L -Diphenylalanine microtubes as a potential drug-delivery system: Characterization, release kinetics, and cytotoxicity

Article abstract of DOI:10.1021/la4019162

Microtubes obtained from the self-assembly of l-diphenylalanine (FF-MTs) were evaluated as potential vehicles for drug delivery. The biological marker Rhodamine B (RhB) was chosen as a model drug and conjugated to the peptide arrays during self-organization in the liquid phase. Microscopy and X-ray studies were performed to provide morphological and structural information. The data revealed that the cargo was distributed either in small aggregates at the hydrophobic surface of the FF-MTs or homogeneously embedded in the structure, presumably anchored at polar sites in the matrix. Raman spectroscopy revealed notable shifts of the characteristic RhB resonance peaks, demonstrating the successful conjugation of the fluorophore and peptide assemblies. In vitro assays were conducted in erythrocytes and fibroblast cells. Interestingly, FF-MTs were found to modulate the release of the load. The release of RhB from the FF-MTs followed first-order kinetics with a steady-state profile, demonstrating the potential of these carriers to deliver drugs at constant rates in the body. Cytotoxicity investigations revealed high cell viability up to concentrations of 5 mg mL^-1, demonstrating the low toxicity of the FF-MTs.

Full text of DOI:10.1021/la4019162

Article
pubs.acs.org/Langmuir
L‑Diphenylalanine Microtubes As a Potential Drug-Delivery System:
Characterization, Release Kinetics, and Cytotoxicity
Rondes F. Silva,† Daniele R. Araujo,† Emerson R. Silva,† Romulo A. Ando,‡ and Wendel A. Alves*,†
́
̂
†Centro de Cien
̂ ́
cias Naturais e Humanas, Universidade Federal do ABC, 09210-170 Santo Andre, SP, Brazil
‡Instituto de Química, Universidade de Sao Paulo, C.P. 26077, 05513-970 Sao Paulo, SP, Brazil
̃
̃
ABSTRACT: Microtubes obtained from the self-assembly of
L-diphenylalanine (FF-MTs) were evaluated as potential
vehicles for drug delivery. The biological marker Rhodamine
B (RhB) was chosen as a model drug and conjugated to the
peptide arrays during self-organization in the liquid phase.
Microscopy and X-ray studies were performed to provide
morphological and structural information. The data revealed
that the cargo was distributed either in small aggregates at the
hydrophobic surface of the FF-MTs or homogeneously
embedded in the structure, presumably anchored at polar
sites in the matrix. Raman spectroscopy revealed notable shifts
of the characteristic RhB resonance peaks, demonstrating the
successful conjugation of the fluorophore and peptide
assemblies. In vitro assays were conducted in erythrocytes
and fibroblast cells. Interestingly, FF-MTs were found to
modulate the release of the load. The release of RhB from the FF-MTs followed first-order kinetics with a steady-state profile,
demonstrating the potential of these carriers to deliver drugs at constant rates in the body. Cytotoxicity investigations revealed
high cell viability up to concentrations of 5 mg mL−1, demonstrating the low toxicity of the FF-MTs.
groups at the respective side-chains and/or their ability to
specifically interact with proteins embedded in biological
membranes. Thus, the binding of these peptides to the cell
membrane would be assisted either by electrostatic attraction to
anionic lipids in the membrane or via amino acid pairing.10,11
The utilization of RK amino acids is also largely inspired by
their relative abundance in some cell-penetrating peptides
(CCPs), such as the transactivator of transcription gene (TAT)
found in the genome of the human immunodeficiency virus
(HIV), and Penetratin, a 16-amino acid sequence derived from
the Antennapedia protein.12,13 In addition to these TAT
mimetic sequences, ionic complementary self-assembling
peptides have also been investigated, particularly for the
transportation of hydrophobic drugs. In ref 14, Keyes-Baig et
al. present a proof of concept using a system involving the
EAK16 peptide and pyrene as a model drug. Later, this same
sequence was successfully used to carry the anticancer
compound ellipticine through the plasma membrane of cancer
cell lines.10,15
1. INTRODUCTION
The bottom-up assembly of supramolecular structures has
attracted considerable interest due to the potential applications
of these structures in a variety of fields, including biomedicine.
For example, micro- and nanoscaffolds can be employed in
therapeutics, where they are excellent candidates for vectoriza-
tion in gene,1,2 protein,3 and drug delivery.4 These synthetic
carriers possess interesting characteristics with respect to
traditional routes of administration, particularly oral and
injection methods.5 Rational drug design, cell-specific targeting,
and controlled release of the load are among the properties of
these assemblies that enable reduced systemic side effects and
improved pharmacological efficiency. In this context, peptides
are among the most promising materials for the fabrication of
molecular carriers; they possess high chemical diversity and
intrinsic biocompatibility, providing a broad approach for
designing polymorphic arrays endowed with suitable cargo
affinity and targeting abilities.6−8
In recent years, several studies have described Trojan
peptides for intracellular delivery.9 A large number of these
studies employed hybrid systems in which the cargo was
conjugated to sequences of amino acids, which were often rich
in arginine (R) and lysine (K) and formed wrapped structures
that protected the load during transport and enabled breaching
of the cytoplasmic barrier. Although the detailed mechanisms of
internalization remain unclear, the successful use of these
amino acids has been attributed to the presence of cationic
The homodimer L-diphenylalanine (FF) is the most
investigated building block for the construction of peptide
self-assemblies since the discovery of the first FF nanotubes by
Reches and Gazit in 2003.16−19 Typically, self-assembly takes
Received: May 21, 2013
Revised: July 19, 2013
Published: July 23, 2013
© 2013 American Chemical Society
10205
dx.doi.org/10.1021/la4019162 | Langmuir 2013, 29, 10205−10212

Langmuir
Article
Figure 1. Schematic representation of the multiscale self-assembly of the FF-MTs21 and their conjugation to RhB. Stacked FF hexamers form
honeycomb-like arrays, which give rise to nanotubes. Subsequently, these nanotubes cluster into larger microtubes. The inner surfaces of the
nanotubes exhibit both hydrophobic and hydrophilic groups, with the latter being able to trap polar species.
place in a liquid environment and is driven by hydrophobic
forces. A variety of polymorphs has been reported, although
tubular structures organized according to a multiscale frame-
work appear most frequently. In these arrays, six FF units form
cyclic hexamers. Subsequently, the hexamers are stacked to
produce narrow channels with a van der Waals diameter of
approximately 10 Å.20 The amine and carboxyl groups of the
amino acids surround the inner core of the tubules. This
arrangement is advantageous because it provides appropriate
sites for anchoring polar molecules to the peptide matrix, which
is characterized by numerous benzene units from the FF side-
chains. At the upper level, the narrow channels self-associate in
a hexagonal packing formation to produce sheets.21 The coiling
of the sheets generates nanoscale tubes that exhibit hydro-
phobic external walls. Due to steric effects during packing,
hydrophobic/hydrophilic groups remain exposed on the inner
surface of the nanotubes.20,21 Finally, these nanotubes can self-
assemble on a larger scale to form bundles comprising
microscale tubular arrangements. This assembly process is
illustrated in Figure 1. In this study, we investigate the use of
these arrays for the transportation of a model hydrophilic
compound to demonstrate the potential of FF-based assemblies
as intracellular delivery vehicles.
exerting a strong effect on the electronic properties.22 FF
micro/nanotubes, henceforth designated FF-MTs, exhibit
several interesting characteristics, such as high length
homogeneity and, more importantly, high stability in severe
thermal and enzymatic conditions.23,24
Despite their numerous advantages, research on peptide
micro/nanotubes as delivery systems is seldom reported in the
literature and remains almost completely unexplored for
exclusively FF-based compounds. In fact, only very recent
studies have explored the capabilities of FF-containing for
molecular transportation.25−28 As a rule, these studies have
used peptides conjugated with other compounds to host and
transport hydrophobic drugs.25 Moreover, they have focused on
the physicochemical aspects of supramolecular ordering and
used spectroscopic measurements to provide insight into the
interactions responsible for stabilizing the arrays. In our current
work, we investigated the utilization of FF-MTs as molecular
carriers and used a wide set of experimental techniques to
provide information on the location of the drug mimick within
the peptide matrix. Rhodamine B was chosen as a model drug
due to its excellent characteristics as a biological marker. In
addition, the hydrophilic nature of the probe allowed us to
demonstrate the ability of FF-MTs to host polar species.
In the following sections, we describe the experimental
methods used to prepare our system as well as the
physicochemical characterization of the resulting cargo-
conjugated arrays. Detailed structural data were obtained
through X-ray diffraction and microscopic techniques, namely,
electron microscopy, atomic force microscopy, and confocal
These self-assembled structures are held together by a
complex interplay of backbone−backbone hydrogen bonds and
π···π interactions between the aromatic rings of the side-chains.
In addition, residual H2O molecules embedded in the
hydrophilic channels of the peptide matrix are thought to be
protagonists in stabilizing the structure by mediating H-bonds,
10206
dx.doi.org/10.1021/la4019162 | Langmuir 2013, 29, 10205−10212

Langmuir
Article
Figure 2. Micrographs of the FF-MT/RhB samples: (a) fluorescence image displaying peptide needles with high aspect ratios. The homogeneous
fluorescence suggests the presence of RhB across the entire assembly at the microscale; (b) colocalization micrograph exhibiting the fluorescence
from both RhB (in red) and ZcPc (false-colored in green); (c) detail of the region marked by a white dashed square in panel b, indicating the outer
location of the hydrophobic dye; (d) SEM image depicting the open ends of the FF-MTs and the surface grooves related to the multiscaled assembly
of the tubes; (e) topological AFM image displaying the finer details of the FF-MT surface; and (f) detail of the region marked by a red square in
panel e with enhanced local contrast allowing the observation of nanometric RhB aggregates (indicated by red arrows).
For the colocalization experiments, RhB-labeled FF-MTs were
dispersed in dimethyl sulfoxide (DMSO) containing zinc phthalocya-
nine (ZcPc) at a concentration of 1.0 × 10−4 mol L−1. ZcPc is a
polyaromatic fluorophore that exhibits strong hydrophobic behavior.
Thus, it is expected to adhere to nonpolar sites on the FF-MTs,
whereas the highly hydrophilic RhB should exhibit an affinity with the
polar groups available at the peptide polymorph.
microscopy. Raman spectroscopy was performed to provide
information about intermolecular interactions. The results of in
vitro assays are also discussed. Erythrocyte and fibroblast cells
were used to evaluate the cytotoxicity of the FF-MTs and the
release kinetics of the load. A clear modulation effect induced
by the FF assemblies was observed, indicating that these arrays
have the potential to be used as molecular carriers.
2.3. Electron Microscopy, Atomic Force Microscopy, and
Confocal Microscopy. Scanning electron microscopy (SEM) was
conducted using an LV-SEM JEOL microscope (JSM 5900LV) at
LME/LNano (Laboratory of Electron Microscopy of the Brazilian
Nanotechnology National Laboratory in Campinas). Micrographs
were obtained with magnifications between 500× and 5000×. Atomic
force microscopy (AFM) was performed with a Digital Instruments
Nanoscope III at MTA/LNNano (Scanning Probe Microscopy
platform of the LNNano). The FF-MTs/RhB conjugates were
redispersed in water, and droplets from these solutions were deposited
onto glass plates and dried under vacuum overnight. The imaging
process was conducted by registering scans of 4096 × 4096 pixels
covering areas of 1 and 15 μm2. The tip was operated in tapping mode
with a driving frequency of 250 kHz. Fluorescence and qualitative
colocalization analyses were performed on a Leica TCS SP5 II
confocal laser scanning microscope, hosted at the Department of
Microbiology of the Federal University of Sao Paulo. Samples were
prepared by sandwiching droplets of FF-MT/RhB and FF-MT/RhB/
ZcPc solutions between microscope cover slides. The light source was
provided by Ar/HeNe lasers. To probe the fluorescence emission from
RhB and ZcPc, the illumination wavelengths were tuned respectively
2. EXPERIMENTAL SECTION
2.1. Self-Assembly Process. Analytical-grade FF and 1,1,1,3,3,3,-
hexafluor-2-propanol (HFP) were purchased from Sigma-Aldrich
(MO, USA) and used as received. Micro- and nanotubes were
obtained through the self-assembly of FF in the liquid phase; first, we
dissolved the dipeptide in HFP to a concentration of 100 mg mL−1.
Subsequently, ultrapure water (resistivity >18 MΩ cm−1 at room
temperature) was added to provide stock solutions with a final peptide
concentration of 10 mg mL−1. The addition of water led to the
spontaneous formation of FF-MTs in a medium with neutral pH.
Fresh solutions were prepared before each experiment to avoid
undesired aggregation.
2.2. Fluorescent Labeling. To evaluate the potential of the FF-
MTs as drug-delivery systems, rhodamine B (RhB) was used as a
probe. The incorporation of RhB was conducted during the FF self-
assembly. RhB at concentration of 10−3 mol L−1 was added to the FF +
H2O mixture, leading to the spontaneous accommodation of the dye
within the microtubes. Following conjugation, the solvent was left to
dry overnight at room temperature, and the assemblies were then
cleaned with ultrapure water several times to eliminate residual RhB.
10207
dx.doi.org/10.1021/la4019162 | Langmuir 2013, 29, 10205−10212

Langmuir
Article
to 543 and 633 nm. The observation window was adjusted to a range
of 560−640 nm for RhB and 645−800 nm for ZcPc.
The materials were characterized by performing microscopy
assays. First, fluorescence trials were conducted using a confocal
microscope to investigate the conjugation of RhB to the FF-
MTs. Figure 2a presents a typical image from these
experiments. A clear formation of tubular needles can be
observed with high aspect ratios (typically >50). Although there
was considerable polydispersity in the diameters, with values of
approximately 0.7−10 μm or more, the majority of the
structures exhibited diameters within a narrow range averaging
2.4. X-ray Analysis. X-ray diffraction (XRD) patterns of powdered
samples were recorded at room temperature on a D8 Focus Discover
diffractometer at the experimental multiuser platform at UFABC.
Measurements were collected in the Bragg−Brentano configuration.
The beam was provided by a Cu-target source, λ = 0.154 nm,
operating at 40 kV/30 mA. The 2θ range was scanned between 3° and
40° with a step of 0.007°. The raw data were fitted using the General
Structure Analysis System (GSAS) software. Structural models
previously obtained for the FF-MTs were used as inputs for Le Bail
refinements.29,30
2.2
1.0 μm. These values are in agreement with the sizes
commonly observed for FF-MTs prepared under similar
conditions.28,29,33 The RhB fluorescence appeared to be
homogeneously distributed across the needles, suggesting
uniform conjugation with the peptide structures at the
micrometer scale.
2.5. Raman Spectroscopy. The Raman spectra of powdered
samples were obtained on an FT-Raman Bruker RFS 100/S using the
1064 nm excitation of a Nd/YAG laser, typically with 25 mW of
output power and a spectral resolution of 2.0 cm−1, by accumulating
512 scans.
To obtain further information about the location of RhB
within the conjugates, we performed a colocalization analysis by
labeling the FF-MTs simultaneously with ZcPc, a highly
hydrophobic compound. A typical imaging result is displayed in
Figure 2b,c. We observed that the nonpolar dye, false-colored
in green in Figure 2b,c, whether the nonpolar dye was
colocated with RhB. This result suggests that the RhB was
attached to the inside of the structures and not only to the
external surfaces of the arrays, likely intercalated within the
inner core of the nanotubes or, less conceivably, inside the
narrow channels created by the stacking of the FF hexamers.
Significantly, this observation has important implications for
drug delivery applications because it is highly desirable that the
load be hosted inside the vector to avoid degradation in the
intercellular medium.
To probe the distribution of the FF-MTs in greater detail, we
also obtained SEM and AFM images. The micrographs
collected from these experiments are displayed in Figure 2d,f.
They reveal the multiple length scales of the self-assembly and
the presence of nanometric RhB aggregates on the surface of
the tubes. This behavior is expected because the hydrophilic
nature of RhB does not allow for its homogeneous binding to
the hydrophobic surface.
SEM and AFM are only able to provide information about
the external surface of the FF-MTs. XRD was performed to
provide insight on the inner structure of the systems. Figure 3
presents XRD profiles from polycrystalline samples of bare FF-
MTs and FF-MT/RhB conjugates. We observed that the
addition of RhB does not alter the crystallographic symmetry of
the peptide assemblies. The reflections in both the FF-MTs and
FF-MT/RhB systems are perfectly indexed to the hexagonal
space group P61. Although the organization of the crystals
remained unchanged, a small shift in the peak positions toward
smaller angles was observed in the FF-MT/RhB diffractogram,
suggesting that the lattice parameters suffered a slight alteration
upon conjugation with the load. In fact, Le Bail refinements30
(red lines in Figure 2) indicate that the unit cell parameters
increased from a = b = 24.157 0.005 Å and c = 5.463 0.005
Å for bare FF-MTs to a = b = 24.198 0.005 Å and c = 5.598
0.005 Å for FF-MT/RhB conjugates. These findings provide
evidence for the intercalation of RhB in the interstices of the
peptide matrix. Similar behavior has been observed previously
in FF-MTs hosting guest molecules.28,29
2.6. In Vitro Release Assays. In vitro release assays were
performed using an automatized FDA-approved vertical Franz-type
diffusion cell with 1.76 cm2 permeation area (Microette Plus, Hanson
Res., USA) containing an artificial membrane (cellulose acetate sheets
with 1000 Da MWCO) to separate the donor from the receptor
compartments (see details in Figure 5a). The donor compartment was
filled with 1 mL of the FF-NTs formulation or Rh B and the receptor
compartment with 7 mL of 20 mM HEPES buffer at pH 7.4 and 37 °C
under constant magnetic stirring (350 rpm). At predetermined time
intervals, aliquots were withdrawn (1 mL) from the acceptor
compartment at regular intervals and the Rh B concentration
monitored by UV−vis spectrophotometry (543 nm, y = −0.02934 +
0.10172x, R2 = 0.99975, analytical curve previously obtained). Data
were expressed as percentage of Rh B released for each sample (n = 3
replicates/experiment). Release constant kinetics values were analyzed
by two-tailed unpaired t test and the statistical significance was defined
as p < 0.05.
2.7. Cell Viability Studies. Male Wistar albino rats (250−280 g)
were obtained from CEMIB-UNICAMP (Centro de Bioterismo, State
University of Campinas). The protocol was approved by the
UNICAMP Institutional Animal Care and Use Committee (protocol
1961-1/2246-1), which follows the recommendations of the Guide for
the Care and Use of Laboratory Animals. Animals were anesthetized
with 2% halothane, and blood was collected by cardiac puncture.
Following centrifugation (3500 rpm for 5 min), red blood cells (0.15%
hematocrit) were treated with FF-MTs (0.01−5 mg mL−1 at 37 °C for
15 min). The amount of hemoglobin released was determined by
UV−vis spectroscopy at 412 nm and expressed as the percent of
hemolysis.31
Cell viability was assessed by the tetrazolium reduction (MTT) test.
MTT (1 mg/mL) was incubated for 3 h with 3T3 mouse fibroblasts
(37 °C). Cells were treated with FF-MT solutions (2 × 104 cells in
culture dishes with 96 wells, concentrations ranging from 0.25 to 5 mg
mL−1) and incubated for 2 h at 37 °C. The number of viable cells was
determined by measuring the amount of MTT converted to formazan
by mitochondrial dehydrogenases. The resulting formazan crystals
were dissolved in a 1 N HCl-isopropyl alcohol mixture (1:24 v/v) and
shaken for 20 min. Subsequently, the dye-containing solution was
removed, and the sample absorbance was determined at 570 nm.32
3. RESULTS AND DISCUSSION
3.1. Characterization. The self-assembly of FF-MTs is
multiscaled, with the microscale tubes comprising an assembly
of smaller nanotubes. The external and internal walls of the FF-
MTs are strongly hydrophobic because they are composed of
aromatic rings from the FF side-chains. In contrast, in the
peptide matrix, there are polar sites available to host hydrophilic
molecules, primarily in the inner core of the nanotubes.20,21 In
Figure 1, we provide a schematic depiction of the multiscaled
self-assembly of the peptides as well as our proposed
mechanism for binding RhB to the FF arrays.
To further characterize the FF-MT/RhB system, Raman
spectra were obtained. Figure 4 displays the FT-Raman spectra
of the FF-MTs, RhB, and the FF-MT/RhB systems in the
spectral range (1400−1700 cm−1) encompassing the most
intense bands of RhB at 1510 and 1529 cm−1. Based on
10208
dx.doi.org/10.1021/la4019162 | Langmuir 2013, 29, 10205−10212

Langmuir
Article
the electronic delocalization and affecting the vibrational
frequencies of the above-mentioned modes.
3.2. In Vitro Release Assays. The schematic drawing of
our release assays is shown in Figure 5a. Figure 5b reveals that
the incorporation of RhB into the FF-MTs significantly reduced
its release rate compared to RhB in the absence of FF-MTs.
The total release (100%) of the bare RhB was achieved at 60
min, whereas only 35% of the RhB had been released at 60 min
in the presence of the FF-MTs, indicating that the
incorporation of RhB into the FF-MTs slowed the RhB
permeation across the membrane. Furthermore, using the
release profiles, the flux was calculated as the slope of the linear
regression line in the time interval of 10−60 min (Figure 5c).
Analysis of the release curves provided release rate constant
values (K) for RhB and the FF-MT/RhB conjugates of 1.45
0.095 (R2 = 0.9915) and 0.41 0.062% min−1 (R2 = 0.9565),
respectively (p < 0.001). The area under the curve (AUC0−60
min) values were also determined as 33 300 83 for bare RhB
and 24 997 21 for the FF-MT/RhB. These findings indicate
that the release was significantly slower for incorporated RhB
(p < 0.001).
Figure 3. XRD data from the FFMTs/RhB (top) and bare FFMTs
(bottom). Red lines correspond to the Le Bail refinements indexing a
P61 space group (unit cell parameters given in the text). The shift in
the peak positions toward smaller angles for RhB-containing
microtubes suggests the intercalation of guest molecules in the
interstice of the peptide matrix. Blue lines exhibit the difference
between the experimental data and fitted models.
The release of RhB from the FF-MTs followed first-order
kinetics, indicating a constant release rate profile and the ability
of FF-MTs to deliver drugs at constant levels in the body.
Although these findings were produced from an in vitro assay,
our results demonstrate that the differential release behavior for
bare RhB and FF-MT/RhB makes FF-MTs a promising new
carrier system for hydrophilic molecules.
Our results for the FF-MTs are comparable to those found
for other promising nanosystems for use as drug transporters,
such as unilamellar liposomes,34 cyclodextrin inclusion
complexes,35 chitosan nanoparticles,36 and poly(ε-caprolac-
tone) nanospheres;37 these systems also allowed for the release
of 20−30% of the model drug in approximately 60 min.
3.3. Hemolytic Assays: Membrane Interaction. We also
performed in vitro studies to monitor hemolytic behavior at FF-
MT concentrations ranging from 0.1 to 5 mg mL−1. The
concentration of the FF-MTs at the onset of hemolysis or
membrane saturation (Csat) and membrane solubilization
(Csol) were 1.9 and 4 mg mL−1, respectively. In addition, the
concentration at which a 50% hemolytic effect was observed
was 2.8 mg mL−1 (Figure 6).
The concentration-effect curve revealed that the hemolytic
profile was similar to well-described low-toxicity carrier systems,
such as polymeric nanocapsules38 and cyclodextrins.34
Hemolytic assays can be used to explore the mechanisms of
interaction between new drug-carrier candidates and biological
membranes.39 Specifically, we suggest that hemolytic assays can
provide information about a possible mechanism of interaction
between FF-MTs and biological membranes. The comparison
with cyclodextrins is significant because the hemolytic
mechanism invoked by cyclodextrins involves changes to the
membrane fluidity as a result of the uptake of lipid membranes
and cholesterol extraction into the hydrophobic cavity of the
carriers.40,41 In this context, we believe that the presence of
external diphenylalanine residues can induce changes to the
membrane fluidity by insertion of these residues into the
erythrocyte membrane bilayers, possibly forming an “FF-MT
shell” structure (as a surface effect) and causing lysis at high
concentrations of FF-MTs. However, to confirm this
hypothesis, additional assays must be performed, such as
quantification of the lipid components and SEM for the
evaluation of morphological changes to the red blood cells.
Figure 4. FT-Raman spectra of FF-MTs, RhB, and the FF-MT/RhB
systems in the spectral range (1400−1700 cm-1) encompassing the
most intense bands of RhB at 1510 and 1529 cm-1. These vibrations
are associated to CC and CN bonds of fused rings and
diethylamine substituents, respectively. In the FF-MT/RhB Raman
spectrum, they appear shifted to lower wavenumbers pointing to the
presence of RhB inside the structure of the FF-MTs since RhB is most
likely to interact with the polar groups of the matrix.
literature data and DFT calculations, these bands can be
assigned to the vibrational modes that involve the CC and
CN stretching of fused rings and diethylamine substituents,
respectively. These two bands are clearly shifted to lower
wavenumbers in the FF-MT/RhB Raman spectrum. These
results support the hypothesis that RhB is present inside the
structure of the FF-MTs because RhB is most likely to interact
with the polar groups of the matrix, causing a perturbation of
10209
dx.doi.org/10.1021/la4019162 | Langmuir 2013, 29, 10205−10212

Langmuir
Article
Figure 5. (a) Schematic picture of the Franz diffusion cell used in our experiments. (b) In vitro release assays for RhB from the FF-MTs (pH 7.4 at
37 °C). Data express the Rh B percentage release from the donor compartment across the artificial membrane. (c) First-order kinetics and release
constants calculated from the in vitro release percentage for RhB at the time interval from 10 to 60 min. Data shown as the mean SD (n = 3
replicates/experiment).
Figure 7. Cell viability percentage, evaluated by MTT reduction test,
after treatment of the 3T3-fibroblasts cell line with various
concentrations of FF-NTs (37 °C and pH 7.4). Data expressed as
the mean SD (n = 6 replicates/experiment). Gray line: a guide for
the eye highlighting the general trend of viability across the evaluated
concentration range.
Figure 6. Concentration-dependent hemolytic effect of the FF-NTs
(0.15% hematocrit and 15 min under incubation for FF-NTs and cells,
37 °C, and pH 7.4). Data expressed as the mean
replicates/experiment).
SD (n = 6
because the investigation of cell viability provides information
about the biocompatibility of the materials for a variety of
applications. In terms of cell viability, our results were similar to
those obtained for a variety of carrier systems, including a
dipeptide hydrogel,42 poly(pluronic-co-L-lactide micelles,43 L-
arginine-functionalized beta-cyclodextrin-modified quantum
dots,44 liposomes,34 cyclodextrins,45 and solid lipid nano-
particles,46 confirming the potential biocompatibility of FF-
3.4. Cytotoxicity Evaluation: MTT Reduction Test. The
cell culture assays revealed no significant reduction in fibroblast
viability because the percentage of viable cells was greater than
50% for all concentrations tested (Figure 7), indicating the low
toxicity of the FF-MTs as a carrier system.
The cytotoxicity evaluation of a potential new drug carrier is
an essential prerequisite for its use as a therapeutic system
10210
dx.doi.org/10.1021/la4019162 | Langmuir 2013, 29, 10205−10212

Langmuir
Article
Notes
MTs. Furthermore, biocompatibility is influenced by particle
size,47,48 as observed for other carrier systems, including
liposomes, nanoparticles, micelles, and carbon nanotubes;
particles with large dimensions induce membrane damage
and are recognized by the reticulo-endothelial system, reducing
the efficiency of the carrier system following parenteral
application, for example.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful for the financial support provided by Fundaca
de Amparo a
̧
o
̃
̀
Pesquisa do Estado de Sao Paulo (FAPESP, Grant
̃
08/53576-9) and from Conselho Nacional de Desenvolvimento
́
́
Cientifico e Tecnologico (CNPq, Grant 472197/2012-6). This
4. CONCLUSIONS AND PERSPECTIVES
work was also supported by INCT in Bioanalytics (FAPESP,
Grant 08/57805-2, and CNPq, Grant 573672/2008-3). We are
thankful to LNano for the use of their SEM and AFM facilities.
In this work, we investigated the potential of L-diphenylalanine
microtubes as molecular carriers and, in particular, as vehicles
for the delivery of a hydrophilic compound. The biological
marker RhB was used as a model drug. Microscopy, XRD, and
Raman measurements indicated that the hydrophilic load was
conjugated to the self-ordered structure in the form of small
aggregates that were either located at the outer surface of the
tubes or embedded in the interstices of the assembly,
presumably anchored at polar sites in the peptide matrix.
This information about the location of the load in the peptide
matrix is an important issue for further developments and
demonstrates the ability of FF-MTs to protect polar molecules
during transport across an intercellular medium. Toxicity assays
demonstrated that the damage caused by the microtubes was
relatively low, with cells presenting high viability rates at
concentrations of up to 5 mg mL−1. This value is comparable to
those found for other promising self-assemblies intended for
use as molecular carriers.42−44,49 Significantly, our study
demonstrated that FF-MTs are able to modulate the release
of cargo. After 60 min, only approximately 35% of the RhB load
had been released. The modulation is characterized by first-
order kinetics with a steady-state regime.
́
The MTA staff (Raul Freitas, Evandro Lanzoni, Vinicius
Pimentel, and Christoph Deneke) at LNano is kindly
acknowledged for helpful discussions and assistance during
AFM experiments. R.F.S. and E.R.S. are grateful to CAPES for
the provided doctoral and postdoctoral fellowships, respectively
(PNPD 23038007044201108l).
REFERENCES
■
(1) Fichter, K. M.; Ingle, N. P.; McLendon, P. M.; Reineke, T. M.
Polymeric Nucleic Acid Vehicles Exploit Active Interorganelle
Trafficking Mechanisms. ACS Nano 2013, 7, 347.
(2) Srinivasachari, S.; Fichter, K. M.; Reineke, T. M. Polycationic
beta-cyclodextrin ″Click Clusters″: Monodisperse and versatile
scaffolds for nucleic acid delivery. J. Am. Chem. Soc. 2008, 130, 4618.
(3) Murthy, N.; Thng, Y. X.; Schuck, S.; Xu, M. C.; Frechet, J. M. A
novel strategy for encapsulation and release of proteins: hydrogels and
microgels with acid-labile acetal cross-linkers. J. Am. Chem. Soc. 2002,
124, 12398.
(4) Discher, D. E.; Eisenberg, A. Polymer vesicles. Science 2002, 297
(5583), 967−973.
(5) Hughes, G. A. Nanostructure-mediated drug delivery. Nanomed.:
Nanotechnol., Biol., Med. 2005, 1, 22.
In summary, our results support the proposed use of these
peptide arrays to release therapeutic compounds at constant
rates. Since the carrier is composed exclusively from amino acid
building blocks, FF-MTs are thought to exhibit good
biocompatibility.50 This characteristic, together with their
good stability in solution,51 demonstrates their potential as
drug carriers. In this context, our findings extend their
biomedical applications, specially for carrying hydrophilic
species. Due to their double architecture (hydrophilic core
and hydrophobic shell), FF-MTs are self-assembled via the
hydrophobic effect, and in water, they present visco-elastic
aspect behaving like a hydrogel at the concentration used here
(10 mg/mL). Since the visco-elastic behavior of FF-MTs
carriers is probably load-dependent, a rheological character-
ization should be conducted for each specific drug in order to
obtain a detailed visco-elastic picture of the system. Although
such specific studies are still needed for the FF-MTs/RhB
system presented here, results obtained elsewhere for
suspensions of bare microtubes made from similar FF-
derivatives points to the shear modulus varying from 102 to
103 Pa,52 which would allow application under biological
conditions.25 Besides, the reduced dimensions of FF-MTs are
interesting for the development of an injectable local delivery
system or a deposit delivery system. However, it should be
noted that in vivo pharmacological assays will be necessary in
order to determine pharmacokinetic parameters such as half-life
and mean residence time in live organisms.
(6) Gazit, E. Self-assembled peptide nanostructures: the design of
molecular building blocks and their technological utilization. Chem.
Soc. Re.v 2007, 36, 1263.
(7) de la Rica, R.; Matsui, H. Applications of peptide and protein-
based materials in bionanotechnology. Chem. Soc. Rev. 2010, 39, 3499.
(8) Schwartz, J. J.; Zhang, S. Peptide-mediated cellular delivery. Curr.
Opin. Mol. Ther. 2000, 2, 162.
(9) Huang, Y.; Jiang, Y.; Wang, H.; Wang, J.; Shin, M. C.; Byun, Y.;
He, H.; Liang, Y.; Yang, V. C. Curb challenges of the “Trojan Horse”
approach: Smart strategies in achieving effective yet safe cell-
penetrating peptide-based drug delivery. Adv. Drug Delivery Rev.
2013, DOI: 10.1016/j.addr.2012.11.007.
(10) Fung, S.-Y.; Yang, H.; Sadatmousavi, P.; Sheng, Y.; Mamo, T.;
Nazarian, R.; Chen, P. Amino Acid Pairing for De Novo Design of
Self-Assembling Peptides and Their Drug Delivery Potential. Adv.
Funct. Mater. 2011, 21, 2456.
(11) Binder, H.; Lindblom, G. A molecular view on the interaction of
the trojan peptide penetratin with the polar interface of lipid bilayers.
Biophys. J. 2004, 87, 332.
(12) Brooks, H.; Lebleu, B.; Vives, E. Tat peptide-mediated cellular
delivery: back to basics. Adv. Drug Delivery Rev. 2005, 57, 559.
(13) Vives, E. Present and future of cell-penetrating peptide mediated
delivery systems: “is the Trojan horse too wild to go only to Troy?”. J.
Controlled Release 2005, 109, 77.
(14) Keyes-Baig, C.; Duhamel, J.; Fung, S. Y.; Bezaire, J.; Chen, P.
Self-assembling peptide as a potential carrier of hydrophobic
compounds. J. Am. Chem. Soc. 2004, 126 (24), 7522.
(15) Fung, S. Y.; Yang, H.; Chen, P. Sequence Effect of Self-
Assembling Peptides on the Complexation and In Vitro Delivery of
the Hydrophobic Anticancer Drug Ellipticine. PLoS One 2008, 3,
e1956.
(16) Reches, M.; Gazit, E. Designed aromatic homo-dipeptides:
formation of ordered nanostructures and potential nanotechnological
applications. Phys. Biol. 2006, 3, S10.
AUTHOR INFORMATION
■
Corresponding Author
*Tel.: +55 11 4996 0193. Fax: +55 11 4996 3166. E-mail:
wendel.alves@ufabc.edu.br.
10211
dx.doi.org/10.1021/la4019162 | Langmuir 2013, 29, 10205−10212

Langmuir
Article
(17) Huang, R.; Su, R.; Qi, W.; Zhao, J.; He, Z. Hierarchical,
interface-induced self-assembly of diphenylalanine: formation of
peptide nanofibers and microvesicles. Nanotechnology 2011, 22,
245609.
(18) Yan, X.; Zhu, P.; Li, J. Self-assembly and application of
diphenylalanine-based nanostructures. Chem. Soc. Rev. 2010, 39, 1877.
(19) Reches, M.; Gazit, E. Casting metal nanowires within discrete
self-assembled peptide nanotubes. Science 2003, 300, 625.
system for the anti-Alzheimer drug tacrine. Nanomed.: Nanotechnol.,
Biol., Med. 2010, 6, 144.
(37) Campos, E. V. R.; de Melo, N. F. S.; Guilherme, V. A.; de Paula,
E.; Rosa, A. H.; de Araujo, D. R.; Fraceto, L. F. Preparation and
characterization of poly(e-caprolactone) nanospheres containing the
local anesthetic lidocaine. J. Pharm. Sci. 2013, 102, 215.
(38) Alves, C. F.; de Melo, N. F. S.; Fraceto, L. F.; de Araujo, D. R.;
Napimoga, M. H. Effects of 15d-PGJ(2)-loaded poly(D,L-lactide-co-
glycolide) nanocapsules on inflammation. Br. J. Pharmacol. 2011, 162,
623.
(39) Bender, E. A.; Adorne, M. D.; Colome, L. M.; Abdalla, D. S. P.;
Guterres, S. S.; Pohlmann, A. R. Hemocompatibility of poly(epsilon-
caprolactone) lipid-core nanocapsules stabilized with polysorbate 80-
lecithin and uncoated or coated with chitosan. Int. J. Pharm. 2012, 426,
271.
(40) Motoyama, K.; Toyodome, H.; Onodera, R.; Irie, T.; Hirayama,
F.; Uekama, K.; Arima, H. Involvement of Lipid Rafts of Rabbit Red
Blood Cells in Morphological Changes Induced by Methylated beta-
Cyclodextrins. Biol. Pharm. Bull. 2009, 32, 700.
(41) Irie, T.; Uekama, K. Pharmaceutical applications of cyclodextrins
0.3. Toxicological issues and safety evaluation. J. Pharm. Sci. 1997, 86,
147.
(42) Mahler, A.; Reches, M.; Rechter, M.; Cohen, S.; Gazit, E. Rigid,
self-assembled hydrogel composed of a modified aromatic dipeptide.
Adv. Mater. 2006, 18, 1365.
(43) Guo, Q.; Li, X.; Ding, Q.; Li, D.; Zhao, Q.; Xie, P.; Tang, X.;
Luo, F.; Qian, Z. Preparation and characterization of poly(pluronic-co-
l-lactide) nanofibers for tissue engineering. Int. J. Biol. Macromol. 2013,
58, 79.
(44) Zhao, Y.; Tanaka, M.; Kinoshita, T.; Higuchi, M.; Tan, T.
Nanofibrous scaffold from self-assembly of beta-sheet peptides
containing phenylalanine for controlled release. J. Controlled Release
2010, 142, 354.
(45) de Paula, E.; Cereda, C. M. S.; Fraceto, L. F.; de Araujo, D. R.;
Franz-Montan, M.; Tofoli, G. R.; Ranali, J.; Volpato, M. C.; Groppo, F.
C. Micro and nanosystems for delivering local anesthetics. Exp. Op.
Drug Delivery 2012, 9, 1505.
(20) Gorbitz, C. H. Nanotube Formation by Hydrophobic
̈
Dipeptides. Chem.Eur. J. 2001, 7, 5153.
(21) Gorbitz, C. H. The structure of nanotubes formed by
diphenylalanine, the core recognition motif of Alzheimer’s [small
beta]-amyloid polypeptide. Chem. Commun. 2006, 2332.
(22) Andrade-Filho, T.; Ferreira, F. F.; Alves, W. A.; Rocha, A. R.
The effects of water molecules on the electronic and structural
properties of peptide nanotubes. Phys. Chem. Chem. Phys. 2013, 15,
7555.
(23) Ryu, J.; Park, C. B. High stability of self-assembled peptide
nanowires against thermal, chemical, and proteolytic attacks.
Biotechnol. Bioeng. 2010, 105, 221.
(24) Adler-Abramovich, L.; Reches, M.; Sedman, V. L.; Allen, S.;
Tendler, S. J. B.; Gazit, E. Thermal and chemical stability of
diphenylalanine peptide nanotubes: Implications for nanotechnolog-
ical applications. Langmuir 2006, 22, 1313.
(25) Huang, R.; Qi, W.; Feng, L.; Su, R.; He, Z. Self-assembling
peptide-polysaccharide hybrid hydrogel as a potential carrier for drug
delivery. Soft Matter 2011, 7, 6222.
(26) Gour, N.; Kedracki, D.; Safir, I.; Ngo, K. X.; Vebert-Nardin, C.
Self-assembling DNA-peptide hybrids: morphological consequences of
oligonucleotide grafting to a pathogenic amyloid fibrils forming
dipeptide. Chem. Commun. 2012, 48, 5440.
(27) Koley, P.; Pramanik, A. Multilayer vesicles, tubes, various porous
structures and organo gels through the solvent-assisted self-assembly
of two modified tripeptides and their different applications. Soft Matter
2012, 8, 5364.
(28) Souza, M. I.; Jaques, Y. M.; de Andrade, G. P.; Ribeiro, A. O.; da
́
́
Silva, E. R.; Fileti, E. E.; Avilla, E. d. S.; Pinheiro, M. V. B.; Krambrock,
K.; Alves, W. A. Structural and Photophysical Properties of Peptide
Micro/Nanotubes Functionalized with Hypericin. J. Phys. Chem. B
2013, 117, 2605.
(46) Barbosa, R. M.; Silva, C. M. G. d.; Bella, T. S.; Araujo, D. R. d.;
́
́
Marcato, P. D.; Duran, N.; Paula, E. d. Cytotoxicity of solid lipid
nanoparticles and nanostructured lipid carriers containing the local
anesthetic dibucaine designed for topical application. Journal of
Physics. Conf. Ser. 2013, 429, 012035.
(29) Cipriano, T. C.; Takahashi, P. M.; de Lima, D.; Oliveira, V. X.;
Souza, J. A.; Martinho, H.; Alves, W. A. Spatial organization of peptide
nanotubes for electrochemical devices. J. Mater. Sci. 2010, 45, 5101.
(30) Le Bail, A. Whole powder pattern decomposition methods and
applications: A retrospection. Powder Diffrac. 2005, 20, 316.
(31) Malheiros, S. V. P.; Pinto, L. M. A.; Gottardo, L.; Yokaichiya, D.
K.; Fraceto, L. F.; Meirelles, N. C.; de Paula, E. A new look at the
hemolytic effect of local anesthetics, considering their real membrane/
water partitioning at pH 7.4. Biophys. Chem. 2004, 110, 213.
(32) Denizot, F.; Lang, R. Rapid colorimetric assay for cell growth
and survival. Modifications to the tetrazolium dye procedure giving
improved sensitivity and reliability. J. Immunol. Methods 1986, 89, 271.
(33) Amaral, H. R.; Kogikoski, S., Jr.; Silva, E. R.; Souza, J. A.; Alves,
W. A. Micro- and nano-sized peptidic assemblies prepared via solid-
vapor approach: Morphological and spectroscopic aspects. Mater.
Chem. Phys. 2012, 137, 638.
(34) de Araujo, D. R.; Cereda, C. M.; Brunetto, G. B.; Vomero, V. U.;
Pierucci, A.; Neto, H. S.; de Oliveira, A. L.; Fraceto, L. F.; Braga, Ade
F.; de Paula, E. Pharmacological and local toxicity studies of a
liposomal formulation for the novel local anaesthetic ropivacaine. J.
Pharm. Pharmacol. 2008, 60, 1449.
(35) de Araujo, D. R.; Tsuneda, S. S.; Cereda, C. M. S.; Carualho, F.
D. G. F.; Prete, P. S. C.; Fernandes, S. A.; Yokaichiya, F.; Franco, M. K.
K. D.; Mazzaro, I.; Fraceto, L. F.; Braga, A. d. F. A.; de Paula, E.
Development and pharmacological evaluation of ropivacaine-2-
hydroxypropyl-beta-cyclodextrin inclusion complex. Eur. J. Pharm.
Sci. 2008, 33, 60.
(47) Wei, Z.; Hao, J.; Yuan, S.; Li, Y.; Juan, W.; Sha, X.; Fang, X.
Paclitaxel-loaded Pluronic P123/F127 mixed polymeric micelles:
Formulation, optimization and in vitro characterization. Int. J.
Pharm. 2009, 376, 176−185.
(48) Shimizu, K.; Uchiyama, A.; Yamashita, M.; Hirose, A.;
Nishimura, T.; Oku, N. Biomembrane damage caused by exposure
to multi-walled carbon nanotubes. J. Toxicol. Sci. 2013, 38, 7.
́
́ ́
adi, J.; Fenyvesi, E.; Ivanyi,
(49) Kiss, T.; Fenyvesi, F.; Bac
́
skay, I.; Var
́
R.; Szente, L.; Tos
́
aki, A.; Vecsernyes, M. Evaluation of the cytotoxicity
́
of β-cyclodextrin derivatives: Evidence for the role of cholesterol
extraction. Eur. J. Pharm. Sci. 2010, 40, 376.
(50) Ulijn, R. V.; Smith, A. M. Designing peptide based
nanomaterials. Chem. Soc. Rev 2008, 37, 664.
(51) Andersen, K. B.; Castillo-Leon, J.; Hedstrom, M.; Svendsen, W.
E. Stability of diphenylalanine peptide nanotubes in solution.
Nanoscale 2011, 3, 994.
(52) Smith, A. M.; Williams, R. J.; Tang, C.; Coppo, P.; Collins, R. F.;
Turner, M. L.; Saiani, A.; Ulijn, R. V. Fmoc-Diphenylalanine self
assembles to a hydrogel via a novel architecture based on pi-pi
interlocked beta-sheets. Adv. Mater. 2008, 20, 37.
(36) Wilson, B.; Samanta, M. K.; Santhi, K.; Kumar, K. P. S.;
Ramasamy, M.; Suresh, B. Chitosan nanoparticles as a new delivery
10212
dx.doi.org/10.1021/la4019162 | Langmuir 2013, 29, 10205−10212