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Fabrication of ellagic acid incorporated self-assembled peptide microtubes and their applications
Colloids and Surfaces B: Biointerfaces 95 (2012) 154–161
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Fabrication of ellagic acid incorporated self-assembled peptide microtubes and their applications Stacey N. Barnaby a , Karl R. Fath b,c , Areti Tsiola b,1 , Ipsita A. Banerjee a,∗ a b c
Department of Chemistry, Fordham University, 441 East Fordham Road, Bronx, NY 10458, United States Biology Department, Queens College, The City University of New York, 6530 Kissena Boulevard, Flushing, NY 11367, United States The Graduate Center, The City University of New York, 365 Fifth Avenue, New York, NY 10016, United States
a r t i c l e
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Article history: Received 1 December 2011 Received in revised form 20 February 2012 Accepted 21 February 2012 Available online 1 March 2012 Keywords: Peptides Bolaamphiphiles Self-assembly Ellagic acid Threonine
a b s t r a c t Ellagic acid (EA), a plant polyphenol known for its wide-range of health benefits was encapsulated within self-assembled threonine based peptide microtubes. The microtubes were assembled using the synthesized precursor bolaamphiphile bis(N-␣-amido threonine)-1,5-pentane dicarboxylate. The self-assembly of the microstructures was probed at varying pH. In general, tubular formations were observed at a pH range of 4–6. The formed microtubes were then utilized for fabrication with EA. We probed the ability of the microtubes as drug release vehicles for EA as well as for antibacterial applications. It was found that the release of EA was both pH and concentration dependent. The biocompatibility as well as cytotoxicity of the EA-fabricated microtubes was examined in the presence of mammalian normal rat kidney (NRK) cells. Finally the antibacterial effects of the EA incorporated peptide microtubes was examined against Escherichia coli and Staphylococcus aureus. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Over the past decade, there has been a tremendous surge in the development of nanomaterials to mimic and target biological systems for various biomedical as well as biosensor applications [1–3]. Specifically, a plethora of nanomaterials have been explored for their potential in drug delivery applications. For example, Yang et al. [4] designed polymer vesicles from heterofunctional amphiphilic triblock copolymers for targeted anticancer drug delivery. In a separate study, lipid–polymer hybrid nanoparticles were designed for targeting of mammalian cells or tissues [5]. Polymer nanoparticles consisting of PFO (polyfluorene) and poly(l-glutamic acid) conjugated with doxorubicin have also been fabricated [6]. Amphiphilic block copolymer micelles have been utilized as hydrophobic drugs within films [7]. Polymeric dendrimers have also been fabricated using poly(l-glutamic acid) [8], and its subsequent conjugation with DOX allowed for a targeted drug delivery system. Gao et al. [9] have designed nanoparticles based on polyacrylamide that could avoid being up taken by macrophages. Furthermore, Ding et al. [10] designed a system of cisplatin
∗ Corresponding author. Tel.: +1 718 817 4445; fax: +1 718 817 4432. E-mail address: [email protected] (I.A. Banerjee). 1 Current address: Department of Biological Sciences and Geology, Queensboro Community College, 222-05 56th Avenue, Bayside, NY 11364, United States. 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2012.02.031
(CDDP)-loaded gelatin/poly(acrylic acid) nanoparticles for transtissue drug delivery. In addition to various synthetic structures, natural drug delivery systems have also been investigated, such as chitosan [11–13], pullulan [14–17], alginates [18–23], and hyaluronic acid [24–26]. For example, chitosan-functionalized graphene oxide hybrid nanosheets were fabricated for the controlled release of Ibubrofen and 5-fluorouracil [27]. The biopolymer pullulan has been modified and its ability to self-assemble into hydrogels for drug delivery has been explored. Pullulan hydrogels have been found to serve as a coating for stents needed in local arterial therapy. Furthermore, pullulan/DOX conjugated nanoparticles were utilized as drug delivery vehicles for pH-controlled release. Several hyaluronic acid (HA) conjugates have also been synthesized and allowed to self-assemble into nanoparticles that were shown to target tumors [26]. Alginate based copolymers composed of 1–4 linked ␣-l-guluronic acid and -d-mannuronic acid have been functionalized with proteins and utilized for the controlled release of TGF- 1, basic fibroblast growth factor (bFGF), tumor necrosis factor receptor (TNFR), as well as angiogenesis factor, epidermal growth factor (EGF), and urogastrone. In a separate study, DOX was released from alginate embedded magnetic nanoheaters. Peptide nanotubes and microtubes are a relatively new class of biomaterials that have garnered much interest because of their facile self-assembly and ability to be functionalized, as well as their biocompatibility [28–33]. Nanotubes grown from cationic
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dipeptide (H Phe Phe NH2 ·HCl) precursors were efficiently bound to negatively charged single-stranded DNA (ssDNA) and utilized as delivery vehicles into HeLa cells [34]. Phenylalanine-based nanotubes were utilized in drug release studies for insulin [35]. Liu et al. [36] have shown that cyclic peptide nanotubes (CPNs) composed of (Trp-d-Leu)4-GLN-d-Leu can be utilized as drug delivery vectors for the antitumor drug 5-fluorouracil. Peptide nanotubes have also been utilized in other biological applications. For example, peptide nanotubes were coated with antibodies, and assembled onto device platforms, in order to detect pathogens [37,38]. In a recent study, we have demonstrated the ability of bis(N-␣amido-threonine)-1,3-propane dicarboxylate to self-assemble into microtubes, which could be efficiently conjugated with mucin [39]. In the work presented here, we have studied the formation of peptide nanotubules from a newly synthesized bolaamphiphile, bis(N-␣-amido-threonine)-1,5-pentane dicarboxylate, which was fabricated with the plant phytochemical ellagic acid (EA). Ellagic acid is a polyphenol found in fruits and nuts that possesses a multitude of biomedical properties [40–49]. Various drug delivery systems for EA have been reported, such as in situ gelling systems composed of chitosan-glycerol phosphate, composite films of chitosan/EA, as well as liposomes and poly(lactide-co-glycolide) (PLGA) and polycaprolactone (PCL) nanoparticles loaded with EA [50–53]. It has also been reported that EA and coenzyme Q10 co-encapsulated nanoparticles reduced lipid peroxidation and dyslipidemia in diabetic rats [54]. However, to our knowledge, EA has never been fabricated on peptide nanotubes or microtubes, neither has the potential biological applications of such nanoconjugates been explored. In this work, we have examined the encapsulation efficiency of EA in selfassembled threonine based peptide microtubes and investigated its in vitro release at varying pH values. We found that the release was concentration and pH dependent. Further, the materials were found to not only be biocompatible, by investigating the cellular toxicity of EA incorporated microtubes in the presence of NRK (normal rat kidney) cells, but it was also found that the bioconjugate not only retains, but also enhances, its antibacterial properties when compared to EA by itself. 2. Methods 2.1. Materials N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide (EDAC), lthreonine-methyl ester hydrochloride, pimelic acid, hydroxybenzotriazole (HOBT), triethylamine, DMF, methanol, acetone, and DMSO-d6 with 0.1% (v/v) TMS, were purchased from Sigma–Aldrich and used as received. Buffer solutions of various pH values, hydrochloric acid, ellagic acid, and sodium hydroxide pellets were purchased from Fisher Scientific. Normal rat kidney cells (American Type Culture Collection CRL-6509) were maintained in Dulbecco’s Minimal Essential Medium (Lonza, Walkersville, MD) supplemented with 10% fetal calf serum (Hyclone, Logan, UT), 100 units/ml penicillin, and 100 g/ml streptomycin. 2.2. Synthesis of bis(N-˛-amido-threonine)-1,5-pentane dicarboxylate The bolaamphiphile bis(N-␣-amido-threonine)-1,5 pentane dicarboxylate was synthesized via modification of previously established methods [55,56]. Pimelic acid (0.15 g, 0.94 mmol) was dissolved in DMF. To the solution, EDAC (0.05 g, 0.32 mmol) and 1-hydroxybenzotriazole (0.05 g, 0.37 mmol) were added. The mixture was cooled to 5 ◦ C and shaken for an hour. To the mixture, the protected threonine methyl ester hydrochloride (0.3 g, 1.8 mmol),
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and triethylamine (5 l) were added. The contents were stirred at 5 ◦ C for 24 h, at which point the solution was allowed to reach room temperature and the intermediate was rotary evaporated. The oily yellow intermediate obtained was placed on ice and treated with citric acid (0.15 M, 3 mmol), upon which the precipitate was washed with sodium bicarbonate (0.1 M, 2 mmol). The intermediate was then dissolved in DMF, and refluxed for 4 h at 85 ◦ C, in order to allow the protecting group to be removed via base hydrolysis in the presence of NaOH (0.1 M). Upon completion of base hydrolysis, the reaction mixture was rotary evaporated to obtain the product, which was washed with methanol and then recrystallized, to obtain a product that was orange/brown in appearance. The final product was confirmed by 1 H NMR and FTIR spcectroscopy. FTIR: = 3527( OH), 3361(N H amide), 1732 (C O, acid), 1652 (C O, amide). NMR: ı (8.2) (d, 2H, NH doublet); ı (4.5) (2H, m); ı (2.0) (4H, m); ı (1.51) (4H, m); ı (1.1) (6H, d). Elemental analysis (calculated): C 49.6; H 7.2; N: 7.82; O: 35.38. Observed: C: 49.1; H: 7.02; N: 8.01; O:35.87. 2.3. Self-assembly Individual stock solutions of the synthesized bolamphiphile in the range of 0.5–3.5 mM were prepared in buffer solutions ranging in pH values 4–6. In general, the solutions were allowed to assemble for 2–3 weeks. The formed assemblies were washed twice with deionized water and the morphologies were analyzed by TEM and SEM. The assemblies remained stable at room temperature for over 6 months. 2.4. Preparation of EA-loaded microtubules Varying concentrations of EA in the range of 0.02–0.2 mM were incubated with the nanotubes under ambient conditions for 24 h, at which point they were centrifuged and washed thrice in denionized water prior to analysis in order to remove any un-entrapped EA. 2.5. Measurement of entrapment efficiency In order to determine the amount of EA loaded into the peptide microtubes, the difference between the total amount of EA added into the microtubes and the amount of EA present in the supernatant after the loading process was calculated. The loaded microtubes were centrifuged under ambient conditions at 20 × g. The entrapment efficiency was calculated using the following equation: (X1 − X2 )/X1 × 100%, where X1 is the total concentration of EA and X2 is the concentration of the unloaded EA. Therefore, entrapment efficiency of EA in microtubules =
total amount of EA used − free EA in supernatant × 100 total amount of EA used
(1)
2.6. Drug release studies of EA The microtubes were loaded with EA at a concentration of 0.06 mM concentration and allowed to incubate for 2 h at room temperature, at which point the assemblies were centrifuged and the supernatant was removed. Absorbance spectroscopy readings were taken for 3 h at 37 ◦ C. In order to test the effect of pH, 500 l of the respective buffer solutions (pH 1–8) were added to the washed microtubes. Measurements were carried out as described above. 2.7. Cytotoxicity studies NRK cells were grown as monolayers in 25 or 75 cm2 culture flasks under a humidified atmosphere of 5% CO2 in air at 37 ◦ C.
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Cells were seeded at densities of 2 × 104 cells/ml and harvested when they reached 80–90% confluence. To study the effects of EA loaded microtubes on cell proliferation, cells were seeded at densities of 2 × 104 cells/ml in 12-well plates. EA loaded microtubes at a concentration of 0.6 mM (20 l) were added to each well. At the designated time point (24 h, 48 h, 4 days) after plating, cell proliferation was calculated. The medium from each well was collected. Live cells were harvested after applying 500 l 1× trypsin EDTA (Cellgro, Manassas, VA). Trypsinization was blocked with 500 l medium and the total volume was added to the centrifuge tubes. The cell suspension was centrifuged for 2 min and the pellet was resuspended in 500 l of medium. 45 l of the latter cell suspension was added to 45 l of 0.4% Trypan Blue Stain (Gibco) and the density of live and dead cells were calculated using a hemocytometer. All studies were done in triplicate. 2.8. Anti-bacterial studies Late log phase Escherichia coli (E. coli; DH5a strain) or Staphalococcus aureus (S. aureus) were diluted in LB medium to 104 colony forming units (cfu)/ml as determined using dilution plating. We evaluated the bacteriostatic properties of the EA loaded microtubes by determining the relative number of bacteria in the cultures by measuring the turbidity of 200-l aliquots of diluted bacteria plus 10 l of the EA loaded microtubes in 96-well plates using 630nm light in a BioTek ELx 800 microplate reader. Readings were taken every hour for 10 h as the bacteria were incubated in aerobic conditions at 37 ◦ C. To compare the growth characteristics of bacteria in the presence of different additives (with differing optical densities), the starting OD630 of each well was subtracted from subsequent readings from that well. As diluent controls, we added the appropriate volumes of ultrapure water in the absence of EA loaded microtubes to wells containing bacteria. Some wells contained only LB medium to assay for the growth of any contaminating environmental bacteria. To assay the bacteriocidal activity of the EA loaded microtubes, late log phase bacteria were diluted in LB medium to 104 cfu/ml and 200 l was mixed with 10 l of the EA loaded microtubes and incubated overnight at 37 ◦ C with shaking. After 24 h, the number of live bacteria was determined by serial dilution in LB broth not containing EA loaded microtubes, and by counting the number of colonies growing from these dilutions when spread on LB agar plates. The final dilutions (107 –108 ) greatly reduced the free EA loaded microtubes concentration so that any subsequent bacteria growth was essentially in the absence of EA loaded microtubes. Single live bacteria were identified by the formation of macroscopic bacterial colonies. Images of the plates were captured using a Kodak Gel Logic 440 imaging system and the grayscale values were inverted in Photoshop to make the colonies more visible. 2.9. Statistical analysis Statistical analysis of the data was done using student ‘t’ test; p < 0.05 was considered significant compared to controls. The results are expressed as mean ± standard deviation (SD). 2.10. Characterization 2.10.1. FTIR-spectroscopy Analyses were performed using Matteson Infinity IR equipped with DIGILAB, ExcaliBur HE Series FTS 3100 software. The washed samples were vacuum dried and mixed with KBr to form pellets. The measurements for the samples were carried out at 400–4000 cm−1 .
2.10.2. Absorbance spectroscopy Absorbance spectroscopy was carried out using a Thermo Scientific NanoDrop 2000. Readings were taken at a wavelength range of 190–800 nm. All samples were repeated in triplicates. 2.10.3. Transmission electron microscopy Samples were washed twice with nanopure water and air-dried onto carbon-coated copper grids for characterization by transmission electron microscopy (JEOL 1200 EX) operated at 100 kV. 2.10.4. Scanning electron microscopy The morphologies of the samples were further analyzed using SEM (Hitachi S-2600N) operated between 15 and 25 kV. A few drops of the sample solution were pipetted out onto MCE filter paper (5 mm), which were air-dried. Portions of the stained MCE filter paper were then cut out and mounted on SEM sample stubs with double sided tape and carbon coated using an SPI-module carbon coater before analysis. 3. Results and discussion 3.1. Microtube self-assembly Bolaamphiphiles have served as an attractive means for forming various supramolecular structures via self-assembly [57–60] such as nanofibers and nanotubes [61–65], vesicles [66,67], and crystals [68–70]. The fabrication of peptide assemblies has proven advantageous because of their ability to mimic biological systems [71–77]. Non-covalent interactions such as – stacking interactions, as well as hydrophobic interactions and intra and inter-molecular hydrogen bonding play a key role in the formation of supramolecular assemblies. Although peptide microtubes are synthetic-derived molecules, they form similar supramolecular structures as those found in nature [78]. In previous work, we have reported the self-assembly of the bolaamphiphile HOOC Thr NH CO (CH2 )3 NH CO Thr COOH [28], which formed a high propensity of tubular structures with a diameter of 500 nm–2 m in the pH range of 4–6, whereas at higher pH values, globular structures were formed. In this study, we have increased the alkyl spacer between the head groups to a pentyl group, forming HOOC Thr NH CO (CH2 )5 NH CO Thr COOH, as shown in Fig. 1a. Fig. 1b shows the proposed inter and intramolecular hydrogen bonding interactions between the carboxyl groups, the NH and C O groups as well as the hydroxyl groups between the bolaamphiphiles that result in the formation of the supramolecular structures. We examined the self-assembly at a pH range of 4–6. At a pH value of 4, we observed a relatively low yield with a few short, truncated nanotubular structures (Fig. 2a). At a pH value of 5, short proto-fibrillar structures were observed (Fig. 2b). However, at a pH value of 6, we observed a high yield of tubular structures, with an average diameter of 500 nm–1 m (Fig. 2c). The microtubes appear to be smooth on the surface, and are seen to form an interlocking mesh. Previous studies examining the growth of microtubes have shown that the self-assembly occurred via both acid-anion dimers and hydrogen bonding networks [79]. We expect similar forces to be involved in the assembly. When analyzing the effect of the alkyl chain length on self-assembly, it is seen that in terms of diameter, the chain length has little effect. However it appears that increasing the chain length leads to higher yields of the tubular structures, and results in a mesh of microtubes. The assemblies formed due to extensive hydrogen bonding interactions between the hydroxyl group and the backbone of the molecule, as well as between the C O and NH groups, aids in the self-assembly of the molecule [80] thus allowing for the formation of tubular structures. This is consistent with cyclic peptide assemblies, where nanotubes are formed
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Fig. 3. FTIR spectrum of the self-assembled microtubes formed at pH 6.
Fig. 1. (a) Chemical structure of the synthesized bolaamphiphile; (b) threedimensional model showing the proposed hydrogen bonding interactions between the bolaamphiphiles.
by spontaneous stacking of molecules on top of one another, which results in hollow nanotube structures [81]. Further, the addition of two alkyl groups allows for enhanced hydrophobic interactions. The mechanism of formation of the tubules was further explored using FTIR-spectroscopy. 3.2. FTIR-spectroscopy In order to determine the underlying forces allowing for the selfassembly of the microstructures, we analyzed FTIR spectra of the self-assembled microtubes formed. As shown in the supplementary data, Fig. 3 shows the FTIR spectrum of the self assembled microtubes in the range of 1300–2000 cm−1 . In the C O region, two peaks were observed, one at 1730 cm−1 due to hydrogen bonded carboxylic groups and another at 1645 cm−1 due to the amide I peak after self-assembly. The peak at 1730 cm−1 is significantly quenched compared to the as synthesized bolaamphiphile due to H-bonding interactions, while the amide I peak is shifted compared to the as synthesized bolaamphiphile which was seen at 1652 cm−1 (data not shown). These results indicate the strong role
that the carboxyl and amide groups play in the self-assembly. The short peak seen at 1558 cm−1 was assigned to asymmetric COO− vibration indicating that at pH 6, some of the carboxyl groups are deprotonated and thus acid-anion dimers are formed. An additional peak at 1539 cm−1 was also observed due to amide II N H bending vibration after self-assembly. A strong peak at 1420 cm−1 was also observed due to CH3 bending vibrations from the threonine moieties. Furthermore, spectral changes were observed in the OH stretching region, where we observed an intense broad peak at 3454 cm−1 after self-assembly due to hydrogen bonding interactions (range not shown in figure). 3.3. EA entrapment Peptide conjugated nanoparticles [82], vesicles [83], lipid–polymer hybrids [84], conjugated polymer nanoparticles, vesicles and micelles [85,6,4,86,87], gold nanorods [88], and gels [89] are commonly employed as drug delivery vehicles. Peptide-based systems represent an ingenious option for delivering drugs [90], because they are biocompatible, and they can be tailored for specific environments through functionalization methods. Herein, we explored the potential use of Thr based microtubes to serve as a carrier of EA. It was found that loading of EA into the microtubes is concentration dependent as shown in Fig. 4 and that at a concentration range of 0.06–0.1 mM, the entrapment was significantly higher (p < 0.05) in comparison to other concentrations. We found that the encapsulation efficiency is optimal at 0.06 mM, such that the EA is completely encapsulated within the microtubes. Fig. 5 shows the entrapment of EA within the microtubes. Fig. 5a
Fig. 2. Growth of nano and microtubular structures (a) at pH 4 (scale bar = 500 nm), (b) pH 5 (scale bar = 2 m) and (c) pH 6 (scale bar = 2 m).
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Fig. 4. EA entrapment efficiency at varying concentrations. All experiments were carried out in triplicate. Bars indicate SD of the mean (n = 3). The asterisks represent statistical difference p < 0.05.
represents the microtubular structure, without the incorporation of EA, while Fig. 5b shows the incorporation after 0.06 mM concentration of EA as represented by the darker sections of the tubules [91]. At higher concentrations (Fig. 5c), (0.09 mM), EA was found to deposit in the form of aggregates on the outside of the microtubes. EA has been known to form discotic liquid crystal assemblies upon aggregation in water [92]. Thus, it appears that at low concentrations, in the presence of the threonine based peptide microtubes, EA efficiently inserts into the tubules by capillary action. However, at higher concentrations, EA appears to aggregate forming assemblies on its own, hence encapsulation is relatively lesser. Thus, in general, for drug release studies, tubules encapsulated with EA at 0.06 mM concentration were selected. 3.4. Release of EA from microtubes We examined the release of EA at varying pH values. As shown in Fig. 6, the release of EA from the microtubes was found to be pH dependent. At low pH, (pH 1) we observed that the release was slowest, while under near neutral conditions (pH 6 and 7), the drug released at a higher steady rate and the % release was found to be 50% after over 2 h. Under basic conditions, EA released fastest and after 2 h, we observed a decrease in the percentage release. We attribute the different rates of EA release based on pH to be a result of the binding interactions between EA and the microtubes. Under highly acidic conditions (pH 1), EA is strongly bound to the microtubes because of the strong hydrogen bonding as EA is completely protonated under those conditions. However as pH increases, hydrogen bonding interactions are less due to
Fig. 6. Release of EA from the microtubes at varying pH. All experiments were carried out in triplicate. Error bars are based on three parallel samples and represent standard deviations.
deprotonation process of EA [93] resulting in faster release and under basic conditions, due to the negative charge of EA, it is likely that there is repulsion between the EA molecules, leading to higher rate of release. 3.5. Anti-bacterial studies EA has well characterized anti-bacterial properties against E. coli [94], oral bacteria [95], Propionibacterium acnes, S. aureus, and Staphylococcus epidermidis [96]. We wished to determine whether the incorporation of microtubes fabricated with EA could enhance its antibacterial properties. For antibacterial studies, 0.1 mM EA was incorporated onto the microtubes, because at those concentrations EA is present on the outside surface of the microtubes as well, and hence would allow for higher contact with bacteria. To assay the bacteriostatic properties of the microtubes, we added the microconstructs to freshly-diluted stationary phase cultures containing 104 cfu/ml of either the Gram-negative bacteria E. coli or the Gram-positive bacteria S. aureus. The cultures were incubated at 37 ◦ C and the relative number of bacteria in the cultures was determined by measuring the turbidity (optical density with 630nm light) of the cultures every hour for a total of 10 h. We found that, after the typical lag phase with little detected growth, both E. coli and S. aureus incubated in the solvent control wells (ultrapure water) grew rapidly during the course of the experiment. The growth of Gram-negative E. coli was inhibited 63% by EA and 84% by EA integrated with microtubes. The percent inhibition was
Fig. 5. TEM images of EA loaded microtubes. (a) At 0 mM concentration of EA; (b) at 0.06 mM concentration of EA; and (c) at 0.09 mM concentration of EA.
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Fig. 7. Growth curves of E. coli (a) and S. aureus (b) in LB medium inoculated with 104 cfu/ml of bacteria in 96-well plates in the presence of EA or microtubes entrapped with EA. The extent of growth was determined by change in turbidity using a plate reader and 630-nm light. The solvent control (water) indicates the growth of bacteria without additives. Error bars are based on three parallel samples and represent standard deviations; and (c) bactericidal properties of EA-fabricated microtubes. Bacteria (E. coli and S. aureus) at an initial concentration of 104 cells/ml were incubated with the EA, microtubes loaded with EA or water (solvent control) for 24 h.
determined by the differences in culture turbidity at the 10-h time point (Fig. 7a). By comparison, the growth of Gram-positive S. aureus was not significantly inhibited by EA and 50% by EA incorporated microtubes (Fig. 7b). Control wells containing Luria Broth alone did not change in turbidity during the incubation period, which suggested that there was no contamination from environmental bacteria. We found that E. coli was more susceptible than S. aureus to growth-inhibition by EA. Further, the entrapment of EA into peptide microtubes enhanced its bacteriostatic properties. This is most likely because threonine on its own has been reported to display antibacterial properties [97,98], thus a blend of threonine based microtubes with EA, enhanced the antibacterial effects. In general, turbidity-based assays are commonly used to assess compounds for antibacterial properties. While these assays are relatively rapid and easy to perform, they are limited in that a dead bacterium can also scatter light. Further, usually these assays follow bacteria growth in the continual presence of the treatment agent. Thus we also determined whether our treated bacteria at the end of the incubation period were alive and capable of growth in the absence of EA bound microtubes. Therefore we incubated bacteria as before in the turbidity assays, diluted the treated bacteria in LB (107 - or 108 -fold) and then determined the number of viable bacteria on LB agar plates (Fig. 7c). We found that EA alone had relatively less bacteriocidal activity for both E. coli and S. aureus, but EA bound peptide microtubes decreased the number of alive E. coli by 96% and the number of alive S. aureus by 66% after a 24-h treatment as shown in Table SI (supplementary information). These results indicate that threonine microtubes integrated with EA enhances bacteriocidal activity.
indicated that the number of cells had quadrupled and were significantly higher (p < 0.05) in comparison with the initial number of cells. These results indicate that the EA loaded microtubes are non-toxic to mammalian cells and can be potentially utilized as a drug delivery system.
3.6. Biocompatibility studies To determine whether the EA incorporated microtubes could be utilized for potential biomedical applications, we investigated their biocompatibility by conducting in vitro cytotoxicity and cellproliferation studies with cultured NRK cells. Trypsinized cells were allowed to attach and spread in 12-well plates and 20 l of microtubular solutions were added to the culture media. We observed no cell lysis or changes in cell morphology in the presence of EA loaded microtubes. The cells continued to be well spread when bound to the microtubes (Fig. 8a). Cytotoxicity analysis using regular MTT assays revealed that the cells are capable of dividing and growing in the presence of the EA loaded microtubes (Fig. 8b). We found that the number of cells more than doubled after 24 h. We also examined the samples after a period of 96 h and the results
Fig. 8. (a) Photomicrograph of NRK cells after 48 h in the presence EA loaded microtubes (concentration of EA = 0.09 mM); (b) cytotoxicity studies showing the growth and proliferation of NRK cells over time in the presence of EA loaded microtubes (bars indicate SD of the mean, n = 3). The asterisk indicates statistical difference p < 0.05.
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In conclusion, we have synthesized a new peptide bolaamphiphile, bis(N-␣-amido threonine)-1,5-pentane dicarboxylate, which formed microtubular assemblies at room temperature. It was found that the growth of the microtubes was pH dependent, and a pH value of six is optimum for growth of the microtubular structures. We have examined the potential of the microtubes as vehicles for drug release in the presence of ellagic acid. It was found that the release of EA was pH dependent. We also investigated the anti-bacterial properties of the microconjugates, and found that they were effective against E. coli and S. aureus bacteria, with the most potent activity in the presence of the Gram-negative E. coli. The microconjugates in this study were found to be biocompatible with mammalian cells, and hence may be used as potential drug delivery vehicles. Acknowledgments This work was conducted in part using equipment from the Queens College Facilities for Imaging, Cell and Molecular Biology. The authors thank Dr. Patrick Brock and Dr. Barbara Balestra from the Geology Department at Queens College for the use of the scanning electron microscope. These studies were funded in part by the Fordham Dean’s Office Summer Science Internship Program (S.B), Fordham University Faculty Research Grant (I.B.), and grants from the PSC-CUNY Research Award Program (K.F.). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.colsurfb.2012.02.031. References [1] C.-W. Kuo, J.-J. Lai, K.H. Wei, R. Chen, Adv. Funct. Mater. 17 (2007) 3707. [2] E. Katz, I. Willner, Angew. Chem. Int. Ed. 43 (2004) 6042. [3] (a) I. Willner, B. Willner, Nano Lett. 10 (2010) 3805; (b) A.J. Hilmer, M.S. Strano, Nat. Nanotechnol. 5 (2010) 481. [4] X. Yang, J.J. Grailer, I.J. Rowland, A. Javadi, S.A. Hurley, V.Z. Matson, D.A. Steeber, S. Gong, ACS Nano 4 (2010) 6805. [5] L. Zhang, J.M. Chan, F.X. Gu, J.-W. Rhee, A.Z. Wang, A.F. Radovic-Moreno, F. Alexis, R. Langer, O.C. Farokhzad, ACS Nano 2 (2008) 1696. [6] X. Feng, F. Lv, L. Liu, H. Tang, C. Xing, Q. Yang, S. Wang, ACS Appl. Mater. Interfaces 2 (2010) 2429. [7] B.-S. Kim, S.W. Park, P.T. Hammond, ACS Nano 2 (2008) 382. [8] H. Yuan, K. Luo, Y. Lai, Y. Pu, B. He, G. Wang, Y. Wu, Z. Gu, Mol. Pharm. 7 (2010) 953. [9] D. Gao, H. Xu, M.A. Philbert, R. Kopelman, Nano Lett. 8 (2008) 3320. [10] D. Ding, Z. Zhu, R. Li, X. Li, W. Wu, X. Jiang, B. Liu, ACS Nano 5 (2011) 2520. [11] E. Lee, J. Lee, I.H. Lee, M. Yu, H. Kim, S.Y. Chae, S. Jon, J. Med. Chem. 51 (2008) 6442. [12] A.N. Zelikin, ACS Nano 4 (2010) 2494. [13] K.Y. Cai, Y. Hu, Z. Luo, T. Kong, M. Lai, X.J. Sui, Y.L. Wang, L. Yang, L.H. Deng, Angew. Chem. Int. Ed. 47 (2008) 7479. [14] T.D. Leathers, Appl. Microbiol. Biotechnol. 62 (2003) 468. [15] K. Kuroda, K. Fujimoto, J. Sunamoto, K. Akiyoshi, Langmuir 18 (2002) 3780. [16] A. San Juan, M. Bala, H. Hlawaty, P. Portes, R. Vranckx, L.J. Feldman, D. Letourneur, Biomacromolecules 10 (2009) 3074. [17] D. Lu, X. Wen, J. Liang, Z. Gu, X. Zhang, Y. Fan, J. Biomed. Mater. Res. B. Appl. Biomater. 89 (2009) 177. [18] W.R. Gombotz, D.K. Pettit, Bioconjug. Chem. 6 (1995) 332. [19] R.J. Mumper, A.S. Hoffman, P.A. Puolakkainen, L.S. Bouchard, W.R. Gombotz, J. Control. Release 30 (1994) 241. [20] E.R. Edelman, E. Mathiowitz, R. Langer, M. Klagsbrun, Biomaterials 12 (1991) 619. [21] S. Wee, W.R. Gombotz, Proc. Int. Symp. Control. Release Bioact. Mater. 1 (1994) 730. [22] E.C. Downs, N.E. Robertson, T.L. Riss, M.L. Plunkett, J. Cell. Physiol. 152 (1992) 422. [23] G. Orive, A.M. Carcaboso, R.M. Hernández, A.R. Gascón, J.L. Pedraz, Biomacromolecules 6 (2005) 927. [24] G. Orive, R.M. Hernández, A.R. Gascón, R. Calafiore, T.M.S. Chang, P. De Vos, G. Hortelano, D. Hunkeler, I. Lacîk, A.M.J. Shapiro, J.L. Pedraz, Nat. Med. 2 (2003) 104. [25] P.U. Rokkaben, Ann. Med. 23 (1991) 109. [26] S.E. Burke, C.J. Barrett, Biomacromolecules 6 (2005) 1419.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Fabrication of ellagic acid incorporated self-assembled peptide microtubes and their applications Stacey N. Barnaby a , Karl R. Fath b,c , Areti Tsiola b,1 , Ipsita A. Banerjee a,∗ a b c
Department of Chemistry, Fordham University, 441 East Fordham Road, Bronx, NY 10458, United States Biology Department, Queens College, The City University of New York, 6530 Kissena Boulevard, Flushing, NY 11367, United States The Graduate Center, The City University of New York, 365 Fifth Avenue, New York, NY 10016, United States
a r t i c l e
i n f o
Article history: Received 1 December 2011 Received in revised form 20 February 2012 Accepted 21 February 2012 Available online 1 March 2012 Keywords: Peptides Bolaamphiphiles Self-assembly Ellagic acid Threonine
a b s t r a c t Ellagic acid (EA), a plant polyphenol known for its wide-range of health benefits was encapsulated within self-assembled threonine based peptide microtubes. The microtubes were assembled using the synthesized precursor bolaamphiphile bis(N-␣-amido threonine)-1,5-pentane dicarboxylate. The self-assembly of the microstructures was probed at varying pH. In general, tubular formations were observed at a pH range of 4–6. The formed microtubes were then utilized for fabrication with EA. We probed the ability of the microtubes as drug release vehicles for EA as well as for antibacterial applications. It was found that the release of EA was both pH and concentration dependent. The biocompatibility as well as cytotoxicity of the EA-fabricated microtubes was examined in the presence of mammalian normal rat kidney (NRK) cells. Finally the antibacterial effects of the EA incorporated peptide microtubes was examined against Escherichia coli and Staphylococcus aureus. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Over the past decade, there has been a tremendous surge in the development of nanomaterials to mimic and target biological systems for various biomedical as well as biosensor applications [1–3]. Specifically, a plethora of nanomaterials have been explored for their potential in drug delivery applications. For example, Yang et al. [4] designed polymer vesicles from heterofunctional amphiphilic triblock copolymers for targeted anticancer drug delivery. In a separate study, lipid–polymer hybrid nanoparticles were designed for targeting of mammalian cells or tissues [5]. Polymer nanoparticles consisting of PFO (polyfluorene) and poly(l-glutamic acid) conjugated with doxorubicin have also been fabricated [6]. Amphiphilic block copolymer micelles have been utilized as hydrophobic drugs within films [7]. Polymeric dendrimers have also been fabricated using poly(l-glutamic acid) [8], and its subsequent conjugation with DOX allowed for a targeted drug delivery system. Gao et al. [9] have designed nanoparticles based on polyacrylamide that could avoid being up taken by macrophages. Furthermore, Ding et al. [10] designed a system of cisplatin
∗ Corresponding author. Tel.: +1 718 817 4445; fax: +1 718 817 4432. E-mail address: [email protected] (I.A. Banerjee). 1 Current address: Department of Biological Sciences and Geology, Queensboro Community College, 222-05 56th Avenue, Bayside, NY 11364, United States. 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2012.02.031
(CDDP)-loaded gelatin/poly(acrylic acid) nanoparticles for transtissue drug delivery. In addition to various synthetic structures, natural drug delivery systems have also been investigated, such as chitosan [11–13], pullulan [14–17], alginates [18–23], and hyaluronic acid [24–26]. For example, chitosan-functionalized graphene oxide hybrid nanosheets were fabricated for the controlled release of Ibubrofen and 5-fluorouracil [27]. The biopolymer pullulan has been modified and its ability to self-assemble into hydrogels for drug delivery has been explored. Pullulan hydrogels have been found to serve as a coating for stents needed in local arterial therapy. Furthermore, pullulan/DOX conjugated nanoparticles were utilized as drug delivery vehicles for pH-controlled release. Several hyaluronic acid (HA) conjugates have also been synthesized and allowed to self-assemble into nanoparticles that were shown to target tumors [26]. Alginate based copolymers composed of 1–4 linked ␣-l-guluronic acid and -d-mannuronic acid have been functionalized with proteins and utilized for the controlled release of TGF- 1, basic fibroblast growth factor (bFGF), tumor necrosis factor receptor (TNFR), as well as angiogenesis factor, epidermal growth factor (EGF), and urogastrone. In a separate study, DOX was released from alginate embedded magnetic nanoheaters. Peptide nanotubes and microtubes are a relatively new class of biomaterials that have garnered much interest because of their facile self-assembly and ability to be functionalized, as well as their biocompatibility [28–33]. Nanotubes grown from cationic
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dipeptide (H Phe Phe NH2 ·HCl) precursors were efficiently bound to negatively charged single-stranded DNA (ssDNA) and utilized as delivery vehicles into HeLa cells [34]. Phenylalanine-based nanotubes were utilized in drug release studies for insulin [35]. Liu et al. [36] have shown that cyclic peptide nanotubes (CPNs) composed of (Trp-d-Leu)4-GLN-d-Leu can be utilized as drug delivery vectors for the antitumor drug 5-fluorouracil. Peptide nanotubes have also been utilized in other biological applications. For example, peptide nanotubes were coated with antibodies, and assembled onto device platforms, in order to detect pathogens [37,38]. In a recent study, we have demonstrated the ability of bis(N-␣amido-threonine)-1,3-propane dicarboxylate to self-assemble into microtubes, which could be efficiently conjugated with mucin [39]. In the work presented here, we have studied the formation of peptide nanotubules from a newly synthesized bolaamphiphile, bis(N-␣-amido-threonine)-1,5-pentane dicarboxylate, which was fabricated with the plant phytochemical ellagic acid (EA). Ellagic acid is a polyphenol found in fruits and nuts that possesses a multitude of biomedical properties [40–49]. Various drug delivery systems for EA have been reported, such as in situ gelling systems composed of chitosan-glycerol phosphate, composite films of chitosan/EA, as well as liposomes and poly(lactide-co-glycolide) (PLGA) and polycaprolactone (PCL) nanoparticles loaded with EA [50–53]. It has also been reported that EA and coenzyme Q10 co-encapsulated nanoparticles reduced lipid peroxidation and dyslipidemia in diabetic rats [54]. However, to our knowledge, EA has never been fabricated on peptide nanotubes or microtubes, neither has the potential biological applications of such nanoconjugates been explored. In this work, we have examined the encapsulation efficiency of EA in selfassembled threonine based peptide microtubes and investigated its in vitro release at varying pH values. We found that the release was concentration and pH dependent. Further, the materials were found to not only be biocompatible, by investigating the cellular toxicity of EA incorporated microtubes in the presence of NRK (normal rat kidney) cells, but it was also found that the bioconjugate not only retains, but also enhances, its antibacterial properties when compared to EA by itself. 2. Methods 2.1. Materials N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide (EDAC), lthreonine-methyl ester hydrochloride, pimelic acid, hydroxybenzotriazole (HOBT), triethylamine, DMF, methanol, acetone, and DMSO-d6 with 0.1% (v/v) TMS, were purchased from Sigma–Aldrich and used as received. Buffer solutions of various pH values, hydrochloric acid, ellagic acid, and sodium hydroxide pellets were purchased from Fisher Scientific. Normal rat kidney cells (American Type Culture Collection CRL-6509) were maintained in Dulbecco’s Minimal Essential Medium (Lonza, Walkersville, MD) supplemented with 10% fetal calf serum (Hyclone, Logan, UT), 100 units/ml penicillin, and 100 g/ml streptomycin. 2.2. Synthesis of bis(N-˛-amido-threonine)-1,5-pentane dicarboxylate The bolaamphiphile bis(N-␣-amido-threonine)-1,5 pentane dicarboxylate was synthesized via modification of previously established methods [55,56]. Pimelic acid (0.15 g, 0.94 mmol) was dissolved in DMF. To the solution, EDAC (0.05 g, 0.32 mmol) and 1-hydroxybenzotriazole (0.05 g, 0.37 mmol) were added. The mixture was cooled to 5 ◦ C and shaken for an hour. To the mixture, the protected threonine methyl ester hydrochloride (0.3 g, 1.8 mmol),
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and triethylamine (5 l) were added. The contents were stirred at 5 ◦ C for 24 h, at which point the solution was allowed to reach room temperature and the intermediate was rotary evaporated. The oily yellow intermediate obtained was placed on ice and treated with citric acid (0.15 M, 3 mmol), upon which the precipitate was washed with sodium bicarbonate (0.1 M, 2 mmol). The intermediate was then dissolved in DMF, and refluxed for 4 h at 85 ◦ C, in order to allow the protecting group to be removed via base hydrolysis in the presence of NaOH (0.1 M). Upon completion of base hydrolysis, the reaction mixture was rotary evaporated to obtain the product, which was washed with methanol and then recrystallized, to obtain a product that was orange/brown in appearance. The final product was confirmed by 1 H NMR and FTIR spcectroscopy. FTIR: = 3527( OH), 3361(N H amide), 1732 (C O, acid), 1652 (C O, amide). NMR: ı (8.2) (d, 2H, NH doublet); ı (4.5) (2H, m); ı (2.0) (4H, m); ı (1.51) (4H, m); ı (1.1) (6H, d). Elemental analysis (calculated): C 49.6; H 7.2; N: 7.82; O: 35.38. Observed: C: 49.1; H: 7.02; N: 8.01; O:35.87. 2.3. Self-assembly Individual stock solutions of the synthesized bolamphiphile in the range of 0.5–3.5 mM were prepared in buffer solutions ranging in pH values 4–6. In general, the solutions were allowed to assemble for 2–3 weeks. The formed assemblies were washed twice with deionized water and the morphologies were analyzed by TEM and SEM. The assemblies remained stable at room temperature for over 6 months. 2.4. Preparation of EA-loaded microtubules Varying concentrations of EA in the range of 0.02–0.2 mM were incubated with the nanotubes under ambient conditions for 24 h, at which point they were centrifuged and washed thrice in denionized water prior to analysis in order to remove any un-entrapped EA. 2.5. Measurement of entrapment efficiency In order to determine the amount of EA loaded into the peptide microtubes, the difference between the total amount of EA added into the microtubes and the amount of EA present in the supernatant after the loading process was calculated. The loaded microtubes were centrifuged under ambient conditions at 20 × g. The entrapment efficiency was calculated using the following equation: (X1 − X2 )/X1 × 100%, where X1 is the total concentration of EA and X2 is the concentration of the unloaded EA. Therefore, entrapment efficiency of EA in microtubules =
total amount of EA used − free EA in supernatant × 100 total amount of EA used
(1)
2.6. Drug release studies of EA The microtubes were loaded with EA at a concentration of 0.06 mM concentration and allowed to incubate for 2 h at room temperature, at which point the assemblies were centrifuged and the supernatant was removed. Absorbance spectroscopy readings were taken for 3 h at 37 ◦ C. In order to test the effect of pH, 500 l of the respective buffer solutions (pH 1–8) were added to the washed microtubes. Measurements were carried out as described above. 2.7. Cytotoxicity studies NRK cells were grown as monolayers in 25 or 75 cm2 culture flasks under a humidified atmosphere of 5% CO2 in air at 37 ◦ C.
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Cells were seeded at densities of 2 × 104 cells/ml and harvested when they reached 80–90% confluence. To study the effects of EA loaded microtubes on cell proliferation, cells were seeded at densities of 2 × 104 cells/ml in 12-well plates. EA loaded microtubes at a concentration of 0.6 mM (20 l) were added to each well. At the designated time point (24 h, 48 h, 4 days) after plating, cell proliferation was calculated. The medium from each well was collected. Live cells were harvested after applying 500 l 1× trypsin EDTA (Cellgro, Manassas, VA). Trypsinization was blocked with 500 l medium and the total volume was added to the centrifuge tubes. The cell suspension was centrifuged for 2 min and the pellet was resuspended in 500 l of medium. 45 l of the latter cell suspension was added to 45 l of 0.4% Trypan Blue Stain (Gibco) and the density of live and dead cells were calculated using a hemocytometer. All studies were done in triplicate. 2.8. Anti-bacterial studies Late log phase Escherichia coli (E. coli; DH5a strain) or Staphalococcus aureus (S. aureus) were diluted in LB medium to 104 colony forming units (cfu)/ml as determined using dilution plating. We evaluated the bacteriostatic properties of the EA loaded microtubes by determining the relative number of bacteria in the cultures by measuring the turbidity of 200-l aliquots of diluted bacteria plus 10 l of the EA loaded microtubes in 96-well plates using 630nm light in a BioTek ELx 800 microplate reader. Readings were taken every hour for 10 h as the bacteria were incubated in aerobic conditions at 37 ◦ C. To compare the growth characteristics of bacteria in the presence of different additives (with differing optical densities), the starting OD630 of each well was subtracted from subsequent readings from that well. As diluent controls, we added the appropriate volumes of ultrapure water in the absence of EA loaded microtubes to wells containing bacteria. Some wells contained only LB medium to assay for the growth of any contaminating environmental bacteria. To assay the bacteriocidal activity of the EA loaded microtubes, late log phase bacteria were diluted in LB medium to 104 cfu/ml and 200 l was mixed with 10 l of the EA loaded microtubes and incubated overnight at 37 ◦ C with shaking. After 24 h, the number of live bacteria was determined by serial dilution in LB broth not containing EA loaded microtubes, and by counting the number of colonies growing from these dilutions when spread on LB agar plates. The final dilutions (107 –108 ) greatly reduced the free EA loaded microtubes concentration so that any subsequent bacteria growth was essentially in the absence of EA loaded microtubes. Single live bacteria were identified by the formation of macroscopic bacterial colonies. Images of the plates were captured using a Kodak Gel Logic 440 imaging system and the grayscale values were inverted in Photoshop to make the colonies more visible. 2.9. Statistical analysis Statistical analysis of the data was done using student ‘t’ test; p < 0.05 was considered significant compared to controls. The results are expressed as mean ± standard deviation (SD). 2.10. Characterization 2.10.1. FTIR-spectroscopy Analyses were performed using Matteson Infinity IR equipped with DIGILAB, ExcaliBur HE Series FTS 3100 software. The washed samples were vacuum dried and mixed with KBr to form pellets. The measurements for the samples were carried out at 400–4000 cm−1 .
2.10.2. Absorbance spectroscopy Absorbance spectroscopy was carried out using a Thermo Scientific NanoDrop 2000. Readings were taken at a wavelength range of 190–800 nm. All samples were repeated in triplicates. 2.10.3. Transmission electron microscopy Samples were washed twice with nanopure water and air-dried onto carbon-coated copper grids for characterization by transmission electron microscopy (JEOL 1200 EX) operated at 100 kV. 2.10.4. Scanning electron microscopy The morphologies of the samples were further analyzed using SEM (Hitachi S-2600N) operated between 15 and 25 kV. A few drops of the sample solution were pipetted out onto MCE filter paper (5 mm), which were air-dried. Portions of the stained MCE filter paper were then cut out and mounted on SEM sample stubs with double sided tape and carbon coated using an SPI-module carbon coater before analysis. 3. Results and discussion 3.1. Microtube self-assembly Bolaamphiphiles have served as an attractive means for forming various supramolecular structures via self-assembly [57–60] such as nanofibers and nanotubes [61–65], vesicles [66,67], and crystals [68–70]. The fabrication of peptide assemblies has proven advantageous because of their ability to mimic biological systems [71–77]. Non-covalent interactions such as – stacking interactions, as well as hydrophobic interactions and intra and inter-molecular hydrogen bonding play a key role in the formation of supramolecular assemblies. Although peptide microtubes are synthetic-derived molecules, they form similar supramolecular structures as those found in nature [78]. In previous work, we have reported the self-assembly of the bolaamphiphile HOOC Thr NH CO (CH2 )3 NH CO Thr COOH [28], which formed a high propensity of tubular structures with a diameter of 500 nm–2 m in the pH range of 4–6, whereas at higher pH values, globular structures were formed. In this study, we have increased the alkyl spacer between the head groups to a pentyl group, forming HOOC Thr NH CO (CH2 )5 NH CO Thr COOH, as shown in Fig. 1a. Fig. 1b shows the proposed inter and intramolecular hydrogen bonding interactions between the carboxyl groups, the NH and C O groups as well as the hydroxyl groups between the bolaamphiphiles that result in the formation of the supramolecular structures. We examined the self-assembly at a pH range of 4–6. At a pH value of 4, we observed a relatively low yield with a few short, truncated nanotubular structures (Fig. 2a). At a pH value of 5, short proto-fibrillar structures were observed (Fig. 2b). However, at a pH value of 6, we observed a high yield of tubular structures, with an average diameter of 500 nm–1 m (Fig. 2c). The microtubes appear to be smooth on the surface, and are seen to form an interlocking mesh. Previous studies examining the growth of microtubes have shown that the self-assembly occurred via both acid-anion dimers and hydrogen bonding networks [79]. We expect similar forces to be involved in the assembly. When analyzing the effect of the alkyl chain length on self-assembly, it is seen that in terms of diameter, the chain length has little effect. However it appears that increasing the chain length leads to higher yields of the tubular structures, and results in a mesh of microtubes. The assemblies formed due to extensive hydrogen bonding interactions between the hydroxyl group and the backbone of the molecule, as well as between the C O and NH groups, aids in the self-assembly of the molecule [80] thus allowing for the formation of tubular structures. This is consistent with cyclic peptide assemblies, where nanotubes are formed
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Fig. 3. FTIR spectrum of the self-assembled microtubes formed at pH 6.
Fig. 1. (a) Chemical structure of the synthesized bolaamphiphile; (b) threedimensional model showing the proposed hydrogen bonding interactions between the bolaamphiphiles.
by spontaneous stacking of molecules on top of one another, which results in hollow nanotube structures [81]. Further, the addition of two alkyl groups allows for enhanced hydrophobic interactions. The mechanism of formation of the tubules was further explored using FTIR-spectroscopy. 3.2. FTIR-spectroscopy In order to determine the underlying forces allowing for the selfassembly of the microstructures, we analyzed FTIR spectra of the self-assembled microtubes formed. As shown in the supplementary data, Fig. 3 shows the FTIR spectrum of the self assembled microtubes in the range of 1300–2000 cm−1 . In the C O region, two peaks were observed, one at 1730 cm−1 due to hydrogen bonded carboxylic groups and another at 1645 cm−1 due to the amide I peak after self-assembly. The peak at 1730 cm−1 is significantly quenched compared to the as synthesized bolaamphiphile due to H-bonding interactions, while the amide I peak is shifted compared to the as synthesized bolaamphiphile which was seen at 1652 cm−1 (data not shown). These results indicate the strong role
that the carboxyl and amide groups play in the self-assembly. The short peak seen at 1558 cm−1 was assigned to asymmetric COO− vibration indicating that at pH 6, some of the carboxyl groups are deprotonated and thus acid-anion dimers are formed. An additional peak at 1539 cm−1 was also observed due to amide II N H bending vibration after self-assembly. A strong peak at 1420 cm−1 was also observed due to CH3 bending vibrations from the threonine moieties. Furthermore, spectral changes were observed in the OH stretching region, where we observed an intense broad peak at 3454 cm−1 after self-assembly due to hydrogen bonding interactions (range not shown in figure). 3.3. EA entrapment Peptide conjugated nanoparticles [82], vesicles [83], lipid–polymer hybrids [84], conjugated polymer nanoparticles, vesicles and micelles [85,6,4,86,87], gold nanorods [88], and gels [89] are commonly employed as drug delivery vehicles. Peptide-based systems represent an ingenious option for delivering drugs [90], because they are biocompatible, and they can be tailored for specific environments through functionalization methods. Herein, we explored the potential use of Thr based microtubes to serve as a carrier of EA. It was found that loading of EA into the microtubes is concentration dependent as shown in Fig. 4 and that at a concentration range of 0.06–0.1 mM, the entrapment was significantly higher (p < 0.05) in comparison to other concentrations. We found that the encapsulation efficiency is optimal at 0.06 mM, such that the EA is completely encapsulated within the microtubes. Fig. 5 shows the entrapment of EA within the microtubes. Fig. 5a
Fig. 2. Growth of nano and microtubular structures (a) at pH 4 (scale bar = 500 nm), (b) pH 5 (scale bar = 2 m) and (c) pH 6 (scale bar = 2 m).
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Fig. 4. EA entrapment efficiency at varying concentrations. All experiments were carried out in triplicate. Bars indicate SD of the mean (n = 3). The asterisks represent statistical difference p < 0.05.
represents the microtubular structure, without the incorporation of EA, while Fig. 5b shows the incorporation after 0.06 mM concentration of EA as represented by the darker sections of the tubules [91]. At higher concentrations (Fig. 5c), (0.09 mM), EA was found to deposit in the form of aggregates on the outside of the microtubes. EA has been known to form discotic liquid crystal assemblies upon aggregation in water [92]. Thus, it appears that at low concentrations, in the presence of the threonine based peptide microtubes, EA efficiently inserts into the tubules by capillary action. However, at higher concentrations, EA appears to aggregate forming assemblies on its own, hence encapsulation is relatively lesser. Thus, in general, for drug release studies, tubules encapsulated with EA at 0.06 mM concentration were selected. 3.4. Release of EA from microtubes We examined the release of EA at varying pH values. As shown in Fig. 6, the release of EA from the microtubes was found to be pH dependent. At low pH, (pH 1) we observed that the release was slowest, while under near neutral conditions (pH 6 and 7), the drug released at a higher steady rate and the % release was found to be 50% after over 2 h. Under basic conditions, EA released fastest and after 2 h, we observed a decrease in the percentage release. We attribute the different rates of EA release based on pH to be a result of the binding interactions between EA and the microtubes. Under highly acidic conditions (pH 1), EA is strongly bound to the microtubes because of the strong hydrogen bonding as EA is completely protonated under those conditions. However as pH increases, hydrogen bonding interactions are less due to
Fig. 6. Release of EA from the microtubes at varying pH. All experiments were carried out in triplicate. Error bars are based on three parallel samples and represent standard deviations.
deprotonation process of EA [93] resulting in faster release and under basic conditions, due to the negative charge of EA, it is likely that there is repulsion between the EA molecules, leading to higher rate of release. 3.5. Anti-bacterial studies EA has well characterized anti-bacterial properties against E. coli [94], oral bacteria [95], Propionibacterium acnes, S. aureus, and Staphylococcus epidermidis [96]. We wished to determine whether the incorporation of microtubes fabricated with EA could enhance its antibacterial properties. For antibacterial studies, 0.1 mM EA was incorporated onto the microtubes, because at those concentrations EA is present on the outside surface of the microtubes as well, and hence would allow for higher contact with bacteria. To assay the bacteriostatic properties of the microtubes, we added the microconstructs to freshly-diluted stationary phase cultures containing 104 cfu/ml of either the Gram-negative bacteria E. coli or the Gram-positive bacteria S. aureus. The cultures were incubated at 37 ◦ C and the relative number of bacteria in the cultures was determined by measuring the turbidity (optical density with 630nm light) of the cultures every hour for a total of 10 h. We found that, after the typical lag phase with little detected growth, both E. coli and S. aureus incubated in the solvent control wells (ultrapure water) grew rapidly during the course of the experiment. The growth of Gram-negative E. coli was inhibited 63% by EA and 84% by EA integrated with microtubes. The percent inhibition was
Fig. 5. TEM images of EA loaded microtubes. (a) At 0 mM concentration of EA; (b) at 0.06 mM concentration of EA; and (c) at 0.09 mM concentration of EA.
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Fig. 7. Growth curves of E. coli (a) and S. aureus (b) in LB medium inoculated with 104 cfu/ml of bacteria in 96-well plates in the presence of EA or microtubes entrapped with EA. The extent of growth was determined by change in turbidity using a plate reader and 630-nm light. The solvent control (water) indicates the growth of bacteria without additives. Error bars are based on three parallel samples and represent standard deviations; and (c) bactericidal properties of EA-fabricated microtubes. Bacteria (E. coli and S. aureus) at an initial concentration of 104 cells/ml were incubated with the EA, microtubes loaded with EA or water (solvent control) for 24 h.
determined by the differences in culture turbidity at the 10-h time point (Fig. 7a). By comparison, the growth of Gram-positive S. aureus was not significantly inhibited by EA and 50% by EA incorporated microtubes (Fig. 7b). Control wells containing Luria Broth alone did not change in turbidity during the incubation period, which suggested that there was no contamination from environmental bacteria. We found that E. coli was more susceptible than S. aureus to growth-inhibition by EA. Further, the entrapment of EA into peptide microtubes enhanced its bacteriostatic properties. This is most likely because threonine on its own has been reported to display antibacterial properties [97,98], thus a blend of threonine based microtubes with EA, enhanced the antibacterial effects. In general, turbidity-based assays are commonly used to assess compounds for antibacterial properties. While these assays are relatively rapid and easy to perform, they are limited in that a dead bacterium can also scatter light. Further, usually these assays follow bacteria growth in the continual presence of the treatment agent. Thus we also determined whether our treated bacteria at the end of the incubation period were alive and capable of growth in the absence of EA bound microtubes. Therefore we incubated bacteria as before in the turbidity assays, diluted the treated bacteria in LB (107 - or 108 -fold) and then determined the number of viable bacteria on LB agar plates (Fig. 7c). We found that EA alone had relatively less bacteriocidal activity for both E. coli and S. aureus, but EA bound peptide microtubes decreased the number of alive E. coli by 96% and the number of alive S. aureus by 66% after a 24-h treatment as shown in Table SI (supplementary information). These results indicate that threonine microtubes integrated with EA enhances bacteriocidal activity.
indicated that the number of cells had quadrupled and were significantly higher (p < 0.05) in comparison with the initial number of cells. These results indicate that the EA loaded microtubes are non-toxic to mammalian cells and can be potentially utilized as a drug delivery system.
3.6. Biocompatibility studies To determine whether the EA incorporated microtubes could be utilized for potential biomedical applications, we investigated their biocompatibility by conducting in vitro cytotoxicity and cellproliferation studies with cultured NRK cells. Trypsinized cells were allowed to attach and spread in 12-well plates and 20 l of microtubular solutions were added to the culture media. We observed no cell lysis or changes in cell morphology in the presence of EA loaded microtubes. The cells continued to be well spread when bound to the microtubes (Fig. 8a). Cytotoxicity analysis using regular MTT assays revealed that the cells are capable of dividing and growing in the presence of the EA loaded microtubes (Fig. 8b). We found that the number of cells more than doubled after 24 h. We also examined the samples after a period of 96 h and the results
Fig. 8. (a) Photomicrograph of NRK cells after 48 h in the presence EA loaded microtubes (concentration of EA = 0.09 mM); (b) cytotoxicity studies showing the growth and proliferation of NRK cells over time in the presence of EA loaded microtubes (bars indicate SD of the mean, n = 3). The asterisk indicates statistical difference p < 0.05.
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In conclusion, we have synthesized a new peptide bolaamphiphile, bis(N-␣-amido threonine)-1,5-pentane dicarboxylate, which formed microtubular assemblies at room temperature. It was found that the growth of the microtubes was pH dependent, and a pH value of six is optimum for growth of the microtubular structures. We have examined the potential of the microtubes as vehicles for drug release in the presence of ellagic acid. It was found that the release of EA was pH dependent. We also investigated the anti-bacterial properties of the microconjugates, and found that they were effective against E. coli and S. aureus bacteria, with the most potent activity in the presence of the Gram-negative E. coli. The microconjugates in this study were found to be biocompatible with mammalian cells, and hence may be used as potential drug delivery vehicles. Acknowledgments This work was conducted in part using equipment from the Queens College Facilities for Imaging, Cell and Molecular Biology. The authors thank Dr. Patrick Brock and Dr. Barbara Balestra from the Geology Department at Queens College for the use of the scanning electron microscope. These studies were funded in part by the Fordham Dean’s Office Summer Science Internship Program (S.B), Fordham University Faculty Research Grant (I.B.), and grants from the PSC-CUNY Research Award Program (K.F.). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.colsurfb.2012.02.031. References [1] C.-W. Kuo, J.-J. Lai, K.H. Wei, R. Chen, Adv. Funct. Mater. 17 (2007) 3707. [2] E. Katz, I. Willner, Angew. Chem. Int. Ed. 43 (2004) 6042. [3] (a) I. Willner, B. Willner, Nano Lett. 10 (2010) 3805; (b) A.J. Hilmer, M.S. Strano, Nat. Nanotechnol. 5 (2010) 481. [4] X. Yang, J.J. Grailer, I.J. Rowland, A. Javadi, S.A. Hurley, V.Z. Matson, D.A. Steeber, S. Gong, ACS Nano 4 (2010) 6805. [5] L. Zhang, J.M. Chan, F.X. Gu, J.-W. Rhee, A.Z. Wang, A.F. Radovic-Moreno, F. Alexis, R. Langer, O.C. Farokhzad, ACS Nano 2 (2008) 1696. [6] X. Feng, F. Lv, L. Liu, H. Tang, C. Xing, Q. Yang, S. Wang, ACS Appl. Mater. Interfaces 2 (2010) 2429. [7] B.-S. Kim, S.W. Park, P.T. Hammond, ACS Nano 2 (2008) 382. [8] H. Yuan, K. Luo, Y. Lai, Y. Pu, B. He, G. Wang, Y. Wu, Z. Gu, Mol. Pharm. 7 (2010) 953. [9] D. Gao, H. Xu, M.A. Philbert, R. Kopelman, Nano Lett. 8 (2008) 3320. [10] D. Ding, Z. Zhu, R. Li, X. Li, W. Wu, X. Jiang, B. Liu, ACS Nano 5 (2011) 2520. [11] E. Lee, J. Lee, I.H. Lee, M. Yu, H. Kim, S.Y. Chae, S. Jon, J. Med. Chem. 51 (2008) 6442. [12] A.N. Zelikin, ACS Nano 4 (2010) 2494. [13] K.Y. Cai, Y. Hu, Z. Luo, T. Kong, M. Lai, X.J. Sui, Y.L. Wang, L. Yang, L.H. Deng, Angew. Chem. Int. Ed. 47 (2008) 7479. [14] T.D. Leathers, Appl. Microbiol. Biotechnol. 62 (2003) 468. [15] K. Kuroda, K. Fujimoto, J. Sunamoto, K. Akiyoshi, Langmuir 18 (2002) 3780. [16] A. San Juan, M. Bala, H. Hlawaty, P. Portes, R. Vranckx, L.J. Feldman, D. Letourneur, Biomacromolecules 10 (2009) 3074. [17] D. Lu, X. Wen, J. Liang, Z. Gu, X. Zhang, Y. Fan, J. Biomed. Mater. Res. B. Appl. Biomater. 89 (2009) 177. [18] W.R. Gombotz, D.K. Pettit, Bioconjug. Chem. 6 (1995) 332. [19] R.J. Mumper, A.S. Hoffman, P.A. Puolakkainen, L.S. Bouchard, W.R. Gombotz, J. Control. Release 30 (1994) 241. [20] E.R. Edelman, E. Mathiowitz, R. Langer, M. Klagsbrun, Biomaterials 12 (1991) 619. [21] S. Wee, W.R. Gombotz, Proc. Int. Symp. Control. Release Bioact. Mater. 1 (1994) 730. [22] E.C. Downs, N.E. Robertson, T.L. Riss, M.L. Plunkett, J. Cell. Physiol. 152 (1992) 422. [23] G. Orive, A.M. Carcaboso, R.M. Hernández, A.R. Gascón, J.L. Pedraz, Biomacromolecules 6 (2005) 927. [24] G. Orive, R.M. Hernández, A.R. Gascón, R. Calafiore, T.M.S. Chang, P. De Vos, G. Hortelano, D. Hunkeler, I. Lacîk, A.M.J. Shapiro, J.L. Pedraz, Nat. Med. 2 (2003) 104. [25] P.U. Rokkaben, Ann. Med. 23 (1991) 109. [26] S.E. Burke, C.J. Barrett, Biomacromolecules 6 (2005) 1419.
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