A co-loaded bifunctional nanogel of an antibacterial peptide and a prebiotic and its application

Abstract

The present invention provides a co-loaded bifunctional nanogel of an antimicrobial peptide and a prebiotic and its application. First, the present invention realizes the encapsulation of the prebiotic by the secondary desolvation method, and then realizes the encapsulation of the antimicrobial peptide by the swelling method to obtain the co-loaded bifunctional nanogel of the antimicrobial peptide and the prebiotic. The preparation method of the present invention is simple and easy to implement and is easy to promote. The nanogel prepared by the present invention has the characteristics of bacterial specificity and time-controlled release, effectively exerting the biological effects of both. It can not only first locally slowly release OH-CATH 30 to kill pathogenic bacteria such as Staphylococcus aureus, and secondly, the inulin slowly released subsequently can promote the growth of probiotics in the vagina to a certain extent. The nanogel of the present invention can release the antimicrobial peptide and the prebiotic successively, thereby effectively killing pathogenic bacteria at the infection site and promoting the growth of probiotics, and can be applied to the preparation of a biodegradable preparation for anti-bacterial infection and promoting the rapid recovery of the lactic acid bacteria microecology.

Description

Technical Field

[0001] The invention relates to the technical field of nanomaterials, and in particular to a co-loaded bifunctional nanogel of antimicrobial peptides and prebiotics and applications thereof. Background Art

[0002] Aerobic vaginitis (AV) is a gynecological disease that has been plaguing many women. This disease increases the risk of abortion in pregnant women. The main flora in AV is a variety of aerobic bacteria, such as Escherichia coli (E. coli), group B streptococci and Staphylococcus aureus (S. aureus). The diversity of bacterial species disrupts the vaginal flora microecology, making the survival of the original beneficial bacteria Lactobacillus in the microenvironment at risk.

[0003] At present, the treatment of aerobic vaginitis is the use of antibiotics or probiotic supplementation. However, traditional antibiotics will indiscriminately kill pathogens and native vaginal beneficial bacteria (such as Lactobacillus crispatus), which may cause the vaginal microbiome to become unbalanced again. Exogenous probiotic supplementation is also difficult to effectively colonize in the body. Therefore, how to selectively eliminate pathogens and effectively protect the proliferation of native lactobacilli is an urgent problem to be solved in the treatment and prevention of aerobic vaginitis. In order to solve the problem of bacterial selectivity, previous studies have shown that narrow-spectrum antibiotics or selective antibiotics are promising options. Among them, antimicrobial peptide antibiotics of natural origin or continuous optimization have strong antibacterial, antiviral and antifungal activities, and low drug resistance, and are gradually attracting attention. However, the vaginal flora is diverse, complex and dynamic, and the intervention of any treatment method will produce a series of complex dynamic changes. Indiscriminate sterilization and probiotic supplementation each have their shortcomings, and it is inevitable to lose one while focusing on the other in the regulation of the flora. Therefore, a new type of nanogel with bacterial specificity and time-controlled release is needed. Summary of the invention

[0004] The purpose of the present invention is to provide a dual-function nanogel co-loaded with antimicrobial peptides and prebiotics and its application in aerobic bacterial vaginitis. The provided nanogel has the characteristics of bacterial specificity and time-controlled release. First, the antimicrobial peptide OH-CATH 30 can be slowly released in situ to kill pathogenic bacteria, and then the prebiotic inulin is released to promote the growth of vaginal probiotics. The present invention uses gelatin as a drug carrier to achieve specific cleavage under the action of gelatinase produced by pathogenic bacteria, thereby achieving the time-sequential release of OH-CATH30 and inulin.

[0005] In order to solve the above problems, the present invention provides the following technical solutions:

[0006] A dual-functional nanogel containing antimicrobial peptides and prebiotics, wherein the nanogel is prepared by the following steps:

[0007] (1) Prepare gelatin aqueous solution, antibacterial peptide OH-CATH 30 aqueous solution, and inulin aqueous solution separately, and filter them;

[0008] (2) Mix the gelatin aqueous solution and the inulin aqueous solution, stir evenly, and then add acetone; after stirring, add glutaraldehyde, continue to stir to obtain a mixed solution, then add glycine, stir, centrifuge, take the lower layer to obtain gelatin-inulin nanogel, wash it, and freeze-dry it to obtain CNGs freeze-dried powder;

[0009] (3) Mix the CNGs freeze-dried powder with the antibacterial peptide OH-CATH 30 aqueous solution, let it stand, centrifuge, take the lower layer, wash it, and pipette it evenly to obtain a co-loaded bifunctional nanogel of antibacterial peptide and prebiotic.

[0010] The mature antibacterial peptide OH-CATH 30 extracted from king cobra has the sequence Lys-Phe-Phe-Lys-Lys-Leu-Lys-Asn-Ser-Val-Lys-Arg-Ala-Lys-Lys-Phe-Phe-Lys-Pro-Arg-Val-Ile-Gly-Val-Ser-Ile-Pro-Phe (KFFKKLKNSVKRAKKFFKPRVIGVSIPF). It has the lowest hemolytic activity and the strongest antibacterial activity, especially against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA).

[0011] Furthermore, the gelatin used to prepare the gelatin aqueous solution in step (1) is high-molecular-weight gelatin prepared by the desolvation method.

[0012] Furthermore, the pH of the gelatin aqueous solution in step (1) is 5.8 - 6.2.

[0013] Furthermore, in step (2), the concentration of the gelatin aqueous solution is 8 mg / mL - 12 mg / mL; the concentration of the inulin aqueous solution is 20 mg / mL - 100 mg / mL, and the gelatin aqueous solution and the inulin aqueous solution are mixed in equal volumes.

[0014] Furthermore, the concentration of the gelatin aqueous solution is 10 mg / mL.

[0015] Furthermore, the concentration of the inulin aqueous solution is 40 mg / mL - 80 mg / mL.

[0016] Furthermore, the concentration of the inulin aqueous solution is 60 mg / mL.

[0017] Inulin, as a prebiotic, can produce short-chain fatty acids (acetic acid, propionic acid, and butyric acid) in the body, lower the environmental pH, promote the growth of Bifidobacterium and Lactobacillus, and has the function of improving the intestinal microenvironment. The characteristics of inulin to restore the acidic microenvironment and promote the proliferation of probiotics can well balance the microenvironment in the vagina.

[0018] Further, in step (2), after mixing the gelatin aqueous solution and the inulin aqueous solution, the pH is adjusted to 5.9 - 6.5.

[0019] Further, in step (2), after mixing the gelatin aqueous solution and the inulin aqueous solution, the pH is adjusted to 6.0. At this time, the obtained nanogel has a stable negative charge.

[0020] Further, the concentration of glycine added in step (2) is 90 mmol / L - 110 mmol / L.

[0021] Further, the concentration of glycine added in step (2) is 100 mmol / L.

[0022] Further, the volume of glycine added in step (2) is the same as that of the mixed solution.

[0023] Further, the stirring conditions in step (2) are: at room temperature, 600 - 1000 rpm, wherein, after adding glutaraldehyde, stir for 2 - 4 h, and after adding glycine, stir for 1 - 2 h.

[0024] Further, the stirring conditions after adding glycine in step (2) are 600 - 1000 rpm at room temperature for 1 h - 2 h.

[0025] Further, the centrifugation conditions in step (2) are 10000 - 22000 rpm for 30 min, 2 - 3 times, preferably 10000 rpm for 30 min, 2 times.

[0026] Further, the centrifugation conditions in step (2) are 10000 rpm for 30 min.

[0027] Further, the concentration of the antimicrobial peptide OH-CATH30 solution in step (3) is 3.0 mg / mL - 9.0 mg / mL.

[0028] Further, the mass of the CNGs freeze-dried powder is 10 mg.

[0029] Further, the concentration of the antimicrobial peptide OH-CATH 30 aqueous solution is 3.0 mg / mL - 9.0 mg / mL, the volume is 500 - 1000 μL, preferably 5 mg / mL - 7 mg / mL, more preferably 7 mg / mL, and the volume is 1000 μL.

[0030] Further, the standing condition in step (3) is to stand at room temperature for 20 h to 30 h.

[0031] Further, the standing condition in step (3) is to stand at room temperature for 24 h.

[0032] Further, the morphology of the CNGs is: quasi-circular; the CSNGs have a core-shell structure.

[0033] The present invention also provides an application of the co-loaded bifunctional nanogel of the antimicrobial peptide and the prebiotic in the preparation of a biodegradable preparation for anti-bacterial infection and promoting the growth of probiotics.

[0034] The present invention discovers that by means of the sequential responsive drug release performance of the nano-drug delivery system, the "elimination of pathogenic bacteria" and "restoration of Lactobacillus" are fused in terms of time and space, and the mechanism of action of the controllable release of drugs on regulating the dynamics of the flora is explored, so as to promote the clinical application of the nano-drug delivery technology in infectious diseases such as aerobic vaginitis.

[0035] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0036] (1) The present invention provides a co-loaded bifunctional nanogel of an antimicrobial peptide and a prebiotic. Through nanotechnology, the antimicrobial peptide OH-CATH 30 and inulin can be encapsulated to construct a co-loaded bifunctional nanogel. The nanogel has a core-shell structure and bacterial specificity. The nanogel disclosed in the present invention firstly realizes the encapsulation of the prebiotic by the secondary desolvation method, and then realizes the encapsulation of the antimicrobial peptide by the swelling method. The preparation method of the nanogel provided by the present invention is simple and easy to implement, and is easy to popularize and use.

[0037] (2) The nanogel prepared by the present invention has the characteristics of bacterial specificity and time-controlled release, effectively exerts the biological effects of both, and can not only firstly locally slow-release OH-CATH 30 to kill pathogenic bacteria such as Staphylococcus aureus, but secondly, the inulin slowly released subsequently can promote the growth of probiotics in the vagina to a certain extent. The nanogel of the present invention can release the antimicrobial peptide and the prebiotic successively, thereby effectively killing pathogenic bacteria at the infection site and promoting the growth of probiotics, and can be applied to the preparation of a biodegradable preparation for anti-bacterial infection and promoting the rapid recovery of the lactic acid bacteria microecology. It is used for the treatment of intestinal and vaginal inflammations of humans and animals, and has good market application prospects. Description of the Drawings

[0038] Figure 1 It is the encapsulation efficiency and drug loading of inulin in CSNGs.

[0039] Figure 2 It is the encapsulation efficiency and drug loading of OH-CATH 30 in CSNGs.

[0040] Figure 3 are the particle sizes and zeta potentials of CNGs and CSNGs.

[0041] Figure 4 are the transmission electron microscope images of CNGs and CSNGs.

[0042] Figure 5 are the infrared spectroscopy analysis diagrams of CNGs and CSNGs.

[0043] Figure 6 are the respective cumulative release amounts of OH-CATH 30 in CSNGs in a simulated infection environment.

[0044] Figure 7 are the respective cumulative release amounts of inulin in CSNGs in a simulated infection environment.

[0045] Figure 8 is the comparison of the bactericidal effects of CSNGs and clindamycin against Staphylococcus aureus.

[0046] Figure 9 is the comparison of the bactericidal effects of clindamycin under the condition of co-culturing beneficial bacteria and pathogenic bacteria.

[0047] Figure 10 is the comparison of the bactericidal effects of CSNGs under the condition of co-culturing beneficial bacteria and pathogenic bacteria.

[0048] Figure 11 are the cytotoxicity results of raw materials inulin, gelatin, OH-CATH 30, and CSNGs on macrophage RAW 264.7.

[0049] Figure 12 is the cytotoxicity of CSNGs on epithelial cells HK-2.

[0050] Figure 13 are the results of the erythrocyte hemolysis experiment of CSNGs at different concentrations.

[0051] Figure 14 are the colony counts of Staphylococcus aureus and Lactobacillus crispatus in the vagina of rats after the establishment of the infection model and after treatment with each drug.

[0052] Figure 15 is the detection of the levels of inflammatory factors in tissues after 7 days of treatment.

[0053] Figure 16 are the results of HE staining of histopathology after 7 days of treatment.

[0054] Figure 17 is the fluorescence probe in-situ labeling display diagram of bacteria in tissues after 7 days of treatment. Specific implementation manners

[0055] The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0056] Example 1 Preparation of a co-loaded bifunctional nanogel of an antimicrobial peptide and a prebiotic

[0057] Materials: Gelatin (Type B) was purchased from Beijing Solarbio Science & Technology Co., Ltd. Inulin, which is polymerized from fructose molecules, was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. OH-CATH 30 was synthesized by GL Biochem (Shanghai) Ltd. OH-CATH30 is a mature antimicrobial peptide extracted from king cobra in the Cathelicidin family, and its amino acid sequence is: KRFKKFFKKLKNSVKKRAKKFFKKPRVIGVSIPF.

[0058] Macrophage RAW 264.7 was provided by the Shandong Provincial Laboratory of Protein and Polypeptide Drugs for preservation; Staphylococcus aureus ATCC6538 was provided by the American Type Culture Collection. SD rats were provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd. (SCXK (Lu) 20190003) and were approved by the WFMU Animal Research Ethics Committee.

[0059] A method for preparing a co-loaded bifunctional nanogel of an antimicrobial peptide and a prebiotic, comprising the following steps:

[0060] (1) First, high molecular weight gelatin was prepared by the standard desolvation method and reserved. Then, 2%, 4%, 6%, 8%, 10% (w / v) inulin aqueous solutions were respectively prepared for preparing nanogels with different inulin contents.

[0061] A 1% (w / v) high molecular weight gelatin aqueous solution was prepared and the pH was adjusted to 6.0.

[0062] (2) 20 mL of the inulin solution and 20 mL of the gelatin solution were uniformly mixed. Acetone was added dropwise to the mixed solution and stirred until the solution showed a faint blue turbidity and then stopped. After continuing to stir for 10 min, 0.4 mL of glutaraldehyde (25%, w / v) was added and stirred at 1000 rpm for 2 h; subsequently, 100 mmol / L glycine with the same total volume as the above solution was added and stirred at 1000 rpm for 1 h, and then centrifuged at 10000 rpm and 25 °C for 30 min. The nanogel was centrifuged and washed twice with pure water, and the supernatant and nanoparticles were collected for freeze-drying.

[0063] (3) Preparation of Bifunctional Nanogels (CSNGs) Co-loaded with OH-CATH 30 and Inulin

[0064] Add 1.0 mL of OH-CATH30 aqueous solutions with concentrations of 3.0 mg / mL, 5.0 mg / mL, 7.0 mg / mL, and 9.0 mg / mL to 10 mg of CNGs respectively. After swelling for 24 h, centrifuge at 5000 g for 30 min, twice. Collect the supernatant and CSNGs for freeze-drying.

[0065] Example 2 Performance Determination of Co-loaded Bifunctional Nanogels (CSNGs) of Antibacterial Peptide and Prebiotic

[0066] Use the product prepared in Example 1 for performance determination

[0067] 1. Determination of Encapsulation Efficiency and Drug Loading of Inulin in CSNGs

[0068] Dissolve phenol at 60 °C and prepare 6% (w / v) phenol aqueous solution, shake well. Take dried glucose to prepare 0.1 mg / mL glucose stock solution, shake well.

[0069] Standard curve determination: Take 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, and 1.2 mL of glucose stock solution into test tubes respectively, and add double-distilled water to make up to 2 mL. Take 1 mL from each test tube and add 1 mL of 6% (w / v) phenol solution and 5 mL of concentrated sulfuric acid. After shaking well, let stand at room temperature for 20 min, and cool to room temperature with running water to measure OD 490nm , and draw the standard curve.

[0070] Determination of inulin encapsulation efficiency: Take 1 mL of the supernatant collected during the preparation of the nanogel, dilute it to 10 mL with distilled water and then take 1 mL and add 1 mL of distilled water. After mixing well, take 1 mL from each sample well and add 1 mL of 6% (w / v) phenol solution and 5 mL of concentrated sulfuric acid. After mixing well, let stand at room temperature for 20 min, and cool to room temperature with running water. Use the test tube with glucose concentration of 0 as the blank control to measure OD 490nm . Repeat the experiment 3 times.

[0071] Encapsulation efficiency (%) = (mass of inulin input - mass of inulin in supernatant) / mass of inulin input × 100%

[0072] Determination of inulin drug loading: Take 1 mg of nanoparticles and dilute it to 10 mL. Take 1 mL and add 1 mL of double-distilled water respectively. After mixing well, take 1 mL from each sample well and add 1 mL of 6% (w / v) phenol solution and 5 mL of concentrated sulfuric acid. After mixing well, let stand at room temperature for 20 min, and cool to room temperature with running water. Use the test tube with glucose concentration of 0 as the blank control to measure OD 490nm . Repeat the experiment 3 times.

[0073] Drug loading (%) = mass of inulin in nanoparticles / mass of nanoparticles × 100%

[0074] The results are as Figure 1 shown. Among the drug loading and encapsulation efficiency of inulin, when 6% of inulin is added, it is the optimal ratio.

[0075] 2. Determination of encapsulation efficiency and drug loading of OH-CATH30 in CSNGs

[0076] The Cy3 fluorescence labeling method was used to detect the encapsulation efficiency and drug loading of OH-CATH30.

[0077] The labeling method is as follows: First, prepare a 1 mol / L sodium bicarbonate solution and adjust its pH to 8.5 - 9.5 with 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide. Secondly, dissolve 100 mg of OH-CATH 30 in 10 mL of PBS buffer (pH 7.2 - 7.4), and then adjust the pH of the PBS solution to 8.5 - 9.5 with the prepared sodium bicarbonate. In addition, prepare 10 mg / mL Cy3 in DMSO (protected from light), mix well, take 10 μL and add it to the prepared OH-CATH30 solution, shake and react at 37 °C for 1 h. After the reaction, use a salt precipitation column to remove excess salt ions. Freeze-dry the collected Cy3-labeled OH-CATH 30. Prepare the fluorescently labeled OH-CATH 30.

[0078] Determination of the standard curve: Prepare freeze-dried Cy3-labeled OH-CATH 30 with different concentrations (2000 μg / mL, 1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL), and take 200 μL respectively to measure the absorbance at an excitation wavelength of 550 nm and an emission wavelength of 570 nm.

[0079] Encapsulation efficiency detection: Dissolve 10 mg of CNGs in 1 mL of an aqueous solution of fluorescently labeled OH-CATH 30 (3 mg / mL, 5 mg / mL, 7 mg / mL, 9 mg / mL). After homogenization, swell and adsorb at room temperature for 24 h, and then centrifuge twice (5000 g, 30 min). Collect the supernatant and freeze-dried CSNGs respectively. After diluting the supernatant to 4 mL, take 200 μL each to measure the absorbance at an excitation wavelength of 550 nm and an emission wavelength of 570 nm, and calculate the encapsulation efficiency according to the standard curve data. Each concentration has three replicate wells.

[0080] Drug loading detection: Weigh 1 mg of freeze-dried fluorescently labeled CSNGs at each concentration and dissolve it in 1 mL of purified water. After thorough mixing, take 200 μL from each solution and measure the absorbance at an excitation wavelength of 550 nm and an emission wavelength of 570 nm. Calculate the drug loading according to the standard curve data, with three replicate wells for each concentration.

[0081] As Figure 2 shown, at 7 mg / mL, the drug loading and encapsulation efficiency of OH-CATH 30 are the highest, representing the optimal ratio.

[0082] 3. Particle size, zeta potential, and transmission electron microscopy characterization

[0083] Inject 1 mL of CSNGs into the particle size cup and zeta potential cup, and use a Malvern laser particle size analyzer to measure the particle size distribution and zeta potential of the nanoparticles. This experiment was repeated 3 times. As Figure 3 shown, the hydrodynamic diameter of CNGs is approximately: 168.8 nm with a negative charge; the hydrodynamic diameter of CSNGs is approximately: 221.6 nm with a positive charge.

[0084] Drop the CNGs and CSNGs solutions onto a 200-mesh copper grid respectively. After standing for 3 min, remove the excess solution with filter paper, stain with 3% uranyl acetate for 5 min, then remove the excess liquid with filter paper, and observe and take pictures under a transmission electron microscope. As Figure 4 shown, the morphology of CNGs is approximately round with a diameter of about 77.8 nm, while CSNGs have a larger diameter, about 116.2 nm.

[0085] 4. Infrared spectroscopy characterization

[0086] Use the solid potassium bromide tablet method for characterization. Take an appropriate amount of potassium bromide powder and the sample to be tested, place them in an infrared drying oven to dry, then thoroughly grind the potassium bromide in a mortar until it becomes a fine powder. Use a jack and mold to prepare a transparent potassium bromide tablet, measure the infrared spectrum, and set it as the background. Mix the dried gelatin, inulin, GNPs, CNGs, and CSNGs with potassium bromide powder at a ratio of 1:100 respectively, and thoroughly grind until homogeneous, then measure the infrared spectrum. The detection wavenumber is 4000 - 500 cm -1 , and the result of each detection is the average of 36 detections. The results show that ( Figure 5 ), compared with the amide I band showing C=N and C=O stretching vibrations at 1637 cm -1 , once glutaraldehyde crosslinks to form nanogels of GNGs, the C=O stretching vibration slightly shifts to 1649 cm -1 , indicating that glutaraldehyde crosslinks with two amino groups to form an imine. Secondly, the characteristic peaks of inulin include the stretching vibration peak of CH2 at 2926 cm -1 and the stretching vibration peak of C-O-C at 1026 cm -1The bending vibration peak at . After the inulin was co-assembled and cross-linked into CNGs, these two characteristic peaks remained unchanged in the CNGs at 2931 cm -1 and 1026 cm -1 . Two strong stretching vibration peaks of C-O and C-N of OH-CATH 30 in CSNGs also appeared at 1205 cm -1 and 1136 cm -1 . At the same time, the absorption peaks of multiple in-plane bending vibration peaks of CH2 near 722 cm -1 might be shielded during the formation of the nanogel. These results indicate that inulin and OH-CATH 30 were successfully loaded into the nanogel to form CSNGs.

[0087] 5. Detection of the release behavior of CSNGs

[0088] The cumulative release amount of OH-CATH 30 in CSNGs was determined by centrifugation and fluorescence methods. Accurately weigh 4 portions of 7 mg of Cy3 fluorescently labeled CSNGs, mix them thoroughly with 1 mL of distilled water, and centrifuge at 5000 g for 20 min. After collecting the supernatant, take 7 mL of simulated vaginal fluid (SVF) with gelatinase concentrations of 0 U, 1 U, 5 U, and 10 U, and resuspend the nanoparticles. The release system was stirred at 100 rpm in a 37 °C constant temperature water bath, and 300 μL of the release medium was taken at 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 18 h, and 24 h, respectively. After the release solution was centrifuged at 5000 g for 20 min, 200 μL of the supernatant was taken to measure the fluorescence intensity, and 200 μL of fresh simulated vaginal fluid was added to the remaining 100 μL of the release solution. Take 300 μL of the fresh release culture medium, mix it evenly, and return it to the 7 mL release system. Record the fluorescence intensity at each time point and calculate the cumulative release amount. Each group was repeated 3 times. It can be seen from Figure 6 that gelatinase eroded the outer shell of CSNGs in a dose-dependent manner within the first 10 hours to rapidly release OH-CATH 30. The cumulative release of OH-CATH 30 was 63% within 10 hours, and the release amount remained at about 70% within 24 hours.

[0089] The cumulative release amount of inulin in CSNGs was determined by centrifugation and the phenol-sulfuric acid method. Four portions of 7 mg CSNGs were accurately weighed into 1 mL of distilled water, thoroughly mixed, centrifuged at 5000 g for 20 min, and the supernatant was collected. The nanoparticles were resuspended in 7 mL of simulated vaginal fluid (SVF) with gelatin concentrations of 0 U, 1 U, 5 U, and 10 U. The release system was stirred at 100 rpm in a constant temperature water bath at 37 °C, and 300 μL of the release medium was taken at 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 18 h, and 24 h, respectively. After centrifuging the release solution at 5000 g for 20 min, 200 μL of the supernatant was taken to measure the fluorescence intensity, and 200 μL of fresh simulated vaginal fluid was added to the remaining 100 μL of the release solution. 300 μL of the fresh release culture medium was mixed evenly and returned to the 7 mL release system. The phenol-sulfuric acid method was used to determine the inulin content at each time point, and the cumulative release amount was calculated. Each group was repeated 3 times. From Figure 7 It can be seen that compared with OH-CATH 30, inulin was slightly released within the first 10 hours, and the maximum release amount was less than 35%. Under the action of gelatinase for more than 10 hours, the inulin in the core of CSNGs showed the characteristics of accelerated release. When incubated for 24 h, the cumulative release amount was as high as over 73%. The difference in the release time of OH-CATH 30 and inulin in CSNGs in the gelatinase buffer (10 U) provided a good cascade release system for the treatment of aerobic vaginitis (AV).

[0090] 6. Verification of the selective bactericidal effect of CSNGs

[0091] Clindamycin (10 mg / ml, 5 mg / ml, 2.5 mg / ml, 1.6 mg / ml, 0.8 mg / ml, 0.4 mg / ml) and CSNGs (400 μg / ml, 200 μg / ml, 100 μg / ml, 50 μg / ml, 25 μg / ml, 12.5 μg / ml) were prepared with sterile water respectively, and were incubated with Staphylococcus aureus culture solution (1×10 6 CFU / mL per well) at 37 °C for 2 h, 6 h, and 12 h. At different times, the culture solutions diluted 10 2 , 10 3 , 10 4 times were taken respectively, and 100 μL was evenly spread on the mannitol solid medium and incubated for counting. The experiment was set with 3 replicates in total. The results are as Figure 8 shown. Clindamycin had a slow bactericidal rate at the MBC concentration (2500 μg / mL), and no colony regeneration occurred after 6 h. Different from the action of antibiotics, CSNGs could kill most bacterial cells within 2 h, and the MBC (100 μg / mL) was much lower than that of clindamycin.

[0092] A co-culture model was established to simulate vaginal microbiota imbalance and verify the effects of CSNGs on pathogenic and beneficial bacteria under environmental imbalance. Staphylococcus aureus (1×10 6 CFU / mL) and Lactobacillus crispatus (1×10 5 CFU / mL) were co-cultured in LB liquid medium. After treatment with CSNGs, clindamycin, and a mixture of clindamycin and inulin respectively, the changes in bacterial viability over time were measured at 2 h, 6 h, 12 h, and 24 h. As Figure 9 and Figure 10 shown, at concentrations of 0.4 - 10 mg / mL, clindamycin gradually inhibited the growth of Staphylococcus aureus and Lactobacillus crispatus with increasing concentration gradient. Even though inulin was initially added to promote the proliferation of Lactobacillus crispatus, the results showed no significant increase in the number of bacteria. For the CSNGs we prepared, at the MIC (50 μg / mL) and MBC (100 μg / mL), Staphylococcus aureus was inhibited or killed, with a survival rate of less than 10%. Meanwhile, at 24 h, the colony number of Lactobacillus crispatus increased significantly. With the complete elimination of Staphylococcus aureus in CSNGs (100 μg / mL), the delayed release of inulin more effectively promoted the proliferation of Lactobacillus crispatus, with a proliferation rate of over 50% at 24 h. The results indicate that CSNGs can effectively and selectively kill the pathogenic bacterium Staphylococcus aureus and then slowly support the growth of the beneficial bacterium Lactobacillus crispatus under in vitro simulated vaginal microbiota disorders.

[0093] 7. In vitro cytotoxicity and biocompatibility of CSNGs

[0094] The cell viability of macrophages RAW264.7 (representative of immune cells) and renal tubular epithelial cells (HK-2 cells, representative of normal cells) was detected using the MTT method. First, macrophages (RAW 264.7) and human renal tubular epithelial cells (HK-2) (8000 cells / well) were cultured in a 96-well plate for 19 hours. RAW 264.7 cells were incubated with OH-CATH 30, CNGs, and CSNGs respectively, and HK-2 cells were co-incubated with CSNGs. Using a mixture of clindamycin and inulin as a positive control, after incubation in a CO2 incubator at 37 °C for 24 h, MTT solution (10 μL) was added to the 96-well plate and incubated for 4 hours, and then removed from the medium. 150 μL of DMSO was added to each well, and after mixing, the absorbance was measured at 490 nm.

[0095] The cell viability calculation formula is as follows: Cell viability (%) = OD 剂量组 / OD 对照组 ×100%

[0096] where OD 剂量组OD values for CSNGs, clindamycin, and clindamycin + inulin groups, and the OD value of the OD control group is the OD value of DMSO.

[0097] The results are as Figure 11 shown. First, after RAW264.7 was co-incubated with Cath30, CNGs, and CSNGs for 24 h at concentrations of 0.25×, 0.5×, and 1× MBC (100 μg / ml), the survival rate of RAW264.7 cells was above 80%. In contrast, clindamycin and the mixture of clindamycin and inulin showed significant dose-dependent cytotoxicity, and the cytotoxicity was less than 20% when treated at its MBC concentration against Staphylococcus aureus. As Figure 12 shown, even when the concentration of CSNGs was as high as 4× MBC, there was no obvious cytotoxicity to HK-2, while the group of clindamycin and the mixture of clindamycin and inulin showed cytotoxicity to HK-2 cells even at 0.5× MBC.

[0098] An erythrocyte hemolysis experiment was performed on the prepared CSNGs to detect its biosafety. Blood was collected from the ear vein of Japanese white rabbits, centrifuged at 1000 rpm for 5 min, and a 2% erythrocyte suspension was prepared with physiological saline. 500 μL of CSNGs with different concentrations (6.25, 12.5, 25, 50, 100, 200 μg / ml) was prepared with physiological saline and mixed with 500 μL of erythrocyte suspension, and physiological saline and Triton X-100 (1%, v / v) were used as negative and positive controls. After incubation at 37 °C for 3 h, a photo was taken, centrifuged at 3000 rpm for 10 min, and the absorbance of the supernatant was measured at 570 nm. All experiments were repeated 3 times. The results are as Figure 13 shown. Even at 200 μg / mL, CSNGs had good biocompatibility.

[0099] 8. Verification of the in vivo effect of CSNGs

[0100] Using rats with aerobic bacterial vaginitis as a model, the dual-functional effect of CSNGs nanogel was evaluated to study its therapeutic effect. After the model was successfully established, SD rats were divided into 4 groups (10 rats in each group). They were administered with physiological saline, CSNGs, clindamycin, and clindamycin + inulin respectively. For 7 consecutive days, once a day. After the model was successfully established and 7 days after administration, vaginal mucus was taken for bacterial culture and counting. The results are as Figure 14 shown in a, 14b. After modeling, the number of Staphylococcus aureus in the vagina was twice that of Lactobacillus crispatus, while 7 days after administration, after treatment with our nanodrug and the mixture of clindamycin and inulin, Staphylococcus aureus was significantly eliminated, which was one-tenth of the infection situation, and returned to the normal level of the vaginal microenvironment ( Figure 14c). It should be noted that based on the time-controlled release of inulin by CSNGs, the number of Lactobacillus crispatus significantly proliferated to more than 2200 CFU / mL, which was more than 2.7 times that of the infection condition. On the contrary, clindamycin and the mixed group showed that even in the presence of inulin, the probiotics were inhibited( Figure 14 d).

[0101] Inflammatory response detection was carried out as Figure 15 shown. After 7 days of treatment, the vaginal tissues of rats were ground and centrifuged to collect the supernatant, and the detection was carried out according to the instructions of the enzyme-linked immunosorbent assay kit. It was found that CSNGs could reduce the inflammatory levels of IL-6 and TNF-α in the vaginal tissues. The regulatory effect of CSNGs on IL-6 in the infected tissues was better than that on TNF-α, and the regulatory effect on TNF-α was better than that of the free clindamycin group and the clindamycin and inulin mixed group, indicating that CSNGs had a significant regulatory effect on the body's immune system stimulated by bacterial antigens. The histological inflammation of the vagina treated with CSNGs hydrogel was further evaluated by hematoxylin and eosin (H&E) staining( Figure 16 ). It was shown that compared with the CSNGs group, the clindamycin group, and the clindamycin + inulin group, the control group had inflammatory infiltration, characterized by a large number of small round dark blue-stained inflammatory cells, the shedding of its tissue epithelial mucosa, and epithelial keratinization. The thickness of the multi-layered squamous epithelium of the mucosa in the clindamycin treatment group was significantly thinned, and the mucosa was completely shed. The CSNGs group had fewer inflammatory cells and a normal multi-layer density of the mucosa.

[0102] The results of the bacterial content in the tissue were as Figure 17 shown. We used fluorescence in situ hybridization technology to label the mRNA of Staphylococcus aureus (green fluorescence) and Lactobacillus crispatus (red fluorescence) in the vaginal tissues. As expected, compared with other groups, there were a large number of red fluorescences and a small number of green fluorescences in the tissues of the CSNGs group, and a large number of green fluorescences and a small number of red fluorescences in the control group. This conclusion was consistent with the conclusion of the previous vaginal fluid bacterial count after treatment. Through the above verification at the animal level, the CSNGs designed by us had good effects in vivo.

[0103] In summary, the present invention prepared a co-loaded bifunctional nanogel of an antibacterial peptide and a prebiotic through a series of experiments. The prepared nanogel had significant bacterial specificity and time-controllable release characteristics, and could release the antibacterial peptide and the prebiotic successively; at the same time, the prepared nanogel could be applied to the preparation of a biodegradable preparation for anti-bacterial infection and promoting the rapid recovery of lactic acid bacteria microecology.

[0104] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions required to be protected by the present invention.

Claims

1. A co-loaded bifunctional nanogel of an antimicrobial peptide and a prebiotic, characterized in that, The nano-gel is prepared by the following steps: (1) Prepare an aqueous gelatin solution, an aqueous solution of antibacterial peptide OH-CATH 30, and an aqueous inulin solution respectively, and filter them; the sequence of the antibacterial peptide OH-CATH 30 is KFFKKLKNSVKRAKKFFKPRVIGVSIPF; (2) Mix the aqueous gelatin solution and the aqueous inulin solution, stir evenly, and then add acetone; after stirring, add glutaraldehyde, continue stirring to obtain a mixed solution, then continue to add glycine, stir, centrifuge, take the lower layer to obtain gelatin-inulin nano-gel, wash it and then freeze-dry it to obtain a freeze-dried powder of CNGs; (3) Mix the freeze-dried powder of CNGs with the aqueous solution of antibacterial peptide OH-CATH 30, let it stand, centrifuge, take the lower layer, wash it and then pipette it evenly to obtain a co-loaded bifunctional nano-gel of antibacterial peptide and prebiotic; The concentration of the aqueous gelatin solution is 8 mg / mL - 12 mg / mL; the concentration of the aqueous inulin solution is 20 mg / mL - 100 mg / mL, and the aqueous gelatin solution and the aqueous inulin solution are mixed in equal volumes; The concentration of the antibacterial peptide OH-CATH30 solution is 3.0 mg / mL - 9.0 mg / mL.

2. The co-loaded dual-functional nanogel of the antimicrobial peptide and the prebiotic according to claim 1, wherein The gelatin used to prepare the aqueous gelatin solution in step (1) is a high molecular weight gelatin prepared by a desolvation method.

3. The co-loaded bifunctional nanogel of the antimicrobial peptide and prebiotic according to claim 1, characterized in that, The pH of the aqueous gelatin solution in step (1) is 5.8 - 6.

2.

4. The co-loaded bifunctional nanogel of the antimicrobial peptide and prebiotic according to claim 1, wherein The concentration of glycine added in step (2) is 90 mmol / L - 110 mmol / L; the volume of glycine added is the same as the volume of the mixed solution.

5. The co-loaded bifunctional nanogel of the antimicrobial peptide and prebiotic according to claim 1, characterized in that, The standing condition in step (3) is to stand at room temperature for 20 h - 30 h.

6. The co-loaded bifunctional nanogel of the antimicrobial peptide and prebiotic according to claim 1, wherein The morphology of the CNGs is: round; the CSNGs have a core-shell structure.

7. Use of the co-loaded bifunctional nano-gel of antibacterial peptide and prebiotic according to any one of claims 1 - 6 in the preparation of a biodegradable preparation for anti-bacterial infection and promoting the growth of probiotics.

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