Bacteriocidal and aggregating bifunctional photosensitizer aggregates and methods for their preparation

Abstract

The application relates to a bacterial aggregation and killing bifunctional photosensitizer aggregate and a preparation method thereof. An AIE photosensitizer and a glycosyl amphiphilic molecule are added into a phosphate buffer solution according to a certain molar ratio, stirred uniformly, and then left to form the photosensitizer aggregate spontaneously. The preparation process of the photosensitizer aggregate constructed by the method is simple, convenient to operate, and has good repeatability. Experimental results prove that the constructed aggregate can induce bacterial aggregation, realize efficient killing of pathogenic bacteria, and further improve the sterilization efficiency when light is applied.

Description

[0001] The present application relates to the field of biological application materials, and particularly relates to a bacterial aggregation and killing bifunctional photosensitizer aggregate and a preparation method thereof. BACKGROUND

[0002] In recent years, with the large use of antibiotics, many pathogenic bacteria have developed serious drug resistance to existing antibiotics, and especially the problem of multiple drug-resistant gram-negative bacterial infection has become one of the main factors threatening human health, so it is urgent to develop new antibacterial methods which are efficient and not easy to develop drug resistance.

[0003] Photodynamic therapy (PDT) is one of the most promising therapies at present, which uses photosensitizer to produce reactive oxygen species (ROS) under light, oxidatively destroys surrounding biomolecules such as lipids, proteins and nucleic acids, and then kills pathogenic microorganisms. PDT has the advantages of non-invasiveness and broad-spectrum antibacterial property. It shows good efficacy for infections caused by bacteria, fungi and viruses, especially drug-resistant bacteria.

[0004] Gram-negative bacteria have a double-membrane structure compared with gram-positive bacteria. The outer membrane is composed of a layer of dense negatively charged lipopolysaccharide and lipoprotein, which forms a strong permeability barrier and prevents photosensitizer from penetrating the cell wall. Especially with the emergence of bacterial drug resistance, it further hinders the penetration of photosensitizer into bacteria and reduces the effect of photodynamic therapy. Therefore, how to develop new strategies to improve the effect of photodynamic therapy has attracted widespread attention. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application constructs an AIE photosensitizer aggregate rich in polysaccharide groups on the surface based on a simple co-assembly method, which is expected to achieve the dual functions of aggregating and killing bacteria and achieve high killing efficiency.

[0006] To achieve the above object, the present application provides the following technical solutions:

[0007] The co-assembly aggregate of the present application is an aggregate formed by the non-covalent forces π-π stacking, hydrophobic interaction, etc. of AIE photosensitizer and glycosyl amphiphilic molecules.

[0008] The AIE photosensitizer is a type II photosensitizer that can generate singlet oxygen (O2). 1

[0009] The glycosyl amphiphilic molecule is an alkyl glucosamine that can specifically recognize the bacterial surface agglutinin.

[0010] Further, the structural formula of the glycosyl amphiphilic molecule is​

[0011]

[0012] Further, the AIE photosensitizer has a structural formula

[0013]

[0014] The molar ratio of the AIE photosensitizer to the glycosyl amphiphilic molecule is 1:0-1:5.

[0015] The AIE photosensitizer aggregate of the present application is prepared by the following method:

[0016] The AIE photosensitizer and the glycosyl amphiphilic molecule are added to a phosphate buffer solution (PBS) in a certain ratio, stirred uniformly, and then left to form aggregates spontaneously.

[0017] The molar ratio of the IQ-Cm / DGal aggregate is 1:0-1:1, which is added to PBS in a certain ratio, stirred uniformly, and then left to form aggregates spontaneously.

[0018] The molar ratio of the TPyEt / DGal aggregate is 1:0-1:5, which is added to PBS in a certain ratio, stirred uniformly, and then left to form aggregates spontaneously.

[0019] The present application further provides a bacterial aggregation and killing bifunctional photosensitizer aggregate prepared by the preparation method.

[0020] The present application has the following beneficial effects: In the present application, AIE photosensitizer and glycosyl amphiphilic molecule are used as building blocks, and AIE photosensitizer aggregate with a surface rich in glycosyl groups is constructed by a simple co-assembly strategy. The preparation process is simple and easy to operate, and has good repeatability. The glycosyl groups on the surface of the aggregate can specifically recognize the bacterial surface lectin, and can synergistically induce bacterial aggregation with the electrostatic interaction provided by the cationic groups of the AIE photosensitizer, thereby shortening the interaction distance between the photosensitizer and the bacteria. The constructed aggregate has dark toxicity to bacteria, and can generate reactive oxygen species under light. Combined with the experimental results, it is proved that the aggregate constructed in the present application can induce bacterial aggregation and achieve efficient photodynamic killing of bacteria. The method provided in the present application can enable photosensitizers with low self-antibacterial activity to achieve efficient killing of bacteria by introducing glycosyl co-assembly into aggregates. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the specific embodiments or prior art of the present application, the drawings required to be used in the description of the specific embodiments or prior art will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0022] Figure 1 (a) UV-Vis absorption spectra of different ratio IQ-Cm / DGal aggregates in PBS solution; (b) fluorescence spectra of different ratio IQ-Cm / DGal aggregates in PBS solution.

[0023] Figure 2 (a) TEM images of different ratio IQ-Cm / DGal aggregates in PBS solution; (b) particle size distribution of different ratio IQ-Cm / DGal aggregates in PBS solution.

[0024] Figure 3 Fluorescence imaging and bright field images of P. aeruginosa after 30 min of interaction with different ratio IQ-Cm / DGal aggregates are shown.

[0025] Figure 4 Size distribution of different ratio IQ-Cm / DGal aggregates in PBS solution with P. aeruginosa for 0 min (a) and 30 min (b) are shown.

[0026] Figure 5 The active oxygen production performance of different ratio IQ-Cm / DGal aggregates under white light irradiation (20 mW / cm 2 ) is shown using ABDA as a probe.

[0027] Figure 6 (a) The antibacterial performance of different ratio IQ-Cm / DGal aggregates against P. aeruginosa in the dark or under light is shown; (b) photographs of colonies of P. aeruginosa grown on agar plates in the dark or under light with different ratio IQ-Cm / DGal aggregates.

[0028] Figure 7 (a) The antibacterial performance of different ratio TPyEt / DGal aggregates against P. aeruginosa in the dark or under light is shown; (b) photographs of colonies of P. aeruginosa grown on agar plates in the dark or under light with different ratio TPyEt / DGal aggregates. DETAILED DESCRIPTION

[0029] The technical solutions of the present application will be described clearly and completely below in conjunction with the drawings. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the protection scope of the present application.

[0030] In addition, the technical features involved in the different embodiments of the present application described below can be combined with each other as long as they do not conflict with each other.

[0031] Example 1: Preparation of IQ-Cm / DGal aggregates with different proportions

[0032] IQ-Cm and DGal were added into PBS respectively, and the final concentration of IQ-Cm was controlled to be 20 μM, and the molar ratio of IQ-Cm to DGal was controlled to be 1:0, 1:0.2, 1:0.5, 1:0.8 and 1:1. After mixing evenly, the aggregates were obtained by standing for half an hour.

[0033] <Photophysical property characterization of IQ-Cm / DGal aggregates with different proportions>

[0034] As shown in the UV-Vis absorption spectrum of a, the maximum absorption of IQ-Cm / DGal aggregates with different proportions was at 450 nm. Figure 1 As shown in the fluorescence spectrum of b, the maximum emission wavelength of IQ-Cm / DGal aggregates with different proportions was about 650 nm. Figure 1

[0035] <Morphology characterization of IQ-Cm / DGal aggregates with different proportions>

[0036] The particle size and potential of IQ-Cm / DGal aggregates with different proportions were measured by dynamic light scattering, and the morphology was observed by transmission electron microscopy. As shown in a, the particle size of the aggregates formed after the co-assembly of IQ-Cm / DGal with different proportions was about 600 nm. As shown in b, the morphology of the aggregates formed by the co-assembly of IQ-Cm / DGal with different proportions was spherical. Figure 2 Figure 2

[0037] <Interaction of IQ-Cm / DGal aggregates with different proportions with bacteria>

[0038] After the solution of IQ-Cm / DGal aggregates with different proportions was cultured with Pseudomonas aeruginosa with OD 600 =1.0 in the dark for 30 min, the supernatant was removed by centrifugation, 10 μL of PBS was added to homogenize the precipitate, 2 μL of the homogenate was taken onto a clean glass slide, and a cover glass was covered to observe.

[0039] ​​​The interaction of different proportions of IQ-Cm / DGal aggregates with P. aeruginosa was observed by fluorescence microscopy. As shown in Figure 3 , with the increase of DGal, the ability of aggregates to aggregate bacteria increased. The IQ-Cm / DGal aggregates emitted orange fluorescence, and it could be observed that the bacteria aggregated around the aggregates, and the colonies were evenly distributed and fewer in the absence of aggregates. The fluorescence imaging results showed that aggregates formed by different proportions of IQ-Cm / DGal could adhere to P. aeruginosa and cause aggregation.

[0040] <Assessment of the ability of aggregates of different proportions of IQ-Cm / DGal to aggregate bacteria>

[0041] Dynamic light scattering was used to measure the particle size of different proportions of IQ-Cm / DGal aggregates after 0 min and 30 min of interaction with bacteria, as shown in Figure 4 a, the particle size of P. aeruginosa was 1110 nm, and when different proportions of IQ-Cm / DGal aggregates interacted with bacteria for 0 min, the size did not change significantly; as shown in Figure 4 b, after different proportions of IQ-Cm / DGal aggregates interacted with bacteria for 30 min, the particle size increased significantly; the DLS results showed that different proportions of IQ-Cm / DGal aggregates could induce bacterial aggregation, and the ability to aggregate bacteria increased with the increase of the proportion of DGal added.

[0042] <Assessment of the singlet oxygen production performance of aggregates of different proportions of IQ-Cm / DGal>

[0043] The ability of different proportions of IQ-Cm / DGal aggregates to produce singlet oxygen was detected using the commercial probe 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA). ABDA can be oxidized and degraded to internal peroxide in the presence of ROS, and its ultraviolet absorbance at 400 nm will decrease, so the ability of singlet oxygen production can be evaluated according to the degree of decrease in the characteristic absorption peak of ABDA. Different proportions of IQ-Cm / DGal aggregate PBS solutions were taken, and ABDA probe was added, and irradiated with 20 mW / cm 2 white light, and the absorbance value was measured every 10 s to explore the change of absorbance at 379 nm with time. As shown in Figure 5 , the A / A0 value of ABDA and DGal remained basically unchanged, indicating that no reactive oxygen species were produced. However, after adding different proportions of IQ-Cm / DGal aggregates, the A / A0 value decreased rapidly, and at 60 s it was basically reduced to zero, indicating that the aggregates could efficiently produce singlet oxygen, and the singlet oxygen production efficiency was 1:0.2>1:0.8>1:0.5>1:1>1:0.

[0044] <Assessment of the antibacterial activity of different proportions of IQ-Cm / DGal aggregates>

[0045] The experiment takes Pseudomonas aeruginosa as an example, and uses traditional flat plate method to evaluate the antibacterial effect of different proportions of IQ-Cm / DGal aggregates in the dark and under light. The monoclonal colonies are placed in 10 mL of LB liquid medium, and cultured at 37°C, 180 rpm in a shaking incubator for 6-8 h. A certain volume of bacterial solution is taken, centrifuged to remove the culture medium (7100 rpm, 2 min), washed twice with PBS, finally suspended in PBS and adjusted to OD 600 = 1.0. The dark group is to make the bacterial solution (~ 10 8 CFU / mL) act with different concentrations of IQ-Cm / DGal aggregates at 37°C for 40 min; the light group is to make the bacterial solution (~ 10 8 CFU / mL) act with different concentrations of IQ-Cm / DGal aggregates at 37°C for 10 min, and then irradiated with 60 mW / cm 2 white light for 30 min. Then dilute 10 4 times with PBS, take 100 μL of diluted bacterial solution and evenly coat on LB solid medium (solid medium plate specification is 90 mm), each concentration is carried out in triplicate, and incubate at 37°C for 8-10 h. The number of bacterial colonies is counted. The antibacterial activity (IR) of different proportions of IQ-Cm / DGal aggregates is calculated according to the following formula: wherein A is the colony forming unit (cfu) of the control group, and B is the colony forming unit (cfu) of the experimental group.

[0046] As shown in Figure 6 , the dark toxicity of different proportions of IQ-Cm / DGal aggregates to Pseudomonas aeruginosa increases with the increase of the proportion of DGal, and can reach 50% of the bactericidal efficiency. When further irradiated with 60 mW / cm 2 white light for 30 min, the antibacterial activity of different proportions of aggregates is further improved compared with that of 40 min incubation in the dark, and the antibacterial effect of IQ-Cm / DGal = 1:0.8 aggregate reaches the optimum, and can reach nearly 100% of the killing activity. The experimental results show that under the dual action of aggregating bacteria and light generated singlet oxygen, AIE photosensitizer aggregates rich in sugar groups on the surface can achieve high-efficiency sterilization.

[0047] Example 2: Antibacterial activity evaluation of different proportions of TPyEt / DGal aggregates

[0048] The IQ-Cm photosensitizer was replaced by TPyEt, and the mixing molar ratio of TPyEt and DGal was changed to assemble TPyEt / DGal aggregates. The antibacterial activity of the aggregates was evaluated to verify the universality of the method. As shown in Figure 7 The dark toxicity of TPyEt / DGal aggregates with different ratios to P. aeruginosa increased with the increase of the ratio of DGal for 40 min of dark action. When the ratio reached 1:5, the antibacterial activity reached 90.28%, which was significantly higher than 16.38% of TPyEt itself. When further irradiated with 20 mW / cm 2 of white light for 30 min, the antibacterial activity of the aggregates with different ratios was higher than that of the dark action for 40 min. The killing efficiency of the aggregates with a ratio of 1:5 reached 97.35% under light irradiation.

[0049] Although the embodiments of the present application have been disclosed as above, they are not limited only to the application listed in the specification and embodiments, and can be fully applied to various fields suitable for the present application, and additional modifications can be easily made by those skilled in the art, and therefore the present application is not limited to specific details and the figures shown and described herein, without departing from the general concept defined by the claims and the equivalent scope.

Claims

1. A method for preparing photosensitizer aggregates with dual functions of bacterial aggregation and killing, characterized in that, The steps are as follows: AIE photosensitizer and amphiphilic glycosylated molecules are added to phosphate buffer solution in a certain molar ratio, and the two spontaneously form photosensitizer aggregates; Wherein, the AIE photosensitizer is a type II photosensitizer that can generate singlet oxygen, and the glycosyl amphiphilic molecule is dodecyl galactosamine; The molar ratio of the AIE photosensitizer to the glycosyl amphiphilic molecule is 1:0.8; The structural formula of the AIE photosensitizer is: 。 2. The photosensitizer aggregate with dual functions of bacterial aggregation and killing, prepared by the method described in claim 1.

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