Separable microneedles for near-infrared light triggered transdermal

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Controlled Release and Delivery Systems

Separable microneedles for near-infrared light triggered transdermal delivery of metformin on diabetic rats Yang Zhang, Danfeng Wang, Mengyue Gao, Bin Xu, Jiangyin Zhu, Weijiang Yu, Depeng Liu, and Guohua Jiang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00642 • Publication Date (Web): 20 Jun 2018 Downloaded from http://pubs.acs.org on June 21, 2018

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ACS Biomaterials Science & Engineering

Separable microneedles for near-infrared light triggered transdermal delivery of metformin on diabetic rats

Yang Zhang,†,§,ǁ Danfeng Wang,‡ Mengyue Gao,† Bin Xu,†,§,ǁ Jiangyin Zhu,†,§,ǁ Weijiang Yu,†,§,ǁ Depeng Liu,†,§,ǁ and Guohua Jiang†, §,

†

⊥,

ǁ, *

Department of Polymer Materials, Zhejiang Sci-Tech University, Hangzhou, Zhejiang

310018, China ‡

Department of Gynecology and Obstetrics, Tonglu Maternal and Child Health Care Hospital,

Tonglu, Zhejiang 311500, China §

National Engineering Laboratory for Textile Fiber Materials and Processing Technology

(Zhejiang), Hangzhou 310018, China ⊥

Key Laboratory of Advanced Textile Materials and Manufacturing Technology (ATMT),

Ministry of Education, Hangzhou, Zhejiang 310018, China ǁ

Institute of Smart Fiber Materials, Zhejiang Sci-Tech University, Hangzhou, Zhejiang

310018, China

Corresponding author: Guohua Jiang, E-mail:[email protected], Tel: 86-571-86843527

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ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract: The near-infrared (NIR) light-triggered and separable segmented microneedles (MNs), consisted of lauric acid and polycaprolactone (LA/PCL) arrowheads and polyvinyl alcohol and polycaprolactone (PVA/PVP) supporting bases, have been fabricated. The hypoglycemic drug (metformin) and photothermal conversion factor (Cu7S4 nanoparticles) are capsulated into LA/PCL arrowheads. Owing to the dissolution of soluble supporting bases after absorption of tissue fluid, the separable MNs arrowheads can be embedded into skin after insertion. Under the near-infrared (NIR) light irradiation, the LA/PCL arrowheads exhibit an excellent thermal ablation change with low amount of Cu7S4 nanoparticles (0.1 wt%) due to the low melting point of LA and PCL, thus enabling the release behavior of encapsulated model drug to be photothermal triggered. Compared with hypodermic injection of metformin, a thermal ablation of separable MNs triggered with NIR irradiation in current research exhibit an excellent hypoglycemic effect in vivo. It suggests that the NIR-induced thermal ablation MNs is a prospective transdermal drug delivery system for precise control of timing and dosage of the drug dependent on a NIR administration.

Keywords: microneedles, transdermal delivery, diabetes, hypoglycemic effect, near-infrared (NIR) light

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INTRODUCTION Microneedles (MNs) are sharp, needle like structure with a diameter of micron and a length of less than 1 millimeter. MNs can create temporary mechanical channels on the skin surface and subsequently transport encapsulated drug across the skin barrier with painless due to a restricted penetration depth of typically less than 500 µm.1 Compared with conventional hypodermic needles, they represent an alternative technical approach to across stratum corneum for a better drug permeation in a minimally invasive,2 and they allow patient to safely treat by self-administration with a minimal risk of bleeding, injury, or infection. In particular, MNs administration brings convenience and saves costs for the patients, such as diabetes, who need to frequent medication.3-6

Many kinds of MNs have been developed for transdermal drug delivery in previous reports, including insoluble MNs,7 dissolvable MNs,8-10 coating MNs,11 and hollow MNs.12,13 Usually, insoluble MNs are made from metal,14 silicon,7,15 or undissolving polymers.16 They can construct micro channels on the skin surface after insertion, and the encapsulated drugs are gently released from the surface of MNs into skin tissue through these micro channels. Nevertheless, the contraction and occlusion of these micro channels may reduce the drug delivery efficiency of MNs.17 Hollow MNs are mainly used to deliver drug solution into body, however congestion of the micro channels maybe impede the transportation of drugs.17 The penetration ability and drug administration of coating MNs would subject to the coating formulation and procedures as well as coating capacity.18 Dissolvable MNs made from biocompatible and biodegradable polymers could deliver the payload gradually by a dissolution of the MN materials in skin tissue without a necessary disposal on polymers and 3



an infection possibility of skin.4

Noticeable progress has achieved in dissolvable MNs as a potential choice for targeted delivery percutaneously in recent years. Types of biocompatible and hydrosoluble polymers have been used for fabrication of dissolvable MNs for an efficient drug transportion.19-23 But there are still many problems which limit their future application: (1) The drug delivery efficiency is rely on the solubility of dissolvable MNs. The poor solubility may cause an insufficient drug release and a potential cutaneous Infections. (2) The toxicity generated with overdose or inefficacy caused by underdose would emerge on patients owing to a passive manner of most dissolvable MNs drug delivery regardless of physiological changes in treatment.

Thus, drug released on demand is contributed to balance the treatment efficacies and side-effects. The drug release rate could be controlled in biodegradable nano-particles which loaded in dissolvable MNs arrays.24-26 Numerous studies for triggered drug release by ultrasound, magnetic-field or light have been carried out.27-30 Recently, a MNs transdermal delivery system combined with H2O2-triggered mesoporous silica nanoparticles (MSNs) loaded with insulin has been developed in our group. Antidiabetic drug (insulin) and glucose oxidase (GOx) were loaded in the MSNs whose surface modified by host-guest complexation between 4-(imidazoyl carbamate) phenylboronic acid pinacol ester (ICBE) and a-cyclodextrin (a-CD). Glucose would translate into gluconic acid and create H2O2 by GOx in the MSNs. The phenylboronic ester bonds on the surface of MSNs would be broken due to the oxidation of H2O2, which leaded to a destruction and disassembly of host-guest complexation and followed a release behavior of preloaded insulin.31 Lately, a NIR-responsive segmented MNs 4


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system were developed in our group. In that system, prussian blue nanoparticles (PB NPs) was as a photothermal conversion factor while metformin was as an antidiabetic drug. Both of them were encapsulated into a polycaprolactone (PCL) MNs arrowheads. These MNs arrowheads were supported on dissoluble polymer basements and showed preeminent photothermal conversion efficiencies by adjusting the amount of PB NPs in MNs. Under NIR irradiation, the drug-loaded arrowheads phase state translated from solid to flow due to the rapid thermal ablation, thus causing the release of encapsulated metformin from MNs.32

Cu7S4 nanocrystals (NPs) is a novel-innovative type of photothermal conversion agent exhibiting an excellent photothermal conversion property and low cytotoxicity.33,34 They have been developed as NIR-driver photothermal agents for efficient photothermal ablation (PTA) of cancer cells.35 Normal cells of human body can withstand at the hyperthermia temperature range of 45~50 °C.36 The phase-change material (PCM) formulated from Lauric acid (LA) (m.p. = ~44 °C) and polycaprolactone (PCL) (m.p. = ~50 °C) exhibits a permissible melting range of 46~48 °C.37 Metformin is an anti-diabetic drug that mainly administered orally but with a biological utilization rate of about 55%, mostly absorbed through the small intestine.38 In this research, the hypoglycemic drug (metformin) and photothermal conversion factor (Cu7S4 nanoparticles) are capsulated into LA/PCL arrowheads that are supported on a solid PVA/PVP substrate. And the MNs arrowheads are left in the skin after insertion, followed the thermal ablation of MNs arrowheads for release of loaded drugs attribute to conversion from NIR irradiation (Scheme 1A). The NIR-responsive release of encapsulated drug into the skin tissue to research the hypoglycemic effect on diabetic Sprague-Dawley (SD) rats has been carried out (Scheme 1B). 5



Scheme 1. Schematics of thermal ablation behavior of separable MNs (A) and NIR triggered transdermal drug delivery on diabetic SD rats (B).

MATERIALS AND METHODS Materials Sulfur (99.99%), copper (II) nitrate trihydrate (Cu(NO3)2.3H2O, AR, 99%), oleyla mine (OM, 80~90%), 1-octadecene (ODE, >90% GC) and streptozocin (STZ) were obtained from Macklin (Shanghai, China). Lauric acid (LA, AR, 98%), polycaprolactone (PCL, MW=70~90 kDa), polyvinyl alcohol (PVA, type 1688), polyvinylpyrrolidone (PVP, MW = 100 kDa) and 6


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metformin were purchased from Aladdin (Shanghai, China). Polydimethylsiloxane (PDMS, Sylgard 184) gained from Dow Corning (Midland, MI) is poured onto the PMMA master mold (Patch size: 8 mm × 8 mm, Array Size: 10 × 10, Needle Height: 700 µm, Needle Base: 300 µm, Needle Pitch: 500 µm. Micropoint Technologies Pte Ltd, Singapore) to make a MNs mould. Male SD rats were provided from Zhejiang Academy of Medical Sciences (Hangzhou, China). All chemicals were used in experiments without any further aftertreatment. Cu7S4 NPs were prepared according to the previous reports.39,40

Characterization and MTT analysis of Cu7S4 NPs To confirm the preparation of Cu7S4 NPs, we had done the XRD analysis (ARL XTRA, Thermo ARL, Switzerland). The morphology and composition of Cu7S4 NPs were characterized by TEM (JEM-2100, JEOL, Japan). To evaluated the cytotoxicity of Cu7S4 NPs, the methyl thiazolyl tetrazolium (MTT) assay on the HeLa cells.41 About 6,000/well cells with 180 µL Dulbecco’s modified eagle medium (DMEM) were cultured into a 96-well plate in a CO2 (5%) humidified incubator at 37 °C for 48 h, and 5 mg/mL MTT solution of 20 µL was then added to each well. After that, these cells were exposed to 5, 10, 20, 40, 80 µg/mL of Cu7S4 NPs for 24 h, and 150 µL DMSO was added into each well after the DMEM were removed from wells. The absorbance of cells solutions each well were measured with microplate reader (Multiskan MK3, Thermo Electron Corporation) to assess the cytotoxicity.

Fabrication of phase-change materials To fabricate low phase transition temperature materials, LA and PCL were dissolved in methanol with different mass ratios (1:2, 1:1, 2:1), and then dried in vacuum oven after 30 7



min of agitation. The melting temperature range of PCM were analyzed by DSC (Q2000, America).

Fabrication of MNs and mechanical strength test The PDMS female MNs mold was prepared by a PMMA master mold for fabricating MNs.42 The metformin-loaded LA/PCL MNs with dissolvable PVA/PVP supporting substrate were then fabricated via a two-step molding technology. Firstly, 0.6 g of PCM was dissolved in 3 mL of dichloromethane (DCM) and 3 mL of methanol (ME) to obtain a 10% (w/v) uniform PCM solution with stirring. A homogeneous Cu7S4 NPs/PCM/metformin solution was prepared by dissolving Cu7S4 NPs and metformin (97%, Alladin) into the previous PCM solution and then stirring for 20 min. About 0.1 mL of the mixture was added into the qualitative filter mesh (pore diameter: 80-120 µm) loaded MNs mold then centrifuged (10,000 rpm) for 15 min to prepare the MNs tips loaded with metformin, and the treated mold was followed placed at 60 °C for 10 min to wipe off the residuary DCM and ME solvents with the filter paper was removed. Second step, a 30 wt% PVA hydrogel and a 30 wt% PVP hydrogel were mixed at a weight ratio of 1:2. Approximately half a milliliter of the composite hydrogel was placed onto the treated mold loaded MNs tips via another centrifugation (8,000 rpm) for 5 min to generate dissolvable supporting bases. Then the obtained LA/PCL-PVA/PVP MNs were peeled from the mold gently after desiccated in a room temperature overnight and characterized by SEM (Vltra55, Zeiss, German) and macroscope (BST500xUSB, Baisite, China). Three kinds of separable MNs with a different weight ratios of LA/PCL tips (2:1, 1:1, 1:2) were prepared to analyze the transdermal strength of them by a material testing machine (5943, Instron Co. Ltd., America). To analyze the morphology changes of the separable MNs 8


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after mechanical test, a macroscope (BST500xUSB, BAISITE, China) was used to observe at different displacement.

To measure the drug release quantity in vitro, an inseparable MNs with PCL supporting substrate can be extracted out from puncture sites was fabricated. In detail, 0.1 g PCL pellets were placed on the surface of MN tips loaded mold followed the whole mold was put in 60 °C oven for 10 min. Then the mold was again centrifuged (8000 rpm) for 5 min after PCL melt. The obtained inseparable MNs can be peeled from mold immediately.

To visualize the drug release behaviors and skin penetration studies in vitro, a Rhodamine 6G (R6G, Macklin, Shanghai, China) was used as the micromolecule to replace metformin. In brief, R6G (1 mg/mL) was added to a uniform Cu7S4 NPs/PCM solution in the first step of MNs fabrication with the second step was unchanging. An inseparable MNs was prepared with R6G-loaded MN tips and PCL supporting bases to observe the melting behavior of MN tips under different NIR irradiation time (0, 1 and 3 min) after extracted out from puncture sites.

NIR-light-responsive properties of MNs To determine the applicable density of Cu7S4 NPs in the MNs, MNs with different concentrations (0, 0.1, 1.0 and 3.0 wt%) of Cu7S4 NPs in MN tips were irradiated with a NIR light (808 nm, 1600 mW, 0.4 W/cm2) continuously to obtain the temperature curve and observe the melting behavior. For the MNs (0.1 wt%), 5 cycles of laser on/off were applied to evaluate the repeatability of the NIR-triggered photothermal conversion. During each cycle, the MNs were irradiated with NIR for 2 min (laser on) and NIR laser was subsequently 9



switched off until MNs temperature decreased to indoor temperature (laser off). The thermal images and real-time temperatures of MNs samples were recorded with a NIR thermal imager (Ti400, Fluke, America) and the brightfield macroscopic images were recorded by a macroscope.

Skin penetration in vitro To analyze the thermal ablation behavior of MNs tips in skin, stripped skin samples were penetrated by R6G-loaded inseparable MNs exposed an NIR irradiation for 0, 1 or 3 min. A macroscope was used to photograph the morphology of puncture sites and treated MNs extracted from skin. R6G-loaded separable MNs were penetrated to stripped skin then irradiated with NIR laser for 0, 1 or 3 min respectively to research the in vitro skin insertion property and drug release behaviors of separable MNs. A confocal laser scanning microscope (CLSM, Axio Observer A1, Carl Zeiss, Germany) was used to characterize the treated skin samples by reconstructing a three-dimensional (3-D) images of R6G diffusion before histological sections analysis.

Thermal damage assay To assess the skin tissue thermal injury of MNs system, several densities of Cu7S4 NPs (0.1, 1.0 and 3.0 wt%) were applied on skin samples under different NIR irradiation duration (0, 1, 3 and 5 min) for followed histological studies with hematoxylin and eosin (H&E) staining. The skin samples without insertion but exposed to NIR irradiation were used as a control.

Drug release behavior in vitro 10


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To analyze the in vitro NIR-responsive behavior, metformin-loaded (1 mg) inseparable MNs were inserted to skin samples followed extracted out to dissolved in a 10 mL of dichloromethane/methanol (w/w = 1:1) solution completely after irradiated with NIR light for 2 min (short period) or 4 min (long period) each cycle. And nonexposure for 2 h with NIR laser off after that. Several laser on/off cycles were iterated to assess NIR-responsive release behavior. The concentrations of residual metformin were determined by analyzing the solutions with an UV-Vis spectrophotometer (U3900H, Hitachi, Japan).

Hypoglycemic effect in vivo The SD rats weighed 200±20 g were used for preparation of a tape 2 diabetic mouse model by streptozotocin (STZ) according to previous literature.43 Five experimental groups (n = 5 for each group) were obtained from diabetic rats to analyze hypoglycemic effects in vivo: (1) Healthy group, with non-treatment on healthy rats; (2) Control group, with non-treatment on diabetic rats; (3) Injection group, with a single dose of metformin subcutaneous injection on diabetic rats (~1 mg) ; (4) MNs group (Low dose), with ~0.5 mg metformin-loaded MNs applied on diabetic rats under three cycles of 4 min NIR irradiation; (5) MNs group (High dose), with ~1 mg metformin-loaded MNs on diabetic rats under three cycles of 4 min NIR irradiation. The MNs groups were inserted into rats’ skin with assistance of a spring applicator obtained from Micropoint Technologies Pte Ltd (Singapore). To manifest the NIR-light-triggered property of MN system in vivo, some other diabetic rats in MNs groups were fed for 30 min after each cycle, and the next cycles were started at 30 min after fed. The blood glucose levels (BGLs) were monitored in real time by a blood glucose meter (Sinocare Inc., Changsha, China). 11



Acute toxicity assay and biodistribution To evaluate the acute toxicity of MNs system, treated rats from each MNs group were selected randomly and sacrificed for histology analysis.44,45 Paraffin-embedded sectioning of major organs (heart, liver, spleen, lung and kidney) fixed in 10% neutral buffered formalin were then treated with H&E staining and observed by an inverted fluorescence microscope (Ts2, Nikon). All of procedures above were conducted by HuaAn Biotechnology Co., Ltd (Hangzhou, China).

RESULTS AND DISCUSSION Preparation of NIR triggered and separable MNs Cu7S4 nanoparticle is a relatively novel type of photo-thermal conversion factor with an excellent photo-thermal ablation performance. Here, Cu7S4 nanoparticles have been selected as the photo-thermal conversion factors because of their biosafety and simple synthesis process. Cu7S4 nanoparticles are firstly prepared from Cu(NO3)2/oleylamine (OM) and sulfur/1-octadecene (ODE) solutions at 180 °C for 30 min (Figure S1).33 Owing to the superior photo-thermal conversion efficiency and low cytotoxicity of them (Figure S2), Cu7S4 nanoparticles are utilized to prepare NIR-induced thermal ablation MNs. A two-step micromolding process is conducted to fabricate our MNs, as shown in Figure S3. Usually, MNs are not easily inserted into skin from top to bottom for its high elasticity.46,47 To address to this issue, the MNs arrowheads consisted of Cu7S4 nanoparticles, metformin and LA/PCL PCM are supported on dissolvable PVA /PVP base to as a mechanical spacer for elimination of skin deformation. After the as-fabricated MNs insertion into skin, the drug-loaded MNs 12


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arrowheads can be imbedded into skin tissue owing to a solvation of PVA/PVP sustentacular substrate after absorption of interstitial fluid. Under NIR irradiation, the LA/PCL arrowheads can be melt, thus releasing the carrying drugs from embedded MNs.

The MNs prepared from pure LA and PCL are unsatisfactory due to the low mechanical strength of LA and high melting point of PCL (~50 oC). According to previous reports,32,48-50 instantaneous tissue thermal damage generates when skin is exposed to temperature overtop 50 oC. By adjusting the composition of LA and PCL, the MNs prepared with weight ratio of LA and PCL at 1:2 exhibit the appropriate initial melting range of 46~48 oC. And their mechanical strength is 0.18 N/needle at displacement of 400 µm, which make them have enough strength for insertion into skin (Figure S4). The morphology alterations of MNs with displacement at 400 µm can be observed after mechanical compression what are different from their initial state. The needle tip of MNs shown a bend without fracture and separation demonstrates the excellent flexible and toughness of the prepared MNs (inset in Figure S4B). In following study, MNs fabricated with weight ratio of LA and PCL at 1:2 are chosen as a research model for evaluation of the photo-thermal ablation property and applications in vivo and in vitro.

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Figure 1 Digital images of as-fabricated NIR triggered and separable MNs in this study (A), SEM images of separable MNs without (B) and with the photo-thermal conversion agent (C).

Figure 1A show the typical pyramid-shape of as-prepared MNs under microscope with an arrangement of 10×10 placed on a patch of 1 cm2. Further exact information of MNs on arrays can obtained from SEM images. As shown in Figure 1B and 1C, these MNs reveal an obvious double-section structure with clear edges. The upper-layered arrowhead is ~400 µm in height. These arrowheads are supported on a solid basement with an uncovered length of 200 µm. The separation distance between two MNs is about 600 µm while the substrate width 14


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of each MN is approximately 300 µm (Figure 1B-a and 1C-a). The roof of as-prepared MNs is a quadrangle with a siding of ~10 µm (Figure 1B-b and 1C-b). EDS analysis has further confirmed the successful embedding of Cu7S4 nanoparticles into MNs (Figure 1B-c and 1C-c).

Thermal ablation properties of MNs The Cu7S4 nanoparticles display a strong NIR absorption under irradiation.34,51 Their photo-thermal conversion efficiency is higher than 77.0%.33 In the NIR-responsive MNs system, the release rate of encapsulated drugs and thermal damage degree of skin tissue may be influenced by the loaded amount of Cu7S4 nanoparticles in MNs to as a critical parameter, which possibly affects the photothermal transformation from NIR light. To evaluation the photothermal conversions and thermal ablation properties of MNs, varying contents of Cu7S4 nanoparticles were housed into MNs arrowheads under a consecutive NIR irradiation (808 nm) with an intensity of 0.4 W/cm2.

Figure 2A reveals the time-temperature distribution of MNs arrowheads with different contents of Cu7S4 nanoparticles under a NIR irradiation. All MNs display a rapid augment of temperature except the pure polymer MNs. Compared to MNs loaded with 0.1 wt% of Cu7S4 nanoparticles, more Cu7S4 nanoparticles (1.0 wt% or 3.0 wt%) utilized MNs exhibit a faster elevation, which can up to more than 50 oC of temperature within 30s and may take a thermal damage to skin tissues. While the temperature of MNs with a lower content of Cu7S4 nanoparticles (0.1 wt%) will be maintained around 48 oC under irradiation within more than 50 s, which may be suitable for drug delivery through skin tissue. By contrast, there is 15



negligible temperature variation of MNs without Cu7S4 nanoparticles. Repetitive intermittent NIR irradiation experiment (5 cycles) is further implemented on lesser amount Cu7S4 nanoparticles (0.1 wt%) loaded MNs to analyze a circulating photothermal conversion property. As shown in Figure 2B, these temperatures rapidly rise to exceed 45 oC with NIR laser turned on by 1 min. And the temperature quickly drops to room temperature after the NIR laser is switched off. This result demonstrates that the as-fabricated MNs show a rapidly stabilize photothermal transformation behavior caused by NIR light. The surface infrared thermal images at different exposure times are photographed with a NIR thermal imager (Fluke Ti400, USA). As shown in Figure 2C, there is almost no change in temperature of these MNs with an absence of Cu7S4 nanoparticles. In sharp contrast, the temperature rises rapidly from the initial ~20 to ~48 oC within the first 20 s with loading of 0.1 wt% of Cu7S4 nanoparticles in MNs.

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Figure 2 Time-temperature curves of MNs loaded varying amounts of Cu7S4 nanoparticles (A) and the lower amount of Cu7S4 nanoparticles (0.1 wt%) under repetitively intermittent NIR light for 5 cycles (B). Infrared thermal images of MNs prepared with and without Cu7S4 nanoparticles under a continuous NIR irradiation in the initial 40 seconds (C).

A morphology change of MNs tips after NIR-light irradiation also can be observed under microscope. As shown in Figure S5, the needle tips with the absence of Cu7S4 nanoparticles can keep their initial shape even for a long time NIR irradiation (80 s). However, the MN arrowheads are easily to be melted in the existence of Cu7S4 nanoparticles. The MNs tips become dull at NIR irradiation for 60, 10 and 1 s with loading of 0.1, 1.0 and 3.0 wt% of Cu7S4 nanoparticles, respectively. Thus, MNs system loaded Cu7S4 nanoparticles is a photo-response with thermal ablation of arrowheads by NIR irradiation.52 17



Skin insertion in vitro MNs encapsulated red fluorochrome (R6G) to as a model drug for evaluation of skin insertion performance and NIR-triggered release behavior of as-prepared MNs in vitro.53,54 Figure 3A exhibits a typical morphology of R6G loaded MNs in the initial. After insertion, these MNs are imbedded into skin tissues, as shown in Figure 3B. Figure 3C shows the MNs images removed from the skin at a specific irradiation time. Light-triggered melting behaviors are also can be observed when the MNs are embedded, demonstrating that NIR light can penetrate MNs patch and skin tissue. A melting of MNs arrowheads can be revealed due to the absorption of NIR light by Cu7S4 nanoparticles. More time for NIR irradiation, more notable phase transition behaviors (i.e., melting) of MN tips can be observed.

Figure 3 Bright field micrographs of R6G-loaded MNs (A) and it corresponding image of the punctured skin (B). The R6G-loaded MNs images with MNs removed from the punctured skin under continuous irradiation for 0, 1 and 3 min (C).

To analyze the NIR responsive release behavior of drugs, the R6G-loaded MNs have 18


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penetrated the stratum corneum (SC). A confocal laser scanning microscope is used to characterize the histological sections which embedded red fluorescence (R6G) for easily detection in skin tissues. As shown in Figure 4A, the white MNs arrowheads (white arrows) can be detected in the skin tissues, which indicates the MNs arrowheads have been separated with dissolving PVA/PVP sustentive basements caused by absorption of interstitial fluid. The red fluorescent signal only can be observed in the MNs area while there is no red fluorescence in skin tissues. The red fluorescence displays a typical pyramid-shaped morphology under original condition of MNs arrowheads, indicating that R6G fluorochromes are embedded in the MNs arrowheads with no fluorochromes permeated into skin tissues. After NIR exposing, a fusion behavior of these MNs arrowheads can be observed obviously and more R6G can be diffused into internal tissue after 3 min NIR irradiation (Figure 4B and 4C). As shown in the 3D reconstruction images, the initial pyramid-shaped morphology of MNs fade away gradually, suggesting that R6G fluorochromes are released only when MNs exposed with NIR irradiation.

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Figure 4 Fluorescence and merged histological sections of R6G-loaded MNs applied skin without irradiation (A) and under continuous irradiation for 1 (B) or 3 (C) min in vitro and corresponding 3-D reconstruction images.

Thermal damages of skin When exposed to a temperature above physiological temperature with a long irradiation, the skin tissues will undergo a thermal damage according to previous reports.48-50 MNs loaded with different amounts of Cu7S4 nanoparticles are utilized to investigate the thermal damage on skin tissues under NIR irradiation of 0.4 W/cm2 by histological analysis with conventional H&E staining.55,56 Typical H&E stained images of normal skin tissues with a NIR irradiation for 0~5 min exhibit the attributive character of normal dermis stratum as shown in Figure 5A. In the case of MNs with 0.1 wt% of Cu7S4 nanoparticles, almost no any tissue damage can be founded on the skin samples except MNs holes on the skin surface. The epidermis as well as the dermis are unmarred even after the NIR exposure for 5 min (Figure 5B). Increasing the 20


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amount of Cu7S4 nanoparticles in the MNs, skin damage can be extended from epidermis to the dermis. Tissue necrosis in dermis can be founded when MNs loaded 1.0 wt% of Cu7S4 nanoparticles exposed for 3 min and 3.0 wt% of Cu7S4 nanoparticles exposed for 1 min (Figure 5C and 5D). Therefore, the MNs encapsulated Cu7S4 nanoparticles of 0.1 wt% have been selected as a research model for analysis of drug release behavior in vivo.

Figure 5 Bright field micrographs of histologic sections with H&E staining on control skin (A) and skins applicated with MNs loaded Cu7S4 nanoparticles of 0.1 wt% (B), 1.0 wt% (C) and 3.0 wt% (D) under NIR irradiations for 0, 1, 3 and 5 min. Scale bar: 500 µm.

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NIR triggered drug release behavior in vitro and in vivo The MNs system with several NIR irradiation on/off cycles are further implemented to assess the on-demand drug triggered release behavior. A NIR irradiation for constant exposure duration (2 or 4 min) are applied on skin samples embedded inseparable metformin-loaded MNs. And the release amount of system can be obtained by subtracting the amount of residuary metformin from original amount loaded in MNs. Figure 6A shows a release behavior of metformin with NIR laser on/off. The pink regions represent laser on state, while white regions represent laser off state. A consistent and rapid release of metformin (~30.5% and 14.6% of metformin is released after each irradiation for 4 and 2 min, respectively) can be observed during the laser-on state. Additionally, there is no significant drug diffusion from MNs when NIR laser is turned off. In the case of each NIR exposure for 4 min, ~90.1% loading metformin can be released from MNs after 3 NIR irradiation on/off cycles. And the same released level can be reached after 6 NIR laser on/off cycles under each NIR exposure for 2 min. The release curve exhibits a stepped and consistent release behavior under a NIR exposure. In contrast, there is no significant drug release without NIR irradiation due to an unmelt of LA/PCL. These results imply that NIR can penetrate sufficiently deep to cause the MN to melt, thus activating the drug release from the implanted drug warehouse.57,58

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Figure 6 Drug release profiles of metformin-loading MNs in vitro after exposure to an intermittent laser with a constant irradiation time of 2 or 4 min (A). Blood glucose levels of diabetic groups applied with MNs loaded metformin of 0.5 and 1.0 mg under NIR irradiation cycles compared to hypodermic injection group with 1.0 mg of metformin and health group (B). Decline speeds of Blood glucose levels (each hour) on diabetic rats applied with MNs loaded metformin of 0.5 and 1.0 mg (C). Blood glucose levels of diabetic rats after feeding and NIR exposure (D).

Different doses of metformin-loaded MNs (0.5 mg and 1.0 mg) are applied on type 2 diabetic rats caused by STZ for further evaluation of hypoglycemic effect in vivo. The longer NIR irradiation period (4 min) is selected for responsive release of metformin from MNs due to a negligible thermal damage on skin tissues. Figure 6B shows the blood glucose level of diabetic rats (initial blood glucose levels are ~402.8±10.5 mg/dL) after application of 23



metformin-loaded MNs and 1.0 mg of subcutaneous injection. In the subcutaneous injection group, the blood glucose levels are decreased quickly and reached their minimum (~82.3±6.5 mg/dL) at 2 h and returned to high blood glucose state. However, in the MNs groups, SD rat dorsal skins are applied with repetitive NIR irradiations for triggered release of metformin in vivo. The blood glucose levels exhibit a slow decrease tendency depended on the drug-loading dose in MNs and reach their respective minimum (~180.7±8.2 and 125.2±4.7 mg/dL) at 6 h. Additionally, the MNs groups can maintain their low blood glucose levels for a longer time compared with the subcutaneous injection groups. MNs loaded more metformin (1.0 mg) reveal a better hypoglycemic effect to control blood glucose levels. And the blood glucose levels of them can be reduced to below half-level (200 mg/dL) within 3.0 h, following the blood glucose levels are maintained at a low level for more than 4.5 h compared with ~3 in the subcutaneous injection groups. Metformin released from MNs can be quickly absorbed and utilized, with a bioavailability of at least 92.8±4.4% relative to the subcutaneous injection (Table S1).59 These results demonstrated that the released metformin can be diffused across skin and then permeate into systemic circulation.

Figure 6C shows the descent rate of blood glucose levels calculated by relevant data from Figure 6B in MNs groups. Under NIR irradiation, the descent rate of blood glucose levels rapidly increases, and then maintain the same level after the first 1.0 h. These results demonstrate a certain time lag for the descent rate of blood glucose levels due to the release and diffusion of metformin after a NIR irradiation and the MNs arrowheads gradually solidify for mitigation of drug release after removing of NIR irradiation. Repeated exposure to the NIR light result in a similar descent rate of blood glucose levels, demonstrating the stability 24


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and reproducibility of the NIR-triggerable MNs. These results also indicate that MNs system triggered by NIR irradiation show a continuous and initiative manner metformin-release behavior and an effective hypoglycemic activity on diabetic rats.60, 61

Normally, the blood glucose concentration can be increased after feeding on rats. To analyze the hypoglycemic behavior of MNs system after feeding, the blood glucose levels are monitored in real time. As shown in Figure 6D, the blood glucose levels are degressive gradually with an application of metformin-loading MNs system under 4 min of NIR irradiation while they are returned to initial levels cause by a sufficient food and water on diabetic rats after 2 h. However, the blood glucose levels are declined again when rat samples are exposed to another NIR irradiation for response of metformin release from MNs. Suchlike NIR-responsive MNs system is relatively feasible for suppling a rapid hypoglycemic action to sporadic blood glucose rising and preventing an autonomous event after usual mealtime.

25



Figure 7 H&E-staining images of major organs from diabetic samples applied with 0.5 and 1.0 mg metformin-loading MNs while untreated diabetic samples as a control. Scale bar: 100 µm.

Cu7S4 nanoparticles may be accumulated in liver and spleen along with many phagocytes in vivo but would metabolized through the liver and spleen mainly.39,62,63 Histologic section with H&E staining on representative organs (heart, liver, spleen, lung, and kidney) in MNs groups are also administrated to evaluate whether the NIR-responsive MNs with Cu7S4 nanoparticles cause any possible tissue damage. As shown in Figure 7, negligible tissue damage can be observed compared to control group, indicating that as-prepared MNs system is nontoxic for potential clinical application.64

CONCLUSION In summary, the novel NIR-induced photo-triggerable MNs have been fabricated that can be implanted into the skin as a depot for the on-demand delivery of anti-diabetic drug such as metformin. These implantable MNs exhibited photo-thermal conversion properties using low amount of photo-thermal conversion agent (0.1 wt% of Cu7S4 nanoparticles). When exposed to a NIR laser, light-induced heating can trigger metformin release from the melted MNs. We demonstrated that the proposed devices could release metformin in a pulsatile or programmed manner, with low drug leakage in the off state. Such as-prepared NIR-responsive MNs system is relatively convenient for supplying a continuous and initiative manner of metformin release behavior and an effective hypoglycemic activity on diabetic rats.

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■ ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.XXXXX.

TEM, high-resolution TEM images, selected area electron diffraction, EDS and XRD patterns of Cu7S4 nanoparticles, cell viability of Cu7S4 nanoparticles in vitro after incubation with Hela cells, the change of melting point of LA and PCL composites and the compression test of MNs with different compositions, morphology changes of as-prepared MNs with different contents of Cu7S4 nanoparticles under a continuous 0.4 W/cm2 NIR irradiation, pharmacodynamic parameters of blood glucose levels on diabetic rats treated with injection and MNs system.

■ AUTHOR INFORMATION

Corresponding Author *Tel.: +86-571-8684-3527. E-mail: [email protected].

ORCID Guohua Jiang: 0000-0003-3666-8216

Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGEMENTS 27



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This work was financially supported by the Zhejiang Provincal Natural Science Foundation of China (LY18E03006), the National Natural Science Foundation of China (51373155) and “521 Talents Training Plan” in Zhejiang Sci-Tech University (ZSTU). We also gratefully acknowledge Hua-An Biotechnology Co., Ltd. (Hangzhou, China) for histological experiments.

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Scheme 1. Schematics of thermal ablation behavior of separable MNs (A) and NIR triggered transdermal drug delivery on diabetic SD rats (B). 160x144mm (300 x 300 DPI)



Figure 1 Digital images of as-fabricated NIR triggered and separable MNs used in this study (A), SEM images of separable MNs without (B) and with the photo-thermal conversion agent (C). 162x147mm (300 x 300 DPI)


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Figure 2 Time-temperature curves of MNs loaded varying amounts of Cu7S4 nanoparticles (A) and the lower amount of Cu7S4 nanoparticles (0.1 wt%) under repetitively intermittent NIR light for 5 cycles (B). Infrared thermal images of MNs prepared with and without Cu7S4 nanoparticles under a continuous NIR irradiation in the initial 40 seconds (C). 133x100mm (300 x 300 DPI)



Figure 3 Bright field micrographs of R6G-loaded MNs (A) and it corresponding image of the punctured skin (B). The R6G-loaded MNs images with MNs removed from the punctured skin under continuous irradiation for 0, 1 and 3 min (C). 89x44mm (300 x 300 DPI)


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Figure 4 Fluorescence and merged histological sections of R6G-loaded MNs applied skin without irradiation (A) and under continuous irradiation for 1 (B) or 3 (C) min in vitro and corresponding 3-D reconstruction images. 98x54mm (300 x 300 DPI)



Figure 5 Bright field micrographs of histologic sections with H&E staining on control skin (A) and skins applicated with MNs loaded Cu7S4 nanoparticles of 0.1 wt% (B), 1.0 wt% (C) and 3.0 wt% (D) under NIR irradiations for 0, 1, 3 and 5 min. Scale bar: 500 µm. 163x150mm (300 x 300 DPI)


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Figure 6 Drug release profiles of metformin-loading MNs in vitro after exposure to an intermittent laser with a constant irradiation time of 2 or 4 min (A). Blood glucose levels of diabetic groups applied with MNs loaded metformin of 0.5 and 1.0 mg under NIR irradiation cycles compared to hypodermic injection group with 1.0 mg of metformin and health group (B). Decline speeds of Blood glucose levels (each hour) on diabetic rats applied with MNs loaded metformin of 0.5 and 1.0 mg (C). Blood glucose levels of diabetic rats after feeding and NIR exposure (D). 136x104mm (300 x 300 DPI)



Figure 7 H&E-staining images of major organs from diabetic samples applied with 0.5 and 1.0 mg metformin-loading MNs while untreated diabetic samples as a control. Scale bar: 100 µm. 110x68mm (300 x 300 DPI)


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Table of Contents

Separable microneedles for near-infrared light triggered transdermal delivery of metformin on diabetic rats

Yang Zhang,†,§,‖ Danfeng Wang,‡ Mengyue Gao,† Bin Xu,†,§,‖ Jiangyin Zhu,†,§,‖ Weijiang Yu,†,§,‖ Depeng Liu,†,§,‖ and Guohua Jiang†, §,

⊥,

‖, *


Separable microneedles for near-infrared light triggered transdermal

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