CN114767631A

CN114767631A - Preparation method and application of Pickering emulsion for resisting bacteria and inflammation and promoting wound healing

Info

Publication number: CN114767631A
Application number: CN202210417181.2A
Authority: CN
Inventors: 玄光善; 李斌; 裴津泽
Original assignee: Qingdao University of Science and Technology
Current assignee: Qingdao University of Science and Technology
Priority date: 2022-04-20
Filing date: 2022-04-20
Publication date: 2022-07-22
Anticipated expiration: 2042-04-20
Also published as: CN114767631B

Abstract

The invention discloses a preparation method of a Pickering emulsion for resisting bacteria and inflammation and promoting wound healing. The emulsion comprises chitosan/Arabic gum (CS/GA) nanoparticles, tea tree oil and vitamin E, wherein the composition ratio is that the ratio of chitosan to Arabic gum is 0.25-4.0, the ratio of an oil phase to a water phase is 0.2-0.8, and the content of the vitamin E in the oil phase is 5-60 mg/mL. The chitosan and Arabic gum nanoparticles combined by electrostatic interaction are irregular oval or spherical, and the stability of the emulsion can be improved. Compared with the traditional tea tree oil emulsion, the Pickering emulsion provided by the invention enhances the antibacterial activity of tea tree oil on staphylococcus aureus, escherichia coli, pseudomonas aeruginosa and candida albicans; can improve the ultraviolet irradiation stability of vitamin E and promote wound healing.

Description

Preparation method and application of Pickering emulsion for resisting bacteria and inflammation and promoting wound healing

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to preparation and application of an antibacterial, anti-inflammatory and wound healing promotion Pickering emulsion.

Background

Wound healing is a complex process that generally involves four sequential processes of hemostasis, inflammation, neovascularization, and maturation of new tissue to rapidly restore the integrity of the skin barrier, limit dehydration, and prevent infection from occurring. This requires a synergistic effect of antibacterial, anti-inflammatory and fibroblast proliferation promoting substances. The wound surface generally refers to the intact destruction of the skin and the loss of damage to normal tissues after the normal skin or tissues are damaged by internal or external factors. Because the wound surface is directly contacted with the external environment and is very easy to infect, the wound surface nursing urgently needs to develop the advanced wound surface dressing with the antibacterial function and the healing promoting effect.

The wound repair material is a biomedical polymer material which covers the surface of the window and plays a role in promoting the healing of skin wounds. The existing wound repair materials comprise hemostatic gauze, hemostatic sponge, gel products and the like, and can play a role in protecting wounds to a certain extent. However, the materials have single functions, hemostatic gauze and hemostatic sponge have good hemostatic effects, but the wound healing promotion function is poor, the dressing change is required regularly, secondary bleeding and infection are easily caused, and gel products have certain wound repair effects, but have general hemostatic performance, do not have antibacterial effects and are very easy to infect bacteria. In addition, the gel products contain many tiny particles, which are easy to enter blood along with wounds to trigger thrombus. Therefore, the development of a wound repair material with functions of hemostasis and promotion of wound repair has long-acting antibacterial effect, is safe and reliable for human bodies, and is very important.

Pickering emulsions are emulsions stabilized by solid particles. The multicomponent system consisting of particles, aqueous phase and oil phase enables the Pickering emulsion to be loaded with both hydrophilic and hydrophobic drugs to improve its effectiveness. In addition, the Pickering emulsion stabilized by solid particles and composed of natural biodegradable high molecular materials has higher biosafety and biocompatibility than the classical emulsion stabilized by the traditional surfactant. When Pickering emulsion is used as a wound dressing in direct contact with defective skin, it avoids the hemolytic, irritating and toxic properties of the surfactant. Meanwhile, the special structure of the solid particle shell can enable the Pickering emulsion to have a slow release effect.

The natural essential oil tea tree oil has high antibacterial, analgesic and scar inhibiting effects, and when used as an antibacterial agent, the natural essential oil does not cause any drug resistance, and is safer than the traditional antibiotics. Chitosan is a natural polymer, has the storage capacity second to cellulose in nature, and has excellent adhesiveness, biocompatibility, hemostatic property, antibacterial property and wound healing promoting capability of accelerating tissue regeneration. The acacia has good film forming property, can protect skin from lipid peroxidation caused by continuous exposure to ultraviolet rays, and has the effect of preventing premature aging of skin.

Disclosure of Invention

Aiming at the defects and shortcomings of the prior art and products, the invention aims to provide a preparation method of a Pickering emulsion for resisting bacteria, diminishing inflammation and promoting wound healing.

The invention also aims to provide the CS/GA nano-particles prepared by the method.

Still another object of the present invention is to provide a Pickering emulsion for wound healing promotion, anti-inflammatory and antibacterial, which is loaded with vitamin E, prepared by the above method.

The object of the present invention is achieved by at least one of the following technical means.

An antibacterial, anti-inflammatory and wound healing promoting Pickering emulsion comprises an oil phase, a water phase and a stabilizer, wherein the stabilizer is CS/GA nanoparticles, and the oil phase contains a fat-soluble active ingredient and an antioxidant active ingredient.

Furthermore, the fat-soluble active ingredient is tea tree oil, and the antioxidant active ingredient is vitamin E.

Furthermore, the mass ratio of CS to GA in the CS/GA nano particles is 0.25-4.0.

The preparation method of the Pickering emulsion comprises the following steps:

(1) accurately weighing a certain amount of chitosan in an acetic acid solution, and stirring until the chitosan is dissolved. Dropwise adding a gum arabic aqueous solution with a certain concentration into a chitosan solution, and stirring to obtain a series of CS/GA nanoparticle solutions with different concentrations and proportions.

(2) Pouring a certain volume of chitosan/Arabic gum (CS/GA) nanoparticle solution into a flask, adding an oil phase, putting into a rotor, magnetically stirring until the mixture is uniformly mixed, and putting into a high-pressure homogenizer for homogenizing.

(3) Accurately weighing vitamin E, uniformly mixing the vitamin E in the oil phase, and preparing the emulsion containing VE Pickering according to the method (2).

Further, in the step (1), the concentration of the acetic acid solution is 1%, the stirring time is 30-60 minutes, and the mass ratio of CS/GA is 0.25-4.0.

Further, in the step (2), the oil phase is tea tree oil, the proportion of the oil phase is 0.2-0.8, the homogenization time is 3-10 minutes, and the pressure is 200-600 MPa.

Further, in the step (3), the concentration of the vitamin E is 5-60 mg/mL.

The invention also comprises other oil phases and oil phases with biological activity, such as olive oil, clove oil and the like, and antibacterial and anti-inflammatory active ingredients, such as resveratrol, curcumin and the like.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) the prepared CS/GA nano particles with stable biocompatibility and good wettability can be used as a stability example of a Pickering emulsion for resisting bacteria, diminishing inflammation and promoting wound healing, the Pickering emulsion has good storage stability, temperature stability and irradiation stability, and a fat-soluble active ingredient loaded in the Pickering emulsion can play a synergistic bactericidal effect with the CS/GA nano particles, so that a new opportunity is provided for the application of the Pickering emulsion in the field of pharmacy.

(2) The invention relates to a preparation method of Pickering emulsion loaded with vitamin E, wherein the vitamin E has good anti-inflammatory effect, can reduce the generation of inflammatory factors, accelerate wound healing and expand the application space of the Pickering emulsion in the pharmaceutical field.

Drawings

FIG. 1 is a graph showing the results of measuring the contact angle of the CS/GA nanoparticles obtained in example 1;

FIG. 2 is a transmission electron microscope result diagram of the CS/GA nanoparticles obtained in example 1;

FIG. 3 is a graph comparing the bacteriostatic ratio of the emulsion encapsulating VE Picking obtained in example 4

FIG. 4 is a graph of wound healing indications obtained from the encapsulated VE packaging emulsion obtained in example 6;

Detailed Description

The present invention will be described in further detail with reference to the following examples and drawings, but the present invention is not limited thereto.

Example 1

The preparation method of the CS/GA nano-particle comprises the following specific steps:

accurately weighing a certain amount of chitosan in a 1% acetic acid solution, and stirring until the chitosan is dissolved. Dropwise adding the Arabic gum aqueous solution with a certain concentration into the chitosan solution, and stirring for 30-60 minutes to obtain the CS/GA nanoparticle solution with the mass ratio of 0.25-4.0. According to contact angle measurement, the contact angle of the nanoparticles obtained under different CS/GA mass ratios changes from 73 to 94 degrees, particularly the mass ratio is 1: the contact angle of the nanoparticles obtained under the condition of 1 is close to 90 degrees (figure 1), and the scanning electron microscope observation forms round or oval aggregates with uniform particle size, and the average particle size is about 100nm (figure 2).

Example 2

The preparation method of the antibacterial Pickering emulsion provided by the embodiment comprises the following specific steps:

pouring a certain volume of chitosan/Arabic gum (CS/GA) nanoparticle solution with the mass ratio of 0.25-4.0 into a 250mL flask, adding tea tree oil to make the total volume 100mL, stirring until the mixture is uniformly mixed, and placing the mixture into a high-pressure homogenizer for homogenizing for 5min (400 MPa).

The storage stability, the emulsion index and the emulsion particle size were determined for the antimicrobial Pickering emulsion obtained in the above example. The emulsion emulsification index and the rate of change in particle size at different oil phase ratios are shown in table 1. The results in Table 1 show that the obtained Pickering emulsion is stable, and the CS/GA nanoparticles play a role in stabilizing the emulsion.

Table 1 Emulsification index,particle size and particle size change rate of Pickering emulsion within 30days

Example 3

The preparation method of the Pickering emulsion for promoting wound healing by resisting bacteria and inflammation comprises the following specific steps:

pouring 60mL of chitosan/Arabic gum (CS/GA) nanoparticle solution with the mass ratio of 0.25-4.0 into a 250mL flask, weighing 2g of vitamin E, uniformly mixing in 40mL of oil phase, mixing with water phase, stirring until uniform mixing, and homogenizing for 5min under 400MPa in a high-pressure homogenizer.

Content measurement and irradiation stability tests are carried out on the anti-bacterial, anti-inflammatory and wound healing promoting Pickering emulsion obtained in the above embodiment, the encapsulation efficiency and the drug loading rate of the Pickering emulsion in the embodiment are obviously higher than those of a common emulsion, and the stability effect on protecting vitamin E is better than that of a common emulsion stabilized by Tween 80 (figure 3).

Example 4

The in vitro bacteriostasis experiment of the Pickering emulsion for resisting bacteria, diminishing inflammation and promoting wound healing provided by the embodiment comprises the following specific steps:

the culture dish is made of escherichia coli, staphylococcus aureus, pseudomonas aeruginosa and candida albicans. Clamping an Oxford cup by using tweezers to punch on a solid culture medium, respectively sucking 80 mu L of tea tree oil, common emulsion, edible olive oil, CS/GA nanoparticles and Pickering emulsion solution into the holes by using a liquid transfer gun, culturing for 18-24 h in a constant-temperature incubator, adjusting the temperature according to the appropriate temperature of each strain, taking out and placing on a black background table, and measuring the size of an antibacterial ring by using a vernier caliper.

The experimental results of the embodiment and the comparative example 1 are shown in fig. 4, that there is no bacteriostatic zone in the edible olive oil, and after the tea tree oil is respectively made into a common emulsion taking tween 80 as a surfactant and a Pickering emulsion stabilized by CS/GA nanoparticles, the size of the bacteriostatic zone of 4 kinds of bacteria is not significantly changed in the common emulsion compared with that of pure tea tree oil, and the bacteriostatic zone of the Pickering emulsion is significantly increased, which indicates that the CS/GA nanoparticles and the tea tree oil can exert a synergistic bacteriostatic action in the emulsion.

Example 5

The in vivo anti-inflammatory experiment of the anti-bacterial anti-inflammatory and wound healing promoting Pickering emulsion provided by the embodiment comprises the following specific steps:

(1) selecting 20-22 g Kunming mice, randomly dividing the Kunming mice into 5 groups, wherein each group comprises 5 mice, namely a blank control group, a compound dexamethasone acetate cream positive control group (2.4g/kg), a high (4.8g/kg) emulsion containing VE Pickering, a medium (2.4g/kg) emulsion containing VE Pickering and a low dose group (1.2 g/kg).

(2) After the depilatory treatment of the back of the mice, the administration was continued for 7 days, 1 time per day. After the last administration for 2h, the tail of the mouse was subjected to hot compress, and 0.5% Evans blue (0.1mL/10g) was injected as a peritoneal capillary permeability marker.

(3) After 10min, 1.2% glacial acetic acid (0.1mL/10g) was injected intraperitoneally.

(4) After 20min, the mice were sacrificed by dislocation, the abdomen was dissected open, the abdominal cavity was washed several times with physiological saline, the washing solution was aspirated with a pipette, and the washing solutions were combined.

(5) Adding physiological saline to 5mL, centrifuging at 3000rpm/min for 15min, and measuring absorbance at 610nm with ultraviolet spectrophotometer.

(6) The content of evans blue in the peritoneum of each mouse was calculated according to the standard curve. The inhibition rate of the abdominal cavity exudation of the mice in the administration group was calculated by taking the exudation amount of the dye in the mice in the blank group as 100%. Permeability is measured as inhibition value.

The results are shown in table 2, where the Pickering emulsion group (64.13%) was lower than the control group (71.16%) at the same dose. The VE Pickering-containing emulsion has the function of obviously reducing the increase of the permeability of capillary vessels in the abdominal cavity of a mouse caused by acetic acid and has certain acute anti-inflammatory effect.

Table 2 Date of acetic acid induced Evans blue peritoneal permeability

Example 6

The wound healing promotion test of the antibacterial anti-inflammatory wound healing promotion Pickering emulsion provided by the embodiment comprises the following specific steps:

(1) 4% chloral hydrate solution is prepared. 0.2g of chloral hydrate is precisely weighed, and the volume of distilled water is adjusted to 5 mL.

(2) Feeding Kunming mice in cages for 12 hours day and night at the temperature of 22 +/-2 ℃ and the humidity of 50-60% for 1 week, injecting 4% chloral hydrate (0.1mL/10g) into the abdominal cavity, determining whether the mice are in an anesthetic state after about 10min, and supplementing 0.05mL chloral hydrate if the mice are not completely anesthetized.

(3) A 5mm wound was made on the back of the mouse with sterilized scissors.

(4) The preparation method comprises the following steps of randomly dividing the raw materials into 5 groups, wherein each group comprises 5 raw materials, namely a Pickering emulsion group, a common emulsion group, a CS/GA nanoparticle group, a tea tree oil group and a blank group. The medicine is smeared twice a day according to the amount of 2.4g/kg for 10 days continuously, the red swelling and the pus forming conditions of the wound are observed, and the diameter of the wound is measured by a vernier caliper and recorded.

The results of the experiment are shown in table 3, the area of the wound surface treated with Pickering emulsion is minimal at day 5 and healing is complete at day 10. The healing effect of the Pickering emulsion is superior to that of a single CS/GA nanoparticle group and a single tea tree oil group, and the synergistic effect of the tea tree oil, the vitamin E and the CS/GA nanoparticles is embodied.

Table 3 Wound healing rate of different media

^****Indicates an extremely significant difference(P<0.001)

Although the embodiments have been described, a person skilled in the art can make other variations and modifications to these embodiments once having learned the basic inventive concept, so that the above description is only an embodiment of the present invention, and is not intended to limit the scope of the present invention, but rather, the present invention is applicable to other related fields, either directly or indirectly, or through equivalent structures or equivalent processes performed in the specification and drawings of the present invention.

Claims

1. The Pickering emulsion is characterized by consisting of chitosan/Arabic gum (CS/GA) nanoparticles, tea tree oil and vitamin E, wherein chitosan and Arabic gum are combined through static electricity to form a nanoparticle serving as a stabilizing agent of the emulsion, the ratio of chitosan to Arabic gum is 0.25-4.0, an oil phase consists of tea tree oil and vitamin E, the content of the vitamin E in the oil phase is 5-60mg/mL, and the ratio of the oil phase to a water phase is 0.2-0.8.

2. The anti-bacterial, anti-inflammatory and wound-healing Pickering emulsion according to claim 1, comprising the steps of:

(1) and dissolving chitosan in an acetic acid solution, dropwise adding a gum arabic aqueous solution with a certain concentration into the chitosan solution, and stirring for 30-60 minutes to obtain a CS/GA nanoparticle solution with a certain concentration and proportion.

(2) Pouring a certain volume of chitosan/Arabic gum (CS/GA) nanoparticle solution into a 250mL flask, adding an oil phase to make the total volume 100mL, stirring until the mixture is uniformly mixed, and placing the mixture into a high-pressure homogenizer for homogenization.

(3) Accurately weighing vitamin E, uniformly mixing the vitamin E in the oil phase, and preparing the Pickering emulsion containing VE 5-60mg/mL according to the method (2).

Further, in the step (2), the oil phase is tea tree oil, the proportion of the oil phase is 0.2-0.8, the homogenization time is 2-10 minutes, and the pressure is 200-600 MPa.

Further, in the step (3), the content of vitamin E in the oil phase is 5-60 mg/mL.

3. The anti-microbial, anti-inflammatory and wound-healing Pickering emulsion of claim 1, wherein the contact angle is closest to 90 ° at a CS/GA mass ratio of 1: 1.

4. The anti-bacterial, anti-inflammatory and wound-healing Pickering emulsion according to claim 1, wherein the concentration of CS/GA nanoparticles is 1.5%.

5. The anti-bacterial, anti-inflammatory and wound-healing Pickering emulsion according to claim 1, wherein the oil phase is 40% by weight.

6. The anti-bacterial, anti-inflammatory and wound-healing Pickering emulsion according to claim 1, wherein the vitamin E content in the oil phase of the emulsion is 20 mg/mL.