CN111643481A

CN111643481A - Nano-medicine, preparation method and application in treatment of pancreatic duct cancer

Info

Publication number: CN111643481A
Application number: CN202010331200.0A
Authority: CN
Inventors: 赵蔚; 吴均; 殷晓煜
Original assignee: National Sun Yat Sen University
Current assignee: Sun Yat Sen University; National Sun Yat Sen University
Priority date: 2020-04-24
Filing date: 2020-04-24
Publication date: 2020-09-11

Abstract

The invention discloses a nano-drug, a preparation method and application thereof in treating pancreatic duct cancer. In particular, the nano material (NP) Cys-8E entrapped compound UNC0638 used in the invention is prepared into nano drug NP_UNC0638The tumor part of pancreatic cancer can be targeted, and the compound has better drug delivery capacity for pancreatic ductal adenocarcinoma cells which cannot be acted by traditional drugs, and the treatment effect of the compound UNC0638 is obviously improved. The nano-drug NP_UNC0638Can induce apoptosis of pancreatic ductal adenocarcinoma cells, and nanometer drug NP_UNC0638Can avoid UNC0638 acting on normal cells, and reduce its toxic and side effects. The nano-drug NP of the invention_UNC0638Has the advantages of targeting pancreatic ductal adenocarcinoma, small side effect and the like, and has good application prospect and wide development space in the field of clinical treatment of pancreatic ductal adenocarcinoma.

Description

Nano-medicine, preparation method and application in treatment of pancreatic duct cancer

Technical Field

The invention belongs to the technical field of biological medicines. More particularly, relates to a nano-drug, a preparation method and application thereof in treating pancreatic duct cancer.

Background

Pancreatic cancer mainly includes adenocarcinoma derived from exocrine cells and neuroendocrine carcinoma derived from islet cells, of which pancreatic ductal adenocarcinoma is one of the most aggressive tumors. The early clinical symptoms are atypical, the diagnosis is difficult, the prognosis is extremely poor, the incidence rate is nearly equal to the death rate of diseases, the 5-year survival rate of American patients is still lower than 7%, and the 5-year survival rate of China is still lower than 5%. The current treatment means is mainly surgical excision and is assisted by chemotherapy means. However, nearly 80% of patients have local infiltration and even distant organ metastasis during treatment, and the chance of surgical resection is lost, so that how to improve the effective rate of chemotherapy, reduce the drug resistance of tumors and reduce the recurrence and metastasis of pancreatic ductal adenocarcinoma is important.

It has been shown that ductal adenocarcinoma of the pancreas is composed of a sub-population of tumor cells with various functions, a small fraction of which have the characteristics of tumor stem cells, such as self-renewal and multipotent differentiation capacity, which make the ductal adenocarcinoma stem cells play a decisive role in the proliferation, invasion, recurrence, metastasis and resistance of ductal adenocarcinoma of the pancreas. The discovery of the pancreatic ductal adenocarcinoma stem cells provides a new visual field for understanding the biological characteristics of the pancreatic ductal adenocarcinoma, but at present, no treatment scheme capable of targeting the pancreatic ductal adenocarcinoma stem cells is successfully applied to clinical treatment, so that a treatment means capable of effectively killing tumor stem cells and reducing the proportion of the tumor stem cells is found, and the method is an important way for improving the treatment effect.

Meanwhile, nanotechnology has rapidly entered the field of tumor therapy, and active or passive targeted therapy is performed by using a nanomaterial as a carrier, and even gene therapy can reduce toxic and side reactions caused by nonspecific distribution of chemotherapeutic drugs, target tumor stem cells, resist multidrug resistance and improve the anticancer efficacy of drugs. The preparation of a stimuli-responsive polymer system (including temperature, light, oxidation reduction and pH response) for nano-carriers is widely applied to the field of biomedicine, wherein the reduction-responsive controlled-release system can respond to higher glutathione concentration in a tumor microenvironment so as to be widely applied to delivery of anti-cancer drugs and tumor treatment because the polymer molecular structure of the system has special reduction-sensitive functional groups such as disulfide bonds and the like. The polyester polymer is an important biomedical nano material, has the advantages of good biocompatibility, excellent biodegradability, no toxicity, harmlessness, low price, wide range and the like, and has good application prospect and wide development space in the field of drug delivery. The direct polycondensation method is one of the main methods for synthesizing the polyester macromolecules in the prior art, but the direct polycondensation method still has the defects that the conditions such as reaction temperature, pressure, time, vacuum degree and the like are difficult to control. Therefore, the research on the polyester material which has simple synthesis method, good controllability of synthesis conditions and certain excellent properties of a synthetic product is one of the important conditions for promoting the polymer to be applied to the tumor treatment.

In this study, EHMT2 (also known as G9a or KMT1C) is the second reported histone methyltransferase containing the SET domain, which catalyzes mainly the dimethylation of H3K9 (H3K9me2) and regulates the transcriptional repression of multiple genes. Research shows that EHMT2 is not only involved in cell differentiation, gene transcription and embryonic development and other biological processes, but also is overexpressed in various cancers, and is closely related to poor prognosis of cancer patients. It has been reported that high levels of EHMT2 correlate with poor clinical outcomes in pancreatic cancer patients. Inhibition of EHMT2 in pancreatic cancer may overcome drug resistance. UNC0638 is a potent EHMT2 inhibitor, selectively acts on a broad spectrum of epigenetic and non-epigenetic targets, can reduce the global level of H3K9me2 in various tumor cell lines, obviously inhibits the formation of tumor cell clones, and has the effect almost equal to the effect of knocking down the expression level of EHMT2 in cells by using shRNA. In mouse embryonic stem cells, UNC0638 activates EHMT 2-silenced genes and retroviral reporter genes in a concentration-dependent manner without promoting differentiation. Recent studies have shown that UNC0638 is highly toxic to normal cells. Therefore, a nano-drug which has good biocompatibility and can target tumor cells is developed based on UNC0638 research and has important significance in pancreatic ductal adenocarcinoma treatment.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects and shortcomings of the existing UNC0638, provide a novel nano-drug and realize the loading of drug NP by a nano-precipitation method_UNC0638Preparing nano medicine carrying system with GSH responsiveness and having the function of NP_UNC0638The application prospect of the nano-medicine is wide. The NP_UNC0638The nano-drug can realize high-efficiency loading of UNC0638, has strong sensitivity and removal capacity to GSH in tumor environment, and can effectively deliver the drug to pancreatic duct adenocarcinoma cells and pancreatic duct adenocarcinoma stem cells, NP_UNC0638Can be quickly released in tumor cells to induce the apoptosis of the tumor cells, and opens up a new way for the effective treatment of pancreatic ductal adenocarcinoma.

In order to achieve the purpose, the invention adopts the following technical scheme:

a nanometer medicinal preparation, the nanometer medicinal preparation is NP_UNC0638Wherein, the NP is_UNC0638Having UNC0638, and a polymer Cys-8E for use in the UNC0638 delivery vehicle in the preparation of the nano-drug.

Further, the drug NP_UNC0638Has disulfide bond and ester bond, and has sensitivity response to glutathione.

Further, the nano-drug is a GSH sensitive nano-drug.

Further, the drug NP_UNC0638The chemical formula of (A) is as follows:

the invention also provides a preparation method of the nano-drug, which comprises the following steps:

dissolving UNC0638 and Cys-8E and stabilizer, distearoyl phosphatidyl ethanolamine-polyethylene glycol in water-soluble organic solvent to form oil phase. Then slowly dropping the oil phase into the water phaseIn the method, the nano-drug NP with uniform and stable particle size is obtained_UNC0638。

Further, the nano-drug NP_UNC0638The particle size range is 50nm-300 nm.

Further, the particle size is 120 nm.

The invention further provides an application of the nano-drug, and the nano-drug NP_UNC0638The application in treating pancreatic ductal adenocarcinoma is provided.

The invention has the beneficial effects that the nano-particles with controllable particle size are prepared by using the polyester polymer with good biocompatibility and reduction response to encapsulate the anti-cancer drug, and can be accumulated in tumor tissues through high permeability and high retention (EPReffect) of the tumor tissues, thereby realizing the passive targeting effect. Then, the high-concentration glutathione function of the tumor tissue is utilized, so that the disulfide bond in the nano system starts to break, the release of the drug is accelerated, the effect of treating cancer is achieved, and the clinical application value is very high.

Drawings

FIG. 1A shows a Dynamic Light Scattering (DLS) plot of NP nanopharmaceuticals of the present invention encapsulating drug UNC 0638;

FIG. 1B shows a Transmission Electron Microscope (TEM) photograph of the blank nanoparticles and NPUNC0638 nanomedicine of the present invention.

FIG. 2 shows the uptake of nano-drugs by pancreatic ductal adenocarcinoma cells in an example of the present invention.

FIG. 3 shows the enrichment of the nano-drug at the tumor site, wherein FIG. 3A shows the change of the interval of time after the injection of DiR dye into tumor-bearing naked tail vein and the encapsulation of DiR dye by nano-material Cys-8E; FIGS. 3B and 3C show the distribution of DiR dye in organs and tumors and the radiation efficiency

FIG. 4 shows cell viability assay, with the Nanoparticulate NPUNC0638 of the present invention inhibiting PDAC cell proliferation.

FIG. 5 shows the Nanopharmaceutical NPUNC0638 inhibiting PDAC formation of tumor spheres, wherein FIG. 5A shows three-dimensional tumor sphere formation; figure 5B shows the number of tumor spheres per well.

FIG. 6 shows the in vivo growth inhibition of PDAC animal models by nanomedicines, wherein 6A is a plot of tumor size versus tumor size; and 6B is a tumor growth plot.

Detailed Description

The present invention will be further described with reference to the accompanying drawings, and it should be noted that the following examples are provided to illustrate the detailed embodiments and specific operations based on the technical solutions of the present invention, but the scope of the present invention is not limited to the examples.

Example 1

Preparation of GSH response type drug-loaded UNC06838 nano system

When preparing drug-loaded Cys-8E nanoparticles, Cys-8E was dissolved in DMSO to form oil phase 1 at a concentration of 20 mg/ml. UNC06838 drug was dissolved in oil phase 1 at a drug concentration of 9 mg/ml. Stabilizer DSPE-PEG2000 was dissolved in DMSO at a concentration of 4mg/ml to form oil phase 2.

The appropriate volume of 1, 2 phase oil was mixed thoroughly to make DSPE-PEG2000 Cys-8E, about 20 wt% of the total mass of UNC06838, and added drop by drop to deionized water stirred at 2000 r/m. The volume ratio of the oil phase to the water phase is 1: 9. Centrifuging three times with ultrafiltration tube with molecular weight cutoff of 100,000Da, and removing

Free organic solution DMSO, finally resuspended with PBS. DLS detects the particle size of the drug-loaded nanoparticles to be 100-150 nm; the nano-particles can be observed to be in a uniformly dispersed spherical structure by a TEM image.

Example 2

Drug delivery capability of Polymer Cys-8E

NP was obtained from NPs loaded with fluorescent coumarin 6(2.0 ug/ml)_{Coumarin compound}6。

(1) BxPC-3(ATCC number CRL-1687, human origin, epithelial-like adherent growth) PANC-1 cells (ATCC number CRL-1469, human origin, epithelial-like adherent growth) in logarithmic growth phase were digested with trypsin, blown up into a single cell suspension, counted, and inoculated into 6-well culture plates at 1.5 × 510 per well⁵And (4) cells. Culturing in an incubator for 24h, adding pure coumarin 6 solution and NP coumarin 6 solution respectively after the cells adhere to the wall, incubating for 30 min at 37 ℃, fixing with 4% paraformaldehyde, and staining with DAPI. Using a fluorescence microscopeAnd recording the fluorescence intensity by a flow cytometer.

(2) BxPC-3 and PANC-1 cells in a logarithmic growth phase are inoculated in a low-adhesion 24-pore plate, after the cells are cultured in an incubator at 37 ℃ for 5 days, a single, compact and regular-form tumor sphere (stem cell) is selected for experiment, and a pure coumarin 6 solution and an NP coumarin 6 solution are respectively added to ensure that the final concentration is 200 ng/ml. After 30 minutes of co-incubation at 37 ℃, fixation with 4% paraformaldehyde was carried out for 15 minutes. The fluorescence intensity was recorded using a fluorescence microscope and flow cytometer.

(3) The experiment adopts 4-week-old BALB/c male nude mice, purchased from Nanjing university model animal research institute, with the weight of 18-20 g, and the mice are raised in cages without special pathogenic bacteria (SPF), alternately illuminated for 12h and fed freely. Digested with 5% trypsin and the cells washed 2 times with normal saline. After washing, the cells were resuspended in physiological saline, adjusted to 107/ml, and prepared for subcutaneous tumorigenesis. Nude mice were randomly divided into two groups of 3 mice each, numbered individually, and injected subcutaneously into the anterior axilla with 100mL of 107/mL cell suspension. Regularly monitoring the size of the tumor until the tumor grows to 500mm³100mL of physiological saline, 100mL of physiological saline containing 10g of near infrared Dye (DiR) dissolved therein, and an equal amount of NPDiR were injected through the tail vein of the mouse, respectively. Mice were anesthetized with the IVIS imaging system and fluorescent signals were captured at 1 hour, 4 hours, 8 hours, 12 hours, and 24 hours post-injection, respectively. After observation, mice were sacrificed and the major organs (heart, lung, liver, spleen, kidney) and tumor tissues were imaged in vitro.

The experimental results are as follows:

the tumor ball and animal models derived from the pancreatic ductal adenocarcinoma cells, whether the cell lines are cell lines, show that the fluorescence intensity of the tumor part after the nano-encapsulation effect is obviously enhanced (fig. 3 and 4), and show that the Cys-8E nano-vector has better capability of targeting the pancreatic ductal adenocarcinoma.

Example 3

NP_UNC06838And comparing the influence of the nano-drug on the proliferation of pancreatic ductal adenocarcinoma cells.

Detection of different concentrations of NP by cell viability assay_UNC06838Nanoparticle and UNC06838 free drug-para-pancreasThe effects of adenoductal adenocarcinoma cell proliferation and pancreatic ductal adenocarcinoma stem cell formation into tumor nodules.

(1) BxPC-3 and PANC-1 cells in exponential growth phase are digested by pancreatin, blown into single cell suspension, counted and inoculated into 96-well culture plate. Culturing in incubator for 24 hr, adding NP of different concentrations after cell adherence_UNC06838The nanoparticles and UNC06838 free drug were cultured in an incubator for 48h, and cell viability was assessed according to CellTiter-Glo luminescent cell viability assay kit instructions.

(2) BxPC-3 and PANC-1 cells in a logarithmic growth phase are inoculated in a low-adhesion 96-well plate, cultured in an incubator at 37 ℃ for 5 days, a single, compact and regular tumor ball is selected for experiment, DMSO, free NP with different concentrations and NP with equal concentration are respectively added_UNC06838A nanometer medicinal preparation. After 48 hours of action, the number of tumor nodules was determined by counting under a microscope.

The experimental results are as follows:

as shown in figures 4 and 5, NP_UNC06838The nano-drug has obviously better inhibition effect on the proliferation of tumor cell from pancreatic ductal adenocarcinoma cell line and pancreatic ductal adenoma stem cell than UNC06838 free drug. The calculation method of the action relationship of the medicine comprises the following steps: survival rate ═ experimental group number-blank number)/(control group number-blank number)

Example 4

NP_UNC06838Inhibition effect of nano-drug on pancreatic duct adenocarcinoma stem cells in animal model

Culturing ductal adenoma pancreas SJSA1 cell to obtain ductal adenoma pancreas stem cell, culturing for 5 days, and making into 1 × 10 with PBS⁷Single cell suspension per mL was injected subcutaneously at the left underarm position of male nude mice, 0.2mL each. The condition of the mice was observed every day, and the major diameter (a) and the minor diameter (b) of the tumor were measured with a vernier caliper according to the formula V (mm)³)＝(a×b²) The tumor volume was calculated. When the tumor volume reaches 80-100 mm³At the time, animals were randomly divided into 6 groups of 5 animals each. Respectively, control group PBS; nanomaterial blank control group NPs; equal concentrations of UNC06838 free drug; equal concentrationDegree NP_UNC06838The drug is administered 5 times every other day through the tail vein. After 24 days, the mice were sacrificed by cervical dislocation, and the tumors were peeled off, weighed, and compared for the size of the tumor growth.

The experimental results are as follows:

results show NP_UNC06838The nano-drug has obviously better effect on inhibiting the growth of tumors derived from pancreatic ductal adenoma stem cells than other experimental groups, and shows that NP (human tumor necrosis factor)_UNC06838The nano-drug also has the effect of inhibiting the self-renewal capacity of the pancreatic ductal adenoma stem cells in vivo.

Various corresponding changes and modifications can be made by those skilled in the art based on the above technical solutions and concepts, and all such changes and modifications should be included in the protection scope of the present invention.

Claims

1. The nano-drug is characterized in that the nano-drug is NP_UNC0638Wherein, the NP is_UNC0638Having UNC0638, and a polymer Cys-8E for use in the UNC0638 delivery vehicle in the preparation of the nano-drug.

2. The nano-drug of claim 1, wherein the drug NP is_UNC0638Has disulfide bond and ester bond, and has sensitivity response to glutathione.

3. The nano-drug of claim 1 or 2, wherein the nano-drug is a GSH sensitive nano-drug.

4. The nano-drug of claim 3, wherein the drug NP is_UNC0638The chemical formula of (A) is as follows:

5. a method for preparing a nano-drug according to claim 1, 2 or 3, comprising the steps of:

dissolving UNC0638 and Cys-8E and stabilizer, distearoyl phosphatidyl ethanolamine-polyethylene glycol in water-soluble organic solvent to form oil phase. Then slowly dripping the oil phase into the water phase to obtain the nano-drug NP with uniform and stable particle size_UNC0638。

6. The method for preparing nano-drug of claim 5, wherein the nano-drug NP is_UNC0638The particle size range is 50nm-300 nm.

7. The method of claim 5, wherein the particle size is 120 nm.

8. The use of the nanomedicine of claim 1, wherein the nanomedicine NP is administered_UNC0638The application in treating pancreatic ductal adenocarcinoma is provided.