CN113072582B - A kind of lipid derivative containing π-π conjugated pyridyl, preparation method and application - Google Patents

Abstract

The invention provides a π-π conjugated pyridyl lipid derivative, a preparation method and application thereof; the synthesis method of the π-π conjugated pyridyl lipid derivative of the present invention is simple, the cost of raw materials is low, and the reaction reagents are easy to use. The method has no pollution and mild reaction conditions; the method of the invention has good popularization and is beneficial to large-scale production; The π-π stacking effect is used to significantly increase the drug loading of the doxorubicin nanoliposome and improve the stability of the preparation, realize the oxidatively stimulated release of the drug to the tumor tissue, and enhance the curative effect; the π-π conjugated pyridyl lipid provided by the present invention When the doxorubicin pyridyl functionalized liposome prepared by the derivative is used for in vivo test, it has no toxic and side effects on the kidney and liver of mice, and will not affect the normal growth of mice, and has high bioavailability.

Description

A kind of lipid derivative containing π-π conjugated pyridyl, preparation method and application

technical field

The invention belongs to the field of biomedicine; in particular, it relates to a π-π conjugated pyridyl lipid derivative, a preparation method and an application.

Background technique

Nano-drugs refer to drugs with a size of 20-200 nm formed by encapsulating anti-tumor drugs into carriers through certain physical encapsulation or chemical bonding methods. Compared with traditional small-molecule chemotherapeutics, nanodrugs have the following advantages: 1) improve drug solubility and stability, and prolong their blood circulation time; 2) use enhanced high flux and retention effect (EPR) to passively target tumors to tumors 3) Active targeting molecules mediated by antigen-antibody and receptor-ligand can be designed to achieve high drug uptake and high penetration into tumor tissue, reduce toxic and side effects, and achieve better therapeutic effects; 4) Introducing stimuli responses (exogenous and endogenous stimuli) to regulate the controlled release of the loaded drug to further improve the curative effect. Based on this, nanomedicine will significantly improve the therapeutic effect of tumor-related diseases.

As a nano-drug carrier, liposomes have been widely used to deliver small molecule drugs and nucleic acids, and a variety of liposome nano-drugs have been approved for clinical treatment of tumors, such as doxorubicin liposomes, daunorubicin Liposomes and Cytarabine Liposomes. However, clinical results show that the therapeutic effect of nanomedicine is still not completely satisfactory, which is mainly attributed to its low drug loading (<10%), poor stability, insufficient endocytosis into tumor cells and slow intracellular release. and other internal problems. Therefore, how to seek advantages and avoid disadvantages, promote strengths and avoid weaknesses, and maximize the detoxification and efficiency enhancement of liposome nanomedicines is of great significance for tumor drug treatment and has great clinical application prospects.

Aiming at the functional challenges that the existing liposome nano-drugs are difficult to meet the high drug loading and high stability, and at the same time can be released in response to the intracellular functionalization, the present invention provides a disulfide compound containing a π-π conjugated pyridyl group. Phosphatidylcholine and its application in doxorubicin nanomedicine. The strong π-π stacking interaction between pyridyl lipid materials and doxorubicin drug molecules can effectively achieve stable and efficient drug loading. However, redox degradation can occur in the tumor microenvironment, and the encapsulated drug can be rapidly released. Efficacy. The dithiopyridyl lipid derivative and the doxorubicin liposome prepared by the present invention have the characteristics of high drug loading and high stability, and to a certain extent solve the problem of the doxorubicin liposome currently used in clinical practice. The existing low dosage and safety hazards are of great significance for the development of functional carrier materials with my country's independent intellectual property rights.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a lipid derivative containing π-π conjugated pyridyl, a preparation method and an application.

The present invention is achieved through the following technical solutions:

In the first aspect, the present invention relates to a lipid derivative containing a π-π conjugated pyridyl, and its chemical structure is represented by the general formula (I):

in,

N is an integer from 5 to 8;

The π-π conjugated lipid derivative uses a biocompatible pyridyl group as the hydrophobic part of the lipid molecule, provides a π-π stacking effect, and is non-covalently bonded to a small molecule hydrophobic drug with an aromatic structure to enhance the Stability and drug loading during drug delivery.

In the second aspect, the present invention also relates to a method for synthesizing the aforementioned π-π conjugated pyridyl lipid derivatives, the specific steps are as follows:

Step 1, the mercaptoalkanoic acid (I-1) is dissolved in anhydrous dichloromethane (or chloroform, or toluene), and the dichloromethane solution of triphenylchloromethane is added dropwise under stirring, and the two charge moles. The ratio is 1:1-1:5, and the reaction at room temperature is 4-12h. After the reaction, dichloromethane (or chloroform, or toluene) was removed by rotary evaporation; after concentration, recrystallized with ethyl acetate (or acetonitrile or petroleum ether) to obtain triphenyl ether-protected mercaptoalkanoic acid (I- 2), the chemical reaction equation of this step is as follows:

Step 2, dissolve the triphenyl ether-protected mercaptoalkanoic acid (I-2) in dimethyl sulfoxide (or dimethyl sulfoxide/dichloromethane mixture v/v=1:1), add under stirring Activator CDI, the molar ratio of the two materials is 1:1-1:2.5, and the temperature is raised to 35-45°C for reaction for 2-4h. After the activation was completed; GPC and catalyst DBU were added, and the reaction was continued for 12-14 h; after the reaction was completed, the reaction solution was precipitated with an ether solution containing 10% glacial acetic acid, and after concentrating, chloroform/methanol/water (the volume ratio in the figure was: v /v/v=65:25:4) column chromatography to obtain bistriphenyl ether protected mercaptoalkanoic acid glycerophosphatidylcholine (I-3), the chemical reaction equation of this step is as follows:

Step 3, dissolving bistriphenyl ether protected mercaptoalkanoic acid glycerophosphatidylcholine (I-3) in trifluoroacetic acid/dichloromethane solution (volume ratio is v/v=1:1), deprotection reagent and The molar ratio of the long carbon chain phosphatidylcholine protected by the bis-thiol group is 5:1-10:1, and the deprotection time is 1.5-3h; no further post-treatment is required, and the deprotection reagent is removed by rotary evaporation at 50 °C; The obtained deprotected bisthiol long carbon chain phosphatidylcholine is dissolved in dichloromethane (or methanol or trichloromethane or dimethylformamide), and dithiodipyridine is added under stirring, and the two molar ratios are 1:2.5- 1:6, heat up to 35-45°C, and react for 24-48h; after the reaction, concentrate the reaction solution, use chloroform/methanol/water (volume ratio is v/v/v=65:25:4) column layer analysis to obtain bis-mercaptopyridyl glycerol phosphatidylcholine (I-4), and the chemical reaction equation of this step is as follows:

The present invention provides a method for synthesizing the above-mentioned π-π conjugated pyridyl lipid derivatives. The synthesis method is efficient and fast, has good versatility, high yield, low synthesis cost, and is environmentally friendly in the synthesis process, and is suitable for industrial scale-up production.

The present invention also provides the application of the π-π conjugated pyridyl lipid derivative described in the above technical solution in preparing functionalized blank liposomes and in drug carriers.

In the third aspect, the functionalized blank liposome prepared by the π-π conjugated pyridyl lipid derivative provided by the present invention has an average particle diameter of 120-300 nm, is spherical and uniform in size;

Commonly used phospholipids include one or more of lecithin, soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, distearoyl phosphatidyl choline, distearoyl phosphatidyl glycerol, dipalmitoyl lecithin, etc.;

Preferred are soybean lecithin, egg yolk lecithin and hydrogenated soybean lecithin;

The molar ratio of the phospholipid, cholesterol and DSPE-PEG2000 is 50-60:30-45:32-45:4-8, preferably 56:35:38:6;

The hydration temperature of the blank liposome is 50-60°C, and the hydration time is 1-3h;

In the fourth aspect, the present invention also provides a preparation method of the doxorubicin-loaded functionalized liposome, wherein the preparation is prepared by an ammonium sulfate gradient method.

The preparation method of the doxorubicin pyridyl functionalized liposome is as follows:

1) Preparation of blank liposomes: Dissolve disulfide π-π conjugated pyridyl phospholipids, soybean lecithin, cholesterol and DSPE-PEG2000 in chloroform/methanol (volume ratio is v/v=4: 1), after rotating at 40-50°C to form a film, hydrate with 100-300mmol/L ammonium sulfate for 1-3h;

2) Prepare ammonium sulfate gradient blank liposomes by dialysis method: place the blank liposome liquid described in step 1) in a dialysis bag (molecular weight cut-off 3500D), and use phosphate buffered saline (PBS, pH=7.4) as a dialysis medium , dialysis was performed for 4-6 h; liposome vesicles passed through polycarbonate membranes with diameters of 800 nm, 450 nm and 220 nm in turn, and finally formed ammonium sulfate gradient blank liposomes;

3) Mix the ammonium sulfate gradient blank liposome in step 2) with the doxorubicin aqueous solution, and incubate at 40-50° C. for 40 min to obtain doxorubicin pyridyl functionalized liposome.

Wherein, the drug-to-lipid mass ratio of doxorubicin and phospholipid is 1:5-1:20, preferably 1:8-1:14;

The doxorubicin drug pyridyl liposome provided by the invention has an average particle diameter of 150-300 nm, is spherical and uniform in size;

The fifth aspect, the use of the above-mentioned adriamycin liposome provided by the present invention in the treatment of tumors; the tumors include leukemia (lymphocytic and granulocytic), malignant lymphoma, breast cancer, bronchial lung cancer (undifferentiated small cellular and non-small cell), ovarian cancer, soft tissue sarcoma, osteoblastic sarcoma, rhabdomyosarcoma, Ewing sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, testis cancer, stomach or liver cancer.

The present invention has the following advantages:

1) The synthesis method of the π-π conjugated pyridyl lipid derivatives of the present invention is simple, the cost of raw materials is low, the reaction reagents are easily obtained without pollution, and the reaction conditions are mild; the method of the present invention has good generalizability and is conducive to large-scale production. ;

2) Compared with the traditional liposome preparation, the π-π conjugated pyridyl liposome provided by the present invention can significantly increase the drug loading of the doxorubicin nanoliposome and improve the π-π stacking effect. The stability of the preparation can realize the oxidatively stimulated release of the drug to the tumor tissue and enhance the curative effect;

3) When the doxorubicin pyridyl functionalized liposome prepared by the π-π conjugated pyridyl lipid derivative provided by the present invention is used in the in vivo test, it has no toxic and side effects on the kidney and liver of mice, and will not affect Normal growth of mice, high bioavailability;

4) A novel π-π conjugated pyridyl lipid derivative with high drug loading and high stability provided by the present invention will become a novel carrier platform for clinical chemotherapy treatment.

Description of drawings

Fig. 1 is blank pyridyl liposome preparation chemical characterization results of the present invention: a) particle size distribution diagram; b) transmission electron microscope diagram;

Fig. 2 is the in vitro release result figure of doxorubicin pyridyl liposome of the present invention;

Fig. 3 is the influence figure of the doxorubicin pyridyl liposome of the present invention on the survival rate of MCF-7 cells;

Fig. 4 is the influence figure of the doxorubicin pyridyl liposome of the present invention on A549 cell viability;

Fig. 5 is the influence figure of the doxorubicin pyridyl liposome of the present invention on the survival rate of HepG-2 cells;

Fig. 6 is the in vivo tumor growth curve diagram of the doxorubicin preparation of the present invention against MCF-7 tumor-bearing nude mice

Figure 7 is a graph showing the in vivo body weight change of the doxorubicin preparation of the present invention against MCF-7 tumor-bearing nude mice.

FIG. 8 is a reaction schematic diagram of the synthesis method of the π-π conjugated pyridyl lipid derivative according to the present invention.

Detailed ways

The present invention will be described in detail below with reference to specific embodiments. It should be noted that the following examples are only further descriptions of the present invention, but the protection scope of the present invention is not limited to the following examples.

Example 1

The synthesis of triphenyl ether protected mercaptoundecanoic acid, the chemical structure is as follows:

1 g/4.58 mmol of mercaptoundecanoic acid was dissolved in 20 mL of anhydrous dichloromethane, and a solution of 2.55 g/9.16 mmol of triphenylchloromethane in dichloromethane was added dropwise while stirring, and the reaction was carried out at room temperature for 6 h. After the reaction, the reaction solution was concentrated and recrystallized twice with 200 mL of petroleum ether to obtain 2.01 g of a white solid with a yield of 95.2%.

¹ H NMR (500MHz, CDCl ₃ ): δ 7.36-7.23(m, 9H), 2.59(s, 1H), 2.27(s, 1H), 1.63(s, 1H), 1.47(s, 1H), 1.40 (d, J=7.7Hz, 2H), 1.31 (dd, J=18.0, 5.5Hz, 5H); ¹³ C NMR (125MHz, CDCl ₃ ): δ 177.13 (s, 1H), 145.34 (s, 7H) ,129.38(s,14H),129.13(s,15H),127.79(s,3H),64.64(s,2H),34.64(s,2H),30.23(s,5H),28.95(t,J=1.6 Hz,15H),24.81(s,2H).HRMS,ESI ⁺ ,m/z:Calcd for C ₃₀ H ₃₆ O ₂ S[MH] ^- : 459.24; found 459.24.

Example 2

The synthesis of glycerol phosphatidylcholine protected by bistriphenyl ether, the chemical structure is as follows:

0.46 g/0.99 mmol of triphenyl ether protected mercaptoundecanoic acid and 0.24 g/1.49 mmol of CDI were dissolved in 15 mL of anhydrous dimethyl sulfoxide, and activated at 35 °C for 2 h. To the above reaction system, 0.10 g/0.39 mmol glycerophosphocholine and 0.23 g/1.49 mmol DBU were continuously added, and the reaction was carried out overnight at 45°C. After the reaction was completed, the reaction solution was precipitated with an ether solution containing 10% glacial acetic acid, and after concentration, chloroform/methanol/water (v/v/v=65:25:4) column chromatography was used to obtain 0.64g of bistriphenylene Glycerol phosphatidylcholine protected by ether-protected mercaptoalkanoic acid, yield: 56.4%.

¹ H NMR (500 MHz, CDCl ₃ ): δ 7.40-7.34 (m, 14H), 7.34-7.25 (m, 16H), 5.43 (s, 1H), 4.51 (s, 1H), 4.29 (d, J= 8.0Hz, 3H), 3.99(d, J=30.1Hz, 2H), 3.80(s, 2H), 3.24(s, 9H), 2.52(s, 2H), 2.47–2.38(m, 6H), 1.74( s, 1H), 1.73–1.64 (m, 6H), 1.49–1.41 (m, 6H), 1.41–1.13 (m, 35H). ¹³ C NMR (125MHz, CDCl ₃ ): δ174.55 (s, 1H) ,174.36(s,2H),145.34(s,14H),129.38(s,28H),129.13(s,29H),127.79(s,14H),69.29(s,2H),68.20(s,2H), 66.81(s, 2H), 64.64(s, 5H), 64.25(s, 2H), 60.53(s, 1H), 54.72(s, 5H), 34.09(d, J＝15.0Hz, 4H), 30.23(s ,10H),29.07–28.74(m,47H),25.33(s,5H).HRMS,ESI ⁺ ,m/z:Calcd forC ₆₈ H ₈₈ NO ₈ PS ₂ [M+H] ⁺ :1142.57; found 1142.57.

Example 3

Synthesis of bismercaptoundecylglycerol phosphatidylcholine, the chemical structure is as follows:

Dissolve 0.2g/0.17mmol of bistriphenyl ether-protected mercaptoalkanoic acid glycerophosphatidylcholine in 10mL trifluoroacetic acid/dichloromethane (v/v=1:1) solution, react at room temperature for 4h; at 50°C , using rotary evaporation to remove the deprotection reagent of trifluoroacetic acid; to the above system, add 0.15g of 0.68mmol dithiodipyridine in dichloromethane solution (10mL), and continue to react at room temperature for 24h; Methane/methanol/water (v/v/v=65:25:4) column chromatography to obtain 0.13 g of dimercaptoundecylglycerol phosphatidylcholine, yield: 86.3%.

¹ H NMR (500 MHz, CDCl ₃ ): δ 8.26 (dd, J=7.5, 1.4 Hz, 3H), 7.57-7.52 (m, 3H), 7.35 (dd, J=7.5, 1.4 Hz, 3H), 7.20 –7.15(m,3H), 5.45(s,1H), 4.63–4.42(m,2H), 4.42–4.38(m,1H), 4.33(s,4H), 4.01–3.97(m,1H), 3.80 (s, 3H), 3.24 (s, 12H), 2.56–2.52 (m, 5H), 2.50 (s, 3H), 2.42 (s, 3H), 1.69 (d, J=5.9Hz, 5H), 1.63– 1.60 (m, 4H), 1.54 (dd, J=47.8, 3.9Hz, 2H), 1.60–1.16 (m, 34H). ¹³ C NMR (125MHz, CDCl ₃ ): δ 174.55 (s, 2H), 174.36 (s,1H),160.01(s,3H),145.72(s,3H),139.66(s,3H),121.37(s,3H),119.67(s,1H),69.29(s,1H),68.20( s, 1H), 66.81(s, 1H), 64.25(s, 1H), 60.53(s, 1H), 54.72(s, 5H), 38.03(s, 3H), 34.09(d, J=15.0Hz, 3H ),28.93(dd,J＝6.7,5.0Hz,19H),25.33(s,1H).HRMS,ESI ⁺ ,m/z:Calcd forC ₄₀ H ₆₆ N ₃ O ₈ PS ₂ [M+H] ⁺ : 876.35; found 876.35.

Example 4

The synthesis of triphenyl ether protected mercaptohexadecanoic acid, the chemical structure is as follows:

1 g/3.47 mmol of mercaptoundecanoic acid was dissolved in 20 mL of anhydrous toluene, and a toluene solution of 1.45 g/5.19 mmol of triphenylchloromethane was added dropwise while stirring, and the reaction was carried out at room temperature for 6 h. After the reaction, the reaction solution was concentrated and recrystallized twice with 200 mL of petroleum ether to obtain 1.67 g of a white solid with a yield of 93.1%.

¹ H NMR (500 MHz, CDCl ₃ ): δ 7.37-7.26 (m, 7H), 7.22-7.18 (m, 2H), 2.54 (s, 1H), 2.28 (s, 1H), 1.65 (d, J= 17.9Hz, 2H), 1.46(s, 1H), 1.43–1.30(m, 10H). ¹³ C NMR (125MHz, CDCl ₃ ): δ 177.13(s, 1H), 145.34(s, 7H), 129.38( s, 14H), 129.13(s, 15H), 127.79(s, 7H), 64.64(s, 2H), 34.64(s, 2H), 30.23(s, 5H), 29.07–28.74(m, 24H), 24.81 (s,2H).HRMS, ESI ⁺ ,m/z: Calcd for C ₃₄ H ₄₄ O ₂ S[MH] ^- : 515.34; found 515.34.

Example 5

Synthesis of bistriphenyl ether-protected hexadecanoic acid glycerol phosphatidylcholine, the chemical structure is as follows:

0.52/1.0 mmol of triphenyl ether protected mercaptohexadecanoic acid and 0.24 g/1.5 mmol of CDI were dissolved in 15 mL of anhydrous dimethyl sulfoxide, and activated at 35 °C for 2 h. To the above reaction system, 0.10 g/0.40 mmol glycerophosphocholine and 0.24 g/1.5 mmol DBU were continuously added, and the reaction was carried out at 45°C overnight. After the reaction was completed, the reaction solution was precipitated with an ether solution containing 10% glacial acetic acid, and after concentration, chloroform/methanol/water (v/v/v=65:25:4) column chromatography was used to obtain 0.67g of bistriphenylene Glycerol phosphatidylcholine protected with thioalkanoic acid by ether, yield: 53.8%.

¹ H NMR (500 MHz, CDCl ₃ ): δ 7.40-7.34 (m, 14H), 7.34-7.25 (m, 16H), 5.43 (s, 1H), 4.51 (s, 1H), 4.29 (d, J= 8.0Hz, 3H), 3.99(d, J=30.1Hz, 2H), 3.80(s, 2H), 3.24(s, 9H), 2.52(s, 2H), 2.47–2.38(m, 6H), 1.74( s, 1H), 1.73–1.64 (m, 6H), 1.49–1.41 (m, 6H), 1.41–1.13 (m, 35H). ¹³ C NMR (125MHz, CDCl ₃ ): δ174.55 (s, 1H) ,174.36(s,2H),145.34(s,14H),129.38(s,28H),129.13(s,29H),127.79(s,14H),69.29(s,2H),68.20(s,2H), 66.81(s, 2H), 64.64(s, 5H), 64.25(s, 2H), 60.53(s, 1H), 54.72(s, 5H), 34.09(d, J＝15.0Hz, 4H), 30.23(s ,10H),29.07–28.74(m,47H),25.33(s,5H).HRMS,ESI ⁺ ,m/z:Calcd for C ₇₆ H ₁₀₄ NO ₈ PS ₂ [M+H] ⁺ :1254.69; found 1254.69.

Example 6

Synthesis of bis-mercaptohexadecyl glycerol phosphatidylcholine, the chemical structure is as follows:

0.2 g/0.16 mmol of bistriphenyl ether protected mercaptoalkanoic acid glycerophosphatidylcholine was dissolved in 10 mL of trifluoroacetic acid/dichloromethane (v/v=1:1) solution, and reacted at room temperature for 4 h. Without further workup, the trifluoroacetic acid deprotection reagent was removed by rotary evaporation at 50°C. To the above system, a methanol solution (10 mL) of 0.15 g/0.68 mmol of dithiodipyridine was added, and the reaction was continued at room temperature for 24 h. After the completion of the reaction, chloroform/methanol/water (v/v/v=65:25:4) was used for column chromatography after concentration to obtain 0.15g of bis-mercaptohexadecylpyridyl glycerophosphatidylcholine, yield: 91.3 %.

¹ H NMR (500 MHz, CDCl ₃ ): δ 8.33-8.19 (m, 3H), 7.58-7.53 (m, 2H), 7.45-7.31 (m, 3H), 7.23-7.18 (m, 2H), 5.36 ( s, 1H), 4.74(s, 1H), 4.35(s, 1H), 4.27(s, 2H), 3.94(d, J=0.8Hz, 2H), 3.80(s, 2H), 3.24(s, 11H ), 2.64–2.60(m, 5H), 2.50(s, 3H), 2.46(s, 2H), 1.72(d, J=13.9Hz, 5H), 1.65–1.56(m, 5H), 1.50–1.45( m, 5H), 1.42–1.28 (m, 45H). ¹³ C NMR (125MHz, CDCl ₃ ): δ 174.55 (s, 2H), 174.36 (s, 1H), 160.01 (s, 1H), 145.72 (s) ,3H),139.66(s,3H),121.37(s,3H),119.67(s,3H),69.29(s,1H),68.20(s,1H),66.81(s,1H),64.25(s, 1H), 60.53(s, 1H), 54.72(s, 5H), 38.03(s, 3H), 34.09(d, J=15.0Hz, 3H), 28.93(dd, J=6.7, 5.0Hz, 30H), 25.33(s,1H).HRMS,ESI ⁺ ,m/z:Calcd for C ₄₈ H ₈₂ N ₃ O ₈ PS ₄ [M+H] ⁺ : 988.47; found988.47.

Example 7

Preparation and characterization of blank pyridyl-functionalized liposomes

Blank pyridyl functionalized liposomes were prepared by thin film dispersion method. Weigh the disulfide π-π conjugated pyridyl phospholipid and lecithin, cholesterol and DSPE-PEG2000 in Example 1 according to the molar ratio of 56:35:38:6, and use 15 mL of chloroform/methanol (v/v=4: 1) Dissolve, the lipid solution is uniformly dispersed under _N2 atmosphere to form a film, and vacuum dried at 35°C for 10-14h. Use deionized water or phosphate buffer (pH=7.4) for lipid hydration, the hydration temperature is 50-60°C, and the hydration time is 1-3h. After ultrasonic treatment and 0.22μm microporous membrane filtration, the concentration of 1.5-4mg/mL π-π conjugated pyridyl functionalized liposomes was obtained, which was stored at 4 °C for future use;

Take 1 mL of the above blank pyridyl liposome suspension diluted 20 times with normal saline (pH=7.4), and then use a laser particle size analyzer (DLS) to measure its particle size and particle size distribution, as shown in Figure 1a, the blank pyridyl liposome The average particle size of the liposomes was 165±13 nm.

Take 10 μL of the 20-fold diluted liposome suspension, drop it onto a copper mesh supported by a 300-mesh carbon membrane, and stain with 2% phosphotungstic acid for 5 min. After drying at room temperature, the nanomorphology of the liposomes was observed by transmission electron microscopy (TEM), as shown in Figure 1b, and the liposomes were spherical and uniform in size.

Example 8

Preparation and characterization of doxorubicin-pyridyl-functionalized liposomes.

Doxorubicin-pyridyl-functionalized liposomes were prepared by ammonium sulfate gradient method. Weigh the disulfide π-π conjugated pyridyl phospholipid and lecithin, cholesterol and DSPE-PEG2000 in Example 1 according to the molar ratio of 56:35:38:6, and use 15 mL of chloroform/methanol (v/v=4: 1) Dissolve, the lipid solution is uniformly dispersed under _N2 atmosphere to form a film, and vacuum dried at 35°C for 10-14h. Hydrate with 100-300mmol/L ammonium sulfate for 1-3h;

Further, the above-mentioned blank liposome liquid was placed in a dialysis bag (molecular weight cut-off 3500D), and phosphate buffered saline (PBS, pH=7.4) was used as the dialysis medium, and the dialysis time was 4-6h. The liposome vesicles passed through polycarbonate membranes with diameters of 800 nm, 450 nm and 220 nm in turn to form ammonium sulfate gradient blank liposomes. Finally, the ammonium sulfate gradient blank liposome was mixed with an aqueous solution of doxorubicin, in which the drug-lipid mass ratio of doxorubicin and phospholipid was 1:12 or 1:20, respectively. Functionalized liposomes.

The traditional doxorubicin liposome was used as the control group, and the preparation method was the same as above, except that the traditional lecithin was used instead of the disulfide π-π conjugated pyridyl phospholipid. The particle size and potential of the two types of liposomes are shown in Table 1:

Table 1

Example 9

Doxorubicin Encapsulation Efficiency and Drug Loading Determination

Precisely pipette 1 mL of doxorubicin solution, freeze-dry it, and fully dissolve it with chromatography-grade methanol. The content of doxorubicin in the samples was measured by (UV/Vis) ultraviolet-visible spectrophotometry. Combined with the known free drug doxorubicin/water standard curve, the corresponding drug loading (DLC%) and encapsulation efficiency (DLE%) were calculated. Its formula is as follows:

Encapsulation efficiency (DLE%) = W _total - W _swimming / W _total × 100%

Among them, W _always represents the total drug content, and _W represents the free drug content

Drug loading (DLC%)=W _total- W _swim /Wc×100%

Wherein, Wc is the mixed lipid dosage

The encapsulation efficiency of different modified liposomes is shown in Table 2 below:

Table 2

As shown in Table 2 above, the encapsulation efficiency gradually increased with the decrease of the drug-to-lipid ratio. At the same drug-lipid ratio of 1:12 or 1:20, compared with traditional liposomes, pyridyl liposomes and doxorubicin have a stronger π-π stacking effect, showing better encapsulation efficiency. Its encapsulation rate reaches 85.62%, realizing stable and efficient encapsulation of drugs.

Example 10

In vitro response release assay

In vitro responsive release rates of doxorubicin-pyridyl-functionalized liposomes were determined by dialysis. 1.0 mL of doxorubicin pyridyl liposome was transferred to a cellulose ester dialysis bag (MWCO 3500 Da), and placed in 20 mL of dialysate containing different concentrations of 0.5 mM or 1 mM GSH at 37°C for dialysis. At a predetermined time point, 2 mL of the release medium was sampled and an equal amount of fresh PBS dialysate was added, and the drug release concentration at different time points was determined by HPLC. The formula for calculating the release rate (%) is: release rate (%)=(Wn/W)×100%. Wherein, Wn is the cumulative release amount of the liposome at a certain time point; W is the total amount of doxorubicin encapsulated in the liposome.

As shown in Figure 2, in the dialysate containing different concentrations of 0.5mM or 1mM GSH, the doxorubicin pyridyl liposome showed a high release rate, and the cumulative release of doxorubicin within 12h reached 85%, with obvious redox Responsiveness.

Example 11

Cytotoxicity test

1) Cell culture

Breast cancer cells MCF-7, lung cancer cells A549 and liver cancer cells HepG-2 were purchased from the Cell Bank of the Chinese Academy of Sciences in Shanghai. Take the above frozen cells and quickly place them in a 37°C water bath to thaw. The same volume of RPMI-1640 medium containing 10% fetal bovine serum was added, pipetted, dispersed and centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. Continue to add 1 mL of culture medium and transfer it to a cell culture flask, and incubate at a constant temperature under 5% CO ₂ and 37°C. After the density of the cultured cells grows to 80% confluence, they are digested with trypsin-EDTA digestion solution, passaged or subjected to further experiments.

2) MTT detection

Take log-phase MCF-7 or A549 or HepG-2 cells, plate them in a 96-well plate at a cell density of 1×10 ⁴ per well, and incubate overnight at 37°C, 5% CO ₂ . 200 μL of doxorubicin pyridyl liposomes at different concentrations were added to the experimental group, and 200 μL of blank medium was added to the control group. Each experimental group was set up with 6 test concentrations and each test concentration was set up with 6 replicate wells. Continue to culture for 24 hours after adding the drug, and add pre-prepared MTT solution (20 μL, 5 mg/mL) to each well for 4 hours in the dark. After that, the upper medium was discarded, 150 μL of biological grade DMSO was added to each well, and the absorbance value at 570 nm was detected by a microplate reader. Cytotoxicity was determined as the percentage of the absorbance value of each well after adding the sample to the absorbance value of the control group as the survival rate.

As shown in Figure 3 (MCF-7 cells), Figure 4 (A549 cells), and Figure 5 (HepG-2 cells), the toxicity of doxorubicin pyridyl liposomes to the three tumor cells was consistent, and the cytotoxicity increased with loading increased with increasing liposome concentration. Compared with free doxorubicin, pyridyl liposomes show stronger cell killing effect and have good application prospects in the field of drug delivery.

Example 12

In vivo pharmacodynamic experiments

1) Establishment of MCF-7 nude mouse xenograft tumor model

Female Balb/c nude mice, 5 weeks old, 16-18 g body weight, were provided by Jinan Pengyue Laboratory Animal Company. The cultured human breast cancer MCF-7 cell suspension was collected at a concentration of 1×10 ⁷ g/mL, and 0.1 mL each was subcutaneously inoculated into the right axilla of nude mice.

2) Pharmacodynamic experiments

After 14 days of inoculation, when the tumors grew to 100-150 mm, the animals ^were randomly divided into the negative control group with normal saline, and the groups administered with free doxorubicin and doxorubicin pyridyl liposome. It was administered by tail vein injection at a dose of 5 mg/kg, once every two days. Using vernier calipers to measure tumor width (W) and length (L), the anti-tumor effects of the test samples were dynamically observed.

In vivo pharmacodynamic evaluation:

1) Tumor volume growth curve: Before administration, measure the tumor growth width (W) and length (L) of each experimental group, calculate the tumor volume according to the following formula, and draw the trend of the tumor volume with the administration time, that is, the tumor volume growth curve:

Tumor volume=(L×W ² )/2

2) Tumor growth inhibition rate: After 24 days of administration, the nude mice were sacrificed, and the tumor mass was surgically removed and weighed and photographed. The tumor growth inhibition rate was calculated according to the following formula:

Tumor growth inhibition rate=(average tumor weight of administration group-average tumor weight of model group)/average tumor weight of model group×100 (Formula VI)

3) In vivo toxicity evaluation:

Body weight change of nude mice: Before administration, the body weight of nude mice in each experimental group was weighed, and the change curve of body weight with administration time was drawn to evaluate the in vivo safety of the given nanomedicine.

As shown in Figure 6, compared with the normal saline control group, traditional doxorubicin liposomes and doxorubicin pyridyl liposomes can largely inhibit the growth of tumor volume, and pyridyl liposomes (tumor inhibitory liposomes) Index 78.9±3.65%) was better than traditional liposomes (59.0±4.32%) in tumor inhibition in vivo, which was consistent with in vitro cell experiments, indicating that its oxidative responsiveness played an important role.

As shown in Figure 7, the weight change curve of mice shows that the free doxorubicin experimental group has the slowest weight gain and obvious toxic and side effects; the weight change curve of the mice in the pyridyl doxorubicin liposome group is similar to the normal saline group, which proves that , Nano-administration has the effect of reducing toxicity in vivo.

The derivative synthesized for the first time in the present invention has good safety, and the derivative is a redox pyridyl lipid derivative, which can be used in doxorubicin nanomedicine; the present invention is designed according to the complex aromatic ring structure of small molecule medicine Containing pyridyl dithioglycerol phosphatidyl choline as a drug carrier, the doxorubicin drug is prepared into a stable, high drug-loading and redox-responsive liposome nano-dosage form, avoiding the nano-formulation by the endothelial reticulum system ( RES), thereby prolonging blood circulation and improving the therapeutic effect.

The newly synthesized drug carrier pyridyl functionalized phospholipid structure of the present invention is as follows:

Moreover, the redox-responsive phospholipids prepared by the present invention undergo the following degradation reactions under the action of high-concentration GSH:

Because the redox-responsive pyridyl phospholipid derivative of the present invention has balanced amphiphilicity, it can self-assemble with cholesterol and DSPE-PEG2000 to form nanoliposomes through hydrophilic-hydrophobic and π-π stacking interactions to realize the anti-doxorubicin drug When the liposome is targeted to the tumor oxygen microenvironment, the phospholipid disulfide structure responds to rupture in the tumor cells with high GSH expression, and the liposome nanostructure is destroyed, thus releasing doxorubicin in response.

Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various variations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims (10)

1. a kind of containing π-π conjugated pyridyl lipid derivative, is characterized in that, has following structure:

. 2. a synthetic method containing π-π conjugated pyridyl lipid derivatives as claimed in claim 1, is characterized in that, comprises the steps: Step 1, dissolve the mercaptoalkanoic acid in the reaction solvent, add the dichloromethane solution of the protective reagent dropwise with stirring, the molar ratio of the protective reagent to the mercaptoalkanoic acid is 1:1-1:5, and the reaction at room temperature is 4- 12 h; after the reaction was completed, dichloromethane was removed by rotary evaporation, after concentration, recrystallization and purification were performed to obtain the mercaptoalkanoic acid protected by triphenyl ether; In step 2, the triphenyl ether-protected mercaptoalkanoic acid is dissolved in the reaction solvent, and a coupling reagent is added under stirring, the molar ratio of the two is 1:1-1:2.5, and the activation reaction is performed at 35-45 ° C for 2-4 h; Add glycerophosphocholine and catalyst, and react for 12-14h; sediment the reaction solution with ether solution containing 10% glacial acetic acid, after concentration, use the elution solvent chloroform/methanol/water volume ratio: v/v/v=65 : 25:4 column chromatography to obtain bistriphenyl ether protected mercaptoalkanoic acid glycerophosphatidylcholine; Step 3, dissolving the bistriphenyl ether-protected mercaptoalkanoic acid glycerophosphatidylcholine in the deprotection reagent, the deprotection time is 1.5-3 h; at 50 °C, remove the deprotection reagent by rotary evaporation; The deprotected bis-mercapto long-carbon chain phosphatidylcholine is dissolved in the reaction solvent, and the dithiodipyridine is added under stirring. The molar ratio of the deprotected bis-mercapto long-carbon chain phosphatidylcholine and the dithiodipyridine is 1:2.5- 1:6, heat up to 35-45°C, and react for 24-48 h; after the reaction is over, use the elution solvent chloroform/methanol/water volume ratio after concentration: v/v/v=65:25:4 column Chromatography to obtain lipid derivatives containing π-π conjugated pyridyl groups. 3. the synthetic method that contains π-π conjugated pyridyl lipid derivatives as claimed in claim 2, is characterized in that, in step 1, described protective reagent is triphenylchloromethane; Described mercaptoalkanoic acid is mercaptoundecanoic acid, mercaptododecanoic acid or mercaptohexadecanoic acid; the reaction solvent is dichloromethane, toluene, chloroform or dimethylformamide; the recrystallization solvent in the recrystallization purification is ethyl acetate ester, acetonitrile or petroleum ether. 4. the synthetic method that contains π-π conjugated pyridyl lipid derivatives as claimed in claim 2, is characterized in that, in step 2, described reaction solvent is dimethyl sulfoxide, methylene chloride or dimethyl A mixture of sulfoxide and dichloromethane; the coupling reagent is N,N'-carbonyldiimidazole or 1,8-diazabicyclo[5.4.0]undec-7-ene; purified by silica gel column chromatography , to obtain a long carbon chain phosphatidylcholine protected by a white waxy solid bis-mercapto group, and the elution solvent is a mixture of chloroform, methanol, and water in a volume ratio of 65:25:4. 5. the synthetic method that contains π-π conjugated pyridyl lipid derivatives as claimed in claim 2, is characterized in that, in step 3, described deprotection reagent is that trifluoroacetic acid, methyl chloride solution volume ratio is 1 : 1 mixture; the molar ratio of the deprotection reagent and the long carbon chain phosphatidylcholine protected by bisthiol is 5:1-10:1, and the reaction is carried out at 50°C; the reaction solvent is dichloromethane, methanol, trichloromethane Chloromethane or dimethylformamide; silica gel column chromatography to obtain a white waxy solid, and the elution solvent is a mixture of chloroform, methanol, and water in a volume ratio of 65:25:4. 6. A blank functionalized liposome prepared by the π-π conjugated pyridyl lipid derivative according to claim 1, wherein the liposome has an average particle diameter of 120-300 nm, which is Spherical, uniform in size. 7. the preparation method of the blank functionalized liposome prepared by containing π-π conjugated pyridyl lipid derivatives as claimed in claim 6, is characterized in that, described method comprises the following steps: 1) Dissolve the π-π conjugated pyridyl lipid derivative and co-phospholipid in a solution of chloroform and methanol in a volume ratio of 4:1 according to a certain molar ratio, containing π-π conjugated pyridyl lipid The concentration of the lipid derivative is 1.5-4 mg/mL, and the molar ratio of the lipid derivative containing π-π conjugated pyridyl to soybean lecithin, cholesterol and DSPE-PEG2000 is 50-60:30-45:32 -45:4-8; 2) The lipid solution is uniformly dispersed to form a film under N ₂ atmosphere, and dried under vacuum at 35°C for 10-14 h; 3) Use deionized water or phosphate buffer for lipid hydration, hydrate at 50-60 °C for 1-3 h; after ultrasonic treatment and 0.22 μm microporous membrane filtration, the concentration of 1.5-4 mg/mL is obtained. The π-π conjugated pyridyl functionalized liposome can be stored at 4°C. 8. a method for preparing doxorubicin pyridyl-functionalized liposomes from a blank functionalized liposome prepared by containing π-π conjugated pyridyl lipid derivatives as claimed in claim 6, wherein the The method includes the following steps: 1) Preparation of blank liposomes: Dissolve π-π conjugated pyridyl lipid derivatives and co-phospholipids in a solution of chloroform and methanol with a volume ratio of 4:1 according to a certain molar ratio, 40-50 Rotate to form a film at ℃; hydrate with 100-300 mmol/L ammonium sulfate for 1-3 h; 2) Prepare ammonium sulfate gradient blank liposomes by dialysis: place the blank liposome fluid in a dialysis bag, and use phosphate buffer as a dialysis medium for dialysis for 4-6 hours; liposome vesicles pass through diameters in turn 800 nm, 450 nm and 220 nm polycarbonate membranes, finally forming ammonium sulfate gradient blank liposomes; 3) Mix the ammonium sulfate gradient blank liposome and the doxorubicin aqueous solution according to the mass ratio of 1:5-1:20, and incubate at 40-50 °C for 40 min to obtain the doxorubicin pyridyl functionalized liposome. 9 . The application of the doxorubicin pyridyl functionalized liposome as claimed in claim 8 in the preparation of a medicament for treating tumors. 10 . 10. The application of the doxorubicin pyridyl functionalized liposome as claimed in claim 9 in the preparation of a medicament for the treatment of tumors, wherein the tumors include leukemia, malignant lymphoma, breast cancer, bronchial lung cancer, ovary Carcinoma, soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, testicular cancer, stomach cancer or liver cancer.

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