Reducible Responsive Curcumin Water-Soluble Nanoparticles and Their Applications

Abstract

The present invention belongs to the field of biomedical technology, and specifically relates to a reduction-responsive curcumin water-soluble nanoparticle and its application. First, curcumin and 3,3'-dithio / selenodipropionic acid are synthesized into a 3,3'-dithio / selenodipropionic acid-curcumin conjugate through an esterification reaction, and then the 3,3'-dithio / selenodipropionic acid-curcumin conjugate is dehydrated and condensed with polyethylene glycol to obtain a polyethylene glycol-curcumin conjugate linked by a disulfide / selenium bond. The polyethylene glycol-curcumin conjugate synthesized in the present invention can self-assemble into nanoparticles in an aqueous solution, can significantly improve the water solubility of curcumin, has good colloidal stability and the property of releasing curcumin in a reduction-responsive manner, and can effectively induce apoptosis of tumor cells, and has an application prospect in the field of tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a reduction-responsive curcumin water-soluble nanoparticle and its application. Background Art

[0002] Curcumin is a plant polyphenol extracted from turmeric, and it exerts its anti-cancer effect by differentiating tumor cells, inducing apoptosis of tumor cells and inhibiting the growth of tumors at all stages. It has a wide range of clinical applications. However, free curcumin has low solubility, poor stability and low absorption rate, resulting in low bioavailability. Therefore, various solutions have been proposed to overcome the above defects.

[0003] Improving the tumor targeting of curcumin through a nano-drug delivery system is a current research hotspot. It can increase the water solubility of the drug, prevent drug degradation, prolong the blood circulation time, and can easily enter tumor tissues through the EPR effect. Therefore, self-assembly to form nanoparticles can significantly improve the drug transport performance of curcumin in vivo and in vitro, and to a certain extent improve the tumor targeting of curcumin. Summary of the Invention

[0004] One of the purposes of the present invention is to provide a reduction-responsive curcumin water-soluble nanoparticle, which can significantly improve the water solubility of curcumin and can effectively induce apoptosis of tumor cells.

[0005] The reduction-responsive curcumin prodrug nanoparticle has the following chemical structural formula:

[0006]

[0007]

[0008] Among them, the preparation reaction formula of the polyethylene glycol-curcumin conjugate (Compound 1) connected by a disulfide bond is as follows:

[0009]

[0010] The specific preparation method is carried out according to the following steps:

[0011] (1) Preparation of 3,3'-dithiodipropionic acid-curcumin conjugate (Compound 4)

[0012] Using curcumin (Compound 3) and 3,3'-dithiodipropionic acid (DTDPA) as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) as a condensing agent, 4-dimethylaminopyridine (DMAP) as a catalyst, and dichloromethane as a solvent, heat to 50 °C and reflux for 24 hours. Evaporate the organic solvent using a rotary evaporator, cool to room temperature, then add 5 mL of distilled water, dialyze the above dispersion in distilled water for three days using a dialysis membrane, and freeze-dry to obtain 3,3'-dithiodipropionic acid-curcumin conjugate (Compound 4).

[0013] Among them, the molar ratio of curcumin, 3,3'-dithiodipropionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 1:1~2:2~4:0.5~1.

[0014] (2) Preparation of disulfide bond-linked polyethylene glycol-curcumin conjugate (Compound 1)

[0015] Using 3,3'-dithiodipropionic acid-curcumin conjugate (Compound 4) and polyethylene glycol as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) as a condensing agent, 4-dimethylaminopyridine (DMAP) as a catalyst, and dichloromethane as a solvent, heat to 50 °C and reflux for 24 hours. Evaporate the organic solvent using a rotary evaporator, cool to room temperature, then add 5 mL of distilled water, dialyze the above dispersion in distilled water for three days using a dialysis membrane, and freeze-dry. The freeze-dried product is separated and purified by column chromatography to obtain the disulfide bond-linked polyethylene glycol-curcumin conjugate (Compound 1).

[0016] Among them, the molar ratio of 3,3'-dithiodipropionic acid-curcumin conjugate, polyethylene glycol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 1:1:4~8:1~2, and the molecular weight of polyethylene glycol is 4000 or 1500.

[0017] The preparation reaction formula of the diselenide bond-linked polyethylene glycol-curcumin conjugate (Compound 2) is as follows:[[]]END]]

[0018]

[0019] The specific preparation method is carried out according to the following steps:

[0020] (1) Using curcumin (Compound 3) and 3,3'-diselenodipropionic acid (DSePA) as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) as a condensing agent, 4-dimethylaminopyridine (DMAP) as a catalyst, and dichloromethane as a solvent, heat to 50 °C and reflux for 24 hours. Evaporate the organic solvent using a rotary evaporator, cool to room temperature, then add 5 mL of distilled water, and dialyze the above dispersion in distilled water for three days using a dialysis membrane. Freeze-dry to obtain 3,3'-diselenodipropionic acid-curcumin conjugate (Compound 5).

[0021] Among them, the molar ratio of curcumin, 3,3'-diselenodipropionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 1:1~2:2~4:0.5~1.

[0022] (2) Preparation of diselenide bond-linked polyethylene glycol-curcumin conjugate (Compound 2)

[0023] Using 3,3'-diselenodipropionic acid-curcumin conjugate (Compound 5) and polyethylene glycol as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) as a condensing agent, 4-dimethylaminopyridine (DMAP) as a catalyst, heat to 50 °C in dichloromethane, condense and reflux, and react for 24 hours under a closed and light-shielded condition. Evaporate the organic solvent using a rotary evaporator, cool to room temperature, then add 5 mL of distilled water, and dialyze the above dispersion in distilled water for three days using a dialysis membrane. Add it to a dialysis bag and dialyze in ultrapure water for three days. Freeze-dry, and purify the freeze-dried product by column chromatography to obtain diselenide bond-linked polyethylene glycol-curcumin conjugate (Compound 2).

[0024] Among them, the molar ratio of 3,3'-diselenodipropionic acid-curcumin conjugate, polyethylene glycol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 1:1:4~8:1~2, and the molecular weight of polyethylene glycol is 4000 or 1500.

[0025] The advantages of the present invention are as follows:

[0026] The curcumin nanoparticles provided by the present invention improve the water solubility of curcumin, have the characteristics of reducing-responsive drug release, and can effectively induce apoptosis of tumor cells. Description of the Drawings

[0027] Figure 1 It is the nuclear magnetic resonance hydrogen spectrum of the 3,3'-dithiopropionic acid-curcumin conjugate prepared in Example 1 of the present invention and the disulfide bond-linked polyethylene glycol-curcumin conjugate prepared in Example 2.

[0028] Figure 2 1H NMR spectra of the 3,3'-diselenodipropionic acid-curcumin conjugate prepared in Example 3 of the present invention and the diselenide bond-linked polyethylene glycol-curcumin conjugate prepared in Example 4.

[0029] Figure 3 IR spectrum of the disulfide bond-linked polyethylene glycol (molecular weight of polyethylene glycol 4000)-curcumin conjugate prepared in Example 2 of the present invention.

[0030] Figure 4 IR spectrum of the disulfide bond-linked polyethylene glycol (molecular weight of polyethylene glycol 1500)-curcumin conjugate prepared in Example 2 of the present invention.

[0031] Figure 5 IR spectrum of the diselenide bond-linked polyethylene glycol (molecular weight of polyethylene glycol 4000)-curcumin conjugate prepared in Example 4 of the present invention.

[0032] Figure 6 IR spectrum of the diselenide bond-linked polyethylene glycol (molecular weight of polyethylene glycol 1500)-curcumin conjugate prepared in Example 4 of the present invention.

[0033] Figure 7 Thermal analysis images of the curcumin prodrug nanoparticles prepared in Examples 2 and 4 of the present invention.

[0034] Figure 8 Images of the curcumin prodrug nanoparticles prepared in Examples 2 and 4 of the present invention after standing for 30 days. Detailed Description of the Invention

[0035] The present invention will be further described below with reference to specific examples.

[0036] Example 1: Preparation of 3,3'-dithiobispropionic acid-curcumin conjugate

[0037] 20 mL of dichloromethane was added to a 50 mL round-bottom flask, and a magnetic stirrer was added. Curcumin (36.8 mg, 0.1 mmol) and 3,3'-dithiobispropionic acid (21 mg, 0.1 mmol) were added, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (57.6 mg, 0.3 mmol) and 4-dimethylaminopyridine (12.2 mg, 0.1 mmol) were dissolved by stirring at room temperature. The mixture was heated to 50 °C, refluxed under condensation, and reacted for 24 hours under a closed and light-shielded condition. After the reaction was completed, the organic solvent was removed by a rotary evaporator, cooled to room temperature, then 5 mL of distilled water was added, and the above dispersion was dialyzed in distilled water for three days using a dialysis membrane (cut-off molecular weight 3500 Da). The dialyzed liquid was lyophilized, and the lyophilized product was separated and purified by column chromatography to obtain 56 mg of a yellow powdery solid with a yield of 82%.

[0038] The retention time of the 3,3'-dithiobis(propionic acid)-curcumin conjugate was measured to be 15.011 min with a purity of 85% by high performance liquid chromatography (mobile phase: acetonitrile / water = 90:10, v / v; flow rate: 1 mL / min; detection wavelength: 360 nm). The molecular weight of the 3,3'-dithiobis(propionic acid)-curcumin conjugate was measured to be 559.1275 g / mol by liquid chromatography-mass spectrometry. The 3,3'-dithiobis(propionic acid)-curcumin conjugate was characterized by 1H nuclear magnetic resonance spectroscopy. As Figure 1 shown, compared with curcumin, characteristic peaks of the methylene groups in 3,3'-dithiobis(propionic acid) appeared in the 1H NMR spectrum of the 3,3'-dithiobis(propionic acid)-curcumin conjugate (16 - 17), indicating the successful synthesis of the 3,3'-dithiobis(propionic acid)-curcumin conjugate. The chemical shifts of the 3,3'-dithiobis(propionic acid)-curcumin conjugate were as follows: 1H NMR (400 MHz, DMSO-d 6 ) δ 7.78–7.45 (m, 3H), 7.45–7.26 (m, 2H), 7.16 (td, J = 7.8, 3.0 Hz, 2H), 6.97 (td, J = 13.3, 12.3, 7.1 Hz, 1H), 6.89–6.66 (m, 2H), 6.26–5.97 (m, 1H), 3.84 (s, 6H), 3.00 (dddt, J = 43.3, 21.5, 13.8, 7.4 Hz, 8H).

[0039] Example 2: Preparation of Disulfide-Linked Polyethylene Glycol-Curcumin Conjugates (PEG4000-SS-CUR, PEG1500-SS-CUR)

[0040] 20 mL of dichloromethane was added to a 50 mL round-bottom flask, and a magnetic stirrer was added. Polyethylene glycol with a molecular weight of 4000 or 1500 (400 / 150 mg, 0.1 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (153 mg, 0.8 mmol), 4-dimethylaminopyridine (20 mg, 0.16 mmol), and 3,3'-dithiobis(propionic acid)-curcumin conjugate (56 mg, 0.1 mmol) were added and stirred until dissolved at room temperature. The mixture was heated to 50 °C, refluxed under condensation, and reacted for 24 hours under a closed and light-shielded condition. After the reaction was completed, the organic solvent was removed by a rotary evaporator, cooled to room temperature, then 5 mL of distilled water was added, and the above dispersion was dialyzed against distilled water for three days using a dialysis membrane (cut-off molecular weight 3500 Da). The dialyzed liquid was freeze-dried, and the freeze-dried product was separated and purified by column chromatography to obtain a yellow powdery solid of 432 / 189 mg (purity 90 / 93%), with a yield of 95 / 92%.

[0041] The synthesized disulfide - linked polyethylene glycol - curcumin conjugate was characterized by infrared spectroscopy. As Figure 3 , 4 shown, the absorption peaks at 1629 and 1510 cm -1 are attributed to the skeletal vibrations of the aromatic rings in curcumin, and the absorption peak at 1735 cm -1 is attributed to the stretching vibration of the ester bond. The synthesized disulfide - linked polyethylene glycol - curcumin conjugate was characterized by proton nuclear magnetic resonance spectroscopy. As Figure 1 shown, compared with the 3,3'-dithiodipropionic acid - curcumin conjugate, characteristic peaks of the methylene groups in polyethylene glycol appeared in the proton nuclear magnetic resonance spectrum of the disulfide - linked polyethylene glycol - curcumin conjugate (18 - 19), indicating the successful synthesis of the disulfide - linked polyethylene glycol - curcumin conjugate.

[0042] Example 3: Preparation of 3,3'-diselenodipropionic acid - curcumin conjugate

[0043] 20 mL of dichloromethane was added to a 50 mL round - bottom flask, a magnetic stirrer was added and stirred. Curcumin (36.8 mg, 0.1 mmol) and 3,3'-diselenodipropionic acid (30.5 mg, 0.1 mmol), 1-(3 - dimethylaminopropyl)-3 - ethylcarbodiimide hydrochloride (57.6 mg, 0.3 mmol), 4 - dimethylaminopyridine (12.2 mg, 0.1 mmol) were added. The mixture was stirred and dissolved at room temperature, heated to 50 °C, refluxed under condensation, and reacted for 24 hours under closed and light - shielded conditions. After the reaction was completed, the organic solvent was removed by a rotary evaporator, cooled to room temperature, then 5 mL of distilled water was added, and the above - mentioned dispersion was dialyzed in distilled water for three days using a dialysis membrane (cut - off molecular weight 3500 Da). The dialyzed liquid was freeze - dried, and the lyophilized product was separated and purified by column chromatography to obtain 54 mg of a yellow powdery solid with a yield of 80%.

[0044] The retention time of the disulfide - containing curcumin conjugate measured by high - performance liquid chromatography (mobile phase acetonitrile / water = 90:10, v / v; flow rate 1 mL / min; detection wavelength 360 nm) was 2.614 min, and the purity was 88%. The molecular weight of the 3,3'-diselenodipropionic acid - curcumin conjugate measured by liquid chromatography - mass spectrometry (acetonitrile / water = 90:10, v / v; flow rate 1 mL / min; detection wavelength 360 nm) was 655.9917 g / mol. The 3,3'-diselenodipropionic acid - curcumin conjugate was characterized by proton nuclear magnetic resonance spectroscopy and high - performance liquid chromatography. As Figure 2As shown, compared with free curcumin, characteristic peaks of methylene in 3,3'-diselenodipropionic acid appeared in the 1H NMR spectrum of 3,3'-diselenodipropionic acid-curcumin conjugate (16 - 17), indicating the successful synthesis of 3,3'-diselenodipropionic acid-curcumin conjugate. The chemical shift of 3,3'-diselenodipropionic acid-curcumin conjugate is: 1HNMR(300MHz, DMSO-d 6 ) δ7.85–7.45(m, 2H), 7.45–7.22(m, 2H), 7.22–7.06(m, 2H), 7.06–6.92(m, 1H), 6.94–6.64(m, 2H), 6.64–6.29(m, 1H), 6.29–5.94(m, 1H), 3.84(s, 6H), 3.59–2.60(m, 8H).

[0045] Example 4: Preparation of Diselenide Bond-Linked Polyethylene Glycol-Curcumin Conjugates (PEG4000-SeSe-CUR, PEG1500-SeSe-CUR)

[0046] Add 20 mL of dichloromethane to a 50 mL round-bottom flask, add a magnetic stirrer, and add polyethylene glycol with a molecular weight of 4000 or 1500 (400 / 150 mg, 0.1 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (153 mg, 0.8 mmol), 4-dimethylaminopyridine (20 mg, 0.16 mmol) and 3,3'-diselenodipropionic acid-curcumin conjugate (65.6 mg, 0.1 mmol). Stir and dissolve at room temperature, then heat to 50 °C, condense and reflux, and react for 24 hours under closed and light-shielded conditions. After the reaction is completed, evaporate the organic solvent using a rotary evaporator, cool to room temperature, then add 5 mL of distilled water, and dialyze the above dispersion in distilled water for three days using a dialysis membrane (cut-off molecular weight 3500 Da). Freeze-dry the dialyzed liquid, and purify the lyophilized product by column chromatography to obtain a yellow powdery solid 432 / 187 mg (purity 93 / 89%), and the yield is 93 / 87%.

[0047] The synthesized diselenide bond-linked polyethylene glycol-curcumin conjugate was characterized by infrared spectroscopy. As Figure 5 、 6 shown, the absorption peaks at 1629 and 1510 cm -1 are attributed to the skeletal vibration of the aromatic ring in curcumin, and the absorption peak at 1735 cm -1 is attributed to the stretching vibration of the ester bond. The synthesized diselenide bond-linked polyethylene glycol-curcumin conjugate was characterized by 1H NMR spectroscopy. As Figure 2As shown, characteristic peaks of methylene in polyethylene glycol appeared in the 1H NMR spectrum of the diselenide bond-linked polyethylene glycol-curcumin conjugate (18 - 19), indicating the successful synthesis of the diselenide bond-linked polyethylene glycol-curcumin conjugate.

[0048] Example 5: Preparation and Particle Size and Zeta Potential Evaluation of Curcumin Prodrug Nanoparticles

[0049] Weigh 2 mg each of the disulfide bond-linked polyethylene glycol-curcumin conjugate prepared in Example 2 of the present invention and the diselenide bond-linked polyethylene glycol-curcumin conjugate prepared in Example 4, dissolve them in 2 mL of dimethyl sulfoxide to prepare a 1 mg / mL solution, then add 5 mL of distilled water, and dialyze the above dispersion in distilled water for three days using a dialysis membrane (cut-off molecular weight 3500 Da). Collect the solution in the dialysis membrane to obtain curcumin prodrug nanoparticles PEG4000-SS-CUR, PEG1500-SS-CUR, PEG4000-SeSe-CUR, and PEG1500-SeSe-CUR, as Figure 8 shown.

[0050] Take 1 mL each of curcumin prodrug nanoparticles PEG4000-SS-CUR, PEG1500-SS-CUR, PEG4000-SeSe-CUR, and PEG1500-SeSe-CUR, dilute them with 1 mL of ultrapure water respectively, and ultrasonically disperse for 5 min. Take 1 mL of the aqueous solutions of PEG4000-SS-CUR, PEG1500-SS-CUR, PEG4000-SeSe-CUR, and PEG1500-SeSe-CUR, and measure the particle size and zeta potential of curcumin prodrug nanoparticles PEG4000-SS-CUR, PEG1500-SS-CUR, PEG4000-SeSe-CUR, and PEG1500-SeSe-CUR using dynamic light scattering. As can be seen from Table 1, the average particle sizes of curcumin prodrug nanoparticles PEG4000-SS-CUR, PEG1500-SS-CUR, PEG4000-SeSe-CUR, and PEG1500-SeSe-CUR are 90.7, 122.3, 112.3, and 181.6 nm respectively. And with the increase of the drug loading, the particle size of the samples increases, and the absolute value of the zeta potential also increases. The polydispersity index of the samples is less than 0.3, indicating good dispersion of the samples.

[0051] Table 1. Particle Size and Zeta Potential of the Prepared Curcumin Prodrug Nanoparticles

[0052] Sample Name Diameter (nm) Dispersity Index Potential (mV) PEG4000-SS-CUR 90.7±1.3 0.251±0.022 6.61±0.56 PEG1500-SS-CUR 122.3±4.5 0.212±0.002 3.19±0.44 PEG4000-SeSeCUR 112.3±4.6 0.097±0.009 16.1±1.47 PEG1500-SeSe-CUR 181.6±4.9 0.282±0.008 16.3±2.45

[0053] Example 6: Drug Loading Evaluation of Curcumin Prodrug Nanoparticles

[0054] The drug loading of curcumin in the prepared nanoparticles was detected by the microplate reader method. For the aqueous solutions of curcumin prodrug nanoparticles PEG4000-SS-CUR, PEG1500-SS-CUR, PEG4000-SeSe-CUR, and PEG1500-SeSe-CUR, 100 μL of the supernatant was taken after centrifugation, dissolved in 1 mL of acetonitrile, filtered through a 0.22-μm filter membrane, and then the curcumin content was analyzed in a microplate reader, and the absorbance at 420 nm was measured. As can be seen from Table 2, the drug loadings of curcumin prodrug nanoparticles PEG4000-SS-CUR, PEG1500-SS-CUR, PEG4000-SeSe-CUR, and PEG1500-SeSe-CUR were 12.8%, 15.10%, 15.2%, and 16.3% respectively. As can be seen from Tables 1 and 2, with the increase of the drug loading, the particle size of the sample increased accordingly. The calculation formula for the drug loading of curcumin is:

[0055]

[0056] Table 2. Drug loadings of PEG4000-SS-CUR, PEG1500-SS-CUR, PEG4000-SeSeCUR, and PEG1500-SeSe-CURNPs

[0057] Sample Name Drug Loading (%) PEG4000-SS-CUR 12.8 PEG1500-SS-CUR 15.10 PEG4000-SeSeCUR 15.20 PEG1500-SeSe-CUR 16.30

[0058] Example 7: Evaluation of the stability of curcumin prodrug nanoparticles

[0059] The particle size distribution and zeta potential of the nanoparticles were characterized by dynamic light scattering (DLS), and the evaluation was carried out using the mean ± standard deviation (SD). The DLS method was also used to determine the stability of the aqueous dispersions of curcumin prodrug nanoparticles PEG4000-SS-CUR, PEG1500-SS-CUR, PEG4000-SeSe-CUR, and PEG1500-SeSe-CUR in PBS buffer at pH 7.4 for 5 days. As Figure 8 shown, the polydispersity indices of the samples were all less than 0.3, indicating good stability and dispersibility of the samples.

[0060] Table 3. Changes in the polydispersity indices of PEG4000-SS-CUR, PEG1500-SS-CUR, PEG4000-SeSe-CUR, and PEG1500-SeSe-CURNPs in PBS buffer (pH 7.4) for 5 days

[0061]

[0062] Example 8: Evaluation of the ultraviolet absorption and thermal analysis of curcumin and curcumin prodrug nanoparticles

[0063] Weigh 1 mg of curcumin, curcumin prodrug nanoparticles PEG4000-SS-CUR, PEG1500-SS-CUR, PEG4000-SeSe-CUR, and PEG1500-SeSe-CUR separately and dissolve them in dimethyl sulfoxide. Use a UV-visible spectrophotometer to measure the absorbance changes of curcumin, curcumin prodrug nanoparticles PEG4000-SS-CUR, PEG1500-SS-CUR, PEG4000-SeSe-CUR, and PEG1500-SeSe-CUR in the range of 300 - 900 nm. As shown in Table 4, the maximum absorption wavelength of free curcumin is at 435 nm, and the maximum absorption wavelengths of curcumin prodrug nanoparticles PEG4000-SS-CUR, PEG1500-SS-CUR, PEG4000-SeSe-CUR, and PEG1500-SeSe-CUR are 406, 410, 407, and 414 nm respectively. This indicates that there are varying degrees of blue shifts in the UV-visible absorption of curcumin prodrug nanoparticles PEG4000-SS-CUR, PEG1500-SS-CUR, PEG4000-SeSe-CUR, and PEG1500-SeSe-CUR.

[0064] Weigh 5 mg of curcumin, curcumin prodrug nanoparticles PEG4000-SS-CUR, PEG1500-SS-CUR, PEG4000-SeSe-CUR, and PEG1500-SeSe-CUR separately and place them in a crucible. Use a differential scanning calorimeter to perform thermal analysis on the samples. As Figure 7 shown, for curcumin prodrug nanoparticles PEG4000-SS-CUR, PEG1500-SS-CUR, PEG4000-SeSe-CUR, and PEG1500-SeSe-CUR, only the melting endothermic peaks of the corresponding polyethylene glycols appear, and there is no melting endothermic peak of curcumin crystals, indicating that curcumin in curcumin prodrug nanoparticles PEG4000-SS-CUR, PEG1500-SS-CUR, PEG4000-SeSe-CUR, and PEG1500-SeSe-CUR is in an amorphous state. This result further demonstrates the successful preparation of curcumin prodrug nanoparticles PEG4000-SS-CUR, PEG1500-SS-CUR, PEG4000-SeSe-CUR, and PEG1500-SeSe-CUR.

[0065] Table 4. Absorbance changes of test solutions of each compound at 300 - 900 nm

[0066]

[0067]

[0068] Example 9: In vitro Release Evaluation of Curcumin Prodrug Nanoparticles

[0069] Disperse PEG4000-SS-CUR, PEG1500-SS-CUR, PEG4000-SeSe-CUR, and PEG1500-SeSe-CUR in pH 7.4 phosphate buffer (10 mM) (Release Solution I) or pH 7.4 phosphate buffer (10 mM) containing 10 mM reduced glutathione (Release Solution II), and then place them on a shaker for shaking at a rotation speed of 160 rpm and a temperature of 37 °C. Take out 0.5 mL of the release solution at 1, 2, 4, 8, 12, 24, and 48 time points respectively. The CUR release experiments in each release solution were repeated in parallel three times. The in vitro release experiment was carried out under light-proof conditions throughout. Mix the taken-out release solution with 0.5 mL of acetonitrile, and determine the concentration of the released curcumin derivative by HPLC and calculate the cumulative release amount. As can be seen from Table 5, PEG4000-SS-CUR, PEG1500-SS-CUR, PEG4000-SeSe-CUR, and PEG1500-SeSe-CUR all have the characteristics of reduction-responsive drug release. Among them, the reduction-responsive drug release ability of PEG4000-SS-CUR is slightly lower than that of the other three prodrugs, and PEG4000-SeSe-CUR has the best reduction-responsive drug release characteristics.

[0070] Table 5. Cumulative Drug Release Amounts of PEG4000-SS-CUR, PEG1500-SS-CUR, PEG4000-SeSe-CUR, and PEG1500-SeSe-CUR Nanoparticles

[0071]

[0072] Example 10: Apoptosis Evaluation of Curcumin Prodrug Nanoparticles in Breast Cancer Cells

[0073] Culture the cells with a medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution. Among them, RPMI 1640 medium is used for 4T1 cells. Set the temperature of the incubator to 37 °C and the volume ratio of carbon dioxide to 5.0%. Seed 4T1 cells at a density of 1×10 per well 5The cells were seeded into a 12-well plate at a certain cell number and transferred to a carbon dioxide incubator for continued culture for 24 h. Then, the cells were incubated with free curcumin, curcumin prodrug nanoparticles PEG4000-SS-CUR, PEG1500-SS-CUR, PEG4000-SeSe-CUR, and PEG1500-SeSe-CUR diluted with the corresponding complete medium for 24 h. Then, the medium in each well plate was aspirated with a pipette, 0.5 mL of EDTA-free trypsin was added to each well for digestion, and after 3 min, the medium was added to terminate the digestion. The supernatant was discarded by centrifugation. Subsequently, 1 mL of PBS buffer was added for washing once, the supernatant was discarded, 100 μL of 1×Binding Buffer was added to disperse the cells, and the cells were filtered through a 400-mesh sieve. For the Control group (unstained), FITC / PI double-staining group, FITC / PI single-staining group, and drug group, 5 μL of Annexin V-FITC and 5 μL of PI were added, gently mixed, and incubated in the dark for 10 min. Then, 400 μL of 1×Binding Buffer was added to disperse and mix well, and the cells were tested on a flow cytometer. The apoptosis rates of breast cancer cells induced by curcumin, curcumin prodrug nanoparticles PEG4000-SS-CUR, PEG1500-SS-CUR, PEG4000-SeSe-CUR, and PEG1500-SeSe-CUR were 32.4%, 15.6%, 34.7%, 57.6%, and 36.2% respectively. This indicates that PEG4000-SS-CUR has little effect on cell apoptosis, but compared with curcumin, PEG1500-SS-CUR, PEG1500-SeSe-CUR, and PEG4000-SeSe-CUR enhanced late apoptosis of cells by 2.3%, 25.2%, and 3.8% respectively. This indicates that PEG1500-SS-CUR, PEG1500-SeSe-CUR, and PEG4000-SeSe-CUR have a significant promoting effect on late apoptosis of breast cancer cells.

[0074] Table 6. Results of apoptosis of breast cancer cells induced by free CUR, PEG4000-SS-CUR, PEG1500-SS-CUR, PEG4000-SeSe-CUR, and PEG1500-SeSe-CUR nanoparticles

[0075]

[0076]

[0077] As mentioned above, only the preferred specific embodiments of the present invention are described, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, should be covered by the protection scope of the present invention.

Claims

1. A class of reduction-responsive curcumin water-soluble nanoparticles, characterized in that, the nanoparticles are self-assembled from a disulfide bond-linked polyethylene glycol-curcumin conjugate Compound 1 or a diselenide bond-linked polyethylene glycol-curcumin conjugate Compound 2 in an aqueous solution; ， Compound 1 ， Compound 2 wherein, in the disulfide bond-linked polyethylene glycol-curcumin conjugate, the polyethylene glycol is PEG1500, and in the diselenide bond-linked polyethylene glycol-curcumin conjugate, the polyethylene glycol is PEG1500 or PEG4000.

2. The reduction-responsive curcumin water-soluble nanoparticles according to claim 1, characterized in that, the preparation steps of Compound 1 are as follows: (1) Add curcumin and 3,3'-dithiobis(propionic acid) to a solvent, use 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride as a condensing agent and 4-dimethylaminopyridine as a catalyst, heat to 50 °C in an organic solvent and react for 24 hours. Evaporate the organic solvent by a rotary evaporator, cool to room temperature, then add 5 mL of distilled water to obtain a dispersion. Dialyze the above dispersion in distilled water for three days using a dialysis membrane. Finally, freeze-dry the dialyzed liquid at -80 °C for 24 hours. The freeze-dried product is separated and purified by column chromatography to obtain 3,3'-dithiobis(propionic acid)-curcumin conjugate; (2) Add 3,3'-dithiobis(propionic acid)-curcumin conjugate and polyethylene glycol to a solvent, use 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride as a condensing agent and 4-dimethylaminopyridine as a catalyst, heat to 50 °C in an organic solvent and react for 24 hours. Evaporate the organic solvent by a rotary evaporator, cool to room temperature, then add 5 mL of distilled water, dialyze the above dispersion in distilled water for three days using a dialysis membrane. Finally, freeze-dry the dialyzed liquid at -80 °C for 24 hours. The freeze-dried product is separated and purified by column chromatography to obtain the disulfide bond-linked polyethylene glycol-curcumin conjugate Compound 1.

3. The reduction-responsive curcumin water-soluble nanoparticles according to claim 1, characterized in that, the preparation steps of Compound 2 are as follows: (1) Add curcumin and 3,3'-diselenobis(propionic acid) to a solvent, use 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride as a condensing agent and 4-dimethylaminopyridine as a catalyst, heat to 50 °C in an organic solvent and react for 24 hours. Evaporate the organic solvent by a rotary evaporator, cool to room temperature, then add 5 mL of distilled water to obtain a dispersion. Dialyze the above dispersion in distilled water for three days using a dialysis membrane. Finally, freeze-dry the dialyzed liquid at -80 °C for 24 hours. The freeze-dried product is separated and purified by column chromatography to obtain 3,3'-diselenobis(propionic acid)-curcumin conjugate; (2) Add 3,3'-diselenodipropionic acid-curcumin conjugate and polyethylene glycol into a solvent, use 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride as a condensing agent, 4-dimethylaminopyridine as a catalyst, heat to 50 °C in an organic solvent and react for 24 hours. After evaporating the organic solvent by a rotary evaporator and cooling to room temperature, then add 5 mL of distilled water, dialyze the above dispersion in distilled water for three days, and finally freeze-dry the dialyzed liquid at -80 °C for 24 hours. The freeze-dried product is separated and purified by column chromatography to obtain the diselenide bond-linked polyethylene glycol-curcumin conjugate compound 2.

4. The reducible-responsive curcumin water-soluble nanoparticles according to claim 2, characterized in that in the step (1), the molar ratio of curcumin, 3,3'-dithiobipropionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 1:1-2:2-4:0.5-1, and dichloromethane is used as a solvent.

5. The reducible-responsive curcumin water-soluble nanoparticles according to claim 2, characterized in that in the step (2), the molar ratio of the disulfide bond-linked polyethylene glycol-curcumin conjugate, polyethylene glycol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 1:1:4-8:1-2, dichloromethane is used as a solvent, and the molecular weight of polyethylene glycol is 1500.

6. The reducible-responsive curcumin water-soluble nanoparticles according to claim 3, characterized in that in the step (1), the molar ratio of curcumin, 3,3'-diselenodipropionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 1:1-2:2-4:0.5-1, and dichloromethane is used as a solvent.

7. The reducible-responsive curcumin water-soluble nanoparticles according to claim 3, characterized in that in the step (2), the molar ratio of the 3,3'-diselenodipropionic acid-curcumin conjugate, polyethylene glycol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 1:1:4-8:1-2, dichloromethane is used as a solvent, and the molecular weight of polyethylene glycol is 4000 or 1500.

8. The reducible-responsive curcumin water-soluble nanoparticles according to claim 1, characterized in that the nanoparticles have colloidal stability; have reducible-responsive drug release characteristics; can induce apoptosis of tumor cells.

9. The application of the reducible-responsive curcumin water-soluble nanoparticles according to claim 1, characterized in that the nanoparticles are used for preparing a drug for treating breast cancer.

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