Preparation method and application of nano pearl for tumor treatment

Abstract

The invention discloses a preparation method and application of nano pearls for tumor treatment, and belongs to the technical field of material preparation. The preparation method comprises the following steps: dissolving ferric salt and 2-aminoterephthalic acid in dimethylformamide, after thorough dissolution, transferring into a high-pressure kettle with a Teflon lining for sealed reaction, and after the reaction, centrifugally collecting a product MIL-101; the preparation method comprises the following steps: adding mPEG-COOH into anhydrous dimethyl sulfoxide containing EDC (Ethylene Dichloride) and NHS (N-Hydroxysuccinimide), activating at room temperature, then adding polyaspartic acid, vigorously stirring to react, and dialyzing with ultrapure water to obtain an mPEG-PASP (Polyaspartic Acid) solution; uniformly mixing the MIL-101 ethyl acetate solution with the PICA ethyl acetate solution, and stirring at room temperature; then, an mPEG-PASP solution and a COOH-PEG-DNs ethyl acetate solution are added into the mixed solution obtained after stirring, and stirring is conducted at the room temperature; and finally, washing with ethyl acetate to obtain PICA (at) MIL(at) DNs / PASP composite nanoparticles, namely the nano pearl. The composite nano-particles are regular in shape, the size is about 200-210 nm, and the composite nano-particles have good dispersity, regular crystal structure and remarkable anti-tumor effect.

Description

Technical Field

[0001] The present invention relates to a method for preparing a nano-composite material, and in particular to a method for preparing and applying a nano-pearl for treating tumors, which can be applied in the fields of biology and medicine. Background Art

[0002] Cancer remains one of the most serious threats to human health. Due to its high morbidity and mortality rates, it poses a major threat to human well-being and socioeconomic progress. Its high mortality rate stems from the uncontrolled proliferation and metastasis of cancer cells and the limitations of traditional treatments (such as surgery, chemotherapy, and radiotherapy) - these methods are often accompanied by systemic toxicity, drug resistance, and damage to normal tissues. Therefore, there is an urgent need to explore innovative and transformative cancer treatment strategies to effectively address these challenges. In recent years, the emergence of new tumor treatment strategies has provided hope for addressing these challenges, including targeted therapy, immunotherapy, photodynamic therapy (PDT), sonodynamic therapy (SDT), and chemodynamic therapy (CDT). Compared with other treatment modalities, CDT utilizes the chemical properties of the drug itself to achieve therapeutic effects. In addition, as a minimally invasive therapy, CDT generates cytotoxic substances in situ through tumor-specific chemical reactions, which has the advantages of high selectivity and low side effects.

[0003] The core principle of chemodynamic therapy is to use the metal ion-mediated Fenton or Fenton-like reaction to convert endogenous hydrogen peroxide (H2O2) into hydroxyl radicals (·OH) (typical reaction scheme: Fe 2+ +H2O2→Fe 3+ +·OH+OH - These highly reactive oxygen species (ROS) can induce oxidative stress, leading to inflammatory infiltration of neutrophils, increased secretion of proteases, and the production of a large number of oxidative intermediates, which can trigger oxidative stress reactions and induce cancer cell death. However, CDT mainly relies on the Fenton reaction to function, and the key step of the Fenton reaction is Fe 2+ Reacts with H2O2 to generate ·OH. Compared with divalent iron ions, the more stable trivalent iron ions are more likely to precipitate in a neutral environment. Therefore, the efficient Fenton reaction depends on acidic pH. Studies have shown that by adding chelating ligands to adjust the reactivity of iron ions, Fe 3+The solubility of picolinic acid is extended to higher pH values, thereby improving the efficiency of the Fenton reaction. Picolinic acid (PICA) is a metabolite of tryptophan that acts as a bidentate ligand in the absorption of zinc and other divalent and trivalent metal ion nutrients. It can also be effectively absorbed by microorganisms such as Arthrobacter and Streptomyces. The role of picolinic acid (PICA) in Fenton chemistry was first reported in organic synthesis and involves the ability of Gif(IV), GoAgg(III) and related systems in pyridine-acetic acid solution to selectively oxidize hydrocarbons. It was originally believed that the reaction was only carried out by Fe 3+ -OOH complexes or iron-based substances, but later it was proved that ·OH was involved. The coordination reaction of PICA with iron ions makes Fe 3+ The solubility of Fe 3+ to Fe 2+ conversion, producing more ·OH and improving the efficiency of the Fenton reaction.

[0004] To enhance the effectiveness of CDT, overexpression of glutathione (GSH) in the TME is also a key factor to consider. GSH is one of the most important endogenous antioxidants in the human body, playing a key role in maintaining cellular redox balance, immune regulation, and metabolic processes. In tumor cells, high levels of GSH can scavenge ROS, thereby reducing the effectiveness of CDT. Therefore, reducing GSH content in the TME is particularly important to improve the efficiency of CDT. It has been reported that 2,4-dinitrobenzenesulfonamide (DNs) compounds can effectively deplete GSH and responsively release SO2. SO2 is a colorless, transparent gas with a pungent odor and is a signaling molecule in normal physiological processes. Increasing evidence indicates that SO2 can disrupt intracellular redox balance, inhibit the function of the intracellular antioxidant system, and further reduce GSH content in tumor cells. Therefore, SO2 generated by DNs consuming GSH can further promote cellular redox imbalance and cause apoptosis. This is both a source of SO2 toxicity and an opportunity for its anti-tumor activity, creating an opportunity for its use in tumor gas therapy.

[0005] Metal-organic frameworks (MOFs) are a class of porous hybrid materials composed of organic ligands and metal ions / clusters through coordination bonds. In recent years, their unique advantages, including porosity, high surface area, structural and functional diversity, biocompatibility, and biodegradability, have led to their increasing application in the biomedical field. On the one hand, MOFs possess abundant pores and functional groups, which facilitate the loading and delivery of various drug molecules. On the other hand, their abundant metal centers can be directly used to catalyze the Fenton reaction. Furthermore, their large surface area facilitates sufficient contact between reactants (such as O2 and H2O2) and the catalyst, thereby promoting the generation of ROS. From these perspectives, biocompatible MOF nanomaterials are promising candidates for the construction of multifunctional CDT nanomedicines for the combined treatment of malignant tumors. Summary of the Invention

[0006] The present invention aims to overcome the deficiencies of the prior art and provide a method for preparing and applying nano-pearls for tumor treatment. The nano-pearls are PICA@MIL@DNs / PASP composite nanoparticles. These nano-pearls can deplete GSH overexpressed in cancer cells through DNs, reducing GSH's consumption of OH. The generated SO2 further triggers cellular redox imbalance, causing cell apoptosis and achieving the effect of increasing CDT. The method is simple and stable and can be widely applied in the biological and medical fields.

[0007] Specifically, this application is implemented through the following solutions:

[0008] A method for preparing nano-pearls for tumor chemokinetic / gas synergistic therapy, comprising the following steps:

[0009] 1) dissolving a trivalent iron salt and 2-aminoterephthalic acid (BDC-NH2) in dimethylformamide (DMF); mixing the resulting mixed solution under stirring, then sealing it in a Teflon-lined autoclave, subjecting it to a high-temperature and high-pressure reaction, centrifuging it, and washing it to obtain MIL-101;

[0010] 2) 1-Ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are dissolved in anhydrous dimethyl sulfoxide (DMSO); methoxypolyethylene glycol carboxyl groups (mPEG-COOH) are added for activation at room temperature, followed by the addition of polyaspartic acid (PASP) with vigorous stirring to complete the reaction. The reaction product is dialyzed against ultrapure water in a dialysis tubing to obtain an mPEG-PASP solution;

[0011] 3) The ethyl acetate solution of MIL-101 and the ethyl acetate solution of PICA were mixed uniformly; stirred at room temperature, and then the mPEG-PASP solution and the ethyl acetate solution of carboxyl-polyethylene glycol-2,4-dinitrobenzenesulfonamide (COOH-PEG-DNs) were added to the stirred mixed solution and stirred at room temperature, and washed to obtain PICA@MIL@DNs / PASP composite nanoparticles, i.e., the nanopearls.

[0012] In the above scheme, the obtained composite nanoparticles have regular shapes, an average particle size of 180 to 200 nm, and good dispersibility. The composite material has a good porous structure and can be degraded in an acidic tumor microenvironment.

[0013] Furthermore, as a preferred embodiment of the present invention, the trivalent iron salt in step 1) is ferric chloride hexahydrate; the mass ratio of ferric chloride hexahydrate to 2-aminoterephthalic acid is 25:9; the stirring time of the mixed solution in step 1) is 10-20 minutes, the reaction temperature is 90-100°C, and the reaction time is 2-3 hours.

[0014] As a preferred embodiment of the present invention, the molar ratio of EDC and NHS in step 2) is 1:1; the molar ratio of mPEG-COOH to polyaspartic acid (PASP) is 6:5, the activation time is 1.5-2.5 h, the stirring time is 1.5-2.5 h, the dialysis time is 48-54 h, and the dialysis molecular weight cutoff (MWCO) is 5000-6000 Da.

[0015] As a preferred embodiment of the present invention, in step 3), the volume ratio of MIL-101 ethyl acetate solution to PICA ethyl acetate solution is 1:2; the concentration of MIL-101 ethyl acetate solution is 400-800 μg mL -1 The concentration of PICA ethyl acetate solution is 400-800 μg mL -1 , keep the concentrations of the two the same when administering the medicine.

[0016] As a preferred embodiment of the present invention, the concentration of the mPEG-PASP solution in step 3) is 400-800 μg mL -1 The concentration of COOH-PEG-DNs ethyl acetate solution was 400-800 μg mL -1 , keep the concentrations of the two the same when administering; the volume ratio of MIL-101 ethyl acetate solution to mPEG-PASP solution is 1:1; the volume ratio of COOH-PEG-DNs ethyl acetate solution to MIL-101 ethyl acetate solution is 1:2.

[0017] Furthermore, the washing agent used in step 3) is ethyl acetate.

[0018] The present invention further provides PICA@MIL@DNs / PASP composite nano-pearls prepared by the aforementioned preparation method.

[0019] The present invention further provides the use of the PICA@MIL@DN / PASP composite nano-pearls in the preparation of anti-tumor drugs.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) MIL-101, with its porous structure, can serve as a carrier for both PICA and COOH-PEG-DNs;

[0022] (2) When PICA@MIL-101@COOH-EDG-DNs / PASP acts on tumor cells, it will be degraded in the acidic tumor microenvironment, leading to the release of iron ions and PICA;

[0023] (3) The released PICA can keep Fe(III) soluble at a neutral pH, promote the conversion of Fe(III) to Fe(II), and produce additional ·OH and a more selective oxidant;

[0024] (4) In the presence of endogenous H2O2, divalent iron ions continuously generate ·OH through the Fenton reaction, and DNs can consume the overexpressed GSH in cancer cells, reducing the consumption of ·OH by GSH. At the same time, the generated SO2 can induce cellular redox imbalance, causing cell apoptosis and improving the effect of CDT.

[0025] (5) The present invention has low requirements for experimental instruments, the method is simple and easy to operate, and the obtained nano-pearls are uniform in shape and size and have good dispersibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a scanning electron microscope image of the product obtained in Example 1.

[0027] Figure 2 This is a transmission electron microscope image of the product obtained in Example 1.

[0028] Figure 3 This is a transmission electron microscope image of the product obtained in Example 5.

[0029] Figure 4 The UV-VIS absorption spectra of the products obtained in Examples 1, 2 and 5 are shown.

[0030] Figure 5 The X-ray diffraction data (XRD) of the products obtained in Examples 1 and 5 are shown.

[0031] Figure 6 The particle size data analysis chart of the products obtained in Examples 1 and 5.

[0032] Figure 7 This is a charge data analysis diagram of the products obtained in Examples 1 and 5.

[0033] Figure 8 The live-death data of MIL@PASP, PICA@MIL@PASP and PICA@MIL@DNs / PASP under different pH conditions.

[0034] Figure 9 HE and TUNEL sections of animal tumors of MIL@PASP, PICA@MIL@PASP and PICA@MIL@DNs / PASP. DETAILED DESCRIPTION

[0035] The following examples provide those of ordinary skill in the art with information on how to make and evaluate the present invention, and the examples are intended to be illustrative of the present disclosure and are not intended to limit the scope thereof. Although every effort has been made to ensure the accuracy of the values (e.g., amounts, temperatures, etc.), some errors and deviations should be considered. Unless otherwise stated, temperatures are in ° C or at ambient temperature.

[0036] Example 1

[0037] In this embodiment, MIL-101 is prepared as follows:

[0038] Dissolve ferric chloride hexahydrate (FeCl3·6H2O, 0.25 g) and 2-aminoterephthalic acid (BDC-NH2, 0.090 g) in 20 mL of dimethylformamide (DMF). Mix the mixture under magnetic stirring for 10 minutes. Once completely dissolved, seal the mixture in a Teflon-lined stainless steel autoclave. After reacting at 90°C for 2 hours, centrifuge (8000 rpm, 10 minutes) to obtain a brown product, which is then rinsed three times with DMF and ethanol.

[0039] The obtained product was tested, as shown in Figure 1 、 Figure 2 As shown. Figure 1 The scanning electron microscope image shows that the obtained MIL-101 has a regular three-dimensional structure, which proves the successful preparation of the material. Figure 2 Transmission electron microscopy images show that the prepared MIL-101 has good dispersibility and a porous structure, and can be used as a carrier for subsequent synthetic materials.

[0040] Example 2

[0041] The preparation of mPEG-PASP in this example is as follows:

[0042] mPEG-PASP was prepared by coupling polyaspartic acid (PASP MW 5000) with mPEG-COOH (MW 5000) using EDC / NHS. First, 0.6 mmol of mPEG-COOH was added to 10 mL of anhydrous dimethyl sulfoxide (DMSO) containing 0.6 mmol of EDC and 0.6 mmol of NHS and activated at room temperature for 2 hours. Then, 0.5 mmol of polyaspartic acid was added, and the reaction was completed by vigorous stirring at 25°C for 2 days. The product was then dialyzed against ultrapure water in dialysis tubing for 48 hours with a molecular weight cutoff (MWCO) of 5000 Da.

[0043] Example 3

[0044] In this example, MIL@PASP was prepared, and the specific process was as follows:

[0045] 4 mL of MIL-101 solution (500 μg mL -1 ) and 4 L mPEG-PASP solution (500 μg mL -1 ) were mixed evenly and stirred at room temperature for 24 h (500 rpm). Finally, the mixture was washed three times with ethyl acetate to remove excess mPEG-PASP to obtain MIL@PASP.

[0046] Example 4

[0047] This example is used to prepare PICA@MIL@PASP. The specific process is as follows:

[0048] 4 mL of MIL-101 solution (500 μg mL -1 ) and 8 mL of PICA solution (500 μg mL -1 ) were mixed evenly and stirred at room temperature for 24 h (500 rpm). Then 4 mL of mPEG-PASP solution (500 μg mL -1 ), stirred at room temperature for 24 h (500 rpm), and then washed three times with ethyl acetate to remove excess PICA and mPEG-PASP, obtaining PICA@MIL@PASP.

[0049] Example 5

[0050] This example is used to prepare PICA@MIL@DNs / PASP. The specific process is as follows:

[0051] 4 mL of MIL-101 solution (500 μg mL -1 ) and 8 mL of PICA solution (500 μg mL -1) were mixed evenly and stirred at room temperature for 24 h (500 rpm). Then 4 mL of mPEG-PASP solution (500 μg mL -1 ) and 8 mL of COOH-PEG-DNs solution (500 μg mL -1 ), stirred at room temperature for 24 h (500 rpm), and then washed three times with ethyl acetate to remove excess PICA, COOH-PEG-DNs, and mPEG-PASP, yielding PICA@MIL@DNs / PASP.

[0052] Transmission electron microscopy of the product obtained in Example 5 Figure 3 As shown, Figure 4 is the UV-visible absorption curve of Examples 1-5, Figure 5 、 Figure 6 and Figure 7 The X-ray diffraction data (XRD), particle size and potential diagram of Example 1 and Example 5 are shown in FIG. Figure 3 It can be seen that compared with MIL-101, the size of PICA@MIL@DNs / PASP has increased significantly, and an obvious layered multi-porous structure can be seen, proving the integrity of the structure. Figure 4 The UV-visible absorption graph shows that PICA, COOH-PEG-DNs and mPEG-PASP were successfully loaded onto MIL-101, proving the successful preparation of PICA@MIL@DNs / PASP nanopearls. Figure 5 The XRD data further proves that the material can still maintain its intact crystal structure and physical structure after long-term stirring. Figure 6 and Figure 7 The changes in the particle size and surface potential of the material are shown, indicating that the PICA@MIL@DNs / PASP nanopearls were successfully prepared.

[0053] Figure 8 Different materials were co-cultured with mouse colon cancer cells at different pH values for 24 hours, followed by live-dead staining. The results showed that at the same pH value, the PICA@MIL@DNs / PASP group had a better killing ability against mouse colon cancer cells; even at a neutral pH of 7.4, the PICA@MIL@DNs / PASP group still had a significant killing ability against mouse colon cancer cells.

[0054] Figure 9 These are H&E and TUNEL sections of animal tumors made of different materials. As can be seen from the figure, compared with other groups, the PICA@MIL@DNs / PASP group caused the highest level of apoptosis and necrosis in tumor tissue, convincingly confirming the excellent anti-cancer efficacy of PICA@MIL@DNs / PASP nanopearls.

[0055] The above-described embodiments merely illustrate several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. Persons skilled in the art will readily appreciate that variations and modifications may be made without departing from the scope of the present invention, all of which fall within the scope of protection of the present invention.

Claims

1. A method for preparing nano-pearls for tumor treatment, characterized in that: The steps include: 1) dissolving a trivalent iron salt and 2-aminoterephthalic acid (BDC-NH2) in dimethylformamide (DMF); mixing the resulting mixed solution under stirring, then sealing it in a Teflon-lined autoclave, subjecting it to a high-temperature and high-pressure reaction, centrifuging it, and washing it to obtain MIL-101; 2) 1-Ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are dissolved in anhydrous dimethyl sulfoxide (DMSO); methoxypolyethylene glycol carboxyl groups (mPEG-COOH) are added for activation at room temperature, followed by the addition of polyaspartic acid (PASP) with vigorous stirring to complete the reaction. The reaction product is dialyzed against ultrapure water in a dialysis tubing to obtain an mPEG-PASP solution; 3) The MIL-101 ethyl acetate solution and the PICA ethyl acetate solution were mixed uniformly; the mixture was stirred at room temperature, and then the mPEG-PASP solution and the carboxyl-polyethylene glycol-2,4-dinitrobenzenesulfonamide (COOH-PEG-DNs) ethyl acetate solution were added to the stirred mixed solution and stirred at room temperature, and washed to obtain PICA@MIL@DNs / PASP composite nanoparticles, i.e., the nanopearls.

2. The preparation method according to claim 1, characterized in that The trivalent iron salt in step 1) is ferric chloride hexahydrate; the mass ratio of ferric chloride hexahydrate to 2-aminoterephthalic acid is 25:9; the stirring time of the mixed solution in step 1) is 10-20 minutes, the reaction temperature is 90-100° C., and the reaction time is 2-3 hours.

3. The preparation method according to claim 1, characterized in that The molar ratio of EDC and NHS in step 2) is 1:1; the molar ratio of mPEG-COOH to polyaspartic acid (PASP) is 6:5, the activation time is 1.5-2.5 hours, the stirring time is 1.5-2.5 hours, the dialysis time is 48-54 hours, and the dialysis molecular weight cutoff (MWCO) is 5000-6000 Da.

4. The preparation method according to claim 1, characterized in that In step 3), the volume ratio of MIL-101 ethyl acetate solution to PICA ethyl acetate solution is 1:2; the concentration of MIL-101 ethyl acetate solution is 400-800 μg mL -1 The concentration of PICA ethyl acetate solution is 400-800 μg mL -1 , keep the concentrations of the two the same when administering the medicine.

5. The preparation method according to claim 1, characterized in that The concentration of the mPEG-PASP solution in step 3) is 400-800 μg mL -1 The concentration of COOH-PEG-DNs ethyl acetate solution was 400-800 μg mL -1 , keep the concentrations of the two the same when administering the medicine.

6. The preparation method according to claim 5, characterized in that The volume ratio of MIL-101 ethyl acetate solution to mPEG-PASP solution was 1:1; the volume ratio of COOH-PEG-DNs ethyl acetate solution to MIL-101 ethyl acetate solution was 1:

2.

7. The preparation method according to claim 1, characterized in that The washing agent used in the step 3) is ethyl acetate.

8. PICA@MIL@DNs / PASP composite nanopearls prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the PICA@MIL@DN / PASP composite nano-pearls according to claim 8 in the preparation of anti-tumor drugs.

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