Molecules 2018, 23, 568
16 of 19
is based on two fluorescent dyes: Calcein AM (showing a green color for live cells) and ethidium
homodimer-1 (EthD-1, which stains dead cells red). The ‘live/dead’ test was performed according to
the manufacturer’s instructions. Briefly, the endothelial cells were cultured in the presence of peptides
for 48 h. The samples were then washed twice with phosphate-buffered saline (PBS) and incubated in a
◦
solution of dyes (0.6
µM Calcein AM1 and 1.5
µ
M EthD-1) for 30 min at 37 C. The stain was removed
by washing with PBS. Microscopic observations were then made using a fluorescence microscope
(Olympus GX71, Olympus, Tokyo, Japan).
The live and dead cells were counted using the Image J program. The results have been presented
as a mean
±
standard deviation (SD). The statistical analysis of the obtained results was carried
out using one-way analysis of variance (ANOVA). Results with p > 0.05 were considered to be
statistically significant.
3
.5.3. Cytotoxicity Test
Cell Culture: The mouse fibroblast cell line L929 (ATCC, American Type Culture Collection
2
(
ATCC), Manassas, VA, USA) was expanded in 75 cm tissue culture flasks with Eagle’s Minimum
◦
Essential Medium supplemented with 10% horse serum (ATCC, Manassas, VA, USA) at 37 C in a
humidified, 5% CO atmosphere. The medium was changed every 3 days until a 70% confluent cell
2
monolayer developed. The cells were then detached from the culture flasks using 5% Trypsin-EDTA
(
GE Healthcare—HyClone Laboratories Inc., Logan, UT, USA). After flushing and centrifugation,
4
the cells were concentrated to 5
×
10 cells/mL in the culture medium. Next, 200
µL of the cell
suspension was placed into wells in 96-well culture plates (ThermoSci, Nunc, Roskilde, Denmark).
The cells were allowed to adhere for 24 h, before 10 L of peptide suspension was added to each well.
The peptides and 7-ent, in the form of powder, were resuspended in PBS (10 g/mL). The study was
conducted for 3 and 7 days. The TCPS plates served as a positive control.
µ
7
µ
Cell Morphology: Three cultures of L929 cells were stained with 0.01% acridine orange (AO) for
min. The samples were next rinsed in PBS, observed and photographed under an Olympus CX41
1
(Olympus, Tokyo, Japan) fluorescence microscope to evaluate their morphology and attachment to the
examined materials.
4
. Conclusions
Understanding the complex relations between the structure of di- and tripeptides and their
susceptibility for self-assembly remains a challenge. Self-aggregating fibrous structures could be
used as scaffolds in regenerative medicine. However, they require careful design and sequencing,
taking into account the multidimensionality of the scaffold structure, its mechanical parameters,
cytocompatibility, toxicity, and susceptibility to degradation under physiological conditions into
bioneutral metabolites. In this study, aggregation susceptibility tests were conducted on a pool of
2
0 di- and tripeptides, composed of aromatic amino acids and cysteine/methionine. Twelve of the
compounds formed amyloid-like fibrous structures. The selected short peptides, composed of or
amino acids, were tested with regard to their usefulness as scaffolds, taking into account the form,
hydrophilicity, and toxicity of the aggregates.
The cytotoxicity of the peptides was tested on the endothelial cell line EA.hy 926. The peptides
that did not fulfil the standards were: H–TrpTrpMet–OH (14), H–TrpTrp–OH ( ), H–TrpCysTrp–OH
11), and H–PhePheMet–OH (13). Those that showed potential for use in regenerative
medicine were H– Phe Phe–OH (1-ent), H– Cys Phe Phe–OH (4-ent), H–CysTyrTyr–OH ( ) and
H– Phe Phe Cys–OH (7-ent). This group may be expanded to include H–TyrTyrMet–OH (15) and
L
D
2
(
D
D
D
D
D
6
D
D
D
H–TyrMetTyr–OH (18), in which cysteine is replaced with methionine residue. Significantly, none of
the selected materials were derived from tryptophan peptides. The influence of peptide aggregates
on the morphology of L919 fibroblast cells was also studied. Preliminary findings suggest that the
stereochemistry of the peptide had no effect on either the morphology or viability of the cells. This is
particularly important because, in the case of peptides consisting of
D amino acids, increased resistance