A kind of intelligent antibacterial hydrogel and preparation method thereof

Abstract

The invention discloses an intelligent antibacterial hydrogel and a preparation method thereof, wherein the specific steps of the preparation method are as follows: mixing a tannic acid aqueous solution with 3-formylphenylboronic acid, adjusting the pH value to 8.0-9.0, obtaining a first solution, then adding an aqueous solution of tobramycin of equal volume to the first solution, and standing for 5-30 minutes to form a TA / BA / TOB hydrogel. The TA / BA / TOB hydrogel of the invention is a bifunctional anchoring structure composed of an aldehyde group and boric acid, and gelled with natural structural monomers (tobramycin and tannic acid), to construct a fully small molecule dynamic covalent intelligent hydrogel, which not only embodies the traditional advantages of the hydrogel, but also has soft and adjustable mechanical properties, solution stability, good biocompatibility, controllable drug release and in vivo antibacterial activity.

Description

Technical Field

[0001] The present invention relates to the field of hydrogel technology, and in particular to an intelligent antibacterial hydrogel and a preparation method thereof. Background Art

[0002] Smart hydrogels have multi-stimulus responsiveness and special functions, and are gaining increasing attention in biomedical applications. The construction of smart hydrogels using reversible dynamic covalent bonds has been widely reported. However, these multi-stimulus responsive hydrogels often require complex macromolecular gel presynthesis and strict preparation conditions. In our early work, many all-small molecule dynamic covalent smart hydrogels have been successfully prepared to overcome these problems. They have the advantages of simple preparation, multi-stimulus responsiveness, high drug loading and strong in vitro antibacterial activity, but have disadvantages such as hardness, fragility and instability in solution, which limits its application. Summary of the invention

[0003] The purpose of the present invention is to provide an intelligent antibacterial hydrogel and a preparation method thereof in view of the deficiencies in the prior art.

[0004] To achieve the above object, the technical solution adopted by the present invention is:

[0005] The first aspect of the present invention is to provide a method for preparing an intelligent antibacterial hydrogel, which specifically comprises the following steps: mixing an aqueous tannic acid solution with 3-formylphenylboronic acid, adjusting the pH value to 8.0-9.0 to obtain a first solution, then adding an aqueous tobramycin solution having an equal volume to the first solution, and standing for 5-30 minutes to form a TA / BA / TOB hydrogel.

[0006] Furthermore, the molar concentration of the tannic acid aqueous solution is 0.05-5 mol / L.

[0007] Furthermore, the molar ratio of the tannic acid to 3-formylphenylboronic acid is 1:(10000-20000).

[0008] Furthermore, the molar ratio of the tannic acid to 3-formylphenylboronic acid is 1:14000.

[0009] Further, the pH value was adjusted to 8.2.

[0010] Furthermore, the molar concentration of the tobramycin aqueous solution is 0.14-0.5 mol / L.

[0011] More preferably, the ratio of the molar concentration of the tannic acid to the molar concentration of tobramycin is 5:(10-30).

[0012] Furthermore, the ratio of the molar concentration of the tannic acid to the molar concentration of tobramycin is 5:14.

[0013] The second aspect of the present invention is to provide an intelligent antibacterial hydrogel prepared by the above preparation method.

[0014] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0015] The TA / BA / TOB hydrogel of the present invention is a bifunctional anchoring structure composed of aldehyde groups and boric acid, and is gelled with natural structural monomers (tobramycin and tannic acid) to construct an all-small molecule dynamic covalent smart hydrogel. It not only embodies the traditional advantages of this type of hydrogel, but also has soft and adjustable mechanical properties, solution stability, good biocompatibility, controllable drug release and in vivo antibacterial activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a study on the structure of TA / BA / TOB hydrogel in one embodiment of the present invention; wherein, Figure 1 a is the chemical structure diagram of TA / BA / TOB; Figure 1 b and Figure 1 c 11B NMR and X-ray photoelectron spectra of TA / BA / TOB, respectively; Figure 1 d is the N 1s resolution spectrum of TA / BA / TOB hydrogel; Figure 1 e is the N 1s resolution spectrum of TA / 2-BA / TOB mixture; Figure 1 f is the N 1s resolution spectrum of TA / 4-BA / TOB complex; Figure 1 g is the N 1s resolution spectrum of the TA / 4-BA / TOB mixture when the solvent was replaced by DMSO.

[0017] Figure 2 The results of the study on the physical properties of TA / BA / TOB hydrogel in one embodiment of the present invention are as follows; wherein: Figure 2 ac are rheological results of the rheological properties of TA / BA / TOB hydrogel; Figure 2 de, Figure 2 h is the response behavior study results of TA / BA / TOB hydrogel.

[0018] Figure 3 is a diagram for adjusting the mechanical properties (G′ and G″) of TA / BA / TOB hydrogel in one embodiment of the present invention; wherein, Figure 3 a is the effect of TOB concentration on the mechanical properties of TA / BA / TOB hydrogel; Figure 3 b: Effect of TA concentration on the mechanical properties of TA / BA / TOB hydrogel.

[0019] Figure 41 is the effect of adding HCl, NaOH or H2O2 on the peak of catechol-borate (10.8 ppm) in one embodiment of the present invention.

[0020] Figure 5 ac is the in vitro antibacterial activity of TA / BA / TOB hydrogel in one embodiment of the present invention; Figure 5 df is the degradation of TA / BA / TOB hydrogel in vivo in one embodiment of the present invention.

[0021] Figure 6 This is the volume of gel after the mice injected with TBT gel in one embodiment of the present invention were heated once a day for 6 consecutive days.

[0022] Figure 7 The therapeutic effect of TBT gel on a mouse Staphylococcus aureus infection model in one embodiment of the present invention is demonstrated; wherein, Figure 7 a is a model diagram of TBT gel treatment of Staphylococcus aureus infection; Figure 7 b shows the changes in body temperature of mice treated with PBS or TBT gel during the heating cycle; Figure 7 c is an image of mice infected with Staphylococcus aureus implanted with TBT gel and heated, with unheated TBT gel, free TOB and PBS as controls (n=6); Figure 7 di is the number of surviving bacteria in the skin tissue and major organs after treatment.

[0023] Figure 8 The present invention relates to an inflammatory analysis of infected skin tissue after TBT gel treatment of a mouse Staphylococcus aureus infection model; wherein, RT-PCR is used to detect the levels of proinflammatory cytokines TNF-α (a) and IL-6 (b) in the skin tissue after treatment; (c) Gram staining and immunohistochemical staining of TNF-α and IL-6 in the skin tissue after treatment.

[0024] Fig. 9 This is a biocompatibility test of TBT gel in one embodiment of the present invention; wherein, Fig. 9 ab are Masson staining results showing that TBT gel does not cause obvious inflammation and fibrosis reactions; Fig. 9 cd: There was no significant difference in hematological factors between the mice in the TBT gel group and the mice in the PBS group, indicating that TBT gel had no obvious hematotoxicity and hepato-renal toxicity. DETAILED DESCRIPTION

[0025] The following will be combined with the embodiments of the present invention and the accompanying drawings to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0027] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but they are not intended to limit the present invention.

[0028] 1. Materials

[0029] Tobramycin (TOB), tannic acid (TA), 2-formylphenylboronic acid (2-BA), 3-formylphenylboronic acid (BA), 4-formylphenylboronic acid (4-BA), tris(hydroxymethyl)methylaminomethane solution, and malachite green. Hydrogen peroxide (H2O2), sodium hydroxide (NaOH), and hydrochloric acid (HCl). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Agar. Acetonitrile and 2,4-dinitrofluorobenzene. Tryptic soy broth was purchased from Oxoid (Basingstoke, UK). Staphylococcus aureus (USA300) was from ATCC. H&E staining reagents, immunohistochemical staining reagents, TNF-α antibody, and IL-6 antibody were purchased from Servicebio, Wuhan, China.

[0030] Example 1

[0031] Tannic acid aqueous solution (0.05 mol / L, 1 mL) was mixed with 3-formylphenylboronic acid (0.7 mol), and the pH value was adjusted to 8.2 to obtain a first solution. Then, an equal volume of tobramycin aqueous solution (0.14 mol / L) was added to the first solution, and the TA / BA / TOB hydrogel (TBT gel) was formed after standing for 10 minutes.

[0032] Example 2

[0033] Tannic acid aqueous solution (0.05 mol / L, 1 mL) was mixed with 3-formylphenylboronic acid (0.5 mol), and the pH value was adjusted to 8.0 to obtain a first solution. Then, an equal volume of tobramycin aqueous solution (0.10 mol / L) was added to the first solution, and the TA / BA / TOB hydrogel (TBT gel) was formed after standing for 10 minutes.

[0034] Example 3

[0035] Tannic acid aqueous solution (0.05 mol / L, 1 mL) was mixed with 3-formylphenylboronic acid (1.0 mol), and the pH value was adjusted to 9.0 to obtain a first solution. Then, an equal volume of tobramycin aqueous solution (0.30 mol / L) was added to the first solution, and the TA / BA / TOB hydrogel (TBT gel) was formed after standing for 10 minutes.

[0036] 2. Detection

[0037] A TA / 2-BA / TOB mixture and a TA / 4-BA / TOB mixture were prepared by the method of Example 1, and compared with the TA / BA / TOB hydrogel prepared in Example 1, the following tests were performed.

[0038] 2.1 Rheological study

[0039] The rheological properties of TBT gels under different molar ratio formulas were tested using a rheometer (TA Instruments, USA) at an angular frequency of 1 rad / s and a strain of 2%. The angular frequency-dependent rheological properties were studied when the angular frequency was swept from 0.0628 rad / s to 628 rad / s and the oscillation force was set to 2%. The thixotropic properties were measured by alternating the dye solution to 2% or 200% for 3 cycles with the angular frequency set to 10 rad / s.

[0040] 2.2 Response behavior of gel

[0041] The response behavior of TBT gel was investigated by adding 100 μL of irritant solution (1 mol / L HCl, 1 mol / L NaOH or 10% (w / w) H2O2) to a glass bottle containing 200 μL TBT gel. After incubation at room temperature for 30 min, the glass bottle was inverted to evaluate the gel reactivity. The temperature response was investigated by irradiating 200 μL TBT gel in the bottle under an incandescent lamp (75 W) for 5 min. The dimension of the vertical irradiation was set to 20 cm, and the temperature was maintained between 40°C and 45°C for 5 min. At the same time, a digital temperature sensor (Temperature Recording Minggao, Beijing, China) was used. The plate temperature was set from 25°C to 55°C, and the measurement method of temperature-dependent rheology was studied.

[0042] 2.3 Nuclear magnetic resonance studies

[0043] The NMR experiments were carried out using an AVANCEIII 500 MHz NMR spectrometer.

[0044] All chemical shifts were referenced to an external standard of 0.0 ppm BF3·Et2O. Samples prepared in deuterated water or ethanol were placed in polytetrafluoroethylene tubes. The boron atomic concentration in the sample was maintained at 0.35 mol / L.

[0045] 2.4 X-ray Photoelectron Spectroscopy (XPS)

[0046] TBT gel, TA / 4-BA / TOB gel and TA / 2-BA / TOB mixture were prepared and freeze-dried as described above. Then the samples were measured by X-ray photoelectron spectrometer (XPS, Escalab250Xi, ThermoFisher).

[0047] 2.5 In vitro drug release

[0048] 1 ml of TBT gel was added to each bottle, and 5 ml of PBS (pH 5), PBS (pH 7.4) and PBS (pH 5) were added and incubated at 25°C, 25°C and 45°C, respectively. At the specified time intervals, 200 μL of the supernatant was collected to analyze the released TOB, and an equal volume of PBS was added. A high performance liquid chromatography (HPLC, Agilent 1200) instrument was used, equipped with a C18 column (4.6*150 mm, diameter 5 mm), and 10 μL of sample was injected (pre-column derivatization, Sanger reaction) to determine the TOB concentration. The mobile phase was 33% tris (hydroxymethyl) methylaminomethane (0.25% wt) and 67% acetonitrile, pumped at a flow rate of 1.0 mL / min. The detection wavelength was 365 nm, and the standard curve was y=34531x-47.6 (x is TOB concentration, mg / mL, y is peak area, R2=0.999)

[0049] 2.6 In vitro antibacterial test

[0050] Staphylococcus aureus was cultured to the logarithmic phase for antibacterial test. The optical density (OD) of the bacterial solution at a wavelength of 600nm was measured using a Thermo Fisher microplate to calculate the number of bacteria. 30μLTBT gel and 180μL PBS were placed under a 75W incandescent lamp for 5min, with a vertical illumination size of 20cm and a heating terminal temperature of 45°C. This process was defined as a heating cycle. Centrifuge at 5000rpm for 3min, collect 60uL of the supernatant, and add it to a 96-well plate containing 100μL of Staphylococcus aureus (108CFU / mL). After incubation at 37°C for 24h, dilute and place on a TSB plate. The inoculated Staphylococcus aureus was cultured overnight at 37°C and the number of colonies was counted. The TBT gel was not heated, and TOB, TA, and BA were also carried out as described above, with the same concentrations as the relevant TBT gel. Each sample was repeated three times. The agar plate diffusion test was used to further evaluate the antibacterial effect of heat-stimulated TBT gel on Staphylococcus aureus. Mix 19.8 mL of sterile TSB medium, including 1.2% wt agar, with Staphylococcus aureus suspension (108 CFU / mL, 200 μL). Then pour the mixture into a plate, and after the plate cools, open a hole in the center of the TSB plate. Add 5 μL of TBT gel to each well and heat 1-3 times respectively. Culture at 37°C for 16 hours and measure the diameter of the growth inhibition zone of Staphylococcus aureus.

[0051] 2.7 Biocompatibility test

[0052] Female Balb / c mice (6-8 weeks, 20 g) were purchased from East China Normal University (Shanghai, China). All animals approved by the Ethics Committee of East China Normal University (No. m20210701) were cared for in accordance with the guidelines of the National Institutes of Health. To evaluate the biocompatibility of TBT gel in vivo, 50 μL of TBT gel or PBS was injected into the back of Balb / c mice (n=5). All mice were killed two weeks later and fresh blood was collected for routine blood tests. At the same time, the skin tissue around the injection area was collected and fixed with 4% paraformaldehyde for H&E staining.

[0053] 2.8 Gel degradation in vivo

[0054] To evaluate the degradation of the gel in vivo, 50 μL of TBT gel (containing malachite green for observation) was weighed and injected into the back of Balb / c mice. The TBT gel injection area was subjected to one heating cycle. The vertical illumination size was still set to 20 cm. Different groups (n=4) were subjected to 0 to 3 heating cycles. All mice were then killed, and the injected gel was removed and weighed. Another group was injected with 50 μL of TBT gel and heated once a day for 6 consecutive days. The skin around the injection area was photographed every day.

[0055] 2.9 In vivo antibacterial assay

[0056] Twenty-four female Balb / c mice (6-8 weeks, 20 g) were anesthetized and randomly divided into four groups. Balb / c mice were injected with 50 μL TBT gel, 50 μL TBT gel, free TOB and PBS on the back (n=6). After 2 h, Staphylococcus aureus (80 μL, 108 CFU / mL) was directly injected into the treated area. The TBT gel-treated group and the PBS-treated group were subjected to one heating cycle respectively (see above for operation details). All mice were killed after 48 h. The heart, liver, spleen, lung, kidney and surrounding skin of the treatment area of ​​each group were obtained for bacterial count and histological analysis. q RT-PCR was used to detect proinflammatory cytokines such as TNF-α and IL-6 (n=6), and H&E staining and immunostaining of TNF-α and IL-1β in tissues were performed according to standard procedures.

[0057] 3. Experimental Results

[0058] 3.1 As Figure 1 As shown in a, BA is used as a functional anchor, which acts like a bridge to connect TA and TOB through dynamic chemistry. The proposed hydrogel design has several advantages: 1) the use of natural functional small molecule building blocks in the gel formation process; 2) the gel is made multi-stimulus responsive through dynamic covalent and hydrogen bonding interactions; 3) the controllable release of TOB and good antibacterial effects in vitro and in vivo are achieved by designing thermal response tests.

[0059] Firstly, the formation mechanism of TBT gel in aqueous solution was studied using 11B NMR and X-ray photoelectron spectroscopy (XPS). Figure 1 As shown in b, only BA has a sharp peak at 29.8. Under the condition of pH 8.2, the addition of TA molecules forms a new peak at 10.8, which is the formed catechol-boronate. It is worth noting that the TA / BA / TOB mixture has another new peak at 5.7, which is probably due to the formation of an imine bond to form another form of catechol-boronate. XPS scan ( Figure 1 c) and the N1s resolution spectrum of TBT gel showed the formation of imine bonds, with the proportion of imine bonds being approximately 28% ( Figure 1 d). In addition, we also investigated the gelation of BA (2-BA and 4-BA) analogs linked to TA and TOB. Figure 1 e and Figure 1As shown in Figure 5, the TA / 2-BA / TOB mixture cannot form a hydrogel, while the TA / 4-BA / TOB mixture can form a yellow hydrogel with poor transparency. Compared with the TBT gel, the n1s resolution spectrum shows that their imine bond ratios are all low. The large steric hindrance of 2-BA hinders the formation of catechol-borate and imine bonds, resulting in insufficient cross-linking. The poor water solubility and small steric hindrance of 4-BA lead to uneven cross-linking, limited cross-linking, but fast cross-linking speed, resulting in poor transparency of the hydrogel. However, when the solvent was replaced by DMSO, due to the higher solubility of 4-BA, the proportion of imine bonds increased to 35% ( Figure 1 g).

[0060] 3.2 Rheological properties of TBT gel Rheological results are shown in Figure 2 As shown in a, the gel time of TBT gel is about 200s through the intersection time point of the loss modulus (G″) and storage modulus (G'). Figure 3 As shown in the figure, the mechanical properties (G′ and G″) of TBT gel can be significantly adjusted by changing the concentrations of TOB and TA. When the TOB concentration increased from 0.04M to 0.18M, ​​the loss modulus (G″) and storage modulus (G') of TBT hydrogel increased. When the TA concentration increased from 0.025M to 0.32M, the modulus (G' and G″) showed a trend of first increasing and then decreasing. Assuming that there are 5 binding sites on each TA molecule, 0.35M BA theoretically reacts with 0.07M TA to form catechol-boronate. Therefore, excess TA may be unfavorable for the formation of intermediate gelling agents with multiple boronate bonds, thereby reducing the crosslinking density in the hydrogel and resulting in reduced stiffness. TBT gel also exhibits thixotropy ( Figure 2 b), shear thinning, plasticity, and self-healing behavior, and most hydrogels ( Figure 2 c).

[0061] Study on the response behavior of TBT gel. Figure 2 As shown in (d), TBT gel exhibited rapid degradation in the presence of HCl, NaOH, and H2O2 due to the breakage of dynamically reversible chemical bonds (i.e., catechol-boronate and imine bonds) and hydrolysis / oxidation of TA. Figure 4 The results of 11B NMR provide further evidence. The peak of catechol-borate (10.8ppm) disappears or weakens with the addition of HCl, NaOH or H2O2. It is worth noting that the TBT gel shows a strong temperature sensitivity. The modulus (G′ and G″) decreases sharply with increasing temperature, and a gel-sol phase transition with G′≤G″ occurs around 40°C. In addition, the thermal images recorded by the digital temperature sensor show that the TBT gel has dissolved at around 44.5°C ( Figure 2 e). Figure 2As shown in h, TBT gel was immersed in heated PBS at pH 5 for 30 days and showed sustained drug release.

[0062] 3.3 We further studied the in vitro antibacterial activity of TBT gel. It is worth noting that bacterial suspension can create an acidic microenvironment, which helps TBT gel release TOB to kill bacteria. Figure 5 As shown in a, TBT gel and TA had weak inhibitory effects on Staphylococcus aureus, which was due to the inherent antibacterial properties of TA and the trace release of TBT gel, which was insufficient to achieve a good antibacterial effect. When TBT gel was assisted by an additional heating cycle, the number of surviving bacteria dropped sharply after treatment, indicating a good bacterial effect, which was consistent with the in vitro drug release behavior. In addition, the results of the agar plate diffusion test showed that the longer the TBT gel was heated within 15 minutes, the better the antibacterial effect ( Figure 5 bc).

[0063] The degradation of TBT gel in vivo was further studied. Figure 5 As shown in d, mice were subjected to a heating cycle. Mice injected with TBT gel were heated once a day for 6 consecutive days, and a significant decrease in gel volume was observed, indicating that TBT gel was degraded ( Figure 6 The residual gel mass after 0, 2, and 3 heating cycles also showed significant differences. After 3 consecutive heating cycles, the degradation rate of TBT gel implanted in mice was about 35% ( Figure 5 d-5f). These results indicate that appropriate thermal stimulants can improve the drug release and control the degradation of CT gel in vivo without damaging the mouse skin.

[0064] 3.4TBT gel has good antibacterial activity and thermal-responsive controlled release behavior, which prompted us to explore its in vivo antibacterial effect on Staphylococcus aureus ( Figure 7 a). Figure 7 As shown in b, the TBT gel group and the PBS group were subjected to heating cycles. The mice treated with TBT gel without heating, TOB and PBS heating had severe ulcers around the infected area, while the mice treated with TBT gel heating had healthy and normal skin ( Figure 7 c). The number of viable bacteria around the skin in the TBT gel heating group was 1000 times lower than that in the PBS heated once, and two orders of magnitude lower than that in the TBT gel and TOB treatments ( Figure 7 d). In addition, compared with other groups, the bacterial detection rate in the heart, liver, spleen, lung, kidney and other organs of the TBT gel heating group was also significantly reduced ( Figure 7 e).

[0065] 3.5 When bacterial infection occurs, many immune cells and macrophages are recruited to the infected area and produce pro-inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) to attack bacteria. Therefore, the expression of inflammatory cytokines may cause inflammatory infiltration, which is also a factor in assessing the severity of bacterial infection. Figure 8 As shown in ab, the expression of IL-6 and TNF-α in the TBT gel and heating groups was significantly lower than that in the other groups. Immunohistochemical staining also showed similar phenomena, which was consistent with the above results. Gram staining of the skin around the infected area confirmed that the heated TBT gel had a high antibacterial activity in vivo ( Figure 8 c).

[0066] Finally, the in vivo toxicity of TBT gel was evaluated. Fig. 9 c and Fig. 9 d, there was no significant difference in the hematological factors between the TBT gel group and the PBS group, indicating that TBT gel has minimal systemic toxicity and hepato-renal toxicity. In addition, Masson staining results also showed that TBT gel did not cause significant inflammatory and fibrotic reactions ( Fig. 9 a, b).

[0067] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an intelligent antibacterial hydrogel, characterized in that: The specific steps are: mixing a tannic acid aqueous solution with 3-formylphenylboronic acid, adjusting the pH value to 8.0-9.0 to obtain a first solution, then adding a tobramycin aqueous solution of an equal volume to the first solution, and standing for 5-30 minutes to form a TA / BA / TOB hydrogel; wherein the molar concentration of the tannic acid aqueous solution is 0.05-5 mol / L, and the molar ratio of the tannic acid to the 3-formylphenylboronic acid is 1:(10000-20000); the molar concentration of the tobramycin aqueous solution is 0.14-0.5 mol / L, and the ratio of the molar concentration of the tannic acid to the molar concentration of the tobramycin is 5:(10-30).

2. The preparation method according to claim 1, characterized in that The molar ratio of the tannic acid to the 3-formylphenylboronic acid is 1:14000.

3. The preparation method according to claim 1, characterized in that Adjust pH to 8.

2.

4. An intelligent antibacterial hydrogel prepared according to the preparation method according to any one of claims 1 to 3.

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