Composition for treating bacterial infection wound and preparation method thereof

Abstract

The invention discloses a composition for treating bacterial infection wounds and a preparation method of the composition. The composition comprises hydrogel and lipidosome nano bubbles which are loaded on the hydrogel and used for adsorbing MRSA toxins and responding to light treatment, and the lipidosome nano bubbles are loaded on the hydrogel and used for adsorbing MRSA toxins and responding to light treatment. The fat extract is loaded on the hydrogel and is used for promoting wound repair; the method comprises the following steps: S1, preparing lipidosome nanobubbles NB with phototherapy response; s2, preparing a fat extract FE; and S3, loading NB and FE onto the hydrogel to complete the preparation. According to the scheme, the problem that the light treatment efficiency is reduced due to diffusion of lipidosome nano bubbles can be solved, the problem that healthy tissue is damaged due to light treatment can be relieved, and meanwhile wound healing can be assisted.

Description

Technical Field

[0001] The invention relates to biomedicine, in particular to a composition for treating bacterial infection wounds and a preparation method thereof. Background Art

[0002] Methicillin-resistant Staphylococcus aureus (MRSA) is a common pathogen in clinical practice that can cause a series of organ-specific infections. In addition, MRSA can also spread to blood-borne infections through local wound infections, causing bone and joint infections, pneumonia, sepsis, infective endocarditis, and even septic shock. However, the multidrug resistance of MRSA makes it impossible to obtain good treatment results with a series of antibiotics in clinical practice, and the development speed of new antibiotics is much lower than the resistance speed of MRSA, so the development of non-antibiotic therapies has become a new option for MRSA treatment.

[0003] Liposome nanobubbles developed based on MRSA virulence factor adsorption and phototherapy have shown good prospects in the treatment of MRSA infection: through virulence factor adsorption, liposome nanobubbles can reduce the level of MRSA exotoxins at the infection site, promote the host immune system to recognize bacteria and improve the clearance ability; further, under external near-infrared light, liposome nanobubbles generate photothermal and photodynamic reactive oxygen species (ROS) to directly kill bacteria. However, liposome nanobubbles also have some shortcomings in the treatment of local MRSA infection, which mainly include three points: (1) The essence of liposome nanobubbles is nanoparticles. After local administration at the MRSA infection site, they will diffuse rapidly due to size problems, thereby affecting the efficiency of phototherapy; (2) The use of phototherapy to eliminate bacteria generally requires higher photothermal and photodynamic doses, which can easily cause off-target damage to healthy tissues around the infection. (3) In addition to removing bacteria, rapid healing of bacterially infected wounds is also an important factor in the treatment of infection. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a composition for treating bacterially infected wounds and a preparation method thereof, which can improve the problem of reduced phototherapy efficiency caused by the diffusion of liposome nanobubbles, alleviate the problem of healthy tissue damage caused by phototherapy, and assist wound healing.

[0005] To achieve the above object, the present invention provides the following technical solution: a composition for treating bacterial infection wounds, comprising

[0006] Hydrogel,

[0007] Liposome nanobubbles loaded on hydrogels and used to adsorb MRSA toxins and respond to light therapy;

[0008] Fat extract, loaded on hydrogel, is used to promote wound repair.

[0009] As a further improvement of the present invention, the hydrogel is a temperature-sensitive F-127 hydrogel.

[0010] Also provided is a method for preparing a composition for treating bacterially infected wounds, comprising the following steps:

[0011] S1. Preparation of liposome nanobubbles NB with phototherapy response;

[0012] S2. Preparation of fat extract FE;

[0013] S3. Load NB and FE onto the hydrogel to complete the preparation.

[0014] As a further improvement of the present invention, the hydrogel in S3 is F-127 hydrogel.

[0015] As a further improvement of the present invention, S3 includes dissolving F-127 powder in the NB and FE mixed solution to form a hydrogel G (NB / FE) loaded with NB and FE.

[0016] As a further improvement of the present invention, the ratio of the NB and FE mixed solution to the F-127 powder is 2 mL:234.6 mg, and the F-127 ratio in the obtained hydrogel G (NB / FE) is 19% (w / v), the NB lipid concentration is 2.5 mg / mL, and the FE is 0.32 mg / mL.

[0017] As a further improvement of the present invention, the fat extract FE in S2 is prepared by the following steps:

[0018] S21. Obtain human fat tissue and wash it to remove red blood cells;

[0019] S22. centrifuging the human adipose tissue and collecting the middle fat layer for mechanical emulsification;

[0020] S23. The product after mechanical emulsification is frozen and thawed, and separated by centrifugation;

[0021] S24. Collect the third layer product to complete the preparation of fat extract FE.

[0022] The beneficial effect of the present invention is that liposome nanobubbles are loaded into the hydrogel to reduce the diffusion of nanobubbles after injection at the infected site, ensuring that the adsorption of virulence factors and phototherapy achieve the best efficiency; at the same time, the fat extract loaded in the hydrogel is used to reduce nonspecific damage during phototherapy and accelerate the repair of damaged tissue. Therefore, the hydrogel system loaded with phototherapy nanobubbles and lipid extracts can simultaneously achieve the purpose of treating and repairing bacterial infection wounds, and the effect of reducing diffusion can promote the improvement of treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The hydrodynamic size, surface potential and transmission electron microscopy characterization diagram of the NB of the present invention;

[0024] Figure 2 The scanning electron microscope characterization images of G(NB) and G(NB / FE) of the present invention;

[0025] Figure 3 The rheological properties and FE cumulative release diagram of G(NB / FE) of the present invention;

[0026] Figure 4 The photothermal and photodynamic performance diagrams of G(NB / FE) of the present invention;

[0027] Figure 5 This is a diagram showing the in vitro bactericidal effect of G(NB / FE) combined with NIR irradiation of the present invention;

[0028] Figure 6 This is a diagram showing the neutralization effect of G(NB / FE) of the present invention on hemolytic toxins and cytotoxic toxins in sMT;

[0029] Figure 7 This is a graph showing the photodynamic antibacterial efficacy of sMT-enhanced G(NB / FE) of the present invention;

[0030] Figure 8 This is a graph showing the ability of G(NB / FE) of the present invention to alleviate oxidative stress;

[0031] Fig. 9 This is a graph showing the long retention of G(NB / FE) of the present invention at the infected part of the skin;

[0032] Fig.10 This is a diagram of the present invention's G(NB / FE) treating and repairing MRSA skin infection under NIR irradiation. DETAILED DESCRIPTION

[0033] The present invention will be further described below in detail with reference to the embodiments shown in the accompanying drawings.

[0034] Reference Figure 1-10 As shown,

[0035] 1. Preparation and Characterization of Liposome Nanobubbles (NB)

[0036] IR780 was dissolved in 10 mL of chloroform at a ratio of 1 wt% to 2 mg of sphingomyelin. The chloroform was then evaporated using a rotary evaporator and resuspended in 10% (w / v) sucrose. PFC was then added (5 μL of PFC per mg of lipid) and dispersed by sonication in an ice bath for 10 min using a probe sonicator (80 W, 2 seconds on, 3 seconds off). 20 mL of oxygen (O2) was then injected into the NB. The prepared NB was subjected to basic physical characterization, including determination of the hydrodynamic size and surface potential ( Figure 1 -a) and the surface morphology was observed using a transmission electron microscope (TEM) ( Figure 1 -b). The results showed that NB exhibited a core-shell structure, with a hydrodynamic size of about 265nm and a surface potential of about 15mV.

[0037] 2. Obtaining Lipid Extract (FE) and Determining Its Protein Concentration

[0038] Human adipose tissue was collected from cesarean section scars. The adipose tissue was first rinsed with saline to remove red blood cells, and then the middle fat layer was collected by centrifugation at 1200×g for 3 min and mechanically emulsified. The emulsification process was performed by two 10 cm 3 The syringe was connected by a female-to-female Luer-Lok connector and passed back and forth 30 times. Subsequently, the emulsified fat was frozen at -80°C / thawed at 37°C to further disrupt the adipose tissue. After one freeze-thaw cycle, the adipose tissue was centrifuged again at 1200×g for 5 min. At this time, the adipose tissue was separated into 4 layers, and the third layer, the fat extract (FE), was collected. Finally, FE was passed through a 0.22μm filter. FE was stored at -20°C until use. The protein concentration of FE was determined using the Pierce BCA protein assay kit.

[0039] 3. Construction and characterization of thermosensitive F-127 hydrogels co-loaded with NB and FE (G(NB / FE))

[0040] 234.6 mg of F-127 powder was dissolved in 2 mL of NB / FE mixture and dissolved overnight at 4°C to form G(NB / FE) hydrogel (the corresponding F-127 ratio was 19% (w / v), of which NB (lipid concentration) was 2.5 mg / mL and FE was 0.32 mg / mL). The preparation of G(NB) or G(FE) hydrogel was also carried out in the same way. Subsequently, a series of characterizations of G(NB / FE) hydrogel were performed, including: (1) After freeze-drying the hydrogel, the basic morphology of G(NB / FE) hydrogel and G(FE) hydrogel was observed by scanning electron microscopy. (2) The rheological properties of G(NB / FE) hydrogel were detected by hybrid rheometer. (3) The release law of FE from G(NB / FE) hydrogel was determined. The scanning electron microscopy results showed that NB particles existed in G(NB / FE) hydrogel, while G(FE) hydrogel only showed the void structure of hydrogel without NB particle distribution ( Figure 2 The rheological properties of G(NB / FE) hydrogels show that the hydrogels have good temperature sensitivity and can transform from liquid to gel at 37°C, which meets the requirements of subsequent applications ( Figure 3 -a). The results of FE in vitro release experiments showed that FE can be released in time during phototherapy to exert its biological effects ( Figure 3 -b).

[0041] 4. Phototherapy function of G(NB / FE) hydrogel

[0042] First, the photothermal properties of G(NB / FE) hydrogel were measured. 2 The G(NB / FE) hydrogel was irradiated with a 300W NIR light source (power, 15min) and the temperature change was monitored every 1min using Fotric 322pro thermal imaging. The PBS+NIR irradiation group served as the experimental control group. The results showed that the G(NB / FE) hydrogel maintained a good temperature under NIR irradiation, while the PBS group had no obvious photothermal effect. Figure 4 -a)

[0043] Secondly, the photodynamic properties of G(NB / FE) hydrogel were determined. The present invention uses a ROS indicator (DCFH-DA) to determine the ROS generation ability of G(NB / FE) hydrogel under NIR light. The general process is: use 200μL of DCFH-DA (10μM) to measure the ROS generation of G(NB / FE) hydrogel containing 10μg / mL (IR780 concentration) under irradiation of 808nm laser (1min). NB+NIR laser alone, G(NB / FE) hydrogel alone and NB alone were used as control groups. The results show that G(NB / FE) hydrogel has higher photodynamic properties than NB nanobubbles alone ( Figure 4 -b)

[0044] 5. The killing effect of G(NB / FE) hydrogel on MRSA bacteria in vitro

[0045] 2×107 CFU / mL of MRSA dispersed in TSB medium was mixed with G(NB / FE) hydrogel (containing 10μg / mL of IR780) to 200μL, and then irradiated with 808nm laser at 1.0W / cm2 for 3min. The obtained 10μL bacterial suspension was plated on TSB agar plate, and the colonies were counted after incubation at 37°C for 24h. MRSA treated with G(FE)+NIR, NB+NIR or PBS+NIR laser was used as control. The results showed that G(NB / FE) hydrogel had a stronger antibacterial effect due to its higher photothermal and photodynamic properties ( Figure 5 ).

[0046] 6. G(NB) hydrogel alleviates sMT-induced hemolysis and cytotoxicity

[0047] First, collect the toxins (sMT) secreted by MRSA: inoculate the culture fluid of MRSA (USA300) onto TSB agar plates and incubate at 37°C for 24 hours. Then, transfer a single colony to 4 mL of TSB and continue incubating at 37°C for 12 hours. Then, add 200 mL of fresh TS and culture at 37°C at 180 rpm / min for 24 hours. Subsequently, centrifuge the bacterial solution at 5000 × g for 10 minutes, filter the supernatant with a filter membrane (0.2 μm) and collect it. Finally, freeze-dry the collected filtrate and reconstitute it with water to 1 / 10 of the initial volume (i.e., sMT).

[0048] Subsequently, the adsorption and neutralization effect of G(NB) hydrogel on sMT was tested. In the red blood cell (RBC) hemolysis protection experiment, whole blood of ICR mice was obtained by mandibular puncture, centrifuged at 1000×g for 5 minutes, and RBC was separated. Then, 4.7vol% sMT was pre-incubated with G(NB) hydrogel (containing 2mg / mL NB) and added to 100μL of RBC (5vo1%) to a final volume of 150μL, and incubated at 37°C for 30min. Subsequently, the sample was centrifuged at 12000×g for 5min, and the supernatant was collected, and hemoglobin was detected at a wavelength of 540nm. NB liposomes of the same concentration were used as a control. The results show that G(NB / FE) hydrogel can effectively adsorb hemolytic toxins in sMT and alleviate hemolysis of red blood cells ( Figure 6 -a).

[0049] In the cytotoxicity protection experiment, HUVECs, L929 and VK-2 cells were first cultured in a 96-well plate to 80% confluence. At the same time, 5 vol% sMT was mixed with G(NB) hydrogel (containing 2 mg / mL NB) to 100 μL culture medium, and the mixture was allowed to stand at 37°C for 1 hour and then centrifuged at 12000×g for 10 minutes. The supernatant was collected and added to the three cell lines respectively and incubated for 6 hours, and then the cell viability was determined using the CCK-8 kit. Cells treated with culture medium alone (media), sMT only, and NB+sMT were used as the control groups. The results showed that G(NB / FE) hydrogel can effectively adsorb the relevant toxins in sMT and alleviate cytotoxicity ( Figure 6 -b).

[0050] 7. Regulating the photodynamic bactericidal effect of G(NB / FE) hydrogel using sMT

[0051] 2×10 7 After mixing MRSA with CFU / mL and G(NB / FE) hydrogel (containing 10 μg / mL IR780), 8 μL of sMT was added to 200 μL and the mixture was sterilized by 808 nm laser at 1.0 W / cm 2 Irradiate for 3 minutes. Then, 10 μL of bacterial suspension was plated on TSB agar and incubated at 37°C for 24 hours. Finally, the number of bacterial colonies was counted. MRSA treated with PBS+NIR, sMT+NIR and G(NB / FE) hydrogel+NIR were used as controls. The results showed that sMT can improve the MRSA killing effect of G(NB / FE) hydrogel ( Figure 7 The main reason why sMT improves the bactericidal effect of phototherapy is that sMT can be adsorbed by NB nanobubbles in G(NB / FE) hydrogel, thereby responsively increasing the release of oxygen and photosensitizer, improving the photodynamic effect under the irradiation of external light, and improving the bactericidal efficiency.

[0052] 8. Resistance of G(NB / FE) hydrogel to cellular oxidative stress

[0053] First, tBHP (100 μm) was used to induce HUVECs (70% confluence in a 96-well plate) into an oxidative stress state, and then G(NB / FE) hydrogel was added and incubated for 4 hours. After incubation, the ROS generation capacity of G(NB / FE) hydrogel under NIR light was measured using a ROS indicator (DCFH-DA). Then, the green signal was detected using a fluorescence microscope. Image J software was used for analysis. At the same time, after the oxidative stress protection treatment, the cells were stained with Calcein-AM. Finally, the fluorescence intensity was quantitatively determined. In both tests, only culture medium, only FE, and G(NB) hydrogel were added after tBHP treatment of HUVECs as controls ( Figure 8 ). The results showed that G(NB) hydrogel delivery retained the pro-angiogenic activity of FE and the ability to prevent cell damage caused by oxidative stress.

[0054] 9. Long-term retention of G(NB / FE) hydrogel in MRSA skin infection

[0055] Subcutaneously inject 100 μL of 5 × 10 7 CFU / mL of MRSA was injected into the right back of mice to establish a skin infection model. After 24 hours, G(NB / FE) hydrogel (containing 100μg / kg of NB) was subcutaneously injected into the infection site, and the IVIS system (PerkinElmer, IVIS lumina III) was immediately used to detect the IR780 signal at different excitation / emission time points (0, 1, 3, 6min). 100μg / kg of NB was used as a control. The results showed that compared with NB, G(NB / FE) hydrogel can better retain NB in ​​MRSA skin infection sites and prolong the local concentration of NB ( Fig. 9 ).

[0056] 10.G(NB / FE) hydrogel for treating MRSA skin infection under NIR irradiation

[0057] To test the ability of G(NB / FE) hydrogel to treat MRSA infection in vivo, 100 μL of 5×10 7 CFU / mL, and a MRSA skin infection model was established. 24 h after infection, NB, G(NB) hydrogel or G(NB / FE) hydrogel (all containing 0.65 mg / kg IR780) were given in situ, and then NIR laser (808 nm, 1.0 W / cm 2 , 5min) were treated respectively. Mice treated with PBS+NIR irradiation were used as controls. The size of skin lesions in each group of mice was detected at different times (1, 2, 4, 6, 8, 10, 12, and 14d). On the 14th day, all mice were euthanized, and the collected lesions were fixed with 4% paraformaldehyde for bacterial counts. The results showed that the MRSA bacterial content in the skin infected tissue treated with G(NB / FE) hydrogel was the lowest, and the skin lesions recovered better ( Fig.10 -a). In order to better study the dynamic repair effect of G(NB / FE) hydrogel on MRSA-infected wounds over time, we studied inflammatory factors, new blood vessels, and collagen deposition on the 3rd and 14th days. The results showed that G(NB / FE) hydrogel showed the lowest IL-6 expression and the highest VEGF expression on the 3rd day after treatment ( Fig.10-b), while at day 14, the G(NB / FE) hydrogel treatment group also showed lower collagen deposition, the lowest TNF-α expression, and the highest VEGF expression ( Fig.10 -c). In summary, under NIR irradiation, G(NB / FE) hydrogel can effectively kill bacteria and accelerate wound healing.

[0058] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A composition for treating bacterial infection wounds, characterized in that: include Hydrogel, Liposome nanobubbles loaded on hydrogels and used to adsorb MRSA toxins and respond to light therapy; Fat extract, loaded on hydrogel, is used to promote wound repair.

2. The composition for treating bacterial infection wounds according to claim 1, characterized in that: The hydrogel is a temperature-sensitive F-127 hydrogel.

3. A method for preparing a composition for treating bacterially infected wounds, characterized in that: The steps include: S1. Preparation of liposome nanobubbles NB with phototherapy response; S2. Preparation of fat extract FE; S3. Load NB and FE onto the hydrogel to complete the preparation.

4. The method for preparing the composition for treating bacterial infection wounds according to claim 3, characterized in that: The hydrogel in S3 is F-127 hydrogel.

5. The method for preparing the composition for treating bacterial infection wounds according to claim 4, characterized in that: S3 includes dissolving F-127 powder in a mixed solution of NB and FE to form a hydrogel G (NB / FE) loaded with NB and FE.

6. The method for preparing the composition for treating bacterial infection wounds according to claim 5, characterized in that: The ratio of the NB and FE mixed solution to the F-127 powder was 2 mL:234.6 mg, and the F-127 ratio in the obtained hydrogel G (NB / FE) was 19% (w / v), the NB lipid concentration was 2.5 mg / mL, and the FE was 0.32 mg / mL.

7. The method for preparing the composition for treating bacterial infection wounds according to claim 3, 4, 5 or 6, characterized in that: The fat extract FE in S2 is prepared by the following steps: S21. Obtain human fat tissue and wash it to remove red blood cells; S22. centrifuging the human adipose tissue and collecting the middle fat layer for mechanical emulsification; S23. The product after mechanical emulsification is frozen and thawed, and separated by centrifugation; S24. Collect the third layer product to complete the preparation of fat extract FE.

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