CN114949206A

CN114949206A - Novel antibacterial therapy for photodynamic treatment of bacterial endophthalmitis

Info

Publication number: CN114949206A
Application number: CN202210454134.5A
Authority: CN
Inventors: 彭琛; 于靖; 李亭亭; 吴岩; 蔡雯婷
Original assignee: Shanghai Tenth Peoples Hospital
Current assignee: Shanghai Tenth Peoples Hospital
Priority date: 2022-04-27
Filing date: 2022-04-27
Publication date: 2022-08-30

Abstract

The invention discloses a novel antibacterial therapy for photodynamic treatment of bacterial endophthalmitis, which comprises the following steps: selecting a TTPy molecule; dissolving TTPy in DMSO to obtain 5mM mother liquor; diluting the mother solution by adopting a PBS solution; injecting the diluted mother solution into rat eyes; the injection part is illuminated under certain conditions to realize effective in vivo antibiosis. The invention relates to the technical field of photodynamic therapy exploration applied to bacterial endophthalmitis, and particularly provides an application which can avoid potential bacterial drug resistance, can solve the defect of fluorescence aggregation quenching of a traditional photosensitizer, can achieve a good sterilization effect through a low administration concentration, can realize the protection of a retina and effective in vivo antibiosis, and particularly successfully utilizes AIEgens to carry out photodynamic antibacterial research, verifies the safety and effectiveness of TTPy at a cell and animal level, and can lay a foundation for similar antibacterial application.

Description

Novel antibacterial therapy for photodynamic treatment of bacterial endophthalmitis

Technical Field

The invention relates to the technical field of photodynamic therapy exploration applied to bacterial endophthalmitis, in particular to a novel antibacterial therapy for photodynamic therapy of bacterial endophthalmitis.

Background

Bacterial Endophthalmitis (BE) is a serious ocular infection, and the disease progresses rapidly, belonging to clinical and ophthalmological emergencies. Although the incidence of endophthalmitis after cataract surgery is about 0.1%, irreversible vision loss, even eyeball loss, can result if effective treatment is not available in a timely manner. At present, BE is mainly treated by empirical intravitreal injection of vancomycin in combination with other antibiotics, and severe cases require vitrectomy. However, in recent years, abuse of antibiotics has led to the frequent development of resistance by pathogens, even the appearance of superbacteria. Moreover, antibiotics have a certain toxic effect on the retinal tissue in the eye, which may lead to hemorrhagic obliterative retinal vasculitis, once it occurs, with the patient's vision being extremely poor. In addition, BE treatment is long in hospital stay, the treatment effect is difficult to guarantee, and patients need to bear high time and money cost. Therefore, it is urgent and necessary to research alternative antibacterial therapies of BE to solve the above problems.

Photodynamic therapy (PDT) has shown great potential in the treatment of drug-resistant bacterial infections. Compared with the traditional therapy, PDT generates Reactive Oxygen Species (ROS) to kill pathogens through Photosensitizers (PSs) after illumination, and has the advantages of low cytotoxicity, low drug resistance, space-time accuracy and the like. More importantly, PDT is well suited for the treatment of ocular infections due to the good light transmission of the eyeball. However, most conventional PSs have fluorescence quenching problems in the aggregate state, which greatly limits their clinical applications.

Disclosure of Invention

Aiming at the situation and overcoming the defects of the prior art, the invention provides a novel antibacterial therapy for photodynamic therapy of bacterial endophthalmitis, which can avoid the potential problem of bacterial drug resistance, can also solve the defect of aggregation fluorescence quenching of the traditional photosensitizer, can achieve good sterilization effect through lower administration concentration, can protect retina and effectively resist bacteria in vivo, and has better effect.

The technical scheme adopted by the invention is as follows: the invention relates to a novel antibacterial therapy for photodynamic treatment of bacterial endophthalmitis, which comprises the following steps:

the method comprises the following steps: selecting solid TTPy molecular powder;

step two: dissolving TTPy molecular powder by DMSO to obtain mother liquor with the concentration of 5 mM;

step three: diluting the mother solution by adopting a PBS solution;

step four: injecting the diluted mother solution into rat eyes;

step five: the injection part is irradiated under certain conditions, ROS is generated quickly and efficiently, and effective in-vivo antibiosis is realized on the premise of ensuring no cytotoxicity.

Further, in the first step, the TTPy molecule is synthesized and purified by using a TTPy molecule with positive charge and medium hydrophobicity, and rapid identification of bacteria and normal cells can be realized under the driving of electrostatic and hydrophobic interaction.

Preferably, the illumination of the certain condition in the step five is that the illumination power is 20mW/cm ² For 15min under white light.

Preferably, the novel antimicrobial therapy does not affect the normal eyeball when TTPy is injected into the vitreous cavity alone. In practical application, the photodynamic antibacterial activity of TTPy leads to early innate immune response, and neutrophils are recruited to the infection part in the vitreous cavity earlier, thereby not only limiting the spread of infection, but also protecting retina from tissue damage related to inflammation.

The invention relates to a novel antibacterial therapy for photodynamic therapy of bacterial endophthalmitis, which has the following beneficial effects by adopting the scheme:

1. rapid identification of bacteria and normal cells is achieved using aiegens (ttpy) carrying a positive charge and moderate hydrophobicity, driven by electrostatic and hydrophobic interactions.

2. TTPy can rapidly and efficiently generate ROS under a certain illumination condition, selectively kill staphylococcus aureus (S.aureus), realize effective in-vivo antibiosis on the premise of ensuring no cytotoxicity, have no obvious toxicity on normal cells of eyes, show the high-efficiency antibacterial potential of TTPy, and overcome the potential problem of bacterial drug resistance.

3. TTPy is used for exploring BE for treating staphylococcus aureus infection, and compared with a commercial photosensitizer Rose Bengal which is commonly used clinically, retina protection and effective in vivo antibiosis are realized, and a new strategy is provided for treatment of drug-resistant and refractory BE.

4. Efficient sterilization of TTPy triggers an early innate immune response that recruits neutrophils earlier to the site of infection in the vitreous chamber, limiting the spread of infection, while inhibiting the occurrence of fulminant inflammation and protecting the retina from tissue damage associated with inflammation.

5. TTPy can protect retina structure better when effectively disinfecting, has kept visual function to the utmost extent.

Drawings

FIG. 1 is a schematic diagram showing the change in fluorescence color under an ultraviolet lamp and PL spectrum in example 1;

FIG. 2 is a schematic representation of the ROS production assay of example 1;

FIG. 3 is a schematic diagram of Zeta potential measurement in example 1;

FIG. 4 is a graph showing the activity of ARPE-19 cells detected by CCK-8 in example 2;

FIG. 5 is a graph showing the activity of HCE-T cells detected by CCK-8 in example 2;

FIG. 6 is a schematic diagram of the cytotoxicity assay of Calcein AM/PI in example 2; (Scale: 100 μm)

FIG. 7 shows the expression of Bcl-2 and Bax in ARPE-19 cells in the detection of apoptosis rate and apoptotic protein in example 2;

FIG. 8 is a graph of gray scale analysis of Bcl-2 in the detection of apoptosis rate and apoptotic protein in example 2;

FIG. 9 is a graph of gray scale analysis of Bax in the detection of apoptosis rate and apoptotic protein in example 2;

FIG. 10 is a schematic diagram of flow cell apoptosis in the detection of apoptosis rate and apoptotic proteins in example 2;

FIG. 11 is a graph showing the statistical analysis of the apoptosis rate of ARPE-19 cells in the detection of apoptosis rate and apoptotic protein in example 2;

FIG. 12 is a staining chart of pathological examination of important organs (heart, liver, spleen, lung, kidney) of the rat in example 2;

FIG. 13 is a graph showing statistical analysis of serum electrolyte levels in rats in example 2;

FIG. 14 is a diagram showing the structural and morphological changes of the eyeball of the rat in example 2;

FIG. 15 is a graph showing the bactericidal effect of agar plate plating at different light powers and times in example 3;

FIG. 16 shows 20mW/cm in example 3 ² S.aureus colony count histogram at power, 0.05 μ M TTPy in 10min of light;

FIG. 17 shows 20mW/cm in example 3 ² S.aureus colony count histogram at power, 0.05 μ M TTPy in 15min of light;

FIG. 18 is a graph showing a morphological change of S.aureus under an electron microscope in example 3;

FIG. 19 is a schematic diagram of a photograph of the eye surface in example 3;

FIG. 20 is a graph of clinical inflammation scores in example 3;

FIG. 21 is a graph showing the measurement of the concentration of aqueous humor protein in example 3;

FIG. 22 is a vitreous pathology diagram according to example 3;

FIG. 23 is a retinal pathology map of example 3;

FIG. 24 is a diagram showing the detection of the contents of four common factors for endophthalmitis in the vitreous cavity by ELISA using CXCL1 in example 3;

FIG. 25 is a graph showing the content of four common factors for endophthalmitis in the vitreous cavity measured by ELISA using TNF- α in example 3;

FIG. 26 is a diagram showing the detection of the contents of four common factors for endophthalmitis in the vitreous cavity by ELISA for IL-1. beta. in example 3;

FIG. 27 is a diagram showing the content of four common factors for endophthalmitis in the vitreous cavity measured by IFN-. gamma.ELISA in example 3;

FIG. 28 is a staining pattern of neutrophils in example 3;

FIG. 29 is a graph of neutrophil counts in example 3;

FIG. 30 is a diagram showing the Transwell neutrophil count in example 3.

In the drawings, fig. 13: (A) AST (B) ALT (C) TG (D) T-CHO (E) Ca ²⁺ (F)K ⁺ (G)Cl ^- (H)CRE。

In FIG. 14, the cornea (A-C): normal group; ttpy + Light group; ttpy + Dark group; retina (D-F): normal group; ttpy + Light group; ttpy + Dark group.

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

As shown in fig. 1-30, the present invention relates to a novel antibacterial therapy for photodynamic treatment of bacterial endophthalmitis, comprising the steps of:

step three: diluting the mother solution by adopting a PBS solution;

step four: injecting the diluted mother solution into rat eyes;

The implementation of this solution is verified by the following examples.

Example 1, staphylococcus aureus (s. aureus) was selected as the recognition target. The TTPy molecular powder was dissolved in DMSO to give a 5mM stock solution, which was diluted with PBS for further experiments.

S1, preparation of bacterial liquid:

diluting Staphylococcus aureus, shaking Staphylococcus aureus (S.aureus) at 37 deg.C overnight for 18h, centrifuging to remove supernatant, resuspending, measuring with microplate reader, adjusting OD 600 to 1.0, and diluting to 10 ═ OD 600 ⁷ CFU/ml is used for later use, and bacterial liquids with different concentrations are obtained;

s2, ultraviolet absorption and photoluminescence spectrum determination:

respectively and uniformly mixing bacterial solutions with different concentrations with TTPy molecular solutions with different concentrations in a glass tube, incubating for 15min, shooting and recording fluorescence under the irradiation of a 365nm ultraviolet lamp in a dark room environment, uniformly mixing the TTPy molecular solutions with different concentrations with the bacterial solutions, and then measuring a PL spectrum by using a fluorescence spectrometer;

TTPy was diluted to different concentrations (0, 0.02, 0.05, 0.1, 0.2. mu.M) to analyze its photophysical properties.

As shown in FIG. 1A, under 365nm UV light, pure PBS and TTPy solutions showed no significant fluorescence. After the s.aureus and the TTPy are incubated for 15min, the mixed solution can be observed by naked eyes to show obvious light red fluorescence, and the fluorescence intensity is increased along with the increase of the concentration, which indicates that the TTPy has good affinity and biological imaging capability to the s.aureus.

As shown in FIG. 1B, when a single TTPy solution is irradiated by 489nm excitation light, the maximum emission wavelength is 610nm, and no significant difference exists between PL intensities at different concentrations. PL intensity increased significantly after 15min incubation with s.aureus, and the maximum emission wavelength exhibited a concentration-dependent red-shift.

S3, Zeta potential, active oxygen determination:

uniformly mixing TTPy molecular solutions with different concentrations with a bacterial solution, incubating for 15min, measuring the Zeta potential of S.aureus bacterial solution, carrying out whole-process dark operation, diluting DCFH-DA, adding the diluted DCFH-DA into TTPy molecular solutions with different concentrations, and dividing the diluted DCFH-DA into a PBS group, a DCFH-DA group and a DCFH-DA + TTPy group; adding the solution into 96-well blackboard in sequence, each well has 100 μ L, and measuring initial value I before illumination ₀ Giving illumination (power 20 mW/cm) ² ) Measuring absorbance I every 1min for 15min _t Record I _t /I ₀ 。

ROS yield was measured by DCFH-DA kit for 0.1. mu.M TTPy solution after white light irradiation. The results are shown in FIG. 2, under white light illumination (20 mW/cm) ² ) Obvious ROS generation can be detected after 1 min; the ROS yield of 15min of irradiation can reach about 80 times of that before irradiation.

In order to verify the affinity of TTPy to s.aureus, Zeta potentials of s.aureus mixtures after incubation with different concentrations of TTPy were measured and the results are shown in fig. 3, with the Zeta potential values of the s.aureus solution alone being-11.20 ± 0.15mV and the potentials decreasing after incubation with different concentrations of TTPy (0.02, 0.05 and 0.1 μ M) with statistical differences between the groups.

In particular embodiments, materials used include, but are not limited to: DMEM/F12 culture medium, fetal bovine serum, ampicillin-streptomycin double antibody, PBS (20X), epidermal growth factor, human recombinant insulin, 0.25% Trypsin-EDTA, serum-free cell cryopreservation solution, flow apoptosis kit, SDS-PAGE protein loading buffer solution, BCA protein concentration determination kit, PAGE gel rapid preparation kit, Bax antibody, Bcl-2 antibody, Action antibody, PMSF, Tween-20, Marker, secondary antibody (Rabbit, Rat), cell protein lysate, BSA, primary antibody, secondary antibody diluent, CCK-8 detection kit, Calcein-PI cell activity and cytotoxicity detection kit, 4% paraformaldehyde, pentobarbital sodium, eyeball fixative solution, 20X PBS, proparacaine hydrochloride eye drops, compound tropicamide eye drops, DAPI, CRE kit, TG kit, T-CHO kit, and the like, K ⁺ 、Cl ^- 、Ca ²⁺ Ion kit, ALT, AST kit, MPO antibody and BCA testKit, rat neutrophilic granulocyte separating kit, CXCL1, TNF-alpha, IL-1 beta and IFN-gamma Elisa kit.

Example 2 investigation of the safety of TTPy at the cellular and animal level.

(1) Cell activity assay:

cells in logarithmic growth phase were digested, resuspended, plated. After the cells adhered to the wall, TTPy solutions of different concentrations were added and incubated at room temperature for 15 min.

Group a white light (20 mW/cm) ² ) Irradiating for 30 min; dark treatment of group b at room temperature for 30min, and replacement of fresh culture medium for culture; group c was directly placed in the incubator and incubated in the dark.

After 24h, 100. mu.L of the mixed stock medium and 10. mu.L of CCK-8 solution were added to each well, and the mixture was incubated in a constant temperature incubator for 2 h. And measuring the absorbance value by adopting a 450nm wavelength microplate reader.

The results of CCK-8 assays for cell activity after intervention in ARPE-19 and HCE-T cells by TTPy at different concentrations are shown in FIGS. 4-5.

As shown in FIGS. 4 and 5, after incubation for 24h in the dark with light for 30min, TTPy had no significant effect on ARPE-19 cell activity at 0.5. mu.M or less, and cell activity of group a was significantly decreased at 1.0. mu.M, while cell activity of groups b and c was not significantly decreased. While HCE-T cells showed significant activity decrease in three groups a, b and c at 0.2. mu.M TTPy, the activity value was still above 80%.

(2) Calcein/PI live/dead cell staining

Add 10. mu.L of Calcein AM and PI to 10mL of assay buffer and mix well. After 24h of drug intervention on cells seeded in 6-well plates, the supernatant was discarded. Adding 1mL of Calcein AM/PI detection working solution, covering the monolayer cells, incubating for 30min at 37 ℃ in the dark, and collecting images under a fluorescence microscope.

The toxic effect of TTPy on intraocular cells was further examined by Calcein AM/PI cytotoxic staining. As shown in FIG. 6, TTPy at different concentrations had no significant cytotoxicity to ARPE-19 cells in three groups (same group) of a, b and c, and the ratio of live cells to dead cells was not statistically different in each group.

(3) Flow cytometry for detecting apoptosis

The cells were treated and disrupted as described above, and each group of cells was collected in a flow tube, centrifuged at 4 ℃ in a centrifuge, and the supernatant was discarded. After washing with PBS, 195. mu.L of Annexin V-FITC binding solution, 5. mu.L of Annexin V-FITC, and 10. mu.L of propidium iodide staining solution were added to each flow tube and gently mixed. Incubated for 15min in dark and detected by an up-flow cytometer.

The influence of TTPy on ARPE-19 apoptosis state is evaluated by detecting the expression of apoptosis protein by Western Blot and detecting the apoptosis rate by a flow cytometer.

As shown in FIGS. 7-9, there was no significant change in the expression level by statistical analysis of the expression of the two apoptosis-related proteins Bcl-2 and Bax.

FIGS. 10-11 reflect the apoptosis rates of ARPE-19 in each group, and the results show that there is no significant statistical difference between groups, so that TTPy does not cause ARPE-19 apoptosis in a certain concentration range.

(4) H & E staining for histopathological changes of each organ

Rats were sacrificed 7d after treatment, and the major organs of heart, liver, spleen, lung, and kidney were harvested and fixed in 4% paraformaldehyde for 24 h.

The fixed tissues are respectively put into gradient ethanol (50%, 70%, 80%, 95% and 100%) to be dehydrated for 2 h. Placing the dehydrated tissue in xylene for 2 times and 15 min/time to be transparent. The transparent sample tissue was placed in an embedding cassette and wax soaked for 2h (60 ℃). Paraffin section xylene 2 times dewaxing, gradient ethanol rehydration. Staining was performed with hematoxylin and eosin. Dehydrating with gradient ethanol, sealing with neutral gum after xylene is transparent, and collecting image under optical microscope.

After injecting the bacterial liquid and TTPy 7d into the vitreous cavity, respectively picking up important organs and venous blood of each group for detection, and observing whether the TTPy damages the structures and functions of the heart, the liver, the spleen, the lung and the kidney. As a result of H & E staining, it was found that the structure and morphology of each organ were not significantly abnormal as compared with those of normal rats, as shown in fig. 12. There was no significant difference between the serological electrolyte levels of each group, as shown in figure 13. The above results indicate that TTPy has excellent biological safety without affecting the structure, morphology and function of each important organ. As shown in fig. 14, there was no significant change in the cornea and retina structure of rat eyeball, and the normal eyeball was not affected by the injection of TTPy in the vitreous cavity alone.

Example 3 study of the effectiveness of TTPy at cellular and animal levels.

(1) Traditional plating for observing bacterial proliferation

Diluting the bacterial liquid to 10 degrees ⁵ CFU/ml, preparing TTPy solutions with different concentrations, mixing with bacterial solutions with different concentrations respectively, and incubating for 15 min. After the incubation is finished, pipetting the solution into a 24-well plate, and illuminating the Light group for a specific time; the Dark group continued incubation at room temperature in the Dark. After diluting 500 times, the mixture was spread evenly on a solid agar plate and incubated overnight in an incubator at 37 ℃. Colony counts were performed by ImageJ.

The photodynamic bactericidal effect of TTPy on s.aureus was evaluated by the traditional plating method, and as shown in fig. 15, the bactericidal effect was explored at different lighting powers and times to determine the concentration and lighting conditions for the best effective sterilization.

At a power of 40mW/cm ² At a TTPy concentration of 0.05. mu.M in 15min of light, no significant S.aureus proliferating colonies were visible on the agar plates.

Further at lower power (20 mW/cm) ² ) And excellent bactericidal effect was also observed with longer light exposure time (30 min).

Then at 20mW/cm ² At power, 0.05 μ M TTPy was effectively killed by both s.aureus groups at 10 and 15min in light, as shown in fig. 16 and 17, with no apparent viable s.aureus colonies seen in both groups. In order to obtain the best sterilizing effect, the illumination power is 20mW/cm ² Subsequent studies were performed by irradiation for 15 min.

(2) Bacteria and cell co-culture

ARPE-19 cells were plated on confocal culture dish at 37 ℃ in 5% CO ₂ And (5) allowing the culture box to stay overnight and adhere to the wall. Different concentrations (0.02. mu.M, 0.05. mu.M, 0.1. mu.M) of TTPy and S.aureus were added to the dishes and incubated for 15 min. Fluorescence images were taken by confocal laser scanning microscopy.

When TTPy concentration was 0 μ M and incubated in the dark (as shown in fig. 18), s. When s.aureus was disrupted to varying degrees by light intervention with different concentrations of TTPy, the cells appeared to be irregularly shaped. The bacterial morphology under an electron microscope proves that TTPy plays an antibacterial role by destroying cell membranes of S.aureus, and realizes a high-efficiency bactericidal effect on S.aureus.

(3) Scanning electron microscope for observing sterilization efficiency of TTPy molecules

The above-mentioned bacterial solution was resuspended in PBS and then dispensed into centrifuge tubes, 100. mu.L/tube. The bacterial solution and TTPy molecular solution with different concentrations are mixed and incubated for 15min, the Light group is placed in a 24-well plate and is irradiated for 15min, and the Dark group is placed in a room temperature and Dark place for 15 min.

After the illumination is finished, collecting the bacterial liquid in a centrifuge tube, centrifuging to remove the supernatant, and fixing with 4% paraformaldehyde overnight. After fixation, the supernatant is discarded by centrifugation, and the ethanol is dehydrated in a gradient way for 6min each time. After resuspending with 100% ethanol, 2 μ L of the suspension was dropped onto aluminum foil paper, dried at room temperature, and characterized after gold spraying treatment.

(4) Establishment of bacterial endophthalmitis animal model

All rats were injected with 10 μ L of s.aureus solution (100CFU) in the eyes to establish a rat endophthalmitis model, which was randomly divided into four groups for subsequent experiments, and the intervention treatment was performed 1h after s.aureus bacterial solution injection.

The grouping is as follows:

control group: incubating 10 μ L of 1 × PBS for 15min, and illuminating for 30 min;

RB + Light group: incubating for 15min with 10 μ L and 0.1 μ M RB, and illuminating for 30 min;

TTPy + Dark group: 10 μ L, 0.1 μ M TTPy, incubated for 15min, dark for 30 min;

TTPy + Light group: 10 μ L, 0.1 μ M TTPy, incubated for 15min, illuminated for 30 min.

(5) Ocular surface photography, clinical inflammation scoring

Slit lamp photographs were taken at various time points (6h, 12h, 1d, 3d, and 7d) after intervention, ocular infections and inflammatory manifestations were recorded, and Clinical inflammation scores were performed (CIS).

The ocular surface condition after treatment was recorded by slit lamp to observe and quantify s.

(6) Determination of Total protein concentration in aqueous humor

After rats are anesthetized and killed at different time points after intervention treatment, the eyeballs are quickly picked up and placed on dry ice for quick freezing. The frozen eye ball was cut on ice along the limbus, and aqueous humor was collected in a centrifuge tube and stored at-80 ℃. The collected aqueous humor was centrifuged for 5min, and the supernatant was diluted 30-fold. And (3) determining the total protein concentration of the aqueous humor by using a BCA method, and determining the OD value of the pore plate under the 540nm of an enzyme-labeling instrument. Protein concentration values are expressed as mean ± standard deviation.

(7) HE staining of eyeball pathological section

Rats were sacrificed at various time points and the eyes removed and fixed in ocular fixative overnight. Dehydration, clearing, embedding, sectioning, deparaffinization, staining were performed as described above, and the anterior chamber, ciliary body, vitreous body, retinal pathological changes were observed under an optical microscope.

All the eyeballs were visibly white, with cellulose exudation and pupil adhesion visible at 12h (fig. 19).

As shown in figure 20, where the clinical inflammation scores were higher for the TTPy + Light group and the RB + Light group, the differences were statistically significant.

Obvious corneal edema, massive exudation of anterior chamber cellulose and pupil adhesion can be seen at 24h after treatment. Among them, the TTPy + Light group showed the lightest inflammation, and the RB + Light group showed the heaviest inflammation. There was still a small amount of unabsorbed cellulose in the anterior chamber after 7d in the Control group and the TTPy + Dark group. The intravitreal inflammation was consistent with the anterior segment expression (see fig. 22), at 12h, significant inflammatory cell infiltration occurred in the vitreous of all rats, and the inflammatory cell counts of the TTPy + Light group and the TTPy + RB group were significantly increased, with strong inflammation; at 24h, inflammatory cell counts were significantly reduced in the TTPy + Light group, infection and inflammation were controlled, retinal structures were effectively protected during infection (as shown in fig. 23), and the retinas of the other three groups were destroyed to varying degrees.

(8) ELISA for detecting inflammatory factors

After rats are anesthetized and the neck is removed to death at different time points after intervention treatment, the eyeballs are quickly picked up and placed on dry ice for quick freezing. After the crystal is removed, collecting vitreous humor, and detecting the contents of four common factors of endophthalmitis in the vitreous cavity by an ELISA detection kit of TNF-alpha, IL-1 beta, CXCL1 and gamma-IFN.

As shown in fig. 24, CXCL1 peaked earliest, with TTPy + Light and RB + Light groups significantly higher than the other groups at 3-6h, which recruited more neutrophils to the site of infection.

As shown in fig. 25, higher TNF- α levels were also observed in the TTPy + Light group of vitreous before 12h, which resulted in earlier, more infiltration of neutrophils and monocytes into the vitreous cavity. Also, CXCL1 and TNF- α levels recovered more rapidly in the TTPy + Light group, indicating a faster recovery of the blood retinal barrier and a reduction in exuding neutrophils.

As shown in FIG. 26, IL-1. beta. was significantly elevated in the Control group and TTPy + Dark group at 12h and 1d, probably due to the secretion of large amounts of alpha-toxin by the bacteria proliferating in the vitreous cavity.

Furthermore, as shown in figure 27, the TTPy + Light group showed lower IFN- γ levels, indicating less macrophage and lymphocyte infiltration in the later stages.

As shown in figure 28, the numbers of neutrophils were significantly greater in the TTPy + Light and RB + Light groups at 12h than in the other groups, consistent with previous results, and again it was verified that the antibacterial activity of TTPy induced a faster innate immune response. However, in the 24h Control and TTPy + Dark groups, a large number of neutrophils infiltrated the vitreous cavity, causing severe inflammation and tissue damage.

As shown in fig. 29 and fig. 30, the in vitro transwell assay results indicated that neutrophils from the TTPy and RB groups migrated into the sub-chamber earlier at 1d after light exposure.

The results of the above studies indicate that photodynamic anti-bacterial action of TTPy may lead to an early innate immune response with earlier recruitment of neutrophils to the site of infection in the vitreous chamber, which not only limits the spread of infection, but also protects the retina from tissue damage associated with inflammation.

According to the above embodiments, the summary and effects are as follows:

1. the low-concentration TTPy can selectively kill staphylococcus aureus (S.aureus) under the illumination condition, has no obvious toxicity to normal eye cells, shows the high-efficiency antibacterial potential of the TTPy, and overcomes the potential problem of bacterial drug resistance in the prior art.

2. Efficient sterilization of TTPy triggers an early innate immune response that recruits neutrophils earlier to the site of infection in the vitreous chamber, limiting the spread of infection, while inhibiting the occurrence of fulminant inflammation and protecting the retina from tissue damage associated with inflammation.

3. RB causes serious retinal damage after sterilization, and inevitably causes the loss of the visual function of a rat, while the TTPy provided by the scheme can effectively sterilize and better protect the retinal structure, and the visual function of the rat is greatly reserved.

The novel antibacterial therapy provided by the scheme not only successfully utilizes AIEgens to carry out photodynamic antibacterial research, but also verifies the safety and effectiveness of TTPy in cell and animal levels, and lays a foundation for the next antibacterial application of the molecules.

Compared with the conventional commonly used method for treating bacterial endophthalmitis by injecting antibiotics into a vitreous cavity clinically, the method can solve the problems of potential bacterial drug resistance and retinal toxicity in the prior art, has better treatment effect and shorter treatment time and treatment cost, can also solve the defect of aggregated fluorescence quenching of the traditional photosensitizer, and can achieve good sterilization effect through lower administration concentration. High antibacterial capacity and low cytotoxicity are very suitable vehicles for photodynamic therapy and also provide a new therapeutic strategy for bacterial endophthalmitis.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

The present invention and its embodiments have been described above, and the description is not intended to be limiting, and the drawings are only one embodiment of the present invention, and the actual structure is not limited thereto. In summary, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A novel antibacterial therapy for photodynamic treatment of bacterial endophthalmitis comprising the steps of:

the method comprises the following steps: selecting aggregation-induced emission molecules as a base material;

step two: dissolving aggregation-induced emission molecules by using DMSO (dimethyl sulfoxide) to obtain a mother solution with the concentration of 5 mM;

step three: diluting the mother solution by adopting a PBS solution;

step four: injecting the diluted mother solution into rat eyes;

2. A novel antibacterial therapy for photodynamic treatment of bacterial endophthalmitis according to claim 1, wherein: in the first step, the aggregation-induced emission molecule is a solid TTPy molecule with reddish brown color, and the TTPy molecule is a TTPy molecule with positive charge and medium hydrophobicity.

3. A novel antibacterial therapy for photodynamic treatment of bacterial endophthalmitis according to claim 1, characterized in that: the illumination under certain conditions in the step five is that the illumination power is 20mW/cm ² For 15min under white light.

4. A novel antibacterial therapy for photodynamic treatment of bacterial endophthalmitis according to claim 1, characterized in that: the novel antibacterial therapy does not affect normal eyeballs when TTPy is injected into a simple vitreous cavity.

5. A novel antibacterial therapy for photodynamic treatment of bacterial endophthalmitis according to claim 1, characterized in that: the safety and effectiveness of the novel antibacterial therapy is verified at the cellular and animal level.

6. A novel antibacterial therapy for photodynamic treatment of bacterial endophthalmitis according to claim 1, characterized in that: in practical applications, the photodynamic anti-bacterial activity of TTPy results in an early innate immune response, with earlier recruitment of neutrophils to the site of infection in the vitreous chamber, limiting the spread of infection and protecting the retina from tissue damage associated with inflammation.