Secondary dual-targeting nano-polymer vesicle containing folic acid and cell-penetrating peptide TAT as well as preparation method and application of secondary dual-targeting nano-polymer vesicle

Abstract

The invention belongs to the field of biological medicine, and relates to a nano polymer drug carrier with a cell membrane and cell nucleus secondary dual-targeting function. The invention discloses a preparation method of a drug carrier modified by double ligands of folic acid FA and cell penetrating peptide TAT and a research result of the drug carrier on OVCAR-3 cell toxicity. The FA and TAT double-ligand modified nano-polymer vesicle has a cell membrane and cell nucleus secondary double-targeting function, can overcome various physiological barriers and deliver drugs into cell nucleuses and release the drugs, so that the drug effects of adriamycin and other anti-cancer drugs acting on the cell nucleuses are improved, the toxic and side effects of the drugs are reduced and avoided, and the FA and TAT double-ligand modified nano-polymer vesicle has a good application prospect. In addition, the drug carrier disclosed by the invention has good biocompatibility and biodegradability.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a cell membrane and cell nucleus two-level dual-targeting nano polymer drug carrier. Background Art

[0002] At present, the main methods for treating tumors in clinic include surgery, radiotherapy and chemotherapy, among which chemotherapy is the most commonly used method. Doxorubicin is one of the most effective and commonly used chemotherapy drugs for treating breast cancer, lung cancer and other cancers in clinic. However, due to its lack of tumor targeting, it will also have toxic side effects on normal tissue cells while killing cancer cells. Moreover, the target of doxorubicin (DOX) is located in the cell nucleus. After being blocked by multiple biological barriers inside and outside the cell, only a small part of the drug can enter the cell nucleus to play a role, which greatly reduces the therapeutic effect of doxorubicin. Targeted nano drug delivery systems have attracted extensive attention from researchers because of their good biocompatibility and the ability to selectively deliver anticancer drugs to tumor cells, thereby improving the therapeutic effect of drugs and avoiding their toxic side effects. At present, many targeted nano drug delivery systems have been developed for tumor treatment. However, most of the research on targeted nano drug delivery systems focuses on targeting the cell membrane surface of tumor cells, and there are not many studies on cell nucleus targeted nano drug delivery systems. For many anticancer drugs such as doxorubicin that act on the cell nucleus, the nucleus-targeted nanodrug delivery system is expected to help them enter the nucleus of cancer cells more efficiently and directly, thereby improving their anti-cancer effects.

[0003] Polymer materials are widely used in the study of carriers for targeted nano-drug delivery systems because of their advantages such as biocompatibility, biodegradability, modifiability, ability to encapsulate anticancer drugs and increase drug stability. Connecting one or more targeting ligands, such as folic acid, transferrin, peptides, and antibodies, to polymer materials is one way to enable polymer drug carriers to target tumor cells and their subcellular organelles. Folic acid (FA) can achieve nanocarrier targeting of tumor cells by specifically binding to cancer cells that overexpress folate receptors on the cell membrane surface (such as human ovarian cancer cells OVCAR-3). The transactivator of transcription (TAT) derived from human immunodeficiency virus (HIV-1) is a cell-penetrating peptide with extremely strong cell penetration, which can help nanocarriers penetrate the cell membrane and then enter the cell nucleus through the nuclear pore complex, thereby achieving nuclear targeting of nanocarriers. There are many ways to connect targeting ligands to drug carriers, one of which is to use biotin-avidin bridging technology. There is a strong non-covalent bond and biological interaction between biotin and avidin. An avidin has four action sites that can bind to biotin. The use of biotin-avidin technology to connect ligands such as TAT ​​can effectively avoid the inactivation of TAT that may occur when the TAT polypeptide is connected to the polymer chain through chemical reactions. Pluronic materials have been approved by the US Food and Drug Administration as food additives and pharmaceutical ingredients due to their good biocompatibility and biodegradability. Polylactic acid (PLA) is a type of hydrophobic segment that is often selected. Because of its good biocompatibility and biodegradability, it can be degraded into small molecules in the human body and is easily excreted from the body. It is widely used in the field of biomedicine. Polylactic acid (PLA) is modified at the hydroxyl end of Pluronic, which is conducive to the formation of stable nano-polymer vesicles. Summary of the invention

[0004] The present invention aims to prepare a novel dual-targeting ligand containing folic acid FA and cell-penetrating peptide TAT with secondary dual-targeting function of cell membrane and cell nucleus by using biotin-avidin bridging technology, a nano polymer vesicle FA(TAT)-F127-PLA composed of Pluronic F127 and PLA, so that the nano polymer vesicle can be used as a drug carrier to deliver the anti-tumor drug doxorubicin into the cell nucleus of tumor cells, thereby improving its drug effect and reducing its toxic side effects, and at the same time providing a preparation method of the polymer vesicle.

[0005] The preparation of the secondary dual-targeted nanodrug carrier FA(TAT)-F127-PLA / DOX encapsulated with doxorubicin and its in vitro cytotoxicity and quantitative cell uptake study on human ovarian cancer cells OVCAR-3 (folate receptor overexpression) with overexpression of folate receptor are as follows: (1) Weigh a certain amount of polymer material and doxorubicin and fully dissolve them in N,N-dimethylformamide (DMF) solvent, then slowly disperse them drop by drop into a stirring phosphate buffer solution. After the dispersion operation is completed, dialyze to remove DMF, transfer the solution in the dialysis bag to a centrifuge tube and centrifuge for a period of time, remove the precipitate to obtain a nanovesicle solution encapsulating DOX. Then, Avidin / PBS and biotinylated TAT (Biotin-TAT) / PBS solutions are added to the solution in sequence, and finally a secondary dual-targeted FA (TAT)-F127-PLA / DOX nanopolymer vesicle solution is prepared.

[0006] (2) The toxic effects of different drug carriers on OVCAR-3 cells were detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl [3-(4,5-Dimethyl-2-Thiazolyl)-2,5-Diphenyl, MTT] colorimetric method. OVCAR-3 cells with stable growth were taken, washed with PBS, and then digested with trypsin containing EDTA. After adding complete medium to terminate digestion, centrifugation was performed, the cell pellet was resuspended and the cell concentration was adjusted. Then, the same volume of cell suspension was added to a 96-well cell plate and cultured in a 37°C, 5% CO2 incubator. After culturing the cells for a period of time, the culture medium was aspirated, and then the same volume of different drug-loaded nano-polymer vesicles and free DOX solution were added to each well. The same amount of complete medium was used as a blank control. Five replicate wells were set up in each group and incubated with OVCAR-3 cells for 24 h and 48 h, respectively. The nanoparticle solution, free drug and complete medium were replaced every 24 h. Dilute the MTT solution with basal culture medium, absorb the culture medium on the 96-well plate, add the diluted MTT solution, wrap it with tin foil and incubate it in an incubator for 4 hours. After that, absorb the MTT solution, add a certain amount of DMSO, and detect the OD value at 562 nm with an enzyme marker. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 The results of the in vitro cytotoxicity study of the dual-targeted drug carrier of the present invention on OVCAR-3 cells: (A) and (B) are the cell viability of the drug-loaded nanovesicles and free DOX groups after incubation with cells for 24 h and 48 h, respectively, *p<0.05, **p<0.01.

[0008] Table 1 IC50 values ​​of doxorubicin nanovesicles with different loadings on OVCAR-3 cells after 24 h and 48 h of treatment.

[0009] Samples IC50 (mg / L, 24 h) IC50 (mg / L, 48 h) 5%: 5% FA(TAT)-F127-PLA / DOX 3.00±0.37 0.36±0.06 5% FA-F127-PLA / DOX 3.60±0.68 0.44±0.07 5% TAT-F127-PLA / DOX 3.97±0.57 0.57±0.07 PLA-F127-PLA / DOX 4.24±0.55 0.62±0.08 Free DOX 4.75±0.34 0.87±0.08 DETAILED DESCRIPTION

[0010] 1. Weigh 0.43 mg FA-F127-PLA, 0.42 mg Biotin-F127-PLA, 4.15 mg PLA-F127-PLA block polymer and 2 mg DOX and dissolve them in 1 ml DMF. Then disperse the mixture dropwise in 5 ml ultrapure water under stirring. Then put the suspension into a dialysis bag (MWCO 12-14KDa) and dialyze it in ultrapure water for 8 h. During this period, the ultrapure water was changed every 1 h to remove excess DMF. After the dialysis is completed, the suspension is centrifuged at 3000 rpm for 5 min to remove the precipitate, thereby obtaining FA (Biotin) -F127-PLA / DOX nanovesicles. Then, 460 μL of Avidin / PBS (1 mg / ml) solution was incubated with FA(Biotin)-F127-PLA / DOX solution at room temperature for 40 min, and then 63 μL of Biotin-TAT / PBS (1 mg / ml) solution was added to the mixed solution. After further incubation at room temperature for 40 min, a FA(TAT)-F127-PLA / DOX nanopolymer vesicle solution with a ligand molar content ratio of 5%: 5% was finally obtained.

[0011] 2. The MTT colorimetric method was used to detect the toxicity of the drug-loaded nanovesicle group and the free DOX group on OVCAR-3 cells. The different concentration groups were incubated with OVCAR-3 cells for 24 and 48 h, respectively. After reaching the time point, the culture medium was discarded, washed with PBS buffer, and an appropriate concentration of MTT and fresh culture medium were added to each well, and incubated in the dark for 4 h. After the incubation was completed, the culture medium containing MTT was discarded, and an appropriate amount of dimethyl sulfoxide (DMSO) was added. The absorbance value was detected at a wavelength of 562 nm using an enzyme reader.

[0012] from Figure 1 The results and basis Figure 1 The calculated IC50 values ​​in Table 1 show that the degree of toxicity to OVCAR-3 cells is: secondary dual-targeted nanovesicle formulation > single-targeted nanovesicle formulation > non-targeted nanovesicle formulation > Free DOX. This shows that the three types of targeted nanoformulations are beneficial to the sustained release of doxorubicin. More importantly, the secondary dual-targeted nanovesicle formulation of FA(TAT)-F127-PLA / DOX is more toxic to OVCAR-3 cells than the two single-targeted and non-targeted nanovesicle formulations.

[0013] The nano polymer vesicles provided by the present invention have a cell nucleus targeting function, can overcome multiple physiological barriers, deliver drugs to the cell nucleus and release them, thereby improving drug efficacy while avoiding drug toxicity and side effects, and have good application prospects. In addition, the drug carrier of the present invention has good biocompatibility and biodegradability.

Claims

1. A novel dual targeting ligand containing folate FA and cell-penetrating peptide TAT with secondary dual targeting function to cell membrane and cell nucleus, nanopolymer vesicle FA(TAT)-F127-PLA composed of Pluronic F127 and PLA.

2. The nano-polymer vesicle according to claim 1, characterized in that: The preparation method of the drug carrier is as follows: (1) Weigh 0.43 mg FA-F127-PLA, 0.42 mg BT-F127-PLA, 4.15 mg PLA-F127-PLA block polymer and 2 mg DOX and dissolve them in 1 ml DMF. (2) Disperse the mixture dropwise in 5 ml ultrapure water under stirring, then place the suspension in a dialysis bag (MWCO 12-14KDa) and dialyze it in ultrapure water for 8 h, changing the ultrapure water every 1 h to remove excess DMF. Disperse the mixture dropwise in 5 ml ultrapure water under stirring, then place the suspension in a dialysis bag (MWCO 12-14KDa) and dialyze it in ultrapure water for 8 h, changing the ultrapure water every 1 h to remove excess DMF. (3) After dialysis, the suspension was centrifuged at 3000 rpm for 5 min to remove the precipitate, thereby obtaining FA(Biotin)-F127-PLA / DOX nanovesicles. (4) Then, 460 μL of Avidin / PBS (1 mg / ml) solution was incubated with FA(Biotin)-F127-PLA / DOX solution at room temperature for 40 min, and then 63 μL of Biotin-TAT / PBS (1 mg / ml) solution was added to the mixed solution. After further incubation at room temperature for 40 min, a FA(TAT)-F127-PLA / DOX nanopolymer vesicle solution with a ligand molar content ratio of 5%:5% was finally obtained.

3. Use of the polymer according to claim 1 as an anti-tumor drug carrier.

4. The use according to claim 3, characterized in that: The prepared FA(TAT)-F127-PLA / DOX secondary dual-targeting nanovesicle preparation has anti-tumor effect.

Images