Preparation method of acid-responsive water-soluble fullerene nanoparticles

Abstract

This invention discloses a method for preparing acid-responsive water-soluble fullerene nanoparticles. The method includes the following steps: S1, performing a Prato reaction on a fullerene material, a glycine derivative, and a carboxyl-containing compound to obtain a fullerene derivative with carboxyl-terminated ends; S2, performing an esterification reaction on the fullerene derivative with carboxyl-terminated ends and a water-soluble polymer in an organic solvent B under the action of a catalyst to obtain a water-soluble fullerene derivative; S3, performing a condensation reaction on an amino acid and a compound with aldehyde-terminated ends under alkaline conditions to obtain an amino acid derivative with acid-responsive properties; S4, mixing the water-soluble fullerene derivative and the amino acid derivative with acid-responsive properties in water to composite them, obtaining acid-responsive water-soluble fullerene nanoparticles. This method has advantages such as suitable size, stable properties, good biosafety, and simple preparation method, and can be applied to the preparation of antitumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials technology, and particularly relates to a method for preparing acid-responsive water-soluble fullerene nanoparticles. Background Technology

[0002] Fullerenes are a series of atomic clusters composed of different carbon atoms, possessing a cage-like fused ring structure. They exhibit advantages such as high stability, high strength, and large specific surface area. In the biomedical field, fullerenes, due to their unique large conjugated electron structure, can capture nearby free radicals and promote their recombination quenching, playing an important role in anti-inflammatory, antioxidant, and other related diseases. Furthermore, fullerenes can regulate the body's immunity, showing excellent therapeutic prospects in the field of tumor immunotherapy.

[0003] The pure carbon cage structure of fullerenes, while providing stability, also results in poor water solubility, thus limiting their application in the biomedical field. Therefore, it is necessary to modify the water solubility of fullerene materials to better enable their application in biomedical settings. Tumor sites typically exhibit a slightly acidic physiological characteristic; designing fullerene materials with a slightly acidic response would be beneficial for achieving better anti-tumor therapeutic effects. However, current methods for preparing acid-responsive fullerene materials are still insufficient. Summary of the Invention

[0004] To address the above-mentioned shortcomings of existing technologies, the present invention aims to provide a method for preparing acid-responsive water-soluble fullerene nanoparticles. This method utilizes the Prato reaction and esterification reaction to prepare water-soluble fullerene materials; condenses amino acids with compounds containing aldehyde terminals under alkaline conditions to generate acid-responsive amino acid derivatives; then, the obtained water-soluble fullerene is compounded with the acid-responsive amino acid derivatives to obtain an acid-responsive water-soluble fullerene-amino acid composite system. This nano-co-delivery system has advantages such as suitable size, stable properties, good biosafety, and simple preparation method, and can be applied to the preparation of antitumor drugs.

[0005] In a first aspect, the present invention provides a method for preparing acid-responsive water-soluble fullerene nanoparticles, comprising the following steps:

[0006] S1. Fullerene material, glycine derivative and carboxyl-containing compound are subjected to Prato reaction in organic solvent A to obtain fullerene derivative with carboxyl terminus.

[0007] S2. The fullerene derivative containing a carboxyl terminus is esterified with a water-soluble polymer in an organic solvent B under the action of a catalyst to obtain a water-soluble fullerene derivative.

[0008] S3. The amino acid and the compound with the aldehyde terminal are condensed in an organic solvent C under alkaline conditions to obtain an amino acid derivative with acid-responsive properties.

[0009] S4. The water-soluble fullerene derivative and the amino acid derivative with acid-responsive properties are mixed in water to combine them, thereby obtaining the acid-responsive water-soluble fullerene nanoparticles.

[0010] In the above-described method for preparing acid-responsive water-soluble fullerene nanoparticles, the fullerene material includes one or more of hollow fullerenes and metallofullerenes; preferably, the hollow fullerene includes C 60 Or C 70 Preferably, the metallofullerene is selected from Gd@C 82 、Lu@C 82 Y2@C 82 Gd2@C 82 and Lu2@C 82 Any one or more of the following;

[0011] The carboxyl-containing compound includes one or more of 3-aldehyde phenylacetic acid, 3-aldehyde benzoic acid, 4-aldehyde phenylacetic acid, and 4-aldehyde benzoic acid;

[0012] The glycine derivatives include one or more of glycine, N-ethylglycine, and N-methylglycine;

[0013] The molar ratio of the fullerene material, the glycine derivative, and the carboxyl-containing compound is 1:(0.8-1.2):(0.8-1.2), preferably 1:1:0.9.

[0014] In the above-mentioned method for preparing acid-responsive water-soluble fullerene nanoparticles, the Prato reaction is carried out in an anhydrous inert gas atmosphere, such as a nitrogen atmosphere.

[0015] The Prato reaction is carried out at a temperature of 80–120°C for 2–8 hours, or by reflux and stirring at 115°C for 3–6 hours (e.g., 4 hours).

[0016] The organic solvent A is one or more of toluene, benzene, and o-xylene;

[0017] In step S1, after the Prato reaction is completed, the method further includes the following post-processing steps: after the reactants are cooled to room temperature, the solvent is removed by rotary evaporation and column chromatography is performed to remove unreacted fullerene material, the ethyl acetate concentration is increased to 20% by volume, and a deep purple-red liquid is obtained by rotary evaporation.

[0018] In the above-mentioned method for preparing acid-responsive water-soluble fullerene nanoparticles, the water-soluble polymer is selected from one or more of polyethylene glycol (such as PEG600), polyethyleneimine, hyaluronic acid, and polyamino acids.

[0019] The molar ratio of the fullerene derivative containing the carboxyl terminus to the water-soluble polymer is 1:(1-3), preferably 1:2;

[0020] The esterification reaction is carried out under normal temperature and pressure with stirring, and the reaction time is 12 to 48 hours, such as 12 hours.

[0021] The catalysts are 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP);

[0022] The organic solvent B is one or more of tetrahydrofuran, dichloromethane, and trichloromethane;

[0023] The method further includes the following post-processing steps after the esterification reaction is completed: after stopping the reaction, the solvent is removed by rotary evaporation, the product is dissolved by adding solvent, and the product is dialyzed in water using a 3500 Da dialysis bag. The solution in the bag is collected to obtain a clear, yellowish-brown fullerene aqueous solution, which is then filtered through a 450 nm filter membrane and freeze-dried.

[0024] In the above-mentioned method for preparing acid-responsive water-soluble fullerene nanoparticles, the amino acid is selected from one or more of glycine, alanine, arginine, serine, lysine, tryptophan, and aspartic acid.

[0025] The aldehyde-terminated compound is selected from one or more of benzaldehyde, phenylacetaldehyde, terephthalaldehyde, p-formaldehyde benzoic acid, and p-formaldehyde phenylacetic acid;

[0026] The molar ratio of the amino acid to the compound with the aldehyde terminal is 1:(0.5-2), preferably 1:1.

[0027] In the above-mentioned method for preparing acid-responsive water-soluble fullerene nanoparticles, the alkaline conditions are provided by one or more alkaline reagents selected from sodium hydroxide, potassium hydroxide, ammonia, and triethylamine.

[0028] The molar ratio of the alkaline reagent to the amino acid is 1:(0.5-2), preferably 1:1;

[0029] The condensation reaction is carried out at a temperature of 40–80°C for 4–12 hours, such as at 65–75°C for 4 hours.

[0030] The organic solvent C is one or more of ethanol, methanol, and acetone;

[0031] In step S3, the method further includes the following post-processing steps: cooling the solid-liquid mixture obtained from the reaction with ice water, then filtering it, washing the solid with ethanol and / or methanol, and vacuum drying the resulting yellow solid powder at 45°C.

[0032] In the above-mentioned method for preparing acid-responsive water-soluble fullerene nanoparticles, the molar ratio of the water-soluble fullerene derivative to the amino acid derivative with acid-responsive properties is 1:(1-10), such as 1:1.

[0033] In a specific embodiment of the present invention, when the mass ratio of the water-soluble fullerene derivative to the amino acid derivative with acid-responsive properties is 1:3, there is no or only a weak inhibitory effect on L02 cells, while there is a significant inhibitory effect on 4T1 tumor cells.

[0034] In the above-mentioned method for preparing acid-responsive water-soluble fullerene nanoparticles, the mixing process can be carried out under ultrasonic conditions.

[0035] In step S4, the method further includes filtering the mixed system with a 220nm filter membrane after mixing to remove undissolved large particulate impurities.

[0036] Secondly, the present invention provides acid-responsive water-soluble fullerene nanoparticles obtained by any of the preparation methods described above. The acid-responsive water-soluble fullerene nanoparticles of the present invention exhibit acid-responsive characteristics in an acidic range such as pH = 5.5–6.5.

[0037] Thirdly, the present invention provides the application of the acid-responsive water-soluble fullerene nanoparticles in the preparation of antitumor drugs.

[0038] In the above applications, the tumor may be breast cancer; as an example, the anti-tumor drug may specifically be a drug that inhibits the activity of 4T1 tumor cells.

[0039] The present invention has the following beneficial effects:

[0040] 1. Suitable size distribution. The acid-responsive water-soluble fullerene nanoparticles obtained by this invention have a hydrated particle size of 109 nm and a homogeneous solution system.

[0041] 2. Stable properties. The aqueous solution of nanoparticles obtained by this invention is stable and will not precipitate even after prolonged standing.

[0042] 3. Excellent acid response properties. The nanoparticles obtained in this invention are stable under neutral or slightly alkaline conditions and spontaneously decompose in weakly acidic environments with pH = 6.5 and below, exhibiting suitable acid response characteristics.

[0043] 4. Good biocompatibility. The fullerene materials, water-soluble polymer materials, amino acids, etc. contained in this invention have good biocompatibility and have little impact on the physiological functions of normal cells.

[0044] 5. The raw materials are widely available and the preparation method is economical and simple. The raw materials involved in this invention are all relatively common and have low costs. The reaction steps involved are simple and do not require special conditions such as high temperature and high pressure, making it environmentally friendly and safe. Attached Figure Description

[0045] Figure 1 The time-of-flight mass spectra of the C70-PEG(600) molecules prepared in Example 1 are shown.

[0046] Figure 2 The infrared absorption spectrum of the Arg-Ter molecule prepared in Example 1 is shown.

[0047] Figure 3 The DLS particle size distribution and potential of the acid-responsive C70@Arg nanoparticles prepared in Example 1 are shown.

[0048] Figure 4 The state of the fullerene-arginine nanocomposite C70@Arg prepared in Example 1 in different pH buffers (12 hours) is shown.

[0049] Figure 5 The effect of different proportions of C70@Arg on L02 cell viability in Example 2 is shown (24h).

[0050] Figure 6 The effect of different concentrations of C70@Arg on the viability of 4T1 and L02 cells in Example 2 is shown (24h). Detailed Implementation

[0051] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0052] Unless otherwise specified, the methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0053] Fullerene C in the following examples 70 Purchased from Beijing Funakang Biotechnology Co., Ltd., product CAS number 115382-22-7, item number F001.

[0054] Example 1: Preparation of acid-responsive water-soluble fullerene-arginine nanocomposite

[0055] This embodiment provides a method for preparing acid-responsive water-soluble fullerene-arginine nanocomposites, including:

[0056] (1) Preparation of water-soluble fullerene derivative C70-PEG(600)

[0057] 100mg C 70 12 mg N-ethylglycine and 18 mg 3-aldehyde phenylacetic acid were dissolved in 100 mL of toluene solution and refluxed at 115 °C for 4 hours under a nitrogen atmosphere. After the reaction mixture cooled to room temperature, a large amount of solvent was evaporated, and column chromatography was performed. Unreacted C70 molecules were removed with pure toluene solvent, and the concentration of ethyl acetate was increased to 20% by volume to obtain a deep purple-red liquid. After evaporation to dryness, 40 mg of deep red solid C70-FA was obtained.

[0058] Add 36.5 mg EDCI, 7.8 mg DMAP, and 150 mg anhydrous magnesium sulfate to the above dark red solid, and dissolve in 10 mL of dried tetrahydrofuran while stirring. Then weigh 46 mg of PEG600 and dissolve it in 10 mL of dichloromethane, slowly adding it dropwise to the reaction system. React at room temperature in air for at least 12 hours. After stopping the reaction, remove the solvent by rotary evaporation, dissolve the product in tetrahydrofuran, and dialyze through a 3500 Da dialysis bag. Collect the purified sample from the dialysis bag to obtain a clear, yellowish-brown fullerene aqueous solution, filter through a 450 nm filter membrane, and lyophilize to obtain 15 mg of dark red solid.

[0059] Depend on Figure 1 It can be seen that the molecular weight distribution of the obtained product is around 1600 Da, which verifies the successful synthesis of C70-PEG(600).

[0060] (2) Preparation of acid-responsive arginine derivatives

[0061] 250 mg of arginine, 200 mg of terephthalaldehyde, and 60 mg of sodium hydroxide were placed in a 100 mL single-necked flask, and approximately 40 mL of ethanol was added. The mixture was heated at 65-75 °C for 4 h in air. The resulting solid-liquid mixture was cooled with ice water and then filtered. The solid was washed with a large amount of ethanol. The resulting yellow solid powder was dried under vacuum at 45 °C, and approximately 300 mg of a bright yellow Arg-Ter solid was collected.

[0062] Depend on Figure 2 The infrared spectrum of the obtained product shows a characteristic C=N stretching vibration peak, which verifies the successful synthesis of Arg-Ter.

[0063] (3) Preparation of acid-responsive water-soluble fullerene-arginine nanocomposite C70@Arg

[0064] As needed, C70-PEG600 and Arg-Ter are dissolved in ultrapure water at a molar ratio of 1:1, mixed by simple ultrasonication, filtered through a 220nm filter membrane, and allowed to stand to obtain a water-soluble fullerene-arginine nanocomposite C70@Arg with acid-responsive characteristics.

[0065] Depend on Figure 3 It can be seen that the obtained C70@Arg nanoparticles have a size of about 110 nm and good dispersion.

[0066] Figure 4 The state of the obtained C70@Arg nanoparticles in buffer solutions at pH 5.5, 6.5, and 7.5 is shown. The results indicate that C70@Arg exhibits good stability in the buffer solution at pH 7.5, while it shows acid-responsive properties in buffer solutions at pH 5.5 and 6.5, validating the rationality of the design scheme.

[0067] Example 2: Investigation of safety at the cellular level

[0068] To investigate the optimal ratio of Arg-Ter to C70-PEG(600) at the cellular level, we designed the following cell experiments.

[0069] Prepare a series of concentrations of C70@Arg culture medium:

[0070] 2 mg Arg-Ter + 0 mg C70-PEG(600) were dissolved in 1 mL of ultrapure water, filtered through a sterile filter membrane, and diluted to a total concentration of 200 ug / mL.

[0071] 1.5 mg Arg-Ter + 0.5 mg C70-PEG(600) were dissolved in 1 mL of ultrapure water, filtered through a sterile filter membrane, and diluted to a total concentration of 200 ug / mL.

[0072] 1 mg Arg-Ter + 1 mg C70-PEG(600) was dissolved in 1 mL of ultrapure water, filtered through a sterile filter membrane, and diluted to a total concentration of 200 ug / mL.

[0073] 0.5 mg Arg-Ter + 1.5 mg C70-PEG(600) were dissolved in 1 mL of ultrapure water, filtered through a sterile filter membrane, and diluted to a total concentration of 200 ug / mL.

[0074] 0 mg Arg-Ter + 2 mg C70-PEG(600) was dissolved in 1 mL of ultrapure water, filtered through a sterile filter membrane, and diluted to a total concentration of 200 ug / mL.

[0075] L02 cells were seeded into 96-well plates at a density of 10,000 cells / well, with 6 wells in each group. A blank control group containing ordinary culture medium was also included. The culture time was 1 day, and then CCK-8 cell viability was measured.

[0076] Figure 5 The results showed that higher Arg-Ter concentrations significantly inhibited the growth of L02 cells, and the growth rate of L02 cells gradually increased as the Arg-Ter concentration decreased. Considering that the cell inhibitory effect of 25% Arg-Ter was greatly reduced, this proportion was selected for further cell safety experiments.

[0077] Experimental groups were set up with C70@Arg concentrations of 0, 12.5, 25, 50, 100, and 200 ug / mL, respectively. Cell-level safety was verified for 4T1 and L02 cells. The cell concentration was 10,000 cells / well, with 6 wells in each group. The culture time was 1 day, and then CCK-8 cell viability was detected.

[0078] Figure 6 The results showed that within the normal physiological range (<=100ug / mL), C70@Arg had no or only a weak inhibitory effect on L02 cells, but a significant inhibitory effect on 4T1 cells. This indicates that the material has little effect on normal cells, but a significant inhibitory effect on rapidly growing 4T1 tumor cells, demonstrating good potential for anti-tumor biological applications.

[0079] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for preparing acid-responsive water-soluble fullerene nanoparticles, characterized in that, Includes the following steps: S1. Fullerene material, glycine derivative and carboxyl-containing compound are subjected to Prato reaction in organic solvent A to obtain fullerene derivative with carboxyl terminus. S2. The fullerene derivative containing a carboxyl terminus is esterified with a water-soluble polymer in an organic solvent B under the action of a catalyst to obtain a water-soluble fullerene derivative. S3. The amino acid and the compound with the aldehyde terminal are condensed in an organic solvent C under alkaline conditions to obtain an amino acid derivative with acid-responsive properties. S4. The water-soluble fullerene derivative and the amino acid derivative with acid-responsive properties are mixed in water to combine them, thereby obtaining the acid-responsive water-soluble fullerene nanoparticles.

2. The method for preparing acid-responsive water-soluble fullerene nanoparticles according to claim 1, characterized in that: The fullerene material includes one or more of hollow fullerenes and metallic fullerenes; preferably, the hollow fullerene includes C 60 Or C 70 Preferably, the metallofullerene is selected from Gd@C 82 、Lu@C 82 Y2@C 82 Gd2@C 82 and Lu2@C 82 Any one or more of the following; The carboxyl-containing compound includes one or more of 3-aldehyde phenylacetic acid, 3-aldehyde benzoic acid, 4-aldehyde phenylacetic acid, and 4-aldehyde benzoic acid; The glycine derivatives include one or more of glycine, N-ethylglycine, and N-methylglycine; The molar ratio of the fullerene material, the glycine derivative, and the carboxyl-containing compound is 1:(0.8-1.2):(0.8-1.2), preferably 1:1:0.

9.

3. The method for preparing acid-responsive water-soluble fullerene nanoparticles according to any one of claims 1-2, characterized in that: The Prato reaction is carried out in an anhydrous inert gas atmosphere; The Prato reaction is carried out at a temperature of 80–120°C for a reaction time of 2–8 hours. The organic solvent A is one or more of toluene, benzene, and o-xylene.

4. The method for preparing acid-responsive water-soluble fullerene nanoparticles according to any one of claims 1-3, characterized in that: The water-soluble polymer is selected from one or more of polyethylene glycol, polyethyleneimine, hyaluronic acid, and polyamino acids; The molar ratio of the fullerene derivative containing the carboxyl terminus to the water-soluble polymer is 1:(1-3), preferably 1:2; The esterification reaction was carried out under normal temperature and pressure with stirring, and the reaction time was 12–48 h. The catalyst is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine; The organic solvent B is one or more of tetrahydrofuran, dichloromethane, and trichloromethane.

5. The method for preparing acid-responsive water-soluble fullerene nanoparticles according to any one of claims 1-4, characterized in that: The amino acid is selected from one or more of glycine, alanine, arginine, serine, lysine, tryptophan, and aspartic acid; The aldehyde-terminated compound is selected from one or more of benzaldehyde, phenylacetaldehyde, terephthalaldehyde, p-formaldehyde benzoic acid, and p-formaldehyde phenylacetic acid; The molar ratio of the amino acid to the compound with the aldehyde terminal is 1:(0.5-2), preferably 1:

1.

6. The method for preparing acid-responsive water-soluble fullerene nanoparticles according to any one of claims 1-5, characterized in that: The alkaline conditions are provided by an alkaline reagent consisting of one or more of sodium hydroxide, potassium hydroxide, and triethylamine. The molar ratio of the alkaline reagent to the amino acid is 1:(0.5-2), preferably 1:1; The condensation reaction is carried out at a temperature of 40–80°C for 4–12 hours. The organic solvent C is one or more of ethanol, methanol, and acetone.

7. The method for preparing acid-responsive water-soluble fullerene nanoparticles according to any one of claims 1-6, characterized in that: The molar ratio of the water-soluble fullerene derivative to the acid-responsive amino acid derivative is 1:(1-10).

8. The method for preparing acid-responsive water-soluble fullerene nanoparticles according to any one of claims 1-7, characterized in that: The mixing is performed under ultrasonic conditions; In step S4, the method further includes, after mixing, filtering the mixed system with a 220nm filter membrane to remove undissolved large particulate impurities.

9. Acid-responsive water-soluble fullerene nanoparticles obtained by the preparation method according to any one of claims 1-8.

10. The use of the acid-responsive water-soluble fullerene nanoparticles according to claim 9 in the preparation of antitumor drugs.

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