CN103142481A - Drug-loaded liposome overcoming tumor drug resistance, preparation method and application thereof - Google Patents

Abstract

The invention provides a drug-loaded liposome, which includes a long-circulation liposome and a pharmaceutically active component encapsulated therein, and the pharmaceutically active component is paclitaxel and resveratrol. The present invention also provides a preparation method of drug-loaded liposomes, the method comprising, using thin film-ultrasonic method, liposome source, distearoylphosphatidylethanolamine-methoxypolyethylene glycol 2000 and drug activity The components are made into the drug-loaded liposome, and the active components of the drug are paclitaxel and resveratrol. And provide the drug-loaded liposome prepared by the above method. In addition, the present invention also provides the application of the above-mentioned liposome in the preparation of antitumor drugs and in the treatment of tumors. The drug-loaded liposome of the present invention contains paclitaxel and resveratrol, not only has the activity of inhibiting the development of drug-sensitive tumors, but also can effectively inhibit the development of chemotherapeutic drug-resistant tumors, and has great clinical application value.

Description

A drug-loaded liposome for overcoming tumor drug resistance and its preparation method and application

technical field

The invention relates to a drug-loaded liposome for overcoming tumor drug resistance, a preparation method of the drug-loaded liposome, and an application of the drug-loaded liposome in preparing antitumor drugs and treating tumors.

Background technique

1. Tumor drug resistance

At present, in the treatment of many tumors, the poor effect of conventional chemotherapy and poor prognosis are important clinical problems, and tumor multidrug resistance (MDR) is the key factor for the failure of tumor chemotherapy. After treatment, the remaining tumor stem cells develop drug resistance, which often leads to decreased sensitivity to certain drugs, and causes tumor recurrence or even metastasis. The drug resistance of human malignant tumors to chemotherapy can be divided into natural drug resistance (nature resistance) and acquired drug resistance (acquired resistance); Drug resistance (multidrug resistance, MDR). PDR is only resistant to the induced original drug, and does not produce cross-resistance in the face of other drugs; MDR is induced by one drug, but at the same time produces cross-resistance to other anticancer drugs with different structures and mechanisms of action .

There are various mechanisms of drug resistance, such as:

Enhancement of DNA repair ability: Chemotherapy drugs cause DNA damage, and the enhancement of dihydrofolate reductase (DHFR) and DNA damage repair-related enzyme activity (MGMT) can increase its resistance to chemotherapy drugs.

P-glycoprotein expression: P-glycoprotein is an energy-dependent drug excretion pump that can bind to some antineoplastic drugs and also has ATP binding sites. Once P-glycoprotein combines with anti-tumor drugs and provides energy through ATP, the drug can be pumped out of the cell, so that the concentration of the drug in the cell is continuously reduced, and the cytotoxic effect is weakened until it is lost, and drug resistance appears. Phenomenon. P-glycoprotein is expressed in normal bile ducts, kidneys, small intestines, adrenal glands, and hematopoietic stem cells, and is responsible for physiological functions such as hormone transport and excretion of toxins. Tumor patients with high expression of P-glycoprotein are often accompanied by poor prognosis, such as low remission rate, high recurrence rate, and short survival period, which can be used as prognosis evaluation indicators.

Glutathione S-transferase: Glutathione is a cysteine-containing tripeptide, which is the main non-protein sulfhydryl group in cells. Glutathione S-transferase can catalyze the combination of electrophilic compounds and GSH in the body, increase the water solubility of toxic compounds, reduce toxicity, and finally excrete the cells.

2. Paclitaxel

Paclitaxel is a mitosis inhibitor or spindle toxin, which is a diterpenoid compound extracted from the bark of Pacific yew tree. It can cause the rapidly dividing tumor cells to be firmly fixed in the mitotic stage, so that the replication of cancer cells is blocked and they die. It was approved by the US FDA in 1992. It is currently clinically very important for ovarian cancer, breast cancer and non-small First-line and second-line treatment of cancers such as cell lung cancer. It can also be used in the treatment of head and neck cancer, esophageal cancer, seminoma, and recurrent non-Hokin's lymphoma. Its adverse reactions include: allergic reactions; bone marrow suppression; neurotoxicity; cardiovascular toxicity; local reactions such as alopecia and inflammation.

Because paclitaxel is poorly soluble in water, the preparation is formulated with 50% ethanol and 50% surfactant polyoxyethylene castor oil derivatives to increase the water solubility of paclitaxel. The solvent also has toxic and side effects, such as allergic reactions, toxic kidney damage, neurotoxicity, and cardiovascular toxicity.

Docetaxel Injection (Aisu, Docetaxel, Docetaxel Injection) is a semi-synthetic paclitaxel analogue synthesized from the extract of the leaves of Taxus chinensis. The mechanism of action is to strengthen tubulin polymerization and inhibit microtubule Depolymerization, resulting in the formation of stable non-functional microtubule bundles, thereby disrupting mitosis in tumor cells. Its intracellular concentration is 3 times higher than that of paclitaxel, and its residence time in cells is longer, which is an important reason why it has greater antitumor activity than paclitaxel in vitro. In the in vivo test, it is effective against colon cancer, breast cancer, lung cancer, ovarian tumor transplantation, etc. in mice. The side effects are similar to paclitaxel, but its myelosuppression is more toxic. Docetaxel is generally formulated from polysorbate 80.

Liposomes are biological membrane-like bilayer structures composed of phospholipids and cholesterol. In 1971, Rymen et al. began to use it as a drug carrier. Compared with other carriers, liposomes have the following advantages: cell affinity and targeting; sustained release; reduced drug toxicity and improved drug stability. Paclitaxel liposome for injection (lipusu) is a new type of drug developed by Jiangsu Provincial Institute of Materia Medica, Nanjing Cisco Pharmaceutical Co., Ltd., and Jiangsu Provincial Liposomal Drug Engineering Technology Research Center. It was launched in 2004 and promoted. It is the first liposome drug approved by my country's Food and Drug Administration, and it is also the first international marketed paclitaxel liposome drug for injection.

Lipusu encapsulates paclitaxel, which is insoluble in water, in a new drug carrier-liposome phospholipid bilayer, which solves the solubility problem of paclitaxel. Compared with ordinary paclitaxel, lipusu has the following advantages:

(1) The risk of hypersensitivity caused by solvents is removed

Paclitaxel liposome fundamentally eliminates the mixed solvent of polyoxyethylated castor oil and absolute ethanol that must be used in ordinary paclitaxel injections, and eliminates the risk of hypersensitivity reactions. And pretreatment is not required, which provides convenience for clinical medication and saves patients from the adverse effects of large doses of hormones.

(2) The toxic and side effects of paclitaxel liposome are significantly less than those of paclitaxel injection

Due to the unique pharmacokinetic properties of liposome drugs, extended half-life in vivo, and small fluctuations in blood drug concentration, compared with paclitaxel injection, paclitaxel liposomes have less impact on the blood system, blood pressure and liver function. Drug toxicity is lower. In addition, the effect of paclitaxel liposome on blood pressure was significantly lower than that of paclitaxel injection, and the toxic reactions of peripheral blood and liver were significantly weakened.

(3) Significantly improve the body's tolerance to paclitaxel

The acute toxicity test showed that the LD ₅₀ of paclitaxel liposome was twice as large as that of paclitaxel injection, indicating that its tolerance was significantly improved, which provided room for increasing the dose of clinical medication and improving the curative effect.

(4) Extended half-life, highlighting the sustained-release effect

Preclinical pharmacokinetic tests showed that the half-life of paclitaxel liposome was more than double that of paclitaxel injection.

(5) Has the characteristics of targeted drug delivery

The concentration of paclitaxel liposome in liver, lung, lymphoid tissue and other tissues and organs was significantly increased. Follow-up detection does not increase the toxicity of this organ, which is beneficial to the treatment of local lesions.

Through laboratory research and clinical observation, it is found that paclitaxel liposome solves the solubility problem of paclitaxel; improves the stability of paclitaxel in solution; avoids allergic reactions without affecting antitumor activity; in addition, paclitaxel liposome also has intraperitoneal , the possibility of intrathoracic administration. Therefore, paclitaxel liposome has certain advantages compared with paclitaxel.

However, with the widespread application of tumor chemotherapy drugs in clinical treatment, the problem of tumor drug resistance has become more and more prominent, which has become one of the main obstacles to the effective treatment of tumors. However, the above-mentioned various forms of paclitaxel drugs have poor curative effects on drug-resistant tumors.

3. Resveratrol

In the 1980s, the World Health Organization found that although the French prefer high-fat foods such as cheese, the incidence and mortality of coronary heart disease are lower than those in other Western countries. The reason may be that the French often drink alcoholic beverages containing resveratrol Wine related. Since then, resveratrol has attracted much attention. So far, resveratrol has been found in at least 72 species of plants in 21 families and 31 genera. Resveratrol is mainly derived from the dried rhizome and root of Polygonum cuspidatum Sieb.et Zucc., the skin and seeds of Vitis vinifera fruit in the family Polygonaceae, and the seeds of Arachis hypogaea in Fabaceae.

Resveratrol is a natural antioxidant that can reduce blood viscosity, inhibit platelet aggregation and vasodilation, keep blood flowing, prevent the occurrence and development of cancer, and have anti-atherosclerosis, coronary heart disease, ischemia Sexual heart disease, prevention and treatment of hyperlipidemia.

In addition, resveratrol also has cardiovascular protection, anti-oxidation, anti-free radical, anti-inflammatory, antibacterial and other effects, and there are many clinical examples.

Contents of the invention

The purpose of the present invention is to overcome the defects that various forms of chemotherapeutic drugs in the prior art have poor curative effect on drug-resistant tumors, and provide a drug-loaded liposome with better curative effect on drug-resistant tumors and its preparation method and application.

The invention provides a drug-loaded liposome, which is characterized in that the drug-loaded liposome includes a long-circulating liposome and a pharmaceutically active component encapsulated therein, and the long-circulating liposome is a liposome surface coated Distearoylphosphatidylethanolamine-methoxy polyethylene glycol 2000 modified liposome, the active ingredients of the drug are paclitaxel and resveratrol.

The present invention also provides a preparation method of drug-loaded liposome, which is characterized in that the method comprises, using thin film-ultrasonic method, liposome source, distearoylphosphatidylethanolamine-methoxypolyethylene glycol 2000 and pharmaceutical active components to prepare the drug-loaded liposome, the pharmaceutical active components are paclitaxel and resveratrol. And provide the drug-loaded liposome prepared by the above method.

In addition, the present invention also provides the application of the above-mentioned drug-loaded liposome in the preparation of antitumor drugs and in the treatment of tumors.

The drug-loaded liposome of the present invention contains paclitaxel and resveratrol, in addition to having inhibitory activity on the development of drug-sensitive tumors, it can also effectively inhibit the development of chemotherapeutic drug-resistant tumors, so it has great clinical applications. Value.

Description of drawings

The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the description, together with the following specific embodiments, are used to explain the present invention, but do not constitute a limitation to the present invention. In the attached picture:

Fig. 1a and Fig. 1b are the transmission electron micrographs of the drug-loaded liposome of an embodiment of the present invention;

Figure 2a shows the result after 24 hours of drug-loaded liposome treatment of MCF-7 sensitive cells according to one embodiment of the present invention, and Figure 2b shows the result of this drug-loaded liposome treatment of MCF-7 resistant cells for 24 hours ;

Figure 3a shows the result after 48 hours of drug-loaded liposome treatment of MCF-7 sensitive cells according to one embodiment of the present invention, and Figure 3b shows the result of this drug-loaded liposome treatment of MCF-7 resistant cells for 48 hours ;

Figure 4a shows the result after 72 hours of drug-loaded liposome treatment of MCF-7 sensitive cells according to one embodiment of the present invention, and Figure 4b shows the result of this drug-loaded liposome treatment of MCF-7 resistant cells for 72 hours ;

Figure 5 shows the results of tumor changes after treatment of drug-resistant breast cancer tumor-bearing nude mice with drug-loaded liposomes according to an embodiment of the present invention.

Detailed ways

The invention provides a drug-loaded liposome, which is characterized in that the drug-loaded liposome includes a long-circulating liposome and a pharmaceutically active component encapsulated therein, and the long-circulating liposome is a liposome surface coated Distearoylphosphatidylethanolamine-methoxy polyethylene glycol 2000 (DSPE-mPEG2000) modified liposome, the active ingredients of the drug are paclitaxel and resveratrol.

In the present invention, the molar ratio of the long-circulation liposome and the active ingredient of the drug can be the same as that of the liposome carrier and the active ingredient of the drug in the prior art. The molar ratio of active components is 1:0.001-0.5, more preferably 1:0.01-0.3, most preferably 1:0.02-0.1.

According to the present invention, in the active ingredient of the drug, the molar ratio of paclitaxel and resveratrol can be changed within a wide range, for example, the molar ratio of paclitaxel and resveratrol can be 1:0.01-100 The inventors of the present invention have found that within a certain concentration range, as the amount of resveratrol increases, its synergistic effect with paclitaxel is also more and more significant, preferably, the molar ratio of paclitaxel and resveratrol 1:1-80, more preferably 1:3-40, considering the synergistic effect of resveratrol and the cytotoxicity of paclitaxel, the most preferably 1:5-20. The inventors of the present invention found that using paclitaxel and resveratrol within the above preferred ratio range can obtain drug-loaded liposomes with higher therapeutic effects.

According to the present invention, the long-circulation liposome can be the same as the conventional long-circulation liposome in the prior art, wherein the source of liposome is the same as distearoylphosphatidylethanolamine-methoxypolyethylene glycol 2000 The molar ratio can be changed in a wide range, for example, it can be 1:0.01-1, preferably 1:0.02-0.2, more preferably 1:0.05-0.1.

The invention provides a method for preparing drug-loaded liposomes, which is characterized in that the method comprises, using thin film-ultrasonic method, liposome source, distearoylphosphatidylethanolamine-methoxypolyethylene glycol 2000 The drug-loaded liposome is prepared with a drug active component, and the drug active component is paclitaxel and resveratrol.

Wherein, the molar ratio of the liposome source, distearoylphosphatidylethanolamine-methoxy polyethylene glycol 2000 and the active ingredient of the drug can be the ratio of the conventional liposome carrier to the active ingredient of the drug, Preferably, the molar ratio of the liposome source, distearoylphosphatidylethanolamine-methoxy polyethylene glycol 2000 and the active pharmaceutical component is 1:0.01-1:0.001-0.5, more preferably 1: 0.02-0.2:0.01-0.3, most preferably 1:0.05-0.1:0.02-0.1.

According to the method of the present invention, in the pharmaceutically active component, the molar ratio of paclitaxel and resveratrol can be adjusted as needed, preferably, the molar ratio of paclitaxel and resveratrol is 1:0.01-100, More preferably 1:1-80, more preferably 1:3-40, most preferably 1:5-20.

According to the present invention, the liposome source can be any conventional substance in the art capable of producing liposome carriers, including but not limited to at least one of soybean lecithin, hydrogenated soybean lecithin, egg yolk lecithin, preferably Soy Lecithin.

The liposome source, distearoylphosphatidylethanolamine-methoxypolyethylene glycol 2000, paclitaxel and resveratrol can all be obtained commercially.

In the present invention, the specific steps of the thin film-ultrasonic method are well known to those skilled in the art, that is, various raw materials are dissolved and the solvent is removed to form a thin film, and then particles are obtained by ultrasonic crushing.

Preferably, the ultrasonic conditions in the thin film-ultrasonic method include frequency of 200-800W, preferably 300-500W; ultrasonic time of 5-30S, preferably 10-20S; interval of 2-20S, preferably 5 -10S; the number of cycles is 5-30 times, preferably 10-20 times.

Specifically, the thin film-ultrasonic method may include the following steps,

(1) mixing liposome source, distearoylphosphatidylethanolamine-methoxy polyethylene glycol 2000, paclitaxel, resveratrol and the first organic solvent to obtain the first mixed solution,

(2) evaporation removes the first organic solvent in the first mixed solution to form a thin film,

(3) The film is mixed with pure water or saline to obtain a second mixed solution, and the drug-loaded liposome is obtained by ultrasonication.

Wherein, the first organic solvent may be a solvent capable of dissolving various raw materials, preferably at least one of absolute ethanol, chloroform and methanol, most preferably absolute ethanol.

In the present invention, the evaporation method is a conventional method in the art, as long as the first organic solvent can be removed, for example, it can be performed in a rotary evaporator.

According to the present invention, the addition amount of the first organic solvent can be conventionally selected in the art, preferably, in the first mixed solution, the concentration of the paclitaxel is 1×10 ^-4 mol/L-10 mol/L.

The amount of pure water or physiological saline can also be a conventional choice in this field. Preferably, in the second mixed solution, the concentration of paclitaxel is 1×10 ^-5 mol/L-10 mol/L, more preferably 1×10 ^-3 mol/L-1 mol/L, most preferably 5×10 ^-2 mol/L-0.5 mol/L.

The present invention is not particularly limited to the mixing method of the liposome source, distearoylphosphatidylethanolamine-methoxy polyethylene glycol 2000, paclitaxel, resveratrol and the first organic solvent, preferably, first The liposome source, distearoylphosphatidylethanolamine-methoxypolyethylene glycol 2000 and the first organic solvent were mixed, and then paclitaxel and resveratrol were added. Wherein, for liposome source, distearoyl phosphatidylethanolamine-methoxy polyethylene glycol 2000 and first organic solvent, there is no particular limitation on the order of addition of the three, for the addition of paclitaxel and resveratrol The addition order is also not particularly limited. The inventors of the present invention found that, through the above preferred method, drug-loaded liposomes with better therapeutic effects can be obtained.

The invention provides the drug-loaded liposome prepared by the above method.

The present invention also provides the application of the above drug-loaded liposome in the preparation of antitumor drugs. The drug-loaded liposome of the present invention is particularly suitable for the preparation of drugs against drug-resistant tumors.

The present invention also provides the application of the above drug-loaded liposome in tumor treatment. The drug-loaded liposome of the present invention is particularly suitable for treating drug-resistant tumors.

Such tumors and drug-resistant tumors include, but are not limited to, ovarian cancer, breast cancer, non-small cell cancer, head and neck cancer, esophageal cancer, seminoma, liver cancer, lung adenocarcinoma, prostate cancer, and non-Hokin's lymphoma, and at least one of their drug-resistant tumors.

The present invention is illustrated in more detail by the following examples. In the embodiment of the present invention, the soybean lecithin used was purchased from Shanghai Huyu Biochemical Reagent Co., Ltd., the commodity number is LE030A, and the CAS number is 8002-43-5; the egg yolk lecithin used was purchased from Shanghai Huyu Biochemical Reagent Co., Ltd., commodity The No. is P0051; the distearoylphosphatidylethanolamine-methoxypolyethylene glycol 2000 used is purchased from genzyme company, the product number is LP-R4-039, and the CAS No. is 147867-65-0; the paclitaxel (PTX) used is purchased from From Beijing Nuorui Pharmaceutical Technology Co., Ltd., CAS No. 33069-62-4; the resveratrol (Res) used was purchased from Hubei Xingyinhe Chemical Co., Ltd., CAS No. 501-36-0.

Example 1

This example is used to prepare drug-loaded liposome: PTX+Res liposome-1.

(1) Mix 4×10 ^-2 mol soybean lecithin, 2×10 ^-3 mol distearoylphosphatidylethanolamine-methoxypolyethylene glycol 2000 with 150ml of absolute ethanol, dissolve and add 3×10 ^-4 mol paclitaxel and 1.2×10 ^-3 mol resveratrol to obtain the first mixed solution,

(2) evaporation removes the absolute ethanol in the first mixed solution to form a thin film,

(3) The film was mixed with 5ml of physiological saline, and the drug-loaded liposome was obtained by ultrasonication, that is, PTX+Res liposome-1.

The conditions of the ultrasound include: the frequency is 350W, the ultrasound is 18s, the interval is 8s, and the ultrasound is 15 times.

Example 2

This example is used to prepare drug-loaded liposome: PTX+Res liposome-2.

(1) Mix 4×10 ^-2 mol soybean lecithin, 2×10 ^-3 mol distearoylphosphatidylethanolamine-methoxypolyethylene glycol 2000 with 200ml of absolute ethanol, dissolve and add 3×10 ^-4 mol paclitaxel and 3.6×10 ^-3 mol resveratrol to obtain the first mixed solution,

(2) evaporation removes the absolute ethanol in the first mixed solution to form a thin film,

(3) The film was mixed with 4ml of physiological saline, and the drug-loaded liposome was obtained by ultrasonication, that is, PTX+Res liposome-2.

The conditions of the ultrasound include: the frequency is 400W, the ultrasound is 15s, the interval is 5s, and the ultrasound is 10 times.

Comparative example 1

Paclitaxel-loaded liposomes (PTX liposomes) were prepared according to the method of Example 2, except that resveratrol was not added.

Comparative example 2

Resveratrol-loaded liposomes (Res liposomes) were prepared according to the method in Example 2, except that no paclitaxel was added.

Comparative example 3

According to the method of Example 2, 4×10 ^-2 mol soybean lecithin and 2×10 ^-3 mol distearoylphosphatidylethanolamine-methoxypolyethylene glycol 2000 were mixed with 200ml of absolute ethanol to prepare Long-circulating liposomes.

Example 3

This example is used to prepare drug-loaded liposome: PTX+Res liposome-3.

(1) Mix 8×10 ^-2 mol of soybean lecithin, 2×10 ^-3 mol of distearoylphosphatidylethanolamine-methoxypolyethylene glycol 2000 and 100ml of chloroform, dissolve and add 3×10 ^-4 mol paclitaxel and 6×10 ^-3 mol resveratrol to obtain the first mixed solution,

(2) evaporation removes the chloroform in the first mixed solution to form a thin film,

(3) The film was mixed with 3ml of pure water, and the drug-loaded liposome was obtained by ultrasonication, that is, PTX+Res liposome-3.

The conditions of the ultrasound include: the frequency is 300W, the ultrasound is 20s, the interval is 10s, and the ultrasound is 20 times.

Example 4

This example is used to prepare drug-loaded liposome: PTX+Res liposome-4.

(1) Mix 0.4mol egg yolk lecithin, 2×10 ^-2 mol distearoylphosphatidylethanolamine-methoxypolyethylene glycol 2000 and 100ml absolute ethanol, add 3×10 ^-4 mol paclitaxel after dissolving and 1.2×10 ^-2 mol resveratrol to obtain the first mixed solution,

(2) evaporation removes the absolute ethanol in the first mixed solution to form a thin film,

(3) Mix the film with 2ml of physiological saline, and obtain drug-loaded liposomes by ultrasonication, that is, PTX+Res liposome-4.

The conditions of the ultrasound include: the frequency is 500W, the ultrasound is 10s, the interval is 5s, and the ultrasound is 12 times.

test case 1

Detect PTX+Res liposome-3 with a transmission electron microscope (model is Tecnai G2 F20 U-TWIN), the results are shown in Figure 1a and Figure 1b, as can be seen from Figure 1a and Figure 1b, the drug-loaded liposome Relatively uniform particles with a diameter of 70-100 nm were formed.

test case 2

This test example is used to detect the activity of the drug-loaded liposome of the present invention at the cellular level.

Cell experiment 1: Inoculate MCF-7 sensitive cells (human breast cancer cells) in a 96-well plate with an inoculation volume of 5000 cells/well, add drugs after the cells adhere to the wall, and divide them into 4 groups according to different drugs, namely:

(1) Normal saline group;

(2) liposome group (separately add the long circulation liposome that comparative example 3 makes);

(3) Paclitaxel liposome group (adding the PTX liposome that comparative example 1 makes), wherein, the final concentration of paclitaxel in the culture medium is 5 μ g/ml;

(4) The liposome group (PTX+Res liposome) of encapsulating paclitaxel and resveratrol simultaneously, wherein, comprise 4 groups, add the PTX+Res liposome-1 that embodiment 1-4 makes respectively , PTX+Res liposome-2, PTX+Res liposome-3 and PTX+Res liposome-4, wherein, in each group, the final concentration of paclitaxel in the medium was 5 μg/ml, while the The final concentrations of reverthol were 5, 15, 25, and 50 μg/ml, respectively, with 6 replicate wells for each concentration;

Wherein, the amount of liposome added in the group of adding PTX+Res liposome-2 in (2) and (4) is all the same. 24h, 48h, and 72h after adding the drug, the OD value was detected with the MTS kit.

Cell experiment 2: detection was performed according to the same method as cell experiment 1, except that MCF-7 paclitaxel-resistant cells were inoculated.

The results of Cellular Experiment 2 are shown in Figures 2a-2b, 3a-3b and 4a-4b.

Figure 2a and Figure 2b are the results after 24 hours of treatment, wherein the vertical axis represents the measured OD value, which reflects the number of surviving cells, and the histograms of each group reflect the normal saline group, liposome group, paclitaxel lipid body group, and a group of test results in PTX+Res liposome-1 to PTX+Res liposome-4 (the horizontal axis represents the final concentration of resveratrol in the medium, from left to right is Test results of PTX+Res Liposome-1 to PTX+Res Liposome-4). Figure 2a is the detection result of MCF-7-sensitive cells, and Figure 2b is the detection result of paclitaxel-resistant breast cancer cells (MCF-7-resistant cells). In Figure 2a, the inhibitory activity of PTX+Res liposome-1 to PTX+Res liposome-4 with different concentrations of resveratrol on breast cancer cells was close to that of paclitaxel alone, even when adding low When the concentration of resveratrol, the inhibitory activity of PTX+Res liposome-1 to PTX+Res liposome-4 is worse than the effect of paclitaxel alone. But as can be seen from Figure 2b, paclitaxel liposomes almost no longer have inhibitory activity for paclitaxel drug-resistant tumor cells, while drug-loaded liposomes PTX+Res liposome-1 to PTX+Res liposomes according to the present invention Plastid-4 has obvious cancer cell inhibitory activity. And, as the concentration of resveratrol increased, its inhibitory activity became more and more significant.

Figure 3a and Figure 3b are the results after 48 hours of treatment, wherein Figure 3a is the detection result of MCF-7 sensitive cells, and Figure 3b is the detection result of MCF-7 resistant cells. Figure 3a and Figure 3b are similar to the results of Figure 2a and Figure 2b, the inhibitory activity of PTX+Res liposome-1 to PTX+Res liposome-4 with different concentrations of resveratrol on breast cancer cells was comparable to that of The inhibitory activity of paclitaxel was close, but paclitaxel liposomes almost no longer had inhibitory activity for paclitaxel-resistant tumor cells, while PTX+Res liposome-1 to PTX+Res liposome-4 according to the present invention all Has obvious cancer cell inhibitory activity.

Figure 4a and Figure 4b are the results after treatment for 72 hours, wherein Figure 4a is the detection result of MCF-7 sensitive cells, and Figure 4b is the detection result of MCF-7 resistant cells. Figure 4a and Figure 4b are also similar to the results of the above two groups of figures. Compared with Figure 3a and Figure 3b, the number of surviving cells in Figure 4a and Figure 4b further decreased.

It can be seen from the above results that the drug-loaded liposomes of the present invention are particularly suitable for inhibiting the development and propagation of paclitaxel-resistant tumor cells, and have obvious concentration- and time-dependence. After 72 hours of treatment, there is still obvious The antitumor activity of the drug-loaded liposome of the present invention shows that the antitumor activity of the drug-loaded liposome is durable.

Test case 3

This test example is used to detect the inhibitory activity of the drug-loaded liposome of the present invention on paclitaxel-resistant breast cancer at the animal level.

Animal experiment: MCF-7 resistant cells were inoculated into 16g Balb/c nude mice by subcutaneous injection, 10 ⁷ cells/mouse, and when the tumor grew to 3mm, the drug was administered. There were 5 groups in total, namely:

(1) normal saline group,

(2) liposome group (liposomes prepared in injection comparative example 3),

(3) Paclitaxel liposome group (injection of the PTX liposome prepared in Comparative Example 1, the injection amount is 8 mg/kg body weight),

(4) Resveratrol liposome group (res liposomes prepared in injection comparative example 2, the injection amount is 20mg/kg body weight),

(5) The liposome group (PTX+Res liposome that injection embodiment 2 makes) of wrapping paclitaxel and resveratrol simultaneously, intraperitoneal injection every day, paclitaxel injection amount is 8mg/kg body weight, resveratrol injection The amount is 20mg/kg body weight.

Wherein, the amount of liposome injection in group (2) is the same as that in group (5). Two weeks after the injection, the tumors were taken out and photographed, and the results are shown in Figure 5 (three parallel experiments for each group).

As can be seen from Figure 5, compared with the normal saline group, using paclitaxel liposomes alone and resveratrol liposomes alone has been unable to produce inhibitory effects on paclitaxel-resistant breast cancer cells, but using the present invention PTX+Res liposome-2, the drug-loaded liposome combined with resveratrol and paclitaxel, can significantly inhibit paclitaxel-resistant breast tumors.

The results of animal experiments further prove that the drug-loaded liposome of the present invention has strong inhibitory activity on drug-resistant tumors.

In addition, various combinations of different embodiments of the present invention can also be combined arbitrarily, as long as they do not violate the idea of the present invention, they should also be regarded as the disclosed content of the present invention.

Claims (17)

1. a kind of drug-loaded liposome, it is characterized in that, this drug-loaded liposome comprises long-circulation liposome and the pharmaceutically active component encapsulated therein, and described long-circulation liposome is liposome surface covered with two Stearoyl phosphatidylethanolamine-methoxy polyethylene glycol 2000 modified liposome, the drug active components are paclitaxel and resveratrol. 2. The drug-loaded liposome according to claim 1, wherein the molar ratio of the long-circulating liposome to the active pharmaceutical component is 1:0.001-0.5. 3. The drug-loaded liposome according to claim 1, wherein, in the active pharmaceutical component, the molar ratio of paclitaxel and resveratrol is 1:0.01-100. 4. The drug-loaded liposome according to any one of claims 1-3, wherein, in the long-circulation liposome, the liposome and distearoylphosphatidylethanolamine-methoxypolyethylene glycol The molar ratio of alcohol 2000 is 1:0.01-1. 5. a preparation method of drug-loaded liposome, is characterized in that, the method comprises, utilizes film-ultrasonic method, liposome source, distearoyl phosphatidylethanolamine-methoxy polyethylene glycol 2000 and The drug-loaded liposome is prepared from the drug-active components, and the drug-active components are paclitaxel and resveratrol. 6. method according to claim 5, wherein, the mol ratio of described liposome source, distearoyl phosphatidylethanolamine-methoxy polyethylene glycol 2000 and pharmaceutically active component is 1: 0.01-1 : 0.001-0.5. 7. The method according to claim 5, wherein, in the active pharmaceutical component, the molar ratio of paclitaxel and resveratrol is 1:0.01-100. 8. The method according to claim 5, wherein the liposome source is at least one of soybean lecithin, hydrogenated soybean lecithin, egg yolk lecithin. 9. The method according to claim 5, wherein the ultrasonic conditions in the thin film-ultrasonic method include that the frequency is 200-800W, the ultrasonic time is 5-30S, the interval time is 2-20S, and the number of cycles is 5-30S. 30 times. 10. The method according to any one of claims 5-9, wherein the thin film-ultrasonic method comprises the steps of, (1) mixing liposome source, distearoylphosphatidylethanolamine-methoxy polyethylene glycol 2000, paclitaxel, resveratrol and the first organic solvent to obtain the first mixed solution, (2) evaporation removes the first organic solvent in the first mixed solution to form a thin film, (3) The film is mixed with pure water or saline to obtain a second mixed solution, and the drug-loaded liposome is obtained by ultrasonication. 11. The method according to claim 10, wherein the first organic solvent is at least one of absolute ethanol, chloroform and methanol. 12. The method according to claim 10, wherein, in the second mixed solution, the concentration of the paclitaxel is 1×10 ^-5 mol/L-10 mol/L. 13. The drug-loaded liposome prepared by the method according to any one of claims 5-12. 14. The application of the drug-loaded liposome described in any one of claims 1-4 and 13 in the preparation of antitumor drugs. 15. The use according to claim 14, wherein the tumor is a drug-resistant tumor. 16. The application of the drug-loaded liposome according to any one of claims 1-4 and 13 in tumor therapy. 17. The use according to claim 16, wherein the tumor is a drug-resistant tumor.

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