CN111317720A

CN111317720A - Leukocyte-simulated pluronic-lipid nano hybrid drug delivery carrier and application thereof

Info

Publication number: CN111317720A
Application number: CN201811526773.8A
Authority: CN
Inventors: 沙先谊; 陈沁月; 何文秀
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2018-12-13
Filing date: 2018-12-13
Publication date: 2020-06-23

Abstract

The invention belongs to the field of biomedicine, relates to a tumor-targeted biomimetic hybrid nano drug delivery carrier, and particularly relates to a leucocyte-simulating biomimetic hybrid pluronic-lipid bilayer membrane serving as a drug delivery carrier of a chemotherapeutic drug, which can be used for targeted treatment of metastatic breast cancer and inhibition of metastasis of metastatic breast cancer to distal organs. In vivo and in vitro experimental results show that the drug delivery carrier can stably entrap chemotherapeutic drugs, simultaneously retain the biological function and structure of membrane protein, effectively adhere inflammatory endothelial cells, remarkably inhibit the invasion and migration capacity of a metastatic breast cancer cell model, and down-regulate the expression of matrix metalloproteinase-9, so that the drug delivery carrier is accumulated in a tumor site in a targeted manner, prolongs the in vivo circulation time, reduces the toxicity of the chemotherapeutic drugs, has a good tumor targeted killing effect, avoids the far-end escape of tumor cells, and further inhibits the formation of a metastatic focus.

Description

Leukocyte-simulated pluronic-lipid nano hybrid drug delivery carrier and application thereof

Technical Field

The invention belongs to the field of biomedicine, relates to a tumor-targeted biomimetic hybrid nano drug delivery carrier, and particularly relates to a leukocyte-simulated pluronic-lipid nano hybrid drug delivery carrier and application thereof.

Background

The prior art discloses that breast cancer becomes the malignant tumor with the highest morbidity which is harmful to the health of women at present and becomes the death reason of the sixth malignant tumor of women in China. The current treatment of breast cancer faces a series of problems of serious toxicity brought by chemotherapeutic drugs, drug resistance of tumor cells, high infiltration and metastasis capacity of breast cancer cells and the like, and the design of a drug delivery system is challenged.

It is known in the art that one of the objectives in constructing a delivery system is to achieve targeted accumulation of carriers and drugs at the tumor site. Research shows that the tumor microenvironment is an important living environment for influencing the growth and metastasis of tumors, and one of the characteristics is infiltration of immune cells. Immune cells including leukocytes and T cells are directionally accumulated to tumor tissue parts along the concentration gradient of chemotactic factors under the stimulation of the chemotactic factors secreted by tumor cells, endothelial cells and the like; inflammatory endothelial cells and tumor cells at the tumor site correspondingly up-regulate the expression of adhesion factors such as ICAM-1, which specifically bind to receptors on the immune cell membrane such as LFA-1, allowing immune cells to cross endothelial cells into the tumor microenvironment and interact with tumor cells up-regulated by adhesion factors under ligand-receptor mediation; in this regard, researchers have focused on designing immune cell-like vectors that exploit their directed adhesion to tumor cells to achieve drug delivery vehicle accumulation at the tumor site.

Research is carried out on red cell membrane coating or neutral particle membrane coating of the nanoparticles, the circulation time of the nanoparticles in vivo is prolonged, and the accumulation probability of the nanoparticles at tumor sites is increased through the high permeability and retention effect (EPR effect) of tumors; or the ligand on the cell membrane is used as a preparation target head to realize the targeted delivery of the nanoparticles to the tumor part; in addition, the research combines membrane protein of immune cells and inorganic material or phospholipid material to form a carrier similar to white blood cells, and the experiment verifies that the bionic carrier accumulates in blood vessels at the ear inflammation part; the design of the bionic drug delivery carrier based on the cell membrane opens up a new idea for tumor drug delivery.

Researchers are concerned that the microenvironment is also an important factor affecting tumor cell metastasis, for example, infiltrating macrophages and neutrophils are the major sources of MMPs of the matrix metalloproteinase family; MMPs almost degrade various protein components in the extracellular matrix (ECM), disrupt the histological barrier to tumor cell invasion, affect tissue remodeling, promote breast cancer infiltration, metastasis and angiogenesis, and play a critical role in tumor invasion and metastasis, thus reducing the amount of MMPs in the microenvironment can inhibit tumor metastasis.

In addition, in the mechanism research aiming at the anti-tumor metastasis, P85 and P123 in the pluronic material are found to be effective in reducing the expression of MMP-9 in a breast cancer cell line; pluronic as an amphiphilic macromolecular biomaterial has an excellent self-assembly function and good biocompatibility, and is often used as a carrier for delivering chemotherapeutic drugs in a micelle form, but reports show that the integrity of micelles after entering the body is not good, 80% of micelles are depolymerized after entering the body, and the targeted delivery efficiency of the drugs is reduced.

Based on the basis of the prior art and research background, the inventor of the application intends to provide a leukocyte-mimicking pluronic-lipid nano hybrid drug delivery carrier, which integrates leukocyte membrane proteins from natural sources into an artificially constructed pluronic-lipid hybrid membrane. The biomimetic hybrid drug delivery platform can effectively retain the function of leukocyte membrane protein, exerts the characteristic of specific combination of the leukocyte membrane protein and tumor cell adhesion factors, and realizes targeted delivery of chemotherapeutic drugs to tumor parts; in addition, the Pluronic P123 in the hybrid membrane plays a role in reducing MMP-9, protects the basement membrane from the degradation of MMP-9 at the tumor part with high efficiency, and prevents tumor cells from escaping from the original position to a distal organ to form a metastasis, thereby realizing the metastasis inhibition and treatment effect on the breast cancer.

Disclosure of Invention

The invention aims to design a leukocyte-simulating pluronic-lipid nano hybrid drug delivery carrier designed based on a bionics principle based on the basis of the prior art and the research background, and provides the leukocyte-simulating pluronic-lipid nano hybrid drug delivery carrier. The bionic hybrid drug delivery carrier realizes the targeted accumulation of the carrier at a tumor site by utilizing the specific combination of an adhesion factor receptor in leukocyte membrane protein and the tumor site, and accurately plays a role in killing the tumor of chemotherapeutic drugs; on the other hand, the MMP-9 down-regulation effect of the Pluronic P123 component in the hybrid membrane is utilized to protect the cell matrix of the tumor part from being damaged, prevent the tumor cells from escaping from the tumor part to a distal organ to form a metastasis, and effectively inhibit the occurrence of metastasis.

The model drug adopted by the invention is Paclitaxel (PTX) which is a classical chemotherapy drug and is encapsulated in the hydrophobic region of the hybrid membrane by a thin-film dispersion method.

The hybrid membrane adopted by the invention has good stability and higher encapsulation efficiency when encapsulating the chemotherapeutic drug PTX. The preparation method is a film dispersion method.

The invention constructs a pluronic-lipid nanometer hybrid drug delivery carrier simulating leucocytes by the following method:

① film-forming Pluronic and phospholipid by thin film dispersion method, gradient centrifuging ② mouse mononuclear macrophage J774A.1 cell line cultured in vitro to obtain cell membrane protein, ③ co-incubating Pluronic-phospholipid hybrid film with macrophage cell membrane protein, extruding the film, and fusing the film protein into hybrid film to form bionic hybrid nano-carrier.

The main components of the hybrid membrane material of the invention are a phosphatidylcholine structure and cholesterol, and the addition ratio of DPPC: DSPC: DOPC: cholesterol is 5:1:3: 1; in addition, the pluronic P123 is embedded therein, and the adding proportion is P123: the molar ratio of the phospholipid component was 1: 10.

The leucocyte membrane protein loaded by the bionic hybrid carrier is added in the proportion of membrane protein: the mass ratio of the membrane components is 1:30, and the expression of an adhesion factor receptor LFA-1 on the membrane protein is characterized by adopting a Western blot method.

The average particle size of the bionic hybrid carrier is 170-180nm, and compared with a micron-scale cell, the specific surface area of the bionic hybrid carrier is larger, so that the effect of a membrane structure can be exerted more efficiently; meanwhile, the particle size is proper, and the particles can be accumulated in a tumor part in vivo through an EPR effect.

The invention provides a preparation method of the drug delivery carrier, and provides pharmacodynamic evaluation of the drug delivery result of the carrier and an investigation result of the action mechanism of the preparation.

According to the invention, the mouse-derived metastatic breast cancer cell line 4T1 is used as a model to evaluate the effects of the bionic hybrid nano-carrier in inhibiting cell dynamics, invasion and migration and reducing MMP-9 expression; the uptake efficiency of the bionic hybrid nano-carrier in tumor cells and normal macrophages is investigated; the in-vivo imaging result of the in-situ breast cancer model proves that the carrier is specifically delivered to a tumor site in a targeted mode, and the directional accumulation and the penetration of the preparation at the tumor site are realized; in-vivo pharmacodynamic evaluation shows that compared with free drugs, the drug-loaded preparation can remarkably improve the killing effect of chemotherapeutic drugs on tumor cells, inhibit the development of in-situ tumors and simultaneously remarkably inhibit the formation of far-end lung metastases; the result shows that the prepared drug delivery carrier has good drug targeted delivery effect, can remarkably increase the accumulation and penetration of the drug at the tumor part so as to improve the tumor killing effect, and can effectively prevent the metastasis of the tumor.

Drawings

FIG. 1. characterization of biomimetic hybrid nano drug delivery vehicle LPL, wherein,

(A) the particle size distribution diagram (a), the particle size (b) and the zeta potential (c) of the three nano drug delivery carriers, and the control group of the biomimetic hybrid carrier (LPL) comprises a common Blank Liposome (BL) and a common hybrid carrier (BPL);

(B) transmission electron microscope photographs of three nano drug delivery carriers, (a) common blank liposome, namely BL, (b) common hybrid carrier, namely BPL, and (c) biomimetic hybrid carrier, namely LPL. A scale: 50 nm;

(C) SDS-PAGE characterizes the total protein component. LMP is leukocyte membrane protein (Leucocytesmembrane protein);

(D) the characterization result of the adhesion factor receptor LFA-1 by Western blot;

(E) the characterization result of the infrared spectrum on the pluronic P123 and the membrane protein of the carrier, (a) BL, (b) LMP,

(c)P123，(d)BPL，(e)LPL。

FIG. 2 evaluation of cellular uptake of the biomimetic hybrid nano-drug delivery vehicle LPL, wherein,

(A) uptake of BL, BPL and LPL by 4T1 cells and j774a.1 cells;

(B) flow cytometry quantification 4 hours after BL, BPL and LPL uptake by 4T1 cells;

(C) flow cytometry quantification of j774a.1 cell uptake BL, BPL and LPL 4 hours later.

FIG. 3 evaluation of cellular level anti-metastasis of the biomimetic hybrid nano drug delivery vehicle LPL, wherein,

(A) the qualitative and quantitative results of BL, BPL and LPL on the healing capacity of 4T1 cell scratch,

(B) qualitative and quantitative results of the effect of BL, BPL and LPL on the invasion and migration ability of 4T1 cells,

(C) the effect of BL, BPL and LPL on MMP-9 expression levels in 4T1 cells.

(D) Quantification of the effect of BL, BPL and LPL on the invasive capacity of 4T1 cells.

FIG. 4. evaluation of in vivo distribution of biomimetic hybrid nano drug delivery carrier LPL, wherein,

(A) the distribution of BPL and LPL in the tumor within 24h and the distribution of two nanometer drug delivery carriers in the viscera after 24h,

(B) quantification of BPL and LPL distribution in tumors over 24h,

(C) the quantitative result of the distribution condition of the two nano drug delivery carriers in the viscera after 24 hours,

(D) the penetration of BPL and LPL in the tumor tissue after 24 h.

FIG. 5. evaluation of the in vivo efficacy of biomimetic hybrid nano-drug delivery vehicle delivering paclitaxel (PTX-LPL), wherein,

(A) the growth size of each group of tumors during multiple dosing trends,

(B) mean weight results for each group of tumors after multiple dosing,

(C) the results of the body weight changes in each group of tumor-bearing mice after multiple dosing,

(D) in vitro photographs of each group of tumors after multiple dosing,

(E) after multiple times of administration, the lung tumor metastasis rate of each group of tumor-bearing mice is counted,

(F) and (3) carrying out pathological section staining (HE staining) on lung tissues of each group of tumor-bearing mice after multiple dosing.

Detailed Description

Example 1: preparation and characterization of bionic hybrid nano drug delivery carrier LPL

DPPC, DSPC, DOPC and cholesterol are weighed according to a molar ratio of 5:1:3:1, and dissolved by a mixed solvent of chloroform and methanol (chloroform: methanol volume ratio of 3: 1). Simultaneously, according to the formula of pluronic: p123 was added at a molar ratio of phospholipid 1:10 and the residual solvent was removed by rotary evaporation at 40 ℃ for 30 min. Then 1mL of Phosphate Buffered Saline (PBS) was added for hydration, and the mixture was shaken at 120 rpm at 45 ℃ for 30min to obtain a solution with a whitish opalescence. Adding the extracted leukocyte membrane protein into the solution, incubating at 37 ℃ for half an hour, extruding the whole solution system by using an extruder to allow the solution system to pass through a 200nm polycarbonate membrane to form a pluronic-lipid nano drug delivery carrier (LPL) simulating leukocytes, measuring the particle size and zeta potential of the nanoparticles by using a Malvern particle size meter, and observing the form of the nanoparticles by using a transmission electron microscope. In addition, protein components of LPL and specific adhesion factor receptors are characterized by SDS-PAGE and Western blot technology, and finally, the existence of membrane protein and pluronic P123 on the carrier is preliminarily proved by infrared spectrum.

The results show that: the particle sizes of BL, BPL and LPL are 149.4 +/-2.4 nm, 152.8 +/-1.9 nm and 175.4 +/-1.1 nm respectively, the electron micrograph of (c) of figure 1B shows that the hybrid carrier after the biomimetic has a layer of protein membrane of about 20-25nm outside, the electric potential of which is reduced by about 5mV compared with the electric potential of BPL, and the results of figure 1(A) and figure 1(B) show that compared with the common lipid membrane without pluronic embedding, the particle size, the electric potential and the form of the hybrid membrane formed after pluronic embedding the lipid are not obviously changed, which shows that the pluronic embedding does not influence the surface physical property of the membrane;

the SDS-PAGE and Western blot results of FIG. 1 show that the protein components of the biomimetic hybrid vector and the extracted leukocyte membrane protein component (LMP) are basically consistent, and the expression of the adhesion factor receptor LFA-1 can be detected, thereby proving that the extracted leukocyte membrane protein is successfully integrated on the hybrid membrane;

the results of infrared spectroscopy showed characteristic peaks of phospholipid component, pluronic and membrane protein, as shown in FIG. 1(E), the peak of stretching vibration characteristic of the phosphoryl group in the phosphatidylcholine molecule was located at 1736cm^-1The asymmetric vibration characteristic peak of the tertiary amino is positioned at 970cm^-1(ii) a The characteristic absorption of the C-O-C long chain in the pluronic P123 macromolecule is positioned at 1090cm^-1(ii) a Two characteristic absorption peaks of carbonyl of amido bond in protein molecule are provided, which are respectively 1631cm^-1and 1551cm^-1The characteristic absorption peak of phosphatidylcholine molecules and the characteristic absorption peak of pluronic can be respectively detected in the hybrid carrier; and absorption peaks of the three components can be detected in the bionic hybrid vector, and the results further prove the successful mosaic of the pluronic in the hybrid vector and the successful fusion of the membrane protein.

Example 2: evaluation of cellular uptake of Biochemical hybrid NanoDriver vectors LPL

Method 4T1 cells and J774A.1 cells grown to log phase were treated with 5 × 10⁴The density of individual cells/well was seeded in 24-well plates. After the cell density in the wells is about 80%, the medium is discarded and serum-free medium containing 0.2mg/mL of the DiD dye-labeled BL, BPL and LPL is added. After incubation for 1h, 2h and 4h, aspirating the upper layer culture medium of the cells, rinsing the cell surface with precooled PBS for three times, then fixing with 4% paraformaldehyde at room temperature for 15min, and taking pictures with a fluorescence microscope;

flow cytometry was used to quantify the uptake of fluorescently labeled vector 4T1 cells and J774A.1 cells grown to log phase at 2 × 10⁵The density of individual cells/well was seeded in 6-well plates. The medium was discarded until the cell density in the wells reached about 80% and serum-free medium containing 0.2mg/mL of DiD dye-labeled BL, BPL and LPL was added. After incubation for 4h, cells were collected by digestion centrifugation and resuspended in PBS for assay detection by flow cytometry;

the result is shown in fig. 2(a), in 4T1 cells and j774a.1 cells, as the incubation time is prolonged, the red fluorescence becomes stronger, that is, the amount of the taken nanocarrier is increased, but when the incubation time is the same, the biomimetic hybrid vector can be taken up by 4T1 cells more and less by j774a.1 than other nanocarriers, which indicates that the uptake efficiency of tumor cells is significantly improved by the biomimetic nanocarriers, and the phagocytic effect of macrophages is avoided, and the flow quantitative data shown in fig. 2(B) and (C) further confirm the result, and after four hours of incubation, the red fluorescence intensity after 4T1 cells take up LPL is 4 times that after BPL, and the red fluorescence intensity after j774a.1 cells take up BPL is 2 times that after LPL, which is consistent with the qualitative result shown in fig. 2 (a).

Example 3: cell-level anti-metastasis evaluation of biomimetic hybrid nano drug delivery carrier LPL

Scratch test 4T1 cells at 1 × 10⁵The density of each cell/well was seeded in 12-well plates, the medium was discarded until the cell density in the wells reached about 90%, and a vertical scratch was made in the middle of the culture well with a 200. mu.L pipette tip, followed by washing off floating cell debris with PBS and adding serum-free medium containing 0.2mg/mL of BL, BPL and LPL for 24h of incubation. Photographing is carried out after incubation for 0h and 24h, the width of the scratch is compared, and the scratch inhibition rate is calculated;

transwell experiment 4T1 cells were previously cultured in complete medium containing 0.2mg/mL of BL, BPL and LPL for 24h, digested and resuspended in medium containing 1% Fetal Bovine Serum (FBS), and then seeded on the upper part of a Transwell chamber, in a migration experiment, a quantity of 100. mu.L of 1 × 10 cells were seeded per chamber⁶In invasion experiments, the upper part of the chamber was first filled with matrigel gel to mimic the extracellular matrix in tumor tissue, followed by inoculation of 2 × 10⁵Immersing the lower part of the chamber in 500 mu L of complete culture medium, inoculating for 24h, wiping the cells on the upper part of the chamber with a cotton swab, washing the cells on the lower part with PBS, fixing for 20min at the temperature of precooled methanol-20 ℃, then staining the cells on the lower chamber with 0.5% crystal violet solution, washing the residual crystal violet solution after 30min, taking a picture and recording the number of the cells on the lower chamber;

western blot experiment, 1 × 10 for 4T1 cells⁶Inoculating the cells/hole in a 6-hole plate at a density of about 90%, removing the supernatant culture medium, adding a serum-free culture medium containing 0.2mg/mL DiD dye-labeled BL, BPL and LPL, incubating for 24h, washing the cells in each hole with PBS, lysing the cells with RIPA lysate containing protease inhibitor, destroying the cell membrane structure, measuring the protein content of the lysed cell sample, quantifying the protein in each hole, separating the cell sample with the same protein content with 10% separation gel, transferring the cell sample to a polyvinylidene fluoride (PVDF) membrane, and using 5% milk. Finally, the sealed membrane is incubated overnight at 4 ℃ by using primary antibody of MMP-9, then incubated at room temperature by using secondary antibody labeled by horseradish peroxidase, and after the incubation is finished, the imaging photographing is carried out on the protein band;

as shown in fig. 3(a), in the scratch test, the healing degrees of scratches of cells treated by BL, BPL and LPL were 11.36 ± 3.02%, 58.23 ± 4.67%, 80.85 ± 2.67%, respectively, and the healing degree of scratches of 4T1 cells treated by LPL was minimal, while the scratches of 4T1 cells treated by BL were almost healed, which proves that P123 has a very good restraining effect on cell motility, and the biochemical design further enhances the uptake of the vector by the cells, thereby improving the action efficiency of P123;

FIG. 3(B) shows that the number of cells migrating to the lower Transwell chamber was minimal for 4T1 cells treated with LPL, while the number of cells migrating to the lower Transwell chamber was similar for 4T1 cells treated with BL compared to the control untreated 4T1 cells; the migration inhibition rates of BL, BPL and LPL on 4T1 cells are 8.99 +/-3.06%, 70.15 +/-4.37% and 84.70 +/-4.16% through quantitative calculation; the corresponding invasion inhibition rates are 0.94 +/-5.36%, 58.15 +/-12.07% and 90.93 +/-3.87%. The result is consistent with the scratch experiment result, and the migration and invasion capacity of the 4T1 cells is effectively reduced by the presence of P123 in the nano carrier;

the expression level of MMP-9 in 4T1 cells treated by different nano-carriers is evaluated by Western blot experiment, as shown in figure 3(C), MMP-9 bands of 4T1 cells treated by LPL are very shallow, the expression rate of MMP-9 relative to internal reference β -action is calculated by quantitative results, the expression rate of MMP-9 treated by LPL is only 10.31 +/-0.41%, and MMP-9 is taken as a main component for destroying tumor extracellular matrix and effectively inhibits the tumor extracellular matrix to protect the integrity of the tumor extracellular matrix so as to avoid escape and metastasis of the tumor cells.

Example 4: evaluation of in vivo distribution of biomimetic hybrid nano drug delivery carrier LPL

The method comprises the steps of firstly establishing an in-situ breast cancer model by Balb/c mice, taking 4T1 cells in logarithmic growth phase, digesting the cells by pancreatin, centrifuging and collecting the cells, washing the cells twice by PBS after centrifugation, counting the cells, and adjusting the concentration of the cells to be 1 × 10⁶Placing each cell/mL in an ice box for later use, taking a female Balb/c white mouse with 4-6 weeks, depilating the right lower abdomen of the female Balb/c mouse, exposing a breast pad, sucking 100 mu L of tumor cell suspension by using a syringe, subcutaneously feeding a needle from the right rear limb, pushing all the cell suspension in the syringe into the lower part of the breast pad after the needle reaches the breast pad part, slowly moving the needle out after waiting for 5s to avoid carrying out tumor cells, and inoculating the tumor cells for 5 days until the tumor volume is about 50mm³Carrying out an experiment;

examining the distribution situation of the nano-carriers in vivo, randomly dividing tumor-bearing mice into 2 groups, wherein each group comprises 3 mice, injecting BPL and LPL (DiR concentration is 0.2mg/kg) marked by DiR into tail veins, performing fluorescence imaging photographing for 1h, 2h, 4h, 8h, 12h and 24h after injection by using a small animal living body imaging system, anesthetizing and killing the tumor-bearing mice after 24h of injection, separating main organ tissues and tumor tissues and performing in-vitro photographing;

examining the tumor penetration capacity of the nano-carrier, randomly dividing tumor-bearing mice into 2 groups, wherein each group comprises 3 mice, injecting DiD-labeled BPL and LPL (DiD concentration is 0.2mg/kg) into tail veins, anesthetizing and killing the tumor-bearing mice 24h after injection, separating tumor tissues, and performing frozen section and staining observation;

as shown in FIG. 4(A), accumulation of DiR-labeled LPL and BPL at the tumor sites was significantly increased with time, but the fluorescence intensity at the tumor sites of the mice with tumor was significantly stronger in the LPL-treated group than in the BPL-treated group at the same time point after 8 hours, and as shown in the quantitative results in FIG. 4(B), the fluorescence intensity at the tumor sites of the LPL group was 1.47 times, 1.66 times and 2.06 times that of the BPL group at 8 hours, 12 hours and 24 hours. In addition, the imaging of main organs and tumors of tumor-bearing mice can find that the fluorescence intensity of the liver of the LPL group mice is only 49.7 percent of that of the BPL group, which indicates that the LPL can be obviously accumulated at the tumor part in the tumor-bearing mice and has good tumor targeting property; meanwhile, the cleaning function of a net system (RES system) including the liver can be avoided, and the circulation time in the body is prolonged;

FIG. 4(C) shows cryo-section staining of 24 h-isolated tumor tissue, blue fluorescence indicating nuclear results, and red fluorescence indicating DID-labeled LPL or BPL, showing that BPL is distributed only in the periphery of tumor tissue and rarely penetrates into the tumor, while LPL is distributed in large amounts from the periphery to the interior of tumor, indicating that LPL can effectively penetrate into the tumor tissue and is beneficial for the drug loaded therein to exert its pharmacological effect.

Example 5: in vivo pharmacodynamic evaluation of biomimetic hybrid nano drug delivery carrier LPL

The method comprises the following steps: firstly, Paclitaxel (PTX) is loaded into nano-carriers by a thin film dispersion method to form drug-loaded nano-carriers, namely PTX-BL, PTX-BPL, PTX-LBL (biomimetic drug-loaded liposome without P123 heterozygote) and PTX-LPL. Randomly dividing the breast cancer in-situ tumor-bearing nude mice into 6 groups, injecting paclitaxel injection (Taxol), PTX-BL, PTX-BPL, PTX-LBL and PTX-LPL (PTX 10mg/kg) once every two days, totally administering 5 times, administering PBS with the same volume as a control group, monitoring the tumor volume and the tumor-bearing mice weight once every two days in the administration process, after the administration period is finished, carrying out anesthesia and sacrifice on the mice in each group, taking out main organs for paraffin section and HE staining, and taking out tumor tissues for photographing and weighing;

the results are shown in the tumor volume curve of fig. 5(a), all the drug-loaded preparations can inhibit the growth of tumor tissues compared with free drugs, but the inhibition effect of the leucocyte biochemical nano drug delivery carriers LBL and LPL can inhibit the growth of tumors most obviously, and the ex-vivo tumor photographing and weighing results of fig. 5(B) and (D) also show that after the administration is finished, the tumor tissues of tumor-loaded mice treated by the biochemical drug delivery carriers (PTX-LBL and PTX-LPL) are smallest in form and lightest in tumor weight, which indicates that the drug delivery carriers designed by biochemical simulation can directionally accumulate and further permeate into the tumor tissues due to good tumor targeting, so that the chemotherapeutic drugs can more efficiently exert the tumor killing effect to inhibit the growth of tumors obviously; fig. 5(C) shows that the tumor-bearing mice of each administration group did not change particularly significantly in body weight, demonstrating that the administration dose of the free drug is less toxic to mice per se, and that the four nanocarriers are relatively safe for in vivo use as a drug delivery system; FIGS. 5(E) and (F) show the preliminary evaluation of the inhibition of metastasis in vivo by drug delivery vehicle, according to the slicing results of HE in lung, with the lung structure of healthy mice as reference, the tumor-bearing mice in PBS group, PTX group and PTX-BL group form a very large area of metastasis in lung, the lung is very dense as a whole, the lung tissue is squeezed by the tumor and loses the original vacuolar structure, compared with the three groups, the degree of metastasis in lung of LBL group is slightly better, a certain alveolar structure can be observed, but a large number of tumor cells are still infiltrated around the alveoli, probably because PTX-LBL can effectively kill in situ tumor cells, and the metastasis of in situ cells to far end is preliminarily inhibited; but MMP-9 in the tumor part is still highly expressed, thereby destroying the extracellular matrix and still providing an opportunity for escape of tumor cells; compared with other groups, the lung metastasis conditions of the BPL group and the LPL group are remarkably relieved, and the pluronic P123 is proved to be capable of remarkably inhibiting the metastasis of tumor cells in vivo; the leucocyte biochemical imitating behavior of LPL is favorable for the heterozygosis drug delivery carrier to accumulate more at the tumor site, and the anti-metastasis effect of P123 is exerted more fully.

The results show that the leukocyte biomimetic pluronic-phospholipid hybrid nano-carrier PTX-LPL has better safety, good in-vivo tumor inhibition effect and most obvious tumor metastasis blocking effect.

Claims

1. A Pluronic-lipid nanometer hybrid drug delivery carrier simulating white blood cells is characterized in that a plurality of artificial lipid materials are used for constituting main components of a membrane, Pluronic P123 is embedded into the membrane and forms a hybrid membrane together with a plurality of phospholipid components, meanwhile, a natural leukocyte membrane protein component is loaded on the membrane, and a chemotherapeutic drug taxol is coated on the membrane, so that the Pluronic-lipid nanometer hybrid drug delivery carrier simulating white blood cells is prepared.

2. The leukocyte-mimicking pluronic-lipid nano-hybrid drug delivery vehicle according to claim 1, wherein said pluronic P123 is embedded in the bimolecular lipid membrane by hydrophilic-lipophilic amphiphilic properties to form a hybrid membrane.

3. The leukocyte-mimicking pluronic-lipid nano-hybrid drug delivery vehicle according to claim 1, wherein a leukocyte membrane protein that retains a bioactive function is incorporated in the hybrid membrane.

4. The leukocyte-mimicking pluronic-lipid nano-hybrid drug delivery vehicle according to claim 1, wherein said plurality of artificial lipid components constituting the hybrid membrane are a class of phosphocholine structured lipids and cholesterol, including Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylcholine (DSPC), 1, 2-Dioleoylphosphatidylcholine (DOPC) and cholesterol.

5. The leukocyte-mimicking pluronic-lipid nano-hybrid drug delivery vehicle according to claim 1, wherein said hybrid membrane component pluronic P123, which is an amphiphilic macromolecule, has good biocompatibility, can be embedded in a bimolecular membrane structure composed of amphiphilic phospholipid molecules to down-regulate the expression of MMP-9 in metastatic breast cancer cells.

6. The leukocyte-mimicking pluronic-lipid nano-hybrid drug delivery vehicle according to claim 1, wherein said naturally extracted leukocyte membrane proteins are extracted from mouse monocyte macrophage j774a.1 cell line after in vitro amplification, collection, disruption.

7. The leukocyte-mimicking pluronic-lipid nano-hybrid delivery vehicle according to claim 1, wherein the chemotherapeutic drug Paclitaxel (PTX) is used as a model drug and is entrapped in a bilayer membrane by a membrane dispersion method.

8. The leukocyte-mimicking pluronic-lipid nano-hybrid drug delivery vehicle according to claim 4, wherein the plurality of artificial lipid components and cholesterol are proportioned and added to form a membrane by a thin-film dispersion method.

9. The leukocyte-mimicking pluronic-lipid nano-hybrid drug delivery vehicle according to claim 5, wherein said pluronic P123 is embedded in a bilayer membrane composed of phospholipid molecules, and is prepared by co-drying pluronic P123 and a plurality of phospholipid components and then hydrating and extruding the same during vacuum drying to form a membrane.

10. The leukocyte-mimicking pluronic-lipid nano-hybrid drug delivery vehicle according to claim 6, wherein said naturally-extracted leukocyte membrane proteins, including LFA-1 adhesion factor receptors, are prepared by gradient centrifugation of mouse monocyte macrophage j774a.1 membrane proteins.

11. The leukocyte-mimicking pluronic-lipid nano-hybrid drug delivery vehicle according to claim 1, wherein said drug delivery vehicle is a highly leukocyte-mimicked artificial hybrid nano-drug delivery vehicle having a particle size of 170nm, being homogeneous and stable, having a long circulation time in vivo, and capable of targeted binding to tumor cells via adhesion factor receptor-ligand mediated specific binding.