Microneedles Integrated with ZnO Quantum-Dot-Capped Mesoporous

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Controlled Release and Delivery Systems

Microneedles integrated with ZnO quantum dots capped mesoporous bioactive glasses for glucose-mediated insulin delivery Bin Xu, Qinying Cao, Yang Zhang, Weijiang Yu, Jiangying Zhu, Depeng Liu, and Guohua Jiang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00626 • Publication Date (Web): 12 Jun 2018 Downloaded from http://pubs.acs.org on June 13, 2018

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ACS Biomaterials Science & Engineering

Microneedles integrated with ZnO quantum dots capped mesoporous bioactive glasses for glucose-mediated insulin delivery Bin Xu,†, §, ǁ Qinying Cao,‡ Yang Zhang,†, §, ǁ Weijiang Yu,†, §, ǁ Jiangying Zhu,†, §, ǁ Depeng Liu, †, §, ǁ and Guohua Jiang†, §,

†

⊥,

ǁ, *

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

Zhejiang 310018, China ‡

Shijiazhuang Obstetrics and Gynecology Hospital, Shijiazhuang, Hebei 050011,

China §

National Engineering Laboratory for Textile Fiber Materials and Processing

Technology (Zhejiang), Hangzhou, Zhejiang 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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Abstract: A self-responsive insulin delivery system has highly desirable due to its high sensitivity dependent on blood glucose levels. Herein, a smart pH-triggered and glucose-mediated transdermal delivery system, insulin-loaded and ZnO quantum dots (ZnO QDs) capped mesoporous bioactive glasses (MBGs) integrated with microneedles (MNs), was developed to achieve control and painless administration. ZnO QDs as a promise pH-responsive switch were employed to cap the nanopores of MBGs via electrostatic interaction. The drug (insulin) and glucose-responsive factor (glucose oxidase/catalase, GOx/CAT) were sealed into the pores of MBGs. GOx/CAT in the MBGs could catalyze glucose to form gluconic acid, resulting decrease in the local pH. The ZnO QDs on the surface of the MBGs could be dissolved in the acidic condition, leading to dis-assembly of the pH-sensitive MBGs and then release of preloaded insulin from the MBGs. As a result of administration in diabetic model, an excellent hypoglycemic effect and lower hypoglycemia risk were obtained. These results indicate that as-prepared pH-triggered and glucose-mediated transdermal delivery systems have hopeful applications in the treatment of diabetes. Keywords: transdermal delivery; glucose-mediated; insulin; microneedles; diabetes

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INTRODUCTION Diabetes, is a group of metabolic disorders in which there are high blood glucose levels and insulin deficiency over a prolonged period.1-3 Although insulin doesn't cure diabetes, it is almost impossible for people to survive suffering from severe diabetics. However, oral administration of pure insulin has low bioavailability owing mainly to the gastric pH, enzymatic and physical barriers of the intestinal tract.4 Daily hypodermic injections of insulin is a standard treatment of most diabetic patients, especially for type I diabetes. Compared with oral administration, hypodermic injections provide a more common and rapid way to deliver the insulin into circulatory system, reducing or avoiding the elimination of insulin in the intestine, stomach or other extreme conditions.5 Unfortunately, such administration needs regular and bear pain at the same time that may increase the risk of trauma and inflammation, causing the low compliance and dissatisfaction from patient.1,4,6-8 Furthermore, this also can raise the risk of complications such as hypoglycemia, which can cause syncope in critically ill patients and can have dire consequence even in otherwise healthy patients.7,9 Efforts are being made to develop new insulin delivery systems, particularly for those employing delivery routes with minimal invasive, painless and controlled release of insulin responsive to blood glucose levels.10 Microneedle drug delivery system (MDDS) is an attractive and effective method to provide a painless and non-invasive self-administration pathway. Microneedles (MNs) can create micro-channels and enable medications to be

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penetrate or across the cuticle of the skin into the systemic circulation.10-12 MNs also can improve the low compliance of patient with medication regimen due to its not stimulate nerves that can not cause pain.10,11 Despite the great progress and different kind of MNs available (solid MNs,13,14 dissolving MNs,15-18 coated MNs,19-21 and hollow MNs22,23), there still face enormous dilemma and hard to break through several technical bottlenecks in practical applications. These include high-cost and precision machining, poor biocompatibility of the MNs materials, and difficulty of controlling the drug release of transdermal delivery. The controllable or programmable MNs systems are promising therapy for diabetics that enable controlled drug delivery according to the blood sugar concentration in the body and avoid or reduce the risk of hypoglycemia due to excessive drug release, thereby avoiding side effects on the tissue structure and circulation system of the skin.16,24-27 Many near-infrared-, light-, magnetic-field-, ultrasound-, or pH-responsive materials are often used to controlled drug release.28-31 Although these efforts have been applied to enhance the efficiency of drug penetrating the skin in a positive way，it is still a great challenge to choose a mild and physicologically triggered route for transdermal administration.32 Glucose oxidase (GOx) utilized an enzyme catalyst can induce glucose to form gluconic acid in the presence of oxygen (O2), thereby lowering the local pH value.3,10 In particularly, in the presence of catalase (CAT), it can lead to lower microenvironmental pH, which can reach 5.5 within 45 min under the synergistic effect of CAT.33-36 Bioactive glasses (BGs) are promising used in many

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biomedical applications on account of their remarkable bioactive, absorbable and osteogenic properties.37,41 Over the past decades, remarkable progress in their synthesis methods and processing technology have enabled preparation of mesoporous bioactive glasses (MBGs) with larger surface areas and adapted nanoporosity. The novel structural characteristics of the MBGs have also bring new possibilities, such as new materials as controlled drug delivery carriers.42,43 Recently, multifunctional MBGs have been developed by doping various therapeutic ions,44,45 embedding metal or metal oxide nanoparticles37,39,46 surface modification of organic ligand,47 and combining with other organic or inorganic materials.40,43 Due to their uniform and dense mesoporous channel structures and excellent surface area and pore volume, MBGs are the promising carriers for loading and efficient delivery of drugs. The incorporation of biomolecules in MBGs can enhance the therapeutic effects. ZnO quantum dots (ZnO QDs) are considered to be relatively low toxic to cells and environmentally harmlessly, compared with other types of QDs. Furthermore, ZnO QDs can be used as “gatekeepers” to cap the nanopores of drug carriers to prevent drug leakage and allow preloaded drugs to be released at acid environment due to their peculiar acid degradation properties that rapid degradation under acidic conditions with pH less than 5.5 but slow degradation or stable under physiological conditions (pH≈7.4).48-50 Herein, multifunctional MBGs as drug carriers have been firstly prepared by post aggregation of ZnO QDs onto MBGs via electrostatic interactions. Furthermore, the synthesized

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hybrid materials combining porous structure of MBGs and pH-sensibility of ZnO QDs, have been used as drug carriers for loading of insulin, CAT and GOx. Finally, the smart MNs integrated with these as-prepared pH-responsive MBGs was fabricated by duplicating model method. After penetrating the skin and participating the body circulation, CAT and GOx in the MBGs can catalyze glucose to form gluconic acid, leading to decrease the local pH in MBGs. The ZnO QDs on the surface of MBGs could be dissolved, resulting in the opening of nanopores on MBGs and then release of the encapsulated insulin. The hypoglycemic effect on diabetic rats was evaluated as well after transcutaneous administration (Figure 1).

Figure 1. Schematic preparation of glucose-mediated microneedles integrated with ZnO quantum dots capped MBGs for transdermal delivery of insulin.

MATERIALS AND METHODS Materials Dodecylamine (DDA), polyvinylpyrrolidone (PVP, Mw = 1,300 KDa), 6


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triethylphosphate (TEP), hydrochloric (HCl), 3-aminopropyltriethoxysilane (APTES), calcium nitrate tetrahydrate (CN), tetraethyl orthosilicate (TEOS), glucose oxidase (GOx, from Aspergillus niger), fluorescein isothiocyanate (FITC), zinc acetate dihydrate, porcine insulin (30 IU/mg, from porcine pancreas), sodium hydroxide (NaOH), catalase (CAT) and streptozotocin (STZ) were all purchased from Shanghai Aladdin Chemical Reagent Co. (China). SAKURA Tissue-Tek ® O. C. T. Compound (O. C. T.) was provided by Hua-An Biotechnology Co., Ltd. Poly(methyl methacrylate) microneedle molds were purchased from Micropoint Technologies Pte, Ltd (Singapore). Male Sprague Dawley (SD) rats (weight of 200～210 g) were provided from the Experimental Animal Center of Zhejiang Academy of Medical Sciences. Preparation of MBGs MBGs were synthesized according to previous method with slight modification.37,39,43 Briefly, DDA (2.23 g) as template and catalyst was firstly dissolved in a mixture solution of ethyl alcohol (80 mL) and deionized water (40 mL) at room temperature. Then, TEOS (16 mL) was added dropwise into solution with moderate stirring for 30 min, followed by added CN (3.39 g) and TEP (1.22 mL) into mixture. The solution was continuously reacted for 3 h. The crude product was obtained by centrifugation (10,000 rpm, 10 min) and the product was washed 3 time with ethyl alcohol and deionized water before it dried in a freeze-drying to obtained MBGs. In order to remove residues and templates, the MBGs was heated at 650oC in air conditions for 3 h with the heating rate of

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2oC/min and then cooled down to room temperature. The composition of MBGs (mole fraction) was SiO2/CaO/P2O5 =80/16/4. Synthesis of ZnO QDs Zinc oxide quantum dots (ZnO QDs) were synthesized using previous method with slight modification.50,51 Briefly, zinc acetate dihydrate (0.41 g) was dissolved in anhydrous ethanol (10 mL) under refluxes at 80oC for 1.5 h. After the solution was completely dissolved and then cooled down 30 min, KOH (0.23 g) dissolved in anhydrous ethanol (5 mL) was dropwise added. After an hour of reaction, (3-aminopropyl)-triethoxysilane (APTES) (1 mL) mixed in anhydrous ethanol (5 mL) and deionized water (0.5 mL) was added. The solution was reacted for 2 h with stirred at room temperature. Finally, ZnO QDs was collected by centrifugation (12,000 rpm, 5 min) and washed 3 times with anhydrous ethanol before it dispersed in deionized water or freeze-drying obtained ZnO QDs. Drug loading The insulin solution (4.0 mg/mL) was prepared as follows: the insulin was added to HCl solution (0.01 M, pH = 2.0) to make it completely dissolved and then the pH of insulin solution was adjusted by NaOH solution (1 M) to 7.0. Afterwards, the as-prepared MBGs (40 mg) was dispersed in the above solution (2.0 mL) followed by the addition of the catalase (CAT, 0.20 mg) and glucose oxidase (GOx, 0.80 mg). The solution was moderate stirred for 48 h to absorb insulin into the nanopores before the addition of as-prepared ZnO QDs aqueous

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solution (10.0 mg/mL) dropwise. After stirring for 6 h, the insulin-loaded complexes (Ins+GOx/CAT-MBGs/ZnO) were collected by centrifugation (12,000 rpm, 30 min), washed 3 times with deionized water and following a freeze-drying. The drug-free carriers (MBGs/ZnO) and insulin-loaded hybrid complexes without GOx and CAT (Ins-MBGs/ZnO) were using the same method to be prepared. Preparation of MNs MNs were prepared according to our previous study.8,16,17 Briefly, the mold consist of 100 pyramid shaped microneedles with the same height of each microneedle (the height of the microneedle is 550 µm that excepted pedestal). The

hybrid

complex

(Ins+GOx/CAT-MBGs/ZnO,

Ins-MBGs/ZnO

and

MBGs/ZnO) was dispersed in deionized water (1 mL), and then PVP (0.4 g) was added to form the viscous paste. The as-prepared MNs fabricated by a two-step molding process was described in our previous report.16 These MNs were labelled as Ins+GOx/CAT-MBGs/ZnO MNs, Ins-MBGs/ZnO MNs and MBGs/ZnO MNs, respectively. Characterization The diameter size and ξ-potential of MBGs particles were characterized by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). The diameter and morphology of MBGs was observed by transmission electron microscopy (TEM, JEOL, JSM-2100, Japan) and scanning electron microscopy (SEM, Zesis, ULTRA-55, Germany). The drug loading capacity (DLC) and encapsulation efficiency (EE) of insulin were

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measured using the UV spectrophotometry method (λ = 276 nm, Aucy, UV1901PC, Shanghai).16,52 The absorbance was recorded and then the content of insulin from the supernatant was calculated by standard curve of insulin (Fig. S1). The DLC and EE were calculated using the formula (1) and (2): Drug loading capacity (DLC) =

total amount of insulin added - free insulin × 100% weight of MBGs

(1) Encapsulat ion efficiency (EE) =

total amount of insulin added - free insulin × 100 % total of amount of insulin added

(2)

Insulin release in vitro The

content

of

insulin

release

in

vitro

from

the

composite

(Ins+GOx/CAT-MBGs/ZnO or Ins-MBGs/ZnO) was evaluated in PBS solution (8 mg/mL) with different pH values (pH = 2.0, 5.0 and 7.4) and different concentrations of glucose (5 mM and 20 mM) on orbital shaker at the room temperature. At the predetermined time points, test samples were collected and removed from the supernatant. The content of insulin in supernatant was determined by absorbance measurement and using the standard curve of insulin. To evaluate the pH-triggered property of MBGs, different medium solutions include PBS (pH 5.0) and glucose (5 mM) were used as responsive mediators with different time periods at the same condition. The percent of insulin released from the drug-loaded MBGs was evaluated by calculating the ratio of the content of insulin from the supernatant to the initial content added.

Mechanical strength test 10


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The mechanical strength test of the MNs was using a dynamic tensile compression system (5943 single column materials testing system, Instron Co. Ltd USA).16 To distinguish the mechanical property, the various MNs (Ins+GOx/CAT-MBGs/ZnO MNs and PVP MNs) against a stainless-steel plate were used to record the force-displacement curves.

Skin penetration test To assess the skin penetration property of the MNs in vitro, FITC labelled insulin loaded MBGs/ZnO MNs were sticking on separated and naked rat skins. To observe the penetration of the microneedles into the skin and their dissolution on the skin, the FITC-Ins+GOx/CAT-MBGs/ZnO MNs were applied onto the SD rat skin. The as-prepared samples were observed by a confocal laser scanning microscope (CLSM, C2, Nikon Corporation, Japan) to assess their dissolution and dispersion of FITC after penetration. To further evaluate the depth of the penetration and the release of drug from microneedle patches, histological specimens for microneedles insertion were prepared using a scalpel to excise the SD rat skins. The histological specimens was embedded in an O. C. T. compound and using a cryotome (CryoStar NX50, Thermo Fisher Scientific, USA) for histological sectioning to form multiple frozen OCT-skin samples with a 7 µm-thick to be observed using CLSM. The penetration depth, the dissolution of microneedles and the drug released from MBGs could be directly observed from the fluorescence images and merged images.

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Transdermal delivery of insulin on diabetic rats The diabetic rats of experiment were carried out according to the regulations of use for laboratory animal, provided by the Experimental Animal Center of Zhejiang Academy of Medical Sciences, China. Diabetic rat model was induced in SD rats according to previously described.16,53 The blood samples were taken from tail vein of diabetic rats to measure the concentrations of blood glucose by using a blood glucose meter (Sinocare Inc., Changsha, China). The level of blood glucose for diabetic rats were continuously monitored for 3 days after intraperitoneal injection to ensure that their blood glucose levels reach a stable hyperglycemic state before the experiments. Six experimental groups (the SD rats were divided randomly and three diabetic rats for each group) were as follow designed: diabetes SD rats treated with SC injection (20 IU/Kg), diabetes rats for transcutaneously treated with blank MNs (MBGs/ZnO) and diabetes rats for transcutaneously treated with insulin loaded MNs (Ins-MBGs/ZnO MNs) (20 IU/Kg) or (Ins+GOx /CAT-MBGs/ZnO MNs) (20 IU/Kg). In addition, diabetic rats treated with insulin loaded MNs (PVP MNs) (30 IU/Kg) and diabetes rats treated with insulin loaded MN (Ins+GOx/CAT-MBGs/ZnO MN) (30 and 60 IU/Kg) in order to certify this drug delivery system has a controllable and triggered capacity. The levels of blood glucose for each diabetic rat group were monitored over time until the return initial state of hyperglycemia. To further testify the controllable and triggered capacity of drug delivery system, the MNs administered again when the hyperglycemia had reached. More remarkably, the

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diabetic SD rats were feed after the lowest possible levels of blood glucose levels has reached. Then, the blood glucose levels were monitored as above. To test the concentration of plasma insulin, the serum from tail vein blood samples were collected by centrifugation (3,000 rpm, 5 min) and then measured using an insulin ELISA Kit (DuMa Biotechology Co. Ltd, China). Area above the curve (AAC) and area under the curve (AUC) were calculated from the curve of blood glucose levels and the plasma insulin concentration, respectively. To evaluate the biological activity of the insulin-loaded MBGs, the pharmacological activity (RPA%) and bioavailability (RBA%) were calculated relative to the subcutaneous administration of insulin according to the following equations:

(3)

(4) Additionally, the organs of diabetic rats including the heart, liver, spleen, lung and kidney in vivo were excised and inserted in OCT compound for histological sectioning after 2 h treatment. All the procedures were operated and handled in Hua-an Biotechnological Co., Ltd (Hangzhou, China).

RESULTS AND DISCUSSION Preparation of MBGs/ZnO In this study, MBGs are firstly prepared by a sol-gel processing.36,38,42 The morphology of MBGs is observed by TEM. As shown in Figure 2A, the obtained MBGs have well defined and slightly aggregated nanospheres, mostly ranging

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between 200 and 450 nm. The HR-TEM image shows the MBGs possess a ‘wormhole-like’ porous structure, which is uniformly observed throughout the inner structure. The N2 absorption isotherms of the MBGs is shown in Figure S2, revealing a type Ⅳ isothermal curve.16 The Brunauer-Emmett-Teller (BET) surface area and pore diameter size are 338 m2 g-1 and 3.4 nm, respectively. The APTES stabled ZnO QDs exhibit an excellent water-dispersivity. As shown in Figure S3, The ZnO QDs show uniform size about 3 nm and the lattice fringes at 0.28 nm. After adding of ZnO QDs into MBGs solution, the positive charged ZnO QDs (20.7 mV) are aggregated around the negative charged MBGs (-19.7 mV) to form MBGs/ZnO complexes (14.8 mV), characterized by ξ-potential analysis (Figure 2C). The resulting MBGs/ZnO complexes are highly dispersed and spherical, as shown in Figure 2B. The morphologies of MBGs have no significantly change after coating of ZnO QDs. As also can be seen in TEM image, MBGs/ZnO display a dense structure on the surface of MBGs, since ZnO QDs are capped the nanopores of MBGs as nanolids and the nanoporosity on surface of MBGs is eliminated as well. The mean size of MBGs/ZnO is around at 253 nm that measured by DLS (Figure S4). To further verify that ZnO QDs have successfully been coated on the surface of MBGs, the phase structure and elemental analysis of the surface of MBGs are analyzed by X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS). As shown in Figure 2D, the XRD pattern for MBGs shows a broad peak at 2θ = 24o, indicating the amorphous nature of MBGs.37 ZnO QDs show the feature

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peaks at 2θ = 31.77, 34.42, 36.25, 47.53, 56.61, 62.86 and 67.96. These diffraction peaks can be indexed to the (100), (002), (101), (102), (110), (103) and (112) crystal planes of hexagonal structure of ZnO (JCPDS card no. 36-1451).54 The peaks assigned to ZnO QDs still can be observed in the XRD pattern of MBGs/ZnO, suggesting the presence of ZnO QDs in MBGs/ZnO. The similar results also can be found in XPS patterns of MBGs/ZnO (Figure S4) The XRD pattern of MBGs/ZnO not only show the amorphous structure of MBGs but also the feature peaks of ZnO, indicating ZnO QDs are present on the surface of MBGs. The high-resolution XPS pattern of Zn 2p exhibit two energy peaks appeared at 1044.7 and 1021.7 eV, corresponding to Zn 2p 1/2 and Zn 2p 3/2 ,

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Figure 2. TEM images of MBGs (A) and MBGs/ZnO (B); ξ-potential of ZnO QDs, MBGs and MBGs/ZnO (C); and XRD patterns of ZnO QDs, MBGs and MBGs/ZnO (D).

respectively. The splitting of the 2p doublet of ZnO is 23.0 eV, demonstrating the formation of ZnO.55 The EDS mapping results (Figures S5 and S6) indicate that ZnO QDs have distributed on the surface of the MBGs.

Insulin release in vitro To investigate the insulin release behaviors in vitro, the insulin release profiles are evaluated against in PBS solution with different pH and glucose concentration. The average drug loading capacity (DLC) and encapsulation efficiency (EE) of Ins+GOx/CAT-MBGs/ZnO are 9.8% and 48.8%, respectively (Table S1). Usually, it is a well-recognized indicator of elevated risk of future type 2 diabetes with the impaired glucose tolerance (IGT) for the 2 h plasma glucose concentration >7.8 mM during the oral glucose tolerance test (OGTT). 16,56

Therefore, different glucose solution (5 and 20 mM) to be selected for the

evaluation of the insulin release behaviors. Figure 3A shows the insulin release from Ins+GOx/CAT-MBGs/ZnO at different concentration of glucose. In the PBS solution (pH = 7.4), only about 19.4% of preloaded insulin can been released after incubating for 12 h. Increasing the glucose concentration to 5 and 20 mM, more than 66.8% and 40.2% of insulin to be released after 12 h. The possible is that more gluconic acid can be produced by the enzyme catalysis of GOx, resulting in lowering the local pH value and accelerating the degradation of ZnO QDs. It is confirmed by the fluorescence analysis of ZnO QDs under different pH

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solution (Figure S7A). ZnO QDs and MBGs/ZnO exhibit stronger fluorescent with peak centered around 550 nm. In the solution with pH at 5.0, no fluorescent signals can be founded for both. For investigation the effect of pH on insulin release, the insulin loaded carriers (Ins+GOx/CAT-MBGs/ZnO) are incubated in PBS solution with different pH values. As shown in Figure 3B, only about 19.2% of insulin can be released from drug carriers after incubating for 12 h. A prominently rapid release can be observed in the solution with lower pH. About of 42.4% and 80.6% of insulin can be released with the pH of solution at 5.0 and 2.0, indicating the pH-dependent performance of the obtained hybrid complexes.

Figure 3. The profiles of insulin release against different concentrations of glucose (A) and pH (B) of Ins+GOx/CAT-MBGs/ZnO; the insulin release profiles from Ins+GOx/CATMBGs/ZnO by adjusting pH from 7.4 to 2.0 (C) and the insulin release profiles from

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Ins+GOx/CAT-MBGs/ZnO and Ins-MBGs/ZnO by adjusting concentration of glucose and pH in solution (D).

In order to further evaluate the synergistic effect of GOx and CAT for the release of insulin, the insulin loaded carriers (Ins+GOx/CAT-MBGs/ZnO) are firstly dispersed in PBS solution (pH =7.4). As shown in Figure 3C, after incubating for 3 h, about 13.4% of insulin can be released. Increasing the pH to 5.0, ~40.2% of insulin can be released for further incubating for 4 h. Furthermore, the release amount of insulin can be reached ~79.3% by incubating in the solution with pH at 2.0 for 4 h. Interestingly, the release rate of insulin is lower than that in the solution by only adding of glucose. As shown in Figure 3D, about 12.5% of insulin can be released after incubating in PBS solution for 3 h from both carriers. After increasing the concentration of glucose to 5 mM, the released insulin can be improved to 34.4%. In contrast, there is a slight increase (~17.8%) in the release of insulin from Ins-MBGs/ZnO. Decreasing the pH for the both solution of Ins+GOx/CAT-MBGs/ZnO and Ins-MBGs/ZnO, the release rate of insulin increased correspondingly. The content of released insulin can be improved to about 52.6% and 37.2% after 4 h, respectively. These results imply that CAT has a promoting effect with the assistance of GOx. The pH is an important role for the release of insulin, exhibiting a pH-triggered release behaviors.

Preparation and mechanical strength test of MNs The insulin loaded carriers (Ins+GOx/CAT-MBGs/ZnO) can be used as the depots due to the pH-triggered opening switch of nanoporosity. These carriers

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loaded with different amount of insulin can be embedded into MNs by a two-step micromolding process (Table S2). Figure 4A shows a typical digital image of MNs, indicating the successfully replicating from the mold. SEM images provide more detail information of MNs on the arrays. The shape of these MNs is pyramid-type with the height of about 550 µm. The distance of needle-tip between two MNs is ~550 µm and the width of base for each MNs is ~250 µm. The needle-tip of the MNs with a width of ~20 µm (Figure 4B-D). Under the UV-light irradiation, bright tips can be observed after embedding MBGs/ZnO into the MNs due to the excellent photoluminescence properties (Figure S7B).

Figure 4. Digital images of as-fabricated Ins+GOx/CAT-MBGs/ZnO MNs used in this study (A); SEM images of the as-fabricated MNs with top-view (B) and side-view (C and D) under different magnification; the mechanical strength of MNs measured using a dynamic tensile compression machine (E), and the static state of MNs after compression by a 500 g weight for 5 min (F). 19



In order to investigate the mechanical properties of the MNs, the resistances of MNs to the pressure from the stainless-steel plate are tested. As shown in Figure 4E, the MNs embedded with carriers exhibit better mechanical performance compared with that of PVP MNs. A failure force for Ins+GOx/CAT-MBGs/ZnO MNs at ～0.30 N per needle with a displacement of 0.5 mm can be observed. However, only ～0.12 N per needle can be obtained for the PVP MNs at the same displacement. The MNs have no obviously break against a static force (500 g) for 5 min, indicating their excellent toughness and mechanical strength (Figure 4F). These results imply that the strength and tenacity of the MNs can be prominently promoted by filling of rigid inorganic particles into polymer matrix. This can be attributed to the physical structure of MBGs, which combining its rigidity with the toughness of PVP to promote MNs mechanical properties.16 According to the previous reports,17,23,57 a MN with the needle-tip size of 20 um needs a maximum force ( >0.15 N) to penetrate into the skin. Therefore, the as-fabricated MNs have enough strength to insert the skin in this work.

Skin penetration test To further assess the skin insertion property in vitro and the feasibility of triggered release from the MNs, FITC-labelled insulin is chosen as a model drug. The confocal laser-scanning microscopy (CLSM) images in Figure 5A-C show the separated skin attached with FITC-Ins+GOx/CAT-MBGs/ZnO MNs for less than 5 min on healthy rats. The strong green fluorescence can be seen from the

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MNs that penetrated into skin (Figure 5A). After penetration for 30 min, the weak green fluorescence can be found around the area of the skin insert sites (Figure 5B). Further extension the insertion time to 60 min, the stronger green fluorescence can be seen in around the area of skin penetrate sites (Figure 5C). Nevertheless, the green fluorescence is barely visible in deeper skin tissues. In the diabetic rats group, no significant difference can be found for skin insertion time less than 5 min (Figure 5D). However, a stronger green fluorescence can be found in around the area of the skin puncture sites after insertion for 30 min, as shown in Figure 5E. After skin insertion 60 min, the intense fluorescence could be seen in the deeper skin tissues and penetrated in the space between the MNs (Figure 5F), demonstrating the infiltration and diffusion of more insulin from MBGs after MNs absorption of the interstitial fluid with high concentration of glucose. Similar results also can be founded in the histological sections with different depths from the surface of skin at the perpendicular direction and their 3D-reconstruction images (Figure S8).

21



Figure 5. Fluorescence and merged histological sections of FITC-labelled insulin-loaded MNs applied on healthy (A, B and C) and diabetic rats (D, E and F) for ~5, 30 and 60 min in vivo (Scale bar = 200 µm).

Hypoglycemic effect in vivo To investigate the hypoglycemic effect of pH-triggered MNs in vivo, different doses of insulin-loaded MNs are applied on diabetic rats (the the blood glucose levels are (520±10.5 mg dL-1). Figure 6A shows the blood glucose levels (BGLs) of insulin-loaded MNs (Ins+GOx/CAT-MBGs/ZnO and Ins-MBGs/ZnO) after the transdermal administration, compared with subcutaneous (S.C.) injection and blank groups. In the subcutaneous injection group, the BGLs are decreased quickly and reached their minimum (72±12.5 mg dL-1) at 2 h and returned to high BGLs state afterwards. However, in the Ins+GOx/CAT- MBGs/ZnO MNs groups, the BGLs exhibit a slower reduction trend, and reach its low level for period of 3 h. In the case of Ins-MBGs/ZnO MNs group, a slight decrease of the BGLs can be founded in the first 5 h, indicating the hard to release insulin from MNs. The plasma insulin levels (PILs) are showed a similar trend, as shown in Figure 6B. The PILs of S.C. injection group reaches its maximum value after administration for 2 h compared with 3~5.5 h for Ins+GOx/CAT-MBGs/ZnO MNs treatment, and then slowly declines to their initial state.

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Figure 6. The profiles of blood glucose levels (A) and plasma insulin concentrations (B) after application of insulin-loaded MNs or SC injection of insulin onto diabetic rats (n = 3). Data are represented as the mean ± standard deviation (*P < 0.05, **P < 0.01).

Table 1 shows the RPA% and RPB% are 95.3% and 96.2% for Ins+GOx/CAT-MBGs/ZnO MNs groups compared with that of the S.C. injection. The results indicate the insulin loaded MNs almost completely dissolved after insertion in the skin and then preloaded insulin can be easily released from MNs without any impairment on activity.3, 10, 11, 16

Table 1. Pharmacokinetic parameters of diabetic rats after administration of pH-triggered MNs integrated with insulin-loaded MBGs for glucose-mediated transdermal delivery of insulin (n=3).

-1

Dose (IU Kg ) AAC AUC RPA% RPB%

Insulin solution (S.C)

Ins+GOx/CAT-MBGs/ZnO MNs

20 2708.5 ± 132.5 1706.9 ± 129.7 100 100

20 2581.7 ± 158.7 1642.7 ± 147.6 95.3 ± 2.8 96.2 ± 3.6

AUC: area under the insulin plasma concentration-time curve. AAC: area above the curve of the blood glucose level.

Usually, the BGLs can be increased after feeding. To examine the hypoglycemic effect of MNs after feeding, the BGLs are monitored at various 23



time intervals. Figure 7A shows the schematic process of MNs treatment on diabetic rats. Firstly, the diabetic rats are treated by Ins+GOx/CAT-MBGs/ZnO MNs with different doses of insulin. The BGLs can be decreased after the administration and maintained its lower level (< 200 mg dL-1) for 2 and 6 h dependent on the doses of insulin in the MNs, indicating that a better hypoglycemic effect can be obtained after administration with high doses of insulin loaded in MNs (Figure 7B). Afterwards, BGLs is returned gradually to its initial state after 16~18 h. However, in the insulin MNs group, the BGLs of diabetic rats are rapidly decreased from (515 ± 11.2 mg mL-1) to around (92±13.4 mg mL-1) within 2 h and then returned to the state of hyperglycemic after 8 h. Then, the new MNs are attached on these diabetic rats again (state 2) after 3 h, and then the sufficient water and food are feed to diabetic rats (state 3). The BGLs can be re-retained after the administration and risen back to initial state, as shown in Figure 7B. Interestingly, the maintenance time of BGLs below the initial state is shorter than that in the first cycle. It is attribute to the glucose supplementation after feeding. Nevertheless, it can be prolonged the resistance time using the MNs with loading high doses of insulin.

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Figure 7. Schematic process of diabetic rats treated by MNs and feeding (A); BGLs of diabetic rats treated by MNs and feeding (B, n = 3); BGLs of diabetic rats treated by insulin MNs and Ins+GOx/CAT-MBGs/ZnO MNs (C) and their corresponding hypoglycemia index (D).

In order to evaluate the risk of hypoglycemia, the insulin MN and Ins+GOx/CAT-MBGs/ZnO MNs are administered to the healthy rats. As shown in Figure 7C, The BGLs of rats treated with insulin MNs can be decreased from (128±10.5 mg dL-1) to (60±8.7 mg dL-1). However, in the Ins+GOx/CATMBGs/ZnO MNs group, the BGLs of rats have no significant reduction although same doses of insulin being applied, indicating reduce the risk of hypoglycemia treated by as-fabricated MNs. The corresponding hypoglycemia index is shown in Figure 7D, which defined as the difference between the initial and nadir blood glucose readings divided by the time at which the nadir was reached.1,3,7 The

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MNs integrated with insulin-loaded and pH-triggered MBGs displayed a lower hypoglycemic index compared to insulin-loaded MNs. In addition, to explore the biodistribution of drug in rats, the organs including heart,

liver,

spleen,

lung,

and

kidney

are

excised

after

treat

by

FITC-Ins+GOx/CAT- MBGs/ZnO MNs (30 IU Kg -1) for 2 h. The histological sectionings are prepared by embedding of organs into an OCT compound using a cryotome. The separated organs are made into histological specimens. The frozen OCT-organ specimens are observed using an inverted fluorescence microscope. As shown in Figure 8, the strongest green fluorescence has presented in liver and kidney, but other organs have no fluorescence can be observed, indicating that most insulin are absorbed by liver and kidney. These results are similar with previous reports that the main organs for capturing and metabolizing foreign proteins are liver and kidney.58, 59

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Figure 8. The histological sections of heart, liver, spleen, lung, and kidney of rats after treated by FITC-Ins+GOx/CAT-MBGs/ZnO MNs (Scale bar = 100 µm).

CONCLUSION In summary, the MNs integrated with insulin-loaded and ZnO QDs capped MBGs, were designed to achieve painless and controlled administration. GOx/CAT in the MBGs could catalyze glucose to form gluconic acid, leading to

27



decrease local pH in MBGs. The ZnO QDs on the surface of MBGs could be dissolved in the acidic condition, resulting the dis-assembly of the pH-sensitive MBGs and then release of preloaded insulin from the MBGs. The as-fabricated MNs exhibited a well mechanical strength compared with the pure PVP MNs due to the filling of rigid inorganic particles into polymer matrix. As a result of transcutaneous administration on diabetic rats, the as-fabricated MNs demonstrated a glucose-mediated release characteristics. This work indicates the as-fabricated MNs integrated with MBGs-ZnO complexes have promising application in the treatment of diabetes. In addition, this glucose-mediated and pH-triggered transdermal delivery system as a promising delivery system also can be delivered different therapeutic drugs to treat other diseases.

■ ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.XXXXX. Insulin standard curve, nitrogen absorption isotherms and pore size distribution, TEM images, DLS distribution, XPS spectra, element mappings, the fluorescent spectra, digital images of MNs, confocal micrographs and 3D reconstruction images, DLC and EE in MNs.

■ AUTHOR INFORMATION Corresponding Author

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

ORCID Guohua Jiang: 0000-0003-3666-8216

Notes The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS 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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Figure 1. Schematic preparation of glucose-mediated microneedles integrated with ZnO quantum dots capped MBGs for transdermal delivery of insulin. 102x58mm (300 x 300 DPI)



Figure 2. TEM images of MBGs (A) and MBGs/ZnO (B); ξ-potential of ZnO QDs, MBGs and MBGs/ZnO (C); and XRD patterns of ZnO QDs, MBGs and MBGs/ZnO (D). 149x125mm (300 x 300 DPI)


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Figure 3. The insulin release profiles against different concentrations of glucose (A) and pH (B) of Ins+GOx/CAT-MBGs/ZnO; the insulin release profiles from Ins+GOx/CAT- MBGs/ZnO by adjusting pH from 7.4 to 2.0 (C) and the insulin release profiles from Ins+GOx/CAT-MBGs/ZnO and Ins-MBGs/ZnO by adjusting concentration of glucose and pH in solution (D). 135x102mm (300 x 300 DPI)



Figure 4. Digital images of as-fabricated Ins+GOx/CAT-MBGs/ZnO MNs used in this study (A); SEM images of the as-fabricated MNs with top-view (B) and side-view (C and D) under different magnification; the mechanical strength of MNs measured using a dynamic tensile compression machine (E), and the static state of MNs after compression by a 500 g weight for 5 min (F). 114x73mm (300 x 300 DPI)


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Figure 5. Fluorescence and merged histological sections of FITC-labelled insulin-loaded MNs applied on healthy (A, B and C) and diabetic rats (D, E and F) for ~5, 30 and 60 min in vivo (Scale bar = 200 µm). 97x53mm (300 x 300 DPI)



Figure 6. The profiles of blood glucose levels (A) and plasma insulin concentrations (B) after application of insulin-loaded MNs or SC injection of insulin onto diabetic rats (n = 3). Data are represented as the mean ± standard deviation (*P < 0.05, **P < 0.01). 66x24mm (300 x 300 DPI)


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Figure 7. Schematic process of diabetic rats treated by MNs and feeding (A); BGLs of diabetic rats treated by MNs and feeding (B, n = 3); BGLs of diabetic rats treated by insulin MNs and Ins+GO x /CAT-MBGs/ZnO MNs (C) and their corresponding hypoglycemia index (D). 124x87mm (300 x 300 DPI)



Figure 8. The histological sections of heart, liver, spleen, lung, and kidney of rats after treated by FITCIns+GOx/CAT-MBGs/ZnO MNs (Scale bar = 100 µm). 219x270mm (300 x 300 DPI)


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Table of Contents

Microneedles integrated with ZnO quantum dots capped mesoporous bioactive glasses for glucose-mediated insulin delivery Bin Xu,†, §, ‖ Qinying Cao,‡ Yang Zhang,†, §, ‖ Weijiang Yu,†, §, ‖ Jiangying Zhu,†, §, ‖ Depeng Liu, †, §, ‖ and Guohua Jiang†, §,

⊥,

‖, *


Microneedles Integrated with ZnO Quantum-Dot-Capped Mesoporous

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