Parvovirus-like drug carrier targeting tumor stem cells and preparation method and application thereof

Abstract

The application discloses a virus-mimic drug carrier targeting tumor stem cells and a preparation method and application thereof. The drug carrier can improve cell uptake efficiency by simulating physical surface characteristics of a biological system and can directly target tumor stem cells. In addition, the drug carrier can protect healthy tissues due to the targeting property, significantly reduce side effects of high-dose single use of a loaded drug such as PTC209, effectively enhance the response to tumor stem cells, inhibit tumor proliferation and metastasis, and reduce drug resistance of combined use of drugs such as cisplatin.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to a viral-like drug carrier targeting tumor stem cells, its preparation method, and its application. Background Technology

[0002] Biological macromolecules, such as DNA, siRNA, and proteins, as well as many small molecule drugs, struggle to enter target cells due to their poor stability, low solubility, and / or lack of targeting. This results in drug molecules failing to exert their therapeutic effects and potentially causing systemic toxicity. Various microbially derived natural carriers and chemically synthesized carriers have been widely used in drug delivery research. Compared to viral carriers, non-viral carriers, such as nanoparticles, are safer delivery tools. These nanoparticle systems have been widely used as carriers for antitumor drugs, enhancing their antitumor effects while minimizing adverse reactions. Existing nanoparticles primarily achieve targeted accumulation within tumor tissue through enhanced permeability and retention (EPR) effects caused by abnormal vascular leakage and impaired lymphatic drainage in the tumor region. However, relying solely on EPR-mediated passive targeting is insufficient 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, developing non-viral drug delivery systems with target specificity and high delivery efficiency remains a significant challenge.

[0004] The information in the background section is merely intended to illustrate the general background of the invention and should not be construed as an admission or implication in any way that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0005] To address at least some of the aforementioned technical problems, this invention designs a viral-like drug carrier with specific targeting capabilities, which can improve targeting of tumor stem cells, thereby enhancing drug delivery, particularly local delivery efficiency after systemic administration. Compared to currently known particles, the drug carrier of this invention has a larger surface area and superior drug loading capacity, and can be used as a carrier for many systems. This invention uses inorganic materials as the core particle, while incorporating biomimetic spike structures on its surface, making the core particle surface rough. Furthermore, a biomimetic envelope, particularly a covalently bound hyaluronic acid (HA) membrane, is further provided on the surface of the drug carrier to further enhance its targeting ability to tumor stem cells and its biocompatibility. Moreover, this invention can also adsorb drugs, particularly small molecule drugs, such as, but not limited to, PTC209, into the porous structure of the core particle of the drug carrier, thereby achieving a more effective therapeutic effect. Specifically, this invention includes the following:

[0006] In a first aspect, the present invention provides a viral-like drug carrier for targeting tumor stem cells, comprising a nuclear particle, spikes, and a membrane, wherein the nuclear 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 disposed on the surface of the nuclear particle in a manner that substantially does not close the open porous structure, and the membrane is a hydrophilic layer that wraps around the surfaces of the nuclear particle and the spikes.

[0007] In some embodiments, the viral drug carrier targeting tumor stem cells according to the present invention includes, but is not limited to, inorganic materials, and the envelope layer includes, but is not limited to, organic materials.

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

[0009] In some embodiments, the viral drug carrier targeting tumor stem cells according to the present invention, wherein the envelope is wrapped around the surface of the nucleus and spike by an active group selected from at least one of amino, quaternary ammonium salt, polyethyleneimine, imidazole, guanidine, and pyridyl groups.

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

[0011] In some embodiments, the viral drug carrier for targeting tumor stem cells according to the present invention has a membrane having a porous structure that allows drugs to pass through.

[0012] A second aspect of the invention provides a pharmaceutical composition comprising a viral drug carrier targeting tumor stem cells as described in this invention and a drug, particularly a small molecule drug, loaded on said carrier. Preferably, the pharmaceutical composition is a systemic administration composition.

[0013] A third aspect of the present invention provides a method for preparing a viral-like drug carrier targeting tumor stem cells, comprising the following steps:

[0014] (1) Prepare core particles with open porous structures and perform a first functionalization modification on the core particles to obtain core particles containing active groups;

[0015] (2) Prepare a spike precursor suspension, mix it with the suspension of the core particle, generate spikes on the surface of the core particle under the condition that the surface opening of the core particle is not substantially closed, and perform a second functionalization modification on the core particle containing spikes to obtain nanoparticles containing active groups.

[0016] (3) An organic polymer with reactive groups on its surface is covalently attached to the surface of the nanoparticles containing active groups to obtain the virus-like drug carrier.

[0017] In some embodiments, the method for preparing a viral drug carrier targeting tumor stem cells according to the present invention further includes (4) the step of encapsulating a drug within the viral drug carrier.

[0018] In some embodiments, according to the method for preparing a viral drug carrier targeting tumor stem cells according to the present invention, step (2) includes:

[0019] (1') Emulsify the catalyst that can provide an alkaline environment with an organic solvent, add the spike precursor and stir to obtain the spike precursor suspension;

[0020] (2') The spike precursor suspension is added to the nuclear particle suspension for reaction.

[0021] A fourth aspect of the invention provides the use of the viral-mimicking drug carrier according to the invention in the preparation of antitumor drug compositions.

[0022] In some embodiments, according to the application described in the invention, the pharmaceutical composition is a systemic administration composition.

[0023] In some embodiments, according to the application described in the invention, the application includes combined use with other therapeutic agents.

[0024] This invention develops a targeted biomimetic nanomedicine carrier that simultaneously addresses issues such as tumor proliferation and metastasis, side effects associated with small molecule drugs, and resistance to combination therapies like cisplatin. In some embodiments, addressing the high incidence of side effects from the clinical use of PTC209 at high doses alone, this invention provides a viral-like drug carrier targeting tumor stem cells. This carrier enhances cellular uptake efficiency by mimicking the physical surface properties of biological systems and directly targets tumor stem cells via its envelope. Furthermore, it protects healthy tissue while effectively enhancing the response to tumor stem cells, inhibiting tumor proliferation and metastasis, and mitigating resistance to combination therapies like cisplatin. This highly efficient drug carrier platform provides a novel biomimetic strategy for targeting tumor stem cells. Attached Figure Description

[0025] Figure 1 Synthesis and characterization of PTC209@VNP-HA. (a) Transmission electron microscopy (TEM) images showing the nanomorphic forms of (i) MSN, (ii) MSN-NH2, (iii) VNP, (iv) VNP-NH2, and (v) VNP-HA. Scale bar: 50 nm. (b) Dynamic light scattering analysis of hydrodynamic diameter and (c) potential in PBS. (d) Nitrogen uptake 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 at different pH values.

[0026] Figure 2 Virus mimicry morphology promotes CSC uptake. (a) TEM and (b) SEM images show the nanomorphic forms of (i) MSN-HA and (ii) VNP-HA. Scale bar: 50 nm. (c) Fourier transform infrared (FTIR) spectra of MSN-NH2, MSN-HA, VNP-NH2, VNP-HA, and HA. (d) HA concentrations of MSN-HA and VNP-HA. (e) Biolayer interferometry (BLI) assessment of CD44 binding kinetics with (i) MSN-HA and (ii) VNP-HA. (fh) Confocal microscopy images showing uptake of MSN-HA and VNP-HA by cells (red), nuclei (blue), and membranes (green). Scale bar: 40 μm. Data represent the proportion of nanoparticles uptake 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) p-H2A.X immunofluorescence imaging and quantification (red), cell nuclei stained with DAPI (blue). (g, h) Images and quantification of DNA comets (10 cells per group) in CAL27 and SCC15 cells treated with free PTC209, PTC209@MSN-HA, or PTC209@VNP-HA. Scale bar: 100 μm. (i, j) Apoptosis in CAL27 and SCC15 cells 24 h after treatment, detected by TUNEL staining. Scale bar: 50 μm. **P<0.01.

[0028] Figure 4 PTC209@VNP-HA inhibits stem cell characteristics in head and neck squamous cell carcinoma (HNSCC). (a) Western blot analysis shows stem cell markers after PTC209@VNP-HA treatment. (b) High aldehyde dehydrogenase (ALDH) activity. high CAL27 and (c) ALDH high Quantification and visualization of tumor spheroid formation in SCC15 cells treated with PTC209@VNP-HA 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 (a) Time-sharing of CSC mice during treatment and sacrifice. (b) Tumor images (circled areas indicate lesions). (c) Tumor volume quantification. (d) Hematoxylin and eosin (H&E) staining of HNSCCs. The upper and lower columns 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) Quantification of lymph node metastasis areas. *P<0.05, **P<0.01.

[0030] Figure 6 PTC209@VNP-HA overcomes cisplatin resistance in a cisplatin-resistant xenograft mouse model. (a) Schematic diagram of SCC-R tumor model establishment and treatment. (b) MTT analysis of cisplatin-treated SCC15 cells and SCC15 cisplatin-resistant cells, and (c) IC50 analysis. 50 (d) Image of tumor sample. Scale bar: 1 cm. (e) Tumor volume and growth. (f) Tumor weight. *P<0.05, **P<0.01.

[0031] Figure 7 PTC209@VNP-HA combined with cisplatin therapy effectively inhibited HNSCC by clearing CSCs. (a) Administration of tamoxifen-labeled tomato + BMI1 + Schematic diagram of CSCs. (b, c) Image of tongue tumor; circled area is the lesion. Scale bar: 2 mm. (d) Representative image of HNSCCs stained with H&E. The upper and lower columns show low-magnification (scale bar 500 μm) and high-magnification (scale bar 100 μm) images, 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 metastatic lymph nodes (h) percentage and (i) area. (j, k) Immunofluorescence image and quantification of cells (p-H2A.X) (red) and cell nuclei (DAPI, blue). n = 12. Scale bar: 25 μm. (l, m) Bmil + Tomato + CSC images and quantification of HNSCC. n = 12. White dashed line represents the tumor-stromal boundary. Scale bar: 25 μm. *P<0.05, **P<0.01.

[0032] Figure 8 (a) Histological analysis of organs from 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 routine blood and blood biochemical analysis of serum samples from nude mice after different treatments.

[0033] Figure 9 The surface morphology changes were observed when the feed volume of the shell and particle 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.

[0034] Figure 10(a) Characterization of nitrogen adsorption isotherms (b) (i) Characterization of pore size distribution of MSN and (ii) VNP.

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

[0036] Figure 12 (a) TGA curves of MSN, MSN-NH2, and MSN-HA. (b) TGA spectra of VNP, VNP-NH2, and VNP-HA. The amounts of MSN-coupled amino groups and HA were approximately 4.59% and 3.32%, respectively. The contents of VNP-coupled amino groups and HA were approximately 4.64% and 3.42%, respectively.

[0037] Figure 13 BLI evaluated the binding performance of CD44 protein (mouse) to (a) MSN-HA and (b) VNP-HA, with CD44 protein immobilized on the biosensor.

[0038] Figure 14 (a) MSN-NH2, MSN-HA, PTC209@MSN-HA, (b) Hemolytic effect of VNP-NH2, VNP-HA, PTC209@VNP-HA on erythrocytes of ICR mice.

[0039] Figure 15 (a) Ex vivo imaging of organs and tumors in mice after injection of (i) MSN-HA and (ii) VNP-HA. (b) Quantitative accumulation in tumors at different time intervals. Detailed Implementation

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

[0041] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0042] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0043] Viral drug carrier

[0044] In one aspect, the present invention provides a viral-like drug carrier for targeting tumor stem cells (sometimes referred to herein as "the drug carrier of the present invention"), comprising a nuclear particle, spikes, and a capsule, wherein the nuclear 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 disposed on the surface of the nuclear particle in a manner that substantially does not close the open porous structure, and the capsule is a hydrophilic layer that wraps around the surfaces of the nuclear particle and the spikes.

[0045] In this invention, the core particles serve as the main drug carrier. Unlike traditional viral-like carriers, the core particles in this invention are inorganic material core particles. The inorganic materials are not particularly specific, and examples include, but are not limited to, clay, calcium carbonate, hydroxyapatite, graphene oxide, boron nitride, and silicon dioxide. This invention may use one or more of the above materials, or a combination of two or more of them. When used in combination, the proportions of each material are not specific and can be freely set by those skilled in the art as needed.

[0046] In this invention, the diameter of the nucleus particle is generally 50-400 nm, preferably 60-350 nm, such as 70-300 nm, 80-250 nm, 90-200 nm, 100-150 nm, 100-140 nm, and more preferably 110-140 nm, such as 110-135 nm, 115-135 nm, 120-135 nm, 125-135 nm, such as 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135 nm.

[0047] In this invention, the nuclear particle must have an open porous structure. Here, "open" means that at least one end of the pore in the porous structure extends to the surface of the nuclear particle. Both ends of the porous structure can extend to the surface and open, thereby forming a porous structure that penetrates the nuclear particle. The porous structure can also extend to the surface at only one end to form an opening, thereby forming a porous structure that is open at one end and closed at the other. The average pore size of the pore is generally 0.5-8 nm, preferably 3-8 nm, and even more preferably 3.5-8 nm (e.g., 3.5-7.5 nm, 3.5-7 nm, 3.5-6.5 nm, 3.5-6 nm, 3.5-5.5 nm), for example 3.5 nm, 4 nm, 4.5 nm, 5 nm, 5.5 nm, 6 nm, 6.5 nm, 7 nm, 7.5 nm, 8 nm. The pore volume is not less than 0.5 cm³ / g, preferably not less than 0.6 cm³ / g, and even more preferably not less than 0.7 cm³ / g. The specific surface area is not less than 350 m² / g, preferably not less than 400 m³ / g, even more preferably not less than 450 m³ / g, and even more preferably not less than 500 m³ / g. In a specific embodiment, the average pore size of the core particles is 5.5 nm, the pore volume is 0.710363 cm³ / g, and the specific surface area is 505.7690 m² / g.

[0048] In this invention, the spikes of the viral drug carrier refer to protruding structures extending from the surface of the core particle. Preferably, the protruding structure of the spikes resembles the rough surface (shell particle) of a virus. In some embodiments, unlike viruses, the spikes of this invention are composed of inorganic materials. Here, inorganic materials are not particularly limited, and examples include, but are not limited to, clay, calcium carbonate, hydroxyapatite, graphene oxide, boron nitride, and silicon dioxide. This invention may use one or more of the above materials, or a combination of two or more of the above materials. 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. It should be noted that both the spikes and the core particle of this invention can be inorganic materials, and the spike material and the core particle material can be the same or different.

[0049] In this invention, spikes are used to increase or enhance the roughness of the surface of nuclear particles, thereby promoting the adsorption or endocytosis of carriers or drugs by cells. The shape and size of the spikes are not particularly limited as long as this objective is achieved. To achieve the above objective, spikes are typically 1-60 nm in size, such as 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 8 nm, 10 nm, 12 nm, 14 nm, 16 nm, 18 nm, 20 nm, 22 nm, 24 nm, 26 nm, 28 nm, 30 nm, 32 nm, 34 nm, 36 nm, 38 nm, 40 nm, 45 nm, 50 nm, etc. Furthermore, spikes typically need to be smaller than the size of the nucleus, for example, 1 / 10 to 1 / 600 of the nucleus 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 increasing or improving roughness. On the other hand, if the spike size is too small, it tends to close the openings of the porous structure, which is not conducive to drug loading and also not conducive to increasing or improving roughness. In addition, spikes typically need to be larger than the pore size of the porous structure to avoid the spikes closing the surface openings of the nucleus.

[0050] In this invention, the number of spikes needs to be controlled to avoid excessive spikes that could cause the closure of the surface openings of the nucleus particle. The number of spikes should ideally occupy more than 1 / 30 of the surface area of ​​the nucleus particle, for example, more than 1 / 25, 1 / 20, 1 / 15, or 1 / 10. If the number is too small, it will affect the surface roughness, thereby affecting the absorption of the carrier or drug by the cell. On the other hand, the number of spikes should ideally occupy less than 3 / 4 of the surface area of ​​the nucleus particle, for example, less than 1 / 2, 1 / 4, 1 / 5, 1 / 6, 1 / 7, or 1 / 8. If the number is too large, it will affect the surface openings of the nucleus particle, thereby affecting drug loading and subsequent drug release.

[0051] In this invention, the bonding between the core particle and the spike is not limited, for example, through non-covalent or covalent bonds. Preferably, the core particle and the spike are tightly bonded. For example, bonding can be achieved through a connection method selected from electrostatic adsorption, hydrogen bonding, and covalent bonding. More preferably, the core particle carries an active group, said active group being selected from at least one of amino, quaternary ammonium salt, polyethyleneimine, imidazole, guanidine, and pyridinyl groups, and more preferably amino, i.e., the core particle has an amino modification.

[0052] In this invention, the capsule is the outermost layer of the drug carrier. The capsule of this invention not only provides a hydrophilic surface layer, improving biocompatibility, but also provides specific targeting to tumor stem cells. For this purpose, the capsule of this invention is preferably prepared from organic materials. Organic materials include hydrophilic materials, preferably organic materials containing reactive groups (such as carboxyl groups) and groups or molecules targeting tumor stem cells, and more preferably, excluding lipid materials. Examples of hydrophilic materials that can be used in this invention include, but are not limited to, hyaluronic acid, which has the property of targeting proteins on the surface of tumor stem cells, particularly the CD44 receptor, a protein widely and highly expressed on the surface of tumor stem cells.

[0053] In this invention, preferably, the coating is covalently bonded to the surface of the spikes and nuclei. To achieve covalent bonding, preferably, the molecules in the coating are bonded to the surface of the spikes and nuclei via active groups. Here, the active groups are not particularly specific, and examples include, but are not limited to, amino, quaternary ammonium salt, polyethyleneimine, imidazole, guanidine, and pyridyl groups. This invention may use one or more combinations of the above-mentioned active groups. In some embodiments, this invention preferably uses an amino group as the active group.

[0054] In this invention, the molecular weight of the organic material is not particularly limited as long as the objective of the invention can be achieved. Generally, the molecular weight of organic materials such as hyaluronic acid is 5 kDa or less, preferably 3 kDa or less, even more preferably 2 kDa or less, and further preferably 1 kDa or less. If the molecular weight is too large, it is not conducive to drug loading or drug release after administration. On the other hand, the molecular weight of organic materials such as hyaluronic acid is generally 50 Da or more, preferably 100 Da or more, such as 150 Da or more, 200 Da or more, 250 Da or more, etc. If the molecular weight is too small, the resulting drug carrier or drug targeting will be poor. In a preferred embodiment, the molecular weight of hyaluronic acid is 500-1000 Da.

[0055] In this invention, the source of hyaluronic acid is not particularly limited, and can be derived from hyaluronic acid or its salts with a molecular weight greater than oligomers, including but not limited to hyaluronic acid or its salts extracted from animal tissues, hyaluronic acid or its salts obtained through microbial fermentation, or hyaluronic acid or its salts obtained through artificial synthesis. It also includes degraded hyaluronic acid or its salts obtained through further processing or treatment of hyaluronic acid or its salts from the above sources. Such processing includes enzymatic hydrolysis, chemical decomposition, and ultrasonic treatment.

[0056] In this invention, the relative molecular mass (sometimes simply referred to as "molecular weight") of hyaluronic acid can be determined using known methods. An exemplary 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)℃ to measure the outflow time, using the one-point method to determine the intrinsic viscosity, and calculating the relative molecular mass according to the empirical formula: viscosity = 3.6×10Mr.

[0057] In this invention, the amount of organic material is generally 0.5-10%, preferably 1-8%, and even more preferably 2-6% (e.g., 2-5%, 2-4%), such as 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6%. Excessive amounts are detrimental to drug loading and release after drug administration. Conversely, insufficient amounts result in poor tissue compatibility and targeting. In this invention, the quantification of organic material is determined using 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 coating is typically calculated. For example, MSN, MSN-NH2, VNP, VNP-NH2, and VNP-HA are first dispersed in deionized water. Then, DMSO is mixed at 90°C for 20 minutes to induce a colorimetric reaction. After cooling the mixture to room temperature, ethanol is added to stop the reaction, and the absorbance at 570 nm is recorded using a microplate reader. A standard curve is plotted using a glycine solution. After grafting HA onto amino-modified nanoparticles, the remaining amino content was calculated via the ninhydrin reaction. The amount of HA grafted was determined by comparing the remaining amino content with the amino content before HA grafting.

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

[0059] Pharmaceutical Composition

[0060] A second aspect of the present invention provides a pharmaceutical composition comprising the pharmaceutical carrier described in the first aspect and a drug. The drug is adsorbed or embedded within and / or on the surface of the pharmaceutical carrier.

[0061] In this invention, the drug is not limited and any known drug can be used. Preferably, the drug of this invention is a small molecule drug, such as an antitumor drug. The drug carrier of this invention needs to contact cells to promote cell absorption. Simultaneously, since the drug needs to be loaded onto the carrier, hydrophilic drugs, especially small molecule hydrophilic drugs, are more conducive to achieving the objectives of this invention. In addition to the above, the inventors have discovered that the drug carrier of this 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] To address the ongoing challenges in the preparation of viral-mimicking nanomedicines, this invention, in its third aspect, provides a method for preparing a viral-mimicking drug carrier targeting tumor stem cells. The inventors have conducted extensive research on the preparation process and discovered that the viral-mimicking nanomedicine prepared using the method of this invention possesses excellent properties (including but not limited to drug loading, targeting, and therapeutic properties). Specifically, the preparation method of this invention includes:

[0064] (1) Prepare core particles with open porous structure, and perform a first functionalization modification on the core particles to obtain a core particle suspension containing active groups;

[0065] (2) Prepare a spike precursor suspension, mix it with the core particle suspension, generate spikes on the surface of the core particle under the condition that the surface opening of the core particle is not substantially closed, and perform a second functionalization modification on the core particle containing spikes to obtain nanoparticles containing active groups.

[0066] (3) An organic polymer with reactive groups on its surface is grafted onto the surface of the nanoparticles containing active groups to obtain the virus-like nanoparticles.

[0067] In step (1) of the present invention, by way of example, organosilicon compounds (e.g., MTES, TEOS, etc.) and cationic surfactants (e.g., CTAB, CTAC, etc.) are used as mesoporous template agents, organic solvents (e.g., aliphatic hydrocarbon solvents, such as pentane, cyclohexane, octane, cycloheptane, etc.) are used as oil phases, and basic compounds (e.g., ethanolamine, diethanolamine, etc.) are used as catalysts and pH adjusters to obtain core particles with open porous structures.

[0068] In this invention, the core particles undergo a first functionalization modification, thereby enabling them to carry a positive charge on their surface through surface modification. Preferably, the core particles from step (1) are dispersed in an organic solvent, an amino-containing compound is added, and the mixture is heated under reflux and then centrifuged 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 combinations thereof. Furthermore, compounds containing both amino groups and polymerizable functional groups, such as vinyl and propenyl groups, can also be used in this invention, thereby introducing an amino group while simultaneously forming a dendritic structure on the surface of the core particles through a polymerization reaction.

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

[0070] Step (2) of this invention involves preparing a spike precursor suspension and forming spikes on the core particle. To achieve spikes on the surface of the core particle in a manner that substantially does not close the open porous structure, the inventors conducted extensive research and discovered that the addition steps and volume ratios of the core particle suspension and the spike precursor suspension can affect the closure of the pores by the spikes. First, a catalyst capable of providing an alkaline environment is emulsified with an organic solvent, and the particle precursor is added and stirred to obtain the spike precursor suspension. The catalyst providing the alkaline environment is not particularly limited, but includes, for example, L-arginine, alkanolamine compounds, pyridine, imidazole, quaternary ammonium salts, etc. The organic solvent includes, but is not limited to, aliphatic hydrocarbon solvents, such as pentane, cyclohexane, octane, cycloheptane, etc. The particle precursor refers to any suitable compound capable of providing particulate form after the reaction, such as a precursor capable of providing, but not limited to, nanoparticles of calcium carbonate, graphene oxide, boron nitride, silicon dioxide, etc., which are known to those skilled in the art. The particulate precursors used in this invention include, but are not limited to, ethyl silicate (e.g., tetraethyl orthosilicate), sodium silicate, silica powder, diatomaceous earth, and silica.

[0071] In the above steps, the weight-to-volume ratio (m / v, mg / mL) of the catalyst and the particulate precursor is 50-200:1, preferably 100-200:1, and even more preferably 150-200:1. An aliphatic hydrocarbon solvent is added to an aqueous solution containing the catalyst and stirred to emulsify. Then, the particulate precursor is added and stirred at 40-80°C (preferably 45-75°C, even more preferably 50-70°C, and further preferably 55-65°C) for 1-10 h, preferably 1-5 h, 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 nuclear particle suspension was added to the spike precursor suspension for reaction, the spikes formed on the surface of the nuclear particle blocked the mesoporous structure of the nuclear particle. This further affected the entry of small molecules into the mesopores, thereby reducing the drug loading and ultimately affecting the therapeutic or tumor-improving effect. However, when the spike precursor suspension was added to the nuclear particle suspension for reaction, the spikes were set on the surface of the nuclear particle in a substantially open, porous structure. This allowed all or almost all of the small molecule drugs to be loaded onto the nuclear particle, greatly increasing the drug loading and further ensuring the therapeutic or tumor-improving effect. In some embodiments, when the nuclear particle suspension was added to the spike precursor suspension for reaction, the spikes formed on the surface of the nuclear particle blocked the mesoporous structure of the nuclear particle. The pore size was measured to be less than 3 nm, the pore volume less than 0.55 cm³ / g, and the specific surface area less than 375 m² / g. In one specific implementation, when the nuclear particle suspension was added to the spike precursor suspension for reaction, it was determined that the pore size was less than 3 nm, the pore volume was 0.508043 cm³ / g, and the specific surface area was 374.7986 m² / g.

[0073] In a preferred embodiment, the concentration of amino-modified nuclear particles (sometimes referred to herein as MSN-NH2) in the buffer solution is 0.1-10 mg / ml, preferably 0.1-8 mg / ml, even more preferably 0.1-6 mg / ml, further preferably 0.5-4 mg / ml, and more preferably 1-4 mg / ml, for example 1, 1.5, 2, 2.5, 3, 3.5, 4 mg / ml.

[0074] To obtain better particle morphology, that is, to maintain the complete morphology of the virus-like nanoparticles so as to facilitate subsequent organic polymer grafting, the volume ratio of the core particle suspension to the spike precursor suspension can be controlled in the range of 2.5-6:1, such as 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, and 6:1.

[0075] To carry out the grafting reaction in the subsequent step (3), a compound containing an active group (preferably amino) is grafted onto the particles prepared in step (2) to form a bioactive surface that can be used to graft organic polymers. First, the stirrer is heated to 100-200°C (preferably 110-180°C, more preferably 120-160°C, and even more preferably 130-150°C), while 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-containing compound is added dropwise. 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 combinations thereof. Furthermore, compounds containing both an amino group and polymerizable functional groups, such as vinyl or propenyl groups, can also be used in this invention, thereby introducing an amino group while simultaneously forming a dendritic structure on the surface of the particles prepared in step (2) through a polymerization reaction.

[0076] In the modification process described above, the volume ratio of the organic solvent to the amino-containing compound is 0.1-10:1, preferably 0.1-5:1, and even more preferably 0.1-2:1. The mixture is then stirred in a stirrer at the temperature mentioned above for 1-36 h (preferably 5-24 h, even more preferably 8-20 h, and even more preferably 10-15 h). The mixture is then centrifuged, washed with alcohol, and stored.

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

[0078] In step (3), at room temperature, an organic polymer containing carboxyl groups (preferably hyaluronic acid) is activated in a buffer solution using EDC as a catalyst for 0.1-5 h, preferably 0.1-2 h, and even more preferably 0.1-1 h. The weight ratio of catalyst to organic polymer is 1:1-10, preferably 1:1-8, even more preferably 1:1-6, and even more preferably 1:1-5. NHS is then added, and the mixture is stirred for another 0.1-5 h (preferably 0.1-2 h, even more preferably 0.1-1 h). The weight ratio of EDC to NHS is 1:1-5, preferably 1:1-2, and even 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. The weight ratio of the nanoparticles containing active groups to the organic polymer is 1:1-10, preferably 1:1-8, more preferably 1:1-6, and even more preferably 1:1-5. The reaction time is 0.1-10 h, preferably 0.1-5 h, and even more preferably 1-5 h.

[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, even more preferably 0.1-2 mg / ml, and 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 includes step (4), which is the step of encapsulating a small molecule drug within the viral-like drug carrier. Known methods in the art can be used for the post-loading process of small molecules, such as, but not limited to, using rotary evaporation to load small molecule drugs, such as PTC209, onto the prepared viral-like drug carrier.

[0081] application

[0082] A fourth aspect of the invention provides the use of a viral-like drug carrier in the preparation of an antitumor drug composition. The drug or composition of the invention is used to treat or improve tumors in a subject in need, 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 systemic drug delivery composition and targets the tumor site, particularly tumor stem cells.

[0084] In a preferred embodiment, the tumor is a solid tumor, particularly 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), or tongue cancer.

[0085] In this invention, the term "treatment or improvement" refers to therapeutic or preventative measures aimed at slowing (reducing) undesirable physiological changes or disorders, such as the occurrence, development, or worsening of a tumor. Beneficial or desired clinical outcomes include, but are not limited to, the following, whether detectable or undetectable: symptom relief, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and reduction (whether partial or complete). Those requiring treatment include individuals who already have head and neck tumors or head and neck tumor-related diseases, or those who require prevention or improvement of head and neck tumors or head and neck tumor-related diseases.

[0086] In this 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 the volume and / or weight of tumor cells;

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

[0091] (5) Extend survival time.

[0092] This invention further provides the use of biomimetic drug carriers or pharmaceutical compositions comprising them in the preparation of medicaments for the combined treatment of tumors (e.g., head and neck tumors) with other drugs. In this invention, "combination therapy" refers to the use of the biomimetic drug carriers or pharmaceutical compositions comprising them of this invention as part of a specific treatment regimen, intended to produce a beneficial (additive or synergistic) effect through the combined action of the biomimetic drug carriers or pharmaceutical compositions comprising them with one or more other drugs. The beneficial effects of the combination include, but are not limited to, the pharmacokinetic or pharmacodynamic synergistic effects resulting from the combination of the biomimetic drug carriers or pharmaceutical compositions comprising them with other drugs. The combined administration of the biomimetic drug carriers or pharmaceutical compositions comprising them with other drugs is typically carried out over a prescribed period of time (typically minutes, hours, days, or weeks, depending on the chosen combination). "Combination therapy" includes the sequential administration of the biomimetic drug carriers or pharmaceutical compositions comprising them with other drugs (i.e., each drug is administered at different times), as well as the substantially simultaneous administration of these biomimetic drug carriers or pharmaceutical compositions comprising them with other drugs, or they may be formulated as a single co-formulated pharmaceutical composition containing two components.

[0093] In this invention, other drugs or therapeutic agents are not particularly limited, but include, but are not limited to: EGFR inhibitors (e.g., cetuximab), immune checkpoint inhibitors (PD-1 / PD-L1, e.g., pembrolizumab, nivolumab), EGFR-TKIs (tyrosine kinase inhibitors, e.g., afatinib), PI3K / mTOR pathway inhibitors (e.g., everolimus), antibody-drug conjugates (ADCs, Datopotamab Deruxtecan (Dato-DXd)), PARP inhibitors (olaparib), and 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 layering method. Tetraethyl orthosilicate (TEOS) was used as the silicon source, hexadecyltrimethylammonium chloride (CTAC) was used as the template agent for the mesoporous structure, and cyclohexane was used as the oil phase to promote the formation of a stable two-phase system. Triethanolamine was used as a catalyst and pH adjuster. 0.36 g of triethanolamine, 48 mL of 25 wt% hexadecyltrimethylammonium chloride, and 72 mL of deionized water were added to a round-bottom flask. 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 dissolved in cyclohexane was added to the reaction system along the wall of the flask. After slow stirring at 60°C for 24 hours, a white reaction product was obtained.

[0097] After transferring the reaction solution to a separatory funnel, the milky white aqueous phase containing MSN was separated and centrifuged at 20000×g for 60 minutes to obtain MSN precipitate. Subsequently, MSN was washed at 60°C with acidic methanol (37% hydrochloric acid and methanol mixed at a volume ratio of 1:10) to remove residual impurities and CTAC from the mesopores. Finally, after washing with anhydrous ethanol, the MSN was dispersed in anhydrous ethanol for later use, with an average particle size of 110 nm.

[0098] 2. MSN Modification

[0099] Amino modification of MSN resulted in positively charged nanoparticles. Specifically, 250 mg of MSN was dispersed in 150 ml of xylene, and 2.5 ml of an aminosilane source, 3-aminopropyltriethoxysilane (APTES), was added. After heating under reflux for 12 hours, the mixture was centrifuged at 20000×g for 30 minutes to obtain MSN-NH2, which was then repeatedly washed with anhydrous ethanol. Finally, MSN-NH2 was dispersed in anhydrous ethanol for later use.

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

[0101] VNP consists of peripheral particles and an MSN core. To prepare the peripheral particles, L-arginine (87 mg) was dissolved in deionized water (69.5 mL), and octane (5.23 mL) was added followed by high-speed stirring to emulsify. After adding TEOS (0.5 mL), the mixture was stirred at 60°C for 3 hours to obtain a suspension containing peripheral particles with a particle size of approximately 10 nm. Subsequently, MSN-NH2 (100 mg) was dispersed in carbonate buffer (50 mL, pH 9.5, Ct[CO3]). 2- VNPs were obtained by reacting the ions in a solution of 50 mmol / L with a suspension of 16 mL of peripheral particles, reacting at room temperature for 8 hours, and washing with anhydrous ethanol.

[0102] The steps for amino-modifying VNP included grafting an aminosilane onto the VNP shell particles to form a bioactive surface suitable for HA grafting. The stirrer was heated to 145°C while VNP (250 mg) was added to 150 mL of xylene. Then, 2.5 mL of APTES was added dropwise, and the mixture was stirred at 145°C for 12 hours using a rectifier with a heated stirrer. The VNP-NH2 was centrifuged at 20000×g for 30 minutes 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 VNPs to endow them with the ability to target and recognize tumor stem cells. At room temperature, the carboxyl groups of HA (32 mg) were activated for 0.5 h in phosphate-buffered saline (PBS, pH 7.4, 25 mL) using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 12 mg) as a catalyst. N-hydroxysuccinimide (NHS, 14 mg) was added, and the mixture was stirred for another 0.5 h. Then, MSN-NH2 and VNP-NH2 (100 mg) were dispersed separately in PBS (pH 7.4, 25 mL) and condensed with the carboxyl-activated HA for 3 h. 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 small molecule drug PTC209 was loaded into mesoporous structures using a solvent evaporation method for antitumor drug delivery. 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, then mixed. The DMF was completely evaporated under a nitrogen atmosphere at room temperature to obtain dry PTC209@VNP-HA and PTC209@MSN-HA powders, respectively.

[0107] Example 2

[0108] I. Experimental Methods

[0109] 1. Characterization of nanoparticles

[0110] The samples were measured using transmission electron microscopy (TEM, 100 kV). A small amount of sample was dispersed in ethanol and then transferred to a copper grid. Images of the PTC209@MSN-HA and PTC209@VNP-HA samples were acquired using high-resolution transmission electron microscopy (HRTEM) and energy-dispersive X-ray spectroscopy (EDX). 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. Zetasizer Pro analysis system was used to measure zeta potential and dynamic light scattering to determine surface charge and particle size distribution, respectively. Nitrogen adsorption-desorption measurements were performed at -196 °C using an ASAP 2020 analyzer to obtain porosity data, including specific surface area, pore size distribution, and total pore volume. Fourier transform infrared (FTIR) spectroscopy analysis was performed on MSN-NH2, MSN-HA, VNP-NH2, VNP-HA and HA samples to confirm that HA had been successfully attached to 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 profiles of PTC209 from PTC209@VNP-HA were evaluated using PBS dialysis 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 2000 Da to ensure that only smaller molecules could diffuse freely, while larger nanoparticles were confined within the bag. The dialysis bag was placed in a shaker at 37°C and stirred at 100 rpm to promote diffusion. 1.5 mL of the released culture medium was collected, and an equal volume of PBS was added to the dialysis system to maintain a constant volume and ensure continuous diffusion. Each sample was centrifuged at 20000×g for 10 min to separate the supernatant containing dissolved PTC209. 1 mL of the supernatant was 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 235 nm. The concentration of PTC209 in each sample was determined by comparing the measured absorbance values ​​with the calibration curve. These concentrations were then plotted as release curves over time.

[0113] 3. The binding specificity of VNP-HA to CD44 protein

[0114] Biolayer interferometry (BLI) was performed using the Octet RED96e system to assess the affinity of HA for CD44 protein. Specifically, His-CD44 protein (25 μg / mL) was loaded onto a trionitrile acetate biosensor (ForteBio) and held for 600 s. The biosensor was then placed in a binding buffer containing 500 ng / L VNP-HA for 200 s. VNP (500 ng / L) served as a control.

[0115] 4. Cell sorting and culture

[0116] The HNSCC cell lines CAL27 and SCC15 were obtained from the ATCC Biostandard Resource Center in the United States. Cells with high aldehyde dehydrogenase activity were screened from CAL27 and SCC15 cells using the ALDEFLUOR assay kit. ALDH was screened by flow cytometry. high Cells were compared with a control treated with diethylamine benzaldehyde (DEAB) in terms of fluorescence intensity. ALDH high Cells were cultured in Dulbecco's Modified Eagle medium (DMEM) supplemented with 1% antibiotics and 10% fetal bovine serum (FBS) in a cell culture incubator at 37°C and 5% CO2.

[0117] To establish a cisplatin-resistant SCC15-R cell line, SCC15 cells were gradually exposed to increasing concentrations of cisplatin, eventually maintaining 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] 96-well microplate, approximately 1×10⁻⁶ oz per well 4 Cells were cultured for 12 h. Afterward, nanoparticles at concentrations of 1-200 μg / mL and PTC209 at concentrations of 0.25-10 μmol / L were cultured for 24 h each. Then, 10 μL of 5 mg / mL methylthiazolium tetrazolium (MTT) solution was added to each well, and the cells were incubated at 37°C for 4 h. MTT was aspirated, and 100 μL of dimethyl sulfoxide was added to solubilize the purple methylthiazolium crystals. Cell viability was assessed by measuring the optical density at 570 nm using a microplate reader. The control group maintained 100% cell viability in untreated medium. Both the control and experimental concentrations were repeated three times to ensure statistical reliability. Cell viability was calculated as follows:

[0120] .

[0121] 6. Cell delivery experiments

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

[0123] 7. Transwell invasion experiment

[0124] CAL27 and SCC15 cells were treated with PTC209@VNP-HA, and invasion assays were performed by counting invasive clones that passed through a polycarbonate membrane containing 8 μm pores. The well plates were coated with matrix. Approximately 3 × 10⁶ cells were collected. 5 Cells were suspended in serum-free DMEM and seeded into the upper cavity, while an appropriate amount of DMEM was added to the lower cavity. Subsequently, PTC209@VNP-HA (containing 2.5 μg / mL PTC209) was added to the upper cavity. 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 h, the matrix on the upper surface of the polycarbonate membrane was scraped off, and the cells on the membrane were fixed with paraformaldehyde solution, stained with crystal violet, and observed and photographed under a microscope. Cells in nine random regions of the images were counted using ImageJ software.

[0125] 8. Comet detection

[0126] DNA damage was detected using a single-cell gel electrophoresis (SCGE) comet detection kit (ADI-900-166). Cells were embedded in agarose, lysed, and electrophoresed to separate DNA into “comet tail” fragments. In this analysis, the length and intensity of the comet tail indicate the level of damage in individual cells to determine the impact of environmental and therapeutic factors on DNA integrity. Cell suspensions were mixed with agarose solution. 75 μL of cell suspension from each sample was loaded onto preheated slides at 37°C. Slides were incubated in pre-chilled lysis buffer at 4°C and then transferred to alkaline solution at room temperature. To separate DNA fragments, slides were electrophoresed in Tris / borate / ethylenediaminetetraacetic acid buffer. Slides were stained with CYGREEN nucleic acid dye until DNA was visible and then allowed to stand at room temperature to allow the agarose gel to form a planar image. At least 100 cells per sample were re-evaluated using the CASPv1.2.2 analysis tool.

[0127] 9. TUNEL apoptosis detection

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

[0129] 10. Tumor stem cell spheroidization experiment

[0130] In the tumor stem cell spheroidization experiment, ALDH was used. high and ALDH low Cells were added to ultra-low adhesion plates and cultured in serum-free DMEM / F12 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. After 12 days of PTC209@VNP-HA treatment, spheroids 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 cell lysis, the lysed proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane. Non-specific protein binding was blocked with 5% skim milk for 1 hour. Primary antibodies included anti-BMI1, anti-ALDH1, anti-SOX2, and anti-GAPDH, followed by labeling with corresponding fluorescently 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 conducted in accordance with the animal experimental protocols approved by the Peking University Biomedical Ethics Committee. Female nude mice were prepared and diluted for transplantation. ALDH high SCC15 CSCs were treated with PTC209@VNP-HA and digested into single-cell suspensions. Equal volumes of matrix were mixed with varying numbers of treated cells, and the mixtures were injected into the left axilla of nude mice. Four weeks later, tumors in all mice were photographed and recorded. Data were analyzed using ELDA software.

[0135] 13. In vivo experiments

[0136] (1) In situ tumor model

[0137] SCC15-ALDH high CSC(1×10 6The drug was inoculated onto the tongue of female BALB / c nude mice. Approximately one week later, all mice were randomly assigned to groups (n=6) and administered 200 μL of control solution, 5 mg / kg PTC209, 60 mg / kg PTC209, 20 mg / kg PTC209@MSN-HA, or 20 mg / kg PTC209@VNP-HA via tail vein injection every 3 days for 3 weeks. After sacrifice, the tongue and cervical lymph nodes (3-4 per mouse) were removed. In a human cancer cisplatin-resistant cell line model, SCC15 cisplatin-resistant cells (5 × 10⁻⁶) were used... 6 The tumor was injected into the left axilla of a nude mouse. After one week of growth, the tumor volume reached approximately 100 mm. 3 Mice were randomly divided into 5 groups (n=6), receiving either 200 μL of 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, the mice were sacrificed, tumors were removed, and the mice were 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 A spontaneous head and neck squamous cell carcinoma model was established in C57 mice (6-8 weeks old) at 18 weeks of age. At 22 weeks, mice were randomly divided into five groups (n=12), receiving either 200 μL of control solution, 3 mg / kg cisplatin, 20 mg / kg PTC209@VNP-HA, or a combination of 3 mg / kg cisplatin and 20 mg / kg PTC209@VNP-HA. Administration was administered via tail vein injection every 3 days for 3 weeks. Tamoxifen-labeled Bmi1+CSCs were administered before sacrifice; after euthanasia, the tongue and cervical lymph nodes (3-4 per mouse) were excised, 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] Antigen retrieval was performed on paraffin sections in citrate buffer. To facilitate binding to target molecules, horseradish peroxidase-labeled polymers were incubated with the tissue at room temperature, using 3,3'-diaminobenzidine as a chromogenic agent 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 overnight incubation with p-H2A.X antibody at 4°C. Color development was performed using Cy2-labeled secondary antibody. Cell nuclei were then stained with DAPI solution.

[0142] II. Experimental Results

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

[0144] This invention develops a drug delivery nanoplatform (PTC209@VNP-HA) comprising a PTC209-loaded MSN core and HA-modified virus-like protrusions. First, microporous microspheres were synthesized using a single-pot hydrothermal process. The negatively charged MSN surface was modified with amino groups using APTES (MSN-NH2). Shell-like nanoparticles were prepared using a unique synthetic method, enabling them to electrostatically adsorb onto MSN-NH2. VNPs were synthesized through a series of methods, assembling a virus-like structure including multiple branched nanostructures. VNP-NH2 was formed through VNP amino modification, and HA was covalently bonded to the shell particles to form VNP-HA. Then, PTC209 was adsorbed onto the mesoporous core to obtain PTC209@VNP-HA.

[0145] TEM images showed that both MSN and MSN-NH2 maintained a uniform morphology and dendritic structure, with no significant change in size (approximately 110 nm). VNPs were formed by attaching oriented particles to the surface of MSN-NH2; these oriented particles had a narrow size range (approximately 10 nm, Figure 1a). APTES-modified VNPs (VNP-NH2, approximately 130 nm) and HA-modified VNP-NH2 (VNP-HA, approximately 130 nm) did not alter their viral mimicry morphology, nor did they cause significant size changes. Dynamic light scattering analysis was used to analyze the structure of VNP-HA (…). Figure 1(b) The results showed that the particle size distribution trend was similar to that of the TEM images. PTC209@VNP-HA and other nanoparticles exhibited good monodispersity in PBS. Due to the colloidal stability conferred by 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 amino functionalization of the MSN (-26.58) and VNP (10.40) surfaces, the zeta potentials of MSN-NH2 and VNP-NH2 were positive (14.03 and 19.53 mV, respectively), while after chemical linkage with HA, the zeta potential of VNP-HA was negative (-15.29 mV). Figure 1 c). Traditional silica microspheres and solid silica nanoparticles are non-biodegradable due to their silica content, while dendritic silica microspheres exhibit excellent biodegradability due to their thin-walled pores 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 core. By day 28, approximately 56% of the VNP-HA in PBS had been degraded. Achieving optimal biodegradability is crucial for prolonging the treatment duration of VNP-HA and reducing systemic toxicity, which is essential for future research and clinical applications.

[0146] This embodiment underwent multiple tests to verify that PTC209 was fully loaded into PTC209@VNP-HA. TEM images and nitrogen adsorption experiments showed that, compared to the original MSN (>6 nm), the simulated virus morphology formed by previous synthesis methods reduced the core MSN mesopore diameter (<3 nm). TEM images (Figure 1a) also show that, after synthesis using the method of this invention, the shell particles are firmly attached to MSN-NH2, forming VNPs.

[0147] To further determine whether the virus-mimicking nanoparticles could carry drugs, nitrogen adsorption experiments were conducted. To verify that VNPs could carry target drugs, nitrogen adsorption experiments confirmed that the mesopores of VNPs were slightly smaller than those of MSNs (approximately 5.5 nm), and demonstrated that the mesopores of the finally synthesized VNP-HA were further reduced by approximately 5 nm compared to VNPs. Figure 1 d, e, Figure 10 SEM images showed that the drug-loaded PTC209@VNP-HA had comparable effects to the mixture of PTC209 powder and VNP-HA powder, and no drug crystals were observed in the PTC209@VNP-HA group, indicating that PTC209 was completely loaded into the nanoparticles. Figure 11The elemental mapping profile provides clear and direct evidence for PTC209 loading. VNP-HA exhibits a background signal, while PTC209@VNP-HA shows 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] Controlled drug release at the tumor site is a key performance indicator for nanoparticles in cancer therapy. This example evaluates the release of PTC209@VNP-HA at three pH conditions (7.4, 6.5, and 5.5). Figure 1 g). After 24 hours of incubation in a neutral environment (pH=7.4), PTC209@VNP-HA exhibited remarkable stability, releasing only about 20% of the loaded PTC209 drug. This low level of drug leakage is highly advantageous as it significantly reduces potential toxicity to healthy tissues. In contrast, in acidic environments at pH 6.5 and 5.5, the PTC209 release rate of PTC209@VNP-HA increased sharply to 64% and 83% after 24 h, and gradually increased to 70% and 91% after 72 h.

[0149] This invention further explores the morphological adjustment of viral-mimicking drug carriers. After preparing a virus-mimicking drug carrier by mixing capsids and nucleosomes, its morphological changes were tracked using TEM images. The results are as follows... Figure 9 As shown, adjusting the feed volume of the capsid solution from (a) 1.2 mL to (b) 0.8 mL, then to (c) 0.4 mL, and finally to (d) 0.2 mL resulted in significant changes in surface morphology. The best sample quality was obtained with a feed volume of 0.8 mL. Scale bar: 50 nm. It should be noted that insufficient capsid quantity can lead to loss of viral morphology. Figure 9 In this context, 0.8 mL corresponds to 16 mL of shell stock solution in Example 1, 1.2 mL corresponds to 24 mL of shell stock solution, 0.4 mL corresponds to 8 mL of shell stock solution, and 0.2 mL corresponds to 4 mL of shell stock solution.

[0150] 2. Virus mimicry 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 images clearly show that the MSN-HA and VNP-HA mesoporous structures have different external surface morphologies. FTIR spectral analysis was performed to confirm the successful binding of HA. MSN-HA and VNP-HA (3522 cm⁻¹)-1 The stretching vibration of the hydroxyl group in ) is stronger than that of VNP-NH2 (3441 cm). -1 Carboxyl groups were detected in both HA and nanoparticles (1648 cm⁻¹). -1 Tensile vibration () Figure 2 c). Furthermore, TGA results showed that the conjugation rates of HA on MSN and VNP were approximately 3.41% and 3.42%, respectively. Figure 12 Using the ninhydrin reaction, the amount of HA carried by MSN-HA and VNP-HA was calculated. Statistical analysis showed that both had the same number of HA grafts. Figure 2 d) indicates that HA was successfully attached to the nanoparticles.

[0152] Further experimental confirmation is needed to determine whether CD44 protein can target VNP-HA. Therefore, the binding affinity of VNP-HA to CD44 protein was evaluated in a BLI assay. To observe the interaction between CD44 protein and MSN-HA and VNP-HA, CD44 protein was immobilized on a biosensor chip. Compared to nanoparticles without HA, MSN-HA and VNP-HA exhibited stronger BLI signals through specific interactions with CD44 protein, indicating that HA-bound nanoparticles can target CD44 protein. Figure 2 e Figure 13 ).

[0153] Furthermore, the intracellular uptake efficiency and distribution of nanoparticles were determined using fluorescence microscopy. Rhodamine B was added to the nanoparticles as a fluorescent label for tracking and imaging to facilitate detection. SCC15 cells were seeded in 24-well culture plates and incubated with MSN-HA and VNP-HA media for 4 hours. Cellular uptake of VNP-HA was significantly higher than that of MSN-HA. The fluorescence produced by VNP-HA was approximately 6 times that of MSN-HA. Figure 2 Therefore, VNP-HA holds promise for improving the delivery efficiency of anticancer drugs and can be used as a drug carrier in subsequent experiments.

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

[0155] To assess the potential adverse effects of PTC209@VNP-HA, the cytotoxicity of different concentrations of empty nanoparticles was first evaluated in vitro. Nanoparticles (MSN, MSN-NH2, MSN-HA, VNP, VNP-NH2, and VNP-HA) were incubated with HNSCC or human umbilical vein endothelial cells (HUVECs) for 24 hours. Cell viability results showed a decreasing trend with increasing dose, especially above 100 μg / mL. 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) Due to the limited number of CD44 receptors on the surface of normal cells, their absorption of HA-modified nanoparticles is slower, 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 different concentrations (0.25, 0.5, 1, 2.5, 5, 7.5, and 10 μmol / L of free PTC209, PTC209@MSN-HA, and PTC209@VNP-HA). Subsequently, MTT assays were performed, and the half-maximal inhibitory concentration (IC50) was calculated. 50 IC50 value. Compared with the PTC209@MSN-HA (10 μM) and PTC209 (>10 μM) treatment groups, PTC209@VNP-HA showed a higher IC50 value at 24 h (5 μM). 50 The value decreased significantly ( Figure 3 b). These results indicate that PTC209@VNP-HA inhibits tumor cell growth. In subsequent in vitro experiments, cells were treated with 5 μM PTC209 (lower IC50 and free PTC209 than PTC209@MSN-HA) for 24 h. Following intravenous injection, the nanoparticles initially interacted with erythrocytes. To test the hemolytic effect of the nanoparticles, mouse erythrocytes were incubated with the nanoparticles MSN-NH2, MSN-HA, PTC209@MSN-HA, VNP-NH2, VNP-HA, and PTC209@VNP-HA, and 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-NH2 or VNP-NH2, with a hemolysis rate <4%. Figure 14 This reduction may be attributed to the HA-modified surface, which increases hydrophilicity and reduces potential harm to erythrocytes.

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

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

[0158] 4. PTC209@VNP-HA inhibits the characteristics of HNSCC stem cells.

[0159] To demonstrate the effects of VNP-HA on in vitro CSCs, ALDH was isolated from CAL27 and SCC15 cells using flow cytometry. high Further experiments were conducted on CSC. Several experiments were performed to determine whether PTC209@VNP-HA could remove SSC. First, ALDH treated with PTC209@VNP-HA was tested. high CAL27 and ALDH high The levels of various CSC markers were assessed in SCC15 cells. The levels of BMI1, ALDH1, and SOX2 proteins decreased after treatment. Figure 4 a) Since spheroid formation is an indicator of CSC self-renewal capacity, a tumor spheroidization experiment was conducted to further determine whether BMI1 can modulate tumorigenic potential. The results showed that PTC209@VNP-HA effectively reduced ALDH. high The number of cell spheres ( Figure 4(b, c) The potent antitumor activity observed in vitro by PTC209@VNP-HA is conducive to further exploring the inhibitory effect of PTC209@VNP-HA on CSCs in vivo. First, the distribution of the nanoparticles in vivo was observed. ALDH... high SCC15 cell-induced tumor mice were treated with control solution (PBS), MSN-HA, or VNP-HA via tail vein injection. The biodistribution of nanoparticles was monitored in vivo at 1, 12, and 24 h after treatment. Fluorescent signals corresponding to VNP-HA were detected at the tumor site after 12 h, and the fluorescence signal significantly increased after 24 h. Figure 15 Next, in vivo dilution experiments were performed to confirm the tumorigenic changes in CSCs after PTC209@VNP-HA treatment. 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 exhibiting CSC characteristics, frequently migrate and proliferate in cervical lymph nodes. To determine whether PTC209VNP-HA impairs stem cell and metastasis in mouse orthotopic HNSCC, a mouse orthotopic HNSCC model was used. ALDH was then applied... high SCC15 control solution, PTC209 (low dose, 5 mg / kg), PTC209@MSN-HA (PTC209, dose 5 mg / kg), and CSC were inoculated onto the tongue of mice. Mice were then treated every 3 days with PTC209VNP-NA (PTC209, dose 5 mg / kg) and PTC209 (high dose, 60 mg / kg) for 3 weeks. Figure 5 a). The results showed that PTC209@MSN-HA reduced in situ tumor growth, while low-dose PTC209 alone did not inhibit tumor growth, similar to the control group. Mice treated with nanomedicine exhibited virus-like morphology, with reduced tumor volume compared to mice treated with nanomedicine that exhibited smooth morphology. PTC209@VNP-HA showed stronger inhibitory effects than low-dose PTC209 and PTC209@MSN-HA. Figure 5 (bd). Anti-PCK immunostaining was performed on cervical lymph nodes to specifically detect lymph node metastasis. 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. highThe extent of CSC lymph node metastasis is greater than that of PTC209@MSN-HA. Figure 5 (e, f) The dose of PTC209 used in the in vivo experiments was 5 mg / kg, significantly lower than the dose in previous studies (60 mg / kg). Furthermore, the in vivo antitumor effects of PTC209@VNP-HA and high-dose PTC209 on HNSCCs were compared. The results showed that the high-dose PTC209 group and the PTC209@VNP-HA group had similar effects, indicating that the proposed VNP design strategy can reduce the drug dosage and thus increase the potency of PTC209 by 12-fold.

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

[0163] Cisplatin chemotherapy is the primary treatment strategy for patients with advanced HNSCC. Despite its widespread use and effectiveness, cisplatin resistance has become a significant challenge. Increasing evidence suggests that SSCs are associated with resistance to cancer treatment and relapse or recurrence. However, effectively eradicating SSCs and overcoming chemoresistant cancer remains a critical challenge. A cisplatin resistance model was established using a previously described approach. SCC15 cisplatin-resistant cells (SCC15-R) were implanted into the left axilla of nude mice; one week later, the mice were treated for 3 weeks with control solution, cisplatin, PTC209@MNS-HA, PTC209@VNP-HA, or PTC209@VNP-HA combined with cisplatin, respectively. Figure 6 a). The results showed that, compared with SCC15 cells, SCC15-R cells had enhanced proliferation capacity and IC50. 50 The value was higher. This confirms the successful establishment of the SCC15-R cell line. Figure 6 (b, c) The results showed that although the cisplatin-resistant model was unresponsive to cisplatin treatment, the viral mimicry morphology promoted the anti-cancer effect of PTC209 to a greater extent than the smooth morphology in the cisplatin-resistant model. 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 in vivo. Figure 6 df).

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

[0165] A 4NQO-induced HNSCC mouse model (Rosa) was previously developed. tdTomato The model is based on Bmil. CreERCharacterized by allowing uninterrupted tracking of CSC lineages in vivo after administration. Tumor-bearing mice were treated with control solution, cisplatin, PTC209@VNP-HA, or a combination of PTC209@VNP-HA and cisplatin for 4 weeks after administration of the 4NQO inducer, followed by treatment with deionized water. One day before euthanasia, the mice were given tamoxifen-labeled tomato. + BMI1 + CSC Figure 7 a) Both the cisplatin group and the PTC209@VNP-HA group showed a reduction in tumor lesion area compared to the control group. Notably, the reduction in lesion area achieved by combining PTC209@VNP-HA with cisplatin was significantly greater than that achieved 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 to the control group. When PTC209@VNP-HA was combined with cisplatin, the number, size, and invasiveness of HNSCC tumors were significantly reduced compared to 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 to the control group. PTC209@VNP-HA combined with cisplatin effectively eliminated most lymph node metastases in HNSCC (df). Figure 7 Immunostaining showed that, compared with mice treated with cisplatin and PTC209@VNP-HA alone, the p-H2A.X level in mice treated with the combination therapy was significantly increased. Figure 7 j, k). Subsequently, BMI1 was labeled in vivo. + CSC was used to evaluate the effect of combination therapy on eliminating these cells. Results showed that cisplatin increased BMI in HNSCC. + The number of CSCs. In vivo markers showed that the number of CSCs in HNSCC increased after cisplatin treatment compared to the control group. Unlike cisplatin alone, PTC209@VNP-HA combined with cisplatin reduced BMI by 1. + The number of CSCs. Compared to using cisplatin or PTC209@VNP-HA alone, the combination of PTC209@VNP-HA and cisplatin also significantly improved CSC clearance. Figure 7 (l, m).

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

[0167] III. Discussion

[0168] CSCs (cell cytokines) are a subset of tumor cells with self-renewal and differentiation potential, playing a crucial role in several key aspects of tumor growth, invasion, metastasis, and chemotherapy resistance. CSCs drive tumor formation and sustained expansion, and promote malignant tumor progression through complex molecular mechanisms. The small molecule inhibitor PTC209 can disrupt the self-renewal of CSCs. Although PTC209, alone or in combination with other therapies, significantly inhibits tumor progression, challenges related to immunogenicity, off-target gene effects, and dose-limiting toxicities hinder its widespread application in vivo.

[0169] In this embodiment, VNP-HA was designed for delivery of PTC209 as a highly effective SCS-targeted therapy for HNSCC. Although viral vectors have been used to treat cancer, concerns remain regarding immunogenicity and mutagenic risks. Therefore, the development of non-viral vectors with virus-like morphologies may create promising tools for achieving an appropriate balance between safety and efficacy. VNP-HA offers several advantages in enhancing 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 VNP-HA exhibits negligible cytotoxicity. Hemolysis assays indicate that VNP-HA has virtually no toxicity to blood cells during in vivo circulation. Factors such as nanoparticle size and surface charge are also important to ensure that VNP-HA can easily reach tumor tissues via blood circulation. The size of VNP-HA is approximately 130 nm, which appears ideal for accumulation and selective retention in HNSCC utilizing the EPR effect. VNP-HA carries a negative charge and binds relatively weakly to blood proteins, which facilitates its long-term circulation in the blood. Furthermore, the viral mimicry enhanced the cellular uptake efficiency of the nanoparticles, improving cell delivery performance. In an in situ tumor model, the viral mimicry of PTC209@VNP-HA promoted cellular uptake and enhanced the therapeutic efficacy of PTC209 compared to PTC209@MSN-HA. Finally, through HA modification, the VNP surface was easily functionalized, thereby improving CSC targeting precision. Analysis showed that hyaluronic acid was successfully grafted onto the nanoparticles. Molecular affinity experiments demonstrated that VNP-HA successfully targeted the CD44 receptor protein.

[0170] Previous studies have shown that cisplatin treatment primarily eliminates proliferating cells, rendering CSCs unaffected. This invention demonstrates, both in vitro and in vivo, that PTC209@VNP-HA clears CSCs through the DNA damage / apoptosis pathway, effectively inhibiting tumor growth and metastasis. While PTC209@VNP-HA inhibits the growth and metastasis of HNSCC, previous studies have shown that specific cytokines or growth factors can lead to the reprogramming or redifferentiation of non-stem cell tumor cells into CSCs. Combination therapy of PTC209@VNP-HA with cisplatin effectively clears both CSCs and non-CSCs, achieving optimal therapeutic effects.

[0171] In summary, VNP-HA possesses multifaceted functions, high stability, low toxicity, good biodegradability, and biocompatibility. In vitro and in vivo experiments demonstrate that PTC209@VNP-HA effectively eliminates cancer cell sclerosis (CSCs) in HNSCC, prevents metastasis, and overcomes cisplatin resistance, exhibiting significant research value and clinical translational potential. The virus mimicry strategy proposed in this invention will provide guidance for designing innovative bionanomaterial drug delivery platforms and offer promising therapeutic approaches for the 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, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a viral-like drug targeting tumor stem cells and loaded with PTC209, characterized in that, Includes the following steps: (1) Preparation of mesoporous silica nanoparticles (MSN) 0.36 g of triethanolamine and 48 mL of 25 wt% hexadecyltrimethylammonium chloride were placed in a round-bottom flask, and 72 mL of deionized water was added. 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 dissolved in cyclohexane was added to the reaction system along the wall of the reaction flask. After stirring slowly at 60°C for 24 hours, a white reaction product was obtained. After transferring the reaction solution to a separatory funnel, the milky white aqueous phase containing MSN was separated and centrifuged at 20000×g for 60 minutes to obtain MSN precipitate. Then, MSN was washed with acidic methanol at 60°C to remove residual impurities and CTAC in the mesopores. The acidic methanol was obtained by mixing 37% hydrochloric acid and methanol at a volume ratio of 1:

10. Finally, after washing with anhydrous ethanol, MSN was dispersed in anhydrous ethanol for later use, with an average particle size of 110 nm. (2) MSN Modification 250 mg of MSN was dispersed in 150 ml of xylene, and 2.5 ml of 3-aminopropyltriethoxysilane was added. After heating and refluxing for 12 hours, MSN-NH2 was obtained by centrifugation at 20000×g for 30 minutes. MSN-NH2 was repeatedly washed with anhydrous ethanol and finally dispersed in anhydrous ethanol for later use. (3) Preparation and modification of nanoparticles with enveloped virus morphology 87 mg of L-arginine was dissolved in 69.5 mL of deionized water, and 5.23 mL of octane was added followed by high-speed stirring to emulsify. 0.5 mL of TEOS was added, and the mixture was stirred at 60 °C for 3 hours to obtain a suspension containing peripheral particles. 100 mg of MSN-NH2 was dispersed in 50 mL of pH 9.5 Ct[CO3] 2- VNPs were obtained by adding 16 mL of a suspension of peripheral particles to a carbonate buffer solution of 50 mmol / L, reacting at room temperature for 8 hours, and washing with anhydrous ethanol. The steps for amino modification of VNP include: heating the stirrer to 145°C, adding 250 mg of VNP to 150 mL of xylene, then adding 2.5 mL of 3-aminopropyltriethoxysilane dropwise, and stirring at 145°C for 12 hours using a rectifier with a heated stirrer. The resulting VNP-NH2 is centrifuged at 20000×g for 30 minutes and washed three times with ethanol to remove xylene and residual 3-aminopropyltriethoxysilane. The product is stored in ethanol for subsequent use. (4) VPN surface grafting Hyaluronic acid (HA) was grafted onto the surface of VNP to endow it with the ability to target and recognize tumor stem cells. At room temperature: 32 mg of HA carboxyl groups were activated in 25 mL of phosphate-buffered saline at pH 7.4 using 12 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride as a catalyst for 0.5 hours. 14 mg of N-hydroxysuccinimide was added and stirring was continued for 0.5 hours. Then, 100 mg of VNP-NH2 was dispersed in 25 mL of PBS at pH 7.4 and condensed with carboxyl-activated HA for 3 hours. After repeated washing with deionized water and anhydrous ethanol, VNP-HA was obtained and stored at 4°C. (5) Loading of small molecule drugs 2.5 mg of PTC209 was dissolved in 0.25 ml of dimethylformamide and 7.5 mg of VNP-HA was ultrasonically dispersed. They were then mixed and the DMF was completely evaporated under a nitrogen gas flow at room temperature to obtain dry PTC209@VNP-HA powder, which is a biomimetic drug.

2. The method for preparing the viral drug targeting tumor stem cells and loaded with PTC209 according to claim 1, characterized in that, The molecular weight of the hyaluronic acid is 500 Da-5 kDa.

3. A viral-like drug targeting tumor stem cells, characterized in that, It is obtained by the preparation method described in claim 1 or 2.

4. The viral-like drug targeting tumor stem cells according to claim 3, characterized in that, The drug is a systemic drug.

5. The use of the viral-mimicking drug targeting tumor stem cells according to claim 3 or 4 in the preparation of an antitumor drug composition, characterized in that, The tumor is a squamous cell carcinoma of the head and neck.

6. The application according to claim 5, characterized in that, Further use in combination with small molecule drugs including cisplatin, 5-fluorouracil, carboplatin, nedaplatin and paclitaxel.

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