GSH (glutathione) response type nano-drug carrier as well as preparation method and application thereof

Abstract

The invention discloses a GSH (glutathione) response type nano-drug carrier as well as a preparation method and application thereof, the nano-drug carrier is a levofloxacin loaded BSA (bovine serum albumin) and PAMAM (polyamidoamine) compound, and is obtained by coupling BSA to a levofloxacin loaded PAMAM nano-drug by a disulfide bond-containing compound; the invention further discloses application of the levofloxacin loaded BSA and PAMAM compound in an antibiotic controlled release drug delivery system. According to the present invention, the drug can be effectively sealed, the non-specific release of the drug under the normal physiological condition can be prevented, the controllable release of the drug can be initiated in the presence of the GSH, the on-demand drug delivery can be achieved, and the drug delivery scheme can be optimized;

Description

Technical Field

[0001] The present invention relates to the technical field of drug delivery systems, and specifically relates to a GSH-responsive nano-drug carrier, a preparation method thereof, and an application thereof. Background Art

[0002] In clinical applications, the selection of antibiotics must be tailored to specific pathogens to ensure efficacy and specificity. Antibacterial drugs mainly inhibit or eliminate pathogens by interfering with bacterial metabolism, growth, and reproduction. To achieve the best therapeutic effect, it is necessary to maintain a sufficient drug concentration in the blood, making appropriate and sufficient doses essential. In addition, antibiotic treatment must continue for a period of time to ensure complete eradication of pathogens and reduce the risk of drug resistance. However, long-term use may cause adverse reactions, potentially damaging overall health. Therefore, once the infection is controlled, antibiotic treatment should be stopped immediately to minimize drug-related side effects. Therefore, the construction of intelligent antibiotic release systems provides a feasible strategy for improving the safety and effectiveness of antibacterial therapy.

[0003] Traditional antibiotics (such as levofloxacin) are mainly administered orally or by injection. However, these administration methods are difficult to maintain the optimal drug concentration, resulting in low bioavailability and potential toxic side effects. Moreover, the main disadvantages of traditional small molecule drugs used in the prior art are the lack of targeting, systemic distribution of drugs, and easy damage to healthy tissues (such as the toxicity of chemotherapy drugs to bone marrow and digestive tract). In addition, there are also problems such as easy generation of drug resistance, limited treatment of complex diseases, difficulty in delivering macromolecules, and narrow therapeutic windows.

[0004] The present invention utilizes the unique radial branching structure of dendrimers to provide abundant drug-loading sites for the encapsulation of levofloxacin. At the same time, by using the stability of albumin in blood circulation, the biocompatibility and delivery performance are enhanced through surface modification. Specifically, the present invention uses an NHS-activated disulfide compound as a linker between albumin and dendrimers. Through the efficient coupling reaction between NHS ester and amine groups, rapid and efficient modification of albumin is achieved, while levofloxacin is encapsulated in the inner cavity of dendrimers. Since disulfide bonds can be reductively cleaved in the presence of glutathione in vivo, at this time albumin detaches from the surface of dendrimers, exposing the dendrimer core, which can promote the release of levofloxacin, thereby realizing glutathione-responsive controlled drug release. This strategy is expected to improve the accuracy and therapeutic effect of antibiotic administration, while reducing the risk of drug resistance, providing a new method for the development of intelligent antibiotic delivery systems. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a GSH-responsive nano-drug carrier, a preparation method thereof, and an application thereof.

[0006] The technical solution of the present invention is: a GSH-responsive nano-drug carrier, and the nano-drug carrier is levofloxacin-loaded BSA@PAMAM composite, which is obtained by coupling BSA to the PAMAM nano-drug loaded with levofloxacin by a disulfide-containing compound, and the disulfide-containing compound is NHS-activated disulfide compound; the raw materials of the nano-drug carrier include: 0.34-3.4 g of NHS-activated disulfide compound, 0.05-0.5 g of PAMAM powder, 0.05-0.5 g of levofloxacin, 0.1-1.0 g of BSA, and 10-100 mL of deionized water;

[0007] Explanation: In the present invention, BSA is coupled to the PAMAM nano-drug loaded with levofloxacin by a disulfide-containing compound, which can effectively seal the drug and prevent its non-specific release under normal physiological conditions. At the same time, in the presence of GSH, the disulfide bond is cleaved, triggering the controlled release of the drug, realizing drug delivery on demand, maintaining the effective concentration, and optimizing the dosing regimen. PAMAM is mainly used for the loading of antibiotics. Due to its divergent dendritic structure, it has a large loading space for hydrophobic drugs; BSA is mainly used to improve the hydrophilicity of the surface of PAMAM, as well as the stability in an aqueous solution environment; NHS-activated disulfide compound is mainly used to couple BSA and PAMAM;

[0008] Among them, the coupling reaction equation of BSA and PAMAM through NHS-activated disulfide compound is as follows:

[0009] NHS-O-CO-CH2CH2-S-S-CH2CH2-CO-O-NHS + BSA-NH2 + PAMAM-NH2 →

[0010] BSA-NH-CO-CH2CH2-S-S-CH2CH2-CO-NH-PAMAM + 2NHS;

[0011] Two active ester groups (-CO-O-NHS) of the NHS-activated disulfide compound (bis-NHS ester) respectively undergo nucleophilic substitution reactions with the primary amino groups (-NH2) on the surfaces of BSA and PAMAM to form stable amide bonds, and at the same time, two molecules of NHS (N-hydroxysuccinimide) are released; 1 molecule of NHS-activated disulfide compound needs to react with 2 molecules of amino groups (1 molecule from BSA and 1 molecule from PAMAM); the by-product only releases non-toxic NHS, and no additional purification steps are required;

[0012] BSA (Bovine Serum Albumin) and PAMAM (Polyamidoamine Dendrimer) serve as coupling sites. Among them, BSA mainly participates in the reaction through the ε-amino group of lysine (Lys) residues, and the surface of PAMAM is rich in primary amino groups (-NH2); the middle -S-S- bond (disulfide bond) acts as a cleavable linker and can be broken in a reducing environment (such as intracellular GSH) to release the coupled molecule (if a dynamic release function is required).

[0013] Furthermore, the preparation method of the NHS-activated disulfide compound is as follows: Take 3,3'-dithiodipropionic acid, NHS, and EDCI and mix them to obtain a mixture. Then dissolve the mixture in dichloromethane, stir overnight at 20 - 28 °C, evaporate the solvent by rotary evaporation once to obtain a crude product. Use ethyl acetate:methanol with a volume ratio of 48 - 52:1 as the mobile phase, purify it by silica gel column chromatography, collect the target fraction, and perform rotary evaporation for the second time to obtain a white solid, which is the NHS-activated disulfide compound;

[0014] The molar ratio of 3,3'-dithiodipropionic acid, NHS, and EDCI is 1:2 - 5:2 - 5; the ratio of the mixture to dichloromethane is 10 mmol:10 - 50 mL.

[0015] Note: Since both ends of 3,3'-dithiodipropionic acid need to be activated, their ratio must be greater than 1:2. The excess reagent solution water can be removed by dialysis. Through the NHS-activated disulfide compound, BSA can be efficiently coupled to the surface of PAMAM, improving the stability of the antibiotic loaded on the PAMAM carrier, preventing drug leakage at will, and secondly improving the stability of the entire drug carrier;

[0016] The synthesis reaction equation of the NHS-activated disulfide compound is as follows:

[0017] HOOC-CH2CH2-S-S-CH2CH2-COOH + 2NHS + 2EDCI → NHS-O-CO-CH2CH2-S-

[0018] S-CH2CH2-CO-O-NHS + 2EDU·HCl;

[0019] The two carboxylic acids (-COOH) of 3,3'-dithiodipropionic acid (containing two carboxylic acid groups) are activated; NHS (N-Hydroxysuccinimide) serves as an activating reagent to convert the carboxylic acid into an active ester (-CO-O-NHS); EDCI (Carbodiimide Condensing Agent) promotes the esterification reaction of the carboxylic acid and NHS and is itself converted into a urea derivative (EDU·HCl);

[0020] Each carboxylic acid group consumes 1 molecule of NHS and 1 molecule of EDCI, so the total molar ratio is 1:2:2 (carboxylic acid group:NHS:EDCI). The optimal molar ratio (1:2.2:2.2) provided in this application is the actual feeding ratio, slightly in excess to ensure complete reaction; EDCI first combines with the -OH of the carboxylic acid to form an active intermediate, and then NHS attacks to form an NHS ester, while releasing EDU·HCl. The double carboxylic acid activation of dithiodipropionic acid finally generates a symmetric double NHS ester product.

[0021] Furthermore, the parameters of the first rotary evaporation are: the vacuum degree is -0.09 to -0.1 MPa, the temperature is 25 - 30 °C, the rotation speed is 300 - 500 rpm, and the time is 30 - 60 min; the parameters of the second rotary evaporation are: the vacuum degree is -0.08 to -0.09 MPa, the temperature is 45 - 70 °C, the rotation speed is 700 - 1000 rpm, and the time is 20 - 30 min;

[0022] Note: The parameter settings of the first evaporation can enable the efficient evaporation and rapid removal of dichloromethane under mild vacuum, and at the same time, it can also avoid the destruction of the activity of the NHS ester at high temperature, reduce the hydrolysis or side reactions of the NHS ester; the parameter settings of the second rotary evaporation can completely remove ethyl acetate / methanol, and the temperature of 45 - 70 °C can also avoid the decomposition of the NHS ester; the first evaporation quickly removes the main solvent dichloromethane, and the second evaporation is for fine purification, improving the total yield and efficiently preparing a highly active NHS-activated disulfide compound.

[0023] A preparation method of a GSH-responsive nanomedicine carrier includes the following steps:

[0024] S1. Dissolve PAMAM powder in deionized water, stir until completely dissolved, then add levofloxacin, and continue stirring for 20 - 28 h to obtain a first composite solution. Sequentially add BSA and NHS-activated disulfide compound to the first composite solution, and stir for 10 - 14 h until the reaction is complete to obtain a second composite solution;

[0025] S2. Transfer the second composite solution into a dialysis bag with a MWCO of 100 kDa, then dialyze with deionized water for 20 - 28 h, and change the deionized water every 6 h. After dialysis, lyophilize the obtained product to obtain a white powder, which is the levofloxacin-loaded BSA@PAMAM complex.

[0026] Note: First, load levofloxacin into the PAMAM carrier, and then modify BSA on the surface of PAMAM through the NHS-activated disulfide compound to improve the encapsulation stability and hydrophilicity of PAMAM; dialysis can effectively remove free PAMAM, BSA, NHS-activated disulfide compound, etc.

[0027] Further, it also includes: using 2,4,5,6-tetra(9-carbazolyl) isophthalonitrile as a photocatalyst to perform photoinduced catalysis on the first composite solution and the NHS-activated disulfide compound respectively under alkaline conditions with pH = 7 - 7.2;

[0028] The method of the photoinduced catalysis is as follows: add a photocatalyst accounting for 3 - 5 mol% of the first composite solution to the first composite solution, stir in the dark for 20 - 30 min, and then irradiate with the first visible light for 8 - 10 h, with a power of 3 - 5 mW / cm 2 ;

[0029] Before adding the NHS-activated disulfide compound to the first composite solution, dissolve 0.08 - 0.12 mmol of the NHS-activated disulfide compound and 0.1 - 0.14 mmol of diphenyl disulfone in 4 - 6 mL of 1,4-dioxane, add a photocatalyst accounting for 4 - 5 mol% of the NHS-activated disulfide compound, stir in the dark for 20 - 30 min, and under the conditions of a nitrogen atmosphere and 25 - 28 °C, irradiate and react with the second visible light for 1.5 - 2 h. Using ethyl acetate: petroleum ether with a volume ratio of 50:1 as the mobile phase, purify by silica gel column chromatography.

[0030] Note: The synergistic effect of the carbazolyl group and the nitrile group of the catalyst can simulate the photosensitive behavior of thiolate. First, perform mild photocatalysis on the PAMAM-levofloxacin composite solution to enhance the drug-loading capacity; then photoactivate the NHS disulfide compound alone to generate sulfur radicals, which can achieve more efficient construction of GSH-responsive disulfide bonds when mixed with BSA later, avoid premature oxidation of the thiol groups of BSA, and ensure the precise release of drugs in high GSH.

[0031] Further, both the first visible light and the second visible light are LED lights. The wavelength of the first visible light is 450 nm; the wavelength of the second visible light is 390 nm, and the power is 5 - 7 mW / cm 2 ;

[0032] Note: The first visible light can perform photocatalytic treatment on the PAMAM-levofloxacin composite solution, promote the non-covalent binding of levofloxacin and the PAMAM dendrimer, enhance the drug-loading stability, and avoid the photodegradation of levofloxacin and reduce the drug efficacy; the photocatalysis of the second visible light can avoid the reduction of the coupling efficiency caused by the hydrolysis of the NHS ester.

[0033] Further, in S1, the stirring parameters are all: stir at 22 - 28 °C and 100 - 1000 rpm until uniform;

[0034] Note: Under the above stirring parameters, the PAMAM powder, deionized water, levofloxacin, BSA, and the NHS-activated disulfide compound can be effectively mixed, thereby improving the loading effect of levofloxacin.

[0035] Further, in S2, the freeze-drying method is: drying at a temperature of -30 to -60 °C for 24 - 100 h;

[0036] Explanation: Low-temperature freeze-drying enables the complex to form a porous network structure, reducing the leakage of levofloxacin, while maintaining the dynamic reversibility of disulfide bonds and enhancing the drug release efficiency triggered by GSH.

[0037] Application of a GSH-responsive nanodrug carrier in an antibiotic controlled-release drug delivery system.

[0038] The beneficial effects of the present invention are as follows:

[0039] (1) In the present invention, BSA is conjugated to PAMAM nanodrugs loaded with levofloxacin through a disulfide bond-containing compound, which can effectively seal the drug and prevent its non-specific release under normal physiological conditions. At the same time, in the presence of GSH, the disulfide bond is cleaved, triggering the controlled release of the drug, achieving on-demand drug delivery, maintaining an effective concentration, and optimizing the drug delivery regimen; this method ensures the therapeutic effect while reducing the overuse of antibiotics and related adverse reactions, reducing the risk of drug resistance, and minimizing damage to liver and kidney functions, the hematopoietic system, and the nervous system.

[0040] (2) The NHS-activated disulfide compound prepared by the specific method of the present invention, the activated ester (NHS-SS-COOH) formed by the reaction of 3,3'-dithiobis(propionic acid) with NHS / EDCI can specifically bind to the free sulfhydryl groups (such as Cys-34) of BSA to form a stable disulfide bond bridging structure. The disulfide bond in the complex can be rapidly reduced and cleaved in high-concentration GSH to release levofloxacin, while maintaining a stable function in normal tissues. The linear structure of the NHS-activated disulfide compound can reduce the steric hindrance of the PAMAM-BSA complex compared to cyclic disulfides, promoting drug loading and release kinetics, and further optimizing the drug response effect to GSH.

[0041] (3) The nanodrugs obtained in the present invention have the following advantages compared to traditional small molecule drugs: First, improve drug solubility. The nanocarrier can encapsulate hydrophobic drugs, improving their solubility and bioavailability; Second, enhance targeting and reduce side effects. Surface modification with antibodies and ligands can precisely recognize the receptors of diseased cells and accumulate in the lesion site through the "enhanced permeability and retention effect (EPR effect)" of inflamed tissues; Third, control the drug release rate, reduce the dosing frequency, maintain a stable blood drug concentration, and avoid the peak-valley effect. Description of the Drawings

[0042] Figure 1 is the standard calibration curve of levofloxacin;

[0043] Figure 2 is the 1 HNMR spectrum of the NHS-activated disulfide compound in Example 1 of the present invention;

[0044] Figure 3 is the 13 C NMR spectrum of the NHS-activated disulfide compound in Example 1 of the present invention;

[0045] Figure 4 is the drug release behavior of levofloxacin BSA@PAMAM in normal saline and normal saline containing 5 mM GSH in Example 1 of the present invention. Detailed implementation manners

[0046] The present invention will be further described in detail below in combination with the detailed implementation manners to better reflect the advantages of the present invention.

[0047] Example 1: A GSH-responsive nano-drug carrier, the nano-drug carrier is a levofloxacin-loaded BSA@PAMAM complex, which is obtained by coupling BSA to a levofloxacin-loaded PAMAM nano-drug with a disulfide-containing compound, and the disulfide-containing compound is an NHS-activated disulfide compound; the raw materials of the nano-drug carrier include: 0.68 g of NHS-activated disulfide compound, 0.1 g of PAMAM powder, 0.1 g of levofloxacin, 0.2 g of BSA, and 20 mL of deionized water;

[0048] The preparation method of the NHS-activated disulfide compound is: take 2.37 mmol of 3,3'-dithiodipropionic acid, 5.23 mmol of NHS, and 5.23 mmol of EDCI and mix them to obtain a mixture, then dissolve the mixture in 20 mL of dichloromethane, stir overnight at 24 °C, remove the solvent by rotary evaporation once to obtain a crude product, use ethyl acetate:methanol with a volume ratio of 50:1 as the mobile phase, purify by silica gel column chromatography, collect the target fraction, and perform rotary evaporation again to obtain a white solid, which is the NHS-activated disulfide compound;

[0049] The parameters of the first rotary evaporation are: the vacuum degree is -0.095 MPa, the temperature is 27 °C, the rotation speed is 400 rpm, and the time is 45 min; the parameters of the second rotary evaporation are: the vacuum degree is -0.085 MPa, the temperature is 55 °C, the rotation speed is 800 rpm, and the time is 20 - 30 min;

[0050] Based on the above preparation method of a GSH-responsive nano-drug carrier, it includes the following steps:

[0051] S1. Dissolve PAMAM powder in deionized water, stir at a temperature of 25 °C and a condition of 500 rpm until completely dissolved, then add levofloxacin, and continue to stir at a temperature of 25 °C and a condition of 500 rpm for 24 h to obtain a first composite solution. Sequentially add BSA and NHS-activated disulfide compound to the first composite solution, and stir at 25 °C and 600 rpm for 12 h until the reaction is complete to obtain a second composite solution;

[0052] S2. Transfer the second composite solution into a dialysis bag with MWCO: 100 kDa, then dialyze with deionized water for 24 h, and change the deionized water every 6 h. After dialysis, dry the obtained product at a temperature of -45 °C for 48 h until dry to obtain a white powder, which is the levofloxacin-loaded BSA@PAMAM complex;

[0053] Application of a GSH-responsive nanodrug carrier in an antibiotic controlled-release drug delivery system, including:

[0054] Weigh 0.1 g of freeze-dried levofloxacin BSA@PAMAM powder, dissolve it in 100 ml of 10 mM GSH solution, ultrasonicate it at 50 kHz for 30 min, then let it stand for 30 min, and continuously stir overnight to ensure complete drug release. Take a part of the solution, centrifuge it with an Amicon centrifugal filter tube (MWCO: 3 kDa), and collect the filtrate. Use ultraviolet-visible spectroscopy to measure the absorbance of levofloxacin in the filtrate, and compare it with the standard calibration curve ( Figure 1 ) to calculate the drug loading amount, and the drug loading amount is 15.2%.

[0055] Example 2: Different from Example 1, the raw materials of the nanodrug carrier include: 0.34 g of NHS-activated disulfide compound, 0.05 g of PAMAM powder, 0.05 g of levofloxacin, 0.1 g of BSA, and 10 mL of deionized water;

[0056] In the preparation method of the NHS-activated disulfide compound, stir overnight at 20 °C, remove the solvent by rotary evaporation once to obtain a crude product, use ethyl acetate: methanol with a volume ratio of 48:1 as the mobile phase, purify it by silica gel column chromatography, collect the target fraction, and perform rotary evaporation for the second time to obtain a white solid, which is the NHS-activated disulfide compound.

[0057] Example 3: Different from Example 1, the raw materials of the nanodrug carrier include: 3.4 g of NHS-activated disulfide compound, 0.5 g of PAMAM powder, 0.5 g of levofloxacin, 1.0 g of BSA, and 100 mL of deionized water;

[0058] In the preparation method of the NHS-activated disulfide compound, stir overnight at 28°C, remove the solvent by rotary evaporation once to obtain the crude product. Use ethyl acetate:methanol with a volume ratio of 52:1 as the mobile phase, purify by silica gel column chromatography, collect the target fraction, and perform rotary evaporation again to obtain a white solid, which is the NHS-activated disulfide compound.

[0059] Example 4: Different from Example 1, the molar ratio of 3,3'-dithiodipropionic acid, NHS, and EDCI is 1:2:2; the ratio of the mixture to dichloromethane is 10 mmol:10 mL.

[0060] Example 5: Different from Example 1, the molar ratio of 3,3'-dithiodipropionic acid, NHS, and EDCI is 1:5:5; the ratio of the mixture to dichloromethane is 10 mmol:50 mL.

[0061] Example 6: Different from Example 1, the parameters of the first rotary evaporation are: vacuum degree of -0.09 MPa, temperature of 25°C, rotation speed of 300 rpm, and time of 30 min; the parameters of the second rotary evaporation are: vacuum degree of -0.08 MPa, temperature of 45°C, rotation speed of 700 rpm, and time of 20 min.

[0062] Example 7: Different from Example 1, the parameters of the first rotary evaporation are: vacuum degree of -0.1 MPa, temperature of 30°C, rotation speed of 500 rpm, and time of 60 min; the parameters of the second rotary evaporation are: vacuum degree of -0.09 MPa, temperature of 70°C, rotation speed of 1000 rpm, and time of 30 min.

[0063] Example 8: Different from Example 1, in S1, dissolve the PAMAM powder in deionized water, stir at a temperature of 22°C and a condition of 100 rpm until completely dissolved, then add levofloxacin, and continue to stir at a temperature of 22°C and a condition of 100 rpm for 20 h to obtain the first composite solution. Add BSA and the NHS-activated disulfide compound to the first composite solution in sequence, and stir at 22°C and 100 rpm for 10 h until the reaction is complete to obtain the second composite solution.

[0064] Example 9: Different from Example 1, in S1, dissolve the PAMAM powder in deionized water, stir at a temperature of 28°C and a condition of 1000 rpm until completely dissolved, then add levofloxacin, and continue to stir at a temperature of 28°C and a condition of 1000 rpm for 28 h to obtain the first composite solution. Add BSA and the NHS-activated disulfide compound to the first composite solution in sequence, and stir at 28°C and 1000 rpm for 14 h until the reaction is complete to obtain the second composite solution.

[0065] Example 10: Different from Example 1, in S2, the second composite solution was transferred into a dialysis bag with MWCO: 100 kDa, and then dialyzed with deionized water for 20 h, and the deionized water was changed every 6 h. After dialysis, the obtained product was dried at -30 °C for 24 h until dry, and the white powder obtained was the levofloxacin-loaded BSA@PAMAM complex.

[0066] Example 11: Different from Example 1, in S2, the second composite solution was transferred into a dialysis bag with MWCO: 100 kDa, and then dialyzed with deionized water for 28 h, and the deionized water was changed every 6 h. After dialysis, the obtained product was dried at -60 °C for 100 h until dry, and the white powder obtained was the levofloxacin-loaded BSA@PAMAM complex.

[0067] Example 12: Different from Example 1, it further includes: using 2,4,5,6-tetra(9-carbazolyl) isophthalonitrile as a photocatalyst to perform photoinduced catalysis on the first composite solution and the NHS-activated disulfide compound respectively under alkaline conditions with pH = 7.1;

[0068] The method of photoinduced catalysis is: adding a photocatalyst accounting for 4 mol% of the first composite solution to the first composite solution, stirring in the dark for 25 min, and then irradiating with LED light at a wavelength of 450 nm for 9 h, with a power of 4 mW / cm 2 ;

[0069] Before adding the NHS-activated disulfide compound to the first composite solution, 0.1 mmol of the NHS-activated disulfide compound and 0.12 mmol of diphenyl disulfone were dissolved in 5 mL of 1,4-dioxane, adding a photocatalyst accounting for 4.5 mol% of the NHS-activated disulfide compound, stirring in the dark for 25 min, and irradiating and reacting with LED light for 1.8 h under the conditions of a nitrogen atmosphere, 27 °C, a wavelength of 390 nm, and a power of 6 mW / cm 2 The purification was performed by silica gel column chromatography using ethyl acetate: petroleum ether with a volume ratio of 50:1 as the mobile phase.

[0070] Example 13: Different from Example 12, it further includes: using 2,4,5,6-tetra(9-carbazolyl) isophthalonitrile as a photocatalyst to perform photoinduced catalysis on the first composite solution and the NHS-activated disulfide compound respectively under alkaline conditions with pH = 7.

[0071] Example 14: Different from Example 12, it further includes: using 2,4,5,6-tetra(9-carbazolyl) isophthalonitrile as a photocatalyst to perform photoinduced catalysis on the first composite solution and the NHS-activated disulfide compound respectively under alkaline conditions with pH = 7.2.

[0072] Example 15: Different from Example 12, a photocatalyst accounting for 3 mol% of the first composite liquid was added to the first composite liquid, stirred in the dark for 20 min, and then irradiated with LED light at a wavelength of 450 nm for 8 h with a power of 5 mW / cm 2 .

[0073] Example 16: Different from Example 12, a photocatalyst accounting for 5 mol% of the first composite liquid was added to the first composite liquid, stirred in the dark for 30 min, and then irradiated with LED light at a wavelength of 450 nm for 10 h with a power of 3 mW / cm 2 .

[0074] Example 17: Different from Example 12, 0.08 mmol of NHS-activated disulfide compound and 0.1 mmol of diphenyl disulfone were dissolved in 4 mL of 1,4-dioxane, a photocatalyst accounting for 4 mol% of the NHS-activated disulfide compound was added, stirred in the dark for 20 min, and irradiated with LED light for a reaction of 1.5 h under the conditions of a nitrogen atmosphere, 25 °C, a wavelength of 390 nm, and a power of 5 mW / cm 2 .

[0075] Example 18: Different from Example 12, 0.12 mmol of NHS-activated disulfide compound and 0.14 mmol of diphenyl disulfone were dissolved in 6 mL of 1,4-dioxane, a photocatalyst accounting for 5 mol% of the NHS-activated disulfide compound was added, stirred in the dark for 30 min, and irradiated with LED light for a reaction of 2 h under the conditions of a nitrogen atmosphere, 28 °C, a wavelength of 390 nm, and a power of 7 mW / cm 2 .

[0076] Experimental Example: Method for Evaluating Drug Release Behavior: Take 0.1 g of freeze-dried levofloxacin BSA@PAMAM powder and dissolve it in 5 mL of normal saline (pH = 7.4) solution and 5 mL of normal saline (pH = 7.4) + 5 mM GSH solution respectively. Transfer the prepared solution into a dialysis bag (MWCO: 3 kDa), and immerse the dialysis bag in 95 mL of the corresponding release medium (normal saline or normal saline + 5 mM GSH). Place the sample in a thermostatic shaker at 37.5 °C with a rotation speed of 100 - 300 rpm for incubation, continuously shake well, extract 1 mL of dialysis fluid every 1 h within 0 - 24 h for analysis, extract 1 mL of dialysis fluid every 4 h within 24 - 36 h for analysis, and then extract 1 mL of dialysis fluid at 48 h for analysis; Use ultraviolet-visible spectroscopy to measure the concentration of levofloxacin in the dialysis fluid to evaluate the drug release behavior. Keep the volume constant by replenishing an equal volume of fresh release medium after each sampling to ensure stable experimental conditions. This experimental design evaluates the release behavior of BSA@PAMAM-loaded levofloxacin nanoparticles under physiological conditions (normal saline) and GSH-triggered conditions (normal saline + GSH), providing reference data for antibacterial applications.

[0077] Conclusion: Figure 1 is the standard calibration curve of levofloxacin; Figure 2 is the 1H NMR spectrum of NHS-activated disulfide, Figure 3 is the 13C NMR spectrum of NHS-activated disulfide, indicating that the structure of the NHS-activated disulfide designed in the present invention is correct.

[0078] It can be seen from Figure 4 that the in vitro drug release behavior of the levofloxacin-loaded BSA@PAMAM system in different solution environments. When using normal saline to simulate normal body fluids, during 0 - 50 h, the drug release rate remains extremely slow, indicating that the nano-drug effectively inhibits the non-specific release of the drug. While in normal saline containing 5 mM GSH, the release rate of levofloxacin increases significantly, especially within 0 - 20 h, and the drug is rapidly released into the simulated GSH solution. This phenomenon proves the GSH-responsive property of the BSA@PAMAM nano-drug. These results show that by conjugating BSA to levofloxacin-loaded PAMAM nano-drug through a disulfide compound, the drug can be effectively sealed to prevent its non-specific release under normal physiological conditions. However, in the presence of GSH, the disulfide bond is cleaved, triggering the controlled release of the drug. Therefore, conjugating BSA to PAMAM nano-drug using NHS-activated disulfide compounds not only improves its stability but also successfully realizes the GSH-triggered drug release mechanism. This design strategy has great potential for achieving precise drug delivery in glutathione-rich microenvironments.

[0079] In physiological saline containing 5 mM GSH, the GSH-responsive nanodrug carriers prepared in Examples 1 to 18 and Control Groups 1 to 3 were placed, and the release percentage of levofloxacin at the 48-hour node was measured; to explore the effect of photoinduced catalysis on the release percentage of levofloxacin, the results are as follows:

[0080] Control Group 1: Different from Example 12, 2,4,5,6-tetra(9-carbazolyl) isophthalonitrile was replaced with tetrabutylammonium decatungstate.

[0081] Control Group 2: Different from Example 12, the first composite solution was not subjected to photoinduced catalysis.

[0082] Control Group 3: Different from Example 12, stirring before the photoreaction was not protected from light.

[0083] Table 1 Release percentage of levofloxacin from the GSH-responsive nanodrug carriers prepared in Examples 1 to 18 and Control Groups 1 to 3 at 48 h

[0084]

[0085] Conclusion: From the comparison of Examples 1 to 18, it can be seen that adding photoinduced catalysis to the first composite solution and NHS-activated disulfide compounds in Examples 12 to 18 can further optimize the response effect of the nanodrug to GSH. This is mainly because the photocatalyst in the first composite solution promotes the stabilization of hydrogen bonds and hydrophobic interactions between PAMAM and levofloxacin under visible light irradiation, reducing the premature leakage of the drug in the blood circulation and keeping it stable in normal tissues; at the same time, the photocatalytic effect can make the internal cavity of PAMAM more suitable for the size of levofloxacin, increasing the drug loading capacity, thereby optimizing the high response effect under GSH conditions; considering comprehensively, Example 12 was determined as the further optimized scheme for Examples 1 to 11;

[0086] From the comparison of Examples 12 to 14 and Control Group 1, replacing the catalyst with tetrabutylammonium decatungstate will weaken the response effect of the nanodrug carrier to GSH. This is because 2,4,5,6-tetra(9-carbazolyl) isophthalonitrile can generate long-lived triplet excited states through visible light excitation, selectively catalyze the dynamic exchange of disulfide bonds, and generate a thioether-sulfone hybrid structure that is easily cleaved by GSH, and it has no destructive effect on PAMAM and levofloxacin under mild conditions, and the generated disulfide bond derivatives respond rapidly to GSH; while tetrabutylammonium decatungstate is a polyoxometalate, and the piperazine ring and carboxyl group of levofloxacin are easily attacked by the reactive oxygen species generated by tetrabutylammonium decatungstate, resulting in structural fracture or the formation of quinolone impurities, leading to a decrease in the release percentage of the drug in tumor tissues (high GSH environment);

[0087] From the comparison of Examples 12 to 14 and Control Group 2, it can be seen that without photoinduced catalysis of the first composite solution, the response effect of the nano-drug carrier to GSH will deteriorate. This is because by skipping the photocatalysis step, the disulfide bond cannot be efficiently converted into a GSH-sensitive structure, resulting in a decrease in the cleavage rate of the carrier in the tumor microenvironment; and during the photocatalysis stage, through the coupling reaction of the NHS ester group with the surface amine group of PAMAM, the stability of the carrier can be enhanced. Without catalysis, the coupling efficiency decreases, and the carrier is prone to premature dissociation in the blood circulation.

[0088] From the comparison of Example 12, Examples 15 to 16 and Control Group 3, it can be seen that light avoidance treatment after adding the photocatalyst can effectively improve the GSH response effect. This is because if the first composite solution and the photocatalyst are directly exposed to visible light, it will immediately excite them to generate triplet excited states, resulting in the ineffective consumption of the catalyst before it fully contacts the substrate, insufficient concentration of the active catalyst in the subsequent formal photocatalysis stage, and a decrease in the dynamic exchange efficiency of the disulfide bond, thereby reducing the GSH response effect. Therefore, considering comprehensively, Example 12 is selected as the optimal solution.

Claims

1. A GSH-responsive nanomedicine carrier, characterized in that, The nano-drug carrier is levofloxacin-loaded BSA@PAMAM complex, which is obtained by conjugating BSA to the PAMAM nano-drug loaded with levofloxacin by a disulfide bond-containing compound. The disulfide bond-containing compound is NHS-activated disulfide compound. The raw materials of the nano-drug carrier include: 0.34 - 3.4 g of NHS-activated disulfide compound, 0.05 - 0.5 g of PAMAM powder, 0.05 - 0.5 g of levofloxacin, 0.1 - 1.0 g of BSA, and 10 - 100 mL of deionized water.

2. The GSH-responsive nanomedicine carrier according to claim 1, characterized in that, The preparation method of the NHS-activated disulfide compound is as follows: 3,3'-dithiobispropionic acid, NHS, and EDCI are mixed to obtain a mixture, and then the mixture is dissolved in dichloromethane, stirred overnight at 20 - 28 °C, and the solvent is removed by rotary evaporation once to obtain a crude product. Using ethyl acetate:methanol with a volume ratio of 48 - 52:1 as the mobile phase, it is purified by silica gel column chromatography, the target fraction is collected, and rotary evaporation is carried out for the second time to obtain a white solid, which is the NHS-activated disulfide compound. The molar ratio of 3,3'-dithiobispropionic acid, NHS, and EDCI is 1:2 - 5:2 - 5; the ratio of the mixture to dichloromethane is 10 mmol:10 - 50 mL.

3. The GSH-responsive nano-drug carrier according to claim 2, wherein The parameters of the first rotary evaporation are: the vacuum degree is -0.09 to -0.1 MPa, the temperature is 25 - 30 °C, the rotation speed is 300 - 500 rpm, and the time is 30 - 60 min; the parameters of the second rotary evaporation are: the vacuum degree is -0.08 to -0.09 MPa, the temperature is 45 - 70 °C, the rotation speed is 700 - 1000 rpm, and the time is 20 - 30 min.

4. The preparation method of a GSH-responsive nano-drug carrier according to any one of claims 1-3, characterized in that, It includes the following steps: S1. Dissolve the PAMAM powder in deionized water, stir until completely dissolved, then add levofloxacin, and continue to stir for 20 - 28 h to obtain a first composite solution. Sequentially add BSA and NHS-activated disulfide compound to the first composite solution, and stir for 10 - 14 h until the reaction is complete to obtain a second composite solution. S2. Transfer the second composite solution into a dialysis bag with MWCO:100 kDa, then dialyze it with deionized water for 20 - 28 h, and change the deionized water every 6 h. After dialysis, the obtained product is freeze-dried to obtain a white powder, which is the levofloxacin-loaded BSA@PAMAM complex.

5. The preparation method of a GSH-responsive nano drug carrier according to claim 4, characterized in that, It also includes: Using 2,4,5,6-tetra(9-carbazolyl) isophthalonitrile as a photocatalyst, photoinduced catalysis is carried out on the first composite solution and the NHS-activated disulfide compound respectively under alkaline conditions with pH = 7 - 7.

2. The method of photoinduced catalysis is as follows: adding a photocatalyst accounting for 3-5 mol% of the first composite liquid to the first composite liquid, stirring in the dark for 20-30 min, and then irradiating with first visible light for 8-10 h, with a power of 3-5 mW / cm 2 ; Before adding the NHS-activated disulfide compound to the first composite solution, 0.08 - 0.12 mmol of the NHS-activated disulfide compound and 0.1 - 0.14 mmol of diphenyl disulfone are dissolved in 4 - 6 mL of 1,4-dioxane, a photocatalyst accounting for 4 - 5 mol% of the NHS-activated disulfide compound is added, and the mixture is stirred in the dark for 20 - 30 min. Under the conditions of a nitrogen atmosphere and 25 - 28 °C, it is irradiated with the second visible light for reaction for 1.5 - 2 h, and ethyl acetate:petroleum ether with a volume ratio of 50:1 is used as the mobile phase, and purified by silica gel column chromatography.

6. The GSH-responsive nanomedicine carrier according to claim 5, wherein Both the first visible light and the second visible light are LED lights. The wavelength of the first visible light is 450 nm and the power is 3 - 5 mW / cm 2 ; the wavelength of the second visible light is 390 nm and the power is 5 - 7 mW / cm 2 .

7. The preparation method of a GSH-responsive nanomedicine carrier according to claim 4, characterized in that, In S1, the stirring parameters are all: stirring at 22 - 28 °C and 100 - 1000 rpm until uniform.

8. The preparation method of a GSH-responsive nano drug carrier according to claim 4, characterized in that, In S2, the freeze-drying method is: drying at a temperature of -30 to -60 °C for 24 - 100 h.

9. Use of a GSH-responsive nanodrug carrier according to any one of claims 1 - 3 in an antibiotic controlled-release drug delivery system.

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