CN113855816B - EGFR-targeting nanoparticle drug-loading system and preparation method thereof - Google Patents

EGFR-targeting nanoparticle drug-loading system and preparation method thereof Download PDF

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CN113855816B
CN113855816B CN202111136070.6A CN202111136070A CN113855816B CN 113855816 B CN113855816 B CN 113855816B CN 202111136070 A CN202111136070 A CN 202111136070A CN 113855816 B CN113855816 B CN 113855816B
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lung cancer
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CN113855816A (en
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黄来强
刘昱宏
蒋盛威
刘可为
冯春燕
陈华清
胡洋
王坤
过冬冬
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Shenzhen International Graduate School of Tsinghua University
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Abstract

The application discloses an EGFR-targeting nanoparticle drug-carrying system and a preparation method thereof. The preparation method comprises the following steps: and modifying the anti-EGFR nucleic acid aptamer on the surface of the nanoparticle by using EDC/NHS amide forming reaction. The EGFR-targeting nanoparticle drug-loading system has the advantages of actively targeting lung cancer cells with high expression of EGFR protein, improving the drug effect of the drug, reducing the toxic and side effects, and being widely applied in the fields of medicine and biology.

Description

EGFR-targeting nanoparticle drug-loading system and preparation method thereof
Technical Field
The invention belongs to the technical fields of biotechnology and biological medicine, and relates to a nanoparticle drug-loading system targeting EGFR and a preparation method thereof.
Background
Lung cancer is a malignant tumor that occurs in the bronchial epithelial mucosa or alveoli, and is the leading part of cancer death cases in china and even worldwide. With the expansion of the population of smokers in recent years, lung cancer has become the first worldwide morbidity and mortality in men, and the second most frequently in women to breast cancer. By morphological and histological analysis of lung cancer tissue, we initially divided lung cancer into Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC occupies 75% -80% of lung cancer cases, mainly including lung Adenocarcinoma (AD) and lung Squamous Cell Carcinoma (SCC).
In the clinical treatment of lung cancer at the present stage, surgical excision is still the first treatment means, and then chemotherapy and radiotherapy are carried out according to different pathological tissue conditions. The surgical excision is suitable for patients without distant metastasis of cancer cells, and the radiotherapy and the chemotherapy have relieving effects on early small cell lung cancer, but have obvious curative effect differences according to different disease positions and physical conditions of the patients, have no specificity to killing the cancer cells, and can cause a plurality of side effects such as liver and kidney injury and the like to the bodies of the patients. In recent years, with the continuous progress of information technology and biotechnology, basic research on cancer cell gene level is perfected by combining samples and experiences of clinical treatment under big data statistics, and personalized treatment such as targeted treatment and immunotherapy of lung cancer is receiving attention. However, the prior art has low drug delivery efficiency for treating lung cancer, so that development of a drug delivery system is urgently needed to solve the above problems.
Disclosure of Invention
In order to solve the problems set forth in the background art, the present application is directed to a nanoparticle drug delivery system targeting EGFR and a preparation method thereof. The EGFR-targeting nanoparticle drug-carrying system has the advantages of actively targeting lung cancer cells with high expression of EGFR protein, improving the drug effect of the drug, reducing toxic and side effects, and being widely applied in the fields of medicine and biology.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: in one aspect, the invention provides an EGFR-targeting nanoparticle drug delivery system, comprising nanoparticles, wherein the surfaces of the nanoparticles are modified with anti-EGFR nucleic acid aptamers.
Further, the nanoparticle is a chitosan nanoparticle.
Further, the preparation method of the chitosan nanoparticle comprises the following steps: mixing and stirring sodium tripolyphosphate (sodium tripolyphosphate, TPP) solution and chitosan solution, then performing ultrasonic dispersion, and then performing low-temperature centrifugation to obtain the chitosan nanoparticle.
Further, the concentration of the sodium tripolyphosphate solution is 2-5mg/mL, and the concentration of the chitosan solution is 0.5-3mg/mL;
the volume ratio of the sodium tripolyphosphate solution to the chitosan solution is 1:3-1:5.
Further, the nucleotide sequence of the anti-EGFR nucleic acid aptamer is shown as SEQ ID NO. 1. anti-EGFR aptamer sequence (DNA Single Strand) ,76nt)：5′-TAC CAG TGC GAT GCT CAG TGC CGT TTC TTC TCT TTC GCT TTT TTT GCT TTT GAG CAT GCT GAC GCA TTC GGT TGA C-3′.
In another aspect, the invention provides a method for preparing an EGFR-targeted nanoparticle drug delivery system according to any one of the above, comprising the steps of: and modifying the anti-EGFR nucleic acid aptamer on the surface of the nanoparticle by using EDC/NHS amide forming reaction.
In another aspect, the invention provides a medicament for treating lung cancer, comprising any of the above nanoparticle drug delivery systems and system-encapsulated drugs targeting EGFR.
Further, the system-entrapped drug comprises EGFR tyramine inhibitor drugs and/or autophagy pathway related drugs.
Further, the EGFR tyramine inhibitor comprises gefitinib, afatinib and erlotinib.
Further, the autophagy pathway related drugs include rapamycin, 3MA.
Furthermore, the medicine for treating lung cancer can be prepared into various forms such as oral liquid or injection, and various dosage forms can be prepared according to a conventional method in the pharmaceutical field.
The beneficial effects of the invention are as follows:
(1) The invention provides a drug delivery carrier targeting EGFR (epidermal growth factor receptor) -high-expression lung cancer cells (namely an EGFR-targeting nanoparticle drug delivery system), which can specifically combine with EGFR to realize high-efficiency positioning of cancer cells, specifically combine with EGFR to realize drug delivery, and improve drug delivery efficiency.
(2) The invention can improve the drug effect and reduce the toxic and side effects through targeted delivery of the drug. Can be widely applied in the fields of medicine and biology, and can generate huge social and economic benefits.
(3) The nanoparticle drug-loading system for targeting EGFR has good encapsulation rate on various drugs, and has great application value in the aspect of targeted treatment of lung cancer cells with EGFR high expression.
Drawings
Fig. 1 (a) and (b) are respectively particle size distribution diagrams of chitosan nanoparticle NP prepared in the embodiment of the present invention and chitosan nanoparticle NP-Apt with anti-EGFR nucleic acid aptamer surface-modified, fig. 1 (c) is a potential diagram of chitosan nanoparticle prepared in the embodiment of the present invention and chitosan nanoparticle with anti-EGFR nucleic acid aptamer surface-modified, and fig. 1 (d) is a transmission electron microscope diagram of chitosan nanoparticle with anti-EGFR nucleic acid aptamer surface-modified prepared in the embodiment of the present invention.
FIG. 2 is a graph showing uptake of chitosan nanoparticles with anti-EGFR aptamer modified on the surface of target cells (H1975 cells) and non-target cells (293T cells) photographed by a confocal microscope according to an embodiment of the present invention; wherein blue is a nucleus, green is a nanoparticle, and red is a target cell transfected with mCherry red fluorescent protein (H1975 cell); the Merge plot is a combination of the other three plots, showing both red and blue as target cells, with the green nanoparticles having significant aggregation around the target cells.
FIG. 3 is a graph showing the effect of each treatment group on proliferation of target cells H1975 and non-target cells 293T according to an embodiment of the present invention, wherein each column is sequentially shown from left to right as control, NP-Apt, gefitinib+rapamycin, NP (gefitinib+rapamycin), and NP-Apt (gefitinib+rapamycin).
Detailed Description
For a clearer understanding of the present invention, the present invention will now be further described with reference to the following examples and drawings. The examples are for illustration only and are not intended to limit the invention in any way.
In the examples, each of the starting reagent materials is commercially available, and the experimental methods without specifying the specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.
Example 1 nanoparticles were entrapped with two drugs, gefitinib and rapamycin, and their efficacy was tested in target cells H1975 and non-target cells 293T
The method comprises the following steps:
1) Synthesizing chitosan nanoparticles, chitosan nanoparticles with anti-EGFR nucleic acid aptamer modified on the surface, gefitinib and rapamycin-coated chitosan nanoparticles with anti-EGFR nucleic acid aptamer modified on the surface (NP is the chitosan nanoparticles, NP-Apt is the chitosan nanoparticles with anti-EGFR nucleic acid aptamer modified on the surface, NP (gefitinib+rapamycin) is the gefitinib and rapamycin-coated chitosan nanoparticles, and NP-Apt (gefitinib+rapamycin) is the anti-EGFR nucleic acid aptamer-coated chitosan nanoparticles
Synthesizing gefitinib and rapamycin-coated chitosan nanoparticles and anti-EGFR nucleic acid aptamer-surface-modified chitosan nanoparticles coated with gefitinib and rapamycin:
(1) Gefitinib (added at a concentration of 10 mM) or rapamycin (added at a concentration of 10 mM) was dissolved in 10mL of chitosan solution (chitosan solution at a concentration of 2mg/mL, solvent 1% v/v acetic acid solution, pH=5) and stirred at room temperature for 5min to allow them to be thoroughly mixed.
(2) 3ML of TPP solution was added dropwise (2 mg/mL, deionized water as solvent) to the solution of step (1) while stirring magnetically at 800rpm for 30min at room temperature.
(3) And (3) performing ultrasonic dispersion on the solution obtained in the step (2) for 3min, and centrifuging in a low-temperature high-speed centrifuge for 15min at the temperature of 4 ℃ and the speed of 10,000rpm to obtain a precipitate, namely the gefitinib and rapamycin-entrapped chitosan nanoparticle.
(4) The chitosan nanoparticle precipitate coated with gefitinib and rapamycin was dissolved with 3mL of DEPC water and stored at low temperature.
(5) To the resulting 3mL of gefitinib and rapamycin-entrapped chitosan nanoparticle solution was added an anti-EGFR aptamer (nucleotide sequence shown as SEQ ID NO: 1) (addition concentration 3 nM) and stirred at room temperature.
(6) EDC and sulfo-NHS are prepared into a high-concentration stock solution, and the stock solution is added into the reaction system of the step (5) to respectively obtain the final concentration of 50mM and 5mM, and the mixture is stirred for 3 hours at room temperature.
(7) And (3) performing ultrasonic dispersion on the solution obtained in the step (6) for 10min, and centrifuging in a low-temperature high-speed centrifuge for 15min at a temperature of 4 ℃ and a speed of 10,000rpm.
(8) Discarding the supernatant, dissolving the chitosan nanoparticle coated with gefitinib and rapamycin and surface-modified with anti-EGFR aptamer in PBS, and preserving at low temperature.
The chitosan nanoparticle and the chitosan nanoparticle with the anti-EGFR aptamer modified on the surface are synthesized only by not adding gefitinib and rapamycin in the step (1).
2) Characterization of nanoparticles
The particle size and Zeta potential of the nanoparticles (NP and NP-Apt) were measured by a Markov particle size analyzer. Dispersing NP-Apt in ultrapure water, then dripping on a micro-grid copper net for vacuum drying, and observing the microscopic morphology by using a transmission electron microscope. As a result, as shown in FIG. 1, it can be seen from FIG. 1 that the water and particle size of NP-Apt are distributed at 100-200nm, the electropositive property is achieved, and the actual particle size under electron microscopy is 80-90nm.
3) Targeted uptake of nanoparticles
EGFR-highly expressed lung cancer cell H1975 and 293T cells without EGFR expression are used as research objects.
First, a target cell H1975 with red fluorescence was obtained by cell transformation.
(1) The 293T cells are inoculated in a 60mm dish, the pooling rate of the cells is ensured to reach 50% -60%, and plasmid transfection is carried out 12-18h after inoculation.
(2) Fresh complete medium was changed prior to transfection and 3mL was added to a 60mm dish.
(3) The preparation of virus packaging liquid, the virus packaging using a three-plasmid packaging method, using core plasmid (plasmid with EGFR objective protein gene, 4. Mu.g), packaging plasmid and envelope plasmid (PSPAX. Mu.g, PMD-2G 1. Mu.g), transfection reagent (PEI 24. Mu.L), in the order of RPMI 1640, PEI, plasmid, respectively, and finally, pre-warmed serum-free RPMI 1640 to a total volume of 200. Mu.L.
(4) Standing the liquid obtained in the step (3) at room temperature for 30min to fully polymerize PEI and plasmid DNA, then slightly adding the transfection system dropwise into the small dishes in the corresponding step (2), and continuously culturing in a carbon dioxide incubator at 37 ℃ after shaking gently.
(5) And 4 times of virus stock solutions (namely cell culture solution in a small dish) are continuously collected at 24 hours, 48 hours, 72 hours and 96 hours after transfection, 3mL of fresh complete culture solution is supplemented in the small dish after each time of virus collection, the virus stock solution which is firstly harvested is sealed by a sealing film and then is temporarily stored in a refrigerator at 4 ℃, and the collected viruses for 4 times are mixed together so as to ensure the uniformity of the viruses, and the collected virus stock solution is stored at 4 ℃.
(6) Centrifugation at 1000rpm for 5min to remove cell debris, and filtration with a 0.45 μm filter to remove cell debris and other impurities.
(7) H1975 cells to be infected are seeded one day in advance in the 6-well plate, preferably at a seeding density of 40-50%.
(8) Before virus infection, 1mL of fresh culture medium is used for changing liquid, 2 mu L of polybrene (Polybreen, 10mg/mL and the final working concentration is 10 mu g/mL) is added into the virus liquid obtained in the step (6) and is uniformly mixed, and then the mixture is uniformly dropped into a six-hole plate hole dropwise, and the mixture is gently shaken uniformly.
(9) The liquid was changed 48h after infection, and the infection efficiency was identified under a fluorescence microscope after the liquid change.
(10) After stable expression of red fluorescence was observed, puromycin puromycin,1 μg/mL) was added for screening to remove untransfected cells, obtaining H1975 cells stably expressing mCherry protein.
Next, nanoparticles with green fluorescence were prepared. The NP-Apt prepared in 1) is dissolved in FITC (solvent is PBS) solution with the concentration of 3mM, after incubation for 3 hours at the low temperature of 4 ℃, the supernatant is removed by centrifugation (100000 rpm,4 ℃ for 10 min), the nano particles are collected, and the nano particles are washed by PBS for multiple times, so that the green fluorescent stable modified nano particles are finally obtained.
Next, target cell H1975 with red fluorescence was combined with non-target cell 293T at 1:1 (cell number ratio), and after the cells are attached, adding FITC green fluorescence modified nano particles, and continuously culturing for 3 hours.
Finally, the cells were removed, washed with PBS multiple times to remove the medium, fixed with 4% paraformaldehyde for 10min, stained with DAPI for 5min, and observed under confocal microscope. The target uptake results are shown in fig. 2, and as can be seen from fig. 2, blue fluorescence and red fluorescence are simultaneously used as target cells in the Merge graph, and green nanoparticles are aggregated around the target cells, so that the nanoparticles have the target recognition characteristic, and the targeted drug delivery can be realized.
4) Cytotoxicity of nanoparticles
The NP-Apt is the chitosan nanoparticle with the anti-EGFR nucleic acid aptamer modified on the surface, the NP (gefitinib+rapamycin) is the chitosan nanoparticle with the gefitinib and rapamycin coated, and the NP-Apt (gefitinib+rapamycin) is the chitosan nanoparticle with the anti-EGFR nucleic acid aptamer modified on the surface.
293T cells were selected as negative control cells that did not express EGFR protein. H1975 cells and 293T cells were inoculated into 96-well plates, 5000 cells/well, serum-free medium, NP, NP-Apt, gefitinib+rapamycin, NP (gefitinib+rapamycin), NP-Apt (gefitinib+rapamycin), were added to each group after cell attachment, the concentrations of gefitinib and rapamycin were 2.5. Mu.M and 5. Mu.M, respectively, and the drug concentration entrapped in the nanoparticles was consistent with the individual drug concentration calculated from entrapment rate. After 48h of treatment with each drug, the cell culture medium was removed, 100 μl/well of fresh serum-free medium was added, simultaneously CCK8 solution (10 μl/well) was added, and after further incubation at 37 ℃ for 1h, the absorbance at 450nm was detected with a microplate reader, and cell viability was calculated.
Cytotoxicity of each treatment group on target cells H1975 and non-target cells 293T is shown in fig. 3, and it can be seen from the graph that the killing effect of NP-Apt (gefitinib+rapamycin) group on H1975 in the target cells H1975 is obviously enhanced compared with other groups, which indicates that targeted delivery of gefitinib and rapamycin-coated chitosan nanoparticles with anti-EGFR nucleic acid aptamer modified on the surfaces can enhance the drug effect. Compared with a direct administration treatment group, the killing effect of the NP-Apt (gefitinib+rapamycin) group on 293T cells in a non-target cell 293T graph is obviously reduced, which indicates that the targeted delivery of chitosan nano particles coated with gefitinib and rapamycin, the surfaces of which are modified with anti-EGFR nucleic acid aptamer, can reduce the injury of drugs on the non-target cells.
SEQUENCE LISTING
<110> Shenzhen International research institute at Qinghua university
<120> Nanoparticle drug delivery system targeting EGFR and preparation method thereof
<130> CP121010566C
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 76
<212> DNA
<213> Artificial sequence
<400> 1
taccagtgcg atgctcagtg ccgtttcttc tctttcgctt tttttgcttt tgagcatgct 60
gacgcattcg gttgac 76

Claims (4)

1. A medicament for treating lung cancer, which is characterized by comprising a nanoparticle medicament carrying system for targeting EGFR and a medicament carried by the system;
the nanoparticle drug-loading system for targeting EGFR comprises nanoparticles, wherein the surfaces of the nanoparticles are modified with anti-EGFR nucleic acid aptamer, and the nucleotide sequence of the anti-EGFR nucleic acid aptamer is shown as SEQ ID NO. 1;
the nano particles are chitosan nano particles;
the system-entrapped medicine comprises EGFR tyrosin inhibitor medicines and autophagy pathway related medicines, wherein the EGFR tyrosin inhibitor medicines are gefitinib, and the autophagy pathway related medicines are rapamycin.
2. The medicine for treating lung cancer according to claim 1, wherein the preparation method of the chitosan nanoparticle is as follows: mixing and stirring the sodium tripolyphosphate solution and the chitosan solution, then performing ultrasonic dispersion, and then performing low-temperature centrifugation to obtain the chitosan nanoparticle.
3. The drug for treating lung cancer according to claim 2, wherein the concentration of the sodium tripolyphosphate solution is 2-5mg/mL, and the concentration of the chitosan solution is 0.5-3mg/mL;
the volume ratio of the sodium tripolyphosphate solution to the chitosan solution is 1:3-1:5.
4. The medicine for treating lung cancer according to claim 1, wherein the preparation method of the nanoparticle drug delivery system targeting EGFR comprises the following steps: and modifying the anti-EGFR nucleic acid aptamer on the surface of the nanoparticle by using EDC/NHS amide forming reaction.
CN202111136070.6A 2021-09-27 2021-09-27 EGFR-targeting nanoparticle drug-loading system and preparation method thereof Active CN113855816B (en)

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US10953059B2 (en) * 2015-06-03 2021-03-23 Chang Gung Medical Foundation Keelung Chang Gung Memorial Hospital Methods and compositions for treating non-small cell lung cancer
CN106821985B (en) * 2017-02-28 2020-04-10 福州大学 Aptamer-modified oxygen-carrying and drug-carrying multifunctional liposome compound
WO2019152957A1 (en) * 2018-02-02 2019-08-08 Arizona Board Of Regents On Behalf Of Arizona State University Dna-chimeric antigen receptor t cells for immunotherapy

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JP2017079635A (en) * 2015-10-27 2017-05-18 国立大学法人 熊本大学 Carrier for capturing tumor cell

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"EGFR-targeted photodynamic therapy by curcumin-encapsulated chitosan/TPP nanoparticles";Wen-Hsuan Tsai et al.;《International Journal of Nanomedicine》;第第2018卷卷(第第13期期);第903–916页 *

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