A Novel Cell-Penetrating Peptide Derived from Human Eosinophil Cationic Protein

Cell-penetrating peptides (CPPs) are short peptides which can carry various types of molecules into cells; however, although most CPPs rapidly penetrate cells in vitro, their in vivo tissue-targeting specificities are low. Herein, we describe cell-binding, internalization, and targeting characteristics of a newly identified 10-residue CPP, denoted ECP32–41, derived from the core heparin-binding motif of human eosinophil cationic protein (ECP). Besides traditional emphasis on positively charged residues, the presence of cysteine and tryptophan residues was demonstrated to be essential for internalization. ECP32–41 entered Beas-2B and wild-type CHO-K1 cells, but not CHO cells lacking of cell-surface glycosaminoglycans (GAGs), indicating that binding of ECP32–41 to cell-surface GAGs was required for internalization. When cells were cultured with GAGs or pre-treated with GAG-digesting enzymes, significant decreases in ECP32–41 internalization were observed, suggesting that cell-surface GAGs, especially heparan sulfate proteoglycans were necessary for ECP32–41 attachment and penetration. Furthermore, treatment with pharmacological agents identified two forms of energy-dependent endocytosis, lipid-raft endocytosis and macropinocytosis, as the major ECP32–41 internalization routes. ECP32–41 was demonstrated to transport various cargoes including fluorescent chemical, fluorescent protein, and peptidomimetic drug into cultured Beas-2B cells in vitro, and targeted broncho-epithelial and intestinal villi tissues in vivo. Hence this CPP has the potential to serve as a novel vehicle for intracellular delivery of biomolecules or medicines, especially for the treatment of pulmonary or gastrointestinal diseases.


Introduction
Cell-penetrating peptides (CPPs) are peptides derived from proteins that can transport cargo such as nanoparticles, low molecular weight compounds, other peptides, proteins, and nucleic acids into cells [1]. CPPs may potentially be used during clinical procedures such as gene therapy and cancer treatment, and thus substantial efforts have been made to discover CPPs with suitable carrier properties [1,2].
CPPs are usually divided into two groups [1], synthetic peptides such as oligoarginines which penetrate 293T cells [8,9], and peptides derived from natural proteins such as TAT [47][48][49][50][51][52][53][54][55][56][57] (GRKKRRQRRRP) from nuclear transcription activator Tat protein (TAT) of human immunodeficiency virus-1, which penetrates various cell types [10]. In the past two decades, 52 CPPs derived from natural proteins that can translocate across cell membranes have been reported [1,11]. Twenty-eight of these CPPs including 15 viral protein-derived peptides, 7 animal modulator-derived peptides, 3 antimicrobial peptides, and 3 toxin-derived peptides have been demonstrated or predicted to interact with cell-surface HS before penetrating plasma membranes [1,11,12]. Most of these heparin-binding CPPs possess consensus heparin-binding motifs XBBXB or XBBBXXBX, where B is a basic amino acid and X is any amino acid. These peptides are further classified as cationic or amphipathic peptides [13]. Heparin-binding CPPs not only requires electrostatic interactions, but also uses aromatic residues for hydrophobic interactions with target cells [14]. However, little is known about how sequential aromatic and cationic residues affect the interactions of CPPs with cell-surface molecules.

Cytotoxic Effects of ECP 32-41
To get a comprehensive analysis of toxic profiles induced by ECP 32-41 , cytotoxic and membrane disruptive properties of ECP 32-41 were analysed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) and lactate dehydrogenase (LDH) leakage assay, respectively. Beas-2B was treated with ECP 32-41 up to 100 mM at 37uC for 24 h. No sign of any negative effects in cell viability were observed after treatment with ECP 32-41 ( Figure 5A) and no significant changes (P.0.05) in LDH levels were found between ECP 32-41 treated and untreated cells ( Figure 5B). These results demonstrated that treatment of cells with ECP 32-41 had no effects on cytotoxicity and membrane disruption.

Tissue Targeting of ECP 32-41 in an Animal Model
GAG expression is related to cell differentiation and growth [32], and specific HPSGs are differentially expressed in different cell types [33]. To delineate tissue targeting by ECP [32][33][34][35][36][37][38][39][40][41] in vivo and to develop potential applications, eGFP-ECP 32-41 and eGFP were separately injected into the circulatory system of specificpathogen-free rats through tail veins. The tissues were immunohistochemically stained with anti-eGFP antibody. Interestingly, 1 h after injection, significant eGFP-ECP 32-41 signals were detected in broncho-epithelial and intestinal villi tissues ( Figure 7A, 7C), which was quite similar to tissue distribution as ECP [23]. eGFP alone was not detected in these tissues ( Figure 7B, 7D). As is known that mammalian mucosal cells are rich in HSPGs, ECP 32-41 may potentially be used for in vivo targeting of broncho-epithelial and intestinal villi tissues.

Discussion
CPPs are a class of peptides differing in sequence, size, and charge that can translocate across plasma membranes. In this study, a newly identified CPP corresponding to residues 32-41 of human ECP (ECP 32-41 ) was characterized. ECP 32-41 delivered a small, fluorescent compound (Figure 1), a recombinant protein (Figure 6A), and a peptidomimetic drug ( Figure 6D) into Beas-2B cells, and targeted specific rat tissues in vivo (Figure 7), showing that it can act as a delivery vehicle in both types of environments.
Previous studies have emphasized that the use of CPPs should improve drug delivery to cells, although CPPs usually target cells promiscuously [46]. Most CPPs have high internalization rates in vitro but low target specificity in vivo [47]. Certain peptides, denoted cell-targeting peptides, specifically target a certain type(s) of cell(s) and bind to their target(s) strongly [48]. CPP fusion with cell-targeting peptide might therefore, prove useful as drug delivery systems, although however, TAT linked to antibody did not retain the cell-targeting ability of the antibody [46]. Nevertheless, Kuniyasu and colleagues, using phage display technology, isolated the peptide, CAYHRLRRC that contained a lymph nodehoming sequence (CAY) and a cell-penetrating motif (RLRR) [49], which selectively penetrated leukaemia and lymphoma cells in vitro. Notably, we found that ECP 32-41 could penetrate cells in vitro and selectively penetrate broncho-epithelial and intestinal villi tissues in vivo (Figure 7). ECP 32-41 targets specific cells and tissues effectively and thus may be used in the development of innovative biomaterials for molecular detection and diagnosis purposes.
In summary, ECP 32-41 is not cytotoxic and can be covalently coupled to many different molecules, it has a substantial cargo delivery potential as an attractive candidate for intracellular delivery of therapeutic molecules. GAG-mediated internalization may be the major pathway for ECP [32][33][34][35][36][37][38][39][40][41] internalization. Finally, ECP 32-41 is a human-derived peptide and specifically targets certain tissues, we expect that, with or without modification, it can be useful as a drug delivery system.

Peptide Design and Synthesis
Peptides with or without an N-terminally conjugated fluorescein isothiocyanate (FITC) group (Table 1) were synthesised by Genemed Synthesis Inc. and their purities (.90%) were assessed by analytical high-performance liquid chromatography. FITC was conjugated to N-terminus of ECP 32-41 through a 5-carbon linker, which gave a spacer of approximately 10 angstroms in length. Peptide sequences were confirmed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry in Genemed Synthesis Inc.

Cell-based ELISA
Cells (2610 4 /well) were seeded into 96-well black plate and incubated under a 5% CO 2 atmosphere at 37uC for 24 h. Each well was then washed with 150 ml ice-cold PBS. To prevent nonspecific antibody binding, BSA was used as a blocking agent, and PBS containing 2% (w/v) BSA was added to each well at 4uC and incubated for 1 h. The wells were then washed with 100 ml icecold PBS. FITC-conjugated peptides were diluted to 10, 20, or 100 mM in PBS and then, medium (95 ml) and a peptide/PBS solution (5 ml) were gently mixed, added into a well, and the plate was placed on ice for 1 h. Each well was then washed with 100 ml PBS and the FITC fluorescent intensity for each sample was measured using a fluorescence spectrophotometer (Wallac Victor II, Perkin Elmer, USA) and excitation and emission wavelengths of 485 nm and 521 nm respectively.

Flow Cytometry
Cells (3.0610 5 /well) were added into six-well plates and cultured in the indicated medium. After 24 h, each FITC-peptide, dissolved in medium, was added into a well and the samples incubated for 1 h. Cells were then harvested, washed, and suspended in PBS. The fluorescent intensities of the cell samples were measured using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ) and excitation and emission wavelengths of 488 nm and 515-545 nm respectively. The relative internalization of each peptide is reported as the mean uorescent signal for 10,000 cells.

Confocal Laser-scanning Microscopy (CLSM)
Cells were cultured on coverslips (1.0610 4 /coverslip) in indicated medium. After 24 h, cell samples were each incubated at 37uC for 1 h with an FITC-peptide. The cells were then washed twice with PBS, fixed with 2% (w/v) paraformaldehyde and incubated, first in PBS for 15 min, then with 50 mM NH 4 Cl in PBS for 10 min, and finally permeabilised with 0.5% (v/v) Triton-X-100 at 25uC for 5 min. Nuclei were stained with Hoechst 33342 Fluorescent Stain, (Sigma-Aldrich) during the final 5 min of incubation. Cells were then washed twice with 0.05% Triton-X-100, once with PBS, and the coverslips were mounted in a Vectashield anti-fade mounting medium (Vector Labs). CLSM was performed using LSM510 META (Carl Zeiss, Göttingen, Germany) to assess the distribution of the FITC-peptides in the cells.

Heparinase and Chondroitinase ABC Depletion of GAGs
Beas-2B cells (3.0610 5 /well) were incubated with RPMI 1640 medium overnight in six-well plates and then treated with 5 U/ml of heparinase I, 2.4 U/ml of heparinase III, or 10 U/ml of chondroitinase ABC (Sigma-Aldrich) at 37uC for 2 h. After a PBS wash, the cells were incubated with FITC-ECP 32-41 and assayed for internalization of the peptide by flow cytometry as described above.

Membrane Disruption Assay
Lactate dehydrogenase (LDH) was used to quantify membrane disruption. The release of LDH from cells was measured by Promega CytoTox-ONE assay (Promega, USA). Cells (1.0610 4 / well) were seeded into the wells of 96-well plates and incubated overnight. Cell samples were then exposed to different concentrations of ECP [32][33][34][35][36][37][38][39][40][41] . After 24 h, 100 ml of extracellular medium was transferred to a black 96-well plate containing 100 ml of CytoTox-ONE reagent, incubated at RT for 10 min. Fluorescent intensity for each sample was measured using a fluorescence spectrophotometer (Wallac Victor II, Perkin Elmer, USA) and excitation and emission wavelengths of 540 nm and 590 nm respectively. LDH released from cells lysed with 0.1% Triton X-100 in medium was defined as 100% leakage and LDH released from untreated cells was set as 0% leakage.

Subcellular Fractionation
Beas-2B cells (8610 5 /dish) were cultured in 10 cm dish for 24 h, followed by incubation with 20 mM eGFP or eGFP-ECP 32-41 at 4uC for 1 h. The cells were washed twice with PBS and then incubated at 37uC for 1 h, 2 h, 3 h and 4 h, separately. Cells were then homogenized and fractionated by floatation in Percoll gradients (GE Healthcare, USA) separating cytoplasm and endosomes [51]. In brief, cells were scraped off in 1 ml PBS with a rubber policeman and pelleted at 3006g for 5 min. The pellet was resuspended in 1 ml homogenization buffer (0.25 M sucrose, 3 mM Imidazole and 0.5 mM EDTA, pH 7.3) and pelleted again at 8006g for 7 min. The pellet was resuspended in 100 ml homogenization buffer with a syringe until the cells were broken but the nuclei were still intact as observed by light microscopy. The homogenate was diluted to a total volume of 1 ml with homogenization buffer. After homogenization, the gold-filled fraction was pelleted together with the nuclei at 8006g for 7 min. The pellet was resuspended in 650 ml 17% Percoll and loaded onto a 500 ml 64% sucrose cushion in a 2 ml Beckman ultracentrifuge tube. The samples were centrifuged for 90 min at 27,0006g in a Beckman SW55Ti rotor with fast acceleration to distribute the nuclear fraction at the top and the endosome-filled organelles at the bottom of the sucrose cushion. The pellet was resuspended in 100 ml homogenization buffer and referred to endosomal fraction in the results.

Western Blotting
Protein concentration from each fraction was estimated by BCA protein assay kit (Thermo). Proteins were resolved as reported in 12% SDS-PAGE and blotted to BioTrace TM polyvinylidene fluoride Membrane (Pall Life Sciences, USA). The membrane was incubated in blocking solution (5% nonfat dry milk in PBS) for 1 h. Blots were incubated with antibodies for anti-actin (Novus Biologicals, CO), anti-lysosomal-associated membrane protein 1 (LAMP-1) (Santa Cruz Biotechnology, CA) and anti-His (Clontech Laboratories, CA) in PBS with 0.1% Tween 20 (TPBS) for 1 h. After wash with TPBS for 10 min three times, the membrane was incubated with horseradish peroxidase-conjugated anti-mouse IgG in TPBS at 25uC for 1 h. After wash with TPBS for 10 min three times, the protein on membrane was detected using chemiluminescent detection kit (ECL, Amersham Life Science) and chemiluminescence was measured by Kodak X-Omat film. The blotted signal was quantitated using NIH ImageJ software.

Immunohistochemical Staining
Adult female specific-pathogen-free Sprague-Dawley rats (Narl:SD) with body weights between 200 and 300 g were purchased from, and maintained at, the National Laboratory Animal Center, Taiwan. The rats were separated into two groups and injected with 5 nmol of enhanced green fluorescence protein (eGFP) or eGFP-ECP 32-41 through their tail veins. All animals were asphyxiated with CO 2 , 1 h after injection. All major organs including brain, heart, lung, trachea, kidney, liver, spleen and intestine were removed and immediately fixed in 10% neutralbuffered formaldehyde. The tissue samples were processed by standard methods to prepare paraffin wax-embedded block samples [25]. The blocks were sectioned into 6 mm slices and were examined using a Super Sensitive Non-Biotin HRP Detection System (BioGenex Laboratories, San Ramon, CA) as previously described [25]. All these slices were then observed by using light microscope (Zeiss-Axioplan, Germany).

Statistical Analysis
Each result is reported as the mean 6 standard deviation (SD), where n is the number of experiments. To compare two means, statistical analysis was performed using the unpaired Student's ttest in GraphPad Prism v4.02 (GraphPad Software, USA). Oneway analysis of variance (ANOVA), followed by Dunnett's test, was used to test for differences among multiple treatments. A P value ,0.05 was considered to be statistically significant.