阅读说明：本技术 角鲨烯化西达本胺前药自组装纳米粒及制备方法与应用 (Squalene Sidapamide prodrug self-assembly nanoparticles and preparation method and application thereof ) 是由 谷满仓 陈凯迪 赵山 石燕 于 2021-09-13 设计创作，主要内容包括：本发明公开了一种角鲨烯化西达本胺前药自组装纳米粒及制备方法与应用,首先将角鲨烯酸和西达本胺分别溶解于甲基甲酰胺中,以N-羟基琥珀酰亚胺以及1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸为催化剂,将两者混合反应制备角鲨烯化西达本胺前药分子；然后将角鲨烯酸和叶酸-聚乙二醇-氨基或对甲氧基苯甲酰胺-聚乙二醇-氨基分别溶解于甲基甲酰胺中,以N-羟基琥珀酰亚胺(NHS)以及1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸(EDCI)为催化剂,将两者混合反应得小分子配体,将小分子配体以及精氨酸-甘氨酸-天冬氨酸和角鲨烯化西达本胺前药分子在水中自组装形成纳米粒；本发明可有效解决西达本胺在实体肿瘤治疗方面存在的渗透率低、靶向性差、毒副作用大的缺点。(The invention discloses a squalene based self-assembly nanoparticle of a precursor of cidandamine, a preparation method and application thereof, wherein firstly, squalene and cidandamine are respectively dissolved in methyl formamide, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride are used as catalysts, and the squalene based self-assembly nanoparticle and the cidadamine precursor are mixed and reacted to prepare a squalene based prodrug molecule; then, respectively dissolving squalene acid and folic acid-polyethylene glycol-amino or p-methoxybenzamide-polyethylene glycol-amino in methyl formamide, mixing N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) as catalysts, reacting to obtain a small molecular ligand, and self-assembling the small molecular ligand and arginine-glycine-aspartic acid and squalene-cidalimide prodrug molecules in water to form nanoparticles; the invention can effectively solve the defects of low permeability, poor targeting property and large toxic and side effects of the cidentamine in the aspect of solid tumor treatment.)

1. A preparation method of squalene-based self-assembled Sida benamine prodrug nanoparticles is characterized by comprising the following steps:

(1) preparation of squalene-based cidaproamine (SQ-CHI) prodrug molecule: dissolving squalene acid (SQ-COOH) in methyl formamide (DMF) to prepare a first SQ-COOH stock solution with the concentration of 100-200 mg/mL, adding 30-60 mg of N-hydroxysuccinimide (NHS) and 50-100 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) into each milliliter of SQ-COOH stock solution, and reacting at room temperature for 30-120 min to obtain a first SQ-COOH reaction solution. Dissolving xidalamine (CHI) in methyl formamide (DMF) to prepare a first CHI stock solution with the concentration of 50-100 mg/mL, mixing the SQ-COOH reaction solution and the CHI stock solution according to the volume ratio of 1-2: 1, reacting for 48-96 h under the condition of room temperature nitrogen protection, and purifying by using a flash column chromatography after the reaction is finished to obtain the squalene xidalamine (SQ-CHI) prodrug molecule; the eluent is dichloromethane: and (5) 85-495: 5 methanol.

(2) Preparing a ligand-modified pegylated squalene molecule: dissolving SQ-COOH in DMF to prepare second SQ-COO with the concentration of 1000-2000 mg/mLAnd (3) adding 30-60 mg of NHS and 50-100 mg of EDCI into each milliliter of SQ-COOH stock solution, and reacting at room temperature for 30-120 min to obtain a second SQ-COOH reaction solution. Another folic acid-polyethylene glycol-amino (FA-PEG-NH) is selected₂) P-methoxybenzamide-polyethylene glycol-amino (AEAA-PEG-NH2) or (arginine-glycine-aspartic acid-polyethylene glycol-amino) RGD-PEG-NH₂Dissolving the mixture in DMF to prepare a second DMF stock solution with the concentration of 50-100 mg/mL, mixing the second SQ-COOH reaction solution and the second DMF stock solution according to the volume ratio of 1-2: 1, and reacting for 48-96 h under the condition of nitrogen protection at room temperature. And (3) after the reaction is finished, centrifuging the mixture at a high speed of 10000-14000 rpm for 30-60 min by using an ultrafiltration centrifugal tube with the molecular cut-off of 3Kda, and removing free SQ-COOH to obtain the ligand modified polyethylene glycol squalene molecule.

(3) Preparing ligand modified squalene cidadamine prodrug nanoparticles. Dissolving the squalene-based cidentamine (SQ-CHI) prodrug molecule prepared in the step 1 in 95-100% ethanol by volume concentration to prepare a third stock solution with the concentration of 3.32-6.64 mg/mL. And (3) dissolving the ligand-modified polyethylene glycol squalene molecule prepared in the step (2) in 95-100% ethanol by volume concentration to prepare a fourth stock solution with the concentration of 0.664-3.32 mg/mL. And mixing the third stock solution and the fourth stock solution according to the volume ratio of 1:1, wherein the mass ratio of the squalene-based cidentamine (SQ-CHI) prodrug molecule to the ligand-modified polyethylene glycol squalene molecule in the obtained mixed solution is 1: 0.1 to 1. Taking the mixed solution as an oil phase, taking the deionized water as a water phase, and setting the volume ratio of the oil phase to the water phase to be 1: 1-20. And dropwise adding the oil phase into the water phase, stirring for 5-10 min, performing reduced pressure evaporation to remove ethanol, and performing ultrasonic treatment on the mixture in an ultrasonic instrument for 30-60 min. Thus obtaining the ligand modified squalene cidalimine prodrug nanoparticles.

2. A squalene-based cidaliamine prodrug self-assembled nanoparticle prepared according to the method of claim 1.

3. The use of the squalene-based cidentamine prodrug self-assembled nanoparticles of claim 2 in the preparation of a medicament for the anti-solid tumor treatment.

4. The application of the squalene based cidentamine prodrug as claimed in claim 3, wherein the preparation is prepared from squalene based cidentamine prodrug self-assembly nanoparticles, and the preparation can improve targeting property of the cidentamine to solid tumors and tumor microenvironment penetration, and enhance curative effect of the cidentamine on malignant tumors such as pancreatic cancer, colorectal cancer, gastric cancer, esophageal cancer, breast cancer and lung cancer.

Technical Field

The invention relates to the technical field of medicines, in particular to squalene and cidalimide prodrug self-assembly nanoparticles, a preparation method and application thereof.

Background

Pancreatic cancer is a malignant tumor of the digestive tract, the mortality rate is 94% of the morbidity rate, and the tumor heterogeneity is high. Due to the lack of early diagnosis markers, the prognosis effect is extremely poor, and the survival rate of 5 years is only 8-9% at present. Pancreatic cancer has a very complex tumor microenvironment, including multiple types of inflammatory cells. In addition, the tumor-associated fibroblasts form a dense extracellular matrix, which can inhibit drug delivery and simultaneously compress the space of blood vessels, so that small-molecule drugs can hardly enter the tumor to kill tumor cells. The treatment of pancreatic cancer is mainly based on traditional surgical resection, however, the proportion of patients meeting surgical conditions is low, and the recurrence rate after resection is high.

The xidalbenamine is a new molecular entity drug independently designed and synthesized by Shenzhen Micronuclear Biotechnology Limited liability company, has novel mechanism, is a first subtype selective Histone Deacetylase (HDAC) inhibitor in the world, belongs to epigenetic regulators and can be used for treating various diseases such as cancers and the like. Clinical studies have shown that xidapamide has a definite therapeutic effect on various lymphomas including peripheral T cell lymphoma, and is the first approved oral drug for the treatment of peripheral T cell lymphoma to produce an effect by inhibiting the biological activity of HDAC, and thus produce alterations in gene expression (i.e., epigenetic alterations) of multiple signaling pathways directed against tumorigenesis. The latest preclinical research results show that the xidapipramine has good anti-tumor effect on solid tumors including breast cancer, pancreatic cancer and the like, and the comprehensive index is better than the contemporary research results of the drugs with the same action mechanism. However, the cydarifamide is a hydrophilic small molecular compound and has the defects of low targeting property, high blood toxicity, low tumor microenvironment infiltration efficiency and the like on solid tumors such as pancreatic cancer and the like. These disadvantages severely limit the utility of xidapamide in solid tumors.

Squalene is a precursor of natural synthetic cholesterol lipid, has strong hydrophobicity and good biological safety, and can be widely applied to the fields of medicines, cosmetics, health products and the like. Many research results show that squalene has certain anti-tumor biological activity, and the action mechanism of squalene is to inhibit the growth of tumor cells and enhance the immunity of an organism, so that the resistance to tumors is enhanced. At present, squalene is introduced into carboxyl through structural modification and is covalently linked with small molecular drugs such as gemcitabine and the like to form a prodrug molecule, the prodrug molecule effectively improves the disadvantage of short half-life of gemcitabine, improves antitumor activity, is easier to self-assemble in water to form nanoparticles, and discloses that the squalene prodrug has good prospect of being applied to tumor targeted therapy. However, no published report on the application of the angular squalene prodrug in improving the microenvironment permeability of pancreatic cancer and responding to drug release through pancreatic cancer cells exists at present.

Disclosure of Invention

The invention aims to provide squalene-based self-assembled nanoparticles of a cidentamine prodrug, a preparation method and application, aiming at the defects of poor targeting property, low permeability of a tumor microenvironment and the like of the cidentamine in treating solid tumors. The invention improves the biocompatibility of the cidentamine by linking the cidentamine and squalene to form a prodrug molecule, and prepares a novel dual-targeting self-assembled nanoparticle by taking small molecule ligands such as folic acid, p-methoxybenzamide or RGD as targets. The self-assembled nanoparticles can obviously improve the osmosis effect of the sidapamide on the pancreatic cancer tumor microenvironment, and have stronger pancreatic cancer tumor targeting and tumor cell growth inhibition effects.

The purpose of the invention is realized by the following technical scheme: a preparation method of squalene-based cidaliamine prodrug self-assembly nanoparticles comprises the following steps:

(1) preparation of squalene-based cidaproamine (SQ-CHI) prodrug molecule: dissolving squalene acid (SQ-COOH) in methyl formamide (DMF) to prepare a first SQ-COOH stock solution with the concentration of 100-200 mg/mL, adding 30-60 mg of N-hydroxysuccinimide (NHS) and 50-100 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) into each milliliter of SQ-COOH stock solution, and reacting at room temperature for 30-120 min to obtain a first SQ-COOH reaction solution. Dissolving xidalamine (CHI) in methyl formamide (DMF) to prepare a first CHI stock solution with the concentration of 50-100 mg/mL, mixing the SQ-COOH reaction solution and the CHI stock solution according to the volume ratio of 1-2: 1, reacting for 48-96 h under the condition of room temperature nitrogen protection, and purifying by using a flash column chromatography after the reaction is finished to obtain the squalene xidalamine (SQ-CHI) prodrug molecule; the eluent is dichloromethane: and (5) 85-495: 5 methanol.

(2) Preparing a ligand-modified pegylated squalene molecule: and dissolving SQ-COOH in DMF to prepare a second SQ-COOH stock solution with the concentration of 1000-2000 mg/mL, adding 30-60 mg of NHS and 50-100 mg of EDCI into each milliliter of SQ-COOH stock solution, and reacting at room temperature for 30-120 min to obtain a second SQ-COOH reaction solution. Another folic acid-polyethylene glycol-amino (FA-PEG-NH) is selected₂) P-methoxybenzamide-polyethylene glycol-amino (AEAA-PEG-NH2) or (arginine-glycine-aspartic acid-polyethylene glycol-amino) RGD-PEG-NH₂Dissolving the mixture in DMF to prepare a second DMF stock solution with the concentration of 50-100 mg/mL, mixing the second SQ-COOH reaction solution and the second DMF stock solution according to the volume ratio of 1-2: 1, and reacting for 48-96 h under the condition of nitrogen protection at room temperature. And (3) after the reaction is finished, centrifuging the mixture at a high speed of 10000-14000 rpm for 30-60 min by using an ultrafiltration centrifugal tube with the molecular cut-off of 3Kda, and removing free SQ-COOH to obtain the ligand modified polyethylene glycol squalene molecule.

(3) Preparing ligand modified squalene cidadamine prodrug nanoparticles. Dissolving the squalene-based cidentamine (SQ-CHI) prodrug molecule prepared in the step 1 in 95-100% ethanol by volume concentration to prepare a third stock solution with the concentration of 3.32-6.64 mg/mL. And (3) dissolving the ligand-modified polyethylene glycol squalene molecule prepared in the step (2) in 95-100% ethanol by volume concentration to prepare a fourth stock solution with the concentration of 0.664-3.32 mg/mL. And mixing the third stock solution and the fourth stock solution according to the volume ratio of 1:1, wherein the mass ratio of the squalene-based cidentamine (SQ-CHI) prodrug molecule to the ligand-modified polyethylene glycol squalene molecule in the obtained mixed solution is 1: 0.1 to 1. Taking the mixed solution as an oil phase, taking the deionized water as a water phase, and setting the volume ratio of the oil phase to the water phase to be 1: 1-20. And dropwise adding the oil phase into the water phase, stirring for 5-10 min, performing reduced pressure evaporation to remove ethanol, and performing ultrasonic treatment on the mixture in an ultrasonic instrument for 30-60 min. Thus obtaining the ligand modified squalene cidalimine prodrug nanoparticles.

The squalene-based cidaliamine prodrug self-assembly nanoparticles prepared by the method.

An application of the squalene-based cidaliamine prodrug self-assembly nanoparticles in preparation of drugs for treating solid tumors. The squalene-based self-assembly nanoparticle of the precursor of the xidalbenamine can be prepared into a preparation, the preparation can improve the targeting property of the xidalbenamine to solid tumors and the tumor microenvironment penetration, and enhance the curative effect of the xidalbenamine on treating malignant tumors such as pancreatic cancer, colorectal cancer, gastric cancer, esophageal cancer, breast cancer, lung cancer and the like.

The invention is further investigated below:

1. determination of drug loading

The squalene-based self-assembled nanoparticles of the precursor of the cidalidamine are centrifuged at a high speed, supernatant is taken, the content of CHI in the supernatant is determined by adopting ultra-performance liquid chromatography (UPLC), and the corresponding concentration can be calculated according to a standard curve of the cidalidamine, so that the drug-loading rate is calculated. The drug loading of the final nanoparticles is 50-80%.

2. In vitro drug delivery

The invention utilizes the high expression trypsin level of pancreatic cancer cells to construct a squalene-based cidentamine prodrug carrying pancreatin response link molecules. The prodrug can release the xidapamide in a pancreatic enzyme environment in a response mode, and the release amount of the xidapamide in an environment without the pancreatic enzyme is low. In addition, squalene is a lipid compound, can improve the lipophilicity of the drug by being linked with the drug, solves the problems of poor permeability of the tumor microenvironment of the xidabenamine molecule and the like, and has wide application prospect.

Putting 2mL of fresh CHI stock solution or CHI-SQ-PEG-FA NPs into a dialysis bag (3KDa), dialyzing with a dialysis medium containing 0.1-0.25% of pancreatin, sampling 1mL after 0.1-72 h, adding an equivalent isothermal release medium, and determining the drug release amount by using a UPLC method. And finally, the cumulative release rate of the nanoparticles for releasing CHI in 72h under the 0.1% pancreatin environment is 60-70%, the cumulative release rate of the nanoparticles for releasing CHI in 72h under the 0.25% pancreatin environment is 70-90%, and the cumulative release rate of the nano-drug for releasing CHI in a medium without trypsin is only 30-50%.

3. Determination of stability of squalene-based cidaliamine prodrug self-assembled nanoparticles in PBS

The squalene based self-assembled nanoparticles of the Xidabenamine prodrug prepared by the invention are mixed with PBS, and are placed in constant temperature oscillation of 37 +/-0.5, and the particle size and the Zeta potential of the squalene based self-assembled nanoparticles of the Xidabenamine prodrug are measured within 0-96 h. The particle diameter change and the Zeta potential change are used as evaluation indexes. The particle size of the final nanoparticle is 170-210 nm after 96 hours, and the Zeta potential is-25 to-10 mV.

4. MTS method for detecting cytotoxicity

The method adopts an MTS method to detect the inhibition effect of the blank nanoparticles on a Sidaparinine sensitive pancreatic cancer cell line (PSN-1) and a Sidaparinine resistant pancreatic cancer cell line (CFPAC-1). And (3) taking the culture solution containing the blank nano-carrier for incubation, measuring the OD value by using an enzyme-labeling instrument, and calculating the cell survival rate. Finally, after PSN-1 and CFPAC-1 cells are treated by blank nanoparticles with different concentrations (3.125-100 mu M), the cell survival rate is 93-97%.

5. Cell potency assay

(1) Single layer cell culture model pharmacodynamic experiment

The method constructs PSN-1 and CFPAC-1 single-layer cell models, respectively adds culture solution containing 0.15625-50 mu M nanoparticles for incubation for 96h, and adopts an enzyme-labeling instrument to measure the OD value. Finally, after the PSN-1 and CFPAC-1 are treated by 0.15625-50 mu M nanoparticles, the survival rates are 90-5% and 95-10% respectively.

(2) Co-culture tumor sphere model drug effect experiment

The invention mixes PSN-1 or CFPAC-1 and HPSC cell suspension in logarithmic phase according to the ratio of 2: 7 proportion mixing, and placing 5000 cells per hole in an ultra-low adsorption cell plate to form an in-vitro co-culture tumor sphere model. After different nano-drugs and tumor balls are incubated for 48-144 h, equal volume of CellTiter-Glo reagent is added, and a multifunctional microplate reader is adopted to detect the luminescence signal value at 562 nm. Finally, in PSN-1/HPSC and CFPAC-1/HPSC three-dimensional tumor sphere models, the cell survival rate of the nanoparticles after 48-144 h treatment is 70-20% and 60-10%

6. Cell uptake assay

(1) Monolayer cell model uptake assay

The invention firstly prepares the coumarin 6 marked self-assembly nano-particle. The monolayer cell model was constructed as in step (1) of step 5. And respectively adding a serum-free culture solution containing nanoparticles for treatment for 24h, and measuring the fluorescence intensity of the coumarin 6 by using a fluorescence microscope. Finally, the uptake of the nanoparticles is a slow process, the fluorescence intensity reaches peak values respectively after 12 hours after administration, and the elimination speed of the fluorescence signal is slow, so that the nanoparticles can be well taken up by tumor cells, and a certain slow release effect is realized.

(2) Quantitative analysis of internalization and internalization

The monolayer cell model was constructed as in step (1) of step 5. After the model is constructed, culture solutions containing CHI-SQ-FA/C6 NPs, CHI-SQ-AEAA/C6 NPs and CHI-SQ-RGD/C6 NPs are respectively added into a cell culture box for incubation for 4-24 h, and the cell uptake condition is quantitatively analyzed by using a flow cytometer. Finally, the fluorescence intensity of each nanoparticle reaches a peak within 12h, and the fluorescence intensity of each nanoparticle in PSN-1 cells within 12h is 400-800 multiplied by 10³The fluorescence intensity of CFPAC-1 cells for 12h is 800-1000 multiplied by 10³The same result as in step (1) was obtained.

(3) Co-culture tumor ball model uptake experiment

The co-culture tumor sphere model construction method is the same as the step (2) in the step 5. After the model construction is completed, the NPs containing CHI-SQ-FA/C6, CHI-SQ-AEAA/C6 and CHI-SQ-RGD/C6 are respectively added, 0.2 mu g/mL DAPI 0.5mL is added after the NPs are cultured in a serum-free culture solution for 12 hours for incubation, and the distribution condition of the drug in the tumor spheres is observed by adopting a laser confocal microscope. Finally, each nanoparticle has a remarkable fluorescence signal.

The invention has the beneficial effects that:

1. the squalene-based cidentamine prodrug molecule constructed by the invention has the characteristic of lipophilicity, so that the biocompatibility of a hydrophilic small molecule drug cidentamine is obviously improved, the permeability of the cidentamine to a pancreatic cancer tumor microenvironment is greatly improved, and the drug can enter tumor cells to play a drug effect.

2. The squalene-based cidentamine prodrug molecule constructed by the invention is linked by a pancreatin response bond, can release a large amount of cidentamine in a trypsin environment, and greatly improves the release of the cidentamine in a pancreatic cancer tumor microenvironment.

3. The preparation method prepares the squalene-based self-assembled prodrug nanoparticles of the cidaproamine by a reverse solvent evaporation method, and is simple and easy to operate.

4. The squalene based cidentamine prodrug self-assembly nanoparticles realize active targeting of pancreatic cancer tumor cells by modifying small molecular ligands on the surfaces, have the EPR effect of the nanoparticles, have dual targeting, can specifically deliver more therapeutic drugs to tumor parts to play the drug effect, and have remarkable therapeutic advantages.

Drawings

FIG. 1 is a scanning electron microscope image of self-assembled nanoparticles prepared in example 7;

FIG. 2 is a graph of the particle size distribution of the self-assembled nanoparticles prepared in example 7;

FIG. 3 is a scanning electron microscope image of self-assembled nanoparticles prepared in example 8;

FIG. 4 is a graph of the particle size distribution of the self-assembled nanoparticles prepared in example 8;

FIG. 5 is a drug release curve diagram of the Xidabenamine-squalene-folic acid nanoparticles in a (0-0.25)% concentration trypsin environment; wherein (a) is a drug release curve chart of the sinediamine-squalene-folic acid nanoparticles in a 0% trypsin environment; (b) is a drug release curve chart of the Xidabenamine-squalene-folic acid nano-particle under the environment of 0.1 percent concentration trypsin; (c) is a drug release curve chart of the Xidabenamine-squalene-folic acid nano-particle under the environment of 0.25 percent concentration trypsin;

FIG. 6 is a drug release curve diagram of the xidabenamine-squalene-p-methoxybenzamide nanoparticles in a (0-0.25)% concentration trypsin environment; wherein (a) is a drug release curve chart of the sinediamine-squalene-p-methoxybenzamide nanoparticles in a 0% trypsin environment; (b) is a drug release curve chart of the xidabenamine-squalene-p-methoxybenzamide nanoparticles in a 0.1% trypsin environment; (c) is a drug release curve chart of the xidabenamine-squalene-p-methoxybenzamide nanoparticles in a 0.25% trypsin environment;

FIG. 7 is a drug release profile of the nanoparticle of sindbylamine-squalene-arginine-glycine-aspartic acid under the condition of (0-0.25)% concentration trypsin; wherein (a) is a drug release curve chart of the sidabenamine-squalene-arginine-glycine-aspartic acid nanoparticles in a 0% trypsin environment; (b) is a drug release curve chart of the Xidabenamine-squalene-arginine-glycine-aspartic acid nanoparticles in the environment of 0.1 percent concentration trypsin; (c) is a drug release curve chart of the Xidabenamine-squalene-arginine-glycine-aspartic acid nanoparticles in the environment of 0.25 percent concentration trypsin;

FIG. 8 is a graph showing the change in particle size and zeta potential at 96h after mixing of nanoparticles with PBS;

FIG. 9 is a graph of a blank nanocarrier-treated PSN-1 cytotoxicity assay;

FIG. 10 is a graph of a blank nanocarrier-treated CFPAC-1 cytotoxicity assay;

FIG. 11 is a histogram of cell proliferation after different concentrations of nanoparticles treated a monolayer of PSN-1 cells for 96 h;

FIG. 12 is a bar graph of cell proliferation after 96h treatment of CFPAC-1 monolayers with varying concentrations of nanoparticles;

FIG. 13 is a graph of tumor cell proliferation after 144h in the PSN-1/HSPC three-position tumor sphere model treated with nanoparticles;

FIG. 14 is a graph of tumor cell proliferation after 144h in a nanoparticle-treated CFPAC-1/HSPC three-position tumor sphere model;

FIG. 15 is a histogram of fluorescence intensity after nanoparticle treatment of PSN-1 monolayers;

FIG. 16 is a bar graph of fluorescence intensity after nanoparticle treatment of CFPAC-1 monolayers.

Detailed Description

In order to facilitate understanding of the present invention, the technical solutions of the present invention will be further described with reference to the following embodiments, but the present invention is not limited thereto.

Example 1

A process for preparing a squalene-based prodrug molecule of xidabenamine (SQ-CHI), comprising the steps of:

weighing 100mg of 1,1 ', 2-trisqualenoic acid (1, 1', 2-Tris-norsqualenoyl acid, SQ-COOH), adding 1ml DMF, dissolving uniformly, gradually adding 30mg of N-hydroxysuccinimide (NHS) and 100mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) into the reaction system, and reacting for 30min under the condition of nitrogen protection at room temperature. Accurately weighing 100mg of cydapamide, dissolving the cydapamide in 1mLDMF, dropwise adding the cydapamide into the reaction system, continuously stirring and reacting for 48 hours under the condition of room temperature nitrogen protection, and purifying by using a flash column chromatography (eluent is dichloromethane: methanol 99: 1; 95: 5) after the reaction is finished. As a result, the SQ-CHI prodrug molecule was successfully prepared.

Example 2

A process for preparing a squalene-based prodrug molecule of xidabenamine (SQ-CHI), comprising the steps of:

weighing 1,1 ', 2-trisqualenoic acid (1, 1', 2-Tris-norsqualenoyl acid, SQ-COOH)200mg, adding 1mLDMF to dissolve uniformly, gradually adding N-hydroxysuccinimide (NHS)60mg and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI)50mg to the reaction system, and reacting for 120min under the condition of nitrogen protection at room temperature. Accurately weighing 100mg of cydapamide, dissolving the cydapamide in 1mLDMF, dropwise adding the cydapamide into the reaction system, continuously stirring and reacting for 96h under the condition of room temperature nitrogen protection, and purifying by using a flash column chromatography (eluent is dichloromethane: methanol: 95: 5; 85: 5) after the reaction is finished. As a result, the SQ-CHI prodrug molecule was successfully prepared.

Example 1 and example 2 both successfully prepared SQ-CHI prodrug molecules, preferably the prodrug molecule prepared in example 1 for subsequent study.

Example 3

The preparation process of the folic acid-polyethylene glycol-squalene (FA-PEG-SQ) micromolecule ligand comprises the following steps:

accurately weighing 100mg of SQ-COOH, adding 100 mu of LDMF, dissolving uniformly, gradually adding 30mg of NHS and 100mg of EDCI into a reaction system, and reacting for 30min under the condition of room temperature and nitrogen protection. Accurately weighing FA-PEG-NH₂100mg of the powder was dissolved in 100. mu.L of DMF, and the resulting solution was added dropwise to the above reaction system, followed by stirring at room temperature under nitrogen for 48 hours. After the reaction, the mixture was centrifuged at 10000rpm for 30min in an ultrafiltration centrifugal tube (molecular cut-off 3 kDa). As a result, the SQ-PEG-FA complex was successfully prepared.

Example 4

The preparation process of the folic acid-polyethylene glycol-squalene (FA-PEG-SQ) micromolecule ligand comprises the following steps:

200mg of SQ-COOH is accurately weighed and added with 100 mu of LDMF to be dissolved uniformly, 60mg of NHS and 50mg of EDCI are gradually added into a reaction system, and the reaction is carried out for 120min under the condition of room temperature nitrogen protection. Accurately weighing FA-PEG-NH₂100mg of the powder was dissolved in 100. mu.L of DMF, and the resulting solution was added dropwise to the above reaction system, followed by stirring at room temperature under nitrogen for 96 hours. After the reaction, the mixture was centrifuged at 14000rpm for 30min in an ultrafiltration tube (molecular cut-off 3 kDa). As a result, the SQ-PEG-FA complex was successfully prepared.

Example 5

The preparation process of the p-methoxybenzamide-polyethylene glycol-squalene (AEAA-PEG-SQ) micromolecule ligand comprises the following steps:

accurately weighing 100mg of SQ-COOH, adding 100 mu of LDMF, dissolving uniformly, gradually adding 30mg of NHS and 100mg of EDCI into a reaction system, and reacting for 30min under the condition of room temperature and nitrogen protection. Accurately weighing AEAA-PEG-NH₂100mg of the powder was dissolved in 100. mu.L of DMF, and the resulting solution was added dropwise to the above reaction system, followed by stirring at room temperature under nitrogen for 48 hours. After the reaction, the mixture was centrifuged at 10000rpm for 30min in an ultrafiltration centrifugal tube (molecular cut-off 3 kDa). As a result, the SQ-PEG-AEAA complex was successfully prepared.

Example 6

The preparation process of arginine-glycine-aspartic acid-polyethylene glycol-squalene (RGD-PEG-SQ) small molecule ligand comprises the following steps:

200mg of SQ-COOH is accurately weighed and added with 100 mu of LDMF to be dissolved uniformly, 60mg of NHS and 50mg of EDCI are gradually added into a reaction system, and the reaction is carried out for 120min under the condition of room temperature nitrogen protection. Accurately weighing RGD-PEG-NH₂100mg of the powder was dissolved in 100. mu.L of DMF, and the resulting solution was added dropwise to the above reaction system, followed by stirring at room temperature under nitrogen for 96 hours. After the reaction, the mixture was centrifuged at 14000rpm for 30min in an ultrafiltration tube (molecular cut-off 3 kDa). As a result, the SQ-PEG-RGD complex was successfully prepared.

Examples 3-6 were all successful in preparing small molecule ligand complexes, and the FA-PEG-SQ small molecule ligand prepared in example 3 was preferred for subsequent nanoparticle preparation, but the invention is not limited thereto.

Example 7

The preparation process of the squalene based self-assembled prodrug of the xidabenamine comprises the following steps: 6.64mg of SQ-CHI prepared in example 1 was weighed out accurately, and 2mL of 95% ethanol was added and mixed well to prepare a 3.32mg/mL SQ-CHI stock solution. 6.64mg of SQ-PEG-FA prepared in example 3 was weighed out accurately, added with 2mL of 95% ethanol and mixed well to prepare 3.32mg/mL SQ-PEG-FA stock solution. And (3) mixing the SQ-CHI stock solution and the SQ-PEG-FA stock solution uniformly in a volume ratio of 1:1, wherein the mass ratio of the SQ-CHI to the SQ-PEG-FA in the mixed solution is 1: 1. the mixed solution of SQ-CHI and SQ-PEG-FA is used as oil phase, and deionized water is used as water phase. Dropwise adding the oil phase into 1mL of deionized water under stirring to make the volume ratio of the oil phase to the water phase 1:20, stirring for 5min, evaporating under reduced pressure to remove ethanol, and ultrasonic treating the mixture in an ultrasonic instrument for 60min to obtain CHI-SQ-FA NPs suspension. Detecting the particle size distribution and zeta potential of the nanoparticles, and observing the appearance of the nanoparticles by using a transmission electron microscope. As a result, the FA-SQ-CHI nanoparticles obtained as shown in FIGS. 1 and 2 have uniform particle size, typical "core-shell" structure, particle size of 173.3 + -1.5 nm, PDI of 0.2 + -0.2, and zeta potential of-13.1 + -0.9 mV.

Example 8

The preparation process of the squalene based self-assembled prodrug of the xidabenamine comprises the following steps: 6.64mg of SQ-CHI prepared in example 1 was weighed out accurately, and 1mL of 100% ethanol was added thereto and mixed well to prepare 6.64mg/mL of SQ-CHI stock solution. 6.64mg of SQ-PEG-AEAA prepared in example 3 was weighed out accurately and mixed with 10mL of 100% ethanol to prepare a 0.664mg/mL SQ-PEG-AEAA stock solution. And (3) mixing the SQ-CHI stock solution and the SQ-PEG-FA stock solution uniformly in a volume ratio of 1:1, wherein the mass ratio of the SQ-CHI to the SQ-PEG-FA in the mixed solution is 1: 0.1. the mixed solution of SQ-CHI and SQ-PEG-FA is used as oil phase, and deionized water is used as water phase. Dropwise adding the oil phase into 1mL of deionized water under stirring to make the volume ratio of the oil phase to the water phase 1:1, stirring for 10min, evaporating under reduced pressure to remove ethanol, and subjecting the mixture to ultrasonic treatment in an ultrasonic instrument for 30min to obtain CHI-SQ-AEAA NPs suspension. Detecting the particle size distribution and zeta potential of the nanoparticles, and observing the appearance of the nanoparticles by using a transmission electron microscope. As a result, the obtained CHI-SQ-AEAA nanoparticles have uniform particle size, typical core-shell structure, particle size of 168.2 +/-0.7 nm, PDI of 0.4 +/-0.1 and zeta potential of-7.2 +/-1.1 mV, as shown in FIGS. 3 and 4.

Example 9: drug loading measurement

Respectively re-dissolving the prepared CHI-SQ-FA NPs, the prepared CHI-SQ-AEAA NPs and the prepared CHI-SQ-RGD NPs, centrifuging at 12000rpm for 30min at high speed, filtering the filtrate with a 0.22 mu m filter membrane to obtain a subsequent filtrate, measuring the content of the CHI in the subsequent filtrate by adopting a UPLC method, freeze-drying to obtain the total mass of the nanoparticles, and calculating the drug loading (DL%) of the nanoparticles according to an equation. Chromatographic conditions are as follows: the chromatographic column is ACQUITYBEH C18 column (2.1X 50mm,1.7 μm); the volume flow is 0.2 mL/min; column temperature 23; the sample injection amount is 5 mu L; the detection time is 10 min; mobile phase 0.1% formic acid solution: acetonitrile (75: 25); the detection wavelength is 258 nm.

The result is calculated according to a CHI standard curve, and the drug loading of the CHI-SQ-FA NPs is 74.1 +/-0.5 percent; the drug loading of CHI-SQ-AEAA NPs is 59.8 +/-0.4%; the drug loading of the CHI-SQ-RGD NPs is 63.2 +/-0.8%.

Example 10: release of Sidapamide under different trypsin environments

2mL of the CHI-SQ-FA NPs, CHI-SQ-AEAA NPs and CHI-SQ-RGD NPs suspensions prepared in example 7 were measured out accurately, and placed in a pre-treated dialysis bag (3KDa), 0.1% of pancreatin was placed in the dialysis bag and in 40mL of release medium, respectively, and shaken at a constant temperature of 37. + -. 0.5 (75rpm) for 0.1, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 30, 36, 48, and 72 hours, 1mL was sampled, and then the same amount of release medium was added, and after all samples were filtered through a 0.22 μm microporous filter, the drug content was measured by UPLC, and the cumulative drug release rate (Q%) of the CHI was calculated based on the CHI standard curve.

Example 11: release of Sidapamide under different trypsin environments

2mL of the CHI-SQ-FA NPs suspension prepared in example 7 was measured accurately, placed in a pre-treated dialysis bag (3KDa), 0.25% of pancreatin was placed in the dialysis bag, respectively placed in 40mL of release medium, 1mL was sampled at a constant temperature of 37. + -. 0.5 with shaking (75rpm), respectively at 0.1, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 30, 36, 48, 72 hours, then 1mL was supplemented with an equal amount of release medium at the same temperature, all samples were filtered through 0.22 μm microporous filter membrane and then assayed for drug content using UPLC, and the CHI cumulative drug release rate (Q%) was calculated from the CHI standard curve.

The release of CHI-SQ-AEAA NPs and CHI-SQ-RGD NPs in different trypsin environments is determined by the same method as that of CHI-SQ-FA.

As shown in FIGS. 5-7, when the release medium does not contain trypsin, the cumulative release rate of each nanoparticle for 72h is only 41.0 +/-2.0% (CHI-SQ-FA NPs); 39.2. + -. 1.7% (CHI-SQ-AEAA NPs); 35.1. + -. 1.3% (CHI-SQ-RGD NPs). When the release medium contains 0.1 percent of trypsin, the accumulative release rate of the nanoparticles for 72 hours reaches 68.2 +/-3.8 percent; 65.2. + -. 0.7% (CHI-SQ-AEAA NPs); 62.1. + -. 2.5% (CHI-SQ-RGD NPs). When the release medium contains 0.25 percent of trypsin, the cumulative drug release rate of the nanoparticles for 72 hours reaches 80.2 +/-4.0 percent and 79.2 +/-3.4 percent (CHI-SQ-AEAA NPs); 85.1 + -0.3% (CHI-SQ-RGD NPs). Trypsin can effectively promote the release of nanoparticles.

Example 12: determination of the stability of CHI-SQ-FA NPs in PBS

CHI-SQ-FA NPs prepared in example 7 were mixed with PBS (pH7.4) in a volume ratio of 1:20 after mixing, the mixture was placed at a constant temperature of 37. + -. 0.5 (100rpm) and the particle size and Zeta potential of the CHI-SQ-FA NPs were measured at 1, 3, 6, 12, 18, 24, 30, 36, 42, 48, 72, 96h, respectively. The change in particle size and Zeta potential was used as an index for stability evaluation.

As shown in FIG. 8, the particle size of CHI-SQ-FA NPs increased slightly after 96h to 205.6. + -. 3.2nm, and the Zeta potential reached-20.8. + -. 0.9 mV.

The stability determination methods of the CHI-SQ-AEAA NPs and the CHI-SQ-RGD NPs are the same as those of the CHI-SQ-FA NPs. The result shows that the particle size of CHI-SQ-AEAA NPs reaches 196.6 +/-3.8 nm after 96 hours, and the Zeta potential reaches-10.3 +/-1.2 mV; after CHI-SQ-AEAA NPs are used for 96 hours, the particle size reaches 188.5 +/-1.5 nm, and the Zeta potential reaches-3.4 +/-0.2 mV. Overall, the nanoparticles have better stability within 96 h.

Example 13: MTS method for detecting cytotoxicity

Taking cells of the Xidaaniline sensitive pancreatic cancer cell line (PSN-1) and the Xidabenamine resistant pancreatic cancer cell line (CFPAC-1) in the logarithmic growth phase to perform cell culture in a ratio of 4-6 multiplied by 10⁴The cell suspension of each/mL was inoculated into a 96-well plate, cultured for 24 hours at 37, and then added with 3.125, 6.25, 12.5, 25.0, 50.0, 100.0. mu. mol. L^-1And (3) continuing culturing SQ-PEG NPs for 96h, absorbing the drug-containing culture solution, adding 100 mu L of MTS-containing working solution into each hole, continuing incubating in the incubator for 4h, taking out and shaking for 14s, measuring the OD value at 490nm by using an enzyme-labeling instrument, and calculating the cell survival rate.

The results are shown in fig. 9-10, after blank nanoparticle treatment, the average survival rates of PSN-1 and CFPAC-1 cells are 95.4 ± 0.7% and 93.7 ± 0.5%, respectively, and the survival rates of PSN-1 and CFPAC-1 cells at various concentrations are greater than 93%, so that the method has good safety.

Example 14: in vitro cell potency assay

1. Single layer cell culture model pharmacodynamic experiment

PSN-1 and CFPAC-1 cells in logarithmic growth phase are taken and digested by 0.25 percent of pancreatin to prepare 4-6 multiplied by 10⁴Cell suspension of one/ml, then seeded at 100. mu.L per well in 96 wellsAnd (4) placing the cell plate in a 37-thermostat incubator for culturing for 2h to allow the cells to adhere to the wall. Adding 100 μ L of the mixture containing 0.15625, 1.5625, 3.125, 6.25, 12.5, 25, and 50 μmol · L into each well^-1And (3) transferring the cell plate into an incubator to continue culturing for 96h, discarding the original culture solution, adding 100 mu L of MTS working solution (MTS stock solution/cell culture solution is 1:5) into each hole, continuing transferring into the incubator to culture for 4h, taking out, shaking on a shaking bed for 15 s, and measuring the absorbance (OD value) of each hole at 490nm by using a microplate reader to calculate the proliferation rate of the tumor cells.

The study on the drug effect of CHI-SQ-AEAA NPs and CHI-SQ-RGD NPs on single-layer PSN-1 and CFPAC-1 cells was the same as that of CHI-SQ-FA NPs.

As shown in FIGS. 11 to 12, the concentration of the drug reached 3.125. mu. mol. L^-1When the concentration reaches 50 mu mol.L, the free CHI and the nanoparticles can both obviously inhibit the growth of tumor cells^-1In the meantime, the survival rate of the PSN-1 cells of the CHI-SQ-FA NPs group is only 10.1 +/-3.7%, the survival rate of the CFPAC-1 cells is 21.1 +/-0.9%, the survival rate of the PSN-1 cells of the CHI-SQ-PEG NPs group is 15.7 +/-4.0%, and the survival rate of the CFPAC-1 cells is 25.3 +/-1.8%. The results of CHI-SQ-AEAA NPs and CHI-SQ-RGD NPs were consistent with those of CHI-SQ-FA NPs. Wherein, the PSN-1 cell survival rate of the CHI-SQ-AEAA NPs group is only 18.7 +/-4.2 percent, and the CFPAC-1 cell survival rate is 28.7% +/-1.7 percent; the survival rate of the PSN-1 cells of the CHI-SQ-RGD NPs group is 16.21% + -3.2%, and the survival rate of the CFPAC-1 cells is 14.7% + -1.5%.

2. Co-culture tumor sphere model drug effect experiment

PSN-1 and HPSC cell suspensions at log phase were mixed as 2: 7 proportion mixing, and placing 5000 cells per hole in an ultra-low adsorption cell plate to form an in-vitro co-culture tumor sphere model. Incubating the culture solution containing CHI-SQ-FA NPs and the tumor ball for 48, 96 and 144 hours, adding an equal volume of CellTiter-Glo reagent, placing the cell culture plate on an orbital shaker for shaking for 15min, placing the cell culture plate at room temperature in a dark place for 30min, absorbing cell lysate into a black 96 plate hole, and detecting a luminescence signal value (562nm) by using a multifunctional microplate reader. The CFPAC-1/HPSC co-culture cell tumor sphere treatment method is the same as the PSN-1/HPSC co-culture cell tumor sphere.

The results are shown in FIGS. 13-14, and the cell survival rate of the CFPAC-1/HPSC co-cultured cell tumor cell model after being treated by CHI-SQ-FA NPs for 144h is 39.8 +/-9.0%; after the PSN-1/HPSC co-culture cell tumor sphere model is treated by CHI-SQ-FA NPs for 144h, the cell survival rate is 28.6 +/-4.6%. After the CFPAC-1/HPSC co-culture cell tumor cell model is treated by CHI-SQ-PEG NPs for 144h, the cell survival rate is 45.8 +/-7.1%; after the PSN-1/HPSC co-culture cell tumor sphere model is treated by CHI-SQ-PEG NPs for 144h, the cell survival rate is 53.7 +/-3.2%. The efficacy results of the CHI-SQ-AEAA NPs and the CHI-SQ-RGD NPs on the co-culture tumor sphere model are the same as that of the CHI-SQ-AEAA NPs, namely the cell survival rate is 23.7 +/-2.3% after the CHI-SQ-AEAA NPs process the CFPAC-1/HPSC co-culture cell tumor sphere model for 144h and the cell survival rate is 50.3 +/-5.1% after the CHI-SQ-AEAA NPs process the CFPAC-1/HPSC co-culture cell tumor sphere model for 144 h; the cell survival rate of the CHI-SQ-RGD NPs after processing the CFPAC-1/HPSC co-cultured cell tumor sphere model for 144h is 35.1 +/-1.3%, and the cell survival rate of the CHI-SQ-RGD NPs after processing the PSN-1/HPSC co-cultured cell tumor sphere model for 144h is 44.3 +/-3.1%.

Example 15: in vitro cell uptake assay

1. Monolayer cell culture model uptake assay

1mg of coumarin 6 is accurately weighed and uniformly dissolved in absolute ethyl alcohol to form 200 mug/mL coumarin 6 stock solution. 10 mu L of coumarin 6 and CHI-SQ-FA prepared in example 7 are uniformly mixed at a ratio of 1:0.7, 0.1mL of mixed solution is dropwise added into 1mL of deionized water under stirring by a syringe, the mixture is stirred for 5min, ethanol is removed by evaporation under reduced pressure, and the mixture is placed in an ultrasonic instrument and subjected to ultrasonic treatment for 30 min. And then, transferring the coated coumarin 6 nanoparticles into a 3KDa ultrafiltration tube, and centrifuging at 12000rpm for 20min at high speed to remove the uncoated coumarin 6, thus obtaining the CHI-SQ-FA/C6 NPs.

PSN-1 or CFPAC-1 cells in logarithmic growth phase are taken and treated with 10⁶one/mL of the cells was inoculated in a 6-well plate, cultured routinely for 24 hours to adhere, added with a culture medium of CHI-SQ-FA/C6 NPs, CHI-SQ-AEAA/C6 NPs or CHI-SQ-RGD/C6 NPs to the cell culture chamber and incubated for 4 hours, 12 hours and 24 hours, then the drug-containing medium was discarded, the cell uptake process was terminated by adding ice-cold PBS (pH7.4), and washed 3 times with 1mL of PBS (pH7.4), then the cells were digested from the 6-well plate with 0.05% trypsin, and digested with fresh fine cellsStopping digestion of the cell culture solution, centrifuging, removing supernatant, collecting cells, and counting the cells to make the number of the cells be 3-5 × 10⁵Cells were washed 3 times in PBS (pH7.4) and cell uptake was quantified using flow cytometry.

As shown in FIGS. 15 to 16, in PSN-1 and CFPAC-1 cells, the fluorescence intensities of CHI-SQ-FA NPs, CHI-SQ-AEAA NPs and CHI-SQ-RGD NPs all reached a peak at 12 hours, and the fluorescence intensities of the CHI-SQ-FA NPs were 549.6 + -17.5X 10³(PSN-1) and 620.7. + -. 30.0X 10³(CFPAC-1), CHI-SQ-AEAA NPs 613.3 + -12.7X 10³(PSN-1) and 810.7. + -. 34.1X 10³(CFPAC-1), CHI-SQ-RGD NPs 533.5 + -13.5X 10³(PSN-1) and 792.5. + -. 21.1X 10³(CFPAC-1). Compared with free coumarin 6, the tumor cells can slowly and massively take the self-assembled nanoparticles prepared by the invention.

2. Co-culture tumor ball model uptake experiment

The CFPAC-1/HPSC and PSN-1/HPSC three-dimensional tumor sphere model culture method was the same as in example 14, step (3). And (3) after 24h of culture, removing the culture solution by aspiration, washing the culture solution by PBS for 3 times, adding the CHI-SQ-FA/C6 NPs prepared in the step (1) to the culture solution in a serum-free culture solution for culture, removing the culture solution by aspiration after 12h, and rinsing the precooled PBS for 3 times. Fixing with 4% paraformaldehyde for 15min, discarding, rinsing with PBS 3 times, adding 0.5mL DAPI (0.2 μ g/mL), incubating for 12h, discarding, rinsing with PBS 3 times, transferring the treated tumor spheres to laser confocal four-fold small dish, and observing the distribution of the drug in the tumor spheres by using a laser confocal microscope.

The results show that after the CHI-PEG-FA NPs prepared by the invention are treated for 12 hours, an obvious fluorescence signal is detected in a tumor sphere model, and the fluorescence signal is stronger than that of free coumarin 6 and the nanoparticles of the Sidapamide-squalene-polyethylene glycol. Has good permeability in a tumor sphere model.

The embodiment shows that the invention creatively links squalene and xidabenamine through a pancreatin response bond through chemical modification to form amphipathic squalene-xidabenamine prodrug molecules, the prodrug molecules can obviously improve the permeability of the xidabenamine to a pancreatic cancer tumor microenvironment, and the xidabenamine prodrug molecules are easy to break under the high-expression trypsin environment of pancreatic cancer cells so as to be released, so that the pancreatic cancer targeted drug release is realized. The squalene-based cidalimide prodrug can form nanoparticles through self-assembly in an aqueous solution, so that the retention time of prodrug molecules in vivo and the passive targeting property of a pancreatic cancer microenvironment are remarkably improved. Meanwhile, the invention takes folic acid, p-methoxybenzamide or RGD and other small molecular ligands as target heads, and can further obviously improve the active targeting effect of the xidapipramine on pancreatic cancer cells. In a word, the small-molecule ligand target-modified squalene cidaliamine prodrug self-assembly nanoparticles are innovatively prepared, and have the characteristics of enhanced permeability of a pancreatic cancer microenvironment, pancreatic cancer cell response drug release property, small-molecule ligand-mediated pancreatic cancer cell active targeting property, tumor tissue passive targeting property of the self-assembly nanoparticles, large drug loading capacity, good stability and simple and easy operation method. The invention provides a new strategy for the anti-pancreatic cancer treatment of the Sida benamine from the preparation angle.