Article pubs.acs.org/Langmuir
L‑Diphenylalanine
Microtubes As a Potential Drug-Delivery System: Characterization, Release Kinetics, and Cytotoxicity
Rondes F. Silva,† Daniele R. Araújo,† Emerson R. Silva,† Rômulo A. Ando,‡ and Wendel A. Alves*,† †
Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, 09210-170 Santo André, SP, Brazil Instituto de Química, Universidade de São Paulo, C.P. 26077, 05513-970 São 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.
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 vectorization 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 © 2013 American Chemical Society
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 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 10205
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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.
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 cargoconjugated arrays. Detailed structural data were obtained through X-ray diffraction and microscopic techniques, namely, electron microscopy, atomic force microscopy, and confocal
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 framework 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 sidechains. 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 hydrophobic 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 selfassemble 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. 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
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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 phthalocyanine (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. 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
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. 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 FFMTs as drug-delivery systems, rhodamine B (RhB) was used as a probe. The incorporation of RhB was conducted during the FF selfassembly. 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
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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. 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 2.5. 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. 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
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 FFMTs. 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 2.2 ± 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. 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 FFMTs 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 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
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 FFMTs 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. 10208
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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). 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-caprolactone) 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 FFMT 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 nanocapsules 38 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 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.
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 CC and CN 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 CC and CN 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
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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 ± SD (n = 6 replicates/experiment).
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 Larginine-functionalized beta-cyclodextrin-modified quantum dots,44 liposomes,34 cyclodextrins,45 and solid lipid nanoparticles,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
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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.
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ACKNOWLEDGMENTS We are grateful for the financial support provided by Fundaçaõ de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Grant 08/53576-9) and from Conselho Nacional de Desenvolvimento ́ e Tecnológico (CNPq, Grant 472197/2012-6). This Cientifico 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. ́ 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).
4. CONCLUSIONS AND PERSPECTIVES 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 firstorder kinetics with a steady-state regime. 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 characterization 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 FFderivatives 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.
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REFERENCES
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dx.doi.org/10.1021/la4019162 | Langmuir 2013, 29, 10205−10212
