CN110151994B - Bacteriophage and application thereof in preparation of photodynamic preparation for inactivating bacteria - Google Patents

Bacteriophage and application thereof in preparation of photodynamic preparation for inactivating bacteria Download PDF

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CN110151994B
CN110151994B CN201910480938.0A CN201910480938A CN110151994B CN 110151994 B CN110151994 B CN 110151994B CN 201910480938 A CN201910480938 A CN 201910480938A CN 110151994 B CN110151994 B CN 110151994B
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photosensitizer
bacteriophage
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CN110151994A (en
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牛忠伟
田野
高偲嘉
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Technical Institute of Physics and Chemistry of CAS
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    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
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    • C12N2795/14011—Details ssDNA Bacteriophages
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Abstract

The invention discloses a bacteriophage and application thereof in preparing a photodynamic preparation for inactivating bacteria, and relates to the technical field of new nano materials. The surface of the phage is connected with a photosensitizer through weak interaction, the weak interaction can be electrostatic interaction or hydrophilic-hydrophobic interaction, and the photosensitizer can specifically target a certain bacterium through specific interaction between the phage and the bacterium.

Description

Bacteriophage and application thereof in preparation of photodynamic preparation for inactivating bacteria
Technical Field
The invention relates to the technical field of new nano materials. More particularly, it relates to a bacteriophage and its use in the preparation of a photodynamic preparation for inactivating bacteria.
Background
Bacterial infections pose a serious threat to human health, and the problem is exacerbated by the development of bacterial resistance caused by the abuse of antibiotics. Due to the characteristics of non-invasiveness, high space-time selectivity and the like, photodynamic sterilization attracts wide attention. The photodynamic sterilization consists of three components, namely a photosensitizer, a light source and oxygen, wherein the three components are nontoxic in the presence of the photosensitizer and the light source. The photosensitizer can transfer energy or electrons to oxygen under the irradiation of light source, thereby generating active oxygen species such as singlet oxygen (C)1O2) Superoxide anion (O)2 ·-) And hydroxyl radical (. OH), etc. The active oxygen species are highly oxidative and can cause irreversible damage to many important biomolecules, such as phospholipid bilayers, proteins, DNA, and the like. Photodynamic therapy can therefore be effective in killing bacteria, including drug resistant bacteria. Meanwhile, the photodynamic sterilization is not easy to cause drug resistance of bacteria. However, photosensitizers often lack targeting to bacteria, which can cause serious side effects on normal mammalian cells. The existence of the outer membrane of gram-negative bacteria greatly weakens the photodynamic bactericidal effect compared with gram-positive bacteria.
Specifically targeting photosensitizers to bacteria is a key problem in photodynamic disinfection. The general method comprises: photosensitizers are encapsulated with nanocarriers such as polymers or MOFs, etc., and released at specific sites by stimulus-responsiveness (Mao D, et al, metal-Organic-Framework-associated in vivo bacterial labeling and preparation antibiotic therapy [ J ] Adv Mater 2018,2018,1706831.). The carrier used in the method may have reduced biological safety due to non-degradability and the like. Another approach is to covalently cross-link segments of targeted bacteria, such as sugars, polypeptides, etc., with photosensitizers to target them to bacteria (Xiao F, et al. Pathologen-specific polymeric antibodies with a symmetrical membrane displacement and enhanced photodynamic large to inhibition high molecular optropic bacterial strain [ J ] ACS Nano 2019,13, 1511.). The preparation steps of the method are relatively complicated, and the introduction of organic solvents in chemical synthesis can increase the biological toxicity of the method.
Phage (phase) is a virus that attacks bacteria, contains proteins and nucleic acids as main components, is biologically safe, and is non-toxic to humans and animals. The phage exists widely in nature and is easy to obtain. Because the adsorption organ of the phage and the receptor molecule on the surface of the receptor bacterium have complementarity, the phage has strict host specificity. In this patent, we adsorbed photosensitizers to phage surfaces targeting gram-negative bacteria, such as M13, by weak interactions. Through specific interactions between the phage and the bacteria, the photosensitizer can be specifically targeted to a certain bacteria. Compared with the previously reported methods, the method has the advantages of strong targeting property, simplicity, feasibility and high biological safety.
Disclosure of Invention
The present invention aims to provide a bacteriophage that kills bacteria by photodynamic therapy.
In order to achieve the purpose, the invention adopts the following technical scheme:
a photosensitizer-attached bacteriophage having a photosensitizer attached to the surface of the bacteriophage through a weak interaction.
Further, it is preferable that the weak interaction is an electrostatic interaction or a hydrophobic-hydrophilic interaction.
Furthermore, it is preferred that the bacteriophage is a bacteriophage targeting a gram-negative bacterium;
in addition, preferably, the phage is any one of M13 phage and T1-T7 phage.
Furthermore, it is preferred that the bacteriophage is a bacteriophage targeting e.
In addition, the photosensitizer is preferably any one of a porphyrin photosensitizer, a phthalocyanine photosensitizer, a porphin photosensitizer and a photosensitizer with aggregation-induced emission properties;
in addition, preferably, the photosensitizer with aggregation-induced emission property is a TPE-based photosensitizer;
further, it is preferable that the porphyrin-based photosensitizer is hematoporphyrin, Photofrin or photogomem;
in addition, the phthalocyanine photosensitizer is ZnPc, Photosens, Pc4 or CGP 55847;
in addition, the porphin photosensitizer is Ce6, Foscan, Aptocine or Laserphyrin.
Further, it is preferable that, when the photosensitizer is a positively charged photosensitizer, the method for preparing the photosensitizer-attached phage includes the steps of:
and mixing the phage with the photosensitizer with positive electricity, and obtaining the phage with the photosensitizer adsorbed on the outer surface through electrostatic interaction.
In addition, it is preferable that, when the photosensitizer is a hydrophobic photosensitizer, the method for preparing the photosensitizer-attached phage includes the steps of:
modifying temperature sensitive hydrophobic molecules on the surface of the phage;
mixing the phage modified with the temperature-sensitive hydrophobic molecule with a hydrophobic photosensitizer, and adjusting the temperature to the phase transition temperature of the temperature-sensitive hydrophobic molecule to change the temperature-sensitive hydrophobic molecule from hydrophilic to hydrophobic so as to wrap the photosensitizer.
In addition, the preferable scheme is that the temperature-sensitive hydrophobic molecule is elastin-like protein ELP; the temperature was adjusted to 37 ℃ while the phage was mixed with the photosensitizer.
Further, it is preferable that the bacteriophage and the photosensitizer are mixed in a ratio of the amount of the substance of 1:0.5 to 1: 50.
The invention also provides the use of a bacteriophage as described above for the preparation of a photodynamic preparation for inactivating a bacterium.
The invention has the following beneficial effects:
this patent adsorbs the photosensitizer on the phage surface of targetable bacterium through weak interact, through the specificity interact between phage and the bacterium, makes the photosensitizer can certain bacterium of specificity targeting, and then carries out high efficiency sterilization through photodynamic therapy. Compared with the prior art, the method has the advantages of strong targeting property, simplicity, feasibility, high biological safety and good sterilization effect.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows a TEM micrograph of bacteriophage M13 in example 1 of the present invention.
FIG. 2 shows SEM images of E.coli treated with TPE-Pt-MC @ M13 in test example 1 of the present invention.
Detailed Description
In the prior art, because the photosensitizer lacks targeting, serious side effects can be caused to normal mammalian cells; and compared with gram-positive bacteria, the existence of the gram-negative bacteria outer membrane greatly reduces the photodynamic sterilization effect. If the photosensitizer is coated in a carrier, such as a polymer or MOF, the photosensitizer is released by stimulating a specific site with responsiveness, and the carrier used in the method has low biological safety and is not easy to degrade; if the surface of the photosensitizer is grafted with a segment targeting bacteria, such as sugar or polypeptide, and the like, the targeting property of the photosensitizer to the bacteria is enhanced, the method has complicated steps, and the biosafety is reduced by introducing an organic solvent in the chemical synthesis process.
Based on the problems in the prior art, the invention provides a photosensitizer-attached phage, wherein the surface of the phage is connected with a photosensitizer through weak interaction, and the photosensitizer obtains the capability of targeting bacteria by utilizing strong specific interaction between the phage and the bacteria.
Preferably, in the present invention, the weak interaction is an electrostatic interaction or a hydrophobic-hydrophilic interaction. The two weak interaction forming processes are simple and easy to implement, and the photosensitizer can be effectively attached to the surface of the bacteriophage, so that the bacteriophage can effectively kill bacteria through photodynamic therapy, and in the self-assembly process, no additional chemical synthesis organic solvent is introduced, and no biotoxicity is generated. In the practical operation process, those skilled in the art can also use the design idea provided by the present invention to adopt other types of weak interactions such as metal coordination, host-guest interaction, receptor-ligand interaction, etc. according to the specific types of photosensitizers and phages.
The presence of the outer membrane of gram-negative bacteria greatly reduces the effectiveness of photodynamic disinfection compared to gram-positive bacteria. Preferably, the bacteriophage is a bacteriophage targeting gram-negative bacteria, and further, the bacteriophage is a bacteriophage targeting escherichia coli e.coli; the phage is any one of M13 phage and T1-T7 phage.
In a preferred embodiment of the present invention, the photosensitizer is any one of a porphyrin photosensitizer, a phthalocyanine photosensitizer, a porphin photosensitizer, and a photosensitizer with aggregation-induced emission properties. The TPE-Pt-MC in the photosensitizer with aggregation-induced emission properties has positive charges and can be attached to the surface of the phage through electrostatic interaction.
Preferably, the photosensitizer with aggregation-induced emission property is a TPE-based photosensitizer, such as TPE-Pt-MC; the porphyrin photosensitizer is hematoporphyrin, Photofrin or photom; the phthalocyanine photosensitizer is ZnPc, Photosens, Pc4 or CGP 55847; the porphin photosensitizer is Ce6, Foscan, Aptocine or Laserphyrin.
In a preferred embodiment of the present invention, when the photosensitizer is a photosensitizer which is itself positively charged (e.g., TPE-Pt-MC), the weak interaction between the photosensitizer and the bacteriophage may be an electrostatic interaction, and in this case, the method for preparing the photosensitizer-attached bacteriophage includes the following steps: and mixing the phage with the photosensitizer with aggregation-induced emission property, and obtaining the phage with the photosensitizer adsorbed on the outer surface through electrostatic interaction. Further preferably, the bacteriophage and the photosensitizer are mixed in a ratio of the amount of the substances of 1:0.5 to 1: 50.
When the weak interaction between the photosensitizer and the phage is hydrophilic-hydrophobic interaction and the photosensitizer is a hydrophobic photosensitizer (such as ZnPc, Ce6, hematoporphyrin and the like), the preparation method of the phage attached with the photosensitizer comprises the following steps:
modifying temperature sensitive hydrophobic molecules on the surface of the phage; mixing the phage modified with the temperature-sensitive hydrophobic molecule with a hydrophobic photosensitizer, and adjusting the temperature to the phase transition temperature of the temperature-sensitive hydrophobic molecule to change the temperature-sensitive hydrophobic molecule from hydrophilic to hydrophobic so as to wrap the photosensitizer. Further preferably, the bacteriophage is mixed with the photosensitizer according to the ratio of the amount of the substances of 1:0.5-1: 50; the specific temperature to be adjusted is determined according to the type of the thermo-sensitive hydrophobic molecule, for example, when the surface of M13 phage is modified with elastin-like protein (ELP), the temperature can be adjusted to 37 ℃.
The invention also provides the application of the phage in preparing a photodynamic preparation for inactivating bacteria, and different kinds of bacteria can be killed by photodynamic therapy through selecting different kinds of phage.
In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Example 1
The M13 phage was obtained by a prior art method and its TEM photograph is shown in FIG. 1. Mixing M13 bacteriophage with the mass ratio of 1:2 and TPE-based photosensitizer (TPE-Pt-MC) with aggregation-induced emission property, and obtaining M13 bacteriophage with the photosensitizer TPE-Pt-MC adsorbed on the outer surface through electrostatic interaction, namely TPE-Pt-MC @ M13. The TPE-Pt-MC @ M13 was used for M13-specific targeting of bacteria for antibacterial experiments.
Example 2
The T2 bacteriophage is obtained by the method in the prior art, the T2 bacteriophage with the mass ratio of 1:1 and a TPE-based photosensitizer (TPE-Pt-MC) with aggregation-induced emission properties are mixed, and the T2 bacteriophage with the photosensitizer TPE-Pt-MC adsorbed on the outer surface, namely TPE-Pt-MC @ T2, is obtained through electrostatic interaction. The TPE-Pt-MC @ T2 was used for T2-specific targeting of bacteria for antibacterial experiments.
Example 3
M13 phage was obtained by a method of the prior art, and elastin-like protein (ELP) was modified on the surface of M13 phage to obtain ELP-M13. The polypeptide is temperature sensitive, i.e., a hydrophilic to hydrophobic transition can occur with increasing temperature. ELP-M13 phage with the mass ratio of 1:50 is mixed with a photosensitizer ZnPc at 37 ℃, and the M13 phage with the photosensitizer ZnPc adsorbed on the outer surface is obtained through hydrophilic-hydrophobic interaction, namely ZnPc @ M13. The ZnPc @ M13 was used for M13-specific targeted bacteria for antibacterial experiments.
Test example 1
The TPE-Pt-MC @ M13 prepared in example 1 was used in E.coli for antibacterial experiments.
The antibacterial experiment steps are as follows:
(1) individual e.coli colonies were picked into 50mL of trypticase medium and incubated overnight at 37 ℃ on a shaker at 200 rpm.
(2) The overnight-cultured broth was treated with 10mM pH 7.4K2HPO4-KH2PO4Diluting the buffer solution by a certain multiple to obtain 105CFU/mL of bacterial liquid.
(3) Taking phage (TPE-Pt-MC @ M13) (concentration is 20 mu M TPE-Pt-MC +10 mu M M13) adsorbing photosensitizer and 10 mu M M135CFU/mL of the bacterial solution were mixed and allowed to interact in the dark for 15 min.
(4)20mW/cm2Irradiating the bacteria liquid interacted with the TPE-Pt-MC @ M13 by a 420nm light source (such as an LED lamp, a laser and the like) for 10-20 min. The light wavelength is selected according to the ultraviolet absorption of the photosensitizer, for example, the light wavelength of TPE-Pt-MC @ M13 is 420 nm.
(5) Sucking 100. mu.L of the irradiated bacterial liquid, placing the bacterial liquid on an MH solid culture medium, evenly coating, reversely placing the bacterial liquid in an incubator at 37 ℃, and counting after overnight.
Coli treated with TPE-Pt-MC @ M13 SEM pictures as shown in FIG. 2, and bacterial film rupture was observed.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.

Claims (7)

1. Use of a photosensitizer-attached bacteriophage for the preparation of a photodynamic preparation for inactivating bacteria, wherein the surface of said bacteriophage is linked to a photosensitizer by weak interaction, said photosensitizer being TPE-Pt-MC.
2. Use according to claim 1, wherein said weak interaction is an electrostatic interaction.
3. Use according to claim 1, wherein the bacteriophage is a bacteriophage targeting a gram negative bacterium.
4. The use according to claim 1, wherein the bacteriophage is any one of M13 bacteriophage and T1-T7 bacteriophage.
5. Use according to claim 1, wherein the bacteriophage is a bacteriophage targeting e.
6. Use according to claim 1, wherein the photosensitizer-attached phage is prepared by a method comprising the steps of:
and mixing the phage with the photosensitizer with positive electricity, and obtaining the phage with the photosensitizer adsorbed on the outer surface through electrostatic interaction.
7. The use according to claim 6, wherein the bacteriophage and the photosensitizer are mixed in a ratio of the amount of the substance of 1:0.5 to 1: 50.
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US11944682B2 (en) * 2020-01-24 2024-04-02 The Hong Kong University of Science and Technologyy Aggregation induced emission-bacteriophage bioconjugates
CN111956817A (en) * 2020-08-10 2020-11-20 武娟 Indoor environment photodynamic broad spectrum sterilization and disinfection method

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