WO2022077830A1 - Use of artemisinin and derivative thereof in preparation of sensitizer for thermodynamic therapy - Google Patents

Abstract

A new use of artemisinin and a derivative thereof. The artemisinin or the derivative thereof is directly used as a sensitizer for thermodynamic therapy, and is used for thermodynamic therapy of tumors; or a liposome nanocomposite prepared from the artemisinin or the derivative thereof, and indocyanine green is used as a sensitizer for thermodynamic therapy or a photothermal agent for photothermal therapy, and is used for thermodynamic therapy and/or photothermal therapy of tumors. The artemisinin or the derivative thereof and the liposome nanocomposite show an excellent tumor-targeting capability and anti-tumor activity of synergistic photothermal therapy and thermodynamic therapy.

Description

Application of artemisinin and its derivatives in the preparation of thermodynamic therapy sensitizers

This application requires a Chinese patent application with the application number CN202011111545.1 and the invention titled "Application of artemisinin and its derivatives in the preparation of thermodynamic therapy sensitizers" to be submitted to the China Patent Office on October 16, 2020. Priority, the entire contents of which are incorporated herein by reference.

technical field

The invention belongs to the field of functional materials and medicines, and particularly relates to the application of artemisinin derivatives, artemisinin derivatives and indocyanine green liposome nanomaterials in the preparation of tumor thermodynamic therapy sensitizers.

Background technique

Photodynamic therapy (PDT) is a treatment for cancer that uses light to excite photosensitizers, which then undergo a photochemical reaction in the presence of oxygen to generate reactive oxygen species (ROS) that induce cancer cells and tissue damage. PDT has the advantages of being minimally invasive and non-drug-resistant, and thus has been widely developed, but the therapeutic effect of PDT on hypoxic tumors is limited. Recently, a new approach, thermodynamic therapy (TDT), has emerged. It uses heat as an energy source to activate sensitizers, which then generate ROS or other free radicals for cancer treatment. Compared to light, TDT can provide heat in more ways, such as chemical reactions, light, ultrasound, radiation and microwaves. Therefore, TDT is a novel and promising cancer therapy. In addition, in TDT, active species can be directly obtained from thermodynamic sensitizers after heating without oxygen dependence, so TDT can overcome the deficiency of PDT's limited efficacy on hypoxic tumors. However, related reports are still rare, and a few thermally labile azobisisobutyronitrile derivatives can decompose after thermal activation and lead to the generation of free radicals, thus serving as chemothermographic agents for cancer therapy.

Indocyanine green (ICG) is currently the only near-infrared imaging reagent approved by the U.S. Food and Drug Administration (FDA) for clinical use. ICG is a three-carbocyanine dye with a maximum emission wavelength between 795 and 845 nm. It is a drug molecule with an amphiphilic structure. ICG can absorb light energy and convert it into heat energy or generate singlet oxygen, which can be used for photothermal therapy (PTT) or photodynamic therapy (PDT). However, it is easily decomposed in the light environment, which brings difficulties to the preservation and subsequent application of the drug. In addition, ICG is unstable in aqueous solution, and has a fast clearance rate in biological tissues, such as plasma (with a half-life of 2-4 min), which limits its application in diagnosis and treatment. The modification and transformation through nanometers can effectively improve its photostability and thermal stability, as well as its water stability.

Artemisinin, a sesquiterpene lactone containing an endoperoxide bridge, is a well-known antimalarial drug. As an antimalarial drug, artemisinin has shown high safety. Due to its unique structure, artemisinin and its derivatives have various other uses besides antimalarial. Recently, it has been reported that the peroxide bridge of artemisinin peroxide can be activated by Fe ^ions to generate reactive free radicals for cancer therapy. Unlike the ROS generated during PDT, the ROS generated by artemisinin does not depend on the oxygen content of the surrounding environment, which is particularly beneficial for the treatment of hypoxic tumor tissues. It is well known that the storage of Fe ²⁺ ions in blood is relatively high due to the presence of hemoglobin. However, it did not show a specific distribution in tumor tissue, and the Fe ²⁺ ion level in tumor tissue was too low to activate artemisinin, thus limiting the development of artemisinin in clinical application. At present, there is no report on the use of artemisinin's thermal sensitivity to generate active substances for tumor therapy. The use of artesunate and indocyanine green entrapped in liposomes to prepare nanomaterials for photothermal therapy and thermodynamic therapy of tumors has not yet been reported.

SUMMARY OF THE INVENTION

The object of the present invention is to provide an application of artemisinin and its derivatives in the preparation of tumor thermodynamic therapy sensitizers, which not only have extremely high anti-tumor activity of synergistic photothermal therapy and thermodynamic therapy, but also show It has excellent tumor targeting and has significant advantages as an anticancer drug.

To achieve the above object, the present invention adopts the following technical solutions:

An application of artemisinin and its derivatives in the preparation of a sensitizer for tumor thermodynamic therapy. Or its derivatives are directly used as thermodynamic therapy sensitizers for thermodynamic therapy of tumors; or artemisinin or its derivatives and indocyanine green are made into liposome nanocomplexes as thermodynamic therapy sensitizers Photothermal agents or photothermal agents for thermodynamic therapy and/or photothermal therapy of tumors;

Described artemisinin derivative comprises any one in artemether, dihydroartemisinin and artesunate;

The thermal effect condition is to raise the temperature to ≥48°C by direct or indirect heating technology; the heating technology includes heating, laser, ultrasound or microwave.

Specifically, when artesunate is used as an artemisinin derivative, the artesunate and indocyanine green are encapsulated in a lipid bilayer to form a liposome nanocomplex of microvesicles. The preparation method includes the following steps:

1) Dipalmitoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol 2000, cholesterol hemisuccinate, artesunate and indocyanine green in a molar ratio of 12:1.5:9:3 : 13 was added to the mixed solution of chloroform and methanol (the volume ratio of chloroform and methanol was 1:1), so that it was completely dissolved, and then ultrasonically treated for 1 to 5 min;

2) After ultrasonication, the solution is rotated and evaporated to dryness, and then deionized (DI) water is used to peel off the wall to form a liposome suspension;

3) Use an ultrasonic cell crusher to pulverize the liposome suspension at 0 to 20 °C for 5 to 10 minutes, and then dialyze the liposome suspension with deionized water at 4 to 25 °C for 12 to 48 hours (molecular weight cut-off is 10000) to remove Free indocyanine green and artesunate are used to obtain the liposome nanocomposite with an average particle size of 100-200 nm.

The obtained indocyanine green-artesunate liposome nanocomposite is used as a sensitizer or a photothermal agent in antitumor therapy with a laser wavelength of 808 nm and an intensity of 0.3-0.8 W·cm ^-2 .

The beneficial effects and outstanding advantages of the present invention are:

(1) Artemisinin derivatives are the main drugs for the clinical treatment of malaria with high safety, and their human safety and pharmacokinetic properties have been widely evaluated. The invention provides a new use of artemisinin derivatives, that is, as a sensitizer or a photothermal agent (wherein, artesunate has significant thermosensitive activity), because the drug is a relatively safe clinical application Therefore, it is more conducive to clinical use and promotion.

(2) Indocyanine green is a reagent approved by the US Food and Drug Administration (FDA) for clinical use, with high safety and strong photodynamic and photothermal therapeutic effects. The present invention also provides a liposome nanocomposite composed of an artemisinin derivative and indocyanine green, which directly utilizes the photothermal effect of indocyanine green to provide an in-situ heat source for artesunate without an external heat source, The overall design is clever.

(3) Indocyanine green-artesunate liposome nanocomposite provided by the present invention can produce good photothermal effect under 808nm laser irradiation, and artesunate can produce significant reactive oxygen species Therefore, the nanomaterial has good photothermal and thermodynamic effects at the same time, and can be used as a new anti-tumor drug for thermodynamic therapy and photothermal therapy, with good biocompatibility and high human safety. With excellent biocompatibility (degradability, non-toxicity, non-immunogenicity), liposome preparations of various drugs have been marketed at home and abroad.

(4) The indocyanine green-artesunate liposome nanocomposite provided by the present invention has excellent tumor targeting. After intravenous administration to the tumor of tumor-bearing mice, obvious fluorescence signals and photoacoustic signals were only observed in the tumor part of the mice. Therefore, this liposome nanocomposite can also be used for multimodal diagnosis of tumors.

(5) The indocyanine green-artesunate liposome nanocomposite provided by the present invention has excellent photothermal and thermodynamic synergistic antitumor effects. The tumor basically disappeared on the 10th day of treatment, the tumor inhibition rate reached 100%, and no recurrence occurred until the 14th day. It is indicated that the liposome nanocomposite has excellent anti-tumor efficacy and is a promising anti-tumor drug.

Instruction drawings

Figure 1 is the fluorescence spectrum of DCFH under 808 nm laser (intensity 0.3 W·cm ^-2 , 10 min) in mixed solutions of ARS and ICG with different ratios (ICG fixed at 100 μM).

Figure 2 is a comparison chart of the cytotoxicity of human hepatoma cell HepG2 treated with different nano-drugs under laser irradiation (wherein ICG@NPs, ICG-ARS@NPs are based on the content of ICG, ARS@NPs are based on the content of ARS content as an indicator).

Figure 3 is a graph comparing tumor growth curves of tumor-bearing H22 mice with different treatments within 14 days.

Detailed ways

In order to make the content of the present invention easier to understand, the technical solutions of the present invention will be further described below with reference to specific embodiments, but the present invention is not limited thereto.

Indocyanine green, artemisinin derivatives and raw materials for preparing liposomes used in the examples are all commercially available.

Example 1 Artemisinin derivatives as thermodynamic therapy sensitizers

The 2',7'-dichlorofluorescein diacetate (referred to as DCFH-DA) hydrolyzate 2',7'-dichlorofluorescein diacetic acid (referred to as DCFH) was used as a probe for reactive oxygen species, and the probe excitation wavelength was 488nm, the emission wavelength detection range is 500-600nm. The preparation of the hydrolyzed aqueous solution of DCFH-DA was carried out with reference to existing papers ("J. Immunol. Methods" 1993, 159(1-2), 131-138). By observing the fluorescence of DCFH (at around 524 nm) in the solution containing the sample to be tested with the laser irradiation time, the ability of the sample to generate ROS from heat is determined. The stronger the fluorescence intensity of DCFH, the stronger the ability of heat to generate ROS.

Taking artesunate (ARS) as an example, the specific determination method is to weigh an appropriate amount of artesunate and dissolve it in DMF to obtain a 2mM mother solution. During the test, add 100 μL of 2',7'-dichlorofluoro yellow diacetic acid and 100 μL of artesunate to 1.8 mL of deionized water to obtain a mixed solution (wherein the final concentration of artesunate is 100 μM, 2',7 '-dichlorofluorescein diacetic acid with a final concentration of 10 μM), and then heated the solution in a water bath at 60 °C for 30 min, taking it out every 5 min during this period to test its fluorescence intensity at 500-600 nm, and explore artesunate according to the fluorescence intensity The ability to generate reactive oxygen species when heated determines its thermal sensitization performance.

Using the same method, reactive oxygen species production of dihydroartemisinin, artemether and artemisinin was measured.

Through the analysis results, it was found that, compared with the fluorescence intensity of the simple probe control group, the fluorescence intensity of the artesunate group was 6.475 times that of the control group, the dihydroartemisinin group was 2.813 times that of the control group, and the artesunate group was 2.813 times that of the control group. 0.773 times that of the control group, and 1.164 times that of the control group in the artemisinin group. From the above test results, it is found that artesunate and dihydroartemisinin have obvious heat-sensitizing effects, and can be used for the preparation and research of heat-sensitive nanomedicines.

Example 2

Through the investigation of literature and experiments, it is found that when the concentration of indocyanine green reaches 100 μM, the aqueous solution in which it is located can be heated to 50 °C, which can meet the requirements of artesunate to generate active oxygen when heated. Then, by exploring artesunate and indocyanine It is found that the ratio of artesunate and indocyanine green will show different properties within a certain range. As shown in Figure 1, the molar ratio of artesunate and indocyanine green is 1:1 When the molar ratio of artesunate and indocyanine green is 2:1, the ability to generate reactive oxygen species is the strongest and the intensity is equivalent. Therefore, indocyanine green is selected as the laser irradiation drug to generate heat, and artesunate and indole are used to generate heat. The molar ratio of cyanine green is 1:1 as the optimal drug ratio form.

Example 3 Liposome nanomedicines loaded with indocyanine green and artesunate

Weigh dipalmitoyl phosphatidyl choline, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, cholesterol hemisuccinate, artesunate and indole, respectively, in a molar ratio of 12:1.5:9:3:13 Cyanine green was added with chloroform (10 mL) and methanol (10 mL), dissolved in the mixed solvent, and then sonicated for 5 min. The solution was then rotary evaporated to dryness, and further de-ionized (DI) water was used to de-wall to form a liposome suspension. The liposome suspension was pulverized at 10 °C for 10 min by an ultrasonic cell crusher, and dialyzed with deionized water at 25 °C for 48 h to remove free indocyanine green and artesunate. , the liposome nanocomposite ICG-ARS@NPs was obtained, and the molar ratio of artesunate and indocyanine green in the nanocomposite was 1:1.

Electron microscope analysis showed that the obtained liposome nanocomplexes were relatively uniform spherical or nearly spherical vesicles with a diameter of about 150 nm. Nanoparticle size analyzer also showed that the obtained indocyanine green and artesunate liposome complex had a particle size of about 90-160 nm. The encapsulation efficiency of artesunate in the liposome nanocomposite was analyzed by HPLC, showing that the encapsulation efficiency of indocyanine green and artesunate were 81.4% and 69.0%, respectively.

Comparative Example 1 Indocyanine Green-loaded Liposome Nanomedicine

The liposome nanomedicine ICG@NPs (with a particle size of about 90-160 nm) only added with indocyanine green was prepared according to the method of Example 3.

Comparative Example 2 Packing Artesunate-loaded Liposome Nanomedicine

The liposome nano-drug ARS@NPs (with a particle size of about 90-160 nm) only added with artesunate was prepared according to the method of Example 3.

Example 4

The Example 3 ICG-ARS@NPs were placed in a dialysis bag (MW ₁₀₀₀₀ ) and stirred in a PBS solution (pH 6.5 and pH 7.4) at 120 rpm in a shaker incubator at room temperature. The dialysate was withdrawn at different time points (0 hours, 6 hours, 12 hours, 24 hours, 48 hours and 72 hours) for UV-Vis spectroscopy measurements. The released ICG concentration was calculated by comparing the absorbance at 780 nm with the calibration curve. Drug release was obtained using the following formula:

Drug release (%) = released ICG/total ICG in nanodrugs.

The pH-sensitive efficiency of ICG-ARS@NPs was evaluated in PBS solutions at different pH values by detecting the percentage of ICG released using UV-Vis spectroscopy. The results showed that in PBS solution at pH 6.5, ICG was rapidly released within 24 hours and reached a stable level at 48 hours, with a release percentage of about 80%. However, in PBS solution at pH 7.4, only a small amount of ICG was released, and about 20% was released within 48 hours.

Example 5

The artesunate and indocyanine green liposome nanocomposite of the present invention can effectively generate thermal effect under laser irradiation. The solution temperature can reach 50℃ when irradiated with 808nm laser in aqueous solution for 10min (laser intensity is 0.3～0.8W·cm ^-2 ).

The thermal effect of light can induce artesunate to generate reactive oxygen species. The nanocomposite of artesunate and indocyanine green liposome was irradiated by laser, and the used laser wavelength was 808 nm, and the laser intensity was 0.3 W·cm ^-2 . The electron absorption spectrum of the sample was measured every 1 min of illumination, and the scanning range was 500-600 nm. The experimental results show that artesunate and indocyanine green liposomes can effectively generate fluorescence under laser activation.

Example 6

First, the nanocomplex ICG-ARS@NPs of Example 3, the liposomal nanomedicine ICG@NPs of Comparative Example 1, and the liposomal nanomedicine ARS@NPs of Comparative Example 2 were buffered with water or normal saline or phosphate, respectively. The solution was diluted to a 1 mM solution, and the mixed solution was diluted with a medium to a 100 μM drug-containing medium. In the 96-well plate, when the spreading area of growing cells accounts for more than 75% of the bottom area of the medium, it can be used for drug application. Aspirate the old medium, add 60, 50, 40, 30 μL of drug-containing medium to each well, and supplement the medium to a final volume of 100 μL per well, and set up a blank control group (no light without drug addition and no light without drug addition) , cultured for 2h. The 96-well plate was placed under 808nm laser light (power 0.3W·cm ^-2 ), and each well was illuminated for 5min. After exposure to light, the drug was aspirated and replaced with fresh medium. After culturing for 24 h, 10 μL of 5 g/L MTT was added to each well, and the cells were incubated for another 4 h. Pour out the culture medium in the plate, add 150 μL of DMSO solution to each well, shake for 10 min to fully dissolve the crystals, measure the absorbance (A) value of each well with a microplate reader (wavelength 492 nm), and take the value of A in each group of n wells. The mean was taken as the mean A value of each group. The inhibition rate was calculated according to the following formula:

Inhibition rate (%)=(1-experimental group A/control group A)×100%.

The experimental results are shown in Figure 2. The experimental results show that light alone, or nano-drug alone, has no obvious killing effect on cancer cells. However, under the action of light, when the concentration of ICG in the liposome nanocomposite ICG-ARS@NPs is 40 μM, its inhibition rate against cancer cells can reach 75%, indicating that the composite nanocomposite has significant thermodynamic resistance. cancer effect.

Example 7

H22 tumor-bearing ICR mice were randomly divided into 7 groups: (I) blank (saline), (II) laser alone, (III) ARS@NPs+laser, (IV) ICG-ARS@NPs, (V) free ICG+ Laser, (VI) ICG@NPs+laser, (VII) ICG-ARS@NPs+laser. Mice were injected intravenously with normal saline, free ICG, ICG@NPs, ICG-ARS@NPs and ARS@NPs at a dose of 10 mg·kg ^-1 of ICG or 7.5 mg·kg ^-1 of ARS, respectively. Eight hours after injection, tumors were exposed to 808 nm laser light (0.8 W·cm ⁻² ) for 10 min. During irradiation, infrared thermal images were recorded by an infrared thermal imaging camera (TiX520, Fluke, USA). Body weight and tumor volume of mice were measured every 2 days for a total of 14 days.

As shown in Figure 3, after laser irradiation, the tumor growth of ICG@NPs-treated mice was partially blocked, and the tumor inhibition rate was 75%, which was mainly caused by the photothermal effect; while the irradiated ARS@NPs There was little inhibition of this effect, and tumors grew as fast as in the control group (saline or laser alone). However, in the ICG-ARS@NPs group after laser irradiation, the tumor gradually shrank on the 10th day until it finally disappeared, and did not continue to grow until the 14th day, which proves that the ICG-ARS@NPs has a very effective synergistic effect of PTT and TDT. .

The descriptions of the above embodiments are only used to help understand the method and the core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can also be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (8)

1. An application of artemisinin and its derivatives in the preparation of tumor thermodynamic therapy sensitizers, characterized in that: artemisinin or its derivatives are directly used as thermodynamic therapy sensitizers for the thermodynamic therapy of tumors ; Or use artemisinin or its derivatives and indocyanine green liposome nanocomposite as a sensitizer for thermodynamic therapy or a photothermal agent for photothermal therapy for thermodynamic therapy of tumors and/or or photothermal therapy;

   Described artemisinin derivative includes any one in artemether, dihydroartemisinin and artesunate;

   The thermal effect condition is to raise the temperature to ≥48°C by direct or indirect heating technology; the heating technology includes heating, laser, ultrasound, and microwave.
2. The application of artemisinin and its derivatives in the preparation of tumor thermodynamic therapy sensitizers according to claim 1, characterized in that: using artesunate and indocyanine green as raw materials to prepare liposome nanocomposite The method includes the following steps:

   1) Add dipalmitoyl phosphatidyl choline, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, cholesterol hemisuccinate, artesunate and indocyanine green in proportion to the mixture of chloroform and methanol. In the mixed solvent, it is completely dissolved, and then ultrasonically treated for 1 to 5 minutes;

   2) After ultrasonication, the solution is rotated and evaporated to dryness, and then deionized water is used to de-wall to form a liposome suspension;

   3) Use an ultrasonic cell crusher to pulverize the liposome suspension at 0 to 20 °C for 5 to 10 min, and then dialyze the liposome suspension with deionized water for 12 to 48 h at 4 to 25 °C to remove free indocyanine green and cyanine Artesunate to obtain the liposome nanocomplex.
3. The application of artemisinin and its derivatives in the preparation of tumor thermodynamic therapy sensitizers according to claim 1, characterized in that: dipalmitoyl phosphatidyl choline, distearoyl phosphatidyl choline used in step 1) The molar ratio of ethanolamine-polyethylene glycol 2000, cholesterol hemisuccinate, artesunate and indocyanine green is 12:1.5:9:3:13;

   In the mixed solvent of chloroform and methanol, the volume ratio of chloroform and methanol is 1:1.
4. The application of artemisinin and its derivatives in the preparation of a sensitizer for tumor thermodynamic therapy according to claim 1, characterized in that: in step 3), the dialysis uses a filter membrane with a molecular weight cut-off of 10,000.
5. Liposome nanocomplexes made of artemisia amber and indocyanine green.
6. The preparation method of the described liposome nanocomposite of claim 5 comprises the steps:

   1) Add dipalmitoyl phosphatidyl choline, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, cholesterol hemisuccinate, artesunate and indocyanine green in proportion to the mixture of chloroform and methanol. In the mixed solvent, it is completely dissolved, and then ultrasonically treated for 1 to 5 minutes;

   2) After ultrasonication, the solution is rotated and evaporated to dryness, and then deionized water is used to de-wall to form a liposome suspension;

   3) Use an ultrasonic cell crusher to pulverize the liposome suspension at 0 to 20 °C for 5 to 10 min, and then dialyze the liposome suspension with deionized water for 12 to 48 h at 4 to 25 °C to remove free indocyanine green and cyanine Artesunate to obtain the liposome nanocomplex.
7. The preparation method according to claim 6, wherein the dipalmitoyl phosphatidyl choline, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, cholesterol hemisuccinate, The molar ratio of artesunate and indocyanine green is 12:1.5:9:3:13;

   In the mixed solvent of chloroform and methanol, the volume ratio of chloroform and methanol is 1:1.
8. The preparation method according to claim 6, characterized in that: in step 3), the dialysis adopts a filter membrane with a filtration cutoff molecular weight of 10,000.

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