A drug composition for liver metastasis of pancreatic cancer under the background of metabolic-related fatty liver disease and a preparation method and application thereof

Abstract

The application discloses a kind of drug composition for pancreatic cancer liver metastasis under the background of metabolism-related fatty liver disease and preparation method and application thereof, the drug composition is with double-layer fat molecule spherical liposome as carrier, photosensitizer is loaded in the bilayer of liposome, chemotherapeutic drug gemcitabine is loaded in the cavity of liposome, gene therapy drug SiRNA CD44 is adsorbed on the surface of liposome.The GIR-Lip of the application can realize controllable killing and immune activation of tumor under laser irradiation, efficiently silence the high invasion and migration ability of CD44 tumor cells, combined with the first-line chemotherapeutic drug gemcitabine for pancreatic cancer, can ensure the combined effect, make up the drug resistance and curative effect loss caused by long-term use of gemcitabine.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and pharmaceutical formulation technology, specifically relating to a drug composition for pancreatic cancer liver metastasis in the context of metabolic-related fatty liver disease, its preparation method and application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Pancreatic cancer is known as the "king of cancers," with 80%-85% of cases diagnosed at an advanced stage or already metastatic. Even in a small number of patients who can undergo radical surgery, liver metastasis often occurs shortly after the procedure. Despite advancements in cancer treatment, the five-year survival rate for pancreatic cancer patients with liver metastases remains stagnant at 1% to 3%, showing no significant improvement. Improving the cure rate and survival rate for pancreatic cancer patients with liver metastases is therefore urgently needed.

[0004] Current technologies face significant challenges in treating pancreatic cancer liver metastases, especially in cases complicated by metabolic-associated fatty liver disease (MAFLD), where treatment outcomes are even worse. The main problems are: First, pancreatic cancer liver metastases have an extremely poor prognosis, and traditional chemotherapy (such as gemcitabine) has limited effectiveness and is prone to drug resistance. Second, the liver inflammation, fibrosis, metabolic disorders, and deeply immunosuppressive microenvironment caused by MAFLD not only increase the risk and invasiveness of pancreatic cancer liver metastases but also severely weaken the efficacy of various therapies, including chemotherapy, radiotherapy, and immunotherapy. Specifically: MAFLD alters liver structure and blood flow, and the dense matrix of metastatic lesions makes it difficult for free drugs to be effectively delivered and accumulate at the tumor site; the strong immunosuppressive state jointly promoted by MAFLD and tumors makes it difficult for the immune system to effectively attack the tumor; the high expression of the tumor cell marker CD44 in pancreatic cancer is closely related to drug resistance, recurrence, and metastasis, and current therapies are unable to effectively inhibit the high invasiveness and migration ability of tumors; single therapies (such as chemotherapy, immunotherapy, or photothermal therapy) have limited effectiveness in addressing this complex situation. Currently, there is a particular lack of comprehensive treatment strategies and efficient delivery systems specifically designed for the clinical scenario of pancreatic cancer liver metastasis in the context of MAFLD, which can overcome multiple barriers (drug delivery obstacles, immunosuppression, and high invasiveness and migration ability) and achieve synergistic effects through multiple mechanisms. Summary of the Invention

[0005] To address the treatment challenges of MAFLD complicated with pancreatic cancer liver metastases, this invention designs a novel multifunctional lipid system (Gemcitabine-IR780-siRNA CD44-Liposome, GIR-Lip). The lipid carrier in this system consists of DOTAP and cholesterol. The photosensitizer IR780 is loaded into a lipid bilayer, the chemotherapeutic drug gemcitabine is loaded into the liposome cavity, and the gene therapy drug siRNA CD44 is adsorbed onto the liposome surface. This co-loading of the three drugs aims to overcome drug delivery barriers in the MAFLD liver metastasis microenvironment, reverse the severe immunosuppressive state, effectively reduce the invasion and migration abilities of tumor cells, and improve the treatment efficacy of MAFLD complicated with pancreatic cancer liver metastases.

[0006] The technical solution adopted in this invention is as follows:

[0007] In a first aspect of the invention, a pharmaceutical composition for pancreatic cancer liver metastases in the context of metabolism-related fatty liver disease is provided. The pharmaceutical composition uses spherical liposomes as carriers of bilayer lipid molecules, with a photosensitizer loaded in the liposome bilayer, a chemotherapeutic drug gemcitabine loaded in the liposome cavity, and a gene therapy drug siRNA CD44 adsorbed on the surface of the liposomes.

[0008] In a second aspect of the invention, a method for preparing the pharmaceutical composition for pancreatic cancer liver metastases in the context of metabolism-related fatty liver disease is provided, the method comprising the following steps:

[0009] Globular liposomes containing bilayer lipid molecules and photosensitizers are added to an organic solvent and stirred to form a clear solution. The solution is then evaporated to form a film, hydrated with gemcitabine solution, and then passed sequentially through liposome extruders at 200 nm, 100 nm, and 50 nm to obtain an aqueous solution containing photosensitizers and gemcitabine Gem liposomes (GI-Lip). The aqueous solution of GI-Lip, CD44 siRNA solution, and protamine solution are mixed and incubated to obtain GIR-Lip, which is a drug composition for pancreatic cancer liver metastases in the context of metabolism-related fatty liver disease.

[0010] In one or more embodiments of the present invention, the spherical liposome raw material of the bilayer lipid molecule is phospholipid and accessory phospholipid, and the mass ratio of the phospholipid and accessory phospholipid is 1.5~2:1; preferably, the mass ratio of the phospholipid and accessory phospholipid is 1.81:1.

[0011] The phospholipid is one or more of (1,2-dioleoyloxypropyl)trimethylammonium chloride (DOTAP), 1,2-dioleoyloxy-3-methylammonium propane chloride (DOTMA), and 2-((8-((3β)-cholest-5-en-3-yloxy)octyl)oxy)-N,N-dimethyl-3-((9Z,12Z)-octadec-9,12-dien-1-yloxy)propyl-1-amine)DLin-MC38-DMA, and the auxiliary phospholipid is one or more of cholesterol (Cho) and dioleoylphosphatidylethanolamine (DOPE). Preferably, the phospholipid is DOTAP, and the auxiliary phospholipid is Cho. Experimental verification shows that the combination of DOTAP and Cho has high transfection capacity. Experimental results showed that gemcitabine encapsulation efficiency of up to 40% was obtained by spin-film hydration with DOTAP and Cho as lipid materials at 60℃, which is superior to 10% with phosphatidylcholine PC and Cho as lipid materials and 35% with PLGA polymer as material.

[0012] In one or more embodiments of the present invention, the photosensitizer is one or more of IR780, IR820, and ICG.

[0013] In one or more embodiments of the present invention, the CD44 siRNA sequence includes, but is not limited to, CUCCCCACUAUGACACAUAUTT, GACUCAUGGAUCCGAAUUATT, GGACCGGUUACCAUAACUATT, and CAGGAUGACUCCUUCUUUATT, with the preferred sequence being CAGGAUGACUCCUUCUUUATT.

[0014] In one or more embodiments of the present invention, the ratio of the spherical liposome raw material of the bilayer lipid molecule to the organic solvent is (15~25) mg: (1~5) mL.

[0015] In one or more embodiments of the present invention, the mass ratio of the photosensitizer and gemcitabine is 1:5 to 15; the mass ratio of the aqueous solution of GI-Lip, CD44 siRNA solution and protamine solution is 4 to 16:1:1.

[0016] The ratio of lipids and CD44 siRNA used for transfection was screened in a series of tests, and the optimal ratio of GI-Lip (based on total phospholipid mass), CD44 siRNA and protamine was found to be 8:1:1.

[0017] In one or more embodiments of the present invention, the solution used for hydration is a phosphate buffer solution, a 5% glucose solution, or a 0.9% physiological saline solution.

[0018] In one or more embodiments of the present invention, the organic solvent is selected from one or more of ethanol, chloroform, and diethyl ether.

[0019] In a third aspect, the invention provides the use of the pharmaceutical composition in the preparation of a pharmaceutical formulation for pancreatic cancer liver metastases in the context of metabolic-associated fatty liver disease (MAFLD). This invention is particularly aimed at providing effective therapeutic drugs and strategies for patients with MAFLD and pancreatic cancer liver metastases. Its core application lies in integrating chemotherapy, gene silencing therapy, and photothermal therapy through a specially formulated liposomal nanocarrier for highly efficient co-delivery, aiming to overcome the complex and challenging tumor microenvironment barriers shaped by both MAFLD and pancreatic cancer liver metastases, providing an innovative synergistic treatment option for this clinically challenging condition with extremely poor prognosis. Therefore, this invention is directly applied to the development of effective therapeutic drugs and strategies for patients with MAFLD and pancreatic cancer liver metastases.

[0020] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects:

[0021] (1) The present invention successfully constructed a novel combination therapy system GIR-Lip for MAFLD combined with pancreatic cancer liver metastasis. Liposomes were constructed using DOTAP and cholesterol as carriers. IR780 and gemcitabine were loaded into the bilayer and cavity of the liposomes respectively by thin film dispersion. siRNACD44 was mixed with protamine and then further incubated with GI-Lip to obtain GI-Lip with siRNACD44 adsorbed on the surface, i.e., GIR-Lip.

[0022] (2) The GIR-Lip of the present invention can achieve controlled killing and immune activation of tumors under laser irradiation, and effectively silence CD44 to inhibit the high invasion and migration ability of tumor cells. Combined with gemcitabine, the first-line chemotherapy drug for pancreatic cancer, it can ensure the combined effect and make up for the drug resistance and efficacy loss caused by long-term use of gemcitabine.

[0023] (3) This invention achieves highly efficient inhibition of MAFLD combined with pancreatic cancer liver metastasis. In a mouse model of MALFD combined with KPC liver metastasis, the combined regimen effectively inhibited the growth of liver metastatic tumors compared with the control group.

[0024] (4) The DOTAP and cholesterol concentrations, as well as the loading methods of IR780 and gemcitabine in this invention, are all optimized data, and their therapeutic effects are good. The raw materials used in this invention are simple, safe, and reliable. The carrier materials used, including DOTAP and cholesterol, are safe, non-toxic, and biodegradable, and have good biocompatibility. Attached Figure Description

[0025] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0026] Figure 1 (a) Particle size of GIR-Lip, (b) Potential of GIR-Lip, (c) Electron micrograph of GIR-Lip.

[0027] Figure 2 (a) Temperature curves of GIR-Lip at different concentrations, (b) Temperature curves of GIR-Lip at different powers, (c) CRT expression induced by different formulations.

[0028] Figure 3 (a) The toxicity of combined chemotherapy and gene therapy on KPC cells, (b) The antitumor toxicity after GIR-Lip laser irradiation.

[0029] Figure 4 Different formulations silence CD44 protein expression.

[0030] Figure 5 Antitumor effects of different formulations on a pancreatic cancer liver metastasis model in MAFLD mice. Detailed Implementation

[0031] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0032] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0033] Addressing key deficiencies and unmet clinical needs in the prior art, the core objective of this invention is to provide an innovative pharmaceutical composition and its delivery system for the effective treatment of pancreatic cancer liver metastases in the context of MAFLD. Specifically, this invention aims to creatively combine three active ingredients with different mechanisms of action: the chemotherapeutic drug gemcitabine, siRNA targeting and silencing the key tumor invasion and migration gene CD44, and the near-infrared photosensitizer / photothermal agent IR780, utilizing a specific liposome composed of cationic lipid DOTAP and cholesterol as a co-delivery carrier. Through this design, this invention seeks to achieve the following objectives: efficiently co-encapsulating hydrophilic gemcitabine, negatively charged CD44 siRNA, and hydrophobic IR780 using DOTAP / cholesterol liposomes, protecting the siRNA from degradation, improving the solubility and stability of IR780, and enhancing drug accumulation and tumor cell uptake in the liver and metastatic lesions of MAFLD by leveraging the nanoscale size of the liposomes and the cationic properties of DOTAP. More importantly, this composition achieves a triple synergistic effect: chemotherapy (gemcitabine directly kills tumor cells), gene therapy (CD44 siRNA-specific silencing weakens tumor cell invasion, migration, and immunosuppression), and photothermal / photoimmunotherapy (IR780 generates local high temperatures under near-infrared light irradiation to directly ablate tumors and induce immunogenic cell death). This synergy aims to overcome drug delivery barriers in the liver metastasis microenvironment of MAFLD, reverse the strong immunosuppressive state (transforming "cold" tumors into "hot" tumors), and effectively reduce the invasiveness and migration of tumor cells. The ultimate goal is to significantly improve the treatment efficacy of MAFLD with pancreatic cancer liver metastases, reduce systemic toxicity, and improve patient prognosis.

[0034] The core of this invention is to develop a novel treatment regimen for patients with MAFLD complicated by pancreatic cancer and liver metastases. Based on existing clinical treatments, the regimen integrates phototherapy, chemotherapy and gene therapy. The liposome delivery system can overcome drug delivery barriers and effectively accumulate in the liver tumor. Then, phototherapy, chemotherapy and gene therapy work together to kill the tumor while activating the immune response and enhancing the killing of liver metastases.

[0035] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0036] Example 1: Preparation and Characterization of GIR-Lip

[0037] A liposome GIR-Lip encapsulating an antitumor drug comprises DOTAP, a blank liposome composed of cholesterol, and active substances. The active substances include the antitumor chemotherapy drug gemcitabine, the photosensitizer IR780, and CD44 siRNA.

[0038] The preparation method of GIR-Lip is as follows:

[0039] 13.98 mg DOTAP, 7.72 mg cholesterol, and 1 mg photosensitizer IR780 were added to 2 ml of ethanol and stirred at room temperature to form a clear solution. The organic solvent was removed using a rotary evaporator at 40-60°C, and a film was formed. 2 ml of 5 mg / mL gemcitabine-free aqueous solution was added, and the mixture was hydrated at 50-60°C for 30 min. The film was then passed through a liposome extruder at 200 nm, 100 nm, and 50 nm for 10 times each to obtain an aqueous solution containing IR780 and Gem liposomes (GI-Lip). The aqueous solution of GI-Lip, CD44 siRNA solution (CD44 siRNA sequence 5´-CAGGAUGACUCCUUCUUUATT-3´), and protamine solution (GI-Lip (calculated based on total phospholipid mass), CD44 siRNA, and protamine in a mass ratio of 8:1:1) were mixed and incubated for 30 min to obtain GIR-Lip.

[0040] Detection of particle size, potential and morphology of GIR-Lip ( Figure 1 ).

[0041] Other control liposomes were prepared according to the method described in Example 1.

[0042] Example 2 Evaluation of the photothermal and immune-activating capabilities of GIR-Lip

[0043] The GIR-Lip prepared in Example 1 was diluted with PBS to IR780 concentrations of 1, 5, and 10 μg / mL, at 0.8 W / cm². 2 Under laser irradiation, the temperature rise curve of GIR-Lip was recorded every 30 seconds for 5 minutes to examine the relationship between IR780 concentration and temperature rise. Further evaluation was then conducted at different powers (0.6 W / cm²). 2 0.8 W / cm 2 1.0 W / cm 2 The temperature rise curve of GIR-Lip is shown. The results are as follows: Figure 2 a and Figure 2 As shown in b, the temperature of GIR-Lip increased more significantly with increasing concentration and laser power. Further evaluation of GIR-Lip's ability to activate immune cells followed. After incubation with tumor cells, GIR-Lip was irradiated with laser. Laser irradiation released damage-related molecular patterns that promoted DC cell maturation, thereby activating T cell immunity. Therefore, this example evaluated the expression of damage-related molecular patterns induced by GIR-Lip, such as calreticulin (CRT). The results are as follows... Figure 2 As shown in c, compared to other formulations, GIR-Lip significantly increases CRT expression after laser irradiation.

[0044] Example 3: Evaluation of the chemotherapy effect of GIR-Lip

[0045] The killing effect of GIR-Lip in Example 1 was evaluated using the CCK8 assay on mouse pancreatic cancer cell line KPC. The results are as follows... Figure 3 As shown in figure a, the results indicate that gemcitabine liposomes (G-Lip) can effectively kill tumor cells, and the combined efficacy is even better when combined with siRNACD44. Further evaluation of the antitumor efficacy of GIR-Lip after laser irradiation yielded the following results. Figure 3 b shows that its anti-tumor effect is best after laser irradiation.

[0046] Example 4: Evaluation of CD44 Silencing Effect

[0047] IR780 functions only as a photosensitizer, while gemcitabine functions as a chemotherapeutic agent to kill tumor cells. Therefore, to evaluate the silencing effect of the Lip vector on CD44 siRNA, three groups were set up: free siRNA CD44, Lip, and R-Lip. KPC cells were seeded into 6-well plates and cultured overnight. Then, culture media containing free siRNA CD44, Lip, and R-Lip were added, and incubation was carried out for 48 h. Cells were then collected, lysed using RIPA lysis buffer containing PMSF, and proteins were collected. Western blotting was used to evaluate the efficiency of the formulation in silencing CD44, and the results are shown below. Figure 4 As shown, encapsulating siRNA CD44 in Lip can efficiently silence CD44 protein expression and exert a gene therapy effect.

[0048] Example 5: Evaluation of the antitumor effect of GIR-Lip

[0049] C57BL / 6 mice were first fed a high-fat diet to establish a MAFLD model. After establishment, KPC cells were injected into the spleen to establish a mouse model of pancreatic cancer liver metastasis. The mice were then randomly divided into 5 groups (n=6 per group): 1. NS, 2. R-Lip, 3. GR-Lip, 4. I-Lip+Laser, 5. GIR-Lip+Laser. Intravenous injections were administered every 3 days. After 16 days, the mice were euthanized, and the livers were isolated. The results showed that GR-Lip combined with chemotherapy had a better anti-metastatic effect than R-Lip alone. The single photothermal therapy I-Lip+Laser had a weaker anti-metastatic effect than GR-Lip. However, when photothermal therapy was combined with gene therapy and chemotherapy, GIR-Lip+Laser almost completely inhibited pancreatic cancer liver metastasis. Figure 5 ).

[0050] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A pharmaceutical composition for treating pancreatic cancer liver metastases in the context of metabolism-related fatty liver disease, characterized in that, This pharmaceutical composition uses spherical liposomes of a lipid bilayer as a carrier. A photosensitizer is loaded in the liposome bilayer, the chemotherapeutic drug gemcitabine is loaded in the liposome cavity, and the gene therapy drug CD44 siRNA is adsorbed on the surface of the liposome. The raw materials for the spherical liposomes of the lipid bilayer are one or more of DOTAP and DOTMA, and one or more of cholesterol and DOPE, with the mass ratio of one or more of DOTAP and DOTMA to one or more of cholesterol and DOPE being 1.5~2:

1. The photosensitizer is one or more of IR780, IR820, and ICG, and the CD44 siRNA sequence is CAGGAUGACUCCUUCUUUATT. The method for preparing the pharmaceutical composition includes the following steps: Globular liposomes containing bilayer lipid molecules and photosensitizers are added to an organic solvent and stirred to form a clear solution. The solution is then evaporated to form a film, hydrated with gemcitabine solution, and then passed sequentially through liposome extruders at 200 nm, 100 nm, and 50 nm to obtain an aqueous solution containing photosensitizers and gemcitabine liposomes GI-Lip. The aqueous solution of GI-Lip, CD44 siRNA solution, and protamine solution are mixed and incubated to obtain GIR-Lip, which is a drug composition for pancreatic cancer liver metastases in the context of metabolism-related fatty liver disease.

2. A method for preparing a pharmaceutical composition for pancreatic cancer liver metastases in the context of metabolism-related fatty liver disease as described in claim 1, characterized in that, The method includes the following steps: Globular liposomes containing bilayer lipid molecules and photosensitizers are added to an organic solvent and stirred to form a clear solution. The solution is then evaporated to form a film, hydrated with gemcitabine solution, and then passed sequentially through liposome extruders at 200 nm, 100 nm, and 50 nm to obtain an aqueous solution containing photosensitizers and gemcitabine liposomes GI-Lip. The aqueous solution of GI-Lip, CD44 siRNA solution, and protamine solution are mixed and incubated to obtain GIR-Lip, which is a drug composition for pancreatic cancer liver metastases in the context of metabolism-related fatty liver disease.

3. The method for preparing the pharmaceutical composition for pancreatic cancer liver metastases in the context of metabolism-related fatty liver disease as described in claim 2, characterized in that, The ratio of the spherical liposome raw material of the bilayer lipid molecule to the organic solvent is (15~25) mg: (1~5) mL.

4. The method for preparing the pharmaceutical composition for pancreatic cancer liver metastases in the context of metabolism-related fatty liver disease as described in claim 2, characterized in that, The organic solvent is selected from one or more of ethanol, chloroform, and diethyl ether.

5. The use of the pharmaceutical composition of claim 1 or the pharmaceutical composition prepared by any one of claims 2 to 4 in the preparation of a pharmaceutical formulation for pancreatic cancer liver metastases in the context of metabolism-related fatty liver disease.

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