Self-assembled nano particle as well as preparation method and application thereof

Abstract

The invention belongs to the field of food non-thermal sterilization, and relates to a self-assembled nanoparticle. The invention provides a self-assembled nano particle and a preparation method and application thereof, the self-assembled nano particle is combined with photodynamic force to realize photodynamic and physical multifunctional sterilization, and due to amphipathicity, modified chitosan has the capability of forming nano particles through self-assembly. Due to the introduction of phenolic ester, not only is the chitosan endowed with stronger photodynamic activity, but also the lipophilicity of the chitosan is improved, a hydrophobic chain part with a proper length can be effectively combined with bacterial cell membranes in a cuttage manner, efficient sterilization is realized through a physical effect and a photodynamic synergistic mechanism, and the antibacterial effect is remarkably improved. Meanwhile, the excellent AIE characteristic of the material is combined with efficient combination of bacteria, the material can be used for bacterial fluorescence imaging and development of efficient harmful bacteria monitoring means, and the material is completely prepared from food-borne raw materials and has excellent biocompatibility and high safety.

Description

Technical Field

[0001] The present invention belongs to the field of non-thermal sterilization of foods and relates to a self-assembled nanoparticle. Background Art

[0002] Chitosan (CS) is a natural polysaccharide obtained by the deacetylation reaction of chitin and is the second most abundant polysaccharide in nature. The amino groups in deacetylated chitosan ionize to form Chit-NH3 + , and can dissolve in dilute acidic media with a pKa value lower than 7.3. In recent years, chitosan has received extensive attention due to its remarkable antibacterial activity, antioxidant properties, biocompatibility, biodegradability, non-toxicity, low allergenicity, and good film-forming ability, and has been widely used in the field of biomaterials. Its excellent properties have enabled chitosan to be greatly extended in many research fields. However, as a functional biomaterial, chitosan still has certain limitations. For example, due to the lack of hydrogen atom donors in its structure, its antibacterial activity is weak; at the same time, the high swelling property of chitosan also limits its application scope as a material. To overcome these limitations, scientific researchers have carried out a large amount of work dedicated to optimizing the functional properties of chitosan through structural modification. Chitosan molecules have two functional groups, reactive amino and hydroxyl groups, which give it great potential and flexibility in chemical modification.

[0003] Photodynamic inactivation (PDI) is a technology with broad application potential. By irradiating light to induce photosensitizers (PSs) to generate reactive oxygen species (ROS), it can effectively inhibit or eradicate bacteria. In recent years, PDI has received increasing attention due to the following advantages: (I) It has significant antibacterial activity against various bacterial states (such as planktonic and biofilm states); (II) It can rapidly eliminate microorganisms, and due to its unique mechanism, the microbial drug resistance is relatively low. The basic principle of PDI involves three key elements: photosensitizer (PS), light source, and oxygen. The photosensitizer can absorb light of a specific wavelength and be excited to its high-energy triplet state (3PS), and this triplet state reacts with oxygen through electron transfer or energy transfer mechanisms (type I and type II reactions) to produce reactive oxygen species (ROS). These reactive oxygen species have a strong killing effect on microorganisms. Therefore, the PDI technology shows great potential in antibacterial therapy. However, the application efficiency of traditional organic photosensitizers (PSs) in PDI is limited by various factors, mainly reflected in the insufficient interaction with bacteria, resulting in unsatisfactory bacteria eradication effect. These problems are usually related to the short lifespan and limited diffusion distance of ROS, as well as the quenching effect caused by aggregation (ACQ), which not only reduces the imaging sensitivity but also significantly affects the ROS generation efficiency (Advanced Functional Materials, 2023, 33(33), 2301692). In addition, most organic photosensitizers have poor water solubility, which limits their effectiveness in practical applications. Therefore, developing multifunctional synergistic materials to enhance the interaction between photosensitizers and bacteria and overcome the quenching effect has become a key direction to improve the effect of PDI technology.

[0004] Macromolecular materials with aggregation-induced emission (AIE) characteristics of high ROS generation are being rapidly developed for PDI. Chitosan, as a biocompatible polymer, has excellent AIE characteristics ( Carbohydrate Polymers, 2020, 227, 115338). Therefore, grafting natural organic extract phenolic acid ester with photosensitive properties onto chitosan to obtain a novel AIE-type photosensitive material is an attractive preparation scheme for novel antibacterial materials. Phenolic acid ester has a long hydrophobic chain, which can drive the self-assembly of chitosan into a stable structure through hydrophobic interaction. Inspired by this background, grafting phenolic acid ester onto chitosan greatly improves the photodynamic antibacterial activity and hydrophobicity of the material, and can spontaneously assemble into nanoparticles through simple stirring, with nanometer characteristics, and can exert dual-mode bacterial inhibition, including physical membrane damage and photodynamic eradication. There are a large number of positively charged amino groups on the chitosan chain, which can produce strong electrostatic interaction with the bacterial cell membrane. The grafted long hydrophobic chain can be inserted into the bacterial cell membrane, further enhancing the ability to capture bacteria. Combined with light irradiation, PCNPs can generate a large amount of ROS in a short time, damaging the bacterial cell membrane and its contents. The amphiphilic PCNPs with long alkyl chains and positive charges show excellent PDI activity and the ability to efficiently kill bacteria and destroy bacterial biofilms. It is very suitable as a non-toxic green additive for the long-term preservation of fruits and vegetables. Summary of the Invention

[0005] The present invention provides a self-assembled nanoparticle, its preparation method and application. The self-assembled nanoparticle combines with photodynamic to achieve photodynamic and physical multifunctional sterilization. The amphiphilic property enables the modified chitosan to have the ability to self-assemble into nanoparticles.

[0006] The technical solution of the present invention is realized as follows: A self-assembled nanoparticle PCNPs, which is formed by grafting phenolic acid ester with a long hydrophobic chain and photosensitive activity onto chitosan with AIE characteristics and self-assembling into nanoparticles through simple stirring.

[0007] The above-mentioned phenolic acid ester-modified chitosan is obtained by covalent bonding of phenolic acid ester and chitosan through a radical method, in which the hydroxyl group and amino group on the chitosan monomer will combine with the active site and the carbon on the benzene ring of the phenolic acid ester.

[0008] The preparation method of the above-mentioned self-assembled nanoparticle PCNPs comprises the steps of: Dissolve the phenolic acid ester-modified chitosan powder in an acetic acid solution, and self-assemble into nanoparticles during stirring reaction at a speed of 400 - 800 rpm / min, and filter large-particle-size particles through a 0.22 μm needle filter.

[0009] Furthermore, in the modified chitosan acetic acid solution, the mass fraction of the modified chitosan is 0.5 - 2.5%, the volume fraction of the acetic acid solution is 1 - 2%, and the stirring time is 2 - 3 h.

[0010] Preferably, in the modified chitosan acetic acid solution, the mass fraction of modified chitosan is 1%, the volume fraction of acetic acid solution is 1%, and the stirring time is 2.5 h.

[0011] Preferably, the specific steps of this preparation method are as follows: Weigh 0.1 g of modified chitosan and dissolve it in 10 mL of acetic acid solution with a volume fraction of 1%, and stir for 2.5 h. After complete dissolution, filter through a 0.22 μm needle filter.

[0012] Furthermore, when the PCNPs are combined with 300 - 500 nm light irradiation, they show the effect of clearing planktonic bacteria and bacterial biofilms.

[0013] Preferably, when the PCNPs are combined with 420 nm light irradiation, they show the effect of clearing planktonic bacteria and bacterial biofilms.

[0014] The present invention provides the preparation method of the above-mentioned phenolic acid ester - modified chitosan, which includes the following steps: (1) Dissolve chitosan in 1% acetic acid solution and protect it with nitrogen; (2) Dissolve ascorbic acid in hydrogen peroxide and add it to the chitosan acetic acid solution protected by nitrogen, and react for 30 min; (3) Dissolve phenolic acid ester in ethanol, add it to the solution in step (2), and react for 24 h under nitrogen protection in the dark; (4) Adjust the pH of the reaction solution in step (3) to 7, filter by suction to collect the solid precipitate, wash it with ethanol multiple times, and then freeze - dry the solid to remove water to obtain the modified chitosan powder.

[0015] Furthermore, in step (1), the volume fraction of acetic acid solution is 1 - 2%, the mass fraction of chitosan is 0.5 - 2.5%, the molecular weight of chitosan is 100 KDa, and the degree of deacetylation is 90%.

[0016] Preferably, in step (1), the volume fraction of acetic acid solution is 1%, the mass fraction of chitosan is 2.5%, the molecular weight of chitosan is 100 KDa, and the degree of deacetylation is 90%.

[0017] Furthermore, the molar ratio of chitosan monomer to phenolic acid ester is 1 - 5:1, and the molar ratio of ascorbic acid to hydrogen peroxide is 1:5 - 10.

[0018] Preferably, in step (3), the molar ratio of chitosan monomer to phenolic acid ester is 2:1, and the molar ratio of ascorbic acid to hydrogen peroxide is 1:10.

[0019] The specific method of this preparation method is as follows: (1) Dissolve chitosan in acetic acid solution with a volume fraction of 1%, stir for 3 h until clear and transparent, and displace with N2 for 30 min.

[0020] (2) Dissolve 0.5 M ascorbic acid powder in 5 M H2O2, and then slowly add it to the solution in step (1). Continue to stir the reaction under N2 protection in the dark for 30 min.

[0021] (3) First, add anhydrous ethanol to the solution in step (2) and stir for 5 min. Then slowly add the phenolic acid ester solubilized with anhydrous ethanol. React under nitrogen protection in the dark at room temperature for 24 h.

[0022] (4) After the reaction, adjust the pH of the system to 7 with 1 M sodium hydroxide, add an excessive amount of ethanol to precipitate the unmodified chitosan. Wash with ethanol solution and filter by suction to remove the unreacted phenolic acid ester. Lyophilize for 24 h to obtain the modified chitosan powder.

[0023] Further, the present invention provides chitosan modified by any one of alkyl gallate, alkyl caffeate, alkyl chlorogenic acid, and alkyl ferulate.

[0024] The present invention also provides the application of the phenolic acid ester-modified chitosan nanoparticles PCNPs combined with blue light irradiation in antibacterial and anti-biofilm.

[0025] The present invention also provides the application of the phenolic acid ester-modified chitosan nanoparticles PCNPs in grape preservation.

[0026] The present invention has the following beneficial effects: (1) The synthesis method is mild, the operation is simple, and the cost is low: The phenolic acid ester-modified chitosan is synthesized under mild conditions by the green free radical method. This synthesis method not only avoids the use of harsh conditions such as high temperature and high pressure, but also reduces environmental pollution, meeting the current requirements for green chemical synthesis. The amino group of chitosan gives a positive charge and contributes to its hydrophilic property, while the long alkyl chain of the phenolic acid ester acts as a hydrophobic chain. This combination endows the phenolic acid ester-modified chitosan with excellent amphiphilicity. Utilizing this amphiphilic property, in an acetic acid aqueous solution, the phenolic acid ester-modified chitosan molecules are simply stirred, and the hydrophobic interaction induces the hydrophobic part of the phenolic acid ester inside the nanoparticles, while the hydrophilic chitosan chains are assembled on its outer surface. This arrangement increases the intramolecular aggregation and AIE intensity, and increases the water dispersibility of PCNPs. The operation process does not require complex equipment, significantly reducing the production cost. In addition, the raw materials used, chitosan and phenolic acid ester, are both naturally available materials with low cost and wide sources, making this technology more competitive in large-scale production.

[0027] (2) High bacterial affinity and transmembrane ability to achieve excellent antibacterial effects: The PCNPs material prepared in this invention forms a structure with strong bacterial affinity based on the hydrophobic chain of phenolic acid ester and the positively charged amino groups in the chitosan chain. The positively charged amino groups can have strong electrostatic interactions with the negatively charged regions on the bacterial cell membrane, and the hydrophobicity of the phenolic acid ester further enhances the affinity between PCNPs and the bacterial cell membrane, promoting the insertion of nanoparticles into the bacterial cell membrane. During the penetration of the bacterial membrane, PCNPs cause the rupture of the cell membrane and the leakage of internal substances, thereby causing physical damage to bacterial cells. This mechanism enables PCNPs to efficiently achieve antibacterial effects and effectively remove bacterial biofilms, with strong antibacterial effects, and has broad application potential especially in the field of food preservation and other fields.

[0028] (3) Photodynamic synergy enhances antibacterial effects and generates a large amount of ROS: Under blue light (420 nm, 0.213 W / cm²) irradiation, the PCNPs material can significantly improve its peroxidase-like activity. This process generates a large amount of reactive oxygen species (ROS) through photodynamic synergy, such as hydrogen peroxide, singlet oxygen, etc. These ROS can damage important structures such as the bacterial cell membrane, proteins, and DNA, causing oxidative damage to bacteria. Compared with traditional antibacterial methods, photodynamic antibacterial significantly improves the killing efficiency of bacteria. This feature enables the PCNPs material to achieve continuous and efficient antibacterial effects when applied in the food field, especially in low-temperature and food packaging environments.

[0029] (4) Good biocompatibility, green and environmentally friendly, meeting food safety requirements: The PCNPs material prepared in this invention is completely composed of food-derived raw materials and has excellent biocompatibility. Chitosan itself is a natural polysaccharide widely present in organisms, with excellent biodegradability and non-toxicity, and will not cause harm to the human body and the environment. In addition, the green synthesis process of PCNPs does not involve the use of harmful chemicals, so it conforms to the concept of green environmental protection and can be used as a safe and sustainable antibacterial material in the food field. This advantage ensures the safety of PCNPs in food preservation, sterilization treatment, food packaging, etc., to meet the strict requirements of the food industry. Description of the Drawings

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. The drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0031] Figure 1Schematic diagram of the preparation process and antibacterial activity of the phenolic acid ester-modified chitosan nanoparticles prepared in Example 1 of the present invention.

[0032] Figure 2 Fluorescence spectra of the phenolic acid ester-modified chitosan nanoparticles prepared in Example 1 of the present invention and the chitosan solution prepared in Comparative Example 1; the inset is a photograph of the corresponding solution under 365 nm ultraviolet light.

[0033] Figure 3 DLS results of the phenolic acid ester-modified chitosan nanoparticles prepared in Example 1 of the present invention in water; the inset is a TEM image of PCNPs. Scale bar: 200 nm.

[0034] Figure 4 UV absorption spectra of the PCNPs prepared in Examples 1-4, the chitosan solution prepared in Comparative Example 1, and TMB after reaction under blue light irradiation at 400 - 800 nm.

[0035] Figure 5 Composite graph of the grafting rate of phenolic acid esters and the hydrodynamic diameter of the phenolic acid ester-modified chitosan nanoparticles prepared in Examples 1, 5, and 6 of the present invention.

[0036] Figure 6 Dot line graph of the antibacterial effects of the phenolic acid ester-modified chitosan nanoparticles prepared in Example 1 of the present invention and the chitosan solution prepared in Comparative Example 1 against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus at different times under blue light illumination conditions.

[0037] Figure 7 Coating situation of the antibacterial effects of the phenolic acid ester-modified chitosan nanoparticles prepared in Example 1 of the present invention and the chitosan solution prepared in Comparative Example 1 against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus under blue light illumination conditions.

[0038] Figure 8 Generation of hydroxyl radicals, hydrogen peroxide, and singlet oxygen after different times of blue light irradiation of the phenolic acid ester-modified chitosan nanoparticles prepared in Example 1 of the present invention and the chitosan solution prepared in Comparative Example 1.

[0039] Figure 9 Fluorescence images of the phenolic acid ester-modified chitosan nanoparticles prepared in Example 1 of the present invention after co-incubation with Escherichia coli and Staphylococcus aureus for 2 h (scale bar = 10 μm).

[0040] Figure 10 Three-dimensional CLSM images of the clearance effect of the phenolic acid ester-modified chitosan nanoparticles prepared in Example 1 of the present invention on mature biofilms of Escherichia coli in the presence or absence of blue light and blue light alone.

[0041] Figure 11 QCM-D diagram of the interaction between phenolic acid ester-modified chitosan nanoparticles prepared in Example 1 of the present invention and phospholipid bilayers.

[0042] Figure 12 DNA electrophoresis diagram of the effects of PDI mediated by phenolic acid ester-modified chitosan nanoparticles prepared in Example 1 of the present invention on the genomic DNA of Escherichia coli and eDNA in the Escherichia coli biofilm.

[0043] Figure 13 Photographs of the preservation effects of phenolic acid ester-modified chitosan nanoparticles prepared in Example 1 of the present invention and chitosan prepared in Comparative Example 1 on grapes and plate count diagrams of the number of colonies remaining on the grapes on the 8th day. Detailed implementation manners

[0044] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0045] Unless otherwise specified, the test methods used in the following experimental examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0046] Example 1 The preparation method of the self-assembled nanoparticles PCNPs1 of phenolic acid ester (lauryl gallate)-modified chitosan in this example is as follows: (1) Add 0.5 g of chitosan to a 100 ml round-bottom flask and dissolve it in 20 ml of acetic acid solution with a volume fraction of 1%, and continuously stir for 2.5 h under an inert atmosphere of nitrogen until it is completely and transparently dissolved.

[0047] (2) Add 1 mL of hydrogen peroxide solution with a concentration of 5 mM containing 0.5 M ascorbic acid to the solution in step (1), and stir and react at 600 rpm / min at room temperature for 30 min to obtain a mixed solution.

[0048] (3) Prepare 10 mL of an anhydrous ethanol solution containing 1.5 mM lauryl gallate, slowly add it to the mixed solution in step (2), and react for 24 h under nitrogen protection.

[0049] (4) Add 1 M sodium hydroxide solution dropwise to the solution after the reaction in step (3), adjust the pH value to 7, and add an excess of absolute ethanol to precipitate the chitosan conjugate. Collect the precipitate and wash it thoroughly with a mixture of ethanol and water, with the ethanol content gradually increasing from 70% by volume to 100% to remove unreacted lauroyl gallate.

[0050] (5) Lyophilize the washed precipitate in a vacuum freeze dryer to remove water for 24 - 48 h, and grind it to obtain the modified chitosan powder.

[0051] (6) Take 0.1 g of the modified chitosan powder obtained in step (5) and dissolve it in 10 mL of 1% acetic acid solution by volume under magnetic stirring, and stir for 2.5 h to form nanoparticles. Then, filter through a 0.22 μm microporous membrane to remove large aggregates to obtain PCNPs1.

[0052] Figure 1 Taking PCNPs1 prepared in Example 1 of the present invention as an example, the preparation process and antibacterial schematic diagram of phenol - acid - ester - modified chitosan.

[0053] Figure 2 Taking PCNPs1 prepared in Example 1 of the present invention and the chitosan solution prepared in the comparative example as examples, the fluorescence spectrum of the PCNPs solution and the photo of the solution under 365 nm ultraviolet light show that the modified chitosan has AIE properties and can emit stronger fluorescence.

[0054] Figure 3 Taking PCNPs1 prepared in Example 1 of the present invention as an example, the DLS diagram and TEM diagram of PCNPs1 show that self - assembly occurred after stirring to form nanoparticles, and the average particle size is 136.5 nm.

[0055] Example 2 The preparation of self - assembled nanoparticles PCNPs2 of phenol - acid - ester (lauroyl caffeate) - modified chitosan in this example is as follows: (1) Weigh 0.5 g of chitosan in a 100 ml round - bottom flask, dissolve it in 20 ml of 1% acetic acid solution by volume, and continuously stir for 3 h under an inert atmosphere of nitrogen until it is completely and transparently dissolved.

[0056] (2) Add 1 mL of 5 mM hydrogen peroxide solution containing 0.5 M ascorbic acid, and mix at room temperature for 30 min to obtain a mixed solution.

[0057] (3) Prepare a 10 mL absolute ethanol solution containing 1.5 mM lauroyl caffeate, and add it to the mixed solution in step (2). React overnight under a nitrogen atmosphere.

[0058] (4) The chitosan conjugate was precipitated by adjusting the pH of the reaction solution in step (3) to 7 and adding an excess of absolute ethanol. The precipitate was collected and thoroughly washed with an ethanol / water mixture with the ethanol content increased from 70% to 100% by volume to remove unreacted lauroyl caffeate.

[0059] (5) The washed precipitate was freeze-dried under vacuum for 24 h and ground to obtain the modified chitosan powder.

[0060] (6) 0.1 g of the modified chitosan powder obtained in step (5) was dissolved in 10 mL of 1% acetic acid solution by volume under magnetic stirring and stirred for 2.5 h to form nanoparticles. Then, large aggregates were removed by filtration through a 0.22 μm microporous membrane to obtain PCNPs2.

[0061] Example 3 The preparation of the phenolic acid ester (lauroyl chlorogenic acid)-modified chitosan nanoparticles PCNPs3 in this example was as follows: (1) 0.5 g of chitosan was weighed in a 100 ml round-bottom flask and dissolved in 20 ml of 1% acetic acid solution by volume, and continuously stirred for 3 h under an inert atmosphere of nitrogen until completely transparent and dissolved.

[0062] (2) 1 mL of 5 mM hydrogen peroxide solution containing 0.5 M ascorbic acid was added and mixed at room temperature for 30 min to obtain a mixed solution.

[0063] (3) 10 mL of an absolute ethanol solution containing 1.5 mM lauroyl chlorogenic acid was prepared and added to the mixed solution in step (2). The reaction was carried out overnight under a nitrogen atmosphere.

[0064] (4) The chitosan conjugate was precipitated by adjusting the pH of the reaction solution in step (3) to 7 and adding an excess of absolute ethanol. The precipitate was collected and thoroughly washed with an ethanol / water mixture with the ethanol content increased from 70% to 100% by volume to remove unreacted lauroyl chlorogenic acid.

[0065] (5) The washed precipitate was freeze-dried under vacuum for 24 h and ground to obtain the modified chitosan powder.

[0066] (6) 0.1 g of the modified chitosan powder obtained in step (5) was dissolved in 10 mL of 1% acetic acid solution by volume under magnetic stirring and stirred for 2.5 h to form nanoparticles. Then, large aggregates were removed by filtration through a 0.22 μm microporous membrane to obtain PCNPs3.

[0067] Example 4 The preparation of the phenolic acid ester (lauroyl ferulate)-modified chitosan nanoparticles PCNPs4 in this example was as follows: (1) Weigh 0.5 g of chitosan in a 100 ml round-bottom flask and dissolve it in 20 ml of 1% acetic acid solution by volume. Continuously stir for 3 h under an inert atmosphere of nitrogen until it is completely and transparently dissolved.

[0068] (2) Add 1 mL of 5 mM hydrogen peroxide solution containing 0.5 M ascorbic acid and mix at room temperature for 30 min to obtain a mixed solution.

[0069] (3) Prepare 10 mL of an absolute ethanol solution containing 1.5 mM lauryl ferulate and add it to the mixed solution in step (2). React overnight under a nitrogen atmosphere.

[0070] (4) Precipitate the chitosan conjugate by adjusting the pH value of the reaction solution in step (3) to 7 and adding an excess of absolute ethanol. Collect the precipitate and wash it thoroughly with an ethanol / water mixture, with the ethanol content increasing from 70% to 100% by volume to remove the unreacted lauryl ferulate.

[0071] (5) Vacuum freeze-dry the washed precipitate for 24 h and grind it to obtain the modified chitosan powder.

[0072] (6) Dissolve 0.1 g of the modified chitosan powder obtained in step (5) in 10 mL of 1% acetic acid solution by volume under magnetic stirring and stir for 2.5 h to form nanoparticles. Then, remove the large aggregates by filtration through a 0.22 μm microporous membrane to obtain PCNPs4.

[0073] Example 5 The preparation of the phenolic acid ester (lauryl ferulate)-modified chitosan nanoparticles PCNPs1 in this example is as follows: (1) Weigh 0.5 g of chitosan in a 100 ml round-bottom flask and dissolve it in 20 ml of 1% acetic acid solution by volume. Continuously stir for 3 h under an inert atmosphere of nitrogen until it is completely and transparently dissolved.

[0074] (2) Add 1 mL of 5 mM hydrogen peroxide solution containing 0.5 M ascorbic acid and mix at room temperature for 30 min to obtain a mixed solution.

[0075] (3) Prepare 10 mL of an absolute ethanol solution containing 0.5 mM lauryl ferulate and add it to the mixed solution in step (2). React overnight under a nitrogen atmosphere.

[0076] (4) The pH value of the reaction solution in (3) was adjusted to 7 and an excess of absolute ethanol was added to precipitate the chitosan conjugate. The precipitate was collected and thoroughly washed with an ethanol / water mixture with the ethanol content increasing from 70% to 100% by volume to remove unreacted lauroyl ferulate.

[0077] (5) The washed precipitate was freeze-dried under vacuum for 24 h and ground to obtain the modified chitosan powder.

[0078] (6) 0.1 g of the modified chitosan powder obtained in step (5) was dissolved in 10 mL of 1% acetic acid solution by volume under magnetic stirring and stirred for 2.5 h to form nanoparticles. Then, large aggregates were removed by filtration through a 0.22 μm microporous membrane to obtain PCNPs1.

[0079] Example 6 Preparation of the phenolic acid ester (lauroyl ferulate)-modified chitosan nanoparticles PCNPs1 in this example was as follows: (1) 0.5 g of chitosan was weighed in a 100 ml round-bottom flask, dissolved in 20 ml of 1% acetic acid solution by volume, and continuously stirred for 3 h under an inert atmosphere of nitrogen until completely and transparently dissolved.

[0080] (2) 1 mL of a 5 mM hydrogen peroxide solution containing 0.5 M ascorbic acid was added and mixed at room temperature for 30 min to obtain a mixed solution.

[0081] (3) 10 mL of an absolute ethanol solution containing 2.5 mM lauroyl ferulate was prepared and added to the mixed solution in step (2). The reaction was carried out overnight under a nitrogen atmosphere.

[0082] (4) The pH value of the reaction solution in step (3) was adjusted to 7 and an excess of absolute ethanol was added to precipitate the chitosan conjugate. The precipitate was collected and thoroughly washed with an ethanol / water mixture with the ethanol content increasing from 70% to 100% by volume to remove unreacted lauroyl ferulate.

[0083] (5) The washed precipitate was freeze-dried under vacuum for 24 h and ground to obtain the modified chitosan powder.

[0084] (6) 0.1 g of the modified chitosan powder obtained in step (5) was dissolved in 10 mL of 1% acetic acid solution by volume under magnetic stirring and stirred for 2.5 h to form nanoparticles. Then, large aggregates were removed by filtration through a 0.22 μm microporous membrane to obtain PCNPs1.

[0085] Comparative Example 1 Preparation of chitosan solution: Weigh 0.08 g of chitosan powder and dissolve it in 10 mL of 1% acetic acid solution by volume. Stir for 2.5 h until the solution becomes clear and transparent, obtaining a chitosan solution with a mass concentration of 0.8 mg / mL.

[0086] Example of implementation effect 1 The peroxidase-like activity of the PCNPs prepared in Examples 1-4 and the chitosan solution prepared in Comparative Example 1: 3,3',5,5'-Tetramethylbenzidine (TMB) is used as the oxidase substrate. TMB can be oxidized by H2O2 under the catalysis of peroxidase or by O2 under the catalysis of oxidase to generate the blue substance ox-TMB, and the oxidation product has a characteristic absorption peak at OD 652 nm . Add 100 μL of PCNPs1-4 and the chitosan solution to 880 μL of NaAC buffer respectively, and then add 20 μL of TMB ethanol solution to the above mixed solution. The final concentrations of PCNPs1-4 and chitosan are both 0.8 mg / mL, and the final concentration of TMB is 5 mM. Use deionized water to replace PCNPs as the blank control group. Expose the mixed solution to blue light irradiation with a wavelength of 420 nm for 5 min, and the light intensity is 0.213 W / cm 2 . After the irradiation ends, simultaneously record the absorption spectrum of the mixed solution in the wavelength range of 400-800 nm with a UV-visible spectrophotometer.

[0087] Figure 4 Figure shows the UV absorption spectra of the PCNPs prepared in Examples 1-4 and the chitosan solution prepared in Comparative Example 1 after reacting with TMB under blue light irradiation at 400-800 nm. It can be seen from the figure that the chitosan nanoparticles modified with lauryl gallate and lauryl caffeate show characteristic absorption peaks at 652 nm under the synergistic effect of blue light, showing extremely strong peroxidase-like activity. Among them, the chitosan modified with lauryl gallate shows the best photodynamic activity. It shows that the modification of phenolic acid esters with photosensitive activity significantly improves the photodynamic activity of chitosan.

[0088] Example of implementation effect 2 Taking PCNPs1 prepared in Examples 1, 5 and 6 as an example, the differences in the grafting rate of different phenolic acid ester addition amounts and the influence on the particle size of the nanoparticles: (1) Prepare a 1 mg / mL sample. Then, take 1 mL of the 1 mg / mL sample solution and mix it with 1 mL of Folin-Ciocalteu reagent, react in the dark at room temperature for 5 min, add 5 mL of saturated sodium carbonate and mix well, and let it stand and react in the dark at room temperature for 2 h. Finally, measure the absorbance value of the sample solution at a wavelength of 760 nm. And make a standard curve with the absorbance values corresponding to 5 different concentrations (mg / mL) of phenolic acid esters.

[0089] (2)Determine the hydrodynamic diameter of phenolic acid ester-modified chitosan with different grafting rates using a Malvern potentiometer.

[0090] Figure 5 This is a composite graph of the grafting rate and hydrodynamic diameter of the PCNPs prepared in Examples 1, 5, and 6 of the present invention. The amphiphilic property of phenolic acid ester-modified chitosan enables the hydrophobic part of the phenolic acid ester to be encapsulated within the nanoparticles through simple stirring in an acetic acid aqueous solution due to hydrophobic interactions, while the hydrophilic chitosan chains assemble on its outer surface. This arrangement increases the intramolecular aggregation and AIE intensity, and also increases the water dispersibility of phenolic acid ester-modified chitosan. The ratio of hydrophobic groups to hydrophilic groups affects the particle size of the nanoparticles, and thus affects the activity of the nanoparticles. When the addition amount of phenolic acid ester is 1.5 mM and the grafting rate is 189.48 mg / g, the nanoparticles with the smallest particle size can be obtained.

[0091] Example of implementation effect 3 Taking the PCNPs1 prepared in Example 1 and the chitosan solution prepared in Comparative Example 1 as examples, the antibacterial effects on Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were synergistically irradiated with blue light at 420 nm: (1)Overnight culture E. coli , P. aeruginosa the bacterial suspension to the late logarithmic growth phase (~10 9 CFU / mL), and set aside. Dilute the bacterial suspension with LB liquid medium to 2×10 7 CFU / mL. Add 100 μL of PCNPs and chitosan solution to 100 μL of E. coli , P. aeruginosa the bacterial suspension respectively. The final concentrations of PCNPs and chitosan solution are both 0.8 mg / mL, and the concentration of the bacterial suspension is 1×10 7 CFU / mL. Irradiate with blue light at 420 nm or incubate in the dark for 1 - 10 min, and the blue light intensity is 0.213 W / cm 2 . After the incubation, take 100 uL of the mixed liquid, spread it evenly on the technical medium, and after culturing at 37 °C for 24 h, use standard plate counting to determine the colony-forming units.

[0092] (2)Overnight culture S. aureus the bacterial suspension to the late logarithmic growth phase (~10 9 CFU / mL), and set aside. Dilute the bacterial suspension with LB liquid medium to 2×10 6 CFU / mL. Add 100 μL of PCNPs and chitosan solution to 100 μL of E. coli , P. aeruginosaIn the bacterial suspension, the final concentrations of PCNPs and chitosan solution were both 0.8 mg / mL, the concentration of the bacterial suspension was 1×10 6 CFU / mL, irradiated with blue light at 420 nm or incubated in the dark for 1 - 10 min, and the blue light intensity was 0.213 W / cm 2 . After incubation, 100 μL of the mixed liquid was taken and evenly spread on the technical culture medium. After culturing at 37 °C for 24 h, standard plate counting was used to determine the colony - forming units.

[0093] Figure 6 This is a dot - line graph showing the antibacterial effects of the PCNPs prepared in Example 1 of the present invention and the chitosan prepared in Comparative Example 1 on Gram - negative bacteria Escherichia coli and Pseudomonas aeruginosa and Gram - positive bacteria Staphylococcus aureus under conditions of the presence or absence of blue - light irradiation. 100 μL of PBS buffer solution containing PCNPs and chitosan solution with a concentration of 8 mg / mL, and the separate PBS buffer solution was used as a blank control. The pH of the buffer solution was 4.0 and the concentration was 0.2 M. It was added to 900 μL of the bacterial suspension. The concentrations of Escherichia coli and Pseudomonas aeruginosa were 1×10 7 CFU / mL, and the concentration of Staphylococcus aureus was 1×10 6 CFU / mL. It was irradiated with blue light at 420 nm or incubated in the dark for different times, and the blue - light intensity was 0.213 W / cm 2 . After incubation at 37 °C for 24 h, standard plate counting was used to determine the colony - forming units. As shown in the figure, pure blue light had no obvious antibacterial effect. Under the condition of no light, the antibacterial rates of the chitosan solution and PCNPs were about 99%. After blue - light irradiation, the antibacterial ability of the chitosan solution was slightly improved, and the antibacterial ability of PCNPs was greatly improved. Because of electrostatic adsorption and hydrophobic binding, and a large amount of ROS was generated under blue - light irradiation, it could kill 100% of the bacteria.

[0094] Figure 7 This is a coating result graph showing the antibacterial effects of the PCNPs prepared in Example 1 of the present invention and the chitosan solution prepared in Comparative Example 1 on Gram - negative bacteria Escherichia coli and Pseudomonas aeruginosa and Gram - positive bacteria Staphylococcus aureus under blue - light irradiation conditions. The figure shows that under the same conditions, there were still a large number of residual colony counts in the chitosan solution, while PCNPs, due to electrostatic adsorption and hydrophobic binding, and a large amount of ROS was generated under blue - light irradiation, achieved complete killing of bacteria.

[0095] Example of implementation effect 4 Taking the PCNPs1 prepared in Example 1 and the chitosan solution prepared in Comparative Example 1 as an example, the photo - activated peroxidase - like mechanism of phenolic acid - ester - modified chitosan: The ability of PCNPs to generate ROS under blue light irradiation was detected using the HPF hydroxyl radical probe, Aplex Red / HRP hydrogen peroxide probe, and SOSG singlet oxygen fluorescence probe. PCNPs or chitosan solution was added to a 96-well plate and mixed with HPF or SOSG. The concentrations of both PCNPs and chitosan solution were 0.8 mg / mL, and the concentrations of HPF and SOSG were 5 μM. After different irradiation times with blue light, the fluorescence intensity was measured using a microplate reader. For HPF (λ ex / λ em =492 / 515 nm), and for SOSG (λ ex / λ em =504 / 525 nm). To measure the production of hydrogen peroxide, PCNPs or chitosan solution was added to a 96-well plate and mixed with AR / HRP. After incubation for 30 min, after different irradiation times with light, the fluorescence intensity was measured using a microplate reader, with λ ex / λ em =571 / 585 nm. Figure 8 This shows the ability of the PCNPs prepared in Example 1 of the present invention and the chitosan solution prepared in Comparative Example 1 to produce hydroxyl radicals, hydrogen peroxide, and singlet oxygen under blue light irradiation. The HPF experimental results show that there was no obvious change in the chitosan solution over different times, while there was an obvious change in the fluorescence intensity of PCNPs within 1 min, indicating that PCNPs can generate hydroxyl radicals after blue light irradiation. The SOSG and AR / HRP results show that within 3 min, the fluorescence intensity of the chitosan solution increased slightly, indicating that chitosan itself can also produce hydrogen peroxide and singlet oxygen under blue light irradiation, but the fluorescence intensity of PCNPs increased significantly, indicating that grafting lauryl gallate greatly improved the ability to produce hydrogen peroxide and singlet oxygen.

[0096] Example of implementation effect 5 Taking the PCNPs1 prepared in Example 1 and the chitosan solution prepared in Comparative Example 1 as an example, the ability of phenolic acid ester-modified chitosan to capture bacteria and fluorescence imaging: Figure 9 This is the fluorescence image of the PCNPs prepared in Example 1 of the present invention after co-incubation with Escherichia coli and Staphylococcus aureus. Clear blue fluorescence images of Escherichia coli and Staphylococcus aureus were observed under 405 nm laser excitation. When excited with 488 and 561 nm lasers, green and red fluorescence images of the bacteria were observed respectively. These findings indicate that PCNPs can interact with the surface of bacteria to achieve efficient bacterial adsorption and bacterial imaging.

[0097] Example of implementation effect 6 The inhibitory effect of the PCNPs prepared in Example 1 in combination with 420 nm blue light irradiation on Escherichia coli biofilm: To evaluate the biofilm removal ability of PCNPs, 0.2 mL of an Escherichia coli suspension with a concentration of 1×10 9 CFU / mL was mixed with 1.8 mL of TSB in a CLSM quartz culture dish. A mature biofilm was obtained after 12 h. Then, the suspension was removed, and the remaining mature biofilm was carefully rinsed with PBS to remove planktonic bacteria. Subsequently, the biofilm was co-incubated with 0.8 mg / mL of PCNPs under blue light at 420 nm with a light intensity of 0.213 W / cm 2 for 30 min. The treated biofilm was stained using the Live / Dead Baclight Bacterial Viability Kit and incubated in the dark for 15 min to observe the 3D structure of the biofilm by confocal laser scanning microscopy (CLSM). Figure 10 Three-dimensional CLSM images of the clearing effect of the PCNPs prepared in Example 1 of the present invention on mature Escherichia coli biofilms in the presence and absence of blue light and blue light alone. We stained the inflammation after different treatments and observed the death of Escherichia coli cells in the mature Escherichia coli biofilm. As shown in the figure, Escherichia coli in the biofilm showed little death and no red fluorescence when treated with PCNPs alone or blue light irradiation alone. In contrast, Escherichia coli in the biofilm showed a large amount of red fluorescence and a significantly thinner biofilm thickness after treatment with PCNPs plus BL irradiation. These results indicate that PCNPs have excellent anti-biofilm properties when combined with BL irradiation.

[0098] Figure 11 QCM-D diagram of the interaction between the PCNPs prepared in Example 1 of the present invention and the phospholipid bilayer. In the upper right quadrant of the diagram, the negative trend of frequency and the positive change in dissipation are characteristic of binding, indicating that PCNPs initially bind to the surface of the phospholipid bilayer through electrostatic and hydrophobic interactions. When the changes in both Δf and ΔD begin to level off, it indicates that the adsorption of PCNPs on the phospholipid bilayer has reached a saturated state. Subsequently, ΔD decreased significantly and Δf increased, which are characteristic of transmembrane insertion and membrane disruption, revealing that the hydrophobic chains on PCNPs insert into the phospholipid bilayer and disrupt the membrane structure.

[0099] Figure 12DNA electrophoresis pattern of the effect of PCNPs-mediated PDI prepared in Example 1 of the present invention on the genomic DNA of Escherichia coli and eDNA in Escherichia coli biofilms. Agar-gel electrophoresis was used to measure the DNA damage of suspended Escherichia coli and the damage of eDNA in mature Escherichia coli, respectively. As can be seen from the figure, after the treatment of PCNPs combined with blue light irradiation, the DNA intensity decreased significantly, indicating that the treatment of PCNPs combined with blue light irradiation can effectively damage the cell membrane of Escherichia coli cells, resulting in the leakage of contents, and the ROS generated causes macromolecular damage, mainly DNA. eDNA plays an important role in the formation of bacterial biofilms. After the treatment of PCNPs combined with blue light irradiation, the level of eDNA remaining in the biofilm decreased significantly, indicating that PCNPs can achieve excellent anti-biofilm properties by destroying eDNA.

[0100] Example of implementation effect 7 Taking the PCNPs1 prepared in Example 1 and the chitosan solution prepared in Comparative Example 1 as an example, the phenolic acid ester-modified chitosan is used for the preservation of fruits and vegetables: After washing the grapes, select grapes of the same specification and divide them into four groups. Each group is scalded with a flame to form a breakage, and the breakage surface is about 7 cm 2 . Cover the broken part of the grapes with a gauze soaked in an Escherichia coli solution with a concentration of 10 8 CFU / mL for 60 min. After the infection is established, use 200 μL of PCNPs and chitosan solution with a concentration of 0.8 mg / mL to sterilize the broken surface, and irradiate under blue light with a wavelength of 420 nm and an intensity of 0.213 W / cm 2 for 5 min. After storing at room temperature of 25 o °C for 8 days, measure the number of remaining colonies on the grapes. Use the sample treated with deionized water as a control.

[0101] Figure 13 Photographic record of the use of PCNPs1 prepared in Example 1 of the present invention and the chitosan solution prepared in Comparative Example 1 for the preservation of grapes. As can be seen from the figure, for the control and only blue light irradiation treatment, the grape samples began to deteriorate on the second day and showed a large amount of deterioration on the eighth day. The chitosan solution combined with blue light inhibited the deterioration of grapes by harmful bacteria to a certain extent, and the grapes deteriorated on the fourth day. However, the treatment of PCNPs combined with blue light greatly extended the preservation time of the grapes, and the grapes showed slight deterioration on the eighth day. By counting the number of remaining colonies on the grapes on the eighth day, it can also be seen that PCNPs can effectively inhibit and kill the bacteria on the surface of the grapes, achieving the effect of long-term preservation.

[0102] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A self-assembled nanoparticle, characterized in that: The self-assembled nanoparticles are prepared by dissolving chitosan modified with phenolic acid ester in an acetic acid solution, and self-assembling the obtained mixed solution under stirring conditions to obtain nanoparticles with a particle size of 100-150 nm.

2. The self-assembled nanoparticle according to claim 1, characterized in that: The phenolic acid ester modified chitosan is obtained by grafting phenolic acid ester with the activation groups on the chitosan activated by free radicals, and the activation groups are amino groups and hydroxyl groups.

3. The self-assembled nanoparticle according to claim 2, characterized in that: The phenolic acid ester is any one of gallic acid alkyl ester, caffeic acid alkyl ester, chlorogenic acid alkyl ester and ferulic acid alkyl ester.

4. The self-assembled nanoparticle according to any one of claims 1 to 3, characterized in that: The mass fraction of phenolic acid ester modified chitosan in the mixed solution is 0.5-2.5%; the volume fraction of acetic acid solution is 1-2%.

5. The method for preparing the self-assembled nanoparticles according to claim 4, characterized in that: The steps are: (1) Dissolving the modified chitosan in an acetic acid solution and self-assembling to form nanoparticles under stirring conditions; (2) The nanoparticles in step (1) are filtered to obtain self-assembled nanoparticles, i.e., PCNPs.

6. The method for preparing self-assembled nanoparticles according to claim 5, characterized in that: In the step (1), the final concentration of the modified chitosan is 0.5-2.5%wt; the volume fraction of the acetic acid solution is 1-2%; the stirring condition is 400-800 r / min, and the stirring time is 2.5-3 h.

7. The method for preparing self-assembled nanoparticles according to claim 5, characterized in that: The preparation method of the modified chitosan is: a. Adding an acetic acid solution of chitosan to an aqueous solution of ascorbic acid in hydrogen peroxide, after the reaction, adding an ethanol solution of phenolic acid ester to the reaction solution, and protecting the reaction from light to obtain a mixed solution; b. Adjusting the pH of the mixed solution in step a to neutral, collecting the solid, washing and drying it to obtain modified chitosan.

8. The method for preparing self-assembled nanoparticles according to claim 7, characterized in that: The volume fraction of the acetic acid solution in step a is 1-2%; the mass fraction of the chitosan acetic acid solution is 0.5-2.5%; the molecular weight of the chitosan is 100KDa, and the degree of deacetylation is 90%; the concentration of phenolic acid ester in the ethanol solution of phenolic acid ester is 0.5-3 mM; and the molar ratio of chitosan monomer to phenolic acid ester in the mixed solution is 1-5:

1.

9. The method for preparing self-assembled nanoparticles according to claim 7, characterized in that: The ascorbic acid concentration is 0.5-1 M, and the hydrogen peroxide concentration is 5 M.

10. Use of the self-assembled nanoparticles according to any one of claims 1 to 3, characterized in that: The application is selected from any of the following: ① Antibacterial effect combined with blue light irradiation; ② Combined with blue light irradiation to fight biofilm; ③ Fluorescence imaging monitoring of harmful bacteria ④Keep fruits and vegetables fresh.

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