CN114099469A

CN114099469A - Composite nano-drug carrier and preparation method and application thereof

Info

Publication number: CN114099469A
Application number: CN202111472902.1A
Authority: CN
Inventors: 蔡德富; 张琪; 王婧; 刘丹
Original assignee: Qiqihar Medical University
Current assignee: Qiqihar Medical University
Priority date: 2021-12-01
Filing date: 2021-12-01
Publication date: 2022-03-01
Anticipated expiration: 2041-12-01
Also published as: CN114099469B

Abstract

The invention discloses a composite nano-drug carrier and a preparation method and application thereof, in particular to a polysialic acid (PSA) modified zein nano-drug carrier and a drug loading system thereof, in particular to a tumor targeted 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 enhanced tumor accumulation of PSA-zein-HNK in a mouse model carrying 4T1 breast cancer, resulting in ideal anti-tumor efficacy and favorable biosafety, and PSA-zein-HNK significantly inhibits metastasis of breast cancer to the lung and liver. Therefore, the nano-particles are expected to become an effective tumor-targeted 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 medicines, in particular to a composite nano-drug carrier and a preparation method and application thereof, and particularly relates to a polysialic acid (PSA) modified zein nano-drug carrier and a drug carrying system thereof, particularly a tumor targeted drug delivery system, and preparation methods and applications thereof.

Background

With the development of socioeconomic and improvement of the living standard of people, the disease spectrum and death spectrum of people worldwide change remarkably due to the influence of dietary structure change, population aging, urbanization and other factors, and chronic non-infectious diseases become the main cause of death. Among them, malignant tumor is one of the main causes of death worldwide, and has become a large group of diseases which seriously harm human life and health and restrict social and economic development.

Chemotherapy is currently the most commonly used means in tumor therapy, and depends largely on whether the chemotherapeutic agent administered can safely and effectively reach the tumor site, however, many of these chemotherapeutic agents often result in poor efficacy and severe side effects due to their low concentration at the tumor site and similar cytotoxicity to both cancer and healthy cells.

One of the methods to solve these problems is to introduce chemotherapeutic agents into nano-drug carriers, the resulting drug-loaded systems can release the drug continuously, improve the pharmacokinetic profile, and increase tumor accumulation through permeability enhancement and retention (EPR). However, the efficacy of drugs is still limited by drug carriers such as leakage of chemotherapeutic agents, uptake of chemotherapeutic agents by RES organs, insufficient penetration and accumulation of nano-drugs in tumor sites by EPR effect. Therefore, new drug carriers are urgently needed to solve these problems.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a PSA modified zein nano drug carrier and a drug loading system thereof, in particular to a tumor targeted drug delivery system, and preparation methods and applications of the zein nano drug carrier and the drug loading system.

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 cladding layer is formed from PSA.

Specifically, the average molecular weight of PSA is 5000-; in some embodiments of the invention, the PSA has an average molecular weight of 80000 Da.

Specifically, the particle size of the nanoparticle can be 100-200nm (e.g., 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200nm), especially 100-120 nm.

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, 100nm), in particular 10-30 nm.

In particular, the core particle 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, 100nm), in particular 85-95 nm.

In particular, the nanoparticles are spherical.

In particular, the nanoparticles are negatively charged.

In a second aspect of the present invention, there is provided a method for preparing nanoparticles described in the first aspect, comprising the steps of:

(1) forming a core particle;

(2) forming a coating layer;

optionally, (3) removing impurities.

Specifically, the core particles are formed by an anti-solvent precipitation process.

More specifically, step (1) includes: dissolving zein in ethanol water solution, adjusting pH to acidity, and removing ethanol.

Specifically, 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 reagent used to adjust the pH to acidity is a solution of a mineral acid, such as hydrochloric acid.

Specifically, the step (1) further comprises a stirring step; more specifically, the stirring speed may be 500-.

Specifically, step (1) may further include a step of adding a surfactant solution after adjusting the pH to acidity.

Specifically, the surfactant may be a nonionic surfactant, such as a tween, particularly tween 80.

Specifically, the pH of the above 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, the step (2) includes: adding the dispersion of the core particles obtained in the step (1) into a PSA solution.

In particular, the pH of the PSA solution is acidic, for example, pH 2-6 (e.g., 2, 3, 4, 5, 6), pH 4.

Specifically, the concentration of the 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 the PSA solution is 0.1%.

In one embodiment of the invention, the concentration of the PSA solution is 0.1%, wherein the PSA has an average molecular weight of 80000 Da.

Specifically, the step (2) further comprises a stirring step; more specifically, the stirring speed may be 500-.

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 the nanoparticles of the first aspect as a carrier for a drug and in the manufacture of a medicament.

In a fourth aspect of the invention, a drug delivery system is provided, which has sustained release and targeting properties, and is a nanoparticle having a core particle and a coating layer coated on 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 cladding layer is formed from PSA.

Specifically, the average molecular weight of PSA is 5000-; preferably, the PSA has an average molecular weight of 80000 Da.

In particular, the nanoparticles are spherical.

In particular, the nanoparticles are negatively charged.

In one embodiment of the present invention, the above active ingredient is a pharmaceutically active ingredient, particularly an antitumor drug, for example, doxorubicin, epirubicin, pirarubicin, idarubicin; mitoxantrone; topotecan, irinotecan, aminocamptothecin; paclitaxel, docetaxel; gefitinib, imatinib, nilotinib, sunitinib, lapatinib, tofacitinib, crizotinib, masitinib, emtricitinib, ibrutinib, afatinib, flumatinib, erlotinib, lenatinib, erlotinib, apatinib, Talazoparib, loralatinib, TPX-0005; cisplatin, carboplatin, nedaplatin, cycloplatin, oxaliplatin, lobaplatin; vinblastine, vincristine, vinorelbine, berberine, berbamine; honokiol (HNK); uracil mustard, nitrogen mustard, ifosfamide, melphalan, chlorambucil, pipobroman, tritylamine, triethylenethiophosphoramide, busulfan, carmustine, lomustine, streptozocin, dacarbazine, fluorouracil deoxynucleoside, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate; and so on.

Specifically, in the core particle, the weight ratio of zein to active ingredient may be 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 5:1 to 20: 1.

In one embodiment of the invention, the active ingredient is HNK; more specifically, in the core particle, the weight ratio of zein to HNK is 10: 1.

In a fifth aspect of the present invention, there is provided a method for preparing a drug-loaded system according to the fourth aspect, which comprises the following steps:

(1) forming a core particle;

(2) forming a coating layer;

optionally, (3) removing impurities.

More specifically, step (1) includes: dissolving zein and active ingredients (separately or together) in ethanol water solution, adjusting pH to acidity, and removing ethanol.

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, the concentration of the 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 the PSA solution is 0.1%.

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 invention, there is provided a use of the drug delivery system of the fourth aspect in the preparation of a medicament for the prevention and/or treatment of a disease.

Specifically, the disease may be a tumor, an autoimmune disease, an infectious disease, or the like, particularly a tumor.

Specifically, the above-mentioned tumor is a malignant tumor, which includes but is 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, gallbladder cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, prostate cancer, skin cancer, B-cell chronic lymphocytic leukemia, acute lymphocytic leukemia, non-hodgkin lymphoma, acute myelogenous leukemia, diffuse large B-cell lymphoma, multiple myeloma, and the like (including primary tumors and metastatic tumors); in particular, breast cancer, including primary breast cancer and metastatic breast cancer.

In a seventh aspect of the invention, a pharmaceutical composition is provided, which comprises the drug delivery system of the fourth aspect, and one or more pharmaceutically acceptable excipients.

In particular, the pharmaceutically acceptable carrier refers to a pharmaceutical carrier conventional in the pharmaceutical field, in particular to a pharmaceutically acceptable adjuvant for injection, 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 dried, for example, freeze-dried composition, which is suitably formed into an injectable solute by adding sterile water or physiological saline.

Specifically, the pharmaceutical composition may be administered by any suitable route of administration, such as gastrointestinal or parenteral (e.g., intravenous, intramuscular, subcutaneous, intraorgan, intranasal, intradermal, instillation, intracerebral, intrarectal, etc.) route; the above drugs may be in any suitable form, such as a form for administration via the gastrointestinal tract, including, for example, but not limited to, tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, suspensions, and the like; parenteral administration, for example, injection administration: such as injections (e.g., for subcutaneous, intravenous, intramuscular, intraperitoneal), respiratory administration: such as sprays, aerosols, powders, etc., dermal administration forms, such as topical solutions, lotions, ointments, plasters, pastes, patches, etc., mucosal administration forms: such as eye drops, eye ointment, nose drops, gargle, sublingual tablet, etc., and the dosage form of cavity administration: such as suppository, aerosol, effervescent tablet, drop, dripping pill, etc., and can be used for rectum, vagina, urethra, nasal cavity, auditory canal, etc. Preferably, the pharmaceutical composition is an injection.

Specifically, various dosage forms of the pharmaceutical composition can be prepared according to conventional production methods in the pharmaceutical field, for example, a drug-loaded system is mixed with one or more pharmaceutically acceptable auxiliary materials and then the mixture is prepared into a required dosage form.

In an eighth aspect of the present invention, there is provided a method for preventing and/or treating a disease, comprising the step of administering the drug carrier of the fourth aspect or the pharmaceutical composition of the seventh aspect to a subject in need thereof.

In particular, the disease has the definition as defined in the third, sixth aspect of the invention, in particular a malignant tumour, such as breast cancer, including primary breast cancer and metastatic breast cancer.

In one embodiment of the invention, the method is a method of treating a malignant tumor, the therapeutic effect of which includes inhibiting not only the growth of the tumor but also tumor metastasis.

In particular, the subject may be a mammal, e.g. a human, monkey, chimpanzee, dog, mouse, rabbit, etc., in particular a human.

The invention develops polysialic acid (PSA) modified zein core/shell nanoparticles for the first time, which can be used for targeted delivery of drugs (especially anti-tumor drugs such as Honokiol (HNK)), and can realize enhanced drug delivery efficiency, active targeting capability and specific biological distribution at tumor sites, thereby inhibiting growth, migration and invasion of tumors. Taking HNK as an example, experiments demonstrate enhanced tumor accumulation of PSA-zein-HNK in a mouse model carrying 4T1 breast cancer, resulting in ideal anti-tumor efficacy and favorable biosafety, and PSA-zein-HNK significantly inhibits metastasis of breast cancer to the lung and liver. Therefore, the nano-particles prepared by the invention are expected to become an effective tumor-targeted 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 the characterization of PSA-Zein-HNK nanoparticles. (A) Particle size distribution, determined by the DLS method; (B) morphology of PSA-Zein-HNK, detected by TEM; scale bar, 500 nm; (C) HNK release profiles for 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 by SRB after incubation for hours with different HNK preparations, and three bars in each experimental group represent Zein-HNK, PSA-Zein-HNK, and free HNK, respectively. Each bar represents the mean ± SD (n ═ 6),. p <0.05, and. p < 0.01.

FIG. 6 shows the anti-metastatic effects of free HNK, Zein-HNK and PSA-Zein-HNK on 4T1 cells. (A) Representative microscope images and (B, C, D) quantitative analysis of preincubation of 4T1 cells 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 of 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 curves of tumor spheres after various HNK formulation treatments. Each point represents the mean ± SD (n ═ 3) ·, p < 0.01.

FIG. 8 shows the 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 the various formulations; (B) ex vivo fluorescence images of major organs and tumors at 48 hours.

Figure 9 shows the in vivo anti-tumor activity of various HNK formulations in a 4T1 tumor-bearing mouse model. (A) The change profile of tumor volume during treatment; (B) the weight of the tumor mass at the end of the treatment period; (C) images of tumor size observed after sacrifice; (D) h & E staining of tumor sections at the end of treatment period; (E) TUNEL fluorescent staining of apoptotic cells in tumor sections at the end of treatment period; (F) semi-quantitative results of TUNEL fluorescent staining. Data are shown as mean ± SD (n ═ 6), # p <0.05, # p < 0.01.

FIG. 10 shows in vivo anti-metastasis and biosafety studies of a 4T1 tumor-bearing mouse model. (A) Body weight change in 4T1 tumor-bearing mice during treatment; (B) lung metastatic nodule specimens treated with various HNK preparations, with red arrows indicating metastatic nodules; (C) h & E staining of major organ sections at the end of treatment period, green arrows indicate 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 pertains.

The disclosures of the various publications, patents, and published patent specifications cited herein are hereby incorporated by reference in their entirety.

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Some of the reagents, materials, experimental animals used in the experiments of the examples were as follows:

honokiol (HNK, > 98%) was purchased from milan biotechnology limited (gangrene, china). Zein (> 98%) and tween 80 were purchased from carbofuran technologies ltd (beijing, china). Polysialic acid (PSA) was produced by zhenjiang changxing pharmaceuticals limited (zhenjiang, china). Sulfonylrhodamine B sodium Salt (SRB), coumarin-6 (Cou6), Hoechst 33258 and matrigel are all provided by Sigma-Aldrich (Shanghai, China). The Annexin V-FITC/PI apoptosis detection kit is purchased from Biyuntian Biotechnology Ltd (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 obtained from Cell Signaling Technology (Danvers, USA). Rabbit polyclonal antibodies against E-cadherin and vimentin were purchased from Proteitech (Chicago, USA). All other chemicals were used as received unless otherwise stated.

The murine breast cancer cell line 4T1 was obtained from the institute of basic medicine of Chinese academy of medical sciences (Beijing, China) and contained 5% CO at 37 deg.C₂In a humidified atmosphere of (4) in RPMI-1640 medium (Mechen technologies, Inc., Beijing, China) supplemented with 10% fetal bovine serum (Vickers Biotech, Inc., Nanjing, China), 100U/mL penicillin and 100. mu.g/mL streptomycin.

Female BALB/c mice (4-6 weeks old) were purchased from Liaoning laboratory animal resources center (Benxi, China) and bred under specific pathogen-free and temperature-controlled conditions. All animal-related experiments were approved by the ethical committee of the zizaire medical college and fully comply with institutional guidelines for the care and use of laboratory animals.

Statistical analysis:

all experimental results in the examples are expressed as mean ± standard deviation. Statistical differences were determined using student's t-test. p <0.05 was considered significantly different, and p <0.01 was considered very significantly different.

Example 1: examination of molecular weight of PSA

1. Preparation of nanoparticles

PSA with different molecular weights is respectively used for preparing PSA-zein nanoparticles, and the specific steps are as follows:

zein (100mg) 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 stirred magnetically (1000rpm) 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 (5.0mL) was injected into 5.0mL PSA solution (adjusted to pH 4.0) with constant stirring (1000rpm) for 1 hour. The prepared sample was centrifuged at 12000rpm to remove impurities. The resulting sample was a zein/PSA core-shell nanoparticle and was referred to as PSA-zein.

The concentrations of the PSA solutions were all 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, Hitach HT-7700, Japan).

2. Results of the experiment

Experimental results as shown in figure 1, extensive aggregation of nanoparticles occurred when <50kDa and >80kDa PSA was used. Generally, the molecular weight of PSA determines the negative charge provided to each nanoparticle surface during adsorption between PSA and cationic zein. For 50-80kDa PSA, several PSA molecules may be sufficient to reverse the zeta potential of the nanoparticles. Whereas for PSA <50kDa the same adsorption occurs exactly close to the isoelectric point, which reduces electrostatic repulsion, leading to agglomeration of nanoparticles. With >80kDa PSA, it is possible that the larger PSA size leads to adsorption of a single PSA molecule onto the surface of two or more nanoparticles, leading to bridging flocculation.

Example 2: examination of PSA solution concentration

1. Preparation of nanoparticles

PSA solutions with different concentrations are respectively used for preparing the PSA-zein nano-particles, and the specific steps are as follows:

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 PSA solutions were 80k Da.

2. Results of the experiment

Experimental results as shown in fig. 2, when PSA concentration less than 0.1% was used, nanoparticles precipitated rapidly and formed large aggregates, and when PSA concentration was between 0.1% and 1%, nanoparticles (100 nm and 200nm) with good dispersibility and suitable particle size could be obtained, and particle size increased slightly with increasing PSA concentration. However, when the PSA concentration was further increased to more than 1%, the nanoparticle size sharply increased to 1000nm or more. Due to the small size (100-.

Example 3: preparation and characterization of HNK-loaded zein/PSA nanoparticles

1. Preparation of nanoparticles

Zein (100mg) and HNK (10mg) 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 stirred magnetically (1000rpm) 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 (5.0mL) was injected into 5.0mL of a 0.1% PSA (PSA molecular weight 80kDa) (adjusted to pH 4.0) solution with constant stirring (1000rpm) for 1 hour. Freshly prepared samples were centrifuged at 12000rpm to remove unencapsulated HNK. The resulting sample was an HNK-loaded Zein/PSA core-shell nanoparticle and was designated PSA-Zein-HNK (abbreviated PSA-Zein-HNK in the figure).

To prepare Cou6 or DiR loaded nanoparticles, PSA-zein-Cou6 and PSA-zein-DiR were prepared in the same manner as described above for PSA-zein-HNK, replacing HNK with Cou6 or DiR only.

2. Characterization of nanoparticles

The size, structure and morphology of the nanoparticles were confirmed by transmission electron microscopy (TEM, Hitach HT-7700, Japan). A drop of this diluted nanoparticle solution was placed on a copper grid, followed by negative staining with 2% uranyl acetate and drying at room temperature. The images were then observed under TEM at 100 kV. The different nanoparticles were analyzed for average Particle size, polydispersity index (PDI) and zeta potential values by Nicomp 380ZLS Particle Sizing System (PSS, USA). The drug encapsulation efficiency of PSA-zein-HNK was determined by HPLC system. EE is calculated as follows: EE ═ 100% (weight of HNK loaded/weight of HNK fed).

The in vitro release profiles of the different HNK nanoparticles were determined using a dynamic dialysis method in a release medium consisting of PBS with 0.2% tween 80 (pH 7.4). In total, 1mL of each formulation was added to a dialysis bag (MWCO 12000-14000Da) and subsequently dialyzed in 30mL of release medium at 37 ℃ with shaking at a rate level of 100 rpm. At designated time intervals, 1mL volumes of release medium were collected and supplemented with an equal volume of fresh medium accordingly. The HNK content in the external medium was determined using an HPLC system over 48 hours.

3. Results of the experiment

The particle size distribution obtained by dynamic light scattering detection showed that PSA-zein-HNK had a narrow particle size distribution with a mean size of 107.2 ± 10.1nm and a PDI of 0.23 ± 0.05 (fig. 3A). In contrast, the mean size of the unmodified zein-HNK dispersion was 91.4 ± 6.1 nm. The average particle size of the PSA-zein-HNK is slightly larger than the average size of the unmodified zein-HNK, which indicates that the PSA is successfully introduced into the surface of the zein nanoparticles. As shown in fig. 3B, the TEM image of PSA-zein-HNK is clearly spherical with an average size of 103.5nm (the particle size measured by TEM is within a reasonable error range from the particle size measured by the nano-particle sizer), consistent with the experimental results observed for DLS.

The zeta potential of zein-HNK was determined to be 18.2. + -. 1.2 mV. Due to the presence of carboxyl groups in the chemical structure of PSA, the zeta potential of PSA-zein-HNK drops sharply 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 through electrostatic repulsion. Generally, negatively charged nanoparticles may exhibit improved colloidal stability, increased blood circulation time, and reduced toxicity to normal cells as 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-embedded zero-sodium carbonate nanoparticles as effective conductor and transporter. J.Matter. chem.B 2015,3(16),3242-3253, DOI:10.1039/c4tb01920 b).

The above results, including size increase and charge reversal, demonstrate successful modification of PSA on the surface of zein nanoparticles. The HNK loading and encapsulation efficiency in PSA-zein-HNK was 8.6% and 86.3%, respectively, as determined by HPLC. HNK release profiles of zein-HNK and PSA-zein-HNK were studied. As shown in fig. 3C, zein-HNK and PSA-zein-HNK showed sustained HNK release pattern for 48 hours in PBS (pH 7.4) containing 0.2% tween 80, indicating a potential for decreasing dosing frequency. Furthermore, PSA modification did not result in a significant difference between the release profiles between zein-HNK and PSA-zein-HNK. After 12 hours, both nanoparticles released only-50% of the HNK, whereas the HNK increased to about 65% within 24 hours. Nearly 75% of the total HNK was released at 48h, confirming that PSA-zein-HNK had stable loading and transport properties before reaching and accumulating at the tumor site.

Example 4: in vitro cellular uptake

1. Experimental methods

(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)⁵Individual cells/well) and cultured overnight. Then, 100ng/mL PSA-Zein nanoparticles loaded with Cou6 (PSA-Zein-Cou 6, prepared in example 3 and abbreviated as PSA-Zein-Cou6 in the figure) were added and the cells were incubated at 37 ℃. After 1 hour, cells were washed with cold PBS and treated with trypsin. The cells were then centrifuged and redispersed in 0.5mL PBS buffer.

The non-target Zein-Cou6 group and the free Cou6 group are arranged, the PSA-Zein-Cou6 in the experimental conditions is replaced by Zein-Cou6 (abbreviated as Zein-Cou6 in the drawing) and the free Cou6 respectively, and other experimental conditions are the same.

Set PSA + PSA-zein-Cou6 group: 4T1 cells were first cultured with 2.0mg/mL PSA for 1 hour, then PSA-zein-Cou6 for an additional 1 hour, under otherwise identical experimental conditions.

A control group was set, which was treated without any reagent addition, and other experimental conditions were the same.

(2) For the selective receptor competitive inhibition assay, the experimental conditions for each experimental group were as described in section (1), and the cells were then directly tested for mean Cou6 fluorescence intensity by FACScan flow cytometry (BD FACSCalibur, USA).

(3) For confocal laser scanning microscopy studies, 4T1 cells were seeded into glass-bottom dishes and allowed to adhere overnight. After 1 hour incubation with 100ng/mL PSA-zein nanoparticles, cells were washed three times with cold PBS. Subsequently, the cells were fixed with 4% paraformaldehyde for 20min, and then the nuclei were re-stained with 5. mu.g/ml Hoechst 33258 for 20 min. Finally, the treated cells were observed using LSM710 laser confocal microscope (Zeiss, Germany).

A non-targeted zein-Cou6 group, a free Cou6 group, a PSA + PSA-zein-Cou6 group and a control group were set, and the experimental conditions of each experimental group were as described in section (1).

2. Results of the experiment

To determine the targeting effect of PSA and selectin receptors in enhanced uptake, the inventors first compared the tumor targeting ability of different Cou 6-loaded zein nanoparticles. Cellular uptake of different nanoparticles was quantified by flow cytometry. Flow cytometry data showed the order of fluorescence intensity for control < zein-Cou 6< PSA + PSA-zein-Cou 6< PSA-zein-Cou 6< free Cou6 (fig. 4A and B). Cellular uptake levels were nearly doubled in the PSA-zein-Cou6 group compared to the non-targeted zein-Cou6 group. The introduction of PSA is demonstrated to increase the uptake of PSA-zein-Cou6 more than electron adsorption. In addition, competitive inhibition assays were performed to verify PSA-selectin receptor mediated cellular internalization of PSA-zein-Cou 6. Cellular uptake of PSA-zein-Cou6 was significantly reduced after PSA blocking the selectin receptor compared to the control group. These findings provide clear support for internalization of PSA-zein nanoparticles into 4T1 cells via a PSA-selectin receptor mediated pathway.

The images taken by confocal laser scanning microscopy were consistent with quantitative flow cytometry measurements (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, the green fluorescence intensity of the PSA pre-treatment (PSA + PSA-zein-Cou6) group was enhanced, indicating the feasibility and potential of PSA modifications available for targeted drug delivery.

Example 3: in vitro cytotoxicity (SRB) assay

1. Experimental methods

Cell viability of various HNK preparations against 4T1 was determined by SRB colorimetry. Briefly, 4T1 cells were plated at 2X 10³The density of individual cells/well was seeded in 96-well plates and cultured overnight. zein-HNK, PSA-zein-HNK (prepared in example 3) and free HNK solutions were prepared with a range of HNK concentrations (0.3-20 μ g/mL) and incubated with 4T1 cells for 48 h. After an additional 48 hours of incubation, 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 (10mM) was added to dissolve the SRB dye. Finally, the absorbance of the final solution, representing the cell viability, was measured with a microplate reader (Tecan Safire2, Switzerland) at 540 nm.

2. Results of the experiment

As shown in fig. 5, all HNK preparations inhibited cell proliferation in a concentration-dependent manner. In all groups, the antiproliferative effect of free HNK on 4T1 cells was strongest, indicating that more HNK was rapidly transferred into cells by passive diffusion. As expected, PSA-zein-HNK showed much higher cytotoxicity than zein-HNK, probably due to the increased cellular uptake facilitated by PSA modification. IC of PSA-zein-HNK (4.37. mu.g/mL) at 48h₅₀The value is very close to freeHNK (3.99. mu.g/mL) and is significantly higher than zein-HNK (7.74. mu.g/mL). These results strongly confirm the increased cytotoxicity of HNK mediated by PSA-selectin targeted delivery.

Example 4: in vitro cell migration and invasion inhibition assay

1. Experimental methods

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 continuous and involved method for analysis of cell migration in vitro. nat. protocol.2007, 2(2),329 + 333, DOI: 10.1038/nprot.2007.30). 4T1 cells were cultured in 6-well plates overnight until a confluent monolayer was formed. The scratch was then introduced into the confluent monolayer 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 μ g/mL HNK) in fresh serum-free RPMI-1640. The distance between the two wound margins was monitored using an inverted microscope (Zeiss, Axio Observer a1) at 0 hours and 24 hours after treatment. Wound healing rates were calculated as described previously (Zhang, Z.W.; Cao, H.Q.; Jiang, S.J.; Liu, Z.Y.; He, X.Y.; Yu, H.J.; Li, Y.P.Nanoassembly of protocol Enables Novel Therapeutic Efficacy in the prediction of Lung Metastatias of Breast cancer. Small 2014ll, 10(22), 4735-.

The transwell method was performed using 24-well transwell plates (8 μm wells, Costar, USA) to further measure cell migration and invasion. For cell migration assays, 4T1 cells were incubated with different HNK solutions at a concentration of 5.0. mu.g/mL HNK for 24 h. Then, 100. mu.L of RPMI 1640 medium without FBS was pretreated with 5X 10⁴Individual 4T1 cells were seeded in the apical chamber of a 24-well transwell and 600 μ L of medium containing 10% FBS was placed in the basal chamber. After 24 hours, cells migrated to the lower membrane were fixed with 4% paraformaldehyde and stained with 0.4% crystal violet for 30 minutes, and then photographed under an inverted microscope. Subsequently, the cell-bound crystal violet was dissolved with a 33% acetic acid solution for quantitative analysis. The optical density at 570nm was recorded with a microplate reader.

For the invasion test, the test will contain 8X 10⁴100 μ L of serum-free medium of pretreated 4T1 cells were seeded in a top chamber pre-coated with diluted Matrigel (20 μ g/. mu.L; Cornig, USA) and incubated for 36 hours. The following processing steps correspond to the method described above.

2. Results of the experiment

Increased motility is a hallmark of metastatic cells. Wound healing scratch test is used to assess cell motility. All HNK formulations inhibited healing of the scratch to some extent after 24 hours compared to the control group. PSA-zein-HNK showed stronger inhibitory effect, with a wound healing rate of 13.33%, while the zein-HNK group increased wound healing rate to 31.11%. Free HNK had the strongest inhibition of scratch healing with a wound healing rate of 6.67% (fig. 6A and B). In addition, the inventors analyzed 4T1 cells for cell migration in vitro on a Transwell model. Similar inhibition of PSA-zein-HNK treated cells was observed. As shown in fig. 6A and C, the migrating cell number of the PSA-zein-HNK group was significantly lower than that of the zein-HNK group. The cell mobility of PSA-zein-HNK was only 20.05% compared to the control group, whereas the cell mobility of zein-HNK was 54.5%.

Next, the inventors evaluated the effect of PSA-zein-HNK on 4T1 cell invasion in a Transwell plus matrigel assay. Cell invasion is a key biological process of tumor metastasis and provides a prerequisite for initiating tumor metastasis (Wang, J.; Liu D.; Guan, S.; Zhu, W.Q.; Fan, L.; Zhang, Q.; Cai, D.F.Hyurunic acid-modified liposol homo hol nanocarrier: Enhance anti-metastasis and antigen or efficacy against breast cancer caner.Carbohydr.Polymer.2020, 235, DOI: 10.1016/j.carbopol.2020.115981). As shown in fig. 6A and D, the PSA-zein-HNK treated group was significantly less invasive than the zein-HNK group at 4T 1. The cell invasion rates of zein-HNK, PSA-zein-HNK and free HNK are 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 migration and invasion of cells at a higher level than zein-HNK. Therefore, the inventor speculates that PSA-zein-HNK may have excellent inhibitory effect on primary metastasis of tumors.

Example 5: in vitro cytotoxicity to tumor spheroids

1. Experimental methods

The antitumor activity of the different HNK-loaded formulations was further evaluated on 3D tumor spheres (tumor spheres). 3D cell culture and spheroid formation were supported using the pendant drop method as described previously (Del Duca, D.; Werboetski, T.; Del Maestro, R.F. spherical prediction from modification of drops: characterization of a model of a blue tumor invasion. J.neurono. 2004,67(3),295-303, DOI:10.1023/b: neon.0000024220.07063.70). First, agarose (2%, w/v) was dissolved in serum-free RPMI-1640 medium and coated in each well of 48-well plates to prevent cell adhesion. Next, 4T1 cell suspension (1X 10)³20 μ L) droplets were hung on the lid of a 48-well plate. After 48 hours, the formed tumor spheres were placed in each well with 0.9mL of medium. When the size of the tumor spheres is about 100 μm, uniform and dense spheres are used for the subsequent procedure. Tumor spheres were incubated with free HNK, zein-HNK, and PSA-zein-HNK (prepared in example 3) (10 μ g/mL HNK), respectively. At defined time points (1, 3, 5 and 7 days), growth inhibition of tumor spheres was monitored and images were taken using an inverted microscope (Zeiss, Axio Observer a 1).

2. Results of the experiment

As shown in fig. 7A and B, zein-HNK, PSA-zein-HNK, and free HNK were more toxic to the 4T1 tumor sphere than the control, which showed a 13.98-fold increase in volume. Free HNK showed the strongest cytotoxic effect on tumor spheroids 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 have even higher cytotoxicity than zein-HNK. Increased cytotoxicity of PSA-zein-HNK is thought to be associated with PSA modification, which enhances penetration and accumulation of nanoparticles into tumor spheres.

Example 6: in vivo tumor targeting assessment

1. Experimental methods

By mixing 1 × 10⁶A suspension of 4T1 cells (100. mu.L) was injected subcutaneously into the right axilla of BALB/c mice to establish a subcutaneous 4T1 tumor model. The tumor volume reaches 300-400mm³Thereafter, Zein-DiR and PSA-Zein-DiR (prepared in example 3 and abbreviated as Zein-DiR, PSA-Zein-DiR, respectively in the drawing) were injected into subcutaneous tumor-bearing mice via the tail vein at a dose of 500. mu.g/kg DiR. Fluorescence images of anesthetized mice were captured by an in vivo imaging system (IVIS lumine Series iii, PerkinElmer, USA) at predetermined time points. Finally, ex vivo images of the dissected tumor and major organs were taken at 24 hours.

2. Experimental methods

As shown in fig. 8A, the accumulation of zein-DiR in the tumor decreased rapidly and decreased significantly 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 on zein nanoparticles and prolongs their accumulation time in tumors. Finally, tumors and major organs were obtained and then evaluated by ex vivo fluorescence imaging. Ex vivo images show that PSA modification leads to a reduction in the distribution of zein nanoparticles in the lung and enhanced tumor accumulation (fig. 8B). Taken together, these in vivo data indicate that the improved tumor accumulation of such developed PSA-zein nanoparticles would provide a considerable and long-lasting effect of antitumor agents (such as HNK).

Example 7: in vivo anti-tumor study of 4T1 breast cancer model

The subcutaneous 4T 1-loaded breast cancer model is established as described above, when the tumor volume reaches 100mm³Then, it is randomly divided into fourGroup (n ═ 6). These different fractions 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, for a total of 4 times. 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 visually detected metastatic 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 tumor cell apoptosis histologically, tumor samples were also immunofluorescently stained with terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nicked end marker (TUNEL).

2. Results of the experiment

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, the 4T1 breast cancer cells of the saline group grew rapidly during the experiment, while the different HNK formulations significantly inhibited tumor growth. At the end of the experimental period, the mean tumor volume of the saline-treated control group was 1181.9. + -. 115.7mm³Free HNK of 965.9 + -88.3 mm³The zein-HNK is 760.6 +/-65.6 mm³The treatment group of PSA-zein-HNK is 619.1 +/-62.9 mm³. This indicates that encapsulation of HNK in zein nanoparticles is absolutely beneficial compared to administration of free HNK. More importantly, PSA-zein-HNK showed better tumor inhibition than zein-HNK and free HNK treatments, reflecting the advantage 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 demonstrated that PSA-zein-HNK has the highest in vivo anti-tumor efficiency, higher than that of zearalolThe in vivo anti-tumor efficiency of the lysoprotein-HNK is higher 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 anti-tumor effect, histological and immunohistochemical staining was performed to assess changes in major organs and tumors. As shown in fig. 9D, the H & E stained image of the normal saline group tumors was filled with closely packed tumor cells and had an intact morphology, whereas the PSA-zein-HNK group detected tumor necrosis, significant nuclear fragmentation and aggregation. TUNEL staining assay showed that PSA-zein-HNK treatment produced the largest number of apoptotic cells in tumor tissue compared to other formulation treatments (fig. 9E and F), which is consistent with the results for H & E. All these excellent results of PSA-zein-HNK benefit from PSA modification, which leads to higher cellular uptake efficiency, stronger cytotoxicity and more drug accumulation at the tumor site.

To assess possible systemic toxicity associated with different HNK formulation treatments in vivo, body weight changes and H & E sections of major tissues were assessed. As shown in figure 10A, zein-HNK, PSA-zein-HNK and free HNK were well tolerated by mice because they did not exhibit significant weight loss during treatment compared to the saline group. Histological examination of histological sections of major organs also confirmed their biosafety in vivo, as there were no significant pathological changes in mice treated with different HNK preparations (fig. 10C). These data indicate that PSA-modified zein core/shell nanoparticles for targeted delivery of HNK exhibit excellent biosafety and can be used as safe formulations for antitumor therapy.

Metastasis usually occurs in patients with advanced breast cancer. Breast cancer is well known for lung metastasis and results in rapid death of the patient (Liu, M.T.; Ma W.J.; Zhao, D.; Li, J.J.; Li, Q.R.; Liu Y.H.; Hao, L.Y.; Lin Y.F. enhanced peptide of a temporal Framework Nucleic Acid by Modification with iRGD for DOX-Targeted Delivery to Triple-Negative Breast cancer. ACS. Appl. mater. interfaces 2021,13(22),25825 25835, DOI: 10.1021/acquisition.1c07297). Encouraged by these excellent anti-tumor efficacy, the inventors further examined the anti-metastatic efficacy of PSA-zein-HNK nanoparticles. Images of the lungs are taken and metastatic nodules are calculated at the end of the process. As shown in fig. 10B, many metastatic nodules were detected in the saline group, indicating that the breast tumor had metastasized to the lung. However, a significant reduction in the number of lung metastatic nodules was observed in mice treated with different HNK formulations. Free HNK and zein-HNK treatment showed moderate inhibition of lung metastasis. While almost no 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 trend of metastatic nodules on the lung surface, H & E stained images of lung and liver sections in the saline group showed many tumor cell infiltrates (with large nuclei). Whereas tumor cell infiltration was significantly less in the free HNK and zein-HNK treated groups than in the normal saline group. More importantly, metastatic lesions were controlled to a minimum by PSA-zein-HNK without tumor cell infiltration. These results indicate that PSA-zein-HNK can not only effectively inhibit the growth of primary tumors, but also inhibit the formation of tumor metastases.

In conclusion, the inventor successfully prepares a PSA modified zein nanoparticle for targeted delivery of HNK by using an anti-solvent precipitation and electrostatic deposition technology for the first time. PSA modifications stabilize zein nanoparticles and confer their active targeting properties in breast cancer therapy. PSA-zein-HNK is a biologically safe and biocompatible nanomedicine delivery system that exhibits specific binding and selective toxicity to selectin-positive breast cancer cells. After intravenous injection, PSA-zein-HNK nanoparticles showed ideal tumor suppression efficiency and did not cause significant systemic toxicity throughout the treatment, 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 inhibit both the invasive growth of primary tumors and metastases.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and the like that are within the spirit and principle of the present invention are included in the present invention.

The foregoing embodiments and methods described in this disclosure may vary based on the abilities, experience, and preferences of those skilled in the art.

The mere order in which the steps of a method are listed in the present invention does not constitute any limitation on the order of the steps of the method.

Claims

What is claimed is: 1. A nanoparticle comprising a core particle and a coating layer covering the outer surface of the core particle, wherein the core particle comprises zein, and the coating layer comprises PSA.

2. The nanoparticle according to claim 1, wherein the average molecular weight of the PSA is 5000-120000 Da, preferably 50000-80000 Da, more preferably 80000 Da.

3. The nanoparticle according to claim 1, wherein the particle size of the nanoparticle is 100-200 nm, and the thickness of the coating layer is 1-100 nm;

Preferably, the particle size of the nanoparticles is 100-120 nm;

Preferably, the thickness of the coating layer is 10-30 nm;

Preferably, the nanoparticles are negatively charged.

4. A drug-loading system, which is a nanoparticle, the nanoparticle has a core particle and a coating layer coated on the outer surface of the core particle, wherein the core particle comprises zein, and the coating The coating comprises PSA, and the core particles are loaded with one or more active ingredients.

5. The drug-carrying system according to claim 4, wherein the average molecular weight of the PSA is 5000-120000 Da, preferably 50000-80000 Da, more preferably 80000 Da.

6. The drug-carrying system according to claim 4, wherein the particle size of the nanoparticles is 100-200 nm, and the thickness of the coating layer is 1-100 nm;

Preferably, the particle size of the nanoparticles is 100-120 nm;

Preferably, the thickness of the coating layer is 10-30 nm;

Preferably, the nanoparticles are negatively charged.

7. The drug-carrying system according to claims 4-6, wherein the active ingredient is an antitumor drug;

Preferably, the antitumor drug is selected from: doxorubicin, epirubicin, pirarubicin, idarubicin; mitoxantrone; topotecan, irinotecan, ixitecan, amino Camptothecin; Paclitaxel, Docetaxel; Gefitinib, Imatinib, Nilotinib, Sunitinib, Lapatinib, Tofacitinib, Crizotinib, Masitinib , entrectinib, ibrutinib, afatinib, flumatinib, erlotinib, neratinib, alectinib, apatinib, Talazoparib, lorlatinib, TPX-0005; Platinum, carboplatin, nedaplatin, cycloplatin, oxaliplatin, lobaplatin; vinblastine, vincristine, vinorelbine, berberine, berberamine; and honokiol; uracil mustard, Nitrogen mustard, ifosfamide, melphalan, chlorambucil, piperidine, tritamide, triethylene phosphoramide, busulfan, carmustine, lomustine, streptavidin Zocin, dacarbazine, fluorouracil deoxynucleoside, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate;

More preferably, the active ingredient is honokiol.

8 . The drug-carrying system according to claim 7 , wherein, in the core particles, the weight ratio of zein and active ingredients is 1:1 to 100:1. 9 .

9. the preparation method of the drug-carrying system described in any one of claim 4-8, it comprises the steps:

(1) forming core particles;

(2) forming a coating layer;

Preferably, step (1) includes: dissolving the zein and the active ingredient in an aqueous ethanol solution, adjusting the pH to acidity, and removing the ethanol;

Preferably, step (2) comprises: adding the dispersion of core particles obtained in step (1) into the PSA solution;

More preferably, after adjusting pH to acidity, step (1) further comprises the step of adding surfactant solution;

More preferably, the concentration of the PSA solution is 0.05-10%, preferably 0.1-1%, more preferably 0.1-0.25%.

10. A pharmaceutical composition, comprising the drug-carrying system according to any one of claims 4-8, and one or more pharmaceutically acceptable excipients.