RIPL peptide (IPLVVPLRRRRRRRRC)-conjugated liposomes for enhanced intracellular drug delivery to hepsin-expressing cancer cells.

European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx

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European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb

Research paper

RIPL peptide (IPLVVPLRRRRRRRRC)-conjugated liposomes for enhanced intracellular drug delivery to hepsin-expressing cancer cells Min Hyung Kang a, Min Jung Park a, Hyun Joon Yoo a, Kwon Yie hyuk a, Sang Gon Lee a, Sung Rae Kim a, Dong Woo Yeom a, Myung Joo Kang b, Young Wook Choi a,⇑ a b

College of Pharmacy, Chung-Ang University, Seoul, Republic of Korea College of Pharmacy, Dankook University, Cheonan-Si, Republic of Korea

a r t i c l e

i n f o

Article history: Received 11 December 2013 Accepted in revised form 11 March 2014 Available online xxxx Keywords: Liposome Cell penetrating/homing peptide Intracellular delivery Polyarginine IPL Targeting Hepsin

a b s t r a c t Background: To facilitate selective drug delivery to hepsin (Hpn)-expressing cancer cells, the RIPL peptide (IPLVVPLRRRRRRRRC; 16mer; 2.1 kDa) was synthesized as a novel cell penetrating/homing peptide (CPHP) and conjugated to a liposomal carrier. Methods: RIPL peptide-conjugated liposomes (RIPL-Lipo) were prepared by conjugating RIPL peptides to maleimide-derivatized liposomal vesicles via the thiol-maleimide reaction. Vesicle size and zeta potential were examined using a Zetasizer. Intracellular uptake specificity of the RIPL peptide, or RIPL-Lipo, was assessed by measuring mean fluorescence intensity (MFI) after treatment with a fluorescent marker in various cell lines: SK-OV-3, MCF-7, and LNCaP for Hpn(+); DU145, PC3, and HaCaT for Hpn(). FITC-dextran was used as a model compound. Selective translocational behavior of RIPL-Lipo to LNCaP cells was visualized by fluorescence microscopy and confocal laser scanning microscopy. Cytotoxicities of the RIPL peptide and RIPL-Lipo were evaluated by WST-1 assay. Results: RIPL peptides exhibited significant Hpn-selectivity. RIPL-Lipo systems were of positively charged nanodispersion (165 nm in average; 6–24 mV depending on RIPL conjugation ratio). RIPL-Lipo with the conjugation of 2300 peptide molecules revealed the greatest MFI in all cell lines tested. Cellular uptake of RIPL-Lipo increased by 20- to 70-fold in Hpn(+) cells, and 5- to 7-fold in Hpn() cells, compared to the uptake of FITC-dextran. Cytosolic internalization of RIPL-Lipo was time-dependent: bound instantly; internalized within 30 min; distributed throughout the cytoplasm after 1 h. Cytotoxicities of RIPL peptide (up to 50 lM) and RIPL-Lipo (up to 10%) were minor (cell viability >90%) in LNCaP and HaCaT cells. Conclusion: By employing a novel CPHP, the RIPL-Lipo system was successfully developed for Hpn-specific drug delivery. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Abbreviations: Hpn, hepsin; IPL, IPLVVPLC, Hepsin specific sequence; R8, RRRRRRRRC, Octa arginines sequence; RIPL, IPLVVPLRRRRRRRRC, Hepsin specific and cell penetrating sequence; RIPL-Lipo, RIPL peptide-conjugated liposomes; CL, conventional liposomes; CLSM, confocal laser scanning microscopy; CPHP, cell penetrating homing peptide; CPP, cell penetrating peptide; D.W., distilled water; DSPE-PEG2000-mal, distearoyl phosphatidyl ethanolamine-polyethylene glycolmaleimide; DTNB, 5,50 -dithio-bis(2-nitrobenzoic acid); EE, entrapment efficiency; FITC-dextran, fluorescein dextran isothiocyanate; Fmoc SPPS, 9-fluorenylmethyloxycarbonyl solid phase peptide synthesis; MFI, mean fluorescence intensity; MMP, matrix metalloproteases; MW, molecular weight; pArg, polyarginine; PBS, phosphate buffer saline; PC, phosphatidylcholine; PDI, polydispersity index; RIPL-FITC, fluorescence-tagged RIPL peptide; uPa, Urokinase plasminogen activator. ⇑ Corresponding author. College of Pharmacy, Chung-Ang University, 221 Heuksuk-dong, Dongjak-gu, Seoul 156-756, Republic of Korea. Tel.: +82 2 820 5609; fax: +82 2 826 3781. E-mail address: [email protected] (Y.W. Choi).

The poor selectivity of chemotherapeutic agents for specific target sites or cells is one of the major obstacles in chemotherapy [1–3]. Surface-functionalized delivery systems including liposomal nanocarrier have been widely applied to target specific cancer cells and efficiently transfer a cargo to cytoplasm through diverse mechanisms [4–8]. Active targeting can be accomplished by molecular recognition of the diseased cells by various specific molecules overexpressed at the site of disease via ligand-receptors or antigen–antibody interactions [9], e.g., folate receptor as a target protein to distinguish ovarian and cervical cancer cells from normal cells [8,10]. Prostate-specific membrane antigen (PSMA) has been widely used for prostate cancer targeting [11–13],

http://dx.doi.org/10.1016/j.ejpb.2014.03.016 0939-6411/Ó 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: M.H. Kang et al., RIPL peptide (IPLVVPLRRRRRRRRC)-conjugated liposomes for enhanced intracellular drug delivery to hepsin-expressing cancer cells, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.03.016

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M.H. Kang et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx

however, hepsin has recently been addressed as a biomarker to detect early prostate cancer [14]. Hepsin (Hpn) belongs to the hepsin/TMPRSS/enteropeptidase subfamily within the class of type II transmembrane serine proteases [15]. This extracellular protease has been detected at significant levels in many different types of mammalian cells, including human hepatoma cells (HepG2), peripheral nerve cells (PC12), and prostate cancer cells (LNCaP) [16]. Notably, Hpn is consistently up-regulated in cancer cells, but is either absent or expressed at very low levels in normal prostate and benign prostatic hypertrophy [14]. Based on this difference, IPLVVPL (IPL) has been introduced as an Hpn-specific peptide possessing both high affinity and high selectivity for Hpn. The employment of cell penetrating peptides (CPPs) is a useful approach for enhanced cytosolic drug delivery [17]. Among CPPs, polyarginine (pArg) peptides have been widely used in studies aiming to enhance the cellular uptake of various drugs in vitro and in vivo. For example, the octaArg (R8)-doxorubicin conjugate and R8 functionalized liposomes effectively suppressed tumor proliferation without any significant weight loss in mice [18–20], and pArg-conjugated cationic liposomes exhibited effective intracellular delivery of small interfering RNA [21,22]. Nevertheless, the application of CPPs for enhanced intracellular drug delivery has been limited, because the majority of known CPPs are non-selective for specific cells or tissue. Many attempts to assign selectivity to CPPs by the conjugation of a homing peptide have been made. For instance, by the conjugation of PEGA (CPGPEGAGC; a breast vasculature-specific peptide) with pVEC (LLIILRRRIRKQAHAHSK; a CPP), PEGA-pVEC showed both cell-specific and cell-penetrating properties [23]. Attachment of DV3 (LGASWHRPDKG; CXC chemokine receptor 4 ligand) to TAT peptide (GRKKRRQRRRPQ; a CPP) resulted in an enhancement of tumor cell killing compared with treatment with non-targeted parenteral peptides [24]. These chimeric peptides are called ‘‘cell penetrating homing peptides’’ (CPHP) and have been successfully used for the targeted delivery of drug molecules, nanoparticles, and liposomes to various tissues including tumors [25]. Therefore, in the present study, RIPL peptide (IPLVVPLRRRRRRRRC) was synthesized as a novel CPHP and conjugated to a liposomal carrier to facilitate selective drug delivery. Conformational and physical properties of different types of RIPL peptide-conjugated liposomes were characterized in terms of the extent of surface modification, vesicle size and zeta potential. Using the various cell lines including LNCaP, SK-OV-3, and MCF-7 as Hpn-expressing cells and DU-145, PC-3, and HaCaT as Hpn-non-expressing cells, intracellular uptake behaviors of RIPL peptide-conjugated liposomes containing fluorescence-labeled macromolecules as a model probe were observed by fluorescence microscopy, confocal laser scanning microscopy (CLSM), and flow cytometry. Cytotoxicities of the RIPL peptide and the peptideconjugated liposomal carriers were also evaluated.

purchased from Millipore (Billerica, MA, USA). Phosphate buffer saline (PBS; pH 7.4, 10x) and cell culture materials including Roswell Park Memorial Institute medium (RPMI) 1640 medium, fetal bovine serum, penicillin–streptomycin, and trypsin-EDTA (0.25%) were obtained from Invitrogen (Carlsbad, CA, USA). NUNC CC2 chamber slides were purchased from Nalgene Nunc International (Rochester, NY, USA). Human prostate cancer cell lines (LNCaP, DU145 and PC3), an ovarian carcinoma cell line (SK-OV-3), a breast cancer cell line (MCF-7), and a human keratinocyte cell line (HaCaT) were purchased from the Korean Cell Line Bank (Seoul, Korea). All other chemicals and reagents purchased from commercial sources were of analytical or cell culture grade. 2.2. Synthesis of RIPL peptide Peptides were synthesized by Fmoc SPPS (9-fluorenylmethyloxycarbonyl solid phase peptide synthesis) and purified by reverse phase HPLC. Amino acid units were coupled one by one from the C-terminal using an automated peptide synthesizer (ASP48S, Peptron Inc.). S-trityl-L-cysteine-2-chlorotrityl resin was used to attach the first amino acid of the C-terminal to a resin. All the amino acids used in the peptide synthesis were those protected by trityl, t-butyloxycarbonyl, t-butyl, and the like, whereby the N-terminal is protected by Fmoc, and residues are all removed in acid. As a coupling reagent, 2-(1H-benzotriazol-1-y1)-1, 1, 3, 3-tetramethyluroniurn hexafluorophosphate/hydroxyl-benzotriazole/N-methylmorpholine was used. An elution in preparative HPLC (Shimadzu, Kyoto, Japan) purification was carried out on a Vydac Everest C18 column (250 22 mm; 10 lm) with a water–acetonitrile linear gradient (3–40% v/v of acetonitrile) containing 0.1% (v/v) trifluoroacetic acid. Molecular weights of the purified peptide were confirmed using LC/ MS (Agilent HP1100 series). The settings for the LC/MS, operated in the electrospray positive ion mode, with a liquid chromatography flow of 0.4 mL/min were as follows: drying gas flow, 8 L/min; nebulizer pressure, 35 psig; nebulizer temperature, 350 °C; capillary voltage, 4.0 kV. 2.3. Structure prediction and model construction of RIPL peptide Secondary structure prediction was carried out to determine the structural significance of targeting sequences using PSIPRED, which is based on a dictionary of protein secondary structure (DSSP) [26]. Three-dimensional models of the RIPL peptide were constructed by MODELLER for the selection of the best model with the highest confidence score. The above-mentioned tools were accessed through the Local Meta-Threading Server (http://zhanglab.ccmb. med.umich.edu/LOMETS/) [27]. The structure of the complex between the RIPL peptide and Hpn is predicted and described by ZDOCK, a protein-docking algorithm (http://zlab.umassmed.edu/ zdock/) [28]. 2.4. Preparation of RIPL-conjugated liposomes

2. Materials and methods 2.1. Materials Soya phosphatidylcholine (PC) and distearoyl phosphatidyl ethanolamine–polyethylene glycol–maleimide (DSPE-PEG2000-mal) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Tween 80, cystein hydrochloride anhydrous, 5,50 -dithio-bis(2-nitrobenzoic acid) (DTNB) and fluorescein dextran isothiocyanate (FITC-dextran, 4 kDa) were purchased from Sigma (St. Louis, MO, USA). RIPL peptides (IPLVVPLRRRRRRRRC, 16mer), octa-arginine (R8; RRRRRRRRC), and IPL (IPLVVPLC) were synthesized by Peptron Co. (Daejeon, Korea). The polyethersulfone ultrafiltration membrane was

The RIPL-conjugated liposomes (RIPL-Lipo) were prepared by conjugating RIPL peptides to maleimide-derivatized liposomal vesicles via a thiol-maleimide reaction as previously reported [29]. All liposomal vesicles were prepared by the thin film hydration method. First, PC and Tween80 were dissolved at a molar ratio of 9:1 in chloroform–methanol mixture (1:1) in a round bottom flask. After adding DSPE-PEG2000-maleimide at 0.2–4.0% of the total molar concentration, the organic solvent was removed by rotary vacuum evaporation at 35 °C, above the phase transition temperature of the phospholipid and Tween80, and solvent traces were removed under nitrogen gas streaming. Thin lipid film was then hydrated with 1 mL distilled water containing 10 mg/mL of FITC-dextran. The total molar concentration of the liposomal



constituents was 7.16 mM initially. Prepared liposomal solution was extruded on Mini-Extruder with 20 passes through a 200 nm polyethersulfone membrane for homogenous size distribution and efficient entrapment. Finally, the RIPL peptide solution was added to the maleimide-derivatized liposomal solution and allowed to react for 12 h at room temperature. RIPL-Lipo were purified from un-reacted RIPL peptide and un-entrapped FITC-dextran using a cellulose ester dialysis membrane (100 kDa MWCO) against distilled water for 48 h. After dialysis, liposomal stock solutions were prepared by replenishing the purified liposomes with distilled water to 5 mL, finally resulted in total molar concentration of the liposomal constituents as 1.432 mM. Based on the composition ratio of conjugated RIPL peptide on liposomal surface as 0.1, 0.35, 1.0, and 2.0 mole ratio, the liposomal stock solutions were designated as RIPL(0.1)-Lipo, RIPL(0.3)-Lipo, RIPL-Lipo, and RIPL(2.0)-Lipo, respectively. For comparison, conventional liposomes (CL) were separately prepared with PC and Tween80 as described above, excluding the addition of DSPE-PEG2000-maleimide and RIPL peptide. And empty liposomes were separately prepared by the same procedure except for hydration with distilled water under the absence of FITC-dextran. 2.5. Conformational characterization of RIPL-Lipo The number of external maleimide groups and the conjugation rate of RIPL peptides on maleimide-derivatized liposomes were calculated indirectly by determining the amount of unreacted cysteine in an Ellman’s reaction [4]. To block the unreacted maleimide residue, maleimide-derivatized liposomes were incubated at room temperature for 30 min with a three-fold molar amount of cysteine hydrochloride anhydrous. A known amount of DTNB (0.1 mg/mL) was added to react with the unreacted cysteine, leading to the formation of a cysteine-TNB (5-thio-2-nitrobenzoic acid) adduct to 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. The conjugation rate of RIPL peptides on maleimide-derivatized liposomes was calculated as described above by determining unreacted maleimide of RIPL-Lipo after dialysis. The HPLC systems consisted of a pump (W2690/5, Waters, USA), UV detector (W2489, k = 412 nm, Waters, USA), a data station (Empower3, Waters, USA), and a C18 column (Shiseido, Japan) at the flow rate of 1.0 mL/min. The mobile phase was composed of a mixture of methanol and 10 mM ammonium formate solution (5:95 v/v).

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of 1.0 mL/min. The FITC-dextran peak was separated with a retention time of 1.3 min. Prior to the assay, liposomal samples were subjected to pre-treatment procedure by the addition of 2% triton-X and sonication to break up the vesicle entirely. Separately, entrapment efficiency (EE) of FITC-dextran was determined by centrifugation. Aliquot of unpurified solution of RIPL-Lipo was diluted 4000 times with distilled water and centrifuged (12,000g, 30 min), and the amount of FITC-dextran in the supernatant ([FITC]supernatant) was determined by HPLC. EE was calculated by the following equation:

EEð%Þ ¼

½FITCinitial ½FITCsupernatant 100 ½FITCinitial

where [FITC]initial is the amount of FITC-dextran initially added in the film hydration process. 2.8. Cell culture All cell lines used in this study, including human prostate cancer cells (LNCaP, DU145 and PC3), ovarian carcinoma cells (SKOV-3), breast cancer cells (MCF-7), and human keratinocyte cells (HaCaT), were grown in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 units/ml penicillin G, and 100 lg/mL streptomycin. 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. 2.9. In vitro cell uptake specificity of RIPL peptide Cell specificity and cell penetration efficiency of the synthesized peptides were estimated by determining the mean fluorescence intensity (MFI) of FITC or FITC-dextran via flow cytometry (FACSCalibur; Becton Dickinson, New Jersey, USA). To analyze the cell specificity of the RIPL peptide, various cell lines including LNCaP, SK-OV-3, MCF-7, DU145, PC3 and HaCaT were seeded in growth media at a density of 1 106 per well in a 6-well plate. After reaching 70–80% confluence, the cells were incubated with 1 lM RIPL peptide-conjugated FITC (RIPL-FITC) in RPMI medium for 2 h at 37 °C, washed with PBS three times, and subjected to flow cytometry for the quantification of MFI values via acquisition of 10,000 events per histogram. To evaluate the cell penetration efficiency, LNCaP cells were co-incubated with FITC-dextran (28 lg/mL) and 3 lM of R8, IPL, or RIPL peptides for 2 h at 37 °C. Cells were then harvested and washed with PBS, followed by the measurement of MFI values as described above.

2.6. Size and zeta potential of RIPL-Lipo 2.10. In vitro cell uptake study of RIPL-Lipo Liposomal stock solutions (10 lL) were diluted to 800 lL of 1 mM KCl solution and were examined for size distribution and PDI (polydispersity index) using a dynamic light scattering particle size analyzer (Zetasizer Nano-ZS; Marlvern Instrument, Worcestershire, UK) equipped with a 50 mV laser at a scattering angle of 90°. Zeta potential measurements were taken with disposable capillary cells and the M3-PALS measurement technology, built into the Zetasizer system. All measurements were carried out in triplicate under ambient conditions. 2.7. Determination of FITC-dextran and encapsulation efficiency The concentration of FITC-dextran in liposomal stock solutions was measured by HPLC with fluorescence detection at an excitation wavelength of 485 nm and emission wavelength of 520 nm. Chromatography was carried out on a C18 column (Shiseido, Japan) with methanol–10 mM phosphate buffer (5:95 v/v) at the flow rate

The uptake of liposomes into cultured cells was examined by determining the MFI of the probe using a FACS, and visualized by monitoring the cell association using a fluorescence microscope and CLSM. Briefly, LNCaP, SK-OV-3, MCF-7, DU145, PC3, and HaCaT cells were seeded in growth media at a density of 1 106 cells per well into NUNC CC2 chamber slides. After reaching 70–80% confluence, the cells were incubated for 2 h at 37 °C in RPMI media (2 mL) containing liposomal stock solutions (0.1 mL) of CL and RIPL-Lipo, in which the concentration of FITC-dextran was 28 lg/ mL. In order to measure MFI values by flow cytometry, the cells were treated as previously described. For microscopic observation, the cells were visualized using a fluorescence microscope under 200 magnification (Motic, Beijing, China). Separately, LNCaP cells treated as described above were washed three times with PBS, mounted onto slides without fixation process, and the fluorescence of the probes delivered to the cells was monitored using a Zeiss


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LSM 510 Meta confocal microscope with Z-sectioning mode under 400 magnification (Carl Zeiss, Oberkochen, Germany). Confocal image of live cells was obtained with different chamber slides at predetermined time points of 10 min, 30 min, 1 h, and 2 h. Z stacks were created at 1 lm intervals throughout the 20 lm of the sections with a guard region of 2 lm excluded from top and bottom of the Z stack. 2.11. Cytotoxicity assessment The cytotoxicities of RIPL peptides and RIPL-Lipo were evaluated in LNCaP and HaCaT cells by WST-1 assay as previously reported [4,30]. Based on the cleavage of WST-1 into formazan by mitochondrial dehydrogenases in viable cells, a colorimetric assay for quantifying cell proliferation and cell viability was carried out. LNCaP and HaCaT cells were seeded in growth medium at a density of 1 104 per well into a 96-well plate. After reaching 70–80% confluence, the cells were incubated at 37 °C for 2 h in RPMI medium containing RIPL peptide or empty RIPL-Lipo at different concentrations. In positive and negative controls, methanol and distilled water (D.W.) were used to replace the sample treatment, respectively. Cells were then incubated with WST-1 reagent at 37 °C for 2 h, and the absorbance of WST formazan dye was measured at 450 nm using a microplate reader. Cell viability was calculated as the percentage of viable cells relative to the untreated sample. 2.12. Statistical analysis Values were processed using Microsoft Excel 2010 software and presented as mean ± standard deviation (n = 3). Statistical significance was determined by Student’s t-test and considered to be significant at P < 0.05. 3. Results 3.1. Synthesis and identification of peptides R8, IPL, RIPL peptide, and RIPL-FITC were synthesized by Fmoc SPPS using automated peptide synthesizer and purified with preparative HPLC. The molecular weight (MW) of a synthesized peptide was calculated by first, summing every MW of all amino acids in the relevant peptide sequence, then, by subtracting from that sum the number of peptide bonds multiplied by the MW of water, since the formation of peptide bonds is always accompanied by the loss of one water molecule. The molecular ion peaks of R8, IPL, RIPL peptide, and RIPL-FITC were measured as 1370, 853, 2102, and 2619, respectively. As listed in Table 1, calculated MWs of all peptides closely corresponded to the MWs of the purified peptides observed by LC/MS analysis. 3.2. Cell uptake specificity of RIPL peptide Cellular uptake specificity of the RIPL peptide by Hpn-expressing cells was assessed by measuring MFI after various cell lines (SK-OV-3, MCF-7, and LNCaP for Hpn(+); DU145, PC3, and HaCaT for Hpn()) were treated with fluorescent macromolecules. FITC-dextran was used as a model compound because it is minimally transported through cell membranes. As shown in Fig. 1, MFI values of FITC-dextran alone were less than those of RIPL-FITC in all tested cell lines. The fluorescent marker itself was poorly transported into the cells, regardless of cell type. In comparison, the uptake of RIPL-FITC was remarkably high. Significant differences, at P < 0.05, were found in all Hpn(+) cell lines, but not in Hpn() cell lines. Particularly in the LNCaP cell line, the uptake

of RIPL-FITC was 8.3-fold greater than that of FITC-dextran, suggesting a selective interaction of the RIPL peptide with the specific cell line. For further evaluation how the RIPL peptide enhances the uptake of FITC-dextran, comparative uptake studies in LNCaP cell were performed with equivalent peptides (R8, IPL, RIPL) or in the absence of a peptide (Control). As depicted in Fig. 2, when compared to the control, R8 and RIPL significantly increased the MFI values, but IPL did not. The MFI value representing R8 uptake was greater than that of RIPL, possibly due to the reduced mole fraction of pArg in the RIPL peptide. These results suggested that pArg and IPL played important roles in the enhancement of cell penetration and cell homing function, respectively.

3.3. Characteristics of RIPL-Lipo RIPL-Lipo systems were successfully prepared with PC, Tween 80, DSPE-PEG2000-mal, and RIPL peptides, and characterized for physical and conformational properties (Table 2). We investigated the physical characteristics of liposomal nanocarrier systems in terms of vesicular size, polydispersity index, zeta potential, and EE. The average size of all systems was observed to be about 160 nm by dynamic light scattering. Low polydispersity indices, below 0.07, indicated a narrow and homogenous size distribution. Compared to the slight negative charge of CL, all RIPL-Lipo systems were positively charged. RIPL(0.1)-Lipo, RIPL(0.3)-Lipo, RIPL-Lipo, and RIPL(2.0)-Lipo showed zeta potentials of about 6, 16, 24, and 27 mV, respectively, indicating the proportions of liposome to the amount of attached RIPL peptide. This peptide contains positively charged amino acid (Arg) molecules in the sequence. The concentration of FITC-dextran in liposomal stock solutions was measured as 0.56 ± 0.02 mg/mL for all RIPL-Lipo. Conjugation of RIPL peptides to liposomal surfaces did not affect EE, revealing about 28% of initially added amount of FITC-dextran on average for all formulations. Conformational aspects of RIPL-Lipo were characterized by determining the external amounts of maleimide groups and bound peptide molecules. The molar amount of external maleimide groups acting as a substrate for peptide conjugation was determined by Ellman’s reaction. The molar amount of external maleimide [Mal]external, was calculated as [Cys]initial [TNB]detected, where [Cys]initial is the molar amount of cysteine initially added and [TNB]detected is the molar amount of TNB detected by HPLC. Ellman’s assay revealed that approximately 51 ± 2.3% of the initially added DSPE-PEG-mal was oriented externally at the liposomal surface, regardless of the amount of DSPE-PEG-mal added (0.2–4.0%). The number of external maleimide groups per vesicle was then calculated as follows: [N]vesicle [Mal]external/[Lipid]total, where [N]vesicle is the number of lipid molecules that formed a vesicle, [Mal]external is the molar amount of external maleimide as described above, and [Lipid]total is the molar amount of total lipids initially added. [N]vesicle was estimated by the following formula [31]: 4pr2 2/A, where r and A refer to the radius of the liposomes (80 nm) and the cross-sectional area of the PC head group (0.72 nm2), respectively. From these equations, we calculated that the vesicle was formed by approximately 223,000 lipid molecules. The extent of RIPL peptide conjugation on maleimide-derivatized liposomes was demonstrated by determining the amount of unreacted maleimide after dialysis of the product. The conjugation reaction was completed voluntarily, yielding no detectable, residual TNB. As a result, RIPL peptide molecules located on the external surface of the vesicle proportionally increased with the amount of peptide added: approximately 230, 800, 2300, and 4550 peptide molecules per vesicle for RIPL(0.1)-Lipo, RIPL(0.3)-Lipo, RIPL-Lipo, and RIPL(2.0)-Lipo, respectively.


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M.H. Kang et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx Table 1 Sequence and molecular weight of peptides used. Abbreviation

Sequence

Calculated MW

Observed MW

Description

R8 IPL RIPL RIPL-FITC

RRRRRRRRC IPLVVPLC IPLVVPLRRRRRRRRC IPLVVPLRRRRRRRRCK-FITC

1369.83 852.51 2101.32 2618.80

1370 853 2102 2619

Known as cell penetrating sequence [36] Known as hepsin-specific sequence [14] A novel peptide for cell penetrating and hepsin targeting Used as a fluorescent maker by RIPL peptide binding

Fig. 1. Cell specificity of RIPL peptide in Hpn(+) and Hpn() cell lines. Various cells were incubated with 1 lM RIPL-FITC or FITC-dextran alone at 37 °C and pH 7.4 for 2 h. Mean fluorescence intensity (MFI) was measured as cellular uptake by flow cytometry. Values represent mean ± S.D. (n = 3). Statistical analysis was performed using the Student’s t-test (*P < 0.05 versus paired group; **P < 0.005 versus paired group).

Fig. 3. Effect of the number of conjugated RIPL peptides per vesicle on the cellular uptake of RIPL-Lipo. LNCaP cells were treated with RIPL(0.1)-Lipo, RIPL(0.3)-Lipo, RIPL-Lipo, and RIPL(2.0)-Lipo containing equivalent FITC-dextran (28 lg/mL). Values represent mean ± S.D. (n = 3).

3.4. Selection of the optimized RIPL-Lipo system Surface modification of the vesicular nanocarrier could be a crucial factor for cell interaction. Therefore, when preparing the four RIPL-Lipo systems, it was necessary to screen the RIPL-Lipo constructs for further experiments. We analyzed the effect of the conjugated RIPL peptide ratios on the cellular uptake of RIPL-Lipo, by measuring MFI after treatment in LNCaP cell line. As shown in Fig. 3, cellular uptake was dependent on the number of RIPL peptides. MFI values increased proportionally up to 2300 molecules of the conjugated peptides, but, afterward, the value plateaued. Therefore, RIPL-Lipo, which has 2275 peptide molecules, was selected and used thereafter as the optimized system. 3.5. In vitro cellular uptake evaluation of RIPL-Lipo

Fig. 2. Effect of admixed peptides for FITC-dextran uptake in LNCaP cell. Cells were treated with FITC-dextran (6.2 lM) and different peptides (3 lM) for 2 h, while control was treated with FITC-dextran alone. Values represent mean ± S.D. (n = 3). Statistical significances were compared using the Student’s t-test (*P < 0.05 versus paired group; **P < 0.005 versus paired group).

Cellular uptake efficiency of RIPL-Lipo was assessed by flow cytometry in various cell lines such as LNCaP, DU145, and PC3 for human prostate cancer, MCF-7 for breast cancer, SK-OV-3 for ovarian carcinoma, and HaCaT as a reference. Cells were treated with different samples of FITC-dextran alone, FITC-dextran-loaded CL or RIPL-Lipo. As shown in Fig. 4 (upper panel), a greater shift of

Table 2 Physical and conformational characteristics of liposomal nanocarriers. Formulations

CL RIPL(0.1)-Lipo RIPL(0.3)-Lipo RIPL-Lipo RIPL(2.0)-Lipo *

Composition (mol ratio)

Conformational characteristics*

Physical characteristics a

b

PC

Tween80

DSPE-PEG-Mal

Peptide

Size (nm)

PDI

ZP (mV)

EE (%)

Total maleimides/vesicle

Peptide molecules/vesicle

90 89.8 89.4 88.2 86.4

10 10 9.9 9.8 9.6

– 0.2 0.7 2.0 4.0

– 0.1 0.35 1.0 2.0

160.3 ± 5.4 162.7 ± 4.3 164.5 ± 2.4 164.2 ± 2.7 165.9 ± 3.9

0.038 0.048 0.052 0.065 0.070

2.4 ± 3.2 6.1 ± 0.8 16.2 ± 1.1 24.2 ± 2.7 27.2 ± 0.9

29.3 ± 2.7 28.4 ± 1.3 29.0 ± 1.7 28.3 ± 2.1 27.7 ± 2.4

– 446 1589 4460 8920

– 227 810 2274 4549

Values calculated as described in the text. Zeta potential. b Entrapped efficiency for FITC-dextran. a


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Fig. 4. Flow cytometry results for FITC-dextran uptake in Hpn(+) and Hpn() cell lines incubated for 2 h. Upper panel shows the treatment effect: untreated cells (black); cells treated with FITC-dextran alone (red), FITC-dextran loaded CL (green), and FITC-dextran loaded RIPL-Lipo (blue). Lower panel indicates the relative ratio of MFI values in different treatments: (a) FITC-dextran loaded CL versus FITC-dextran alone; (b) FITC-dextran loaded RIPL-Lipo versus FITC-dextran alone; (c) FITC-dextran loaded RIPL-Lipo versus FITC-dextran loaded CL. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

the FI graph was observed after RIPL-Lipo treatment, whereas little to no shift was observed after treatments with CL or FITC-dextran alone. The MFI values of RIPL-Lipo were counted in the order of

SK-OV-3 (419) > LNCaP (214) > MCF-7 (153) > PC3 (87) > DU145 (33) > HaCaT (21), indicating selective binding and cellular uptake of RIPL-Lipo due to cell penetrating and homing function of the



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Fig. 5. Fluorescence microscopy images of various cell lines incubated with RIPL-Lipo at 37 °C for 2 h. Concentration of FITC-dextran was 28 lg/mL. Scale bar indicates 100 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

conjugated RIPL peptide. In terms of cell specificity, the relative ratio of MFI values in different treatments was calculated for further analysis (lower panel in Fig. 4). Compared to FITC-dextran alone, CL revealed less than 3-fold increase in all cell types tested, whereas RIPL-Lipo showed 5- to 7-fold increase in Hpn() cells (DU145, PC3, and HaCaT) and approximately 20- to 70-fold increase in Hpn(+) cells (SK-OV-3, MCF-7, and LNCaP). Likewise, in comparison with CL, RIPL-Lipo increased the MFI values by as much as 2- to 5-fold in Hpn() cells and 9- to 26-fold in Hpn(+) cells. Therefore, in summary, cellular uptake of the macromolecule was increased by liposomal formulation, and the uptake was further enhanced by RIPL peptide modification of the liposomal surface. Interestingly, Hpn-specific intracellular delivery was feasible with RIPL-Lipo.

translocational behavior of RIPL-Lipo in LNCaP cells was further investigated by CLSM with orthogonal views of Z stacks (Fig. 6). Immediately after the treatment (10 min), green fluorescence with mild intensity in XY plane was observed in the vicinity of cell membranes, indicating selective binding of RIPL-Lipo to the extracellular membrane of Hpn(+) cells. In comparison, the intensity in Z directions (XZ and YZ planes) was very weak or negligible. After 30 min, green spots with moderate intensity were observed in the interior compartment of the cell, exhibiting evidence of internalization of RIPL-Lipo. After 1 h, in all orthogonal planes, green spots became intensified and were distributed throughout the cell structure. This intense response was maintained for 2 h after the treatment. Cytosolic internalization of RIPL-Lipo was time-dependent. 3.7. Cytotoxicity of RIPL peptide and RIPL-Lipo

3.6. Selective binding and internalization of RIPL-Lipo to Hpn(+) cell To better understand the selectivity and translocational behavior of RIPL-Lipo, it was visualized by fluorescence microscopy and CLSM. Hpn-favoring RIPL-Lipo was added to a suspension of Hpn(+) or Hpn() cells and microscopic monitoring took place at appropriate time points. First, in the fluorescence microscopic observation (Fig. 5), the addition of RIPL-Lipo revealed a cell-type dependency: prominent green spots were visible in Hpn(+) cells, whereas only weak or faint marks were visible in Hpn() cells. This observation was consistent with the results of flow cytometry analysis, indicating RIPL-Lipo selectively binds to and interacts with Hpn-expressing cells. In the case of CL, regardless of cell type, fluorescence spots were invisible (data not shown). Meanwhile, the

The toxicities of RIPL peptide and RIPL-Lipo were examined using LNCaP and HaCaT cells. The cells were treated with varying amounts of peptide or liposomes, and cell viability was examined by WST-1 assay. Cell viability in the untreated group was deemed to be 100%. As shown in Fig. 7A, toxicity was minor (cell viability >90%) with the addition of up to 50 lM of RIPL peptide in both cells. However, cell viability decreased to less than 70% at concentrations above 100 lM for LNCaP or 500 lM for HaCaT cells. Additionally, as depicted in Fig. 7B, RIPL-Lipo revealed no toxicity when added in concentrations less than 10% (cell viability >90%) in both cell lines, though cell viability decreased as the concentration increased over 10%. However, the concentration range used in this experiment was very low: less than 5 lM or 5% for RIPL peptide


8


Fig. 6. Confocal images with orthogonal views of LNCaP cells incubated with RIPL-Lipo at 37 °C at pH 7.4. Concentration of FITC-dextran was 28 lg/mL. The positions of the section plane were shown by colored lines; XY plane (blue), XZ plane (green), YZ plane (red). Scale bar indicates 50 lm. (A) Immediately after the treatment, a small amount of RIPL-Lipo strongly bound to extracellular membrane because of Hpn selectivity; (B) after 30 min, RIPL-Lipo were translocated into cells owing to endocytosis and cell penetration; (C) after 1 h, florescence intensity of RIPL-Lipo in cytosol was increased overall; (D) after 2 h, intense fluorescence was maintained. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

and RIPL-Lipo, respectively. Cytotoxicity of CL was negligible in both cell types (data not shown). Therefore, we suggest that RIPL-Lipo did not cause any significant toxicity in either cell type.

4. Discussion Various approaches for selective ligand tagging of molecular cargo and drug carriers have been tested to enhance the cellular uptake specificity of therapeutic drugs and/or imaging agents. In particular, much attention has been paid to the use of cell-specific and cell-penetrating peptides or proteins, which may result in significant increases in drug concentration at target site or cell, therefore dramatically reducing unwanted side effects [32]. Given that the cell membrane is a major biological barrier for the delivery of therapeutic drugs and particulate carriers [32], successful employment of efficient intracellular target moieties, which selectively bind to cell surface receptors and promote selective uptake of attached cargos, is a point of focus in emerging medicine, especially for cancer chemotherapy. In previous research, CPPs have been successfully employed for enhanced intracellular drug delivery [4,33,34]. CPP-conjugated nanoparticles could be accumulated in cancer tissues by the enhanced permeability and retention effect termed ‘‘passive targeting.’’ Then, the nanoparticles would be internalized into cancer cells by CPP-mediated translocation. However, because of their non-selectivity, the application of CPPs has been very limited. In recent years, several CPHPs have been successfully employed for targeted drug delivery. For example, ErbB2-binding peptide (homing sequence), TAT-derived peptide

(CPP), and STAT3BP (signal transducers and activators of transcription proteins) were serially connected, demonstrating selective inhibition of the growth of ErbB2-overexpressing cells in vitro and a greater reduction in tumor growth in xenograft models in vivo [35]. In the present study, we synthesized a novel CPHP, which was named RIPL peptide (16mer; 2.1 kDa), and developed a RIPL-Lipo system for Hpn-specific drug targeting with enhanced cellular uptake. RIPL peptide was successfully synthesized by Fmoc SPSS, an automated peptide synthesizer, and its secondary and tertiary structures were verified with relevant software programs. This novel CPHP carries two essential domains of IPL for targeting action and R8 for cell penetrating action. IPL analogues were randomly selected by phage display in Hpn-transfected PC3 cells, due to a shared homology in secondary structure of protein sequences [14]. To investigate structural similarity between RIPL peptide and IPL analogues, we developed a prediction for the secondary structure of the RIPL peptide through use of the PSIPRED method, in which secondary structure is designated as C (coil), E (strand), and H (helix). The sequence of IPL analogues has a connection of coil and strand structure (CCEE) in common (Table 3). In the RIPL peptide, the C-terminus of the IPL peptide was connected to the R8 peptide: Despite the pArg linkage, this common secondary structure was retained; therefore, its Hpn targeting property would not be hampered. We demonstrated that fluorescence-tagged RIPL peptide (RIPL-FITC) exhibited greater cellular uptake in Hpn(+) cells than the uptake of FITC-dextran alone (Fig. 1), indicating the retention of Hpn affinity, regardless of R8 linkage.



Fig. 7. Cytotoxicities of RIPL peptide (A) and empty RIPL-Lipo (B) in LNCaP and HaCaT cells by WST-1 assay. For positive and negative controls, methanol (MeOH) and distilled water (D.W.) were used to replace the sample treatment, respectively. The bars represent the concentration range used in this study. Data are mean ± SD (n = 3).

Table 3 Secondary structure of peptides predicted by PSIPRED method.

a

Peptide sequence

Secondary structurea

IPL analogues IPLVVPL IPLWVPL IPLVLVPL IPLVVPLGGSCK

CCEECCC CCEECCC CCEEEECC CCEEEECCCCCC

RIPL peptide IPLVVPLRRRRRRRRC

CCEEEHHHHHHHHCCC

Represented by C (coil), E (strand), and H (helix).

Cell penetration efficiency of Arg-rich peptides is related to the number of Args in peptide sequence. When the number of Args was increased by 16, R8 peptides exhibited optimal translocation into mouse macrophage RAW 264.7 cells [36]. In this regard, the RIPL peptide was considered to include the optimal number of Args. The cell penetrating capability of the RIPL peptide could be estimated with a CPP prediction method [37], in which the value intervals of CPPs are described using a set of five descriptors (Z1–Z5) that represent a composite of the physical characteristics of the amino acids. Among the five descriptors, the most relevant are Z1 (lipophilicity), Z2 (steric bulk), and Z3 (polarity). The value intervals for efficient CPPs range as follows: Z1 ranges from 1.25 to 1.92; Z2 ranges from 1.22 to 1.29; Z3 ranges from 0.5 to 1.94. The numerical calculation of average Z values for amino acids constituting RIPL was obtained as 0.48 (Z1), 0.73 (Z2), and 2.5 (Z3): lipophilicity and steric bulk were satisfactory, but polarity was not. Nevertheless, the inclusion of this novel peptide allowed for significantly better cell penetration than the

9

control or IPL (Fig. 2). In comparison with R8, however, cellpenetrating efficiency of the RIPL peptide was somewhat reduced. This reduction was commonly presented in artificial CPHP, possibly due to a decreased molar fraction of relevant peptides in the conjugate. For instance, the cell penetration efficiency of pVECPEGA (LLIILRRRIRKQAHAHSK-CPGPEGAGC), a combination of pVEC (CPP) and PEGA (aminopeptidase binding peptide), was 0.25-fold less than that of the constituting peptides [23]. Moreover, instead of PEGA, when a linear breast tumor homing peptide (CREKA) was conjugated to pVEC, the efficiency was decreased to one-third of the constituting peptides [38]. These observations may be attributed to the reduced molar fraction of positively charged amino acids like Arg and lysine. In addition, because the cell surface is negatively charged, a reduction in positivity could reduce the electrostatic interaction between peptide and cell, thereby decreasing the cell-penetrating effect [39]. Cellular binding and the internalization of ligand-modified liposomes may depend on the extent of conjugation of the targeting ligand and CPPs to the liposomal surface [34,40]. For the successful development of RIPL-Lipo, the architecture of RIPL conjugation to the liposomal surface should be configured in linear or brush conformation aspects: When the distance (D) between PEG molecules on the nanoparticle surface is shorter than Rf, which defines the Flory diameter of the space occupied by a PEG molecule, the lateral pressure between the overcrowded PEG linkers will force the extension of the PEG chains into a linear conformation. This extension of ligand moieties away from the surface into more linear conformations contributes to the interaction between ligand and target molecule [2,41]. The minimal number of PEG linkers needed for a linear conformation (nPEG) was calculated as follows: nPEG = 4pr2/D2, where r refers to the radius of liposomes and D value is equal to the Rf of the linker molecules. The RIPL-Lipo system was measured to be 80 nm in radius and used PEG2000, whose Rf value is approximately 5.6 nm in solution, as a linker [2]. Therefore, the nPEG needed in order to adopt a linear conformation was calculated to be about 2500. In this study, as shown in Fig. 3, cellular uptake of RIPL-Lipo was maximized at the conjugation of 2300 peptide molecules. This result is in agreement with the theoretical calculation, since RIPL peptide was conjugated to PEG linkers. In addition, the number of conjugated CPPs on the liposomal surface for optimal system efficiency is dependent on the type of target ligand, liposomal size, and cell type. In previous studies, the translocation of liposomes modified with the TAT peptide or penetratin was proportional to the number of peptide molecules attached to the liposomal surface [34]. The penetratin-conjugated liposomal system showed strong cell-association and internalization at the conjugation level of 120 peptide molecules [42]. In contrast, the HVGGSSV peptide, a tumor vasculature-targeting peptide selected by phage display as an IPL peptide without cell penetrating property, was attached at a level of 2% of total lipid (about 1700 peptide molecules) to enhance tumor accumulation [43]. To the best of our knowledge, our study is the first documented attempt to optimize the extent of CPHP conjugation on a liposomal surface. As illustrated in Fig. 8B, we propose that RIPL-Lipo might be endocytosed after selective binding to Hpn(+) cells and temporarily entrapped in endosomes, later escaping into the cytosol. RIPLLipo may selectively bind to the cell surface due to Hpn recognition by IPL domain, then, it may undergo proteolytic cleavage by serine protease exposing the R8 peptide and triggering cell penetration. It has been generally recognized that arginine-rich, peptide-conjugated cargos are translocated by endocytosis [44–47], even though the cellular uptake mechanism of arginine-rich peptides was thought to undergo non-endocytic translocation [48]. Therefore, liposomes carrying cleaved pArg may be translocated by the endocytic pathway, which leads to lysosomal delivery and subsequent degradation or escape to cytosol compartment. Similar


10


Fig. 8. Schematic diagram describing the tertiary structures of the RIPL peptide and Hpn (A) and uptake mechanism and intracellular pathways of RIPL-Lipo in Hpn(+) and Hpn() cells (B). RIPL-Lipo selectively bound to Hpn, therefore, less interacted with Hpn() cells. Protease cleaved polyarginine of RIPL sequence. The uptake of liposomes by endocytosis would cause temporary entrapment in endosome, however, liposomes could escape into cytosol or be degraded by lysozyme. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

mechanisms for translocation of the iRGD peptide (CRGDKGPDC)modified liposomes were reported: The RGD motif of the iRGD peptide recognizes the vb3/avb5 integrins on tumor endothelial cells; iRGD is then cleaved by proteases to expose the cryptic CendR element, RGDK/R, at the C-terminus, and eventually the CendR element mediates binding to neuropilin-1 to induce vascular and tissue penetration [49]. There are some arguments as to whether or not the endosomally-entrapped liposomes could avoid lysosomal breakage and travel to cytosol while remaining intact [17]. In the case of RIPL-Lipo, due to CPP functioning of R8, the liposomes could escape from the endosome being delivered to cytosol. It was evident that pH-sensitive formulations, which were designed to hide a Tat moiety at physiological pH but expose the CPP moiety in an acidic endosomal environment, could translocate through the endosomal membrane, passing by lysosomal degradation, and subsequently increasing the cytotoxicity of cancer cells [50,51]. Our observation with CLSM also supported this behavior, as shown in Fig. 6, resulting in the scattered dot-like fluorescence throughout the cytoplasm even after 2 h. Cancer-associated proteases including Hpn, matrix metalloproteases (MMP), and urokinase plasminogen activator (uPa) have been focused on targeted drug delivery: MMP was used to selectively activate a CPP moiety with MMP-sensitive linker in tumor sites [52]; uPa was applied to uPa-sensitive polymer-caged liposomes using a protease function [53]. However, there has been little application of Hpn protease in drug delivery. We firstly demonstrated that Hpn protease functionality could be applied to developing strategies of drug targeting. Instead of PSMA, Hpn could be used as a diagnostic tool for prostate cancer, in which metastasis accompanies Hpn overexpression [54]. By the application of the RIPL peptide and RIPL-Lipo, we also proved that Hpn was a selectable target molecule for breast and ovarian cancer cells. In particular, RIPL-Lipo would be a promising tool for Hpnselective targeting for various drugs covering cell membrane impermeable macromolecules, protein drugs, diagnostics, and chemotherapeutics.

5. Conclusion In the present study, we synthesized a novel CPHP, which we have named RIPL peptide (16mer; 2.1 kDa), and successfully developed a RIPL-Lipo system for Hpn-specific drug delivery, revealing enhanced selectivity and cellular uptake compared to conventional

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