ADR by Anti-miR-21

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Reverse multidrug resistance in human HepG2/ADR by anti-miR-21 combined with hyperthermia mediated by functionalized gold nanocages Weiping Wang, Shengnan Huang, Jinxiu Yuan, Xin Xu, Huili Li, Zhanwei Lv, Wei Yu, Shaofeng Duan, and Yurong Hu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00046 • Publication Date (Web): 02 Jul 2018 Downloaded from http://pubs.acs.org on July 4, 2018

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Molecular Pharmaceutics

Reverse multidrug resistance in human HepG2/ADR by

anti-miR-21

combined

with

hyperthermia

mediated by functionalized gold nanocages Weiping Wang,‡,† Shengnan Huang,‡,† Jinxiu Yuan,† Xin Xu,† Huili Li,† Zhanwei Lv,† Wei Yu,† Shaofeng Duan,*,§,⊥Yurong Hu*,†,#

†

Henan Province Key Laboratory of Targeting Therapy and Diagnosis for Critical Diseases, School of Pharmaceutical Sciences, Zhengzhou University, No. 100 Science Avenue, Zhengzhou, Henan 450001, P. R. China.

§

College of Pharmacy, Henan University, Jin Ming Avenue, Kaifeng, Henan 475004, P. R. China.

⊥

Department of Orthopedics, the First Affiliated Hospital of Zhengzhou University, 1 Construction Road, Zhengzhou 450052, P. R. China.

#

Key Laboratory of Key Technology of Drug Preparation, Ministry of Education, Institute of Drug Discovery & Development, Zhengzhou University, No. 100 Science Avenue, Zhengzhou 450001, P. R. China.

ABSTRACT: Multidrug resistance (MDR) remains a formidable challenge to effective clinical cancer therapy. Herein, a non-viral gene delivery system HA/anti-miR-21/PPAuNCs to overcome MDR was reported. This system could condense microRNA-21 inhibitor (anti-miR21) into hyaluronic acid (HA)-conjugated and polyethyleneimine (PEI)-modified PEGylated

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gold nanocages (AuNCs) and had good stability. In vitro studies demonstrated that HA/anti-miR21/PPAuNCs could enhance intracellular DOX accumulation in DOX resistant HCC cells (HepG2/ADR cells) and increase the sensitivity to DOX of HepG2/ADR cells through upregulating PTEN protein expression mediated by anti-miR-21 and upregulating P-gp protein expression mediated by the hyperthermia of HA/PPAuNCs upon mild near infrared (NIR) irradiation. Furthermore, the therapeutic effects had been enhanced due to the combination of chemotherapy, gene therapy, and photothermal therapy (PTT). Besides, HA/anti-miR21/PPAuNCs had good biocompatibility. These findings would provide new insights and strategies for the treatment of cancers with MDR. KEYWORDS: multidrug resistance; microRNA-21 inhibitor; gold nanocages; PTEN protein; Pgp protein

1 INTRODUCTION Hepatocellular carcinoma (HCC) has been reported as one of the most malignant cancers with high incidence and mortality in humans.1,2 HCC continues to remain one of the major global health challenges because of multidrug resistance (MDR) of chemotherapeutic agents, which is considered to be a major impediment of successful chemotherapy.3-5 Therefore, a new treatment model that could reverse multidrug resistance is urgently needed for HCC treatment.

The combination of hyperthermia and chemotherapy usually leads to an improved anticancer efficiency.6-9 Hyperthermia increases the sensitivity of therapeutic drugs against cancer cells so that it could be beneficial to cure tumors with resistance to chemotherapeutic agents.10,11 However, due to the limitation of the available technology, very few satisfactory results have


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been obtained.12,13 Tremendous studies have shown that the initial exposure to chemotherapeutic agents such as Taxol could induce the apoptosis, and even the death of most cancer cells; however, with the extension of treatment time, cancer cells could become antagonistic to chemotherapeutic drugs.10,14,15 Hence, multiple administration may cause cancer cells to lose sensitivity not only to Taxol but also to various other chemotherapeutic agents, which is called multidrug resistance (MDR).16,17 Considerable research work has been reported that Pglycoprotein (P-gp) was overexpressed in diverse MDR cell lines and could employ the energy of ATP hydrolysis to export the unrelated hydrophobic molecules out of the cell membrane, which is the main reason for the decrease of accumulation of chemotherapeutic drugs in MDR cells.18,19 It has also been reported that hyperthermia could reduce the expression of P-gp.20

Much work so far has demonstrated that MicroRNAs (miRNAs) acted as regulators of Pgp gene expression in various cancer types and played an important role in reversing MDR.21-23 MiRNAs served as a kind of endogenous single stranded RNA molecules, which are corrected with the progresses of cytodifferentiation, proliferation, apoptosis, anti-viral defense of cancer.24,25 Recently, it has been reported that downregulation of miRNA expression exerted essential roles in inhabiting tumor progression and invasion.23,26 They could also have significant effect on the sensitivity of tumor cells to chemotherapeutic drugs.27 Among them, microRNA-21 (miR-21), an important crucial oncogenic miRNA, had the advantages of promoting cell invasion by regulating various genes, including programmed cell death 4 (PDCD4) gene, tissue inhibitor of metallopeptidase inhibitor 3 (TIMP3), phosphatase and tensin homolog (PTEN) gene in a variety of tumors, such as lung cancer, glioblastoma, and breast cancer.28,29 Recent studies have reported that the expression level of PTEN was related to miR-21, which facilitated the activation of phosphatidylinositol 3-kinase (PI3K)/Akt pathway by restraining PTEN,30-32 while


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the high activity of Akt and its downstream factors induced insensitivity of drug-resistant cells to chemotherapy drugs.33,34 As aforementioned, the sensitivity of cells to chemotherapeutic drugs may be increased by miRNA-21 inhibitor (anti-miR-21). In this study, we developed a novel gene delivery vector, hyaluronic acid (HA)-modified, polyethylenimine (PEI)-conjugated PEGylated gold nanocages (AuNCs), which was designated as HA/PPAuNCs. This system possessed relative high photothermal conversion efficiency upon near infrared (NIR) laser irradiation, low cytotoxicity, and high targeted delivery ability. Moreover, anti-miR-21 could be condensed by PEI onto the surface of HA/PPAuNCs and then delivered into target cells. Therefore, HA/anti-miR-21/PPAuNCs might effectively overcome DOX resistance of HCC and provide new insights for cancer treatment.

2 MATERIALS AND METHODS 2.1 Materials Chloroauric acid (HAuCl4·3H2O, 99.9%), polyethyleneimine (PEI, MW ≈ 1,800), 4', 6diamidino-2-phenylindole (DAPI), 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), Silver nitrate (AgNO3, exceeding 99%) , poly vinyl pyrrolidone (PVP, Mw ≈ 55,000) and 11-mercaptoundecanoic acid (MUA) were all purchased from Sigma-Aldrich (Boston, MA, USA). P-gp Rabbit mAb, PTEN Rabbit mAb, beta actin (β-actin) antibody were purchased from You Ningwei Biological Technology Co., Ltd (Shanghai, China). Lipofectamine 2000 (Life Technologies Corporation). microRNA inhibitor negative control (anti-miR-NC, 5’CAGUACUUUUGUGUAGUACAA-3’)

and

MicroRNA-21

inhibitor

(anti-miR-21,

5’-

UCAACAUCAGUCUGAUAAGCUA-3’) were purchased from Gene Pharma Co., Ltd (Shanghai, China).


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2.2 Synthesis and characterization of HA/miRNA/PPAuNCs According to the previous reports,35,36 we first prepared AuNCs and then conjugated functionalized agents onto the nanoparticles to obtain PEI-modified PEGylated gold nanocages (PPAuNCs). Next, PPAuNCs were mixed with miRNAs (anti-miR-21 or anti-miR-NC) in an equivalent volume with different molar ratios (AuNCs/miRNA = 1:4000, 1:3600, 1:3200, 1:2800, 1:2400 and 1:2000), followed by incubation at 37 ℃ for 30 min to afford miRNA/PPAuNCs, which were then performed with agarose gel electrophoresis assay to achieve the optimum molar ratio. Then HA solution (0.25 mg/mL) was added to the reaction mixture based on the molar ratio of PPAuNCs to HA as 1:2 and the incubation was continued for another 30 min to acquire HA/miRNA/PPAuNCs (HA/anti-miR-21/PPAuNCs or HA/miR-NC/PPAuNCs). The morphology of the HA/ miRNA/PPAuNCs was characterize by Transmission electron microscopy (TEM, FEI Tecnai 20, Netherlands), and the optical properties of the nanocomplexes were examined by employing an UV-vis-NIR spectrometer. The mean particle diameter and Zeta potential of the HA/miRNA/PPAuNCs complexes were determined on a Malvern Nano ZS90 instrument with DTS software (Malvern, Worcestershire, UK) using the DTS (nano) program. To investigate the photo-thermal conversion efficiency of PPAuNCs, PPAuNCs solutions at 0.01 and 0.03 nM were treated with 808 nm NIR laser irradiation at 1.25, 1.5 and 2.0 W/cm2 for 10 min respectively. Meanwhile, the temperature changes were monitored by a digital thermometer. Ultrapure water upon 808 nm NIR irradiation with the power density of 2.0 W/cm2 for 10 min was used as a control. Hemolysis assay: Red blood cells (RBCs) were extracted from heparin sodium-stabilized


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human whole blood by centrifugation. We re-disperse the pure RBCs in 10 mL PBS for the following test. Thereafter, we mixed 200 µL of the 2% RBCs solution with 800 µL of HA/miRNA/AuNCs NPs at different concentrations (0.005, 0.01, 0.02 and 0.05 nM) and incubated at 37℃ for 4 h in swing bed with shaking at a speed of 150 rpm. Meanwhile, the PBS was considered as negative control and water were employed as positive control respectively. After incubation, the solution was centrifuged at 4,000 rpm for 5 min and 200 µL of supernatant was transferred to a 96-well plate. Then, we applied a Tecan microplate reader (Dynex, USA) to detect absorbance of the supernatant at 540 nm and calculated the percent hemolysis using the following formula:

For investigating the stability, the HA/miRNA/PPAuNCs NPs were stored in water at 4℃ for 15 days . At each time point (0, 3, 5 and 15 d), the zeta potential and the mean hydrodynamic particle size of the nanocomplexes were tested in triplicate using DLS. 2.3 Cell culture DOX resistance of human hepatocellular carcinoma (HepG2/ADR) cells and HepG2 cells were provided by the Shanghai Institute of Biochemistry and Cell Biology, CAS (Shanghai, China). The cells were cultured in DMEM containing 10% fetal bovine serum (FBS). We added DMEM medium containing DOX (1.0 µg/mL) to preserve the drug resistance of HepG2/ADR cells. 2.4 Cell uptake of DOX HepG2/ADR cells and HepG2 cells with a density of 6.0 × 105 cells/well were inoculated a


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6-well plate respectively and cultured for 24 h with fresh complete medium prior to transfection. Then, the HepG2 and HepG2/ADR cells were divided into four groups: (1) blank control (culture medium without DOX) group, (2) negative control (free DOX) group, (3) HA/anti-miR21/PPAuNCs + DOX group and (4) HA/anti-miR-21/PPAuNCs + NIR + DOX group. The cells treated with HA/anti-miR-21/PPAuNCs or HA/anti-miR-21/PPAuNCs with NIR laser irradiation were exposed to DOX (2.85 µg/mL) for 6 h. The final concentrations of PPAuNCs and anti-miR21 were 0.02 nM and 48 nM respectively. The NIR laser irradiation (1.25 W/cm2, 10 min) was performed at 4 h post-incubation. After that, the cells were washed three times with PBS and 1 mL culture medium was added before images were taken under a fluorescence microscope (Nikon Eclipse 50i POL, Japan). Quantitative analysis was performed as well. The cells were collected and lysed with 200 µL cell lysates after incubation for 6 h. Then we determinated the total protein concentration of 25 µL cell segments from each well by a BCA assay. The rest of the cell lysates were collected for HPLC assay of Dox concentration. The intracellular DOX was finally described as the DOX amount per unit cell protein (µg DOX/mg cell protein). 2.5 Cell viability assay HepG2/ADR cells and HepG2 cells with a density of 1.0 ×104 cells/well were inoculated in a 96well plate, respectively. Then, the HepG2 cells and HepG2/ADR cells were divided into five groups after incubation for 24 h: (1) blank control (culture medium without DOX) group , (2) DOX solution (free DOX) group, (3) HA/anti-miR-21/PPAuNCs group, (4) HA/anti-miR21/PPAuNCs + DOX group and

(5) HA/anti-miR-21/PPAuNCs + NIR + DOX group,

respectively. The cells incubated with HA/anti-miR-21/PPAuNCs or HA/anti-miR-21/PPAuNCs with NIR laser irradiation were exposed to DOX (2.85 µg/mL) for 6 h. The final concentrations


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of PPAuNCs and anti-miR-21 were 0.02 nM and 48 nM respectively. The NIR laser irradiation (0.75 W/cm2, 10 min) was performed at 4 h post-incubation. The cells were incubated with MTT solution for another 4 h after incubation for 48 h. Thereafter, DMSO was added to lysis the resultant formazan crystals when the cell culture medium was discarded. The absorbance value of each group was measured at 490 nm. Cell viability (%) = experimental group absorbance value/control group absorbance value × 100%. 2.6 Cells apoptosis assay HepG2/ADR cells and HepG2 cells with a density of 6.0 × 105 cells/well were inoculated at 6well plate respectively and cultured for 24 h prior to transfection. Experimental grouping and processing were the same as those of 2.4. After incubation for 48 h, flow cytometry was applied to detect the apoptosis of cells and CFlow Plus software was used to perform the data analysis. Besides, a fluorescence microscopy was employed to investigate the cells after different treatments. 2.7 Western blotting assay for P-gp and PTEN protein P-gp assay: HepG2/ADR cells and HepG2 cells with a density of 4.0×105 cells/well were inoculated in a 6-well plate respectively. After incubation for 24 h, the cells were rinsed three times with PBS. Experimental grouping and processing were the same as those of 2.4. After incubation for 48 h, the cells were rinsed three times with PBS and then homogenized and centrifuged to remove the debris. Proteins were then analyzed on soldium dodecyl sulfatepolyacrylamide (SDS-PAGE) gels, and electrophoresis was transformed to cellulose nitrate filter membranes (NC). NC were blocked with 5% (w/v) evaporated skimmed milk in the TBST buffer at 37 ℃ for 2 h, followed by incubation with the cardinal antibody named P-gp Rabbit mAb


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overnight. The corresponding secondary Abs (goat anti-mouse) was then incubated with the NC in the dark at 37 ℃ for 2 h. They were both exposed to enhanced chemiluminescence (ECL) and imaged software was used to detect the band density and normalized to that of β-actin from the same cells. PTEN protein assay: HepG2 cells and HepG2/ADR cells were inoculated in a 6-well plate and incubated for 24 h, respectively. Experimental grouping and processing were the same as those of 2.4. After incubation for 48 h, the subsequent western blotting assay was conducted in the same way as mentioned above except that PTEN Rabbit mAb was used as the cardinal antibody. 2.8 In vivo therapeutic effect of HA/anti-miR-21/PPAuNCs All animal experiments satisfied requirements of the "Regulations on management of national animal experiment (Chinese 1988)", "animal treatment guidelines (2006 China)" and the Zhengzhou Life Science Ethics Committee Review Board (ERB) rendered a license. Firstly, the back of the female BALB/c nude mouse (20 ± 2 g) was inoculated with 5.0 × 106 HepG2/ADR cells in 100 µL PBS containing 0.2% BSA. Fourteen days later, we randomly divided the mice into five groups (n = 6 per group): DOX solution, HA/anti-miR-21/PPAuNCs, DOX + HA/anti-miR-21/PPAuNCs, DOX + HA/anti-miR-21/PPAuNCs + NIR and normal saline (control). The DOX + HA/anti-miR-21/PPAuNCs + NIR group received i.v. administration at an anti-miR-21 dose of 1.6 mg/kg (~0.032 mg per animal) and a DOX dose of 5 mg/kg (~0.10 mg per animal) every three days for a total of six doses in each nude mouse. At 6 h post injection, the tumor was irradiated by laser (0.75 W/cm2, 808 nm, 10 min). The localized temperature changes of the tumor were recorded by an NIR thermal camera during the NIR


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irradiation. The free DOX, HA/anti-miR-21/PPAuNCs and DOX + HA/anti-miR-21/PPAuNCs groups received i.v. injections at the same doses of anti-miR-21 and DOX every three days for 18 days. The volume of tumor and weight of mice were measured and recorded respectively. The tumor volume was computed on the basis of the formula: V = (L×W2)/2 (L and W, on behalf of the long diameter and short diameter of the subcutaneous tumors respectively). 2.9 Statistical analysis All researches were carried out in triplicate unless otherwise indicated. Statistical analysis was conducted using GraphPrism 5.0 software. The statistically significant difference(s) between two or more groups were assessed by Students’t-test. p value of 0.05 was selected as the cut off for statistical significance.

3 RESULTS AND DISCUSSION 3.1 Preparation and characterization of HA/miRNA/PPAuNCs AuNCs were synthesized with the method described previously.35,37 The particle diameter of AuNCs was 50 nm confirmed by TEM image analysis (Figure 1A). The slight red-shift of the UV-vis-NIR spectrum of HA/anti-miR-21/PPAuNCs in comparison with those of AuNCs and MUA-AuNCs demonstrated the changes of the surface of AuNCs and the successful construction of HA/anti-miR-21/PPAuNCs (Figure 1B). MiRNAs were condensed by electronic interaction and agarose gel electrophoresis was used to evaluated the condensation efficiency. As shown in Figure 1C, when the molar ratio of the nanoparticle to gene reached 1: 2400 and 1:2000, the miRNAs were completely condensed by the nanoparticle. And 1: 2400 was selected as the optimum molar ratio of PPAuNCs to miRNAs for the following experiments. Finally, the


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diameters and zeta potentials of the complexes were determined on a Malvern Nano ZS90 instrument (Figure 1D, E), showing that the average hydrodynamic size and the surface charge of the HA/miRNA/PPAuNCs were 150.45 ± 2.4 nm and -12.11 ± 0.3 mV, respectively. The successful formation of a layer-by-layer assembled HA miRNA /PPAuNCs complex was supported by all these results. Subsequently, we tested the photo-thermal conversion property of HA/anti-miRNC/PPAuNCs of two different concentrations (0.01 and 0.03 nM) upon NIR irradiation with three different power densities (1.25, 1.5 and 2.0 W/cm2). As seen in Figure 1F, the temperatures of HA/anti-miR-NC/PPAuNCs solutions upon NIR laser irradiation at 1.25 W/cm2 elevated from 25 °C to 39.9 °C (∆T = 14.9 °C, 0.01 nM) and 46.1 °C (∆T = 21.1 °C, 0.03 nM). When the density was 1.5 W/cm2, the temperatures increased to 41.8 °C (∆T = 16.8 °C, 0.01 nM) and 48.0 °C (∆T = 23.0 °C, 0.03 nM). The temperatures reached 45.2 °C (∆T = 20.2 °C, 0.01 nM) and 51.7 °C (∆T = 26.7 °C, 0.03 nM) when the density was 2.0 W/cm2. In sharp comparison, the temperature of nanoparticle-free ultrapure water only increased by 2.0 °C even after 10 min irradiation. HA/anti-miR-NC/PPAuNCs exerted superior photothermal conversation property, which supplied a good basis for the subsequent PTT . Hemolysis is a crucial toxicological factor that should be considered for the in vivo study of the HA/miRNA/PPAuNCs, so we performed hemolysis assay in this study. As shown in Figure S1, HA/miRNA/PPAuNCs NPs of different concentrations exhibited almost negligible hemolysis which were lower than 0.2% hemolysis. The results demonstrated non-hemolytic properties of the HA/miRNA/PPAuNCs NPs and their safety for in vivo application. The hemocompatibility of the HA/miRNA/PPAuNCs NPs may be ascribed to their negative surface charge and the good biocompatibility of both PEG and HA.


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Stability of HA/miRNA/PPAuNCs NPs is very important for efficient drug delivery, so we tested the average zeta potential and hydrodynamic size of HA/miRNA/PPAuNCs NPs storing at 4 °C for different time. The results were shown in Table S1 and demonstrated good stability at each time point (0, 3, 5 and 15 d).

Figure 1. Physicochemical characterization of the HA/miRNA/PPAuNCs nanocomplexes. (A) Representative TEM image of the HA/miRNA/PPAuNCs. (B) UV–Vis spectra of the AuNCs, AuNCs-MUA and HA/antimiR-21/PPAuNCs. (C) Gel electrophoresis assay of the condensation efficiency of HA/PPAuNCs to anti-miR21 at different molar ratio. Lane 1: naked anti-miR21 (control), Lane 2 to Line 7: molar ratio of the anti-miR-


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21 to HA/PPAuNCs = 4000: 1, 3600:1, 3200:1, 2800:1, 2400:1 and 2000:1. (D) Hydrodynamic size of the HA/miRNA/AuNCs. (E) Zeta potential of HA/miRNA/AuNCs nanoparticles. (F) Temperature changes of HA/anti-miR-NC/PPAuNCs suspensions under NIR irradiation for 10 min and water upon NIR irradiation at the density of 2.0 W/cm2 was used as a control.

3.2 DOX cell uptake The results of the cellular uptake and internalization of DOX in HepG2 cells and HepG2/ADR cells suggested that the fluorescence intensity of DOX in HepG2 cells increased obviously in group b, c and d. However, in comparison with HepG2 cells, HepG2/ADR cells almost had no fluorescent signal when they treated with free DOX, respectively. Nevertheless, after HepG2/ADR cells were treated with HA/anti-miR-21/PPAuNCs or HA/anti-miR21/PPAuNCs + NIR, the DOX cellular uptake was gradually improved. The reason is possibly that HA/anti-miR-21/PPAuNCs could moderately reverse the drug resistance of the HepG2/ADR cells, and hyperthermia also improved the reverse efficiency. Accordingly, HA/anti-miR21/PPAuNCs upon NIR irradiation was more conducive to the uptake of DOX (Figure 2). We performed quantitative analysis to further investigate the cellular uptake of DOX in the presence of HA/anti-miR-21/PPAuNCs with or without NIR laser irradiation. As seen in Figure S2, after incubation with DOX for 6 h, the intracellular DOX concentration in the HepG2/ADR cells was less than half of that in the HepG2 cells, which confirmed the presence of MDR in HepG2/ADR cells. HA/anti-miR-21/PPAuNCs improved the intracellular DOX accumulation in the HepG2/ADR cells, which was nearly 2.5 times that of the DOX group. However, HA/antimiR-21/PPAuNCs had not significant effect on the HepG2 cells. Interestingly, the intracellular DOX concentrations in both the HepG2/ADR cells and HepG2 cells were enhanced by the treatment of HA/anti-miR-21/PPAuNCs upon irradiation in comparison with the DOX group.


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The concentration of DOX in the HepG2/ADR cells treated with the HA/anti-miR-21/PPAuNCs + NIR + DOX group was nearly 5 times that of the DOX group. In contrast, the concentration of DOX of the HA/anti-miR-21/PPAuNCs + NIR + DOX group was only 1.9 times that of the DOX group in the HepG2 cells. All of these results demonstrated that HA/anti-miR21/PPAuNCs could reverse MDR in HepG2/ADR cells and HA/anti-miR-21/PPAuNCs upon irradiation was more effectively for reversing MDR in HepG2/ADR cells.

Figure 2. DOX uptake was detected in HepG2 cells and HepG2/ADR cells (magnification: 200). After the cells were treated for 6 h with (a) culture medium (blank control), (b) free DOX, (c) HA/anti-miR-21/PPAuNCs + DOX, (d) HA/anti-miR-21/PPAuNCs + DOX + NIR (1.25 W/cm2, 10 min). The fresh complete medium or DOX (2.85 µg/mL) were added to (a) or (b) (c) (d) respectively and incubated for 6 h. The concentrations of anti-miR-21 and PPAuNCs were 48 nM and 0.02 nM, respectively.

3.3 Cell viability The anti-proliferative effect of the HA/anti-miR-21/PPAuNCs and hyperthermia on HepG2/ADR cells and HepG2 cells was measured using MTT assay. After treatment with free DOX of the same concentration, the cell viabilities of HepG2/ADR cells and HepG2 cells were 73.07 ± 3.87% and 56.32 ± 5.02% respectively, suggesting that HepG2/ADR cells were less


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sensitive to free DOX than HepG2 cells (Figure 3A). But on the contrary, the HepG2/ADR cells and HepG2 cells treated with HA/anti-miR-21/PPAuNCs showed similar cell viabilities (75.98 ± 2.94% and 73.70 ± 3.87%, p = 0.38). In addition, the HepG2/ADR cells treated with HA/antimiR-21/PPAuNCs + DOX achieved a much better anti-tumor effect with a cell viability of 45.65 ± 3.86% than those of free DOX and HA/anti-miR-21/PPAuNCs groups. However, the HepG2/ADR cells treated with HA/anti-miR-21/PPAuNCs + DOX showed a higher cell viability than that of HepG2 cells (30.74 ± 1.53%). The anti-cancer efficiency of HA/anti-miR21/PPAuNCs+ NIR + DOX was further enormously enhanced against either HepG2 cells (30.11 ± 1.22%) or HepG2/ADR cells (40.11 ± 1.11%). This result may be owing to the synergistic effect of anti-miR-21(gene therapy) ， DOX (chemotherapy) and AuNCs (hyperthermia mediated) by HA/anti-miR-21/PPAuNCs under NIR (photothermal therapy) and reversal of the DOX resistance in the HepG2/ADR cells . To test the anti-tumor efficiency of different therapies on HepG2/ADR cells and HepG2 cells, the cell morphology was further investigated. In HepG2 cells, there was no remarkable discrepancy in cell morphology between the HA/anti-miR-21/PPAuNCs group and the control group, indicating the moderate cytotoxicity of anti-miR-21. Compared with the control group, the cell morphology of the HA/anti-miR-21/PPAuNCs + DOX and HA/anti-miR-21/PPAuNCs + DOX + NIR groups was significantly changed (Figure 3B). Moreover, the density of the cells treated with HA/anti-miR-21/PPAuNCs + NIR + DOX was much lower than that of the control group, which was not contradicting the result of the cell viability test. Therefore, the HA/anti-miR-21/PPAuNCs not only were successfully delivered into drugresistant cells, but also reduced the drug resistance in them. More interestingly, in the


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HepG2/ADR cells, the hyperthermia mediated by HA/anti-miR-21/PPAuNCs under mild NIR improved the cellular uptake of DOX and synergistically reversed the drug resistance.


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Figure 3. (A) Cell viability of HepG2/ADR cells and HepG2 cells treated with different ways. **p＜0.01 and *

p＜0.05 represented significant fifferences between HepG2/ADR cells and HepG2 cells, ##p＜0.01 and #p＜

0.05 represented significant distinction among the groups in HepG2/ADR cells. (B) Representative images of HepG2/ADR cells and HepG2 cells treated with different ways (magnification: 200). (C) Apoptosis analysis of HepG2/ADR cells and HepG2 cells treated with different ways. In group of A, B and C: (a) culture medium (blank control), (b) free DOX, (c) HA/anti-miR-21/PPAuNCs, (d) HA/anti-miR-21/PPAuNCs + DOX, (e) HA/anti-miR-21/PPAuNCs + NIR + DOX. The concentrations of DOX, PPAuNCs and anti-miR-21 were 2.68 µg/mL, 0.02 nM and 48 nM, respectively. The NIR condition was 808 nm, 2.0 W/cm2 for 10 min.

3.4 Apoptosis analysis To assess the therapeutic effect of anti-miR-21 on HepG2/ADR cells and HepG2 cells, the cells were treated with DOX solution (free DOX), culture medium (blank control), HA/anti-miR21/PPAuNCs, HA/anti-miR-21/PPAuNCs + DOX, HA/anti-miR-21/PPAuNCs + NIR + DOX, respectively (Figure 3C). The experimental results showed that the apoptosis rates of HepG2 cells cultured with free DOX or HA/anti-miR-21/PPAuNCs were 45.1% and 26.5% respectively, while the apoptosis rates of HepG2 cells treated with HA/anti-miR-21/PPAuNCs + DOX and HA/anti-miR-21/PPAuNCs + DOX + NIR significantly increased to 70.6% and 74.9% respectively. These results implied that combination of DOX with anti-miR-21 effectively improved cell apoptosis, namely, the gene therapy combined with the chemotherapy showed a synergistic effect. It’s well known that cell resistance is mainly reflected in two aspects: one is the high expression of p-glycoprotein (p-gp) on the surface of the cell membrane that can discharge the chemotherapeutic agent. The other one is that the intracellular apoptosis pathways are disturbed and the cell apoptosis is prevented. For example, PTEN protein with low expression contributes


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to cell proliferation. The apoptosis rates of HepG2/ADR and HepG2 cells treated with free DOX were 32.1% and 45.1% respectively. As for the HA/anti-miR-21/PPAuNCs + DOX and HA/antimiR-21/PPAuNCs + NIR + DOX groups, the apoptosis rates of HepG2/ADR cells increased to 55.4% and 62.3%, respectively, verifying the synergistic effect of the combined therapy. 3.5 Western blot analysis As shown in Figure 4A, the expression of p-gp in HepG2 cells (64.63 ± 5.07%) was lower than that in HepG2/ADR cells (101.78 ± 6.96%), which further confirmed the resistance of HepG2/ADR cells. Moreover, in HepG2/ADR cells, the irradiation with 808 nm NIR enormously downregulated the expression of p-gp protein to 30.28 ± 1.64%, but not in HepG2 cells (72.93 ± 7.11%), proving that hyperthermia could reverse the sensitivity of drug-resistant cells to DOX (Figure 4C). AuNCs upon mild NIR laser irradiation downregulated the expression of drugresistance-related P-gp, resulting in enhanced intracellular DOX accumulation, which was in agreement with the results reported by Wan, Dong, Wang and so on.38-40 In order to further demonstrate the growth inhibition effect induced by DOX and HA/antimiR-21/PPAuNCs with or without NIR in HepG2/ADR cells and HepG2 cells, the protein expression levels of PTEN were evaluated. The experimental results showed that the protein level of PTEN was lower in HepG2/ADR cells than that in HepG2 cells. The treatment with free DOX had no obvious effect on the protein levels of PTEN both in HepG2/ADR cells and HepG2 cells. As anticipated, HA/anti-miR-21/PPAuNCs moderately upregulated the levels of PTEN both in HepG2/ADR cells and HepG2 cells, indicating that anti-miR-21 executed its biological function by upregulating the expression of PTEN protein. However, the levels of PTEN protein of the HepG2/ADR cells and HepG2 cells treated with HA/anti-miR-21/PPAuNCs + DOX


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showed no remarkable difference, compared with those treated with HA/anti-miR-21/PPAuNCs. More importantly, the treatment with HA/anti-miR-21/PPAuNCs + NIR + DOX significantly upregulated the level of PTEN both in HepG2/ADR cells and HepG2 cells in comparison with any other group. That is, the hyperthermia mediated by HA/anti-miR-21/PPAuNCs under NIR might have enhanced the transfection efficiency or improved the regulating effect of anti-miR21.

Figure 4. (A) The expression levels of p-gp protein in HepG2/ADR cells and HepG2 cells were analyzed by western blot, cells treated with or without NIR irradiation, respectively. (B)The expression levels of PTEN protein in HepG2/ADR cells and HepG2 cells with different treatments were analyzed by western blot. (a) culture medium (Control group), (b) free DOX group, (c) HA/anti-miR-21/PPAuNCs group, (d) HA/anti-miR21/PPAuNCs + DOX group and (e) HA/anti-miR-21/PPAuNCs+ NIR + DOX group in HepG2 cells. (f) culture medium (Control group), (g) free DOX group, (h) HA/anti-miR-21/PPAuNCs group, (i) HA/anti-miR21/PPAuNCs + DOX group and (j) HA/anti-miR-21/PPAuNCs+ NIR + DOX group in HepG2/ADR cells. (C) Semi-quantification of the western blots for the p-gp levels by Image J (a software for gradient analysis of Western blot bands). The p-gp levels were presented as the ratio of p-gp to β-actin. Data were mean ± SD


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(n=3). There was no significant (ns) differences between the groups of without and with NIR irradiation in HepG2 cells. Significant differences (***p

ADR by Anti-miR-21

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