CN110917339A - Lysostaphin gel and application thereof in MRSA infected wound surface - Google Patents

Abstract

The invention discloses a lysostaphin gel and a preparation method thereof, wherein the gel comprises lysostaphin and a gel matrix, and the gel matrix comprises 25-40% of poloxamer, 5-25% of glycerol and water. The gel provided by the invention is stable, maintains the activity of lysostaphin, is used for treating MRSA infected wounds, and has an obvious effect.

Description

Lysostaphin gel and application thereof in MRSA infected wound surface

Technical Field

The invention belongs to the field of medicines, and particularly relates to a lysostaphin gel and application thereof in treatment of MRSA infected wounds.

Background

With the widespread, high-volume use of antibiotics, more and more staphylococcus aureus produces penicillinase, resulting in the emergence of a large number of methicillin-resistant staphylococcus aureus (MRSA). MRSA has now become one of the major highly toxic pathogenic bacteria of nosocomial infections. Patients with systemic or local MRSA infections in burn areas, ICU, respiratory wards, hematology and pediatrics become one of the most problematic problems for clinical treatment.

Rebuilding or restoring the skin barrier is the ultimate goal of wound healing, and a wound cover with excellent performance can temporarily play a part of the role of the skin barrier function, and provides an environment beneficial to wound healing. The ideal wound dressing should effectively prevent bacterial infection, especially methicillin-resistant staphylococcus aureus (MRSA), improve the success rate of skin grafting and accelerate wound healing, and simultaneously should have good biocompatibility, control and absorb wound seepage, be suitable for gas and vapor permeation, protect new tissues, accelerate wound healing and other characteristics. The existing dressings are various and are roughly divided into three types: 1. inert dressings are traditional dressings, such as gauze, but gauze cannot keep the wound moist for a long time, the wound is possibly delayed to heal, and soaked dressings are easy to pass pathogens to cause secondary infection. In addition, dressing fiber is easy to fall off, foreign body reaction is caused, and the healing of the wound surface is influenced. 2. A dual-phase acting dressing of the type which can promote wound healing by virtue of the local environment it creates: (1) semi-permeable membrane (plastic dressing), an absorbent membrane made of polyurethane casein material, wherein one side of the dressing is added with adhesive material; (2) polyurethane foam; (3) hydrogel dressing: is a three-dimensional net-shaped water-absorbing polymer consisting of gelatin, polysaccharide, various electrolyte compounds and methacrylic resin. The double-phase acting dressing generally has the defects of poor water absorption, poor adhesion capability with the wound surface and poor anti-infection capability. 3. Bioactive dressing (sealing dressing) is also called as sealing dressing because the dressing can be closely adhered with the wound surface to prevent the wound surface from drying. It is a dressing that maintains a moist environment in the wound, and its absorbency is related to its structure. It can act with locally applied medicine and endogenous molecules of the body to promote wound healing, but has poor bactericidal effect on bacterial infected wound surface, and cannot effectively treat the wound surface.

The dressing has the defects that pathogenic bacteria causing wound infection cannot be effectively killed, or wound seepage cannot be effectively controlled and absorbed, and the like. Therefore, the development of an external preparation for treating and repairing infected wounds, which can effectively sterilize, especially MRSA, and simultaneously can effectively control and absorb the wound exudate, protect the new tissues and accelerate the wound healing, is very urgent.

The vancomycin has a definite treatment effect on systemic infection, and for local wound infection, the effect of controlling infection can be increased by jointly using the lysostaphin on the basis of using the vancomycin, but the rapid degradation and inactivation of the lysostaphin in vitro become a great problem of the lysis of wound MRSA.

CN101721691A A biological preparation for treating and repairing infected wound and its preparation method. The biological preparation consists of lysostaphin 0.1-30 wt%, chitosan or chitosan derivative 3-50 wt% and collagen 30-95 wt%. The biological agent uses collagen and chitosan as gel auxiliary materials, has higher manufacturing cost, higher content of lysostaphin and uneconomical product.

Aiming at the MRSA-infected osteomyelitis, artificial bones made of nano hydroxyapatite and chitosan are designed as carriers to adsorb lysostaphin, so that the lysostaphin is slowly released in the MRSA-infected osteomyelitis, and a continuous sterilization effect is achieved. For MRSA infectious wound surfaces after wounds such as burns, a slow release carrier suitable for popularization is not reported at present. The inventor aims to develop a disinfectant preparation which is used on wound surfaces, can keep the activity of enzyme to a certain degree, continuously and slowly releases lysostaphin, has low manufacturing cost and is easy to popularize. After a large amount of research, experiments and screening, the combination of poloxamer and glycerol is finally found to prepare a temperature-sensitive novel gel preparation, and the lysostaphin is attached to the gel preparation, so that the lysostaphin can be slowly released in vitro and stably exert a sterilization effect.

Disclosure of Invention

The invention aims to provide a staphylococcus lysozyme preparation which is stable in vitro, in particular to a staphylococcus lysozyme gel. The results of in vitro experiments and mouse MRSA infection wound surface experiments prove that the novel gel preparation can stabilize the activity of lysostaphin, maintain the curative effect of the lysostaphin on cracking MRSA in vivo and in vitro, has obvious effect and has high clinical application value.

The staphylococcus lysozyme gel provided by the invention comprises staphylococcus lysozyme and a gel matrix, wherein the gel matrix comprises poloxamer and glycerol.

Preferably, the lysostaphin gel provided by the invention comprises 25-40% of poloxamer, 5-25% of glycerol and the balance of water. More preferably, the gel matrix consists of 30-35% poloxamer, 10-25% glycerol and the balance water, most preferably poloxamer 18835%, glycerol 10% and water.

If the pH of the gel is not suitable, a small amount of acid or base may be added to adjust the pH to around 6.8.

In another embodiment, the lysostaphin gel of the present invention has a lysostaphin content of 2.5 to 20U/ml, and preferably, a lysostaphin content of 10U/ml. Most preferably, the lysostaphin gel is poloxamer 18835% (mass fraction) + glycerol 10% (volume fraction) + pure water + lysostaphin 10U/ml.

The application of the lysostaphin gel in preparing the medicament for treating MRSA infected wound surfaces.

The invention also provides a method for preparing the staphylococcus lysozyme gel, which comprises the following steps:

(1) weighing poloxamer, adding glycerol and double distilled water (namely pure water), shaking, mixing uniformly, and standing at low temperature to form a gel solution;

(2) adjusting the pH value of the gel solution after low-temperature standing to about 6.8;

(3) adding appropriate amount of lysostaphin, and heating in 37 deg.C water bath;

(4) and cooling after uniform mixing to obtain the lysostaphin gel.

In the method of the present invention, the poloxamer is poloxamer 188.

Drawings

FIG. 1. Effect of different ratios of gels on lysostaphin activity;

FIG. 2 OD in different time points of gel-enzyme, disinfectant 1, disinfectant 2 and simple gel (-) on MRSA in vitro lysis test₆₀₀Comparing the numerical values;

FIG. 3 in vitro lysis effects of gel-enzyme, disinfectant 1, disinfectant 2, and 75% medical alcohol and simple gel on MRSA and clinical isolates;

FIG. 4. effect of lysostaphin gel on the action of lysostaphin;

FIG. 5 shows the therapeutic effect of lysostaphin gel of the present invention on mouse MRSA-infected wounds.

Detailed Description

The following examples are merely representative for understanding and illustrating the nature of the invention, and are not intended to limit the scope of the invention in any way.

Materials and methods of the following examples:

animal and main reagent and instrument

18 SPF-grade Balb/c female mice, with a week age of 8-10 weeks and a body weight of 20-22g, were purchased from Tianqin Biotechnology Ltd, Changsha, license number SCXK (Xiang) 2019-. Yeast extract and tryptone were obtained from Oxoid, UK, agar powder from Changsheng biotechnology, Inc., Beijing ancient cooking, and sodium chloride from Biotechnology engineering (Shanghai) Inc. Preparing a liquid LB culture medium: 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, adding double distilled water to 1000mL, sterilizing by high pressure steam, preparing an LB liquid culture medium, and storing at 4 ℃; semi-solid LB medium: adding 7.5g of agar powder into 1000ml of LB liquid culture medium, sterilizing by high-pressure steam, and storing in an oven at 55 ℃; solid LB medium: 15g of agar powder is added into 1000ml of LB liquid culture medium, and the mixture is sterilized by high-pressure steam and then stored in an oven at 4 ℃.

Bacterial strains and lysostaphin sources:

the MRSA standard strain was given by the institute for burn of the general institute of burn, southwest hospital of third military medical science, and 12 clinical isolates were isolated from the clinical laboratory of the hospital affiliated to Zunyi medical university, which contained ATCC29213 and ATCC 25923. Lysostaphin was provided by professor luchim, the microbiological laboratory of Shandong university (provided by Jining Baishi microbial technology, Inc.).

EXAMPLE 1 screening of gel matrices

New gel prepared from poloxamer (188) and glycerol

The formula of the gel suspension is as follows:

A. 10ml of 10% glycerol, 35% poloxamer (188) and 10ml of double distilled water;

B. 10ml of 10% glycerol, 40% poloxamer (188) and 10ml of double distilled water;

C. 25% glycerol + 30% poloxamer (188) + 10ml double distilled water;

D. 20% of glycerol, 35% of poloxamer (188) and 10ml of double distilled water;

E. 30% glycerol, 25% poloxamer (188) and 10ml double distilled water;

F. 30% glycerol + 30% poloxamer (188) + 10ml double distilled water.

The preparation process comprises the following steps:

weighing poloxamer (188) and glycerol according to mass percent, adding 10ml of double distilled water, mixing to obtain a suspension, shaking up to obtain a suspension under vigorous shaking, adjusting the pH value to about 6.8 with acid or alkali, and storing in a refrigerator at 4 ℃ overnight (>12 h). The suspension was observed to be in a fluid gel form every other day, and was placed in a 37 ℃ water bath with the water line exceeding the suspension. The time of the different constituent gels and the texture of the gels were observed. The gel was left at room temperature, and the state of the suspension at room temperature was observed. We obtained the best ratio of the effect of the 3-component glue.

TABLE 1 observation of gel quality phenomena for each formulation group

The results in table 1 found that 3 gel-like compositions were the most ideal, and are respectively a formulation: 10% glycerol + 35% poloxamer (188), C formula 25% glycerol + 30% poloxamer (188), D formula: 20% glycerol + 35% poloxamer (188), followed by formula B: 10% glycerol + 40% poloxamer (188). Formulations E and F were difficult to gel.

EXAMPLE 2 Staphylolytic enzyme gels

The A, B, C, D, E gel matrix of example 1 was prepared by adding 900ul of gel into 1.5ml EP tube at 4 deg.C, adding 100ul of lysostaphin at 100U/ml, making the concentration of the gel-enzyme to be 10U/ml, preparing 5U/ml and 2.5U/ml gel-enzyme preparations by the same method, pouring MRSA double agar plate, and adding 3 groups of gel-enzyme preparations at final concentrations of 10U/ml, 5U/ml and 2.5U/ml, respectively, selecting the optimum ratio of the enzyme cleavage effect for the subsequent experiments according to the transparency and diameter of the zone, observing the size and transparency of the zone, finding that the gel ratio of the gel effect and the lysis activity is ①②③, wherein the lysostaphin gel of group ③, 10% glycerol and 35% poloxamer (188) was the most stable and the best.

① 20% glycerol + 35% poloxamer (188) + lysostaphin 10%;

② 25% glycerol + 30% poloxamer (188) + lysostaphin 10%;

③ 10% glycerol + 35% poloxamer (188) + lysostaphin 10%;

FIG. 1 shows that the gel proportion of ①②③, ①% of glycerol, 35% of poloxamer (188) + lysostaphin 10%, ②% of glycerol, 30% of poloxamer (188) + lysostaphin 10%, ③% of glycerol, 35% of poloxamer (188) + lysostaphin 10% and ③, which shows that the bacteriostatic circle has the largest diameter, the highest transparency and the best bacteriostatic effect, according to FIG. 1.

EXAMPLE 3 lysostaphin-188 in vitro lysis assay

Taking 1.5ml of MRSA bacterial liquid mixed by shaking of 4 tubes of single bacterial colonies per tube,16000g, centrifuge for 2min, add 900ul PBS per tube to resuspend the bacteria, label separately: enzyme-188, xiao 1, xiao 2, (-) -; 100ul of enzyme-188 is added into the enzyme-188 tube, the final concentration is 10U/ml, and 100ul of disinfectant 1, disinfectant 2 and PBS are respectively added into the disinfectant 1, disinfectant 2 and (-) tubes. Placing into a shaking table at 37 ℃ for incubation; taking the mixed liquid for 0-60 min to detect OD₆₀₀The value is obtained. As shown in FIG. 2, OD of enzyme-188 group₆₀₀The drop over time is significantly better than the other groups.

Example 4 gel-in vitro cleavage of MRSA and clinical isolates

Single colonies of MRSA and 12 clinical isolates were picked up in 2ml liquid LB medium at 180RPM, 37 ℃ and shake-cultured overnight. And (3) respectively sucking 200ul of the subnatal bacterial liquid which is turbid into the test tubes, adding 3-5ml of semisolid culture medium, and uniformly mixing on a vortex oscillator for 10s to prepare a double-layer agar plate. The best gel ratio of the schizomycete effect obtained from the previous experiment is 10% glycerol + 35% poloxamer (188), and the following gels are all used in the ratio. 10ul of gel-enzyme preparation with the concentration of 10U/ml, disinfectant 1, disinfectant 2, gel without enzyme and 75% medical alcohol are respectively dripped into different areas of the double-layer agar plate of the strain. Two clinically common skin disinfectants were used for the positive control: the disinfectant 1 is gel hand sanitizer (the components are 0.22 to 0.26 percent of trichloro hydroxyl diphenyl ether and 50 to 60 percent of ethanol), and the disinfectant 2 is liquid sanitary hand sanitizer (the components are 0.25 to 0.30 percent of chlorhexidine acetate, 70 to 75 percent of ethanol and 0.04 to 0.06 percent of benzalkonium bromide); negative controls used a gel of 75% alcohol and non-solubilized staphylococcal enzyme. As a result, it was found that the gel-enzyme was able to cleave MRSA as well as 12 clinical isolates, and the cleavage effect was more complete than that of disinfectant 1. As shown in fig. 3. In this test, the concentration of lysostaphin was increased to 100U/ml, and the rest was kept constant, and 10ul of the gel-enzyme lysis solution was added dropwise to the MRSA double-layer agar plate in the same manner as described above, and it was found that the gel-enzyme lysis effect was more complete. The results are shown in figure 3, the in vitro lysis effect of gel-enzyme, disinfectant 1, disinfectant 2, 75% medical alcohol and simple gel on MRSA and clinical isolates is shown in the upper left graph, which is a graph of the effect of gel-enzyme lysis MRSA strains with the concentration of 10U/ml; the upper right graph is a graph of the effect of gel-enzyme lysis of MRSA strains at a concentration of 100U/ml; ATCC29213 is shown in the lower left, middle and right panels, respectively; staphylococcus aureus 1201; ATCC 25923. As a result, it was found that the gel-enzyme was able to cleave MRSA as well as 12 clinical isolates (only part of the picture is shown) with a more complete cleavage effect than disinfectant 1.

EXAMPLE 5 Effect of the novel gel on the stability of lysostaphin

To examine whether the novel gel has an influence or a protective effect on the lysostaphin lysis effect, the following four experiments were performed on the following lysostaphin gel (hereinafter referred to as "gel-enzyme") prepared from 10% glycerol + 35% poloxamer (188) + 10U/ml lysostaphin.

Firstly, 200ul of MRSA bacterial liquid is added into 3-5ml of semisolid culture medium, a double-layer agar plate is poured, gel-enzyme and lysostaphin (the concentration is 10U/ml) are respectively dripped, and the size and the transparency of two groups of antibacterial zones are observed. The results showed that the zone of inhibition was comparable in size and clarity with no significant difference between the two groups, indicating that the gel-enzyme did not reduce the lytic potency of the lysostaphin (see FIG. 4A)

Then gel-enzyme and lysostaphin (both at a concentration of 10U/ml) were placed at room temperature, 4 ℃ and-20 ℃ for 12h, respectively. Adding 200ul of MRSA bacterial liquid into 3-5ml of semi-solid culture medium, and pouring into a double-layer agar plate. Dripping 10ul of lysostaphin and gel-enzyme at different temperatures in different areas, and observing the diameter and the transparency of the inhibition zone after 4-6 h. Gel-enzyme and lysostaphin (both at 10U/ml) were taken 15ul separately in EP tubes, 3 tubes per group, and treated at 37 deg.C, 40 deg.C and 50 deg.C for 15 min. Then 10ul of the treated enzyme and enzyme products are dripped on an MRSA double-layer agar plate, and the size and the brightness of the inhibition zone are observed after 4-6 h. The results are shown in FIG. 4C, after treatment at 37 deg.C, 40 deg.C and 50 deg.C for 15min, the inhibition zone of the lysostaphin group after temperature treatment is turbid, the inhibition zone can still be seen at 37 deg.C, and the inhibition zone after treatment at 40 deg.C and 50 deg.C is quite turbid, and only faint spots can be seen with naked eyes; in the gel-enzyme group, clear inhibition zones can still be seen after treatment at different temperatures. Therefore, the novel gel is used as a support for lysostaphin, so that the thermal stability of the lysostaphin can be strongly increased, and the activity of the lysostaphin in a certain temperature range is ensured to be less influenced.

And (3) dropwise adding the equivalent gel-enzyme and lysostaphin subjected to repeated freeze thawing on an MRSA double-layer agar plate, and observing the sizes and the transparencies of the two groups of inhibition zones, wherein freeze thawing is usually performed in the use process of the enzyme. Repeated freeze-thaw experiments: storing gel-enzyme and lysostaphin in refrigerator at 4 deg.C and-20 deg.C respectively, simulating repeated freeze thawing during use, taking out from 4 deg.C and-20 deg.C, standing at room temperature for 5-10min, storing at 4 deg.C and-20 deg.C for more than 6h, taking out again to room temperature, standing for 5-10min, repeating freeze thawing for 3-5 times, and detecting their enzyme activities. As shown in fig. 4D, the bacteria-splitting effect of the gel-enzyme group is not significantly reduced after repeated freeze thawing, while the transparency of the zone of inhibition is reduced after repeated freeze thawing of lysostaphin, which indicates that the enzyme-splitting efficiency is affected during repeated freeze thawing. Therefore, the novel gel has the function of protecting the enzyme activity in the repeated freezing and thawing process

To examine the influence of the gel on the enzyme, an equivalent amount of a lysostaphin gel preparation (hereinafter referred to as gel-enzyme) and lysostaphin at a concentration of 10U/ml was dropped onto each of the MRSA double-layer plates.

The gel-enzyme and the lysostaphin are stored at different temperatures in a short time to simulate the conditions of transportation and short-term storage, two groups of enzyme preparations containing the same amount of enzyme are stored at room temperature, 4 ℃ and-20 ℃ for 12 hours respectively, and then the two groups of enzyme preparations with the same amount are dripped into corresponding grids on an MRSA double-layer agar plate, so that the results show that the diameters of two groups of inhibition zones are equivalent, but the boundaries of the inhibition zones processed under different conditions of the gel-enzyme groups are clearer than those of the lysostaphin groups, the boundaries of the inhibition zones of the lysostaphin groups are fuzzy, and surrounding bacteria tend to expand towards the middle of the inhibition zones (fig. 4B). Therefore, under the same short-term storage condition, the addition of the novel gel of the invention is beneficial to stabilizing the activity of the enzyme. The results in FIG. 4 show that: FIG. 4A gel-enzyme and lysostaphin, both of which are 10U/ml, are respectively added dropwise to an MRSA double-layer agar plate, and the sizes and transparencies of the inhibition zones are equivalent; FIG. 4B shows that two groups of enzyme preparations containing equal amounts of enzymes are stored at room temperature, 4 ℃ and-20 ℃ for 12h respectively, and then the two groups of enzyme preparations with equal amounts are dripped into corresponding grids on an MRSA double-layer agar plate, and the diameters of the inhibition zones are equal, but the boundaries of the inhibition zones of a gel-enzyme (i.e. the lysostaphin gel of the invention) group processed under different conditions are clearer than those of the lysostaphin group, the boundaries of the inhibition zones of the lysostaphin group are fuzzy, and peripheral bacteria tend to expand towards the middle of the inhibition zones; FIG. 4C shows that after treatment at 37 deg.C, 40 deg.C and 50 deg.C for 15min, the inhibition zone of the lysostaphin group after temperature treatment is turbid, the inhibition zone can still be seen at 37 deg.C, and the inhibition zone is very turbid after treatment at 40 deg.C and 50 deg.C, and only invisible spots can be seen; in the gel-enzyme group, clear inhibition zones can still be seen after treatment at different temperatures; fig. 4d shows that the cracking effect of the gel-enzyme group is not significantly reduced and the transparency of the lysostaphin zone is reduced after repeated freeze-thawing.

Example 6 therapeutic Effect of the novel lysostaphin gel on mouse MRSA-infected wounds

The therapeutic effect of the novel lysostaphin gel of the present invention on mouse MRSA-infected wounds was examined with the example of a gel-enzyme gel (hereinafter referred to as "gel-enzyme") containing 10% glycerol + 35% poloxamer (188) + lysostaphin 10U/ml.

Selecting single colony obtained by cutting with MRSA bacterial liquid in advance, culturing 3-5ml MRSA bacterial liquid, and measuring OD of bacterial liquid₆₀₀And counting bacteria, and then diluting the bacterial titer to 1 x 107cfu/ml for standby; 6C 57 mice were divided into gel-enzyme treated group, disinfectant 1 group, disinfectant 2 group and 75% medical alcohol group. The mouse uses isoflurane to continuously inhale and anaesthetize, and the back moults, warm saline water washing, and tweezers gently lift back central line skin, use external diameter 5m 5mm circular hole puncher two places of punching, the circular surface of a wound of four symmetrical 5mm size appears promptly in the mouse back. Each wound surface was dripped with 5ul1 × 10⁷cfu/ml bacterial liquid, 5ul bacterial liquid is dripped again after 6-8h, and the wound surface is covered by sterile gauze. The infection symptoms appeared on the mouse wound after about 8 h: the wound is red and swollen and the wound surface is exuded. After the wound surface is successfully infected, the negative control hole is not treated; 5ul of gel-enzyme (concentration 10U/ml) is dripped into the gel-enzyme treatment hole; gel hand-eliminating hole 5ul of gel hand-eliminating is dripped into each wound surface, and 5ul of 75% medical wine is dripped into 75% medical alcohol holeJing, 1 time a day, the wound healing effect was observed. The results are shown in FIG. 5, in which well No. 1 is the novel lysostaphin gelling group, well No. 2 is the disinfectant 1 group, well No. 3 is the disinfectant 2 group, and well No. 4 is the 75% alcohol group. The results show that: the wound healing effect of the hole 1 wound is better than or equal to that of other groups 10 days after the treatment by adding different treatment factors. Therefore, the novel lysostaphin gel disclosed by the invention has a good treatment effect on MRSA infected wounds.

Claims (9)

1. A lysostaphin gel comprising a lysostaphin and a gel matrix comprising a poloxamer and glycerol.

2. The gel of claim 1, wherein the gel base comprises 25-40% poloxamer, 5-25% glycerol, and water.

3. The gel of claim 1, wherein the gel base comprises 30-35% poloxamer, 10-25% glycerol, and water.

4. The gel of claim 3, wherein the gel matrix consists of poloxamer 35%, glycerin 10%, and the balance water.

5. A gel according to any one of claims 1 to 4, which has a lysostaphin content of from 2.5 to 20U/ml.

6. The gel according to claim 5, wherein the content of lysostaphin is 10U/ml.

7. The gel of any one of claims 1 to 4, wherein the poloxamer is poloxamer 188.

8. A process for preparing the gel of claims 1-6 comprising the steps of:

(1) weighing poloxamer, adding glycerol and double distilled water, shaking, mixing, and standing at low temperature to obtain gel solution;

(2) adjusting the pH value of the gel solution after low-temperature standing to about 6.8;

(3) adding appropriate amount of lysostaphin, and heating in 37 deg.C water bath;

(4) and cooling after uniform mixing to obtain the lysostaphin gel.

9. Use of a lysostaphin gel according to claims 1 to 7 in the manufacture of a medicament for the treatment of MRSA infected wounds.

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