CN118634357A - Kaolin-based antibacterial hemostatic powder and preparation method thereof - Google Patents

Abstract

The invention discloses a kaolin-based antibacterial hemostatic powder and a preparation method thereof. The invention relates to the technical field of hemostatic powder. The preparation method comprises the steps of stirring, freeze-drying and grinding a kaolin solution modified with an ε-polylysine solution and a polyethyleneimine solution to obtain the kaolin-based antibacterial hemostatic powder. The kaolin-based antibacterial hemostatic powder of the invention can not only effectively stop bleeding, but also realize the rapid transformation of "powder glue", effectively avoid the detachment of kaolin powder during hemostatic application, and reduce the risk of thrombosis during the application of kaolin for hemostasis; in addition, the risk of bacterial infection at the bleeding wound is also greatly reduced, the problem of wound being susceptible to bacterial infection after kaolin powder hemostasis is solved, and the thrombosis hazard that may exist during the application of kaolin for hemostasis is avoided.

Description

Kaolin-based antibacterial hemostatic powder and preparation method thereof

Technical Field

The invention relates to the technical field of hemostatic powder, in particular to a kaolin-based antibacterial hemostatic powder and a preparation method thereof.

Background Art

Uncontrolled bleeding is the main cause of serious injury or even death, which is a problem that needs to be solved urgently in the field of wound care medicine. The main hemostatic products on the market include gauze, sponges, bandages, hemostatic powders and new excipients. Compared with gauze and bandage hemostatic materials, powdered hemostatic materials can be used for irregular wounds and act on deep bleeding sites. At present, the hemostatic powders on the market include zeolite hemostatic powder, chitosan hemostatic powder, Yunnan Baiyao and starch hemostatic powder. For example, a new chitosan composite hemostatic powder is published in the Chinese patent (CN201410131189.8). The patent adopts the alkaline coprecipitation method to prepare chitosan and calcium salt by centrifugation, washing and dehydration to obtain chitosan composite hemostatic powder. The coagulation time of the hemostatic powder in the rabbit femoral artery bleeding model is 70±6 s, which has excellent hemostatic performance. However, the strong base sodium hydroxide reagent is introduced in the preparation process of chitosan composite hemostatic powder, which has certain safety hazards. A Chinese patent (CN200810063282.4) discloses a natural zeolite hemostatic agent, in which the pore size of the selected natural zeolite is 0.3~2.0 nm, and the blocky natural zeolite is crushed, modified and ion exchanged to prepare zeolite hemostatic powder for emergency hemostasis. However, the natural zeolite hemostatic agent has the problem of thermal burns during the hemostasis process, and the preparation of the hemostatic agent requires ion exchange and high temperature treatment, and the process is relatively complicated. In addition, the skin tissue is traumatized and bleeding, and it is susceptible to infection by pathogens, leading to more serious harm. Currently, the hemostatic materials on the market are mainly hemostatic, with a single function and no antibacterial properties. Therefore, there is a practical application demand for the development of new antibacterial hemostatic products.

Summary of the invention

The purpose of the present invention is to provide a kaolin-based antibacterial hemostatic powder and a preparation method thereof in view of the above-mentioned deficiencies in the prior art.

The invention discloses a method for preparing kaolin-based antibacterial hemostatic powder. The kaolin-based antibacterial hemostatic powder comprises the steps of stirring, freeze-drying and grinding a kaolin solution modified with epsilon-polylysine, a polyacrylic acid solution and a polyethyleneimine solution respectively.

Furthermore, the preparation method of ε-polylysine modified kaolin is: uniformly mix the ε-polylysine solution and the kaolin solution, freeze-dry and grind into powder to prepare the ε-polylysine modified kaolin.

Furthermore, the ε-polylysine modified kaolin solution is added to the polyacrylic acid solution or the polyethyleneimine solution and mixed evenly. Subsequently, the polyethyleneimine solution or the polyacrylic acid solution is added dropwise to the above mixed solution under stirring, and the mixture is freeze-dried and ground into powder to obtain the kaolin-based antibacterial hemostatic powder.

Furthermore, in step S1, the mass ratio of ε-polylysine to kaolin is 1:3-2.

Furthermore, in step S2, the concentration of the ε-polylysine modified kaolin solution is 0.05-1.6 g/mL.

Furthermore, in step S2, the concentration of the polyethyleneimine solution is 5-15 wt%.

Furthermore, in step S2, the concentration of the polyacrylic acid solution is 5-15 wt%.

Furthermore, in step S2, the mass ratio of ε-polylysine-modified kaolin, polyethyleneimine, and polyacrylic acid is 3-8:5:5.

A method for preparing kaolin-based antibacterial hemostatic powder prepared by the above-mentioned preparation method.

Kaolin [Al ₂ Si ₂ O ₅ (OH) ₄ ] is a 1:1 type layered aluminosilicate nanoclay, composed of octahedral Al(OH) ₃ stacked on tetrahedral SiO ₄ sheets, with a typical two-dimensional sheet structure, a negatively charged surface and high hydrophilicity. Kaolin has good biological safety, surface negative charge and high hydrophilicity, making it an efficient hemostatic material. ε-polylysine (ε-PL) is a natural cationic antibacterial polypeptide with good biocompatibility and broad-spectrum antibacterial properties. The present invention utilizes the mutual combination of positive and negative charges and uses ε-polylysine to physically modify negatively charged kaolin, giving it excellent antibacterial properties, breaking the limitations of kaolin powder in previous hemostatic applications.

The present invention conducts stirring, freeze drying, grinding and other steps on the kaolin solution modified by ε-polylysine, respectively with polyacrylic acid solution and polyethyleneimine solution, and prepares a kaolin-based composite hemostatic powder with excellent adhesion, hemostatic performance and antibacterial performance. When the powder adheres to the bleeding wound, it absorbs the blood components of the wound and quickly forms a gel, and the stress of the gel increases with the increase of strain, and the mechanical strength is significantly improved. In addition, kaolin has the effect of concentrating blood cells and platelets, further stimulating the coagulation cascade reaction, and promoting rapid hemostasis at the bleeding wound. At the same time, the positively charged ε-polylysine adsorbs negatively charged bacteria and destroys its cell membrane through electrostatic action, killing bacteria, and polyacrylic acid and polyethyleneimine also have certain antibacterial properties, and there is a synergistic antibacterial effect with the kaolin modified by ε-polylysine.

The kaolin-based antibacterial hemostatic powder of the present invention can not only effectively stop bleeding, but also realize the rapid transformation of "powder-glue", which can effectively avoid the shedding of kaolin powder during hemostatic application, and reduce the risk of thrombosis caused by the application of kaolin hemostatic powder; in addition, it also greatly reduces the risk of bacterial infection at bleeding wounds, solves the problem that wounds are susceptible to bacterial infection after kaolin powder hemostasis, and avoids the thrombosis hazards that may exist during the application of kaolin hemostatic powder.

The kaolin-based antibacterial hemostatic powder prepared by the present invention does not require additional chemical reagents, does not require special protection, is environmentally friendly, has a simple preparation process, is easy to industrialize, has no exothermic phenomenon, and can expand the application range of kaolin-based hemostatic materials.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is an XRD diagram of the kaolin-based antibacterial hemostatic powder prepared in Example 1;

FIG2 is a Zeta potential diagram of the kaolin-based antibacterial hemostatic powder prepared in Example 1;

FIG3 is a graph showing the powder-to-rubber conversion performance of the kaolin-based antibacterial hemostatic powder prepared in Example 1;

FIG4 is a stress-strain diagram of the kaolin-based antibacterial hemostatic powder prepared in Example 1;

FIG5 is a graph showing the compression modulus of the kaolin-based antibacterial hemostatic powder prepared in Example 1;

FIG6 shows the antibacterial properties of the kaolin-based antibacterial hemostatic powder prepared in Example 1;

FIG7 is the in vitro coagulation time of the kaolin-based antibacterial hemostatic powder prepared in Example 1;

FIG8 is an in vitro coagulation index of the kaolin-based antibacterial hemostatic powder prepared in Example 1;

FIG. 9 is a hemolysis rate experiment of the kaolin-based antibacterial hemostatic powder prepared in Example 1.

DETAILED DESCRIPTION

The following are specific embodiments of the present invention and the accompanying drawings to further describe the technical solution of the present invention, but the present invention is not limited to these embodiments.

Embodiment 1:

A method for preparing a kaolin-based antibacterial hemostatic powder is specifically completed according to the following steps: first, ε-polylysine and kaolin are mixed uniformly in a solvent at a mass ratio of 1:2, freeze-dried and ground into powder for standby use. Then, a 0.05 g/mL Kao@ε-PL solution, 5 wt% polyethyleneimine and 5 wt% polyacrylic acid solution are prepared. Subsequently, the prepared Kao@ε-PL solution is added to the polyacrylic acid solution and mixed uniformly. Under stirring conditions, the polyethyleneimine solution is added dropwise to the above mixed solution. After freeze-drying, it is ground into powder for standby use. The final product is recorded as PP-Kao@ε-PL.

Example 2

A method for preparing a kaolin-based antibacterial hemostatic powder is specifically completed according to the following steps: first, ε-polylysine and kaolin are mixed uniformly in a solvent at a mass ratio of 1:3, freeze-dried and ground into powder for standby use. Then, a 0.8 g/mL Kao@ε-PL solution, 10 wt% polyethyleneimine and 10 wt% polyacrylic acid solution are prepared. Subsequently, the prepared Kao@ε-PL solution is added to the polyacrylic acid solution and mixed uniformly. Under stirring conditions, the polyethyleneimine solution is added dropwise to the above mixed solution. After freeze-drying, it is ground into powder for standby use. The final product is recorded as PP-Kao@ε-PL.

Example 3

A method for preparing a kaolin-based antibacterial hemostatic powder is specifically completed according to the following steps: first, ε-polylysine and kaolin are mixed uniformly in a solvent at a mass ratio of 1:2, freeze-dried and ground into powder for standby use. Then, a 1.6 g/mL Kao@ε-PL solution, 15 wt% polyethyleneimine and 15 wt% polyacrylic acid solution are prepared. Subsequently, the prepared Kao@ε-PL solution is added to the polyacrylic acid solution and mixed uniformly. Under stirring conditions, the polyethyleneimine solution is added dropwise to the above mixed solution. After freeze-drying, it is ground into powder for standby use. The final product is recorded as PP-Kao@ε-PL.

Comparative Example 1

5 wt% polyethyleneimine and 5 wt% polyacrylic acid solution were mixed evenly, freeze-dried and ground into powder for later use. The final product was recorded as PP.

The powder-to-gel conversion performance, antibacterial performance and in vitro hemostatic performance of the antibacterial kaolin powder were verified. The specific steps are as follows:

As for its powder-to-gel conversion performance, 0.1-0.2 mL of anticoagulated rabbit blood was dripped onto the surface of PP-Kao@ε-PL powder to observe its powder-to-gel conversion phenomenon. In addition, 50 mg of PP-Kao@ε-PL was weighed and added to 50 µL of an aqueous solution containing rhodamine 6G. After immersion for 3 minutes, the PP-Kao@ε-PL gel was manually stretched and observed using pointed tweezers to evaluate the powder-to-gel conversion effect of the antibacterial kaolin powder.

For its mechanical properties, the stress and compression modulus of the two gels of PP and PP-Kao@ε-PL were tested by rheometer. 0.5 g of PP and PP-Kao@ε-PL powder were weighed into 10 mL sample bottles and 1 mL of deionized water was added to obtain antibacterial kaolin hemostatic gel. The strain sweep and frequency sweep of all prepared PP and PP-Kao@ε-PL gels were measured at 37 ° C. The plate diameter was 40 mm, and the interval between the plate and the sample was 1000 μm. Before the measurement, the gel was balanced at 37 ° C for 180 s to make it stable. The stress-strain curve was obtained under the condition of 0.01-1000% strain range and 1 Hz. For the compression modulus, the compression modulus of the prepared gel was tested with frequency under the condition of 0.01-100 Hz frequency range and 1% strain.

For its antibacterial properties, Escherichia coli ( E.coli ) and Staphylococcus aureus ( S.aureus ) were used as bacterial models. First, the bacteria were activated. Single E.coli and Staphylococcus aureus colonies were taken and placed in a 50 mL centrifuge tube containing 10 mL of liquid culture medium. The activated culture was then diluted 1×10 ³ times, 100 µL of the diluted activated culture was added to 10 mL of liquid culture medium, and 10 mg of Kao, PP and PP-Kao@ε-PL samples were added and cultured for 4~6 h. Subsequently, the cultured culture was diluted 1×10 ⁴ , and 100 µL of the diluted culture was spread on the surface of the agar plate. It was placed in a shaker at 37 ℃ and 180 r/min for 10~14 h, and then photographed and recorded. The experiment was repeated 3 times for each group of samples.

Among them, photo counting: use ImageJ to count the colonies in the photos of each experimental group, count the number of colonies on the agar plate, and calculate the colony forming unit (CFU).

CFU = (bacterial colony count × dilution factor) / coating volume

For the in vitro hemostasis experiment, a certain mass of sample was weighed and added to a 2 mL centrifuge tube, and then 50 μL of anticoagulated rabbit blood was added, followed by 5 μL of 0.1 M CaCl ₂ solution. After the above system was incubated at 37 °C for 5 min, 2 mL of deionized water was slowly added. The supernatant was then centrifuged (6000 rpm, 5 min). The supernatant of each group was collected, and the absorbance value of the supernatant of each group at 540 nm was detected by an ELISA instrument. Deionized water was used as the positive control group, PBS was used as the negative control group, and each group of samples was repeated 3 times. For the in vitro coagulation time experiment, 10 mg of sample was added to a 2 mL centrifuge tube, and then 50 μL of anticoagulated rabbit blood was added, followed by 5 μL of 0.1 M CaCl ₂ solution. The above system was incubated at 37 °C, and the system was inverted at fixed intervals to observe whether a blood clot was formed, and the coagulation time was recorded. The experiment was repeated 3 times for each group of samples. For the hemolysis experiment, a certain amount of whole blood was centrifuged and washed 3 times with PBS to collect blood cells. Then 10 mg of the sample was added to a 2 mL centrifuge tube, and the collected blood cell suspension was added to the centrifuge tube. Deionized water was used as the positive control group, and PBS was used as the negative control group. The above system was placed at 37 ° C for 1 h, and then the supernatant of each group was collected. The absorbance value of each group of samples at 540 nm was detected by an enzyme marker, and the experiment was repeated 3 times for each group of samples.

See Figure 1, which is an XRD diagram of the kaolin-based antibacterial hemostatic powder prepared in Example 1. The results show that polyethyleneimine and polyacrylic acid exist in an amorphous state, and the XRD characteristic peaks of Kao@ε-PL, PP-Kao@ε-PL and Kao are consistent, indicating that the antibacterial kaolin-based hemostatic powder is successfully prepared.

See Figure 2, the Zeta potential diagram of the kaolin-based antibacterial hemostatic powder prepared in Example 1. The results show that the potential of Kao powder is -10.07 mV, and after modification with ε-polylysine, the potentials of Kao@ε-PL and PP-Kao@ε-PL powders are 27.1 mV and 46.32 mV, respectively. After the positively charged ε-polylysine and amino-containing polyethyleneimine are compounded with the negatively charged kaolin, the Zeta potential of the kaolin-based antibacterial hemostatic powder is changed.

See Figure 3 for the powder-to-gel conversion performance of the kaolin-based antibacterial hemostatic powder prepared in Example 1. From the experimental results, it can be observed that after the Kao@ε-PL powder is modified by polyethyleneimine and polyacrylic acid, the powder exhibits excellent blood absorption performance, and the powder completely absorbs anticoagulated rabbit whole blood and forms a gel within 4 to 6 seconds. In addition, the powder has excellent viscoelasticity after absorbing the solution to form a gel.

See Figure 4, which is a stress-strain diagram of the kaolin-based antibacterial hemostatic powder prepared in Example 1. The results show that after adding Kao@ε-PL, as the strain increases, the stress of the PP gel is 5.8 kPa when the strain is 165%, while the stress of the PP-Kao@ε-PL gel is 8.9 kPa. In addition, the stress of the PP-Kao@ε-PL gel increases with the increase of strain.

See Figure 5, which is a compression modulus diagram of the kaolin-based antibacterial hemostatic powder prepared in Example 1. The experimental results show that after adding Kao@ε-PL, the compression modulus of PP and PP-Kao@ε-PL both increase with the increase of frequency, but the compression modulus of PP-Kao@ε-PL is significantly higher than that of PP gel, indicating that after adding Kao@ε-PL, the mechanical strength of PP-Kao@ε-PL gel is significantly improved.

See Figure 6 for the antibacterial properties of the kaolin-based antibacterial hemostatic powder prepared in Example 1. The results show that compared with the control group, Kao powder has no bacterial inhibition phenomenon (Figure 6a). Positively charged PP powder can electrostatically bind to negatively charged bacteria and then destroy the cell membrane of bacteria, which has a certain bacterial inhibition effect but is not obvious. PP-Kao@ε-PL powder has obvious antibacterial effects on both E.coli and S.aureus bacteria, and the CFU of both bacteria is 0 (Figure 6b and c). It shows that PP-Kao@ε-PL powder has excellent antibacterial properties.

See Figure 7, the in vitro coagulation time of the kaolin-based antibacterial hemostatic powder prepared in Example 1. As shown in the figure, the coagulation time of the control group is 424±23 s, while the in vitro coagulation times of Kao, PP, Kao@ε-PL and PP-Kao@ε-PL are 135±6 s, 178±32 s, 181±35 s and 132±7 s, respectively. The experimental results show that PP-Kao@ε-PL has obvious in vitro coagulation performance.

See Figure 8, the in vitro coagulation index (BCI) of the kaolin-based antibacterial hemostatic powder prepared in Example 1. The results show that the in vitro coagulation indexes of Kao, PP, Kao@ε-PL and PP-Kao@ε-PL are 7.5±1.6%, 40.27±1.15%, 37.47±4.19% and 25.02±4.0%, respectively. The BCI value of PP-Kao@ε-PL is lower than that of PP and Kao@ε-PL groups, further indicating that the hemostatic powder has excellent coagulation performance.

See Figure 9, the hemolysis rate experiment of the antibacterial kaolin hemostatic powder prepared in Example 1. The experimental results show that the hemolysis rate of the control group is 96±0.09%, while the hemolysis rates of Kao, PP, Kao@ε-PL and PP-Kao@ε-PL are 27.58±7.81%, 0.43±4.0%, 4.79±0.90% and 2.77±0.48%, respectively. The hemolysis rate of PP-Kao@ε-PL is less than 5%, indicating that the hemostatic powder has excellent blood compatibility.

For matters not mentioned above, the prior art applies.

Although some specific embodiments of the present invention have been described in detail through examples, those skilled in the art should understand that the above examples are for illustration only and are not intended to limit the scope of the present invention. Those skilled in the art to which the present invention belongs may make various modifications or supplements to the specific embodiments described or replace them in a similar manner, but will not deviate from the direction of the present invention or exceed the scope defined by the attached claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc. made to the above embodiments based on the technical essence of the present invention should be included in the protection scope of the present invention.

Claims (9)

1. A preparation method of kaolin-based antibacterial hemostatic powder is characterized by comprising the following steps of: stirring, freeze-drying and grinding the epsilon-polylysine modified kaolin solution with a polyacrylic acid solution and a polyethyleneimine solution respectively to obtain the kaolin-based antibacterial hemostatic powder.

2. The method of manufacturing according to claim 1, wherein: the preparation method of the epsilon-polylysine modified kaolin comprises the following steps: uniformly mixing the epsilon-polylysine solution and the kaolin solution, freeze-drying and grinding the mixture to powder to prepare the epsilon-polylysine modified kaolin.

3. The method of manufacturing according to claim 1, wherein: and adding the epsilon-polylysine modified kaolin solution into the polyacrylic acid solution or the polyethyleneimine solution, uniformly mixing, then dripping the polyethyleneimine solution or the polyacrylic acid solution into the mixed solution under the stirring condition, and grinding the mixture to be powdery after freeze drying to obtain the kaolin-based antibacterial hemostatic powder.

4. The method of manufacturing as claimed in claim 2, wherein: in the step S1, the mass ratio of epsilon-polylysine to kaolin is 1:3-2.

5. A method of preparation as claimed in claim 3, wherein: in the step S2, the concentration of the epsilon-polylysine modified kaolin solution is 0.05-1.6 g/mL.

6. A method of preparation as claimed in claim 3, wherein: in the step S2, the concentration of the polyethyleneimine solution is 5 to 15wt%.

7. A method of preparation as claimed in claim 3, wherein: in step S2, the concentration of the polyacrylic acid solution is 5-15 wt%.

8. The method of manufacturing according to claim 1, wherein: in the step S2, the mass ratio of the epsilon-polylysine modified kaolin to the polyethyleneimine to the polyacrylic acid is 3-8:5:5.

9. A process for preparing a kaolin-based antibacterial hemostatic powder prepared by the process of any one of claims 1-8.