Polymeric Microneedles Integrated with Metformin-Loaded and PDA

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Polymeric Microneedles Integrated with MetforminLoaded and PDA/LA-Coated Hollow Mesoporous SiO2 for NIR-triggered Transdermal Delivery on Diabetic Rats Yang Zhang, Guohua Jiang, Wenjie Hong, Mengyue Gao, Bin Xu, Jiangying Zhu, Gao Song, and Tianqi Liu ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00470 • Publication Date (Web): 23 Nov 2018 Downloaded from http://pubs.acs.org on November 25, 2018

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ACS Applied Bio Materials

Polymeric Microneedles Integrated with Metformin-Loaded and PDA/LA-Coated Hollow Mesoporous SiO2 for NIR-triggered Transdermal Delivery on Diabetic Rats

Yang Zhang,†,‡,§,‖ Guohua Jiang,†,‡,§,‖,* Wenjie Hong,† Mengyue Gao,† Bin Xu,†,‡,§,‖ Jiangying Zhu,†,‡,§,‖ Gao Song,†,‡,§,‖ and Tianqi Liu†,‡,§,‖

†

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

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, Zhejiang 310018, China ‖

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

310018, China

Corresponding author: E-mail:[email protected] (G. Jiang)

1

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Abstract: Herein, a NIR-responsive polymeric microneedles (MNs) system incorporated with metformin-loaded and polydopamine/lauric acid (PDA/LA)-coated hollow mesoporous SiO2 have been developed for transdermal delivery of antidiabetic drug (metformin). Firstly, an anti-diabetic drug was firstly loaded within hollow mesoporous SiO2 nanoparticles (HMSN) by a diffusion method. Then, PDA as photothermal conversion agent and lauric acid (LA) as phase change material (PCM) were coated onto the HMSN to form NIR-responsive drug nanocarriers.

Finally,

these

metformin-Loaded

and

PDA/LA-coated

HMSN

were

encapsulated into poly(vinylpyrrolidone) (PVP) MNs. After insertion into skin tissue, LA could be melt with the photothermal conversion of PDA under NIR-light, and thus enabling to release encapsulated metformin from MNs. The in vivo release behavior of metformin from MNs into skin was further studied to investigate its hypoglycemic effect on diabetic rats. Compared with the subcutaneous injection of metformin, the bioavailability of MNs-NIR groups was 95.8±2.7%. The antidiabetic drug can be precisely released by adjustment of exposure time and power densities of NIR-light. Keywords: microneedles; diabetes; NIR irradiation; transdermal delivery; hypoglycemic

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1. INTRODUCTION Millions of people in the worldwide have been plagued by diabetes, which has become the third most threatening condition affecting public health following the cardiovascular diseases and malignant tumors at present.1-3 Hyperglycemia (high blood sugar) will lead to a malfunction of the pankreatin or a resistance of body to the hormone.2,4-7 At present, subcutaneous (SC) injection of insulin using hypodermic needles is a most effective and commonly method to inhibit the hyperglycemia for diabetic patients. However, the pain caused by SC injections has a poor compliance to patients and thus impeding the progress of insulin treatment.8 As a novel alternative, microneedles (MNs) with length less than 1 mm can pierce into the skin’s stratum corneum to form microchannels.9 These microchannels can allow the drug to penetrate or across skin epidermis into the system’s circulation. Therefore, it is a more effective and attractive way for drug delivery with high compliance of patients.10-13 To overcome the corneum barrier of skin in transdermal drug delivery system, MNs have been primarily fabricated by microelectronics technologies with raw materials of silicon,14 metals15 or polymers.16 Although silicon is a common material for fabrication of substrate with extensive processing experience from microelectronics industry, silicon is relatively expensive and brittle material for MNs with unproven biocompatibility.14,17 Compared with silicon, metal materials are cheaper and stronger, enabling them to be more attractive for MNs with enough structural strength.15,18 However, low biocompatibility and high processing cost limit the wide applications of metal MNs. In recent year, polymeric MNs have obtained more and more attention because they can be made of widely available polymeric materials with inexpensive, biocompatible and biodegradable properties.16-20 And the dissoluble MNs can be 3



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soften and/or dissolved in skin tissues, and thus generating the release of encapsulated drugs. Despite the great progress has been made on polymeric MNs, there is still an enormous dilemma for breaking through some technical bottlenecks in the practical application. Low release dosing/rate may result in a restricted treatment effect and corresponding high treatment expenditure. However, overdosing of drugs may lead to the toxicity on the body.21 Most traditional drug delivery systems using MNs are passive administrations, regardless of the changes of patient’s physical condition. It is usually hard to avoid the side-effects induced by over dosage or ineffectiveness.22 These impediments have stimulated interest in developing a controllable or programmable MNs system, which can directed respond external stimuli to release encapsulated drugs in MNs, thus avoiding or reducing the risk of hypoglycemia caused by overdose drug release and side effects on skin tissue structure and blood circulatory system. At present, some “artificial pancreatic enzymes” have been developed as on-demand based positive manner for drug release on diabetic patients.21 They can respond to external stimuli, e.g. light-, heat-, ultrasound- or magnetic-field, realizing the controllable drug delivery.

For

example,

3,5-dimethoxybenzoin

to

ibuprofen form

the

as

a

model

drug,

photo-responsive

can

ibuprofen

be

conjugated conjugates.

to The

photo-responsive conjugates are further encapsulated in a polymeric MN arrays by micromolding. These MNs can provide up to three doses of ibuprofen (50 mg) under an application of optical trigger (15 W UV lamp at 365 nm) with a long period of time (160 h).22 In order to avoid the utilization of UV light and multiple chemical reaction for conjugation of drugs, some NIR-triggered transdermal drug delivery systems have been established by 4


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encapsulated NIR-responsive nanosized absorbers and drug molecules in biodegradable polymeric MNs.23 NIR light is attractive for transdermal delivery particularly due to its high penetrability with a minimal photodamage on skin tissues. NIR light can be absorbed by the NIR-responsive nanosized absorbers and converted into heat, causing a phase transition of the polymeric MNs, and, thus, stimulating the release of loaded drugs from the MNs. These systems would make dosages to be adjustable and realizes the controlled release of drugs. A wide number of NIR-responsive nanosized absorbers including lanthanum hexaboride (LaB6) nanoparticles (NPs),23,24 Au NPs,25 Bi quantum dots,26 carbon dots,27 or metal sulfides,28,29 have been studied in theranostic applications. However, most typical NIR-responsive nanosized absorbers are inorganic substance with non-biodegradable, non-specific biological distribution, and poor characterized bioretention, resulting in a toxic effect potentially.30 Organic NIR-responsive absorbers have been attracted attention because of their high molar absorptivity, good photostability. In addition, organic NIR-responsive absorbers usually can be biodegraded, leading to be excreted with short time after injection and thus reducing the concerns about long-term toxicity as well.31 For example, prussian blue (PB) nanoparticles approved by FDA in clinical treatment, have been developed as a NIR-responsive absorber in MNs transdermal delivery system by our group.32 The NIR-triggered release of drugs can be precisely controlled with an adjustment of NIR exposure time and amount of encapsulated PB NPs. Although lots of attempts have been made to improve the efficiency of drug delivery across skin with a positive and controllable manner, it is still an enormous challenge for choosing a mild transdermal administration. Decomposition and biocompatible polymers 5



provide broad prospects for the development of recyclable photothermal conversion materials. Polydopamine (PDA) as an organic polymeric photothermal conversion agent, is biocompatible in vivo and suitable for application with a strong NIR absorptive capacity and a high photothermal conversion efficiency of ~40%.33 Herein, the polymeric MNs integrated with metformin-loaded and PDA/LA-coated hollow mesoporous SiO2 had been prepared for transdermal delivery of antidiabetic drug (metformin) on diabetic rats under NIR irradiation (Scheme 1). Firstly, the anti-diabetic drug (metformin) was loaded within hollow mesoporous SiO2 nanoparticles (HMSN) to as drug warehouse by a diffusion method. Then, PDA as photothermal conversion agent and lauric acid (LA) (m.p. ≈ 44-46 °C) as phase change material (PCM) were coated onto the HMSN to form NIR-responsive drug nanocarriers. These metformin-loaded and PDA/LA-coated hollow mesoporous SiO2 nanocarriers were further encapsulated into poly(vinylpyrrolidone) (PVP) MNs. After insertion of MNs into skin tissue, these metformin-loaded nanocarriers were diffused into skin issue from MNs. Under NIR irradiation, LA could be melted and the holes on HMSN could be opened as well due to the photothermal conversion of PDA, and thus prompting the release of metformin from HMSN into skin tissue. The NIR-responsive release of loaded metformin in vivo has also been investigated to achieve the hypoglycemic effect on diabetic rats.

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Scheme 1. Schematics for preparation of metformin-loaded and PDA/LA-coated hollow mesoporous SiO2 nanocomposites and NIR-responsive release of loaded metformin on diabetic rats by the transdermal delivery method. 2. EXPERIMENTAL 2.1 Materials Lauric acid (LA, AR, 98%), Poly(vinyl pyrrolidone) (PVP, MW = 130 kDa), tetraethyl orthosilicate (TEOS), anhydrous sodium carbonate (Na2CO3), cetyltrimethyl ammonium bromide (CTAB), methyl thiazolyl tetrazolium (MTT), streptozo ticin (STZ) and metformin (MET) were purchased from Aladdin Chemistry Co. Ltd. (Shanghai, China) and without any further treatment. Poly(dimethyl silicone) (PDMS) microneedle (MN) molds and spring applicator were obtained from Micropoint Technologies Pte. Ltd. in Singapore. The derionized (DI) water was used in all studies. About 200 g of male SD rats were offered by Zhejiang Academy of Medical Sciences Animal Experimental Center (Hangzhou, China). 7



2.2 Preparation of metformin-loaded and PDA/LA-coated HMSN A three-step method was used to fabricate the drug-loaded and PDA/LA-coated hollow mesoporous SiO2 nanocomposites according to the previous studies.34-36 Briefly, 4 mL of ammonia (37-38%) mixed with 3 mL of TEOS was added in a mixture of 100 mL ethanol and 8 mLDI water to obtain solid SiO2 nanoparticles (sSiO2 NPs) by stirring at 30 °C for 6 h. A uniform ethanol solution (water : ethanol = 220 mL : 10 mL) containing CTAB (1200 mg) was poured into as-prepared sSiO2 NPs suspension and TEOS (1.075 mL) was subsequently added in above mixture. The obtained dispersion continued to be stirred for 12 h and followed centrifuged at 8,000 rpm and washed by water to collect core-shell SiO2 NPs. A selective etching method was subsequently used to prepare HMSN.37,38 Namely, the as-prepared core-shell SiO2 NPs were redispersed in Na2CO3 solution (0.4 M, 150 mL) and then mixed for 2 h at 50 °C to remove sSiO2 cores.39 A solution of methanol/HCL (50 mL:3 mL) mixed with crude products was further refluxed at 80 °C for one day to remove CTAB micelles on the mesoporous shell and afterwards final HMSN could be obtained via centrifugation and lavation with water for 3 cycles.40 Loading antidiabetic drug (metformin) into HMSN was performed by a diffusion route. Typically, 50 mg of HMSN was added into an aqueous solution (25 mL) containing metformin (50 mg). And the above mixture was further stirred for 24 h at ambient temperature to obtain metformin-loaded HMSN (Met/HMSN). The final products were obtained via centrifuge and lavation with DI water for 5 times for removal of the drug on the surface of HMSN. The drug loading percentage was measured by subtracting the amount of supernate from total addition of metformin with an ultraviolet and visible spectrophotometer 8


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(UV-Vis, U3900H, Hitachi, Japan). The metformin-loaded and photothermal-responsive HMSN were prepared according to a previous approach.41-43 The as-prepared Met/HMSN (50 mg) was suspended into a mixture containing 20 mL water and 2 mL ethanol. 2 mL of LA ethanol solution (5 mg/mL or 10 mg/mL) was poured into above mixture and stirred for about 5 min. 50 μL of ammonium hydroxide was then added in above mixture and 60 mg of dopamine (DA) was added and stirred for 16 h to form PDA/LA shell on the surface of Met/HMSN (PDA/LA-Met/HMSN). The final metformin-loaded and NIR-responsive HMSN were obtained via centrifuge and abstersion with DI water for 3 cycles for removing any unpolymerized DA and dried in vacuo at 25 °C for future utilization. Above nanocomposites prepared with 5 mg/mL LA ethanol solution was denoted as PDA/LA-Met/HMSN (LA coating Ⅰ) while 10 mg/mL LA ethanol solution was denoted as PDA/LA-Met/HMSN (LA coating Ⅱ). Rhodamine 6G (R6G, Macklin, Shanghai, China) as a fluorescent model drug was used to replace metformin for visualization of drug diffusion behaviors in skin tissue. The same preparation process was performed to prepare R6G-loaded and photothermal-responsive HMSN

(PDA/LA-R6G/HMSN).

With

no

drug

loading

HMSN

represented

as

PDA/LA-HMSN was used for comparative analysis. 2.3 Characterization A scanning electron microscopy (SEM, Vltra55, Zeiss, German) and a transmission electron microscope (TEM, JEM-2100, JEOL, Japan) were used to analyze the morphologies of HMSN with or without drug loading. A N2 adsorption investigation (3H-2000PS1/2 static 9



volume method, Beijing, China) was utilized for analysis of pore structure and superficial area of HMSN. The particle sizes of as-prepared drug carriers were investigated with a Dynamic optical scatterometer (Malvern Instruments, Worcestershire, UK) and every sample was measured by triplicate. Infrared spectra were recorded to analyze the surface chemistry and physical structure of these nanoparticles by a Fourier transform infrared (FTIR, Nicolet 5700, Thermo Electron Co., USA) and all samples were prepared as KBr pellets with a spectral width ranging from 4000-400 cm−1. The melting temperature area of PDA/LA-Met/HMSN was confirmed by a differential scanning calorimeter (DSC, Q2000, America). An UV-Vis spectrophotometer was used to analyze the drug loading in HMSN with absorbance range of 200-1000 nm. The thermal stability was assessed with a thermogravimetric analysis (TGA, Pyris Diamond I, PerkinElmer Co.) which was conducted at a temperature ranging of 25-800 °C (20 °C/min) under dynamic N2 atmosphere. 2.4 MTT assay of PDA/LA-HMSN An MTT assay was conducted for evaluating the cytotoxicity of PDA/LA-HMSN.7 Various PDA/LA-HMSN concentrations (12, 25, 50, 100 and 200 μg/mL) were incubated with 3T3-L1 cells in a 96-well plate at CO2 humidified incubator (5%, 37 °C) for 48 h. Another incubating for 4 h after a MTT solution (20 μL, 5 mg/mL) was added into wells. Afterwards, DMSO (150 μL) was continued to add to wells with a removal of MTT solution from each well. And the absorbance of above solution in every well was measured via an ELIASA (Multiskan MK3, Thermo Electron Corporation). And each concentration of prepared PDA/LA-HMSN samples was conducted with 5 replicates.

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2.5 Photothermal properties assay The photothermal conversion properties of PDA/LA-HMSN were investigated by NIR irradiation with 808 nm diode laser.44,45 The PDA/LA-HMSN particles (100 μg/mL) were dispersed into a DI water (1.0 mL) and the obtained dispersions were irradiated with varied power intensity (0.5, 1.0, 1.5, 2.0 and 2.5 W/cm2) of 808 nm NIR laser radiation. The real-time temperature of PDA/LA-HMSN solution were monitored by an infrared imaging device (Ti400, Fluke, America). To further assess the photothermal stability and repeatability of PDA/LA-HMSN, recycling temperature variations of PDA/LA-HMSN suspensions were monitored with a 1.0 W/cm2 of irradiation for 6 min with the state of laser on and followed naturally cooled to indoor temperature with the state of laser off for 5 laser on/off cycles. 2.6 Fabrication of MNs The PDMS microneedle mold with 600 μm of height and 300 μm of base (Patch Size: 1 cm × 1 cm, Array Size: 15 × 15) was used to prepare MNs.16 Briefly, 56.5 mg of PDA/LA-Met/HMSN or PDA/LA-R6G/HMSN was uniformly dispersed into 1 mL of water to obtain a uniform solution. And a composite hydrogel was formed by adding 0.4 g PVP into the above solution. The viscous hydrogel (~100 mg) was poured onto the surface of MNs mould under a centrifugation at 8,000 rpm for 10 min for formation of drug-loaded MN bodies. Untreated PVP matrix solution was continued to be placed on the upper cavity of obtained mold with another identical centrifugation to form drug-free supporting substrates and patch layers. The final MNs were demolded after natural withering overnight and deposited in dry condition for future use. And the morphology of MNs was characterized by a 11



SEM and digital macroscope. 2.7 Mechanical properties testing The mechanical strength of as-prepared MNs was evaluated by a universal mechanical tester (5943, INSTRON Co. LTD., America), in which the stainless-steel plate against MN arrays at an initial distance of 1 mm and a platform motion velocity of 0.5 mm/min.10 The displacement and corresponding force of the steel platform were recorded each 0.1 s when contacted with MN arrays to obtain a force-displacement curve. The mechanical strength of pure PVP MNs was also measured for comparation. A digital macroscope was used to observe the morphology changes of MNs after mechanical testing. 2.8 Skin penetration test in vitro The MNs with loading of PDA/LA-R6G/HMSN were prepared to analyze the skin penetration capability in vitro owe to the fluorescence properties of R6G.24 The separated skin samples cleaned by alcohol (75%) were applied with as-prepared MNs. To study the NIR-responsive drug release behaviors, 1.0 W/cm2 of NIR irradiations were conducted for 6 min after insertion of MNs into isolated skin for 5 min. The treated skin was characterized by a laser confocal scanning microscope (LCSM, Axio Observer A1, Carl Zeiss, Germany) to rebuild a three-dimensional image of fluorescent region. Histological sections characterization was carried out for further study of the solvation of MNs and the diffusion of drugs with a cryostat microtome (CryoStar NX50, Thermo Fisher Scientific, USA). 2.9 Thermal damage assay

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The MNs with PDA/LA-HMSN as the fillers were irradiated by a 1.0 W/cm2 of NIR laser irradiation (808 nm) with different exposure time (0, 6 and 12 min) for the thermal damage assessment. The tissue sections from skin samples were treated with hematoxylin and eosin (H&E) staining before histological characterization analysis to compare from untreated normal skin tissues (Control Group). 2.10 Drug release behavior in vitro The release behaviors of metformin from HMSN with or without NIR irradiation were performed to analyze the NIR-responsive behavior in vitro. In brief, 100 mg of PDA/LA-Met/HMSN were distracted into a PBS solution (10 mL, pH 7.4) at 37 °C under shaking. These solutions were irradiated by a 1.0 W/cm2 of NIR irradiation (808 nm) for 6 min at desired time point. After same intervals, the supernatants were obtained by a centrifugation for analysis of released metformin with an UV-Vis spectral spectrum according to previous standard calibration curve. The PDA/LA-Met/HMSN suspension without NIR radiation was conducted as a control. 2.11 Hypoglycemic effect in vivo The in vivo hypoglycemic activity of MNs with PDA/LA-Met/HMSN as fillers were performed on tape 2 diabetic rats (200 ± 20 g) induced by STZ.46 Four treatment groups (five rats for each group) were used for following treatment with MNs or native metformin: (1) Control group with non-treatment; (2) Injection group with subcutaneous injection of metformin on a single dose (1.0 mg); (3) MNs group (loaded with 1.0 mg of metformin in Met/HMSN without PDA/LA coating) with no NIR exposure; (4) MNs group (loaded with 13



1.0 mg of metformin in PDA/LA-Met/HMSN) with no NIR exposure; (5) MNs group (loaded with 1.0 mg of metformin in PDA/LA-Met/HMSN) with 6 min of NIR irradiation (1.0 W/cm2) at desired time (0, 2, 4 and 6 h). MNs were pressed firmly onto skin with a spring applicator for 5 min to be dissolved by absorption of tissue liquid. The blood glucose levels (BGLs) of tail vein blood samples from treated rats were monitored with a blood glucometer. In addition, a consecutive MNs administration with same NIR irradiation cycles was conducted to analyze the control capability of plasma glucose of prepared MNs.47 2.12 System acute toxicity experiment For acute toxicity assessment in vivo, SD rats from MNs group were randomly selected as the treatment group while SD rats with non-treatment were used as a control group. The representative organs included kidney, spleen and liver of experimental rats in each group were harvested for paraffin-embedded sectioning. Histological examinations were conducted by a fluorescence inversion microscope (Ts2, Nikon) after H&E staining.48,49 3. RESULTS AND DISCUSSION 3.1 Synthesis and characterization of PDA/LA-HMSN The particle size and surface properties of the drug carriers play important roles in drug release and cellular uptake.50 Herein, a selective etching method is used to prepare HMSN as drug carriers.37,38 The HMSN exhibits a symmetrical spherical and hollow structure with diameter at ~280-320 nm and shell thickness at ~30-40 nm, as shown in Figure 1A-a,b. In the case of PDA/LA-HMSN, a light color coating with a layer thickness of 10-20 nm can be found on the surface of HMSN particles. The size of PDA/LA-HMSN displays an average 14


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diameter range of 300-350 nm (Figure 1B-a,b). Due to the coated of organic layer on the surface of HMSN, the as-prepared PDA/LA-HMSN exhibits a rougher surface that confirmed by the SEM results (Figure 1A-c and Figure 1B-c). After dispersed in a 1.0 mL of ultrapure water and irradiated with a 1.0 W/cm2 of NIR radiation, some of the LA coating on PDA/LA-HMSN are disappeared while some LA coating still existing on HMSN, as shown in the surface morphology on TEM images (Figure 1C-a,b) and SEM photograph (Figure 1C-c). Above results indicate that the microchannels may be formatted contribute to an ablation of LA coating but blocked again thanks to a solidification of LA coating.

Figure 1. TEM images of HMSN with low (A-a) and high magnification (A-b). TEM images of PDA/LA-HMSN before NIR irradiation with low (B-a) and high magnification (B-b). TEM images of PDA/LA-HMSN with low (C-a) and high magnification (C-b) after NIR (1.0 15



W/cm2) irradiation for 5 min. SEM images of HMSN (A-c) and PDA/LA-HMSN before (B-c) and after (C-c) NIR (1.0 W/cm2) irradiation for 5 min.

The sizes of HMSN and PDA/LA-HMSN are further determined with a DLS. The diameters of the HMSN and PDA/LA-HMSN were 300 and 336 nm, respectively, as shown in Figure 2A. The specific area of HMSN is 924.4 m2/g according to a BET method (inset in Figure 2A), and the corresponding Barrett Joyner Halenda (BJH) pore diameter is ~4.2 nm (Figure S1). In addition, a rapid decline near the middle of relative pressure on the desorption curve implies as-prepared HMSN has open-ended mesopores.16 Due to the large pore size and open-ended mesopores properties, HMSN is a potential drug carriers for loading of drugs. The FT-IR and EDS analysis are further conducted to confirm the formation of PDA on the surface of HMSN. As shown in Figure 2B, the band that appeared at 1510 cm-1 and 1619 cm-1 are owe to N-H stretching and C=C resonance vibration from PDA, respectively,51-53 indicating the successfully formation of PDA shell on HMSN. The appearing of N peak in EDS spectrum of PDA/LA-HMSN further confirms the existence of PDA shell (Figure S2). For the confirmation of phase change behaviors of PDA/LA coating on the surface of HMSN, DSC analysis was conducted and the results are shown in Figure 2C. Compared to PDA/LA composites with a heat absorption peak at ~46 oC, the PDA/LA-HMSN shows a little difference at ~45 oC. However, no melting behavior can be observed for PDA-HMSN with range from 20-100 oC, demonstrating the acceptable thermal phase change behaviors of PDA/LA coating. TGA results are confirmed that the 71.1% weight maintenance in PDA/LA-HMSN, while 91.0% in HMSN during heating process. The disparity in weight loss 16


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of them are mainly attributed to the PDA/LA organic coating (inset in Figure 2C).

Figure 2. DLS and Nitrogen adsorption-desorption isotherm (inset) of HMSN and PDA/LA-HMSN (A); FTIR spectra of HMSN and PDA/LA-HMSN (B); DSC curves of PCM, PDA-HMSN and PDA/LA-HMSN (C, TGA curves of HMSN and PDA/LA-HMSN inset in C); Cytotoxicity of PDA/LA-HMSN incubating with 3T3-L1 cells (D).

The entrapment efficiency (EE) and loading content (LC) of metformin in PDA/LA-HMSN are calculated to be 78.1% and 32.2%, respectively. From the UV-Vis spectra analysis, a strong absorption peak at 235 nm can be found in PDA/LA-Met/HMSN, indicating that metformin is successfully loaded in HMSN (Figure S3). To further visualize the thermosensitive property of PDA/LA coating, PDA/LA-R6G/HMSN is chosen to 17



dispersion in PBS solution. For comparison, R6G is spread out quickly from the carriers with little temperature interference. However, in the case of PDA/LA-R6G/HMSN, darker brownish red can be observed when placed them in 46 oC for 1 day compared with them in 37 oC,

which may owe to the release of R6G (Figure S4). To estimate the biocompatibility of

PDA/LA-HMSN, a MTT assay is investigated in vitro. The cell viabilities of 3T3-L1 after incubated with diverse concentrations of PDA/LA-HMSN are shown in Figure 2D. The result confirmed the PDA/LA-HMSN is biocompatible with toxicity barely, even the concentration is up to 200 μg/mL. 3.2 Evaluation of NIR-light-responsive photothermal properties PDA is an excellent photothermal conversion agent in many drug delivery system, in which the irradiation intensity may was a critical parameter for drug release efficiency.54,55 To estimate the NIR-light-responsive photothermal properties of PDA/LA-HMSN, various power intensity of NIR irradiation (808 nm) from 0.5 to 2.5 W/cm2 are applied on suspension of PDA/LA-HMSN. The temperature of solutions measured by IR imaging senor is shown in Figure 3A. All test samples are displayed a temperature rise within 6 min. The temperatures of solutions quickly exceed 50 oC with irradiation intensity at 2.5 and 2.0 W/cm2, and it also can be reached ~50 oC after irradiation for 10 min with power intensity at 1.5 W/cm2. However, it only can be reached ~40 oC with power intensity at 0.5 W/cm2 even irradiation for more than 10 min. Only the suspension with 1.0 W/cm2 of irradiation could maintain a temperature of about 46 oC. A thermal damage on skin tissues may be caused when imposed temperature up to 50 oC for a long time.56 Therefore, 1.0 W/cm2 is selected as the suitable power intensity for the following investigations. The photothermal repeatability assay is 18


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further carried out with NIR (1.0 W/cm2) irradiation for 6 min and then laser was switched off instantly. As shown in Figure 3B, the temperatures of solutions can be rose to ~46 oC with NIR laser on and then dropped to room temperature with NIR laser off. Almost no differentia can be observed after repetition for 5 cycles. Therefore, PDA/LA-HMSN exhibits an excellent NIR-light-responsive photothermal properties with good photostability.

Figure 3. Photothermal curve of aqueous suspensions dispersed with PDA/LA-HMSN under NIR irradiation (808 nm) at varied power densities (A); Photothermal curve of PDA/LA-HMSN suspension liquid for five laser on/off cycles under a 1.0 W/cm2 of NIR irradiation (B).

3.3 Fabrication of MNs As shown in Figure 4A, the as-fabricated MNs displayed a typical pyramid-shape morphology with a 15×15 array. Further morphology characterizations via SEM analysis are shown in Figure 4B and 4C. The MNs are ~200 μm in width and ~600 μm in height while ~500 μm in interval. A mechanics performance testing of MNs is carried out for evaluating the mechanical strength property of as-prepared MNs. The nanoparticles loaded in MNs 19



would distribute uniformly at the front part of MNs contribute to the centrifugation, leading an enhancement of MNs’ mechanical properties.12,17 As shown in Figure 4D-a, compared with pure PVP MNs, the composite MNs exhibit an excellent mechanical performance. The mechanical strength is about 0.175 N/needle while PVP MNs is roughly 0.097 N/needle with displacement at 400 μm. It indicates the existence of inorganic nanoparticles will enhance their mechanical property. And the they can be penetrated into skin due to their enough mechanical strength according previous studies.17,57 Figure 4D-b,c shows the morphology comparison diagram of MNs before and after compression test. The tips of MNs are changed with a severe inflection but no fracture compared with their original state. It further confirms that composite MNs possess an excellent flexibility and toughness.

Figure 4. Digital microscope images of NCs-PVP MNs under low (A-a, inset shows the top 20


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view of MNs patch) and high magnification (A-b). SEM images of side elevation at low (B-a) and high magnification (B-b), vertical view at low (C-a) and high magnification (C-b) of NCs-PVP MNs. Mechanical strength assays (D-a) and microscope images of NCs-PVP MNs before (D-b) and after (D-c) mechanical properties test.

3.4 Skin penetration in vitro R6G is chosen as a fluorescent dye to replace metformin for analysis of skin penetration property and visualization of drug release behaviors from MNs in vitro. The separated skin samples applied with R6G-loaded MNs are carried out and characterized with a CLSM. As shown in Figure 5, the dead slots (~350 μm deep) left on skin surface (white arrows), which come near the deepness of dermis layer. In consequence, the as-prepared MNs possess an excellent penetration performance, which can pierce skin stratum corneum without significant damage on skin tissues.58,59 The red fluorescent signal exhibits a typical pyramid-shaped morphology, which can be hardly detected in deep skin tissues but slightly observed around puncture sites after application of R6G-loaded MNs for 1 min (Figure 5A). There is no obviously change of fluorescent signal in skin after insertion of R6G-loaded MNs for 5 min but the pyramid-shaped morphology is starting to fade (Figure 5B) due to the degradation of PVP. However, an intense fluorescence can be obviously detected in the deeper tissue after a

21



Figure 5. Fluorescence 3D reconstruction images (a) and corresponding histological sections of skin samples (b: Bright-filed images; c: Fluorescent images; d: Merged images) applied with R6G-loaded MNs for 1 min (A) and 5 min (B), and next under NIR irradiation for 6 min (C).

1.0 W/cm2 of NIR irradiation (808 nm) for 6 min (Figure 5C). Meanwhile, the pyramid-shaped morphology of MNs are disappeared from the 3-D reconstruction image, which suggesting that the as-prepared MNs possess a NIR-light-responsive ability. 3.5 Thermal damages on skin A thermal damage on skin tissue will be caused when contacted to temperatures above physiological temperature for a long time.60 To estimate the thermal damage on skin, skin samples were applied with MNs under a 1.0 W/cm2 of NIR irradiation (808 nm) before their histological analysis using H&E staining. The skin sample with non-treatment is chosen as a 22


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control. As shown in Figure 6, compared with control, there is no histological damage can be observed nearly for the samples with MNs under NIR irradiation with 6 min. Even NIR exposure prolonged for 12 min, there is still no obvious tissue lesion can be observed in epidermis and dermis of skin. Therefore, the as-prepared MNs system exhibits a good security capability in drug release without any tissue damage under NIR exposure.

Figure 6. Bright field photographs of skin histological sections with application of MNs under NIR irradiation for different time before H&E staining. The skin sample with nontreatment is chosen as a control.

3.6 Drug release with NIR irradiation in vitro and in vivo The entrapment efficiency and loading content of metformin in PDA/LA-Met/HMSN have been calculated to be 78.1% and 32.2% according to UV-Vis assay. For analyzing the drug release behavior from PDA/LA-Met/HMSN with NIR irradiation in vitro, the release profile of metformin from PDA/LA-Met/HMSN are performed in PBS buffer solution with or without NIR exposure. The release amount of encapsulated metformin is ~16.3% and ~12.1% consistently for each cycle after 6 min of NIR irradiation (1.0 W/cm2) for PDA/LA-Met/HMSN (LA coating Ⅰ) and PDA/LA-Met/HMSN (LA coating Ⅱ) respectively, as shown in Figure 7A. And no obviously drug diffusion can be detected when 23



the NIR laser switch off. About 72.5% and 60.7% of metformin can be released from PDA/LA-Met/HMSN (LA coating Ⅰ) and PDA/LA-Met/HMSN (LA coating Ⅱ) after five NIR laser on/off cycles. However, only ~17.8% of loaded-metformin can be released from PDA/LA-Met/HMSN without NIR irradiation by a concentration diffusion route. Above results demonstrate that the more content of LA in PDA/LA coating, the lower drug release rate from Met/HMSN with a 1.0 W/cm2 of NIR irradiation. The melting of PDA/LA coating induced by a NIR exposure may generate micro-channels on the surface of PDA/LA-Met/HMSN, which increase the release of metformin and form an effective drug release behavior controlled with NIR irradiation.41 The hypoglycemic effect analyses are performed on type Ⅱ diabetic SD rats produced by STZ in vivo. The diabetic rats applied with a disposable dose of metformin (1 mg) by subcutaneous injection are as contrast while the diabetic rats with non-treatment as control. As shown in Figure 7B, the initial blood glucose levels (BGLs) in each group are ~401.2±6.8 mg/dL. And the blood glucose concentrations in injection group are rapidly declined and followed depress to their minimum value (81.8±4.5 mg/dL) after 2 h. While in MNs group loaded with Met/HMSN without PDA/LA coating, the hypoglycemic rate is slower compared with subcutaneous injection of a single dose, and the BGLs are declined to minimum value (122.2±3.7 mg/dL) after about 3 h. However, the BGLs of experimental rats applied by MNs administration with repetitive and discontinuous NIR irradiation (1.0 W/cm2) in MNs group (loaded with PDA/LA-Met/HMSN) are deceased slightly compared with that of the injection group. And the minimum value of MNs administration group is ~167.7±5.2 mg/dL at 8 h. In addition, the blood glucose levels can be maintained below 200 mg/dL for around ~4.0 h in 24


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MNs administration group compared with ~2.5 h for subcutaneous injection group and ~3.0 h for MNs group loaded with Met/HMSN without PDA/LA. Relative to the group of subcutaneous injection, the MNs-NIR exhibits a bioavailability of 95.8±2.7% (Table S1). No obviously hypoglycemia effect can be observed for the MNs administration group (loaded with PDA/LA-Met/HMSN) without NIR irradiation, which demonstrating that the beginning of drug release can be caused by a NIR irradiation, and then diffused into skin tissue and systemic circulation. The corresponding hourly decline speeds of blood glucose levels in MNs administration group (loaded with PDA/LA-Met/HMSN) with NIR irradiation are shown in Figure 7C. The first hour descent rate after NIR irradiation in each NIR laser on/off cycle is faster than that of the second hour without NIR irradiation. It also indicates the low drug leakage can be formed without NIR exposure. Furthermore, a consecutive MNs administration with same NIR irradiation (1.0 W/cm2) cycles is performed to analyze the repeatable plasma glucose control capability of MNs. The blood glucose concentrations are maintained in a narrow range from 160 to 280 mg/dL and no hypoglycemia effect can be observed during the treatment time, as shown in Figure 7D. Therefore, the as-prepared MNs system exhibits an initiative manner for drug release and an efficient hypoglycemic ability on diabetic rats with triggered by NIR irradiation.

25



Figure 7. In vitro drug release profiles of PDA/LA-Met/HMSN with LA coating Ⅰ (prepared with 5 mg/mL of LA ethanol solution) and LA coating Ⅱ (prepared with 10 mg/mL of LA ethanol solution) in PBS solution with and without NIR irradiation for 6 min at specific time (A). Blood glucose concentrations of diabetic rats treated with hypodermic injection drug-loaded MNs (with and without PDA/LA coating) with and without NIR irradiation for 6 min at specific time. The diabetic rat sample without any treatment is used as a control (B). Corresponding decline rates of blood glucose concentrations hourly calculated from MNs groups (C). The BGLs of diabetic rats which applied by metformin-loaded MNs cycles at specific time. The diabetic rats in each MNs cycle are applied with uniformly spaced NIR irradiation for 4 times (D). The black arrows mean the addition of NIR irradiation while the red arrows mean the addition of MNs administration.

To analyze whether there are tissue damages in MNs system, a histologic section assay of 26


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representative organs include liver, spleen and kidney from treated rats in MNs-NIR group after H&E staining is carried out. The rats from control group is as a control. The results are shown in Figure 8. No obvious tissue damage is detected compared with control, which indicating that MNs system is secure with no toxicity for a potential clinic application.

Figure 8. H&E-stained histologic images of major organs from experimental rats after treated with by MNs and NIR irradiation. The untreated diabetic samples were as control.

4. CONCLUSION In conclusion, the polymeric MNs combined with metformin-loaded and PDA/LA-coated hollow mesoporous SiO2 had been prepared by a template method. Herein, PDA on the surface of HMSN could be used as a photothermal transfer agent while LA as a thermal responsive agent. A hypoglycemic drug (metformin) was encapsulated into HMSN. Under NIR irradiation, LA coating exhibited an obvious thermal ablation due to the photothermal transfer of PDA, and so promoting the release of encapsulated metformin from drug 27



warehouse with photothermal trigger. Furthermore, the proposed drug delivery system could release loaded metformin in an intermittent cycle administration with NIR laser on/off cycles contribute to few drug revelations when laser off. Therefore, the prepared devices display an excellent photothermal-responsive capacity with well metformin loading efficiency and no toxicity in vivo. There would be a well hypoglycemic effect and a low risk of hypoglycemia at the same time when above MNs system applied on diabetic rats. Such NIR-triggered MNs transdermal delivery system exhibits an excellent drug release behavior and efficient hypoglycemic activity on diabetic models with a relatively convenient and initiative manner. ■ ASSOCIATED CONTENT Supporting Information Pore size distribution of HMSN; EDS spectra on PDA/LA-HMSN; UV-Vis spectra of Met, HMSN, Met/HMSN and PDA/LA-Met/HMSN; photographs of various NCs dispersion at different temperature; pharmacodynamic parameters of BGLs on experimental rats which applied with MNs-NIR system and subcutaneous injection. ■ AUTHOR INFORMATION Corresponding Author *Tel.: +86-571-8684-3527. E-mail: [email protected]. ORCID Guohua Jiang: 0000-0003-3666-8216 Notes 28


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The authors declare no competing financial interest. ■ ACKNOWLEDGEMENTS This work was financially supported by the Natural Science Foundation of Zhejiang Province (LY18E03006), the National Natural Science Foundation of China (51373155, 51873194) 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. ■ REFERENCES (1) Wang, S.; Wen, X.; Han, X.; Wang, Y.; Shen, M.; Fan, S.; Zhuang, J.; Xu, W.; Zhang, Z.; Shan, Q.; Li, M.; Hu, B.; Sun, C.; Wu, D.; Lu, J.; Zheng, Y. Microrna-30d Preserves Pancreatic Islet Β-Cell Function through Negative Regulation of the JNK Signaling Pathway via Socs3 in Mice with Streptozotocin-Induced Diabetes Mellitus. J. Cell. Physiol. 2018, 233 (9), 7343-7355. DOI: 10.1002/jcp.26569. (2) American Diabetes Association, Diagnosis and Classification of Diabetes Mellitus. Diabetes Care, 2010, 33 (Suppl. 1), S62-S69. DOI: 10.2337/dc10-S062. (3) Xu, Y.; Wang, L.; He, J.; Bi, Y.; Li, M.; Wang, T.; Wang, L.; Jiang, Y.; Dai, M.; Lu, J.; Xu, M.; Li, Y.; Hu, N.; Li, J.; Mi, S.; Chen, C.-S.; Li, G.; Mu, Y.; Zhao, J.; Kong, L.; Chen, J.; Lai, S.; Wang, W.; Zhao, W.; Ning, G. Prevalence and Control of Diabetes in Chinese Adults. JAMA. 2013, 310 (9), 948-959. DOI: 10.1001/jama.2013.168118. (4) Gong, L.; Liu, F.; Wang, J.; Wang, X.; Hou, X.; Sun, Y.; Qin, W.; Wei, S.; Zhang, Y.; Chen, L.; Zhang, M. Hyperglycemia Induces Apoptosis of Pancreatic Islet Endothelial Cells via Reactive Nitrogen Species-Mediated Jun N-Terminal Kinase Activation. BBA-Mol. Cell Res. 2011, 1813 (6), 1211-1219. DOI: 10.1016/j.bbamcr.2011.03.011. (5) Yu, W.; Jiang, G.; Zhang, Y.; Liu, D.; Xu, B.; Zhou, J. Polymer Microneedles Fabricated 29



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TOC (Table of Contents) 88x53mm (300 x 300 DPI)


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Scheme 1. Schematics for preparation of metformin-loaded and PDA/LA-coated hollow mesoporous SiO2 nanocomposites and NIR-responsive release of loaded metformin on diabetic rats by the transdermal delivery method. 177x102mm (300 x 300 DPI)



Figure 1. TEM images of HMSN with low (A-a) and high magnification (A-b). TEM images of PDA/LA-HMSN before NIR irradiation with low (B-a) and high magnification (B-b). TEM images of PDA/LA-HMSN with low (C-a) and high magnification (C-b) after NIR (1.0 W/cm2) irradiation for 5 min. SEM images of HMSN (A-c) and PDA/LA-HMSN before (B-c) and after (C-c) NIR (1.0 W/cm2) irradiation for 5 min. 177x136mm (300 x 300 DPI)


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Figure 2. DLS and Nitrogen adsorption-desorption isotherm (inset) of HMSN and PDA/LA-HMSN (A); FTIR spectra of HMSN and PDA/LA-HMSN (B); DSC curves of PCM, PDA-HMSN and PDA/LA-HMSN (C, TGA curves of HMSN and PDA/LA-HMSN inset in C); Cytotoxicity of PDA/LA-HMSN incubating with 3T3-L1 cells (D). 177x134mm (300 x 300 DPI)



Figure 3. Photothermal curve of aqueous suspensions dispersed with PDA/LA-HMSN under NIR irradiation (808 nm) at varied power densities (A); Photothermal curve of PDA/LA-HMSN suspension liquid for five laser on/off cycles under a 1.0 W/cm2 of NIR irradiation (B). 177x69mm (300 x 300 DPI)


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Figure 4. Digital microscope images of NCs-PVP MNs under low (A-a, inset shows the top view of MNs patch) and high magnification (A-b). SEM images of side elevation at low (B-a) and high magnification (B-b), vertical view at low (C-a) and high magnification (C-b) of NCs-PVP MNs. Mechanical strength assays (D-a) and microscope images of NCs-PVP MNs before (D-b) and after (D-c) mechanical properties test. 177x124mm (300 x 300 DPI)



Figure 5. Fluorescence 3D reconstruction images (a) and corresponding histological sections of skin samples (b: Bright-filed images; c: Fluorescent images; d: Merged images) applied with R6G-loaded MNs for 1 min (A) and 5 min (B), and next under NIR irradiation for 6 min (C). 177x108mm (300 x 300 DPI)


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Figure 6. Bright field photographs of skin histological sections with application of MNs under NIR irradiation for different time before H&E staining. The skin sample with non- treatment is chosen as a control. 177x41mm (150 x 150 DPI)



Figure 7. In vitro drug release profiles of PDA/LA-Met/HMSN with LA coating Ⅰ (prepared with 5 mg/mL of LA ethanol solution) and LA coating Ⅱ (prepared with 10 mg/mL of LA ethanol solution) in PBS solution with and without NIR irradiation for 6 min at specific time (A). Blood glucose concentrations of diabetic rats treated with hypodermic injection drug-loaded MNs (with and without PDA/LA coating) with and without NIR irradiation for 6 min at specific time. The diabetic rat sample without any treatment is used as a control (B). Corresponding decline rates of blood glucose concentrations hourly calculated from MNs groups (C). The BGLs of diabetic rats which applied by metformin-loaded MNs cycles at specific time. The diabetic rats in each MNs cycle are applied with uniformly spaced NIR irradiation for 4 times (D). The black arrows mean the addition of NIR irradiation while the red arrows mean the addition of MNs administration. 177x136mm (300 x 300 DPI)


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Figure 8. H&E-stained histologic images of major organs from experimental rats after treated with by MNs and NIR irradiation. The untreated diabetic samples were as control. 177x92mm (300 x 300 DPI)


Polymeric Microneedles Integrated with Metformin-Loaded and PDA

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