mAb MDR1-modified chitosan nanoparticles overcome acquired EGFR-TKI resistance through two potential therapeutic targets modulation of MDR1 and autophagy
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Tyrosine kinase inhibitors (TKIs) that act against the epithelial growth factor receptor (EGFR) were once widely used in chemotherapy for many human cancers. However, acquired chemoresistance occurred in almost all patients, limiting the clinical application of EGFR-TKI. Thus far, no effective methods existing can resolve this problem. Designing a therapeutic treatment with a specific multi-target profile has been regarded as a possible strategy to overcome acquired EGFR-TKI resistance.
MDR1 antibody-modified chitosan nanoparticles loading gefitinib and autophagy inhibitor chloroquine were prepared by ionic crosslinking and electrostatic attracting method. MTT assay, flow cytometry analysis and western blot assay were all performed to confirm the effect of different formulations of gefitinib on the proliferation of SMMC-7721/gefitinib cells. The preparations demonstrated their multi-target potential to achieve both tumor-targeting selectivity and the desired antitumor effects by blocking cell-surface MDR1 and inhibiting autophagy.
mAb MDR1-modified CS NPs, when combined with the co-delivery of gefitinib and chloroquine, showed targeting and therapeutic potential on enhancing the delivery of anticancer drugs and inducing significant cell apoptosis against acquired EGFR-TKI resistance through the modulation of autophagy and while blocking the activity of the MDR1 receptor.
KeywordsEGFR Tyrosine kinase inhibitor Nanoparticles Gefitinib Autophagy Chloroquine
tyrosine kinase inhibitors
epithelial growth factor receptor
The epidermal growth factor receptor (EGFR) is a membrane-surface protein with tyrosine kinase activity. Studies have shown that it is highly expressed in most cancer patients, and that abnormal EGFR signaling pathways play an important role in tumorigenesis, tumor progression, and metastasis. Tyrosine kinase inhibitors (TKIs) that act against the EGFR (EGFR-TKIs), such as gefitinib, the first selective EGFR-TKI domain, can effectively prevent tumor growth, metastasis [1, 2, 3], and angiogenesis, and promote tumor cell apoptosis [4, 5, 6]. EGFR-TKIs are typically successful in the treatment of malignancies, especially for non-small cell lung cancer [7, 8, 9, 10]. However, after a certain period of drug exposure, tumor cells gradually become insensitive to EGFR-TKIs, ultimately surviving following exposure to chemotherapy drugs. In this way, cells develop acquired chemoresistance, thus significantly reducing the therapeutic effect of EGFR-TKIs and limiting their clinical applications [11, 12, 13, 14].
The occurrence of acquired resistance is very complicated and many reports demonstrate that the overexpression of MDR1 protein and the upregulation of autophagy are mainly attributed to acquired resistance. The MDR1 protein, also known as resistant protein, is primarily located in the cell membrane and its overexpression excretes extracellular chemotherapeutic drugs in tumor cells, resulting in reduced chemotherapeutic effects and insensitivity of drugs to tumor cells. Therefore, the inhibition of MDR1 could prevent the efflux of drugs and improve the efficacy of chemotherapy [15, 16, 17, 18, 19].
In autophagy, autophagosomes are lysed with lysosomes to form autolysosomes that degrade damaged and deformed macromolecules and organelles in the cytoplasm for normal cell survival. A large number of studies have shown that the augment of cell autophagy promoted tumor cell resistance and autophagy inhibition would be a potential target for reversing drug resistance [20, 21, 22, 23]. HSF-1 upregulated Atg7 expression by directly binding to the ATG7 promoter which, in turn, activated autophagy and promoted tumor cell resistance . Activation of reactive oxygen species (ROS)/ERK-mediated protective cell autophagy blocked the occurrence of apoptosis and ultimately led to tumor cell proliferation and a reduction in their sensitivity toward drugs .
Chitosan (CS) with the excellent biocompatibility, low toxicity and higher bioadhesion is a kind of natural cationic polymers, and especially suitable for building nanoparticle system to pass some molecules such as drug compounds, vaccines into cells. The cationic electricity allows CS to combine with some other functional substances having negatively charged ion and results in direct and effective delivery of drugs through the cell surface. Hence, we prepared CS nanoparticles (NPs) conjugated with the monoclonal antibody against MDR1 (mAb MDR1), which is capable of entrapping the anticancer drug, gefitinib, and chloroquine (CQ)—a known inhibitor of autophagolysosome formation—to explore whether EGFR-TKI resistance could be reversed in EGFR-TKI-resistant cancer cells. We used an excellent nanoparticulate drug-delivery system against multiple antitumor targets. The mAb MDR1 modified NPs loaded with gefitinib and CQ (gefitinib/CQ mAb MDR1-NPs) combined with MDR1 receptors which were situated at the surface of SMMC-7721/gefitinib cells (established gefitinib resistant) and they effectively enhanced drug accumulation in these cells, owing to the specific binding between mAb MDR1 and the MDR1 receptor. In addition, when compared with single-treatment therapy that targeting either MDR1 or autophagy, the combination of blocking MDR1 at the cell surface and inhibiting autophagy increased the intracellular accumulation of drugs and restored the cells’ sensitivity to the drugs, thereby reversing acquired EGFR-TKI resistance. Taken together, an excellent nanoparticulate drug-delivery system against multiple antitumor targets was a possible strategy to overcome acquired EGFR-TKI resistance.
Gefitinib was purchased from Eastbang Pharmaceutical Co., Ltd (Guangzhou, People’s Republic of China); Chloroquine, acetic acid and sodium tripolyphosphate (TPP) were obtained from Sigma (St Louis, USA). CS with the deacetylation degree of 80% and molecular weight of approximately 400 kDa was purchased from Haixin Biological Product Co., Ltd (Ningbo, People’s Republic of China). PBS and FBS were purchased from Thermo Fisher Scientific (Shanghai, China). Albumin Bovine V was got from Solarbio Technology Co., Ltd (Beijing, China) and Annexin V-FITC/PI Apoptosis Detection Kit was obtained from BestBio Technology Co., Ltd (Shanghai, China). The antibody used for the research such as MDR1/ABCB1 (E1Y7B) Rabbit mAb (mAb MDR1 we used), p Glycoprotein 1(MDR1), LC3A/B rabbit mAb, and cleaved-caspase3 Rabbit mAb were purchased from Cell Signaling Technology (Boston, USA) and bax, β-actin, cleaved-PARP, goat anti-rabbit lgG secondary antibody HRP were from AbSci (Maryland, USA). Human hepatocellular carcinoma cell lines SMMC-7721/gefitinib cells (established gefitinib resistant) were obtained from Jinzhou Medical University and were maintained at 37 °C in a humidified atmosphere of 5% CO2, 95% air. And other chemicals purchased such as MTT, Rhodamine B, sodium polyphosphate, SDS and glycine were of analytical grade and obtained from Sigma-Aldrich.
Preparation and identification of gefitinib/CQ mAb MDR1-NPs
According to our previous reports [26, 27], CS NPs were prepared by an ionic crosslinking method. CS was dissolved in a solution of glacial acetic acid. When sodium polyphosphate (TPP) was added into the solution, positively charged CS was aggregated around the negatively charged TPP to form NPs. Once any residual was removed by washing the solution with distilled water and centrifugation, the prepared NPs were resuspended and free mAb MDR1 was added. As the CS NPs were positively charged and the mAb MDR1 was negatively charged, mAb MDR1 was modified to the NPs surface by electrostatic attraction. The obtained NPs were precipitated by centrifugation at 16,000 rpm for 20 min, then the NPs were separated and washed with PBS for three times to remove the free antibody. The green fluorescence emitted by mAb MDR1 was observed using a laser confocal microscope to determine whether the mAb MDR1 was conjugated on the surface of the CS NPs. The size, morphology, and zeta potential were characterized by transmission electron microscope (TEM) (JEM-1200EX; JEOL, Tokyo, Japan) and a Zetasizer (Nano ZS90; Malvern Instruments, Malvern, UK). The encapsulation ratios of gefitinib and CQ were measured with an ultraviolet–visible (UV–Vis) spectrometer and the in vitro drug-release behavior in the NPs was studied using a dialysis method with a Mw cutoff 1000, and each experiment was replicated for 3 times.
Distribution and the cellular uptake of NPs
According to our previous reports , before the preparation of NPs, Rhodamine B (RhoB) was dissolved in the CS solution, and with the addition of TPP, RhoB was encapsulated in the core of the NPs to label their traction. A drug-resistant cell line SMMC-7721/gefitinib in a logarithmic growth cycle was seeded into the confocal dishes to reach a cell density of 5 × 105 cells/mL. RhoB-labeled NPs and RhoB-labeled mAb MDR1 NPs were passed through a 0.22 μm filter and added into the confocal dish, and the SMMC-7721/gefitinib cells were incubated with the NPs. The location and distribution of RhoB-labeled NPs in the cells were visualized by confocal laser scanning microscopy (FluoView FV10i; Olympus Corporation, Tokyo, Japan) at given time intervals. The relative fluorescence ratio of NPs (RFR, %) was quantified by calculating the percentage ratio of fluorescence intensity emitted by internalized RhoB-labeled NPs cells to the initial fluorescence intensity from the total added RhoB-labeled NPs. And the process was: cells in full growth media were seeded in a 96-well plate (5 × 104 cells/well) followed by the addition of RhoB-labeled NPs. The fluorescence intensity of the initially added RhoB-labeled NPs was determined by checking the fluorescence intensity of RhoB in each well. At different time interval, cold PBS was used to wash cells to remove the uninternalized NPs, while quantification of intracellular NPs was detected using a microplate reader (Synery-2; BioTek Instruments) by checking the fluorescence intensity of RhoB.
An endocytosis inhibition test was also performed by preincubating the tumor cells with chlorpromazine, genistein, cytochalasin D, and sodium azide for 2 h, followed by the treatment of RhoB-labeled NPs for continuous incubation. The relative uptake ratio was determined by comparing the relative fluorescence ratio of NPs treated with inhibitors with the relative fluorescence ratio of NPs treated with non-inhibitors.
Cells were harvested by trypsin digestion and adjusted to 5 × 105 cells/mL, and they were uniformly added into 96-well plates at a concentration of 100 μL cells per well and placed in a cell incubator for 24 h until they were adhered for extension. After that, according to the protocol of our previous study , free gefitinib; gefitinib and mAb MDR1; gefitinib and CQ; a mixture of gefitinib, CQ, and mAb MDR1; gefitinib NPs; gefitinib/CQ NPs; gefitinib mAb MDR1-NPs and gefitinib/CQ mAb MDR1-NPs, using the same gradient concentration of gefitinib that of 5, 10, 15, 30, 40 μg/mL, were used to treat SMMC-7721/Gefitinib cells for 24 h at 37 °C for further analysis. In all, 10 µL of the 12 mM MTT stock solution was added to each well and incubated for 6 h at 37 °C under 5% CO2 and 95% O2. Then, 50 µL of dimethyl sulfoxide was added into each well and mixed thoroughly with a pipette and incubated at 37 °C for 10 min. The absorbance of the solution was quantified using a BioTek Synergy-2 microplate reader to measure absorbance at 490 nm.
Cell apoptosis evaluated by flow cytometry
An Annexin V (AV)–FITC/propidium iodide (PI) staining assay was performed, and apoptotic and necrotic cells were quantified by flow cytometry. According to the kit introduction and the protocol of our previous study , free gefitinib; gefitinib and mAb MDR1; gefitinib and CQ; a mixture of gefitinib, CQ, and mAb MDR1; gefitinib NPs; gefitinib/CQ NPs; gefitinib mAb MDR1-NPs and gefitinib/CQ mAb MDR1-NPs, featuring the same concentration of gefitinib that of 25 μg/mL, were used to treat SMMC-7721/gefitinib cells for 24 h at 37 °C for further analysis.
Western blot assay
To further evaluate the cell apoptosis effects induced by free drug and drug-loaded NPs with the same concentration of gefitinib, western blot was used as a widely employed analytical technique to detect the expression levels of related proteins. Proteins in the cells were solubilized by employing buffers to promote cell lysis; the proteins were subsequently separated using gel electrophoresis. Further, proteins were transferred from the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF) followed by the blocking of non-specific binding. The film was put into the small box, and 15 mL of 1% BSA was added for the continous incubation for 90 min on the shaking bed. After incubation with a primary antibody and a secondary antibody, enhanced chemiluminescence was stained and the levels of the targeted proteins were photographed and analyzed using a UVP gel analysis system (iBox Scientia 600; UVP, LLC, Upland, CA, USA).
Preparation and determination of the characteristics of gefitinib/CQ mAb MDR1-NPs
The physical and chemical properties of gefitinib/CQ mAb MDR1-NPs, such as particle size, appearance, encapsulation efficiency, and in vitro release, were investigated. The results show that mAb MDR1-NPs exhibited a spherical morphology with a narrow size distribution. The average particle size of the CS NPs and the mAb MDR1-NPs was valued at 96.3 ± 6.3 and 103.8 ± 11.3 nm, respectively. The average zeta potentials were 22.6 ± 4.4 and 15.4 ± 6.4 mV, respectively. The encapsulation efficiency of gefitinib and CQ in mAb MDR1-NPs was 85.6% ± 9.2% and 88.7% ± 5.8%, respectively. The polydispersity index of the mAb MDR1-NPs was at about 0.14 ± 0.06. The in vitro release of gefitinib and CQ in both the CS NPs and mAb MDR1-NPs, as conducted by the dialysis bag method, showed a similar biphasic release. The accumulative release rates of gefitinib in both CS NPs and mAb MDR1-NPs had increased gradually from approximately 25.6 and 22.3%, respectively, in the initial 4 h to over 87.6 and 83.4% at 24 h and, finally, they were slightly enhanced to 97.6 and 95.4% at 48 h. The CQ encapsulated in the NPs depended on the variation of time required to control the release. The in vitro release percentage of CQ in the CS NPs and mAb MDR1-NPs were 18.9 and 16.7%, respectively, within the first 4 h, and about 74.3 and 71.3% of CQ were completely released within 24 h. Finally, more than approximately 82.3 and 79.8% of the total CQ slowly leaked out from the CS NPs and mAb MDR1-NPs into the medium within 48 h.
Distribution and the cellular uptake of NPs
Quantitative analysis of the cellular uptake of NPs
Cell apoptosis and necrosis
Western blot assay
Gefitinib/CQ mAb MDR1-NPs were successfully prepared and characterized by their smaller particle size, positive zeta potential, higher drug encapsulation, and controllable release pattern. With the mediation of MDR1 receptors overexpressed on the cell surface as a specific targeting site, mAb MDR1 located at the surface of the NPs quickly bound to the MDR1 receptor to promote the internalization of NPs and to enhance the intracellular concentration of the drugs. The RFR (%) of both the CS NPs and mAb MDR1-NPs had increased gradually from approximately 26.7 and 35.7%, respectively, in the initial 3 h to over 56.2 and 69.8% at 6 h. Furthermore, by blocking MDR1 by mAb MDR1, the activity of MDR1 was inhibited and the efflux of the drug via MDR1 was reduced; therefore, gefitinib/CQ mAb MDR1-NPs greatly facilitated gefitinib uptake by effectively transporting NPs into the cells. The results further confirmed that as autophagy was inhibited by downregulating the ratio of LC3 II and LC3 I in the presence of CQ-loaded NPs, cell viability was significantly decreased further, and the greatest cell apoptosis effects were induced through the efficient prohibition of autophagy. Taken together, gefitinib/CQ mAb MDR1-NPs induced the strongest cell inhibition effects. The IC50 value in gefitinib/CQ mAb MDR1-NPs-treated, SMMC-7721/gefitinib cells within 24 h was 22.3 μg/mL and the ratio of AV positive cells treated with gefitinib/CQ mAb MDR1-NPs was 56.0%. Western blot assay was also performed to determine the level of related proteins to clarify the role of autophagy and the expression of MDR1 proteins on overcoming acquired drug resistance. The results showed that after being treated with gefitinib/CQ mAb MDR1-NPs, mAb MDR1 efficiently suppressed the expression of the MDR1 protein, while CQ significantly inhibited autophagy by downregulating the ratio of LC3 II and LC3 I; therefore, the expression of apoptosis-related proteins such as bax, cleaved caspase-3, and cleaved parp was significantly up-regulated. All of these results showed that a higher level of autophagy—as a protective mechanism—and the overexpression of MDR1 could participate in the induction of tumor-acquired resistance in tumor cells. To determine whether the overexpression of MDR1 and the augmentation of autophagy contributed to cell death, we manipulated autophagic activity using an autophagy inhibitor (CQ) and mAb MDR1 targeting MDR1. We found that the reversal activity of gefitinib-acquired resistance was significantly exacerbated in the presence of CQ, which inhibited autophagy and blocked the expression of MDR1. These results suggested that CQ was capable of reversing MDR; the induction of autophagy represented a defense mechanism, and inhibiting this process may be an effective strategy to augment the reversal activity on overcoming acquired EGFR-TKI resistance. In addition, mAb MDR1 greatly facilitated gefitinib uptake by effectively transporting NPs into cells and enhancing the cytotoxic effect in SMMC-7721/gefitinib cells upon mediation of mAb MDR1 to suppress MDR1 expression.
Briefly, we found that mAb MDR1-modified CS NPs, when combined with the co-delivery of gefitinib and CQ, showed targeting and therapeutic potential on enhancing the delivery of anticancer drugs and inducing significant cell apoptosis against acquired EGFR-TKI resistance through the modulation of autophagy and blocking the activity of MDR1. This finding implied that autophagy and MDR1 may serve as dual targets to overcome acquired resistance in SMMC-7721/gefitinib cells, and the clinical application of autophagy and MDR1 inhibition might be one of the important strategies for overcoming acquired EGFR-TKI resistance in SMMC-7721/gefitinib cells.
YZ and CS performed the preparation and characteristics of the NPs; and CS helped with the biological study. LZ and YS supervised the whole work and helped in the analysis of biological data. All authors read and approved the final manuscript.
This work was supported by the National Natural Science Foundation of China (No. 81641167). English-language editing of this manuscript was provided by Journal Prep.
The authors declare that they have no competing interests.
Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.
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This work was supported by the National Natural Science Foundation of China (No. 81641167) in the design of the study and analysis of data.
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