Peptide-Chaperone-Directed Transdermal Protein Delivery Requires

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Peptide-Chaperone-Directed Transdermal Protein Delivery Requires Energy Renquan Ruan,†,‡ Peipei Jin,§ Li Zhang,§ Changli Wang,§ Chuanjun Chen,∥ Weiping Ding,*,†,‡ and Longping Wen*,§ †

Center for Biomedical Engineering, ‡Department of Electronic Science and Technology, and §Hefei National Laboratory for Physical Sciences at Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China ∥ Department of Oral and Maxillofacial Surgery, The Third Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230027, China

ABSTRACT: The biologically inspired transdermal enhanced peptide TD1 has been discovered to specifically facilitate transdermal delivery of biological macromolecules. However, the biological behavior of TD1 has not been fully defined. In this study, we find that energy is required for the TD1-mediated transdermal protein delivery through rat and human skins. Our results show that the permeation activity of TD1-hEGF, a fusion protein composed of human epidermal growth factor (hEGF) and the TD1 sequence connected with a glycine-serine linker (GGGGS), can be inhibited by the energy inhibitor, rotenone or oligomycin. In addition, adenosine triphosphate (ATP), the essential energetic molecule in organic systems, can effectively facilitate the TD1 directed permeation of the protein-based drug into the skin in a dose-dependent fashion. Our results here demonstrate a novel energy-dependent permeation process during the TD1-mediated transdermal protein delivery that could be valuable for the future development of promising new transdermal drugs. KEYWORDS: transdermal drug delivery, peptide chaperon, transdermal peptide, ATP, energy

1. INTRODUCTION Transdermal drug delivery system is a route of administration for local and systemic distribution wherein active ingredients are delivered across the skin.1 Transdermal drug delivery has a variety of advantages compared with other types of drug delivery such as oral, intravenous, and intramuscular.2,3 It can avoid not only the significant first-pass effect through the liver where drugs can be prematurely metabolized, but also the frequent hypodermic injections that are painful and generate dangerous medical waste and disease transmission by needle reuse, especially in less developed countries.4 In addition, transdermal drug delivery system is noninvasive, can be easily self-administered and can maintain long periods of drug release. Recently, proteins and peptides are making up a significant and growing fraction of approved therapeutics.5 For injection, the effects of drugs are experienced very quickly; however, the repeated administration may be necessary because of the short half-life of peptides.1 For oral delivery, the gastrointestinal degradation of protein and peptide molecules makes it © 2014 American Chemical Society

infeasible. Thus, almost without exception, the approved proteins are administered by injection,6 underscoring the significant need for transdermal delivery of protein drugs. Although skin, the largest organ in the human body, provides a convenient and painless interface for systemic delivery,6 its basic functions limit its utility for this purpose because the skin functions are mainly to protect the body from external insults.7 The outermost layer of the epidermis, the stratum corneum, provides the extreme tough barrier to diffusion. It only permits the entry of small and lipophilic drugs and uniformly prevents the penetration of large hydrophilic molecules, rendering the transdermal delivery of protein drugs extremely challenging.8 Over the years, a variety of chemical and physical approaches has been developed to enhance skin permeability.9 Some Received: Revised: Accepted: Published: 4015

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the best of our knowledge, our work for the first time confirms that the peptide-chaperone-directed transdermal protein delivery requires energy. Our results presented here can not only indirectly explain the mechanism that there exist some specific interactions between TD1 and unknown skin components but also provide a new strategy to enhance and optimize the TD1-mediated transdermal delivery of protein drugs for clinical use.

chemical enhancers, such as azone (1-dodecylazacycloheptan-2one) and propylene glycol,10 can insert amphiphilic molecules into the highly ordered lipid bilayers to disorganize the bilayer structure in stratum corneum. In addition, liposomes, dendrimers, and microemulsions have also been used as chemical enhancers to form supramolecular structure that can increase not only skin permeability but also drug solubility in formulation and drug partitioning into the skin.11 Even so, one challenge of these approaches is how to reduce skin irritation. Some enhancers without irritation have been successfully used to deliver small molecules;12 however, the impact of these enhancers on delivering hydrophilic compounds or macromolecules is limited. Many physical or mechanical approaches are also designed to enhance drug delivery through the skin, such as iontophoresis,13,14 electroporation,15,16 cavitational ultrasound,16,17 and more recently, microneedles,18,19 thermal ablation,20 and microdermabrasion.21 These approaches are successful because they target their effects to the stratum corneum. However, most of these techniques function primarily by altering or breaching the structure of the stratum corneum aggressively using shear force.22 Biologically inspired transdermal enhancers, such as cell-penetrating peptides,23,24 SPACE peptide,25 and antimicrobial peptide magainins,26 have emerged in recent years and represent an alternative approach for transdermal drug delivery.27 Cell-penetrating peptide, which is covalently attached to cyclosporine, increases topical absorption of cylosporine that inhibits cutaneous inflammation.28 SPACE peptide coadministered with small interfering RNA (siRNA) can increase skin permeation for in vivo therapy.25 In our previous work, we have performed a novel highthroughput screen based on phage display and successfully identified an 11-amino acid peptide, TD1 (ACSSSPSKHCG), that ferries insulin across intact rat skin upon coadministration.29 Subsequent work conducted in other groups also demonstrates the ability of TD1 to enhance the transdermal delivery of macromolecules. For example, the TD1 peptide can effectively enhance the delivery of siRNA across the rat skin,30 the delivery of growth hormone across the porcine abdominal skin,31 and the delivery of botulinum neurotoxin type A (BoNT-A) across the rat skin.32 In the literature, the preliminary studies indicate that TD1 exhibits high sequence specificity29,33 and no physiological response in vivo,32 shows a saturating dose−response curve, opens the skin barrier transiently, and brings protein molecules deep into hair follicles to effect its transdermal enhancing activity.29 In addition, egg phosphatidylcholine is able to form a noncovalent complex with TD1, which implies an interaction between TD1 and the negatively charged cell lipids.31 Although the discovery of the enhancer TD1 is exciting, its mechanism to promote the transdermal protein delivery is still unclear. Some evidence suggested that TD1 overcomes the skin barrier by a distinct mechanism that most likely involves specific interactions between TD1 and unknown skin components, rather than modification of electrostatic interactions by changing pH34 or only cationic group mediated delivery across biomembrane by loosening the tight junction in epithelial cell.33,35,36 In this work, our objective is to investigate the energy dependence of the TD1-mediated transdermal protein delivery. Our hypothesis is that the mediation process of TD1 is an active, energy consumptive transport rather than only a passive diffusion without energy in most routes of administration. To

2. MATERIALS AND METHODS 2.1. Ethics Statement. All samples were collected in accordance with the ethical guidelines mandated by the University of Science and Technology of China (USTC) as approved by the Medical Ethics Committee. The Declaration of Helsinki Principles was followed. Fresh human skins were obtained from plastic surgery patients in the Third Affiliated Hospital of Anhui Medical University, China. Sprague−Dawley (SD) rats were obtained from Slaccas Company, Shanghai, China. All individuals were over 18 years of age, and all studied participants who agreed to participate in this study were informed in detail in writing and required to sign consent forms. All participants provided written consent. All rats were bred and housed in specific pathogen-free conditions ranging in weight from 200 to 300 g at the Association of Laboratory Care, an approved animal facility at the school of Life Science. Each rat was sedated with a dose of 1 mL of 20% urethane solution before experiments. All animal procedures were approved by the Medical Ethics Committee. 2.2. Materials. The energy inhibitors rotenone and oligomycin were purchased from Sigma (R8875, Sigma, USA). They were dissolved in methanol with 1.0 mol/L, and continually diluted 1:100 with HEPES buffer (20 mM HEPES and 150 mM NaCl, pH 7.5). ATP, ADP, AMP, and GTP were purchased from A. Johnson Matthey (Alfa Aesar, USA). These energy materials were dissolved with HEPES buffer and adjusted to pH 7.5. The human EGF antibody was purchased from Wuhan Boster (EK0325, Boster, China). Luminescent ATP Detection Assay Kit was purchased from Beyotime Company, China. 2.3. TD1-hEGF Purification. TD1-hEGF, a fusion protein composed of human epidermal growth factor (hEGF) and the TD1 sequence connected with a glycine-serine linker (GGGGS), was purified (hEGF is a representative protein drug as a cargo across skin). The construction of the plasmid for expressing TD1-hEGF protein using the GST system has been described in detail.37 The E. coli strain harboring the expression plasmid was cultured at 37 °C overnight in LB medium containing 100 μg/mL of ampicillin. The cultures were diluted 1:100 in TB medium containing 0.05% glucose and 1 mM IPTG, and then incubated at 16 °C for 24 h with rigorous shaking. After harvesting and resuspending the cell pellet in PBS buffer, the soluble protein was extracted from the bacterial cells using high-pressure crashing. The cell lysate (10 mL) was incubated with 1 mL of equilibrated GST resin (GE, USA) at 4 °C overnight. The resin was washed with 5 times the incubation volume with PBS buffer to reduce nonspecific binding, followed by treatment with 15 μL of TEV protease (Promega; diluted in 1 mL PBS buffer) for 2 h. After centrifugation at 5000 × g for 10 min, the cleaved recombinant protein was further purified by gel filtration chromatography (Sephadex G75, GE Health, US) on AKTA purifier machine. The G75 column (Φ16 × 700 mm) was washed with PBS (pH7.5) at a rate of 1 mL/min for 1 h. Then, the crude protein 4016

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Figure 1. Structure prediction of TD1-hEGF. The SWISS-MODEL was used to automatically analyze the TD1-hEGF structure (http://swissmodel. expasy.org). (A,B) The 3D structure of TD1-hEGF (TD1 and hEGF motif do not interfere with each other). (C) Purity of TD1-hEGF was analyzed by HPLC. (D) ATP stability in HEPES buffer with pH 7.5 during experiments (sampling times: 1, 2, 4, 12, and 24 h).

compartments. The receptor compartments were filled with normal saline maintained at 37 °C (low temperature experiments were at 4 °C), and each cell was equipped with a stirring magnet. Five hundred microliters of the freshly prepared drug formulations containing the indicated protein and various concentrations of ATP was added to each donor compartment. Two hundred microliter solutions from the receptor compartment were withdrawn at various times, and the same volume of normal saline was replaced. The permeated protein in the receptor wells was determined using enzyme linked immunosorbent assay (ELISA). 2.5. Permeated Protein Determination. One hundred microliters of permeated solution was added to wells of ELISA plate coated with the hEGF antibody and incubated at 37 °C for 2 h. Then, the plate was washed with 200 μL of PBS buffer for three times. The sample was incubated with hEGF antibody diluted (1:200 in PBS) at 37 °C for 1 h. After washing three times, HRP-conjugated antibody was added to each well of the plate at 37 °C for 30 min. Finally, 100 μL of TMB reagent was treated to generate sufficient color development. The reaction with was stopped with stop solution (2 M H2SO4) and the absorbance of each well were read using 450 nm. 2.6. Skin Deactivation. The skin of SD rats was obtained according to the method mentioned above. Four percent paraformaldehyde prepared in PBS was used to fix the isolated skin at 4 °C for 12 h, and then the deactivated skin was washed with PBS 3 times (30 min each time) to remove the paraformaldehyde before use. 2.7. Stability Test of TD1-hEDF. The isolated skin (100 ± 5 mg) was incubated with 500 μL of TD1-hEGF solution in the 24-well plate for 36 h at room temperature. The supernatant was collected at 0, 8, 16, and 36 h and then centrifuged with 5000 × g for 10 min. The concentration of TD1-hEGF in the

was loaded (1 mL/min). The retention time of the protein of interest was approximately 30 min. Finally, the eluent was collected according to the ultraviolet absorption (280 nm). The protein concentration was determined with a BCA assay (Beyotime, China). The purity of the protein was verified using high performance liquid chromatography (Agilent 1200LC, Santa Clara, DE, USA) with a stainless steel column (TSK-GEL C18 Φ4.6 × 150 mm). The parameters were set as follows: the flow rate was 1 mL/min; the ultraviolet wavelength was 280 nm; and the sample loaded was 20 μL. The chromatogram was developed by running a linear gradient from 10% acetonitrile/ 90% H2O/0.1% trifluoroacetic acid to 60% acetonitrile/40% H2O/0.1% trifluoroacetic acid within 20 min at room temperature. 2.4. Skin Permeation Studies. The skin used in the transdermal permeation studies was obtained from the abdomen of SD rats (200 ± 10 g) or the fresh outer skin of a male plastic surgery patient. Each rat was sedated with a dose of 1 mL of 20% urethane solution. The hair on the abdomen of these rats was removed with a razor, and the exposed skin was excised from the body. Then, the subcutaneous fat of the isolated rat skin or human skin was removed. The obtained skin was immersed in normal saline at room temperature and carefully inspected visually for any defects before the skin permeation experiments were performed. The transdermal delivery system was the same as the one in our previous work.37 Franz diffusion cells (PermeGear, USA) with 5 mL receptor compartments and 0.5 cm2 diffusion areas were used during the permeation studies. The isolated rat or human skin was carefully mounted on the lower half of the Franz diffusion cell with the epidermis facing upward. The upper and lower parts of the cell were fastened together using a clamp, with the epidermis acting as a seal between the donor and receptor 4017

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does not change (Figure 2B). Therefore, TD1-hEGF is stable in our skin permeation tests. 3.3. Effects of Energy Inhibitors and Temperature on the TD1-Mediated Transdermal Delivery. We tested the effects of two energy inhibitors, rotenone and oligomycin, on the transdermal penetration of TD1-hEGF in vitro based on the assumption that these small molecules could permeate into the skin and inhibit the energy-mediated transport process.38,39 Rotenone interferes with the electron transport chain in mitochondria, and oligomycin inhibits ATP synthase by blocking its proton channel (F0 subunit). The two inhibitors were mixed with 100 μg of TD1-EGF in HEPES buffer during the experiment. After 4 h of permeation, rotenone and oligomycin at a concentration of 10 mM inhibit TD1-hEGF’s transdermal activity by 76% and 81%, respectively, in the rat skin experiments (Figure 3). Obviously, the permeation of

supernatant was determined by BCA assay, and the molecular weight of TD1-hEGF was detected by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). 2.8. Statistical Analysis. All the data were expressed as mean ± SEM and analyzed by the two-tailed unpaired student’s t tests. *P < 0.05. **P < 0.01.

3. RESULTS AND DISCUSSION 3.1. Protein Preparation and Structure Prediction. We chose the TD1-hEGF transdermal delivery system to test the above hypothesis. TD1-hEGF is a fusion protein composed of human epidermal growth factor (hEGF) and the TD1 sequence connected with a glycine-serine linker (GGGGS) (Figure 1A). Its structure was automatically predicted and analyzed using the SWISS-MODEL (Figure 1B). Our previous work shows that the TD1 fusion protein can enhance the transdermal delivery of hEGF by more than 10-fold compared to the delivery of hEGF without TD1, but the TD1 motif does not interfere with hEGF function, which is consistent with the results predicted here. In the meantime, TD1-hEGF accumulates in the skin compared to the control, as measured by the skin section.37 In this study, we expressed TD1-hEGF and hEGF as controls in E. coli and purified the proteins using the GST purification system to more than 95% purity as judged by high performance liquid chromatography (HPLC) (Figure 1C). All experiments were performed on Franz diffusion cells using intact abdomen skin from Sprague−Dawley rats and fresh human skin. 3.2. Stability of TD1-hEGF in the Skin. We tested the stability of TD1-hEGF in the rat skin in 36 h. Our results show that the concentration of TD1-hEGF almost remains unchanged in the solution after 8 h (Figure 2A, the decrease in the concentration of TD1-hEGF in the first 8 h may be due to the binding of TD1-hEGF to the skin). In addition, according to SDS-PAGE, the linker between TD1 and hEGF is also quite stable because the molecular weight of TD1-hEGF

Figure 3. Effect of energy inhibitors on the permeation of TD1-hEGF. The concentration of hEGF or TD1-hEGF was 0.2 mg/mL. The concentration of ATP was 20 mM. The solution volume was 500 μL. Purified hEGF or TD1-hEGF was assayed for in vitro permeation through rat skin at 37 °C with or without 10 mM of rotenone or oligomycin, and at the end of 4 h, the amount of protein that permeated the skin was determined with ELISA. Results are depicted as the mean ± SEM, n ≥ 5. **P < 0.01 compared to the TD1-hEGF group.

TD1-hEGF is dependent on energy consumption. Most drugs permeate into skin by diffusion, which is one of several naturally occurring transport phenomena with time-dependence and dose-dependence.40 However, our previous research shows that as the concentration of TD1-hEGF increases, a maximum amount in the transdermal penetration is reached, rather than exhibiting a continuous linear increase.37 Therefore, we think that the transport behavior of TD1-hEGF is not only diffusion. Here, it should be noted that the permeation of TD1hEGF, although significantly higher than hEGF, is still low. The reason is that the permeation of TD1-hEGF is mainly dependent on the drug concentration and the skin area when the volume of the drug solution and the administration time are kept constant. In this study, the donor concentration of TD1hEGF was low. For the given skin area, the low concentration of TD1-hEGF means not only the low donor concentration of hEGF but also the low donor concentration of TD1 that causes the incomplete opening of the skin barriers and thus the low skin permeability. In addition, the area of the used skin was also small. Therefore, the amount of permeated TD1-hEGF was low.

Figure 2. Stability of TD1-hEGF in the rat skin. (A) The concentrations of TD1-hEGF in the supernatant at 0, 8, 16, and 36 h. The solution volume was 500 μL. The control group only consisted of PBS and skin. Results are depicted as the mean ± SEM, n ≥ 5. (B) The molecular weight of TD1-hEGF in the supernatant detected by SDS-PAGE. 4018

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Temperature generally affects the activity of organisms. We further verified that lowering the temperature of transdermal delivery system from 37 to 4 °C could also result in a significant decrease in the transdermal penetration of TD1-hEGF, whereas it has little effect on the penetration of hEGF (Figure 4).

Figure 5. Effect of ATP on the permeation of hEGF or TD1-hEGF across rat skin. The concentration of ATP was 20 mM. The concentration of hEGF or TD1-hEGF was 0.1 mg/mL. The solution volume was 500 μL. The amount of permeated protein at various time points after administration was determined by ELISA. Results are depicted as the mean ± SEM, n ≥ 9. *P < 0.05 and **P < 0.01.

Figure 4. Effect of temperature on the permeation of TD1-hEGF. The hEGF or TD1-hEGF concentration was 0.2 mg/mL. The solution volume was 500 μL. Purified hEGF or TD1-hEGF was assessed for the permeation across the rat skin at 37 or 4 °C, and the amount of protein across the skin was determined at 4 h. The hEGF group was the control. Results are depicted as the mean ± SEM, n ≥ 3. *P < 0.05.

Diffusion is a temperature-dependent process: a decrease in temperature will cause a decrease in diffusion ability of drugs and thus a decrease in the drug permeation rate. However, in this study, the penetration decrease of TD1-hEGF resulting from the temperature-induced diffusion decrease was only about 11%, whereas the total decrease caused by the temperature decrease was almost 50%. Therefore, this result is consistent with the notion that the transdermal protein delivery mediated by the peptide requires energy. Here, the temperature-induced diffusion decrease was roughly estimated according to the expressions:41 De = fsD0 and D0= kBT/(6πηr), where De is the effective diffusion coefficient of drugs, fs is a function related to the skin structure; D0 is the free diffusion coefficient of drugs, T is the temperature, kB is the Boltzmann constant, η is the viscosity, and r is the molecular radius. 3.4. Effect of ATP on the TD1-Mediated Transdermal Delivery. To further confirm the energy dependence of the TD1-mediated transdermal delivery, we assessed the effect of direct ATP addition on the permeation of TD1-hEGF across rat skin. ATP is an unstable acidic molecule in unbuffered water or at extreme pH because it is hydrolyzed to ADP; however, it is quite stable in solutions between pH 6.8 and 7.4. To make sure of the stability of ATP in our drug system, we tested the concentration of ATP dissolved in HEPES buffer with pH 7.5 for 24 h. As shown in Figure 1D, ATP only degrades less than 5% in 24 h. Therefore, ATP is stable during the transdermal experiments. In the study on the effects of ATP on the permeation of TD1-hEGF, dramatically increased levels of TD1-hEGF were detected in the receptor well at both 4 and 16 h, with the addition of 20 mM ATP, whereas ATP did not significantly affect the skin permeation of hEGF (Figure 5). Meanwhile, the effect of ATP on enhancing the transdermal permeation of TD1-hEGF is dose-dependent, with significant effects observed at 10 mM and the maximum effect observed at 20 mM (Figure 6). However, the same amount of the ATP addition does not enhance the transdermal permeation of hEGF over the entire concentration range. Our results also indicate that the ATP-mediated transdermal mechanism of

Figure 6. Effect of ATP concentrations on the permeation of hEGF or TD1-hEGF across rat skin. The concentration of hEGF or TD1-hEGF was 0.1 mg/mL. The solution volume was 500 μL. TD1-hEGF or hEGF was assayed for permeation across rat skin in the presence of the indicated concentrations of ATP at 16 h, and the amount of permeated protein was determined by ELISA. n ≥ 3.

TD1-hEGF might be an enzymatic reaction, and ATP is the key energy supplier. The effect of ATP is specific, as 20 mM GTP, ADP, and AMP all fail to enhance the transdermal permeation of TD1-hEGF (50 μg) (Figure 7). Even if the energy inhibitor is used, the addition of ATP still can enhance the transdermal permeation of TD1-hEGF (Figure 3). In addition, we confirmed the ATP-dependent effect in human skin freshly isolated from adipose tissue. The permeation of TD1-hEGF across the intact human skin, although significantly higher than hEGF, is still quite low, but the addition of 20 mM ATP can generate an approximately 5-fold enhancement (Figure 8). 3.5. Effect of Skin Activity on the TD1-Mediated Transdermal Delivery. To assess the effect of skin activity, we studied the permeation of TD1-hEGF across two kinds of skin: the deactivated skin and the stale skin (Figure 9; the permeation across the deactivated skin is the control group for Figure 5). Here, the deactivated skin was prepared by fixing the fresh rat skin in 4% paraformaldehyde, and the stale skin was prepared by placing the fresh rat skin in a humidified box at room temperature for 8 or 16 h before use. Our results show that due to the decrease in skin activity, the permeation of 4019

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TD1-hEGF decreases with time. However, as long as the skin is active, the energy dependence of the TD1-mediated transdermal delivery exists (Figure 9). In our experiments, the rat skin maintained a high level of activity in 20 h. Once the skin was deactivated or lost its activity, the peptide chaperone would no longer facilitate the delivery of hEGF across the skin, and ATP would fail to enhance the delivery process. The results presented here indicate again that the hEGF delivery medicated by TD1 is an energy-consumptive biological process.

4. CONCLUSIONS In this work, we investigated the energy dependence of the TD1-mediated transdermal protein delivery and confirmed for the first time that this delivery process requires energy. Our results show that in the TD1-hEGF delivery through the rat skin, when the energy inhibitor, rotenone or oligomycin, is added, the permeation activity of TD1-hEGF decreases dramatically. Lowering the temperature can also result in a significant decrease in the transdermal penetration of TD1hEGF, whereas it has little effect on the penetration of hEGF. Moreover, the direct ATP addition can benefit the permeation of TD1-hEGF across skin, and its effect is dose-dependent. Skin activity significantly affects the permeation of TD1-hEGF across skin, and the deactivation of skin may turn off the pathway of the transdermal macromolecular delivery mediated by TD1. The stratum corneum consists of corneocytes that are biologically dead but still active;42,43 therefore, the stratum corneum is not as a dead tissue, but rather as possessing multiple types of catalytic activity.42,44 This makes it possible for peptides such as SPACE and TD1 to enhance the transdermal delivery of not only macromolecules (up to 100 kDa)25,32,45 but also phages.25,29 To date, the molecular mechanism that these peptides enhance the transdermal protein delivery remains unclear. Current studies only showed that these peptides might provide multiple pathways for drugs into the viable skin layers, such as energy-dependent endocytosis, energy-independent direct translocation across cell membranes, and cell junction change.23 According to the preliminary results in this work, our hypothesis is that a potential binding of the peptide molecules to some certain components in the skin exists, possibly resulting in the temporary junction change of corneocytes and thus the temporary open of the stratum corneum barrier. The peptide TD1 can permeate the skin no matter whether hair follicles exist.30 Therefore, the peptide-mediated permeation of macromolecular drugs across the skin may be mainly via the paracellular pathway. Our results also show the transdermal protein delivery mediated by the peptide is an energydependent process. The work presented here provides some indirect evidence for the interaction between TD1 and skin components by quantifying the TD1-hEGF transport. Further research is needed to elucidate the molecular mechanism underlying this intriguing finding. Transdermal delivery is an attractive route of administration for peptides and proteins. Biologically inspired enhanced technology for further transdermal drug development is an exciting advance. Clarifying the mechanism of the novel energy-dependent permeation process might promote the development of a new transdermal formula or patches of therapeutic proteins.

Figure 7. Effect of ATP analogues on the permeation of hEGF or TD1-hEGF across rat skin. The concentrations of ATP analogues were all 20 mM. The concentration of TD1-hEGF was 0.1 mg/mL. The solution volume was 500 μL. The amount of permeated protein was determined by ELISA at 4 h. Results are depicted as the mean ± SEM, n ≥ 3. *P < 0.05 and **P < 0.01 compared to the ATP group.

Figure 8. Effect of ATP on the permeation of TD1-hEGF across human skin. The concentration of hEGF or TD1-hEGF was 0.1 mg/ mL. The solution volume was 500 μL. The concentration of ATP was 20 mM. TD1-hEGF or hEGF was assayed for permeation across freshly isolated human skin, and the amount of permeated protein was determined at 16 h.

Figure 9. Effect of skin activity on the permeation of TD1-hEGF across rat skin. The deactivated skin was prepared by fixing the fresh rat skin in 4% paraformaldehyde, and the stale skin was prepared by placing the fresh rat skin in a humidified box at room temperature for 8 or 16 h before use. The hEGF or TD1-hEGF concentration was 50 μg/mL. The solution volume was 500 μL. The concentration of ATP was 20 mM. The amount of permeated protein was determined at 4 h (1 h, fresh skin separated from rat; 8 h, stale skin placed for 8 h; 16 h, stale skin placed for 16 h). Results are depicted as the mean ± SEM, n ≥ 5. 4020

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AUTHOR INFORMATION

Corresponding Authors

*(L.W.) E-mail: [email protected]. *(W.D.) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the Chinese Ministry of Sciences 973 Program (2007CB935800 and 2010CB912804), the National Natural Science Foundation of China (#30721002, #31071211, #30830036, #31170966, and #31101020), the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-YW-R-139), the Fundamental Research Funds for the Central Universities (WK2070000008 and WK2100000001), the Scientific and Technological Major Special Project (2009ZX09103-650), the Specialized Research Fund for the Doctoral Program of Higher Education of China (WJ2100230004), and the Natural Science Foundation of Anhui Province (BJ2100230008).

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