflammatory damage caused by excessive NETs release.
9. The use according to claim 8, characterized in that: The tissue inflammatory damage diseases caused by excessive NETs release include periodontitis, nephritis, and arthritis.