Endoplasmic reticulum-targeted fluorescent nanocarrier, preparation method and application thereof

Abstract

The present application provides a fluorescent nanocarrier, abbreviated as NPs-NAP-FA-SS, which is prepared by grafting pullulan with pullulan as the hydrophilic part, and the fluorescent small molecules (E)-3-((3-(2-(6-(4-morpholinostyryl)-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethoxy)-3-oxopropyl)disulfonyl)propionic acid and folic acid as the hydrophobic part through esterification reaction. The fluorescent nanocarrier prepared in the present application has good targeting to the endoplasmic reticulum in cell experiments, with a Pearson coefficient R=0.943, and can encapsulate drugs, with a drug loading rate DLC and an encapsulation rate DLE of 10.1% and 78.9% respectively; it also takes into account excellent biosafety and is safe for living cells.

Description

Technical Field

[0001] The present application relates to the technical field of fluorescent nanomaterials, and more specifically, to an endoplasmic reticulum-targeted fluorescent nanocarrier, a preparation method and an application thereof. Background Art

[0002] In recent years, fluorescent nano-micelles have been considered as ideal tools for bioimaging and drug delivery. Fluorescent nano-micelles have attracted extensive attention due to their good water solubility, superior biocompatibility and excellent cell permeability. The characteristics of cancer biology tell us that nano-carriers can exhibit passive targeting ability to cancer cells, promoting the accumulation of nano-carriers in cancer cells through the enhanced permeability and retention (EPR) effect. The use of functional nano-fluorescent polymer carriers for cancer treatment is the main research direction of researchers, because small molecule drugs have strong side effects, low stability, poor water solubility, unsatisfactory biodistribution and pharmacokinetics. Despite the recent progress in the research of functional nano-fluorescent polymer carriers for cancer diagnosis and treatment, there are still great challenges to visualize and treat cancer with sufficient sensitivity and spatial resolution, unique stability and high therapeutic effect. The development of a new method for preparing endoplasmic reticulum-targeted functional nano-fluorescent polymer carriers is of great significance for the effective treatment of cancer.

[0003] Pullulan is a non-toxic, non-immunogenic hydrophilic polysaccharide that is degraded into monosaccharide units by a range of enzymes and has been widely used in drug delivery. Modified pullulan has been widely used in the biomedical field, which utilizes various stimulus responses such as temperature, pH, charge, redox, and light. Redox-responsive bonds, such as diselenides and disulfides, have been incorporated to prepare different redox-responsive polymer nanosystems. Various bioactive molecules, such as plasmid DNA, bioproteins, drugs, and siRNA, have also been explored in the recent advances of redox-sensitive polymer therapeutic nanosystems. It is known that folate receptor (FR) is overexpressed in the vast majority of cancer cells and activated macrophages involved in various inflammatory and autoimmune diseases, while its expression is low in healthy tissues. For example, disulfide-crosslinked folate-modified pullulan nanoparticles were developed to target the asialoglycoprotein receptor overexpressed in liver cancer cells.

[0004] The endoplasmic reticulum is an important subcellular organelle that plays an important role in cell metabolism. Endoplasmic reticulum dysfunction can lead to many diseases, such as cardiovascular disease, Alzheimer's disease, and cancer. Precision medicine involves the delivery of anticancer drugs to diseased tissues and cells, which often leads to the accumulation of drugs in healthy tissues, which not only reduces the efficacy but also causes serious side effects without the desired therapeutic effect. With the continuous improvement of medical level, the requirements for drug delivery are getting higher and higher. In addition to cell targeting, the research focus is on subcellular localization. Organelle targeting can deliver drugs more accurately, maximize drug efficacy, and reduce toxic side effects. Nowadays, nanotechnology is widely used in the treatment and diagnosis of diseases, and many functionally modified nanomaterials are designed for subcellular localization, which targets lysosomes, mitochondria, cytoplasm, and nucleus. Many studies have reported subcellular targeted nanodiagnostics, therapeutics, and drug therapy delivery systems, such as mitochondria, nuclei, and lysosomes. In recent years, endoplasmic reticulum-targeted nanotherapy and diagnosis has attracted increasing attention. Commonly used nano drug carriers include inorganic nanomaterials (such as mesoporous silica, alumina nanorods, etc.), liposomes, nano micelles, polymers, supramolecular assemblies, etc. Common preparation strategies for endoplasmic targeted drug carriers include endoplasmic reticulum-targeted small molecule modified nanocarriers, endoplasmic reticulum-targeted peptide-modified nanoparticles, liposomes with ER membrane fusion properties, viral vector-mediated endoplasmic reticulum targeting, and strategies involving intracellular antibodies. However, there are almost no reports on strategies for preparing drug carriers by grafting hydrophilic polymers with lipid small molecules to achieve endoplasmic reticulum targeting. Therefore, the preparation of a nano drug carrier that can accurately target the endoplasmic reticulum in cancer cells is of great significance for cancer treatment. Summary of the invention

[0005] In response to the above problems, the present application provides a fluorescent nanocarrier NPs-NAP-FA-SS for endoplasmic reticulum targeted drug delivery, which uses pullulan as the hydrophilic part, and fluorescent small molecules (E)-3-((3-(2-(6-(4-morpholinostyryl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethoxy)-3-oxopropyl)disulfonyl)propionic acid and folic acid as hydrophobic links to achieve the following invention objectives: targeting the endoplasmic reticulum and cancer cells for endoplasmic reticulum targeted drug delivery; high biosafety; high drug encapsulation rate.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] An endoplasmic reticulum-targeted fluorescent nanocarrier, the structural formula of the nanocarrier is as follows:

[0008] ;

[0009] NPs-NAP-FA-SS

[0010] The preparation method of the fluorescent nanocarrier comprises the following steps:

[0011] (1) 4-bromo-1,8-naphthalene dicarboxylic anhydride and ethanolamine are heated under reflux in anhydrous ethanol to prepare 6-bromo-2-(2-hydroxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione;

[0012] (2) Using potassium tert-butoxide as a catalyst and tetrahydrofuran as a solvent, methyltriphenylphosphonium bromide and 4-(4-morpholino)benzaldehyde are reacted in an anhydrous and oxygen-free environment at room temperature for 11-13 hours to obtain 4-(4-morpholino)styrene;

[0013] (3) Using palladium acetate and tri(2-methylphenyl)phosphine as catalysts and acetonitrile and triethylamine as a mixed solvent, 6-bromo-2-(2-hydroxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione and 4-(4-morpholino)styrene are reacted under heating conditions in an anhydrous and oxygen-free environment to prepare (E)-2-(2-hydroxyethyl)-6-(4-morpholinophenylvinyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione;

[0014] (4) Using 4-dimethylaminopyridine as a catalyst, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride as a dehydrating agent and dichloromethane as a solvent, (E)-2-(2-hydroxyethyl)-6-(4-morpholinostyryl)-1H-benzo[de]isoquinoline-1,3(2H)-dione and 3,3'-dithiodipropionic acid were reacted at 25°C for 48 h to prepare (E)-3-((3-(2-(6-(4-morpholinostyryl)-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethoxy)-3-oxopropyl)disulfonyl)propionic acid;

[0015] (5) Using DMSO as solvent, 4-dimethylaminopyridine as catalyst and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride as dehydrating agent, folic acid and (E)-3-((3-(2-(6-(4-morpholinostyryl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethoxy)-3-oxopropyl)disulfonyl)propionic acid were reacted with pullulan at 60°C for 48 h to obtain fluorescent nanocarriers.

[0016] In the step (1), the molar ratio of 4-bromo-1,8-naphthalene dicarboxylic anhydride to ethanolamine is 1:4; and the mass volume ratio of 4-bromo-1,8-naphthalene dicarboxylic anhydride to anhydrous ethanol is 1 g:3.5-3.7 mL.

[0017] The temperature of the heating reflux reaction in step (1) is 98-102° C. and the time is 1.8-2.2 h.

[0018] In the step (2), the molar ratio of methyltriphenylphosphonium bromide to 4-(4-morpholine)benzaldehyde is 1.6:1; the molar ratio of methyltriphenylphosphonium bromide to potassium tert-butoxide is 1:1; and the molar volume ratio of 4-(4-morpholine)benzaldehyde to tetrahydrofuran is 1 mmol:0.9-1.1 mL.

[0019] In the step (3), the molar ratio of 6-bromo-2-(2-hydroxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione to 4-(4-morpholine)styrene is 1:1.5; the molar ratio of 4-(4-morpholine)styrene to palladium acetate is 7.5:1; the molar ratio of palladium acetate to tri(2-methylphenyl)phosphine is 1:1; the volume ratio of acetonitrile to triethylamine is 3:1; and the molar volume ratio of 4-(4-morpholine)styrene to acetonitrile is 1 mmol:1.8-2.2 mL.

[0020] In the step (3), the heating condition is heating under reflux at 105° C. for 12 h.

[0021] In the step (4), the molar ratio of (E)-2-(2-hydroxyethyl)-6-(4-morpholinostyryl)-1H-benzo[de]isoquinoline-1,3(2H)-dione to 3,3'-dithiodipropionic acid is 1:4; the molar ratio of 3,3'-dithiodipropionic acid to 4-dimethylaminopyridine is 2:1; the molar ratio of 4-dimethylaminopyridine to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1; and the molar volume ratio of 3,3'-dithiodipropionic acid to dichloromethane is 1 mmol:6-6.5 mL.

[0022] In the step (5), the molar ratio of folic acid, (E)-3-((3-(2-(6-(4-morpholinostyryl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethoxy)-3-oxopropyl)disulfonyl)propionic acid to pullulan is 0.0005:0.002:1; the molar ratio of DMAP to folic acid is 8:1; the molar ratio of DMAP to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1; and the molar volume ratio of pullulan to DMSO is 2 mmol:1 mL.

[0023] The fluorescent nanocarrier is used in endoplasmic reticulum targeted drug delivery.

[0024] The structural formula of 4-(4-morpholine)styrene in step (2) is as follows:

[0025] ;

[0026] The structural formula of (E)-2-(2-hydroxyethyl)-6-(4-morpholinostyryl)-1H-benzo[de]isoquinoline-1,3(2H)-dione in step (3) is as follows:

[0027] ;

[0028] The structural formula of (E)-3-((3-(2-(6-(4-morpholinostyryl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethoxy)-3-oxopropyl)disulfonyl)propanoic acid in step (4) is as follows:

[0029] .

[0030] Compared with the prior art, the present invention achieves the following beneficial effects:

[0031] (1) The present application discloses a fluorescent nano-micelle NPs-NAP-FA-SS, which has pullulan as the hydrophilic part, and fluorescent small molecules (E)-3-((3-(2-(6-(4-morpholinostyryl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethoxy)-3-oxopropyl)disulfonyl)propionic acid and folic acid as hydrophobic segments, which respectively play the role of targeting the endoplasmic reticulum and cancer cells.

[0032] (2) The critical micelle concentration (CMCC) value of the fluorescent nanomicelles NPs-NAP-FA-SS prepared by the present invention is 5 μg / mL, the particle size is 219.2 nm, and the shape is spherical.

[0033] (3) The preparation method of the fluorescent nanomicelle NPs-NAP-FA-SS disclosed in the present application not only has good ER targeting properties for ER imaging, but also has excellent biosafety. The cytotoxicity test of the fluorescent nanomicelle NPs-NAP-FA-SS shows that the fluorescent nanomicelle NPs-NAP-FA-SS is safe for living cells. When the final concentration of the fluorescent nanomicelle NPs-NAP-FA-SS loaded with DOX and not loaded with DOX in the co-culture medium reaches 100 μg / mL, the activity of HeLa cells is 87.65% and 91.39%, respectively.

[0034] (4) After the fluorescent nanomicelles NPs-NAP-FA-SS disclosed in the present application were loaded with the drug DOX, the drug loading DLC ​​and the encapsulation efficiency DLE were 10.1% and 78.9%, respectively. After loading DOX, the fluorescent nanomicelles NPs-NAP-FA-SS still had excellent targeting to the endoplasmic reticulum, and could be used for endoplasmic reticulum targeted drug delivery and endoplasmic reticulum targeted therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1a-1d (E)-3-((3-(2-(6-(4-morpholinostyryl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethoxy)-3-oxopropyl)disulfonyl)propionic acid, folic acid, pullulan and the fluorescent nano micelle NPs-NAP-FA-SS described in the present application 1 H NMR spectrum;

[0036] in Figure 1a (E)-3-((3-(2-(6-(4-morpholinostyryl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethoxy)-3-oxopropyl)disulfonyl)propanoic acid 1 H NMR spectrum; Figure 1b For folic acid 1 H NMR spectrum; Figure 1c Pullulan 1 H NMR spectrum; Figure 1d For the fluorescent nanomicelles NPs-NAP-FA-SS 1 H NMR spectrum.

[0037] Figure 2 The product 4-(4-morpholine)styrene in step (2) of the preparation of fluorescent nano-micelles NPs-NAP-FA-SS in Example 1 is 1 H NMR spectrum.

[0038] Figure 3 The product E)-2-(2-hydroxyethyl)-6-(4-morpholinostyryl)-1H-benzo[de]isoquinoline-1,3(2H)-dione in step (3) of the preparation of fluorescent nano-micelles NPs-NAP-FA-SS in Example 1 is 1 H NMR spectrum.

[0039] Figure 4 The product (E)-3-((3-(2-(6-(4-morpholinostyryl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethoxy)-3-oxopropyl)disulfonyl)propionic acid in step (4) of the preparation of fluorescent nano-micelles NPs-NAP-FA-SS in Example 1 is 1 H NMR spectrum.

[0040] Figure 5 The critical micelle concentration CMCC value and particle size diagram of the fluorescent nanomicelle NPs-NAP-FA-SS prepared in this application;

[0041] 5a is the test graph of the critical micelle concentration CMCC value of fluorescent nanomicelles NPs-NAP-FA-SS;

[0042] 5b is the particle size distribution diagram of fluorescent nanomicelles NPs-NAP-FA-SS.

[0043] Figure 6 This is the transmission electron microscopy morphology of the fluorescent nanomicelle NPs-NAP-FA-SS (magnified 15,000 times).

[0044] Figure 7 Graph showing the change in particle size distribution of the fluorescent nanomicelles NPs-NAP-FA-SS prepared in the present application over time in the presence of GSH.

[0045] Figure 8 This is the cytotoxicity diagram of the fluorescent nanomicelle NPs-NAP-FA-SS prepared in this application.

[0046] Fig. 9 This is a co-localized cell imaging image of the fluorescent nanomicelles NPs-NAP-FA-SS prepared in this application and the commercial probe ER-Tracker-Blue-White-DPX;

[0047] 9a, 9b, 9c, 9d and 9e are the cell imaging images of ER-Tracker-Blue-White-DPX, the cell imaging images of NPs-NAP-FA-SS without drug encapsulation, the overlapping cell images, bright field images and colocalization scatter plots of ER-Tracker-Blue-White-DPX and NPs-NAP-FA-SS, respectively.

[0048] Fig.10 This is a co-localized cell imaging image of the drug-encapsulated fluorescent nanomicelle NPs-NAP-FA-SS described in the present application and the commercial probe ER-Tracker-Blue-White-DPX; 10a, 10b, 10c, 10d and 10e are respectively the cell imaging image of ER-Tracker-Blue-White-DPX, the cell imaging image of drug-encapsulated NPs-NAP-FA-SS, the overlapping cell image, bright field image and co-localized scatter plot of ER-Tracker-Blue-White-DPX and NPs-NAP-FA-SS. DETAILED DESCRIPTION

[0049] The present application is described in detail below in conjunction with the preparation method of fluorescent nanomicelle NPs-NAP-FA-SS, product performance testing and application. This is only used to specifically describe the present application, rather than to limit the scope of protection of the present application. Those skilled in the art may make some non-essential improvements and adjustments to the present application based on the above content, which still fall within the scope of protection of the present application.

[0050] Example 1: Fluorescent nanomicelles NPs-NAP-FA-SS and their preparation.

[0051] Fluorescent nano-micelles NPs-NAP-FA-SS are based on pullulan as the hydrophilic part, and fluorescent small molecules (E)-3-((3-(2-(6-(4-morpholinostyryl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethoxy)-3-oxopropyl)disulfonyl)propionic acid and folic acid as hydrophobic segments to target the endoplasmic reticulum and cancer cells respectively. The structural formula is as follows:

[0052] ;

[0053] The fluorescent nanomicelles NPs-NAP-FA-SS were prepared by the following steps:

[0054] (1) Preparation of 6-bromo-2-(2-hydroxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione

[0055] 4-Bromo-1,8-naphthalene dicarboxylic anhydride and ethanolamine were heated to reflux at 100°C in a solvent for 2 hours, and after cooling, the pure product 6-bromo-2-(2-hydroxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione was obtained by suction filtration.

[0056] The molar ratio of 4-bromo-1,8-naphthalene dicarboxylic anhydride to ethanolamine is 1:4, specifically: 4-bromo-1,8-naphthalene dicarboxylic anhydride 20 mmol, 5.541 g; ethanolamine 80 mmol, 4.838 ml;

[0057] The solvent is anhydrous ethanol, and the added amount is 20 mL.

[0058] (2) Preparation of 4-(4-morpholino)styrene

[0059] Methyltriphenylphosphonium bromide and solvent tetrahydrofuran were added to a flask and nitrogen was bubbled for 15 minutes. Potassium tert-butoxide catalyst was added and nitrogen was bubbled for 30 minutes. Then 4-(4-morpholine)benzaldehyde was added. The reaction temperature was room temperature and the reaction time was 12 hours. After stopping the reaction, the crude product was diluted with n-hexane and filtered and dried to obtain a crude product, which was then purified by column chromatography to obtain a white crystalline solid, namely 4-(4-morpholine)styrene.

[0060] The molar ratio of methyltriphenylphosphonium bromide to 4-(4-morpholine)benzaldehyde is 1.6:1, and the specific amounts of each substance used are: methyltriphenylphosphonium bromide 32 mmol, 11.335 g; 4-(4-morpholine)benzaldehyde 20 mmol, 3.825 g; potassium tert-butoxide catalyst 32 mmol, 3.591 g; solvent tetrahydrofuran 20 mL.

[0061] like Figure 2 As shown, the hydrogen nuclear magnetic resonance spectrum of 4-(4-morpholine)styrene is characterized as follows: 1 H NMR (400 MHz, CDCl3) δ 7.35 (s, 2H), 6.91 (s, 2H), 6.66 (s, 1H), 5.65 (s, 1H), 5.11 (s,1H), 3.90 (s, 4H), 3.21 (s, 4H).

[0062] (3) Synthesis of (E)-2-(2-hydroxyethyl)-6-(4-morpholinostyryl)-1H-benzo[de]isoquinoline-1,3(2H)-dione

[0063] 6-Bromo-2-(2-hydroxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione and 4-(4-morpholino)styrene were added to an eggplant-shaped bottle, dissolved in a mixed solvent of acetonitrile and triethylamine with palladium acetate and tri(2-methylphenyl)phosphine as catalysts, and heated under reflux at 105°C for 12 h in an anhydrous and oxygen-free nitrogen environment to obtain a crude product. After purification by column chromatography, the pure product, acid-responsive fluorescent small molecule (E)-2-(2-hydroxyethyl)-6-(4-morpholinophenylvinyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione, was obtained.

[0064] The molar ratio of 6-bromo-2-(2-hydroxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione and 4-(4-morpholino)styrene is 1:1.5, and the specific amounts of each substance used are 6-bromo-2-(2-hydroxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione 1.0 mmol, 0.3201 g; 4-(4-morpholino)styrene 1.5 mmol, 0.2839 g; palladium acetate 0.2 mmol, 0.0450 g; tri(2-methylphenyl)phosphine 0.2 mmol, 0.0610 g; acetonitrile 3 mL; and triethylamine 1 mL.

[0065] like Figure 3As shown, (E)-2-(2-hydroxyethyl)-6-(4-morpholinostyryl)-1H-benzo[de]isoquinoline-1,3(2H)-dione is characterized by its hydrogen nuclear magnetic resonance spectrum as follows: 1 H NMR (400 MHz, DMSO) δ 8.96 (s, 1H), 8.52 (s,1H), 8.45 (s, 1H), 8.20 (s, 1H), 7.98 (s, 1H),7.87 (s, 1H), 7.73 (s, 2H),7.55 (s, 1H), 7.02 (s, 2H), 4.84 (s, 1H), 4.16 (s, 2H), 3.76 (s, 4H), 3.64 (s, 2H), 3.22 (s, 4H).

[0066] (4) Synthesis of (E)-3-((3-(2-(6-(4-morpholinostyryl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethoxy)-3-oxopropyl)disulfonyl)propionic acid

[0067] (E)-2-(2-hydroxyethyl)-6-(4-morpholinostyryl)-1H-benzo[de]isoquinoline-1,3(2H)-dione and 3,3'-dithiodipropionic acid were added to an eggplant flask. They were dissolved in dichloromethane using 4-dimethylaminopyridine (DMAP) as a catalyst and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) as a dehydrating agent, and the esterification reaction was carried out at room temperature for 48 hours. After purification by column chromatography, (E)-3-((3-(2-(6-(4-morpholinostyryl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethoxy)-3-oxopropyl)disulfonyl)propionic acid was obtained.

[0068] The molar ratio of the (E)-2-(2-hydroxyethyl)-6-(4-morpholinophenylvinyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione and 3,3'-dithiodipropionic acid is 1:4, and the specific amounts of each substance used are (E)-2-(2-hydroxyethyl)-6-(4-morpholinophenylvinyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione 0.2 mmol, 0.0857 g; 3,3'-dithiodipropionic acid 0.8 mmol, 0.1682 g; 4-dimethylaminopyridine 0.4 mmol, 0.0490 g; 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride 0.4 mmol, 0.0767 g, and the amount of dichloromethane used is 5 mL.

[0069] like Figure 4As shown, the nuclear magnetic resonance hydrogen spectrum of (E)-3-((3-(2-(6-(4-morpholinostyryl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethoxy)-3-oxopropyl)disulfonyl)propionic acid is characterized as follows: 1 H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 8.59 (s, 2H), 7.98 (s, 1H), 7.75 (d, J = 25.1Hz, 2H), 7.57 (d, J = 8.6 Hz, 2H), 7.33 (s, 1H), 6.95 (s, 2H), 4.50 (s, 4H), 3.90 (s, 4H), 3.27 (s, 4H), 2.91 (s, 4H), 2.75 (s, 4H).

[0070] (5) Preparation of fluorescent nanomicelles NPs-NAP-FA-SS

[0071] Using DMSO as solvent, 4-dimethylaminopyridine as catalyst and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) as dehydrating agent, folic acid and (E)-3-((3-(2-(6-(4-morpholinostyryl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethoxy)-3-oxopropyl)disulfonyl)propionic acid were reacted with pullulan at 60°C for 48 h to obtain fluorescent nanomicelles NPs-NAP-FA-SS.

[0072] The molar ratio of folic acid and (E)-3-((3-(2-(6-(4-morpholinostyryl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethoxy)-3-oxopropyl)disulfonyl)propionic acid to pullulan is 0.0005:0.002:1, and the specific amount of each substance used is pullulan 20 mmol, 3.2432 g; (E)-3-((3-(2-(6-(4-morpholinostyryl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethoxy)-3-oxopropyl)disulfonyl)propionic acid 0.04 mmol, 0.0254 g; DMAP 0.08 mmol, 0.01 g; EDC.HCl 0.08 mmol, 0.0153 g; folic acid 0.01 mmol, 0.0044 g; DMSO is 10 mL.

[0073] Example 2 Physical characteristics of NPs-NAP-FA-SS micelles

[0074] The fluorescent nanomicelles NPs-NAP-FA-SS in Example 1 were tested by Malvern laser particle size analyzer and transmission electron microscope.

[0075] Figure 5 a is the critical micelle concentration CMCC value test of the fluorescent nano-micelles NPs-NAP-FA-SS prepared in this application, and the CMCC value is 5 μg / mL, Figure 5 b is the particle size of the fluorescent nanomicelle NPs-NAP-FA-SS prepared in this application, Figure 6 This is the morphology of the fluorescent nanomicelles NPs-NAP-FA-SS prepared in this application. The particle size is 219.2 nm and the shape is spherical.

[0076] Example 3 Effect of GSH on fluorescent nanomicelles NPs-NAP-FA-SS

[0077] GSH was added to the aqueous solution of 40 μg / mL fluorescent nanomicelle NPs-NAP-FA-SS (the final concentration of GSH in the mixed solution was 10 mM), and the particle size distribution and size change of the fluorescent nanomicelle NPs-NAP-FA-SS at different time periods were tested.

[0078] Figure 7 The particle size distribution and size change of the fluorescent nanomicelles NPs-NAP-FA-SS disclosed in the present application over time. As time goes by, the particle size distribution becomes wider and the particle size becomes larger. The particle size reaches the maximum value at 32 h and small particles are produced, indicating that the GSH response process has been completed and the fluorescent nanomicelles NPs-NAP-FA-SS have been completely dissociated.

[0079] Example 4 Cytotoxicity test of fluorescent nanomicelles NPs-NAP-FA-SS

[0080] The aqueous solution of fluorescent nano-micelles NPs-NAP-FA-SS and the HeLa cell suspension were mixed to obtain a co-culture solution. The density of HeLa cells in the co-culture solution was controlled to be 10 4 After culturing in a carbon dioxide incubator for 24 h, the standard MTT method was used to verify the cytotoxicity of the fluorescent nanomicelles NPs-NAP-FA-SS. Figure 8As shown, the oblique line bar graph represents the change in the activity of HeLa cells with the increase in the final concentration of NPs-NAP-FA-SS micelles in the co-culture medium, and the horizontal line bar graph represents the change in the activity of HeLa cells with the increase in the final concentration of NPs-NAP-FA-SS micelles loaded with DOX (nano micelles preassembled by the method of Example 6) in the co-culture medium. The results show that when the final concentration of the fluorescent nanomicelles NPs-NAP-FA-SS loaded with DOX and not loaded with DOX in the co-culture medium reaches 100 μg / mL, the activities of HeLa cells are 87.65% and 91.39%, respectively.

[0081] Therefore, the fluorescent nanomicelles NPs-NAP-FA-SS disclosed in the present application are safe for living cells and thus safe for the human body, and can be safely used for endoplasmic reticulum imaging and drug delivery.

[0082] Example 5 is applied to intracellular endoplasmic reticulum imaging.

[0083] The fluorescent nanomicelles NPs-NAP-FA-SS in Example 1 were applied to endoplasmic reticulum imaging during cell experiments.

[0084] Fig. 9 The fluorescent nanomicelles NPs-NAP-FA-SS disclosed in the present application (40 μg / mL, final concentration) and the commercial blue endoplasmic reticulum targeting probe (ER-Tracker-Blue-White-DPX, 5 nmol / L, final concentration) were cultured in HeLa cells (HeLa cell density was 10 4 The cells were cultured in a carbon dioxide incubator at 40 °C for 0.5 h in a culture medium containing 10% FA-1 cells / ml and imaged using a Leica SP8 fluorescence confocal microscope. The Pearson coefficient R was 0.923, indicating that the fluorescent nanomicelles NPs-NAP-FA-SS had excellent targeting to the endoplasmic reticulum and could be used for endoplasmic reticulum imaging.

[0085] Example 6 Application to endoplasmic reticulum targeted drug delivery.

[0086] The fluorescent nanomicelles NPs-NAP-FA-SS in Example 1 were used to encapsulate the cancer drug doxorubicin (DOX) into NPs-NAP-FA-SS micelles by dialysis. In short, 0.0185 g of DOX·HCl and 0.01 mL of TEA were dissolved in 2 mL of DMSO. The hydrochloride was removed by continuous stirring (5.5 h) to obtain a DOX solution; then 0.3693 g of NPs-NAP-FA-SS was dissolved in 6 mL of DMSO. The dissolved NPs-NAP-FA-SS solution was then mixed and stirred with the DOX solution for 12 h, and then added dropwise to 20 mL of PBS for pre-assembly to form nanomicelles. The dialysis bag was dialyzed in PBS for 48 h with a 1000 molecular weight dialysis bag, and the PBS solution outside the dialysis bag was taken to determine the drug loading, and analyzed by UV---Vis. The drug loading (DLC) and drug loading efficiency (DLE) were calculated using the following formulas:

[0087] ;

[0088] Doxorubicin (DOX) was used as a model drug to evaluate the drug loading performance of NPs-NAP-FA-SS micelles. NPs-NAP-FA-SS micelles loaded with Dox were prepared by dialysis method. The DLC and DLE were 10.1% and 78.9%, respectively.

[0089] Fig.10 After the cells were incubated with 40 μg / mL fluorescent nanomicelle NPs-NAP-FA-SS encapsulated with the drug DOX disclosed in the present application and a commercial blue endoplasmic reticulum targeting probe (ER-Tracker-Blue-White-DPX, 5 nmol / L), imaging was performed using a Leica SP8 fluorescence confocal microscope. The Pearson coefficient R=0.893, indicating that the fluorescent nanomicelle NPs-NAP-FA-SS still has excellent targeting to the endoplasmic reticulum after encapsulating DOX, and can be used for endoplasmic reticulum targeted drug delivery.

[0090] The above is only a preferred implementation of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present application. These improvements and modifications should also be regarded as the scope of protection of the present application.

Claims

1. A method for preparing an endoplasmic reticulum-targeted fluorescent nanocarrier, characterized in that: The preparation method of the fluorescent nanocarrier comprises the following steps: (1) 4-bromo-1,8-naphthalene dicarboxylic anhydride and ethanolamine are heated under reflux in anhydrous ethanol to prepare 6-bromo-2-(2-hydroxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione; (2) Using potassium tert-butoxide as a catalyst and tetrahydrofuran as a solvent, methyltriphenylphosphonium bromide and 4-(4-morpholino)benzaldehyde are reacted in an anhydrous and oxygen-free environment at room temperature for 11-13 hours to obtain 4-(4-morpholino)styrene; (3) Using palladium acetate and tri(2-methylphenyl)phosphine as catalysts and acetonitrile and triethylamine as a mixed solvent, 6-bromo-2-(2-hydroxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione and 4-(4-morpholino)styrene are reacted under heating conditions in an anhydrous and oxygen-free environment to prepare (E)-2-(2-hydroxyethyl)-6-(4-morpholinophenylvinyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione; (4) Using 4-dimethylaminopyridine as a catalyst, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride as a dehydrating agent and dichloromethane as a solvent, (E)-2-(2-hydroxyethyl)-6-(4-morpholinostyryl)-1H-benzo[de]isoquinoline-1,3(2H)-dione and 3,3'-dithiodipropionic acid were reacted at 25°C for 48 hours to prepare (E)-3-((3-(2-(6-(4-morpholinostyryl)-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethoxy)-3-oxopropyl)disulfonyl)propionic acid; (5) Using DMSO as solvent, 4-dimethylaminopyridine as catalyst and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride as dehydrating agent, folic acid and (E)-3-((3-(2-(6-(4-morpholinostyryl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethoxy)-3-oxopropyl)disulfonyl)propionic acid were reacted with pullulan at 60°C for 48 h to obtain fluorescent nanocarriers.

2. The preparation method according to claim 1, characterized in that: In the step (1), the molar ratio of 4-bromo-1,8-naphthalene dicarboxylic anhydride to ethanolamine is 1:4; and the mass volume ratio of 4-bromo-1,8-naphthalene dicarboxylic anhydride to anhydrous ethanol is 1 g:3.5-3.7 mL.

3. The preparation method according to claim 1, characterized in that: The temperature of the heating reflux reaction in step (1) is 98-102° C. and the time is 1.8-2.2 h.

4. The preparation method according to claim 1, characterized in that: In the step (2), the molar ratio of methyltriphenylphosphonium bromide to 4-(4-morpholine)benzaldehyde is 1.6:1; the molar ratio of methyltriphenylphosphonium bromide to potassium tert-butoxide is 1:1; and the molar volume ratio of 4-(4-morpholine)benzaldehyde to tetrahydrofuran is 1 mmol:0.9-1.1 mL.

5. The preparation method according to claim 1, characterized in that: In the step (3), the molar ratio of 6-bromo-2-(2-hydroxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione to 4-(4-morpholine)styrene is 1:1.5; the molar ratio of 4-(4-morpholine)styrene to palladium acetate is 7.5:1; the molar ratio of palladium acetate to tri(2-methylphenyl)phosphine is 1:1; the volume ratio of acetonitrile to triethylamine is 3:1; and the molar volume ratio of 4-(4-morpholine)styrene to acetonitrile is 1 mmol:1.8-2.2 mL.

6. The preparation method according to claim 1, characterized in that: In the step (3), the heating condition is heating under reflux at 105° C. for 12 h.

7. The preparation method according to claim 1, characterized in that: In the step (4), the molar ratio of (E)-2-(2-hydroxyethyl)-6-(4-morpholinostyryl)-1H-benzo[de]isoquinoline-1,3(2H)-dione to 3,3'-dithiodipropionic acid is 1:4; the molar ratio of 3,3'-dithiodipropionic acid to 4-dimethylaminopyridine is 2:1; the molar ratio of 4-dimethylaminopyridine to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1; and the molar volume ratio of 3,3'-dithiodipropionic acid to dichloromethane is 1 mmol:6-6.5 mL.

8. The preparation method according to claim 1, characterized in that: In the step (5), the molar ratio of folic acid, (E)-3-((3-(2-(6-(4-morpholinostyryl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethoxy)-3-oxopropyl)disulfonyl)propionic acid to pullulan is 0.0005:0.002:1; the molar ratio of 4-dimethylaminopyridine to folic acid is 8:1; the molar ratio of 4-dimethylaminopyridine to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1; and the molar volume ratio of pullulan to DMSO is 2 mmol:1 mL.

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