CN115340571A - Preparation method and application of a novel artemisinin derivative and liposome - Google Patents

Abstract

The invention discloses a preparation method and application of novel artemisinin derivatives and liposomes. This method first synthesizes the artemisinin derivative TPP‑SS‑ATS which is sensitive to glutathione GSH and has mitochondrial targeting effect; then prepares the new artemisinin derivative liposome TPP‑SS by thin film dispersion method ‑ATS‑LS. The liposome novel artemisinin derivative liposome TPP-SS-ATS-LS prepared by the present invention can realize dual-targeted drug delivery to tumor tissue and tumor cell mitochondria, and significantly improves the anti-inflammatory effect of artemisinin drugs. Tumor efficacy.

Description

Preparation method and application of a novel artemisinin derivative and liposome

technical field

The invention belongs to the field of traditional Chinese medicine preparations, and in particular relates to a preparation method of novel artemisinin derivatives, liposomes and antitumor effects.

Background technique

The mechanism of artemisinin and its derivatives in anti-tumor applications is very different from other drugs. Some chemotherapy drugs exert anti-tumor effects by intervening in the biosynthesis of tumor cell nucleic acid and destroying DNA function (refer to the literature: Fang Miao , Wang Degang, Liu Peiqing. Study on the Mechanism of Antitumor Action of Natural Medicines[J]. Food and Drugs, 2022,24(2):167-171; Progress [J]. Medical Review, 2020, 26(18): 3707-3711, 3716; Xu Jiao, Meng Linghua, Qing Chen. Current status and development of clinical application of traditional antineoplastic drugs [J]. Acta Pharmaceutica Sinica, 2021, 56(6):1551-156); and artemisinin drugs release oxygen free radical ROS, initiate mitochondrial apoptosis program, and combine oxidative damage of cell model structure to produce anti-tumor effect (references: Zhou Y, Li W ,Xiao Y.Profiling of multiple targets of artemisinin activated by heminincancer cell proteome[J].ACS Chem Biol,2016,11:882–888;Liu Qingqing,Yang Zhenhua.Anti-tumor research progress of artemisinin and its derivatives[J] ].Life Science, 2020,32(1):62-69). Due to the unique anti-tumor mechanism of artemisinin drugs, these drugs have become very attractive anti-tumor drug candidates and have received extensive international attention. However, artemisinin drugs have problems such as poor solubility, low bioavailability, and lack of tumor targeting, which restrict their anti-tumor efficacy. In summary, improving the solubility and targeting of artemisinin drugs is the key to increasing their antitumor efficacy.

The invention uses the tumor cell membrane transport protein GLUT1 as the target to construct tumor-targeted liposomes, and realizes the tumor-targeted delivery of artemisinin drugs; and synthesizes artemisinin based on triphenylphosphine mitochondrial targeted migration Mitochondria-targeted prodrug further realizes mitochondria-targeted delivery of liposomes. The present invention improves the anti-tumor efficacy of artemisinin drugs through dual-target drug delivery of tumor and tumor cell mitochondria.

Contents of the invention

Aiming at the problems existing in the prior art, the object of the present invention is to provide a kind of preparation method and application of novel artemisinin derivatives and liposomes. After artemisinin derivatives enter tumor cells, their common structural peroxo bridging group will break and generate a large number of free radicals, causing oxidative stress and mitochondrial function damage in cancer cells. Based on this, the present invention designs and prepares a novel artemisinin derivative liposome TPP-SS-ATS-LS with dual targeting of tumor cells and their mitochondria.

Technical scheme of the present invention is:

A novel artemisinin derivative, characterized in that the structure of the novel artemisinin derivative is as follows:

A preparation method of a novel artemisinin derivative liposome, the steps comprising: first synthesizing the artemisinin derivative TPP-SS-ATS which is sensitive to glutathione GSH and has a mitochondrial targeting effect; A new artemisinin derivative liposome TPP-SS-ATS-LS was prepared by film dispersion method.

Further, the particle size of the novel artemisinin derivative liposomes is normally distributed, and observed under a transmission electron microscope is a regular spherical shape.

Further, the method for synthesizing the artemisinin derivative TPP-SS-ATS is:

(1) Weigh artesunate and 2,2-dithiodiethanol and dissolve them in an organic solvent, wherein the ratio of artesunate and 2,2-dithiodiethanol is 1:50 to 50:1, React at -20 to 40°C for 0.1-96 hours. After the reaction, use a rotary evaporator to recover the organic solvent at 20-100°C to obtain the crude SS-ATS; use petroleum ether: ethyl acetate solution as the mobile phase, and use a silica gel column The intermediate SS-ATS crude product is purified to obtain the intermediate SS-ATS;

(2) Weigh the intermediate SS-ATS and 4-carboxybutyl triphenylphosphine bromide TPP and dissolve them in an organic solvent, wherein the intermediate SS-ATS and 4-carboxybutyl triphenylphosphine bromide TPP are used in proportion 1:50 to 50:1, react at -20 to 40°C for 0.1-96 hours, use a rotary evaporator to recover the organic solvent at 20-100°C after the reaction, and obtain the crude product of TPP-SS-ATS; Methane:methanol solution was used as the mobile phase, and the crude product was purified by a silica gel column to obtain a new artemisinin derivative TPP-SS-ATS.

Further, the specific method for preparing the new artemisinin derivative liposome TPP-SS-ATS-LS by thin film dispersion method is as follows: phospholipid, alkyl glycoside, new artemisinin derivative TPP-SS-ATS, Cholesterol and dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 2000DSPE-PEG2000 were dissolved in an organic solvent; then the resulting solution was transferred to an evaporating flask of a rotary evaporator, and concentrated under reduced pressure at 20-100°C until the organic solvent was completely Volatilize, add ultra-pure water to a rotary evaporator, hydrate at 20-80°C, hydration time 0.1-3.0 hours, and then pass through ultrasonic vibration and microporous membrane filtration in turn to prepare a new artemisinin derivative liposome TPP -SS-ATS-LS.

Further, the weight percentage of phospholipid is 0.2-5.0%, the weight percentage of cholesterol is 0.01-0.5%, the weight percentage of alkyl glycoside is 0.2-3.0%, dipalmitoylphosphatidylethanolamine-methoxy polyethylene glycol 2000 The weight percentage of the new artemisinin derivative TPP-SS-ATS is 0.02-5.0%, and the weight percentage of the purified water is 73.5-99.0%.

Further, the phospholipids are one or more of soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin and PEGylated phospholipids.

Further, the alkyl glucoside is one or more of the alkyl glucosides with an alkyl carbon number of 2-30 and a glucose molecular fragment number of 1-10 in each molecule of the alkyl glucoside.

Further, the ratio of petroleum ether: ethyl acetate ranges from 10:1 to 2:1; the ratio of dichloromethane: methanol ranges from 500:1 to 50:1; a silica gel column of 50 to 500 meshes is used for purification.

The application of a novel artemisinin derivative liposome in the preparation of antitumor drugs.

The present invention selects artesunate as a raw material to synthesize a novel artemisinin derivative TPP-SS-ATS that is sensitive to glutathione GSH and has a mitochondrial targeting effect. After confirming its structure by NMR and mass spectrometry, it uses A new artemisinin derivative liposome TPP-SS-ATS-LS was prepared by film dispersion method.

A preparation method of a novel artemisinin derivative liposome TPP-SS-ATS-LS, the steps comprising: first synthesizing an artemisinin derivative TPP that is sensitive to glutathione GSH and has a mitochondrial targeting effect -SS-ATS; then the new artemisinin derivative liposome TPP-SS-ATS-LS was prepared by thin film dispersion method.

The synthetic method of novel artemisinin derivative TPP-SS-ATS is: (1) Weigh artesunate and 2,2-dithiodiethanol and dissolve in organic solvent, wherein artesunate and 2,2 - The ratio of the amount of dithiodiethanol is 1:50 to 50:1, react at -20 to 40°C for 0.1-96 hours, and use a rotary evaporator to recover the organic solvent at 20-100°C after the reaction to obtain SS-ATS Crude product; using petroleum ether: ethyl acetate solution as the mobile phase, the intermediate SS-ATS crude product is purified by a silica gel column to obtain the intermediate SS-ATS. (2) Weigh the intermediate SS-ATS and 4-carboxybutyl triphenylphosphine bromide TPP and dissolve them in an organic solvent, wherein the intermediate SS-ATS and 4-carboxybutyl triphenylphosphine bromide TPP are used in proportion 1:50 to 50:1, react at -20 to 40°C for 0.1-96 hours, use a rotary evaporator to recover the organic solvent at 20-100°C after the reaction, and obtain a new artemisinin derivative TPP-SS- The crude product of ATS; using dichloromethane:methanol solution as the mobile phase, the crude product was purified by silica gel column to obtain the novel artemisinin derivative TPP-SS-ATS.

In the synthetic step (1) of novel artemisinin derivatives TPP-SS-ATS, the particle size of column chromatography silica gel used for purifying intermediate SS-ATS is between 50-500 mesh, and the eluent sherwood oil: ethyl acetate The ratio range is 10:1～2:1.

In the synthesis step (2) of the novel artemisinin derivatives TPP-SS-ATS, the particle size of the column chromatography silica gel used for purifying the intermediate SS-ATS is between 100-500 mesh, and the eluent dichloromethane:methanol The ratio range is 500:1～50:1.

The specific method of preparing artemisinin derivative liposome TPP-SS-ATS-LS by thin film dispersion method is as follows: (1) phospholipid, alkyl glycoside, novel artemisinin derivative TPP-SS-ATS, cholesterol and dipalmitoylphosphatidylethanolamine-methoxy polyethylene glycol 2000DSPE-mPEG2000 are dissolved in an organic solvent; (2) then the gained solution is transferred to the evaporating flask of a rotary evaporator, concentrated under reduced pressure until the organic solvent is completely volatilized, Add ultrapure water into a rotary evaporator flask, hydrate, and then pass through ultrasonic vibration and microporous membrane filtration in sequence to prepare the new artemisinin derivative liposome TPP-SS-ATS-LS.

The amount of auxiliary materials used in the step (1) and step (2) of preparing the novel artemisinin derivative liposome TPP-SS-ATS-LS is (percentage by weight): phospholipid 0.2-5.0%, cholesterol 0.01-0.5% , alkyl glycoside 0.2-3.0%, dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 2000 0.02-5.0%, artemisinin derivatives TPP-SS-ATS, 0.02-3.0%, purified water 73.5- 99.0%.

The phospholipid used in the step (1) of preparing the novel artemisinin derivative liposome TPP-SS-ATS-LS is one or more of soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin and PEGylated phospholipid.

The alkyl glycoside used in the step (2) of preparing the novel artemisinin derivative liposome TPP-SS-ATS-LS is: each molecule of the alkyl glucoside contains an alkyl carbon number of 2-30, the number of glucose molecular fragments One or more of 1-10 alkyl glucosides.

In the step (2) of preparing the novel artemisinin derivative liposome TPP-SS-ATS-LS, the temperature for removing the organic solvent by using a rotary evaporator under reduced pressure is 20-100° C.; the liposome hydration temperature is 20-80° C. ℃, the hydration time is 0.1-3.0 hours.

The advantages of the present invention are as follows:

(1) The new artemisinin derivative TPP-SS-ATS synthesized by the present invention has a triphenylphosphine structural fragment in its structure, and the derivative can utilize the mitochondrial targeting property of triphenylphosphine to realize the mitochondrial target of the drug Delivery to the target, improve mitochondrial killing ability, thereby enhancing the anti-tumor performance of artemisinin drugs.

(2) Liposome TPP-SS-ATS-LS, a novel artemisinin derivative liposome prepared by the present invention, can realize dual-targeted drug delivery to tumor tissue and tumor cell mitochondria, and significantly improves the artemisinin class. Antitumor efficacy of drugs.

(3) The present invention adopts two-step condensation-esterification reaction to synthesize TPP-SS-ATS, does not need heating, and reaction speed is fast, and raw material cost is cheap, and synthesis process is mature.

(4) The present invention selects conventional auxiliary materials to prepare liposome TPP-SS-ATS-LS, the process is simple, the energy consumption is low, and the production cost can be effectively controlled.

(5) The liposome TPP-SS-ATS-LS prepared by the present invention has excellent pharmaceutical properties and high biological safety.

Description of drawings

Fig. 1 is a synthetic route diagram of the novel artemisinin derivative TPP-SS-ATS.

Figure 2 is a chemical structure diagram of the intermediate SS-ATS.

Fig. 3 Mass spectrum of intermediate SS-ATS.

Figure 4 is the proton nuclear magnetic resonance spectrum of the intermediate SS-ATS.

Fig. 5 is a chemical structure diagram of a novel artemisinin derivative TPP-SS-ATS.

Fig. 6 is the mass spectrum of the novel artemisinin derivative TPP-SS-ATS.

Fig. 7 is the hydrogen nuclear magnetic resonance spectrum of the novel artemisinin derivative TPP-SS-ATS.

Fig. 8 is the carbon nuclear magnetic resonance spectrum of the novel artemisinin derivative TPP-SS-ATS.

Fig. 9 is the nuclear magnetic resonance HSQC spectrum of the novel artemisinin derivative TPP-SS-ATS.

Figure 10 is the chemical structure and nuclear magnetic resonance HMBC related signal diagram of artemisinin derivative TPP-SS-ATS;

(a) Chemical structure of TPP-SS-ATS;

(b) NMR HMBC-related signal map of TPP-SS-ATS.

Fig. 11 is a particle size distribution diagram of liposome TPP-SS-ATS-LS.

Fig. 12 is a transmission electron microscope image of liposome TPP-SS-ATS-LS.

Figure 13 is a differential scanning calorimetry curve of liposome TPP-SS-ATS-LS and blank liposome Blank LS.

Fig. 14 is the stability data graph of liposome TPP-SS-ATS-LS;

(a) Particle size and polydispersity coefficient of TPP-SS-ATS-LS;

(b) Encapsulation efficiency and zeta potential (zeta potential) of TPP-SS-ATS-LS.

Figure 15 is the mitochondrial targeting imaging of liposome TPP-SS-ATS-LS.

Fig. 16 is an in vivo imaging diagram of tumor targeting of liposome TPP-SS-ATS-LS.

Figure 17 is a graph showing the results of in vitro anticancer activity evaluation of liposome TPP-SS-ATS-LS on three breast cancer cells.

Fig. 18 is a schematic diagram of the administration scheme of the in vivo anti-tumor experiment.

Figure 19 is a picture of the anti-breast cancer effect of the drug (A: tumor appearance of mice in each administration group; B: effect of drug on tumor weight; C: body weight of mice in each group during the experiment).

Fig. 20 is a diagram of pathological analysis (H&E staining) of mouse organs and tissues after administration.

Fig. 21 is a picture of the test results of blood routine and liver and kidney function indexes of mice after administration (A: routine blood biochemical indexes; B: blood biochemical indexes of liver and kidney function).

Detailed ways

The present invention is described in further detail below in conjunction with accompanying drawing, and the examples given are only used to explain the present invention, and are not intended to limit the scope of the present invention.

1. Synthesis and structure identification of artemisinin derivatives

1. Synthesis of Intermediate SS-ATS

Weigh 5g of artesunate and 3.6g of dicyclohexylcarbodiimide DCC and dissolve them in 500ml of dichloromethane, stir at 350rpm for 1h at room temperature to promote the complete dissolution of the three; add 3g of 2,2-dithiodiethanol HEDS, 0.5 Add g p-dimethylaminopyridine DMAP to dichloromethane solution, stir at 350 rpm for 3 hours at room temperature (Figure 1); after the reaction, use a rotary evaporator to recover dichloromethane at 35°C to obtain the intermediate SS-ATS Crude. Using petroleum ether: ethyl acetate (10:1-2:1) as the mobile phase, the silica gel (200-300 mesh) column was used for purification to obtain the intermediate SS-ATS. Its structure was confirmed by MS and NMR.

2. Synthesis of new artemisinin derivatives TPP-SS-ATS

Weigh 1.6g of intermediate SS-ATS conjugate, 3.2g of 1-ethyl-3(3-dimethylpropylamine) carbodiimide EDCI dissolved in 50ml of dichloromethane, stir at 350rpm for 1h at room temperature, and dissolve completely to obtain After clarifying the solution, add 3.2g of 4-carboxybutyltriphenylphosphine bromide TPP and 0.6g of p-dimethylaminopyridine DMAP into the methylene chloride solution, stir at room temperature at 350rpm, and continue the reaction for 12h (Figure 1); the reaction is over Finally, dichloromethane was recovered under reduced pressure at 35° C. using a rotary evaporator to obtain a crude product of TPP-SS-ATS. Using dichloromethane:methanol (500:1~50:1) as mobile phase, the silica gel (200~300 mesh) column was used for purification to obtain the artemisinin derivative TPP-SS-ATS. Determine the product's one-dimensional, two-dimensional NMR spectrum and molecular weight data to determine its chemical structure.

3. Structural identification of intermediate SS-ATS and novel artemisinin derivative TPP-SS-ATS

(1) Structural identification of intermediate SS-ATS

The SS-ATS conjugate is an oil, and the mass spectrometry data shows that its quasi-molecular ion peak m/z: 559.1[M+K] ⁺ , combined with NMR spectral analysis, it is determined that the molecular formula of the compound is C ₂₃ H ₃₆ O ₉ S ₂ (Fig. 2, Figure 3). ¹ H NMR (600MHz, CDCl ₃ ) showed the characteristic proton signal of the SS-ATS conjugate: δ _H 2.37 (1H, brt, J = 13.5Hz, H-4α), 2.03 (1H, brd, J = 14.5, H-4β),1.43-1.52(1H,m,H-5α),1.88-1.94(1H,overlapped,H-5β),1.27-1.30(1H,m,H-5a), 1.34-1.36(1H, m, H-6), 0.96-1.05 (1H, m, H-7α), 1.72 (1H, overlapped, H-7β), 1.36-1.38 (1H, overlapped, H-8α), 1.78 (1H, brd, J＝13.3Hz, H-8β), 1.62 (1H, br d, J＝13.9Hz, H-8a), 2.53-2.59 (1H, m, H-9), 5.78 (1H, d, J＝9.8Hz ,H-10),5.44(1H,s,H-12),1.43(3H,s,H-14),0.96(3H,d, J＝5.0Hz,H-15),0.86(3H,d, J=6.6Hz, H-16). The above ¹ H NMR and MS spectra suggested that SS-ATS was successfully synthesized ( FIG. 4 ).

(2) Structural identification of a novel artemisinin derivative TPP-SS-ATS

The TPP-SS-ATS conjugate is a white amorphous solid, and the mass spectrometry data shows that its quasi-molecular ion peak m/z: 851.4[M-Br] ⁺ , combined with NMR spectral analysis, the molecular formula of the compound is C ₄₅ H ₅₆ BrO ₁₀ PS ₂ (Figures 5, 6). ¹ H NMR (600MHz, CDCl ₃ ) and HSQC spectrum (Fig. 7, 9) show the characteristic proton signal of the artesunate structural fragment: δ _H 2.37 (1H, td, J=14.0, 3.8Hz, H-4α), 2.03(1H,dt,J=14.5,3.8Hz,H-4β),1.43-1.52(1H,m,H-5α),1.87-1.92(1H,m,H-5β),1.27-1.30(1H, m, H-5a), 1.33-1.34 (1H, m, H-6), 0.96-1.07 (1H, m, H-7α), 1.72 (1H, dd, J＝13.4, 3.0Hz, H-7β) ,1.37(1H,dd,J＝13.5,3.3Hz,H-8α),1.78(1H,dd,J＝13.6,3.6Hz,H-8β),1.62(1H,dt,J＝13.7,4.4Hz, H-8a), 2.54-2.56(1H, m, H-9), 5.78(1H, d, J＝9.8Hz, H-10), 5.31(1H, s, H-12), 1.41(3H, s ,H-14),0.96(3H,d,J＝6.1Hz,H-15),0.85(3H,d,J＝7.1Hz,H-16); characteristics of 2,2-dithiodiethanol structural fragment Proton signal: δ _H 4.32 (4H, dd, J = 15.9, 6.6Hz, H-2′and 7′) and 2.90-2.91 (4H, m, H-3′and 6′); 4-carboxybutyltri The characteristic proton signals of the structural fragment of phenylphosphine bromide: δH 2.92-2.94 (2H, m, Hb), 1.93-1.99 (2H, m, Hc), 4.00-4.05 (2H, m, Hd) and 7.70-7.90 ( 15H, m,H-TPP arene protons).

The characteristic signals of the ¹³ C NMR (150MHz, CDCl ₃ ) spectrum (Figure 8) are as follows: δC 171.1 ( _C -17, the ester carbonyl of the artesunate structural fragment), 171.9 (C-20, the artesunate structural fragment ester carbonyl), 62.5and 62.6(C-2′or C-7′), 36.9 and 37.0(C-3′or C-6′), 172.9(Ca), 117.9-135.1(Cf, triphenylphosphine Aromatic carbons of structural fragments) (Figure 9). In the HMBC spectrum (Fig. 10), the long-range correlation signal of δ _H 4.32 (H-2′) and δ _C 171.9 (C-20) confirmed the C-20 linkage to the artesunate structural fragment at the 4-carboxy C-2′ position of butyltriphenylphosphine bromide structural fragment; δ _H 4.32 (H-7′) and δ _C 172.9 (Ca), δ _H 1.93-1.99 (Hc) and δ _C 172.9 (Ca) The long-range correlation signal confirms that the C-7' of the 2,2-dithiodiethanol structural fragment is connected to the Ca position of the 4-carboxybutyltriphenylphosphine bromide structural fragment. The above spectral features show that the new artesunate derivative TPP-SS-ATS has been successfully synthesized.

2. Preparation of liposomes

1. Preparation of Liposome TPP-SS-ATS-LS

Novel artemisinin derivative liposome of the present invention and preparation method thereof are as shown in Figure 1, adopt film dispersion method to prepare artemisinin derivative liposome, specific method is as follows (the total weight of each adjuvant and medicine is 100 %): 1.43% by weight of soybean lecithin, 0.14% by weight of n-octyl-β-D-glucopyranoside, and 0.19% by weight of artemisinin derivative TPP -SS-ATS, 0.07% by weight of cholesterol and 0.17% by weight of dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 2000 (DSPE-mPEG2000) were dissolved in 20ml of dichloromethane, and Transfer the solution to a rotary evaporator bottle, concentrate under reduced pressure at 40°C until the dichloromethane is completely volatilized, add ultra-pure water with a weight ratio of 98% to the rotary evaporator bottle, and hydrate at 40°C and 150rpm for 25 minutes , and then ultrasonically oscillated for 3 minutes at a power of 250W, and filtered through a 0.22 μm microporous membrane to obtain the artemisinin derivative liposome TPP-SS-ATS-LS, which is a translucent liquid with a light blue milky appearance. Light.

2. Preparation of Blank Liposomes

The preparation method of blank liposome is basically the same as liposome TPP-SS-ATS-LS. The only difference between the two is that the blank liposome prescription does not contain the new artemisinin derivative TPP-SS-ATS.

3. Formulation evaluation of liposome TPP-SS-ATS-LS

1. Determination of liposome TPP-SS-ATS-LS particle size, Zeta potential distribution, and PDI particle size dispersion coefficient

(1) Experimental method

Get an appropriate amount of liposome TPP-SS-ATS-LS sample and place it in a test dish, and use a Malvern Zetasizer NanoZS nanometer particle size analyzer to measure liposome particle size, Zeta potential distribution and polydispersity index PDI. Measuring method: the test temperature is 25°C, the test times are 3 times, and the average value of the data is taken for result analysis.

(2) Experimental results

The particle size of liposome TPP-SS-ATS-LS is normally distributed (Figure 11), and its average particle size is measured to be 87.60±1.65nm, Zeta potential and PDI polydispersity coefficient are 31.4±1.7mV, 0.241± 0.013; the particle size, Zeta potential and PDI of blank liposomes were 94.83±0.71nm, -36.9±1.2mV, 0.200±0.008, respectively (Table 1).

Table 1 is the Zeta potential, particle size, polydispersity index PDI and encapsulation efficiency of liposomes

2. Determination of Encapsulation Efficiency (EE) of Liposome TPP-SS-ATS-LS

(1) Experimental method

1) Determination of the content of TPP-SS-ATS in liposome TPP-SS-ATS-LS (M1): add an appropriate amount of liposome TPP-SS-ATS-LS into a volumetric flask, ultrasonically extract at room temperature for 15 minutes, and use HPLC method Determination of the content of TPP-SS-ATS (M1).

2) Determination of free TPP-SS-ATS content (M2): add an appropriate amount of liposome TPP-SS-ATS-LS to a 4ml ultrafiltration tube (molecular weight cut-off: 10kDa), centrifuge at 5000rpm for 120min at 4°C, collect by ultrafiltration solution, and constant volume, the content (M2) of free TPP-SS-ATS was measured by high performance liquid chromatography. Results were analyzed using the mean of data from three parallel experiments. Calculate the encapsulation efficiency (EE) of artemisinin derivative liposome according to the following formula:

(2) Experimental results

The encapsulation efficiency of the liposome TPP-SS-ATS-LS is 95.2 ± 0.3%, suggesting that the liposome prepared by the present invention can efficiently entrap the mitochondria targeting conjugate TPP-SS-ATS.

3. TEM morphology observation of liposome TPP-SS-ATS-LS

(1) Experimental method

Liposome TPP-SS-ATS-LS, diluted 5 times with ultrapure water, was dropped on a 200-mesh copper grid, then the excess sample was removed with filter paper, and after natural drying, the liposome TPP-SS-LS was observed with a H7650 transmission electron microscope. Microscopic morphology of ATS-LS.

(2) Experimental results

The liposome TPP-SS-ATS-LS was observed by transmission electron microscope as a regular spherical shape (Figure 12).

4. Differential Scanning Calorimetry (DSC) Analysis of Liposome TPP-SS-ATS-LS

(1) Experimental method:

The present invention adopts NANO DSC differential scanning calorimeter to detect the phase transition temperature of liposome TPP-SS-ATS-LS and blank liposome; min; during the experiment, the pressure of the sample cell was 3 atmospheres, and ultrapure water was used as the control solution.

(2) Experimental results

The phase transition temperature of the liposomes before and after drug loading decreased from 67.29°C to 57.75°C (Figure 13), suggesting that the new artemisinin derivative TPP-SS-ATS was loaded in the lipid membrane structure of the liposomes, and had a strong effect on liposomes. The phase transition temperature of the film structure has an influence. This structural feature of liposome TPP-SS-ATS-LS is conducive to the recognition of the triphenylphosphine structural fragment (TPP) by the mitochondria, thereby pulling the liposomes to migrate to the mitochondria and realizing the mitochondrial targeting of the liposomes.

5. Stability evaluation of liposome TPP-SS-ATS-LS

(1) Experimental method

After the liposome TPP-SS-ATS-LS was prepared, it was sealed and stored at 4°C. At 0, 7, 14, 28 and 42 days after sample preparation, the particle size, encapsulation efficiency, Zeta potential and PDI were measured.

(2) Experimental results

The RSD% of liposome TPP-SS-ATS-LS particle size and encapsulation efficiency measured on the 0, 7, 14, 28 and 42 days after sample preparation were 1.60% and 0.96% respectively; and in the stability study Among them, the Zeta potential of liposome TPP-SS-ATS-LS was always greater than 26mV, indicating that liposome TPP-SS-ATS-LS had good stability at 4°C (Fig. 14, A: particle size, PDI; B: Encapsulation efficiency, Zeta potential).

4. Targeting evaluation of liposome TPP-SS-ATS-LS

1. Mitochondrial targeting evaluation

(1) Experimental method

MDA-MB-231 cells were inoculated into 96-well culture dishes at a cell density of 2000 cells/well, and cultured for 12 h; then mitochondria were stained with the mitochondrial red fluorescent probe (Mito Tracker Red CMXros), and 2'-[4 -Ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5'-bi-1H-benzimidazole trihydrochloride trihydrate (Hoechst33342 blue fluorescent probe) on Nuclei were stained. After washing with PBS, fluorescein PEG phospholipid-labeled liposome FITC-TPP-SS-ATS-LS (green fluorescence) was added. The cells were continuously monitored for 48 hours with PE Perkin Elmer Operetta CLSTM (Perkinlemer, USA) to observe the mitochondrial targeting.

(2) Experimental results

A high-content imaging system was used to monitor the mitochondrial targeting of liposome TPP-SS-ATS-LS. During the experiment, at the 6th hour after administration, fluorescently labeled liposome TPP-SS-ATS-LS It was found in the mitochondria (Figure 15), and then the fluorescently labeled liposome TPP-SS-ATS-LS gradually accumulated in the mitochondria; the related results indicated that TPP-SS-ATS-LS had mitochondrial targeting.

2. In vivo tumor targeting evaluation

(1) Experimental method

Wash the 4T1 cells in the axillary tumor tissue of the tumor-bearing mice, resuspend them with normal saline, adjust the density to 1×107 cells/ml, inoculate the cells with 0.2ml of the cell suspension, and inject 1,1-28 Alkyl-3,3,3,3-tetramethylindocyanine iodide DiR-labeled liposome TPP-SS-ATS-LS. At 4, 8, 12, 24, 36, 48, 60, 72, 96, 120, 144 and 168 hours after drug injection, the mice were anesthetized with isoflurane and detected using the IVIS Lumina III imaging system (Perkinlemer, USA) Distribution of fluorescently labeled liposomes in vivo.

(2) Experimental results

The results of in vivo imaging showed that liposome TPP-SS-ATS-LS has good tumor targeting, as shown in Figure 16. In this study, the liposome TPP-SS-ATS-LS was injected intraperitoneally into tumor model animals, the fluorescence intensity of the tumor site gradually increased with time, while the fluorescence intensity of the administration site (peritoneal cavity) gradually weakened ( Figure 16). The fluorescent signal intensity at the tumor site reached the highest level 24-60 hours after administration, and then gradually decreased. By intraperitoneal injection, the possible process of liposome TPP-SS-ATS-LS targeting tumor tissue is as follows: after administration, liposome enters the systemic circulation through the capillaries and lymphatic vessels in the peritoneum, and then the liposome is gradually absorbed by the tumor. Tissue capture to achieve the effect of tumor-targeted delivery.

5. Efficacy and safety evaluation of liposome TPP-SS-ATS-LS

1. CCK-8 method to detect the cytotoxic effect of drugs on various breast cancer cells

(1) Experimental method

1) Plating: Observe the state of the cells, digest them when the cells adhere to the wall and grow to 90%, count the cells on a counting plate, inoculate them in a 96-well plate at a cell density of 3000-5000/well, and culture them in a 5% CO2 incubator at 37°C for 24 hours .

2) Dosing: When the cell density is observed under the microscope to be about 60%-70%, the administration begins. The groups are the blank liposome Blank-LS group and the new artemisinin derivative liposome TPP-SS-ATS -LS group, artemisinin derivatives TPP-SS-ATS group and artesunate original drug ATS group. The dosing concentration was set at 2.5 μM-160 μM.

3) Add CCK-8: Observe the cell state after adding the drug for 12h, 24h, and 48h respectively, add CCK-8 to the 96-well plate and put it in the incubator for further incubation for 2h.

4) Detection: use a microplate reader to measure the OD value at 450 nm, and calculate the survival rate.

(2) Experimental results

The effect of drugs on the viability of breast cancer cells is shown in Figure 17. Liposomes TPP-SS-ATS-LS, TPP-SS-ATS, and ATS can inhibit the cell viability of breast cancer cells after administration for 12h, 24h, and 48h, respectively. , and was dose-dependent. Liposome TPP-SS-ATS-LS and TPP-SS-ATS have similar inhibitory effects, and both can significantly inhibit the growth of different types of breast cancer cells. Under the same concentration conditions, starting from 20 μM, compared with the original drug ATS, liposome The anti-tumor effect of plastid TPP-SS-ATS-LS is more obvious. The IC50 value of liposome TPP-SS-ATS-LS is 17.69μM (4T1 cells, 48h) ~ 67.48μM (MCF-7 cells, 12h); the IC50 value of ATS is 33.50μM (4T1 cells, 48h) ~ 797.5μM (MCF-7 cells, 12h).

2. In vivo evaluation of antitumor activity of liposomes

(1) Experimental method

The 4T1 cells in the axillary tumor tissue of the tumor-bearing mice were washed with normal saline and resuspended, and the density was adjusted to 1×107/ml, and the mice were inoculated with 0.2ml of the cell suspension. Tumor-bearing mice were randomly divided into six groups, namely control group, ATS low-dose group, ATS high-dose group, liposome TPP-SS-ATS-LS low-dose group, liposome TPP-SS-ATS-LS high-dose group, dose group, gemcitabine (GEM) positive control group. The drug doses of the low-dose and high-dose groups were 15 mg/kg or 30 mg/kg, and were injected intraperitoneally from the 4th day of inoculation, and injected once every two days; the drug dose of the GEM group was 30 mg/kg, injected once every 4 days; the control group Mice were given saline. The dosing regimen is shown in Figure 18.

Material collection and tumor inhibition rate calculation: weigh the weight of the mice before the material collection, and fix the mice in the supine position on the mouse board after euthanasia, dissect the heart, liver, kidney, spleen, brain and tumor tissues, and record the weight; according to the following The formula calculates the tumor growth inhibition rate TGI of breast cancer mice:

Where "T" represents the average mass of tumor tissue in the experimental group, and "C" represents the average mass of tumor tissue in the control group.

(2) Experimental results

The present invention evaluates the anti-cancer effect of liposome TPP-SS-ATS-LS on 4T1 breast cancer tumor-bearing mice by recording the change of the body weight of the mice with the intervention time and the tumor weight of the mice in each group after the materials are collected. The results showed that compared with the model group, the weight of breast cancer tumor tissue in the low and high dose groups of artesunate original drug was significantly reduced; while the tumor weight in the low and high dose groups of liposome TPP-SS-ATS-LS was significantly reduced. Many; the tumor growth inhibition rate (TGI) of artesunate former drug under the same administration concentration 30mg/kg condition is 37.7%, and the tumor growth inhibition rate (TGI) of liposome TPP-SS-ATS-LS group of equal dose ) is close to 56.4%. Compared with the model group, the gemcitabine group had the lowest tumor weight, and the liposomal TPP-SS-ATS-LS high-dose group was close to it. Each administration group had little effect on the body weight of tumor-bearing mice (Fig. 19).

3. Safety evaluation of new artemisinin derivative liposomes

The present invention further studies the organic toxicity of liposome TPP-SS-ATS-LS to other non-tumor organs. After drug intervention, the mouse heart, liver, kidney, spleen, and brain tissues are taken for pathological analysis, and small tumors are detected at the same time. Blood routine and biochemical indicators of liver and kidney function in mice.

(1) Pathological analysis

1) Experimental method

In order to evaluate the safety of liposome TPP-SS-ATS-LS, the present invention conducts pathological analysis on the main tissues and organs (heart, liver, kidney, spleen, brain) of mice after administration. The main steps of making pathological sections are as follows: tissue fixation in 10% formalin, embedding in paraffin, sectioning and H&E staining (hematoxylin-eosin staining method), etc.; after making pathological sections, observe them under a microscope and take pictures for preservation.

2) Experimental results

Pathological tissue section observation found that the heart, liver, spleen, kidney and brain tissues of the liposome TPP-SS-ATS-LS treatment group mice were similar to those of the normal saline group, and no pathological changes caused by liposomes were seen. The results were as follows: As shown in Figure 20: (1) Heart: The morphology and structure of cardiomyocytes in the blank control group, the model group and each drug-administered group were normal, the cardiomyocytes were arranged neatly and regularly, the horizontal lines were clear, the cell membrane was complete, and the staining was uniform. (2) Liver: The vacuolar degeneration of hepatocytes in the gemcitabine GEM group was more obvious, and the pathological changes of liver tissue in the other administration groups were similar to those in the model group, indicating that liposomal TPP-SS-ATS-LS and artesunate ATS would not aggravate the model Liver injury in mice.

(3) Kidney: the renal tubular epithelial cells of mice in blank group, model group and each administration group were neatly arranged, the distribution of mesangial matrix did not increase, and the structure of glomerulus was clear and uniform in size. (4) Spleen: The cortex and medulla of the spleen in the blank group, the model group and each administration group were clearly connected, and there were densely distributed lymphocytes in the white pulp and red pulp, showing that the spleen was not affected. (5) Brain: The neurons and glial cells in the brain tissue of the mice in each group were closely arranged without cell swelling. The analysis of the above results indicated that the liposome TPP-SS-ATS-LS had no obvious toxicity to each organ of mice.

(2) Blood biochemical analysis

1) Experimental method

Let the collected blood stand overnight at 4°C, centrifuge at room temperature at 3500rpm for 15min, and draw the supernatant, and use an automatic biochemical analyzer to detect blood routine indicators of mice, including white blood cell WBC, red blood cell RBC, platelet PLT, hemoglobin HGB, hematocrit HCT, and mean platelet volume MPV, mean hemoglobin MCH, mean corpuscular volume MCV, platelet distribution width PDW, liver and kidney function indicators serum creatinine CREA, blood urea nitrogen BUN, alanine aminotransferase ALT, aspartate aminotransferase AST, alkaline phosphatase ALP levels.

2) Experimental results

The blood routine test results are shown in Figure 20. Compared with the model group, all the indicators of the drug intervention group were normal, and there was no statistically significant difference between the two groups (P>0.05), suggesting that liposome TPP-SS-ATS- LS intervention did not cause obvious damage and inflammation to the mice. Liver and kidney function blood biochemical indicators showed that compared with the model group, there was no significant difference in each treatment group, and the data had no statistical difference (P>0.05), indicating that liposome TPP-SS-ATS-LS had no effect on mouse liver and kidney function. Obvious effect (Figure 21). Description Liposome TPP-SS-ATS-LS has the advantages of safety and low toxicity.

6. Statistical analysis of data

The experimental results are expressed as mean ± standard deviation (mean ± SD), using Graphpad Prism software, using one-way analysis of variance for overall and comparative analysis between two groups, if the variance is not homogeneous, Dunnett's test is used. P<0.05 indicated that the difference was statistically significant.

The anti-breast cancer effects of new artemisinin derivative liposomes are shown in Figures 18-21.

Although specific embodiments of the present invention are disclosed for the purpose of illustration, the purpose is to help understand the content of the present invention and implement it accordingly. Those skilled in the art can understand that: without departing from the spirit and scope of the present invention and the appended claims Inside, various substitutions, changes and modifications are possible. Therefore, the present invention should not be limited to the disclosed content of the preferred embodiment, and the protection scope of the present invention should be defined by the claims.

Claims (10)

1. A novel artemisinin derivative is characterized in that the structure of the novel artemisinin derivative is shown as the following formula:

2. a preparation method of novel artemisinin derivative liposome comprises the following steps: firstly synthesizing artemisinin derivative TPP-SS-ATS which is sensitive to glutathione GSH and has a mitochondrion targeting effect; then preparing the novel artemisinin derivative liposome TPP-SS-ATS-LS by adopting a film dispersion method.

3. The method as claimed in claim 2, wherein the novel artemisinin derivative liposomes have normal distribution of particle size and regular spherical shape observed by transmission electron microscopy.

4. The method of claim 2, wherein the artemisinin derivative TPP-SS-ATS is synthesized by:

(1) Weighing artesunate and 2, 2-dithiodiethanol, dissolving in an organic solvent, wherein the dosage ratio of the artesunate to the 2, 2-dithiodiethanol is 1 to 50, reacting for 0.1-96 hours at-20 to 40 ℃, and recovering the organic solvent at 20-100 ℃ by using a rotary evaporator after the reaction is finished to obtain a crude product of SS-ATS; purifying the intermediate SS-ATS crude product by a silica gel column by taking petroleum ether and ethyl acetate solution as a mobile phase to obtain an intermediate SS-ATS;

(2) Weighing an intermediate SS-ATS and 4-carboxybutyltriphenylphosphonium bromide TPP, dissolving in an organic solvent, wherein the dosage ratio of the intermediate SS-ATS to the 4-carboxybutyltriphenylphosphonium bromide TPP is 1; and (3) taking dichloromethane-methanol solution as a mobile phase, and purifying the crude product by adopting a silica gel column to obtain the novel artemisinin derivative TPP-SS-ATS.

5. The method as claimed in claim 2, wherein the thin film dispersion method is used to prepare the novel artemisinin derivative liposome TPP-SS-ATS-LS by the following steps: dissolving phospholipid, alkyl glycoside, novel artemisinin derivative TPP-SS-ATS, cholesterol and dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 2000DSPE-PEG2000 in organic solvent; and transferring the obtained solution into an evaporation bottle of a rotary evaporator, concentrating at 20-100 ℃ under reduced pressure until the organic solvent is completely volatilized, adding ultrapure water into the rotary evaporation bottle, hydrating at 20-80 ℃ for 0.1-3.0 hours, and then sequentially performing ultrasonic oscillation and microfiltration membrane filtration to obtain the novel artemisinin derivative liposome TPP-SS-ATS-LS.

6. The method as claimed in claim 5, wherein the weight percentage of phospholipid is 0.2-5.0%, the weight percentage of cholesterol is 0.01-0.5%, the weight percentage of alkyl glycoside is 0.2-3.0%, the weight percentage of dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 2000 is 0.02-5.0%, the weight percentage of novel artemisinin derivative TPP-SS-ATS is 0.02-3.0%, and the weight percentage of purified water is 73.5-99.0%.

7. The method according to claim 5 or 6, wherein the phospholipid is one or more of soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin and PEGylated phospholipid.

8. The method as claimed in claim 5 or 6, wherein the alkyl glycoside is one or more of alkyl glycoside containing 2-30 carbon atoms in alkyl group and 1-10 fragment number of glucose molecule.

9. The method according to claim 4, wherein the ratio of petroleum ether to ethyl acetate is in the range of 10; the ratio range of dichloromethane to methanol is 500-50; purifying by adopting a 50-500 mesh silica gel column.

10. Use of the novel artemisinin derivative liposome prepared by the method of claim 2 in preparation of antitumor drugs.

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