Inhalable pemetrexed-functionalized tumor-microenvironment-targeted drug delivery system for treating lung cancer

Abstract

The present invention relates to an inhalable tumor-microenvironment-targeted drug delivery system for treating lung cancer. Specifically, the present invention provides a lipid-polymer hybrid nanoparticle comprising: a poly(lactic-co-glycolic acid) (PLGA) polymer core that encapsulates a first therapeutic agent such as paclitaxel (PTX); and a lipid shell functionalized with pemetrexed (PEM) that performs a dual function as both an anticancer agent and a target ligand. The nanoparticles are directly delivered to the lungs through inhalation, simultaneously target folate receptor alpha (FR-α) of lung cancer cells and folate receptor beta (FR-β) of immunosuppressive tumor-associated macrophages (M2-TAMs) through a PEM ligand, and can repolarize M2-TAMs into an immunoactive M1 phenotype, while exhibiting a direct cancer cell killing effect by PTX and PEM.

Description

Pemetrexid-functionalized tumor microenvironment-targeted inhaled drug delivery system for lung cancer treatment

[0001] The present invention relates to a drug delivery system for the treatment of lung cancer, and more specifically, to an inhalable hybrid nanoparticle targeting the tumor microenvironment and a pharmaceutical composition containing the same.

[0002] Lung cancer is a leading cause of cancer-related deaths worldwide and is an intractable disease with a high mortality rate. Current standard treatments for lung cancer include surgery, radiation therapy, chemotherapy, targeted therapy, and immunotherapy. Among these, chemotherapy plays a pivotal role in the treatment of the majority of patients, including those with advanced or metastatic non-small cell lung cancer (NSCLC). Chemotherapy regimens containing drugs such as paclitaxel (PTX) and pemetrexed (PEM) are primarily administered systemically via intravenous (IV) injection. However, this method of systemic administration has serious limitations. Anticancer drugs cannot distinguish between cancer cells and normal cells and are distributed nonspecifically, causing severe systemic side effects such as bone marrow suppression, gastrointestinal disorders, and neuropathy. This toxicity not only reduces the patient's quality of life but also limits therapeutic doses and leads to treatment discontinuation, posing a significant obstacle to achieving optimal therapeutic effects. Furthermore, prolonged chemotherapy leads to the development of drug resistance, which is a major cause of treatment failure.

[0003] Recent therapeutic paradigms are shifting toward recognizing the importance of the Tumor Microenvironment (TME)—a complex ecosystem that actively supports tumor growth, invasion, and metastasis—rather than just the cancer cells themselves. The TME is composed of immune cells, fibroblasts, blood vessels, and the extracellular matrix; in particular, Tumor-Associated Macrophages (TAMs) are a core component of the TME. TAMs primarily exist as the M2 subtype, which performs immunosuppressive functions; they contribute to cancer progression and treatment resistance by promoting tumor cell proliferation, inducing angiogenesis, and suppressing anti-tumor immune responses. Consequently, there is a growing recognition that it is difficult to achieve a sustained therapeutic response without effectively controlling the functions of the TME, particularly M2-TAMs. Strategies to reorganize the TME from an immunosuppressive state to an immuno-active state—such as repolarizing M2-TAMs into the M1 subtype with anti-tumor functions—are emerging as promising approaches for new cancer therapies.

[0004] To address these issues, inhalation therapy is gaining attention as a local treatment method for delivering drugs directly to the lungs. The inhalation route is non-invasive and can deliver high concentrations of drugs to the lung tumor site without going through systemic circulation, offering the potential to minimize systemic side effects and maximize local therapeutic effects. Furthermore, nanoparticle technology plays a crucial role in protecting drugs, controlling release, and enhancing delivery efficiency to target cells. In particular, nanoparticles made from the biodegradable polymer poly(lactic acid-co-glycolic acid) (PLGA) are widely used in the development of drug delivery systems as a safe material approved by the U.S. Food and Drug Administration (FDA).

[0005] The academic literature Nano Lett. 2018;18(6):3571-3579 discloses a technique for repolarizing M2-TAMs into the M1 subtype using hyaluronic acid-based nanoparticles. This aligns in part with the objective of the present invention to modulate the tumor microenvironment. However, the administration route is limited to intraperitoneal injection.

[0006] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of lung cancer comprising lipid-polymer hybrid nanoparticles for inhalation administration.

[0007] Another objective of the present invention is to provide a method for treating lung cancer comprising the step of administering the pharmaceutical composition by inhalation to a subject.

[0008] Another objective of the present invention is to provide a method for preparing the hybrid nanoparticles by a single-step nanoprecipitation method.

[0009] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by a person skilled in the art from the description below.

[0010] To solve the above problem, the present invention provides a pharmaceutical composition comprising lipid-polymer hybrid nanoparticles for inhalation administration, wherein

[0011] The above nanoparticles are,

[0012] (a) a polymer core encapsulating a first therapeutic agent inside; and

[0013] (b) a lipid shell disposed on the surface of the polymer core; comprising,

[0014] The present invention provides a pharmaceutical composition for the prevention or treatment of lung cancer, characterized in that the above lipid shell functions as a second therapeutic agent and is functionalized with pemetrexed, which functions as a target ligand for folate receptors.

[0015] According to one aspect, the polymer of the polymer core may be any one selected from the group consisting of poly(lactic acid-co-glycolic acid) (PLGA), polylactic acid (PLA), polycaprolactone (PCL), and polyglycolic acid (PGA).

[0016] According to one side, the first therapeutic agent may be any one selected from the group consisting of paclitaxel, docetaxel, vincristine, and vinblastine.

[0017] According to one side, the lipid shell may additionally contain a pulmonary surfactant.

[0018] According to one aspect, the pemetrexid can be covalently bonded to the lipid shell through a DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol)]) linker.

[0019] According to one aspect, the nanoparticles may simultaneously bind to folate receptor alpha (FR-α) expressed in lung cancer cells and folate receptor beta (FR-β) expressed in tumor-associated macrophages (TAMs).

[0020] According to one aspect, the nanoparticle may repolarize the tumor-associated macrophage from an immunosuppressive M2 subtype to an immunoactive M1 subtype.

[0021] According to one aspect, the average particle size of the nanoparticles may be 100 nm to 200 nm.

[0022] According to another embodiment of the present invention, a method for treating lung cancer is provided, comprising the step of administering the pharmaceutical composition in a therapeutically effective amount to a subject other than a human by inhalation.

[0023] According to another embodiment of the present invention, an inhaled lung cancer treatment comprising the above pharmaceutical composition is provided.

[0024] According to another embodiment of the present invention, (a) a step of synthesizing a DSPE-PEG-PEM conjugate by reacting pemetrexed or a derivative thereof with DSPE-PEG-amine;

[0025] (b) a step of preparing a core solution by dissolving a hydrophobic polymer and a first therapeutic agent in an organic solvent;

[0026] (c) a step of preparing a lipid layer solution by dispersing the DSPE-PEG-PEM conjugate synthesized in step (a) and a pulmonary surfactant in an aqueous solution; and

[0027] (d) a method for manufacturing hybrid nanoparticles for drug delivery, comprising the step of injecting the core solution of step (b) into the lipid layer solution of step (c) to form hybrid nanoparticles by single-step nanoprecipitation.

[0028] According to one side, the synthesis of step (a) above may be carried out through a DCC (Dicyclohexylcarbodiimide) coupling reaction.

[0029] According to one aspect, the nanoparticle formation in step (d) above may be characterized by having a final weight ratio of lipids to polymers of 1:1 to 1:3.

[0030] The pemetrexid-functionalized inhaled drug delivery system targeting the tumor microenvironment according to the present invention exhibits synergistic anticancer efficacy. In addition to the direct cytotoxic effects of paclitaxel (PTX) and pemetrexid (PEM), the nanoparticles of the present invention provide an immunotherapeutic effect that repolarizes immunosuppressive M2-TAM into an immunoactive M1 subtype. This allows for direct attack on cancer cells while simultaneously targeting the tumor microenvironment.

[0031] The effects of the present invention are not limited to those mentioned above, and other unmentioned effects will be clearly understood by a person skilled in the art from the description below.

[0032] Figure 1 shows the results of the preparation and characterization of pulmonary surfactant-based hybrid nanoparticles for inhalation therapy. (A) is a schematic diagram of the composition and structure of PEM-PSNP@PTX, (B) and (C) are the results of size and zeta potential measurements, respectively, (D) is a representative transmission electron microscope (TEM) image of PEM-PSNP@PTX, (E) to (G) show the drug loading rate, drug encapsulation efficiency, and release profile of PEM-PSNP@PTX compared with PLGA@PTX, (H) and (I) show the size and zeta potential stability before and after nebulization, and (J) shows the in vitro stability over time.

[0033] Figure 2 shows the results of in vitro cell uptake and cytotoxicity evaluation. (A) and (B) are Western blot results showing FR-α expression in H1793 and A549 cells, (C) is a confocal microscopy image of the cytotoxicity of H1793 and A549 cancer cells after PEM-PSNP@PTX treatment, and (D) is a confocal microscopy image of the cell uptake ability of labeled PEM-PSNP@Dil.

[0034] Figure 3 shows the results of the evaluation of the targeting and repolarization efficacy of tumor-associated macrophages (TAMs) in vitro. (A) is an overview of M1 and M2 macrophage differentiation, (B) is the flow cytometry results showing cell surface expression of CD86 and CD206 in M0, M1, and M2 populations, (C) and (D) are Western blot results showing FR-β expression in M0, M1, and M2, (E) is a confocal microscopy image of the cellular uptake ability of labeled PEM-PSNP@Dil in M2 macrophages, and (F) is the flow cytometry results for CD86 (M1) and CD206 (M2) expression after PEM-PSNP@PTX treatment.

[0035] Figure 4 shows the results of in vivo inhalation delivery and distribution confirmation. (A) and (B) are time-dependent biodistribution images and signal-to-background ratio analysis results in major organs after PEM-PSNP@Dil inhalation, and (C) and (D) are bioluminescence imaging results for establishing a mouse lung cancer model and confirming nanoparticle absorption in the lungs.

[0036] Figure 5 shows the results of the in vivo inhalation efficacy evaluation of PEM-PSNP@PTX in a mouse model. (A) is a schematic diagram of the experimental schedule, (B) and (C) are bioluminescent images and average luminescence intensity of tumors over time in each group, (D) to (F) are in vitro observation results of lung tissue excised after treatment, and (G) is the measurement result of the average luminescence intensity of lung tissue.

[0037] Figure 6 shows the in vivo tumor microenvironment modulating effect of PEM-PSNP@PTX in a lung cancer mouse model. (A) is the result of measuring inflammatory cytokine levels in alveolar lavage fluid, and (B) is the result of immunohistochemical (IHC) staining of lung tissue.

[0038] Figure 7 shows the results of the in vivo inhalation toxicity evaluation of PEM-PSNP@PTX in mice. (A) is the blood chemistry test result, and (B) is the hematoxylin and eosin (H&E) staining image of major organs.

[0039] Figure 8 shows an overview of the formulation process of PEM-PSNP@PTX.

[0040] Figure 9 shows the Fourier transform infrared (FT-IR) spectra of pemetrexid, DSPE-PEG-NH2, and PEM-DSPE-PEG.

[0041] Figure 10 shows the optimization results of inhaled nanoparticles according to the polymer-to-lipid ratio.

[0042] Figure 11 shows the results of a competitive cell uptake test of PEM-PSNP@Dil with added folic acid (FA).

[0043] Figure 12 shows data on in vivo inhalation therapy of PEM-PSNP@PTX in mice.

[0044] Figure 13 is a survival curve comparing the survival rates of mice in each treatment group.

[0045] Figure 14 is the result of a flow cytometry analysis showing a decrease in CD206 expression in lung tissue after inhalation of PEM-PSNP@PTX.

[0046] Figure 15 shows the results of flow cytometry analysis after PEM-PSNP@PTX treatment, which shows that the proportion of M2 macrophages (CD206) among total macrophages (F4 / 80) in lung tissue is reduced.

[0047] Figure 16 shows a schematic diagram of the present invention.

[0048] The inventors intend to provide an invention by integrating four functions—(1) cancer cell death, (2) immune cell death, (3) cancer cell targeting, and (4) immune cell targeting—into a single system through a single molecule called pemetrexid, implementing this as a combination therapy with paclitaxel, and completing it as an inhaled formulation.

[0049] To solve the above problem, the present invention provides a pharmaceutical composition comprising lipid-polymer hybrid nanoparticles for inhalation administration, wherein

[0050] The above nanoparticles are,

[0051] (a) a polymer core encapsulating a first therapeutic agent inside; and

[0052] (b) a lipid shell disposed on the surface of the polymer core; comprising,

[0053] The present invention provides a pharmaceutical composition for the prevention or treatment of lung cancer, characterized in that the above lipid shell functions as a second therapeutic agent and is functionalized with pemetrexed, which functions as a target ligand for folate receptors.

[0054] As used herein, the "lipid-polymer hybrid nanoparticle" refers to a nano-sized particle comprising a core formed of a polymer material and a shell formed of lipid molecules. This structure combines the advantages of the structural stability and controlled drug release capabilities of polymer nanoparticles with the excellent biocompatibility and ease of surface functionalization of liposomes.

[0055] "Pemetrexed (PEM)" is a pyrrolopyrimidine-based multi-target antifolate that inhibits nucleotide synthesis and stops cell replication by inhibiting enzymes involved in folate-dependent metabolic pathways, such as thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyl transferase (GARFT). In addition to its function as a therapeutic agent, it simultaneously performs the function of a target ligand that binds to folate receptors due to its structural similarity to folate.

[0056] Paclitaxel (PTX) is a taxane-class anticancer drug that induces apoptosis by inhibiting the depolymerization of microtubules and abnormally stabilizing them to form microtubule bundles, thereby arresting the cell cycle at the G2 / M phase. It is also known to exert positive effects on the tumor microenvironment through its immunomodulatory functions.

[0057] Pulmonary surfactant (PS) is a complex of lipids and proteins present at the gas-liquid interface of alveoli, playing a physiological role in preventing alveolar collapse during exhalation by lowering surface tension. It is primarily composed of phospholipids, such as dipalmitoylphosphatidylcholine (DPPC), and specific surfactant proteins (SP-A, SP-B, SP-C, SP-D). In this invention, it is used as a biomimetic material to enhance the biocompatibility, stability, and distribution of nanoparticles within the lungs.

[0058] The "Folate Receptor (FR)" is a glycoprotein located on the cell surface that mediates the intracellular uptake of folate and its analogs. Among the subtypes of FR, folate receptor alpha (FR-α) is overexpressed in various epithelial cancer cells, such as ovarian cancer and lung cancer, whereas its expression is limited in normal tissues. Folate receptor beta (FR-β) is known to be highly expressed primarily in activated macrophages, particularly in M2 subtype tumor-associated macrophages (M2-TAMs) within the tumor microenvironment. The present invention can target both of these subtypes simultaneously.

[0059] The present invention relates to an inhaled drug delivery system based on multifunctional lipid-polymer hybrid nanoparticles designed to maximize the efficiency of lung cancer treatment and minimize side effects. The core of the present invention is to (1) induce direct cancer cell death by simultaneously delivering anticancer agents of different mechanisms (PTX, PEM) through a single nanoparticle platform, (2) simultaneously target cancer cells (FR-α) and immunosuppressive cells (M2-TAMs, FR-β) by utilizing the drug itself (PEM) as a target ligand, and (3) reprogram the immunosuppressive tumor microenvironment into an immune-activating environment.

[0060] The nanoparticles of the present invention have a unique core-shell structure. This is an intentional design for efficiently compartmentalizing and loading hydrophobic drugs, hydrophilic drugs, or molecules for surface functionalization.

[0061] The core of the nanoparticle is composed of a biodegradable and biocompatible polymer, preferably PLGA. In the present invention, a hydrophobic anticancer agent can be encapsulated within this PLGA core, preferably paclitaxel. The PLGA matrix physically traps PTX to enhance stability and enables the continuous release of the drug as the polymer gradually degrades. This allows the drug to maintain an effective concentration at the tumor site for an extended period, thereby maximizing the anticancer effect.

[0062] The surface of the PLGA core is surrounded by a lipid layer. This lipid shell enhances the biocompatibility of nanoparticles, evades recognition by the immune system, and provides a platform for surface functionalization. The lipid shell is mainly composed of phospholipids and PEG derivatives.

[0063] In the DSPE-PEG-PEM conjugate, DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine) is a type of phospholipid that acts as an anchor, with its hydrophobic tail portion stably inserted into the lipid shell. PEG (polyethylene glycol) is a hydrophilic polymer that imparts a stealth effect to the nanoparticle surface, preventing opsonization and extending the residence time in the blood or tissues. Pemetrexid (PEM), an anticancer agent and target ligand, can be stably attached to the PEG terminus via covalent bonding. This conjugate exposes PEM to the nanoparticle surface, enabling it to perform targeting functions against FR-α and FR-β.

[0064] Another feature of the present invention is that a lung surfactant (PS) is incorporated into the lipid shell. By introducing PS, which coats the inner wall of the alveoli, onto the surface of the nanoparticles, the following effects can be achieved: (1) avoiding the inhaled nanoparticles being recognized as foreign substances and removed by alveolar macrophages, thereby increasing the residence time in the lungs; (2) improving the distribution of nanoparticles by effectively spreading them along the surface of the alveoli due to the surface activity of PS; and (3) maximizing biocompatibility by minimizing the risk of local irritation or inflammatory response by using bio-derived materials.

[0065] In the present invention, the term "prevention" refers to any act of suppressing or delaying the occurrence, spread, or recurrence of lung cancer by administering the composition of the present invention, and the term "treatment" refers to any act of improving or beneficially altering the symptoms of lung cancer by administering the composition of the present invention.

[0066] In the present invention, the term “pharmaceutical composition” means one prepared for the purpose of preventing or treating a disease, and may be formulated in a form particularly suitable for inhalation administration. For example, it may be formulated in the form of a sterile solution or suspension for a nebulizer, or a fine powder for a dry powder inhaler (DPI).

[0067] In the present invention, "included as an active ingredient" means that the corresponding ingredient is included in an amount necessary or sufficient to realize the desired biological or therapeutic effect. In actual application, the effective amount is determined as an amount for treating lung cancer, taking into account factors that do not cause other toxicities. For example, it may vary depending on various factors such as the type and stage of the tumor being treated, the form of the composition administered, the patient's age or weight, or the severity of the disease. A person skilled in the art to which the present invention pertains can empirically determine the therapeutically effective amount of an individual composition by considering these factors without excessive experimentation.

[0068] In addition, the pharmaceutical composition of the present invention may include one or more pharmaceutically acceptable carriers in addition to the active ingredients described above, depending on each formulation.

[0069]

[0070] The above-mentioned pharmaceutically acceptable carrier must be suitable for inhalation administration, and for example, for nebulizer formulations, saline solution, sterile water, Ringer's solution, or buffer solution may be used. Additionally, for dry powder inhaler (DPI) formulations, carriers such as lactose and mannitol may be included, and other conventional additives such as preservatives, stabilizers, dispersants, or propellants may be further included as needed. Such formulations may preferably be prepared according to each component using methods conventional in the art or methods disclosed in Remington's Pharmaceutical Science.

[0071] The composition of the present invention must be administered in a pharmaceutically effective amount according to the intended method. The term “pharmaceutically effective amount” in the present invention means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause serious side effects. The effective dose level may be determined based on factors including the patient’s health status, the severity of lung cancer, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, drugs used concomitantly or concurrently, and other factors well known in the medical field.

[0072] Accordingly, lung cancer can be prevented or treated by administering the pharmaceutical composition of the present invention to an individual. In the present invention, the term “individual” is not limited to mammals such as livestock or humans requiring prevention or treatment of the disease, but preferably may be a human.

[0073] The pharmaceutical composition of the present invention is preferably formulated for inhalation administration, which is the primary route of administration. For example, it may be prepared as an isotonic aqueous solution or suspension for a nebulizer, or in the form of a micronized active ingredient mixed with a suitable carrier for a dry powder inhaler.

[0074] The pharmaceutical composition according to the present invention may be administered via parenteral routes, including intravenous, subcutaneous, and intramuscular routes, in addition to the primary route of administration, inhalation, as needed, and the dosage of the active ingredient may be appropriately selected according to various factors such as the route of administration, the patient's age, gender, body weight, and severity of the patient. Furthermore, the composition of the present invention may be administered in combination with other known lung cancer treatments that can enhance the desired effect.

[0075] In the pharmaceutical composition of the present invention, the total effective amount of the hybrid nanoparticles, which are the active ingredients, can be administered to a patient as a single dose, or administered by a fractional treatment method in which multiple doses are administered over a long period. The content of the active ingredients in the pharmaceutical composition of the present invention may vary depending on the severity of the disease, but the effective dose can be finally determined by considering various factors such as the patient's age, weight, health status, gender, severity of the disease, diet, and excretion rate, as well as the route of administration and the number of treatments.

[0076] According to one aspect, the polymer of the polymer core may be any one selected from the group consisting of poly(lactic acid-co-glycolic acid) (PLGA), polylactic acid (PLA), polycaprolactone (PCL), and polyglycolic acid (PGA), and preferably may be PLGA.

[0077] According to one aspect, the first therapeutic agent may be any one selected from the group consisting of paclitaxel, docetaxel, vincristine, and vinblastine, and preferably paclitaxel.

[0078] According to one side, the lipid shell may additionally contain a pulmonary surfactant.

[0079] According to one aspect, the pemetrexid can be covalently bonded to the lipid shell through a DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol)]) linker.

[0080] According to one aspect, the nanoparticles may simultaneously bind to folate receptor alpha (FR-α) expressed in lung cancer cells and folate receptor beta (FR-β) expressed in tumor-associated macrophages (TAMs).

[0081] According to one aspect, the nanoparticle may repolarize the tumor-associated macrophage from an immunosuppressive M2 subtype to an immunoactive M1 subtype.

[0082] According to one aspect, the average particle size of the nanoparticles may be 100 nm to 200 nm.

[0083] According to another embodiment of the present invention, a method for treating lung cancer is provided, comprising the step of administering the pharmaceutical composition in a therapeutically effective amount to a subject other than a human by inhalation.

[0084] According to another embodiment of the present invention, an inhaled lung cancer treatment comprising the above pharmaceutical composition is provided.

[0085] According to another embodiment of the present invention, (a) a step of synthesizing a DSPE-PEG-PEM conjugate by reacting pemetrexed or a derivative thereof with DSPE-PEG-amine;

[0086] (b) a step of preparing a core solution by dissolving a hydrophobic polymer and a first therapeutic agent in an organic solvent;

[0087] (c) a step of preparing a lipid layer solution by dispersing the DSPE-PEG-PEM conjugate synthesized in step (a) and a pulmonary surfactant in an aqueous solution; and

[0088] (d) a method for manufacturing hybrid nanoparticles for drug delivery, comprising the step of injecting the core solution of step (b) into the lipid layer solution of step (c) to form hybrid nanoparticles by single-step nanoprecipitation.

[0089] According to one side, the synthesis of step (a) above may be carried out through a DCC (Dicyclohexylcarbodiimide) coupling reaction.

[0090] According to one aspect, the nanoparticle formation in step (d) above may be characterized by having a final weight ratio of lipids to polymers of 1:1 to 1:3, and preferably 1:2.

[0091]

[0092] The terms used in the embodiments are for illustrative purposes only and should not be interpreted as intended to be limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as "comprising" or "having" are intended to indicate the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0093] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the embodiments pertain. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0094] In describing the components of the embodiments, terms such as first, second, A, B, (a), (b), etc., may be used. These terms are intended merely to distinguish the components from other components, and the nature, order, or sequence of the components is not limited by these terms. Where it is stated that a component is "connected," "combined," or "joined" to another component, it should be understood that the component may be directly connected or joined to the other component, but that another component may also be "connected," "combined," or "joined" between each component.

[0095]

[0096] Hereinafter, embodiments are described in detail with reference to the attached drawings. However, various modifications may be made to the embodiments, and thus the scope of the patent application is not limited or restricted by these embodiments. It should be understood that all modifications, equivalents, and substitutions to the embodiments are included within the scope of the rights.

[0097] In addition, when describing with reference to the attached drawings, identical components are assigned the same reference numeral regardless of drawing symbols, and redundant descriptions thereof are omitted. In describing the embodiments, if it is determined that a detailed description of related prior art could unnecessarily obscure the essence of the embodiments, such detailed description is omitted.

[0098]

[0099] The present invention is capable of various modifications and may have various embodiments. Specific embodiments are illustrated in the drawings and described in detail in the detailed description below. However, this is not intended to limit the present invention to specific embodiments, and it should be understood that it includes all modifications, equivalents, and substitutions that fall within the spirit and scope of the present invention. In describing the present invention, detailed descriptions of related prior art are omitted if it is determined that such detailed descriptions may obscure the essence of the present invention.

[0100]

[0101] Example 1. Preparation and Characterization of Nanoparticles

[0102] In this study, hybrid nanoparticles (hereinafter PEM-PSNP@PTX) smaller than 200 nm were designed by combining a hydrophobic core made of PLGA with ester ends and a lipid layer composed of a DSPE-PEG-PEM conjugate and waste surfactant (PS) (Fig. 1A). This single-step nanoprecipitation method produced nanoparticles with excellent pharmacokinetic properties by combining the advantages of polymer nanoparticles and liposomes (Fig. 8).

[0103] DSPE-PEG-PEM was synthesized via a DCC chemical reaction by linking the amine groups of DSPE-PEG-amine to the carboxyl groups of PEM. The conjugation of PEM and DSPE-PEG-amine was confirmed by Fourier Transform Infrared Spectroscopy (FTIR) analysis (Fig. 9). Nanoparticles with optimal sizes (100–150 nm) and ζ potentials (-35–-60 mV) suitable for drug delivery were generated when the lipid / polymer weight ratio was 0–50% (Fig. 9). PEM-PSNP@PTX achieved optimal size and charge when the lipid / polymer weight ratio was 50%, exhibiting a particle size of 135.30 ± 4.98 nm and a surface charge of -53.13 mV (Figs. 1B and C).

[0104] The nanoparticle structure was analyzed using negative staining transmission electron microscopy (TEM) with uranyl acetate. Uranyl acetate selectively stained the PS and PEG-bound lipids, increasing electron density and forming a faint ring around the PLGA core (Fig. 1D). The thickness of this ring was approximately 16.75 nm, which was consistent with the expected thickness of the PS and PEG lipid shells.

[0105] Next, the drug loading (DL), encapsulation efficiency (EE), and release profile of lipid-polymer hybrid NPs (PEM-PSNP@PTX) were investigated by comparing them with PLGA NPs (PLGA@PTX). The PTX EE% for PEM-PSNP@PTX and PLGA@PTX were approximately 82.07±3.91% and 58.31±2.51%, respectively (Fig. 1E). The DL% for PEM-PSNP@PTX and PLGA@PTX were approximately 17.60±0.19% and 15.31±1.51%, respectively (Fig. 1F). Furthermore, PTX encapsulated in PEM-PSNP@PTX exhibited a sustained-release profile, with approximately 50% released over 40 hours and gradual release up to 120 hours. On the other hand, PLGA@PTX released more than 50% of its contents within 5 hours (G in Fig. 1).

[0106] Subsequently, the spray stability of PEM-PSNP@PTX was evaluated. Aerosol particles capable of reaching the distal alveolar cavity were generated from the PEM-PSNP@PTX solution using ultrasonic spraying. This process can destabilize the nanoparticles. However, measurements of particle size and surface charge before and after spraying confirmed that no significant changes were observed (H and I in Fig. 1). This suggests that ultrasonic spraying does not impair the stability of PEM-PSNP@PTX. Furthermore, the nanoparticles maintained a stable state even under storage conditions (J in Fig. 1).

[0107]

[0108] Example 2. Evaluation of in vitro cell uptake and cytotoxicity

[0109] The cellular uptake and cytotoxicity of PEM-PSNP@PTX were evaluated using Western blot and confocal laser scanning microscopy (CLSM). In the total protein expression analysis, FR-α expression was significantly increased in H1793 cells compared to A549 cells. Therefore, uptake studies were conducted using H1793 cells as a FR-positive cell line and A549 cells as a FR-negative cell line (Figure 2A and B).

[0110] Next, folate-mediated cytotoxicity was evaluated by treating FR-positive and FR-negative cell lines with PEM-PSNP@PTX. H1793 and A549 cell lines were treated with PEM-PSNP@PTX for 2 hours, followed by an additional 22 hours of culture in fresh medium to evaluate cytotoxicity. Less than 10% cytotoxicity was observed in FR-negative A549 cells, whereas more than 40% cytotoxicity was observed in FR-positive H1793 cells (Fig. 2C).

[0111] In addition, PEM-PSNP@DiI was prepared using the fluorescent dye DiI instead of PTX. Free DiI or PEM-PSNP@DiI was treated to FR-positive and FR-negative cells in the same manner. The signals of free DiI and PEM-PSNP@DiI decreased rapidly in A549 cells but remained bound in H1793 cells (Fig. 2D). To further confirm FR-mediated targeting, H1793 cells were pretreated with an excess of free folic acid (FA) before exposure to PEM-PSNP@DiI. When FR was blocked by FA pretreatment, the DiI signal was significantly reduced (Fig. 11). These findings support the FR-mediated targeting potential of PEM-PSNP@PTX in FR-positive cells.

[0112]

[0113] Example 3. Evaluation of in vitro tumor-associated macrophage (TAM) targeting and repolarization efficacy

[0114] To verify the validity of TAM repolarization, human THP-1 monocytes were first differentiated into PMA to establish M0 macrophages, and then M1 macrophages were polarized with LPS / IFN-γ and M2 macrophages with IL-4 / IL-13 (Fig. 3A). The M1 and M2 polarized macrophages were identified through flow cytometry, and the expression of CD86 in M1 macrophages and CD206 in M2 macrophages increased, respectively (Fig. 3B).

[0115] Western blot analysis showed that FR-β expression was significantly increased in M2 macrophages compared to M0 and M1 macrophages (Fig. 3C and D). Subsequently, the cellular uptake efficiency of PEM-PSNP@DiI in FR-β-positive M2 macrophages was evaluated. When treated with PEM-PSNP@DiI, FR-β-positive M2 macrophages showed co-localization of FR-β expression with PEM-PSNP@DiI (Fig. 3E), which supports the potential for FR-mediated targeting of FR-β-positive TAMs by PEM-PSNP@PTX.

[0116] To evaluate the repolarization effect of PEM-PSNP@PTX, THP-1 monocytes were differentiated and polarized into M0, M1, and M2 macrophages, treated with nanoparticles, and analyzed by flow cytometry (Fig. 3F). When M2 macrophages were treated with PEM-PSNP@PTX, the proportion of M2 marker CD206-positive cells decreased to 61.8%, and the proportion of M1 marker CD86-positive cells increased to 63.1%. These results demonstrate that PEM-PSNP@PTX can effectively repolarize TAMs from an immunosuppressive M2 phenotype to an immunoactive M1 phenotype without inducing apoptosis.

[0117]

[0118] Example 4. Confirmation of in vivo inhalation delivery and distribution

[0119] To evaluate the organ biodistribution of inhaled PEM-PSNP@DiI, major organs (lungs, heart, liver, kidneys, and spleen) were sampled, and DiI signals were measured at various time points for 48 hours after inhalation. It was observed that the signal-to-background ratio (SBR) in the lungs remained high at 2 or higher for up to 48 hours after inhalation (Figures 4A and 4B). This indicates that PEM-PSNP@DiI was continuously accumulated in the lungs, and non-specific distribution to other organs was very low.

[0120] After constructing a mouse lung cancer model using LLC-luc cells and confirming tumor formation via bioluminescent imaging, the delivery efficiency of PEM-PSNP@DiI in the tumor-containing lungs was further investigated (Fig. 4C). Mice were classified into mild and severe models based on the degree of tumor progression, and PEM-PSNP@DiI was administered via inhalation (Fig. 4D and Fig. 12). Optical and fluorescence imaging of the lungs revealed that the DiI signal was clearly co-localized with the tumor site.

[0121]

[0122] Example 5. Evaluation of in vivo anticancer efficacy

[0123] The in vivo therapeutic effect of inhaled PEM-PSNP@PTX was evaluated in a mouse lung cancer model. The drug was administered by inhalation three times a week for three weeks, and tumor size was tracked by measuring luminescence after luciferin injection (Fig. 5A).

[0124] Bioluminescent imaging analysis during inhalation treatment showed that PEM-PSNP@PTX inhalation significantly inhibited tumor growth compared to the free PTX and PLGA@PTX administration groups (Figs. 5B and C, Fig. 13) and significantly increased survival time (Fig. 14). These results were reconfirmed by bioluminescent imaging and histological analysis of the lungs excised on day 25 (Figs. 5D to G). Photographs and H&E staining of the lungs treated with PEM-PSNP@PTX showed a significant reduction in the size and number of tumor nodules.

[0125] To further confirm the regulatory effects of PEM-PSNP@PTX on TAM, bronchoalveolar lavage (BAL) fluid was collected from normal mice, tumor mice, and tumor mice treated with PEM-PSNP@PTX (Fig. 6A). As a result of measuring the levels of cytokines (IL-6, IL-10, IL-12, TNF-α, and TGF-β) associated with M1 and M2 macrophages, a transition to an immune-activated environment was confirmed in mice treated with PEM-PSNP@PTX, with significantly increased levels of IL-12 and TNF-α, which promote anti-tumor immune responses, and decreased levels of TGF-β, which is associated with immunosuppression.

[0126] Next, lung tissue from tumor-bearing mice was harvested and analyzed using immunohistochemistry (IHC). While high expression of the M2 marker (CD206) was observed in the control group, inhalation of PEM-PSNP@PTX resulted in decreased CD206 expression and increased expression of the M1 marker (CD86) (Fig. 6B). Additionally, flow cytometry analysis of the proportion of M2 macrophages (CD206) within the total macrophage population (F4 / 80) revealed a significant decrease in the proportion of M2 macrophages in lungs treated with PEM-PSNP@PTX (Fig. 15). All of the above results demonstrate that PEM-PSNP@PTX inhalation therapy not only directly kills cancer cells but also effectively repolarizes M2 macrophages to the M1 phenotype, thereby improving the tumor microenvironment.

[0127]

[0128] Example 6. In vivo toxicity evaluation

[0129] Toxicity evaluation of inhaled PEM-PSNP@PTX was performed on all treatment groups. Analysis of liver function indicators (ALT, AST) and kidney function indicators (BUN, CREA) via blood chemistry revealed no significant toxicity in any treatment group compared to the control group (Fig. 7A). Furthermore, H&E staining of major organs (lungs, heart, liver, kidneys, spleen) showed no significant histopathological changes in cell number or morphology compared to the control group (Fig. 7B). These results support the high biological safety of inhaled PEM-PSNP@PTX in mice, demonstrating that local delivery via the inhalation route can effectively minimize systemic toxicity.

[0130]

[0131] Although the embodiments have been described above with reference to the limited drawings, those skilled in the art can apply various technical modifications and variations based on the above. For example, suitable results may be achieved even if the described techniques are performed in a different order than described, and / or if the components of the described system, structure, device, circuit, etc. are combined or assembled in a form different from described, or replaced or substituted by other components or equivalents.

[0132] Therefore, other implementations, other embodiments, and equivalents to the claims also fall within the scope of the claims set forth below.

[0133] The present invention was carried out with the support of national research and development projects. Specifically, it was completed with the support of the "University Technology Management Promotion (TLO Innovation Type)" project organized by the Ministry of Science and ICT (Project No. RS-2024-00459242, Project No. 2710075009) and the "Korea-US Innovation Performance Creation R&D" project organized by the Ministry of Health and Welfare, specifically the "Korea-US Innovative Research on Precision Cancer Treatment Using Theranostic Convergence Imaging Platform" (Project No. RS-2024-00436472, Project No. 2460003955).

Claims

1. A pharmaceutical composition comprising lipid-polymer hybrid nanoparticles for inhalation administration, The above nanoparticles are, (a) a polymer core encapsulating a first therapeutic agent inside; and (b) a lipid shell disposed on the surface of the polymer core; comprising, A pharmaceutical composition for the prevention or treatment of lung cancer, characterized in that the above lipid shell is functionalized with pemetrexed, which functions as a second therapeutic agent and simultaneously functions as a target ligand for folate receptors.

2. A pharmaceutical composition according to claim 1, wherein the polymer of the polymer core is selected from the group consisting of poly(lactic acid-co-glycolic acid) (PLGA), polylactic acid (PLA), polycaprolactone (PCL), and polyglycolic acid (PGA).

3. A pharmaceutical composition according to claim 1, characterized in that the first therapeutic agent is selected from the group consisting of paclitaxel, docetaxel, vincristine, and vinblastine.

4. A pharmaceutical composition according to claim 1, characterized in that the lipid shell further comprises a pulmonary surfactant.

5. A pharmaceutical composition according to claim 1, characterized in that the pemetrexid is covalently bonded to the lipid shell through a DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol)]) linker.

6. A pharmaceutical composition according to claim 1, characterized in that the nanoparticles simultaneously bind to folate receptor alpha (FR-α) expressed in lung cancer cells and folate receptor beta (FR-β) expressed in tumor-associated macrophages (TAMs).

7. A pharmaceutical composition according to claim 1, wherein the nanoparticles repolarize the tumor-associated macrophages from an immunosuppressive M2 subtype to an immunoactive M1 subtype.

8. A pharmaceutical composition according to claim 1, characterized in that the average particle size of the nanoparticles is 100 nm to 200 nm.

9. A step of administering a pharmaceutical composition of any one of claims 1 to 8 to a subject other than a human by inhalation in a therapeutically effective amount; comprising a method for treating lung cancer.

10. An inhaled lung cancer treatment comprising a pharmaceutical composition of any one of claims 1 to 8. 11.(a) A step of synthesizing a DSPE-PEG-PEM conjugate by reacting pemetrexed or a derivative thereof with DSPE-PEG-amine; (b) a step of preparing a core solution by dissolving a hydrophobic polymer and a first therapeutic agent in an organic solvent; (c) a step of preparing a lipid layer solution by dispersing the DSPE-PEG-PEM conjugate synthesized in step (a) and a pulmonary surfactant in an aqueous solution; and (d) a method for preparing hybrid nanoparticles for drug delivery, comprising the step of injecting the core solution of step (b) into the lipid layer solution of step (c) to form hybrid nanoparticles by single-step nanoprecipitation.

12. A manufacturing method according to claim 4, characterized in that the synthesis of step (a) is performed through a DCC (Dicyclohexylcarbodiimide) coupling reaction.

13. A manufacturing method according to claim 4, wherein the nanoparticle formation in step (d) is characterized by having a final weight ratio of lipids to polymers of 1:1 to 1:3.

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