Double-responsiveness nano-drug controlled release system based on polydopamine as well as preparation and application of double-responsiveness nano-drug controlled release system

Abstract

According to the ROS / enzyme dual-responsive nano-drug controlled release system and the preparation method and application thereof, under the dark condition, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 4-dimethylaminopyridine are dissolved in dichloromethane respectively, protoporphyrin is added, stirring activation is performed, and the ROS / enzyme dual-responsive nano-drug controlled release system is obtained; placing the activation system in an ice-water bath, slowly dropwise adding a hydroxytyrosol solution, transferring to room temperature after dropwise adding, stirring for reaction, and purifying to obtain an intermediate product PPIX-DE; and dissolving the intermediate product PPIX-DE in N, N-dimethylformamide (DMF), mixing with a Tris buffer solution containing dopamine hydrochloride, adjusting the pH value of the system to 8.5, carrying out a stirring reaction at room temperature in a dark place, and after the reaction is finished, carrying out purification treatment to obtain the final product PDAP nanoparticles. Polydopamine (PDA) is used as a carrier and is copolymerized with protoporphyrin (PPIX) modified by hydroxytyrosol (DE) to obtain the polydopamine protoporphyrin nanoparticles (PDAP), so that the stability, the water solubility and the pathogen targeting property of the photosensitizer drug protoporphyrin are improved.

Description

Technical Field

[0001] This invention belongs to the field of pesticide technology, specifically relating to a dual-responsive nanomedicine controlled-release system and its preparation and application. Background Technology

[0002] During the growth, transportation, and storage of fruits, pathogen infection often leads to fruit rot and spoilage, causing huge economic losses. To combat bacterial contamination, large amounts of antibacterial agents are typically sprayed onto the fruit surface. However, due to the drug resistance of harmful microorganisms, global food quality and safety still face serious challenges. Furthermore, traditional antibacterial agents, lacking the ability to target bacteria, significantly reduce the utilization efficiency of their active ingredients. Therefore, developing a novel antibacterial system with low toxicity, a novel bactericidal mechanism, and high bacterial targeting ability to improve drug utilization is of great promise in the field of fruit protection. Photodynamic inactivation (PDI), as an emerging bacterial inactivation strategy, has recently shown great potential, rapidly and efficiently eliminating bacteria without inducing drug resistance. Unlike traditional antibacterial agents, ROS-mediated photoinduced bactericidal action exhibits a unique multi-target mechanism, significantly reducing the likelihood of target microorganisms developing drug resistance.

[0003] Protoporphyrin (PPIX) belongs to the second-generation photosensitizers and is the most reactive molecule among porphyrin photosensitizers. It has good singlet oxygen yield and low phototoxicity, and is also a precursor for chlorophyll synthesis in plants. This not only indicates that protoporphyrin has high efficiency in killing bacteria and pathogens, but also that it has certain benefits for plants after appropriate absorption. These characteristics are sufficient to demonstrate its suitability for plant protection. However, currently, research and application of protoporphyrin in fruit protection are limited.

[0004] The reasons why protoporphyrin is rarely used in the field of fruit protection can be attributed to the following points: (1) Poor photostability. The ROS generated by the protoporphyrin under light can oxidize its own structure, resulting in photodynamic inactivation and failing to achieve long-term efficacy; (2) Poor solubility. Most drugs are water-soluble pesticides rather than organic solvents, but protoporphyrin is prone to aggregate in water, which leads to fluorescence quenching, and this will also cause it to lose photodynamic activity; (3) Lack of pathogen targeting. The indiscriminate killing effect of ROS will harm normal plant cells. Summary of the Invention

[0005] The purpose of this invention is to provide a ROS / enzyme dual-responsive nanomedicine controlled release system based on polydopamine. Polydopamine (PDA), which has good biocompatibility, strong fluorescence quenching effect and good dispersibility in water, is used as a carrier and copolymerized with protoporphyrin (PPIX) modified with hydroxytyrosol (DE) to obtain polydopamine-based protoporphyrin nanoparticles (PDAP). This improves the stability, water solubility and targeting of the photosensitizer drug protoporphyrin to pathogens.

[0006] To achieve the above objectives, the following technical solution is adopted: A method for preparing a polydopamine-based ROS / enzyme dual-responsive nanomedicine controlled-release system includes the following steps: S1. Under light-protected conditions, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) were dissolved in dichloromethane (DCM), and protoporphyrin (PPIX) was added and stirred for activation. Then, the activated system was placed in an ice-water bath and hydroxytyrosol (DE) solution was slowly added dropwise. After the addition was completed, the system was transferred to room temperature and stirred for reaction. The intermediate product PPIX-DE was obtained after purification. S2. Dissolve the intermediate product PPIX-DE in N,N-dimethylformamide (DMF), mix it with a Tris buffer solution containing dopamine hydrochloride (DA·HCl), adjust the pH of the system to 8.5, stir the reaction at room temperature in the dark, and after the reaction is completed, purify the product to obtain the final product PDAP nanoparticles.

[0007] According to the above scheme, in step S1, the molar ratio between 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, hydroxytyrosol and protoporphyrin is (5~6):(5~6):(1~1.5):1.

[0008] According to the above scheme, the total proportion of solute in the activation system in step S1 is 3.0~3.5wt%; the concentration of the hydroxytyrosol solution is 0.40~0.45 M; after the addition is complete, the mixture is transferred to room temperature and stirred for 40~48 h.

[0009] According to the above scheme, the purification process in step S1 includes: washing the crude product with deionized water and anhydrous ethanol, dispersing it in water after complete washing, and then freeze-drying it to obtain the intermediate product PPIX-DE.

[0010] According to the above scheme, the mass ratio of dopamine hydrochloride to intermediate product DE-PPIX in step S2 is (100~110):1.

[0011] According to the above scheme, the concentration of the intermediate product PPIX-DE in DMF in step S2 is 0.015~0.020 M; the concentration of dopamine hydrochloride in Tris buffer solution is 0.12~0.15 M; and the reaction is carried out under light-protected conditions at room temperature with stirring for 20~24 h.

[0012] According to the above scheme, the Tris buffer solution in step S2 is prepared using the following method: Mix 50 mL of 0.1 M Tris alkaline solution with 14.7 mL of 1 M HCl, adjust the volume to 100 mL, and finally fine-tune the pH to 8.5 with 1 M HCl / 1 M NaOH.

[0013] According to the above scheme, the purification process in step S2 includes: transferring the mixture after the reaction into a 3500 Da dialysis bag and dialyzing it with an N,N-dimethylformamide aqueous solution. After dialysis, centrifuging is performed to collect the lower layer product, which is then washed multiple times with DMSO. Finally, it is dispersed in deionized water and freeze-dried to obtain the final product, PDAP nanoparticles. The volume ratio of the N,N-dimethylformamide aqueous solution to water in the dialysis process is 2:1.

[0014] This invention also provides a polydopamine-based ROS / enzyme dual-responsive nanomedicine controlled-release system, prepared using the above-described method. Furthermore, the application of this dual-responsive nanomedicine controlled-release system in plant protection is also provided.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes polydopamine as a base, exhibiting strong hydrophilicity, biocompatibility, adhesion, and powerful fluorescence quenching ability. First, protoporphyrin is modified using hydroxytyrosol via ester condensation, then copolymerized with dopamine to form nanoparticles. These nanoparticles improve the water solubility of protoporphyrin; the strong fluorescence quenching ability significantly enhances the photostability of protoporphyrin. Due to the unique structure of polydopamine, the light energy absorbed by excited-state PPIX can be efficiently transferred to the PDA carrier, ultimately released as heat energy. This means that the ability of this energy to transfer to surrounding oxygen molecules to generate singlet oxygen is significantly reduced. The chemical structure of polydopamine is similar to that of mussel byssal protein (rich in dopamine), exhibiting strong bioadhesion. Furthermore, the dopamine molecule contains abundant catechol and amino groups, enabling the nanomedicine to tightly bind to the waxy layer on the plant surface through hydrogen bonding. This is one of the advantages of polydopamine in plant protection. Attached Figure Description

[0016] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. In particular, the accompanying drawings described below are only some embodiments of the present invention.

[0017] Figure 1 The infrared absorption spectrum of the intermediate product PPIX-DE prepared in Example 3 is shown.

[0018] Figure 2 The 1H NMR spectrum of the intermediate product PPIX-DE prepared in Example 3.

[0019] Figure 3The UV-Vis absorption spectra of the intermediate product PPIX-DE prepared in Example 3 and the PDA-DE-PPIX (PDAP) nanoparticles prepared in Example 3.

[0020] Figure 4 TEM images of PDAP nanoparticles and PDA nanoparticles prepared in Example 3.

[0021] Figure 5 This is a schematic diagram showing the retention rate of PPIX in the PDAP nanoparticles prepared in Example 3 under ultraviolet light irradiation during the photostability experiment.

[0022] Figure 6 The cumulative drug release rate trend of the PDAP nanoparticles prepared in Example 3 under different stimulus conditions in the stimulus response investigation experiment.

[0023] Figure 7 This is a schematic diagram showing the change of DPBF cumulative oxidation rate with light exposure time in the photoactivity experiment of PDAP nanoparticles prepared in Example 3.

[0024] Figure 8 The antibacterial effects of the PDAP nanoparticles prepared in Example 3 against Staphylococcus aureus and Escherichia coli are shown in the figure.

[0025] Figure 9 -11 is a schematic diagram showing the inhibition rates of PDAP nanoparticles prepared in Example 3 against Staphylococcus aureus and Escherichia coli compared to different control groups. Specifically, Figure 9 The absorbance of Staphylococcus aureus and Escherichia coli cells in different experimental groups; Figure 10 The inhibition rate of Staphylococcus aureus in different experimental groups in plate culture antibacterial experiment; Figure 11 The inhibition rates of PDAP and control PPIX against Staphylococcus aureus and Escherichia coli after light treatment are shown.

[0026] Figure 12-15 The protective ability of PDAP nanoparticles prepared in Example 3 against fruit was tested. Specifically, Figure 12 Image of the appearance of grapes; Figure 13 The percentage of grape quality loss; Figure 14 A schematic diagram illustrating pH changes in grapes; Figure 15 This represents the sugar loss rate of grapes. Detailed Implementation

[0027] To facilitate understanding of the present invention, a clearer and more comprehensive description is provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough understanding of the disclosure of the present invention. Unless otherwise defined, all technical and scientific terms used in this invention are part of the scope of this invention.

[0028] A specific embodiment provides a ROS / enzyme dual-responsive nanomedicine controlled-release system based on polydopamine. The intermediate product PPIX-DE is obtained by linking hydroxytyrosol (DE) and protoporphyrin (PPIX) via ester bonds. This intermediate product is then copolymerized with dopamine in a Tris buffer solution at pH 8.5 to obtain the final product, PDAP nanoparticles. At the site of plant pathogen infection, pathogens secrete excessive ROS and various hydrolytic enzymes to further damage the plant. Overexpressed ROS disrupts and degrades the structure of the nanoparticles. Simultaneously, under the specific activation of esterases, the ester bonds are broken, releasing photosensitizing drugs to kill the pathogens. This nanomedicine controlled-release system exhibits superior pathogen targeting and avoids damage to healthy plants, showing promising application prospects in plant protection and photodynamic antibacterial applications.

[0029] A specific embodiment provides a method for preparing the above-mentioned dual-responsive nanomedicine controlled release system: S1: Under dark conditions, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) were dissolved in 20 mL of dichloromethane (DCM) to obtain a mixed solution. A certain amount of protoporphyrin was then weighed and added to the system, and the mixture was activated at room temperature for 6 h under N2 atmosphere. A certain amount of hydroxytyrosol was then dissolved in N,N-dimethylformamide solution. After activation, the solution was slowly added dropwise to the above reaction system in an ice-water bath. The reaction was continued to be stirred at room temperature for 40-48 h. After the reaction was completed, the intermediate product PPIX-DE was obtained.

[0030] Preferably, the total proportion of hydroxytyrosol in the mixed solution is 3%, and the concentration of hydroxytyrosol in the N,N-dimethylformamide solution is 0.43 M.

[0031] Specifically, the purification process includes: evaporating the reaction solution under reduced pressure using a rotary evaporator, washing it three times with ethanol and deionized water respectively, dispersing it in deionized water after complete washing, and freeze-drying it to obtain the intermediate product PPIX-DE.

[0032] Preferably, the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, hydroxytyrosol, and protoporphyrin is 5:5:1.2:1.

[0033] S2: Under dark conditions, the intermediate product PPIX-DE was first dissolved in 10 mL of N,N-dimethylformamide solution, and then dopamine was dissolved in 10 mL of Tris buffer solution with pH=8.5. The two solutions were then mixed evenly, and the pH was adjusted to 8.5 with 1 M NaOH. The reaction was carried out at room temperature for 24 h to obtain the final product PDAP nanoparticles.

[0034] Specifically, the purification process includes: after the reaction is completed, the reaction solution is transferred to a dialysis bag with a cutoff of 3500 and dialyzed with DMF aqueous solution. After dialysis, the supernatant is discarded by centrifugation, and the lower layer product is washed three times with DMSO. After washing, the solution is dispersed in deionized water and freeze-dried to obtain the final product PDAP.

[0035] Preferably, the concentration of PPIX-DE in N,N-dimethylformamide is 3 mM, and the concentration of dopamine in Tris buffer solution is 1.3 M, wherein the mass ratio of PPIX-DE to dopamine is 1:100. The Tris buffer solution is prepared as follows: 50 mL of 0.1 M Tris alkaline solution is mixed with 14.7 mL of 1 M HCl, the volume is adjusted to 100 mL, and finally the pH is finely adjusted to 8.5 with 1 M HCl / 1 M NaOH.

[0036] The above method prepared ROS / enzyme dual-responsive photodynamic nanomedicines. Polydopamine, a highly hydrophilic and safe non-toxic substrate, was used as the base and copolymerized with a modified photosensitizer, protoporphyrin, to obtain particles. These nanoparticles can inhibit the absorption of photons by PPIX, causing a transition from the ground state to the excited state, thus suppressing the photosensitizer protoporphyrin's photosensitivity and improving its photosensitizer photosensitizer photosensitizer photosensitizer photosensitizer photosensitizer photosensitizer photosensitizer photosensitizer. At the site of plant pathogen infection, this system can respond to the highly expressed ROS environment at the infection site and the esterases within the pathogens and pests, disrupting the nanoparticle structure, releasing the photosensitizer drug, and restoring the photosensitizer drug's photodynamic activity, achieving targeted bactericidal and insecticidal effects.

[0037] The present invention will be further described below with reference to the embodiments, but is not limited to the embodiments described below.

[0038] Example 1 The preparation method of ROS / enzyme dual-responsive photodynamic nano-antibacterial system 1 includes the following steps: (1) Preparation of PPIX-DE: Under light-protected conditions, EDCI (0.34 g, 1.78 mmol) and DMAP (0.217 g, 1.78 mmol) were dissolved in 15 mL of dichloromethane, and PPIX (0.2 g, 0.355 mmol) was dissolved in 5 mL of dichloromethane. The PPIX solution was then slowly added to the above system, mixed thoroughly, and then protected with N2. The mixture was stirred at room temperature for 6 h. After complete activation, DE (0.066 g, 0.43 mmol) was completely dissolved in 1 mL of DMF and slowly added dropwise to the above activation system under an ice-water bath atmosphere. The reaction was then continued for 40 h. After the reaction was completed, the reaction solution was evaporated under reduced pressure using a rotary evaporator. The solution was then washed three times with ethanol and deionized water, respectively. After complete washing, the solution was dispersed in deionized water and freeze-dried to obtain the PPIX-DE sample.

[0039] (2) Preparation of PDAP1 nanoparticles: Under light-protected conditions, DA·HCl (0.2 g, 1.3 mmol) was dissolved in 10 mL of Tris buffer solution with pH=8.5, and PPIX-DE (0.02 g, 0.03 mmol) was dissolved in 10 mL of DMF solution. Then, the DMF solution of PPIX-DE was slowly added dropwise to the Tris buffer solution containing DA·HCl. After the addition was complete, the pH was adjusted to 8.5 with 1M NaOH. The reaction was carried out at room temperature for 24 h under light-protected conditions. After the reaction was completed, the reaction solution was transferred to a dialysis bag with a cutoff of 3500 mL and dialyzed with DMF aqueous solution for 1 day. After dialysis, the solution was centrifuged at 10000 rpm for 10 min, the supernatant was discarded, and the lower layer product was washed several times with DMSO and water until all unreacted PPIX-DE, DA and excess impurities were removed. Then, the product was dispersed in deionized water and freeze-dried to obtain the final product PDAP1 nanoparticles.

[0040] Example 2 The preparation method of ROS / enzyme dual-responsive photodynamic nano-antibacterial system 2 includes the following steps: (1) Preparation of PPIX-DE: Under light-protected conditions, EDCI (0.34 g, 1.78 mmol) and DMAP (0.217 g, 1.78 mmol) were dissolved in 15 mL of dichloromethane, and PPIX (0.2 g, 0.355 mmol) was dissolved in 5 mL of dichloromethane. The PPIX solution was then slowly added to the above system, mixed thoroughly, and then protected with N2. The mixture was stirred at room temperature for 6 h. After complete activation, DE (0.066 g, 0.43 mmol) was completely dissolved in 1 mL of DMF and slowly added dropwise to the above activation system under an ice-water bath atmosphere. The reaction was then continued for 40 h. After the reaction was completed, the reaction solution was evaporated under reduced pressure using a rotary evaporator. The solution was then washed three times with ethanol and deionized water, respectively. After complete washing, the solution was dispersed in deionized water and freeze-dried to obtain the PPIX-DE sample.

[0041] (2) Preparation of PDAP2 nanoparticles: Under light-protected conditions, DA·HCl (0.2 g, 1.3 mmol) was dissolved in 10 mL of Tris buffer solution at pH 8.5, and PPIX-DE (0.004 g, 0.006 mmol) was dissolved in 10 mL of DMF solution. Then, the DMF solution of PPIX-DE was slowly added dropwise to 10 mL of Tris buffer solution containing DA·HCl (0.2 g, 1.3 mmol). After the addition was complete, the pH was adjusted to 8.5 with 1 M NaOH, and the reaction was carried out at room temperature for 24 h under light-protected conditions. After the reaction was completed, the reaction solution was transferred to a dialysis bag with a cutoff of 3500 mL and dialyzed with DMF aqueous solution for 1 day. After dialysis, the solution was centrifuged at 10000 rpm for 10 min, the supernatant was discarded, and the lower layer product was washed several times with DMSO and water until all unreacted PPIX-DE, DA and excess impurities were removed. Then, the product was dispersed in deionized water and freeze-dried to obtain the final product PDAP2 nanoparticles.

[0042] Example 3 The preparation method of ROS / enzyme dual-responsive photodynamic nano-antibacterial system 3 includes the following steps: (1) Preparation of PPIX-DE: Under light-protected conditions, EDCI (0.34 g, 1.78 mmol) and DMAP (0.217 g, 1.78 mmol) were dissolved in 15 mL of dichloromethane, and PPIX (0.2 g, 0.355 mmol) was dissolved in 5 mL of dichloromethane. The PPIX solution was then slowly added to the above system, mixed thoroughly, and then protected with N2. The mixture was stirred at room temperature for 6 h. After complete activation, DE (0.066 g, 0.43 mmol) was completely dissolved in 1 mL of DMF and slowly added dropwise to the above activation system under an ice-water bath atmosphere. The reaction was then continued for 40 h. After the reaction was completed, the reaction solution was evaporated under reduced pressure using a rotary evaporator. The solution was then washed three times with ethanol and deionized water, respectively. After complete washing, the solution was dispersed in deionized water and freeze-dried to obtain the PPIX-DE sample.

[0043] (2) Preparation of PDAP3 nanoparticles: Under light-protected conditions, DA·HCl (0.2g, 1.3mmol) was dissolved in 10mL of Tris buffer solution with pH=8.5, and PPIX-DE (0.002g, 0.003mmol) was dissolved in 10mL of DMF solution. Then, the DMF solution of PPIX-DE was slowly added dropwise to 10mL of Tris buffer solution containing DA·HCl (0.2g, 1.3mmol). After the addition was completed, the pH was adjusted to 8.5 with 1M NaOH. The reaction was carried out at room temperature for 24h under light-protected conditions. After the reaction was completed, the reaction solution was transferred to a dialysis bag with a cutoff of 3500 and dialyzed with DMF aqueous solution for 1 day. After dialysis, the solution was centrifuged at 10000 rpm for 10 min, the supernatant was discarded, and the lower layer product was washed with DMSO and water several times until all unreacted PPIX-DE, DA·HCl and excess impurities were removed. The product was then dispersed in deionized water and freeze-dried to obtain the final product PDAP3 nanoparticles.

[0044] Taking Example 3 as an example, unless otherwise specified, the nanoparticles used in subsequent experiments were all PDAP3 nanoparticles prepared in Example 3. To verify the successful synthesis of the PPIX-DE and PDAP nanoparticles prepared in Example 3, we used infrared spectroscopy, nuclear magnetic resonance characterization, ultraviolet analysis, and photoelectron spectroscopy to determine the structure of the PPIX-DE and PDAP nanoparticles.

[0045] Figure 1 The image shows the infrared absorption spectrum of the PPIX-DE intermediate prepared in Example 3. Compared with the infrared spectrum of PPIX, the modified PPIX-DE shows a new absorption peak at 1150 cm⁻¹. This peak is attributed to the stretching vibration peak of COC on the ester group. The appearance of this absorption peak proves that a new ester bond has appeared in the compound structure, indicating that PPIX and DE have been successfully grafted.

[0046] Figure 2 The 1H NMR spectrum of the PPIX-DE intermediate prepared in Example 3. PPIX-DE and PPIX... 1 ¹H-NMR (all solvents were DMSO-d6), PPIX 1 The NMR peak assignments in the 1H-NMR spectrum are as follows: 12.27 ppm (2H, -COOH), 10.18 ppm (4H, meso-H), 8.45 ppm (2H, -CH=CH2), 6.2-6.4 ppm (4H, -CH=CH2), 4.33 ppm (4H, -C-CH2-CO), 3.60~3.69 ppm (12H, -CH3). Comparison reveals that PPIX-DE... 1The disappearance of the signal peak at 12.27 ppm (2H, -COOH) in the H-NMR spectrum is attributed to the esterification of the carboxyl group of PPIX; the newly added doublet at 8.1~8.2 ppm is attributed to the dihydroxyl groups on the DE benzene ring; 6.6 The newly added three peaks at 6.8 ppm are attributed to the three hydrogens on the DE benzene ring.

[0047] Figure 3 The UV-Vis absorption spectra of the intermediate product PPIX-DE prepared in Example 3 and the PDA-DE-PPIX (PDAP) nanoparticles prepared in Example 3. Figure 3 The mid-UV-Vis absorption spectrum shows characteristic absorption peaks at 280 nm and 405 nm, attributed to the B-band of the benzene ring in the DE molecule and the Solette band of PPIX, respectively, the latter originating from the π-π* transition in the conjugated system. Simultaneously, the Q-band absorption peak of PPIX appears in the 500-650 nm range. The UV-Vis absorption spectrum of PDAP exhibits distinct B-band and Q-band absorptions at 280 nm and 500-650 nm, with a certain degree of redshift in the Q-band. This is attributed to the extension of the conjugated system caused by intermolecular π-π stacking interactions in PPIX, which lowers the energy of the π→π* transition. The significant weakening and redshift of the Solette absorption band at 405 nm further confirms the alteration of the porphyrin ring conjugated structure. These results further demonstrate the successful synthesis of PDAP.

[0048] Taking Example 3 as an example, unless otherwise specified, the nanoparticles used in subsequent experiments were all PDAP3 nanoparticles prepared in Example 3. In order to observe the morphology and morphological differences of the PDAP and PDA prepared above, the morphology of PDA and PDAP was observed using transmission electron microscopy.

[0049] Figure 4 TEM images of PDAP nanoparticles and PDA nanoparticles prepared in Example 3. Figure 4 (a) shows a TEM image of the PDA, which exhibits a spherical structure with a particle size of approximately 150 nm. Figure 4 (b) and (c) are TEM images of PDAP. It can be seen that the size of the copolymerized nanoparticles is mostly below 100 nm. Some nanoparticles have a spherical structure, while others have irregular spheres.

[0050] Taking Example 3 as an example, unless otherwise specified, the nanoparticles used in subsequent experiments are all PDAP3 nanoparticles prepared in Example 3. The performance of PDAP is evaluated below.

[0051] (1) Investigation on the photostability of ROS / enzyme dual-response PDAP nanoparticles: The photosensitizer PPIX exhibits photodynamic activity under light irradiation, generating a large amount of ROS, which can not only oxidize the target organism but also degrade itself. Therefore, improving the photosensitizer PPIX is particularly important. Based on this, we studied the photosensitizer PPIX in PDAP nanoparticles by measuring the change in fluorescence absorption intensity of PPIX.

[0052] The specific procedure is as follows: A certain amount of PDAP solution was irradiated with a fluorescent lamp, with an equal dose of PPIX solution used as a control. The irradiation conditions were a 10W fluorescent lamp (wavelength 365 nm) and a distance of 20 cm between the lamp and the sample. During the exposure, 3 mL of sample solution was added to a quartz cuvette at fixed intervals, and the fluorescence emission spectrum of the sample solution at 405 nm was measured. The PPIX retention rate (RR) in the sample solution can be calculated using the following formula:

[0053] In the formula: F0 This indicates the fluorescence intensity of the sample at the maximum emission wavelength before illumination. Ft The fluorescence intensity of the sample at the maximum emission wavelength is represented by the illumination time t.

[0054] Figure 5 This diagram illustrates the retention rate of PPIX in the PDAP nanoparticles prepared in Example 3 under UV irradiation during a photostability experiment. Under illumination, the PPIX dispersion exhibited a retention rate of only 2.6% after 8 hours of light exposure, while the PDAP dispersion maintained a PPIX retention rate as high as 94.6%. In contrast, PDAP effectively prevents PPIX photodegradation and demonstrates excellent photostability.

[0055] (2) Investigation of drug release from ROS / enzyme dual-response PDAP nanoparticles: The drug release of PDAP under different conditions was investigated using dialysis. Four groups of identical PDAP samples were dispersed in PBS at pH 7.4. One group served as a blank control group, while the other three groups underwent the following treatments: incubation with 0.1 mM H₂O₂ for 16 h; incubation with 60 U / mL esterase for 16 h; and incubation with both 0.1 mM H₂O₂ and 60 U / mL esterase for 16 h. The concentration of the PDAP dispersions in all these cases was 0.1 mg / mL. Then, 10 mL of each solution was added to a dialysis bag with a molecular weight cutoff of 1000, immersed in 20 mL of dialysate, and shaken in a constant-temperature shaking incubator. At predetermined time intervals, 3 mL samples were taken and 3 mL of fresh dialysate was added. The PPIX content in the dialysate was calculated using a UV spectrophotometer. The cumulative release rate (CR) of PPIX was calculated using the following formula: *100% In the formula ：M0 This is the initial drug content (ug) of the nanoparticles. Mt It is the total amount of drug released at time t (ug), where t is the sampling time.

[0056] Figure 6 This is a trend graph showing the cumulative drug release rate of the PDAP nanoparticles prepared in Example 3 under different stimulus conditions in a stimulus response investigation experiment. Figure 6 As shown, under no stimuli, the cumulative release rate of PPIX at 120 h was only 6.6%, indicating that PDAP has good stability and almost no PPIX release without stimulation. After incubation with 60 U / mL esterase for 16 h, the cumulative release rate reached 55.3% at 48 h, slowly increasing to 65.5% within 120 h, which also indicates that esterase can stimulate PDAP to accelerate the release of PPIX. However, it is worth noting that after incubation with 0.1 mM H2O2 alone for 16 h, the cumulative release rate at 120 h was only 14.6%, while the cumulative release rate increased to 85.4% after incubation with both 60 U / mL esterase and 0.1 mM H2O2 for 16 h. This result shows that H2O2 does not stimulate PDAP to release PPIX, but it can synergize with esterase to more effectively stimulate the release of PPIX from nanoparticles, further demonstrating the ROS / enzyme response capability of PDAP.

[0057] (3) Investigation on the photoactivity of ROS / enzyme dual-response PDAP nanoparticles: 1,3-Diphenylisobenzofuran (DPBF) 1 O is highly selective and can interact with 1 The oxidation reaction at O ​​produces dibenzoylbenzene (DBB), which has no UV absorption, resulting in a decrease in its UV absorption intensity at 408 nm. Therefore, the oxidation rate of DPBF can be used to monitor this process. 1 The generation of O.

[0058] The specific procedures are as follows: Blank DPBF solution, PPIX solution and PDAP dispersion were used as control groups. PDAP dispersion incubated with 60 U / mL esterase for 16 h and PDAP dispersion incubated with 0.1 mM H2O2 and 60 U / mL esterase for 16 h were used as experimental groups. The concentration of DPBF solution was 20 ug / mL (solvent was DMSO), the concentration of PPIX solution was 10 ug / mL and the concentration of PDAP dispersion was 100 ug / mL. Specifically, to investigate the photodynamic activity of PPIX, PPIX solution was mixed with an equal dose of 20 μg / mL DPBF solution and an equal dose of blank DPBF solution, respectively, and the UV absorption spectrum was measured every 10 seconds under a 23W white light lamp. To investigate the effect of nanoparticles on photoactivity, PDAP dispersion and PPIX solution were added to an equal dose of 20 μg / mL DPBF solution, mixed thoroughly, and the above operation was repeated. To investigate the photodynamic activity induced by esterase stimulation and the synergistic stimulation of ROS and esterase, PDAP dispersion incubated with 60 U / mL esterase for 16 h, PDAP dispersion incubated with 0.1 mM H2O2 and 60 U / mL esterase for 16 h, and PDAP dispersion were added to an equal dose of 20 μg / mL DPBF solution, mixed thoroughly, and the above operation was repeated.

[0059] Figure 7 This is a schematic diagram showing the cumulative oxidation rate of DPBF with light exposure time in the photoactivity experiment of PDAP nanoparticles prepared in Example 3. In five different DBPF solutions, after 80 s of light exposure, the final cumulative oxidation rates of DPBF in the blank DPBF solution group, the DPBF group with added PPIX solution, the DPBF group with added PDAP dispersion, the DPBF group with added 0.1 mM H2O2 incubated for 16 h, the DPBF group with added 60 U / mL esterase incubated for 16 h, and the DPBF group with added 0.1 mM H2O2 and 60 U / mL esterase co-incubated for 16 h were 12.6%, 59.4%, 17.2%, 19.7%, 48.3%, and 56.7%, respectively. During illumination, comparing the cumulative oxidation rates of blank DPBF solution and DPBF solution with added PPIX solution, it was found that the cumulative oxidation rate increased from 12.6% to 59.4% after adding PPIX solution, indicating that PPIX can generate a large amount of oxidation products under illumination. 1 The cumulative oxidation rate of DPBF in an equal dose of PDAP dispersion was 17.2%, almost identical to that of the blank DPBF solution, but significantly lower than that of the DBPF group with added PPIX. This is because DPBF itself does not release PPIX. 1O or PPIX resulted in a relatively low oxidation rate of DPBF, which inhibited the photodynamic activity of PPIX and improved its photostability. The oxidation rate of DPBF in PDAP dispersion after hydrogen peroxide incubation was 19.7%, while the oxidation rates of DPBF in PDAP dispersion after esterase incubation and co-incubation with esterase and hydrogen peroxide increased to 48.3% and 56.7%, respectively. This indicates that esterase stimulation induced the release of a large amount of PPIX from the nanoparticles and generated a large amount of PPIX. 1 O, and hydrogen peroxide can synergistically enhance the photodynamic activity of esterases. (4) Investigation of the antibacterial activity of ROS / enzyme dual-response PDAP nanoparticles: The antibacterial activity of PDAP nanoparticles was evaluated using plate counting and cell density methods. The specific method is as follows: Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) strains were selected as antibacterial evaluation models. First, both strains were cultured in Luria-Bertham (LB) liquid medium for a certain period, and the initial absorbance (OD600) at a wavelength of 600 nm was measured until it reached an absorbance value of 0.5. The experiment was divided into 8 groups: control group, 0.1 mM H2O2 group, PDA group, PDAP group, PDAP group containing 0.1 mM H2O2, PDAP group containing 60 U / mL esterase, PDAP group containing both 0.1 mM H2O2 and 60 U / mL esterase, and free PPIX group. All experimental group samples were pretreated; samples containing esterase or H2O2 were incubated for 16 hours; all experimental groups were treated under the light conditions described in Section 2.7 above for 3 hours; the sample concentration was 0.1 mg / ml. The sample solution (0.1 mg / mL) was mixed with the bacterial suspension (volume ratio 1:1) and incubated at 37°C in the dark on a shaker (200 rpm) for 24 hours. After incubation, 3 mL of sample was taken and analyzed by UV-Vis spectrophotometry (OD). 600 The bacterial growth was determined. In addition, Staphylococcus aureus was used for a plate test. Staphylococcus aureus (OD) was placed on a plate. 600 =0.5) Perform 10 saturations with sterile PBS (pH 7.0). 5 Dilute 100 μl of the diluted suspension with the pretreatment solution, spread it onto LB agar plates, and incubate at 37°C for 24 hours. Spread the mixture evenly onto LB solid medium and incubate at 37°C for another 24 hours. Assess the antibacterial activity of PDAP by counting bacterial colonies. To assess the effect of light on the antibacterial activity of PDAP, under the aforementioned light conditions, irradiate 0.1 mg / mL PDAP dispersion for 3 hours, then add 0.1 mM H2O2 and 60 U / mL esterase, incubate for 16 hours, and set up a control group without light. Perform the same antibacterial experiment and compare the results with the above experimental results.

[0060]

[0061] Where: CFU Sample The number of colonies (CFU) on plates after different samples were exposed to light. Blank It is the colony count after plating the untreated blank sample.

[0062] Figure 8 The images show the antibacterial effects of the PDAP nanoparticles prepared in Example 3 against Staphylococcus aureus and Escherichia coli compared with different control groups. (a) is an agar plate image of different samples against Staphylococcus aureus; (b, c) are agar plate images of Staphylococcus aureus (b) and Escherichia coli (c) under PDAP treatment with or without light.

[0063] Figure 9 , 10 Figures 1 and 11 show the inhibition rates of PDAP nanoparticles prepared in Example 3 against Escherichia coli and Staphylococcus aureus compared to different control groups. Figure 9 The absorbance of Staphylococcus aureus and Escherichia coli cells after treatment; Figure 10 The inhibition rate of Staphylococcus aureus in different experimental groups in plate culture antibacterial experiment; Figure 11 The inhibition rates of PDAP and control PPIX against Staphylococcus aureus and Escherichia coli after light treatment are shown. The OD values ​​of Escherichia coli in the experimental groups treated with PDA, PDAP, PDAP mixed with H2O2, PDAP mixed with esterase, PDAP mixed with esterase and H2O2, and PPIX are also shown. 600 The values ​​decreased to 0.34, 0.31, 0.29, 0.2, 0.06, and 0.01, respectively, while the OD600 values ​​of Staphylococcus aureus decreased to 0.23, 0.21, 0.20, 0.12, 0.05, and 0, respectively. These results indicate that esterase treatment significantly enhances the antibacterial effect of PDAP, and dual treatment with esterase and hydrogen peroxide further enhances its inhibitory level. In the plate experiment, the colony growth trend of each experimental group and the OD600 value were... 600 The data were consistent. After light treatment, the inhibition rates of *Escherichia coli* and *Staphylococcus aureus* in the PDAP group were both 100%. In contrast, the bacterial inhibition rates in the PPIX group were 41.4% and 47.8%, respectively. After light exposure, the antibacterial activity of PPIX significantly decreased, but PPIX released via PDAP still maintained high antibacterial activity upon stimulation. This result is attributed to the photodegradation instability of PPIX, while the PDAP carrier significantly enhanced the photostability of PPIX.

[0064] (5) Fruit protection ability test of ROS / enzyme dual-response PDAP nanoparticles: Grapes were used as the fruit model to evaluate the fruit protection ability of PDAP nanoparticles: The specific method is as follows: First, the surface of the grapes is pre-disinfected with 75% ethanol for 10 minutes, then divided into four groups. All groups are immersed in E. coli (10... 8 Grape samples were immersed in a solution containing CFU / mL for 30 minutes. After treatment, the four groups of samples were uniformly sprayed with sterile PBS, 0.1 MPPIX solution, 0.1 M PDAP solution, and 0.1 M PDAP solution, respectively. Subsequently, the grape samples treated with PPIX solution and one of the PDAP solutions were exposed to light for 30 minutes (irradiation conditions are described in Section 2.7). All operations were performed under sterile conditions. After the samples were transferred to sterile bottles, they were incubated in a 25°C shaker incubator. Morphological changes, mass loss, sugar loss, and pH changes were monitored at preset time points.

[0065] Figure 12 , 13 14 and 15 show the physicochemical changes that occurred in grapes during the test of the protective ability of PDAP nanoparticles prepared in Example 3 against fruit. Figure 12 Image of the appearance of grapes; Figure 13 The percentage of grape quality loss; Figure 14 A schematic diagram illustrating pH changes in grapes; Figure 15 The sugar loss rate of grapes was measured. Grapes treated with sterile PBS, PDAP, and PPIX showed visible hyphae and wrinkles on the surface by day 5, and significant pulp collapse by day 7. In contrast, the PDAP group under light only showed slight shrinkage from day 10. Under the same light conditions, the antibacterial activity of PPIX on the grape surface was significantly lower than that of PDAP, attributed to the weak photostability and insufficient bacterial targeting of PPIX. This phenomenon also demonstrates that PPIX in PDAP possesses excellent photostability and bacterial targeting ability. In addition to morphological monitoring, physicochemical parameters—including mass loss, sugar content, and pH—were analyzed. After 14 days, the mass loss and sugar loss in the blank control group were 55.0% and 81.9%, respectively. After light treatment, the mass loss and sugar loss in the PDAP group decreased to 20.3% and 23.0%, respectively. Furthermore, the pH of the grapes showed an overall upward trend. Specifically, the pH of the blank control group rose to 3.72, while the pH of the PDAP-treated group only rose to 3.44. Under light conditions, the pH increase in the PDAP group was significantly lower than that in the control group. These physicochemical parameter trends indicate that grapes treated with PDAP and exposed to light suffered the least damage. This is attributed to the photodynamic antibacterial properties of PDAP, which eliminated most of the infecting bacteria. In conclusion, PDAP's superior photodynamic antibacterial properties can effectively prevent bacterial infections in grapes.

[0066] It should be noted that the above content is merely a preferred embodiment of the present invention and the technical principles applied thereto. Those skilled in the art should understand that the present invention is not limited to the specific embodiments described herein—for those skilled in the art, various adaptive adjustments and equivalent substitutions can be made to the embodiments without departing from the scope of protection of the present invention. Therefore, although the present invention has been described in detail through the above embodiments, the scope of protection of the present invention is not limited to these specific embodiments; while maintaining the core concept of the present invention, it can also cover many other equivalent implementation methods, and the specific scope of protection of the present invention should be determined by the content of the appended claims.

Claims

1. A method for preparing a ROS / enzyme dual-responsive nanomedicine controlled-release system, characterized in that... Includes the following steps: S1. Under light-protected conditions, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine were dissolved in dichloromethane, and protoporphyrin was added and stirred to activate the mixture. Then, the activated system was placed in an ice-water bath and hydroxytyrosol solution was slowly added dropwise. After the addition was completed, the mixture was transferred to room temperature and stirred to react. The intermediate product PPIX-DE was obtained after purification. S2. Dissolve the intermediate product PPIX-DE in N,N-dimethylformamide, mix it with a Tris buffer solution containing dopamine hydrochloride, adjust the pH of the system to 8.5, stir the reaction at room temperature in the dark, and after the reaction is completed, purify the product to obtain the final product PDAP nanoparticles.

2. The preparation method of the ROS / enzyme dual-responsive nanomedicine controlled release system as described in claim 1, characterized in that... In step S1, the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, hydroxytyrosol and protoporphyrin is (5~6):(5~6):(1~1.5):

1.

3. The preparation method of the ROS / enzyme dual-responsive nanomedicine controlled release system as described in claim 1, characterized in that... The total solute content in the activation system described in step S1 is 3.0~3.5 wt%; the concentration of the hydroxytyrosol solution is 0.40~0.45 M. After the addition is complete, the mixture is transferred to room temperature and stirred for 40-48 hours.

4. The preparation method of the ROS / enzyme dual-responsive nanomedicine controlled release system as described in claim 1, characterized in that... The purification process in step S1 includes washing the crude product with deionized water and anhydrous ethanol. After complete washing, the product is dispersed in water and then freeze-dried to obtain the intermediate product PPIX-DE.

5. The preparation method of the ROS / enzyme dual-responsive nanomedicine controlled release system as described in claim 1, characterized in that... The mass ratio of dopamine hydrochloride to intermediate product DE-PPIX in step S2 is (100~110):

1.

6. The preparation method of the ROS / enzyme dual-responsive nanomedicine controlled release system as described in claim 1, characterized in that... The concentration of the intermediate product PPIX-DE in DMF in step S2 is 0.015~0.020 M; the concentration of dopamine hydrochloride in Tris buffer solution is 0.12~0.15 M; and the reaction is carried out under light-protected conditions at room temperature with stirring for 20~24 h.

7. The preparation method of the ROS / enzyme dual-responsive nanomedicine controlled release system as described in claim 1, characterized in that... The Tris buffer solution mentioned in step S2 is prepared using the following method: Mix 50 mL of 0.1 M Tris alkaline solution with 14.7 mL of 1 M HCl, adjust the volume to 100 mL, and finally fine-tune the pH to 8.5 with 1 M HCl / 1 M NaOH.

8. The preparation method of the ROS / enzyme dual-responsive nanomedicine controlled release system as described in claim 1, characterized in that... The purification process in step S2 includes: transferring the mixture after the reaction to a 3500 Da dialysis bag and dialyzing it with an aqueous solution of N,N-dimethylformamide. After dialysis, centrifuging is performed to collect the lower layer product, which is then washed multiple times with DMSO. Finally, the product is dispersed in deionized water and freeze-dried to obtain the final product, PDAP nanoparticles. The volume ratio of the aqueous solution of N,N-dimethylformamide to water in the dialysis process is 2:

1.

9. A ROS / enzyme dual-responsive nanomedicine controlled release system, prepared by the preparation method of the ROS / enzyme dual-responsive nanomedicine controlled release system according to any one of claims 1-8.

10. The application of the ROS / enzyme dual-responsive nanomedicine controlled release system of claim 9 in plant protection.

Images