The Relation between Thermodynamic and Structural Properties and Cellular Uptake of Peptides Containing Tryptophan and Arginine

Introduction Transfer of cargos across cellular membranes is a major issue in cell biology and medicine. During the past few decades, significant progress has been made in focusing on the manufacture of new carriers for cargo delivery into cells. Cell-penetrating peptides or protein transduction domains (PTDs) 1 are new carriers that have been widely used to promote the uptake of various macromolecules. CPPs have been shown to efficiently improve transportation of PMO (phosphorodiamidate morpholinos), 2 PNA (peptide nucleic acids), 3 DNA,


Introduction
Transfer of cargos across cellular membranes is a major issue in cell biology and medicine.During the past few decades, significant progress has been made in focusing on the manufacture of new carriers for cargo delivery into cells.Cell-penetrating peptides or protein transduction domains (PTDs) 1 are new carriers that have been widely used to promote the uptake of various macromolecules.CPPs have been shown to efficiently improve transportation of PMO (phosphorodiamidate morpholinos), 2 PNA (peptide nucleic acids), 3 DNA, 4 siRNA (short interfering RNA), 5 drug, 6 and nanoparticles 7 across biological membranes.Recently several classes of CPPs have been introduced.Model amphipathic peptides (MAP) are one of the most common classes of CPPs that have been used in scientific research. 8Amphipathic peptides contain hydrophobic and cationic residues in the backbone, this class of peptides have the ability to enter cells and can have antimicrobial properties. 91][12] Tryptophan has an aromatic side chain 13 and can participate in hydrophobic interactions to the lipid hydrocarbon chains of the cell membrane. 14Arginine is a hydrophilic and basic amino acid; 15 the guanidino groups of arginine tend to bind with phosphate in the cell membrane lipids. 16In this study, four amphipathic peptides containing tryptophan and arginine (RRWWWWRR, RRRWWWWRRR, WWRRRRWW and WWWRRRRWWW) were designed and synthesized in order to explore the relationships between the structural/physicochemical properties and penetration into the MCF-7 cells.

Peptide synthesis
The synthesis of peptides was carried out manually with Rink amide resin using the solid-phase synthesis method.O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU) and N,Ndiisopropylethylamine (DIPEA) in dimethylformamide (DMF) were used as coupling and activating reagents, respectively.Fmoc protecting group was removed from the resin and amino acids by addition of a solution of 20% (v/v) Piperidine/DMF.The Kaiser test was performed to monitor deprotection of peptides in solid phase peptide synthesis.A cleavage cocktail (TFA/H 2 O/TIS/EDT: 94 /2.5 /2.5 /1) was used for sidechain deprotection and the final cleavage of the peptide from the Rink amide resin.Crude peptides were precipitated by cold diethyl ether under vigorous stirring.The peptides containing the cleavage cocktail and Et 2 O were centrifuged at 4000 rpm for three minutes, followed by decantation in order to obtain the solid peptide precipitate. 17,18TC Labeling of Peptides A solution of 1.1 equivalent of FITC in pyridine/DMF/DCM (12:7:5) was prepared and incubated with the deprotected peptide for 12 h under gentle mixing conditions.19 The completion of the reaction was confirmed by the Kaiser test.The obtained FITC-peptide was cleaved from resin with the cleavage cocktail and was precipitated by diethyl ether.

Cytotoxicity analysis
The Mcf-7 cells were seeded in 200 µL of medium per well in 96-well plates 24 h prior to the experiment.The old medium was replaced by different concentrations of peptides synthesized in serum containing medium and plates incubated for 24-72 h at 37°C in a humidified atmosphere of 5% CO2.Cell viability was then determined by measuring the color intensity of the formazan solution at 570nm using an ELISA reader (Bioteck Instruments, Wmooski, VT, USA).The percentage of inhibition of proliferation was calculated as follows:

Fluorescent microscopy
The human breast cancer cell line MCF-7 was cultured in 6-well plates (200×10 3 cells/well) for 24 h in an incubator at 37°C in a 5% CO 2 environment.The Cells were treated with 15 μM concentration of R2W4R2-FITC, W 2 R 4 W 2 -FITC, W 3 R 4 W 3 -FITC and R 3 W 4 R 3 -FITC.After 1 h incubation at 37 °C, the medium containing the peptide was removed.The cells were washed with PBS and examined with an emission filter of 530 nm BP (green emission) using an Olympus IX81 fluorescence microscope (Olympus Optical Co., Ltd., Tokyo, Japan).

Flow cytometry assay
Flow cytometry was used to determine the percentage of labelled cells and compared with control cells.MCF-7 cells were treated with 5 μM, 15 μM and 25 μM concentration of peptides in separate experiments for 1 h at 37°C and the cells were washed twice with phosphate buffered saline.However, because flow cytometry does not discriminate between cell membrane-bound and internalized FITC-peptides, the cells were treated with trypsin/EDTA (0.25% trypsin and 0.02% EDTA for 5 min) to minimize any contribution of peptides attached to the outer surface of the cell membrane. 20an fluorescence ratios were determined for 10,000 cells sampled after the addition of peptides.Fluorescence ratios were calculated in arbitrary units set to a value of 100 for MCF-7 cells.The results were expressed as a percentage of gated populations and data were analysed using control cells.Flow cytometry was carried out using FACSCalibur (Becton Dickinson, San Jose, CA, USA) applying an argon laser.Excitation occurred at 488 nm for FITC.

Peptide secondary structure
Structure prediction of synthesized peptides was performed to determine their secondary structure.We applied the GOR V algorithm in order to explain the different levels of uptake by MCF-7 cells.This method has high accuracy (Q3=73.5%)with a wide range of amino acid residues for predicting the secondary structures of peptides. 21It is based on the use of information theory and Bayesian statistics for predicting the secondary structure of peptides and proteins, 22 by analysis and comparison of an input sequence with 513 proteins, to calculate the helix, sheet and coil probabilities at each residue position. 23The GOR V server uses the FASTA sequence format and calculates the probability helix (α-helix), extended (β-sheet) and Peptides uptake and thermodynamic and structural properties Advanced Pharmaceutical Bulletin, 2015, 5(2), 161-168 coil structures.The probabilities are then normalized to 0 and 1 using the following equation: 23,24 P H +P E +P C =1 The GOR V server is available online at http://gor.bb.iastate.edu(the Plant Sciences Institute at Iowa State University).

Helical wheel projection and thermodynamic parameters
Helical wheel projection illustrates the correct angular displacement from one amino acid to another amino acid in a cylindrical plot of the α-helix structure. 25The Wimley-White is one of the most common scales that can be used for free energy calculation of peptides.This value is provided by the free-energy contributions of the side chains and backbone of peptides. 26The free energy of peptides may be used to ascertain the feasibility of peptide transport across the cell membrane.Helical wheel projection, estimation of the transfer free energy and the total hydrophobic moment of peptides were performed by Membrane Protein Explorer (MPEx 3.2), free software provided by the Stephen White Laboratory at the University of California (Irvine, CA).

Cytotoxicity in cell culture
The cell viability of the human breast cancer cell line MCF-7 are summarized in Figure 1.Compared to control, the cells underwent reduced proliferation following treatment with R 2 W 4 R 2 .All of the peptides had cytotoxic effects at concentrations of 25µM and 50 µM, following a 72-hour incubation period.The cell toxicity was dose and time dependent.

Cellular Uptake and labeling
Increasing the concentration of all peptides resulted in an increased population of FITC-labelled cells (Figure 2).This effect was relatively linear in the concentration range of 5, 15 and 25μM of the peptides.The major difference was observed when comparing the two peptides containing four and six tryptophans at both ends.Accumulation of both R 2 W 4 R 2 and R 3 W 4 R 3 in MCF-7 was suitable.However the mean cellular uptake of W 2 R 4 W 2 and W 3 R 4 W 3 in 15μM concentrations of peptides was 2.52 and 7.18-fold less than that of R 3 W 4 R 3 respectively.According to the results, there was no significant difference between penetration of R 2 W 4 R 2 and R 3 W 4 R 3 sequences.It therefore appears that the location of tryptophan and arginine residues have a dramatic effect on cellular uptake.It seems that the number of tryptophan residues in W 2 R 4 W 2 and W 3 R 4 W 3 sequences affects the cellular uptake (Figure 2 and 3).

Secondary structure prediction
The results of prediction of secondary structure and the graphical model are shown in Table 1.According to the results, for W 2 R 4 W 2 sequence, coil structure is dominant with no α-helix structure.However, in other sequences, a high probability of α-helix formation was indicated.The amphipathic α-helical is important for the effective transportation of peptides across bilayer lipid membranes.It has been suggested that this structure leads to pore formation followed by peptide insertion into the cell membrane. 27No relevance between the αhelix structure and cellular uptake of W 2 R 4 W 2 and W 3 R 4 W 3 sequences was observed.

Helical wheel projection and total hydrophobic moment
It has been suggested that the cellular uptake efficiency of peptide is dependent on the regular arrangement of hydrophobic and hydrophilic amino acid residues in one side of the helical wheel projection. 11,28The helical wheel projection for R 2 W 4 R 2 , R 3 W 4 R 3 and W 3 R 4 W 3 sequences are shown in (Figure 4).According to this projection, these peptides do not show any regular structure.Therefore, it cannot be used to explain differences in cellular uptake of the peptides.The hydrophobic moment was performed to explain the high permeability of some irregular hydrophobic and hydrophilic residues in the α-helix structure, such as the amphiphilic peptide melittin. 29The results of the total hydrophobic moment (μH) are shown in Table 2. Despite the fact that these peptides had the same value for the total hydrophobic moment, W 3 R 4 W 3 had much lower cellular uptake activity than the other peptides studied in this study.

Wimley-White scales
It is believed that difference between octanol hydrophobicity scale and interfacial hydrophobicity scale can be an effective parameter to predict behavior of peptides to penetrate the cell membrane.According to the research conducted in this field, peptides with ∆G°O ct-w ≤20 kcal/mol -1 would be good candidates for penetrating the cell membrane. 30Based on the values obtained from the W.W scale (Table 2) and flow cytometry data, ∆G°O ct-w does not appear to be a suitable parameter for predicting cell penetrating peptides, since ∆G°O ct-w of all peptides were less than 20 kcal/mol -1 .Another parameter used to predict the penetration efficiency of peptides is the transfer free energy values of peptide from water to membrane.The amount of energy required for transferring a random-coil and α-helix insertion into the hydrophobic core of the cell membrane is +1.15 kcal mol -1 (∆G Oct ≥ +1.15 kcal mol -1 ). 31 According to the data in Table 2, R 2 W 4 R 2 , R 3 W 4 R 3 and W 2 R 4 W 2 have enough energy for penetration from water to membrane.The data also shows that W 3 R 4 W 3 with lowest cellular uptake has not sufficient free energy value.It seems that uptake efficiency and transfer free energy values correlate with each other and this parameter may be used to explain differences in uptake of the peptides into the MCF-7 cells (Figure 5).

Conclusion
In this study, four amphipathic peptides containing tryptophan and arginine amino acid residues were synthesized to assess the importance of different parameters in their cell uptake.The flow cytometry results showed different cellular uptake efficiency.Various thermodynamic and structural parameters that might influence peptide transport through the cell membrane were investigated.The secondary structure and the helical wheel projection failed to describe the differences in cellular uptake of the peptides.On the other hand it seems that the uptake efficiency correlates well with the transfer free energy values and this parameter may be used to describe the different uptake efficiency of the cell penetrating peptides into MCF-7 cells.

Figure 2 .
Figure 2. Cellular uptake of FITC-labeled peptides in live MCF-7 cells after incubation for 1 h at 37 °C.The uptake values of intensity are seen for R2W4R2 (A) and R3W4R3 (B), followed by, in order of decreasing intensity, W 2R4W 2 (C), W3R4W3 (D).(E) A comparison of the cellular uptake of peptides in 5, 15, and 25μM concentrations.The results are expressed as a percentage of gated populations.Each value is the mean ± SD of two separate determinations.

Figure 5 .
Figure 5.Effect of thermodynamic parameters for the transfer of peptide from water to the intracellular space.