CN109745326B

CN109745326B - Pharmaceutical composition containing gefitinib and histone deacetylase inhibitor, liposome preparation of pharmaceutical composition and pharmaceutical application of liposome preparation

Info

Publication number: CN109745326B
Application number: CN201711063958.5A
Authority: CN
Inventors: 黄永焯; 彭会歌
Original assignee: Shanghai Institute of Materia Medica of CAS
Current assignee: Shanghai Institute of Materia Medica of CAS
Priority date: 2017-11-02
Filing date: 2017-11-02
Publication date: 2021-03-12
Anticipated expiration: 2037-11-02
Also published as: CN109745326A

Abstract

The present invention relates to a pharmaceutical composition comprising gefitinib and a histone deacetylase inhibitor, liposomal formulations thereof and their use in the preparation of a medicament for the treatment of EGFR^T790MThe application of the medicine for treating the non-small cell lung cancer with mutant drug resistance, wherein the histone deacetylase inhibitor is preferably vorinostat and TMP 195. Specifically, the invention also relates to a double-targeting co-drug-loaded liposome, which is a liposome with the surface modified by herceptin and mannose and comprises the pharmaceutical composition. The double-targeting co-drug-loaded liposome can be used for regulating macrophage polarization and treating the tumor cells with EGFR^T790MMutant non-small cell lung cancer.

Description

Pharmaceutical composition containing gefitinib and histone deacetylase inhibitor, liposome preparation of pharmaceutical composition and pharmaceutical application of liposome preparation

Technical Field

The invention belongs to the field of biomedicine, and more particularly relates to a pharmaceutical composition containing gefitinib and a histone deacetylase inhibitor, a liposome preparation thereof and application thereof in preparing medicines for treating EGFR (epidermal growth factor receptor)^T790MThe application of the mutant drug-resistant non-small cell lung cancer medicine.

Background

Lung cancer is the highest incidence and mortality class of cancer in china and even worldwide. With the aggravation of industrialization and urbanization in China, air pollution is serious, the smoking rate is high, and the harm of lung cancer is further aggravated. Among all malignant tumors, lung cancer is a relatively deep research on pathogenesis, and the main cause of the onset of non-small cell lung cancer (NSCLC) is the deletion of the 19 th exon of Epidermal Growth Factor Receptor (EGFR) or the mutation of the 858 th amino acid from leucine (L) to arginine (R) on the 21 st exon, which results in the abnormal activation of EGFR and canceration of cells. Aiming at the molecular characteristics of the mutated EGFR, a first generation molecular targeted Tyrosine Kinase Inhibitor (TKI) -gefitinib is developed, and the gefitinib is combined with the phosphorylation site of the EGFR to inhibit the phosphorylation activation of the EGFR so as to inhibit the tumor growth.

The appearance of the molecular targeted inhibitor greatly improves the treatment efficiency of tumors, greatly reduces toxic and side effects due to the specific molecular targeting property, and improves the median survival time of Chinese patients with advanced lung adenocarcinoma from 14.1 months to 33.5 months since gefitinib is marketed. However, 50% of patients will undergo threonine (T) to methionine (M) mutation at amino acid 790 in exon 20 after treatment, resulting in a significant increase in EGFR binding to ATP, resistance to gefitinib, and failure of molecular targeted therapy. Although the second generation of TKI afatinib, which is an irreversible inhibitor, was later developed, it was not effective in controlling EGFR due to its narrow therapeutic window^T790MResistance due to mutation, and limited clinical benefit. The third generation TKI Oxitinib in 3 months in 2017 is approved to be listed in China and aims at EGFR^T790MThe drug-resistant patients have remarkable clinical benefit, however, the EGFR can generate C797S mutation after the patients receive the oxitinib treatment, so that the oxitinib is resistant to the drug. Throughout the history of development of EGFR-TKI, the development of one generation and the next generation of inhibitors consumes a lot of time and manpower and material investment, however, the new drug-resistant mutation sites of EGFR are unpredictable.

Therefore, it appears to be crucial to go on to reverse the resistance of molecular targeted therapies from other aspects. Extensive combined medication experiments are also developed clinically, and can be roughly divided into: the molecular targeting drug is combined with a chemotherapeutic drug, an anti-angiogenesis drug, an immunotherapy drug and the like, so that the tumor microenvironment plays a role in the current tumor therapy. The tumor microenvironment is an important component of tumorigenesis, tumor progression, and tumor metastasis, and is the "soil" for tumor growth. The tumor inflammation microenvironment is an important component of the tumor microenvironment, and tumor-associated macrophages (TAMs) account for more than 50% of all inflammatory cells in tumor tissues and play an important role in the tumor growth process. Reference T_HT of cells_H1-T_HPolarization of type 2, giantThe phenotype of phagocytic cells can be divided into two extreme types, namely, classically activated anti-inflammatory M1 type and selectively activated pro-inflammatory M2 type, M1 type shows stronger antigen presenting capacity, and has cytotoxicity and anti-tumor capacity, and M2 type macrophage is mainly involved in immune regulation and tissue repair, and promotes tumor growth and immune escape.

Under the induction of tumor cells, TAM is mainly polarized in M2 type, and clinical experiments show that the marker molecules CD68 and CD163 of M2 type macrophages in all types of non-small cell lung cancer tissues are remarkably upregulated, which indicates that the main phenotype of TAM in the non-small cell lung cancer tissues is M2 type, while the number of M1 type macrophages in the non-small cell lung cancer tissues is positively correlated with the survival period of patients and the treatment efficiency of drugs. The type of macrophage polarization is altered by external stimuli, e.g., histone deacetylase inhibitors can modulate the conversion of TAM to a type that is anti-neoplastic and pro-inflammatory, inhibiting the growth and metastasis of breast cancer (Guerriro J L, Sotayo A, Ponichtera H E, et al. class IIa HDAC inhibition processes breast cancer and tumors through anti-tumor macroantigens [ J ] Nature,2017,543(7645): 428-432). Therefore, a new strategy can be provided for the drug resistance of molecular targeted therapy by regulating the polarization of TAM.

As the hyperproliferation of tumor cells needs a large amount of energy, compared with normal tissues, the tumor tissues have rich blood vessels, loose arrangement of vascular endothelium, poor structural integrity and lymphatic return loss, and are beneficial to more material exchange. This feature also results in the selective high permeability and retention of macromolecular and lipid particles, a phenomenon known as high permeability and retention Effect (EPR) of solid tumor tissues, and according to reports (He C, HuY, Yin L, et al. effects of particulate size and surface charge on particulate and biological nanoparticles, 2010,31(13):3657-3666), nanoparticles with a particle size around 150nm have better passive targeting due to the EPR effect. In addition, in order to improve the accumulation of nanoparticles in tumor cells and improve the efficiency of nanoparticles penetrating cell membranes, cell penetrating peptides and specific targeted modification mediation are also widely studied. Moreover, if the two drugs are loaded into the same nanoparticle in an accurate compatibility ratio, the two drugs can be synchronously delivered to a target site, enter tumor cells in a compatible dosage, maximize the synergistic effect and achieve the similar effect in vitro experiments (Goldman A, Kulkarnia, Kohand M, et al.

However, the combined use of molecular targeted drugs has less research on the modification of dosage forms in pharmacy, and no research on the modification of the monoclonal antibody herceptin as a targeted ligand on the surface of a drug delivery system by using HER2 as a target has been reported.

Disclosure of Invention

Technical purpose

The technical purpose of the invention is to provide a method for overcoming the defect caused by EGFR^T790MA non-small cell lung cancer molecule target treatment drug-resistant medicinal preparation caused by mutation and a preparation method thereof.

Technical scheme

In one aspect, the invention provides a pharmaceutical composition comprising gefitinib and a histone deacetylase inhibitor.

In one embodiment, the histone deacetylase inhibitor may be selected from vorinostat, TMP195, preferably, the histone deacetylase inhibitor is vorinostat.

In one embodiment, the mass ratio of gefitinib to histone deacetylase inhibitor (preferably vorinostat) in the pharmaceutical composition is 1: 0.12-1.

In one aspect, the present invention provides a liposome comprising the above pharmaceutical composition.

In one aspect, the present invention provides a surface-modified liposome, which is a liposome whose surface is modified by one or more selected from polyethylene glycol, mannose, polypeptide and protein, and which comprises the above pharmaceutical composition.

In one aspect, the present invention provides a dual targeting co-drug loaded liposome, which is a liposome having a surface modified with herceptin and mannose and comprising the above pharmaceutical composition.

In one embodiment, the dual-targeted co-drug-loaded liposomes have a particle size of 186.9nm, a polydispersity index of 0.11nm, and an encapsulation efficiency for gefitinib of greater than 80% and for a histone deacetylase inhibitor (preferably vorinostat) of greater than 80%. For example, in the present application, the encapsulation efficiency for gefitinib can reach 88.6%, and the encapsulation efficiency for vorinostat can reach 83.8%.

On the other hand, the invention provides a preparation method of the double-targeting co-drug-loaded liposome, which comprises the following steps:

(a) reacting mannose isothiocyanate with PEG having an amino group to prepare mannose-modified PEG;

(b) adding a pharmaceutical composition comprising gefitinib and a histone deacetylase inhibitor, preferably vorinostat, to lecithin, cholesterol, the mannose-modified PEG prepared in step (a), and PEG-NHS ester to prepare a liposome encapsulating the pharmaceutical composition;

(c) adding herceptin into the liposome which is prepared in the step (b) and wraps the pharmaceutical composition to carry out a light-shielding reaction;

(d) and centrifuging to obtain the double-targeting co-drug-loaded liposome.

In one embodiment, the PEG with amino group in step (a) and the PEG-NHS ester in step (b) may have a molecular weight in the range of 2000-5000 Da, in particular the PEG with amino group is DSPE-PEG₂₀₀₀-NH₂The PEG-NHS ester is DSPE-PEG₂₀₀₀-NHS。

In one embodiment, the step (b) may be performed by a thin film dispersion hydration method, an ethanol injection method, a reverse phase evaporation method, an ammonium sulfate gradient method, or the like, and preferably, the step (b) may be performed by a thin film dispersion hydration method.

In one aspect, the invention provides a use of the above pharmaceutical composition or the above liposome containing the pharmaceutical composition or the above dual-targeting co-drug-loaded liposome in the preparation of a medicament for regulating macrophage polarization or serving as a macrophage immunomodulator.

In another aspect, the present invention provides an article as described aboveApplication of pharmaceutical composition or liposome containing pharmaceutical composition or double-targeting co-drug-loaded liposome in preparation of medicine for treating non-small cell lung cancer, especially EGFR^T790MThe application of the mutant drug-resistant non-small cell lung cancer medicine.

Advantageous effects

The invention has the following beneficial effects:

(1) the histone deacetylase inhibitor (preferably vorinostat) is combined with gefitinib, and can down-regulate the expression of a marker molecule of M2 type macrophages under the condition that the macrophages are cultured alone or are co-cultured with tumor cells, and up-regulate the expression of the marker molecule of the M1 type macrophages, namely regulate the differentiation of the macrophages to M1 type.

(2) In the invention, the herceptin is modified on the surface of the liposome and is used for targeting HER2 positive non-small cell lung cancer, and mannose modification is carried out on the liposome and is targeted to M2 type macrophages. The inventor researches and discovers that in vitro cell lines and tumor-bearing mice, the distribution of the liposome in tumor cells and M2 type macrophages and the accumulation of the liposome at the tumor site can be obviously improved by modifying herceptin and mannose on the surface of the liposome, the macrophage is reprogrammed, and the EGFR (epidermal growth factor receptor) is resisted^T790MThe mutation results in a molecule targeted therapy resistant to the drug. The specific mechanism is that the liposome (hereinafter also referred to as 'double-targeting co-drug-loaded liposome') prepared by the invention regulates M2 type macrophages to differentiate into M1 type macrophages, further increases the levels of active oxygen and superoxide anions in tumor cells, breaks the balance between NOX3 and MsrA, leads to the oxidative metabolism of methionine on 790 th position of 20 th exon of EGFR, namely EGFR occurs^T790MDegrading and restoring the sensitivity of the non-small cell lung cancer containing the T790M drug-resistant mutation to gefitinib, thereby overcoming EGFR^T790MThe mutation results in a molecule targeted therapy resistant to the drug.

(3) The medicine encapsulated by the liposome has pH dependency in vitro release, and is suitable for delivering antitumor medicines.

Drawings

Fig. 1 is a schematic diagram of molecular targeted therapy drug resistance reversal by regulating polarization of tumor-associated macrophages to M1 type macrophages according to the double-targeted co-drug-loaded liposome of the invention.

FIG. 2 shows a polypeptide with EGFR^T790MThe mutant non-small cell lung cancer cell line H1975 and the expression of HER2 in the subcutaneous transplanted tumor tissue of a nude mouse and the expression of mannose receptor after the differentiation of bone marrow-derived macrophages.

Fig. 3a shows the particle size distribution of the dual targeting co-drug loaded liposomes prepared according to preparation example 1 of the present invention.

Fig. 3b shows the morphology of the dual targeting co-loaded liposomes prepared according to preparation example 1 of the present invention under transmission electron microscope.

Fig. 4 shows the stability of the dual targeting co-drug loaded liposome prepared according to preparation example 1 of the present invention when stored at 4 ℃.

Fig. 5 shows the release of gefitinib drug in liposomes under different pH conditions from the dual-targeted co-drug loaded liposomes prepared according to preparation example 1 of the present invention.

FIG. 6 shows that the double targeted co-loaded liposomes and unmodified co-loaded liposomes prepared according to Experimental example 1 of the present invention are in HER2 and EGFR^T790MPositive non-small cell lung cancer cell line H1975 and uptake on mannose receptor positive M2-type macrophages.

FIG. 7 shows that the histone deacetylase inhibitors vorinostat and gefitinib are used alone or in combination in experimental example 2 of the present invention, and the dual-targeting co-drug-loaded liposome comprising vorinostat and gefitinib are used in combination with HER2 and EGFR^T790MCytotoxicity of positive non-small cell lung cancer cell line H1975.

FIG. 8 shows that the histone deacetylase IIa inhibitors TMP195 and gefitinib, used alone or in combination in experimental example 2 of the present invention, act on HER2 and EGFR^T790MCytotoxicity of positive non-small cell lung cancer cell line H1975.

FIG. 9 shows the morphology of macrophages induced in preparative example 3 of the present invention.

FIG. 10 shows the characterization of macrophage polarization pattern by qRT-PCR when macrophages were cultured alone in Experimental example 3 of the present invention.

FIG. 11 shows the characterization of macrophage polarization type by qRT-PCR when macrophages were co-cultured with tumor cells H1975 in Experimental example 3 of the present invention.

FIG. 12 shows the characterization of macrophage polarization pattern by Western blot in experimental example 3 of the present invention.

FIG. 13 shows the characterization of macrophage polarization pattern by ELISA in co-culture with tumor cells H1975 in Experimental example 3 of the present invention.

FIG. 14 shows the cytotoxicity of Gefitinib to H1975 cells in culture medium with macrophage of M1 type or M2 type and in normal culture medium in Experimental example 4 of the present invention.

FIG. 15 shows apoptosis of H1975 after treatment with H1975 drug alone, in coculture with M1-type or M2-type macrophages, in experimental example 4 of the present invention.

FIG. 16 shows the activation of the intracellular signaling pathway protein of H1975 in the case of tumor cells H1975 cultured alone or co-cultured with M2-type macrophages in Experimental example 4 of the present invention after the treatment of simultaneous administration of tumor cells and macrophages.

FIG. 17 shows the intracellular reactive oxygen species levels of H1975 cells treated with tumor cells H1975 alone or together with macrophages of M2 type under the condition of co-culture of the tumor cells H1975 in Experimental example 5 of the present invention.

FIG. 18 shows superoxide anion (O) in H1975 cells treated by co-administration of tumor cells and macrophages under the conditions of culturing the tumor cells H1975 alone or co-culturing the tumor cells with M2-type macrophages in Experimental example 5 of the present invention₂ ^-) And (4) horizontal.

FIG. 19 shows EGFR and EGFR in H1975 cells treated by co-administration of tumor cells and macrophages under the conditions of culturing the tumor cells H1975 alone or co-culturing the tumor cells with M2-type macrophages in Experimental example 5 of the present invention^T790MThe level of expression.

FIG. 20 shows the expression levels of anti-apoptotic proteins and NOX3 and MsrA proteins in H1975 cells treated by co-administration of tumor cells and macrophages under the conditions of culturing the tumor cells H1975 alone or co-culturing the tumor cells with M2-type macrophages in Experimental example 5 of the present invention.

FIG. 21 shows apoptosis of H1975 cells in experimental example 5 of the present invention after treatment of tumor cells H1975 with co-administration of macrophages under the condition of culturing the tumor cells H1975 alone or co-culturing the tumor cells with M2-type macrophages.

FIG. 22 shows that the double-targeting co-drug-loaded liposome and the unmodified co-drug-loaded liposome in HER2 and EGFR in Experimental example 6 of the present invention^T790MThe time-dependent distribution of the drugs on the nude mouse subcutaneous transplantation tumor model of the positive non-small cell lung cancer cell line H1975 and the distribution of the drugs in each organ at the experimental end point.

FIG. 23 shows that the double-targeting co-drug-loaded liposome and the unmodified co-drug-loaded liposome in HER2 and EGFR in Experimental example 6 of the present invention^T790MCo-localization of liposomes with the receptors HER2 and mannose receptor in tumor tissues of the nude mouse subcutaneous transplantation tumor model of the positive non-small cell lung cancer cell line H1975.

FIG. 24 shows the change in tumor volume during the administration of gefitinib-resistant non-small cell lung cancer mouse graft tumor model in Experimental example 7 of the present invention.

FIG. 25 shows the double targeting co-drug loaded liposome pair HER2 and EGFR in Experimental example 7 of the present invention^T790MTumor suppression efficiency and tumor redistribution in nude mouse subcutaneous transplantation tumor model of positive non-small cell lung cancer cell line H1975.

FIG. 26 shows the double targeting co-drug loaded liposome pair HER2 and EGFR in Experimental example 7 of the present invention^T790MPhotographs of tumors in each treatment group at the endpoint of the nude mouse subcutaneous transplantation tumor model treatment of the positive non-small cell lung cancer cell line H1975.

FIG. 27 shows macrophage distribution and polarization patterns in tumor tissues after administration of Gefitinib-resistant non-small cell lung cancer mouse graft tumor model in Experimental example 7 of the present invention.

FIG. 28 is a graph showing the reactive oxygen species levels in tumor tissues after 4 hours of administration to a Gefitinib-resistant non-small cell lung cancer mouse graft tumor model in Experimental example 7 of the present invention.

FIG. 29 is a graph showing the protein expression levels in tumor tissues after administration of a Gefitinib-resistant non-small cell lung cancer mouse graft tumor model in Experimental example 7 of the present invention.

FIG. 30 shows the double-targeted co-drug-loaded liposome pair HER2 and EGFR in Experimental example 7 of the present invention^T790MApoptosis of cells in tumor tissues of each treatment group at the end of treatment of the nude mouse subcutaneous transplantation tumor model of the positive non-small cell lung cancer cell line H1975.

Detailed Description

The invention will now be further illustrated by the following examples, which are set forth in the following description by way of illustration only. These examples are not meant to impose any limitation on the invention. It will be apparent to those skilled in the art that various changes and modifications can be made in the present invention within the scope and spirit of the invention. It is to be understood that the present invention is intended to cover various changes and modifications encompassed in the appended claims.

Reagent and medicine

Yolk lecithin PC-98T, DSPE-PEG₂₀₀₀、DSPE-PEG₂₀₀₀-NHS purchased from oshmaro pharmaceutical science ltd; gefitinib, vorinostat, coumarin-6 were purchased from gangrenum biotechnology limited; herceptin was purchased from roche; mannose isothiocyanate was purchased from Kaisenlai medicine science and technology, Inc., Beijing; DIR and DID were purchased from Shanghai Byza Biotech, Inc.; chromatographic grade methanol and acetonitrile were purchased from carbofuran technologies ltd; primary anti-EGFR, phospho-EGFR (Tyr1068), Erk1/2, phospho-Erk1/2(Thr202/Tyr204), Akt, phospho-Akt (Ser473), HER2, HDAC2, TGF- β were purchased from CST, internal reference primary anti-GAPDH, β -Actin were purchased from Sigma-Aldrich, primary anti-CD 206(MR), NOX3, methionine sulfoxide reductase a (methionine sulfoxide reductase a, MsrA) were purchased from Abcam, HRP-coupled anti-mouse and goat-rabbit secondary antibodies were purchased from shanghai bi yutian biotechnology limited; the total RNA extraction reagent Trizol is purchased from Tiangen Biochemical technology (Beijing) limited, and the genome DNA removal reverse transcription kit and SYBR Green Supermix are purchased from Bio-Rad; the TNF-alpha ELISA kit is purchased from David Biotech Co., Ltd; active oxygen, O₂ ^-The detection kit is purchased from Shanghai BinYuntian biotechnology limited; other reagents used were from the national pharmaceutical group (Shanghai) chemical reagents company. The human NSCLC cell line H1975 was purchased from cell banks of Shanghai Biopsis, Chinese academy of sciences. RPMI 1640 cell culture medium dry powder, fetal bovine serum were purchased from Gibco. Trypsin and diabody

(penicilin-streptomycin) was purchased from Shanghai Binyan Biotech limited. Co-culture 12-well cell culture plates and Transwell chambers were purchased from Corning.

Preparation of example 1

Preparation of double-targeting co-drug-loaded liposome

(a) Weighing appropriate amount of mannose isothiocyanate, dissolving with dichloromethane, adding appropriate amount of triethylamine to adjust pH to alkalinity, and mixing with mannose isothiocyanate/DSPE-PEG₂₀₀₀-NH₂Adding appropriate amount of DSPE-PEG at a molecular ratio of 3/1₂₀₀₀-NH₂Reacting at room temperature for 4 hours, removing organic solvent by rotary evaporation, adding appropriate amount of water, sufficiently shaking, centrifuging at 15000r for 5 minutes, taking supernatant, and lyophilizing to obtain mannose-modified DSPE-PEG₂₀₀₀It is called DSPE-PEG₂₀₀₀-Man。

(b) Weighing yolk lecithin (PC 98T), cholesterol, and DSPE-PEG₂₀₀₀、DSPE-PEG₂₀₀₀Man and DSPE-PEG₂₀₀₀NHS was dissolved in chloroform and added to the round bottom flask in a molecular ratio of 10/2/1/0.1/0.1. Weighing gefitinib and vorinostat, dissolving the gefitinib and vorinostat in methanol, adding the dissolved gefitinib and vorinostat into a round-bottom flask according to the mass ratio of 30/1/0.12, and uniformly mixing. The round bottom flask was placed on a rotary evaporator and the organic solvent was removed by vacuum rotary evaporation and a thin film was visible on the bottom of the round bottom flask.

(c) Weighing a proper amount of anhydrous glucose, and preparing a hydration solution with the mass volume ratio of 5% by using ultrapure water. Adding a proper amount of hydration liquid into a round-bottom flask, and carrying out water bath rotary evaporation for hydration for 10min to form a milky transparent solution. Incubate at 37 ℃ for 30 min.

(d) The prepared liposomes were passed through 400nm and 200nm polycarbonate membranes using an Avanti's liposome Mini extruder, each extruded at 20 nm.

(e) Adding herceptin into the liposome prepared in (d), and shaking-reacting for 8 hours at room temperature in a dark place.

(f) Centrifuging at 15000rpm/min for 3min to remove free drug, and storing the prepared target liposome at 4 deg.C.

Characterization of Dual-targeting Co-loaded liposomes

(1) Characterization of particle size morphology

The particle size distribution of the prepared double-target co-drug-loaded liposome is shown in figure 3a by measuring the particle size of the prepared double-target co-drug-loaded liposome by using a dynamic light scattering method, and the average particle size is about 186 nm. The liposomes prepared by observation under transmission electron microscope are in the form of uniform spheroidal particles as shown in FIG. 3 b.

(2) Stability of double-targeting co-drug-loaded liposomes

The double-targeting co-drug-loaded liposome prepared in preparation example 1 was stored at 4 ℃, and the particle size distribution of the liposome was examined within one week. The experimental result is shown in fig. 4, the particle size of the liposome is not obviously changed within one week, which indicates that the double-targeting co-drug-loaded liposome has good stability.

(3) Encapsulation efficiency

The invention adopts a high performance liquid chromatograph to carry out qualitative and quantitative analysis on the drug entrapped in the prepared liposome. The liquid phase method used is as follows:

the HPLC is obtained from Agilent technologies (China) and has an instrument model of 1200, and the chromatographic column is Zorbax Elipse XDB C₁₈Column (5 μm, 4.6X150mm) with column temperature set at 40 ℃ using an ultraviolet detector.

Gefitinib: phase a is water containing 0.1% triethylamine, pH adjusted to 2.8 with phosphoric acid; and the phase B is pure acetonitrile. The mobile phase ratio A/B was 75/25, the flow rate was 1mL/min, and the detection wavelength was 344 nm. The peak-out time was about 3.3 min.

Vorinostat: phase a is water containing 0.1% phosphoric acid; and the phase B is pure acetonitrile. The mobile phase ratio A/B was 73/27, the flow rate was 1mL/min, and the detection wavelength was 241 nm. The peak-off time was approximately 6.7 min.

Accurately weighing 20 μ L of the prepared double-targeting co-drug-loaded liposome, adding 180 μ L of methanol, vortexing for 5min, centrifuging at 15000rpm for 5min, and placing the supernatant in a liquid-phase sampling bottle for quantitatively detecting the drug content. The detected encapsulation rate of the double-targeting co-drug-loaded liposome to the gefitinib is 88.6 percent, and the encapsulation rate to the vorinostat is 83.8 percent. The liposome has similar entrapment rate to the two medicines, and is the basis for loading the two medicines in the same liposome in accurate compatible dosage.

(4) In vitro release

The in-vitro release characteristics of the double-targeting co-drug-loaded liposome to the encapsulated drug are inspected by a dialysis method. 400 μ L of the above prepared dual-targeting co-drug loaded liposome was placed in a dialysis bag (molecular weight cut-off 8000Da), sealed and tightened, placed in a 50mL centrifuge tube containing 40mL of PBS containing 0.1% tween 80, the pH of the release medium was adjusted to 4.7, 5.5, 6.3 and 7.4, respectively, mimicking lysosomes, endosomes, tumor microenvironment and physiological conditions, each of which was set up in three parallel experiments. The centrifuge tubes were incubated at 150rpm on a shaker at 37 ℃ and 500. mu.L of release medium samples were taken at 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 24h and supplemented with an equal volume of fresh release medium. Shaking and mixing uniformly after 24h, cutting off the dialysis bag, and taking 200 mu L as a reference for complete release. After all the samples taken were centrifuged at 15000rpm, the supernatant was placed in a liquid phase sampling flask, and the in vitro release of the drug was measured according to the method for quantifying the drug as described above.

As shown in fig. 5, the release of the drug in the liposome is pH dependent, and the release of the drug is less than 40% after 24 hours at pH 6.3 and 7.4, whereas the release of the drug has reached about 70% at pH 4.7 and 5.5 at hour 8. The double-targeting co-drug-loaded liposome is proved to have certain stability under the conditions of physiology and tumor microenvironment, and the drug can be rapidly released under the conditions of lysosomes and endosomes, so that the double-targeting co-drug-loaded liposome is particularly important for the delivery of anti-tumor drugs, the systemic toxicity of the drug can be reduced, and the treatment efficiency of the drug can be improved.

Experimental example 1

In vitro cellular uptake

When the liposome is prepared, a green fluorescent probe coumarin 6 is introduced according to the mass ratio of gefitinib to coumarin 6 of 10/1, the double-targeting co-drug-loaded liposome and the unmodified co-drug-loaded liposome carrying coumarin 6 are respectively prepared, a fluorescence spectrophotometer is adopted to quantify the coumarin 6 carried in the liposome under the condition that the excitation light wavelength is 466nm and the emission wavelength is 504nm, and the cell entry efficiency of the liposome is characterized by the amount of the coumarin 6 entering cells. The method comprises the following specific steps:

HER2 positive cell line H1975 and M2 type macrophages at 8x10 per well⁴The individual cells were seeded in 12-well cell culture plates at 37 ℃ with 5% CO₂Culturing for 24h under the condition, adding the double-targeting co-drug-loaded liposome and the unmodified co-drug-loaded liposome into each hole by 0.1 mu g of coumarin 6, and collecting cells for detection by a flow cytometer after the liposome is treated for 4 h. To examine the effect of targeted modification on liposome entry efficiency, tumor cells were treated with 1mg of herceptin 2h prior to dosing to shield them from HER2, followed by addition of dual-targeted co-drug-loaded liposomes containing 0.1 μ g of coumarin 6 and unmodified co-drug-loaded liposomes to each well, followed by collection of cells after treatment and flow cytometry. Macrophages of type M2 were treated in the same way with mannose.

The experimental result is shown in fig. 6, the uptake of coumarin 6 in cells can be remarkably improved by the double-targeting co-drug-loaded liposome, and after HER2 or mannose receptor shielding, the uptake of the double-targeting co-drug-loaded liposome and unmodified co-drug-loaded liposome cells is not different; no effect on uptake of unmodified co-loaded liposomes was obtained by HER2 or lack of mannose receptor shielding. This result suggests that targeted modification of liposomes can increase the efficiency of liposome entry through receptor mediation.

Experimental example 2

In vitro antitumor Activity

The in vitro toxicity of the drug and the double-targeting co-drug-loaded liposome on HER2 positive H1975 cells is detected by an MTT method to characterize the influence of targeted modification and co-loading on the synergistic efficiency of the combined drug. The specific experiment is as follows:

at 5x10³Density of cells/well H1975 cells were seeded in 96-well plates at 37 ℃ with 5% CO₂Culturing under the condition for 24h, and adding gefitinib with different concentrations (the mass ratio of vorinostat to gefitinib is 0.12:1, and the mass ratio of TMP195 to gefitinib is 1:1) Cells were treated and 6 secondary wells were set for each concentration, while negative controls without drug were set. After 48 hours of incubation, 20. mu.L of 5mg/mL MTT solution was added to each well, followed by 4 hours of incubation, the liquid was removed from each well by a syringe, 200. mu.L of dimethyl sulfoxide was added to each well, the absorbance of each well was measured at 490nm and 570nm using a microplate reader, and the cell viability of each administered group was calculated as the ratio of the average absorbance of each administered group to the average absorbance of the negative control group. The experimental results are shown in fig. 7 and 8, the synergistic effect of the histone deacetylase inhibitor and gefitinib is enhanced along with the increase of the drug concentration, and the synergistic efficiency of the double-targeting co-loaded liposome is remarkably improved compared with the combined use of free drugs.

Experimental example 3

Polarization and regulation of mouse bone marrow-derived macrophages

(1) Culture and induction of mouse bone marrow-derived macrophages (BMDM)

Soaking 3-5 weeks old Balb/C mouse in 75% ethanol for 5min after euthanasia, separating femur and tibia of mouse, soaking in 75% ethanol for 5min, washing twice with PBS for 5min each time, cutting bone ends, sucking serum-free culture medium with sterile syringe, and washing bone marrow from one end until bone marrow is completely washed out. Centrifuging at 1500rpm for 5min, discarding supernatant, and collecting bone marrow cells. The bone marrow cells were resuspended in RPMI-1640 medium containing 20ng/mL M-CSF and 10% fetal bovine serum at 37 ℃ in 5% CO₂Culturing for 3-4 days under the condition. The macrophages are polarized into M1 type by using 100ng/mL LPS and 20ng/mL IFN-gamma for inducing 24 h; macrophage polarization to M2 type was induced by IL-4 at 40ng/mL for 24 h. The morphology of macrophages after induction is shown in figure 9.

(2) Characterization of BMDM polarization by qRT-PCR and regulation thereof

As 1x10⁵Concentration per well BMDM was plated in 12-well cell culture plates and treated with the corresponding induction factor for 24h, followed by treatment of M2-type macrophages with gefitinib (5 μ M), vorinostat (1 μ M), a combination of two drugs or dual targeting co-loaded liposomes for 24h, with simultaneous stimulation of drug treatment and induction factor. Total RNA in each macrophage group was extracted using Trizol according to the instructions for the reagentsExtracting; then preparing cDNA from each group of RNA according to the instructions of the reverse transcription kit; a20. mu.L PCR reaction was prepared according to SYBR Green instructions. And (3) quantitatively analyzing the PCR result by using a delta C T-T method by taking BMDM as a negative control. The experimental results are shown in fig. 10: after 24 hours of LPS and IFN-gamma induction, mRNA of M1 type macrophage marker molecules CD86, IL-12p40, iNOS and TNF-alpha in macrophages is obviously up-regulated; after 24 hours of IL-4 induction, mRNA of M2 type macrophage marker molecules Arginase I, IL-10 and CD206 in macrophages is obviously up-regulated; indicating that the macrophage model is successfully constructed. However, after the combination of vorinostat, gefitinib and vorinostat and the treatment of the double-targeting co-drug-loaded liposome, the expression level of the M1 type marker molecule in the M2 type macrophage is obviously up-regulated, and the expression level of the M2 type marker molecule is obviously down-regulated, which indicates that the polarization type of the macrophage is converted from M2 type to M1 type after the M2 type macrophage is re-educed. The double-targeting co-drug-loaded liposome has active targeting capability on M2 type macrophages, so that the regulation capability on the macrophages is remarkably enhanced.

Tumor cells H1975 were co-cultured with BMDM in a 0.4 μm Transwell co-culture chamber, with H1975 in the lower chamber and BMDM in the upper chamber, with only material and no cell exchange between cells. The qRT-PCR experiment was performed in the same manner as that of BMDM alone, and the results are shown in FIG. 11, in which the expression level of the M1 type marker molecule was significantly increased in M2 type macrophages, and the expression level of the M2 type marker molecule was significantly decreased, similar to that of BMDM alone. Shows that under the co-culture condition, after the combination of vorinostat, gefitinib and vorinostat and the treatment of the double-targeting co-drug-loaded liposome, the polarization type of macrophage is converted from M2 type to M1 type.

(3) Western blot experiment characterization of BMDM polarization and regulation and control thereof

As 1x10⁵Concentration per well BMDM was plated in 12-well cell culture plates and treated with the corresponding induction factor for 24h, followed by treatment of M2-type macrophages with gefitinib (5 μ M), vorinostat (1 μ M), a combination of two drugs or dual targeting co-loaded liposomes for 24h, with simultaneous stimulation of drug treatment and induction factor. After cell lysis, CD206 (mannose) was detected by western blot assaySugar receptor, MR), HDAC2, TGF- β and β -Actin proteins.

The experimental result is shown in fig. 12, after the combination of vorinostat, gefitinib and vorinostat and the treatment of the dual-targeting co-drug-loaded liposome, the expression levels of CD206 and TGF-beta in M2 type macrophages are obviously reduced, and meanwhile, the expression level of HDAC2 is also obviously reduced, which indicates that the combination of vorinostat, gefitinib and vorinostat and the dual-targeting co-drug-loaded liposome can regulate the polarization of macrophages by regulating the expression and activation of HDAC 2. Macrophages were also co-cultured with tumor cells, and the expression of CD206, HDAC2, TGF-. beta.and β -Actin proteins in M2-type macrophages also tended to be similar after the same treatment.

(4) Characterization of BMDM polarization and its regulation by ELISA experiment

According to 6x10⁴Concentration per well BMDM is planted in a 24-well cell culture plate, the upper chamber of a co-culture system is co-cultured for 24 hours, then a corresponding induction factor is used for treating for 24 hours, M2 type macrophages are treated for 24 hours by gefitinib (5 mu M), vorinostat (1 mu M) and combined or double-targeting co-drug-loaded liposome of two drugs, a culture medium is collected, the drug treatment and the induction factor are simultaneously stimulated, and each group is provided with 3 auxiliary wells. The content of TNF-alpha in the culture medium was determined according to the instructions of the ELISA kit.

The results of the experiment are shown in FIG. 13: m1 type macrophages secrete a large amount of TNF-alpha, M2 type macrophages do not secrete TNF-alpha, however, after the combination of vorinostat, gefitinib and vorinostat and the treatment of double-targeting co-drug-loaded liposome, the amount of TNF-alpha secreted by M2 type macrophages is remarkably improved, and the change of macrophage polarization types is indicated.

By means of qRT-PCR, western blot and ELISA experimental characterization, under the condition that macrophages are cultured independently or co-cultured with tumor cells, the combination of vorinostat, gefitinib and vorinostat and the double-targeting co-drug-loaded liposome can regulate the re-differentiation of M2 type macrophages into M1 type macrophages.

Experimental example 4

Effect of macrophages on tumor cells

(1) Cytotoxicity test

And (3) placing the mouse bone marrow cells in a culture dish, respectively inducing the cells into M1 type macrophages and M2 type macrophages after the cells are completely attached to the wall, and collecting cell culture media after 24 hours.

H1975 cells were cultured at 5X10³The concentration of each well is planted in a 96-well plate, after 24 hours, gefitinib with different concentrations is prepared by using a normal culture medium, an M1 type macrophage culture medium or an M2 type macrophage culture medium respectively, tumor cells are treated, and after 48 hours of drug treatment, the survival rate of the tumor cells is detected by adopting an MTT method.

The experimental results are shown in fig. 14: compared with the normal medium group, the sensitivity of the cells of the M1 type macrophage medium group H1975 to gefitinib is obviously enhanced, while the sensitivity of the M2 type macrophage medium group to gefitinib is worse. This indicates that EGFR inclusion can be improved by modulating macrophage polarization patterns^T790MThe sensitivity of the drug-resistant mutant tumor cells H1975 to gefitinib further reverses the drug resistance.

(2) Apoptosis assay

At 8x10⁴Density of each well H1975 cells were seeded in 12-well plates, and after 24H, gefitinib (Gef, 5 μ M), vorinostat (Vor, 1 μ M), a combination of two drugs (GV) or double targeting co-drug loaded liposomes (tLGV) were administered for 24H, the cells were collected by digestion, subjected to Annexin V-FITC/PI staining, and subjected to flow cytometry to detect apoptosis. In addition, tumor cells were co-cultured with macrophages, the tumor cells were inoculated into the upper chamber of the co-culture system, the macrophages were induced into M1 type or M2 type before administration to only the tumor cells, and 24 hours later, the cells were collected and stained in the same manner to detect apoptosis.

The experimental result is shown in fig. 15, the combined use of gefitinib and vorinostat can obviously improve the drug effect, and when two drugs are entrapped in the same liposome, the liposome has active targeting capability to tumor cells, and the synergistic effect is obviously strengthened. When the tumor cells are co-cultured with M1 type macrophages, the sensitivity of the tumor cells to all drugs is obviously improved, and when the tumor cells are co-cultured with M2 type macrophages, the sensitivity of the tumor cells to the drugs is not obviously different from that of the tumor cells cultured under the condition of independent culture.

(3) Activation of Signal proteins

At 8x10⁴H1975 cells are inoculated on a 12-hole plate or an upper chamber of a co-culture system at a density of each hole, after 24 hours, macrophages are induced into M2 type, tumor cells and M2 type macrophages are simultaneously and respectively given with gefitinib (5 mu M), vorinostat (1 mu M), combination of two drugs, unmodified co-drug-loaded liposome or double-targeting co-drug-loaded liposome, after 10nM of oritinib is used as a positive control for treatment for 12 hours, H1975 is cracked to extract intracellular proteins, after the concentration of each group of proteins is quantitatively adjusted to be consistent through BCA, expression conditions of EGFR, Erk, Akt and phosphorylated proteins thereof are detected through a Western blot experiment.

The experimental results are shown in fig. 16, and under the condition of H1975 single culture and co-culture with M2 type macrophages, oxitinib can effectively inhibit the activation of EGFR, and further block the activation of downstream signal molecules Erk and Akt. Whereas gefitinib failed to inhibit activation of EGFR in both H1975 alone and M2-type macrophages, gefitinib, although synergistic with vorinostat and enhanced when both drugs were co-loaded in the same liposome, failed to completely inhibit activation of EGFR as well as Erk and Akt. Under the condition of co-culture of H1975 and M2 type macrophages, when two cells simultaneously receive gefitinib and vorinostat in a combined mode and unmodified co-drug-loaded liposomes, particularly double-targeting co-drug-loaded liposomes, activation of EGFR in H1975 cells is fully inhibited, activation of Erk and Akt is completely blocked, and activity of a molecular targeting inhibitor is recovered.

Experimental example 5

Mechanism of macrophage effect on tumor cells

(1) Reactive Oxygen Species (ROS) levels

At 8x10⁴(ii) cell/well Density H1975 cells were seeded in 12-well plates or in the upper chamber of co-culture system, 24H later, macrophages were first induced to M2 type, tumor cells and M2 type macrophages were simultaneously administered with gefitinib (5. mu.M), vorinostat (1. mu.M), combination of two drugs or dual targeting co-drug-loaded liposome treatmentAnd 6h, removing the culture medium in the tumor cells, and staining the tumor cells according to the instruction of the active oxygen determination kit. And detecting the active oxygen level in the tumor cells by using a flow cytometer.

The experimental result is shown in fig. 17, after gefitinib, gefitinib and vorinostat are combined and double-targeting co-drug-loaded liposome is treated with H1975, the reactive oxygen species level in the tumor cells is obviously increased, while the reactive oxygen species level in the tumor cells treated with vorinostat is not changed greatly; however, when co-cultured with M2-type macrophages, the level of reactive oxygen species decreases. When tumor cells and macrophages are co-cultured and administered simultaneously, the reactive oxygen species level is improved after vorinostat treatment compared with that of H1975 alone, and the reactive oxygen species level of H1975 cells is improved more remarkably after double-targeting co-drug-loaded liposome treatment.

(2) Superoxide anion (O)₂ ^-) Level of

At 8x10⁴(ii) cell/well Density H1975 cells were seeded in 12-well plates or in the upper chamber of co-culture system, 24H later, macrophages were first induced to M1 type or M2 type, tumor cells and M2 type macrophages were treated with gefitinib (5 μ M), vorinostat (1 μ M), combination of two drugs or dual targeting co-drug loaded liposomes for 6H at the same time, respectively, the medium in tumor cells was removed, and the cells were cultured according to O₂ ^-The instructions of the assay kit stained the tumor cells. Flow cytometry for detecting O in tumor cells₂ ^-And (4) horizontal.

The results are shown in FIG. 18, H1975 intracellular O₂ ^-The level changes are similar to the changes in reactive oxygen species, briefly, O in tumor cells when H1975 is co-cultured with M1-type macrophages₂ ^-Increased levels of O when co-cultured with M2-type macrophages₂ ^-The level is reduced, the polarization type of macrophages is changed after the macrophages of H1975 and M2 types are co-cultured and simultaneously treated by double-targeting co-drug-loaded liposome, and H1975 intracellular O₂ ^-The level is also significantly increased.

(3) EGFR and EGFR^T790MmRNA level of

At 8x10⁴(ii) cell/well Density H1975 cells were inoculated into 12-well plates or in the upper chamber of a co-culture system, 24H later, macrophages were induced to M2 type, tumor cells and M2 type macrophages were simultaneously treated with gefitinib (5. mu.M), vorinostat (1. mu.M), oxitinib (10nM), gefitinib and vorinostat or double-targeting co-drug-loaded liposomes for 6H, total RNA of H1975 cells was extracted, and qRT-PCR experiments were performed to examine EGFR and EGFR^T790MThe mRNA level of (a).

As shown in FIG. 19, the results of the experiments showed that EGFR and EGFR among the treatment groups were observed in the case of H1975 alone or in the case of co-culture with M2-type macrophages^T790MDoes not vary much in the mRNA level of (A).

(4) Changes in protein expression

At 8x10⁴H1975 cells are inoculated in a 12-well plate or an upper chamber of a co-culture system at a density of one well, macrophages are induced into M2 type 24H later, the tumor cells and the M2 type macrophages are simultaneously and respectively treated with gefitinib (5 mu M), vorinostat (1 mu M), oxitinib (10nM), gefitinib and vorinostat or double-targeting co-drug-loaded liposome for 6H, total protein in the tumor cells is collected by cracking H1975, and the expression conditions of anti-apoptotic proteins Bcl-2 and Bcl-XL are detected by adopting a western blot experiment. It has been shown that H1975 intracellular ROS are especially O₂ ^-When the level is increased, the balance between NOX3 and MsrA can be broken, so that the oxidative metabolism of M at 790 th position of EGFR is caused, and the EGFR is caused^T790M(iii) restoration of H1975 sensitivity to gefitinib, overcoming resistance (see, Leung E L H, Fan X X, Wong M P, et al, targeting tyrosine kinase inhibitor-resistant non-small cell containing cancer by inducing polypeptide growth factor receptor degradation of vitamin A methyl 790 oxidation [ J].Antioxidants&redox signaling,2016,24(5): 263-279). Based on this we tested the expression levels of NOX3 and MsrA in H1975 cells.

The results are shown in FIG. 20, and show that following administration, the active oxygen and O in tumor cells₂ ^-The increase of the level, the expression of the anti-apoptosis proteins Bcl-2 and Bcl-XL in the tumor cells is inhibited, and the expression amount of the proteins, active oxygen and O₂ ^-The levels are inversely proportional. However, only the active oxygen and O of the double-targeting co-drug-loaded liposome group are generated when the tumor cells are co-cultured with M2 type macrophages₂ ^-The elevated level was sufficient to break the balance between NOX3 and MsrA, restoring the sensitivity of H1975 to gefitinib.

(5) H1975 sensitivity to drugs

At 8x10⁴Density per well H1975 cells were seeded in 12-well plates or in the upper chamber of co-culture systems and 24H later macrophages were induced to M2 type, tumor cells and M2 type macrophages were treated with gefitinib (Gef, 5 μ M), vorinostat (Vor, 1 μ M), ocitinib (Osi, 10nM), gefitinib in combination with vorinostat (GV) or dual targeting co-loaded liposomes (tLGV), respectively, for 24H. H1975 cells are collected, and after Annexin V-FITC/PI staining, the apoptosis condition is detected by a flow cytometer.

The experimental result is shown in fig. 21, compared with the case of co-culture with M2-type macrophages, when co-culture with M2-type macrophages is not sensitive to gefitinib and oxitinib treatment, but the response degree to vorinostat is increased, when gefitinib and vorinostat are combined, the H1975 apoptosis rate under co-culture conditions is obviously increased, while the dual-targeting co-drug-loaded liposome group is obviously increased, which indicates that vorinostat, gefitinib and vorinostat are combined, and the dual-targeting co-drug-loaded liposome improves the drug treatment efficiency by regulating the polarization of M2-type macrophages to M1-type macrophages.

Experimental example 6

In vivo targeting efficiency of herceptin-modified liposomes

(1) In vivo imaging

During the preparation of the liposome, a red fluorescent probe DIR is introduced according to the mass ratio of gefitinib to DIR of 10/1, and a double-targeting co-drug-loaded liposome carrying DIR and an unmodified co-drug-loaded liposome are respectively prepared. And (3) quantifying the DIR entrapped in the liposome by using a fluorescence spectrophotometer under the condition of the excitation light wavelength of 748nm and the emission wavelength of 780nm, and representing the time course of the liposome in vivo through the time distribution of the DIR.

Will be 1x10⁶Planting H1975 cells in SPF grade nude of 3-5 weeks oldThe back of the mouse is close to the lateral subcutaneous part of the right hind leg until the tumor reaches 200mm³And then performing in vivo imaging. The method comprises the following specific steps:

tail vein injection of double-target co-drug-loaded liposome or unmodified co-drug-loaded liposome containing 20 mug DIR, 1% sodium pentobarbital is anesthetized by intraperitoneal injection and then photographed at the 2h, 4h, 8h and 24h after injection respectively under a living body imaging instrument of the small animal, and the distribution of the liposome in the body of the small animal is observed. After 24h of injection, the mice are euthanized, and the heart, liver, spleen, lung, kidney and tumor of the mice are taken to take pictures under a small animal living body imaging instrument, the distribution condition of the liposome is quantitatively indicated according to the mean value of the fluorescence radiation efficiency of each tissue organ, and the accumulation condition of the liposome in each main organ is detected.

The experimental result is shown in fig. 22, compared with the unmodified co-drug-loaded liposome, the double-targeting co-drug-loaded liposome significantly improves the distribution of the liposome at the tumor site, which indicates that the herceptin significantly improves the active targeting property of the liposome.

(2) Co-localization of dual-targeting co-drug-loaded liposomes with HER2 and mannose receptor

During preparation of the liposome, a red fluorescent probe DID is introduced according to the mass ratio of gefitinib to DID of 10/1, and the DID-loaded double-targeting co-drug-loaded liposome and the unmodified co-drug-loaded liposome are respectively prepared. And quantifying the DID entrapped in the liposome by using a fluorescence spectrophotometer under the condition of exciting light wavelength 644nm and emission wavelength 665nm, and observing the co-localization of the DID and HER2 through immunofluorescence staining to characterize the active targeting property of the liposome at a tumor part.

Will be 1x10⁶Planting H1975 cells on the back of a 3-5-week SPF-level nude mouse close to the subcutaneous side of the right hind leg until the tumor reaches 200mm³And then used for co-localization experiments. The method comprises the following specific steps:

injecting a double-target co-drug-loaded liposome containing 20 mu g of DID and an unmodified co-drug-loaded liposome into tail vein, euthanizing a mouse after 4h, quickly taking out tumor tissue, placing the tumor tissue on ice, freezing and slicing after OTC embedding, fixing the slice for 20min by acetone precooled at the temperature of-20 ℃, and observing co-location of the DID-labeled liposome and HER2 or mannose receptor under a Leica confocal microscope after staining and flaking.

The experimental result is shown in fig. 23, the double-targeting co-drug-loaded liposome is distributed in a large amount at the tumor position for 4 hours, and has obvious co-localization with HER2 or mannose receptor, the DID fluorescence at the tumor position of the unmodified co-drug-loaded liposome group is obviously weaker than that of the herceptin modified co-drug-loaded liposome group, and the DID fluorescence is rarely co-localized with HER2 or mannose receptor, which indicates that the targeting modification obviously improves the active targeting property of the liposome.

Experimental example 7

In vivo antitumor Activity of Herceptin-modified liposomes

(1) Tumor growth inhibition

Taking H1975 cells in logarithmic growth phase, and mixing 1x10⁶Planting the seeds on the back of a 3-5-week SPF-level nude mouse close to the subcutaneous side of the right hind leg until the tumor reaches 100mm³And then used for in vivo antitumor activity research. The method comprises the following specific steps:

dividing tumor-bearing mice into six groups randomly, wherein each group comprises 6 mice, injecting physiological saline, gefitinib (10mg/kg), gefitinib and vorinostat together (the dose of vorinostat is 1.2mg/kg), oxitinib (2mg/kg), double-targeting co-drug-loaded liposome and unmodified co-drug-loaded liposome respectively in tail vein, administrating every other day for eight times, recording the weight change of the mice after each administration, measuring the major diameter (L, unit: mm) and the minor diameter (S, unit: mm) of the tumor, and calculating the tumor volume: v (unit: mm)³)＝L×S²/2. After the administration, the nude mice were dislocated to sacrifice the tumor, the tumor was removed, blood and envelope on the tumor surface were carefully removed, weighed and photographed.

The experimental results are shown in fig. 24, fig. 25 and fig. 26, because the binding ability of gefitinib, vorinostat and serum protein is strong and easy to remove, the combination efficiency of free drugs of gefitinib and vorinostat is limited, the synergistic effect efficiency of two drugs is improved by the unmodified liposome due to the passive targeting and the accurate dose, the synergistic effect efficiency is remarkably improved by the double-targeting co-drug-loaded liposome due to the good active targeting, the tumor inhibition efficiency reaches 72.1%, and the tumor inhibition efficiency of the positive control drug Osi is only 68.2%.

(2) Polarized types of macrophages in tumor cells

One mouse was randomly selected from each group 24 hours after the 4 th and last administrations, the mice were euthanized, tumor tissues were removed, and frozen sections were immediately taken with a section thickness of 8 μm. The tissue sections were then stained by immunofluorescence, using CD206 for M2-type macrophages and CD80 for M1-type macrophages. And (3) observing the distribution and polarization type of macrophages in the tumor tissue by using a Leica super-resolution confocal microscope after dyeing.

The experimental results are shown in fig. 27, macrophages in the normal saline and gefitinib groups are mainly distributed in M2 type in the whole administration process, and the recruitment of M2 type macrophages is increased along with the growth of tumors, while the macrophages in the oxitinib, gefitinib and vorinostat groups, unmodified co-drug-loaded liposome groups are less distributed and mostly distributed in M2 type, while the macrophages in the double-targeting co-drug-loaded liposome group are mostly distributed in M1 type, and the macrophages in the M1 type are also increased along with the increase of treatment times. This indicates that the dual-targeting co-drug-loaded liposome improves tumor resistance by re-differentiating the recruited TAM into M1 type.

(3) Reactive oxygen species levels in tumor tissue

Taking H1975 cells in logarithmic growth phase, and mixing 1x10⁶Planting the seeds on the back of a 3-5-week SPF-level nude mouse close to the subcutaneous side of the right hind leg until the tumor reaches 200mm³And then, randomly taking two mice, injecting physiological saline into a group of tail veins, injecting a double-targeting co-drug-loaded liposome into a group of tail veins, and adding a red fluorescent probe DID into the prescription when preparing the double-targeting co-drug-loaded liposome, wherein the mass ratio of the DID to the gefitinib is 1/10. Injecting active oxygen labeled probe DCFH-DA into tumor 4h after tail vein injection, the dose is 2.5mg/kg, immediately euthanizing the mouse after 10min, taking down the tumor tissue of the mouse, freezing and slicing, the thickness of the slice is 8 μm, and observing the active oxygen level in the tumor tissue by using a Leica super-resolution confocal microscope after DAPI stains the tumor cells.

The experimental result is shown in fig. 28, and the distribution of a large amount of double-targeting co-drug-loaded liposomes in the tumor tissue leads to the significant increase of the reactive oxygen level in the tumor tissue.

(4) Protein expression levels in tumor tissue

In tumor tissues taken at the end of the tumor growth inhibition experiment, one tumor tissue is randomly taken from each group, a piece of cracked extracted protein is cut, and the expression conditions of EGFR, Erk, Akt and phosphorylated proteins thereof as well as NOX3 and MsrA in the tumor tissues are detected by adopting a western blot experiment.

The experimental result is shown in fig. 29, only the phosphorylation of EGFR, Erk and Akt of the ocitinib and the dual-targeting co-drug-loaded liposome group is fully inhibited, the expression level of NOX3 of the dual-targeting co-drug-loaded liposome group is remarkably up-regulated, and the expression level of MsrA is inhibited, so that the total EGFR protein level of the dual-targeting co-drug-loaded liposome group is remarkably lower than that of other groups. The results show that the double-targeting co-drug-loaded liposome can be accumulated in tumor tissues due to good active targeting property, and can regulate TAM to be differentiated to M1 type, so that the active oxygen level in the tumor tissues is improved, the balance between NOX3 and MsrA is broken, and EGFR occurs^T790MDegrading and restoring the sensitivity of the drug-resistant tumor to the drug.

(5) Apoptosis in tumor tissue

One of the tumor tissues taken at the end of the tumor growth inhibition experiment was randomly selected from each group, paraffin-embedded sections were stained with TUNEL, and apoptosis in the tumor tissues was detected by fluorescence microscopy.

The experimental results are shown in fig. 30, and the group of dual-targeted co-drug-loaded liposomes exhibited the most apoptosis.

Claims

1. A method of preparing a dual-targeted co-drug-loaded liposome comprising the steps of:

(b) adding a pharmaceutical composition comprising gefitinib and vorinostat to lecithin, cholesterol, DSPE-PEG, the mannose-modified PEG prepared in step (a), and PEG-NHS ester to prepare a liposome encapsulating the pharmaceutical composition, wherein the mass ratio of gefitinib to vorinostat is 1: 0.12-1;

(c) adding herceptin into the liposome which is prepared in the step (b) and wraps the pharmaceutical composition to carry out a light-shielding reaction; and

(d) centrifuging to obtain the double-targeting co-drug-loaded liposome,

wherein, the molecular weight range of the PEG with the amino in the step (a) and the DSPE-PEG and PEG-NHS ester in the step (b) is 2000-5000 Da.

2. The method of claim 1, wherein the first and second light sources are selected from the group consisting of,

wherein, the step (b) is carried out by a thin film dispersion hydration method, an ethanol injection method, a reversed phase evaporation method or an ammonium sulfate gradient method.

3. The method of claim 2, wherein the PEG with an amino group is DSPE-PEG₂₀₀₀-NH₂The PEG-NHS ester is DSPE-PEG₂₀₀₀-NHS。

4. The method of claim 2, wherein step (b) is performed using a thin film dispersion hydration method.

5. A dual-targeted co-drug loaded liposome prepared according to the method of any one of claims 1-4.

6. The double-targeted co-drug-loaded liposome of claim 5, wherein the surface of the double-targeted co-drug-loaded liposome is modified by herceptin and mannose.

7. The dual-targeting co-drug-loaded liposome of claim 5 or 6, wherein the particle size of the dual-targeting co-drug-loaded liposome is 186.9nm, the polydispersity index is 0.11nm, the encapsulation efficiency on gefitinib is more than 80%, and the encapsulation efficiency on vorinostat is more than 80%.

8. Use of a dual-targeted co-drug loaded liposome according to any of claims 5 to 7 in the preparation of a medicament for the treatment of EGFR^T790MThe application of the mutant drug-resistant non-small cell lung cancer medicine.

9. The use of claim 8, wherein the dual-targeted co-drug-loaded liposomes reverse molecularly targeted drug resistance by modulating macrophage polarization or acting as a macrophage immunomodulator.