CN113661988B

CN113661988B - Ti-PA-Ply self-assembly sterilization material and preparation method and application thereof

Info

Publication number: CN113661988B
Application number: CN202111102701.2A
Authority: CN
Inventors: 何晓东; 徐立群
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2021-09-21
Filing date: 2021-09-21
Publication date: 2022-04-22
Anticipated expiration: 2041-09-21
Also published as: CN113661988A

Abstract

The invention discloses a Ti-PA-Ply self-assembly sterilization material, preparation and application thereof, wherein the preparation method comprises the following steps: taking a titanium sheet as a substrate, and co-modifying by using natural biological compounds Phytic Acid (PA) and epsilon-polylysine (Ply) antibacterial peptide. PA and Ply form coatings on the surface of the titanium sheet through their inherent affinity and ionic bonding between positron and electronegative groups. Co-deposition of PA-Ply shows nonspecific surface modification. And the physical and chemical characteristics of PA-Ply on the surface of the titanium sheet are characterized, and polymer decoration, surface elements and microscopic morphology are disclosed. The Ti-PA-Ply self-assembly sterilization material prepared by the invention has the advantages of simplified reaction steps, mild reaction conditions, good stability and the like, has potential application in sterilization, and also has the function of resisting bacterial infection.

Description

Ti-PA-Ply self-assembly sterilization material and preparation method and application thereof

Technical Field

The invention belongs to the field of antibacterial materials, and relates to a Ti-PA-Ply self-assembled multifunctional material, and a preparation method and application thereof.

Background

Implant-related infections (IAI) caused by bacterial biofilms are very common in implant replacement surgery, and in severe cases require further surgical removal of the implant or amputation. Implants infected with planktonic bacteria may grow and colonize the surface further in the form of a biofilm, threatening post-implantation surgery. In addition, the formation of biofilm on implant surfaces can hinder bone regeneration, and biodiverse bacterial pathogens are the only causes of major fatal diseases such as tuberculosis, pneumonia and food-borne diseases, which remain serious concerns for human survival. To address this problem, implant surface modification is a common and effective strategy.

At present, people research various functional antibacterial coatings and divide the coatings into two types, namely antifouling type and sterilization type. The anti-fouling coating is passive, protecting the implant surface from bacterial adhesion and infection, but does not kill microorganisms. The antifouling applications of hydrophilic polymers, such as polyethylene glycol (PEG) and zwitterionic polymers, are widely explored. On the other hand, the antibacterial coating directly kills attached planktonic bacteria, and is an effective strategy for inhibiting bacterial colonization. A number of payloads have been developed in antimicrobial coatings, including antimicrobial peptides, enzymes, ammonium compounds, metal nanoparticles, and photoactive materials. In most cases, the anti-fouling coating can protect the implant from bacterial infection. However, such an active covering layer only protects the implant from bacterial infections and not the surrounding tissue, which limits the clinical transforming effect of the implant. The modification of the surface of the substrate by the coating is a complex process, and a rapid, effective and biosafety surface modification method needs to be researched to promote the application of the biomaterial.

The PA-Ply is surface modified by a simplified method by utilizing natural molecules of PA and natural antibacterial peptides of Ply. PA natural molecules are extracted from plants and are fermented by microorganisms, and the PA natural molecules have obvious biocompatibility to human bodies. The rich phosphate groups in the PA can be covalently connected with the surfaces of metal oxides such as TiO2 and the like, and the PA can also be used for crosslinking functional materials and substrates. Ionic and hydrogen bonds between the electronegative PA and the positive layer lead to intricate connections between PA-Ply. Due to the affinity of PA, PA- ply can be modified on different surfaces. Titanium has been successfully used for artificial implantation in dentistry and orthopedics for decades, and it is an interesting matter to study the surface modification of titanium. Ti-PA-Ply was subjected to surface morphology, elemental analysis and hydrophilicity studies. In the study, Ti-PA-Ply showed good antibacterial performance both in vitro and in vivo. And analyzed the cell growth and biocompatibility of the PA-Ply coating. The results show that PA-Ply is a multifunctional coating with antimicrobial properties.

Disclosure of Invention

1. A Ti-PA-Ply self-assembly sterilization material is characterized in that a titanium sheet is taken as a substrate, natural biological compounds phytic acid PA and epsilon-polylysine Ply antibacterial peptide carry out self-assembly reaction co-modification on the surface of the titanium sheet, and a PA-Ply modified self-assembly multifunctional coating is obtained on the surface of the titanium sheet; the reaction refers to the complex connection between PA-Ply caused by ionic bonds and hydrogen bonds between negative electronegative PA and positive electronegative Ply, and the co-deposited coating is carried out on the surface of the titanium sheet; and co-deposition of PA-Ply appears non-specific; abundant phosphate groups in PA and TiO existing on the surface of titanium sheet₂Forming a covalent linkage.

2. The preparation method of the Ti-PA-Ply self-assembly sterilization material is characterized by comprising the following steps of:

1) mixing PA and Ply in 6mL deionized water to obtain PA-Ply suspension;

2) fully washing the titanium sheet in deionized water by ultrasonic and vortex, and drying in a drying oven at 60 ℃;

3) placing the titanium sheet in the step 2) in a 5 cm culture dish, and soaking the titanium sheet in the PA-Ply suspension in the step 1) for static deposition; the suspension becomes a transparent solution within 30 min;

4) standing for 4 hours at room temperature, washing the suspension and the substrate with deionized water, and then naturally drying to form a PA-Ply coating on the titanium sheet to obtain the Ti-PA-Ply self-assembled sterilization material.

Further, in the step 1), the molar ratio of PA to Ply is one of 1:1, 1:1, 1:3 and 1: 4.

Further, in the step 1), the concentration of PA was 2 mg/mL.

3. The use of the Ti-PA-Ply self-assembled bactericidal material of claim 1 for inhibiting bacterial adhesion and bacterial growth, and for corrosion and fog prevention.

The invention has the beneficial effects that: the Ti-PA-Ply sterilization material prepared by the invention not only has good biocompatibility, but also has good sterilization effect and anti-corrosion and anti-fog performance. The method has the advantages of simple operation, mild reaction conditions, safety, environmental protection, good stability and the like.

Drawings

In order to make the purpose, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings:

FIG. 1 is a schematic diagram of the synthesis of Ti-PA-Ply self-assembled bactericidal material.

FIG. 2 is a graph of the effect of Ti-PA-Ply self-assembled bactericidal material on Escherichia coli and Staphylococcus aureus and a graph of the effect under a scanning electron microscope.

FIG. 3 is a graph showing the number of bacteria surviving on the surface of Escherichia coli and Staphylococcus aureus after they were contacted with Ti-PA-Ply self-assembled bactericidal material.

FIG. 4 is a fluorescence confocal microscope and fluorescence intensity diagram of biofilm formation by Escherichia coli and Staphylococcus aureus on the surface of Ti-PA-Ply self-assembled sterilization material.

FIG. 5 is a graph showing in vitro evaluation of photothermal antibacterial performance of Ti-PA-Ply self-assembled antibacterial material against MRSA (methicillin-resistant Staphylococcus aureus).

FIG. 6 is a graph showing the evaluation of the in vivo antibacterial performance of the Ti-PA-Ply self-assembled bactericidal material by the rat subcutaneous infection model.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

The Ti-PA-Ply self-assembly sterilization material is prepared by the reaction shown in figures 1(a) - (b), a titanium sheet is taken as a substrate, and PA-Ply is coated on the titanium sheet, wherein the PA-Ply is prepared by combining natural biological compounds Phytic Acid (PA) and epsilon-polylysine (Ply) antimicrobial peptide, the combination refers to that ionic bonds and hydrogen bonds formed between negatively charged PA and positively charged Ply are independently responsible for the complex connection and formation of a PA-Ply complex, and phosphate groups rich in PA can be combined with TiO₂And covalently connecting the surfaces of the metal oxides to form a stable bonding layer. In the method, Staphylococcus aureus (S.) (S. aureus) And Escherichia coli (E. coli) The bactericidal effect of Ti-PA-Ply was tested as a representative of gram-positive and negative bacteria.

Example 1

The preparation of the Ti-PA-Ply self-assembled sterilizing material is shown in FIGS. 1(a) - (b), and the specific contents are as follows:

1) mixing PA and Ply according to different ratios (m/m, 1:1, 1:2, 1:3, 1:4 and PA are 2mg/mL) in 6mL deionized water to obtain PA-Ply suspension;

2) the Ti pieces were thoroughly washed by sonication and vortexing in deionized water and dried in an oven at 60 ℃.

3) Placing the Ti sheet in the step 2) in a 5 cm culture dish, and soaking the Ti sheet in the PA-Ply suspension in the step 1) for static deposition;

4) and standing for 4 hours at room temperature, washing the suspension and the substrate with deionized water, and naturally drying to obtain the stable Ti-PA-Ply bactericidal material.

Example 2

The contact sterilization performance of the Ti-PA-Ply self-assembled sterilization material is observed and evaluated by a scanning electron microscope, and the specific contents are as follows:

PA-Ply coating of titanium surfaces is an effective antibacterial strategy to combat planktonic bacterial pathogens. Ti and the modified substrate were treated in PBS to evaluate antibacterial performance, and colonies on the modified and unmodified substrates were compared. Bacteria attached to the Ti-PA-Ply substrate are killed by Ply. The antibacterial mechanism is presumed to be the disruption of the bacterial membrane by amide groups on the Ply chain. To confirm this, SEM micrographs of treated Ti and Ti-PA-Ply were captured.

As can be seen from FIGS. 2(a) - (c), the Ti-PA-Ply surface bacteria showed the morphology of E.coli and S.aureus compared to intact cell membranes on pure titanium. This experiment clearly demonstrates that the Ti-PA-Ply surface has contact bactericidal properties.

Example 3

Evaluating the bacteria adhesion inhibition performance of the surface of the Ti-PA-Ply self-assembled sterilization material by using a coating plate counting method, which comprises the following specific contents:

1. and (3) culturing bacteria: the frozen tube containing the strain was removed from the-20 ℃ freezer, thawed, inoculated into TSB medium, and shake-cultured at 37 ℃ for future use.

2. The method comprises the following specific steps of (sterilizing the equipment and liquid in advance):

1) bacterial suspension was diluted to 1X 10 concentration with PBS buffer⁷ cells/mL；

2) Placing unmodified or modified titanium plate (pristine Ti or Ti-PA-Ply) with specification of 1 cm × 1 cm in a 24-well plate, adding 1 mL of bacterial solution into each well, and culturing for 5 hours in a constant temperature incubator at 37 ℃;

3) sucking out the bacterial solution, adding 1 mL of PBS buffer solution along the hole wall to clean the surface of the titanium sheet, and repeating the steps for three times;

4) adding 3 mL of PBS buffer solution into a 15 mL centrifuge tube, putting the cleaned titanium sheet into the centrifuge tube, and carrying out ultrasonic treatment for 5 minutes to strip bacteria adhered to the surface;

5) adding 900 mu L of PBS buffer solution into a 1.5 mL centrifuge tube, sucking 100 mu L of the ultrasonic stripped bacterial solution, uniformly mixing by oscillation and carrying out gradient dilution;

6) pipette 100. mu.L of the solution after gradient dilution onto TSB agar plates, spread with a spreading rod until the bacterial solution is completely absorbed, incubate the plates at 37 ℃ for 24 hours, and count by photography.

FIGS. 3(a) - (e) are graphs of the number of bacteria surviving on the surface after exposure of Ti-PA-Ply samples to E.coli and S.aureus at different ratios of PA-Ply and different standing times: as shown in fig. 3(a) and 3(b), the modified Ti has different antibacterial properties compared to the original Ti. Ti-PA-Ply (1:1) has a weak antibacterial property against E.coli (4.9 X 10^5 cfu/cm) and Staphylococcus aureus (3.6 X 10^5 cfu/cm). With the increase of the layering rate, the sterilization efficiency is also increased. The surface area of the Ti-PA-Ply (1:2) is 6.9 X 10^4 cfu/cm and 1.1 X 10^4 cfu/cm for Escherichia coli and Staphylococcus aureus, respectively. When the thickness ratio is further increased to 1:3 and 1:4, E.coli and S.aureus have negligible bacterial survival of ≦ 10^2 cfu/cm on both Ti-PA-Ply (1:3) and Ti-PA-Ply (1: 4). Thus, the antimicrobial properties are related to the ply ratio of the coating.

Meanwhile, in order to better understand the antibacterial performance of Ti-PA-Ply, the substrate is stored for several days under normal conditions and is measured by a coating method. The antibacterial performance of the substrate (Ti-PA-Ply, 1:4) is still maintained after the substrate is placed for 1, 2, 3, 4 and 12 weeks (the killing rate is more than 99 percent)

Example 4

The inhibition capacity of the Ti-PA-Ply self-assembled bactericidal material on the formation of bacterial biofilms is evaluated by a fluorescence imaging method, and the specific contents are as follows:

1) bacterial suspension was diluted to 1X 10 concentration with TSB medium and PBS buffer at a volume ratio of 1:1⁵cells/mL；

2) Placing unmodified or modified titanium sheets (Pristine T or Ti-PA-Ply) with the specification of 1 cm multiplied by 1 cm into a 24-hole plate, adding 1 mL of bacterial solution into each hole respectively, and placing the 24-hole plate into a constant-temperature incubator at 37 ℃ for culturing for 24 hours;

3) sucking out the bacterial solution, adding 1 mL of PBS buffer solution along the hole wall to clean the surface of the titanium sheet, and repeating the steps twice;

4) dripping 15 mu L of SYTO9 and ammonium iodide (PI) combined dye onto a titanium plate, and coloring for 15 minutes;

5) add 1 mL of distilled water to a 24-well plate and carefully wash away excess fluorescent dye from the surface of the titanium plate.

FIG. 4 is a fluorescence confocal microscope and fluorescence intensity plot of biofilm formation by E.coli and Staphylococcus aureus on Ti-PA-Ply surfaces: in CLSM observations, live bacteria showed green fluorescence, while dead bacteria showed red fluorescence. As expected, the results were similar to the plate method. The green fluorescence of the original Ti surface and the Ti-PA-Ply surface are similar, while the red fluorescence is negligible. Whereas in CLSM images, green fluorescence decreased and red fluorescence increased with increasing thickness ratio. This also demonstrates that the bacteria killed at the substrate surface are not released and that the reduction in Ti-PA-Ply over the original Ti-PA-Ply is not because Ti-PA-Ply inhibits the adhesion of the bacteria but is killed.

Example 5

The Ti-PA-Ply photothermal antibacterial performance of the MRSA (methicillin-resistant Staphylococcus aureus) used was evaluated in vitro as follows:

1. MRSA was cultured for use.

1) PBS solution diluted MRSA bacterial concentration to 10^6 cfu/mL,

2) 24-well plates containing original Ti or Ti-PA-Ply substrate were co-cultured at 37 ℃ for 5 hours with 1 mL of bacterial suspension per well.

3) Ti and Ti-PA-Ply were stained with a combination of SYTO 9 and PI dyes, observed in CLSM, and colonies were counted by spread plate method.

FIGS. 5(a) - (c) are graphs of in vitro evaluation of photothermal antibacterial performance of Ti-PA-Ply on MRSA (methicillin-resistant Staphylococcus aureus): green fluorescence was observed on the original Ti surface, while the level of red fluorescence observed on the Ti surface was negligible; in contrast, only red fluorescence was observed with Ti-PA-Ply. The results show that the modified substrate can remove the adhered bacteria and inhibit the formation of MRSA biological membrane.

The antibacterial performance was calculated by the plate spreading method. On TSB-Agar plates of the same dilution, it was observed that the bactericidal effect was better when the Ti-PA-Ply was increased to 1:3 and 1:4 (PA: Ply, m/m). The plates were counted for further dilutions and the results were similar to those of E.coli and S.aureus as shown in FIG. 5 b. Ti-PA-Ply has a killing effect on >99% of MRSA when the ratio of PA and Ply is 1:3 and 1:4, respectively.

Example 6

A rat subcutaneous infection model is adopted to evaluate the in vivo antibacterial performance of the Ti-PA-Ply self-assembled bactericidal material, and the specific contents are as follows:

1. SD rats (200-220g) were used for the experiments. The experimental part was divided into 2 groups (n = 6) implanted in rats for the original Ti and Ti-PA-Ply matrices, respectively.

2. The specific procedure was (all animal experiments below were performed according to the animal Experimental guidelines approved by the university of southwestern Committee for animal use and Care university (UCUCUCA))

1) Rats were anesthetized in the operative part and shaved and disinfected on the backs of the rats.

2) The skin was incised and the original Ti and Ti-PA-Ply matrix (10 mm x 10 mm) was implanted under the skin. The implants were inoculated with Staphylococcus aureus at a concentration of 1 X 10 CFU/mL (10 . mu.L) and the skin was sutured.

3) After 5 d, the rats were sacrificed and the implants removed.

4) Adherent bacteria on the implants were counted using the plate method.

5) The immunopathological changes of heart, liver, spleen, lung, kidney and skin of transplanted and healthy rats were examined by H & E staining.

FIGS. 6(a) - (e) are graphs for evaluation of in vivo antibacterial performance of Ti-PA-Ply self-assembled bactericidal material by rat subcutaneous infection model: after 5 d, the implant was removed and the matrix tested for bacteria by plating (FIG. 6 f). Appropriate dilutions were made (FIGS. 6d and e) and the number of colonies on the Ti and Ti-PA-Ply surfaces was counted. The number of Ti surface bacteria is 2.9 multiplied by 10^6 cfu, and the number of Ti-PA-Ply surface bacteria is only 5.8 multiplied by 10^3 cfu. Tissue inflammation causing large area bacterial infection was analyzed. After dissection, the spleen of the rats in the Ti group was observed to be larger than that in the Ti-PA-Ply group (FIG. 6b, c), presumably due to inflammation caused by bacterial infection resulting in spleen swelling.

In addition, pathological tissue of rat skin to which the implant was attached was examined by H & E and Masson staining. Under the microscope, more inflammatory cells were visible in the Ti group than in the Ti-PA-Ply group in FIG. 6g (black arrows). Masson staining (FIG. 6h) showed more inflammatory cells (yellow circles) and collagen fibers (blue) in the Ti group compared to the Ti-PA-Ply group, indicating that the PA-Ply coating had anti-bacterial infection and reduced bacterial-induced inflammation.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims

1. A Ti-PA-Ply self-assembly sterilization material is characterized in that a titanium sheet is taken as a substrate, natural biological compounds phytic acid PA and epsilon-polylysine Ply antibacterial peptide carry out self-assembly reaction co-modification on the surface of the titanium sheet, and a PA-Ply modified self-assembly multifunctional coating is obtained on the surface of the titanium sheet; the reaction refers to the complex connection between PA-Ply caused by ionic bonds and hydrogen bonds between negative electron groups in PA and positive electron groups in Ply, and the co-deposited coating is carried out on the surface of the titanium sheet; and co-deposition of PA-Ply appears non-specific; abundant phosphate groups in PA and TiO existing on the surface of titanium sheet₂Forming a covalent linkage;

the preparation method of the Ti-PA-Ply self-assembly sterilization material is characterized by comprising the following steps of:

1) mixing PA and Ply in 6mL deionized water to obtain PA-Ply suspension;

4) standing for 4 hours at room temperature, washing the suspension and the substrate with deionized water, and then naturally drying to form a PA-Ply coating on the titanium sheet to obtain the Ti-PA-Ply self-assembled sterilization material;

in the step 1), the molar ratio of PA to Ply is 1:3 or 1: 4; in the step 1), the concentration of PA is 2 mg/mL.

2. The use of a Ti-PA-Ply self-assembled bactericidal material according to claim 1 for inhibiting bacterial adhesion and bacterial growth.