CN114099469B - Composite nano-drug carrier and preparation method and application thereof - Google Patents

Composite nano-drug carrier and preparation method and application thereof Download PDF

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CN114099469B
CN114099469B CN202111472902.1A CN202111472902A CN114099469B CN 114099469 B CN114099469 B CN 114099469B CN 202111472902 A CN202111472902 A CN 202111472902A CN 114099469 B CN114099469 B CN 114099469B
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psa
zein
hnk
tumor
drug
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CN114099469A (en
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蔡德富
张琪
王婧
刘丹
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Qiqihar Medical University
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    • 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
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • 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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • 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
    • A61K47/56—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Abstract

The invention discloses a composite nano-drug carrier and a preparation method and application thereof, in particular to a Poly Sialic Acid (PSA) modified zein nano-drug carrier and a drug carrying system thereof, especially a tumor targeting drug delivery system, and a preparation method and application thereof. The drug carrier can realize enhanced drug delivery efficiency, active targeting capability and specific biological distribution at tumor sites, thereby inhibiting the growth, migration and invasion of tumors. Taking HNK as an example, experiments demonstrate that PSA-zein-HNK increased tumor accumulation in a mouse model carrying 4T1 breast cancer, resulting in desirable anti-tumor efficacy and favorable biosafety, and that PSA-zein-HNK significantly inhibits metastasis of breast cancer to lung and liver. Therefore, the nano-particles are expected to become an effective tumor targeting drug carrier for drug development and disease treatment.

Description

Composite nano-drug carrier and preparation method and application thereof
Technical Field
The invention relates to the technical field of biological medicine, in particular to a composite nano-drug carrier and a preparation method and application thereof, and especially relates to a Poly Sialic Acid (PSA) -modified zein nano-drug carrier and a drug carrying system thereof, especially a tumor targeting drug delivery system, and a preparation method and application thereof.
Background
Along with the social economic development and the improvement of the living standard of people, the worldwide disease spectrum and the death spectrum are obviously changed under the influence of factors such as dietary structure change, population aging, urbanization and the like, and chronic non-infectious diseases become main causes of death. Among them, malignant tumor is one of the main causes of death worldwide at present, and has become a major disease which seriously endangers human life and health and restricts social and economic development.
Chemotherapy is currently the most common means in tumor treatment, and depends largely on whether the chemotherapeutic agent administered can safely and effectively reach the tumor site, however, many of these agents often result in poor efficacy and serious side effects due to their low concentration at the tumor site and similar cytotoxicity to both cancer cells and healthy cells.
One approach to solving these problems is to incorporate chemotherapeutic agents into nano-drug carriers, the resulting drug-loaded systems can sustain drug release, improve pharmacokinetic profiles, and enhance tumor accumulation through permeability enhancement and retention (EPR). However, the efficacy of drugs is still limited by some limitations of drug carriers, such as leakage of chemotherapeutic agents, uptake of chemotherapeutic agents by RES organs, insufficient penetration and accumulation of nanomedicines in tumor sites by EPR effect. Thus, new drug carriers are urgently needed to solve these problems.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a PSA modified zein nano-drug carrier and a drug carrying system thereof, in particular to a tumor targeting drug carrying system, and a preparation method and application thereof.
In a first aspect of the present invention, there is provided a nanoparticle having a core-shell structure, which has a core particle and a coating layer coated on an outer surface of the core particle, wherein the core particle comprises zein and the coating layer comprises PSA.
In one embodiment of the invention, the core particle is formed from zein.
In one embodiment of the invention, the cover is formed from a PSA.
In particular, the average molecular weight of the PSA is from 5000 to 120000Da (for example 5000, 10000, 20000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 110000, 120000 Da), in particular from 50000 to 80000Da; in some embodiments of the invention, the average molecular weight of the PSA is 80000Da.
In particular, the nanoparticle may have a particle size of 100-200nm (e.g., 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200 nm), particularly 100-120nm.
In particular, the thickness of the coating in the nanoparticle may be 1-100nm (e.g. 1, 5, 10, 12, 14, 15, 16, 18, 20, 22, 24, 25, 26, 28, 30, 40, 50, 60, 70, 80, 90, 100 nm), in particular 10-30nm.
In particular, the core particles in the nanoparticle may have a particle size of 50-100nm (e.g., 50, 60, 70, 80, 85, 86, 88, 90, 92, 94, 96, 98, 100 nm), particularly 85-95nm.
Specifically, the nanoparticle is spherical.
Specifically, the nanoparticle is negatively charged.
In a second aspect of the present invention, there is provided a method for preparing the nanoparticle according to the first aspect, comprising the steps of:
(1) Forming core particles;
(2) Forming a coating layer;
optionally, (3) removing impurities.
Specifically, the core particles are formed by an antisolvent precipitation method.
More specifically, step (1) comprises: zein is dissolved in aqueous ethanol, the pH is adjusted to acidity, and ethanol is removed.
In particular, the aqueous ethanol solution may be 75-95% (v/v) aqueous ethanol solution, particularly 85% aqueous ethanol solution.
Specifically, adjusting the pH to acidic is adjusting the pH to 4.5-6.5 (e.g., 4.5, 5, 5.2, 5.5, 5.7, 6, 6.5).
Specifically, the agent used to adjust the pH to be acidic is a mineral acid solution, such as hydrochloric acid solution.
Specifically, the step (1) further comprises a stirring step; more specifically, the stirring speed may be 500 to 5000rpm (e.g., 500, 1000, 2000, 3000, 4000, 5000 rpm), and the stirring time may be 10 to 60 minutes (e.g., 10, 20, 30, 40, 50, 60 minutes).
Specifically, after adjusting the pH to be acidic, the step (1) may further include a step of adding a surfactant solution.
Specifically, the surfactant may be a nonionic surfactant such as tween, particularly tween 80.
Specifically, the pH of the surfactant solution is acidic, for example, pH 3.5 to 4.
Specifically, the concentration of the above surfactant solution is 0.01 to 1% (e.g., 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%), particularly 0.01 to 0.1%.
In one embodiment of the present invention, step (1) comprises: dissolving zein in ethanol water solution, stirring, adjusting pH to acidity, stirring the obtained solution, adding surfactant, and removing ethanol.
Specifically, step (2) includes: adding the dispersion of core particles obtained in step (1) to a PSA solution.
Specifically, the PSA solution is acidic in pH, for example, pH 2-6 (e.g., 2, 3, 4, 5, 6), pH 4.
In particular, the concentration of PSA solution may be 0.05-10% (w/v) (e.g., 0.01%, 0.05%, 0.075%, 0.1%, 0.2%, 0.25%, 0.4%, 0.5%, 0.6%, 0.8%, 1%, 2.5%, 5%, 7.5%, 10%), particularly 0.1-1%, more particularly 0.1-0.25%; in some embodiments of the invention, the concentration of PSA solution is 0.1%.
In particular, the average molecular weight of the PSA is from 5000 to 120000Da (for example 5000, 10000, 20000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 110000, 120000 Da), in particular from 50000 to 80000Da; in some embodiments of the invention, the average molecular weight of the PSA is 80000Da.
In one embodiment of the invention, the concentration of the PSA solution is 0.1% with an average molecular weight of 80000Da.
Specifically, the step (2) further comprises a stirring step; more specifically, the stirring speed may be 500 to 5000rpm (e.g., 500, 1000, 2000, 3000, 4000, 5000 rpm), and the stirring time may be 0.5 to 5 hours (e.g., 0.5, 1, 2, 3, 4, 5 hours).
Specifically, step (3) may include: centrifuging the system obtained in the step (2) to remove impurities.
In a third aspect of the invention there is provided the use of a nanoparticle according to the first aspect as a carrier for a medicament and in the manufacture of a medicament.
In a fourth aspect of the invention, a drug delivery system is provided having sustained release and targeting properties, which is a nanoparticle having a core particle and a coating layer coating the outer surface of the core particle, wherein the core particle comprises zein, the coating layer comprises PSA, and the core particle is loaded with one or more active ingredients.
In one embodiment of the invention, the core particle is formed from zein and an active ingredient.
In one embodiment of the invention, the cover is formed from a PSA.
In particular, the average molecular weight of the PSA is from 5000 to 120000Da (for example 5000, 10000, 20000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 110000, 120000 Da), in particular from 50000 to 80000Da; preferably, the average molecular weight of the PSA is 80000Da.
In particular, the nanoparticle may have a particle size of 100-200nm (e.g., 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200 nm), particularly 100-120nm.
In particular, the thickness of the coating in the nanoparticle may be 1-100nm (e.g. 1, 5, 10, 12, 14, 15, 16, 18, 20, 22, 24, 25, 26, 28, 30, 40, 50, 60, 70, 80, 90, 100 nm), in particular 10-30nm.
In particular, the core particles in the nanoparticle may have a particle size of 50-100nm (e.g., 50, 60, 70, 80, 85, 86, 88, 90, 92, 94, 96, 98, 100 nm), particularly 85-95nm.
Specifically, the nanoparticle is spherical.
Specifically, the nanoparticle is negatively charged.
In one embodiment of the invention, the above active ingredient is a pharmaceutically active ingredient, in particular an antitumor drug, e.g. doxorubicin, epirubicin, pirarubicin, idarubicin; mitoxantrone; topotecan, irinotecan, and aminocamptothecin; paclitaxel, docetaxel; gefitinib, imatinib, nilotinib, sunitinib, lapatinib, tofacitinib, crizotinib, masitinib, emtrictinib, ibrutinib, afatinib, flumatinib, erlotinib, lenatinib, ai Leti, apatinib, talazoparib, lorlatinib, TPX-0005; cisplatin, carboplatin, nedaplatin, cycloplatin, oxaliplatin, lobaplatin; vinblastine, vincristine, vinorelbine, berberine, berbamine; honokiol (HNK); uracil nitrogen mustard, ifosfamide, melphalan, chlorambucil, pipobromine, trolamine, triethylthiophosphamide, busulfan, carmustine, lomustine, streptozocin, dacarbazine, fluorouracil deoxynucleoside, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate; etc.
In particular, the weight ratio of zein to active ingredient in the core particle may be from 1:1 to 100:1 (e.g., 1:1, 5:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1), particularly from 5:1 to 20:1.
In one embodiment of the invention, the active ingredient is HNK; more specifically, the weight ratio of zein to HNK in the core particle is 10:1.
In a fifth aspect of the present invention, there is provided a method for preparing the drug delivery system of the fourth aspect, comprising the steps of:
(1) Forming core particles;
(2) Forming a coating layer;
optionally, (3) removing impurities.
Specifically, the core particles are formed by an antisolvent precipitation method.
More specifically, step (1) comprises: zein and the active ingredient (separately or together) are dissolved in aqueous ethanol, the pH is adjusted to acidity, and ethanol is removed.
In particular, the aqueous ethanol solution may be 75-95% (v/v) aqueous ethanol solution, particularly 85% aqueous ethanol solution.
Specifically, adjusting the pH to acidic is adjusting the pH to 4.5-6.5 (e.g., 4.5, 5, 5.2, 5.5, 5.7, 6, 6.5).
Specifically, the agent used to adjust the pH to be acidic is a mineral acid solution, such as hydrochloric acid solution.
Specifically, the step (1) further comprises a stirring step; more specifically, the stirring speed may be 500 to 5000rpm (e.g., 500, 1000, 2000, 3000, 4000, 5000 rpm), and the stirring time may be 10 to 60 minutes (e.g., 10, 20, 30, 40, 50, 60 minutes).
Specifically, after adjusting the pH to be acidic, the step (1) may further include a step of adding a surfactant solution.
Specifically, the surfactant may be a nonionic surfactant such as tween, particularly tween 80.
Specifically, the pH of the surfactant solution is acidic, for example, pH 3.5 to 4.
Specifically, the concentration of the above surfactant solution is 0.01 to 1% (e.g., 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%), particularly 0.01 to 0.1%.
In one embodiment of the present invention, step (1) comprises: dissolving zein and active ingredient (such as HNK) in ethanol water solution, stirring, adjusting pH to acidity, stirring the obtained solution, adding surfactant, and removing ethanol.
Specifically, step (2) includes: adding the dispersion of core particles obtained in step (1) to a PSA solution.
Specifically, the PSA solution is acidic in pH, for example, pH 2-6 (e.g., 2, 3, 4, 5, 6), pH 4.
In particular, the concentration of PSA solution may be 0.05-10% (w/v) (e.g. 0.01%, 0.05%, 0.075%, 0.1%, 0.15%, 0.2%, 0.25%, 0.4%, 0.5%, 0.6%, 0.8%, 1%, 2.5%, 5%, 7.5%, 10%), particularly 0.1-1%, more particularly 0.1-0.25%; in some embodiments of the invention, the concentration of PSA solution is 0.1%.
In particular, the average molecular weight of the PSA is from 5000 to 120000Da (for example 5000, 10000, 20000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 110000, 120000 Da), in particular from 50000 to 80000Da; in some embodiments of the invention, the average molecular weight of the PSA is 80000Da.
In one embodiment of the invention, the concentration of the PSA solution is 0.1% with an average molecular weight of 80000Da.
Specifically, the step (2) further comprises a stirring step; more specifically, the stirring speed may be 500 to 5000rpm (e.g., 500, 1000, 2000, 3000, 4000, 5000 rpm), and the stirring time may be 0.5 to 5 hours (e.g., 0.5, 1, 2, 3, 4, 5 hours).
Specifically, step (3) may include: centrifuging the system obtained in the step (2) to remove impurities such as unencapsulated active ingredients.
In a sixth aspect of the present invention there is provided the use of a drug delivery system according to the fourth aspect in the manufacture of a medicament for the prophylaxis and/or treatment of a disease.
In particular, the disease may be a tumor, an autoimmune disease, an infectious disease, etc., in particular a tumor.
Specifically, the tumors are malignant tumors, which include but are not limited to: bladder cancer, breast cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer (including small cell lung cancer, non-small cell lung cancer), head and neck cancer, esophageal cancer, gall bladder cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, prostate cancer, skin cancer, B-cell chronic lymphocytic leukemia, acute lymphoblastic leukemia, non-hodgkin's lymphoma, acute myelogenous leukemia, diffuse large B-cell lymphoma, multiple myeloma, and the like (including primary and metastatic tumors); in particular breast cancer, including primary and metastatic breast cancer.
In a seventh aspect of the invention, there is provided a pharmaceutical composition comprising the drug delivery system of the fourth aspect, and one or more pharmaceutically acceptable excipients.
Specifically, the pharmaceutically acceptable carrier refers to a pharmaceutical carrier conventional in the pharmaceutical field, in particular a pharmaceutically acceptable injection adjuvant, such as isotonic sterile saline solution (sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, etc., or a mixture of the above salts), or a dry, e.g., lyophilized composition, which forms an injectable solute by adding sterile water or physiological saline as appropriate.
In particular, the pharmaceutical composition may be administered by any suitable route of administration, such as gastrointestinal or parenteral (e.g., intravenous, intramuscular, subcutaneous, intra-organ, intranasal, intradermal, instillation, intracerebral, intrarectal, etc.) route; the medicament may be in any suitable dosage form, such as a dosage form for parenteral administration, including, for example, but not limited to, tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, suspensions, and the like; parenteral dosage forms, for example, injection dosage forms: such as injections (e.g., for subcutaneous, intravenous, intramuscular, intraperitoneal injection), respiratory administration forms: such as spray, aerosol, powder spray, etc., skin administration dosage forms, such as topical solution, lotion, ointment, plaster, paste, patch, etc., mucosal administration dosage forms: such as eye drops, eye ointments, nasal drops, gargle, sublingual tablets, etc., and dosage forms for oral administration: such as suppository, aerosol, effervescent tablet, drop, dripping pill, etc., for treating rectum, vagina, urethra, nasal cavity, auditory canal, etc. Preferably, the pharmaceutical composition is an injection.
Specifically, the various dosage forms of the pharmaceutical composition may be prepared according to conventional production methods in the pharmaceutical arts, for example by mixing the drug-carrying system with one or more pharmaceutically acceptable excipients, and then formulating it into the desired dosage form.
In an eighth aspect of the invention, there is provided a method of preventing and/or treating a disease comprising the step of administering to a subject in need thereof a drug delivery system according to the fourth aspect or a pharmaceutical composition according to the seventh aspect.
In particular, the diseases have the definitions according to the third and sixth aspects of the invention, in particular malignant tumors, such as breast cancer, including primary and metastatic breast cancer.
In one embodiment of the invention, the method is a method of treating a malignancy, the therapeutic effect of which includes not only inhibiting tumor growth, but also inhibiting tumor metastasis.
In particular, the subject may be a mammal, such as a human, monkey, chimpanzee, canine, murine, rabbit, etc., particularly a human.
The invention develops the polysialic acid (PSA) modified zein core/shell nano-particles for the first time, which can be used for targeted drug delivery (particularly antitumor drugs such as Honokiol (HNK)), and can realize enhanced drug delivery efficiency, active targeting capability and specific biodistribution at tumor sites so as to inhibit the growth, migration and invasion of tumors. Taking HNK as an example, experiments demonstrate that PSA-zein-HNK increased tumor accumulation in a mouse model carrying 4T1 breast cancer, resulting in desirable anti-tumor efficacy and favorable biosafety, and that PSA-zein-HNK significantly inhibits metastasis of breast cancer to lung and liver. Therefore, the nano-particles prepared by the invention are expected to become an effective tumor targeting drug carrier for drug development and disease treatment.
Drawings
Figure 1 shows the effect of different PSA molecular weights on nanoparticle size.
Figure 2 shows the effect of different PSA solution concentrations on nanoparticle size.
FIG. 3 shows characterization of PSA-Zein-HNK nanoparticles. (a) particle size distribution, as determined by DLS method; (B) morphology of PSA-Zein-HNK, detected by TEM; scale bar, 500nm; (C) HNK release curves of different formulations.
FIG. 4 shows cellular uptake of zein and PSA-zein nanoparticles in 4T1 cells. (A) Cou6 fluorescence intensity of 4T1 cells, measured by flow cytometry, incubated for 1 hour; (B) quantitative analysis of Cou6 uptake based on flow cytometry; (C) Confocal microscopy images of 4T1 cells after 1 hour incubation; scale bar, 20 μm. Data are shown as mean ± SD (n=3), p <0.05, p <0.01.
FIG. 5 shows the cell viability of 4T1 cells incubated with different HNK preparations by SRB, three bars for each experimental group representing Zein-HNK, PSA-Zein-HNK, and free HNK, respectively. Each bar represents mean ± SD (n=6), p <0.05, p <0.01.
FIG. 6 shows the anti-metastatic effects of free HNK, zein-HNK and PSA-Zein-HNK on 4T1 cells. (A) Typical microscopy images and (B, C, D) quantitative analysis of 4T1 cells preincubated with all groups in wound healing, migration and invasion assays. Data are shown as mean ± SD (n=3), × p <0.01.
FIG. 7 shows an in vitro growth inhibition assay for 4T1 tumor spheres. (A) Representative images of tumor spheres treated with free HNK, zein-HNK and PSA-Zein-HNK, tumor spheres cultured in RPMI-1640 medium as controls, scale bar, 100 μm; (B) Growth curve of tumor spheres after treatment with various HNK formulations. Each dot represents the mean ± SD (n=3), × p <0.01.
FIG. 8 shows the in vivo biodistribution and in vivo tumor targeting properties of DiR-labeled PSA-Zein nanoparticles in 4T1 tumor-bearing mice. (A) In vivo fluorescence images of 4T1 tumor-bearing mice at various time points after administration of various formulations; (B) Ex vivo fluorescence images of major organs and tumors at 48 hours.
FIG. 9 shows the in vivo antitumor activity of various HNK formulations in a 4T1 tumor-bearing mouse model. (a) a profile of tumor volume change during treatment; (B) the weight of the tumor mass at the end of the treatment period; (C) tumor size images observed after sacrifice; (D) H & E staining of tumor sections at the end of the treatment period; (E) TUNEL fluorescent staining of apoptotic cells in tumor sections at the end of the treatment period; (F) semi-quantitative results of TUNEL fluorescent staining. Data are shown as mean ± SD (n=6), p <0.05, p <0.01.
Figure 10 shows in vivo anti-metastasis and biosafety studies in a 4T1 tumor-bearing mouse model. (a) weight change of 4T1 tumor-bearing mice during treatment; (B) Lung metastatic nodule specimens treated with various HNK preparations, red arrows indicating metastatic nodules; (C) H & E staining of major organ sections at the end of treatment period, green arrow indicates metastatic lesions, scale bar, 50 μm.
Detailed Description
Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates.
Various publications, patents, and published patent specifications cited herein are incorporated by reference in their entirety.
The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The reagents, materials and experimental animals used in the experiments of the examples were as follows:
honokiol (HNK, > 98%) was purchased from melons biotechnology limited (dalton, china). Zein (> 98%) and tween 80 were purchased from belvedere technologies limited (beijing, china). Polysialic acid (PSA) was produced by zhenjiang changxing pharmaceutical limited (zhenjiang, china). Sulfonyl rhodamine B sodium Salt (SRB), coumarin-6 (Cou), hoechst 33258 and matrigel are all supplied by Sigma-Aldrich (Shanghai, china). Annexin V-FITC/PI apoptosis assay kit was purchased from Biyun Biotechnology Inc. (Beijing, china). Rabbit polyclonal anti-human Bcl-2, mouse polyclonal anti-human Bax, rabbit polyclonal anti-GAPDH, rabbit anti-mouse immunoglobulin G (IgG) and goat anti-rabbit IgG were all obtained from Cell Signaling Technology (Danvers, USA). Rabbit polyclonal antibodies against E-cadherin and vimentin were purchased from Proteintech (Chicago, USA). All other chemical reagents were used as received unless otherwise indicated.
Murine breast cancer cell line 4T1 was obtained from the institute of basic medicine of the national academy of sciences of medicine (Beijing, china) and contained 5% CO at 37 ℃C 2 Is cultured in RPMI-1640 medium (Michaelis scientific Co., beijing, china) supplemented with 10% fetal bovine serum (Viton Biotechnology Co., nanj, china), 100U/mL penicillin and 100. Mu.g/mL streptomycin.
Female BALB/c mice (4-6 weeks old) were purchased from Liaoning laboratory animal resource center (Benxi, china) and kept under specific pathogen-free and temperature-controlled conditions. All animal-related experiments were approved by the ethical committee of zizikhar medical college and fully met institutional guidelines for care and use of experimental animals.
Statistical analysis:
all experimental results in the examples are expressed as mean ± standard deviation. Statistical differences were determined using student t-test. p <0.05 was considered significant, and p <0.01 was considered extremely significant.
Example 1: investigation of the molecular weight of PSA
1. Preparation of nanoparticles
PSA-zein nanoparticles were prepared using PSA of different molecular weights, respectively, as follows:
zein (100 mg) was dissolved in 5.0mL ethanol/water (85:15, v/v). After stirring at 1000rpm for 1 hour, the above mixed solution was adjusted to pH 5.7 using 1mol/L HCl and magnetically stirred (1000 rpm) for 30min while being added dropwise to 20.0mL of a 0.05% Tween 80 solution (adjusted to pH 4.0). The remaining ethanol was then removed by rotary evaporator at 37 ℃.
For the method of coating zein nanoparticles with PSA, the resulting zein nanoparticle dispersion (5.0 mL) was injected into 5.0mL of PSA solution (adjusted to pH 4.0) and stirring (1000 rpm) was continued for 1 hour. The prepared sample was centrifuged at 12000rpm to remove impurities. The resulting sample was zein/PSA core-shell nanoparticles and was referred to as PSA-zein.
The concentrations of the PSA solutions were 0.1%, wherein the molecular weights of the PSA were 5, 10, 25, 40, 50, 60, 70, 80, 90, 120 kDa, respectively.
The size, structure and morphology of the nanoparticles were confirmed by transmission electron microscopy (TEM, hitachi HT-7700, japan).
2. Experimental results
Experimental results as shown in figure 1, nanoparticles undergo extensive aggregation when using PSA of <50kDa and >80 kDa. In general, the molecular weight of the PSA determines the negative charge provided to the surface of each nanoparticle during adsorption between the PSA and the cationic zein. For a 50-80kDa PSA, several PSA molecules may be sufficient to reverse the zeta potential of the nanoparticle. Whereas for a PSA of <50kDa, the same adsorption occurs just near the isoelectric point, which reduces electrostatic repulsion, resulting in agglomeration of nanoparticles. With respect to PSA of >80kDa, the possible reason is that the larger PSA size results in adsorption of a single PSA molecule to the surface of two or more nanoparticles, leading to bridging flocculation.
Example 2: investigation of the concentration of the PSA solution
1. Preparation of nanoparticles
PSA-zein nanoparticles were prepared using PSA solutions of different concentrations, respectively, as follows:
zein (100 mg) was dissolved in 5.0mL ethanol/water (85:15, v/v). After stirring at 1000rpm for 1 hour, the above mixed solution was adjusted to pH 5.7 using 1mol/L HCl and magnetically stirred (1000 rpm) for 30min while being added dropwise to 20.0mL of a 0.05% Tween 80 solution (adjusted to pH 4.0). The remaining ethanol was then removed by rotary evaporator at 37 ℃.
For the method of coating zein nanoparticles with PSA, the resulting zein nanoparticle dispersion (5.0 mL) was injected into 5.0mL of PSA solution (adjusted to pH 4.0) and stirring (1000 rpm) was continued for 1 hour. The prepared sample was centrifuged at 12000rpm to remove impurities. The resulting sample was zein/PSA core-shell nanoparticles and was referred to as PSA-zein.
The concentrations of the PSA solutions were 0.05%, 0.075%, 0.1%, 0.25%, 0.5%, 1%, 2.5%, 5%, 7.5%, and 10%, respectively, and the molecular weights of the PSAs used in the respective PSA solutions were 80 kDa.
The size, structure and morphology of the nanoparticles were confirmed by transmission electron microscopy (TEM, hitachi HT-7700, japan).
2. Experimental results
Experimental results as shown in fig. 2, when the PSA concentration of less than 0.1% is used, the nanoparticles will rapidly precipitate and form large agglomerates, and when the PSA concentration is between 0.1% and 1%, nanoparticles (100-200 nm) having good dispersibility and suitable particle size can be obtained, and the particle size slightly increases as the PSA concentration increases. However, when the PSA concentration is further increased to more than 1%, the nanoparticle size increases sharply to 1000nm or more. Because the nano particles with small particle size (100-200 nm) are smaller than the aperture of vascular leakage and have poor lymphatic return, ideal treatment conditions (Ossipov, D.A. nanostructured hyaluronic acid-based materials for active delivery to cancer. Expert Opinion on Drug Delivery,2010,7 (6): 681-703) can be provided for the aggregation and positioning of the nano particles at the tumor site of a patient.
Example 3: preparation and characterization of HNK-loaded zein/PSA nanoparticles
1. Preparation of nanoparticles
Zein (100 mg) and HNK (10 mg) were dissolved in 5.0mL ethanol/water (85:15, v/v). After stirring at 1000rpm for 1 hour, the above mixed solution was adjusted to pH 5.7 using 1mol/L HCl and magnetically stirred (1000 rpm) for 30min while being added dropwise to 20.0mL of a 0.05% Tween 80 solution (adjusted to pH 4.0). The remaining ethanol was then removed by rotary evaporator at 37 ℃.
For the method of coating zein-HNK nanoparticles with PSA, the resulting zein-HNK nanoparticle dispersion (5.0 mL) was injected into 5.0mL of 0.1% PSA (PSA molecular weight 80 kDa) (adjusted to pH 4.0) solution and stirring (1000 rpm) was continued for 1 hour. Freshly prepared samples were centrifuged at 12000rpm to remove unencapsulated HNK. The resulting sample was HNK-loaded Zein/PSA core-shell nanoparticles and was referred to as PSA-Zein-HNK (abbreviated PSA-Zein-HNK in the figures).
To prepare nanoparticles loaded with Cou6 or DiR, PSA-zein-Cou and PSA-zein-DiR were prepared in the same manner as described above for PSA-zein-HNK, substituting only Cou6 or DiR for HNK.
2. Characterization of nanoparticles
The size, structure and morphology of the nanoparticles were confirmed by transmission electron microscopy (TEM, hitachi HT-7700, japan). One drop of this diluted nanoparticle solution was placed on a copper-loaded mesh, followed by negative staining with 2% uranyl acetate and drying at room temperature. The image was then observed under TEM at 100 kV. The average particle size, polydispersity index (PDI) and zeta potential values of the different nanoparticles were analyzed by Nicomp 380ZLS Particle Sizing System (PSS, USA). The drug encapsulation efficiency of PSA-zein-HNK was determined by HPLC system. The formula of EE is as follows: ee= (weight of HNK loaded/weight of HNK fed) ×100%.
The in vitro release profile of the different HNK nanoparticles was determined using a dynamic dialysis method in a release medium consisting of PBS (pH 7.4) with 0.2% tween 80. In total, 1mL of each formulation was added to a dialysis bag (mwco=12000-14000 Da), followed by shaking at 37 ℃ at a rate level of 100rpm, dialyzing against 30mL of release medium. At designated time intervals, a 1mL volume of release medium was collected and an equal volume of fresh medium was replenished accordingly. The HNK content in the external medium was determined using an HPLC system over 48 hours.
3. Experimental results
The particle size distribution obtained by dynamic light scattering detection showed that PSA-zein-HNK had a narrow particle size distribution with an average size of 107.2±10.1nm and a pdi of 0.23±0.05 (fig. 3A). In contrast, the average size of the unmodified zein-HNK dispersion was 91.4±6.1nm. The average particle size of PSA-zein-HNK was slightly larger than the average size of unmodified zein-HNK, indicating that PSA was successfully incorporated into the surface of zein nanoparticles. As shown in FIG. 3B, the TEM image of PSA-zein-HNK was clearly spherical with an average size of 103.5nm (the particle size measured by TEM was within a reasonable error range from the particle size measured by a nanoparticle sizer), consistent with the experimental results observed by DLS.
The zeta potential of zein-HNK was measured to be 18.2±1.2mV. Due to the carboxyl groups present in the chemical structure of PSA, the zeta potential of PSA-zein-HNK drops dramatically to-33.5±3.1mV after modification with PSA. The relatively high zeta potential is a key factor in improving the stability of nanoparticles in aqueous media by electrostatic repulsion. In general, negatively charged nanoparticles may exhibit improved colloidal stability, increased blood circulation time, and reduced toxicity to normal cells compared to positively charged nanoparticles (Liang, H.S., huang, Q.R., zhou, B., he, L., lin, L.F., an, Y.P., li, Y., liu, S.L., chen, Y.J., li, B.self-assembled zein-sodium carboxymethyl cellulose nanoparticles as An effective drug carrier and transporter J.Mater. Chem. B2015,3 (16), 3242-3253, DOI:10.1039/c4tb01920 b).
The above results, including size increase and charge reversal, confirm successful modification of PSA on the surface of zein nanoparticles. HNK loading and encapsulation efficiency in PSA-zein-HNK were 8.6% and 86.3%, respectively, as determined by HPLC. HNK release curves for zein-HNK and PSA-zein-HNK were studied. As shown in fig. 3C, zein-HNK and PSA-zein-HNK showed sustained HNK release in PBS (pH 7.4) containing 0.2% tween 80 for 48 hours, indicating a potential decrease in dosing frequency. Furthermore, PSA modification did not result in a significant difference between the release profile between zein-HNK and PSA-zein-HNK. After 12 hours, both nanoparticles released only-50% of HNK, whereas HNK increased to about 65% within 24 hours. Near 75% of total HNK was released at 48h, which confirms that PSA-zein-HNK has stable load and transport properties before reaching and accumulating at the tumor site.
Example 4: in vitro cellular uptake
1. Experimental method
(1) To demonstrate that PSA-zein nanoparticles enhance cellular internalization through ligand-receptor recognition, 4T1 breast cancer cells overexpressing selectin (selectin) were utilized and studied using flow cytometry. 4T1 cells were seeded in 6-well plates (1X 10) 5 Individual cells/well) and cultured overnight. Then, 100ng/mL of PSA-Zein nanoparticle loaded with Cou (PSA-Zein-Cou, prepared in example 3, abbreviated as PSA-Zein-Cou6 in the drawings) was added and the cells were incubated at 37 ℃. After 1 hour, the cells were washed with cold PBS and treated with trypsin. The cells were then centrifuged and re-dispersed in 0.5mL PBS buffer.
The non-targeted Zein-Cou group and the free Cou group are arranged, the PSA-Zein-Cou6 in the experimental conditions is replaced by Zein-Cou6 (called Zein-Cou6 for short in the drawing) and the free Cou6 respectively, and other experimental conditions are the same.
psa+psa-zein-Cou group: 4T1 cells were first incubated with 2.0mg/mL PSA for 1 hour, then with PSA-zein-Cou6 for an additional 1 hour, with the other experimental conditions being identical.
A control group was set, which was treated without any additional reagents, and the other experimental conditions were the same.
(2) For the selective receptor competitive inhibition assay, the experimental conditions of each experimental group were as described in section (1), and then the average Cou fluorescence intensity of the cells was directly tested by FACScan flow cytometer (BD FACSCalibur, USA).
(3) For confocal laser scanning microscopy studies, 4T1 cells were seeded in glass bottom dishes and allowed to adhere overnight. After 1 hour incubation with 100ng/mL PSA-zein nanoparticles, the cells were washed three times with cold PBS. Subsequently, the cells were fixed with 4% paraformaldehyde for 20min, followed by re-staining the nuclei with 5. Mu.g/ml Hoechst 33258 for 20min. Finally, the treated cells were observed using an LSM710 laser confocal microscope (Zeiss, germany).
Non-targeted zein-Cou, free Cou, psa+psa-zein-Cou 6 and control groups were set up, and the experimental conditions of each experimental group were as described in section (1).
2. Experimental results
To determine the targeting effect of PSA and selectin receptors in enhanced uptake, the inventors first compared the tumor targeting ability of different Cou loaded zein nanoparticles. Cellular uptake of the different nanoparticles was quantitatively determined by flow cytometry. Flow cytometry data showed that the order of fluorescence intensity was control < zein-Cou 6< psa+psa-zein-Cou 6< PSA-zein-Cou 6< free Cou (fig. 4A and B). The cellular uptake levels in the PSA-zein-Cou group were approximately doubled compared to the non-targeted zein-Cou group. Indicating that the introduction of PSA increased uptake of PSA-zein-Cou 6 more than electroadsorption. In addition, competitive inhibition assays were performed to verify PSA-selectin receptor-mediated internalization of PSA-zein-Cou 6. The cellular uptake of PSA-zein-Cou 6 was significantly reduced after blocking the selectin receptor by PSA compared to the control group. These findings provide clear support for PSA-zein nanoparticle internalization into 4T1 cells via PSA-selectin receptor mediated pathways.
The captured image collected by confocal laser scanning microscopy was consistent with quantitative flow cytometry determination (fig. 4C), with a significant increase in green fluorescence in the PSA-modified zein nanoparticle group, but a significant decrease in Cou6 internalization in the zein nanoparticle group. However, PSA pretreatment (psa+psa-zein-Cou 6) group green fluorescence intensity enhancement, indicating the feasibility and potential of PSA modification for targeted drug delivery.
Example 3: in vitro cytotoxicity (SRB) assay
1. Experimental method
Cell viability of the various HNK preparations on 4T1 was determined by SRB colorimetry. Briefly, 4T1 cells were plated at 2X 10 3 The density of individual cells/wells was seeded in 96-well plates and cultured overnight. zein-HNK, PSA-zein-HNK (prepared in example 3) and free HNK solutions with a range of HNK concentrations (0.3-20 μg/mL) were prepared and incubated with 4T1 cells for 48h. After further culturing for 48 hours, the cells were fixed with 10% cold trichloroacetic acid, air-dried, and stained with 0.4% SRB dye, and then 150. Mu.L of Tris base solution (10 mM) was added to dissolve the SRB dye. Finally, the absorbance of the final solution representing cell viability was measured with a microplate reader (Tecan Safire2, switzerland) at 540 nm.
2. Experimental results
As shown in fig. 5, all HNK preparations inhibited cell proliferation in a concentration-dependent manner. Of all groups, free HNK has the strongest antiproliferative effect on 4T1 cells, indicating that more HNK is rapidly transferred into cells by passive diffusion. As expected, PSA-zein-HNK exhibited much higher cytotoxicity than zein-HNK, possibly due to the increased cellular uptake facilitated by PSA modification. IC of PSA-zein-HNK (4.37. Mu.g/mL) at 48h 50 The values were very close to free HNK (3.99. Mu.g/mL) and significantly higher than zein-HNK (7.74. Mu.g/mL). These results strongly demonstrate the increase in cytotoxicity of HNK mediated by PSA-selectin targeted delivery.
Example 4: in vitro cell migration and invasion inhibition assay
1. Experimental method
The effect on 4T1 cell migration was analyzed using a simple 2D scratch wound healing scratch test (Liang, C.C.; park, A.Y.; guan, J.L.In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat. Protoc.2007,2 (2), 329-333, DOI: 10.1038/nprot.2007.30). 4T1 cells were cultured overnight in 6-well plates until confluent monolayers were formed. The scratches were then introduced into the confluent monolayers with a sterile pipette tip and washed with PBS to remove floating cells. The cells were then incubated with zein-HNK, PSA-zein-HNK (prepared in example 3) and free HNK (5. Mu.g/mL HNK) in fresh serum-free RPMI-1640. The distance between the two wound edges was monitored 0 and 24 hours after treatment using an inverted microscope (Zeiss, axio Observer A1). Wound healing rates were calculated as described (Zhang, z.w.; cao, h.q.; jiang, s.j.; liu, z.y.; he, x.y.; yu, h.j.; li, y.p. nanoassembly of Probucol Enables Novel Therapeutic Efficacy in the Suppression of Lung Metastasis of Breast cancer.small 2014,10 (22), 4735-4745, doi: 10.1002/smll.201400799).
The transwell method was performed using a 24-well transwell plate (8 μm well, costar, USA) to further measure cell migration and invasion. For the cell migration assay, 4T1 cells were incubated with different HNK solutions at a concentration of 5.0. Mu.g/mL HNK for 24h. Then 100. Mu.L of the pretreated 5X 10 medium in RPMI 1640 medium without FBS 4 Individual 4T1 cells were seeded into the top chamber of a 24-well transwell and 600 μl of 10% fbs-containing medium was placed in the bottom chamber. After 24 hours, the cells migrating to the lower membrane were fixed with 4% paraformaldehyde and stained with 0.4% crystal violet for 30 minutes, then photographed under an inverted microscope. Subsequently, the cell-bound crystal violet was dissolved with 33% acetic acid solution for quantitative analysis. The optical density at 570nm was recorded with a microplate reader.
For invasive tests, will contain 8X 10 4 100. Mu.L of serum-free medium of pretreated 4T1 cells was inoculated into a top chamber pre-coated with diluted Matrigel (20. Mu.g/. Mu.L; cornig, USA) and incubated for 36 hours. The following processing steps correspond to the method described above.
2. Experimental results
Increased motility is a marker of metastatic cells. Wound healing scratch tests were used to assess cell motility. All HNK formulations inhibited the healing of scratches to some extent after 24 hours compared to the control. PSA-zein-HNK showed a stronger inhibition, with a wound healing rate of 13.33%, whereas zein-HNK group increased the wound healing rate to 31.11%. Free HNK had the strongest effect of inhibiting scratch healing with a wound healing rate of 6.67% (fig. 6A and B). In addition, the inventors analyzed in vitro cell migration of 4T1 cells on a Transwell model. Similar inhibition of PSA-zein-HNK treated cells was observed. As shown in fig. 6A and C, the number of migrating cells was significantly lower in the PSA-zein-HNK group than in the zein-HNK group. The cell mobility of PSA-zein-HNK was only 20.05% compared to the control, whereas that of zein-HNK was 54.5%.
Next, the inventors evaluated the effect of PSA-zein-HNK on 4T1 cell invasion in a Transwell matrigel assay. Cell invasion is a key biological process for tumor metastasis, providing a prerequisite for initiation of tumor metastasis (Wang, j.; liu, d.; guan, s.; zhu, w.q.; fan, l.; zhang, q.; cai, d.f. hyaluronic acid-modified liposomal honokiol nanocarrier: enhanced anti-metastasis and antitumor efficacy against breast cancer.Carbohydr.Polym.2020,235, DOI:10.1016/j. As shown in fig. 6A and D, the 4T1 cell invasion of PSA-zein-HNK treated groups was significantly lower than that of zein-HNK groups. Cell invasion rates of zein-HNK, PSA-zein-HNK and free HNK were 35.81%, 16.59% and 14.79%, respectively. These results demonstrate that PSA-zein-HNK has 2.15 times greater resistance to attack than zein-HNK. Based on the above results, HNK treatment can inhibit migration and invasion of 4T1 cells, and targeting PSA-zein-HNK inhibits higher levels of migration and invasion of cells than zein-HNK. Thus, the inventors speculate that PSA-zein-HNK may have excellent inhibitory effects on primary metastasis of tumors.
Example 5: in vitro cytotoxicity to tumor spheroids
1. Experimental method
The antitumor activity of the different HNK loaded formulations was further evaluated on 3D tumor spheres (tumor spheres). Suspension drop support was used as described previously (Del Duca, D.; werbowetski, T.; del Maestro, R.F. Sphereid preparation from hanging drops: characterization of a model of brain tumor division. J. Neuroncol. 2004,67 (3), 295-303, DOI:10.1023/b: neon.00000244220.07063.70)3D cell culture and spheroid formation. First, agarose (2%, w/v) was dissolved in serum-free RPMI-1640 medium and coated in each well of a 48-well plate to prevent cell adhesion. Next, 4T1 cell suspension (1X 10 3 20 μl) of droplets were suspended from the lid of the 48-well plate. After 48 hours, the formed tumor spheres were placed in each well, each well containing 0.9mL of medium. When the tumor sphere size was about 100 μm, a uniform and dense sphere was used for the subsequent procedure. Tumor spheres were incubated with free HNK, zein-HNK and PSA-zein-HNK (prepared in example 3) (10. Mu.g/mL HNK), respectively. At defined time points (1, 3, 5 and 7 days), tumor sphere growth inhibition was monitored and images were taken using an inverted microscope (Zeiss, axio o server A1).
2. Experimental results
As shown in fig. 7A and B, zein-HNK, PSA-zein-HNK, and free HNK were more toxic to 4T1 tumor spheres than the control, showing a 13.98-fold increase in volume. Free HNK shows the strongest cytotoxic effect on tumor spheres due to the diffusion effect. In addition, the sphere volume in the PSA-zein-HNK and zein-HNK groups was reduced to 23.18% and 44.23%, respectively. Thus, PSA-zein-HNK was shown to be even more cytotoxic than zein-HNK. Increased cytotoxicity of PSA-zein-HNK is thought to be associated with PSA modification, which enhances nanoparticle penetration and accumulation into tumor spheres.
Example 6: in vivo tumor targeting assessment
1. Experimental method
By mixing 1X 10 6 A4T 1 cell suspension (100. Mu.L) was subcutaneously injected into the right armpit of BALB/c mice to establish a subcutaneous 4T1 tumor model. The tumor volume reaches 300-400mm 3 After that, zein-DiR and PSA-Zein-DiR (prepared in example 3, abbreviated as Zein-DiR, PSA-Zein-DiR, respectively in the figures) were injected into subcutaneous tumor-bearing mice via the tail vein at a dose of 500 μg/kg DiR. At predetermined time points, anesthetized mice are captured by an in vivo imaging system (IVIS Lumina Series III, perkinElmer, USA) Fluorescent image. Finally, in vitro images of the dissected tumor and major organs were taken at 24 hours.
2. Experimental method
As shown in fig. 8A, zein-DiR accumulation in the tumor decreased rapidly and was significantly attenuated at 48 hours. In contrast, PSA-zein-DiR showed strong fluorescence in tumors from 4 hours up to 48 hours. Clearly, PSA-zein-DiR showed much higher tumor accumulation than non-targeted zein-DiR, confirming that PSA modification confers tumor targeting effects on zein nanoparticles and extends their accumulation time in tumors. Finally, tumors and major organs were obtained and then assessed by ex vivo fluorescence imaging. Ex vivo images showed that PSA modification resulted in reduced distribution of zein nanoparticles in the lung and enhanced tumor accumulation (fig. 8B). Taken together, these in vivo data indicate that improved tumor accumulation of such developed PSA-zein nanoparticles would provide a substantial and long lasting effect of anti-tumor agents (e.g., HNK).
Example 7: in vivo anti-tumor study on 4T1 breast cancer model
Subcutaneous lotus 4T1 breast cancer model was established as described above when tumor volume reached 100mm 3 At this time, it was randomly divided into four groups (n=6). These different components were injected intravenously with physiological saline, free HNK, zein-HNK and PSA-zein-HNK (prepared in example 3) at a dose of 15mg/kg HNK, once every two days, 4 times total. Tumor volume and mouse body weight were measured every other day. After 24 days of treatment, tumors and major organs were collected. Tumors were weighed and imaged to compare tumor growth inhibition. Lungs were fixed with 4% paraformaldehyde for 48 hours, and macroscopic metastasis nodules were counted to assess the inhibition of cancer metastasis by different HNK formulations. Major organs and tumors were fixed with 4% paraformaldehyde followed by H&E (hematoxylin and eosin) staining was used for further histological analysis. To further study the apoptosis of tumor cells histologically, tumor samples were also subjected to immunofluorescent staining with the terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end marker (TUNEL).
2. Experimental results
In vivo anti-tumor effects of zein-HNK, PSA-zein-HNK and free HNK were tested in BALB/c mice bearing 4T1 breast cancer. The antitumor effect was demonstrated by the relative tumor volume, tumor weight and tumor image during and after treatment with different HNK formulations. As shown in fig. 9A, 4T1 breast cancer cells in the saline group grew rapidly during the experiment, while different HNK formulations significantly inhibited tumor growth. At the end of the experimental period, the average tumor volume of the normal saline-treated control group was 1181.9.+ -. 115.7mm 3 Free HNK of 965.9 + -88.3 mm 3 zein-HNK is 760.6 + -65.6 mm 3 PSA-zein-HNK treatment group was 619.1.+ -. 62.9mm 3 . This indicates that it is absolutely beneficial to encapsulate HNK in zein nanoparticles compared to administration of free HNK. More importantly, PSA-zein-HNK showed better inhibition of tumors than zein-HNK and free HNK treatments, reflecting the advantages of PSA modification for targeted drug delivery. Similar results were observed in tumor weights (fig. 9B) and images (fig. 9C) after treatment with different HNK formulations. All measurements prove that the PSA-zein-HNK has the highest in vivo anti-tumor efficiency, which is higher than that of zein-HNK, and the zein-HNK has higher in vivo anti-tumor efficiency than that of free HNK and normal saline. Taken together, these results further confirm the specific and optimal antitumor effects of the proposed PSA-zein-HNK.
To further investigate the antitumor effect, histological and immunohistochemical staining was performed to assess changes in major organs and tumors. As shown in fig. 9D, H & E stained images of saline tumors filled with tightly packed tumor cells and had intact morphology, whereas tumor necrosis was detected in PSA-zein-HNK group, with obvious nuclear fragmentation and aggregation. TUNEL staining assay showed that PSA-zein-HNK treatment produced the greatest number of apoptotic cells in tumor tissue compared to other formulation treatments (fig. 9E and F), consistent with the H & E results. All these excellent results of PSA-zein-HNK benefit from PSA modification, which results in higher cellular uptake efficiency, greater cytotoxicity and more drug accumulation at the tumor site.
To assess the possible systemic toxicity associated with different HNK formulation treatments in vivo, body weight changes and H & E sections of the major tissues were assessed. As shown in fig. 10A, zein-HNK, PSA-zein-HNK, and free HNK were well tolerated by mice because they did not show significant weight loss during treatment compared to the saline group. Histological examination of histological sections of the major organs also confirmed their in vivo biosafety, as there were no obvious pathological changes in mice treated with different HNK preparations (fig. 10C). These data demonstrate that PSA-modified zein core/shell nanoparticles for targeted delivery of HNK exhibit excellent biosafety and can be used as a safe formulation for anti-tumor therapy.
Metastasis usually occurs in patients with advanced breast cancer. Breast cancer is well known for lung metastasis, resulting in rapid patient death (Liu, m.t.; ma, w.j.; zhao, d.; li, j.j.; li, q.r.; liu, y.h.; hao, l.y.; lin, y.f. enhanced Penetrability of a Tetrahedral Framework Nucleic Acid by Modification with iRGD for DOX-Targeted Delivery to Triple-Negative Breast cancer acs appl. Mate. Interfaces 2021,13 (22), 25825-25835, doi:10.1021/acsami.1c 07297). Inspired by these excellent antitumor efficacy, the inventors further examined the anti-metastatic efficacy of PSA-zein-HNK nanoparticles. An image of the lung is taken and at the end of the process, a metastatic nodule is calculated. As shown in fig. 10B, a number of metastatic nodules were detected in the normal saline group, indicating that the breast tumor had metastasized to the lung. However, a significant reduction in the number of pulmonary metastatic nodules was observed in mice treated with different HNK formulations. Free HNK and zein-HNK treatment showed moderate inhibition of lung metastasis. While few lung nodules were found in PSA-zein-HNK treatment, indicating that PSA-zein-HNK is superior to all other HNK formulations in inhibiting breast cancer metastasis. Similar to the tendency of metastatic nodules on the lung surface, H & E staining images of lung and liver sections in the saline group showed many tumor cell infiltrates (with large nuclei). Whereas the tumor cell infiltration of the free HNK and zein-HNK treated groups was significantly less than that of the saline group. More importantly, the metastatic lesions were controlled to a minimum by PSA-zein-HNK without tumor cell infiltration. These results indicate that PSA-zein-HNK is not only effective in inhibiting the growth of primary tumors, but also in inhibiting the formation of tumor metastases.
In summary, the inventors have successfully prepared a PSA-modified zein nanoparticle for targeted delivery of HNK using anti-solvent precipitation and electrostatic deposition techniques for the first time. PSA modifications stabilize zein nanoparticles and confer them active targeting properties in breast cancer treatment. PSA-zein-HNK is a biosafety and biocompatible nano-drug delivery system exhibiting specific binding and selective toxicity to selectin-positive breast cancer cells. PSA-zein-HNK nanoparticles showed desirable tumor inhibition efficiency after intravenous injection and did not cause significant systemic toxicity throughout the treatment period, probably due to increased nanoparticle accumulation at the tumor site. PSA modification plays an important role in improving HNK accumulation at tumor sites. More importantly, PSA-zein-HNK also showed greater inhibition of breast cancer lung metastasis in vitro and in vivo models, further contributing to improved breast cancer treatment. This work provides a promising nano-platform for breast cancer treatment with enhanced drug delivery efficiency that can simultaneously inhibit invasive growth of primary tumors and metastases.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is to be construed as including any modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
The foregoing embodiments and methods described in this invention may vary based on the capabilities, experience, and preferences of those skilled in the art.
The listing of the steps of a method in a certain order in the present invention does not constitute any limitation on the order of the steps of the method.

Claims (11)

1. Use of a drug delivery system for the preparation of a pharmaceutical composition for the treatment of breast cancer, said drug delivery system being a nanoparticle having a core particle and a coating layer coating the outer surface of said core particle, wherein said core particle comprises zein, said coating layer comprises PSA, and said core particle is loaded with honokiol, said PSA has an average molecular weight of 50000-80000Da, and said nanoparticle has a particle size of 100-200nm;
the pharmaceutical composition comprises the drug carrying system and one or more pharmaceutically acceptable auxiliary materials;
the treatment includes inhibiting the growth of breast cancer and inhibiting the metastasis of breast cancer;
The preparation method of the drug-carrying system comprises the following steps:
(1) Forming core particles: dissolving zein and active ingredients in ethanol water solution, adjusting pH to acidity, and removing ethanol;
(2) Forming a coating layer: adding the dispersion of core particles obtained in step (1) to a PSA solution; the concentration of the PSA solution is 0.1-1%.
2. The use according to claim 1, wherein the PSA has an average molecular weight of 80000Da.
3. The use according to claim 1, wherein the nanoparticle has a particle size of 100-120nm.
4. The use according to claim 1, wherein the thickness of the coating is 1-100nm.
5. The use according to claim 4, wherein the coating has a thickness of 10-30nm.
6. The use of claim 1, wherein the nanoparticle is negatively charged.
7. The use according to claim 1, wherein the weight ratio of zein to honokiol in the core particles is from 1:1 to 100:1.
8. The use according to claim 1, wherein the pH is adjusted to be acidic to a pH of 4.5-6.5.
9. The use according to claim 1, wherein the PSA solution is acidic in pH.
10. The use according to claim 1, wherein step (1) further comprises the step of adding a surfactant solution after adjusting the pH to acidic.
11. The use according to claim 1, wherein the concentration of the PSA solution is 0.1 to 0.25%.
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