Polypeptide hydrogel preparation method based on carbodiimide coupled micro-fluidic chip

Abstract

The invention relates to the cross technical field of microfluidic technology, chemical synthesis and biomedicine, and discloses a carbodiimide coupled microfluidic chip-based polypeptide hydrogel preparation method, which comprises: (1) constructing a carbodiimide coupled microfluidic chip, forming a sample flow channel on a chip substrate, and forming a sample flow channel on the chip substrate, the channel comprises three inlets and an outlet; and (2) preparing polypeptide hydrogel, namely introducing the gel material containing carboxyl or amino into the first inlet, introducing the EDC / MES solution into the second inlet, introducing the polypeptide solution into the third inlet, and collecting the prepared polypeptide hydrogel at the outlet. According to the invention, through the constructed micro-fluidic chip, on the premise that a stabilizer NHS is not used, a micro-fluidic laminar flow mixing effect is adopted, so that the obstruction of high-viscosity hydrogel on EDC diffusion is avoided, the carboxyl activation efficiency is improved, and then polypeptide is chemically modified to a carboxyl or amino-containing gel material efficiently, so that the polypeptide hydrogel preparation is obtained.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of microfluidics, chemical synthesis and biomedicine, and specifically to a method for preparing polypeptide hydrogels based on carbodiimide-coupled microfluidic chips. Background Technology

[0002] Bacterial infections currently pose a serious threat to human life and health. Antimicrobial peptides exert their bactericidal effect by disrupting bacterial cell membranes, showing significant killing effects against highly drug-resistant pathogens such as Staphylococcus aureus. However, these antimicrobial peptides suffer from strong hemolytic side effects, severely limiting their application. Antimicrobial peptide delivery based on biocompatible materials can reduce hemolysis while maintaining antimicrobial efficacy. Hydrogels are the most commonly used drug carriers, protecting antimicrobial peptides from blood influence until release. Compared to physical adsorption or encapsulation of antimicrobial peptides, immobilizing them on hydrogels through chemical covalent coupling can improve their stability and better control their concentration / density and residence time at the target site.

[0003] Carbodiimide coupling is a commonly used method for chemical covalent coupling. The carboxyl groups of the hydrogel are activated and stabilized using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide), which then form amide bonds with the primary amine groups on the antimicrobial peptide. However, the high viscosity of the hydrogel hinders the diffusion of EDC / NHS, requiring mechanical stirring or ultrasonic assistance, which affects the stability of the antimicrobial peptide. Secondly, activation and coupling are pH-competitive; the activation stage requires a lower pH to prevent EDC hydrolysis and improve utilization. Coupling requires a higher pH to deprotonate the amino group (-NH2), restoring nucleophilicity and attacking the activated carboxylic acid intermediate, thus improving coupling efficiency. Therefore, the entire preparation process is time-consuming and labor-intensive (approximately 12 hours). Furthermore, chemical cross-linking agents, such as NHS, are cytotoxic, requiring complex separation and purification after preparation.

[0004] Therefore, there is an urgent need to develop a peptide chemical coupling platform to efficiently prepare ready-to-use antimicrobial agents, providing a scalable engineering paradigm to solve the paradoxical problem of "highly effective antimicrobial activity - safe application" of antimicrobial peptides. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a method for preparing polypeptide hydrogels based on carbodiimide-coupled microfluidic chips.

[0006] To achieve the above objectives, the technical solution of the present invention is: a method for preparing polypeptide hydrogels based on carbodiimide-coupled microfluidic chips, comprising the following steps:

[0007] (1) Construct a carbodiimide-coupled microfluidic chip, the microfluidic chip including a chip substrate, a sample flow channel opened on the chip substrate, the channel including a carboxyl activation region and a polypeptide coupling region, the carboxyl activation region having two inlets at its head end, namely a first inlet and a second inlet, the polypeptide coupling region having a third inlet at its head end and an outlet at its tail end, the tail end of the carboxyl activation region being connected to the head end of the polypeptide coupling region;

[0008] (2) Prepare a polypeptide hydrogel by introducing a gel material containing carboxyl or amino groups into the first inlet, introducing an EDC / MES solution into the second inlet, and introducing a polypeptide solution into the third inlet. The three inlets are introduced with equal volumes and the flow rates of each inlet are the same. The prepared polypeptide hydrogel is collected at the outlet.

[0009] Furthermore, the channel has a continuous arched structure, with a total length of 75mm, a height of 0.15mm, a maximum width of 0.3mm, and a minimum width of 0.04mm. The arch is a semicircle with a radius of 0.26mm.

[0010] Furthermore, the carboxyl activation region is 30 mm long and includes 62 arched channels, and the polypeptide coupling region is 45 mm long and includes 75 arched channels.

[0011] Furthermore, the gel material containing carboxyl or amino groups is an alginate hydrogel.

[0012] Furthermore, the preparation method of the alginate hydrogel is as follows: sodium alginate is dissolved in 0.1M MES to obtain the alginate hydrogel, wherein the mass-volume ratio of sodium alginate to MES is (10-30) mg: 1 mL.

[0013] Further; the EDC / MES solution is prepared by dissolving EDC in 0.1M MES to obtain an EDC / MES solution, wherein the mass-to-volume ratio of EDC to MES is 15mg:(1-3)mL.

[0014] Further, the method for preparing the polypeptide solution is to dissolve the polypeptide in a 0.1M PBS solution with a pH of 9.5 to obtain the polypeptide solution, wherein the mass-to-volume ratio of the polypeptide to the PBS solution is (100-300) μg:1mL.

[0015] Furthermore, the polypeptide is selected from one of the following: antimicrobial peptides, collagen mimic peptides, antitumor peptides, enzyme-regulated peptides, vaccine peptides, hormone peptides, and cytokine peptides.

[0016] Furthermore, the flow rates at the first, second, and third inlets are all 50 μL / min.

[0017] The beneficial effects of this invention are as follows: This invention constructs an integrated carbodiimide-coupled microfluidic chip. Without using the stabilizer NHS, it utilizes the microfluidic laminar mixing effect to avoid the obstruction of EDC diffusion by high-viscosity hydrogels, improving carboxyl activation efficiency and thus efficiently chemically modifying peptides onto gel materials containing carboxyl or amino groups to obtain peptide hydrogel formulations. The preparation time for 6 mL of peptide hydrogel formulation is only 20 min. The antimicrobial peptide hydrogel formulation prepared by this invention reduces the hemolytic activity of antimicrobial peptides while maintaining good antibacterial effects. Animal experiments show that the antimicrobial peptide hydrogel reduces the hemolysis rate from 72.4% of free peptides to 2.4%, while maintaining a strong killing effect (99.33% bactericidal rate) against methicillin-resistant Staphylococcus aureus (MRSA), and shortens the wound healing time to 7.2 ± 0.8 days. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the structure of the carbodiimide-coupled microfluidic chip of the present invention;

[0019] Figure 2 This is a schematic diagram of the carboxyl activation region structure in the channel of the present invention;

[0020] Figure 3 (A) Ultraviolet absorption spectra of antimicrobial peptide, alginate hydrogel and antimicrobial peptide hydrogel; (B) Fourier transform infrared spectra of alginate hydrogel and antimicrobial peptide hydrogel.

[0021] Figure 4 These are XPS images of alginate hydrogel and antimicrobial peptide hydrogel;

[0022] Figure 5 (A) SEM image of alginate hydrogel; (B) SEM image of antimicrobial peptide hydrogel;

[0023] Figure 6 This is a fluorescence intensity diagram of antimicrobial peptide hydrogels containing different concentrations of antimicrobial peptides;

[0024] Figure 7 This is an antimicrobial colony diagram of antimicrobial peptides, alginate hydrogels, and antimicrobial peptide hydrogels against Staphylococcus aureus.

[0025] Figure 8 This is a graph showing the effect of antimicrobial peptides, alginate hydrogels, and antimicrobial peptide hydrogels on erythrocyte solubility.

[0026] Figure 9 The study investigated the cytotoxicity of antimicrobial peptides, alginate hydrogels, and antimicrobial peptide hydrogels against human liver cancer cells HepG2 and human lung adenocarcinoma epithelial cells A549.

[0027] Figure 10The amount of bacteria in the skin of MRSA-infected mice treated with antimicrobial peptides, alginate hydrogels, and antimicrobial peptide hydrogels;

[0028] Figure 11 These are photos of MRSA-infected mice on days 1-7 after treatment with antimicrobial peptides, alginate hydrogels, and antimicrobial peptide hydrogels.

[0029] Figure 12 H&E staining images of skin wound tissue sections from MRSA-infected mice treated with antimicrobial peptides, alginate hydrogels, and antimicrobial peptide hydrogels.

[0030] Figure 13 (A) Residual amount of antimicrobial peptide hydrogel at different times; (B) Release amount of antimicrobial peptide at different times (w / w%); (C) Antimicrobial inhibition rate of antimicrobial peptides released in different pH environments;

[0031] Figure 14 This is a photograph of the carbodiimide-coupled microfluidic chip of this invention.

[0032] In the diagram: 1. Chip substrate; 2. Channel; 3. Arched channel; 4. First entrance; 5. Second entrance; 6. Third entrance; 7. Exit. Detailed Implementation

[0033] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. It should be noted that, unless otherwise specified, the raw materials involved in the present invention are all purchased through conventional commercial channels.

[0034] Example 1:

[0035] A method for preparing antimicrobial peptide hydrogels based on carbodiimide-coupled microfluidic chips includes the following steps:

[0036] (1) Construction of carbodiimide-coupled microfluidic chip

[0037] The microfluidic chip includes a chip substrate 1, on which sample flow channels 2 are formed. The channels 2 have a continuous arched structure, comprising 137 arched channels 3. The total length of channel 2 is 75 mm, the height is 0.15 mm, the widest point (L) is 0.3 mm, and the narrowest point is 0.04 mm. The arches are semicircles with a radius (R) of 0.26 mm. The continuous arched structure of channel 2 forms a smooth fluid guiding path, effectively reducing flow resistance. Channel 1 includes a carboxyl activation region and a polypeptide coupling region. The carboxyl activation region is 30 mm long and includes 62 arched channels 3, while the polypeptide coupling region is 45 mm long and includes 75 arched channels 3. The carboxyl activation region has two inlets at its head, namely a first inlet 4 and a second inlet 5. The polypeptide coupling region has a third inlet 6 at its head and an outlet 7 at its tail. The tail end of the carboxyl activation region connects to the head end of the polypeptide coupling region (e.g., ...). Figure 1 , Figure 2 and Figure 14 ).

[0038] (2) Preparation of antimicrobial peptide hydrogels using carbodiimide-coupled microfluidic chips

[0039] 40 mg of sodium alginate (AR, 90%, M / G = 1:2, Maklin, Shanghai) was dissolved in 2 mL of 0.1 M MES (2-(N-morpholino)ethanesulfonic acid, pH = 4.5, ST370, Beyotime, Shanghai) under stirring and sonication to prepare 2% (w / w) alginate hydrogel.

[0040] 400 μg of antimicrobial peptide (using antimicrobial peptide AMP-20 from patent CN202210392287.1) was dissolved in 2 mL of 0.1 M PBS (pH = 9.5) to prepare antimicrobial peptide AMP-20 solution.

[0041] Two mL of the 2% (w / w) alginate hydrogel prepared above was introduced into the first inlet 4. 15 mg of EDC was dissolved in 2 mL of 0.1 M MES (pH = 4.5) and introduced into the second inlet 5. Two mL of the antimicrobial peptide AMP-20 solution prepared above was introduced into the third inlet 6. All three inlets were introduced simultaneously at a flow rate of 50 μL / min. The prepared antimicrobial peptide hydrogel was collected at the outlet 7. The carboxyl activation pH was 4.5, and the antimicrobial peptide coupling pH was 6.5. The working principle is as follows: Figure 1 .

[0042] Example 2: Characterization

[0043] The antimicrobial peptide AMP-20 (HfAMP), alginate hydrogel, and antimicrobial peptide hydrogel (HfAMP@Alginate) from Example 1 were subjected to ultraviolet absorption spectroscopy measurements. The results are shown in the figure. Figure 3 (A) Fourier transform infrared spectroscopy analysis was performed on the antimicrobial peptide AMP-20 (HfAMP) and the antimicrobial peptide hydrogel (HfAMP@Alginate). The results are shown in Figure 1. Figure 3 (B) XPS and SEM analyses were performed on the alginate hydrogel and antimicrobial peptide hydrogel (HfAMP@Alginate), respectively. The results are shown in Figures [1]. Figure 4 and Figure 5 .

[0044] like Figure 3 As shown in (A), the UV peak of the antimicrobial peptide is around 220 nm. The alginate gel (HfAMP@Alginate) modified with the antimicrobial peptide also shows a peak at the same location. Figure 3 As shown in (B), with the doping of antimicrobial peptides, the sample at 3384 cm⁻¹... -1 The OH absorption peak near 3345 cm⁻¹ -1 The redshift in the vicinity is due to the enhanced intermolecular hydrogen bonding caused by the large number of NH groups introduced into the polypeptide. 1607cm -1 The increase in the relative intensity of the absorption peak is related to the C=O stretching vibration mode of the amide group, indicating that the activated alginate reacts with the amine group to form an amide structure, and the antimicrobial peptide is successfully modified onto the surface of the alginate gel. Figure 4 XPS spectral analysis showed that the antimicrobial peptide was linked to alginate via an amide bond, with a significant N 1s peak indicating successful amide bond introduction. Furthermore, while alginate is sulfur-free, the antimicrobial peptide contains sulfur amino acids, resulting in a peak in the S2p region of the alginate gel containing the antimicrobial peptide.

[0045] In conventional carboxyl activation methods, the molar ratio of added EDC to carboxyl groups in the alginate hydrogel is typically 1:1 to 5:1. This is because some EDC undergoes hydrolysis in conventional methods, requiring the addition of excess EDC to ensure that most carboxyl groups are activated. Since the microfluidic chip of this invention allows for precise pH control, there is no need to worry about the hydrolysis of EDC and intermediates. Figure 6As shown, the antimicrobial peptide hydrogels modified with different concentrations of FITC-AMP-20 (prepared by GL Biochem, Shanghai, China) were characterized by fluorescence spectrophotometry. The preparation method of the antimicrobial peptide hydrogels containing different concentrations of antimicrobial peptide was as follows: Following the method in Example 1, 2 mL of 2% (w / w) alginate hydrogel was introduced into the first inlet 4; 15 mg of EDC was dissolved in 2 mL of 0.1 M MES (pH = 4.5) and introduced into the second inlet 5; 400 μg, 280 μg, 160 μg, and 40 μg of the antimicrobial peptide FITC-AMP-20 were dissolved in 2 mL of 0.1 M PBS (pH = 9.5) and introduced into the third inlet 6. All three inlets were introduced simultaneously at a flow rate of 50 μL / min. Antimicrobial peptide hydrogels with surface-modified FITC-AMP-20 concentrations of 100 μg / mL, 70 μg / mL, 40 μg / mL, and 10 μg / mL were obtained at outlet 7, respectively. The concentration of the modified antimicrobial peptide could be increased in increments of 30 μg / mL. Therefore, antimicrobial peptide hydrogels modified with different concentrations of antimicrobial peptide can be easily obtained by controlling the amount of antimicrobial peptide added, without worrying about the completeness of the reaction and avoiding reagent waste.

[0046] Example 3: Treatment of mice with bacterial infection using the antimicrobial peptide hydrogel prepared in Example 1

[0047] In vitro experiments

[0048] To demonstrate that the antimicrobial peptide hydrogel prepared based on a carbodiimide-coupled microfluidic chip is a good antimicrobial agent, its bactericidal and therapeutic effects in vitro and in vivo were studied. For example... Figure 7 The colony count experiment on the agar plate shown was performed by washing methicillin-resistant Staphylococcus aureus (MRSA) in the mid-log phase with phosphate-buffered saline (PBS, pH 7.4) and then resuspending it in PBS. The Staphylococcus aureus suspension was then diluted in Mueller-Hinton broth (MHB) to obtain a concentration of 1 × 10⁻⁶. 6 CFU / mL bacterial suspension. Take a 96-well plate and add 50 μL of the antimicrobial peptide AMP-20 solution (HfAMP), antimicrobial peptide hydrogel (HfAMP@Alginate), alginate hydrogel, and PBS (pH 7.4, Blank) from Example 1 to each well. To achieve a final plasma concentration of 50%, add 100 μL of human plasma (provided by the Affiliated Hospital of Guizhou Medical University) to each well, followed by 50 μL of the above-prepared 1×10⁻⁶ CFU / mL bacterial suspension. 6CFU / mL bacterial suspension. Incubate at 37°C for 24 hours, then take 20 μL of the mixture from each well, add 180 μL of PBS (pH 7.4), and place on an LB agar plate. Incubate overnight at 37°C. Figure 7 As shown, the antimicrobial peptide hydrogel and the antimicrobial peptide AMP-20 have similar antimicrobial capabilities, both of which can significantly reduce the number of colonies on agar plates, while the alginate gel has little effect on the number of colonies.

[0049] Antimicrobial peptide hydrogels (HfAMP@Alginate) containing 100 μg / mL, 70 μg / mL, 40 μg / mL, and 10 μg / mL AMP-20, as well as solutions of the antimicrobial peptide AMP-20 (HfAMP) and alginate hydrogels containing the above concentration gradients, were prepared according to Example 1.

[0050] Hemolytic assay

[0051] Human red blood cells (provided by the Affiliated Hospital of Guizhou Medical University) were collected in an anticoagulant tube at 5 mL. The cells were centrifuged at 2000 rpm for 5 min. The supernatant was washed three times with 20 times the volume of PBS solution (pH 7.4), and then resuspended and diluted with 20 times the volume of PBS solution (pH 7.4) to prepare a 4% hRBCs resuspension. 100 μL of each concentration gradient of the prepared HfAMP@Alginate, HfAMP, and Alginate were added to each well of a 96-well plate, followed by 100 μL of the 4% hRBCs resuspension and incubation for 1 h. The positive control contained 100 μL of human red blood cell suspension and 100 μL of 0.1% Triton X-100; the negative control contained 100 μL of human red blood cell suspension and 100 μL of PBS (pH 7.4, 10 mM). One hour later, the sample was removed, centrifuged at 1000g for 10 minutes, and the supernatant was transferred to a 96-well plate. The OD was measured using a microplate reader. 570 Calculate the absorbance value and the percentage of hemolysis. For example... Figure 8 As shown, the antimicrobial peptide exhibited a high hemolysis rate, reaching 74.2% upon exposure to 100 μg / mL, compared to 2.4% for the antimicrobial peptide hydrogel. This system reduced the hemolysis rate from 72.4% for the free peptide to 2.4%.

[0052] Cytotoxicity assay

[0053] Human hepatocellular carcinoma cells HepG2 (provided by the Affiliated Hospital of Guizhou Medical University) and human lung adenocarcinoma epithelial cells A549 (human alveolar basal epithelial cells for lung cancer, provided by the Affiliated Hospital of Guizhou Medical University) were used to evaluate the cytotoxicity of antimicrobial peptides on mammalian cells.

[0054] Human hepatocellular carcinoma HepG2 cells and human lung adenocarcinoma epithelial cells A549 were respectively added to 1 mL of DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin sulfate to prepare 2×10⁻⁶ cells / mL of DMEM medium. 5 HepG2 cell suspension at a concentration of 2×10 cells / mL 5 A549 cell suspension at cells / mL.

[0055] 100 μL of HepG2 cell suspension and A549 cell suspension were added to 96-well plates, respectively, to a final concentration of 2 × 10⁻⁶. 4 Cells / well. 100 μL of HepG2 cell suspension and 100 μL of LA549 cell suspension were used as positive controls, and 100 μL of DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin sulfate was used as a negative control. Cells were cultured at 37°C for 24 h. Then, 100 μL of antimicrobial peptide hydrogel containing 10 μg / mL AMP-20 (HfAMP@Alginate) and 10 μg / mL antimicrobial peptide AMP-20 (HfAMP) solution and 10 μg / mL alginate hydrogel were added to each cell suspension, respectively, and the cells were cultured at 37°C for 24 h. After culture, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for another 4 h. The absorbance was measured at 450 nm using a microplate reader, and the percentage of viable cells was calculated. When exposed to an antimicrobial peptide containing 10 μg / mL, the survival rates of the antimicrobial peptide and the antimicrobial peptide hydrogel against HepG2 cells were 32.90% and 114.59%, respectively, and the survival rates against A549 cells were 86.66% and 89.18%, respectively. Figure 9 ).

[0056] In vivo experiments

[0057] Sixteen BALB / c mice (6-8 weeks, 18-20 g) (provided by the Animal Experiment Center of Guizhou Medical University) were randomly divided into four groups: a control group, a group treated with antimicrobial peptide AMP-20 (HfAMP) 1 mg / kg, a group treated with antimicrobial peptide hydrogel (HfAMP@Alginate) (calculated based on HfAMP content at 1 mg / kg), and a group treated with the same concentration of alginate hydrogel. First, the mice were dehaired on their backs the day before the experiment using a depilatory cream; after dehairing, 200 μL of 1×10⁻⁶ HfAMP@Alginate was subcutaneously injected into the left and right buttocks. 8Methicillin-resistant Staphylococcus aureus (MRSA) solution at CFU / mL was administered. Treatment began 2 hours after infection, while the control group received 0.9% NaCl (physiological saline). Treatment was administered once daily for 7 consecutive days. Mice were euthanized 24 hours after the last administration. Skin tissue from the infection site was collected, homogenized, diluted with PBS (pH 7.4), and inoculated onto LB agar plates. After incubation at 37°C for 24 hours, colony counts were recorded. Simultaneously, the infected skin tissue was dehydrated, embedded, and stained with hematoxylin and eosin (HE) to observe tissue changes. The inhibition rate was calculated as follows:

[0058]

[0059] The antimicrobial peptide hydrogel exhibits good cell biocompatibility. A mouse model of subcutaneous MRSA infection was established to evaluate the in vivo antimicrobial activity of the antimicrobial peptide and the antimicrobial peptide hydrogel. The bacterial load in the skin tissue of the negative control group was 1.12 × 10⁻⁶. 9 The bacterial load in the skin tissue of the antimicrobial peptide and antimicrobial peptide hydrogel treatment groups was 3.26 × 10⁻⁶ CFU / g, while the bacterial load in the skin tissue of the antimicrobial peptide and antimicrobial peptide hydrogel treatment groups was 3.26 × 10⁻⁶ CFU / g, respectively. 7 CFU / g, 5.70×10 6 CFU / g ( Figure 10 This indicates that antimicrobial peptides and antimicrobial peptide hydrogel treatment can significantly reduce the bacterial load on skin tissue. Based on experimental data, the antimicrobial peptide hydrogel group showed the highest bactericidal rate (99.33%), significantly superior to free antimicrobial peptides (88.57%). Figure 11 As shown, ulcerative pustules caused by Staphylococcus aureus, after treatment with antimicrobial peptide hydrogel, showed significant reduction in skin inflammation, and the healing time of infected wounds was shortened to 7.2 ± 0.8 days. Under treatment with antimicrobial peptide hydrogel, the number of neutrophils in mouse skin tissue sections decreased, and the inflammatory response was reduced. In contrast, untreated mice, particularly those in the Staphylococcus aureus (MRSA) carrier group, showed a large number of neutrophils in their skin tissue sections, indicating a significant inflammatory response. Figure 12 ).

[0060] Example 4: Validation of Controlled Sustained Release of Antimicrobial Peptide Hydrogel under Inflammatory Environment

[0061] An antimicrobial peptide hydrogel loaded with 100 μg / mL antimicrobial peptide was prepared using the method described in Example 1 of this invention. MRSA was inoculated into TSB medium and cultured at 37°C until the logarithmic growth phase (OD600≈0.6). The MRSA culture medium was adjusted to pH 5.5 and dispensed into 6-well plates (5 mL per well). The antimicrobial peptide hydrogel sample (5 mm in diameter, 1 mm in thickness) was immersed in acidic culture medium containing MRSA (pH 5.5) and a neutral control (MRSA culture medium adjusted to pH 7.4). Three replicates were set for each group. Samples were taken at 0, 2, 4, 8, 12, and 24 hours, the gel was removed, and the residual mass of the gel was measured. After washing with PBS (pH 7.4), the gel was freeze-dried, weighed, and the residual rate was calculated (see Example 1). Figure 13 ).

[0062] Simultaneous sampling of culture supernatant (1 mL each time, replenished with an equal volume of fresh culture medium). Antimicrobial peptide concentration detection: HPLC method: chromatographic conditions (C18 column, mobile phase: acetonitrile-0.1% TFA, gradient elution), quantitative analysis of antimicrobial peptides. Antimicrobial activity verification: the released antimicrobial peptide solution (diluted to the minimum inhibitory concentration) was co-incubated with MRSA for 6 hours. Antimicrobial inhibition rate determination: plating onto TSA plates, counting colonies (CFU / mL). Calculation of inhibition rate:

[0063]

[0064] In an acidic environment, ion exchange in alginate gels accelerates, initiating hydrolysis. Amide bonds are highly sensitive to hydrolysis; during gel hydrolysis, these bonds may break, releasing AMP-20. The inflammatory environment induced by MRSA is typically acidic (pH 5.5), such as... Figure 13 As shown in Figure (A), the degradation of alginate gel was significantly accelerated under acidic conditions of pH 5.5, with a residual rate of 15% after 24 hours and a release of 95% of AMP-20 after 24 hours. Figure 13 (B) In acidic environments, due to rapid release and localized high concentration, the released antimicrobial peptides exhibit stronger antibacterial activity. Figure 13 (C)

[0065] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications may be made without departing from the technical solutions described in the claims.

[0066] In the description of this invention, it should be understood that the terms indicating orientation or positional relationship are based on the orientation or positional relationship shown in the drawings and are only for the convenience of describing the invention and simplifying the description, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention.

Claims

1. A method for preparing peptide hydrogels based on carbodiimide-coupled microfluidic chips, characterized in that, Includes the following steps: (1) Construct a carbodiimide-coupled microfluidic chip, the microfluidic chip including a chip substrate, a sample flow channel opened on the chip substrate, the channel including a carboxyl activation region and a polypeptide coupling region, the carboxyl activation region having two inlets at its head end, namely a first inlet and a second inlet, the polypeptide coupling region having a third inlet at its head end and an outlet at its tail end, the tail end of the carboxyl activation region being connected to the head end of the polypeptide coupling region; (2) Prepare a polypeptide hydrogel by introducing a gel material containing carboxyl or amino groups into the first inlet, introducing an EDC / MES solution into the second inlet, and introducing a polypeptide solution into the third inlet. The three inlets are introduced with equal volumes and the flow rates of each inlet are the same. The prepared polypeptide hydrogel is collected at the outlet.

2. The method for preparing polypeptide hydrogels based on carbodiimide-coupled microfluidic chips according to claim 1, characterized in that: The channel has a continuous arched structure, with a total length of 75mm, a height of 0.15mm, a maximum width of 0.3mm, and a minimum width of 0.04mm. The arch is a semicircle with a radius of 0.26mm.

3. The method for preparing polypeptide hydrogels based on carbodiimide-coupled microfluidic chips according to claim 2, characterized in that: The carboxyl activation region is 30 mm long and includes 62 arched channels, while the polypeptide coupling region is 45 mm long and includes 75 arched channels.

4. The method for preparing polypeptide hydrogels based on carbodiimide-coupled microfluidic chips according to claim 1, characterized in that: The gel material containing carboxyl or amino groups is an alginate hydrogel.

5. The method for preparing polypeptide hydrogels based on carbodiimide-coupled microfluidic chips according to claim 4, characterized in that: The preparation method of the alginate hydrogel is as follows: sodium alginate is dissolved in 0.1M MES to obtain the alginate hydrogel, wherein the mass-volume ratio of sodium alginate to MES is (10-30) mg:1 mL.

6. The method for preparing polypeptide hydrogels based on carbodiimide-coupled microfluidic chips according to claim 1, characterized in that: The EDC / MES solution is prepared by dissolving EDC in 0.1M MES to obtain the EDC / MES solution, wherein the mass-to-volume ratio of EDC to MES is 15mg:(1-3)mL.

7. The method for preparing polypeptide hydrogels based on carbodiimide-coupled microfluidic chips according to claim 1, characterized in that: The polypeptide solution is prepared by dissolving the polypeptide in a 0.1M PBS solution with a pH of 9.5, wherein the mass-to-volume ratio of the polypeptide to the PBS solution is (100-300) μg:1mL.

8. The method for preparing polypeptide hydrogels based on carbodiimide-coupled microfluidic chips according to claim 1, characterized in that: The polypeptide is selected from one of the following: antimicrobial peptides, collagen mimic peptides, antitumor peptides, enzyme-regulated peptides, vaccine peptides, hormone peptides, and cytokine peptides.

9. The method for preparing polypeptide hydrogels based on carbodiimide-coupled microfluidic chips according to claim 1, characterized in that: The flow rates at the first, second, and third inlets are all 50 μL / min.

Images