Recyclable cationic micro-nano photosensitizer, preparation method and application

Abstract

The invention relates to the technical field of photodynamic sterilization, in particular to a recyclable cationic micro-nano photosensitizer and a preparation method and application thereof.The recyclable cationic micro-nano photosensitizer comprises a magnetic core, the surface of the magnetic core is coated with a shell layer, the shell layer is composed of a silane coupling agent conjugated with photosensitizer molecules and quaternary ammonium molecules, and a stable inorganic-organic hybrid shell layer is formed; the photosensitizer and quaternary ammonium molecules are firmly embedded into the shell layer in a covalent mode, and release or falling of the photosensitizer and the quaternary ammonium molecules in subsequent application is effectively avoided. The arrangement of the magnetic nucleus endows the recoverable cationic micro-nano photosensitizer with external magnetic field response capability, the recoverable cationic micro-nano photosensitizer can be rapidly recovered from a reaction system through a simple magnetic separation technology, and the recovery method is simple and high in efficiency. The quaternary ammonium molecules endow the recoverable cation micro-nano photosensitizer with stable electropositivity in a wide pH value range, the electrostatic adsorption capacity of the micro-nano photosensitizer and the bacterial surface is remarkably enhanced, the sterilization effect is enhanced, and the problem that in the prior art, a photosensitizer is difficult to recover in liquid food is solved.

Description

Technical Field

[0001] This invention relates to the field of photodynamic sterilization technology, specifically to a recyclable cationic micro / nano photosensitizer, its preparation method, and its application. Background Technology

[0002] Foodborne pathogens (such as *S. aureus*, *E. coli*, *L. monocytogenes*, and *S. typhimurium*) are major culprits in liquid food contamination and foodborne illnesses. Photodynamic inactivation (PDI) is a non-thermal physical sterilization technology that utilizes the interaction of photosensitizers, oxygen, and light of specific wavelengths to generate reactive oxygen species (ROS), thereby killing microorganisms. It boasts advantages such as being environmentally friendly, gentle in operation, controllable in process, not reliant on expensive equipment, and having minimal impact on food quality, and has been widely applied in the sterilization of solid foods. However, because most photosensitizers possess a superhydrophobic, surface-aroma structure, they have poor water solubility. In liquid foods, insufficiently water-soluble photosensitizers are difficult to disperse uniformly, affecting sterilization effectiveness. Furthermore, photosensitizers are difficult to completely remove from liquid foods, posing a high risk of residue and making separation and recovery from food challenging. Residual photosensitizers not only adversely affect food safety and sensory quality but also increase sterilization costs, thus limiting their application in liquid foods.

[0003] Currently, some studies have attempted to improve the water solubility of photosensitizers by encapsulating them with cyclodextrin and to assist magnetic recovery through the biotin-streptavidin system. However, this approach has limited effectiveness in inactivating Gram-negative bacteria. Furthermore, while cationic micro and nano carriers have demonstrated good affinity for Gram-negative bacteria in biomedical PDIs, there has been no systematic research on cationic micro and nano photosensitizers in the food field, especially in liquid foods.

[0004] Nanotechnology can solve the problem of water dispersibility of photosensitizers and improve the killing efficiency against Gram-negative bacteria by combining cationic surface modification. However, the problems of residues after sterilization in liquid food and reuse have not yet been solved, resulting in safety risks and high application costs. Summary of the Invention

[0005] To address the problem of difficulty in recovering photosensitizers from liquid foods in existing technologies, this invention provides a recyclable cationic micro / nano photosensitizer, its preparation method, and its application.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] The present invention provides a recyclable cationic micro / nano photosensitizer, comprising a magnetic core, wherein the surface of the magnetic core is coated with a shell layer, the shell layer being composed of a silane coupling agent conjugated with photosensitizer molecules and quaternary ammonium molecules.

[0008] Optionally, the magnetic core is iron(III) oxide nanoparticles or microparticles.

[0009] Optionally, the photosensitive molecule includes one or more of dihydroporphyrin e6, curcumin, Bengal rose red, mercaptoporphyrin, IR-783 near-infrared dye derivative, protoporphyrin IX, and riboflavin.

[0010] Optionally, the quaternary ammonium molecule includes one or more of N-(3-trimethoxysilylpropyl)-N,N,N-trimethylammonium chloride, N-(3-triethoxysilylpropyl)-N,N,N-trimethylammonium chloride, (3-aminopropyl)trimethylammonium chloride, N-(2-hydroxyethyl)-N,N,N-trimethylammonium chloride, 2,3-epoxypropyltrimethylammonium chloride, (3-carboxypropyl)trimethylammonium chloride, N-(carboxymethyl)-N,N,N-trimethylammonium chloride, N-(mercaptoethyl)-N,N,N-trimethylammonium chloride, N-(acryloyloxyethyl)-N,N,N-trimethylammonium chloride, and 2-methacryloyloxyethyltrimethylammonium chloride.

[0011] Optionally, the silanized quaternary ammonium molecule is N-(3-trimethoxysilylpropyl)-N,N,N-trimethylammonium chloride, N-(3-triethoxysilylpropyl)-N,N,N-trimethylammonium chloride, or a product covalently bonded to a quaternary ammonium molecule by a silane coupling agent.

[0012] Optionally, the silane coupling agent includes one or more of 3-aminopropyltriethoxysilane, 3-mercaptopropyltrimethoxysilane, 3-isocyanatepropyltriethoxysilane, and 3-glycidoxypropyltrimethoxysilane.

[0013] This invention provides a method for preparing a recyclable cationic micro / nano photosensitizer as described above, comprising:

[0014] Preparation of silanized photosensitive molecules and silanized quaternary ammonium molecules;

[0015] A magnetic core dispersion is prepared, and silanized photosensitizing molecules, silanized quaternary ammonium molecules, and a catalyst are added to the magnetic core dispersion to react and obtain a recyclable cationic micro / nano photosensitizer; wherein the catalyst is ammonia, NaOH, or KOH.

[0016] Optionally, the solvent of the magnetic nucleus dispersion is an organic mixed solution, and the concentration of magnetic nuclei in the magnetic nucleus dispersion is 0.1–200 mg / mL;

[0017] The organic mixed solution is an aqueous solution of ethanol, an aqueous solution of methanol, an aqueous solution of methanol and ethanol, or a mixture of alkane solvent and nonionic surfactant. The alkane solvent includes one of cyclohexane, isooctane, n-hexane, paraffin oil, and octane. The nonionic surfactant is one or more of Igepal CO-520, Riton X-100, Brij 30, Span 80, and Tween 80. The molar ratio of silanized quaternary ammonium molecules to silanized photosensitive molecules added to the magnetic core dispersion is 1:10 to 200:1, and the total molar concentration of silanized quaternary ammonium molecules and silanized photosensitive molecules is 1 to 200 mM.

[0018] Optionally, before adding silanized photosensitive molecules, silanized quaternary ammonium molecules and catalyst to the magnetic core dispersion, tetramethyl silicate or tetraethyl silicate is also added to the magnetic core dispersion, and the reaction is carried out for 0.5 to 24 hours to generate a pre-prepared silica layer on the surface of the magnetic core.

[0019] Such as the application of recyclable cationic micro / nano photosensitizers in the sterilization of liquid food.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention discloses a recyclable cationic micro / nano photosensitizer, comprising a magnetic core coated with a shell. The shell is composed of a silane coupling agent conjugated with photosensitizer molecules and quaternary ammonium molecules, forming a stable inorganic-organic hybrid shell. This ensures the photosensitizer and quaternary ammonium molecules are firmly embedded in the shell via covalent bonding, effectively preventing release or detachment during subsequent applications. Simultaneously, the shell environment enhances the photosensitizer's stability to external stimuli such as light and heat. The magnetic core endows the recyclable cationic micro / nano photosensitizer with external magnetic field responsiveness, allowing for rapid recovery from the reaction system using simple magnetic separation technology. The recovery method is simple, efficient, and yields a high recovery rate. The quaternary ammonium molecules impart stable positive charge to the recyclable cationic micro / nano photosensitizer over a wide pH range, enabling efficient adsorption of negatively charged target substances via electrostatic interactions. This significantly improves the photosensitizer's targeted enrichment efficiency in complex systems and enhances its electrostatic adsorption capacity with bacterial surfaces. Consequently, it improves the effectiveness of reactive oxygen species (ROS) generated during photodynamic sterilization, thus strengthening the sterilization effect. Therefore, the recyclable cationic micro / nano photosensitizer of the present invention, through the synergistic design of magnetic core and functional molecules, has high recyclability, targeted adsorption and reusability. When used in liquid food, it can not only play a better sterilization role, but also be easy to separate, recycle and leave no residue, thus having significant technical and economic value.

[0022] The magnetic core is made of iron oxide nanoparticles or microparticles. The iron oxide nanoparticles or microparticles exhibit superparamagnetism at room temperature, that is, they have no remanence when there is no external magnetic field, thus avoiding aggregation; when a magnetic field is applied, they can be magnetized instantly, which is beneficial for the rapid separation of photosensitizers.

[0023] The silanized quaternary ammonium molecule is N-(3-trimethoxysilylpropyl)-N,N,N-trimethylammonium chloride and / or N-(3-triethoxysilylpropyl)-N,N,N-trimethylammonium chloride. In use, it can be directly covalently modified with the surface of the silica layer without the need for silanization treatment by a silane coupling agent, which can further simplify the preparation process of recyclable cationic micro / nano photosensitizer.

[0024] The silane coupling agent includes one or more of 3-aminopropyltriethoxysilane, 3-mercaptopropyltrimethoxysilane, 3-isocyanatepropyltriethoxysilane, and 3-glycidyl etheroxypropyltrimethoxysilane. The molecular structure of the silane coupling agent contains both hydrolyzable groups and organic functional groups. The hydrolyzable groups form stable chemical bonds with the silica layer surface; the organic functional groups bind to the photosensitizer molecules through covalent or hydrogen bonds, thereby constructing a "molecular bridge" at the inorganic-organic interface. This significantly enhances interfacial bonding, effectively prevents the photosensitizer from detaching during the recovery process, and avoids adverse effects on food safety and sensory quality due to photosensitizer residue.

[0025] A method for preparing a recyclable cationic micro / nano photosensitizer, as described above, involves preparing silanized photosensitizing molecules and silanized quaternary ammonium molecules; preparing a magnetic core dispersion; and adding the silanized photosensitizing molecules, silanized quaternary ammonium molecules, and a catalyst to the magnetic core dispersion for reaction, thereby obtaining the recyclable cationic micro / nano photosensitizer. In this preparation method, the introduction of a magnetic core endows the recyclable cationic micro / nano photosensitizer with superparamagnetism, enabling rapid recovery under an applied magnetic field. The silanized photosensitizer and silanized quaternary ammonium molecules undergo a hydrolysis-condensation reaction with a silane coupling agent, forming a stable inorganic-organic hybrid shell in situ on the surface of the magnetic core. This structural design ensures that the photosensitizer and quaternary ammonium molecules are firmly embedded in the shell through covalent bonding, effectively preventing their release or detachment during subsequent applications. Simultaneously, the shell environment enhances the stability of the photosensitizer to external stimuli such as light and heat. The positively charged quaternary ammonium molecules can rapidly adsorb negatively charged bacterial cell walls, synergistically achieving a dual antibacterial effect of "physical destruction + chemical killing" through the reactive oxygen species generated by the photosensitizer. This preparation method, through synergistic innovation in core-shell structure design, silanization modification, and alkaline condition control, achieves efficient loading of the photosensitizer, stable recovery of magnetic materials, and synergistic optimization of broad-spectrum antibacterial performance, providing a high-performance, recyclable solution for liquid food sterilization, medical disinfection, and environmental remediation.

[0026] Before adding silanized photosensitizing molecules, silanized quaternary ammonium molecules, and catalysts to the magnetic core dispersion, tetramethyl silicate or tetraethyl silicate is also added to the magnetic core dispersion. The reaction is carried out for 0.5–24 h, forming a pre-prepared silica layer on the surface of the magnetic core. The surface of the pre-prepared silica layer is rich in hydroxyl groups, providing active sites for subsequent silanization modification. This ensures that the photosensitizer and quaternary ammonium molecules are stably bound by covalent bonds, avoiding the problem of desorption caused by physical adsorption. At the same time, the pre-prepared silica layer, as a stable carrier, has excellent chemical inertness, which can protect the magnetic core from the corrosion of reactive oxygen species generated by the photosensitizer, avoid the self-quenching effect caused by direct contact between the photosensitizer and the matrix, ensure that the photosensitizer maintains high efficiency after multiple cycles of use, and ensure the long-term safety of the material.

[0027] The above-mentioned application of recyclable cationic micro / nano photosensitizers in the sterilization of liquid foods exemplifies this. Under light irradiation, these recyclable cationic micro / nano photosensitizers generate singlet oxygen, directly disrupting bacterial cell membranes and DNA. The cationic quaternary ammonium groups penetrate the cell wall through electrostatic adsorption. This dual action effectively enhances sterilization efficiency and offers advantages such as good safety, excellent recyclability, and reusability. This provides the liquid food industry with an efficient, safe, environmentally friendly, and economical sterilization solution, possessing broad market application prospects. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the preparation method of a recyclable cationic micro / nano photosensitizer according to the present invention.

[0029] Figure 2 This is a synthetic route diagram of the silanized photosensitive molecule Ce6-APTES and the silanized quaternary ammonium molecule EPTAC-APTES in Example 2 of the present invention.

[0030] Figure 3 This is a schematic diagram of the preparation process of the recyclable cationic nanophotosensitizer CMNP in Example 2 of the present invention, as well as the structure of the surface photosensitizer molecule Ce6 and the quaternary ammonium molecule.

[0031] Figure 4The figures shown are test results of the recyclable cationic nanophotosensitive agent CMNP in Example 2 of the present invention. A is a transmission electron microscope image of the recyclable cationic nanophotosensitive agent CMNP; B is a comparison of the photodynamic efficiency of the recyclable cationic nanophotosensitive agent CMNP and Ce6; C is the hysteresis curve of the recyclable cationic nanophotosensitive agent CMNP; D is the particle size distribution of the newly prepared recyclable cationic nanophotosensitive agent CMNP; E is the particle size distribution of the recyclable cationic nanophotosensitive agent CMNP after standing for one week; F is the surface zeta potential data of the newly prepared and after standing for one week recyclable cationic nanophotosensitive agent CMNP; G is the absorption spectrum of the newly prepared recyclable cationic nanophotosensitive agent CMNP; and H is the absorption spectrum of the recyclable cationic nanophotosensitive agent CMNP after standing for one week.

[0032] Figure 5 The graph shows the photodynamic killing effect of the recyclable cationic photosensitizer CMNP prepared in Example 2 on typical foodborne pathogens in drinking water. In Figure A, CMNP (Ce6 equivalent concentration of 5 μg / mL) shows the change in photodynamic sterilization efficiency against Escherichia coli in drinking water at different light doses. Figure B shows the effect at 30 J / cm³. 2 The photodynamic sterilization efficiency of CMNP at different Ce6 equivalent concentrations against Escherichia coli in drinking water was observed under varying light doses. C represents the photodynamic sterilization efficiency of CMNP (Ce6 equivalent concentration of 5 μg / mL) against Salmonella in drinking water at different light doses. D represents the photodynamic sterilization efficiency at 30 J / cm². 2 The photodynamic sterilization efficiency of CMNP at different Ce6 equivalent concentrations on Salmonella in drinking water under different light dose conditions, where E represents the photodynamic sterilization efficiency of CMNP at a Ce6 equivalent concentration of 5 μg / mL at 30 J / cm². 2 Photodynamic sterilization efficiency of Staphylococcus aureus and Listeria monocytogenes in drinking water under light dose conditions.

[0033] Figure 6 The graph shows the sterilization test results of the recyclable cationic photosensitizer CMNP prepared in Example 2 in apple juice. A represents the change in photodynamic sterilization efficiency of CMNP (Ce6 equivalent concentration of 10 μg / mL) against Escherichia coli in clear apple juice without light dose; B represents the change in photodynamic sterilization efficiency of CMNP (Ce6 equivalent concentration of 10 μg / mL) against Salmonella in clear apple juice without light dose; and C represents the sterilization efficiency of CMNP with a Ce6 equivalent concentration of 10 μg / mL at 90 J / cm². 2 Photodynamic sterilization efficiency of Staphylococcus aureus and Listeria monocytogenes in clear apple juice under light dose conditions.

[0034] Figure 7The image shows the magnetic adsorption test results of the recyclable cationic nanophotosensitizer CMNP prepared in Example 2 in clarified apple juice. In the image, A is the iron content in the clarified apple juice at 2, 4 and 6 h of magnetic adsorption. The dashed line represents the iron content in the untreated apple juice. B is a photograph of the untreated clarified apple juice after adding CMNP and after 6 h of magnetic adsorption.

[0035] Figure 8 The image shows the antibacterial test results of the recyclable cationic photosensitizer CMNP prepared in Example 2, where A represents CMNP with a Ce6 equivalent concentration of 10 μg / mL at 90 J / cm². 2 The image shows the sterilization efficiency of five repeated photodynamic sterilizations of clarified apple juice containing Escherichia coli under light dose conditions. b represents the sterilization efficiency of CMNP at a Ce6 equivalent concentration of 10 μg / mL at 90 J / cm². 2 Under light dose conditions, the sterilization efficiency of each of the five repeated photodynamic sterilizations of clarified apple juice containing Listeria monocytogenes is shown in the graph. C shows the particle size distribution of CMNPs recovered after the fifth cycle (compared with freshly prepared CMNPs). D shows the surface potential of CMNPs recovered after the fifth cycle (compared with freshly prepared CMNPs). E shows the iron content in apple juice after the fifth photodynamic sterilization and magnetic recovery of CMNPs in clarified apple juice, compared with the iron content in untreated apple juice. Detailed Implementation

[0036] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0037] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0038] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0039] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0040] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0041] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0042] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications conventional in the art. In this specification and the following examples, unless otherwise specified, "%" refers to weight percentage, "parts" refers to parts by weight, and "ratio" refers to weight proportion.

[0043] The present invention will be further described in detail below with reference to specific embodiments. These descriptions are for explanation purposes only and are not intended to limit the scope of the invention.

[0044] This invention discloses a recyclable cationic micro / nano photosensitizer, comprising a magnetic core, the surface of which is coated with a shell, the shell being composed of a silane coupling agent conjugated with photosensitizer molecules and quaternary ammonium molecules.

[0045] The magnetic core is made of iron(III) oxide nanoparticles or microparticles.

[0046] The photosensitizer includes one or more of dihydroporphyrin e6 (Ce6), curcumin (Cur), Bengal rose red (RB), mercaptoporphyrin (Por), IR-783 near-infrared dye derivative, protoporphyrin IX, and riboflavin (RF).

[0047] The quaternary ammonium molecule includes one or more of the following: N-(3-trimethoxysilylpropyl)-N,N,N-trimethylammonium chloride, N-(3-triethoxysilylpropyl)-N,N,N-trimethylammonium chloride, (3-aminopropyl)trimethylammonium chloride, N-(2-hydroxyethyl)-N,N,N-trimethylammonium chloride, 2,3-epoxypropyltrimethylammonium chloride, (3-carboxypropyl)trimethylammonium chloride, N-(carboxymethyl)-N,N,N-trimethylammonium chloride, N-(mercaptoethyl)-N,N,N-trimethylammonium chloride, N-(acryloyloxyethyl)-N,N,N-trimethylammonium chloride, and 2-methacryloyloxyethyltrimethylammonium chloride.

[0048] The silane coupling agent includes one or more of 3-aminopropyltriethoxysilane, 3-mercaptopropyltrimethoxysilane, 3-isocyanatepropyltriethoxysilane, and 3-glycidoxypropyltrimethoxysilane.

[0049] When the silanized quaternary ammonium molecule is N-(3-trimethoxysilylpropyl)-N,N,N-trimethylammonium chloride, N-(3-triethoxysilylpropyl)-N,N,N-trimethylammonium chloride, or a product covalently bonded to a quaternary ammonium molecule by a silane coupling agent, it can be directly used for covalent modification of the silica layer surface without silanization by a silane coupling agent.

[0050] See Figure 1 This invention provides a method for preparing the recyclable cationic micro / nano photosensitizer as described above, comprising:

[0051] S1: Preparation of silanized photosensitive molecules and silanized quaternary ammonium molecules, specifically:

[0052] Photosensitizer molecules and quaternary ammonium compounds contain reactive groups such as carboxyl, amino, alkynyl, azide, hydroxyl, mercapto, olefin, and isothiocyanate. Optional photosensitizers include: dihydroporphyrin E6, curcumin (Cur), Bengal rose red, mercaptoporphyrin, IR-783 near-infrared dye derivatives, protoporphyrin IX, riboflavin, etc. Optional quaternary ammonium molecules include one or more of the following: N-(3-trimethoxysilylpropyl)-N,N,N-trimethylammonium chloride, N-(3-triethoxysilylpropyl)-N,N,N-trimethylammonium chloride, (3-aminopropyl)trimethylammonium chloride, N-(2-hydroxyethyl)-N,N,N-trimethylammonium chloride, 2,3-epoxypropyltrimethylammonium chloride, (3-carboxypropyl)trimethylammonium chloride, N-(carboxymethyl)-N,N,N-trimethylammonium chloride, N-(mercaptoethyl)-N,N,N-trimethylammonium chloride, N-(acryloyloxyethyl)-N,N,N-trimethylammonium chloride, and 2-methacryloyloxyethyltrimethylammonium chloride.

[0053] The photosensitizer molecules are covalently bonded through a silane coupling agent via amidation, esterification, isothiocyanate-amine coupling reaction, epoxy-amine addition reaction, epoxy-alcohol addition reaction, epoxy-thiol addition reaction, etc., to form a silanized photosensitizer. Optional silane coupling agents include one or more of the following: 3-aminopropyltriethoxysilane (APTES), 3-mercaptopropyltrimethoxysilane (MPTMS), 3-isocyanatepropyltriethoxysilane (IPTES), and 3-glycidyl etheroxypropyltrimethoxysilane (GPTMS).

[0054] When silane coupling agents are covalently combined with the aforementioned photosensitizers and quaternary ammonium molecules through amidation, esterification, isothiocyanate-amine coupling reaction, epoxy-amine addition reaction, epoxy-alcohol addition reaction, epoxy-thiol addition reaction, etc., the solvent used is an organic solvent, including: dimethyl sulfoxide (DMSO), dimethylformamide (DMF), acetonitrile, methanol, ethanol, tetrahydrofuran (THF), dichloromethane (DCM), pyridine, toluene, and isopropanol (IPA). The catalysts required for amidation and esterification reactions include: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and its hydrochloride (EDC and EDC·HCl), N,N'-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate (HBTU) or O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethylurea tetrafluoroborate (TBTU), with reaction temperatures ranging from 0℃ to 100℃. The catalysts required for the reactions of epoxy groups and hydroxyl and mercapto groups include triethylamine (TEA) and imidazole, with reaction temperatures ranging from 0℃ to 100℃.

[0055] When the above reactions of photosensitizer and silane coupling agent, and quaternary ammonium molecules and silane coupling agent are carried out, the molar ratio of photosensitizer and quaternary ammonium molecules to silane coupling agent is 1:1 to 1:3. The molar concentration of silane coupling agent in the reaction system is 0.01 to 1.0 M. The catalyst is added in catalytic equivalent. After the reaction is completed, the product is not purified. The reaction solution can be removed by vacuum distillation or directly used for the next step. The concentration of the obtained silanized photosensitizer or silanized quaternary ammonium molecule is calculated according to the concentration of photosensitizer and quaternary ammonium molecule used in the reaction.

[0056] S2: Prepare a magnetic core dispersion, and add silanized photosensitizing molecules, silanized quaternary ammonium molecules, and a catalyst to the magnetic core dispersion. The reaction yields a recyclable cationic micro / nano photosensitizer, specifically:

[0057] The magnetic nuclei are dispersed in an organic mixed solution to obtain a magnetic nuclei dispersion with a concentration of 0.1–200 mg / mL; the organic mixed solution is an aqueous solution of ethanol, an aqueous solution of methanol, or an aqueous solution of a mixture of methanol and ethanol, wherein the volume ratio of water to the organic solvent methanol or ethanol is 1:100–50:1; pure methanol, ethanol, or a mixture of the two can also be used.

[0058] Silanized photosensitizing molecules, silanized quaternary ammonium molecules, and a catalyst are added to a magnetic core dispersion. The mixture is stirred or shaken at 20°C–50°C for 1–48 hours. After stirring, the mixture is centrifuged or magnetically adsorbed to obtain a precipitate. The precipitate is washed 2–5 times with ethanol, DMF, or DMSO, and then washed 2–5 times with pure water or buffer solution to obtain a recyclable cationic micro / nano photosensitizer. In the magnetic core dispersion containing the silanized photosensitizing molecules, silanized quaternary ammonium molecules, and a catalyst, the concentration of the catalyst is 0.01–1.0 M, the molar ratio of silanized quaternary ammonium molecules to silanized photosensitizer is 1:10–200:1, and the total molar concentration of silanized quaternary ammonium molecules and silanized photosensitizing molecules is 1–200 mM.

[0059] In some optional embodiments, before adding silanized photosensitizing molecules, silanized quaternary ammonium molecules, and catalyst to the magnetic core dispersion, tetramethyl silicate or tetraethyl silicate is added to the magnetic core dispersion to generate a pre-prepared silica layer on the magnetic core surface. This reduces the amount of silanized quaternary ammonium molecules and silanized photosensitizer used, while further reducing the quenching effect of the core iron oxide particles on the photosensitizer performance. The concentration of tetramethyl silicate or tetraethyl silicate is 5–500 mM.

[0060] For magnetic nanoparticles of iron oxide prepared by thermal decomposition as the magnetic core, the preferred preparation method is the reverse microemulsion method. Specifically:

[0061] Magnetic nanoparticles of iron oxide (Fe3O4) at a concentration of 1–200 mg / mL were dispersed in cyclohexane, and 5%–30% (by volume) of Igepal CO-520, ammonia, NaOH, or KOH were added. Simultaneously, silanized quaternary ammonium molecules and a silanized photosensitizer were added. The mixture was stirred or shaken at 20–50 °C for 6–48 h. Ethanol was then added to break the emulsion, and the precipitate was obtained by centrifugation or magnetic adsorption. The precipitate was washed 2–5 times with ethanol, DMF, or DMSO, followed by 2–5 washes with pure water or buffer solution to obtain a recyclable cationic micro / nano photosensitizer.

[0062] In the above process, cyclohexane and Igepal CO-520 are preferred reagents. Cyclohexane can be replaced by isooctane, n-hexane, paraffin oil, octane, or a mixture of two or more of these solvents. Igepal CO-520 can be replaced by Riton X-100, Brij 30, Span 80, Tween 80, or a mixture of two or more of these solvents. The amount of ammonia, NaOH, or KOH added as a catalyst is 0.01–0.3 M; the molar ratio of silanized quaternary ammonium molecules to silanized photosensitizer is 1:10–200:1; and the total molar concentration of both is 1–200 mM. Similar to the sol-gel method, in some preferred embodiments, before adding silanized quaternary ammonium molecules and silanized photosensitizers, tetramethyl silicate or tetraethyl silicate (TEOS) is added first. After addition, the molar concentration of tetramethyl silicate or tetraethyl silicate (TEOS) is 5-500 mM, and the reaction is carried out under the same conditions for 6-24 h. After centrifugation or magnetic adsorption, the precipitate is obtained and then dispersed in the solvent system. Silanized quaternary ammonium molecules and silanized photosensitizers are added to continue the reaction. Alternatively, silanized quaternary ammonium molecules and silanized photosensitizers can be added directly after the reaction for 6-24 h to continue the reaction, thereby obtaining a recyclable cationic micro / nano photosensitizer.

[0063] Example 1

[0064] Under nitrogen protection, 184 mg (0.5 mmol) of Cur and 234 μL (1.0 mmol) of 3-isocyanate propyltriethoxysilane (IPTES) were added sequentially to a clean, dry 50 mL round-bottom flask. Then, 10 mL of anhydrous DMF was added to fully dissolve the reactants, followed by 3 μL of triethylamine. The mixture was stirred at 50 °C for 12 h. After the reaction was complete, the product obtained was the silanized photosensitizer Cur-IPTES (silanized photosensitizer). The product did not require further purification and was used directly in subsequent reactions.

[0065] The silanized quaternary ammonium molecule can be directly made using N-(3-triethoxysilylpropyl)-N,N,N-trimethylammonium chloride without the need for silanization.

[0066] Magnetic iron oxide nanoclusters (MNPs) were prepared using a solvothermal method to serve as magnetic cores. Specifically, FeCl3·6H2O was used to prepare the magnetic cores. 0.375g of sodium citrate, 120mg of sodium citrate, 12mL of ethylene glycol, and 0.45g of sodium acetate were placed together in a brown sample bottle. A magnetic stirrer was added, and the mixture was completely dissolved in 1-2 hours. 70mL of this solution was then added to a hydrothermal reactor and kept at 205℃ for 16 hours. After the reactor cooled to room temperature, the mixture was centrifuged and washed three times each with anhydrous ethanol, water, and anhydrous ethanol via magnetic adsorption to obtain MNPs. The MNPs dispersed in anhydrous ethanol were transferred to a clean sample bottle for quantification (quantification method: three clean 0.5mL centrifuge tubes were labeled 1, 2, and 3, weighed, and then 50µL of the MNPs from the sample bottle was added to each tube. The ethanol was then magnetically removed, and the tubes were dried at 60℃. The tubes were then weighed again, and the total mass of the tubes and nanoparticles minus the weight of the empty tube was taken as the average value, which is 50µL). The mass of the MNPs (multiplied by 20, which is the mass of each milliliter of MNPs in the sample vial) should be marked and stored at -20°C.

[0067] Take a 50 mL flask and disperse 30 mg of MNPs in a mixture of 30 mL ethanol and 10 mL water. After thorough ultrasonic dispersion, add 0.38 mL ammonia, 0.05 mmol cur-IPTES and 0.5 mmol N-(3-triethoxysilylpropyl)-N,N,N-trimethylammonium chloride. Stir at room temperature for 8 h, then perform magnetic separation and wash three times with ethanol and pure water respectively to obtain a cationic magnetic photosensitizer loaded with curcumin (a recyclable cationic micro / nano photosensitizer).

[0068] Example 2

[0069] Under nitrogen protection, 298.3 mg (0.5 mmol) of Ce6, 351 μL (1.5 mmol) of 3-aminopropyltriethoxysilane (APTES), 475 mg (3.0 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), and 345 mg (3.0 mmol) of N-hydroxysuccinic anhydride (NHS) were added sequentially to a dry, clean 50 mL round-bottom flask. Then, 10 mL of anhydrous N,N-dimethylformamide (DMF) was added to fully dissolve the reactants. The mixture was stirred at 40 °C for 24 h. After the reaction was complete, the solvent was removed by rotary evaporation. The resulting product did not require further purification and was directly used in subsequent reactions to obtain the silanized photosensitizer (silanized photosensitizer Ce6-APTES).

[0070] 242.6 mg (1.6 mmol) of 2,3-epoxypropyltrimethylammonium chloride (EPTAC) was weighed into a 10 mL round-bottom flask, and 187.2 μL (0.8 mmol) of APTES was added. Under nitrogen protection, 3 mL of anhydrous ethanol was added to dissolve the reactants, and the mixture was refluxed at 60 °C for 24 h. After the reaction was complete, the solvent was removed by rotary evaporation. The obtained product did not require further purification and was used directly for subsequent applications, yielding silanized quaternary ammonium molecules (silanized quaternary ammonium molecules EPTAC-APTES). See the synthetic route diagram below. Figure 2 .

[0071] Magnetic iron oxide nanoparticles (IONPs) were synthesized via thermal decomposition to serve as magnetic cores. The specific method is as follows: 1.0595 g of iron triacetylacetone (Fe(acac)3) and 3.876 g of 1,2-hexadecanediol were weighed, added to 2.853 mL of oleic acid and 2.907 mL of oleylamine, and dissolved in 30 mL of diphenyl ether. The solution was then transferred to a clean 100 mL round-bottom flask. Under nitrogen protection, the mixture was magnetically stirred and heated to 200 °C for 2 h, then the temperature was further increased to 300 °C and maintained for 1 h. After the reaction was complete, the mixture was allowed to cool naturally to room temperature. The resulting product was washed five times with anhydrous ethanol (using magnetic separation) and finally dispersed in cyclohexane for storage, yielding the magnetic iron oxide nanoparticles (IONPs).

[0072] 5.4 mg of IONPs were dispersed in 0.3 mL of cyclohexane, and 10 mL of cyclohexane, 1.0 mL of Gepal CO-520, and 0.3 mL of 6.5% ammonia were added. After magnetic stirring and sonication for 2 min, 70 μL of tetraethoxysilane (TEOS) was added, and the mixture was reacted at 25 °C with shaking for 24 h to obtain silica-coated IONPs (SiO2@IONPs). Subsequently, 40 μmol of Ce6-APTES and EPTAC-APTES in a molar ratio of 1:50 were added to the system, and the reaction was again carried out at 25 °C for 24 h. The emulsion was broken by adding 50 mL of anhydrous ethanol, and the precipitate was collected by magnetic separation and washed five times with anhydrous ethanol to obtain a recoverable cationic micro / nano photosensitizer (a cationic magnetic photosensitizer loaded with dihydroporphyrin e6 (Ce6), CMNP). See also Figure 3 In this embodiment, TEOS is first used to form a silica layer without photosensitizer and quaternary ammonium molecules on the surface of iron oxide nanoparticles (IONPs) through hydrolysis-condensation. Then, the same process is used to coat this layer with an organic-inorganic hybrid layer containing photosensitizer Ce6 and quaternary ammonium molecules. The intermediate silica layer can maintain a certain distance between the photosensitizer and the iron oxide core, reducing the adverse effects of IONPs on the photosensitizer molecules in the photodynamic process. Finally, the CMNP surface layer covalently binds photosensitizer Ce6 molecules and quaternary ammonium molecules, ensuring its relatively constant surface positive charge and excellent photodynamic performance.

[0073] To further illustrate the beneficial effects of this invention, the colloidal and structural stability and photodynamic efficacy of the CMNP prepared in this embodiment were tested. The test method was as follows: 2.0 mL of freshly prepared CMNP ethanol stock solution was taken, centrifuged, and the precipitate was redispersed in pure water. Transmission electron microscopy (TEM), a nanoparticle size and zeta potential analyzer, and an enzyme-linked immunosorbent assay (ELISA) reader were used to analyze its structure, morphology, hydrated particle size, zeta potential, and UV-Vis absorption spectrum. After storing the above dispersion at room temperature for one week, the same tests were repeated to evaluate its colloidal dispersibility and structural stability. To test its superparamagnetic properties and magnetic response, the hysteresis loop of the CMNP was measured using a vibrating sample magnetometer (VSM). The test results are shown in [reference needed]. Figure 4 ,Depend on Figure 4 Transmission electron microscopy (TEM) images of the recyclable cationic nanophotosensitive agent CMNP show that the prepared recyclable cationic nanophotosensitive agent CMNP has very uniform particle size and core-shell structure characteristics.

[0074] To evaluate the reactive oxygen species (ROS) generation capacity of the recyclable cationic photosensitizer CMNP prepared in this embodiment under photoirradiation, the diphenylbenzofuran (DPBF) photobleaching method was used for testing. CMNP equivalent to 5.0 μg / mL Ce6 was dispersed in 3.0 mL of a mixed solvent of DMF:H2O (volume ratio 9:1), and 0.1 mM DPBF was added simultaneously. The reaction system was continuously irradiated with a 660 nm LED light source for 10 min under stirring. At irradiation times of 0, 1, 2, 4, 6, 8, and 10 min, 120 μL of the reaction solution was taken out, and after centrifugation at 22,000 × g to remove CMNP, the absorbance of DPBF at 415 nm was measured. The DPBF degradation curve was plotted with irradiation time on the x-axis and the percentage of DPBF absorbance at each time point relative to the initial value at 0 min on the y-axis. As a control, ROS generation capacity was tested using 5.0 μg / mL free Ce6 under the same conditions, but without centrifugation. See [link to results]. Figure 4 ,Depend on Figure 4 As shown in B, analysis using the DPBF quenching method indicates that, within a specific time period, the Ce6 loaded on the prepared recyclable cationic nanophotosensitive agent CMNP exhibits comparable photodynamic properties to an equivalent amount of free Ce6, suggesting that the magnetic nucleus, under the influence of the silica layer, did not affect the photosensitizing activity of Ce6. Figure 4As shown in C, D, E, F, G, and H, the recyclable cationic photosensitizer CMNP possesses superparamagnetism, enabling rapid separation and recovery under an applied magnetic field. Furthermore, the recyclable cationic photosensitizer CMNP prepared in this embodiment exhibits excellent colloidal and structural stability, ensuring that the chemical structure of the photosensitizer Ce6 remains intact during storage or recycling, and that key parameters such as its absorption spectrum and singlet oxygen yield remain stable, adapting to various environmental conditions.

[0075] The photodynamic killing effect of the CMNP prepared in this embodiment on typical foodborne pathogens was tested in drinking purified water and clear apple juice. The specific method was as follows:

[0076] (1) Strains and culture conditions

[0077] Preserved strains of Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Salmonella typhimurium were revived by shaking culture in nutrient broth (NB) at 37±1℃ for 24 h. To prepare the experimental bacterial suspension, the bacterial suspension was continuously passaged into fresh NB every 24 h to maintain bacterial viability. Before the experiment, 5.0 mL of culture medium was centrifuged at 4℃, 4000×g for 5 min to collect the bacterial cells. The cells were then washed twice with PBS buffer and resuspended in sterile purified drinking water or sterile clear apple juice to achieve an initial bacterial concentration of approximately 10⁻⁶. 7 CFU / mL. The resulting bacterial suspension was used for subsequent experimental testing.

[0078] (2) Photodynamic sterilization and sterilization effect analysis of CMNP

[0079] Take 0.5 mL of bacterial suspension and 0.5 mL of CMNP (Ce6 equivalent concentration of 0–20 μg / mL), and resuspend them together in 0.5 mL of sterile purified drinking water or clear apple juice. Incubate at 37°C with shaking for 30 min. Then, aliquot the mixed suspension into 48-well plates and irradiate them under a 660 nm LED light source for 0–30 min, continuously shaking to mix during the irradiation process.

[0080] A light control group (LightControl, LC, light only), a dark control group (Dark Control, DC, photosensitizer only), and a blank control group (no photosensitizer and no irradiation) were set up. Light dose (J / cm²) 2 Based on optical power density (W / cm²) 2 Calculation of the product of ) and irradiation time (s).

[0081] The photodynamic sterilization effect was evaluated using the plate count method. In this method, samples from each treatment group were serially diluted tenfold, and 100 μL of each diluted sample was spread evenly onto PCA medium using a sterile spreader. After incubation at 37°C in the dark for 24 hours, colonies were counted to calculate the photodynamic sterilization efficiency.

[0082] Sterilization efficiency is expressed as the log reduction of bacterial colonies, and the calculation formula is as follows:

[0083] Log reduction = log 10 (N0 / N)

[0084] Where N0 is the total number of colonies (CFU / mL) in the blank control group, and N is the total number of colonies (CFU / mL) in the treatment group.

[0085] See test results Figure 5 ,Depend on Figure 5 From A and C, we know that at 30 J / cm 2 and 60J / cm 2 At light irradiation doses, CMNP at an equivalent concentration of 5 μg / m³Ce6 showed significant bactericidal effects against both Escherichia coli and Salmonella, but the difference between the two was not statistically significant, indicating that 30 J / cm³... 2 It has essentially reached saturation. (By...) Figure 5 As shown in B and D, the sterilization effect increases with increasing Ce6 concentration, reaching its optimal level at 10 μg / mL, followed by a slight decrease, possibly related to particle aggregation or light shielding. Figure 5 As shown in E, compared to Gram-negative bacteria, CMNP exhibits higher sterilization efficiency against Staphylococcus aureus and Listeria monocytogenes, indicating that its positively charged surface structure is more conducive to binding with Gram-positive bacterial membranes, enhancing the localized action efficiency of reactive oxygen species. Overall, CMNP demonstrates good photodynamic inactivation ability against all four model bacteria in pure water.

[0086] Testing the Recovery and Reuse Efficiency of CMNP after Photodynamic Sterilization in Clarified Apple Juice

[0087] (1) Removal and residual detection of CMNP

[0088] CMNP with an equivalent Ce6 concentration of 10.0 μg / mL was added to clarified apple juice and thoroughly mixed. The mixture was then magnetically separated at 4°C for 6 hours using a commercially available N35 grade neodymium iron boron magnet. An equal volume of 38% hydrochloric acid was added to the juice sample, and after mixing, the mixture was digested at 100°C for 1 hour. The digested samples, along with iron ion standard solutions of different concentrations treated in the same manner, were analyzed using atomic absorption spectrometry (AAS). The instrument automatically calculated the iron ion concentration in the samples.

[0089] In the CMNP residue detection, untreated clear apple juice was used as a blank control group.

[0090] (2) Test on the repeated use of CMNP in clarified apple juice

[0091] E. coli and L. monocytogenes were selected as representative Gram-negative and Gram-positive strains, respectively, and subjected to five consecutive photodynamic sterilization (PDI) cycles. Each treatment used CMNP at a Ce6 equivalent concentration of 10.0 μg / mL, with an irradiation dose of 90 J / cm². 2 (660nm LED light source). After each round of PDI, the antibacterial effect was evaluated using the plate count method.

[0092] After each PDI cycle, CMNPs used for sterilization were magnetically adsorbed and recovered from the sterilized apple juice at 4°C for 6 hours. The recovered CMNPs were washed three times by centrifugation with phosphate buffer (pH 2.4) and then twice with ultrapure water before being used in the next PDI cycle.

[0093] After five rounds of PDI treatment and recycling, the reused CMNPs were redispersed in ultrapure water, and their hydration particle size and zeta potential were measured to assess whether their colloidal stability was maintained. Simultaneously, the final batch of clarified apple juice after magnetic separation was tested for iron residue, and the residual CMNP content was analyzed using AAS.

[0094] See test results Figure 6 and Figure 7 Based on the test results Figure 6 As shown in A and B, with increasing light dose, the photodynamic sterilization effect of CMNP on Escherichia coli and Listeria monocytogenes is significantly enhanced, especially when the light dose reaches 90 J / cm². 2 At that time, the log sterilization values ​​for both were 3.36±0.13 and 2.14±0.05, respectively. Figure 6 As shown in C, the log bactericidal values ​​of Staphylococcus aureus and Salmonella typhimurium were 3.13±0.02 and 5.17±0.15, respectively. Overall, CMNP still has good photodynamic sterilization activity in clarified apple juice.

[0095] The method for detecting the recovery and reuse effect of the recyclable cationic photosensitizer CMNP prepared in this embodiment after photodynamic sterilization in clarified apple juice is as follows:

[0096] (1) Removal and residual detection of CMNP

[0097] CMNP with an equivalent Ce6 concentration of 10.0 μg / mL was added to clarified apple juice and thoroughly mixed. The mixture was then magnetically separated at 4°C for 6 hours using a commercially available N35 grade neodymium iron boron magnet. An equal volume of 38% hydrochloric acid was added to the juice sample, and after mixing, the mixture was digested at 100°C for 1 hour. The digested samples, along with iron ion standard solutions of different concentrations treated in the same manner, were analyzed using atomic absorption spectrometry (AAS). The instrument automatically calculated the iron ion concentration in the samples.

[0098] In the CMNP residue detection, untreated clear apple juice was used as a blank control group.

[0099] (2) Test on the repeated use of CMNP in clarified apple juice

[0100] E. coli and L. monocytogenes were selected as representative Gram-negative and Gram-positive strains, respectively, and subjected to five consecutive photodynamic sterilization (PDI) cycles. Each treatment used CMNP at a Ce6 equivalent concentration of 10.0 μg / mL, with an irradiation dose of 90 J / cm². 2 (660nm LED light source). After each round of PDI, the antibacterial effect was evaluated using the plate count method.

[0101] After each PDI cycle, CMNPs used for sterilization were magnetically adsorbed and recovered from the sterilized apple juice at 4°C for 6 hours. The recovered CMNPs were washed three times by centrifugation with phosphate buffer (pH 2.4) and then twice with ultrapure water before being used in the next PDI cycle.

[0102] After five rounds of PDI treatment and recycling, the reused CMNPs were redispersed in ultrapure water, and their hydration particle size and zeta potential were measured to assess whether their colloidal stability was maintained. Simultaneously, the final batch of clarified apple juice after magnetic separation was tested for iron residue, and the residual CMNP content was analyzed using AAS.

[0103] See test results Figure 7 and Figure 8 ,Depend on Figure 7 It can be seen that under the influence of an external magnetic field, CMNPs can be completely magnetically separated and recovered from clarified apple juice within 6 hours. Figure 8 As can be seen from A and B, the recyclable cationic micro / nano photosensitizer CMNP exhibits outstanding reusability. Figure 8 From C and D, it can be seen that after five cycles, CMNP still has very good structural stability. Figure 8As can be seen from E, after the CMNP was used for the fifth time to clarify apple juice through photodynamic sterilization and magnetic recycling, the iron content in the apple juice did not change compared with the iron content in the untreated apple juice, indicating that the CMNP still has excellent magnetic separation characteristics after repeated recycling.

[0104] Example 3

[0105] 95 mg (0.25 mmol) of mercaptoporphyrin (Por-SH) was dissolved in 10 mL of anhydrous ethanol, and 120 μL (0.5 mmol) of 3-glycidyl etheroxypropyltrimethoxysilane (GPTMS) and 35 μL (0.25 mmol) of triethylamine (TEA) were added. The mixture was stirred at 40 °C for 18 h under nitrogen protection. The solvent was removed by rotary evaporation, and the resulting product was directly used for modification to obtain the silanized photosensitive molecule (Por-GPTMS).

[0106] 30 mg of MNPs were dispersed in a mixture of 30 mL ethanol and 10 mL water. After ultrasonic dispersion, ammonia (final concentration 0.1 M), 0.05 mmol of Por-GPTMS, and 0.5 mmol of N-(3-trimethoxysilylpropyl)-N,N,N-trimethylammonium chloride were added, and the mixture was stirred at room temperature for 24 h. After centrifugation and washing, a cationic magnetic photosensitizer loaded with mercaptoporphyrin was obtained (a recyclable cationic micro / nano photosensitizer).

[0107] Example 4

[0108] IR-783 (39.4 mg, 0.05 mmol) and N-(2-aminoethyl)maleimide (approximately 30.5 mg, 0.2 mmol) were dissolved in 2 mL of anhydrous DMF. The mixture was stirred at 40 °C for 16 h under nitrogen protection to promote nucleophilic substitution of the chloromethyl and amino groups on the IR-783 molecule, yielding maleimide-modified IR-783 molecules (IR-783-Mal). IR-783-Mal (0.05 mmol) and MPTMS (50 μL, approximately 0.24 mmol) were dissolved in 2 mL of anhydrous DMSO. The mixture was stirred at 40 °C for 12 h in the presence of triethylamine (5 μL) to generate the silanized photosensitive molecule IR-783-MPTMS. The product, after solvent removal by rotary evaporation, can be directly used for subsequent material preparation or surface modification reactions.

[0109] APTES (0.5 mmol, 117 μL) was dissolved in anhydrous DMF, and IPTES (1.0 mmol, 234 μL) and triethylamine (3 μL) were added. The mixture was reacted at 50 °C for 12 h under nitrogen protection to obtain silanized quaternary ammonium molecules (APTES-IPTES).

[0110] 30 mg of MNPs were dispersed in ethanol:water = 9:1, and IR-783-MPTMS (0.05 mmol) and APTES-IPTES (0.5 mmol) were added. NaOH (0.1 M) was used as a catalyst, and the reaction was carried out at room temperature for 24 h to obtain a cationic magnetic photosensitizer (CMNP-NIR783) loaded with IR-783-MPA.

[0111] Example 5

[0112] 100 mg (0.18 mmol) of protoporphyrin IX was added to pyridine along with 100 μL of APTES, 10 mg of HOAt, and 5 mg of DMAP. The mixture was reacted at 60 °C for 12 h to obtain the silanized photosensitizing molecule (protoporphyrin IX-APTES).

[0113] 45 μL (3-carboxypropyl)trimethylammonium chloride (0.25 mmol), 58 μL APTES (0.25 mmol), 0.05 mmol EDC and 0.05 mol NHS were dissolved together in 1 mL DMF. After stirring at room temperature for 12 h, the solvent was removed by rotary evaporation to obtain silanized quaternary ammonium molecules (CPTAC-APTES), which were directly used in subsequent experiments.

[0114] After pre-coating 5 mg of IONPs with TEOS (300 mM), protoporphyrin IX-APTES (0.025 mmol) and CPTAC-APTES (0.25 mmol) were added, and the mixture was reacted at 25 °C for 24 h. After demulsification and washing, cationic magnetic photosensitizer loaded with protoporphyrin IX was obtained (recoverable cationic micro / nano photosensitizer).

[0115] Example 6

[0116] Bengal rose red (20 mg, approximately 0.031 mmol) and HBTU (11.7 mg, 0.031 mmol) were dissolved in 2 mL of anhydrous DMSO. Then, APTES (50 μL, approximately 0.226 mmol) and DIPEA (16 μL, approximately 0.093 mmol) were added. The mixture was stirred at room temperature for 18 h under nitrogen protection. The product obtained after the reaction was the silanized photosensitizer RB-APTES. 0.5 mmol of Ce6, 351 μL (1.5 mmol) of 3-aminopropyltriethoxysilane (APTES), 475 mg (3.0 mmol) of EDC·HCl, and 345 mg (3.0 mmol) of NHS were added, followed by 10 mL of anhydrous N,N-dimethylformamide (DMF) to fully dissolve the reactants. The mixed solution was stirred at 40°C for 24 hours. After the reaction was completed, the solvent was removed by rotary evaporation. The product obtained did not require further purification and was directly used in subsequent reactions to obtain silanized photosensitive molecules (Ce6-APTES).

[0117] After pre-coating 5 mg of IONPs with TEOS, Ce6-APTES (0.01 mmol), RB-APTES (0.01 mmol), and N-(3-triethoxysilylpropyl)-N,N,N-trimethylammonium chloride (0.5 mmol) were added. The mixture was reacted at 25 °C for 24 h, and then washed to obtain a recyclable cationic micro / nano photosensitizer (CMNP-Ce6-RB).

[0118] Example 7

[0119] Riboflavin (30 mg, approximately 0.08 mmol) and GPTMS (60 μL, approximately 0.25 mmol) were dissolved in 2 mL of anhydrous DMF, followed by the addition of triethylamine (5 μL). The reaction system was stirred at 50 °C for 12 h under nitrogen protection to promote the nucleophilic addition reaction between the hydroxyl groups of the riboflavin sugar chain and the epoxy groups on the GPTMS molecules, generating β-hydroxy ether-linked silanized photosensitizer molecules (RF-GPTMS). After the reaction, the solvent was removed by rotary evaporation, and the product was directly used in the next step. 30 mg of MNPs was then dissolved in an ethanol:water ratio of 9:1, along with RF-GPTMS (0.08 mmol), N-(3-trimethoxysilylpropyl)-N,N,N-trimethylammonium chloride (0.3 mmol), and NaOH (0.1 M). The mixture was reacted at 40 °C for 5 h, centrifuged, and washed three times with ethanol to obtain the retrievable cationic micro / nano photosensitizer (CMNP-RF).

[0120] The application of the aforementioned recyclable cationic micro / nano photosensitizer in the sterilization of liquid foods involves using the prepared recyclable cationic micro / nano photosensitizer for photodynamic killing of Gram-positive and Gram-negative pathogens in liquid foods. Applicable liquid foods include: apple juice, orange juice, grape juice, pear juice, pineapple juice, lemon juice, mango juice (clarified), passion fruit juice, cherry juice, strawberry juice, blueberry juice, hawthorn juice, loquat juice, lychee juice, pomegranate juice, raspberry juice, mixed fruit juice beverages (clarified), fruit juice-based functional beverages, fruit enzyme beverages, low-alcohol fruit wine, white wine, red wine, sparkling wine, non-alcoholic wine, fruit-flavored rice wine, fruit-flavored beer, fruit-flavored sparkling wine, fruit vinegar beverages, purified drinking water, mineral water, soda water, and other clarified liquid foods. Specific usage methods are as follows:

[0121] A cationic micro / nano photosensitizer with a loaded photosensitizer concentration of 1–100 μM was added to the liquid food. After stirring or standing for 30 min, a light source with a characteristic excitation wavelength corresponding to the photosensitizer (optical power density 0.01–20 W / cm²) was used. 2 The liquid food system was irradiated with a stirring agent for 10–90 min, and then the cationic micro-nano photosensitizer in the liquid food system was recovered using a permanent magnet or electromagnet (continuously in a sterile low-temperature environment for 4–100 h).

[0122] The recovered cationic micro / nano photosensitizer can be reused after being centrifuged or magnetically washed 2-3 times in a buffer system (non-aqueous liquid food) with a pH of 2.5-3.5. For purified water, mineral water, soda water, etc., this washing step can be omitted and the product can be reused directly.

[0123] This recyclable cationic micro-nano photosensitizer generates singlet oxygen under light irradiation, directly destroying bacterial cell membranes and DNA. The cationic quaternary ammonium groups penetrate the cell wall through electrostatic adsorption. This dual action effectively improves sterilization efficiency and has good safety, excellent recyclability, and reusability. It provides the liquid food industry with an efficient, safe, environmentally friendly, and economical sterilization solution with broad market application prospects.

[0124] In summary, this invention provides a recyclable cationic micro / nano photosensitizer, its preparation method, and its application. This recyclable cationic micro / nano photosensitizer utilizes micro / nano technology to solve the problem of reduced or even lost photodynamic efficiency due to aggregation of hydrophobic photosensitizers in liquid foods. By covalently modifying the surface of the micro / nano photosensitizer with quaternary ammonium molecules, a stable positive charge state is maintained on the surface of the nano-photosensitizer, achieving broad-spectrum photodynamic inactivation against both Gram-negative and Gram-positive bacteria. Using superparamagnetic iron oxide micro / nano particles as the core of the nano-photosensitizer allows it to be completely removed from the liquid matrix magnetically after photodynamic sterilization in liquid foods, avoiding the impact of photosensitizer residue on the quality of liquid foods and mitigating safety risks. The recyclable cationic micro / nano photosensitizer of this invention combines photosensitizer, quaternary ammonium molecules, and a magnetic core through covalent bonding, exhibiting outstanding colloidal and structural stability, allowing for multiple reuses, reducing the application cost of photodynamic sterilization technology, and meeting the needs of green and sustainable development.

[0125] The above description is merely a preferred embodiment of the present invention and is not intended to limit the technical solution of the present invention in any way. Those skilled in the art should understand that, without departing from the spirit and principles of the present invention, the technical solution can be modified and replaced in several simple ways, and these modifications and replacements are all within the scope of protection covered by the claims.

Claims

1. A recyclable cationic micro / nano photosensitizer, characterized in that, It includes a magnetic core, the surface of which is covered with a shell, the shell being composed of a silane coupling agent conjugated with photosensitizer molecules and quaternary ammonium molecules.

2. The recyclable cationic micro / nano photosensitizer according to claim 1, characterized in that, The magnetic core is made of iron(III) oxide nanoparticles or microparticles.

3. The recyclable cationic micro / nano photosensitizer according to claim 1, characterized in that, The photosensitive molecules include one or more of dihydroporphyrin e6, curcumin, Bengal rose red, mercaptoporphyrin, IR-783 near-infrared dye derivative, protoporphyrin IX, and riboflavin.

4. The recyclable cationic micro / nano photosensitizer according to claim 1, characterized in that, The quaternary ammonium molecule includes one or more of the following: N-(3-trimethoxysilylpropyl)-N,N,N-trimethylammonium chloride, N-(3-triethoxysilylpropyl)-N,N,N-trimethylammonium chloride, (3-aminopropyl)trimethylammonium chloride, N-(2-hydroxyethyl)-N,N,N-trimethylammonium chloride, 2,3-epoxypropyltrimethylammonium chloride, (3-carboxypropyl)trimethylammonium chloride, N-(carboxymethyl)-N,N,N-trimethylammonium chloride, N-(mercaptoethyl)-N,N,N-trimethylammonium chloride, N-(acryloyloxyethyl)-N,N,N-trimethylammonium chloride, and 2-methacryloyloxyethyltrimethylammonium chloride.

5. The recyclable cationic micro / nano photosensitizer according to claim 1, characterized in that, The silanized quaternary ammonium molecule is N-(3-trimethoxysilylpropyl)-N,N,N-trimethylammonium chloride, N-(3-triethoxysilylpropyl)-N,N,N-trimethylammonium chloride, or a product covalently bonded to a quaternary ammonium molecule by a silane coupling agent.

6. The recyclable cationic micro / nano photosensitizer according to claim 1, characterized in that, The silane coupling agent includes one or more of 3-aminopropyltriethoxysilane, 3-mercaptopropyltrimethoxysilane, 3-isocyanatepropyltriethoxysilane, and 3-glycidoxypropyltrimethoxysilane.

7. A method for preparing a recyclable cationic micro / nano photosensitizer as described in any one of claims 1-6, characterized in that, include: Preparation of silanized photosensitive molecules and silanized quaternary ammonium molecules; A magnetic core dispersion is prepared, and silanized photosensitizing molecules, silanized quaternary ammonium molecules, and a catalyst are added to the magnetic core dispersion to react and obtain a recyclable cationic micro / nano photosensitizer; wherein the catalyst is ammonia, NaOH, or KOH.

8. The method for preparing the recyclable cationic micro / nano photosensitizer according to claim 7, characterized in that, The solvent of the magnetic nucleus dispersion is an organic mixed solution, and the concentration of magnetic nuclei in the magnetic nucleus dispersion is 0.1-200 mg / mL; The organic mixed solution is an aqueous solution of ethanol, an aqueous solution of methanol, an aqueous solution of methanol and ethanol, or a mixture of an alkane solvent and a nonionic surfactant. The alkane solvent includes one of cyclohexane, isooctane, n-hexane, paraffin oil, and octane. The nonionic surfactant is one or more of Igepal CO-520, Riton X-100, Brij 30, Span 80, and Tween 80. The molar ratio of silanized quaternary ammonium molecules to silanized photosensitive molecules added to the magnetic core dispersion is 1:10 to 200:1, and the total molar concentration of silanized quaternary ammonium molecules and silanized photosensitive molecules is 1 to 200 mM.

9. The method for preparing the recyclable cationic micro / nano photosensitizer according to claim 7, characterized in that, Before adding silanized photosensitive molecules, silanized quaternary ammonium molecules and catalyst to the magnetic core dispersion, tetramethyl silicate or tetraethyl silicate is also added to the magnetic core dispersion, and the reaction is carried out for 0.5 to 24 hours to generate a pre-prepared silica layer on the surface of the magnetic core.

10. The application of the recyclable cationic micro / nano photosensitizer as described in any one of claims 1-6 in the sterilization of liquid food.

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