Design of Multifunctional Liposomal Nanocarriers for Folate Receptor

Article pubs.acs.org/molecularpharmaceutics

Design of Multifunctional Liposomal Nanocarriers for Folate Receptor-Specific Intracellular Drug Delivery Min Hyung Kang,† Hyun Joon Yoo,† Yie Hyuk Kwon,† Ho Yub Yoon,† Sang Gon Lee,† Sung Rae Kim,† Dong Woo Yeom,† Myung Joo Kang,‡ and Young Wook Choi*,† †

College of Pharmacy, Chung-Ang University, 221 Heuksuk-dong, Dongjak-gu, Seoul 156-756, Korea College of Pharmacy, Dankook University, Cheonan-Si, Chungnam 330-714, Korea

‡

S Supporting Information *

ABSTRACT: As a novel carrier for folate receptor (FR)-targeted intracellular delivery, we designed two types of targetable liposomal systems using Pep-1 peptide (Pep1) and folic acid as a cellpenetrating peptide (CPP) and target molecule, respectively. Folate-linked Pep1 (Fol-Pep1) was synthesized by solid phase peptide synthesis (SPPS) and verified using 1H NMR and farultraviolet (UV) circular dichroism (CD). The chimeric ligand (Fol-Pep1)-modified liposome (cF-P-L) was prepared by coupling Fol-Pep1 to maleimide-derivatized liposomes at various ratios. The dual ligand (folate and Pep1)-modified liposome (dF/P-L) was prepared by separately attaching both ligands to the liposomal surface via a short (PEG2000) or long (PEG3400) linker. The physical and conformational characteristics including vesicle size, zeta potential, and the number of conjugated ligands were determined. Intracellular uptake specificities of various fluorescent probe-containing cF-P-L and dF/P-L systems were assessed using FR-positive HeLa and FR-negative HaCaT cells. Cellular uptake behavior was visualized by confocal laser scanning microscopy (CLSM). Internalization was time-dependent. Fol-Pep1 and Pep-1 cytotoxicities were negligible up to 25 μM in FR-positive and FR-negative cells. Empty cF-P-L and dF/P-L were nontoxic at the concentration used. The optimized dF3/P2(450/90) system carrying 450 PEG3400-linked folate and 90 PEG2000-linked Pep1 molecules could be a good candidate for FR-specific intracellular drug delivery. KEYWORDS: liposome, cell penetrating peptide, drug targeting, Pep-1, dual-ligand, folic acid, intracellular delivery

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INTRODUCTION Liposome systems have received much attention as drug carriers owing to their biocompatibility and applicability. The functional properties of a liposome can be regulated by surface modification including polymeric substitution, attachment of monoclonal antibodies, and incorporation of charged lipid derivatives, proteins, or peptide seer. Cell-penetrating peptides (CPPs) have been introduced to achieve efficient intracellular drug delivery. CPPs are relatively short peptides (less than 30 amino acids) with a net positive charge that translocates across the plasma membrane of eukaryotic cells, thereby successfully delivering not only small molecular drugs but also large molecular therapeutics and particulate carriers into cells.1−3 Pep-1 peptide (Pep1; 21mers, KETWWETWWTEWSQPKKKRKV) is one of the most common CPPs including TAT, polyarginine, penetratin, and transportan. This peptide efficiently facilitates the delivery and intracellular localization of a range of peptides, proteins, genes, and nanoparticles into a broad spectrum of cell lines via the nonendocytic pathway. In addition, it has several advantages, such as rapid and highly efficient intracellular delivery, stability in physiological buffer, lack of toxicity, and lack of sensitivity to serum. Previously, we have demonstrated that the Pep1-conjugated © XXXX American Chemical Society

liposome system successfully translocated entrapped macromolecules into cells.4 However, CPP-mediated intracellular translocations are not cell or tissue specific. To overcome this drawback, several strategies have been implemented to generate specificity by introducing targeting ligands that can distinguish cancer from normal cells. For targeted delivery to tumors, the cellular target often consists of a tumor-associated biomarker that is membrane-bound and overexpressed in tumor cells. Targeted drug delivery is attributed to the interaction between target molecules and receptors. Folic acid has been extensively used as a targeting molecule of the folate receptor (FR), which is restrictively expressed in normal cells but has high expression in various malignant tumors of epithelial origin.5 Folate recognition by FR facilitates the delivery of folic acid-tethered substances via receptor-mediated endocytosis. For example, folate-conjugated carboplatin, vinca alkaloid, and paclitaxel were developed as folate-conjugated chemotherapeutic Received: May 21, 2015 Revised: October 22, 2015 Accepted: October 29, 2015

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DOI: 10.1021/acs.molpharmaceut.5b00399 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics agents.6−8 In addition, various types of drug carriers such as liposomes, lipid nanoparticels, micelles, and dendrimers have been folate-conjugated to induce endocytosis of cargoes into FR-expressing cells.9 Recently, the combination of targeting moieties and CPPs into a single system has been studied for successful drug targeting. Such strategies include multifunctional drug delivery systems, which are fabricated with multiple ligands or a chimeric ligand. The cyclic RGD peptide, a specific ligand with affinity for integrin αvβ3, and octaarginine were coupled to a liposome vesicle to enhance intracellular delivery with improved cell selectivity.10 The cell-penetrating homing peptide (CPHP) is a common type of chimeric ligand, formed by the linear linkage of CPP and a targeting moiety.11 The use of CPHP to enhance specific intracellular drug delivery has been introduced. In our previous study, the RIPL peptide (IPLVVPLRRRRRRRRC) was developed as a hepsin-selective CPHP and conjugated to liposomes in various ligand ratios, revealing that cellular uptake was optimized at 1% conjugation with RIPL peptides.12 In this study, we designed two types of targetable liposomal systems using Pep1 and folic acid as a CPP and a target molecule, respectively. First, folate-linked Pep1 (Fol-Pep1) was synthesized as a chimeric ligand and conjugated to liposomes via a thio-maleimide reaction to prepare a chimeric ligand (Fol-Pep1)-modified liposome (cF-P-L), in which varying amounts of Fol-Pep1 molecules were attached to the liposomal surface. Second, Pep1 was conjugated to folate-tethered liposomes to prepare a dual ligand (folate and Pep1)-modified liposome (dF/P-L), in which both ligands were separately attached to the liposomal surface via a short or long linker. Liposomal systems were optimized in terms of linker length and number of ligands. In addition, the translocation efficiency and specificity of various cF-P-L and dF/P-L systems were investigated using FR-positive HeLa and FR-negative HaCaT cells.

RPMI 1640 medium, penicillin−streptomycin, fetal bovine serum, and trypsin−EDTA (0.25%) were purchased from Invitrogen (Carlsbad, CA, USA). LysoTracker Red DND-99 was purchased from Molecular Probes, Inc. (Eugene, OR, USA). All other reagents and chemicals obtained from commercial sources were of cell culture or analytical grade. Synthesis of Pep1 and Fol-Pep1. Pep1 was synthesized by Fmoc solid phase peptide synthesis (SPPS) using ASP48S (Peptron, Daejeon, Korea). For synthesis from the C-terminus of Pep1, NH2-Cys(Trt)-2-chlorotrityl resin was used as the first amino acid. Fmoc-Val-OH was added in excess under the presence of a coupling reagent, which was composed of HBTU/ HOBt/NMM (1:1:2 molar ratio) in DMF. The mixture was reacted for 2 h at room temperature and was washed with DMF, MeOH, and DMF in serial order. For the Fmoc deprotection, 20% piperidine in DMF was added to the reactant, reacted for 5 min, and washed with DMF, methanol, and DMF in series. The remainder of the peptide sequence was synthesized by repeating the above procedure one by one. The completed peptide backbone was cleaved from the resin by adding TFA/ EDT/thioanisole/TIS/H2O (90:2.5:2.5:2.5:2.5 in molar ratio). Synthesized Pep1 was purified by reverse phase high-performance liquid chromatography (HPLC) using a C18 column (250 mm × 22 mm, 10 μm; Grace Vydac, Hesperia, CA, USA). Gradient elution was performed with 0.1% (v/v) trifluoroacetatecontaining acetonitrile from 10% to 75% (v/v). The molecular weights (MW) of the peptides were determined using liquid chromatography/mass spectrometry (LC/MS; Agilent HP1100 series, Palo Alto, CA, USA), which operated in the electrospray positive ion mode with the following settings: LC flow, 0.4 mL/min; drying gas flow, 8 L/min; nebulizer pressure, 35 psig; nebulizer temperature, 350 °C; capillary voltage, 4.0 kV. Fol-Pep1 was synthesized separately by coupling glutamic acid and pteroic acid to Pep1 (Scheme 1). Fmoc-Glu(OH)-OtBu (Fmoc-L-glutamic acid 5-tert-butyl ester) was reacted with Pep1attached resin by the same coupling procedure as described above. After conjugation of glutamic acid, the N-terminus’ Fmoc group was released with 20% piperidine in DMF. Pteroic acid was added in excess in the presence of the coupling reagent HBTU/HOBt/NMM. The procedures for the subsequent backbone cleavage from the resin complex, peptide isolation by HPLC, and MW determination by LC/MS were the same as described above. Characterization of the Peptides. The presence of Pep1, Fol-Pep1, and folic acid in dimethyl sulfoxide-d6((CD3)2SO) were confirmed by 600 MHz 1H NMR spectroscopy (Bruker; New Orleans, LA, USA), and the results were analyzed by the MestRenova software (Mestrelab Research, Santiago de Compostela, Spain). Far-ultraviolet (UV, 190−260 nm) circular dichroism (CD) spectra were obtained from the peptides to analyze their secondary structure. The peptide concentration was 1 mg/mL in a cuvette of 0.1 cm path length, and the spectra were recorded at room temperature using a CD detector (Chirascan-plus, Applied Photophysics, Leatherhead, U.K.) with a bandwidth of 1 nm. Data were expressed in molar ellipticity values and plotted against the scanned wavelength. Secondary structural contents were estimated from the ellipticities by K2D3 protein secondary structure prediction (http://k2d3.ogic.ca/).13 The concentrations of the peptides were determined by HPLC. The HPLC system consisted of a Waters UV detector (W2489), pump (W2690/5), and data station (Empower3) (Waters; Milford, MA, USA). The chromatographic separation was performed using a C18 column (Shiseido, Tokyo, Japan) at 4 °C

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EXPERIMENTAL SECTION Materials. Soy phosphatidylcholine (PC), polysorbate80 (Tween 80), fluorescein dextran isothiocyanate (FITC-dextran, 4 kDa), and 4-(2-hydroxyethyl)-1-piperazineethanesul-fonic acid (HEPES) were purchased from Sigma-Aldrich (St Louis, MO, USA). The Pep-1 peptide (Pep1; KETWWETWWTEWSQPKKKRKVC, 22mer) and folate-linked Pep1 peptide (Fol-Pep1; folate-KETWWETWWTEWSQPKKKRKVC) were synthesized by Peptron Co. (Daejeon, Korea). All amino acids (FmocLys(Bco)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Glu(OH)-OtBu), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), N-hydroxybenzotriazole (HOBt), 4-methylmorpholine (NMM), dimethylformamide (DMF), trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT), thioanisole, and triisopropylsilane (TIS) were purchased from Anaspec (San Jose, CA, USA). A poly(ether sulfone) ultrafiltration membrane was purchased from EMD Millipore (Billerica, MA, USA). Distearoylphosphatidyl ethanolaminepolyethylene glycol-maleimide (DSPE-PEG-Mal: DSPEPEG2000-mal, DSPE-PEG3400-mal) and distearoylphosphatidyl ethanolamine-polyethylene glycol-folate (DSPE-PEG-Fol: DSPE-PEG2000-fol, DSPE-PEG3400-fol) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). For cell culture experiments, the human uterine cervical cancer cell line (HeLa) and human keratinocyte cells (HaCaT) were purchased from the Korean Cell Line Bank (Seoul, Korea). B


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Molecular Pharmaceutics Scheme 1. Synthesis Procedure of Folate-Linked Pep-1 Peptide (Fol-Pep1)a

To protect the α-carboxyl group in folic acid, pteroic acid was conjugated with glutamic acid-derivatized Pep1-resin via the γ-carboxyl group. Abbreviations: HBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOBt, N-hydroxybenzotriazole; NMM, 4-methylmorpholine (NMM); DMF, dimethylformamide; TFA, trifluoroacetic acid; EDT, 1,2-ethanedithiol; TIS, triisopropylsilane. a

with a flow rate of 1.0 mL/min and a mobile phase of 0.1% TFA in water (eluent A) and 0.1% TFA in acetonitrile (eluent B). The eluent gradient ramped from 10 to 60% B in 50 min and subsequently back to 10% B over 5 min. Twenty microliters of each sample were injected into the column followed by UV detection at 220 nm. Preparation of the Pep1-Conjugated Liposome (P-L). The Pep1-conjugated liposome (P-L) was prepared by conjugating Pep1 to vesicles via the thiol-maleimide reaction. Briefly, liposomes were prepared by the thin film hydration method.14 PC, Tween80, and DSPE-PEG2000-maleimide were dissolved at a molar ratio of 90:10:0.1 in methanol and chloroform (1:1) in a round-bottom flask. The organic solvent was removed using a rotary vacuum evaporator at a temperature higher than that of the lipid transition, and solvent traces were completely removed under nitrogen gas. The lipid film was subsequently hydrated with 1 mL of pH 7.0 phosphate-buffered saline (PBS) containing 10 mg/mL of FITC-dextran, resulting in 7.16 mM of total molar initial concentration of the liposomal constituents. The liposomal solution was extruded with an Avanti Mini-Extruder (Alabaster, AL, USA) with 10 passes through a 200 nm poly(ether sulfone) membrane for efficient entrapment and homogeneous size distribution. Excess amount of Pep1 was added and allowed to react for 12 h at room temperature. P-L was isolated from untrapped FITC-dextran and free Pep1 by dialysis against distilled water using a cellulose ester dialysis membrane (50,000 MWCO) for 48 h. After dialysis, final total molar concentration of the liposomal stock solutions was controlled to 1.432 mM by replenishing the purified liposomes with PBS. For comparison, elastic liposomes (EL) were prepared as described above, excluding the addition of DSPE-PEG2000-maleimide and Pep1. Preparation of the Folate-Tethered Liposome (F-L). The folate-conjugated liposome (F-L) was prepared by the film hydration and size-extrusion process as described above. PC, Tween80, and DSPE-PEG2000-fol were dissolved (90:10:0.5) in methanol and chloroform (1:1). The following steps were the same as that described for the P-L preparation method, excluding the addition of Pep1. Preparation of the Chimeric Ligand-Modified Liposome (cF-P-L). As illustrated in Figure 1, four types of cF-P-L were prepared by varying the number of Fol-Pep1 ligands: cF-P2(9), cF-P2 (45), cF-P2 (90), and cF-P2 (450), where the subscript “2” represents the length of the linker used (PEG2000),

and the numeric value in parentheses the number of conjugated ligands (Fol-Pep1). Briefly, PC and Tween80 (90:10 molar ratio) were dissolved in a mixture of methanol and chloroform (1:1) in a round bottom flask. To control the number of maleimide groups, DSPE-PEG2000-mal was added at 0.01−0.5% of the liposomal constituents. Film hydration and extrusion were performed as described above. Subsequently, Fol-Pep1 was added in excess and allowed to react for 12 h at room temperature. Purification and further steps were performed by the same procedure as described for the P-L preparation above. Preparation of the Dual Ligand-Modified Liposome (dF/P-L). By controlling the linker length of folate and Pep1, four types of dF/P-L were prepared: dF2/P2(450/90), dF3/ P2(450/90), dF3/P3(450/90), and dF2/P3(450/90) (Figure 1). Moreover, by controlling the number of conjugated Pep1 ligands, three types of dF/P-L were prepared additionally: dF3/P2(450/9), dF3/P2(450/450), and dF3/P2(450/900), where the subscript “2” and “3” represent the length of linker used (PEG2000 or PEG3400) and the numeric ratios in parentheses represent the number of ligands as the folate to Pep1 ratio. For example, the liposome containing 450 folate molecules with the PEG2000 linker and 90 Pep1 molecules with the PEG3400 linker was designated as dF2/ P3(450/90). Briefly, PC, Tween80, DSPE-PEG-Fol, and DSPEPEG-MAL were dissolved at a molar ratio of 90:10:0.5:0.1−1 in a mixture of methanol and chloroform (1:1). The evaporation, hydration, and extrusion processes were performed as described above. The Pep1 peptide was subsequently conjugated to the maleimide group for 12 h, and the number of conjugations was controlled by adjusting the amount of DSPE-PEG-Mal that was initially added. Purification and further steps were performed by the same procedure as described for the P-L preparation above. Prepared samples were stored under ambient condition and used for the experiments within a week. Conformational Characterization of Ligand Conjugation. The extent of ligand conjugation was determined by HPLC assay as previously reported.4,15 For Pep1 and Fol-Pep1 conjugation, indirect quantification was applied by determining the amount of uncoupled Pep1 and Fol-Pep1 during liposome preparation. Briefly, 3-fold molar amount of cysteine was added to block the unreacted maleimide groups on liposomal vesicle. After 30 min incubation, unreacted cysteine was reacted with Ellman’s reagent (5,5′-dithiobis(2-nitrobenzoic acid)), leading to the formation of a cysteine-TNB (5-thio-2-nitrobenzoic acid) adduct to C


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Molecular Pharmaceutics

particle size analyzer (Zetasizer Nano-ZS; Marlvern Instrument, Worcestershire, U.K.) equipped with a 50 mV laser at a scattering angle of 90°. Zeta potential (ZP) measurements were recorded with disposable capillary cells and the M3-PALS measurement technology, built into the Zetasizer system. All measurements were performed in triplicate under ambient conditions. Determination of the FITC-Dextran and the Encapsulation Efficiency. The concentration of FITC-dextran in the liposomal stock solutions was measured by HPLC with fluorescence detection at an excitation and emission wavelength of 485 and 520 nm, respectively. Chromatography was performed on a C18 column (Shiseido, Tokyo, Japan) with methanol− 10 mM PBS (5:95 v/v) at a flow rate of 1.0 mL/min. Prior to the assay, liposomal samples were pretreated with 2% Triton X-100 and sonication to disintegrate the vesicles entirely. The entrapment efficiency (EE) of FITC-dextran was determined separately by centrifugation. An aliquot of the unpurified liposomal solution was diluted 4000 times with the mobile phase, centrifuged at 12,000 × g for 30 min, and the amount of FITC-dextran in the supernatant ([FITC]supernatant) was determined by HPLC. The EE (%) was calculated by the following equation: (([FITC]initial − [FITC]supernatant)/[FITC]initial) × 100, where [FITC]initial is the amount of FITC-dextran initially added to the film hydration process. Cell Culture. Human uterine cervical carcinoma (HeLa) and human keratinocyte (HaCaT) cells were grown in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 μg/mL streptomycin, and 100 units/mL penicillin G. Cultures were maintained at 37 °C in a humidified 5% CO2 incubator. The cells were subcultured every 2−4 days and were used for experiments at passages 5−20. Determination of FR Expression. To determine the level of expression of FR protein, Western blotting was carried out as previously reported.16 Cell lysates were prepared from HeLa cells (FR-positive) and HaCaT cells (FR-negative) using RIPA lysis buffer (NCI9900KR, Thermo Scientific, Loughborough, UK) with 1× protease inhibitor cocktail (ThermoFisher Scientific, Waltham, MA, USA) and were loaded on a 12.5% SDSpolyacrylamide gel. After SDS-PAGE and electroblotting, membranes were treated for 1 h with TBS-Tween 20 buffer containing 5% skim milk to block nonspecific binding and then incubated for 2 h at room temperature with a monoclonal mouse FR-α primary antibody (ALX-804-439, Enzo Life Sciences, Farmingdale, NY, USA). To detect FR expression, the membranes were incubated in blocking buffer with a goat antimouse IgGheavy and light chain antibody HRP conjugated (A90-116P, Bethyl, Montgomery, TX, USA). Protein bands were visualized by X-ray film using a chemiluminescence (Thermo Scientific, Loughborough, U.K.). Equal loading of protein was verified using β-actin (A2228, Sigma-Aldrich, St. Louis, MO, USA). In Vitro Cell Uptake Study. The intracellular delivery of the liposomes was determined by measuring the mean fluorescence intensity (MFI) of FITC-dextran entrapped in the liposome using flow cytometry (FACSCalibur; Becton Dickinson, Franklin Lakes, NJ, USA). For the cellular uptake study, cells were seeded at a density of 3 × 105 cells per well in a 6-well plate with growth media. At 75−80% confluence, the cells were incubated for 2 h at 37 °C in RPMI media (2 mL) with FITC-dextran (28 μg/mL)-entrapped liposomal stock solutions (100 μL). Following incubation, the cells were washed two times with PBS (0.01 M, pH 7.4) to remove the traces of liposomal vesicles left in the wells, detached by trypsin EDTA, and suspended in 1 mL

Figure 1. Schematic illustration of the various cF-P-L and dF/P-L systems synthesized. The number of ligands on the liposomal vesicles does not represent the actual but the relative number in each formulation. Abbreviations: Pep1, Pep-1 peptide; Fol-Pep1, folate-linked Pep-1 peptide; cF-P-L, chimeric ligand (Fol-Pep1)-modified liposome; dF/P-L, dual ligand (folate and Pep1)-modified liposome; cF-P2(N), cF-P-L with N molecules of PEG2000-linked Fol-Pep1; dF2/P2(N/n), dF/P-L with N molecules of PEG2000-linked folate and n molecules of PEG2000-linked Pep1; dF3/P2(N/n), dF/P-L with N molecules of PEG3400-linked folate and n molecules of PEG2000-linked Pep1; dF3/P3(N/n), dF/P-L with N molecules of PEG3400-linked folate and n molecules of PEG3400-linked Pep1; dF2/P3(N/n), dF/P-L with N molecules of PEG2000-linked folate and n molecules of PEG3400-linked Pep1; PEG, polyethylene glycol.

cause the concomitant release of an equivalent of free TNB. The amount of liberated TNB was analyzed by HPLC to estimate the amount of cysteine used for adduct formation under the assumption that every external maleimide was blocked stoichiometrically by a cysteine addition. HPLC conditions were same as described for peptide determination, and Pep1 and FolPep1 peaks were separated with a retention time of 7.192 and 7.08 min, respectively. Separately, in the case of Fol conjugation, direct quantification of liposomal folate content was performed after disrupting the Fol-conjugated liposomes with 10% Triton X-100. A C18 column (Shiseido, Tokyo, Japan) was used with a mobile phase of methanol and 10 mM sodium phosphate buffer (pH 7.0, 92:8 v/v). The column eluent was monitored at 285 nm, and the DSPE-PEG2000-folate peak was separated with a retention time of 2.1 min. Size and Zeta Potential Measurements. Liposomal stock solutions were diluted approximately 80-fold in a 1 mM KCl solution and were examined for their size distribution and polydispersity index (PDI) using a dynamic light scattering D


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Figure 2. Instrumental analyses of peptide synthesis. (A) LC/MS spectrum of Fol-Pep1 and Pep1 representing the molecular ion peaks. (B) HPLC chromatogram of Fol-Pep1. (C) 1H NMR spectra of the Pep-1 peptide (Pep1), folate-linked Pep-1 peptide (Fol-Pep1), and folic acid. P1, P2, and P3 ranges correspond to the signals derived from the methyl group, alpha proton (Hα), and amino proton (NH) of Pep1, respectively. Peaks designated as 1, 2, and 3 correspond to the folic acid spectrum. The inset box shows CD spectra of Fol-Pep1 and Pep1 in 10 mM phosphate buffer (pH 7.4) at 20 °C. CD spectra were converted and are displayed in molar residue ellipticity [θ].

of PBS. The suspended cells were introduced into a flow cytometer equipped with a 488 nm argon ion laser. For the quantification of the MFI value, 1 × 104 designated cells were collected per histogram. In addition, to examine the role of FR binding on the liposomal uptake, a competitive binding assay was performed with the selected liposomal formulations, cF-P2(9) and dF3/P2(450/90), as previously reported.17 Briefly, 1 mM of free folic acid was added to the incubation media in the FR blocking group 1 h before cell treatment. After incubation for 2 h at 37 °C, cells were then washed two times with PBS to remove unbound liposomes and excess folic acid. The following steps were performed by the same procedure as described above. The uptake behavior of liposomes in FR-positive cells was visualized using confocal laser scanning microscopy (CLSM). Before the liposomal treatment, HeLa cells were incubated with 50 nM LysoTracker Red DND-99, which accumulates and fluoresces in late endosomes and lysosomes in live cells. After

lysosomal staining for 30 min, HeLa cells treated as described above were washed thrice with PBS and mounted onto slides without fixation to prevent the potential artifactual uptake of liposomes.18 The fluorescence of the probes was monitored at 400× magnification using a Zeiss LSM 510 Meta confocal microscope in Z-sectioning mode (Carl Zeiss, Oberkochen, Germany). Confocal images of live cells were obtained at 10 min and 2 h. Z stack images were created with 1 μm intervals throughout the sections with a guard region of 2 μm excluded from the top and bottom of the Z stack. Cytotoxicity Assessment. The cytotoxicity of the peptides and liposomal carriers was evaluated in HeLa and HaCaT cells by the WST-1 assay as previously reported.19 HeLa and HaCaT cells were seeded in growth medium at a density of 1 × 104 cells per well in a 96-well plate. At 70−80% confluence, the cells were incubated at 37 °C for 2 h in RPMI medium containing the peptides at different concentrations or empty E


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Molecular Pharmaceutics Table 1. Characteristics of the Pep-1 Peptide (Pep1) and Folate-Linked Pep-1 Peptide (Fol-Pep1)a secondary structure composition (%)

a

ligands

sequence

calcd MW

obsd MW

α-helix

β-strand

random coil

function

Fol-Pep1 Pep1

folate-KETWWETWWTEWSQPKKKRKVC KETWWETWWTEWSQPKKKRKVC

3373.23 2949.41

3374 2951

32.05 30.29

6.02 5.24

61.93 64.47

targeting and cell penetrating cell penetrating

Abbreviations: MW, molecular weight; Fol-Pep1, folate-linked Pep-1 peptide; Pep1, Pep-1 peptide.

Conformational Characterization of the Liposomal Nanocarriers. Various liposomal formulations of F-L, P-L, cF-P-L, and dF/P-L were successfully prepared with the predetermined composition and characterized for conformational and physical properties (Table 2). The conformational aspects of the liposomes were characterized by determining the number of peptide and folic acid ligand molecules that were located on the liposomal surface. Based on a previous report,12 we calculated the number of ligands by the following formula: [N]vesicle × ([Ligand]external/[Lipid]total), where [N]vesicle is the calculated number of lipid molecules per vesicle, [Lipid]total is the molar amount of the total lipids initially added, and [Ligand]external is the molar amount of external ligands of DSPEPEG-Mal or DSPE-PEG-Fol, which was reported to be approximately 51% of the initially added ligand molecules oriented externally at the liposomal surface. [N]vesicle was estimated by the following formula: 4πr2 × 2/A, where r and A refer to the radius of the liposomes and the cross-sectional area of the PC headgroup (0.72 nm2), respectively. As a result, we could confirm that the liposomes carrying a different number of ligands per vesicle were prepared successfully. Next, the folate orientation on the liposomal surface was determined because FR recognition is the most important factor for this kind of receptor-mediated intracellular delivery. For cF-P-L, the chimeric ligand (Fol-Pep1) was conjugated to the preformed liposomes via the thio-maleimide reaction between Pep1 and the maleimide group of DSPE-PEG-Mal. Thus, the folate ligand could be located at the terminal end, orienting outward. For dF/P-L, the outermost orientation of the specific ligand could have been dependent on the length of the linker, regardless of the number of ligands. For example, in the dF3/P2 system the folate ligand resided outward since PEG2000 and PEG 3400 were used as a linker for Pep1 and folate, respectively. Physical Characteristics of the Liposomal Nanocarriers. The physical characteristics of the liposomal systems were investigated in terms of their vesicular size, polydispersity index, ZP, and EE. The average size of the liposome preparations was about 140−170 nm as measured by dynamic light scattering. EL, F-L, and P-L systems had a smaller size compared with that of cF-P-L and dF/P-L. Liposomes with a PEG3400 linker were somewhat larger than liposomes with a PEG2000 linker, which might be attributed to the increased hydrodynamic diameter including the ligand and PEGylated environment around a particle. All formulations showed a low PDI below 0.3, indicating a narrow and homogeneous size distribution. There were no changes in size distribution until the use for the experiment. Surface modification with chimeric or double ligands did not affect the EE of FITC-dextran, which was on average about 28% (0.56 ± 0.02 mg/mL) for all formulations. Surface modification with folate and Pep1 ligands changed the ZP of liposomes since these ligands carry negative and positive charges inherently. EL and F-L were negatively charged, but P-L was positively charged. All cF-P-L systems showed

liposomal carriers at the same concentration as the cell uptake study. After incubation, cells were washed with fresh medium and then incubated with 10% WST-1 reagent for 30 min. The absorbance of the WST-1 formazan dye was measured at 450 nm using a microplate reader (FlexStation 3; Molecular Devices, CA, USA). Cell viability was calculated as the percentage of viable cells relative to that of the untreated sample. Statistical Analysis. Values were processed using the Microsoft Excel 2010 software and presented as means ± standard deviation (n = 3). Statistical significance was determined by the Student’s t-test, and data were considered significant at p < 0.05.

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RESULTS Synthesis of Pep1 and Fol-Pep1. Pep1 and Fol-Pep1 were synthesized by Fmoc SPPS using an automated peptide synthesizer and were subsequently purified with preparative HPLC. The MW of each synthesized peptide was calculated by the summation method as reported earlier.12 The molecular ion peaks of Pep1 and Fol-Pep1 were 2951 and 3374, respectively, as measured by LC/MS (Figure 2A). The calculated MW of the peptides closely corresponded to the observed MW of the purified peptides (Table 1). In HPLC analysis, Fol-Pep1 was separated by a single peak with a retention time of 7.08 min, indicating the absence of other impurities (Figure 2B). Characterization of Fol-Pep1. The newly synthesized FolPep1 was characterized by 1H NMR spectroscopy (Figure 2C), demonstrating the successful coupling of folate to Pep1. In the spectrum of Fol-Pep1, the signals at 8.7, 7.7, and 6.7 ppm (designated as 1, 2, and 3) correspond to those of folic acid; the signals at 0.8−1.5, 4.0−5.0, and 8.0−8.5 ppm (designated as P1, P2, and P3) correspond to the methyl, α-hydrogen, and α-amine groups of Pep1, respectively. In addition, far-UV CD (190−260 nm) was used to compare the secondary structure contents of Pep1 and Fol-Pep1 (Figure 2C). The CD spectra show that the secondary structures of both peptides are very similar, exhibiting minima at ∼205 nm, a pronounced shoulder at ∼221 nm, and a maximum at 190 nm, which are typical characteristics of a helical conformation. The ellipticity ratios of Pep1 and Fol-Pep1 at 208 and 222 nm, which were used to distinguish between the 310 and α-helix structures in the secondary structure, were 0.56 and 0.42, respectively, indicating the characteristics of the 310 structure by the criteria described by [θ]222/[θ]208 ratio P-L > cF-P2(9) > F-L > EL. In HaCaT cells, a significant peak shift was observed after treatment with P-L and dF3/P2(450/90). The MFI values in HaCaT cells were in the order of P-L > dF3/P2(450/90) > EL > cFP2(9) > F-L. To compare the relative cellular uptake in HeLa and HaCaT cells, the relative ratio of the MFI values of the liposomal formulations against that of FITC-dextran alone was plotted (Figure 4C). Compared with FITC-dextran treatment alone, EL and P-L enhanced the cellular uptake by approximately 3-fold and 5.2-fold, respectively, in both cell lines. In contrast, F-L, cF-P2(9), and dF3/P2(450/90) exhibited selective translocation in FR-expressing HeLa cells. Both F-L and cF-P2(9) increased the uptake by about 4.2-fold in HeLa cells and 2-fold in HaCaT cells. In particular, dF3/P2(450/90) showed a 9.2-fold greater fluorescence intensity than FITCdextran alone in HeLa cells, while showing only a 3.9-times H


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Figure 4. FR expression and comparison of the liposomal cell uptake in FR-positive HeLa and FR-negative HaCaT cells. (A) Western blot for FRexpression. (B) Flow cytometry results for treatment effect. (C) The relative MFI values of the liposomal systems compared with those of FITCdextran alone. (D) Competitive assays of liposome internalization under FA-untreated (FA−) or FA-pretreated (FA+) condition. Values represent means ± SD (N = 3). Statistical analysis was performed using the Student’s t test (*, p < 0.05 versus the paired group; **, p < 0.005 versus the paired group). Abbreviations: FR, folate receptor; EL, elastic liposome; F-L, folate-tethered liposome; P-L, Pep1-conjugated liposome; cF-P2(9), cF-P-L with 9 molecules of PEG2000‑linked Fol-Pep1; dF3/P2(450/90), dF/P-L with 450 molecules of PEG3400-linked folate and 9 molecules of PEG2000linked Pep1; MFI, mean fluorescence intensity; FITC, fluorescein isothiocyanate; FA, folic acid.

interaction between folate and FR. For P-L, the relatively weak green fluorescence was distributed throughout the cytosol. In particular, dF3/P2(450/90) exhibited a rapid and considerably increased intracellular fluorescence compared with that of other liposomal systems. After the 2 h treatment, all liposomal systems had enhanced fluorescence intensity regardless of system type. Cellular uptake was time-dependent, which was further proven by comparing the three-dimensional view with the Z direction since the fluorescence intensities in both the XZ and YZ planes were also greatly increased. In cells treated with F-L and cF-P2(9), cytosolic fluorescence slightly increased, while the occurrence of green fluorescent dots on the cell membrane significantly increased. P-L increased the fluorescence throughout the cytosol. dF3/P2(450/90) showed the most intensified fluorescence in both cytosol and cell surface. Additionally, to further understand the rapid translocation of dF3/P2(450/90) after 10 min of treatment, lysosomes in HeLa cells were stained with the LysoTracker Red probe. As shown in Figure 5B, the green and red fluorescence was distributed independently in the cytoplasm. Very weak or faint spots in the green fluorescence channel corresponded closely with the location of the red-labeled lysosome. In the merged image (Figure 5B), the red color tone of the lysosomal fraction is, however, maintained, indicating that the fluorescence of the green fluorescent spots and that of the lysosomes did not overlap. Thus, we could establish that dF3/P2(450/90) bypassed the endosomelysosomal pathway at an early translocation stage.

higher translocation efficiency in normal FR-negative cells. As a result, F-L, cF-P2(9), and dF3/P2(450/90) confirmed their FRspecificity, which was significantly different (p < 0.05) between HeLa and HaCaT cells. Separately, a competition assay with cF-P2(9) and dF3/P2(450/90) was performed to confirm the effect of FR blocking on the cellular uptake of the proposed liposomal system. As shown in Figure 4D, cellular uptake of both systems was suppressed by folic acid (FA)-pretreatment. In HeLa cells, the cellular uptake of dF3/P2(450/90) and cF-P2(9) was greatly suppressed by FR blocking, revealing significant difference at p < 0.005 and p < 0.05, respectively. However, in HaCaT cells, both cF-P2(9) and dF3/P2(450/90) showed insignificant difference at p < 0.05 between FA-untreated and FA-pretreated groups. The results demonstrated a reasonable assumption that, in FR-positive cells, free folic acid competes with the folate conjugates and inhibits the FR-mediated internalization. Binding and Translocation of Liposomal Systems to FR-Positive Cells. To compare the translocation behavior of liposomal systems, fluorescently marked liposomes were visualized by CLSM in orthogonal mode (Figure 5A). FR-positive HeLa cells were treated with various F-L, P-L, cF-P2(9), and dF3/ P2(450/90) liposomes. To verify the intracellular fluorescence signals, optical sections were taken through the midline of the cells at 10 min and 2 h after treatment. Immediately after treatment (10 min), F-L and cF-P2(9) were found to be bound on the HeLa cell surface, indicating the selective liposomal binding due to the I


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Figure 5. Confocal images of the intracellular translocation of various liposomes in FR-positive HeLa cells. Cells were treated with liposomes containing equivalent concentrations of FITC-dextran (28 μg/mL) and were observed by CLSM without fixation. (A) Orthogonal views of the treatment with F-L, P-L, cF-P2(9), and dF3/P2(450/90) at 10 min and 2 h after treatment. The colored lines show the positions of the section planes: XY plane (blue), XZ plane (green), and YZ plane (red). Scale bar: 20 μm. (B) Merged image of the dF3/P2(450/90) treatment at 10 min. LysoTracker-stained cells in the red channel were overlaid with the green channel images to assess colocalization of the fluorescent probes. Scale bar: 20 μm.

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Cytotoxicity of the Peptides and Liposomal Systems. The toxicities of the peptides and liposomal formulations were examined using HeLa and HaCaT cells. The cells were treated with varying amounts of peptide, and the cell viability was assessed by the WST-1 assay. Cell viability in the untreated group was 100%. As shown in Figure 6A,B, cytotoxicity of Fol-Pep1 and Pep1 was negligible up to 25 μM in both the FR-positive and FR-negative cells. However, at a peptide concentration >50 μM, significant differences in cell viability between Fol-Pep1-treated and Pep1-treated groups were found in both cell lines. HeLa cells were more sensitive to both Pep1 and Fol-Pep1 than HaCaT cells were: the inhibitory concentration of 50% cell viability (IC50) of Fol-Pep1 and Pep1 in HeLa and HaCaT cells was 124 and 84 μM and 210 and 160 μM, respectively. Regardless of the cells’ FR expression, Fol-Pep1 was less cytotoxic than Pep1. The liposomal formulations used in this study were prepared separately without fluorescent probe and their cytotoxicities were assessed at the concentration used in the cellular uptake experiment (Figure 6B). None of the liposomes caused any significant cytotoxicity to both cell lines. In comparison with the control group (100%), all treatment groups were within the range of 97% to 102% cell viability. Therefore, we believe that all liposomal systems used in this study were noncytotoxic to either cell type.

DISCUSSION Recently, site-specific targeting with functional moieties such as monoclonal antibodies, folic acid, transferrin, integrins, and peptides for efficient intracellular delivery has received more attention. Various CPPs including arginine-rich peptides, penetratin, transportan, TAT, and Pep-1 peptide were introduced as potential moieties.20,21 These peptides could deliver and facilitate cell-impermeable drug or cargoes across the cell membrane into the cytoplasm. However, CPP-mediated delivery cannot distinguish target cells from nontarget cells. To solve this problem, a complementary strategy combining a target concept to CPP-mediated delivery is suggested. In this study, liposomes were used as a platform to accommodate both functions because of the ease of surface modification and biocompatibility. The approach for liposomal surface modification with both targeting and CPP moieties can be divided into chimeric ligand-modified or dual ligand-modified systems. The cell-penetrating tumor-homing peptide (CPHP) has been synthesized as a chimeric ligand by directly linking target molecules with CPPs for intracellular delivery to specific cells. In the current study, Fol-Pep1 was designed as a novel CPHP using a modular approach. For efficient targeting, the folate moiety should be placed in front of Pep1, which is possible in a linear conjugation form between the carboxyl group of folate and the N-terminus of Pep1. This approach retains the high affinity for J


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Figure 6. Cytotoxicities of Pep1, Fol-Pep1, and various empty liposomes in HeLa and HaCaT cells determined by the WST-1 assay. (A) Cells were treated with a varying amount of peptides. (B) Cells were treated with empty liposomes at the same concentration used in the cell uptake study. Control cells were untreated and considered having 100% cell viability. Data are expressed as means ± SD (N = 3). Abbreviations: Pep1, Pep-1 peptide; Fol-Pep1, folate-linked Pep-1 peptide; EL, elastic liposome; F-L, folate-tethered liposome; P-L, Pep1-conjugated liposome; cF-P2(9), cF-P-L with 9 molecules of PEG2000‑linked Fol-Pep1; dF3/P2(450/90), dF/P-L with 450 molecules of PEG3400-linked folate and 9 molecules of PEG2000linked Pep1; Pep1, Pep-1 peptide; Fol-Pep1, folate-linked Pep-1 peptide; CTL, control.

in the positive charge of the peptide. A decrease in charge could reduce the electrostatic interaction between Fol-Pep1 and the negatively charged cell surface, resulting in less cytotoxicity, but possibly also less efficient cell penetration.12 In this study, cellular uptake of cF-P-L was dependent on the number of ligands (Figure 3A). However, the uptake was maximized in FR-positive cells treated with cF-P2(9) having 0.005% folate ligand and diminished to an ineffective level at higher amounts of ligand. This result is consistent with those of an earlier report that folate-targeted liposomes containing low amounts (0.01−0.03%) of folate-conjugated PEG molecules exhibited the greatest binding to FR-expressing cells, while the liposomes containing a higher amount (>0.5%) of ligand progressively decreased the FR selectivity.29 These phenomena could be attributed to the interaction between surface-located ligands. Increasing the number of ligands on the liposomal surface would cause dimerization or multimerization of adjacent ligands, resulting in significant loss of affinity for the relevant receptor. Moreover, the low Fol-Pep1 ratio in the optimized cF-P-L did not yield a sufficient CPP concentration at which endocytosisindependent translocation occurs. Indeed, nonendosomal translocation is associated with the ability of Pep-1 to interact with membrane lipids, depending on the local concentration of Pep-1.30 Cellular uptake of Pep-1 and cargo complexes is directly correlated

the FR binding when folate is covalently linked to a foreign molecule via its γ-carboxyl group.22 Therefore, to avoid formation of amide bonds via the α-carboxyl group in folic acid, glutamic acid and pteroic acid were sequentially coupled with Pep1 as shown in Scheme 1, instead of a direct coupling reaction between Pep1 and folic acid.23 For amphipathic CPPs including Pep1, the secondary structure of CPPs is a major factor in the cell-penetrating mechanism. Although the translocation mechanism of Pep1 is still controversial, it is demonstrated that the helical structure of Pep1 helps its insertion into the cellular membrane and is directly responsible for the efficiency of its cellular uptake.24−26 For example, most amphipathic peptides such as TP10, MAP, EB1, and M918 strongly interact with lipid membranes when they exist in the helix form only.27 In this study, both Fol-Pep1 and Pep1 had similar CD spectra and secondary structure composition, which were interpreted as helical structures. This result is consistent with those of other studies on structure analysis using TOCSY and NOESY 2D 1H NMR.28 The Fol-Pep1’s secondary, helical structure was retained after conjugation with folic acid, satisfying the structural prerequisite for its cell-penetrating function. In terms of cytotoxicity, CPPs could damage cell membranes by inducing transient pores or perturbation. The cytotoxicity of Fol-Pep1 was negligible in both HeLa and HaCaT cells, which might be related to the reduction K


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Figure 7. Illustrations assuming the intracellular translocation pathway of various liposomal nanocarriers in FR-expressing cells. EL can pass through the cell membrane in a narrow form. F-L and cF-P-L selectively bind to the FR and are then entrapped by FR-mediated endocytosis. Some of the endocytosed F-L and cF-P-L diffuse into the cytoplasm, while the other liposomes undergo endosome-lysosomal degradation or exocytosis by FR recycling. P-L translocates into the cytoplasm via instant perturbation of the cell membrane by Pep1. dF/P-L selectively bind to FR, then follow the endosomal pathway similar to F-L. Simultaneously, during the early translocation stage, most of the bound dF/P-L could escape into the cytoplasm like P-L via the instant cell-penetrating action of Pep1. Abbreviations: Pep1, Pep-1 peptide; FR, folate receptor; EL, elastic liposome; F-L, folatetethered liposome; P-L, Pep1-conjugated liposome; cF-P-L, chimeric ligand (Fol-Pep1)-modified liposome; dF/P-L, dual ligand (folate and Pep1)modified liposome.

different types of dF/P-L, in which PEG2000 and a phenylbutyryl group were used as long and short linkers for folate and Pep1, respectively, efficient cellular uptake was not obtained.15 This phenomenon might be due to the effects of both steric hindrance and restricted mobility of the conjugated ligand. Short linker length could not support the intimate contact between Pep1 and the cell membrane, resulting in less cellular uptake. Meanwhile, in the aspect of the number of conjugated ligands, dF3/P2(450/90) was considered as an optimized system for enhanced cellular uptake with high selectivity. Presumably, decreased cellular uptake in dF3/P2(450/450) and dF3/P2(450/900) resulted from limited flexibility of the ligands in overtagged liposome, while the decrease in dF3/P2(450/9) was caused by insufficient concentration of CPP at the outermost space. Based on the comparison of the cellular uptake behavior of various liposomes in this study, we assume a cellular uptake mechanism of liposomal nanocarriers as illustrated in Figure 7. EL could translocate through the cell membrane because of Tween80, which was embedded in the lipid bilayer as an elastic modulator. However, the cellular uptake of EL was passive and nonselective to FR-expressing cells. Therefore, the cellular uptake of F-L apparently depends on FR expression. It has been shown that F-L selectively binds to the FR expressed by target cells, which is followed by FR-mediated endocytosis. Once the FR is saturated, translocation through FR-mediated endocytosis would be limited.34 Although some of the endocytosed F-L could be safely delivered into the cytoplasm, major parts of F-L undergo endosome-lysosomal degradation or are expelled into the extracellular fluid by FR recycling. cF-P2(9) was expected to exhibit targeting and cell-penetrating functions simultaneously since it is an optimized cF-P-L. However, its cellular uptake level was similar to that of F-L, possibly due to the cellpenetrating dysfunction and steric hindrance of adjacent ligands as discussed above. Therefore, FR-mediated endocytosis would be suggested as a major pathway for cF-P-L uptake. P-L could

with the structure of the nanoparticles that results in localized high concentration of peptides at the cell surface.31,32 Therefore, a low level of Fol-Pep1 on the liposomal surface resulted in less cellular association and translocation of cF-P-L compared with those of P-L. P-L was superior to F-L and cF-P-L in terms of cellular uptake in FR-negative HaCaT cells (Figure 4C,D). Nevertheless, these three liposomal systems revealed the same level of cellular uptake in FR-positive HeLa cells, indicating the complementary FR binding of the folate ligand. However, for the cellular uptake of dF/P-L, the relative linker length of the ligands would be more important than the number of ligands. The linker length controls the ligand protrusion from the liposomal surface, which determines the extent of ligand function. Thus, we investigated linker optimization for dF/P-L with PEG2000 and/or PEG3400 for the FR specificity and cell membrane interaction. In this study, dF3/P2(450/90), which used PEG2000 as a linker for Pep1 and PEG3400 for folic acid, exhibited the highest translocation in HeLa cells. The difference in linker length between folate and Pep1 would reduce the steric hindrance of the ligands, and the adequate projection of folate molecules at the outermost space would efficiently translocate the liposomal nanocarrier into FR-positive cells. This principle that targeting ligands should be located at the outermost space compared with CPP is well consistent with other multifunctional systems reported. For example, PLGA−PEG nanoparticles were modified with folic acid and nona-arginine simultaneously, in which nona-arginine and folic acid were conjugated with the short and relatively long PEG linkers, respectively. The multifunctional nanoparticles demonstrated superior cellular uptake and selectivity to either folate- or nona-arginine-modified nanoparticles.33 In addition, the dual ligand system composed of the RGD peptide and octa-arginine, in which the RGD peptide was conjugated to the PEG2000 linker as the targeting ligand and octaarginine was directly linked to the liposomal bilayer, also exhibited similar results.10 However, in our previous study with L


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(3) Stewart, K. M.; Horton, K. L.; Kelley, S. O. Cell-penetrating peptides as delivery vehicles for biology and medicine. Org. Biomol. Chem. 2008, 6, 2242−2255. (4) Kang, M. J.; Kim, B. G.; Eum, J. Y.; Park, S. H.; Choi, S. E.; An, J. J.; Jang, S. H.; Eum, W. S.; Lee, J.; Lee, M. W. Design of a Pep-1 peptide-modified liposomal nanocarrier system for intracellular drug delivery: Conformational characterization and cellular uptake evaluation. J. Drug. Target. 2011, 19, 497−505. (5) Leamon, C. P.; Low, P. S. Folate-mediated targeting: from diagnostics to drug and gene delivery. Drug Discovery Today 2001, 6, 44−51. (6) Aronov, O.; Horowitz, A. T.; Gabizon, A.; Gibson, D. Folatetargeted PEG as a potential carrier for carboplatin analogs. Synthesis and in vitro studies. Bioconjugate Chem. 2003, 14, 563−574. (7) Majoros, I. J.; Myc, A.; Thomas, T.; Mehta, C. B.; Baker, J. R. PAMAM dendrimer-based multifunctional conjugate for cancer therapy: synthesis, characterization, and functionality. Biomacromolecules 2006, 7, 572−579. (8) Reddy, J. A.; Dorton, R.; Westrick, E.; Dawson, A.; Smith, T.; Xu, L. C.; Vetzel, M.; Kleindl, P.; Vlahov, I. R.; Leamon, C. P. Preclinical evaluation of EC145, a folate-vinca alkaloid conjugate. Cancer Res. 2007, 67, 4434−4442. (9) Lee, R. J.; Low, P. S. Delivery of liposomes into cultured KB cells via folate receptor-mediated endocytosis. J. Biol. Chem. 1994, 269, 3198−3204. (10) Kibria, G.; Hatakeyama, H.; Ohga, N.; Hida, K.; Harashima, H. Dual-ligand modification of PEGylated liposomes shows better cell selectivity and efficient gene delivery. J. Controlled Release 2011, 153, 141−148. (11) Ruoslahti, E.; Duza, T.; Zhang, L. Vascular homing peptides with cell-penetrating properties. Curr. Pharm. Des. 2005, 11, 3655− 3660. (12) Kang, M. H.; Park, M. J.; Yoo, H. J.; Lee, S. G.; Kim, S. R.; Yeom, D. W.; Kang, M. J.; Choi, Y. W. RIPL peptide (IPLVVPLRRRRRRRRC)-conjugated liposomes for enhanced intracellular drug delivery to hepsin-expressing cancer cells. Eur. J. Pharm. Biopharm. 2014, 87, 489−499. (13) Louis-Jeune, C.; Andrade-Navarro, M. A.; Perez-Iratxeta, C. Prediction of protein secondary structure from circular dichroism using theoretically derived spectra. Proteins: Struct., Funct., Genet. 2012, 80, 374−381. (14) Nagarsenker, M.; Londhe, V. Y.; Nadkarni, G. Preparation and evaluation of liposomal formulations of tropicamide for ocular delivery. Int. J. Pharm. 1999, 190, 63−71. (15) Kang, M. J.; Park, S. H.; Kang, M. H.; Park, M. J.; Choi, Y. W. Folic acid-tethered Pep-1 peptide-conjugated liposomal nanocarrier for enhanced intracellular drug delivery to cancer cells: conformational characterization and in vitro cellular uptake evaluation. Int. J. Nanomed. 2013, 8, 1155−1165. (16) Puligujja, P.; McMillan, J.; Kendrick, L.; Li, T.; Balkundi, S.; Smith, N.; Veerubhotla, R. S.; Edagwa, B. J.; Kabanov, A. V.; Bronich, T. Macrophage folate receptor-targeted antiretroviral therapy facilitates drug entry, retention, antiretroviral activities and biodistribution for reduction of human immunodeficiency virus infections. Nanomedicine 2013, 9, 1263−1273. (17) Xiang, G.; Wu, J.; Lu, Y.; Liu, Z.; Lee, R. J. Synthesis and evaluation of a novel ligand for folate-mediated targeting liposomes. Int. J. Pharm. 2008, 356, 29−36. (18) Richard, J. P.; Melikov, K.; Vives, E.; Ramos, C.; Verbeure, B.; Gait, M. J.; Chernomordik, L. V.; Lebleu, B. Cell-penetrating peptides A reevaluation of the mechanism of cellular uptake. J. Biol. Chem. 2003, 278, 585−590. (19) Francoeur, A.; Assalian, A. Microcat: A novel cell proliferation and cytotoxicity assay based on WST-1. Biochemica 1996, 3, 19−25. (20) Green, M.; Loewenstein, P. M. Autonomous functional domains of chemically synthesized human immunodeficiency virus tat transactivator protein. Cell 1988, 55, 1179−1188.

attach to the cell membrane nonspecifically via electrostatic interactions between Pep1 and the cell surface and be taken up in the cytosol through membrane structure perturbation by virtue of Pep1. This route could bypass endosome entrapment and lysosomal degradation. Compared with other systems, dF3/P2(450/90), as an optimized dF/P-L, showed a considerably enhanced cellular uptake and rapid translocation in FRpositive cells. We assumed that this superiority probably results from the synergistic effect of folic acid and Pep1. Thus, the cellular uptake mechanism of dF/P-L would be considered as a combined mechanism of F-L and P-L uptake. Once FR binding of dF/P-L occurrs, dF/P-L follows the endosomal pathway similar to F-L. However, especially for the early translocation stage, the confocal results exhibited immediate distribution of dF/P-L into cytosol within 10 min, possibly suggesting that the majority of dF/P-L may bypass the endosome-lysosomal pathway due to the direct cell-penetrating action of Pep1. This might be a good reason that dF/P-L could be an excellent nanocarrier for FR-targeted intracellular drug delivery.

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CONCLUSION Various types of cF-P-L and dF/P-L were successfully developed for FR-targeted delivery using folic acid and Pep1. As a novel CPHP, Fol-Pep1 was synthesized and characterized in terms of its chemical property and secondary structure. cF-P(9) liposomes exhibited a similar cellular uptake to that of F-L, while dF3/P2(450/90) liposomes had a significantly increased cellular uptake with high FR selectivity, possibly owing to the synergistic effect of folate and Pep1. The uptake mechanism and intracellular pathway related to both liposome systems have been proposed, suggesting a useful approach for designing multifunctional systems for FR-selective drug targeting.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut. 5b00399.

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DLS data and TEM images (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +82 2 820 5609. Fax: +82 2 826 3781. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science, and Technology (2011-0009876).

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REFERENCES

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Design of Multifunctional Liposomal Nanocarriers for Folate Receptor

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