CN112826796B

CN112826796B - Application of nanoparticles in preparation of medicine for treating gallbladder-related diseases

Info

Publication number: CN112826796B
Application number: CN202110039449.9A
Authority: CN
Inventors: 王瑞兵; 张相军; 王一涛
Original assignee: University of Macau
Current assignee: University of Macau
Priority date: 2021-01-12
Filing date: 2021-01-12
Publication date: 2023-01-13
Anticipated expiration: 2041-01-12
Also published as: CN112826796A

Abstract

The invention discloses an application of nanoparticles in preparing a medicament for treating gallbladder-related diseases, wherein the nanoparticles are nanoparticles transported through liver and gallbladder, and the nanoparticles subjected to intravenous injection can be distributed to gallbladder by a liver and gallbladder system transport method, so that the aim of targeted dissolution of gallstone is fulfilled. Based on the liver and gall system transportation way, various nano-drug forms can be applied to the treatment of gallstones in an intravenous injection mode. Meanwhile, various gallbladder and bile duct related diseases can be treated by using the nano-medicament which is subjected to intravenous injection and passes through the transportation way.

Description

Application of nanoparticles in preparation of medicine for treating gallbladder-related diseases

Technical Field

The invention relates to the technical field of treatment of gallbladder-related diseases, in particular to application of nanoparticles in preparation of a medicine for treating gallbladder-related diseases.

Background

Gallbladder-related diseases include gallstones, cholecystitis, cholangitis, biliary cysts, biliary tumors, gallbladder cancer, mirizzi syndrome, and the like. Among them, gallstones, also called cholelithiasis, are crystalline deposits in the biliary tract or gallbladder, with prevalence rates of up to 10-20% in adults worldwide, with the highest socioeconomic costs among all digestive diseases. In addition to biliary and pancreatic duct obstruction, gallstone disease also leads to other medical complications, such as cholecystitis, pancreatitis, gallbladder and Gastrointestinal (GI) cancer. Although the pathogenesis is not completely understood, gallstone formation is currently generally attributed to an imbalance in the secretion of bile salts, bilirubin, phospholipids and cholesterol. The supersaturated state of cholesterol in bile causes nucleation of Cholesterol Crystals (CC), which is considered to be the first step to occur in the formation of cholesterol-type gallstones (the most common type of gallstone).

Currently, cholecystectomy is mainly used clinically to treat symptomatic gallstones. However, this is an invasive procedure and may cause various side effects such as bile duct damage, fat intolerance, metabolic abnormalities and post-cholecystectomy syndromes. Bile that continues to flow into the intestine after surgery may also increase the incidence of intestinal cancer. In addition, residual calculus is often found after surgery and recurrence of bile duct stones occurs. Drugs aimed at regulating cholesterol absorption and metabolism, such as ursodeoxycholic acid (UDCA), ezetimibe, statins and nuclear receptor agonists/antagonists, are able to inhibit the formation of cholesterol-type gallstones in preclinical and clinical studies. However, these drugs are all administered systemically, with non-specific distribution in the individual organs and rapid clearance from the human body. Therefore, only a limited amount of drugs can reach the target site, and thus high doses and long-term administration of these drugs are required to treat gallstone diseases, thus causing various side effects.

Therefore, there is a need for a drug that can effectively treat gallstones with little side effects. Correspondingly, other gallbladder-related diseases such as cholecystitis, cholangitis, biliary cyst, biliary tract tumor, gallbladder cancer, mirizzi syndrome and the like also need a medicament with good treatment effect and small side effect.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide application of nanoparticles in preparation of a medicine for treating gallbladder-related diseases so as to improve the technical problem.

The invention is realized by the following steps:

in a first aspect, the invention provides an application of nanoparticles in preparing a medicament for treating gallbladder-related diseases, wherein the nanoparticles are nanoparticles transported through liver and gallbladder. The nanoparticles in the medicine can be transported to the biliary tract and gallbladder through the liver and gallbladder system after intravenous injection, thereby realizing the treatment of gallbladder diseases.

In a second aspect, the invention also provides a specific embodiment of treating gallbladder diseases by transporting nano-drugs through liver and gall, namely an application of the beta-cyclodextrin derivative nano-particles in preparing drugs for treating gallstones.

In a third aspect, the invention also provides an application of the nanoparticles in preparing a medicament for treating gallstones. For example, the nanoparticle is a beta-cyclodextrin derivative nanoparticle. The beta-cyclodextrin derivative nanoparticle is mainly prepared by the following steps: chemically bonding 4-hydroxy-2, 6-tetramethylpiperidine-1-oxyl free radical and 4- (hydroxymethyl) phenylboronic acid pinacol ester to beta-cyclodextrin to obtain a beta-cyclodextrin derivative, and preparing the beta-cyclodextrin derivative into beta-cyclodextrin derivative nanoparticles.

In a fourth aspect, the invention also provides an application of the nanoparticles in preparing an intravenous injection for promoting cholesterol dissolution and inhibiting cholesterol crystallization. For example, the nanoparticle is the above-described β -cyclodextrin derivative nanoparticle.

In a fifth aspect, the invention also provides an application of the nanoparticle in preparing an inhibitor for inhibiting neutrophil-mediated inflammation. For example, the nanoparticle is the above-mentioned β -cyclodextrin derivative nanoparticle.

In a sixth aspect, the invention also provides an application of the nanoparticle in preparing an intravenous injection for reducing the generation of active oxygen of the neutrophil induced by cholesterol crystallization. For example, the nanoparticle is the above-described β -cyclodextrin derivative nanoparticle.

In a seventh aspect, the invention also provides an application of the nanoparticle in preparing an intravenous injection for reducing formation of a neutrophil trapping net induced by cholesterol crystallization. For example, the nanoparticle is the above-described β -cyclodextrin derivative nanoparticle.

The technical scheme of the invention has the beneficial effects that: a beta-cyclodextrin derivative obtained by chemically bonding a 4-hydroxy-2, 6-tetramethylpiperidine-1-oxyl radical and a 4- (hydroxymethyl) phenylboronic acid pinacol ester to beta-cyclodextrin, and further preparing nanoparticles based on the beta-cyclodextrin derivative, the beta-cyclodextrin derivative nanoparticles being capable of reaching the gallbladder via the hepatobiliary system after intravenous injection; the degradation product of the beta-cyclodextrin derivative nanoparticles, namely free beta-cyclodextrin, can directly dissolve CC, so that the growth of gallstones is delayed. In addition, the beta-cyclodextrin derivative nanoparticles can also inhibit neutrophil-mediated inflammation by relieving the oxidative stress of neutrophils, so that the development of gallstone diseases is further inhibited. Similarly, other types of nano-drugs can be used for treating other types of gallbladder diseases after being transported through the liver and gallbladder system.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and those skilled in the art can also obtain other related drawings based on the drawings without inventive efforts.

FIG. 1 is a schematic illustration of liver and gall bladder transport and treatment of gallstones with TPCD NP;

FIG. 2 is a schematic representation of TPCD NP preparation;

FIG. 3 is a graph of TPCD NP distribution in DI water as determined by DLS;

FIGS. 4 and 5 are SEM and TEM images of TPCD NP, respectively;

FIG. 6 is a MALDI-TOF mass spectrum of TPCD and Cy 5-TPCD;

FIG. 7 is a Fourier-infrared spectrum of beta-CD and TPCD;

FIG. 8 shows TPCD NP at/without 1mM H ₂ O ₂ Degradation in PBS solution of (a);

figure 9 is an IVIS image showing fluorescence of cy7.5 loaded TPCD NP in the gallbladder at different time points after i.v. injection in normal and GS mice;

FIG. 10 is a graph of mean fluorescence intensity of gallbladder at various time points after intravenous injection of Cy7.5-loaded TPCD NP in mice;

FIG. 11 is an image of Cy5-TPCD NP uptake by CLSM on LO2 cells;

FIG. 12 is a CLSM image showing exocytosis of Cy5-TPCD NP in LO2 cells;

FIG. 13 is a CLSM panel showing exocytosis of HUVEC cells on Cy5-TPCD NP;

FIG. 14 is a schematic representation of the Transwell method for determining the transport efficiency of Cy5-TPCD NP by hepatocytes and endothelial cells;

FIGS. 15 and 16 are graphs showing, in order, the transport rates of LO2 cells and HUVEC cells for Cy5-TPCD NP;

FIG. 17 is a schematic representation of a treatment regimen for the TPCD NP inhibition of the development of gallstones;

FIGS. 18, 19 and 20 are gallbladder pictures of each group of mice, gallbladder sizes of different groups of mice and gallstone pictures collected from the gallbladder of each group of mice respectively;

FIGS. 21 and 22 show the cumulative size of gallstones in each mouse and the incidence of gallstones in each group in sequence;

FIG. 23, panel A is IVIS diagram showing Cy5-TPCD NP distribution in gallbladder and free Cy5- β -CD distribution in bile; b is the mean fluorescence intensity of free Cy5- β -CD dissolved in bile calculated from a (mean ± SE, n = 3); c is imaging co-localization of Cy5- β -CD and CC collected in gall bladder with CLSM, scale =25 μm; d is co-localization of Cy5- β -CD and CC collected from gall bladder by FCM analysis; e is the Mean Fluorescence Intensity (MFI) and F ratio (mean ± SE, n = 3) of the CC bound to Cy5- β -CD calculated from D; g is Cy5-TPCD NP (100. Mu.g/mL) with/without 1mM H ₂ O ₂ Degradation profile in bile (mean ± SE, n = 3); h is the solubility of cholesterol in bile containing TPCD NP or β -CD (mean ± SE, n = 3); i is the nucleation of crystal of CC in bile containing TPCD NP or β -CD (n = 5);

FIG. 24 is a FCM analysis of the time dependence of LO2 cells on Cy5-TPCD NP uptake;

FIG. 25 is a graph of FCM analysis of the concentration dependence of LO2 cells on Cy5-TPCD NP uptake;

in FIG. 26, A is the particle size distribution of TPCD NP incubated in serum-free DMEM medium at different times, and B is the particle size distribution of TPCD NP transported to the lower chamber by LO2 cells at different times;

FIG. 27 is the nucleation of crystal of CC in bile with/without TPCD NP/β -CD;

FIG. 28, A is the Cy5-TPCD NP distribution in blood and liver neutrophils; b, FCM analysis shows the ROS content of the peritoneal neutrophils; c is FCM analysis showing formation of neutrophils NETs, P <0.05,. P <0.001; d is a CLSM image which shows the inhibition effect of TPCD NP on the formation of neutrophil NETs in the abdominal cavity stimulated by CCs;

FIG. 29 is the body weight and organ index of mice;

figure 30 is biomarkers of liver function in serum, data represent mean ± SE, P <0.05, P <0.01 and P <0.001 compared to model group;

fig. 31 is a blood routine for mice including WBCs, red Blood Cells (RBCs), platelets (PLTs), hemoglobin (HGB), data representing mean ± SE, × P <0.05, compared to model group;

fig. 32 is a H & E staining picture of mouse organ sections, scale =100 μm.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.

The application of the nanoparticles transported by the liver and the gallbladder in preparing the medicine for treating gallbladder diseases, namely the application of the beta-cyclodextrin derivative nanoparticles in treating gallstones, is specifically explained below.

The inventor finds that the existing medicine for treating gallstone has no action principle of directly interacting with cholesterol or dissolving CC. Therefore, a drug that can directly dissolve CC will be effective in treating gallstones. It has further been found that drugs which inhibit neutrophil-mediated inflammation by reducing oxidative stress can inhibit the formation of gallstones, but this strategy does not solubilize CC or alter the supersaturation of cholesterol in the bile, and therefore the development of drugs which can solubilize CC and also inhibit neutrophil-mediated inflammation may have a synergistic effect in the treatment of gallstone diseases. Based on this, the following technical solutions are proposed.

Some embodiments of the present invention provide an application of a beta-cyclodextrin derivative nanoparticle in preparing a drug for treating gallstones, wherein the beta-cyclodextrin derivative nanoparticle is mainly prepared by the following steps: chemically bonding 4-hydroxy-2, 6-tetramethylpiperidine-1-oxyl free radical (Tempol, defined as Tpl) and 4- (hydroxymethyl) phenylboronic acid pinacol ester (PBAP) to beta-cyclodextrin to obtain beta-cyclodextrin derivatives (TPCD), and preparing the TPCD into the beta-cyclodextrin derivative nanoparticles.

Beta-cyclodextrin (beta-CD) and its derivatives (beta-CDs) have been widely used as adjuvants for various preparations to enhance solubility and stability of drug molecules due to their cavitating effect, however, it is well known that intravenously injected beta-CDs can be rapidly excreted together with urine by glomerular filtration, and almost no beta-CDs molecules can be transferred from systemic circulation to bile. On the other hand, nanoparticles accumulate in the liver in large quantities after injection and can be gradually transferred to the bile duct via the hepatobiliary system, while the efficiency of hepatobiliary transport is very high (up to 100%) in some cases. Therefore, the inventor creatively proposes a beta-cyclodextrin derivative TPCD obtained by chemically bonding Tpl and PBAP to beta-cyclodextrin, and further prepares nanoparticles based on the beta-cyclodextrin derivative, referring to fig. 1, after the beta-cyclodextrin derivative nanoparticles are injected intravenously, the nanoparticles can reach the gall bladder through the liver and gall system; the degradation product of the beta-cyclodextrin derivative nanoparticle, namely free beta-CD, can directly dissolve CC, and the growth of gallstones is delayed. In addition, the beta-cyclodextrin derivative nanoparticles can also inhibit neutrophil-mediated inflammation by relieving the oxidative stress of neutrophils, so that the development of gallstone diseases is further inhibited.

It should be noted that the preparation of the beta-cyclodextrin derivative nanoparticles is the prior art. The preparation process is shown in figure 2. Specifically, the preparation of the beta-cyclodextrin derivative into the beta-cyclodextrin derivative nanoparticles comprises the following steps: adding an ethanol solution containing lecithin and DSPE-mPEG2000 into water, uniformly mixing to obtain a first mixed solution, dropwise adding an alcohol solution of TPCD into the first mixed solution, uniformly mixing, and concentrating to obtain TPCD NP, or dropwise adding a mixed alcohol solution of TPCD and Cy7.5NHS ester into the first mixed solution, uniformly mixing, and concentrating to obtain TPCD NP loaded with Cy7.5, or dropwise adding a mixed alcohol solution of TPCD and TPCD (Cy 5-TPCD) loaded with Cy5 into the first mixed solution, uniformly mixing, and concentrating to obtain Cy5-TPCD NP. Namely, the beta-cyclodextrin derivative nanoparticles can be TPCD NP, TPCD NP loaded with Cy7.5 or Cy5-TPCD NP. Cy7.5-loaded TPCD NP or Cy5-TPCD NP was used for fluorescent tracing of TPCD NP in vitro and in vivo.

In some embodiments, the ratio of lecithin to DSPE-mPEG2000 is 5 to 7:8 to 10. In some embodiments, the concentration is a vacuum rotary evaporation concentration. In some embodiments, the gallstone in the above medicament for treating gallstone is a cholesterol-type gallstone. Further, in some embodiments, the drug is an intravenous drug. In some embodiments, treating gallstones is promoting cholesterol dissolution and inhibiting cholesterol crystallization or inhibiting neutrophil-mediated inflammation.

In some embodiments, the chemical structure of TPCD is:

chemical Structure of Cy5-TPCD

Wherein R in both chemical structures ₁ Is H or

R ₂ Is H or

Cy5 is

Some embodiments of the invention also provide application of the beta-cyclodextrin derivative nanoparticles in preparing intravenous injection for promoting cholesterol dissolution and inhibiting cholesterol crystallization. Some embodiments of the invention also provide application of the beta-cyclodextrin derivative nanoparticles in preparing an inhibitor for inhibiting neutrophil-mediated inflammation. Some embodiments of the present invention also provide the use of the above-described beta-cyclodextrin derivative nanoparticles for the preparation of an intravenous injection for reducing active oxygen production of cholesterol crystallization-induced neutrophils.

Some embodiments of the invention also provide application of the beta-cyclodextrin derivative nanoparticles in preparing intravenous injections for reducing formation of neutrophil trapping nets induced by cholesterol crystallization.

The features and properties of the present invention are described in further detail below with reference to examples.

Examples

Materials: beta-cyclodextrin (beta-CD), 4-hydroxy-2, 6-tetramethylpiperidin-1-oxyl radical (Tempol, defined as Tpl), 4- (hydroxymethyl) phenylboronic acid pinacol ester (PBAP), N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride, 4- (dimethylamino) pyridine (DMAP) and anhydrous N, N-Dimethylformamide (DMF) were purchased from Sigma-Aldrich (USA). Anthocyanidin dye 5 carboxylic acid, anthocyanidin dye 5NHS ester and anthocyanidin dye 7.5NHS ester were purchased from Lumiprobe (usa). Soybean lecithin was purchased from TCI (shanghai, china). DSPE-mPEG2000 was purchased from Siiranxi Biotechnology Ltd (Siam, china). Gallstone feed: the feed with 10% fat, 1% cholesterol and 0.5% cholate was provided by Nantong Temilion feed science and technology Co., ltd., nantong, china. Hochest33342 and DAPI staining solutions were purchased from Beyotime (shanghai, china). Anti-mouse CD11b-FITC and anti-mouse Ly-6G-PE antibodies were purchased from BD biosciences. Sytox Green was purchased from Thermo Fisher Scientific (USA). Ursodeoxycholic acid (UDCA) was purchased from alatin (shanghai, china).

1. Preparation of TPCD NP, cy7.5-Supported TPCD NP and Cy5-TPCD NP

1. Synthesis of TPCD: TPCD is synthesized by bonding Tpl and PBAP to beta-CD according to the reported existing method, and the chemical structure and the synthesis process of TPCD can be seen in FIG. 2.

2. Synthesis of Cy5-TPCD: 145.5mg of TPCD (. About.0.05 mmol), 5mg of the anthocyanin dye 5 carboxylic acid (. About.0.01 mmol), 10mg of EDC (. About.0.05 mmol) and 5mg of DMAP (. About.0.04 mmol) were dissolved together in 10mL of anhydrous DMF and reacted with light at 40 ℃ for 48 hours. Deionized water was then added to the above solution, and 8000g was centrifuged for 10 minutes to collect a precipitate. The resulting precipitate was then washed 3 times with acetone and dried under vacuum at 60 ℃.

It should be noted that characterization of TPCD and Cy5-TPCD can be performed using matrix assisted laser Desorption/ionization time of flight (MALDI-TOF) Mass Spectrometry (Microflex LT/SH system). Fourier transform infrared (FT-IR) spectra were measured using a Thermo Nicolet iS10FT-IR spectrometer. The UV/Vis spectra were determined using a DR6000 UV-Vis Spectrophotometer (Hach). The fluorescence spectra were measured using a Thermo Scientific Lumina fluorescence spectrometer.

3. Preparation of TPCD NP: a1 mL ethanol solution containing 6mg lecithin and 9mg DSPE-mPEG2000 was first added to 10mL water and stirred at 65 ℃ for more than 30 minutes. 2mL of methanol containing TPCD (50 mg) was added dropwise slowly to the above solution with stirring, and stirring was continued at room temperature for 2h. The suspension was then concentrated by rotary evaporation in vacuo.

4. Preparation of Cy7.5 loaded TPCD NP: a1 mL ethanol solution containing 6mg lecithin and 9mg DSPE-mPEG2000 was first added to 10mL water and stirred at 65 ℃ for more than 30 minutes. 1mg of Cy7.5NHS ester and 50mg of TPCD were co-dissolved in a methanol solution, and the resulting methanol solution was slowly added dropwise to the above solution with stirring, and stirring was continued at room temperature for 2 hours. The suspension was then concentrated by rotary evaporation in vacuo.

5. Preparation of Cy5-TPCD NP: a1 mL ethanol solution containing 6mg lecithin and 9mg DSPE-mPEG2000 was first added to 10mL water and stirred at 65 ℃ for 30 minutes or more. 5mg of Cy5-TPCD and 45mg of TPCD are dissolved in a methanol solution, and the obtained methanol solution is slowly dropped into the solution under stirring, and stirring is continued for 2h at room temperature. The suspension was then concentrated by rotary evaporation in vacuo.

Characterization of tpcd NP: the Dynamic Light Scattering (DLS) size distribution and Zeta potential of TPCD NPs were characterized using ZetasizenonoZS (Malvern). TPCD NP was measured to be 105.2. + -. 1.2nm in diameter (as shown in FIG. 3) by dynamic laser light scattering (DLS) analysis, a polydispersity index (PDI) of 0.11. + -. 0.01, and a zeta potential of-36.7. + -. 0.7mV.

The morphology of TPCD NPs was characterized using transmission electron microscopy (TEM, TECNAI-10 microscope, philips, the Netherlands) and scanning electron microscopy (SEM, crossbeam 340, zeiss) and the results are shown in FIGS. 4 and 5. Figures 4 and 5 show that TPCD NPs are spherical.

The mass spectra of TPCD and Cy5-TPCD MALDI-TOF are shown in FIG. 6. The Fourier-infrared spectra of the beta-CD and TPCD are shown in FIG. 7.

Degradation of TPCD NP: TPCD NP (2 mg/mL) was placed with or without 1mM H ₂ O ₂ In PBS (10 mM, pH7.4), and incubated at 37 ℃. The light transmittance of the solution at 500nm was measured at 0, 5, 15, 30, 45, 60, 90, 120, 150 and 180 minutes to calculate the degradation ratio. The results are shown in FIG. 8, and it can be seen from FIG. 8 that the concentration is 1mM H ₂ O ₂ After 1 hour incubation in solution, about 100% of TPCD NP was degraded. The TPCD NP remained relatively stable after incubation in Phosphate Buffered Saline (PBS) at 37 ℃ for 3 h.

2. Distribution of TPCD NP in gallbladder after intravenous injection

The distribution of intravenous TPCD NP in the gallbladder was first studied. Cy7.5-loaded TPCD NP was administered intravenously to C57BL/6 mice at a dose of 1mg Cy7.5/kg body weight. At 0, 0.5, 1, 6, 18, 30, 48, 72 and 96 hours, the gallbladder was collected and imaged with IVIS in vivo imaging system (PerkinElmer, usa), respectively. The fluorescence intensity of Cy7.5 was then measured. C57BL/6 mice were subjected to gallstone diet for 6 weeks as gallstone model mice (GS mice), and the biodistribution of TPCD NP in the GS mice was evaluated by the same method as described above. As a result, as shown in FIGS. 9 and 10, fluorescence of Cy7.5 in the gallbladder of normal mice and GS mice was observed at 0.5 hour after administration, indicating that TPCD NP was rapidly transported through the hepatobiliary system after intravenous injection. In FIG. 10, the left column indicates normal mice, and the right column indicates GS mice. Fluorescence in the gallbladder gradually increased over 30 hours of administration and thereafter gradually decreased, but was detectable until 4 days post-administration, indicating a longer duration of hepatobiliary transport and longer retention of NP in the gallbladder.

NPs of a size less than 150-200nm may pass from the hepatic sinus through the hepatic sinus window into the Diels's cavity. After being taken up by hepatocytes, the nanoparticles can be excreted into bile canaliculi (composed of endothelial cells) together with bile, and then transferred into bile ducts and gall bladder. The distribution of TPCD NP in the gallbladder may be based on this transport pathway. To test this hypothesis, human hepatocytes (LO 2) and endothelial cells (HUVEC) were used to study the in vitro transport process of TPCD NP. The method comprises the following specific operations: LO2 cells were cultured in Dulbecco's modified Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin (PS), and HUVEC cells were cultured in F-10K medium containing 10% FBS,1% PS,0.1mg/mL heparin and 30 μ g/mL Endothelial Cell Growth Supplement (ECGS). Cells were incubated with 100. Mu.g/mL Cy5-TPCD NP for 1 hour. After washing, cells were stained with Hochest33342 and imaged with a confocal laser scanning microscope (CLSM, leica TCS SP 8). To determine the time-dependent uptake of Cy5-TPCD NP by LO2 cells, cells were incubated with 100. Mu.g/mL Cy5-TPCD NP for various times and analyzed by flow cytometry (FCM, cytoflex, beckman) after washing. To determine the concentration-dependent uptake of Cy5-TPCD NP by LO2 cells, cells were incubated with various concentrations of Cy5-TPCD NP for 2 hours and analyzed with FCM after washing.

As shown in FIGS. 11 and 12, cy5-TPCD NP was efficiently taken up by LO2 cells after 1 hour incubation of LO2 cells with 100. Mu.g/mL Cy5-TPCD NP. The uptake of Cy5-TPCD NP by LO2 cells was time and concentration dependent. The exocytosis of the NP by the cells can then be observed by Confocal Laser Scanning Microscopy (CLSM). As shown in fig. 12, cy5-TPCD NPs taken up by LO2 cells were successfully exocytosed from the cells when incubation was continued for 22 to 38 minutes after washing the cell supernatant and replacing with fresh medium. Similar exocytosis of Cy5-TPCD NP was also observed in HUVEC cells (as shown in FIG. 13). Transwell was then used to examine the rate of transfer of NP from the upper chamber to the lower chamber (as shown in figure 14). The transport rate of LO2 cells to Cy5-TPCD NP was only 1.3% after 1 hour, but increased rapidly to 18.3% and 24.0% after 2 and 4 hours of incubation, respectively (as shown in figure 15). After 6 hours of incubation, the transport process reached a saturation point. A similar transport process was also observed in HUVEC cells, and the rate of transport of Cy5-TPCD NP by HUVEC cells was relatively lower than that of LO2 cells (as shown in fig. 16). Based on in vivo profiling studies, these results further indicate that TPCD NP can be transported via the hepatobiliary transport pathway through hepatocytes and endothelial cells into the gallbladder.

3. TPCD NP can inhibit occurrence and development of gallstone diseases

Mice fed a gallstone diet were given a high dose (10 mg/kg), a medium dose (2 mg/kg) and a low dose (0.4 mg/kg) of TPCD NP by intravenous injection once every 4 days for 6 consecutive weeks. Meanwhile, mice fed on gallstone diet were given physiological saline (100. Mu.L/20 g as GS model control group), tpl (1.5 mg/kg as antioxidant control group, corresponding to the dose of Tpl in 10mg/kg TPCD NP), and UDCA (60 mg/kg as positive drug control group) orally by intravenous injection, respectively, with the same frequency and duration of administration (as shown in FIG. 17). The other group of mice fed on a normal diet served as a normal control group. Mice were sacrificed at the end of 6 weeks, the gallbladder was collected, photographed and the size of the gallbladder was recorded. As shown in FIGS. 18 and 19, in GS model mice fed with gallstone diet, the size of gallbladder was from-132 mm in normal control group mice ² Increased to 322mm ² . GS mice treated with high dose (10 mg/kg) TPCD NP had a significantly reduced gallbladder size compared to the model group, approximately 190mm ² . In the groups dosed with Tpl, UDCA and low dose TPCD NP, the mouse gall bladder size was much smaller than the GS model mice, although still significantly larger than the normal control mice.

Gallstones in the gallbladder were then collected for analysis. As shown in fig. 20, no gallstone formation was observed in normal mice. However, a large number of gallstones were observed in the gall bladder of GS model mice given physiological saline, and the cumulative size of gallstones was about 32.5mm per mouse ² (as shown in fig. 21). Meanwhile, most mice in this group had gallstones greater than 1.0mm in diameter (as shown in figure 22). After the treatment of antioxidant Tpl, the size of gallstone accumulated in each mouse is reduced to 16.2mm ² Meanwhile, the existence rate of gallstones with the diameter of more than 1.0mm is also obviously reduced. Very importantly, a 10mg/kg dose is administeredVery few gallstones were observed in the gall bladder of TPCD NP-treated mice, with an accumulated gallstone size of only-2.1 mm per mouse ² Gallstones were reduced by approximately 15.5-fold and 8.0-fold compared to GS model mice and Tpl treated mice, respectively (fig. 20 and 21). While gallstones with diameters greater than 1.0mm were not observed in this TPCD NP-treated group, gallstones with diameters between 0.5 and 1.0mm were detected in only 14.3% of the mice (fig. 22). Indeed, treatment of GS mice with medium and low doses of TPCDNP also had significant inhibition of gallstone development and progression (fig. 20-22). Notably, oral UDCA treatment at the 60mg/kg dose only ameliorated gallstone formation to a small extent (figures 20-22), meaning that oral bile acids had a very limited therapeutic effect on gallstones. Thus, the above results demonstrate that intravenous TPCD NP is effective in reducing the growth of mouse gallstones and inhibiting the development of gallstone disease.

4. Fate of TPCD NP in process of transferring to gallbladder

After 4 hours of intravenous injection of Cy5-TPCD NP into GS model mice, the gallbladder was separated, bile was collected and separated by centrifugation, followed by IVIS imaging. As shown in a and B in fig. 23, after centrifugation at 16000g for 10 minutes, significant Cy5 fluorescence was shown in the upper bile solution, indicating that Cy5-TPCD NP had been degraded to free β -CD during hepatobiliary transport. In addition, laser confocal microscopy (CLSM) images of CC collected from GS mouse gall bladder clearly showed co-localization of β -CD and CC (C in fig. 23). Fluorescence of Cy5 in CC (isolated directly from gallbladder or after sonication of gallstones) was also detected by FCM, showing that about 37.3% of CC had fluorescently labeled β -CD binding (D to F in FIG. 23). The above studies indicate that TPCD NP can be rapidly transported into bile through hepatobiliary pathway, and free beta-CD as a degradation product of TPCD NP can effectively bind CC and gallstones in gallbladder.

Next, it was continuously investigated whether the degradation of TPCD NP was carried out during transport or in bile after transport. As shown in FIGS. 12 and 24-25, intracellular and exocytosis of NPs were aggregated together (as a typical marker of degradation of NPs), indicating that the nanoparticles were already interacting with hepatocytes and HUVEC cellsDegradation occurs. Subsequently, the particle size distribution of nanoparticles transported from the upper chamber of the transwell via LO2 cells to NPs in the lower chamber was determined. The specific operation is as follows: will be 5X 10 ⁴ LO2 or HUVEC cells were added to the upper chamber of a transwell chamber with a pore size of 8 μm and incubated at 37 ℃ for 1 hour. Cy5-TPCD NP was then added to the upper chamber and the final concentration of NP was set to 100. Mu.g/mL, respectively. After 1, 2, 4 and 6 hours, 200. Mu.L of the medium in the lower chamber was collected and mixed by adding 800. Mu.L of methanol. The content of Cy5-TPCD NP transferred to the lower chamber was measured by a fluorescence spectrophotometer after sonication for 10min and centrifugation. Then 200 μ L of fresh medium was replenished into the lower chamber. The transport rate of TPCD NP was calculated at different time points. The particle size distribution of TPCD NPs in the transfer to the lower chamber was determined by changing the cell culture medium to DMEM without FBS. TPCD NP was added to the upper chamber at a final concentration of 100. Mu.g/mL and incubated for 1, 2, 4, and 6 hours. The media in the lower chamber was collected and the particle size distribution of TPCD NP following transport was determined by DLS analysis.

TPCD NP in DMEM was 204.4. + -. 0.3nm in diameter and 0.29. + -. 0.01 in PDI as measured by DLS. When incubated in DMEM at 37 ℃ for 6h, there was little change in particle size and distribution (as shown in a in fig. 26). In contrast, TPCD NP transported by LO2 cells after 1 hour of incubation had a diameter of 957.6. + -. 114.6nm, a PDI of 0.78. + -. 0.07, and a particle size distribution varying from-100 nm to-10 μm (as shown in B in FIG. 26). Particle size increases were also observed in NPs after 2, 4 and 6 hours of transport by LO2 cells. Therefore, accumulation of TPCD NPs may already occur during transport.

To examine the degradation of TPCD NP in bile, cy5-TPCD NP was incubated in bile at 37 ℃ for various times. The specific operation is as follows: fresh bile from purchased pigs was stored at 4 ℃. Before use, bile was centrifuged at 16000g for 10 min. Bile suspensions containing 0, 0.02, 0.05, 0.1, 0.2, 0.5 and 1mg/mL Cy5-TPCD NP were prepared. mu.L of Cy5-TPCD NP/bile suspension was mixed with 900. Mu.L of methanol and centrifuged at 16000g for 10 min. The fluorescence intensity of the supernatant was measured, and then a standard curve of concentration-fluorescence intensity was prepared. To determine the degradation of TPCD NP in bile, 100. Mu.g of Cy5-TPCD NP was mixedSuspended in 1mL of bile (with/without 1mM H) ₂ O ₂ ) And incubated at 37 ℃ with shaking at 100 rpm. 0. After 0.5, 1, 2, 4, 8 and 24 hours, the suspension was centrifuged at 16000g for 10 minutes. mu.L of the supernatant was mixed with 900. Mu.L of methanol, sonicated for 10min and centrifuged, and then the fluorescence was measured using a fluorescence spectrophotometer. The concentration of free Cy5- β -CD in the bile was then calculated.

TPCD NP was rapidly degraded in bile, and after 8h incubation, the concentration of free β -CD in bile supernatant was approximately 35 μ G/mL (G in FIG. 23). The hydrolysis of TPCD NP may be caused by hydrogen peroxide (H) in bile ₂ O ₂ ) As a result, it is capable of degrading TPCD to water-soluble β -CD. And adding H into bile ₂ O ₂ Further increasing the degradation rate of TPCD NP (G in figure 23). It is important to note that the rate of degradation of TPCD NP may be faster than that of Cy5-TPCD NP (fig. 8), since the binding of Cy5 to β -CD may reduce the water solubility of β -CD to some extent. Thus, TPCD NP can be transported into the gallbladder via the hepatobiliary route after intravenous administration, with its degradation occurring both during transport and after entering the bile.

5. TPCD NP increases cholesterol dissolution and inhibits precipitation of CC in bile

Since the precipitation of CC in bile is the first step in gallstone formation, it was first assessed whether the β -CD of TPCD NP and its degradation products could increase the solubility of CC in bile. First, CC was prepared: prepare a 2mg/mL cholesterol/ethanol solution and add 1.5 volumes of deionized water. The precipitated CC was then dried under vacuum. CC was suspended in PBS and sonicated for 10min prior to use.

The cholesterol solubility was determined by the following method: 5mg CC was added to 500. Mu.L of bile or bile containing 50. Mu.g/mL beta-CD, 100. Mu.g/mL beta-CD, 50. Mu.g/mL TPCD NP or 100. Mu.g/mL TPCD NP. After sonication at 37 ℃ for 20 minutes, the suspension was centrifuged at 16000g for 10 minutes. Then 100. Mu.L of the supernatant was mixed with 900. Mu.L of methanol and centrifuged. The concentration of cholesterol in the supernatant was determined by High Performance Liquid Chromatography (HPLC).

When 5mg/mL CC was suspended in bile and sonicated at 37 ℃ for 20 minutes, the dissolved cholesterol concentration was approximately 845. Mu.g/mL (H in FIG. 23). However, when 50 and 100. Mu.g/mL TPCD NP were added to the CC bile suspension for incubation, respectively, the cholesterol concentration increased to-938 and 1028. Mu.g/mL. Free β -CD also significantly promoted the dissolution of CC in bile as a degradation product of TPCD NP (H in fig. 23). Thus, TPCD NP promotes the solubilization of CC in bile, probably due to the degradation of TPCD NP to free β -CD.

Excess CC was mixed with bile and sonicated at 37 ℃ for 20 minutes to prepare bile with saturated cholesterol. After centrifugation at 16000g for 10 minutes, the supernatant was filtered through a 0.22 μm filter and 50 μ g/mL β -CD,100 μ g/mL β -CD,50 μ g/mL TPCD NP,100 μ g/mL TPCD NP, or deionized water (deionized water equivalent to the β -CD and TPCD NP groups) was added, respectively. Then 200 μ L of bile from a different set (n = 5) was added to the 96-well plate and 200 μ LPBS was added to the remaining blank wells. The well plate was then sealed and incubated at 37 ℃ in the dark. The nucleation of the crystals of CC was then recorded daily using a microscope.

In the observation for 21 days, the precipitation of CC crystals was significantly inhibited by adding TPCD NP to bile (fig. 23, I and 27), and no CC crystals were observed when free β -CD was added to bile. The results show that the free beta-CD can promote the dissolution of cholesterol and inhibit the crystallization of CC as a degradation product of TPCD NP in bile, which is probably one of the action mechanisms that TPCD NP can inhibit the generation and development of mouse gallstone.

6. Inhibition of neutrophil-mediated inflammation by TPCD NP

Neutrophil-mediated inflammation, such as neutrophil ROS production and NETs formation, can contribute to the development of gallstone disease. TPCD NP can clear ROS, relieve oxidative stress and show excellent treatment effect in various inflammatory diseases. The distribution of Cy5-TPCD NP in mouse neutrophils after intravenous injection was first evaluated. The specific operation is as follows: cy5-TPCD NP (10 mg/kg mouse body weight) was injected intravenously into mice, and then sacrificed 2 hours later. Placing the liver in0.5mg/mL collagenase IV was sheared and incubated at 37 ℃ for 40 minutes. The suspension was then passed through a 70 μm filter and 50g was separated for 2 minutes. The collected supernatant suspension was then centrifuged at 420g for 5 minutes. After washing, the cells were stained with antibody at 4 ℃ for 30 minutes and analyzed with FCM. 200 μ L of blood was collected and analyzed by antibody staining after lysing erythrocytes with ACK lysis solution. Wherein CD11b ⁺ Ly-6G ⁺ The double-expressing cells were neutrophils.

As shown in a of fig. 28, the neutrophils isolated from liver and blood 2 hours after NP administration showed Cy5 fluorescence, indicating that Cy5-TPCD NP was efficiently taken up by neutrophils in vivo. Neutrophils were further isolated from the abdominal cavity of mice and whether TPCD NP could inhibit neutrophil-mediated inflammation was evaluated. As shown by B in fig. 28, when neutrophils were incubated with 200 μ g/mL CC for 1h, ROS production in neutrophils significantly increased. In contrast, ROS production remained at normal levels after pre-incubation of neutrophils with TPCD NP at concentrations of 50 and 100. Mu.g/mL for 1h, followed by additional incubation with 200. Mu.g/mL CC for 1h.

2 x 10 to ⁵ Neutrophils were incubated with TPCD NP media at concentrations of 0, 20, 50, and 100. Mu.g/mL, respectively. After 1 hour, cells were washed with PBS and incubated with CC (200. Mu.g/mL) for 1 hour. Cells were then stained with ROS fluorescent probe (DCFHDA) and analyzed with FCM. To determine the formation of NETs, neutrophils were first incubated with various concentrations of TPCD NP for 1h. After washing, cells were incubated with CC (200. Mu.g/mL) for 1 hour, stained with Sytox Green (1. Mu.M) for 10 minutes, and washed for detection of extracellular DNA by FCM.

As shown by C and D in fig. 28, extracellular DNA staining indicated a significant increase in the formation of NETs when neutrophils were incubated with CC for 1 hour. In contrast, when stimulated with CC, extracellular DNA content was significantly reduced after the first 1h incubation of neutrophils with TPCD NPs, demonstrating significant inhibition of NETs formation (C and D in fig. 28). Thus, TPCD NP can effectively distribute into neutrophils after intravenous injection and inhibit neutrophil-mediated inflammation, such as reducing CC-induced neutrophil ROS production and inhibiting NETs formation, and thus, TPCD NP may play a role in delaying the development of gallstones through this mechanism of action.

7.TPCD NP in vivo safety evaluation

The body weights of mice receiving gallstone diet feeding and various treatment groups were recorded every 4 days. After 6 weeks, mice were sacrificed and blood was collected to determine blood routine parameters. Serum was isolated to determine biomarkers of liver function. Major organs collected included heart, liver, spleen, lung and kidney. It should be noted that: the number of Whole Blood Cells (WBC), red Blood Cells (RBC), platelets (PLT) and hemoglobin was measured with an automatic hematology analyzer (Sysmex KX-21, sysmex Co., japan). Liver function biomarkers, including alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST), were detected with a Roche Cobas C501 (switzerland) instrument.

The body weight of the GS model group mice remained relatively low and increased less all the time, which was probably due to the cholelithiasis (a in fig. 29). In the other administration groups, the body weight of the mice was somewhat reduced at the time of administration and gradually recovered in the next several days. Different administration groups had little effect on the organ index of the heart (B in fig. 29). However, compared to normal diet-fed mice, the liver organ index was significantly increased in gallstone diet-fed mice, while Tpl, UDCA and TPCD NP treatment failed to reverse the above effects (C in fig. 29). In addition, the organ index of the spleen of mice fed with gallstone diet was also significantly increased compared to the mice fed with normal diet, indicating that systemic inflammation may be caused (D in fig. 29). Both UDCA and TPCD NP-administered groups reduced the spleen organ index to levels of normal control mice. The organ indexes of the lung and kidney in the GS model group were not significantly different from those of the mice in the other treatment groups (E and F in fig. 29).

Meanwhile, serum levels of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) as biomarkers of liver function were significantly increased in GS control group compared to normal group (as shown in fig. 30), indicating that the liver was damaged after receiving gallstone diet. While ALT levels were significantly reduced, and AST levels were slightly reduced after antioxidant Tpl treatment compared to GS model mice (B in fig. 30). And after TPCD NP is treated, ALT and AST of the TPCD NP are reduced to the level of normal mice, which shows that the TPCD NP has excellent liver protection effect besides the effect of treating gallstone diseases. The White Blood Cell (WBC) number was slightly increased in the GS model mouse compared to the normal control group (a in fig. 31). WBC counts in mice were reduced to levels in normal control mice after treatment with high doses of TPCD NP. While the numbers of Red Blood Cells (RBC), platelets (PLT) and Hemoglobin (HGB) of the GS model group were not significantly different from those of the other treatment groups (B to D in fig. 31).

Hematoxylin-eosin (H & E) section staining: tissues were fixed in 4% paraformaldehyde for at least 48 hours, then sections were prepared and H & E stained by wuhan seiver biotechnology limited (china). Image acquisition was performed using a microscope (Nikon) and analyzed using BR 4.5 software. In hematoxylin-eosin (H & E) stained liver tissue sections of GS model mice, a large number of intracytoplasmic lipid droplets were observed in hepatocytes (indicated by arrows in fig. 32). In other treatment groups, changes in the level of lipid droplets within the liver cytoplasm were also observed. No detectable lesions were observed in the heart, spleen, lung and kidney tissue sections of all groups of mice (fig. 32). Therefore, the research shows that the TPCD NP can effectively inhibit the growth of gallstones and has good safety.

In conclusion, the TPCD NP is proved to be effectively distributed into a gall bladder through a liver and gall system, and can effectively inhibit the occurrence and the development of gallstone in a mouse model. Mechanistically, free beta-CD is taken as a degradation product of TPCD NP, is effectively combined with CC and gallstones in a gallbladder, and increases the solubility of cholesterol and inhibits the crystallization of the CC. In addition, TPCD NP can significantly alleviate neutrophil-mediated inflammation, such as based on CC-induced ROS production and NETs formation. The nano-drug transported by the hepatobiliary system is disclosed for the first time for treating hepatobiliary/gastrointestinal diseases. Therefore, not only is a new strategy provided for effectively preventing and treating gallstone diseases, but also an important new idea is provided for designing and developing a new therapy which can treat other digestive system diseases by using the same transport route.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The application of the nanoparticles in preparing the intravenous injection for treating gallbladder-related diseases is characterized in that the nanoparticles are transported through liver and gallbladder, and the nanoparticles in the intravenous injection are distributed to gallbladder by a liver and gallbladder system transport method after intravenous injection, so that the aim of targeted treatment of the gallbladder-related diseases is fulfilled;

the nanoparticle is based on a supramolecular macrocyclic compound, the supramolecular macrocyclic compound has the effect of dissolving cholesterol, and the supramolecular macrocyclic compound is a derivative based on beta-cyclodextrin;

the nanoparticle is mainly prepared by the following steps: chemically bonding 4-hydroxy-2, 6-tetramethylpiperidine-1-oxyl free radical and 4- (hydroxymethyl) phenylboronic acid pinacol ester to beta-cyclodextrin to obtain a beta-cyclodextrin derivative, and preparing the beta-cyclodextrin derivative into beta-cyclodextrin derivative nanoparticles;

the method for preparing the beta-cyclodextrin derivative into the beta-cyclodextrin derivative nanoparticles comprises the following steps:

adding an ethanol solution containing lecithin and DSPE-mPEG2000 into water, and uniformly mixing to obtain a first mixed solution;

dropwise adding the alcoholic solution of the beta-cyclodextrin derivative into the first mixed solution, uniformly mixing, and concentrating, or dropwise adding the mixed alcoholic solution of the beta-cyclodextrin derivative and Cy7.5NHS ester into the first mixed solution, uniformly mixing, and concentrating, or dropwise adding the mixed alcoholic solution of the beta-cyclodextrin derivative and the Cy 5-loaded beta-cyclodextrin derivative into the first mixed solution, uniformly mixing, and concentrating.

2. Use according to claim 1, wherein the ratio between lecithin and DSPE-mPEG2000 is from 5 to 7:8 to 10.

3. Use according to claim 2, wherein the concentration is a vacuum rotary evaporation concentration.

4. Use according to claim 2, characterized in that the chemical structure of the β -cyclodextrin derivative is:

chemical structure of Cy 5-loaded beta-cyclodextrin derivative

Wherein R in the chemical structures of the beta-cyclodextrin derivative and the Cy 5-loaded beta-cyclodextrin derivative ₁ Is H or

R ₂ Is H or

Cy5 is

5. The use according to claim 1, wherein the gallbladder-related disease is any one of gallstones, cholecystitis, gallbladder microbial infection, gallbladder cancer, bile duct cancer, intrahepatic bile duct cancer and cholestasis.

6. The use of claim 5, wherein said gallstones comprise cholesterol gallstones, bile pigment gallstones and melanin-type gallstones.

7. The use of claim 5, wherein the gallstones are cholesterol-type gallstones.

8. The use according to any one of claims 1 to 7, wherein the treatment of gallstones is the promotion of cholesterol dissolution and the inhibition of cholesterol crystallization or the inhibition of neutrophil mediated inflammation.

9. Use of nanoparticles for the preparation of an intravenous injection for promoting cholesterol dissolution and inhibiting cholesterol crystallization, wherein the nanoparticles are as defined in any one of claims 1 to 8.

10. Use of a nanoparticle for the preparation of an intravenous injection for inhibiting neutrophil-mediated inflammation, wherein the nanoparticle is according to any one of claims 1 to 8.

11. Use of a nanoparticle for the preparation of an intravenous injection for reducing the reactive oxygen species production by cholesterol crystallization-induced neutrophils, wherein the nanoparticle is according to any one of claims 1 to 8.

12. Use of a nanoparticle for the preparation of an intravenous injection for reducing the formation of neutrophil trap networks induced by cholesterol crystallization, wherein said nanoparticle is as defined in any one of claims 1 to 8.