Cell-Penetrating Peptide-Patchy Deformable Polymeric Nanovehicles

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Cell-Penetrating Peptide-Patchy Deformable Polymeric Nanovehicles with Enhanced Cellular Uptake and Transdermal Delivery Daehwan Park, Jin Yong Lee, Heui Kyoung Cho, Woo Jin Hong, Jisun Kim, Hyemyung Seo, Ikjang Choi, Youngbok Lee, Juhyeon Kim, Sun-Joon Min, So-Hyun Yoon, Jae Sung Hwang, Kwang Jin Cho, and Jin Woong Kim Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00292 • Publication Date (Web): 30 May 2018 Downloaded from http://pubs.acs.org on May 30, 2018

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Cell-Penetrating Peptide-Patchy Deformable Polymeric Nanovehicles with Enhanced Cellular Uptake and Transdermal Delivery Daehwan Parka, Jin Yong Leea, Heui Kyoung Chob, Woo Jin Hongb, Jisun Kimc, Hyemyung Seoc,*, Ikjang Choia, Youngbok Leea,d, Juhyeon Kime,f, Sun-Joon Mind, So-Hyun Yoong, Jae Sung Hwangg,*, Kwang Jin Choh, Jin Woong Kima,d,* a

Department of Bionano Technology, Hanyang University, Ansan 15588, Republic of

Korea; b Cosmetic Research Center, Coway Co. Ltd., Seoul 08502, Republic of Korea;

c

Department of Molecular & Life Sciences, Hanyang University, Ansan 15588, Republic of Korea; d Department of Chemical and Molecular Engineering, Hanyang University, Ansan 15588, Republic of Korea;

e

Center for Neuro-Medicine, Korea Institute of

Science and Technology (KIST), Seoul 02792, Republic of Korea;

f

Department of

Chemistry, Korea University, Seoul 02841, Republic of Korea; g Department of Genetic Engineering, Kyung Hee University, Yongin 17104, Republic of Korea;

h

Damy

Chemical Co., Material Science Research Institute, Seoul 08501, Republic of Korea.

ABSTRACT We herein propose a polymeric nanovehicle system that has the ability to remarkably improve cellular uptake and transdermal delivery. Cell-penetrating peptide-patchy deformable polymeric nanovehicles were fabricated by tailored co-assembly of amphiphilic poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-b-PCL), mannosylerythritol lipid (MEL), and YGRKKRRQRRR-Cysteamine (TAT)-linked MEL. Using X-ray diffraction, differential scanning calorimetry, and nuclear magnetic resonance analyses, we revealed that the incorporation of MEL having an asymmetric alkyl chain configuration, was responsible for the deformable phase property of the vehicles. We also discovered that the nanovehicles were mutually attracted, exhibiting a gel-like fluid characteristic due to the dipole-dipole interaction between the hydroxyl group of MEL and the methoxy group of PEO-b-PCL. Co-

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assembly of TAT-linked MEL with the deformable nanovehicles significantly enhanced cellular uptake due to macropinocytosis and caveolae-/lipid raft-mediated endocytosis. Furthermore, the in vivo skin penetration test revealed that our TAT-patchy deformable nanovehicles remarkably improved transdermal delivery efficiency. KEYWORDS: Transdermal Delivery, Polymeric Nanovehicles, Controlled Phase Property, Cell-Penetrating Peptide

Introduction Transdermal delivery is a method of administering active ingredients through the skin.1,2 For the target active drugs to penetrate deeply into the skin, the barrier functions of the stratum corneum must be overcome. In terms of skin safety and delivery efficiency, one of the reasonably accessible transdermal delivery systems involves the use of nano-sized carriers designed

using amphiphiles,

including

biocompatible

surfactants3-5, lipids6-8, and

biopolymers9-11. To maximize the diffusivity of carriers through the skin layers, the particle size should be controlled within the range of nanometer length scales.12 In addition, in order for drug carriers to pass through the intercellular lipid membrane, it is important that they have a deformable or fluidizable phase.13 Therefore, liposome-based vesicle systems that meet these requirements are now widely used. However, as liposome vesicles are composed of a low-molecular-weight lipid surfactant, one fatal disadvantage is that it is difficult to maintain structural stability when a surfactant has a packing parameter of less than 0.5 or under an extreme solution environment.14,15 To overcome these limitations, many researchers, in recent years, have manipulated the phase crystallinity, flexibility, and deformability of nano-sized vehicles so that they could readily pass through the intercellular lipid layer of the stratum corneum.16-18 For example, deformable liposomes with a highly flexible lipid membrane structure can squeeze through the pores of the stratum corneum.19-21


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Advanced technologies employing active and passive delivery methods, such as microneedling22, iontophoresis23, electroporation24, and chemical enhancement25, have been developed to promote drug delivery throguh skin layers. Although chemical enhancement does not involve a high amount of drug delivery, it is still considered because it is not only safe for the skin but also does not require the use of complicated devices. In addition, if a chemical enhancement technique with active delivery capability is developed, the amount of drug delivery can be increased. Recent advances in transdermal delivery were achieved by exploitation of cell-penetrating peptides (CPPs).26-28 CPPs are short peptides that facilitate cellular uptake of DNA29, genes30, small chemicals31, and nanoparticles32. Although the entry mechanism of individual CPPs and CPP conjugates are still debatable33, CPPs have been successfully used to transport cargo to intracellular compartments of the cell. In particular, transactivating transcriptional activator (TAT) peptide has been widely studied and shown to efficiently penetrate cells.34-37 As TAT has positive charges stemming from arginine and lysine residues, it can easily reach the cell membrane due to electrostatic interactions.38 Taking advantage of this phenomenon, the patching of TAT on nanocarriers can lead to enhanced transdermal delivery.39,40 Through this study, we introduce a polymeric micelle-based nanovehicle system with enhanced cellular uptake and transdermal delivery efficiency. The polymeric nanovehicles were fabricated by tailored co-assembly of amphiphilic poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-b-PCL) and mannosylerythritol lipid (MEL). As MEL has an asymmetric molecular geometry, its incorporation endowed the polymeric nanovehicles with flexible and deformable phase properties. In order to enhance cellular uptake, the polymeric nanovehicles were also conjugated with CPP. For this purpose, MEL was chemically modified to yield a peptide linker moiety. Through thiol-maleimide reaction, TAT peptides containing a cysteamide at the terminal sequence formed covalent bonds at the



surface of the nanovehicles. Finally, in vitro cell internalization and in vivo transdermal delivery tests revealed that the polymeric nanovehicles exhibited significantly enhanced transdermal delivery efficiency and cellular uptake.

Experimental Materials. Poly (ethylene oxide)-b-poly (ε-caprolactone) copolymers (PEO-b-PCL) were kindly supplied from ACT (Korea). Mannosylerythritol lipids (MEL) was received from Damy Chemical (Korea). TAT peptiede (YGRKKRRQRRR-Cysteamide) was purchased from Peptron Co. (Daejeon, Korea). N, N-diisopropylethylamine (TCI, Japan) was used as a base. 4-nitrophenylchloroformate, methylene chloride, ammonium chloride (NH4Cl), Magnesium sulfate (MgSO4), acetonitrile, N-(2-aminoehtyl) maleimide trifluoroacetate salt, ethyl acetate (EtOAc), n-hexane were all purchased from Sigma-Aldrich (USA). Tetrahydrofuran (>98%, TCI, Japan) was used as a removable solvent. Retinol was used as a model drug (Retinol 50C, 50% mixture in Tween 20, BASF, Germany). For all experiments, deionized double distilled water was used. Synthesis of a MEL-based peptide linker. For conjugation of CPP to polymeric nanovehicles, MEL with a peptide linker moiety was synthesized by using two-step organic reaction. In the first step, a round-bottomed flask was charged with MEL (1 g), and was then purged with nitrogen. Anhydrous methylene chloride (32 mL) and pyridine (367 µL) and N, N-diisopropylethylamine (87 µL) were sequentially added to the flask. A solution of 4nitrophenylchloroformate (551 mg) in anhydrous methylene chloride (4 mL) was added to the mixture via syringe and the resulting solution was allowed to stir for 2 min at room temperature. To the reaction was added saturated (aq) NH4Cl, and the layers were separated. The aqueous layer was extracted twice with methylene chloride. The organic layers were


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combined, dried with MgSO4, filtered, and concentrated using a rotary evaporator. The resulting residue was purified by flash column chromatography on silica gel (EtOAc:nhexane=1:1) to afford orthoformate compound 1 (750 mg) as a colorless oil. In the second step, a round-bottom flask was charged with orthoformate compound 1 (100 mg). The flask was evaporated and refilled with nitrogen. Acetonitrile (2.8 mL) was added, followed by N, N-diisopropylehtylamine (73 µL). N-(2-aminoehtyl) maleimide trifluoroacetate salt (53 mg) was added in a single portion, and the reaction was allowed to stir for 12 h at room temperature. The solvent was the removed under reduced pressure. The residue was dissolved in methylene chloride and then concentrated using rotary evaporator. The resulting residue was purified by flash column chromatography on silica gel (EtOAc:n-hexane=1:2) to afford maleimide 2 (56 mg) as a colorless oil. Fabrication of TAP-patchy polymeric nanovehicles. To produce polymeric nanovehicles, PEO-b-PCL, MEL, and MEL-based peptide linker, were completely dissolved in THF via sonication for 10 min at 40 ˚C. Subsequently, DI water was added dropwise to the mixture while vigorously stirring. In this process, we used a syringe pump (Pump 11Elite, Harvard Apparatus, USA) with a flow rate of 100 µL min-1. The nanovehicles were produced by selfassembly of PEO-b-PCL, MEL, and MEL-based peptide linker. The concentration of PEO-bPCL and MEL were kept constant at 3 w/v% relative to the total volume of the nanovehicle dispersion. The concentration of MEL-based peptide linker was set to 200 µM. THF was completely removed from the dispersion by evaporation at 40 ˚C. Then, controlled amounts of TAT were added to the nanodispersion while gently stirring at room temperature for 12 h. The synthetic procedure is illustrated in Figure 1. Characterization of nanovehicles. The crystallinity of PEO-b-PCL/MEL mixtures was identified with an X-ray diffractometer (XRD, Rigaku, Japan) equipped with a twotheta/theta scanning mode, a 40kV/100mA X-ray, and a RINT 2000 wide angle goniometer.



In the measurement, 2θ was varied 5 ˚ to 40 ˚. The thermal property of PEO-b-PCL/MEL mixtures was measured by using a differential scanning calorimetry (DSC, Universal V4.3A, TA Instrument, USA). The heating rate was adjusted to 10 ˚C/min in the range from -50 ˚C to 100 ˚C under the nitrogen flow. The melting temperature for each sample was assigned by determining the heat flow. The particle size and zeta potential of polymeric nanovehicles were measured by dynamic light scattering (DLS, ELS-Z, Otsuka electronics, Japan) at 25 °C. To avoid any multiple light scattering, all the nanovehicle samples were diluted to 0.3 wt% of dispersion solid concentration in water before the measurement. The average particle size and standard deviation were obtained after the measurement was repeated three times. The morphology of emulsion drops was analyzed with a transmission electron microscope (TEM, Energy-Filtering Transmission Electron Microscope, LIBRA 120, Carl Zeiss, Germany) operating at 120 kV. Before TEM observation, all test samples were negatively stained with 1 wt% uranyl acetate. Dense suspension rheology measurements. The particle-to-particle interaction was observed via dense suspension rheology. For this, a DHR-1 rheometer (TA instrument, USA) was used in the stress-control mode with a cone-plate geometry, of which diameter was 40 mm and the angle was 2˚. The concentration of polymeric nanovehicles in water was adjusted to 20 w/v% by evaporating water. Before operation of the rheometer, the dispersion was equilibrated before the measurement for 10 min. To prevent any evaporation of water from the dispersion system, a solvent trap was equipped around the test sample. The suspension rheology was measured by running flow sweep, oscillation amplitude. The flow sweep measurement was conducted at a shear rate ranging from 0.001 to 100. A strain range of oscillation amplitude was 0.01-100 %. NMR analyses. All of the NMR measurements were performed using a Bruker 400 MHz NMR spectrometer equipped with a broadband probe containing three pulsed field gradients


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(Bruker Biospin, Billerica, MA) at a temperature of 298 K. The NMR data were processed using the TOPSPIN 3.5 program (Bruker Biospin, Billerica, MA). Chemical shifts were calibrated with an internal standard (trimethylsilyl propanoic acid (TMSP) signal at 0 ppm). T2 relaxation analysis was conducted to characterize structural organization of nanovehicles composed of PEO-b-PCL, MEL, and D2O. We carried out nuclear overhauser effect spectroscopy (NOESY) to identify the spatial distance between hydrogens. Using diffusion ordered spectroscopy (DOSY), we also determined the diffusion coefficients of water molecules in the continuous phase and nanovehicles in the dispersion system (see ESI for more experimental details). Fibroblast cell culture and nanovehicle treatment. Human skin fibroblast cell GM01651 was provided from Coriell Cell Repository (Camden, NJ) and cultured in Minimum Essential Medium with Earle’s (MEM/EBSS; Hyclone, Logan, UT) supplemented with 15% fetal bovine serum (FBS; Gibco, Rockville, MD), 2mM L-glutamine (Gibco, Rockville, MD), 0.1mM nonessential amino acid (Sigma, St. Louis, Mo), and 1% penicillin-streptomycin at 37°C under 5% CO2 condition. All cell culture plates or cover-glasses were coated with 0.1% gelatin prior to experimental cell plating. Nanovehicles were diluted using PBS solution before use. For the treatment of nanoparticle to cultured fibroblast cells, media were removed from cell culture plates and then cells were incubated in 200 µl of nanovehicle solution for 1 hour in 96 well plates (3.5 x 103 cells) or 48 well plates (8.2 x 103 cells). After 2 times of washing with Dulbecco’s Phosphate-Buffered Saline (DPBS; Welgene, Korea), fluorescence was measured at 552nm using microplate reader (BioTek, Winooski, VT). For immunocytochemistry, cover-glasses of cultured cells were fixed with 3.7% formaldehyde for 15 min and washed twice using DPBS solution. Fluorescence images were taken using Zeiss LSM800 Confocal microscope (ZEISS, Oberkochen, Germany).



In vivo transdermal delivery study. Confocal Raman spectroscopy set-up was conducted for evaluation of in vivo transdermal delivery performance in a manner similar to that previously reported.11 The topical delivery experiments were performed on the volar forearms of four female subjects with age ranging from 24 to 40 years under standardized conditions. The experiments were conducted according to the Good Clinical Practices guidelines and regulations on the Human Study of Health & Functional Food from the Ministry of Food and Drug Safety in Korea. Three test samples were prepared; propylene glycol/ethanol (1/1, v/v), PEO-b-PCL vehicles only, and TAT-patchy PEO-b-PCL/MEL nanovehicles (ϕMEL=0.2, TAT=100 µM). All the sample formulations contained 0.3 w/v% of retinol. The concentration of nanovehicles in the dispersion was set to 3 w/v%. At exactly 30 min after topical application, the Raman spectra were collected from the skin surface to 24 µm below with 3 µm intervals and seven scans were collected for each treatment site at each time point. Detecting the characteristic Raman band of retinol at 1594 cm-1, we determined the retinol intensity values from the relative area of this band from the surface to 12 µm deep in the skin.

Results and discussion For the fabrication of polymeric nanovehicles with a deformable phase property and cellpenetrating capability, the core phase of polymeric nanovehicles was softened by incorporation of MEL and their surface was tagged with TAT peptide linkers, as schematically illustrated in Figure 1. MEL has an asymmetric molecular geometry from two hydrophobic alkyl chains with different carbon numbers.18 Because of this unique molecular structure, co-assembly of MEL with PEO-b-PCL effectively blocks the molecular affinity of the PCL block, thus making the hydrophobic core deformable. In addition, to introduce TAT on the nanovehicles, we synthesized a MEL-based peptide linker by modifying a hydroxy


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group of MEL with a maleimide (Figure 2A). Successful synthesis of the MEL-based peptide linker was confirmed by proton nuclear magnetic resonance (1H-NMR) analysis (Figure 2B– C). The product yield reached ~34.2%. As the peptide linker still retained the amphiphilic properties, and the amount added was less than 200 µM, it had little effect on the formation of the polymer nanovehicles, which was evident as there was no difference in particle size and zeta potential before and after the introduction of the peptide linker (Figure S1). Then, the TAT peptide, with cysteamide group at the terminus, was added into the dispersion of peptide linker-decorated polymeric nanovehicles, allowing the thiol-maleimide condensation reaction to occur and consequently producing positively charged, TAT-conjugated polymeric nanovehicles (the zeta potential shifted to ~7 mV, see Figure S1).

Figure 1. Schematic illustration of the fabrication of polymeric nanovehicles for transdermal delivery by co-assembly of amphiphilic copolymer with biocompatible lipid (MEL) and CPP.

The particle size of the polymeric micellar vehicles decreased from ~80 nm to ~40 nm with an increased weight fraction of MEL in the PEO-b-PCL/MEL mixture, ϕMEL, of 0.2 (Figure 3A). In fact, MEL lowered the interfacial tension weakly, but effectively, because of its surfactant-like molecular structure (Figure S2A). When ϕMEL > 0.2, the particle size rather



increased, which means that there must have been some changes in the colloidal state. In order to exactly confirm this, we observed the particle morphology with varying ϕMEL using transmission electron microscopy (TEM). The bare PEO-b-PCL nanovehicles showed a spherical particle shape with an average particle diameter of ~75 nm (Figure 3B). Maintaining the spherical particle shape means that it formed a solid phase enough to withstand the strain coming from the capillary force in the process of drying. Considering the hydration thickness, the particle size obtained from the TEM image was similar with the particle size obtained using dynamic light scattering method. The introduction of MEL not only led to the formation of smaller vehicles with a homogeneous phase but also to deformed particle shape (Figure 3C), implying that the vehicle phase has become more flexible so that it could not withstand the capillary force. Interestingly, when excess MEL was introduced (ϕMEL = 0.5), new particles with a diameter of 40–80 nm were observed (Figure 3D). Their phase contrast and particle shape were different from those of the vehicles produced at ϕMEL < 0.2. It appears that excess MEL formed new nanoscale droplets of MEL. In order for such particle-drop splitting phenomenon to occur, the spreading coefficient (S) must have a negative value. For the three-phase system consisting of PEO-b-PCL/MEL/water, the S value was measured to be -29.3 mN/m, which directly explains why new MEL droplets were generated (Figure S2B).


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1

Figure 2. (A) Synthesis of a MEL-based peptide linker. H-NMR spectra (B) before and (C) after incorporation of maleimide peptide linker to MEL.



Figure 3. (A) Hydrodynamic particle size of polymeric nanovehicles prepared with varying ϕMEL against total mass of PEO-b-PCL/MEL. TEM images of polymeric nanovehicles; (B) ϕMEL = 0, (C) ϕMEL = 0.2, and (D) ϕMEL = 0.5.

To investigate how the introduction of MEL affected the phase property of the vehicles in the concentration regime where a morphologically homogeneous phase was observed from the TEM images, we carried out X-ray diffraction (XRD) and differential scanning calorimetry (DSC) analyses (Figure 4A–B). In the XRD spectra of the PEO-b-PCL sample,


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the characteristic crystal peaks of PCL blocks were detected at 2θ = 21.3° and 23.4°.41,42 Strong peaks at 2θ = 19.0° and 23.2° from methoxy-terminated PEO were also observed.41,42 The peaks at 23.2° and 23.4° were superimposed. As ϕMEL increased, the peak intensity was gradually weakened. The crystallinity, calculated from XRD spectra, was noticeably reduced from 38% to 16% when ϕMEL increased to 0.4. In the DSC spectra, the melting point of the PCL block shifted from 56.6°C to 54.3°C and the PEO block from 52.5°C to 47.3°C,42 which were consistent with the XRD results. The heating enthalpy of the PEO-b-PCL/MEL with ϕMEL = 0.3 decreased by 67.8% compared with that of PEO-b-PCL. An interpretation of these results is that considerably looser molecular assembly of PEO-b-PCL by virtue of asymmetric molecular geometry of MEL could be obtained with increasing ϕMEL. In order to identify the phase property of the nanovehicles in the suspension state, we also conducted 1H-NMR T2 relaxation analysis, which allows us to obtain structural and dynamic information on polymeric nanomicellar phases in an atomic resolution (Figure 4C, see also ESI). The T2 relaxation time of the PEO-b-PCL/MEL vehicles increased linearly up to ϕMEL = 0.3. It was obvious from this tendency that the presence of MEL in the micellar core dissociated the PCL block domain, thus making it deformable. However, when ϕMEL > 0.3, the T2 relaxation time rather decreased due to the generation of new MEL drops, which is the same result as that shown by TEM. These results suggest that the nanovehicles can be successfully softened by incorporating a controlled amount of MEL into the homogeneously mixed PEO-b-PCL/MEL phase.



Figure 4. (A) XRD spectra and (B) DSC curves of PEO-b-PCL/MEL mixtures with varying ϕMEL; (a) ϕMEL = 0, (b) ϕMEL = 0.05, (c) ϕMEL = 0.1, (d) ϕMEL = 0.2, and (e) ϕMEL = 0.3. These experiments proceeded in the bulk film state. (C) T2 relaxation time of PEO-b-PCL/MEL nanovehicles with varying ϕMEL.

We also investigated how the incorporation of MEL affected vehicle-to-vehicle interactions through dense suspension rheology studies. In the absence of MEL, the dispersion of bare PEO-b-PCL nanovehicles showed a shear rate-independent viscosity. Conversely, the co-assembly of MEL with PEO-b-PCL increased the viscosity of the dispersion by approximately 10 fold at the low shear rate regime, while exhibiting typical shear thinning at the high shear rate regime (Figure 5A). This implies that the vehicles interacted with each other to form a particular gel structure in the low shear rate regime. However, as the shear rate increased, the gel bonds were broken, resulting in a gel-to-sol transition. For further clarification, we carried out oscillatory rheology measurements (Figure 5B). The storage (G') and loss (G'') moduli of the dispersion of PEO-b-PCL/MEL vehicles were much higher than those of the bare PEO-b-PCL vehicles. It was also remarkable that the dispersion of PEO-b-PCL/MEL vehicles exhibited a typical elastic fluid behavior, as G' > G''. In the absence of MEL, a typical viscous fluid behavior, corresponding to G' < G'', was


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observed. Again, these results demonstrated well that the MEL at the surface of the vehicles induced a mutual attraction between them, thus forming a gel-analogous dispersion fluid.

Figure 5. Rheological behaviors of concentrated PEO-b-PCL/MEL vehicles. (A) Viscosity behavior as a function of shear rate. (B) Storage modulus, G' (closed symbol), and loss modulus, G'' (open symbol), as a function of oscillation strain. Concentration of PEO-bPCL/MEL vehicles was set to 20 wt%.

To quantify the fluidity of the continuous phase and that of the nanovehicles, we tracked the protons of the water molecule and the PEO block in the vehicle, respectively, using diffusion-ordered spectroscopy (DOSY) NMR analysis. This approach allowed us to obtain the diffusion coefficients of the continuous phase and of the nanovehicles. The diffusion coefficient (D) is expressed by the Stokes-Einstein equation,43 D = KT / 6πηa, where K is the Boltzmann constant, T is the temperature, η is the viscosity, and a is the particle radius. The diffusion coefficient of water molecules decreased as ϕMEL increased (Figure 6A) because the nanovehicles acted as impediments.44 In the case of the nanovehicles, however, the diffusion



coefficient did not show any significant change until ϕMEL = 0.3, but fell sharply after 0.3. Based on the Stokes-Einstein equation, it is reasonable to say that the incorporation of ϕMEL, which decreased the particle size of the nanovehicles (see Figure 3A), may increase the diffusion coefficient. However, there was no change in the diffusion coefficient as the viscosity of the dispersion system itself was increased to a similar level. To further clarify the fundamental driving force of this unique fluidity behavior, we characterized the spatial distance between hydrogens in the dispersion of the nanovehicles using two-dimensional 1H1

H nuclear Overhauser effect spectroscopy (NOESY) NMR analysis (Figure 7). The result

represented that several intra- and/or inter-molecular NOEs of vehicle-to-vehicle were noticeable detected, especially NOE between the methoxy group of PEO-b-PCL (3.70 ppm) and the hydroxyl group of MEL (0.81 ppm). This implies that the spatial distance between the hydrogens in the hydroxyl group of MEL and the methoxy end group of PEO was less than ~5 Å that is a close enough distance to induce the attraction between PEO-b-PCL/MEL nanovehicles due to the dipole-dipole interaction. This interaction did not seriously affect the long-term dispersion stability of nanovehicles because it occurs not permanently but temporarily (see Figure S3).


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Figure 6. DOSY NMR analysis for the determination of the diffusion coefficient of (A) water molecules and (B) PEO-b-PCL/MEL vehicles with varying ϕMEL. In these observations, the vehicles were diluted to 3% w/v in water.



Figure 7. 2D 1H-1H NOESY spectra of (A) PEO-b-PCL nanovehicles and (B) PEO-bPCL/MEL nanovehicles. NOEs of (C) PEO-b-PCL nanovehicles and (D) PEO-b-PCL/MEL nanovehicles. Only for PEO-b-PCL/MEL nanovehicles, inter- and/or intra-particular NOEs were detected between the methoxy group of PEO-b-PCL and the hydroxyl group of MEL.

After introducing a small amount of MEL-based peptide linker and a fluorescent probe (Nile red) into the deformable PEO-b-PCL/MEL nanovehicles, TAT, with cysteamide group at the


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terminus, was conjugated with the linker to produce TAT-patchy deformable nanovehicles (Figure 8A, Figure S4). Then, their ability to be internalized into living cells was measured using in vitro cultured human skin fibroblasts. The deformable nanovehicle treatment showed an increased level of cellular uptake (>2 folds) compared to bare nanovehicle treatment (Figure 8B-C). This result indicated that treatment using the nanovehicles with soft phase property increased the cell permeability of the nanovehicles. The treatment with 100 µM TAT showed a 30-fold increase in cellular uptake compared to that observed with bare nanovehicles,

suggesting

that the potential cellular induction mechanisms

were

macropinocytosis and caveolae-/lipid raft-mediated endocytosis.45 However, treatment with 200 µM TAT showed reduced levels of cellular uptake compared to treatment with 100 µM TAT, which can be explained by cellular toxicity, the threshold limit of peptide translocation, or the inhibition of clathrin-mediated endocytosis.46 Based on these in vitro cell internalization results, we optimized the concentration of the TAT treatment to 100 µM for the next experiment.



Figure 8. (A) Chemical reaction of cysteamide ended TAT with MEL linker. (B) Cellular uptake of nanovehicles containing Nile red fluorescent probe. The value of Nile red assay obtained by flow cytometry was expressed with changing compositions of nanovehicles; (a) ϕMEL = 0, (b) ϕMEL = 0.2, ϕMEL = 0.2 with TAT at (c) 50 µM, (d) 100 µM, and (e) 200 µM. (C) Fluorescence microscopy image of fibroblast cells treated with 100 µM TAT. (D) Decay constant associated with the absorption coefficient of the retinol drug into the skin; (a) propylene glycol/ethanol, (b) bare PEO-b-PCL nanovehicles (ϕMEL = 0), and (c) TAT-patchy deformable PEO-b-PCL/MEL nanovehicles (ϕMEL = 0.2, TAT = 100 µM).

In order to explore more practical applications of the CPP-patchy deformable nanovehicles, we evaluated their transdermal delivery performance by measuring human skin topical permeability in vivo using confocal Raman spectroscopy. Three test samples were prepared: 1) propylene glycol/ethanol controls, 2) bare nanovehicles, and (3) TAT-patchy deformable nanovehicles. These samples contained the same amount of retinol as a probe drug


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Biomacromolecules

(0.3 wt% in the test formulation). In the Raman spectra collected from the skin surface at 24µm depth with the 3-µm depth intervals, the depth profiles of the retinol probe were obtained. Then, the decay constant was determined from the exponential Arrhenius function47,

y = × ⁄ + , where A1 is the amplitude corresponding to the quantity of product on the skin surface and t1 is the decay constant associated with the absorption coefficient of the retinol during penetration into the skin (Figure 8D, Figure S5). The t1 of bare vehicles was higher than that of the propylene glycol/ethanol control, implying that small nanovehicles of a size of ~80 nm have a sufficiently high diffusivity enabling them to exhibit better transdermal delivery performance than the chemical enhancing effect. It is remarkable that TAT-patchy deformable vehicles showed an increase in t1 of more than 110% over the propylene glycol/ethanol control, and 60% over the bare vehicles (Table S1). These results indicated directly that these nanovehicles with deformable phase characteristics and TATmediated intercellular adhesion capability serve as an excellent alternative for transdermal delivery.

Conclusion In summary, this study introduced an approach to fabricate polymeric nanovehicles for enhanced cellular uptake and transdermal delivery. The key strategy used in this study was the softening of the vehicle phase and patching of CPP on the surface of the vehicles. We successfully showed that the incorporation of an appropriate amount of MEL into the PEO-bPCL vehicles was effective not only in reducing the tight molecular association of the vehicle core phase, but also in forming a gel-like dispersion fluid due to the intervehicular association by dipole-dipole attractions. Furthermore, we observed that patching TAT onto the deformable nanovehicles dramatically increased the cellular uptake, which was attributed to



both macropinocytosis and caveolae-/lipid raft-mediated endocytosis. From in vivo skin permeation tests using confocal Raman spectroscopy, we showed that our nanovehicles remarkably improved transdermal delivery performance. These results highlight the practical applications of the CPP-patchy deformable polymeric nanovehicles in the cosmetics and dermal medicine industries.

ASSOCIATED CONTENT Supporting Information. Full experimental details about 1H NMR analysis and measurement of dynamic interfacial tension for characterizing structural organization of nanovehicles. More information and details about zeta potential of nanovehicles, interfacial tension of polymer mixtures, 1H NMR spectra for characterizing peptide conjugation, and Raman spectra for transdermal delivery performance. AUTHOR INFORMATION Corresponding Author *email: [email protected] *email: [email protected] *email: [email protected]

ACKNOWLEDGMENT This research was supported by Coway Co. Ltd. and by National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIP) (2016R1A2B2016148, 2018M3A9G2057170).

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