Cis-platinum-polyphenol compound lipidosome for tumor treatment and preparation method of cisplatin-polyphenol compound lipidosome

Abstract

The invention discloses a cisplatin-polyphenol compound liposome for tumor treatment. The cisplatin-polyphenol compound liposome comprises a cisplatin-polyphenol compound and a liposome, the cis-platinum-polyphenol compound is a compound formed by cis-platinum and polyphenol mainly through ionic bonds and coordinate bonds; the cisplatin-polyphenol compound liposome is prepared from a cisplatin-polyphenol compound, phospholipid and cholesterol through a reverse evaporation method. Compared with the cis-platinum with the solubility of about 2mg / mL in water, the solubility of the cis-platinum in the water can be remarkably improved by virtue of complexation of the cis-platinum and the polyphenol, so that the cis-platinum is wrapped in the hydrophilic core of the liposome, and the problem that the cis-platinum liposome is difficult to prepare is effectively solved. The cisplatin-polyphenol compound liposome disclosed by the invention has good stability, can enhance the drug uptake of tumor cells, and has a good anti-tumor effect in vivo and in vitro. Meanwhile, the cisplatin-polyphenol compound liposome disclosed by the invention has good safety and can be used for remarkably reducing serious toxic and side effects generated by cisplatin.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical preparations and relates to a cisplatin-polyphenol complex for tumor treatment and a preparation method and application thereof. Background Art

[0002] Cancer is the second leading cause of death in humans, with nearly 10 million people dying of cancer each year worldwide. Chemotherapy is currently one of the most commonly used methods for treating cancer in clinical practice, and it is also one of the most effective means of treating cancer. Since the American scientist Rosenberg discovered the anti-tumor activity of cisplatin in 1969, the research and application of platinum anticancer drugs have developed rapidly. Currently, six platinum compounds have been approved for marketing. Cisplatin, carboplatin and oxaliplatin were approved for marketing by the US Food and Drug Administration in 1978, 1989 and 2002, respectively. Nedaplatin, heptaplatin and lobaplatin were approved for marketing in Japan, South Korea and China in 1995, 1999 and 2005, respectively. To this day, platinum drugs are still one of the most widely used chemotherapy drugs. According to statistics, chemotherapy regimens based on or with platinum anticancer drugs account for 70% to 80% of all clinical chemotherapy regimens.

[0003] Platinum drugs have good clinical efficacy on a variety of solid tumors such as testicular cancer, ovarian cancer, bladder cancer, lung cancer, leukemia and gastric cancer, but their serious side effects, such as nephrotoxicity, neurotoxicity and ototoxicity, greatly limit the clinical efficacy and application of platinum drugs. In order to obtain new anticancer drugs with high efficiency and low toxicity, researchers have synthesized and screened thousands of platinum compounds with anti-tumor activity. Although more than 30 platinum compounds have entered clinical trials, most of them have failed at this stage. At the same time, drug delivery technology has developed rapidly in recent decades and has received widespread attention. Ten platinum-based nanodrug preparations have entered the clinical trial stage. At present, the main research direction to improve the shortcomings of platinum chemotherapy has focused on the optimization of delivery strategies.

[0004] As a classic drug carrier, liposomes have the advantages of high efficiency, low toxicity, passive targeting, etc., and have obvious advantages in improving the efficacy of platinum drugs and reducing side effects. However, the low water solubility and lipid solubility of cisplatin have always been a challenge in the preparation of cisplatin liposomes. This physical and chemical characteristic makes the bioavailability of traditional cisplatin liposomes limited in tumor treatment. The drug-lipid molar ratio of liposome preparations is relatively low, and it is impossible to deliver enough drugs to tumors. Summary of the invention

[0005] The purpose of the present invention is to construct a cisplatin-polyphenol complex liposome, wherein cisplatin and polyphenol form a complex through coordination bonds and ionic bonds, which can improve the water solubility of cisplatin. The cisplatin-polyphenol complex is used to prepare liposomes, thereby improving the permeability and retention effect of the drug in solid tumors, thereby improving the therapeutic effect of cisplatin and reducing the toxicity of the drug, so that it can be better applied to the treatment of tumors.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] A cisplatin-polyphenol complex liposome for tumor treatment comprises a cisplatin-polyphenol complex and a liposome.

[0008] The cisplatin-polyphenol complex is a complex formed by cisplatin and polyphenol mainly through ionic bonds and coordination bonds.

[0009] The polyphenol is one of compounds with a polyhydroxy structure such as tannic acid, gallic acid, epigallocatechin gallate (CAS: 989-51-5), and resveratrol; preferably, the polyphenol is epigallocatechin gallate.

[0010] The cisplatin-polyphenol complex liposome is prepared by using the cisplatin-polyphenol complex, phospholipid and cholesterol through a reverse evaporation method.

[0011] The phospholipid is selected from soybean lecithin, egg yolk lecithin, hydrogenated lecithin, etc.; preferably, the phospholipid is soybean lecithin.

[0012] Another object of the present invention is to provide a method for preparing the cisplatin-polyphenol complex liposome, comprising the following steps:

[0013] Step (1), dissolving cisplatin and polyphenol in water, adjusting the pH of the reaction system to 7-10 with a tris buffer solution, and reacting in a dark environment;

[0014] Step (2), the reaction solution is ultrafiltered using an ultrafiltration tube to purify the cisplatin-polyphenol complex solution;

[0015] Step (3), dissolving phospholipids and cholesterol in anhydrous ethanol to obtain an organic phase; using the cisplatin-polyphenol complex solution obtained in step (2) as the aqueous phase, slowly dropping the cisplatin-polyphenol complex solution into the organic phase; after the addition is completed, performing ultrasonic emulsification;

[0016] Step (4), after ultrasonic emulsification, the solvent in the solution is removed by evaporation under reduced pressure, physiological saline is added for hydration, and ultrasonication is performed to obtain cisplatin-polyphenol complex liposomes.

[0017] In step (1), the molar ratio of cisplatin to polyphenol is 2:1 to 5:1, preferably 5:1.

[0018] The mass volume ratio of cisplatin to water is 10:1 to 1:1 mg / mL, preferably 5:1 mg / mL.

[0019] Cisplatin and polyphenol are dissolved in water, and the solution is acidic. Under acidic conditions, there will be competition for proton hydrogen, which is not conducive to the formation of cisplatin-polyphenol complexes. Therefore, the present invention adjusts the general reaction system to be neutral or alkaline. Preferably, the pH of the reaction system is adjusted to 7.4 with a tris buffer solution.

[0020] The reaction is carried out in a water bath shaker; the reaction temperature is 30-45°C, preferably 37°C; the reaction time is 18-30h, preferably 24h; the rotation speed of the water bath shaker is 80-120rpm, preferably 100rpm.

[0021] In step (2), the ultrafiltration molecular weight cut-off (MWCO) of the ultrafiltration tube is 3KD. The ultrafiltration time is 8 to 15 minutes, preferably 10 minutes, and the ultrafiltration speed is 2500 to 3500 r / min, preferably 3000 r / min.

[0022] In step (3), the molar ratio of phospholipid to cholesterol is 2:1 to 4:1, preferably 2.5:1.

[0023] Specifically, the molar ratio of phospholipid to cholesterol is 2:1, 2.5:1, 3.6:1, or 4:1.

[0024] The molar ratio of the phospholipid to cisplatin is 11:1 to 4:1, preferably 6:1 to 4:1, and more preferably 4:1.

[0025] Specifically, the molar ratio of the phospholipid to cisplatin is 11:1, 9:1, 6:1, or 4:1.

[0026] The volume ratio of the organic phase to the aqueous phase is 2:1 to 4:1, preferably 2:1.

[0027] The ultrasonic emulsification equipment is an ultrasonic cell crusher.

[0028] The power of the ultrasonic emulsification is 80-150W, preferably 100W, and the time of the ultrasonic emulsification is 3-10 minutes, preferably 5 minutes.

[0029] The reduced pressure evaporation was carried out at a pressure of -0.1 MPa, a temperature of 37°C and a time of 20 min.

[0030] In step (4), the mass volume ratio of cisplatin to water for hydration is 1:1-2.5:1 mg / mL, the hydration temperature is 20-30° C., and the hydration time is 20-60 min, preferably 30 min.

[0031] The ultrasonic device is an ultrasonic cell crusher. The ultrasonic power is 150-250W, preferably 200W, and the ultrasonic time is 8-15min, preferably 10min.

[0032] Another object of the present invention is to provide the use of the cisplatin-polyphenol complex liposome in preparing drugs for treating tumors.

[0033] The tumors are nasopharyngeal cancer, testicular cancer, ovarian cancer, bladder cancer, lung cancer, leukemia, and gastric cancer.

[0034] Beneficial effects of the present invention:

[0035] Polyphenol compounds are a class of natural compounds with a polyhydroxy structure that can coordinate and complex with a variety of metal ions including platinum to form metal-polyphenol complexes. This type of complex has good water solubility. Compared with the solubility of cisplatin in water of about 2 mg / mL, cisplatin can significantly improve its solubility in water by complexing with polyphenols (the solubility of CDDP-EGCG can reach about 5 mg / mL), so that it can be encapsulated in the hydrophilic core of liposomes, thereby effectively overcoming the difficulty in preparing cisplatin liposomes.

[0036] The cisplatin-polyphenol complex liposome of the present invention has good stability, can enhance the uptake of drugs by tumor cells, and has good anti-tumor effects both in vivo and in vitro. At the same time, the cisplatin-polyphenol complex liposome of the present invention has good safety and can significantly reduce the serious toxic side effects produced by cisplatin. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Transmission electron microscopy image of CDDP-EGCG complex.

[0038] Figure 2 This is the infrared spectrum of CDDP-EGCG complex.

[0039] Figure 3 This is the UV spectrum of CDDP-EGCG complex.

[0040] Figure 4 This is a diagram for judging the binding force of the CDDP-EGCG complex.

[0041] Figure 5 This is the transmission electron microscopy image of CDDP-EGCG Lips.

[0042] Figure 6 This is the particle size distribution diagram of CDDP-EGCG Lips.

[0043] Figure 7 This is the stability experimental results of CDDP-EGCG Lips.

[0044] Figure 8 This is the in vitro release result of CDDP-EGCG Lips.

[0045] Fig. 9 The figure is the hemolytic safety result of CDDP-EGCG Lips; A is the hemolysis of red blood cells, from left to right are the positive control group, the negative control group, CDDP-EGCG Lips concentrations of 50μg / mL, 100μg / mL 200μg / mL, 500μg / mL, 1000μg / mL; B is the hemolysis rate of red blood cells.

[0046] Fig.10 The graphs show the in vitro cytotoxicity results of each drug.

[0047] Fig.11 The graphs show the experimental results of cell uptake of each drug; compared with the FITC group, ***P<0.001, ****P<0.0001.

[0048] Fig.12 The graphs are the tumor growth curves after drug administration in each group.

[0049] Fig.13 The figures are for the tumor weights of each group after drug administration.

[0050] Fig.14 Actual pictures of mouse tumors after drug administration in each group.

[0051] Fig.15 The graph shows the changes in body weight of mice after drug administration in each group. DETAILED DESCRIPTION

[0052] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0053] Abbreviations: CDDP: cisplatin; GA: gallic acid; TA: tannic acid; EGCG: epigallocatechin gallate; FITC: fluorescein isothiocyanate.

[0054] Example 1

[0055] Preparation of CDDP-polyphenol complex

[0056] 5 mg CDDP and an appropriate amount of polyphenols (GA, TA, EGCG) were dissolved in 1 mL of water at a molar ratio of 5:1, and the pH of the system was adjusted to 7.4 with a tris buffer solution. The mixture was reacted at 100 rpm in a 37°C water bath shaker in a dark environment for 24 hours. The reaction solution was transferred to an ultrafiltration tube (MWCO: 3KD) and purified by ultrafiltration using a centrifuge (3000 r / min, 10 min) to obtain CDDP-polyphenol complexes (CDDP-GA, CDDP-TA, CDDP-ECGC).

[0057] The CDDP-polyphenol complex was taken and the PDI of the CDDP-polyphenol complex was measured by a dynamic light scattering nanoparticle size analyzer. As shown in Table 1, the PDI of the CDDP-polyphenol compound was less than 0.3, which showed good uniformity.

[0058] Table 1: PDI values ​​of CDDP-polyphenol complexes

[0059]

[0060]

[0061] Example 2

[0062] Preparation and formulation optimization of CDDP-polyphenol complex liposomes

[0063] (I) Investigation of the encapsulation efficiency of different types of CDDP-polyphenol complex liposomes

[0064] 50 mg of soybean lecithin and 10 mg of cholesterol were dissolved in 2 mL of anhydrous ethanol to obtain an organic phase; 1 mL of CDDP-polyphenol complex (CDDP-GA, CDDP-TA, CDDP-ECGC prepared in Example 1) solution was taken as an aqueous phase, wherein the concentration of CDDP in the CDDP-GA complex solution was 2.09 mg / mL, the concentration of CDDP in the CDDP-TA complex solution was 4.03 mg / mL, and the concentration of CDDP in the CDDP-EGCG complex solution was 4.51 mg / mL; the aqueous phase was slowly added dropwise to the organic phase; after the addition was completed, an ultrasonic cell crusher was used, its probe was inserted into the material, ultrasonic emulsification was performed at a power of 100 W for 5 min, and the material was transferred to a pear-shaped flask, and the organic solvent was removed by evaporation at a constant temperature of 37° C. under reduced pressure (-0.1 MPa) for 20 minutes to obtain a semisolid colloid; 5 mL of normal saline was added and hydrated at room temperature for 30 min. Then, an ultrasonic cell disruptor was used, and its probe was inserted into the material, and probe ultrasonication was performed at a power of 200 W for 10 minutes to obtain liposome preparations (CDDP-EGCG Lips, CDDP-GA Lips, CDDP-TA Lips).

[0065] To measure the liposome encapsulation efficiency, the liposomes were placed in an ultrafiltration tube with a MWCO of 10KD and centrifuged at 3000r / min for 10min. The free drug passed through the ultrafiltration membrane under the action of centrifugal force. The liposome encapsulation efficiency (EE) was determined by atomic absorption spectrophotometry.

[0066] EE%=(W 总 -W 游 ) / W 总 ×100%

[0067] Wherein, Wtotal represents the total amount of cisplatin in the liposomes, and Wfree represents the amount of free cisplatin.

[0068] Table 2: Encapsulation efficiency of different types of CDDP-polyphenol complex liposomes (n=3)

[0069]

[0070] The results are shown in Table 2, which show that CDDP-EGCG Lips has obvious advantages, and its encapsulation efficiency can reach 56.60%, which is far higher than other CDDP-polyphenol liposomes. Therefore, EGCG was selected for the construction of CDDP-polyphenol liposome system.

[0071] (II) Screening of the molar ratio of CDDP and EGCG in the composite:

[0072] Referring to Example 1 "Preparation of CDDP-polyphenol complex", the molar ratio of CDDP to EGCG was changed (2:1, 3:1, 5:1) to prepare CDDP-ECGC complexes with different drug ratios, and their drug loading rates and drug loading amounts were measured to screen out the optimal molar ratio of CDDP to EGCG.

[0073] The results are shown in Table 3. The change in the molar ratio of CDDP to EGCG did not significantly affect the drug loading rate of the complex. When the molar ratio of CDDP:EGCG was 5:1, the drug loading of the complex was the highest, which was 51.47%. Therefore, the molar ratio of CDDP:EGCG was selected as 5:1 as the optimal ratio for preparing CDDP-EGCG complex.

[0074] Table 3: Drug loading efficiency of CDDP-EGCG complex

[0075]

[0076] (III) Screening of auxiliary material proportions:

[0077] The liposome formulation was optimized with encapsulation efficiency as the evaluation index.

[0078] Referring to the embodiment "(i) Investigation of the encapsulation efficiency of different types of CDDP-polyphenol complex liposomes", the amount of cholesterol was fixed at 10 mg, the molar ratio of soybean lecithin to cholesterol was adjusted to 2:1, 2.5:1, and 3.6:1, and other conditions were kept unchanged. The encapsulation efficiency of CDDP-EGCG Lips prepared under different ratios was compared. The results are shown in Table 4.

[0079] Referring to the embodiment "(i) Investigation of the encapsulation efficiency of different types of CDDP-polyphenol complex liposomes", the amount of the drug (i.e., CDDP, the same below) was fixed (i.e., the CDDP-ECGC solution was fixed to 1 mL), the molar ratio of soybean lecithin to the drug was adjusted to 11:1, 9:1, and 4:1, and other conditions were unchanged. The encapsulation efficiency of CDDP-EGCG Lips prepared under different ratios was compared, and the results are shown in Table 5.

[0080] Referring to the embodiment "(i) Investigation of the encapsulation efficiency of different types of CDDP-polyphenol complex liposomes", the volume of the aqueous phase was fixed to 1 mL, the volume ratio of the organic phase to the aqueous phase was adjusted to 2:1, 3:1, and 4:1, and other conditions were kept unchanged. The encapsulation efficiency of CDDP-EGCG Lips prepared under different ratios was compared. The results are shown in Table 5.

[0081] According to Table 4, Table 5 and Table 6, the optimal molar ratio of soybean lecithin to cholesterol is 2.5:1, and the encapsulation efficiency of the prepared liposome is 58.41%. The optimal molar ratio of soybean lecithin to drug is 4:1, and the encapsulation efficiency of the prepared liposome is 62.75%. The optimal volume ratio of organic phase to aqueous phase is 2:1, and the encapsulation efficiency of the prepared liposome is 52.42%.

[0082] Therefore, the optimal preparation process of CDDP-EGCG Lips is: the molar ratio of CDDP and EGCG is 5:1, the volume ratio of organic phase and aqueous phase is 2:1, the molar ratio of soybean lecithin and drug (CDDP) is 4:1, and the molar ratio of soybean lecithin and cholesterol is 2.5:1. The specific method is: CDDP and EGCG are dissolved in 1mL water at a molar ratio of 5:1, the concentration of CDDP is 5mg / mL, and the pH of the system is adjusted to 7.4 with tris buffer solution, and the reaction is carried out at a speed of 100rpm in a 37°C water bath shaker in a dark environment for 24 hours; the reaction solution is transferred to an ultrafiltration tube (MWCO: 3KD), and ultrafiltration purification is performed by a centrifuge (3000r / min, 10min) to obtain a CDDP-ECGC complex; 50mg soybean lecithin and 10mg cholesterol are dissolved in 2mL anhydrous ethanol to obtain an organic phase; 1mL The CDDP-ECGC complex solution was used as the aqueous phase, wherein the concentration of CDDP in the CDDP-EGCG complex solution was 4.51 mg / mL. The aqueous phase was slowly added dropwise to the organic phase. After the addition was completed, an ultrasonic cell crusher was used to ultrasonically emulsify for 5 minutes at a power of 100 W, and then transferred to a pear-shaped flask, and the organic solvent was evaporated for 20 minutes at a constant temperature of 37°C under reduced pressure (-0.1 MPa) to obtain a semisolid colloid; 5 mL of physiological saline was added, and the mixture was hydrated at room temperature for 30 minutes. Then, a 200 W probe type ultrasound was used for 10 minutes to obtain CDDP-EGCG Lips.

[0083] Table 4: Encapsulation efficiency of liposomes with different phospholipid / cholesterol ratios

[0084]

[0085] Table 5: Encapsulation efficiency of liposomes with different phospholipid / drug ratios

[0086]

[0087] Table 6: Encapsulation efficiency of liposomes with different organic phase / aqueous phase ratios

[0088]

[0089] In order to investigate the reproducibility of the preparation, three batches of CDDP-EGCGLips were prepared according to the best formula and process obtained from the above experiment, and characterized, and the results are shown in Table 7. The average particle size of the sample was (182.74±9.22) nm, the average PDI was (0.257±0.018), the average encapsulation efficiency was (54.09±3.89)%, and the average drug loading was (4.16±0.30)%, indicating that the preparation had good reproducibility.

[0090] Table 7: Characterization of different batches of cisplatin liposome formulations

[0091]

[0092] Example 3

[0093] Characterization of CDDP-EGCG complex

[0094] 1. Observation of the morphology of CDDP-EGCG complex: CDDP-EGCG complex was prepared according to the method of Example 1, and the complex sample was observed by transmission electron microscopy. Figure 1 As shown, the CDDP-EGCG complex has a uniform morphology.

[0095] 2. A CDDP-EGCG complex was prepared according to the method of Example 1, and CDDP and EGCG solid powders were mixed at a CDDP to EGCG molar ratio of 5:1 to obtain a physical mixture of CDDP and EGCG. The ultraviolet spectra and infrared spectra of CDDP, EGCG, CDDP-EGCG complex and the physical mixture of CDDP and EGCG were measured by ultraviolet spectroscopy and infrared spectroscopy, respectively, to characterize the complex.

[0096] Figure 2 From the infrared spectrum, we can see that: EGCG has a pyrogallol structure, and the hydrogen bonds within or between molecules cause the stretching vibration peak of OH to red shift. In the CDDP-EGCG complex, the metal platinum and phenolic hydroxyl groups are complexed, and the free phenolic hydroxyl groups will blue shift on the original basis. 1040cm -1 The stretching vibration of CO on the benzene ring produces a color enhancement effect, and the absorption intensity is significantly enhanced. It may be that the complexation of CDDP with the adjacent benzene ring enhances the coplanar effect of the two benzene rings, thereby enhancing the absorption intensity. Figure 3 From the UV spectrum, we can see that the E2 band of the benzene ring of the CDDP-EGCG complex is red-shifted and the absorption intensity increases. This may be because the phenolic hydroxyl group of EGCG forms a coordination bond with the metal ion, which enhances the p-π conjugation effect formed by the lone pair of electrons of the phenolic hydroxyl group on the benzene ring and the large π electron system on the benzene ring, causing the absorption band to red-shift and the absorption intensity to increase.

[0097] 3. Determination of binding force of CDDP-EGCG complex:

[0098] The CDDP-EGCG complex was prepared according to the method of Example 1. 2 mL of CDDP-EGCG complex solution was taken and 100 μL of 10 mM KNO 3 Aqueous solution, Tween 20 aqueous solution, urea (CH 4 N 2 O) aqueous solution and EDTA aqueous solution were used to destroy ionic bonds, hydrophobic interactions, hydrogen bonds and coordination bonds, respectively, and the samples were scanned by UV light after 1 h.

[0099] The results are as follows Figure 4 As shown, EDTA and KNO were added to the complex 3 After adding CH 4 N 2 O and Tween 20, the complex did not change, indicating that the binding force of CDDP-EGCG is mainly ionic bond and coordination bond.

[0100] Example 4

[0101] Characterization of cisplatin-polyphenol complex liposomes

[0102] 1. Liposome morphology observation

[0103] CDDP-EGCG Lips were prepared according to the optimal preparation process of CDDP-EGCG Lips in Example 2. A liposome preparation diluted with a small amount of pure water was dripped onto a copper mesh, excess liquid was absorbed with filter paper, and negatively stained with 2% phosphotungstic acid solution for 2 minutes. The excess dye was adsorbed with filter paper, and the liposome morphology was observed under a transmission electron microscope after the liquid on the mesh evaporated.

[0104] The results are as follows Figure 5 As shown, it can be seen that CDDP-EGCG Lips are regular circles with uniform size and a particle size of about 160 nm.

[0105] 2. Determination of particle size, PDI value and Zeta potential

[0106] CDDP-EGCG Lips were prepared according to the optimal preparation process of CDDP-EGCG Lips in Example 2, and the liposome solution diluted with pure water was taken to measure the particle size, PDI value and Zeta potential of CDDP-EGCG Lips using a dynamic light scattering nanoparticle size analyzer.

[0107] Figure 6 The particle size distribution diagram of CDDP-EGCG Lips shows that the average particle size of CDDP-EGCG Lips is (182.74±9.22) nm, which is normally distributed. The PDI and Zeta potential of CDDP-EGCG Lips are shown in Table 8. CDDP-EGCG Lips exhibit strong negative charge. Electrostatic repulsion can prevent aggregation between nanoparticles, which is beneficial to the long-term storage of nanoparticles.

[0108] Table 8: PDI and zeta potential of CDDP-EGCG Lips

[0109]

[0110] Example 5

[0111] Study on the stability of cisplatin-polyphenol complex liposomes

[0112] CDDP-EGCG Lips were prepared according to the optimal preparation process of CDDP-EGCG Lips in Example 2, and the prepared liposomes were stored at 4°C to investigate their storage stability. Three parallel samples were placed under each storage condition. Samples were taken on the first day (recorded as day 0), the 7th day, the 14th day, the 21st day and the 28th day of the sample to observe whether stratification or precipitation occurred, and samples were taken to measure the particle size and encapsulation efficiency of the liposomes.

[0113] The results are as follows Figure 7 As shown in the figure, the particle size and encapsulation efficiency of CDDP-EGCG Lips remained stable within 28 days under 4°C environmental conditions, and the sample did not show stratification or precipitation.

[0114] Example 6

[0115] In vitro release of cisplatin-polyphenol complex liposomes

[0116] The CDDP-EGCG complex was prepared according to the method of Example 1, and the concentration of the drug (i.e., CDDP) in the CDDP-EGCG complex solution was adjusted to 0.1 mg / mL by pure water; CDDP-EGCG Lips were prepared according to the optimal preparation process of CDDP-EGCG Lips in Example 2, and the concentration of the drug (i.e., CDDP) in the CDDP-EGCG Lips solution was adjusted to 0.1 mg / mL by physiological saline; 0.3 mg of CDDP was weighed and dissolved in 3 mL of pure water to obtain a 0.1 mg / mL CDDP solution.

[0117] 1 mL CDDP solution, CDDP-EGCG complex solution, and CDDP-EGCG Lips solution were accurately drawn and added to dialysis bags (molecular weight cutoff: 3500D), respectively. After clamping at both ends, they were placed in 30 mL of release media with different pH values ​​(pH 7.4, 6.5, 5.0 PBS buffer) preheated to 37°C, and continuously shaken at a rate of 100 r / min in a 37°C constant temperature shaker. 1 mL of dialysate sample was taken at predetermined time points (0, 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h), and an equal volume of phosphate buffer was added in time. The obtained samples were measured for CDDP concentration by atomic absorption spectrophotometer, and the cumulative release rate of CDDP was calculated.

[0118] The results are as follows Figure 8As shown in the figure, CDDP-EGCG complex and CDDP-EGCG Lips release CDDP faster in PBS at pH = 5.0 compared with pH = 7.4, because acidic conditions can break the balance of the coordination bond formed by platinum and polyphenols, thereby releasing CDDP. The acid-responsive release of CDDP-EGCG Lips helps the drug to be released under the slightly acidic conditions of the tumor and exert its anti-cancer effect.

[0119] Example 7

[0120] Hemolytic safety of cisplatin-polyphenol complex liposomes

[0121] The drug is directly injected into the systemic circulation through the tail vein, so it is necessary to investigate whether the preparation is hemolytic to ensure the safety of the preparation. The eyeballs of the mice were removed with curved forceps to collect blood from the eye sockets, and the blood was collected in a 1.5mL EP tube moistened with 1% sodium heparin in advance. 0.5mL of plasma was taken, 0.5mL of PBS (pH 7.4) was added, mixed, centrifuged at 3000rpm for 5min, the supernatant was discarded, and 0.5mL of PBS was added to resuspend, and repeated 3 times, the supernatant was discarded, 100μL of red blood cell pellet was taken, 4.9mL of PBS was added, and mixed to obtain a red blood cell suspension.

[0122] An experimental group, a positive control group, and a negative control group were set up. Positive control group: take the above 500 μL red blood cell suspension, add 100 μL 2% TritonX 100-water solution, and mix; negative control group: take the above 500 μL red blood cell suspension, add 100 μL PBS solution, and mix. Experimental group: CDDP-EGCG Lips were prepared according to the optimal preparation process of CDDP-EGCG Lips in Example 2, and adjusted to a series of CDDP-EGCG Lips with concentration gradients using physiological saline: 50 μg / mL, 100 μg / mL 200 μg / mL, 500 μg / mL, 1000 μg / mL (concentration is calculated as CDDP); take the above 500 μL red blood cell suspension, add 100 μL of a series of CDDP-EGCG Lips with concentration gradients, and mix. After mixing, incubate at 37°C in an incubator for 2 hours, centrifuge at 3000 rpm for 5 minutes, place the EP tube on black cardboard and take a picture ( Fig. 9 A). Take 200 μL of supernatant and measure the absorbance at 540 nm. Calculate the hemolysis rate of the preparation ( Fig. 9 B).

[0123] Hemolysis rate (%) = OD (sample) - OD (negative) / OD (positive) - OD (negative)

[0124] like Fig. 9As shown, under the condition of 500 μg / mL high concentration preparation, there is no hemolysis of red blood cells, and the hemolysis rate is below 5%, which has good safety.

[0125] Example 8

[0126] In vitro anticancer activity of cisplatin-polyphenol complex liposomes

[0127] CNE-2 and 5-8F nasopharyngeal carcinoma cells were cultured at 37°C and 5% CO 2 The cells were cultured in an incubator using RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% double antibodies (streptomycin and penicillin).

[0128] Preparation of blank liposomes: Weigh 50 mg of soybean lecithin and 10 mg of cholesterol and dissolve them in 2 mL of anhydrous ethanol to obtain an organic phase; take 1 mL of pure water as the aqueous phase and slowly drop it into the organic phase, act under 100 W probe ultrasound for 5 minutes, transfer to a pear-shaped flask, and evaporate under reduced pressure (-0.1 MPa) at 37°C for 20 minutes to remove the organic solvent to obtain a semi-solid colloid; add 5 mL of physiological saline and hydrate at room temperature for 30 minutes. Then use 200 W probe ultrasound for 10 minutes to obtain blank liposomes.

[0129] CDDP-EGCG was prepared according to the method of Example 1.

[0130] CDDP-EGCG Lips were prepared according to the optimal preparation process of CDDP-EGCG Lips in Example 2.

[0131] The blank liposomes, CDDP, CDDP-EGCG, and CDDP-EGCG Lips solutions were diluted with culture medium to 6 concentrations (0.1, 1, 5, 10, 50, and 100 μg / mL, with the concentration calculated as CDDP).

[0132] Take CNE-2 and 5-8F cells from the same batch of passages in the logarithmic growth phase and adjust the cell concentration to 4×10 4 The cells were seeded into a 96-well culture plate and placed in a 37°C, 5% CO 2Culture in the incubator for 24 hours to allow the cells to grow attached to the wall. Remove the culture plate, discard the supernatant of each well, and add different concentrations of drugs (6 concentrations of blank liposomes, CDDP, CDDP-EGCG, CDDP-EGCG Lips) to the corresponding wells of the experimental group, 20μL per well, complete culture medium as blank control, and untreated cell fluid as negative control, all incubated in the incubator for 24 hours. Add 10μL of newly prepared 5.0mg / L MTT solution to each well and continue to culture for 4 hours. After 4 hours, terminate the culture, discard the supernatant, add 100μL DMSO to each well, and shake at a low speed for 5-10 minutes to fully dissolve the crystals. Use an enzyme marker to measure the absorbance value of each well at a wavelength of 570nm.

[0133] like Fig.10 As shown in the figure, within 24 hours, the killing rate of CDDP, CDDP-EGCG, and CDDP-EGCG Lips on CNE-2 and 5-8F cells gradually increased with the increase of concentration. With the increase of concentration, the cell survival rate of the blank liposome group did not change significantly, indicating that the liposome material itself has no cytotoxicity and the cell killing ability comes entirely from the drug. CDDP-EGCG Lips has the same cytotoxicity to CNE-2 and 5-8F cells as CDDP and CDDP-EGCG, indicating that the preparation does not affect the activity of CDDP.

[0134] Example 9

[0135] Study on the Uptake of Cisplatin-Polyphenol Complex Liposomes

[0136] Preparation of CDDP-EGCG-FITC: CDDP-EGCG was prepared according to the method of Example 1, 800 μL CDDP-EGCG was mixed with 200 μL FITC solution (5 mg / mL), and the mixture was incubated in the dark for 12 h to obtain CDDP-EGCG-FITC.

[0137] Preparation of CDDP-EGCG-FITC Lips: Weigh 50 mg of soybean lecithin and 10 mg of cholesterol and dissolve them in 2 mL of anhydrous ethanol to obtain an organic phase; take 1 mL of CDDP-EGCG-FITC solution as the aqueous phase, slowly drop it into the organic phase, act under 100 W probe ultrasound for 5 minutes, transfer it to a pear-shaped flask, and evaporate at a constant temperature of 37 ° C -0.1 MPa under reduced pressure for 20 minutes to remove the organic solvent to obtain a semi-solid colloid; add 5 mL of physiological saline and hydrate at room temperature for 30 minutes. Then use 200 W probe ultrasound for 10 minutes to obtain CDDP-EGCG-FITC Lips.

[0138] CNE-2 cells were cultured at 37°C and 5% CO 2Incubate in the incubator at 4 °C for 1 h. When in the logarithmic growth phase, digest with trypsin and adjust the cell concentration to 4 × 10 5 / mL, 2mL of the above cell solution was inoculated into a sterile 6-well plate, and cultured in an incubator for 24h. After the cells were completely attached to the wall, the original culture medium was aspirated, and FITC group, CDDP-EGCG-FITC group, and CDDP-EGCG-FITC Lips group were set up, and 2μg / mL of FITC, CDDP-EGCG-FITC, and CDDP-EGCG-FITC Lips (concentration in terms of FITC) solutions were added respectively, and incubated for 4h in the dark (n=3); the culture medium was discarded, washed once with PBS, digested with trypsin, centrifuged, and resuspended with 1mL PBS. The flow cytometer FSC voltage was adjusted to 360V, the SSC voltage was 250V, and the FITC voltage was 230V, and the flow cytometer FITC channel analysis was performed.

[0139] The experimental results are as follows Fig.11 As shown, compared with the FITC and CDDP-EGCG-FITC groups, the cell uptake in the CDDP-EGCG-FITC Lips group was significantly increased, indicating that CDDP-EGCG-FITC Lips is more easily taken up by tumor cells, can effectively improve the permeability and retention effect of drugs in solid tumors, exert anti-cancer effects, and thus reduce damage to normal cells.

[0140] Embodiment 11

[0141] Anticancer effect of cisplatin-polyphenol complex liposomes in vivo

[0142] CNE-2 cells were cultured at 37°C and 5% CO 2 The cells were cultured in an incubator with RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% double-antibody (streptomycin and penicillin). When the cells reached the logarithmic growth phase, they were digested with trypsin, centrifuged, and washed once with PBS to remove residual serum. The cells were resuspended in PBS to obtain a final concentration of 5×10 7 Cell suspension of 100 / mL. Under sterile conditions, 75% alcohol was used to disinfect the skin under the left armpit of the mouse. A 1mL syringe was used to extract the cell suspension and inoculated into the left armpit of the mouse. 0.1mL of cell suspension was inoculated into each mouse. After inoculation, the mice were kept in the animal breeding room. When the tumor volume was about 100mm, the mouse was inoculated with 0.1mL of cell suspension. 3When the tumor size was about 1.5, the mice were divided into 4 groups according to the principle of even distribution of the tumor size, namely the control group, free CDDP group, CDDP-EGCG complex group (CDDP-EGCG solution prepared according to the method of Example 1), CDDP-EGCG Lips group (CDDP-EGCG Lips solution prepared according to the optimal preparation process of CDDP-EGCG Lips in Example 2), 6 mice in each group. The control group mice were injected with normal saline by tail vein, 0.2mL each time, and the three drug groups were administered with a dosage of CDDP 5mg / kg, 0.2mL each time, once every other day, and administered 4 times continuously. The body weight and tumor volume of the mice were recorded every other day from the first administration. After the end of the administration, the nude mice were observed for one week and the nude mice were killed. The tumors were collected and the tumor tissues were weighed. The tumor growth curve was drawn according to the recorded tumor volume.

[0143] The experimental results are as follows Fig.12 , Fig.13 , Fig.14 As shown, CDDP, CDDP-EGCG, and CDDP-EGCG Lips can effectively inhibit tumor growth, and CDDP and CDDP-EGCG Lips have better inhibitory effects on tumors. Fig.15 The weight changes of mice showed that the weight of mice in the CDDP group dropped rapidly after administration and they became listless. CDDP has serious systemic toxicity, while the weight of mice in the CDDP-EGCG and CDDP-EGCG Lips were normal and their mental state was good after administration, which can effectively reduce the toxic side effects of CDDP.

[0144] In summary, the cisplatin-polyphenol complex liposomes of the present invention, especially CDDP-EGCG Lips, have good stability, can enhance the uptake of drugs by tumor cells, and have good anti-tumor effects both in vivo and in vitro. At the same time, the cisplatin-polyphenol complex liposomes of the present invention have good safety and can significantly reduce the serious toxic side effects of cisplatin.

Claims

1. A cisplatin-polyphenol complex liposome for tumor treatment, characterized in that: Includes cisplatin-polyphenol complexes and liposomes.

2. The cisplatin-polyphenol complex liposome according to claim 1, characterized in that: The cisplatin-polyphenol complex is a complex formed by cisplatin and polyphenol mainly through ionic bonds and coordination bonds; the polyphenol is one of tannic acid, gallic acid, epigallocatechin gallate and resveratrol.

3. The cisplatin-polyphenol complex liposome according to claim 1 or 2, characterized in that: The polyphenol is epigallocatechin gallate.

4. The cisplatin-polyphenol complex liposome according to claim 1, characterized in that: The cisplatin-polyphenol complex liposome is prepared by using cisplatin-polyphenol complex, phospholipid and cholesterol through reverse evaporation method; the phospholipid is selected from soybean lecithin, egg yolk lecithin and hydrogenated lecithin.

5. The cisplatin-polyphenol complex liposome according to claim 4, characterized in that: The phospholipid is soybean lecithin.

6. A method for preparing the cisplatin-polyphenol complex liposome according to claim 1, characterized in that: The following steps are involved: Step (1), dissolving cisplatin and polyphenol in water, adjusting the pH of the reaction system to 7-10 with a tris buffer solution, and reacting in a dark environment; Step (2), the reaction solution is ultrafiltered using an ultrafiltration tube to purify the cisplatin-polyphenol complex solution; Step (3), dissolving phospholipids and cholesterol in anhydrous ethanol to obtain an organic phase; using the cisplatin-polyphenol complex solution obtained in step (2) as the aqueous phase, slowly dropping the cisplatin-polyphenol complex solution into the organic phase; after the addition is completed, performing ultrasonic emulsification; Step (4), after ultrasonic emulsification, the solvent in the solution is removed by evaporation under reduced pressure, physiological saline is added for hydration, and ultrasonication is performed to obtain cisplatin-polyphenol complex liposomes.

7. The method for preparing cisplatin-polyphenol complex liposome according to claim 6, characterized in that: In step (1), the molar ratio of cisplatin to polyphenol is 2:1 to 5:1, preferably 5:1; The mass volume ratio of cisplatin to water is 10:1 to 1:1 mg / mL; The pH of the reaction system was adjusted to 7.4 with tris buffer solution. The reaction is carried out in a water bath shaker; the reaction temperature is 30-45°C, preferably 37°C, and the reaction time is 18-30h, preferably 24h; In step (2), the ultrafiltration cut-off molecular weight of the ultrafiltration tube is 3KD.

8. The method for preparing cisplatin-polyphenol complex liposome according to claim 6, characterized in that: In step (3), the molar ratio of phospholipid to cholesterol is 2:1 to 4:1, preferably 2.5:1; The molar ratio of the phospholipid to cisplatin is 11:1 to 4:1, preferably 6:1 to 4:1, more preferably 4:1; The volume ratio of the organic phase to the aqueous phase is 2:1 to 4:1, preferably 2:1; The power of the ultrasonic emulsification is 80-150W, preferably 100W, and the time of the ultrasonic emulsification is 3-10min, preferably 5min; In step (4), the mass volume ratio of cisplatin to water for hydration is 1:1-2.5:1 mg / mL, the hydration temperature is 20-30° C., and the hydration time is 20-60 min; The power of the ultrasound is 150-250W, preferably 200W, and the time of the ultrasound is 8-15min, preferably 10min.

9. Use of the cisplatin-polyphenol complex liposome according to any one of claims 1 to 5 in the preparation of drugs for treating tumors.

10. The use according to claim 9, characterized in that: The tumors are nasopharyngeal cancer, testicular cancer, ovarian cancer, bladder cancer, lung cancer, leukemia, and gastric cancer.

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