Application of slow-release biological composite nano-film in resistance to pseudomonas aeruginosa infection

Abstract

The invention discloses an application of a slow-release biological composite nano-film in resisting pseudomonas aeruginosa infection, and relates to the technical field of medicines.The technical key points are as follows: step S1, preparing a nano-emulsion with the concentration of 1% (w / v) by adopting a phase transformation method, the nano-emulsion is prepared from the following components: chlorhexidine acetate, a Tween 80 surfactant, a propylene glycol solvent and isopropyl myristate, s2, adding polyvinyl alcohol into the nano-emulsion until the final concentration reaches 10% (w / v), stirring for several times, and continuously stirring for 6 hours at 200rpm until the nano-emulsion is completely swelled; s3, adding chitosan into the nano-emulsion containing 5% of PVA at the concentration of 1% (w / v), and stirring for 10 minutes at the speed of 200rpm to obtain a mixed matrix; and step S4, adding a Glucocone 600CS UP solution under slight stirring until a clear and transparent fluid film forming system is formed. The antibacterial agent has a faster and better antibacterial effect, and can better promote healing and inhibit inflammation in a pseudomonas aeruginosa wound infected mouse model.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to the application of a sustained-release bio-composite nano-film in the treatment of Pseudomonas aeruginosa infection. Background Art

[0002] Pseudomonas aeruginosa (PA), also known as Pseudomonas aeruginosa, is a Gram-negative bacillus of the genus Pseudomonas. It is an opportunistic pathogen that causes severe drug-resistant infections in critically ill and elderly patients, and has currently become one of the drug-resistant bacteria with a relatively high infection rate in high-risk populations such as critically ill and elderly patients. When patients have low immunity due to various reasons, it is more likely to cause infections of the skin, respiratory tract, etc.

[0003] Wound infections caused by multi-drug resistant (MDR) Pseudomonas aeruginosa are a severe challenge facing global public health. The widespread existence of antibiotic resistance and the transient nature of antibiotic activity in wounds urgently require innovative treatment strategies with strong antibacterial synergy and sustained-release formulations. Summary of the Invention

[0004] The purpose of the present invention is to solve the above problems and provide the application of a sustained-release bio-composite nano-film in the treatment of Pseudomonas aeruginosa infection.

[0005] To achieve the above purpose, the technical solution of the present invention is as follows:

[0006] The present invention provides a method for preparing a sustained-release bio-composite nano-film, comprising the following steps:

[0007] Step S1: Prepare a nano-emulsion with a concentration of 1% (w / v) by the phase inversion method. The nano-emulsion contains the following components: chlorhexidine acetate, Tween 80 surfactant, propylene glycol solvent, and isopropyl myristate;

[0008] Step S2: Add polyvinyl alcohol (PVA) to the nano-emulsion to make its final concentration reach 10% (w / v). After stirring in portions, continuously stir at 200 rpm for 6 hours until the nano-emulsion is completely swollen;

[0009] Step S3: Add chitosan to the nano-emulsion containing 5% PVA at a concentration of 1% (w / v), and stir at 200 rpm for 10 minutes to obtain a mixed matrix;

[0010] Step S4: Add Glucopone 600CS UP solution under gentle stirring until a clear and transparent fluid film-forming system is formed.

[0011] The present invention is further provided that in step S2, the degree of polymerization of polyvinyl alcohol is 600 - 800, the viscosity is 4 - 6 mPa·s, and the degree of alcoholysis is 88% ± 2%.

[0012] The present invention is further configured such that in step S3, the degree of acetylation of chitosan is 85%, the viscosity is 20 - 200 mPa·s, and the molecular weight is 50 - 190 kDa.

[0013] The present invention also provides the application of the sustained-release biocomposite nanofilm in the treatment of Pseudomonas aeruginosa infection.

[0014] Compared with the prior art, the beneficial effects of the present solution are as follows: In the present invention, a novel nanofilm system CNF is designed and developed based on the nanoemulsion and CS-PVA liquid film system, which has high stability and good quality characteristics. In addition, the nanofilm can continuously release the loaded drug in simulated wound fluid. In vitro and in vivo results show that the toxicity of the nanofilm is reduced. Importantly, we found that this novel sustained-release system not only has a faster and better antibacterial effect, but also can better promote healing and inhibit inflammation in a mouse model of Pseudomonas aeruginosa wound infection. These results indicate that this novel multifunctional nanofilm system with improved antibacterial effect and continuous drug release has good clinical application prospects. Description of the Drawings

[0015] Figure 1 It is a schematic diagram of the design of a novel antibacterial nano-membrane system for accelerating wound healing in the embodiment of the present invention;

[0016] Figure 2 It shows the film-forming and physical properties of the nanofilm system in the embodiment of the present invention. (A) Appearance, (B) Film-forming ability, (C) Size distribution, (D) Zeta potential distribution, (E) SEM observation of the surface morphology, 1–3 correspond to magnification factors of 7.5, 15, and 30k respectively;

[0017] Figure 3 It is the morphology of the novel nanofilm system observed by transmission electron microscopy in the embodiment of the present invention. (A, C) Magnification factor of 15k, (B, D) Magnification factor of 30k, (C, E) Magnification factor of 60k for Q22;

[0018] Figure 4 It is the structure and stability characterization of the novel nanofilm system in the embodiment of the present invention. (A) DSC, (B) TG. (C) FITR, (D) Size change (n = 3), (E) PdI change (n = 3), (F) Zeta potential change (n = 3);

[0019] Figure 5 It is the drug release curve, antibacterial effect against Pseudomonas aeruginosa, and toxicity test of the novel nanofilm system in the embodiment of the present invention. (A) Release efficiency in simulated wound fluid; (B) Minimum inhibitory concentration, (C) Time-bacteriostatic curve, (D) Acute toxicity test in L929 cell line;

[0020] Figure 6 It is the in vivo toxicity evaluation of the nano-film system in the embodiments of the present invention. Images of H&E-stained skin tissues (200×) after 14 days of treatment;

[0021] Figure 7 It is the cytokine levels in Pseudomonas aeruginosa-infected wounds in the embodiments of the present invention. Detection of the levels of IL-β (A, E), TNF-α (B, F), IL-6 (C, G), and IL-10 (D, H) in serum 12 and 24 h after bacterial inoculation (n = 3);

[0022] Figure 8 It is the in vivo antibacterial activity of the nano-film system against Pseudomonas aeruginosa-infected skin wounds. (A) Results of local wound bacterial colonization (n = 5), (B) wound healing curve (n = 5), (C) illustration of wound size changes and H&E staining results (100×). Results of continuous treatment for 0, 1, 3, 5, 7, and 14 days are presented, and H&E staining images of skin tissues after 7 days of continuous treatment. Detailed implementation manners

[0023] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments and drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0024] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0025] Embodiment:

[0026] 1. Materials

[0027] 1.1 Strains, animals, and ethical statements

[0028] Pseudomonas aeruginosa PAO1 was purchased from ATCC, USA and stored at -80 °C. It was cultured using LB liquid culture dishes, which were purchased from China Aoboxing Biotechnology Co., Ltd. CFU was determined by the plate counting method on LB nutrient agar (China Aoboxing Biotechnology Co., Ltd.). SPF-grade BALB / c mice (6 - 8-week-old females) were purchased from Beijing Huafukang Biotechnology. The animals were housed in the SPF laboratory of the Third Military Medical University (24 °C, 50% humidity, 12 h light-dark cycle). Before the experiment, the animals were anesthetized with sodium pentobarbital and sacrificed by CO2 asphyxiation. The animal experiment was approved by the Animal Ethics Committee of the Third Military Medical University (AMUWEC2020973).

[0029] 1.2 Design and Preparation of the Novel Nanomembrane System

[0030] The 1% (w / v) nanoemulsion was prepared by the phase inversion method, containing chlorhexidine acetate (CHX, Jinzhou Jiutai Pharmaceutical), Tween 80 (Sinopharm Group), propylene glycol (Sinopharm Group), and isopropyl myristate (IPM, BASF). Subsequently, polyvinyl alcohol (PVA, polymerization degree 600 - 800, Chongqing Meijiameng Technology) was added at a ratio of 10% (w / v), and it was stirred in cold water multiple times until completely swollen. 1% chitosan (CS, Sigma, deacetylation degree 85%) was added to the mixture of 5% PVA and 1% nanoemulsion, and it was stirred at 200 rpm for 10 minutes. Auxiliary materials were gradually added to form a transparent and flowing film-forming system. The blank nanomembrane (BNF) did not contain drugs.

[0031] 1.3 Observation of the Film-Forming Characteristics of the Nanomembrane

[0032] The transparency and color of the 5 mg / mL CHX nanomembrane (CNF) were observed under natural light on a white background. 50 μL of the sample was dropped onto a glass slide and dried at 37 °C to evaluate the film-forming ability. The stability of the solution over time and the film thickness were observed. After diluting the sample with 1% aqueous solution, the hydrodynamic size distribution, PdI, and zeta potential were measured using a Malvern Nano ZS. The sample diluted 100-fold with water was observed for its morphology using a FEI Tecnai 10 transmission electron microscope (120 kV) and a Hitachi S-3400N scanning electron microscope.

[0033] 1.4 Physicochemical Stability of the Nanomembrane System

[0034] The physicochemical stability was evaluated. TG / DSC analysis was performed at a rate of 10 °C / min in a nitrogen atmosphere (TA Q600). The characteristic absorption peaks were detected using a Perkin Elmer Lambda 950 FTIR spectrometer (400 - 4000 cm-1) after pressing the sample into a KBr tablet. Unstable phenomena such as precipitation and turbidity were observed through 6 centrifugation cycles (3000×g, 10 minutes, alternating between 4 - 25 °C). The particle size, PdI, and zeta potential were measured after the sample was stored at room temperature for 0, 6, 12, and 24 months.

[0035] In vitro release kinetics of 1.5 nm film

[0036] HPLC (Waters E2695) was used to determine the drug release in simulated wound fluid (SWF). The CNF and CHX solutions (containing 5 mg / mL of the drug) were placed in a dialysis bag (MWCO 12 kDa) and immersed in 50 mL of SWF (pH 7.4), and shaken at 37 °C and 100 rpm. Samples were taken at regular intervals and an equal volume of medium was replenished, and the released amount was detected by HPLC.

[0037] In vitro antibacterial effect of 1.6 nm film

[0038] The minimum inhibitory concentration (MIC) was determined by the microdilution method. The PAO1 bacterial suspension (106 CFU / mL) and a series of concentrations of CNF / CHX (final concentration 50 - 3.125 μg / mL) were added to each well of a 96-well plate. After incubation at 37 °C for 16 h, the absorbance at 600 nm (OD < 0.05 indicates no visible growth) was measured. The positive control was PAO1, and the blank controls were BNF and LB. According to the MIC value (2x MIC - 0.5x MIC)

[26] , the time-kill experiment was carried out to detect the bactericidal effect of 3.12 - 12.5 μg / mL CNF / CHX at different time points (1 - 640 minutes). Samples were taken, diluted and spread on LB plates, and counted using an automatic colony counter (Hangzhou Shengsheng Technology). BNF was used as the blank control.

[0039] In vitro cytotoxicity of 1.7 nm film

[0040] Referring to ISO10993-5, L929 cells were seeded in a 96-well plate (1×104 / well), and medium containing CHX / CNF (1000 - 15.625 μg / mL) was added respectively. After culturing for 24 / 48 h, the cell viability was determined by the CCK-8 method.

[0041] In vivo toxicity of 1.8 nm film

[0042] Referring to the ISO10993 guidelines, a 1 cm skin incision was made on the back of mice, which were randomly divided into the CHX (5 mg / mL), CNF (5 mg / mL) and control groups (n = 5). 100 μL of the drug was administered locally every day for 14 days. The heart, liver, spleen, lung, kidney and skin tissues were taken, fixed with 4% paraformaldehyde, embedded in paraffin, stained with H&E, and observed under an optical microscope (Olympus BX53).

[0043] 1.9 Wound infection model and treatment

[0044] Twenty-four mice were randomly divided into the PA infection group, CHX group, CNF group and BNF group (n = 6). After anesthesia with pentobarbital, the hair on the back was removed, and a 1×1 cm 2Full-thickness incision. All mouse wounds were inoculated with 200 μL of PAO1 bacterial suspension (2×109 CFU / mL) for 30 seconds, and 100 μL of the treatment was administered locally 10 minutes later for 30 seconds.

[0045] 1.10 Cytokine levels in infected wounds

[0046] Mice were sacrificed at 12 / 24 h after drug administration (n = 3), blood was collected to isolate serum, and the levels of IL-1β, TNF-α, IL-6, and IL-10 were detected using an ELISA kit (Beijing Dakwei Biotechnology).

[0047] 1.11 Wound bacterial load

[0048] On the 4th day, the wound surface was wiped 10 times with a sterile cotton swab and placed in a saline centrifuge tube for shaking. After gradient dilution of the sample, it was spread on LB agar and incubated for 16 - 20 h to count CFU.

[0049] 1.12 Wound healing rate and pathological changes

[0050] The wound areas on the 1st, 4th, 7th, 10th, and 14th days were recorded (analyzed by ImageJ). On the 7th day, the wound tissues of randomly selected mice were fixed with 10% formaldehyde for 24 h, embedded in paraffin, sectioned (5 μm), and stained with H&E to evaluate skin morphology and collagen synthesis.

[0051] 1.13 Statistical analysis

[0052] Data analysis was performed using GraphPad Prism 8.0.1. Data are expressed as mean ± SD or SEM, and t-tests or one-way ANOVA (Newman-Keuls test) were used for intergroup comparisons. *P < 0.05, **P < 0.01, ***P < 0.001.

[0053] 2. Results

[0054] 2.1 Design and preparation of the novel nanofilm system

[0055] Using CHX as the active drug, CNF was successfully developed based on the nanoemulsion and PVA-CS film system, endowing the nanofilm with antibacterial and wound-healing functions ( Figure 1 ).

[0056] 2.2 Appearance and film-forming property of the nanofilm

[0057] CNF (0.5%, 5 mg / mL) was clear ( Figure 2 A), and the formed film was complete without defects ( Figure 2 B). The film-forming time was 2 minutes in an oven at 37°C and could be maintained for 5 hours.

[0058] 2.3 Nanoparticle size and zeta potential of the nanofilm

[0059] The average particle size of CNF measured by Nano ZS was 59.71 nm (PdI = 0.266), with a single main peak at 43.2 nm( Figure 2 C). The zeta potential was -15.2 mV, pH was 6.30, conductivity was 0.131 mS / cm, and electrophoretic mobility was -1.211 μm / v( Figure 2 D).

[0060] 2.4 Surface Morphology of the Nanomembrane

[0061] SEM showed that the nanoemulsion particles aggregated and were wrapped by a polymer sheath, and the average pore size of the macroporous structure was 1 - 100 nm( Figure 2 E). At 7500 times magnification, the ridged structure on the particle surface was shown( Figure 2 E-1), and the particles were clearer at 15000 and 30000 times magnification( Figure 2 E-2,3).

[0062] 2.5 TEM Morphology of the Nanomembrane

[0063] TEM showed that after 100-fold dilution of the CNF aqueous solution, the drug appeared as black particles at 15000 times magnification( Figure 3 A,D), and the fibrous structure was shown at 30000 times magnification( Figure 3 B,E), and the network structure was presented at 60000 times magnification( Figure 3 C,F), indicating that the particles were evenly dispersed.

[0064] 2.6 Structural Characterization of the Nanomembrane

[0065] DSC showed that the CHX aqueous solution had a characteristic peak at 74.43 °C, and the peak of CNF shifted at 88.61 °C( Figure 4 A). TG analysis showed that the maximum weight loss rate temperatures of CHX and CNF were 73.81 °C and 92.56 °C respectively( Figure 4 B). FTIR showed that CNF had characteristic peaks of O-H stretching, C-H stretching and amide I at 3420, 2080, 1640 nm( Figure 4 C), confirming the successful loading of the drug.

[0066] 2.7 Stability of the Nanomembrane

[0067] After 6 centrifugation cycles (3000×g, 10 minutes, alternating at 4 - 25 °C), no flocculation, delamination or precipitation occurred. The particle size was 48.99 - 66.51 nm after 24 months of storage at room temperature( Figure 4 D), PdI was 0.14 - 0.24( Figure 4 E), and the zeta potential was -14.9~-10.49 mV( Figure 4 F), indicating good stability (P>0.05).

[0068] 2.8 In Vitro Release Kinetics of the Nanomembrane

[0069] The release of CNF in SWF is slow, and the release amount in 0.5 h is < 25%, while the release of CHX reaches 90% in 0.5 h (P < 0.001)( Figure 5 A), showing the significant sustained-release characteristics of CNF.

[0070] Antibacterial effect of 2.9-nanometer film in vitro

[0071] The MIC of CNF is 5 μg / mL, which is better than that of CHX (10 μg / mL)( Figure 5 B). The time-killing curve shows that 12.5 μg / mL CNF can completely kill bacteria in 45 minutes, while the same concentration of CHX takes 2 hours( Figure 5 C). 6.25 μg / mL CNF can completely kill bacteria in 8 hours, and 3.13 μg / mL CNF can inhibit bacteria for 16 hours, which is significantly better than CHX.

[0072] Cytotoxicity of 2.10-nanometer film in vitro

[0073] CNF has no obvious toxicity to L929 cells when ≤ 62.5 μg / mL, and the cell survival rate of 125 μg / mL CNF (48.32%) is significantly higher than that of CHX (76.21%) (P < 0.001)( Figure 5 D), indicating that the nanofilm system improves biocompatibility.

[0074] Toxicity of 2.11-nanometer film in vivo

[0075] H&E staining shows that there is no significant toxicity in the skin tissue of the CNF group, and no pathological changes are seen in the main organs (heart, liver, spleen, lung, kidney)( Figure 6 ), suggesting that the nanofilm components have a protective effect.

[0076] Changes in cytokines of infected wounds

[0077] CNF significantly reduces the levels of serum IL-1β, TNF-α, and IL-6 at 12 / 24 h after infection (P < 0.05) and increases the level of IL-10( Figure 7 ), showing that the anti-inflammatory effect is better than that of CHX.

[0078] Wound bacterial load and healing rate

[0079] CNF (5 mg / mL) significantly reduces the bacterial load on the wound surface( Figure 8 A). The starting time of scab formation (2.66 days) is shortened by 1.75 times compared with that of CHX (4.66 days), the complete scab formation time (7 days) is shortened by 1.26 times, and the starting time of scab shedding (9.8 days) is shortened by 1.4 times (P < 0.05)( Figure 8 B).

[0080] 2.14 Pathological evaluation of wound healing

[0081] The wound area in the CNF group was the smallest on the 3rd / 5th day, significantly reduced on the 7th day, and completely healed on the 14th day( Figure 8 C). H&E showed a significant reduction in inflammatory infiltration in the dermis of the CNF group, while severe inflammatory reactions and skin damage occurred in the CHX group.

[0082] The above specific embodiments are only explanations of the present invention, and they are not limitations to the present invention. After reading this specification, those skilled in the art can make modifications without creative contributions to this embodiment as needed, but as long as it is within the scope of the claims of the present invention, it is protected by the patent law.

Claims

1. A preparation method of a sustained-release biological composite nanofilm, characterized in that, It includes the following steps: Step S1: Prepare a nanoemulsion with a concentration of 1% (w / v) by the phase inversion method. The nanoemulsion contains the following components: chlorhexidine acetate, Tween 80 surfactant, propylene glycol solvent, and isopropyl myristate; Step S2: Add polyvinyl alcohol to the nanoemulsion to make its final concentration reach 10% (w / v). After stirring in batches, continuously stir at 200 rpm for 6 hours until the nanoemulsion is completely swollen; Step S3: Add chitosan to the nanoemulsion containing 5% PVA at a concentration of 1% (w / v), and stir at 200 rpm for 10 minutes to obtain a mixed matrix; Step S4: Add Glucopone 600CS UP solution under gentle stirring until a clear and transparent fluid film-forming system is formed.

2. The preparation method of a sustained-release biological composite nanofilm according to claim 1, characterized in that: In step S2, the degree of polymerization of polyvinyl alcohol is 600–800, the viscosity is 4-6 mPa·s, and the degree of alcoholysis is 88% ± 2%.

3. The preparation method of a sustained-release biological composite nanofilm according to claim 1, characterized in that: In step S3, the degree of acetylation of chitosan is 85%, the viscosity is 20-200 mPa·s, and the molecular weight is 50-190 kDa.

4. Use of the sustained-release bio-composite nanofilm according to any one of claims 1-3 in the treatment of Pseudomonas aeruginosa infection.

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