Preparation method and application of sound-sensitive nano-particles MIL-125 atI3C based on titanium-based metal-organic framework

Abstract

The invention relates to the technical field of titanium-based metal-organic framework materials, in particular to a preparation method and application of sound-sensitive nano-particles MIL-125 at-I3C based on a titanium-based metal-organic framework. Titanium and 2-amino terephthalic acid H2ATA organic ligand are bridged, defects are regulated and controlled through high-temperature calcination, and the sound-sensitive nano-particles MIL-125 at-I3C are obtained. The preparation method comprises the following steps: preparing a titanium-based metal-organic framework material MIL-125 with high sound-sensitive activity and good biocompatibility, and covalently binding a metabolic regulator indoletricarboxaldehyde I3C with the MIL-125 through Schiff base reaction to generate MIL-125-at-I3C. According to the invention, MIL-125 at-I3C is effectively accumulated in the tumor through the high permeability of the solid tumor and the retention effect EPR effect, and the sonodynamic therapy of the tumor part is realized under ultrasonic irradiation. The preparation method disclosed by the invention is simple, high in efficiency and low in cost, and meanwhile, the prepared nanoparticles release I3C at a tumor site to enhance the generation of immune factors, can generate a large amount of reactive oxygen species (ROS) under ultrasonic irradiation to induce tumor immunogenic death, and has a wide application prospect in the field of integrated tumor treatment.

Description

Technical Field

[0001] The invention relates to the technical field of titanium-based metal-organic framework materials, and in particular to a preparation method of titanium-based metal-organic framework-based sonosensitive nanoparticles MIL-125@I3C and applications thereof. Background Art

[0002] Cancer has become one of the major diseases threatening human health in the world. Although traditional chemotherapy drugs have achieved anti-cancer efficacy to a certain extent, they are usually limited in efficacy and produce serious toxic side effects due to their rapid clearance in human blood circulation and poor targeting of tumors. For many years, the development of new drug delivery systems with stable in vivo metabolic properties, good targeting and high anti-cancer efficiency has been the focus of anti-tumor research. In recent years, therapeutic strategies based on ROS generation have achieved great success in cancer treatment. Sonodynamic therapy (SDT) stands out for its potential advantages, including minimal damage, deep tissue penetration and spatiotemporal effectiveness. The strong pressure fluctuations caused by ultrasound (US) waves in SDT can cause the periodic expansion and rupture of gas or bubbles in the liquid, resulting in cavitation. Sonoluminescence is subsequently triggered, showing properties similar to those of light of different wavelengths. Light emission can activate specific sonosensitizers to produce cytotoxic ROS, thereby inducing irreversible damage to liposomes and DNA, and ultimately destroying tumor cells. In sonodynamic therapy, the selection of appropriate photosensitizers is particularly important to improve the therapeutic effect of SDT. In recent years, many organic and inorganic sonosensitizers have been reported. Organic sonosensitizers are mostly porphyrin derivatives and other lipid-soluble small molecules. These compounds have poor water solubility and US stability, and are quickly cleared from the body, resulting in low concentrations in tumor sites, further affecting the efficacy of SDT. They also have certain phototoxicity and skin sensitivity, which seriously restricts their application in tumor treatment. Compared with organic sonosensitizers, inorganic sonosensitizers, such as titanium dioxide nanoparticles (TiO2NPs), have good chemical stability and low phototoxicity and can be used for tumor SDT. However, due to the electron (e) of TiO2NPs, - )-hole (h + ) due to the fast recombination (50±30ns) of the ionomer, resulting in a very low quantum yield of ROS. Therefore, the development of new, safer and more effective titanium-based sonosensitizers is of great significance for enhancing the sonodynamic therapy of tumors.

[0003] In addition, cancer metabolism has pleiotropic effects in the initiation and evolution of tumors, and intervention targeting metabolism is considered to be a fundamental way to achieve tumor regulation. There are many potential therapeutic targets in the huge metabolic network of tumors, and there are intricate connections between them and signal networks such as immunity, metastasis, and drug resistance. Based on the characteristics of the tumor microenvironment, constructing a drug delivery system to multi-level regulate the tumor microenvironment has been proven to be an effective strategy to achieve multi-dimensional control of tumors. Tumor metabolism can effectively promote key biological processes such as tumor cell proliferation, growth, migration, and invasion. By inhibiting the metabolic pathways of tumor cells, the occurrence and development of tumors can be effectively prevented. Targeting tumor metabolism has become a promising anti-cancer strategy. For example, reactivating anti-tumor immunity through immune checkpoint inhibitor treatment is the key to current tumor treatment. Studies have shown that the aryl hydrocarbon receptor agonists indole-3-carboxaldehyde (I3C) and CD8 released by probiotics + Microbiota-host interactions between T cells can effectively enhance anti-tumor immunity.

[0004] With the continuous development of various anti-tumor strategies, the combination therapy of two or more drugs can target different medicinal mechanisms and different cancer pathways, and is expected to take advantage of the advantages of multiple anti-cancer methods and make up for their shortcomings, thereby achieving better anti-tumor therapeutic effects. However, there is still a lack of reliable solutions. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method and application of sonosensitive nanoparticles MIL-125@I3C based on titanium-based metal-organic framework, aiming to obtain nanoparticles combining MIL-125 and I3C through a simple, green and safe method, and apply them to the sonodynamic and metabolic regulation of tumors to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A method for preparing a titanium-based metal-organic framework-based sonosensitive nanoparticle MIL-125@I3C, which comprises bridging titanium and 2-aminoterephthalic acid (H2ATA) organic ligands, and regulating defects by high-temperature calcination to prepare a titanium-based metal-organic framework material (MIL-125) with high sonosensitive activity and good biocompatibility, and covalently combining a metabolic regulator indoletricarboxaldehyde (I3C) with MIL-125 through a Schiff base reaction to generate the sonosensitive nanoparticle MIL-125@I3C; the preparation method comprises the following steps:

[0008] Step 1: Accurately weigh tetrabutyl phthalate and 2-aminoterephthalic acid, and stir with a mixture of N,N-dimethylformamide (DMF) and methanol (MeOH) until completely dissolved;

[0009] Step 2: The stirred mixture was ultrasonically treated, and the obtained suspension was transferred to a reactor and heated at 150°C for 48 hours; when the reactor was naturally cooled to room temperature, the suspension was washed with DMF and ethanol to remove the residual reagents, and then freeze-dried to obtain MOF (Ti) powder;

[0010] Step 3: Transfer the dried MOF (Ti) nanoparticles to a muffle furnace and calcine them under high temperature; after the reaction is completed, take out the brown powder MIL-125 for quantitative analysis;

[0011] Step 4: Stir different concentrations of I3C and MIL-125 in an appropriate amount of methanol solvent for 24 hours, then centrifuge and wash with methanol for multiple times and then dry to obtain the desired sonogenic nanoparticles MIL-125@I3C. Preferably, in step 1, the mass ratio of tetrabutyl phthalate to 2-aminoterephthalic acid is 1:1-3.

[0012] Preferably, in step 1, the volume ratio of DMF to MeOH is 9:1.

[0013] Preferably, in step 1, the stirring temperature is room temperature, the stirring time is 20-30 min, and the stirring speed is 1000 rpm.

[0014] Preferably, in step 2, the ultrasonic temperature is maintained at room temperature, and the ultrasonic time is 15-25 min.

[0015] Preferably, in step 4, the concentration of I3C is 1 mg / mL-2 mg / mL, and the concentration of MIL-125 is 1 mg / mL.

[0016] Preferably, in step 4, the amount of methanol solvent is not too much, and the amount of methanol solvent is 0.5-1.5 mL.

[0017] The present invention also provides an application of the titanium-based metal-organic framework-based sonosensitive nanoparticles MIL-125@I3C prepared by the above-mentioned preparation method in the preparation of tumor sonodynamic therapy drugs.

[0018] The present invention also provides an application of the titanium-based metal-organic framework-based sonosensitive nanoparticles MIL-125@I3C prepared by the above-mentioned preparation method in the preparation of a drug carrier preparation.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The sonosensitive nanoparticles based on titanium-based metal-organic framework obtained by the present invention have uniform size and good dispersibility, and the synthesis method is simple and fast, which is convenient for production.

[0021] 2. The titanium-based metal-organic framework-based sonosensitive nanoparticles obtained by the present invention have good biosafety and high stability, can generate a large amount of active oxygen under acoustic irradiation, and can be used for sonodynamic therapy of tumors.

[0022] 3. The titanium-based metal-organic framework-based sonogenic nanoparticles prepared by the present invention can activate CD8 through I3C + T cells, prompting them to produce IFNγ and granzyme B, thereby enhancing the tumor suppressor effect.

[0023] 4. The present invention synthesizes titanium-based metal-organic framework-based sonosensitive nanoparticles MIL-125@I3C with high biosafety / stability through a one-step method. The obtained MIL-125@I3C has a high quantum yield of reactive oxygen species and has excellent biocompatibility and dispersibility, and has great potential in sonodynamic therapy of tumors and regulation of tumor metabolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is an electron microscope image of the titanium-based metal-organic framework-based sonosensitive nanoparticles MIL-125@I3C obtained in an embodiment of the present invention;

[0025] Figure 2 This is a particle size distribution diagram of the titanium-based metal-organic framework-based sonosensitive nanoparticles MIL-125@I3C obtained in an embodiment of the present invention;

[0026] Figure 3 This is an infrared image of the titanium-based metal-organic framework-based sonosensitive nanoparticles MIL-125@I3C obtained in an embodiment of the present invention;

[0027] Figure 4 This is an XRD pattern of the titanium-based metal-organic framework-based sonosensitive nanoparticles MIL-125@I3C obtained in an embodiment of the present invention;

[0028] Figure 5 This is a DPBF degradation diagram of the titanium-based metal-organic framework-based sonosensitive nanoparticles MIL-125@I3C obtained in an embodiment of the present invention;

[0029] Figure 6 This is the MB degradation diagram of the titanium-based metal-organic framework-based sonosensitive nanoparticles MIL-125@I3C obtained in an embodiment of the present invention;

[0030] Figure 7 This is a diagram of cellular uptake of the titanium-based metal-organic framework-based sonosensitive nanoparticles MIL-125@I3C obtained in an example of the present invention;

[0031] Figure 8This is an ICD effect diagram of the titanium-based metal-organic framework-based sonosensitive nanoparticle MIL-125@I3C obtained in an embodiment of the present invention;

[0032] Fig. 9 This is a graph showing the tumor treatment effect of the titanium-based metal-organic framework-based sonosensitive nanoparticles MIL-125@I3C on melanoma mice according to an embodiment of the present invention.

[0033] Fig.10 This is a quantitative graph showing the effect of the titanium-based metal-organic framework-based sonosensitive nanoparticles MIL-125@I3C on the cytokine content in the tumor according to an embodiment of the present invention. DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Example:

[0036] See also Figure 1-10 A method for preparing sonographically sensitive nanoparticles MIL-125@I3C based on titanium-based metal-organic frameworks is disclosed. Titanium-based metal-organic framework materials (MIL-125) with high sonographic activity and good biocompatibility are prepared by bridging titanium and 2-aminoterephthalic acid (H2ATA) organic ligands and regulating defects by high-temperature calcination. The metabolic regulator indoletricarboxaldehyde (I3C) is covalently bonded to MIL-125 through a Schiff base reaction to generate sonographically sensitive nanoparticles MIL-125@I3C.

[0037] The preparation method of the sonosensitive nanoparticles MIL-125@I3C specifically comprises the following steps:

[0038] Step 1: Accurately weigh 0.26 mL of tetrabutyl phthalate and 0.543 g of 2-aminoterephthalate, mix with 9 mL of DMF and 1 mL of MeOH, and stir at room temperature for 30 min until completely dissolved;

[0039] Step 2: The stirred mixture was ultrasonically treated for 20 min, and the obtained suspension was transferred to a 50 mL reactor and heated at 150 ° C for 48 h; when the reactor was naturally cooled to room temperature, the suspension was washed three times with DMF and ethanol to remove the residual reagents, and then freeze-dried to obtain MOF (Ti) powder;

[0040] Step 3: Transfer the dried MOF (Ti) nanoparticles to a muffle furnace and calcine at 400°C; after the reaction is completed, take out the brown powder MIL-125 for quantitative measurement;

[0041] Step 4: Stir 2 mg / mL of I3C and 1 mg / mL of MIL-125 in 1 mL of methanol solvent for 24 h, then centrifuge and wash with methanol for multiple times and dry to obtain the desired nanoparticles MIL-125@I3C.

[0042] Among them, the morphology of the prepared sonosensitive nanoparticles MIL-125 is as follows Figure 1 As shown, the particle size of the sonosensitive nanoparticles MIL-125 is Figure 2 shown.

[0043] The amino group in MIL-125 and the carbonyl group in I3C will undergo a Schiff base reaction to produce a carbon-nitrogen double bond. Figure 3 The infrared spectrum shows that in the infrared spectrum of MIL-125@I3C, the amino and carbonyl groups are weakened, and the characteristic peaks of carbon-nitrogen double bonds are newly generated. Figure 4 In the figure, the X-ray diffraction phase pattern (XRD) of MIL-125@I3C shows the successful synthesis of MIL-125 and I3C.

[0044] 5 μg / mL MIL-125@I3C nanoparticles and 1 μg / mL 1,3-diphenylisobenzofuran (DPBF) were dissolved in 3.0 mL of dimethyl sulfoxide (DMSO) and ultrasonicated (ultrasonic frequency 1 MHZ, duty cycle 50%, power -1.0 w / cm 2 ) at different times, and the UV absorption of the solution was tested. DPBF is a probe for detecting reactive oxygen species. As reactive oxygen species are generated, the absorption of its characteristic peak at 410 nm will gradually decrease. Figure 5 It can be seen that the in vitro active oxygen generation performance of calcined MIL-125 nanoparticles is improved.

[0045] 5 μg / mL MIL-125@I3C nanoparticles and 1 μg / mL methylene blue (MB) were dissolved in 3.0 mL of ultrapure water and sonicated at a frequency of 1 MHZ, a duty cycle of 50%, and a power of -1.0 w / cm 2 ) at different times, and the UV absorption of the solution was tested. MB is a probe of ·OH. With the generation of ·OH, the absorption of its characteristic peak at 665nm will gradually decrease. Figure 6 It can be seen that the in vitro ·OH generation performance of calcined MIL-125 nanoparticles is improved.

[0046] The sonodynamic performance of the above nanoparticles in vitro is as follows Figure 5 and Figure 6As shown; with the extension of ultrasound time or illumination time, it can be observed that the absorption values ​​of the characteristic peak of DPBF at 410nm and the characteristic peak of MB at 665nm gradually decrease, indicating that the titanium-based MOF-based sonogenic nanoparticles MIL-125@I3C will produce a large amount of reactive oxygen under ultrasound conditions, and have good sonodynamic and photodynamic performance in vitro.

[0047] BODIPY-labeled nanoparticles were incubated with B16OVA melanoma cells to reflect their endocytosis. Figure 7 As shown, with the extension of co-incubation time, the green fluorescence increased, indicating that the sonosensitizing nanoparticles MIL-125@I3C can be effectively taken up by B16OVA cells.

[0048] Laser confocal microscopy was used to detect that the sonographic nanoparticles MIL-125@I3C induced calreticulin (CRT) externalization and high-mobility group protein 1 (HMGB1) externalization after ultrasound. Figure 8 After incubation with MIL-125@I3C, the tumor cell membranes that were ultrasonically treated with MIL-125@I3C showed obvious CRT protein distribution, indicating that MIL-125@I3C treatment induced the excretion of CRT in tumor cells. HMGB1 is a protein located in the cell nucleus. When tumor cells are stimulated, HMGB1 protein will be excreted from the cell nucleus. Under normal circumstances, HMGB1 is located in the cell nucleus. After treatment with MIL-125@I3C, the fluorescence of HMGB1 protein in tumor cells is weakened, indicating that the excretion of HMGB1 in the nucleus of tumor cells is increased after MIL-125@I3C ultrasonic treatment.

[0049] The tumor treatment curve of the titanium-based metal-organic framework-based sonogenic nanoparticle MIL-125@I3C on melanoma mice is shown in the figure below: Fig. 9 As shown; B16OVA melanoma mice (tumor size: ~50mm 3 ) were randomly divided into 5 groups (5 in each group):

[0050] Group (1) was the control group (control), which was injected with PBS only;

[0051] Group (2) was the material group (I3C), which was injected with I3C via the tail vein only;

[0052] Group (3) was the material group (MIL-125@I3C), which was only injected with MIL-125@I3C via the tail vein;

[0053] The fourth group was the material (MIL-125) ultrasound group, with tail vein injection of MIL-125 + ultrasound conditions (ultrasound frequency 1 MHZ, duty cycle 50%, power -1.0 w / cm 2 )5min / each;

[0054] The fifth group was the material (MIL-125@I3C) ultrasound group, with tail vein injection of MIL-125@I3C + ultrasound conditions (ultrasound frequency 1MHZ, duty cycle -80%, power -1.0w / cm 2 )5min / each.

[0055] 12 h after tail vein injection, the tumor was treated with ultrasound irradiation. Tumor volume was measured and recorded every day. Tumor volume was determined by the following equation: Volume = length × width 2 / 2.

[0056] Compared with the control group, groups (2) and (3) showed certain tumor inhibition effects, indicating that I3C metabolic regulation can play a therapeutic role; the tumor inhibition effect of group (4) was more obvious, indicating that SDT had a significant killing effect on the tumor; the growth of the tumor in group (5) was significantly improved by tail vein injection of titanium-based metal-organic framework-based sonogenic nanoparticles MIL-125@I3C + ultrasound irradiation group compared with other groups, indicating that the nanoparticles can combine sonodynamics and metabolic regulation in tumor treatment to achieve a synergistic therapeutic effect.

[0057] Then we tested the content of related cytokines in tumor tissues through Elisa. Fig.10 It can be seen that MIL-125@I3C after ultrasound can activate tumor CD8+T cells and increase the production of granzyme B and interferon γ.

[0058] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

[0059] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A method for preparing acoustic sensitive nanoparticles MIL-125@I3C based on titanium-based metal-organic framework, characterized in that: Through the bridging of titanium and 2-aminoterephthalic acid H2ATA organic ligands and the regulation of defects by high-temperature calcination, a titanium-based metal-organic framework material MIL-125 with high sonogenic activity and biocompatibility is prepared, and the metabolic regulator indole tricarboxaldehyde I3C is covalently bound to MIL-125 through a Schiff base reaction to generate sonogenic nanoparticles MIL-125@I3C; the preparation method comprises the following steps: Step 1: Accurately weigh tetrabutyl phthalate and 2-aminoterephthalic acid, and stir with a mixture of N,N-dimethylformamide DMF and methanol MeOH until completely dissolved; Step 2: The stirred mixture was ultrasonically treated, and the obtained suspension was transferred to a reactor and heated at 150°C for 48 hours; when the reactor was naturally cooled to room temperature, the suspension was washed with DMF and ethanol to remove the residual reagents, and then freeze-dried to obtain MOF (Ti) powder; Step 3: Transfer the dried MOF (Ti) nanoparticles to a muffle furnace and calcine them under high temperature; after the reaction is completed, take out the brown powder MIL-125 for quantitative analysis; Step 4: Stir different concentrations of I3C and MIL-125 in an appropriate amount of methanol solvent for 24 hours, then centrifuge and wash with methanol for multiple times and then dry to obtain the desired sonographic nanoparticles MIL-125@I3C.

2. The method for preparing the titanium-based metal-organic framework-based acoustic sensitive nanoparticles MIL-125@I3C according to claim 1, characterized in that: In step 1, the mass ratio of tetrabutyl phthalate to 2-aminoterephthalic acid is 1:1-3.

3. The method for preparing the titanium-based metal-organic framework-based sonosensitive nanoparticles MIL-125@I3C according to claim 1, characterized in that: In step 1, the volume ratio of DMF to MeOH was 9:

1.

4. The method for preparing the titanium-based metal-organic framework-based sonosensitive nanoparticles MIL-125@I3C according to claim 1, characterized in that: In step 1, the stirring temperature is room temperature, the stirring time is 20-30 min, and the stirring speed is 1000 rpm.

5. The method for preparing the titanium-based metal-organic framework-based sonosensitive nanoparticles MIL-125@I3C according to claim 1, characterized in that: In step 2, the ultrasonic temperature is maintained at room temperature, and the ultrasonic time is 15-25 minutes.

6. The method for preparing the titanium-based metal-organic framework-based sonosensitive nanoparticles MIL-125@I3C according to claim 1, characterized in that: In step 4, the concentration of I3C is 1 mg / mL-2 mg / mL, and the concentration of MIL-125 is 1 mg / mL.

7. The method for preparing the titanium-based metal-organic framework-based sonosensitive nanoparticles MIL-125@I3C according to claim 1, characterized in that: In step 4, the methanol solvent is 0.5-1.5 mL.

8. Use of titanium-based metal-organic framework-based sonosensitive nanoparticles MIL-125@I3C prepared by the preparation method described in any one of claims 1 to 7 in the preparation of tumor sonodynamic therapy drugs.

9. Use of titanium-based metal-organic framework-based sonosensitive nanoparticles MIL-125@I3C prepared by the preparation method according to any one of claims 1 to 7 in the preparation of drug carrier preparations.

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