WO2022188046A1 - Chemotherapeutic drug-/photosensitizer-loaded nanomicelle, preparation method therefor, and application thereof - Google Patents

Chemotherapeutic drug-/photosensitizer-loaded nanomicelle, preparation method therefor, and application thereof Download PDF

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WO2022188046A1
WO2022188046A1 PCT/CN2021/079844 CN2021079844W WO2022188046A1 WO 2022188046 A1 WO2022188046 A1 WO 2022188046A1 CN 2021079844 W CN2021079844 W CN 2021079844W WO 2022188046 A1 WO2022188046 A1 WO 2022188046A1
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photosensitizer
solution
chemotherapeutic drug
loaded
drug
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Chinese (zh)
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杨红
张米娅
陈华兵
陈亮
柯亨特
邓益斌
庞海燕
朱洁
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苏州大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles

Definitions

  • the invention relates to nano-medicine technology, in particular to micellar nano-particles with dual therapeutic effects of encapsulating chemotherapeutic drugs (such as doxorubicin) and photosensitizers (such as indocyanine green) and preparation thereof.
  • chemotherapeutic drugs such as doxorubicin
  • photosensitizers such as indocyanine green
  • Chemotherapy drugs are the main treatment methods for cancer, but they have problems such as high toxicity, poor selectivity and strong side effects. Even with the use of drug combination therapy or the combination of surgery and chemotherapy, the curative effect is still poor. By encapsulating the drug with new drug delivery systems such as liposomes and micelles, its curative effect on tumors can be effectively improved.
  • Doxorubicin is a widely used antitumor chemotherapy drug, but it has problems such as cardiotoxicity.
  • Phototherapy is a new type of tumor treatment method in recent years, including photothermal therapy (PTT) and photodynamic therapy (PDT). Thus play a role in killing cancer cells.
  • Indocyanine green (ICG) is currently the only FDA-approved photosensitizer, and light can produce both PTT and PDT therapeutic effects. In the absence of light, the toxicity is extremely low, but it is unstable in water and is easily eliminated in the body.
  • the preparation process of these nanoparticles is relatively complicated, the carrier for biological purification requires complex extraction and purification, the chemically synthesized polymer has potential toxicity and complex synthesis steps, and the inorganic nanoparticles have poor biocompatibility and certain toxicity. .
  • micellar drug delivery systems are significant. It can improve the bioavailability of poorly soluble drugs, reduce toxic and side effects, improve in vivo stability, prolong circulation time, and have sustained release and passive targeting to tumor cells.
  • polyethylene glycol-b-polycaprolactone PEG-PCL as the carrier material to prepare micelles, is conducive to further development and application in clinical practice.
  • ICG/DOX micelles with a particle size of 221 nm were prepared at 37°C. However, there are the following defects: 1) The particle size is too large. Usually ⁇ 200nm is more conducive for micelles to avoid the interference of the endothelial reticulum system RES and target the tumor site; 2) The preparation temperature is high, and ICG requires low temperature storage, which will affect the stability.
  • the purpose of the present invention is to provide a chemotherapeutic drug/photosensitizer-loaded micelle and its preparation method and application. It is based on the fact that after the chemotherapeutic drug DOX enters the cell, under the synergistic effect of the photosensitizer ICG, the lysosome escapes and enters the nucleus to exert the chemotherapeutic effect. Therefore, nanoparticles encapsulating DOX and ICG are designed.
  • the present invention adopts the commercially available carrier material PEG-PCL, and realizes the preparation of ICG/DOX co-encapsulated micelles (ID-M) with a particle size of ⁇ 100 nm at room temperature (25°C), which has the advantages of simple preparation process and preparation conditions.
  • a chemotherapeutic drug/photosensitizer-carrying nanomicelle is obtained by encapsulating the chemotherapeutic drug and photosensitizer in a polymer; the hydrated particle size of the chemotherapeutic drug/photosensitizer-carrying nanomicelle is 10 ⁇ 160 nm; preferably, the chemotherapeutic drug is doxorubicin, the photosensitizer is indocyanine green, and the polymer is PEG-PCL.
  • the preparation method of the above chemotherapeutic drug/photosensitizer-loaded nanomicelles includes the following steps: mixing chemotherapeutic drug solution, photosensitizer solution, and polymer solution, and then ultrasonically treating to obtain solution A; adding solution A dropwise to water, and ultrasonically treating the solution A. , to obtain solution B; solution B is subjected to dialysis, ultrafiltration and centrifugation to obtain nanomicelles loaded with chemotherapeutic drugs/photosensitizers.
  • the invention discloses the application of the above-mentioned chemotherapeutic drug/photosensitizer-loaded nanomicelles in the preparation of tumor therapeutic drugs; the polymer PEG-PCL is selected as a carrier, and the chemotherapeutic drugs and photosensitizers are simultaneously loaded to realize the combination on a nanometer drug-loading platform.
  • the dual targeted therapy of chemotherapy toxicity and phototoxicity on tumors plays the role of chemotherapy and/or photodynamic therapy and/or photothermal therapy.
  • the preparation of the chemotherapeutic drug/photosensitizer-loaded nanomicelles is carried out at room temperature.
  • the solvents are all DMSO (dimethyl sulfoxide).
  • the mass ratio of photosensitizer, chemotherapeutic drug and polymer is (0.1-2.0): (0.1-2.0) (0.8-16), preferably (0.5-1.5): (0.5-1.5): (4- 12), most preferably, the mass ratio of photosensitizer to chemotherapeutic drug is 1:1; for example, the mass ratio of photosensitizer, chemotherapeutic drug and polymer is 1:1:6-10.
  • the volume ratio of solution A and water is 1:5-15.
  • the ultrasonic treatment time is 5-10 minutes.
  • the concentration of the chemotherapeutic drug solution is 1 mg/200 ⁇ L; the concentration of the photosensitizer solution is 1 mg/200 ⁇ L; the concentration of the polymer solution is 6-10 mg/200 ⁇ L, preferably 8 mg/200 ⁇ L.
  • the rotational speed of the ultrafiltration centrifugation is 3500-6000 r ⁇ min -1 .
  • DOXHCl is dissolved in a DMSO solution at room temperature, triethylamine is added for desalting, and the DMSO solution of DOX is formed by ultrasonic treatment;
  • ICG is dissolved in DMSO, and the DMSO solution of ICG is formed by ultrasonic treatment;
  • the DMSO solution was mixed with the DMSO solution of DOX and the DMSO solution of ICG, added dropwise to water after ultrasonic treatment, and then ultrasonically treated to obtain a mixed solution containing micelles. After ultrafiltration and concentration, micelles containing chemotherapeutic drugs and photosensitizers were obtained.
  • Solution ID-M the DMSO solution at room temperature
  • triethylamine is added for desalting
  • the DMSO solution of DOX is formed by ultrasonic treatment
  • ICG is dissolved in DMSO
  • the DMSO solution of ICG is formed by ultrasonic treatment
  • the DMSO solution was mixed with the DMSO solution of DOX and the DM
  • desalted doxorubicin and indocyanine green are co-encapsulated in the hydrophobic cavity of the micelle to obtain micelles with a particle size of not more than 160 nm, which can be used as a new anti-tumor dosage form to enhance the toxicity and target of the drug to tumors.
  • tropism play a synergistic effect, reduce systemic side effects, so as to realize the combined treatment of tumors.
  • the chemotherapeutic drug is doxorubicin
  • the photosensitizer is indocyanine green
  • the structural formulas of doxorubicin (DOX) and indocyanine green (ICG) are respectively as follows: .
  • the micelle prepared by using the commercially available material PEG-PCL in the present invention is ideal in particle size, light stability, release of the micelle and its killing effect on tumor cells, and has a great application prospect in tumor treatment.
  • the mechanism of anti-tumor effect was investigated at the cellular level, and it was confirmed from different aspects such as photothermal, singlet oxygen, cytotoxicity, intracellular drug distribution, etc.
  • micellar nanoparticle prepared by the invention has high uptake by tumor cells, has a large killing effect on tumor cells, and has a synergistic effect when combined with chemotherapy/photodynamic therapy under the condition of near-infrared light. Photodynamic therapy damages cells and enhances the effect of chemotherapy. It has high-efficiency, low-toxicity and anti-tumor effects with both chemotherapy and photodynamic therapy, and is a safe and effective new nano preparation.
  • Figure 1 Morphological characterization of nanoparticles: TEM image of 1-A micelles; dynamic particle size and distribution of 1-B micelles; Zeta potential map of 1-C micelles.
  • Figure 8 Fluorescence images of drug entry into the nucleus before and after micellar irradiation.
  • micellar nanoparticles carrying chemotherapeutic drugs/photosensitizers of the present invention are referred to as "micelles" for short.
  • the carrier of the present invention is PEG-PCL, specifically PEG 115 - b -PCL 60 , which is commercially available or prepared according to existing conventional methods, such as the article "Research on Environmentally Responsive Nanoparticles for Tumor Imaging and Treatment”; ultrasonic device It is KQ-100DE, Kunshan Ultrasonic Instrument Co., Ltd.
  • the ultrasonic power of the embodiment is 100W.
  • Example 1 Weigh 8 mg of PEG 115 - b -PCL 60 and dissolve it in 200 mL of DMSO, and ultrasonically for 5 minutes to obtain a polymer solution; weigh 1 mg of ICG, add 200 mL of DMSO, and ultrasonically dissolve for 5 minutes to obtain a photosensitizer solution; Another 1 mg of DOX ⁇ HCl was weighed and dissolved in 200 mL of DMSO, 3 mL of triethylamine was added, and sonicated for 5 minutes to obtain a desalted doxorubicin solution.
  • the micelles were prepared by liquid dispersion method. Mix the above three solutions and continue to sonicate for 5 min to obtain a homogeneous solution A. Another 6 mL of deionized water was taken, and solution A was added dropwise to the water (2 drops per second) while sonicating. The dropwise addition was completed, and the initial micellar solution B was obtained by continuing to sonicate for 10 minutes. The entire operation was at room temperature (25 °C). . The solution B was dialyzed (the intercepted molecular weight of the dialysis bag was MWCO: 3500 Da) overnight, and the deionized water was changed every 2 h for dialysis .
  • the micelle solution ID-M co-encapsulated with ICG and DOX was obtained, which was a nanomicelle solution loaded with chemotherapeutic drugs/photosensitizers, and was placed for 48 hours without turbidity.
  • the encapsulation efficiencies were: ICG 96.1 ⁇ 1.4%, DOX 93.9 ⁇ 3.2%, and the drug loadings were: ICG 17.5 ⁇ 2.4%, DOX 9.1 ⁇ 1.8%.
  • the results show that the prepared micelles have a unimodal distribution, the average hydrated particle size is 80.4 ⁇ 14.3 nm (this is the particle size including the surface hydration layer), and the polydispersity coefficient (PDI) is 0.252 ⁇ 0.015, which is beneficial to avoid endothelial reticulum
  • the micelles were intercepted to form a long circulation in vivo and played a passive targeting role; as shown in Figure 1-C, the results showed that the micelles were negatively charged, and the Zeta potential was -17.23 ⁇ 0.66 mV. It is beneficial to the circulation of micelles in the body and is not easy to be removed.
  • Figure 3 is a graph of cumulative release profiles.
  • Table 1 is the zero-order equation, the first-order equation, the Higuchi equation and the Weibull equation, respectively, to fit the data related to the cumulative release of DOX, and obtain the ID-M and free I/D in pH 5.0 PBS and pH 7.4 PBS, respectively. Release behavior results: The cumulative release of free (Free) I/D in pH 7.4 PBS for 24 h was 74.8 ⁇ 7.0 ⁇ g, and in pH 5.0 PBS for 24 h, the cumulative release was 77.3 ⁇ 2.6 ⁇ g; ID-M in pH 7.4 The cumulative release in PBS for 24 h was 23.1 ⁇ 5.2 ⁇ g, and the cumulative release in pH 5.0 PBS for 24 h was 47.5 ⁇ 4.7 ⁇ g.
  • Cytotoxicity study of micelles To investigate the toxicity of micelles to tumor cells , logarithmically growing 4T1 cells were seeded in 96-well plates (cell density 5 ⁇ 10 4 ⁇ mL -1 ) and incubated at 37 o C, 5% CO 2 , different concentrations of I/DM and Free I/D were added. After 24 hours of administration, the medium was changed. The light group was treated with a 785 nm laser (1.5 W ⁇ cm -2 , 3 min). After continuing to incubate for 24 h, the medium was replaced, 20 mL of 5 mg ⁇ mL -1 MTT solution was added, and the cells were incubated for 4 h.
  • IC 50 (mg ⁇ mL -1 ): IM group 4.72 ⁇ DM group 1.83 ⁇ Free I/D group 1.21 ⁇ ID-M group 0.72.
  • the cytotoxicity of ID-M was significantly increased after light exposure, the cell viability was 3.6 ⁇ 0.3 mg ⁇ mL -1 at high concentration (IC 50 decreased by 46.67%), and the cell viability of Free I/D was 15.7 ⁇ 0.2 mg ⁇ mL -1 .
  • the cytotoxicity of ID-M was statistically investigated, except that it was different from IM under non-illumination, there was no significant difference with other groups (P>0.05).
  • ID-M (>1 mg ⁇ mL -1 ) was significantly different from each group (P ⁇ 0.01), and the anti-tumor effect was significantly enhanced. Moreover, the calculated synergy index CI of ID-M is 0.62, which meets the requirement of the literature that CI ⁇ 0.8 has a significant synergistic therapeutic effect. tumor synergy.
  • the non-fluorescent DHE can be oxidized by ROS to ethidium oxide in cells, and ethidium oxide is incorporated into DNA to generate red fluorescence. amount of ROS.
  • PBS group 0.4 ⁇ DM group 0.5 ⁇ Free I/D group 1.4 ⁇ IM group 2.4 ⁇ ID-M group 2.6. This indicated that ID-M had the strongest ability to promote ROS production in tumor cells and had the greatest phototoxicity.
  • nuclei were stained with 1 mg ⁇ mL -1 Hoechest 33342, incubated and washed three times with PBS, lysosomes were stained with 50 mM Lysotracker DND-26, incubated and washed, added 1 mL of medium, and confocal with laser Microscopy to observe the state of cells in a confocal dish.
  • DOX can enter the nucleus of tumor cells to inhibit the synthesis of DNA and RNA, and the effect of chemotherapy can be judged by the amount of DOX entering the nucleus.
  • Figure 8 shows the results of DOX entry in ID-M. Nuclei were stained blue, lysosomes were stained green, and DOX showed red fluorescence. The DOX in the nucleus was reflected by the red-blue fluorescence co-localization rate, the DOX in the lysosome was reflected by the red-green fluorescence co-localization rate, and the remaining DOX was in the cytoplasm.
  • the high co-localization rate of red and blue indicates that DOX enters the nucleus more and has good anti-tumor effect; the high co-localization rate of red and green indicates that DOX is abundant in lysosomes and has poor effect.
  • the co-localization rate it was found that before light exposure, 92.5% of DOX was in the lysosome, 2.7% in the nucleus, and 4.8% in the cytoplasm. 82.7% of the total DOX entered the nucleus at 12 h after illumination, increasing the amount of the drug in the nucleus by 29.6 times. Therefore, the cytotoxicity of ID-M is enhanced by light exposure, which is related to the increase of the amount of drug entering the nucleus, thus confirming that ID-M effectively inhibits tumor cell growth, and there is a synergistic effect of chemotherapy and phototherapy.
  • Comparative Example 1 Weigh 8 mg of PEG 115 - b -PCL 60 and dissolve it in 200 mL of DMSO, and ultrasonicate for 5 minutes to obtain a polymer solution; weigh 1 mg of ICG, add 200 mL of DMSO, and ultrasonically dissolve for 5 minutes to obtain a photosensitizer solution; Another 1 mg of DOX ⁇ HCl was weighed and dissolved in 200 mL of DMSO, 3 mL of triethylamine was added, and sonicated for 5 minutes to obtain a desalted doxorubicin solution.
  • the micelles were prepared by liquid dispersion method. Mix the above three solutions, drop directly into 6 mL of deionized water without ultrasonication, dropwise under ultrasonication (2 drops per second), complete the dropwise addition, and continue to ultrasonicate for 10 minutes to obtain initial micelle solution B. The entire operation process to room temperature (25°C). The solution B was dialyzed (the intercepted molecular weight of the dialysis bag was MWCO: 3500 Da) overnight, and the deionized water was changed every 2 h for dialysis . After filtration and centrifugation for 10 minutes, the color of the supernatant is very light, and the lower layer has a lot of sediment, and the color is very dark, indicating that the drug is precipitated and the encapsulation fails.
  • the micelles were prepared by liquid dispersion method. Mix the above three solutions and continue to sonicate for 5 min to obtain a homogeneous solution A. Take another 6 mL of deionized water, add solution A to the water while sonicating (complete addition within 1 second, not dropwise), and continue to sonicate for 10 minutes to obtain initial micelle solution B. The entire operation is at room temperature (25 °C). The solution B was dialyzed (the intercepted molecular weight of the dialysis bag was MWCO: 3500 Da) overnight, and the deionized water was changed every 2 h for dialysis . After filtration and centrifugation for 10 minutes, the color of the supernatant is very light, and the lower layer has a lot of sediment, and the color is very dark, indicating that the drug is precipitated and the encapsulation fails.
  • the micelles were prepared by liquid dispersion method. The whole operation process is room temperature (25 °C), the above three solutions are mixed, and the sonication is continued for 5 min to obtain a uniform solution A. Take another 6 mL of deionized water, add solution A dropwise to the water (2 drops per second) while ultrasonicating, and complete the direct dialysis without ultrasonication (dialysis bag intercepted molecular weight is MWCO: 3500 Da) overnight, every 2 h Change the deionized water once for dialysis, put it in an ultrafiltration centrifuge tube after dialysis, and centrifuge it with ultrafiltration at a speed of 5000 r ⁇ min -1 for 10 minutes. Precipitation, package loading failed.
  • the encapsulation effect of the comparative method (3) is better than that of the comparative method (1) and (2), but the three comparative methods are much worse than the drug-carrying effect of the present invention.
  • method after ultrafiltration, there is very little sediment in the lower layer.
  • micell ID-M can also be prepared by thin film dispersion method.
  • the specific scheme is as follows: ICG, DOX and PEG-PCL are co-dissolved in dichloromethane, ultrasonically mixed in a distillation flask, and spin on a rotary evaporator. After drying, distilled water was added dropwise under ultrasonic conditions to dissolve the film on the wall of the distillation flask. After centrifugation at 1500 r ⁇ min -1 for 10 min, the micelle solution was obtained by passing through a 220 nm filter membrane. The particle size of the micellar solution prepared by this method is 35.1 ⁇ 2.8 nm, but it is prone to turbidity. After 10 minutes, the micellar solution appears turbid, indicating instability.
  • Comparative Example 3 Dissolve 8 mg of PEG 115 - b -PCL 60 , 1 mg of ICG, and 1 mg of DOX ⁇ HCl in 600 mL of DMSO, add 3 mL of triethylamine, and sonicate for 5 minutes to obtain a homogeneous solution A. Another 6 mL of deionized water was taken, and solution A was added dropwise to the water (2 drops per second) while sonicating. The dropwise addition was completed, and the initial micellar solution B was obtained by continuing to sonicate for 10 minutes. The entire operation was at room temperature (25 °C). .
  • the solution B was dialyzed (the intercepted molecular weight of the dialysis bag was MWCO: 3500 Da) overnight, and the deionized water was changed every 2 h for dialysis . Filtration and centrifugation for 10 minutes to obtain the micellar solution ID-M co-encapsulated with ICG and DOX.
  • the particle size was analyzed by a laser scattering particle size analyzer, and the average hydrated particle size was 212.4 ⁇ 12.7 nm.
  • the existing technology has the problems of high preparation temperature and large particle size of nano-drugs.
  • the present invention is designed to prepare particle size at room temperature not exceeding 37 ° C.
  • the PEG-PCL smaller than 200nm encapsulates ICG/DOX micelles, giving full play to the synergistic effect of the two treatments, laying the foundation for the further development of new formulations with high efficiency and low toxicity.
  • the invention relates to nano-medicine technology, in particular to micellar nanoparticles encapsulating the dual therapeutic effects of chemotherapeutic drugs (such as doxorubicin) and photosensitizers (such as indocyanine green) and preparation thereof, and as a new anti-tumor dosage form, enhancing the The toxicity and targeting of the drug to the tumor can play a synergistic effect, reduce the systemic toxicity and side effects, so as to realize the combined treatment of the tumor.
  • chemotherapeutic drugs such as doxorubicin
  • photosensitizers such as indocyanine green

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Abstract

A chemotherapeutic drug-/photosensitizer-loaded nanomicelle, a preparation method therefor, and an application thereof, aiming at combining functions of phototherapy and chemotherapy to prepare an indocyanine green (ICG) and doxorubicin (DOX) co-loaded micelle (ID-M), characterizing the prepared micelle, and investigating an in vitro anti-tumor effect of the micelle at the cellular level. The prepared micelle nanoparticles are mild in condition, small in particle size, uniform in dispersion, round in morphology, and good in chemical stability and illumination stability, and have high reactive oxygen generation capability under the irradiation of near-infrared light. In addition, in a cell experiment, the nanoparticles have strong cytotoxicity and good cell uptake for tumor cells, exert a synergistic effect, reduce toxic and side effects, and implement phototherapy and chemotherapy combined treatment of tumors.

Description

载化疗药物/光敏剂的纳米胶束及其制备方法和应用Nanomicelles loaded with chemotherapeutic drugs/photosensitizers and preparation methods and applications thereof 技术领域technical field
本发明涉及纳米药物技术，具体为包载化疗药物（如阿霉素）和光敏剂（如吲哚菁绿）的双重治疗效果的胶束纳米粒及其制备。The invention relates to nano-medicine technology, in particular to micellar nano-particles with dual therapeutic effects of encapsulating chemotherapeutic drugs (such as doxorubicin) and photosensitizers (such as indocyanine green) and preparation thereof.
背景技术Background technique
癌症是全球性的重大疾病，化疗药物是癌症的主要治疗手段，但其具有毒性大，选择性差，副作用强等问题。即使采用药物联合治疗或是手术与化疗联合应用等方法，疗效仍旧欠佳。通过将药物用脂质体、胶束等新型给药体系包载可有效改善其对肿瘤的疗效。Cancer is a major global disease. Chemotherapy drugs are the main treatment methods for cancer, but they have problems such as high toxicity, poor selectivity and strong side effects. Even with the use of drug combination therapy or the combination of surgery and chemotherapy, the curative effect is still poor. By encapsulating the drug with new drug delivery systems such as liposomes and micelles, its curative effect on tumors can be effectively improved.
阿霉素（DOX）是广泛应用的抗肿瘤化疗药物，但有心脏毒性等问题。光治疗是近年来新型的肿瘤治疗方法，包括光热治疗（photothermal therapy, PTT）和光动力治疗（photodynamic therapy, PDT），其主要原理是光敏剂受激发光照射后，会产生热和活性氧，从而起到杀伤癌细胞的作用。吲哚菁绿（ICG）是目前唯一被FDA批准上市的光敏剂，光照可同时产生PTT和PDT治疗作用。而在无光照时毒性极低，但其在水中不稳定，在体内易被清除。Doxorubicin (DOX) is a widely used antitumor chemotherapy drug, but it has problems such as cardiotoxicity. Phototherapy is a new type of tumor treatment method in recent years, including photothermal therapy (PTT) and photodynamic therapy (PDT). Thus play a role in killing cancer cells. Indocyanine green (ICG) is currently the only FDA-approved photosensitizer, and light can produce both PTT and PDT therapeutic effects. In the absence of light, the toxicity is extremely low, but it is unstable in water and is easily eliminated in the body.
现有研究运用不同类型的载体对ICG和DOX进行共包载，包括：生物提纯的载体（如人血清白蛋白、红细胞膜等）、经过化学合成的载体（响应性聚合物、修饰靶头的聚合物等）以及无机纳米粒载体（介孔二氧化硅、金纳米笼、MOF结构等）形成不同的给药系统，粒径大多数在100-200 nm之间，能在一定的条件下达到理想的释放效果以及对肿瘤细胞的杀伤的作用。但是，这些纳米粒的制备工艺相对复杂，生物提纯的载体需要复杂的提取和纯化、化学合成的高分子聚合物会有潜在毒性且合成步骤复杂、无机纳米粒生物相容性差且有一定的毒性。Existing studies have used different types of carriers to co-encapsulate ICG and DOX, including: biologically purified carriers (such as human serum albumin, red blood cell membranes, etc.), chemically synthesized carriers (responsive polymers, modified target heads, etc.) polymers, etc.) and inorganic nanoparticle carriers (mesoporous silica, gold nanocages, MOF structures, etc.) to form different drug delivery systems, and most of the particle sizes are between 100-200 nm, which can be achieved under certain conditions. Ideal release effect and killing effect on tumor cells. However, the preparation process of these nanoparticles is relatively complicated, the carrier for biological purification requires complex extraction and purification, the chemically synthesized polymer has potential toxicity and complex synthesis steps, and the inorganic nanoparticles have poor biocompatibility and certain toxicity. .
胶束给药系统优势显著。它能够提高难溶性药物的生物利用度，降低毒副作用，改善体内稳定性，延长循环时间，具有缓释作用和对肿瘤细胞的被动靶向作用。运用合成工艺成熟且已经上市的聚合物材料，聚乙二醇-b-聚己内酯PEG-PCL作为载体材料制备胶束，有利于进一步研发运用于临床。现有技术在37℃条件下制得粒径221nm的ICG/DOX胶束。但是，存在以下缺陷：1）粒径偏大。通常<200nm更有利于胶束避开内皮网状系统RES的干扰，靶向到达肿瘤部位；2）制备温度偏高，ICG要求低温保存，该温度会影响稳定性。The advantages of micellar drug delivery systems are significant. It can improve the bioavailability of poorly soluble drugs, reduce toxic and side effects, improve in vivo stability, prolong circulation time, and have sustained release and passive targeting to tumor cells. Using the polymer materials with mature synthesis technology and already on the market, polyethylene glycol-b-polycaprolactone PEG-PCL as the carrier material to prepare micelles, is conducive to further development and application in clinical practice. In the prior art, ICG/DOX micelles with a particle size of 221 nm were prepared at 37°C. However, there are the following defects: 1) The particle size is too large. Usually <200nm is more conducive for micelles to avoid the interference of the endothelial reticulum system RES and target the tumor site; 2) The preparation temperature is high, and ICG requires low temperature storage, which will affect the stability.
技术问题technical problem
为解决以上技术问题，本发明的目的是提供一种载化疗药物/光敏剂的胶束及其制备方法和应用。它基于化疗药物DOX进入细胞后，在光敏剂ICG的协同作用下，实现溶酶体逃逸，进入到细胞核内发挥化疗效果，故设计包载DOX、ICG的纳米粒，1）在纳米粒的测定中，采用荧光分光光度计测定DOX的含量，紫外-可见分光光度计测定ICG的含量，保证结果可靠；2）体外和细胞实验，用游离药物以及单独包载ICG和DOX的胶束作对照，仍可得到可靠的结果。因此，本发明采用市售载体材料PEG-PCL，实现了在室温（25℃）制备出粒径≤100 nm的ICG/DOX共包载胶束（ID-M），具有制备工艺简单、制备条件温和、粒径可控、生物相容性好、肿瘤靶向性和滞留性好等优势，整合了光化疗治疗手段实现高效杀伤肿瘤细胞、有效结合光治疗促进化疗药物进核发挥疗效的纳米胶束的制备和应用。In order to solve the above technical problems, the purpose of the present invention is to provide a chemotherapeutic drug/photosensitizer-loaded micelle and its preparation method and application. It is based on the fact that after the chemotherapeutic drug DOX enters the cell, under the synergistic effect of the photosensitizer ICG, the lysosome escapes and enters the nucleus to exert the chemotherapeutic effect. Therefore, nanoparticles encapsulating DOX and ICG are designed. 1) Determination of nanoparticles Fluorescence spectrophotometer was used to determine the content of DOX, and UV-visible spectrophotometer was used to determine the content of ICG to ensure reliable results; 2) In vitro and cell experiments, free drug and micelles containing ICG and DOX alone were used as controls. Still get reliable results. Therefore, the present invention adopts the commercially available carrier material PEG-PCL, and realizes the preparation of ICG/DOX co-encapsulated micelles (ID-M) with a particle size of ≤100 nm at room temperature (25°C), which has the advantages of simple preparation process and preparation conditions. The advantages of mildness, controllable particle size, good biocompatibility, tumor targeting and retention, etc., integrate photochemotherapy to achieve efficient killing of tumor cells, and effectively combine phototherapy to promote chemotherapeutic drugs into the nucleus. Beam preparation and application.
技术解决方案technical solutions
本发明采用如下技术方案：一种载化疗药物/光敏剂的纳米胶束，由聚合物包载化疗药物和光敏剂得到；所述载化疗药物/光敏剂的纳米胶束的水合粒径为10~160 nm；优选的，化疗药物为阿霉素，光敏剂为吲哚菁绿，聚合物为PEG-PCL。The present invention adopts the following technical scheme: a chemotherapeutic drug/photosensitizer-carrying nanomicelle is obtained by encapsulating the chemotherapeutic drug and photosensitizer in a polymer; the hydrated particle size of the chemotherapeutic drug/photosensitizer-carrying nanomicelle is 10 ~160 nm; preferably, the chemotherapeutic drug is doxorubicin, the photosensitizer is indocyanine green, and the polymer is PEG-PCL.
上述载化疗药物/光敏剂的纳米胶束，其制备方法包括以下步骤，将化疗药物溶液、光敏剂溶液、聚合物溶液混合，然后超声处理，得到溶液A；将溶液A滴加入水中，超声处理，得到溶液B；溶液B经过透析、超滤离心，得到载化疗药物/光敏剂的纳米胶束。The preparation method of the above chemotherapeutic drug/photosensitizer-loaded nanomicelles includes the following steps: mixing chemotherapeutic drug solution, photosensitizer solution, and polymer solution, and then ultrasonically treating to obtain solution A; adding solution A dropwise to water, and ultrasonically treating the solution A. , to obtain solution B; solution B is subjected to dialysis, ultrafiltration and centrifugation to obtain nanomicelles loaded with chemotherapeutic drugs/photosensitizers.
本发明公开了上述载化疗药物/光敏剂的纳米胶束在制备肿瘤治疗药物中的应用；选用聚合物PEG-PCL作为载体，同时包载化疗药物和光敏剂，实现在一个纳米载药平台结合化疗毒性和光毒性对肿瘤的双重靶向治疗作用，起到化疗和/或光动力治疗和/或光热治疗的作用。The invention discloses the application of the above-mentioned chemotherapeutic drug/photosensitizer-loaded nanomicelles in the preparation of tumor therapeutic drugs; the polymer PEG-PCL is selected as a carrier, and the chemotherapeutic drugs and photosensitizers are simultaneously loaded to realize the combination on a nanometer drug-loading platform. The dual targeted therapy of chemotherapy toxicity and phototoxicity on tumors plays the role of chemotherapy and/or photodynamic therapy and/or photothermal therapy.
本发明中，载化疗药物/光敏剂的纳米胶束的制备在室温下进行。In the present invention, the preparation of the chemotherapeutic drug/photosensitizer-loaded nanomicelles is carried out at room temperature.
 本发明中，化疗药物溶液、光敏剂溶液、聚合物溶液中，溶剂都是DMSO（二甲基亚砜）。In the present invention, in the chemotherapeutic drug solution, the photosensitizer solution and the polymer solution, the solvents are all DMSO (dimethyl sulfoxide).
 本发明中，光敏剂、化疗药物、聚合物的质量比为（0.1～2.0）∶（0.1～2.0）（0.8～16），优选为（0.5～1.5）∶（0.5～1.5）∶（4～12），最优选的，光敏剂、化疗药物的质量比为1∶1；比如，光敏剂、化疗药物、聚合物的质量比为1∶1∶6～10。In the present invention, the mass ratio of photosensitizer, chemotherapeutic drug and polymer is (0.1-2.0): (0.1-2.0) (0.8-16), preferably (0.5-1.5): (0.5-1.5): (4- 12), most preferably, the mass ratio of photosensitizer to chemotherapeutic drug is 1:1; for example, the mass ratio of photosensitizer, chemotherapeutic drug and polymer is 1:1:6-10.
本发明中，溶液A、水的体积比为1∶5～15。In the present invention, the volume ratio of solution A and water is 1:5-15.
本发明中，超声处理的时间为5～10分钟。In the present invention, the ultrasonic treatment time is 5-10 minutes.
本发明中，化疗药物溶液的浓度为1mg/200μL；光敏剂溶液的浓度为1mg/200μL；聚合物溶液的浓度为6～10mg/200μL，优选8mg/200μL。In the present invention, the concentration of the chemotherapeutic drug solution is 1 mg/200 μL; the concentration of the photosensitizer solution is 1 mg/200 μL; the concentration of the polymer solution is 6-10 mg/200 μL, preferably 8 mg/200 μL.
本发明中，超滤离心的转速为3500~6000 r·min -1。 In the present invention, the rotational speed of the ultrafiltration centrifugation is 3500-6000 r·min -1 .
本发明在室温下，将DOXŸHCl溶于DMSO溶液中，加入三乙胺脱盐，超声处理形成DOX的DMSO溶液；将ICG溶于DMSO中，超声处理，形成ICG的DMSO溶液；将PEG-PCL的DMSO溶液与DOX的DMSO溶液、ICG的DMSO溶液混合，超声处理后滴加入水中，再超声处理，得到含胶束的混合溶液，经超滤浓缩后得到包载化疗药物和光敏剂的胶束溶液ID-M。本发明中将脱盐阿霉素和吲哚菁绿共同包载在胶束的疏水性空腔中，得到粒径不超过160nm的胶束，作为抗肿瘤新药剂型，增强药物对肿瘤的毒性和靶向性，发挥协同增效作用，降低全身毒副作用，从而实现肿瘤的联合治疗。In the present invention, DOXHCl is dissolved in a DMSO solution at room temperature, triethylamine is added for desalting, and the DMSO solution of DOX is formed by ultrasonic treatment; ICG is dissolved in DMSO, and the DMSO solution of ICG is formed by ultrasonic treatment; The DMSO solution was mixed with the DMSO solution of DOX and the DMSO solution of ICG, added dropwise to water after ultrasonic treatment, and then ultrasonically treated to obtain a mixed solution containing micelles. After ultrafiltration and concentration, micelles containing chemotherapeutic drugs and photosensitizers were obtained. Solution ID-M. In the present invention, desalted doxorubicin and indocyanine green are co-encapsulated in the hydrophobic cavity of the micelle to obtain micelles with a particle size of not more than 160 nm, which can be used as a new anti-tumor dosage form to enhance the toxicity and target of the drug to tumors. tropism, play a synergistic effect, reduce systemic side effects, so as to realize the combined treatment of tumors.
本发明中，化疗药物为阿霉素，光敏剂为吲哚菁绿；阿霉素(DOX)、吲哚菁绿(ICG)结构式分别如下：
Figure 414349dest_path_image001
。 In the present invention, the chemotherapeutic drug is doxorubicin, and the photosensitizer is indocyanine green; the structural formulas of doxorubicin (DOX) and indocyanine green (ICG) are respectively as follows:
Figure 414349dest_path_image001
.
本发明采用市售材料PEG-PCL制备的胶束，粒径、光照稳定性、胶束的释放以及其对肿瘤细胞的杀伤作用，均较为理想，在肿瘤治疗中具有较大的应用前景。并且在细胞层面考察抗肿瘤效应的机制，从光热、单线态氧、细胞毒性、胞内的药物分布等不同方向证实胶束包载两种药物，具有抗肿瘤协同作用。The micelle prepared by using the commercially available material PEG-PCL in the present invention is ideal in particle size, light stability, release of the micelle and its killing effect on tumor cells, and has a great application prospect in tumor treatment. In addition, the mechanism of anti-tumor effect was investigated at the cellular level, and it was confirmed from different aspects such as photothermal, singlet oxygen, cytotoxicity, intracellular drug distribution, etc.
有益效果beneficial effect
本发明制备的胶束纳米粒肿瘤细胞摄取量高，对肿瘤细胞的杀伤作用大，在近红外光照条件下联合化疗/光动力治疗具有协同作用，光动力治疗损伤细胞并增强了化疗效果，展示了兼具化疗和光动力治疗的高效低毒抗肿瘤作用，是安全有效的纳米新制剂。The micellar nanoparticle prepared by the invention has high uptake by tumor cells, has a large killing effect on tumor cells, and has a synergistic effect when combined with chemotherapy/photodynamic therapy under the condition of near-infrared light. Photodynamic therapy damages cells and enhances the effect of chemotherapy. It has high-efficiency, low-toxicity and anti-tumor effects with both chemotherapy and photodynamic therapy, and is a safe and effective new nano preparation.
附图说明Description of drawings
图1纳米粒的形态表征图：1-A胶束的透射电镜图；1-B胶束的动态粒径及其分布图；1-C胶束的Zeta电位图。Figure 1. Morphological characterization of nanoparticles: TEM image of 1-A micelles; dynamic particle size and distribution of 1-B micelles; Zeta potential map of 1-C micelles.
图2胶束的光照稳定性图。Figure 2 Light stability graph of micelles.
图3胶束的药物累计释放量图。Figure 3. The cumulative drug release profile of micelles.
图4乳腺癌细胞4T1对胶束的摄取量图。Figure 4. Uptake of micelles by breast cancer cells 4T1.
图5胶束对4T1细胞的细胞毒性结果图。Figure 5. Results of cytotoxicity of micelles on 4T1 cells.
图6胶束光照促进细胞内产生活性氧荧光图。Figure 6. Fluorescence image of micellar light for promoting the generation of reactive oxygen species in cells.
图7胶束光照促进细胞溶酶体膜破坏图。Figure 7. Micellar light irradiation promotes cell lysosomal membrane destruction.
图8胶束光照前后药物入核情况荧光图。Figure 8. Fluorescence images of drug entry into the nucleus before and after micellar irradiation.
本发明的实施方式Embodiments of the present invention
下面结合附图和实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不局限于此。本发明载化疗药物/光敏剂的胶束纳米粒，简称“胶束”。本发明的载体为PEG-PCL，具体为PEG 115- b-PCL 60，市售或者根据现有常规方法制备，比如“用于肿瘤成像与治疗的环境响应性纳米粒的研究”一文；超声装置为KQ-100DE，昆山市超声仪器有限公司，实施例超声功率为100W。 The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and embodiments. The following examples illustrate the present invention, but are not limited thereto. The micellar nanoparticles carrying chemotherapeutic drugs/photosensitizers of the present invention are referred to as "micelles" for short. The carrier of the present invention is PEG-PCL, specifically PEG 115 - b -PCL 60 , which is commercially available or prepared according to existing conventional methods, such as the article "Research on Environmentally Responsive Nanoparticles for Tumor Imaging and Treatment"; ultrasonic device It is KQ-100DE, Kunshan Ultrasonic Instrument Co., Ltd. The ultrasonic power of the embodiment is 100W.
实施例一：称取8 mg PEG 115- b-PCL 60溶于200 mL DMSO中，超声5分钟得到聚合物溶液；称取ICG 1 mg，加200 mL DMSO，超声5分钟溶解得光敏剂溶液；另称取1 mg DOX·HCl溶于200 mL DMSO中，加3 mL三乙胺，超声5分钟得到脱盐阿霉素溶液。 Example 1: Weigh 8 mg of PEG 115 - b -PCL 60 and dissolve it in 200 mL of DMSO, and ultrasonically for 5 minutes to obtain a polymer solution; weigh 1 mg of ICG, add 200 mL of DMSO, and ultrasonically dissolve for 5 minutes to obtain a photosensitizer solution; Another 1 mg of DOX·HCl was weighed and dissolved in 200 mL of DMSO, 3 mL of triethylamine was added, and sonicated for 5 minutes to obtain a desalted doxorubicin solution.
胶束的制备：采用液中分散法制备胶束。将上述三份溶液混合，继续超声5 min得均匀的溶液A。另取6 mL去离子水，边超声边将溶液A逐滴加到水中（每秒2滴），滴加完成，继续超声10分钟得胶束初溶液B，整个操作过程为室温（25 ℃）。将溶液B透析（透析袋拦截分子量为MWCO：3500 Da）过夜，每隔2 h换一次去离子水进行透析，透析完成后置于超滤离心管中，以5000 r·min -1的转速超滤离心10分钟，得到ICG及DOX共包载的胶束溶液ID-M，为载化疗药物/光敏剂的纳米胶束溶液，放置48小时，未见混浊。 Preparation of micelles: The micelles were prepared by liquid dispersion method. Mix the above three solutions and continue to sonicate for 5 min to obtain a homogeneous solution A. Another 6 mL of deionized water was taken, and solution A was added dropwise to the water (2 drops per second) while sonicating. The dropwise addition was completed, and the initial micellar solution B was obtained by continuing to sonicate for 10 minutes. The entire operation was at room temperature (25 °C). . The solution B was dialyzed (the intercepted molecular weight of the dialysis bag was MWCO: 3500 Da) overnight, and the deionized water was changed every 2 h for dialysis . After filtration and centrifugation for 10 minutes, the micelle solution ID-M co-encapsulated with ICG and DOX was obtained, which was a nanomicelle solution loaded with chemotherapeutic drugs/photosensitizers, and was placed for 48 hours without turbidity.
根据常规方法计算载药量与包封率，比如载药量(drug loading, DL)依据公式DL%=(游离药物的质量+包裹药物的质量)/(游离药物的质量+包裹药物的质量+载体的质量)x100%进行计算，包封率(entrappment efficiency, EE)依据公式EE%=(包载药物的质量)/(游离药物的质量+包载药物的质量)x100%计算。包封率分别为：ICG 96.1±1.4%，DOX 93.9±3.2%，载药量分别为：ICG 17.5±2.4%，DOX 9.1±1.8%。The drug loading and encapsulation efficiency are calculated according to conventional methods, such as drug loading (DL) according to the formula DL%=(mass of free drug + mass of encapsulated drug)/(mass of free drug + mass of encapsulated drug + The mass of the carrier)×100% is calculated, and the encapsulation efficiency (entrappment efficiency, EE) is calculated according to the formula EE%=(the quality of the encapsulated drug)/(the mass of the free drug+the mass of the encapsulated drug)×100%. The encapsulation efficiencies were: ICG 96.1±1.4%, DOX 93.9±3.2%, and the drug loadings were: ICG 17.5±2.4%, DOX 9.1±1.8%.
另外，上述方法中不加ICG、不加DOX或者不加ICG/DOX，同法制备得到单包载ICG胶束溶液(I-M)、单包载DOX胶束溶液(D-M)及空白胶束溶液(B-M)。In addition, no ICG, no DOX or no ICG/DOX was added in the above method, and the single-encapsulated ICG micelle solution (I-M), the single-encapsulated DOX micelle solution (D-M) and the blank micelle solution ( B-M).
胶束的形态表征：（ 1 ）胶束的透射电镜表征：取20 μL胶束溶液ID-M滴到铜网碳支持膜上，放入干燥器中挥干水分后用120 kV透射电镜（TEM）观察形态。结果如图1-A。结果表明，制备的纳米粒为规整的圆形，粒径为41.0±5.9 nm。 Morphological characterization of micelles: ( 1 ) Transmission electron microscope characterization of micelles: Take 20 μL of micelle solution ID-M and drop it on the copper mesh carbon support film, put it in a desiccator to evaporate the water, and use 120 kV transmission electron microscope (TEM) ) to observe the shape. The results are shown in Figure 1-A. The results showed that the prepared nanoparticles were regular circles with a particle size of 41.0±5.9 nm.
（ 2 ）胶束的粒径及其分布表征：取新制胶束溶液ID-M，常规稀释后，超声10 min混匀，取1 mL采用激光散射粒径仪对其粒径及分布进行分析。结果如图1-B。结果表明制得的胶束为单峰分布，平均水合粒径为80.4±14.3 nm（此为包含表面水化层的粒径），多分散系数（PDI）为0.252±0.015，有利于避免内皮网状细胞拦截，形成体内长循环，发挥被动靶向作用；如图1-C，结果表明胶束带有负电荷，Zeta电位为-17.23±0.66 mV。有利于胶束在体内的循环，不易被清除。 ( 2 ) Characterization of the particle size and distribution of micelles: Take the newly prepared micelle solution ID-M, after routine dilution, ultrasonically mix for 10 minutes, and take 1 mL to analyze its particle size and distribution with a laser scattering particle size analyzer. The results are shown in Figure 1-B. The results show that the prepared micelles have a unimodal distribution, the average hydrated particle size is 80.4±14.3 nm (this is the particle size including the surface hydration layer), and the polydispersity coefficient (PDI) is 0.252±0.015, which is beneficial to avoid endothelial reticulum The micelles were intercepted to form a long circulation in vivo and played a passive targeting role; as shown in Figure 1-C, the results showed that the micelles were negatively charged, and the Zeta potential was -17.23±0.66 mV. It is beneficial to the circulation of micelles in the body and is not easy to be removed.
胶束的光热稳定性考察：取ICG浓度为10 mg×mL -1的ID-M胶束溶液0.5 mL，以785 nm激光器（1.5 W×cm -2）光照5 min，每30 s记录一次溶液温度。关闭激光器，待溶液冷却到室温后，以相同条件再次照射5 min，如此光照去光照反复5次。结果如图2，前三次升温稳定达到36.5±1.1 oC（温差：11.5±1.1 oC），光热稳定性良好。 Study on the photothermal stability of micelles: Take 0.5 mL of ID-M micelle solution with an ICG concentration of 10 mg×mL -1 and irradiate it with a 785 nm laser (1.5 W×cm -2 ) for 5 min, and record every 30 s solution temperature. The laser was turned off, and after the solution was cooled to room temperature, it was irradiated again for 5 min under the same conditions, and the irradiation and de-illumination were repeated 5 times. The results are shown in Figure 2. The first three temperature rises were stable at 36.5±1.1 o C (temperature difference: 11.5±1.1 o C), and the photothermal stability was good.
胶束的体外释放行为考察：用透析法考察。分别将80 mg×mL -1DOX浓度的ID-M、游离 I/D溶液置于透析袋（MWCO：3500 Da）后，放入模拟生理环境和肿瘤细胞溶酶体内环境的pH 7.4 PBS、pH 5.0 的PBS中，在37 oC、120 r×min -1的恒温震荡仪中透析。分别于不同时间点取释放液，并补充等体积新鲜缓冲液，用荧光法检测缓冲液中DOX的含量，计算累积释放量、考察释放规律。游离ICG/DOX（游离 I/D）是先将两者混溶于一定体积的DMSO中，在超声条件下注入去离子水形成澄清的体系，DMSO在水中的含量为5%。 Investigation of the release behavior of micelles in vitro: investigation by dialysis. The ID-M and free I/D solutions with a concentration of 80 mg×mL -1 DOX were placed in a dialysis bag (MWCO: 3500 Da), and then placed in pH 7.4 PBS, pH 7.4 simulating the physiological environment and the tumor cell lysosomal environment. 5.0 PBS, dialyzed in a constant temperature shaker at 37 o C, 120 r×min -1 . The release solution was taken at different time points and supplemented with an equal volume of fresh buffer solution, the content of DOX in the buffer solution was detected by fluorescence method, the cumulative release amount was calculated, and the release law was investigated. Free ICG/DOX (free I/D) is firstly mixed with a certain volume of DMSO and injected into deionized water under ultrasonic conditions to form a clear system. The content of DMSO in water is 5%.
图3为累积释放曲线图。表1为分别采用零级方程、一级方程、Higuchi方程和Weibull方程，对DOX累计释放量相关数据进行拟合，得到ID-M、游离I/D分别在pH 5.0 PBS、pH 7.4 PBS中的释放行为结果：游离（Free） I/D在pH 7.4 PBS中24 h的累计释放量为74.8±7.0 μg，pH 5.0 PBS中24 h的累计释放量为77.3±2.6 μg；ID-M在pH 7.4 PBS中24 h的累计释放量为23.1±5.2μg，pH 5.0 PBS中24 h的累计释放量为47.5±4.7 μg。胶束药物和游离药物的释放规律拟合后都符合Weibull方程，药物经胶束包载后缓释作用显著，在模拟正常生理环境的pH 7.4 PBS中的释放非常缓慢，而模拟肿瘤细胞溶酶体内环境pH 5.0 PBS使释放加快，T 1/2从105.36 h缩短至22.85 h，缩短了78.3%。说明ID-M可将DOX靶向输送至肿瘤细胞溶酶体处释放，并发挥作用，而在达到靶部位前药物泄漏少。 Figure 3 is a graph of cumulative release profiles. Table 1 is the zero-order equation, the first-order equation, the Higuchi equation and the Weibull equation, respectively, to fit the data related to the cumulative release of DOX, and obtain the ID-M and free I/D in pH 5.0 PBS and pH 7.4 PBS, respectively. Release behavior results: The cumulative release of free (Free) I/D in pH 7.4 PBS for 24 h was 74.8±7.0 μg, and in pH 5.0 PBS for 24 h, the cumulative release was 77.3±2.6 μg; ID-M in pH 7.4 The cumulative release in PBS for 24 h was 23.1±5.2 μg, and the cumulative release in pH 5.0 PBS for 24 h was 47.5±4.7 μg. After fitting, the release laws of both micellar drug and free drug conform to the Weibull equation. After the drug is encapsulated in micelles, the sustained release effect is remarkable. In vivo pH 5.0 PBS accelerated the release, and T 1/2 shortened from 105.36 h to 22.85 h, a shortening of 78.3%. It shows that ID-M can deliver DOX to tumor cell lysosomes for release and play a role, and the drug leaks less before reaching the target site.
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细胞摄取量的考察：取对数生长的4T1细胞接种于6孔板中（细胞密度 5´10 5个/孔），置于细胞培养箱中（37 oC，5% CO 2）孵育24 h后，分别加入ICG浓度为8 mg×mL -1的ID-M、对照用游离ICG+DOX混合物（Free I/D），孵育24 h并用PBS清洗细胞后，将细胞吹打分散在PBS中并计数；超声（500 W，120 s）粉碎细胞，取细胞粉碎液加入DMSO，细胞摄入的ICG和DOX的浓度分别用紫外分光光度计和荧光分光光度计来检测。图4显示药物经胶束包载后的ID-M，可使细胞摄取ICG的量为1.72±0.02 μg/10 6个细胞，摄取DOX的量为1.38±0.11 μg/10 6个细胞，显著高于游离ICG摄取量0.82±0.02 μg/10 6个细胞，游离DOX的量0.54±0.03μg/10 6个细胞；DOX的摄取量增加了2.0倍，ICG的摄取量增加了1.6倍。而且细胞摄取量ID-M组与D-M组、I-M组的对应药物几乎一致，ID-M保持了各单包载胶束的优势。 Investigation of cell uptake: 4T1 cells with logarithmic growth were seeded in 6-well plates (cell density 5´10 5 cells/well), placed in a cell culture incubator (37 o C, 5% CO 2 ) and incubated for 24 h Then, ID-M with an ICG concentration of 8 mg×mL -1 was added, and a free ICG+DOX mixture (Free I/D) was added for control, incubated for 24 h, and the cells were washed with PBS. The cells were pipetted and dispersed in PBS and counted. ; Ultrasound (500 W, 120 s) to crush cells, take the cell crushing solution and add DMSO, and the concentrations of ICG and DOX taken up by cells were detected by UV spectrophotometer and fluorescence spectrophotometer, respectively. Figure 4 shows that ID-M after the drug was encapsulated in micelles can make the amount of ICG uptake by cells to be 1.72±0.02 μg/10 6 cells, and the amount of DOX uptake is 1.38 ± 0.11 μg/10 6 cells, which is significantly higher When the free ICG uptake was 0.82±0.02 μg/10 6 cells, the free DOX amount was 0.54±0.03 μg/10 6 cells; the uptake of DOX was increased by 2.0 times, and the uptake of ICG was increased by 1.6 times. Moreover, the cellular uptake of the corresponding drugs in the ID-M group was almost the same as that of the DM group and the IM group, and the ID-M maintained the advantages of each single-encapsulated micelle.
胶束的细胞毒性考察：为考察胶束对肿瘤细胞的毒性 ，取对数生长的4T1细胞接种于96孔板中（细胞密度5´10 4个×mL -1）孵育37 oC，5% CO 2，加入不同浓度的I/D-M、Free I/D，给药24 h后，更换培养基，光照组用785 nm激光器（1.5 W×cm -2、3 min）。继续孵育24 h后，更换培养基，加入20 mL 5 mg×mL -1 MTT溶液，孵育4 h。吸出孔板中的液体，加入DMSO，振荡混匀，用酶标仪检测490 nm处的吸光度值（Abs）。计算细胞存活率（%）= Abs 实验组/Abs 对照组´100%；将各组细胞存活率及给药浓度输入IC 50计算器，计算得到IC 50值；并且，计算协同指数CI，CI=+，当CI < 0.8，协同治疗效果显著。 Cytotoxicity study of micelles: To investigate the toxicity of micelles to tumor cells , logarithmically growing 4T1 cells were seeded in 96-well plates (cell density 5´10 4 ×mL -1 ) and incubated at 37 o C, 5% CO 2 , different concentrations of I/DM and Free I/D were added. After 24 hours of administration, the medium was changed. The light group was treated with a 785 nm laser (1.5 W×cm -2 , 3 min). After continuing to incubate for 24 h, the medium was replaced, 20 mL of 5 mg×mL -1 MTT solution was added, and the cells were incubated for 4 h. Aspirate the liquid in the well plate, add DMSO, shake and mix, and use a microplate reader to detect the absorbance value (Abs) at 490 nm. Calculate the cell survival rate (%) = Abs experimental group /Abs control group´100%; input the cell survival rate and drug concentration of each group into the IC 50 calculator, and calculate the IC 50 value; and calculate the synergy index CI, CI = +, when CI < 0.8, the synergistic treatment effect is significant.
从图5的细胞存活率可知，空白胶束对细胞几乎没有毒性。通过计算，得到结果为，光照前，只有DOX化疗作用，ID-M组高浓度下细胞存活率为24.8±2.2 mg×mL -1，Free I/D的细胞存活率为34.5±8.5 mg×mL -1，计算得到IC 50（mg×mL -1）：I-M在给药浓度范围内几乎没有毒性<<Free I/D组3.02<<D-M组1.44≈ID-M组1.35。光照后，可兼有DOX化疗和ICG光毒性作用，IC 50（mg×mL -1）：I-M组4.72<<D-M组1.83<Free I/D组1.21<<ID-M组0.72。光照后ID-M的细胞毒性显著增加，高浓度下细胞存活率为3.6±0.3 mg×mL -1（IC 50降低了46.67%），Free I/D的细胞存活率为15.7±0.2 mg×mL -1。同时，ID-M的细胞毒性经统计学考察，非光照下除与I-M有差异外，与其余各组均无显著性差异（P>0.05）。但光照下ID-M（>1 mg×mL -1）与各组均有极为显著的差异（P<0.01），抗肿瘤作用增效显著。并且，计算得到ID-M的协同指数CI为0.62，符合文献对CI<0.8即有显著协同治疗效果的要求，说明本文联合两种治疗手段，ICG和DOX共包载ID-M具有显著的抗肿瘤协同作用。 It can be seen from the cell viability in Figure 5 that the blank micelles have little toxicity to cells. By calculation, the result is that, before light irradiation, only DOX chemotherapy has the effect, the cell viability rate of ID-M group at high concentration is 24.8±2.2 mg×mL -1 , and the cell viability rate of Free I/D is 34.5±8.5 mg×mL -1 , calculated IC 50 (mg×mL -1 ): IM has almost no toxicity in the administration concentration range<<Free I/D group 3.02<<DM group 1.44≈ID-M group 1.35. After irradiation, DOX chemotherapy and ICG phototoxicity can be combined, IC 50 (mg×mL -1 ): IM group 4.72<<DM group 1.83<Free I/D group 1.21<<ID-M group 0.72. The cytotoxicity of ID-M was significantly increased after light exposure, the cell viability was 3.6±0.3 mg×mL -1 at high concentration (IC 50 decreased by 46.67%), and the cell viability of Free I/D was 15.7±0.2 mg×mL -1 . At the same time, the cytotoxicity of ID-M was statistically investigated, except that it was different from IM under non-illumination, there was no significant difference with other groups (P>0.05). However, under light irradiation, ID-M (>1 mg×mL -1 ) was significantly different from each group (P<0.01), and the anti-tumor effect was significantly enhanced. Moreover, the calculated synergy index CI of ID-M is 0.62, which meets the requirement of the literature that CI<0.8 has a significant synergistic therapeutic effect. tumor synergy.
促进细胞内产生活性氧的考察：将对数生长期的4T1细胞接种于12孔板中，加入用培养基稀释的ICG浓度为4 mg×mL -1的I-D、D-M、ID-M、Free I/D，孵育24 h，用无血清培养基洗三次，光照组（785 nm，1.5 W×cm -2）与非光照组一起加入DHE孵育30 min。用荧光显微镜观察红色荧光。ICG促进产生活性氧（ROS）是其发挥杀伤细胞作用的重要指标。以荧光探针二氢乙啶DHE检测ROS，利用没有荧光的DHE可在细胞内能被ROS氧化成氧化乙啶，氧化乙啶掺入DNA中产生红色荧光，用荧光显微镜观察荧光强弱判断胞内ROS的量。如活性氧ROS荧光检测结果(图6)可见，荧光强度（a.u.），光照前：PBS组0.2=Free I/D组0.2=I-M组0.2<D-M组0.3=ID-M组0.3，各组几乎都只有非常微弱的荧光。光照后：PBS组0.4<D-M组0.5<<Free I/D组1.4<<I-M组2.4<ID-M组2.6。说明ID-M促进肿瘤细胞内产生ROS的能力最强，光毒性最大。 Investigation of promoting the production of reactive oxygen species in cells: 4T1 cells in logarithmic growth phase were seeded in 12-well plates, and ID, DM, ID-M, Free I diluted with culture medium were added at a concentration of 4 mg×mL -1 of ICG /D, incubated for 24 h, washed three times with serum-free medium, and incubated with DHE for 30 min in the light group (785 nm, 1.5 W×cm -2 ) and the non-light group. Red fluorescence was observed with a fluorescence microscope. ICG promotes the production of reactive oxygen species (ROS), which is an important indicator of its ability to kill cells. The fluorescent probe dihydroethidium DHE is used to detect ROS. The non-fluorescent DHE can be oxidized by ROS to ethidium oxide in cells, and ethidium oxide is incorporated into DNA to generate red fluorescence. amount of ROS. As can be seen from the reactive oxygen species ROS fluorescence detection results (Figure 6), the fluorescence intensity (au), before light: PBS group 0.2=Free I/D group 0.2=IM group 0.2<DM group 0.3=ID-M group 0.3, each group almost All have only very weak fluorescence. After illumination: PBS group 0.4<DM group 0.5<<Free I/D group 1.4<<IM group 2.4<ID-M group 2.6. This indicated that ID-M had the strongest ability to promote ROS production in tumor cells and had the greatest phototoxicity.
破坏细胞溶酶体膜促进药物进入细胞核的考察：用吖啶橙（AO）荧光探针法检测，将4T1细胞接种于24孔板中，分别加入用培养基稀释的ICG浓度为4 mg×mL -1的ID-M、Free I/D，I-M，D-M溶液孵育24 h后，光照组（785 nm，1.5 W×cm -2）与非光照组一起加入10 mg×mL -1的AO孵育30 min。用荧光显微镜观察孔板内细胞的不同荧光。吖啶橙（AO）荧光探针可透过活细胞膜，进入细胞内偏酸性的溶酶体中被质子化产生红色荧光，而在细胞核等偏中性的环境中则呈现绿色荧光。从图7荧光显微镜观察结果可知，光照前各组破膜率比较接近。光照后含有ICG的样品组都有红色荧光减弱，绿色荧光增强的现象。统计后发现，光照后30 min各组的溶酶体破膜率分别为：PBS组1.3% < D-M组5.3% < Free I/D组41.3% < I-M组100% = ID-M组100%。因此，光照后ICG产生ROS，促使溶酶体破膜，进而促进ID-M中的DOX进入细胞核发挥杀死肿瘤细胞的作用。 The investigation of destroying the cell lysosomal membrane to promote the drug entering the nucleus: using acridine orange (AO) fluorescent probe method for detection, 4T1 cells were seeded in 24-well plates, and ICG diluted with culture medium was added at a concentration of 4 mg×mL After 24 hours of incubation in ID-M, Free I/D, IM and DM solutions of -1 , the light group (785 nm, 1.5 W×cm -2 ) and the non-light group were added with 10 mg×mL -1 of AO to incubate for 30 hours. min. Fluorescence microscopy was used to observe the different fluorescence of cells in the well plate. The acridine orange (AO) fluorescent probe can penetrate the living cell membrane, enter the acidic lysosome in the cell, and be protonated to produce red fluorescence, while in the neutral environment such as the nucleus, it exhibits green fluorescence. It can be seen from the observation results of fluorescence microscope in Fig. 7 that the membrane rupture rate of each group is relatively close before illumination. After illumination, the red fluorescence was weakened and the green fluorescence was enhanced in the sample groups containing ICG. After statistics, it was found that the lysosomal membrane rupture rates of each group 30 minutes after light were as follows: PBS group 1.3% < DM group 5.3% < Free I/D group 41.3% < IM group 100% = ID-M group 100%. Therefore, after illumination, ICG generates ROS, which promotes the rupture of lysosomes, which in turn promotes the entry of DOX in ID-M into the nucleus to kill tumor cells.
光照促进化疗药物进入细胞核的协同作用考察：将4T1细胞接种于共聚焦培养皿中，加入用培养基稀释的ICG浓度为4 mg×mL -1的ID-M溶液，孵育6 h后，光照（785 nm，1.5 W×cm -2）后继续孵育6 h。与非光照组一起，用1 mg×mL -1 Hoechest 33342染核，孵育并用PBS洗三次，用50 mM的Lysotracker DND-26染溶酶体，孵育洗涤，加入1 mL培养基，用激光共聚焦显微镜观察共聚焦培养皿中的细胞状态。 Study on the synergistic effect of light on promoting the entry of chemotherapeutic drugs into the nucleus: 4T1 cells were seeded in a confocal petri dish, and ID-M solution with a concentration of 4 mg×mL -1 of ICG diluted with culture medium was added. After incubation for 6 h, light ( 785 nm, 1.5 W×cm -2 ) and continue to incubate for 6 h. Together with the non-light group, nuclei were stained with 1 mg × mL -1 Hoechest 33342, incubated and washed three times with PBS, lysosomes were stained with 50 mM Lysotracker DND-26, incubated and washed, added 1 mL of medium, and confocal with laser Microscopy to observe the state of cells in a confocal dish.
DOX可进入肿瘤细胞核抑制DNA与RNA的合成，以其进核量判断化疗作用。图8为ID-M中DOX进核结果。细胞核均被染成蓝色，溶酶体被染成绿色，DOX显红色荧光。细胞核中的DOX由红蓝荧光共定位率反映，溶酶体中的DOX由红绿荧光共定位率反映，余下的DOX则在胞浆中。红蓝共定位率高，说明DOX进入细胞核多，抗肿瘤效果好；红绿共定位率高，说明DOX在溶酶体中多，效果差。结果，考察共定位率发现，光照前，DOX有92.5%在溶酶体中，2.7%在细胞核中，4.8%在胞浆中；光照后不同时间，DOX从溶酶体逃逸进入细胞核的量逐渐提升，到光照后12 h占总量82.7%的DOX进入细胞核，使核内药量增加29.6倍。因此，ID-M的细胞毒性因光照而提高，与进入细胞核的药量增加有关，从而证实了ID-M高效抑制肿瘤细胞生长，存在化疗和光治疗的协同作用。DOX can enter the nucleus of tumor cells to inhibit the synthesis of DNA and RNA, and the effect of chemotherapy can be judged by the amount of DOX entering the nucleus. Figure 8 shows the results of DOX entry in ID-M. Nuclei were stained blue, lysosomes were stained green, and DOX showed red fluorescence. The DOX in the nucleus was reflected by the red-blue fluorescence co-localization rate, the DOX in the lysosome was reflected by the red-green fluorescence co-localization rate, and the remaining DOX was in the cytoplasm. The high co-localization rate of red and blue indicates that DOX enters the nucleus more and has good anti-tumor effect; the high co-localization rate of red and green indicates that DOX is abundant in lysosomes and has poor effect. As a result, investigating the co-localization rate, it was found that before light exposure, 92.5% of DOX was in the lysosome, 2.7% in the nucleus, and 4.8% in the cytoplasm. 82.7% of the total DOX entered the nucleus at 12 h after illumination, increasing the amount of the drug in the nucleus by 29.6 times. Therefore, the cytotoxicity of ID-M is enhanced by light exposure, which is related to the increase of the amount of drug entering the nucleus, thus confirming that ID-M effectively inhibits tumor cell growth, and there is a synergistic effect of chemotherapy and phototherapy.
对比例一：称取8 mg PEG 115- b-PCL 60溶于200 mL DMSO中，超声5分钟得到聚合物溶液；称取ICG 1 mg，加200 mL DMSO，超声5分钟溶解得光敏剂溶液；另称取1 mg DOX·HCl溶于200 mL DMSO中，加3 mL三乙胺，超声5分钟得到脱盐阿霉素溶液。 Comparative Example 1: Weigh 8 mg of PEG 115 - b -PCL 60 and dissolve it in 200 mL of DMSO, and ultrasonicate for 5 minutes to obtain a polymer solution; weigh 1 mg of ICG, add 200 mL of DMSO, and ultrasonically dissolve for 5 minutes to obtain a photosensitizer solution; Another 1 mg of DOX·HCl was weighed and dissolved in 200 mL of DMSO, 3 mL of triethylamine was added, and sonicated for 5 minutes to obtain a desalted doxorubicin solution.
（1）采用液中分散法制备胶束。将上述三份溶液混合，不进行超声直接滴入6 mL去离子水中，滴加在超声下进行（每秒2滴），滴加完成，继续超声10分钟得胶束初溶液B，整个操作过程为室温（25 ℃）。将溶液B透析（透析袋拦截分子量为MWCO：3500 Da）过夜，每隔2 h换一次去离子水进行透析，透析完成后置于超滤离心管中，以5000 r·min -1的转速超滤离心10分钟，上清颜色很浅，下层沉淀很多，颜色很深，表明药物析出，包载失败。 (1) The micelles were prepared by liquid dispersion method. Mix the above three solutions, drop directly into 6 mL of deionized water without ultrasonication, dropwise under ultrasonication (2 drops per second), complete the dropwise addition, and continue to ultrasonicate for 10 minutes to obtain initial micelle solution B. The entire operation process to room temperature (25°C). The solution B was dialyzed (the intercepted molecular weight of the dialysis bag was MWCO: 3500 Da) overnight, and the deionized water was changed every 2 h for dialysis . After filtration and centrifugation for 10 minutes, the color of the supernatant is very light, and the lower layer has a lot of sediment, and the color is very dark, indicating that the drug is precipitated and the encapsulation fails.
（2）采用液中分散法制备胶束。将上述三份溶液混合，继续超声5 min得均匀的溶液A。另取6 mL去离子水，边超声边将溶液A加入到水中（1秒内加完，非滴加），继续超声10分钟得胶束初溶液B，整个操作过程为室温（25 ℃）。将溶液B透析（透析袋拦截分子量为MWCO：3500 Da）过夜，每隔2 h换一次去离子水进行透析，透析完成后置于超滤离心管中，以5000 r·min -1的转速超滤离心10分钟，上清颜色很浅，下层沉淀很多，颜色很深，表明药物析出，包载失败。 (2) The micelles were prepared by liquid dispersion method. Mix the above three solutions and continue to sonicate for 5 min to obtain a homogeneous solution A. Take another 6 mL of deionized water, add solution A to the water while sonicating (complete addition within 1 second, not dropwise), and continue to sonicate for 10 minutes to obtain initial micelle solution B. The entire operation is at room temperature (25 °C). The solution B was dialyzed (the intercepted molecular weight of the dialysis bag was MWCO: 3500 Da) overnight, and the deionized water was changed every 2 h for dialysis . After filtration and centrifugation for 10 minutes, the color of the supernatant is very light, and the lower layer has a lot of sediment, and the color is very dark, indicating that the drug is precipitated and the encapsulation fails.
（3）采用液中分散法制备胶束。整个操作过程为室温（25 ℃），将上述三份溶液混合，继续超声5 min得均匀的溶液A。另取6 mL去离子水，边超声边将溶液A逐滴加到水中（每秒2滴），滴加完成不超声直接透析（透析袋拦截分子量为MWCO：3500 Da）过夜，每隔2 h换一次去离子水进行透析，透析完成后置于超滤离心管中，以5000 r·min -1的转速超滤离心10分钟，上清颜色很浅，下层沉淀很多，颜色很深，表明药物析出，包载失败。 (3) The micelles were prepared by liquid dispersion method. The whole operation process is room temperature (25 °C), the above three solutions are mixed, and the sonication is continued for 5 min to obtain a uniform solution A. Take another 6 mL of deionized water, add solution A dropwise to the water (2 drops per second) while ultrasonicating, and complete the direct dialysis without ultrasonication (dialysis bag intercepted molecular weight is MWCO: 3500 Da) overnight, every 2 h Change the deionized water once for dialysis, put it in an ultrafiltration centrifuge tube after dialysis, and centrifuge it with ultrafiltration at a speed of 5000 r·min -1 for 10 minutes. Precipitation, package loading failed.
相对而言，第（3）种对比方法的包载效果较（1）（2）对比方法要好，但是这三个对比方法都较本发明的载药效果差得多，本发明实施例一的方法，超滤后，下层沉淀非常少。Relatively speaking, the encapsulation effect of the comparative method (3) is better than that of the comparative method (1) and (2), but the three comparative methods are much worse than the drug-carrying effect of the present invention. method, after ultrafiltration, there is very little sediment in the lower layer.
对比例二：采用薄膜分散法也可以制备胶束ID-M，具体方案如下：将ICG、DOX以及PEG-PCL共同溶解在二氯甲烷中，超声混合在蒸馏烧瓶中，在旋转蒸发仪上旋干，后在超声条件下逐滴加入蒸馏水，将蒸馏瓶壁上的薄膜溶解，1500 r·min -1离心10 min后，过220 nm滤膜得到胶束溶液。此方法制备的胶束溶液粒径为35.1±2.8 nm，但易产生浑浊，放置10分钟，胶束溶液出现浑浊，说明不稳定。 Comparative example 2: Micellar ID-M can also be prepared by thin film dispersion method. The specific scheme is as follows: ICG, DOX and PEG-PCL are co-dissolved in dichloromethane, ultrasonically mixed in a distillation flask, and spin on a rotary evaporator. After drying, distilled water was added dropwise under ultrasonic conditions to dissolve the film on the wall of the distillation flask. After centrifugation at 1500 r·min -1 for 10 min, the micelle solution was obtained by passing through a 220 nm filter membrane. The particle size of the micellar solution prepared by this method is 35.1±2.8 nm, but it is prone to turbidity. After 10 minutes, the micellar solution appears turbid, indicating instability.
对比例三：将8 mg PEG 115- b-PCL 60、1 mg ICG、1 mg DOX·HCl溶于600 mL DMSO中，加3 mL三乙胺，超声5分钟得到均匀的溶液A。另取6 mL去离子水，边超声边将溶液A逐滴加到水中（每秒2滴），滴加完成，继续超声10分钟得胶束初溶液B，整个操作过程为室温（25 ℃）。将溶液B透析（透析袋拦截分子量为MWCO：3500 Da）过夜，每隔2 h换一次去离子水进行透析，透析完成后置于超滤离心管中，以5000 r·min -1的转速超滤离心10分钟，得到ICG及DOX共包载的胶束溶液ID-M，采用激光散射粒径仪对其粒径进行分析，平均水合粒径为212.4±12.7nm。 Comparative Example 3: Dissolve 8 mg of PEG 115 - b -PCL 60 , 1 mg of ICG, and 1 mg of DOX·HCl in 600 mL of DMSO, add 3 mL of triethylamine, and sonicate for 5 minutes to obtain a homogeneous solution A. Another 6 mL of deionized water was taken, and solution A was added dropwise to the water (2 drops per second) while sonicating. The dropwise addition was completed, and the initial micellar solution B was obtained by continuing to sonicate for 10 minutes. The entire operation was at room temperature (25 °C). . The solution B was dialyzed (the intercepted molecular weight of the dialysis bag was MWCO: 3500 Da) overnight, and the deionized water was changed every 2 h for dialysis . Filtration and centrifugation for 10 minutes to obtain the micellar solution ID-M co-encapsulated with ICG and DOX. The particle size was analyzed by a laser scattering particle size analyzer, and the average hydrated particle size was 212.4±12.7 nm.
      现有技术存在制备温度高、纳米药物粒径偏大的问题，为更好地实现光敏剂ICG和化疗药DOX的联合应用，本发明设计在不超过37 oC的室温条件下，制备粒径小于200nm的PEG-PCL包载ICG/DOX胶束，充分发挥两种治疗手段的协同作用，为进一步研发高效低毒新型制剂奠定基础。本发明涉及纳米药物技术，具体为包载化疗药物（如阿霉素）和光敏剂（如吲哚菁绿）的双重治疗效果的胶束纳米粒及其制备，并作为抗肿瘤新药剂型，增强药物对肿瘤的毒性和靶向性，发挥协同增效作用，降低全身毒副作用，从而实现肿瘤的联合治疗。 The existing technology has the problems of high preparation temperature and large particle size of nano-drugs. In order to better realize the combined application of photosensitizer ICG and chemotherapeutic drug DOX, the present invention is designed to prepare particle size at room temperature not exceeding 37 ° C. The PEG-PCL smaller than 200nm encapsulates ICG/DOX micelles, giving full play to the synergistic effect of the two treatments, laying the foundation for the further development of new formulations with high efficiency and low toxicity. The invention relates to nano-medicine technology, in particular to micellar nanoparticles encapsulating the dual therapeutic effects of chemotherapeutic drugs (such as doxorubicin) and photosensitizers (such as indocyanine green) and preparation thereof, and as a new anti-tumor dosage form, enhancing the The toxicity and targeting of the drug to the tumor can play a synergistic effect, reduce the systemic toxicity and side effects, so as to realize the combined treatment of the tumor.

Claims (10)

  1. 一种载化疗药物/光敏剂的纳米胶束，其特征在于，由聚合物包载化疗药物和光敏剂得到；所述载化疗药物/光敏剂的纳米胶束的水合粒径为10~160 nm。A chemotherapeutic drug/photosensitizer-carrying nanomicelle, characterized in that it is obtained by encapsulating a chemotherapeutic drug and a photosensitizer by a polymer; the hydrated particle size of the chemotherapeutic drug/photosensitizer-carrying nanomicelle is 10-160 nm.
  2. 根据权利要求1所述载化疗药物/光敏剂的纳米胶束，其特征在于，化疗药物为阿霉素，光敏剂为吲哚菁绿，聚合物为PEG-PCL。The chemotherapeutic drug/photosensitizer-loaded nanomicelle of claim 1, wherein the chemotherapeutic drug is doxorubicin, the photosensitizer is indocyanine green, and the polymer is PEG-PCL.
  3. 根据权利要求1所述载化疗药物/光敏剂的纳米胶束，其特征在于，将化疗药物溶液、光敏剂溶液、聚合物溶液混合，然后超声处理，得到溶液A；将溶液A滴加入水中，超声处理，得到溶液B；溶液B经过透析、超滤离心，得到载化疗药物/光敏剂的纳米胶束。The chemotherapeutic drug/photosensitizer-loaded nanomicelle of claim 1, wherein the chemotherapeutic drug solution, photosensitizer solution, and polymer solution are mixed, and then ultrasonically treated to obtain solution A; solution A is added dropwise to water, Ultrasonic treatment is performed to obtain solution B; solution B is subjected to dialysis, ultrafiltration and centrifugation to obtain nanomicelles loaded with chemotherapeutic drugs/photosensitizers.
  4. 权利要求1所述载化疗药物/光敏剂的纳米胶束在制备肿瘤治疗药物中的应用。The application of the chemotherapeutic drug/photosensitizer-loaded nanomicelle of claim 1 in the preparation of a tumor therapeutic drug.
  5. 权利要求1所述载化疗药物/光敏剂的纳米胶束的制备方法，其特征在于，包括以下步骤，将化疗药物溶液、光敏剂溶液、聚合物溶液混合，然后超声处理，得到溶液A；将溶液A滴加入水中，超声处理，得到溶液B；溶液B经过透析、超滤离心，得到载化疗药物/光敏剂的纳米胶束。The preparation method of the nanomicelle carrying chemotherapeutic drug/photosensitizer according to claim 1, it is characterized in that, comprises the following steps, mixing chemotherapeutic drug solution, photosensitizer solution, polymer solution, then ultrasonic treatment, obtain solution A; Solution A is added dropwise to water, and ultrasonically treated to obtain solution B; solution B is subjected to dialysis, ultrafiltration and centrifugation to obtain nanomicelles loaded with chemotherapeutic drugs/photosensitizers.
  6. 根据权利要求5所述载化疗药物/光敏剂的纳米胶束的制备方法，其特征在于，载化疗药物/光敏剂的纳米胶束的制备在室温下进行；光敏剂、化疗药物、聚合物的质量比为（0.1～2.0）∶（0.1～2.0）∶（0.8～16）。The method for preparing a chemotherapeutic drug/photosensitizer-loaded nanomicelle according to claim 5, wherein the chemotherapeutic drug/photosensitizer-loaded nanomicelle is prepared at room temperature; The mass ratio is (0.1-2.0): (0.1-2.0): (0.8-16).
  7. 根据权利要求5所述载化疗药物/光敏剂的纳米胶束的制备方法，其特征在于，溶液A、水的体积比为1∶5～15。The method for preparing a chemotherapeutic drug/photosensitizer-loaded nanomicelle according to claim 5, wherein the volume ratio of solution A and water is 1:5-15.
  8. 根据权利要求5所述载化疗药物/光敏剂的纳米胶束的制备方法，其特征在于，超声处理的时间为5～10分钟。The method for preparing a chemotherapeutic drug/photosensitizer-loaded nanomicelle according to claim 5, wherein the ultrasonic treatment time is 5-10 minutes.
  9. 根据权利要求5所述载化疗药物/光敏剂的纳米胶束的制备方法，其特征在于，超滤离心的转速为3500~6000 r·min -1。 The method for preparing a chemotherapeutic drug/photosensitizer-loaded nanomicelle according to claim 5, wherein the ultrafiltration centrifugation rotates at a speed of 3500-6000 r·min -1 .
  10. 根据权利要求5所述载化疗药物/光敏剂的纳米胶束的制备方法，其特征在于，化疗药物为阿霉素，光敏剂为吲哚菁绿，聚合物为PEG-PCL。The method for preparing a chemotherapeutic drug/photosensitizer-loaded nanomicelle according to claim 5, wherein the chemotherapeutic drug is doxorubicin, the photosensitizer is indocyanine green, and the polymer is PEG-PCL.
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