Near-Infrared Light-Activatable Microneedle ... - ACS Publications

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Near-Infrared Light-Activatable Microneedle System for Treating Superficial Tumors by Combination of Chemotherapy and Photothermal Therapy Mei-Chin Chen,* Zhi-Wei Lin, and Ming-Hung Ling Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan 70101 S Supporting Information *

ABSTRACT: Because of the aggressive and recurrent nature of cancers, repeated and multimodal treatments are often necessary. Traditional cancer therapies have a risk of serious toxicity and side effects. Hence, it is crucial to develop an alternative treatment modality that is minimally invasive, effectively treats cancers with low toxicity, and can be repeated as required. We developed a light-activatable microneedle (MN) system that can repeatedly and simultaneously provide photothermal therapy and chemotherapy to superficial tumors and exert synergistic anticancer effects. This system consists of embeddable polycaprolactone MNs containing a photosensitive nanomaterial (lanthanum hexaboride) and an anticancer drug (doxorubicin; DOX), and a dissolvable poly(vinyl alcohol)/polyvinylpyrrolidone supporting array patch. Because of this supporting array, the MNs can be completely inserted into the skin and embedded within the target tissue for locoregional cancer treatment. When exposed to near-infrared light, the embedded MN array uniformly heats the target tissue to induce a large thermal ablation area and then melts at 50 °C to release DOX in a broad area, thus destroying tumors. This light-activated heating and releasing behavior can be precisely controlled and switched on and off on demand for several cycles. We demonstrated that the MN-mediated synergistic therapy completely eradicated 4T1 tumors within 1 week after a single application of the MN and three cycles of laser treatment. No tumor recurrence and no significant body weight loss of mice were observed. Thus, the developed light-activatable MN with a unique embeddable feature offers an effective, user-friendly, and low-toxicity option for patients requiring long-term and multiple cancer treatments. KEYWORDS: cancer therapy, locoregional treatment, superficial tumor, synergistic effect, transdermal drug delivery, triggered release

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being tested in clinical trials for treating superficial head and neck cancers and lung cancer by Nanospectra Biosciences, Inc.2 In PTT, plasmonic nanoparticles are delivered to tumors either intravenously or intratumorally; the tumors are subsequently laser irradiated at the nanoparticle resonant energy to generate considerable heat for ablation. After irradiation, the nanoparticles are cleared from the tumor sites and accumulated in the liver, spleen, and other organs because of the leaky nature of the tumor vasculature.3−6 Treatments are often repeated because some tumor cells survive and continue to grow after the initial treatment. Furthermore, because of rapid clearance from the tumors, nanoparticles should be reinjected to achieve an adequate concentration within the tumors for the following PTT. However, multiple and frequent

ancer is the most common life-threatening illness and a leading cause of death, and the standard treatments for cancer include surgery, chemotherapy, and radiotherapy. However, these treatment regimens are used in cases of limited efficacy and are associated with toxicity and side effects. Therefore, alternative therapeutic approaches have been developed for treating cancer. Among these approaches, photothermal therapy (PTT), a minimally invasive local treatment, is an attractive alternative to conventional cancer therapies because of its remote controllability, easy applicability, and low systemic toxicity and side effects. PTT involves employing plasmonic nanoparticles localized in tumors as exogenous energy absorbers that convert laser energy into heat, causing irreversible cellular damage and subsequent tumor destruction. This heat generation can also cause coagulation within the tumor vasculature, thus enhancing the effects of other targeted therapeutics.1 In preclinical studies, PTT has efficiently treated superficial tumors, and it is currently © XXXX American Chemical Society

Received: August 13, 2015 Accepted: November 20, 2015

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DOI: 10.1021/acsnano.5b05043 ACS Nano XXXX, XXX, XXX−XXX

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Figure 1. Schematic illustrations of combination of chemotherapy and photothermal therapy using near-infrared (NIR) light-activatable microneedles (MNs). When inserted into a tumor from the skin surface, the supporting array patch can be quickly dissolved by the interstitial fluid, thus leaving the MNs in the target tissue for repeated and locoregional cancer therapies. Upon exposure to NIR light, the embedded MN array can uniformly heat the tumor to induce a large thermal ablation area and then melt to release doxorubicin in a broad area, thus destroying the tumor.

injections of these nanoparticles may cause adverse side effects, leading to patient discomfort. In this study, we developed a near-infrared (NIR)-lightactivatable microneedle (MN) system that repeatedly produces heat and simultaneously releases encapsulated anticancer drugs into the tumor when activated by the NIR laser (Figure 1). This system consists of embeddable polycaprolactone (PCL) MNs containing plasmonic nanoparticles (lanthanum hexaboride; LaB6) and an anticancer drug (doxorubicin; DOX), and a dissolvable poly(vinyl alcohol)/polyvinylpyrrolidone (PVA/ PVP) supporting array patch (Figure 2a). LaB6 nanomaterials were excellent absorbers of NIR irradiation and have been served as alternative plasmonic materials for thermal ablation of cancer cells.7−9 We encapsulated LaB6 nanoparticles into MNs to prevent their rapid clearance from the tumor, thus allowing repeated dosing and PTT from a single administration. When irradiated with NIR light, the LaB6 absorbs the laser energy and converts it into heat, inducing the MN melting at 50 °C, thus triggering DOX release from the MNs to the tumor. Once the light source is switched off, the temperature quickly returned to its initial value and the drug release is stopped. The light-activatable heating and releasing behavior of the proposed MNs can be used to produce a synergistic effect of chemotherapy and PTT in treating superficial tumors. Furthermore, the heat and drug dose delivered to the tumors can be well controlled by adjusting the irradiation parameters. In this MN system, the dissolvable PVA/PVP supporting array, connected to the base of the PCL MNs (Figure 2a), can provide an extended length for counteracting skin deformation during MN puncture and offer mechanical strength for completely inserting the MNs into the skin. On insertion, the supporting array is quickly dissolved by the interstitial fluid, thus embedding the MNs into the tissue for locoregional and repeated treatments (Figure 1). Because of this embeddable design of the MN, it is possible to combat the tumor from within, while sparing surrounding healthy tissue. Moreover, rapid dissolution of the supporting array can substantially

Figure 2. Characterization of NIR light-activatable MN system. This system consists of embeddable polycaprolactone (PCL) MNs containing photosensitive nanoparticles (lanthanum hexaboride; LaB6) and an anticancer drug (doxorubicin; DOX), and a dissolvable poly(vinyl alcohol)/polyvinylpyrrolidone (PVA/PVP) supporting array patch. (a) Schematic illustrations of the MN system. Bright-field micrographs of (b and b1) stainless steel MN master structure and (c and c1) DOX-loaded PCL MN system: b and c, low magnification, b1 and c1: high magnification. The inset in (a) shows the MN specifications.

minimize the patch wearing time and prevent uncomfortable feeling and skin irritation caused by long-term contact with transdermal adhesive or patches. In this study, to examine the modifiable on−off heating behavior, the infrared thermal images of MNs were taken when irradiated with NIR laser intermittently. DOX was then loaded in the MNs to assess the feasibility of light-modulated drug release. Finally, the in vivo antitumor efficacy of combined PTT B

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ACS Nano and chemotherapy using the NIR-light-activatable MNs was compared with that of PTT or chemotherapy alone in tumorbearing mice.

RESULTS AND DISCUSSION Characterization of Microneedles. MNs are a minimally invasive drug delivery platform, designed for efficient transdermal drug delivery by creating micropores in the stratum corneum. They have been considered as a more comfortable alternative to injections in delivering drugs, vaccines, and cosmetics into the skin.10−19 However, because of the inherent elasticity of the skin, considerable skin deformation occurs during insertion, which markedly reduces the insertion depth of MNs.20−22 Incomplete insertion of MNs may lead to reduced dose delivery and cause wastage of valuable medicines. In this study, we developed a completely insertable MN system with a dissolvable PVA/PVP supporting array design (Figure 2a) with an extended length for counteracting skin deformation during puncture. Figure 2b,b1 presents bright-field micrographs of stainless steel MN master structures. Here, we used a two-step casting process for localizing DOX (red) and LaB6 nanoparticles in the MNs to ensure that all drugs can be delivered into the target tissue, with no waste in the supporting array. As shown in Figure 2c,c1, the DOX-loaded MNs equipped with the PVA/PVP supporting array patch were successfully fabricated. This patch consisted of 81 (9 × 9) MNs that had a tip-to-tip distance of 1000 μm. The base width and height of the MNs and supporting array were 300 and 600 μm, respectively (Figure 2a, inset). To confirm whether the developed MNs enabled complete insertion into the skin, samples were applied to a porcine cadaver skin. After insertion for 5 min, the skin surface was wiped clean for removing the dissolved PVA/PVP supporting array patch. The skin surface revealed a complete array of red spots (9 × 9) that were corresponded to the MN puncture sites (Figure 3a,a1), showing that all MNs were inserted into the skin. Histological sections clearly demonstrated that DOXloaded MNs (red) were completely inserted and embedded within the tissue with a maximum insertion depth of 700−800 μm (Figure 3b,c). For sustained transdermal delivery, patients must wear a patch for a long time, thus causing inconvenience, an uncomfortable feeling, and skin irritation in some people. In our system, the PVA/PVP supporting array facilitates complete insertion of the MNs and is quickly dissolved by the skin interstitial fluid to reduce patch-induced adverse effects. Such a design is unique and particularly attractive and useful for patients requiring long-term transdermal patch therapy. Near-infrared-light-activatable Property of Microneedles. To evaluate if MN heating behavior could be activatable by NIR light, they were irradiated with laser for five on/off cycles. During each cycle, the MNs were exposed to NIR light until their temperature raised to 50 °C for 3 min; subsequently, the light source was switched off until the temperature dropped to room temperature. The morphological and temperature changes of the MNs were recorded in real time by using infrared thermal video. As shown in Video 1 and Figure 4a, NIR light rapidly elevated the MN temperature to 50 °C within 20 s because of the light-to-heat transduction of the encapsulated LaB6 nanoparticles. When the temperature reached 50 °C, the PCL MNs started melting and gradually became blunt (Figure 4b). Once the irradiation was switched off, MNs quickly cooled and turned into a solidified state (Video 1). Such heating and

Figure 3. Skin insertion ability of MNs. (a and a1) Porcine cadaver skin after insertion of DOX-loaded MNs and (b and c) their corresponding histological sections: a, low magnification; a1, high magnification; b, bright-field image; c, fluorescence image.

melting behavior can be repetitively activated by NIR light over several cycles. Moreover, a gradual melting of the MNs was found when the number of on/off cycles increased (Figure 4b). These results indicated that the proposed MNs are lightresponsive devices and their phase transition can be remotely triggered by NIR light. Near-Infrared-Light-Activatable Release of Anticancer Drug. An ideal drug delivery system enables users to adjust the dose regimens according to their physiological response and clinical requirements.23−25 However, most triggerable systems have drug leakage problems under a nontriggering condition and lack repeatability over multiple release cycles.26 To demonstrate the repeatability of our light-activatable system, the DOX-loaded MNs were applied onto a porcine cadaver skin and subsequently irradiated with laser light intermittently. During the laser-on state, the temperature was controlled at 50 °C for 3 (short period) or 6 min (long period); subsequently, the light source was turned off for 3 h. In the control group, the MNs were applied onto the skin but without NIR irradiation for 21 h. Figure 5 presents DOX release profiles of MNs that were (short period, square; long period, triangle) and were not (no NIR, circle) subjected to NIR light. Drug release occurred in a triggered and stepwise manner, where the laser-on state induced a steep increase in drug release, and the laser-off state permitted only negligible drug release. The DOX release can be repeatedly activated by NIR light, and consistent dosing can be observed after every irradiation period (11%−12% release per short-period irradiation; 22%−24% release per longperiod irradiation). Because of the highly hydrophobic and slow hydrolytic mechanism of the PCL, almost no DOX leakage was observed in the no NIR group. These results demonstrated that the NIR-activatable MN system enabled precise and controlled release of anticancer drugs, and the drug release can be C

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Figure 4. Light-activated heating behavior of MNs. (a) Temperature changes of MNs after irradiation with NIR light for 5 irradiation cycles. During each irradiation, MNs were exposed to laser until their temperature rises to 50 °C for 3 min and light source was then switched off until the temperature decreased to room temperature. (b) Thermal images of MNs at initial irradiation stage, and at the end of each laser-on state.

the skin. These results indicate that NIR laser deeply penetrates the skin tissue and is absorbed by the LaB6 encapsulated in the MN to generate heat, thus causing a phase transition in the PCL MNs. A solid-to-liquid transition of the polymer MN may enhance the molecular mobility, thus triggering DOX release. In Vivo Combination of Chemotherapy and Photothermal Therapy for Breast Cancer. To investigate the anticancer effects of MN-mediated chemotherapy and PTT in the severe combined immunodeficient (SCID) mice bearing 4T1 breast tumors, we compared the tumor volume, body weight, and survival rates of mice exposed to three cycles of short-period laser after inserting DOX-loaded MNs (the DOXloaded MN + NIR group) through the skin into the tumor with those of the untreated control, DOX, and MN + NIR groups. For the MN + NIR group, mice underwent insertion of LaB6loaded MNs (without DOX) into the tumor from the skin surface and subsequent irradiation using the same procedure employed for the DOX-loaded MN + NIR group. For the DOX group, mice were only intratumorally injected with a single high dose of DOX.27,28 After the treatment, the tumor volumes of the DOX, MN + NIR, and DOX-loaded MN + NIR groups were significantly lower than that of the untreated group (Figure 7a1). The tumor inhibition effects were markedly higher in the MN + NIR group than in the DOX group (Figure 7a1), implying that repeated PTT is beneficial for slowing tumor growth. Although most studies on hyperthermia have focused on the thermal effects on tumors at 42−44 °C, higher temperatures may increase the therapeutic benefits.29−32 A previous study reported that progressive necrosis of tumors occurs at temperatures >45 °C.29 In the MN + NIR group, tumor growth was completely inhibited until Day 14 (Figure 7a1), and no significant body weight loss was observed after the PTT period (initial 6 days) until Day 14 (Figure 7b), revealing that MN-mediated PTT at

Figure 5. Light-activated release behavior of MNs. DOX release profiles of MNs that were (short period, square; long period, triangle) and were not (no NIR, circle) subjected to intermittent NIR light (n = 5 for each group). During the laser-on state, the temperature was controlled at 50 °C for 3 (short period) or 6 min (long period); subsequently, the light source was switched off for 3 h. In the no NIR group, MNs were inserted into the skin but without NIR irradiation for 21 h. The DOX loading amount was 1013 ± 43 μg per patch (n = 5).

modulated by simply adjusting the time of laser exposure and number of on/off cycles. To visualize the morphological changes of the samples within the skin after NIR light irradiation, the MN puncture sites were excised and subjected to confocal microscopic analysis and histological examination. Figure 6 presents confocal 3D reconstruction images and histological sections of the MNinserted skin after exposure to short-period laser for zero, three, and five cycles. The embedded DOX-loaded MNs (red) decreased in size with an increase in the number of irradiation cycles. Furthermore, the NIR-induced melting behavior remained apparent even when the MNs were embedded within D

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Figure 6. Light-activated melting behavior of MNs. Confocal 3-D reconstruction images and their corresponding histological sections of the MN-inserted skin after exposure to short-period laser for zero, three, and five on/off cycles.

Figure 7. In vivo anticancer effect of MN-mediated chemotherapy and photothermal therapy in the SCID mice bearing 4T1 breast tumors. Changes of (a and a1) tumor volume, (b) body weight, and (c) survival rate of mice after treatments: untreated mice (black), mice injected with DOX intratumorally (blue), mice treated with MNs plus NIR light (green), and mice treated with DOX-loaded MNs plus NIR light (red) (n = 7 for each group). a1: the tumor volume changes for the first 14 days after treatment.

50 °C effectively destroys cancer cells and may not induce acute or severe toxicity. As shown in Figure 8, the PTT-treated tumor sites shrank and gradually turned into a black scar (arrow) on Day 20. DOX, a chemotherapeutic drug, is commonly used for treating breast cancer and other cancers. However, multiple and high doses of DOX are generally required for obtaining clinically evident anticancer efficacy.31 Moreover, directly injecting the DOX solution into the tumors may cause drug leakage into the neighboring healthy tissues, thus reducing the therapeutic efficacy and leading to toxicity concerns.33 Figure 7b presents initial body weight loss on the initial 3 days in the DOX group that may have occurred because of DOX-induced

toxicity. Chemotherapy or PTT alone slowed tumor growth; however, tumor growth continued after the cessation of therapy (Figures 7a,a1 and 8). This observation indicated that injecting a single high dose of DOX or administering PTT three times cannot completely eradicate tumors. In both the DOX and the MN + NIR groups, tumor recurrence led to gradual weight loss and death in the mice. Body weight loss and death occurred earlier and were more rapid in the DOX group than in the MN + NIR group (Figure 7b,c), indicating that MN-mediated PTT induces less toxicity than single, high-dose chemotherapy and prolongs the life span of mice. In the DOX-loaded MN + NIR group, the tumors gradually shrank and disappeared within 7 days of treatment (Figure E

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Figure 8. Photographs of untreated mice, mice injected with DOX intratumorally (chemotherapy), mice treated with MNs plus NIR light (photothermal therapy), and mice treated with DOX-loaded MNs plus NIR light (synergistic therapy) at Day 0, Day 10, and Day 20 after treatment. All the untreated mice died before Day 16.

United States.35 The primary treatment modality is surgical resection, followed by radiotherapy. However, patients undergoing extensive excision of the oral cavity experience severe speech, eating, and psychological disabilities. Moreover, radiotherapy is associated with several side effects, such as xerostomia, chronic dental decay, and risk of mandibular osteonecrosis, considerably affecting patient quality of life.36 Therefore, the development of an alternative therapy that is safe, repeatable, and has less adverse effects is required. In this study, we demonstrated the application of an NIRlight-activatable MN system for treating superficial tumors in a mice model. MNs were inserted into the tumor from the skin surface. This procedure is nonsurgical and minimally invasive. The mean skin thickness of mice used here was only 330−370 μm. Figure 3b shows that the proposed MNs can be fully inserted into the porcine skin at a depth of 700−800 μm, indicating that they can penetrate through the mice skin and into the subcutaneous tumor for local chemotherapy and PTT. According to this insertion depth, the MNs may locate between the hypodermis and the tumor tissue. After treatment, no tumor recurrence was observed in the mice (Figures 7 and 8), demonstrating that the MN-mediated combined therapy is effective and might be applicable for treating superficial tumors, particularly those in the skin or head and neck region. Figure S1 (Supporting Information) shows that after MN insertion, the holes created on the rat skin quickly heal, and the skin barrier function recovers within 6 h. This observation indicates that for treating oral cavity cancer, the developed system has the potential to substantially reduce postoperative oral disabilities and the wound care period, thus considerably increasing patient compliance. Moreover, the NIR-activatable MNs ensure convenient drug regimens for patients with cancer.

7a1). The tumors were gradually replaced by a hard scab by Day 10 (arrow in Figure 8) that eventually flaked off, and the skin completely healed by Day 20 (dotted circle in Figure 8). No tumor recurrence was observed until Day 50 (Figure 7a), revealing that the MN-mediated synergistic therapy completely eradicated the tumors. In all NIR groups, the short-period laser was given once every 3 days for a total of three cycles. During each irradiation, the drug release amount of the DOX-loaded MN + NIR group was only approximately one-third of the dose given by the DOX group. Reducing the DOX dose during each treatment may lower the drug toxicity to normal organs. Figure 7b shows that no considerable body weight loss was observed in the DOX-loaded MN + NIR group, and the body weight increased by 11% on Day 50. Furthermore, no abnormal behaviors or pathological signs were observed in all treated mice, demonstrating that the combined cancer therapy using MNs was tolerated well. Notably, the DOX-loaded MN + NIR group had a 100% survival rate 50 days after the treatment, whereas all mice in the control, DOX, and MN + NIR groups died between 16 and 38 days (Figure 7c). PTT combined with chemotherapy using DOX-loaded MNs exhibited anticancer efficacy superior to that of PTT or chemotherapy alone. This observation may be attributed to PTT-induced cytoplasm shrinkage of the tumors and increased space between the tumor cells, enabling DOX to spread throughout the tumors.33,34 Conventional hypodermic injections often poorly distribute DOX in tumors, thus only partially damaging tumor tissues.33 Moreover, drug leakage from the injection site may cause systemic toxicity (Figure 7b) and reduce the drug concentration at the tumor sites. In the proposed MN system, because of the array configuration, embedded MNs can uniformly deliver DOX and generate heat in a broad area to destroy more cancer cells and induce a large thermal ablation area. Compared with nanosized carriers, MNs are easily retained in target tissues because of their microscale size, thus enabling long-term and multiple chemotherapy and PTT through a single administration of MNs and preventing adverse side effects caused by multiple injections of anticancer drugs or plasmonic nanoparticles. Oral cavity cancer is common in the head and neck area, accounting for more than 17000 new cases annually in the

CONCLUSIONS This study revealed that the developed MN system effectively produces heat and simultaneously releases the encapsulated DOX into the tumor sites when activated by NIR light. Because the supporting array dissolves by the interstitial fluid, the MN array can be left within the target tissue for repeated, locoregional cancer treatments. These embedded MNs can uniformly deliver heat and anticancer drugs to the tumor, thus F

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To determine the loading and residual amounts of DOX in the MNs, the samples before and after insertion were separately dissolved in acetone and sonicated at 100 W for 1 h for DOX extraction. The amount of DOX extracted from the MNs was determined on the basis of the fluorescence intensity in the extract by using spectrofluorometer. Cell Culture. The mouse breast cancer cell line 4T1 (ATCC CRL2539) was kindly provided by Prof. Chao-Liang Wu (Department of Biochemistry and Molecular Biology, National Cheng Kung University, Tainan, Taiwan). The cells were cultured in Dulbecco’s modified essential medium supplemented with antibiotics containing 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% fetal bovine serum. The cells were then incubated in a humidified atmosphere containing 5% CO2 at 37 °C. Synergistic Effect of Chemotherapy and Photothermal Therapy. Eight-week-old SCID mice (CB17/Icr-Prkdcscid/CrlNarl, National Laboratory Animal Center, Taiwan) were maintained in microisolators under specific pathogen-free conditions and were fed sterile chow and chlorinated water. Subcutaneous tumor xenografts were established by injecting cell suspensions (105 cells) into the backs of the SCID mice. When the subcutaneous tumors grew to 70−100 mm3 (8−10 days after injecting the cells), the mice were randomized into four groups: (1) the control group, which received no treatment; (2) the DOX group, in which the mice were intratumorally injected with a single dose of DOX (300 μg) in phosphate-buffered saline (150 μL); (3) the MN + NIR group, in which the mice underwent insertion of LaB6-loaded MNs (without DOX) into the tumor from the skin surface and subsequent irradiation with short-period laser (5 W/cm2), which was administered once on Days 0, 3, and 6 and discontinued when the temperature of the MNs raised to 50 °C for 3 min; and (4) the DOX-loaded MN + NIR group, in which the mice underwent insertion of DOX-loaded MNs into the tumor from the skin surface and subsequent irradiation using the same procedure employed for the MN + NIR group. The mouse body weight and tumor volume (1/2 × length × width2) were recorded every 2 days.39 The tumor size was determined using caliper measurements, and the mice were euthanized when the tumors were ≥10% of the body weight.

causing tumor necrosis in a large area. We demonstrated that MN-mediated PTT and chemotherapy completely eradicated the tumors within 1 week after a single administration of MNs. As per our review of relevant literature, this study is the first to use polymeric MN devices for cancer therapy. These results suggest that NIR-light-activatable MNs exert a synergistic effect on tumor inhibition and are an effective, convenient, and tolerable treatment option for treating superficial tumors.

MATERIALS AND METHODS Preparation of Lanthanum Hexaboride Nanoparticles. LaB6 nanoparticles were prepared according to previous reports.7,37 Fabrication of a Microneedle System. An MN master structure was fabricated using precision electrical discharge machining technology (Hong Da Precise Industry Co., Ltd., Taiwan). A silicone polymer (polydimethylsiloxane, PDMS) is poured onto the stainless steel master structure to make an inverse MN mold.38 A two-step casting process was used for fabricating MNs with a dissolvable supporting array. First, a 25% (w/v) PCL solution was prepared by dissolving 2.4 g of PCL in 9.6 mL of acetone and stirring under 60 °C for 3 h. A LaB6 solution (4 mg/mL) and DOX (80 mg) were added to this PCL solution and then well mixed to obtain a homogeneous solution. One milliliter of the mixture was poured onto a filter paper that was covered onto the mold surface and then centrifuged at 3880g for 1.5 h. The filter paper was then removed and the mold was placed in a 70 °C oven overnight to form DOX-loaded MNs (i.e., the first layer). To form the dissolvable supporting array, a 60 wt % PVA/PVP aqueous solution prepared at a weight ratio of 2:1 was used to fill the PDMS mold again. One milliliter of the PVA/PVP solution was placed on the dried first layer, followed by further centrifugation at 5100 rpm and 30 °C for 30 min. The filled mold was air-dried at room temperature for 1 day and then placed in an oven at 37 °C for 1 day. Finally, the MN patches were peeled from the PDMS mold gently. An MN system with a nondissolvable supporting array was prepared for studying in vitro drug release. To develop this array, after being filled with the first layer, a PCL flake (0.5 g, 1 × 1 cm2) was put onto the mold surface and then placed into a vacuum oven set at −70 kPa for 6 h at 70 °C. After being cooled to 25 °C, the sample was taken from the PDMS mold. In Vitro Skin Insertion Test. To evaluate the in vitro skin insertion ability of DOX-loaded MNs, the MNs were inserted into porcine cadaver skin for 5 min until the supporting array completely dissolved in the skin. The inserted skin was selected and then prepared for histological specimens.37 Near-Infrared-Light-Activatable Property of Microneedles. To demonstrate the light-activatable ability, the prepared MN was irradiated with an 808 nm laser light (830 mW) intermittently at a power density of 5 W/cm2. During laser irradiation, the temperature was controlled at 50 °C for 3 min; the light source was then switched off until the temperature decreased to room temperature. The on/off cycle was repeated five times. During irradiation, thermal images of samples were recorded with an infrared thermal camera.37 In Vitro Near-Infrared-Light-Activated Release of Anticancer Drug. To assess the NIR-light-activatable DOX release ability of the MNs, the MNs were irradiated with laser for several cycles. The amount of released DOX was measured by subtracting the amount remaining in MNs embedded within the skin after NIR irradiation from the amount initially loaded in MNs. However, measuring the drug remaining in the MNs that are embedded within the skin is difficult. Therefore, the MN system with a nondissolvable supporting array was prepared for studying in vitro drug release. DOX-loaded MNs with the nondissolvable PCL supporting array were applied onto the skin and secured to it. Subsequently, the skin was irradiated with laser light, and the temperature was controlled at 50 °C for 3 (short period) or 6 min (long period); subsequently, the light source was switched off for 3 h. The laser on/off switch was repeated several times to evaluate the laser-dependent release. The samples without NIR irradiation were used as a control.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.5b05043. Real-time thermographic video of microneedles that were irradiated with an intermittent laser for 5 cycles (AVI) Skin resealing and recovory after microneedle treatment; materials; ethics statement; statistical analysis (PDF)

AUTHOR INFORMATION Corresponding Author

*M.-C. Chen. E -mail: [email protected]. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors would like to thank Prof. Dong-Hwang Chen from the Department of Chemical Engineering at the National Cheng Kung University in Taiwan for assisting with preparation of lanthanum hexaboride nanoparticles. This work was supported by grants from the Ministry of Science and Technology (MOST 103-2221-E-006-083-MY3 and MOST 103-2622-E-006-034-CC2). REFERENCES (1) Park, J. H.; von Maltzahn, G.; Xu, M. J.; Fogal, V.; Kotamraju, V. R.; Ruoslahti, E.; Bhatia, S. N.; Sailor, M. J. Cooperative Nanomaterial G

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DOI: 10.1021/acsnano.5b05043 ACS Nano XXXX, XXX, XXX−XXX