阅读说明：本技术 一种共载吴茱萸碱及磷酸氯喹的复方脂质体的制备方法 (Preparation method of compound liposome carrying evodiamine and chloroquine phosphate together ) 是由 曹勇 杨成莉 文志鹏 郑志昌 李明 樊兴华 白洋 李琳 于 2021-08-04 设计创作，主要内容包括：本发明涉及医药技术领域,公开了一种共载吴茱萸碱及磷酸氯喹的复方脂质体的制备方法,包括EVO-Lips的制备和EVO-CQ-Lips的制备；还公开了复方脂质体及应用。本发明将CQ及EVO共同包载于脂质体内制成复方脂质体,该复方脂质体在体内可高效准确的作用于乳腺癌细胞,提高了EVO在体内的生物利用度,降低了CQ对正常组织细胞的杀伤作用,副作用小,在癌症部位可协同发挥持久的作用,协同杀死肿瘤细胞,使治疗作用更加迅速、高效。(The invention relates to the technical field of medicines, and discloses a preparation method of a compound liposome carrying evodiamine and chloroquine phosphate together, which comprises the steps of preparing EVO-Lips and preparing EVO-CQ-Lips; also discloses compound liposome and application thereof. The CQ and the EVO are jointly encapsulated in the liposome to prepare the compound liposome, the compound liposome can efficiently and accurately act on breast cancer cells in vivo, the bioavailability of the EVO in vivo is improved, the killing effect of the CQ on normal tissue cells is reduced, the side effect is small, the compound liposome can cooperatively play a lasting effect on cancer parts, tumor cells are cooperatively killed, and the treatment effect is quicker and more efficient.)

1. A preparation method of a compound liposome carrying evodiamine and chloroquine phosphate together is characterized by comprising the following specific steps:

s1: preparation of EVO-Lips

Weighing HSPC, CHOL and EVO, wherein the mass of the HSPC, CHOL and EVO is 7mg, 2mg and 1mg respectively, dissolving the HSPC, CHOL and EVO in chloroform by ultrasonic waves, adding the dissolved solution into a 20mL round-bottom flask, and rotating the flask on a reduced-pressure rotary evaporator to remove an organic solvent chloroform to obtain a stable liposome membrane; adding the prepared liposome membrane into 1.5mL of ammonium sulfate buffer solution with the concentration of 1M for hydration for 1h, then transferring the liposome membrane into a 4mL EP tube, and carrying out ultrasonic treatment by using an ultrasonic cell disruption instrument;

s2: preparation of EVO-CQ-Lips

And (S1) after the ultrasound in the step (S1) is finished, adding the solution after the ultrasound into a dialysis bag, dialyzing for 12h by using PBS buffer solution with the pH value of 7.4, adding 200uL chloroquine phosphate with the concentration of 50mg/mL after the dialysis for 12h, and incubating for 30min at the temperature of 65 ℃ on a constant-temperature water bath kettle to obtain the compound liposome EVO-CQ-Lips carrying both the evodiamine and the chloroquine phosphate.

2. The method for preparing the evodiamine and chloroquine phosphate co-loaded compound liposome of claim 1, wherein in step S1, ddH is firstly used by an ultrasonic time-varying horn₂Performing O ultrasonic for 30s, and performing absolute ethyl alcohol ultrasonic for 30s, wherein the ultrasonic power is 40% during ultrasonic liposome treatment, and the ultrasonic time is set as 5s ultrasonic pause for 5 s.

3. The method for preparing the evodiamine and chloroquine phosphate co-loaded compound liposome of claim 1, wherein the concentration of the PBS buffer solution in the step S2 is 0.01moL, and the volume is 1L.

4. A complex liposome prepared by the preparation method of any one of claims 1-3.

5. The use of the compound liposome of claim 4 in the preparation of a medicament for treating breast cancer.

Technical Field

The invention relates to the technical field of medicines, in particular to a preparation method of a compound liposome carrying evodiamine and chloroquine phosphate together.

Background

Breast cancer is the second largest cancer that seriously compromises female health, with an average of about 20 thousands of women diagnosed with breast cancer worldwide each year, and about 4 thousands of women dying. Chemotherapy remains one of the main methods for its treatment, but the efficacy is not optimal due to the lack of suitable carriers for the accurate and efficient delivery of chemotherapeutic drugs to cancer sites to kill cancer cells. Therefore, the excellent carrier is researched to carry the chemotherapeutic drug, so that the carrier can enter the cancer part as efficiently and accurately as possible and play a lasting role, and the carrier has important significance for improving the treatment effect of the chemotherapeutic drug.

Evodiamine (EVO) is tryptamine indole alkaloid and is a main active ingredient of traditional Chinese medicine evodia. Modern pharmacological research results show that the polypeptide can regulate the expression of NF-kappa B, P53 protein and has an anticancer effect. Research shows that in the late stage of chemical drug treatment, based on a cell self-repair mechanism, the tumor cells excessively activate an autophagy mechanism to perform damaged cell self-repair, so that the chemotherapy effect is reduced. Therefore, the combined use of the autophagy inhibitor can inhibit the up-regulation of the autophagy at the late stage of the tumor cells, and can play a synergistic anti-tumor effect. Chloroquine phosphate (CQ) has the effect of inhibiting autophagy of cells, is generally considered to play an autophagy inhibition mechanism by blocking the fusion process of autophagosomes and lysosomes, and can regulate and control tumor suppressor protein P53 genes and enhance the anticancer activity of chemotherapeutic drugs.

Based on the theoretical research basis, the chloroquine phosphate and the evodiamine are jointly used for synergistic antitumor. Because EVO is a fat-soluble medicine, has small water solubility and low in-vivo bioavailability, and CQ has a stronger killing effect on normal tissue cells, how to deliver EVO and CQ into tumor parts efficiently and accurately becomes a key problem to be solved by the invention.

Disclosure of Invention

Based on the problems, the invention provides the preparation method of the compound liposome carrying the evodiamine and the chloroquine phosphate together, and the compound liposome can efficiently and accurately act on breast cancer cells, so that the treatment effect is quicker and more efficient.

In order to solve the technical problems, the invention provides a preparation method of a compound liposome carrying evodiamine and chloroquine phosphate together, which comprises the following specific steps:

s1: preparation of EVO-Lips

Weighing HSPC, CHOL and EVO, wherein the mass of the HSPC, CHOL and EVO is 7mg, 2mg and 1mg respectively, dissolving the HSPC, CHOL and EVO in chloroform by ultrasonic waves, adding the dissolved solution into a 20mL round-bottom flask, and rotating the flask on a reduced-pressure rotary evaporator to remove an organic solvent chloroform to obtain a stable liposome membrane; adding the prepared liposome membrane into 1.5mL of ammonium sulfate buffer solution with the concentration of 1M for hydration for 1h, then transferring the liposome membrane into a 4mL EP tube, and carrying out ultrasonic treatment by using an ultrasonic cell disruption instrument;

s2: preparation of EVO-CQ-Lips

And (S1) after the ultrasound in the step (S1) is finished, adding the solution after the ultrasound into a dialysis bag, dialyzing for 12h by using PBS buffer solution with the pH value of 7.4, adding 200uL chloroquine phosphate with the concentration of 50mg/mL after the dialysis for 12h, and incubating for 30min at the temperature of 65 ℃ on a constant-temperature water bath kettle to obtain the compound liposome EVO-CQ-Lips carrying both the evodiamine and the chloroquine phosphate.

Further, in step S1, the ultrasonic time-varying horn is first applied with ddH₂Performing O ultrasonic for 30s, and performing absolute ethyl alcohol ultrasonic for 30s, wherein the ultrasonic power is 40% during ultrasonic liposome treatment, and the ultrasonic time is set as 5s ultrasonic pause for 5 s.

Further, the concentration of the PBS buffer solution in step S2 was 0.01moL, and the volume was 1L.

In order to solve the technical problems, the invention also provides the compound liposome.

In order to solve the technical problems, the invention also provides application of the compound liposome in preparing a medicine for resisting breast cancer.

Compared with the prior art, the invention has the beneficial effects that: the CQ and the EVO are jointly encapsulated in the liposome to prepare the compound liposome, the compound liposome can efficiently and accurately act on breast cancer cells in vivo, the bioavailability of the EVO in vivo is improved, the killing effect of the CQ on normal tissue cells is reduced, the side effect is small, the compound liposome can cooperatively play a lasting effect on cancer parts, tumor cells are cooperatively killed, and the treatment effect is quicker and more efficient.

Drawings

FIG. 1 is a transmission electron microscope result of an optimal prescription EVO-CQ-Lips of an embodiment of the present invention;

FIG. 2 is a graph of the particle size distribution (nm) of the EVO-CQ-Lips for optimal prescription according to an embodiment of the present invention;

FIG. 3 is a graph of the results of the EVO-CQ-Lips storage stability test for the optimal recipe of the present invention (n is 3);

fig. 4 is a graph showing the results of comparing the release rates of EVO and CQ in examples of the present invention under different pH conditions (n-3);

fig. 5 is a graph of the inhibition rate of MCF-7 cell growth in examples of the present invention (n-6);

FIG. 6 is a graph showing the results of viable and dead staining of MCF-7 cells by different concentrations of EVO-CQ-Lips in examples of the present invention (. mu.M).

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.

Example (b):

in order to jointly use chloroquine phosphate and evodiamine for synergistically resisting breast cancer tumors, the inventor researches how to efficiently and accurately deliver EVO and CQ to tumor parts and overcomes the defects that the bioavailability of EVO in vivo is low and CQ has stronger killing effect on normal tissue cells.

The apparatus used in this example was as follows: SW-CJ-2F clean bench (Sujing group Antai Co.), BB15 cell culture chamber (U.S. Thermo Co.), IX73-U inverted fluorescence microscope (Olympus Co., Ltd., Japan), ELX800 Universal enzyme-labeling instrument (U.S. Bio-tek Co., Ltd.), Milli-Q Elix 15Advantage ultra-pure water system (U.S. Millipore Co., Ltd.), RE-2000A rotary evaporator (Shanghai Kangshi Biochemical instruments Co., Ltd.), 81-2 type constant temperature magnetic stirrer (Shanghai Spanish instruments Co., Ltd.), TG16W high speed centrifuge (Changshan Xiangzhi instruments Co., Ltd.), PSAC12R-120 type pH meter (Togada Macro electronics Co., Ltd.), ultrasonic cell crusher (Ningbo New Biotech Co., Ltd.), 720 type ultraviolet spectroscopy (Beijing Punju analytical instruments Co., Ltd.), ultrasonic cell crusher (Ningbo New Biotech Co., Ltd.), f-380 fluorescence spectrophotometer (Tianjin Hongkong science and technology development Co., Ltd.).

The reagents were as follows: cholesterol (Cholesterol, CHOL, batch No.: 111618-200301, Melphalan), hydrogenated soybean Lecithin (Lecithin hydrogenated, HSPC, batch No.: 525600-.

The cells used in this example were human breast cancer cells (MCF-7) purchased from Wuhan Protech Life technologies, Inc.

Selection of measurement wavelength: accurately weighing 0.25mg and 5mg of EVO and CQ bulk drugs dried to constant weight, adding 1mL of methanol for dissolving, placing in a 100mL volumetric flask, adding distilled water for diluting to scale, shaking uniformly to prepare EVO with mass concentration of 2.5 mu g/mL and CQ control solution with mass concentration of 2.5 mu g/mL, using distilled water as blank, and carrying out wavelength scanning within the range of 200-600nm, wherein the result shows that the maximum absorption peak exists at the position where lambda is 225nm and the CQ is 343nm when EVO is lambda is; and (3) respectively taking blank liposome solutions after demulsification, and scanning the EVO-CQ-Lips liposome solutions after demulsification by the same method, wherein the results show that the sample solutions respectively have maximum absorption at 225nm and 343nm, and the blank liposome solutions have no absorption at the two wavelengths, which indicates that the auxiliary materials and the solvent have no interference on the drug determination.

Drawing a standard curve: accurately weighing a certain mass of EVO, accurately configuring the concentration (mu g/mL) to be 1.0, 1.5, 2.0, 2.5 and 3.0 mu g/mL by using a volumetric flask, measuring the absorbance (A value) of the EVO at the lambda of 225nm by using a 720-type ultraviolet spectrophotometer, and linearly regressing the absorbance (A value) to the concentration (C) to obtain a regression equation: a ═ 0.1744C +0.0504 (R)²0.9991), the result shows that the EVO mass concentration is in the range of 1-3 mug/mL, and the linear fitting degree with the absorbance is better;

accurately weighing a certain mass of CQ, accurately configuring the concentration (mu g/mL) to be 10, 15, 20, 25 and 30 by using a volumetric flask, measuring the absorbance (A value) of the CQ by using a 720-type ultraviolet spectrophotometer at the lambda of 343nm, and linearly regressing the absorbance (A value) to the concentration (C) to obtain a regression equation: a ═ 0.0212C +0.0962 (R)²0.9994), the result shows that the CQ mass concentration is better in linear fitting degree with the absorbance in the range of 1-3 mug/mL.

Precision experiment, daily precision: measuring the absorbances of 5 groups of EVO (1.0, 2.0 and 3.0 mu g/mL) and CQ (10.0, 20.0 and 30.0 mu g/mL) in parallel in one day, calculating the Relative Standard Deviation (RSD) of the absorbances, and the precision RSD of EVO and CQ in the day is less than 5.0 percent, and the result shows that the precision of EVO and CQ in the day is good; precision in the daytime: the absorbances of 5 groups were measured in parallel for EVO (1.0, 2.0, 3.0. mu.g/mL) and CQ (10.0, 20.0, 30.0. mu.g/mL) over 1, 2, 3, 4, 5 days, respectively, and the Relative Standard Deviation (RSD) of the absorbances was calculated, and the daytime RSD of the absorbances of EVO and CQ was less than 5.0%, indicating that the daytime precision of EVO and CQ was good.

Sample recovery rate experiment: the percent recovery (%) of EVO (1.0, 2.0, 3.0 mug/mL) and CQ (10.0, 20.0, 30.0 mug/mL) was calculated respectively, the percent recovery (%) of EVO and CQ was between 95% and 105%, and the encapsulation efficiency accuracy of EVO and CQ measured by the method was good.

The preparation method of EVO-CQ-Lips comprises the following steps:

s1: preparation of EVO-Lips

Weighing EVO, HSPC and CHOL according to the prescription proportion, dissolving EVO, HSPC and CHOL with appropriate amount of chloroform under ultrasonic wave, adding into 20mL round bottom flask, and performing vacuum rotary evaporation on the flaskRotating to remove organic solvent chloroform to form stable liposome membrane; adding the prepared liposome membrane into 1.5mL ammonium sulfate buffer solution for hydration for 1h, transferring the hydrated solution into a 4mL EP tube, and performing ultrasonic treatment with an ultrasonic cell disruptor using ddH as an ultrasonic time-varying horn₂Performing O ultrasonic for 30s, performing absolute ethyl alcohol ultrasonic for 30s, setting ultrasonic power according to a prescription during ultrasonic liposome, and setting ultrasonic time to be ultrasonic 5s and pause for 5 s;

s2: preparation of EVO-CQ-Lips

After completion of the sonication in step S1, the solution was added to a dialysis bag and dialyzed against PBS buffer (pH 7.4) for 12 hours in a volume of 0.01 moL/L (2.38 g/L) of PBS buffer (1L/L)₂HPO₄·12H₂O+0.38gNaH₂PO₄ddH addition to 2L₂O), dialyzing for 12h, adding 200uL chloroquine phosphate (50mg/mL), and incubating on a constant-temperature water bath kettle for 30min according to the incubation temperature (65 ℃) to obtain EVO-CQ-Lips.

In this example, the optimal formula of EVO-CQ-Lips was screened according to the above preparation method, and the solvent screening and results are as follows: according to the properties of HSPC, CHOL and EVO, firstly, screening a solvent according to a basic prescription (HSPC: CHOL: EVO is 6:3:1), screening the solvent which has better and uniform film formation and does not bubble under vacuum reduced pressure, and obtaining a result shown in attached table 1, wherein the film formation is uniform and does not bubble easily when the solvent is chloroform;

screening results of solvent for prescription in attached Table 1

Note: HSPC, CHOL and EVO are in mass ratio. Good for +++ good, + normal, -poor

Screening the optimal prescription proportion and obtaining the results: hydrogenated soy lecithin: cholesterol: the proportion of evodiamine (HSPC: CHOL: EVO), ultrasonic power (%), ammonium sulfate concentration (pH 4.5) are influence factors, and influence of the evodiamine on the particle size (nm) and encapsulation efficiency (%) of cholesterol and hydrogenated soybean lecithin is discussed; taking a set basic prescription (HSPC: CHOL: EVO is 6:3:1, ammonium sulfate concentration (pH 4.5) is 3M, and ultrasonic power is 40%) as a reference, incubating at 65 deg.C for 3min as a fixed value, and screening the prescription as shown in the attached table 2;

prescription screening index of attached table 2

Note: HSPC: CHOL: EVO is mass ratio

Then, 1-9 prescriptions of EVO-CQ-Lips were prepared in a prescription ratio using chloroform as a prescription solvent, and the particle size (nm), PDI, EVO encapsulation efficiency% (EE%), CQ encapsulation efficiency% (EE%) were measured, and the results are shown in the following Table 3;

prescription screening of attached Table 3

Note: HSPC: CHOL: EVO is mass ratio

The results of the above 9 prescriptions are combined, so that prescription 2 and prescription 5 both have good particle sizes, and the final optimal prescription is obtained by orthogonal screening, namely HSPC: CHOL: EVO is 7: 2: 1 (exact mass of 7mg, 2mg, 1mg), ammonium sulfate concentration (pH) of 1M, ultrasonic power of 40% as the optimum formulation ratio, and the particle size, potential and encapsulation efficiency of the nanoparticles prepared with the optimum formulation are shown in table 4.

TABLE 4 optimum prescription nanoparticle characteristics

In this example, the morphological particle size and distribution of EVO-CQ-Lips were measured. TEM is used for observing appearance morphology, and a phosphotungstic acid negative dyeing method is adopted: preparing EVO-CQ-Lips according to an optimal prescription, dripping the mixture on a special copper wire after double-distilled water is used for resuspension, standing and air-drying the mixture, dripping 2% (w/v) phosphotungstic acid for negative dyeing for 2min, and observing the appearance form of the mixture under a Transmission Electron Microscope (TEM); as shown in the attached figure 1, the EVO-CQ-Lips is irregular sphere-like due to the coating of a layer of cholesterol and soybean phosphatidylcholine components; the particle size (nm) and the distribution thereof are measured by a particle sizer, the particle size distribution is shown in attached figures 1 and 2, and the particle size distribution of the EVO-CQ-Lips prepared by the optimal formula is more uniform within 170 +/-6.24 nm.

In the embodiment, an ultracentrifugation method is adopted to measure the liposome encapsulation efficiency, the prepared liposome suspension is taken, filtered by a 0.8-micron filter head to remove EVO and CQ which are not encapsulated in the liposome, the filtered liposome solution is added into a 1.5-mL centrifuge tube, centrifuged for 90min at 1300r/min, the liposome is collected, a proper amount of methanol is added for ultrasonic demulsification, the volume is diluted by PBS to a proper concentration, then the absorbance is measured, and the drug content W in the liposome is calculated_EVOAnd W_CQSetting the total dosage as M_EVO，M_CQ. The encapsulation efficiency was calculated as follows: EE%_EVO/CQ/M_EVO/CQX 100% EVO-CQ-Lips was digested with methanol to release encapsulated EVO and CQ, and the encapsulation efficiencies of EVO and CQ were measured with an ultraviolet spectrophotometer at (λ max (EVO) 225nm and λ max (CQ) 343nm), respectively, and the optimum formulation was selected based on the measured particle diameter and encapsulation efficiency. In the present embodiment, the ultracentrifugation method sample addition recovery rate is examined, namely, raw materials of EVO and CQ are precisely weighed and placed in a measuring flask, after methanol is dissolved, distilled water is added to dilute the raw materials into solutions with EVO concentrations of 1.0, 2.0 and 3.0 mu g/mL, CQ concentrations of 10, 20 and 30 mu g/mL and 10.0mg/mL, 0.1mL of drug solutions with different mass concentrations and 0.2mL of blank liposomes are respectively uniformly mixed, the mixture is centrifugally treated and placed in an ultrafiltration tube by the same method, filtrate is collected after centrifugal ultrafiltration, absorbance is measured after dilution, and the recovery rate is calculated. The encapsulation efficiency was measured to be ee (evo)% > 60.03%, ee (cq)% > 9.31%.

This example also investigated the stability and in vitro release of EVO-CQ-Lips, stability studies: preparing 8 parts of liposome according to an optimal prescription, evenly dividing into A, B groups, standing group A at 4 ℃, standing group B at room temperature of 25 ℃, measuring the particle size (nm) of the prepared liposome by a particle size analyzer after 0, 0.5, 1, 2, 4, 8, 16, 28 and 32(d), wherein the stability result of the particle size (nm) is shown in figure 3, the particle size (nm) of the optimal prescription liposome is stable at 4 ℃ and 25 ℃ for 0-2d, the particle size (nm) fluctuates at 2-8d but the change of the particle size (nm) is not large, probably because the 90plus PALS particle size analyzer tends to be stable, the stability of the optimal prescription EVO-CQ-Lips is good when the optimal prescription EVO-CQ-Lips is stored at 4 ℃ and 25 ℃ for 0-32 d; EVO-CQ-Lips are smooth sphere-like shapes and are dispersed very unevenly, and the EVO-CQ-Lips are irregular sphere-like shapes because a layer of cholesterol and soybean phosphatidylcholine components is wrapped; the EVO-CQ-Lips is connected with a CQ hydrophilic layer due to the outer shell, so that a dark shadow layer can be seen, and the shape is stable due to high cholesterol content.

In vitro release results investigation: the liposome prepared by the optimal formula is placed in a constant temperature shaking table with 37 ℃ and 120rpm after being resuspended in 1mL phosphate buffered saline (PBS, group A is pH 4.0, group B is pH 7.4), the release rate is measured at preset time points (0h, 0.5h, 2h, 4h, 6h, 18h, 1d, 2d, 4d, 7d, 14d, 21d and 28d), the result of the release rate curve is shown in figure 4, the in vitro release result shows that CQ is released faster than EVO at low pH, the release rates are equivalent when the pH is close to neutral, and the release rates can reach more than 80%.

In this example, the in vitro anti-tumor activity of the EVO-CQ-Lips cells was studied, and the MTT method was used to determine the in vitro anti-MCF-7 proliferation effect of EVO-CQ-Lips: counting MCF-7 cells in logarithmic growth phase with a cell counting plate, mixing with culture medium, spreading in 4 96-well plates at a density of 4000/mL per well, and adding CO₂And sucking out the culture medium in a 96-well plate 24h after the incubator, sequentially adding 200uL of EVO-CQ-Lips with serial concentration medicines and free EVO and CQ medicine culture medium solutions with corresponding concentrations into each 96-well plate, continuously culturing for 48h, adding 20 mu of LMTT solution (5mg/mL), taking out the 96-well plate from the incubator after 4h, sucking out the liquid, adding 150uL of dimethyl sulfoxide (DMSO) solution, and immediately measuring the light absorption value at 570nm by using a microplate reader. The results are shown in FIG. 5, in whichFIG. 5A shows that free EVO has an inhibitory effect on the growth of MCF-7 cells, FIG. 5B shows that free CQ has an inhibitory effect on the growth of MCF-7 cells, and FIG. 5C shows that EVO-CQ-Lips has an inhibitory effect on the growth of MCF-7 cells; the results of IC50 calculated by the inhibition ratio of free EVO group, free CQ group and EVO-CQ-Lips group are shown in the attached table 5, and the results show that EVO-CQ-Lips have stronger in vitro anti-breast cancer cell proliferation capability than free EVO and CQ, and the difference has statistical significance (P)<0.05). Compared with free EVO and CQ, EVO-CQ-Lips has stronger in-vitro breast cancer proliferation inhibition effect, and may have a significant relation with the significant improvement of drug solubility after liposome encapsulation.

TABLE 5 IC50 values of free EVO, free CQ and EVO-CQ-Lips for Breast cancer cells

"" differences compared to EVO-CQ-Lips group had statistical significance P <0.05

This example also investigated the anti-cancer effect of viable and dead cells, using MCF-7 cells grown in log phase at 8X 10⁴Inoculating the mixture into a 24-hole cell culture plate, and adding 0.05, 5 and 100 mu M of EVO-CQ-Lips nanoparticle cell culture solution with final concentration gradient after 24 hours; three multiple wells are arranged for each concentration, only fresh culture medium is replaced in a blank well, and the prepared Calcein-AM/PI staining solution is added after 24 hours; incubating at 37 ℃ for 30min, sucking away the staining working solution, terminating the incubation, adding 10 mu L of an anti-fluorescence quenching blocking solution, repeating cell climbing and nail polish blocking, observing yellow-green living cells at an excitation wavelength of 490nm, and observing red dead cells at an excitation wavelength of 545nm, wherein the result is shown in figure 6, and as the concentration of the nanoparticle drug increases, the quantity of calcein staining cells is obviously reduced, which shows that the living cells are sharply reduced, while the fluorescence of PI stained apoptotic cell nuclei is gradually enhanced, which shows that the quantity of the dead cells is obviously increased. Therefore, the nanoparticles have obvious killing effect on macrophage MCF-7.

In conclusion, the optimal formula of the embodiment can efficiently and accurately act on the breast cancer cells, can synergistically play a lasting role in the cancer part, and synergistically kill the tumor cells, so that the treatment effect is quicker and more efficient. The invention adopts the nano drug-carrying system to carry the small evodiamine traditional Chinese medicine molecules for the first time and uses the small evodiamine traditional Chinese medicine molecules for the research of the anticancer effect, adopts the nano drug-carrying system to carry the antimalarial drug chloroquine phosphate for the research of the anticancer effect for the first time, has small side effect, and further promotes the further application and development of the evodiamine and the antimalarial drug chloroquine phosphate.

The above is an embodiment of the present invention. The embodiments and specific parameters in the embodiments are only for the purpose of clearly illustrating the verification process of the invention and are not intended to limit the scope of the invention, which is defined by the claims, and all equivalent structural changes made by using the contents of the specification and the drawings of the present invention should be covered by the scope of the present invention.