Upconversion nanodelivery system inducing efficient ferroptosis effect for targeted killing of brain glioma and preparation method thereof

Abstract

The application discloses an up-conversion nano drug delivery system for inducing a high-efficiency ferroptosis effect to target and kill brain glioma and a preparation method thereof, and belongs to the field of biological medicines. The method comprises the following steps: preparing up-conversion nanoparticles by using a modified thermal decomposition method; wrapping the up-conversion nanoparticles with mesoporous silica and connecting hydrophilic enzymes through electrostatic adsorption; and loading hydrophobic chemotherapeutic drugs by using a thin film hydration method to obtain an up-conversion nano composite drug delivery system. The composite drug delivery system uses the photoreduction of the up-conversion nanoparticles to enhance the content of ferrous ions, combines with glucose oxidase to enhance the Fenton reaction, mediates a high-efficiency ferroptosis effect, and targets and kills brain glioma cells. The application provides a new idea for the future clinical treatment of brain glioma and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an upconversion nanodelivery system that induces highly efficient ferroptosis to target and kill gliomas and its preparation method. Background Technology

[0002] Upconversion nanoparticles, a type of nanoparticle, have attracted widespread attention due to their advantages such as low background fluorescence, deeper tissue penetration, and tunable emission wavelength, making them more suitable for the treatment of deep tumors. Sorafenib is the only FDA-approved drug for advanced liver cancer, exhibiting excellent anti-cancer effects. However, due to its poor water solubility, conventional oral formulations have very low bioavailability and significant side effects. Glucose oxidase is an endogenous redox enzyme that uses β-D-glucose as a substrate. Its good biocompatibility and high efficiency and specificity for glucose have led to its widespread application in the biomedical field. Glucose oxidase produces hydrogen peroxide by consuming glucose, mediating tumor starvation therapy and increasing local oxidative stress in the tumor, initiating the Fenton reaction, and enhancing the killing effect on tumor cells. However, free glucose, when used in vivo, is difficult to use directly for tumor treatment due to its poor stability, short in vivo half-life, known immunogenicity, and systemic toxicity. Encapsulating target drugs with suitable carriers to improve their solubility and stability in systemic circulation, reduce toxic side effects, avoid or minimize immune clearance, and enable targeted delivery to the tumor site for greater anti-tumor effects is currently a hot research topic. Mesoporous silica has attracted attention due to its controllable morphology, excellent biocompatibility, and high functionalized surface area. The abundant silanol groups on its surface make it easier to modify and provide good encapsulation for hydrophilic drugs, although most chemotherapy drugs are currently hydrophobic. Gliomas are common intracranial tumors, and traditional treatments are ineffective, making the search for more effective treatments urgent. Given the high content of unstable iron pools in glioma cells, inducing ferroptosis in glioma cells has become an effective way to eliminate gliomas. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention proposes an upconversion nanodelivery system that induces highly efficient ferroptosis to target and kill gliomas, and its preparation method. The objective of this invention can be achieved through the following technical solutions:

[0004] A first aspect of the present invention relates to an upconversion nanodelivery system for targeting and killing gliomas by inducing a highly efficient ferroptosis effect, and a method for preparing the same, comprising the following steps:

[0005] Upconversion nanoparticles are encapsulated in mesoporous silica.

[0006] Electrostatic adsorption is used to adsorb hydrophilic drugs onto mesoporous silica.

[0007] A lipid bilayer is wrapped around the outside of mesoporous silica and loaded with hydrophobic drugs.

[0008] A second aspect of the present invention provides a drug delivery carrier that induces a highly efficient ferroptosis effect to target and kill glioma cells, comprising:

[0009] Upconversion nanoparticles;

[0010] Mesoporous silica encapsulated outside the upconversion nanoparticles is used to carry hydrophilic drugs; and a lipid bilayer encapsulated outside the mesoporous silica is used to carry hydrophobic drugs.

[0011] A third aspect of the present invention provides a drug delivery system comprising: a drug component and the drug delivery carrier described above.

[0012] In a fourth aspect, the present invention provides specific applications of the above-described drug delivery carrier and drug delivery system:

[0013] The use of the above-described drug delivery carrier in the preparation of a medicament for treating the glioma; and the use of the above-described drug delivery system in the preparation of a medicament for treating the glioma.

[0014] The beneficial effects of this invention are:

[0015] The self-made upconversion nanocomposite drug delivery system, simultaneously loading hydrophobic chemotherapeutic drugs and hydrophilic enzymes, exhibited spherical, uniform size and regular morphology under transmission electron microscopy (TEM), with an average particle size of 83 nm. Scanning electron microscopy confirmed the presence of Y, Yb, Tm, Nd, F, Na, O, C, and Cl elements in the nanocomposite drug delivery system. This invention solves the technical problem of simultaneously encapsulating hydrophilic enzymes and hydrophobic drugs in most nanosystems. Furthermore, the nanocomposite drug delivery system in this invention has a relatively small particle size, allowing for significant accumulation in the tumor site after entering the body. It also demonstrates good biocompatibility, no significant killing effect on normal cells, and good safety. The nanocomposite drug delivery system exhibited a highly efficient ferroptosis effect in glioma cells, effectively killing them. Compared with the use of GOx and SRF alone, the combined effect significantly enhanced the killing effect on glioma cells. With the assistance of NIR, the antitumor effect was further improved, resulting in satisfactory antitumor efficacy. Nanotechnology has developed rapidly in recent years. Upconversion nanocomposite drug delivery systems loaded with hydrophobic chemotherapeutic drugs and hydrophilic enzymes will play an increasingly important role in the treatment of tumors. This invention solves the dilemma that the treatment of glioma is ineffective in the face of traditional methods, provides a new idea for clinical treatment, and also provides a theoretical basis for its future clinical transformation. Attached Figure Description

[0016] The invention will now be further described with reference to the accompanying drawings.

[0017] Figure 1 This is a schematic diagram of a composite upconversion nanomedicine delivery system;

[0018] Figure 2 A schematic diagram of the synthesis of a composite upconversion nanodelivery system;

[0019] Figure 3 Transmission electron microscopy image of composite upconversion nanoparticles;

[0020] Figure 4 Transmission electron microscopy image of mesoporous silicon encapsulated by composite upconversion nanoparticles;

[0021] Figure 5 Transmission electron microscopy image of a composite upconversion nanodelivery system;

[0022] Figure 6 A diagram illustrating the in vitro killing of glioma cells by a composite upconversion nanomedicine delivery system;

[0023] The components corresponding to the numbers in the diagram are as follows:

[0024] 1. Upconversion nanoparticles, 2. Mesoporous silica layer, 3. Glucose oxidase, 4. Sorafenib, 5. Lipid bilayer, 6. Upconversion nanoparticles encapsulating mesoporous silica to adsorb glucose oxidase (UCNP-mSiO2-GOx), 7. Composite system simultaneously loading glucose oxidase and sorafenib (UCNP-mSiO2-GOx / SRF), A. Electrostatic adsorption method, B. Thin film hydration method. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Preparation of upconversion nanomaterials:

[0027] Preparation of upconversion nanoparticles by thermal decomposition

[0028] UCNPs were prepared by thermal decomposition. First, 1 mmol of the core structure was prepared by weighing YCl3·6H2O (0.1942 g), YbCl3·6H2O (0.0558 g), and TmCl3·6H2O (0.0061 g), measuring 6 mL of oleic acid and 14 mL of 1-octadecene, and placing them in a 100 mL three-necked round-bottom flask. The reaction system was then purged with sufficient Ar2, magnetically stirred, and heated to 150 °C for 30 min to remove moisture and impurities.

[0029] Cool to 50°C, introduce NaOH (0.1g) and NH4F (0.1475g) dispersed in 10mL methanol using a syringe, seal for 30min, then introduce Ar2 and heat to 100°C and maintain for 30min to remove methanol;

[0030] Heat to 160℃ and maintain for 30 min, then continue to heat to 300℃ and maintain for 1 h to promote nanocrystal growth;

[0031] Cool to room temperature, add 30 mL of ethanol to precipitate, centrifuge at 12000 rpm for 10 min, wash three times with n-hexane / ethanol (1:3), and disperse in 10 mL of n-hexane for later use.

[0032] To prepare a 1 mmol shell structure, weigh YCl3·6H2O (0.1942 g), YbCl3·6H2O (0.093 g), and NdCl3 (0.006 g), measure 6 mL of oleic acid and 14 mL of 1-octadecene, and place them in a 100 mL three-necked round-bottom flask. Then, pass sufficient Ar2 into the system, stir magnetically, and heat to 150 °C for 30 min to remove moisture and impurities.

[0033] Cool to 50°C, introduce 10 ml of the pre-prepared UCNP core structure n-hexane solution using a syringe and seal for 30 min, then introduce NaOH (0.1 g) and NH4F (0.1475 g) dispersed in 10 mL of methanol and seal for 30 min;

[0034] Ar2 was introduced and the temperature was raised to 100℃ and maintained for 30 min to remove methanol and n-hexane. Then the temperature was raised to 160℃ and maintained for 30 min. Finally, the temperature was raised to 300℃ and maintained for 1 h to promote the growth of nanocrystals.

[0035] Cool to room temperature, add 30 mL of ethanol to precipitate, centrifuge at 12000 rpm for 10 min, wash three times with n-hexane / ethanol (1:3), and disperse in 10 mL of n-hexane for later use.

[0036] The present invention employs an upconversion nanoparticle preparation method, but other methods can also be used to prepare it. Upconversion nanoparticles have the characteristics of deep penetration and adjustable emission wavelength. Therefore, the preparation method and steps do not constitute a limitation on the scope of protection of the present invention.

[0037] Preparation of upconversion nanoparticle-encapsulated mesoporous silica UCNP-mSiO2 (UMSNs):

[0038] Mix UCNP dispersed in n-hexane with 0.2g CTAB and 20mL pure water, sonicate for 30min, then stir vigorously and heat the mixture to 70℃ until a transparent CTAB-UCNP solution is obtained.

[0039] Cool the solution to room temperature, take 10 mL of CTAB-UCNP solution and sonicate it for more than 30 min, then immediately add it dropwise to the system (20 mL of water, 2 mL of ethanol, 100 μL of 2M NaOH). Sonicate the system for 30 min before stirring for 2 h, and continue to sonicate it in the ice-water mixture for 30 min to make the UCNP monodisperse.

[0040] The reaction system was heated to 70°C and stirred for 2 hours. 150 μL of TEOS and 1 mL of ethanol mixture were added over 30 minutes. The reaction was then carried out for 8 hours. After that, 500 μL of APTES-ethanol mixture containing 20% ​​was added and the reaction was carried out overnight.

[0041] Wash three times with ethanol / water (1:1), then disperse in 5 ml of ethanol and add to 20 mL of 0.5 g NH4NO3. Stir magnetically and purify at 60 °C for 6 h. Wash three times and disperse in 10 mL of pure water.

[0042] 3) Preparation of glucose oxidase UCNP@mSiO2-GOx (UMSNs-GOx) by electrostatic adsorption on mesoporous silica:

[0043] 10 mg of UMSNs-NH2 was dispersed in 10 mL of glacial acetic acid-sodium acetate buffer at pH 5.5, and 1 mg of glucose oxidase (GOx) was added and stirred overnight to obtain UCNP@mSiO2-GOx.

[0044] 4) Preparation of a nanocomposite drug delivery system simultaneously loaded with hydrophilic enzymes and hydrophobic drugs using a thin-film hydration method:

[0045] ① Weigh out 20 μmol of the lipid system with a DPPC / cholesterol / DSPE-PEG-mal molar ratio of 77.5 / 20 / 2.5, which is equivalent to weighing out 11.377 mg DPPC, 1.55 mg cholesterol, and 1 mg DSPE-PEG. 2000 Add 0.2 mg SRF to 2 mL of chloroform and sonicate for 5 min to mix thoroughly.

[0046] ② Use a rotary evaporator at 33°C under vacuum, rotate slowly to allow the chloroform to evaporate completely and form a uniform lipid film, then place it in a vacuum drying oven overnight to allow the chloroform to evaporate fully;

[0047] ③ Add 10 mg of UCNP@mSiO2-GOx and disperse it in 10 ml of glacial acetic acid-sodium acetate buffer solution, and hydrate it at 45 °C for 0.5 h;

[0048] ④ Sonicate in a water bath for 10 minutes, then sonicate with a probe for 6 minutes at a power of 100W. After standing at room temperature for 4 hours, centrifuge at 8000 rpm for 5 minutes and wash three times with glacial acetic acid-sodium acetate buffer.

[0049] 5) The CCK8 assay was used to detect the killing effect of different nano-formulations on G422 cells:

[0050] G422 cells were seeded at a density of 5000 cells per well in 100 μL of 1640 medium in 96-well plates. After incubation at 37°C for 24 h until complete cell adhesion, different nano-preparations were added, and the groups were as follows: PBS, UMSNs-GOx, UMSNs-SRF, UMSNs-GOx / SRF, and NIR irradiation. The concentration of GOx in each group was 20 ng / mL, the concentration of sorafenib was 12 μM, and the NIR irradiation group was 2.0 W / cm². 2 Incubate for 5 minutes. After 24 hours, discard the supernatant, add 100 μL of culture medium containing 10% CCK8 detection solution, and continue incubation for 1-4 hours. Detect the OD value at 450 nm using a microplate reader.

[0051] like Figure 6 As shown, compared with the use of GOx and SRF alone, the combination of the two significantly enhanced the killing effect on glioma cells. After adjuvant NIR, the anti-tumor effect was further improved, and the anti-tumor effect was satisfactory.

[0052] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0053] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A method for preparing an upconversion nanodelivery system that induces highly efficient ferroptosis to target and kill gliomas, comprising the following steps: Upconversion nanoparticles are encapsulated in mesoporous silica. Electrostatic adsorption is used to adsorb hydrophilic drugs onto mesoporous silica. A lipid bilayer is wrapped around the outside of mesoporous silica and loaded with hydrophobic drugs; The hydrophilic drug is glucose oxidase, and the hydrophobic drug is sorafenib. The method for preparing the upconversion nanoparticles includes the following steps: Weigh out YCl3·6H2O, YbCl3·6H2O, and TmCl3·6H2O, measure out oleic acid and 1-octadecene, and pass in sufficient Ar2, stir magnetically and heat to 150°C to remove moisture and impurities. Cool to 50°C, introduce NaOH and NH4F dispersed in methanol using a syringe, seal for 30 min, then introduce Ar2 and heat up to maintain for 30 min to remove methanol; Heat to 160℃, maintain this temperature, and then continue to increase the temperature. Cool to room temperature, add ethanol to precipitate, centrifuge, wash three times with n-hexane / ethanol, and disperse in n-hexane for later use; Weigh out YCl3·6H2O, YbCl3·6H2O, NdCl3, measure out oleic acid and 1-octadecene, place them in a three-necked round-bottom flask, then pass sufficient Ar2 into the system, stir magnetically and heat to maintain the temperature to remove moisture and impurities. After cooling to 50°C, a pre-prepared UCNP core structure hexane solution was introduced using a syringe and then sealed. NaOH and NH4F dispersed in methanol were then introduced and the solution was sealed again. Ar2 was introduced and the temperature was raised to maintain the desired level to remove methanol and n-hexane; Cool to room temperature, add ethanol to precipitate, centrifuge, and wash with n-hexane / ethanol; The process of coating the lipid bilayer onto the outside of mesoporous silicon includes the following steps: Weigh out DPPC, cholesterol, and DSPE-PEG at a molar ratio of 77.5 / 20 / 2.

5. 2000 -mal and sorafenib were dispersed in chloroform and sonicated to mix thoroughly. Rotary evaporator, vacuum, rotation to completely evaporate chloroform and form a uniform lipid film, then placed in vacuum drying oven overnight to allow chloroform to fully evaporate; Add UCNP@mSiO2-GOx and disperse in glacial acetic acid-sodium acetate buffer, then hydrate. The mixture was subjected to water bath sonication, supplemented by probe sonication. After standing at room temperature, it was centrifuged and washed with glacial acetic acid-sodium acetate buffer.

2. The preparation method of the upconversion nanodrug delivery system according to claim 1, characterized in that, The process of encapsulating upconversion nanoparticles in mesoporous silica includes the following steps: Hexane-dispersed upconversion nanoparticles were mixed with CTAB and pure water, first sonicated, then stirred, and the temperature of the mixture was raised until a transparent CTAB-UCNP solution was obtained. The solution was cooled to room temperature. The CTAB-UCNP solution was first sonicated, and then immediately added dropwise to a system containing water, ethanol and 2M NaOH. The system was pre-sonicated and stirred, and then sonicated in an ice-water mixture to make the upconversion nanoparticles monodisperse. Heat the reaction system to a stirring temperature, add a mixture of TEOS and ethanol, then add a mixture of APTES and ethanol containing 20%, and react overnight. Wash with ethanol / water, then disperse in ethanol and add to a solution containing NH4NO3. Stir magnetically, purify, wash three times, and disperse in pure water. UMSNs-NH2 was dispersed in glacial acetic acid-sodium acetate buffer, glucose oxidase was added and stirred overnight to obtain UCNP@mSiO2-GOx.

3. The drug delivery system prepared by the method of claim 1 for inducing efficient ferroptosis-targeting and killing upconversion nanodelivery systems for glioma.

4. Use of the drug delivery system of claim 3 in the preparation of a medicament for treating the glioma.

Images