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Controlled Release and Delivery Systems

Fabrication of dissolving microneedles with thermal-responsive coating for NIR-triggered transdermal delivery of metformin on diabetic rats Depeng Liu, Yang Zhang, Guohua Jiang, Weijiang Yu, Bin Xu, and Jiangying Zhu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00159 • Publication Date (Web): 17 Apr 2018 Downloaded from http://pubs.acs.org on April 18, 2018

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ACS Biomaterials Science & Engineering

Fabrication

of

dissolving

microneedles

with

thermal-

responsive coating for NIR-triggered transdermal delivery of metformin on diabetic rats Depeng Liu,†,ǁ Yang Zhang,†,ǁ Guohua Jiang,*,†,‡,§,⊥ Weijiang Yu,† Bin Xu,†and Jiangyin Zhu†

† Department of Polymer Materials, Zhejiang Sci-Tech University, Hangzhou, 310018, 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, 310018, China ⊥ Institute of Smart Fiber Materials, Zhejiang Sci-Tech University, Hangzhou, 310018, China

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ABSTRACT: This study described a near-infrared (NIR) light-responsive polymer-nanodots composite microneedles (MNs) used for on-demand transdermal drug delivery. Bismuth (Bi) nanodots stabilized by poly(vinylpyrrolidone) (PVP) as photothermal conversion agent and metformin as anti-diabetic drug were introduced into the dissolving MNs that coated with lauric acid (LA). When the MNs were irradiated with NIR light, light-to-heat transduction induced by the Bi nanodots caused the LA to melt. As a result, the polymer matrix was dissolved after absorbing the interstitial fluid, and enabling the encapsulated metformin release from the MNs into skin tissue. Compared with subcutaneous injection of metformin, the administration using the Bi nanodots-induced NIR responsive MNs developed in current research exhibited a remarkable hypoglycemic effect in vivo. This work indicates that the as-fabricated Bi nanodots-induced NIR responsive and LA-coated MNs have the potential applications in diabetes treatment. Additionally, this artificial MNs also have a promising platform for delivering other therapeutic drugs.

KEYWORDS: Bi nanodots, microneedles, transdermal delivery, triggered release, diabetes

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1. Introduction The microneedle drug delivery system (MDDS) is the attractive and effective method to delivery of various drugs as an alternative to invasive and painful hypodermic injections.(1) Usually, MNs are micron-sized structures with a length of less than 1 mm which can be used to overcome the skin barrier located in the top layer of the skin. The micro-channels can be created by piercing MNs onto the skin using which may increase the permeability and absorption of the drugs.(2-4) As same time, as these MNs do not penetrate to the depths where nerve endings reside, the MNs enables minimally-invasive and pain-free delivery of drugs into skin tissues.(5) Several types of MNs have been developed for delivering drugs percutaneously in recent years, including solid MNs,(6) dissolving MNs,(7-9) coated MNs, (10-12) and hollow MNs.(13,14) Despite the many advances and diverse types of MNs available, most drug delivery based MNs is a passive manner regardless of the changing physiological circumstances of patients. It is often difficult to completely avoid the toxicity caused by overdose or the inefficacy attributable to underdose.

Near-infrared (NIR) responsive photothermal therapy (PTT) is an attractive alternative to combine with chemotherapy as it could cause membrane damage, cell injury, and protein denaturation as a systemic effect. Importantly, the wavelength of NIR located at “biological window”, which is minimally absorbed by the blood and soft tissues, leads to the deep penetration.(15,16) Diabetes mellitus characterized by imbalance of blood glucose level regulation mechanisms is becoming a worldwide public health problem.(17,18) Recently, a near-infrared light triggered and separable MNs system were fabricated by our group for

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on-demand treatment on diabetic rats. The photothermal conversion agent (Prussian blue nanoparticles, PB NPs) and hypoglycemic drug (metformin) were embedded into the separable polycaprolactone (PCL) MNs arrowheads which were capped on dissolving polyvinyl alcohol/polyvinyl pyrrolidone (PVA/PVP) solid supporting substrate. Due to the excellent photothermal conversion of PB NPs in MNs arrowheads, the as-fabricated MNs underwent a rapid thermal ablation from a solid to a liquid state under NIR irradiation, thus enabling the release of encapsulated metformin into skin tissue.(19) It allowed on-demand control of timing and dose of the drug released in an active manner, thus improving treatment efficiency.

As known, Bi is the only nontoxic heavy metal and has relatively inexpensive features to further expand its prospects. Moreover, its light absorption can extend to NIR region, which could lead to photothermal imaging. Bismuth is a well-known “green metal” that is nontoxic and inexpensive.(20-22) Lauric acid (LA) as a natural food-grade phase change material (PCM) exhibits a permissible melting range (m. p. = ~ 44 °C).(23,24) In this study, a new coated microneedle system has been developed by proposing photothermal triggered drug release from the MNs together with enhanced permeation of the therapeutics upon NIR illumination. The anti-diabetic drug (metformin) and photo-thermal conversion agent (Bi nanodots) are encapsulated into PVP MNs. The LA was coated on the surface of MNs by a spray method. When the MNs were irradiated with NIR light, light-to-heat transduction mediated by the Bi nanodots caused the lauric acid to melt and enable drug release from the MNs. The NIR triggered release of encapsulated payload into the skin to explore the hypoglycemic effect on

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diabetic rats has been investigated.

2. Materials and methods

2.1 Materials Poly(vinyl pyrrolidone) (PVP, MW=130,000), anhydrous calcium chloride (CaCl2), anhydrous sodium carbonate (Na2CO3), bismuth nitrate pentahydrate (Bi(NO3)3·5H2O), sodium borohydride (NaBH4), lauric acid (LA), sodium citrate tribasic dihydrate, citric acid, dimethyl sulfoxide (DMSO), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), streptozocin (STZ), metformin were purchased from Aladdin Chemistry Co. Ltd. (Shanghai, China). All chemicals were obtained from commercial sources and used directly without further purification. Forty-day-old male Sprague Dawley (SD) rats weighing 200 ± 20 g were supplied by Experimental Animal Center of Zhejiang Academy of Medical Sciences. The rats were housed at a room temperature of 22 °C and a relative humidity of 50%. The deionized water was used throughout all experiments. Bi nanodots stabilized by PVP were synthesized according to a previous literature.(25) CaCO3 nanoparticles with average diameter 200 - 250 nm were prepared by a coprecipitation method.(26)

2.2 The in vitro cell viability of microneedles composite

The in vitro cell viability of microneedles composite was tested using MTT assay towards MCF-7 cells. In detail, MCF-7 cells were seeded in 96-well plates at a density of 5 × 103 cells per well and were cultured at 37oC and 5% CO2 for 24 h in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS. Then, the culture medium of each 5



well was replaced by 200 µL of fresh culture medium containing series dilutions of microneedles composite with different concentrations (0, 200, 400, 600, 800, and 1000 µg mL-1). After incubated for another 24 h at the same condition, 20 µL of MTT solution (5 mg mL-1) was added to each well and incubated for an additional 4 h. After removing the culture media, the purple formazan crystal was dissolved in 150 µL of dimethyl sulfoxide (DMSO). The absorbance of the solution was read at 490 nm by using a microplate reader (SpectraMax Plus 384, Molecular Devices, USA) to determine the OD values.

2.3 Fabrication of LA-coated MNs The PMMA master mold (Patch size: 8 × 8 mm, Array Size: 10 × 10, Needle Height: 500 µm, Needle Base: 250 µm, Needle Pitch: 500 µm) was used to prepare an inverse MNs mold with PDMS.(27) A two-step molding process was used to prepare the metformin-loaded and LA-coated MNs (Figure S1). Firstly, the mixture with different amount of PVP, metformin, CaCO3 and Bi nanodots (Table S1) was added into PDMS mold and followed centrifuged (10,000 rpm) for 15 min to form MN tips. Then, the mold was dried in a 60 °C oven for 10 min to remove the residual water. PVP (40 wt%) solution was placed onto the MN tips loaded mold with another centrifugation (8,000 rpm) for 5 min to form supporting patch. After dried in a vacuum chamber at room temperature, the as-prepared MNs (Bi-CaCO3/PVP MNs) were carefully separated from the mold. Finally, lauric acid (0.25 g mL-1 in ethyl acetate) was spray-coated on the surface of MNs to obtain LA-coated MNs (LA/Bi-CaCO3/PVP MNs) using a commercial spray water compensator (XL001, Shenzhen Yuanlian Communication Co. Ltd, China) for 5 seconds at room temperature with a 10 cm of 6


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distance between spray nozzle and MN patch. To characterize the thickness of LA coating, 0.25 g mL-1 LA ethyl acetate solution mixed with fluorescein isothiocyanate isomer Ⅰ (FITC, MW 389.38 Da) (0.1 wt%) was used to obtain the fluorescent LA-coated microneedles with the same method. The three-dimensional (3-D) images of FITC-mixed LA-coated MNs were obtained by a confocal laser scanning microscope to analyze the LA coating.

2.4 Mechanical compression tests The mechanical compression tests of MNs were measured with a stress-strain gauge by pressing the needle against a stainless-steel plate on a low-force mechanical testing system (5943 MicroTester, Instron, USA). A microneedle patch was placed on the flat rigid surface of a stainless steel plate. The initial gauge was set as 2.00 mm between the microneedle patches tip and the stainless steel plate, with 10.00 N as the cell loading capacity. The speed of the top stainless-steel plate moving toward the MNs was set as 0.1 mm s-1. The force was measured when the moving sensor touched the uppermost point of the microneedle array. The testing machine subsequently recorded the force required to move the mount as a function of MNs displacement. And the morphology analysis of LA/CaCO3/PVP MNs before and after mechanical test was characterized by a macroscope (BST500xUSB, BAISITE, China).

2.5 Near-infrared light responsive properties To evaluate the NIR light responsive property of MNs, the composite MNs with different content of Bi nanodots were exposed to the 808 nm NIR laser light (1.6 W) continuously at a 7



power density of 1.6 W cm-2 until the temperature was steady. For intermittent irradiation, the MNs were repeatedly exposed to the laser for 1.5 min (laser on). Subsequently, the laser was turned off and the system temperature was cooled naturally to the ambient temperature for 1 min (laser off). This cycle was repeated four times to assess the repeatability of the NIR-responsive property. The maximal temperature of the MNs was recorded every second and plotted as a function of the exposure time. The thermographic images of MNs were captured using an infrared thermography system (Ti400, Fluke, USA).

2.6 Skin insertion in vitro To observe the melting behaviors of MNs and permeation and diffusion of drugs in the skin, metformin replaced by R6G (1.0 wt%) MNs were prepared by the same method mentioned above. R6G-loaed MNs were applied onto SD rat skin samples which cleaned with 75% alcohol and irradiated by NIR laser for 0 or 1.5 min. The treated MNs extracted out from puncture sites were photographed by a microscope. To evaluate the skin penetration ability and drug release behaviors of MNs in vitro, R6G-loaded separable MNs were inserted into the skin samples followed exposed to NIR irradiation for 0 or 1.5 min, respectively. The puncture sites were imaged by a microscope before irradiation. And the treated skin samples were characterized with a confocal laser scanning microscope (CLSM, Axio Observer A1, Carl Zeiss, Germany) to reconstruct the histological sections analysis. To prepare histological specimens, the insertion sites of MNs were excised carefully from dorsum of rats. The skin tissue was embedded immediately in optimal cutting temperature (O.C.T.) compound (Tissue-Tek, Sakura, USA). Then, the frozen samples were sliced into 5-µm-thick section 8


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using a cryotome (CryoStar NX50 freezing microtome, Thermo Scientific, USA) and observed by a confocal laser scanning microscope (C2, Nikon, Japan).

2.7 NIR-triggered drug release in vitro To investigate the NIR-light-triggered drug release behaviors in vitro, metformin-loaded (1 mg) LA-coated MNs were penetrated into a detached skin and exposed to NIR irradiation for 1.5 min each cycle with an interval of 2h. Several laser on/off cycles were repeated to analyze the triggered release behaviors. And then the exposed MNs were pulled out and immersed into an ethyl acetate solution to wipe off the LA coating followed dissolved in a 10 mL of deionized water completely. The drug concentrations of above solution were analyzed by an UV-Vis spectrophotometer (U3900H, Hitachi, Japan) according to a previous standard calibration curve to determine the residual metformin.

2.8 NIR-triggered drug release in vivo The in vivo efficacy of MNs was evaluated on STZ-induced type II diabetic rats. Here, type II diabetic rats were induced by an injection of streptozotocin (45 mg kg-1) citrate buffer (10 mM, pH 4.5) as previously method.(28-30) The blood glucose level (BGL) of rats was monitored from tail vein blood using a glucose meter (Sinocare Inc., China). Rats were considered to be diabetic when their fasting glycemia was higher than 16.0 mM. The rats with stable hyperglycemic state were divided into four groups (three rats for each group), and fasted overnight before administration. Diabetic rats were subcutaneous injection with metformin solution at a dose of 5 mg kg-1 (~ 1.0 mg of metformin). Other groups were selected to be treated on the dorsum of rats by MNs (5 mg kg-1) with or without NIR 9



irradiation. The BGLs of rats were continuously monitored every hour until the stable hyperglycemia returned.

2.9 Acute toxicity in rats For the in vivo acute toxicity studies, healthy SD rats were randomly divided into two groups: the MNs and control groups. The MNs group was applied with MNs (without drug) and then irradiated with NIR light. The rat without any treatment was used as the blank control. All animals were fed with normal chows and water ad libitum. After designated time periods, the rats were sacrificed to perform histological analysis. For histological examinations, the main organs of the rats (liver, spleen, and kidney) were harvested. Then, the tissue samples were embedded in an OCT compound, sliced, and stained with hematoxylin and eosin (H&E). The histological sections were observed under an optical microscope. All the procedures above were guided with HuaAn Biotechnology Co., Ltd. (Hangzhou, China).

3. Results and discussion

3.1 Preparation of MNs The Bi nanodots stabled by PVP are firstly synthesized by a simple reducing method according to the previous report.(25) Representative transmission electron microscopy (TEM) images reveal the obtained Bi nanodots with uniform distribution and size less than 10 nm (Figure S2A and S2B). The high-resolution TEM (HRTEM) shows a spacing of 0.326 nm, corresponding to that of the Bi (012) plane (Figure S2B).(31) The selected area electron 10


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diffraction (SAED) pattern shown in Figure S2C revealed that the obtained Bi nanodots are highly crystalline.(25) Only the intense peaks corresponding to Bi element are detected in the EDS analysis (Figure S2D) besides C and O elements from PVP. The presence of PVP on the Bi nanodots can be further conformed by Fourier transform infrared (FT-IR) spectroscopy (Figure S2E). The XRD pattern (Figure S2F) suggests that all the diffraction peaks can be well indexed to Bi (rhombohedral, JCPDS card No. 44-1246), indicating the high purity.(32)

Due to their strong and broadband NIR absorbance, high photothermal conversion efficiency and low toxicity, Bi nanodots have the highly promising as a PTT therapeutic nanoagent.(33,34) Herein, Bi nanodots have been chosen as the photothermal conversion agent to be encapsulated into the MNs. To assess the safety of the composite microneedles for applying in animal experiments, we firstly used the MTT assay to test the cell viability of the composite microneedles materials. As shown in Figure S3, the cell viability of MCF-7 cells was not hindered after 24 h of incubation with the composite microneedles, and the cell viability was over 90% even at a high concentration of 1000 µg mL-1. Figure 1 shows the typical scanning electron microscope (SEM) images of MNs arranged in a 10 × 10 array in a 1 cm2 patch, indicating the successful replication of the matter mold. Figures 1A-D show the SEM images of MNs with Bi nanodots and without LA coating. These MNs are in the pyramid-shape with the height around 450 µm. The tip-to-tip distance between two MNs is ~ 600 µm. The base for each MNs is ~ 250 µm in width. From the Figures 1C and 1D, the as-fabricated MNs display a smooth surface with clear edges. Figures 1E-H show the SEM images of MNs with LA coating. They reveal that coating of LA onto the surface of the MNs

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do not result in any changes in MNs morphology. However, the surface of MNs become rough, indicating the LA layer has been coated on MNs, as shown in Figures 1G and 1H.

To investigate the thickness of LA coating, we have used a confocal laser scanning microscope to reconstruct the three-dimensional (3-D) images of fluorescent LA-coated MNs. As shown in Figure S4A, a bright green fluorescent ring with an average thickness of about 5 µm can be founded around a dark hole on microneedles at each depth. Figure S4B shows the 3-D reconstruction images for the FITC-mixed LA-coated MNs at different perspective that revealed the LA coating on MNs surface obviously.

The mechanical strength of MNs prepared from pure PVP is 0.16 N/needle with displacement at 500 µm. For further improve the mechanical strength of MNs, CaCO3 nanoparticles as a filling material have been added into the PVP to form the MNs matrix. The mechanical strength can be increased to 0.75 N/needle with displacement at 500 µm. Although there is a slight drop in mechanical strength after coating of LA on the surface MNs, they still have enough strength (0.47 N/needle) to penetrate into the skin (Figure S5A).(35,36) The morphology changes of microneedles can be observed visibly compared with the initial state (Figure S5B-a) after mechanical experiment (Figure S5B-b). The tip of MNs shown a severe bending with no break indicates the excellent flexible and toughness of the LA/CaCO3/PVP MNs.

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Figure 1. The SEM images of Bi-CaCO3/PVP (A-D) and LA/Bi-CaCO3/PVP (E-H). 3.2 Photothermal properties The Bi nanodots have a strong absorption in the NIR region with high photo-thermal conversion efficiency.(25) In the NIR triggerable MNs, the loading amount of Bi nanodots in MNs is an important parameter, which probably efficiently induces the conversion of heat from NIR light to melt the MNs, causing the release of loaded drugs from MNs. Simultaneously, the tissues should suffer minimal damage caused by the produced heat. Different amounts of Bi nanodots are encapsulated into MNs to investigate the photo-thermal transformations under a continuous 808 nm-NIR-light irradiation with power at 0.4 W cm2.

Figure 2A shows the time-temperature curves of MNs with different amount of Bi nanodots. MNs with loading of Bi nanodots exhibit a rapid elevation of temperature under a NIR-light irradiation. However, a faster increase in temperature can be observed with more Bi nanodots encapsulated. For the MNs fabricated with 0.089 wt% or 0.177 wt% of Bi nanodots, the temperature of MNs increases rapidly to more than 50 °C within 30 s. The

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temperature of MNs can be maintained around 47 °C (> melting point of LA) even NIR irradiation for more than 90s with a lower amount of Bi nanodots (0.044 wt%). In contrast, the temperature change of MNs is negligible with absence of Bi nanodots. Because the temperature of normal human body is about 37 °C, the skin tissues could be quickly heated over 50 °C after laser irradiation at high concentration of Bi nanodots, which can induce the damage of skin.(37,38) However, there is only a small increase in temperature (~ 32 °C) with the lowest amount of Bi nanodots (0.022 wt%), which can’t induce the melting of LA layer on the surface of MNs. Only the concentration of Bi nanodots controlled in a suitable range in MNs, the LA layer can be induced to melt, and caused minimal thermal effect on skin as well.

Repeating intermittent NIR light (4 cycles) is applied on these MNs with loading of 0.044 wt% of Bi nanodots. As shown in Figure 2B, the temperature of MNs rapidly elevates to more than 47 °C within 1.5 min when the NIR laser is switched on. Once the NIR laser is switched off, the temperature quickly drops to room temperature. This demonstrates that MNs have a fast and stable response to NIR light. NIR thermal imager is further used to record the surface infrared thermal images at different times. As shown in Figure 2C, only the temperature within MNs patch region can be rapidly risen to ~ 47 °C within the first 60 s.

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Figure 2. Temperature-time curves with different amount of Bi nanodots in MNs (A), temperature-time curves of MNs with 0.044 wt% Bi nanodots under intermittently NIR light for 4 cycles (B). Thermal infrared camera photos of MNs fabricated with 0.044 wt% Bi nanodots under continuous 0.4 W cm-2 NIR irradiation in the first 90 seconds (C).

3.3 Thermal damage Thermal damage occurs when tissues are exposed to temperatures higher than physiological temperature for extended periods of time.(39-43) To evaluate thermal damage on skin tissue in this study, MNs with different amounts of Bi nanodots are used on the skin surface of rats under NIR irradiation (0.4 W/cm2) using conventional hematoxylin and eosin (H&E) staining.(39,40) Figure 3A shows an H&E-stained image of normal skin tissue, revealing the characteristic features of normal dermis stratum. The Bi nanodots used in MNs is varied to determine the effect on skin damage. We stuck the MNs with 0.022 wt% Bi 15



nanodots on the skin, and then illuminated the NIR laser for 1.5 min. The blank domain among collagen fibers (yellow arrows) is the tissue fluid in natural tissue. Compared with the normal skin issue, almost no damage can be observed in the excised skin samples besides the needle holes on the surface of the skin tissue (Figure 3B). However, the histology analysis of skin treated by MNs with loaded by 0.08 wt% of Bi nanodots confirmed the establishment of a damage reaching the subcutaneous tissue underneath the dermis (Figure 3C).(41) The regions denoted by the blue arrows represent collagen fibers. When skin temperature rises above a critical value (~ 50 oC), denaturation occurs.(42) The resultant effect of collagen fiber denaturation is the shrinkage of skin.(43) As shown in Figure 3C, significant change in collagen fiber configuration in dermis can be observed. The collagen fibers melt and the blank regions between collagen fibers decreased when the thermal damage occurs. Therefore, the MNs with 0.044 wt% of Bi nanodots have been chosen as an ideal model for evaluation of photothermal properties and drug release behaviors in the following study.

Figure 3. Bright-field microscopic images of skin sections after H&E staining control skin (A); and skin with MNs (0.044 wt% and 0.089 wt% Bi nanodots for B and C) application after 1.5 min NIR irradiation. Scale bar: 300 µm.

3.4 Skin insertion 16


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To evaluate the skin insertion capability in vitro and the feasibility of triggered release from the MNs, the fluorescent dye (R6G) was chosen as a model drug and encapsulated into them.(44,45) The images of all R6G loaded MNs exhibit typical pyramid-shape at the initial state. Figure 4A shows the morphology change of MNs without LA coating before and after skin insertion for ~ 60 min. The height and morphology of MNs are changed due to the swelling and degradation of PVP in the skin tissue. No obvious differences in appearance are found between the MNs with LA coating before and after insertion and without NIR irradiation, suggesting that LA layer on the surface of MNs delay the swelling and degradation of PVP matrix in MNs, as shown in Figure 4B. However, it is obvious that the MNs get shorter after insertion in skin under NIR irradiation and this result demonstrated that the MNs with LA coating are mostly dissolved (Figure 4C). It implies NIR light can be generated heat to cause melting of LA coating by Bi nanodots and thus promoting the swelling and degradation of PVP in skin tissue.

Figure 4. The morphology changes of MNs without LA coating before and after inserting for 60 min (A), MNs with LA coating before and after inserting for 60 min (B) and MNs with LA 17



coating before and after inserting for 60 min with assistant of NIR irradiation (C). Scale bar: 500 µm.

The inserting depth of the MNs and drug distribution in the skin are further confirmed by the fluorescence images. The histological examination reveal that in vitro insertion depth of MNs into the porcine skin is approximately 200 ~ 400 µm. The R6G in the MNs can be permeated and diffused into skin tissue from the MNs without LA coating and NIR irradiation (Figure 5A). However, only a little of R6G can be founded at the piercing positions for the MNs with LA coating, and no R6G can be observed under the deeper tissue due to the blocking of LA coating (Figure 5B). In the case of MNs with LA coating with assistant of NIR irradiation, a large amount of red fluorescence can be observed under skin tissue (Figure 5C). These results further confirm the NIR-triggered release features of the fabricated MNs.

Figure 5. Fluorescence images after insertion of MNs without LA coating (A), MNs with LA 18


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coating (B) and MNs with LA coating with assistant of NIR irradiation (C). Scale bar: 200 µm.

3.5 NIR-triggered drug release in vitro and hypoglycemic effect in vivo To confirm the NIR-triggered drug release behavior in vitro, several laser on/off cycles are further investigated. First, an ethyl acetate solution is used to wash the LA coating of MNs which are exposed to NIR light for a fixed exposure duration (1.5 min). Then, the treated MNs are dissolved into DI water for determine the residual dose by an UV-Vis spectrophotometer. As shown in Figure 6A, a switchable on/off release of metformin can be found. During a laser-on state, the system shows a consistent and rapid metformin release behavior and there is no obvious drug leakage from MNs when laser off. About 91.12% loaded metformin can be released from the carrier after three NIR laser cycles (First cycle: ~ 41.01%; Second cycle: ~ 34.09%; Third cycle: ~ 15.13%). These results indicate the MNs can be triggered to release of drugs in subcutaneous with assistance of NIR irradiation.

To evaluate the hypoglycemic effect of MNs in vivo, metformin-loaded MNs are applied on streptozotocin (STZ) induced diabetic rats (the blood glucose levels are (380 ± 25.5 mg dL-1). Figure 6B shows the blood glucose levels (BGLs) after the administration of metformin-loaded MNs, compared with subcutaneous injection and blank groups. We found that the blood glucose levels (BGLs) of diabetes mice are stable without any drug administration.(46) A maximum decrease in BGLs of approximately 74.0% of its initial value can be appeared after 2 h of subcutaneous injection of metformin (~ 1.0 mg). MNs group resulted in a hypoglycemic effect at the same dose of metformin, with a maximum decrease 19



in BGLs of approximately 69.4% of its initial value, 6 h post-administration under NIR irradiation. While, only approximately 9.4% of its initial value can be obtained for the MNs groups without NIR irradiation. These results also demonstrate that the MNs displayed an on-demand for drug release on hypoglycemic administration. The relative pharmacological activity (RPA) for metformin-loaded MNs groups is approximately 97.4% that associated with the subcutaneous administration (Table S2). It also indicates that the metformin loaded in MNs released slowly and was absorbed from the skin into the systemic circulation, resulting in a hypoglycemic effect.

Figure 6. NIR triggered release of metformin from MNs In vitro (A). Drug-release profiles of 20


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metformin-loaded MNs and hypodermic injection on diabetic mice In vivo: blood glucose levels versus time in a treatment cycle (B) and blood glucose levels versus time with after feeding (C).

Figure 6C shows the BGLs versus time with 3 times interval feeding in 12 h. The BGLs can be decreased after the administration of metformin-loaded MNs under NIR exposure for 1.5 min. After 2 h, the sufficient food and water are provided to diabetic rats and the blood glucose levels can be returned to initial level. Then, the MNs are exposed under NIR light again to trigger the metformin released from MNs, and thereupon the blood glucose levels are also dropped. Thus, the stability and accuracy of the drug delivered using the as-fabricated MNs is confirmed.(47) Such NIR-triggerable MNs are convenient for use to provide a rapid onset of temporary blood glucose rising and to promote early prevention of spontaneous events of the usual feeding time.

At the same time, to determine whether the NIR-triggered MNs give rise to any possible tissue damage, H&E staining of the representative organs containing heart, spleen, and kidney in different groups were also performed. As shown in Figure 7, no noticeable tissue lesion took place and the results confirmed the hypotoxicity and safety of as-fabricated MNs for potential clinical application.

21



Figure 7. H&E stained images of major organs from 1.0 mg metformin-loaded MNs administration, and untreated model samples as control. Scale bar: 100 µm.

4. Conclusion In this study, we have developed novel coated and dissolving MNs with NIR-triggered drug release and high efficiency in transdermal drug delivery. The LA layer on the surface of MNs can be melted by the absorption of NIR light due to the high photothermal conversion efficiency of Bi nanodots in the MNs. And thus the as-fabricated MNs enable drug sequencing and sustained delivery. This study demonstrates that the coated and dissolving MNs with NIR-triggered drug release feature is an attractive alternative for conventional subcutaneous injection and not only provides a convenient operation but also allows fast and efficient hypoglycemic control. They have the potential to combine important features of sustained/controlled drug delivery in clinical applications.

22


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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 images, FTIR spectra and XRD patterns of Bi nanodots, cell viability of composite MNs in vitro after incubation with MCF-7 cells, confocal micrographs of the FITC-mixed LA-coated microneedles at varying depths and their 3-D reconstruction images, mechanical strength of microneedles measured by a dynamic tensile compression machine, The composition for preparation of MNs and Pharmacokinetic parameters after administration of NIR-triggered MNs for transdermal delivery of metformin on diabetic rats.

■ AUTHOR INFORMATION

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

ORCID Guohua Jiang: 0000-0003-3666-8216

Author Contributions

ǁ D.L. and Y.Z. contributed equally to this work.

Notes

23



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The authors declare no competing financial interest.

■ ACKNOWLEDGEMENTS

This work was supported by Natural Science Foundation of Zhejiang Province (LY18E030006), National Natural Science Foundation of China (51373155) and “521 Talents Training Plan” in Zhejiang Sci-Tech University (ZSTU). We also gratefully acknowledge HuaAn Biotechnology Co., Ltd. (Hangzhou, China) for histological experiments.

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Acta

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TABLE OF CONTENTS

Fabrication of dissolving microneedles with thermalresponsive

coating

for

NIR-triggered

transdermal

delivery of metformin on diabetic rats Depeng Liu,†,ǁ Yang Zhang,†,ǁ Guohua Jiang,*,†,‡,§, ⊥ Weijiang Yu,† Bin Xu,†and Jiangyin Zhu†

For Table of Contents Use Only



Figure 1. The SEM images of Bi-CaCO3/PVP (A-D) and LA/Bi-CaCO3/PVP (E-H). 73x30mm (300 x 300 DPI)


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Figure 2. Temperature-time curves with different amount of Bi nanodots in MNs (A), temperature-time curves of MNs with 0.044 wt% Bi nanodots under intermittently NIR light for 4 cycles (B). Thermal infrared camera photos of MNs fabricated with 0.044 wt% Bi nanodots under continuous 0.4 W cm-2 NIR irradiation in the first 90 seconds (C). 99x55mm (300 x 300 DPI)



Figure 3. Bright-field microscopic images of skin sections after H&E staining control skin (A); and skin with MNs (0.044 wt% and 0.089 wt% Bi nanodots for B and C) application after 1.5 min NIR irradiation. Scale bar: 300 µm. 44x11mm (300 x 300 DPI)


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Figure 4. The morphology changes of MNs without LA coating before and after inserting for 60 min (A), MNs with LA coating before and after inserting for 60 min (B) and MNs with LA coating before and after inserting for 60 min with assistant of NIR irradiation (C). Scale bar: 500 µm. 78x35mm (300 x 300 DPI)



Figure 5. Fluorescence images after insertion of MNs without LA coating (A), MNs with LA coating (B) and MNs with LA coating with assistant of NIR irradiation (C). Scale bar: 200 µm. 169x84mm (192 x 192 DPI)


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Figure 6. NIR triggered release of metformin from MNs In vitro (A). Drug-release profiles of metforminloaded MNs and hypodermic injection on diabetic mice In vivo: blood glucose levels versus time in a treatment cycle (B) and blood glucose levels versus time with after feeding (C). 144x117mm (300 x 300 DPI)



Figure 7. H&E stained images of major organs from 1.0 mg metformin-loaded MNs administration, and untreated model samples as control. Scale bar: 100 µm. 92x48mm (300 x 300 DPI)


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