CN113425841B

CN113425841B - ICG-Ga nano material with laser-driven loose bonding and preparation method and application thereof

Info

Publication number: CN113425841B
Application number: CN202110374034.7A
Authority: CN
Inventors: 周民; 李杨杨; 祁宇宸; 谢婷婷
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2020-07-13
Filing date: 2021-04-07
Publication date: 2022-09-16
Anticipated expiration: 2041-04-07
Also published as: CN113425841A

Abstract

The invention provides an ICG-Ga nano material with laser-driven loose bonding, which comprises six elements of Ga, C, O, N, S and H, wherein the valence state of the Ga element comprises Ga ³⁺ And Ga ^δ+ (ii) a The bonding mode between Ga element and ICG in the nano material is laser-driven loose bonding, wherein the laser-driven loose bonding refers to a weak bonding mode which is easy to activate by laser and is formed between Ga element and ICG, and Ga element is rapidly released under the irradiation of near-infrared laser. The invention introduces Ga element which can disturb the iron metabolism process of bacteria into an ICG molecular structure with photodynamic antibacterial function to form antibacterial material with multiple antibacterial modes, and has the advantages of wide spectrum, high sterilization efficiency and difficult generation of drug resistance. The preparation method has the characteristics of simple production process, high yield, good reproducibility and low cost and large-scale production, and does not generate secondary pollution to the environment in the preparation process. The ICG-Ga nano material can obviously inhibit the growth of drug-resistant bacteria, has excellent antibacterial effect and has potentialIs developed into a novel non-antibiotic antibacterial drug for treating multiple drug-resistant bacterial infection.

Description

ICG-Ga nano material with laser-driven loose bonding and preparation method and application thereof

Technical Field

The invention belongs to the field of medical materials, and particularly relates to an ICG-Ga nano material with laser-driven loose bonding, and a preparation method and application thereof.

Background

Suppurative liver abscess is an invasive bacterial infection, and suppurative liver abscess (PLA) is a suppurative infection of liver parenchyma, and may cause serious complications. The prevalence of PLA is on the whole rising with the increase in the number of patients with diabetes and biliary diseases. Clinically available antibiotics are the primary method of treating liver infections. However, due to the inappropriate use of early antibiotics, more and more bacteria show lower sensitivity to antibiotics, the variety and number of resistant bacteria is gradually increasing, and even multi-drug resistant (MDR) bacteria and highly virulent pathogenic bacteria are emerging. Thus, this treatment has not been able to completely eradicate the bacteria at the site of infection.

In addition, the development of new drugs is increasingly difficult, and despite the enormous effort to find new antibiotics, the development of new drugs lags far behind the development of antibiotic resistance. Scientists have hardly discovered new antibiotics in the last two decades, and the treatment of diseases caused by drug-resistant bacterial infection is becoming a big problem facing the human society, so that the search for novel high-efficiency antibacterial agents is important, and the development of high-efficiency non-antibiotic-based strategies for resisting bacterial liver abscess is urgently needed.

Over the past few decades, some non-antibiotic antimicrobials, such as antimicrobial peptides, polymers and inorganic Nanoparticles (NPs), have shown potential as antimicrobial materials. Among them, the rapid development of inorganic non-antibacterial agents including silver, copper and gold nanoparticles has been widely used in the field of wound dressings and surgical instruments, which exhibit effective antibacterial therapeutic properties. However, inorganic antibacterial agents reported so far are mainly applied to the field of external wound infection. Whether these metal-based antibacterial agents (especially silver-based nanoparticles) can be used to fully exert the antibacterial ability against internal organ infections still presents some biosafety issues. To date, there have been few reports of the treatment of bacterial liver abscesses using any non-antibiotic drug, including nanoparticles.

Recent research shows that the medicine gallium nitrate clinically used for treating hypercalcemia has certain antibacterial performance. Some researches show that the gallium ions can inhibit the growth of various pathogens in experimental animals, and effectively prevent and treat bacterial infection. The pharmacological basis of the antibacterial and bactericidal activity of gallium ions is different from that of antibiotics, and the gallium ions rely on the chemical mimicry of gallium elements and iron elements which are highly similar to each other to confuse an identification system of organisms, so that the gallium ions replace the iron elements in thalli. Although many studies on gallium have been made on the ionic form, gallium ions still have a problem of hydrolysis into various forms of hydroxy compounds in physiological environments, and this is one of the important factors that prevent the wide application thereof in vivo. The physiological environment refers to the biological, physiological or environment around cells, and is generally a condition of blood or lymph circulation in an organism that affects the life and function of cells. In experiments, the simulation is often performed with Phosphate Buffered Saline (PBS), Fetal Bovine Serum (FBS), or the like. The hydrolysis is a process in which a gallium ion in an inorganic salt of gallium is combined with a hydroxide ion generated by water ionization to generate a hydroxyl compound of gallium, and further precipitation may occur, and at this time, the particle size of the compound increases, and the dissolution property deteriorates. On the one hand, due to the low efficiency of delivery to infected tissues, targeted therapy in vivo of gallium compounds is limited, and gallium is most abundant in the kidneys after injection of gallium nitrate (David, P, Kelsen, et al pharmaceutical of gallium nitrate man [ J ]. Cancer,1980), and therefore is not suitable for targeted therapy of liver diseases; on the other hand, inorganic salts of gallium are easily hydrolyzed in physiological environment liquid to form precipitates, for example, gallium nitrate in Fetal Bovine Serum (FBS) is hydrolyzed to become larger in particle size and worse in uniformity (see fig. 17), and the precipitates may be generated in physiological environment, thereby affecting the delivery in vivo, not only limiting the antibacterial performance, but also limiting the function of targeted therapy. Indocyanine green (ICG) is an FDA-approved near-infrared (NIR) fluorescent dye that can act as a photosensitizer that generates Reactive Oxygen Species (ROS) under irradiation of near-infrared laser light, useful for photodynamic therapy (PDT). ICG molecules have strong absorption in near-infrared light wave bands, and can generate active oxygen free radicals under the irradiation of near-infrared laser, so that bacteria are oxidized and damaged to play an antibacterial role. However, ICG is metabolized very rapidly in vivo, and generally about 97% of normal persons are eliminated from blood after intravenous injection for 20 minutes, and insufficient residence time in the affected area greatly limits its sustained antibacterial effect. Even if gallium is combined with some ligands to be prepared into a nano preparation, the stability of the gallium in an aqueous solution is difficult to ensure, and micro bubbles need to be constructed outside to maintain the solubility of the gallium (Chinese patent CN108578696A), so that the release of gallium ions is further influenced, and the antibacterial performance and the function of targeted therapy of the gallium ions are influenced. To date, there is no report that gallium nanoagents can target treatment of bacterially infected tissues.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a brand-new ICG-Ga nano material, a preparation method and application thereof. By controlling the formation of laser-driven loose bonding in the ICG-Ga nano material, the defects of over-rapid ICG metabolism in vivo and easy hydrolysis and precipitation in a gallium ion physiological environment are overcome, the ICG-Ga nano material with uniform size and good stability is synthesized, and the ICG-Ga nano material has broad-spectrum antibacterial performance and can realize targeted delivery to the liver.

Unexpectedly, the present invention has been developed to comprise clinically recognized gallium (III) (Ga) ³⁺ ) And indocyanine green (ICG) molecules by self-assembly in aqueous solutionThe engineered, biocompatible non-antibiotic loosely binds ICG-Ga nanomaterials to broadly combat multidrug-resistant (MDR) bacteria. The ICG-Ga nano material has excellent stability, and can keep good dispersibility in various solution systems (see figure 2).

The invention synthesizes a special ICG-Ga-containing laser-driven loosely-bonded nano material by a one-step method through self-assembly, which has good stability in aqueous solution (see figure 2) and has a targeting function to liver (see figure 5). The material has excellent antibacterial effect, 99.5 percent of bacteria can be killed by 25 mu g/mL of the material under the irradiation of 808nm laser (see figure 3), the defects of ICG and Ga in antibacterial application in the prior art are overcome, and the synergistic better effect is generated. Due to the use of gallium and ICG with high biocompatibility, the obtained material has excellent biocompatibility, and the survival rate of cells in vitro experiments reaches 80% when the concentration reaches 266 mu g/mL (see figure 10), so that the concentration required by treatment is completely met, and the safety of an organism is basically not influenced.

The ICG-Ga nano material can break the laser loose bonding between ICG and Ga elements by utilizing ICG molecules under the irradiation of near-infrared laser, and the induced photodynamic effect destroys a bacterial membrane and further promotes the endocytosis of gallium ions; the gallium ions then replace the iron in the bacterial cells, disrupting the metabolism of the bacterial iron and showing a synergistic bacterial killing and biofilm disruption effect. The ICG-Ga nano material shows excellent broad-spectrum antibacterial effect on bacteria in vitro and in vivo, and the excellent liver targeting function of the ICG-Ga nano material also provides an effective strategy for resisting liver abscess infected by MDR bacteria.

Specifically, the technical scheme of the invention is as follows:

an ICG-Ga nano material with laser-driven loose bonding, which comprises six elements of Ga, C, O, N, S and H, wherein the valence state of Ga element comprises Ga ³⁺ And Ga ^δ+ (ii) a The combination mode between Ga element and ICG in the nano material is laser-driven loose bonding, wherein the laser-driven loose bonding means that laser is easily formed between the Ga element and the ICGThe activated weak bonding mode can quickly release Ga element under the irradiation of near-infrared laser. The nano-materials can be Nanoparticles (NPs) or Nanoclusters (NCs).

Preferably, the detection method of the laser-driven loose bonding is that under the irradiation of near-infrared laser, the nano material quickly releases Ga element, and the mass proportion of the Ga element released within 10min is 0.1-3%; preferably, the wavelength of the near-infrared laser is 750-825 nm; preferably, the wavelength is 808 nm.

Preferably, the nano material has a safe antibacterial function, and the detection method comprises the following steps: at 1.0W/cm ² Under the condition of 808nm laser irradiation for 10min, the Minimum Inhibitory Concentration (MIC) is 25 mug/mL, the MIC is the concentration of a drug for inhibiting the propagation of pathogenic microorganisms at the minimum, and the survival rate of cells is more than 90 percent under the MIC concentration; preferably, the survival rate of the cells is more than 95%; more preferably, the survival rate of the cells is more than 99%. The antibacterial effect can be effectively exerted under the condition of ensuring that the material does not generate toxic or side effect on normal tissues.

Preferably, the nano material can maintain stability in any one of water, Phosphate Buffered Saline (PBS) and Fetal Bovine Serum (FBS), and the detection method comprises: measuring the hydrodynamic diameter of the nano material in various solutions for 7 consecutive days, comparing the hydrodynamic diameter of the nano material from 2 to 7 days with that of the nano material from 1 day, and the variation amplitude is not more than 20%. The stability refers to that the nano material in any one of water, PBS and FBS can not react with components in the solution to generate new components with changed sizes or dispersivity, can not generate precipitation or flocculation to influence the solubility of the material in the solution, and can not influence the delivery process of the nano material in vivo due to the change of the shape of the nano material in the solution in a physiological environment.

Preferably, the nano material is efficiently and rapidly targeted to the liver part and can be metabolized and discharged within 12 h. The detection method comprises the following steps: the mice injected with the nano material into the tail vein are continuously detected through living fluorescence imaging, within 15min after the materials are injected, the abdominal cavity area of the mice can detect a strong fluorescence signal and reach a peak value, the subsequent signal is gradually weakened within 0.5-8 h, and the signal disappears after 12 h.

Preferably, the particle size range of the nano material is 1-10 nm.

Preferably, the material has an average particle size of 5 nm.

Preferably, the hydrodynamic diameter of the nano material is 1-20 nm.

Preferably, the nanomaterial has a hydrodynamic diameter of 11.32 ± 1.21nm and a Polymer Dispersity Index (PDI) of less than 0.2.

Preferably, the mass proportion of the Ga element in the material is 1-50%; preferably, the Ga element accounts for 10-20% of the material by mass; more preferably, the Ga element content in the material is 14.5% by mass. Preferably, the Ga element accounts for 14.5% by mass of the material.

Preferably, the nano material has a maximum absorption peak at the position of 785nm of laser wavelength, and the absorption peak is not shifted due to the change of the mixture ratio of reactants.

Preferably, the material generates Reactive Oxygen Species (ROS) under the irradiation of near infrared laser, and the intensity of the ROS is in positive correlation with the power and the irradiation time of the laser. Preferably, the laser wavelength is 808 nm. Preferably, the material is formed by reacting Ga ions and indocyanine green.

The invention also provides a method for preparing the ICG-Ga material with the laser-driven loose bonding, the material is generated by the reaction of Ga ions and ICG, and the nano material is synthesized in the aqueous solution through a self-assembly one-step method without the assistance of adding other solvents.

Specifically, the preparation method comprises the following steps: 1) mixing the aqueous solution containing Ga ions and the aqueous solution of ICG in sequence without adding to obtain a mixed solution; 2) and continuously stirring the mixed solution for reacting for a certain time, and then filtering and washing to obtain the material.

Preferably, the reaction temperature in the step 2) is 4-50 ℃, and the reaction time is not less than 1 h; preferably, the reaction temperature is 20-30 ℃; preferably, the reaction temperature is25 ℃; preferably, the reaction time is 2-4 h; preferably, the reaction time is 3 h. Preferably, the Ga ions are derived from an inorganic gallium salt. Preferably, the inorganic gallium salt is gallium chloride (GaCl) ₃ ). Preferably, the inorganic gallium salt is gallium bromide (GaBr) ₃ )。

Preferably, the molar ratio of Ga ions to ICG is 0.1-100: 1; preferably, the molar ratio of Ga ions to ICG is 2-6: 1; more preferably, the molar ratio of Ga ions to ICG is 3.07: 1.

Preferably, the aqueous solution of Ga ions and the aqueous solution of indocyanine green are not sequentially mixed under light-shielding conditions.

The invention relates to application of a material containing ICG-Ga in any one or more fields of medicine, antibiosis and sterilization.

Preferably, the material is used as an antibacterial agent in and/or outside the human and/or animal body.

Preferably, the material is used as a targeted therapy of the liver of humans and/or animals.

The invention has the beneficial effects that: the invention designs an ICG-Ga nano material with laser-driven loose bonding. The material can be kept stable in various solution systems, especially in physiological environments, and cannot generate dimensional change or precipitate in the solution; unexpectedly, ICG-Ga is activated to generate ROS with antibacterial effect under the irradiation of near-infrared laser, meanwhile, the loose bonding of ICG-Ga is opened, ICG and Ga synergistically generate the antibacterial effect of non-antibiotics, the defects that ICG is excessively fast metabolized in vivo and gallium ions are unstable and easy to hydrolyze in a physiological environment are overcome, and the synergistic antibacterial effect of the non-antibiotics is not easily resisted by drug-resistant bacteria, so that drug-resistant pathogens can be effectively inhibited, the medicine can be used for treating diseases caused by drug-resistant bacterial infection, and the dosage of the medicine is 1.0W/cm ² The Minimum Inhibitory Concentration (MIC) of the sample was 25. mu.g/mL when the sample was irradiated with the 808nm laser for 10 min. And when the concentration of the material is lower than 133 mug/mL, the killing rate to normal cells is lower than 10%, so that the MIC of the material is far lower than the concentration at which the material can generate toxicity to normal tissues, and the material has the characteristic of safety and antibiosis. In addition, the material has liver targeting function and can be used for treating diseasesSystemic toxicity to other organ tissues during the procedure is almost negligible. Thus, the ICG-Ga NPs of the present invention are highly potential internal antimicrobials.

Drawings

FIG. 1 is a graph of the characterization of ICG-Ga NPs, wherein, graph A: transmission Electron Microscope (TEM) images of the synthesized ICG-Ga NPs; and B, drawing: water and kinetic Diameter (DLS) profiles of ICG-Ga NPs suspended in aqueous solution; and (C) diagram: ultraviolet-visible absorption spectra of ICG-Ga NPs and ICG solutions; and (D) diagram: energy dispersive X-ray spectroscopy of ICG-Ga NPs; E. and (F) diagram: x-ray photoelectron spectroscopy (XPS) of ICG-Ga NPs and Ga3d orbital spectroscopy of the binding energy of ICG-Ga NPs and fitting curves.

FIG. 2 is a schematic representation of the stability of ICG-Ga NPs in water, Phosphate Buffered Saline (PBS) and Fetal Bovine Serum (FBS). Wherein, A represents the photographs of ICG-Ga NPs at different time points in one week in three solutions; panel B represents the initial hydrodynamic diameter distribution of ICG-Ga NPs in three solutions; the C-plot represents the hydrodynamic diameter statistics of ICG-Ga NPs at different time points in one week in the three solutions.

FIG. 3 is a graph showing the in vitro antibacterial activity of ICG-Ga NPs against ESBL E.coli. Wherein, A is as follows: viability of ESBL E.coli after 24 hours incubation with different concentrations (0, 3.125, 6.25, 12.5 and 25. mu.g/mL) of ICG-Ga NPs in LB medium at 37 ℃; and B, drawing: after incubation with 25. mu.g/mL ICG-Ga NPs, at different power intensities (0, 0.1, 0.25, 0.5, 0.75 and 1W/cm) ² ) Then the survival rate of ESBL colibacillus is kept for 24 hours in LB culture medium at 37 ℃ under the laser irradiation of 808nm for 10 min; and (C) diagram: after incubation with 25. mu.g/mL ICG-Ga NPs or ICG for 2h, with or without 1.0W/cm ² The 808nm laser is irradiated for 10 minutes, and the survival rate of ESBL escherichia coli is 24 hours after the culture; and (D) diagram: optical photographs of bacterial colonies formed by treated ESBL e coli in all groups; e, drawing: the corresponding CFU count; and (F) diagram: after incubation with 25. mu.g/mL ICG-Ga NPs or ICG, the cells were irradiated with 808nm laser light (1W/cm) ² 10min), fluorescence image of ESBL e.coli stained with DCFH-DA; and (G) diagram: flow cytometric analysis of ESBL e.coli by staining with DCFH-DA to quantify ROS production; and (H) diagram: staining with SYTO9 and Propidium Iodide (PI) dyeFluorescence image of the treated ESBL E.coli of (1). (. p < 0.05)

FIG. 4 is a graph showing in vitro antibacterial effect against ESBL E.coli and biofilm formation inhibition of ICG-Ga NPs. Wherein, A is as follows: SEM images of ESBL escherichia coli after different treatments; and B, drawing: ga element scanning images of SEM images of escherichia coli subjected to different treatments; and (C) diagram: TEM images of the treated ESBL e coli; and (D) diagram: 3D confocal laser scanning microscope images (size: 630 μm. times.630 μm) of ESBL E.coli biofilms after different treatments, and viable bacteria with green fluorescence can be observed when the biofilms are stained with SYTO 9.

FIG. 5 is a graph representing the in vivo therapeutic effect of ICG-Ga NPs. Wherein, A is as follows: biodistribution images of ICG-Ga NPs post-injection in a mouse model of liver abscesses. Fluorescence images of ESBL E.coli infected mouse models at different times (0, 0.25, 0.5, 0.75, 1,2, 4, 6, 8 and 12h) after intravenous injection of ICG-Ga NPs. (the circled part is a region with higher ICG-Ga content); and B, drawing: ex vivo fluorescence images of major organs (heart, lung, liver, spleen and kidney) in ESBL e.coli infected mice after ICG-Ga NPs 2h (n ═ 3) injection; and (C) diagram: survival curve of infected mice after treatment (n-10); and (D) figure: optical images of bacterial colonies formed on LB agar plates on

days

1 and 3 after each treatment; e, drawing: the corresponding CFU counts of ESBL e.coli of day 1 bacterial colonies, (n ═ 3); and (F) diagram: the corresponding CFU count of ESBL e.coli of the bacterial colonies on day 3, (n ═ 3); and (G) diagram: gram stained images of liver sections on day 1 and day 3 after each treatment. (. p <0.05,. p <0.01,. p <0.001)

FIG. 6 is a graph representing the in vivo therapeutic effect of ICG-Ga NPs. Wherein, A is as follows: photographs of livers and liver sections H & E, IL-6 and IL-1b stained images on day 1 after different treatments; photographs of livers and liver sections H & E, IL-6 and IL-1b stained images on day 3 after different treatments.

FIG. 7 is a preliminary in vivo toxicity profile of ICG-Ga NPs. Wherein, A is as follows: h & E stained section images of major organs of mouse heart, liver, spleen, lung and kidney; and B, drawing: body weight change in mice following ICG-Ga NPs injection; C-M diagram: blood biochemical and hematological examination index analysis maps including White Blood Cells (WBC), Red Blood Cells (RBC), Hemoglobin (HGB), Mean Cell Volume (MCV), mean red blood cell hemoglobin (MCH), mean red blood cell hemoglobin concentration (MCHC), Platelets (PLT), alanine transferase (ALT), aspartate transferase (AST), Blood Urea Nitrogen (BUN), Creatinine (CREA).

FIG. 8 is a schematic diagram showing the mechanism of synthesis and antibacterial activity of ICG-Ga NPs

FIG. 9 TG curves of ICG and ICG-Ga NPs.

FIG. 10 is a graph depicting cytotoxicity in vitro. Wherein, A is as follows: viability of Hepl hepatocytes incubated for 24h with nanomaterials (ICG-Ga or Ag NPs) at different concentrations (0, 4.16, 8.32, 16.6, 33.28, 66.56, 133.12, 266.24. mu.g/mL); and B, drawing: viability of HEK 293 cells incubated for 24h with nanomaterial (ICG-Ga or Ag NPs) at different concentrations (0, 4.16, 8.32, 16.6, 33.28, 66.56, 133.12, 266.24 μ g/mL); and (C) diagram: viability of HUVEC cells incubated for 24h with different concentrations (0, 4.16, 8.32, 16.6, 33.28, 66.56, 133.12, 266.24 μ g/mL) of nanomaterial (ICG-Ga or Ag NPs); and (D) diagram: viability of HaCaT cells incubated at different concentrations (0, 4.16, 8.32, 16.6, 33.28, 66.56, 133.12, 266.24 μ g/mL) of nanomaterial (ICG-Ga or Ag NPs) for 24 h.

FIG. 11 is a graph of flow cytometry quantitative analysis for detecting ROS production in the presence of ICG-Ga NPs by ESBL E.coli under different laser irradiation conditions. Wherein, A is as follows: comparing ROS generation efficiency graphs of different laser powers under irradiation for 10 minutes; and B, drawing: the laser power is 1W/cm ² Comparison of ROS generation efficiency for different irradiation times.

FIG. 12100. mu.g/mL of PBS solution of ICG-Ga NPs at a power density of 1.0W/cm with or without ² The emission profile of Ga ions under 808nm laser irradiation.

FIG. 13 is a graph showing the photothermal effect of ICG-Ga NPs. Wherein, A is as follows: under 808nm laser irradiation, a series of power intensities (0.1, 0.25, 0.5, 0.75 and 1W/cm) ² ) Thermal images of ICG-Ga NPs in LB media for 10 minutes; b, drawing: corresponding temperature profile.

FIG. 14 bar graph of the ROS production positivity of ESBL E.coli under different treatment regimes. (NS: no significance, p < 0.001).

FIG. 15.A is a diagram: ex vivo fluorescence images of dissected major organs (heart, lung, liver, spleen and kidney) at 0.25 and 6 hours (n ═ 3); and B, drawing: quantification of the Total fluorescence intensity of ICG-Ga NPs in dissected major organs.

FIG. 16 is a schematic diagram showing the calculation of the synergistic effect of disrupting iron metabolism and photodynamic therapy in ICG-Ga NPs.

FIG. 17 Ga (NO) ₃ ) ₃ Characterization of the stability when dissolved in FBS. Wherein, A is as follows: ga (NO) ₃ ) ₃ Photograph dissolved in FBS; and B, drawing: ga (NO) ₃ ) ₃ Hydrodynamic diameter profile dissolved in FBS.

FIG. 18 Ga (NO) ₃ ) ₃ Characterization chart of in vitro antibacterial performance of ESBL escherichia coli. Wherein, A is as follows: different concentrations of Ga (NO) ₃ ) ₃ Survival rate 24h after ESBL colibacillus treatment; and B, drawing: corresponding photograph of 24h treated bacteria.

Detailed Description

The invention is further illustrated with reference to the following examples and figures; in this example, the antibacterial effect was analyzed by ICG-Ga antibacterial material preparation and in vitro antibacterial data.

Experimental Material

Indocyanine green (ICG) and gallium chloride (GaCl) ₃ ) Chemicals were purchased from Sigma-Aldrich. Phosphate Buffered Saline (PBS), physiological saline (NaCl) solution and Dulbecco's Modified Eagle's Medium (DMEM) were purchased from national drug-controlled chemical Co. 3- (4, 5-Dimethyl-2-thiazyl) -2, 5-diphenyl-2-tetrazolium bromide (MTT) assay kit, LIVE/DEAD BAC BII kit and DCFH-DA kit were purchased from Thermo Fisher Scientific (China). Lysogenic Broth (LB) broth medium was purchased from Biomerieux. Fetal Bovine Serum (FBS) was purchased from Gibco. Hepl hepatocytes, human embryonic kidney 293(HEK 293) cells, Human Umbilical Vein Endothelial (HUVEC) cells and immortalized keratinocytes (HaCaT) cells were obtained from the American type culture Collection. Coli (ESBL e. coli) producing β -lactamase was obtained from american type culture collection (ATCC 25922).

Characterization instrument

The size and morphology of ICG-Ga NPs were characterized by field emission scanning electron microscopy (Hitachi SU-70, Japan). The size distribution of the ICGGa NPs was measured using a Malvern Zetasizer Nano-ZS90 (Malvern, UK). UV-Vis spectra were recorded using a UV-2600 spectrophotometer (Shimadzu, Japan).

Example 1:

synthesis of ICG-Ga NPs (nanoparticles)

10mg of ICG powder was dissolved in 100mL of water to form a homogeneous ICG solution (100 mg/L). Subsequently, 1.4mL of gallium chloride (GaCl) ₃ ) The solution (5mg/mL) was slowly added to the above ICG solution. The mixed solution was further stirred at room temperature for 3 hours while keeping out of light. Finally, ICG-Ga NPs were obtained by centrifugal filtration through a 30kDa MWCO Amicon filter (Sigma-Aldrich) and stored at 4 ℃ for further characterization.

Detection of laser-driven Loose bonding in ICG-Ga NPs

5mL of 100. mu.g/mL ICG-Ga NPs in PBS was put into a 8000Da dialysis bag. The dialysis bag was placed in a beaker containing 95mL of PBS solution and was stirred continuously. Respectively in the presence or absence of power density of 1.0w/cm ² When the solution in the dialysis bag was irradiated with the laser beam at 808nm, 1mL of the solution was taken out from the beaker at 2-min intervals for 10 min. The taken-out solution was digested with concentrated nitric acid, and then the content of Ga element therein was detected by inductively coupled plasma mass spectrometry (ICP-MS). The material can rapidly release Ga element in a system under the irradiation of near-infrared laser, and the mass percentage of the Ga element released in 10min is 0.1-3%.

Detection of stability of ICG-Ga NPs

ICG-GaNPs were dissolved in 1mL of water, PBS and FBS, respectively, at a concentration of 200. mu.g/mL, wherein PBS and FBS were used to simulate physiological conditions. The three solutions were placed in cuvettes and photographs taken daily and the hydrodynamic diameter measured (fig. 2) for 7 consecutive days. As can be seen from the photographs, the three solutions in the cuvette remained clear at all times, no precipitate formed, and the hydrodynamic diameter remained essentially unchanged with a magnitude of less than 20% from day 1 to day 7. Therefore, the ICG-GaNPs can be kept stable in various solution systems, especially in physiological environments, can effectively maintain the shape in vivo and play an antibacterial function.

4. Trait characterization

ICG-Ga NPs were prepared by a one-step metal coordination-assisted self-assembly procedure (fig. 8). Transmission Electron Microscopy (TEM) showed that the shapes of the synthesized ICG-Ga NPs had a uniform size distribution, and the average size of the ICG-Ga NPs was about 5nm (FIG. 1A). However, the hydrodynamic diameter of the synthesized nanoparticles as measured by Dynamic Light Scattering (DLS) was 11.32 ± 1.21nm (fig. 1B), the size of which was higher than that in TEM results, and probably mainly some degree of agglomeration in aqueous solution. The ultraviolet visible spectrum shows that the ICG-GaNPs have strong absorption at 600-900 nm, and the ICG-Ga NPs have overlapped absorption peaks (figure 1C) at about 800nm and are overlapped with the absorption peak positions of pure ICG molecules, so that the ICG-Ga NPs can realize photodynamic activity under the irradiation of near-infrared laser. Furthermore, Energy Dispersive Spectroscopy (EDS) analysis indicated that ICG-Ga NPs contained C, O, N, S and Ga elements (fig. 1D). In addition, X-ray photoelectron spectroscopy (XPS) measurements were also performed to further confirm the composition of the elements, which showed XPS spectra of ICG-Ga NPs and detailed information of characteristic peaks of O1, N1, C1, S2p and O1. Ga3d (FIG. 1E) that is very consistent with the composition of ICG-Ga NPs. In particular, the Ga3d peak can be deconvoluted into two components. It can be observed that the two peaks are located at 20.3 and 19.6eV, respectively (FIG. 1F), which can be attributed to Ga ³⁺ And Ga ^δ+ Species of the species. Finally, a Thermogravimetric (TG) test was performed to further confirm that the ICG molecule has successfully coordinated with the Ga ion (fig. 9).

5. Cell compatibility assays

Cytotoxicity of ICG-Ga NPs was studied compared to silver nanoparticles (Ag NPs) using four normal human cells, including human embryonic kidney 293(HEK 293) cells, Hepl hepatocytes, Human Umbilical Vein Endothelial (HUVEC) cells, and human immortalized keratinocytes (HaCaT) cells (fig. 10). The relative cell viability of the ICG-Ga NPs group remained above 80% as the concentration of NPs (relative to Ga) increased from 0 to about 266 μ g/mL. However, when the concentration of Ag NPs was examined in the range of 0 to about 266. mu.g/mL, cell viability showed significant cytotoxicity when the concentration of Ag NPs reached 33.28. mu.g/mL. When the concentration of Ag NPs reaches about 266. mu.g/mL, about 99% of the test cells can be killed. The above results reflect the excellent cell compatibility of ICG-Ga NPs.

6. Characterization of antibacterial Properties

The Minimum Inhibitory Concentration (MIC) is the concentration of the drug which can inhibit the propagation of pathogenic microorganisms at the minimum, generally is the concentration of the drug which can inhibit the growth of pathogenic bacteria in a culture medium after bacteria are cultured in vitro for 18 to 24 hours, and is an index for measuring the antibacterial activity of the antibacterial drug.

After the drug-resistant Escherichia coli is incubated with ICG-Ga NPs with different concentrations (3.125, 6.25, 12.5 and 25 mu g/mL) for 24 hours, the ICG-Ga materials with 3.125 mu g/mL and 6.25 mu g/mL do not show obvious antibacterial effect. However, as the material concentration increased, 12.5. mu.g/mL and 25. mu.g/mL of ICG-Ga showed killing of about 30% and 65% of bacteria, respectively, exhibiting concentration-dependent antibacterial effects (FIG. 2A). Bacteria were incubated with 25. mu.g/mL ICG-Ga NPs for 2h at different power densities (0.1, 0.25, 0.5, 0.75, 1, 1.5W/cm) ² ) After 808nm laser irradiation for 10min, the bacteria were cultured for 24h, and the results showed that: compared with a complete control group (a pure bacteria group without material addition and laser irradiation), the antibacterial efficiency of 25 mu g/mL ICG-Ga NPs is still about 65% under the condition of no laser irradiation. The antibacterial effect is gradually enhanced along with the increase of the laser power, and when the power density is 0.75W/cm ² Can kill 90% of bacteria, especially at laser power of 1W/cm ² Or more than 1W/cm ² When the bacteria were killed by ICG-Ga over 99.5%, the bacteria exhibited extremely strong antibacterial performance (FIG. 2B). At the binding of 1W/cm ² The MIC of ICG-Ga was 25. mu.g/mL when subjected to near-infrared laser. Furthermore, the temperature change of the bacterial suspension may vary slightly at the applied laser power intensity (fig. 13). Even at 1W/cm ² The maximum temperature of the bacterial suspension did not exceed 42 c, indicating that the superior antibacterial performance may not be due to photothermal effects, but rather due primarily to gallium ions and ICG binding molecules. Meanwhile, the above results show that the power intensity of laser irradiation has a significant influence on the antibacterial activity.

Subsequently, the radiation power intensity and concentration of ICG-Ga NPs were fixed at 1W/cm ² And 25. mu.g/mL to study the antibacterial activity of the combination. The bacteria received or did not receive 1W/cm after 2h incubation with 25. mu.g/mL ICG-Ga NPs and ICG molecules ² The survival rate after 808nm laser irradiation. The results show that: compared with a control group, the pure photodynamic group (ICG + Laser) can kill 30% of bacteria, and the combination of photodynamic antibiosis and gallium ion antibiosis (ICG-Ga NPs + Laser) can effectively kill more than 95% of bacteria. Under the condition of not giving 808nm laser irradiation, pure ICG does not show an antibacterial effect, but the ICG-GaNPs group can still kill about 60 percent of bacteria, which shows that the ICG-Ga NPs have a strong combined bactericidal effect. In the experiment, ICG-Ga NPs with low dose can combine multiple ways such as photodynamic action and ionic action to realize high-efficiency antibiosis (figure 3C). After the bacteria are incubated with 25 mu g/mL ICG-Ga material and ICG molecules for 2 hours, the existence of the bacteria is 1W/cm ² The plate is coated with the bacteria after 808nm laser irradiation and the corresponding colony count is counted. Compared with the control group, the number of bacteria in the ICG group alone has no obvious difference, which indicates that the pure ICG has no antibacterial effect. The colony number of the ICG + Laser group is slightly reduced, but still is obviously higher than that of the ICG-Ga group and the ICG-GaNPs + Laser group, wherein ICG-GaNPs can inhibit bacteria by more than 60%, and ICG-GaNPs + Laser can inhibit bacteria by more than 90%, which shows that the ICG-GaNPs and the ICG-Ga NPs + Laser have obvious bacteriostatic action and the combined antibacterial effect is better than that of a pure material (figure 16), and the corresponding colony number statistics also show the same results (figure 3D and figure 3E).

In addition, the antimicrobial activity of ICG or ICG-Ga NPs under laser irradiation may be due to toxic ROS. Therefore, DCFH-DA was used to study ROS production after different treatments on ESBL E.coli. As shown in fig. 3F, G and fig. 11, bacteria treated with ICG or ICG-Ga NPs plus laser irradiation exhibited significant fluorescence signals, demonstrating the rapid generation of ROS under laser irradiation. In addition, live/dead staining assays were performed to assess bacterial survival by detecting fluorescence. As shown in FIG. 3H, the live/dead staining images of ESBL E.coli of the control group and the ICG group showed almost no red fluorescence signal, indicating that the bacteria were still alive. In contrast, some red fluorescence appeared in the ICG NPs + Laser and ICG-Ga NPs groups, while more red fluorescence signal was observed in the ICG-Ga NPs + Laser group. These results indicate that ICG-Ga NPs have excellent antibacterial properties at 808nm radiation, which is consistent with colony formation analysis (FIGS. 3D, E).

7. Detection of antibacterial mechanism

To further study the antibacterial mechanism, the morphological changes of the ESBL e coli after different treatments were observed by Scanning Electron Microscope (SEM), and the ESBL e coli of the control group and the ICG group alone were in a normal rod shape with a complete and smooth surface. However, when the bacteria were irradiated with 808nm laser after treatment with ICG or ICG-Ga NPs, the original morphology distorted and showed wrinkled bacterial cell walls with clear lesions and holes (FIG. 4A). Notably, SEM correlation analysis showed enhanced Ga ³⁺ It accumulated on the disrupted bacterial membrane (FIG. 4B), which may promote Ga ³⁺ Endocytosis of (a). In addition, the internal morphology of the bacteria was observed by Transmission Electron Microscopy (TEM). After treatment with ICG plus laser irradiation or ICG-Ga NPs, the shape of ESBL E.coli became irregular and part of the outer membrane was damaged (FIG. 4C). In particular, after treatment with ICG-Ga NPs plus laser irradiation, the innermost contents of the bacteria disappeared and the membrane was partially destroyed, indicating a loss of structural integrity of the cell wall. Bacterial biofilms are a major cause of drug resistance. Thus, the biofilm-eliminating effect of ICG-Ga NPs was further evaluated. As shown in fig. 4D, green fluorescence indicates live bacteria in the biofilm. Compared with a control group and an ICG group, the growth of an ESBL escherichia coli biomembrane can be effectively inhibited by adding gallium. The biofilm was almost completely eliminated and could hardly be formed in the ICG-Ga NPs + Laser mode. Thus, laser treated ICG-GaNPs revealed a synergistic antibacterial effect of Ga-based therapy and photodynamic therapy, which resulted in severe destruction of bacterial biofilms.

8. Characterization of in vivo Combined antimicrobial Effect

In view of exciting in vitro antibacterial results, a series of examinations were performed using ESBL E.coli infected mouse liver abscess model to investigate the in vivo combined antibacterial effect of ICG-Ga NPs. Initially, the near infrared absorption and fluorescence emission properties of ICG molecules were used to monitor their biodistribution in order to observe and analyze the biodistribution and metabolic behavior of ICG-Ga NPs in vivo. Subsequently, 200. mu.L of IGG-Ga NPs (100. mu.g/mL) were administered to ESBL E.coli infected mice and imaged by an optical imaging system at different time points (FIG. 5A). Within the first 0.25 hours after administration, a strong fluorescence signal was detected in the abdominal region of the mice, which reached a maximum shortly thereafter. Thus, the signal gradually decreased at time points of 0.5-8 hours and finally disappeared at 12 hours. The change in fluorescence intensity may be caused by the biodistribution behavior of ICG-Ga NPs in mice. Mice were sacrificed at time points of 0.25, 2 and 6 hours to harvest heart, liver, spleen, lung and kidney. The fluorescence signals of these major organs were detected by the same imaging system (fig. 5B and fig. 15). In vivo fluorescence images showed that ICG-Ga NPs accumulated rapidly in liver tissue and peaked within 0.25 h. Subsequently, it is rapidly removed from the body of the mouse, resulting in a reduction in accumulation due to the clearance characteristics of liver and kidney metabolism. As expected, a higher fluorescence signal of the liver was observed consistent with the whole-body fluorescence image. Thus, these results indicate that ICG-GaNPs can be localized to the liver site to treat bacterial infections.

After administration of 200. mu.L PBS, ICG (100. mu.g/mL) or ICG-Ga NPs (100. mu.g/mL), the mixture was irradiated with a laser at 808nm at 1W/cm ² The survival rate of the power intensity irradiated infected mice for 10 minutes) were each carefully studied, as shown in fig. 5C. Mice in the control group and ICG group died continuously from day 1 to day 3 or day 4, respectively. In particular, the death time of both groups of mice was concentrated on day 1 and day 2, indicating that ICG alone is not effective in treating acute liver abscess caused by ESBL e. In the ICG + Laser and ICG-Ga NPs groups, the survival rates were 20% and 50%, respectively, indicating that the therapeutic effect was gradually enhanced. Encouraging, the final survival rate for the ICG-Ga + Laser group was 70%, significantly higher than for all other groups. The above results directly reflect the excellent ESBL E.coli killing performance of PDT-combined ICG-Ga NPs. Thus, liver tissue was further excised by using standard plate methods to quantify the bacterial numbers after these different treatments. As shown in FIGS. 5D-F, compared to other treatment methods, the second time after the injection of saline or ICG aloneThe highest number of bacteria was found in the livers of infected mice at day 1 and day 3, while the highest number of bacteria was found in all groups at day 3, which was significantly increased compared to day 1. In addition, the number of bacteria in the ICG + Laser and ICG-Ga NPs groups decreased slightly on

days

1 and 3, indicating an enhanced in vivo killing efficiency against ESBL E.coli. The ICG-Ga + Laser treatment group has the least bacteria number and shows the most obvious antibacterial efficiency. Similar experimental results were observed by gram staining (fig. 5G). This is in good agreement with the in vitro results, thus clearly demonstrating that ICG-Ga + Laser treatment has the best effective capacity against ESBL e.

9. Pathological changes and verification index characterization of infected liver after different therapies

The present invention evaluates pathological changes in the infected liver and associated inflammatory indicators after various treatments. Mice were sacrificed and livers were collected on day 1 and day 3 for hematoxylin and eosin (H & E) staining as well as inflammatory immunohistochemical staining. Representative photographs of infected livers showed significant differences between the various treatments (FIG. 5). The livers of mice in the control, ICG and ICG + Laser groups had distinct abscess regions (highlighted by yellow dashed lines) on day 1 after ESBL E.coli infection. H & E staining of these livers also showed extensive necrosis and inflammatory cell infiltration. Particularly on day 3 post-infection, the liver tissue necrosis became more severe in the control, ICG and ICG + Laser groups, with some degree of dissolution and liquefaction. In addition, the expression levels of inflammatory factors (IL-6 and IL-1b) were significantly higher in these groups than in other groups, including the ICG-Ga NPs group and the ICG-Ga + Laser group. The presence of distinct foci of inflammation and necrotic regions indicate a typical inflammatory response. It is noted that by receiving laser irradiation at 808nm followed by injection of ICG-Ga NPs, the degree of inflammation can be suppressed and the histopathological conditions including reduction of abscess region, local suppuration, mild necrosis, reduction of inflammatory cell infiltration and expression levels of IL-6 and IL-1b can be significantly alleviated. Notably, after ICG-Ga + Laser treatment, the liver abscess sites were substantially restored to normal, suggesting that combining Ga with ICG-mediated photodynamic therapy is most effective for histopathological improvement in ESBL e.

Toxicity evaluation of ICG-Ga

Although cytotoxicity evaluation showed that the ICG-Ga NPs of the present invention have excellent biocompatibility, biosafety studies are still worth the attention of the present invention. The primary toxicity of ICG-Ga NPs was evaluated. Histological analysis of the major organs (fig. 7A) was performed on day 14 after intravenous injection of therapeutic doses of ICG-Ga NP, indicating that normal tissue architecture is free of significant inflammatory injury or organ damage. It was also found that the body weight of mice in all groups remained similar, indicating no significant acute toxicity of ICG-Ga NPs (fig. 7B). Likewise, there were no significant differences in blood biochemical and hematological parameters, indicating normal liver and kidney function (fig. 7C-M). The above results demonstrate that ICG-Ga NPs with reliable biosafety in vivo can be used as an excellent antimicrobial agent, inhibiting bacterial growth at the tested doses, with negligible toxicity.

The following are experimental materials and experimental methods for each experiment.

11. Cell culture

All cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% diabody (penicillin-streptomycin) and placed in a medium containing 95% O ₂ And 5% CO ₂ At 37 ℃. Cells were harvested by centrifugation (1000rpm3 min) and subcultured with 0.25% trypsin until the degree of fusion reached about 80%.

12. Cytotoxicity assessment

Cytotoxicity of ICG-Ga on Hepl cells, HEK 293 cells, HUVEC cells and HaCaT cells was tested by standard MTT assay. Cells were seeded in 96-well plates (100. mu.L/well, 8.0X 10) ⁴ cells/mL) and cultured in an incubator at 37 ℃ for 12 hours. The medium was then removed and the cells were treated with ICG-Ga NPs dispersed in fresh medium at various concentrations (0, 4.16, 8.32, 16.6, 33.28, 66.56, 133.12, 266.24. mu.g/mL) and incubated for an additional 24 hours. Furthermore, MTT solution (20. mu.L, 5mg/mL) was added instead of the medium, followed by incubation for 4 hours. The medium was discarded again and 150. mu.L of dimethyl sulfoxide (DMSO) was added to each well to dissolveAnd (4) the formazan is decomposed, and the formazan is shaken for at least 15 minutes. The corresponding spectral absorption at a wavelength of 570nm was recorded with a microplate reader (SpectraMax, MD M5, USA).

In vitro antimicrobial efficiency detection of ICG-Ga NPs

EBSL E.coli was selected to evaluate the synergistic antibacterial effect of ICG-Ga NPs. After mixing 8mL of LB broth, 20. mu.L of E.coli in logarithmic growth phase was inoculated into a test tube in a biochemical incubator. Subsequently, 100. mu.L of the bacterial suspension was transferred to 96-well plates after 24 hours of treatment with different concentrations (0, 3.125, 6.25, 12.5 and 25. mu.g/mL) of ICG-Ga NP. Absorbance at 600nm (OD600) was measured with a multimode microplate reader (SpectraMax, MD M5, USA) to indicate the bacterial concentration. Each concentration group was placed in parallel wells without bacteria (OD600 ═ OD600 experiment-OD 600 background) to subtract out background absorption of the substance.

14. Evaluation of photodynamic antibacterial efficiency of ICG-Ga NPs in vitro

EBSL E.coli is excited by a 808nm laser at a range of light intensities (0, 0.1, 0.25, 0.5, 0.75 and 1W/cm ² ) After 10 minutes of irradiation, the cells were inoculated with 25. mu.g/mL ICG-Ga NP for 2h and then incubated in a biochemical incubator at 37 ℃ for a further 24 h. 100 μ L of the bacterial suspension of the treated EBSL E.coli was transferred to a 96-well plate to measure OD600, and the bacterial concentration was indicated by a multimode microplate reader (SpectraMax, MD M5, USA) to further assess viability. Each concentration group was placed in parallel wells without bacteria (OD600 ═ OD600 experiment-OD 600 background) to subtract out background absorption of the substance.

In vitro synergistic antimicrobial efficiency detection of ICG-Ga NPs

After seeding with gradient concentration of ICG-Ga NPs or ICG 2 times, using 808nm laser at 1W/cm ² The EBSL E.coli was irradiated for 10 minutes. Further cultured in a shaking incubator at 37 ℃ for 24 hours. OD600 was measured by a microplate reader (SpectraMax, MD M5, USA). Each concentration set was placed in parallel wells without bacteria to subtract out background absorption of the substance (OD600 ═ OD600 experiment-OD 600 background). Meanwhile, 10. mu.L of the bacterial suspension of EBSL E.coli described above was serially diluted 10 according to the double dilution method described above ⁶ Is more than that of LB broth culture medium.The number of diluted bacterial solutions was determined by standard plate counting methods. The cloning plates were photographed and counted. Each group had three replicates.

LIVE/DEAD staining for checking bacterial viability assay

After inoculation with 25. mu.g/mL ICG-Ga NP or ICG, the resulting mixture was diluted with 10 ⁷ 1.5mL of EBSL E.coli in WFU at 1W/cm ² Receives laser radiation of 808nm at the light intensity of (1). After 2 hours and further 2 hours in EBSL E.coli at 37 ℃ in a shaking incubator, the cells were collected and washed twice with NaCl solution. SYTO9 and propidium iodide (50. mu.L 30. mu.M) dyes were used for staining for 15 min. The samples were observed under an inverted fluorescence microscope (Leica DMI 4000B, germany).

17. Reactive Oxygen Species (ROS) detection in bacteria

Oxidative oxygen produced by various treatments in bacterial cells was detected using 2', 7' -dichlorodihydrofluorescein (DCFH-DA, Sigma-Aldrich). Specifically, 25. mu.g/mL ICG-Ga NPs or ICG 2h were inoculated in a shaking incubator at 37 ℃ and then irradiated with a laser beam of 808nm at 1W/cm ² The EBSL E.coli was irradiated for 10 minutes. The treated ESBL E.coli was then immediately stained for 30 minutes by adding DCFH-DA dye (1: 1000 dilution) to the bacterial suspension. The bacterial suspension was centrifuged (8000rpm, 5 minutes) and then redispersed in PBS. The green fluorescence of ROS was observed and quantified using fluorescence microscopy (Leica DMI 4000B, Germany) and flow cytometry (Cytoflex Beckman America), respectively.

18. Bacterial morphology detection

2mL of 10 ⁶ ESBL E.coli/CFU was inoculated into a test tube in a biochemical incubator, and then 2mL of LB broth was mixed and exposed to 2mLICG-Ga NPs or ICG at a concentration of 25. mu.g/mL under laser irradiation at 808nm for 2h at 1W/cm ² Is irradiated for 10 minutes. The bacterial suspension was collected by centrifugation (8000rpm, 5 minutes) and fixed in a 2.5% glutaraldehyde solution for 12 hours, then fixed again with a 1% osmium tetroxide solution for 1-2 hours. After each fixation, the bacteria were washed 3 times with PBS. Subsequently, dehydration was carried out for 15 minutes using a concentration gradient of ethanol (30%, 50%, 70%, 80%, 90%, 95% and 100%). Critical point drying in a critical point dryer (day)Stereo HCP-type 2, Japan). Finally, the sample was observed by a scanning electron microscope (Japanese Hitachi SU-8010). For TEM, the dehydrated sample was further embedded in a Spur embedding medium and sectioned with a cutter (Leica EM UC7, Germany). The sample sections were stained with a citric acid solution and uranyl acetate for 10 minutes and observed by a transmission electron microscope (HitachiH-7650, japan).

19. Biofilm inhibition function assay

Biofilms were formed on coverslips horizontally suspended in 6-well plates. Each cover slip was inoculated to 2X 10 in LB broth medium in a biochemical incubator at 37 deg.C ⁶ CFU/mL of ESBL E.coli. The medium was changed daily until biofilm formation. The supernatant in each well was removed and 3mL of LB medium containing ICG-Ga NPs or ICG at a concentration of 25. mu.g/mL was added to each well of the 6-well plate until ESBL E.coli biofilm was formed. After 2 hours incubation with ICG-Ga NPs or ICG, 1.0W/cm with a 808nm laser ² The holes were irradiated for 10 minutes. The treated biofilm was stained with SYTO9 solution for 15 minutes. For 3D Confocal Laser Scanning Microscope (CLSM) image observation, 3D images of stained biofilms were obtained by laser confocal scanning microscope (Leica TCS SP8, germany) and the data were analyzed using LAS X software.

Detection of in vivo antibacterial efficiency of ICG-Ga NPs

All animal experiments were approved by the animal protection and use committee of the medical college of Zhejiang university. The therapeutic effect of ICG-Ga NPs was evaluated in a well-established liver abscess model. Briefly, 8 week old female Balb/c mice were purchased from Shanghai SLAC animal experiments, Inc. Mice were injected with 50 μ lesble in the liver parenchyma (1.0 × 10) ⁷ CFU/mL) formed abscesses, randomized into five groups for different treatments (n-5, each group). Respectively. After infection with bacteria, mice were injected intravenously with 200. mu.LICG and ICG-Ga NPs (100. mu.g/mL) at 1.0W/cm ² The power density of (2) was irradiated with a 808nm laser for 10 minutes. As a control, saline was injected into infected mice. After each treatment on

days

1 and 3, the liver was harvested and disrupted with a tissue prep disintegrator, and the bacteria were thoroughly dispersed with PBS.The number of bacteria was determined by standard plate counting methods. Meanwhile, liver tissue of each group was collected and fixed with 4% formalin solution to perform hematoxylin and eosin (H)&E) Staining, immunohistochemistry (IL-6 and IL-1b) staining and gram staining. The staining results were observed using an Olympus IX71 microscope and photographs of infected livers were taken. 200 μ L of 100 μ g/mL ICG-Ga NPs were injected in ESBL E.coli infected model mice and photographed with IVIS Lumina LT series (Perkinelmer) at different time points (0, 0.5, 1,2, 4) for 8, 12 hours. Mice were sacrificed and their major organs, including heart, liver, spleen, lung and kidney, were also harvested and photographed. The data for each photograph was analyzed using the Living Image 4.5 software (Perkin Elmer). All experimental procedures involving animals were performed according to the guidelines of the ethical committee of the institutional animal testing center of university at zhejiang.

In vivo preliminary toxicity testing of ICG-Ga NPs

Body weights of mice were also recorded when administered with 200. mu.L of 100. mu.g/mL ICG-Ga NPs. Mice were sacrificed on day 14 and blood was collected for blood routine, alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), Blood Urea Nitrogen (BUN) and Creatinine (CREA) examination. Major organs including heart, liver, spleen, lung and kidney were collected for H & E staining. Sections were obtained with an Olympus IX71 microscope.

Examples 2-13 various ICG-Ga NPs were synthesized with reference to the synthesis and detection methods of example 1, and the contents are detailed in table 1.

TABLE 1 preparation conditions and characterization of the Properties of ICG-GaNPs under different reaction conditions

Serial number	Reaction temperature/. degree.C	Reaction time/h	Ga ion: ICG molar ratio	Hydrodynamic diameter (nm)	Ga element mass ratio (%)	MIC(μg/mL)
							Example 2	4	3	3.07：1	12.48±1.56	14.6	25
Example 3	20	3	3.07：1	11.95±1.32	14.6	25
							Example 4	30	3	3.07：1	11.66±1.25	14.5	25
Example 5	50	3	3.07：1	12.77±1.78	14.3	25
							Example 6	25	1	3.07：1	10.89±2.05	13.7	25
Example 7	25	2	3.07：1	11.27±1.34	14.2	25
							Example 8	25	4	3.07：1	11.79±1.20	14.6	25
Example 9	25	5	3.07：1	12.30±1.15	14.7	25
							Example 10	25	3	0.1：1	19.88±3.86	1	100
Example 11	25	3	2：1	11.35±1.31	10	35
							Example 12	25	3	6：1	11.38±1.24	20	30
Example 13	25	3	100：1	1.81±0.67	50	60

The laser-driven loose bonding performance, stability, biocompatibility detection and other properties of the ICG-GaNPs prepared in the embodiments 2-13 are detected and characterized, and the results are similar to those of the embodiment 1.

Comparative example 1: see Chinese patent CN108578696A

(1) Detection of optimal Ga by Isotermal transfer calorimetry (Isothermal titration calorimetry, ITC) ³⁺ : ICG molar ratio. Specifically, 167mL Hepes buffer (10mM, pH 6.8) was added with 70.5mL CH ₃ OH、11.5mL CHCl ₃ And 1mL of n-hexane and standing for 24h, preparing gallium ions and ICG into 0.05mM and 1mM solutions respectively by using the prepared buffer, and dripping the ICG into the gallium ion solution at 20 ℃, wherein the ratio of the gallium ions to the ICG is 1: 2;

(2) preparing methanol-water solvent (such as 10:1,5:1,3:1,2:1,1:1,1:2,1:3,1:5,1:10) with different volume ratios. Dissolving 1mg ICG with 5mL of different methanol-water solvents respectively, dripping gallium ion solution (5mL) prepared from the same methanol-water solvent according to the optimal molar weight of gallium ions obtained in the step (1) into the solution, and stirring the solution at room temperature for 2 hours; the nanometer size of the self-assembly system formed under different methanol-water solvents is detected by using a particle size analyzer, and the solvent with the ratio of the methanol to the water solvent being 2:1 is the final use solvent.

(3) Preparing gallium ion/ICG @ Microbublless composite microbubbles: preparing 25ml (total) of the optimal Ga-ICG mixture (ICG,2.5mg) in the step (1) by using the final solvent in the step (2), weighing 5mg of soybean lecithin, 2.7mg of cholesterol and 0.7mg of DSPE-PEG2000, dissolving by using 1ml of trichloromethane respectively, adding the solution into the stirred Ga-ICG mixture, removing the trichloromethane solvent and the methanol solvent by using a rotary evaporator under the programmed temperature rise, and fixing the volume of the final mixture to 10 ml; adding C under sealed condition in 65 ℃ water bath ₃ H ₈ Intermittently ultrasonically bubbling the gas side by using an ultrasonic probe (pulse: 10s/10 s; 750W, 20kHz, 33 percent) to obtain the final Ga-ICG-loaded liposome microbubble.

In this comparative example, the obtained Ga-ICG encapsulated in liposome microbubbles was reacted in a methanol aqueous solution using an ultrasound-activated material to generate ROS. The difference between this comparative example and example 1 is that the morphology and structure of the obtained material are different, and the Ga-ICG is wrapped in liposome microbubble in this comparative example to maintain the uniformity in the solutionAnd when ROS are activated by using ultrasonic waves, the release of Ga ions is limited, and an ideal antibacterial effect cannot be exerted. When excited by laser irradiation, the ROS generation efficiency was inferior to that of example 1, and the stability was also affected. When 100. mu.g/mL of comparative example 1 was at 1W/cm ² Under the irradiation of 808nm laser, the inhibition rate of the compound on bacteria is lower than 10%, the antibacterial effect is basically not shown, and the MIC of the compound is far higher than that of the compound in the example 1 under the same condition. Whereas ICG-Ga in example 1 formed a separate laser driven loosely bonded structured nanomaterial. Thus, the material can be excited by a near-infrared laser. Under the irradiation of near-infrared laser, embodiment 1 can generate a large amount of ROS, and simultaneously, Ga ions in the material are rapidly released, so that the function of destroying the metabolism of bacterial iron is achieved, and the two synergistically exert a good antibacterial effect. In addition, example 1 can be prepared in aqueous solution by a one-step process without the addition of other solvents, the resulting material can be stable in water, PBS, FBS, does not undergo particle size change or precipitation due to hydrolysis, and can be well delivered in vivo.

Comparative example 2: see the paper ma chao. study of antibacterial properties of inorganic gallium compounds [ D ]. southwest university of transportation, 2015.

Gallium nitrate was dissolved in deionized water to give a gallium ion concentration of 0.01 mol/L. Putting gallium nitrate water solutions with different volumes of 0.01mol/L and sodium acetate water solutions with different concentrations into a reaction kettle, sealing the reaction kettle under different experimental conditions (pH values), reacting at a constant temperature of 170 ℃ for 10 hours, and naturally cooling to room temperature. The reaction product was filtered, and the precipitate was washed repeatedly with water and anhydrous ethanol. And (3) carrying out vacuum drying at low temperature to obtain an intermediate product GaOOH, placing the obtained intermediate product on a shelf in a muffle furnace, roasting for 3h at the temperature of 600 ℃, and carrying out annealing treatment for 5h in the air to finally obtain gallium oxide powder.

In this comparative example, a rod-like gallium oxide nanocrystal with a length of 2 μm, a width of 1 μm, and a thickness of 300nm was obtained without modification by other ligands. This comparative example differs from example 1 in the morphology of the resulting material. The size of the gallium oxide nanocrystal in the comparative example reaches the micron level, the solubility of the gallium oxide nanocrystal in the aqueous solution is poor, and the gallium oxide nanocrystal can only be used for the antibacterial coating on the surface of an article, but not be used for in vivo antibacterial in the field of medicine. The material in the embodiment 1 is nano-particles with the average size of about 5nm, and the obtained material can keep stable in a physiological environment and has good in-vivo application prospect. On the other hand, the gallium oxide nanocrystals in this comparative example have good crystallinity, and are not prone to release gallium ions with antibacterial effect, but the Ga ions and the ICG in example 1 form a loosely bonded composite structure driven by laser, so that example 1 can be excited to generate ROS under the irradiation of near-infrared laser, and the ROS can play an antibacterial effect of photodynamic therapy, and simultaneously, the Ga ions are efficiently released to play a role in destroying iron metabolism, and the two are synergistic, so that the antibacterial effect is far superior to that of this comparative example.

Comparative example 3: gallium nitrate (Ga (NO) ₃ ) ₃ ) Detection of stability and antibacterial Properties of

(1) And (3) detecting the stability of gallium nitrate: a solution of gallium nitrate in FBS was prepared by diluting 0.1mL of a 2mg/mL solution of gallium nitrate in 1mL FBS, placing the above solution in a cuvette, taking a photograph and measuring the hydrodynamic diameter (FIG. 17).

(2) And (3) detecting the in-vitro antibacterial property of gallium nitrate: configuration of gradient concentration Ga (NO) ₃ ) ₃ Solutions (0, 1.6, 3.1, 6.3, 12.5, 25, 50, 100, 200, 400. mu.g/mL). After incubation for 24h with ESBL E.coli, the images were taken and absorbance at 600nm was measured with a microplate reader (FIG. 18).

Ga (NO) was tested in this comparative example ₃ ) ₃ Stability in FBS and its in vitro antibacterial properties. As can be seen from graph A of FIG. 17, flocculent precipitates were generated immediately after the addition of the gallium nitrate solution to FBS. From the hydrodynamic diameter distribution (fig. 17B), the size exhibits a non-uniform multimodal distribution. In example 1, the ICG-Ga nanomaterial can maintain the particle size in various solution systems for a long time, and can better maintain the morphological structure in vivo, thereby achieving effective delivery of drugs. As can be seen from FIG. 18, the in vitro antibacterial performance of gallium nitrate is poor, and the MIC of gallium nitrate is more than 400 mug/mL. In example 1, the MIC of ICG-Ga was 25. mu.g/mL efficiency under 808nm laser irradiationThe fruit is better.

The above examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Claims

1. An ICG-Ga nano material with laser-driven loose bonding, which is characterized in that the nano material comprises six elements of Ga, C, O, N, S and H, wherein the valence state of the Ga element comprises Ga ³⁺ And Ga ^δ+ (ii) a The bonding mode between Ga element and ICG in the nano material is laser-driven loose bonding, wherein the laser-driven loose bonding refers to a weak bonding mode which is easy to activate by laser and is formed between Ga element and ICG, and Ga element is rapidly released under the irradiation of near-infrared laser;

the detection method of the laser-driven loose bonding comprises the following steps that under the irradiation of near-infrared laser, the nano material quickly releases Ga element, and the mass proportion of the Ga element released within 10min is 0.1-3%;

the nano material is generated by reaction of Ga ions and ICG, and is synthesized in aqueous solution through a self-assembly one-step method.

2. Nanomaterial according to claim 1, characterized in that the nanomaterial morphology is nanoparticles and/or nanoclusters; the diameter of the nano particles is 1-100 nm; the nanoclusters have a diameter of less than 1 nm.

3. The nanomaterial according to claim 1, wherein the near-infrared laser wavelength is 750-825 nm.

4. The nanomaterial according to claim 1, wherein the near infrared laser wavelength is 808 nm.

5. The nano-meter of claim 1The material is characterized in that the nano material has a safe antibacterial function, and the detection method comprises the following steps: at 1.0W/cm ² The minimum inhibitory concentration of the 808nm laser beam of (1) is 25. mu.g/mL for 10min, and the survival rate of the cells at this concentration is 90% or more.

6. The nanomaterial according to claim 5, wherein the survival rate of the cells is 95% or more.

7. The nanomaterial according to claim 5, wherein the survival rate of the cells is 99% or more.

8. The nanomaterial according to claim 1, wherein the nanomaterial can maintain stability in any one of water, phosphate buffer and fetal bovine serum, and the detection method comprises: measuring the hydrodynamic diameter of the nano material in various solutions for 7 consecutive days, comparing the hydrodynamic diameter of the nano material from 2 to 7 days with that of the nano material from 1 day, and the variation amplitude is not more than 20%.

9. The nanomaterial of claim 8, wherein the amplitude of the variation is no more than 10%.

10. The nanomaterial according to claim 1, wherein the nanomaterial is efficiently and rapidly targeted to a liver part and can be metabolically excreted within 12h, and the detection method comprises the following steps: continuously detecting a mouse injected with the nano material through living fluorescence imaging, wherein a fluorescence signal can be detected in an abdominal cavity area of the mouse within 15min after the material is injected, the fluorescence signal reaches a peak value, the signal is gradually weakened in the following 0.5-8 h, and the signal disappears after 12 h.

11. The nanomaterial according to claim 1, wherein the nanomaterial has a particle size ranging from 1 to 10 nm.

12. Nanomaterial according to claim 1, characterized in that the nanomaterial has an average particle size of 5 nm.

13. The nanomaterial according to claim 1, wherein the nanomaterial has a hydrodynamic diameter of 1 to 20 nm.

14. The nanomaterial of claim 1, wherein the nanomaterial has a hydrodynamic diameter of 11.32 ± 1.21nm and a polymer dispersibility index of less than 0.2.

15. The nanomaterial according to claim 1, wherein the mass proportion of the Ga element in the nanomaterial is 1-50%.

16. The nanomaterial according to claim 1, wherein the content of Ga in the nanomaterial is 10-20% by mass.

17. The nanomaterial according to claim 1, wherein the nanomaterial comprises 14.5% by mass of Ga.

18. The nanomaterial of claim 1, wherein the nanomaterial has a maximum absorption peak at a laser wavelength of 785nm, and the absorption peak is not shifted by a change in the reactant ratio.

19. The nanomaterial according to any one of claims 1 to 18, wherein the nanomaterial generates reactive oxygen species under irradiation of a near-infrared laser, and the intensity thereof is positively correlated with the power of the laser and the irradiation duration.

20. The nanomaterial of claim 19, wherein the laser wavelength is 808 nm.

21. A method for preparing the nanomaterial of claim 1, wherein the method comprises the following steps:

(1) mixing the aqueous solution containing Ga ions and the aqueous solution of ICG in sequence without adding to obtain a mixed solution;

(2) continuously stirring the mixed solution for reacting for a certain time, and then filtering and washing to obtain the nano material;

the reaction time is not less than 1 h.

22. The method according to claim 21, wherein the reaction temperature in the step (2) is 4 to 50 ℃.

23. The method of claim 22, wherein the reaction temperature is 20 ℃ to 30 ℃.

24. The method of claim 22, wherein the reaction temperature is 25 ℃.

25. The method of claim 22, wherein the reaction time is 2 to 4 hours.

26. The method of claim 25, wherein the reaction time is 3 hours.

27. The method according to claim 22, wherein the Ga ions are derived from a water-soluble inorganic gallium salt.

28. A method according to claim 27, wherein said inorganic gallium salt is selected from the group consisting of gallium chloride and gallium bromide.

29. The production method according to any one of claims 22 to 28, wherein the molar ratio of Ga ions to ICG is 0.1 to 100: 1.

30. The method according to any one of claims 22 to 28, wherein the molar ratio of Ga ions to ICG is 2 to 6: 1.

31. The production method according to any one of claims 22 to 28, wherein the molar ratio of Ga ions to ICG is 3.07: 1.

32. An antibacterial material, characterized in that: the antibacterial material comprises a nanomaterial according to any one of claims 1 to 20.

33. An antibacterial drug, which is characterized in that: the antibacterial agent comprising the nanomaterial according to any one of claims 1 to 20.

34. Use of a nanomaterial according to any of claims 1 to 20 in the manufacture of an antibacterial medicament.

35. Use of the nanomaterial of any of claims 1 to 20 in the preparation of a liver-targeted drug.