A compound with hemostatic and persistent bactericidal effect and a method for its preparation

Abstract

The application relates to a compound with hemostatic and persistent bactericidal effects and a preparation method thereof, and belongs to the technical field of medical products, which is a CS-ICG / AuNCs compound formed by sequentially immersing a chitosan sponge carrier in ICG solution and AuNCs solution, wherein the ICG loading is achieved by sequentially immersing arginine-modified CS in low-concentration ICG solution and high-concentration ICG solution for immersion loading. In the application, the modified CS is gradient-loaded with ICG and then loaded with AuNCs, so that the loading amount, loading depth and loading uniformity of ICG and AuNCs in the sponge are effectively improved, the loading efficiency of ICG and AuNCs reaches 99.7% and 67.4% respectively, the synergistic bactericidal effect of ICG and AuNCs is improved, and the inhibition of the hemostatic effect of CS is reduced, so that the compound has the dual advantages of rapid hemostasis and long-acting bacteriostasis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical products, in particular to a compound with hemostatic and persistent sterilization effect and a preparation method thereof. BACKGROUND

[0002] In the field of intersection of trauma medicine and microbiology, wound infection has always been a key problem affecting patient prognosis, prolonging treatment cycle and increasing medical cost. Wound infection is difficult to cure due to its bacterial drug resistance, mixed bacterial infection and biofilm formation. Methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli as important pathogenic bacteria of wound infection have attracted extensive attention. As a gram-positive bacterium, MRSA is significantly more difficult to treat due to its resistance to methicillin and other β-lactam antibiotics. As a gram-negative bacterium, Escherichia coli has a unique lipopolysaccharide (LPS) outer membrane structure. After the release of LPS, it can activate the body's strong inflammatory response, causing systemic inflammatory syndrome. Escherichia coli can also produce various toxins to destroy tissue integrity, and synergize with other pathogenic bacteria to exacerbate the complexity of wound infection. Therefore, it is of great significance to prepare an ideal wound repair material to promote tissue regeneration and improve wound healing.

[0003] CS sponge is considered to be the most promising wound healing material due to its high porosity and good mechanical properties. CS sponge has a porous structure with high porosity, which is beneficial to absorb exudate from the wound surface. At the same time, the hemostatic properties of this sponge enable it to stop the continuous bleeding of the wound. ICG (indocyanine green) can generate singlet oxygen and other active oxygen under light excitation, which can accurately destroy the cell membrane, proteins and nucleic acids of bacteria, achieving efficient sterilization. Compared with traditional antibiotics, bacteria are difficult to develop resistance to ICG photodynamic sterilization, opening up a new path to solve the difficult problem of bacterial resistance, and having great application potential in the medical field.

[0004] Gold nanoclusters (AuNCs) exhibit unique dual advantages in antibacterial and biological catalysis fields. Its antibacterial properties are derived from its special nanostructure, which can interact with bacterial cell walls and cell membranes, destroying the integrity of the membranes; at the same time, gold nanoclusters have the characteristics of hydrogen peroxide enzyme, which can simulate the function of natural hydrogen peroxide enzyme, synergize with drug antibacterial effect, and more effectively resist bacterial infection. In the prior art, Au nanoparticles have been combined with ICG, which has significant synergistic sterilization efficacy. Therefore, loading ICG and AuNCs onto CS sponge with hemostatic efficacy to obtain a compound with hemostatic and sterilization dual efficacy is of great significance to promote tissue regeneration and improve wound healing. SUMMARY

[0005] The application aims to provide a complex CS-ICG / AuNCs with hemostatic and long-lasting bactericidal effects.

[0006] The application further aims to provide a preparation method of the CS-ICG / AuNCs complex.

[0007] The application aims to achieve the above-mentioned purposes by the following technical solutions.

[0008] The CS-ICG / AuNCs complex is characterized in that it is a CS-ICG / AuNCs complex formed by sequentially immersing arginine-modified chitosan sponges in ICG solution and AuNCs solution, and the ICG is loaded by sequentially immersing the CS sponges in low-concentration ICG solution and high-concentration ICG solution for gradient immersion treatment.

[0009] Further, the gradient immersion treatment is to immerse the CS sponges in ICG solution with a concentration of 100-200 μg / mL for 1-2 h, centrifuge, and then immerse the CS sponges in ICG solution with a concentration of 500-600 μg / mL for 40-60 min.

[0010] Further, the AuNCs solution is prepared by mixing HAuCl4 solution, 6-mercaptoacetic acid solution and NaOH solution in deionized water, adding NaBH4 solution until the solution turns yellow-brown, and then collecting the filtrate after standing for 2-3 h.

[0011] Further, the concentration of the HAuCl4 solution is 18-22 mmol / L, the concentration of the 6-mercaptoacetic acid solution is 4-6 mmol / L, the concentration of the NaOH solution is 0.8-1.2 mol / L, and the concentration of the NaBH4 solution is 18-20 mmol / L.

[0012] Further, the volume ratio of the deionized water, the HAuCl4 solution, the 6-mercaptoacetic acid solution, the NaOH solution and the NaBH4 solution is 4.8-5:0.4-0.5:4-4.5:0.4-0.5:0.2.

[0013] Further, the chitosan sponge is prepared by weighing chitosan (CS), adding acetic acid to prepare a CS solution, freezing the CS solution, adding the frozen CS solution into a mold, freeze-drying the CS solution into a CS sponge, immersing the CS sponge in acetic acid solution for 1-2 h, and then washing the CS sponge to neutral.

[0014] Further, the mass-volume concentration of the acetic acid is 1-2%, and the concentration of the prepared CS solution is 20-22 mg / mL.

[0015] Further, the freezing treatment temperature is -15--20℃, and the freezing time is 10-12 h.

[0016] A preparation method of a compound having hemostatic and persistent bactericidal effects, characterized in comprising the following steps:

[0017] (1) After mixing HAuCl4 solution, 6-mercaptoacetic acid solution and NaOH solution in deionized water, NaBH4 solution is added until the solution turns yellow-brown, and then the solution is left to stand for 2-3 h, and the filtrate is collected by filtration to obtain AuNCs solution;

[0018] (2) Cystose (CS) is weighed, acetic acid is added to prepare a CS solution, the CS solution is subjected to freezing treatment, the CS solution is then added to a mold, and the CS sponge is obtained by freeze-drying, the CS sponge is immersed in NaOH solution for 1-2 h, and then washed to neutral;

[0019] (3) The CS in step (2) is immersed in arginine solution;

[0020] (4) The CS sponge is immersed in ICG solution for gradient immersion treatment;

[0021] (5) The CS sponge is immersed in AuNCs solution for ultrasonic treatment, and then freeze-dried to obtain a CS-ICG / AuNCs compound.

[0022] Further, in step (1), the concentration of the HAuCl4 solution is 18-22 mmol / L, the concentration of the 6-mercaptoacetic acid solution is 4-6 mmol / L, the concentration of the NaOH solution is 0.8-1.2 mol / L, and the concentration of the NaBH4 solution is 18-20 mmol / L.

[0023] Further, the volume ratio of the deionized water, the HAuCl4 solution, the 6-mercaptoacetic acid solution, the NaOH solution and the NaBH4 solution is 4.8-5:0.4-0.5:4-4.5:0.4-0.5:0.2.

[0024] Further, in step (2), the concentration of the prepared CS solution is 20-22 mg / mL, and the mass concentration of the NaOH solution is 2-3%.

[0025] Further, the freezing treatment temperature is -15--20℃, and the freezing time is 10-12 h.

[0026] Further, in step (3), the mass fraction of the arginine solution is 2.5-3.5%, and the immersion time is 30-45 min.

[0027] Further, the gradient soaking treatment in step (4) is first soaking in an ICG solution with a concentration of 100-200 μg / mL for 1-2 h, and then soaking in an ICG solution with a concentration of 500-600 μg / mL for 40-60 min after centrifugal treatment.

[0028] Further, the ultrasonic power in the ultrasonic treatment in step (5) is 0.8-1.2 kw, and the ultrasonic treatment time is 30-60 min.

[0029] Most specifically, a preparation method of a composite with hemostatic and persistent bactericidal effects, characterized in that, comprising the following steps:

[0030] (1) adding HAuCl4 solution with a concentration of 18-22 mmol / L, 6-mercaptoacetic acid solution with a concentration of 4-6 mmol / L and NaOH solution with a concentration of 0.8-1.2 mol / L in deionized water, mixing, adding NaBH4 solution with a concentration of 18-20 mmol / L until the solution turns yellow-brown, standing for 2-3 h, and then filtering to collect the filtrate to obtain AuNCs solution, wherein the volume ratio of the deionized water, HAuCl4 solution, 6-mercaptoacetic acid solution, NaOH solution and NaBH4 solution is 4.8-5:0.4-0.5:4-4.5:0.4-0.5:0.2;

[0031] (2) weighing chitosan (CS), adding acetic acid with a mass concentration of 1-2% to prepare a CS solution, placing the CS solution in a mold, freezing at -15 to -20 ℃ for 10-12 h, freeze-drying to obtain a CS sponge, immersing the CS sponge in 2-3% NaOH solution with a mass concentration for 1-2 h, and then washing to neutral;

[0032] (3) immersing the CS sponge in arginine solution with a mass fraction of 2.5-3.5% for 30-45 min;

[0033] (4) first immersing the CS sponge treated in step (3) in ICG solution with a concentration of 100-200 μg / mL for 1-2 h, and then immersing in ICG solution with a concentration of 500-600 μg / mL for 40-60 min after centrifugal treatment;

[0034] (5) immersing the CS sponge in AuNCs solution with an ultrasonic power of 0.8-1.2 kw for 30-60 min, and then freeze-drying to obtain a CS-ICG / AuNCs composite.

[0035] In the process of loading ICG and AuNCs by soaking chitosan sponge, the problems of low loading amount of ICG and AuNCs, poor loading uniformity, and serious agglomeration of AuNCs on the surface of the sponge, which not only reduces the bactericidal effect, but also inhibits the hemostatic effect of the CS sponge, so that the finally prepared composite material cannot achieve the effect of rapid hemostasis and long-acting antibiosis. In the process of co-loading, ICG can cause AuNCs to agglomerate more seriously on the surface of the chitosan sponge.

[0036] In the present application, the positively charged arginine chitosan sponge is soaked in different concentrations of ICG solution in sequence. The low concentration of ICG solution has low viscosity and small penetration resistance, so that small molecule ICG can quickly penetrate into the deep part of the sponge for binding. Then, the ICG that is not firmly combined with the sponge is removed by centrifugation. In the higher concentration of ICG solution, the loading of ICG on the surface of the sponge is further enhanced. In this way, the gradient loading of ICG drives the deep penetration of ICG in the sponge, improves the dispersion of ICG in the sponge, preserves the binding sites of AuNCs in the sponge, and also reduces the blockage of the pores of the sponge by small molecule ICG, thereby reserving channels for the subsequent deep penetration of AuNCs. Secondly, as ICG penetrates into the deep part of the sponge, the charge density of the sponge is gradually reduced by electrostatic neutralization, which weakens the electric field strength of the sponge, thereby improving the adsorption of AuNCs on the surface of the chitosan sponge and the movement of AuNCs in the deep part of the sponge, reducing the agglomeration of AuNCs caused by the enrichment of AuNCs on the surface of the chitosan sponge, and finally achieving the uniform adsorption of AuNCs on the surface and in the deep part of the chitosan sponge. Through the above loading method, the loading amount and uniformity of ICG and AuNCs in the chitosan sponge are effectively improved, and the synergistic antibacterial effect of ICG and AuNCs is effectively improved. Through the synergistic therapeutic effect of the long-acting antibacterial property of AuNCs and the rapid antibacterial effect of reactive oxygen species (ROS), the long-acting antibacterial effect is achieved.

[0037] The present application has the following technical effects:

[0038] In the present application, the CS sponge is modified by arginine, and then loaded with ICG in a gradient step-by-step manner, and then loaded with AuNCs to prepare a CS-ICG / AuNCs composite. The loading amount, loading depth, and loading uniformity of ICG and AuNCs in the sponge are effectively improved, and the loading efficiency of ICG and AuNCs reaches 99.7% and 67.4%, respectively. Therefore, the synergistic bactericidal effect of ICG and AuNCs is improved, and the inhibition of the hemostatic effect of CS is reduced, so that the composite material achieves the dual advantages of rapid hemostasis and long-acting antibiosis. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1A scanning electron microscope image of the chitosan sponge prepared in Example 1 of the present application.

[0040] Figure 2 A scanning electron microscope image of the chitosan sponge prepared in Example 1 of the present application after adsorbing red blood cells.

[0041] Figure 3 Zeta potential changes of the CS system in each step of Example 1 of the present application.

[0042] Figure 4 A live and dead staining image of E. coli after being treated with different CS systems.

[0043] Figure 5 A live and dead staining image of MRSA after being treated with different CS systems.

[0044] Figure 6 ROS effect images of different CS systems. DETAILED DESCRIPTION

[0045] The present application will be described in detail below through examples. It is necessary to point out here that the following examples are only used to further illustrate the present application and cannot be understood as limiting the protection scope of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application according to the above content of the present application.

[0046] Example 1

[0047] A preparation method of a composite CS-ICG / AuNCs having hemostatic and persistent bactericidal effects, comprising the following steps:

[0048] (1) After mixing a HAuCl4 solution with a concentration of 20 mmol / L, a 6-mercaptoacetic acid solution with a concentration of 5 mmol / L and a NaOH solution with a concentration of 1.0 mol / L in deionized water, a NaBH4 solution with a concentration of 20 mmol / L is added until the solution turns yellow-brown, and after standing for 2.5 h, the filtrate is collected by filtration to obtain an AuNCs solution, and the volume ratio of the deionized water, the HAuCl4 solution, the 6-mercaptoacetic acid solution, the NaOH solution and the NaBH4 solution is 4.8:0.5:4:0.5:0.2;

[0049] (2) Chitosan (CS) is weighed and a CS solution is prepared by adding acetic acid with a mass volume concentration of 1%, and the CS solution is frozen at-15℃ for 12 h, then the CS solution is added to a mold and freeze-dried into a CS sponge, and the CS sponge is immersed in a 2.5% NaOH solution with a mass concentration for 1.5 h and then washed to neutral.

[0050] (3) The CS sponge is soaked in an arginine solution with a mass fraction of 3% for 40 min, and then vacuum dried;

[0051] (4) The CS sponge treated in step (3) is first soaked in an ICG solution with a concentration of 150 μg / mL for 1.5 h, centrifuged, and then soaked in an ICG solution with a concentration of 550 μg / mL for 50 min;

[0052] (5) The CS sponge is immersed in an AuNCs solution for ultrasonic treatment, the ultrasonic power is 1.0 kw, the ultrasonic treatment time is 50 min, and then the CS-ICG / AuNCs composite is obtained by freeze-drying.

[0053] Example 2

[0054] A preparation method of a composite CS-ICG / AuNCs with hemostatic and persistent bactericidal effects, comprising the following steps:

[0055] (1) A HAuCl4 solution with a concentration of 22 mmol / L, a 6-mercaptoacetic acid solution with a concentration of 6 mmol / L, and a NaOH solution with a concentration of 1.2 mol / L are added to deionized water and mixed, then a NaBH4 solution with a concentration of 20 mmol / L is added until the solution turns yellow-brown, and the solution is left to stand for 3 h, and then the filtrate is collected by filtration to obtain an AuNCs solution, wherein the volume ratio of the deionized water, the HAuCl4 solution, the 6-mercaptoacetic acid solution, the NaOH solution, and the NaBH4 solution is 5:0.45:4.5:0.45:0.2;

[0056] (2) Chitosan (CS) is weighed and added to an acetic acid solution with a mass concentration of 1.5% to prepare a CS solution, the CS solution is frozen at -18℃ for 12 h, the CS solution is added to a mold, and the CS sponge is obtained by freeze-drying, the CS sponge is immersed in a 2% NaOH solution for 2 h, and then washed to neutral;

[0057] (3) The CS sponge is soaked in an arginine solution with a mass fraction of 2.5% for 45 min, and then vacuum dried;

[0058] (4) The CS sponge treated in step (3) is first soaked in an ICG solution with a concentration of 100 μg / mL for 2 h, centrifuged, and then soaked in an ICG solution with a concentration of 600 μg / mL for 40 min;

[0059] (5) The CS sponge is immersed in an AuNCs solution for ultrasonic treatment, the ultrasonic power is 0.8 kw, the ultrasonic treatment time is 30 min, and then the CS-ICG / AuNCs composite is obtained by freeze-drying.

[0060] Example 3

[0061] A preparation method of a complex CS-ICG / AuNCs with hemostatic and persistent bactericidal effects, comprising the following steps:

[0062] (1) After mixing a HAuCl4 solution with a concentration of 18 mmol / L, a 6-mercaptoacetic acid solution with a concentration of 4 mmol / L and a NaOH solution with a concentration of 0.8 mol / L in deionized water, a NaBH4 solution with a concentration of 18 mmol / L is added until the solution turns yellow-brown, and after standing for 2 h, the filtrate is collected by filtration to obtain an AuNCs solution, wherein the volume ratio of the deionized water, the HAuCl4 solution, the 6-mercaptoacetic acid solution, the NaOH solution and the NaBH4 solution is 5:0.4:4.5:0.4:0.2;

[0063] (2) A chitosan (CS) is weighed, and a CS solution is prepared by adding acetic acid with a mass concentration of 1-2%, and the CS solution is frozen at-20℃ for 10 h, and then the CS solution is added to a mold and freeze-dried into a CS sponge, and the CS sponge is immersed in a 3% NaOH solution with a mass concentration for 1 h, and then washed to neutral;

[0064] (3) The CS sponge is immersed in an arginine solution with a mass fraction of 3.5% for 30 min, and then vacuum dried;

[0065] (4) The CS sponge treated in step (3) is first immersed in an ICG solution with a concentration of 200 μg / mL for 1 h, and then treated by centrifugation, and then immersed in an ICG solution with a concentration of 500 μg / mL for 60 min;

[0066] (5) The CS sponge is immersed in an AuNCs solution and treated by ultrasonic, the ultrasonic power is 1.2 kw, and the ultrasonic treatment time is 60 min, and then freeze-dried to obtain a CS-ICG / AuNCs complex.

[0067] The scanning electron microscope image of the chitosan (CS) sponge prepared in Example 1 is shown in Figure 1 , and the scanning electron microscope image of the chitosan sponge after attaching red blood cells is shown in Figure 2 .

[0068] The Zeta potential change of the CS-ICG / AuNCs prepared in Example 1 is tested by a Zeta potential analyzer, as shown in Figure 3As shown in the Zeta potential diagram, the Zeta potential of CS after arginine soaking is +16 mV, and the Zeta potential of CS-ICG0 after soaking in low concentration ICG in step (4) is +10 mV. After further soaking in high concentration ICG, the potential flips to -7 mV. After loading AuNCs, the potential value increases. The obvious changes in potential at each stage indicate that ICG and AuNCs loading is successful.

[0069] The live and dead E. coli staining of different CS systems prepared according to Example 1 is as follows: Figure 4 As shown, LIVE / DEAD® BacLight TM The bacterial viability kit uses green to represent surviving bacteria and red to represent dead bacteria. As you can see, the CS-ICG / AuNCs kit has the most red bacteria, indicating that the most E. coli died. The CS-ICG group is less effective than the CS-ICG / AuNCs kit, but it still has a relatively high number of deaths. The other groups have fewer E. coli deaths.

[0070] The live and dead staining of methicillin-resistant Staphylococcus aureus in different CS systems prepared according to Example 1 is as follows: Figure 5 As shown, LIVE / DEAD® BacLight TM In the bacterial viability kit, green represents surviving bacteria and red represents dead bacteria. It can be seen that the CS-ICG / AuNCs group has the most red, indicating that Staphylococcus aureus died the most. The CS / ICG group is weaker than CS-ICG / AuNCs, but it also has more deaths. The other groups have fewer deaths.

[0071] The ROS intensity produced by different CS systems was measured using flow cytometry, and the results are as follows: Figure 6 As shown, the higher the proportion of red in the P2 region, the higher the positive rate and the more ROS produced. It can be seen that the ROS intensity of the CS-ICG / AuNCs group prepared in Example 1 of this invention is higher. This is due to the photosensitizing effect of ICG deeply distributed in CS and the enhanced ROS production by surface plasmon resonance (SPR) of AuNCs on the surface.

[0072] Comparative Example 1:

[0073] Compared to Example 1, AuNCs are loaded first, followed by ICG loading, with the remaining steps being the same as in Example 1.

[0074] Comparative Example 2

[0075] Compared with Example 1, in the process of loading ICG and AuNCs, a one-step loading of high-concentration ICG (directly soaking in 555ug / ml ICG for 1h) was adopted, followed by loading AuNCs, and the remaining steps were the same as in Example 1.

[0076] Comparative Example 3

[0077] Compared with Example 1, chitosan sponge was soaked in the mixed solution of ICG and AuNCs for simultaneous loading.

[0078] The Zeta potential of the CS-ICG / AuNCs prepared in each comparative example was tested by a Zeta potential analyzer. The Zeta potential of the CS-ICG / AuNCs prepared in Example 1 was -23 mV, the Zeta potential of the CS-ICG / AuNCs prepared in Comparative Example 1 was -19 mV, the Zeta potential of the CS-ICG / AuNCs prepared in Comparative Example 2 was -32 mV, and the Zeta potential of the CS-ICG / AuNCs prepared in Comparative Example 3 was -37 mV. It can be seen that different soaking and loading steps can cause obvious differences in the loading and distribution of ICG and AuNCs on CS, and also affect the subsequent performance of the material, such as functional synergy and stability.

[0079] The loading efficiency of ICG and AuNCs directly determines the ROS intensity and also directly affects the antibacterial and antibacterial effects during use. High loading efficiency can significantly improve the ROS yield and improve the combined antibacterial and antibacterial effect. The loading efficiency of ICG and AuNCs in the CS-ICG / AuNCs synthesized in Example 1 and Comparative Examples 1-3 was calculated according to the following formula by using the effective ingredient mass before soaking in steps (4) and (5) and the residual amount of effective ingredient in the solution after soaking.

[0080]

[0081] After calculation, the ICG loading efficiency of the CS-ICG / AuNCs prepared in Example 1 and Comparative Examples 1-3 was 99.7%, 51.6%, 54.4% and 48.6% respectively, and the AuNCs loading efficiency was 67.4%, 67.2%, 46.9% and 51.2% respectively. The above differences are due to the fact that in Comparative Example 1, AuNCs preferentially enrich on the surface of CS, hindering the inward migration of ICG, resulting in a significant decrease in ICG loading efficiency. In Comparative Example 2, high concentrations of ICG and AuNCs are enriched on the surface of CS in succession. Due to the decrease in the inward migration efficiency of ICG to CS, ICG is concentrated on the surface, resulting in a decrease in loading efficiency. At the same time, the occupation of the surface enrichment sites leads to an unsatisfactory loading efficiency of the subsequent AuNCs. In Comparative Example 3, the simultaneous loading of ICG and AuNCs causes competition for sites, and the strong negative AuNCs preferentially enrich, thereby hindering the further loading of ICG.

[0082] Hemostatic test:

[0083] 100ug of whole blood was dropped on the sponge, each group was incubated at 37℃ for 90s. Subsequently, 15mL of deionized water was added to dissolve hemoglobin from free red blood cells. Finally, the supernatant was taken out of the container and the absorbance was measured at 540 nm using a UV spectrophotometer. Gelatin sponge (GS) was used as a control. The above detection was repeated 3 times for each group.

[0084] 100ug of whole blood was dropped on the sponge, each group was incubated at 37℃ for 90s. Subsequently, 15mL of deionized water was added to dissolve hemoglobin from free red blood cells. Finally, the supernatant was taken out of the container and the absorbance was measured at 540 nm using a UV spectrophotometer, and the BCL value (BCL value refers to the relative content of uncoagulated blood in the system after the blood contacts the test material under certain conditions (such as 37℃, physiological pH) for a certain time, accounting for the percentage of the total initial blood volume). The above detection was repeated 3 times for each group, and it was calculated that the BCL value of the CS sponge was only 20.0%, while the BCL of the cotton ball was almost 3 times that of the CS sponge, indicating that the CS sponge had good procoagulant ability. On the basis of CS, Example 1, Comparative Examples 1-3 further loaded ICG and AuNCs, and the BCL values of CS-ICG / AuNCs were 22.4%, 41.2%, 44.7% and 51.3%. It can be seen that by soaking in ICG of different concentrations and then loading AuNCs, the inhibition of the loading of ICG and AuNCs on the procoagulant effect of CS is effectively reduced. ICG / AuNCs itself has no procoagulant activity, and hemostasis depends on the adsorption of negatively charged platelets by CS to activate coagulation factors and start the coagulation process. The loading of ICG / AuNCs reduces the positive potential of CS, resulting in a decrease in platelet adsorption performance. If ICG and AuNCs are concentrated on the surface of CS, a negative electric barrier is formed on the surface, which is more unfavorable for the adsorption of platelets. In Example 1 of the present application, ICG and AuNCs penetrate into the interior of CS and are distributed, thereby reducing the barrier effect of the surface of CS and reducing the inhibition of the loading of ICG and AuNCs on the procoagulant effect of CS.

[0085] Antibacterial ability test:

[0086] (1) Microbial penetration test:

[0087] The microbial penetration test is used to evaluate the resistance of the test materials prepared in Example 1, Comparative Examples 1-3 to the diffusion of microorganisms from the environment to the top surface of the wound, and the barrier durability of the test materials. The same volume of test materials (including Example 1, Comparative Examples 1-3) was placed in an open vial containing nutrient broth. The negative control was a closed vial with a screw cap, and the positive control was an open vial. The test vials were placed in an open environment for 10 days and observed. Any turbidity of the nutrient solution in the vial was recorded as microbial contamination.

[0088] During the observation process, the positive control group vials appeared serious turbidity on the 4th day, while the negative control group and each experimental group did not appear turbidity, indicating that the CS sponge structure was stable and had good resistance to the spread of microorganisms to the wound top surface.

[0089] (2) Continuous contact antibacterial test:

[0090] Staphylococcus aureus (ATCC 6538) Staphylococcus aureus was inoculated in LB broth and cultured at 37°C on a shaker until OD 600 = 0.6~0.8, and the viable cell count was 10 8 CFU / mL. The test bacteria solution was diluted with sterile normal saline to 10 6 CFU / mL.

[0091] In a 24-well sterile culture plate, the test bacteria solution and test material (Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3) were added and shaken to ensure complete contact between the test material and the test bacteria solution. The blank control group was the test bacteria solution alone. The test was activated by NIR light with a wavelength of 808 nm for 10 min. The same light exposure was performed at the same time every day for 10 min. At 0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h, 0.5 mL of bacteria solution was taken from each test group, gradient diluted, and plated to count the viable cell count (CFU / mL) and calculate the antibacterial efficiency.

[0092]

[0093] The longer the antibacterial efficiency is ≥ 90%, the stronger the bactericidal persistence. The antibacterial efficiency results are shown in Table 1.

[0094] Table 1:

[0095]

[0096] In Example 1, the antibacterial efficiency of the CS-ICG / AuNCs group remained above 90% during the 7-day test period, indicating strong bactericidal persistence. In Comparative Example 1, the antibacterial efficiency was significantly lower than 90% on the 4th day, and by the 7th day, the bactericidal efficiency decreased to 71.6%, indicating poor bactericidal persistence compared to Example 1. In Comparative Example 2, the antibacterial efficiency was lower than 90% on the 5th day, and by the 7th day, the antibacterial efficiency also decreased to 81.3%. In Comparative Example 3, the antibacterial efficiency was already lower than 90% on the 3rd day, and by the 7th day, the antibacterial efficiency had decreased significantly to 60.1%, indicating extremely poor bactericidal persistence.

Claims

1. A method for preparing a complex with hemostatic and long-lasting bactericidal effects, characterized in that, Includes the following steps: (1) Add HAuCl4 solution, 6-mercaptoacetic acid solution and NaOH solution to deionized water and mix them. Then add NaBH4 solution until the solution turns yellowish-brown. After standing for 2-3 hours, filter and collect the filtrate to obtain AuNCs solution. (2) Weigh chitosan, add acetic acid to prepare CS solution, freeze-dry the CS solution into a mold, freeze-dry the CS sponge, immerse the CS sponge in NaOH solution for 1-2 hours, and then wash until neutral. (3) Soak the CS from step (2) in an arginine solution; (4) Immerse the CS sponge in ICG solution for gradient soaking treatment. The gradient soaking is to first soak in ICG solution with a concentration of 100~200μg / mL for 1~2h, centrifuge, and then soak in ICG solution with a concentration of 500~600μg / mL for 40~60min. (5) The CS sponge was immersed in AuNCs solution and ultrasonically treated, and then freeze-dried to obtain CS-ICG / AuNCs sponge.

2. The method for preparing a complex with hemostatic and long-lasting bactericidal effects as described in claim 1, characterized in that: In step (1), the concentration of HAuCl4 solution is 18~22 mmol / L, the concentration of 6-mercaptoacetic acid solution is 4~6 mmol / L, the concentration of NaOH solution is 0.8~1.2 mol / L, and the concentration of NaBH4 solution is 18~20 mmol / L.

3. A method for preparing a complex with hemostatic and long-lasting bactericidal effects as described in claim 1 or 2, characterized in that: The volume ratio of the deionized water, HAuCl4 solution, 6-mercaptoacetic acid solution, NaOH solution, and NaBH4 solution is 4.8~5:0.4~0.5:4~4.5:0.4~0.5:0.

2.

4. The method for preparing a complex with hemostatic and long-lasting bactericidal effects as described in claim 3, characterized in that: The concentration of the CS solution prepared in step (2) is 20~22 mg / mL, and the mass concentration of the NaOH solution is 2~3%.

5. The method for preparing a complex with hemostatic and long-lasting bactericidal effects as described in claim 4, characterized in that: In step (3), the mass fraction of the arginine solution is 2.5-3.5%, and the soaking time is 30-45 min.

6. A method for preparing a complex with hemostatic and long-lasting bactericidal effects, characterized in that, Includes the following steps: (1) Add HAuCl4 solution with a concentration of 18~22 mmol / L, 6-mercaptoacetic acid solution with a concentration of 4~6 mmol / L, and NaOH solution with a concentration of 0.8~1.2 mol / L to deionized water, mix them, add NaBH4 solution with a concentration of 18~20 mmol / L until the solution turns yellowish-brown, let it stand for 2~3 hours, filter and collect the filtrate to obtain AuNCs solution. The volume ratio of the deionized water, HAuCl4 solution, 6-mercaptoacetic acid solution, NaOH solution and NaBH4 solution is 4.8~5:0.4~0.5:4~4.5:0.4~0.5:0.2; (2) Weigh chitosan CS, add acetic acid with a mass-volume concentration of 1~2% to prepare CS solution, freeze at -15~-20℃ for 10~12h, add CS solution into mold, freeze dry to form CS sponge, immerse CS sponge in NaOH solution with a mass-volume concentration of 2~3% for 1~2h, and then wash until neutral; (3) First, soak the CS sponge in an ICG solution with a concentration of 100~200μg / mL for 1~2h, centrifuge it, and then soak it in an ICG solution with a concentration of 500~600μg / mL for 40~60min. (4) Immerse the CS sponge in AuNCs solution and sonicate for 30-60 min, then freeze dry to obtain CS-ICG / AuNCs complex.

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