Multiple hydrogen bond cross-linked outer membrane inner core type norcantharidin lipid nanoparticles, preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a multi-hydrogen bond cross-linked outer membrane inner core type norcantharidin lipid nanoparticle and a preparation method and application thereof. In order to improve the encapsulation rate of norcantharidin (Nor), solve the problems of poor water solubility, insufficient targeting, low stability and strong toxic and side effects of the existing Nor preparation, the application provides a multi-hydrogen bond cross-linked outer membrane inner core type norcantharidin lipid nanoparticle (Man-Nor-NLCs). Compared with the existing nanoparticles for delivering Nor, the Man-Nor-NLCs of the application not only has enhanced structural stability, but also can realize precise targeting and intelligent controlled release, and has specific, rapid and efficient accumulation capacity in tumor tissues. Meanwhile, the preparation raw material is biodegradable and non-toxic, and is easy to industrialize, so the application has wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a multi-hydrogen bond cross-linked outer membrane core type of norcantharidin lipid nanoparticles, its preparation method, and its application. Background Technology

[0002] Norcantharidin (Nor, CAS No.: 5442-12-6) is a compound synthesized from the active ingredient extracted from the traditional Chinese medicine blister beetle. Its core mechanism of action includes inhibiting cancer cell DNA synthesis, blocking the cell cycle in the G2 / M phase, and disrupting the cytoskeleton and ultrastructure. It also enhances its anti-tumor effect through immunomodulatory effects such as increasing white blood cell count and inhibiting hepatitis B virus replication, making it one of the commonly used anti-tumor drugs in clinical practice. However, Nor has significant inherent limitations that severely restrict its clinical application value: First, its extremely poor water solubility makes it difficult to evenly disperse in topical or injectable formulations, directly reducing drug absorption efficiency; second, its low bioavailability means it is easily metabolized and eliminated rapidly after entering the body, making it difficult to form an effective therapeutic concentration in target tissues; third, it has potential irritant effects on the digestive and urinary systems, posing a certain toxicity risk. Existing Nor formulation technologies also suffer from numerous bottlenecks: limited formulation types, primarily consisting of ordinary tablets and injections, lacking efficient drug delivery systems to fundamentally improve solubility and targeting; insufficient formulation stability, with drugs prone to precipitation or degradation during storage, reducing efficacy and potentially increasing adverse reactions; lack of targeting leading to widespread drug distribution in the body, resulting in low concentrations at the target site, limited efficacy, and exacerbated side effects; and immature manufacturing processes, making it difficult to balance drug solubility, stability, and biocompatibility, and lacking standardized, efficient preparation methods. Therefore, developing a Nor delivery system that combines high stability, precise targeting, intelligent controlled release capabilities, and excellent biocompatibility has become an urgent technical challenge.

[0003] Nanostructured lipid carriers, as an improved type of lipid nanoparticle, are a second-generation solid lipid nanoparticle developed based on solid lipid nanoparticles, and represent a novel nanostructured delivery system. Nanostructured lipid carriers are prepared by adding liquid oils with significantly different chemical properties to solid lipids, allowing the nanoparticles to exist in a crystalline defect or amorphous structure. Mixed lipids are commonly used as carrier materials, with the liquid lipids being mixed into the solid lipids. The main preparation methods for nanostructured lipid carriers include melt-emulsification, high-pressure homogenization, solvent diffusion, and thin-film dispersion-ultrasonic methods. Due to the poor water solubility of norcantharidin, the encapsulation efficiency of nanostructured lipid carriers prepared by conventional methods is low. Existing methods for preparing nanostructured lipid carriers are not suitable for norcantharidin. The applicant previously developed a norcantharidin nanostructured lipid carrier (patent CN115054578 A), with the following raw material components by weight ratio: norcantharidin 0.1-6 parts, polyethylene glycol-400 5-15 parts, polyoxyethylene castor oil 8-16 parts, ethyl oleate 20-50 parts, glyceryl tripalmitate 0.4-2 parts, phospholipid 1-5 parts, glyceryl monostearate 2-5 parts, Tween-80 1-5 parts, and aqueous phase 72-308 parts. However, the stability of the previously prepared norcantharidin nanostructured lipid carrier still needs improvement, and the safety of norcantharidin needs further enhancement. Passive targeting solely through the tumor EPR effect suffers from individual variability and limitations in tumor site targeting efficiency. Summary of the Invention

[0004] To improve the encapsulation efficiency of norcantharidin (Nor) and address the problems of poor water solubility, insufficient targeting, low stability, and strong toxic side effects in existing Nor formulations, this invention provides a multi-hydrogen bond cross-linked outer membrane core-type norcantharidin lipid nanoparticle (Man-Nor-NLCs) to achieve efficient, precise, and safe drug delivery.

[0005] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In a first aspect, the present invention provides a multi-hydrogen bond cross-linked outer membrane core-type norcantharidin lipid nanoparticle (Man-Nor-NLCs), the structure of which is norcantharidin lipid nanoparticles (Nor-NLCs) as the core and mannan (Man) as the outer membrane coating, and the core and outer membrane are cross-linked by multiple hydrogen bonds (MHBs).

[0006] In some embodiments, the raw materials for preparing the core Nor-NLCs include, by weight, 0.1 to 1 part of norcantharidin, 4.5 to 12 parts of polyethylene glycol-400, 15 to 40 parts of ethyl oleate, 6 to 15 parts of polyoxyethylene castor oil, 0.5 to 1.5 parts of tripalmitic acid glyceride, 1 to 3 parts of phospholipids, 1 to 4 parts of glyceryl monostearate, 1 to 3.5 parts of Tween-80, and 50 to 200 parts of water.

[0007] In some embodiments, the Nor-NLCs are prepared by first preparing a norepinephrine nanoemulsion and then dissolving it with a lipid film.

[0008] In some embodiments, the Man-Nor-NLCs have a particle size of 160–180 nm.

[0009] In some implementation schemes, the dosage of Man is 0.5 to 8 parts.

[0010] In some preferred embodiments, the concentration of the Man solution is 20 mg / mL.

[0011] In some embodiments, the volume ratio of Man solution to Nor-NLCs is 2–10:0.1–1. Preferably, the volume ratio of Man solution to Nor-NLCs is 2–3:1.

[0012] Secondly, the present invention provides a method for preparing the above-mentioned Man-Nor-NLCs, comprising the following steps: (1) Preparation of kernel Nor-NLCs: The norcantharidin nanoemulsion was obtained by mixing 0.1-1 parts of norcantharidin, 4.5-12 parts of polyethylene glycol-400, 15-40 parts of ethyl oleate, and 6-15 parts of polyoxyethylene castor oil in an aqueous phase of 2-6 parts. Then, 0.5 to 1.5 parts of tripalmitoyl glyceride, 1 to 3 parts of phospholipid, and 1 to 4 parts of glyceryl monostearate are mixed in an organic phase, and the organic phase is removed to obtain a uniform lipid film. Then, the norcantharidin nanoemulsion and lipid film were dissolved and mixed in an aqueous solution containing 1 to 3.5 parts of Tween-80 to obtain Nor-NLCs. (2) Preparation of Man-Nor-NLCs by active modification with Man: Man aqueous solution is mixed with Nor-NLCs prepared in step (1) to obtain Man-Nor-NLCs.

[0013] In some implementations, the amount of water used in the Man aqueous solution is the same as the amount used in the Tween-80 aqueous solution.

[0014] In some preferred embodiments, the amount of water used in the Man aqueous solution and the amount of Tween-80 aqueous solution are both 70 to 200 parts.

[0015] In some implementation schemes, the dosage of Man is 0.5 to 8 parts.

[0016] In some embodiments, the volume ratio of Man solution to Nor-NLCs is 2–10:0.1–1. Preferably, the volume ratio of Man solution to Nor-NLCs is 2–3:1.

[0017] In some embodiments, the organic phase for preparing the lipid film is preferably selected from one or any combination of anhydrous ethanol, chloroform, acetone, dichloromethane, and ethyl acetate.

[0018] In some embodiments, the norcantharidin nanoemulsion is obtained by adding water droplets to a mixture of norcantharidin, polyethylene glycol-400, ethyl oleate, and polyoxyethylene castor oil.

[0019] In some embodiments, the Man aqueous solution is obtained by first fully swelling Man in water and then stirring to mix thoroughly. In some preferred embodiments, the concentration of the Man solution is 20 mg / mL.

[0020] In some implementations, Man-Nor-NLCs are obtained by adding an aqueous solution of Man dropwise to norcantharidin lipid nanoparticles while stirring.

[0021] Thirdly, the present invention provides the use of the above-mentioned Man-Nor-NLCs in the preparation of drugs for the prevention and / or treatment of fibrotic diseases, liver or tumors.

[0022] In some embodiments, the tumor is preferably selected from liver cancer, bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastoma, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct carcinoma, leiomyosarcoma, and lipoma. Liposarcoma, nasopharyngeal carcinoma, neuroendocrine carcinoma, ovarian cancer, salivary gland carcinoma, metastatic tumors caused by spindle cell carcinoma, anaplastic large cell lymphoma, undifferentiated thyroid carcinoma, non-Hodgkin lymphoma, Hodgkin lymphoma, glioma, and malignant hematologic disorders such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), or any combination thereof.

[0023] In some embodiments, the fibrotic disease is preferably one or any combination of liver fibrosis, pulmonary fibrosis, pancreatic fibrosis, renal fibrosis, cardiac fibrosis, endometrial fibrosis, ocular fibrosis, splenic fibrosis, myelofibrosis, and cutaneous fibrosis.

[0024] Beneficial Effects: This invention constructs a targeted drug delivery system based on an integrated design concept of "lipid core-multiple hydrogen bond crosslinks-mannan outer membrane." Its structure uses Nor-NLCs as the core and Man as the outer membrane coating, with the core and outer membrane crosslinked via MHBs. Compared with existing norcantharidin lipid nanoparticles, the Man-Nor-NLCs of this invention have the following advantages: (1) Significantly improves drug loading and dissolution efficiency: The "lipid core" compound system, combined with the molecular anchoring effect of MHBs, enables the drug to be dispersed in an amorphous or molecular state, with an encapsulation rate of over 87.41%, effectively solving the problem of poor water solubility; (2) Enhanced structural stability and circulation efficiency: MHBs mildly crosslinks to enhance the binding between the outer membrane and the inner core. After 6 months of storage, the encapsulation retention rate is >90%, while reducing plasma protein adsorption and clearance by the mononuclear macrophage system, thus prolonging the in vivo circulation time. (3) Achieving precise targeting and intelligent controlled release: The dual mode of "active targeting + passive targeting" enhances the drug accumulation in the tumor site, and MHBs dynamically dissociates in the acidic microenvironment of the tumor to achieve intelligent sustained release, significantly reducing off-target toxicity; (4) Optimize biocompatibility and safety: The carrier material is biodegradable, MHBs do not require toxic cross-linking agents, have no residual toxicity and do not cause significant damage to major organs, thus improving patient tolerance; (5) Clear industry prospects: The carrier is suitable for multiple administration routes such as oral and intravenous injection, and the preparation process is suitable for large-scale production, which expands the clinical application scenarios and industrialization potential.

[0025] (6) The Man-Nor-NLCs prepared in this invention serve as drug carriers, combining the characteristics of drug-containing microemulsions, liposomes, and ligand modification. This allows the nanoparticles to exist in a crystal defect or amorphous structure, increasing the excipients' capacity to contain the drug and avoiding leakage of the drug during storage and the resulting decrease in encapsulation efficiency. At the same time, ligand modification is used to exert the active targeting effect of the drug, achieving active targeting through receptor-mediated endocytosis, significantly increasing the drug enrichment at the tumor site, and reducing the drug distribution in normal organs.

[0026] (7) By using microemulsion as a drug delivery method, the present invention can encapsulate the drug in liquid lipids to the maximum extent. Then, by using the thin film dispersion-ultrasound method, some of the drug-containing liquid lipids and free drugs are mixed in solid lipids, which increases the proportion of irregular crystal forms of norcantharidin in the nanoparticle structure, increases the space capacity for carrying lipid-soluble drugs, thereby improving the drug loading capacity of the carrier. Man-Nor-NLCs with an encapsulation rate of (87.41±0.16)% can be prepared, which prevents Nor from leaking during storage and enhances the stability of the formulation.

[0027] (8) Man, as a ligand modifier, has a hydrophilic polysaccharide structure that can reduce plasma protein adsorption, reduce the recognition and clearance of the mononuclear macrophage system, prolong the in vivo circulation time of the carrier and enhance its structural stability in the blood environment. This alleviates the shortcomings of simple nanoemulsions, such as short in vivo circulation and easy clearance. Man's active targeting effect can enable the drug to be accurately enriched at the tumor site, reduce the drug exposure in normal tissues, significantly reduce off-target toxicity, and improve the problem of obvious toxic side effects of simple NLC. In addition, Man is derived from natural polysaccharides, has good biocompatibility and low immunogenicity. Its modification process does not introduce toxic substances, which can further improve the biocompatibility of NLC, reduce the irritation of the formulation to blood vessels or tissues, and supplement the optimization space of simple NLC in terms of biocompatibility details.

[0028] (9) The MHBs crosslinked Man-Nor-NLCs prepared in this invention have specific, rapid and efficient accumulation ability in liver cancer tumor tissue, providing a novel delivery system for norcantharidin, which is of great significance; the preparation process is simple, the cost is low, it is easy to control and easy to industrialize, and it has broad application prospects.

[0029] As can be seen, the MHBs network formed between the Man and Nor-NLCs surface modification groups in this paper constructs a physically cross-linked "outer membrane-core" structure. This design has three major advantages: (1) Enhanced structural stability: The MHBs-mediated Man network forms a dense outer membrane, which significantly improves the stability of nanomedicines in blood circulation and prolongs their half-life; (2) Intelligent response drug release: MHBs are controllable and can trigger drug release under weakly acidic or high glutathione (GSH) conditions in the tumor microenvironment; (3) Dual active targeting basis: Man can specifically recognize the mannose receptor (MR / CD206) highly expressed on the surface of liver cancer cells and M2 type TAMs, achieving dual-cell targeted enrichment. Attached Figure Description

[0030] Figure 1 The particle size distribution of Man-Nor-NLCs prepared in Example 1 of this invention is shown in the diagram. Figure 2 Transmission electron microscopy image of Man-Nor-NLCs prepared in Example 1 of this invention; Figure 3 The infrared spectra of Man-Nor-NLCs, Man-Nor-NLCs physical mixture, Nor-NLCs physical mixture, Man, and Nor prepared in Example 1 of this invention are shown. Figure 4The DSC diagrams of Man-Nor-NLCs, Man-Nor-NLCs physical mixtures, Nor-NLCs physical mixtures, Man, and Nor prepared in Example 1 of the present invention are shown. Figure 5 XRD patterns of Man-Nor-NLCs, Man-Nor-NLCs physical mixture, Nor-NLCs physical mixture, Man, and Nor prepared in Example 1 of this invention; Figure 6 Release curves (B) of Man-Nor-NLCs, Nor-NLCs, and Nor prepared in Example 1 of this invention in pH 1.2 HCl (A) and pH 6.8 PBS release media. Figure 7 The blood drug concentration-time curves of Man-Nor-NLCs, Nor-NLCs, and Nor prepared in Example 1 of this invention in rats; Figure 8 Man-Nor-NLCs, Nor-NLCs, and Nor-induced H2O prepared in Example 1 of this invention 22 Line graph of survival rate of tumor-bearing mice; Figure 9 The Man-Nor-NLCs, Nor-NLCs, and Nor inhibitors prepared in Example 1 of this invention are effective against H1N1 hepatocellular carcinoma. 22 Inhibition of cell proliferation (n=6), (A: 24 h B: 48 h C: 72 h) Figure 10 Man-Nor-NLCs, Nor-NLCs, and Nor-induced H2O prepared in Example 1 of this invention 22 Fluorescence detection image of cells; Figure 11 Man-Nor-NLCs, Nor-NLCs, and Nor-induced hepatocellular carcinoma H2O prepared in Example 1 of this invention 22 Tumor images of tumor-bearing mice; compared with the model group. # P <0.01, ## P <0.01, ### P <0.001; compared with Nor, *P <0.01, **P <0.01, ***P <0.001; compared with Nor-NLCs, $ P <0.01, $$ P <0.01, $$$P <0.001; Figure 12 HE staining images of Man-Nor-NLCs, Nor-NLCs, and Nor prepared in Example 1 of this invention; Figure 13 Safety evaluation diagrams of Man-Nor-NLCs, Nor-NLCs, and Nor prepared in Example 1 of this invention; compared with the normal group, @ P <0.01, @@ P <0.01, @@@ P <0.001; compared with the model group, # P <0.01, ## P <0.01, ### P <0.001; compared with Nor, *P <0.01, **P <0.01, ***P <0.001; compared with Nor-NLCs, $ P <0.01, $$ P <0.01, $$$ P <0.001. Detailed Implementation

[0031] To make the technical problems, solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with the embodiments. Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art.

[0032] In this document, the terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., meaning “including but not limited to”).

[0033] In this document, “and / or” means and includes any and all possible combinations of one or more of the associated listed items. For example, “the composition contains A and / or B” can be interpreted as the composition contains A, the composition contains B, or the composition contains both A and B.

[0034] In this paper, the term "norcantharidin" or "Nor" refers to a compound synthesized artificially from the active ingredient extracted from the traditional Chinese medicine blister beetle. It is primarily used to treat liver cancer and various solid tumors, exhibiting low side effects and ease of clinical application. However, it has extremely poor water solubility and low bioavailability, and poses potential irritation to the digestive and urinary systems, indicating a certain toxicity risk. Therefore, there is a need to develop a Nor delivery system that combines high stability, precise targeting, intelligent controlled release capability, and excellent biocompatibility for the efficient, precise, and safe delivery of Nor to relevant targets.

[0035] To address the aforementioned needs, the applicant has developed the targeted drug delivery system described in this invention based on an integrated design concept of "lipid core-multiple hydrogen bond crosslinking-mannan outer membrane": a multi-hydrogen bond crosslinked outer membrane core-type norcantharidin lipid nanoparticles (Man-Nor-NLCs), the structure of which uses norcantharidin lipid nanoparticles as the core and mannan (Man) as the outer membrane coating, with the core and outer membrane crosslinked by multiple hydrogen bonds.

[0036] The Man-Nor-NLCs design provided by this invention achieves end-to-end optimization of "efficient drug loading - stable delivery - precise targeting - synergistic therapy" through the functional synergy and hierarchical coupling of each structural unit. The lipid core, as the core functional unit for drug loading and sustained release, directly determines the solubility improvement effect and in vivo delivery efficiency of Nor through its material selection and structural design. In some examples, biocompatible and biodegradable lipid materials (such as phospholipids, solid lipids such as tripalmitoyl glycerol and monostearate) are selected to construct the core. Relying on the hydrophobic domains of lipid molecules to form hydrophobic interactions with Nor, efficient encapsulation of poorly soluble Nor can be achieved, significantly improving drug solubility and dispersion uniformity, thus solving the formulation dispersion problem from the source. Simultaneously, the lipid core possesses natural sustained-release properties, which can delay the metabolic clearance rate of Nor in vivo, prolong the drug's action time, and ensure the sustained maintenance of effective drug concentration at the target site. Furthermore, the amphiphilic nature of lipid materials makes them easy to self-assemble into nanoscale particle structures. The hydrophilic groups of their surface phospholipid molecules can serve as sites for subsequent cross-linking modifications, providing stable structural support for the cross-linking of multiple hydrogen bonds (MHBs) and the construction of the outer membrane of Man. This is the core foundation for carrying drugs and connecting outer functional units in integrated design.

[0037] The term "hydrogen bonds (HB)" used in this article refers to chemical bonds that are widely present in nature and are of great significance to the development of nature. The basic structure of HB can be represented as XH…Y, mainly composed of two parts: XH as a proton donor and Y as a proton acceptor. Qualitative analysis of HB can be performed using analytical methods such as FT-IR spectroscopy, with research focusing primarily on HBs composed of a single donor and a single acceptor. Based on the number of HBs formed by the two structural units, they can be classified as single, double, triple, quadruple, and MHBs. Single and double HBs have relatively weak strength and cannot form strong three-dimensional cross-linked networks when used as physical cross-linking points, resulting in relatively weak material mechanical strength. However, when MHBs coexist, the superposition of HBs can generate strong interaction forces, leading to a more stable three-dimensional cross-linked network with excellent mechanical properties when used as physical cross-linking points. As widely existing weak interactions in nature, their synergistic effect endows materials with excellent structural stability and dynamic responsiveness, serving as a key bridge connecting the lipid core and the manganese outer membrane: MHBs promote the formation of multi-scale structures containing ultra-strong hydrogen bonds and nanoscale domains by forming a uniform network. MHBs can improve interchain friction and energy dissipation; the uniform network can evenly distribute stress and avoid stress concentration; the high-strength HB domains restrict the movement of polymer chains and can dissipate energy through configurational transformation, reducing stress concentration, and can also hinder macroscopic crack propagation, causing crack deflection and bifurcation, thereby further dissipating energy; the uniformity at the macroscopic scale ensures that the material has no macroscopic defects. Therefore, materials constructed by physical cross-linking of MHBs possess extremely excellent mechanical properties, achieving both high toughness and high rigidity, surpassing many synthetic materials. Assembling various nanostructures based on physical cross-linking of MHBs is a topic of great interest and is considered an important approach to constructing nanostructures "from the bottom up," and this structure can be regulated by environmental conditions. Due to the wide variety of HBs, the constructed material structures exhibit diversity. Recently, many studies have focused on cross-linking MHBs to form adhesives, elastomers, gels, and two-dimensional assemblies, but few have covered targeted drug delivery systems. There are no research reports on the application of physical cross-linking of MHBs to construct drug delivery systems with special structures.

[0038] Based on the above approach, the applicant introduced the ligand Man into Nor-NLCs via physical cross-linking with MHBs. In the integrated Man-Nor-NLCs design described herein, MHB cross-linking is mainly formed through two pathways: first, intermolecular hydrogen bonds are formed between the hydrophilic groups (such as hydroxyl and amino groups) of the phospholipid molecules on the lipid core surface and the hydroxyl and carboxyl groups on the Man molecular chain; second, intramolecular hydrogen bonds are formed between the polar groups within the Man molecular chain. This dual cross-linking effect enables a tight bond between the lipid core and the Man outer membrane, significantly improving the overall structural stability of the carrier and effectively preventing premature drug leakage during storage or in vivo circulation, thus addressing the problem of insufficient stability in existing formulations. Simultaneously, MHBs possess mild and reversible properties, without compromising the drug activity of Nor or the biocompatibility of the carrier. Furthermore, they can respond to stimuli such as pH changes and enzymatic hydrolysis in the tumor microenvironment to achieve dynamic dissociation of the cross-linked network, thereby regulating intelligent drug release and enabling precise drug delivery at the target site. In addition, the uniform nanodomain structure formed by MHB cross-linking enhances the mechanical toughness and rigidity of the carrier, strengthening its shear resistance during in vivo circulation and ensuring targeted delivery efficiency.

[0039] The term "mannan" or "Man" used in this article, as a natural polysaccharide, possesses inherent advantages such as good biocompatibility, low immunogenicity, and mild modification processes, making it an ideal material for constructing actively targeted outer membranes. Its modification process does not damage the lipid core structure or the drug activity of Nor, exhibiting broad compatibility. More importantly, Man can specifically recognize and bind to the Man receptor highly expressed on the surface of tumor cells, achieving precise drug delivery to the tumor target site through receptor-mediated endocytosis. This increases targeting efficiency by 2–5 times compared to unmodified carriers, significantly reducing drug distribution in normal tissues and lowering off-target toxicity. Simultaneously, Man possesses unique immunomodulatory activity, forming a synergistic anti-tumor effect with Nor through a dual mechanism: on the one hand, Man can participate in the tumor cell glycolysis pathway, directly inhibiting tumor cell proliferation and synergistically responding to the chemotherapy effect of Nor; on the other hand, it can activate dendritic cells to induce IL-10 production and promote CD8 activation. + T immune cells recognize and kill cancer cells, while activating macrophages to produce TNF-α and NO, regulating the body's immune microenvironment. This effect coincides with the immunomodulatory effect of NO in increasing white blood cells, jointly constructing a chemotherapy / immunotherapy synergistic treatment system to further enhance anti-tumor efficacy.

[0040] In some examples in this paper, the raw materials for preparing core Nor-NLCs, by weight, include 0.1 to 1 part of norcantharidin, 4.5 to 12 parts of polyethylene glycol-400, 15 to 40 parts of ethyl oleate, 6 to 15 parts of polyoxyethylene castor oil, 0.5 to 1.5 parts of tripalmitic acid glyceride, 1 to 3 parts of phospholipids, 1 to 4 parts of glyceryl monostearate, 1 to 3.5 parts of Tween-80, and 50 to 200 parts of water.

[0041] The proportions of each component in the raw materials for preparing the core norcantharidin lipid nanoparticles in this paper were further optimized based on the proportions in patent CN115054578 A. Compared with the norcantharidin lipid nanoparticles obtained previously, the norcantharidin lipid nanoparticles in this paper have a smaller molecular weight distribution index (PDI), which is 0.618 ± 0.068 in patent CN 115054578 A. vs This paper (PDI=0.18±0.02) shows a more uniform distribution.

[0042] In some examples described herein, the volume ratio of Man solution to Nor-NLCs is 2–10:0.1–1. Preferably, the volume ratio of Man solution to Nor-NLCs is 2–3:1.

[0043] Preparation method of Man-Nor-NLCs This article also provides a method for preparing Man-Nor-NLCs, including the following steps: (1) Preparation of kernel Nor-NLCs: The norcantharidin nanoemulsion was obtained by mixing 0.1-1 parts of norcantharidin, 4.5-12 parts of polyethylene glycol-400, 15-40 parts of ethyl oleate, and 6-15 parts of polyoxyethylene castor oil in an aqueous phase of 2-6 parts. Then, 0.5 to 1.5 parts of tripalmitoyl glyceride, 1 to 3 parts of phospholipid, and 1 to 4 parts of glyceryl monostearate are mixed in an organic phase, and the organic phase is removed to obtain a uniform lipid film. Then, the norcantharidin nanoemulsion and lipid film were dissolved and mixed in an aqueous solution containing 1 to 3.5 parts of Tween-80 to obtain Nor-NLCs. (2) Preparation of Man-Nor-NLCs by active modification with Man: Man aqueous solution is mixed with Nor-NLCs prepared in step (1) to obtain Man-Nor-NLCs.

[0044] The preparation method of the core norepinephrine lipid nanoparticles described in this article is as described in patent CN 115054578 A. Based on the existing patent CN 115054578 A, the component ratios of each raw material are adjusted to obtain norepinephrine lipid nanoparticles with higher uniformity of distribution, which is more conducive to the sustained release of norepinephrine drug in vivo. The core of this Man-Nor-NLCs preparation method lies in the first-ever deep integration of MHBs crosslinking technology with outer membrane-core structure design, constructing norcantharidin lipid nanoparticles with both stable delivery and precise targeting functions. This breaks through the limitations of traditional single-structure lipid nanoparticles, constructing a "Man outer membrane-lipid core" composite system. The outer membrane is tightly crosslinked with the core through MHBs, forming a functionally integrated structure of "stable coating-responsive dissociation," solving the technical pain points of ligand modification detachment and insufficient structural stability. It abandons toxic chemical crosslinking agents, utilizing the synergistic effect of MHBs to achieve mild crosslinking, ensuring both the compactness of the carrier structure and endowing it with tumor microenvironment (acidic) responsiveness, filling the technical gap in the application of MHBs in norcantharidin lipid nanoparticles. Integrating the dual modes of "EPR effect passive targeting + mannose receptor active targeting," the enhanced structural stability from MHBs crosslinking prolongs in vivo circulation time, providing a guarantee for targeted delivery and overcoming the bottleneck of limited single-targeting efficiency. Furthermore, the cross-linking of MHBs ensures that the encapsulation retention rate of the carrier is >90% and the particle size change rate is <8% after 6 months of storage. Simultaneously, hydrogen bond dissociation in the acidic tumor microenvironment enables sustained and controlled drug release, avoiding burst release and off-target toxicity. The "lipid core" compound system, combined with the spatial containment effect of MHBs, achieves an encapsulation rate of over 87.41% for poorly soluble norcantharidin. The outer membrane protects the drug from degradation, while the core promotes transmembrane transport, resulting in a 5-8 times increase in drug accumulation at the tumor site compared to traditional carriers. Both carrier materials (lipids and manganese) are biodegradable, and the MHBs cross-linking leaves no toxic residues. There is no significant damage to major organs in vivo, and off-target toxicity is significantly reduced, greatly improving patient tolerance. This treatment specifically addresses the clinical pain points of Norcantharidin, such as poor water solubility, low bioavailability, and strong toxic side effects, achieving a tumor inhibition rate of 80.80% and significantly prolonging the survival of tumor-bearing mice. It provides a novel "highly effective, low-toxicity, and precise" formulation for the treatment of liver cancer and other tumors, broadening its clinical application scenarios.

[0045] In some examples, the crosslinking of MHBs is achieved by mixing an aqueous solution of Man with core-derived norcantharidin lipid nanoparticles at a volume ratio of 2–10:0.1–1. The amount of water in the Man aqueous solution is the same as that in the Tween-80 aqueous solution. In some preferred examples, the amount of water in the Man aqueous solution and the amount of Tween-80 aqueous solution are both 70–200 parts.

[0046] In some preferred examples, the volume ratio of the Man solution to Nor-NLCs is 2 to 3:1.

[0047] In some specific embodiments, the preparation method of Man-Nor-NLCs includes the following steps: (1) Construction of “core” Nor-NLCs: First, Nor was mediated using nanoemulsion. The nanoemulsion consisted of an oil phase (ethyl oleate), an emulsifier (polyoxyethylene castor oil), and a co-emulsifier (polyethylene glycol-400). After stirring in a water bath at 40°C in the dark for 3-4 h, the mixture was stirred in a water bath at 20°C while adding the aqueous phase dropwise to obtain the Nor-mediated nanoemulsion. Second, the nanoemulsion, surfactant (Tween-80), solid lipids (glyceryl monostearate and phospholipids), and liquid lipids (glyceryl tripalmitate) were added to an organic solution. A white film was formed by vacuum evaporation, and finally, the “core” Nor-NLCs were obtained by hydration, cooling, and ultrasonic treatment.

[0048] (2) Construction of "outer membrane-core" Man-Nor-NLCs by physical cross-linking of MHBs: Man-Nor-NLCs were constructed by cross-linking the ligand Man with MHBs through a non-chemical synthesis pathway (temperature-driven swelling and contraction). The procedure was as follows: A suitable amount of Man was weighed and dissolved in ultrapure water, and the mixture was magnetically stirred at 40°C for 3-4 h. After full swelling, the mixture was shrunk by stirring in an ice bath for 30 min, and then stirred in a water bath at room temperature. Nor-NLCs were added dropwise while stirring, and stirring was continued for 3-5 h to obtain Man-Nor-NLCs. A method for treating fibrotic diseases, liver or tumors Based on the integrated design presented in this paper, the constructed Man-Nor-NLCs achieve functional synergy among their structural units: the lipid core addresses the core challenge of Nor's poor water solubility, MHBs crosslinking ensures carrier stability and intelligent controlled release capability, and the Man outer membrane enables precise targeting and immune synergy. This system not only significantly improves Nor's bioavailability and reduces toxic side effects but also enhances therapeutic efficacy through the synergistic effect of chemotherapy and immunotherapy, expanding Nor's clinical application scenarios. The research findings not only provide technical support for upgrading the clinical application of norcantharidin but also accumulate experimental data for the application of MHBs crosslinking technology in the field of nanomedicine, providing a reference paradigm for the development of delivery systems for other poorly soluble antitumor drugs, possessing significant academic value and industrial transformation potential. Therefore, this paper provides a method for treating liver, fibrotic diseases, or tumors using the constructed Man-Nor-NLCs, which involves administering an effective amount of the Man-Nor-NLCs of this invention to a subject.

[0049] As used herein, the term "effective amount" means the amount of a compound that is sufficient to achieve such treatment or prevention when administered to a subject for the treatment or prevention of a disease. "Effective amount" can vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject being treated. "Therapeutic effective amount" refers to an effective amount for therapeutic treatment. "Prophylactic effective amount" refers to an effective amount for prophylactic treatment.

[0050] As used herein, the term "administration" means the physical introduction of a drug agent into a subject using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration include at least one of the following: intradermal single-point injection, intradermal multiple-point injection, subcutaneous single-point injection, subcutaneous multiple-point injection, intravenous injection, peritumoral injection, intratumoral injection, intrapleural injection, intraperitoneal injection, subarachnoid injection, intra- or peri-lymphatic injection, or intramuscular injection.

[0051] As used herein, the terms “subject,” “individual,” and “patient” are well-known in the art and are used interchangeably herein to refer to any subject requiring treatment, including mammals and non-mammals. Mammals refer to any member of the mammalian class, including but not limited to: humans; non-human primates such as chimpanzees and other ape and monkey species; farm animals such as cattle, horses, sheep, goats, and pigs; livestock such as rabbits, dogs, and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs; and so on. Examples of non-mammals include, but are not limited to, birds. The term “object” is not limited to a specific age or sex. In some embodiments, the object is a person.

[0052] The tumors mentioned in this article are preferably selected from liver cancer, bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastoma, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, and liposarcoma. Nasopharyngeal carcinoma, neuroendocrine carcinoma, ovarian cancer, salivary gland carcinoma, metastatic tumors caused by spindle cell carcinoma, anaplastic large cell lymphoma, undifferentiated thyroid carcinoma, non-Hodgkin lymphoma, Hodgkin lymphoma, glioma, and malignant hematologic diseases such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML) – one or any combination thereof.

[0053] The fibrotic diseases mentioned in this article are preferably selected from one or any combination of liver fibrosis, pulmonary fibrosis, pancreatic fibrosis, renal fibrosis, cardiac fibrosis, endometrial fibrosis, ocular fibrosis, splenic fibrosis, myelofibrosis, and skin fibrosis.

[0054] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. The advantages and features of this invention will become clearer with this description. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions were performed under conventional conditions in the art or as recommended by the manufacturer. Unless otherwise stated, the experimental materials and reagents used in the following embodiments are commercially available.

[0055] In practice, 1 part raw material = 100 mg, 1 part water = 0.1 mL = 100 mg.

[0056] Example 1: Preparation of mannan-modified, multi-hydrogen-bonded outer membrane core-type norcantharidin lipid nanoparticles (Man-Nor-NLCs) (1) Preparation of core-derived norcantharidin lipid nanoparticles (Nor-NLCs): One part of norcantharidin, 4.5 parts of polyethylene glycol-400, 16.5 parts of ethyl oleate, and 6 parts of polyoxyethylene castor oil were stirred in a 40°C water bath in the dark for 3 hours. Then, the mixture was stirred in a 20°C water bath while slowly adding 3 parts of ultrapure water (5-7 drops / min). After stirring for 15 minutes, the norcantharidin nanoemulsion was obtained. 0.5 parts of tripalmitic acid glyceride, 1 part of phospholipid, and 1.25 parts of glyceryl monostearate were placed in a round-bottom flask, and 25 mL of anhydrous ethanol was added to dissolve them. The organic solvent was removed by rotary evaporation in a 40ºC water bath, forming a uniform lipid film on the inner wall of the round-bottom flask. Then, the prepared norcantharidin nanoemulsion was added. Separately, 1.25 parts of Tween-80 and 50 parts of ultrapure water were taken and sonicated for 15 min to fully dissolve them, forming a Tween-80 solution. The Tween-80 solution was added to the round-bottom flask containing the norcantharidin nanoemulsion, and the flask was placed in an ice bath and sonicated for 10 min to remove the film from the inner wall of the round-bottom flask, thus obtaining the norcantharidin lipid nanoparticles.

[0057] (2) Preparation of outer membrane-coated norepinephrine lipid nanoparticles (Man-Nor-NLCs) by active modification of mannan (Man) Dissolve 1 part of Man in 50 parts of ultrapure water 3, and stir magnetically at 40 ºC for 3 h. After full swelling, stir in an ice bath for 30 min to shrink, and then stir in a water bath at room temperature to obtain a Man aqueous solution with a concentration of 20 mg / mL. Add the norcantharidin lipid nanoparticles obtained in step (1) dropwise while stirring. The volume ratio of Man solution to norcantharidin lipid nanoparticles is 2:1. Stir in a water bath at 25 ºC for 5 h to obtain Man-Nor-NLCs.

[0058] Example 2: Preparation of Man-Nor-NLCs (1) Preparation of kernel Nor-NLCs: 0.6 parts of norcantharidin, 7 parts of polyethylene glycol-400, 27 parts of ethyl oleate, and 10 parts of polyoxyethylene castor oil were stirred in a 40ºC water bath in the dark for 4 hours. Then, the mixture was stirred in a 20°C water bath while slowly adding 3 parts of ultrapure water (5-7 drops / min). After stirring for 20 minutes, the norcantharidin nanoemulsion was obtained. 1.5 parts of tripalmitic acid glyceride, 3 parts of phospholipid, and 4 parts of monostearate glyceride were placed in a round-bottom flask, and 20 mL of chloroform was added to dissolve them. The organic solvent was removed by rotary evaporation in a 40ºC water bath, forming a uniform lipid film on the inner wall of the round-bottom flask. Then, the prepared norcantharidin nanoemulsion was added. Separately, 3 parts of Tween-80 and 70 parts of ultrapure water were taken and sonicated for 30 min to fully dissolve them, forming a Tween-80 solution. The Tween-80 solution was added to the round-bottom flask containing the norcantharidin nanoemulsion, and the flask was placed in an ice bath and sonicated for 15 min to remove the film from the inner wall of the round-bottom flask, thus obtaining the norcantharidin lipid nanoparticles.

[0059] (2) Preparation of Man-Nor-NLCs by active modification with Man Dissolve 3 parts of Man in 70 parts of ultrapure water, and stir magnetically at 40 ºC for 3 h. After full swelling, stir in an ice bath for 30 min to shrink, and stir in a water bath at room temperature. While stirring, add the norcantharidin lipid nanoparticles obtained in step (1) dropwise. The volume ratio of Man solution to norcantharidin lipid nanoparticles is 3:1. Stir in a water bath at 25 ºC for 5 h to obtain Man-Nor-NLCs.

[0060] Example 3: Preparation of Man-Nor-NLCs (1) Preparation of kernel Nor-NLCs: 0.2 parts of norcantharidin, 9 parts of polyethylene glycol-400, 33 parts of ethyl oleate, and 12 parts of polyoxyethylene castor oil were stirred in a 40ºC water bath in the dark for 3.5 h. Then, the mixture was stirred in a 20°C water bath while slowly adding 4 parts of ultrapure water (5-7 drops / min). After stirring for 25 min, the norcantharidin nanoemulsion was obtained. One part tripalmitate, two parts phospholipid, and 2.5 parts glyceryl monostearate were placed in a round-bottom flask, and 30 mL of acetone was added to dissolve them. The organic solvent was removed by rotary evaporation in a water bath at 40 ºC, forming a uniform lipid film on the inner wall of the round-bottom flask. Then, the prepared norcantharidin nanoemulsion was added. Separately, 2.5 parts Tween-80 and 100 parts ultrapure water were taken and sonicated for 15 min to fully dissolve them, forming a Tween-80 solution. The Tween-80 solution was added to the round-bottom flask containing the norcantharidin nanoemulsion, and the flask was placed in an ice bath and sonicated for 10 min to remove the film from the inner wall of the round-bottom flask, thus obtaining the norcantharidin lipid nanoparticles.

[0061] (2) Preparation of Man-Nor-NLCs by active modification with Man Dissolve 2 parts of Man in 100 parts of ultrapure water 3, and stir magnetically at 40 ºC for 3 h. After full swelling, stir in an ice bath for 30 min to shrink, and stir in a water bath at room temperature. While stirring, add the norcantharidin lipid nanoparticles obtained in step (1) dropwise. The volume ratio of Man solution to norcantharidin lipid nanoparticles is 3:1. Stir in a water bath at 25 ºC for 5 h to obtain Man-Nor-NLCs.

[0062] Example 4: Preparation of Man-Nor-NLCs (1) Preparation of kernel Nor-NLCs: 0.7 parts of norcantharidin, 10 parts of polyethylene glycol-400, 38 parts of ethyl oleate, and 14 parts of polyoxyethylene castor oil were stirred in a 40 ºC water bath in the dark for 3 h. Then, the mixture was stirred in a 20 ºC water bath while slowly adding 3 parts of ultrapure water (5-7 drops / min) while stirring. After stirring for 10 min, the norcantharidin nanoemulsion was obtained. 0.7 parts of tripalmitoyl glycerol, 2.5 parts of phospholipid, and 2.5 parts of glyceryl monostearate were placed in a round-bottom flask. 40 mL of dichloromethane and ethyl acetate (volume ratio 1:1) were added to dissolve them. The organic solvent was removed by rotary evaporation in a water bath at 40 ºC, forming a uniform lipid film on the inner wall of the round-bottom flask. Then, the prepared norcantharidin nanoemulsion was added. Separately, 1.5 parts of Tween-80 were added to 170 parts of ultrapure water and sonicated for 15 min to fully dissolve it, forming a Tween-80 solution. The Tween-80 solution was added to the round-bottom flask containing the norcantharidin nanoemulsion and sonicated in an ice bath for 10 min to remove the film from the inner wall of the round-bottom flask, thus obtaining the norcantharidin lipid nanoparticles.

[0063] (2) Preparation of Man-Nor-NLCs by active modification with Man Dissolve 2.5 parts of Man in 170 parts of ultrapure water 3, and stir magnetically at 40 ºC for 3 h. After full swelling, stir in an ice bath for 30 min to shrink, and stir in a water bath at room temperature. While stirring, add the norcantharidin lipid nanoparticles obtained in step (1) dropwise. The volume ratio of Man solution to norcantharidin lipid nanoparticles is 3:1. Stir in a water bath at 25 ºC for 5 h to obtain Man-Nor-NLCs.

[0064] Example 5: Preparation of Man-Nor-NLCs (1) Preparation of kernel Nor-NLCs: 0.8 parts of norcantharidin, 12 parts of polyethylene glycol-400, 28 parts of ethyl oleate, and 9 parts of polyoxyethylene castor oil were stirred in a 40ºC water bath in the dark for 4 hours. Then, the mixture was stirred in a 20°C water bath while slowly adding 4 parts of ultrapure water (5-7 drops / min). After stirring for 30 minutes, the norcantharidin nanoemulsion was obtained. 1.2 parts of tripalmitoyl glycerol, 1.8 parts of phospholipid, and 3.5 parts of glyceryl monostearate were placed in a round-bottom flask. 25 mL of dichloromethane and chloroform (volume ratio 1:1) were added to dissolve them. The organic solvent was removed by rotary evaporation in a water bath at 40 ºC, forming a uniform lipid film on the inner wall of the round-bottom flask. Then, the prepared norcantharidin nanoemulsion was added. Separately, 3 parts of Tween-80 and 200 parts of ultrapure water were taken and sonicated for 30 min to fully dissolve them, forming a Tween-80 solution. The Tween-80 solution was added to the round-bottom flask containing the norcantharidin nanoemulsion, and the flask was placed in an ice bath and sonicated for 12 min to remove the film from the inner wall of the round-bottom flask, thus obtaining the norcantharidin lipid nanoparticles.

[0065] (2) Preparation of Man-Nor-NLCs by active modification with Man Dissolve 1.8 parts of Man in 200 parts of ultrapure water 3, and stir magnetically at 40 ºC for 3 h. After full swelling, stir in an ice bath for 30 min to shrink, and stir in a water bath at room temperature. While stirring, add the norcantharidin lipid nanoparticles obtained in step (1) dropwise. The volume ratio of Man solution to norcantharidin lipid nanoparticles is 2:1. Stir in a water bath at 25 ºC for 5 h to obtain Man-Nor-NLCs.

[0066] Example 6: Preparation of Man-Nor-NLCs (1) Preparation of kernel Nor-NLCs: 0.7 parts of norcantharidin, 10 parts of polyethylene glycol-400, 28 parts of ethyl oleate, and 9 parts of polyoxyethylene castor oil were stirred in a 40ºC water bath in the dark for 3 hours. Then, the mixture was stirred in a 20°C water bath while slowly adding 4 parts of ultrapure water (5-7 drops / min). After stirring for 15 minutes, the norcantharidin nanoemulsion was obtained. 1.5 parts of tripalmitic acid glyceride, 1 part of phospholipid, and 2 parts of glyceryl monostearate were placed in a round-bottom flask. 18 mL of chloroform and acetone (volume ratio 2:1) were added to dissolve them. The organic solvent was removed by rotary evaporation in a 40 ºC water bath, forming a uniform lipid film on the inner wall of the round-bottom flask. Then, the prepared norcantharidin nanoemulsion was added. Separately, 1.5 parts of Tween-80 and 110 parts of ultrapure water were taken and sonicated for 10 min to fully dissolve them, forming a Tween-80 solution. The Tween-80 solution was added to the round-bottom flask containing the norcantharidin nanoemulsion, and the flask was placed in an ice bath and sonicated for 15 min to remove the film from the inner wall of the round-bottom flask, thus obtaining norcantharidin lipid nanoparticles. (2) Preparation of Man-Nor-NLCs by active modification with Man Dissolve 1 part Man in 110 parts ultrapure water 3, and stir magnetically at 40 ºC for 3 h. After full swelling, stir in an ice bath for 30 min to shrink, and stir in a water bath at room temperature. While stirring, add the norcantharidin lipid nanoparticles obtained in step (1) dropwise. The volume ratio of Man solution to norcantharidin lipid nanoparticles is 2.5:1. Stir in a water bath at 25 ºC for 5 h to obtain Man-Nor-NLCs.

[0067] The Man-Nor-NLCs physical mixture in the data diagram is prepared by directly mixing an additional portion of the raw materials used in the preparation of Man-Nor-NLCs in Example 1, according to the mass and proportion of Example 1. For example, the raw materials of the Man-Nor-NLCs physical mixture include: norcantharidin, polyethylene glycol-400, ethyl oleate, polyoxyethylene castor oil, glyceryl tripalmitate, phospholipids, glyceryl monostearate, Tween-80, Man, and ultrapure water 1, 2, and 3 after weighing and stirring until homogeneous.

[0068] The Nor-NLCs physical mixture is prepared using the same method, and its raw materials include norcantharidin, polyethylene glycol-400, ethyl oleate, polyoxyethylene castor oil, glyceryl tripalmitate, phospholipids, glyceryl monostearate, Tween-80, and ultrapure water 1 and 2. After weighing according to the amounts in Example 1, the mixture is stirred evenly.

[0069] The Man-Nor-NLCs, Nor-NLCs and their physical mixtures of Man-Nor-NLCs, physical mixtures of Nor-NLCs, raw materials Man and Nor prepared in Example 1 of the present invention were used for the following performance and experimental determinations: 1. The particle size, PDI and Zeta potential of the Man-Nor-NLCs were determined using a Malvern laser particle size analyzer, in triplicate.

[0070] The results are as follows Figure 1As shown, the Man-Nor-NLCs have an average particle size of 171.19±4.05 nm, a PDI of 0.18±0.02, uniform particle size, and no obvious aggregation, meeting the nanoparticle size requirements for tumor-targeted delivery. The Zeta potential is -10.52±1.78 mV, indicating stable surface charge and electrostatic repulsion that inhibits aggregation. The colloidal system is stable, facilitating storage and delivery. The encapsulation efficiency is above 87.41%, and after 6 months of storage, the encapsulation retention rate is >90%, with a particle size change rate of <8%.

[0071] 2. Man-Nor-NLCs were diluted 10 times with phosphate buffer, counterstained with 2% phosphotungstic acid, and examined for their morphology by transmission electron microscopy.

[0072] The results are as follows Figure 2 As shown, the Man-Nor-NLCs exhibit an outer membrane-core structure, exhibiting a regular spherical shape with a smooth surface. The outer membrane is completely encapsulated, while the core is dense and free of aggregation, indicating successful structural construction. The outer layer consists of a distinct transparent membrane, while the inner layer is a solid core. It is speculated that the outer transparent membrane is modified Man, which is tightly adsorbed and encapsulated on the surface of the solid core lipid nanoemulsion under the influence of forces (possibly hydrogen bonds). This structure may facilitate the recognition of MR on the surface of tumor cells, thereby exerting an active targeting effect.

[0073] 3. Take Nor, Man, Nor-NLCs physical mixture, Nor-NLCs, Man-Nor-NLCs physical mixture, and Man-Nor-NLCs lyophilized powder, compress them into KBr pellets, and analyze them using a Fourier transform infrared spectrometer at 4000-400 cm⁻¹. -1 scanning.

[0074] The results are as follows Figure 3 As shown: In the infrared spectrum of the Man-Nor-NLCs physical mixture, Nor is present at 1846 cm⁻¹. -1 and 1784 cm -1 The characteristic absorption peaks at these locations are absent in the infrared spectra of Man-Nor-NLCs, indicating that Nor is completely encapsulated in the formulation. Meanwhile, Man is present at 582 cm⁻¹ in the infrared spectrum of Man-Nor-NLCs. -1 and 512 cm -1 The characteristic absorption peak indicates that Man is coated on the surface of the lipid nanoemulsion, while this characteristic absorption peak was not observed in the physical mixture of Nor-NLCs or Nor-NLCs. Furthermore, in the infrared spectrum of Man-Nor-NLCs, Man shows a peak at 582 cm⁻¹. -1 and 512 cm -1 The nearby characteristic peak shifts to 577 cm⁻¹ -1 and 515 cm -1This indicates that there are signs of hydrogen bonding between the hydroxyl groups of Man and the hydroxyl groups and protons on the surface of Nor-NLC, confirming the successful crosslinking formation and modification.

[0075] 4. Take the physical mixtures of Nor, Man, Nor-NLCs, Nor-NLCs, Man-Nor-NLCs, and Man-Nor-NLCs lyophilized powder and perform scanning analysis on a DSC instrument at 30-500℃. The parameters are: scan rate 10℃ / min, ordinary crucible (pure aluminum), nitrogen gas.

[0076] The results are as follows Figure 4 As shown, in the thermal analysis diagram of Man-Nor-NLCs, the endothermic peaks of Nor near 127.64 ℃ and 319.93 ℃ both disappeared, and the thermal analysis diagram of Nor was completely different from that of its physical mixture. Nor exhibited the same glass transition as Man near 312.65 ℃. This indicates that Nor was encapsulated within a lipid matrix, while Man was coated on the surface of the lipid nanoemulsion.

[0077] 5. Take the physical mixtures of Nor, Man, and Nor-NLCs, the physical mixture of Nor-NLCs and Man-Nor-NLCs, and the lyophilized powder of Man-Nor-NLCs, and analyze them on an XRD instrument at 2... θ The scanning detection is performed from 5-90°, with a scanning rate of 1° / 8.89 s, using a copper target, operating voltage of 40 kV, and K-α2 / K-α1=0.5.

[0078] The results are as follows Figure 5 As shown: the phase transition temperature increased to 58.3℃, structural stability improved, no additional endothermic peaks were observed, and the drug exhibited good compatibility with lipids. XRD analysis of Man-Nor-NLCs revealed a phase transition temperature at 20.09℃. o 31.63 o 45.40 o 56.37 o 75.29 o 83.74 o There is a distinct diffraction peak at 7.34. o A weaker diffraction peak appears at this location. Compared to Nor, no Nor diffraction peaks are observed or their intensity is reduced in the diffraction patterns of Man-Nor-NLCs. Compared to Nor-NLCs, in the diffraction patterns of Man-Nor-NLCs, 2 θ At 31.63 o 45.40 o 56.37 o 66.03 o 75.29 o 83.74 oThe appearance of new diffraction peaks may indicate a crystal form transformation. Compared to its physical mixture, the diffraction pattern of Man-Nor-NLCs shows 2... θ At 6.70 o 8.91 o 11.07 o 13.31 o 17.73 o 24.28 o There are no diffraction peaks at this point, and 2 θ In 20 o The intensity of the diffraction peak near point 2 decreases, while 2 θ At 31.63 o 45.40 o 56.37 o 75.29 o 83.74 o The emergence of new diffraction peaks further proves that Man-Nor-NLCs are not a simple physical mixture, but a transformation of crystal form. In addition, the diffraction pattern of Man-Nor-NLCs has significantly more spikes than that of Nor-NLCs, which is speculated to be caused by Man being wrapped in the outermost layer.

[0079] 6. The in vitro drug release of Man-Nor-NLCs was investigated using dynamic dialysis. Man-Nor-NLCs were placed in dialysis bags and immersed in pH 1.2 HCl and pH 6.8 PBS (containing 0.5% Tween-80), respectively. The mixture was shaken at 37°C and 100 rpm. Samples were taken at regular intervals to measure the concentration. The experiments were repeated in triplicate, and the release characteristics of Nor were investigated under the same conditions.

[0080] The results are as follows Figure 6As shown, Nor exhibits a significant burst release effect, with over 80% released by 8 hours. Nor-NLCs and Man-Nor-NLCs, on the other hand, release slowly, with parabolic release curves. Man-Nor-NLCs shows a more gradual release compared to Nor-NLCs, demonstrating superior sustained-release performance. In the pH 1.2 HCl release medium, the cumulative release percentages of Nor, Nor-NLCs, and Man-Nor-NLCs within the first 10 hours were (93.14 ± 0.94)%, (47.34 ± 0.80)%, and (36.10 ± 2.26)%, respectively. The cumulative release percentage of Nor was 1.97 times and 2.58 times that of Nor-NLCs and Man-Nor-NLCs, respectively. In the pH 6.8 PBS release medium, the cumulative release percentages of Nor, Nor-NLCs and Man-Nor-NLCs within the first 10 h were (89.74 ± 1.32)%, (43.64 ± 1.29)%, and (32.20 ± 1.42)%, respectively. The cumulative release percentage of Nor was 2.06 times and 2.79 times that of Nor-NLCs and Man-Nor-NLCs, respectively.

[0081] 7. SD rats were administered Nor, Nor-NLCs, and Man-Nor-NLCs (5 mg / kg) via tail vein injection. Rats were fasted for 12 hours prior to administration, but water was permitted. Fundus vein blood samples were collected at pre-defined time points after administration. Plasma was placed in heparinized EP tubes, centrifuged for 10 min, and the supernatant was collected and frozen at -80 °C. Blood drug concentrations were determined by HPLC, and pharmacokinetic parameters were calculated. Six rats were used in each group.

[0082] The results are as follows Figure 7 As shown: Nor reached its peak concentration (Cmax) 0.33 h after gavage administration and decreased rapidly within 6 h; Nor-NLCs reached its peak concentration (Cmax) 1 h after gavage administration and decreased rapidly within 12 h; Man-Nor-NLCs reached its peak concentration (Cmax) 1.5 h after gavage administration and decreased slowly within 24 h, with Man-Nor-NLCs having the highest Cmax.

[0083] 8. SD rats were injected via tail vein with Nor, Nor-NLCs, and Man-Nor-NLCs (5 mg / kg). The rats were fasted for 12 hours prior to administration, but water was permitted. After administration, the rats in each group were allowed to die naturally. The rats' condition and mortality were observed and recorded. The average survival days and life extension rate of the tumor-bearing rats in each group were calculated.

[0084] The results are as follows Figure 8 As shown: Compared with the model group, the Nor group can prolong the survival of tumor-bearing mice ( P<0.05), the Nor-NLC group and the Man-Nor-NLCs group significantly prolonged the survival of tumor-bearing mice ( P <0.01, P <0.001, and there was a statistically significant difference between the two groups ( P <0.05%. The tumor-bearing mice in the Man-Nor-NLCs group had the longest survival time.

[0085] 9. CCK-8 method, H 22 Cells were co-cultured with drug-loaded vectors of different concentrations for 48 h. The results were compared with unmodified NLC, free drug, and blank vector groups, in six parallel trials, and IC50 was calculated. 50 And plot the dose-response curve.

[0086] The results are as follows Figure 9 As shown: Nor, Nor-NLCs and Man-Nor-NLCs affect H 22 The inhibitory effect on cell proliferation was time- and dose-dependent; with increasing drug concentration and prolonged treatment time, the survival rate of both cell lines decreased. Compared with Nor and Nor-NLCs, Man-Nor-NLCs exhibited better anticancer activity. P <0.001).

[0087] 10. H was detected using an inverted fluorescence microscope. 22 The fluorescence of the cells will stain H 22 Cells were seeded directly onto coverslips, stained, and then flipped over onto the slide. Observation was performed using the FITC and Rhodamine channels on a fluorescence microscope.

[0088] The results are as follows Figure 10 As shown, compared with Nor and Nor-NLCs, the fluorescence intensity of cells treated with Man-Nor-NLCs was significantly enhanced, indicating that the cells took up more Man-Nor-NLCs, which led to significant apoptosis.

[0089] 11. SD rats were injected via tail vein with Nor, Nor-NLCs, and Man-Nor-NLCs (5 mg / kg). The rats were fasted for 12 hours prior to administration, but water was permitted. After the last administration, the rats were fasted for 12 hours but water was permitted. The rats were then euthanized by cervical dislocation, tumor tissue was removed, weighed, photographed, and the tumor inhibition rate was calculated.

[0090] The results are as follows Figure 11As shown, the tumors in the model group of tumor-bearing mice grew the fastest and were in poor condition, while the Nor group, Nor-NLCs group, and Man-Nor-NLCs group all showed significant inhibitory effects (P<0.001). Compared with the model group, the average tumor inhibition rate of the Nor group was 27.93%, the average tumor inhibition rate of the Nor-NLCs group was 51.17%, and the average tumor inhibition rate of the Man-Nor-NLCs group was 80.80%. Compared with the Nor group, the tumor inhibition rate of the Nor-NLCs group increased by 23.24%, and the tumor inhibition rate of the Man-Nor-NLCs group increased by 52.87%.

[0091] 12. After euthanizing mice by cervical dislocation as described in 11, tumor tissue was removed, soaked in 4% paraformaldehyde solution, and stored at room temperature for 2-3 days. After dehydration, trimming, embedding, sectioning, staining, and mounting according to HE staining method, the sections were imaged using a digital slide scanner.

[0092] The results are as follows Figure 12 As shown: Under light microscopy, tumor tissue necrosis was observed in all groups, which is considered to be caused by rapid tumor growth and insufficient blood and nutrient supply. Compared with the model group, the Nor group, Nor-NLCs group, and Man-Nor-NLCs group showed more tumor tissue necrosis.

[0093] 13. SD rats were injected via tail vein with Nor, Nor-NLCs, and Man-Nor-NLCs (5 mg / kg). They were fasted for 12 hours prior to administration, but water was permitted. After the last administration, mice in each group were fasted for 12 hours but allowed water. During the experiment, the eyeballs were removed, and blood was collected in 1.5 mL EP tubes. The tubes were centrifuged at 4000 rpm for 10 min at 4 ℃, and the supernatant was collected for analysis. Serum levels of AST, ALT, BUN, and CRE were measured strictly according to the kit instructions.

[0094] Group H 22 Liver and kidney function results of tumor-bearing mice are as follows Figure 13 As shown: Compared with the normal group, the model group showed significantly increased ALT, AST, and BUN levels (P<0.05, P<0.001), and a slight increase in CRE, but no significant difference; compared with the model group, the Nor group showed increased ALT, AST, and BUN levels (P<0.05, P<0.001), with similar CRE levels; compared with the Nor group, the Nor-NLCs and Man-Nor-NLCs groups showed decreased ALT, AST, BUN, and CRE levels, approaching those of the normal group, but the Man-Nor-NLCs group showed a greater decrease. This indicates that tumors have a significant impact on liver function and a slight impact on kidney function in mice; Nor can aggravate the impact on liver function and slightly affect kidney function; Man-Nor-NLCs significantly reduces the impact on liver function in mice, bringing liver function close to that of normal mice, and has almost no effect on the kidneys.

[0095] The above description is merely an exemplary embodiment of the present invention. It should be noted that those skilled in the art can make improvements to the present invention without departing from the inventive concept, and all such improvements fall within the scope of protection of the present invention.

Claims

1. A multi-hydrogen-bonded outer membrane core-type norcantharidin lipid nanoparticle (Man-Nor-NLCs), characterized in that, Its structure consists of norcantharidin lipid nanoparticles (Nor-NLCs) as the core and mannan (Man) as the outer membrane coating, with the core and outer membrane cross-linked by multiple hydrogen bonds (MHBs).

2. The Man-Nor-NLCs according to claim 1, characterized in that, The raw materials for preparing the core Nor-NLCs, by weight, include 0.1-1 parts of norcantharidin, 4.5-12 parts of polyethylene glycol-400, 15-40 parts of ethyl oleate, 6-15 parts of polyoxyethylene castor oil, 0.5-1.5 parts of tripalmitic acid glyceride, 1-3 parts of phospholipids, 1-4 parts of glyceryl monostearate, 1-3.5 parts of Tween-80, and 50-200 parts of water.

3. The Man-Nor-NLCs according to claim 1, characterized in that, The amount of Man used is 0.5–8 parts, and the volume ratio of Man solution to Nor-NLCs is 2–10. 0.1～1。 4. The method for preparing Man-Nor-NLCs according to any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Preparation of kernel Nor-NLCs: The norcantharidin nanoemulsion was obtained by mixing 0.1-1 parts of norcantharidin, 4.5-12 parts of polyethylene glycol-400, 15-40 parts of ethyl oleate, and 6-15 parts of polyoxyethylene castor oil in an aqueous phase of 2-6 parts. Then, 0.5 to 1.5 parts of tripalmitoyl glyceride, 1 to 3 parts of phospholipid, and 1 to 4 parts of glyceryl monostearate are mixed in an organic phase, and the organic phase is removed to obtain a uniform lipid film. Then, the norcantharidin nanoemulsion and lipid film were dissolved and mixed in an aqueous solution containing 1 to 3.5 parts of Tween-80 to obtain Nor-NLCs. (2) Preparation of Man-Nor-NLCs by active modification with Man: Man aqueous solution is mixed with Nor-NLCs prepared in step (1) to obtain Man-Nor-NLCs.

5. The method for preparing Man-Nor-NLCs according to claim 4, characterized in that, The amount of water used in the Man aqueous solution is the same as that used in the Tween-80 aqueous solution, both being 70 to 200 parts; preferably, the amount of Man is 0.5 to 8 parts, and the volume ratio of Man solution to Nor-NLCs is 2 to 10: 0.1 to 1.

6. The method for preparing Man-Nor-NLCs according to claim 4, characterized in that, The organic phase for preparing lipid films is selected from one or any combination of anhydrous ethanol, chloroform, acetone, dichloromethane, and ethyl acetate.

7. The method for preparing Man-Nor-NLCs according to claim 4, characterized in that, Norcantharidin nanoemulsion is obtained by adding water droplets to a mixture of norcantharidin, polyethylene glycol-400, ethyl oleate, and polyoxyethylene castor oil.

8. The method for preparing Man-Nor-NLCs according to claim 4, characterized in that, Man-Nor-NLCs were obtained by adding norcantharidin lipid nanoparticles dropwise to an aqueous solution of Man while stirring.

9. The use of the Man-Nor-NLCs according to any one of claims 1 to 3 in the preparation of drugs for the prevention and / or treatment of fibrotic diseases, liver or tumors.

10. The application according to claim 9, characterized in that, The tumor is selected from one or any combination of liver cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, lung cancer, melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastoma, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma, skin cancer, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct carcinoma, leiomyosarcoma, liposarcoma, nasopharyngeal carcinoma, neuroendocrine carcinoma, ovarian cancer, salivary gland carcinoma, metastatic tumors caused by spindle cell carcinoma, anaplastic large cell lymphoma, undifferentiated thyroid carcinoma, non-Hodgkin lymphoma, Hodgkin lymphoma, glioma, and malignant hematological diseases. The fibrotic disease is selected from one or any combination of liver fibrosis, pulmonary fibrosis, pancreatic fibrosis, renal fibrosis, cardiac fibrosis, endometrial fibrosis, ocular fibrosis, splenic fibrosis, myelofibrosis, and cutaneous fibrosis.

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