Preparation method and application of antibacterial flushing fluid applied to vacuum sealing drainage device

Abstract

The invention relates to the technical field of biological medicine, in particular to a preparation method and application of antibacterial flushing fluid applied to a vacuum sealing drainage device. The invention provides application of quaternary ammonium salt chitin in preparation of antibacterial flushing fluid. The antibacterial flushing fluid provided by the invention is simple and feasible in method, economical in material and suitable for industrial production, and the flushing fluid has the characteristics of safety, no toxicity and good biocompatibility. The antibacterial agent has a good antibacterial effect on gram-positive bacteria, gram-negative bacteria, anaerobic bacteria and other bacteria, can effectively control wound infection, can prevent generation of drug-resistant bacteria, and also can improve the inflammation condition of wounds.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular, to a preparation method and application of an antibacterial irrigation solution for a vacuum sealing drainage device. Background Art

[0002] Vacuum sealing drainage (VSD) technology is to seal the wound surface by using a bio-semipermeable membrane, connect an external negative pressure to form a closed negative pressure environment on the wound surface, and at the same time use an irrigation solution to drain necrotic tissues, exudates, and pus from the patient's wound surface to the outside of the body. VSD is widely used in the treatment of complex and refractory wound surfaces such as suppurative infections on the body surface, soft tissue defects, diabetic ulcers, and postoperative incision infections.

[0003] Currently, the irrigation solution used based on the VSD device in clinical practice is mainly 0.9% normal saline, and some doctors will also use antibiotics such as penicillins for irrigation according to the infection situation of the patient's wound surface. However, the wound surfaces treated with VSD technology usually have a complex situation of infection with various bacteria such as Gram-positive bacteria, Gram-negative bacteria, anaerobic bacteria, and fungi. Using normal saline or normal saline + antibiotics for irrigation has problems such as a single effect and a narrow antibacterial spectrum, and there is a risk of inducing the generation of drug-resistant bacteria. In addition, whether it is normal saline or antibiotics as the irrigation solution, they can only play a single irrigation or limited antibacterial role, and cannot achieve broad-spectrum antibacterial and promote the effects of wound surface angiogenesis, collagen deposition, fibroblast growth and migration, etc. The treatment effect on wound surfaces with severe infection and slow healing is limited.

[0004] Hyaluronic acid (HA) is a natural polysaccharide and one of the important components of the extracellular matrix (ECM). It exists in biological tissues such as the skin and connective tissues and plays an important role in the process of tissue repair and regeneration. Chitin is the second largest natural polymer compound after cellulose. Chitin-based materials have characteristics such as hemostasis, antibacterial, biodegradability in vivo, and biocompatibility, and are polymer materials with great development and application prospects. Summary of the Invention

[0005] The purpose of the present invention is to provide a quaternary ammonium chitin irrigation solution for use with a VSD device for use in the cleaning and repair of complex and refractory wound surfaces.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides the application of quaternary ammonium chitin in the preparation of an antibacterial irrigation solution.

[0008] Preferably, the degree of substitution of the quaternary ammonium chitosan is 0.42 to 0.56.

[0009] Preferably, the quaternary ammonium chitosan is mixed and crosslinked with hyaluronic acid to obtain an antibacterial irrigation solution; the antibacterial irrigation solution is an irrigation solution for vacuum sealing drainage.

[0010] Preferably, the volume ratio of the quaternary ammonium chitosan to hyaluronic acid is 1 to 3:1 to 3; the crosslinking time is 2.5 to 3.5 h.

[0011] Preferably, the preparation method of the quaternary ammonium chitosan is as follows:

[0012] (1) Dissolve chitosan in a KOH / urea system to obtain a chitosan solution;

[0013] (2) Mix and react the chitosan solution with 2,3-epoxypropyltrimethylammonium chloride to obtain quaternary ammonium chitosan.

[0014] Preferably, it further includes: dialyzing the quaternary ammonium chitosan using a semipermeable membrane dialysis bag with a molecular weight cut-off of 8000 to 14000 Da, and then freeze-drying at -20 to -30 °C for 40 to 60 h.

[0015] Preferably, the degree of deacetylation of the chitosan in step (1) is 40% to 55%; the concentration of chitosan in the chitosan solution is 1.5% to 2.5%.

[0016] Preferably, in the KOH / urea system of step (1), the mass concentration of KOH is 14% to 18%, and the mass concentration of urea is 6% to 10%.

[0017] Preferably, the molar ratio of the chitosan solution to 2,3-epoxypropyltrimethylammonium chloride in step (2) is 1:5 to 7.

[0018] Preferably, the reaction time in step (2) is 20 to 28 h.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The irrigation solution material provided by the present invention is economical, suitable for industrial production, and the irrigation solution has the characteristics of being safe, non-toxic, and having good biocompatibility. It has good antibacterial effects against bacteria such as Gram-positive bacteria, Gram-negative bacteria, and anaerobic bacteria, can effectively control wound surface infections, prevent the generation of drug-resistant bacteria, and improve the inflammatory conditions of the wound. The irrigation solution also has the effects of promoting angiogenesis, collagen deposition, and fibroblast growth and migration on the wound surface, can accelerate the healing of the wound surface, and has good use effects for the treatment of complex and difficult-to-heal wound surfaces and the use scenarios in plateau areas.

[0021] Generally speaking, the higher the degree of substitution of the quaternary ammonium salt, the stronger the antibacterial property, but the impact on cells will also be more significant. Therefore, we select quaternary ammonium salt chitin with a low degree of substitution on the premise of maintaining the same antibacterial property. This range of degree of substitution (0.42 - 0.56) can better ensure antibacterial property while taking into account biological safety. Moreover, in the present invention, quaternary ammonium salt chitin and hyaluronic acid are used in combination. Physical cross-linking reaction occurs between quaternary ammonium salt chitin and hyaluronic acid, and electrostatic attraction occurs between charges to form ionic bonds. The addition of hyaluronic acid improves the affinity of the irrigation solution for the wound and enhances the use effect of the irrigation solution. Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0023] Figure 1 It is a detection chart of blood white blood cells, CRP, PCT, IL-6, α-SMA, and VEGF-α indicators for the control group and the experimental group.

[0024] Figure 2 It is a bacterial culture result chart for the control group and the experimental group on the 7th day of irrigation.

[0025] Figure 3 It is a wound anatomical chart for the control group and the experimental group.

[0026] Figure 4 It is an MRI examination of the wound tract of the wounds of the animals in the control group and the experimental group. Detailed Embodiments

[0027] The present invention provides the application of quaternary ammonium salt chitin in the preparation of an antibacterial irrigation solution.

[0028] In the present invention, the degree of substitution of the quaternary ammonium salt chitin is 0.42 - 0.56; preferably 0.46 - 0.52; further preferably 0.48 - 0.50; more preferably 0.49.

[0029] In the present invention, quaternary ammonium salt chitin and hyaluronic acid are mixed and cross-linked to obtain an antibacterial irrigation solution; the antibacterial irrigation solution is an irrigation solution for vacuum sealing drainage.

[0030] In the present invention, the volume ratio of the quaternary ammonium salt chitin to the hyaluronic acid is 1 - 3:1 - 3; preferably 2:1 - 3; further preferably 1:3.

[0031] In the present invention, the cross-linking time is 2.5 - 3.5 h; preferably 3 h.

[0032] In the present invention, the preparation method of the quaternary ammonium chitosan is as follows:

[0033] (1) Dissolve chitosan in a KOH / urea system to obtain a chitosan solution;

[0034] (2) Mix the chitosan solution with 2,3-epoxypropyltrimethylammonium chloride and react to obtain quaternary ammonium chitosan.

[0035] In the present invention, it further includes: dialyzing the quaternary ammonium chitosan using a semipermeable membrane dialysis bag with a molecular weight cut-off of 8000 - 14000 Da, and then freeze-drying at -20 to -30 °C for 40 - 60 h; preferably, dialyzing the quaternary ammonium chitosan using a semipermeable membrane dialysis bag with a molecular weight cut-off of 9000 - 13000 Da, and then freeze-drying at -22 to -28 °C for 44 - 56 h; more preferably, dialyzing the quaternary ammonium chitosan using a semipermeable membrane dialysis bag with a molecular weight cut-off of 10000 - 12000 Da, and then freeze-drying at -24 to -26 °C for 48 - 52 h; most preferably, dialyzing the quaternary ammonium chitosan using a semipermeable membrane dialysis bag with a molecular weight cut-off of 11000 Da, and then freeze-drying at -25 °C for 50 h.

[0036] In the present invention, for step (1), the degree of deacetylation of the chitosan is 40% - 55%; preferably 44% - 51%; more preferably 46% - 49%; most preferably 48%.

[0037] In the present invention, for step (1), the concentration of chitosan in the chitosan solution is 1.5 - 2.5%; preferably 1.7 - 2.3%; more preferably 1.9 - 2.1%; most preferably 2%.

[0038] In the present invention, for step (1), in the KOH / urea system, the mass concentration of KOH is 14 - 18%, and the mass concentration of urea is 6 - 10%; preferably, in the KOH / urea system, the mass concentration of KOH is 15 - 17%, and the mass concentration of urea is 7 - 9%; more preferably, in the KOH / urea system, the mass concentration of KOH is 16%, and the mass concentration of urea is 8%.

[0039] In the present invention, for step (2), the molar ratio of the chitosan solution to 2,3-epoxypropyltrimethylammonium chloride is 1:5 - 7; preferably 1:6.

[0040] In the present invention, for step (2), the reaction time is 20 - 28 h; preferably 21 - 27 h; more preferably 22 - 26 h; most preferably 24 h.

[0041] The technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0042] Example 1

[0043] (1) Purify chitin: Use a 1.2% (v / v) glacial acetic acid solution to dissolve 3.5 w / t% of chitin powder with a deacetylation degree of 40%. Use KOH or NaOH solution to adjust the pH value of the solution to 7.2 to precipitate chitin.

[0044] (2) Under negative pressure suction, rinse chitin with ultrapure water, and then freeze-dry it under low-temperature vacuum to obtain purified chitin. Dissolve chitin in a KOH / urea system (KOH mass concentration 18%, urea mass concentration 9%, and the final mass concentration of chitin is 2.3%).

[0045] (3) Mix the chitin solution with 2,3-epoxypropyltrimethylammonium chloride in a molar ratio of 1:6 and react to obtain liquid quaternary ammonium salt chitin with a quaternary ammonium salt substitution degree of 0.42. Then, dialyze it using a dialysis bag with a molecular weight cut-off of 8000 Da semi-permeable membrane, change deionized water at a frequency of 4 times a day, and continue dialysis for 7 days. The product obtained by dialysis is freeze-dried at -27°C under low-temperature vacuum for 50 h to obtain solid quaternary ammonium salt chitin.

[0046] (4) Mix quaternary ammonium salt chitin with hyaluronic acid molecules (the volume ratio of the two is 1:2, and the mass concentration of both substances is 0.8%), adjust the pH to 7, and stir and crosslink for 3.5 hours to obtain hyaluronic acid-quaternary ammonium salt chitin.

[0047] Example 2

[0048] (1) Purify chitin: Use a 0.9% (v / v) glacial acetic acid solution to dissolve 2.5 w / t% of chitin powder with a deacetylation degree of 40%-55%. Use KOH or NaOH solution to adjust the pH value of the solution to 7 to precipitate chitin.

[0049] (2) Under negative pressure suction, rinse chitin with ultrapure water, and then freeze-dry it under low-temperature vacuum to obtain purified chitin. Dissolve chitin in a KOH / urea system (KOH mass concentration 14%, urea mass concentration 7%, and the final mass concentration of chitin is 2%).

[0050] (3) Mix the chitin solution with 2,3-epoxypropyltrimethylammonium chloride in a molar ratio of 1:6 and react to obtain liquid quaternary ammonium salt chitin with a quaternary ammonium salt substitution degree of 0.56. Then, dialyze it using a dialysis bag with a semipermeable membrane having a molecular weight cut-off of 14,000 Da, replace the deionized water at a frequency of 4 times a day, and continue dialysis for 7 days. The product obtained by dialysis is freeze-dried at -25 °C under low-temperature vacuum for 43 h to obtain solid quaternary ammonium salt chitin.

[0051] (4) Mix quaternary ammonium salt chitin with hyaluronic acid molecules (the volume ratio of the two is 2:1, and the mass concentration of both substances is 0.8%), adjust the pH to 7, and stir and crosslink for 2.5 hours to obtain hyaluronic acid-quaternary ammonium salt chitin.

[0052] Example 3

[0053] (1) Purify chitin: Use a 1% (v / v) glacial acetic acid solution to dissolve chitin powder with a deacetylation degree of 40%-55% at 3 w / t%. Use KOH or NaOH solution to adjust the pH value of the solution to 7 to precipitate chitin.

[0054] (2) Under negative pressure suction, rinse chitin with ultrapure water, and it is the purified chitin after freeze-drying under low-temperature vacuum. Dissolve chitin in the KOH / urea system (the mass concentration of KOH is 16%, the mass concentration of urea is 8%, and the final mass concentration of chitin is 2%).

[0055] (3) Mix the chitin solution with 2,3-epoxypropyltrimethylammonium chloride in a molar ratio of 1:6 and react to obtain liquid quaternary ammonium salt chitin with a quaternary ammonium salt substitution degree of 0.42 - 0.56. Then, dialyze it using a dialysis bag with a semipermeable membrane having a molecular weight cut-off of 8,000 - 14,000 Da, replace the deionized water at a frequency of 4 times a day, and continue dialysis for 7 days. The product obtained by dialysis is freeze-dried at -25 °C under low-temperature vacuum for 48 h to obtain solid quaternary ammonium salt chitin.

[0056] (4) Mix quaternary ammonium salt chitin with hyaluronic acid molecules (the volume ratio of the two is 1:1, and the mass concentration of both substances is 0.8%), adjust the pH to 7, and stir and crosslink for 3 hours to obtain hyaluronic acid-quaternary ammonium salt chitin.

[0057] (5) Prepare a 1 μg / mL concentration of hyaluronic acid-quaternary ammonium salt chitin rinsing solution using pure water.

[0058] Experimental Example 1 Construction of an animal model of a multiple bacterial infection and complex and difficult-to-heal wound

[0059] 1. Experimental animals: Panamanian miniature pigs (body weight 20 - 25 kg); all pathogenic bacterial strains were purchased from the American Type Culture Collection (ATCC), including: Escherichia coli (E. coli, ATCC8739), Staphylococcus aureus (S. aureus, ATCC25923), Candida albicans (C. albicans, ATCC10231), and the anaerobic bacterium Bacteroides fragilis (B. fragilis, ATCC25285).

[0060] 2. Model construction: After anesthetizing the Panamanian miniature pigs with the anesthetic sodium pentobarbital (3%, 30 mg / Kg), an animal anesthesia ventilator was used to maintain stable respiration, and meloxicam analgesic was injected at a dose of 0.2 mg / kg. A drill was used to penetrate the muscle in the right hindlimb thigh muscle of the Panamanian miniature pig, and a 1 mL syringe was used to evenly inject 0.3 mL each of S. aureus, E. coli, C. albicans, and B. fragilis (anaerobic bacterium) at a concentration of 2×10 8 CFU / mL. Then, the two entrances and exits of the wound surface were sealed with 3M semi-permeable membrane, and a wound animal model with multiple bacterial infections could be formed after 24 hours.

[0061] 3. Experimental grouping:

[0062] Control group (VSD group): The wound surface was irrigated with 0.9% normal saline combined with a VSD device (manufacturer: Wuhan VSD Medical Technology Co., Ltd., model and specification: VSD-B-2-15×10×1).

[0063] Experimental group (Qc-VSD group): The wound surface was irrigated with the irrigation solution prepared in Example 3 at a concentration of 1 μg / mL combined with a VSD device.

[0064] The specific method is as follows: The wound surface of the Panamanian miniature pig was thoroughly debrided, foreign bodies on the wound surface were removed, necrotic tissues were removed, and the blood supply of the surrounding tissues of the wound was ensured. The VSD foam dressing was designed according to the shape characteristics of the wound tract of the wound surface, and the trimmed VSD foam dressing was placed on the wound surface tissue and completely covered. A supporting biological semi-permeable membrane was used for local sealing, and special attention was paid to the tightness at the uneven parts of the wound surface and the drainage pipeline. The VSD device was connected to external negative pressure, and an appropriate negative pressure suction (-175 mmHg - -275 mmHg) was set. The wound surface was irrigated for 2 hours every day after the operation. After the wound surface was fully irrigated, the negative pressure was turned off for 2 - 4 hours, and then the negative pressure was restored to continue drainage. The VSD device was removed after 7 days of irrigation.

[0065] On the 0th, 2nd, 4th, and 7th days of using the irrigation solution of Example 1, blood samples were collected from the experimental animals in each group to detect inflammatory indicators such as blood white blood cells, CRP (C-reactive protein), PCT (procalcitonin), and IL-6 (interleukin-6), and the degree of wound inflammation was analyzed; indicators such as α-SMA (α-smooth muscle actin) and VEGF-α (vascular endothelial growth factor-α) were detected, and the angiogenesis in the wound surface was analyzed. On the 7th day of using the irrigation solution of Example 1, wound secretion specimens were collected from the wounds of each group for bacterial smear culture. LB agar plates were used to detect the conventional bacterial infections of the wound surface, and blood agar plates were used to culture in an anaerobic environment to detect the anaerobic bacterial infections of the wound surface.

[0066] On the 7th day of the experiment, MRI (magnetic resonance imaging) examinations were performed on the hind limb wounds of the Bama miniature pigs in each group, and the healing conditions of the wound tracts in each group were analyzed from the imaging perspective. Finally, the animals in each group were euthanized, and the hind limb wound tracts of the Bama miniature pigs were dissected to visually observe the healing conditions of the wound tracts.

[0067] The results are as follows:

[0068] As Figure 1 shown in a-d, starting from the second day, the inflammatory indicators such as blood white blood cells, CRP, PCT, and IL-6 in the control group increased more significantly compared to the experimental group, and the inflammatory indicators in the experimental group remained at a relatively low level throughout. Until the 7th day of using the irrigation solution of Example 1, the inflammatory indicators basically returned to normal, indicating that the irrigation solution of Example 1 can effectively control the wound inflammation.

[0069] As Figure 1 shown in e-f, the detected values of α-SMA and VEGF-α in the experimental group were always higher than those in the control group after using the irrigation solution of Example 1, indicating the significant effect of the irrigation solution of Example 1 in promoting wound angiogenesis.

[0070] As Figure 2 shown in a-b, on the 7th day of using different irrigation solutions respectively, wound secretions were collected for bacterial smear culture. The bacterial loads of conventional bacteria and anaerobic bacteria on the wound surface in the control group were significantly higher than those in the experimental group, and the multiple bacterial infections in the experimental group had been effectively controlled. This reflects the significant effect of the irrigation solution of Example 1 in broad-spectrum antibacterial (a shows the situation of conventional bacterial infection; b shows the situation of anaerobic bacterial infection).

[0071] As Figure 3 shown, after the dissection of the wound surface in the control group, it could be visually observed that there were obvious purulent membranes on the wound surface, more exudates, severe infections, and there were still large gaps in the wound tissue, and there was no sign of healing. After the dissection of the wound surface in the experimental group, it could be visually observed that there was no pus on the wound surface, and the wound tissue had basically healed.

[0072] As Figure 4As shown, MRI examinations were performed on the wound tracts of two groups of animals. In the control group, the wound tract had a larger gap, and the wound tract from the skin to the deep muscle tissue could still be clearly distinguished. However, the MRI examination of the wound tract in the experimental group indicated that the wound surface had basically healed and no obvious gap was seen. Therefore, the irrigation solution provided by the present invention has a significant effect on promoting complex infected wound surfaces.

[0073] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Application of quaternary ammonium chitosan in the preparation of antibacterial flushing solution.

2. The use according to claim 1, characterized in that: The substitution degree of the quaternary ammonium salt chitosan is 0.42-0.

56.

3. The use according to claim 1 or 2, characterized in that: The quaternary ammonium salt chitosan and hyaluronic acid are mixed and cross-linked to obtain an antibacterial flushing liquid; the antibacterial flushing liquid is a flushing liquid for negative pressure sealing drainage.

4. The use according to claim 3, characterized in that: The volume ratio of the quaternary ammonium salt chitosan to the hyaluronic acid is 1-3:1-3; and the cross-linking time is 2.5-3.5 hours.

5. The use according to claim 1 or 2, characterized in that: The preparation method of the quaternary ammonium salt chitosan is: (1) dissolving chitosan in a KOH / urea system to obtain a chitosan solution; (2) The chitosan solution is mixed with 2,3-epoxypropyltrimethylammonium chloride to obtain quaternary ammonium salt chitosan.

6. The use according to claim 5, characterized in that: Also includes: The quaternary ammonium salt chitosan is dialyzed using a semipermeable membrane dialysis bag with a molecular weight cutoff of 8000 to 14000 Da, and then freeze-dried at -20 to -30°C for 40 to 60 hours.

7. The use according to claim 5, characterized in that: The deacetylation degree of the chitosan in step (1) is 40% to 55%; the concentration of the chitosan in the chitosan solution is 1.5 to 2.5%.

8. The use according to claim 5, characterized in that: In the KOH / urea system of step (1), the mass concentration of KOH is 14-18%, and the mass concentration of urea is 6-10%.

9. The use according to claim 5, characterized in that: The molar ratio of the chitosan solution to 2,3-epoxypropyltrimethylammonium chloride in step (2) is 1:5-7.

10. The use according to claim 5, characterized in that: The reaction time of step (2) is 20 to 28 hours.

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