Intelligent bacteriophage as well as preparation method and application thereof

Abstract

The invention discloses an intelligent bacteriophage as well as a preparation method and application thereof. The intelligent bacteriophage is a bacteriophage coupled with an I-type photosensitizer probe. The smart bacteriophage provided by the invention increases the binding of bacteria and the permeability of extracellular polymeric substances, thereby simplifying the removal of multi-drug-resistant bacteria and biological membranes thereof. Besides, the eye drops containing the intelligent bacteriophage formula show good non-invasive healing ability in a mouse bacterial keratitis model, promote cornea healing without scars, protect eye tissues, recover visual functions and prevent long-term discomfort and pain, and have potential application value in treatment of bacterial eye disease infection.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an intelligent bacteriophage and a preparation method and application thereof. Background Art

[0002] Phage therapy has many advantages, including: (1) good specificity. Phages have strong specificity and generally only infect pathogens of a specific species without destroying normal flora; (2) Phages have strong proliferation ability and can be used as therapeutic agents to continuously expand their efficacy; (3) No secondary contamination. Phages are not metabolized after entering the body and will not cause secondary contamination of the body. Phages only act at the site of bacterial infection and decrease with the death of pathogens until they disappear; (4) The phage preparation process is simple, the R&D cycle is short, and the cost is low.

[0003] Photodynamic therapy has many advantages in clinical practice, mainly including: (1) non-invasive treatment, no need for anesthesia, low toxicity and side effects, little impact on liver and kidney function, short treatment time, no need for hospitalization, can be completed in outpatient clinics, and high comfort; (2) no need for long-term medication, not affected by patient compliance, strong controllability, and can assist mechanical treatment; (3) good selectivity, low incidence of drug resistance, and effective killing of pathogenic microorganisms without damaging host tissues.

[0004] Bacterial keratitis is a common eye disease and the main cause of blindness caused by infectious corneal diseases, accounting for about 90% of infectious keratitis. It develops and progresses rapidly, and can lead to corneal suppuration, perforation, and even blindness. It is also accompanied by severe infection and inflammation. The severity of the infection is closely related to the type and virulence of the infecting strain and the host's immune response to the infecting bacteria. If bacterial keratitis is not diagnosed and treated in time, it is easy to develop into corneal perforation or even endophthalmitis, and severe patients require enucleation. The treatment of bacterial keratitis is mainly based on antibacterial therapy. When the vision of the treated patient is limited, appropriate anti-inflammatory treatment can be given, but most patients will still form corneal scars.

[0005] Therefore, it is very important to develop a drug that can specifically and efficiently kill multidrug-resistant Pseudomonas aeruginosa in vitro and fight bacterial keratitis. Summary of the invention

[0006] The purpose of the present invention is to provide a smart bacteriophage and its preparation method and application, by combining a molecule with alkyl iodide and a bacteriophage with a thiol functional group through a nucleophilic substitution reaction, to obtain a new type of smart bacteriophage probe with excellent photodynamic therapy combined with phage therapy. Smart bacteriophages can screen out corresponding phages according to different pathogens, thereby forming a diversified treatment plan to ensure higher efficacy and safety. This customized treatment method not only improves the efficacy, but also reduces the drug resistance problem caused by the abuse of antibiotics.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] In the first aspect of the present invention, a type I photosensitizer probe is provided, and the structural formula of the type I photosensitizer probe is as follows:

[0009]

[0010] In the second aspect of the present invention, a method for preparing the type I photosensitizer probe is provided, the method comprising:

[0011] N-bromosuccinimide was added to a DMF solution containing 2-thiopheneacetonitrile (compound 1) and stirred at room temperature. DMF was removed by vacuum distillation, and the crude product was purified to obtain compound 2;

[0012] Compound 2, 4-pyridineboronic acid, K2CO3 and Pd(PPh3)4 are added to tetrahydrofuran and water to obtain a mixture, the mixture is heated to reflux and stirred, cooled to room temperature and then vacuum distilled to remove most of THF, and then purified to obtain compound 3;

[0013] Compound 3, 4-dimethylaminobenzaldehyde (compound 4) and ethanol were respectively mixed, and then tert-butyl disodium was added and stirred at room temperature. After filtering, it was washed with cold ethanol and dried under reduced pressure to obtain ACR-DM.

[0014] ACR-DM was dissolved in acetonitrile, and then 1,3-diiodopropane was added. The reaction was heated under N2 protection, cooled to room temperature, and the solvent was removed by distillation under reduced pressure. The crude product was then purified to obtain a type I photosensitizer probe, referred to as ACR-DMT.

[0015] In the third aspect of the present invention, a smart bacteriophage is provided, wherein the smart bacteriophage is: a bacteriophage coupled with the type I photosensitizer probe;

[0016] The bacteriophage is a bacteriophage whose head coat protein contains a thiol functional group, and the alkyl iodide of the type I photosensitizer probe undergoes nucleophilic reaction with the thiol functional group on the bacteriophage coat protein for coupling.

[0017] In a fourth aspect of the present invention, a method for preparing a smart bacteriophage is provided, the method comprising:

[0018] The type I photosensitizer probe ACR-DMT is added to the phage solution and incubated in the dark to obtain a mixed solution, i.e., the smart phage;

[0019] Preferably, the bacteriophage is a bacteriophage corresponding to bacteria.

[0020] As a specific embodiment, the bacteriophage includes at least one of Pseudomonas aeruginosa phage, Escherichia coli phage, Salmonella typhimurium phage, Streptococcus pneumococcus phage, and Staphylococcus aureus phage.

[0021] Preferably, when the smart bacteriophage is used for antibacterial purposes, the concentration of the type I photosensitizer probe ACR-DMT is ≥2 μM; when the smart bacteriophage is used for imaging, the concentration of the type I photosensitizer probe ACR-DMT is ≥10 μM.

[0022] In the fifth aspect of the present invention, the use of the smart bacteriophage in the preparation of antibacterial drugs or imaging products is provided.

[0023] In the application, the smart bacteriophage is mixed with bacterial liquid.

[0024] When used for preparing antibacterial drugs, the final concentration of the type I photosensitizer probe ACR-DMT in the mixed smart phage is ≥1 μM.

[0025] When used for imaging, the final concentration of the type I photosensitizer probe ACR-DMT in the mixed smart phage is ≥5 μM.

[0026] In the sixth aspect of the present invention, there is provided the use of the intelligent bacteriophage in the preparation of a drug for treating bacterial keratitis.

[0027] In the seventh aspect of the present invention, there is provided the use of the smart bacteriophage in the preparation of a drug for preparing an anti-Pseudomonas aeruginosa drug.

[0028] The present invention has the following advantages and beneficial effects:

[0029] 1. The smart bacteriophage of the present invention is highly targeted and can combine molecules with alkyl iodide and bacteriophages with thiol functional groups through nucleophilic substitution reactions to obtain a new type of smart bacteriophage probe with excellent photodynamic therapy combined with phage therapy, thereby increasing bacterial binding and the permeability of extracellular polymers, thereby simplifying the removal of multi-drug resistant bacteria and their biofilms. Smart bacteriophages can screen out corresponding phages according to different pathogens, thereby forming a diversified treatment plan to ensure higher efficacy and safety. This customized treatment method not only improves the efficacy, but also reduces the drug resistance problem caused by the abuse of antibiotics.

[0030] 2. The smart bacteriophage of the present invention can be prepared in various dosage forms, such as eye drops, ointments, sprays, oral solutions, patches, microneedles, etc.

[0031] 3. The smart bacteriophage of the present invention is widely applicable to various bacterial infection diseases, such as blepharitis, meibomian gland inflammation, dacryocystitis, styes, conjunctivitis, keratitis (including corneal ulcers), and aseptic chemotherapy during ophthalmic surgery. This wide applicability makes the smart bacteriophage have good application prospects in a variety of clinical scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0033] Figure 1 For ACR-DMT 1 H NMR spectrum.

[0034] Figure 2 For ACR-DMT 13 C NMR spectrum.

[0035] Figure 3 The graph shows the measurement of ROS production by ACR-DMT at different time points after white light irradiation.

[0036] Figure 4 Figure 2 is a graph of the decomposition rate of ABDA with and without ACR-DMT or RB.

[0037] Figure 5 This is the OH production rate diagram of ACR-DMT at different time points.

[0038] Figure 6 Schematic diagram of smart phage eye drops targeting multidrug-resistant Pseudomonas aeruginosa.

[0039] Figure 7 This is a plate coating image showing the inactivation of multidrug-resistant Pseudomonas aeruginosa by smart phage eye drops using photodynamic combined phage therapy.

[0040] Figure 8 Crystal violet staining of smart phage eye drops inactivating multidrug-resistant Pseudomonas aeruginosa biofilm using photodynamic combined phage therapy.

[0041] Fig. 9 This is a slit lamp and sodium fluorescein staining image of the cornea during the treatment of bacterial keratitis with smart bacteriophage eye drops.

[0042] Fig.10 This is a preparation route map for the type I photosensitizer probe. DETAILED DESCRIPTION

[0043] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0044] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0045] The general idea of ​​the present invention is as follows:

[0046] According to a typical embodiment of the present invention, a type I photosensitizer probe is provided, and the structural formula of the type I photosensitizer probe is as follows:

[0047]

[0048] According to another typical embodiment of the present invention, the preparation route is as shown in the figure below, which provides a method for preparing the type I photosensitizer probe, and the preparation method comprises:

[0049] Step S1, adding 7.58 g of N-bromosuccinimide to a DMF solution containing 2-thiopheneacetonitrile (compound 1, 5 g, 40.0 mmol), stirring at room temperature, removing DMF by vacuum distillation, and then purifying the crude product to obtain compound 2;

[0050] Step S2, adding 202 mg of compound 2, 160 mg of 4-pyridineboronic acid, 1380 mg of K2CO3 (10 mmol) and 5.6 mg of Pd(PPh3)4 to tetrahydrofuran and water to obtain a mixture, heating the mixture to reflux with stirring, cooling to room temperature and vacuum distilling to remove most of THF, followed by purification to obtain compound 3;

[0051] Step S3, respectively take 600 mg of compound 3 (1.0 mmol), 447 mg of 4-dimethylaminobenzaldehyde (compound 4, 1.0 mmol) and ethanol, mix them evenly, then add disodium tert-butyl and stir at room temperature, filter, wash with cold ethanol, and dry under reduced pressure to obtain ACR-DM.

[0052] Step S4: 70 mg ACR-DM (0.211 mmol) was dissolved in 5 mL acetonitrile, and then 312.2 mg 1,3-diiodopropane (1.055 mmol) was added. The reaction was heated under N2 protection, cooled to room temperature, and the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column chromatography using DCM / MeOH as eluent to obtain a type I photosensitizer probe, referred to as ACR-DMT.

[0053] According to a typical embodiment of the present invention, a method for preparing a smart bacteriophage is provided, the method comprising:

[0054] Adding the type I photosensitizer probe ACR-DMT to the phage solution and incubating in the dark to obtain a mixed solution, thereby obtaining a smart phage;

[0055] The intelligent phage of the present invention can screen out corresponding phages (Escherichia coli phage, Salmonella typhimurium phage, Streptococcus pneumoniae phage, Staphylococcus aureus phage, etc.) according to different pathogens, thereby forming a diversified treatment plan to ensure higher efficacy and safety.

[0056] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0057] The present application will be described in detail below with reference to embodiments and experimental data.

[0058] Example 1. Type I photosensitizer probe and preparation method thereof

[0059] A type I photosensitizer probe that targets the thiol functional group on the phage capsid protein.

[0060]

[0061] The structural formula of the type I photosensitizer probe is as follows:

[0062] The preparation method of the type I photosensitizer probe with targeted phage is as follows:

[0063] (1) Compound 2: Add N-bromosuccinimide to a solution of 2-thiopheneacetonitrile (Compound 1) in N,N-dimethylformamide (DMF). Stir at room temperature, remove DMF by vacuum distillation, and purify the crude product by silica gel column chromatography using petroleum ether / ethyl acetate as eluent to obtain Compound 2.

[0064] (2) Compound 3: Compound 2, 4-pyridineboronic acid, K2CO3 (10 mmol) and Pd(PPh3)4 were added to a double-necked round-bottom flask filled with tetrahydrofuran (THF) and water, and equipped with a condenser under N2 protection. The mixture was heated to reflux and stirred overnight. The mixture was cooled to room temperature, vacuum distilled to remove most of the THF, and extracted three times with dichloromethane (DCM). The organic phase was collected, washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed in vacuo, and purified by silica gel column chromatography with DCM / ethyl acetate eluent to obtain compound 3.

[0065] (3) Compound 3 was characterized by NMR and mass spectrometry: 1 H NMR(400MHz, CDCl3)δ.8.60(d,J=4.0Hz,2H),7.41(d,J=4.0Hz,2H),7.37(d, J=3.2Hz,1H),7.086(d,J=2.8Hz,1H),3.95(s,2H),4.24(s,3H),4.18(s,3H). 13 C NMR (100MHz, CDCl3) δ.150.6,141.9,140.8,132.9,128.7,125.6,119.8,116.5,19.1.

[0066] (4) ACR-DM: Compound 3, 4-dimethylaminobenzaldehyde (compound 4) and ethanol were added to a round-bottom flask. Sodium tert-butylate was added to the above solution. Stir at room temperature, filter, wash with a small amount of cold ethanol, and dry under reduced pressure to obtain ACR-DM.

[0067] (5) Characterization of ACR-DM by NMR and mass spectrometry: 1 H NMR (400MHz, CDCl3) δ.8.58 (d, J = 4.0Hz, 2H), 7.82 (d, J = 6.8Hz, 2H), 7.44-7.43 (m, 3H), 7.27-7.25 (m, 2H), 6.70 (d, J = 6.8Hz, 2H), 3.07 (s, 6H). 13C NMR (100MHz, CDCl3) δ.151.8,150.4,142.4,141.2,140.7,139.4,131.4,126.2,126.0,120.8,119.4,117.9,111.7,98.5,40.0.

[0068] (6) ACR-DMT: ACR-DM was dissolved in acetonitrile, and then 1,3-diiodopropane was added. The reaction was heated under N2 protection, cooled to room temperature, and the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column chromatography using DCM / MeOH as eluent to obtain ACR-DMT.

[0069] (7) Characterization of ACR-DMT by NMR and mass spectrometry: 1 H NMR (600MHz, DMSO-d6) δ.8.92(d,J=7.2Hz,2H),8.32(d,J=7.2Hz,2H),8.29(d,J=4.0Hz,2H),8.29(d,J=4.0Hz,2H),7.90(d,J=9.2Hz,2H), 7.86 (s, 1H), 7.60 (d, J = 4.0Hz, 1H), 6.86 (d, J = 9.2Hz, 2H), 4.54 (t, J = 7.2Hz, 2H), 3.27 (t, J = 7.2Hz, 2H), 3.08 (s, 6H), 2.46 (t, J = 7.2Hz, 2H). 13 C NMR(600MHz,DMSO-d6)δ.152.6,148.0,147.5,144.8,143.9,134.9,133.7,132.0,1 26.2,121.9,119.9,118.1,111.9,95.2,60.3,40.1,39.7,33.9.HRMS(ESI):m / z[MI - ] + Calculate for C 23 H 23 IN3S:500.0652; found 500.0622.

[0070] Example 2: Intelligent bacteriophage and preparation method thereof

[0071] A method for preparing an intelligent phage formed by combining the type I photosensitizer probe obtained in Example 1 with a phage.

[0072] 1. When used for antibacterial purposes, the method comprises:

[0073] Preparation of antibacterial and anti-biofilm solution: 1 μL of ACR-DMT (2 mM) was added to 999 μL of phage solution and incubated at 37°C in the dark for 20 minutes. The final concentration of ACR-DMT was 2 μM. After the incubation, the mixed solution was mixed with an equal volume of bacterial solution. The final concentration of ACR-DMT was 1 μM.

[0074] 2. When used for imaging, the method comprises:

[0075] Preparation of staining solution: Add 1 μL of ACR-DMT (10 mM) to 999 μL of phage solution and incubate at 37°C in the dark for 20 minutes. The final concentration of ACR-DMT is 10 μM. After incubation, mix the mixed solution with an equal volume of bacterial culture solution. The final concentration of ACR-DMT is 5 μM.

[0076] Example 3: Performance measurement of smart bacteriophage

[0077] 1. Determination of target molecule reactive oxygen species (indicator H2DCF-DA)

[0078] The ROS yield of the compound ACR-DMT in the solution was tested using 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA) as the detection probe. In order to convert H2DCF-DA into 2,7-dichlorodihydrofluorescein (H2DCF), 0.25 mL of H2DCF-DA ethanol solution (1 mM) was added to 1 mL of NaOH (10 mM) aqueous solution, and then stirred at room temperature for 30 min. The solution was then adjusted with 5 mL of PBS solution (pH 7.4) to obtain a frozen solution for later use. The DMSO solution of the compound was added to the above solution to obtain a final concentration of 2 μM. The sample was placed in a fluorescence spectrometer and the fluorescence intensity (λ ex :488nm).

[0079] The results are as follows Figure 3 As shown in the figure, after continuous irradiation for 100s, the fluorescence intensity of H2DCF remained unchanged. 10μMACR-DMT can enhance the fluorescence of H2DCF by 48.4 times. In addition, under hypoxic conditions (0.1% O2), the ROS generation efficiency was almost unchanged compared with that under normoxic conditions (21% O2). This experiment confirms that this type of compound can effectively and quickly generate ROS under light irradiation and is not limited by oxygen content.

[0080] 2. Singlet oxygen yield determination (indicator ABDA)

[0081] The singlet oxygen generation performance of the photosensitizer ACR-DMT and the commercially available photosensitizer Bengal Rose Red (RB) under light irradiation was investigated using ABDA (9,10-anthryl-bis(methylene)dimalonic acid) as an indicator. 1 During the O2 reaction, ABDA is oxidized to a peroxy bridge structure, resulting in a decrease in the absorbance of ABDA at 378 nm. The rate of decrease can indirectly reflect the photosensitizer under light. 1 O2 production rate.

[0082] The results are as follows Figure 4 As shown in Figure 2, the absorbance of ABDA at 378 nm remained essentially unchanged from 0 to 540 s. However, after the addition of RB, the absorbance steadily decreased to 24.3% of the original value. In the presence of ACR-DMT, only 29.2% of ABDA was degraded, indicating that ACR-DMT can generate a small amount of 1 O2.

[0083] 3. Hydroxyl free radical determination (indicator HPF)

[0084] Hydroxyphenylfluorescein was used as an indicator to investigate the generation of ·OH. HPF and PS (photosensitizer) were mixed in PBS and irradiated with white light. The fluorescence intensity of HPF at 515 nm was recorded.

[0085] The results are as follows Figure 5 As shown in Figure 2, after 100 s of illumination, ACR-DMT significantly increased the fluorescence intensity of HPF under both normoxic conditions (21% O2) and hypoxic conditions (0.1% O2), with negligible differences. In contrast, in the absence of PS, little change was observed, suggesting that PS can effectively generate ·OH through a type I mechanism.

[0086] 4. Bacterial fluorescence imaging to explore the specific recognition of host bacteria by intelligent phage eye drops

[0087] Multidrug-resistant Pseudomonas aeruginosa was cultured in LB medium at 37°C and 180 rpm overnight, centrifuged at 10,000 × g for 1 min, and washed twice with PBS. PBS was diluted to OD 600 =0.6, then the selected concentration of intelligent phage eye drops and multidrug-resistant Pseudomonas aeruginosa bacterial solution were mixed in equal volumes, and the ability of intelligent phage eye drops to specifically identify host bacteria was determined using a laser confocal microscope (CLSM). First, multidrug-resistant Pseudomonas aeruginosa was stained green with a green nucleic acid dye NucGreen, and then multidrug-resistant Pseudomonas aeruginosa was co-cultured with intelligent phage eye drops and incubated in a 37°C water bath in the dark for 20 minutes. For fluorescent tracing of bacteria, the samples were kept in the dark. Fluorescent images were recorded using a CLSM equipped with a 100x oil-immersion objective.

[0088] The results are as follows Figure 6 As shown, the intelligent phage eye drops can clearly target multidrug-resistant Pseudomonas aeruginosa, indicating that the intelligent phage eye drops have a highly efficient and specific targeting effect.

[0089] 5. Plate count method to investigate the in vitro antibacterial activity of intelligent phage eye drops

[0090] Multidrug-resistant Pseudomonas aeruginosa was cultured overnight in LB medium at 37°C and 180 rpm, and the cultured bacteria were harvested and the medium was removed. The bacteria were washed twice with PBS. PBS was diluted to OD 600 =0.6, the intelligent phage eye drops were mixed with an equal volume of multidrug-resistant Pseudomonas aeruginosa and incubated in a 37°C water bath in the dark for 30 min, followed by exposure to / without white light (60 mW cm -2 ) for 20 minutes. Then for fluorescent staining, the bacteria Live&Dead TM The culture was stained with the assay kit. Dead bacteria were stained red, while whole bacteria were stained green. Fluorescence images were recorded with a CLSM equipped with a 100x oil immersion objective.

[0091] The results are as follows Figure 7 As shown, in the intelligent phage eye drops illumination group, almost all multidrug-resistant Pseudomonas aeruginosa fluorescence appeared red, proving that the intelligent phage eye drops have a good ability to kill multidrug-resistant Pseudomonas aeruginosa.

[0092] 6. Crystal violet staining method to investigate the in vitro inhibition of biofilm activity of intelligent phage eye drops

[0093] Multidrug-resistant Pseudomonas aeruginosa was cultured in LB medium at 37°C and 180 rpm overnight, and the OD 600 = 0.1 multidrug-resistant Pseudomonas aeruginosa was pre-suspended in TSB liquid medium. The bacterial suspension and intelligent phage eye drops were added to a 96-well microtiter plate and a 40 mm culture dish, incubated at 37°C for 30 minutes, and white light (60 mW cm -2 ) for 30 minutes. After incubation for 16 hours, the medium was gently replaced with PBS three times. The biofilm in the culture dish was then photographed and stained with crystal violet to determine the ability of the intelligent phage eye drops to inhibit biofilm growth.

[0094] The results are as follows Figure 8 As shown, in the intelligent phage eye drops light-irradiated group, the biofilm of multidrug-resistant Pseudomonas aeruginosa could hardly form, and there was no residual crystal violet, proving that the intelligent phage eye drops have excellent ability to inhibit the biofilm of multidrug-resistant Pseudomonas aeruginosa.

[0095] Example 4: Establishment of a bacterial keratitis disease model and evaluation of the therapeutic effect of intelligent phage eye drops

[0096] The animal experiment of the intelligent phage eye drops obtained in Example 2 on the bacterial keratitis mouse model is as follows:

[0097] Eight-week-old male C57BL / 6 mice were selected. Multidrug-resistant Pseudomonas aeruginosa was inoculated on both corneas of the mice. The mice were intraperitoneally injected with 5% chloral hydrate at a dose of 0.01 mL g -1 The cornea was then inoculated with 2 μL of a multidrug-resistant Pseudomonas aeruginosa suspension (approximately 1.0 × 10 8 CFU mL -1 )Inoculation of mechanically injured mouse cornea.

[0098] 24 hours after inoculation, mice with eye infection were randomly divided into 6 groups (n=5):

[0099] (1) Uninfected or untreated (control group);

[0100] (2) No treatment group after successful modeling (no treatment group);

[0101] (3) 2 μL of Pseudomonas aeruginosa phage (2.7×10 4 PFU mL -1 ), giving 60mWcm -2 White light exposure, 20 minutes each time, twice a day, for 6 consecutive days (phage+L);

[0102] (4) 2 μL of ACR-DMT (type I photosensitizer probe) (1 μM) of Example 1 was perfused into the cornea of ​​the model mice, and 60 mW cm -2 White light exposure for 20 minutes (ACR-DMT+L), twice a day for 6 days;

[0103] (5) 2 μL of the intelligent phage eye drops (1 μM) of Example 2 were perfused into the cornea of ​​the model mice twice a day and left in the dark for 20 minutes for 6 consecutive days;

[0104] (6) 2 μL of the intelligent phage eye drops (1 μM) of Example 2 were perfused into the cornea of ​​the model mice, and 60 mW cm -2 White light irradiation, 6 consecutive days, twice a day (treatment group).

[0105] The results are as follows Fig. 9As shown, the corneal epithelium in the treatment group recovered significantly, and the corneal clarity was very close to that of the control group. Turbidity and edema gradually disappeared, no new blood vessels were generated, corneal transparency increased, and the intelligent phage eye drops showed better healing ability for bacterial keratitis.

[0106] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0107] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0108] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A type I photosensitizer probe, characterized in that: The structural formula of the type I photosensitizer probe is as follows:

2. A method for preparing the type I photosensitizer probe according to claim 1, characterized in that: The method comprises: N-bromosuccinimide was added to a solution of 2-thiopheneacetonitrile in N,N-dimethylformamide and stirred at room temperature, and DMF was removed by vacuum distillation, and the crude product was purified to obtain compound 2; Compound 2, 4-pyridineboronic acid, K2CO3 and Pd(PPh3)4 are added to tetrahydrofuran and water to obtain a mixture, the mixture is heated to reflux and stirred, cooled to room temperature and then vacuum distilled to remove most of THF, and then purified to obtain compound 3; Compound 3, 4-dimethylaminobenzaldehyde and ethanol were mixed respectively, and then tert-butyl disodium was added and stirred at room temperature, filtered, washed with cold ethanol, and dried under reduced pressure to obtain ACR-DM; ACR-DM was dissolved in acetonitrile, and then 1,3-diiodopropane was added. The reaction was heated under N2 protection, cooled to room temperature, and the solvent was removed by distillation under reduced pressure. The crude product was then purified to obtain a type I photosensitizer probe, referred to as ACR-DMT.

3. A smart bacteriophage, characterized in that: The smart bacteriophage is: a bacteriophage coupled with the type I photosensitizer probe according to claim 1; The bacteriophage is a bacteriophage whose head coat protein contains a thiol functional group, and the alkyl iodide of the type I photosensitizer probe undergoes nucleophilic reaction with the thiol functional group on the bacteriophage coat protein for coupling.

4. A method for preparing the smart bacteriophage according to any one of claim 3, characterized in that: The preparation method comprises: The type I photosensitizer probe ACR-DMT described in claim 1 is added to the phage solution and incubated in the dark to obtain a mixed solution, namely, the smart phage.

5. The preparation method according to claim 4, characterized in that: The bacteriophage is the bacteriophage corresponding to bacteria.

6. The preparation method according to claim 4, characterized in that: When the smart bacteriophage is used for antibacterial purposes, the concentration of the type I photosensitizer probe ACR-DMT is ≥2 μM; when the smart bacteriophage is used for imaging, the concentration of the type I photosensitizer probe ACR-DMT is ≥10 μM.

7. Use of the smart bacteriophage described in claim 3 in the preparation of antibacterial drugs or imaging products.

8. The use according to claim 3, characterized in that: In the application, the smart bacteriophage described in any one of claims 1 to 3 is mixed with a bacterial culture liquid for the preparation of an antibacterial drug, and the final concentration of the type I photosensitizer probe ACR-DMT in the mixed smart bacteriophage is ≥1 μM, and when used for imaging, the final concentration of the type I photosensitizer probe ACR-DMT in the mixed smart bacteriophage is ≥5 μM.

9. Use of the smart bacteriophage according to claim 3 in the preparation of a drug for treating bacterial keratitis.

10. Use of the smart bacteriophage according to claim 3 in the preparation of a drug for use in preparing an anti-Pseudomonas aeruginosa drug.

Images