CN114432248A

CN114432248A - Targeted composite carrier, drug-loading system, preparation method and application thereof

Info

Publication number: CN114432248A
Application number: CN202210103200.4A
Authority: CN
Inventors: 武敬亮; 王方卿; 张波; 董金萍; 李成垒; 李娜
Original assignee: Weifang University of Science and Technology
Current assignee: Weifang University of Science and Technology
Priority date: 2022-01-28
Filing date: 2022-01-28
Publication date: 2022-05-06
Anticipated expiration: 2042-01-28
Also published as: CN114432248B

Abstract

The invention discloses a targeted composite carrier, a drug-loading system, a preparation method and application thereof. The composite carrier comprises a first targeting carrier and a second targeting carrier, wherein the first targeting carrier comprises the following components: dioleoyl phosphatidylethanolamine, lecithin, cholesterol succinate monoester, cholesterol, imidazolyl cholesterol, and DSPE-PEG-HA; the DSPE-PEG-HA is prepared by passing DSPE-PEG-NH₂Is prepared by mixing hyaluronic acid; the imidazolyl cholesterol is prepared from chloroformic acid cholesterol and N- (3-aminopropyl) imidazole; the second targeting vector comprises the following components: a compound phospholipid and glycocholic acid, wherein the compound phospholipid comprises lecithin and DSPE-PEG-GA; the particle size of the first targeting vector is 150 nm-200 nm; the particle size of the second targeting carrier is 25-50 nm. The invention controls the tumor microenvironment and kills the tumor cells in a targeted way by loading the drugs through different targeted carriers, achieves the effect of synergistic anti-tumor and solves the problem of tumor drug resistance generated by long-term treatment of a single drug.

Description

Targeted composite carrier, drug-loading system, preparation method and application thereof

Technical Field

The invention relates to a delivery carrier and a targeted drug carrier, in particular to a targeted composite carrier, a drug-carrying system, a preparation method and application thereof.

Background

Hepatocellular carcinoma (HCC) is one of the most common malignancies and is the leading cause of cancer-related death. Currently, surgical treatment and liver transplantation are the main methods for clinical treatment of liver cancer, but overall survival rates are low, mainly due to malignant proliferation and systemic metastasis. With the intensive research on cancer, Tumor Microenvironment (TME) provides the necessary soil conditions for the growth, invasion and metastasis of tumor cells. Therefore, targeting tumor microenvironment strategies is a key to tumor therapy.

TME consists of stromal cells, tumor vessels, extracellular matrix (ECM) and various types of factors. Tumor-associated fibroblasts (CAFs) have received much attention as an important component of TME. Among them, CAFs can not only secrete collagen to deposit ECM, but also establish cross-talk with liver cancer cells to induce drug resistance and metastasis of liver cancer cells. In addition, a large amount of ECM can not only form a physical barrier, but also press tumor blood vessels, limit the penetration of the drug in the tumor and reduce the bioavailability of the drug. Therefore, inhibition of CAFs activation and ECM deposition is crucial to improving the therapeutic effect of liver cancer.

Capsaicin (CAP) is a pungent alkaloid extracted from red pepper, and has anticancer, antiproliferative and antioxidant effects. Recent research shows that CAP effectively inhibits fibroblast activation and treats hepatic fibrosis. Furthermore, Telmisartan (TEL) is an angiotensin ii receptor 1 (AT 1R) antagonist that inhibits secretion of ECM by CAF through the transforming growth factor- β (TGF- β) signaling pathway.

Disclosure of Invention

The purpose of the invention is as follows: in order to solve the problems of the prior art, the invention provides a targeted composite carrier, a drug-loading system, a preparation method and application thereof.

The technical scheme is as follows: the composite carrier comprises a first targeting carrier and a second targeting carrier, wherein the first targeting carrier comprises the following components: dioleoyl phosphatidylethanolamine, lecithin, cholesterol succinate monoester, cholesterol, imidazolyl cholesterol, and DSPE-PEG-HA; the DSPE-PEG-HA is prepared by passing DSPE-PEG-NH₂Is prepared by mixing hyaluronic acid; the imidazolyl cholesterol is prepared from chloroformic acid cholesterol and N- (3-aminopropyl) imidazole; the second targeting vector comprises the following components: compound phospholipid and glycocholic acid, wherein the compound phospholipid comprises lecithin and DSPE-PEG-GA; the particle size of the first targeting vector is 150 nm-200 nm; the particle size of the second targeting carrier is 25-50 nm.

In a preferred embodiment of the present invention, the first targeting vector comprises dioleoyl phosphatidylethanolamine, lecithin, cholesterol succinate monoester, cholesterol, imidazolyl cholesterol and DSPE-PEG-HA in a mass ratio of 10-15: 5-10: 1-5.

As a specific embodiment of the invention, the first targeting vector comprises dioleoyl phosphatidylethanolamine, lecithin, cholesterol succinic acid monoester, cholesterol, imidazolyl cholesterol and DSPE-PEG-HA in a mass ratio of 14:6:3:2:2: 1.

In a preferred embodiment of the invention, in the second targeting vector, the mass ratio of lecithin to DSPE-PEG-GA is 15-20: 1; the mass ratio of the composite phospholipid to glycocholic acid is 1.4-3.5: 1.

In one embodiment of the present invention, in the second targeting vector, the mass ratio of lecithin to DSPE-PEG-GA is 20: 1; the mass ratio of the composite phospholipid to glycocholic acid is 1.7: 1.

In a preferred embodiment of the present invention, the imidazolyl cholesterol is synthesized by chloroformic acid cholesterol and N- (3-aminopropyl) imidazole in the presence of triethylamine.

As a preferred embodiment of the present invention, the DSPE-PEG-NH₂The molecular weight of the hyaluronic acid is 2000-5000 Da, and the molecular weight of the hyaluronic acid is 2000-5000 Da.

As a specific embodiment of the invention, the DSPE-PEG-NH₂Has a molecular weight of 2000Da and the molecular weight of the hyaluronic acid is 5000 Da.

The drug carrier comprises dioleoyl phosphatidylethanolamine, lecithin, cholesterol succinic acid monoester, cholesterol, imidazolyl cholesterol and DSPE-PEG-HA in a mass ratio of 10-15: 5-10: 1-5; the DSPE-PEG-HA is prepared by passing DSPE-PEG-NH₂Is prepared by mixing hyaluronic acid; the imidazolyl cholesterol is prepared from chloroformic acid cholesterol and N- (3-aminopropyl) imidazole; the drug loaded by the drug loading system is capsaicin and telmisartan.

The drug carrier or the drug carrier loaded with adriamycin is characterized by comprising composite phospholipid and glycocholic acid in a mass ratio of 1.4-3.5: 1, wherein the composite phospholipid comprises lecithin and DSPE-PEG-GA in a mass ratio of 15-20: 1; the mass ratio of the drug adriamycin loaded by the drug carrier to the lecithin is 1: 15-20.

The preparation method of the composite carrier comprises the following steps:

(S1) preparation of first targeting vector

(S11) Synthesis of DSPE-PEG-HA: DSPE-PEG-NH₂Activating EDC and NHS, and then performing crosslinking reaction with hyaluronic acid in a nitrogen environment to obtain DSPE-PEG-HA;

(S12) Synthesis of Imidazoylcholesterol: synthesizing imidazolyl cholesterol from chloroformic acid cholesterol and N- (3-aminopropyl) imidazole under the catalytic action of triethylamine;

(S13) adding dioleoyl phosphatidylethanolamine, lecithin, cholesterol succinate monoester, cholesterol, imidazolyl cholesterol and DSPE-PEG-HA into chloroform, removing the chloroform by using a rotary evaporator, adding a PBS solution for hydration when a layer of uniform film is formed, then carrying out ultrasonic treatment in an ice bath, and filtering to obtain a first targeting vector;

(S2) preparation of a second targeting vector: mixing lecithin and DSPE-PEG-GA in ethanol, slowly dripping into glycocholic acid solution, evaporating ethanol, performing ultrasonic treatment in ice bath, and filtering to obtain a second targeting vector.

The drug loading system comprises a first drug loading system and a second drug loading system, wherein the first drug loading system comprises a first targeting carrier, and capsaicin and telmisartan which are loaded by the first targeting carrier; the first targeting vector comprises dioleoyl phosphatidylethanolamine, lecithin, cholesterol succinate monoester, cholesterol, imidazolyl cholesterol and DSPE-PEG-HA; the DSPE-PEG-HA is prepared by passing DSPE-PEG-NH₂Is prepared by mixing hyaluronic acid; the imidazolyl cholesterol is prepared from chloroformic acid cholesterol and N- (3-aminopropyl) imidazole; the second drug carrier system comprises a second targeting carrier and adriamycin loaded through the second targeting carrier; the second targeting carrier comprises a compound phospholipid and glycocholic acid, and the compound phospholipid comprises lecithin and DSPE-PEG-GA; and/or in the first drug loading system, the drug loading rate of capsaicin is 2-3%, and the drug loading rate of telmisartan is 2-3%; in the second medicine carrying system, the medicine carrying amount of the adriamycin is 2-3%.

The preparation method of the medicine carrying system comprises the following steps:

(1) preparation of the first targeting vector

(1a) Synthesis of DSPE-PEG-HA: DSPE-PEG-NH₂Activating EDC and NHS, and performing crosslinking reaction with hyaluronic acid in nitrogen environment to obtain DSPE-PEG-HA；

(1b) Synthesis of imidazolyl cholesterol: synthesizing imidazolyl cholesterol from chloroformic acid cholesterol and N- (3-aminopropyl) imidazole under the catalytic action of triethylamine;

(1c) adding dioleoyl phosphatidylethanolamine, lecithin, cholesterol succinate monoester, cholesterol, imidazolyl cholesterol, DSPE-PEG-HA, capsaicin and telmisartan into chloroform, removing the chloroform by using a rotary evaporator, adding a PBS solution for hydration when a layer of uniform film is formed, and then carrying out ultrasonic treatment in an ice bath and filtering to obtain a first medicine carrying system;

(2) preparation of the second targeting vector: mixing lecithin and DSPE-PEG-GA in ethanol, slowly dripping into a solution containing glycocholic acid and adriamycin, evaporating the ethanol, performing ultrasonic treatment in an ice bath, and filtering to obtain a second drug-loading system.

The drug carrier system is applied to the preparation of liver cancer drugs.

The pharmaceutical composition comprises capsaicin, telmisartan and adriamycin.

Has the advantages that: (1) the invention provides a composite carrier (lipid large spheres and nano-spheres) preparation for treating liver cancer, which carries TEL and CAP together through lipid Large Spheres (CTHLs), HAs liver cancer targeting and deep permeability, and can be modified by HA and CD44⁺Active intake of CAFs; DOPE in the vector promotes fusion of liposome and inclusion bodies; the proton sponge effect of AIM-Chol promotes TEL and CAP to escape from inclusion bodies, blocks the activation of CAFs, inhibits ECM deposition, and improves the permeation efficiency of DGNs; the nano-Spheres (DGNs) load DOX, have double pH sensitivity and CAFs targeting, inhibit the development of liver cancer through the synergistic effect of CTHLs and DGNs (2) in the composite carrier disclosed by the invention, and the uptake efficiency of liver cancer cells is improved through the DGNs modified by Glycyrrhetinic Acid (GA); (3) according to the invention, by changing the proportion of the composite phospholipid to the glycocholic acid hydrate, the micelle with the particle size of 30nm is prepared, thereby being beneficial to the delivery of DOX into liver cancer tissues; (4) the invention evaluates the anti-liver cancer effect of the composite carrier lipid macro-sphere and nano-sphere mixed preparation by establishing a novel in vivo and in vitro research model, simulates the liver cancer environment at the cellular level, and establishes a new in vivo and in vitro research modelCo-culture model of EL-7402 and hepatic stellate cell (LX-2): BEL-7402+ LX-2, evaluating the targeting ability of CTHLs and DGNs through an in vitro uptake experiment, detecting the penetrating ability of DGNs by adopting a 3D tumor ball, and analyzing the antiproliferative and anti-migration characteristics of the mixed preparation. Establishing an H22+ m-HSCs co-implantation mouse model, an in-situ liver cancer model and a lung metastasis model, and evaluating the antiproliferation and metastasis effects of the mixed preparation; (5) the invention realizes the combined medication of capsaicin, telmisartan and adriamycin, the combined medication of TEL and CAP inhibits the activation of CAFs and the deposition of ECM, the TME is effectively regulated, and on the basis of the regulation of the TME, chemotherapeutic drug adriamycin (DOX) is added to block cross-talk between CAFs and liver cancer cells and inhibit the drug resistance and transfer of hepatocellular carcinoma; (6) the invention overcomes the obstacles faced by the treatment of DOX combined with TEL and CAP through the preparation of the targeting carrier, solves the defects of weak targeting property and large toxic and side effects of the traditional combined preparation, and solves the problems that the action targets of DOX, TEL and CAP on different cells are different, the pharmacokinetics and tissue distribution in vivo are different and the synergistic action is difficult to be played through the targeting carrier; (7) the drug delivery system solves the problem that the physical barrier formed by ECM in the prior art seriously hinders the drug from penetrating deep into the tumor to cause liver cancer relapse, and realizes high-efficiency drug delivery, synergistic action and deep penetration of the drug through a new drug delivery system.

Drawings

FIG. 1 is a schematic diagram of the preparation of a vector of the present invention;

FIG. 2 is a synthesis route and nuclear magnetic characterization map of DSPE-PEG-HA carrier material, wherein, A is the synthesis route, and a in B is HA characteristic peak B is PEG characteristic peak;

FIG. 3 shows the particle size change of lipid globules with different phospholipid feed ratios at acidic pH;

FIG. 4 is a graph representing DGNs and CTHLs, wherein A is a particle size distribution graph of DGNs and B is a particle size distribution graph of CTHLs; the C picture is a TEM image of the DGNs under the pH7.4 condition; d is TEM image of CTHLs under pH7.4 condition; e is a TEM image of CTHLs under the condition of pH5.0; panel F is the 7-day stability of DGNs; graph G is the 7 day stability of ctlls; graph H is an in vitro release profile of DGNs; graph I is an in vitro release profile of ctlls at different pH conditions;

FIG. 5 shows the in vitro uptake and retention of DGNs, wherein Panel A is the in vitro confocal image of DGNs; panel B is the quantification of DGNs uptake by flow cytometry; panel C is flow cytometry to examine the effect of various agents on DGNs retention; d picture is the quantitative result of flow cytometry for detecting the intake of various preparations;

FIG. 6 shows the in vitro uptake results of LX-2 for CTHLs, wherein, Panel A is the in vitro laser confocal images of CTHLs; panel B is flow cytometry quantification of ctlls uptake;

FIG. 7 shows the 3D multicellular spheroid results of DGNs, wherein graph A is a laser confocal graph of DOX, DNs and DGNs on the 3D multicellular spheroid; panel B shows fluorescence quantification of each group at a depth of 60 μm;

FIG. 8 shows cytotoxicity evaluation of DGNs in combination with CTHLs, wherein, A is a diagram of BEL-7402 model alone and LX-2/BEL-7402 coculture model; panel B shows MTT assay of BEL-7402 model alone; the C picture is the MTT detection of LX-2/BEL-7402 coculture model; d is the live and dead staining result of LX-2/BEL-7402 coculture model;

FIG. 9 shows the effect of DGNs in combination with CTHLs on tumor cell migration inhibition, wherein, A is a diagram of a BEL-7402 model alone and an LX-2/BEL-7402 co-culture model; panel B is the cell migration result of the BEL-7402 model alone; panel C shows the results of cell migration in the LX-2/BEL-7402 co-culture model; d is the cell migration inhibition rate of BEL-7402 model alone; e is the cell migration inhibition ratio of LX-2/BEL-7402 coculture model;

FIG. 10 shows the results of the research on cell migration mechanism by DGNs in combination with CTHLs on transwell (A) LX-2/BEL-7402 coculture model (B) the quantification of transwell by LX-2/BEL-7402 coculture model (C) Western blot analysis of E-Cadherin and Vimentin; (D) E-Cadherin and Vimentin quantitative analysis;

FIG. 11 is biodistribution of DGNs and CTHLs in tumor bearing mice (A) NIR images of DGNs and CTHLs at different time points (B) fluorescence images of different organs and tumors (C) fluorescence intensity analysis of different tissues at DID wavelength (D) fluorescence intensity analysis of different tissues at DIR wavelength (E) tumor sections of free DID-treated group at different depths (F) tumor sections of DID-treated group at different depths;

FIG. 12 is a quantification of CTHLs on the results of in vivo penetration of DGNs (A) tumor slice images from different treatment groups (B) tumor slice fluorescence intensity;

FIG. 13 is a photograph of antitumor evaluation of DGNs in combination with CTHLs on H22 subcutaneous transplanted tumors (A) a schematic representation of H22 subcutaneous transplanted tumors (B) tumor volume change of different treatment groups (C) body weight change of different treatment groups (D) tumor suppression rate of different treatment groups (E);

FIG. 14 is an H & E image of tumors and major organs of different treatment groups;

FIG. 15 is a photograph of tumor evaluation of DGNs in combination with CTHLs on m-HSC/H22 subcutaneous transplanted tumors (A) m-HSC/H22 subcutaneous transplanted tumors (B) tumor volume change of different treatment groups (C) body weight change of different treatment groups (D) tumor suppression rate of different treatment groups (E);

FIG. 16 is a Western blot analysis (G) E-Cadherin and Vimentin quantitative analysis of E-Cadherin and Vimentin tissues for the evaluation of the therapeutic effect of DGNs in combination with CTHLs on m-HSC/H22 subcutaneous transplants (A) tumor H & E panels for different treatment groups (B) tumor Masson panels for different treatment groups (C) α -SMA immunohistochemistry panels for tumors for different treatment groups (D) CD31 immunohistochemistry panels for tumors for different treatment groups (E) number of tumor microvasculature (F);

fig. 17 is a schematic of DGNs in combination with ctlls for evaluation of treatment effect on liver engraftment (a) liver engraftment of liver engraftment (B) liver photographs of different treatment groups (C) tumor H & E photographs of different treatment groups (D) tumor Masson photographs of different treatment groups (E) tumor α -SMA immunohistochemistry photographs of different treatment groups (F) CD31 immunohistochemistry photographs of tumors of different treatment groups;

fig. 18 shows the evaluation of the effect of DGNs in combination with ctlls on lung metastasis treatment (a) a schematic representation of lung metastasis (B) weight change in different treatment groups (C) lung nodule count in different treatment groups (D) lung photographs in different treatment groups (E) H & E pictures in different treatment groups.

Detailed Description

A source of material

1.1 materials

Egg phosphatidylcholine (SPC), 1, 2-dioleoyl-sn-glycerol-3Phosphoethanolamine (DOPE) and cholesterol were both purchased from a.v.t. pharmaceutical limited (china, shanghai). DOX, CAP, TEL were purchased from Dalian America Biotechnology, Inc., China. Glycocholic Acid (GAH) and Cholestyril Hemisuccinate (CHEMS) were purchased from Macklin Biochemical Co., Ltd., China, Shanghai. DSPE-PEG₂₀₀₀-NH₂Purchased from sienna millennium biotechnology limited (china, sienna). Antibodies against CD31, E-Cadherin and the like were purchased from Abcam corporation (USA, MA, USA). Other chemical reagents are analytical or chromatographic grade.

1.2 cells and animals

The human liver cancer cell line (BEL-7402, HepG2), the human hepatic stellate cell (LX-2), the mouse hepatic carcinoma cell (H22) and the mouse hepatic cell (m-HSCs) are all from Beijing institute of biotechnology. BALB/c mice (female, 18-22 g) were from the center of the experimental animals of Jinanpunyue, China. All animal experiments were approved by the Ethics Committee of animal experiments of the Weifang medical college (WFMC, 2019-.

Second, sample preparation

Example 1: synthesis of DSPE-PEG-HA

By DSPE-PEG₂₀₀₀-NH₂And cross-linking reaction with HA (MW 5000) to synthesize a novel functional phospholipid: DSPE-PEG-HA. 100mg HA was dissolved in distilled water, and 46.5mg carbodiimide (EDC) and 34.5mg N-hydroxysuccinimide (NHS) were added for activation. Mixing 68mg DSPE-PEG₂₀₀₀-NH₂Add to HA solution and stir slowly. And reacting for 24 hours at normal temperature in a nitrogen environment. Purifying the obtained solution, lyophilizing, and mixing with water¹H NMR analysis of the sample structure showed in FIG. 2, which confirmed DSPE-PEG by hydrogen nuclear magnetic resonance spectroscopy₂₀₀₀The structure of the HA polymer, the absorption band at 2.0ppm belonging to the methyl group of HA and the characteristic peak at 3.6ppm being DSPE-PEG₂₀₀₀-NH₂A PEG group of (2). DSPE-PEG₂₀₀₀HA peaks at both 3.6ppm and 2.0ppm indicated that HA was successfully modified to DSPE-PEG₂₀₀₀-NH₂The above.

Example 2: preparation of Co-loaded CAP and TEL lipid globules (CTHLs)

(1) AIM-Chol Synthesis

Cholesterol chloroformate (0.45g, 1mmol) was dissolved in dichloromethane (10 mL). N- (3-aminopropyl) imidazole (0.6mL, 4mmol) was added dropwise, 200. mu.L of triethylamine was added, and the mixture was stirred in an ice bath for 10 hours, after the reaction was completed, the solvent was removed by vacuum distillation. And purifying the product by column chromatography. Under the catalytic action of triethylamine, cholesteryl chloroformate and N- (3-aminopropyl) imidazole generate AIM-Chol.

(2) The membrane evaporation method was used to make pH sensitive lipid macrospheres (fig. 1). Firstly, determining optimized proportion of Dioleoylphosphatidylethanolamine (DOPE) and lecithin (SPC), and adding cholesterol succinate (CHEMS), cholesterol, imidazolyl-cholesterol (AIM-Chol) and DSPE-PEG according to the proportion of 1:3, 1:1 and 7:3₂₀₀₀HA prepares liposome, detects the change of particle size of three kinds of liposome under the condition of acid pH (pH5.5), and determines the charging ratio of phospholipid. The co-loaded lipid globules were prepared by adding Capsaicin (CAP) and Telmisartan (TEL).

And (4) analyzing results: three kinds of liposomes were prepared by controlling the charge ratio (mass ratio) of DOPE to SPC, DOPE: SPC being 7:3, DOPE: SPC being 1:1 and DOPE: SPC being 1: 3. The change of the particle size of different liposomes at an acidic pH value (pH5.5) is detected, the feeding ratio of the phospholipids is determined, and the result is shown in figure 3, when DOPE: SPC is 7:3 under the condition of pH5.0, the particle size of the liposomes is gradually increased along with the incubation time, and the feeding ratio has obvious pH sensitivity. Therefore, we chose DOPE: SPC ═ 7:3 as the formulation for the preparation of acid sensitive liposomes.

(3) The final optimization process of CTHLs is as follows:

dioleoylphosphatidylethanolamine (DOPE)70mg, lecithin (SPC)30mg, cholesteryl succinate (CHEMS)15mg, cholesterol 15mg, imidazolyl-cholesterol (AIM-Chol)10mg, DSPE-PEG-HA5mg, Capsaicin (CAP)5mg, and Telmisartan (TEL)5mg were dissolved in 5mL of chloroform. Chloroform was removed with a rotary evaporator and conditions of rotary evaporation: 40 ℃ and 20 rpm. When a uniform film was formed, 5mL of PBS (pH 7.4) was added and hydrated for 1 hour. Finally, the CTHLs were obtained by sonication for 5min in an ice bath and sequentially through 0.45 μm and 0.22 μm polyethersulfone membranes.

Example 3: preparation of DOX-loaded liver-targeting nanospheres (DGNs)

The DOX-loaded nanospheres were prepared by ethanol injection (FIG. 1). Firstly, GAH is dissolved in PBS, and the pH is adjusted to 5.5-6.0 by sodium hydroxide solution. Next, SPC and DSPE-PEG-GA (synthesized by the method of example 1 of CN 2018113191397) ethanol solution (DSPE-PEG-GA/SPC ratio of 1:20) were added dropwise to the GAH solution at different ratios of GAH to phospholipid (SPC) (1.2,1.4,1.7,2.0,3.5), and DOX was added. After evaporation of the ethanol, it was sonicated in an ice bath and passed through 0.45 μm and 0.22 μm polyethersulfone membranes in sequence.

The content of glycocholic acid is changed by fixing the content of SPC and DSPE-PEG-GA (20:1), and the influence of the mass ratio of the composite phospholipid/glycocholic acid on the size of the DGNs is examined. The results are shown in Table 1. The particle size of DGNs tends to increase as glycocholic acid content decreases. Wherein when the mass ratio of the composite phospholipid to the glycocholic acid is 1.7, the prepared nanoparticles are about 32 nm. The particle size micelle is beneficial to the penetration of the DGNs at a tumor part, and the DGNs are reduced to be metabolized by the kidney during extracorporeal circulation.

TABLE 1 Effect of different ratios of complex phospholipids and Glycocholic acid on DGNs particle size and PDI

The optimization process of the DGNs comprises the following steps:

SPC, DOX (SPC: DOX ═ 20:1) and DSPE-PEG-GA were mixed in ethanol (GAH/phospholipid complex ratio 1:1.7, DSPE-PEG-GA/SPC ratio 1:20) and then slowly instilled into the GAH solution. After evaporation of ethanol, DGNs were sonicated in an ice bath for 5min and passed through 0.45 μm and 0.22 μm polyethersulfone membranes in sequence to obtain DGNs.

Third, result analysis

3.1 analytical methods

3.1.1 characterization of CTHLs and DGNs

The particle size distribution and zeta potential of CTHLs and DGNs were measured using a Malvern Zetasizer Nano ZS90, respectively. The changes in the particle size of CTHLs and DGNs in PBS or RPMI 1640 were measured after 7 days of storage at 4 ℃. The morphology of CTHLs and DGNs was observed with a Transmission Electron Microscope (TEM). The DOX content of DGNs was determined by UV spectrophotometry at 480nm, and the CAP and TEL content in CTHLs was determined by High Performance Liquid Chromatography (HPLC) at 280 and 295 nm. The drug Loading (LE) and Encapsulation Efficiency (EE) are calculated as follows:

3.1.2 in vitro delivery of CTHLs and DGNs

(1) Drug release of ctlls was evaluated in PBS solutions at different pH values (pH 7.4,6.5 or 5.5). 1mL of the CTHLs was added to a dialysis bag (MWCO. RTM. 3500) and the release medium was 30mL (PBS, 0.5% Tween-80). The release system was then placed in a 37 ℃ incubator. At the set time point, 2mL of release medium was removed and then supplemented with 2mL of release medium, and the amounts of CAP and TEL released were determined by high performance liquid chromatography at 280nm and 295nm, respectively. (2)1mL of DGNs were added to a dialysis bag (MWCO. RTM. 3500), release medium (30mL of PBS solution, pH7.4, 0.5% Tween-80), and the release system was then placed in a 37 ℃ incubator. At the set time point, 2mL of release medium was removed and then 2mL of release medium was replenished and the absorbance of DOX was measured at 480nm using an ultraviolet spectrophotometer.

3.1.3 in vitro Co-culture research model establishment for simulating microenvironment

In the liver cancer microenvironment, there are not only liver cancer cells, but also matrix cells such as Hepatic Stellate Cells (HSCs), and the HSCs promote proliferation and metastasis of liver cancer. To simulate a real microenvironment, we created a co-culture model consisting of BEL-7402 and LX-2 cells. BEL-7402 and LX-2 cells were added to the plate at a ratio of 5:1, and cultured for 24 hours to form a co-cultured cell model. To distinguish between the two cells, we pretreated LX-2 cells with CFSE, a novel dye that fluorescently labeled live cells, to C-LX-2, followed by mixed culture to prepare a distinguishable BEL-7402/C-LX-2 co-culture model.

3.1.4 cellular uptake and drug retention assays

(1) DOX loaded nanospheres (DNs) were prepared by mixing SPC and DOX (SPC: DOX ═ 20:1) in ethanol (GAH/SPC ratio 1:1.7) and then slowly instilled into the GAH solution. After evaporation of the ethanol, DNs was sonicated in an ice bath for 5min and sequentially through 0.45 μm and 0.22 μm polyethersulfone membranes to afford DNs.

(2) Since both CAP and TEL drugs do not have fluorescence, hydrophobic fluorescein C6 was used instead of both drugs in cell uptake experiments to prepare C6-loaded lipid nanospheres (C6-Ls) and C6-loaded lipid nanospheres (C6-HLs). Preparation of C6-loaded lipid globules (C6-Ls): dioleoylphosphatidylethanolamine (DOPE)70mg, lecithin (SPC)30mg, cholesteryl succinate (CHEMS)15mg, cholesterol 15mg, imidazolyl-cholesterol (AIM-Chol)10mg, and coumarin 6(C6) 5mg were dissolved in chloroform 5 mL. Chloroform was removed with a rotary evaporator and conditions of rotary evaporation: 40 ℃ and 20 rpm. When a uniform film was formed, 5mL of PBS (pH 7.4) was added and hydrated for 1 hour. Finally, sonication was carried out for 5min in an ice bath, and sequentially through 0.45 μm and 0.22 μm polyethersulfone membranes, to obtain C6-loaded lipid globules (C6-Ls). Preparation of C6-loaded lipid globules (C6-HLs): dioleoylphosphatidylethanolamine (DOPE)70mg, lecithin (SPC)30mg, cholesteryl succinate (CHEMS)15mg, cholesterol 15mg, imidazolyl-cholesterol (AIM-Chol)10mg, DSPE-PEG-HA5mg, 5mg coumarin 6(C6) was dissolved in 5mL of chloroform. Chloroform was removed with a rotary evaporator and conditions of rotary evaporation: 40 ℃ and 20 rpm. When a uniform film was formed, 5mL of PBS (pH 7.4) was added and hydrated for 1 hour. Finally, sonication was performed for 5min in an ice bath, and sequentially through 0.45 μm and 0.22 μm polyethersulfone membranes, to obtain C6-loaded lipid macrospheres (C6-HLs). Ingestion experiment: BEL-7402 and LX-2 were inoculated into cell culture dishes (5X 10)⁴Cell/culture dish), cell uptake was detected. BEL-7402 cells were cultured for 12h, free DOX (DOX concentration 10. mu.g/mL), DOX-loaded nanospheres (DNs) (DOX concentration 10. mu.g/mL), and GA-modified nanospheres (DGNs) (DOX concentration 10. mu.g/mL) were added, and incubated for 0.5 h. LX-2 cells were cultured for 12h, and free coumarin 6(C6, concentration of C6 is 2 μ g/mL) and C6-loaded lipid microspheres (C6-Ls, concentration of C6 is 2%μ g/mL), HA-modified C6-loaded lipid microspheres (C6-HLs, C6 at 2 μ g/mL) in DMEM medium without fetal bovine serum for 0.5 h. After three PBS washes, DAPI staining was performed for 10 min. Subsequently, the images were photographed with a confocal laser scanning microscope and quantified by AccuriC6 Plus flow cytometer.

Retention experiments: BEL-7402/LX-2 cocultured cells were seeded in 6-well plates (cell inoculum size was 50 ten thousand/well per well), DOX (DOX concentration 2. mu.g/mL), DOX + CAP + TEL (DOX concentration 2. mu.g/mL, CAP concentration 2. mu.g/mL, TEL concentration 2. mu.g/mL), DGNs (DOX concentration 2. mu.g/mL), DGNs + CTHLs (DOX concentration 2. mu.g/mL, CAP concentration 2. mu.g/mL, TEL concentration 2. mu.g/mL) were added, and incubated for 12 hours. Cells were collected and assayed for intracellular drug amounts on an AccuriC6 Plus flow cytometer.

3.1.5 in vitro antitumor experiments

To better evaluate the role of drugs in the tumor microenvironment, we established two cell models: (1) BEL-7402 cells; (2) "BEL-7402 + LX-2" co-cultured cells. The two cell models were first seeded separately in 96-well plates, 6X 10 cells were added to each well³The cells were cultured at 37 ℃ for 24 hours. Adding DOX, CAP + TEL, DOX + CAP + TEL (the concentration ratio of DOX to CAP to TEL is 1:1:1), CTHLs, DGNs and DGNs + CTHLs (the concentration ratio of DOX to CAP to TEL is 1:1:1), setting the concentration of DOX to be 0.01-10 mu g/mL, and culturing for 48 h. Then, MTT solution was added to each well and incubated for an additional 4 hours. The medium was removed and 150 μ l LDMSO was added per well. OD (490nm) was measured using a microplate reader (ELX800, BioTek, USA), and cytotoxicity of each group was calculated. And (3) detecting the staining characteristics of the live and dead cells of the BEL-7402 cell and LX-2 cell co-culture model by adopting a Calcein-AM/PI double staining kit.

3.1.6 cell migration assay

Cell migration and invasion characteristics were analyzed using scratch test and transwell experiments. (1) Cell scratch test. Respectively mixing BEL-7402 cells and BEL-7402/C-LX-2 mixed cells at 6 × 10⁵Each well was inoculated into 6-well plates and incubated at 37 ℃ for 24 hours. Cells were scratched with 200. mu.L pipette tips and cultured in serum-free medium with different drugs (drug final concentration in cells is 2. mu.g/mL) for 24 hours. In thatThe scratched wounds were monitored by Nikoneclipse Ti-S microscope at 0 and 24 hours and photographed. Gap closure rates were measured using Image J software. (2) Cell invasion was analyzed using Transwell. The BEL-7402/C-LX-2 cocultured cells (5X 10)⁴) And inoculating in the upper chamber. After 24h of incubation, the cells attached to the lower chamber were fixed with 4% paraformaldehyde and then stained with crystal violet. Subsequently, cells were captured using a Nikoneclipse Ti-S microscope.

3.1.73D tumor sphere drug penetration analysis

And (3) constructing a 3D tumor sphere model by using a liquid covering method. Adding 1.5% low melting point agarose (50 μ L) into 96-well plate, sterilizing by ultraviolet irradiation for 30min, and mixing 2 × 10³A suspension of BEL-7402 cells was plated in a well plate and incubated for 10 days to grow into spheres. The medium was removed and DOX (DOX concentration 10. mu.g/mL), DNs (DOX concentration 10. mu.g/mL) and DGNs (DOX concentration 10. mu.g/mL) were added, respectively. After incubation for 8h, the distribution characteristics of the drug in the tumor ball are detected by using a confocal laser scanning microscope.

3.1.8 Western blot analysis of anti-hepatoma mechanism

Western blot is adopted to detect the expression conditions of proteins such as E-cadherin, Vimentin and the like in the liver cancer cells. Tumor cells and liver cancer tissues were lysed to extract proteins, spotted on 10% or 12% SDS-PAGE gels, and transferred to PVDF membrane. The PVDF membrane was then sealed with 5% emulsion TBST for 2 hours at room temperature, and then incubated with primary antibody overnight at 4 ℃. Subsequently, a secondary antibody was added and incubated at room temperature for 1 h. Detecting protein expression by hypersensitive ECL chemiluminescence solution.

3.1.9 in vivo imaging and tissue infiltration analysis

The preparation method of DID-GNs comprises the following steps: SPC and DID (DID: SPC ═ 1:30) and DSPE-PEG-GA were mixed in ethanol (GAH/SPC ratio 1:1.7, DSPE-PEG-GA/SPC ratio 1:20) and then slowly instilled into the GAH solution. After evaporation of the ethanol, DNs was sonicated in an ice bath for 5min and sequentially through 0.45 μm and 0.22 μm polyethersulfone membranes to obtain DID-GNs.

The preparation method of the DIR-HLs comprises the following steps: dioleoylphosphatidylethanolamine (DOPE)70mg, lecithin (SPC)30mg, cholesteryl succinate (CHEMS)15mg, cholesterol 15mg, imidazolyl-cholesterol (AIM-Chol)10mg, DSPE-PEG-HA5mg, and DIR 2mg were dissolved in 5mL of chloroform. Chloroform was removed with a rotary evaporator and conditions of rotary evaporation: 40 ℃ and 20 rpm. When a uniform film was formed, 5mL of PBS (pH 7.4) was added and hydrated for 1 hour. Finally, ultrasound was performed for 5min in an ice bath, and DIR-HLs was obtained sequentially through 0.45 μm and 0.22 μm polyethersulfone membranes.

Two fluorescent substances, namely DID and DIR, are selected to replace drugs, and the biological distribution of CTHLs and DGNs in H22 tumor-bearing mice is detected. 2 x 10 to⁶H22 cell suspension was injected subcutaneously into BALB/c mice to establish H22 tumor-bearing model. When the tumor volume is about 200mm³Mice were then divided into DID, DIR, DID-GNs, DIR-HLs and DID-GNs + DIR-HLs. The different formulations were diluted with sterile PBS (pH 7.4) to give concentrations of DID and DIR of 60 μ g/mL, and injected at 0.1mL per 10g tail vein per mouse body weight. The biodistribution of DID and DIR in mice was monitored at different time points using a near infrared fluorescence imaging system (NIRF). Organs and tumors were harvested after 48h and the fluorescence intensity was analyzed ex vivo.

To assess the permeability of DGNs, a H22 tumor-bearing mouse model was established. When the tumor volume is about 200mm³When, mice were divided into 5 groups: DID, DID-GNs (TEL pretreatment), DID-GNs (TEL + CAP pretreatment), DID-GNs (CTHLs pretreatment). All pretreatment groups were treated continuously via tail vein for 3 days, i.v. drug dose: DOX (3mg/kg), CAP (3mg/kg), TEL (3 mg/kg). Mice were sacrificed 12 hours after DID injection and tumors were isolated. The tumor tissue was then cut into sections 10 μm thick. Tumor sections were stained with DAPI and then examined using a confocal laser scanning microscope (TCSSP 8; come card).

3.1.10 to build 3 in vivo tumor-inhibiting models for overall evaluation of tumor-inhibiting effect

(1) Tumor model with single load. Female BALB/c mice were selected and inoculated subcutaneously on the right side at 2X 10⁶H22 cells, and the tumor volume is about 150mm³At that time, mice were randomly grouped (n ═ 5) and drug treatment was performed.

(2) Co-implantation of the model. Female BALB/c mice were selected, mixed with H22 and m-HSC cells at a 5:1 ratio, and inoculated subcutaneously 2X 10 on the right⁶Individual cell, equal tumor volume increase to about 150mm³Then, the patients were randomly grouped (n is 5) and medication was performed.

(3) In situ transplantation of tumor models. We established two subcutaneous tumor models (H22 tumor-bearing model, m-HSC/H22 co-implantation model). BALB/c mice were anesthetized with 5% chloral hydrate and the abdominal cavity was cut longitudinally to expose the liver. Injecting the liver leaf into 5 × 10 with sterile syringe⁵H22 cells (PBS,25 μ L Matrigel), and abdominal wounds were sutured.

3.1.11 evaluation of tumor inhibition of subcutaneous transplantable tumors

In order to evaluate the liver cancer treatment effect of CTHLs and DGNs, an H22 subcutaneous tumor-bearing mouse model is established, and the tumor volume is about 150mm³And is divided into 7 groups (n is 5 groups): (1) physiological saline; (2) CAP + TEL; (3) DOX; (4) DOX + CAP + TEL; (5) CTHLs; (6) DGNs; (7) DGNs + CTHLs.

Establishing m-HSC/H22 co-implantation model when the tumor reaches about 150mm³Divided into 8 groups (n ═ 5): (1) h22 control group; (2) m-HSC/H22 control group; (3) CAP + TEL; (4) DOX; (5) DOX + CAP + TEL; (6) CTHLs; (7) DGNs; (8) DGNs + CTHLs.

Intravenous drug dose: DOX (3mg/kg), CAP (3mg/kg), TEL (3mg/kg), 7 times in total. Mice were monitored for tumor size and body weight every other day after drug treatment. After two weeks, the mice were sacrificed and the tumors were harvested and weighed.

4% paraformaldehyde fixes tumor and important organs. The tumor sections were then subjected to H & E staining, immunohistochemical analysis, Masson trichrome staining and western blot analysis.

3.1.12 evaluation of tumor inhibition of in situ transplanted tumor

To further evaluate the tumor suppression effect of DGNs and ctlls, we constructed an HCC in situ tumor model. After 2 weeks, mice were divided into 5 groups: (1) physiological saline; (2) DOX + CAP + TEL; (3) CTHLs (4) DGNs (5) DGNs + CTHLs. DGNs (DOX concentration 300. mu.g/mL) and CTHLs (CAP concentration 300. mu.g/mL, TEL concentration 300. mu.g/mL) were diluted with sterile PBS (pH 7.4) and injected at 0.1mL per 10g of mouse body weight. Mice were injected with different formulations every 2 days. After 14 days, all animals were euthanized and organs were isolated for analysis.

3.1.13 Lung metastasis assay

Tail vein injection 2X 10⁶H22 cells to construct a liver cancer lung metastasis model. Then, the mice were randomly divided into 7 groups (n ═ 3): (1) physiological saline; (2) CAP + TEL; (3) DOX; (4) DOX + CAP + TEL; (5) (ii) CTHLs; (6) DGNs; (7) DGNs + CTHLs. DGNs (DOX concentration 300. mu.g/mL) and CTHLs (CAP concentration 300. mu.g/mL, TEL concentration 300. mu.g/mL) were diluted with sterile PBS (pH 7.4) and injected at 0.1mL per 10g of mouse body weight. Mice were injected with different formulations every 2 days. Mice were sacrificed after 14 days. Lungs were removed and fixed with 4% paraformaldehyde. The number of metastatic nodules was then measured and lung tissue sections were H&And E, dyeing.

3.1.14 statistical analysis

Data are presented as mean ± standard deviation. Data mapping and statistical analysis were performed using Prism 8.0 (GraphPad). The difference between the two groups is measured by student's t test or variance analysis, and the difference with P <0.05 is statistically significant.

3.2 results and discussion

3.2.1 analysis of the physicochemical Properties of DGNs and CTHLs

The characterization of CTHLs and DGNs is shown in Table 2. The average particle size of DGNs is about 32nm (shown in a diagram in figure 4), so that the kidney metabolism caused by too small particle size is avoided, and the DGNs are favorably infiltrated to the deep part of a tumor. The zeta potential of DGNs is negative. The encapsulation efficiency and drug loading of DOX were 88.9% and 2.38%, respectively. The CTHLs have an average particle diameter of 160nm (shown in B in FIG. 4), and have a potential characteristic of a negative potential. The encapsulation efficiency and drug loading of CAP were 83.7% and 2.79%, respectively, and the encapsulation efficiency and drug loading of TEL were 83.1% and 2.77%, respectively. TEM images showed that DGNs and CTHLs were regularly spherical at pH7.4, while CTHLs increased in particle size and irregular in shape at pH5.0 (FIG. 4, panels C-E). Next, the stability of DGNs and CTHLs in PBS and RPMI 1640 medium was investigated. As shown in FIG. 4, panels F and G, the results indicate that DGNs and CTHLs remain stable for 7 days.

The release of DOX from DGNs is shown in graph H of fig. 4. The low in vitro cumulative release rate of DGNs over 8h compared to free DOX indicates that DGNs are long circulating. In addition, the sensitivity to pH was verified by measuring the release rate of ctlls at different pH conditions. As shown in fig. 4, panel I, the cumulative release rate of CAP over 48h at pH5.0 was 72.5%, significantly higher than pH 6.5 (54.3%) and pH7.4 (42.5%), indicating that DOPE has an effect of enhancing drug release under acidic conditions. The cumulative release rate of TEL is significantly lower than pH7.4 (69.2%) at pH5.0 (10.4%) and pH 6.5 (42.8%), the main reason being its weak acidity. Thus, the rapid release of TEL from lysosomes into the cytoplasm by pH-sensitive liposomes facilitates its action.

TABLE 2 characterization of DGNs and CTHLs

3.2.2 cellular uptake and drug Retention assays

Research shows that factors such as particle size, potential, surface modification and the like of the nano-carrier play an important role in the uptake of tumor cells. Nanoparticles smaller than 20nm are rapidly cleared by the kidney in the blood circulation in vivo, whereas nanoparticles larger than 100nm are not favorable for cellular uptake. In this study, we prepared 32nm nanospheres. As shown in the graph A in FIG. 5, the fluorescence signal of DNs group is stronger than that of the free DOX group, indicating that the nano-micelle with smaller particle size is more easily absorbed by the cell. In addition, the DGNs group showed stronger fluorescence signals than DNs, indicating that DGNs may increase DOX uptake by HCC cells through GA-mediated endocytosis. The fluorescence intensity of each group was quantified by flow cytometry, and the results were consistent with the confocal laser results (panel B in fig. 5). We also examined the uptake of CTHLs by LX-2, and the cells treated with the non-targeted liposome group showed a stronger green color, as shown in Panel A-B in FIG. 6. This is probably due to the overexpression of the CD44 receptor on the surface of LX-2 cells, and the HA-modified liposomes could promote receptor-mediated endocytosis.

Research shows that CAFs in the microenvironment of liver cancer can induce the drug efflux of tumor cells and reduce the sensitivity of tumors to drugs. We established LX-2/BEL-7402 co-culture model, simulated in vivo environment, and tested the drug retention effect of different preparation groups (shown in C in FIG. 5). The fluorescence signal of the LX-2/BEL-7402 group was weaker than that of BEL-7402 alone, indicating that CAFs can reduce DOX accumulation in tumor cells. Interestingly, after CAP and TEL were added to the co-culture model, the fluorescence signals of each group were significantly enhanced, indicating that CAP and TEL were effective in inhibiting drug efflux. Intracellular DOX was measured by flow cytometry (shown in panel D of FIG. 5), consistent with the CLSM assay results.

3.2.3 detection of the penetration of DGNs in 3D tumor spheres

To evaluate the penetration of DGNs, a 3D tumor sphere model was established and Z-stack scans of CLSM were used to monitor the penetration of different formulations. As shown in panel a-B of fig. 7, DOX is distributed primarily at the periphery of the 3D tumor sphere, rather than internally. In contrast, two nanoparticle groups can penetrate inside the sphere. The fluorescence intensity of the DGNs in the deep tumor is obviously higher than that of the DNs group, which shows that the modification of glycyrrhetinic acid improves the penetration effect of the nanoparticles.

3.2.4 CTHLs enhance cytotoxicity of DGNs

The occurrence and development of tumors are closely related to TME, and CAFs in TME can improve the drug resistance of tumor cells. However, the traditional in vitro study model is tumor cell and cannot truly simulate TME. In the study, on the basis of liver cancer cells (BEL-7402), a BEL-7402/LX-2 co-culture cell model is newly established, TME is simulated, and in-vitro drug toxicity of different dosage forms is detected (shown in A picture in figure 8). As shown in the B-C diagram in FIG. 8, each group had a significant concentration dependence. The IC50 was significantly reduced in BEL-7402/LX-2 co-cultured cells in the free drug group compared to BEL-7402 cells alone. Wherein the IC50 of free DOX was 7.13. mu.g/mL higher than BEL7402 cells for the co-culture system by 6.4 times. This is because activation of LX-2 decreases the sensitivity of BEL-7402 to drugs, resulting in tumor resistance. Meanwhile, the DGNs + CTHLs group had higher toxicity than the free DOX + CAP + TEL group. This is probably due to the increased drug uptake of DGNs through GA receptor mediated effects, which was also demonstrated in cellular uptake studies. Live/dead staining results also indicated that the DGNs + CTHLs group had a stronger pro-apoptotic effect than the other agent groups (shown in panel D in FIG. 8).

3.2.5 CTHLs can inhibit migration of hepatocarcinoma cells by blocking EMT

To investigate the effect of different preparations on cell migration, we established a BEL-7402 single cell model and a BEL-7402/LX-2 co-culture model (shown in A in FIG. 9). As shown in the B and D panels of FIG. 9, the mobility of BEL-7402/LX-2 co-cultured cells (49.3%) was significantly higher than that of BEL-7402 cells alone (39.97%), indicating that LX-2 promoted cell migration. Furthermore, the group of DGNs + CTHLs was effective in inhibiting cell migration of BEL-7402 and BEL-7402/LX-2 alone (FIGS. 9C and E), probably because treatment with DGNs in combination with CTHLs effectively inhibited cross-talk between LX-2 and BEL-7402, thereby effectively inhibiting cell migration. In addition to cell scratch studies, we used a transwell experiment to further examine cell invasion capacity. As shown in a-B diagram in fig. 10, the number of cells affected was less in the DGNs + ctlls group compared to the other groups, indicating that the lipid globule + nanosphere mixture formulation effectively inhibited tumor invasion and migration.

Studies have shown that CAFs promote tumor migration by inducing tumor cells EMT. We examined the expression of EMT-associated proteins by western blot experiments. As shown in FIG. 10, panels C-D, the expression of Vimentin and E-Cadherin in LX-2/BEL-7402 co-cultured systems was significantly up-regulated and down-regulated, compared to BEL-7402 cells, indicating that CAFs promote EMT in tumor cells. In addition, compared with a control group, the expression of Vimentin in the CAP + TEL group is obviously reduced, and the expression of E-Cadherin is obviously increased, which indicates that CAP combined with TEL can effectively inhibit CAFs-mediated EMT and inhibit tumor cell migration.

3.2.6 tissue distribution studies of DGNs and CTHLs in vivo

To assess the tissue distribution of nanospheres and lipid macrospheres in vivo, DID and DIR were encapsulated in DGNs and ctlls, respectively, and the biodistribution of DID-GNs and DIR-HLs in vivo was observed by NIRF technique. As shown in FIG. 11A, DID-GNs were observed to aggregate at the tumor site at the DID excitation wavelength, since modification of glycyrrhetinic acid improved the liver cancer targeting ability of DID-GNs. At the DIR excitation wavelength, DIR-HLs was also observed to be distributed at the tumor site, probably due to the increased accumulation of DIR in the tumor region as a result of the EPR effect of the liposomes. In addition, it was observed at the tumor site

Resonance Energy Transfer (FRET) effect. At the DID excitation wavelength, the DID fluorescence intensity (excitation peak is 648 nm; emission peak is 670nm) of the DID-GNs group is obviously enhanced compared with the DID-GNs + DIR-HLs group. On the contrary, at the excitation wavelength of DIR, the fluorescence intensity of DIR (FRET acceptor: excitation peak, 750 nm; emission peak, 782nm) of DIR-HLs group is obviously reduced compared with DID-GNs + DIR-HLs group, which indicates that DID-GNs and DIR-HLs can be gathered at the tumor site, and is favorable for exerting the synergistic antitumor effect of the dosage form. Meanwhile, the in vitro fluorescence quantification results also confirmed the FRET effect (panels B-D in FIG. 11). In addition, we frozen-sectioned DID-GNs group tumors, the DID-GNs group showed stronger fluorescence intensity at deep tumor sites (panels E and F in FIG. 11) compared to free DID group, indicating that small size nanospheres are more favorable for tissue penetration.

3.2.7 CTHLs enhance deep penetration of DID-GNs into tissues

Recent studies have shown that CAFs and ECM in the tumor microenvironment inhibit drug penetration in tumor tissues, reducing the tumor therapeutic efficacy. Therefore, the CTHLs can inhibit the activation of CAFs, reduce ECM in a tumor microenvironment and effectively increase the deep penetration of the drug. To confirm this approach, a model of co-implantation of H22/m-HSCs into tumors was constructed. After 3 days of treatment, the drug permeation was observed for each treatment group. As shown in Panel A-B of FIG. 12, the DID-GNs group showed stronger fluorescence intensity compared to free DID, which is consistent with the experimental results of 3D tumor spheres. In addition, after the TEL or the TEL + CAP is added, the fluorescence intensity in the deep part of the tumor is enhanced, which shows that the TEL and the CAP can promote the micro-environment to be loose and improve the permeability of the medicine. And after the CTHLs are added, the fluorescence inside the tumor tissue is stronger, which shows that the CTHLs have stronger microenvironment regulation effect than free TEL + CAP, and the deep penetration of DID-GNs in the liver cancer tissue is effectively enhanced.

3.2.8 GNs combined with CTHLs inhibit H22 subcutaneous graft tumor development

A H22 tumor-bearing mouse model was established to evaluate the antitumor activity of DGNs in combination with CTHLs (FIG. 13, panel A). As shown in panels B and D of fig. 13, the drug-treated groups showed different degrees of tumor growth inhibition effects compared to the saline group, in which the tumor volumes of the DGNs + CTHLs group were much smaller than those of the DGNs or CTHLs group, indicating that the combination of DGNs and CTHLs could improve the antitumor effect. Histological analysis results show that in the DGNs + CTHLs group, tumor tissues have obvious nucleolysis and vacuolization of cytoplasm, and the combined treatment group has better anti-tumor curative effect (shown in a picture A in figure 13). The body weight measurements showed that all the nanoformulation groups were similar to the control group, while the free DOX group had lower body weight (panel C in figure 13). In addition, the organ H & E results (shown in a in fig. 14) show that after the nano-preparation treatment, the cell nuclei were uniform in size, the structure was not significantly changed, the myocardial muscle fibers were tightly arranged and the morphology was regular, whereas the myocardial nuclei of mice in the DOX treatment group were firmly contracted or ruptured, and the myocardial arrangement was disorderly and loosely, and significantly dissolved and ruptured. The results show that the nanometer preparation effectively reduces the toxic and side effects of DOX.

3.2.9 CTHLs promote the inhibitory effect of DGNs on m-HSC/H22 co-implanted tumor-bearing models

The liver cancer microenvironment comprises a plurality of stromal cells such as hepatic stellate cells, immune cells and the like, and the stromal cells play an important role in the occurrence and development of primary tumors. It has been reported that CAFs in the tumor microenvironment promote tumor cell proliferation and migration. Therefore, to mimic TME, we mixed murine HSCs (m-HSCs) with H22 cells, co-implanted into the right dorsal side of mice, established an m-HSC/H22 co-implanted tumor-bearing model, and validated the in vivo anti-tumor effect of each formulation (shown in panel A in FIG. 15).

As shown in panels B and D of FIG. 15, the tumor volume of the m-HSC/H22 tumor-bearing mouse model was significantly increased compared to the conventional H22 tumor-bearing mouse model. This is because the CAFs in TME promote tumor proliferation. In addition, compared with the free drug group, the tumor volumes of the DGNs or the CTHLs are reduced to different degrees, which shows that the lipid globules and the nano globules can obviously improve the anti-tumor curative effect. H & E staining showed that apoptotic regions of DGNs or CTHLs were significantly increased compared to the free drug group (shown in panel A in FIG. 16), while the DGNs + CTHLs combination treatment group showed the strongest inhibition of tumor growth. This is probably due to the fact that CTHLs release CAP and TEL, inhibit the activation of CAFs, and enhance the liver cancer treatment effect of DGNs. Masson staining and α -SMA immunohistochemistry experiments showed (panels B-C in FIG. 16) that the DGNs + CTHLs group had less collagen deposition and smaller α -SMA positive regions than the DGNs group, indicating that CTHLs were effective in inhibiting CAF activation and reducing collagen production. In addition, CD31 protein immunohistochemistry experiment shows that DGNs + CTHLs group tumor microvessels are obviously reduced compared with free drugs, indicating that the nano-carrier based combination treatment strategy can inhibit tumor angiogenesis (figures D-E in figure 16).

To analyze the anti-tumor mechanism of DGNs + CTHLs, we performed Western blot detection. As shown in FIG. 16, panels F-G, the expression of E-Cadherin was decreased in mHSC/H22 mice compared to H22 mice, and the expression of Vimentin was reversed. This suggests that the addition of m-HSCs cells promotes the EMT of HCC cells. After the medicine is added, the E-Cadherin expression of the DGNs + CTHLs group is obviously increased, and the Vimentin expression is obviously reduced, so that the DGNs and the CTHLs are combined for treatment, and the EMT of HCC can be effectively reversed.

3.2.10 DGNs combined with CTHLs for inhibiting in situ tumors

In order to evaluate the anti-HCC effect of DGNs and CTHLs, an orthotopic liver cancer transplantation tumor model (shown in a picture in figure 17) is established. As shown in panels B-C of FIG. 17, compared to the free DOX + CAP + TEL treatment group, the tumor area of the DGNs + CTHLs treatment group was significantly reduced, and the number of tumor apoptosis regions was increased, indicating that the combination of DGNs and CTHLs could effectively improve the anti-hepatoma effect. Masson and immunohistochemistry (shown in panels D-F of fig. 16) showed that the DGNs + CTHLs treated group had smaller α -SMA positive areas, less collagen deposition and reduced neovascularization compared to the DGNs treated group. This indicates that CTHLs regulate and control the tumor microenvironment, effectively inhibit CAFs activation in the microenvironment, reduce ECM deposition, and reduce angiogenesis.

3.2.11 DGNs combined with CTHLs for inhibiting lung metastasis

Liver cancer metastasis is a major cause of poor prognosis of liver cancer. In order to test the efficacy of DGNs combined with CTHLs in inhibiting liver cancer lung metastasis, a liver cancer lung metastasis model was established by intravenous injection of H22 cells (shown in A in FIG. 18). Panel B in figure 18 shows no significant weight change in the nanoformulation group compared to the control group. In FIG. 18, panels C-D show the status of lung metastasis nodules, and the number of nodules in the DGNs + CTHLs group is smaller than that in the other groups, showing stronger lung metastasis inhibition effect. The H & E stained images of the lungs (shown in E in fig. 17) showed that there were different degrees of lung metastases in each treatment group, while the lung metastases were small in the DGNs + CTHLs group, suggesting that DGNs in combination with CTHLs effectively inhibited liver cancer metastasis.

Claims

1. A composite vector, comprising a first targeting vector and a second targeting vector, wherein the first targeting vector comprises the following components: dioleoyl phosphatidylethanolamine, lecithin, cholesterol succinate monoester, cholesterol, imidazolyl cholesterol, and DSPE-PEG-HA; the DSPE-PEG-HA is prepared by passing DSPE-PEG-NH₂Is prepared by mixing hyaluronic acid; the imidazolyl cholesterol is prepared from chloroformic acid cholesterol and N- (3-aminopropyl) imidazole; the second targeting vector comprises the following components: compound phospholipid and glycocholic acid, wherein the compound phospholipid comprises lecithin and DSPE-PEG-GA; the particle size of the first targeting vector is 150 nm-200 nm; the particle size of the second targeting carrier is 25-50 nm.

2. The composite carrier of claim 1, wherein the first targeting carrier comprises dioleoyl phosphatidylethanolamine, lecithin, cholesterol succinate monoester, cholesterol, imidazolyl cholesterol, and DSPE-PEG-HA in a mass ratio of 10-15: 5-10: 1-5.

3. The composite carrier of claim 1, wherein the mass ratio of lecithin to DSPE-PEG-GA in the second targeting carrier is 15-20: 1; the mass ratio of the composite phospholipid to glycocholic acid is 1.4-3.5: 1.

4. The composite carrier according to claim 1, wherein the imidazolyl cholesterol is synthesized by chloroformic acid cholesterol and N- (3-aminopropyl) imidazole in the presence of triethylamine; and/or said DSPE-PEG-NH₂The molecular weight of the hyaluronic acid is 2000-5000 Da, and the molecular weight of the hyaluronic acid is 2000-5000 Da.

5. A drug carrier is characterized in that the drug carrier comprises 10 to E15: 5-10: 1-5 dioleoylphosphatidylethanolamine, lecithin, cholesterol succinic acid monoester, cholesterol, imidazolyl cholesterol and DSPE-PEG-HA; the DSPE-PEG-HA is prepared by passing DSPE-PEG-NH₂Is prepared by mixing with hyaluronic acid; the imidazolyl cholesterol is prepared from chloroformic acid cholesterol and N- (3-aminopropyl) imidazole; the drug loaded by the drug carrier is capsaicin and telmisartan; and/or the drug carrier comprises composite phospholipid and glycocholic acid in a mass ratio of 1.4-3.5: 1, the composite phospholipid comprises lecithin and DSPE-PEG-GA in a mass ratio of 15-20: 1, and the drug loaded on the drug carrier is adriamycin.

6. The method for preparing the composite carrier according to claim 1, comprising the steps of:

(S1) preparation of first targeting vector

(S12) Synthesis of Imidazoylcholesterols: synthesizing imidazolyl cholesterol from chloroformic acid cholesterol and N- (3-aminopropyl) imidazole under the catalytic action of triethylamine;

7. The drug carrier system is characterized by comprising a first drug carrier system and a second drug carrier system, wherein the first drug carrier system comprises a first targeting carrier, capsaicin loaded by the first targeting carrier andtelmisartan; the first targeting vector comprises dioleoyl phosphatidylethanolamine, lecithin, cholesterol succinate monoester, cholesterol, imidazolyl cholesterol and DSPE-PEG-HA; the DSPE-PEG-HA is prepared by passing DSPE-PEG-NH₂Is prepared by mixing hyaluronic acid; the imidazolyl cholesterol is prepared from chloroformic acid cholesterol and N- (3-aminopropyl) imidazole; the second drug carrier system comprises a second targeting carrier and adriamycin loaded through the second targeting carrier; the second targeting carrier comprises a compound phospholipid and glycocholic acid, and the compound phospholipid comprises lecithin and DSPE-PEG-GA; and/or in the first drug loading system, the drug loading rate of capsaicin is 2-3%, and the drug loading rate of telmisartan is 2-3%; in the second medicine carrying system, the medicine carrying amount of the adriamycin is 2-3%.

8. A method of preparing the drug delivery system of claim 7, comprising the steps of:

(1) preparation of the first targeting vector

(1a) Synthesis of DSPE-PEG-HA: DSPE-PEG-NH₂Activating EDC and NHS, and then performing crosslinking reaction with hyaluronic acid in a nitrogen environment to obtain DSPE-PEG-HA;

9. The use of the drug delivery system of claim 7 in the preparation of a liver cancer drug.

10. A pharmaceutical composition, which is characterized by comprising capsaicin, telmisartan and adriamycin.