CN110917339B - Lysostaphin gel and application thereof in MRSA (tissue-specific respiratory tract infection) 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 glycerin and water. The gel of the invention is stable, keeps the activity of lysostaphin, is used for treating MRSA infection wound surface, and has obvious effect.

Description

Lysostaphin gel and application thereof in MRSA (tissue-specific respiratory tract infection) wound surface

Technical Field

The invention belongs to the field of medicines, and in particular relates to a lysostaphin gel and application thereof in treating MRSA infection wound surfaces.

Background

With the widespread and large use of antibiotics, an increasing number of staphylococcus aureus produces penicillinase, leading to the emergence of a large array of methicillin-resistant staphylococcus aureus (MRSA). MRSA has now become one of the major, more virulent pathogens for nosocomial infections. Patients with systemic or local MRSA infections in burn lesions, ICU, respiratory wards, hematological and pediatric settings are one of the most troublesome problems in clinical treatment.

Rebuilding or restoring the skin barrier is the final goal of wound healing, a good performing wound covering can temporarily function as part of the skin barrier function, providing an environment that is conducive to wound healing. The ideal wound dressing should firstly effectively prevent bacterial infection, especially infection of methicillin-resistant staphylococcus aureus (MRSA), improve the success rate of skin grafting and accelerate wound healing, and simultaneously have the characteristics of good biocompatibility, control and absorption of wound exudate, suitability for permeation of gas and water vapor, protection of new tissues, acceleration of wound healing and the like. The types of existing dressings are numerous and broadly divided into three types: 1. inert dressing, i.e. traditional dressing such as gauze, but gauze does not permanently keep the wound moist, which may cause delayed healing of the wound, and saturated dressing is prone to passage of pathogens, which causes secondary infections. In addition, dressing fiber is easy to fall off, foreign body reaction is caused, and wound healing is affected. 2. Biphasic active dressings of this type may be used to promote wound healing using the local environment created by the dressing: (1) A semi-permeable film (plastic dressing), an absorbent film made of polyurethane casein material, wherein one side of the dressing is provided with an adhesive material; (2) a polyurethane foam; (3) hydrogel dressing: is a three-dimensional net-shaped water-absorbing polymer composed of gelatin, polysaccharide, a plurality of electrolyte complexes and methacrylic resin. Biphasic active dressings generally suffer from the disadvantage of poor water absorption, poor adhesion to the wound surface and, more importantly, lack of anti-infective ability. 3. Bioactive dressing (airtight dressing) can be tightly adhered to the wound surface to prevent the wound surface from drying. It is a dressing that maintains the moist environment of the wound, its absorbency being related to its structure. It can act with locally applied medicine and endogenous molecules of organism to promote wound healing, but the bactericidal effect on bacterial infectious wound surface is poor, and the wound surface can not be effectively treated.

The dressing has the common defects that pathogenic bacteria causing wound infection cannot be effectively killed, wound seepage cannot be effectively controlled and absorbed, and the like. Therefore, development of an external infection wound treatment and repair preparation which can effectively sterilize, especially MRSA, effectively control and absorb wound seepage, protect new tissues and accelerate wound healing is urgent.

For systemic infection, vancomycin has definite treatment effect, and for local wound infection, the combination of the vancomycin and lysostaphin can increase the effect of controlling the infection, but the rapid degradation and inactivation of the lysostaphin in vitro becomes a great difficulty in the development of the MRSA of the cracked wound.

CN101721691a biological agent for treating and repairing infection wound and a preparation method thereof. The biological preparation comprises (by weight) lysostaphin 0.1-30%, chitosan or chitosan derivative 3-50%, and collagen 30-95%. The biological preparation uses collagen and chitosan as gel auxiliary materials, the manufacturing cost is high, the content of lysostaphin is also high, and the product is uneconomical.

Aiming at the osteomyelitis infected by MRSA, artificial bones made of nano hydroxyapatite and chitosan are designed as carriers to adsorb lysostaphin, so that the lysostaphin is slowly released in the osteomyelitis infected by MRSA, and the effect of continuous sterilization is achieved. For MRSA infectious wound surfaces after wounds such as burns and the like, a slow release carrier suitable for popularization is not reported at present. The inventor aims at developing a disinfection preparation which is used on a wound surface, can keep the activity of enzyme to a certain extent, continuously and slowly releases lysostaphin, has lower cost and is easy to popularize. Through a great number of researches, experiments and screening, the novel temperature-sensitive gel preparation is finally prepared by combining poloxamer and glycerin, and the lysostaphin is attached to the gel preparation, so that the lysostaphin can be slowly released in vitro and can stably exert a sterilizing effect.

Disclosure of Invention

The invention aims to provide a lysostaphin preparation stable in vitro, in particular to a lysostaphin gel. The in vitro experiment and the mouse MRSA infection wound experiment result prove that the novel gel preparation can stabilize the activity of the lysostaphin, maintain the curative effect of the lysostaphin for in vivo and in vitro pyrolysis of the MRSA, has obvious effect and has high clinical application value.

The lysostaphin gel provided by the invention comprises lysostaphin and a gel matrix, wherein the gel matrix comprises poloxamer and glycerin.

Preferably, the lysostaphin gel of the present invention, the gel matrix comprises 25-40% poloxamer, 5-25% glycerol and the balance water. More preferably, the gel matrix is composed of 30-35% poloxamer, 10-25% glycerin and the balance water, most preferably 18835% poloxamer, 10% glycerin 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, preferably a lysostaphin content of 10U/ml. Most preferably, the lysostaphin gel is poloxamer 18835% (mass fraction) +glycerin is 10% (volume fraction) +pure water+lysostaphin 10U/ml.

The invention relates to application of lysostaphin gel in preparing medicaments for treating MRSA infection wound surfaces.

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

(1) Weighing poloxamer, adding glycerol and double distilled water (i.e. pure water), shaking, mixing, and standing at low temperature to obtain gel solution;

(2) Regulating the PH value of the gel solution after standing at low temperature to about 6.8;

(3) Adding a proper amount of lysostaphin, and heating in a water bath at 37 ℃;

(4) And (5) uniformly mixing and cooling to obtain the lysostaphin gel.

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

Drawings

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

FIG. 2 gel-enzyme, disinfectant 1, disinfectant 2 and simple gel (-) in vitro lysis assay of MRSA at different time OD ₆₀₀ Comparing the numerical values;

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

FIG. 4 effect of lysostaphin gel on lysostaphin action;

FIG. 5. Therapeutic effect of lysostaphin gel of the present invention on wound surface of MRSA infection in mice.

Detailed Description

The following examples are merely representative for the understanding and elucidation of the essence of the invention, but do not limit the scope of the invention in any way.

Materials and methods of the following examples:

animal and main reagent and instrument

18 SPF-class Balb/c female mice, 8-10 weeks old, weighing 20-22g, purchased from Changsha Gekko Biotechnology Inc., license number SCXK (Hunan) 2019-0014. Yeast extract and tryptone were purchased from Oxoid company, england, agar powder from Beijing Ding Guo Changchun biotechnology Co., ltd, and sodium chloride from biological engineering (Shanghai) Co., ltd. 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 medium, and preserving at 4 ℃; semisolid LB medium: 7.5g of agar powder is added into 1000ml of LB liquid medium, and the mixture is stored in a 55 ℃ oven after high-pressure steam sterilization; solid LB medium: 15g of agar powder was added to 1000ml of LB liquid medium, and the mixture was sterilized by high-pressure steam and stored in an oven at 4 ℃.

Bacterial strain and lysostaphin source:

the MRSA standard strain was given by the third army university southwest hospital full army burn institute, and 12 clinical isolates were isolated from the compliance university affiliated hospital clinical laboratory, including ATCC29213 and ATCC25923. Lysostaphin was supplied by the university of shandong microbiological laboratory Lu Xuemei (supplied by Jining Baishi microbiological technologies Co., ltd.).

Example 1 screening of gel matrices

Novel gel prepared from poloxamer (188) and glycerin

Gel suspension formulation:

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

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

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

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

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

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

The preparation process comprises the following steps:

weighing poloxamer (188) and glycerin in percentage by mass, adding 10ml of double distilled water, mixing to obtain a suspension, shaking vigorously until the suspension is obtained, adjusting the pH value of the suspension to about 6.8 by using acid or alkali, and refrigerating overnight (> 12 h) in a refrigerator at 4 ℃. The suspension was observed to be fluid, gel-like every other day, and was placed in a 37 ℃ water bath with a water line above the suspension. The time of the different composition gums and the texture of the gels were observed. The gel was left at room temperature and the state of the suspension was observed at room temperature. We obtain the optimal proportion of the 3-component adhesive.

TABLE 1 observations of gel quality for the formulation groups

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

EXAMPLE 2 lysostaphin gel

The gel matrix of A, B, C, D, E of example 1 was prepared by adding 900ul of gel to a 1.5ml EP tube at 4℃and adding 100ul of lysostaphin at a concentration of 100U/ml to give a gel-enzyme concentration of 10U/ml. Gel-enzyme preparations at concentrations of 5U/ml and 2.5U/ml were prepared in the same manner. The double-layer agar plates of MRSA were inverted and 3 gel-enzyme preparations were added dropwise to the plates at final concentrations of 10U/ml, 5U/ml and 2.5U/ml, respectively. And selecting the ratio with the best enzyme cracking effect according to the transparency and the diameter of the inhibition zone for subsequent experiments. The size and transparency of the inhibition zone are observed, and the gel proportion with good gel effect and schizophrenic activity is found to be three groups (1) (2) and (3), wherein the lysostaphin gel with the concentration of 10% glycerol and 35% poloxamer (188) in the group (3) is the most stable, and the inhibition effect is the best. As in fig. 1.

(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: the size and transparency of the inhibition zone are observed, and the gel ratio with good gel effect and schizophrenic activity is found to be as shown in three groups of figures (1), (2) and (3): (1) 20% glycerol+35% poloxamer (188) +lysostaphin 10%; (2) 25% glycerol+30% poloxamer (188) +lysostaphin 10%; (3) 10% glycerol+35% poloxamer (188) +lysostaphin 10%. As can be seen from FIG. 1, (3) the diameter of the inhibition zone is the largest, the transparency is the highest, and the inhibition effect is the best.

Example 3 in vitro lysis assay of lysostaphin-188

Taking 4 tubes of single colony shaking mixed MRSA bacterial liquid, centrifuging for 2min at 1.5ml per tube and 16000g, adding 900ul PBS per tube to resuspend bacteria, and respectively marking: enzyme-188, digest 1, digest 2, (-); 100ul of enzyme-188 was added to the enzyme-188 tube at a final concentration of 10U/ml, and 100ul of disinfectant 1, disinfectant 2 and PBS were added to the disinfectant 1, disinfectant 2 and (-) tubes, respectively. Placing into a shaking table at 37 ℃ for incubation; taking the mixed solution of 0 min-60 min to detect OD ₆₀₀ Values. As shown in FIG. 2, the enzyme-188 group OD ₆₀₀ The decrease over time is significantly better than the other groups.

EXAMPLE 4 gel-enzymatic in vitro cleavage of MRSA and clinical isolates

Single colonies of MRSA and 12 clinical isolates were picked separately on 2ml liquid LB medium, 180RPM,37℃and shake cultured overnight. The next day bacterial liquid is turbid, 200ul bacterial liquid is respectively sucked into test tubes, then 3-5ml of semi-solid culture medium is added, and the mixture is uniformly mixed for 10s on a vortex oscillator, so as to prepare a double-layer agar plate. The gel with the best schizomycete effect obtained by the previous experiment is prepared from 10 percent of glycerin and 35 percent of poloxamer (188), and the following gel is prepared by the following steps. 10ul of gel-enzyme preparation with 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 positive control: the disinfectant 1 is gel hand-disinfection (the components are 0.22 to 0.26 percent of trichlorohydroxydiphenyl ether and 50 to 60 percent of ethanol), and the disinfectant 2 is liquid toilet hand-disinfection (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); the negative control used a gel of 75% alcohol and undissolved staphylokinase. As a result, it was found that gel-enzyme can cleave MRSA and 12 clinical isolates, and the cleavage effect was more thorough than that of disinfectant 1. As shown in fig. 3. In the test, the concentration of lysostaphin is increased to 100U/ml, the rest is unchanged, 10ul of lysostaphin is dripped into an MRSA double-layer agar plate by the method, and the gel-enzyme cleavage effect is more thorough when the concentration of the lysostaphin is increased. The results are shown in FIG. 3, wherein the gel-enzyme, disinfectant 1, disinfectant 2, 75% medical alcohol and simple gel have in vitro lysis effects on MRSA and clinical isolates, and the upper left graph shows the effect of gel-enzyme lysis on MRSA strains at a concentration of 10U/ml; the upper right panel shows the effect of gel-enzyme lysis of MRSA strains at a concentration of 100U/ml; lower left, middle, right panels are ATCC29213, respectively; staphylococcus aureus 1201; ATCC25923. As a result, it was found that gel-enzymes can cleave MRSA as well as 12 clinical isolates (the pictures only show part), and the cleavage effect is more thorough than that of disinfectant 1.

Example 5 Effect of novel gels on stability of lysostaphin

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

Firstly, 200ul of MRSA bacterial liquid is taken and added with 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 transparency of two groups of inhibition zones are observed. The results showed that the two groups had comparable inhibition zone sizes and transparency, and no significant difference, indicating that the gel-enzyme did not reduce the lytic efficacy of lysostaphin (FIG. 4A)

Then, the gel-enzyme and lysostaphin (concentration of 10U/ml) were placed at room temperature, 4℃and-20℃for 12 hours, respectively. 200ul of MRSA bacteria solution is added into 3-5ml of semisolid culture medium, and a double-layer agar plate is poured. And dripping 10ul of lysostaphin and gel-enzyme which are placed at different temperatures into different areas, and observing the diameter and the penetration brightness of the inhibition zone after 4-6 hours. Gel-enzyme and lysostaphin (concentration of 10U/ml) were each taken in 15ul of EP tubes, 3 tubes of each group, and they were treated at 37℃at 40℃and 50℃for 15min, respectively. Then, 10ul of the treated enzyme and enzyme products are dripped on an MRSA double-layer agar plate, and the size and the penetration brightness of the bacteriostasis zone are observed after 4-6 hours. As shown in FIG. 4C, after 15min of treatment at 37 ℃, 40 ℃ and 50 ℃, the bacteriostasis zone of the lysostaphin group after temperature treatment is turbid, the bacteriostasis zone is still visible at 37 ℃, and after treatment at 40 ℃ and 50 ℃, the bacteriostasis zone is very turbid, and only invisible spots can be seen by 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 an depending carrier of the 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.

Gel-enzyme and lysostaphin which are repeatedly frozen and thawed in equal quantity are dripped on an MRSA double-layer agar plate, and the sizes and the brightness of two groups of inhibition zones are observed, so that the enzyme is frozen and thawed in the use process. Repeated freeze thawing experiments: storing gel-enzyme and lysostaphin refrigerator at 4deg.C and-20deg.C respectively, simulating repeated freeze thawing in the use process, taking out from 4deg.C and-20deg.C, standing at room temperature for 5-10min, then respectively storing at 4deg.C and-20deg.C for more than 6 hr, taking out again, standing at room temperature for 5-10min, repeating freeze thawing for 3-5 times, and detecting enzyme activity. As shown in FIG. 4D, the effect of the gel-enzyme group on the split bacteria is not significantly reduced after repeated freeze thawing, but the transparency of the inhibition zone of the lysostaphin is reduced after repeated freeze thawing, and thus the cleavage efficiency of the lysostaphin 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 investigate the effect of gel on enzyme, equal amounts of lysostaphin gel preparation (hereinafter referred to as gel-enzyme) and lysostaphin were added dropwise to MRSA bilayer plates at a concentration of 10U/ml, respectively.

The gel-enzyme and lysostaphin are preserved at different temperatures in a short time to simulate the conditions of transportation and short-term storage, two groups of enzyme preparations containing equal enzymes are preserved at room temperature, 4 ℃ and-20 ℃ for 12 hours respectively, and then equal amounts of the two groups of enzyme preparations are dripped into corresponding lattices on an MRSA double-layer agar plate, and the results show that the diameters of the two groups of inhibition zones are equivalent, but the boundary of the inhibition zone under different conditions of the gel-enzyme group is clearer than that of the lysostaphin group, the boundary of the inhibition zone of the lysostaphin group is fuzzy, and surrounding bacteria have a tendency to expand towards the middle of the inhibition zone (figure 4B). The addition of the novel gel of the invention is therefore advantageous for stabilizing the activity of the enzyme under comparable short-term storage conditions. The results in fig. 4 show that: FIG. 4A shows that gel-enzyme and lysostaphin which are respectively 10U/ml in concentration are dripped on an MRSA double-layer agar plate, and the size and transparency of the bacteriostasis circles of the two groups are equivalent; FIG. 4B. Two groups of enzyme preparations containing equal amount of enzyme are respectively stored at room temperature, 4 ℃ and-20 ℃ for 12 hours, and then equal amount of the two groups of enzyme preparations are dripped into corresponding grids on an MRSA double-layer agar plate, wherein the diameters of the two groups of inhibition zones are equal, but the boundary of the inhibition zone under different conditions of the gel-enzyme group (namely the lysostaphin gel of the invention) is clearer than that of the lysostaphin group, the boundary of the inhibition zone of the lysostaphin group is fuzzy, and surrounding bacteria tend to expand towards the middle of the inhibition zone; FIG. 4C, after 15min of treatment at 37, 40 and 50 ℃, the zone of inhibition of the lysostaphin group after temperature treatment is cloudy, the zone of inhibition is still visible at 37, and the zone of inhibition after treatment at 40 and 50 ℃ is very cloudy, with only visible spots to the naked eye; in the gel-enzyme group, clear inhibition zones can still be seen after treatment at different temperatures; FIG. 4D shows that after repeated freeze thawing, the gel-enzyme group shows no significant decrease in the schizophrenic effect and the lysostaphin shows a decrease in the transparency of the zone of inhibition.

Example 6 therapeutic Effect of novel lysostaphin gel on wound surfaces infected with mouse MRSA

The novel lysostaphin gel of the present invention was examined for its therapeutic effect on wound surfaces infected with MRSA in mice, using 10% glycerol+35% poloxamer (188) +10U/ml gel-enzyme gel of lysostaphin (hereinafter referred to as "gel-enzyme").

Selecting single colony obtained by pre-plating with MRSA bacterial liquid, culturing 3-5ml MRSA bacterial liquid, and measuring OD of bacterial liquid ₆₀₀ Counting bacteria, and then diluting the bacterial titer to 1 x 107cfu/ml for later use; 6C 57 mice were grouped into gel-enzyme treatment group, disinfectant 1 group, disinfectant 2 group and 75% medical alcohol group. The mice were continuously inhaled with isoflurane for anesthesia, the backs were dehaired, warm saline was used for cleaning, forceps were used to gently lift the back midline skin, two punches were used with a 5mm circular punch with an outer diameter, and four symmetrical 5mm circular wounds were present on the backs of the mice. 5ul1 x 10 is dripped into each wound surface ⁷ cfu/ml bacteria liquid, dripping 5ul bacteria liquid again after 6-8h, and covering the wound surface with sterile gauze. After about 8 hours the wound of the mice shows symptoms of infection: red and swelling wound, exudation of wound surface. After the wound surface is infected successfully, the negative control hole is not treated; gel-enzyme treatment Kong Dijia ul gel-enzyme (concentration 10U/ml); gel hand-eliminating holes are dripped with 5ul gel hand-eliminating holes and 75% medical alcohol Kong Dijia ul75% medical alcohol for each wound surface, 1 time a day, and the healing effect of the wound surface is observed. As shown in FIG. 5, well 1 is a novel lysostaphin gel action group, well 2 is a disinfectant 1 group, well 3 is a disinfectant 2 group, and well 4 is a 75% alcohol group. The results show that: the healing effect of Kong Chuangmian No. 1 was superior or equal to the other groups 10 days after treatment with different treatment factors. Therefore, the novel lysostaphin gel of the invention has good therapeutic effect on the wound surface of MRSA infection.

Claims (6)

1. A lysostaphin gel consists of lysostaphin and a gel matrix, wherein the gel matrix consists of 30-35% of poloxamer, 10-25% of glycerin and double distilled water, and the poloxamer is poloxamer 188.

2. The gel of claim 1, wherein the gel matrix consists of 35% poloxamer, 10% glycerin, and the balance double distilled water.

3. A gel according to any one of claims 1-2, having a lysostaphin content of 2.5-20U/ml.

4. A gel according to claim 3, having a lysostaphin content of 10U/ml.

5. A method of preparing the gel of any one of claims 1-4, comprising the steps of:

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

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

(3) Adding a proper amount of lysostaphin, and heating in a water bath at 37 ℃;

(4) And (5) uniformly mixing and cooling to obtain the lysostaphin gel.

6. Use of a lysostaphin gel as claimed in any one of claims 1 to 4 in the manufacture of a medicament for the treatment of MRSA infection wounds.

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