Photothermal Combined Therapy of Superficial Skin Tumors

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Au Nanocage-Strengthed Dissolving Microneedles for ChemoPhotothermal Combined Therapy of Superficial Skin Tumors Liyun Dong, Yuce Li, Zhao Li, Nan Xu, Pei Liu, Hongyao Du, Yamin Zhang, Yuqiong Huang, Jinjin Zhu, Guichao Ren, Jun Xie, Ke Wang, Yajie Zhou, Chen Shen, Jintao Zhu, and Juan Tao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18293 • Publication Date (Web): 01 Mar 2018 Downloaded from http://pubs.acs.org on March 2, 2018

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ACS Applied Materials & Interfaces

Au Nanocage-Strengthed Dissolving Microneedles for ChemoPhotothermal Combined Therapy of Superficial Skin Tumors Liyun Dong,†,# Yuce Li,‡,# Zhao Li,‡ Nan Xu,† Pei Liu,‡ Hongyao Du,† Yamin Zhang, † Yuqiong Huang,† Jinjin Zhu,† Guichao Ren,† Jun Xie,‡ Ke Wang,‡ Yajie Zhou,† Chen Shen,† Jintao Zhu,‡,* Juan Tao†,* †

Department of Dermatology, Union Hospital, Tongji Medical College, Huazhong University of

Science and Technology (HUST), Wuhan 430022, China ‡

Key Laboratory of Material Chemistry for Energy Conversion and Storage (HUST), Ministry of

Education, School of Chemistry and Chemical Engineering, HUST, Wuhan 430074, China #

These authors contributed equally to this work

*

Corresponding authors, E-mail: [email protected] (J. Zhu); [email protected] (J. Tao)

1 Environment ACS Paragon Plus

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ABSTRACT: For superficial skin tumors with high incidence, surgery and systemic therapy are relatively invasive and possible to cause severe side effect, respectively. Yet, topical therapy is confronted with the limited transdermal capacity due to the stratum corneum barrier layer of skin. Therefore, it is crucial to develop a highly effective and minimally invasive alternative transdermal approach for treating superficial skin tumors. Here, we developed gold nanocage (AuNC)- and chemotherapeutic drug doxorubicin (DOX)-loaded hyaluronic acid dissolving microneedle (MN) arrays. The loaded AuNCs are not only reinforcers to enhance mechanical strength of the MNs, but also effective agents for photothermal therapy to obtain effective transdermal therapy for superficial skin tumors. The resultant MNs can effectively penetrate the skin, dissolve in the skin and release cargoes within the tumor site. Photothermal effect of AuNCs initiated by near-infrared (NIR) laser irradiation combined with the chemotherapy effect of DOX destroyed tumors synergistically. Moreover, we verified the potent anti-tumor effects of the DOX/AuNCs loaded MNs after four administrations to superficial skin tumor-bearing mice without obvious side effect. Therefore, the drugs-loaded AuNC-loaded dissolving microneedle system provides a promising platform for effective, safe, minimally invasive combined treatment of superficial skin tumors. KEYWORDS:

Dissolving

microneedles,

Transdermal

Photothermal therapy, Superficial skin tumors


drug

delivery,

Chemotherapy,

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1. INTRODUCTIOON Superficial skin tumors (SST), including benign tumors (e.g., hemangioma, sebaceous nevus, seborrheic keratosis, etc.), precancerosis (e.g., actinic keratosis, etc.) and malignant tumors (e.g., squamous cell carcinoma, malignant melanoma, Bowen's disease, extramammary Paget's disease, mycosis fungoides, etc.), are the most common tumors in humans with increasing incidences worldwide.1 People from all communities and ages were influenced by the SST, which seriously threaten the health and life of human beings.2 For treating the SST, surgery is quite invasive which needs long recovery time for patients, and remains risk of tumor recurrence if the tumor tissues are removed incompletely or scarring when they located on the face. Some of the patients could not tolerate surgery due to the complications with other diseases, such as heart failure. On the other hand, chemotherapy, one of the most classic anti-tumor therapies, however, usually showed unsatisfied efficacy in the malignant melanoma due to the poor response duration and survival of patients. Some reports demonstrated that combined therapies are usually more effective than single chemotherapy.3-5 Photothermal therapy could ablate the tumor cells by converting light to thermal energy under laser irradiation, so that especially suitable for the treatment of SST.6 The combination of photothermal therapy and chemotherapy can obtain intensive anti-tumor effects at lower drug dosages than conventional single treatments, thus avoiding the unpleasant side effects.7-9 To achieve combined therapeutic effects, chemotherapeutic drugs and photothermal agents usually need to be delivered precisely into tumor site simultaneously. Systemic administration such as intravenous injection usually need high administration doses to guarantee local drug amounts in the tumor sites, which will damage the normal cells and cause severe side effects to human body.10 Compared with other administration routes, direct injection into the tumor site is



more effective. Although traditional intra-tumor injection of drugs in solution could relatively reduce the dosage, it may cause the drug leakage to neighbor normal tissues since there is no enough spaces intra the solid tumor interstitium for the liquids, resulting in toxicity to the normal tissues and loss of effective drug in tumor area.11 Topical therapy is safe and convenient, while the weak penetrability of drugs to get through the stratum corneum barrier layer of skin limited its further applications,12 even with the help of some penetration enhancers. In addition, the intratumoral drug amount delivered by passive diffusion is limited as well. Therefore, an effective, safe and minimally invasive transdermal approach for treating SST is highly needed. Microneedles (MNs) are needle arrays with micrometer dimensions that could effectively penetrate the skin barriers of the stratum corneum and create pathways for drug delivery into the skins.13 Due to the significant advantages such as easy operation, excellent skin-penetration ability and painless, the MNs have drawn more and more attentions in the past decades as promising multi-drug co-delivery platforms.14 So far, research and application of MNs mainly focus on delivering vaccines, small molecules, biotherapeutics, anti-cancer agents, siRNA, aesthetic medicines, and collecting fluid samples for biomarker testing.15-21 Differ from the other types of MNs (i.e., solid, coated and hollow MNs14), dissolving MNs fabricated from soluble, biocompatible and biodegradable materials could penetrate into skin, self-dissolve in the skin and release the payloads without causing adverse effects.22 Owing to the advantages of simple mass production, limited drug loss during absorption process, patch-free, precise dosing, rapid drug release, good biocompatibility and painless, dissolving MNs are suitable for drug delivery to the superficial tissues, especially to the SST. Nowadays, several groups performed dissolving MNs for treating SST on animal models, and some inspiring results were obtained. For example, Gu et al. reported a self-dissociation MN patch for the sustained delivery of anti-PD1 antibody


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with enhanced antitumor efficacy.23 Chen et al. developed a dissolving MN system co-loading with lanthanum hexaboride and anticancer drug, which displayed effective synergistic chemophotothermal therapy of superficial tumors.7 Mostly reported dissolving MNs for in vivo applications are fabricated from biopolymers, such as polysaccharides (e.g., hyaluronic acid (HA), carboxymethyl cellulose (CMCs) and chitosan), due to their excellent biocompatibility, biodegradability and solubility.24-26 Despite the promising features, weak mechanical properties of the polysaccharides limited their applications.12,

27

Increasing polymer concentration in the preparing procedure is efficacious,

however, it makes the manufacturing process more difficult because of the significantly enhanced viscosity of the polymer solution.28 Meanwhile, crosslinking endows MNs with enhanced strength along with decreased solubility as well.29 Recently, Chen et al. reported that incorporation of nanoparticles (e.g., layered double hydroxides) could increase the elastic modulus of carboxymethylcellulose MNs,27 potentially useful for generation of MNs with sufficient mechanical strength for in vivo applications. Among various photothermal materials, gold nanocages (AuNCs) stand out because of their chemical stability, ease of synthesis, good biocompatibility, photoluminescence characteristic, and excellent photothermal effect.30, 31 Compared to the other kind of Au nanoparticles (e.g., nanorod), AuNCs possess higher extinction coefficient so that they can induce same photothermy at lower mass concentration and less particle number,32 allowing the useage of less nanoparticles to achieve the same therapeutic effect. Herein, we developed Au nanocage-loaded HA microneedle arrays to load chemotherapeutic drug doxorubicin (DOX) (Scheme 1). Mechanical strength, determined by nanoindentation, was significantly improved after the incorporation of AuNCs. The transdermal effect and drug release behavior of the MNs were



further investigated. We also evaluated the biodistribution and clearance of the loaded agents in vivo. Moreover, we established tumor-bearing mice model to evaluate the in vivo anti-tumor effect of this drug delivery platform through the combination of chemotherapy and photothermal therapy. 2. EXPERIMENTAL SECTION 2.1. Materials: Polydimethylsiloxane (PDMS) were purchased from Dow Corning Corporation (Midland, MI, USA), HA and DOX was purchased from Meilunbio® (Dalian, China), while the other chemicals were supplied by Sinopharm Chemical Reagent Co., Ltd (Beijing, China). Gold nanocages were prepared as previously reported procedure.33 Briefly, CH3COOAg (0.8 mL, 62.4 g/L in glycol) was rapidly added into the mixed solution of NaSH (0.03 mmol/L), HCl (0.25 mmol/L) and polyvinyl pyrrolidone (PVP) (4.0 g/L) in glycol (10 mL) at 150 °C, and remained stirring for 1 h. The resultant solution was then cooled by ice-bath and treated with 20 mL of acetone, followed by centrifugation at 8000 rpm for 10 min and washed with Mili-Q water (Millipore, Billerica, MA, USA) twice. The obtained Ag nanocubes were redispersed in Mili-Q water (100 mL) containing 0.15 g of PVP, and HAuCl4 aqueous solution (14.0 mL, 1.0 mmol/L) was then dropwise added at 90 °C and stirring for 30 min. After cooling to room temperature, saturated NaCl solution was supplied to remove the generated AgCl. The purified AuNCs were obtained after centrifuging at 8000 rpm for 10 min and washing with Mili-Q water twice. 2.2. Characterization of AuNCs. Morphology of AuNCs were observed by transmission electron microscope (TEM, FEI TecnaiG2 F20, Eindhoven, the Netherlands) at 200 kV. ζ-potential, size and size distribution of AuNCs were measured with a dynamic laser scattering (DLS, Malvern Nano-ZS90, Malvern, UK). UV–vis absorption measurement were performed on a UV–vis spectrophotometer (UV-1800, Shimadzu Inc., Kyoto, Japan). X-Ray Diffraction was performed


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on an X-ray diffractometer (XRD, EMPYREAN, Almelo, the Netherlands) with Cu radiation target. Luminescent property of AuNCs were detected by an Edinburgh FLS920 spectrofluorometer (Edinburgh Instruments Ltd., Livingston, UK). 2.3. Preparation of the MNs: MNs were prepared through a two-step micromolding method. Briefly, PDMS was firstly casted on a steel microneedle master mold (Micropoint Technologies Pte Ltd, Singapore) to obtain a flexible PDMS female mold. HA aqueous solution (300 g/L) was then poured into the PDMS female mold under vacuum and the sample was allowed to dry in a sealed desiccator overnight, followed by peeling off from the molds and storing in a sealed desiccator at room temperature under nitrogen. To prepare drug-loaded MNs, excess aqueous solutions containing DOX and/or AuNCs at certain concentrations were firstly covered and filled in the MN mold under vaccum. Thereafter, redundant solution was removed to make sure each cavity was filled with same volume of drug solution. Then, the solution was allowed evaporation for a short period to concentrate the solution to the MN tips before HA was covered on the mold to form the base. 2.4. Characterization of the MNs: Morphologies of MNs were observed by a dermoscope (STL1-500X, Gaosuo, Shenzhen, China) and scanning electron microscope (SEM, Siron 200, FEI, Eindhoven, the Netherlands). To determine drug loading efficiency, the MNs were firstly dissolved in a certain volume of Mili-Q water, and then DOX and AuNC contents were detected by UV–vis absorption spectra and inductively coupled plasma mass spectrometry (ICP-MS, ELAN DRC-e, PerkinElmer, Norwalk, CT, USA) using standard curve methods, respectively. Modulus of MNs was determined by a nanoindenter (Piuma Chiaro, Optics11, Amsterdam, The Netherlands) with a sphere silicon indenter. Indenter was pressed into MNs with constant strain rate (0.05 s−1) from the tip of the MNs till the maximal load reach 700 mN. Data were



analyzed by the Piuma software (Piuma Chiaro, Optics11, Amsterdam, The Netherlands) using the Hertzian contact theory. 2.5. Cell Culture and In Vitro Cellular Uptake: Mouse melanoma cell line B16F10 were purchased from American Type Culture Collection (ATCC), and were cultured in Dulbecco’s modified eagle’s medium (DMEM, Gibco, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, USA) and 1% (v/v) penicillin-streptomycin (Hyclone, USA). Cells were cultured at 37 °C in a humidified incubator (Heraeus, Germany) with 5% CO2. The solution of AuNCs (0.1 mg/mL) and DOX (33 µg/mL) were incubated with B16F10 melanoma cells for 0 min, 10 min, 1 h, 24 h, and 48 h, respectively. After that, the cells were washed with phosphate buffer solution (PBS, 0.1 M, pH 7.4, Guge, China) to remove residual free AuNCs in media, and fixed with 4% paraformaldehyde (Dafeng Inc., China) for 20 min at room temperature. The cellular nuclei were stained with Hochest 33258 (Sigma-Aldrich, USA) for 5 min, followed by washing for three times with PBS to remove free Hochest molecules. Then, the cells were observed by confocal laser scanning microscopy (CLSM, Leica TCSNT1, Wetzlar, Germany). 2.6. In Vitro Photothermal Effect of AuNCs: Photothermal effect of AuNCs was performed by recording the time-dependent temperature variation profiles under a NIR laser (1.0 W/cm2, 808 nm) using a K-type thermocouple (Model SC-GG-K-30-36, Omega Engineering Inc., Stamford, CT, USA). To verify whether photothermal effect of AuNCs could kill tumor cells in vitro, B16F10 cells were incubated with normal saline (NS) or AuNCs solution for 24 h and exposed to NIR laser (Changchun New Industries Optoelectronics Tech. Co. Ltd, MDL-III-808nm-2.5W13120010, China) at a power density of 2.5 W/cm2 for 5 min. After 45 min incubation, the CCK-


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8 assay (Cell Counting Kit-8, Dojindo, Japan) was performed to evaluate the cell viabilities of each group according to manufacture instructions. The experiments were performed in triplicate. 2.7. Insertion Capability: To assess the skin insertion capability, AuNCs-loaded MNs were applied onto the dorsal skin of a hair-removed mouse for 5 min. The MNs were removed subsequently, and the skin surface at the insertion sites was imaged by the dermoscope. The frozen sections of the samples were viewed using the fluorescence microscope (Nikon Eclipseci, Tokyo, Japan). To visualize the pathways made by MNs into mice skin and distribution of the loaded AuNCs in the pathways, CLSM images were taken every 10 µm in depth with total depth of 300 µm. To detect the time-dependent dissolution of MNs in vitro, the tips of MNs were treated with 30% water/ethanol (v/v) solution, which is close to the water content in skin, and the morphologies of MNs were observed by a dermoscope (STL1-500X, Gaosuo, China). The in vivo dissolution of MNs were observed by dermoscope and SEM after being treated with mice skin for 5 min. 2.8. Animals: Six-week-old C57 mice were purchased from Institute of Laboratory Animal Science (Beijing, China), and maintained in standard specific pathogen-free (SPF) conditions in the Laboratory Animal Center of Huazhong University of Science and Technology. All experiments were conducted according to the guidelines of the Laboratory Animal Center. During the animal experiments, the local Ethical Committee Quantita Protocol and Chinese law was followed. 2.9. In Vivo Photothermal Effect: Subcutaneous melanoma mice model was establish by injecting 1× 106 of B16F10 cells at the dorsal flank of C57 mice. When the subcutaneous tumors grew to 4–6 mm in diameter (7–10 days after injecting the cells), dorsal hair around the tumor site was removed. Tumor sites with or without DOX/AuNCs-loaded MNs treatment were



exposed to NIR laser (power density of 1 W/cm2 for 1 min), and the infrared thermal images of the tumors were taken by infrared fusion thermal imager (Ti95, Fluke, China) immediately after the laser exposure. 2.10. In Vivo Distribution and Biocompatibility: C57 mice were injected with 1× 106 of B16F10 cells at the dorsal flank to obtain subcutaneous melanoma mice model. When the subcutaneous tumors grew to 4–6 mm in diameter (7–10 days after injecting the cells), dorsal hair around the tumor site was removed. The mice were randomly divided into six groups: (1) no treatment; (2) 1 h after being treated with DOX/AuNCs@MN; (3) 6 h after being treated with DOX/AuNCs@MN; (4) 24 h after being treated with DOX/AuNCs@MN; (5) 48 h after being treated with DOX/AuNCs@MN; (6) 72 h after being treated with DOX/AuNCs@MN. To determine the retention time of the loaded DOX and AuNCs in tumors and their bio-distribution in mice, fluorescence signals at tumor sites on the tumor-bearing mice were immediately investigated using live fluorescence imaging system (IVIS Lumina XR, Caliper, Mountain View, CA, USA) when the mice were sacrificed. Then, the ex vivo tumors and organs (tumor-draining lymph node, heart, liver, spleen, lungs and kidneys) were obtained by dissecting the mice, and were observed using the live fluorescence imaging system to get their fluorescence signals. The excitation/emission wavelengths (λex/λem) for AuNC and DOX are 690/750 and 488/580 nm, respectively. To investigate the biocompatibility of DOX/AuNCs-loaded MNs system, C57 mice were randomly divided into two groups: (1) Normal mice group, in which the mice were not injected with tumor cells; (2) Tumor-bearing mice group, in which the mice were injected with 1× 106 of B16F10 cells at their dorsal flank to obtain subcutaneous melanoma mice model. Both groups were received the same DOX/AuNCs@MN+NIR laser treatment. For the normal mice, we


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applied DOX/AuNCs@MN+NIR laser on their dorsal flank normal skin. For the tumor-bearing mice model, we provided DOX/AuNCs@MN+NIR laser on their tumor sites. The treatments were given on day 0, 3, 6 and 9, and all mice were sacrificed on day 12 and major organs (heart, liver, spleen, lung and kidney) were obtained for further pathological investigation. 2.11. In Vivo Anti-tumor Effect: C57 mice were seeded with B16F10 cells at a density of 1 × 106 tumor cells/100 µL PBS into the dorsal flank to establish the subcutaneous melanoma mice model. When the subcutaneous tumors reached 4–6 mm in diameter (7–10 days after injecting the cells), the mice were randomized into seven groups: (1) control group, in which the mice received no treatment; (2) NIR group, in which the mice were only exposed to NIR laser (power density of 1 W/cm2 for 1 min); (3) MN group, in which the mice tumors underwent insertion of hyaluronic acid MNs without drug; (4) DOX-loaded MN group, in which the mice were treated with DOX-loaded MNs at the tumor site for 5 min; (5) AuNCs-loaded MN + NIR group, in which the mice were treated with AuNCs-loaded MNs at the tumor site and subsequently exposed to NIR laser (1 W/cm2 for 1 min); (6) DOX and AuNCs solution + NIR group, in which the mice received intra-tumor injection of DOX and AuNCs solution and subsequent NIR laser irradiation (1 W/cm2 for 1 min); (7) DOX and AuNCs-loaded MN + NIR group, in which the mice were treated with DOX and AuNCs-loaded MN and subsequently exposed to NIR laser (1 W/cm2 for 1 min). Amount of AuNCs and DOX in solution were the same as that in MNs. The NIR laser was applied 30 min after the MN insertion or the intra-tumor injection treatment. All the groups received the treatments on day 0, 3, 6, and 9. The mice were sacrificed on day 12, and the tumors were harvested to get tumor weight. Body weights and tumor volumes (0.5 × Length × Width2) of the mice were recorded every 2 days, n = 6. Same processes were carried out for survival rates to day 45, n = 6.



3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of AuNCs. Preparation of AuNCs were illustrated in Figure 1a. Briefly, well-shaped Ag Nanocubes were firstly prepared through reduction of Ag (I) by glycol in the presence of PVP and NaSH (Figure 1b). AuNCs were then prepared by the galvanic replacement reaction of HAuCl4 and Ag nanocubes. Our results indicated that the formed AuNCs have uniform shape and narrow size distribution with negative surface charge (−15.4 ± 0.2 mV) (Figure 1c, 1d). The average hydrodynamic diameter obtained by DLS (129.4 nm) were slightly larger than that from TEM investigation (59.2 nm in side length). The UV-vis absorbance peak of Ag nanocubes and AuNCs were 441 and 792 nm, respectively, which is similar to previous report (Figure 1e).33 The diffraction peaks in XRD pattern (Figure 1f) located at 38.2°, 44.4°, 64.5°, 77.5°, and 81.7° can be attributed to the (111), (200), (220), (311), and (222) planes of the face-centered cubic (fcc) structure of gold (JCPDS No. 04-0784), respectively, which agreed well with the previous report,34 indicating the successful synthesis of nanostructures of AuNCs. Other small peaks were assigned to the traced residues of AgCl (JCPDS No. 31-1238).35 The excitation spectrum (λem 750 nm) and emission spectrum (λex 690 nm) of AuNCs indicated that AuNCs possessed good luminescence properties (Figure 1g, 1h). 3.2. Preparation of the MNs: The MNs were fabricated through a two-step micromolding method, as described in the experimental section (Scheme 2). The needles were well arranged with uniform sizes and morphologies (Figure 2). As shown in the SEM images (Figure 2a and 2b), each of the pyramid were 450 µm in height, 200 µm in base width, and were arranged into a 10 × 10 microneedle array with a 500 µm in center-to-center distance, which were exactly accordant with the master mold. All of the microneedles displayed sharp tips, which is necessary to ensure the insertion capability of MNs in the skin.


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The drug loading capacities of the MNs were determined by UV–vis spectrophptometer and ICP-MS investigation, respectively (see Figure S1 and S2 in the Supporting Information (SI)). Loading amount of both DOX and AuNCs in the MNs can be precisely regulated by adjusting their feeding concentration of the aqueous solutions. Generally, distribution of the loaded drugs in the MNs correlates with the drug delivery depth in skin. Thus, dermoscope investigation on the drug-loaded MNs were performed (Figure 2c and 2d) to determine the location of the loaded drugs in the MNs. Compared with the control MNs, black tips of AuNC-loaded MNs in the dermoscope image indicated that most of the loaded AuNCs are concentrated on tips of the pyramid in the MNs, as indicated by the arrows in Figure 2d. Therefore, HA MNs with loaded AuNCs at the tips of the needles and controlled structures were successfully fabricated through our technique. 3.3. In Vitro Cellular Uptake and Photothermal Effect of AuNCs: To confirm whether tumor cells could efficiently uptake the AuNCs, the B16F10 melanoma cells were incubated with the AuNCs solution for different time periods. In general, AuNCs display fluorescence signal under laser irradiation due to localized surface plasmon resonance.30 As shown in Figure 3a, red fluorescent signals of the AuNCs around the nuclei, which were stained in blue, were observed after incubation with the cells for 10 min, indicating that the AuNCs could be rapidly internalized by the B10F10 cells. With the extension of incubation time, a significant timedependent increase of fluorescence intensity around the nuclei was observed, implying that total amounts of intracellular AuNCs increased with incubation time. After incubation for 24 and 48 h, intense red fluorescence signal from AuNCs was observed around the nuclei, and clearly reflected the outlines of the cells. We also investigated the cellular uptake efficiency of DOX,



and we reduced the concentration of DOX to 33 µg/mL due to its toxicity. The intracellular red fluorescence intensity of DOX exhibited a time-dependent increase manner (Figure S3). Photothermal effect of AuNCs in DOX/AuNCs@MN was confirmed by a time-dependent temperature variation of DOX/AuNCs@MN solutions under NIR irradiation at different AuNC concentrations (Figure S4). The solution temperature increased significantly within 5 min, and the temperature increased with the increase of AuNC content. To evaluate the cytotoxicity induced by photothermal effect in vitro, B16F10 cells were incubated with the AuNCs for 24 h with or without NIR laser irradiation. Clearly, the AuNCs did not show any significant cytotoxicity in absence of NIR laser irradiation, indicating the good biocompatibility of the AuNCs (Figure 3b). When exposed to NIR laser, viability of the AuNCs treated cells decreased to 55% while cell viability of NS group was 88%, indicating the significant difference in cell viability between the two groups. This result indicated that the NIR laser exposure at certain power density and time could cause slight decrease of cell viability, and the AuNCs could largely enhance the damage of the tumor cells due to the photothermal effect. 3.4. Mechanical Strength and Skin Penetration of the MNs: The stratum corneum is the first protective barrier of skin with much higher mechanical strength than the corium layer. Effective skin puncture can be achieved by the sharp needles only if the modulus of the needle tips is higher than 35 MPa.36, 37 Thus, Young’s moduli of the prepared MNs were measured through nanoindentation approach (Figure 4 and Figure S5, S6). The nanoindentation cycle consists of three periods: loading-holding-unloading. Young’s modulus was calculated by the loading periods through the Hertzian contact theory. Loading forces were increased at constant velocity and the nanoindenter tip sank into materials during the loading period. In general, stronger materials require higher forces to achieve the same penetration depth during the loading period.


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Although effective Young’s moduli of MNs could be manipulated by increasing the HA concentration (Figure S6), increase of HA concentration will lead to significant rise in the viscosity of HA solution, making it difficult to generate uniform and well-shaped MNs through our technique. For example, when HA concentration was 400 mg/mL, some resultant MNs were obtuse and defective (Figure S7). The MNs containing AuNCs displayed significantly enhanced resistance to indentation, and thus have much higher Young’s moduli than the control MNs at the same HA concentration (Figure 4). The lower HA concentration and viscosity make it easier for the generation of uniform and well-shaped MNs (Figure 2). With the increase of AuNC contents from 0 to 4 µg/patch, the effective Young’s moduli of the MNs increased from 68.9 to 224.9 MPa. Incorporation of AuNCs significantly enhanced the mechanical strength of the MNs, which could be attributed to the reinforcement of Au nanoparticles to the polymeric materials.27,38 When loaded with DOX, Young’s modulus of the MNs decreased from 68.9 to 38.4 MPa (Figure 4). For DOX/AuNC loaded MNs, Young’s moduli of the MNs also displayed similar AuNC concentration-depended behavior. These results indicated that AuNCs were effective reinforcers for the HA-based MNs. Based on these results, the AuNCs-loaded MNs were used to assess the skin penetration capacities of the AuNCs-strengthened MNs. After pressing the MNs into the dorsal skin of a hair-removed mouse at a constant force, the skin surface exhibited an array of black spots which correspond to the inserting sites of the AuNCs-strengthened MNs (Figure 5a), indicating that the MNs could effectively penetrate the stratum corneum. The result was further confirmed by the histological frozen section examination, exhibiting the shape of MNs penetration pathways and the insertion depth of ~ 220 µm (Figure 5b and 5c). In comparison, the MNs without AuNCs displayed weak skin penetration capacities due to their relatively lower elastic modulus (Figure



S8). Therefore, the loaded AuNCs were helpful to improve the mechanical strength of the HA dissolving MNs and the penetration of needles through the stratum corneum. To confirm the dissolving time of MN tips after penetrating the stratum corneum, we performed the time-dependent dissolving images of MNs (Figure S9). Since HA is highly water soluble, the MN tips dissolved rapidly within 3 min after being treated with 30% water/ethanol (v/v) solution, which is close to water content in skin. Moreover, the in vivo dissolving experiments also indicated that the MN tips can be well dissolved in mouse skin after being treated for 5 min, which were observed by both dermatoscope and SEM (Figure S10). These results demonstrated that the MNs could be disintegrated rapidly and released the drugs into the skin. To visualize AuNCs release in the vertical direction of the mouse skin, the punctured sites of skin were observed at increasing depths from the skin surface using CLSM (Figure 5d). Red fluorescent array of AuNCs spots was clearly detected in the skin (Figure 5d). During the total depth of 300 µm of scanning from the skin surface, intensity of AuNCs fluorescence signal at the inserting sites increased gradually from skin surface to the depth of ~150 µm, and kept strong between 150 µm and 230 µm, and then reduced slowly as the scanning went deeper. This result was consistent with the insertion depth and drug distribution results of the MNs where most of the AuNCs were loaded in the needle tips (~ 1/5 of the needle height). As shown in Figure 5e, the red spots in the CLSM image and black spots in bright field image could almost overlap in the merged image, indicating that the released AuNCs were right inside the insertion pathways and only a few portions spread to the surrounding tissues. 3.5. In Vivo Photothermal Effect of AuNCs: The in vivo photothermal effect of AuNCs was evaluated using the infrared thermal imaging system by applying the DOX and AuNCs loaded


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MNs and subsequently NIR laser irradiation at tumor sites in subcutaneous melanoma mice model (Figure S11). Compared to the base temperature (33.6 °C) of mice tumor sites without MNs and NIR laser irradiation, local temperature of tumor with only NIR laser irradiation without MNs was 39.4 °C. Comparatively, for the mice treated with DOX and AuNCs loaded MNs followed by the same NIR laser irradiation, temperature at tumor site rose up to 55.0 °C. Generally, irreversible cellular damage will occur when temperature at heated tissues reaches critical value (e.g., hyperthermia temperature at 42 °C).39 For the NIR group mice, temperature at tumor site increased slightly and limited inhibition on tumor growth was achieved. However, for the DOX/AuNCs-loaded MN + NIR group, the photothermal effect of AuNCs was triggered by NIR laser and then elicited direct destruction of tumor cells in vivo. 3.6. Distribution and Biocompatibility of DOX and AuNCs Loaded MNs in Vivo: To investigate the duration time of loaded DOX and AuNCs in MNs at the tumor sites, we have investigated fluorescence signal variation from DOX and AuNCs in B16F10 melanoma-bearing mice at different time points through a live fluorescence imaging system (Figure 6). Figure 6a, 6b presented the fluorescence images of DOX and AuNCs respectively at the tumor sites 0, 1, 6, 24, 48 and 72 h after MNs insertion. The AuNCs exhibited the highest fluorescence intensity at 24 h and displayed a significantly decreased intensity at 72 h. Moreover, the major organs (tumor-draining lymph node, heart, liver, spleen, lung and kidney) of these MNs treated mice were separated at different time periods and their fluorescence images were captured (Figure 6c, 6d). The DOX and AuNCs were mostly distributed in liver, then kidney and lung, and both DOX and AuNCs peaked at 24 h. The diminished fluorescence signals of both DOX and AuNCs at 72 h indicated that most of them could be excreted in 72 h after the administration of the



DOX/AuNCs-loaded MNs. Taking this into consideration, the MNs were administrated every 3 days in the following experiments. To demonstrate the biocompatibility of the DOX and AuNCs Loaded MNs systems, we performed the pathological images of major organs (heart, liver, spleen, lung and kidney) in normal mice and in tumor-bearing mice which both applied with DOX/AuNCs@MN+NIR laser (see Figure S12). The pathological images showed no obvious abnormal histologic changes in the organs of either group, demonstrating the good biocompatibility and safety profile of the dissolving MNs delivery system. 3.7. In Vivo Anti-tumor Effect of Combination of Chemotherapy and Photothermal Therapy with the MNs: To investigate in vivo anti-tumor effect of combination of chemotherapy and photothermal therapy with the MNs, different treatments were carried out using the subcutaneous B16F10 melanoma-bearing mice model. Following treatments were performed on the subcutaneous melanoma-bearing mice, respectively: only NIR laser irradiation, HA MNs, DOX-loaded MNs, AuNCs-loaded MNs with subsequent NIR laser, intra-tumor injection of DOX and AuNCs solution with NIR laser, DOX/AuNCs-loaded MNs with subsequent NIR laser. The tumor volumes, tumor weights, and body weights were recorded to compare the anti-tumor efficiency among the groups. The tumor-bearing mice without any treatment were chosen as the negative control. Clearly, after 4 rounds of treatments, the tumor volumes of the DOX-loaded MN group, AuNCs-loaded MN + NIR group, intra-tumor injection of DOX/AuNCs + NIR group, and DOX/AuNCs-loaded MN + NIR group were significantly lower than the control group (Figure 7a). The inhibition effect on tumor volumes of intra-tumor injection of DOX/AuNC + NIR group and DOX/AuNCs-loaded MN + NIR group were notably higher than DOX-loaded MNs group


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and NIR group, suggesting that the chemo-phototermal combined therapies had stronger inhibition capability on tumor growth. The AuNCs-loaded MN + NIR group exhibited certain tumor inhibition effect before day 10; however, the tumors began to grow more rapidly than the combined therapy groups after day 10. As a result, on day 12 when the mice were sacrificed, the final tumors volumes and tumor weights in AuNCs-loaded MN + NIR group were relatively higher than that in DOX/AuNCs-loaded MN + NIR group. Moreover, the control HA MNs had little impacts on the tumor growth due to their great biocompatibility. Although NIR laser irradiation can inhibit tumor growth at certain level, it is not enough to kill the tumor cells. Similar results were also found in the photographs of ex vivo tumors in Figure S13a. As shown in Figure 7b, compared to the tumor weight 1.73 ± 0.98 g for no treatment group, tumor weight for intra-tumor injection of DOX/AuNCs + NIR group and DOX/AuNCs-loaded MN + NIR group were 0.27 ± 0.12 g and 0.30 ± 0.18 g, respectively, which were significantly lighter. In addition, these combined therapy groups demonstrated much lighter tumor weights than NIR group and DOX-loaded MN group. The tumor weight of AuNCs-loaded MN + NIR group was 0.44 ± 0.14 g, and no statistically significant differences were detected in comparison to the combined therapy groups. Yet, as shown in Figure S13a, the AuNCs-loaded MN + NIR group gave larger ex vivo tumor sizes than the combined therapy groups. These results were consistent with the ones for tumor volume in Figure 7a. Body weights of the control group and MN group increased rapidly due to the speedy tumor growth, and slightly decreased owing to cachexia at the advanced stage (Figure 7c). In the intratumor injection of DOX/AuNCs + NIR group, the body weight slightly decreased in the midterm of the treatment due to the mild toxicity of leakage of DOX to surrounding normal tissues, while rise again in the late period because the mice recovered from the mild toxicity. In



comparison, the body weight of the DOX/AuNCs-loaded MN + NIR group was stable, indicating the safety of MN arrays owing to the uniform drug delivery and no leakage to surrounding tissues. Survival curves indicated that 67% mice of the intra-tumor injection of DOX/AuNCs + NIR group and DOX/AuNCs-loaded MN + NIR group survived for more than 45 days (Figure 7d), which is significant higher than survival rate of the control group. For AuNCs-loaded MN + NIR group, although the tumor volumes and tumor weights showed no significant difference with the DOX/AuNCs-loaded MN + NIR group, the survival rate began to drop since day 23 and all mice were dead on day 33, indicating the insufficient anti-tumor effect of AuNCs-loaded MN + NIR group. No recurrence of the tumor was observed in the intra-tumor injection of DOX/AuNCs + NIR group and DOX/AuNCs-loaded MN + NIR group (Figure S13b), and the residual mice in these two groups survived for 30 to 40 days bearing slowly growing tumors. In the other five groups, no mice survived more than 33 days, and the control group and the MN group displayed the shortest survival times. The above results indicated that the combination of chemotherapy and photothermal therapy of the DOX/AuNCs loaded MNs exhibited stronger and more sustained inhibition effect on tumors than single therapy groups. 4. CONCLUSIONS In summary, we have successfully fabricated the AuNCs- and DOX-loaded dissolving HA MN arrays with enhanced mechanical strength. The resultant MNs could efficiently penetrate the skin barrier, dissolve and release the loaded drugs into the skin. The released DOX and AuNCs gradually accumulated in liver and finally were excreted out of the mice body 72 h after administration by the MN. The AuNCs with photothermal effect in MNs converted luminous energy to thermal energy to kill tumor cells initiated by NIR laser exposure, in addition to the


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directly damage to tumor cells by DOX. Moreover, the anti-tumor effects by the combination of chemotherapy and photothermal therapy of the DOX/AuNCs loaded MNs was verified using a tumor-bearing mice model in vivo. These results suggested that our combined treatment MNs systems exhibit significant inhibition on tumor growth and are promising effective drug delivery approach for superficial skin tumor therapy. ASSOCIATED CONTENT Supporting Information Available: Additional figures showing the drug loading capacity, nanoindendation test, in vivo photothermal effect of AuNCs and tumors size for different experimental groups. This material is available free of charge via the internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (J. Z.) and [email protected] (J. T.) Notes The authors declare no competing financial interest. Acknowledgement: This work was supported by National Natural Science Foundation of China (81502367, 81573047, 81602760 and 51703074), Clinical Research Physician Program of Tongji Medical College, HUST (5001530014), the China Postdoctoral Science Foundation (2017M612454) and the Scientific Research Training Program for Young Talents of Wuhan Union Hospital. We also thank the HUST Analytical and Testing Center for allowing us to use its facilities.



Figures:

Scheme 1. Schematic illustration showing drug/Au NPs-loaded dissolving hyaluronic acid microneedle system for the combination of chemotherapy and photothermal therapy of treating melanoma.


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Figure 1. Synthesis and characterization of AuNCs. (a) Schematic illustration showing the synthesis route of AuNCs: 1) Replacement reaction between Ag and HAuCl4, and the formation of a partially hollow nanostructures; 2) Formation of nanoboxes with a uniform, smooth, homogeneous wall composed of Au/Ag alloy. (b) TEM images of Ag nanocubes; (c) TEM images of AuNCs; (d) DLS, (e) UV–vis spectra, (f) XRD, (g) excitation spectrum (λem 750 nm) and (h) emission spectrum (λex 690 nm) of the formed AuNCs. The scale bar in (c) can be applied in (b).



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Remove redundant Short solution Vacuum evaporation

Drug solution

HA solution Peel off

Vacuum & dry

Scheme 2. Schematic illustration showing the preparation of drug-loaded MNs.


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Figure 2. (a, b) SEM images of the formed microneedles in drying state, (c, d) Bright field optical microscopy images of the formed microneedles. The arrows in (d) pointed to the black tips of AuNC-loaded MNs, indicating that most of the loaded AuNCs were concentrated on the tips of the needles.



Figure 3. (a) Fluorescence microscopy images of AuNCs uptaken by B16F10 melanoma cells in vitro. AuNCs (red) were incubated with B16F10 melanoma cells for different time. Cell nucleus was stained with Hoechst (blue). (b) Photothermal effect of AuNCs to B16F10 melanoma cells after 24 h incubation in vitro. These experiments were carried out in triplicate and the results were shown as the mean ± SD. *, P < 0.05；**, P < 0.01 (n=3).


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Figure 4. Effective Young’s modulus of the MNs with different AuNCs or DOX/AuNCs loading. Error bars represent the standard deviation.



Figure 5. Skin insertion ability of AuNCs-loaded microneedles. (a) Dermoscope image of hairremoval mice dorsal skin after microneedles insertion; (b and c) The fluorescence microscopy images from frozen section of mice ear skin penetrated by microneedles. The arrows in (b) indicate the microneedles insertion sites. (d) CLSM image of AuNCs-loaded microneedles applied mouse dorsal skin at varying depths; (e) CLSM image, bright field dermoscope image and merged image. Red fluorescence signal in CLSM image indicated the penetration of AuNCs in the skin indicated while black dots in the bright-field image indicated the microneedles insertion sites.


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Figure 6. In vivo fluorescence microscopy images of the B16F10 tumors treated with DOX/AuNCs loaded microneedles. The fluorescence intensity indicated relative amount of (a) DOX and (b) AuNCs at each time points. Fluorescence imaging of ex vivo major organs from B16F10 melanoma-bearing mice treated with DOX/AuNCs-loaded microneedles after various time periods. The fluorescence intensity indicated relative amount of (c) DOX and (d) AuNCs.



Figure 7. In vivo anticancer effect of microneedles in subcutaneous melanoma-bearing C57 mice by photothermal therapy and chemotherapy. (a) Tumor volume of each group with increasing time for twelve days. (b) Tumor weight of each group on day 12. (c) Body weight. (d) KaplanMeier survival curves. There were six mice per group. The results (a, b and c) were shown as the mean ± SD. Statistical significance was calculated by t-test. Comparisons of survival curves were made using the long-rank test. *, P < 0.05；**, P < 0.01; ***, P < 0.001. Same color in (bd) represented the same group shown in (a).


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Photothermal Combined Therapy of Superficial Skin Tumors

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