Ultrasonic-responsive oxygen-carrying traditional Chinese medicine microcapsule, preparation method thereof and application of ultrasonic-responsive oxygen-carrying traditional Chinese medicine microcapsule in treatment of infected wounds

Abstract

The invention belongs to the field of biological materials, and particularly relates to an ultrasonic responsive oxygen-carrying and traditional Chinese medicine microcapsule and a preparation method and application thereof in treatment of infected wounds, two glass tubes with different inner diameters are coaxially sleeved and fixed, the glass tube with the larger inner diameter is used as an outer phase channel, and the glass tube with the smaller inner diameter is used as an inner phase channel; the preparation method comprises the following steps: dissolving sodium alginate and curcumin into deionized water to prepare a mixed pre-gel solution; oxygen is introduced into the liquid fluorocarbon, and oxygen-enriched fluorocarbon liquid is obtained after treatment; the prepared mixed pre-gel solution and the oxygen-enriched fluorocarbon liquid are simultaneously introduced into the external phase channel and the internal phase channel respectively, electric field force is applied to outlets of the channels, and the mixed pre-gel solution and the oxygen-enriched fluorocarbon liquid fall into a collection liquid after being subjected to electronic injection to obtain the oxygen-carrying and traditional Chinese medicine microcapsule. The loaded traditional Chinese medicine curcumin and oxygen dissolved in the fluorocarbon achieve simultaneous delivery of traditional Chinese medicine and oxygen, and controllable oxygen release and sonodynamic therapy can be achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomaterials, and particularly relates to an ultrasound-responsive oxygen-loaded and traditional Chinese medicine microcapsule, a preparation method thereof and application thereof in treating infected wounds. BACKGROUND

[0002] Wound treatment is a very important issue in clinical practice. The wound healing process can be affected by many factors, among which inflammation, bacterial infection and hypoxia are the most important. In terms of relieving inflammation, many traditional Chinese medicines perform well, such as curcumin, berberine and emodin, etc. These traditional Chinese medicines can inhibit the production and release of inflammatory mediators, and interfere with their binding to receptors. At the same time, traditional Chinese medicines can also resist oxidative stress, and interact with multiple signaling pathways to activate the immune system to relieve inflammation. It is worth noting that recent studies have found that some traditional Chinese medicines with ultrasound-responsive characteristics have great potential in promoting sonodynamic therapy against bacteria. On the other hand, in order to reverse the hypoxic state of the wound, the current methods mainly focus on improving the air permeability of wound dressings or applying hyperbaric oxygen therapy. Although some progress has been made, these oxygen supply methods often require complex equipment and lack controllability. In addition, given the multiple factors affecting wound healing, it is inevitable to require multiple administrations and different doses, which increases the challenge and difficulty of chronic wound treatment. Therefore, new treatment strategies need to be developed to achieve controllable anti-inflammatory drugs and oxygen supply to promote the treatment of infected wounds. SUMMARY

[0003] The technical problem to be solved by the present application is to provide a controllable oxygen and traditional Chinese medicine delivery method to achieve better results in the sonodynamic treatment of infected wounds.

[0004] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: A preparation method of an ultrasound-responsive oxygen-loaded and traditional Chinese medicine microcapsule, comprising the following steps: S1, two glass tubes with different inner diameters are coaxially sleeved and fixed, the glass tube with larger inner diameter is used as the outer phase channel, and the glass tube with smaller inner diameter is used as the inner phase channel; S2, sodium alginate and curcumin are dissolved in deionized water to obtain a mixed pre-gel solution; oxygen is introduced into liquid fluorocarbon compound to obtain oxygen-rich fluorocarbon liquid after treatment; S3, the mixed pre-gel solution and the oxygen-rich fluorocarbon liquid prepared in step S2 are introduced into the outer phase channel and the inner phase channel of step S1 respectively at the same time, and an electric field force is applied at the outlet of the channel, and the oxygen-loaded and traditional Chinese medicine microcapsule is obtained after electric spraying and falling into the collection liquid.

[0005] Specifically, in step S1, the outer phase channel has a diameter of 450-500 microns; and the inner phase channel has a diameter of 100-150 microns.

[0006] Specifically, in step S2, the concentration of sodium alginate in the mixed pre-gel solution is 2-2.5 wt%, and the concentration of curcumin is 0.01-0.02 M.

[0007] Preferably, in step S2, the oxygen content in the oxygen-rich fluorocarbon liquid is 30-40 mg / L.

[0008] Preferably, in step S3, the flow rate of the mixed pre-gel solution into the outer phase channel is 40-80 μL / min; and the flow rate of the oxygen-rich fluorocarbon liquid into the inner phase channel is 10-15 μL / min.

[0009] Preferably, in step S3, the electric field applied at the outlet of the channel is 8-12 kV.

[0010] Preferably, in step S3, the collection liquid is a calcium chloride solution with a concentration of 1.5-2 wt%.

[0011] Further, the ultrasound-responsive oxygen-loaded and traditional Chinese medicine microcapsules prepared by the above preparation method are also within the protection scope of the present application.

[0012] Further, the present application also claims the use of the above ultrasound-responsive oxygen-loaded and traditional Chinese medicine microcapsules in the preparation of a drug for treating infected wounds.

[0013] Specifically, the oxygen-loaded and traditional Chinese medicine microcapsules are continuously stimulated by therapeutic ultrasound waves to release the active ingredients of the drug, the therapeutic ultrasound waves have a frequency of 0.8-1.0 MHz and a power of 1.5 W cm -2 , and the stimulation time is 10-20 min.

[0014] The present application combines microfluidic electrospraying technology to realize the preparation of hydrogel microcapsules loaded with oxygen and traditional Chinese medicine, and realizes controllable oxygen release and sonodynamic therapy under the action of therapeutic ultrasound waves, relieves the anoxic environment of infected wounds, and kills bacteria at the same time, and the released traditional Chinese medicine further plays an anti-inflammatory role, thereby providing a new strategy for the treatment of infected wounds.

[0015] Compared with the prior art, the present application has the following beneficial effects: (1) The present application is based on the advantages of microfluidic electrospraying technology in preparing micron-sized materials, combined with curcumin, a traditional Chinese medicine with anti-inflammatory and sonosensitive properties, and fluorocarbons, which have excellent oxygen-carrying capacity, to construct an intelligent traditional Chinese medicine microcarrier with structural design and ultrasonic responsiveness. In the mixed pre-gel solution, the concentration of sodium alginate is 2-2.5 wt%, the concentration of curcumin is 0.01-0.02 M, the dissolved oxygen content in fluorocarbons is 30-40 mg / L, and the collection liquid is a calcium chloride solution with a concentration of 1.5-2 wt%. The sodium alginate, curcumin and fluorocarbons used have good biological safety and stability, and the use of microfluidic electrospraying technology can obtain oxygen-carrying and traditional Chinese medicine microcapsules with core-shell structure and ultrasonic responsiveness.

[0016] (2) The ultrasonic-responsive oxygen-carrying and traditional Chinese medicine microcapsules prepared in the present application achieve simultaneous delivery of traditional Chinese medicine and oxygen based on the traditional Chinese medicine curcumin loaded in the calcium alginate hydrogel shell and the oxygen dissolved in the fluorocarbons, and can achieve controlled oxygen release and sonodynamic therapy under the action of therapeutic ultrasound, effectively killing bacteria in the wound surface. In addition, the loaded curcumin further plays an anti-inflammatory role through sustained release, promoting the healing of infected wounds.

[0017] (3) The ultrasonic-responsive oxygen-carrying and traditional Chinese medicine microcapsules prepared in the present application further verify that under the action of external therapeutic ultrasound, they can effectively kill bacteria, produce oxygen to alleviate the hypoxic microenvironment at the wound surface, and long-term release of curcumin inhibits the inflammatory response, promoting the healing of infected wounds. Therefore, the ultrasonic-responsive oxygen-carrying and traditional Chinese medicine microcapsules of the present application can achieve the co-delivery of oxygen and important traditional Chinese medicine and promote the healing of infected wounds through sonodynamic therapy. BRIEF DESCRIPTION OF DRAWINGS

[0018] The above and / or other aspects of the present application will become apparent from the following detailed description of the application taken in conjunction with the accompanying drawings.

[0019] Figure 1 The process of generating microdroplets using microfluidic electrospraying technology (a), the obtained oxygen-carrying and traditional Chinese medicine microcapsules (b), the particle size distribution of the microcapsules (c), the electron microscope images of the microcapsules (d-f), the effects of adjusting the flow rate and voltage of the inner and outer phases on the particle size and core-shell ratio of the microcapsules (g-i).

[0020] Figure 2 The schematic diagram of the ultrasonic-responsive changes of the ultrasonic-responsive oxygen-carrying and traditional Chinese medicine microcapsules prepared in the present application (a), the changes of the microcapsules under the action of ultrasound taken by light microscope (b-e), the characterization of controlled oxygen release (f), and the characterization of reactive oxygen species produced by the microcapsules under the action of ultrasound (g, h).

[0021] Figure 3 Colony formation of E. coli and S. aureus after treatment with the oxygen-loaded and traditional Chinese medicine microcapsules prepared according to the present application (a), electron scanning microscope images of E. coli and S. aureus before and after treatment (b), and effect of the oxygen-loaded and traditional Chinese medicine microcapsules on inducing macrophages to change from M1 type (c) to M2 type (d).

[0022] Figure 4 Healing process of infected wounds in mice after treatment with the oxygen-loaded and traditional Chinese medicine microcapsules prepared according to the present application (a), wound change characterization (b), hematoxylin-eosin staining images of the skin of each group at the wound site (c), wound area change with days (d), final wound area comparison (e), and wound width comparison based on tissue section statistics (f).

[0023] Figure 5 Collagen staining images of infected wounds in mice after treatment with the oxygen-loaded and traditional Chinese medicine microcapsules prepared according to the present application, and IL-6, TNF-a, and HIF-1a immunostaining images (a), and IL-6, TNF-a, and HIF-1a quantification statistics (b-d).

[0024] Figure 6 Staining images of the kidney, lung, heart, liver, and spleen of mice after treatment with the oxygen-loaded and traditional Chinese medicine microcapsules prepared according to the present application. DETAILED DESCRIPTION

[0025] The present application can be better understood according to the following examples.

[0026] Example 1: Preparation of the oxygen-loaded and traditional Chinese medicine microcapsules The mixed pre-gel solution contains 2-2.5 wt.% sodium alginate (low viscosity) and 0.01-0.02 M curcumin. Oxygen-rich fluorocarbon liquid is prepared by continuously adding oxygen in fluorocarbon compound for 15 min, and the collection liquid contains 1.5-2 wt.% calcium chloride. Two syringe pumps are used to push the two-phase liquid at a constant speed. Different flow rates and voltages are set, and the relationship between the core and shell sizes of the microcapsules and the flow rate of the external phase, the flow rate of the internal phase, and the voltage is statistically analyzed, as shown in Figure 1 .

[0027] Under the action of liquid laminar flow and external voltage, the mixed pre-gel liquid and oxygen-rich fluorocarbon liquid form core-shell structure microdroplets at the outlet of the capillary, as shown in Figure 1 a. In the calcium chloride collection liquid, calcium ions replace sodium ions in sodium alginate to form a calcium alginate hydrogel shell, and finally obtain morphologically stable core-shell structure microcapsules, as shown in Figure 1b. By adjusting the flow rate of the inner and outer phases and the voltage, we found that the inner core diameter became smaller as the outer phase flow rate increased; the inner core diameter became larger as the inner phase flow rate increased; and the microcapsule particle size became smaller as the voltage increased, as shown in Figure 1 g-i. Through experiments, we found that the microcapsules were most stable when the outer phase microchannel flow rate was 40-80 μL / min, the inner phase microchannel flow rate was 10-15 μL / min, and the outlet electric field force was 8-12 kV, and the particle size was 385.28 ± 23.04 microns, as shown in Figure 1 c. After freeze-drying the obtained microcapsules, scanning electron microscopy was performed, and the complete shell and clear core-shell structure were observed, as shown in Figure 1 d-f.

[0028] Example 2: Functional characterization of ultrasound-responsive oxygen-loaded and traditional Chinese medicine microcapsules: (1) The microcapsules were placed in a room temperature phosphate buffer solution, and a therapeutic ultrasound wave was used to give a continuous external stimulus (1.0 MHz, 1.5 W cm -2 ). The morphology of the microcapsules after 0, 20, 40, and 60 s of stimulation was recorded under a stereomicroscope, as shown in Figure 2 We found that the microcapsules were initially complete and stable, and under the action of ultrasound for 20 s, the oxygen-rich fluorocarbon liquid gradually underwent a liquid-gas phase transition to form a gas core; under the action of ultrasound for 40 s, the gas core gradually increased in size; and under the action of ultrasound for 60 s, the inner core completely vaporized and broke the hydrogel shell due to the increase in volume, achieving the release of oxygen, as shown in Figure 2 b-e. By monitoring the oxygen content in the solution using a dissolved oxygen meter, we found that compared with the passive release of oxygen, the stimulation of ultrasound could cause a high-level release of oxygen from the microcapsules, as shown in Figure 2 f.

[0029] (2) The oxygen-loaded and traditional Chinese medicine microcapsules were continuously stimulated by a therapeutic ultrasound wave (1.0 MHz, 1.5 W cm -2 ) for 0, 5, 10, 15, and 20 min, and the supernatant was extracted at different time intervals and mixed with a singlet oxygen detection solution to measure the fluorescence intensity of different groups. We found that the generation of singlet oxygen was significantly more efficient under the oxygen-loaded condition, confirming that our microcapsules could enhance the sonodynamic efficiency of curcumin, as shown in Figure 2 g, h.

[0030] Example 3: Antibacterial and anti-inflammatory properties of ultrasound-responsive oxygen-loaded and traditional Chinese medicine microcapsules: (1) Escherichia coli and Staphylococcus aureus were co-cultured with oxygen-loaded and traditional Chinese medicine microcapsules, and were divided into ultrasound stimulation and non-ultrasound stimulation groups. After co-culture, plating and colony formation were performed. The results are shown in Figure 3As shown in Figure 2, Escherichia coli and Staphylococcus aureus co-cultured with oxygen-carrying and Chinese medicine microcapsules and subjected to therapeutic ultrasound stimulation were almost killed, confirming the efficient antibacterial ability of ultrasound-responsive oxygen-carrying and Chinese medicine microcapsules in synergistic ultrasound treatment. Figure 3 We further observed Escherichia coli and Staphylococcus aureus by scanning electron microscopy and found that the bacteria showed obvious membrane damage after ultrasound-responsive oxygen carrier and Chinese medicine microcapsule synergistic ultrasound treatment, confirming its killing effect on bacteria. Figure 3 As shown in b.

[0031] (2) Ultrasound-responsive oxygen carriers and Chinese herbal medicine microcapsules were co-cultured with lipopolysaccharide-induced inflammatory macrophages and ultrasonic stimulation was applied. Immunofluorescence staining was used to observe the polarization of macrophages. The results showed that the inflammatory macrophages after ultrasound-responsive oxygen carriers and Chinese herbal medicine microcapsules synergistic ultrasound treatment showed significant M2 polarization, confirming its anti-inflammatory properties. The results are as follows Figure 3 As shown in d.

[0032] Example 4: Application of Ultrasound-Responsive Oxygen Carrier and Traditional Chinese Medicine Microcapsules in the Treatment of Infected Wounds: (1) After anesthetizing the mice, an 8 mm diameter wound was made on their backs and Staphylococcus aureus liquid was dripped to construct an infection wound model. The mice were randomly divided into four groups, namely: control group, P / C-MCs group, P / C-MCs + US group and PO / C-MCs + US group. The changes in the wound surface were recorded by taking photos during the different treatment processes, such as Figure 4 shown.

[0033] Among them, the control group did not receive any treatment intervention.

[0034] The P / C-MCs group contained microcapsules encapsulating curcumin and applied to the infected wound.

[0035] In the P / C-MCs + US group, microcapsules encapsulating curcumin were applied to the infected wound under ultrasound irradiation.

[0036] In the PO / C-MCs + US group, the microcapsules carrying oxygen and curcumin acted together on the infected wound under ultrasound irradiation.

[0037] We found that compared with the other groups, the wounds in the PO / C-MCs + US group healed faster after ultrasound-responsive oxygen carrier and Chinese medicine microcapsule synergistic ultrasound treatment, and the redness and swelling around the wounds were significantly reduced compared with the other groups. Figure 4 The skin of the wounds in each group was further stained with hematoxylin-eosin, and it was found that the wound tissue repair was more complete and the inflammatory cells were less after ultrasound-responsive oxygen carrier and Chinese medicine microcapsule synergistic ultrasound treatment. Figure 4c, f.

[0038] (2) Further collagen, IL-6, TNF-alpha, HIF-1 alpha immunostaining of wound tissue and quantitative statistics, we found that the wound tissue after ultrasound responsive oxygen and traditional Chinese medicine microcapsule synergistic ultrasound treatment of collagen deposition more, IL-6, TNF-alpha, HIF-1 alpha positive less, indicating that the wound repair after treatment is better, the inflammatory response is reduced, the hypoxic state is relieved, the results are shown in Figure 5 Meanwhile, the main organs of mice, kidney, lung, heart, liver, spleen were stained with hematoxylin-eosin, and there was no significant difference between each group, confirming that the ultrasound responsive oxygen and traditional Chinese medicine microcapsule synergistic ultrasound treatment is biosafety, and the results are shown in Figure 6

[0039] The present application provides a kind of ultrasound responsive oxygen and traditional Chinese medicine microcapsule and its preparation method and the application of thought and method in treating infected wound, the method and approach for specifically realizing this technical scheme are many, above-mentioned only preferred embodiment of the present application, it should be pointed out, for the ordinary skilled person in the prior art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application. The components not explicitly described in the embodiment can be realized by existing technology.​

Claims

1. A method for preparing ultrasound-responsive oxygen-carrying and traditional Chinese medicine microcapsules, characterized in that: The steps include: S1. Coaxially connect and fix two glass tubes with different inner diameters. The glass tube with the larger inner diameter serves as the outer phase channel, and the glass tube with the smaller inner diameter serves as the inner phase channel. S2, dissolving sodium alginate and curcumin in deionized water to prepare a mixed pre-gel solution; introducing oxygen into the liquid fluorocarbon compound to obtain an oxygen-enriched fluorocarbon liquid after treatment; S3. The mixed pre-gel solution and oxygen-rich fluorocarbon liquid prepared in step S2 are respectively introduced into the external phase channel and the internal phase channel of step S1 at the same time, and an electric field force is applied at the channel outlet. After electrospraying, the mixed pre-gel solution and the oxygen-rich fluorocarbon liquid fall into the collection liquid to obtain oxygen-carrying and traditional Chinese medicine microcapsules.

2. The method for preparing the ultrasound-responsive oxygen-carrying and traditional Chinese medicine microcapsules according to claim 1, wherein: In step S1, the outer phase channel is a capillary with an inner diameter of 450-500 microns; the inner phase channel is a capillary with an inner diameter of 100-150 microns.

3. The method for preparing the ultrasound-responsive oxygen-carrying and traditional Chinese medicine microcapsules according to claim 1, wherein: In step S2, the concentration of sodium alginate in the mixed pre-gel solution is 2-2.5 wt %, and the concentration of curcumin is 0.01-0.02 M.

4. The method for preparing the ultrasound-responsive oxygen-carrying and traditional Chinese medicine microcapsules according to claim 1, wherein: In step S2, the dissolved oxygen content in the oxygen-enriched fluorocarbon liquid is 30-40 mg / L.

5. The method for preparing the ultrasound-responsive oxygen-carrying and traditional Chinese medicine microcapsules according to claim 1, wherein: In step S3, the flow rate of the mixed pregel solution into the external phase channel is 40-80 μL / min; the flow rate of the oxygen-rich fluorocarbon solution into the internal phase channel is 10-15 μL / min.

6. The method for preparing the ultrasound-responsive oxygen-carrying and traditional Chinese medicine microcapsules according to claim 1, wherein: In step S3, the electric field force applied at the channel outlet is 8-12 kV.

7. The method for preparing the ultrasound-responsive oxygen-carrying and traditional Chinese medicine microcapsules according to claim 1, characterized in that: In step S3, the collected liquid is a calcium chloride solution with a concentration of 1.5-2 wt%.

8. Ultrasound-responsive oxygen-carrying and traditional Chinese medicine microcapsules prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the ultrasound-responsive oxygen carrier and traditional Chinese medicine microcapsules according to claim 8 in preparing medicines for treating infected wounds.

10. The use according to claim 9, characterized in that Therapeutic ultrasound is used to continuously stimulate the oxygen-carrying and Chinese medicine microcapsules to achieve the release of active pharmaceutical ingredients. The therapeutic ultrasound has a frequency of 0.8-1.0 MHz and a power of 1.5 W cm -2 , the stimulation time is 10-20min.

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