Journal of Controlled Release 199 (2015) 168–178

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Peptides as skin penetration enhancers: Mechanisms of action Sunny Kumar a,b, Michael Zakrewsky a,b,1, Ming Chen a,b,1, Stefano Menegatti a,b, John A. Muraski c,⁎, Samir Mitragotri a,b,⁎ a b c

Center for Bioengineering, University of California, Santa Barbara, CA, United States Department of Chemical Engineering, University of California, Santa Barbara, CA, United States Convoy Therapeutics, 405 W Cool Drive, Suite 107, Oro Valley, AZ 85704, United States

a r t i c l e

i n f o

Article history: Received 5 October 2014 Accepted 8 December 2014 Available online 9 December 2014 Keywords: Cell penetrating peptide Cyclosporine Keratin Transporter Topical Transcellular

a b s t r a c t Skin penetrating peptides (SPPs) have garnered wide attention in recent years and emerged as a simple and effective noninvasive strategy for macromolecule delivery into the skin. Although SPPs have demonstrated their potential in enhancing skin delivery, they are still evolving as a new class of skin penetration enhancers. Detailed studies elucidating their mechanisms of action are still lacking. Using five SPPs (SPACE peptide, TD-1, polyarginine, a dermis-localizing peptide and a skin penetrating linear peptide) and a model hydrophobic macromolecule (Cyclosporine A, CsA), herein we provide a mechanistic understanding of SPPs. To evaluate the mechanism and safety of SPPs, their effects on skin lipids, proteins and keratinocyte cells were evaluated. Three SPPs (SPACE, Polyarginine and TD-1) significantly enhanced CsA penetration into the skin. SPPs did not alter the skin lipid barrier as measured by skin resistance, transepidermal water loss (TEWL) and Fourier transform infrared (FTIR) spectroscopic analysis. In contrast, SPPs interacted with skin proteins and induced changes in skin protein secondary structures (α-helices, β-sheet, random coils and turns), as evaluated by FTIR analysis and confirmed by in-silico docking. SPPs enhanced CsA skin penetration, via a transcellular pathway, enhancing its partitioning into keratin-rich corneocytes through concurrent binding of SPP with keratin and CsA. Interaction between SPP and keratin best correlated with measured CsA skin transport. Many SPPs appeared to be safe as shown by negligible effect on skin integrity, nominal skin irritation potential and cytotoxicity. Among the peptides tested, SPACE peptide was found to be least toxic to keratinocytes, and among the most effective at delivering CsA into the skin. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The skin is the largest and most easily accessible organ of the human body for drug delivery [1]. Drug delivery through the skin offers numerous advantages and has great implications for pharmaceutical and cosmetic products. However, drug penetration into and across the skin is a serious challenge [1–3]. The skin serves as a natural protective barrier from the external environment and its low permeability severely limits the transport of most pathogens, toxins and drug molecules [4]. The main transport barrier resides in the outermost layer of the skin, the

Abbreviations: SPP, skin penetrating peptide; SPACE, skin penetrating and cell entering; SDS, sodium dodecyl sulfate; Poly-R, poly-arginine; CsA, Cyclosporine A; SC, stratum corneum; FTIR, Fourier transform infrared; Da, Dalton; CPE, chemical penetration enhancer; PBS, phosphate buffer saline; FDC, Franz diffusion cells; TEWL, transepidermal water loss; HEKa, human epidermal keratinocytes; LP-12, Linear Peptide-12mer; DLP, dermis localizing peptide ⁎ Corresponding authors. E-mail addresses: [email protected] (J.A. Muraski), [email protected] (S. Mitragotri). 1 Equal contribution.

http://dx.doi.org/10.1016/j.jconrel.2014.12.006 0168-3659/© 2014 Elsevier B.V. All rights reserved.

stratum corneum (SC), which is comprised of keratin-rich cells embedded in multiple lipid bilayers [4]. To penetrate through the SC, drugs must navigate through the tortuous lipid pathways surrounding the keratin-rich cells, or repeatedly partition between the aqueous, keratin-rich phase and the lipid phase [5]. Therefore, only potent drugs with optimal physicochemical properties (molecular weight b500 Da, high hydrophobicity, and adequate solubility in aqueous and non-aqueous solvents) can be passively transported through the SC [4,6]. Numerous skin penetration enhancement strategies have been evolved to promote drug delivery across the SC including active and passive methods [7]. Active skin penetration enhancers generally include devices, which are effective but may be difficult to use on large skin areas [7]. In contrast, passive methods such as chemical permeation enhancers (CPEs) are simpler to use and can be applied to large skin areas. Most CPEs enhance skin permeation by affecting the lipid region of the SC either through lipid extraction, fluidization or introduction of other modes of structural reorganization [2,8]. Although CPEs offer high potential in overcoming the skin barrier to enhance transport of drug molecules, their safety as skin penetration enhancers is a potential concern; a balance must often be sought between transport

S. Kumar et al. / Journal of Controlled Release 199 (2015) 168–178

enhancement and skin irritation [2]. A major reason behind CPEassociated skin toxicity is that a large number of CPEs (such as azone derivatives, fatty acids, alcohols, esters, sulphoxides, pyrrolidones, glycols, surfactants and terpenes) are small molecules (b500 Da) that can penetrate the skin in significant quantities and can cause skin irritation, cytotoxicity or irreversibly alter the skin barrier [8,9]. To realize the benefits of transdermal/dermal delivery in the clinic, skin penetration enhancers must not only overcome the barrier properties of the skin but also meet safety and patient-compliance requirements. Hence, the search continues for novel skin penetration enhancers that are effective, non-invasive, non-toxic and nonirritating to the skin. Recently, small peptides (1000–1500 Da) have been identified as safer alternatives to enhance the delivery of small and large molecules into and across the skin [2–11]. Introduced just a decade ago, skin-penetrating peptides (SPPs) are still evolving as a class of skin penetration enhancers [4]. The use of SPPs for transdermal drug delivery is particularly intriguing because the SC presents a formidable, non-specific barrier to the penetration of peptides (N500 Da) [10–14]; hence, the ability of peptides to act as penetration enhancers is unexpected. Several studies have reported the use of SPPs [10–23], and a few have hypothesized potential mechanisms ranging from transient pore formation in appendages [10] to interactions with skin lipids or keratin in the skin [11–13]; however, a comprehensive investigation of the primary underlying mechanisms responsible for SPP-mediated skin penetration enhancement has yet to be undertaken for several reported SPPs in a single concerted study. Herein, we report the first study aimed at understanding the mechanism by which SPPs, as a group, mediate skin penetration enhancement of macromolecules. To this end, we evaluated, using five different peptides (Fig. 1, Table 1), skin penetration enhancement of a model macromolecule (Cyclosporine A, CsA), structural changes in the microscopic domains of the skin SC barrier, and cellular toxicity. Of the five peptides studied here, three have been previously reported in

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the literature and shown to enhance skin delivery (SPACE peptide [11, 17,18], polyarginine (Poly-R) [14,20,22,23] and TD-1 [10,15,16,19,21]). One peptide has been reported before but not well-studied (dermis localizing peptide, DLP [11]) and one peptide is reported here for the first time (Linear Peptide-12 mer, LP-12). Molecular properties of all five peptides are reported in Table 1. The results of this study suggest that SPPs, as a class, enhance transport of CsA through a single mechanism: transcellular partitioning of drug through binding of SPPs to keratin. Moreover, SPPs appeared to interact with keratin without causing irritation. Taken together, this study suggests that transport of hydrophobic macromolecules may be enhanced by pairing with a keratin-binding peptide and further establishes that SPPs are an advantageous alternative delivery strategy for topical and transdermal products. 2. Materials and methods 2.1. Chemicals The peptides were chemically synthesized by Genscript Inc. (Piscataway, NJ, USA). Cyclosporine A (CsA) was purchased from Abcam (Cambridge, MA, USA). 3H-Cyclosporine A was purchased from PerkinElmer (Waltham, MA, USA). All other common chemicals required for the experiments were obtained from Fisher Scientific (Fair Lawn, NJ, USA). 2.2. In-vitro skin penetration study Full thickness porcine skin was procured (Lampire Biological Laboratories, Pipersville, PA, USA) and processed as reported in our earlier studies [17]. The skin integrity was determined by measuring the skin conductivity [24]. In-vitro skin penetration studies were performed using Franz diffusion cells (FDCs) under occlusive condition at 37 ± 1 °C. The effective penetration area and receptor cell volume were 1.77 cm2

Fig. 1. Structures of skin penetrating peptides (SPPs). 3D conformations of SPPs predicted using Pep-Fold 1.5: (i) SPACE, (ii) DLP, (iii) LP-12, (iv) TD-1, and (v) Poly-R.

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Table 1 Molecular properties of SPPs. Name

Sequence

Isoelectric point

Charge at pH 7

Hydrophilicity

Hydrophobicity

Mol. wt.

SPACE Poly-R TD-1 DLP LP-12

ACTGSTQHQCG RRRRRRR ACSSSPSKHCG ACKTGSHNQCG HIITDPNMAEYL

7.26 13.28 8.24 8.24 4.18

0 7 1 1 −1.9

9% 100% 18% 18% 25%

9% – 18% 9% 50%

1092.18 1111.33 1063.18 1105.22 1416.62

and 12.0 mL, respectively. The receptor compartment was filled with pH 7.4 phosphate buffered saline (PBS). Test formulations used in the study were CsA alone (5 mg/mL) or CsA (5 mg/mL) with a SPP (25 mg/mL) dissolved in (45%, v/v) ethanol/PBS solution. Penetration of CsA was measured in the presence of SPPs. SPACE, DLP and TD-1 formulations were prepared using pH 8.0 PBS (50 mM). Penetration data for CsA alone (control) and CsA with SPACE peptide (25 mg/mL) was taken from Ref. [25]. Penetration of CsA in the presence of Poly-R, TD1 and LP-12 was measured in this study. Poly-R and LP-12 peptide were prepared using at pH 7.0 PBS (50 mM) and pH 10.0 PBS (50 mM), respectively. LP-12 peptide was discovered by performing a phage display peptide library screening on full thickness porcine skin, following a previously published method [11], using a 12-mer phage display peptide library (PhD12 library, New England Biolabs, Ipswich, MA). Test formulations were spiked with 3H-CsA (25 μCi/mL) for the purpose of quantitation. Test formulations (100 μL) were loaded into the donor compartment of FDCs (n = 3) and incubated for 24 h at 37 °C with moderate stirring. After 24 h of incubation, the amount of CsA that penetrated into different layers of the skin and the amount of CsA that permeated across the skin were quantified. To measure CsA penetration into different layers (SC, epidermis and dermis) of the skin, the tape-stripping technique was used as described in our earlier studies [17]. To measure CsA that permeated across the skin, 3 mL of sample was withdrawn from the receptor compartment. Further, tissue and receptor chamber samples were then incubated with 5 mL of aqueous based tissue solubilizer, (Soluble, PerkinElmer, Inc., Walthan, MA) overnight at 60 °C. The following day, 5 mL of liquid scintillation cocktail (Ultima Gold, PerkinElmer, Inc., Walthan, MA), was added and 3H-CsA in each sample was quantified using a liquid scintillation counter (TRI-CARB 2100TR, Packard Instrument Company, Downers Grove, IL). 2.3. Change in skin resistance and transepidermal water loss (TEWL) The TEWL and skin resistance were measured before and after SPP treatment. To measure the TEWL (gm/m2/h), a vapometer (Delfin Technologies, Kuopio, Finland) was placed on the donor chamber of FDCs [26]. For measuring the skin resistance, an electric potential was applied using a constant power supply unit (Phoresor II, Iomed, Inc., Salt Lake City, UT) through platinum electrodes (Fisher Scientific). The current (I) across the skin was measured using a multimeter (Fluke, Everett, WA) and the skin resistance (R) was calculated from Ohm's law (V = IR) [26,27]. The ratios representing change in skin resistance and TEWL were calculated respectively by dividing skin resistance and water loss values obtained after SPP treatment with values obtained before SPP treatment. 2.4. Fourier transform infrared (FTIR) spectroscopy measurements Full thickness porcine skin was purchased from Lampire Biological Laboratories (Pipersville, PA) and stored at − 80 °C. Skin was thawed at room temperature for 30 min prior to use. Hair was clipped using scissors and the skin was submerged in a 60 °C water bath for 90 s to loosen the epidermis from the dermis. The epidermis was then separated from the dermis using forceps. Next, the epidermis-SC was floated on 0.25% trypsin solution with the SC facing up. Trypsin digestion was allowed to occur for 24 h at room temperature. This step has been

shown sufficient to digest the epidermis without disrupting the SC microstructure [28]. Isolated SC was then washed in PBS and allowed to dry at room temperature for 72 h. The SC was then cut into individual 15 mm diameter samples and a pre-treatment FTIR spectrum was collected for each sample. SC samples were then incubated with 200 μL of test formulation (25 mg/mL peptide dissolved in 45% v/v ethanol in PBS) in a 24-well culture plate (Corning Inc., Corning, NY) for 24 h. 45% v/v ethanol in PBS was also tested as a negative control, and each test condition and control condition were performed in quadruplicate. After treatment, the SC was thoroughly washed with PBS and allowed to dry for 72 h. Post-treatment FTIR spectra were collected for each sample and compared to the spectra before treatment to assess the effects of SPPs on skin microstructure. Spectra were collected using a Nicolet Magna 850 spectrometer with a resolution of 2 cm−1 and averaged over 100 scans. Spectra were baseline corrected, smoothed, and analyzed in Origin Pro. 2.5. In-silico docking analysis The coordinate files for the skin-penetrating peptides (SPPs) were generated using the structure prediction server PEP-FOLD. The coordinate file for the 2B regions from the central coiled-coil domains of human keratin5 and keratin14, expressed in the keratinocytes of epidermis, was obtained from the RCSB Protein Data Bank (PDB, 3TNU). The solvent accessible residues on keratin were defined as “active” and used as target for ligand docking. All active residues exhibit a relative solvent accessibility higher than 40%, as defined by the program NACCESS. Molecular modeling was performed using the program HADDOCK (version 2.1). Default HADDOCK parameters (e.g., temperatures for heating/ cooling steps, and number of molecular dynamics sets per stage) were used in the docking procedure. The resulting docked structures were grouped in clusters by assigning a minimum cluster size of 4 and an RMSD (root-mean-square-distance) lower than 2.5 Å using the program ProFit (http://www.bioinf.org.uk/software/profit/). All the clusters selected for each sequence based on visual inspection of the lowest energy docked solution were analyzed according to the in-silico binding score (HPScore, HMScore, HSScore, −log(Kd), and DiG). The structures used for analysis were the most energetically favored docked pairs from each cluster. Clusters were analyzed using built-in scoring functions, which comprise empirical scoring functions that estimate the free energy of binding, and hence the affinity, of a given protein–ligand complex of known three-dimensional structure. These functions account for van der Waals interactions, hydrogen bonding, deformation penalty, and hydrophobic effects, atomic contact energy, softened van der Waals interactions, partial electrostatics, and additional estimations of the binding free energy, and dipole–dipole interactions. The rankings were then compiled, each listing the sequences ordered based on the scoring value obtained according to the respective function. These rankings were finally totaled and averaged to obtain a final list of sequences, where lower (more negative) score indicates higher affinity. The coordinate files for the SPPs, as obtained using the server PEP-FOLD, and the coordinates for Cyclosporine A (PDB, 1CsA) were used to generate the respective SPP/CsA couples using the docking program HADDOCK. All residues of both peptides were defined as active for this docking. These dipeptide couples were in turn docked against human keratin (PDB, 3TNU) to obtain SPP/CsA/keratin complexes. Docking parameters

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and scoring functions adopted for both simulations were as described above. 2.6. Mass spectroscopy SPPs (25 mg/mL), CsA (5 mg/mL) and their mixture were prepared in 45% (v/v) ethanol/water for mass spectroscopic analysis using Micromass QTOF (Waters Corporation, Beverly, MA) with an electrospray ion source. Samples were diluted with acetonitrile/water containing 0.1% formic acid and then introduced via a Harvard Apparatus syringe pump at 10 μL/min flow rate. The capillary was held at 3.5 kV. Nitrogen was used as nebulizer, desolvation, and cone gas. 2.7. Cell culture and cytotoxicity assessment Human adult epidermal keratinocytes (HEKa cells) and all cell culture materials were acquired from Life Technologies (Grand Island, NY). HEKa cells were cultured in 1× keratinocyte serum-free medium supplemented with 25 U/mL penicillin, 25 μg/mL streptomycin, and 50 μg/mL neomycin. Cultures were grown at 37 °C with 5% CO2. The cytotoxicity of SPPs was assessed using the MTT Cell Proliferation Assay (ATCC, Manassas, VA). HEKa cells were seeded in 96-well microplates (Corning Inc., Corning, NY) at a density of 5000 cells/well. Cultures were allowed to grow until they reached ~80% confluency. Cells were then incubated with 150 μL of 10, 5, 2.5, or 1.25 mg/mL SPP in media. Media only was used as a negative control, and media without cells was used to subtract background. Cytotoxicity was assessed for 1, 4, and 12 h incubation periods. Viability was determined according to the manufacturer's recommended protocol using a SAFIRE, XFLUOR4, V4.50 microplate reader (Tecan Group Ltd, Morrisville, NY). 2.8. Statistical analysis All experiments were performed in triplicate, unless specified and the results are expressed as mean ± standard deviation (stdev). Student's t-test was used to compare two groups and one-way ANOVA followed by Bonferroni's correction for post-test comparisons was used when more than two groups were compared. The values of p b 0.05, p b 0.01, and p b 0.001 were considered significant with 95%, 99% and 99.9% confidence intervals, respectively. Statistical analyses were performed using GraphPad (Prism version 6) software. 3. Results 3.1. CsA skin penetration enhancement with SPPs Cyclosporine A (CsA) was used as a model macromolecule to assess the skin penetration enhancement abilities of SPPs. Peptide-dependent enhancement of CsA delivery into skin was observed (Fig. 2). SPACE peptide, Poly-R and TD-1 significantly enhanced CsA penetration into the skin compared to the control (45% v/v ethanol, p b 0.001 for SPACE peptide and Poly-R, and p b 0.05 for TD-1) (Fig. 2). In contrast, DLP and LP-12 did not significantly enhance CsA skin penetration compared to the control (p N 0.05) (Fig. 2). 3.2. Effect on skin lipids The ability of SPPs to induce lipid extraction or fluidization was analyzed using FTIR spectroscopy (Fig. 3a–b). A reduction in the area of the deconvoluted peak at 2850 cm−1 indicates extraction of lipids, whereas a positive shift in the peak center and broadening of the full-width at half maximum of CH2 symmetric (2850 cm− 1) or asymmetric (2920 cm−1) stretching contributions, respectively, indicates fluidization of lipids [29,30]. No significant effect of any SPP on extraction or fluidization was noted (Table 2). In fact, SPPs appear to slightly increase the order (i.e. decrease in fluidization) compared to the control solvent

Fig. 2. In-vitro skin penetration of Cyclosporine A (CsA). CsA (5 mg/mL) in 45% (v/v) ethanol/PBS (control) or with SPPs (SPACE, Poly-R, TD-1, DLP and LP-12) in 45% (v/v) ethanol/PBS were applied to the donor compartment of Franz-diffusion cells. The percentage (%) of applied CsA dose entering the skin was calculated by measuring CsA in different skin layers of the skin and receptor medium. Each data point represents mean ± stdev (n = 3). The asterisks (***p b 0.001 and *p b 0.05) indicate significantly higher % CsA skin penetration as compared to control (p b 0.05).

(45% ethanol); although, the change is not statistically significant (p N 0.05). The ratio of the maximum intensity of deconvoluted peaks corresponding to CH2 asymmetric (2920 cm−1) and symmetric (2850 cm−1) stretching is a measure of lateral interactions between acyl chains in the SC and is another indicator of lipid fluidization [31]. An increase in this ratio (Height2920/2850) indicates a shift in lipid microstructure from primarily a lamellar crystalline-state to a more fluid state [31]. However, no significant effect of SPPs on this parameter was found (Table 2). Instead, SPPs appeared to induce a slight increase in the lamellar crystalline lipid phase compared to the ethanol control. Furthermore, no effect of SPPs on methylene scissoring contributions (1480– 1430 cm−1) was noted indicating no effect on acyl chain packing (Supplementary Fig. 1a–b). Finally, CH2 wagging progressions in the “fingerprint” region of the FTIR spectra (1300–1000 cm− 1) were analyzed (Supplementary Fig. 1c) and indicated no significant change in the amounts of lipid disorder [32]. Instead, CH2 wagging progressions after treatment resulted in an increase in the number of peak assignments (Supplementary Fig. 1c), which further indicates that SPP treatment results in a less fluid lipid phase. Taken together, these results indicated that SPPs do not produce a significant fluidization of SC lipids. 3.3. Effect of SPPs on skin resistance and TEWL Effects of SPPs on skin resistance and transepidermal water loss (TEWL) were measured; both carry information about the integrity of SC lipid bilayers [2]. Exposure of the skin to 45% ethanol itself decreased skin resistance and increased TEWL. However, none of the SPPs further increased TEWL or decreased skin resistance compared to the control (45% ethanol) (Fig. 3c–d). Instead, all SPPs improved TEWL and skin resistance with respect to the 45% ethanol, indicating enhanced order of lipid packing/structure compared to 45% ethanol (Fig. 3c–d). 3.4. Effect on skin proteins Effect of SPPs on skin protein structure was evaluated by FTIR spectroscopy. For this purpose, the amide I band (1700–1600 cm−1), corresponding to carbonyl stretching, was compared before and after SPP treatment [29,33–35] (Fig. 4a–b: Example spectra and deconvolution of the 1700–1600 cm− 1 region). There was a statistically significant

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Fig. 3. Effect of SPPs on skin lipids. (a and b) SPPs were incubated with the isolated stratum corneum (SC) for 24 h in 45% (v/v) ethanol/PBS solution. After 24 h of incubation, FTIR spectroscopy was performed to evaluate molecular changes in skin lipid structure. (a) Example spectra before (black) and after (red) SPP treatment and (b) deconvoluted FTIR spectra of skin lipid region (3000–2800 cm−1). (c and d) SPPs were incubated with the skin in donor compartment of FDCs for 24 h in 45% (v/v) ethanol/PBS solution. Changes in (c) skin electrical resistance and (d) transepidermal water loss (TEWL) were measured. Each data point represents mean ± stdev (n = 3–4). The asterisks (***p b 0.001, **p b 0.01 and *p b 0.05) indicate significantly different values as compared to control (45% EtOH). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

increase in % α-helices for SPP treated skin (Table 3). This change was accompanied by an equally proportionate decrease in the percentage of other secondary structures (Table 3). Moreover, the change in % α-

helices for SPP-treated skin correlated closely (r2 = 0.63) with the extent of CsA penetration into the skin (Fig. 4c). 3.5. Peptide binding to keratin

Table 2 Changes in SC lipid microstructure (lipid peak height and area) after treatment with SPPs. Indicators of Indicators of lipid fluidization lipid extraction

Ethanol SPACE Poly-R TD-1 DLP LP-12

Δ Area−1 2850 cm

−1 −1 Δ Center−1 2850 cm Δ FWHM2920 cm Δ Height2920/2850 cm

−0.96 (0.45) −0.94 (2.74) −0.54 (2.58) 0.11 (1.60) −0.36 (1.59) −0.41 (1.22)

−0.08 (0.05) −0.07 (0.06) −0.15 (0.31) −0.06 (0.05) −0.02 (0.07) −0.15 (0.17)

−2.01 (0.82) −0.29 (1.72) −1.01 (3.43) 0.66 (3.07) −0.46 (1.52) −1.28 (3.08)

−0.11 (0.07) −0.12 (0.10) −0.12 (0.08) −0.02 (0.16) −0.14 (0.07) −0.04 (0.01)

Each data point represents mean ± (stdev) for n = 4. No statistically significant (p N 0.05) differences were observed for any of measures shown above.

Keratin is the most abundant protein present in the skin. The SPPs (SPACE, Poly-R, TD-1, DLP and LP-12) were docked in-silico against keratin using the software HADDOCK (version 2.1) (Fig. 5a). The scoring analysis showed that all SPPs bind to keratin protein, although to a markedly different extent (Fig. 5a). In particular, by comparing the different enthalpic contributions, SPACE and DLP appear to bind to keratin predominantly via hydrogen bonding and weak electrostatic interactions, while TD-1 binds mostly through hydrogen bonding. Alternatively, LP-12 binds predominantly via hydrophobic and electrostatic interactions along with minor hydrogen bond interactions. The SPPkeratin binding scores (Fig. 5b) directly correlated with CsA skin penetration enhancement (r2 = 0.44). In addition, the scores directly correlated with changes in skin protein secondary structures (Supplementary Fig. 2a: % α-helix/SPP-keratin binding scores (r2 = 0.82) and Supplementary Fig. 2b: % β-sheet/SPP-keratin binding scores (r2 = 0.91)).

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Fig. 4. Effect of SPPs on skin protein. (a and b) SPPs were incubated with the isolated stratum corneum (SC) for 24 h in 45% (v/v) ethanol/PBS solution. After 24 h of incubation, FTIR spectroscopy was performed to evaluate molecular changes in skin protein structure. (a) Example spectra before (black) and after (red) SPP treatment and (b) deconvoluted FTIR spectra of skin protein amide I region (1700–1600 cm−1). (c) Relationship between percent of CsA skin transport and changes in % alpha-helix skin protein content. Each data point represents mean ± stdev (n = 3–4). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.6. SPP binding to CsA Binding of CsA to SPP was also evaluated in-silico by docking analysis and in-vitro by mass spectroscopy. The in-silico docking analysis showed that all SPPs bind to CsA except for LP-12 (Fig. 5c). Based on the docking results, the molecular interaction between SPPs and CsA mainly comprises hydrogen bonding between the hydrophilic residues of SPPs and the backbone of CsA. Due to the purely aliphatic nature of the residues on CsA, no other interaction was expected, except a mild hydrophobic one with the SPP backbone. These findings were supported and confirmed with mass spectroscopy results as reported in Fig. S3. Mass spectroscopy confirmed that LP-12 does not bind to CsA (Supplementary Fig. 3e). CsA alone does not bind to keratin. Instead, it was found that SPPs operate as binding mediators between keratin and CsA (Fig. 5d), thereby enabling the interaction between the skin protein and the drug.

3.7. SPPs' safety as skin penetration enhancer FTIR spectroscopy and MTT assays were performed to evaluate the safety of SPPs as skin penetration enhancers at the skin and cellular levels, respectively. The ability to disrupt α-helices in the skin, thereby changing their conformation to less rigid secondary structures, is a characteristic feature of protein unfolding and denaturation. In contrast, SPP treatment was found to result in a statistically significant increase in the percentage of α-helices, suggesting that SPPs actually stabilize structural proteins in the skin rather than denature them like most skin penetration enhancers (Table 3) [36]. The nominal irritation potential expected for SPPs was further verified using a previously established method based on FTIR [2]. Irritation potential of SPPs was compared to the

Table 3 Changes in SC protein microstructure (protein secondary structures: % α-helix, % β-sheet, % turns and % random coils) after treatment with SPPs. Each data point represents mean ± (stdev) for n = 4. All the values are statistically significant (p b 0.05) as compared to the ethanol control unless otherwise stated as ‘ns’.

Ethanol SPACE Poly-R TD-1 DLP LP-12

% a-helices

% p-sheets

% turns

% random coils

−0.32 (0.13) 4.12 (0.18) 3.45 (0.15) 0.30 (0.13) −0.79 (0.14) 0.78 (0.20)

0.86 (0.10) −1.42 (0.22) −0.96 (0.41) 0.93 (0.28)ns 2.30 (0.18) 0.63 (0.75)ns

−0.44 (0.15) −4.20 (0.44) −4.96 (0.22) −1.24 (0.17) −1.22 (0.15) −1.89 (0.54)

0.10 (0.20) −1.50 (0.32) −2.47 (0.22) −0.01 (0.21)ns 0.03 (0.14)ns 0.48 (0.63)ns

irritation potential of 5% sodium dodecyl sulfate (SDS), a known skin irritant. The irritation potential of SPP treatment is significantly less than for 5% SDS treatment (Fig. 6a), suggesting that SPPs are not irritating to the skin [2]. The safety of SPPs at the cellular level was evaluated by measuring their cytotoxicity against human adult epidermal keratinocytes (HEKa cells). HEKa cells were incubated for 1, 4, and 12 h with SPPs. Peptide concentrations in the range of 1.25–10 mg/mL were used. The upper limit of this concentration range is lower than that used for transdermal transport studies (25 mg/mL) since the actual concentration experienced by keratinocytes in the epidermis is likely to be far less than that placed on the skin. The highest concentration used in toxicity studies (10 mg/mL) is 40% of the peptide concentration placed on the skin, which is highly likely to exceed the actual concentration experienced by keratinocytes in the epidermis during transdermal transport studies. LP-12 was not soluble beyond 5 mg/mL; hence the highest concentration studied with LP-12 was 5 mg/mL. After incubation for 12 h with SPPs, cell viability was determined using the MTT assay (Fig. 6b). The positive control (5% SDS, not shown) led to complete cell death, while SPACE peptide was least toxic among all the SPPs (Figs. 6b and S5). Similar trends were observed for all incubation times (1, 4 and 12 h) studied (Figs. 6b and S5). The results here indicate that some peptides have an excellent efficacy for CsA delivery into the skin while some have an excellent safety profile. The ultimate utility of an SPP for drug delivery, however, depends on the combined effect of efficacy and safety. To quantify the “efficacy to toxicity” profile of SPPs, we defined a ratio of ‘% CsA delivery’ to ‘% cell death at 12 h after incubation at 5 mg/mL’. While the ratio can be defined in many other ways, the current definition essentially captures the balance of two key attributes of SPPs. The resultant ratio provides a means to compare various peptides and the absolute magnitude of this ratio as such is not relevant to the analysis. DLP and LP-12 were found at the trailing end of the group due primarily to low efficacy of LP-12 and high toxicity of DLP. TD-1 and Poly-R were found to localize in the middle of the group. SPACE peptide offered higher efficacy/toxicity ratio compared to other peptides (p b 0.001) (Fig. 7). 4. Discussion Over the last few years, several SPPs have been reported to deliver macromolecules into the skin in a non-invasive manner [11–14]. Although SPPs constitute an exciting new approach for non-invasive skin penetration enhancement, their mechanisms of action are yet to be known. To this end, the current study provides a generalized

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Fig. 5. In-silico binding analysis of SPP. The in-silico docking figures show SPP in the following order: i) SPACE, ii) DLP, iii) LP-12, iv) TD-1, v) Poly-R. (a) The lowest energy structures from best scoring clusters of SPPs structures docked against human keratin. Shown in gray cartoon format is keratin (PBD code: 3TNU) with peptide structures shown in blue stick format. (b) Relationship between CsA skin transport percent and in-silico SPP-keratin binding score. Each data point represents mean ± stdev (n = 3–4). (c) Structures of CsA (PDB code: 1CsA) and SPPs' interactions modeled using Haddock software. (d) The lowest energy structures from best scoring clusters of SPP-CsA pairs docked against human keratin. Shown in gray cartoon format is keratin (PBD code: 3TNU) with SPP and CsA in purple and blue stick format respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

mechanistic understanding of SPP-mediated skin penetration enhancement of a macromolecule (CsA, Mol. Wt. 1202 Da). The primary objective of the present study was to understand the role of SPP's molecular properties in determining their skin penetration enhancement potential. Five different SPPs (SPACE, TD-1, Poly-R, DLP and LP-12) with different physicochemical properties were used (Fig. 1). We aimed to select SPPs from the literature to cover a range of molecular properties of peptides such as (i) surface charge (negative, neutral and positive), hydrophilicity (9%–100%) and hydrophobicity (0%–50%) as shown in Table 1. While three of these peptides (SPACE, TD-1 and Poly-R) were previously reported in the literature, we added a new peptide (LP-12)

to further diversify the selection. From a mechanistic perspective, SPPs identified in recent years have been reported to interact with SC lipids and proteins [11–13]. However, no detailed understanding and explanation are available to elucidate the mechanisms in detail. A detailed analysis of changes in skin lipid and protein microstructure was performed in order to understand their role in SPPs' mediated skin penetration enhancement. Drug delivery through the skin is mainly limited by the stratum corneum (SC), the top-most skin layer [4,37]. SC is composed of lipidrich intercellular lipid bilayers and keratin filled corneocytes [4,37]. There are mainly three pathways by which a drug molecule can cross

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Fig. 6. SPPs safety at skin and cellular level. (a) Skin irritation potential of SPPs (SPACE, Poly-R, TD-1, DLP and LP-12) was evaluated using a previously established method using FTIR. SDS (5% v/v) was used as a positive control (a known skin irritant). (b) Cellular toxicity with SPPs (SPACE, Poly-R, TD-1, DLP and LP-12) was observed when incubated with human adult epidermal keratinocytes (HEKa cells) for 12 h at different concentrations (1.25, 2.5, 5, and 10 mg/mL). Each data point represents mean ± stdev (n = 4). LP-12 is not soluble at 10 mg/mL and was not tested at this concentration. The asterisks (***p b 0.001) indicate significantly higher values as compared to other peptides; and superscript explains ‘a’ is significant in comparison to all 4 (Poly-R, TD-1, DLP and LP-12) peptides, ‘b’ is significant in comparison to 3 peptides (Poly-R, DLP and LP-12), and ‘c’ is significant in comparison to 1 (Poly-R) peptide.

the intact stratum corneum: (i) via skin appendages (shunt pathway); (ii) through the intercellular lipid pathway; or (iii) via transcellular pathway [3]. Considering that the surface area occupied by skin appendages such as hair follicles and sweat ducts is quite small (typically less than 0.1% of total skin surface area) [38], involvement of shunt pathways in SPP-mediated skin penetration enhancement was not considered. The remaining two pathways, however, were analyzed in detail. The barrier properties of the skin mainly originate from the tortuous lipid bilayers in the SC and the diffusion of molecules through the lipid bilayer represents the intercellular pathway [39]. The intercellular lipid barrier maintains skin integrity, which can be evaluated by measuring skin electrical resistance and transepidermal water loss [40,41]. The perturbation of SC lipids observed with skin penetration enhancers, either through extraction or fluidization, results in altered skin lipid

Fig. 7. SPPs' efficacy and toxicity ratio. The benefit/cost ratio of SPPs is represented as a ratio of potency (% of CsA delivered into the skin) and toxicity (% cellular toxicity) observed with SPP treatment. The ratio was calculated by using the values of % of CsA delivered into the skin with the help of SPPs and dividing these values by the % keratinocyte cell toxicity observed with SPPs at 5 mg/mL concentration during 12 h of incubation. Each data point represents mean ± stdev (n = 3). The asterisks (***p b 0.001) indicate significantly higher % CsA delivery/% toxicity ratio as compared to all other peptides.

structure and is indicated by changes in skin electrical resistance and/ or water loss [2]. Unlike typical organic penetration enhancers such as oleic acid and isopropyl myristate [8,42], SPPs did not change skin's electrical resistance or TEWL compared to the vehicle control (Fig. 3c–d). Further, neither extraction nor fluidization of SC lipids was observed with FTIR spectroscopy after SPP treatment (Fig. 3a–b, Supplementary Fig. 1 and Table 2). Instead, all three independent measurements suggest that SPPs slightly improve skin lipid arrangement and enhance the skin lipid barrier as compared to the control vehicle (45% Ethanol) (Fig. 3). The lack of a direct effect of SPPs on SC lipids suggests that SPPs do not mediate permeation enhancement through classical intercellular lipoidal pathways. Another possible mechanism of drug transport through the skin is through the transcellular pathway [5]. SPP treatment showed a statistically significant, albeit small, change in the percentage of α-helix content in the skin protein secondary structure (Table 3). This change was accompanied by an equally proportionate decrease in the percentage of other secondary structures (Table 3). Moreover, the change in % αhelices for SPP treated skin correlated closely (r2 = 0.63) with the ability to deliver CsA into the skin (Fig. 4c). In-silico docking simulations confirmed binding of SPPs with keratin (Fig. 5a). The ranking of SPPs based upon conventional scoring functions exhibited a strong correlation (r2 = 0.44) with CsA skin transport (Fig. 5b). In addition to suggesting different makeups of the binding mechanisms, these simulations indicate that the aforementioned peptides target keratin through a variety of non-covalent interactions. Binding of Poly-R was found to be dominated by the electrostatic component. This result was expected, since, at the working pH (7.0), Poly-R is positively charged, whereas keratin (pI = 4.0–6.5) is negatively charged. Further, while the docking simulations for the SPACE, DLP, TD-1 and LP-12 indicated a single prominent binding region on keratin, Poly-R was found to possess multiple target spots on the target protein. Hence, while the affinity of Poly-R for each of its binding sites is lower compared to the affinity of the other peptides, the multivalency of this interaction enhances the overall interaction. The combined results of keratin binding and FTIR spectroscopy strongly indicate that SC proteins, especially keratin, play a strong role in CsA transport. Given the strong correlation between CsA transport and keratin binding/structural changes, we postulate that the

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transcellular pathway plays a major role in skin penetration enhancement by SPPs. In principle, drugs permeating through the transcellular pathway have to pass through hydrophilic keratin-packed corneocytes surrounded by multiple hydrophobic lipid bilayers [5]. This process requires a number of partitioning steps (from lipid to protein and protein to lipid), as well as diffusion steps (within the hydrophilic keratin cells and hydrophobic multiple lipid bilayers) [5]. Hence, molecules permeating through this pathway should have an optimal partition coefficient to permeate the hydrophobic lipid bilayer from the donor solution. Considering the high Log P (2.91) of CsA, its partition into the lipid bilayer is expected [43,44]. However, CsA partitioning/penetration into the hydrophilic keratin layer is challenging. We hypothesize that SPPs assist in overcoming this hurdle. Specifically, SPPs exhibit increased partitioning into keratin-rich corneocytes due to their affinity towards keratin. Since CsA also exhibits affinity for SPPs, the SPP–CsA complex exhibits higher partitioning into corneocytes than CsA alone. Current data are consistent with this hypothesis. CsA skin transport enhancement correlates with SPP affinity for keratin (Fig. 5b). The only exception to this correlation, LP-12, which in spite of exhibiting moderate affinity for keratin (Fig. 5a), exhibits poor affinity for CsA, which may explain its lower efficacy in CsA transport. The studies reported here portray a clear mechanistic picture; hydrophobic macromolecules such as CsA exhibit low permeation due to their large size (which limits diffusion in the lipoidal region) and hydrophobic nature (which limits their partitioning into corneocytes). Most SPPs studied here (except for LP-12) associate with CsA and increase their partitioning into corneocytes, which opens an otherwise unaccessible transport pathway for CsA. This hypothesis was further confirmed by in-silico docking analysis of CsA, SPP, and keratin complexes (Fig. 5d). CsA alone did not bind to keratin. Yet, SPPs exhibited a mediating function between CsA and keratin, acting as a bridge between the drug and the protein (Fig. 5d). None of the SPPs molecular properties (Table 2: such as peptide surface charge, hydrophilicity and hydrophobicity), except keratin binding, correlated with skin penetration of CsA (Supplementary Fig. 4a). Fig. S4a appears to indicate a correlation with hydrophobicity; however, skin penetration correlates best with binding to both CsA and keratin. This cannot be displayed in a single figure since binding to CsA is assessed qualitatively. However, a closer look at Fig. 5b reveals that LP-12 is an outlier, likely due to its lack of binding to CsA. Among all the factors, only SPP-keratin binding affinity (with required binding between SPP and CsA) and the change in αhelix directly correlate with enhanced CsA skin transport (Figs. 4c and 5b), which suggests the importance of SPPs' action on skin protein. Although not well studied, the transcellular pathway has been proposed for those molecules that can bypass much of the tortuous lipid channels by favorably partitioning into highly keratinized corneocytes [5]. In principle, utilizing this pathway would provide a much more direct route through the skin [45]. However, the transcellular pathway for skin penetration enhancement has not yet been well appreciated, since chemicals capable of denaturing keratin and opening the transcellular pathway usually result in protein denaturation and subsequent skin irritation [46,47]. In addition, these enhancers cannot limit their activity to the superficial layer of the skin, and eventually diffuse into the viable epidermis where they exert a cytotoxic effect on keratinocytes [4] . As a result, it is challenging to find an optimum balance between the safety and potency of permeation enhancers acting via the transcellular pathway. Interestingly, however, SPPs appear to enhance transport without inducing significant irritation or cytotoxicity (Fig. 6). The current study demonstrates that all five (SPACE, Poly-R, TD-1, DLP, LP-12) peptides investigated in the study had negligible cell toxicity (Fig. 6 and Supplementary Fig. 5). Some peptides indeed show toxicity, although significantly less as compared to 5% SDS solution, which caused complete cell death (data not shown). Among the five peptides tested, SPACE peptide was markedly less toxic to keratinocytes. This trend was observed regardless of the incubation time (Supplementary Fig. 5). Most likely, the relatively weak cytotoxic effect of SPACE peptide

is due to a combination of the net neutral charge and hydrophilicity of SPACE compared to the other SPPs tested. However, there was no observable relationship between cell toxicity and any single SPP molecular property, such as surface charge, hydrophilicity, and hydrophobicity (Supplementary Fig. 4b). Many studies reported in the literature suggest that surface charge [48], amphipathicity, and secondary structure [49] may play a role in peptide mediated cytotoxicity. Therefore, it is expected that each peptide may induce a cytotoxic effect through its own unique combination of molecular properties. Of additional interest, SPPs tested here possessed a CD50 N 2.5 mg/mL. This is substantially higher than values reported in the literature for various other cell lines [50–53]. It is generally accepted that cell type plays a critical role in peptide-mediated cytotoxicity [54]. Therefore, application of peptides for dermal drug delivery may be particularly advantageous because of the reduced cytotoxicity to keratinocytes, the predominant cell type in the epidermis. Alternatively, cytotoxicity may relate to innate internalization ability. Cardozo et al. [50] reported a correlation between uptake of Tat peptide (analog of Poly-R) and cytotoxicity in 208F fibroblasts. In addition, previous studies in this lab have shown differing uptake rates between HUVEC, fibroblast, and keratinocyte cell lines, with keratinocytes having the lowest internalization efficiency [11]. It is unclear what exactly is the cause for the relatively high CD50 values observed in this study and future studies should explore this topic further. Nonetheless, the data strongly suggests that SPPs are relatively safe to use with nominal keratinocyte cytotoxicity. In addition to keratinocyte cytotoxicity, skin irritation potential was assessed through FTIR spectroscopy. FTIR analysis suggests that SPPs do not facilitate enhanced drug transport through the lipid space, but rather through interactions with SC proteins. However, significant interaction with SC proteins has been demonstrated to correlate closely with skin irritation. The ability to disrupt α-helices in the skin, thereby changing their conformation to less rigid secondary structures, is characteristic of protein unfolding and denaturation. In contrast, SPP treatment resulted in a statistically significant increase in % α-helices, suggesting that SPPs actually stabilize structural proteins in the skin rather than denature them like most skin penetration enhancers. The nominal irritation potential expected for SPPs was further verified using a previously established method [2]. Of significant note, the amount of change in secondary structure is small, which indicates stabilization of the dominant mode for that peak, e.g. keratin coiled αhelices, rather than a complete refolding/unfolding from one secondary structure to another, e.g. native β-sheet-rich protein → α-helix dominant structure. Analysis of the effects of SPP treatment on SC protein structure suggests that SPPs may act through a keratin binding mechanism, thereby stabilizing the coiled coil structure of keratin in corneocytes. This would result in the observed increase in proportion of α-helices compared to contributions from less ordered structural modes, and facilitate partitioning of hydrophobic drug into the intracellular space providing a more direct transcellular route for drug delivery, than through the tortuous lipid channels in the SC. While the studies presented here demonstrate a mechanism common to all peptides, clear differences were found in their efficacysafety profiles of studied peptides. Poly-R induced high enhancement of CsA delivery with noticeable toxicity to keratinocytes. DLP was minimally effective in enhancing CsA delivery; yet its toxicity was among the highest of those studied. LP-12 was ineffective in enhancing CsA delivery and its toxicity was moderate. TD-1 exhibited appreciable efficacy and noticeable toxicity. SPACE peptide exhibited minimal toxicity and high enhancement of CsA delivery. A quantitative assessment of the efficacy/toxicity can be found in Fig. 7, which shows the ratio of ‘% CsA delivery’ to ‘% cell death at 12 h after incubation at 5 mg/mL’. SPACE peptide offers a high efficacy to safety ratio. We believe that the net neutral charge, balanced hydrophilicity/hydrophobicity and superior keratin binding affinity contribute to the performance of SPACE peptide in enhancing drug skin penetration and minimal toxicity. Caution should

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be taken while reaching generalized conclusions about the efficacy and safety of SPPs from the results in Fig. 7 since the relative efficacy of peptides in enhancing drug penetration could vary from drug to drug, especially when the drug properties are substantially different compared to CsA. For example, TD-1 has been used to systemically deliver insulin, a molecule significantly different compared to CsA. Further, the ratio of efficacy to toxicity could depend on the concentration of peptides since efficacy and toxicity may depend non-linearly on their concentration. Further, the assessment of toxicity in this study was made based on keratinocyte cultures and the relative toxicity behavior of peptides could be different in vivo. In addition, some of these peptides have been used in different forms, for example, chemical conjugation to cargo in case of Poly-R or ethosomal formulations in case of SPACE peptide. The efficacy/toxicity ratios of peptides could be different for different formulations. 5. Conclusion The current study demonstrates the molecular level understanding of peptides acting as skin penetration enhancers. The SPPs examined in this study appear to act as mediators between CsA and keratin, providing a mechanism for partitioning of drugs into keratin-rich corneocytes through concurrent binding interactions between keratin and SPP, and, SPP and CsA. The SPPs appeared to be safe skin penetration enhancers as shown by negligible effect on skin integrity, nominal skin irritation and cytotoxicity. As observed with in-silico keratin binding and FTIR analysis of α-helix content, SPPs' interactions with skin protein, directly correlated with enhanced drug delivery into the skin. Acknowledgments This work was supported by the Convoy Therapeutics Inc. (SB120129) The MRL Shared Experimental Facilities are supported by the MRSEC Program of the NSF under Award No. DMR 1121053; a member of the NSF-funded Materials Research Facilities Network. SM is a shareholder and scientific advisor of Convoy Therapeutics. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jconrel.2014.12.006. References [1] B.W. Barry, Breaching the skin's barrier to drugs, Nat. Biotechnol. 22 (2004) 165–167. [2] P. Karande, A. Jain, K. Ergun, V. Kispersky, S. Mitragotri, Design principles of chemical penetration enhancers for transdermal drug delivery, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 4688–4693. [3] M.R. Prausnitz, S. Mitragotri, R. Langer, Current status and future potential of transdermal drug delivery, Nat. Rev. Drug Discov. 3 (2004) 115–124. [4] P. Karande, S. Mitragotri, Enhancement of transdermal drug delivery via synergistic action of chemicals, Biochim. Biophys. Acta 1788 (2009) 2362–2373. [5] G.M. El Maghraby, B.W. Barry, A.C. Williams, Liposomes and skin: from drug delivery to model membranes, Eur. J. Pharm. Sci. 34 (2008) 203–222. [6] J. Zhang, M. Liu, H. Jin, L. Deng, J. Xing, A. Dong, In vitro enhancement of lactate esters on the percutaneous penetration of drugs with different lipophilicity, AAPS PharmSciTech 11 (2010) 894–903. [7] M.R. Prausnitz, R. Langer, Transdermal drug delivery, Nat. Biotechnol. 26 (2008) 1261–1268. [8] B.W. Barry, Mode of action of penetration enhancers in human skin, J. Control. Release 6 (1987) 85–97. [9] V.V. Venuganti, O.P. Perumal, Effect of poly(amidoamine) (PAMAM) dendrimer on skin permeation of 5-fluorouracil, Int. J. Pharm. 361 (2008) 230–238. [10] Y. Chen, Y. Shen, X. Guo, C. Zhang, W. Yang, M. Ma, S. Liu, M. Zhang, L.P. Wen, Transdermal protein delivery by a coadministered peptide identified via phage display, Nat. Biotechnol. 24 (2006) 455–460. [11] T. Hsu, S. Mitragotri, Delivery of siRNA and other macromolecules into skin and cells using a peptide enhancer, Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 15816–15821. [12] Y.C. Kim, P.J. Ludovice, M.R. Prausnitz, Transdermal delivery enhanced by magainin pore-forming peptide, J. Control. Release 122 (2007) 375–383.

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