CN107028882B

CN107028882B - Physically-wrapped tumor-targeted nano drug delivery system, and preparation method and application thereof

Info

Publication number: CN107028882B
Application number: CN201710136815.6A
Authority: CN
Inventors: 俞磊; 孙磊; 周靖娥; 彭婷; 李奇龙; 南立娟; 高礼鹏; 闫志强; 王镜; 朱建中
Original assignee: East China Normal University
Current assignee: East China Normal University
Priority date: 2017-03-09
Filing date: 2017-03-09
Publication date: 2020-05-12
Anticipated expiration: 2037-03-09
Also published as: CN107028882A

Abstract

The invention discloses a physically-wrapped tumor targeted nano drug delivery system, a preparation method and application thereof, wherein a low-waist system of the system comprises a targeted element modified by RGERPPR polypeptide, a nano micelle and an anti-tumor drug, and the targeted element is distearoyl phosphatidyl ethanolamine-polyethylene glycol-RGERPPR; the micelle is a polyglutamic acid PGG macromolecular skeleton and a glutamic acid branched chain on the skeleton; the anti-tumor drug is paclitaxel PTX. The tail end of a glutamic acid branched chain of a polyglutamic acid macromolecular skeleton is bonded with an antitumor drug PTX through an ester bond to form PGG-PTX, and then the PTX and distearoyl phosphatidyl ethanolamine in a targeting element wrap the distearoyl phosphatidyl ethanolamine at the hydrophobic end of the targeting element into the PGG-PTX through hydrophobic interaction to form a targeted antitumor nano drug delivery system. The drug delivery system can be used for targeted delivery of tumor treatment drugs, obtains good curative effect on treatment of the brain glioma, and prolongs the median survival time of BALB/C brain glioma nude mice.

Description

Physically-wrapped tumor-targeted nano drug delivery system, and preparation method and application thereof

Technical Field

The invention belongs to the field of pharmaceutical preparations, and particularly relates to a preparation method and application of a targeting element modified in a nano micelle medicament by using a physical encapsulation effect.

Background

Glioma is one of the most common brain tumors, accounts for about 40% of the incidence rate of brain tumor, and has high deterioration degree and postoperative recurrence rate and rapid invasion speed, so that glioma patients have short survival period: clinically diagnosed phase I or II patients have a 6-8 year survival time, phase III patients have a 3 year survival time, and phase IV patients typically have only 12-18 months. As an intracranial tumor, glioma has many unique sites, and its tumor formation and development have complex barriers, such as enzyme barrier, blood-brain barrier (BBB) and tumor blood-brain barrier (BBTB), which prevent the drug or drug delivery system from reaching the tumor site. At present, the methods for treating brain glioma mainly comprise surgery, radiotherapy and chemotherapy. However, brain glioma grows infiltratively and is close to the central nervous system, so that it is difficult to completely remove the tumor by surgery, and the tumor is likely to recur. Radiation therapy is prone to radiation dementia; the conventional chemotherapy drugs have no selectivity to tumor tissues, so that the conventional chemotherapy drugs often cause serious toxic and side effects, have poor treatment effect on glioma, and seriously affect the life quality of patients.

The tumor targeted nano drug delivery system is a drug delivery system which is constructed by utilizing the special physiological and pathological characteristics of tumor tissues and encapsulating tumor diagnosis or treatment drugs by nano carriers and has a targeted positioning function on the tumor tissues. Because the conventional chemotherapy medicament has no targeting on tumor tissues clinically, serious toxic and side effects on normal tissues are often caused, and the administration dosage of the anti-cancer medicament is limited in order to avoid the injury to patients. One of the primary goals of successful tumor therapy is to deliver a sufficient amount of drug to the tumor while minimizing damage to normal tissues. Most chemotherapy drugs cause incomplete tumor response due to sub-optimal doses of the drug to the tumor tissue, which can lead to disease recurrence and drug resistance. However, the tumor targeting nano drug delivery system added with the polypeptide targeting molecule can effectively solve the problem.

To achieve effective drug delivery and reach the tumor site and accumulate, a suitable targeting ligand must be found. Neuropilin-1(NRP-1) is a type I transmembrane glycoprotein on the cell surface, and NRP-1 is expressed on many tumor tissues of human, including prostate cancer, breast cancer, melanoma, pancreatic cancer and glioma, but is not expressed in corresponding epithelial cells of normal tissues. Research shows that NRP-1 is obviously expressed in clinical pathological specimens of human glioma, and the expression quantity of NRP-1 is increased along with the increase of the malignancy degree of the glioma. Recently, researchers screen out a class of polypeptides with tumor vascular permeability and tumor tissue permeability by a polypeptide screening technology, the polypeptides can mediate drug molecules or a nano drug delivery system which is covalently connected with the polypeptides to penetrate through the wall of a tumor vessel and penetrate into the tumor, and the drug molecules or the nano drug delivery system are distributed in the whole tumor tissue, and the polypeptides are called tumor penetrating peptides. Studies have shown that RGERPPR is a specific ligand for NRP-1, exhibiting dual affinity for tumor cells and tumor vessels. The affinity to tumor blood vessels can lead the tumor blood vessels to be targeted to the walls of the tumor blood vessels, the tumor penetrability can lead the tumor blood vessels to penetrate through the walls of the tumor blood vessels and penetrate into the interior of tumor tissues, and the affinity to tumor cells can lead the tumor blood vessels to be specifically combined with the tumor cells, so that the polypeptide has more obvious advantages compared with the common tumor cell targeting molecules as the targeting molecules.

A polymer micelle is a core-shell structure having a hydrophilic outer shell and a hydrophobic inner core. The hydrophobic core is typically composed of a biodegradable polymer that acts as a reservoir for the water-insoluble drug. Since the polymer is required to be non-toxic to cells and metabolised by the kidney. The distribution of the micelle in the organism mainly depends on the property of the shell, meanwhile, the property of the shell plays a decisive role in the stability of the micelle and the interaction of the micelle with plasma protein and cell membranes, and the viscosity of the micelle influences the physical stability of the micelle and the release behavior of the drug.

Disclosure of Invention

The invention aims to construct a PGG-PTX drug delivery system for realizing RGERPPR-PEG-DSPE targeted element modification based on physical encapsulation effect, and preparation and application thereof, and the system is used for realizing targeted drug delivery of tumors. The drug delivery system can be administrated by intravenous injection, acts on a tumor part through RGERPPR mediated targeting, can penetrate through the wall of a tumor vessel and penetrates into the interior of tumor tissues.

The purpose of the invention is realized as follows:

a physically-wrapped tumor-targeted nano drug delivery system is characterized by comprising an RGERPPR polypeptide-modified targeting element, a nano micelle and an anti-tumor drug, wherein the targeting element is distearoyl phosphatidyl ethanolamine-polyethylene glycol-RGERPPR (DSPE-PEG-RGERPPR); the micelle is a polyglutamic acid (PGG) high-molecular skeleton and a glutamic acid branched chain on the skeleton; the anti-tumor drug is Paclitaxel (PTX). The polyglutamic acid macromolecular skeleton and the glutamic acid branched chain are connected through peptide bonds, and the tail end of the glutamic acid branched chain is bonded with the antitumor drug PTX through ester bonds to form PGG-PTX, and the structure of the PGG-PTX is as follows:

then the PTX and the distearoyl phosphatidyl ethanolamine in the targeting element wrap the distearoyl phosphatidyl ethanolamine at the hydrophobic end of the targeting element into PGG-PTX through the hydrophobic interaction, so as to form a targeted anti-tumor nano drug delivery system; wherein R is Na or paclitaxel.

A method of making the above delivery system, the method comprising the steps of:

step 1: preparation of polypeptide-modified targeting elements: the sulfhydryl of the polypeptide RGERPPR reacts with the maleimide group of distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide to form a targeting element through connection. The reaction equation is as follows:

step 2, weighing a, PGG-PTX and b and RGERPPR-PEG-DSPE respectively, wherein the molar ratio of a to b is 98 to 2. Coupling PGG-PTX

The mixture was dissolved in 0.5% sodium cholate solution, and RGERPPR-PEG-DSPE was dissolved in a mixed solution of dichloromethane and acetone at a ratio of 3: 1. Adding the solution b into the solution a, and dripping while vortexing; performing ultrasonic treatment on the cell ultrasonic crusher for 300w and 2s at intervals of 3s in ice bath for 2min to obtain emulsion; 8ml of 1% sodium cholate solution was added to the emulsion, and the mixture was magnetically stirred for 10 min. Removing the organic solvent by rotary evaporation in a water bath at 35 ℃; centrifuging at 21000g for 45min at 4 ℃, discarding the supernatant, and resuspending the precipitate with normal saline to obtain a nanoparticle solution; the sample is separated and purified by a G-50 sephadex column connected with a protein purifier under the ultraviolet detection wavelength of 228 nm. Obtaining a micelle nano anti-tumor drug delivery system which is wrapped with a targeting element and has a physical effect;

and step 3: determining the molecular weight and purity of the polypeptide;

and 4, step 4: determining the targeting element DSPE-PEG-RGERPPR nuclear magnetic resonance spectrum;

and 5: determining the particle size and the shape of the nano drug delivery system;

step 6: determining an in vitro drug release profile of the nano-delivery system;

and 7: determining the uptake of the nanomedicine delivery system into glioma U87MG tumor cells in vitro;

and 8: determining the activity evaluation of the nano drug delivery system on glioma U87MG tumor cells in vitro;

and step 9: determining the in vivo anti-glioma activity effect of said nano drug delivery system.

The cell uptake of the targeted micelle nano-drug and the cell uptake of the non-targeted micelle nano-drug which are loaded with a fluorescent dye cell membrane green fluorescent probe (DIO) by tumor cells are examined through the fluorescent missing nanoparticles, and the result proves that the targeted element RGERPPR-PEG-DSPE modified micelle nano-drug delivery system can enter the tumor cells through the physical wrapping effect.

The delivery system of the present invention has the following features: the particle size is about 100nm and is spherical; the product is stable in phosphate buffer; the drug release characteristics are also improved; the uptake of the drug delivery system by tumor cells in a cell uptake experiment is obviously increased, and the targeting of the drug delivery system to the tumor is better; the drug delivery system remarkably enhances the in-vitro growth inhibition effect of the micelle nano-drug on glioma in-vitro activity evaluation of glioma; the drug delivery system can be used for targeted delivery of tumor treatment drugs, obtains good curative effect on treatment of the brain glioma and prolongs the median life cycle of BALB/C brain glioma nude mice.

Drawings

FIG. 1 is a schematic flow diagram illustrating the preparation of a delivery system according to the present invention;

FIG. 2 is a high performance liquid chromatography HPLC chromatogram of the polypeptide C-RGERPPR of the present invention;

FIG. 3 is a mass MS map of the polypeptide C-RGERPPR of the present invention;

FIG. 4 is nuclear magnetic NMR spectra of MAL-PEG-DSPE and RGE-PEG-DSPE of the present invention;

FIG. 5 is a particle size plot of PGG-PTX and RGE/PGG-PTX of the present invention;

FIG. 6 is a transmission electron micrograph of PGG-PTX and RGE/PGG-PTX of the present invention (A) PGG-PTX, (B) RGE/PGG-PTX;

FIG. 7 is a graph of a long term stability experiment of a nano-delivery system of the present invention;

FIG. 8 is a graph of the drug release profile of the nano-delivery system of the present invention;

FIG. 9 is a qualitative and quantitative uptake chart of U87MG glioma cells of the present invention;

FIG. 10 is a graph of the activity of U87MG cells of the present invention;

FIG. 11 is a graph of survival of glioma nude mice after administration of the present invention.

Detailed Description

The following examples will help to further understand the present invention, but do not limit the content of the present invention.

Referring to fig. 1, a schematic flow chart of the preparation of the drug delivery system of the present invention is shown.

The drug loading of the polymeric prodrug PGG-PTX paclitaxel was 35%.

Example 1

Characterization of the polypeptide C-RGERPPR

And (4) characterizing the purchased pure polypeptide by HPLC and LC-MS methods.

Analytical HPLC method, column: diamonsil C18(5 μm, 200X 4.6 mm); mobile phase A: 0.1% TFA water, mobile phase B: 0.1% TFA acetonitrile; elution procedure: 0-2 min: 5% B, 2-32 min: 5% -65% of B, 32-33 min: 65% -90% of B, 33-36 min: 90% of B; 36-37 min: 90% -5% of B, 37-45 min: 5% of B; flow rate: 0.7 mL/min; column temperature: 40 ℃; wavelength: UV 214, 280 nm. FIG. 2 shows that the purity is greater than 98%; FIG. 3 shows that the molecular weight of the polypeptide is 970.4, and a mass MS spectrum shows that the molecular weight is consistent with theory.

Example 2

Synthesis and characterization of DSPE-PEG-RGERPPR

Dissolving 25mg of pure C-RGERPPR in Ph7.0-7.4 (2 ml of PBS (Ph7.0-7.4, 1 xPBS)) solution, dissolving maleimide-polyethylene glycol-phospholipid complex DSPE-PEG-Mal (molar equivalent is 0.8 times of RGERPPR) in 0.5ml of DMF, adding into polypeptide aqueous solution, ultrasonically removing bubbles, stirring for reaction for about 1 hour, completely reacting the DSPE-PEG-Mal, dialyzing (molecular weight cut-off is 3500Da, dialysis medium is water) to remove excessive C-RGERPPR and DMF, and freeze drying to obtain RGERPPR-PEG-DSPE, H1-NMR. H1-NMR spectrum showed, FIG. 4, a nuclear magnetic spectrum of RGERPPR-PEG-DSPE and maleimide-polyethylene glycol-phospholipid complex (Mal-PEG-DSPE). The RGERPPR-PEG-DSPE nuclear magnetic spectrum disappears at 6.7ppm, namely the characteristic peak of maleimide, which shows that the RGERPPR-PEG-DSPE is successfully synthesized.

Example 3

Preparation and characterization of physical encapsulation RGERPPR-PEG-DSPE modified PGG-PTX nano drug delivery system

Weighing a, PGG-PTX and b and RGERPPR-PEG-DSPE respectively, wherein the molar ratio of a to b is 98 to 2. PGG-PTX was dissolved in 0.5% sodium cholate solution and RGERPPR-PEG-DSPE was dissolved in a mixed solution of dichloromethane: acetone ═ 3: 1. The solution b was added to the solution a, and vortexed while dropping. And (3) carrying out ultrasonic treatment on the cell ultrasonic crusher for 300w for 2s at intervals of 3s in ice bath for 2min to obtain the emulsion. 8ml of 1% sodium cholate solution was added to the emulsion, and the mixture was magnetically stirred for 10 min. Removing the organic solvent by rotary evaporation in a water bath at 35 ℃; centrifuging at 21000g for 45min at 4 ℃, discarding the supernatant, and resuspending the precipitate with normal saline to obtain a nanoparticle solution. The sample is separated and purified by a G-50 sephadex column connected with a protein purifier under the ultraviolet detection wavelength of 228 nm. The micelle nanometer anti-tumor drug delivery system which is wrapped with the targeting element and has the physical effect is obtained.

Preparing 1mg/ml of a high-molecular paclitaxel prodrug (PGG-PTX) and a modified targeted micelle nano drug delivery system (RGE/PGG-PTX), filtering the mixture through a 0.22um filter membrane, and determining the particle size and the particle size distribution of the mixture by using a Malvern laser particle sizer, wherein the particle size distribution of nanoparticles is shown in figure 5, and the average particle sizes of the two nanoparticles, namely the PGG-PTX nanoparticle and the RGE/PGG-PTX nanoparticle, are respectively about 25nm and 109 nm.

The morphology of PGG-PTX and RGE/PGG-PTX was observed by Transmission Electron Microscopy (TEM) using a 1% negative phosphotungstic acid stain to prepare the samples as follows: (1) dropping a drop of sample solution on a copper net; (2) air drying for about 10 minutes, and removing excessive liquid by sucking with filter paper; (3) dropping a drop of 1% phosphotungstic acid on a copper net, drying for 8 minutes at room temperature, and then absorbing the redundant dye solution by using filter paper; (4) and (5) observing the sample under a transmission electron microscope after the sample is completely dried. In FIG. 6, A and B are transmission electron microscope images of two nanoparticles, PGG-PTX and RGE/PGG-PTX, respectively, and the two nanoparticles are relatively regular in morphology and spherical.

Example 4

Stability verification of two nanoparticles, namely PGG-PTX and RGE/PGG-PTX

PGG-PTX and RGE/PGG-PTX were prepared to 1.0mg/ml with 1xPBS solution, particle size and dispersibility index (PDI) were measured with Dynamic Light Scattering (DLS), measured every two days for 3 weeks continuously, and long-term stability of nanoparticles was examined. The results are shown in fig. 7, the stability of the two nanoparticles is good, and the particle size does not change significantly after 3 weeks of standing (p > 0.05).

Example 5

Verification of release performance of two nanoparticle drugs, namely PGG-PTX and RGE/PGG-PTX

Drug Release profiles of PGG-PTX and RGE/PGG-PTX: PGG-PTX and RGE/PGG-PTX were prepared at concentrations in buffer saline at pH 7.4, each in triplicate, and all samples were placed in a 37 ℃ incubator. A group of each sample buffer salt solution is taken at different time points, the released paclitaxel in the solution is extracted by ethyl acetate, and the content of the paclitaxel is determined by a high performance liquid chromatograph (standard curve method). As shown in FIG. 8, the drug release of RGE/PGG-PTX was improved to some extent, and the disadvantage of the slow release of PGG-PTX paclitaxel was improved.

Example 6

Targeting verification of in vitro glioma of nano drug delivery system

Model U87MG cells at 3X 10/well⁴cells/ml are inoculated in a 6-well plate at the density of 24 and each pore volume is 2ml, the culture plate is transferred into a carbon dioxide incubator and cultured for 24 hours under the conditions of 37 ℃, 5 percent of carbon dioxide and saturated humidity, and the adherent growth of the cells is up to 80 percent. Prepared by DMEM culture solution containing 10% fetal calf serumA series of different concentrations of RGE/PGG-PTX and PGG-PTX solutions. The culture broth in the dish was aspirated, and a series of solutions of RGE/PGG-PTX and PGG-PTX were added, while setting the control group, and incubated at 37 ℃ for 2 h. And (4) sucking the supernatant, discarding, slightly washing twice by using a PBS solution, and quantitatively evaluating the cell uptake condition by using a laser confocal qualitative method and a flow cytometer respectively. Nuclei were stained blue with Hochest. Flow cytometry analyzed cells were first washed 2 times with PBS; then digesting, centrifuging and resuspending, repeating for three times; the resuspended cells were detected by flow cytometry. Referring to FIG. 9, in the figure, A is a free fluorescein DIO cell uptake map, B is a PGG-PTX-encapsulated DIO cell uptake map, and C is an RGE/PGG-PTX-encapsulated DIO cell uptake map, and the results of fluorescence micrographs after 2 hours of action with tumor cells at 37 ℃ respectively show that the RGE/PGG-PTX uptake is significantly higher than the uptake of cells to PGG-PTX, which indicates that the RGE/PGG-PTX micelle nano drug delivery system is successfully prepared and has good in vitro targeting to tumor cells.

Example 7

Determination of the Activity evaluation of the Nanoparticulate delivery System on glioma U87MG tumor cells in vitro

In vitro cytotoxicity assay IC was analyzed using CCK-8 kit, using U87MG cells as model₅₀Values to examine the inhibition rate of PGG-PTX and RGE/PGG-PTX and free PTX on cells, specific methods: the density is 2 x 10³cells/ml of the U87MG suspension was inoculated into 96-well plates, placed in a 37 ℃ incubator under 5% CO2, and incubated for 24 hours; adding three drug solutions of Taxol, PGG-PTX and RGE/PGG-PTX with different concentrations into a 96-well plate, and setting a blank control and a negative control (without adding drugs), wherein each group is provided with at least three multiple wells; after adding the drugs, placing the 96-well plate in an incubator at 37 ℃ under the condition of 5% CO2 for further incubation for 48 hours; remove the liquid from the wells and add again 100. mu.l fresh medium and 10. mu.l CCK-8 reagent per well; placing the 96-well plate in a constant-temperature shaking box at 37 ℃ for slight shaking culture for 4 hours; OD at 450nm was measured with a microplate reader.

The survival rate of the cells is calculated by the formula:

cell viability (%) - [ a (dosed) -a (blank) ]/[ a (not dosed) -a (blank) ] × 100%.

In the formula:

a (blank): OD values for wells with media and CCK-8 solution without U87MG cells;

a (dosing): OD of wells with U87MG cells, CCK-8 solution and drug solution;

a (no drug addition): OD of wells with U87MG cells, CCK-8 solution, but no drug solution.

As shown in FIG. 10, the IC50 values of U87MG cells were 0.14. mu.M, 2.39. mu.M and 1.44. mu.M after treatment with Taxol, PGG-PTX and RGE/PGG-PTX for 48 h. It is clear that RGE/PGG-PTX has higher antitumor cell growth activity than that of PGG-PTX without targeting, and that the IC of PGG-PTX₅₀The value is higher than PTX, which shows that the cytotoxicity of the nano-drug formed by connecting the macromolecule with the paclitaxel in a bonding mode is obviously reduced.

Example 8

Evaluation of in vitro and in vivo anti-glioma Activity of the Nanoparticulate delivery System

U87MG cells in logarithmic growth phase were digested, centrifuged, dispersed in PBS phosphate buffer to give single cell suspension, and 1X 106 cells (dispersed in 10ul PBS) were inoculated into 5ul of the right striatum of nude mice using a stereotaxic apparatus. The inoculation position coordinates were located as follows: the front side of bregma of nude mouse is 0.6mm, the right side is 1.8mm, and the depth is 3 mm.

Nude mice were randomly divided into 4 groups of 7 mice each, injected with 100ul of physiological saline, PTX, PGG-PTX, RGE/PGG-PTX on days 11, 15, 18 and 21 after inoculation, respectively, and the cumulative paclitaxel injection dose was 40mg/kg, and the survival time of model nude mice was recorded. Referring to FIG. 11, the results show median survival for each of the saline N.S, free paclitaxel PTX, non-targeted PGG-PTX, and targeted RGE/PGG-PTX groups was 22, 29, 36, and 47 days, respectively. Therefore, the survival time of the tumor-bearing nude mice in the RGE/PGG-PTX group is far longer than that of the PGG-PTX group, which indicates that the modification of CRNGRGPDC can obviously enhance the anti-tumor effect of the adriamycin liposome.

Claims

1. A physically-wrapped tumor-targeted nano drug delivery system is characterized by comprising an RGERPPR polypeptide-modified targeting element, a nano micelle and an anti-tumor drug, wherein the targeting element is distearoyl phosphatidyl ethanolamine-polyethylene glycol-RGERPPR; the micelle is a polyglutamic acid PGG macromolecular skeleton and a glutamic acid branched chain on the skeleton; the anti-tumor drug is paclitaxel PTX; the polyglutamic acid macromolecular skeleton and the glutamic acid branched chain are connected through peptide bonds, and the tail end of the glutamic acid branched chain is bonded with the antitumor drug PTX through ester bonds to form PGG-PTX, and the structure of the PGG-PTX is as follows:

then the PTX and the distearoyl phosphatidyl ethanolamine in the targeting element wrap the distearoyl phosphatidyl ethanolamine at the hydrophobic end of the targeting element into PGG-PTX through hydrophobic interaction to form the physically-wrapped tumor-targeted nano drug delivery system;

wherein, R is Na or paclitaxel;

the molecular weight of the polyethylene glycol in the distearoyl phosphatidyl ethanolamine-polyethylene glycol-RGERPPR is 400-8000.

2. A method of preparing the delivery system of claim 1, comprising the steps of:

step 1: preparation of polypeptide-modified targeting elements: the sulfhydryl of the polypeptide RGERPPR reacts with the maleimide group of distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide to form a targeting element;

step 2, weighing a, PGG-PTX and b, and RGERPPR-PEG-DSPE respectively, wherein the molar ratio of a to b is 98 to 2; dissolving PGG-PTX in 0.5% sodium cholate solution, and dissolving RGERPPR-PEG-DSPE in a mixed solution of dichloromethane, acetone and 3: 1; adding the solution b into the solution a, and dripping while vortexing; performing ultrasonic treatment on the cell ultrasonic crusher for 300w and 2s at intervals of 3s in ice bath for 2min to obtain emulsion; adding 8ml of 1% sodium cholate solution into the emulsion, and magnetically stirring for 10 min; removing the organic solvent by rotary evaporation in a water bath at 35 ℃; centrifuging at 21000g for 45min at 4 ℃, discarding the supernatant, and resuspending the precipitate with normal saline to obtain a nanoparticle solution; separating and purifying the sample through a G-50 sephadex column connected with a protein purifier under the ultraviolet detection wavelength of 228 nm; obtaining the tumor targeted nano drug delivery system which is physically wrapped.

3. Use of a delivery system according to claim 1 for the manufacture of a medicament for the treatment of brain glioma.