CN113908328B

CN113908328B - Antibacterial hemostatic porous microsphere based on sodium alginate and nanocrystalline cellulose

Info

Publication number: CN113908328B
Application number: CN202111061058.3A
Authority: CN
Inventors: 欧阳小琨; 赵丽娟; 凌俊红; 蒋方园; 杨立业
Original assignee: Zhejiang Ocean University ZJOU
Current assignee: Zhejiang Ocean University ZJOU
Priority date: 2021-09-10
Filing date: 2021-09-10
Publication date: 2023-01-24
Anticipated expiration: 2041-09-10
Also published as: CN113908328A

Abstract

The invention provides a preparation method of antibacterial hemostatic porous microspheres with good biocompatibility, nontoxicity and biodegradability, which uses an inverse emulsion method to prepare sodium alginate/cellulose nanocrystalline porous microspheres; the sodium alginate/cellulose nanocrystal loaded epsilon-polylysine porous microspheres prepared by the method are applied to wound hemostasis, can quickly absorb blood, reduce blood flow and promote local aggregation of erythrocytes, blood coagulation factors, blood platelets and the like on wounds, so as to promote hemostasis; in addition, because of its antibacterial property, it can prevent wound infection and promote tissue healing.

Description

Antibacterial hemostatic porous microsphere based on sodium alginate and nanocrystalline cellulose

Technical Field

The invention belongs to the field of high polymer materials, and particularly relates to an antibacterial hemostatic porous microsphere based on sodium alginate and nanocrystalline cellulose.

Background

Many hemostatic agents or hemostatic dressings developed at home and abroad in recent years do not have antibacterial performance, but most products are antibacterial particles such as gold, silver, iodine and the like which are used for carrying and compounding antibiotic medicines or compounding antibacterial action by using materials. However, although the compounding of antibiotic drugs can reduce the dosage of oral or injection antibiotics, the drug resistance problem of long-term use of antibiotics cannot be avoided; more importantly, the hemostatic dressing compounded by the antibacterial particles such as silver, iodine and the like can not be absorbed and degraded by human bodies, is only used on the skin of the body surface and is not suitable for being used as the hemostatic dressing used in vivo, so that the special hemostatic treatment of a plurality of important clinical operations and medical first-aid can not be met.

The natural polymer material cellulose has good biocompatibility and biodegradability, and cellulose-based hemostatic microsphere materials have been reported more. However, the existing microsphere materials have good hemostatic effect but do not have antibacterial performance, and after hemostasis, the microspheres have strong hydrophilic performance, so that bacteria breeding and infection of wounds are easily caused. In addition, a great amount of chemical cross-linking agents such as epichlorohydrin, trimethylolpropane and the like are used in the cellulose-based hemostatic microsphere material prepared at present, and the residue of the cross-linking agents easily causes stronger cytotoxicity and influences the biocompatibility of the microsphere.

Disclosure of Invention

Based on the problems, the invention provides a preparation method of an antibacterial hemostatic porous microsphere with good biocompatibility, nontoxicity and biodegradability, and a preparation method of an antibacterial hemostatic porous microsphere based on sodium alginate and nanocrystalline cellulose, wherein the sodium alginate/nanocrystalline cellulose porous microsphere is prepared by using a reversed phase emulsion method, and the preparation method specifically comprises the following steps:

1) Preparation of oil phase: adding span 80 into liquid paraffin, stirring at 700rpm at 45 ℃ for 1h;

2) Preparation of the aqueous phase: dissolving sodium alginate and nanocrystalline cellulose in 30ml of deionized water;

3) Preparing microspheres: dropwise adding the water phase into the oil phase, stirring at 800rpm for 2h, dropwise adding 20wt% of 6ml calcium chloride solution, and stirring at 800rpm for 4h; then washing with n-hexane, absolute ethyl alcohol and deionized water for 2 times respectively, centrifuging to remove supernatant, and freeze-drying to obtain sodium alginate/nanocrystalline cellulose SA/NCC porous microspheres; and (3) putting 100mg of the freeze-dried SA/NCC porous microspheres into 20ml of polylysine solution, stirring at 200rpm for 24h, centrifuging, and freeze-drying to obtain the sodium alginate/nanocrystalline cellulose-loaded epsilon-polylysine SA/NCC @ PL porous microspheres.

Preferably, the weight ratio of span 80 to liquid paraffin in the step 1) is 3%.

Preferably, the weight ratio of the sodium alginate to the nanocrystalline cellulose in step 2) is 5:1.

Preferably, the weight ratio of the sodium alginate to the deionized water in the step 2) is 1.25%.

Preferably, the weight ratio of the water phase to the oil phase in step 3) is 1:3.

Preferably, the mass concentration of the calcium chloride solution in the step 3) is 20wt%.

Preferably, the polylysine solution in the step 3) has a mass volume concentration of 6mg/ml.

On the other hand, the invention provides the antibacterial hemostatic porous microsphere based on sodium alginate and nanocrystalline cellulose prepared by the method.

The preparation method of the antibacterial hemostatic porous microspheres containing sodium alginate and nanocrystalline cellulose is simple, the materials are easy to obtain, and the safety coefficient is good; the sodium alginate/nanocrystalline cellulose-loaded epsilon-polylysine porous microspheres prepared by the method are applied to wound surface hemostasis, can quickly absorb blood, reduce blood flow and promote local aggregation of erythrocytes, blood coagulation factors, blood platelets and the like in a wound, thereby promoting hemostasis; in addition, because of its antibacterial property, it can prevent wound infection and promote tissue healing. The porous microspheres prepared by the method have the characteristics of good biocompatibility, antibacterial property, no toxicity and the like, and are expected to show unique advantages in the aspects of wound hemostasis, wound healing and the like.

Drawings

FIG. 1 shows the imbibition rates of SA/NCC @ PL porous microspheres at different ratios.

FIG. 2 is a graph of the porosity of SA/NCC @ PL porous microspheres at different ratios.

FIG. 3 is a graph of the coagulation index of different materials.

Among them, control was negative control Blue crown chitosan powder, SA was sodium alginate, SA/NCC was sodium alginate/nanocrystalline cellulose, a was (4% op 3).

FIG. 4 shows the hemolysis rates of different materials.

N.cont. negative control group, p.cont. positive control group SA sodium alginate, SA/NCC sodium alginate/nanocrystalline cellulose, SA/NCC @ pl porous microspheres (3% op 5.

FIG. 5 is a scanning electron micrograph of SA/NCC porous microspheres and SA/NCC @ PL porous microspheres.

FIG. 6 shows the cell viability of SA/NCC @ PL porous microspheres at different concentrations.

FIG. 7 shows the bacteriostatic rate and the bacteriostatic map of different materials on Escherichia coli, staphylococcus aureus and Pseudomonas aeruginosa.

FIG. 8 is a scanning electron microscope image of SA/NCC @ PL porous microspheres adhering platelets and erythrocytes and intertwining insoluble fibrin. Wherein A, B is a scanning electron microscope image of SA/NCC @ PL porous microsphere adhesion platelets and erythrocytes, and C, D is a scanning electron microscope image of SA/NCC @ PL porous microsphere winding insoluble fibrin.

Fig. 9 shows the time to hemostasis and the amount of bleeding for different materials in a mouse model of liver bleeding.

Figure 10 is the healing capacity of different materials in a mouse wound model.

Fig. 11 is the H & E staining of new tissue at day 10 post-trauma.

Detailed Description

The following examples are intended to further illustrate the present invention, but they are not intended to limit or restrict the scope of the invention.

Example 1

1) Oil phase: adding span 80 (2%, 3%,4%,5%, 6%) in different amounts into 90g of liquid paraffin, 700rpm, and stirring at 45 ℃ for 1h;

2) Water phase: sodium alginate and nanocrystalline cellulose (different mass ratios of both in the aqueous phase: 1,2:1,3.

3) Dropwise adding the water phase into the oil phase (the weight ratio of the water phase to the oil phase is 1:3), stirring at 800rpm for 2h, dropwise adding 6ml of calcium chloride solution (20 wt%), and stirring at 800rpm for 4h; then washing with n-hexane, absolute ethyl alcohol and deionized water for 2 times respectively, centrifuging to remove supernatant, and freeze-drying to obtain SA/NCC porous microspheres; and (3) putting 100mg of the freeze-dried SA/NCC porous microspheres into 20ml of polylysine solution (6 mg/ml), stirring for 24h at 200rpm, centrifuging, and freeze-drying to obtain SA/NCC @ PL porous microspheres prepared under the conditions of different amounts of span 80 (OP) and different amounts of sodium alginate and nanocrystalline cellulose in different proportions.

Example 2

The imbibition and porosity of different amounts of span 80 (OP) prepared in example 1, different amounts of SA/ncc @ pl porous microspheres of sodium alginate and nanocrystalline cellulose in different ratios were measured, and the related results are shown in fig. 1 and fig. 2, resulting in two sets of microspheres preparation conditions a (4% OP 3); b (3% op 5).

And (3) measuring the liquid absorption rate of the porous microspheres: lyophilized SA/NCC @ PL porous microspheres were weighed as W1 and soaked in a centrifuge tube containing deionized water to be sufficiently expanded. Excess liquid was then removed by low speed centrifugation and the wet porous microspheres weighed and reported as W2. Calculating the liquid absorption rate according to the formula:

determination of porosity of porous microspheres: soaking a certain weight of freeze-dried SA/NCC @ PL porous microspheres in 4mL of absolute ethyl alcohol, and ultrasonically oscillating for 10min. Then absolute ethanol was added to 4ml. The weight of the freeze-dried porous microspheres was recorded as Wa. The weight of the absolute ethyl alcohol before soaking in the porous microspheres is recorded as Wb. The total volume of liquid and absolute ethanol supplemented porous microspheres after sonication was recorded as Wc. Wet porous microspheres that absorb ethanol are denoted as Wd. The porosity (P) of the SA/NCC @ PL porous microspheres was calculated by the following formula:

example 3

Comparing the blood coagulation indexes (BCI%) of the different materials with the negative control group and the commercially available blue crown brand chitosan hemostatic powder, the blood coagulation index of the group b (3% OP 5: 1.5%) was the lowest and the blood coagulation effect was the best, and the related results are shown in FIG. 3.

Determination of blood coagulation index: 10mg of SA/NCC @ PL porous microspheres were placed in the bottom of a glass vial and preheated at 37 ℃ for 10min. Then adding 200ul of fresh anticoagulated rabbit blood, mixing uniformly, adding 20ul of CaCl ₂ The solution was mixed and cultured at 37 ℃ for 10min. 5ml of deionized water was added to the glass flask and shaken (50 rpm) for 2min to clean the unsolidified red blood cells. The absorbance of the resulting solution was measured at 540nm with an ultraviolet spectrophotometer. The absorbance of 200ul of unclotted whole blood in 5ml of deionized water was used as a negative control. BCI was calculated according to the following formula:

determination of the hemolysis rate: fresh rabbit anticoagulated blood was mixed with PBS pH =7.4, and erythrocytes in serum were collected by centrifugation. The obtained red blood cells were diluted with PBS. The sample was crushed with agate, PBS was added and the suspension was made ultrasonically for 2h. PBS-diluted erythrocytes were added to PBS suspensions containing different samples and different concentrations of SA/NCC @ PL porous microspheres. Deionized water and PBS were used as positive and negative controls, respectively. All groups were incubated for 3h at 37 ℃ in a shaker, centrifuged and the absorbance measured at 540nm using an ultraviolet spectrophotometer. The percent hemolysis was calculated as follows:

wherein As, aw and Ap are the absorbances of the sample, deionized water and PBS respectively.

Hemolysis rate is a simple and reliable method for assessing the blood compatibility of an implant. The results of the related hemolysis rate are shown in fig. 4, and according to the international standard, only the products with hemolysis rate lower than 5% can meet the blood safety requirement of clinical application. The hemolysis rate of the SA/NCC @ PL group is only 0.99%, which indicates that the SA/NCC @ PL porous microspheres are safe to blood and suitable for being developed as hemostatic materials.

SEM characterization of the prepared SA/NCC and SA/NCC @ PL porous microspheres was performed by using a scanning electron microscope (FEI Quanta 250, japan) to observe the morphological characteristics of the surfaces of the microspheres, and the results are shown in FIG. 5.

The microspheres were found to be around 2 μm in size by SEM characterization. Compared with SA/NCC microspheres, the polylysine-loaded microspheres have denser and more uniform surface pores. The SA/NCC @ PL microsphere with high porosity has better blood absorption capacity when being used for a bleeding wound surface, thereby aggregating platelets and blood cells and promoting the rapid formation of blood clots.

Example 4

Cytotoxicity of SA/NCC @ PL porous microspheres was evaluated by fibroblast (L929) and MTT method. SA/NCC @ PL porous microspheres were sterilized, and then immersed in MEM medium (37 ℃) and incubated for 24 hours. Gradually diluting the leaching solution of the porous microspheres with different concentrations, detecting the cell viability by adopting an MTT method, and taking a blank culture medium without adding the porous microspheres as a negative control group. Relative cell viability was calculated as follows:

the results of the relevant assays are shown in FIG. 6, with results classified according to cell viability (< 50%, toxicity; 51-70%, low cytotoxicity; >70%, no cytotoxicity). Cell viability for SA/NCC @ PL was greater than 80% at all concentrations tested, and relative cell activity was close to 90% especially at SA/NCC @ PL concentrations of 30. Mu.g/ml. Therefore, SA/NCC @ PL porous microspheres are safe for cells in vitro.

Example 5

To combat infection and promote wound healing, we chose staphylococcus aureus (s. Aureus), escherichia coli (e. Coli) and pseudomonas aeruginosa (p. Aeruginosa) for bacteriostatic testing. Chitosan powder, SA/NCC @ PL10mg, containing different concentrations of PL (1.0, 2.0,3.0,4.0,6.0 mg/ml) and 10ml of bacterial suspension (in sterile water, 10 ml) ⁴ CFU/ml) (37 ℃) were incubated for 1h. Then, 50ul of the hatching bacterial suspension was spread evenly on solid agar medium and cultured (37 ℃) for 12 hours. Finally, recording the number of viable bacteria on the solid agar culture medium, and calculating the bacteriostatic rate (Ar,%) of the material. Positive pairThe control group is the commercial blue crown chitosan hemostatic powder, and the negative control group is the bacterial suspension with the same activity without any treatment. The formula for calculating the bacteriostasis rate is as follows:

where Nn is the number of colonies in the negative control group and Ns is the number of colonies in the sample.

The result is shown in FIG. 7, the bacteriostasis rates of the SA/NCC @ PL porous microspheres are close to 100%, and the antibacterial property is remarkable. Especially, the bacteriostasis rate to staphylococcus aureus reaches 98 percent. And the bacteriostasis rate of the material without the loaded PL is lower than 60 percent.

Example 6

The adhesion of SA/NCC @ PL porous microspheres to platelets and erythrocytes and the formation of fibrin were characterized by scanning electron microscopy (FEI Quanta 250, japan). The SA/NCC @ PL porous microspheres were soaked in PBS (37 ℃) for 2h, fresh rabbit whole blood was added, and incubation was carried out at 37 ℃ for 1h. Then washed 3 times with PBS, fixed 2.5% glutaraldehyde at 4 deg.C for 2h, and soaked in 90% and 100% ethanol solutions for 10min, respectively. After freeze-drying for 12h, the porous microspheres are observed to be adhered with platelets, red blood cells and wound fibrin by a scanning electron microscope.

As a result, SA/NCC @ PL showed strong binding ability to red blood cells and platelets, and a large number of red blood cells and platelets adhered and aggregated around the microspheres, as shown in FIG. 8. In particular, it can be seen that platelets surrounding the microspheres exhibit many prosthetic foot deformations and stretches, indicating that the platelets are activated and participate in the hemostatic process. In addition, SA/NCC @ PL microspheres are intertwined with fibrin aggregates, eventually forming a stable blood clot.

Example 7

The liver bleeding model is a common animal model for testing the hemostatic performance. The hemostasis time and amount of bleeding of different materials in a mouse model of liver bleeding were examined. All samples were sterilized with uv radiation for 1h prior to testing. A cross-cut of approximately 1cm was made on the exposed mouse liver. At the end of the experiment, all mice were killed with intravenous air. The clotting time and amount of bleeding from the different materials were recorded and the results are shown in fig. 9.

The data show that after SA/NCC @ PL treatment, the bleeding amount is reduced from 55mg of the negative group to 13mg, and the hemostasis time is shortened from 73s to 48s. The bleeding amount and the bleeding time are similar to those of the commercially available blue crown hemostatic powder. SA/NCC @ PL showed significant advantages in the treatment of bleeding models.

FIG. 10 is the wound healing process observed on the mouse wound model. Wound healing was observed on

days

0,2, 6 and 10. The area of the wound treated with SA/NCC @ PL on day 2 contracted more, and hair grew rapidly around the wound. On day 10, the wound surface of SA/NCC @ PL group was nearly completely healed, and the remaining wound surface area was significantly smaller than that of the other groups. In the wound healing process, the SA/NCC @ PL group is always in the leading position, and the SA/NCC @ PL porous microspheres are proved to remarkably promote the healing of the full-layer defect wound.

To assess the quality of the newly formed skin tissue, further histological analysis was performed by H & E staining on day 10. The results are shown in fig. 11, on day 10 after surgery, the blank group of neodermis showing no hair follicles and sebaceous glands, incomplete keratinization, the other groups showing more hair follicles and sebaceous glands, and the epidermis and dermis layers being significantly stratified. The SA/NCC @ PL group hair follicles had begun to deform, forming an intact dermis similar to normal skin.

Claims

1. A preparation method of antibacterial hemostatic porous microspheres based on sodium alginate and nanocrystalline cellulose is characterized by comprising the following steps: the method for preparing the sodium alginate/nanocrystalline cellulose porous microspheres by using an inverse emulsion method comprises the following steps:

1) Preparation of oil phase: adding span 80 into the liquid paraffin, stirring at 700rpm at 45 ℃ for 1h;

3) Preparing microspheres: dropwise adding the water phase into the oil phase, stirring at 800rpm for 2h, dropwise adding 20wt% of 6ml calcium chloride solution, and stirring at 800rpm for 4h; then washing with normal hexane, absolute ethyl alcohol and deionized water respectively, centrifuging to remove supernatant, and freeze-drying to obtain SA/NCC porous microspheres; putting 100mg of the freeze-dried SA/NCC porous microspheres into 20ml of polylysine solution, stirring at 200rpm for 24h, centrifuging, and freeze-drying to obtain SA/NCC @ PL porous microspheres;

wherein, the weight ratio of span 80 to liquid paraffin in the step 1) is 3 percent; the weight ratio of the sodium alginate to the nanocrystalline cellulose in the step 2) is 5:1; the weight ratio of the sodium alginate to the deionized water in the step 2) is 1.25%.

2. The preparation method of the antibacterial hemostatic porous microsphere based on sodium alginate and nanocrystalline cellulose according to claim 1, characterized in that the weight ratio of the water phase to the oil phase in step 3) is 1:3.

3. The preparation method of the sodium alginate and nanocrystalline cellulose-based antibacterial hemostatic porous microsphere according to claim 1, characterized in that the mass volume concentration of the polylysine solution in the step 3) is 6mg/ml.

4. An antibacterial hemostatic porous microsphere based on sodium alginate, nanocrystalline cellulose prepared by the method of any one of claims 1-3.

5. Use of the sodium alginate, nanocrystalline cellulose-based antibacterial hemostatic porous microspheres of claim 4 in preparation of wound hemostasis and wound healing materials.