Bacteria self-triggering halloysite composite antibacterial material and preparation method thereof

Abstract

The invention discloses a bacterium self-triggering type halloysite composite antibacterial material and a preparation method thereof, and belongs to the field of nonmetallic mineral material and biological material engineering. According to the invention, hexadecyl tributyl phosphonium bromide and copper chloride capable of synergistically resisting bacteria are loaded in a halloysite lumen, and hyaluronic acid is used for wrapping and blocking the halloysite, so that responsive on-demand release and bacteria resistance of the antibacterial agent are realized. When hyaluronidase secreted by bacteria decomposes hyaluronic acid to seal the end, the antibacterial agent in the halloysite lumen is triggered and released, so that the synergistic antibacterial effect is achieved. The preparation method has the technical characteristics that the purity and the length of the halloysite, the loading conditions of the hexadecyl tributyl phosphonium bromide and the copper chloride and the operation parameters of the hyaluronic acid coated and terminated antibacterial halloysite are optimized, so that the bacterium self-triggering type halloysite composite antibacterial material can realize efficient, accurate, controllable and safe on-demand antibacterial effect. The technology can be applied to the fields of intelligent responsive antibacterial products, medical supplies, wound treatment and the like.

Description

Technical Field

[0001] The invention belongs to the field of non-metallic mineral materials and biomaterial engineering, and specifically relates to a hyaluronic acid-wrapped and terminated bacterial self-triggered halloysite composite antibacterial material and a preparation method thereof. Background Art

[0002] As a natural mineral material, halloysite has good biosafety and a through-type tubular structure, making it an excellent drug delivery platform for constructing controlled-release systems for antimicrobial agents. However, the high efficiency and broad-spectrum antibacterial performance of existing halloysite antibacterial materials still need to be further improved, and they lack intelligent responsive antibacterial capabilities, which is not conducive to achieving efficient, precise, controllable and safe on-demand antibacterial.

[0003] Hyaluronic acid is a biocompatible material commonly found in the extracellular matrix of human tissues and has been widely used in biomedical applications such as cosmetics, tissue engineering, and disease treatment. Hyaluronic acid can be degraded by hyaluronidases secreted by a variety of bacteria (such as Gram-positive bacteria: Staphylococcus aureus, Streptococcus, Streptomyces griseus, Propionibacterium acne-prone, Clostridium, etc.; Gram-negative bacteria: Bacteroides vulgaris, Proteus vulgaris, Aeromonas, Vibrio, etc.). Hyaluronidase secreted by bacteria, as a pathogenic factor, destroys the integrity of the tissue matrix and participates in the pathogenic effects of pathogenic bacteria and the infection of diseases. Therefore, by utilizing the specific degradation of hyaluronic acid by hyaluronidase, an intelligent responsive antimicrobial drug release system can be constructed to fight bacterial infections on demand.

[0004] The present invention utilizes hyaluronic acid to encapsulate and cap halloysite loaded with hexadecyltributylphosphonium bromide and cupric chloride to construct a hyaluronidase-responsive antibacterial system, thereby achieving on-demand release of antibacterial agents during the antibacterial process and synergistic enhanced antibacterial effects between organic and inorganic antibacterial agents, so as to respond to bacterial infections in a more efficient, precise, controllable and safe manner. Summary of the invention

[0005] The present invention provides a bacterial self-triggered halloysite composite antibacterial material and a preparation method thereof. The antibacterial material is composed of hexadecyl tributylphosphonium bromide and copper chloride loaded into the halloysite lumen for synergistic antibacterial effects, and the antibacterial halloysite is capped with hyaluronic acid to achieve precise, controllable and on-demand release of the antibacterial agent and high-efficiency antibacterial effects. During the antibacterial process, the antibacterial agent encapsulated in the halloysite is accurately released on demand under the deblocking effect of the hyaluronidase secreted by the bacteria on the hyaluronic acid capping, thereby combating bacterial proliferation and infection.

[0006] The preparation method and process steps of the bacteria self-triggered halloysite composite antibacterial material provided by the present invention are as follows:

[0007] (1) uniformly mixing halloysite and water to obtain a halloysite dispersion;

[0008] (2) mixing the halloysite dispersion in step (1) with hexadecyl tributyl phosphine bromide and cupric chloride, and performing repeated vacuum loading at -0.09 MPa for 15 min three times to obtain antibacterial halloysite;

[0009] (3) uniformly mixing the antibacterial halloysite in step (2) with water to obtain an antibacterial halloysite dispersion;

[0010] (4) The antibacterial halloysite dispersion obtained in step (3) is mixed with hyaluronic acid and stirred in a water bath to obtain a hyaluronic acid-coated and end-capped bacterial self-triggered halloysite composite antibacterial material.

[0011] Preferably, in step (1), the purity of the halloysite is greater than 90%, the length of the halloysite is 100-800 nm, and the mass ratio of the halloysite to water is 1:(20-80).

[0012] Preferably, in step (2), the mass ratio of hexadecyltributylphosphonium bromide to cupric chloride is 3:7 to 7:3, and the total amount of the two is 22.5 to 75.0% of the mass of the halloysite in the halloysite dispersion.

[0013] Preferably, in step (3), the mass ratio of antibacterial halloysite to water is 1:(50-150).

[0014] Preferably, in step (4), the amount of hyaluronic acid used is 3-10% of the mass of the antibacterial halloysite dispersion, the water bath temperature is 50-80° C., and the stirring time is 1-8 hours.

[0015] The halloysite composite antibacterial material prepared by the above method and process steps has the characteristics of bacteria-triggered release of antibacterial agents and a fast response rate. At the same time, the material exhibits excellent synergistic and broad-spectrum antibacterial properties of hexadecyl tributylphosphonium bromide and copper ions, and can be widely used in intelligent responsive antibacterial products and biomedical fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The release curves of copper chloride (Cu) and hexadecyltributylphosphonium bromide (TBHDPB) of Examples 1 to 3 and Comparative Examples 1 to 3 in physiological saline in the (a) absence and (b) presence of hyaluronidase.

[0017] Figure 2 The antibacterial rate curves of Examples 1 to 3 and Comparative Examples 1 to 8 against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli, wherein (a) is the antibacterial rate curve against Staphylococcus aureus, and (b) is the antibacterial rate curve against Escherichia coli.

[0018] Figure 3The kinetic fitting curves of the antibacterial processes of Examples 1 to 3 and Comparative Examples 1 to 3 against (a) Gram-positive Staphylococcus aureus and (b) Gram-negative Escherichia coli are shown. DETAILED DESCRIPTION

[0019] The present invention provides a method for preparing a bacteria self-triggered halloysite composite antibacterial material:

[0020] (1) ultrasonically dispersing halloysite in water to obtain a halloysite dispersion;

[0021] (2) mixing the halloysite dispersion in step (1) with hexadecyl tributyl phosphine bromide and cupric chloride, and performing repeated vacuum loading at -0.09 MPa for 15 min three times to obtain antibacterial halloysite;

[0022] (3) ultrasonically dispersing the antibacterial halloysite in step (2) into water to obtain an antibacterial halloysite dispersion;

[0023] (4) The antibacterial halloysite dispersion obtained in step (3) is mixed with hyaluronic acid and stirred in a water bath to obtain a bacterial self-triggered halloysite composite antibacterial material wrapped and capped with hyaluronic acid.

[0024] The present invention firstly disperses halloysite into water by ultrasonic method to obtain halloysite dispersion liquid.

[0025] In the present invention, the purity of the halloysite is preferably greater than 90%; the length of the halloysite is 100 to 800 nm, more preferably 150 to 400 nm. The present invention does not specifically limit the source of the halloysite of the purity and length, and it can be obtained by a preparation method well known in the art. The present invention limits the purity and length of the halloysite to ensure that hexadecyltributylphosphine bromide and copper chloride can quickly reach an effective antibacterial loading in the halloysite lumen and achieve rapid, accurate, efficient and antibacterial self-triggered antibacterial release of bacteria that meets antibacterial requirements during the antibacterial process.

[0026] The present invention has no particular limitation on the source of the water, and ultrapure water commonly used by those skilled in the art can be used. In the present invention, the mass ratio of the halloysite to water is preferably 1:(20-80), and more preferably 1:(30-50).

[0027] The present invention has no special requirements for the ultrasonic stirring and dispersing operation of halloysite in water, and can be implemented under ultrasonic stirring and dispersing conditions commonly used by those skilled in the art. The present invention improves the dispersion of halloysite through ultrasonic stirring and dispersing, thereby being more conducive to vacuum loading of the antibacterial component in the lumen.

[0028] After obtaining the halloysite dispersion, the present invention uniformly mixes the halloysite dispersion with hexadecyltributylphosphonium bromide and cupric chloride and then performs vacuum loading to obtain antibacterial halloysite.

[0029] In the present invention, the preferred mass ratio of hexadecyl tributyl phosphine bromide and copper chloride is 3:7 to 7:3, more preferably 5:5; the total amount of hexadecyl tributyl phosphine bromide and copper chloride is 22.5 to 75.0% of the mass of the halloysite in the halloysite dispersion, more preferably 45 to 52.5%. The present invention optimizes the ratio and amount of hexadecyl tributyl phosphine bromide and copper chloride to obtain highly effective and broad-spectrum (towards Gram-positive and Gram-negative bacteria) antibacterial halloysite by utilizing the synergistic antibacterial effect between the organic antibacterial agent and the inorganic antibacterial agent.

[0030] Before vacuum loading, the hexadecyl tributyl phosphonium bromide and copper chloride of the present invention are preferably slowly added to the halloysite dispersion under dynamic stirring conditions in the form of an aqueous solution. The present invention has no special restrictions on the preparation and concentration of the hexadecyl tributyl phosphonium bromide and copper chloride solutions, and the preparation methods well known to those skilled in the art are used to prepare and ensure that the amount of each raw material meets the requirements. The present invention has no special restrictions on the stirring operation, and the magnetic stirring operation well known to those skilled in the art can be used at room temperature. The present invention obtains a uniformly mixed loading system by slowly adding the antibacterial agent solution to the halloysite dispersion under dynamic stirring, which is conducive to the subsequent vacuum loading of the antibacterial agent.

[0031] In the present invention, the uniformly mixed hexadecyl tributyl phosphine bromide and cupric chloride halloysite dispersion is placed in a vacuum environment of -0.09MPa at room temperature and vacuum loading is repeated for 3 times for 15 minutes. In the 2 gaps between the 3 vacuum loadings described in the present invention, the mixed hexadecyl tributyl phosphine bromide and cupric chloride halloysite dispersion is restored to normal pressure for 15 minutes and dynamically stirred to regain a uniform loading system. The present invention does not specifically limit the container for the halloysite dispersion mixed with hexadecyl tributyl phosphine bromide and cupric chloride, and a narrow-mouth container commonly used by those skilled in the art is used and the volume requirements during vacuum loading are met. The present invention effectively increases the loading amount of the antibacterial agent in the halloysite lumen by repeated vacuum loading, thereby improving the antibacterial agent release and antibacterial ability of the antibacterial halloysite.

[0032] After the vacuum loading is completed, the present invention preferably filters, washes and dries the antibacterial halloysite in sequence to obtain antibacterial halloysite. The present invention has no special limitation on the filtering and washing operations, and the unloaded antibacterial agent can be removed by filtering and washing operations well known to those skilled in the art. In the present invention, the drying temperature is preferably 40 to 50°C. The present invention has no special limitation on the drying time, and drying to constant weight is sufficient.

[0033] After obtaining the antibacterial halloysite, the present invention ultrasonically disperses the antibacterial halloysite into water to obtain an antibacterial halloysite dispersion. The present invention has no particular limitation on the source of the water, and ultrapure water commonly used by those skilled in the art can be used. In the present invention, the mass ratio of the antibacterial halloysite to water is preferably 1: (50-150), more preferably 1: (80-120).

[0034] In the present invention, the preferred ultrasonic power of the antibacterial halloysite in water is 100-200W, and the preferred ultrasonic time is 5-10min. The present invention preferably uses ultrasonic conditions to disperse the antibacterial halloysite in water to ensure that the antibacterial halloysite is fully dispersed and reduce the impact of ultrasound on the loaded antibacterial agent.

[0035] After obtaining the antibacterial halloysite dispersion, the present invention uniformly mixes the antibacterial halloysite dispersion with hyaluronic acid and dynamically stirs the mixture under water bath heating conditions to obtain a hyaluronic acid-wrapped and terminated bacterial self-triggered halloysite composite antibacterial material.

[0036] In the present invention, the preferred amount of hyaluronic acid is 3-10% of the mass of the antibacterial halloysite dispersion, and more preferably 4-8%. The water bath temperature of the present invention is preferably 50-80°C, and more preferably 60-70°C. The stirring time of the present invention is preferably 1-8h, and more preferably 2-5h. The present invention optimizes the amount of hyaluronic acid and the temperature and time used for encapsulation to ensure that the hyaluronic acid in the antibacterial halloysite dispersion can form an encapsulation end of suitable thickness for the antibacterial halloysite, and construct a bacterial self-triggered halloysite composite antibacterial material that can quickly and accurately realize the on-demand release of antibacterial agents and high-efficiency antibacterial.

[0037] The hyaluronic acid of the present invention is preferably slowly added to the antibacterial halloysite dispersion under dynamic stirring in the form of an aqueous solution. The present invention has no special restrictions on the preparation and concentration of the hyaluronic acid solution, and it can be prepared by a preparation method well known to those skilled in the art and ensure that its dosage meets the requirements.

[0038] After the hyaluronic acid is coated and capped with antibacterial halloysite, the present invention preferably centrifuges and washes and dries the resulting product in sequence to obtain a bacterial self-triggered halloysite composite antibacterial material. The present invention has no special limitation on the centrifugal washing operation, and the free hyaluronic acid can be removed by a centrifugal washing operation well known to those skilled in the art. In the present invention, the drying temperature is preferably 40 to 50°C. The present invention has no special limitation on the drying time, and the hyaluronic acid coated and capped bacterial self-triggered halloysite composite antibacterial material can be obtained by drying to constant weight.

[0039] The present invention uses halloysite as a carrier, and loads organic hexadecyl tributyl phosphonium bromide and inorganic copper chloride that can synergistically enhance antibacterial properties into the through-type lumen of halloysite by a vacuum loading method. Halloysite is mixed with a solution of hexadecyl tributyl phosphonium bromide and copper chloride and then vacuum loaded multiple times to increase the loading amount of the antibacterial agent, thereby preparing antibacterial halloysite. Subsequently, the antibacterial halloysite is mixed and stirred with a hyaluronic acid solution so that it is wrapped and capped with hyaluronic acid, thereby obtaining a bacterial self-triggered halloysite composite antibacterial material that can be triggered by bacteria to secrete hyaluronidase to release hexadecyl tributyl phosphonium bromide and copper chloride for synergistic antibacterial properties.

[0040] In view of the current situation that the existing halloysite antibacterial materials have weak high-efficiency and broad-spectrum antibacterial performance, lack of bacteria self-triggered intelligent response antibacterial ability, and are difficult to adapt to the needs of efficient, precise, controllable and safe on-demand antibacterial, the present invention loads organic hexadecyltributylphosphonium bromide and inorganic cupric chloride that can synergistically enhance antibacterial properties into the through-type lumen of halloysite and uses hyaluronic acid that can be decomposed by bacteria to wrap and seal it, thereby obtaining a bacteria self-triggered halloysite composite antibacterial material that can achieve high-efficiency, broad-spectrum and on-demand antibacterial properties.

[0041] The present invention also provides a bacterial self-triggered halloysite composite antibacterial material prepared by the preparation method described in the above technical solution. The bacterial self-triggered halloysite composite antibacterial material provided by the present invention has the ability of bacterial self-triggered antibacterial, and can achieve high efficiency, broad spectrum and on-demand antibacterial by means of the synergistic strengthening antibacterial effect between organic hexadecyl tributyl phosphonium bromide and inorganic copper chloride. When used, the bacteria release hyaluronidase to decompose the hyaluronic acid encapsulating the end-capped antibacterial halloysite, triggering the halloysite composite antibacterial material to respond rapidly, and release hexadecyl tributyl phosphonium bromide and copper chloride encapsulated in the lumen by hyaluronic acid on demand. Afterwards, the hexadecyl tributyl phosphonium bromide and copper chloride released by the antibacterial halloysite synergistically strengthen antibacterial. Organic hexadecyl tributyl phosphonium bromide can bind to the surface of bacterial cells, interfere with their normal physiological and proliferation processes, destroy the integrity of their wall membrane structure and rupture them. Copper ions can rely on Coulomb force to bind to the surface of bacteria, causing the bacteria to lose their normal physiological functions due to the inactivation of cell surface proteins. At the same time, with the help of hexadecyltributylphosphonium bromide to destroy the bacterial cell wall membrane structure, copper ions can more easily enter the bacterial cells, combine with enzymes, DNA and other substances, and completely inactivate and kill the bacteria.

[0042] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] Example 1

[0044] The preparation method of bacteria self-triggered halloysite composite antibacterial material consists of the following steps:

[0045] (1) 2 g of halloysite (with a purity greater than 90%) having an average length of about 300 nm was ultrasonically dispersed in 80 mL of ultrapure water at a liquid-to-solid ratio of 1:40 to obtain a halloysite dispersion;

[0046] (2) Pre-dissolving 0.5 g of copper chloride and 0.5 g of hexadecyl tributyl phosphonium bromide in water respectively, and then slowly adding the obtained copper chloride solution and hexadecyl tributyl phosphonium bromide solution to the halloysite dispersion obtained in step (1) under dynamic stirring conditions, and then repeating vacuum loading for 3 times for 15 minutes in a vacuum environment of -0.09 MPa at room temperature, restoring normal pressure and dynamically stirring for 15 minutes in 2 intervals between the 3 vacuum loadings to regain a uniform loading system, and after the vacuum loading is completed, filtering, washing, and drying at 45° C. to obtain antibacterial halloysite; wherein the mass ratio of hexadecyl tributyl phosphonium bromide to copper chloride is 5:5, and the total amount of hexadecyl tributyl phosphonium bromide and copper chloride is 50% of the mass of the halloysite;

[0047] (3) 2 g of the antibacterial halloysite obtained in step (2) is dispersed in 200 mL of water by ultrasonic stirring to obtain an antibacterial halloysite dispersion; wherein the mass ratio of acid-impregnated montmorillonite to distilled water is 1:100, the ultrasonic power is 150 W, and the ultrasonic time is 6 min; (4) 8 g of hyaluronic acid is dissolved and added to the halloysite dispersion obtained in step (3), and stirred in a water bath at 70° C. for 4 h. After the antibacterial halloysite is coated and capped, centrifugation, washing, and drying at 45° C. are performed to obtain a hyaluronic acid-coated and capped bacterial self-triggered halloysite composite antibacterial material; wherein the amount of hyaluronic acid used is 4% of the mass of the antibacterial halloysite dispersion.

[0048] Example 2

[0049] The steps are the same as those in Example 1, except that the mass of hyaluronic acid used in step (4) is 12 g, and the amount of hyaluronic acid used is 6% of the mass of the antibacterial halloysite dispersion.

[0050] Example 3

[0051] The steps are the same as those in Example 1, except that the mass of hyaluronic acid used in step (4) is 16 g, and the amount of hyaluronic acid used is 8% of the mass of the antibacterial halloysite dispersion.

[0052] Comparative Example 1

[0053] The steps are the same as those in Example 1, except that the mass of hyaluronic acid used in step (4) is 2 g, and the amount of hyaluronic acid used is 1% of the mass of the antibacterial halloysite dispersion.

[0054] Comparative Example 2

[0055] The steps are the same as those in Example 1, except that the mass of the hyaluronic acid used in step (4) is 30 g, and the amount of the hyaluronic acid used is 15% of the mass of the antibacterial halloysite dispersion.

[0056] Comparative Example 3

[0057] The steps are the same as those in Example 1, except that hyaluronic acid is not added in step (4).

[0058] Comparative Example 4

[0059] The steps are the same as those in Example 1, except that copper chloride and hexadecyltributylphosphine bromide are not added in step (2).

[0060] Comparative Example 5

[0061] The steps are the same as those in Example 1, except that copper chloride and hexadecyltributylphosphonium bromide are not added in step (2); the mass of hyaluronic acid used in step (4) is 12 g, and the amount of hyaluronic acid used is 6% of the mass of the antibacterial halloysite dispersion.

[0062] Comparative Example 6

[0063] The steps are the same as those in Example 1, except that copper chloride and hexadecyltributylphosphonium bromide are not added in step (2); the mass of hyaluronic acid used in step (4) is 16 g, and the amount of hyaluronic acid used is 8% of the mass of the antibacterial halloysite dispersion.

[0064] Comparative Example 7

[0065] The steps are the same as those in Example 1, except that copper chloride and hexadecyltributylphosphonium bromide are not added in step (2); the mass of hyaluronic acid used in step (4) is 2 g, and the amount of hyaluronic acid used is 1% of the mass of the antibacterial halloysite dispersion.

[0066] Comparative Example 8

[0067] The steps are the same as those in Example 1, except that copper chloride and hexadecyl tributyl phosphonium bromide are not added in step (2); the mass of hyaluronic acid used in step (4) is 30 g, and the amount of hyaluronic acid used is 15% of the mass of the antibacterial halloysite dispersion. The triggered release performance of the bacterial self-triggered halloysite composite antibacterial material prepared in Examples 1 to 3 and Comparative Examples 1 to 3 was tested. The Lambert-Beer law shows that the absorbance of a uniform solution is proportional to its concentration, and the total absorbance of a mixed solution containing multiple absorbing substances is additive, based on which the concentration of each component in the mixed solution of copper chloride and hexadecyl tributyl phosphonium bromide can be calculated. When a mixed solution of 1M hydrochloric acid and saline (volume ratio of 1:3) is used as a solvent, the characteristic absorption wavelength of the copper chloride solution is 212 nm, and the characteristic absorption wavelength of the hexadecyl tributyl phosphonium bromide solution is 260 nm. In the concentration range of 0 to 100 mg / L, the concentration (x, mg / mL) of hexadecyl tributylphosphonium bromide solution and its absorbance (y) conform to the following linear relationship:

[0068] 260nm: y = 0.01246 x (R 2 =0.99945)

[0069] 212nm: y = 0.009280 x (R 2 =0.99871)

[0070] The linear relationship between the concentration of copper chloride solution (x, mg / mL) and its absorbance (y) is:

[0071] 260nm: y = 0.00979 x (R 2 =0.99913)

[0072] 212nm: y = 0.01031 x (R 2 =0.99921)

[0073] When conducting the triggered release performance test, the self-triggered halloysite composite antibacterial material was prepared into a 1500ppm suspension in normal saline and ultrasonically treated until there were no obvious particles; at the same time, a 300U / mL hyaluronidase solution was prepared using normal saline; then, the material suspension was mixed with the hyaluronidase solution (or normal saline) in a volume ratio of 1:1 and placed in a 37°C constant temperature oscillator (150r / min) and oscillated every 60m for 10min, 20min, 30min, 60min, 90min, 120min and the following 10h. in sampling and 1M hydrochloric acid in a volume ratio of 3: 1 and centrifugally precipitate mineral particles; test the absorbance of the centrifuge at wavelengths of 212nm and 260nm, and calculate the release of copper chloride and hexadecyl tributyl phosphine bromide according to the linear relationship, wherein Comparative Example 4 is used as the release background of Example 1, Comparative Example 5 is used as the release background of Example 2, Comparative Example 6 is used as the release background of Example 3, Comparative Example 7 is used as the release background of Comparative Example 1, and Comparative Example 8 is used as the release background of Comparative Example 2 to eliminate the influence of hyaluronic acid on the release of copper chloride and hexadecyl tributyl phosphine bromide. In addition, according to the calculation method of the component content of the mixed solution, the loading amount of copper chloride in Examples 1 to 3 and Comparative Examples 1 to 3 is 12.76 to 13.54%, and the loading amount of hexadecyl tributyl phosphine bromide is 13.91 to 15.32%.

[0074] The antibacterial properties of the products prepared in Examples 1 to 3 and Comparative Examples 1 to 8 were tested by colony counting method. Escherichia coli (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacteria) were cultured overnight, and the culture medium was removed by repeated centrifugation and washing with physiological saline and diluted to obtain 6×10 7 CFU / mL of bacterial suspension; the products obtained in the embodiments and comparative examples were formulated into a 40 mg / L suspension and ultrasonically dispersed; 20 mL of the obtained product suspension was mixed with 20 mL of the bacterial suspension in a 100 mL conical flask and sealed with a sealing film; then, the conical flask was placed in a 37°C constant temperature oscillator (150 r / min), 0.9 mL was sampled at 10 min, 20 min, 30 min, 60 min, 90 min and 120 min, respectively, and 0.1 mL was evenly spread on the surface of the nutrient agar medium after several 10-fold dilutions; finally, the culture was carried out at 37°C and a relative humidity greater than 90% for 18 h, and the colonies on the surface of the culture dish were counted.

[0075] The antibacterial rate (Qt) is calculated using the following formula,

[0076]

[0077] The Chick model (pseudo-first-order kinetic model) was used to study the antibacterial kinetics of the product.

[0078]

[0079] Among them, R 0t and R t is the concentration of viable bacteria in the blank and experimental groups at time t (min) (CFU / mL), R0 is the initial concentration of bacteria (CFU / mL), k is the sterilization rate constant (min -1 ).

[0080] Depend on Figure 1 It can be seen that, regardless of whether it is in contact with hyaluronidase, Comparative Example 3, which is not encapsulated and capped by hyaluronic acid, cannot achieve the controlled release of copper chloride and hexadecyl tributyl phosphine bromide on demand, and the antibacterial agent is released quickly and unrestrainedly. Compared with Comparative Example 3, the encapsulation and capping of hyaluronic acid changes the release performance of the antibacterial agent in Comparative Examples 1 to 2. However, Comparative Example 1 with a low degree of encapsulation and capping is not enough to effectively achieve the restrained release of the antibacterial agent on demand, and the antibacterial agent is still released in large quantities in an environment without hyaluronidase; Comparative Example 2, which uses excessive hyaluronic acid to encapsulate and cap, lacks sensitive responsive antibacterial agent release ability, and the opening of the capping requires more hyaluronidase, so the antibacterial agent cannot be effectively released in the same hyaluronidase environment. Compared with Comparative Examples 1 to 3, Examples 1 to 3 with optimized encapsulation and capping of hyaluronic acid have excellent responsive release ability, and the capped hyaluronic acid avoids premature leakage of the antibacterial agent, and the antibacterial agent can only be effectively released on demand when in contact with hyaluronidase. It can be seen that the hyaluronic acid-coated and capped bacterial self-triggered halloysite composite antibacterial material provided by the present invention can realize efficient, accurate and safe on-demand release of responsive antibacterial agents.

[0081] The bacteria-triggered antibacterial ability of the bacteria-triggered halloysite composite antibacterial material provided by the present invention is also proven. Figure 2 , Figure 3 As can be seen from Table 1, Examples 1 to 3 and Comparative Examples 1 to 3 of hyaluronic acid-encapsulated antibacterial halloysite all show bacterial self-triggered antibacterial ability, and Comparative Examples 4 to 8 without copper chloride and hexadecyl tributyl phosphonium bromide do not have antibacterial ability. The uncontrolled rapid release of the antibacterial agent in Comparative Example 3 without encapsulation and the abnormal sensitivity response ability of Comparative Example 1 with low encapsulation and capping degree make it show a slightly faster antibacterial kinetic process; Comparative Example 2 with excessive encapsulation and capping cannot effectively respond to the bacterial environment and fails to release the required amount of antibacterial agent to achieve effective antibacterial. Examples 1 to 3 respond quickly under the stimulation of bacterial secretion of hyaluronidase, release effective amounts of copper chloride and hexadecyl tributyl phosphonium bromide on demand, and achieve complete antibacterial. It can be seen that the bacterial self-triggered halloysite composite antibacterial material encapsulated and capped by hyaluronic acid provided by the present invention can simultaneously respond to the triggering of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli, achieving efficient, broad-spectrum, precise and safe on-demand antibacterial.

[0082] It can be seen from the above comparative examples and embodiments that the bacteria self-triggered halloysite composite antibacterial material prepared by the preparation method provided by the present invention has efficient, broad-spectrum, precise and safe on-demand antibacterial ability triggered by both Gram-positive bacteria and Gram-negative bacteria.

[0083] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

[0084] Table 1 Antibacterial kinetic parameters of Examples 1 to 3 and Comparative Examples 1 to 3 against Staphylococcus aureus and Escherichia coli

[0085]

Claims

1. A bacteria self-triggered halloysite composite antibacterial material, characterized in that: Bacteria secrete hyaluronidase to trigger the hyaluronic acid-coated and capped halloysite to release hexadecyltributylphosphonium bromide and copper chloride encapsulated in the lumen on demand for synergistic antibacterial effect.

2. A method for preparing the bacteria self-triggered antibacterial halloysite composite antibacterial material as claimed in claim 1, comprising the following steps: (1) uniformly mixing halloysite and water to obtain a halloysite dispersion; (2) mixing the halloysite dispersion of step (1) with hexadecyl tributyl phosphine bromide and cupric chloride, and performing repeated vacuum loading at -0.09 MPa for 15 min three times to obtain antibacterial halloysite; (3) uniformly mixing the antibacterial halloysite obtained in step (2) with water to obtain an antibacterial halloysite dispersion; (4) The antibacterial halloysite dispersion obtained in step (3) is mixed with hyaluronic acid and stirred in a water bath to obtain a hyaluronic acid-coated and end-capped bacterial self-triggered halloysite composite antibacterial material.

3. The preparation method according to claim 2, characterized in that: In the step (1), the purity of the halloysite is greater than 90%, the length of the halloysite is 100-800 nm, and the mass ratio of the halloysite to water is 1:(20-80).

4. The preparation method according to claim 2, characterized in that: In the step (2), the mass ratio of hexadecyl tributyl phosphonium bromide to cupric chloride is 3:7 to 7:3, and the total amount of the two is 22.5 to 75.0% of the mass of the halloysite in the halloysite dispersion.

5. The preparation method according to claim 2, characterized in that: In the step (3), the mass ratio of antibacterial halloysite to water is 1:(50-150).

6. The preparation method according to claim 2, characterized in that: In the step (4), the amount of hyaluronic acid used is 3-10% of the mass of the antibacterial halloysite dispersion, the water bath temperature is 50-80° C., and the stirring time is 1-8 hours.

7. A bacteria self-triggered halloysite composite antibacterial material prepared according to the preparation method according to any one of claims 1 to 6.

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