A sialic acid-modified hypoxia-responsive chitosan nitrobenzene graft and preparation and application thereof

Abstract

The application provides a sialic acid modified hypoxia-responsive chitosan nitrobenzene graft and preparation and application, wherein part of the amino groups of chitosan are substituted by nitrobenzene and sialic acid molecules. The application is based on the hypoxia-responsive chitosan nitrobenzene graft, uses double-end amino polyethylene glycol 2000 as a bridging molecule, chemically modifies sialic acid molecules, and efficiently encapsulates the drug ambrisentan. This not only increases the water solubility and in-vivo circulation time of the drug, but also has the ability to actively target the pulmonary artery endothelium through the interaction with the high expression of E-selection of the pulmonary artery endothelial cells in hypoxic pulmonary arterial hypertension, and is transported to the pulmonary artery smooth muscle at the treatment site by the material exchange between the pulmonary artery endothelium and the smooth muscle cells, responds to the high expression of nitroreductase, rapidly depolymerizes and releases the drug, and cooperatively improves the treatment effect of hypoxic pulmonary arterial hypertension, and can be applied in the preparation of hypoxic pulmonary arterial hypertension treatment drugs. The representative structural general formula is:

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical science and relates to a sialic acid-modified chitosan nitrobenzene graft and its synthesis method. In particular, it relates to a sialic acid-modified hypoxia-responsive chitosan nitrobenzene graft and its preparation and application. It is a construction of a sialic acid-modified hypoxia-responsive chitosan nitrobenzene graft that targets the pulmonary artery under hypoxic pulmonary hypertension and its application in the preparation of drugs for hypoxia-induced pulmonary hypertension. Background Technology

[0002] Hypoxic pulmonary arterial hypertension (HPAH) is a progressive disease characterized by pulmonary vascular remodeling and constriction, often leading to right ventricular hypertrophy and right heart failure, endangering life, with poor prognosis and a short median survival. Ambrisentan is a selective endothelin receptor subtype A (ETA) antagonist that blocks endothelin-1 activation of ETA, reducing pulmonary artery smooth muscle cell proliferation and contraction, and is a first-line drug for the treatment of HPAH. Ambrisentan is usually administered orally, but faces challenges such as poor water solubility, short drug half-life, low selectivity, and systemic toxicity. Nanoparticle drug delivery systems can improve therapeutic selectivity by passively or actively targeting and enriching the drug at the target site. However, due to their sustained drug release characteristics, they are prone to toxic side effects and difficulty in accumulating high drug concentrations at the target site. To precisely control drug release, sensitive response drug delivery systems with differentiated drug release between the target site and normal tissues are needed under the premise of targeted drug delivery.

[0003] Related literature reports the emergence of carcinogenic characteristics in HPAH, leading to the concept of carcinoid tumors. Simultaneously, tumor metabolism is predominantly aerobic glycolysis, exhibiting specificity and resulting in the upregulation of various enzymes, including nitroreductase, in the hypoxic microenvironment. Similarly, glycolytic metabolic activity is enhanced in the lungs of HPAH patients. Therefore, we have reason to speculate that HPAH possesses a tumor-like bioreducing hypoxic microenvironment, providing an opportunity for the design of hypoxia-responsive nanomedicine delivery systems.

[0004] Furthermore, related studies have shown that the high blood flow in pulmonary vessels during HPAH results in greater mechanical and shear stress, making it difficult for nanoparticles to effectively remain at the lesion site. Target modification is necessary to enhance the lesion site anchoring ability of nanoparticles. Research indicates that E-selection expression is upregulated on the surface of pulmonary artery endothelial cells in HPAH. Sialic acid is a natural 9-carbon carboxylated monosaccharide that can effectively target E-selection and P-selection on the cell surface. This suggests that sialic acid could be selected for targeted modification to improve the pulmonary artery targeting and aggregation ability of nano-drug delivery systems.

[0005] However, ambrisentan's therapeutic site is pulmonary artery smooth muscle cells. When employing a pulmonary artery endothelial-targeting strategy, it is crucial to confirm that the nanomedicine delivery system can ultimately reach the pulmonary artery smooth muscle. Studies have found that when pulmonary artery endothelial cells are dysfunctional, they secrete large amounts of paracrine factors, inducing pulmonary artery smooth muscle cell proliferation. This phenomenon is particularly pronounced in pulmonary artery endothelial cells derived from HPAH patients. This indicates that during the HPAH process, there is close exchange of substances between the pulmonary artery endothelium and smooth muscle cells, which facilitates drug delivery to the pulmonary artery smooth muscle. Summary of the Invention

[0006] The first objective of this invention is to provide a sialic acid-modified hypoxia-responsive chitosan-nitrobenzene graft, which has the following representative structural formula:

[0007]

[0008] in:

[0009] a represents the number of monocyclic sugars in the sialic acid-modified chitosan-nitrobenzene graft where the amino group is replaced by nitrobenzyl 6-aminohexanoic acid, with a grafting percentage of 5.3%–14.8%; b represents the number of monocyclic sugars in the sialic acid-modified chitosan-nitrobenzene graft where the amino group is not replaced, with a percentage of 75.9%–89.5%; c represents the number of monocyclic sugars in the sialic acid-modified chitosan-nitrobenzene graft where the amino group is replaced by acetyl groups, with a percentage of 5%; d represents the number of monocyclic sugars in the sialic acid-modified chitosan-nitrobenzene graft where the amino group is replaced by polyethylene glycol-modified sialic acid, with a percentage of 0.2%–4.3%.

[0010] The second objective of this invention is to provide a method for preparing sialic acid-modified hypoxia-responsive chitosan-nitrobenzene grafts, which is achieved through the following scheme:

[0011] 1. Preparation of PEGylated sialic acid reaction solution: Weigh 0.08–0.60 mmol of sialic acid, 0.16–1.80 mmol of N-hydroxysuccinimide and 0.16–1.80 mmol of carbodiimide, dissolve in 10–35 mL of dimethyl sulfoxide, place in a 100 mL round-bottom flask, and react at room temperature for 2 h to obtain reaction solution 1. Weigh 0.08–0.60 mmol of diamino-terminated polyethylene glycol 2000, dissolve in 5–30 mL of dimethyl sulfoxide, and slowly add dropwise to reaction solution 1. After reacting at room temperature for 4–12 h, add 0.08–0.60 mmol of N,N′-disuccinimide carbonate, and continue reacting for 6–14 h to obtain the PEGylated sialic acid reaction solution.

[0012] 2. Weigh 4 mmol of chitosan-nitrobenzene grafted material, dissolve it in 15–40 mL of deionized water, and add it to the above polyethylene glycol-modified sialic acid reaction solution. React at 20–60 °C for 8–24 h to obtain the final reaction product. Dialyze the final reaction product in a dialysis bag for 2–3 days to remove dimethyl sulfoxide and water-soluble byproducts, and freeze-dry to obtain a solid powder of sialic acid-modified chitosan-nitrobenzene grafted material.

[0013] The chitosan-nitrobenzene grafting material used in step 2 is covered by the national invention patent "Oxygen-Sensitive Nitrobenzened Chitosan and Its Preparation and Application" (Patent No.: ZL202010127330.2). The weight-average molecular weight of chitosan is 5–18 kDa, and the nitrobenzene grafting rate is 5.3–14.8%.

[0014] A third objective of this invention is to provide the application of sialic acid-modified hypoxia-responsive chitosan-nitrobenzene grafts in the preparation of anti-hypoxia pulmonary hypertension drugs targeting the pulmonary artery. Research results show that sialic acid-modified chitosan-nitrobenzene grafts can be rapidly and extensively taken up by hypoxic pulmonary artery endothelial cells, exhibiting pulmonary artery targeting. Using sialic acid-modified chitosan-nitrobenzene grafts as a targeting carrier for E-selection, encapsulating ambrisentan, the sialic acid-modified chitosan-nitrobenzene graft rapidly releases the drug in a phosphate buffer solution containing 10 μg / mL nitroreductase and 100 μM reduced coenzyme II at 37°C and pH 7.4, demonstrating hypoxia-responsive drug release capability.

[0015] This invention selects the nitrobenzene hypoxia-sensitive graft compound described in ZL202010127330.2 as the parent core, modifies it with polyethylene glycol-modified sialic acid, and constructs a hypoxia-sensitive graft compound targeting the pulmonary artery, efficiently encapsulating ambrisentan. This nano-drug delivery system not only increases the water solubility and in vivo circulation time of ambrisentan, but also targets pulmonary artery endothelial cells in HPAH via sialic acid, and delivers it to the treatment site of pulmonary artery smooth muscle through substance exchange between endothelial and smooth muscle cells. Responding to the highly expressed nitroreductase, it rapidly releases ambrisentan, potentially synergistically enhancing the therapeutic effect of hypoxic pulmonary hypertension.

[0016] The advantages of this invention are as follows: Based on a hypoxia-responsive chitosan-nitrobenzene graft, this invention uses bi-amino-terminated polyethylene glycol 2000 as a bridging molecule to chemically modify sialic acid molecules, constructing a pulmonary artery-targeted hypoxia-responsive graft that efficiently encapsulates the therapeutic drug ambrisentan. This not only increases the water solubility and in vivo circulation time of ambrisentan, but also enhances the uptake of the drug delivery system by pulmonary artery endothelial cells through interaction with the highly expressed E-selection on the surface of pulmonary artery endothelial cells in hypoxic pulmonary hypertension. Furthermore, through substance exchange between pulmonary artery endothelial and smooth muscle cells, the drug is transported to the therapeutic site in pulmonary artery smooth muscle, responding to the highly expressed nitroreductase for rapid depolymerization and drug release, synergistically improving the therapeutic effect of hypoxic pulmonary hypertension. Attached Figure Description

[0017] Figure 1 Synthetic route of sialic acid-modified chitosan nitrobenzene grafts.

[0018] Figure 2 : 1H NMR spectrum of sialic acid-modified chitosan nitrobenzene graft.

[0019] Figure 3 Particle size variation of micelles of sialic acid-modified chitosan nitrobenzene grafts under in vitro hypoxic conditions.

[0020] Figure 4 Morphological changes of micelles of sialic acid-modified chitosan nitrobenzene grafts under in vitro hypoxic conditions.

[0021] Figure 5 In vitro drug release curves of drug-eluting nanoparticles with different nitroreductase concentrations (n=3).

[0022] Figure 6 Flow cytometry was used to evaluate the exchange of substances between pulmonary artery endothelial and smooth muscle cells in a Transwell co-culture model.

[0023] Figure 7 Expression of nitroreductase in pulmonary artery smooth muscle cells under normoxic or hypoxic conditions (n=3, *p<0.05).

[0024] Figure 8 The anti-pulmonary artery smooth muscle cell proliferation efficacy of sialic acid-modified chitosan nitrobenzene grafted drug-loaded nanoparticles (n=3, *p<0.05). Detailed Implementation

[0025] The present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0026] Example 1

[0027] 1. Synthesis of sialic acid-modified chitosan-nitrobenzene grafts

[0028] Using an anaerobic-responsive chitosan-nitrobenzene graft as the parent core and bi-amino-terminated polyethylene glycol 2000 as the bridging molecule, sialic acid molecules were chemically modified to construct a sialic acid-modified chitosan-nitrobenzene graft. The synthetic route is described in [link to synthetic route]. Figure 1 As shown.

[0029] (1) Synthesis of chitosan-nitrobenzene grafts

[0030] According to the national invention patent "Oxygen-Sensitive Nitrobenzened Chitosan and its Preparation and Application" (Patent No.: ZL202010127330.2), a chitosan nitrobenzene grafted product was prepared. The preparation process is as follows:

[0031] Weigh out benzyl p-nitrochloroformate, 0.8–2 molar amounts of 6-aminohexanoic acid, and 3–4 molar amounts of triethylamine. Suspend these in 10–15 mL of dichloromethane and place in a 50 mL dry round-bottom flask. In a nitrogen atmosphere, react in an ice-water bath for 10 min to neutralize the generated hydrogen chloride with triethylamine, ensuring the forward reaction proceeds. Continue reacting at room temperature for 12–24 h. After the reaction is complete, extract with 5 volumes of deionized water and 1 volume of ethyl acetate. Vortex, allow to stand, and after separation, collect the upper ethyl acetate layer. Place in a dry round-bottom flask and remove the solvent by rotary evaporation at 40 °C. Add 10–15 mL of N,N-dimethylformamide, 4–6 molar amounts of carbodiimide (e.g., p-nitrochloroformate), 2–7 molar amounts of chitosan (e.g., p-nitrochloroformate), and an equal volume of deionized water to N,N-dimethylformamide to a flask. React at 50–60 °C for 12–16 h using a magnetic stirrer to obtain the reaction product. Dialyze the reaction product in a dialysis bag (MWCO 3500 Da) for 2–3 days to remove N,N-dimethylformamide and water-soluble byproducts. After dialysis, freeze-dry the reaction product to obtain chitosan-nitrobenzene grafted powder.

[0032] (2) Synthesis of sialic acid-modified chitosan-nitrobenzene grafts

[0033] Weigh 0.08 mmol of sialic acid, 0.16 mmol of N-hydroxysuccinimide, and 0.16 mmol of carbodiimide, dissolve in 10 mL of dimethyl sulfoxide, place in a 100 mL round-bottom flask, and react at room temperature for 2 h to obtain reaction solution 1. Weigh 0.08 mmol of diamino-terminated polyethylene glycol 2000, dissolve in 5 mL of dimethyl sulfoxide, and slowly add dropwise to reaction solution 1. After reacting at room temperature for 4 h, add 0.08 mmol of N,N′-disuccinimide carbonate, and continue reacting for 6 h to obtain polyethylene glycolated sialic acid reaction solution.

[0034] Weigh 4 mmol of chitosan-nitrobenzene grafted material, dissolve it in 15 mL of deionized water, and add it to the above polyethylene glycol-modified sialic acid reaction solution. React at 20 °C for 8 h to obtain the final reaction product. Dialyze the final reaction product in a dialysis bag for 2 days to remove dimethyl sulfoxide and water-soluble byproducts. Freeze-dry to obtain sialic acid-modified chitosan-nitrobenzene grafted material solid powder.

[0035] 2. Determination of the degree of amino substitution, particle size and zeta potential of sialic acid-modified chitosan nitrobenzene grafts

[0036] The degree of amino substitution of the grafted material was determined by the trinitrobenzenesulfonic acid method. 10 mg of chitosan powder was weighed to prepare a 1 mg / mL chitosan stock solution. 0.01–1.0 mL of the chitosan stock solution was accurately measured and diluted with deionized water to 2.0 mL. Then, 2.0 mL each of 4% sodium bicarbonate solution and 0.1% 2,4,6-trinitrobenzenesulfonic acid solution were added sequentially, and the mixture was incubated at 37°C in the dark for 2 hours. 2.0 mL of 2 mol / L hydrochloric acid aqueous solution was added and the mixture was shaken well. The absorbance was measured at 344 nm using a UV spectrophotometer, and a standard curve was plotted. Take 4 mg each of the above-mentioned chitosan nitrobenzene graft and sialic acid-modified chitosan nitrobenzene graft, dissolve them in 2.0 mL of deionized water, and operate in the same way. According to the standard curve, the degree of amino substitution of the chitosan nitrobenzene graft and the sialic acid-modified chitosan nitrobenzene graft are 10.1% and 10.3%, respectively, and the sialic acid modification rate is 0.2%.

[0037] The particle size and surface potential of the above sialic acid modified chitosan nitrobenzene graft were measured to be 276.2±26.6 nm and the zeta potential was 36.9±3.3 mV, respectively.

[0038] Example 2

[0039] 1. Synthesis of sialic acid-modified chitosan-nitrobenzene grafts

[0040] A two-step synthesis of sialic acid-modified chitosan nitrobenzene grafts is described below:

[0041] (1) Synthesis of chitosan-nitrobenzene grafts

[0042] According to the national invention patent "Oxygen-sensitive nitrophenylenedilicate and its preparation and application" (patent number: ZL202010127330.2), a chitosan nitrobenzene grafted product was prepared. The preparation process is as described in Example 1.

[0043] (2) Synthesis of sialic acid-modified chitosan-nitrobenzene grafts

[0044] Weigh 0.60 mmol of sialic acid, 1.50 mmol of N-hydroxysuccinimide, and 1.50 mmol of carbodiimide, dissolve in 20 mL of dimethyl sulfoxide, place in a 100 mL round-bottom flask, and react at room temperature for 2 h to obtain reaction solution 1. Weigh 0.60 mmol of diamino-terminated polyethylene glycol 2000, dissolve in 20 mL of dimethyl sulfoxide, and slowly add dropwise to reaction solution 1. After reacting at room temperature for 8 h, add 0.60 mmol of N,N′-disuccinimide carbonate, and continue reacting for 14 h to obtain polyethylene glycolated sialic acid reaction solution.

[0045] Weigh 4 mmol of chitosan-nitrobenzene grafted material, dissolve it in 30 mL of deionized water, and add it to the above polyethylene glycol-modified sialic acid reaction solution. React at 35 °C for 24 h to obtain the final reaction product. The final reaction product is placed in a dialysis bag and dialyzed for 3 days to remove dimethyl sulfoxide and water-soluble byproducts. It is then freeze-dried to obtain a solid powder of sialic acid-modified chitosan-nitrobenzene grafted material.

[0046] 2. Determination of amino substitution degree, particle size and zeta potential of sialic acid-modified chitosan-nitrobenzene grafts

[0047] The degree of amino substitution of the grafted material was determined by the trinitrobenzenesulfonic acid method. 10 mg of chitosan powder was weighed to prepare a 1 mg / mL chitosan stock solution. 0.01–1.0 mL of the chitosan stock solution was accurately measured and diluted with deionized water to 2.0 mL. Then, 2.0 mL each of 4% sodium bicarbonate solution and 0.1% 2,4,6-trinitrobenzenesulfonic acid solution were added sequentially, and the mixture was incubated at 37°C in the dark for 2 hours. 2.0 mL of 2 mol / L hydrochloric acid aqueous solution was added and the mixture was shaken well. The absorbance was measured at 344 nm using a UV spectrophotometer, and a standard curve was plotted. Take 4 mg each of the above-mentioned chitosan nitrobenzene graft and sialic acid-modified chitosan nitrobenzene graft, dissolve them in 2.0 mL of deionized water, and operate in the same way. According to the standard curve, the degree of amino substitution of the chitosan nitrobenzene graft and the sialic acid-modified chitosan nitrobenzene graft are 10.1% and 14.4%, respectively, and the sialic acid modification rate is 4.3%.

[0048] The particle size and surface potential of the above sialic acid modified chitosan nitrobenzene graft were measured to be 155.7±5.1 nm and the zeta potential was 28.5±1.8 mV, respectively.

[0049] 3. Structural confirmation of sialic acid-modified chitosan-nitrobenzene grafts

[0050] 1The chemical structure of the sialic acid-modified chitosan nitrobenzene graft was confirmed by 1H NMR. 5 mg each of sialic acid and diamino-terminated polyethylene glycol 2000 were dissolved in deuterated dimethyl sulfoxide. Separately, 5 mg each of the chitosan nitrobenzene graft and the sialic acid-modified chitosan nitrobenzene graft were dissolved in heavy water, and both were prepared into 10 mg / mL solutions. Their respective 1H NMR spectra were recorded, and structural analysis was performed to confirm the graft structure. Figure 2 As shown, sialic acid-modified chitosan nitrobenzene grafts 1 The 1H NMR spectrum showed that the proton peak with a chemical shift of 2.0 ppm belonged to the methyl hydrogen of sialic acid, the proton peak with a chemical shift of 3.6 ppm belonged to the -CH2CH2O- of the bi-amino-terminated polyethylene glycol 2000, the proton peaks with chemical shifts of 3.6–3.9 ppm belonged to the chitosan-nitrobenzene graft, and the small proton peaks with chemical shifts of 7.5–8.4 ppm belonged to the hydrogen on the nitrobenzene in the chitosan-nitrobenzene graft, which, after amplification, were consistent with the proton hydrogen peaks of nitrobenzene in the chitosan-nitrobenzene graft. Furthermore, the sialic acid-modified chitosan-nitrobenzene graft... 1 The absence of a hydrogen proton peak for the sialic acid carboxyl group in the 1H NMR spectrum indicates that the sialic acid carboxyl group has reacted with the amino group of polyethylene glycol 2000. These results demonstrate that sialic acid has been linked to polyethylene glycol 2000 via an amide bond, and further linked to the chitosan nitrobenzene graft, yielding a sialic acid-modified chitosan nitrobenzene graft.

[0051] Example 3

[0052] 1. Synthesis of sialic acid-modified chitosan-nitrobenzene grafts

[0053] A two-step synthesis of sialic acid-modified chitosan nitrobenzene grafts is described below:

[0054] (1) Synthesis of chitosan-nitrobenzene grafts

[0055] According to the national invention patent "Oxygen-sensitive nitrophenylenedilicate and its preparation and application" (patent number: ZL202010127330.2), a chitosan nitrobenzene grafted product was prepared. The preparation process is as described in Example 1.

[0056] (2) Synthesis of sialic acid-modified chitosan-nitrobenzene grafts

[0057] Weigh 0.48 mmol of sialic acid, 1.0 mmol of N-hydroxysuccinimide, and 1.0 mmol of carbodiimide, dissolve in 35 mL of dimethyl sulfoxide, place in a 100 mL round-bottom flask, and react at room temperature for 2 h to obtain reaction solution 1. Weigh 0.48 mmol of diamino-terminated polyethylene glycol 2000, dissolve in 15 mL of dimethyl sulfoxide, and slowly add dropwise to reaction solution 1. After reacting at room temperature for 12 h, add 0.48 mmol of N,N′-disuccinimide carbonate, and continue reacting for 10 h to obtain polyethylene glycolated sialic acid reaction solution.

[0058] Weigh 4 mmol of chitosan-nitrobenzene grafted material, dissolve it in 40 mL of deionized water, and add it to the above polyethylene glycol-modified sialic acid reaction solution. React at 60 °C for 16 h to obtain the final reaction product. Dialyze the final reaction product in a dialysis bag for 3 days to remove dimethyl sulfoxide and water-soluble byproducts. Freeze-dry to obtain a solid powder of sialic acid-modified chitosan-nitrobenzene grafted material.

[0059] 2. Determination of amino substitution degree, particle size and zeta potential of sialic acid-modified chitosan-nitrobenzene grafts

[0060] The degree of amino substitution of the grafted material was determined by the trinitrobenzenesulfonic acid method. 10 mg of chitosan powder was weighed to prepare a 1 mg / mL chitosan stock solution. 0.01–1.0 mL of the chitosan stock solution was accurately measured and diluted with deionized water to 2.0 mL. Then, 2.0 mL each of 4% sodium bicarbonate solution and 0.1% 2,4,6-trinitrobenzenesulfonic acid solution were added sequentially, and the mixture was incubated at 37°C in the dark for 2 hours. 2.0 mL of 2 mol / L hydrochloric acid aqueous solution was added and the mixture was shaken well. The absorbance was measured at 344 nm using a UV spectrophotometer, and a standard curve was plotted. Take 4 mg each of the above chitosan nitrobenzene graft and sialic acid-modified chitosan nitrobenzene graft, dissolve them in 2.0 mL of deionized water, and operate in the same way. According to the standard curve, the degree of amino substitution of the chitosan nitrobenzene graft and the sialic acid-modified chitosan nitrobenzene graft are 101% and 13.2%, respectively, and the sialic acid modification rate is 3.1%.

[0061] The particle size and surface potential of the above sialic acid modified chitosan nitrobenzene graft were measured to be 186.7±14.0 nm and the zeta potential was 32.6±2.0 mV, respectively.

[0062] 3. Determination of critical micelle concentration of sialic acid-modified chitosan-nitrobenzene grafts using pyrene fluorescent probe method

[0063] Weigh 2g of pyrene to prepare a 0.0012mg / mL pyrene / acetone solution. Measure 0.5mL of the pyrene / acetone solution and place it in a 10mL glass tube, evaporating the acetone at 50℃. Weigh 10mg of the above sialic acid-modified chitosan-nitrobenzene grafting material and dissolve it in 10mL of deionized water. Sonicate the solution with a probe to obtain a 1.0mg / mL test solution. Dilute with deionized water to prepare 5mL of aqueous solutions of different concentrations of grafting material. Add these solutions to the above pyrene-containing glass tubes and sonicate in a water bath at room temperature for 30min. Set the parameters of the fluorescence spectrophotometer (excitation slit 10nm, emission slit 2.5nm) and measure the fluorescence intensity I of the series of grafting materials at emission wavelengths of 374nm and 385nm under an excitation wavelength of 337nm. 374 and I 385 A graph was plotted with the fluorescence intensity ratio as the ordinate and the logarithmic concentration of the grafted material as the abscissa. The critical micelle concentration of the grafted material was determined to be 49.2 μg / mL.

[0064] 4. In vitro hypoxia response study of sialic acid-modified chitosan-nitrobenzene grafts

[0065] Related literature reports that nitroreductase is specifically highly expressed in hypoxic microenvironments, and 10 μg / mL nitroreductase and 100 μM reduced coenzyme II can simulate hypoxic microenvironments in vitro. 5 mg of sialic acid-modified chitosan-nitrobenzene graft was weighed, dissolved in deionized water to prepare a 1 mg / mL solution, and then incubated with 10 μg / mL nitroreductase and 100 μM reduced coenzyme II. The changes in micelle size were recorded at specific time points of 0, 10, 20, 40, and 60 min using a particle size analyzer, and the changes in micelle morphology were observed at 0 and 1 h using a transmission electron microscope.

[0066] The results of the particle size analyzer are as follows Figure 3 As shown, after incubation of nitroreductase with reduced coenzyme II, the micelle size decreased rapidly over time, and the formed micelles became difficult to detect after 1 hour.

[0067] Transmission electron microscopy results ( Figure 4 The results also showed that, before treatment, the micelles were uniform in size and had a regular spherical shape. After incubation with nitroreductase and reduced coenzyme II, the micelle size decreased rapidly, some micelle structures were significantly destroyed and depolymerized, resulting in irregular aggregation.

[0068] All the above results show that sialic acid-modified chitosan nitrobenzene graft nanoparticles can rapidly depolymerize in response to nitroreductase, and the modification of polyethylene glycol-modified sialic acid does not affect the hypoxia response of the parent chitosan nitrobenzene graft.

[0069] Example 4

[0070] 1. Synthesis of sialic acid-modified chitosan-nitrobenzene grafts

[0071] A two-step synthesis of sialic acid-modified chitosan nitrobenzene grafts is described below:

[0072] (1) Synthesis of chitosan-nitrobenzene grafts

[0073] According to the national invention patent "Oxygen-sensitive nitrophenylenedilicate and its preparation and application" (patent number: ZL202010127330.2), a chitosan nitrobenzene grafted product was prepared. The preparation process is as described in Example 1.

[0074] (2) Synthesis of sialic acid-modified chitosan-nitrobenzene grafts

[0075] Weigh 0.40 mmol of sialic acid, 1.20 mmol of N-hydroxysuccinimide, and 1.20 mmol of carbodiimide, dissolve in 35 mL of dimethyl sulfoxide, place in a 100 mL round-bottom flask, and react at room temperature for 2 h to obtain reaction solution 1. Weigh 0.40 mmol of diamino-terminated polyethylene glycol 2000, dissolve in 30 mL of dimethyl sulfoxide, and slowly add dropwise to reaction solution 1. After reacting at room temperature for 10 h, add 0.40 mmol of N,N′-disuccinimide carbonate, and continue reacting for 12 h to obtain polyethylene glycolated sialic acid reaction solution.

[0076] Weigh 4 mmol of chitosan-nitrobenzene grafted material, dissolve it in 35 mL of deionized water, and add it to the above polyethylene glycol-modified sialic acid reaction solution. React at 25 °C for 24 h to obtain the final reaction product. Dialyze the final reaction product in a dialysis bag for 2 days to remove dimethyl sulfoxide and water-soluble byproducts, and freeze-dry to obtain a solid powder of sialic acid-modified chitosan-nitrobenzene grafted material.

[0077] 2. Determination of amino substitution degree, particle size and zeta potential of sialic acid-modified chitosan-nitrobenzene grafts

[0078] The degree of amino substitution of the grafted material was determined by the trinitrobenzenesulfonic acid method. 10 mg of chitosan powder was weighed to prepare a 1 mg / mL chitosan stock solution. 0.01–1.0 mL of the chitosan stock solution was accurately measured and diluted with deionized water to 2.0 mL. Then, 20 mL each of 4% sodium bicarbonate solution and 0.1% 2,4,6-trinitrobenzenesulfonic acid solution were added sequentially, and the mixture was incubated at 37°C in the dark for 2 hours. 2.0 mL of 2 mol / L hydrochloric acid aqueous solution was added and the mixture was shaken well. The absorbance was measured at 344 nm using a UV spectrophotometer, and a standard curve was plotted. Take 4 mg each of the above-mentioned chitosan nitrobenzene graft and sialic acid-modified chitosan nitrobenzene graft, dissolve them in 2.0 mL of deionized water, and operate in the same way. According to the standard curve, the degree of amino substitution of the chitosan nitrobenzene graft and the sialic acid-modified chitosan nitrobenzene graft are 10.1% and 12.8%, respectively, and the sialic acid modification rate is 2.7%.

[0079] The particle size and surface potential of the above sialic acid modified chitosan nitrobenzene graft were measured to be 173.8±6.3 nm and the zeta potential was 31.3±0.7 mV, respectively.

[0080] 3. Determination of critical micelle concentration of sialic acid-modified chitosan-nitrobenzene grafts using pyrene fluorescent probe method

[0081] Weigh 2g of pyrene to prepare a 0.0012mg / mL pyrene / acetone solution. Measure 0.5mL of the pyrene / acetone solution and place it in a 10mL glass tube, evaporating the acetone at 50℃. Weigh 10mg of the above sialic acid-modified chitosan-nitrobenzene grafting material and dissolve it in 10mL of deionized water. Sonicate the solution with a probe to obtain a 1.0mg / mL test solution. Dilute with deionized water to prepare 5mL of aqueous solutions of different concentrations of grafting material. Add these solutions to the above pyrene-containing glass tubes and sonicate in a water bath at room temperature for 30min. Set the parameters of the fluorescence spectrophotometer (excitation slit 10nm, emission slit 25nm) and measure the fluorescence intensity I of the series of grafting materials at emission wavelengths of 374nm and 385nm under an excitation wavelength of 337nm. 374 and I 385 A graph was plotted with the fluorescence intensity ratio as the ordinate and the logarithmic concentration of the grafted material as the abscissa. The critical micelle concentration of the grafted material was determined to be 44.7 μg / mL.

[0082] 4. Preparation of sialic acid-modified chitosan-nitrobenzene grafted drug-loaded nanoparticles

[0083] Drug-loaded nanoparticles were prepared using the dialysis bag method. 10 mg of sialic acid-modified chitosan-nitrobenzene graft compound was weighed and prepared into a 5 mg / mL micelle solution. An ambrisentan:graft compound = 20% (w / w) solution of dimethyl sulfoxide was slowly added dropwise to the micelle solution. The mixture was stirred at 400 rpm for 2 h at room temperature. The solution was then placed in a dialysis bag (MWCO = 35 kDa) and dialyzed against deionized water for 12 h. The solution from the dialysis bag was collected, centrifuged at 5000 rpm for 10 min, and the supernatant was collected to obtain the drug-loaded nanoparticle solution.

[0084] High-performance liquid chromatography (HPLC) was used to determine the content of ambrisentan. The chromatographic conditions were as follows: Agilent C18 column (4.6 × 250 mm, 5 μm); column temperature 25℃; mobile phase: A: 20 mmol / L potassium dihydrogen phosphate aqueous solution, pH adjusted to 2.5 with phosphoric acid; B: acetonitrile, with a volume ratio of A:B = 45:55 (v / v); flow rate 0.9 mL / min; detection wavelength 262 nm; injection volume 50 μL. The drug loading of the drug-loaded nanoparticles was 15.9%, with an encapsulation efficiency as high as 94.8%. These results indicate that sialic acid-modified chitosan-nitrobenzene graft composite, as a drug carrier with excellent encapsulation properties, can efficiently encapsulate ambrisentan, a drug for treating hypoxic pulmonary hypertension, and also increases its water solubility.

[0085] 5. In vitro hypoxia-responsive release kinetics of drug-loaded nanoparticles

[0086] Using 20 mL of phosphate buffer (pH 7.4) as the release medium, an appropriate amount of drug-loaded nanoparticle solution or ambrisentan dispersion (containing approximately 200 μg of ambrisentan) was transferred and diluted to 1 mL with deionized water. The experimental group consisted of 10 μg / mL nitroreductase and 100 μM reduced coenzyme II, while the control group consisted of no nitroreductase and reduced coenzyme II. Both were placed in dialysis bags (MWCO = 3.5 kDa). The solution was shaken at 37°C for 60 rpm. Samples were taken after a certain time, and the release medium was completely replaced with freshly prepared phosphate buffer. Three parallel groups were sampled. The concentration of ambrisentan at each time point was determined by high-performance liquid chromatography (HPLC), and the cumulative drug release was calculated.

[0087] The results are as follows Figure 5 As shown, under conditions of 0 or 10 μg / mL nitroreductase, the detection rate of free ambrisentan after 8 hours was 100.6% and 100%, respectively, indicating that the dialysis bag had no significant inhibitory effect on drug penetration, and nitroreductase did not affect drug detection. In the drug-loaded nanoparticle group without nitroreductase and reduced coenzyme II, the cumulative drug release after 48 hours was 74.1%. However, in an in vitro simulated hypoxic environment, the drug release from the drug-loaded nanoparticles increased significantly, with a cumulative release rate as high as 96.8% after 48 hours. These results indicate that the sialic acid-modified chitosan-nitrobenzene graft composite loaded with ambrisentan can rapidly release the drug in response to nitroreductase, exhibiting good in vitro hypoxic-responsive drug release properties.

[0088] Example 5

[0089] 1. Synthesis of sialic acid-modified chitosan-nitrobenzene grafts

[0090] A two-step synthesis of sialic acid-modified chitosan nitrobenzene grafts is described below:

[0091] (1) Synthesis of chitosan-nitrobenzene grafts

[0092] According to the national invention patent "Oxygen-sensitive nitrophenylenedilicate and its preparation and application" (patent number: ZL202010127330.2), a chitosan nitrobenzene grafted product was prepared. The preparation process is as described in Example 1.

[0093] (2) Synthesis of sialic acid-modified chitosan-nitrobenzene grafts

[0094] Weigh 0.35 mmol of sialic acid, 180 mmol of N-hydroxysuccinimide, and 1.80 mmol of carbodiimide, dissolve in 25 mL of dimethyl sulfoxide, place in a 100 mL round-bottom flask, and react at room temperature for 2 h to obtain reaction solution 1. Weigh 0.35 mmol of diamino-terminated polyethylene glycol 2000, dissolve in 15 mL of dimethyl sulfoxide, and slowly add dropwise to reaction solution 1. After reacting at room temperature for 9 h, add 0.40 mmol of N,N′-disuccinimide carbonate, and continue reacting for 11 h to obtain polyethylene glycolated sialic acid reaction solution.

[0095] Weigh 4 mmol of chitosan-nitrobenzene grafted material, dissolve it in 25 mL of deionized water, and add it to the above polyethylene glycol-modified sialic acid reaction solution. React at 40 °C for 18 h to obtain the final reaction product. Dialyze the final reaction product in a dialysis bag for 3 days to remove dimethyl sulfoxide and water-soluble byproducts. Freeze-dry to obtain a solid powder of sialic acid-modified chitosan-nitrobenzene grafted material.

[0096] 2. Determination of amino substitution degree, particle size and zeta potential of sialic acid-modified chitosan-nitrobenzene grafts

[0097] The degree of amino substitution of the grafted material was determined by the trinitrobenzenesulfonic acid method. 10 mg of chitosan powder was weighed to prepare a 1 mg / mL chitosan stock solution. 0.01–1.0 mL of the chitosan stock solution was accurately measured and diluted with deionized water to 2.0 mL. Then, 2.0 mL each of 4% sodium bicarbonate solution and 0.1% 2,4,6-trinitrobenzenesulfonic acid solution were added sequentially, and the mixture was incubated at 37°C in the dark for 2 hours. 2.0 mL of 2 mol / L hydrochloric acid aqueous solution was added and the mixture was shaken well. The absorbance was measured at 344 nm using a UV spectrophotometer, and a standard curve was plotted. Take 4 mg each of the above-mentioned chitosan nitrobenzene graft and sialic acid-modified chitosan nitrobenzene graft, dissolve them in 2.0 mL of deionized water, and operate in the same way. According to the standard curve, the degree of amino substitution of the chitosan nitrobenzene graft and the sialic acid-modified chitosan nitrobenzene graft are 10.1% and 12.3%, respectively, and the sialic acid modification rate is 2.2%.

[0098] The particle size and surface potential of the above sialic acid modified chitosan nitrobenzene graft were measured to be 160.9±8.9 nm and the zeta potential was 32.4±1.0 mV, respectively.

[0099] 3. Preparation of sialic acid-modified chitosan-nitrobenzene grafted drug-loaded nanoparticles and fluorescently labeled nanoparticles

[0100] Following the dialysis bag method in Example 4, sialic acid-modified chitosan nitrobenzene grafted drug-loaded nanoparticles were prepared in the same manner. The drug loading of the nanoparticles was 15.2% and the encapsulation rate was 89.9% as detected by high performance liquid chromatography. The nanoparticles were left to stand at room temperature for later use.

[0101] Fluorescein isothiocyanate (FITC) labeled grafted nanoparticles. 6 mg of sialic acid-modified chitosan-nitrobenzene grafted material was weighed and dissolved in a mixed solvent (dimethyl sulfoxide:water = 8:2, v / v) to prepare a 3 mg / mL micelle solution. Under light-protected conditions, the FITC-dimethyl sulfoxide solution was slowly added dropwise to the micelle solution at a dosage ratio of 5% (w / w) of FITC:grafted material. The mixture was stirred at 300 rpm for 16 h. The solution was then transferred to a dialysis bag (MWCO = 3.5 kDa) and dialyzed against deionized water for 24 h. The dialysis bag solution was collected, centrifuged at 5000 rpm for 10 min, and the supernatant was collected to obtain the FITC-labeled sialic acid-modified chitosan-nitrobenzene grafted material. Weigh 6 mg of the chitosan nitrobenzene grafting material prepared according to the national invention patent (patent number: ZL202010127330.2), and proceed with the same method to obtain FITC-labeled chitosan nitrobenzene grafting material. Let stand at room temperature for later use.

[0102] 4. Evaluation of substance exchange between human pulmonary artery endothelial and smooth muscle cells

[0103] The Transwell co-culture model was used to evaluate the exchange of substances between human pulmonary artery endothelial and smooth muscle cells. Cells were cultured at a density of 6 × 10⁶. 6 Human pulmonary artery endothelial cells were seeded at a density of 2 × 10⁶ / mL in the upper chamber of a Transwell (0.4 μm pore size). 5 Human pulmonary artery smooth muscle cells (0.5 mL / mL) were seeded in the lower chamber of a Transwell incubator and cultured for 3 days. Then, they were transferred to a triple-gas hypoxic incubator (1% O2, 5% CO2, 94% N2) for 2 days. 20 μL of the prepared FITC-labeled chitosan-nitrobenzene graft or FITC-labeled sialic acid-modified chitosan-nitrobenzene graft was added to the upper chamber and incubated for 6 hours. The culture medium in both the upper and lower chambers was discarded, and the cells were washed three times with phosphate-buffered saline (PFS), digested with trypsin, collected, resuspended in PFS, and dispersed into a single-cell suspension through a 300-mesh sieve. The mean fluorescence intensity of FITC in human pulmonary artery endothelial and smooth muscle cells was measured by flow cytometry.

[0104] The results are as follows Figure 6 As shown, under hypoxic conditions, the uptake rates of sialic acid-modified chitosan-nitrobenzene grafts by upper pulmonary artery endothelial cells and lower pulmonary artery smooth muscle cells were 18.38% and 56.34%, respectively, both significantly higher than the uptake rates of chitosan-nitrobenzene grafts by individual cell types. These results confirm that, compared to chitosan-nitrobenzene grafts, hypoxic pulmonary artery endothelial cells can take up more sialic acid-modified chitosan-nitrobenzene grafts due to the targeted binding of sialic acid to E-selection, and can better transport the grafts to the therapeutic site of pulmonary artery smooth muscle cells through intercellular exchange, thus treating HPAH.

[0105] 5. Determination of nitroreductase expression in human pulmonary artery smooth muscle cells

[0106] Under hypoxic or normoxic conditions, the expression level of nitroreductase in human pulmonary artery smooth muscle cells was measured using an ELISA kit. Cells were seeded at a density of 1×10⁶ cells per well in 6-well plates. 6 Human pulmonary artery smooth muscle cells were cultured overnight in a cell culture incubator, then transferred to a tri-gas hypoxic incubator (1% O2, 5% CO2, 94% N2) for 2 days. The culture medium was discarded, the cells were washed three times with phosphate-buffered saline, digested with trypsin, and collected. The control group (not transferred to the tri-gas hypoxic incubator) was treated using the same method. Protein quantification was performed using a BCA kit, and the expression level of nitroreductase in the cells was determined according to the standard operating procedure of the ELISA kit.

[0107] The results are as follows Figure 7 As shown, compared with the normoxic group, the content of nitroreductase in human pulmonary artery smooth muscle cells under hypoxic conditions significantly increased to 82.9 pg / mg (p<0.05), which was 91.8% higher than that in the normoxic group. This indicates that hypoxia can induce upregulation of nitroreductase expression in human pulmonary artery smooth muscle cells, enabling them to have a hypoxic reducing microenvironment, which lays the foundation for hypoxia-responsive drug release of intracellular drug-loaded nanoparticles.

[0108] 6. Sialic acid-modified chitosan-nitrobenzene grafted drug-loaded nanoparticles for human pulmonary artery smooth muscle cell cytotoxicity

[0109] The thiazolyl blue colorimetric method was used to evaluate the ability of drug-loaded nanoparticles to inhibit the proliferation of human pulmonary artery smooth muscle cells. Logarithmic growth phase human pulmonary artery smooth muscle cells were collected and analyzed at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 100% into 96-well plates and incubated overnight in a cell culture incubator. A series of concentrations of ambrisentan and sialic acid-modified chitosan-nitrobenzene grafted drug-loaded nanoparticles were added, with untreated blank cells serving as the control group. All cells were transferred to a tri-gas hypoxic incubator (1% O2, 5% CO2, 94% N2) and cultured for 2 days. After incubation, each well was replaced with 200 μL of fresh culture medium, and 20 μL of thiazolyl blue aqueous solution (5 mg / mL) was added for further incubation for 4 hours. The medium was then discarded, and 200 μL of dimethyl sulfoxide was added. The cells were shaken in a gas bath shaker for 0.5 hours, and the absorbance at 570 nm was measured using a microplate reader. The control group (not transferred to the tri-gas hypoxic incubator) was treated using the same method. Cell viability was calculated using the following formula:

[0110] Cell viability (%) = (Experimental group absorbance / Control group absorbance) × 100%

[0111] The results are as follows Figure 8As shown, under both normoxic and hypoxic conditions, the cell viability of the drug-loaded nanoparticle group was lower than that of the free ambrisentan group at the same dosage, and this effect was concentration-dependent, indicating that the drug-loaded nanoparticles had a better inhibitory effect on the proliferation of human pulmonary artery smooth muscle cells compared with free ambrisentan. At an ambrisentan dosage of 10 μg / mL, compared with the drug-loaded nanoparticle group in a normoxic environment, the cell viability of human pulmonary artery smooth muscle cells in the drug-loaded nanoparticle group in a hypoxic environment decreased by 10.4% (p < 0.05), indicating that a hypoxic environment can enhance the inhibitory effect of drug-loaded nanoparticles on the proliferation of human pulmonary artery smooth muscle cells.

Claims

1. The application of a sialic acid-modified hypoxia-responsive chitosan-nitrobenzene graft in the preparation of an anti-hypoxia pulmonary hypertension therapeutic agent targeting the pulmonary artery, characterized in that, The representative structural formulas are as follows: ; in: a represents the number of monocyclic sugar rings in the sialic acid-modified chitosan nitrobenzene graft where the amino group is replaced by nitrobenzyl 6-aminohexanoic acid, with a grafting percentage of 5.3%–14.8%; b represents the number of monocyclic sugar rings in the sialic acid-modified chitosan nitrobenzene graft where the amino group is not replaced, with a percentage of 75.9%–89.5%; c represents the number of monocyclic sugar rings in the sialic acid-modified chitosan nitrobenzene graft where the amino group is replaced by acetyl groups, with a percentage of 5%; d represents the number of monocyclic sugar rings in the sialic acid-modified chitosan nitrobenzene graft where the amino group is replaced by polyethylene glycol-modified sialic acid, with a percentage of 0.2%–4.3%. The preparation method of the sialic acid-modified hypoxia-responsive chitosan nitrobenzene graft is achieved through the following steps: (1) Preparation of polyethylene glycolated sialic acid reaction solution: Weigh 0.08–0.60 mmol of sialic acid, 0.16–1.80 mmol of N-hydroxysuccinimide, and 0.16–1.80 mmol of carbodiimide, dissolve in 10–35 mL of dimethyl sulfoxide, place in a 100 mL round-bottom flask, and react at room temperature for 2 h to obtain reaction solution 1. Weigh 0.08–0.60 mmol of diamino-terminated polyethylene glycol 2000, dissolve in 5–30 mL of dimethyl sulfoxide, and slowly add dropwise to reaction solution 1. After reacting at room temperature for 4–12 h, add 0.08–0.60 mmol of N,N'-disuccinimide carbonate, and continue reacting for 6–14 h to obtain polyethylene glycolated sialic acid reaction solution. (2) Preparation of sialic acid-modified chitosan-nitrobenzene grafts: Weigh 4 mmol of chitosan nitrobenzene grafting material, dissolve it in 15-40 mL of deionized water, add it to the above polyethylene glycol-modified sialic acid reaction solution, and react at 20-60℃ for 8-24 h to obtain the final reaction product. Place the final reaction product in a dialysis bag and dialyze for 2-3 days to remove dimethyl sulfoxide and water-soluble byproducts. Freeze-dry to obtain sialic acid-modified chitosan nitrobenzene grafting material solid powder.

2. The application according to claim 1, characterized in that, The chitosan-nitrobenzene graft used had a chitosan weight-average molecular weight of 5-18. k Da, the grafting rate of nitrobenzene is 5.3~14.8%.

3. The application according to claim 1, characterized in that, The application involves using sialic acid-modified chitosan nitrobenzene grafts as a targeting carrier for E-selection, encapsulating ambrisentan, which is rapidly and extensively taken up by hypoxic pulmonary artery endothelial cells, thus exhibiting pulmonary artery targeting.

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