Hyaluronic Acid Encapsulated CuS Gel-Mediated ... - ACS Publications

Jun 28, 2017 - Beijing Key Lab of Plant Resource Research and Development, School of Science, Beijing Technology and Business University, 11. Fucheng ...
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Research Article pubs.acs.org/journal/ascecg

Hyaluronic Acid Encapsulated CuS Gel-Mediated Near-Infrared LaserInduced Controllable Transdermal Drug Delivery for Sustained Therapy Dongdong Wang,† Haifeng Dong,*,† Meng Li,‡ Xiangdan Meng,† Yu Cao,† Kai Zhang,† Wenhao Dai,† Changtao Wang,*,‡ and Xueji Zhang*,† †

Beijing Key Laboratory for Bioengineering and Sensing Technology, Research Center for Bioengineering and Sensing Technology, School of Chemistry and Bioengineering, University of Science & Technology Beijing, 30 Xueyuan Road, Haidian District, Beijing 100083, P. R. China ‡ Beijing Key Lab of Plant Resource Research and Development, School of Science, Beijing Technology and Business University, 11 Fucheng Road, Haidian District, Beijing 100048, P. R. China S Supporting Information *

ABSTRACT: Efficient transdermal drug delivery to the circulatory system of body has great benefit compared to traditional needle injection and oral administration approach due to avoiding the emotional trauma and pain for the patients. Disrupting the stratum corneum (SC) is crucial for successful transdermal drug delivery, and thermal ablation is emerging as the advanced strategy for it. Herein, hyaluronic acid (HA) encapsulated CuS (HA-CuS) nanoparticles with excellent biocompatibility and remarkable photothermal translation efficacy were synthesized for transdermal drug delivery. The transdermal cargo delivery capability of the proposed HACuS gel was comprehensively investigated. Simply controlling the NIR laser irradiation power or time enabled controllable translocation of the model biomacromolecule of bovine serum albumin (BSA) into skin in vivo. Furthermore, the feasibility of the HA-CuS-mediated transdermal insulin delivery for type 1 diabetes therapy of nude mice was investigated. It presented sustained and efficient transdermal insulin delivery to decrease the blood glucose level for longer time than insulin injection, providing great potential for clinical application. KEYWORDS: Transdermal drug delivery, Stratum corneum, Copper sulfide, Thermal ablation, Sustained therapy

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epithelial layer that serves to continuously renew the SC.4 Nerve endings are situated in the papillary dermis where they can never be destroyed. Therefore, the accompanying pain and patient incompliance by the damage of SC layers may be negligible. Numerous technologies including chemical, electrical, acoustic, mechanical, and thermal methods have been developed to address this issue.1,9,10 Among them, chemical enhancers, iontophoresis, and ultrasound improve the skin permeability by disrupting the SC structure, but SC still remains intact to a certain extent, which limits the transdermal efficiency of hydrophilic macromolecular drugs. The microneedle piercing mediated transdermal delivery appears to have good efficiency, but suffers from invasive disadvantages.1,3,6 Thermal ablation technology represents an attractive approach in recent years.11 It utilizes thermal ablation to generate micrometer-scale perforations in the SC to enhance the

INTRODUCTION Transdermal drug delivery offers unique advantageous features compared to traditional needle injection and oral administration methods.1 It avoids the emotional trauma and pain that is associated with injections, and reduces the risk of needlestick injuries and overdoses. Meanwhile, it displays improved bioavailability and enhanced patient compliance.1−4 Skin is the largest organ in the human body. It roughly accounts for 15% of the total weight and possesses a surface area of 1.5−2.0 m2 with varying thickness.5 Generally, the skin is divided into three layers including the epidermis (EP), the dermis, and the subcutaneous tissues. The EP is the superficial layer of the skin, and the stratum corneum (SC) is the outermost layer of the EP. The SC is 10−20 μm in thickness and affords the main barrier to water loss from the body.6 On the other hand, the SC restricts the permeation of hydrophilic macromolecules into the skin,7,8 which significantly hinders effective transdermal drug delivery. Therefore, disrupting the SC is crucial for successful transdermal delivery. The SC is a nonviable tissue that provides most of the skin’s barrier properties. The viable EP is an © 2017 American Chemical Society

Received: April 5, 2017 Revised: May 31, 2017 Published: June 28, 2017 6786

DOI: 10.1021/acssuschemeng.7b01035 ACS Sustainable Chem. Eng. 2017, 5, 6786−6794

Research Article

ACS Sustainable Chemistry & Engineering

Freda Biochem Co., Ltd. Carbomer 980 was purchased from Guangzhou Qizheng Chemical Technology Co., Ltd. 2, 2, 2tribromoethanol (98.5%) was purchased from J&K Scientific Ltd. BSA-FITC was purchased from Zhongkechenyu Trading Co., Ltd. Human recombinant insulin (19 IU/mg) was purchased from Beijing Solarbio Science & Technology Co., Ltd. Sodium sulfide and cupric chloride were obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Streptozotocin (STZ) and 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (China). All other reagents were of analytical grade. Synthesis of HA-CuS Gel. 150 mg of HA was added to 20 mL of CuCl2 solution (4 mM) and then reacted under stirring for 30 min. 1.6 mL of Na2S (50 mM) and 36 mg of trisodium citrate were then added into the reaction solution successively under stirring. The resulting solution was maintained at 80 °C for 30 min and centrifuged at 12000 rpm to obtain the HA-CuS nanoparticles. CuS nanoparticles were synthesized by the same route but without addition of HA. HA-CuS solution (0.1 mL, 3 mg/mL) was mixed with Carbomer 980 solution (2.9 mL, 10 mg/mL) under magnetic stirring, and then 0.5 mL of butylene glycol was added in the solution to form HA-CuS gel. Blank gel was prepared in the same procedure but without addition of HA-CuS. Cytotoxicity Tests of HA-CuS Nanoparticles.28,29 Normal human dermal fibroblast (NHDF) cells were seeded in a 96-well plate at a density of 104 cells per well for 20 h at 37 °C in 5% CO2. Various concentrations (10, 25, 50, 100, or 200 μg/mL) of HA-CuS nanoparticles were resuspended in fresh Dulbecco’s modified Eagle medium (DMEM), and then the cells were incubated for 4 h. Afterward, the medium was replaced with fresh DMEM (100 μL). MTT solution (20 μL, 5 mg/mL) was then added to each well, and the medium was replaced with dimethyl sulfoxide (DMSO, 100 μL) to solubilize the formazan dye. After shaking (37 °C, 120 rpm) for 15 min, the absorbance of each well was measured using an Anthos 2010 microplate reader (Biochrom Ltd. Cambridge CB4 OFJ England) at 490 nm. The cytotoxicity of HA-CuS nanoparticles was estimated by the percentage of growth inhibition calculated with the following formula: growth inhibition % = (1 − Atest/Acontrol) × 100% In Vivo Experiment of Laser Activation of HA-CuS Gel. Nude mice were divided into 3 groups randomly (n = 3 per group) and were anesthetized by intraperitoneal injection of tribromoethanol (240 mg/ kg). The mice in group 1 received topical application of blank gel, while HA-CuS gel was applied to the mice in group 2 and group 3. The diameter of the applied circled area was 1.5 cm, and the received dosage of the gel was 50 μL. The gel applied on the skin was then evaporated to dryness using a hair dryer for 2 min. The 980 nm NIR laser (LOS-BLD-0980-002W-C/P, Hi-Tech Optoelectronics Co., Ltd.) was used to excite the area covered by the gel for 30 s. The laser power density used in group 2 was 0.24 W/cm2, and 0.58 W/cm2 was used in group 1 and group 3. A thermal imager (TiS65, Fluke Co., Ltd.) was employed to record the temperature in the process and analyzed with Fluke SmartView software. After laser activation, the gel on the skin was removed and the mice were sacrificed. The treated skin was peeled for paraffin slices stained with H&E and frozen slices stained with Nile red. For Nile red staining, a stock solution containing Nile red in acetone (0.05%, w/v) was diluted with glycerol (75%). 50 μL of the glycerol−dye solution (2.5 μg/mL) was applied to each tissue section, and then covered with a coverslip for storage. The paraffin slices were examined under a fluorescent inverted microscope (IX73, Olympus, Japan), and the frozen slices were examined under a confocal laser scanning microscope (FV 1200, Olympus, Japan). In Vivo Experiment of Translocation of BSA-FITC into Skin. BSA was used as a model of hydrophilic biomacromolecule for transdermal delivery. Nude mice were divided into 4 groups (n = 3 per group). Blank gel (50 μL) was applied on the back skin (diameter ∼1.5 cm) of the mice in group 1, while HA-CuS gel (50 μL) was applied on the back skin of the mice in groups 2−4. Afterward, the gel was evaporated to dryness using a hair dryer. The gel-treated skin in groups 1 and 4 was irradiated with a NIR laser at the power intensity of 0.58 W/cm2 for 30 s, while group 3 was irradiated with 0.24 W/cm2

permeability of the skin, while the viable EP and deeper skin tissues do not experience a significant temperature rise to minimize the injury.1,4,11 Various attractive photothermal nanomaterials with strong near-infrared (NIR) absorption, such as organic dyes, noble metal materials, semiconductor compounds, and polymers, have been developed for photothermal agents.11−15 CuS nanostructures are a prospective sort of photothermal agent because of facile preparation, cost-effectiveness, and high stability.16 Importantly, the NIR light absorption of CuS nanostructures derives from the d−d transition of Cu2+ ions, which is robust against the influence of the particles’ shape, size, and surrounding environments.11,17−19 However, the research on biomedical application of CuS nanostructures remains in its infancy stage. CuS nanostructures with advanced photothermal translation efficacy and efficient biocompatibility are continuously being explored.20 Hyaluronic acid (HA) is a linear polysaccharide of Nacetylglucosamine and glucuronic acid in alternating sequence with an average molecular weight of approximately 2 × 105 to 107 Da.21 It is a ubiquitous extracellular matrix component, presenting in skin at high concentrations.22 It provides beneficial effects to the skin such as skin hydration, elasticity regeneration, and improved wound healing. HA is emerging as prospective transdermal drug delivery agent with excellent biocompatibility.21,23 Diabetes mellitus is a metabolic disease resulting from the failure of blood glucose level regulation mechanisms. In 2014 there were 387 million people suffering from diabetes, and the quantity will increase to 592 million by 2035.24 The main treatment of diabetes today is subcutaneous injection of external insulin to control the glucose level at a normal level.25 In the treatment of patients that with insulin-dependent diabetes (type 1) and most patients with non-insulin-dependent diabetes (type 2), the insulin therapy is an essential approach.26 However, multiple daily injections and selfmonitoring of blood glucose levels are required for the insulin therapy, which is inconvenient and diminishes the life quality of the patients.25,27 Transdermal insulin delivery shows great prospect for treatment of diabetes. In this study, we report a simple synthetic route to construct HA encapsulated CuS (HA-CuS) nanoparticles with good biocompatibility and advanced photothermal translation efficacy for controllable transdermal drug delivery. The transdermal delivery capability of the resulting HA-CuS was systematically investigated. Using fluorescein isothiocyanate labeled bovine serum albumin (BSA-FITC) as model, it displayed good capability of tunable translocation of the BSAFITC into skin in vivo by simply controlling the NIR-laser irradiation power or time. Furthermore, it presented sustained and efficient transdermal insulin delivery for type 1 diabetes therapy of nude mice. It maintained the blood glucose level at low level for longer time than insulin injection, providing great potential for clinical application. Our work contributes to design a sustained and efficient transdermal drug delivery HACuS nanosystem, which avoids the emotional trauma and pain for patients and may provide great potential to improve the life quality of patients in clinical application.

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EXPERIMENTAL SECTION

Materials. BALB/c nude mice (both 6−8 weeks, female) were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). HA (molecular mass of 53 kDa) was purchased from 6787

DOI: 10.1021/acssuschemeng.7b01035 ACS Sustainable Chem. Eng. 2017, 5, 6786−6794

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ACS Sustainable Chemistry & Engineering

Figure 1. (a) TEM image and (b) DLS of HA-CuS nanoparticles. Scale bar 20 nm. Inset: HRTEM of HA-CuS nanoparticles. Scale bar 5 nm. (c) FTIR spectra of HA and HA-CuS nanoparticles. (d) UV−visible spectra of HA-CuS nanoparticles and CuS nanoparticles.

CuS nanoparticles at 80 °C for 30 min. The size and morphology of the HA-CuS nanoparticles were characterized by transmission electron microscopy (TEM). Compared to the CuS (Figure S2a), an obscure out-layer assigned to HA was observed on the CuS surface of HA-CuS (Figure 1a), which indicated the successful synthesis of HA-CuS. The highly parallel and ordered lattice fringe in high-resolution TEM (HRTEM) images suggested that the d-spacing of HA-CuS nanoparticles was 0.308 nm, corresponding to the (102) faces of CuS crystals, indicating the successful synthsis of HA-CuS nanoparticles (Figure 1a, inset). The diameters of the CuS nanoparticles and the HA-CuS nanoparticles determined by the dynamic light scattering (DLS) were about 8 nm (Figure S2b) and 10 nm (Figure 1b). The slightly larger aqueous diameter resulted from the out-layer hydrophilic HA. This result further provided evidence of the successful synthesis of HA-CuS. Figure S2c shows the X-ray diffraction (XRD) pattern of these HA-CuS nanoparticles. The well-defined diffraction peaks at 2θ degree of 29.53°, 32.11°, and 47.97° were assigned to the (102), (006), and (110) faces of CuS crystals according to the JCPDS card (No. 06-0464), indicating the successful synthesis of CuS. The Fourier transform infrared spectroscopy (FT-IR) was used to characterize the HA-CuS, as shown in Figure 1c, the pure HA showed the characteristic bands of −OH at 3425 cm−1, the stretching vibration absorption peak of (CO)[COO−] at 1616 cm−1, 1411 cm−1, C−O−C at 1043 cm−1, and −CO−NH− at 1205 cm−1, respectively. All the characteristic bands could be observed in that of HA-CuS nanoparticles, which indicated the successful conjugation of HA on the CuS nanoparticles. The red shift of HA-CuS nanoparticles at 922 nm compared to CuS nanoparticles at 911 nm was due to the assembly of HA on the CuS nanoparticles (Figure 1d), which also indicated that HA was successfully wrapped on CuS nanoparticles.

NIR laser for 30 s. The gel-treated skin of the mice in group 2 received no NIR irradiation. Afterward, BSA-FITC gel (50 μL) consisting of BSA-FITC (0.3 mmol/L) and Carbomer 980 (10 mg/mL) was applied on the pretreated mouse skin and removed after 30 min. The mice were then sacrificed, and the treated skin was peeled for frozen slices and examined under a confocal laser scanning microscope. In Vivo Experiment of Transdermal Insulin Delivery.30 Nude mice were divided into 4 groups (n = 3 per group). All the mice were given STZ by intraperitoneal injections as 100 mg/kg body weight which was dissolved in a 0.01 M citrate buffer at pH 4.5. Seven days after injection of the STZ, the blood glucose levels in mice were stable and higher than normal level (4 mmol/L) and used for study of transdermal insulin delivery in vivo. HA-CuS (50 μL) was applied on the back skin (diameter ∼1.5 cm) of the mice in group 1, and then the gel was evaporated to dryness using a hair dryer. The gel-treated skin was irradiated with NIR laser at the power intensity of 0.24 W/cm2 for 30 s, while the skin of mice in groups 2−4 did not receive any treatment. The human recombinant insulin solution (200 μL, 2 mg/mL) was added to a patch (without any drugs) and then applied to the treated skin area in group 1 and the same location in group 2. The mice in group 3 as a positive control received subcutaneous injections of insulin (2 mg kg−1 body weight). Group 4 was a blank control group. Blood glucose level was measured every 2 h until 10 h by a glucometer.

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RESULTS AND DISCUSSION Synthesis and Characterization of the HA-CuS Nanoparticles. The HA-CuS nanoparticles were synthesized at high temperature. Gel permeation chromatography (GPC) measurements were performed to check the degradation of HA at 80 °C. Figure S1a,b shows the GPC curves of HA before and after 80 °C heating for 30 min detected by laser scattering detector and differential reflective index detector, respectively. Both of the measurements demonstrated that the retention time for HA before and after the heating were almost the same, which indicated that the HA was stable during the synthesis of HA6788

DOI: 10.1021/acssuschemeng.7b01035 ACS Sustainable Chem. Eng. 2017, 5, 6786−6794

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ACS Sustainable Chemistry & Engineering

Figure 2. (a) Temperature elevation of water (control) and HA-CuS solution at different concentrations (25, 50, 100, 500, and 1000 μg/mL) irradiated by an 980 nm laser (0.7 W/cm2) for 600 s. (b) A temperature variation curve of 1 mL of HA-CuS solution during 5 heating/cooling cycles using 980 nm laser at a power density of 0.7 W/cm2.

Figure 3. (a) Cytotoxicity of HA-CuS to NHDF cells measured by MTT. (b) Confocal fluorescence images of NHDF cells treated with different concentrations of HA-CuS and PBS (Control). The cells were all costained by Calcein-AM (live: green) and PI (dead: red). Scale bars, 200 μm.

Figure 4. Infrared thermal images of nude mice exposed to HA-CuS gel and blank gel before and after NIR laser irradiation.

Measurement of Photothermal Performance. To study the photothermal effects of HA-CuS nanoparticles, the

temperature of a series of aqueous solutions containing different concentrations of HA-CuS nanoparticles was carefully 6789

DOI: 10.1021/acssuschemeng.7b01035 ACS Sustainable Chem. Eng. 2017, 5, 6786−6794

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Figure 5. H&E staining of the pretreated skin paraffin slices: (a) HA-CuS gel treatment, (b) blank gel plus NIR laser (0.58 W/cm2), (c) HA-CuS gel plus NIR laser (0.24 W/cm2), and (d) HA-CuS gel plus NIR laser (0.58 W/cm2). Arrow, loss of epidermis. Scale bars, 50 μm.

W/cm2 NIR laser for 30 s with the concentration of HA-CuS nanoparticles at 100 μg/mL. As shown in Figure S3, the cells showed similar cell viability compared to control groups under irradiation for 30 s. It demonstrated the good safety of the short-time laser irradiation for the living cells. Significant cell death was observed when the irradiation time increased to 10 min due to the photothermal effect of the CuS. In Vivo Laser Activation of HA-CuS Gel. The in vivo experiment of laser activation of HA-CuS gel was further performed. The 980 nm NIR laser with different power densities was used to excite the area covered by the HA-CuS gel for 30 s. As shown in Figure 4, the temperature of skin covered with HA-CuS gel increased by 13.2 and 21.0 °C after irradiation by 0.24 W/cm2 and 0.58 W/cm2 NIR laser for 30 s, respectively. As a comparison, the temperature of the control group exposed to blank gel increased by 3.2 °C after irradiation by the high power density (0.58 W/cm2) laser. These results suggested good in vivo laser activation capability of HA-CuS gel. The good photothermal conversion property and excellent biocompatibility of HA-CuS provided promising potential for its biomedical application in transdermal drug delivery. Histological Analysis of the Thermal Ablation Effect. To study the thermal ablation effect of the HA-CuS on the skin of nude mice, histological analysis was performed. Paraffin slices were stained with H&E. As shown in Figure 5a, the SC was intact and undamaged in the case of HA-CuS exposure without NIR laser irradiation. A similar result of SC integrity was also observed for the skin tissues covered with blank gel and irradiated by NIR laser with a power density of 0.58 W/cm2 (Figure 5b). On the contrary, the SC were disarranged and almost removed from skin in the condition of HA-CuS gel exposure coupled with 0.24 W/cm2 NIR laser irradiation, while no damage was displayed for the viable EP (Figure 5c). For the skin of the nude mice coated with HA-CuS gel and having undergone irradiation with higher density power NIR laser

measured after irradiation by a 980 nm laser at a power density of 0.7 W/cm2 for 600 s. As shown in Figure 2a, the temperature of the HA-CuS solution with different concentrations increased by 18.4 °C (25 μg/mL), 26.3 °C (50 μg/mL), 32.7 °C (100 μg/mL), 39.7 °C (500 μg/mL), and 43.4 °C (1000 μg/mL) in 600 s, while the temperature of pure water (conntrol) was only raised by 13.0 °C under the same conditions. These results demonstrated that the HA-CuS nanoparticles could efficiently convert the NIR irradiation into heat. The reliability of photothermal conversion was further investigated. The temperature of HA-CuS nanoparticle (1000 μg/mL) solution was recorded for 5 successive heating/cooling cycles, during which 600 s irradiation and a natural cooling process were applied. As shown in Figure 2b, the peak temperature of each irradiation cycle remained nearly unchanged (69.4 °C, 69.3 °C, 69.1 °C, 69.3 °C, 69.2 °C), which suggested the good stability of HACuS nanoparticles with respect to photothermal conversion. Cytotoxicity of HA-CuS Nanoparticles. HA is a prospective transdermal drug delivery agent with excellent biocompatibility. Before the transdermal drug delivery experiments, cell cytotoxicity experiments of HA-CuS were performed. As shown in Figure 3a, normal human dermal fibroblast (NHDF) cells exhibited superb cell viability with a value of above 90% even at a concentration as high as 200 μg/ mL. MTT analysis demonstrated that this nanoparticle did not have direct toxicity, however, long-term biocompatibility in the human body needs to be further confirmed. Furthermore, the cytotoxicity of the HA-CuS was investigated by Calcein-AM/PI dual stain. As shown in Figure 3b, similar results were obtained that strong green fluorescence of Calcein-AM related to live cells was observed in the NHDF cells with the concentration of nanoparticles at 200 μg/mL, indicating high cell viability. For the sake of providing more evidence to prove the safety of HACuS nanoparticles, the cell viability test under laser irradiation was performed. The NHDF cells were irradiated under the 0.58 6790

DOI: 10.1021/acssuschemeng.7b01035 ACS Sustainable Chem. Eng. 2017, 5, 6786−6794

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Figure 6. Nile red staining of the pretreated skin frozen slices: (a) HA-CuS gel treatment, (b) blank gel plus NIR laser (0.58 W/cm2), (c) HA-CuS gel plus NIR laser (0.24 W/cm2), and (d) HA-CuS gel plus NIR laser (0.58 W/cm2). Arrow, loss of epidermis. Scale bars, 25 μm.

Figure 7. Fluorescence microscopy images of the BSA-FITC-applied skin frozen sections: (a) only HA-CuS gel, (b) blank gel plus NIR laser (0.58 W/cm2), (c) HA-CuS gel plus NIR laser (0.24 W/cm2), and (d) HA-CuS gel plus NIR laser (0.58 W/cm2). Arrow, loss of epidermis. Scale bars, 50 μm.

(0.58 W/cm2), all the SC was removed and a majority of the EP was damaged (Figure 5d). These results suggested that the

HA-CuS gel could effectively and controllably remove the SC for transdermal drug delivery. 6791

DOI: 10.1021/acssuschemeng.7b01035 ACS Sustainable Chem. Eng. 2017, 5, 6786−6794

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Figure 8. (a) The schematic diagram of the experimental process. (b) Blood glucose levels of diabetic mice after treatment with HA-CuS nanoparticles plus NIR laser (0.24 W/cm2), which then received insulin; only received insulin without treatment; diabetic mice given a subcutaneous injection of insulin were used as positive controls; untreated diabetic mice were used as blank controls.

(0.58 W/cm2), the green fluorescence of BSA-FITC was merely observed on the outside of SC (Figure 7a,b). As expected, the green fluorescence was present in the viable EP and a small amount was observed in the dermis layer when the skin was pretreated with HA-CuS gel and NIR laser (0.24 W/cm2) irradiation (Figure 7c), indicating success of transdermal delivery of BSA-FITC. In the case of treatment of HA-CuS gel and higher power NIR laser (0.58 W/cm2) irradiation, strong green fluorescence was observed in the viable EP and the deeper tissues, dermis (Figure 7d), which suggested that the EP was damaged. BSA went through the epidermis by the loss, and a time-dependent diffusion pattern was observed during the process. The concentration around the loss was more than in other places at 5 min (Figure S4), and a uniform distribution of BSA-FITC was observed when the diffusion time was prolonged to 30 min (Figure 7d). Penetration of Insulin through the Skin in Vivo. The feasibility of the HA-CuS mediated transdermal drug delivery for biomedical application was further investigated using insulin as model for type 1 diabetes treatment of nude mice. The HACuS gel (50 μL, 100 μg/mL) was applied on the skin (diameter ∼1.5 cm), and the gel was then evaporated to dryness using a hair dryer. The gel-treated skin was irradiated with NIR laser at the power intensity of 0.24 W/cm2 for 30 s, and the HA-CuS gel was then washed off. The human recombinant insulin solution (200 μL, 2 mg/mL) was added to a patch (without any drugs), and then the patch was applied to the treated skin area. A control group of nude mice was treated with same patch but without the proposed pretreatment. Additionally, nude mice that received subcutaneous injections of insulin and no treatment of insulin were used as positive and negative controls, respectively (Figure 8a). As shown in Figure 8b, the blood glucose level for the groups without proposed pretreatment of HA-CuS gel exposure and NIR laser irradiation and the blank control group maintained a high stable level. The blood glucose level of the mice treated with HA-CuS and NIR laser irradiation was decreased to 85.7% at 2 h and gradually decreased to 84.2% at 8 h, and afterward, the glucose level increased to 99.6% at 10 h. The blood glucose level in the mice that received subcutaneous injections of insulin presented a sudden decrease to 52.7% at 2 h and rapidly increased to a similar level as the HA-CuS gel mediated transdermal delivery in 4 h. The blood glucose level then increased to 93.1% at 6 h and 97.1% at 8 h, and ultimately increased to 105.7% at 10 h. These results suggested that the

In order to provide more assertive evidence to the thermal ablation effect on the skin of nude mice, the frozen slices were made and stained with Nile red for analysis. As shown in Figure 6a, the skin of nude mice was pretreated with HA-CuS gel without NIR laser irradiation. The Nile red stained frozen slices revealed intact and undamaged SC and viable EP layer. Nondamage of SC and viable EP was observed when the mice were treated with blank gel and higher power NIR laser (0.58 W/cm2) irradiation (Figure 6b). In contrast, the skin of nude mice treated with HA-CuS gel and NIR laser (0.24 W/cm2) irradiation displayed significant ablation such that the SC almost disappeared in certain areas (Figure 6c). Furthermore, by increasing the laser power to 0.58 W/cm2, thermal ablation penetrated the viable EP to the epidermal−dermal junction (Figure 6d). The results demonstrated that the HA-CuS had strong NIR absorbance to efficiently convert the NIR irradiation into local heat for SC ablation. HA-CuS gel mediated thermal ablation of the skin induced by the NIR laser (980 nm, 0.24−0.58 W/cm2) to raise the skin temperature at the site of exposure in 30 s NIR irradiation. Transdermal drug delivery using thermal ablation is achieved by heating the skin surface to vaporize tissue, which can locally remove SC at the site(s) of heating. To avoid invasiveness, the laser power intensity and irradiation time should be optimized to ensure efficient perforations in the SC to enhance the permeability of the skin, maintain viable EP, and ensure that deeper skin tissues do not experience a significant temperature rise to minimize the injury. In comparison with 5 s11 or microsecond irradiation,1 a longer irradiation time of 30 s is applied in our system because of the lower laser power intensity of our system (0.24 W/cm2) than that of 1.3 W/cm2 (ref 11) and superheated steam.1 Additionally, the photothermal conversion efficiency and irradiation pattern are significant factors to decide the irradiation time.4 In Vivo Translocation of BSA-FITC into Skin. Using BSA-FITC as a model, the HA-CuS mediated transdermal delivery was investigated. After the skin was treated with HACuS gel and NIR laser irradiation for 30 s, the HA-CuS gel was removed. The BSA-FITC gel (68 kDa, 50 μL, 0.3 mmol/L) was then applied to the specific region and maintained for 30 min. Afterward, the nude mice were sacrificed and the pretreated skin was dissected for cryosectioning. The frozen slices (10 μm thickness) were examined directly under a confocal laser scanning microscope. For both the skin treated with HA-CuS gel alone and the skin treated with blank gel plus NIR laser 6792

DOI: 10.1021/acssuschemeng.7b01035 ACS Sustainable Chem. Eng. 2017, 5, 6786−6794

Research Article

ACS Sustainable Chemistry & Engineering

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HA-CuS-mediated transdermal insulin delivery effectively controlled the blood glucose level at a low level for a longer time than subcutaneous injection, which provided a sustained and efficient therapy of type 1 diabetes.

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CONCLUSIONS In conclusion, we report HA encapsulated CuS nanoparticles for efficient and controllable transdermal drug delivery. The prepared HA-CuS nanoparticles and their photothermal conversion efficacy were comprehensively characterized. It presented excellent biocompatibility and advanced photothermal translation efficacy. The transdermal delivery capability of the HA-CuS gel mediated transdermal drug delivery was systematically investigated. Using BSA as a model, by simply controlling the NIR laser irradiation power or time, it showed good capability of controllable transdermal BSA delivery. Remarkably, HA-CuS-mediated transdermal insulin delivery enabled the blood glucose to remain at a low level for longer time compared to insulin subcutaneous injection for type 1 diabetes therapy of nude mice. The proposed HA-CuSmediated transdermal drug delivery offers great promise for practical application because of its good biocompatibility and controllably sustained therapy.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b01035. GPC curves of HA, TEM image and DLS of CuS nanoparticles, XRD of HA-CuS nanoparticles, MTT analysis of NHDF cells, and fluorescence microscopy images of the BSA-FITC-applied skin (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Haifeng Dong: 0000-0002-6907-6578 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was supported by Special Foundation for State Major Research Program of China (Grant 2016YFC0106602); National Natural Science Foundation of China (Grants 21305008, 21475008, 21275017, 51373024); the Open Research Fund Program of Beijing Key Lab of Plant Resource Research and Development, Beijing Technology and Business University (PRRD-2016-YB2); the Fundamental Research Funds for the Central Universities (FRF-BR-15-020A); and State Key Laboratory of Analytical Chemistry for Life Science (SKLACLS1401).

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REFERENCES

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DOI: 10.1021/acssuschemeng.7b01035 ACS Sustainable Chem. Eng. 2017, 5, 6786−6794

Research Article

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DOI: 10.1021/acssuschemeng.7b01035 ACS Sustainable Chem. Eng. 2017, 5, 6786−6794