A novel indocyanine green delivery system targeting mitochondria of tumor cells and a preparation method thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a new indocyanine green delivery system targeting mitochondria of tumor cells and a preparation method thereof. The new indocyanine green and diamine are used to prepare aminylated new indocyanine green through a substitution reaction, and then the aminylated new indocyanine green is condensed with 3-propylcarboxyl triphenyl phosphonium bromide to prepare a triphenyl phosphonium salt-new indocyanine green conjugate; hydroxyethyl starch and perfluorooctadecanoic acid are used to prepare perfluorooctadecanoic acid hydroxyethyl starch ester through a condensation reaction, and then the perfluorooctadecanoic acid hydroxyethyl starch ester and the triphenyl phosphonium salt-new indocyanine green conjugate are dissolved in dimethyl sulfoxide to prepare the new indocyanine green delivery system targeting mitochondria of tumor cells. The delivery system has the effect of slow release of the triphenyl phosphonium salt-new indocyanine green conjugate, and has a stronger in-vitro photodynamic effect and a stronger photodynamic killing effect on tumor cells than the triphenyl phosphonium salt-new indocyanine green conjugate.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a novel indocyanine green delivery system targeting tumor cell mitochondria and its preparation method. Background Technology

[0002] The mechanism of action of photodynamic therapy (PDT) for tumors involves the reaction of photosensitizers with oxygen under light excitation to generate reactive oxygen species, thereby inducing apoptosis or necrosis. Indocyanine green (ICB) is a near-infrared photosensitizer, but it is easily decomposed in aqueous solution and has poor stability. Neo-indocyanine green (NINCL) is an ICB analogue, and compared to ICB, its stability in aqueous solution is significantly improved, showing potential application value in PDT for tumors. However, the use of NINCL for PDT for tumors still faces limitations due to insufficient tumor targeting and tumor hypoxia.

[0003] Current developments in novel indocyanine green delivery systems employ two approaches: firstly, conjugating neoindocyanine green with hydrophilic polymers or tumor-targeting molecules to enhance its tumor targeting and phototherapy efficacy; and secondly, loading neoindocyanine green onto multifunctional nanoplatforms to improve its photochemical properties and tumor targeting while simultaneously mitigating the hypoxic tumor microenvironment and synergistically enhancing phototherapy effects. These strategies have improved the photodynamic therapy efficacy of neoindocyanine green to some extent, but its therapeutic effect still requires further improvement. The delivery target of the photosensitizer is a crucial factor influencing the efficacy of photodynamic therapy. Mitochondria, as cellular energy factories, are rich in oxygen and are ideal targets for photodynamic therapy. Therefore, the key technical challenge of targeted delivery of neoindocyanine green to tumor cell mitochondria urgently needs to be addressed. Summary of the Invention

[0004] One of the objectives of this invention is to provide a novel indocyanine green delivery system that targets the mitochondria of tumor cells. This system has a strong in vitro photodynamic effect. After being taken up by tumor cells, it can be targeted to the mitochondria and generate a large amount of reactive oxygen species under laser irradiation, thus exhibiting a strong photodynamic killing effect on tumor cells.

[0005] Another object of the present invention is to provide a method for preparing a novel indocyanine green delivery system targeting the mitochondria of tumor cells, which is carried out according to the following steps:

[0006] (1) Preparation of aminated neoindocyanine green (compound 2)

[0007] Using neoindocyanine green (compound 1) and 4,7,10-trioxo-1,13-tetanediamine as raw materials, and methanol as solvent, the reaction was heated to 50°C and stirred for 4 hours. Methanol was then removed by rotary evaporation, and the mixture was dispersed in ultrapure water. After dialyzing in ultrapure water for three days, the resulting product was freeze-dried to obtain amino-modified neoindocyanine green. The reaction formula is as follows:

[0008]

[0009] The molar ratio of neoindocyanine green to 4,7,10-trioxo-1,13-tridecanediamine is 1:5 to 10.

[0010] (2) Preparation of triphenylphosphine salt-neoindocyanine green conjugate (compound 3)

[0011] Using aminated neoindocyanine green and 3-propanoic acid-triphenylphosphine bromide as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) as a condensing agent, 4-dimethylaminopyridine (DMAP) as a catalyst, and N,N-dimethylformamide as a solvent, the reaction was carried out at 50°C with stirring for 24 hours, then cooled to room temperature, dialyzed in ultrapure water for three days, and after freeze-drying, purified by column chromatography to obtain the triphenylphosphine salt-neoindocyanine green conjugate. The reaction formula is as follows:

[0012]

[0013] The molar ratio of amino-modified neoindocyanine green, 3-propanoic triphenylphosphine bromide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 1:2~4:8~16:1~2.

[0014] (3) Preparation of perfluorooctadecaned hydroxyethyl starch ester (compound 5)

[0015] Using hydroxyethyl starch (compound 4) and perfluorooctadecanoate as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) as a condensing agent, 4-dimethylaminopyridine (DMAP) as a catalyst, and dimethyl sulfoxide as a solvent, the reaction was heated to 40°C and stirred for 48 hours. After cooling to room temperature, the mixture was dialyzed in ultrapure water for three days, centrifuged at 8000 rpm for 10 minutes, and the supernatant was freeze-dried to obtain perfluorooctadecanoate hydroxyethyl starch ester. The reaction formula is as follows:

[0016]

[0017] The mass ratio of hydroxyethyl starch to perfluorooctadecanic acid is 5-10:1, and the molar ratio of perfluorooctadecanic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 1:10-20:2-4.

[0018] (4) Preparation of perfluorooctanoic acid hydroxyethyl starch nanoparticles loaded with triphenylphosphine salt-neoindocyanine green coupling compound

[0019] Triphenylphosphine salt-neoindocyanine green coupling compound and perfluorooctanoic acid hydroxyethyl starch ester were added to dimethyl sulfoxide and stirred overnight. The mixture was dialyzed in ultrapure water for three days, centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected and freeze-dried to obtain perfluorooctanoic acid hydroxyethyl starch ester nanoparticles loaded with triphenylphosphine salt-neoindocyanine green coupling compound.

[0020] The mass ratio of triphenylphosphine salt-neoindocyanine green coupling compound to perfluorooctanoic acid hydroxyethyl starch ester is 1:9 to 19.

[0021] The novel indocyanine green delivery system for targeting tumor cell mitochondria prepared by the above method has a hydrated diameter of 100–200 nm and exhibits in vitro photodynamic effects.

[0022] This novel indocyanine green delivery system, which targets the mitochondria of tumor cells, is used to prepare agents that enhance photodynamic therapy for tumors.

[0023] The advantages of this invention are:

[0024] This invention, on the one hand, conjugates the mitochondrial targeting ligand triphenylphosphine salt with neoindocyanine green to enhance the mitochondrial targeting of neoindocyanine green; on the other hand, it constructs a fluorine-containing nanocarrier to deliver the conjugate of triphenylphosphine salt and neoindocyanine green, further enhancing the photodynamic effect of neoindocyanine green through the affinity of oxygen for fluorine.

[0025] The perfluorooctanoic acid hydroxyethyl starch nanoparticles provided by this invention can provide more oxygen for the photosensitization reaction of triphenylphosphine salt-neoindocyanine green conjugate, resulting in a stronger in vitro photodynamic effect. These nanoparticles can slowly release triphenylphosphine salt-neoindocyanine green conjugate. After being taken up by tumor cells, the released conjugate can be targeted to the mitochondria of tumor cells through triphenylphosphine salt. Under laser irradiation, it can induce a significant increase in the reactive oxygen species level of tumor cells, exhibiting a stronger photodynamic killing effect on tumor cells than triphenylphosphine salt-neoindocyanine green conjugate. Attached image description:

[0026] Figure 1 The photon NMR spectrum of the triphenylphosphine salt-new indocyanine green conjugate prepared in Example 3 of this invention is shown.

[0027] Figure 2 This is a high-resolution mass spectrum of the triphenylphosphine salt-new indocyanine green conjugate prepared in Example 3 of the present invention.

[0028] Figure 3 The image shows the 1H NMR spectrum of the perfluorooctanoic acid hydroxyethyl starch ester prepared in Example 5 of this invention.

[0029] Figure 4 The image shows the nuclear magnetic resonance fluorine spectrum of the perfluorooctadecanoic acid hydroxyethyl starch ester prepared in Example 5 of this invention.

[0030] Figure 5 The infrared spectrum of perfluorooctanoic acid hydroxyethyl starch ester prepared in Example 5 of this invention. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments.

[0032] Example 1: Preparation of Aminated Neoindocyanine Green

[0033] Neoindocyanine green (85 mg, 0.1 mmol) and 4,7,10-trioxo-1,13-tetanediamine (220 mg, 1.0 mmol) were added to 10 mL of anhydrous methanol, heated to 50 °C and refluxed with stirring for 4 hours. The methanol was then removed by rotary evaporation, and the mixture was dispersed in 5 mL of ultrapure water. The solution was then added to a dialysis bag (molecular weight cutoff 800 Da) and dialyzed in ultrapure water for three days. The resulting product was freeze-dried to obtain amino-indocyanine green with a yield of 85%.

[0034] Example 2: Preparation of Aminated Neoindocyanine Green

[0035] Neoindocyanine green (85 mg, 0.1 mmol) and 4,7,10-trioxo-1,13-tetanediamine (110 mg, 0.5 mmol) were added to 10 mL of anhydrous methanol, heated to 50 °C and stirred under reflux for 4 hours. The methanol was then removed by rotary evaporation, and the mixture was dispersed in 5 mL of ultrapure water. The solution was then added to a dialysis bag (molecular weight cutoff 800 Da) and dialyzed in ultrapure water for three days. The resulting product was freeze-dried to obtain amino-indocyanine green with a yield of 81%.

[0036] Example 3: Preparation of triphenylphosphine salt-new indocyanine green coupling compound

[0037] The amino-modified neoindocyanine green (52 mg, 0.05 mmol), 3-propanoic acid-triphenylphosphine bromide (43 mg, 0.1 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (192 mg, 1 mmol), and 4-dimethylaminopyridine (12 mg, 0.1 mmol) prepared in Example 1 were added to 5 mL of N,N-dimethylformamide and stirred to dissolve. The mixture was heated to 50 °C and stirred for 24 hours. Then, it was cooled to room temperature and added to a dialysis bag (molecular weight cutoff 1000 Da). The mixture was dialyzed in ultrapure water for three days. After freeze-drying, the triphenylphosphine salt-neoindocyanine green conjugate was obtained by column chromatography with a yield of 67%.

[0038] The synthesized triphenylphosphine salt-novel indocyanine green conjugate was characterized by proton nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. Figure 1As shown, in addition to the characteristic signal of neoindocyanine green, the 1H NMR spectrum of the triphenylphosphine salt-neoindocyanine green conjugate also shows the characteristic signals of triphenylphosphine salt (a, c-e) and the linker arm 4,7,10-trioxo-1,13-tetane (b); Figure 2 As shown, [MH] appeared in the high-resolution mass spectrum of the triphenylphosphine salt-new indocyanine green conjugate. - The characteristic peaks indicate the successful preparation of the triphenylphosphine salt-new indocyanine green conjugate.

[0039] Example 4: Preparation of triphenylphosphine salt-neoindocyanine green coupling compound

[0040] The amino-modified neoindocyanine green (52 mg, 0.05 mmol), 3-propanoic acid-triphenylphosphine bromide (86 mg, 0.2 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (77 mg, 0.4 mmol), and 4-dimethylaminopyridine (6 mg, 0.05 mmol) prepared in Example 1 were added to 5 mL of N,N-dimethylformamide and stirred to dissolve. The mixture was heated to 50 °C and stirred for 24 hours. Then, it was cooled to room temperature and added to a dialysis bag (molecular weight cutoff 1000 Da). The mixture was dialyzed in ultrapure water for three days. After freeze-drying, the triphenylphosphine salt-neoindocyanine green conjugate was obtained by column chromatography with a yield of 62%.

[0041] Example 5: Preparation of perfluorooctadecanohydroxyethyl starch ester

[0042] Hydroxyethyl starch 130 / 0.4 (500 mg), perfluorooctadecanoic acid (100 mg, 0.11 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (422 mg, 2.2 mmol), and 4-dimethylaminopyridine (27 mg, 0.22 mmol) were added to 20 mL of dimethyl sulfoxide and stirred to dissolve. The mixture was heated to 40 °C and stirred for 48 hours. Then, it was cooled to room temperature and added to a dialysis bag (molecular weight cutoff 8000–14000 Da). The mixture was dialyzed in ultrapure water for three days, centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected and freeze-dried to obtain perfluorooctadecanoic acid hydroxyethyl starch ester with a yield of 89%.

[0043] The synthesized perfluorooctanoic acid hydroxyethyl starch ester was characterized by proton nuclear magnetic resonance (NMR) spectroscopy, fluorine spectroscopy, and infrared spectroscopy. Figure 3 As shown, since no new hydrogen is introduced after perfluorooctanoic acid and hydroxyethyl starch are coupled via esterification, its 1H NMR spectrum shows no significant change compared to that of hydroxyethyl starch itself; Figure 4 As shown, the fluorine spectrum of perfluorooctadecanoate hydroxyethyl starch ester showed characteristic fluorine signals of perfluorooctadecanoate (-80 and -121 ppm), indicating successful coupling between perfluorooctadecanoate and hydroxyethyl starch; Figure 5 As shown, the infrared spectrum of perfluorooctadecanohydroxyethyl starch ester exhibits C=O bond stretching vibrations (1690 cm⁻¹) in the ester bond. -1 ) and CF bond stretching vibration (1205cm) -1 The characteristic peaks indicate that perfluorooctadecanic acid and hydroxyethyl starch are successfully bonded via ester bonds. Elemental analysis yielded a mass percentage of 14.8% for perfluorooctadecanic acid.

[0044] Example 6: Preparation of perfluorooctadecanohydroxyethyl starch ester

[0045] Hydroxyethyl starch 130 / 0.4 (1 g), perfluorooctadecanoic acid (100 mg, 0.11 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (211 mg, 1.1 mmol), and 4-dimethylaminopyridine (54 mg, 0.44 mmol) were added to 20 mL of dimethyl sulfoxide and stirred to dissolve. The mixture was heated to 40 °C and stirred for 48 hours. Then, it was cooled to room temperature and added to a dialysis bag (molecular weight cutoff 8000–14000 Da). The mixture was dialyzed in ultrapure water for three days, centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected and freeze-dried to obtain perfluorooctadecanoic acid hydroxyethyl starch ester with a yield of 85%.

[0046] Elemental analysis revealed that the mass percentage of perfluorooctadecanoic acid was 6.2%.

[0047] Example 7: Preparation of perfluorooctanoic acid hydroxyethyl starch nanoparticles loaded with triphenylphosphine salt-neoindocyanine green coupling compound

[0048] The triphenylphosphine salt-neoindocyanine green conjugate (10 mg) prepared in Example 3 and the perfluorooctanoic acid hydroxyethyl starch ester (90 mg) prepared in Example 5 were added to 5 mL of dimethyl sulfoxide and stirred overnight. The mixture was dialyzed in ultrapure water for three days, centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected and freeze-dried to obtain perfluorooctanoic acid hydroxyethyl starch ester nanoparticles loaded with the triphenylphosphine salt-neoindocyanine green conjugate.

[0049] The drug loading of the triphenylphosphine salt-neoindocyanine green conjugate was determined to be 8.6% and the encapsulation efficiency was 77.4% by UV-Vis spectrophotometry.

[0050] Example 8: Preparation of perfluorooctanoic acid hydroxyethyl starch nanoparticles loaded with triphenylphosphine salt-neoindocyanine green coupling compound

[0051] The triphenylphosphine salt-neoindocyanine green conjugate (10 mg) prepared in Example 3 and the perfluorooctanoic acid hydroxyethyl starch ester (140 mg) prepared in Example 5 were added to 5 mL of dimethyl sulfoxide and stirred overnight. The mixture was dialyzed in ultrapure water for three days, centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected and freeze-dried to obtain perfluorooctanoic acid hydroxyethyl starch ester nanoparticles loaded with the triphenylphosphine salt-neoindocyanine green conjugate.

[0052] The drug loading of the triphenylphosphine salt-neoindocyanine green conjugate was determined to be 6.1% and the encapsulation efficiency was 83.3% by UV-Vis spectrophotometry.

[0053] Example 9: Preparation of perfluorooctanoic acid hydroxyethyl starch nanoparticles loaded with triphenylphosphine salt-neoindocyanine green coupling compound

[0054] The triphenylphosphine salt-neoindocyanine green conjugate (10 mg) prepared in Example 3 and the perfluorooctanoic acid hydroxyethyl starch ester (190 mg) prepared in Example 5 were added to 5 mL of dimethyl sulfoxide and stirred overnight. The mixture was dialyzed in ultrapure water for three days, centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected and freeze-dried to obtain perfluorooctanoic acid hydroxyethyl starch ester nanoparticles loaded with the triphenylphosphine salt-neoindocyanine green conjugate.

[0055] The drug loading of the triphenylphosphine salt-neoindocyanine green conjugate was determined to be 4.7% and the encapsulation efficiency was 86.4% by UV-Vis spectrophotometry.

[0056] Comparing Examples 9, 8, and 7, it can be seen that the drug loading of the triphenylphosphine salt-neoindocyanine green conjugate increases with the increase of the ratio of triphenylphosphine salt-neoindocyanine green conjugate to perfluorooctadecanohydroxyethyl starch ester, while the encapsulation efficiency decreases with the increase of the ratio of triphenylphosphine salt-neoindocyanine green conjugate to perfluorooctadecanohydroxyethyl starch ester.

[0057] Example 10: Preparation of perfluorooctanoic acid hydroxyethyl starch nanoparticles loaded with triphenylphosphine salt-neoindocyanine green coupling compound

[0058] The triphenylphosphine salt-neoindocyanine green conjugate (10 mg) prepared in Example 3 and the perfluorooctanoic acid hydroxyethyl starch ester (90 mg) prepared in Example 6 were added to 5 mL of dimethyl sulfoxide and stirred overnight. The mixture was dialyzed in ultrapure water for three days, centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected and freeze-dried to obtain perfluorooctanoic acid hydroxyethyl starch ester nanoparticles loaded with the triphenylphosphine salt-neoindocyanine green conjugate.

[0059] The drug loading of the triphenylphosphine salt-neoindocyanine green conjugate was determined to be 6.4% and the encapsulation efficiency was 58.4% by UV-Vis spectrophotometry. Comparing Examples 7 and 10, it is evident that the perfluorooctanoic acid hydroxyethyl starch ester prepared in Example 5 exhibits better drug loading performance than that prepared in Example 6.

[0060] Comparative Example 1: Preparation of hydroxyethyl starch octadecanoate nanoparticles loaded with triphenylphosphine salt-neoindocyanine green coupling compound

[0061] Following the method of Example 5, perfluorooctadecanoic acid was replaced with n-octadecanoic acid to prepare hydroxyethyl n-octadecanoate starch ester. The triphenylphosphine salt-neoindocyanine green coupling compound (10 mg) and hydroxyethyl n-octadecanoate starch ester (90 mg) prepared in Example 3 were added to 5 mL of dimethyl sulfoxide and stirred overnight. The mixture was dialyzed in ultrapure water for three days, centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected and freeze-dried to obtain hydroxyethyl n-octadecanoate starch ester nanoparticles loaded with the triphenylphosphine salt-neoindocyanine green coupling compound.

[0062] The drug loading of the triphenylphosphine salt-neoindocyanine green conjugate was determined to be 8.9% and the encapsulation efficiency was 80.2% by UV-Vis spectrophotometry.

[0063] Example 11: Measurement of Nanoparticle Size and Potential

[0064] Preparation of test solution: The nanoparticles prepared in Example 7 and Comparative Example 1 were ultrasonically dispersed in ultrapure water to prepare a 5 mg / mL nanoparticle test solution.

[0065] Particle size and potential determination: Take 1 mL of the above-prepared nanoparticle test solution and determine the particle size and potential using a laser particle size and zeta potential analyzer.

[0066] As shown in Table 1, the hydrated diameters of the nanoparticles prepared in Example 7 and Comparative Example 1 are approximately 170 nm and 160 nm, respectively, with similar particle sizes. The polydispersity index is less than 0.3, indicating that the two types of nanoparticles have good particle size distribution. The nanoparticles prepared in Example 7 have a more negative potential, mainly due to the electronegativity of fluorine atoms.

[0067] Table 1. Particle size and potential of nanoparticles prepared in Example 7 and Comparative Example 1

[0068] sample Particle size (nm) polydispersity index zeta potential (mV) Example 7 174.3±6.3 0.214±0.023 -14.6±1.2 Comparative Example 1 162.5±7.6 0.237±0.048 -5.7±0.8

[0069] Example 12 In vitro photodynamic effect

[0070] Preparation of test solutions: Prepare solutions of 1 mM free neoindocyanine green, 1 mM of the triphenylphosphine salt-neoindocyanine green conjugate prepared in Example 3, nanoparticle solutions prepared in Example 7 containing 1 mM of the triphenylphosphine salt-neoindocyanine green conjugate, and nanoparticle solutions prepared in Comparative Example 1 containing 1 mM of the triphenylphosphine salt-neoindocyanine green conjugate. Prepare a 200 μg / mL dimethyl sulfoxide solution of 1,3-diphenylisobenzofuran. Then, using the above-prepared solutions of 1 mM neoindocyanine green and the 200 μg / mL dimethyl sulfoxide solution of 1,3-diphenylisobenzofuran as stock solutions and water as diluent, prepare test solutions containing both 10 μM neoindocyanine green and 20 μg / mL 1,3-diphenylisobenzofuran.

[0071] In vitro photodynamic effect detection: Take 3 mL of each of the different test solutions prepared above, and use 0.5 W / cm² water. 2 The samples were irradiated with a 660nm laser for 0, 1, 2, 3, and 4 minutes. The absorbance of each sample at 405nm was then measured using a UV-Vis spectrophotometer. The absorbance of the sample irradiated for 0 minutes was taken as 100%, and the decrease in absorbance of each sample under laser irradiation was calculated. Three parallel samples were set up for each test group.

[0072] The amount of singlet oxygen produced by each test solution under laser irradiation was studied using the singlet oxygen probe 1,3-diphenylisobenzofuran. After the reaction between 1,3-diphenylisobenzofuran and singlet oxygen, the structure of 1,3-diphenylisobenzofuran was destroyed, and the characteristic absorption at 405 nm decreased. Therefore, the greater the decrease in absorbance at 405 nm of the test sample, the greater the amount of singlet oxygen produced. As shown in Table 2, under 660nm laser irradiation, the triphenylphosphine salt-neoindocyanine green conjugate prepared in Example 3 generated singlet oxygen at a faster rate than free neoindocyanine green in the initial stage. As the irradiation time increased, the amount of singlet oxygen generated gradually became comparable to that of free neoindocyanine green. In comparison, the amount of singlet oxygen generated by the n-octadecanoic acid hydroxyethyl starch nanoparticles loaded with the triphenylphosphine salt-neoindocyanine green conjugate prepared in Example 1 was comparable to that of the triphenylphosphine salt-neoindocyanine green conjugate prepared in Example 3. The perfluorooctadecanoic acid hydroxyethyl starch nanoparticles loaded with the triphenylphosphine salt-neoindocyanine green conjugate prepared in Example 7 generated the most singlet oxygen, indicating that fluorine atoms in the nanoparticles can enrich oxygen and increase the yield of singlet oxygen in the photosensitive reaction.

[0073] Table 2. Changes in absorbance of each test solution at 405 nm under laser irradiation

[0074]

[0075] Example 13 In vitro drug release

[0076] Preparation of pH 7.4 release solution: Weigh 0.1g potassium dihydrogen phosphate, 1.08g disodium hydrogen phosphate dodecahydrate, and 2.5g Tween-80, dissolve them in ultrapure water, and bring the volume to 500mL. Then adjust the pH to 7.4 with dilute hydrochloric acid solution.

[0077] Preparation of pH 5.0 release solution: Weigh 0.1g potassium dihydrogen phosphate, 1.08g disodium hydrogen phosphate dodecahydrate, and 2.5g Tween-80, dissolve them in ultrapure water, and bring the volume to 500mL. Then adjust the pH to 5.0 with dilute hydrochloric acid solution.

[0078] In vitro drug release experiment: 1 mL of the 5 mg / mL nanoparticle test solution prepared in Example 11 was added to a dialysis bag (MWCO: 3500 Da), sealed, and immersed in 50 mL of the above-prepared release solution. The release solution system was then placed in a shaker at 37°C and shaken at 150 rpm. 2 mL of release solution was collected at 1, 2, 4, 8, 12, 24, and 48 hours, and 2 mL of the corresponding blank release solution was added. The release experiment was conducted under light-protected conditions throughout, with three parallel samples for each release experiment. The cumulative release amount of the triphenylphosphine salt-neoindocyanine green conjugate was detected by ultraviolet spectrophotometry.

[0079] As shown in Table 3, the perfluorooctadecanoyl hydroxyethyl starch nanoparticles loaded with triphenylphosphine salt-neoindocyanine green conjugate prepared in Example 7 and the n-octadecanoyl hydroxyethyl starch nanoparticles loaded with triphenylphosphine salt-neoindocyanine green conjugate prepared in Comparative Example 1 showed similar drug release in phosphate buffer at pH 7.4 and pH 5.0, and both exhibited the effect of sustained release of triphenylphosphine salt-neoindocyanine green conjugate.

[0080] Table 3. Release of the triphenylphosphine salt-neoindocyanine green conjugate

[0081]

[0082] Example 14 Intracellular Localization

[0083] Preparation of test solutions: Using the solutions containing 1 mM neoindocyanine green prepared in Example 12 as stock solutions, and using DMEM medium as diluent, the test solutions containing 10 μM neoindocyanine green were diluted to obtain the test solutions.

[0084] Intracellular localization detection: Human liver cancer cells (HepG2) were seeded at a density of 200,000 cells per well in 6-well plates and cultured overnight for adhesion. The culture medium was then replaced with the above-prepared test solution, and the cells were cultured at 37°C for 12 hours. The culture medium was then aspirated, and the cells were washed three times with phosphate-buffered saline (PFS). The mitochondrial green fluorescent probe was diluted with serum-free DMEM medium and added to the 6-well plates for co-culturing with the cells for 30 minutes. The mitochondrial green fluorescent probe was then aspirated, and the cells were washed three times with PFS. The percentage of the overlapping area of ​​the neoindocyanine green-red fluorescence and the mitochondrial green fluorescence was calculated as the proportion of neoindocyanine green localized to the mitochondria using a laser confocal microscope.

[0085] As shown in Table 4, after 12 hours of incubation with cells, only about 14% of the free neoindocyanine green was localized to the mitochondria. The proportion of the triphenylphosphine salt-neoindocyanine green conjugate prepared in Example 3 and the nanoparticles prepared in Example 7 and Comparative Example 1 that were localized to the mitochondria was higher than 60%, indicating that all three had good photosensitizer mitochondrial targeting effects.

[0086] Table 4. Mitochondrial localization ratio of photosensitizer

[0087] sample Mitochondrial localization rate (%) Free neoindocyanine green 14.3±2.2 Example 3 75.7±5.6 Example 7 70.6±6.4 Comparative Example 1 66.4±5.9

[0088] Example 15: Detection of reactive oxygen species levels in tumor cells

[0089] Preparation of test solution: Prepare a 1 mg / mL solution of dimethyl sulfoxide in 2,7-dichlorodihydrofluorescein diacetate, a green fluorescent probe for reactive oxygen species, and then prepare a 10 μg / mL test solution using serum-free DMEM medium as a diluent.

[0090] Tumor cell reactive oxygen species (ROS) level assay: HepG2 cells were seeded at a density of 200,000 cells per well in 12-well plates and cultured overnight for adhesion. The culture medium was then replaced with the test solutions prepared in Example 14 containing 10 μM indocyanine green. The cells were cultured at 37°C for 12 hours. The culture medium was then aspirated, and the cells were washed three times with phosphate-buffered saline (PBFS). 10 μg / mL of the above-prepared test solution was added, and the cells were cultured at 37°C for 20 minutes. The culture medium was then aspirated, and the cells were washed three times with PBFS. The cells were then cultured with 0.5 W / cm² PBFS. 2 Cells were irradiated with a 660nm laser for 4 minutes. After irradiation, cells were digested with trypsin, and the intensity of green fluorescence within the cells was detected by flow cytometry. Cells cultured in blank medium served as a blank control. Three replicates were set up for each group.

[0091] As shown in Table 5, at 0.5 W / cm 2Under 660nm laser irradiation, the triphenylphosphine salt-neoindocyanine green conjugate prepared in Example 3 and the nanoparticles prepared in Comparative Example 1 induced similar increases in reactive oxygen species (ROS) in tumor cells, both exceeding those of free neoindocyanine green. Among them, the nanoparticles prepared in Example 7 induced the highest level of ROS in tumor cells, mainly due to their efficient photodynamic effect and good mitochondrial targeting.

[0092] Table 5. Reactive oxygen species levels in HepG2 cells

[0093] sample Average fluorescence intensity (au) Blank control 12.6±0.3 Free neoindocyanine green 156.2±8.6 Example 3 346.5±16.7 Example 7 402.9±34.5 Comparative Example 1 325.1±29.6

[0094] Example 16: Evaluation of photodynamic killing effect on tumor cells

[0095] Preparation of test solutions: Using the solutions containing 1 mM neoindocyanine green prepared in Example 12 as stock solutions, and using DMEM medium as diluent, test solutions containing 1, 5, 10, 15, and 20 μM neoindocyanine green were obtained by dilution.

[0096] Prepare a 5 mg / mL solution of 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) using phosphate buffer, and filter through a 0.22 μm filter membrane to obtain the MTT solution.

[0097] Photodynamic killing assay: HepG2 cells were seeded at a density of 5000 cells per well in 96-well plates and cultured overnight for adhesion. The culture medium was then replaced with the prepared experimental solutions described above, and the cells were cultured at 37°C for 12 hours. Then, 0.5 W / cm² of medium was used. 2 Cells were irradiated with a 660nm laser for 4 minutes or without laser irradiation, and then cultured in a cell culture incubator at 37°C for 12 hours. 20μL of the prepared MTT solution was added, and the cells were cultured in the incubator at 37°C for 4 hours. The culture medium was then aspirated, and 150μL of dimethyl sulfoxide was added to each well. The cells were shaken to dissolve, and the absorbance at 490nm was measured for each well. Cell viability in the blank culture medium was set as 100%, and the cell viability of each experimental group was calculated. Six replicates were set up for each group.

[0098] Table 6. Cell viability under laser-free conditions

[0099]

[0100] Table 7. Cell viability under laser irradiation conditions

[0101]

[0102] As shown in Tables 6 and 7, under laser-free conditions, free neoindocyanine green, the triphenylphosphine salt-neoindocyanine green conjugate prepared in Example 3, and the nanoparticles prepared in Example 7 and Comparative Example 1 all exhibited good biocompatibility; at 0.5 W / cm 2 Under 660nm laser irradiation, the triphenylphosphine salt-neoindocyanine green conjugate prepared in Example 3 exhibited stronger photodynamic killing activity against tumor cells than free neoindocyanine green. The nanoparticles prepared in Example 1 also showed stronger photodynamic killing activity against tumor cells than free neoindocyanine green, but weaker than the triphenylphosphine salt-neoindocyanine green conjugate prepared in Example 3. The nanoparticles prepared in Example 7 exhibited the strongest photodynamic killing activity against tumor cells. This result is consistent with the increase in reactive oxygen species (ROS) levels induced by laser irradiation, indicating that the highly efficient photodynamic effect and good mitochondrial targeting of the nanoparticles prepared in Example 7 effectively induced tumor cell death by significantly increasing ROS levels in tumor cells.

Claims

1. A novel indocyanine green delivery system targeting mitochondria of tumor cells, characterized in that, The hydration diameter of the delivery system is 100–200 nm; the preparation method of the delivery system is as follows: (1) Using neoindocyanine green and 4,7,10-trioxo-1,13-tetanediamine as raw materials, methanol as solvent, the mixture was heated to 50°C and stirred for 4 hours. Then, methanol was removed by rotary evaporation, and ultrapure water was added for dispersion. The mixture was dialyzed in ultrapure water for three days and then freeze-dried to obtain amino-modified neoindocyanine green. The molar ratio of neoindocyanine green to 4,7,10-trioxo-1,13-tridecanediamine is 1:5 to 10. (2) Using the amino-modified neoindocyanine green and 3-propanecarboxylic triphenylphosphine bromide prepared in step (1) as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride as condensing agent, 4-dimethylaminopyridine as catalyst, and N,N-dimethylformamide as solvent, the mixture was heated to 50°C and stirred for 24 hours, then cooled to room temperature, dialyzed in ultrapure water for three days, and then freeze-dried and purified by column chromatography to obtain the triphenylphosphine salt-neoindocyanine green conjugate. The molar ratio of amino-modified neoindocyanine green, 3-propanoic acid-triphenylphosphine bromide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 1:2~4:8~16:1~2. (3) Using hydroxyethyl starch and perfluorooctadecanoic acid as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride as condensing agent, 4-dimethylaminopyridine as catalyst, and dimethyl sulfoxide as solvent, the mixture was heated to 40°C and stirred for 48 hours, then cooled to room temperature, dialyzed in ultrapure water for three days, centrifuged at 8000 rpm for 10 minutes, and the supernatant was freeze-dried to obtain perfluorooctadecanoic acid hydroxyethyl starch ester; The mass ratio of hydroxyethyl starch to perfluorooctadecanic acid is 5-10:1, and the molar ratio of perfluorooctadecanic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 1:10-20:2-4. (4) The triphenylphosphine salt-new indocyanine green conjugate prepared in step (2) and the perfluorooctanoic acid hydroxyethyl starch ester prepared in step (3) were added to dimethyl sulfoxide and stirred overnight. The mixture was dialyzed in ultrapure water for three days, centrifuged at 8000 rpm for 10 minutes, and the supernatant was taken and freeze-dried to obtain a new indocyanine green delivery system targeting the mitochondria of tumor cells. The mass ratio of triphenylphosphine salt-neoindocyanine green coupling compound to perfluorooctanoic acid hydroxyethyl starch ester is 1:9 to 19.

2. The novel indocyanine green delivery system targeting tumor cell mitochondria according to claim 1, characterized in that, The delivery system exhibits in vitro photodynamic effects.

3. The application of the novel indocyanine green delivery system targeting tumor cell mitochondria according to claim 1, characterized in that, The novel indocyanine green delivery system targeting tumor cell mitochondria is used to prepare agents that enhance tumor photodynamic therapy.

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