Influence of PEG Length on Conformational and Binding Properties of CCK Peptides Exposed by Supramolecular Aggregates Antonella Accardo, Paola Ringhieri, Rosanna Palumbo, Giancarlo Morelli Department of Pharmacy, CIRPeB, University of Naples “Federico II,” and IBB—CNR, Via Mezzocannone 16, 80134 Naples, Italy Received 11 October 2013; revised 4 April 2014; accepted 10 April 2014 Published online 17 April 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22500
ABSTRACT: Five novel peptide amphiphiles (PAs), with common formula (C18)2-PEGx-CCK8 in which the CCK8 peptide and the (C18)2-hydrophobic moiety are spaced by polyethylene linkers of different length (PEG moieties with molecular weights of 700, 1000, 1500, 2000, and 3000 Daltons) are described. They act as potential targetselective nanocarriers towards tumor cells overexpressing
with higher binding properties shown by PA containing PEG with MW of 2000 Dalton. Therefore, PEG2000 can be considered as the best spacer in the formulation of C 2014 nanovectors based on CCK8 peptide amphiphiles. V
Wiley Periodicals, Inc. Biopolymers (Pept Sci) 102: 304– 312, 2014. Keywords: peptide amphiphiles; CCK8; supramolecular aggregates; PEG spacer; fluorescence; circular dichroism
cholecistokynin receptors. PAs self-assemble in supramolecular aggregates, with hydrodynamic radius ranging between 63 and 104 nm, as indicated by DLS measurements. Fluorescence studies suggested that, irrespective
This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [email protected]
from the PEG length, the tryptophan residue located at the center of the CCK8 sequence is completely surrounded by water molecules at high mobility. This result indicates a potential capability of all formulated nanovectors to recognize the overexpressed CCK-2 receptors. CD data suggest that CCK8 peptide, in most of PAs in their aggregate form, adopts a conformation allowing the interaction with the receptor. Anyway, biological data obtained by flow cytometry analysis indicate that the five PAs have a different binding ability towards the CCK-2 receptors,
Additional Supporting Information may be found in the online version of this article. Correspondence to: Giancarlo Morelli, Department of Pharmacy, CIRPeB, University of Naples “Federico II,” & IBB—CNR, Via Mezzocannone 16, 80134 Naples, Italy; e-mail: [email protected] Contract grant sponsor: Italian Minister for Research Contract grant number: PRIN 2009WCNS5C Contract grant sponsor: FIRB “RENAME” Contract grant number: RBAP114AMK C 2014 Wiley Periodicals, Inc. V
304
INTRODUCTION
P
eptide amphiphiles (PAs) are an emergent class of synthetic surfactants in which a peptide moiety with potential biological activity is covalently bound to a hydrophobic moiety, such as one or two alkyl tails. Under specific condition of pH or temperatures, PA molecules are able to self-assemble, in water solution, in well-ordered supramolecular structures with distinctive features with respect to traditional surfactants. They give, spontaneously, monolayers, bilayers, vesicles, elongated worm- or rod-like micelles or spherical micelles.1–6 Moreover, several examples of PAs able to form fibers and gels are also reported. In all these nanostructures, bioactive peptides are disposed at the periphery of the nanostructure perpendicularly to their long axis at near van der Waals density. Peptides, if well-exposed on nanostructure external surfaces, give different chemical and biological functionalities including the ability to form pores in membranes7,8 or to reach, in a selective way, a biological target9 such as membrane receptors
PeptideScience Volume 102 / Number 4
CCK Peptides Exposed by Supramolecular Aggregates
FIGURE 1 Schematic representation of peptide amphiphile (PA) monomers. The amino acid sequence of CCK8 peptide is reported by using the one-letter amino acid code. Ethoxylic linkers used during the synthesis (AdOO, Ahoh, PEG1500 and PEG3000) are also reported.
overexpressed by cancerous cells. Anyway, for all biological functions previously described, peptide availability on nanostructure external surfaces is not the unique requirement for receptor binding. According to the ligand/receptor pathway, the peptide needs to adopt a specific conformation to assure high affinity and selectivity in the binding processes.10 Recently, we reported the synthesis of a set of five peptide amphiphiles with common formula (C18)2-L-CCK8,11 in which (C18)2- is symbolically used to indicate the dioctadecyl hydrophobic moiety; CCK8 is the carboxyl terminal sequence of the peptide hormone cholecystokinin (Asp-Tyr-Met-GlyTrp-Met-Asp-Phe-Amide) able to bind, with high affinity, both CCK1-R and CCK2-R cholecystokinin receptors overexpressed by cancer cells in several human tumors;12 and -L- is here used to indicate a neutral or charged spacer [five ethoxylic linker (AdOO 5 8-amino-3,6-dioxaoctanoic acid) or three lysine residues and an AdOO unit variably positioned]. Physicochemical characterization of the supramolecular aggregates in aqueous solution by dynamic light scattering (DLS) indicated the coexistence of large (60 nm< RH < 90 nm) and small (5 nm < RH < 10 nm) structures. Moreover, peptide conformation, peptide bioavailability on the external aggregate surface and cell binding properties have been assessed. The presence of CCK8 peptide well-exposed on the outer surface of the aggregate has been demonstrated by fluorescence measurements for all systems, anyway, only systems in which the CCK8 peptide presents a structural arrangement in pseudo-a-helix conformation yielded promising binding properties to CCK2R receptors overexpressed by transfected A431 cells. In fact, according to the theory of membrane-bound pathway, CCK8 needs to assume a pseudo-a-helix conformation for the interaction with its G-protein coupled receptors.13–17 Biopolymers (Peptide Science)
305
Here we report the synthesis of five novel peptide amphiphiles (see Figure 1) with similar formula (C18)2-PEGxCCK8. The influence of the PEG length on aggregation behavior and on conformational properties of the peptide fragment is studied by DLS and circular dichroism, respectively. Moreover, the localization of the peptide with respect to the PEG corona has been assessed by fluorescence and proteolitic cleavage. Finally, the binding properties of PA aggregates have been highlighted by in vitro flow cytometry on transfected A431 cells overexpressing CCK2-R receptors. The entire study aims to define the influence of PEG spacers in PA aggregates, containing targeted peptide sequences in which appropriate peptide exposure and conformation are required for selective binding to tumor cells.
MATERIALS AND METHODS Chemicals Protected Na-Fmoc-amino acid derivatives, coupling reagents, and Rink amide MBHA resin were purchased from CalbiochemNovabiochem (Laufelfingen, Switzerland). The Fmoc-21-amino4,7,10,13,16,19-hexaoxaheneicosanoic acid (Fmoc-Ahoh-OH) and the Fmoc-8-amino-3,6-dioxaoctanoic acid (Fmoc-AdOO-OH) were purchased from Neosystem (Strasbourg, France). a-(9-Fluorenylmethyloxycarbonyl) amino-x-carboxy poly(ethylene glycol) derivatives (Fmoc-NH-PEG2000-COOH and Fmoc-NH-PEG3000-COOH) were purchased from Iris Biotech GmbH (Marktredwitz, Germany). The N,N-dioctadecylsuccinamic acid was synthesized according to published methods.18 Fluorescein 5-isothiocyanate (FITC) isomer I, BSA and PBS were purchased from Sigma-Aldrich (Milan, Italy). DMEM, FCS, L-glutamine were purchase from Lonza (Milano, Italy). C18H37CONHLys–(FITC)CONH2 (C18–FITC) was synthesized according to the procedure previously reported.11 All other chemicals were commercially available from Sigma-Aldrich or Fluka (Buchs, Switzerland) or LabScan (Stillorgan, Dublin, Ireland) and were used as received unless otherwise stated. Preparative reverse phase-high performance liquid chromatographies (RP-HPLCs) were carried out on a LC8 Shimadzu HPLC system (Shimadzu Corporation, Kyoto, Japan) equipped with a UV lambda-Max Model 481 detector using a Phenomenex (Torrance, CA) C4 (300 A˚, 250 21.20 mm, 5 mm) column eluted with H2O/0.1% TFA (A) and CH3CN/0.1% TFA (B) from 20% to 95% over 20 minutes at 20 ml min21 flow rate. Purity and identity were assessed by analytical LC-MS analyses by using Finnigan Surveyor MSQ single quadrupole electrospray ionization (Finnigan/Thermo Electron Corporation San Jose, CA), column: C4Phenomenex eluted with H2O/0.1% TFA (A) and CH3CN/0.1% TFA (B) from 20% to 95% over 20 min at 1 ml min21 flow rate. Concentrations of solutions containing peptide surfactant were determined by absorbance measurement on a UV-vis Jasco V-5505 spectrophotometer equipped with a Jasco ETC-505T Peltier temperature controller with a 1 cm quartz cuvette (Hellma).
Peptide Amphiphiles Synthesis The synthesis of Fmoc-Gly-CCK8-amide peptide was carried out in solid-phase under the standard Fmoc strategy19 by using 433A
306
Accardo et al.
Applied Biosystems automatic synthesizer. Rink-amide 4-methylbenzhydrylamine (MBHA) resin (0.78 mmol g21, 0.7 mmol scale, 0.900 g) was used. The elongation of peptides was achieved by sequential addition of Fmoc-AA-OH with benzotriazole-1-yl-oxy trispyrrolidinophosphonium hexafluoro phosphonate/1-hydroxybenzotriazole (PyBOP/HOBt) and N,N-diisopropylethylamine (DIPEA) (1/1/2) as coupling reagents, in dimethylformamide (DMF) in pre-activation mode. All couplings were performed twice for 1 h, by using an excess of 4 equivalents for the single amino acid derivative. Fmoc deprotections were obtained by 30% solution of piperidine in DMF. The peptide-resin was shared in five reactors (each one containing 0.1 mmol of peptide-resin), and each reactor was used to synthetize a peghilated peptide amphiphile (PA-[700], PA-[1000], PA-[1500], PA[2000], and PA-[3000]) by coupling manually the (ethylene glycol) spacer of different length (Fmoc-AdOO-OH, Fmoc-Ahoh-OH; FmocNH-PEG2000-COOH, or Fmoc-NH-PEG3000-COOH) and the N,Ndioctadecylsuccinamic acid mixture according to experimental procedure previously described.10,20,21 PAs were fully deprotected and cleaved from the resin with the trifluoroacetic acid (TFA)/triisopropylsilane (TIS)/H2O (95.5/2/2.5) mixture at room temperature and precipitated with ice-cold water. Purification of the crude products was carried out by RP-HPLC (k 5 280 nm). Final products at high purity degree (>90%) were collected with a yield ranging between 25 and 40% as indicated by LC-MS analysis. PA-[700]: tR 5 25.38 min, MS (ESI1): m/z 2442 calcd. C121H192N17O31S2: [M12H1]/251222.0 PA-[1000]: tR 5 24.66 min, MS (ESI1): m/z 2724 calcd. C136H226N15O37S2: [M12H1]/2 5 1363.0 PA-[1500]: tR 5 24.16 min, MS (ESI1): m/z 3024 calcd. C150H258N13O45S2: [M12H1]/2 5 1513.0 PA-[2000]: tR 5 23.48 min, MS (ESI1): m/z 3733 calcd. C181H310N18O58S2: [M12H1]/2 5 1867.5; [M13H1]/3 5 1245.3 PA-[3000]: tR 5 22.92 min, MS (ESI1): m/z 4643 calcd. C222H405N13O83S2: [M13H1]/3 5 1548.6; [M14H1]/4 5 1161.7
for for for for
173 backscatter detector. Other instrumental settings are: measurement position (mm), 4.65; attenuator, 8; temperature, 25 C; cell, disposable sizing cuvette. DLS samples were prepared at the final concentration of 2 3 1024M and centrifuged at room temperature at 13,000 rpm for 5 min. For each batch, hydrodynamic radii and size distribution were the mean of three measurements and values were calculated as the mean of three different batches. The zeta-potential (f) values of the aggregate surface were collected as the average of 20 measurements.
Circular Dichroism CD spectra were recorded from 195 to 260 nm on a Jasco J-810 spectropolarimeter equipped with a NesLab RTE111 thermal controller unit using a 1 mm quartz cell at 25 C. Circular dichroism measurements were carried out on solutions containing peptides or peptide amphiphiles at concentrations of 2 3 1024M in 10 mM PBS at pH 7.4. Other experimental settings were: scan speed, 10 nm min21; sensitivity, 50 mdeg; time constant, 16 s; bandwidth, 1 nm. Each spectrum was obtained averaging three scans, subtracting contributions from other species in solution, and converting the signal to mean residue ellipticity in units of deg cm2 dmol21 res21.
Fluorescence Microscopy The fluorescence emission spectra were recorded using a Jasco Model FP-750 spectrofluorimeter (Easton, MD) equipped with a Peltier temperature controller in 1.0-cm path length quartz cell at 25 C. Emission spectra of PAs were obtained in 290 to 450 nm range exciting at 280 nm the aggregate solutions at a peptide concentration of 1.0 3 1025M. Equal excitation and emission bandwidths were used throughout the experiments, with a recording speed of 125 nm min21 and automatic selection of the time constant.
for
Cell Culture and Flow Cytometry
All solutions were prepared by weight, buffering the samples at pH 7.4 in 10 mM phosphate buffer. pH measurements were made by using pH-meter MeterLab PHM 220. The pH-meter was calibrated with three standards at pH 4.00, pH 7.00, and pH 10.00. In most cases the samples to be measured were prepared from stock solutions. Concentrations of all solutions were determined by absorbance on a UVvis Jasco (Easton, MD) Model 440 spectrophotometer with a path length of 1 cm using a molar absorptivity (e280) of 6845M21 cm21 for CCK8. This value was calculated according to the Edelhoch method22 taking into account contributions from tyrosine and tryptophan present in the primary structure, which amount to 1215 and 5630M21 cm21, respectively.23 Fluorescent aggregates of PA, containing 1% mol mol21 of C18-FITC fluorescent probe (FITC-PA aggregates), were prepared for in vitro uptake assays. Confirmation of incorporation of the fluorescent probe (1%) into the aggregates was obtained by gel filtration on Sephadex G-50 pre-packed columns (Pharmacia Biotech).
Cellular uptake of PA aggregates was studied by flow cytometry on A431 cells overexpressing the CCK2-R by stable transfection.24 Cells were cultured as exponential growing subconfluent monolayer on 100 mm plates in DMEM supplemented with 10 % FCS, 2 mM L-glutamine and 250 lg/ml of G418 in a humidified atmosphere containing 5% CO2 at 37 C. Cell aliquots (0.5 3 106) were incubated at 4 C in 500 ll final volume. At the end of the incubation, cells were washed, resuspended in PBS/BSA and examined by flow cytometry using a FACScan (Becton Dickinson, CA) equipped with a 488 nm argon laser. A total of 20,000 events per sample were collected. Values of fluorescence intensity were obtained from histogram statistic of CellQuest software. For binding experiments, cells were incubated for 1 h with 22.6 lM FITC-PA aggregates. The fluorescence intensity of cells treated with different not fluorescent aggregates was comparable to untreated cells. Therefore, cells treated with unlabeled PA-[2000] were used as negative control. For competitive binding experiments, cells were pre-incubated with 2.26 mM H-G-CCK8 peptide (100-fold excess with respect to PA concentration) for 30 min followed by the addition of FITC-PA aggregates and an additional incubation of 1 h.
Dynamic Light Scattering (DLS) Measurements
Statistical Analysis
Mean diameter and zeta-potential (f) were measured using a Zetasizer Nano ZS (Malvern Instruments, Westborough, MA) that employs a
Data are represented as means 6 SD from at least three independent experiments. Differences between the fluorescent groups were
Preparation of the Solutions
Biopolymers (Peptide Science)
CCK Peptides Exposed by Supramolecular Aggregates
307
Table I Diffusion Coefficients (D), Hydrodynamic Radii (RH), and the Zeta Potential (f) are obtained from dynamic light Scattering Measurements for the Systems Studied Systems
Dfast 3 10211 (m2 s21)
RH (nm)
Dslow 3 10212 (m2 s21)
RH (nm)
f (mV)
– 3.9 6 0.8 2.6 6 0.8 3.2 6 1.0 1.2 6 0.4
– 5.1 6 1.0 7.8 6 2.3 6.2 6 1.9 17 6 6.4
3.2 6 1.1 2.4 6 1.0 2.6 6 1.1 2.8 6 1.0 1.9 6 0.8
63 6 23 83 6 35 76 6 32 72 6 26 104 6 47
239.7 6 14.0 247.6 6 8.11 242.7 6 6.50 237.4 6 7.53 229.5 6 5.12
PA-[700] PA-[1000] PA-[1500] PA-[2000] PA-[3000]
The terms fast and slow refer to smaller and larger aggregates, respectively.
determined by one way ANOVA using the GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA). Tukey’s posttest was performed for evaluation of differences between the groups. A value of P < 0.05 was considered to indicate a significant difference between groups
Proteolitic Cleavage Limited proteolysis analyses were performed by incubation of 1 3 1023M peptide amphiphile solution (1 ml) with a-chymotrypsin (a-CHT; Sigma-Aldrich) at an enzyme : substrate ratio of 1:100, in 50 ll of a solution containing 100 mM phosphate buffer (pH 7.5) and 20 mM CaCl2 at 25 C. After 2 h, mixture was eluted on pre-equilibrated Sephadex G-50 column. To monitor digestion, each sample was examined by LC-MS spectrometry by using a column Phenomenex Jupiter 4l Proteo 90A˚ (150 3 2.0 mm) eluted with H2O/0.05% TFA (A) and CH3CN/0.05% TFA (B) from 5% to 70% over 20 min at 200 ll min21 flow rate.
RESULTS AND DISCUSSION PA Design, Synthesis, and Aggregate Formulations PAs, reported in Figure 1, contain the CCK8 peptide moiety, a glycine residue at the N-terminus, a polyethylenglycole (PEG) based spacer at different length and a hydrophobic moiety with two C18 alkyl chains. Commonly peptide amphiphiles contain a spacer between the peptide moiety and the hydrophobic portion, which can be a poly-amino acid chain such as poly-alanine or poly-glycine, or a polyethylenglycole moiety. The ethoxylic groups of PEG confer high solubility to the PAs in their monomeric form without altering their net charge and contribute to reduce aggregate clearance through the reticuloendothelial system (RES).25 Moreover, after the aggregation process, the length of the PEG chain can affect dramatically the peptide position on the aggregate surface. In particular, an adequate distancing respect to the hydrophilic corona in micelle is fundamental for peptide that should interact selectively with membrane receptors. Anyway, several studies highlighted that it is not enough to increase the length of the PEG chain to improve the receptor binding.21 Other key aspects Biopolymers (Peptide Science)
have to be considered in the design of target selective supramolecular aggregates: peptide conformation after the aggregation process, and peptide mask by the aggregate forming surfactants. The synthetized PAs contain a PEG spacer with molecular weight ranging between 700 and 3000 Daltons (PEG700– PEG3000). These lengths were selected according to biological results previously obtained with similar aggregates derivatized with well-known targeted peptides such as CCK8,26 octreotide20 or 7–14-bombesin.21 In fact, PEG700 provides the minimal length required to leave the peptide on the aggregate external surface. PAs were synthetized according to standard Fmoc solid phase synthesis protocols. Monomer cleavages were performed using standard trifluoroacetic acid mixtures to allow for complete removal of all protecting groups of the amino acid side chains. The crude products were purified by preparative reversed-phase HPLC on C4 column to a final purity of 90% and were isolated in 25 to 40% yields in lyophilized form. Their molecular masses were determined by ESI mass spectrometry. Self-assembled aggregates were prepared by simply dissolving amphiphiles in 10 mM phosphate buffer at pH 7.4 and physiological ionic conditions (0.9% wt). Peptide concentration in aggregates was determined by UV spectroscopy at 280 nm.
Dynamic Light Scattering (DLS) Characterization Size and zeta potential (f) of aggregates were measured by DLS at h 5 173 on self-assembled PAs at a concentration of 2 3 1024M in 10 mM phosphate buffer at pH 7.4 and containing 0.9% wt of NaCl. All formulations, with the exception of PA-[700], show a bimodal distribution (see Supplementary Information), indicating the presence of two different kinds of aggregates whose apparent translational diffusion coefficients are reported in Table I. At infinite dilution, the hydrodynamic radius, RH may be evaluated by using the Stokes-Einstein equation (see Table I). Hydrodynamic radii of PA-[700]/PA-[2000] are between 63/83 nm, thus indicating that independently of the PEG length the hydrodynamic radii do not dramatically
308
Accardo et al.
FIGURE 3 Fluorescence spectra of tryptophan residue at 25 C and 1.0 3 1025 M PAs concentration. The spectrum was excited at 280 nm and recorded between 290 and 450 nm.
FIGURE 2 Far UV CD spectra of PA supramolecular aggregates in 10 mM TRIS at pH 8.0; concentrations of amphiphilic peptides are 2.0 3 1024M, higher than CMC values, to be certain of the presence of aggregates in solution. In A) CD spectra of PA-[1000] and PA[1500]; in B) CD spectra of PA-[700], PA-[2000], and PA-[3000].
change. On the contrary, PA-[3000] shows a RH of 104 nm with an increase of 30% with respect to the other peptide derivatives. TEM images indicate that the largest aggregates present in all the studied samples correspond to aggregates with a rod-like shape (see Supplementary Information).
Circular Dichroism (CD) Studies It is well-known that Gly-CCK8 nonapeptide assumes a random-coil conformation due to its short peptide sequence.10 It was also reported that when CCK8 is derivatized at the Nterminus with hydrocarbon chains and then anchored to the outer surface of aggregates, structural changes in the peptide conformation can occur. Conformational changes are strictly related to the kind of spacer introduced between the peptide moiety and the alkyl chains.11 Hence, three-dimensional structure of our PA aggregates was here investigated by circular dichroism (CD). Spectra were recorded between 195 and
260 nm on samples in 10 mM phosphate buffer (pH and ionic strength conditions) at 2 3 1024M peptide concentration. The spectrum of PA-[700] (Figure 2B) shows a large negative maximum at 218 nm, indicating that b-sheet structures seem to populate significantly the equilibrium mixture, as previously reported.11 The spectrum of PA-[1000], reported in Figure 2A, shows a shape typical of a-helix peptide conformation, with two negative bands around 208 and 222 nm, respectively. Similar behavior is found for PA-[1500], in which the shape of the spectrum remains unaltered with the negative maximum at 208 nm slightly blu shifted at k 5 205 nm. On the other hand, PA-[2000] and PA-[3000] spectra (see Figure 2B) look like profile of the reference compound Gly-CCK8 in the presence of the membrane-like surfactant DPC.11,27 Thus, PA[2000] and PA-[3000] adopt a conformation similar to that found for the membrane-bound CCK8, responsible for the interaction of the aggregate with the receptor. According to the membrane-bound pathway theory, that indicates CCK8 needs to assume a pseudo-a-helix conformation to give interaction to the CCK1-R and CCK2-R receptors, CD results suggest that all PAs in their aggregate form, with the exception of PA-[700], adopt a conformation allowing the interaction with the receptor.
Fluorescence of Tryptophan The exposure of the bioactive CCK8 portion on the hydrophilic shell of the self-assembled supramolecular aggregates was evaluated by monitoring the fluorescence of the tryptophan residue. Usually, tryptophan fluorophore shows an emission peak centered at 350 to 360 nm in polar solvents, while in hydrophobic solvents the maximum is blue-shifted to Biopolymers (Peptide Science)
CCK Peptides Exposed by Supramolecular Aggregates
FIGURE 4 Flow cytometric analysis of binding of FITC-PA aggregates to A431 cells. Cells at a density of 1 3 106 cells/ml were incubated with 22.6 lM FITC-PA aggregates for 1 h at 4 C. Cells treated with unlabeled PA-[2000] were used as negative control. Values of fluorescence intensity were obtained from histogram statistic of CellQuest software. Date points represent means 6 SD of three independent experiments. *P < 0.01; **P < 0.001.
330 nm.28 Fluorescence emission spectra recorded at 25 C and at a peptide concentration of 1.0 3 1025M are reported in Figure 3. Emission spectra of all PA aggregates show a maximum at 360 nm, which is diagnostic of the hydrophilic environment of the indole group on the tryptophan side-chain. This result suggests that the fluorophore on the PA aggregate surface, irrespective of the PEG length used, is completely surrounded by water molecules of high mobility, and thus potentially bioavailable for receptor binding.
Flow Cytometric Analysis The cell labeling efficiency of each PA formulation was assessed by using flow cytometry analysis based on FITC fluorescence. PA formulations were made fluorescent by insertion of a small amount (1%) of C18-FITC fluorescent probe. The fluorescent formulations are hereinafter named as FITC-PA-[700]/FITCPA-[3000]. Assays were performed on the A431 cell line at 4 C, in order to block non-specific internalization, incubating cells with FITC-PA aggregates for 1 h. Cells treated with unlabeled aggregates were used as negative control. Binding results are shown in Figure 4. Fluorescent FITC-PA-[2000] is found at higher fluorescence values, while FITC-PA-[1000] and FITCPA-[1500] are found in intermediate field. FITC-PA-[3000] is present at low fluorescence values comparable to FITC-PA[700]. This behavior indicates that the PA-[2000] aggregate is more efficient cell-labeling vehicle than PA-[700]. On the other hand, FITC-PA-[700] and FITC-PA-[3000] do not show any effective binding properties towards A431 cells because the bioactive CCK8 peptide is less exposed on the external surface of the aggregates. Moreover, competitive binding experiments on Biopolymers (Peptide Science)
309
FIGURE 5 Inhibition of FITC-PA aggregates binding to A431 cells. Cells were treated with 2.26 mM Gly-CCK8 peptide for 30 min followed by the addition of FITC-PA aggregates and an additional incubation of 1 h at 4 C. Histograms were obtained from a single experiment and were representative of three similar experiments. The fluorescence intensity (FI) for each aggregates is given in the top right corner. Date points represent means 6 SD of three independent experiments. *P < 0.01; **P < 0.001
FITC-PA-[700] and FITC-PA-[2000] were also performed after pre-incubation of cells with a large excess of Gly-CCK8, in order to demonstrate that the uptake is receptor mediated. Results reported in Figure 5 were compared with the binding behavior of fluorescent FITC-PA aggregates alone. When FITC-PA-[700] and FITC-PA-[2000] fluorescence is monitored in the presence of unlabeled Gly-CCK8, less binding occurs, indicating a competition between the two ligands for the receptor.
Proteolitic Cleavage Despite, the fluorescence spectra (Figure 3) indicate that the tryptophan residue of all the PA aggregates is surrounded from water solution, and CD spectra of PA-[2000] and PA-[3000] (Figure 2B) indicate that not significant differences exist from the structural point of view between PEG2000 and PEG3000 derivatives, flow cytometry analysis shown unequivocally a difference in the binding ability of PA-[2000] with respect to PA[3000] towards to the CCK2-R. In order to improve our information about the real position of the peptide with respect to the polyethyleneglycole chain PA aggregates were undergone to the proteolitic cleavage of the peptide sequence by using aChymotrypsin (a-CHT). This enzyme selectively cleaves peptide bonds following aromatic residues.29 The expected CCK8 degradation peaks are listed in Table II, with their
310
Accardo et al.
Table II Hydrolytic Fragments of CCK8-G Peptide Along with Expected Masses After Treatment with a-Chymotrypsin (a-CHT) Peptide Fragment a-Chymotrypsin
MGWMDF GDYMGW MDF MGW GDY
MW (Da) 786.29 728.27 411.15 393.16 354.13
corresponding molecular weights. Self-assembled aggregates of peptide amphiphiles at 1 3 1023M concentration were incubated with the a-CHT enzyme at 25 C for 2h. At the end of the incubation, aggregate solutions were eluted on preequilibrated Sephadex G-50 columns. Digestion products were evaluated by LC-MS spectrometry. The analytical characterization of PA-[2000] and PA-[3000] are reported in Figures 6A and 6B and Figures 6C and 6D, respectively. Chromatogram of
PA-[2000] aggregates shows two peaks well separated in HPLC: a major peak at 15.89 min and a minor one at 12.78 min. These peaks were identified for their molecular masses (MW 826.4 and 411.0 Dalton) and attributed to the MGWMDF esapeptide (K1 salt) and MDF tripeptide, respectively. On the contrary, the HPLC profile of PA-[3000] shows only the peak at Rt 5 12.78 min, corresponding to the MDF tripeptide. These results indicate a major hindrance of the PEG3000 that mask the N-terminal region of CCK8 peptide in the PA-[3000] supramolecular aggregates, preventing enzymatic cleavage of the Tyr-Met bond.
CONCLUSIONS PEG molecules of different molecular weight (700 < PEG < 5000) are extensively used for preparation of sterically stabilized liposomes to increase their circulation time in the blood stream. Studies dealing with the phase behavior of PEG lipid/
FIGURE 6 Analytical characterization by LC-MS of PA-[3000] and PA-[2000] agrregates after proteolitic cleavage with a-chymotrypsin. Panels A) and B) show HPLC profile and MS spectrum of PA-[2000] aggregates, respectively; while panels C) and D) report the analytical characterization of PA-[3000] aggregates.
Biopolymers (Peptide Science)
CCK Peptides Exposed by Supramolecular Aggregates
phospholipid systems highlighted that PEG2000 represents the more appropriate length for in vivo application.30–32 For example, according to these evidences the first liposomal formulation of the cytotoxic drug doxorubicin (DoxilV) approved by FDA is prepared by using DSPE-Peg2000. Five novel peptide amphiphiles with common formula (C18)2-PEGx-CCK8 have been here described as potential target selective nanocarriers towards tumor cells overexpressing cholecystokinin receptors. As indicated by the structural characterization obtained by DLS measurements, PA-[700]/ PA-[2000] self-assemble in water solution at pH 7.4 in supramolecular aggregates with a hydrodynamic radius ranging between 63 and 83 nm. PA-[3000], instead, shows a RH of 104 nm with an increase of 30% with respect to the other peptide derivatives. After the aggregation process, the biological activity of the peptide sequence can be seriously compromised. Hence, several key aspects have to be considered in the design of target selective supramolecular aggregates: (i) the peptide should be well-exposed and surrounded by water molecules in the outer surface of the aggregate; (ii) the peptide conformation required for the binding to the receptor should not be perturbed; (iii) additive surfactants in the formulation and/or spacers used to distance peptide from the aggregate surface should not mask the peptide sequence. Fluorescence studies suggested that, irrespective of the PEG length on the PA, the tryptophan residue located at the center of the CCK8 sequence is completely surrounded by water molecules of high mobility. This result indicates a potential capability of all formulated nanovectors to recognize the over-expressed CCK-2 receptors. Also CD data suggest that CCK8 peptide in all PAs in their aggregate form, with the exception of PA-[700], adopt a conformation allowing the interaction with the receptor. Anyway biological data obtained by flow cytometry analysis indicate that the five PAs with different PEG spacers have a different behavior in the binding ability of PA towards to the CCK2-R, with higher binding properties shown by PA-[2000]. Finally, proteolitic cleavage experiments indicate that in the PA-[2000] supramolecular aggregates the CCK8 peptide presents the Tyr-Met and Trp-Met cleavable amide bonds available for the enzymatic process. At variance, in PA-[3000] the longer PEG moiety masks the active site in CCK8, thus preventing both enzymatic cleavage of the Tyr-Met bond and receptor interaction. Based on these results, PEG2000 is the best spacer for CCK8 peptide amphiphiles. This study can be considered as a first attempt to formulate a more general postulate on the influence of PEG spacers in PA aggregates, containing targeted peptide sequences in which appropriate peptide exposure and conformation is required for selective binding to tumor cells. R
Biopolymers (Peptide Science)
311
We are indebted with Italian Minister for Research (M.I.U.R.) for financial supports under PRIN 2009WCNS5C and FIRB “RENAME” RBAP114AMK projects. The authors thank Mr. L. De Luca for his assistance in editing the manuscript.
REFERENCES 1. Palmer, L.C.; Stupp, S. I. Acc Chem Res 2008, 41, 1674– 1684. 2. Trent, A.; Marullo, R.; Lin, B.; Black, M.; Tirrell M. Soft Matter 2011, 7, 9572–9582. 3. Hamley, I. W. Soft Matter 2011, 7, 4122–4138. 4. Versluis, F.; Marsden, H.R.; Kros, A. Chem Soc Rev 2010, 39, 3434–3444. 5. Zhao, X.; Pan, F.; Xu, H.; Yaseen, M.; Shan, H.; Hauser, C.A.E.; Zhang, S.; Lu, J. R. Chem Soc Rev 2010, 39, 3480–3498. 6. Cui, H.; Webber, M.J.; Stupp, S. I. Biopolymers 2010, 94, 1–18. 7. Paramonov, S. E.; Jun, H. -W.; Hartgerink, J. D. J Am Chem Soc 2006, 128, 7291–7298. 8. Pajewski, R., Ferdani, R., Pajewska, J.; Djedovicˇ, N.; Schlesinger P.H.; Gokel, G. W. Org Biomol Chem 2005, 3, 619–625. 9. Accardo, A.; Tesauro, D.; Roscigno, P.; Gianolio, E.; Paduano, L.; D’Errico, G.; Pedone, C.; Morelli G. J Am Chem Soc 2004, 126, 3097–3107. 10. Accardo, A.; Tesauro, D.; Mangiapia, G.; Pedone, C.; Morelli, G. Biopolymers 2007, 88, 115–121. 11. Accardo, A.; Morisco, A.; Palladino, P.; Palumbo, R.; Tesauro, D.; Morelli, G.; Mol BioSyst 2011, 7, 862–870. 12. Dufresne, M.; Seva, C.; Fourmy, D. Physiol Rev 2006, 86, 805– 847. 13. Pellegrini, M.; Mierke, D.F. Biochemistry 1999, 38, 14775– 14783. 14. Moroder, L., Romano, R., Guba, W.; Mierke, D.F.; Kessler, H.; Delporte, C.; Winand, J.; Christophe, J. Biochemistry 1993, 32, 13551–13559. 15. Lutz, J.; Romano-Goetsch, R.; Escrieut, C.; Fourmy, D.; Math€a, B.; M€ uller, G.; Kessler, H.; Moroder, L. Biopolymers 1997, 41, 799–817. 16. Schwyzer, R. J Mol Recognit 1995, 8, 3–8. 17. Schwyzer, R. Biopolymers 1995, 37, 5–16. 18. Schmitt, L.; Dietrich, C.; Tampe, R. J Am Chem Soc 1994, 116, 8485–8491. 19. Chang, W. C.; White, P. D. In Fmoc Solid Phase Peptide Synthesis; Oxford University Press: New York, 2000. 20. Accardo, A.; Morisco, A.; Gianolio, E.; Tesauro, D.; Mangiapia, G.; Radulescu, A.; Brandt, A.; Morelli, G. J Pept Sci 2011, 17, 154–162. 21. Accardo, A.; Mansi, R.; Morisco, A.; Mangiapia, G.; Paduano, L.; Tesauro, D.; Radulescu, A.; Aurilio, M.; Aloj, L.; Arra, C.; Morelli, G. Mol Biosyst 2010, 6, 878–887. 22. Edelhoch, H. Biochemistry 1967, 6, 1948–1954. 23. Pace, C. N.; Vajdos, F.; Fee, L.; Grimsley, G.; Gray, T. Protein Sci 1995, 4, 2411–2423. 24. Aloj, L.; Panico, M.; Carac o, C.; Del Vecchio, S.; Arra, C.; Affuso, A.; Accardo, A.; Mansi, R.; Tesauro,D.; De Luca, S.; Pedone, C.; Visentin, R.; Mazzi, U.; Morelli, G.; Salvatore, M. Cancer Biother Radiopharm 2004, 19, 91–96.
312
Accardo et al.
25. Torchilin, V. P.; Omelyanenko, V. G.; Papisov, M. I.; Bogdanov, A. A., Jr.; Trubetskoy, V. S.; Herron, J. N.; Gentry, C. A. Biochim Biophys Acta 1994, 1195, 11–20. 26. Accardo, A.; Tesauro, D.; Morelli, G.; Gianolio, E.; Aime, S.; Vaccaro, M.; Mangiapia, G.; Paduano, L.; Schillen K. J Biol Inorg Chem 2007, 12, 267–276. 27. Kumar, M.; Reeve, J. R., Jr.; Hu, W.; Miller, L. J.; Keire, D. A. J Med Chem 2008, 51, 3742–3754. 28. Lackowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983.
29. Creighton, T. E. Proteins: Structures and Molecular Properties; W. H. Freeman: New York, 1993. 30. Klibanov, A. L.; Maruyama, K.; Torchilin, V. P.; Huang, L. FEBS Lett 1990, 268, 235–237. 31. Papahadjopoulos, D.; Allen, T. M.; Gabizon, A.; Mayhew, E.; Matthay, K.; Huang, S. K.; Lee, K. -D.; Woodle, M. C.; Lasic, D. D.; Redemann, C.; Martin, F. J. Proc Natl Acad Sci USA 1991, 88, 11460–11464. 32. Johnsson, M., Hansson, P.; Edwards, K. J Phys Chem B 2001, 105, 8420–8430.
Biopolymers (Peptide Science)
ABSTRACT: Five novel peptide amphiphiles (PAs), with common formula (C18)2-PEGx-CCK8 in which the CCK8 peptide and the (C18)2-hydrophobic moiety are spaced by polyethylene linkers of different length (PEG moieties with molecular weights of 700, 1000, 1500, 2000, and 3000 Daltons) are described. They act as potential targetselective nanocarriers towards tumor cells overexpressing
with higher binding properties shown by PA containing PEG with MW of 2000 Dalton. Therefore, PEG2000 can be considered as the best spacer in the formulation of C 2014 nanovectors based on CCK8 peptide amphiphiles. V
Wiley Periodicals, Inc. Biopolymers (Pept Sci) 102: 304– 312, 2014. Keywords: peptide amphiphiles; CCK8; supramolecular aggregates; PEG spacer; fluorescence; circular dichroism
cholecistokynin receptors. PAs self-assemble in supramolecular aggregates, with hydrodynamic radius ranging between 63 and 104 nm, as indicated by DLS measurements. Fluorescence studies suggested that, irrespective
This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [email protected]
from the PEG length, the tryptophan residue located at the center of the CCK8 sequence is completely surrounded by water molecules at high mobility. This result indicates a potential capability of all formulated nanovectors to recognize the overexpressed CCK-2 receptors. CD data suggest that CCK8 peptide, in most of PAs in their aggregate form, adopts a conformation allowing the interaction with the receptor. Anyway, biological data obtained by flow cytometry analysis indicate that the five PAs have a different binding ability towards the CCK-2 receptors,
Additional Supporting Information may be found in the online version of this article. Correspondence to: Giancarlo Morelli, Department of Pharmacy, CIRPeB, University of Naples “Federico II,” & IBB—CNR, Via Mezzocannone 16, 80134 Naples, Italy; e-mail: [email protected] Contract grant sponsor: Italian Minister for Research Contract grant number: PRIN 2009WCNS5C Contract grant sponsor: FIRB “RENAME” Contract grant number: RBAP114AMK C 2014 Wiley Periodicals, Inc. V
304
INTRODUCTION
P
eptide amphiphiles (PAs) are an emergent class of synthetic surfactants in which a peptide moiety with potential biological activity is covalently bound to a hydrophobic moiety, such as one or two alkyl tails. Under specific condition of pH or temperatures, PA molecules are able to self-assemble, in water solution, in well-ordered supramolecular structures with distinctive features with respect to traditional surfactants. They give, spontaneously, monolayers, bilayers, vesicles, elongated worm- or rod-like micelles or spherical micelles.1–6 Moreover, several examples of PAs able to form fibers and gels are also reported. In all these nanostructures, bioactive peptides are disposed at the periphery of the nanostructure perpendicularly to their long axis at near van der Waals density. Peptides, if well-exposed on nanostructure external surfaces, give different chemical and biological functionalities including the ability to form pores in membranes7,8 or to reach, in a selective way, a biological target9 such as membrane receptors
PeptideScience Volume 102 / Number 4
CCK Peptides Exposed by Supramolecular Aggregates
FIGURE 1 Schematic representation of peptide amphiphile (PA) monomers. The amino acid sequence of CCK8 peptide is reported by using the one-letter amino acid code. Ethoxylic linkers used during the synthesis (AdOO, Ahoh, PEG1500 and PEG3000) are also reported.
overexpressed by cancerous cells. Anyway, for all biological functions previously described, peptide availability on nanostructure external surfaces is not the unique requirement for receptor binding. According to the ligand/receptor pathway, the peptide needs to adopt a specific conformation to assure high affinity and selectivity in the binding processes.10 Recently, we reported the synthesis of a set of five peptide amphiphiles with common formula (C18)2-L-CCK8,11 in which (C18)2- is symbolically used to indicate the dioctadecyl hydrophobic moiety; CCK8 is the carboxyl terminal sequence of the peptide hormone cholecystokinin (Asp-Tyr-Met-GlyTrp-Met-Asp-Phe-Amide) able to bind, with high affinity, both CCK1-R and CCK2-R cholecystokinin receptors overexpressed by cancer cells in several human tumors;12 and -L- is here used to indicate a neutral or charged spacer [five ethoxylic linker (AdOO 5 8-amino-3,6-dioxaoctanoic acid) or three lysine residues and an AdOO unit variably positioned]. Physicochemical characterization of the supramolecular aggregates in aqueous solution by dynamic light scattering (DLS) indicated the coexistence of large (60 nm< RH < 90 nm) and small (5 nm < RH < 10 nm) structures. Moreover, peptide conformation, peptide bioavailability on the external aggregate surface and cell binding properties have been assessed. The presence of CCK8 peptide well-exposed on the outer surface of the aggregate has been demonstrated by fluorescence measurements for all systems, anyway, only systems in which the CCK8 peptide presents a structural arrangement in pseudo-a-helix conformation yielded promising binding properties to CCK2R receptors overexpressed by transfected A431 cells. In fact, according to the theory of membrane-bound pathway, CCK8 needs to assume a pseudo-a-helix conformation for the interaction with its G-protein coupled receptors.13–17 Biopolymers (Peptide Science)
305
Here we report the synthesis of five novel peptide amphiphiles (see Figure 1) with similar formula (C18)2-PEGxCCK8. The influence of the PEG length on aggregation behavior and on conformational properties of the peptide fragment is studied by DLS and circular dichroism, respectively. Moreover, the localization of the peptide with respect to the PEG corona has been assessed by fluorescence and proteolitic cleavage. Finally, the binding properties of PA aggregates have been highlighted by in vitro flow cytometry on transfected A431 cells overexpressing CCK2-R receptors. The entire study aims to define the influence of PEG spacers in PA aggregates, containing targeted peptide sequences in which appropriate peptide exposure and conformation are required for selective binding to tumor cells.
MATERIALS AND METHODS Chemicals Protected Na-Fmoc-amino acid derivatives, coupling reagents, and Rink amide MBHA resin were purchased from CalbiochemNovabiochem (Laufelfingen, Switzerland). The Fmoc-21-amino4,7,10,13,16,19-hexaoxaheneicosanoic acid (Fmoc-Ahoh-OH) and the Fmoc-8-amino-3,6-dioxaoctanoic acid (Fmoc-AdOO-OH) were purchased from Neosystem (Strasbourg, France). a-(9-Fluorenylmethyloxycarbonyl) amino-x-carboxy poly(ethylene glycol) derivatives (Fmoc-NH-PEG2000-COOH and Fmoc-NH-PEG3000-COOH) were purchased from Iris Biotech GmbH (Marktredwitz, Germany). The N,N-dioctadecylsuccinamic acid was synthesized according to published methods.18 Fluorescein 5-isothiocyanate (FITC) isomer I, BSA and PBS were purchased from Sigma-Aldrich (Milan, Italy). DMEM, FCS, L-glutamine were purchase from Lonza (Milano, Italy). C18H37CONHLys–(FITC)CONH2 (C18–FITC) was synthesized according to the procedure previously reported.11 All other chemicals were commercially available from Sigma-Aldrich or Fluka (Buchs, Switzerland) or LabScan (Stillorgan, Dublin, Ireland) and were used as received unless otherwise stated. Preparative reverse phase-high performance liquid chromatographies (RP-HPLCs) were carried out on a LC8 Shimadzu HPLC system (Shimadzu Corporation, Kyoto, Japan) equipped with a UV lambda-Max Model 481 detector using a Phenomenex (Torrance, CA) C4 (300 A˚, 250 21.20 mm, 5 mm) column eluted with H2O/0.1% TFA (A) and CH3CN/0.1% TFA (B) from 20% to 95% over 20 minutes at 20 ml min21 flow rate. Purity and identity were assessed by analytical LC-MS analyses by using Finnigan Surveyor MSQ single quadrupole electrospray ionization (Finnigan/Thermo Electron Corporation San Jose, CA), column: C4Phenomenex eluted with H2O/0.1% TFA (A) and CH3CN/0.1% TFA (B) from 20% to 95% over 20 min at 1 ml min21 flow rate. Concentrations of solutions containing peptide surfactant were determined by absorbance measurement on a UV-vis Jasco V-5505 spectrophotometer equipped with a Jasco ETC-505T Peltier temperature controller with a 1 cm quartz cuvette (Hellma).
Peptide Amphiphiles Synthesis The synthesis of Fmoc-Gly-CCK8-amide peptide was carried out in solid-phase under the standard Fmoc strategy19 by using 433A
306
Accardo et al.
Applied Biosystems automatic synthesizer. Rink-amide 4-methylbenzhydrylamine (MBHA) resin (0.78 mmol g21, 0.7 mmol scale, 0.900 g) was used. The elongation of peptides was achieved by sequential addition of Fmoc-AA-OH with benzotriazole-1-yl-oxy trispyrrolidinophosphonium hexafluoro phosphonate/1-hydroxybenzotriazole (PyBOP/HOBt) and N,N-diisopropylethylamine (DIPEA) (1/1/2) as coupling reagents, in dimethylformamide (DMF) in pre-activation mode. All couplings were performed twice for 1 h, by using an excess of 4 equivalents for the single amino acid derivative. Fmoc deprotections were obtained by 30% solution of piperidine in DMF. The peptide-resin was shared in five reactors (each one containing 0.1 mmol of peptide-resin), and each reactor was used to synthetize a peghilated peptide amphiphile (PA-[700], PA-[1000], PA-[1500], PA[2000], and PA-[3000]) by coupling manually the (ethylene glycol) spacer of different length (Fmoc-AdOO-OH, Fmoc-Ahoh-OH; FmocNH-PEG2000-COOH, or Fmoc-NH-PEG3000-COOH) and the N,Ndioctadecylsuccinamic acid mixture according to experimental procedure previously described.10,20,21 PAs were fully deprotected and cleaved from the resin with the trifluoroacetic acid (TFA)/triisopropylsilane (TIS)/H2O (95.5/2/2.5) mixture at room temperature and precipitated with ice-cold water. Purification of the crude products was carried out by RP-HPLC (k 5 280 nm). Final products at high purity degree (>90%) were collected with a yield ranging between 25 and 40% as indicated by LC-MS analysis. PA-[700]: tR 5 25.38 min, MS (ESI1): m/z 2442 calcd. C121H192N17O31S2: [M12H1]/251222.0 PA-[1000]: tR 5 24.66 min, MS (ESI1): m/z 2724 calcd. C136H226N15O37S2: [M12H1]/2 5 1363.0 PA-[1500]: tR 5 24.16 min, MS (ESI1): m/z 3024 calcd. C150H258N13O45S2: [M12H1]/2 5 1513.0 PA-[2000]: tR 5 23.48 min, MS (ESI1): m/z 3733 calcd. C181H310N18O58S2: [M12H1]/2 5 1867.5; [M13H1]/3 5 1245.3 PA-[3000]: tR 5 22.92 min, MS (ESI1): m/z 4643 calcd. C222H405N13O83S2: [M13H1]/3 5 1548.6; [M14H1]/4 5 1161.7
for for for for
173 backscatter detector. Other instrumental settings are: measurement position (mm), 4.65; attenuator, 8; temperature, 25 C; cell, disposable sizing cuvette. DLS samples were prepared at the final concentration of 2 3 1024M and centrifuged at room temperature at 13,000 rpm for 5 min. For each batch, hydrodynamic radii and size distribution were the mean of three measurements and values were calculated as the mean of three different batches. The zeta-potential (f) values of the aggregate surface were collected as the average of 20 measurements.
Circular Dichroism CD spectra were recorded from 195 to 260 nm on a Jasco J-810 spectropolarimeter equipped with a NesLab RTE111 thermal controller unit using a 1 mm quartz cell at 25 C. Circular dichroism measurements were carried out on solutions containing peptides or peptide amphiphiles at concentrations of 2 3 1024M in 10 mM PBS at pH 7.4. Other experimental settings were: scan speed, 10 nm min21; sensitivity, 50 mdeg; time constant, 16 s; bandwidth, 1 nm. Each spectrum was obtained averaging three scans, subtracting contributions from other species in solution, and converting the signal to mean residue ellipticity in units of deg cm2 dmol21 res21.
Fluorescence Microscopy The fluorescence emission spectra were recorded using a Jasco Model FP-750 spectrofluorimeter (Easton, MD) equipped with a Peltier temperature controller in 1.0-cm path length quartz cell at 25 C. Emission spectra of PAs were obtained in 290 to 450 nm range exciting at 280 nm the aggregate solutions at a peptide concentration of 1.0 3 1025M. Equal excitation and emission bandwidths were used throughout the experiments, with a recording speed of 125 nm min21 and automatic selection of the time constant.
for
Cell Culture and Flow Cytometry
All solutions were prepared by weight, buffering the samples at pH 7.4 in 10 mM phosphate buffer. pH measurements were made by using pH-meter MeterLab PHM 220. The pH-meter was calibrated with three standards at pH 4.00, pH 7.00, and pH 10.00. In most cases the samples to be measured were prepared from stock solutions. Concentrations of all solutions were determined by absorbance on a UVvis Jasco (Easton, MD) Model 440 spectrophotometer with a path length of 1 cm using a molar absorptivity (e280) of 6845M21 cm21 for CCK8. This value was calculated according to the Edelhoch method22 taking into account contributions from tyrosine and tryptophan present in the primary structure, which amount to 1215 and 5630M21 cm21, respectively.23 Fluorescent aggregates of PA, containing 1% mol mol21 of C18-FITC fluorescent probe (FITC-PA aggregates), were prepared for in vitro uptake assays. Confirmation of incorporation of the fluorescent probe (1%) into the aggregates was obtained by gel filtration on Sephadex G-50 pre-packed columns (Pharmacia Biotech).
Cellular uptake of PA aggregates was studied by flow cytometry on A431 cells overexpressing the CCK2-R by stable transfection.24 Cells were cultured as exponential growing subconfluent monolayer on 100 mm plates in DMEM supplemented with 10 % FCS, 2 mM L-glutamine and 250 lg/ml of G418 in a humidified atmosphere containing 5% CO2 at 37 C. Cell aliquots (0.5 3 106) were incubated at 4 C in 500 ll final volume. At the end of the incubation, cells were washed, resuspended in PBS/BSA and examined by flow cytometry using a FACScan (Becton Dickinson, CA) equipped with a 488 nm argon laser. A total of 20,000 events per sample were collected. Values of fluorescence intensity were obtained from histogram statistic of CellQuest software. For binding experiments, cells were incubated for 1 h with 22.6 lM FITC-PA aggregates. The fluorescence intensity of cells treated with different not fluorescent aggregates was comparable to untreated cells. Therefore, cells treated with unlabeled PA-[2000] were used as negative control. For competitive binding experiments, cells were pre-incubated with 2.26 mM H-G-CCK8 peptide (100-fold excess with respect to PA concentration) for 30 min followed by the addition of FITC-PA aggregates and an additional incubation of 1 h.
Dynamic Light Scattering (DLS) Measurements
Statistical Analysis
Mean diameter and zeta-potential (f) were measured using a Zetasizer Nano ZS (Malvern Instruments, Westborough, MA) that employs a
Data are represented as means 6 SD from at least three independent experiments. Differences between the fluorescent groups were
Preparation of the Solutions
Biopolymers (Peptide Science)
CCK Peptides Exposed by Supramolecular Aggregates
307
Table I Diffusion Coefficients (D), Hydrodynamic Radii (RH), and the Zeta Potential (f) are obtained from dynamic light Scattering Measurements for the Systems Studied Systems
Dfast 3 10211 (m2 s21)
RH (nm)
Dslow 3 10212 (m2 s21)
RH (nm)
f (mV)
– 3.9 6 0.8 2.6 6 0.8 3.2 6 1.0 1.2 6 0.4
– 5.1 6 1.0 7.8 6 2.3 6.2 6 1.9 17 6 6.4
3.2 6 1.1 2.4 6 1.0 2.6 6 1.1 2.8 6 1.0 1.9 6 0.8
63 6 23 83 6 35 76 6 32 72 6 26 104 6 47
239.7 6 14.0 247.6 6 8.11 242.7 6 6.50 237.4 6 7.53 229.5 6 5.12
PA-[700] PA-[1000] PA-[1500] PA-[2000] PA-[3000]
The terms fast and slow refer to smaller and larger aggregates, respectively.
determined by one way ANOVA using the GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA). Tukey’s posttest was performed for evaluation of differences between the groups. A value of P < 0.05 was considered to indicate a significant difference between groups
Proteolitic Cleavage Limited proteolysis analyses were performed by incubation of 1 3 1023M peptide amphiphile solution (1 ml) with a-chymotrypsin (a-CHT; Sigma-Aldrich) at an enzyme : substrate ratio of 1:100, in 50 ll of a solution containing 100 mM phosphate buffer (pH 7.5) and 20 mM CaCl2 at 25 C. After 2 h, mixture was eluted on pre-equilibrated Sephadex G-50 column. To monitor digestion, each sample was examined by LC-MS spectrometry by using a column Phenomenex Jupiter 4l Proteo 90A˚ (150 3 2.0 mm) eluted with H2O/0.05% TFA (A) and CH3CN/0.05% TFA (B) from 5% to 70% over 20 min at 200 ll min21 flow rate.
RESULTS AND DISCUSSION PA Design, Synthesis, and Aggregate Formulations PAs, reported in Figure 1, contain the CCK8 peptide moiety, a glycine residue at the N-terminus, a polyethylenglycole (PEG) based spacer at different length and a hydrophobic moiety with two C18 alkyl chains. Commonly peptide amphiphiles contain a spacer between the peptide moiety and the hydrophobic portion, which can be a poly-amino acid chain such as poly-alanine or poly-glycine, or a polyethylenglycole moiety. The ethoxylic groups of PEG confer high solubility to the PAs in their monomeric form without altering their net charge and contribute to reduce aggregate clearance through the reticuloendothelial system (RES).25 Moreover, after the aggregation process, the length of the PEG chain can affect dramatically the peptide position on the aggregate surface. In particular, an adequate distancing respect to the hydrophilic corona in micelle is fundamental for peptide that should interact selectively with membrane receptors. Anyway, several studies highlighted that it is not enough to increase the length of the PEG chain to improve the receptor binding.21 Other key aspects Biopolymers (Peptide Science)
have to be considered in the design of target selective supramolecular aggregates: peptide conformation after the aggregation process, and peptide mask by the aggregate forming surfactants. The synthetized PAs contain a PEG spacer with molecular weight ranging between 700 and 3000 Daltons (PEG700– PEG3000). These lengths were selected according to biological results previously obtained with similar aggregates derivatized with well-known targeted peptides such as CCK8,26 octreotide20 or 7–14-bombesin.21 In fact, PEG700 provides the minimal length required to leave the peptide on the aggregate external surface. PAs were synthetized according to standard Fmoc solid phase synthesis protocols. Monomer cleavages were performed using standard trifluoroacetic acid mixtures to allow for complete removal of all protecting groups of the amino acid side chains. The crude products were purified by preparative reversed-phase HPLC on C4 column to a final purity of 90% and were isolated in 25 to 40% yields in lyophilized form. Their molecular masses were determined by ESI mass spectrometry. Self-assembled aggregates were prepared by simply dissolving amphiphiles in 10 mM phosphate buffer at pH 7.4 and physiological ionic conditions (0.9% wt). Peptide concentration in aggregates was determined by UV spectroscopy at 280 nm.
Dynamic Light Scattering (DLS) Characterization Size and zeta potential (f) of aggregates were measured by DLS at h 5 173 on self-assembled PAs at a concentration of 2 3 1024M in 10 mM phosphate buffer at pH 7.4 and containing 0.9% wt of NaCl. All formulations, with the exception of PA-[700], show a bimodal distribution (see Supplementary Information), indicating the presence of two different kinds of aggregates whose apparent translational diffusion coefficients are reported in Table I. At infinite dilution, the hydrodynamic radius, RH may be evaluated by using the Stokes-Einstein equation (see Table I). Hydrodynamic radii of PA-[700]/PA-[2000] are between 63/83 nm, thus indicating that independently of the PEG length the hydrodynamic radii do not dramatically
308
Accardo et al.
FIGURE 3 Fluorescence spectra of tryptophan residue at 25 C and 1.0 3 1025 M PAs concentration. The spectrum was excited at 280 nm and recorded between 290 and 450 nm.
FIGURE 2 Far UV CD spectra of PA supramolecular aggregates in 10 mM TRIS at pH 8.0; concentrations of amphiphilic peptides are 2.0 3 1024M, higher than CMC values, to be certain of the presence of aggregates in solution. In A) CD spectra of PA-[1000] and PA[1500]; in B) CD spectra of PA-[700], PA-[2000], and PA-[3000].
change. On the contrary, PA-[3000] shows a RH of 104 nm with an increase of 30% with respect to the other peptide derivatives. TEM images indicate that the largest aggregates present in all the studied samples correspond to aggregates with a rod-like shape (see Supplementary Information).
Circular Dichroism (CD) Studies It is well-known that Gly-CCK8 nonapeptide assumes a random-coil conformation due to its short peptide sequence.10 It was also reported that when CCK8 is derivatized at the Nterminus with hydrocarbon chains and then anchored to the outer surface of aggregates, structural changes in the peptide conformation can occur. Conformational changes are strictly related to the kind of spacer introduced between the peptide moiety and the alkyl chains.11 Hence, three-dimensional structure of our PA aggregates was here investigated by circular dichroism (CD). Spectra were recorded between 195 and
260 nm on samples in 10 mM phosphate buffer (pH and ionic strength conditions) at 2 3 1024M peptide concentration. The spectrum of PA-[700] (Figure 2B) shows a large negative maximum at 218 nm, indicating that b-sheet structures seem to populate significantly the equilibrium mixture, as previously reported.11 The spectrum of PA-[1000], reported in Figure 2A, shows a shape typical of a-helix peptide conformation, with two negative bands around 208 and 222 nm, respectively. Similar behavior is found for PA-[1500], in which the shape of the spectrum remains unaltered with the negative maximum at 208 nm slightly blu shifted at k 5 205 nm. On the other hand, PA-[2000] and PA-[3000] spectra (see Figure 2B) look like profile of the reference compound Gly-CCK8 in the presence of the membrane-like surfactant DPC.11,27 Thus, PA[2000] and PA-[3000] adopt a conformation similar to that found for the membrane-bound CCK8, responsible for the interaction of the aggregate with the receptor. According to the membrane-bound pathway theory, that indicates CCK8 needs to assume a pseudo-a-helix conformation to give interaction to the CCK1-R and CCK2-R receptors, CD results suggest that all PAs in their aggregate form, with the exception of PA-[700], adopt a conformation allowing the interaction with the receptor.
Fluorescence of Tryptophan The exposure of the bioactive CCK8 portion on the hydrophilic shell of the self-assembled supramolecular aggregates was evaluated by monitoring the fluorescence of the tryptophan residue. Usually, tryptophan fluorophore shows an emission peak centered at 350 to 360 nm in polar solvents, while in hydrophobic solvents the maximum is blue-shifted to Biopolymers (Peptide Science)
CCK Peptides Exposed by Supramolecular Aggregates
FIGURE 4 Flow cytometric analysis of binding of FITC-PA aggregates to A431 cells. Cells at a density of 1 3 106 cells/ml were incubated with 22.6 lM FITC-PA aggregates for 1 h at 4 C. Cells treated with unlabeled PA-[2000] were used as negative control. Values of fluorescence intensity were obtained from histogram statistic of CellQuest software. Date points represent means 6 SD of three independent experiments. *P < 0.01; **P < 0.001.
330 nm.28 Fluorescence emission spectra recorded at 25 C and at a peptide concentration of 1.0 3 1025M are reported in Figure 3. Emission spectra of all PA aggregates show a maximum at 360 nm, which is diagnostic of the hydrophilic environment of the indole group on the tryptophan side-chain. This result suggests that the fluorophore on the PA aggregate surface, irrespective of the PEG length used, is completely surrounded by water molecules of high mobility, and thus potentially bioavailable for receptor binding.
Flow Cytometric Analysis The cell labeling efficiency of each PA formulation was assessed by using flow cytometry analysis based on FITC fluorescence. PA formulations were made fluorescent by insertion of a small amount (1%) of C18-FITC fluorescent probe. The fluorescent formulations are hereinafter named as FITC-PA-[700]/FITCPA-[3000]. Assays were performed on the A431 cell line at 4 C, in order to block non-specific internalization, incubating cells with FITC-PA aggregates for 1 h. Cells treated with unlabeled aggregates were used as negative control. Binding results are shown in Figure 4. Fluorescent FITC-PA-[2000] is found at higher fluorescence values, while FITC-PA-[1000] and FITCPA-[1500] are found in intermediate field. FITC-PA-[3000] is present at low fluorescence values comparable to FITC-PA[700]. This behavior indicates that the PA-[2000] aggregate is more efficient cell-labeling vehicle than PA-[700]. On the other hand, FITC-PA-[700] and FITC-PA-[3000] do not show any effective binding properties towards A431 cells because the bioactive CCK8 peptide is less exposed on the external surface of the aggregates. Moreover, competitive binding experiments on Biopolymers (Peptide Science)
309
FIGURE 5 Inhibition of FITC-PA aggregates binding to A431 cells. Cells were treated with 2.26 mM Gly-CCK8 peptide for 30 min followed by the addition of FITC-PA aggregates and an additional incubation of 1 h at 4 C. Histograms were obtained from a single experiment and were representative of three similar experiments. The fluorescence intensity (FI) for each aggregates is given in the top right corner. Date points represent means 6 SD of three independent experiments. *P < 0.01; **P < 0.001
FITC-PA-[700] and FITC-PA-[2000] were also performed after pre-incubation of cells with a large excess of Gly-CCK8, in order to demonstrate that the uptake is receptor mediated. Results reported in Figure 5 were compared with the binding behavior of fluorescent FITC-PA aggregates alone. When FITC-PA-[700] and FITC-PA-[2000] fluorescence is monitored in the presence of unlabeled Gly-CCK8, less binding occurs, indicating a competition between the two ligands for the receptor.
Proteolitic Cleavage Despite, the fluorescence spectra (Figure 3) indicate that the tryptophan residue of all the PA aggregates is surrounded from water solution, and CD spectra of PA-[2000] and PA-[3000] (Figure 2B) indicate that not significant differences exist from the structural point of view between PEG2000 and PEG3000 derivatives, flow cytometry analysis shown unequivocally a difference in the binding ability of PA-[2000] with respect to PA[3000] towards to the CCK2-R. In order to improve our information about the real position of the peptide with respect to the polyethyleneglycole chain PA aggregates were undergone to the proteolitic cleavage of the peptide sequence by using aChymotrypsin (a-CHT). This enzyme selectively cleaves peptide bonds following aromatic residues.29 The expected CCK8 degradation peaks are listed in Table II, with their
310
Accardo et al.
Table II Hydrolytic Fragments of CCK8-G Peptide Along with Expected Masses After Treatment with a-Chymotrypsin (a-CHT) Peptide Fragment a-Chymotrypsin
MGWMDF GDYMGW MDF MGW GDY
MW (Da) 786.29 728.27 411.15 393.16 354.13
corresponding molecular weights. Self-assembled aggregates of peptide amphiphiles at 1 3 1023M concentration were incubated with the a-CHT enzyme at 25 C for 2h. At the end of the incubation, aggregate solutions were eluted on preequilibrated Sephadex G-50 columns. Digestion products were evaluated by LC-MS spectrometry. The analytical characterization of PA-[2000] and PA-[3000] are reported in Figures 6A and 6B and Figures 6C and 6D, respectively. Chromatogram of
PA-[2000] aggregates shows two peaks well separated in HPLC: a major peak at 15.89 min and a minor one at 12.78 min. These peaks were identified for their molecular masses (MW 826.4 and 411.0 Dalton) and attributed to the MGWMDF esapeptide (K1 salt) and MDF tripeptide, respectively. On the contrary, the HPLC profile of PA-[3000] shows only the peak at Rt 5 12.78 min, corresponding to the MDF tripeptide. These results indicate a major hindrance of the PEG3000 that mask the N-terminal region of CCK8 peptide in the PA-[3000] supramolecular aggregates, preventing enzymatic cleavage of the Tyr-Met bond.
CONCLUSIONS PEG molecules of different molecular weight (700 < PEG < 5000) are extensively used for preparation of sterically stabilized liposomes to increase their circulation time in the blood stream. Studies dealing with the phase behavior of PEG lipid/
FIGURE 6 Analytical characterization by LC-MS of PA-[3000] and PA-[2000] agrregates after proteolitic cleavage with a-chymotrypsin. Panels A) and B) show HPLC profile and MS spectrum of PA-[2000] aggregates, respectively; while panels C) and D) report the analytical characterization of PA-[3000] aggregates.
Biopolymers (Peptide Science)
CCK Peptides Exposed by Supramolecular Aggregates
phospholipid systems highlighted that PEG2000 represents the more appropriate length for in vivo application.30–32 For example, according to these evidences the first liposomal formulation of the cytotoxic drug doxorubicin (DoxilV) approved by FDA is prepared by using DSPE-Peg2000. Five novel peptide amphiphiles with common formula (C18)2-PEGx-CCK8 have been here described as potential target selective nanocarriers towards tumor cells overexpressing cholecystokinin receptors. As indicated by the structural characterization obtained by DLS measurements, PA-[700]/ PA-[2000] self-assemble in water solution at pH 7.4 in supramolecular aggregates with a hydrodynamic radius ranging between 63 and 83 nm. PA-[3000], instead, shows a RH of 104 nm with an increase of 30% with respect to the other peptide derivatives. After the aggregation process, the biological activity of the peptide sequence can be seriously compromised. Hence, several key aspects have to be considered in the design of target selective supramolecular aggregates: (i) the peptide should be well-exposed and surrounded by water molecules in the outer surface of the aggregate; (ii) the peptide conformation required for the binding to the receptor should not be perturbed; (iii) additive surfactants in the formulation and/or spacers used to distance peptide from the aggregate surface should not mask the peptide sequence. Fluorescence studies suggested that, irrespective of the PEG length on the PA, the tryptophan residue located at the center of the CCK8 sequence is completely surrounded by water molecules of high mobility. This result indicates a potential capability of all formulated nanovectors to recognize the over-expressed CCK-2 receptors. Also CD data suggest that CCK8 peptide in all PAs in their aggregate form, with the exception of PA-[700], adopt a conformation allowing the interaction with the receptor. Anyway biological data obtained by flow cytometry analysis indicate that the five PAs with different PEG spacers have a different behavior in the binding ability of PA towards to the CCK2-R, with higher binding properties shown by PA-[2000]. Finally, proteolitic cleavage experiments indicate that in the PA-[2000] supramolecular aggregates the CCK8 peptide presents the Tyr-Met and Trp-Met cleavable amide bonds available for the enzymatic process. At variance, in PA-[3000] the longer PEG moiety masks the active site in CCK8, thus preventing both enzymatic cleavage of the Tyr-Met bond and receptor interaction. Based on these results, PEG2000 is the best spacer for CCK8 peptide amphiphiles. This study can be considered as a first attempt to formulate a more general postulate on the influence of PEG spacers in PA aggregates, containing targeted peptide sequences in which appropriate peptide exposure and conformation is required for selective binding to tumor cells. R
Biopolymers (Peptide Science)
311
We are indebted with Italian Minister for Research (M.I.U.R.) for financial supports under PRIN 2009WCNS5C and FIRB “RENAME” RBAP114AMK projects. The authors thank Mr. L. De Luca for his assistance in editing the manuscript.
REFERENCES 1. Palmer, L.C.; Stupp, S. I. Acc Chem Res 2008, 41, 1674– 1684. 2. Trent, A.; Marullo, R.; Lin, B.; Black, M.; Tirrell M. Soft Matter 2011, 7, 9572–9582. 3. Hamley, I. W. Soft Matter 2011, 7, 4122–4138. 4. Versluis, F.; Marsden, H.R.; Kros, A. Chem Soc Rev 2010, 39, 3434–3444. 5. Zhao, X.; Pan, F.; Xu, H.; Yaseen, M.; Shan, H.; Hauser, C.A.E.; Zhang, S.; Lu, J. R. Chem Soc Rev 2010, 39, 3480–3498. 6. Cui, H.; Webber, M.J.; Stupp, S. I. Biopolymers 2010, 94, 1–18. 7. Paramonov, S. E.; Jun, H. -W.; Hartgerink, J. D. J Am Chem Soc 2006, 128, 7291–7298. 8. Pajewski, R., Ferdani, R., Pajewska, J.; Djedovicˇ, N.; Schlesinger P.H.; Gokel, G. W. Org Biomol Chem 2005, 3, 619–625. 9. Accardo, A.; Tesauro, D.; Roscigno, P.; Gianolio, E.; Paduano, L.; D’Errico, G.; Pedone, C.; Morelli G. J Am Chem Soc 2004, 126, 3097–3107. 10. Accardo, A.; Tesauro, D.; Mangiapia, G.; Pedone, C.; Morelli, G. Biopolymers 2007, 88, 115–121. 11. Accardo, A.; Morisco, A.; Palladino, P.; Palumbo, R.; Tesauro, D.; Morelli, G.; Mol BioSyst 2011, 7, 862–870. 12. Dufresne, M.; Seva, C.; Fourmy, D. Physiol Rev 2006, 86, 805– 847. 13. Pellegrini, M.; Mierke, D.F. Biochemistry 1999, 38, 14775– 14783. 14. Moroder, L., Romano, R., Guba, W.; Mierke, D.F.; Kessler, H.; Delporte, C.; Winand, J.; Christophe, J. Biochemistry 1993, 32, 13551–13559. 15. Lutz, J.; Romano-Goetsch, R.; Escrieut, C.; Fourmy, D.; Math€a, B.; M€ uller, G.; Kessler, H.; Moroder, L. Biopolymers 1997, 41, 799–817. 16. Schwyzer, R. J Mol Recognit 1995, 8, 3–8. 17. Schwyzer, R. Biopolymers 1995, 37, 5–16. 18. Schmitt, L.; Dietrich, C.; Tampe, R. J Am Chem Soc 1994, 116, 8485–8491. 19. Chang, W. C.; White, P. D. In Fmoc Solid Phase Peptide Synthesis; Oxford University Press: New York, 2000. 20. Accardo, A.; Morisco, A.; Gianolio, E.; Tesauro, D.; Mangiapia, G.; Radulescu, A.; Brandt, A.; Morelli, G. J Pept Sci 2011, 17, 154–162. 21. Accardo, A.; Mansi, R.; Morisco, A.; Mangiapia, G.; Paduano, L.; Tesauro, D.; Radulescu, A.; Aurilio, M.; Aloj, L.; Arra, C.; Morelli, G. Mol Biosyst 2010, 6, 878–887. 22. Edelhoch, H. Biochemistry 1967, 6, 1948–1954. 23. Pace, C. N.; Vajdos, F.; Fee, L.; Grimsley, G.; Gray, T. Protein Sci 1995, 4, 2411–2423. 24. Aloj, L.; Panico, M.; Carac o, C.; Del Vecchio, S.; Arra, C.; Affuso, A.; Accardo, A.; Mansi, R.; Tesauro,D.; De Luca, S.; Pedone, C.; Visentin, R.; Mazzi, U.; Morelli, G.; Salvatore, M. Cancer Biother Radiopharm 2004, 19, 91–96.
312
Accardo et al.
25. Torchilin, V. P.; Omelyanenko, V. G.; Papisov, M. I.; Bogdanov, A. A., Jr.; Trubetskoy, V. S.; Herron, J. N.; Gentry, C. A. Biochim Biophys Acta 1994, 1195, 11–20. 26. Accardo, A.; Tesauro, D.; Morelli, G.; Gianolio, E.; Aime, S.; Vaccaro, M.; Mangiapia, G.; Paduano, L.; Schillen K. J Biol Inorg Chem 2007, 12, 267–276. 27. Kumar, M.; Reeve, J. R., Jr.; Hu, W.; Miller, L. J.; Keire, D. A. J Med Chem 2008, 51, 3742–3754. 28. Lackowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983.
29. Creighton, T. E. Proteins: Structures and Molecular Properties; W. H. Freeman: New York, 1993. 30. Klibanov, A. L.; Maruyama, K.; Torchilin, V. P.; Huang, L. FEBS Lett 1990, 268, 235–237. 31. Papahadjopoulos, D.; Allen, T. M.; Gabizon, A.; Mayhew, E.; Matthay, K.; Huang, S. K.; Lee, K. -D.; Woodle, M. C.; Lasic, D. D.; Redemann, C.; Martin, F. J. Proc Natl Acad Sci USA 1991, 88, 11460–11464. 32. Johnsson, M., Hansson, P.; Edwards, K. J Phys Chem B 2001, 105, 8420–8430.
Biopolymers (Peptide Science)
