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Transdermal Vascular Endothelial Growth Factor Delivery with Surface Engineered Gold Nanoparticles Ying Chen, Yonghui Wu, Jining Gao, Zihao Zhang, Linjie Wang, Xi Chen, Junwei Mi, Yuanjiang Yao, Dongwei Guan, Bing Chen, and Jianwu Dai ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15914 • Publication Date (Web): 23 Jan 2017 Downloaded from http://pubs.acs.org on January 24, 2017

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Transdermal Vascular Endothelial Growth Factor Delivery with Surface Engineered Gold Nanoparticles

‖

‖

‖

Ying Chen†, , Yonghui Wu†, , Jining Gao†, , Zihao Zhang†, Linjie Wang†, Xi Chen†, Junwei Mi†, Yuanjiang Yao†, Dongwei Guan†, Bing Chen†,‡,*, Jianwu Dai†,‡,*

†

Institute of Combined Injury, State Key Laboratory of Trauma, Burns and

Combined Injury, Chongqing Engineering Research Center for Nanomedicine, College of Preventive Medicine, Chongqing Engineering Research Center for Biomaterials and Regenerative Medicine, Third Military Medical University, Chongqing 400038, China ‡

State Key Laboratory of Molecular Developmental Biology, Institute of

Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China

Keywords: VEGF, gold nanoparticles, angiogenesis, transdermal drug delivery, surface engineer

1


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ABSTRACT Skin injuries caused by burns or radiation remain a serious concern in terms of clinical therapy. Due to the damage to the epidermis or dermis, angiogenesis is needed to repair the skin. Vascular endothelial growth factor (VEGF) is one of the most effective factors for promoting angiogenesis and preventing injury progression, but the delivery of VEGF to lesion sites is limited by the skin barrier. Recently, gold nanoparticle (AuNP)-mediated drug delivery into or through the epidermis and dermis has attracted much attention. However, the efficacy of the AuNP-mediated transdermal drug delivery remains unknown. In this study, gold nanoparticles were conjugated with VEGF and generated a surface by carring negative charges, showing an ideal transdermal delivery efficacy for VEGF in wound repair. Our findings may provide new avenues for the treatment of cutaneous injuries. INTRODUCTION Although the potential applications of nanoparticles (NPs) and particulate materials for the percutaneous delivery of drugs have been discussed in a number of excellent studies, the penetration efficacy of topically applying particles to and through the skin is formidable for clinical cosmetic products and dermal drugs.1-3 In the recent decades, NP delivery has been increasingly applied to facilitate local penetration into the skin. Among a variety of NPs, gold nanoparticle (AuNP) is the most potential one to be used in clinic.4-6 Gold nanoparticles exhibit unique properties, including the ease of synthesis, inherent biocompatibility, and the capability of delivering various biological molecules such as small drug molecules, peptides, 2



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proteins, and nucleic acids.7-8 The size and shape of gold nanoparticles can be easily tuned as desired for specific biomedical applications. 9-11 Some researches tried to change the shape of NPs to increase their drug delivery efficacy, but finally found that NPs’ shape had little effect on drug delivery efficacy.12-14 Surface functionality plays a critical role in nanoparticle biological distributions and cell interactions,15-16 yet little is known about how surface charge influences the percutaneous efficiency of nanoparticles in vivo. PEG has been widely used to functionalize NPs to minimize protein corona formation, the introduction of functional groups (e.g. COOH, NH2) on PEG surface coatings can not only endow the nanoparticles with charged moieties but also provide anchor sites for further bioconjugation with biomolecules. Gold nanoparticles have a large surface area, and a variety of thiol-PEG derivatives are available for the surface modification of AuNPs through the well studied Au-S interaction. Skin injuries caused by burns or radiation remain a serious concern in terms of clinical therapy. Due to the damage to the epidermis or dermis, angiogenesis is disordered.17-18 Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis, which triggers a cascade reaction to induce endothelial cell activation, the assembly of new vascular structures, mural cell recruitment, and vessel stabilization.19-21 However, the traditional method cannot make the VEGF reach the epidermis or dermis efficiently and accurately. VEGF transdemals the skin barrier and reach the epidermis or dermis to promote angiogenesis, which may become the efficient way to therapy skin injuries. Here, we applied gold nanoparticles with 3


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different surface charges to evaluate their percutaneous efficiency and AuNPs with the negative surface charge had a high percutaneous efficiency. VEGF conjugated gold nanoparticles still had a surface negative charges, which had a strong ability to delivery VEGF into and through the epidermis and then induce angiogenesis. Our findings may provide new avenues for clinical dermal treatments.

EXPERIMENTAL METHODS Materials Sodium citrate tribasic dehydrate (98%), citric acid monohydrate (98%), and poly (ethylene glycol) methyl ether thiol (HS-PEG-OCH3, Mw=2000 Da) were obtained from Sigma-Aldrich and used as received. N-hydroxysulfosuccinimide (sulfo-NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Thermo Fisher Scientific Inc. (USA). Chloroauric acid (HAuCl4·3H2O, 99.9%) was purchased from Shanghai National Medicine Group Chemical Reagent Co., Ltd. HS-PEG-COOH (Mw=3400 Da) and HS-PEG-NH2 (Mw=3400 Da) were purchased from Laysan Bio, Inc. (USA). Ultrapure water (18.2 MΩ. cm, Millipore) was used for all procedures. The human VEGF165 gene was amplified by polymerase chain reaction from the complementary DNA of human MCF-7 breast tumor cells. Then, it was inserted into the plasmid vector pET-28a (Novagen, Madison, WI, USA), followed by transformation in the BL21 Escherichia coli

strain.

The

protein

was

induced

with

1

mmol/L

isopropyl-D-thiogalactopyranoside for 6 hours. After refolding, the protein containing a 6-His tag was purified using nickel-chelate chromatography and HiTrap heparin HP 4



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columns (GE Healthcare, Chalfont St Giles, United Kingdom). The ELISA kit for human VEGF was purchased from Neobioscience Technology, Co., Ltd. BALB/c 3T3 cells were purchased from ATCC (ATCC® Number: CCL-163™). Instrumentation Dynamic light scattering (DLS) measurements were performed using a Zetasizer system (Nano ZS, Malvern Instruments, UK). Transmission electron microscopy (TEM, JEM-1400, JEOL, Japan) was used to observe the morphology of the AuNPs. The results of the MTT assay and ELISA were detected using an automatic microplate reader (Thermo, Multiskan, FC). All the samples were observed by light microscoy (Carl Zeiss, Jena, Germany). Cryostat sections were generated using a cryostat microtome (Leica, Germany). Preparation of PEGylated AuNPs Citrate-capped AuNPs were synthesized by a previously reported method.22 Then, 200 µL of freshly prepared HS-PEG-COOH, HS-PEG-NH2 and HS-PEG-OCH3 (5 mg/mL) were added to 25 mL solution of AuNPs respectively. The mixtures were incubated for 2 h at room temperature with shaking at 320 rpm and without shaking at 4 ℃ overnight. The functionalized AuNPs were purified by ultra-filtration (3000 rcf, 5 min) and re-dispersed in 25 mL of Milli-Q water. MTT Assay BALB/c 3T3 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, SH30022.01, HyClone, USA) containing 10% fetal bovine serum (16000-044, Gibco, USA), streptomycin (100 µg/mL) and penicillin (100 units/mL) at 37 ℃ in an incubator with 5% CO2. 3T3 cells (cell viability > 99%) in the logarithmic growth phase were used for activity identification. A 1×105 cells /mL 5


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suspension was added to a 96-well plate at 100 µL/well. After the cells were completely adhered, the medium was aspirated. Cultures were prepared with solutions of DMEM containing previously sterilized AuNPs diluted to different concentrations (10 µM, 20 µM, 50 µM). Each well received 100 µL of these suspensions. Eight replicate wells and a blank control (100 µL of medium per well) were prepared. After incubating for 24 h, the supernatant was discarded, and 100 µL of DMEM was added to each well. Then, 10 µL of MTT (ST316, Beyotime, China) solution was added to each well, and the culture was continued for 4 h in the incubator. After 4 h, the culture medium with the MTT solution was aspirated and replaced with 150 µL of dimethyl sulfoxide (0231-4 L, Amresco, USA). The cells were incubated at 37 ℃ for 10 min, followed by shaking at room temperature for 15 min. The absorbance was measured with a microplate reader (570 nm, 630 nm) to calculate the cell proliferation rate. In Vivo Percutaneous Absorption of AuNPs Animal Model Animal experiments were conducted in accordance with the Chinese Ministry of Health guidelines for the care and use of laboratory animals. Fifty BALB/c mice (female, 6-7 weeks old, Laboratory Animal Center, Third Military Medical University, Chongqing, China) were randomly divided into 5 groups with 10 in each group. The mice were anesthetized with 6% chloral hydrate, and a 3 cm × 3 cm area of the back skin was shaved. The exposed skin was covered with gauze soaked in normal saline, hydrated for 10 min, and then dried. Next, 300 µL of the AuNP (citrate-AuNP, AuNP-PEG-OCH3, AuNP-PEG-COOH, and AuNP-PEG-NH2) suspensions (2×1010 /mL, 1.44 µM) was added to a sterile 2 cm × 2 cm piece of gauze, 6



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which was placed on the exposed skin and fixed with tape. During the experiment, the mice were housed in separate cages at 26 ℃ and provided standard food and water. After24 h, the mice were sacrificed by cervical dislocation, and the skin was thoroughly washed with deionized water. TEM of AuNP-treated Skin Once the skin was removed, the tissue was placed on a culture dish and fixed with a 2.5% glutaraldehyde solution at pH 7.2-7.4. Then, it was cut into slices approximately 1 mm × 3 mm in size in the dish, transferred into 2-3 mL of fresh, precooled fixation fluid, and then placed at 4 ℃. The tissue was removed the next day and treated with uranyl acetate for 20 min. After the tissue was dehydrated with an ethanol gradient (30% for 10 min, 50% for 10 min, 75% for 10 min, 95% for 10 min, and 100% for 20 min twice), it was immersed in acetonitrile for 10 min and then polymerized with fresh Spurr resin at 60 ℃ for 16 h. Finally, the tissue was cut into 90 nm/wafer and mounted on 400-mesh copper/palladium grids for TEM observation at 80kV. Silver-enhanced Staining Frozen sections were obtained 24 h after treatment via the methods described above. Skin samples were embedded in OTC (4583, SAKURA Tissue-Tek, Japan) and rapidly cooled on dry ice. Then, the samples were longitudinally cut into sections 6 µm in length, 3 sections per mouse were prepared.. The silver staining (50-22-01, Kirkegaard & Perry Laboratories, USA) and eosin staining were conducted, and then the sections were observed by optical microscopy. The average number of silver-stained particles under five random high-magnification fields (×400) was determined as the density of silver-stained particles per section. The 7


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number of silver-stained particles in each image was calculated using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA). Bioconjugation of AuNP-PEG-COOH with VEGF The concentration of HS-PEG-COOH modified AuNPs (AuNP-PEG-COOH) was adjusted to 2×1011 /mL (14.4 µM). Equal volumes of 60 µL of EDC (1 mg/mL) and NHS-sulfo (1 mg/mL) were added to 5 mL of a AuNP-PEG-COOH solution. The mixture was incubated for 20 min with continuous shaking to activate the carboxyl groups. Then, 2.5 mL of VEGF (His-tagged, 20 µg/mL) was added to the activated AuNP-PEG-COOH solution and incubated for 30 min at room temperature with shaking at 320 rpm and without shaking overnight at 4 ℃. The VEGF bioconjugated AuNP-PEG-COOH was washed three times with ultra-filtration and suspended in 5 mL of phosphate-buffered saline (PBS). The loading amount of VEGF on the gold nanoparticles was quantified by a human ELISA kit with a microplate reader at absorbance of 450 nm. In Vivo Study of the Co-delivery of AuNP-PEG-COOH and VEGF VEGF (His-tagged) Detection The mouse model was established in the same manner as described above. Then, 300 µL AuNP-CONH-VEGF (VEGF, molecular weight of 44 kDa, 20 µg/mL, N=10), 300 µL AuNP-CONH-OVA (ovalbumin, molecular weight of 43 kDa, was bioconjugated with AuNP-PEG-COOH in the same manner as VEGF, 20 µg/mL, control group, N = 10) or 300 µL VEGF (VEGF molecular weight of 44 kDa, 20 µg/mL, control group, N=10) solution was added to the gauze. In the blank control group, the skin was removed without any further treatment (N=10). The mice were sacrificed after 24h of treatment. The skin was fixed in 4% paraformaldehyde 8



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for 24 h, dehydrated and embedded in paraffin. The tissues were longitudinally cut into sections 4 µm in length, conventionally dewaxed, boiled for repair and sealed with normal goat serum working fluid (ZLI-9002, ZSGB-BIO, China). After adding the His antibody (H1029, Sigma-Aldrich, USA), the samples were incubated in a humidified box at 37 ℃ for 60 min and kept at room temperature overnight. Horseradish peroxidase (HRP)-conjugated anti-mouse IgG (pv-6002, ZSGB-BIO, China) was added dropwise the next day; after being washed with PBS, the sections were counterstained with hematoxylin. 3, 3’-Diaminobenzidine (DAB, ZLI-9018, ZSGB-BIO, China) was used for visualization, and distilled water was used to stop the color development, followed by sealing and observation. Vascular Detection Immunohistochemical Staining 7 days after being treated with AuNP-CONH-VEGF, the skin was prepared into paraffin sections, dewaxed, boiled for repair, and sealed using normal goat serum working fluid. The sections were then incubated with an antibody to α smooth muscle actin (α-SMA, bm0002, Boster, China) at 37 ℃ for 60 min and at 4 ℃ overnight. The HRP-conjugated anti-mouse IgG (pv-6002, ZSGB-BIO, China) was added the next day after the sections were washed with PBS. DAB (ZLI-9018, ZSGB-BIO, China) was used for visualization, and distilled water was used to stop color development, followed by sealing and observation. The positive areas per ×20 field were counted using ImageJ (National Institutes of Health, USA). Hematoxylin and Eosin (H&E) Staining The paraffin sections were dewaxed, 9


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stained with H&E and observed by microscopy. The average number of microvessels under five random high-magnification fields (×400) was determined as the density of microvessels per section. Statistical Analysis Statistical differences were determined using the t test or ANOVA. Figures were carried out using GraphPad Software (La Jolla, CA, USA). The nominal level was set at 0.05 in all cases. Results were considered to represent significant differences at P < 0.05.

RESULTS Synthesis and Characterization of AuNPs Figure 1A shows TEM images of citrate-capped AuNPs. Through a statistical analysis of the diameters of 300 particles in each image, the diameter of the AuNPs was calculated to be approximately 11.2 nm±0.1 nm with spherical geometry and a very low polydispersity. Figure 1B shows the DLS analysis of the solutions of AuNPs and AuNPs with different thiol-PEG molecules, and the results showed that the particles had an average hydrodynamic diameter of 15.1 nm±0.1 nm with a very low polydispersity index (PDI) of 0.043. As expected, DLS showed a larger particle size than TEM due to the presence of the hydrated shell. Additionally, the hydrodynamic diameter of AuNPs was increased by approximately 8-10 nm after surface modification; this was attributed to the attachment of the thiol-PEG molecules, which are larger than citrate. In addition, differences in the Zeta potential confirmed the successful surface modification with different thiol-PEG molecules (Figure 1C). 10



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After synthesizing the AuNPs, the UV-vis absorption spectra and an atomic absorption spectrophotometer were used to calculate the concentrations of the AuNP solutions. When the absorbance was 0.5, the concentration of the solution was 0.12 mM (1.67×1012 NPs/mL). Based on the diluted the stock solutions, 2×1010 AuNPs/mL was used in all the experiments, unless otherwise noted. AuNPs Penetration Assessment To verify whether AuNPs can penetrate the skin, we selected BALB/c mice and used their back skin to perform in vivo animal experiments. To prepare TEM slices, the entire thickness of skin were harvested and cut into sections with a thickness of 90 nm. Because these slices were very thin, we did not expect to observe large quantities of AuNPs, and the process of preparing these sections while avoiding contamination is a quite delicate. Figure 2 shows cross-sectional TEM images of AuNPs that penetrated the skin. Each image clearly indicates that a certain quantity of AuNPs entered the skin, demonstrating that the surface charge had no obvious effect on the transdermal properties of AuNPs approximately 10 nm in size. TEM images can only qualitatively demonstrate that AuNPs can pass through the skin and cannot be used to verify the transdermal efficiency of AuNPs with different surface charges. Therefore, we performed silver-enhanced staining on skin samples obtained by the same experimental method to determine the transdermal efficiency of AuNPs with different surface charges. Figure 3 shows that AuNPs passed through the skin barrier in each group and that a certain number of particles reached the dermis. We statistically analyzed the transdermal efficiency of silver-stained AuNPs with different surface 11


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charges.

The

results

showed

the

highest

transdermal

efficiency

in

the

AuNP-PEG-COOH group (P < 0.05). AuNPs Cytotoxicity To investigate the cytotoxicity of AuNP-PEG-COOH, we performed in vitro cytotoxicity assays. As shown in Figure 4, the relative cell proliferation rate was more than 80% at concentrations of 10 µM, 20 µM and 50 µM. According to the relationship between the cell relative proliferation rate and the cytotoxicity grade specified in the United States Pharmacopoeia(USP), when the relative cell growth %(RCG%) ≥ 80%,23 the AuNP-PEG-COOH showed a cytotoxicity grade of 1, indicating satisfactory cytotoxicity results. This test showed that AuNP concentrations within the range of 10 -50 µM had low cytotoxicity and could be used for the next experiment. In Vivo Study of the Co-delivery of AuNP-PEG-COOH and VEGF To verify that AuNPs can carry proteinaceous drugs and achieve co-penetration, AuNP-PEG-COOH

and

VEGF underwent covalent

binding

facilitated

by

EDC/sulfo-NHS (Figure 5A). After conjugation with VEGF, the surface charge of the AuNPs (AuNP-CONH-VEGF) was still negative, and the NPs maintained the improved penetration efficiency. An ELISA kit for human VEGF was used to quantify the conjugation efficiency of the AuNPs with VEGF with a microplate reader at absorbance of 450 nm. The number of VEGF molecules conjugated to each AuNP was approximately 0.165. Because VEGF was labeled with His-tag, the AuNP-CONH-VEGF sample could be subjected to immunohistochemistry after 24 h 12



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of transdermal delivery using the same animal model as described above. As shown in Figure 6, His-tagged VEGF was detected in the skin in the experimental group treated with AuNP-CONH-VEGF but not in the other groups, suggesting that VEGF can permeate the skin after conjugating with AuNP-PEG-COOH. VEGF reach the subcutaneous region through penetrating the skin barrier. To further investigate whether VEGF conjugated with AuNP-PEG-COOH can promote vascular growth after penetrating the skin, we subjected the skin to immunohistochemistry detection after 7 days of the transdermal treatment. As shown in Figure 7, subcutaneous vessels with positive staining were observed in the experimental group (AuNP-CONH-VEGF) and the control group. However, the amount of subcutaneous angiogenesis was significantly higher in the experimental group (skin and dermis) than in the control group. Figure 8 shows the H&E staining results of the skin after 7 days of the transdermal treatment, which were in agreement with the immunohistochemical staining results. The vascular density was significantly higher in the experimental group than in the control group (P < 0.05). In summary, using EDC/sulfo-NHS to connect AuNP-PEG-COOH and VEGF allows them to permeate the skin barrier and induce angiogenesis.

DISCUSSION As the largest human organ, the skin has many important functions; the most important being its barrier function. However, the skin is not completely sealed or impermeable. Certain substances can be absorbed by the dermis through the epidermis, 13


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and this mechanism has become the theoretical basis for the topical treatment of skin diseases in modern dermatology.24 However, due to the natural skin barrier, most drug molecules penetrate the skin poorly. Over the years, researchers have tried to use chemical penetration enhancers and physical mediators to overcome the skin barrier and achieve transdermal delivery.25 These methods have various limitations, such as easily induced skin allergies, ineffectiveness promoting the penetration of macromolecules in drugs, and requiring special equipment and professional operation; in addition, some methods have high costs and are unsuitable for general use. While in recent years, the nanomaterial-based technology for the transdermal drug delivery of large molecules and proteins is very promising and could solve these current problems.26-27 Based on the results of researches in recent years, it was accepted that the preparation of gold nanoparticles was relatively mature, the reagents used in the reaction process were non-toxic and safe, the required equipment was generally available and the experimental techniques were easy to master. Because of the physical characteristics of a small particle size and large surface area, AuNPs can be fully in contact with the skin surface and promote their own transdermal absorption. Our results are consistent with those of previous studies; we further found that AuNPs do have a significant transdermal effect, which lays a foundation for subsequent studies.28, 29 Because of the different mechanisms of interaction between AuNPs and skin, the transdermal efficiency can vary.30-32 Therefore, it is necessary to have a clear understanding of the transdermal effect of a nanoagent to achieve deep drug 14



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penetration and absorption, and this understanding is of great significance to the development

of

transdermal

preparations.

Hence,

we

conducted

surface,

charge-related AuNP treatments and found that the negatively charged NPs were more efficient for transdermal drug delivery than the positively charged or uncharged particles. And our results revealed that the bioconjugated gold nanoparticles also showed a negatively charged surface. However, the work is limited to electric charge, and in the future, we will study other factors, such as the shape, the size, or surface modification of transdermal peptides of NPs, to improve the AuNPs’ efficiency of transdermal delivery drugs. At the same time, as a form of heavy metal, the toxicity of AuNPs to the human body should be a concern;33-34 yet, our results showed that at concentrations less than 50 µM, AuNPs have low cytotoxicity. However, our cytotoxicity results are limited to cellular experiments. In clinical applications, transdermal delivery is a long-term process, and whether the long-term accumulation of metal particles leads to liver toxicity, renal toxicity or nerve damage also needs to be determined with long-term experiments using large numbers of animals.35-37 The results of our current study provide a viable basis for future studies. Due to the low cytotoxicity of AuNPs in transdermal applications, we can presume that the transdermal effect does not rely on cellular damage to the skin barrier but on actual biological activity. Topical angiogenesis is key element for skin repair in clinical therapy.38-40 However, because conventional drug application methods cannot effectively reach the damaged area, transdermal drug delivery has become a more promising method.41-43 15


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In this study, the absorption of VEGF conjugated AuNPs by mouse skin was evaluated. We confirmed that VEGF conjugated AuNP-PEG-COOH were transported into the dermis by detecting the presence of VEGF, which indicated the success of the AuNP-based delivery system. Additionally, evaluating the biological activity of percutaneous VEGF was necessary. Our α-SMA and H&E staining results revealed angiogenesis, indicating that the effect of VEGF was remarkable. Under the premise of not damaging the skin structure, nanocarrier-based transdermal drug delivery systems can enhance transdermal drug absorption efficiency and reduce the side effects of drugs, thereby improving the efficiency of drug use. Ideally, transdermal nanopreparations should (1) protect the drug from damage, (2) have good skin compatibility and cause no damage to the epidermis structure, (3) promote percutaneous penetration and achieve deep diffusion, and (4) be effectively

absorbed

and

degraded

by

the

circulatory

system.

Currently,

nanocarrier-based transdermal drug delivery systems are mostly limited to low doses or small molecule drugs. For the transdermal delivery of high doses or macromolecule drugs, existing nanocarriers need to be combined a high-dose surfactant or with a physical means of promoting drug absorption, such as microneedle ion introduction.44-46 The use of high-dose surfactants will undoubtedly affect the skin compatibility of nanopreparations. Nanoagent modification to render transdermal drug delivery more safe and effective will become an important research direction for improving transdermal delivery technology and delivery efficiency. In our study, the surface modification of negatively charged AuNPs lays a foundation for future 16



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research, in which, further NP modifications, such as changing the NP shape or connecting genes for inducing specific biological activities merit exploration. CONCLUSION This study focused on the transdermal effects of AuNPs with different surface charges. Negatively charged AuNPs showed a higher transdermal efficiency, and after conjugated with VEGF, the AuNPs still had a negative charge. Thus, VEGF conjugated AuNPs could penetrate the skin barrier and allow VEGF exert its biological activity subcutaneously. The binding of protein biological factors to AuNPs can preserve the activity of the protein. This transdermal drug delivery method is non-invasive, efficient, simple and free from environmental restrictions. And it is advantageous for cases requiring non-invasive drug delivery as well as, in the cosmetology field, in which a variety of growth factors are expected to directly penetrate the skin barrier, reach the subcutaneous region and repair the skin.

AUTHOR INFORMATION Corresponding Author *

Jianwu Dai, Phone/fax: 86-010-82614426; E-mail: [email protected]

*

Bing Chen, Phone/fax: 86-010-82614420; E-mail: [email protected]

Author Contributions ‖

Ying Chen, Yonghui Wu, Jining Gao contributed equally to this work.

Notes The authors declare there is no competing financial interest. ACKNOWLEDGMENTS 17


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This work was supported by the National Key Research and Development Plan of China (2016YFC1000801, 2016YFC1000802, 2016YFC1000805) and Chongqing Natural Science Foundation (cstc2013jcyjys10002, cstc2015jcyjys10001).

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Figure 1．(A) TEM image of the citrate-capped gold nanoparticles at a scale of 100 nm (insert image of HRTEM image of AuNP, scale bar is 10 nm.); (B) DLS diagram of the citrate-AuNP and the PEGylated AuNPs; (C) Zeta potential of the PEGylated AuNPs.

Figure 2. TEM images of the skin after transdermal treatment with gold nanoparticles for 24 h. (A) AuNP; (B) AuNP-PEG-OCH3; (C) AuNP-PEG-COOH; (D) AuNP-PEG-NH2. The yellow arrows indicated gold nanoparticles in each group those were located deep in the skin (stratum corneum and epidermis). All image scales were 200 nm. (CF: Collagen Fibers, M: Mitochondrion).

Figure 3．Gold nanoparticles appear as black spots with silver-enhanced staining. Black silver-stained particles accumulated in the stratum corneum, epidermis and dermis

in

figures

(A)

control;

(B)

AuNP;

(C)

AuNP-PEG-OCH3;

(D)

AuNP-PEG-COOH; (E) AuNP-PEG-NH2. (F) The statistical count of the density of silver-stained particles (× 400) at high magnification in the experimental group. Data are expressed as the mean ± SE of at least 6-10 animals per group. * P < 0.05 by ANOVA with Bonferroni’s posttest.

Figure 4. Cytotoxicity study of AuNP-PEG-COOH by MTT assay.

22



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Figure 5. (A) Schematic illustration of synthesis process of AuNPs-COOH with VEGF. (B) Zeta potential after AuNP-PEG-COOH conjugated with VEGF (AuNP-CONH-VEGF).

Figure 6. Immunohistochemical staining of AuNP-CONH-VEGF detecting with His antibody 24 h after transdermal treatment with blank control, VEGF-Applied, AuNP-CONH-VEGF and AuNP-CONH-OVA. Bars=20 µm. The black arrows indicated AuNP-CONH-VEGF.

Figure 7．(A) Immunohistochemical staining was performed to determine α-SMA+ vessels in the skin 7 days after transdermal treatment with blank control, VEGF-Applied, AuNP-CONH-VEGF and AuNP-CONH-OVA. Bars=50 µm. (B) Images were analysed for quantifying α-SMA expression. Data are expressed as the mean ± SE. of at least 6-10 animals per group. * P < 0.05 by ANOVA with Bonferroni’s posttest.

Figure 8. (A) H&E stains were performed in skin cross sections 7 days after transdermal treatment with blank control, VEGF-Applied, AuNP-CONH-VEGF and AuNP-CONH-OVA. Scale bars were 50 µm (top panels) and 20 µm (bottom panels). (B) Images were analysed for quantifying vessels. Data are expressed as the mean ± SE. of at least 6-10 animals per group. * P < 0.05 by ANOVA with Bonferroni’s posttest. 23


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Table of content Graphic 215x224mm (300 x 300 DPI)


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Figure 1．(A) TEM image of the citrate-capped gold nanoparticles at a scale of 100 nm (insert image of HRTEM image of AuNP, scale bar is 10 nm.); (B) DLS diagram of the citrate-AuNP and the PEGylated AuNPs; (C) Zeta potential of the PEGylated AuNPs. 39x27mm (300 x 300 DPI)



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Figure 2. TEM images of the skin after transdermal treatment with gold nanoparticles for 24 h. (A) AuNP; (B) AuNP-PEG-OCH3; (C) AuNP-PEG-COOH; (D) AuNP-PEG-NH2. The yellow arrows indicated gold nanoparticles in each group those were located deep in the skin (stratum corneum and epidermis). All image scales were 200 nm. (CF: Collagen Fibers, M: Mitochondrion). 39x27mm (300 x 300 DPI)


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Figure 3．Gold nanoparticles appear as black spots with silver-enhanced staining. Black silver-stained particles accumulated in the stratum corneum, epidermis and dermis in figures (A) control; (B) AuNP; (C) AuNP-PEG-OCH3; (D) AuNP-PEG-COOH; (E) AuNP-PEG-NH2. (F) The statistical count of the density of silverstained particles (× 400) at high magnification in the experimental group. Data are expressed as the mean ± SE of at least 6-10 animals per group. * P < 0.05 by ANOVA with Bonferroni’s posttest. 39x27mm (300 x 300 DPI)



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Figure 4. Cytotoxicity study of AuNP-PEG-COOH by MTT assay. 39x27mm (300 x 300 DPI)


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Figure 5. (A) Schematic illustration of synthesis process of AuNPs-COOH with VEGF. (B) Zeta potential after AuNP-PEG-COOH conjugated with VEGF (AuNP-CONH-VEGF). 39x27mm (300 x 300 DPI)



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Figure 6. Immunohistochemical staining of AuNP-CONH-VEGF detecting with His antibody 24 h after transdermal treatment with blank control, VEGF-Applied, AuNP-CONH-VEGF and AuNP-CONH-OVA. Bars=20 µm. The black arrows indicated AuNP-CONH-VEGF. 39x27mm (300 x 300 DPI)


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Figure 7．(A) Immunohistochemical staining was performed to determine α-SMA+ vessels in the skin 7 days after transdermal treatment with blank control, VEGF-Applied, AuNP-CONH-VEGF and AuNP-CONH-OVA. Bars=50 µm. (B) Images were analysed for quantifying α-SMA expression. Data are expressed as the mean ± SE. of at least 6-10 animals per group. * P < 0.05 by ANOVA with Bonferroni’s posttest. 39x27mm (300 x 300 DPI)



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Figure 8. (A) H&E stains were performed in skin cross sections 7 days after transdermal treatment with blank control, VEGF-Applied, AuNP-CONH-VEGF and AuNP-CONH-OVA. Scale bars were 50 µm (top panels) and 20 µm (bottom panels). (B) Images were analysed for quantifying vessels. Data are expressed as the mean ± SE. of at least 6-10 animals per group. * P < 0.05 by ANOVA with Bonferroni’s posttest. 39x27mm (300 x 300 DPI)


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Transdermal Vascular Endothelial Growth Factor ... - ACS Publications

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