CN114949235B - Synthesis method of antibacterial repair-promoting electroactive nanoparticles - Google Patents
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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Abstract
The invention relates to the field of nano material preparation methods, in particular to a novel synthesis method of antibacterial repair-promoting electroactive nano particles, which comprises the following steps: s1, immersing BTO nano particles into a NaOH solution; s2, heating and stirring in a water bath; s3, filtering and washing with ethanol and deionized water; s4, vacuum drying at room temperature; s5, adding the BTO/OH nano particles into ethanol solution of succinic anhydride, stirring, washing and drying; s6, dispersing BTO/COOH in an ethanol solution containing Zn (NO 3) 2.6H2O and HCl; s7, adding 50% ethanol solution of 2-methylimidazole and stirring; s8, washing and vacuum drying, namely dispersing the BTO@ZIF-8 in the L-1 ciprofloxacin hydrochloride solution and stirring; s9, washing with absolute ethyl alcohol and drying in vacuum overnight. In the invention, BTO and ZIF-8 show good biocompatibility and can effectively promote wound healing.
Description
Technical Field
The invention relates to the field of nano material preparation methods, in particular to a novel synthesis method of antibacterial repair-promoting electroactive nano particles.
Background
The prior realization scheme of using MOF nano porous material with metal organic metal frame as antibacterial is to compose nano composite material, such as Zhang Yanmei Li Dongdong et al, synthesizing ZIF-8 nano particles at normal temperature, preparing mixed solution of silver nitrate, sodium borohydride and ZIF-8, stirring, centrifuging, vacuum drying to obtain Ag@ZIF-8 nano antibacterial agent with Ag nano particles dispersed on the surface of ZIF-8, wherein the antibacterial agent has high antibacterial effect on bacteria, while in the experimental scheme of promoting tissue repair for piezoelectric material, such as Wu et al, synthesizing Au@BTO composite material by two steps, wherein the first step is to synthesize cubic BaTiO3 by hydrothermal method, and the second step is to chemically reduce AuNPs in cubic BaTiO 3 The Au@BTO composite material with the acoustic dynamic effect is formed on the surface of the material, and the composite material has the piezoeffect under ultrasonic irradiation, so that piezoelectric current can be formed to promote cell migration and tissue repair.
Wound healing is considered a complex biological processComprising three phases: inflammation, new tissue formation and remodeling. Therefore, the development of a multifunctional platform which can not only eliminate wound inflammation, but also accelerate new tissue regeneration has great significance. With development of nanomaterials, the development of a nano-porous material with an organic metal framework such as MOF has been well progressed due to the characteristics of drug loading and antibacterial properties, and the aforementioned MOF and nanoparticles are combined with antibacterial properties or the MOF drug loading is used as a drug release platform for antibacterial properties, but the effect of only single antibacterial treatment is not enough to repair wounds after bacterial infection. Although MOFs release metal ions such as Ag 2+ 、Cu 2+ 、Zn 2+ Is a substance necessary for human body, and may contribute to cell growth and tissue formation, but the result is still unsatisfactory in terms of efficiency and safety as compared with the short-term, high-efficiency, and safe-keeping pursuit. While the piezoelectric material has the effects of cell migration and promoting tissue repair and has a certain antibacterial capability, the piezoelectric material has a single antibacterial mode, and only active oxygen (ROS) generated by the action of redox charges generated by an internal electric field is used for sterilization, and the mode has a certain sterilization capability, but the sterilization effect is limited, if the ROS are too small, the antibacterial effect is weak, and if the ROS are too large, normal cells are possibly damaged to a certain extent. Therefore, the antibacterial and wound tissue repair promotion are performed simultaneously by only using a single piezoelectric material, and the efficiency aspect of the antibacterial and wound tissue repair promotion is still not expected.
Disclosure of Invention
The invention aims at solving the problems in the background technology and provides a synthesis method of novel antibacterial repair-promoting electroactive nanoparticles capable of effectively promoting wound healing.
The technical scheme of the invention is as follows: a novel synthesis method of antibacterial repair-promoting electroactive nanoparticles comprises the following steps:
s1, immersing BTO nano particles into NaOH aqueous solution for surface modification;
s2, heating the mixture obtained in the step S1 in a water bath at 60 ℃, stirring for 2 hours, and then stirring for 17 hours at room temperature;
s3, filtering and washing with ethanol and deionized water for 3 times in sequence;
s4, vacuum drying is carried out for 24 hours at room temperature, and the product of surface hydroxylation is BTO/OH;
s5, adding 2g of BTO/OH nano particles into ethanol solution of succinic anhydride, stirring for 2 hours at room temperature, washing and drying to obtain BTO/COOH nano particles with carboxylated surfaces;
s6, dispersing 0.35g of BTO/COOH nano particles in 20mL of 50% ethanol solution containing 2 mmoles of Zn (NO 3) 2.6H2O and 0.2 mmoles 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;
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 In ciprofloxacin hydrochloride solution, stirring the solution at room temperature for 24 hours;
s9, washing with absolute ethyl alcohol for three times and drying in vacuum overnight to obtain the BTO@ZIF-8/CIP nano particles.
Preferably, in S1, the BTO nanoparticle weight is 2g, the particle diameter is 100nm, and the purity is 99.9%.
Preferably, in S1, the aqueous NaOH solution is 50mmol and the volume is 20ml.
Preferably, in S5, succinic anhydride is 50mL in volume and 3g in weight.
Preferably, in S7, the stirring mode of the mixture is ultrasonic stirring.
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 ZIF-8 can be degraded under a local acidification environment caused by bacteria, so that CIP and Zn are caused 2+ Can be released from ZIF-8 to exert the bactericidal effect. Meanwhile, BTO generates ROS under ultrasonic irradiation, and causes oxidative stress to further inhibit bacterial infection. In addition, the electrical signal generated by the piezoelectric BTO under mechanical load and the 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 biological phaseIs capacitive and can effectively promote wound healing. The experimental result also shows that BTO and ZIF-8 can form a heterostructure and improve the performance of the nano particles. These findings provide new insight into the rational design of an effective wound healing strategy.
Drawings
FIG. 1 is a schematic diagram of an embodiment of the present invention;
FIG. 2 is an antimicrobial schematic;
FIG. 3 is a schematic illustration of wound repair;
FIG. 4 is a schematic diagram showing the effect of the novel drug-loaded nanoparticle on the wound surface in the mouse;
FIG. 5 is a schematic diagram showing the effect of the novel drug-loaded nanoparticle on the wound exudate bacteria in the murine body;
fig. 6 is a schematic representation of histological changes at the wound site of mice.
Detailed Description
Example 1
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, immersing BTO nano particles into NaOH aqueous solution for surface modification; the weight of the BTO nano-particles is 2g, the particle diameter 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 step S1 in a water bath at 60 ℃, stirring for 2 hours, and then stirring for 17 hours at room temperature;
s3, filtering and washing with ethanol and deionized water for 3 times in sequence;
s4, vacuum drying is carried out for 24 hours at room temperature, and the product of surface hydroxylation is BTO/OH;
s5, adding 2g of BTO/OH nano particles into ethanol solution of succinic anhydride, stirring for 2 hours at room temperature, washing and drying to obtain BTO/COOH nano particles with carboxylated surfaces; succinic anhydride is 50mL in volume and 3g in weight;
s6, dispersing 0.35g of BTO/COOH nano particles in 20mL of 50% ethanol solution containing 2 mmoles of Zn (NO 3) 2.6H2O and 0.2 mmoles 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 by ultrasonic waves for 30min;
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 In ciprofloxacin hydrochloride solution, stirring the solution at room temperature for 24 hours;
s9, washing with absolute ethyl alcohol for three times and drying in vacuum overnight to obtain the BTO@ZIF-8/CIP nano particles.
In this example, the synthesized nanoparticle BTO@ZIF-8/CIP was relatively stable under normal physiological conditions, but ZIF-8 was degraded under the bacterial-induced local acidification environment, resulting in CIP and Zn 2+ Can be released from ZIF-8 to exert the bactericidal effect. Meanwhile, BTO generates ROS under ultrasonic irradiation, and causes oxidative stress to further inhibit bacterial infection. In addition, the electrical signal generated by the piezoelectric BTO under mechanical load and the 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. The experimental result also shows that BTO and ZIF-8 can form a heterostructure and improve the performance of the nano particles. These findings provide new insight into the rational design of an effective wound healing strategy.
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, establishing a bacterial infection animal model: male BALB/C mice, average weight 20g, all kept in SPF laboratory animal, experimental standard according to Sichuan university Huaxi animal laboratory ethical committee regulation, mice wound model establishment by cutting the back skin, forming a diameter of about 10mm circular full skin wound, then 50 u L staphylococcus aureus suspension infection wound, staphylococcus aureus suspension 1X 107CFUmL-1;
s2, bacterial culture of exudates: collecting wound exudates at 24h after bacterial infection, marked as day 0 and 4 days after surgery, using a sterile swab, spraying the diluted bacterial suspension on an LB agar plate, culturing for 24h at 37 ℃, and imaging bacterial colonies on the plate for analysis;
s3, in-vivo antibacterial evaluation: successfully infected mice were randomly divided into 6 groups, including a negative control group, a US group, a BTO 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,50% duty cycle, irradiation time was 5min, and the weight and wound size of the mice were recorded daily;
s4, histological analysis: for histological analysis, mice were euthanized at the end of the experiment and the dermal tissue surrounding the original wound was dissected, immersed in 10% solubility formalin, paraffin-embedded, 8 μm pathological sections were taken for hematoxylin eosin staining and Masson trichromatic staining, and the main organs of the five zang organs of mice were also used for H & E staining, and the staining results were analyzed by ImageJ.
Analysis of animal wound antibacterial and repair-promoting experimental results:
as shown in fig. 4, (a) in fig. 4 is an in vivo experimental schematic, (b) is an image of skin wound surface photographed on days 0, 1, 3, 5, 7, 11 respectively in different treatment groups, (c) is a quantitative analysis of wound recovery rate, and (d) is a quantitative analysis of mouse body weight during the experiment.
As shown in fig. 5, (a) in fig. 5 is an antimicrobial picture of the exudates bacteria in the 0 day and 4 day experimental group and control group teams, and (b) is the corresponding bacterial survival rate in (a).
The in vivo antibacterial effect and wound healing effect of the bto@zif-8/cip+us group in a skin wound mouse model of staphylococcus aureus infection were evaluated. After 24 hours of bacterial infection on the back of the circular full-thickness skin wound surface of each group of mice, a large amount of bacteria and severe inflammation phenomenon can be seen on the wound surface, and as shown in (a) of fig. 4, the establishment of a wound model of the mice severely infected by staphylococcus aureus is successful. The wound surface of the infection is subjected to acoustic power treatment (BTO@ZIF-8/CIP+US group), wound surface repair conditions are monitored, and control analysis is carried out on the wound surface repair conditions of a control group (negative control group, US treatment group, BTO group, BTO+US group and BTO@ZIF-8/CIP group). It is noted that the in vivo bactericidal effect of the bto@zif-8/cip+us group was best, almost all bacteria were removed from the wound on day 4, as shown in fig. 5 (a) and (b), and as can be seen from fig. 4 (b), the wound self-healing process of the mice in the negative control group was slow, the larger wound and obvious scar were still visible on day 11, and it was also found that the wound healing rate was the fastest in the bto@zif-8/cip+us group, the wound area was the smallest on day 11, and the therapeutic effects were 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 weight of the mice is slightly increased in the experimental process, which indicates that the animal health condition is good. The experimental results show that BTO@ZIF-8/CIP can release CIP bactericide in an acidic environment of bacterial infection and an electric field generated in acoustic dynamics can generate ROS, the generated ROS also has obvious antibacterial activity, and the synergistic electric field can promote in-vivo wound healing, and in the experimental process, mice stably grow after bacterial infection, so that the novel drug-loaded particles have no influence on the health condition of the mice.
As shown in fig. 6, (a) in fig. 6 is an H & E and Masson stained image of wound tissue after 13 days of treatment of mice, and (b) is an H & E stained image of major organs (heart, liver, spleen, lung and kidney) of mice after bto@zif-8/cip+us treatment. As shown in fig. 6 (a), the experiments analyzed the histological changes of the wounds of mice after 13 days of treatment, and the wound healing activity of the bto@zif-8/cip+us group was better confirmed by analyzing the histological images of hematoxylin and eosin (H & E) and Masson stained wound tissue. From the experimental results, it is evident that treatment of the bto@zif-8/CIP and bto+us groups induced the appearance of dermal tissue and regeneration of strong collagen fibers, indicating that CIP, ROS and electric fields can effectively promote wound healing. Furthermore, the epidermis-dermis interface of the bto@zif-8/cip+us treatment group was clearly intact and collagen fibers were abundant, as shown in (a) of fig. 6, which further confirmed the excellent wound healing effect in synergy with the antibiotic sonokinetics. Further H & E staining of the major organs (heart, liver, spleen, lung and kidney) of mice after BTO@ZIF-8/CIP+US treatment showed no significant change compared to the control group as shown in FIG. 6 (b), which is sufficient to demonstrate that the manner in which the coordinated treatment of wound tissue repair by BTO@ZIF-8/CIP+US does not pose a hazard 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 spirit of the present invention.
Claims (5)
1. The synthesis method of the antibacterial repair-promoting electroactive nanoparticle is characterized by comprising the following steps of:
s1, immersing BTO nano particles into NaOH aqueous solution for surface modification;
s2, heating the mixture obtained in the step S1 in a water bath at 60 ℃, stirring for 2 hours, and then stirring for 17 hours at room temperature;
s3, filtering and washing with ethanol and deionized water for 3 times in sequence;
s4, vacuum drying is carried out for 24 hours at room temperature, and the product of surface hydroxylation is BTO/OH;
s5, adding 2g of BTO/OH nano particles into ethanol solution of succinic anhydride, stirring for 2 hours at room temperature, washing and drying to obtain BTO/COOH nano particles with carboxylated surfaces;
s6, dispersing 0.35g of BTO/COOH nano particles in 20mL of 50% ethanol solution containing 2 mmoles of Zn (NO 3) 2.6H2O and 0.2 mmoles 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;
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 In ciprofloxacin hydrochloride solution, stirring the solution at room temperature for 24 hours;
s9, washing with absolute ethyl alcohol for three times and drying in vacuum overnight to obtain the BTO@ZIF-8/CIP nano particles.
2. The method for synthesizing the antibacterial and repair-promoting electroactive nanoparticles according to claim 1, wherein in S1, the BTO nanoparticles weigh 2g, have a particle diameter of 100nm, and have a purity of 99.9%.
3. The method for synthesizing the antibacterial repair-promoting electroactive nanoparticles according to claim 1, wherein in S1, the aqueous NaOH solution is 50mmol and the volume is 20ml.
4. The method for synthesizing the antibacterial repair-promoting electroactive nanoparticles according to claim 1, wherein in S5, succinic anhydride is 50mL in volume and 3g in weight.
5. The method for synthesizing antibacterial and repair-promoting electroactive nanoparticles according to claim 1, wherein in S7, the stirring mode of the mixture is ultrasonic stirring.
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