An esterase-triggered photodynamic nanopesticide controlled release system and its preparation method

Abstract

The invention discloses an esterase-triggered photodynamic nano pesticide controlled release system, which is characterized in that a nano-micelle with a spherical core-shell structure is prepared by ultrasonically assembling an amphiphilic conjugate in an aqueous solution; the amphiphilic conjugate is formed by connecting a carboxyl polysaccharide and a hematoporphyrin photosensitizer through an ester group; the invention adopts a carboxyl polysaccharide with good water solubility, biodegradability and non-toxicity, and forms a conjugate with the hematoporphyrin photosensitizer through an ester group grafting to improve the water solubility of the hematoporphyrin; the nano-micelle formed by self-assembly of the conjugate in an aqueous solution has good water solubility, and the fluorescence quenching effect caused by the self-aggregation of porphyrin molecules can significantly reduce the photodynamic activity of the hematoporphyrin, thereby improving the photostability of the photosensitizer and optimizing the utilization rate and retention time of the pesticide; when the nano-micelle is absorbed by harmful organisms, the system can respond to enzyme stimulation in the harmful organisms, release the photosensitizer, and trigger high-efficiency photodynamic activity to produce a killing effect.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a photodynamic nanopesticide controlled-release system and a preparation method thereof. Background Art

[0002] Photoactivated pesticides are a new type of pesticide with high activity, low toxicity and no resistance. They have become an important direction for the development of pesticides. During the use of traditional pesticides, due to factors such as leaching, evaporation, deposition, photolysis, and hydrolysis, more than 90% of the active ingredients cannot reach the target site, which increases the resistance of pests. Moreover, the circulation and accumulation of non-degradable toxic components in the environment have caused serious damage to other ecological resources and non-target organisms. The principle of photoactivated pesticides is photodynamic action, that is, the photosensitizer absorbs photons and catalyzes the surrounding oxygen molecules to produce reactive oxygen species (ROS) - including singlet oxygen (1O2), hydroxyl radicals (·OH) and superoxide anions (O2ˉ), which then oxidize biological cell membranes or intracellular lipids, proteins, nucleic acids and other biologically active macromolecules, thereby causing the death of pests. Therefore, compared with traditional pesticides, photoactivated pesticides not only have the advantages of being non-toxic or low-toxic in the absence of light, but also have the advantages of broad spectrum, high activity, and pests are not easy to develop resistance after activation by photodynamic action. Among the photoactivated pesticides discovered so far, porphyrin compounds have been considered to be the most lethal photosensitizers. However, current literature reports show that porphyrin compounds are mostly used as anti-cancer and anti-inflammatory drugs in humans, and are still in the laboratory research stage as photoactivated pesticides.

[0003] The main reasons why porphyrin compounds are difficult to use directly as pesticides are as follows: (1) After being activated by light, porphyrin compounds are very susceptible to photodegradation when efficiently producing ROS, making it difficult for them to remain as pesticides for a long time after spraying; this low photostability reduces the duration of action of the pesticide at the target site and its long-term pest control effect. (2) Many modified porphyrin compounds have poor water solubility, which reduces their utilization rate; and due to the fluorescence quenching effect caused by aggregation, the photodynamic activity is inhibited. Although the retention time is increased, the lethality of the pesticide is reduced. (3) As a plant protection agent, porphyrin molecules may be absorbed by the leaves of the protected crops and other parts during the spraying process, which not only reduces the utilization rate, but also causes phototoxicity to the protected crops.

[0004] CN115462372 A discloses a GSH-responsive photodynamic antibacterial nanosystem, a preparation method and an application thereof, wherein nanomicelles with a spherical core-shell structure are prepared by ultrasonically assembling an amphiphilic conjugate in a phosphate buffer solution, wherein the amphiphilic conjugate is formed by first activating the carboxyl groups of a porphyrin photosensitizer and sodium alginate respectively and then grafting them with cystamine. Summary of the invention

[0005] The purpose of the present invention is to provide an esterase-triggered photodynamic nanopesticide controlled-release system and a preparation method thereof. The system adopts a carboxyl polysaccharide with good water solubility, biodegradability and non-toxicity, and forms a conjugate with a hematoporphyrin photosensitizer through ester grafting to improve the water solubility of the hematoporphyrin. The nanomicelles formed by the self-assembly of the conjugate in an aqueous solution have good water solubility. Due to the fluorescence quenching effect caused by the self-aggregation of porphyrin molecules, the photodynamic activity of the hematoporphyrin can be significantly reduced, thereby improving the photostability of the photosensitizer and optimizing the utilization rate and retention time of the pesticide. When the nanomicelles are absorbed by harmful organisms, the system can respond to enzyme stimulation in the harmful organisms, release the photosensitizer, and trigger high-efficiency photodynamic activity to produce a killing effect.

[0006] In order to achieve the above purpose, the technical solutions adopted are as follows:

[0007] An esterase-triggered photodynamic nanopesticide controlled release system is a nanomicelle with a spherical core-shell structure prepared by ultrasonically assembling an amphiphilic conjugate in an aqueous solution; the amphiphilic conjugate is formed by connecting a carboxylic acid-containing polysaccharide (i.e. a polysaccharide with carboxylic acid) and a hematoporphyrin photosensitizer via an ester group.

[0008] According to the above scheme, the mass content of the hematoporphyrin photosensitizer in the amphiphilic conjugate is 1.23%-11.11%.

[0009] According to the above scheme, the carboxyl polysaccharide is any one of fructose, carboxymethyl starch, N-succinyl chitosan and alginate.

[0010] The preparation method of the above-mentioned esterase-triggered photodynamic nanopesticide controlled release system comprises the following steps:

[0011] 1) dissolving a composite catalyst and carboxyl polysaccharide in water, continuously stirring to catalyze the activation of carboxyl groups, and purifying after the reaction to obtain an intermediate product A; wherein the composite catalyst is composed of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide;

[0012] 2) Under dark conditions, dissolving the hematoporphyrin photosensitizer in a mixed solution of N,N-dimethylformamide and water to obtain a mixed solution, adding the intermediate product A, and then adding a catalytic amount of tetrabutylammonium bromide (TBAB) and dimethylaminopyridine (DMAP) to stir and react, and purifying the obtained product to obtain an amphiphilic conjugate;

[0013] 3) In dark conditions, the amphiphilic conjugate is dispersed in water, and after ultrasonic treatment, it self-assembles into nanoparticles to obtain an enzyme ester-triggered photodynamic nanopesticide system.

[0014] According to the above scheme, the composite catalyst in step 1) is composed of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a molar ratio of 1:1.

[0015] According to the above scheme, the mixed solution of N,N-dimethylformamide and water in step 2) is prepared by mixing N,N-dimethylformamide and water in a volume ratio of 1:1.

[0016] According to the above scheme, in step 2), the hematoporphyrin photosensitizer is added according to the mass ratio of hematoporphyrin photosensitizer to carboxyl polysaccharide (10-50):100.

[0017] According to the above scheme, after the reaction of step 2) is completed, the product is first dialyzed with a mixed solution of N,N-dimethylformamide and water and then dialyzed with deionized water, and then freeze-dried to obtain an amphiphilic conjugate.

[0018] According to the above scheme, the reaction temperature of step 2) is room temperature and the reaction time is 72h.

[0019] According to the above scheme, the concentration of the amphiphilic conjugate dispersed in water in step 3) is 0.2 mg / mL.

[0020] According to the above scheme, the particle size of the nanoparticles in step 3) is 108-350nm.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The conjugate formed by the photosensitizer and carboxyl polysaccharide connected by ester groups can self-assemble into nanomicelles in aqueous media, improving the water solubility of the photosensitizer. The photosensitizer groups aggregated inside the nanomicelles can lead to the fluorescence self-quenching effect, reduce the photodynamic activity of the nanomicelles, and improve the photostability of the photosensitizer in the micelles. Therefore, this nanosystem can not only inhibit the inactivation of pesticides due to photooxidative degradation in non-target environments and increase the retention time, but also avoid the phototoxicity of photosensitizers to non-target organisms. In the infection site of pathogens or in the body of pests, the system can respond to high-concentration esterase stimulation, ester bond degradation to release photosensitizer molecules, eliminate or weaken the aggregated fluorescence quenching effect, restore photodynamic activity, and achieve targeted killing effects.

[0023] The esterase-triggered photodynamic nanopesticide controlled release system provided by the present invention has good water solubility, photostability and targeting, can control the release of pesticides according to whether it is the target environment, significantly improve the utilization rate of photosensitizers, greatly reduce the cost of pesticides, and reduce the harm of pesticides.

[0024] When pathogens infect plant and animal cells, due to the interaction between pathogens and cells, the microenvironment of the cell infection site will change significantly, and the enzymes expressed by pathogenic factors (such as esterase, phosphatase, phospholipase, protease, etc.) will increase significantly, which can specifically open the ester bond of the controlled release system of the present invention to trigger the release of photosensitizers, thereby achieving photoactive pesticides to kill pathogens. The enzyme ester-triggered photodynamic nanopesticide controlled release system obtained by the present invention has good application prospects in plant disease prevention and control and photodynamic antibacterial products. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the PEC-HP3 amphiphilic conjugate prepared in Example 3 of the present invention.

[0026] Figure 2 TEM images of the PEC-HP3 nanoparticles prepared in Example 3 of the present invention before (A) and after (B) incubation with esterase for 24 hours.

[0027] Figure 3 It is the cumulative photodegradation rate of the PEC-HP3 nanoparticles prepared in Example 3 of the present invention.

[0028] Figure 4 This is the drug release kinetics of the PEC-HP3 nanoparticle enzyme spike prepared in Example 3 of the present invention.

[0029] Figure 5 This is the photodynamic activity induced by the nanoparticle enzyme prepared in Example 3 of the present invention.

[0030] Figure 6 The antibacterial effect of the PEC-HP3 nanoparticles prepared in Example 3 of the present invention on Escherichia coli and Staphylococcus aureus.

[0031] Figure 7 The photodynamic antibacterial activity of the PEC-HP3 nanoparticles prepared in Example 3 of the present invention against Staphylococcus aureus and Escherichia coli at different time points. DETAILED DESCRIPTION

[0032] The following examples further illustrate the technical solution of the present invention, but are not intended to limit the scope of protection of the present invention.

[0033] Example 1

[0034] The preparation method of the esterase-triggered light-activated nanopesticide controlled-release system 1 comprises the following steps:

[0035] (1) Preparation of pectin active ester PEC-NHS

[0036] 100 mg of PEC was dissolved in 100 ml of deionized water, and NHS (0.6 mmol, 69.1 mg) and EDC·HCl (0.6 mmol, 115 mg) were added under stirring. After reacting for 4 hours, the mixture was transferred to a dialysis bag and dialyzed against deionized water. The active ester PEC-NHS was obtained by freeze-drying.

[0037] (2) Preparation of the amphiphilic conjugate PEC-HP1

[0038] In the dark, 10 mg of hematoporphyrin was dissolved in a mixed solution of 5 mL of N, N-dimethylformamide and 5 mL of water, 100 mg of the above PEC-NHS was added to the mixed solution, and then a catalytic amount of tetrabutylammonium bromide (TBAB, 30 μmol, 9.7 mg) and dimethylaminopyridine (DMAP, 30 μmol, 3.7 mg) were added and stirred for reaction; after reacting for 72 hours, the mixture was transferred to a dialysis bag (8000D-1400D), first dialyzed with a mixed solution of N, N-dimethylformamide and water and then dialyzed with deionized water, and then freeze-dried to obtain the amphiphilic conjugate PEC-HP1 with a mass content of 1.23%.

[0039] (3) Preparation of esterase-triggered photodynamic antibacterial PEC-HP1 nanoparticle system Under dark conditions, the amphiphilic conjugate prepared in step 2) was dispersed in water to obtain a concentration of 0.2 mg / mL, and then self-assembled to form nanoparticles after ultrasonic treatment, namely, the enzyme ester-triggered photodynamic nanopesticide system PEC-HP NP1. The average particle size of the system particles was 350±4.10 nm.

[0040] Example 2

[0041] The preparation method of the esterase-triggered light-activated nanopesticide controlled-release system 2 comprises the following steps:

[0042] (1) Preparation of pectin active ester PEC-NHS

[0043] 100 mg of PEC was dissolved in 100 ml of deionized water, and NHS (0.6 mmol, 69.1 mg) and EDC·HCl (0.6 mmol, 115 mg) were added under stirring. After reacting for 4 hours, the mixture was transferred to a dialysis bag and dialyzed against deionized water. The active ester PEC-NHS was obtained by freeze-drying.

[0044] (2) Preparation of the amphiphilic conjugate PEC-HP2

[0045] Under dark conditions, 20 mg of hematoporphyrin was dissolved in a mixed solution of 10 mL of N, N-dimethylformamide and 10 mL of water, 100 mg of the above PEC-NHS was added to the mixed solution, and then a catalytic amount of tetrabutylammonium bromide (TBAB, 30 μmol, 9.7 mg) and dimethylaminopyridine (DMAP, 30 μmol, 3.7 mg) were added and stirred for reaction; after reacting for 72 hours, the mixture was transferred to a dialysis bag (8000D-1400D), first dialyzed with a mixed solution of N, N-dimethylformamide and water and then dialyzed with deionized water, and then freeze-dried to obtain the amphiphilic conjugate PEC-HP2 with a drug mass content of 4.10%.

[0046] (3) Preparation of esterase-triggered photodynamic antibacterial PEC-HP2 nanoparticle system

[0047] Under dark conditions, the amphiphilic conjugate prepared in step 2) was dispersed in water to obtain a concentration of 0.2 mg / mL, and then self-assembled to form nanoparticles after ultrasonic treatment, namely, the photodynamic nanopesticide system PEC-HPNP2 triggered by enzyme ester. The average particle size of the system particles was 301±4.10 nm.

[0048] Example 3

[0049] The preparation method of the esterase-triggered light-activated nanopesticide controlled-release system 3 comprises the following steps:

[0050] (1) Preparation of pectin active ester PEC-NHS

[0051] 100 mg of PEC was dissolved in 100 ml of deionized water, and NHS (0.6 mmol, 69.1 mg) and EDC·HCl (0.6 mmol, 115 mg) were added under stirring. After reacting for 4 hours, the mixture was transferred to a dialysis bag and dialyzed against deionized water. The active ester PEC-NHS was obtained by freeze-drying.

[0052] (2) Preparation of amphiphilic conjugate PEC-HP3

[0053] Under dark conditions, 30 mg of hematoporphyrin was dissolved in a mixed solution of 15 mL of N, N-dimethylformamide and 15 mL of water, 100 mg of the above PEC-NHS was added to the mixed solution, and then a catalytic amount of tetrabutylammonium bromide (TBAB, 30 μmol, 9.7 mg) and dimethylaminopyridine (DMAP, 30 μmol, 3.7 mg) were added and stirred for reaction; after reacting for 72 hours, the mixture was transferred to a dialysis bag (8000D-1400D), first dialyzed with a mixed solution of N, N-dimethylformamide and water and then dialyzed with deionized water, and then freeze-dried to obtain the amphiphilic conjugate PEC-HP3 with a drug mass content of 7.07%.

[0054] (3) Preparation of esterase-triggered photodynamic antibacterial PEC-HP3 nanoparticle system

[0055] Under dark conditions, the amphiphilic conjugate prepared in step 2) was dispersed in water to obtain a concentration of 0.2 mg / mL, and then self-assembled to form nanoparticles after ultrasonic treatment, namely, the photodynamic nanopesticide system PEC-HPNP3 triggered by enzyme ester. The average particle size of the system particles was 252±4.10 nm.

[0056] Example 4

[0057] The preparation method of the esterase-triggered light-activated nanopesticide controlled-release system 4 comprises the following steps:

[0058] (1) Preparation of NSC-NHS active ester of butyl chitosan

[0059] 100 mg of N-succinyl chitosan (NSC) was dissolved in 100 ml of deionized water, and NHS (0.6 mmol, 69.1 mg) and EDC·HCl (0.6 mmol, 115 mg) were added under stirring. After reacting for 4 hours, the mixture was transferred to a dialysis bag and dialyzed against deionized water, and freeze-dried to obtain the active ester NSC-NHS.

[0060] (2) Preparation of the amphiphilic conjugate NSC-HP

[0061] Under dark conditions, 40 mg of hematoporphyrin was dissolved in a mixed solution of 20 mL of N, N-dimethylformamide and 20 mL of water, 100 mg of the above PEC-NHS was added to the mixed solution, and then a catalytic amount of tetrabutylammonium bromide (TBAB, 30 μmol, 9.7 mg) and dimethylaminopyridine (DMAP, 30 μmol, 3.7 mg) were added and stirred for reaction; after reacting for 72 hours, the mixture was transferred to a dialysis bag (8000D-1400D), first dialyzed with a mixed solution of N, N-dimethylformamide and water and then dialyzed with deionized water, and then freeze-dried to obtain the amphiphilic conjugate NSC-HP with a drug mass content of 9.61%.

[0062] (3) Preparation of esterase-triggered photodynamic antibacterial NSC-HP nanoparticle system

[0063] Under dark conditions, the amphiphilic conjugate prepared in step 2) was dispersed in water to obtain a concentration of 0.2 mg / mL, and then self-assembled to form nanoparticles after ultrasonic treatment, namely, the enzyme ester-triggered photodynamic nanopesticide system NSC-HPNP. The average particle size of the system particles was 156±4.10 nm.

[0064] Example 5

[0065] The preparation method of the esterase-triggered light-activated nanopesticide controlled-release system 5 comprises the following steps:

[0066] (1) Preparation of pectin active ester PEC-NHS

[0067] 100 mg of PEC was dissolved in 100 ml of deionized water, and NHS (0.6 mmol, 69.1 mg) and EDC·HCl (0.6 mmol, 115 mg) were added under stirring. After reacting for 4 hours, the mixture was transferred to a dialysis bag and dialyzed against deionized water. The active ester PEC-NHS was obtained by freeze-drying.

[0068] (2) Preparation of the amphiphilic conjugate PEC-HP4

[0069] Under dark conditions, 50 mg of hematoporphyrin was dissolved in a mixed solution of 25 mL of N, N-dimethylformamide and 25 mL of water, 100 mg of the above PEC-NHS was added to the mixed solution, and then a catalytic amount of tetrabutylammonium bromide (TBAB, 30 μmol, 9.7 mg) and dimethylaminopyridine (DMAP, 30 μmol, 3.7 mg) were added and stirred for reaction; after reacting for 72 hours, the mixture was transferred to a dialysis bag (8000D-1400D), first dialyzed with a mixed solution of N, N-dimethylformamide and water and then dialyzed with deionized water, and then freeze-dried to obtain the amphiphilic conjugate PEC-HP4 with a drug mass content of 11.11%.

[0070] (3) Preparation of esterase-triggered photodynamic antibacterial PEC-HP4 nanoparticle system

[0071] Under dark conditions, the amphiphilic conjugate prepared in step 2) was dispersed in water to obtain a concentration of 0.2 mg / mL, and then self-assembled to form nanoparticles after ultrasonic treatment, namely, the enzyme ester-triggered photodynamic nanopesticide system PEC-HPNP4. The average particle size of the system particles was 108±1.41 nm.

[0072] Taking Example 3 as an example, the structure of the conjugate obtained in step (2) of this example was characterized by NMR.

[0073] Figure 1 HP in DMSO-d6 and PEC and PEC-HP3 in D2O 1HNMR spectrum. The chemical shifts of HP (see curve A) are assigned as follows: δ10.90 and 10.36 (4H, meso-H), 6.57 (2H, CH(OH)CH3), 4.42 (4H, CH2CH2COO-), 3.70 (12H, cyclo-CH3), 3.19 (4H and CH2CH2COO), 2.12 (6H, CH(OH)CH3); the chemical shifts of PEC (see curve B) are assigned as follows: 5.04, 3.66, 3.96, 4.40 and 4.89 ppm are respectively the H-1, H-2, H-3, H-4 and H-5 signals on the galacturonic acid unit; 3.76 ppm comes from the proton of the methyl ester group (-OCH3) on the galacturonic acid unit in the PEC structure. Compared with curve B, curve C shows new signal peaks at 10.25ppm and 9.73ppm, which belong to the typical meso-H of HP, while 6.64ppm and 2.38ppm are attributed to the proton signals on the secondary carbon and methyl groups of the two side groups (2×CH(OH)CH3) of the HP macrocyclic ring, respectively. All these indicate that HP has been successfully covalently linked to the carboxyl group of PEC.

[0074] Figure 2 (A) shows the TEM image of PEC-HP3-based nanoparticles, which exhibit a spherical core-shell structure.

[0075] Taking Example 3 as an example, the performance of PEC-HP3-based nanoparticles was evaluated.

[0076] 1. Study on the photostability of esterase-triggered photodynamic nanopesticide controlled release system

[0077] The change in UV absorption intensity was used to evaluate the photostability of HP in nanoparticles under light exposure. With HP2HCl as the control, PEC-HP NP3 with the same dose was treated with dark light or irradiation. The irradiation condition was a 23W fluorescent lamp (wavelength range of 430-720nm), and the distance between the lamp and the sample was 10cm. During the light exposure, 3mL of sample solution was taken every 1 hour and placed in a quartz dish to measure the UV-vis absorption spectrum. The photodegradation rate of HP can be calculated by the following formula:

[0078]

[0079] Where A0 is the initial maximum absorption intensity of the sample solution; A t is the maximum absorption intensity when the irradiation time is t; t is the irradiation time.

[0080] The cumulative photodegradation rate of PEC-HP NP3 and HP2HCl is as follows Figure 3As shown in Figure 2, both samples hardly underwent photodegradation in the dark. When exposed to light, HP2HCl degraded by 70.55% within 10 h, while PEC-HP NP3 only degraded by 32.11%, indicating that nano-sizing can effectively inhibit the photodegradation of HP.

[0081] 2 Drug release from esterase-triggered photodynamic nanopesticide controlled release system

[0082] In order to ensure that the HP in NPs obtains the controlled effect of enzyme-stimulated release in practical applications, the drug release behavior of NPs was studied by dialysis. Using 15mL pH 7.4 buffer as a control, 3mg PEC-HP NP3 was dispersed in 15U / mL or 30U / mL esterase buffer solution, then transferred to a dialysis bag, and then immersed in 40mL of the corresponding buffer containing 1% (v / v) Tween 80, and then oscillated at 50rpm in a 37℃ constant temperature water bath oscillator. 3mL of sample solution was taken out at specific time intervals, and then 3mL of fresh buffer was added. The release of HP was determined by ultraviolet spectrophotometry. The cumulative release of HP was calculated as follows:

[0083]

[0084] Among them, m t is the amount of HP released at time t; m0 is the amount of HP in NPs before release; t is the sampling time.

[0085] like Figure 4 In the absence of esterase release medium, the nanoparticles only released 9.5% of HP within 24 hours, and then gradually became flat, indicating that PEC-HP NP3 has good chemical stability. In the solution containing 15U / mL and 30U / mL esterase, the cumulative drug release rate of the nanoparticles in 24 hours reached 26.9% and 52.5%, respectively, which was much higher than the control, indicating that esterase stimulation can lead to HP release rate, and the release rate increases with the increase of enzyme concentration. The morphological bonds of the nanoparticles after incubation in a solution containing 15U / mL esterase for 24 hours Figure 2 (B).

[0086] 3. Photoactivity evaluation of esterase-triggered photodynamic nanopesticide controlled release system

[0087] 9,10-Anthracene dioyl-bis(methylene) dimalonic acid (ABDA) can react with ROS to generate endoperoxide products, resulting in a decrease in its UV-vis absorption intensity at 380nm, so ABDA can be used to monitor the generation of ROS. Take three sample solutions of equal dose (0.05mg / mL of HP): HP2HCl solution, esterase-free PEC-HP NP3 and esterase-containing PEC-HP NP3 solution, add ABDA to a final concentration of 0.15mg / mL, and bubble the sample solution with oxygen for 10 minutes. Use pure ABDA solution as a blank control. Measure the UV-vis absorption spectrum before exposure, then place the sample under the above-mentioned light conditions, and measure the absorption spectrum every 10 minutes.

[0088] Figure 5 The decay curve of ABDA absorption at 380nm with irradiation time is shown. The absorption intensity of the blank ABDA solution changes very little during irradiation and is almost negligible. In the presence of PEC-HP NP3, the absorption intensity of ABDA decreases; however, since NPs themselves do not produce ROS or release HP, the photodegradation rate of ABDA is still low. However, once the enzyme is added to the nanosample solution, the absorption intensity of ABDA decays exponentially, indicating that the enzyme stimulation induces the release of HP from the nanoparticles and catalyzes the generation of a large amount of ROS.

[0089] In addition, compared with the same dose of HP·2HCl, a slightly higher ABDA absorption decay rate was observed in the enzyme-containing nanosample solution after 80 min of irradiation, indicating that the nanosystem can maintain the photosensitivity of HP and thus enhance the ROS generation ability.

[0090] 4. Evaluation of antibacterial activity of esterase-triggered photodynamic nanopesticide controlled release system

[0091] The specific steps are as follows:

[0092] The photodynamic antibacterial effect of PEC-HP NP3 was evaluated. Escherichia coli (representative of Gram-negative bacteria) and Staphylococcus aureus (representative of Gram-positive bacteria) were selected as model bacteria. Since Staphylococcus aureus is sensitive to porphyrin, the concentration of porphyrin to inactivate Staphylococcus aureus should be much lower than that of Escherichia coli to obtain the same killing effect. Therefore, with buffer solution (pH 7.4) as blank control, the following five groups of sample solutions were established for antibacterial experiments: PEC solution, HP·2HCl solution, PEC-HP NP3 solution without esterase, and PEC-HP NP3 solution containing 15U / mL or 30U / mL esterase. Except for the blank solution and PEC solution, the drug (HP) dose for Escherichia coli and the dose for Staphylococcus aureus in the other four groups of samples was 15μg / mL and 0.5μg / mL. In addition, the samples were dispersed in a sterilized buffer solution (pH 7.4) and stored for use.

[0093] Both bacteria were collected, transferred to liquid culture, labeled and cultured in a 37°C shaker for 24 hours to obtain 10 9 CFU / mL bacterial liquid concentration. Six sample solutions (PBS, PEC solution, HP·2HCl solution, PEC-HP NP3 solution without esterase, PEC-HP NP3 solution containing 15U / mL or 30U / mL esterase) were added to a 48-well microplate at a ratio of 1:4 with bacterial liquid (0.1mL) diluted with sterile PBS and shaken at 37°C for 30 minutes to ensure that the mixture was fully in contact, which were six control experimental groups. After 30 minutes, the 48-well microplate was irradiated with the previous experimental conditions. At 20, 40 and 60 minutes of irradiation, 10μL of bacterial liquid was pipetted onto the solid culture medium and evenly coated. The solid culture medium was then incubated in a constant temperature incubator at 37°C for 24 hours. At the end of incubation, the colonies were manually counted using the colony counting method. The antibacterial rate was calculated using the following formula:

[0094]

[0095] CFU Blank and CFU Sample The colony counts of the blank solution and the sample solution are shown in Figure 2. The antibacterial results of each group after exposure for 60 minutes are shown in Figure 2. Figure 6 As shown, the bacterial growth of the PBS group (blank) and the PEC group as controls was almost not inhibited.

[0096] The photodynamic antibacterial activity of each sample solution with antibacterial effect was measured at different time points (20, 40, and 60 min) after mixing, shaking, and exposing to light with Staphylococcus aureus liquid diluted with sterile PBS. Figure 7 (Left) shows the photodynamic antibacterial activity of the treated E. coli liquid diluted with sterile PBS. Figure 7 (right) shown.

[0097] In general, HP·2HCl has a significant inhibitory effect on both bacteria, with antibacterial rates of 95.32% and 89.53% at 20 minutes, respectively. As the light irradiation time increases, the antibacterial rates of both bacteria exceed 99% at 60 minutes, basically achieving a complete inactivation effect.

[0098] For the PEC-HP NP3 group, in the absence of esterase, PEC-HP NP3 showed low antibacterial activity against the two bacteria, with antibacterial rates of 53.04% and 47.28% at 20 minutes, respectively. This indicates that the nanoparticles do not release the photosensitive part of the structure in an environment with less esterase. As the exposure time is extended to 60 minutes, the antibacterial rates increase to 71.70% and 61.70%, respectively, which can be attributed to the gradual increase in the secretion of esterase by bacteria, which stimulates the cleavage of ester bonds in the particles and enhances the antibacterial effect.

[0099] Furthermore, when esterase was added to the culture medium, PEC-HP NP3 showed excellent antibacterial activity, and as the esterase concentration increased from 15U to 30U / mL, the antibacterial rate (%) against Staphylococcus aureus (left) and Escherichia coli (right) increased at different illumination times. When the added esterase concentration was 15U / mL, the antibacterial rate of the nanoparticles against these two bacteria reached 97.78% and 89.31% at 60min, respectively, and when the esterase was further added to its concentration of 30U / mL, its antibacterial rate at 60min was close to HP, which was 99.72% and 99.67%, respectively. This shows that the nanoparticles have esterase response function, and esterase stimulates the nanoparticles to release a large amount of ROS, resulting in photodynamic antibacterial effect.

[0100] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any ordinary technician in the industry can smoothly implement the present invention as shown in the drawings and described above. However, any equivalent changes, modifications and evolutions made by technicians familiar with the profession without departing from the scope of the technical solution of the present invention using the technical content disclosed above are all equivalent embodiments of the present invention. At the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing an esterase-triggered photodynamic nanopesticide controlled release system, characterized in that The following steps are involved: 1) dissolving the composite catalyst and pectin in water, continuously stirring to activate the carboxyl groups, and purifying after the reaction to obtain an intermediate product A; wherein the composite catalyst is composed of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide; 2) Under dark conditions, dissolving a hematoporphyrin photosensitizer in a mixed solution of N,N-dimethylformamide and water to obtain a mixed solution, adding the intermediate product A, and then adding a catalytic amount of tetrabutylammonium bromide and dimethylaminopyridine to stir and react, and purifying the obtained product to obtain an amphiphilic conjugate; the amphiphilic conjugate is formed by connecting pectin and the hematoporphyrin photosensitizer through an ester group, wherein the mass content of the hematoporphyrin photosensitizer is 1.23%-11.11%; 3) In the dark, the amphiphilic conjugate is dispersed in water and self-assembled into nanomicelles with a spherical core-shell structure after ultrasonic fragmentation, thereby obtaining an esterase-triggered photodynamic nanopesticide system.

2. The method for preparing the esterase-triggered photodynamic nanopesticide controlled release system according to claim 1, characterized in that The composite catalyst in step 1) is composed of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a molar ratio of 1:

1.

3. The method for preparing the esterase-triggered photodynamic nanopesticide controlled release system according to claim 1, characterized in that In step 2), the mixed solution of N,N-dimethylformamide and water is prepared by mixing N,N-dimethylformamide and water in a volume ratio of 1:

1.

4. The method for preparing the esterase-triggered photodynamic nanopesticide controlled release system according to claim 1, characterized in that In step 2), the hematoporphyrin photosensitizer is added according to the mass ratio of hematoporphyrin to pectin (10-50):

100.

5. The method for preparing the esterase-triggered photodynamic nanopesticide controlled release system according to claim 1, characterized in that Step 2) After the reaction is completed, the product is dialyzed with a mixed solution of N,N-dimethylformamide and water and then dialyzed with deionized water, and then freeze-dried to obtain an amphiphilic conjugate.

6. The method for preparing the esterase-triggered photodynamic nanopesticide controlled release system according to claim 1, characterized in that Step 2) The reaction temperature is room temperature and the reaction time is 72h.

7. The method for preparing the esterase-triggered photodynamic nanopesticide controlled release system as claimed in claim 1, characterized in that Step 3) The amphiphilic conjugate was dispersed in water at a concentration of 0.2 mg / mL.

8. The method for preparing the esterase-triggered photodynamic nanopesticide controlled release system as claimed in claim 1, characterized in that The particle size of the nanoparticles in step 3) is 108-350nm.

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