Transdermal Gene Delivery by Functional Peptide-Conjugated

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Transdermal Gene Delivery by Functional Peptide Conjugated Cationic Gold Nanoparticle Reverses the Progression and Metastasis of Cutaneous Melanoma Jie Niu, Yang Chu, Yan-Fen Huang, Yee-Song Chong, ZhiHong Jiang, Zheng-Wei Mao, Li-Hua Peng, and Jianqing Gao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b16378 • Publication Date (Web): 02 Mar 2017 Downloaded from http://pubs.acs.org on March 3, 2017

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ACS Applied Materials & Interfaces

Transdermal Gene Delivery by Functional Peptide Conjugated Cationic Gold Nanoparticle Reverses the Progression and Metastasis of Cutaneous Melanoma Jie Niua, Yang Chua, Yan-Fen Huanga, Yee-Song Chonga, Zhi-Hong Jiangb, Zheng-Wei Maoc, Li-Hua Penga,b*, Jian-Qing Gaoa*

a

Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou

310058, PR China b

State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and

Technology, Macau, PR China c

MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer

Science and Engineering, Zhejiang University, Hangzhou 310027, PR China.

*Correspondence authors Li-Hua Peng, Ph.D, Associate Professor. College of Pharmaceutical Sciences, Zhejiang University, 866# Yuhangtang Road, Hangzhou, 310058, P.R. China. Email: [email protected] Tel:+86-571-88208437

Jian-Qing Gao, Ph.D, Professor. College of Pharmaceutical Sciences, Zhejiang University, 866# Yuhangtang Road, Hangzhou, 310058, P.R. China. Email: [email protected]

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ABSTRACT: Permeability barrier imposed by stratum corneum makes an extreme challenge for the topical delivery of plasmid DNA (pDNA), which is widely used in gene therapy. Existing techniques to overcome the skin barrier for biomacromolecules delivery rely on sophisticated mechanical devices. It is still a big challenge to treat the skin cancer, for example, melanoma that initiates in the dermal layer by topical gene therapy. To facilitate the skin penetration of pDNA deeply into the melanoma tissues, we here present a cell penetrating peptide and cationic polymer (PEI) conjugated gold nanoparticle (AuPT) that can compact the pDNAs into cationic nanocomplexes and penetrate through the intact stratum corneum without any additional enhancement used. Moreover, the AuPT is highly efficient in stimulating the intracellular uptake and nuclear targeting of the pDNAs in cells, which guarantees the effective transfection. This study provides evidences for that penetrating peptide conjugated cationic gold nanoparticle offers a promising vehicle for both the skin penetration and transfection of pDNAs, possessing great potential in topical gene therapy.

KEYWORDS: stratum corneum penetration, plasmid DNA, cationic gold nanoparticle, cell penetrating peptide, melanoma

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1. INTRODUCTION Existing techniques to overcome the skin barrier for topical delivery of biomacromolecules, like plasmid DNA (pDNA) and protein, rely on sophisticated mechanical devices,1 such as the ultrasonic apparatus,2 iontophoresis,3 microneedles and electroporation.4 Recently, the utilization of nanoparticles in biomedicine holds great potential for topical drug delivery.5,6 However, most reported topical strategies with nanoparticles still require the combination of photo inducement, thermal ablation, or magnetism to enhance the skin penetration efficiency7,8 so as to induce significant therapeutic effects.9,10 Biomacromolecules are usually hydrophilic with large size and will be mostly blocked by skin. Zheng et al have recently reported the topical delivery of siRNA as effective treatment for skin melisma,11,12 which reminded the potential of topical gene therapy for cutaneous melanoma. As we known, pDNAs are frequently utilized in gene therapy because their higher stability than those of SiRNA, MicroRNA and DNA.13,14 However, until now, topical delivery of pDNAs for the cutaneous melanoma treatment has never been reported elsewhere. Contributing to their small size, general non-toxicity, ease of functionalization and high surface to volume ratio, gold nanoparticles caused increasing attention from non-viral gene delivery and therapy. Recently, Conde et al. investigated gold nanoparticles based triple-combination therapy, consisting of therapeutic gene, drugs and photo based treatments. The triple-combination therapy was demonstrated to efficiently inhibit tumor regression and reversed disease-specific traits to prompt selective and personalized therapies for colon cancer.15 In another study, unimer polyion complex (uPIC) was conjugated on the gold nanoparticles surface. The intravenous injection of uPIC-AuNPs carrying siRNA significantly enhanced the accumulation and penetration of siRNA into solid tumor with longer blood circulation.16 Researchers also invented the first example of reversible ligation of DNA on gold nanoparticles. This conjugation can protect the DNA from degradation and the DNAs can be reversibly released by using light as an external stimulus, which could potentially be widely utilized in drug delivery, catalysis, sensing, and photonics.17 All these evidences reminded the great application potential of gold nanoparticles in gene therapy. Cutaneous melanoma is one of the most deadly cancers in clinic that is still lacking effective therapies.18,19 Advanced treatment options include chemotherapies, anti-programmed death-1and targeted therapies like BRAF inhibitors.20-22 These treatments have provided new options to treat this deadly tumor but also cause serious side effects and easy to be drug-resistant. Gene therapy has emerged as a promising technique for numerous tumor types. In gene therapy, miRNA possesses 3



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the ability to regulate more than one gene, which is different from siRNA and may change multiple tumor-associated molecules simultaneously. Much more evidence has suggested that melanoma is characterized by distinct molecular mutations, which provide unique opportunities for targeted therapy. Recently, microRNA-221 (miRNA-221) was identified to be abnormally expressed in malignant melanoma cells, and it favors the induction of the malignant phenotype through down-modulation of c-Kit receptor and blocking p27 translation.23 In the progression of melanoma, up to 70% of metastases lack the c-Kit receptor and can consequently escape c-Kit-triggered apoptosis.24 Similarly, p27 expression is lost during progression from benign nevi to metastatic cells and its reduction causes the poor survival.25 Because microRNA based short hairpin still suffers from limitations such as stability. DNA-based RNAi drugs on the other hand have the potential of being stably introduced when used in plasmid DNA.26 Antisense sequence of miRNA-221 was constructed and inserted into the plasmid to reduce the expression of miRNA-221, based on the transfection of pDNA into B16F10 cells. It was demonstrated that repression of p27 is a consequence of direct binding of miRNA-221 sites in the 3'UTR.27 It was shown that not only miR-222 but also miRNA-221 was able to reduce viability and induce apoptosis mediated by the KIT, AKT and BCL2 signaling cascade.28 MiRNA-221 has been proposed as a potential tumor suppressor for melanoma therapy. Therefore, the inhibition of miRNA-221 expression that up-regulates the c-Kit receptor and p27 protein is thought to be a novel treatment for advanced melanoma with clinical translation.29,30 Moreover, topical delivery miRNA-221 inhibitor gene can avoid or decrease reticuloendothelial system (RES) uptake, reduce systemic toxicity, and provide targeted gene delivery to the tumor site located at the skin subcutaneous layer. However, stratum corneum always poses a formidable challenge to biomacromolecules penetration. PDNAs, because of their large size, hydrophilic nature, and fast degradation, are normally precluded from percutaneous absorption. To circumvent these problems that confront the current methods, herein, for the first time, we present a novel strategy for the cutaneous melanoma therapy by topical delivery a pDNA encoded with miRNA-221 inhibitor gene through HIV-1 twinarginine translocation peptide (TAT) conjugated catonic gold nanoparticles (AuPT). In this strategy, AuPT acts as not only the topical carrier but also the gene vector for pDNAs. TAT as a vehicle for drug delivery has been thoroughly investigated by many studies.31 It was shown to be non-immunogenic, and its use in cells and animals did not elicit toxic responses.32 By virtue of their strong electrostatic interaction with 4


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anionic membrane surfaces and their sometimes amphipathic nature, TAT peptides offers ‘interfacial activity’ defined as the ability to bind at the bilayer–water interface and perturb membrane structure.33 It can cooperatively disrupt the vertical segregation of hydrophobic and hydrophilic groups in a bilayer and allows the passage of polar molecules across the membrane.34,35 TAT was so suggested as a skin-permeable protein, and has been demonstrated to transport the attached proteins into the skin for strong transcutaneous immunization.36 On the other hand, by transient plasma membrane disruption37 or spontaneous translocation,38 TAT can help the biomacromolecules bypass the endosomal degradative environment.39-41 In our previous study, AuPT was shown to be highly efficient in transfecting pDNAs in both epidermal stem cells and mesenchymal stem cells for their in vitro directed differentiation.42,43

In the present study, using

AuPT as a multifunctional vehicle, the penetration of pDNAs through the different skin layers, and the transfection of these pDNAs in the melanoma cells at the subcutaneous site were investigated. Briefly, both the penetration and distribution of the pDNAs and gold nanoparticles in the different skin layers after the topical application of AuPT/pDNAs were studied in vitro & in vivo. Transfection of the pDNAs encoding miRNA-221 inhibitor gene (Mi221) by AuPT in melanoma cells and melanoma xenograft in mice, as well as its regulation on c-Kit and p27 genes expression of cells and tumor tissues were evaluated. Based on that, the therapeutic effects of AuPT/Mi221 through topical application were evaluated by investigating the tumor cell apoptosis, metastasis and interference in cell cycles. The histology of the tumor tissues was also analyzed by HE, Tunel and EdU staining (see Figure 1A-1D as a schematic design).

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Figure 1. Schematic illustration of the transdermal delivery of pDNAs encoding microRNA-221 inhibitor gene (Mi221) by AuPT nanoparticles for skin cutaneous melanoma treatment. The therapy consists of four major steps, including (A) Preparation of AuPT/Mi221 nanocomplexes; (B) Topical application of AuPT/Mi221 and the skin penetration of AuPT/Mi221; (C) Skin penetration into melanoma, and (D) Gene transfection of AuPT/Mi221 in melanoma cells for tumor therapy.

2. EXPERIMENTALS DETAILS 2.1. Materials. Chloroauric acid (HAuCl4), Sodium borohydride (NaBH4), polyethelyimine (25kD),

amiloride-HCl,

chlorpromazine

(CPZ),

methyl-β-cyclodextrin

(MBC),

4’,6-diamidino-2-phenylindole (DAPI) and Methylthiazoletetrazolium (MTT) were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA). Micro BCA protein assay kit was purchased from Beyotime Biotechnology Inc., China. PDNA encoding luciferase (PGL3) was obtained from Institute of Infectious Diseases, Zhejiang University, China. PDNAs encoding GFP and miRNA-221 inhibitor genes were purchased from Genepharma Company. TAT peptide 6


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(H-Cys-Cys-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-OH, Mw = 1559), FITC-DNA, Cy5.5-pDNA were constructed by Sangon Biotechnology Inc., China. Four-week-old nude mice were supplied by Shanghai SLAC Laboratory Animal Co. Ltd, China. B16F10 cell line was purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (China). Dulbecco's modified Eagle's medium (DMEM), 0.25 wt.% trypsin with 0.02 wt.% ethylene diamine tetraacetic acid, fetal bovine serum (FBS), streptomycin and penicillin were obtained from Gibco BRL (USA). 2.2. Synthesis of AuPT.

HAuCl4 (150 µg/mL) was reduced by NaBH4 (10 mg/mL) in the

presence of PEI (3 mg/mL). The reaction solution was stirred vigorously for 15 minutes at room temperature and stored for at least 1 h to get the AuP suspension. Then AuPT was prepared by adding TAT into the AuP solution under continuous stirring overnight. TAT peptides were mixed with AuP (100 µg/mL) for ligand exchange, with a variable peptide concentration of 0-100 µg/mL. Excess PEI and TAT were removed by dialysis. 2.3. Characterization of AuPT and AuPT/pDNAs Nanocomplexes. To quantify the PEI amount on the AuP nanoparticles, thermogravimetric analysis was carried out for powder samples using a TGA/SDTA851, SWRTZER LAND thermogravimetric analyzer. Samples between 5-15 mg were heated from 30 ℃ to 400 ℃ at a heating rate of 10 ℃/min in air. TGA derivative curves show distinct transitions for different samples between 30 ℃ to 400 ℃. To quantify the TAT amounts on the AuPT nanoparticles, micro-bicinchoninic acid (BCA) assay has been used to detect the amount of TAT peptide on the surface of nanoparticles.44 Briefly, 20 µL of various AuPT aqueous samples containing numerous amounts of peptides were added into 96 well plates. Each well was filled with 200 µL micro-BCA working liquid followed by incubating them at 37 ℃ for 30 min. Absorbance was measured at 570 nm by microplate spectrophotometer. TAT concentration of the sample was determined according to the standard curve based on protein standard solution. The concentration of AuPT was measured by inductively coupled plasma mass spectrometry (ICP-MS) and adjusted to 100 µg/mL. Then different volume of nanoparticle solution was added into a persistent bulk of pDNAs solution (100 µg/mL) to prepare AuPT/pDNAs nanocomplexes. The mixture immediately vortexed for 15s and incubated for 30 min at 37℃. The morphologies of AuPT and AuPT/pDNA nanocomplexes (1:8, w/w) were observed by a JEM-1200EX transmission electron microscope (TEM). The polyplexes diameters were measured by dynamic light scattering (DLS) on a Brookhaven Particle Size Analyzer (90plus) at room temperature. An aqueous dip cell 7



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in the automatic mode (Zetasizer 3000, Malvern Instruments, Southborough, MA) was used to measure the zeta potential of nanoparticles. All measurements were carried out for three times. 2.4. Transfection assay. Reporter gene, pDNA-PGL3 (PGL3) was used for the transfection assay. 5×104 B16F10 cells were seeded on 24-well plate. After cells reached to 80% confluence, the cultural medium was removed and cells were washed with PBS for twice. Each well received 0.5 mL DMEM without FBS. Then different groups of AuPT/PGL3 containing 1 µg of PGL3 and various amounts of AuPT were added to cells. Cells and nanocomplexes were incubated for 6 h at 37 ℃ before the cultural medium was changed by DMEM containing FBS to remove the AuPT/PGL3 nanocomplexes. The cells were incubated for another 18 h and washed with PBS for twice. A luciferase assay kit (Beyotime, China) and a luminometer (Promega, USA) were used to measure the PGL3 luciferase intensity. BCA protein assay reagent kit was used to measure the total protein of each well. Final results were expressed as luciferase intensity of per mg total protein of the tested cells. 2.5. Cellular uptake. B16F10 cells were seeded on 24-well plate with DMEM containing 10% FBS. After cells were incubated overnight reaching 80% confluence, the medium was changed by DMEM without FBS. Then the vector/FITC-pDNAs nanocomplexes or naked FITC-pDNAs were added to each well. Cells were incubated with nanocomplexes for various time periods. Before measuring the cellular uptake of FITC-pDNAs, cells were washed twice with PBS and flow cytometry was used to determine the mean fluorescence intensity per cell. To directly observe the cellular uptake of nanocomplexes at different time points, cy5.5-labeled pDNAs were used and be tracked by CLSM. Briefly, 3×104 B16F10 cells were seeded on 15 mm plate and incubated overnight. Then the cultural solution was replaced by medium without FBS. Naked cy5.5-labeled pDNAs or their nanocomplexes with AuPT were added to the cells and were observed at 1, 3, 6, 9 h, respectively. After that, cells were washed twice and incubated with 4% paraformaldehyde and DAPI for 15 min. Finally, cells were washed with PBS for 3 times and observed by CLSM. 2.6. Intracellular Pathway of AuPT/pDNAs Nanocomplexes. For the illumination of the cellular uptake mechanism, cells were treated with 4 ℃ to clarify energy dependence. Pharmacological inhibitors like 50 µM amiloride-HCl, 10 µg/mL of Chlorpromazine (CPZ) and 10 mg/mL of Methyl-β-cyclodextrin (MBC) have also been utilized for the mechanism studies. Briefly, B16F10 cells were pre-treated with these inhibitors for 30 min at 37 ℃. Cells were then incubated with AuPT/GFP (pDNAs encoded with GFP) nanocomplexes for 6 h then the medium was replaced 8


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by DMEM with 10% FBS. After another culture of 18 h, mean fluorescence intensity per cell was accessed by flow cytometry. 2.7. Analysis of in Vitro & in Vivo Gene Expression. Total RNA was extracted from B16F10 cells or melanoma tissues using DxGeneTM Tissue and Cell Total RNA Extraction Kit (GenePharma, China). MiRNA-221 was quantified by Hairpin-itTM miRNAs qPCR quantitation kit (GenePharma, China). As for the analysis of c-Kit and P27 expression, the primers and glyceraldehyde phosphate dehydrogenase (GAPDH) were synthesized by Sangon Biotech (Shanghai, China). The expressions of miRNA-221, c-Kit and P27 were detected with the CFX-Touch PCR detection system (Bio-Rad Laboratories, CA, USA) complying with the manufacturer’s instructions. 2.8. Anti-proliferation and apoptosis assays. B16F10 cells were seeded on 96-well plate at a density of 1×104 cells/well and cultivated overnight. The medium was replaced by fresh Opti-MEM medium and 10 µL of complexes solution with different concentrations were added to each well cultivating for 6 h. Cells were incubated for 18 h further. Then the medium was replaced with DMEM containing 3-[4, 5-dimethyl-thiazolyl-2]-2, 5-diphenol tetrazolium bromide (MTT, Sigma) (0.5 mg/mL). After 4 h, the supernatant was removed, and 200 µL of dimethyl sulfoxide (DMSO, Sigma) was added to each well. Then the plate was micro-oscillated for 30 s and absorbance was measured at 570 nm. The cell viability was normalized to that of only solvent treated cells. 2.9. Cell cycles analysis. After B16F10 cells were transfected with Mi221 as mentioned above, cells were collected and fixed with 70% ethanol in 4℃ overnight. After washing them twice with PBS, cells were mixed with 100 μL of RNase in 37 ℃ for 30 min. Then 400 μL of Propidium (PI) were added in 4 ℃ in the dark and incubated with the cells for another 30 min. Results were determined by flow cytometry at the wavelength of 488 nm. 2.10. Cells Migration Assay. After different groups of transfection, the cells migration was tested by Transwell assay. B16F10 cells were digested and re-suspended in serum-free medium at a density of 3 × 105 cells/mL and 100 µL of the cells suspension was seeded in the upper chamber. Medium containing 10% FBS was added to the lower chamber. The chambers were incubated at 37°C for 72 h. After the incubation, the medium and cells remained in the upper chamber were removed. Finally, crystal violet (Beyotime, Shanghai, China) was used to stain cells left on the lower side of the membrane for 20 min. Cells were observed and counted by light microscope (Nikon, Japan). 9



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2.11. In vitro skin penetration test. The skins of nude mice (4-week old) were used for in vitro penetration test with Franz diffusion cells. Mice were anesthetized using 10 % chloral hydrate and then the dorsal skin hair of all mice was removed using hair removal cream. The skin was used once it was removed from mice. Skin was cut to a suitable size and mounted on the receptor compartment of the diffusion cell containing PBS buffer (pH 7.4). The receptor was maintained at 37 °C and constantly stirred with a magnetic bar. The amount of total FITC-pDNAs was 10 µg and the ratio between vectors to FITC-pDNAs was 1:8 (w/w). The concentration of vectors was adjusted to 100 µg/mL. Then different vectors solution was added into FITC-pDNAs solution (100 µg/mL), incubating for 30 min before adding into donor cells. PBS was added to the diffusion chamber without bubble. 0.5 mL of vector solution loaded with FITC-pDNAs was added to each donor cell. 100 µL of samples were withdrawn from diffusion chamber at different intervals (1, 3, 6, 9 and 24 h) and fresh PBS was replenished. The samples were extracted onto 96-Well Solid Black Microplate then fluorescence intensity was detected by fluorescence microplate reader. Other skin samples that treated with FITC-pDNAs were fixed to glass cover slips and observed by CLSM at 24h. 2.12. Penetration and Distribution of Vector/pDNAs Nanocomplexes in Xenograft Tumor Tissue. Transdermal and distribution of vector/pDNA in the tumor tissues were imaged with TEM. For the TEM observation, tumor samples were washed twice with PBS then fixed 1 h with 3.5% (v/v) glutaraldehyde. Post-fixation was performed for 1.5 h in 1% (v/v) osmium tetroxide at room temperature. The samples were dehydrated in graded series of ethanol and propylene oxide. Then the samples were embedded in Durcupan (Fluka, Sigma Aldrich). Thickness of section was about 60 µm. Sections were mounted on nickel grids and stained with uranyl acetate before examination under a JEM-1200EX microscope. 2.13. In vivo Transdermal Delivery of Mi221 and Anti-Tumor Effects. Nude mice (4-week old) were purchased from Shanghai SLAC Laboratory Animal Co. Ltd, China. All animal experimental procedures were performed in obedience to guidelines and protocols of the Animal Experimental Ethics Committee of Zhejiang University. 1×106 B16F10 cells were inoculated subcutaneously at right flank of nude mice to set up the melanoma model. The melanoma bearing nude mice were then randomly divided into six groups and each group included six nude mice. For the preparation of AuPT/Mi221, AuP/Mi221 and AuPT/shNC samples, vectors were purified by dialysis to remove excess ligand molecules. The concentrations of AuP and AuPT were adjusted to 10


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100 µg/mL and verified by inductively coupled plasma mass spectrometry (ICP-MS). 12.5 µL of vectors was added into pDNAs solution including 10 µg of pDNAs, vortexed for 15 s and incubated for 20 min prior to applying to the dosal skin of the mice, underwhich the melanoma xenograft was existed. Nude mice were given topical treatment with AuPT/Mi221, AuP/Mi221, AuPT, AuPT/shNC, naked Mi221 or PBS, respectively twice a day. The therapy dose was 100 µL containing 10 µg Mi221 or shNC. The body weights of nude mice were tested every day. All mice were well-tolerated with the tested treatments over the course of research with no noticeable body weight loss or any signs of poisonousness such as diarrhea or edema. 2.14. H&E, TUNEL and EdU staining. On the last day of treatment, tumors were collected and prepared into paraffin and frozen slices in 6 µm thick by standardized protocols. Afterwards, the paraffin slices were analyzed by H&E and TUNEL staining. The processed paraffin slices were imaged and analyzed using a light microscope. Frozen slices were fixed using 4% paraformaldehyde. Consistent with the manufacturer’s instructions (Beyotime, Shanghai, China), fluorimetric TUNEL (TdT-mediated dUTP Nick-End Labeling) staining was used to detect the presence of apoptotic cells. The brief procedure was that frozen sections were treated with 20 µg/mL of proteinase K. Then a nucleotide mixture of fluorescein-12-dUTP and terminal deoxynucleotidyl transferase (TdT) was added to the frozen sections incubating for 90 min. After staining cell nucleus by DAPI (1 µg/mL), fluorescence images of apoptotic cells (red) and cell nuclei (blue) were obtained by CLSM analysis. EdU (keyFluor488 Click-iT EdU Kit, keygen BioTECH, Nanjing, China) was used to label cells nuclei that have undergone S phase of DNA synthesis. Briefly, tumor sections were fixed with 4% paraformaldehyde for 15 min. Next, Click-iT reaction mixture containing CuSO4, keyFluor 488 azide and buffering was prepared according to the instructions of Click-iT EdU Kit. Then the prepared mixture was incubated with each tumor section for 30 min in darkness. Sections were finally washed with PBS twice and observed under CLSM. 2.15. Statistical Analysis. Data are expressed as mean ± standard deviation (SD). A statistically significant value was set as p < 0.05 based on the Student’s t test.

3. RESULTS AND DISCUSSION 3.1. Characterization of AuPT nanoparticles and AuPT/pDNAs polyplexes. PEI was proved to be densely conjugated on the surface of gold nanoparticles (Figure 2A), indicating around 150 11



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PEI molecules were covered on the gold surface (0.8 molecule/nm2). PEI conjugated to the nano-gold could stabilize the hydrophobic gold nanoparticles and kept the nano-gold colloid system stable. TAT peptides could replace PEI on the surface of gold nanoparticles under the reaction of ligand exchange as TAT peptide sequences contain thiol. During ligand exchange process, the capping ligands are displaced by thiol-TAT due to a stronger Au-S linkage and an energy gain associated with the intermolecular interaction. And this stronger Au-S linkage has been proved by many scientists in previous studies.45,46 As the result of ligand exchange with TAT peptide, the amount of TAT on AuPT increased from 0% to 6.7% with the TAT feeding concentration increased (Figure 2B). No significant change was observed for the diameters and zeta potentials of the AuP nanoparticles after the TAT anchoring on them (Figure 2C, Figure 2D). So, AuP nanoparticles with different amounts of TAT conjugation were further tested with transfection efficiency in B16F10 cells to determine the best TAT feeding concentration. It was found that highest fluorescence intensity derived from GFP expression appeared when TAT concentration reached 25 µg/mL (Figure 2E). The results demonstrated that certain amount of TAT could help improve the transfection efficiency while too much TAT may exchange more PEI molecules and reduce the ability of AuPT in gene delivery. As Falk Duchardt et al. had investigated the influence of different kinds of cell penetrating peptides (concentration) including TAT in cellular uptake, it was found that when cells were incubated with the TAT at concentrations of 1-5 µM (equal to 1.5-7.5 µg/mL), the peptide was kept being located only in vesicles which could not enhance the transfection efficiency significantly. However, above a concentration threshold of 2~40 µM, which was nearly equivalent to 3~60 µg/mL, this peptide could internalize predominantly through a process that leads to a rapid distribution of peptides into the cytoplasm and nucleus.47 On the other hand, according to our previous study,42 too much TAT will inhibit the transfection efficiency. Therefore, this concentration ranges of 0-100 µg/mL have been selected for investigation in the present study. On the other hand, according to Figure 2F, diameters of AuPT/pDNAs nanocomplexes decreased with the increase of AuPT. The smallest diameter of AuPT/pDNAs polyplex was 199 ± 7.76 nm, and PDI was 0.27 ± 0.02 when the weight ratio of AuPT to pDNA was 1:8.

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Figure 2. (A) Weight loss of AuPT nanoparticle at 400 ℃ with different TAT feeding concentrations. (B) TAT contents on the AuPT nanoparticles. (C) Diameters and polydispersity indices of the AuPT nanoparticles, (D) Zeta potentials of AuPT nanoparticles. (E) Mean fluorescence intensity per cell (derived from the expressed GFP in transfected cells) of B16F10 cells after transfection at different TAT concentrations, **p< 0.01. (F) Diameters of AuPT/pDNA nanocomplexes at different weight ratios of vector to pDNA. 3.2. In vitro transfection of B16F10 with reporter pDNAs. B16F10 is known as a hard-to-transfect murine tumor cell line.48,49 However, from Figure 3A, transfection efficiency of AuPT in B16F10 reached highest of 1.71×107 RLU/mg total protein when the ratio of vector to pDNA is 1:8 (w/w). The zeta potential of the nanocomplexes with this ration was 16.81±0.56 mV. Along with the priority of nanoparticles with small size in transdermal delivery, this ratio was further investigated in the following study. This transfection efficiency is significantly higher than 13



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those of AuP, PEI and Lipofectamine 2000 (Lipo 2k) with 1.3 folds (p

Transdermal Gene Delivery by Functional Peptide-Conjugated

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