Tumor stem cell targeting virus-like drug carrier and preparation method and application thereof

Abstract

The invention discloses a tumor stem cell targeting virus-like drug carrier as well as a preparation method and application thereof. The drug carrier provided by the invention can improve the cell uptake efficiency by simulating the physical surface characteristics of a biological system, and can directly target tumor stem cells. Besides, the drug carrier has targeting property, so that healthy tissues can be protected, the side effects of the loaded drug such as PTC209 in high-dose independent use are obviously reduced, the reaction to tumor stem cells is effectively enhanced, tumor proliferation and metastasis are inhibited, and the drug resistance of drug combination such as cis-platinum is reduced.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a virus-mimicking drug carrier targeting tumor stem cells and a preparation method and application thereof. Background Art

[0002] Biomacromolecule drugs, such as DNA, siRNA and proteins, as well as many small molecule drugs, are difficult to enter target cells due to their poor stability, low solubility and / or lack of targeting. This results in the inability of drug molecules to exert therapeutic effects and may also cause systemic toxic side effects. Various natural carriers derived from microorganisms and chemically synthesized carriers have been widely used in the study of drug delivery to cells. Compared with viral vectors, non-viral vectors, such as nanoparticles, are safer delivery tools. Such nanoparticle systems have been widely used as carriers of anti-tumor drugs, which enhances the anti-tumor effect of drugs while minimizing adverse reactions. Existing nanoparticles mainly achieve targeted aggregation in tumor tissues through the enhanced permeation and retention (EPR) effect caused by abnormal vascular leakage and impaired lymphatic drainage in the tumor area. However, relying solely on EPR-mediated passive targeting is not enough to achieve highly specific targeted therapy for cancer stem cells (CSCs).

[0003] In summary, the current cell delivery efficiency of nanoparticles is relatively low and they can only perform limited biological functions. Therefore, the development of non-viral drug delivery systems with targeted specificity and high delivery efficiency remains a daunting challenge.

[0004] The information in the background technology is only for illustrating the general background of the present invention and should not be regarded as admitting or suggesting in any form that such information constitutes the prior art known to a person skilled in the art. Summary of the invention

[0005] In order to solve at least part of the above technical problems, the present invention designs a virus-like drug carrier with specific targeting, which can improve the targeting of tumor stem cells, thereby improving drug administration, especially the local delivery efficiency after systemic administration. Compared with currently known particles, the drug carrier of the present invention has a larger surface area and better drug loading capacity, and can be used as a carrier for many systems. The present invention uses inorganic materials as core particles, and combines bionic spike structures on its surface to make the surface of the core particles rough. In addition, a bionic coating is further provided on the surface of the drug carrier, especially a covalently bonded hyaluronic acid (HA) film, to further improve its targeting ability and bio-tissue compatibility for tumor stem cells. Furthermore, the present invention can also adsorb drugs, especially small molecule drugs, such as but not limited to PTC209, into the porous structure of the core particles of the drug carrier, thereby achieving a more effective therapeutic effect. Specifically, the present invention includes the following contents.

[0006] In a first aspect of the present invention, a virus-like drug carrier targeting tumor stem cells is provided, which comprises a core particle, a spike and an envelope, wherein the core particle has a diameter of 50-400 nm and has an open porous structure with a pore size of 0.5-8 nm, the spike is arranged on the surface of the core particle in a manner that does not substantially close the open porous structure, and the envelope is a hydrophilic layer and is wrapped around the surface of the core particle and the spike.

[0007] In certain embodiments, according to the tumor stem cell-targeting viral-like drug carrier of the present invention, the core particle and / or the spike include but are not limited to inorganic materials, and the envelope layer includes but is not limited to organic materials.

[0008] In certain embodiments, according to the tumor stem cell-targeting viral-like drug carrier of the present invention, the inorganic material includes but is not limited to at least one of clay, calcium carbonate, hydroxyapatite, graphene oxide, boron nitride and silicon dioxide.

[0009] In certain embodiments, according to the tumor stem cell-targeting viral drug carrier of the present invention, the envelope is wrapped around the surface of the core particle and the spike through an active group, and the active group is selected from at least one of an amino group, a quaternary ammonium salt group, a polyethyleneimine group, an imidazole group, a guanidine group, and a pyridine group.

[0010] In certain embodiments, according to the tumor stem cell-targeting viral-like drug carrier of the present invention, the organic material is hyaluronic acid with a molecular weight less than 5 kDa.

[0011] In certain embodiments, according to the tumor stem cell-targeting viral drug carrier of the present invention, the envelope has a pore structure that allows drugs to pass through.

[0012] The second aspect of the present invention provides a pharmaceutical composition, which includes the tumor stem cell-targeting viral-like drug carrier of the present invention and a drug loaded on the carrier, especially a small molecule drug. Preferably, the pharmaceutical composition is a systemic administration composition.

[0013] The third aspect of the present invention provides a method for preparing a virus-like drug carrier targeting tumor stem cells, which comprises the following steps:

[0014] (1) preparing a core particle having an open porous structure, and performing a first functional modification on the core particle to obtain a core particle containing an active group;

[0015] (2) preparing a spike precursor suspension, mixing it with the suspension of the core particles, generating spikes on the surface of the core particles under the condition that the surface openings of the core particles are not substantially closed, and performing a second functional modification on the core particles containing the spikes to obtain nanoparticles containing active groups;

[0016] (3) Covalently linking an organic polymer having reactive groups on its surface to the surface of the nanoparticles containing the active groups, thereby obtaining the viral-like drug carrier.

[0017] In certain embodiments, according to the method for preparing a tumor stem cell-targeting viral-like drug carrier of the present invention, the method further comprises the step of (4) embedding the drug in the viral-like drug carrier.

[0018] In certain embodiments, according to the method for preparing a tumor stem cell-targeting viral-like drug carrier of the present invention, step (2) comprises:

[0019] (1') emulsifying a catalyst capable of providing an alkaline environment with an organic solvent, adding a spike precursor and stirring to obtain the spike precursor suspension;

[0020] (2') adding the spike precursor suspension to the core particle suspension for reaction.

[0021] The fourth aspect of the present invention provides use of the virus-mimicking drug carrier according to the present invention in the preparation of an anti-tumor drug composition.

[0022] In certain embodiments, according to the use of the present invention, the pharmaceutical composition is a composition for systemic administration.

[0023] In certain embodiments, the use according to the present invention comprises a combined use with other therapeutic agents.

[0024] The present invention develops a targeted bionic nano drug carrier, which simultaneously solves the problems of tumor proliferation and metastasis, side effects associated with small molecule drugs, and drug resistance to combination drugs such as cisplatin. In certain embodiments, the present invention provides a bionic drug carrier targeting tumor stem cells, which can improve cell uptake efficiency by simulating the physical surface properties of biological systems and directly target tumor stem cells through the capsule. In addition, it can protect healthy tissues while effectively enhancing the response to tumor stem cells, inhibiting tumor proliferation and metastasis, and reducing drug resistance to combination drugs such as cisplatin. The efficient drug carrier platform of the present invention provides a new bionic strategy for targeting tumor stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Synthesis and characterization of PTC209@VNP-HA. (a) Transmission electron microscopy (TEM) images showing (i) MSN, (ii) MSN-NH 2 (iii) VNP, (iv) VNP-NH 2 and (v) nanomorphology of VNP-HA. Scale bar: 50 nm. (b) Dynamic light scattering analysis of hydrodynamic diameter and (c) potential in PBS. (d) Nitrogen adsorption isotherm and (e) pore size distribution of VNP-HA. (f) Elemental mapping of (i) VNP-HA and (ii) PTC209@VNP-HA. (g) Release curves of PTC209 from PTC209@VNP-HA in PBS buffers with different pH values.

[0026] Figure 2 Virus-mimicking morphology promotes CSC uptake. (a) TEM image and (b) SEM image showing the nanomorphology of (i) MSN-HA and (ii) VNP-HA. Scale bar: 50 nm. (c) MSN-NH 2 、MSN-HA、VNP-NH 2 Fourier transform infrared (FTIR) spectra of (a), VNP-HA, and HA. (d) HA concentration of MSN-HA and VNP-HA. (e) Biolayer interferometry (BLI) assessment of CD44 binding kinetics to (i) MSN-HA and (ii) VNP-HA. (fh) Confocal microscopy images showing the uptake of MSN-HA and VNP-HA by cells (red), nuclei (blue), and membranes (green). Scale bar: 40 μm. Data are the proportion of nanoparticles taken up by cells. MFI, mean fluorescence intensity. **P<0.01.

[0027] Figure 3PTC209@VNP-HA inhibits HNSCC invasion and promotes HNSCC apoptosis in vitro. (a) Cytotoxicity of MSN-HA and VNP-HA on (i) CAL27 and (ii) SCC15 cells. (b) Cytotoxicity of free PTC209, PTC209@MSN-HA, and PTC209@VNP-HA on (i) CAL27 and (ii) SCC15 cells. (c, d) Cell invasion after treatment with free PTC209, PTC209@MSN-HA, or PTC209@VNP-HA. Scale bar: 200 μm. (e, f) Immunofluorescence imaging and quantification of p-H2A.X (red), and nuclei were stained with DAPI (blue). (g, h) Images and quantification of DNA comets in CAL27 and SCC15 cells treated with free PTC209, PTC209@MSN-HA, or PTC209@VNP-HA (10 cells per group). Scale bar: 100 μm. (i, j) Apoptosis of CAL27 and SCC15 cells was detected by TUNEL staining 24 h after treatment. Scale bar: 50 μm. **P<0.01.

[0028] Figure 4 PTC209@VNP-HA inhibits stem cell properties of head and neck squamous cell carcinoma (HNSCC). (a) Western blot analysis showing stemness markers after PTC209@VNP-HA treatment. (b) High aldehyde dehydrogenase activity (ALDH high )CAL27 and (c)ALDH high Quantification and visualization of tumor sphere formation in SCC15 cells treated with or without PTC209@VNP-HA. Scale bar: 200 μm. (d, e) In vivo limiting dilution analysis (n=5). Scale bar: 1 cm. **P<0.01.

[0029] Figure 5 VNP-HA can improve the efficacy of PTC209 in preclinical HNSCC models. (a) Injection of ALDH high Treatment and killing time of CSC mice. (b) Tumor images (circled areas are lesions). (c) Tumor volume quantification. (d) Hematoxylin and eosin (H&E) staining of HNSCCs. The upper and lower bars show low-magnification (scale bar: 500 μm) and high-magnification (scale bar: 100 μm) images, respectively. (e) Tumor area quantification. (f) Anti-PCK immunostaining of cervical lymph nodes. Scale bar: 200 μm. (g) Lymph node metastasis rate. (h) Lymph node metastasis area quantification. *P<0.05, **P<0.01.

[0030] Figure 6PTC209@VNP-HA overcomes cisplatin resistance in a cisplatin-resistant xenograft mouse model. (a) Schematic diagram of the establishment and treatment of the SCC-R tumor model. (b) MTT analysis of cisplatin-treated SCC15 and SCC15 cisplatin-resistant cells and (c) IC 50 (d) Tumor sample image. Scale bar: 1 cm. (e) Tumor volume growth. (f) Tumor weight. *P<0.05, **P<0.01.

[0031] Figure 7 PTC209@VNP-HA combined with cisplatin treatment effectively inhibits HNSCC by eliminating CSCs. (a) Administration of tamoxifen labeled Tomato + BMI1 + Schematic diagram of CSC. (b, c) Images of tongue tumors; the circled part is the lesion. Scale bar: 2 mm. (d) Representative images of H&E staining of HNSCCs. The upper and lower bars show images at low magnification (scale bar 500 μm) and high magnification (scale bar 100 μm), respectively. (e, f) Quantification of tumor number and lesion area (n=12). (g) Anti-PCK immunostaining of cervical lymph nodes. Scale bar: 200 μm. Quantification of (h) percentage and (i) area of ​​metastatic lymph nodes. (j, k) Immunofluorescence images and quantification of cells (p-H2A.X, red) and nuclei (DAPI, blue). n=12. Scale bar: 25 μm. (l, m) Bmil + Tomato + CSC images and quantification of HNSCC. n = 12. The white dashed line is the tumor-stroma boundary. Scale bar: 25 μm. *P < 0.05, **P < 0.01.

[0032] Figure 8 (a) Histological analysis of the following five groups of nude mice: control group, cisplatin group, PTC209@MSN-HA group, PTC209@VNP-HA group, and PTC209@VNP-HA group + cisplatin group. H&B staining of the heart, liver, spleen, lung, and kidney showed that each drug had good biocompatibility. Scale bar: 100 μm. (b, c) Changes in blood biochemistry of routine blood and serum samples of nude mice after different treatments.

[0033] Fig. 9 Surface morphology changes when the feed volume of the shell particle solution was adjusted from (a) 1.2 mL to (b) 0.8 mL, to (c) 0.4 mL, and finally to (d) 0.2 mL.

[0034] Fig.10 (a) Nitrogen adsorption isotherm characterization and (b) pore size distribution characterization of (i) MSN and (ii) VNP.

[0035] Fig.11 SEM images of (i) VNP-HA powder mixed with PTC209 powder and (ii) PTC209@VNP-HA. Scale bar: 1 μm.

[0036] Fig.12 (a) MSN, MSN-NH 2 TGA curves of VNP, VNP-NH 2 The TGA spectra of MSN and VNP-HA. The number of coupled amino groups and HA in MSN is about 4.59% and 3.32%, respectively. The content of coupled amino groups and HA in VNP is about 4.64% and 3.42%, respectively.

[0037] Fig.13 BLI evaluation of the binding properties of CD44 protein (mouse) to (a) MSN-HA and (b) VNP-HA, where CD44 protein was immobilized on the biosensor.

[0038] Fig.14 (a) MSN-NH 2 , MSN-HA, PTC209@MSN-HA, (b) VNP-NH 2 , VNP-HA, and PTC209@VNP-HA on the hemolytic effects of red blood cells in ICR mice.

[0039] Fig.15 (a) Ex vivo imaging of organs and tumors after mice were injected with (i) MSN-HA and (ii) VNP-HA. (b) Quantified accumulation in tumors at different time intervals. DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0043] Viral drug delivery system

[0044] According to one aspect of the present invention, there is provided a viral-like drug carrier targeting tumor stem cells (sometimes referred to herein as "the drug carrier of the present invention"), comprising a core particle, spikes and an envelope, wherein the core particle has a diameter of 50-400 nm and has an open porous structure with a pore size of 0.5-8 nm, the spikes are arranged on the surface of the core particle in a manner that does not substantially close the open porous structure, and the envelope is a hydrophilic layer and is wrapped around the surface of the core particle and the spikes.

[0045] In the present invention, the core particle is the main body of the drug carrier. Unlike the traditional virus-like carrier, the core particle of the present invention is an inorganic material core particle. The inorganic material is not particularly specific, and its examples include but are not limited to clay, calcium carbonate, hydroxyapatite, graphene oxide, boron nitride and silicon dioxide. The present invention can use one of the above materials, or two or more of the above materials can be used in combination. In the case of combined use, the ratio of each material is not specific and can be freely set by those skilled in the art as needed.

[0046] In the present invention, the diameter of the core particles is generally 50-400nm, preferably 60-350nm, for example 70-300nm, 80-250nm, 90-200nm, 100-150nm, 100-140nm, further preferably 110-140nm, for example 110-135nm, 115-135nm, 120-135nm, 125-135nm, for example 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135nm.

[0047] In the present invention, the core particles need to have an open porous structure. Here, "open" means that at least one end of the hole in the porous structure extends to the surface of the core particle. The two ends of the porous structure can extend to the surface opening, thereby forming a pore structure that runs through the core particle. The porous structure can also extend to the surface at only one end to form an opening, thereby forming a pore structure with one end open and one end closed. The average pore size of the porous is generally 0.5-8nm, preferably 3-8nm, and also preferably 3.5-8nm (for example 3.5-7.5nm, 3.5-7nm, 3.5-6.5nm, 3.5-6nm, 3.5-5.5nm), for example 3.5nm, 4nm, 4.5nm, 5nm, 5.5nm, 6nm, 6.5nm, 7nm, 7.5nm, 8nm. The pore volume is not less than 0.5cm 3 / g, preferably not less than 0.6cm 3 / g, and preferably not less than 0.7cm 3 / g. Specific surface area not less than 350m 2 / g, preferably not less than 400m 3 / g, preferably not less than 450m 3 / g, preferably not less than 500m 3 In a specific embodiment, the average pore size of the core particles is 5.5 nm and the pore volume is 0.710363 cm 3 / g, specific surface area is 505.7690m 2 / g.

[0048] In the present invention, the spike of the virus-like drug carrier refers to a protruding structure protruding from the surface of the core particle. Preferably, the protruding structure of the spike is similar to the rough surface (shell particle) of the virus. In certain embodiments, unlike the virus, the spike of the present invention is composed of an inorganic material. Here, the inorganic material is not particularly limited, and examples thereof include but are not limited to clay, calcium carbonate, hydroxyapatite, graphene oxide, boron nitride and silicon dioxide. The present invention can use one of the above materials, or two or more of the above materials can be used in combination. In the case of combined use, the ratio of each material is not specific and can be freely set by a person skilled in the art as needed. It should be noted that the spikes and core particles of the present invention can both be inorganic materials, and the spike material and the core particle material can be the same or different.

[0049] In the present invention, the spike is used to enhance or increase the roughness of the surface of the core particle, thereby promoting the adsorption or endocytosis of the carrier or drug by the cell. As long as the purpose can be achieved, the shape and size of the spike are not particularly limited. To achieve the above purpose, the spike is usually 1-60nm, such as 2nm, 3nm, 4nm, 5nm, 6nm, 8nm, 10nm, 12nm, 14nm, 16nm, 18nm, 20nm, 22nm, 24nm, 26nm, 28nm, 30nm, 32nm, 34nm, 36nm, 38nm, 40nm, 45nm, 50nm, etc. In addition, the spikes are usually smaller than the core particle size, for example, 1 / 10-1 / 600 of the core particle size, such as 1 / 40, 1 / 50, 1 / 60, 1 / 70, 1 / 80, 1 / 90, 1 / 100, 1 / 110, 1 / 120, 1 / 130, 1 / 140, 1 / 150, 1 / 160, 1 / 170, 1 / 180, 1 / 190, 1 / 200, 1 / 250, 1 / 300, 1 / 400, 1 / 500, 1 / 600, etc. If the spike size is too large, it is not conducive to the increase or improvement of roughness. On the other hand, if the spike size is too small, it tends to block the opening of the porous structure, which is not conducive to drug loading, and is not conducive to the increase or improvement of roughness. In addition, the spikes are usually larger than the pore size of the porous structure to avoid the surface opening of the core particle being blocked by the spikes.

[0050] In the present invention, it is necessary to control the number of spikes to avoid excessive number of spikes, which causes the closure of the surface opening of the core particle. The number of spikes is preferably more than 1 / 30 of the surface area of ​​the core particle, such as more than 1 / 25, more than 1 / 20, more than 1 / 15, and more than 1 / 10. If the number is too small, the surface roughness is affected, and then the cell absorption of the carrier or drug is affected. On the other hand, the number of spikes is preferably less than 3 / 4 of the surface area of ​​the core particle, such as less than 1 / 2, less than 1 / 4, less than 1 / 5, less than 1 / 6, less than 1 / 7, less than 1 / 8, etc. If the number is too large, the surface opening of the core particle is affected, and then the drug loading and subsequent release of the drug are affected.

[0051] In the present invention, the binding between the core particle and the spike is not limited, for example, it is bound by non-covalent bonds or covalent bonds. Preferably, the core particle and the spike are tightly bound. For example, the binding is carried out by a connection method selected from electrostatic adsorption, hydrogen bonds, and covalent bonds. It is also preferred that the core particle carries an active group, and the active group is selected from at least one of an amino group, a quaternary ammonium salt group, a polyethyleneimine group, an imidazole group, a guanidine group, and a pyridine group, and is further preferably an amino group, that is, the core particle has an amino modification.

[0052] In the present invention, the envelope is the outermost layer of the drug carrier. The envelope of the present invention not only provides a hydrophilic surface layer to improve biocompatibility, but also provides specific targeting for tumor stem cells. For this purpose, the envelope of the present invention is preferably prepared from an organic material. The organic material includes a hydrophilic material, preferably an organic material containing a reactive group (such as a carboxyl group) and a tumor stem cell targeting group or molecule, and further preferably, does not include a lipid material. Examples of hydrophilic materials that can be used in the present invention include, but are not limited to, hyaluronic acid, which has the property of being able to target proteins on the surface of tumor stem cells, and in particular, is able to target and bind to the protein CD44 receptor that is commonly and highly expressed on the surface of tumor stem cells.

[0053] In the present invention, preferably, the envelope is bound to the surface of the spike and core particles by covalent bonds. In order to achieve covalent bonding, preferably, the molecules in the envelope are bound to the surface of the spike and core particles by active groups. Here, the active group is not particularly specific, and examples thereof include but are not limited to amino groups, quaternary ammonium salt groups, polyethyleneimine groups, imidazole groups, guanidine groups and pyridyl groups. The present invention can use one of the above-mentioned active groups, or a combination of multiple groups. In certain embodiments, the present invention preferably uses amino groups as active groups.

[0054] In the present invention, as long as the purpose of the present invention can be achieved, the molecular weight of organic material is not particularly limited. Normally, the molecular weight of organic material such as hyaluronic acid is below 5kDa, preferably below 3kDa, also preferably below 2kDa, further preferably below 1kDa. If the molecular weight is too large, it is unfavorable for the loaded drug, and is also unfavorable for the release after the drug is administered. On the other hand, the molecular weight of organic material such as hyaluronic acid is generally more than 50Da, preferably more than 100Da, such as more than 150Da, more than 200Da, more than 250Da, etc. If the molecular weight is too small, the drug carrier or drug targeting obtained is deteriorated. In a preferred embodiment, the molecular weight of hyaluronic acid is 500-1000Da.

[0055] In the present invention, the source of hyaluronic acid is not particularly limited, and may be hyaluronic acid or its salt having a molecular weight greater than oligomeric hyaluronic acid or its salt, including but not limited to hyaluronic acid or its salt extracted from animal tissue, hyaluronic acid or its salt obtained by microbial fermentation, or hyaluronic acid or its salt obtained by artificial synthesis, and also includes degraded hyaluronic acid or its salt obtained by further treatment or processing of hyaluronic acid or its salt from the above sources. Such treatment includes enzymolysis, chemical decomposition, ultrasonic treatment, etc.

[0056] In the present invention, the relative molecular mass (sometimes referred to as "molecular weight") of hyaluronic acid can be measured by known methods. An exemplary measurement method includes: using an Ubbelohde viscometer with a capillary diameter of 0.788 mm in a constant temperature water bath at (25.0±0.1)°C to measure the outflow time, using a one-point method to measure the intrinsic viscosity, and calculating the relative molecular mass according to the empirical formula: viscosity = 3.6×10 Mr.

[0057] In the present invention, the amount of organic material is generally 0.5-10%, preferably 1-8%, and also preferably 2-6% (e.g., 2-5%, 2-4%), such as 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6%. If the amount is too much, it is not conducive to the release of loaded drugs and drugs after administration. On the other hand, if the amount is too little, the tissue compatibility and targeting deteriorate. In the present invention, the quantitative determination of organic material is carried out by known methods. In an exemplary embodiment, it is determined by comparing the amount of active groups. Specifically, the amount of active groups before and after encapsulation is usually calculated. For example, first, MSN, MSN-NH 2 、VNP、VNP-NH 2 and VNP-HA were dispersed in deionized water, respectively. Then, DMSO was mixed at 90 °C for 20 min for color reaction. After the mixture was cooled to room temperature, ethanol was added to stop the reaction, and the absorbance at 570 nm was recorded using a microplate reader. A standard curve was drawn using glycine solution. After grafting HA on the amino-modified nanoparticles, the remaining amino content was calculated by ninhydrin reaction. The amount of grafted HA was calculated by comparing the remaining amino content with the amino content before HA grafting.

[0058] In the present invention, the particle size of the drug carrier is not limited, and is generally controlled in the range of 60-500nm, preferably 80-400nm, and more preferably 90-300nm. For example, the carrier particles are 50-150nm, preferably 100-150nm, and also preferably 100-120nm, such as 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120nm.

[0059] Pharmaceutical composition

[0060] The second aspect of the present invention provides a pharmaceutical composition, which comprises the drug carrier described in the first aspect and a drug, wherein the drug is adsorbed or embedded in the interior and / or surface of the drug carrier.

[0061] In the present invention, the drug is not limited, and any known drug can be used. Preferably, the drug of the present invention is a small molecule drug, such as an anti-tumor drug. The drug carrier of the present invention needs to contact with cells to promote cell absorption. At the same time, since the drug needs to be loaded into the carrier, hydrophilic drugs, especially small molecule hydrophilic drugs, are more conducive to the realization of the purpose of the present invention. In addition to the above, the inventors found that the drug carrier of the present invention can not only be used to load hydrophilic drugs, but more importantly, it can also load hydrophobic drugs. For example, the small molecule drug is PTC209.

[0062] Preparation method

[0063] In view of the challenging problems that the preparation of current virus-mimicking nanomedicines still faces, the third aspect of the present invention provides a method for preparing a virus-mimicking drug carrier targeting tumor stem cells. The inventors have conducted a lot of research on the preparation process and found that the virus-mimicking nanomedicine prepared by the method of the present invention has excellent properties (including but not limited to drug loading, targeting and therapeutic properties). Specifically, the preparation method of the present invention comprises:

[0064] (1) preparing core particles having an open porous structure, and performing a first functional modification on the core particles to obtain a core particle suspension containing active groups;

[0065] (2) preparing a spike precursor suspension, mixing it with the core particle suspension, generating spikes on the surface of the core particles under the condition that the surface openings of the core particles are not substantially closed, and performing a second functional modification on the core particles containing the spikes to obtain nanoparticles containing active groups;

[0066] (3) Grafting an organic polymer having reactive groups on its surface onto the surface of the nanoparticles containing active groups, thereby obtaining the virus-mimicking nanoparticles.

[0067] In step (1) of the present invention, illustratively, an organosilicon compound (e.g., MTES, TEOS, etc.), a cationic surfactant (e.g., CTAB, CTAC, etc.) is used as a mesoporous template, an organic solvent (e.g., an aliphatic hydrocarbon solvent, such as pentane, cyclohexane, octane, cycloheptane, etc.) is used as an oil phase, and an alkaline compound (e.g., an ethanolamine compound such as ethanolamine, diethanolamine, etc.) is used as a catalyst and a pH regulator to obtain core particles having an open porous structure.

[0068] In the present invention, the core particles are first functionalized so that the surface carries a positive charge through surface modification. Preferably, the core particles of step (1) are dispersed in an organic solvent, an amino-containing compound is added, and centrifugation is performed after heating and refluxing to obtain core particles containing active groups. The amino-containing compound is not particularly limited, as long as it contains an amino group. Examples of such substances include, but are not limited to: 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, γ-aminopropyltriethoxysilane, N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane, N-β-(aminoethyl)-γ-aminopropylmethyldimethoxysilane, N-(2-aminoethyl)-3-aminopropylmethyldimethoxysilane, or a combination thereof. In addition, compounds containing both amino groups and polymerizable functional groups, such as vinyl, propenyl, etc., can also be used in the present invention, so that a dendritic structure is formed on the surface of the core particles by polymerization reaction while introducing an amino group.

[0069] In the first functional modification process, the weight volume ratio (m / v, mg / mL) of the core particle to the amino-containing compound is 50-200: 1, preferably 80-150: 1, and more preferably 80-120: 1. The heating reflux time is 1-36h, preferably 5-24h, more preferably 8-20h, and more preferably 10-15h.

[0070] Step (2) of the present invention is a step of preparing a spike precursor suspension and forming spikes on the core particles. In order to achieve that the spikes are arranged on the surface of the core particles in a manner that the open porous structure is not substantially closed, the inventors have conducted a large number of studies and found that the addition steps and volume ratios of the core particle suspension and the spike precursor suspension can affect the situation where the openings are closed by the spikes. First, a catalyst that can provide an alkaline environment is emulsified with an organic solvent, and a particle precursor is added and stirred to obtain the spike precursor suspension. The catalyst that provides an alkaline environment is not particularly limited, for example, including but not limited to L-arginine, alcohol amine compounds, pyridine, imidazole, quaternary ammonium salts, etc. Organic solvents include but are not limited to aliphatic hydrocarbon solvents, such as pentane, cyclohexane, octane, cycloheptane, etc. Particle precursors refer to any suitable compounds that can provide a particle form after the reaction, for example, precursors that can provide nanoparticles including but not limited to calcium carbonate, graphene oxide, boron nitride, silicon dioxide, etc., and such precursors are known to those skilled in the art. The particle precursor used in the present invention includes, but is not limited to, ethyl silicate (such as tetraethyl orthosilicate), sodium silicate, silicon powder, diatomaceous earth, white carbon black, and the like.

[0071] In the above steps, the weight-to-volume ratio (m / v, mg / mL) of the catalyst and the particle precursor is 50-200: 1, preferably 100-200: 1, and more preferably 150-200: 1. An aliphatic hydrocarbon solvent is added to the aqueous solution containing the catalyst and stirred for emulsification, and then the particle precursor is added and stirred at 40-80°C (preferably 45-75°C, more preferably 50-70°C, and further preferably 55-65°C) for 1-10h, preferably 1-5h, to obtain a spike precursor suspension.

[0072] In step (2), the inventors studied the order of adding the two suspensions and found that when the core particle suspension is added to the spike precursor suspension for reaction, the spikes formed on the surface of the core particles will block the structure of the mesopores of the core particles, which will further affect the entry of small molecules into the mesopores, thereby reducing the drug loading and ultimately affecting the effect of treating or improving tumors. When the spike precursor suspension is added to the core particle suspension for reaction, the spikes are arranged on the surface of the core particles in a substantially unclosed open porous structure, which enables small molecule drugs to be fully or substantially fully loaded on the core particles, greatly improving the drug loading and further ensuring the effect of treating or improving tumors. In certain embodiments, when the core particle suspension is added to the spike precursor suspension for reaction, the spikes formed on the surface of the core particles will block the structure of the mesopores of the core particles. It has been determined that the pore size is less than 3 nm and the pore volume is less than 0.55 cm 3 / g, with a specific surface area of ​​less than 375m 2 In one embodiment, when the core particle suspension is added to the spike precursor suspension for reaction, it is determined that the pore size is less than 3 nm and the pore volume is 0.508043 cm 3 / g, specific surface area 374.7986m 2 / g.

[0073] In a preferred embodiment, the amino-modified core particles (also referred to herein as MSN-NH 2 ) has a concentration in the buffer of 0.1-10 mg / ml, preferably 0.1-8 mg / ml, also preferably 0.1-6 mg / ml, further preferably 0.5-4 mg / ml, more preferably 1-4 mg / ml, for example 1, 1.5, 2, 2.5, 3, 3.5, 4 mg / ml.

[0074] In order to obtain better particle morphology, that is, to maintain the complete morphology of the virus-mimicking nanoparticles and thus facilitate subsequent organic polymer grafting, the volume ratio of the core particle suspension to the spike precursor suspension can be controlled within the range of 2.5-6:1, for example, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1.

[0075] In order to carry out the grafting reaction of the subsequent step (3), a compound containing an active group (preferably an amino group) is grafted onto the particles prepared in step (2) to form a bioactive surface that can be used for grafting an organic polymer. First, the agitator is heated to 100-200° C. (preferably 110-180° C., also preferably 120-160° C., and further preferably 130-150° C.), and the particles prepared in step (2) are added to an organic solvent at a concentration of 0.1-50 mg / mL, preferably 1-10 mg / mL. Then, an amino group-containing compound is added dropwise. The amino group-containing compound is not particularly limited as long as it contains an amino group. Examples of such substances include, but are not limited to, 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, γ-aminopropyltriethoxysilane, N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane, N-β-(aminoethyl)-γ-aminopropylmethyldimethoxysilane, N-(2-aminoethyl)-3-aminopropylmethyldimethoxysilane, or a combination thereof. In addition, compounds containing both an amino group and a polymerizable functional group, such as a vinyl group, a propenyl group, etc., can also be used in the present invention, so that a dendritic structure is formed on the surface of the particles prepared in step (2) by a polymerization reaction while introducing an amino group.

[0076] In the modification of the above steps, the volume ratio of the organic solvent to the amino-containing compound is 0.1-10:1, preferably 0.1-5:1, and more preferably 0.1-2:1, and then stirred in a stirrer at the above-mentioned temperature for 1-36 hours (preferably 5-24 hours, more preferably 8-20 hours, and more preferably 10-15 hours), followed by centrifugation, alcohol washing and storage.

[0077] Step (3) of the present invention is a step of grafting an organic polymer having reactive groups on its surface onto the surface of the nanoparticles containing active groups, thereby obtaining the viral-mimicking drug carrier. In order to complete the grafting reaction, the organic polymer preferably contains reactive groups that can react with the active groups on the surface of the nanoparticles. Therefore, it can be understood that the reactive groups include any suitable groups that can react with at least one of the active groups selected from amino groups, quaternary ammonium salt groups, polyethyleneimine groups, imidazole groups, guanidine groups, and pyridyl groups. In the case where the active group is an amino group, the reactive group is a carboxyl group.

[0078] In step (3), at room temperature, an organic polymer containing a carboxyl group (preferably hyaluronic acid) is activated in a buffer using EDC as a catalyst for an activation time of 0.1-5 hours, preferably 0.1-2 hours, and more preferably 0.1-1 hours. The weight ratio of the catalyst to the organic polymer is 1:1-10, preferably 1:1-8, more preferably 1:1-6, and more preferably 1:1-5. NHS is then added, and the mixture is stirred for a further 0.1-5 hours (preferably 0.1-2 hours, and more preferably 0.1-1 hours). The weight ratio of EDC to NHS is 1:1-5, preferably 1:1-2, and more preferably 1:1-1.5. Then, the nanoparticles containing active groups are dispersed in a buffer and subjected to a condensation reaction with a carboxyl-activated organic polymer, wherein the weight ratio of the nanoparticles containing active groups to the organic polymer is 1:1-10, preferably 1:1-8, further preferably 1:1-6, and further preferably 1:1-5, and the reaction time is 0.1-10h, preferably 0.1-5h, and further preferably 1-5h.

[0079] In a preferred embodiment of step (3) of the preparation method of the present invention, the concentration of the organic polymer (e.g., hyaluronic acid) in the entire reaction system involved in step (3) is 0.1-10 mg / ml, preferably 0.1-5 mg / ml, also preferably 0.1-2 mg / ml, further preferably 0.1-1 mg / ml, for example 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 mg / ml. In a specific embodiment, the concentration of the organic polymer (e.g., hyaluronic acid) in the entire reaction system involved in step (3) is 0.64 mg / ml.

[0080] The preparation method of the present invention further comprises step (4), i.e., a step of embedding the small molecule drug in the said imitation virus drug carrier. The post-drug loading process of the small molecule can be carried out by a known method in the art, for example, but not limited to, loading the small molecule drug, such as PTC209, into the prepared imitation virus drug carrier by rotary evaporation.

[0081] application

[0082] In a fourth aspect of the present invention, a use of a virus-mimicking drug carrier in the preparation of an anti-tumor drug composition is provided, wherein the drug or composition of the present invention is used to treat or improve a tumor in a subject in need thereof, and the treatment or improvement is achieved by administering a therapeutically effective amount of the drug or composition to the subject.

[0083] In a preferred embodiment, the pharmaceutical composition is a systemically administered composition and targets tumor sites, particularly tumor stem cells.

[0084] In a preferred embodiment, the tumor is a solid tumor, in particular a head and neck tumor, including but not limited to tumors in the oral cavity, pharynx, larynx, etc., such as head and neck squamous cell carcinoma (HNSCC), such as tongue cancer, etc.

[0085] In the present invention, the term "treat or improve" refers to a therapeutic treatment or preventive measure, the purpose of which is to slow down (reduce) undesirable physiological changes or disorders, such as the occurrence, development or deterioration of tumors. Beneficial or desired clinical results include, but are not limited to, the following results, whether detectable or undetectable, including relief of symptoms, reduction in disease severity, stabilization of the disease state (i.e., no deterioration), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or complete). Those in need of treatment include those who already have head and neck tumors or head and neck tumor-related diseases or those who need to prevent or improve head and neck tumors or head and neck tumor-related diseases.

[0086] In the present invention, the treatment or improvement includes at least one of the following:

[0087] (1) Inhibit the proliferation or growth of tumor cells;

[0088] (2) Killing tumor stem cells or inducing apoptosis of tumor stem cells;

[0089] (3) reduce or decrease tumor cell volume and / or weight;

[0090] (4) Promote or enhance the tumor-killing function of immune cells;

[0091] (5) Prolong survival time.

[0092] The present invention further provides the use of a viral drug carrier or a pharmaceutical composition comprising the same in the preparation of a drug for treating tumors (e.g., head and neck tumors) in combination with other drugs. In the present invention, "combination therapy" refers to the use of the viral drug carrier of the present invention or a pharmaceutical composition comprising the same as part of a specific treatment regimen, which is intended to produce a beneficial (cumulative or synergistic) effect through the combined action of the viral drug carrier or the pharmaceutical composition comprising the same and one or more other drugs. The beneficial effects of the combination include, but are not limited to, the pharmacokinetic or pharmacodynamic synergy produced by the combination of the viral drug carrier or the pharmaceutical composition comprising the same and other drugs. The combined administration of the viral drug carrier or the pharmaceutical composition comprising the same and other drugs is usually carried out within a prescribed time period (usually minutes, hours, days or weeks, depending on the selected combination). "Combination therapy" includes the sequential administration of the viral drug carrier or the pharmaceutical composition comprising the same and other drugs (i.e., each drug is administered at different times), as well as the substantially simultaneous administration of these viral drug carriers or the pharmaceutical composition comprising the same and other drugs, or they can be formulated as a single co-formulated pharmaceutical composition containing two components.

[0093] In the present invention, other drugs or therapeutic agents are not particularly limited, and include but are not limited to: EGFR inhibitors (such as cetuximab), immune checkpoint inhibitors (PD-1 / PD-L1, such as pembrolizumab, nivolumab), EGFR-TKI (tyrosine kinase inhibitors, such as afatinib), PI3K / mTOR pathway inhibitors (such as everolimus), antibody-drug conjugates (ADC, Datopotamab Deruxtecan (Dato-DXd)), PARP inhibitors (Olaparib), small molecule drugs (cisplatin, 5-fluorouracil, carboplatin, nedaplatin, paclitaxel, etc.).

[0094] Example 1

[0095] 1. Preparation of mesoporous silica nanoparticles (MSN)

[0096] MSNs with mesoporous dendritic morphology were synthesized using a two-phase layered method. The silicon source was tetraethyl orthosilicate (TEOS), hexadecyltrimethylammonium chloride (CTAC) was the template for the mesoporous structure, and cyclohexane was the oil phase to promote the formation of a stable two-phase system. Triethanolamine was used as a catalyst and pH regulator. A round-bottom flask was charged with 0.36 g of triethanolamine, 48 mL of 25 wt% hexadecyltrimethylammonium chloride, and 72 mL of deionized water. The mixture was stirred at 60 °C for at least 1 hour to ensure the formation of CTAC micelles. 40 mL of 20 v / v% tetraethyl orthosilicate was dissolved in cyclohexane and added to the reaction system along the wall of the reaction flask. After slow stirring at 60 °C for 24 hours, a white reaction product was obtained.

[0097] After the reaction solution was transferred to a separatory funnel, the milky white aqueous phase containing MSNs was separated and centrifuged at 20,000 × g for 60 minutes to obtain MSN precipitates. Afterwards, the MSNs were washed with acidic methanol (37% hydrochloric acid and methanol mixed in a volume ratio of 1:10) at 60°C to remove impurities and CTAC remaining in the mesopores. Finally, after washing with anhydrous ethanol, the MSNs were dispersed in anhydrous ethanol for standby use, with an average particle size of 110 nm.

[0098] 2. MSN modification

[0099] After the MSN was amino-modified, the surface of the nanoparticles carried positive charges. Specifically, 250 mg of MSN was dispersed in 150 ml of xylene, and 2.5 ml of amino silicon source, i.e., 3-aminopropyltriethoxysilane (APTES), was added. After heating and refluxing for 12 hours, the MSN-NH 2 and washed repeatedly with anhydrous ethanol. 2 Disperse in anhydrous ethanol for later use.

[0100] 3. Preparation and modification of virus-mimicking nanoparticles (VNPs)

[0101] VNP consists of peripheral particles and MSN core. When preparing peripheral particles, L-arginine (87 mg) was dissolved in deionized water (69.5 mL), octane (5.23 mL) was added and emulsified by high-speed stirring. After adding TEOS (0.5 mL), stirring was carried out at 60 ° C for 3 hours to obtain a suspension containing peripheral particles with a particle size of about 10 nm. After that, MSN-NH 2 (100 mg) was dispersed in carbonate buffer (50 mL, pH 9.5, Ct[CO 3 2- ]=50mmol / L), and the suspension of peripheral particles (16mL) was added thereto, reacted at room temperature for 8 hours and washed with anhydrous ethanol to obtain VNP.

[0102] The steps of amino modification of VNPs include: grafting aminosilane onto the VNP shell particles to form a bioactive surface that can be used to graft HA. The stirrer was heated to 145°C while VNPs (250 mg) were added to 150 mL of xylene. Then, 2.5 mL of APTES was added dropwise and stirred at 145°C for 12 hours using a rectifier with a heated stirrer. VNP-NH 2The mixture was centrifuged at 20,000 × g for 30 min and washed three times with ethanol to remove xylene and residual APTES. The product was stored in ethanol for subsequent use.

[0103] 4. VPN surface grafting

[0104] Hyaluronic acid (HA) was grafted onto the surface of VNP to give it the ability to target and recognize tumor stem cells. The carboxyl groups of HA (32 mg) were activated in phosphate buffered saline (PBS, pH 7.4, 25 mL) at room temperature using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 12 mg) as a catalyst for 0.5 h. N-hydroxysuccinimide (NHS, 14 mg) was added and the mixture was stirred for another 0.5 h. Then, MSN-NH 2 、VNP-NH 2 (100 mg) were dispersed in PBS (pH = 7.4, 25 ml) and reacted with carboxyl-activated HA for 3 hours. After repeated washing with deionized water and anhydrous ethanol, MSN-HA and VNP-HA were obtained and stored at 4°C.

[0105] 5. Loading of small molecule drugs

[0106] The solvent evaporation method was used to load the small molecule drug PTC209 in the mesopores for anti-tumor drug delivery applications. 2.5 mg of PTC209 was dissolved in 0.25 ml of dimethylformamide (DMF), and 7.5 mg of VNP-HA and MSN-HA were ultrasonically dispersed, and then they were mixed. DMF was completely evaporated under nitrogen flow at room temperature to obtain dry PTC209@VNP-HA and PTC209@MSN-HA powders, respectively.

[0107] Example 2

[0108] 1. Experimental Methods

[0109] 1. Characterization of Nanoparticles

[0110] The samples were measured by transmission electron microscopy (TEM, 100 kV). A small amount of the sample was dispersed in ethanol and then transferred to a copper grid. Images of PTC209@MSN-HA and PTC209@VNP-HA samples were obtained by high-resolution transmission electron microscopy (HRTEM) and energy dispersive X-ray (EDX) spectroscopy. Scanning electron microscopy (SEM) was used to observe the surface morphology and encapsulation effect of the nanoparticles. Thermogravimetric analysis (TGA) was performed using a thermogravimetric analyzer-digital scanning calorimeter (Q600) to evaluate the thermal stability and composition of the samples. Zeta potential and dynamic light scattering were measured using a Zetasizer Pro analysis system to determine the surface charge and particle size distribution, respectively. Nitrogen adsorption-desorption measurements were performed using an ASAP 2020 analyzer at -196 °C to obtain porosity data, including specific surface area, pore size distribution, and total pore volume. For MSN-NH 2 、MSN-HA、VNP-NH 2 , VNP-HA, and HA samples were subjected to Fourier transform infrared spectroscopy (FTIR) analysis to confirm that HA was successfully attached to the nanoparticles by identifying specific chemical bonds and functional groups.

[0111] 2. In vitro release curve of PTC209 via PTC209@VNP-HA

[0112] The in vitro release profile of PTC209 from PTC209@VNP-HA was evaluated by dialysis against PBS at pH 7.4, 6.5, and 5.5. The PTC209@VNP-HA dispersion was transferred to a dialysis membrane bag with a molecular weight of 2000Da to ensure that only smaller molecules could diffuse freely, while larger nanoparticles were confined in the membrane bag. The dialysis bag was placed in a shaker at a constant temperature of 37°C and stirred at 100 rpm to promote the diffusion process. 1.5 mL of the released medium was collected and an equal volume of PBS was added to the dialysis system to maintain a constant volume to ensure continuous diffusion. Each sample was centrifuged at 20,000×g for 10 min to separate the supernatant containing dissolved PTC209. 1 ml of the supernatant was taken from the centrifuged supernatant and mixed with 0.5 ml of acetonitrile. The resulting mixture was analyzed using a UV-Vis spectrophotometer. The absorbance of the solution was measured at a wavelength of 235 nm. The concentration of PTC209 in each sample was determined by comparing the measured absorbance values ​​to the calibration curve. These concentrations were then plotted over time as a release curve.

[0113] 3. Binding specificity of VNP-HA and CD44 protein

[0114] Biolayer interferometry (BLI) assay was performed using Octet RED96e system to evaluate the affinity of HA to CD44 protein. Briefly, His-CD44 protein (25 μg / mL) was loaded onto tris(III) nitrile acetic acid biosensor (ForteBio) for 600 s. The biosensor was then placed in binding buffer containing 500 ng / L VNP-HA for 200 s. VNP (500 ng / L) was used as a control.

[0115] 4. Cell sorting and culture

[0116] HNSCC cell lines CAL27 and SCC15 were obtained from the ATCC Biological Standards Resource Center in the United States. CSCs with high aldehyde dehydrogenase activity were screened from CAL27 and SCC15 cells using the ALDEFLUOR detection kit. ALDH was screened using flow cytometry high The fluorescence intensity of cells was compared with that of the control treated with diethylamine benzaldehyde (DEAB). high The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1% antibiotics and 10% fetal bovine serum (FBS) at 37°C and 5% CO 2 cultured in a cell culture incubator.

[0117] To establish the cisplatin-resistant SCC15-R cell line, SCC15 cells were gradually exposed to increasing concentrations of cisplatin and finally maintained at a resistance level of 3 μM cisplatin. When the cells reached 70-80% confluence, PTC209@VNP-HA was introduced into the cell culture.

[0118] 5. Cytotoxicity Assay

[0119] Each well of a 96-well microplate contains approximately 1×10 4 cells. After 12 hours of culture, different concentrations of nanoparticles of 1-200 μg / mL and PTC209 drug concentrations of 0.25-10 μmol / L were cultured for 24 hours, and 10 μL of 5 mg / mL methylthiazole tetrazolium (MTT) solution was added to each well, incubated at 37°C for 4 hours, MTT was aspirated, and 100 μL of dimethyl sulfoxide was added to solubilize the purple tetrazolium crystals. Cell viability was assessed by measuring the optical density at a wavelength of 570 nm using a microplate reader. The control group maintained 100% cell viability in untreated culture medium. The control and test concentrations were repeated three times to ensure statistical reliability. The cell viability was calculated as follows:

[0120]

[0121] 6. Cell delivery experiment

[0122] To track the distribution of nanoparticles, rhodamine B isothiocyanate was covalently bonded to the remaining amine groups of VNP-HA and MSN-HA in ethanol. SCC15 cells were seeded in well plates and cultured, incubated with rhodamine-encapsulated MSN-HA and rhodamine-encapsulated VNP-HA, respectively, and then washed with PBS. Paraformaldehyde fixative was added, and the cells were washed with PBS. Triton X-100 PBS solution was then added, incubated at room temperature, and washed with PBS. Vari Fluor 488-phalloidin working solution was added and stained at room temperature in the dark. The cells were washed with PBS, then sectioned and sealed with confocal laser scanning microscopy sealant.

[0123] 7. Transwell invasion assay

[0124] CAL27 and SCC15 cells were treated with PTC209@VNP-HA and invasion assays were performed by counting the invading clones that passed through a polycarbonate membrane with 8 μm pores. The wells were coated with matrix. Approximately 3×10 5 Cells were suspended in serum-free DMEM and inoculated in the upper chamber, and an appropriate amount of DMEM was added to the lower chamber. Subsequently, PTC209@VNP-HA (containing 2.5μg / mL PTC209) was added to the upper chamber. The drug content of PTC209 in the free PTC209 and PTC209@MSN-HA experimental groups was consistent with that in the PTC209@VNP-HA experimental group. After 24 hours, the surface matrix of the polycarbonate membrane was scraped off, the cells on the membrane were fixed with paraformaldehyde solution, stained with crystal violet, and observed and photographed under a microscope. The cells in 9 random areas in the image were counted using ImageJ software.

[0125] 8. Comet detection

[0126] DNA damage was detected using the Single Cell Gel Electrophoresis (SCGE) Comet Assay Kit (ADI-900-166). Cells were embedded in agarose, lysed, and subjected to electrophoresis to separate DNA into “comet tail” fragments. In this assay, the length and intensity of the comet tails indicate the level of damage in individual cells to determine environmental and therapeutic effects on DNA integrity. The cell suspension was mixed with the agarose solution. 75 μL of the cell suspension per sample was loaded onto pre-heated slides at 37°C. The slides were incubated in pre-cooled lysis buffer at 4°C and then transferred to an alkaline solution for incubation at room temperature. To separate DNA fragments, the slides were electrophoresed in Tris / borate / ethylenediaminetetraacetic acid buffer. The slides were stained with CYGREEN nucleic acid stain until DNA was visible and then left to stand at room temperature to allow the agarose gel to flatten and image. At least 100 cells per sample were evaluated in duplicate using the CASP v1.2.2 analysis tool.

[0127] 9. TUNEL apoptosis detection

[0128] The apoptosis of HNSCC cells was evaluated using TUNEL kit (T2196). Images were obtained under a fluorescence microscope. The TUNEL positivity rate of PTC209@VNP-HA was positive.

[0129] 10. Tumor stem cell sphere formation experiment

[0130] In the tumor stem cell sphere formation experiment, ALDH high and ALDH low Cells were added to ultra-low adhesion well plates and cultured in a medium containing 1% N-2 supplement, 10 ng / mL human recombinant basic fibroblast growth factor, 20 ng / mL human recombinant epidermal growth factor, and 1% B-27 supplement mixed in serum-free DMEM / F12. After 12 days of PTC209@VNP-HA treatment, spheres with a diameter greater than 70 μm were counted under a microscope to evaluate the effect of PTC209@VNP-HA on cell growth and differentiation.

[0131] 11. Western blot analysis

[0132] After lysing the cells, the lysate proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane. Nonspecific protein binding was blocked with 5% skim milk for 1 hour. The primary antibodies included anti-BMI1, anti-ALDH1, anti-SOX2, and anti-GAPDH, and then labeled with the corresponding fluorescent-labeled secondary antibodies.

[0133] 12. Limiting dilution test

[0134] Female nude mice (6-8 weeks old) were obtained from the Department of Zoology, Peking University School of Medicine. All animal experiments were performed in accordance with the animal experiment protocol approved by the Biomedical Ethics Committee of Peking University. Female nude mice were prepared for dilution transplantation. high SCC15 CSCs were treated with PTC209@VNP-HA and digested into single cell suspensions. Equal amounts of matrix were mixed with different numbers of treated cells, and the mixtures were injected into the left axilla of nude mice. After 4 weeks, tumors of all mice were photographed and recorded. Data were analyzed using ELDA software.

[0135] 13. In vivo experiments

[0136] (1) Orthotopic tumor model

[0137] SCC15-ALDH high CSC (1×10 6) was inoculated onto the tongue of female BALB / c nude mice. About 1 week later, all mice were randomly divided into groups (n=6) and treated with 200 μL control solution, 5 mg / kg PTC209, 60 mg / kg PTC209, 20 mg / kg of PTC209@MSN-HA, or 20 mg / kg of PTC209@VNP-HA. The mice were injected once through the tail vein every 3 days for 3 weeks. After the mice were sacrificed, the tongue and cervical lymph nodes (3-4 per mouse) were removed. In the human cancer cisplatin-resistant cell line model, SCC15 cisplatin-resistant cells (5×10 6 ) was injected into the left armpit of nude mice. After one week of growth, the tumor volume reached about 100mm 3 Mice were randomly divided into 5 groups (n=6) and treated with 200 μL control solution, 3 mg / kg cisplatin, 20 mg / kg PTC209@MSN-HA, 20 mg / kg PTC209@VNP-HA, or a combination of 3 mg / kg cisplatin and 20 mg / kg PTC209@VNP-HA. After 3 weeks of treatment, mice were killed, and tumors were removed and weighed.

[0138] (2) Spontaneous head and neck squamous cell carcinoma model

[0139] Bmi1 was fed drinking water containing 4-nitroquinoline N-oxide (4NQO) CreER and Rosa tdTomato Mice (C57 mice, 6-8 weeks old) were cultured for 18 weeks to establish a spontaneous head and neck squamous cell carcinoma model. At 22 weeks, the mice were randomly divided into five groups (n=12) and treated with 200 μL of control solution, 3 mg / kg cisplatin, 20 mg / kg of PTC209@VNP-HA, or 3 mg / kg cisplatin combined with 20 mg / kg of PTC209@VNP-HA. The drugs were injected into the tail vein once every 3 days for 3 weeks. Tamoxifen-labeled Bmi1 was given before sacrifice. + CSC; After euthanasia, the tongue and cervical lymph nodes (3-4 per mouse) were removed and the lesion surface area was calculated. For histological analysis, longitudinal sections of the excised tongue and lymph nodes were fixed overnight in 4% paraformaldehyde. Subsequently, the tissues were dehydrated, embedded in paraffin, and prepared into 5 μm tissue sections.

[0140] 14. Immunostaining

[0141] Paraffin sections were antigen retrieved in citrate buffer. To bind to the target molecule, horseradish peroxidase-labeled polymers were incubated with the tissue at room temperature, using 3,3'-diaminobenzidine as a chromogen to form a visible precipitate at the peroxidase active site. Cell nuclei were stained with hematoxylin for contrast. Antigen retrieval was performed before immunofluorescence, followed by incubation with p-H2A.X antibody overnight at 4°C. Cy2-labeled secondary antibodies were used for color development. Cell nuclei were then stained with DAPI solution.

[0142] 2. Experimental Results

[0143] 1. Synthesis and characterization of PTC209@VNP-HA

[0144] The present invention develops a drug delivery nanoplatform (PTC209@VNP-HA) comprising a PTC209-loaded MSN core and HA-modified virus-mimicking protrusions. First, microporous microspheres were synthesized using a single-pot hydrothermal process. The negatively charged MSN surface was modified with APTES using amino groups (MSN-NH 2 ). A unique synthesis method was used to prepare shell-like nanoparticles, which can be electrostatically adsorbed on MSN-NH 2 VNPs were synthesized by a series of methods that assembled virus-like structures including multiple branched nanostructures. VNP-NH 2 , HA was covalently bound to the shell particles to form VNP-HA, and then PTC209 was adsorbed onto the mesoporous core to obtain PTC209@VNP-HA.

[0145] TEM images show that MSN and MSN-NH 2 The uniform morphology and dendrite structure were maintained, and the size (about 110 nm) did not change significantly. 2 The surface of the VNPs is attached with oriented particles, and the size range of these oriented particles is very narrow (about 10 nm, Figure 1a). 2 , about 130 nm) and HA-modified VNP-NH 2 (VNP-HA, about 130 nm) did not change their virus-mimicking morphology nor cause significant size changes. Dynamic light scattering was used to analyze the VNP-HA structure ( Figure 1b), the results show that the particle size distribution trend is similar to that of the TEM images. PTC209@VNP-HA and other nanoparticles showed good monodispersity in PBS. Due to the colloidal stability conferred by the HA surface modification, VNP-HA showed the best dispersibility among the tested nanoparticles with a polydispersity index of 0.15. The change in potential guided each step of the nanoparticle synthesis process. After the surface amino functionalization of MSN (-26.58) and VNP (10.40), MSN-NH 2 and VNP-NH 2 The zeta potential of VNP-HA was positive (14.03 and 19.53 mV), while after chemical connection with HA, the zeta potential of VNP-HA was negative (-15.29 mV) ( Figure 1 c). Conventional silica microspheres and solid silica nanoparticles are not biodegradable due to their silica composition, whereas dendritic silica microspheres have excellent biodegradability due to their thin pore walls and amorphous structure. The biodegradation of VNP-HA in PBS was evaluated over a 28-day experiment. TEM images showed that VNP-HA degradation proceeded from the outer surface to the inner core. On day 28, the mass of degraded VNP-HA in PBS reached about 56%. Achieving optimal biodegradability is critical to extending the therapeutic time of VNP-HA and reducing systemic toxicity, both for future research and clinical applications.

[0146] This example was tested several times to verify that PTC209 was fully loaded into PTC209@VNP-HA. In the TEM image, nitrogen adsorption experiments showed that the simulated virus morphology formed by the previous synthesis method reduced the core MSN mesopore diameter (<3nm) compared to the original MSN (>6nm). The TEM image (Figure 1a) also shows that after the preparation method of the present invention, the shell particles are firmly attached to the MSN-NH 2 On, a VPN is formed.

[0147] In order to further determine whether the virus-mimicking nanoparticles can carry drugs, nitrogen adsorption experiments were performed. In order to verify that VNP can carry target drugs, nitrogen adsorption experiments were performed, which confirmed that the mesopores of VNP were slightly smaller than those of MSN (about 5.5nm), and proved that the mesopores of the final synthesized VNP-HA were further reduced by about 5nm ( Figure 1 d, e, Fig.10 ). SEM images showed that the drug-loaded PTC209@VNP-HA had the same effect as the mixture of PTC209 powder and VNP-HA powder, and no drug crystals were observed in the PTC209@VNP-HA group, indicating that PTC209 had been completely loaded into the nanoparticles ( Fig.11). The elemental mapping profiles provide clear and direct evidence for PTC209 loading. VNP-HA exhibits a background signal, while PTC209@VNP-HA exhibits a highly concentrated bromine signal with no elemental segregation or phase separation, indicating that bromine atoms are uniformly distributed within the nanostructure ( Figure 1 f).

[0148] The controlled release of drug at the tumor site is a key performance indicator of nanoparticles for cancer treatment. This example evaluates the release of PTC209 at three pH conditions (7.4, 6.5 and 5.5) for PTC209@VNP-HA. Figure 1 g). After incubation in a neutral environment (pH = 7.4) for 24 hours, PTC209@VNP-HA showed remarkable stability, releasing only about 20% of the loaded PTC209 drug. This low level of drug leakage is very advantageous because it significantly reduces potential toxic side effects on healthy tissues. In contrast, in acidic environments of pH 6.5 and 5.5, the PTC209 release rate of PTC209@VNP-HA increased sharply to 64% and 83% after 24 hours, and gradually increased to 70% and 91% after 72 hours.

[0149] The present invention further explores the morphological adjustment of the virus-mimicking drug carrier. After the capsomers and coresomes are mixed to prepare a drug carrier simulating a virus, the morphological changes are tracked by TEM images. The results are as follows Fig. 9 As shown, the surface morphology changed significantly when the feed volume of the capsomere solution was adjusted from (a) 1.2 mL to (b) 0.8 mL, then to (c) 0.4 mL, and finally to (d) 0.2 mL. The sample obtained when the feed volume was 0.8 mL had the best effect. Scale bar: 50 nm. It should be noted that insufficient capsomere quantity will lead to loss of viral morphology. Fig. 9 In the embodiment 1, 0.8 mL corresponds to 16 mL of shell particle stock solution, 1.2 mL corresponds to 24 mL of shell particle stock solution, 0.4 mL corresponds to 8 mL of shell particle stock solution, and 0.2 mL corresponds to 4 mL of shell particle stock solution.

[0150] 2. Virus-mimicking morphology promotes CSC uptake

[0151] To determine the effect of nanoparticle morphology on the effectiveness of drug delivery to CSCs, smooth MSN-HA nanoparticles were prepared under different reaction conditions. TEM ( Figure 2 a) and SEM( Figure 2 b) The image clearly shows that the MSN-HA and VNP-HA mesopores have different external surface morphologies. FTIR spectroscopy analysis was performed to confirm the successful binding of HA. -1) has stronger stretching vibration than VNP-NH 2 (3441cm -1 Carboxyl groups (1648 cm -1 ) tensile vibration ( Figure 2 c). In addition, TGA results showed that the conjugation rates of HA on MSN and VNP were approximately 3.41% and approximately 3.42%, respectively ( Fig.12 ). Using the ninhydrin reaction, the amount of HA carried by MSN-HA and VNP-HA was calculated, and statistical analysis showed that both had the same number of HA grafts ( Figure 2 d), indicating that HA was successfully attached to the nanoparticles.

[0152] It is still necessary to experimentally determine whether CD44 protein can target VNP-HA. Therefore, the binding affinity of VNP-HA to CD44 protein was evaluated in the BLI experiment. In order to observe the interaction of CD44 protein with MSN-HA and VNP-HA, CD44 protein was immobilized on the biosensor chip. Compared with nanoparticles without HA, MSN-HA and VNP-HA showed stronger BLI signals through specific interaction with CD44 protein, indicating that HA-bound nanoparticles can target CD44 protein ( Figure 2 e. Fig.13 ).

[0153] In addition, the absorption efficiency and distribution of the nanoparticles within the cells were determined by fluorescence microscopy. To facilitate detection, rhodamine B was added to the nanoparticles as a fluorescent marker for tracking and imaging. SCC15 cells were seeded in 24-well culture plates and incubated with MSN-HA and VNP-HA culture solutions for 4 hours. The cellular uptake of VNP-HA was significantly higher than that of MSN-HA. The fluorescence generated by VNP-HA was approximately 6 times that of MSN-HA ( Figure 2 fh). Therefore, VNP-HA is expected to improve the delivery efficiency of anticancer drugs, and VNP-HA was used as a drug carrier in subsequent experiments.

[0154] 3. PTC209@VNP-HA inhibits HNSCC invasion and promotes HNSCC apoptosis in vitro

[0155] To evaluate the possible adverse effects of PTC209@VNP-HA, the cytotoxicity of empty nanoparticles at different concentrations was first evaluated in vitro. 2 、MSN-HA、VNP、VNP-NH 2and VNP-HA) were incubated with HNSCC or human umbilical vein endothelial cells (HUVECs) for 24 hours. Cell viability results showed that with the increase of dose, especially above 100 μg / mL, cell viability showed a downward trend. The survival rate of HNSCC cells treated with VNP-HA decreased to below 80%, while the survival rate of HUVEC cells increased ( Figure 3 a). Since normal cells have fewer CD44 receptors on their surface, they absorb HA-modified nanoparticles more slowly, resulting in weaker nanoparticle accumulation and reduced toxicity to normal cells. To investigate whether encapsulated PTC209 has antitumor effects, CAL27 and SCC15 cells were treated with free PTC209, PTC209@MSN-HA, and PTC209@VNP-HA at different concentrations (0.25, 0.5, 1, 2.5, 5, 7.5, and 10 μmol of PTC209 / L), respectively. Subsequently, MTT cell viability assay was performed and the half-maximal inhibitory concentration (IC 50 Compared with the PTC209@MSN-HA (10μM) and PTC209 (>10μM) treatment groups, the IC of PTC209@VNP-HA at 24h (5μM) 50 The value is significantly reduced ( Figure 3 b). These results indicate that PTC209@VNP-HA inhibits the growth of tumor cells. In the following in vitro experiments, a concentration of 5 μM PTC209 (lower than the IC 50 and free PTC209) were treated with cells in vitro for 24 h. After intravenous injection, the nanoparticles initially interacted with red blood cells. To test the effect of the nanoparticles on hemolysis, the nanoparticles MSN-NH 2 , MSN-HA, PTC209@MSN-HA, VNP-NH 2 Incubation of mouse erythrocytes with MSN-HA, VNP-HA, and PTC209@VNP-HA revealed that these nanoparticles induced low hemolysis (<5%). At higher concentrations (100 μg / mL), the hemolysis rates of MSN-HA and VNP-HA were lower than those of MSN-NH 2 or VNP-NH 2 , hemolysis rate <4% ( Fig.14 ). This reduction may be attributed to the HA-modified surface, which increases hydrophilicity and reduces potential damage to red blood cells.

[0156] In vitro invasion assays using PTC209@VNP-HA showed that PTC209@VNP-HA significantly reduced the invasive ability of HNSCC cells compared with free PTC209 and PTC209@MSN-HA ( Figure 3d, e). Immunostaining showed that cells treated with PTC209@VNP-HA displayed significantly higher levels of p-H2A.X, a marker of DNA damage, than those treated with free PTC209 or PTC209@MSN-HA ( Figure 3 f, g). Next, comet assay was performed to detect DNA damage. The results showed that both CAL27 and SCC15 cells treated with PTC209@VNP-HA exhibited significantly longer comet tails compared with cells treated with free PTC209 or PTC209@MSN-HA ( Figure 3 h, i), and damaged cellular DNA was successfully separated from intact DNA.

[0157] Based on the principle that HNSCC cells undergo apoptosis by activating DNA endonucleases that cut genomic DNA between nucleosomes, a TUNEL assay was performed to evaluate the relationship between DNA damage and apoptosis. The results showed that PTC209@VNP-HA promoted apoptosis of CAL27 and SCC15 cells through DNA damage ( Figure 3 j, k).

[0158] 4. PTC209@VNP-HA inhibits HNSCC stem cell characteristics

[0159] To demonstrate the effect of VNP-HA on CSCs in vitro, ALDH was isolated from CAL27 and SCC15 cells using flow cytometry. high Several experiments were performed to determine whether PTC209@VNP-HA could clear SCS. First, ALDH high CAL27 and ALDH high The levels of various CSC markers were evaluated in SCC15 cells. BMI1, ALDH1, and SOX2 protein levels were decreased after treatment ( Figure 4 a). Since sphere formation is an indicator of CSC self-renewal capacity, tumor sphere formation experiments were performed to further determine whether BMI1 can regulate tumorigenic potential. The results showed that PTC209@VNP-HA effectively reduced ALDH high The number of cell spheres ( Figure 4 b, c). The strong antitumor activity of PTC209@VNP-HA observed in vitro is conducive to further exploring the inhibitory effect of PTC209@VNP-HA on CSCs in vivo. First, the distribution of nanoparticles in vivo was observed. highMice bearing tumors induced by SCC15 cells were treated with control solution (PBS), MSN-HA or VNP-HA by tail vein injection, and the biodistribution of nanoparticles was monitored by in vivo imaging system after 1, 12 and 24 hours. The fluorescence signal corresponding to VNP-HA was detected at the tumor site after 12 hours, and the fluorescence signal was significantly enhanced after 24 hours ( Fig.15 ). Next, an in vivo dilution experiment was performed to confirm the changes in the tumorigenicity of CSCs after PTC209@VNP-HA treatment. The results showed that PTC209@VNP-HA significantly inhibited ALDH in vivo high Tumorigenicity of SCC15 cells in nude mice Figure 4 d, e).

[0160] 5. VNP-HA can improve the efficacy of PTC209 in preclinical HNSCC models

[0161] HNSCC cells, especially those that exhibit CSC characteristics, frequently migrate and proliferate in cervical lymph nodes. To determine whether PTC209VNP-HA would impair stemness and metastasis in orthotopic HNSCC in mice, a mouse orthotopic HNSCC model was used. high SCC15 control solution, PTC209 (low dose, 5 mg / kg), PTC209@MSN-HA (PTC209, dose 5 mg / kg), and CSC were inoculated on the tongue of mice, and then the mice were treated with PTC209VNP-HA (PTC209, dose 5 mg / kg) and PTC209 (high dose, 60 mg / kg) every 3 days for 3 weeks ( Figure 5 a). The results showed that PTC209@MSN-HA reduced orthotopic tumor growth, while low-dose PTC209 alone did not inhibit tumor growth, similar to the control group. Mice treated with nanomedicine showed viral-like morphology and reduced tumor volume compared with smooth morphology in mice treated with nanomedicine. PTC209@VNP-HA showed stronger inhibitory effect than low-dose PTC209, PTC209@MSN-HA ( Figure 5 bd). Anti-PCK immunostaining was performed on cervical lymph nodes to specifically detect lymph node metastasis. The results showed that compared with low-dose PTC209 alone, both PTC209@MSN-HA and PTC209@VNP-HA significantly reduced lymph node metastasis, and PTC209@VNP-HA reduced ALDH high The extent of CSC lymph node metastasis was greater than that of PTC209@MSN-HA( Figure 5e, f). The dose of PTC209 used in the in vivo experiment was 5 mg / kg, which is much lower than the dose (60 mg / kg) in previous studies. In addition, the in vivo antitumor effects of PTC209@VNP-HA and high-dose PTC209 on HNSCCs were compared. The results showed that the effects of the high-dose PTC209 group and the PTC209@VNP-HA group were similar, indicating that the proposed VNP design strategy can reduce the drug dose and thus increase the efficacy of PTC209 by 12 times.

[0162] 6. PTC209@VNP-HA overcomes cisplatin resistance in a cisplatin-resistant xenograft mouse model

[0163] Cisplatin chemotherapy is the main treatment strategy for patients with advanced HNSCC. Despite its widespread use and effectiveness, cisplatin resistance has become a major challenge. Increasing evidence suggests that SSCs are associated with resistance to cancer treatment and relapse or recurrence. However, effective eradication of CSCs and overcoming chemoresistance remain key challenges. A cisplatin-resistant model was established according to previously described methods. SCC15 cisplatin-resistant cells (SCC15-R) were implanted into the left axilla of nude mice; 1 week later, the mice were treated with control solution, cisplatin, PTC209@MNS-HA, PTC209@VNP-HA, or PTC209@VNP-HA combined with cisplatin for 3 weeks ( Figure 6 a). The results showed that compared with SCC15 cells, the proliferation ability of SCC15-R cells was enhanced, and IC 50 This confirms the successful establishment of the SCC15-R cell line ( Figure 6 b, c). The results showed that the virus-mimicking morphology promoted the tumor suppressor effect of PTC209 to a greater extent than the smooth morphology in the cisplatin-resistant model, although the model was not responsive to cisplatin treatment. Compared with cisplatin alone, PTC209@VNP-HA combined with cisplatin significantly inhibited the growth of tumor volume and weight, indicating that BMI1 inhibition can overcome cisplatin resistance in HNSCC ( Figure 6 df).

[0164] 7. PTC209@VNP-HA combined with cisplatin effectively inhibits HNSCC by clearing SCS

[0165] A 4NQO-induced HNSCC mouse model was previously developed (Rosa tdTomato ), the model is based on Bmil CreERThe 4NQO inducer was used for 18 weeks and then treated with deionized water for 4 weeks. The tumor-bearing mice were treated with control solution, cisplatin, PTC209@VNP-HA, or a combination of PTC209@VNP-HA and cisplatin. The mice were given tamoxifen-labeled tomato on the day before euthanasia. + BMI1 + CSC( Figure 7 a). The cisplatin group and the PTC209@VNP-HA group both had smaller tumor lesion areas than the control group. It is worth noting that the degree of reduction in lesion area by PTC209@VNP-HA combined with cisplatin was significantly greater than that by using cisplatin or PTC209@VNP-HA alone ( Figure 7 b, c). Histological evaluation showed that PTC209@VNP-HA reduced the number, size, and invasiveness of HNSCC tumors compared with the control group. When PTC209@VNP-HA was combined with cisplatin, the number, size, and invasiveness of HNSCC tumors were significantly reduced compared with PTC209@VNP-HA or cisplatin monotherapy ( Figure 7 df). Next, cervical lymph node metastasis was examined. Anti-PCK immunostaining confirmed that PTC209@VNP-HA reduced lymph node metastasis compared with the control group. PTC209@VNP-HA combined with cisplatin effectively eliminated the majority of HNSCC lymph node metastases ( Figure 7 gi). Immunostaining showed that compared with mice treated with cisplatin and PTC209@VNP-HA alone, the p-H2A.X level in the combined-treatment mice was significantly increased ( Figure 3 j, k) Subsequently, BMI1 was labeled in vivo + CSCs to evaluate the effect of combined treatment to eliminate these cells. The results showed that cisplatin increased BMI1 in HNSCC + The number of CSCs. In vivo labeling showed that the number of CSCs in HNSCC increased after cisplatin treatment compared with the control group. Unlike cisplatin alone, PTC209@VNP-HA combined with cisplatin reduced BMI1 + Compared with cisplatin or PTC209@VNP-HA alone, PTC209@VNP-HA combined with cisplatin also significantly improved the CSC clearance rate ( Figure 7 l, m).

[0166] Next, the safety of PTC209@VNP-HA was evaluated in nude mice. Histopathological examination showed no tissue damage in the heart, liver, spleen, lung, and kidney after administration of PTC209@VNP-HA. Evaluation of standard hematological and blood chemistry parameters showed that mice showed good tolerance to PTC209@VNP-HA ( Figure 8 ).

[0167] Discussion

[0168] CSC tumor cell subpopulation has the potential for self-renewal and differentiation, and plays a vital role in several key aspects of tumor growth, invasion, metastasis and chemotherapy resistance. CSC is able to drive tumor formation and continued expansion, and promote the progression of malignant tumors through complex molecular mechanisms. The small molecule inhibitor PTC209 can disrupt the self-renewal of CSCs. Although PTC209 significantly inhibits tumor progression alone or in combination with other treatments, challenges associated with immunogenicity, off-target gene effects and dose-limiting toxicity have hindered its widespread application in vivo.

[0169] In the embodiment, VNP-HA was designed for delivery of PTC209 as a highly effective SCS-targeted HNSCC treatment. Although viral vectors have been used to treat cancer, there are still concerns associated with immunogenicity and mutagenic risks. Therefore, the development of non-viral vectors with virus-like morphology may create a promising tool for achieving an appropriate balance between safety and efficacy. VNP-HA has multiple advantages in improving the therapeutic effect of PTC209, including its biocompatibility. Silica nanoparticles are widely used in nanocarrier design, and in vivo and in vitro experiments have shown that the cytotoxicity of VNP-HA is almost negligible. Hemolysis tests have shown that VNP-HA has little toxicity to blood cells in the body circulation. In order to ensure that VNP-HA can easily reach tumor tissues through blood circulation, factors such as the size and surface charge of the nanoparticles are also important. The size of VNP-HA is about 130nm, which seems to be ideal for accumulation and selective retention in HNSCC using the EPR effect. VNP-HA is negatively charged and binds relatively weakly to blood proteins, which contributes to its long-term circulation in the blood. In addition, the virus-mimicking morphology enhanced the cellular uptake efficiency of the nanoparticles and improved the cell delivery performance. The orthotopic tumor model showed that the virus-mimicking morphology of PTC209@VNP-HA promoted cellular uptake and enhanced the efficacy of PTC209 compared with PTC209@MSN-HA. Finally, through HA modification, the VNP surface can be easily functionalized to improve CSC targeting accuracy. Analysis showed that hyaluronic acid was successfully grafted onto the nanoparticles. Molecular affinity experiments showed that VNP-HA was able to successfully target the CD44 receptor protein.

[0170] Previous studies have shown that cisplatin treatment mainly eliminates proliferating cells, leaving CSCs unaffected. The present invention has demonstrated both in vitro and in vivo that PTC209@VNP-HA eliminates CSCs via the DNA damage / apoptosis pathway, effectively inhibiting tumor growth and metastasis. Although PTC209@VNP-HA inhibits the growth and metastasis of HNSCC, previous studies have shown that specific cytokines or growth factors can cause non-stem cell tumor cells to reprogram or redifferentiate into CSCs. PTC209@VNP-HA combined with cisplatin can effectively eliminate CSCs and non-CSCs to achieve the best therapeutic effect.

[0171] In summary, VNP-HA has multiple functions, high stability, low toxicity, good biodegradability and biocompatibility. In vitro and in vivo experiments showed that PTC209@VNP-HA can effectively eliminate CSCs, prevent metastasis, and overcome cisplatin resistance in HNSCC, which has important research value and clinical transformation potential. The virus mimicking strategy proposed in this invention will provide guidance for the design of innovative bio-nanomaterial drug delivery platforms and provide promising treatment methods for sustainable elimination of CSCs and tumor regression.

[0172] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A virus-like drug carrier targeting tumor stem cells, characterized in that: It includes a core particle, spikes and a capsule, wherein the core particle has a diameter of 50-400nm and has an open porous structure with a pore size of 0.5-8nm, the spikes are arranged on the surface of the core particle in a manner that does not substantially close the open porous structure, and the capsule is a hydrophilic layer and is wrapped around the surface of the core particle and the spikes.

2. The tumor stem cell-targeting viral drug carrier according to claim 1, characterized in that: The core particle and / or the spikes include an inorganic material, and the envelope includes an organic material.

3. The tumor stem cell-targeting viral drug carrier according to claim 2, characterized in that: The inorganic material includes at least one of clay, calcium carbonate, hydroxyapatite, graphene oxide, boron nitride and silicon dioxide.

4. The tumor stem cell-targeting viral drug carrier according to claim 2, characterized in that: The envelope is wrapped around the surface of the core particle and the spikes through active groups, and the active groups are selected from at least one of amino groups, quaternary ammonium salt groups, polyethyleneimine groups, imidazole groups, guanidine groups and pyridine groups.

5. The tumor stem cell-targeting viral drug carrier according to claim 2, characterized in that: The organic material is hyaluronic acid with a molecular weight less than 5 kDa.

6. The tumor stem cell-targeting viral drug carrier according to claim 1, characterized in that: The envelope has a pore structure that allows the drug to pass through.

7. A pharmaceutical composition, characterized in that The invention comprises the tumor stem cell-targeting virus-mimicking drug carrier according to any one of claims 1 to 6 and a drug loaded on the drug carrier.

8. The pharmaceutical composition according to claim 7, characterized in that It is a composition for systemic administration; Preferably, the drug is a small molecule drug.

9. A method for preparing a virus-like drug carrier targeting tumor stem cells, characterized in that: The following steps are involved: (1) preparing a core particle having an open porous structure, and performing a first functional modification on the core particle to obtain a core particle containing an active group; (2) preparing a spike precursor suspension, mixing it with the suspension of the core particles, generating spikes on the surface of the core particles under the condition that the surface openings of the core particles are not substantially closed, and performing a second functional modification on the core particles containing the spikes to obtain nanoparticles containing active groups; (3) covalently linking an organic polymer having reactive groups on its surface to the surface of the nanoparticles containing the reactive groups, thereby obtaining the viral-like drug carrier; Optionally, the method further comprises (4) embedding a drug in the viral-like drug carrier.

10. Use of the virus-mimicking drug carrier according to any one of claims 1 to 6 in the preparation of an anti-tumor drug composition.

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