Targeted HyaluronateâHollow Gold Nanosphere Conjugate for Anti

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Targeted Hyaluronate - Hollow Gold Nanosphere Conjugate for Anti-Obesity Photothermal Lipolysis Jung Ho Lee, Hyeon Seon Jeong, Dong Hyun Lee, Songeun Beack, Taeyeon Kim, Geon-Hui Lee, Wonchan Park, Chulhong Kim, Ki Su Kim, and Sei Kwang Hahn ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00549 • Publication Date (Web): 27 Oct 2017 Downloaded from http://pubs.acs.org on November 6, 2017

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ACS Biomaterials Science & Engineering

Targeted Hyaluronate - Hollow Gold Nanosphere Conjugate for Anti-Obesity Photothermal Lipolysis

Jung Ho Lee,†,‡ Hyeon Seon Jeong,†,‡ Dong Hyun Lee,¶ Songeun Beack,† Taeyeon Kim,† Geon-Hui Lee,† Won Chan Park,† Chulhong Kim,¶ Ki Su Kim#,§,* and Sei Kwang Hahn†,¶,§,*

† Department of Materials Science and Engineering, Pohang University of Science and Technology (POSTECH), 77 Cheongam-ro, Nam-gu, Pohang, Kyungbuk, 790-784, Korea. ¶ Department of Creative IT Engineering, Pohang University of Science and Technology (POSTECH), 77 Cheongam-ro, Nam-gu, Pohang, Kyungbuk, 790-784, Korea. # Department of Organic Materials Science and Engineering, College of Engineering, Pusan National University, 2 Busandaehak-ro 63 beon-gil, Gumjeong-gu, Busan, 46241, Korea. § PHI BIOMED Co., #613,12 Gangnam-daero 65-gil, Seocho-gu, Seoul, 06612, Korea.

* CORRESPONDING AUTHOR FOOTNOTE Tel.: +82 54 279 2159; Fax: +82 54 279 2399; E-mail : [email protected] (S. K. Hahn) Tel.: +82 51 510 2496; Fax: +82 51 512 8175; E-mail : [email protected] (K.S. Kim)

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ABSTRACT Obesity is associated with the risk of developing several severe diseases, such as metabolic disorder, cancer and heart diseases. Despite wide investigation and trials, a non-invasive obesity therapy is still an important medical unmet need targeting the abnormal adipose tissue. Here, we developed hyaluronate - hollow gold nanosphere - adipocyte targeting peptide (HA-HAuNSATP) conjugates for the photothermal ablation of adipose tissues. The HA-HAuNS-ATP conjugate could be non-invasively delivered into the skin and effectively target to adipocytes in the subcutaneous. With NIR laser illumination, HA-HAuNS-ATP conjugate enabled highly effective photothermal ablation of adipose tissues in C57BL/6 obesity mice. The photoacoustic imaging confirmed the successful transdermal delivery and the photothermal lipolysis of HAHAuNS-ATP conjugate. Taken together, the transdermal HA-HAuNS-ATP conjugate might have a great potential for non-invasive photothermal lipolysis.

[KEYWORDS] Hollow gold nanosphere, Hyaluronate, Adipocyte targeting peptide, Obesity, Photothermal lipolysis


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INTRODUCTION A body mass index (BMI) greater than 30 kg/m2 means obesity, which is the fastest growing health problem in the developed society.1 Obesity can cause various chronic diseases including fatty liver disease,2 stroke,3 type 2 diabetes,4 heart failure5 and hypertension.6 The most typical treatment of obesity is to remove subcutaneous adipose tissues with laser and suction. Although laser-assisted liposuction is one of the powerful methods to remove adipose tissues, this invasive method has several disadvantages, such as discomfort, pain, bleeding, long recovery time and side effect of seromas.7 Recently, obesity treatment has been widely investigated using antiobesity poly(ethylene glycol) (PEG) nanoparticles.8,9 The PEG nanoparticles could effectively control obesity by targeting the blood vessels through white adipose tissues. However, the obesity therapy required the frequent administration of PEG nanoparticles via subcutaneous injection. In addition, the therapeutic agents were non-specifically impaired not only in adipose tissues but in normal tissues. The development of effective lipolysis agents is a strong medical unmet need for the safe and target-specific treatment of obesity. Gold nanoparticles (AuNPs) can be utilized as a therapeutic agent for the obesity therapy. AuNPs have their unique advantages of facile surface modification, great stability, noncytotoxicity and biocompatibility for biomedical applications.10,11 They can absorb the visible and near-infrared (NIR) light, which is converted to heat energy by surface plasmon resonance (SPR).12,13 Due to the efficient photothermal conversion, AuNPs have been widely investigated for various photothermal applications.14 Currently, various types of AuNPs have been investigated for photothermal therapy, among which hollow gold nanospheres (HAuNSs) exhibit the excellent photothermal characteristics.15 The absorption band of HAuNS is in the range of NIR, enabling the biophotonic applications in deep tissues.16 Furthermore, since a hollow



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structure has minimum mass in the same size of other types of AuNPs, HAuNS shows the great photothermal conversion efficiency per unit mass.17 Although AuNPs had been previously applied to the photothermal lipolysis with a high photothermal conversion efficiency,18,19 we tried to develop a new facile nanoplatform of hyaluronate - hollow gold nanosphere - adipocyte targeting peptide (HA-HAuNS-ATP) conjugate for obesity therapy (Figure 1). The transdermal delivery of HA-HAuNS-ATP conjugate can resolve the invasiveness of conventional obesity therapies with the advantages of comfort, painless, less side effect and avoidance of first-pass metabolism.20,21 HA was used as an efficient transdermal delivery carrier, and the enhancer of stability and biocompatibility of HAuNS.22,23 In addition, ATP with a sequence of CKGGRAKDC was used to increase the target-specificity of HAuNSs to adipose tissues, specifically binding to prohibitin proteins on the surface of white adipose tissues.24 The HA-HAuNS-ATP conjugate was prepared by the goldthiol chemistry between HAuNS, thiolated HA (HA-SH) and cysteine of ATP. HA-HAuNS-ATP conjugate can penetrate the skin barrier and target-specifically accumulate in adipocytes (Figure 1). Finally, we carried out the photoacoustic (PA) imaging of HA-HAuNS-ATP conjugate to confirm the successful transdermal delivery and the photothermal lipolysis under NIR light illumination.


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EXPERIMENTAL SECTION Materials. Sodium hyaluronate with a molecular weight (MW) of 10 kDa was obtained from Lifecore Co. (Chaska, MN). Cystamine dihydrochloride was obtained from Tokyo Chemical Industry (Tokyo, Japan). Cobalt (II) chloride hexahydrate, sodium citrate tribasic dehydrate, poly(vinylpyrrolidone) (PVP) with a MW of 55 kDa, sodium borohydride, chloroauric acid (HAuCl4), sodium cyanoborohydride and DL-dithiothreitol (DTT) were purchased from Sigma-Aldrich (St. Louis, MO). Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) was obtained from Alfa Aesar (Haverhill, MA). Hilyte Fluor 647 amine was purchased from AnaSpec (San Jose, CA). PD-10 column was purchased from GE Healthcare (Chicago, IL). The

adipocyte

targeting

peptide

of

Gly-Lys-Gly-Gly-Arg-Ala-Lys-Asp-Gly-Gly-Cys

(GKGGRAKDGGC) was obtained from Peptron Co. (Daejeon, Korea). 3T3-L1 preadipocyte cells of mus musculus were purchased from Korean Cell Line Bank (Seoul, Korea). Dulbecco's modified eagle's medium (DMEM) was purchased from Mediatech (Tewksbury, VA). Fetal bovine serum (FBS) and antibiotics were obtained from Thermo Fisher Scientific (Waltham, MA). Dimethyl sulfoxide (DMSO) and MTT solution were purchased from Georgia Tech Chemistry & Biochemistry (Atlanta, GA). Phosphate-buffered saline (PBS) was purchased from Bioprince (Seoul, Korea). Preparation and Characterization of HAuNS. HAuNS was prepared via the galvanic replacement of cobalt with gold. Cobalt chloride hexahydrate (CoCl2·6H2O, 38.1 mg) and sodium citrate tribasic dihydrate (Na3C6H5O7·2H2O, 47.1 mg) were dissolved in 400 mL of DI water in a round-bottom flask. The solution was purged with nitrogen gas for 40 min to prevent the oxidation of cobalt in the solution. The aqueous solution of PVP (MW = 55 kDa, 1 wt%, 2 mL) was dropped into the degassed solution via a syringe. Then, DI water (120 mL) containing 5 ACS Paragon Plus Environment


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0.1 M of chloroauric acid (HAuCl4, 0.18 mL) was added to 360 mL of the purged solution. To completely oxidize unreacted Co, the solution was allowed to stay for 1 d. HAuNS was purified by the successive centrifugation (8000 rpm, 5 min) and re-dispersion in 0.1 wt% of PVP solution and freeze-dried for 2 d. The prepared HAuNS was analyzed by TEM (JEM - 1011, JEOL products, Tokyo, Japan) operating at 300 kV. The morphology of HAuNS was analized by high resolution (HR) - TEM (JEM - 2100F, JEOL products, Tokyo, Japan) operating at 200 kV. The successful formation of HA-HAuNS-ATP conjugate was analyzed with a UV/Vis spectrophotometer (S-3100, Scinco Co., Seoul, Korea). Photothermal and Photoacoustic Characterization of HAuNS. HAuNS was dispersed in DI water (5 mg/mL) and illuminated with NIR (808 nm) light at a power density of 1.5 W/cm² for 10 min. To observe the solution temperature change by NIR light illumination, a thermocouple probe was inserted into the HAuNS solution and the temperature was recorded every 30 s. DI water was used as a control. To analyze in vitro PA sensitivity and spectrum of HAuNS, 3 silicone tubes were prepared at the concentrations of 0.5, 1, 2 mg/mL, respectively. The laser wavelength was varied from 750 nm to 975 nm for PA excitation with the pulsed laser energy of 15 mJ/cm². DI water was used again as a control. Synthesis and Characterization of End-thiolated HA. HA-SH was synthesized by the reductive amidation reaction. HA (MW = 10 kDa, 50 mg) and cystamine dihydrochloride (30 mg) were dissolved in 5 mL of borate buffer (0.1 M, pH 8.5) with 0.4 M of sodium chloride. Sodium cyanoborohydride (NaBH3CN, 0.2 M) was added to the solution and incubated at 40 ˚C for 5 d. The mixture was dialyzed (MWCO 3.5 kDa) against 0.1 M of sodium chloride solution, 25 vol% of ethanol and water. Then, 0.1 M of DTT was added to the reaction solution and incubated for 12 h to reduce disulfide bonds. After dialysis, the solution was lyophilized for 2 d and stored at 6 ACS Paragon Plus Environment

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20 ˚C. After analysis by 1H nuclear magnetic resonance (NMR), the end-thiolated HA was treated with TCEP to completely cleave disulfide bonds and eluted through PD-10 column before the following experiments. Preparation and Characterization of HA-HAuNS-ATP Conjugate. The end-thiolated HA solution (10 mg/mL, 20 µL) was mixed with the HAuNS solution (10 mg/mL, 1 mL) and stirred for 4 h. Sodium chloride (0.02 M, 1 mL) was added to the solution to prevent the aggregation of HA-HAuNS conjugate. HA-HAuNS conjugate was purified by centrifugation (20,000 g, 20 min, 25 ˚C) and re-dispersed in 1 mL of DI water. After that, 1 µM of ATP in sodium chloride solution (0.02 M, 1 mL) was added to 1 mL of HA-HAuNS conjugate solution. After the mixture stirred for 4 h, the unbound ATP fragment was removed by centrifugation and HA-HAuNS-ATP conjugates were finally re-dispersed in DI water. The hydrodynamic size and zeta potential of HA-HAuNS-ATP conjugate were measured by DLS (Zetasizer Nano, Malvern Instrument Co., UK). The formation of HA-HAuNS-ATP conjugate was analyzed with a TEM operating at 300 kV and a UV/Vis spectrophotometer. The concentration of HAuNS was measured by inductively coupled plasma (ICP, iCAP 6500, Thermo Scientific, Waltham, MA). To quantify the number of HA-SH and ATP on the HAuNS, HiLyte647 amine was conjugated to the carboxyl group of HA and FITC was attached to the amine group of ATP. After binding HiLyte647-HA-SH to HAuNS, the fluorescence of supernatant was measured with a fluorescence spectrophotometer (λEx 584 nm and λEm 650 nm, FP-6500, JASCO, Tokyo, Japan). Then, the number of HA-SH on HAuNS was determined by substracting the concentration of HiLyte647-HA-SH in the supernatant. Using the same method, we quantified the amount of ATP-FITC in the supernatant of HA-HAuNS-ATP conjugate solution. In Vitro Cytotoxicity Test. Human fibroblast (NIH3T3) cells were seeded on a 96-well 7 ACS Paragon Plus Environment


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plate at a density of 5×103 cells/well and cultured in 0.1 mL of DMEM-high glucose medium containing 10 vol% FBS and 1 vol% antibiotics at 37 ˚C and 5% CO2 atmosphere for 1 d. Then, various concentrations of HAuNSs, HA-HAuNS conjugates, HA-HAuNS-ATP conjugates were added to each well from 0 µg/mL to 400 µg/mL and incubated for 1 d. After the cells were washed with PBS twice, the cell culture medium was replaced with a serum-free cell culture medium. MTT solution (2 mg/mL, 20 µL) was added to each well and incubated for 3 h. After removing the solution, 50 µL of DMSO was added to dissolve the formazan in each well. The absorbance at 540 nm was measured with a microplate reader (EMax, Molecular Devices, Sunnyvale, CA). In Vitro Dark-field Imaging for Uptake of HA-HAuNS-ATP Conjugate in Adipocytes. 3T3-L1 preadipocytes were seeded on a 8 chamber glass slide at a density of 2×104 cells/well and incubated in the high glucose DMEM with 10 vol% of FBS and 1 vol% of antibiotics for 2 d. Then, the matured adipocytes were differentiated in 2 weeks. After confirming the formation of lipid droplets within adipocytes by optical microscopy, the medium was replaced with DMEM containing HAuNS, HA-HAuNS conjugate or HA-HAuNS-ATP conjugate, respectively (0.1 µM of free HAuNS equivalent). Then, the cells were incubated for 2 h, washed and fixed with 4% (w/v) of paraformaldehyde solution for 20 min. The uptake of HA-HAuNS-ATP conjugate into adipocyte was assessed by the dark-field imaging. In Vivo Photoacoustic Imaging for Transdermal Delivery of HA-HAuNS-ATP Conjugate. PA imaging was performed to visualize the transdermal delivery of HA-HAuNSATP conjugate. BALB/c nude mice were treated with HA-HAuNS-ATP conjugate (4 mg/mL, 20 µL) for 1 h and the remaining solution was wiped out. A Q-switched Nd:YAG laser (Surelite III10, Continuum, Boston, MA) and a tunable optical parametric oscillator (OPO) laser (Surelite 8 ACS Paragon Plus Environment

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OPO PLUS, Continuum, Boston, MA) were used to generate the PA signals. By tuning the OPO laser, a short laser pulse with a wavelength of 900 nm was illuminated to the target tissues. The PA signals were acquired using a spherically focused ultrasound transducer (V308, Olympus NDT, Center Valley, PA) with a focal length of 25 mm and a center frequency of 5 MHz. Then, the PA signals were amplified using an amplifier (5072PR, Olympus NDT, Center Valley, PA) with a gain of 35 dB and digitized by the data acquisition system (MSO5204, Tektronix, Beaverton, OR) with a sampling frequency of 40 MHz. To obtain volumetric PA images from a whole body of mice, the mechanical raster scanning was performed with a field of view of 60 mm × 40 mm along X and Y directions, respectively. The scanning step size was 0.2 mm and 0.4 mm in X and Y directions. The whole body PA images were reconstructed by the maximum amplitude projection (MAP) method. All animal experiments were performed following the laboratory animal protocol approved by the institutional animal care and use committee of the Pohang University of Science and Technology in accordance with the National Institutes of Health Guide for the Care and Use of Experimental Animals. In Vivo Transdermal and Photothermal Lipolysis. C56BL/6 mice fed with a high-fat diet for 3 months were fully anesthetized by intraperitoneal injection of a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg). HAuNS, HA-AuNS conjugate and HA-HAuNS-ATP conjugate solution (2 mg/mL, 20 µL) were topically applied on the abdominal skin and incubated for 1 h. After wiping the remaining solution, NIR (808 nm) light was illuminated on the skin (1.5 W/cm²) for 10 min. Then, PA imaging was performed using 1,200 nm average power of 220 mW. Statistical Analysis. Statistical analysis was carried out via the t-test using the software of Prism 6.0 (GraphPad Software Inc., La Jolla, CA). The values for *P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant. Data are expressed as means ± standard 9 ACS Paragon Plus Environment


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RESULTS AND DISCUSSION Preparation and Characterization of HAuNS. As schematically shown in Figure 2a, HAuNS was synthesized via the galvanic replacement of Co nanoparticles with gold ions. First PVP was covered on the surface of Co nanoparticle as a stabilizing agent. Then, HAuCl4 on the Co nanoparticle surface initiated the formation of HAuNS. The oxidized Co was escaped from the core template, making the empty space within the gold shell. After complete oxidation of unreacted Co, we could obtain the PVP coated HAuNSs. Figure 2b shows TEM and HR-TEM images of HAuNS with an outer diameter of 45 ± 6.3 nm and a shell thickness of 4.2 ± 0.8 nm. The TEM images revealed that HAuNS had a spherical morphology with an empty core. According to UV/Vis spectrophotometry, the SPR peak of HAuNS appeared in the NIR region of 903 nm (Figure 2c). To assess the photothermal characteristics of HAuNS, the temperature of HAuNS aqueous solution (2 mg/mL) was measured with NIR light illumination. The temperature of HAuNS solution increased more than 20 °C in 10 min under NIR light illumination, compared to the control of DI water (Figure 2d). In addition, in vitro assessment for the PA amplitude of HAuNS at various concentrations of 0.5, 1, 2 mg/mL revealed the highest PA signal of HAuNSs between 925 nm and 950 nm (Figure 2e). These results confirmed that HAuNSs were successfully synthesized and could be exploited for the PA imaging and photothermal therapy. Preparation and Characterization of HA-HAuNS-ATP Conjugate. HA-HAuNS conjugate was synthesized via the coordination chemistry between thiol group of HA-SH and gold atom of HAuNS (Figure 3a).22 The successful synthesis of HA-SH was confirmed by 1H NMR as we previously reported elsewhere.22 After purification of HA-HAuNS conjugate, ATP with a C-terminal amino acid of cysteine could be also attached to HAuNS by the same 11 ACS Paragon Plus Environment


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chemistry (Figure 3a).25 The facile preparation of the HA-HAuNS-ATP conjugate was confirmed by UV/Vis spectrophotometry, DLS and TEM. DLS analysis showed that the hydrodynamic size of HAuNS was ca. 131.8 nm with a narrow PDI of 0.162 and that of HA-HAuNS conjugate was ca. 117.1 nm with a PDI of 0.144. After ATP binding to HA-HAuNS conjugate, the diameter of HA-HAuNS-ATP conjugate was ca. 127.8 nm with a PDI of 0.179 (Figure 3b). When HA-SH was bound to HAuNS, the hydrodynamic size decreased, possibly due to the fall-off of PVP from the surface of HAuNS.26 The surface charge of HAuNS, HA-HAuNS conjugate and HAHAuNS-ATP conjugate was -8.78 ± 0.83 mV, -24.57 ± 0.55 mV and -17.97 ± 1.39 mV, respectively (Figure 3c). HA has a negative charge,27 but ATP has a positive charge due to the cationic amino acids.28 The SPR of HAuNS peak was blue-shifted from 903 nm to 864 nm in accordance with the change in the hydrodynamic diameter of HA-HAuNS conjugate (Figure 3d). The SPR peak of the HA-HAuNS-ATP conjugate was shifted to 872 nm. TEM showed the morphology of HA-HAuNS-ATP conjugate (Figure 3e). In addition, the successful conjugation of HA and ATP onto the HAuNS was confirmed by labelling them with fluorescent dyes. After synthesis, we measured the fluorescence intensity of (HA-HiLyte647)-HAuNS conjugate and HA-HAuNS-(ATP-FITC) conjugate. The molar concentrations of HAuNS, HA-HiLyte and ATP-FITC were calculated to be 8.86 × 10-12 M, 5.19 × 10-10 M and 6.89 × 10-10 M, respectively. From the results, we estimated that 58.6 HA chains and 77.8 ATP peptides were bound to the single HAuNS. Cytotoxicity of HAuNS, HA-HAuNS Conjugate and HA-HAuNS-ATP Conjugate. Before further applications, we investigated the cytotoxicity of HAuNS, HA-HAuNS conjugate and HA-HAuNS-ATP conjugate in the mouse embryonic fibroblast of NIH3T3 cells. Each sample at the concentration from 0 µg/mL to 400 µg/mL was incubated with the cells for 1 d. As 12 ACS Paragon Plus Environment

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shown in Figure 4, there was negligible cytotoxicity for all samples up to a concentration of 200 µg/mL. However, at the concentration of 400 µg/mL, the cytotoxicity of each sample increased significantly. The cytotoxicity of HA-AuNS conjugate and HA-AuNS-ATP conjugate was slightly higher than that of HAuNS at all concentrations due to the enhanced cellular uptake by the HA receptor mediated endocytosis into the NIH3T3 cells. Dark-Field Imaging for Intracellular Uptake to Adipocytes. Dark-field imaging was carried out to investigate the intracellular uptake of HA-HAuNS-ATP conjugates into adipocytes (Figure 5a). Before the imaging, mature adipocytes were incubated with the HAuNS, HAHAuNS conjugate and HA-HAuNS-ATP conjugate for 2 h, respectively. PBS was used as a control. As shown in Figure 5a, HA-HAuNS-ATP conjugates could be up taken to the adipocytes more effectively than the other groups due to the specific binding of ATP to prohibitin located on the surface of adipocytes. The fluorescence intensity of HA-HAuNS-ATP conjugate was the strongest intensity of HA-HAuNS-ATP conjugate was the strongest with the highest ROI values (Figure 5b). From the results, we could confirm the effect of ATP in HA-HAuNS-ATP conjugate on the targeting to adipocytes. Photoacoustic Imaging for Transdermal Delivery on the Dorsal Skin of Mice. PA imaging was performed to investigate the transdermal penetration of HA-HAuNS-ATP conjugate. As we previously reported elsewhere,22,23 HA can facilitate the penetration of nanoparticles into the skin. After topical application of HA-HAuNS-ATP conjugate on the dorsal skin of BALB/c nude mice for 1 h, PA imaging of HAuNS in HA-HAuNS-ATP conjugate was performed using the laser with a wavelength of 950 nm where HAuNS showed the highest PA signal (Figure 2e). Figure 6a and b show the PA MAP images of the dorsal skin in mice before and after the treatment. Compared to the control in Figure 6a, we could observe the significantly enhanced PA 13 ACS Paragon Plus Environment


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signal on the dorsal skin of mice after treatment with HA-HAuNS-ATP conjugate (Figure 6b,c). These results confirmed the effective transdermal penetration of HA-HAuNS-ATP conjugate into the dorsal skin. In Vivo Photothermal Lipolysis. Finally, we assessed the effect of HA-HAuNS-ATP conjugate on the photothermal lipolysis in C56BL/6 obesity mice fed with a high-fat diet for 3 months. After topical treatment of the abdominal region of C56BL/6 obesity mice with PBS as a control, HAuNS, HA-HAuNS conjugate or HA-HAuNS-ATP conjugate, NIR light was illuminated for 10 min for the photothermal lipolysis. After treatment, there was no adverse effect on the skin in mice. PA imaging was carried out to visualize the photothermal lipolysis using a 1200 nm laser. Adipose tissue showed the maximum PA absorption coefficient at 1210 nm.29,30 Figure 7a shows the photograph and the PA image before and after treatment with each sample. Among them, only the PA signal of adipose tissue treated with HA-HAuNS-ATP conjugate decreased remarkably in the dashed area with NIR light illumination. In contrast, there was no significant difference between PA amplitudes before and after treatment with PBS, HAuNS and HA-AuNS conjugate. The PA amplitude quantification clearly confirmed more effective photothermal lipolysis of HA-HAuNS-ATP conjugate than the others (Figure 7b). As reported elsewhere,31 the adipocytes might be necrotized by the photothermal treatment with HA-HAuNS-ATP conjugates. After that, the necrotic and apoptotic cells might be cleared by the phagocytosis of immune systems.32 Taken together, we could confirm the feasibility of HA-HAuNS-ATP conjugate for photothermal lipolysis therapy after non-invasive transdermal delivery. This method can be effectively harnessed to alleviate the problems of laser-assisted liposuction, such as discomfort, pain, bleeding, long recovery time and side effect of seromas. In addition, we believe this facile 14 ACS Paragon Plus Environment

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platform technology of HA-HAuNS-ATP conjugate can be successfully applied to the development of various futuristic photomedicines.

CONCLUSIONS We successfully developed transdermal HA-HAuNS-ATP conjugate for targeted noninvasive photothermal lipolysis. HA-HAuNS-ATP conjugate was synthesized by the coordination chemistry between gold, and thiol groups of HA-SH and ATP-Cys. DLS, UV/Vis spectrophotometry and TEM confirmed the successful preparation of HA-HAuNS-ATP conjugate. In vitro dark field imaging visualized the effective targeted delivery of HA-HAuNSATP conjugate to the adipocytes. In addition, in vivo PA imaging clearly visualized the noticeable transdermal delivery of HA-HAuNS-ATP conjugate. The following photothermal lipolysis with NIR light illumination resulted in the reduction of ca. 20% of the initial lipid. This new HA-HAuNS-ATP conjugate for adipocyte-targeted photothermal lipolysis can be successfully harnessed for non-invasive obesity therapy.



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AUTHOR INFORMATION * Corresponding Author Tel.: +82 54 279 2159; Fax: +82 54 279 2399; E-mail: [email protected] (S. K. Hahn) Tel.: +82 51 510 2496; Fax: +82 51 512 8175; E-mail: [email protected] (K.S. Kim) Author Contributions J.H.L H.S.J. mainly designed and performed experiments, collected samples, analyzed and interpreted data, prepared the figures for the manuscript, and wrote the manuscript. S.K.H., K.S.K. conceived and supervised the project, designed experiments, interpreted data and wrote the manuscript. D.H.L. contributed to the experimental design of PA imaging. S.E.B contributed to preparing and designing the animal experiments. T.K., G.H.L. W.C.P. contributed to preparing manuscript. S.K.H., C.K., K.S.K. made intellectual contributions. All authors contributed to critical reading and revision of this manuscript. ‡These authors contributed equally. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This research was supported by the Nano·Material Technology Development Program (No. 2017M3A7B8065278) and the Basic Science Research Program (2017R1E1A1A03070458) of the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning, Korea. This work was also supported by the World Class 300 Project (R&D) (S2482887) of the Small and Medium Business Administration (SMBA), Korea.


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REFERENCES 1.

Chan, R. S. M.; Woo, J. Prevention of Overweight and Obesity: How Effective Is the Current Public Health Approach. Int. J. Environ. Res. Public Health 2010, 7, 765-783.

2.

Fabbrini, E.; Sullivan, S.; Klein, S. Obesity and Nonalcoholic Fatty Liver Disease: Biochemical, Metabolic and Clinical Implications. Hepatology 2010, 51, 679-689.

3.

Mitchell, A. B.; Cole, J. W.; McArdle, P. F.; Cheng, Y. C.; Ryan, K. A.; Sparks, M. J.; Mitchell, B. D.; Kittner, S. J. Obesity Increases Risk of Ischemic Stroke in Young Adults. Stroke 2015, 46, 1690-1692.

4.

McCarthy, M. L. Genomics, Type 2 Diabetes, and Obesity. N. Engl. J. Med. 2010, 363, 2339-2350.

5.

Kenchaiah, S.; Evans, J. C.; Levy, D.; Wilson, P. W. F.; Benzamin, E. J.; Larson, M. G.; Kannel, W. B.; Vasan, R. S.; Obesity and the Risk of Heart Failure. N. Engl. J. Med. 2002, 347, 305-313.

6.

Re, R. N. Obesity-Related Hypertension. Ochsner J. 2009, 9, 133-136.

7.

Weniger, F. G.; Calvert, J. W.; Newton, E. D. Liposuction of the Legs and Ankles: A Review of the Literature. Plast. Reconstr. Surg. 2004, 113, 1771-1785.

8.

Hossen, M. N.; Kajimoto, K.; Akita, H.; Hyodo, M.; Harashima, H. Vascular-Targeted Nanotherapy for Obesity: Unexpected Passive Targeting Mechanism to Obese Fat for the Enhancement of Active Drug Delivery. J. Control. Release 2012, 163, 101-110.

9.

Won, Y. W.; Adhikary, P. P.; Lim, K. S.; Kim, H. J.; Kim, J. K.; Kim, Y. H. Oligopeptide Complex for Targeted Non-viral Gene Delivery to Adipocytes. Nat. Mater. 2014, 13, 11571164.

10. Cai, W.; Gao, T.; Hong, H.; Sun, J. Applications of Gold Nanoparticles in Cancer 17 ACS Paragon Plus Environment


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

Page 18 of 28

Nanotechnology. Nanotechnol. Sci. Appl. 2008, 1, 17-32. 11. Dykman, L. A.; Khlebtsov, N. G. Gold Nanoparticles in Biology and Medicine: Recent Advances and Prospects. Acta Naturae 2011, 3, 34-55. 12. Amendola, V.; Pilot, R.; Frasconi, M.; Marago, O. M.; Lati, M. A. Surface Plasmon Resonance in Gold Nanoparticles: A Review. J. Phys. Condens. Matter 2017, 29, 1-48. 13. Huang, X.; Jain, P. K.; El-Sayed, I. H.; El-Sayed, M. A. Plasmonic Photothermal Therapy (PPTT) Using Gold Nanoparticles. Lasers Med. Sci. 2008, 23, 217-228. 14. Kennedy, L. C.; Bickford, L. R.; Lewinski, N. A.; Coughlin, A. J.; Hu, Y.; Day, E. S.; West, J. L.; Drezek, R. A. A New Era for Cancer Treatment: Gold-Nanoparticle-Mediated Thermal Therapies. Small 2011, 7, 169-183. 15. Zhang, J. Z. Biomedical Applications of Shape-Controlled Plasmonic Nanostructures: A Case Study of Hollow Gold Nanospheres for Photothermal Ablation Therapy of Cancer. J. Phys. Chem. Lett. 2010, 1, 686-695. 16. Adams, S.; Zhang, J. Z. Unique Optical Properties and Applications of Hollow Gold Nanospheres (HGNs). Coord. Chem. Rev. 2016, 320-321, 18-37. 17. Ren, Q. Q.; Bai, L. Y.; Zhang, X. S.; Ma, Z. Y.; Liu, B.; Zhao, Y. D.; Cao, Y. C. Preparation, Modification, and Application of Hollow Gold Nanospheres. J. Nanomater. 2015, 2015, 1-7. 18. Thovhogi, N.; Sibuyi, N.; Meyer, M.; Onani, M.; Madiehe, A. Targeted Delivery Using Peptide-functionalised Gold Nanoparticles to White Adipose Tissues of Obese Rats. J. Nanopart. Res. 2015, 17, 1-8. 19. Sheng, W.; Alhasan, A. H.; DiBernardo, G.; Almutairi, K. M.; Rubin, J. P.; DiBernardo, B. E.; Almutairi, A. Gold Nanoparticle-assisted Selective Photothermolysis of Adipose Tissue (NanoLipo). Plast. Reconstr. Surg. Glob. Open 2014, 2, 1-8.


Page 19 of 28

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


20. Wiedersberg, S.; Guy, R. H. Transdermal Drug Delivery: 30+ Years of War and Still Fighting!. J. Control. Release 2014, 190, 150-156. 21. Prausnitz, M. R.; Langer, R. Transdermal Drug Delivery. Nat. Biotechnol. 2008, 26, 12611268. 22. Lee, H. W.; Lee, J. H.; Kim, J.; Mun, J. H.; Chung, J.; Koo, H.; Kim, C.; Yun, S. H.; Hahn, S. K. Hyaluronate-Gold Nanorod/DR5 Antibody Complex for Noninvasive Theranosis of Skin Cancer. ACS Appl. Mater. Interfaces 2016, 8, 32202-32210. 23. Jung, H. S.; Kong, W. H.; Sung, D. K.; Lee, M. Y.; Beack, S. E.; Keum, D. H.; Kim, K. S.; Yun, S. H.; Hahn, S. K. Nanographene Oxide - Hyaluronic Acid Conjugate for Photothermal Ablation Therapy of Skin Cancer. ACS Nano 2014, 8, 260-268. 24. Mikhail, G. K.; Pradip, K. S.; Lawrence, C.; Renata, P.; Wadih, A. Reversal of Obesity by Targeted Ablation of Adipose Tissue. Nat. Med. 2004, 10, 625-632. 25. Vallee, A.; Humblot, V.; Pradier, C. M. Peptide Interactions with Metal and Oxide Surfaces. Acc. Chem. Res. 2010, 43, 1297−1306. 26. Carotenuto, G.; Nicolais, L. Size-Controlled Synthesis of Thiol-Derivatized Gold Clusters. J. Mater. Chem. 2003, 13, 1038-1041. 27. Collins, M. N.; Birkinshaw, C. Hyaluronic Acid Based Scaffolds for Tissue Engineering - A Review. Carbohydr. Polym. 2013, 92, 1262-1279. 28. Hossen, M. N.; Kajimoto, K.; Akita, H.; Hyodo, M.; Ishitsuka, T.; Harashima, H. LigandBased Targeted Delivery of a Peptide Modified Nanocarrier to Endothelial Cells in Adipose Tissue. J. Control. Release 2010, 147, 261-268. 29. Lee, M. Y.; Lee, C.; Jung, H. S.; Jeon, M.; Kim, K. S.; Yun, S. H.; Kim, C.; Hahn, S. K. Biodegradable Photonic Melanoidin for Theranostic Applications. ACS Nano 2016, 10, 822-



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Page 20 of 28

831. 30. Allen, T. J.; Hall, A.; Dhillon, A. P.; Owen, J. S.; Beard, P. C. Spectroscopic Photoacoustic Imaging of Lipid-Rich Plaques in the Human Aorta in the 740 to 1400 nm Wavelength Range. J. Biomed. Opt. 2012, 17, 061209. 31. Chu, K. F.; Dupuy, D. E. Thermal Ablation of Tumours: Biological Mechanisms and Advances in Therapy. Nat. Rev. Cancer 2014, 14, 199-208. 32. Poon, I. K.; Hulett, M. D.; Parish, C. R. Molecular Mechanism of Late Apoptotic/necrotic Cell Clearance. Cell Death Differ. 2010, 17, 381-397.


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NIR light HA-HAuNS-ATP Wrinkle

Dermis

Epidermis

stratum corneum

Transdermal delivery

Adipocytes

Subcutaneous

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


Photothermal lipolysis

Figure 1. Schematic illustration of hyaluronate – hollow gold nanosphere – adipocyte targeting peptide (HA-HAuNS-ATP) conjugate for targeted photothermal lipolysis of adipocytes after noninvasive transdermal delivery.



a

AuCl2

AuCl4

-

CoCl2

-

O2

CoO

AuCl4

O2

Co

-

Growth

PVP

b

PVP

Absorbance (A.U.)

c

d

2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 300 400 500 600 700 800 900 1000

W avelength (nm)

e 25

1.0

20

PA Amplitude (A.U.)

DI water 2mg/mL HAuNS

o

Temperature Change ( C)

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

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15 10 5 0

Control (DI) 0.5mg/mL HAuNS 1mg/mL HAuNS 2mg/mL HAuNS

0.8 0.6 0.4 0.2 0.0

0

2

4

6

Time (min)

8

10

750

800

850

900

950

1000

W avelength (nm)

Figure 2. (a) Schematic representation for the synthesis of HAuNS from Co nanoparticle. (b) TEM image (left, scale bar = 50 nm) and HR-TEM image (right, scale bar = 10 nm) of HAuNSs. (c) UV/Vis absorption spectrum of HAuNS. (d) The temperature change of DI water and HAuNS solution (2 mg/mL) with NIR light (1.5 W/cm²) illumination for 10 min. (e) In vitro PA amplitude of HAuNS at different concentrations of 0.5, 1 and 2 mg/mL.


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a

End-thiolated HA

HAuNS

HA-HAuNS conjugate

HA-HAuNS conjugate

ATP

b

HA-HAuNS-ATP conjugate

160

0

Zeta Potential (mV)

Hydrodynamic Size (nm)

c 140 120 100 80 60 S uN HA

d

TP NS -A Au S H N Au HA -H HA

-5 -10 -15 -20 -25 -30

S uN HA

e

TP NS -A Au S H N Au HA -H HA

2.0

Absorbance (A.U.)

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


1.8 1.6

HAuNS HA-HAuNS HA-HAuNS-ATP

1.4 1.2 1.0 0.8 0.6 0.4 300 400 500 600 700 800 900 1000

Wavelength (nm)

Figure 3. (a) Schematic illustration for the synthesis of HA-HAuNS conjugate and HA-HAuNSATP conjugate by the gold-thiol chemistry. (b) The hydrodynamic size, (c) the zeta potential and (d) the UV/Vis spectra of HAuNS, HA-HAuNS conjugate and HA-HAuNS-ATP conjugate (n = 3). (e) TEM image of HA-HAuNS-ATP conjugate (scale bar = 50 nm).



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

PBS

HAuNS

HA-HAuNS

Page 24 of 28

HA-HAuNS-ATP

* ***

**

Figure 4. The cytotoxicity of HAuNS, HA-HAuNS conjugate and HA-HAuNS-ATP conjugate with increasing concentration from 0 µg/mL to 400 µg/mL in NIH3T3 cells (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, versus the control).


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Brightfield

Dark-Field

Merged

HA-HAuNS

HAuNS

Control

a

b

Fluorescence Intensity (A.U.)

HA-HAuNS-ATP

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


300 *

250 200 150 100 50 0 C

tr on

ol

u HA

S TP NS uN -A A S -H uN HA HA HA

Figure 5. (a) Dark-field imaging of mature adipocytes after incubation with PBS as a control, HAuNS, HA-HAuNS conjugate and HA-HAuNS-ATP conjugate (scale bar = 100 µm). (b) The ROI value of the control, HAuNS, HA-HAuNS conjugate and HA-HAuNS-ATP conjugate in mature adipocytes (n = 3, *P < 0.05, versus the control). 25 ACS Paragon Plus Environment


a

Before

b

After 18

Depth (mm) 10 2

Amplitude (AU)

0

c

3.5

PA Amplitude (A.U.)

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

Page 26 of 28

***

3.0 2.5 2.0 1.5 1.0 0.5 0.0 Before

After

Figure 6. (a) before and (b) after transdermal delivery of HA-HAuNS-ATP conjugate (scale bar = 5 mm). (c) The quantification of PA amplitude before and after transdermal delivery of HAHAuNS-ATP conjugate (n = 3, ***P < 0.001).


Page 27 of 28

a

Photograph

Before

After

Control

0

HAuNS HA-HAuNS HA-HAuNS-ATP

1

1.2

PA Amplitude (A.U.)

b

Amplitude (AU)

1 0

Amplitude (AU)

1 0

Amplitude (AU)

1 0

Amplitude (AU)

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


Before After

1.1 1.0 0.9

**

0.8 0.7 0.6 C

tr on

ol

u HA

S TP NS uN -A A S -H uN HA HA HA

Figure 7. (a) The photographs and photoacoustic (PA) images of subcutaneous adipose tissues before and after photothermal lipolysis treatment with PBS as control, HAuNS, HA-HAuNS conjugate and HA-HAuNS-ATP conjugate, respectively (scale bar = 5 mm). (b) The quantification of PA amplitude for subcutaneous adipose tissues before and after transdermal treatment with PBS as the control, HAuNS, HA-HAuNS conjugate and HA-HAuNS-ATP conjugate under NIR light illumination (n = 3, **P < 0.01, vesus the control). 27 ACS Paragon Plus Environment


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– For Table of Contents Use Only –

Targeted Hyaluronate - Hollow Gold Nanosphere Conjugate for Anti-Obesity Photothermal Lipolysis Jung Ho Lee,†,‡ Hyeon Seon Jeong,†,‡ Dong Hyun Lee,¶ Song Eun Beack,† Taeyeon Kim,† GeonHui Lee,† Won Chan Park,† Chul Hong Kim,¶ Ki Su Kim§,* and Sei Kwang Hahn†,¶,§,*

NIR light HA-HAuNS-ATP

Transdermal delivery

Adipocytes

Photothermal lipolysis


Targeted HyaluronateâHollow Gold Nanosphere Conjugate for Anti

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