CN117257917A - Mastonaran M peptide liposome preparation and preparation method and application thereof - Google Patents

Abstract

The invention discloses a Mastoparan M peptide liposome preparation and its preparation method and use, and belongs to the field of medical technology. The present invention selects Mastoparan M peptide, an active peptide isolated from melittin, as the anti-tumor active substance, uses liposomes with good biocompatibility as in vivo delivery carriers, and constructs Lipo@Mastoparan liposome nano-preparations through green synthesis. Lipo@Mastoparan liposome nanoformulation can be used to treat tumor diseases. Compared with other drug delivery systems, the preparation process of the present invention is simple and has better stability, safety and efficiency. It can effectively improve the bioavailability of Mastoparan M peptide in the body and reduce the systemic toxic and side effects of Mastoparan M peptide.

Description

Mastoparan M peptide liposome preparation and its preparation method and use

Technical field

The invention belongs to the field of medicine, and specifically relates to Mastoparan M peptide liposome preparation and its preparation method and use.

Background technique

Currently, due to the acceleration of industrialization and urbanization, the incidence of cancer is increasing year by year, and cancer has become one of the main causes of death among residents in our country. Among the male population in my country, the top three cancers with the highest incidence are: lung cancer, gastric cancer, and liver cancer; among the female population in my country, the top three cancers with the highest incidence are: breast cancer, lung cancer, and colorectal cancer. According to the latest global cancer statistics, breast cancer is the malignant tumor with the highest morbidity and mortality among women in the world. There are approximately 2.26 million new cases every year, accounting for 24.5% of all female malignant tumor incidences, and 680,000 breast cancer deaths. cases, seriously threatening women’s health. With the development of modern life sciences, a variety of new anti-cancer drugs have been widely used in medicine. However, after clinical treatment, it was found that typical anti-cancer drugs such as paclitaxel and vinblastine anti-cancer drugs have serious toxic side effects and cause great damage to the human liver. Therefore, there is an urgent need to develop new anti-tumor drugs with high efficiency and low toxicity for systemic treatment of cancer.

Animal venom is a special natural source for the development of new drugs. In the past few decades, wasp venom has attracted widespread attention from scholars as a medicinal resource because it contains a variety of biologically active ingredients, including small molecule compounds, high Abundant peptides, proteins and allergens have gradually become new hot spots in biotoxin research. Mastoparan M peptide is a tetradecanopeptide active substance isolated and identified from melittin, accounting for 70-80% of the crude extract of bee venom. Studies have shown that at low doses, this peptide has antibacterial, neuroprotective, and improved cerebral ischemia effects. It has a strong active effect. What is more noteworthy is that some researchers have found that Mastoparan M peptide has a cytotoxic effect in vitro and has a significant inhibitory effect on a variety of tumor cells, including liver cancer, colorectal cancer, and melanoma. Mastoparan M peptide is a small molecule substance that is easily metabolized and excreted quickly after entering the body. It has a short blood circulation time and is difficult to effectively enrich and exert its tumor killing effect at the tumor site. Its non-selective mode of action may lead to systemic toxic and side effects. Therefore, how to improve the in vivo biodistribution characteristics of Mastoparan M peptide and achieve a more low-toxicity and efficient anti-tumor effect is also a key scientific issue that needs to be solved.

Liposomes are biomimetic nanoparticles self-assembled by concentric lipid bilayers. Compared with free small molecule drugs, they have longer blood circulation time and can passively accumulate at tumor sites through the EPR effect, significantly improving The pharmacokinetics and pharmacodynamics of the drug, as well as its good biocompatibility and biodegradability, have shown great application value in the field of drug delivery. In recent years, liposomes loaded with chemotherapy drugs have been widely used to treat patients with solid tumors and hematological malignancies. Drugs such as liposomes encapsulating paclitaxel-paclitaxel have been used in the clinical treatment of breast cancer. Therefore, based on the above characteristics of liposomes combined with the new anti-tumor active peptide Mastoparan M, it is expected to develop safe and effective new anti-tumor nanomedicines to achieve effective control of local recurrence and distant metastasis of tumors.

In summary, the present invention is oriented to the clinical need of "developing new anti-tumor disease drugs" and develops new anti-tumor drugs based on Mastoparan M peptide that can be extracted from nature. At the same time, combined with the in vivo delivery advantages of liposomes, it is expected to improve the efficiency of polypeptide drugs. The safety and effectiveness of cancer treatment not only have potential clinical value in improving the survival rate and quality of life of cancer patients, but also have practical significance in reducing patient and social costs.

Contents of the invention

In view of the shortcomings of the existing technology, the present invention provides a Mastoparan M peptide liposome for anti-tumor use.

The technical solution of the present invention is as follows: a nanoliposome for anti-tumor. The liposome is composed of Mastoparan M peptide and phospholipid. The mass ratio of Mastoparan M peptide and phospholipid is 1:1~5, wherein Mastoparan M The amino acid sequence of the peptide is INLKAIAALAKKLL.

In some embodiments, the phospholipid is selected from the group consisting of soy lecithin, egg yolk lecithin, DPPC, DPPE, DSPC and soy lecithin-cholesterol mixture, preferably soy lecithin.

In some embodiments, the mass ratio of the Mastoparan M peptide and phospholipid is 1:2.

The invention also provides a method for preparing nanoliposomes, which includes the following steps: weigh 10 mg of Mastoparan M peptide and 10 to 50 mg of phospholipid and dissolve them in 5 to 20 ml of reaction solvent, and heat and stir at a reaction temperature of 25 to 65°C for 0.5 to 12 hours. , rotary evaporate at 40-60°C to remove the reaction solvent, add 5-20ml ultrapure water for ultrasonic hydration until evenly dispersed, and store at 4°C for later use.

In some embodiments, the phospholipid is selected from the group consisting of soy lecithin, egg yolk lecithin, DPPC, DPPE, DSPC and soy lecithin-cholesterol mixture, preferably soy lecithin.

In some embodiments, the phospholipid is used in an amount of 20 mg.

In some embodiments, the reaction solvent is selected from DMSO, DMF, MeOH, _EtOH , _CH2Cl2 , _CH3Cl , MeOAc or acetone, preferably EtOH.

In some embodiments, the reaction solvent volume is 10 ml.

In some embodiments, the reaction temperature is 55°C

In some embodiments, the reaction time is 2 h.

In some embodiments, the post-treatment step is to remove the reaction solvent by rotary evaporation at 55°C, and add 10 ml of ultrapure water for ultrasonic hydration.

The application of the nanoliposome provided by the present invention in the preparation of drugs for treating tumor diseases, where the tumor is any one selected from the group consisting of breast cancer, liver cancer, colorectal cancer and cervical cancer.

Beneficial effects of the present invention: the active substance Mastoparan M peptide is a natural product. The present invention uses liposomes with good biocompatibility as delivery carriers in the body, and constructs liposome nanomedicine through green synthesis. The liposomes are in vitro It has significant inhibitory effects on various tumor cells including liver cancer, colorectal cancer and melanoma, and significantly inhibits tumor growth in the body. Preparing Mastoparan M peptide into liposomes greatly improves the pharmacological activity of Mastoparan M peptide, improves the targeting and bioavailability of the drug, reduces systemic toxic and side effects, and achieves safer and more efficient anti-tumor treatment.

Description of the drawings

Figure 1 Appearance of Lipo@Mastoparan drug particles

Figure 2 Transmission electron microscope image of Lipo@Mastoparan

Figure 3 Zeta potential of Lipo@Mastoparan

Figure 4 Hydrated particle size of Lipo@Mastoparan

Figure 5 Inhibition of tumor volume in mice after in vivo administration of Lipo@Mastoparan

Detailed ways

The following examples can enable those skilled in the art to understand the present invention more comprehensively, but do not limit the present invention to the scope of the described embodiments.

Example 1 Synthesis of Mastoparan M peptide

The Mastoparan M peptide sequence is INLKAIAALAKKLL, which was synthesized by Shanghai Gill Biochemical Company with a purity of >99%. At the same time, the fluorescein FITC-labeled peptide Mastoparan M-FITC was also synthesized.

Example 2 Prescription optimization and preparation process optimization

2.1 Selection of reaction solvent

First, set the mass ratio of Mastoparan M to egg yolk lecithin to 1:3, the reaction temperature to 45°C, and the reaction time to 4 hours. Remove absolute ethanol by rotary evaporation, and add 8-20 ml of ultrapure water for hydration until uniformly dispersed. 4 °C for later use.

Then we used different reaction solvents such as DMSO, DMF, MeOH, EtOH, CH ₂ Cl ₂ , CH ₃ Cl, MeOAc, and acetone to prepare Lipo@Mastoparan liposomes, and calculated the encapsulation efficiency.

Results: As shown in the table, although the solvent DMF has the best encapsulation rate, compared with absolute ethanol, its encapsulation rate is not much different, and it is toxic and has complicated post-processing, so absolute ethanol was finally chosen as the best reaction solvent.

2.2 Choice of reaction time

Use the best reaction solvent EtOH, use different reaction times (0.5, 1, 2, 3, 4, 6, 8, 12, 24h) in the reaction system, and other conditions are the same as above to prepare Lipo@Mastoparan liposomes, and calculate the package Closure rate.

Results: As shown in the table, when the reaction time is 2h, the liposome encapsulation rate is the highest, so the optimal reaction time is 2h.

2.3 Selection of reaction temperature

Use the best reaction solvent EtOH, use the best reaction time of 2h, and use different reaction temperatures (25, 35, 45, 55, 65°C). Other conditions are the same as above to prepare Lipo@Mastoparan liposomes, and calculate the encapsulation efficiency.

Results: As shown in the table, when the reaction temperature is 55°C, the liposome encapsulation rate is the highest, so the optimal reaction temperature is 55°C.

2.4 Selection of the proportion of reactive drugs and phospholipids

Use the best reaction solvent EtOH, use the best reaction time of 2h, use the best reaction temperature of 55°C, and use different feeding mass ratios of Mastoparan M peptide and egg yolk lecithin (1:1, 1:2, 1:3, 1:4, 1:5), prepare Lipo@Mastoparan liposomes, and calculate the encapsulation efficiency.

Results: As shown in the table, when the ratio of Mastoparan M peptide to phospholipid is 1:2, the liposome encapsulation rate is the highest, so the ratio of Mastoparan M peptide to phospholipid is 1:2.

2.5 Selection of phospholipids

Use the best reaction solvent EtOH, use the best reaction time of 2h, use the best reaction temperature of 55°C, and use the best Mastoparan M peptide and phospholipid feeding ratio. Using different phospholipids (soy lecithin, egg yolk lecithin, DPPC (dipalmitoylphosphatidylcholine), DPPE (dipalmitoylphosphatidylethanolamine), DSPC (distearoylphosphatidylcholine) and soy lecithin - Cholesterol (2:1)) was used to prepare Lipo@Mastoparan liposomes, and the encapsulation efficiency was calculated.

Results: As shown in the table, when the phospholipid is selected from soy lecithin, the liposome encapsulation rate is the highest, so the best phospholipid choice is soy lecithin.

Example 3 Preparation of Lipo@Mastoparan pharmaceutical particles

Weigh 10 mg of Mastoparan M peptide and 20 mg of soy lecithin and dissolve them in 10 ml of ethanol. Heat and stir in an oil bath at 55°C for 2 hours. Remove absolute ethanol by rotary evaporation at 55°C, add 10 ml of water for ultrasonic hydration until evenly dispersed, and store at 4°C for later use.

Test Example 1 Characterization of Lipo@Mastoparan drug particles

General properties of drug particles

After diluting the prepared liposome drug particles Lipo@Mastoparan 10 times with PBS, let it stand to observe its color, clarity, and transparency. The experimental results are shown in Figure 1. Lipo@Mastoparan is a milky white transparent clear liquid.

Morphological appearance of drug particles

The prepared drug particles Lipo@Mastoparan were diluted 20 times with PBS and dispersed using an ultrasonic cleaner for 5 minutes. Use a sterile gun tip to take 10 μL of the diluted ultrasonic sample and drop it on the copper grid to adsorb the liposomes. Use 2% After negative staining with uranium acetate, leave it to dry, place it under a transmission electron microscope to observe its morphological characteristics, select multiple fields of view with appropriate density, take and save the image. As shown in Figure 2, nanoliposome drugs are monodispersed, spherical particles.

Zeta potential of drug particles

Adjust the prepared drug particles Lipo@Mastoparan to a series of concentrations of 10ug/ml, 25ug/ml, 50ug/ml, 100ug/ml, 200ug/ml, and 400ug/ml and then ultrasonic for 15 minutes to disperse evenly. Take 1 mL of the ultrasonic particles. The samples were added to the plastic potential cells one after another, and the Zeta potential of the samples was measured using a nanoparticle size potential analyzer. The preset temperature is 25°C, and the temperature is measured three times in a row, and the average and standard deviation are calculated from the three values.

As shown in Figure 3, the liposome drug particles Lipo@Mastoparan are slightly positively charged.

Hydrated particle size of drug particles

The prepared liposome drug particles Lipo@Mastoparan were diluted with PBS to a series of concentrations of 10ug/ml, 25ug/ml, 50ug/ml, 100ug/ml, 200ug/ml, and 400ug/ml, and then dispersed by ultrasound and added to a quartz cuvette. A dynamic light scattering particle size analyzer was used to measure the particle size. The preset temperature is 25°C and the angle is 90°. Detect three times in a row and calculate the average and standard deviation from the three values.

As shown in Figure 4, the particle size of liposome drug particles Lipo@Mastoparan is approximately 125.6nm.

Encapsulation efficiency of Mastoparan M

Take 10 μL liposome solution, add 1% TritonX-100 to break the membrane, measure the absorption values of different concentration gradient Mastoparan M at 490nm with a UV-visible spectrophotometer and make a standard curve, quantify the loading amount of Mastoparan M polypeptide, and further calculate the encapsulation Rate.

Test Example 2 CCK-8 experiment to detect the cytotoxic effect of Lipo@Mastoparan

i) Cell culture and probe incubation: Hela, HepG2, CT26, and HT29 cells were seeded into a 96-well plate at 1,000 cells/well respectively. After the cells reached the logarithmic phase of growth, Lipo@Mastoparan was added at 1 μg/ml or 10 μg/well. ml, 50 μg/ml, 100 μg/ml, 200 μg/ml, and 300 μg/ml final concentration gradient were added, and incubated for 12, 24, and 48 h.

ii) Color development: Add CCK-8 solution and incubate in the dark for 2 hours. Use a full-wavelength multi-function microplate reader (Varioskan Flash) to measure the absorbance of the sample at 450 nm.

Iii) Calculate cell survival rate:

Cell viability=[(As-Ab)/(Ac-Ab)]×100%

[As: Absorbance of experimental wells (including drugs); Ac: Absorbance of control wells (excluding drugs); Ab: Absorbance of blank wells (excluding cells and drugs)].

The results are shown in the table above. Both Mastoparan M and Mastoparan M liposome administration can inhibit the proliferation of Hela, HepG2, CT26, and HT29 cells. Among them, the administration of Mastoparan M liposome has a significantly stronger inhibitory effect on the proliferation of HepG2, CT26, and HT29 cells. Administer Mastoparan M. Prolonging the action time of Mastoparan M liposome has an obvious effect on Hela, but has no obvious effect on HepG2, CT26, and HT29 cells.

Test Example 3 Anti-breast cancer effect of Lipo@Mastoparan in mice

Construction of breast cancer subcutaneous xenograft tumor model: Take logarithmic growth phase MDA-MB-231-Luc cells (purchased from Beijing Rambolider Biological Co., Ltd.) to prepare a cell suspension with a concentration of 1×10 ⁸ cells/ml; take 6-8 One-week-old BALB/c-Nude female mice were inoculated with 100 μl of cell suspension subcutaneously in the right thigh of nude mice, and were raised in a barrier system to observe tumor growth.

Grouping and administration: 30 mice with the above breast cancer subcutaneous transplant tumor model (tumor volume 50mm3) were randomly divided into: Lipo@Mastoparan treatment group, MastoparanM treatment group, and PBS treatment group. Each group was administered tail vein administration on days 0, 1, 2, and 3.

Monitor tumor size: Use vernier calipers to measure the long diameter L and short diameter W of the tumor every two days, calculate the tumor volume according to the formula "Volume = L × W ² /2", take white light photos, and monitor its dynamic changes.

Observe the behavioral changes, weight changes and dietary changes of the mice, and calculate the survival time of the mice in each group. Death includes natural death, mouse tumor volume >1500mm ³ and a 20% weight loss to determine the death of the mice.

The results are shown in the table above: Lipo@Mastoparan has a good anti-tumor effect, and the therapeutic effect is better than that of the MastoparanM drug treatment group.

Claims (10)

1. A nano-liposome for resisting tumor is prepared from Mastparan M peptide and phosphatide through proportional mixing (1:1-5), and features that its amino acid sequence is INLKAIAALAKKLL.

2. Nanoliposome according to claim 1, characterized in that the phospholipids are selected from the group consisting of soybean lecithin, egg yolk lecithin, DPPC, DPPE, DSPC and soybean lecithin-cholesterol mixture, preferably soybean lecithin; preferably, the mass ratio of the Mastonaran M peptide to the phospholipid is 1:2.

3. The method for preparing nanoliposome of claim 1, comprising the steps of: 10mg of Mastonaran M peptide and 10-50 mg of phospholipid are weighed and dissolved in 5-20ml of reaction solvent, the mixture is heated and stirred for 0.5-12 hours at the reaction temperature of 25-65 ℃, the reaction solvent is removed by rotary evaporation at the temperature of 40-60 ℃, 5-20ml of ultrapure water is added for ultrasonic hydration until the mixture is uniformly dispersed, and the mixture is stored at the temperature of 4 ℃ for standby.

4. A method of preparation according to claim 3, wherein the phospholipid is selected from the group consisting of soy lecithin, egg yolk lecithin, DPPC, DPPE, DSPC and a soy lecithin-cholesterol mixture, preferably soy lecithin.

5. A method of preparation according to claim 3, wherein the amount of phospholipid is 20mg.

6. A process according to claim 3, wherein the reaction solvent is selected from DMSO, DMF, meOH, etOH, CH Cl2, CH3Cl, meOAc or acetone, preferably EtOH.

7. A method of preparation according to claim 3, wherein the reaction solvent volume is 10ml.

8. A method of preparation according to claim 3, wherein the reaction temperature is 55 ℃; the reaction time was 2h.

9. The process according to claim 3, wherein the post-treatment step is a spin-evaporation at 55℃to remove the reaction solvent, and a 10ml ultra-pure water is added for ultrasonic hydration.

10. The use of nanoliposome according to claim 1 for preparing a medicament for treating a tumor selected from any one of breast cancer, liver cancer, colorectal cancer and cervical cancer.