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Research Article www.acsami.org † Novel Approach of Using Near-Infrared Responsive PEGylated Gold Nanorod Coated Poly(L‑lactide) Microneedles to Enhance the Antitumor Eﬃciency of Docetaxel-Loaded MPEG-PDLLA Micelles for Treating an A431 Tumor Ying Hao, and ZhiYong Qian*,† † State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, PR China ‡ Key Laboratory of Optoelectronic Chemical Materials and Devices of Ministry of Education, Jianghan University, Wuhan 430056, PR China *S Supporting Information † YanPeng Jia, MingLing Dong, TaoYe Zhang, JinRong Peng, YiPing Cao, ‡ † ‡ † ABSTRACT: The combination of chemotherapy and photothermal therapy (PTT) plays a signiﬁcant role in synergistic tumor therapy. However, a high dosage of chemotherapy drugs or photothermal agents may cause series side eﬀects. To overcome these challenges, we designed a near-infrared (NIR) responsive PEGylated gold nanorod (GNR-PEG) coated poly(L-lactide) microneedle (PLLA MN) system (GNR-PEG@MN) to enhance antitumor eﬃciency of docetaxel-loaded MPEG-PDLLA (MPEG-PDLLA-DTX) micelles for treating an A431 tumor. The as-made GNR-PEG@MNs contained only 31.83 ± 1.22 μg of GNR-PEG per patch and exhibited excellent heating eﬃcacy both in vitro and in vivo. Meanwhile, GNR-PEG@MN with the height of 480 μm had good skin insertion ability and was harmless to the skin. On the other hand, GNR-PEG@MN had good heating transfer ability in vivo, and the tumor sites could reach 50 °C within 5 min. In comparison with chemotherapy and PTT alone, the combination of low dosage MPEG-PDLLA-DTX micelles (5 mg/kg) and GNR-PEG@MNs completely eradicated the A431 tumor without recurrence in vivo, demonstrating a remarkable synergetic eﬀect. Hence, GNR-PEG@MN could be a promising carrier to enhance the antitumor eﬀect of MPEG-PDLLA-DTX micelles for treating superﬁcial tumors and is expected to have a great potential in clinical translation for human epidermoid cancer therapy. KEYWORDS: photothermal therapy (PTT), GNR-PEG@MNs, MPEG-PDLLA-DTX micelles, A431 tumor, synergetic eﬀect ■ INTRODUCTION In recent decades, cancer has been the most common life- threatening illness to people’s health and has had an upward trend in its morbidity and mortality.1 Among them, human epidermoid cancer is a public health problem which has a growing trend especially in Caucasians.2 Traditional therapies, such as photodynamic therapy,3 may cause skin DNA damage and other skin cancer.4,5 Great eﬀort is needed to be devoted to improve the therapy eﬀect and reduce side eﬀects. Docetaxel (DTX) is a broad-spectrum antitumor drug which has good antitumor eﬀect.6,7 It the half-inhibitory is reported that concentration (IC 50) value of DTX was 6 nM in human epidermoid cancer cells (A431),8 and DTX-loaded MPEG- PDLLA (MPEG-PDLLA-DTX) micelles have entered the clinical stage in South Korea,9 which could improve the eﬃcacy of chemotherapy via enhanced permeability and retention (EPR) eﬀect.10−13 However, it is reported that only a few drugs could arrive at the tumor site through the EPR Received: March 13, 2017 Accepted: April 18, 2017 Published: April 18, 2017 © 2017 American Chemical Society 15317 DOI: 10.1021/acsami.7b03604 ACS Appl. Mater. Interfaces 2017, 9, 15317−15327 Downloaded via BEIJING UNIV OF CHEMICAL TECHNOLOGY on December 17, 2018 at 09:14:26 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

ACS Applied Materials & Interfaces Research Article eﬀect.14,15 In order to increase the eﬃciency in chemotherapy, several synergetic strategies must be designed to further enhance the antitumor eﬃciency. Near-infrared (NIR) responsive photothermal therapy (PTT) is an attractive alternative to combine with chemo- therapy as it could cause membrane damage, cell injury, and protein denaturation as a systemic eﬀect.16−19 What’s more, PTT could induce tumor thermal and improve the accumu- lation of nanodrug in tumor sites.20 Chen et al. in Soochow University have used an imagable and photothermal HSA-ICG- PTX nanoparticle to treat subcutaneous and metastatic breast tumors, and the combination therapy achieved excellent synergistic therapeutic eﬃcacy.21 To date, a variety of NIR- responsive nanostructures were used for photothermal therapy, such as gold nanostar,22 nanocage,23 nanocube,24 nanocluster,25 nanoshell,26 and nanorod.27 Among them, gold nanorod (GNR) was chosen as an ideal photothermal agent, which has several advantages, including easy synthesis, small size, and adjustable aspect ratio to achieve NIR light absorption.28−30 However, in traditional chemotherapy and PTT, chemotherapy drugs or photothermal agents were delivered to tumor sites either intratumorally or intravenously, which not only needed a high dosage of chemotherapy drugs or photothermal agents to achieve better therapeutic eﬃcacy but also caused serious side eﬀects and reduced patients’ quality of life.31 Hence, it is urgent to develop a novel synergetic system of chemotherapy and PTT to enhance the antitumor eﬃciency. Nowadays, a microneedle delivery system is widely used to deliver drugs,32,33 DNA,34 RNA,35 and vaccines36 as the microinjection is a rapid, cost-eﬀective, painless, and direct way for molecule delivery.37,38 Compared with other drug delivery ways, the solid microneedles could pass through the cutin layer of skin and deliver drug to the dermis directly, which could improve the local drug concentration and reduce systemic side eﬀects. Both Gu39−42 et al. in North Carolina State University and Chen43−46 et al. in National Cheng Kung University have made great contributions in the ﬁeld of microneedle delivery systems, which proved that microneedles are ideal vehicles for molecule delivery. Herein, we developed a novel synergetic system of chemotherapy and PTT to treat the A431 tumor by the combination of a NIR-responsive PEGylated gold nanorod (GNR-PEG) coated poly(L-lactide) microneedle (GNR-PEG@ MNs) and DTX-loaded MPEG-PDLLA micelles shown in Figure 1. This system was composed of biodegradable poly(L- lactide) microneedles (PLLA MNs), photothermal agent GNR- PEG, and antitumor nanodrug MPEG-PDLLA-DTX micelles. PLLA has been approved by the Food and Drug Administration (FDA) for clinical use which was suitable as a needle material. GNR-PEG was a harmless photothermal agent that eﬃciently converted the absorbed light energy into heat and made the tumor site thermal.29,30 Meanwhile, MPEG-PDLLA was a safe nanocarrier, and DTX-loaded MPEG-PDLLA-DTX micelles were sensitive to A431 cells.8,9 Last but not least, the above- mentioned systems have not been reported for the combination of chemotherapy and PTT before. We aimed to use this novel NIR responsive GNR-PEG@MN which contained only 31.83 ± 1.22 μg of GNR-PEG per patch and exhibited excellent heating eﬃcacy to enhance the antitumor eﬃciency of low dosage MPEG-PDLLA-DTX micelles (5 mg/kg). In this study, we prepared MPEG-PDLLA-DTX micelles and GNR-PEG via described methods. The GNR-PEG@MNs with the height of 480 μm were prepared by using NIR-responsive Figure 1. Schematic illustration of (A) the preparation of PLLA MNs and GNR-PEG@MNs, (B) the novel synergetic system of chemo- therapy and photothermal therapy to treat A431 tumors by the combination of near-infrared responsive GNR-PEG@MNs and MPEG-PDLLA-DTX micelles. (Step 1: Injected the DTX loaded micelles; Step 2: After the injection, pressed the GNR-PEG@MNs at the tumor sites and under 2 W/cm2 irradiation by 808 nm laser within 5 min.) GNR-PEG adsorbed on PLLA MNs which contained only 31.83 ± 1.22 μg of GNR-PEG per patch. In addition, the GNR- PEG@MN was characterized in terms of scanning electron microscope (SEM), energy spectrum (EDS), skin insertion test, heating transfer experiment, and NIR thermal imaging study. Finally, in vivo antitumor eﬃcacy of combined chemotherapy and PTT was carried out, and the combination of MPEG- PDLLA-DTX micelles and GNR-PEG@MNs was compared with that of chemotherapy or PTT alone in mice bearing an A431 tumor. The histopathological and cell proliferation of the tumors was also observed. ■ RESULTS AND DISCUSSION Characterization of MPEG-PDLLA. The MPEG-PDLLA copolymer (Mn = 3765, 2000−1765) was synthesized via ring- opening method47 by MPEG 2000 and D,L-lactide as described before48 (Figure S1A in the Supporting Information (SI) shows the procedure). The characterization of MPEG-PDLLA by 1H NMR spectra (Varian 400 spectrometer, Varian, USA), Fourier transform infrared spectroscopy (FTIR, NICOLET 200SXV, Nicolet, USA), and gel permeation chromatography (GPC, Agilent 110 HPLC, USA)49,50 conﬁrmed that the MPEG- PDLLA copolymer was synthesized successfully and the molecular weight was 3821 with narrow distribution of 1.08. (Figure S1B−D in the SI) The DTX-loaded MPEG-PDLLA micelles were prepared via a thin-ﬁlm rehydration method.6,9 Dynamic light scattering 15318 DOI: 10.1021/acsami.7b03604 ACS Appl. Mater. Interfaces 2017, 9, 15317−15327

ACS Applied Materials & Interfaces Research Article Figure 2. (A) Particle size of MPEG-PDLLA-DTX micelles (the TEM image inset), (B) UV−vis absorption spectra of GNR and GNR-PEG, and the zeta potential of (C) GNR and (D) GNR-PEG (the TEM image was insetted, respectively). (DLS) and transmission electron microscope (TEM) was used to characterize the DTX-loaded MPEG-PDLLA-DTX micelles (Figure 2A). The particle size of DTX-loaded micelles was about 22.07 ± 0.22 nm, and polydispersity index (PDI) was 0.200 ± 0.002. The drug loading (DL) and encapsulation eﬃciency (EE) of DTX-loaded MPEG-PDLLA micelles were 4.98 ± 0.10% and 99.68 ± 1.96%, respectively, which were determined by a high performance liquid chromatography (HPLC) method as reported before.9 In the morphology study, MPEG-PDLLA-DTX micelles had uniform particle size and distributed as homogeneous in the TEM image (inset into Figure 2A). Meanwhile, we also study the in vitro cellular uptake eﬃciency and cytotoxicity assay of MPEG-PDLLA-DTX micelles (Figure S2). The cellular uptake eﬃciency of the micelles was time-dependent from 0.5 to 4 h (Figure S2A, B), implying that the micelles could be internalized into the cytoplasm of A431 cells. Compared with DTX, the DTX- loaded micelles strongly inhibited the growth of A431 cells with a dose-dependent manner. The IC 50 value was 3.53 ± 0.14 ng/mL after 48 h incubation which was in accordance with the previous study (Figure S2C, D).8 These results demonstrated that DTX was the suitable drug for treating the A431 tumor. Characterization of GNR-PEG. The GNR-PEG was prepared via a ligand exchange method from GNR. In detail, thiol ethylene glycol (PEG-SH) was used to replace the cetyltrimethylammonium bromide (CTAB) surfactant on the GNR surface overnight, which could reduce the toxicity of CTAB.51 We used FTIR and EDS to ensure whether PEG-SH was modiﬁed on GNR. The IR spectra demonstrated that GNR had strong absorption peaks at 2917.85 and 2849.31 cm−1 which belong to C−H stretch vibration, while after PEG-SH modiﬁcation, the peaks at 2914.95 and 1854.32 cm−1 became weaker sharply, conﬁrming the CTAB surfactant was replaced by PEG-SH successfully (Figure S3A). Seen from the results of EDS in Figure S3B, the weight of the Br element was decreased obviously from 17.81% to 0.98% when the PEG-SH was modiﬁed on the surface of GNR (Figure S3C), which suggested the PEG-SH was modiﬁed on GNR. Figure 2B was the UV−vis absorption spectra of GNR and GNR-PEG. Compared with GNR, the maximum absorption of GNR-PEG red-shifted 10 nm; however, it was still about 800 nm, and the absorption intensity did not change which will not aﬀect the photothermal eﬀect. What’s more, the zeta potential ranges from 35.30 ± 1.80 mV to −7.65 ± 0.68 mV, which was suitable for further study as the zeta potential shifted closer to negative (Figure 2C, D) and further proved that PEG- SH was modiﬁed on GNR successfully. The TEM image of GNR (insetted into Figure 2C) and GNR-PEG (insetted into from GNR to GNR-PEG, 15319 DOI: 10.1021/acsami.7b03604 ACS Appl. Mater. Interfaces 2017, 9, 15317−15327

ACS Applied Materials & Interfaces Research Article Figure 3. (A) Photograph of PLLA MNs (left) and GNR-PEG@MNs (right), SEM image of (B1−B2) PLLA MNs and (C1−C2) GNR-PEG@MNs (the black square represents the location of GNR-PEG), and the surface elemental content of (B3) PLLA MNs and (C3) GNR-PEG@MNs (B1,C1, scale bar, 100 μm; B2,C2, scale bar, 10 μm). Figure 2D) revealed that GNR and GNR-PEG had uniform length about 50 nm and good dispersion. Characterization of GNR-PEG@MNs. The NIR-respon- sive GNR-PEG@MNs were obtained from the modiﬁcation of PLLA MNs. First, the PLLA MN was fabricated by melt- molding PLLA on a polydimethylsiloxane (PDMS) template and press-molded to obtain arrays of microneedles.34,52 The NIR-responsive GNR-PEG adsorbed on PLLA MNs via the layer by layer method,53,54 and then we used scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS), FTIR, UV−vis transmittance spectroscopy, and X-ray photo- electron spectroscopy (XPS) to characterize the microneedles. The size of PLLA MNs (left) was 1 cm × 1 cm which consisted of 400 (20 × 20) microneedle tips. When the GNR-PEG absorbed on PLLA MNs to get GNR-PEG@MNs (right), the color of the microneedles turned white to purple (Figure 3A). What’s more, the micrographs of PLLA MNs and GNR-PEG@ MNs shown in Figure S4 presented the bright-ﬁeld micro- graphs of microneedle structures. Figure 3B1 and Figure 3C1 were the SEM image of PLLA MNs and GNR-PEG@MNs, and the microneedle tips were lined up in order. The base width the microneedles were 300 and 480 μm, and height of respectively. The surface of PLLA MNs was clean and smooth (Figure 3B2), while the surface of GNR-PEG@MNs had a lot of rod-shaped objects that were GNR-PEG (Figure 3C2). We used EDS to analyze the elemental content of PLLA MNs and GNR-PEG@MNs (Figure 3B3, C3). The results showed that only C, O elements were on the surface of PLLA MNs, while the surface of GNR-PEG@MNs had C, O, and Au elements, demonstrating that the GNR-PEG absorbed on the surface of PLLA MNs successfully. This is further supported by IR spectra, UV−vis transmittance spectra, and XPS results (Figures S5 and S6). In detail, when polyethylenimine (PEI) modiﬁed PLLA MNs, the absorption peak at 1591.21 cm−1 in the IR spectra was attributed to νNH stretch vibration, and the peak disappeared after GNR-PEG absorbed on PEI-modiﬁed PLLA MNs (Figure S5A), revealing the GNR-PEG was absorbed on PLLA MNs successfully. What’s more, the transmittance measured via UV−vis transmittance spectroscopy declined from 81% to 64% as the modiﬁcation carried on (Figure S5B), which also demonstrated that we obtained GNR- PEG@MNs via a layer by layer method. The surface atomic composition of GNR-PEG@MNs was identiﬁed by XPS analysis (Figure S6). Compared with PLLA MNs, GNR- PEG@MNs had a Au signal, indicating that the GNR-PEG was successfully absorbed on PLLA MNs. In Vitro Skin Insertion Test. Skin insertion ability is the key factor to overcome the skin resistance for drug delivery.43,44 The trypan blue staining method was used to conﬁrm whether the GNR-PEG@MNs could insert into the skin completely. After GNR-PEG@MNs inserted into mice skin for 5 min, the skin treated with trypan blue showed consistent microneedle insertion (Figure 4A). The surface of the skin revealed rows of Figure 4. (A) Photograph, (B) optical micrograph (scale bar, 200 μm), and (C) histological sections (scale bar, 20 μm) of mice skin stained with trypan blue after GNR-PEG@MN application (the black arrow represents the microneedle puncture sites) and H&E-stained mice skin sections (D) after GNR-PEG@MN application (scale bar, 20 μm) (the black dotted line represents the microneedle puncture sites) and (E) 12 h after GNR-PEG@MN application (scale bar, 20 μm). 15320 DOI: 10.1021/acsami.7b03604 ACS Appl. Mater. Interfaces 2017, 9, 15317−15327

ACS Applied Materials & Interfaces Research Article Figure 5. (A) Near-infrared thermal imaging of PLLA MNs and GNR-PEG@MNs under 2 W/cm2 irradiation by an 808 nm laser at diﬀerent time (the black square represents the location of microneedles). (B) Heating curves of PLLA MNs and GNR-PEG@MNs. (C) Temperature changes of PLLA MNs and GNR-PEG@MNs after irradiation with NIR light for 3 cycles. Figure 6. (A) In vivo near-infrared thermal imaging of A431 tumor-bearing mice under 2 W/cm2 irradiation by 808 nm within 5 min (the black circle represents the location of tumors). (B) Heating curves in the tumor sites. “★★” means the P < 0.01. blue spots which corresponded to the GNR-PEG@MN puncture sites (Figure 4B), showing that GNR-PEG@MNs had good skin insertion ability. The histological section (Figure 4C) further clearly demonstrated that GNR-PEG@MNs completely inserted into the skin. We used the H&E staining method to investigate whether the GNR-PEG@MN was harmless to the skin that could recover rapidly to avoid wound infection. From the results in Figure 4D,E, the skin that was treated with GNR-PEG@MNs could be recovered 12 h after indicating that GNR-PEG@MN was harmless to the skin and the damage induced by the GNR- PEG@MNs was reversible. Near-Infrared Thermal Imaging of GNR-PEG@MNs. GNR-PEG has been proved as a harmless and outstanding photothermal agent for PTT.28−30 To ensure whether the GNR-PEG@MNs still had good heating eﬃcacy as GNR-PEG the insertion, for PTT, we used an NIR thermal camera (Fluke Ti32, USA) to investigate the in vitro heating eﬃcacy of PLLA MNs and GNR-PEG@MNs within 5 min (Figure 5A). As the time went on, the temperature of GNR-PEG@MNs quickly reached 55 °C, while the temperature of PLLA MNs was still at room temperature, indicating that GNR-PEG@MNs still had the heating eﬃcacy as well as GNR-PEG. Heating curves and the temperature changes of PLLA MNs and GNR-PEG@MNs after irradiation with NIR light for 3 cycles were shown in Figure 5B, C. Once the irradiation was switched on, the temperature of GNR-PEG@MNs quickly rose to 55 °C, and when the irradiation was switched oﬀ, the temperature of GNR- PEG@MNs rapidly cooled to room temperature, demonstrat- ing that the temperature of GNR-PEG@MNs could be controlled by NIR light. Meanwhile the temperature of PLLA MNs was still at room temperature. Above all, the GNR-PEG@ 15321 DOI: 10.1021/acsami.7b03604 ACS Appl. Mater. Interfaces 2017, 9, 15317−15327

ACS Applied Materials & Interfaces Research Article Figure 7. (A) Heating transfer eﬃcacy in pork under 2 W/cm2 irradiation by an 808 nm laser within 5 min (the black circle represents the location of microneedles, scale bar, 1 cm). (B) Heating curves of the center temperature of the pork. “★★” means the P < 0.01. for MNs that only contained 31.83 ± 1.22 μg of GNR-PEG per patch still had good heating eﬃcacy and was a novel NIR- responsive photothermal agent the combination with chemotherapy. In Vivo near-Infrared Thermal Imaging and Heating Transfer Eﬃcacy Study. An ideal NIR-responsive photo- thermal agent not only had good heating eﬃcacy in vitro, but in vivo, as the tumor tissue could be damaged irreversibly when the temperature of the tumor sites reached 50 °C.55 In vivo thermal imaging study was carried out on female A431 tumor- bearing balb/cA-nu mice (3 mice per group) (Figure 6). To evaluate whether transdermal drug delivery systems had advantages of other drug delivery systems to deliver photo- thermal agent GNR-PEG for PTT, the groups that smeared GNR-PEG and injected GNR-PEG intratumorly were also investigated. The temperature in the tumor sites in the control and the PLLA MNs (transcutaneous) group did not change a lot. The temperature of the group that smeared GNR-PEG at tumor sites raised to 45 °C with ΔT = 8.9 °C within 5 min as the GNR-PEG solution was easier to dry when the 808 laser was on which could decrease the heating eﬃciency of GNR- PEG. The temperature of GNR-PEG (intratumor) could reach 50 °C with ΔT = 13 °C as GNR-PEG could not be evenly distributed in the tumor tissues which also reduced the heating eﬃciency at the tumor sites. The tumor treated with GNR- PEG@MNs transcutaneously increased quickly and reached 50 °C within 1 min with ΔT = 23.8 °C, which were high enough for tumor tissue to be damaged irreversibly. These results demonstrated that the tumor treated with GNR-PEG@MNs had the best heating eﬃcacy among the other two groups. To conﬁrm whether the heating could be transferred to the center of the tumor sites which could induce progressive necrosis of tumors. We further investigated the heating transfer eﬃcacy in pork with the height, length, and width of 2.5, 3, and 3 cm, respectively (Figure 7). Compared with the group of GNR-PEG (smear) and GNR-PEG (subcutaneous), the group treated with GNR-PEG@MNs (transcutaneous) had the best heating transfer eﬃcacy, and the depth of heating almost reached 1.5 cm. Additionally, the heating could be conductive to the middle position of the pork and reached to 55 °C within 5 min, revealing that the heating eﬀect of the GNR-PEG@MNs could be transmitted to the center of tumor tissue in vivo. No signiﬁcant temperature change was observed in the pork treated with PLLA MNs (transcutaneous) and the control group. In summary, compared with other drug delivery systems, the NIR- responsive GNR-PEG@MNs only contained 31.83 ± 1.22 μg of GNR-PEG and had a big contact area for heating transfer to the center of the tumor sites evenly and was a promising carrier to deliver photothermal agent for PTT. In Vivo Antitumor Eﬃcacy. To further study the synergetic eﬀect of chemotherapy and PTT in vivo in comparison with chemotherapy and PTT alone, the tumor- bearing A431 mice were divided into 8 groups randomly (5 mice per group): (1) the saline group, (2) the GNR-PEG@ MNs + laser group, (3) the DTX group (5 mg/kg), (4) the DTX group (10 mg/kg), (5) the DTX (5 mg/kg) + GNR- PEG@MNs + laser group, (6) the MPEG-PDLLA-DTX group (5 mg/kg), (7) the MPEG-PDLLA-DTX group (10 mg/kg), and (8) the MPEG-PDLLA-DTX (5 mg/kg) + GNR-PEG@ MNs + laser group. According to photographs and growth curves of the tumors in Figure 8B, C, mice treated with (1) 15322 DOI: 10.1021/acsami.7b03604 ACS Appl. Mater. Interfaces 2017, 9, 15317−15327

ACS Applied Materials & Interfaces Research Article Figure 8. (A) Schematic illustration of in vivo antitumor eﬃcacy of the combination of GNR-PEG@MNs and MPEG-PDLLA-DTX micelles. (B) Representative photos of mice bearing A431 tumors after treatment for 7, 14, 21, 28, and 35 days (the black circle represents the location of tumors). (C) Growth curves and (D) body weight of the mice in each group. (E) Photograph and (F) weight of subcutaneous tumors in each group (the black circle represents the location of cured tumors). Data are represented as the mean ± standard deviation (n = 5). “★★” and “★” means the P < 0.01 and P < 0.05. (1. Saline, 2. GNR-PEG@MNs + laser, 3. DTX (5 mg/kg), 4. DTX (10 mg/kg), 5. DTX (5 mg/kg) + GNR-PEG@MNs + laser, 6. MPEG-PDLLA-DTX (5 mg/kg), 7. MPEG-PDLLA-DTX (10 mg/kg), 8. MPEG-PDLLA-DTX (5 mg/kg) + GNR-PEG@MNs + laser.) Figure 9. (A) Representative H&E stained images (scale bar, 20 μm), (B) Ki-67 immune histochemical images (scale bar, 20 μm), and (C) Ki-67 LI of tumors in each group. “★★” means the P < 0.01. (1. Saline, 2. GNR-PEG@MNs + laser, 3. DTX (5 mg/kg), 4. DTX (10 mg/kg), 5. DTX (5 mg/ kg) + GNR-PEG@MNs + laser, 6. MPEG-PDLLA-DTX (5 mg/kg), 7. MPEG-PDLLA-DTX (10 mg/kg), 8. MPEG-PDLLA-DTX (5 mg/kg) + GNR-PEG@MNs + laser.) saline had a rapid tumor growth, and then was the group of (2) GNR-PEG@MNs + laser, indicating that the GNR-PEG@MNs + laser group could inhibit tumor slightly which was due to PTT. Additionally, the (5) DTX (5 mg/kg) + GNR-PEG@ MNs + laser group had better inhibit ability than (3) DTX (5 mg/kg) and (4) DTX (10 mg/kg) groups in the early times, while the tumors had a recurrence at day 25 after the treatment, implying that the GNR-PEG@MNs could enhance the antitumor ability of DTX, but the low dosage of DTX could not inhibit tumor growth for a long time. Compared to the DTX group with DTX-loaded MPEG-PDLLA micelles at the same dosage, the MPEG-PDLLA-DTX micelles group had better antitumor ability than the DTX group, which was consistent with the previously reported study.56,57 Whether the DTX group or DTX-loaded MPEG-PDLLA micelles group, the antitumor ability of high dose (10 mg/kg) was better than the low dose (5 mg/kg) group. It was noted that the (8) MPEG- PDLLA-DTX (5 mg/kg) + GNR-PEG@MNs + laser group 15323 DOI: 10.1021/acsami.7b03604 ACS Appl. Mater. Interfaces 2017, 9, 15317−15327

ACS Applied Materials & Interfaces had a more rapid tumor volume reduction than the group of (7) MPEG-PDLLA-DTX (10 mg/kg), and all mice were cured within 14 days after the treatment. In detail, after the combined treatment, the tumors were reduced rapidly and gradually replaced by a scab. Then the scab faded, and the skin was cured at day 20. Notably, no tumor recurrence was observed up to now, demonstrating that the GNR-PEG@MNs could enhance the antitumor ability of MPEG-PDLLA-DTX micelles and reduce the dosage of DTX. Moreover, the photograph of the tumor in the mice (Figure 8E) was consistent with Figure 8B, C. As shown in Figure 8D, the body weight of the mice did not change a lot in the treatment groups. The statistic data of the tumor weight was presented in Figure 8F. What’s more, the major tissues were collected for histopathology study (Figure S7), and no lesions or inﬂammation was found in the hearts, livers, spleens, and kidneys tissues. For lung tissues, it was obvious to see congestion, necrosis, and alveolar deformation in (1) saline and (2) GNR-PEG@MNs + laser group. These results demonstrated that the GNR-PEG@MN was a safer carrier for PTT and could improve the antitumor ability of MPEG-PDLLA-DTX micelles. In short, the synergetic eﬀect of the combination of low dosage MPEG-PDLLA-DTX micelles (5 mg/kg) and GNR- PEG@MNs completely eradicated the A431 tumor without recurrence in vivo. The photothermal agent GNR-PEG@MNs was inserted into the tumor site transcutaneously which was harmless to the skin and minimized the uncomfortable feeling. With the combination of GNR-PEG@MNs, the dosage of MPEG-PDLLA-DTX micelles reduced to 5 mg/kg, which could reduce the occurrence of serious side eﬀects. Compared with other chemo−photothermal therapy systems,12,58 this synergetic system is expected to have great potential in clinical translation for human epidermoid cancer therapy. Tumor cells Histopathological and Proliferation Study. Inspired by the results of in vivo antitumor eﬃcacy, we explored the tumor cells histopathology and proliferation (3 mice per group). Histopathological study of tumor tissues (Figure 9A) revealed that although apoptosis and necroptosis occurred in the tumor site of the (2) GNR-PEG@MNs + laser group there were still some surviving tumor cells in the tumor site. Compared with the group of (3) DTX (5 mg/kg), (4) DTX (10 mg/kg), and (5) DTX (5 mg/kg) + GNR-PEG@ MNs + laser, more tumor cells disappeared in the tumor site in the (7) MPEG-PDLLA-DTX (10 mg/kg) micelles group and (8) MPEG-PDLLA-DTX (5 mg/kg) + GNR-PEG@MNs + laser group, which further indicated that micelles have good antitumor ability and the GNR-PEG@MNs played an important role in enhancing the antitumor eﬃcacy and reducing the dosage of drug for cancer therapy. The proliferation of the tumor cell was investigated by Ki-67 immune histochemical staining (Figure 9B). The saline group has the most Ki-67 immunoreactivity in the treated groups. Compared with the DTX and the MPEG-PDLLA-DTX group, the micelles group had less Ki-67 positive cells in tumor tissues at the same dose, indicating that the micelles could signiﬁcantly inhibit tumor cell proliferation. What’s more, the groups of (5) DTX (5 mg/kg) + GNR-PEG@MNs + laser and (8) MPEG- PDLLA-DTX (5 mg/kg) + GNR-PEG@MNs + laser group had better ability of inhibiting tumor cell proliferation than (3) DTX (5 mg/kg) and (6) MPEG-PDLLA-DTX (5 mg/kg) micelles groups, demonstrating that the GNR-PEG@MNs could enhance the antitumor ability of MPEG-PDLLA-DTX under the irradiation of 808 nm laser and reduce the dosage of Research Article the drug. The quantitative data of the proliferation of tumor cells are shown in Figure 9C. ■ CONCLUSIONS In summary, we successfully prepared MPEG-PDLLA-DTX micelles, GNR-PEG, and PLLA MNs. Then we used the layer by layer method to achieve GNR-PEG@MNs. The GNR- PEG@MNs had good skin insert ability and were harmless to the skin. In addition, the GNR-PEG@MNs had the heating eﬃcacy as well as GNR-PEG, and the temperature of GNR- PEG@MNs could be controlled by NIR light. The in vivo NIR thermal imaging and heating transfer eﬃcacy study proved that the heating eﬀect of the GNR-PEG@MNs could be transmitted to the middle of the tumor tissue and reach 50 °C. The combination of GNR-PEG@MNs and low dosage of MPEG- PDLLA-DTX micelles (5 mg/kg) eﬃciently inhibited the tumor growth and cured all mice without recurrence. Therefore, this novel NIR-responsive GNR-PEG@MN could be a promising strategy to enhance the antitumor eﬀect of the chemotherapy and is expected to have a great potential in clinical translation for human epidermoid cancer therapy. ■ MATERIALS AND METHODS Preparation and Characterization of DTX-Loaded Micelles. In this study, we prepared DTX-loaded MPEG-PDLLA micelles according to a previously published method.6,9 In detail, 5 mg of DTX and 95 mg of MPEG-PDLLA were dissolved together in appropriate anhydrous ethanol. Then the ethanol was removed in vacuum at 37 °C by a rotary evaporator. Finally, the MPEG-PDLLA-DTX micelles were prepared by adding 5 mL of deionized water at 60 °C and ﬁltered with a 220 nm syringe ﬁlter. The DL and EE of DTX-loaded MPEG- PDLLA micelles were determined by the HPLC (Agilent 1260 HPLC, USA) method as reported before.9 DLS (Nano-ZS90, UK) and TEM (H-6009IV, Japan) were used to characterize the DTX-loaded micelles. The cellular uptake eﬃciency and in vitro cytotoxicity of DTX and MPEG-PDLLA-DTX micelles were carried out on A431 cell lines. Preparation and Characterization of GNR-PEG. The GNR was synthesized through a seed-mediated method described before.19 The GNR-PEG was synthesized via a ligand exchange method.51 Brieﬂy, 1 mL of GNR (1 mg/mL) was reacted with 20 mg of PEG-SH dissolved in 19 mL of deionized water and stirred at 25 °C. After reaction for the night, the GNR-PEG was centrifuged at 12 000 rpm for 15 min, and then the the precipitates were collected and dispersed in deionized water. FTIR and EDS (JSM-7500F, JEOL, Japan) were used to conﬁrm whether PEG-SH was modiﬁed on GNR successfully. The morphology, zeta potential distribution, and absorption spectra of GNR and GNR-PEG were measured by TEM, DLS, and UV−vis absorption spectrometry (PE, USA), respectively. Preparation of PLLA MNs. The microneedle master structure which was made from a polydimethylsiloxane (PDMS) template was supplied by Jianghan University. The PLLA MNs were prepared as follows. First, the dried PLLA was placed on a PDMS mold and then press-molded at 170 °C for 15 min on the as-prepared PDMS mold to obtain arrays of PLLA MNs. Preparation and Characterization of GNR-PEG@MNs. The layer by layer method53,54 was used to prepare GNR-PEG@MNs. Brieﬂy, the PLLA MNs were put into 50 mL of PEI solution (0.5 mg/ mL) for 2 h to get positive charge. Then pure water was used to wash the PLLA MNs and blown dry by nitrogen. In addition, PLLA MNs with positive charge were put into GNR-PEG solution (0.5 mg/mL) which had negative charge for 2 h. At last, pure water was used to wash them, and they were blown dry by nitrogen to get GNR-PEG@MNs for which the gold content was 31.83 ± 1.22 μg per patch measured by inductively coupled plasma-atomic emission spectrometry (ICP-AES). FTIR and UV−vis transmittance spectroscopy (PE, USA) were used to ensure whether GNR-PEG absorbed on PLLA MNs to achieve 15324 DOI: 10.1021/acsami.7b03604 ACS Appl. Mater. Interfaces 2017, 9, 15317−15327

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