CN102552928A - Double-stage targeted drug delivery system curing brain tumor and preparation method of same - Google Patents

Abstract

The invention belongs to the technical field of biomedicine, and relates to a targeted drug delivery system for treating brain tumors, in particular to polymer nanoparticles modified by Angiopep-2, a carrier system for drug delivery in brain tumors, and a preparation method thereof. The invention takes the low-density lipoprotein receptor-related protein receptor highly expressed on both the BBB and brain glioma cells as the target, and constructs a dual-stage BBB-targeting and glioma-targeting mechanism mediated by LRP receptors. Targeted nano drug delivery system. The targeted drug delivery system of the present invention can increase the solubility of PTX, prolong the circulation time in the body, and the release of PTX from the carrier has a slow-release effect, and the carrier material is non-toxic, biodegradable and biocompatible, which can effectively solve the problem of The problem of the blood-brain-tumor barrier existing in glioma chemotherapy can be applied to the treatment of glioma.

Description

A dual-stage targeted drug delivery system for treating brain tumors and its preparation method

technical field

The invention belongs to the technical field of biomedicine and relates to a targeted drug delivery system, in particular to a dual-stage targeted drug delivery system for treating brain tumors, especially the carrier system-Angiopep-2 for brain tumor drug delivery Modified polymeric nanoparticles and methods for their preparation.

Background technique

Brain glioma is one of the most common clinical tumors. Studies have shown that it is a tumor derived from the neuroectoderm, accounting for about 40% to 50% of the incidence of intracranial tumors. Because gliomas grow malignantly and infiltrate, and most of them grow in important brain structures, such as the basal ganglia, central sulcus, thalamus, and brainstem, it is not only difficult to completely remove them, but also prone to recurrence after surgery. According to the data of the World Health Organization, malignant glioma is the second cause of death in tumor patients under the age of 34, and the third cause of death in patients aged 35 to 54. Globally, malignant gliomas ruthlessly claim the precious lives of 180,000 to 600,000 young and middle-aged people every year.

Astrocytoma is the most common glioma, accounting for 70-80% of gliomas, and can grow anywhere in the brain or spinal cord. Studies have shown that astrocytomas in adults mostly grow in the brain, while astrocytomas in children often grow in the cerebellum and brainstem. In terms of tumor malignancy, astrocytoma can be divided into the following four grades: Grade I - Pilocytic Astrocytoma, Grade II - Astrocytoma is a low-malignancy tumor, Grade III Grade-Anaplastic Astrocytoma (AA), Grade IV-Multiform Glioblastoma (Glioblastoma Multiform; GBM) is a malignant tumor ^[1] .

The growth characteristic of glioblastoma is infiltrative growth, which has no obvious boundary with normal brain tissue, and most of them are not limited to one lobe of the brain. The average time from the onset of symptoms to seeing a doctor is two years. The malignant tumors grow rapidly and the course of disease is short. Most of them are within 3 months from the onset of symptoms to the time of seeing a doctor, and 70-80% of them are within half a year.

Surgical treatment is based on the growth characteristics of glioma, and it is theoretically impossible to completely remove it. Some tumors growing in important parts such as the brainstem cannot be operated on at all. Therefore, the purpose of surgical treatment can only be limited to reducing tumor volume, reducing the number of tumor cells, Relieve the symptoms of tumor bearing, temporarily reduce intracranial pressure, and complete the four diagnosis and treatment purposes of tumor pathology. However, surgery will activate the tumor cells in the dormant phase to rapidly enter the proliferative phase, resulting in the escalation of tumor malignancy and recurrence in a short period of time after surgery.

Postoperative adjuvant radiotherapy has been proven to be ineffective against malignant gliomas both in theory and in practice, because only when the radiation dose reaches 73-80 Gy can it effectively kill glioma cells, while normal brain tissue The dose that can be tolerated is only 60Gy, which is actually just radiation therapy to the fragile brain tissue itself.

Clinically, chemotherapy is a crucial part of many treatment links after surgical resection of glioma, and its success or failure has a great impact on the quality of life and prognosis of patients. The extensive infiltration and growth of malignant glioma and the existence of tumor cells far away from the primary tumor determine that systemic chemotherapy should be given after surgery, especially when surgery and radiotherapy are difficult to achieve curative effect. Chemotherapy is currently an important adjuvant treatment in clinical practice . At present, the chemotherapy of this cancer has made great progress, but the most prominent problem of postoperative adjuvant transvenous chemotherapy is that the blood flow in the brain is 1/5 of the blood flow in the whole body, and the blood-brain barrier (BBB) in the brain also exists. Due to the special structure, the chemotherapy effect of glioma is not ideal. Therefore, it is particularly important to study targeted therapy methods for glioma.

In normal tissues, the microvascular endothelial space is dense and has a complete structure, and macromolecules and lipid particles are not easy to pass through the vessel wall. However, in solid tumor tissues, there are abundant blood vessels, wide vessel wall spaces, poor structural integrity, and lack of lymphatic drainage, resulting in macromolecules. Substances and lipid particles have selective high permeability and retention. This phenomenon is called the high permeability and retention effect of solid tumor tissue, or EPR effect (enhanced permeability and retention effect) for short. The pathological structural characteristics of solid tumor tissues make macromolecular anticancer drugs have passive targeting or selective characteristics for solid tumors. Targeting; and small molecule anticancer drugs can freely pass through the blood vessel walls of normal tissues and tumor tissues, and the drug distribution in normal tissues and tumor tissues is consistent, which is one of the important reasons for the poor selectivity of anticancer effects and strong side effects First, it does not have a passive targeting effect. Tumor treatment strategies based on the EPR characteristics of solid tumor tissues can increase the targeting of anticancer drugs to a large extent, improve drug efficacy, and reduce toxic and side effects.

Nanoparticles are currently a widely studied drug delivery carrier system for the treatment of brain tumors, mainly because the surface of nanoparticles is easily functionalized (such as PEGylation to prolong the circulation time in vivo), and the EPR effect of tumors is used to carry chemotherapy drugs. The nanoparticles enter the tumor site and enhance the chemotherapy effect. However, it is difficult to improve the chemotherapy effect of glioma by relying solely on the passive targeting effect of this tumor, mainly due to 1) the special physiological environment such as dense cells inside the glioma, high interstitial pressure, hypoxia and other obstacles 2) Glioma grows invasively, and the EPR effect of the tumor infiltrating into normal brain tissue is not obvious, and the abundant BBB hinders the penetration of chemotherapy drugs. In the current clinical treatment, there is an urgent need to solve the problem of blood-brain-tumor barrier (BBTB) existing in glioma chemotherapy.

Studies have found that Angiopep-2 is a small molecule polypeptide with a molecular weight of 2.4K, which can significantly increase BBB transport through the LRP receptor, and its brain penetration ability is 10 times that of Transfferin. The combination of Angiopep-2 and paclitaxel (PTX) ANG1005 has entered the first phase of clinical research in the United States against glioma. However, the conjugate is still poorly water-soluble, and it needs to use polyoxyethylene castor oil and ethanol as solubilizers, just like Taxol, so it still has the disadvantages of sensitization, toxicity, and short circulation time in the body.

Contents of the invention

The purpose of the present invention is to overcome the defects and deficiencies of the prior art, to provide a targeted drug delivery system for the treatment of brain tumors, in particular to a new carrier system for brain tumor drug delivery-Angiopep-2 modified Polymer nanoparticles (abbreviation: ANG-NP-PTX) and a preparation method thereof.

The targeted drug delivery system for the treatment of brain tumors of the present invention takes the low-density lipoprotein receptor-related protein (LRP) receptor highly expressed on both the blood-brain barrier (BBB) and brain glioma cells as the target, Construction of BBB-targeted and glioma-targeted dual-stage targeted nano-drug delivery system mediated by LRP receptors.

Specifically, the present invention provides PEG-PCL-PTX nanoparticles modified by Angiopep-2 (abbreviation: ANG-NP-PTX).

The targeted drug delivery system of the present invention is prepared by the following method:

First, dissolve a certain amount of MePEG-PCL, Male-PEG-PCL and PTX in dichloromethane, add 1% aqueous solution of sodium cholate, 200 W in ice bath, 60 s intermittent ultrasonication to form oil-in-water (O/W ) emulsion; secondly, disperse into 0.5% sodium cholate aqueous solution and magnetically stir for 5 min; again, remove dichloromethane by rotary evaporation at 40°C until there are no bubbles, centrifuge at 12000 rpm at 4°C for 60 min, and discard the supernatant to obtain NP- PTX; then, the prepared NP-PTX was blown and dispersed with a certain amount of HEPES solution (pH 7.0), and the calculated amount of Angiopep-2 was added, and incubated at room temperature with nitrogen gas and protected from light; finally, centrifuged at 12000 rpm at 4°C for 60 min and discarded. The supernatant was removed to obtain ANG-NP-PTX.

In the present invention, PTX and MePEG-PCL: Male-PEG-PCL=4～20:1,

In the present invention, the molar ratio of Male to Angiopep-2 is 1～10:1

The targeting system of the present invention for the treatment of brain tumors has been evaluated in vivo and in vitro:

The Angiopep-2 modified PEG-PCL paclitaxel targeting nanoparticles prepared by the present invention have been carried out in vitro drug release test, cytotoxicity test, cell uptake test, in vivo and in vitro dual-level targeting research, the results show that after being modified by Angiopep-2, It did not significantly change the release behavior of PTX in vitro, but significantly increased the cytotoxicity of U87 MG cells, increased the uptake ability of U87 MG cells, and the results in vivo and in vitro proved to show a dual-level targeting.

1. In vitro release test

ANG-NP-PTX system in vitro release test of the present invention adopts following dialysis method:

Take 1 ml of nanoparticle solution and add it to a pre-swelled dialysis bag with a molecular weight cut-off of 14,000, tie the bag tightly, and put it into 39 mL of 1M sodium salicylate solution at a constant temperature of 37°C and 100 rpm; take 0.2 mL from the release medium at regular intervals, At the same time, an equal amount of 37°C blank release medium was added; the sample PTX concentration was determined by HPLC, and the cumulative release percentage F(t)(%) was calculated; the results are shown in Figure 1.

As shown in Figure 1, the cumulative releases of NP-PTX and ANG-NP-PTX at 12 h were 49.2 % and 50.1 % (P > 0.05), and the cumulative releases at 96 h were 77.2 % and 79.1 % (P > 0.05), it can be seen that the two kinds of nanoparticles all present two-phase release behavior, the first 12 release quickly, and the latter basically show zero-order release, and the drug release behavior is similar.

The results showed that for ANG-NP-PTX, the surface modification of Angiopep-2 did not affect the release behavior of PTX.

2. Cytotoxicity test

The in vitro inhibition test of ANG-NP-PTX on U87 MG cells was determined by MTT method, and the results are shown in Figure 2.

As shown in Figure 2, the IC ₅₀ values of the three formulations of Taxol, NP-PTX and ANG-NP-PTX on U87 MG were 225 ng/mL, 248 ng/mL and 66 ng/mL, the results showed that Angiopep-2 modified The nanoparticles significantly increased the cytotoxic effect of PTX on U87 MG (p<0.01), which was related to the increase of PTX uptake by ANG-NP/PTX through the LRP receptor expressed on the surface of U87 MG.

3. Cellular uptake

The carrier material is fluorescently labeled with rhodamine isothiocyanate (RBITC) to investigate the qualitative uptake of the carrier system by cells,

The results showed (as shown in Figure 4 and Figure 5), within 30-120 min, the fluorescence intensity of the ANG-NP group (B, D, F in Figure 4) was significantly stronger than that of the NP group (A, C in Figure 4 , E), and this uptake was inhibited by 200 μg/ml free Angiopep-2 and Aprotinin (as shown in Figure 5). The results also showed that the uptake of ANG-NP could also be inhibited at 4°C, indicating that Angiopep-2 modification increased the uptake of nanoparticles by U87 MG cells, which was mediated by LRP receptors and energy-dependent.

4. In vitro two-stage targeting evaluation

The BCEC-U87 MG co-culture model was used to simulate the physiological barrier of glioma in vivo, and the dual-level targeting effect of ANG-NP/PTX was evaluated. See the results.

The results showed that (as shown in Figure 5), the inhibitory rate of ANG-NP/PTX on U87 MG cells was significantly greater than that of Taxol and NP/PTX (p<0.01), which was due to the expression of LRP receptors in both BCEC and U87 MG, and ANG-NP /PTX mediates the dual targeting effect of increasing BBB transport and U87 MG uptake through LRP, and this dual targeting effect can be competitively inhibited by 200 μg/mL free Angiopep-2 and Aprotinin LRP receptor ligands (p<0.01) .

5. In vivo dual-stage targeting evaluation

As shown in A in Figure 6, after DIR-labeled nanoparticles were administered through the tail vein, the fluorescence intensity in the brain of the ANG-NP group was significantly stronger than that of the NP group, indicating that the brain tropism of the nanoparticles was increased after ANG modification . The fluorescence distribution of isolated organs after 24 h showed that (see B in Figure 6), the fluorescence intensity of ANG-NP in the brain tumor site was significantly stronger than that of the NP group, and it showed a state of whole brain distribution.

The results showed that 1) ANG-modified nanoparticles increased the permeability of the blood-brain barrier; 2) ANG-modified nanoparticles increased the accumulation and retention at the glioma site; thus, ANG-NP showed a certain dual-level target in vivo tropism.

Experimental results show that the targeting system of the present invention can increase the solubility of PTX and prolong the circulation time in vivo; it has a dual-stage targeting function through the BBB and glioma cells mediated by LRP receptors; moreover, PTX from the carrier The release has a sustained release effect, and the carrier material is non-toxic, biodegradable and biocompatible, which effectively solves the problem of the blood-brain-tumor barrier (Blood Brain Tumor Barrier, BBTB) existing in glioma chemotherapy, and can be applied for the treatment of glioma.

The advantages of the targeted drug delivery system of the present invention are:

1. By adopting nanotechnology, the solubility of PTX is greatly improved, and the safety problems caused by organic solvents during use are avoided.

2. By preparing nanoparticles, the nanometer size effect of nanoparticles and the EPR effect of glioma are used to facilitate the accumulation of PTX in tumor sites.

3. PEGylated long-circulating nanoparticles prolong the circulation time of PTX in the body, increase the chance of PTX accumulating to the tumor site, reduce the phagocytosis of the reticuloendothelial system, and reduce the toxic and side effects of the PTX system.

4. After modification of Angiopep-2, the uptake of tumor cells to nanosystems was increased through LRP mediation, and the apoptotic effect of PTX on tumor cells and the capture ability of G2/M were enhanced.

5. Angiopep-2 modification increases the ability of nanosystems to penetrate the blood-brain barrier through LRP mediation.

6. After Angiopep-2 is modified, it realizes a dual-level targeting of the head group, BBB and tumor through LRP-mediated "one-head-two-target" function.

the

For ease of understanding, the microemulsion drug delivery system of the present invention will be described in detail below with specific drawings and examples. It should be pointed out that the specific examples and accompanying drawings are only for illustration. Obviously, those skilled in the art can make various amendments and changes within the scope of the present invention according to the description herein. These amendments and Modifications are also included within the scope of the present invention.

Description of drawings

Fig. 1 is PTX in vitro release curve figure (n=3) among the present invention.

Fig. 2 is the U87 MG cell survival curve in the present invention (n=3).

Fig. 3 is the uptake fluorescence diagram of U87 MG cells in the present invention, wherein, the action time of Figure A and Figure B is 30 min, the action time of Figure C and Figure D is 60 min, and the action time of Figure E and Figure F is 120 min. A, Figure C, and Figure E are the uptake of NP, and Figures B, D, and F are the uptake of ANG-NP.

Fig. 4 is the uptake fluorescence diagram of U87 MG cells under different inhibition conditions in the present invention.

Figure 5 is a graph of cell viability of U87 MG in the co-culture model of the present invention (n = 3).

Figure 6 is a comparison diagram of the dual-level targeting effect of ANG-NP/PTX in the present invention, wherein, Figure A is the time-lapse fluorescence distribution of mice bearing U87 MG glioma, and Figure B is the isolated mouse after 24 hours Organ fluorescence map.

Detailed ways

Example 1

Add PTX and MePEG-PCL:Male-PEG-PCL= 20:1 into dichloromethane to dissolve , add 1% sodium cholate aqueous solution, put in ice bath at 200 W, 60 s intermittent ultrasonication to form oil-in-water (O/W ) emulsion. Disperse in 0.5% sodium cholate aqueous solution and magnetically stir for 5 min. Remove dichloromethane by rotary evaporation at 40°C until there is no air bubbles, centrifuge at 12,000 rpm at 4°C for 60 min, and discard the supernatant. Use a certain amount of HEPES solution (pH 7.0) to disperse by pipetting, add Angiopep-2 (the molar ratio of Male to Angiopep-2 is 3:1), and incubate for 8 h at room temperature with nitrogen gas and dark. Centrifuge at 12000 rpm at 4°C for 60 min and discard the supernatant to obtain ANG-NP-PTX. The measured particle size is 62.4±4.5, and the number of ANG on each nanoparticle is 7±3.

Example 2

Add PTX and MePEG-PCL:Male-PEG-PCL = 15:1 into dichloromethane to dissolve , add 1% sodium cholate aqueous solution, 200 W in ice bath, 60 s intermittent ultrasonication to form oil-in-water (O/W ) emulsion. Disperse in 0.5% sodium cholate aqueous solution and magnetically stir for 5 min. Remove dichloromethane by rotary evaporation at 40°C until there is no air bubbles, centrifuge at 12,000 rpm at 4°C for 60 min, and discard the supernatant. Use a certain amount of HEPES solution (pH 7.0) to disperse by pipetting, add Angiopep-2 (the molar ratio of Male to Angiopep-2 is 3:1), and incubate for 8 h at room temperature with nitrogen gas and dark. Centrifuge at 12000 rpm at 4°C for 60 min and discard the supernatant to obtain ANG-NP-PTX. The measured particle size is 84.5±6.8, and the number of ANG on each nanoparticle is 25±5.

Example 3

Add PTX and MePEG-PCL:Male-PEG-PCL = 9:1 into dichloromethane to dissolve , add 1% sodium cholate aqueous solution, put 200 W in ice bath, 60 s intermittent ultrasonic to form oil-in-water (O/W ) emulsion. Disperse in 0.5% sodium cholate aqueous solution and magnetically stir for 5 min. Remove dichloromethane by rotary evaporation at 40°C until there is no air bubbles, centrifuge at 12,000 rpm at 4°C for 60 min, and discard the supernatant. Use a certain amount of HEPES solution (pH 7.0) to disperse by pipetting, add Angiopep-2 (the molar ratio of Male to Angiopep-2 is 3:1), and incubate for 8 h at room temperature with nitrogen gas and dark. Centrifuge at 12000 rpm at 4°C for 60 min and discard the supernatant to obtain ANG-NP-PTX. The measured particle size is 124.2±8.2, and the number of ANG on each nanoparticle is 54±9.

Example 4

Add PTX and MePEG-PCL:Male-PEG-PCL = 4:1 into dichloromethane to dissolve , add 1% sodium cholate aqueous solution, 200 W in ice bath, 60 s intermittent ultrasonication to form oil-in-water (O/W ) emulsion. Disperse in 0.5% sodium cholate aqueous solution and magnetically stir for 5 min. Remove dichloromethane by rotary evaporation at 40°C until there is no air bubbles, centrifuge at 12,000 rpm at 4°C for 60 min, and discard the supernatant. Use a certain amount of HEPES solution (pH 7.0) to disperse by pipetting, add Angiopep-2 (the molar ratio of Male to Angiopep-2 is 3:1), and incubate for 8 h at room temperature with nitrogen gas and dark. Centrifuge at 12000 rpm at 4°C for 60 min and discard the supernatant to obtain ANG-NP-PTX. The measured particle size is 614.7±102.8, and the number of ANG on each nanoparticle is 85±23.

Example 5

Add PTX and MePEG-PCL:Male-PEG-PCL = 9:1 to dissolve in dichloromethane, add 1% sodium cholate aqueous solution, 200 W under ice bath, 60 s intermittent ultrasonication to form oil-in-water (O/ W) Emulsion. Disperse in 0.5% sodium cholate aqueous solution and magnetically stir for 5 min. Remove dichloromethane by rotary evaporation at 40°C until there is no air bubbles, centrifuge at 12,000 rpm at 4°C for 60 min, and discard the supernatant. Use a certain amount of HEPES solution (pH 7.0) to disperse by blowing and blowing, add Angiopep-2 (the molar ratio of Male to Angiopep-2 is 10:1), and incubate for 8 h at room temperature under nitrogen and in the dark. Centrifuge at 12000 rpm at 4°C for 60 min and discard the supernatant to obtain ANG-NP-PTX. The measured particle size is 73.4±3.1, and the number of ANG on each nanoparticle is 14±4.

Example 6

Add PTX and MePEG-PCL:Male-PEG-PCL = 9:1 to dichloromethane to dissolve, add 1% sodium cholate aqueous solution, 200 W under ice bath, 60 s intermittent ultrasonication to form oil-in-water (O/W ) emulsion. Disperse into 0.5% sodium cholate aqueous solution and magnetically stir for 5 min. Remove methylene chloride by rotary evaporation at 40°C until there is no air bubbles, centrifuge at 12,000 rpm at 4°C for 60 min, and discard the supernatant. Use a certain amount of HEPES solution (pH 7.0) to disperse by blowing and blowing, add Angiopep-2 (the molar ratio of Male to Angiopep-2 is 5:1), and incubate for 8 h at room temperature with nitrogen gas and in the dark. Centrifuge at 12,000 rpm at 4°C for 60 min and discard the supernatant to obtain ANG-NP-PTX. The measured particle size is 93.5±4.8, and the number of ANG on each nanoparticle is 34±6.

Example 7

Add PTX and MePEG-PCL:Male-PEG-PCL = 9:1 to dichloromethane to dissolve, add 1% sodium cholate aqueous solution, 200 W under ice bath, 60 s intermittent ultrasonication to form oil-in-water (O/W ) emulsion. Disperse into 0.5% sodium cholate aqueous solution and magnetically stir for 5 min. Remove methylene chloride by rotary evaporation at 40°C until there is no air bubbles, centrifuge at 12,000 rpm at 4°C for 60 min, and discard the supernatant. Use a certain amount of HEPES solution (pH 7.0) to disperse by blowing and blowing, add Angiopep-2 (the molar ratio of Male to Angiopep-2 is 3:1), and incubate for 8 h at room temperature with nitrogen gas and in the dark. Centrifuge at 12,000 rpm at 4°C for 60 min and discard the supernatant to obtain ANG-NP-PTX. The measured particle size is 113.5±7.2, and the number of ANG on each nanoparticle is 62±8.

Example 8

Add PTX and MePEG-PCL:Male-PEG-PCL = 9:1 to dichloromethane to dissolve, add 1% sodium cholate aqueous solution, 200 W under ice bath, 60 s intermittent ultrasonication to form oil-in-water (O/W ) emulsion. Disperse into 0.5% sodium cholate aqueous solution and magnetically stir for 5 min. Remove methylene chloride by rotary evaporation at 40°C until there is no air bubbles, centrifuge at 12,000 rpm at 4°C for 60 min, and discard the supernatant. Use a certain amount of HEPES solution (pH 7.0) to disperse by pipetting, add Angiopep-2 (the molar ratio of Male to Angiopep-2 is 1:1), and incubate for 8 h at room temperature with nitrogen gas and dark. Centrifuge at 12,000 rpm at 4°C for 60 min and discard the supernatant to obtain ANG-NP-PTX. The measured particle size is 713.5±172.4, and the number of ANG on each nanoparticle is 79±24.

Example 9

Add PTX and MePEG-PCL:Male-PEG-PCL = 9:1 to dichloromethane to dissolve, add 1% sodium cholate aqueous solution, 200 W under ice bath, 60 s intermittent ultrasonication to form oil-in-water (O/W ) emulsion. Disperse into 0.5% sodium cholate aqueous solution and magnetically stir for 5 min. Remove methylene chloride by rotary evaporation at 40°C until there is no air bubbles, centrifuge at 12,000 rpm at 4°C for 60 min, and discard the supernatant. Use a certain amount of HEPES solution (pH 7.0) to disperse by pipetting, add Angiopep-2 (the molar ratio of Male to Angiopep-2 is 1:1), and incubate for 4 h at room temperature with nitrogen gas and dark. Centrifuge at 12,000 rpm at 4°C for 60 min and discard the supernatant to obtain ANG-NP-PTX. The measured particle size is 74.3±1.1, and the number of ANG on each nanoparticle is 8±2.

Example 10

Add PTX and MePEG-PCL:Male-PEG-PCL = 9:1 to dichloromethane to dissolve, add 1% sodium cholate aqueous solution, 200 W under ice bath, 60 s intermittent ultrasonication to form oil-in-water (O/W ) emulsion. Disperse into 0.5% sodium cholate aqueous solution and magnetically stir for 5 min. Remove methylene chloride by rotary evaporation at 40°C until there is no air bubbles, centrifuge at 12,000 rpm at 4°C for 60 min, and discard the supernatant. Use a certain amount of HEPES solution (pH 7.0) to disperse by blowing and blowing, add Angiopep-2 (the molar ratio of Male to Angiopep-2 is 1:1), and incubate for 6 h at room temperature with nitrogen gas and in the dark. Centrifuge at 12,000 rpm at 4°C for 60 min and discard the supernatant to obtain ANG-NP-PTX. The measured particle size is 91.5±4.7, and the number of ANG on each nanoparticle is 26±7.

Example 11

Add PTX and MePEG-PCL:Male-PEG-PCL = 9:1 to dichloromethane to dissolve, add 1% sodium cholate aqueous solution, 200 W under ice bath, 60 s intermittent ultrasonication to form oil-in-water (O/W ) emulsion. Disperse into 0.5% sodium cholate aqueous solution and magnetically stir for 5 min. Remove methylene chloride by rotary evaporation at 40°C until there is no air bubbles, centrifuge at 12,000 rpm at 4°C for 60 min, and discard the supernatant. Use a certain amount of HEPES solution (pH 7.0) to disperse by pipetting, add Angiopep-2 (the molar ratio of Male to Angiopep-2 is 1:1), and incubate for 8 h at room temperature with nitrogen gas and dark. Centrifuge at 12,000 rpm at 4°C for 60 min and discard the supernatant to obtain ANG-NP-PTX. The measured particle size is 103.2±7.2, and the number of ANG on each nanoparticle is 52±6.

Example 12

Add PTX and MePEG-PCL:Male-PEG-PCL = 9:1 to dichloromethane to dissolve, add 1% sodium cholate aqueous solution, 200 W under ice bath, 60 s intermittent ultrasonication to form oil-in-water (O/W ) emulsion. Disperse into 0.5% sodium cholate aqueous solution and magnetically stir for 5 min. Remove methylene chloride by rotary evaporation at 40°C until there is no air bubbles, centrifuge at 12,000 rpm at 4°C for 60 min, and discard the supernatant. Use a certain amount of HEPES solution (pH 7.0) to disperse by blowing and blowing, add Angiopep-2 (the molar ratio of Male to Angiopep-2 is 1:1), and incubate for 12 h at room temperature with nitrogen gas and in the dark. Centrifuge at 12,000 rpm at 4°C for 60 min and discard the supernatant to obtain ANG-NP-PTX. The measured particle size is 184.3±10.2, and the number of ANG on each nanoparticle is 67±6.

Example 13

Add PTX and MePEG-PCL:Male-PEG-PCL = 9:1 to dichloromethane to dissolve, add 1% sodium cholate aqueous solution, 200 W under ice bath, 60 s intermittent ultrasonication to form oil-in-water (O/W ) emulsion. Disperse into 0.5% sodium cholate aqueous solution and magnetically stir for 5 min. Remove methylene chloride by rotary evaporation at 40°C until there is no air bubbles, centrifuge at 12,000 rpm at 4°C for 60 min, and discard the supernatant. Use a certain amount of HEPES solution (pH 7.0) to disperse by pipetting, add Angiopep-2 (the molar ratio of Male to Angiopep-2 is 1:1), and incubate for 16 h at room temperature with nitrogen gas and dark. Centrifuge at 12,000 rpm at 4°C for 60 min and discard the supernatant to obtain ANG-NP-PTX. The measured particle size is 694.3±102.1, and the number of ANG on each nanoparticle is 88±32.

The results of the above examples show that the targeting system of the present invention for the treatment of brain tumors increases the solubility of PTX and prolongs the circulation time in the body; it has a dual-level targeting system through the BBB and glioma cells mediated by LRP receptors function; moreover, the release of PTX from the carrier has a sustained release effect, and the carrier material is non-toxic, biodegradable and biocompatible, effectively solving the blood-brain-tumor barrier (Blood Brain Tumor Barrier) that exists simultaneously in glioma chemotherapy. , BBTB) problem, can be applied to the treatment of glioma.

Claims (4)

1. a targeting drug delivery system of treating brain tumor is characterized in that, described targeting drug delivery system is the PEG-PCL-PTX nanoparticle that Angiopep-2 modifies, and it prepares through following method:

At first, a certain amount of MePEG-PCL, Male-PEG-PCL and PTX be dissolved in the dichloromethane dissolve, add 1% sodium cholate aqueous solution, 200 W under the ice bath, 60 s are interrupted ultrasonic formation oil-in-water O/W Emulsion;

Secondly, be distributed to 0.5% sodium cholate aqueous solution and magnetic agitation 5 min; Once more, 40 ℃ of rotary evaporations are not removed dichloromethane to there being bubble, and abandoning supernatant gets NP-PTX behind 4 ℃ of centrifugal 60 min of 12000 rpm;

Then, the NP-PTX for preparing is disperseed with a certain amount of HEPES liquid pH 7.0 piping and druming, add the Angiopep-2 of amount of calculation, inflated with nitrogen, lucifuge are hatched under the room temperature;

Disperse with a certain amount of HEPES liquid (pH 7.0) piping and druming, add Angiopep-2 (Male and Angiopep-2 mol ratio are 1 : 1),

At last, abandoning supernatant behind 4 ℃ of centrifugal 60 min of 12000 rpm makes the ANG-NP-PTX nanoparticle.

2. by the targeting drug delivery system of the described treatment brain tumor of claim 1, it is characterized in that, in the described method for preparing, PTX and MePEG-PCL:Male-PEG-PCL=4～20 : 1.

3. by the targeting drug delivery system of the described treatment brain tumor of claim 1, it is characterized in that in the described method for preparing, Male among the described Angiopep-2 and Angiopep-2 mol ratio are 1～10 : 1.

4. the purposes of the targeting drug delivery system of the treatment brain tumor of claim 1 in treatment cerebral glioma medicine.

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