WO2020034696A1 - 一种中药-磁纳米簇化学免疫药物递送系统及其制备方法 - Google Patents

一种中药-磁纳米簇化学免疫药物递送系统及其制备方法 Download PDF

Info

Publication number
WO2020034696A1
WO2020034696A1 PCT/CN2019/087971 CN2019087971W WO2020034696A1 WO 2020034696 A1 WO2020034696 A1 WO 2020034696A1 CN 2019087971 W CN2019087971 W CN 2019087971W WO 2020034696 A1 WO2020034696 A1 WO 2020034696A1
Authority
WO
WIPO (PCT)
Prior art keywords
chinese medicine
traditional chinese
tumor
delivery system
superparamagnetic
Prior art date
Application number
PCT/CN2019/087971
Other languages
English (en)
French (fr)
Inventor
瞿鼎
陈彦
刘玉萍
黄萌萌
刘聪燕
Original Assignee
江苏省中医药研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江苏省中医药研究院 filed Critical 江苏省中医药研究院
Publication of WO2020034696A1 publication Critical patent/WO2020034696A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6939—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5169—Proteins, e.g. albumin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention belongs to the field of medicine, and particularly relates to a traditional Chinese medicine-magnetic nano-cluster chemical immune drug delivery system and a preparation method thereof.
  • Tumor tissue is not a simple aggregate of tumor cells.
  • tumor cells are “seeds” and tumor microenvironment is “soil”, which plays a decisive role in the occurrence, development and metastasis of tumors.
  • the tumor microenvironment mainly includes some immune cells (such as tumor-associated macrophages (TAMs), T cells, dendritic cells, etc.), tumor-associated fibroblasts (TAFs), extracellular matrix, cytokines, and chemokines.
  • TAMs play a central role in the regulation of breast cancer immune networks.
  • TAMs is a diverse cell population, mainly divided into classical activated M1 type and alternative activated M2 type, the former can activate tumor immune clearance ability; the latter can promote tumor tissue development, is the main phenotype in breast cancer TAMs.
  • the mechanism of TAMs in interfering with tumorigenesis and development is mainly manifested in the following aspects:
  • immature TAMs are chemokines (such as cell colony-stimulating factor-1 (CSF-1), chemokine CC domain) in the initial stage of tumors.
  • CSF-1 cell colony-stimulating factor-1
  • TAMs transcription factor signal transduction and transcription activation factor 3 (STAT3) activation;
  • TAMs gradually increase It becomes M2 type and secretes a variety of angiogenic molecules (such as tumor necrosis factor ⁇ (TNF- ⁇ ), metalloproteinase (MMP), vascular endothelial growth factor (VEGF), etc.) to help form vascular structures and gradually infiltrate into tumors Hypoxic areas of tissues develop into mature M2 TAMs; at the same time, M2 TAMs secrete various fibrogenic factors (such as transforming growth factor ⁇ 1 (TGF- ⁇ 1), platelet-derived growth factor (PDGF), VEGF, etc.) to regulate TAFs are activated, which in turn forms collagen deposits, causing increased tumor interstitial pressure and impeding drug delivery.
  • M2 TAMs also help regulatory T cells (Treg cells) and inhibit T by
  • M2 TAMs also secrete EGFR family ligands (such as heparin-binding epidermal growth factor (HB-EGF), etc.) and STAT3 activators (such as interleukin 6 (IL-6), interleukin 10 (IL-10), etc.), Induces the stem effect of tumor cells and resists the killing effect of toxic drugs on tissues. It can be seen that inhibiting the M2 polarization of TAMs is of great significance for antitumor chemoimmunotherapy.
  • EGFR family ligands such as heparin-binding epidermal growth factor (HB-EGF), etc.
  • STAT3 activators such as interleukin 6 (IL-6), interleukin 10 (IL-10), etc.
  • M1 TAMs can recruit granulocytes or clear tumor cells directly through their own phagocytosis; block tumor tissue construction by inhibiting the proliferation of vascular endothelial cells or TAFs; and increase the activity of T lymphocytes by reducing the release of chemokines , Activate the tumor immune microenvironment as a whole.
  • inducing M1 polarization of TAMs is also important for antitumor chemoimmunotherapy. Therefore, TAMs play a central role in the regulatory network of breast cancer development and are an important cause of tumor immunosuppression microenvironment. Relying on reasonable drug interventions to reduce the number of M2 phenotypes and induce M1 polarization. By reactivating the breast cancer immune microenvironment, TAMs have become a "leader" force in eliminating tumors and have become a promising new anti-tumor strategy. .
  • anti-tumor Chinese medicines can activate the immune function of tumor patients, and have good supplementary and synergistic effects on the immunosuppression produced by chemotherapy.
  • certain anti-tumor Chinese medicine components can enhance the phagocytosis of macrophages, increase the activity of natural killer (NK) cells, strengthen the immune clearance of T lymphocytes, improve the efficacy of anti-tumor therapy, and significantly extend the survival of patients. time.
  • NK natural killer
  • ganoderic acid A can effectively resist the activation of macrophage IL-4, significantly reduce the expression of M206 marker CD206, and significantly reduce M2 characteristics
  • the secretion of cytokine IL-10 and down-regulation of M2 type characteristic gene Arg1 indicate that ganoderma acid A can effectively inhibit the M2 polarization of TAMs and is a promising chemical immunotherapeutic agent.
  • TAMs are usually recruited to the deep hypoxic zone away from the tumor blood vessels, while traditional Chinese medicine components such as ganoderma acid A have low water solubility, poor bioavailability, and non-selective biodistribution, reaching the deep layers of the tumor Site doses are limited. Therefore, designing and constructing a drug delivery system that can accurately target TAMs is of great practical significance for the realization of chemoimmunotherapy of traditional Chinese medicine components.
  • SPIONs Superparamagnetic iron oxide nanoparticles
  • the inner orbital iron atoms of the core have the ability to accept electrons.
  • Many molecules with carboxyl and hydroxyl groups (such as dextran, adriamycin) Can be anchored with it, and controlled drug delivery can be achieved under the external magnetic field of Nd-Fe-B.
  • the particle size of SPIONs prepared by the thermal precipitation method can be controlled within 10nm. This ultra-small particle size provides important delivery conditions for deep delivery of SPIONs tumors.
  • the purpose of the present invention is to provide a traditional Chinese medicine-magnetic nano-cluster chemical immunodrug delivery system with stable circulation in the body, efficient tumor aggregation, and targeted TAMs, and a preparation method thereof.
  • the system Under the combined control of "magnetism-reduction trigger-target ligand", the system can deliver traditional Chinese medicine and iron oxide to the TAMs region, induce it to undergo M1-type polarization and inhibit the M2-type conversion process, thereby activating the body itself Immune ability to clear tumors.
  • the present invention adopts the following technical solutions:
  • a traditional Chinese medicine-magnetic nano-cluster chemical immunodrug delivery system comprising superparamagnetic oxide nanoparticles; the surface of the superparamagnetic oxide nanoparticles anchors drugs and targeting ligands of TAMs; superparamagnetic oxide nanoparticles
  • a nanocluster structure is formed by a disulfide bond; the nanocluster structure envelopes a protein.
  • the traditional Chinese medicine-magnetic nano-cluster chemical immunodrug delivery system of the present invention uses traditional Chinese medicine-iron oxide nanoparticles as a combination unit, targets the ligands of tumor-associated macrophages (TAMs) on its peripheral modification, and assembles the nanoparticles into Clusters, as a targeted chemical immunodelivery system, are used in antitumor; specifically, superparamagnetic magnetite nanoparticles (SPIONs) that can induce TAMs and M1 polarization are used as basic carriers, and therapeutic drugs are formulated with Anchorage is anchored on the outer layer of magnetic nanoparticles, targeting ligands are covalently attached on the outer layer of magnetic nanoparticles, and then small-sized nanoparticles are assembled into large-sized nanoclusters by disulfide bonding.
  • TAMs tumor-associated macrophages
  • SPIONs superparamagnetic magnetite nanoparticles
  • Anchorage is anchored on the outer layer of magnetic nanoparticles, targeting ligands
  • the invention aims to reduce the immune clearance in the drug body, improve the aggregation of tumor sites, and improve the ability of TAMs to be directedly delivered, multi-dimensionally enhance the ability of each component to interfere with TAMs and M1 polarization, and achieve anti-tumor chemical immunotherapy.
  • Ganoderma triterpenes (ganoderma acid A, ganoderic acid B, ganoderic acid, ganoderma triterpenes) or other traditional Chinese medicine triterpenes (such as ursolic acid, zidonic acid, coroic acid, triptolide, ginseng triterpenes) Saponin, Bupleurum triterpenoid saponin, Astragalus triterpenoid saponin).
  • the superparamagnetic magnet oxide nanoparticles are assembled into a nano-cluster structure by means of cross-linking;
  • the cross-linking agent is selected from small molecular compounds having a carboxyl group and a mercapto group, such as (di) mercaptosuccinic acid, (di) mercaptosuccinic acid, ( (B) mercaptostearic acid, (di) mercaptoglutarate, and the like.
  • the (di) mercaptosuccinic acid refers to dimercaptosuccinic acid or mercaptosuccinic acid
  • (di) mercaptosuccinic acid, (di) mercaptostearic acid, and (di) mercaptoglutarate are the same as Management.
  • the encapsulated proteins are serum albumin, high density lipoprotein, low density lipoprotein, transferrin, and the like of various animal species.
  • the substrate of the superparamagnetic oxide nanoparticles is Fe 3 O 4 or ⁇ -Fe 2 O 3 .
  • the particle size of the superparamagnetic oxide nanoparticles is in the range of 2 to 10 nm.
  • the nano-cluster particle diameters are all between 120 and 160 nm, and the polydispersity coefficients are all below 0.230.
  • Another object of the present invention is to provide a method for preparing the system.
  • the preparation method of the traditional Chinese medicine-magnetic nano-cluster chemical immune drug delivery system of the present invention includes the following steps:
  • amylopectin and 3-aminopropyltriethoxysilane are used as coating materials to prepare water-soluble aminated SPIONs by a classic thermal co-precipitation method.
  • the targeting ligand is mannose.
  • the classic EDC / NHS condensation method was used to covalently attach mannose to the surface of G-SPIONs to obtain MG-SPIONs.
  • the present invention verifies the size regulation of the targeted magnetic nanoclusters and the infiltration of tumor tissue in vivo and in vitro:
  • 3D tumor sphere cells with a diameter of 500-1500mm are prepared.
  • the magnetic nanocluster labeled with the fluorescent probe was incubated with tumor cell spheres for 1-12 hours. Under the laser confocal microscope, the penetration depth of the tumor spheres was observed layer by layer.
  • Tumor penetration in vivo a nude mouse model bearing subcutaneous tumors was prepared. The tail vein was injected with targeted magnetic nanocluster labeled with FRET fluorescence pair. Tumor tissue was stripped from 1-12h after administration to prepare sections, and the FRET phenomenon and penetration depth were observed under a laser confocal microscope.
  • BSA / MG-SPIONC as the representative magnetic nanocluster (ie, the magnetic nanocluster delivery system of encapsulating protein is bovine serum albumin BSA, targeting ligand is mannose, and drug loading is ganoderma acid A)
  • BSA bovine serum albumin
  • targeting ligand is mannose
  • drug loading is ganoderma acid A
  • Cells represent tumor cells, and their in vivo and in vitro verification methods are as follows:
  • BSA / MG-SPIONC Size change in vitro: BSA / MG-SPIONC was placed in a 0.05 ⁇ 1.0mM GSH solution and incubated for 30 minutes, then the particle size change of BSA / MG-SPIONC was detected with a dynamic light scattering particle size analyzer; BSA / MG- SPIONC was incubated with breast cancer cells for 4-8 hours, and the particle size changes of extracellular and intracellular breast cancer were observed under a transmission electron microscope.
  • Tumor penetration in vivo a nude mouse model of subcutaneous tumor bearing breast cancer was prepared.
  • the tail vein was injected with BSA / MG-SPIONC labeled with FRET fluorescence pair.
  • Tumor tissue was stripped off 2-8 h after drug administration, and sections were prepared and placed under a laser confocal microscope to observe the FRET phenomenon and the depth of penetration.
  • the invention also constructs a TAMs targeting method for evaluating magnetic nanocluster in vivo and in vivo, and an evaluation method of TAMs intervention ability, the steps are as follows:
  • mice Take the intact leg bones of the mice, wash the bone marrow with PBS under sterile conditions for primary bone marrow cell culture, and the cells differentiate to the appearance of the antennae to be primary macrophages.
  • the test group was administered at different concentrations, and the primary macrophage cells were used as model cells.
  • T cells CD3 +
  • NK cells CD3 - CD16 + CD56 +
  • fibroblasts ⁇ -SMA
  • Treg cells FoxP3 +
  • vascular endothelial cells CD31 +
  • mice Take the intact leg bones of the mice, wash the bone marrow with PBS under sterile conditions for primary bone marrow cell culture, and the cells differentiate to the appearance of the antennae to be primary macrophages.
  • the test group was administered at different concentrations, and the primary macrophage cells were used as model cells.
  • T cells CD3 +
  • NK cells CD3 - CD16 + CD56 +
  • fibroblasts ⁇ -SMA
  • Treg cells FoxP3 +
  • vascular endothelial cells CD31 +
  • specific Chinese medicine components / ingredients and mannose are selected as specific target ligands of chemoimmunotherapy agent A and TAMs, and SPIONs with ultra-small particle diameter are used as chemoimmunotherapy agent B and medicine.
  • Transfer carrier Specific classical Chinese medicine components / ingredients are anchored to the surface of SPIONs through the classic electron-rich group-Fe coordination principle, and mannose is covalently bonded to the chemically active site on the outer layer of the particle; meanwhile, dimercaptosuccinic acid, etc.
  • small-sized nanoparticles were cross-linked into larger-sized nanoclusters (SPIONC); finally, bovine serum albumin was encapsulated in the outer layer of SPIONC.
  • the SPIONC of the present invention can better avoid capture by the body's RES system under the driving of protein camouflage and an external magnetic field; after reaching the tumor site, the internal disulfide bonds of the SPIONC are sheared by the GSH in the microenvironment and redistributed into Ultra-small-sized SPIONs penetrate into the deep TAMs area; finally, the traditional Chinese medicine components / components and iron oxide nanoparticles are carried to the TAMs under the guidance of the surface mannose, which inhibits its M2 transition and can also induce the M1 pole ⁇ process.
  • M1 type TAMs not only directly participate in the elimination of tumor cells, but also promote the tumor immune process by inhibiting Treg cell activity, reducing the number of TAFs and vascular endothelial cells, and activating the activity of T cells and NK cells.
  • the traditional Chinese medicine-magnetic nano-cluster chemical immune drug delivery system of the present invention integrates the advantages of magnetic trending, ligand modification, and reduction-sensitive targeting, and is a stable traditional Chinese medicine chemical immune drug delivery with stable tumor circulation, efficient tumor aggregation, and targeted TAMs. system. It is expected to achieve the goal of "completely removing tumors" from the perspective of tumor immune microenvironment. Therefore, this type of drug delivery system has the advantage of providing a precise chemical immunotherapy scheme for various tumors.
  • the innovation of the present invention lies in:
  • a chemoimmunotherapy system that can accurately target TAMs is assembled.
  • This design can not only help the drug delivery system avoid the removal of the RES system and complete the efficient accumulation of tumor areas, but also be re-dissociated by the intratumoral GSH into ultra-small particle size magnetic nanoparticles, and complete the TAMs with the guidance of mannose. Efficient uptake, induction of M1 polarization, and activation of tumor immunity.
  • the project will coordinately complete precise targeted delivery of TAMs through multiple methods of particle size regulation, magnetic trend, and target ligand modification, providing a new type of assembly strategy for the field of targeted nano preparations.
  • Figure 1 is a schematic diagram of BSA / MG-SPIONC step-by-step assembly
  • Figure 4 is an evaluation of the anti-breast cancer advantage of ganoderma acid A nanoparticles combined with TC-MEs.
  • A Changes in tumor growth volume
  • B Tumor index ratio of treatment endpoint and starting point
  • C Body weight changes in each group
  • Figure 6 is a pharmacodynamic study of BSA / MG-SPIONC) in the treatment of breast cancer, A tumor growth curve, B tumor mass after treatment;
  • FIG. 7 is a graph of the particle size change trend of different formulations of BSA / MG-SPIONC.
  • This embodiment takes ganoderma acid A-magnetic nanoparticles (G-SPIONs) as an example to illustrate the preparation method and characterization of traditional Chinese medicine-magnetic nanoparticles.
  • G-SPIONs ganoderma acid A-magnetic nanoparticles
  • a mixed aqueous solution of FeCl 2 and FeCl 3 is used as a raw material, amylopectin is used as a stabilizer, 3-aminopropyltriethoxysilane is used as an amino source, and the solution is prepared by the precipitation of NaOH solution and the pH adjustment of NH 3 ⁇ H 2 O.
  • SPIONs Under magnetic stirring, the ganoderma acid A chloroform solution was slowly dropped into the SPIONs chloroform solution, and the aqueous solution was re-dissolved after spinning, and G-SPIONs were obtained after passing through the membrane. The morphology of G-SPIONs was rounded and the particle size was uniformly distributed by transmission electron microscopy (Figure 2A).
  • ganoderma acid A can also be replaced by ganoderic acid B, ganoderic acid, ganoderma triterpenes, ursolic acid, zidonic acid, coroic acid, triptolide, ginseng triterpenoid saponin, Bupleurum Triterpenoid saponins, astragalus triterpenoid saponins, and the like.
  • This example illustrates the preparation method of mannose-modified ganoderic acid A-magnetic nanoparticles (MG-SPIONs).
  • the G-SPIONs prepared in Example 1 were dispersed in deionized water, and an equimolar amount of succinic acid, 1.1eq of EDC, and 1.3eq of NHS were slowly added at room temperature. After stirring for 2h, an equimolar amount of mannose was added and continued. After stirring at room temperature for 24 hours, unreacted small molecules were removed by dialysis under running water to prepare MG-SPIONs.
  • ganoderma acid A can also be replaced by ganoderic acid B, ganoderic acid, ganoderma triterpenes, ursolic acid, zidonic acid, coroic acid, triptolide, ginseng triterpenoid saponin, Bupleurum Triterpenoid saponins, astragalus triterpenoid saponins, and the like.
  • Succinic acid can also be replaced with malonic acid, glutaric acid, suberic acid, and the like.
  • This embodiment takes ganoderma acid A-magnetic nanoparticles (MG-SPIONC) as an example to illustrate the preparation method and characterization of traditional Chinese medicine-magnetic nanoclusters.
  • MG-SPIONC ganoderma acid A-magnetic nanoparticles
  • the MG-SPIONs prepared in Example 2 were dispersed in 5% dimercaptosuccinic acid, stirred at room temperature for 24 hours, and the free cross-linking agent was removed by running water dialysis to prepare MG-SPIONC.
  • the results of dialysis routine pharmaceutical analysis showed that the encapsulation rate of ganoderic acid A was 80.4 ⁇ 4.4% and the drug loading was 2.2 ⁇ 0.3%; the particle size of MG-SPIONC was 146.4 ⁇ 5.5nm, the PDI was 0.191 ⁇ 0.001, and the zeta potential was -14.45 ⁇ 1.64mV; MG-SPIONC observed significant sedimentation after 7 days of storage.
  • ganoderma acid A can also be replaced by ganoderic acid B, ganoderic acid, ganoderma triterpenes, ursolic acid, zidonic acid, coroic acid, triptolide, ginseng triterpenoid saponin, Bupleurum Triterpenoid saponins, astragalus triterpenoid saponins, and the like.
  • the cross-linking agent may be a small molecule compound having a carboxyl group and a mercapto group such as (di) mercaptosuccinic acid, (di) mercaptosuccinic acid, (di) mercaptostearic acid, and (di) mercaptoglutarate. It should be noted that (di) mercaptosuccinic acid refers to mercaptosuccinic acid or dimercaptosuccinic acid.
  • This embodiment takes bovine serum albumin-encapsulated ganoderic acid A-magnetic nanoparticles (BSA / MG-SPIONC) as an example to illustrate the preparation method and characterization of protein-encapsulated traditional Chinese medicine-magnetic nanoclusters.
  • BSA / MG-SPIONC bovine serum albumin-encapsulated ganoderic acid A-magnetic nanoparticles
  • the MG-SPIONC prepared in Example 3 was slowly dropped into a mixed solution system of 1.1 eq EDC, 1.3 eq NHS, and 1 eq BSA, stirred at room temperature for 24 hours, centrifuged at 6000 rpm for 10 min, and removed the supernatant free BSA solution to prepare BSA / MG- SPIONC.
  • Formulation studies show that the encapsulation rate of ganoderma acid A is 76.3 ⁇ 6.5% and the drug loading is 1.7 ⁇ 0.2%; the particle size of BSA / MG-SPIONC is 163.5 ⁇ 11.2nm, PDI is 0.211 ⁇ 0.002, and zeta potential is ⁇ 25.32 ⁇ 3.13mV; BSA / MG-SPIONC did not observe significant sedimentation after 7 days of storage.
  • ganoderma acid A can also be replaced by ganoderic acid B, ganoderic acid, ganoderma triterpenes, ursolic acid, zidonic acid, coroic acid, triptolide, ginseng triterpenoid saponin, Bupleurum Triterpenoid saponins, astragalus triterpenoid saponins, and the like.
  • the cross-linking agent may be a small molecule compound having a carboxyl group and a mercapto group, such as (di) mercaptosuccinic acid, (di) mercaptosuccinic acid, (di) mercaptostearic acid, and (di) mercaptoglutarate.
  • the encapsulation protein used may be serum albumin, high density lipoprotein, low density lipoprotein, transferrin, etc. of various animal species
  • ganoderic acid A is taken as an example to specifically explain the investigation of the traditional Chinese medicine and traditional Chinese medicine-magnetic nanoparticles to interfere with the polarization process of TAMs.
  • RAW264.7 cells and MCF-7 cells were used as macrophages and tumor cell models, respectively.
  • Cells were co-cultured with the Transwell method.
  • LPS + IFN- ⁇ combined intervention was used as the M1 polarization positive group
  • IL-4 intervention was used as the M2 polarization.
  • Positive group different concentrations of ganoderic acid A were used as the test group.
  • the cells were fixed, blocked and punched, CD206-APC antibody staining, and the expression of M206 marker marker CD206 was detected by flow cytometry.
  • the levels of TNF- ⁇ (M1) and IL-10 (M2) cytokines in cell supernatants after different interventions; RT-PCR was used to quantify the expression of characteristic genes after drug intervention.
  • ganoderma acid A can effectively resist the activation of macrophages IL-4, significantly reduce the expression of M2 marker CD206, significantly reduce the secretion of M2 type cytokines IL-10, and down-regulate the expression of M2 type gene Arg1 (Figure 3A- D), but it has no significant effect on the expression of iNOS and CD86, the characteristic cytokine TNF- ⁇ .
  • the results confirm that ganoderma acid A can effectively inhibit the M2 polarization of TAMs, but has less effect on promoting M1 type polarization.
  • G-SPIONs and SPIONs were used as the test group to test the same index.
  • SPIONs without drugs have a weaker ability to inhibit the conversion of M1 to M2.
  • This example takes ganoderma acid A as an example to specifically explain the pharmacodynamic advantages of traditional Chinese medicine-magnetic nanoparticles combined chemotherapy drugs (TC-MEs) against breast cancer
  • the normal saline group was used as a negative control.
  • the TC-MEs group different doses of ganoderic acid A magnetic nanoparticles (G, 5mg / kg, 10mg / kg, 20mg / kg) + TC-MEs were administered at intervals of 6h and G ( 5mg / kg) + TC-MEs synergetic antitumor effect in the simultaneous administration group.
  • the results showed that compared with the normal saline group, the G-containing administration groups had the effect of inhibiting tumor growth, and the treatment effect was better than that of the TC-MEs group (Fig. 4A).
  • ganoderic acid A may promote the activation of the tumor immune environment, thereby improving the anti-tumor efficacy of TC-MEs, and the drug efficacy ratio of triptolide microemulsion and G-sequence therapy was administered simultaneously. More therapeutic advantages.
  • the physiological saline group was used as the negative control group, and the Celastrol group, Cel + STS group, T / C-L group, and T / CM-L group were compared for the treatment of tumor microenvironment in vivo, and the anti-tumor mechanism of synergism and attenuation was explained.
  • the fluorescence intensity of the T / CM-L group is significantly weaker than that of the positive control and the Celastrol group.
  • T / CM- The morphology of blood vessels in group L slices showed a relatively regular elliptical morphology, and various budding states were significantly reduced, and the vascular states tended to normalize.
  • T / CM-L group may significantly improve the pathological state of blood vessels in tumor tissues; due to the lack of progressive release and passive aggregation performance in the Cel + STS group, the vascular normalization efficiency is obviously not high, which indirectly led to the group's anti-tumor The effect is not ideal; although T / CL transports STS to the tumor site to a certain extent, it lacks the characteristic of progressive release, resulting in the drug not being delivered within the normalization window period, so the antitumor effect has not been optimized (Figure 5A).
  • TAFs tumor-associated fibroblast
  • Cytokines in serum are also an important part of the tumor microenvironment.
  • T / CL and T / CM-L treatment can increase the content of IFN- ⁇ in serum and help improve immune function;
  • T / CL group and T / CL The CCL2 level in the CM-L group was significantly lower than that in the normal saline group (Fig. 5C-F), suggesting that the nano-formulation treatment may reduce the secretion of chemokines and reduce the possibility of tumor growth and metastasis.
  • triptolide can also be replaced by ganoderic acid B, ganoderic acid, ganoderma triterpene, ursolic acid, zidonic acid, coroic acid, ganoderic acid A, ginseng triterpenoid saponin, Bupleurum Triterpenoid saponins, astragalus triterpenoid saponins, and the like.
  • This example illustrates the treatment of breast cancer with the albumin-encapsulated ganoderic acid A-magnetic nanocluster (BSA / MG-SPIONC) prepared in Example 4.
  • BSA / MG-SPIONC albumin-encapsulated ganoderic acid A-magnetic nanocluster
  • Nude mouse pads were inoculated with MCF-7 cells to establish a nude mouse model of in situ breast cancer.
  • the physiological saline group was used as a negative control, and the ganoderic acid A-magnetic nanocluster (MG-SPIONC) and ganoderic acid A-magnetic nanoparticle (MG-SPIONs) were used as a control group to investigate the effect of BSA / MG-SPIONC chemoimmunotherapy.
  • the dosage regimen was from day 0 of vaccination, and was administered every other day at a dose of ganoderma acid A10mg / kg, and was administered until day 28. During the treatment, the weight of nude mice and the tumor growth curve were recorded.
  • ganoderma acid A can also be replaced with ganoderic acid B, ganoderic acid, ganoderma triterpenes, ursolic acid, zidonic acid, coroic acid, triptolide, ginseng triterpenoid saponin, Bupleurum Triterpenoid saponins, astragalus triterpenoid saponins, and the like.
  • Tumor types can also be melanoma, liver cancer, lung cancer, pancreatic cancer, kidney cancer, colon cancer, and the like.
  • This example illustrates an example of an in vitro size regulation study of the albumin-encapsulated ganoderic acid A-magnetic nanoclusters (BSA / MG-SPIONC) prepared in Example 4.
  • MG-SPIONs were prepared in the same manner as in Example 2. Cross-linking agents at different mass fractions have a significant effect on the particle size of MG-SPIONC. The results are shown in Table 1. Regardless of whether MG-SPIONs loaded with ganoderma acid A or SPIONs without drugs, the particle size gradually increased under the action of a cross-linking agent with a mass fraction of 1% to 10%. This cross-linked MG-SPIONC is a reduction-sensitive system, which is easily affected by GSH, dissociates, and forms an initial state with a small particle size. The results are shown in Table 2. The degree of dissociation is different at different GSH concentrations.
  • the particle size of BSA / MG-SPIONC re-dissociates back to the level of MG-SPIONs.
  • MG-SPIONC without BSA encapsulation has the same trend.
  • the particle size of MG-SPIONC without cross-linking was not affected by GSH.
  • ganoderma acid A can also be replaced with ganoderic acid B, ganoderic acid, ganoderma triterpenes, ursolic acid, zidonic acid, coroic acid, triptolide, ginseng triterpenoid saponin, Bupleurum Triterpenoid saponins, astragalus triterpenoid saponins, and the like.
  • the cross-linking agent may be a small molecule compound having a carboxyl group and a mercapto group, such as (di) mercaptosuccinic acid, (di) mercaptosuccinic acid, (di) mercaptostearic acid, and (di) mercaptoglutarate.
  • the encapsulation protein used may be serum albumin, high-density lipoprotein, low-density lipoprotein, transferrin and the like of various animal species.
  • This example illustrates an example of the albumin-encapsulated ganoderic acid A-magnetic nanoclusters (BSA / MG-SPIONC) prepared in Example 4 to enhance tumor targeting by multiple size regulation
  • the composition of BSA / MG-SPIONC is through the assembly of MG-SPIONs, the cross-linking of MG-SPIONC, and the encapsulation steps of BSA / MG-SPIONC. These steps correspond to the implementation of multiple size regulation strategies in the body and the final small-scale combination.
  • Multiple regulation of size in vitro and in vivo is the focus of the present invention.
  • This embodiment verifies the objective basis of the present invention for size regulation and TAM targeting by detecting particle sizes in different formulation states and trigger states. The results are shown in Figure 7.
  • the particle size in the SPIONs state was only below 10 nm, and the particle size of the nanoclusters rapidly increased to about 120 nm after cross-linking. After being encapsulated by BSA, the particle size of the nanoclusters increased again slightly. However, the particle size of BSA / MG-SPIONC dissociates rapidly to the SPIONs state after encountering the triggering of high concentration of GSH in tumor tissues. This size is very conducive to the penetration and targeting of TAM in the hypoxic area of tumors.
  • ganoderma acid A can also be replaced with ganoderic acid B, ganoderic acid, ganoderma triterpenes, ursolic acid, zidonic acid, coroic acid, triptolide, ginseng triterpenoid saponin, Bupleurum Triterpenoid saponins, astragalus triterpenoid saponins, and the like.
  • the cross-linking agent may be a small molecule compound having a carboxyl group and a thiol group such as (di) mercaptosuccinic acid, (di) mercaptosuccinic acid, (di) mercaptostearic acid, (di) mercaptoglutarate, and the like .
  • the encapsulation protein used may be serum albumin, high-density lipoprotein, low-density lipoprotein, transferrin and the like of various animal species.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Nanotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Inorganic Chemistry (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Optics & Photonics (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

本发明涉及一种中药-磁纳米簇化学免疫药物递送系统及其制备方法，包括超顺磁铁氧化物纳米粒；所述超顺磁铁氧化物纳米粒表面锚定药物和TAMs的靶向配体；超顺磁铁氧化物纳米粒通过二硫键形成纳米簇结构；所述纳米簇结构外包封蛋白。本发明的中药-磁纳米簇化学免疫药物递送系统整合了磁力趋向、配体修饰、还原敏感的寻靶优势，是一种体内循环稳定、肿瘤聚集高效、TAMs靶向精准的中药化学免疫药物递送系统。

Description

一种中药-磁纳米簇化学免疫药物递送系统及其制备方法 技术领域
本发明属于医药领域，具体涉及一种中药-磁纳米簇化学免疫药物递送系统及其制备方法。
背景技术
肿瘤组织并非是简单的肿瘤细胞聚集体。根据肿瘤学经典的“种子-土壤”学说，肿瘤细胞是“种子”，肿瘤微环境则是“土壤”，后者在肿瘤的发生、发展、转移进程中扮演着决定性的作用。肿瘤微环境主要包括一些免疫细胞(如肿瘤相关巨噬细胞(TAMs)、T细胞、树突状细胞等)、肿瘤相关成纤维细胞(TAFs)、细胞外基质、细胞因子和趋化因子等。其中，TAMs在调控乳腺癌免疫网络过程中扮演了核心的角色。TAMs是一个多样化的细胞群体，主要分为经典活化的M1型和代替活化的M2型，前者能够激活肿瘤免疫清除能力；后者能够促进肿瘤组织发展，是乳腺癌TAMs中的主要表型。TAMs在干预肿瘤发生发展的机制主要表现为以下几方面：首先，在肿瘤起始阶段未成熟的TAMs被趋化因子(如细胞集落刺激因子-1(CSF-1)、趋化因子C-C结构域配体2(CCL2)等)招募到肿瘤附近，通过转录因子信号转导和转录激活因子3(STAT3)活化作用加速肿瘤细胞的活化和增殖；其次，随着肿瘤发展向前推进，TAMs逐步极化为M2型，并分泌多种促血管生长分子(如肿瘤坏死因子α(TNF-α)、金属蛋白酶(MMP)、血管内皮生长因子(VEGF)等)协助形成血管结构，并逐步浸润到肿瘤组织的乏氧区域发展成为成熟的M2型TAMs；同时，M2型TAMs分泌各种促纤维因子(如转化生长因子β1(TGF-β1)、血小板源性生长因子(PDGF)、VEGF等)调节成TAFs活化，进而形成胶原沉积，造成肿瘤间质压升高，阻碍药物递送；此外，M2型TAMs还通过趋化因子(如CCL17/18/22)帮助调节性T细胞(Treg细胞)、抑制T/NK细胞的活化；最后，M2型TAMs还分泌EGFR家族配体(如肝素结合性表皮生长因子(HB-EGF)等)和STAT3活化物(如白介素6(IL-6)、白介素10(IL-10)等)，诱导肿瘤细胞干性作用，抵抗毒性药物对组织的杀伤作用。由此可知，抑制TAMs的M2极化对于抗肿瘤化学免疫治疗意义重大。与之相反，M1型TAMs能够招募粒细胞或者通过自身的吞噬能力直接清除肿瘤细胞；通过抑制血管内皮细胞或者TAFs增生阻断肿瘤组织的构建；通过减少趋化因子的释放提高T淋巴细胞的活性，整体上激活肿瘤免疫微环境。显然，诱导TAMs的M1极化对于抗肿瘤化学免疫治疗同样重要。因此，TAMs在乳腺癌发展调控网络中扮演核心的角色，是导致肿瘤免疫抑制微环境的重要原因。借助合理的药物干预手段降低M2表型数量、诱导其发生M1极化，通过重新激活乳腺癌免疫微环境的方式使TAMs成为清除肿瘤的“领军”力量，已经成为极具前景的抗肿瘤新策略。
许多抗肿瘤中药可激活肿瘤病人的机体免疫功能，对化疗产生的免疫抑制有较好的补益和协同功效。譬如，某些抗肿瘤中药组分能够增强机体巨噬细胞的吞噬功能、提高自然杀伤(NK)细胞的活性、强化T淋巴细胞的免疫清除能力，提高抗肿瘤治疗的疗效，显著延长患者的生存时间。本团队围绕中药组分/成分(以灵芝酸A为例)做了扎实的工作：灵芝酸A能够有效抵抗巨噬细胞IL-4活化，显著降低M2型标志物CD206表达，明显减少M2型特征细胞因子IL-10分泌，并下调M2型特征基因Arg1表达，表明灵芝酸A可有效抑制TAMs的M2极化，是一种很有前景的化学免疫治疗剂。但是在肿瘤发展过程中，TAMs通常被招募至远离肿瘤血管的深层乏氧区，而诸如灵芝酸A等中药组分的水溶性较低、生物利用度差、生物分布无选择性，到达肿瘤深层部位的剂量有限。因此，设计构建一种可精准靶向TAMs的递药系统对于实现中药组分的化学免疫治疗具有重要的现实意义。
超顺磁氧化铁纳米粒(SPIONs)是一种优良的药物靶向递送载体，其内核铁原子外层空轨道有接纳电子的能力，很多具有羧基、羟基的分子(如葡聚糖、阿霉素等)都可以与之锚定，并在外置Nd-Fe-B的磁场作用下实现可控的药物递送。不仅如此，热沉淀方法制备的SPIONs粒径可控制在10nm以内，这种超小粒径为SPIONs的肿瘤深层递送提供了重要的递送条件。除了具备靶向载药和深层渗透的优势外，我们发现SPIONs还能够诱导M2型TAMs向M1型极化，进而引发Fe离子与M1型TAMs产生的过氧化氢发生Fenton反应。因此，将可诱导TAMs M1极化的中药组分与SPIONs装配成可靶向递送至TAMs的递药系统，有望进一步提高对肿瘤免疫微环境的调控能力，抑制肿瘤的发生和发展。
然而，纳米粒子TAMs靶向递送目前主要面临两个挑战：一，如何躲避血液循环时内皮网状系统(RES)的捕获。有文献显示，迄今为止尚未有一种纳米制剂在肿瘤部位的聚集量超过给药剂量的5％，这就意味着能够被TAMs摄取的剂量更低。现阶段针对减少RES系统摄取的方案多为熟知的聚乙二醇(PEG)修饰、白蛋白包裹等策略，这些技术相对成熟可靠，大量文献显示确可增效减毒。二，如何提高药物靶向递送的精准性。有文献报道，TAMs表面过表达甘露糖(CD206)受体，将其配体修饰到SPIONs上，通过外置磁场干预，有望向TAMs高效递送。但是，小尺寸(<10nm)的纳米粒在血液循环时易被肾脏清除，而较大尺寸(>50nm)的纳米粒又难以向乏氧区渗透，这也是近年来困扰纳米制剂领域的热点问题。通过肿瘤微环境特性调控合适的粒子尺寸，适应不同递送环境的需求是目前常用的策略。显然，设计一种可循环稳定、深层渗透、精准靶向TAMs的药物递送系统是现阶段迫切需要解决的关键问题。
发明内容
本发明的目的是提供一种体内循环稳定、肿瘤聚集高效、TAMs靶向精准的中药-磁纳米簇化学免疫药物递送系统及其制备方法。该系统在“磁趋向-还原触发-靶配体”联合调控下，可将中药和铁氧化物定向递送至TAMs区域，诱导其发生M1型极化的同时抑制M2型转化进程，进而激活机体自身免疫清除肿瘤的能力。以验证中药-磁纳米簇用于化学免疫治疗乳腺癌的优势。
为实现上述技术目的，本发明采用如下技术方案：
一种中药-磁纳米簇化学免疫药物递送系统，包括超顺磁铁氧化物纳米粒；所述超顺磁铁氧化物纳米粒表面锚定药物和TAMs的靶向配体；超顺磁铁氧化物纳米粒通过二硫键形成纳米簇结构；所述纳米簇结构外包封蛋白。
本发明的中药-磁纳米簇化学免疫药物递送系统以中药-铁氧化物纳米粒为组合单元，在其外周修饰上靶向肿瘤相关巨噬细胞(TAMs)的配体，并将纳米粒组装成簇，作为一种靶向性化学免疫递药系统在抗肿瘤中应用；具体来说，采用可诱导TAMs M1极化的超顺磁铁氧化物纳米粒(SPIONs)作为基础载体，将治疗药物以配位方式锚定在磁性纳米粒外层，靶向配体以共价连接的方式锚定在磁性纳米粒外层，再以二硫键方式将小粒径纳米粒组装成大粒径的纳米簇，最后用蛋白作为纳米簇的包封外壳。本发明旨在减少药物体内的免疫清除，提高肿瘤部位的聚集以及改善TAMs定向输送的能力，多维度增强各组分干预TAMs M1极化的能力，实现抗肿瘤化学免疫治疗。
优选灵芝三萜类(灵芝酸A、灵芝酸B、灵芝酸、灵芝三萜)或其他中药三萜类(如熊果酸、齐敦果酸、科罗酸、雷公藤红素、人参三萜皂苷、柴胡三萜皂苷、黄芪三萜皂苷)中的一种或几种。
所述超顺磁铁氧化物纳米粒通过交联手段组装成纳米簇结构；交联剂选用具有羧基和巯基的小分子化合物，如(二)巯丁二酸、(二)巯辛二酸、(二)巯硬脂酸、(二)巯戊二酸等。本发明中，所述的(二)巯丁二酸指二巯基丁二酸或巯基丁二酸，(二)巯辛二酸、(二)巯硬脂酸、(二)巯戊二酸同理。
所述包封蛋白为各种属动物的血清白蛋白、高密度脂蛋白、低密度脂蛋白、转铁蛋白等。
所述超顺磁铁氧化物纳米粒基材为Fe 3O 4或γ-Fe 2O 3等。超顺磁铁氧化物纳米粒的粒径范围为2～10nm。纳米簇粒径均在120～160nm之间，多分散系数均在0.230以下。
本发明的另一目的在于提供上述系统的制备方法。
本发明的中药-磁纳米簇化学免疫药物递送系统的制备方法包括如下步骤：
(1)制备超顺磁铁氧化物纳米粒；
(2)通过配位作用，将药物锚定在超顺磁铁氧化物纳米粒表面；
(3)在载药的超顺磁铁氧化物纳米粒表面修饰TAMs的靶向配体；
(4)将修饰的超顺磁铁氧化物纳米粒通过交联手段组装，获取超顺磁铁氧化物纳米簇；
(5)在超顺磁铁氧化物纳米簇外层包封蛋白。
优选的，所述步骤(1)中，采用支链淀粉和3-氨丙基三乙氧基硅烷为包衣材料，通过经典的热共沉淀法制备水溶性氨基化SPIONs。
所述步骤(3)中，靶向配体选用甘露糖。采用经典的EDC/NHS缩合法将甘露糖共价连接到G-SPIONs表面，获得MG-SPIONs。
本发明对靶向磁纳米簇的尺寸调节和肿瘤组织内渗透进行了体内外验证：
1)体外尺寸变化：将靶向性磁纳米簇置于1.0nM～1.0mM的GSH溶液中，孵育30min后，用动态光散射粒径仪检测磁纳米簇的粒径变化；将靶向性磁纳米簇与肿瘤细胞孵育1～12h，透射电镜下观察细胞外和细胞内磁纳米簇的粒径变化。
2)体外肿瘤渗透：制备直径为500-1500mm的3D肿瘤球细胞。荧光探针标记的磁纳米簇与肿瘤细胞球孵育1～12h，激光共聚焦显微镜下，逐层扫描观察瘤球的渗透深度。
3)体内尺寸变化：制备荷皮下异位瘤肿瘤裸鼠模型。尾静脉精注被FRET荧光对标记的靶向性磁纳米簇，在给药后1-12h，置于近红外活体成像下观察FRET现象。
4)体内肿瘤渗透：制备荷皮下异位瘤肿瘤裸鼠模型。尾静脉精注被FRET荧光对标记的靶向性磁纳米簇，在给药后1-12h，剥离肿瘤组织，制备成切片，置于激光共聚焦显微镜下观察FRET现象以及渗透深度。
以BSA/MG-SPIONC为代表性磁纳米簇(即包封蛋白为牛血清白蛋白BSA，靶向配体为甘露糖，载药为灵芝酸A的磁纳米簇递药系统)，以乳腺癌细胞代表肿瘤细胞，其体内外验证方法如下：
1)体外尺寸变化：将BSA/MG-SPIONC置于0.05～1.0mM的GSH溶液中，孵育30min后，用动态光散射粒径仪检测BSA/MG-SPIONC的粒径变化；将BSA/MG-SPIONC与乳腺癌细胞孵育4～8h，透射电镜下观察细胞外和细胞内乳腺癌的粒径变化。
2)体外肿瘤渗透：制备直径为700-1200mm的3D乳腺癌瘤球细胞。荧光探针标记的BSA/MG-SPIONC与乳腺癌细胞球孵育2～8h，激光共聚焦显微镜下，逐层扫描观察瘤球的渗透深度。
3)体内尺寸变化：制备荷皮下异位瘤乳腺癌肿瘤裸鼠模型。尾静脉精注被FRET荧光对标记的BSA/MG-SPIONC，在给药后2-8h，置于近红外活体成像下观察FRET现象。
4)体内肿瘤渗透：制备荷皮下异位瘤乳腺癌肿瘤裸鼠模型。尾静脉精注被FRET荧光对标记的BSA/MG-SPIONC，在给药后2-8h，剥离肿瘤组织，制备成切片，置于激光共聚焦显微镜下观察FRET现象以及渗透深度。
本发明还构建了靶向磁纳米簇体内外的TAMs靶向以及对TAMs干预能力的评价方法，步骤如下：
1)中药-磁纳米簇诱导TAMs M1极化评价
取小鼠完整的腿骨，无菌条件下PBS冲骨髓进行原代骨髓细胞培养，细胞分化至触角出现即为原代巨噬细胞。选用不同浓度给药组为测试组，原代巨噬细胞细胞为模型细胞，在经典的M1极化诱导剂(LPS+IFN-γ)和经典的M2极化诱导剂IL-4干预下，通过CD206-APC/CD86-APC抗体特异性标记法，流式检测药物干预巨噬细胞后CD206和CD86的表达量；通过ELISA法定量药物干预后细胞上清液中TNF-α和IL-10的含量；通过RT-PCR法定量药物干预后iNOS、CD86、Arg1等基因的表达水平。RAW264.7细胞为平行对照。
2)中药-磁纳米簇巨噬细胞体内外靶向评价
体外：以原代巨噬细胞细胞为模型细胞，将Cy3-NHS标记到中药-磁纳米簇上，流式半定量考察纳米簇细胞摄取，激光共聚焦成像法考察胞内转运机制；
体内：将0.2mL2×10 7/mL MCF-7细胞接种在雌性裸鼠乳垫，建立原位MCF-7荷瘤裸鼠模型。设置(Cy3)中药-磁纳米簇、(Cy3)中药-磁纳米簇和非解离型、(Cy3)中药-磁纳米簇给药组(灵芝酸A剂量为10mg/kg；Cy3剂量为50μg/kg)，通过磁场有无，单次腹腔注射后制备肿瘤切片，用Anti-CD206抗体可视化TAMs，考察纳米簇在TAMs处定位以及磁场、还原环境对靶向性影响。RAW264.7细胞为平行对照。
3)中药-磁纳米簇体外抗肿瘤及体内微环境影响评价
体外：采用transwell小室原代巨噬细胞(上层)与MCF-7(下层)细胞共培养模型，MTT法考察阴性组、中药-磁纳米簇、各种制剂状态的中药-磁纳米簇(包括解离和非解离型)等干预后巨噬细胞清除肿瘤细胞的能力；RAW264.7细胞为平行对照。
体内：设置阴性组，中药-磁纳米簇、各种制剂状态的中药-磁纳米簇(包括解离和非解离型)治疗组，隔天腹腔注射MCF-7原位瘤裸鼠14次，瘤部位设置磁场，记录治疗期间及治疗后的肿瘤体积、存活状态、体重等数据，绘制抑瘤率、生存曲线、肿瘤生长曲线等。治疗结束 后取动物血清和瘤组织，检测细胞裂解液中TNF-α和IL-10水平；特异性抗体标记相关瘤内各免疫/基质细胞：T细胞(CD3 +)、NK细胞(CD3 -CD16 +CD56 +)、成纤维细胞(α-SMA)、Treg细胞(FoxP3 +)、血管内皮细胞(CD31 +)，评价各装配体对微环境影响的贡献。
以BSA/MG-SPIONC为例，其评价方法步骤如下：
1)BSA/MG-SPIONC诱导TAMs M1极化评价
取小鼠完整的腿骨，无菌条件下PBS冲骨髓进行原代骨髓细胞培养，细胞分化至触角出现即为原代巨噬细胞。选用不同浓度给药组为测试组，原代巨噬细胞细胞为模型细胞，在经典的M1极化诱导剂(LPS+IFN-γ)和经典的M2极化诱导剂IL-4干预下，通过CD206-APC/CD86-APC抗体特异性标记法，流式检测药物干预巨噬细胞后CD206和CD86的表达量；通过ELISA法定量药物干预后细胞上清液中TNF-α和IL-10的含量；通过RT-PCR法定量药物干预后iNOS、CD86、Arg1等基因的表达水平。
2)BSA/MG-SPIONC巨噬细胞体内外靶向评价
体外：以原代巨噬细胞细胞为模型细胞，将Cy3-NHS标记到BSA/MG-SPIONC上，流式半定量考察纳米簇细胞摄取，激光共聚焦成像法考察胞内转运机制；
体内：将0.2mL2×10 7/mL MCF-7细胞接种在雌性裸鼠乳垫，建立原位MCF-7荷瘤裸鼠模型。设置(Cy3)MG-SPIONs、(Cy3)BSA/MG-SPIONC和非解离型(Cy3)BSA/MG-SPIONC给药组(灵芝酸A剂量为10mg/kg；Cy3剂量为50μg/kg)，通过磁场有无，单次腹腔注射后制备肿瘤切片，用Anti-CD206抗体可视化TAMs，考察纳米簇在TAMs处定位以及磁场、还原环境对靶向性影响。RAW264.7细胞为平行对照。
3)BSA/MG-SPIONC体外抗肿瘤及体内微环境影响评价
体外：采用transwell小室原代巨噬细胞(上层)与MCF-7(下层)细胞共培养模型，MTT法考察阴性组、G-SPIONs、MG-SPIONs、BSA/MG-SPIONC(包括非解离型)等干预后巨噬细胞清除肿瘤细胞的能力；RAW264.7细胞为平行对照。
体内：设置阴性组，G-SPIONs、MG-SPIONs、BSA/MG-SPIONC(包括非解离型)治疗组，隔天腹腔注射MCF-7原位瘤裸鼠14次，瘤部位设置磁场，记录治疗期间及治疗后的肿瘤体积、存活状态、体重等数据，绘制抑瘤率、生存曲线、肿瘤生长曲线等。治疗结束后取动物血清和瘤组织，检测细胞裂解液中TNF-α和IL-10水平；特异性抗体标记相关瘤内各免疫/基质细胞：T细胞(CD3 +)、NK细胞(CD3 -CD16 +CD56 +)、成纤维细胞(α-SMA)、Treg细胞(FoxP3 +)、血管内皮细胞(CD31 +)，评价各装配体对微环境影响的贡献。
体内抗肿瘤评测指标如下：隔天腹腔注射给药，给药期间记录肿瘤体积和体重，检查血常规、肝功能、TNF-α和IL-6等指标。14天后将动物断颈处死，剥离肿瘤、脾脏、肝脏、胸腺，仔细观察肿瘤大小、形态。用游标卡尺分别测量瘤体的长径(a)和短径(b)，依据公式V＝a·b2/2，计算肿瘤体积，称量体重、记录生存状态和生存时间。动物处死后称脾脏及肝脏重量，计算肿瘤抑制率、肝、脾指数：抑瘤率＝(1-给药组平均肿瘤体积/对照组平均肿瘤体积)×100％；肝指数＝肝脏重量(mg)/裸鼠体重(g)；脾指数＝脾脏重量(mg)/裸鼠体重(g)。常规石蜡包埋、切片、HE染色，并用TUNEL试剂盒观察肿瘤组织的凋亡状态和形态学改变。
本发明对化学免疫治疗制剂的设计，选用特定中药组分/成分和甘露糖分别作为化学免疫治疗剂A和TAMs特异性靶配体，用超小粒径的SPIONs作为化学免疫治疗剂B和药物传输载体。通过经典的富电子基团-Fe配位原理将特定中药组分/成分锚定在SPIONs表面，将甘露糖共价结合在粒子外层的化学活性位点上；同时以二巯基丁二酸等为连接臂将小粒径纳米粒交联成较大粒径的纳米簇(SPIONC)；最后在SPIONC外层包封牛血清白蛋白。
本发明的SPIONC在蛋白的伪装和外置磁场的驱动下，可以较好地躲避机体RES系统的俘获；当到达肿瘤部位后，SPIONC内部二硫键被微环境中的GSH剪切，重新分散成超小尺寸的SPIONs向深层的TAMs区域渗透；最后在表面甘露糖的引导下将携带的中药组分/成分和氧化铁纳米粒共同运送至TAMs，抑制其M2转变的同时还可以诱导发生M1极化过程。M1型TAMs不仅直接参与肿瘤细胞的清除，还通过抑制Treg细胞活性、减少TAFs和血管内皮细胞数量、激活T细胞和NK细胞的活性，共同促进肿瘤免疫进程。
本发明的中药-磁纳米簇化学免疫药物递送系统整合了磁力趋向、配体修饰、还原敏感的寻靶优势，是一种体内循环稳定、肿瘤聚集高效、TAMs靶向精准的中药化学免疫药物递送系统。有望从肿瘤免疫微环境角度实现“完全清除肿瘤”的目标。因此该类的递药系统优势还在于为各类肿瘤提供一种精准的化学免疫治疗方案。
本发明的创新点在于：
1.以超小粒径的G-SPIONs为基础，通过靶向配体修饰、SPIONs交联成簇以及蛋白包封三个装配过程，组装成可精准靶向TAMs的化学免疫治疗系统。这种设计不仅能够帮助给药系统躲避RES系统的清除，完成肿瘤区域的高效聚集，还可被瘤内GSH重新解离成超小粒径的磁纳米粒，伴随甘露糖的指引一同完成TAMs的高效摄取、M1极化的诱导以及肿瘤免疫能力的激活。项目将通过粒径调控、磁力趋向、靶配体修饰多重手段协同完成对TAMs的精准靶向递送，为靶向纳米制剂领域提供了一种新型的组装策略。
2.整合了中药和铁氧化物纳米粒在调控TAMs极化方面的优势，利用二者系统组装的高度匹配性构建一种可精准靶向TAMs的化学免疫递药系统，以激活肿瘤自身免疫微环境的方式帮助中药制剂突破抗肿瘤治疗瓶颈。本发明的思路不同于以往针对肿瘤细胞为靶点设计给药系统，而是打破传统的抗肿瘤治疗思维，从免疫微环境角度出发，尝试将促瘤“元凶”TAMs改造成抑瘤的核心力量，利用免疫清除能力协助中药纳米制剂清除相对顽固的肿瘤细胞，这种“双管齐下”的联合治疗手段为中药多组分抗乳腺癌治疗开拓了新的视野。
本发明内容所涉及的具体制备方法、有效性结果、体内外的抗肿瘤行为均在下列的具体实施例中详细给出。需要指出的是，其他处方组分与药物的组合还有用于其他类型的肿瘤治疗的可能性。
附图说明
图1是BSA/MG-SPIONC逐级装配示意图；
图2是中药-磁纳米粒的(A)形态学，(B)制剂学，(C)释药行为及(D)稳定性初步研究，(
Figure PCTCN2019087971-appb-000001
n＝6)；
图3是中药及中药磁纳米粒对TAMs特征信息考察，(A)基因相对表达量，(B)CD206相对表达量，(C)TAMs上清液TNF-α及(D)IL-10，(
Figure PCTCN2019087971-appb-000002
n＝6)； **P<0.01；
图4是灵芝酸A纳米粒与TC-MEs联合抗乳腺癌优势评价，(A)肿瘤生长体积变化，(B)治疗终点和起点的肿瘤指数比，(C)各组体重变化，(D)各组动物的生存曲线，(
Figure PCTCN2019087971-appb-000003
n＝6)； **P<0.01；
图5是中药组分对肿瘤免疫微环境影响的评价，(A)肿瘤血管免疫染色图片，(B)肿瘤组织成纤维细胞免疫染色图片，(C～F)动物血清IFN-γ、IL-2、CCL2、TGF-β的含量变化，(
Figure PCTCN2019087971-appb-000004
Figure PCTCN2019087971-appb-000005
n＝6)； **P<0.01；
图6是BSA/MG-SPIONC)治疗乳腺癌药效学研究，A肿瘤生长曲线，B治疗后肿瘤质量；
图7是BSA/MG-SPIONC不同制剂状态下的粒径变化趋势图。
具体实施方式
下面结合附图说明和具体实施方式对本发明的技术方案作进一步阐述。
实施例1
本实施例以灵芝酸A-磁纳米粒(G-SPIONs)为例，说明中药-磁纳米粒的制备方法及其表征。
以FeCl 2和FeCl 3混合水溶液为原料，以支链淀粉为稳定剂，3-氨丙基三乙氧基硅烷为氨基源，通过NaOH溶液沉淀、NH 3·H 2O调整pH等步骤制得SPIONs。磁力搅拌下，将灵芝酸A氯仿溶液缓慢滴入SPIONs氯仿溶液中，旋蒸后水溶液复溶，过膜后得G-SPIONs。经透射电镜观察到G-SPIONs形态圆整、粒径分散均匀(图2A)；常规制剂学分析结果表明，当灵芝酸A占氧化铁的质量分数为6％时，其包封率为82.1±3.4％；动态光散射粒径仪检测，G-SPIONs粒径为6.4±0.2nm，PDI为0.041±0.002，zeta电位为-12.42±0.24mV(图2B)；G-SPIONs在pH 7.4的PBS介质中，灵芝酸A的48h累计释放率为57.3±2.4％(图2C)；但存放3天后沉降明显，已经测不到粒径等数据，提示除稳定性外其他制剂学、理化特性均符合后续研究(图2D)。
相同制备和检测方法，灵芝酸A还可以被替代为灵芝酸B、灵芝酸、灵芝三萜、熊果酸、齐敦果酸、科罗酸、雷公藤红素、人参三萜皂苷、柴胡三萜皂苷、黄芪三萜皂苷等。
实施例2
本实施例以说明甘露糖修饰的灵芝酸A-磁纳米粒(MG-SPIONs)的制备方法。
将实施例1制得的G-SPIONs分散在去离子水中，常温下缓慢加入等摩尔量的丁二酸，1.1eq的EDC和1.3eq的NHS，搅拌2h后加入等摩尔量的甘露糖，继续常温搅拌24h，流动水透析除去未反应的小分子，制备得到MG-SPIONs。
相同制备和检测方法，灵芝酸A还可以被替代为灵芝酸B、灵芝酸、灵芝三萜、熊果酸、齐敦果酸、科罗酸、雷公藤红素、人参三萜皂苷、柴胡三萜皂苷、黄芪三萜皂苷等。
丁二酸还可以被替代为丙二酸、戊二酸、辛二酸等。
实施例3
本实施例以以灵芝酸A-磁纳米粒(MG-SPIONC)为例，说明中药-磁纳米簇的制备方法及其表征
将实施例2制得的MG-SPIONs分散在5％的二巯基丁二酸中，常温搅拌24h，流动水透析除去游离的交联剂，制备得到MG-SPIONC。透析常规制剂学分析结果表明，灵芝酸A的包封率为80.4±4.4％，载药量为2.2±0.3％；MG-SPIONC粒径为146.4±5.5nm，PDI为0.191±0.001，zeta电位为-14.45±1.64mV；MG-SPIONC存放7天观察到明显沉降。
相同制备和检测方法，灵芝酸A还可以被替代为灵芝酸B、灵芝酸、灵芝三萜、熊果酸、齐敦果酸、科罗酸、雷公藤红素、人参三萜皂苷、柴胡三萜皂苷、黄芪三萜皂苷等。交联剂可以为(二)巯丁二酸、(二)巯辛二酸、(二)巯硬脂酸、(二)巯戊二酸等具有羧基和巯基的 小分子化合物。需说明的是，(二)巯丁二酸是指巯基丁二酸或二巯基丁二酸。
实施例4
本实施例以牛血清白蛋白包封的灵芝酸A-磁纳米粒(BSA/MG-SPIONC)为例，说明蛋白包封的中药-磁纳米簇的制备方法及其表征。
将实施例3制得的MG-SPIONC缓慢滴入1.1eq EDC、1.3eq NHS和1eq BSA混合溶液体系下，常温搅拌24h，离心机6000rpm 10min，去除上清游离BSA溶液，制备得到BSA/MG-SPIONC。制剂学研究表明，灵芝酸A的包封率为76.3±6.5％，载药量为1.7±0.2％；BSA/MG-SPIONC粒径为163.5±11.2nm，PDI为0.211±0.002，zeta电位为-25.32±3.13mV；BSA/MG-SPIONC存放7天未观察到明显沉降。
相同制备和检测方法，灵芝酸A还可以被替代为灵芝酸B、灵芝酸、灵芝三萜、熊果酸、齐敦果酸、科罗酸、雷公藤红素、人参三萜皂苷、柴胡三萜皂苷、黄芪三萜皂苷等。交联剂可以为(二)巯丁二酸、(二)巯辛二酸、(二)巯硬脂酸、(二)巯戊二酸等具有羧基和巯基的小分子化合物。所使用的包封蛋白可以是各种属动物的血清白蛋白、高密度脂蛋白、低密度脂蛋白、转铁蛋白等
实施例5
本实施例以以灵芝酸A为例，具体说明中药及中药-磁纳米粒干预TAMs极化过程考察
以RAW264.7细胞和MCF-7细胞分别作为巨噬细胞和肿瘤细胞模型，Transwell法共培养细胞，以LPS+IFN-γ组合干预作为M1极化阳性组，以IL-4干预为M2极化阳性组，不同浓度灵芝酸A干预为测试组，共孵育96h后将细胞固定、封闭、打孔，CD206-APC抗体染色，流式检测M2型特征marker CD206的表达情况；利用常规的ELISA法测试不同干预后细胞上清液中TNF-α(M1)和IL-10(M2)细胞因子水平；RT-PCR法定量药物干预后特征基因表达。研究发现，灵芝酸A能够有效抵抗巨噬细胞IL-4活化，显著降低M2型标志物CD206表达，明显减少M2型特征细胞因子IL-10分泌，并下调M2型特征基因Arg1表达(图3A-D)，但对M1型特征基因iNOS、CD86表达，特征细胞因子TNF-α作用不明显。结果证实，灵芝酸A可有效抑制TAMs的M2极化，但促进M1型极化的作用较小。
此外，以G-SPIONs和SPIONs为测试组做相同指标测试，研究发现，G-SPIONs灵芝酸A能够具有M1极化诱导趋势，显著升高M1型标志物CD86表达，降低M2型标志物CD206表达，明显减少M2型特征细胞因子IL-10分泌，促进TNF-α的分泌，不仅能下调M2型特征基因Arg1表达，还能上调M1型特征基因iNOS、CD86表达(图3A-D)。但不载药的SPIONs 抑制M1向M2转化的性能较弱。结果证实，G-SPIONs能够有效抑制TAMs的M2极化，促进其向M1型极化，SPIONs与灵芝酸A在干预TAMs极化作用方面有协同作用。
相同制备和检测方法，将灵芝酸A替换为灵芝酸B、灵芝酸、灵芝三萜、熊果酸、齐敦果酸、科罗酸、雷公藤红素、人参三萜皂苷、柴胡三萜皂苷、黄芪三萜皂苷，得到了相同的结论，SPIONs与具有免疫治疗作用的中药组分在干预TAMs极化作用方面有协同作用
实施例6
本实施例以以灵芝酸A为例，具体说明中药-磁纳米粒联合化疗药物(TC-MEs)抗乳腺癌药效学优势分析
采用生理盐水组为阴性对照，考察TC-MEs组、不同剂量的灵芝酸A磁纳米粒(G，5mg/kg、10mg/kg、20mg/kg)+TC-MEs间隔6h给药组和G(5mg/kg)+TC-MEs同时给药组的协同抗肿瘤效应。结果表明与生理盐水组相比，含G的给药各组均有抑制肿瘤增长的作用，治疗效果优于TC-MEs组(图4A)。通过裸鼠体重变化发现，含雷公藤红素的给药各组均有降低裸鼠体重的效果(图4B)。在同一给药剂量下，先接受G给药，间隔6h后再接受C-MEs给药治疗的小鼠比G和C-MEs同时给药的小鼠生存时间要长，肿瘤指数更低(图4C,D)。结合抗肿瘤机制部分的实验结果分析，灵芝酸A可能促使肿瘤免疫环境活化，进而提高了TC-MEs的抗肿瘤疗效，且雷公藤红素微乳和G次序治疗产生的药效比同时给药更有治疗优势。
实施例7
本实施例以Celastrol(雷公藤红素)为例，说明中药对肿瘤免疫微环境影响评测
以生理盐水组为阴性对照组，比较Celastrol组、Cel+STS组、T/C-L组及T/CM-L组治疗后，对体内的肿瘤微环境变化，阐释增效减毒的抗肿瘤机制。通过对肿瘤组织切片中血管内皮细胞的CD-31marker进行染色，可知T/CM-L组的荧光强度明显弱于阳性对照和Celastrol组，经过STS预先释放以及对微环境的干预，T/CM-L组切片中的血管形态呈较为规则的椭圆形态，各种芽生状态明显变少，血管状态趋于正常化。结果提示T/CM-L组可能显著改善肿瘤组织中血管的病理状态；Cel+STS组由于缺少逐级释放和被动聚集的性能，血管正常化效率明显不高，间接导致了该组的抗肿瘤效果不理想；T/C-L虽然一定程度上运送STS到肿瘤部位，但是缺乏逐级释放的特性，导致药物没有在正常化窗口期内递送，因此抗肿瘤效果没有达到最优化(图5A)。此外，我们还对肿瘤组织中肿瘤相关成纤维细胞(TAFs)膜上的肌动蛋白α-SMA进行抗体标记染色，表征药物干预后瘤组织内TAFs的密度。结果显示，生理盐水组，Celastrol组的荧光强度较高，提示MCF-7异位瘤组织的TAFs密度较高；Cel+STS组给药后，荧光强度 一定程度减弱，暗示STS可能对TAFs的减少有积极作用；T/C-L组及T/CM-L组的肿瘤切片荧光强度相对较低，两者无显著性差异，表明T/C-L组和T/CM-L可能抑制肿瘤组织中TAFs增殖提高雷公藤红素的抗肿瘤效率，且该机制与逐级释放药物特性无关(图5B)。
血清中的细胞因子也是肿瘤微环境的重要组成部分。我们通过ELISA试剂盒对荷瘤小鼠血清中IL-2，CCL2，TGF-β与IFN-γ进行含量测定。结果表明，生理盐水组与各制剂组在IL-2和TGF-β上均无显著差别；但在免疫指标IFN-γ与CCL2上有所差异：Celastrol组的IFN-γ含量显著低于生理盐水组，表明裸药可能存在一定的免疫抑制毒性，但T/C-L和T/CM-L治疗能够提高血清中IFN-γ含量，有助于免疫功能的改善；其次，T/C-L组和T/CM-L组的CCL2水平显著低于生理盐水组(图5C-F)，提示纳米制剂治疗可能减少趋化因子的分泌，降低肿瘤生长和转移的可能性。
相同制备和检测方法，雷公藤红素还可以被替代为灵芝酸B、灵芝酸、灵芝三萜、熊果酸、齐敦果酸、科罗酸、灵芝酸A、人参三萜皂苷、柴胡三萜皂苷、黄芪三萜皂苷等。
实施例8
本实施例说明实施例4制备的白蛋白包封的灵芝酸A-磁纳米簇(BSA/MG-SPIONC)治疗乳腺癌实例
裸鼠乳垫接种MCF-7细胞构建原位乳腺癌裸鼠模型。采用生理盐水组为阴性对照，以灵芝酸A-磁纳米簇(MG-SPIONC)、灵芝酸A-磁纳米粒(MG-SPIONs)为对照组，考察BSA/MG-SPIONC化学免疫治疗效果。给药方案为接种第0天起，隔天给药，剂量为灵芝酸A10mg/kg，给药至第28天，治疗期间记录裸鼠体重，肿瘤生长曲线，给药结束后剥离肿瘤，测量体积和重量。结果如图6A所示，无蛋白包封的纳米簇和纳米粒均可以显著降低肿瘤生长，但白蛋白包封的纳米簇能够更有效地抑制肿瘤的生长。图6B所示，BSA/MG-SPIONC治疗后的肿瘤最轻，提示抗肿瘤效果最明显。
相同制备和研究方法，灵芝酸A还可以被替代为灵芝酸B、灵芝酸、灵芝三萜、熊果酸、齐敦果酸、科罗酸、雷公藤红素、人参三萜皂苷、柴胡三萜皂苷、黄芪三萜皂苷等。肿瘤类型还可是黑色素瘤、肝癌、肺癌、胰腺癌、肾癌、结肠癌等。
实施例9
本实施例说明实施例4制备的白蛋白包封的灵芝酸A-磁纳米簇(BSA/MG-SPIONC)体外尺寸调控研究实例
采用实施例2的方式制备MG-SPIONs。不同质量分数下的交联剂对MG-SPIONC的粒径 影响很大。结果如表1所示，无论是载灵芝酸A的MG-SPIONs还是未载药的SPIONs，粒径均在质量分数为1％～10％的交联剂作用下逐渐增大。这种交联出的MG-SPIONC是一种还原敏感的体系，易受到GSH影响，发生解离，形成小粒径的初始状态。结果如表2所示，在不同GSH浓度下，解离程度有所不同，在1mM GSH时，BSA/MG-SPIONC的粒径重新解离回MG-SPIONs的水平。没有BSA包封的MG-SPIONC也有相同的趋势。没有交联成簇的MG-SPIONC粒径则不受GSH的影响。
相同制备和研究方法，灵芝酸A还可以被替代为灵芝酸B、灵芝酸、灵芝三萜、熊果酸、齐敦果酸、科罗酸、雷公藤红素、人参三萜皂苷、柴胡三萜皂苷、黄芪三萜皂苷等。交联剂可以为(二)巯丁二酸、(二)巯辛二酸、(二)巯硬脂酸、(二)巯戊二酸等具有羧基和巯基的小分子化合物。所使用的包封蛋白可以是各种属动物的血清白蛋白、高密度脂蛋白、低密度脂蛋白、转铁蛋白等。
表1不同交联剂(wt％)下的BSA/MG-SPIONC尺寸
Figure PCTCN2019087971-appb-000006
实施例10
本实施例说明实施例4制备的白蛋白包封的灵芝酸A-磁纳米簇(BSA/MG-SPIONC)多重尺寸调控增强肿瘤靶向性实例
BSA/MG-SPIONC的组成是通过MG-SPIONs的组装，MG-SPIONC的交联，BSA/MG-SPIONC的包封步骤，这些步骤对应的是体内的多重尺寸调控策略实施以及最终的小尺寸联合靶配体介导的TAM靶向化学免疫治疗过程。体内外的尺寸多重调控是本发明的重点。本实施例通过对不同制剂状态以及触发状态下的粒径进行检测，验证本发明可尺寸调控和TAM靶向的客观基础。结果如图7所示，SPIONs状态下的粒径仅为10nm以下，交联后的纳米簇粒径迅速上升至120nm左右，当被BSA包封后，纳米簇的粒径又一次小幅上升。但是 BSA/MG-SPIONC在遭遇到肿瘤组织内高浓度GSH触发后，粒径迅速解离至SPIONs状态，这种尺寸非常利于对肿瘤乏氧区域TAM的渗透和靶向。
相同制备和研究方法，灵芝酸A还可以被替代为灵芝酸B、灵芝酸、灵芝三萜、熊果酸、齐敦果酸、科罗酸、雷公藤红素、人参三萜皂苷、柴胡三萜皂苷、黄芪三萜皂苷等。交联剂可以是交联剂可以为(二)巯丁二酸、(二)巯辛二酸、(二)巯硬脂酸、(二)巯戊二酸等具有羧基和巯基的小分子化合物。所使用的包封蛋白可以是各种属动物的血清白蛋白、高密度脂蛋白、低密度脂蛋白、转铁蛋白等。

Claims (10)

  1. 一种中药-磁纳米簇化学免疫药物递送系统，其特征在于，包括超顺磁铁氧化物纳米粒；所述超顺磁铁氧化物纳米粒表面锚定药物和TAMs的靶向配体；超顺磁铁氧化物纳米粒通过二硫键形成纳米簇结构；所述纳米簇结构外包封蛋白。
  2. 根据权利要求1所述的一种中药-磁纳米簇化学免疫药物递送系统，其特征在于，所述药物为具有免疫治疗作用的中药组分；优选灵芝三萜类或其他中药三萜类。
  3. 根据权利要求1所述的一种中药-磁纳米簇化学免疫药物递送系统，其特征在于，所述超顺磁铁氧化物纳米粒通过交联手段组装成纳米簇结构；交联剂选用具有羧基和巯基的小分子化合物。
  4. 根据权利要求3所述的一种中药-磁纳米簇化学免疫药物递送系统，其特征在于，所述交联剂选用(二)巯丁二酸、(二)巯辛二酸、(二)巯硬脂酸或(二)巯戊二酸。
  5. 根据权利要求1所述的一种中药-磁纳米簇化学免疫药物递送系统，其特征在于，所述包封蛋白为各种属动物的血清白蛋白、高密度脂蛋白、低密度脂蛋白或转铁蛋白。
  6. 根据权利要求1所述的一种中药-磁纳米簇化学免疫药物递送系统，其特征在于，所述超顺磁铁氧化物纳米粒基材为Fe 3O 4或γ-Fe 2O 3。
  7. 根据权利要求1所述的一种中药-磁纳米簇化学免疫药物递送系统，其特征在于，所述超顺磁铁氧化物纳米粒的粒径范围为2～10nm。
  8. 根据权利要求1所述的一种中药-磁纳米簇化学免疫药物递送系统，其特征在于，所述纳米簇粒径范围为120～160nm。
  9. 根据权利要求1～8任一项所述的一种中药-磁纳米簇化学免疫药物递送系统的制备方法，其特征在于，包括如下步骤：
    (1)制备超顺磁铁氧化物纳米粒；
    (2)通过配位作用，将药物锚定在超顺磁铁氧化物纳米粒表面；
    (3)在载药的超顺磁铁氧化物纳米粒表面修饰TAMs的靶向配体；
    (4)将修饰的超顺磁铁氧化物纳米粒通过交联手段组装，获取超顺磁铁氧化物纳米簇；
    (5)在超顺磁铁氧化物纳米簇外层包封蛋白。
  10. 根据权利要求9所述的制备方法，其特征在于，所述步骤(1)中，以支链淀粉和3-氨丙基三乙氧基硅烷为包衣材料，采用热共沉淀法制备水溶性氨基化超顺磁铁氧化物纳米粒。
PCT/CN2019/087971 2018-08-14 2019-05-22 一种中药-磁纳米簇化学免疫药物递送系统及其制备方法 WO2020034696A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810925321.0A CN109045303B (zh) 2018-08-14 2018-08-14 一种中药-磁纳米簇化学免疫药物递送系统及其制备方法
CN201810925321.0 2018-08-14

Publications (1)

Publication Number Publication Date
WO2020034696A1 true WO2020034696A1 (zh) 2020-02-20

Family

ID=64683912

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/087971 WO2020034696A1 (zh) 2018-08-14 2019-05-22 一种中药-磁纳米簇化学免疫药物递送系统及其制备方法

Country Status (2)

Country Link
CN (1) CN109045303B (zh)
WO (1) WO2020034696A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109045303A (zh) * 2018-08-14 2018-12-21 江苏省中医药研究院 一种中药-磁纳米簇化学免疫药物递送系统及其制备方法

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110538195A (zh) * 2019-06-05 2019-12-06 江苏省中医药研究院 一种化学免疫干预剂及应用
CN112426536B (zh) * 2020-11-06 2022-03-18 山东大学 一种磁靶向Fe3O4纳米复合物及其制备方法和应用
CN113975403B (zh) * 2021-11-03 2023-11-17 江苏护理职业学院 转铁蛋白与抗体结合超顺磁性氧化铁纳米粒及其制备方法和应用
CN114668771B (zh) * 2022-03-18 2022-12-06 南京林业大学 一种共载阿霉素和熊果酸的铁蛋白纳米粒的制备方法及应用
CN115368430B (zh) * 2022-09-02 2024-02-02 北京化工大学 一种雷公藤红素金属络合物的制备方法和应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106963724A (zh) * 2017-01-24 2017-07-21 西南科技大学 一种γ‑Fe2O3超顺磁性纳米团簇的制备方法
CN107375235A (zh) * 2017-07-24 2017-11-24 桂林医学院 一种叶酸介导抗肿瘤药物超顺磁肿瘤靶向纳米粒及其制备方法
CN109045303A (zh) * 2018-08-14 2018-12-21 江苏省中医药研究院 一种中药-磁纳米簇化学免疫药物递送系统及其制备方法

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW200631586A (en) * 2005-03-08 2006-09-16 Phytohealth Corp A method for preparing a pharmaceutical compound by way of magnetic carbon nanocapsules
US20130023714A1 (en) * 2008-10-26 2013-01-24 Board Of Regents, The University Of Texas Systems Medical and Imaging Nanoclusters
CN101601695B (zh) * 2009-06-26 2012-02-29 江苏省中医药研究院 灵芝三萜提取物的自微乳化纳米组合物及其制备方法
CN102502858A (zh) * 2011-10-24 2012-06-20 中国科学院理化技术研究所 一种聚丙烯酸修饰的水溶性铁酸盐纳米团簇的制备方法
CN105168134A (zh) * 2015-09-08 2015-12-23 江苏省中医药研究院 一种半乳糖酯修饰的中西药共载口服肝靶向微乳递送系统
CN106729727B (zh) * 2016-12-16 2020-07-31 中国药科大学 靶向配体修饰的还原响应型磁性纳米载体及其制备方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106963724A (zh) * 2017-01-24 2017-07-21 西南科技大学 一种γ‑Fe2O3超顺磁性纳米团簇的制备方法
CN107375235A (zh) * 2017-07-24 2017-11-24 桂林医学院 一种叶酸介导抗肿瘤药物超顺磁肿瘤靶向纳米粒及其制备方法
CN109045303A (zh) * 2018-08-14 2018-12-21 江苏省中医药研究院 一种中药-磁纳米簇化学免疫药物递送系统及其制备方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KOICHIRO HAYASHI ET AL.: "Superparamagnetic Nanoparticle Clusters for Cancer Theranostics Combining Magnetic Resonance Imaging and Hyperthermia Treatment", THERANOSTICS, vol. 6, no. 3, 23 April 2013 (2013-04-23), pages 366 - 376, XP055326901 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109045303A (zh) * 2018-08-14 2018-12-21 江苏省中医药研究院 一种中药-磁纳米簇化学免疫药物递送系统及其制备方法
CN109045303B (zh) * 2018-08-14 2021-10-15 江苏省中医药研究院 一种中药-磁纳米簇化学免疫药物递送系统及其制备方法

Also Published As

Publication number Publication date
CN109045303A (zh) 2018-12-21
CN109045303B (zh) 2021-10-15

Similar Documents

Publication Publication Date Title
WO2020034696A1 (zh) 一种中药-磁纳米簇化学免疫药物递送系统及其制备方法
Pan et al. Urinary exosomes-based engineered nanovectors for homologously targeted chemo-chemodynamic prostate cancer therapy via abrogating EGFR/AKT/NF-kB/IkB signaling
Guo et al. Chemotherapy mediated by biomimetic polymeric nanoparticles potentiates enhanced tumor immunotherapy via amplification of endoplasmic reticulum stress and mitochondrial dysfunction
Bae et al. Chitosan oligosaccharide-stabilized ferrimagnetic iron oxide nanocubes for magnetically modulated cancer hyperthermia
Chen et al. Folic acid-conjugated MnO nanoparticles as a T 1 contrast agent for magnetic resonance imaging of tiny brain gliomas
Feng et al. Fe (III)‐shikonin supramolecular nanomedicine for combined therapy of tumor via ferroptosis and necroptosis
Gao et al. Designing multifunctional cancer-targeted nanosystem for magnetic resonance molecular imaging-guided theranostics of lung cancer
Chung et al. Iron oxide nanoparticles for immune cell labeling and cancer immunotherapy
Elechalawar et al. Dual targeting of folate receptor-expressing glioma tumor-associated macrophages and epithelial cells in the brain using a carbon nanosphere–cationic folate nanoconjugate
AU2018262962B2 (en) Immunomagnetic nanocapsule, fabrication method and use thereof, and kit for treating cancer
Sun et al. A nano “Immune‐Guide” recruiting lymphocytes and modulating the ratio of macrophages from different origins to enhance cancer immunotherapy
Gao et al. A novel nanomissile targeting two biomarkers and accurately bombing CTCs with doxorubicin
WO2021243925A1 (zh) ZIF-8纳米材料在降解广谱突变p53蛋白中的应用
Zhang et al. Human cancer cell membrane-cloaked Fe3O4 nanocubes for homologous targeting improvement
Wang et al. PtNi nano trilobal-based nanostructure with magnetocaloric oscillation and catalytic effects for pyroptosis-triggered tumor immunotherapy
Zhou et al. Nanoparticle‐based MRI‐guided tumor microenvironment heating via the synergistic effect of ferroptosis and inhibition of TGF‐β signaling
Yuan et al. Regulating tumor-associated macrophage polarization by cyclodextrin-modified PLGA nanoparticles loaded with R848 for treating colon cancer
Tang et al. Nanovector assembled from natural egg yolk lipids for tumor-targeted delivery of therapeutics
CN115252828B (zh) 一种负载棉酚的团簇型超小四氧化三铁纳米粒及其制备和应用
Fazilati Anti-neoplastic applications of heparin coated magnetic nanoparticles against human ovarian cancer
US20230233705A1 (en) Gold nanoparticle-containing medicine
CN110448700B (zh) 一种用于靶向诊疗胃癌的纳米载药复合物及制备方法
JP7063727B2 (ja) 抗腫瘍剤、がんの治療及び／又は予防のための医薬組成物
US10736964B2 (en) Immunomagnetic nanocapsule and kit for treating cancer
Iida et al. Preparation of human immune effector T cells containing iron‐oxide nanoparticles

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19849101

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19849101

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 19849101

Country of ref document: EP

Kind code of ref document: A1