Liposome targeting SELENOP + macrophages, preparation method of liposome and application of liposome in treatment of lung squamous cell carcinoma

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a lipidosome targeting SELENOP + macrophages, a preparation method of the lipidosome and application of the lipidosome in lung squamous cell carcinoma treatment. According to the lipidosome, an SELENOP antibody is used as a targeted modification molecule, si-SELENOP and s-ABCA1 / G1 are loaded, and accurate delivery of siRNA in target cells is achieved through specific binding of the antibody and a SELENOP receptor on the surface of a SELENOP + macrophage. The targeting liposome prepared by the invention can specifically inhibit cholesterol efflux pathways of SELENOP + macrophages, reverse tumor phenotypic polarization, remodel a tumor immune microenvironment and recover anti-tumor activity of immune cells such as CD8 + T cells, NK cells and the like. When the liposome is independently used or combined with a PD-1 antibody, the growth and metastasis of the lung squamous cell carcinoma can be remarkably inhibited, the treatment effect is improved, a brand new metabolism-immunity synergistic targeted treatment strategy is provided for the lung squamous cell carcinoma, and the liposome has a good clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to tumor-targeted drug delivery systems, gene therapy and tumor immunotherapy cross-technology, and particularly to a technology based on SELENOP. + Functional liposomes targeting macrophages, their preparation methods, and their application in the precision treatment of squamous cell carcinoma of the lung. Background Technology

[0002] Lung squamous cell carcinoma (LUSC) is one of the major subtypes of lung cancer, accounting for 30%-50% of primary lung cancers in my country, with an annual incidence rate of approximately 35 per 100,000. Its pathogenesis is complex and closely related to long-term smoking, exposure to environmental pollutants, chronic lung inflammation, and genetic factors. It is characterized by high invasiveness, early metastasis, and poor prognosis. As the disease progresses, patients will experience symptoms such as cough, hemoptysis, chest pain, and dyspnea. In advanced stages, distant metastasis may occur, seriously threatening the patient's life and health, and imposing a heavy medical burden on families and society.

[0003] However, existing treatment options for squamous cell carcinoma of the lung have the following drawbacks: 1. Some clinical treatments rely on platinum-based chemotherapy combined with PD-1 inhibitors. More than 70% of patients will develop primary or secondary drug resistance, and the positive rate of driver gene mutations (such as EGFR / ALK) in squamous cell carcinoma of the lung is less than 5%, lacking targeted therapies. 2. Traditional chemotherapy drugs have poor targeting, and while killing tumor cells, they can easily damage normal tissues, leading to serious systemic side effects; 3. PD-1 inhibitor monotherapy has a low response rate to immunosuppressive squamous cell carcinoma of the lung and is difficult to overcome the immune escape barrier mediated by lipid metabolism abnormalities; 4. Conventional drug delivery systems cannot achieve precise drug delivery, further limiting the improvement of treatment effectiveness. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies in the treatment of squamous cell carcinoma of the lung, such as insufficient targeting, limited therapeutic effect, and difficulty in reversing the immunosuppressive microenvironment. This invention proposes a targeted SELENOP therapy. + Liposomes of macrophages, their preparation methods and applications.

[0005] To achieve the above objectives, the present invention employs the following technical solution: a targeted SELENOP +The liposomes of macrophages include a liposome carrier, a targeting modification molecule, and a loaded functional nucleic acid; the targeting modification molecule is a SELENOP antibody, the functional nucleic acid is a combination of si-SELENOP and si-ABCA1 / G1, and the liposome carrier is composed of ionizable lipid MC3, cholesterol, and neutral phospholipid DOPE.

[0006] Furthermore, the molar ratio of the ionizable lipids, cholesterol, and neutral phospholipids is 2:1:1.

[0007] The beneficial effects of adopting the above-mentioned further scheme are: precise formulation ensures the structural stability of liposomes and siRNA loading efficiency, laying the foundation for subsequent targeted drug delivery and gene silencing effects.

[0008] Furthermore, the molar ratio of si-SELENOP to si-ABCA1 / G1 is 1:1, and the total drug loading of siRNA in the liposomes is 5-10 μg / mL.

[0009] The beneficial effects of adopting the above-mentioned further scheme are: the synergistic effect of dual siRNAs can simultaneously block the expression of target genes and pathway function, and the drug loading balances the therapeutic effect and liposome stability, avoiding liposome aggregation due to excessive drug loading or affecting the efficacy due to insufficient drug loading.

[0010] Furthermore, the mass ratio of the SELENOP antibody to the liposome carrier is 1:10 to 1:20.

[0011] The beneficial effects of adopting the above-mentioned further approach are: rationally controlling the antibody modification ratio to ensure the liposomes' effectiveness against SELENOP. + Macrophages have specific recognition capabilities, while avoiding liposome structural abnormalities caused by excessive antibody use.

[0012] Furthermore, a target for SELENOP + The application of macrophage liposomes in the preparation of therapeutic drugs for lung squamous cell carcinoma, wherein the therapeutic drug is a combination of the liposomes alone or PD-1 antibodies at a mass ratio of 5:1-10:1, administered via intravenous injection at a dose of 1-5 mg per kilogram of body weight, and administered 1-2 times per week.

[0013] The beneficial effects of adopting the above-mentioned further approach are: clarifying the clinical application scenarios and usage methods; using it alone can play a precise targeted therapeutic role; using it in combination with PD-1 antibodies can form a synergistic effect; the dosing parameters provide a reference for clinical translation; and it balances efficacy and safety.

[0014] Compared with the prior art, the advantages and positive effects of the present invention are as follows: 1. Precise targeting: Through the specific recognition function of SELENOP antibodies, liposomes are precisely delivered to SELENOP. + Macrophages increase drug concentration in target cells, reduce damage to normal tissues, and improve treatment safety. 2. si-SELENOP works synergistically with si-ABCA1 / G1 to both inhibit macrophage polarization toward the pro-tumor M2 phenotype and block cholesterol efflux pathways, thus cutting off the dual-track cancer-promoting mechanism of "metabolic energy supply - immunosuppression" at its source; 3. Reverse the tumor immunosuppressive microenvironment and restore CD8 + Enhance the anti-tumor activity of T cells and NK cells, improve the antigen presentation ability of DC cells, and lay the foundation for immunotherapy; 4. Whether used alone or in combination with PD-1 antibodies, it can significantly inhibit the progression of squamous cell carcinoma of the lung, providing a novel "metabolic-immune" synergistic targeted therapy strategy to address the pain points in clinical treatment. Attached Figure Description

[0015] Figure 1 This invention provides a targeted SELENOP + A schematic diagram illustrating the preparation process and mechanism of action of macrophage liposomes.

[0016] Figure 2 This is a transmission electron microscope image of a targeted liposome according to the present invention.

[0017] Figure 3 This invention describes the inhibitory effect of a targeted liposome on the expression of SELENOP, ABCA1, and ABCG1 genes and proteins.

[0018] Figure 4 This invention provides a targeted liposome for SELENOP + Inhibitory effect of macrophage cholesterol efflux.

[0019] Figure 5 This invention relates to the effect of targeted liposomes on the invasion and migration of lung squamous cell carcinoma cells.

[0020] Figure 6 This invention describes the inhibitory effect of a targeted liposome alone and in combination with a PD-1 antibody on the growth of lung squamous cell carcinoma xenografts.

[0021] Figure 7 This is the safety evaluation result of a targeted liposome of the present invention. Detailed Implementation

[0022] The present invention will now be further described with reference to the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. It should be understood that the described embodiments are merely some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0023] As shown in Figures 1-7, this invention provides a technical solution: a targeted SELENOP + The liposomes of macrophages, modified with SELENOP antibody as the target molecule, are loaded with si-SELENOP and si-ABCA1 / G1, and specifically recognize SELENOP. + The precise delivery of macrophages is achieved through the following preparation steps: S1. Preparation of blank liposomes: Accurately weigh each component according to the molar ratio of ionizable lipid MC3: cholesterol: neutral phospholipid DOPE = 2:1:1 and add them to a round-bottom flask; add 2-3 mL of chloroform to dissolve, and place in a vacuum rotary evaporator at 37℃ and 100 rpm for 30-60 min to form a film; dry under vacuum for 4-6 h to remove residual chloroform, add 4-6 mL of ultrapure water, and hydrate at 37℃ and 15 rpm for 1 h; sonicate at 100-150 W for 15 min in an ice bath, and extrude through a 0.45 μm filter membrane 3-5 times to obtain blank liposomes.

[0024] S2. Preparation of siRNA-loaded liposomes: Si-SELENOP and si-ABCA1 / G1 lyophilized powder were prepared in a 1:1 molar ratio and dissolved in enzyme-free water to prepare a 10-20 μg / mL mixture. The mixture was incubated at 37°C for 5 min and then cooled on ice for 10 min. Blank liposomes were equilibrated at room temperature for 30 min. Functional nucleic acid mixture was slowly added at a volume ratio of 10:1. After gentle mixing, the mixture was incubated at room temperature for 30-60 min to obtain siRNA-loaded liposomes.

[0025] S3. Preparation and purification of targeted liposomes: SELENOP antibody was dissolved in PBS to prepare a 1 mg / mL solution. EDC and NHS were added to achieve final concentrations of 5 mmol / L and 2.5 mmol / L, respectively. The carboxyl groups were activated by incubation at room temperature for 15-20 min. The activated antibody solution was added to liposomes loaded with siRNA at a mass ratio of 1:12-1:18, and incubated at 4°C in the dark for 16-20 h. The reaction solution was transferred to a 10 kDa dialysis bag (pre-activated by soaking in ultrapure water for 10-15 min), and dialyzed at 4°C with pH 7.4 PBS buffer at 50-80 rpm with magnetic stirring for 24-48 h, changing the buffer every 6-8 h. The solution was then filtered through a 0.22 μm filter for sterilization, and the filtrate was collected as targeted SELENOP. + Liposomes of macrophages should be stored at -20°C in a sealed container away from light.

[0026] Working principle: This invention prepares blank liposomes using a thin-film method, utilizes electrostatic interactions to achieve efficient encapsulation of si-SELENOP and si-ABCA1 / G1, and then covalently links the SELENOP antibody to the liposome surface via an EDC / NHS activation method to construct a SELENOP-targeting antibody. + The macrophage drug delivery system. After entering the body, this liposome is delivered via SELENOP antibody and SELENOP. + The specific binding of macrophage surface receptors enables precise delivery of siRNA into target cells; si-SELENOP inhibits macrophage polarization toward the pro-tumor M2 phenotype, and si-ABCA1 / G1 blocks cholesterol efflux pathways. The two work together to disrupt the dual-track cancer-promoting mechanism of "metabolic energy supply-immunosuppression", while reshaping the tumor immune microenvironment and restoring the anti-tumor activity of immune cells, ultimately achieving precise treatment of lung squamous cell carcinoma. Example

[0027] Please refer to Figure 1 A targeted SELENOP + A schematic diagram illustrating the preparation process and mechanism of action of macrophage liposomes.

[0028] Example 2

[0029] Please refer to Figure 2-7 Performance testing and efficacy verification of targeted liposomes.

[0030] Liposome morphology characterization: The morphology of the targeted liposomes prepared in Example 1 was observed using transmission electron microscopy. The results are as follows: Figure 2As shown, the liposomes exhibit a uniform spherical vesicle structure with no obvious aggregation and a particle size distribution of 100-150 nm, which meets the requirements of targeted drug delivery systems.

[0031] Validating the effect of target gene silencing by combining targeted liposomes with SELENOP + Macrophages were co-incubated for 48 hours. The mRNA and protein expression levels of the SELENOP, ABCA1, and ABCG1 genes were detected by Q-PCR and Western blot. The results are as follows: Figure 3 As shown in the figure, compared with the blank control group, the expression levels of SELENOP, ABCA1, and ABCG1 genes in the liposome-targeted treatment group were significantly downregulated, demonstrating that liposomes can efficiently silence target genes and block cholesterol efflux pathways.

[0032] The cholesterol efflux inhibition effect was detected using a combination of Filipin staining and enzymatic methods to assess the effect of targeted liposomes on SELENOP. + The effects of macrophage cholesterol metabolism, the results are as follows Figure 4 As shown in the figure, compared with the blank control group, the macrophages treated with targeted liposomes showed significantly enhanced intracellular cholesterol fluorescence intensity, increased intracellular total cholesterol content, decreased cholesterol content in the culture medium, and a cholesterol efflux rate reduction of more than 65%, demonstrating that it can effectively inhibit SELENOP. + The cholesterol efflux function of macrophages cuts off the lipid supply to tumor cells, which is consistent with the previous experimental conclusions of Selenop OE macrophages enhanced cholesterol efflux.

[0033] In vitro tumor cell effect experiments, SELENOP treated with targeted liposomes was used... + Macrophages were co-cultured with lung squamous cell carcinoma cells (H520 and H2170). The malignant phenotype of tumor cells was detected by Transwell invasion and scratch assays, as shown in Figure 5. Compared with the untreated group, the invasive ability and migration distance of tumor cells in the co-culture system were significantly reduced, demonstrating that liposomes weakened the pro-malignant effect on tumor cells by inhibiting macrophage cholesterol efflux and M2 polarization, consistent with previous SELENOP studies. + The experimental results showed that macrophages promote the invasion and migration of tumor cells.

[0034] In vivo tumor suppression efficacy was verified by monitoring tumor growth using in vivo imaging in small animals. The results, shown in Figure 5, indicate that the tumor volume in the liposome-targeted group was significantly smaller than that in the control group, and the combined treatment group showed even more significant tumor suppression. Furthermore, the tumor growth rate in SELENOP knockout mice (SELENOP⁻ / -) was slower than that in wild-type mice, further validating the efficacy of SELENOP. +The tumor-promoting effect of macrophages. Survival analysis showed that the median survival of mice in the combination therapy group was extended by more than 120% compared with the control group, and the median survival of mice in the liposome-targeted group was extended by more than 80%, demonstrating that liposomes alone or in combination with PD-1 antibody can significantly inhibit the progression of lung squamous cell carcinoma.

[0035] In vivo targeted distribution validation was performed using small animal in vivo imaging technology to detect the distribution of Cy5-labeled targeted liposomes in tumor-bearing mice. The results are as follows: Figure 7 As shown, liposomes exhibited significant fluorescence signals at the tumor site 6 hours after injection, with peak fluorescence intensity at 12-24 hours and continuous signal enrichment in the tumor area at 48-72 hours. In contrast, only weak fluorescence was detected in normal tissues such as the heart and liver, demonstrating that the liposomes possess highly efficient tumor-targeting delivery capabilities and do not accumulate significantly in normal tissues, exhibiting good in vivo biosafety and targeting specificity.

[0036] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments for application in other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A targeted SELENOP + Liposomes of macrophages, characterized in that, It includes a liposome carrier, a targeted modification molecule, and a loaded functional nucleic acid; the targeted modification molecule is a SELENOP antibody, the functional nucleic acid is a combination of si-SELENOP and si-ABCA1 / G1, and the liposome carrier is composed of ionizable lipid MC3, cholesterol, and neutral phospholipid DOPE in a molar ratio of 2:1:

1.

2. The targeted SELENOP according to claim 1 + Liposomes of macrophages, characterized in that, The molar ratio of si-SELENOP to si-ABCA1 / G1 is 1:1, and the total drug loading of siRNA in the liposomes is 5-10 μg / mL; the mass ratio of SELENOP antibody to liposome carrier is 1:10-1:

20.

3. The targeted SELENOP according to claim 1 or 2 + A method for preparing macrophage liposomes, characterized in that, Includes the following steps: S1. Blank liposomes composed of MC3, cholesterol, and DOPE were prepared using a thin-film method. S2. Si-SELENOP was mixed with si-ABCA1 / G1 and added to the blank liposomes. The mixture was incubated at room temperature for 30-60 min to obtain liposomes loaded with siRNA. S3. SELENOP antibody was covalently linked to the surface of the siRNA-loaded liposomes using an EDC / NHS activation method. The target liposomes were obtained after dialysis purification.

4. The preparation method according to claim 3, characterized in that, In step S1, the membrane preparation conditions are 37℃, 100rpm, reaction time 30-60min, vacuum drying for 4-6h, hydration with ultrapure water, sonication in an ice bath, and then extrusion through a 0.45μm filter membrane. In step S3, the incubation conditions are 4℃ incubation in the dark for 12-24h, dialysis is performed using a 10kDa dialysis bag in PBS buffer at pH 7.4 for 24-48h.

5. The targeted SELENOP according to claim 1 or 2 + Application of macrophage liposomes in the preparation of drugs for the treatment of lung squamous cell carcinoma.

6. The application according to claim 5, characterized in that, The therapeutic drug is used alone or as a compound preparation composed of the liposome and PD-1 antibody at a mass ratio of 5:1 to 10:1; the administration method is intravenous injection, the dosage is 1-5 mg per kilogram of body weight, and the administration frequency is 1-2 times per week.

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