CN114949235A - Synthesis method of novel antibacterial repair-promoting electroactive nanoparticles - Google Patents

Synthesis method of novel antibacterial repair-promoting electroactive nanoparticles Download PDF

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CN114949235A
CN114949235A CN202210545547.4A CN202210545547A CN114949235A CN 114949235 A CN114949235 A CN 114949235A CN 202210545547 A CN202210545547 A CN 202210545547A CN 114949235 A CN114949235 A CN 114949235A
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苟雪
朱子馨
刘来义
罗胜年
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Southwest Jiaotong University
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Abstract

The invention relates to the field of nano material preparation methods, in particular to a synthesis method of novel antibacterial repair-promoting electroactive nano particles, which comprises the following steps: s1, soaking the BTO nano-particles into a NaOH solution; s2, heating in water bath and stirring; s3, filtering and washing with ethanol and deionized water; s4, vacuum drying at room temperature; s5, adding BTO/OH nanoparticles into an ethanol solution of succinic anhydride, stirring, washing and drying; s6, dispersing BTO/COOH in an ethanol solution containing Zn (NO3)2 & 6H2O and HCl; s7, adding a 50% ethanol solution of 2-methylimidazole and stirring; s8, washing and vacuum drying, namely dispersing BTO @ ZIF-8 in an L-1 ciprofloxacin hydrochloride solution and stirring; s9, washing with absolute ethanol and vacuum drying overnight. In the invention, BTO and ZIF-8 show good biocompatibility and can effectively promote wound healing.

Description

Synthesis method of novel antibacterial repair-promoting electroactive nanoparticles
Technical Field
The invention relates to the field of preparation methods of nano materials, in particular to a synthesis method of novel antibacterial repair-promoting electroactive nano particles.
Background
At present, most of the realization schemes related to the application of the metal organic metal framework MOF nano porous material as the antibacterial material are to form a nano composite material, such as the synthetic Ag @ ZIF-8 composite material invented by Zhang Yan Mei Lidong et alThe technical scheme includes that ZIF-8 nano particles are synthesized at normal temperature, then silver nitrate, sodium borohydride and ZIF-8 are prepared into mixed solution, the mixed solution is stirred, centrifuged and dried in vacuum, and an Ag @ ZIF-8 nano antibacterial agent with Ag nano particles dispersed on the surface of ZIF-8 is obtained, the antibacterial agent has high-efficiency antibacterial effect on bacteria, in an experimental scheme of promoting tissue repair of piezoelectric materials, for example, Wu and other people synthesize an Au @ BTO composite material through a two-step method, the first step synthesizes cubic BaTiO3 through a hydrothermal method, and the second step chemically reduces AuNPs to cubic BaTiO 3 The surface of the material has an Au @ BTO composite material with an acoustic dynamic effect, the composite material has a piezoelectron effect under ultrasonic irradiation, and piezoelectric current can be formed to promote cell migration and tissue repair.
Wound healing is considered to be a complex biological process comprising three phases: inflammation, new tissue formation and remodeling. Therefore, it is of great significance to develop a multifunctional platform that can not only eliminate wound inflammation, but also accelerate new tissue regeneration. With the development of nanomaterials, MOF, an organic metal framework, has made good progress due to the characteristics exhibited by its drug-loaded and antibacterial aspects, such as the aforementioned MOF combined with nanoparticles for antibacterial purposes or the MOF-loaded drug used as a drug release platform for antibacterial purposes, but the single antibacterial therapeutic effect is not sufficient for wound repair after bacterial infection. Metal ions such as Ag released although MOF 2+ 、Cu 2+ 、Zn 2+ Are substances essential to the human body and may contribute to cell growth and tissue formation, but the results are still unsatisfactory in terms of time, efficiency and safety compared with those sought. For the piezoelectric material, although the generated electric stimulation has the functions of cell migration and promoting tissue repair, and also has a certain antibacterial capability, the antibacterial mode is single, and active oxygen (ROS) generated only under the action of redox charges generated by a built-in electric field is used for sterilization, although the mode has a certain antibacterial capability, the sterilization effect is limited to a certain extent, if the ROS is too little, the antibacterial effect is weak, and if the ROS is too much, normal cells may be damaged to a certain extent. Therefore, only by a single pressureThe electrical materials simultaneously perform antibacterial and wound tissue repair promotion, and the efficiency aspect thereof is not yet expected.
Disclosure of Invention
The invention aims to provide a synthetic method of a novel antibacterial repair-promoting electroactive nanoparticle capable of effectively promoting wound healing aiming at the problems in the background art.
The technical scheme of the invention is as follows: a synthesis method of novel antibacterial repair-promoting electroactive nanoparticles comprises the following steps:
s1, soaking the BTO nano-particles into NaOH aqueous solution for surface modification;
s2, heating the mixture obtained in the S1 in a water bath at 60 ℃, stirring for 2 hours, and then stirring for 17 hours at room temperature;
s3, filtering and washing for 3 times by using ethanol and deionized water in sequence;
s4, vacuum drying for 24h at room temperature to obtain a product with hydroxylated surface, namely BTO/OH;
s5, adding 2g of BTO/OH nano particles into an ethanol solution of succinic anhydride, stirring for 2h at room temperature, washing and drying to obtain surface carboxylated BTO/COOH nano particles;
s6, dispersing 0.35g of BTO/COOH nanoparticles in 20mL of 50% ethanol solution containing 2mmol of Zn (NO3) 2.6H 2O and 0.2mmol of HCl;
s7, adding 40mL of 50% ethanol solution containing 20mmol of 2-methylimidazole into the suspension in S6, and stirring the obtained mixture at room temperature for 30 min;
s8, washing for 3 times and vacuum drying, collecting to obtain BTO @ ZIF-8 nano particles, and dispersing 30mg of BTO @ ZIF-8 nano particles in 15mL of 2gL -1 Stirring the solution in ciprofloxacin hydrochloride solution at room temperature for 24 hours;
s9, washing the obtained product with absolute ethyl alcohol for three times, and drying the obtained product in vacuum overnight to obtain the BTO @ ZIF-8/CIP nano particle.
Preferably, in S1, the BTO nanoparticles weigh 2g, have a particle diameter of 100nm and a purity of 99.9%.
Preferably, in S1, the NaOH aqueous solution is 50mmol, and the volume is 20 ml.
Preferably, in S5, the succinic anhydride has a volume of 50mL and a weight of 3 g.
Preferably, in S7, the mixture is stirred by ultrasonic wave.
Compared with the prior art, the invention has the following beneficial technical effects: the synthesized nano particle BTO @ ZIF-8/CIP is relatively stable under normal physiological conditions, but the ZIF-8 can be degraded under local acidification environment caused by bacteria, so that CIP and Zn are caused 2+ Can be released from ZIF-8 to exert bactericidal action. Meanwhile, BTO generates ROS under ultrasonic irradiation, which causes oxidative stress, further inhibiting bacterial infection. In addition, the electrical signal generated by piezoelectric BTO under mechanical load and ZIF-8-derived Zn2+ can synergistically promote cell growth and tissue regeneration. In vitro and in vivo experiments show that the unique combination shows good biocompatibility and can effectively promote wound healing. And the experimental result also shows that BTO and ZIF-8 can form a heterostructure and improve the performance of the nano-particle. These findings provide new insights into the rational design of effective wound healing strategies.
Drawings
FIG. 1 is a schematic structural diagram of an embodiment of the present invention;
FIG. 2 is a schematic view of the antimicrobial;
FIG. 3 is a schematic view of wound repair;
FIG. 4 is a schematic diagram showing the effect of novel drug-loaded nanoparticles on the wound surface in a mouse body;
FIG. 5 is a schematic view showing the effect of novel drug-loaded nanoparticles on bacteria in the exudate of a wound in a mouse body;
FIG. 6 is a schematic representation of histological changes at a wound site in a rat.
Detailed Description
Example one
As shown in fig. 1-3, the synthesis method of the novel antibacterial repair-promoting electroactive nanoparticle provided by the invention comprises the following steps:
s1, soaking the BTO nano-particles into NaOH aqueous solution for surface modification; the weight of BTO nano-particles is 2g, the diameter of the particles is 100nm, and the purity is 99.9%; 50mmol of NaOH aqueous solution, and 20ml of NaOH aqueous solution;
s2, heating the mixture obtained in the S1 in a water bath at 60 ℃, stirring for 2 hours, and then stirring for 17 hours at room temperature;
s3, filtering and washing for 3 times by using ethanol and deionized water in sequence;
s4, vacuum drying for 24h at room temperature to obtain a product with hydroxylated surface, namely BTO/OH;
s5, adding 2g of BTO/OH nano particles into an ethanol solution of succinic anhydride, stirring for 2h at room temperature, washing and drying to obtain surface carboxylated BTO/COOH nano particles; the volume of the succinic anhydride is 50mL, and the weight is 3 g;
s6, dispersing 0.35g of BTO/COOH nanoparticles in 20mL of 50% ethanol solution containing 2mmol of Zn (NO3) 2.6H 2O and 0.2mmol of HCl;
s7, adding 40mL of 50% ethanol solution containing 20mmol of 2-methylimidazole into the suspension in S6, and stirring the obtained mixture at room temperature for 30min by ultrasonic waves;
s8, washing for 3 times and vacuum drying, collecting to obtain BTO @ ZIF-8 nano particles, and dispersing 30mg of BTO @ ZIF-8 nano particles in 15mL of 2gL -1 Stirring the solution in ciprofloxacin hydrochloride solution at room temperature for 24 hours;
s9, washing the obtained product with absolute ethyl alcohol for three times, and drying the obtained product in vacuum overnight to obtain the BTO @ ZIF-8/CIP nano particle.
In the example, the synthesized nanoparticles BTO @ ZIF-8/CIP are relatively stable under normal physiological conditions, but ZIF-8 can be degraded under local acidification environment caused by bacteria, resulting in CIP and Zn 2+ Can be released from ZIF-8 to exert bactericidal action. Meanwhile, BTO generates ROS under ultrasonic irradiation, which causes oxidative stress, further inhibiting bacterial infection. In addition, the electrical signal generated by piezoelectric BTO under mechanical load and ZIF-8-derived Zn2+ can synergistically promote cell growth and tissue regeneration. In vitro and in vivo experiments show that the unique combination shows good biocompatibility and can effectively promote wound healing. And the experimental result also shows that BTO and ZIF-8 can form a heterostructure and improve the performance of the nano-particle. These findings provide new insights into the rational design of effective wound healing strategies.
Example two
Based on the embodiment of the synthesis method of the novel antibacterial repair-promoting electroactive nanoparticle, a specific experiment is listed, and the experimental steps are as follows:
s1, establishment of a bacterial infection animal model: male BALB/C mice, with an average body weight of 20g, all were housed in the SPF laboratory, according to the experimental standards specified by the ethical committee for animal experiments in western university of sichuan, the wound model of mice was established by cutting back skin of mice to form a circular full-thickness skin wound of about 10mm in diameter, and then infecting the wound with 50 μ L of a staphylococcus aureus suspension of 1 × 107 CFUmL-1;
s2, bacterial culture of the exudate: 24h after bacterial infection, marking as day 0 and 4 days after operation, collecting wound exudate by using a sterile swab, spraying the diluted bacterial suspension on an LB agar plate, culturing at 37 ℃ for 24h, and imaging bacterial colonies on the plate for analysis;
s3, evaluation of in vivo antibacterial activity: successfully infected mice were randomly divided into 6 groups including a negative control group, a US group, a BTO + US group, a BTO @ ZIF-8/CIP group and a BTO @ ZIF-8/CIP + US group, for the above experiments, the power density of US irradiation was 1.5Wcm-2, 1MHz, a 50% duty cycle, irradiation time was 5min, and the weight and wound size of the mice were recorded every day;
s4, histological analysis: for histological analysis, mice were euthanized at the end of the experiment, dermal tissues around the original wound were dissected, and soaked in 10% solubility formalin, paraffin embedded, 8 μm pathological sections were taken for hematoxylin eosin staining and Masson trichromatic staining, mouse five organs and other major organs were also used for H & E staining, and staining results were analyzed by ImageJ.
Analysis of animal wound antibacterial and repair promotion experiment results:
as shown in fig. 4, (a) in fig. 4 is a schematic diagram of in vivo experiment, (b) is skin wound images taken on days 0, 1, 3, 5, 7 and 11 in different treatment groups, (c) is quantitative analysis of wound recovery rate, and (d) is quantitative analysis of mouse body weight during experiment.
As shown in FIG. 5, (a) in FIG. 5 is the antibacterial picture of the exudate bacteria in the experimental and control groups at day 0 and day 4, and (b) is the corresponding survival rate of the bacteria in (a).
The BTO @ ZIF-8/CIP + US group was evaluated for in vivo antibacterial effects and wound healing effects in a Staphylococcus aureus infected skin wound mouse model. After each group of mice are infected with bacteria on the back of the round full-layer skin wound surface for 24 hours, a large amount of bacteria and serious inflammation phenomena can be seen on the wound surface, and as shown in (a) in fig. 4, the successful establishment of a mouse wound model seriously infected by staphylococcus aureus is demonstrated. The wound repair conditions of the infected wound are monitored by performing sound power treatment on the infected wound (BTO @ ZIF-8/CIP + US group) and are compared with the wound repair conditions of a control group (a negative control group, an US treatment group, a BTO + US group and a BTO @ ZIF-8/CIP group) for analysis. It is noted that the BTO @ ZIF-8/CIP + US group showed the best in vivo bactericidal effect, almost all bacteria were eliminated at several wounds on day 4, as shown in (a) and (b) of FIG. 5, and it can be seen from (b) of FIG. 4 that the wounds of the mice of the negative control group were self-healed slowly, and still large wounds and significant scars were visible on day 11, and it can also be seen that the BTO @ ZIF-8/CIP + US group showed the fastest wound healing speed and the smallest wound area on day 11, and the therapeutic effects were in the BTO @ ZIF-8/CIP group and BTO + US group in this order, except for this group. Meanwhile, (c) in FIG. 4 also shows that the wound closure rate of BTO @ ZIF-8/CIP + US is close to 100% at 11 days, and (d) in FIG. 4 also shows that the body weight of the mice is slightly increased during the experiment, indicating that the health condition of the animals is good. The experimental results show that BTO @ ZIF-8/CIP can release CIP bactericide in the acidic environment of bacterial infection and an electric field generated in acoustodynamics can also generate ROS, the generated ROS also has remarkable antibacterial activity, and the wound healing in vivo can be promoted by the synergistic electric field.
As shown in FIG. 6, (a) is an H & E and Masson stain image of wound tissue after 13 days of treatment of the mouse, and (b) is an H & E stain image of the main organs (heart, liver, spleen, lung and kidney) of the mouse after BTO @ ZIF-8/CIP + US treatment in FIG. 6. As shown in (a) of fig. 6, the experiment analyzed the histological changes of the wounds of the mice after 13 days of treatment, and better confirmed the wound healing activity of the BTO @ ZIF-8/CIP + US group by analyzing histological images of hematoxylin and eosin (H & E) and Masson stained wound tissues. From the experimental results, it is apparent that the treatment of the BTO @ ZIF-8/CIP and BTO + US groups induced the appearance of dermal tissue and the regeneration of strong collagen fibers, indicating that CIP, ROS and electric field can effectively promote wound healing. In addition, the epidermal-dermal junction of the BTO @ ZIF-8/CIP + US treatment group was clearly intact and abundant in collagen fibers, as shown in (a) of fig. 6, which further confirmed the excellent wound healing effect in synergy with the acoustokinetics of antibiotics. Further H & E staining of the major organs (heart, liver, spleen, lung and kidney) of the mice after BTO @ ZIF-8/CIP + US treatment showed no significant change as shown in (b) of FIG. 6, compared to the control group, which is sufficient to demonstrate that the coordinated treatment of wound tissue repair by BTO @ ZIF-8/CIP + US is not detrimental to the health of the organism itself.
The embodiments of the present invention have been described in detail with reference to the drawings, but the present invention is not limited thereto, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention.

Claims (5)

1. A synthesis method of novel antibacterial repair-promoting electroactive nanoparticles is characterized by comprising the following steps:
s1, soaking the BTO nano-particles into NaOH aqueous solution for surface modification;
s2, heating the mixture obtained in the S1 in a water bath at 60 ℃, stirring for 2 hours, and then stirring for 17 hours at room temperature;
s3, filtering and washing for 3 times by using ethanol and deionized water in sequence;
s4, vacuum drying for 24h at room temperature to obtain a product with hydroxylated surface, namely BTO/OH;
s5, adding 2g of BTO/OH nano particles into an ethanol solution of succinic anhydride, stirring for 2h at room temperature, washing and drying to obtain surface carboxylated BTO/COOH nano particles;
s6, dispersing 0.35g of BTO/COOH nanoparticles in 20mL of 50% ethanol solution containing 2mmol of Zn (NO3) 2.6H 2O and 0.2mmol of HCl;
s7, adding 40mL of 50% ethanol solution containing 20mmol of 2-methylimidazole into the suspension in S6, and stirring the obtained mixture at room temperature for 30 min;
s8, washing for 3 times and vacuum drying, collecting to obtain BTO @ ZIF-8 nano particles, dispersing 30mg of the BTO @ ZIF-8 nano particles in 15mL of 2gL-1 ciprofloxacin hydrochloride solution, and stirring the solution at room temperature for 24 hours;
s9, washing the nano particles with absolute ethyl alcohol for three times, and drying the nano particles in vacuum overnight to obtain the BTO @ ZIF-8/CIP nano particles.
2. The method for synthesizing the novel antibacterial repair-promoting electroactive nanoparticles as claimed in claim 1, wherein in S1, the BTO nanoparticles weight is 2g, the particle diameter is 100nm, and the purity is 99.9%.
3. The method for synthesizing the novel antibacterial repair-promoting electroactive nanoparticles as claimed in claim 1, wherein in S1, the volume of NaOH aqueous solution is 50mmol and 20 ml.
4. The method for synthesizing the novel antibacterial repair-promoting electroactive nanoparticle as claimed in claim 1, wherein in the S5, the volume of succinic anhydride is 50mL, and the weight of succinic anhydride is 3 g.
5. The method for synthesizing the novel antibacterial repair-promoting electroactive nanoparticles as claimed in claim 1, wherein the stirring mode of the mixture in S7 is ultrasonic stirring.
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