CN114796112A

CN114796112A - Polymer nano micelle encapsulating two drugs and preparation method and application thereof

Info

Publication number: CN114796112A
Application number: CN202110130398.0A
Authority: CN
Inventors: 孙璐
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2021-01-29
Filing date: 2021-01-29
Publication date: 2022-07-29

Abstract

The invention provides a polymer nano micelle entrapping two drugs, a preparation method and application thereof. The polymer nano micelle is prepared from methoxy polyethylene glycol-polycaprolactone segmented copolymer mPEG-PCL, curcumin and adriamycin, is non-toxic, small in particle size, stable, high in encapsulation efficiency and controllable in drug release behavior, obviously enhances the toxicity and apoptosis promoting effect on 4T1 breast cancer cells compared with a micelle encapsulating a single drug, and can effectively inhibit tumor growth and metastasis and inhibit tumor angiogenesis. The biodegradable methoxy polyethylene glycol-polycaprolactone (mPEG-PCL) polymer nano micelle is used for encapsulating adriamycin and curcumin, the preparation process is simple, no surfactant or additive is used in the preparation process, the safety in clinical application is ensured, and the biodegradable methoxy polyethylene glycol-polycaprolactone polymer nano micelle has an excellent clinical application prospect.

Description

Polymer nano micelle encapsulating two drugs and preparation method and application thereof

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to a polymer nano micelle entrapping two medicines, and a preparation method and application thereof.

Background

Cancer remains a public health concern of widespread concern throughout the world, and has become the second cause of death in the united states, with the expectation that more than cardiovascular disease will become the first cause of death in the next few years. Of these, breast cancer is the leading cause of cancer death in women of 20-59 years of age, and new cases of breast cancer will account for 29% of all new cancer cases in 2015. The chemotherapy is an indispensable part of the breast cancer comprehensive treatment, and can reduce the risk of tumor recurrence and metastasis and improve the overall survival rate of patients by being used as a conventional means for treating the breast cancer. However, most chemotherapy drugs belong to cytotoxic drugs, and can damage normal tissues of the body while killing tumor cells, thereby causing toxic and side effects to different degrees. Meanwhile, some effective chemotherapeutic drugs have poor water solubility and high in vivo metabolism speed, and the administration frequency needs to be increased or the administration dosage needs to be increased so as to maintain the effective blood concentration, so that the effective dosage of the chemotherapeutic drugs is high, and the damage of the drugs to normal tissues is potentially increased. Therefore, how to solve the contradiction between the curative effect and the toxic and side effect is a scientific problem in the current tumor chemotherapy, in other words, how to improve the distribution of the drug in the tumor part and reduce the distribution of the drug in the normal tissues is the key to solve the problem. In order to solve the above problems, various drug delivery systems, such as polymer-drug copolymers, liposomes, micelles, nanogels, microspheres and nanospheres, have been developed in order to increase the drug concentration in tumor tissues and reduce the exposure of the drug to normal host tissues, thereby improving the therapeutic effect and reducing the drug toxicity.

In recent years, biodegradable polymer nano-micelles are used as an important component in the research of nano-drug delivery systems, have great potential in solving the key scientific problems of tumor chemotherapy, provide a new idea for researching innovative drugs, and have received high attention. Moreover, a large number of researches prove that the targeted drug delivery technology of the polymer nano micelle can improve the distribution of chemotherapeutic drugs in tumor parts and obviously improve the anti-tumor effect of the drugs. Polymeric nanomicelles are typically core/shell structures formed by self-assembly of amphiphilic block copolymers. The hydrophobic segment forms a micelle core for entrapping hydrophobic drugs, while the hydrophilic shell is generally composed of polyethylene glycol (PEG) and is intended to increase water solubility, prolong blood circulation time, reduce protein adsorption and recognition of the mononuclear phagocyte system. In addition, one of the most important advantages of polymer nanomicelles is passive targeting of tumor tissues. The role of this passive targeting is dependent on the high Permeability and Retention Effect (EPR) of solid tumors. Unlike the blood vessels in normal tissues, the gap between the adjacent endothelial cells of tumor neovessels reaches 200-1200nm, and the lymphatic circulation of tumor tissues is obstructed, so that our nano-micelles can overflow from the blood vessels and accumulate in the tumor tissues for a long time.

However, due to the heterogeneity of tumor cells, most tumors are difficult to cure with a single therapeutic agent at tolerable doses. The combined application of different medicines can not only enable each medicine to play the maximum cell killing effect within the range allowed by a human body, but also have wider effect on tumor cells with heterogeneity, and can prevent or slow down the generation of drug resistance. Thus, two or more antineoplastic agents are currently included in the standard treatment regimen for most tumors. However, the physicochemical properties of the drugs are different, such as the solubility, and multiple solvents or drug carriers are usually required to be used during the application, so that the drugs cannot reach the tumor tissue at the same time. Therefore, a multifunctional drug delivery system is needed to carry chemotherapeutic drugs with different physicochemical properties, so that the drugs can reach tumor tissues simultaneously to play an anti-tumor role in combination. Although various drug-carrying systems such as micelles, lipids, inorganic nanoparticles and the like have been designed to carry various drugs in recent years, carrying chemotherapeutic drugs with different dissolution characteristics is still not easy to realize, and due to the different characteristics of the drugs, the design and preparation of micelles are often more complicated and complicated, which further increases the difficulty of research.

Therefore, the research on the drug-loaded nano-micelle capable of co-delivering two or more drugs and the synergistic enhancement of the anti-tumor capacity of the anti-tumor drug have important significance.

Disclosure of Invention

The invention aims to provide a polymer nano micelle for encapsulating two drugs. The water-soluble adriamycin and the hydrophobic curcumin are simultaneously encapsulated in the polymer nano micelle, so that the adriamycin and the curcumin can simultaneously reach tumor tissues to jointly play an anti-tumor role.

The invention provides a polymer nano micelle entrapping two drugs, which is characterized by being prepared from the following raw materials: mPEG-PCL, doxorubicin and curcumin; the number average molecular weight of the mPEG-PCL is 3000-5000.

Further, the polymer nano micelle is prepared by feeding adriamycin, curcumin and mPEG-PCL according to the mass ratio of (0.5-1.5) to (1-2) to (50-70); preferably, the mass ratio of the adriamycin, the curcumin and the mPEG-PCL is 1:1.5: 60.

Further, the mPEG-PCL has a number average molecular weight of 4000.

The invention also provides a preparation method of the polymer nano micelle entrapping the two drugs, which comprises the following steps:

(1) weighing curcumin and mPEG-PCL, dissolving in an organic solvent, evaporating to remove the organic solvent, and adding water to dissolve to obtain a Cur-M micelle solution;

(2) and (2) adding a PBS solution into the Cur-M micelle solution obtained in the step (1) to obtain a mixed solution, dropwise adding an adriamycin hydrochloride aqueous solution into the mixed solution while stirring, and stirring to obtain the adriamycin hydrochloride aqueous solution.

Further, the organic solvent in the step (1) is alcohol, preferably ethanol; and/or the concentration of curcumin in the Cur-M micelle solution is 1-2 mg/mL, and the concentration of mPEG-PCL is 50-70 mg/mL; preferably, the concentration of curcumin in the Cur-M micelle solution is 1.5mg/mL, and the concentration of mPEG-PCL is 60 mg/mL.

Further, the evaporation in the step (1) is rotary evaporation at the rotating speed of 100rpm/min under the vacuum condition of 60 ℃; and/or the stirring time is 0.5-1.5 h, preferably 1 h.

Further, the PBS solution of the step (2) has pH of 7.4; the concentration of the doxorubicin hydrochloride aqueous solution is 3-7 mg/mL, preferably 5 mg/mL; and/or the volume ratio of the PBS solution Cur-M micelle solution to the doxorubicin hydrochloride aqueous solution is 1 (5-10) to (1-5), preferably 1:7: 2.

Further, the dropping of the aqueous solution of doxorubicin hydrochloride in the step (2) is completed within 1 minute.

The invention also provides application of the polymer nano micelle entrapping the two drugs in preparation of antitumor drugs.

Furthermore, the anti-tumor drug is a drug for promoting tumor cell apoptosis, and/or inhibiting tumor cell proliferation, and/or inhibiting tumor cell angiogenesis.

The experimental result shows that the polymer nano micelle of the invention encapsulating the two drugs has no toxicity, small particle size, stability, high encapsulation rate and controllable drug release behavior, compared with the micelle encapsulating a single drug, the polymer nano micelle obviously enhances the toxicity and the apoptosis promotion effect on 4T1 breast cancer cells, can effectively inhibit the growth and the metastasis of tumors and inhibit the angiogenesis of the tumors. The biodegradable methoxy polyethylene glycol-polycaprolactone (mPEG-PCL) polymer nano micelle is used for encapsulating adriamycin and curcumin, the preparation process is simple, no surfactant or additive is used in the preparation process, the safety in clinical application is ensured, and the biodegradable methoxy polyethylene glycol-polycaprolactone polymer nano micelle has an excellent clinical application prospect.

The mPEG-PCL refers to a methoxy polyethylene glycol-polycaprolactone block copolymer.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a schematic diagram of the preparation of Dox-Cur-M micelles.

FIG. 2 is a graph showing the particle size distribution of Dox-Cur-M micelles.

FIG. 3 is a transmission electron micrograph of Dox-Cur-M micelles.

FIG. 4 is a sample schematic diagram of blank mPEG-PCL (a), Dox-M (b), Cur-M (c) and Dox-Cur-M (d) micelle solutions.

FIG. 5 is a standard curve of curcumin (A) and doxorubicin (B).

FIG. 6 is the release of curcumin from Cur-M and Dox-Cur-M micelles in PBS solution (A) at pH 7.4 or PBS solution (C) containing 10% FBS; release of Doxorubicin from Dox-M and Dox-Cur-M micelles in PBS solution (B) at pH 7.4 or PBS solution (D) containing 10% FBS.

FIG. 7 is the cytotoxicity of Dox-Cur-M micelles against 4T1 breast cancer cells in vitro. (A) Cell survival rates of Cur-M, Dox-M and Dox-Cur-M micelle samples with different concentration gradients after being acted on 4T1 breast cancer cells for 48 hours. (B) Cell viability of blank mPEG-PCL micelles with different concentration gradients after 48 hours of action on 4T1 breast cancer cells and L929 mouse fibroblasts.

FIG. 8 shows the apoptotic effect of Dox-Cur-M micelles on 4T1 breast cancer cells in vitro. Physiological saline (NS) (A), blank mPEG-PCL micelle (B), Cur-M (C), Dox-M (D) and Dox-Cur-M (E) under a fluorescence microscope, Hoechst33258 nuclear staining pictures of each treatment group of the micelle and the average apoptosis index (F) of each treatment group. Scale bar 100 μm.

FIG. 9 is doxorubicin uptake by 4T1 breast cancer cells in vitro. The free adriamycin group, the free adriamycin and curcumin group, the Dox-M and curcumin free group and the Dox-Cur-M group act on 4T1 breast cancer cells for 2 hours (A) and 4 hours (B), and the tumor cells of each treatment group are quantitatively analyzed by a flow cytometer for 2 hours (C) and 4 hours (D) of the adriamycin-containing positive cell proportion and the average fluorescence intensity of the adriamycin for 2 hours (E) and 4 hours (F).

FIG. 10 is a photograph of doxorubicin uptake by 4T1 breast cancer cells under a fluorescent microscope. After blank mPEG-PCL micelles, Dox-M and Dox-Cur-M micelles act on tumor cells for 2 hours and 4 hours, the nucleus emits blue fluorescence by Hoechst33258 staining, and the tumor cells take up adriamycin of each treatment group and locate in the nucleus to emit red fluorescence. Scale bar 100 μm.

FIG. 11 is a graph of the antitumor effects of saline (control), blank mPEG-PCL micelles, Cur-M, Dox-M, and Dox-Cur-M in a 4T1 subcutaneous model of breast cancer. (A) Pictures of tumors in each treatment group on day 30 (scale ═ 1 cm); (B) tumor growth curves for each treatment group; (C) weight of subcutaneous tumors in each treatment group.

FIG. 12 shows the tumor metastasis inhibition effect of physiological saline (control), blank mPEG-PCL micelles, Cur-M, Dox-M and Dox-Cur-M in the spontaneous lung metastasis model of 4T1 breast cancer. (A) Pictures of spontaneous lung metastases for each treatment group (scale ═ 2 cm); (B) number of tumor metastasis nodules in the lungs of each treatment group; (C) weight of lung in each treatment group.

FIG. 13 is an immunofluorescence photograph of TUNEL staining of tumor tissue in the normal saline (A), blank mPEG-PCL micelles (B), Cur-M (C), Dox-M (D), and Dox-Cur-M groups (E) (scale: 100 μ M); (F) apoptotic index (Apoptotic index) of each treatment group.

Fig. 14 is an immunohistochemical image of Ki-67 staining of tumor tissue in the normal saline (a), blank mPEG-PCL micelles (B), Cur-M (c), Dox-M (d), and Dox-Cur-M group (E) (scale: 100 μ M); (F) ki-67 positivity (Ki-67 LI) for each treatment group.

Fig. 15 is an immunofluorescence picture of CD31 staining of tumor tissue in the normal saline (a), blank mPEG-PCL micelles (B), Cur-M (c), Dox-M (d), and Dox-Cur-M group (E) (scale: 100 μ M); (F) microvascular density (MVD) for each treatment group.

Detailed Description

1. Raw materials and equipment therefor

mPEG-PCL (Mn ═ 4000) synthesized by national focus biotherapy laboratories of university of sichuan;

doxorubicin hydrochloride, zhejiang haizheng pharmaceutical company, china;

curcumin, Sigma, usa;

chromatographically pure methanol, Fisher corporation, usa;

chromatographically pure acetonitrile, Fisher corporation, usa;

ethanol, analytically pure, Chengdu Kelong chemical reagent factory, China;

tween-80, analytically pure, Chengdu Kelong chemical reagent factory, China;

DMSO, analytically pure, metropolis chemical reagent factory, china;

microfiltration, 0.22um, Merck Millipore, germany;

dialysis bag, MD34MM 3500K, usa.

Rotary evaporator, R-201 cheng city new shu west science experimental facilities limited, china;

circulating water type multipurpose vacuum pump, SHB-3, cheng du municipality new shu west experimental facilities limited, china;

a constant temperature water bath, W201D, cheng du shi xinxi branch of academic press, china;

transmission electron microscopy, Hitachi H-600IV, Japan;

particle size/zeta potential meter, Nano2S Malvern, uk;

waters Alliance 2695 high performance liquid chromatography analyzer, usa;

desk centrifuge, TDL-50C, Shanghai' an Tingchan scientific Instrument plant, China.

4T1 mouse breast cancer cells and L929 mouse fibroblasts were from the American Type Culture Collection (ATCC) and were maintained by the national intensive biotherapeutic laboratory of Sichuan university.

DMEM medium and RPMI 1640 medium, Gibco, usa;

fetal bovine serum, Gibco, usa;

MTT reagent (dimethyl thiazole diphenyl tetrazole bromide salt), Sigma company, usa;

hoechst33258 dye, Sigma, usa.

Microplate reader Mutiscan MK3, seimer feishell instruments ltd;

fluorescence microscope Mutiscan MK3, LEICA, Germany;

flow cytometer FACS Calibur model, Becton Dickinson, usa.

BALB/c mice (SPF grade, female, 6-8 weeks, body weight around 18 g) were provided by the Waxi laboratory animal center, Sichuan university.

Apoptosis kit (DeadEnd) ^TM TUNEL fluorescence detection system), Promega corporation, usa;

rabbit anti-mouse Ki-67 antibody, Gene Tech, USA;

biotin-labeled goat anti-mouse immunoglobulin, BD Biosciences Pharmingen, usa;

CD31 antibody, BD Pharmingen TM, USA;

FITC-labeled secondary antibody, Abcam, USA.

Example 1 preparation of the polymer nano-micelle of the present invention encapsulating two drugs simultaneously

The mPEG-PCL polymer nano micelle (Dox-Cur-M) which simultaneously contains the adriamycin and the curcumin is obtained by two steps.

Step 1: preparing mPEG-PCL micelle (Cur-M) entrapping curcumin by a solid dispersion method: curcumin and mPEG-PCL polymer were weighed and added to 5mL of ethanol to be completely dissolved. Then using a rotary evaporator to perform rotary evaporation for 10 minutes at the rotation speed of 100rpm/min and under the negative pressure condition at 60 ℃ to remove ethanol, adding distilled water after forming a transparent film, placing the transparent film in a water bath kettle at 60 ℃ for hydration to obtain a Cur-M micelle solution, wherein the concentration of curcumin in the solution is 1.5mg/mL, and the concentration of mPEG-PCL polymer is 60mg/mL

Step 2: doxorubicin was entrapped in Cur-M micelles by a pH-induced self-assembly method. 0.1mL of PBS (10X, pH 7.4) is added into 0.7mL of Cur-M micelle solution, then 0.2mL of 5mg/mL doxorubicin hydrochloride aqueous solution is added into the solution dropwise under mild stirring, stirring is continued for one hour after doxorubicin is added within 1 minute, and after one hour, the double-drug-loaded mPEG-PCL polymer nano-micelle (Dox-Cur-M) can be obtained (figure 1). The Dox-Cur-M micellar solution is obtained, then is centrifuged at 13000rpm for 10 minutes to remove free curcumin, and then is centrifuged at 20000rpm by a centrifuge tube with a dialysis membrane with the cutoff molecular weight of 3kDa for 5 minutes to separate the unencapsulated adriamycin.

Comparative example 1 preparation of mPEG-PCL Polymer nanomicelle (Dox-M) encapsulating Single drug Adriamycin

Preparation of mPEG-PCL Polymer nanomicelles encapsulating Single drug doxorubicin (Dox-M) the above Cur-M micelle solution was replaced with a blank mPEG-PCL polymer micelle solution (60mg/mL) in accordance with step 2 of example 1, which was obtained by the pH-induced self-assembly method. After the gel bundle is obtained, centrifuging for 5 minutes at 20000rpm by a centrifuge tube with a dialysis membrane with the cutoff molecular weight of 3kDa, and separating the unencapsulated adriamycin.

Comparative example 2 preparation of mPEG-PCL Polymer Nanoglelle (Cur-M) encapsulating curcumin as a monotherapy

Preparation of mPEG-PCL Polymer nanomicelles (Cur-M) entrapping the individual drugs curcumin referring to step 1 of example 1, Cur-M micellar solution was obtained and centrifuged at 13000rpm for 10 minutes to remove free curcumin.

The beneficial effects of the polymer nano-micelle of the invention are demonstrated by the following experimental examples.

Experimental example 1 characterization of physicochemical Properties of Dox-Cur-M micelles of the invention

1. Experimental methods

1.1 determination of Dox-Cur-M micelle diameter

All the following Dox-Cur-M micelles were prepared as in example 1.

The hydrated particle size and distribution of the Dox-Cur-M micelles was measured by Dynamic Light Scattering (DLS) using a laser scattering particle sizer (Nano-ZS, Malvern Instrument, UK); the test temperature of the sample was 25 ℃, and the test was repeated 3 times, and the average value was taken.

1.2 Transmission Electron microscopy to observe the morphology of the real Dox-Cur-M micelle

The morphology of the Dox-Cur-M micelles was observed using a Transmission Electron Microscope (TEM). Before observation, a drop of diluted Dox-Cur-M micelle of example 1 was dropped on a copper mesh, and then negative-stained with phosphotungstic acid solution, dried, and placed under an electron microscope to observe its morphology.

1.3 stability Studies of Dox-Cur-M micelles

The Dox-Cur-M micelle solution is prepared and then is respectively stored at 4 ℃ or 25 ℃ to observe the stability of the solution. If the solution is uniform and bright, the micellar solution is stable, and if precipitation occurs, the micellar solution is unstable.

2. As a result, the

Study on the particle size distribution of Dox-Cur-M micelles using dynamic light scattering is shown in fig. 2, and the results show that the mean particle size of Dox-Cur-M micelles is 25.3 ± 0.2nm, and the particle size distribution of Dox-Cur-M micelles is monodisperse (PDI ═ 0.065 ± 0.011).

The morphology of the Dox-Cur-M micelle is characterized by a transmission electron microscope, and as shown in FIG. 3, the Dox-Cur-M micelle has uniform particle size and an average particle size of about 25 nm. Therefore, the particle size of the Dox-Cur-M micelles observed by a transmission electron microscope was about the same as that observed by dynamic light scattering.

The prepared blank mPEG-PCL, Dox-M, Cur-M and Dox-Cur-M micellar solutions are clear, transparent and uniform as shown in figure 4. The stability of the Dox-Cur-M micellar solution is observed by respectively placing the Dox-Cur-M micellar solution at 4 ℃ or 25 ℃, and the result shows that the Dox-Cur-M micellar solution can be stabilized for about 20 days when placed at 25 ℃, and can be stabilized for more than 1 month without generating precipitates when placed at 4 ℃.

The results show that the polymer nano micelle Dox-Cur-M simultaneously encapsulating curcumin and adriamycin has small particle size, good dispersibility and stability, and is beneficial to cell uptake and long-term storage.

Experimental example 2 establishment of HPLC analysis method and measurement of encapsulation efficiency and drug-loading amount

1. Experimental method

1.1 chromatographic conditions

Waters Alliance 2695 high performance liquid chromatograph, column insetsil/WondaSil C18(4.6x250mm, 5um), column temperature 30 ℃, sample size 20 μ L, flow rate: 1.0mL/min, and detecting that the mobile phase of curcumin is acetonitrile: 1% acetic acid solution (60/40, V/V), detection wavelength 420 nm; the mobile phase for detecting the adriamycin is methanol: acetonitrile: 1% acetic acid solution (40/10/50, V/V), detection wavelength 252 nm.

1.2 Standard Curve for curcumin and Adriamycin

Accurately weighing 10mg curcumin by using an electronic scale, placing the curcumin in a 100mL volumetric flask, and adding 100mL methanol to dissolve the curcumin to obtain a stock solution. Diluting to obtain curcumin methanol standard solutions with different concentrations of 0.1, 0.4, 0.8, 1, 4, 8, 10, 40 and 80 μ g/mL. And (3) carrying out sample injection analysis on the standard solution with the concentration according to the chromatographic condition of 3.1, determining the peak area of the curcumin, and carrying out linear regression on the peak area (A) and the concentration (C) to obtain a standard curve for determining the curcumin content. The same method is adopted to draw a standard curve equation of the adriamycin.

1.3 encapsulation efficiency and drug Loading determination

The Dox-Cur-M micelle mother solution of example 1 (drug concentration 1mg/mL) was prepared according to the above method, and the concentration was measured by using a solution diluted with methanol under the chromatographic conditions of 3.1. The Drug Loading (DL) and the Encapsulation Efficiency (EE) were calculated using the following equations, respectively:

2. results

According to the chromatographic conditions, peak areas of curcumin and adriamycin solutions with different concentrations are measured. By performing linear regression on the peak areas (A) and the concentrations (C), regression equations of curcumin and adriamycin are respectively obtained: a. the _Cur ＝127593C–40969(R＝0.9995)；A _Dox 47010C +7851.3 (R0.9996) shows that curcumin has a good linear relationship in the range of 0.1-80 mug/mL and adriamycin in the range of 0.1-40 mug/mL. The standard curves for curcumin and doxorubicin are shown in figure 5.

After the standard curve is obtained, quantitative analysis is carried out on the drug content of curcumin and adriamycin in the Dox-Cur-M micelle by using HPLC, and the result shows that the encapsulation rate of curcumin in the Dox-Cur-M micelle is 99.32 +/-0.47 percent, and the drug loading rate is 4.97 +/-0.02 percent; the adriamycin entrapment rate is 96.77 +/-0.30%, and the drug loading rate is 4.85 +/-0.01%.

The results show that the polymer nano micelle Dox-Cur-M simultaneously encapsulating curcumin and adriamycin has high drug loading rate and encapsulation efficiency.

Experimental example 3 in vitro Release study of Dox-Cur-M micelles of the invention

1. Experimental methods

First, 1mL of Cur-M (comparative example 2), Dox-M (comparative example 1) and Dox-Cur-M micelles (example 1) were added to a dialysis bag having a cut-off molecular weight of 3.5kDa, respectively, and then the dialysis bag was put into 10mL of a release medium. There are two release media, 0.5 wt% Tween80 or 10% Fetal Bovine Serum (FBS) in PBS at pH 7.4. When the dialysis bag is placed in the release medium, the release medium is preheated to 37 ℃. The release experiment was carried out in a constant temperature shaking chamber at 37 ℃ and a shaking speed of 100 rpm/min. 1mL of the release medium was sampled at 1, 2, 4, 8, 24, 48, 72, 120, 168 hours and stored at-20 ℃ before the remaining release medium was decanted and fresh release medium preheated to 37 ℃ was added. Five replicates of release experiments were performed for each micelle in different release media. The concentrations of curcumin and doxorubicin in the samples were determined using HPLC after the end of the release experiment.

2. Results of the experiment

As shown in fig. 6. Fig. 6, a and C, show that curcumin is released relatively slowly from the Dox-Cur-M micelles, both in neutral PBS and in PBS containing 10% FBS, compared to Cur-M micelles. The analytical reasons may be related to the competitive release of curcumin and doxorubicin from Dox-Cur-M micelles. On day 7, the release rates of curcumin from Cur-M micelles in serum-free and serum-containing release media were approximately 36% and 78%, respectively, while the release rates from Dox-Cur-M micelles in serum-free and serum-containing release media were approximately 17% and 50%, respectively.

As can be seen in B and D of fig. 6, the release of doxorubicin from the Dox-M and Dox-Cur-M micelles exhibited two phases compared to curcumin: the first stage is a relatively rapid burst release, which may be facilitated by the reaction of the ligand of the hydrophilic segment of doxorubicin entrapped in the core of the micelle with the hydrophilic segment of the micelle; the second phase is a relatively slow sustained release, with doxorubicin, which did not react with the hydrophilic segment of the micelle, being competitively released from the Dox-Cur-M micelles with curcumin. The cumulative release rates of doxorubicin from the Dox-Cur-M micelles in the serum-free and serum-containing release media were 49.71 + -3.33% and 50.62 + -0.72%, respectively, while the cumulative release rates of doxorubicin from the Dox-M micelles in the two release media were 57.43 + -3.36% and 70.43 + -0.87%, respectively.

The results show that the polymer nano-micelle Dox-Cur-M simultaneously encapsulating the curcumin and the adriamycin can realize the effective slow release of the adriamycin and the curcumin, and the slow release effect is superior to that of the polymer nano-micelle Dox-M singly loading the adriamycin and the polymer nano-micelle Cur-M singly loading the curcumin.

Experimental example 4 in vitro antitumor study of Dox-Cur-M micelle of the present invention

4.1 cytotoxicity Studies of Dox-Cur-M micelles against in vitro tumor cells

1. Experimental methods

The cytotoxic effect of Dox-Cur-M micelles on 4T1 breast cancer cells was studied by the MTT method. 4T1 breast cancer cells were first seeded into 96-well plates with about 3000 cells at 100. mu.L per well and incubated at 37 ℃ for 24 hours. Then, 100. mu.L of blank mPEG-PCL micelle, Cur-M (comparative example 2), Dox-M (comparative example 1) and Dox-Cur-M micelle (example 1) samples with different concentration gradients were added to each well, and the mixture was incubated at 37 ℃ for 48 hours. Wherein the concentration of adriamycin in Dox-Cur-M and Dox-M is the same, and the concentration of curcumin in Dox-Cur-M and Cur-M is the same. Finally, the viability of the cells was determined by the MTT method. The experiment was repeated six times and the mean value was taken.

Cytotoxicity of blank mPEG-PCL micelles against 4T1 breast cancer cells and L929 mouse fibroblasts was also determined by the MTT method described above.

2. Results

The MTT method is adopted to evaluate whether the cytotoxicity of the polymer nano-micelle loaded with adriamycin and curcumin is enhanced to 4T1 breast cancer cells compared with the polymer nano-micelle loaded with single drug of curcumin or adriamycin. As can be seen in FIG. 7A, the half-lethal concentration (IC) of Dox-Cur-M ₅₀ ) Significantly lower than Dox-M, where the IC50 for Dox-M and Dox-Cur-M were 0.94 and 0.34. mu.g/mL, respectively. However, the IC of Cur-M ₅₀ 9.7. mu.g/mL, the cell activity was still very high when 2.5. mu.g/mL of Cur-M was applied to 4T1 breast cancer cells.

The cytotoxicity of the blank mPEG-PCL micelles on 4T1 breast cancer cells and L929 mouse fibroblasts was also evaluated by the MTT method. As shown in FIG. 7B, 4T1 breast cancer cells and L929 mouse fibroblasts still had a relatively high survival rate when the final concentration of empty mPEG-PCL micelles was 1000. mu.g/mL.

The results show that the mPEG-PCL micelle has good biocompatibility and no toxicity basically, and is a safe drug carrier. Compared with single drug-loaded polymer nano-micelles Cur-M and Dox-M, the Dox-Cur-M micelle provided by the invention has the advantage that the cytotoxicity of tumor cells is obviously enhanced.

4.2 evaluation of the apoptotic Effect of Dox-Cur-M micelles on in vitro tumor cells

1. Experimental methods

The apoptotic effect of Dox-Cur-M micelles (example 1) on 4T1 breast cancer cells was evaluated by observing cell morphology by Hoechst33258 nuclear staining. Sterile coverslips were first placed in 6-well plates and 4T1 breast cancer cells were seeded into 6-well plates, with 1mL per well being approximately 5X 10 ⁴ The cells were incubated overnight at 37 ℃. Physiological saline (NS), blank mPEG-PCL micelle, Cur-M, Dox-M and Dox-Cur-M micelle samples were added to each well for 48 hours the next day. The cell slide was removed, quickly fixed in 70% cold ethanol, washed with PBS, stained with Hoechst33258, coverslip fixed on slide, and cell morphology was observed using fluorescence microscope. 5 equally large fields were randomly selected in each treatment group by double-blind method, and the number of apoptotic cells and the total number of cells in each field were calculated. The apoptosis index (Apoptotic index) (%) is the number of Apoptotic cells/total cell number × 100%, and the average apoptosis index of each group was calculated according to the formula.

2. Results

The apoptosis effect of the polymer nano-micelle loaded with curcumin and adriamycin on 4T1 breast cancer cells is evaluated by Hoechst33258 nuclear staining. When the cell is apoptotic, the cell nucleus is fragmented and the chromosomes are condensed, so that the blue fluorescence of the apoptotic cell is very strong compared with that of the normal cell under a fluorescence microscope. FIG. 8 shows the cell image of physiological saline (NS), blank mPEG-PCL micelles, Cur-M, Dox-M and Dox-Cur-M micelles after 48 hours of action on 4T1 breast cancer cells. From FIG. 8, it was found that apoptotic cells were significantly increased in the Dox-Cur-M group compared with the Dox-M group. The blank mPEG-PCL micelle and the Cur-M group tumor cells emit uniform fluorescence, and almost have few core fragmentations. As shown in FIG. 8F, the Dox-Cur-M micelle group had the highest apoptosis index of 38.30 + -2.26%, which was higher than that of Dox-M (14.13 + -0.28%, P <0.001, ANOVA), Cur-M (3.86 + -0.34%, P <0.001, ANOVA), blank mPEG-PCL micelle (1.86 + -0.10, P <0.001, ANOVA), and normal saline group (2.14 + -0.17%, P <0.001, ANOVA). Furthermore, there was no statistical difference in the apoptosis index between the saline, blank mPEG-PCL micelles and the Cur-M group.

The results show that the apoptosis effect of the Dox-Cur-M polymer nano micelle on the 4T1 tumor cells in vitro is obviously enhanced compared with that of single drug-loaded polymer nano micelle Dox-M and Cur-M.

4.3 Admission Studies of Adriamycin by in vitro tumor cells

1. Experimental methods

Adriamycin uptake by tumor cells in different doxorubicin sample solutions was quantified using flow cytometry. 4T1 breast cancer cells in log phase growth were first seeded into 6-well plates with 1mL per well of approximately 2X 10 ⁵ The cells were cultured at 37 ℃ for 24 hours. The medium in the wells was discarded and then serum free blank mPEG-PCL micelles, free doxorubicin and curcumin, Dox-M (comparative example 1) and free curcumin and Dox-Cur-M (example 1) micelle sample solutions were added to each well. Wherein the concentration of curcumin and adriamycin in the solution is 500 ng/mL. After 2 and 4 hours, cells were harvested, immediately washed 2 times with PBS, and then 1X 10 cells were quantitatively collected by flow cytometry (Coulter Elite Esp, Coulter, Hialeah, FL) per sample ⁴ And (4) detecting red fluorescence of the adriamycin in the cells. The excitation wavelength is 488nm and the emission wavelength is between 564nm and 606 nm.

When Dox-M and Dox-Cur-M micelles acted on tumor cells, the uptake of doxorubicin into the tumor cells was observed using a fluorescence microscope. Sterile coverslips were first placed into 6-well plates and 4T1 breast cancer cells were seeded into 6-well plates, with 1mL per well being approximately 2X 10 ⁵ The cells were cultured at 37 ℃ for 24 hours. Then, blank mPEG-PCL micelle, Dox-M and Dox-Cur-M micelle samples without serum are added into each well for culture. After 2 and 4 hours, the cell slide was removed, quickly fixed in 70% cold ethanol, washed with PBS, stained with Hoechst33258,the coverslip was mounted on a glass slide and the cells were observed using a fluorescence microscope.

2. Results

By flow cytometry analysis, fig. 9 shows the level of doxorubicin uptake by tumor cells when different doxorubicin solutions were applied to the tumor cells. From FIG. 9A, C, E, it was found that after 2 hours of exposure of free doxorubicin to tumor cells, the tumor cells had little to no uptake of doxorubicin. However, tumor cells in the other 4 groups showed accumulation of doxorubicin to varying degrees. Moreover, the Dox-Cur-M group (62.63 + -5.17%, 51.57 + -0.90) was higher than the Dox-M and free curcumin groups (49.44 + -8.40%, 48.73 + -0.90, P <0.05, ANOVA), the Dox-M group (17.32 + -3.44%, 44.66 + -0.77, P <0.001, ANOVA), the free doxorubicin and curcumin groups (3.46 + -0.47%, 41.73 + -0.31, P <0.001, ANOVA) and the free doxorubicin group (0.21 + -0.06%, 41.26 + -0.19, P <0.001, ANOVA) both in the ratio of doxorubicin-containing positive cells and in the mean fluorescence intensity of doxorubicin. As shown in fig. 9B, D, F, the uptake of doxorubicin by the tumor cells was increased to various degrees at 4 hours after the tumor cells were affected in each treatment group compared to 2 hours. However, doxorubicin uptake by tumor cells was greatest in the Dox-Cur-M group compared to the free doxorubicin group, the free doxorubicin and curcumin group, and the Dox-M group. Because the Dox-Cur-M group was the highest compared to the above three groups (P <0.001, ANOVA) regardless of the adriamycin-containing positive cell ratio or the mean fluorescence intensity of adriamycin. Although the Dox-Cur-M group had a higher proportion of adriamycin-containing positive cells and a higher mean fluorescence intensity of adriamycin than the Dox-M and free curcumin groups after 4 hours of culture, there was no uniform chemical difference between the two groups (P >0.05, ANOVA).

FIG. 10 shows the images of the tumor cells of the above treatment groups under a fluorescence microscope after 2 hours and 4 hours of exposure of blank mPEG-PCL micelles, Dox-M and Dox-Cur-M micelles to 4T1 breast cancer cells. The nucleus fluoresces blue by Hoechst33258 dye and the red fluorescence from doxorubicin is also localized in the nucleus as shown in figure 10. After blank mPEG-PCL micelles act for 2 hours and 4 hours, no red fluorescence is emitted by tumor cells. When the Dox-M micelle acts on the tumor cells for 2 hours, the red fluorescence emitted by the adriamycin in the tumor cells is weak, and the red fluorescence emitted by the tumor cells of the Dox-Cur-M micelle group is rapidly enhanced. After the treatment group acts on the tumor cells for 4 hours, the adriamycin in the tumor cells is continuously accumulated, and the emitted red fluorescence is gradually enhanced. Compared with the Dox-M micelle, the red fluorescence emitted by the tumor cells is still stronger in the Dox-Cur-M micelle group.

The above results demonstrate that curcumin in the Dox-Cur-M micelles of the present invention can promote the uptake of doxorubicin by tumor cells.

All experimental data above were processed with SPSS17.0 statistical software using one-way analysis of variance (ANOVA) and the pairwise comparisons between treatment groups were tested using SNK-q. The test level was 0.05 and a P <0.05 considered statistically significant. The data obtained are expressed as mean ± standard deviation.

Test example 5 in vivo antitumor study of Dox-Cur-M micelles of the present invention

5.1 in vivo antitumor Effect study of Dox-Cur-M micelles

1. Experimental methods

A subcutaneous 4T1 breast cancer model was established to study the in vivo anti-tumor effect of the Dox-Cur-M micelles of example 1. On day 0, each 6-8 week female BALB/c mouse was inoculated subcutaneously into the right dorsal side of the mouse 0.1mL of a 4T1 breast cancer cell suspension (containing approximately 5X 10 cells) ⁵ Individual cells). Tumors were palpable near the injection site on day 4, and these tumor-bearing mice were then divided into 5 groups (5 per group) as follows: physiological saline group (Control), blank mPEG-PCL micelle group, Cur-M micelle group of comparative example 2 (curcumin dose of 1mg/kg), Dox-M micelle group of comparative example 1 (doxorubicin dose of 1mg/kg) and Dox-Cur-M micelle group of example 1 (curcumin and doxorubicin doses of 1 mg/kg). On days 4, 7 and 10, administration was by tail vein injection for a total of three times. The size of the tumor was observed daily after the injection of the drug, and the size of the tumor in longitudinal and transverse diameters (unit: mm) was measured and recorded every three days using a vernier caliper. The longest (length) and shortest (width) diameters of the subcutaneous tumors were measured each time, and tumor volumes were calculated according to the following formula: tumor volume (mm) ³ ) 0.52 × length (mm) × width (mm). On day 30, mice in the control group began to die. Kill it by breaking neckMice were sacrificed and immediately removed tumors were weighed. Since 4T1 breast cancer cells were readily vascular-transferred to the lungs and formed metastases in BALB/c mice, the lungs of each treatment group of mice were collected, weighed, and two observers recorded metastatic nodules on each lung by a double-blind method.

2. Results

To compare the in vivo anti-tumor effects between the Dox-Cur-M micelle group and the Dox-M, Cur-M and blank mPEG-PCL micelle group, we established a 4T subcutaneous model of breast cancer. As shown in FIGS. 11A and B, the Dox-Cur-M micelle group showed stronger tumor growth inhibition than the Dox-M and Cur-M micelle groups (P <0.05 and P <0.001, ANOVA). However, the blank mPEG-PCL micelle group had no anti-tumor effect compared to the normal saline group (P >0.05, ANOVA). FIG. 11C shows the tumor weight for each treatment group, and it can be seen that the tumor weight for the Dox-Cur-M micellar group (0.51. + -. 0.22g) is much lower than that for Dox-M (1.27. + -. 0.67g, P <0.05, ANOVA), Cur-M (2.67. + -. 1.10g, P <0.001, ANOVA), empty mPEG-PCL micellar group (4.39. + -. 0.93g, P <0.001, ANOVA), and saline group (4.09. + -. 0.80g, P <0.001, ANOVA).

The results show that the Dox-Cur-M micelle has strong tumor growth inhibition effect, and the effect is superior to that of single drug-loaded polymer nano-micelle Cur-M and Dox-M.

Since 4T1 breast cancer cells were easily transferred to the lung through blood vessels and formed metastases in BALB/c mice, we investigated whether the Dox-Cur-M micelle group had an inhibitory effect on 4T breast cancer spontaneous lung metastasis. As shown in FIGS. 12A and B, the number of tumor metastasis nodules (6.0 + -3.2) in lungs of Dox-Cur-M micelle group was significantly lower than that of Dox-M (15.2 + -4.5, P <0.05, ANOVA), Cur-M (26.4 + -6.2, P <0.001, ANOVA), blank mPEG-PCL micelle group (49.4 + -20.8, P <0.001, ANOVA), and saline group (46.2 + -12.8, P <0.001, ANOVA). As can be seen from fig. 12C, the weight of the lungs was the lightest in the Dox-Cur-M micelle group compared with the remaining four groups.

The results show that the Dox-Cur-M micelle has strong effect of inhibiting tumor metastasis and has better effect than single-drug-loaded polymer nano-micelle Cur-M and Dox-M

5.2 evaluation of Dox-Cur-M micelles for apoptosis of tumor cells in vivo

1. Experimental methods

4T1 mammary carcinoma subcutaneous tumors were collected and fixed in 4% paraformaldehyde, and the tumor tissues were embedded in paraffin. The tissue sections were then stained for deoxyribonucleotide terminal transferase-mediated nicked end labeling (TUNEL) following the procedure on the in situ cell death assay kit. After staining was completed, 2 observers randomly selected 5 equally large fields per treatment group using double blind method under a fluorescent microscope high power microscope (200 ×), and observed and recorded the numbers of apoptotic cells and total cells per field. The apoptosis index (Apoptotic index) (%) is the number of Apoptotic cells/total cell number × 100%, and the average apoptosis index of each group was calculated according to the formula.

2. Results

Whether the Dox-Cur-M micelle has the effect of promoting apoptosis on 4T1 tumor cells in vivo or not is investigated through TUNEL immunofluorescence staining. The cell nuclei were stained green in FIG. 13, indicating apoptosis. Therefore, it is found from FIG. 13 that the cells in the Dox-Cur-M micelle group undergoing apoptosis under the same visual field are significantly more than the Dox-M of comparative example 1, the Cur-M of comparative example 2, the blank mPEG-PCL micelle and the physiological saline group. FIG. 13F shows the Apoptotic index (Apoptotic index) for each treatment group, where the Apoptotic index (21.86 + -2.42%) for the Dox-Cur-M micelle group was greater than that of Dox-M (11.06 + -2.05%, P <0.001, ANOVA), Cur-M (5.86 + -1.28%, P <0.001, ANOVA), blank mPEG-PCL micelles (3.18 + -0.97%, P <0.001, ANOVA), and saline group (2.38 + -0.99%, P <0.001, ANOVA).

The results can show that the Dox-Cur-M micelle has a strong effect of promoting tumor cell apoptosis and the effect is superior to that of single-drug-loaded polymer nano-micelle Cur-M and Dox-M.

5.3 evaluation of the Effect of Dox-Cur-M micelles on tumor cell proliferation in vivo

1. Experimental methods

Ki-67 staining was used to assess the effect of Dox-Cur-M micelles of example 1 on cell proliferation in tumor groups in vivo. Tumor tissue was first embedded in paraffin and cut to 5 μm thickness for Ki-67 staining. After staining was completed under a high power lens (200 ×), 2 observers observed randomly 5 fields per treatment group using double-blind method and the number of Ki-67 positively expressed cells and total cells were recorded. The Ki-67 positive rate (Ki-67 LI) (%) Ki-67 positive cell number/total cell number × 100%, and the average Ki-67 positive rate for each treatment group was calculated according to the formula.

2. Results

Through Ki-67 immunohistochemical staining, whether Dox-Cur-M micelles have the effect of inhibiting proliferation of 4T1 tumor cells or not is researched. The cells with brown nuclei in FIG. 14 were Ki-67 positive cells, and from these, it was found that the Ki-67 positive cells in the Dox-Cur-M micelle group were the least in the treatment group. As can be seen from FIG. 14F, the positive rate (Ki-67 LI) of the Dox-Cur-M micelle group Ki-67 was only 18.6. + -. 6.5%, which is significantly lower than that of Dox-M of comparative example 1 (40.4. + -. 6.7%, P <0.001, ANOVA), Cur-M of comparative example 2 (58.6. + -. 9.8%, P <0.001, ANOVA), blank mPEG-PCL micelle (70.4. + -. 11.32%, P <0.001, ANOVA), and physiological saline group (76.2. + -. 8.0%, P <0.001, ANOVA).

The results show that the Dox-Cur-M micelle has a strong effect of inhibiting the proliferation of tumor cells, and the effect is superior to that of single-drug-loaded polymer nano-micelle Cur-M and Dox-M.

5.4 evaluation of the Effect of Dox-Cur-M micelles on tumor tissue angiogenesis in vivo

1. Experimental methods

To assess the effect of Dox-Cur-M micelles on angiogenesis in tumor tissue, neovasculature in tumor tissue was therefore stained. Tumor tissues of each treatment group were frozen sections, fixed with acetone, incubated with CD31 monoclonal antibody, washed with PBS, incubated with FITC-labeled secondary antibody, and visualized under a fluorescent microscope after staining. Microvessel density (MVD) is the number of microvessels per equilarge field of view, so 2 observers under a high power mirror (200 ×) randomly observed 5 fields of view per treatment group using a double blind method and recorded the number of microvessels, and the average microvessel density per treatment group was calculated.

2. Results

Numerous studies have shown that the growth and metastasis of tumors requires the generation of new blood vessels. Whether Dox-Cur-M micelles inhibited tumor growth by anti-angiogenesis was investigated by CD31 immunofluorescent staining of neovasculature. The nascent blood vessels in FIG. 15 fluoresce red, and the red fluorescence of the tumor tissue of the Dox-Cur-M micelle group can be found to be the weakest. FIG. 15F also reflects that the MVD (17.4 + -6.1) for the Dox-Cur-M micelle set of example 1 is significantly less than the Dox-M (54.2 + -8.5, P <0.001, ANOVA) of comparative example 1, the Cur-M (43.2 + -4.9, P <0.001, ANOVA) of comparative example 2, the blank mPEG-PCL micelles (62.2 + -6.8, P <0.001, ANOVA), and the saline set (63.6 + -7.6, P <0.001, ANOVA).

The results show that the Dox-Cur-M micelle has a strong effect of inhibiting angiogenesis, and the effect is superior to that of single-drug-loaded polymer nano-micelle Cur-M and Dox-M. .

In conclusion, the invention provides the polymer nano micelle encapsulating the adriamycin and the curcumin, the micelle is non-toxic, small in particle size, stable, high in encapsulation efficiency and controllable in drug release behavior, compared with the micelle encapsulating a single drug, the polymer nano micelle obviously enhances the toxicity and the apoptosis promoting effect on 4T1 breast cancer cells, and can effectively inhibit the growth and the metastasis of tumors and inhibit the angiogenesis of the tumors. The biodegradable methoxy polyethylene glycol-polycaprolactone (mPEG-PCL) polymer nano micelle is used for encapsulating adriamycin and curcumin, the preparation process is simple, no surfactant or additive is used in the preparation process, the safety in clinical application is ensured, and the biodegradable methoxy polyethylene glycol-polycaprolactone polymer nano micelle has an excellent clinical application prospect.

Claims

1. The polymer nano micelle entrapping two drugs is characterized by being prepared from the following raw materials: mPEG-PCL, doxorubicin and curcumin; the number average molecular weight of the mPEG-PCL ranges from 3000 to 5000.

2. The two-drug-encapsulating polymer nanomicelle according to claim 1, which is prepared by feeding adriamycin, curcumin and mPEG-PCL in a mass ratio of (0.5-1.5): (1-2): 50-70); preferably, the mass ratio of the adriamycin to the curcumin to the mPEG-PCL is 1:1.5: 60.

3. The two-drug-encapsulating polymeric nanomicelle of claim 1, wherein the mPEG-PCL has a number average molecular weight of 4000.

4. The preparation method of the two-drug-entrapped polymer nanomicelle according to any one of claims 1 to 3, comprising the steps of:

5. The method according to claim 4, wherein the organic solvent in the step (1) is an alcohol, preferably ethanol; and/or the concentration of curcumin in the Cur-M micelle solution is 1-2 mg/mL, and the concentration of mPEG-PCL is 50-70 mg/mL; preferably, the concentration of curcumin in the Cur-M micelle solution is 1.5mg/mL, and the concentration of mPEG-PCL is 60 mg/mL.

6. The method according to claim 4, wherein the evaporation in the step (1) is rotary evaporation at a rotation speed of 100rpm/min under vacuum condition of 60 ℃; and/or the stirring time is 0.5-1.5 h, preferably 1 h.

7. The method according to claim 4, wherein the PBS solution of step (2) has a pH of 7.4; the concentration of the doxorubicin hydrochloride aqueous solution is 3-7 mg/mL, preferably 5 mg/mL; and/or the volume ratio of the PBS solution Cur-M micelle solution to the doxorubicin hydrochloride aqueous solution is 1 (5-10) to (1-5), preferably 1:7: 2.

8. The method according to claim 4, wherein the step (2) of adding the aqueous doxorubicin hydrochloride solution dropwise is performed within 1 minute.

9. The use of the two-drug entrapped polymer nanomicelle according to any one of claims 1 to 3 in the preparation of an antitumor drug.

10. The use according to claim 9, wherein the anti-neoplastic agent is an agent that promotes apoptosis of tumor cells, and/or inhibits proliferation of tumor cells, and/or inhibits angiogenesis of tumor cells.