Photo-responsive titanium dioxide vitamin E succinate nano-particles as well as preparation method and application thereof

Abstract

The invention discloses a photoresponse type titanium dioxide vitamin E succinate nanoparticle and a preparation method and application thereof.The photoresponse type titanium dioxide vitamin E succinate nanoparticle introduces a photoresponse mechanism by co-loading DOX and TiO2NPs to achieve controllable drug release, meanwhile, alpha-TOS is introduced into a liposome component to optimize the physicochemical property of a liposome membrane, meanwhile, the cytotoxicity of a drug is synergistically enhanced, and the drug release effect is improved. The photocatalytic effect of the TiO2NPs is activated by illumination, reactive oxygen species (ROS) is generated in situ to accelerate the rupture of a liposome membrane, and efficient release of drugs in a tumor microenvironment is realized; the preparation method adopts an ultrasonication method to obtain liposome nanoparticles with small particle size (50nm-150nm) and high encapsulation efficiency (greater than 60%), and the liposome nanoparticles have the characteristics of long circulation stability and targeted controlled drug release, can effectively improve the tumor curative effect and reduce the system toxicity, and provides a novel drug delivery platform for combined photocatalytic treatment and chemotherapy.

Description

Technical Field

[0001] This invention relates to nanoparticles, particularly to photoresponsive titanium dioxide vitamin E succinate nanoparticles, and also to the preparation method and application of the above-mentioned nanoparticles. Background Technology

[0002] Breast cancer is one of the most common malignant tumors affecting women worldwide. Currently, the most common treatment strategies include surgery, radiation therapy, and chemotherapy. However, surgery and radiation therapy can only target local tumor masses and cannot address easily metastatic or occult tumor cells, thus only treating the symptoms, not the root cause. In contrast, chemotherapy remains an important clinical treatment. However, traditional chemotherapy drugs such as doxorubicin (DOX) have serious systemic toxicity (such as cardiac damage and bone marrow suppression) and insufficient targeting of tumor tissue, resulting in limited treatment efficacy and poor patient tolerance.

[0003] Current technologies often utilize the cell affinity and tissue compatibility of liposomes to bind chemotherapy drugs, allowing them to adhere to target cells for extended periods, enabling the drugs to fully penetrate the target tissue. Liposomes also enter the cell via endocytosis, and after lysosomal digestion, the drugs are released into the cell, improving organ targeting and effectively achieving the goal of reducing toxicity and increasing efficacy. However, existing liposomes still face the following challenges in breast cancer treatment: 1. The contradiction between stability and release efficiency: Conventional liposomes are easily cleared by the reticuloendothelial system in the bloodstream. Although PEG modification can prolong circulation time, the introduction of PEG can also hinder drug release at the tumor site, and increasing the PEG content exacerbates this obstacle; 2. Insufficient controllable release capability: Traditional liposomes rely on passive diffusion or tumor microenvironment (such as weakly acidic pH, enzymes, etc.) to trigger release, but breast cancer tissue has high heterogeneity and indistinct microenvironmental characteristics, which is insufficient to achieve precise controlled drug release.

[0004] Nano-titanium dioxide (TiO2NPs) has been used in photocatalytic therapy research due to its photocatalytic properties and biocompatibility. The core principle is that TiO2NPs generate reactive oxygen species (ROS) under light irradiation to kill tumor cells. However, using TiO2NPs alone has drawbacks such as limited light penetration depth, small ROS diffusion range, and poor targeting. Meanwhile, the exploration of co-loading TiO2NPs with chemotherapeutic drugs in the same drug delivery system is still in its early stages, especially lacking effective solutions regarding the light-triggered synergistic therapy mechanism and carrier stability design. Existing methods for co-loading TiO2NPs with chemotherapeutic drugs mainly include hollow / mesoporous structure loading, core-shell structure loading, and two-dimensional nanosheet structure loading, but each has its disadvantages. Hollow / mesoporous structure loading methods often have complex preparation processes, poor stability, and a high risk of premature drug leakage. Core-shell structure loading methods have cumbersome preparation processes, and the metal elements constituting the core and shell may pose potential biotoxicity risks. Two-dimensional nanosheet structures involve carriers with excessively large sizes (2-4 μm), resulting in poor dispersibility and easy capture by the reticuloendothelial system, leading to clearance. There is an urgent need to overcome the problems of existing technologies and develop new carrier drugs. Summary of the Invention

[0005] Purpose of the invention: To address the technical problems of insufficient targeting of tumor tissue, poor controllability of drug release, and the contradiction between liposome stability and release efficiency in traditional chemotherapy drug delivery systems, this invention provides titanium dioxide vitamin E succinate nanoparticles that increase the circulation time of liposomes in the blood, avoid premature drug release, and achieve controllable photoresponsive release. The invention also provides the preparation method and application of the above nanoparticles.

[0006] Technical solution: The photoresponsive titanium dioxide vitamin E succinate nanoparticles of the present invention are liposomes co-loaded with chemotherapeutic drugs and titanium dioxide nanoparticles. The liposome carrier with a cavity is assembled with disteaaroyl-sn-glycero-3-phosphocholine (DSPC), D-α-tocopheryl succinate (α-TOS) and disteaaroyl-phosphatidylethanolamine-PEG2000 (DSPE-PEG2000). The chemotherapeutic drugs and titanium dioxide nanoparticles are combined by electrostatic attraction and encapsulated in the liposome carrier.

[0007] The chemotherapy drug is one or more of doxorubicin DOX or its hydrochloride, epirubicin EPI, and pirarubicin THP, and the particle size of the nano-titanium dioxide TiO2NPs is 5-10 nm.

[0008] The molar ratio of distearylphosphatidylcholine, D-α-tocopherol succinic acid, and distearylphosphatidylethanolamine is 60-68.5:30:1.5-10. The structure of the liposome carrier is such that distearylphosphatidylcholine, D-α-tocopherol succinic acid, and distearylphosphatidylethanolamine all possess a hydrophilic head and a hydrophobic tail. Based on this, the hydrophobic tail of D-α-tocopherol succinic acid is connected to the hydrophobic tails of distearylphosphatidylcholine and distearylphosphatidylethanolamine through hydrophobic interactions and van der Waals forces. The hydrophilic head (succinic acid group) of D-α-tocopherol succinic acid is exposed on the hydrophilic outer and inner surfaces of the lipid bilayer, in contact with the aqueous phase, and interacts with water molecules or the polar head of phospholipids through hydrogen bonds. Meanwhile, the polyethylene glycol tail chain of distearate phosphatidylethanolamine-PEG2000 compresses and confines the nanoparticles by increasing the spontaneous curvature of the membrane and forming steric hindrance, thereby reducing the particle size of the nanoparticles and forming a liposome carrier with cavities.

[0009] The mass ratio of the liposome carrier, the chemotherapeutic drug, and the titanium dioxide nanoparticles is 150-750:10-20:1-10.

[0010] The preparation method of the above-mentioned photoresponsive titanium dioxide vitamin E succinate nanoparticles includes the following steps:

[0011] (1) Disteazylophosphatidylcholine, D-α-tocopherol succinate and disteazylophosphatidylethanolamine-PEG2000 were dissolved in anhydrous ethanol, mixed and vortexed to obtain a liposome carrier solution.

[0012] (2) Using anhydrous ethanol as a solvent, prepare a chemotherapy drug-nano titanium dioxide suspension, add the liposome carrier solution prepared in step (1), and vortex mix to obtain a mixture.

[0013] (3) The mixture was transferred to deionized water in a high-speed vortex state using a pipette, and after ultrasonication in an ultrasonic disruptor, photoresponsive titanium dioxide vitamin E succinate nanoparticles were obtained.

[0014] In step (1), the concentrations of distearylphosphatidylcholine, D-α-tocopherol succinate, and distearylphosphatidylethanolamine-PEG2000 are 50-150 mg / mL, preferably 100 mg / mL.

[0015] In step (2), the concentration of chemotherapy drug and nano-titanium dioxide in the anhydrous ethanol suspension is 0.1-0.5 mg / mL.

[0016] In step (3), the transfer involves using a pipette to transfer the mixture into an EP tube containing 500 μL of deionized water in a high-speed vortex state. The pipette uses a long, thin tip (such as the American Sorenson 200 μL extended sample loading tip). The sonication time is 60-90 seconds, and the sonication power is 3%-5%. During the sonication process, it is important to avoid excessively long sonication time (overheating), excessive sonication power, and the sonication probe touching the tube wall. Otherwise, severe foaming will occur, which will cause the liposome structure to collapse and the particle size to become too large.

[0017] The aforementioned photoresponsive titanium dioxide vitamin E succinate nanoparticles can also be used in the preparation of antitumor drugs.

[0018] The tumor in question is either breast cancer, ovarian cancer, or thyroid cancer.

[0019] Invention Principle: The photoresponsive titanium dioxide vitamin E succinate nanoparticles of this invention utilize surface electrostatic adsorption to bind chemotherapy drugs with TiO2NPs. TiO2NPs act as photocatalysts, primarily controlling drug release. DOX is loaded into the cavities of the lipid nanoparticles either adsorbed onto TiO2NPs or in a free form. To avoid premature drug release and increase the circulation time of liposomes in the blood, the formulation of this invention incorporates a PEGylated lipid component, distearate phosphatidylethanolamine-PEG2000. Each PEG content formulation exhibits excellent physical properties. Preferably, a high PEG content (10 mol%) is significantly higher than the PEG percentage in conventional liposomes (0.5 mol%-5 mol%), resulting in the best overall performance in terms of physical properties and release efficiency. Simultaneously, the introduction of TiO2NPs ensures controllable photoresponsive drug release, avoiding the negative hindering effects of increased PEG content. By functionally embedding α-TOS into the phospholipid bilayer, its antioxidant properties enhance liposome stability and drug protection, while its synergistic anticancer effects further enhance the therapeutic efficacy of drugs.

[0020] This invention proposes a photoresponsive mechanism to achieve controllable drug release by co-loading DOX and TiO2NPs. Simultaneously, α-TOS is introduced into the liposome components to optimize the physicochemical properties of the liposome membrane and synergistically enhance drug cytotoxicity. In this system, the PEG ratio can be increased, and the photocatalytic effect of TiO2NPs is activated by light irradiation to generate reactive oxygen species (ROS) in situ, accelerating liposome membrane rupture and achieving efficient drug release in the tumor microenvironment. The preparation method employs ultrasonic disruption, precisely controlling the molar ratio of lipid components to drug-nanomaterials, ultrasonic time, and power to obtain small-sized (50nm-150nm) and high-encapsulation-efficiency (>60%) liposome nanoparticles. This invention combines long-term cycling stability with targeted and controllable drug release, effectively improving tumor treatment efficacy and reducing systemic toxicity, providing a novel drug delivery platform for combined photocatalytic therapy and chemotherapy.

[0021] In the nanoparticle preparation process, DSPC, α-TOS, and DSPE-PEG2000 are introduced into the system as lipid carrier components. They share the characteristic of having a hydrophilic head and a hydrophobic tail. Therefore, in an aqueous environment, they spontaneously form a phospholipid bilayer structure with the hydrophilic head facing the external aqueous phase and the aqueous phase inside the cavity, and the hydrophobic tail connecting them. Lipid composition and content are factors affecting lipid morphology. PEG tail chains can increase the spontaneous curvature of the membrane and create steric hindrance, thereby reducing the nanoparticle size. Therefore, this patent, by appropriately increasing the PEG content in the formulation, forms a nanolipid carrier with a smaller particle size, which is more conducive to drug delivery. Negatively charged TiO2NPs and positively charged DOX are non-covalently combined through electrostatic interactions. The semiconductor material TiO2NPs has a size of only 5-10 nm, and the quantum confinement effect reduces its band gap, red-shifting the absorption range to a wavelength of 500 nm, thus improving the efficiency of photocatalytic production of reactive oxygen species. During ultrasonic disruption, high-frequency ultrasound creates ultrasonic cavitation, generating localized high temperature and pressure. This provides external energy support for the controllable formation of lipid particles. Under suitable duration and power conditions, the lipid components are further uniformly dispersed, ensuring similar nanoparticle composition and concentrated particle size distribution. This solves the problems of irregular shape, uneven size, and uneven component distribution in existing technologies. After cells take up drug nanoparticles through endocytosis, they disrupt the lipid membrane and release the drug via endosome / lysosome escape pathways and photoresponsive pathways. During this process, the drug molecule DOX directly blocks DNA replication and transcription by embedding into the double helix of cancer cell DNA, inhibiting topoisomerase II activity and causing DNA double-strand breaks, thereby inhibiting cancer cell self-repair and proliferation. DOX can also activate the mitochondrial apoptosis pathway, generating reactive oxygen species (ROS) that exacerbate oxidative damage. Meanwhile, the lipid membrane component α-TOS accumulates in the mitochondria of cancer cells due to its lipophilicity. By disrupting redox homeostasis and promoting the oligomerization of BAX protein, it increases the permeability of the outer mitochondrial membrane, triggers irreversible mitochondrial apoptosis, and thus promotes cancer cell apoptosis. When used in combination with DOX, the synergistic effect of the mitochondrial apoptosis pathway brings a more significant anti-cancer effect than that of a single drug.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The nanoparticles of the present invention further encapsulate chemotherapeutic drugs and titanium dioxide nanoparticles through the composite of lipid molecules of liposome nanocarriers with different PEG levels, achieving a nanoparticle size range of 50-150 nm; within this size range, due to the enhanced permeability and retention effect, Retention (EPR), nanoparticles can avoid being cleared by the lymphatic or renal system due to their small particle size, and can also avoid being intercepted by liver sinusoids or phagocytosed by macrophages due to their large particle size; at the same time, as the PEG content increases, the encapsulation rate of chemotherapy drugs increases by 5.42%, further enhancing the efficacy of nanoparticles; (2) The preparation method of nanoparticles in this invention is simple and efficient. Based on the ultrasonic cavitation effect, micron-sized bubbles are generated by high-frequency ultrasonic waves and instantly collapse to bring local high temperature and high pressure, which allows lipid aggregates to be strongly broken and directly form small single-chamber vesicles without the need for extrusion steps. Compared with the common thin film hydration method, this method has many advantages such as controllable particle size, protection of thermosensitive components, and greatly simplified process flow; (3) When applied to anti-tumor drugs, the cytotoxicity against breast cancer cells is significantly improved. The expression of proto-oncogenes (Brca1, Bcl-2) and tumor suppressor genes (Bax, Caspase-3) related to the proliferation of breast cancer cells is downregulated or upregulated, respectively, resulting in significant therapeutic effects. The tumor growth inhibition rate TGI is as high as 70.09%, and there are no serious side effects. Attached Figure Description

[0023] Figure 1 The DLS particle size distribution (A, B) and comparison diagram (C) of DTPL and DTTPL in Examples 1-3 and Comparative Examples 1-3 of the present invention are shown.

[0024] Figure 2 The images are TEM images of DTPL and DTTPL in Embodiment 1 and Comparative Example 1 of the present invention, wherein: DTTPL(A), DTPL(B), DTTPL-Dark(C), and DTTPL-Light(D);

[0025] Figure 3 Comparison of particle size variation curves of DTPL and DTTPL in Examples 1-3 and Comparative Examples 1-3, and DSDCPL and DTSDCPL prepared without α-TOS in Comparative Example 4.

[0026] Figure 4 The infrared absorption spectra of the DOX / TiO2 nanoparticles used in Examples 1-3 of this invention are shown below.

[0027] Figure 5The standard curves used in Examples 1-3 of this invention for measuring DOX concentration by ultra-micro ultraviolet spectrophotometry are shown.

[0028] Figure 6 The release curves of the photoresponsive liposome DTTPL in Examples 1 to 3 of the present invention under light-shielded / light-illuminated conditions are shown, wherein: Low-PEG-Level (A), Med-PEG-Level (B), High-PEG-Level (C);

[0029] Figure 7 The results of electron paramagnetic resonance (EPR) tests on the DTTPL of Embodiment 1 of the present invention under illumination conditions;

[0030] Figure 8 The results of cytotoxicity experiments of free DOX (A), DTPL (B), and DTTPL (C,D) in Example 1 of the present invention are shown.

[0031] Figure 9 The results are RT-qPCR experimental results of the drug delivery system described in Example 1 of the present invention; wherein, Brac1 (A), Bcl-2 (B), Bax (C), and Caspase-3 (D);

[0032] Figure 10 The results of the in vivo tumor treatment experiment in mice using the drug delivery system described in Embodiment 1 of the present invention are as follows: experimental procedure (A), photos of mice and tumors (B), tumor growth curve (C), and mouse weight change curve (D).

[0033] Figure 11 The images show sections and H&E-stained images of tumors and major organs in mice after treatment with the drug delivery system described in Example 1 of this invention.

[0034] Figure 12 The content of various biochemical indicators in the serum of mice after treatment with the drug delivery system described in Example 1 of the present invention is shown; wherein, ALT(A), TG(B), CK(C), LDH(D), AST(E), T-CHO(F), CK-MB(G), LDH1(H). Detailed Implementation

[0035] The technical solution of the present invention will be further described below with reference to the embodiments. The test materials used in the embodiments can all be obtained through conventional means.

[0036] Example 1

[0037] The photoresponsive titanium dioxide vitamin E succinate nanoparticles of the present invention comprise the following components:

[0038] Lipid components: Disteazylophosphatidylcholine (DSPC): purity ≥99%, Tocopheryl succinate (α-TOS): purity ≥98%, Disteazylophosphatidylethanolamine-PEG-2000 (DSPE-PEG2000): purity ≥95%.

[0039] Drug components: 1 mg of doxorubicin (DOX) was weighed and dispersed in 10 mL of anhydrous ethanol to prepare an ethanol solution of 0.1 mg / mL DOX for later use; 1 mg of nano titanium dioxide (TiO2NPs, 5-10 nm) and 1 mg of doxorubicin (DOX) were weighed and dispersed together in 10 mL of anhydrous ethanol to prepare an ethanol suspension of 0.1 mg / mL DOX / TiO2 for later use;

[0040] Solvents and equipment: anhydrous ethanol, deionized water, vortex mixer, ultrasonic disruptor (probe diameter 3mm, maximum power 150W), and pipettes of various sizes.

[0041] The nanoparticles are prepared by the following steps:

[0042] (1) Disteaaroyl-sn-glycero-3-phosphocholine (DSPC), D-α-tocopheryl succinate (α-TOS) and disteaaroyl-phosphatidylethanolamine-PEG2000 (DSPE-PEG2000) were dissolved in anhydrous ethanol to prepare 100 mg / mL solutions. The lipid components were transferred to the same EP tube in a molar ratio of DSPC:α-TOS:DSPE-PEG2000 = 60:30:10 and mixed with a vortex mixer.

[0043] (2) Add 100 μL of DOX / TiO2 suspension in anhydrous ethanol to the EP tube and mix it by vortexing.

[0044] (3) Add 500 μL of deionized water to a clean EP tube. Transfer the mixture in the EP tube described in S3 to the EP tube using a pipette (equipped with a sample loading tip with certain characteristics). During this process, the latter needs to be kept in a high-speed vortex state. Transfer the EP tube to an ultrasonic disruptor for ultrasonication. After continuous ultrasonication for 90 seconds, photoresponsive titanium dioxide vitamin E succinate nanoparticles are obtained, denoted as High-PEG-DTTPL.

[0045] Example 2

[0046] The photoresponsive titanium dioxide vitamin E succinate nanoparticles of the present invention are prepared by the following steps:

[0047] (1) Disteaaroyl-sn-glycero-3-phosphocholine (DSPC), D-α-tocopheryl succinate (α-TOS) and disteaaroyl-phosphatidylethanolamine-PEG2000 (DSPE-PEG2000) were dissolved in anhydrous ethanol to prepare 100 mg / mL solutions; the lipid components were transferred to the same EP tube in a molar ratio of DSPC:α-TOS:DSPE-PEG2000 = 65:30:5 and mixed with a vortex mixer;

[0048] (2) Add 100 μL of DOX / TiO2 suspension in anhydrous ethanol to the EP tube and mix it by vortexing.

[0049] (3) Add 500 μL of deionized water to a clean EP tube. Transfer the mixture in the EP tube in S3 to a new EP tube containing deionized water using a pipette (equipped with a sample loading tip with certain characteristics). During this process, the latter needs to be kept in a high-speed vortex state. Transfer the EP tube to an ultrasonic disruptor for ultrasonication. After continuous ultrasonication for 90s, photoresponsive titanium dioxide vitamin E succinate nanoparticles are obtained, denoted as Med-PEG-DTTPL.

[0050] Example 3

[0051] The photoresponsive titanium dioxide vitamin E succinate nanoparticles of the present invention are prepared by the following steps:

[0052] (1) Disteaaroyl-sn-glycero-3-phosphocholine (DSPC), D-α-tocopheryl succinate (α-TOS) and disteaaroyl-phosphatidylethanolamine-PEG2000 (DSPE-PEG2000) were dissolved in anhydrous ethanol to prepare 100 mg / mL solutions; the lipid components were transferred to the same EP tube in a molar ratio of DSPC:α-TOS:DSPE-PEG2000 = 68.5:30:1.5 and mixed with a vortex mixer;

[0053] (2) Add 100 μL of DOX / TiO2 suspension in anhydrous ethanol to the EP tube and mix it by vortexing.

[0054] (3) Add 500 μL of deionized water to a clean EP tube. Transfer the mixture in the EP tube in S3 to a new EP tube containing deionized water using a pipette (equipped with a sample loading tip with certain characteristics). During this process, the latter needs to be kept in a high-speed vortex state. Transfer the EP tube to an ultrasonic disruptor for ultrasonication. After continuous ultrasonication for 90 seconds, photoresponsive titanium dioxide vitamin E succinate nanoparticles are obtained, denoted as Low-PEG-DTTPL.

[0055] Comparative Example 1

[0056] A composite nanoparticle, compared with Example 1, is obtained by replacing the DOX / TiO2 suspension with anhydrous ethanol in step (2) with a DOX solution with anhydrous ethanol, while keeping everything else unchanged. The nanoparticle is denoted as High-PEG-DTPL.

[0057] The physicochemical properties of Example 1 and Comparative Example 1 are characterized as follows:

[0058] Particle size and dispersibility: The average particle size determined by dynamic light scattering DLS was 53.92 nm for High-PEG-DTPL and 73.82 nm for High-PEG-DTTPL.

[0059] Encapsulation efficiency: The encapsulation efficiency of DOX was measured by ultra-micro UV spectrophotometry to be 77.84% for High-PEG-DTPL and 61.28% for High-PEG-DTTPL.

[0060] Stability: High-PEG-DTPL and High-PEG-DTTPL were stored in a refrigerator at 4°C, and the particle size was measured every 3 days.

[0061] Comparative Example 2

[0062] A composite nanoparticle, compared with Example 2, is obtained by replacing the DOX / TiO2 suspension with anhydrous ethanol in step (2) with a DOX solution with anhydrous ethanol, while keeping everything else unchanged. The nanoparticle is denoted as Med-PEG-DTPL.

[0063] The physicochemical properties of Example 2 and Comparative Example 2 are characterized as follows:

[0064] Particle size and dispersibility: The average particle size determined by dynamic light scattering (DLS) was 54.17 nm for Med-PEG-DTPL and 76.52 nm for Med-PEG-DTTPL.

[0065] Encapsulation efficiency: The encapsulation efficiency of DOX was 74.87% for Med-PEG-DTPL and 52.98% for Med-PEG-DTTPL, as determined by ultra-micro UV spectrophotometry.

[0066] Stability: Med-PEG-DTPL and Med-PEG-DTTPL were stored in a refrigerator at 4°C, and the particle size was measured every 3 days.

[0067] Comparative Example 3

[0068] A composite nanoparticle, compared with Example 3, is obtained by replacing the DOX / TiO2 suspension with anhydrous ethanol in step (2) with a DOX solution with anhydrous ethanol, while keeping everything else unchanged. The nanoparticle is denoted as Low-PEG-DTPL.

[0069] The physicochemical properties of Example 3 and Comparative Example 3 are characterized as follows:

[0070] Particle size and dispersibility: The average particle size determined by dynamic light scattering DLS was: Low-PEG-DTPL 58.58 nm, Low-PEG-DTTPL 87.82 nm.

[0071] Encapsulation efficiency: The encapsulation efficiency of DOX was measured by ultra-micro UV spectrophotometry as 71.8% for Low-PEG-DTPL and 46.7% for Low-PEG-DTTPL.

[0072] Stability: Low-PEG-DTPLL and Low-PEG-DTTPL were stored in a refrigerator at 4°C, and the particle size was measured every 3 days.

[0073] Table 1. Particle size and encapsulation efficiency of DTPL and DTTPL in Examples 1-3 and Comparative Examples 1-3

[0074]

[0075] As shown in Table 1 and Figure 1As shown, the particle size and encapsulation efficiency of DTPL and DTTPL in Examples 1-3 and Comparative Examples 1-3 are as follows: with the increase of PEG content (1.5% to 10%), the nanoparticle size gradually decreases. This is because when the PEGylated lipid (DSPE-PEG2000) inserts into the lipid bilayer, its large-volume hydrophilic groups (PEG chains) force adjacent phospholipid molecules to arrange themselves more closely (the intermolecular distance between phospholipids can decrease from 0.9 nm to 0.6 nm), leading to an increase in the spontaneous curvature of the membrane and a decrease in membrane permeability, thereby reducing the nanoparticle size and preventing drug leakage. Simultaneously, the PEG chains form a dense polymer brush on the liposome surface, generating strong steric hindrance. When liposomes approach each other, the PEG layer compression must overcome entropy reduction resistance, significantly increasing the fusion energy barrier and the transmembrane diffusion free energy barrier, thus preventing the fusion of different nanoparticles and hindering drug molecule penetration through the lipid bilayer. Therefore, it can be seen from the table that the higher the PEG content, the stronger the above two effects, resulting in a gradual decrease in nanoparticle size and an increase in encapsulation efficiency. Since the encapsulated TiO2NPs occupy a limited internal aqueous phase space, the encapsulation volume available for the drug is indirectly reduced. Furthermore, TiO2NPs may cause slight disturbance to the integrity of the lipid membrane during the preparation process. The nanoparticles of the present invention effectively inhibit the fusion between nanoparticles while maintaining the encapsulation efficiency, and achieve high tumor inhibition and therapeutic effects.

[0076] Comparative Example 4

[0077] A composite nanoparticle, free of α-TOS compared to the examples; lipid components: 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC): purity ≥99%; 1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoate (SM-102): purity ≥98%; cholesterol (CHOL): purity ≥98%; distearoylphosphatidylethanolamine-polyethylene glycol-2000 (DSPE-PEG2000): purity ≥95%; preparation method:

[0078] (1) Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-octylnonyl-8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octyl ester (1-octylnonyl) 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoate (SM-102) and distearate phosphatidylethanolamine-PEG2000 (DSPE-PEG2000) were dissolved in anhydrous ethanol to prepare 100 mg / mL solutions; cholesterol (CHOL) was dissolved in anhydrous ethanol to prepare 25 mg / mL solutions. The lipid components described in S1 were transferred to the same EP tube in a molar ratio of SM-102:DSPC:CHOL:DSPE-PEG2000 = 51.5:3.5:38:10 and mixed using a vortex mixer.

[0079] (2) Add 100 μL of DOX / TiO2 suspension in anhydrous ethanol to the EP tube and mix it by vortexing.

[0080] (3) Add 500 μL of deionized water to a clean EP tube. Transfer the mixture in the EP tube in S3 to a new EP tube containing deionized water using a pipette (equipped with a sample loading tip with certain characteristics). During this process, the latter needs to be kept in a high-speed vortex state. Transfer the EP tube to an ultrasonic disruptor for ultrasonication. After continuous ultrasonication for 90 seconds, High-PEG-DTSDCPL is obtained.

[0081] Comparative Example 5

[0082] A composite nanoparticle, compared with Comparative Example 4, has its lipid components in step (1) changed to a molar ratio of SM-102:DSPC:CHOL:DSPE-PEG2000 = 51.5:8.5:38:5 to obtain Med-PEG-DTSDCPL.

[0083] Comparative Example 6

[0084] A composite nanoparticle, compared with Comparative Example 4, has its lipid components in step (1) changed to a molar ratio of SM-102:DSPC:CHOL:DSPE-PEG2000 = 51.5:12:38:1.5 to obtain Low-PEG-DTSDCPL.

[0085] Comparative Example 7

[0086] A composite nanoparticle, compared to Example 1, involves adding chemotherapy drugs via ethanol injection after ultrasonic disruption. The preparation method includes the following steps:

[0087] (1) Disteaaroyl-sn-glycero-3-phosphocholine (DSPC), D-α-tocopheryl succinate (α-TOS) and disteaaroyl-phosphatidylethanolamine-PEG2000 (DSPE-PEG2000) were dissolved in anhydrous ethanol to prepare 100 mg / mL solutions. The lipid components were transferred to the same EP tube in a molar ratio of DSPC:α-TOS:DSPE-PEG2000 = 60:30:10 and mixed with a vortex mixer.

[0088] (2) Add 500 μL of deionized water to a clean EP tube. Transfer the mixture in the EP tube described in S2 to a new EP tube containing deionized water using a pipette (equipped with a sample loading tip with certain characteristics). During this process, the latter needs to be kept in a high-speed vortex state. Transfer the EP tube described in S3 to an ultrasonic disruptor for ultrasonication and continue ultrasonication for 90 seconds.

[0089] (3) Quickly inject 100 μL of DOX / TiO2 suspension in anhydrous ethanol into the EP tube and vortex mix it to obtain High-PEG-DTTPL-Injection.

[0090] Comparative Example 8

[0091] A composite nanoparticle, compared with Comparative Example 6, was obtained by adding chemotherapy drugs after ultrasonic disruption, replacing the DOX / TiO2 suspension in step (3) with a DOX solution in anhydrous ethanol, and keeping everything else unchanged, to obtain High-PEG-DTPL-Injection.

[0092] Comparative Example 9

[0093] A composite nanoparticle was prepared by changing the ultrasonic time in step (3) to 30 s, while keeping other steps unchanged, compared to Example 1. High-PEG-DTTPL-30s and High-PEG-DTPL-30s nanoparticles were obtained respectively.

[0094] Comparative Example 10

[0095] A composite nanoparticle, compared to Example 1, was prepared using PBS as the solvent. 1 mg of doxorubicin (DOX) was weighed and dispersed in 10 mL of PBS to prepare a 0.1 mg / mL DOX PBS solution. 1 mg each of nano-titanium dioxide (TiO2NPs, 5-10 nm) and doxorubicin (DOX) were weighed and dispersed together in 10 mL of PBS to prepare a 0.1 mg / mL DOX / TiO2 PBS suspension. High-PEG-DTPL-PBS and High-PEG-DTPL-PBS nanoparticles were obtained, respectively.

[0096] Table 2. Particle sizes of DTPL and DTTPL in the examples and comparative examples

[0097]

[0098]

[0099] As shown in Table 2, the nanoparticles are often too large in size and have a highly irregular distribution, even exhibiting flocculation under certain conditions, rendering them completely unusable in practice. Specifically, without the addition of α-TOS as a lipid component, DSDCPL and DTSDCPL exhibit very poor stability, beginning to settle, flocculate, coalesce, and disintegrate after being stored at 4°C for more than 3 days. In contrast, α-TOS plays a significant role in maintaining particle stability. Figure 3 ).

[0100] Adjustments to the preparation methods, including ethanol injection, 30s sonication, and PBS solvent, failed to yield ideal nanoparticles. The reasons are as follows: Ethanol injection is more suitable for the binding of nucleic acid drugs to ionizable lipids. Under high-speed injection, the negatively charged nucleic acid drug and the positively charged lipid carrier are tightly bound together by electrostatic attraction, while the drug and lipid carrier involved in this invention do not rely on a positive-negative potential binding mode. 30s sonication leads to insufficient dispersion of lipid aggregates, resulting in excessively large and uneven particle sizes. Furthermore, using PBS as a solvent for DOX or DOX / TiO2 often results in a gel-like structure in the drug system. This is because in PBS, DOX and negatively charged TiO2 NPs form a cross-linked network through electrostatic bridging, and the high ionic environment of PBS exacerbates the aggregation of liposomes and hydrophobic components (α-TOS / DOX), collectively constructing a three-dimensional gel structure. In anhydrous ethanol, however, electrostatic interactions are eliminated (DOX / TiO2 charge shielding) and all components are fully dissolved (hydrophobic interactions disintegrate), causing the lipid membrane to become fluid and preventing network formation.

[0101] According to Examples 1-3 and Comparative Examples 1-3, the lipid particles with various PEG contents (1.5 mol%-10 mol%) involved in the formulations proposed in this application all exhibit good physical properties, among which the high PEG content (10 mol%) shows the best overall performance in terms of particle size, encapsulation efficiency, and light-controlled release effect.

[0102] The photoresponsive titanium dioxide vitamin E succinate nanoparticles of this invention were used to verify their photoresponsive drug release and anticancer therapeutic capabilities:

[0103] 1. TEM images of DTPL and DTTPL

[0104] TEM uses a high-energy electron beam to penetrate ultrathin samples and magnifies the image through electromagnetic lenses to reveal the atomic-scale structure, composition, and defects within the material. During the imaging process, electrons are scattered by atoms; the stronger the scattering, the darker the area, and the weaker the scattering, the brighter the area, thus forming a bright-dark image that reflects the microscopic morphology of the sample.

[0105] Figure 2 The images show TEM images of DTTPL (A), DTPL (B), DTTPL-Dark (C), and DTTPL-Light (D). Comparing B and C, it can be seen that the lipid particle size of DTTPL is slightly larger due to the introduction of TiO2NPs. Comparing C and D, it can be seen that the lipid membrane of the nanoparticles ruptures after light exposure, allowing the drug to be released.

[0106] 2. Infrared spectroscopy test of DOX / TiO2

[0107] Dry DOX, TiO2 NPs, and DOX / TiO2 composites were prepared using the KBr pellet method, and characterized by infrared absorption spectra using Fourier transform infrared spectroscopy. Figure 4 It can be seen that the absorption peak of DOX / TiO2 is consistent with that of DOX and TiO2NPs, and no new absorption peak is generated. That is, the two do not form a covalent bond and will not interfere with the release of DOX.

[0108] 3. Photoresponsive in vitro release experiment:

[0109] Experimental setup: DTTPL in 460nm visible light (intensity 50Mw / cm²) 2 The control group was irradiated for 30 minutes under light-protected conditions, while the DTTPL group was left to stand for 30 minutes in the dark. Then, both groups of DTTPL were placed in dialysis bags, using sufficient deionized water as the dialysate. The absorbance of DOX in the dialysis bags was measured at 0h, 0.5h, 1h, 2h, 4h, and 19h, and analyzed using a concentration standard curve. Figure 5 The corresponding concentration of DOX was calculated, and the drug release rate curves of DTTPL under light and dark environments were obtained.

[0110] Result: According to Figure 6 It can be seen that at a high PEG content (10 mol%), the release rate of DTTPL-Light is 20% higher than that of DTTPL-Dark, showing the greatest difference among nanoparticles with various PEG contents and exhibiting the best light control effect. The direct reason for this is that under illumination, valence band electrons in the semiconductor material TiO2NPs absorb photons and transition to the conduction band, leaving holes in the valence band. After the electron / hole pairs transfer to the surface of TiO2NPs, they undergo redox reactions with water and oxygen molecules in the surrounding environment, producing ·OH and ·O2. - Free radicals attack lipid membranes, thereby promoting the efficient release of drug molecules.

[0111] 4. Electron paramagnetic resonance testing

[0112] Electron paramagnetic resonance (EPR) is a magnetic resonance technique originating from the magnetic moments of unpaired electrons. It can be used to qualitatively and quantitatively detect unpaired electrons in atoms or molecules and explore the structural characteristics of their surrounding environment. Free radicals, once generated, often disappear rapidly due to instability. DMPO is a commonly used scavenger for free radicals such as hydroxyl radicals and superoxide anion radicals. By binding with these free radicals, DMPO can prolong their existence time, facilitating testing.

[0113] Result: Passed Figure 7 It can be seen that significant O2 was observed in the DTTPL-Light system during the EPR test. - The presence of ·OH signals, while DTPL-Dark and DTTPL-Dark showed no significant signals, directly demonstrating that DTTPL only generates a large amount of ·O2 under illumination. - and ·OH.

[0114] 5. CCK-8 assay for cytotoxicity

[0115] The main components of the CCK-8 reagent are WST-8 and PMS. Under the action of PMS, WST-8 is reduced by dehydrogenases in the mitochondria of cells to a highly water-soluble yellow-orange formazan product, which produces a characteristic absorbance signal at OD 450nm, thus characterizing the relative amount of live cells.

[0116] Experimental groups were set up with free DOX, DTPL, and DTTPL, and cell viability was obtained using CCK-8 reagent. The specific implementation method was as follows: MCF-7 cells were seeded in brand new 96-well plates at a density of 5000 cells / well. After culturing for 24 hours, the cells adhered and were then treated with a series of concentration gradients of 0 nM, 5 nM, 25 nM, 50 nM, 125 nM, and 500 nM. The cells were then cultured for another 72 hours, after which the original culture medium was discarded and replaced with fresh culture medium containing 10% CCK-8 reagent. The cells were then incubated at 36.5℃ for 1-4 hours. Experimental results ( Figure 8 It was pointed out that free DOX, DTPL, DTTPL-Dark, and DTTPL-Light had an effect on the IC50 of MCF-7 cells. 50 The values ​​were 23.8 nM, 13.9 nM, 11.9 nM, and 6.05 nM, respectively, indicating that the drug's toxicity to MCF-7 cells was significantly improved by liposome encapsulation and light treatment.

[0117] 6. RT-qPCR gene expression experiment

[0118] When drugs induce apoptosis in cancer cells, they often achieve this by affecting gene expression within the cancer cells. Real-time quantitative PCR (RT-qPCR) is a molecular biology technique used for real-time amplification and detection of specific DNA or RNA sequences. This method utilizes fluorescent dyes or probes that emit signals when they bind to amplified DNA or RNA molecules, thereby quantifying the target sequence.

[0119] Gapdh is a gene that is stably expressed in all cells, and therefore is often used as an internal reference gene to characterize the relative expression level of target genes. According to Figure 9 The results showed that, in cells treated with DOX, Brca1 expression was downregulated, and the DNA self-repair ability of tumor cells was inhibited. Simultaneously, Bcl-2 expression was downregulated and Bax expression was upregulated, which is attributed to the introduction of α-TOS, which permeates the mitochondrial membrane in MCF-7 cells. The synergistic effect of these two factors led to enhanced activation and upregulation of Caspase-3 expression. Comparing the treatment results of each drug group, DTTPL-Light treatment clearly exhibited the strongest effect in downregulating proto-oncogene expression and upregulating tumor suppressor gene expression.

[0120] 7. In vivo therapeutic experiments in mice

[0121] 0.1 mL of MDA-MB-231 cell suspension (5 × 10⁻⁶ cells) was subcutaneously injected into the axilla of BALB / c-nu mice. 6 A tumor model was constructed using individual cells. When the tumor volume reached 80-100 mm², the model was further developed. 3Mice were randomly divided into five groups (n=6 per group): NC, DOX, DTPL, DTTPL-Dark, and DTTPL-Light (dose: 2 mg / kg). Each mouse received a tail vein injection every three days for a total of five treatments. Tumor size and body weight were measured every two days over three weeks. One week after the last treatment, mice were sacrificed and blood, major organ samples, and tumors were collected for various tests and analyses.

[0122] Figure 10 The results showed that after treatment, tumor growth in the DTTPL-Light group mice was significantly inhibited, with a tumor growth inhibition rate (TGI) of 70.09%, significantly higher than the 48.26% in the free DOX group. Simultaneously, the body weight of mice in each group did not fluctuate abnormally, increasing by 2-3g during the experiment, which is within the normal growth range, indicating that the treatment process was safe and effective. H&E staining results of tumor sections showed that compared to the saline group and the free DOX treatment group, the tumor volume in mice treated with DTTPL-Light was significantly smaller, and the number of tumor cells was also reduced. H&E staining results of sections from major organs showed that the treatment process did not cause serious damage to the major organs of the mice. Figure 11 The comparison of various biochemical indicators in serum confirmed the above results. It was noted that after DTTPL treatment, aspartate aminotransferase (AST), triglycerides (TG), total cholesterol (T-CHO), and lactate dehydrogenase isoenzyme 1 (LDH1) levels were almost identical to those in the saline group, while alanine aminotransferase (ALT) was slightly elevated. In contrast, compared to the free DOX group, lactate dehydrogenase (LDH) and creatine kinase-MB isoenzyme (CK-MB) levels were decreased. Figure 12 In summary, the above changes indicate that the mice were in good overall health after treatment. Compared to direct treatment with free DOX, DTTPL even reduced cardiac damage, while possibly causing a mild inflammatory response in the liver.

Claims

1. A photoresponsive titanium dioxide vitamin E succinate nanoparticle, characterized in that, The nanoparticles are liposomes co-loaded with chemotherapeutic drugs and titanium dioxide nanoparticles. The liposome carrier with cavities is assembled from distearylphosphatidylcholine, D-α-tocopherol succinate and distearylphosphatidylethanolamine. The chemotherapeutic drugs and titanium dioxide nanoparticles are combined by electrostatic attraction and encapsulated in the liposome carrier.

2. The nanoparticles according to claim 1, characterized in that, The chemotherapy drug is one or more of doxorubicin DOX or its hydrochloride, epirubicin EPI, and pirarubicin THP, and the particle size of the nano-titanium dioxide TiO2NPs is 5-10 nm.

3. The nanoparticles according to claim 1, characterized in that, The molar ratio of distearylphosphatidylcholine, D-α-tocopherol succinic acid, and distearylphosphatidylethanolamine is 60-68.5:30:1.5-10. The structure of the liposome carrier is as follows: the hydrophobic tail of D-α-tocopherol succinic acid is connected to the hydrophobic tails of distearylphosphatidylcholine and distearylphosphatidylethanolamine through hydrophobic interactions and van der Waals forces; the hydrophilic head of D-α-tocopherol succinic acid is exposed on the hydrophilic outer and inner surfaces of the lipid bilayer, in contact with the aqueous phase, and interacts with water molecules or the polar head of phospholipids through hydrogen bonds.

4. The nanoparticles according to claim 1, characterized in that, The mass ratio of the liposome carrier, chemotherapeutic drug, and titanium dioxide nanoparticles is 150-750:10-20:1-10.

5. A method for preparing the photoresponsive titanium dioxide vitamin E succinate nanoparticles according to claim 1, characterized in that, Includes the following steps: (1) Distearylphosphatidylcholine, D-α-tocopherol succinate and distearylphosphatidylethanolamine-PEG2000 were dissolved in anhydrous ethanol, mixed and vortexed to obtain a liposome carrier solution. (2) Using anhydrous ethanol as a solvent, prepare a chemotherapy drug-nano titanium dioxide suspension, add the liposome carrier solution prepared in step (1), and vortex mix to obtain a mixture. (3) The mixture was transferred to deionized water in a high-speed vortex state using a pipette, and after ultrasonication in an ultrasonic disruptor, photoresponsive titanium dioxide vitamin E succinate nanoparticles were obtained.

6. The manufacturing method according to claim 5, characterized in that, In step (1), the concentrations of distearylphosphatidylcholine, D-α-tocopherol succinate, and distearylphosphatidylethanolamine-PEG2000 are 50-150 mg / mL.

7. The manufacturing method according to claim 5, characterized in that, In step (2), the concentration of chemotherapy drug and nano-titanium dioxide in the anhydrous ethanol suspension is 0.1-0.5 mg / mL.

8. The manufacturing method according to claim 5, characterized in that, In step (3), the ultrasound time is 60s-90s and the ultrasound power is 3%-5%.

9. The use of the photoresponsive titanium dioxide vitamin E succinate nanoparticles according to claim 1 in the preparation of antitumor drugs.

10. The application according to claim 9, characterized in that, The tumor is breast cancer, ovarian cancer, or thyroid cancer.

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