Microbially degradable polymer embedded with aggregation-induced emission molecules and method of using same for bacterial detection and antibiotic screening

Abstract

The present invention provides a composition, comprising a shell layer, wherein the shell layer comprises a microbially degradable polymer and a plurality of aggregation-induced emission molecules. The present invention also provides a method for detecting the presence of bacteria, comprising contacting a sample with the described composition. Since bacteria cause a decrease in the fluorescence intensity of the composition, the presence or absence of bacteria in the sample can be determined on the basis of change in the fluorescence intensity. The present invention further provides an antibiotic screening method, comprising contacting the described composition with bacteria, then adding an antibiotic to be tested, and evaluating the effectiveness of said antibiotic on the basis of changes in the fluorescence intensity of the composition.

Description

Microbial degradable polymers embedded with molecules possessing aggregation-induced luminescence and their application in bacterial detection and antibiotic screening. Technical Field

[0001] This invention relates to a composition comprising a shell layer containing a microbial-degradable polymer and a plurality of aggregation-induced emission molecules. Because bacteria degrade the microbial-degradable polymer in the composition, the plurality of aggregation-induced emission molecules are released, ultimately leading to a decrease in fluorescence intensity. Therefore, the presence of bacteria and the effectiveness of antibiotics can be assessed by observing changes in the fluorescence intensity emitted by the composition. Background Technology

[0002] Nanographene (nanoG) possesses excellent aggregation-induced emission (AIE) properties. Therefore, aggregating dispersed nanoG can result in enhanced fluorescence. Furthermore, the hydrophobic nature of nanoG can be utilized to achieve aggregation by taking advantage of its solubility differences in different solvents; however, this is limited to organic solution systems and cannot be applied to the biomedical field.

[0003] Poly(lactic-co-glycolic acid) (PLGA) is a highly biodegradable and biocompatible material that can also be used to produce surgical sutures. Polymer vesicles composed of PLGA exhibit excellent sustained-release drug capsule applications, prepared via a double emulsification process. In this process, the first emulsification step forms a water-in-oil PLGA microsphere, and the second emulsification step produces water-in-oil PLGA polymer vesicles, which feature a hydrophilic hollow core and a hydrophilic vesicle surface. Besides being able to load hydrophilic drugs, the entire vesicle is also water-soluble. Furthermore, the PLGA shell of the vesicle exhibits hydrophobic properties, making it suitable for loading hydrophobic drugs or materials. Previous studies have shown that loading magnetic iron oxide nanoparticles and gold nanoparticles can impart magnetic and optical properties to the vesicles. Summary of the Invention

[0004] This invention prepares hydrophobic cyanide-modified nanographene (nanoG) with excellent aggregation-induced emission (AIE) effect. The nanoG is embedded in the hydrophobic shell of a PLGA polymer to form nanoG-embedded PLGA polymer vesicles. The large-scale aggregation of nanoG sandwiched between the inner and outer shells of the PLGA results in significantly enhanced AIE luminescence. Furthermore, by utilizing the rapid degradation of PLGA polymers by live bacteria, the nanoG is released from the PLGA polymer shell, thereby reducing the aggregation state of the nanoG and causing the AIE effect to decrease with increasing bacterial count.

[0005] Simultaneously, by pre-treating the bacterial mixture with a suitable antibiotic, excellent selectivity for the detection of methicillin-resistant Staphylococcus aureus (MRSA) was achieved. Therefore, taking the screening of antibiotics for unknown infections as an example, the screening method of this invention is as follows: This nanoG-embedded PLGA polymer vesicle is integrated into a 96-well plate, and a sample solution containing bacteria and the antibiotic to be tested are added. The groups of antibiotics that do not show fluorescence decay are then observed to identify the most effective antibiotic. Although this method cannot identify the type of bacteria, it can identify the most suitable antibiotic within a 1-hour detection time. This rapid screening method for effective antibiotics will effectively alleviate and control the patient's infection symptoms. The nanoG-embedded PLGA polymer vesicle of this invention features ease of operation, low cost, observable luminescence changes, and rapid detection (approximately 1 hour), making it applicable to pathogen rapid screening reagents, precision medicine, and in vitro diagnostic analysis platforms.

[0006] The present invention provides a composition comprising: a shell layer, wherein the shell layer comprises a microbial degradable polymer and a plurality of molecules having aggregation-induced emission.

[0007] In some respects, the plurality of aggregation-induced emission molecules are encapsulated or dispersed within the microbial degradable polymer. When bacteria degrade the microbial degradable polymer, the plurality of aggregation-induced emission molecules are released.

[0008] In this invention, the outer shell layer has a spherical structure. In some aspects, the outer shell layer can be divided into three layers, sequentially from the inside out: a first shell layer, an intermediate layer, and a second shell layer. Therefore, the intermediate layer is located between the first shell layer and the second shell layer. Thus, the intermediate layer covers the first shell layer, and the second shell layer covers the intermediate layer. In one specific embodiment, the outer shell layer includes a first shell layer, an intermediate layer, and a second shell layer, with the intermediate layer located between the first shell layer and the second shell layer.

[0009] In one embodiment, the first shell and the second shell comprise the microbial degradable polymer. In another embodiment, the intermediate layer comprises the plurality of molecules with aggregation-induced emission. Thus, the plurality of molecules with aggregation-induced emission are sandwiched between the microbial degradable polymer.

[0010] In some aspects, the materials of the outer shell, the first shell, and the second shell comprise the microbial degradable polymer, particularly a polymer that can be degraded or broken down by bacteria. In this invention, when the composition comes into contact with bacteria, the bacteria degrade the microbial degradable polymer in the outer shell (or the outermost second shell) of the composition, thereby releasing the plurality of molecules exhibiting aggregation-induced emission. In one specific embodiment, the microorganism comprises bacteria. In a preferred specific embodiment, the microbial degradable polymer is a bacterial degradable polymer.

[0011] In this invention, the microbial degradable polymer is both hydrophilic and hydrophobic. In another specific embodiment, the microbial degradable polymer comprises polylactic-glycolic acid (PLGA), polycaprolactone (PCL), polylactic acid (PLA), polybutylene succinate (PBS), PBS-based amphiphilic copolymers, polyethylene glycol-PLGA (PEG-PLGA), PLA-PEG, galactose-PLA, poly(globalide-co-ε-caprolactone) (PGlCL), polyhydroxyalkanoates (PHAs), polyaspartic acid (Poly(aspartic acid)), cellulose, galactomannan, alginate, chitosan, or starch. In a preferred specific embodiment, the microbial degradable polymer comprises PLGA.

[0012] Aggregation-induced emission (AIE) refers to the phenomenon of enhanced luminescence in organic compounds in the condensed state. In solution, these compounds exhibit weak fluorescence emission due to the non-radiative decay of excited-state energy caused by intramolecular rotation; in the aggregated state, rotation is hindered, suppressing non-radiative energy conversion and causing the excited-state energy to be released radiatively, producing fluorescence. Therefore, aggregation-induced emission can refer to fluorescence triggered by molecular aggregation. Common molecules exhibiting AIE effects include silylopyropentadiene-type, cyclic polyene-type, polyaromatic substituted ethylene-type, nitrile-substituted stilbene-type, and pyran-type molecules, with tetraphenylethylene being a commonly used AIE molecule. In another specific embodiment, the molecule exhibiting aggregation-induced emission comprises molecules possessing AIE properties. In a preferred embodiment, the molecule exhibiting aggregation-induced emission comprises tetraphenylethene (TPE), hexaphenylsilole (HPS), triphenylamine derivatives, TPE-functionalized polymers, AIE-active conjugated polymers, AIE-active metal-organic frameworks, carbon dots, graphene quantum dots, silicon dots, cyclodextrin-AIE complexes, Au nanoclusters, Ag nanoclusters, graphene oxide, reduced graphene oxide doped with fluorophores, or cyanide-modified graphene nanoparticles. In a more preferred embodiment, the molecule exhibiting aggregation-induced emission comprises cyanide-modified graphene nanoparticles.

[0013] In this invention, the presence of bacteria is detected by changes in the luminescence / fluorescence intensity emitted by molecules possessing AIE properties. Herein, the luminescence intensity can be determined from the magnitude of fluorescence measured by a fluorescence spectrometer or fluorescence microscope. According to the design principles of the invention, bacteria degrade the outer shell or the second shell, thereby releasing the plurality of aggregation-induced luminescence molecules in the intermediate layer. Therefore, the aggregation state of the plurality of aggregation-induced luminescence molecules located in the intermediate layer decreases, leading to a decrease in the luminescence intensity of the AIE. In some aspects, the luminescence intensity of the composition decreases with bacterial concentration.

[0014] In this invention, the outer shell layer contains a hydrophilic core. Therefore, the outer shell layer covers the outer surface of the hydrophilic core; or the first shell layer covers the outer surface of the hydrophilic core. The hydrophilic core is composed of a hydrophilic polymer (e.g., polyvinyl alcohol (PVA)). In this invention, the composition has a spherical structure. In some aspects, the structure of the composition, from the inside out, is: the core, the first shell layer, the intermediate layer, and the second shell layer. In one specific embodiment, the outer shell layer contains a hydrophilic core. In a preferred specific embodiment, the material of the hydrophilic core or the hydrophilic polymer comprises polyvinyl alcohol or gelatin.

[0015] In some respects, the composition is a polymer vesicle. The composition can be prepared using either monoemulsification or double emulsification.

[0016] This invention provides a single-emulsion preparation method for a composition, comprising: preparing an oil phase solution comprising a microbially degradable polymer and a plurality of aggregation-induced emission molecules; preparing an aqueous phase solution comprising a hydrophilic polymer; mixing the oil phase solution and the aqueous phase solution to form an oil-in-water emulsion, and forming a plurality of the composition within the oil-in-water emulsion; and collecting and purifying the plurality of the composition. In the composition prepared by the single-emulsion method, the plurality of aggregation-induced emission molecules are encapsulated within the microbially degradable polymer (oil phase) and exhibit a relatively loose distribution. The microbially degradable polymer (such as PLGA) molecules have hydrophilic and hydrophobic ends. Therefore, in aqueous solution, the hydrophilic end of the microbially degradable polymer is on the outside and the hydrophobic end is on the inside, forming polymer vesicles.

[0017] This invention further provides a dual emulsification method for preparing compositions, comprising: preparing an oil phase solution comprising a microbially degradable polymer; preparing a first aqueous phase solution comprising a hydrophilic polymer; mixing the oil phase solution and the first aqueous phase solution to form a first water-in-oil emulsion; preparing an organic solution comprising a plurality of molecules having aggregation-induced emission; mixing the first water-in-oil emulsion and the organic solution to form a second water-in-oil emulsion; preparing a second aqueous phase solution comprising the hydrophilic polymer; mixing the second water-in-oil emulsion and the second aqueous phase solution to form an oil-in-water emulsion, and forming a plurality of the compositions in the oil-in-water emulsion; and collecting and purifying the plurality of the compositions. Therefore, the composition is a dual-emulsification core-shell nanostructure. In the dual emulsification method, the surface of the dual-emulsification core-shell nanostructure is hydrophilic, the core (PVA) is also hydrophilic, and the shell (such as PLGA) is hydrophobic. Therefore, the multiple molecules with aggregation-induced emission are sandwiched between the two shells to form the intermediate layer, and exhibit a relatively compact aggregation state.

[0018] In this invention, the molecule possessing AIE properties needs to be hydrophobic. This is also to ensure that the molecule with aggregation-induced emission (AIE) is encapsulated within the microbial biodegradable polymer during emulsification. Because the molecules of the microbial biodegradable polymer have both hydrophilic and hydrophobic ends, the microbial biodegradable polymer contacts the molecule with AIE properties and hydrophobicity through its hydrophobic end. This invention utilizes the above principle to encapsulate the molecule with AIE properties within the microbial biodegradable polymer. Furthermore, the molecule with AIE properties and hydrophobicity is sandwiched within the interface of the hydrophobic shell of the microbial biodegradable polymer. In one specific embodiment, the molecule with aggregation-induced emission is hydrophobic.

[0019] The present invention also provides a method for detecting the presence of bacteria, comprising: (1) providing the composition described in the present invention; (2) contacting a sample with the composition; (3) observing the luminescence intensity emitted by the composition in step (2); and (4) comparing the luminescence intensity of the composition in step (3) with the luminescence intensity of a composition not in contact with the sample, wherein if the luminescence intensity of the composition in step (3) decreases, it indicates that bacteria are present in the sample.

[0020] In some respects, the sample comprises a biological sample. This biological sample is typically obtained from mammals, such as humans or non-human primates. In this invention, the sample is derived from an individual. Hereinafter, the term "individual" can refer to any mammal, preferably a human. In other respects, the sample can be any sample, comprising liquid or solid samples. In one specific embodiment, the sample comprises tissue biopsy, blood, plasma, urine, saliva, tears, sputum, feces, bile, pleural fluid, ascites, synovial fluid, cerebrospinal fluid, extracellular fluid, cultured cells, culture medium, or waste tissue.

[0021] In this invention, the luminescence property of the composition originates from the molecule exhibiting aggregation-induced emission, and this molecule can be excited by ultraviolet light to emit fluorescence. In one specific embodiment, the luminescence of the composition is generated by ultraviolet light excitation.

[0022] When bacteria are present in the sample, they degrade the microbial-degradable polymer in the outer shell of the composition, releasing multiple molecules with aggregation-induced emission, leading to a decrease in emission intensity. Conversely, if no bacteria are present in the sample, the emission intensity of the composition will not decrease or change. Therefore, the emission intensity is negatively correlated with bacterial concentration. Thus, the emission intensity emitted by the composition before and after contact with the sample can be compared to determine the presence of bacteria in the sample. Furthermore, this method for detecting bacteria can also use a composition that has not been in contact with the sample as a control group for comparison, and compare the emission intensity of the composition in contact with the sample with the emission intensity of the control group to determine the presence of bacteria in the sample.

[0023] Therefore, the fluorescence intensity of the composition can be observed under ultraviolet light before contact with the sample. If the fluorescence intensity decreases or quenches after contact with the sample, it indicates the presence of bacteria in the sample. In this document, the term "observation" includes both visual observation and instrumental measurement. In some aspects, the instrument includes a fluorescence spectrometer or a fluorescence microscope. Under ultraviolet light irradiation, the change in the luminescence intensity emitted by the composition before and after contact with the sample can be directly observed with the naked eye. Therefore, this invention enables the visual detection of bacteria.

[0024] In this document, the term "bacteria" includes both Gram-positive and Gram-negative bacteria. In some aspects, the Gram-positive bacteria include Bacillus, Listeria, Staphylococcus, Streptococcus, and Enterococcus. In one specific embodiment, the Staphylococcus includes Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA). In other aspects, the Gram-negative bacteria include Shigella, Salmonella typhi, Escherichia coli, Proteus, Pseudomonas aeruginosa, Bordetella pertussis, and Vibrio cholerae. In a preferred specific embodiment, the bacteria include MRSA or Escherichia coli.

[0025] The present invention provides a kit for detecting bacteria, comprising a composition described herein.

[0026] The method of using this kit is to contact the sample to be tested with the composition to initiate a reaction. This reaction can be carried out in wells, plates, tubes, chambers, droplets, flow cells, glass slides, chips, or on a solid substrate. Under ultraviolet light irradiation, the change in the intensity of the light emitted by the composition after contact with the sample is observed. When the intensity of the light decreases, it indicates the presence of bacteria in the sample. Therefore, this kit provides a rapid and visual method for detecting bacteria.

[0027] Based on the detection principle of this invention, when bacteria are present, the aggregation-induced emission molecule in the composition is released, leading to a decrease or quenching of the fluorescence intensity of AIE. Therefore, the detection method of this invention can rapidly detect the presence of bacteria. Consequently, the detection method of this invention can be further applied to antibiotic screening platforms to rapidly assess the effectiveness of antibiotics.

[0028] The present invention further provides a method for screening antibiotics, comprising: (a) contacting a sample containing at least one bacterium with the composition of the present invention to form a mixture; (b) adding the antibiotic to be tested to the mixture of step (a); (c) observing the luminescence intensity emitted by the composition of step (b); and (d) comparing the luminescence intensity of the composition of step (c) with the luminescence intensity of a control group, wherein the presence of the at least one bacterium in the sample causes a decrease in the luminescence intensity of the composition; therefore, if the luminescence intensity of the composition is higher than that of the control group, it indicates that the antibiotic to be tested is effective against at least one bacterium in the sample, wherein the control group is the composition of the present invention in contact with the sample, but without the antibiotic to be tested.

[0029] In this invention, the composition possessing AIE properties can be used for high-throughput antibiotic screening applications. When an effective antibiotic is present, bacterial growth is inhibited; however, when an ineffective antibiotic is present, bacteria grow rapidly. Since bacterial concentration is negatively correlated with the luminescence intensity of the composition's AIE, the effectiveness of the antibiotic can be evaluated based on the luminescence intensity.

[0030] For example, the test results of this antibiotic screening method may show three possibilities:

[0031] Scenario 1: The antibiotic being tested can inhibit or kill all bacteria in the sample. Therefore, in the observation results, the luminescence intensity of the composition will not decrease, indicating that the antibiotic being tested is the most effective antibiotic.

[0032] Scenario 2: The antibiotic being tested can only inhibit or kill some of the bacteria in the sample. Since the sample may contain various types of bacteria, it's difficult to kill all of them with a single antibiotic; therefore, some attenuation of luminescence intensity will still occur. However, if an effective antibiotic is used, the growth of some bacteria can still be inhibited, thus suppressing some of the attenuation of luminescence intensity. Therefore, in the observed results, compared to the control group (without any antibiotics), the luminescence intensity of this composition will still be higher than that of the control group, indicating that the antibiotic is still effective.

[0033] The third scenario: The antibiotic being tested is unable to inhibit or kill some of the bacteria in the sample. Therefore, in the observation results, the luminescence intensity of the composition will be the same as that of the control group or quenched, indicating that the antibiotic is ineffective.

[0034] In the presence of ineffective antibiotics, bacterial growth is not inhibited, and bacterial concentrations are high. However, in the presence of effective antibiotics, bacterial growth is inhibited, and bacterial concentrations are very low. When co-cultured with a composition containing molecules with AIE, bacterial concentration is negatively correlated with fluorescence intensity. The effectiveness of the antibiotic is determined by whether the attenuation of fluorescence intensity is inhibited.

[0035] In some respects, the control group can be replaced by a group containing only the composition, but without any sample or antibiotic. Therefore, the control group is not exposed to bacteria, and thus its fluorescence intensity will not decrease. If, after contact with the sample, the composition exhibits the same or similar fluorescence intensity as the control group (composition only) upon the addition of antibiotic, then the antibiotic is considered effective.

[0036] In some respects, this method for screening antibiotics can also be used to determine bacterial resistance. For example, when the species of bacteria in the sample are known, it can be used to test which antibiotic the bacteria are resistant to. For instance, when fluorescence is quenched, it indicates that the bacteria cannot be inhibited or killed by that antibiotic, and therefore the bacteria are resistant to that antibiotic. Therefore, the steps of this method for screening antibiotics can also be used to assess bacterial resistance to antibiotics.

[0037] In this invention, the effectiveness of an antibiotic can be rapidly assessed by observing changes in the luminescence intensity of the composition. Furthermore, the test results are available in a short time (approximately 1 hour). Since the sample is from an individual, once the antibiotic's effectiveness is confirmed, the individual can be treated. Therefore, this antibiotic screening method can quickly identify effective antibiotics for treating patients.

[0038] By embedding nanoG into the hydrophobic interface of a microbially degradable PLGA shell, this invention successfully prepared a novel AIE-characteristic polymer vesicle for microbial detection. This novel heterostructure, with nanoG sandwiched within the polymer interstices of the vesicle shell, exhibits a stronger AIE effect compared to typical solvent-induced aggregation methods. Detailed characterization of the nanoG-embedded PLGA polymer vesicles revealed that both Gram-negative and Gram-positive bacteria can disrupt the microbially degradable PLGA vesicles, leading to nanoG leakage and inhibiting the AIE effect. Further research showed that this unique microbial response to AIE silencing is concentration- and time-dependent, demonstrating that AIE vesicles can be used not only for visual thin-film sensing but also for precise liquid chromatography quantitative analysis. Through kanamycin pre-screening, this AIE-characteristic biosensor successfully achieved excellent specificity against methicillin-resistant Staphylococcus aureus (MRSA). Furthermore, the feasibility of using an AIE-characteristic biosensor for rapid, non-invasive detection of MRSA-infected wounds was also demonstrated. The PLGA polymer vesicles embedded with nanoG of the present invention have flexible applications and good potential for specific detection of drug-resistant pathogens in a variety of environments. Attached Figure Description

[0039] Figure 1 shows the preparation process of the nano-graphene of the present invention.

[0040] Figure 2 shows the structure of a PLGA polymer vesicle embedded with nanoG. This PLGA polymer vesicle has a water-in-oil-in-water structure, with nanoG sandwiched within the interface of its hydrophobic shell.

[0041] Figure 3 shows the fluorescence spectra of nanoG-embedded PLGA polymer vesicles in deionized water and nanoG in dichloromethane. The left panel shows the difference in fluorescence intensity between nanoG-embedded PLGA polymer vesicles and simple nanoG aggregates, while the right panel shows the blue fluorescence intensity emitted by nanoG-embedded PLGA polymer vesicles and simple nanoG aggregates under UV excitation.

[0042] Figure 4 shows the structure of PLGA polymer vesicles embedded with nanoG under a microscope. The left image shows the PLGA polymer vesicles embedded with nanoG under a high-magnification scanning electron microscope (SEM). The right image shows the fluorescence image of the PLGA polymer vesicles embedded with nanoG under a conjugate focal microscope, which shows the blue light emitted by the nanoG in the PLGA polymer shell.

[0043] Figure 5 shows the case where PLGA polymer vesicles embedded with nanoG are degraded by bacteria, leading to nanoG release and AIE attenuation.

[0044] Figure 6 shows that the AIE decay of PLGA polymer vesicles embedded with nanoG is time-dependent and depends on bacterial concentration. Figure 6A shows the AIE decay of PLGA polymer vesicles embedded with nanoG at a concentration of 1.2 × 10⁻⁶. 8 Fluorescence spectra of MRSA at CFU / mL incubated at 37 °C for different times (0, 1, 3, 5, 7, and 24 hours). Figure 6B shows the fluorescence intensity of MRSA at 430 nm over time. Figure 6C shows the fluorescence intensity of MRSA at 1.2 × 10⁻⁶ CFU / mL with nanoG-embedded PLGA polymer vesicles. 8 Fluorescence spectra of *E. coli* CFU / mL incubated at 37°C for different times (0, 1, 3, 5, 7, and 24 hours). Figure 6D shows the change in fluorescence intensity of *E. coli* at 430 nm over time. Figure 6E shows the fluorescence intensity of PLGA polymer vesicles embedded with nanoG and at a concentration of 1.2 × 10⁻⁶ CFU / mL. 8 The fluorescence spectrum of MRSA at CFU / mL incubated at 37°C for 1 hour. Figure 6F shows the fluorescence intensity of MRSA at 430 nm over time. Figure 6G shows the fluorescence spectrum of PLGA polymer vesicles embedded with nanoG at a concentration of 1.2 × 10⁻⁶ CFU / mL. 8 The fluorescence spectrum of CFU / mL *E. coli* incubated at 37°C for 1 hour. Figure 6H shows the change in fluorescence intensity of *E. coli* at 430 nm over time. The excitation wavelength of the fluorescence spectrum was fixed at 330 nm. All measurements were performed independently three times.

[0045] Figure 7 shows the use of AIE probes prepared from PLGA polymer vesicles embedded with nanoG for antibiotic screening. Figure 7A shows a pre-screening strategy for antibiotics that involves using the antibiotic to eliminate bacteria in a bacterial mixture sensitive to that antibiotic, thereby retaining the target bacteria for further AIE detection. Figure 7B shows the use and non-use of 4.5 μg mL -1 Bacterial growth curves of methicillin-resistant Staphylococcus aureus and Escherichia coli at 0.1 OD for kanamycin.

[0046] Figure 8 shows that after reacting the sample and antibiotics with an AIE probe prepared by adding PLGA polymer vesicles containing embedded nanoG for 1 hour, the fluorescence intensity of each antibiotic group was used to determine which antibiotic had the best bactericidal effect on the bacteria in the sample. Detailed Implementation

[0047] The embodiments of the present invention may have different implementations and are not limited to the examples given below. The following embodiments only represent various aspects and features of the present invention.

[0048] Experimental methods:

[0049] 1. Preparation methods of nano-graphene

[0050] As shown in Figure 1, the preparation steps of nanographene are as follows:

[0051] (1) Synthesis of compound S1: 4-bromobenzonitrile (4.97 g, 27.3 mmol), triphenylphosphine (PPh3) (0.17 g, 2.5 mol%), PdCl2(PPh3)2 (0.96 g, 5 mol%), cuprous iodide (CuI) (0.17 g, 2.5 mol%), and trimethylsilylacetylene (4.05%, 41.2 mmol) were added to a two-necked flask. The flask was degassed and filled with nitrogen. A mixture of tetrahydrofuran (THF) (60 mL) and triethylamine (Et3N) (5.9 mL) was added using a syringe, and the mixture was reacted at room temperature for 15 hours. A yellow solid was obtained. The residue was separated using MeOH / K2CO3, purified by silica gel column chromatography, and extracted with CH2Cl2-hexane to give a colorless solid compound S1 (2.67 g, 77%).

[0052] (2) Synthesis of compound S2: Compound S1 (1 g, 7.87 mmol), 4-bromobenzonitrile (1.43 g, 7.87 mmol), PPh3 (41 mg, 0.16 mmol), Pd(PPh3)2Cl2 (110 mg, 0.16 mmol), and Et3N (30 mL) were added to a flask. The mixture was stirred for 10 minutes while argon (Ar) was bubbled into the solution, followed by the addition of cuprous iodide (60 mg, 0.31 mmol). The mixture was heated under nitrogen and refluxed for 2 hours and then filtered. The filtrate was purified by silica gel column chromatography (hexane / CH2Cl2, 3:7) to give the white solid product compound S2 (1.3 g, 72%).

[0053] (3) Synthesis of compound S3: 8.0 g (40.0 mmol) of pyrene and 200 mL of tert-butyl chloride solution were added to 8.0 g (60.0 mmol) of powdered aluminum trichloride at 0 °C. After stirring the reaction mixture at room temperature for 3 hours, it was poured into a large volume of ice / water mixture and extracted with CH2Cl2 (twice, 250 mL each time). The combined CH2Cl2 extracts were washed with water (twice, 200 mL each time) and dried over magnesium sulfate. The solvent was evaporated under vacuum to leave the residue, which was then washed with alcohol to give a colorless solid, compound S3. The yield was 78%.

[0054] (4) Synthesis of compound S4: Compound S3 (1.6 mmol) was prepared and dissolved in a solution of CH2Cl2 (6.4 mL) and CH3CN (6.4 mL). Sodium periodate (NaIO4) (2.8 g, 13.086 mmol), H2O (8 mL), and RuCl3·xH2O (0.04 g, 0.192 mmol) were added to the solution. The dark brown suspension was heated overnight at 30–40 °C. The reaction mixture was poured into 500 mL of water and extracted with CH2Cl2. The combined extracts were washed with water and brine, dried over anhydrous magnesium sulfate, and the solvent was evaporated under vacuum. The crude product was subjected to column chromatography to obtain the desired compound S4, bright orange crystals, in a yield of 40%.

[0055] (5) Synthesis of compound S5: Iron pentacarbonyl (0.92 mL, 6.83 mmol) was added to a reflux mixture containing 4-tert-butylbenzyl bromide (2.414 g, 13.14 mmol), sodium hydroxide (2.27 g), benzyltriethylammonium chloride (0.1 g), deionized water (1.3 mL), and dichloromethane (31.5 mL). The reaction mixture was refluxed overnight with vigorous stirring and extracted twice with CH2Cl2 (250 mL each time). The combined CH2Cl2 extracts were washed with water twice (200 mL each time), dried over magnesium sulfate, and the solvent was evaporated under vacuum. The resulting solid was purified by silica gel column chromatography using a mixture of dichloromethane and petroleum ether (3:1) as the extractant to give the desired compound S5. The yield was 65%.

[0056] Synthesis of compound S6: Under argon atmosphere, DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) (0.18 mL, 1.21 mmol) was added dropwise to a suspension of compound S4 (118 mg, 0.32 mmol) and compound S5 (204 mg, 0.68 mmol) (65 mL) dissolved in anhydrous ethanol. The mixture was stirred at 78 °C for 40 min. The solid formed was collected by vacuum filtration, washed twice with cold ethanol (2 mL each time), and dried under vacuum to give a pale green solid, compound S6. The yield was 70%.

[0057] Synthesis of compound nanoG: Compound S6 (500 mg, 0.528 mmol) and compound S2 (250 mg, 1.05 mmol) were added to a dry Schlenk flask equipped with a magnetic stir bar, and 2 mL of diphenyl ether was added. The reaction mixture was refluxed at 260 °C for 6 hours. The crude product was subjected to column chromatography (using dichloromethane / hexane = 2:3 as the extractant) to obtain compound nanoG. The yield of nanoG was 38%. The nanographene of this invention is modified with cyanide.

[0058] 2. Preparation method of PLGA polymer vesicles embedded with nanoG

[0059] PLGA polymer vesicles embedded with nanoG can be prepared using either a single-emulsion method or a double-emulsion method.

[0060] Preparation method of single emulsion

[0061] First, an oil phase solution containing 0.8 mL dichloromethane, 2 mg nanoG, and 0.8 mg PLGA was prepared. 40 mg polyvinyl alcohol (PVA) was dissolved in 4 mL deionized water to obtain an aqueous phase solution. Then, the oil phase solution was slowly added dropwise to the aqueous phase solution, and the mixture was ultrasonically treated in an ice bath for 1 hour to form nanoG-loaded water-in-oil PLGA microspheres. The resulting product was aliquoted into centrifuge tubes and centrifuged (4000 rpm, 4 °C, 1 min). The precipitate was dispersed with deionized water. The above collection and washing process was repeated at least three times to obtain purified polymer particles.

[0062] Preparation method of double emulsification

[0063] First, an oil phase solution containing 4 mL of dichloromethane and 40 mg of PLGA was prepared. 8 mg of PVA was dissolved in 0.8 mL of deionized water to obtain an aqueous phase solution. For the first emulsification, the aqueous phase solution was slowly added dropwise to the oil phase solution, and the mixture was ultrasonically treated in an ice bath for 1 hour to form water-in-oil PLGA microspheres. Next, 500 mL of nanoG (2 mg) dissolved in dichloromethane was added to the first emulsified solution. This solution was then transferred to another 12 mL aqueous phase solution containing 120 mg of PVA for a second emulsification, and stirred in an ice bath using a homogenizer for 20 minutes to form water-in-oil PLGA polymer vesicles embedded with nanoG. The resulting products were then centrifuged separately in tubes (1000 rpm, 4 °C, 1 min). The precipitate was dispersed using deionized water. These collection and washing processes were performed at least three times to obtain purified polymer vesicles.

[0064] This invention uses nanoG-embedded PLGA polymer vesicles prepared by a double emulsification method for subsequent testing experiments. Plain PLGA polymer vesicles are also obtained using the same double emulsification method, but without the addition of nanoG.

[0065] Liquid phase bacterial detection

[0066] 1500 μL of nanoG-embedded PLGA polymer vesicles were mixed with 500 μL of bacterial sample solutions containing different concentrations of methicillin-resistant Staphylococcus aureus (MRSA) or Escherichia coli (MRSA: 6.7 × 10⁻⁶).3 6.7×10 4 6.7×10 5 6.7×10 6 6.7×10 7 1.7×10 8 3.4×10 8 5.0×10 8 6.7×10 8 CFU / mL; Escherichia coli was 1.2 × 10⁻⁶. 4 1.2×10 5 1.2×10 6 1.2×10 7 1.2×10 8 3.0×10 8 6.0×10 8 9.0×10 8 and 1.0×10 9 The solutions (CFU / mL) were incubated at 37°C for 1, 3, 5, 7, or 24 hours. The control group was prepared similarly, but without the addition of bacteria. The emission spectra of the resulting solutions were then measured directly using a fluorescence spectrometer.

[0067] Fabrication of AIE thin-film sensors and visualization of bacterial detection

[0068] 10 μL of deionized water containing polymer vesicle samples was dropped onto a glass slide and allowed to dry at room temperature to form a circular PLGA film on the slide. To detect bacteria using this film sensor, 10 μL of bacterial samples were dropped directly onto the AIE film and incubated for specific times (1, 3, 5, or 24 hours). The sensor's luminescence was triggered by direct UV irradiation using a handheld UV lamp (BD-TECK INSTRUMENTS, BD405CAF). A high-pressure autoclave was used to detect a concentration of 6.7 × 10⁻⁶. 8 CFU / mL and 1.2×10 9 Sterilize the MRSA solution and E. coli solution at CFU / mL to obtain dead cell samples.

[0069] Evaluation of specific MRSA detection

[0070] Equal amounts of MRSA and Escherichia coli were mixed at 2.7 × 10⁻⁶. 8 and 4.8×10 8CFU / mL was mixed to prepare a two-in-one bacterial solution. 500 μL of kanamycin solution (4.5 μg / mL) was added to this bacterial solution to kill kanamycin-sensitive *E. coli* while preserving kanamycin-resistant *MRSA*. The resulting 1000 μL solution was then directly mixed with 1000 μL of nanoG-embedded PLGA polymer vesicles and incubated for 1 hour, followed by emission spectroscopy measurements and evaluation. Separately, 10 μL of the bacterial solution was dropped onto the AIE thin-film sensor and incubated for 1 hour, followed by visual evaluation.

[0071] Antibiotic screening test

[0072] NanoG-embedded PLGA polymer vesicles in deionized water were pre-dropped into the wells of a 96-well plate, and the kit was prepared by both natural drying and freeze-drying. Both methods were expected to form probe films within the wells, dividing the plate into control and test areas (Figure 8). Subsequently, specimens from various clinically acquired infection symptoms were used for further testing, including blood, serum, urine, saliva, and wound samples. Specimens were directly dropped into the positive control well in the control area, while phosphate-buffered saline (PBS) solution was added to the negative control well. Simultaneously, in the test wells, different pre-tested antibiotics were sequentially added in addition to the specimen solution. After 1 hour of incubation at room temperature, the 96-well plate was directly irradiated with a handheld UV lamp, and the results were recorded by photography, or fluorescence intensity values ​​were obtained using a fluorescence spectrometer for statistical analysis.

[0073] Experimental results

[0074] As shown in Figure 1, this invention prepares hydrophobic cyanide-modified nanographene (nanoG) with excellent AIE (autocorrelation effect). This invention further embeds nanoG into the hydrophobic shell interface of a microbially degradable PLGA polymer using a double emulsification method (as shown in Figure 2). The double emulsification process is as follows: First, an oil phase solution containing dichloromethane and PLGA and an aqueous PVA solution are prepared. During the first emulsification, the aqueous PVA solution is slowly added dropwise to the oil phase solution, and the mixture is ultrasonically treated in an ice bath for 1 hour to form water-in-oil PLGA microspheres. At this point, the main shell component is the PLGA polymer, and PVA can adhere to the hydrophilic interface of the PLGA polymer shell to increase shell stability. Then, a dichloromethane solution containing nanoG is added to the first emulsion, and the entire solution is transferred to another aqueous PVA solution. The mixture is then stirred using a homogenizer in an ice bath for a second emulsification, thereby forming water-in-oil-in-water PLGA polymer vesicles embedded with nanoG. At this point, some PVA also adheres to the surface of the polymer vesicles, increasing shell stability and ultimately forming this novel AIE polymer material (physically sandwiching AIE-enabled nanoG between PLGA polymer shells). Interestingly, compared to nanoG aggregation induced by organic solvents (dichloromethane), the nanoG within the shells of these PLGA polymer vesicles exhibits a tightly aggregated state, thus demonstrating a more significant AIE effect, and its fluorescence brightness can be easily interpreted with the naked eye (as shown in Figure 3). As shown in Figure 4, the spherical morphology of these nanoG-embedded PLGA polymer vesicles can be observed under an electron microscope, and further observation using a fluorescence microscope reveals that the AIE-effect graphene nanoparticles are indeed present within the shells of each polymer vesicle (the fluorescence image shows a blue ring-like morphology, indicating the presence of graphene nanoparticles within the spherical shells).

[0075] This invention tests the efficacy of nanoG-embedded PLGA polymer vesicles in detecting bacteria. As shown in Figure 5, embedding nanoG into the hydrophobic shell of a PLGA polymer to form nanoG-embedded PLGA polymer vesicles effectively enhances the inherent AIE effect of nanoG. Due to the rapid degradation of PLGA polymers by live bacteria, the aggregated nanoG can be released from the PLGA polymer shell, leading to a decrease in the AIE effect. Therefore, the AIE effect decreases with the number of bacteria. Furthermore, the change in fluorescence intensity emitted by this AIE effect is visible to the naked eye. Based on the principle of the AIE effect, these nanoG-embedded PLGA polymer vesicles have great potential in bacterial detection and related applications.

[0076] This invention selects one Gram-negative bacterium (Escherichia coli) and one Gram-positive bacterium (MRSA), and mixes nanoG-embedded PLGA polymer vesicles with the bacteria. Changes in fluorescence intensity are measured using a fluorescence spectrometer. As shown in Figures 6A to 6D, at 37°C, with fixed concentrations of MRSA and E. coli, after culturing the nanoG-embedded PLGA polymer vesicles for different times (1, 3, 5, 7, and 24 hours), a decrease in fluorescence signal can be observed. This experimental result further confirms the design principle of this invention, namely, the shell of the nanoG-embedded PLGA polymer vesicles is degraded by bacteria, thereby releasing the nanoG and reducing the AIE effect of nanoG. It is noteworthy that the most significant fluorescence intensity decay is observed within a 1-hour incubation period, implying that the measurement time for bacterial detection using this invention can be effectively controlled within 1 hour. Therefore, the present invention also mixes PLGA polymer vesicles embedded with nanoG with bacterial sample solutions containing different concentrations of MRSA and Escherichia coli and lets them stand for 1 hour to obtain a fluorescence quenching phenomenon that is bacterial concentration-dependent (as shown in Figures 6E to 6H).

[0077] Although the degradation of PLGA polymer vesicles lacks bacterial selectivity, making it impossible to achieve selective detection directly by culturing bacteria with nanoG-embedded PLGA polymer vesicles, this invention achieves excellent selectivity for specific bacteria (MRSA in this test) by pre-treating the bacterial mixture with a suitable antibiotic. As shown in Figures 7A and 7B, the antibiotic kanamycin is added to a solution containing MRSA and E. coli. It is known that kanamycin can kill E. coli, but it is ineffective against MRSA. Therefore, after this pre-treatment, only MRSA remains in the solution. At this point, the AIE probe prepared from nanoG-embedded PLGA polymer vesicles will only be affected by MRSA and degrade, resulting in fluorescence attenuation only influenced by the concentration of MRSA, thus achieving selective detection. This AIE probe offers advantages such as ease of operation, low cost, visually observable fluorescence changes, and rapid detection, making it suitable as a novel pathogen sensor. Furthermore, its non-invasive detection applications can be further developed.

[0078] Taking the screening of antibiotics to treat unknown infections as an example, AIE probes prepared from nanoG-embedded PLGA polymer vesicles can be integrated onto rapid screening strips (or 96-well plates) to screen suitable antibiotics. As shown in Figure 8, physiological saline and sample solution are added to the control area of ​​the 96-well plate as negative and positive control groups, respectively. The positive control group contains bacteria, which will cause the fluorescence emitted by the AIE probe to decrease. In the test area, sample solution and the test antibiotics a, b, c, and d can be added to the corresponding areas. Under this experimental design, the probes in the negative control group's wells will show AIE luminescence (as a reference for sterility), while the probes in the positive control group's wells should show AIE quenching (indicating the presence of bacteria). If the fluorescence intensity of the positive and negative control groups is the same, it means that the sample does not contain bacteria. On the other hand, in the various antibiotic groups in the test area, if the fluorescence attenuation of that antibiotic group is similar to that of the positive control group or if fluorescence quenching occurs, it means that the antibiotic is ineffective. However, if the fluorescence attenuation of this antibiotic group is inhibited compared to the positive control group, it indicates that the antibiotic can still kill some bacteria in the sample, thus indicating its effectiveness. Furthermore, if the antibiotic group emits the same fluorescence intensity as the negative control group, it indicates that this antibiotic is the optimal choice, meaning it can effectively eliminate all bacteria and maintain the probe's AIE effect. As shown in Figure 8, the fluorescence intensity of antibiotic groups a, c, and d is the same as the positive control group, indicating that antibiotics a, c, and d are ineffective. However, the fluorescence intensity of antibiotic group b is the same as the negative control group, indicating that this antibiotic can effectively kill the bacteria in the sample.

[0079] Therefore, by observing the antibiotic groups that do not show fluorescence decay through the above screening method, the most effective antibiotic can be identified. Although this method cannot identify the type of bacteria, it can identify the most suitable antibiotic within a one-hour testing period. This method of rapidly screening for effective antibiotics will allow patients' infection symptoms to be effectively relieved and controlled by appropriate antibiotic treatment in a short time, thus achieving the goal of rapid and accurate medication.

[0080] The present invention may be practiced under any requirements or limitations not specifically disclosed herein. The terminology used in the description is not intended to be limiting. There is no difference in expression and description using these terms and any equivalents thereof, but it should be understood that modifications are possible within the scope of the invention. Therefore, while embodiments and other aspects of the invention have been described, the content disclosed herein can be modified and varied by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention.

Claims

1. A composition comprising: an outer shell layer, wherein the outer shell layer comprises a microbially degradable polymer and a plurality of molecules having aggregation-induced emission of light.

2. The composition of claim 1, wherein the outer shell layer comprises a first shell layer, an intermediate layer, and a second shell layer, and the intermediate layer is positioned between the first shell layer and the second shell layer.

3. The composition of claim 2, wherein the first shell layer and the second shell layer comprise the microbially degradable polymer, and the intermediate layer comprises the plurality of molecules having aggregation-induced emission of light.

4. The composition of claim 1, wherein the microbially degradable polymer comprises polylactic-co-glycolic acid (PLGA), polycaprolactone (PCL), polylactic acid (PLA), polybutylene succinate (PBS), a PBS-based amphiphilic copolymer, polyethylene glycol-PLGA (PEG-PLGA), PLA-PEG, galactose-PLA, poly(oxa-cyclohexadecene-2-ketone-co-caprolactone) (PGlCL), polyhydroxyalkanoate (PHAs), polyaspartic acid, cellulose, galactomannan, alginate, chitosan, or starch.

5. The composition of claim 1, wherein the molecules having aggregation-induced emission of light are hydrophobic.

6. A method for detecting the presence of bacteria, comprising: (1) providing the composition of claim 1; (2) contacting a sample with the composition; (3) observing the light emission intensity of the composition in step (2); and (4) comparing the light emission intensity of the composition in step (3) with that of the composition not contacted with the sample, wherein a decrease in the light emission intensity of the composition in step (3) indicates the presence of bacteria in the sample.

7. The method of claim 6, wherein the light emission of the composition is induced by ultraviolet light.

8. A kit for detecting bacteria, comprising the composition of claim 1.

9. A method for screening antibiotics, comprising: (a) contacting a sample containing at least one bacteria with the composition of claim 1 to form a mixture; (b) adding a test antibiotic to the mixture of step (a); (c) observing the light emission intensity of the composition in step (b); and (d) comparing the light emission intensity of the composition in step (c) with that of a control group, wherein the presence of the at least one bacteria in the sample causes a decrease in the light emission intensity of the composition; thus, if the light emission intensity of the composition is higher than that of the control group, it indicates that the test antibiotic is effective against the at least one bacteria in the sample, wherein the control group is the composition contacted with the sample but without the addition of the test antibiotic.

10. The method of claim 9, wherein the bacteria comprise methicillin-resistant Staphylococcus aureus or Escherichia coli.

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