An NIR-activated Spatiotemporally Controllable Nanoagent for

Jun 17, 2019 - An NIR-activated Spatiotemporally Controllable Nanoagent for Achieving Synergistic Gene-Chemo-Photothermal Therapy in Tumor Ablation ...
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Article Cite This: ACS Appl. Bio Mater. 2019, 2, 2994−3001

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NIR-Activated Spatiotemporally Controllable Nanoagent for Achieving Synergistic Gene-Chemo-Photothermal Therapy in Tumor Ablation Xue-Jiao Yang, Xiang-Ling Li,* Hong-Yuan Chen, and Jing-Juan Xu* State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China Downloaded via GUILFORD COLG on July 19, 2019 at 07:12:14 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

S Supporting Information *

ABSTRACT: A rational combination of different therapeutic modalities within one single nanostructure is promising to enhance the therapeutic response, especially to achieve a synergistic therapeutic efficacy for tumor treatment. Herein, a near-infrared (NIR) photothermally activated nanoagent, which could achieve a spatially controllable codelivery of different curative molecules and a temporally controlled responsive release, was designed to perform effective genechemo-photothermal therapy of malignant tumors. The nanoagent consisted of a gold nanorod (AuNR) functionalized with mPEG, DNA, and small interfering RNA (siRNA). With the aid of aptamer AS1411-mediated recognition and endocytosis, the nanoagents were selectively delivered into cancer cells; subsequently, the photothermal conversion of AuNRs happened while under NIR irradiation, which successfully achieved an effective photothermal therapy, induced dehybridization of DNA duplexes, and simultaneously released doxorubicin (DOX) and siRNA. Then, the released siRNA silenced the expression of the multidrug resistance associated protein 1 (MRP1), the primary cause of the undesirable expelling of DOX in PC-3 cells, yielding a remarkable improvement in the efficiency of gene-chemo therapy. All of the results of in vitro and in vivo studies revealed that our prepared nanoagents exhibited an excellent performance in synergistic gene-chemo-photothermal therapy and successfully inhibited tumor growth. This work provides an interesting concept of a nanoscale therapeutic agent in achieving a dramatically enhanced therapeutic ability for tumor ablation, which benefits from the spatiotemporally controllable properties and the synergistic combination of chemotherapy, gene therapy, and photothermal therapy in one single nanoagent. KEYWORDS: AuNRs, targeted codelivery, controlled release, synergistic therapy, tumor ablation

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INTRODUCTION Chemotherapy remains the dominant treatment for a variety of cancers. However, as relevant researches have revealed, it is hardly possible to realize the desired therapeutic efficacy via chemotherapy alone, due to limited tissue penetration,1 nontargeted drug delivery,2 drug resistance,3,4 etc. Recently, synergistic therapy instead of monotherapy is becoming the trend of cancer treatment, especially in personalized medication. In this regard, gene therapy as a novel molecular medicine has shown remarkable therapeutic benefits in combating various diseases, such as infectious diseases, cancer, Parkinson’s, and Alzheimer’s disease.5,6 Typically, siRNA, which could silence a sequence-specific gene via targeting and cleaving the corresponding mRNA, is regarded as a revolutionary tool in gene therapy.7,8 More fortunately, the specific siRNA is also capable of targeting the drug-resistance gene, knocking down the corresponding expression through the action of the so-called RNA-induced silencing complex (RISC),9 and restoring the drug sensitivity of cancer cells, which synergistically enhance the efficacy of chemotherapy.10−12 Therefore, it is highly meaningful to rationally © 2019 American Chemical Society

combine siRNA-based gene therapy and chemotherapy within one single nanoparticle while combating a malignant tumor. Furthermore, it is urgently needed and desirable to develop tumor-targeted therapeutic nanoagents to achieve a spatially controlled codelivery of nucleic acid molecules and drugs into cancer cells, as a low intratumoral accumulation is the major limitation of nanomaterials for tumor treatments. To this end, a variety of ligands and biomolecules that selectively attach to receptors overexpressed on cancer cell membranes are conjugated to drug delivery materials as targeting molecules, of which aptamers and antibodies are the most widely investigated.13−18 Another major barrier of therapeutic nanomaterials for tumor treatments is that the nanomaterials would react with the external microenvironment and induce an undesired release of cargos during the delivery process before reaching the tumor site. Stimulus-responsive approaches have been integrated in these nanomaterials for enhanced Received: April 17, 2019 Accepted: June 17, 2019 Published: June 17, 2019 2994

DOI: 10.1021/acsabm.9b00329 ACS Appl. Bio Mater. 2019, 2, 2994−3001

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chemotherapy because siRNA can only work in the cytoplasm and DOX owns a higher lethality to cancer cells when it enters the nucleus. On the other hand, the photothermal effect itself can destroy cancer cells to some extent through hyperthermia.41 Afterward, the developed nanoagent not only achieves a precise targeted delivery and light-controlled release but also performs the relative gene regulation, thus significantly improving therapeutic efficacy and reducing adverse effects. During in vivo experiments, the nanoagent exhibits a satisfactory inhibition of tumor growth and negligible systemic cytotoxicity. The powerful therapeutic nanoagent explores a promising strategy for the precise medicine.

therapeutic outcomes since it could take advantage of the controlled-responsive release and avoid the aforementioned shortcomings of nanomaterials in cancer nanotechnology. Thus, many triggering stimuli have been involved in the intelligent delivery systems, including various internal or external stimuli such as pH, 19,20 biomolecules, 21−24 light,25−31 ultrasound,32 magnetic field,33 etc. Near-infrared (NIR) light as one of the external stimuli has become a more attractive trigger, owing to its noninvasive and deep tissue penetration. In particular, many photothermal conversion materials can absorb the photo energy of NIR light, effectively convert into heat, quickly increase the intracellular temperature, and then successfully achieve the NIR-activated controllable release for curative molecules34 and photothermal therapy (PTT) for irreversible cancer cell damage. Thus, it is promising to incorporate a targeting molecule and curative molecules into one single photothermal converted nanoparticle for realizing the spatially controllable targeting delivery, temporally controllable-responsive release, and synergistically therapeutic outcomes. Herein, we have successfully designed and constructed a spatiotemporally controllable NIR-activated nanoagent (AuNR/siRNA-DOX) for synergistic gene-chemo-photothermal therapy of malignant tumors (Scheme 1). Gold nanorods

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RESULTS AND DISCUSSION Preparation and Characterization of the Nanoagents (AuNR/siRNA-DOX). The AuNRs were first prepared according to the classical seed-mediated and surfactant-assisted method.35 The TEM image in Figure 1A showed that the

Scheme 1. Schematic Illustration of the NIR-Activated Nanoagent for Synergistic Gene-Chemo-Photothermal Therapy Figure 1. (A) TEM image of the prepared AuNRs. (B) UV−vis absorption spectra of CTAB-coated AuNRs (black line) and dsDNAfunctionalized AuNRs (red line).

prepared AuNRs possessed a good monodispersity with the average length and width of 44.3 ± 5.1 nm and 11.5 ± 1.3 nm (the aspect ratio was ∼3.8), respectively. After functioning with mPEG and DNA sequences, the results of the UV−vis measurements revealed that the longitudinal surface plasmon resonance (LSPR) was blue-shifted from 769 to 748 nm due to a smaller aspect ratio. Meanwhile, a characteristic absorption peak of DNA appeared at about 260 nm (Figure 1B). Besides, ζ potential analysis (Figure S1) indicated a change from 27.6 to −9.9 mV, further confirming that the mPEG and DNA successfully assembled on the surface of AuNRs. To accurately quantify the amount of oligonucleotide sequences loaded on each AuNR, the classic ME-displacement fluorescent method42,43 and fluorophore modified oligonucleotides (termed as Spacer-Fluor) were utilized. According to the fluorescent quantitative analysis, the ratio of oligonucleotide sequences to AuNR was estimated to be 278:1 (Figure S2). Additionally, polyacrylamide gel electrophoresis results (Figure S3) showed that there were significant changes in mobility between sequences in different lanes, verifying the successful hybridization of Apt-SH sequences and Spacer sequences (Spacer-SH sequences and Sense sequences). Then in order to quantify the amount of DOX loaded on each AuNR, UV−vis absorption spectra of a series of DOX were measured to establish a calibration curve (Figure S4A). After the loading process and centrifugation separation, the absorption intensity of DOX in the supernatant was measured (Figure S4B) and the concentration was calculated to be 3.51 μM according to the calibration curve. Thus, as the total

(AuNRs) are chosen as the skeleton units, as they can be readily synthesized with various aspect ratios,35 which enables intense absorption in the NIR region and excellent photothermal conversion performance. Then AuNRs were functionalized with mPEG and oligonucleotides to provide excellent biocompatibility, targeting delivery, and natural binding sites for DOX, a potent antitumor drug that can preferably intercalate dsDNA into double-stranded CG or GC sequences.36 After aptamer-mediated targeting and endocytosis,37,38 the AuNRs heated the surroundings via photothermal conversion, resulting in dehybridization of the DNA duplexes and release of DOX and siRNA. In addition, NIR light irradiation can also break up the lysosomal membranes and contribute to lysosomal escape of the nanoagents,39,40 which ensures the possibility of subsequent gene silencing and 2995

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ACS Applied Bio Materials amount of drug used in the loading process was 5 μM, the concentration of DOX retained in the nanoagents was 1.49 μM and the amount of DOX loaded on each AuNR was estimated to be 2099. Photothermal Properties of AuNRs and the Developed Nanoagents. To evaluate the photothermal effect of AuNRs and the nanoagents (AuNR/siRNA-DOX) under continuous wave (CW) NIR light irradiation, the temperature changes of different solutions were monitored with an infrared thermal imaging camera. Once exposed to NIR light irradiation (808 nm), the temperature of AuNRs in water sharply increased from 25 to 50 °C in 5 min. The temperature elevation analysis also showed that the change in photothermal conversion ability before and after modification was negligible (Figure 2A,B). In addition, the cyclic irradiation experiments

the confocal microscopy. As shown in Figure 3A, an obvious red fluorescence signal of DOX was detected in the experimental group (top in Figure 3A), while there was only an extremely weak red fluorescence in the two control groups. Obviously, with the aid of aptamer AS1411, the nanoagent could keep away from normal cells and specifically enter cancer cells, which achieved the spatially targeted delivery, thereby improving the treatment efficiency and reducing the systemic cytotoxicity. To examine the light-controllable curative molecules release in living cells, PC-3 cells were incubated with the nanoagents for 4 h and then exposed to NIR laser irradiation, followed by another 1 h incubation. Afterward, the cells were stained with nuclear dye Hoechst 33342 to assist in locating DOX. Under NIR laser irradiation, the thermal images revealed that, in the test group, the temperature of cells incubated with the nanoagents increased rapidly to 47 °C in 5 min (Figure S7), which was higher than the Tm value of the DNA duplexes assembled on the surface of AuNRs. As illustrated in the Figure 3B, in the control group without laser irradiation, the red fluorescence signal of DOX was widely distributed in the cytoplasm. In comparison, the results of the test group were completely different. There was a perfect colocalization of DOX and Hoechst 33342 after NIR irradiation, demonstrating that DOX could be released from the nanocarrier, escape from lysosomes, and enter the nucleus. The above results indicated that the nanosystem possessed a good dark stability and NIRactivated drug/siRNA release capability in cells, which can achieve the temporally stimulus-responsive release in tumor treatment. Meanwhile, the gene regulation via the released siRNA was assessed by detecting the expression levels of multidrug resistance protein MRP1 after various treatments. Particularly, PC-3 cells were divided into four groups: (a) incubated with DMEM medium (control group); (b) incubated with the nanoagent AuNR/siRNA-DOX; (c) transfected free siRNA with Lipofectamine 3000 reagent; (d) incubated with free chemotherapeutic drug DOX. Western blot analysis demonstrated that, compared with the control group, an apparent increase in the MPR1 expression was observed, while cells were treated with a high concentration of DOX (group d, Figure 3C), consistent with the mechanism of multidrug resistance. In contrast, while cells were cultured with the therapeutic nanoagents, the nanoagents could knock down MRP1 expression to 67%, which achieved similar results to that of the group transfected free siRNA. We also studied the effect of nanoagents on MRP1 expression in DOX-resistant prostate cancer cells. The results (Figure S8) showed that our nanoagents successfully reduced the protein expression both in general PC-3 cells and DOX-resistant PC-3 cells. The above results verified that the prepared nanoagent could restore the drug sensitivity of PC-3 cells, thereby improving the synergistic efficacy of gene-chemotherapy. Considering the above results, the therapeutic efficacy of the smart nanoagent AuNR/siRNA-DOX for combating cancer cells was investigated by flow cytometry analysis. The late apoptosis rate of AuNR/siRNA-DOX treated cells was up to 84.4%, while AuNR/DNA and DOX treated cells showed a much lower late apoptosis rate (Figure 3D), indicating that synergistic gene-chemo-photothermal therapy could induce a much more severe apoptosis than two separate treatments in PC-3 cells. Furthermore, the staining experiments with calceinAM/PI were performed to visually observe apoptosis. As

Figure 2. (A) Temperature elevation of different solutions as a function of time under irradiation (808 nm, 1.5 W/cm2) and (B) the corresponding thermal images before (top) and after (bottom) NIR irradiation. (C) Temperature−time curve and (D) thermal images of the nanoagent during the irradiation cycle experiment. Every cycle was composed of 5 min irradiation (808 nm, 1.5 W/cm2) followed by a 6 min cooling phase.

exhibited the excellent photothermal stability of AuNRs (Figure S5) and AuNR/siRNA-DOX (Figure 2C,D) during five heating and cooling cycles, proving that the synthesized nanoagents could perform well as photothermal nanomaterials in subsequent experiments. Then the photoresponsive drug release properties of the smart nanoagents were investigated. As shown in Figure S6, the amount of DOX released from the nanoagents could reach 88% within 6 min, while irradiated with 808 nm laser at the power density of 1.5 W/cm2. In contrast, the leakage of DOX in the control group without NIR irradiation could be negligible. Thus, the release of the drug was intelligently activated by NIR laser irradiation. NIR-Activated Gene-Chemo-Photothermal Therapy in Vitro. Before performing it for therapy in cancer cells, the targeted delivery of the nanoagents was investigated with laser scanning confocal microscopy (LSCM). The prostate cancer cells (PC-3) and normal prostate epithelial cells (RWPE-1) were incubated with the proposed nanoagent (+) or control nanoagent (−), which was not modified with aptamer AS1411, a guanine-rich oligonucleotide specifically targeting nucleolin overexpressed on the cell membranes of tumor cells.35 Subsequently, cells were washed with PBS and imaged by 2996

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Figure 3. (A) The CLSM images of PC-3 cells and RWPE-1 cells incubated with nanoagents (+) or control nanoagents (−), which were not modified with aptamer AS1411. Scale bar: 25 μm. (B) Confocal images of PC-3 cells incubated with nanoagents and Hoechst 33342 with or without 808 nm laser irradiation. Scale bar: 10 μm. (C) Western blot analysis of relative MRP1 expression levels in PC-3 cells after different treatments. (D) Flow cytometric analysis of cells apoptosis after different treatments using Annexin V-FITC/PI staining. (E) The CLSM live/dead cell images of PC-3 cells stained by Calcein-AM/PI. The cells were preincubated with or without the developed nanoagents for 48 h. Scale bar: 100 μm. (F) Cell viability of PC-3 cells after different treatments. (a) Control; (b) DOX (2 μM) (chemotherapy); (c) DOX (2 μM) + siRNA (genechemotherapy); (d) AuNR/DNA + NIR (photothermal therapy); (e) AuNR/siRNA + NIR (gene-photothermal therapy); (f) AuNR/DNA-DOX + NIR (chemo-photothermal therapy); (g) AuNR/siRNA-DOX + NIR (gene-chemo-photothermal therapy); (h) AuNR/siRNA-DOX. Groups d− g were exposed to NIR irradiation (1.5 W/cm2, 5 min). Error bars indicate the standard deviation of four tests.

Figure 4. Therapeutic effect of the nanoagents in nude mice. (A) In vivo photothermal images of the control group with the PBS injection and the experimental group with the nanoagent injection before (−) and after (+) 5 min of NIR irradiation (808 nm, 1.0 W/cm2). (B) Relative tumor growth curves and (C) body weight change curves of mice in different groups during various treatments. Error bars indicate standard deviations, n = 4, (**p < 0.01, *p < 0.05). (D) H&E staining of major organs from mice in 5 groups after different treatments. Scale bar: 40 μm. (E) Representative photographs of the mice before (0 day) and after (15 day) being subjected to different treatments. (F) Photographs of tumors after treatments for 15 days. Group 1: PBS. Group 2: AuNR/siRNA-DOX without NIR irradiation. Group 3: DOX (2 μM). Group 4: AuNR/DNADOX with NIR irradiation. Group 5: AuNR/siRNA-DOX with NIR irradiation.

shown in Figure 3E, there was only strong green fluorescence signal of calcein-AM generated by viable cells in three control groups, revealing that just NIR irradiation or the nanoagent without NIR irradiation caused negligible cell injury. However, an obvious red fluorescence signal suggesting a high cell

mortality was observed in the group where cells had been sequentially treated with AuNR/siRNA-DOX and NIR irradiation. The results showed that the nanoagent owned a temporally controllable-responsive property and exhibited an effective cytotoxicity only when activated by an 808 nm laser. 2997

DOI: 10.1021/acsabm.9b00329 ACS Appl. Bio Mater. 2019, 2, 2994−3001

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effective dose of DOX might cause systemic toxicity. Additionally, the hematoxylin and eosin (H&E) staining analysis of major organs was performed. Severe liver tissue damage was observed after injection with free DOX solution (Figure 4D). Apart from this, no other experimental groups showed any pathological abnormality when compared with the control group. Taken together, our developed nanoagent possessed the perfect biocompatibility and negligible systemic toxicity in biomedical applications.

Finally, to further evaluate the efficiency of multimodal therapy, the viability of cancer cells after incubation under different conditions was studied using the Cell Counting Kit-8 (CCK-8). As shown in Figure 3F, PC-3 cells maintained over 91.9% of cell viability when only cultured with AuNR/siRNADOX, indicating the good biocompatibility of the nanoagent before activation by NIR irradiation. On the other hand, the rational combination of gene-chemo-photothermal therapy induced a much higher mortality than the single-modality treatments and any dual-modal combined therapy. Moreover, the cell viability tests of other cancer cells treated with or without the therapeutic nanoagent were also performed (Figure S9). The outcomes revealed that without irradiation, the nanomaterial AuNR/siRNA-DOX exhibited negligible effects on the growth of cells, and the cell viability of all cell lines was up to 90%. After irradiation with an NIR laser, the rational combination therapy induced a high mortality of cancer cells. All results of in vitro experiments demonstrated that the nanoagents could not only efficiently beat cancer cells but also maximally avoid the side effects caused by curative molecules leakage. Synergistic Therapeutic Effect of the Nanoagent in Vivo. Motivated by the above desirable experimental results, the in vivo therapeutic potential of the nanoagents was evaluated using a PC-3 tumor xenograft model in nude mice. First, to monitor the PTT effect of the nanoagents in vivo, the 808 nm laser power was optimized to be 1 W/cm2, and the temperature change of the tumor site under laser irradiation was recorded. The thermal images showed that the temperature of the tumor in the nanoagent-injected mouse rapidly increased to 48 °C in 5 min, sufficient to cause cell damage and trigger release of DOX and siRNA, while only a gentle heating effect was observed in the control group (Figure 4A). For assessing the therapeutic efficiency of the nanoagents in vivo, all of the tumor-bearing mice were randomly divided into five groups (four mice per group) when the tumor volumes grew up to ∼70 mm3 and then received different treatments: injection with PBS (group 1), injection with AuNR/siRNADOX but no laser irradiation (group 2), injection with DOX solution (group 3), injection with AuNR/DNA-DOX and NIR laser irradiation (group 4), injection with AuNR/siRNA-DOX and NIR laser irradiation (group 5). Relative tumor sizes of all mice were recorded every 3 days during the 15 day treatment. The results of the tumor sizes (Figure 4B,E) indicated that the tumor of mice in group 1 grew as quickly as that of the control group, suggesting that the inactivated nanoagent owned an excellent biocompatibility and had a negligible cytotoxicity. In contrast, mice treated with AuNR/siRNA-DOX and laser irradiation experienced a nearly complete tumor regression (Figure S10), which was much better than the effects of chemotherapy alone in group 3 and chemo-photothermal therapy in group 4. After treatments, the tumors were harvested for ex vivo imaging (Figure 4F), and the results further confirmed the superior synergistic therapeutic effect of the nanoagent on malignant tumors. Note that one mouse in group 1 and group 2 died during the experiments, possibly due to the oversized tumors. To further investigate the possible toxicity of the injected substances, body weights of all mice were monitored during the 15 day treatment. No obvious change was observed in four of the groups, suggesting the low side effect of the nanoagents. However, the free DOX solution caused a slight weight loss compared with other groups (Figure 4C), indicating that an

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CONCLUSION In summary, a smart NIR light-triggered nanoagent composed of a gold nanorod as the photothermal converter, aptamers as the targeting units, and siRNA sequences as the gene regulators was developed to achieve the targeted codelivery and controlled release of curative molecules, while realizing a rational combination of gene regulation and chemo-photothermal therapy. Modification of aptamer AS1411, which can target nucleolin overexpressed on tumor cell membranes, significantly promoted the accumulation of nanoagents at tumor sites as well as reducing adverse effects on normal cells. Subsequently, NIR laser irradiation enabled nanocarriers to successfully escape from lysosomes and release DOX and siRNA to the cytoplasm, which markedly improved the efficiency of chemotherapy and gene silencing. Due to the spatially and temporally controlled delivery, the nanoagent exhibited a desirable biocompatibility and synergistic treatment effect both in vitro and in vivo. Furthermore, the tumors in PC3 tumor-bearing nude mice were almost completely eliminated after the 15 day collaborative treatment of gene-chemophotothermal therapy, far exceeding the therapeutic outcome of several other treatments. Therefore, the study provides an interesting concept of the NIR-activated therapeutic nanoagent, which not only showed a spatiotemporally controllable delivery and release but also achieved an improved therapeutic efficacy via gene-chemo-photothermal therapy. Moreover, with the appropriate design and functionalization, more materials can be applied to this versatile method to fabricate a multimodal therapeutic system for future clinical applications.

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EXPERIMENTAL SECTION

Synthesis of Gold Nanorods (AuNRs). The AuNRs were fabricated based on the classical seed-mediated method.35,44,45 First, the nanoparticle seeds were prepared. The gold salt HAuCl4 (0.5 mM, 5 mL) was mixed with the stabilizer CTAB (0.2 M, 5 mL), and then the reducing agent, ice-cold NaBH4 (0.01 M, 0.6 mL), was added to the above solution with stirring. After the solution changed from yellow to brownish yellow, the mixture was still stirred for another 2 min. Then the seed solution was aged at room temperature for 0.5 h prior to the further experiment. Next, the growth solution for AuNRs was prepared. Specifically, 4.8 mL of 4 mM AgNO3 was added to 100 mL of an aqueous solution containing CTAB (1.8 g) and 5-BrSA (0.22 g), and then the mixture was kept undisturbed at 30 °C for 15 min. After that, 50 mL of HAuCl4 solution (1 mM) was added to the above solution, followed by gently stirring for 15 min. Subsequently, 0.4 mL of 0.064 M AA was added to the resulting solution, and the solution was gently stirred for 30 s. While the solution turned colorless, 0.16 mL of the prepared seed solution was injected into the growth solution. Then the mixture was stirred for 30 s and left undisturbed at 30 °C overnight for AuNR growth. Excess CTAB and 5-BrSA were removed by centrifuging at 8500 rpm, and the AuNRs were redispersed in pure water. The obtained nanoparticles were characterized by transmission electron microscopy (TEM) and UV− vis measurements, and the concentration was calculated on the basis of the Beer−Lambert Law.46 2998

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ACS Applied Bio Materials Preparation of the AuNR/siRNA-DOX Nanoagents. AuNRs were modified based on the reported literature.47 The synthesized AuNR solution (1 mL) was centrifuged, and the supernatant was removed. Then mPEG-SH (100 μM, 70 μL) was added to the precipitate, followed by vigorous vortexing for 20 s. Subsequently, Tween 20 (0.01 wt %, 4 mL) was added to suspend the AuNRs. The above processes were repeated at least twice to thoroughly displace the absorbed CTAB. The newly activated Spacer-SH sequences (100 μM, 70 μL) and DNA duplexes (50 μM, 28 μL) of Apt-SH and Spacer were added to the above mixture. After shaking for 3 h, the above solution was gradually aged with 1.0 M NaCl solution and the final salt concentration was 0.1 M. Then the resulted solution was centrifuged to remove the unreacted DNA, and the precipitate was dispersed in PBS. Subsequently, the siRNA sequences (10 μM, 120 μL) and excess DOX were sequentially added to the AuNR/DNA solution. After the mixture stirred for 3 h, the AuNR/siRNA-DOX nanoagents were centrifuged, washed, redispersed in 1 mL of PBS, and then kept at 4 °C for use. Cell Culture and Laser Scanning Confocal Microscopy Imaging. PC-3 cells, HeLa cells, and 4T1 cells were cultured in DMEM medium supplemented with 10% fetal calf serum, penicillin (80 U/mL), and streptomycin (0.08 mg/mL). MCF-7 cells were cultured in RPMI-1640 supplemented with fetal calf serum, penicillin, and streptomycin. RWPE-1 cells were culture in a keratinocyte medium supplemented with keratinocyte growth supplement, penicillin, and streptomycin. All of cells were cultured in the standard incubator at 37 °C with an atmosphere containing 5% CO2. For confocal imaging, cells of diverse cell lines were, respectively, seeded in several Petri dishes and cultured for 48 h. Then the cells were incubated with a fresh medium containing nanoagents or control nanoagents for 4 h. After that, the cells were washed completely and cultured in a fresh medium. Some cells were irradiated under an 808 nm laser at the power density of 1.5 W/cm2 for 5 min, and all of the cells were cultured for another 1 h. Finally, the cells were stained with Hoechst 33342 for 15 min, washed three times with PBS, and then detected for the imaging analysis by confocal microscopy. Establishment of DOX-Resistant Prostate Cancer Cell Lines. A DOX-resistant prostate cancer cell line was established from PC-3 cells by stepwise selection in gradually increasing concentrations of DOX from 10 to 100 nM. Initially, PC-3 cells were cultured in a cell medium containing DOX (10 nM) for 48 h, and then survival cells were continuously cultured under the above condition for another 9 days. After the screening process, survival cells were treated with new conditions in turn, which contained higher concentrations of DOX (50 nM, 100 nM). Over one month, DOX-resistant PC-3 cells were established and further cultured in the medium with 100 nM DOX. Western Blot Analysis of MRP1 Expression. Initially, PC-3 cells (5 × 105 per well) in 6-well plates were incubated at 37 °C for 24 h. Then all of the cells were randomly divided into four groups and treated differently. Cells in the control group were not specially treated. Cells in the second group were incubated with the nanoagents for 4 h and then irradiated under 808 nm laser with the power density of 1.5 W/cm2 for 5 min after washing three times with PBS. Subsequently, cells were cultured in fresh media for 48 h. The MRP1 expression in the third group was modulated by transfecting antisense strands into PC-3 cells according to a standard protocol of the Lipofectamine 3000 transfection reagent. Finally, all of the cells were washed thoroughly with ice-cold PBS and then lysed in RIPA lysis buffer containing 1 mM PSMF on ice. The lysates were centrifuged at 4 °C with 12 000 rpm for 5 min. The relative expression levels of MRP1 were determined by Western blotting, in which GAPDH acted as the internal reference. Cell Apoptosis Experiments. PC-3 cells were seeded in 6-well plates for 24 h, and then cells were incubated with DMEM, DOX (2 μM), AuNR/DNA, and AuNR/siRNA-DOX for 4 h, respectively. Then the supernatant was changed with fresh DMEM medium, and 808 nm laser irradiation (1.5 W/cm2, 5 min) was conducted in the last two groups. After the above processes, the cells were incubated in the fresh culture medium for 48 h. Then the cells were harvested, washed twice with PBS, and stained according to the manufacturer’s

instructions of the Annexin V-FITC/PI apoptosis detection kit. Then the samples in PBS were analyzed with a flow cytometer by counting 10 000 events. In Vitro Cytotoxicity. The antiproliferative effect of different treatments was assessed by a CCK-8 assay. PC-3 cells were seeded in two 96-well plates (104 cells/well) and cultured overnight. Then the cells were divided into eight groups: cultured in fresh media (control, group a); treated with free DOX (2 μM, group b), DOX (2 μM), and siRNA (transfected with Lipofectamine 3000, group c), AuNR/DNA (AuNRs modified with Apt-SH and Spacer-SH, group d), AuNR/ siRNA (AuNRs modified with Apt-SH/Spacer and Spacer-SH/siRNA duplexes, group e), AuNR/DNA-DOX (DOX loaded, DNA sequence-conjugated AuNR, group f), AuNR/siRNA-DOX (DOX loaded, DNA sequence and siRNA duplex-conjugated AuNR, group g), AuNR/siRNA-DOX (group h) at 37 °C for 4 h, respectively. Next, cells were washed thoroughly with PBS and cultured with fresh media. For PTT, the cells in groups d−g were exposed to NIR laser irradiation (808 nm, 1.5 W/cm2) for 5 min. After incubation for another 24 or 48 h, the supernatant was replaced with 100 μL of fresh medium containing 10 μL of CCK-8, followed by 1.5 h incubation. Finally, the absorption intensity at 450 nm was recorded. All of the experiments were repeated four times. In Vivo Tumor Suppression Analysis. A xenograft PC-3 tumorbearing mouse model was established by subcutaneously injecting PC3 cells (∼1 × 107) into female nude mice. The tumor volume was monitored by a caliper and calculated according to the formula: V = (length × width2)/2. After the tumor sizes grew up to ∼70 mm3, all of the mice were randomly divided into five groups (n = 4 each group) and then intravenously injected with different agents: injection with PBS (group 1), injection with AuNR/siRNA-DOX but no NIR laser irradiation (group 2), injection with DOX solution (group 3), injection with AuNR/DNA-DOX and laser irradiation (group 4), injection with AuNR/siRNA-DOX and then laser irradiation (group 5). The tail vein injection was conducted every 3 days during the 15 day treatment, and tumor volumes and body weights were measured before every injection. In groups 4 and 5, tumors on the mice were exposed to 808 nm laser irradiation (1.0 W/cm2, 5 min) on the second day after injection with the agents. All animal experiments were conformed to the institutional animal use and care guidelines approved by the Model Animal Research Center of Nanjing University (MARC). Histological Analysis. After 15 day treatments, all mice were euthanized for collecting the major organs including the heart, liver, spleen, lung, and kidney. The organs were fixed in 10% neutralbuffered formalin overnight, embedded in paraffin, and then cut with a microtome. Next, the sections were stained with hematoxylin and eosin (H&E), and the images were obtained with an optical microscope. Statistical Analysis. Data were given as mean ± standard deviation. The t test was adopted to execute statistical analysis with the software SPSS. Statistical significance was denoted by an asterisk (*p < 0.05, **p < 0.01).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.9b00329. Detailed description of the materials, apparatus, sequences and additional experimental results, ζ potentials of the synthesized nanomaterials; calculation for the ratio of DNA to AuNR; polyacrylamide gel electrophoresis analysis; calculation for the ratio of DOX to AuNR; irradiation cycle experiment of nanomaterials; photoresponsive release profiles of the nanoagents; the photothermal images of PC-3 cells; Western blot analysis of relative MRP1 expression levels; cell viability of several cancer cell lines; photograph of mice treated with nanoagents (PDF) 2999

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Tel: +86-25-89687924. ORCID

Jing-Juan Xu: 0000-0001-9579-9318 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21327902, 21535003, and 21605072).

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DOI: 10.1021/acsabm.9b00329 ACS Appl. Bio Mater. 2019, 2, 2994−3001