Journal of Science: Advanced Materials and Devices (2017) 178e182 Contents lists available at ScienceDirect Journal of Science: Advanced Materials and Devices journal homepage: www.elsevier.com/locate/jsamd Original Article Spheroidal and nanocrystal structures created from carbodiimide crosslinking reaction with RADA16 Jorge Monreal*, Robert Hyde Department of Physics, University of South Florida, Tampa, FL 33620, USA a r t i c l e i n f o a b s t r a c t Article history: Received May 2017 Received in revised form 16 May 2017 Accepted 17 May 2017 Available online 20 May 2017 RADA16 is a widely studied polypeptide known for its ability to self-assemble into b-sheets that form nanofibers Here we show that it is possible to self-crosslink the molecule via 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (EDC) as aqueous solutions The product results in a mix of nanocrystals and near micron-size spherules SEM and TEM pictures provide a view of the structures and nano tracking analysis gives their size distributions FTIR analysis provides evidence for the existence of a crosslinking reaction © 2017 The Authors Publishing services by Elsevier B.V on behalf of Vietnam National University, Hanoi This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) Keywords: RADA16 Crosslinking EDC Spherules Introduction The ability of RADA16 to self-assemble into nanofibers has been studied extensively for use as cell culture scaffolding and drug delivery [1e3] It is known that RADA16 conforms into b-sheets and self-assembles into nanofibers with diameters in the range of 3e10 nm [4,5] forming hydrogels when dissolved in water Selfassembly produces two distinctive sides: one hydrophobic due to alanine (A), the other hydrophilic due to arginine (R) and aspartic acid (D) [6] One group has crosslinked a peptide made of a combination of RADA16-Bone morphogenic protein with poly(lactic-coglycolic acid) via EDC for bone regeneration [7] Here we study selfcrosslinking of the RADA16 peptide via EDC which could lead to an entirely new range of possible designed peptides with a myriad of functional characteristics We find the formation of nanocrystals as a result of the crosslinking reaction The methods for nanocrystal formation described here, particularly for drug delivery applications, are highly desirable as they constitute simple wet chemistry reactions at room temperature Such simplicity seems advantageous to current nanocrystal production methods such as milling, precipitation with colloidal stabilization, and homogenization for medical and clinical applications [8] * Corresponding author E-mail address: jmonreal@alum.mit.edu (J Monreal) Peer review under responsibility of Vietnam National University, Hanoi RADA16 studied here is acetylated with an amine N-terminus Ac-[RADA]4-NH2 The arginine and aspartic acid amino acid residues are positively and negatively charged, respectively Side chains in aspartic acid provide carboxyl groups on the hydrophilic side available for crosslinking by a carbodiimide reaction mechanism and the N-terminus primary amine is also available for crosslinking It is not expected that the guanidinium group in arginine will crosslink EDC is a zero-length crosslinker which reacts with carboxyl groups to form amine reactive intermediates These react with amino groups to form peptide bonds An N-substituted urea forms when the intermediate fails to react with the amine [9] N-acylurea could also form as a side reaction during crosslinking However, the reaction is limited to carboxyls in hydrophobic regions of a protein or polypeptide Given that alanine, which forms the hydrophobic region of RADA16 and only contains eCH3, the side reaction was not expected to occur here Experimental RADA16 was obtained from 3D Matrix as a lyophilized powder prepared by exchanging TFA for HCl [10] The arginine had a chlorine counterion and the aspartic acid was protonated It was reconstituted in deionized water at a nominal 2.0% (w/v) to give a solution with pH z 2e3 EDC was obtained from TCI America (USA) as a hydrochloride with a MW ¼ 191.70 g molÀ1 and of 98.0% purity It was dissolved in http://dx.doi.org/10.1016/j.jsamd.2017.05.008 2468-2179/© 2017 The Authors Publishing services by Elsevier B.V on behalf of Vietnam National University, Hanoi This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) J Monreal, R Hyde / Journal of Science: Advanced Materials and Devices (2017) 178e182 deionized water to obtain a nominal 20% (w/v) solution with pH z 7.68 as measured with a Sensorex polymer electrode The RADA16 e EDC reaction proceeded as follows To 100 mL of 2% (w/v) RADA16 gel we added 50 mL of 20% (w/v) EDC The mixture was shaken vigorously for approximately on a Vortex Genie mixer at setting then placed in a lab bench Fisher-Scientific centrifuge for The mixture sat for 24 h and the resulting solution had a pH ¼ 3.53 Mixing was carried out at 22 C Sample preparation for viewing under SEM consisted of 250 mL of 70% (w/v) ethanol added to reactant mixture Approximately 100 mL of the solution was placed on a coverglass that had previously been cleaned by immersion in ethanol and sonicated for 10 The product solution on the coverglass was evaporated for about on top of a hotplate set at 90 C A 10 nm layer of AuePd was deposited on top of the dried RADA16/EDC film with a Denton sputtering system Preparation of samples for viewing under TEM required nominal dilution factors (DF) ¼ 1000 Samples were vacuum dried at 45 C and negatively died Nanoparticle Tracking Analysis (NTA) equipment, Malvern Instruments Nanosight LM10 with capability of tracking particles in the size range of e2000 nm, required volumes in the range of 0.8e1 mL We used DF ¼ 1000 in deionized water to study the distribution of particle sizes in our sample FTIR studies on RADA16 were conducted at room temperature, 22 C on a Jasco FT/IR 4100, at cmÀ1 resolution, equipped with a multi-reflection Attenuated Total Reflectance (ATR) accessory equipped with a ZnSe crystal Spectra for EDC and RADA16 þ EDC product were measured on a Bruker Vertex 70 spectrometer with a single pass ATR accessory 179 A JEOL JSM-63900LV SEM equipped with an energy-dispersive X-ray spectroscopy (EDS) detector from Oxford Instruments gave SEM pictures and material composition data TEM data was obtained in collaboration with the Microscopy Core Facility Results and discussion Fig 1a shows a SEM picture of the resulting product from a reaction between RADA16 hydrogel and EDC prepared as detailed in the Experimental section, both previously dissolved in deionized water Nanoparticles of approximately 70e80 nm are readily visible and randomly dispersed throughout the film surface To rule out contamination from NaCl or other types of salts, we measured elemental X-ray dispersion with the EDS detector on a mm  mm field of view at four different sample locations In addition to elements typical of organic compounds EDS measurements showed significant traces of chlorine No other elements were found We attribute the presence of chlorine to counterions in the RADA16 arginine amino acid residues as well as the hydrochloride from EDC Fig 1b shows the sample viewed under TEM at 28.7 kX magnification and exhibits a similar nanoparticle monodispersity as seen under SEM It is readily apparent that nanoparticles appear to be crystalline in nature and randomly dispersed Fig 1c shows a nanocrystal at 824 kX TEM magnification This particle appears to have either an orthorhombic or tetragonal crystal structure Studies of additional TEM pictures, led us to believe there is a preponderance of orthorhombic nanocrystal structures Mixed with the nanocrystals, and somewhat hidden in Fig 1b are larger sized spherules Fig 1d presents these spherules, which in general tend to be >0.5 mm Interestingly, one could also observe the presence of Fig (a) SEM picture of 2% w/v RADA16 reacted with 20% EDC at 10 kX Monodisperse particles seen throughout sample EDS showed presence of Cl and organic compounds only; (b) Same sample viewed under TEM at 28.7 kX TEM Monodisperse orthorhombic nanocrystals visible; (c) TEM close-up view of a z70 nm nanocrystal at 824 kX; (d) TEM view of spherules at 10.9 kX Crosslinked RADA16 nanofibers in process of agglomeration are visible in the middle of picture and lower left corner 180 J Monreal, R Hyde / Journal of Science: Advanced Materials and Devices (2017) 178e182 crosslinked RADA16 nanofibers in process of agglomeration in Fig 1d at the middle and lower left corner of the picture To ensure the nanocrystals were not due to unreacted EDC, we measured particle distribution of the reactant using NTA on a sample at DF ¼ 1000 in deionized water The same dilution sample was viewed under TEM at 78.7 kX magnification, Fig 2a TEM shows that there are “plate-like” square particles or flakes within the EDC solution NTA showed particles to be in the range of 46e300 nm, and less probable sizes >500 nm, Fig 2b A visual comparison of Fig 2a with Fig 2c, which shows product nanocrystals, reveals different crystal morphologies Whereas crystals in EDC are “plate-like” flakes at various stages of dissolution, product nanocrystals are solid, wellformed orthorhombic-like structures NTA quantified size distributions of nanocrystal and spherule mix in product solution Fig 2d presents data obtained for one set of measurements from a sample of product solution diluted in deionized water at DF ¼ 1000 and measured at 25 C Particles in the 100e600 nm range are likely to be nanocrystals Sizes >900 nm are likely spherules Indeed, in a representative area covered with spherules, 2e, a manual count of N ¼ 13 spherules yielded an average size D ¼ 987 nm with standard error ¼ 59 nm The 95% confidence interval in this region is [859, 1115] nm Therefore, we attribute the size distribution peaking at 902 nm in Fig 2d to spherules Such distribution of sizes did not appear in NTA measurements of EDC To gather further evidence that the spherules and nanocrystals were not just a result of desegregated RADA16 hydrogel and unreacted EDC, respectively, FTIR measurements were conducted FTIR measurements were obtained for RADA and EDC alone as well as RADA ỵ EDC after reaction Fig 3a is an FTIR plot of RADA16 hydrogel prior to reaction with EDC It shows the distinctive b-sheet peak at 1621 cmÀ1 [11] Fig 3b shows FTIR data in magenta for EDC prior to reaction with EDC Of particular importance are the peaks at 2130 and 1702 cmÀ1 as these distinctive peaks for EDC disappear after the crosslinking reaction with RADA16 The peak at 2130 cmÀ1 is attributed to the N]C]N bonds of EDC [12] We attribute the peak at 1702 cmÀ1 to stretching of the cumulated C]N bonds since Fig (a) TEM view of plate-like crystals present in 20% EDC solution at 78.7 kX; (b) NTA measurement of 20% EDC, DF ¼ 1000, in deionized water measured at 21 C; (c) TEM view of 2% RADA16 þ 20% EDC solution at 78.7 kX; (d) NTA measurement of 2% w/v RADA16 ỵ 20% EDC solution, DF ẳ 1000, in deionized water at 25 C; (e) TEM view of spherules from different location than Fig 1d at 28.7 kX; (f) Spherule size distribution statistics of (e) as measured with the TEM measuring tool J Monreal, R Hyde / Journal of Science: Advanced Materials and Devices (2017) 178e182 181 Fig (a) FTIR spectra of 2% RADA16 The significant peak at 1636 cmÀ1 is due to the stable b-sheets (b) Overlaid FTIR spectra of unreacted 20% EDC (magenta) and RADA16 ỵ EDC (black) after reaction N]C]N bonds in EDC produce two distinctive peaks at 2130 and 1702 cmÀ1, respectively, which disappear after crosslinking reaction C is an sp hybridized carbon It is expected that these bonds would no longer be present after reaction of the primary amine with the unstable intermediate O-acylisourea That is in fact what we found Fig 3b shows the RADA16 ỵ EDC product in black Peaks at 2130 and 1702 cmÀ1 are conspicuously absent, confirming that a crosslinking reaction indeed took place The b-sheet peak disappears after crosslinking Evidently, the stable b-sheet structure of RADA16 has been disrupted by the crosslinking mechanism FTIR data, thus, lends support to the existence of a proposed crosslinking reaction of RADA16 activated by EDC It is likely that crosslinking proceeds through EDC activation of the carboxyl groups present in the aspartic acid amino acid residues The unstable, amine-reactive O-acylisourea intermediate that results from activation of the carboxyl groups then reacts with available primary amines Primary amines available for reaction either come from the N-terminus or the guanidinium group of the arginine subgroup 182 J Monreal, R Hyde / Journal of Science: Advanced Materials and Devices (2017) 178e182 While the guanidinium cation is highly stable in an aqueous solution, reactions stemming from a combination of both the N-terminus and possibly guanidinium groups cannot be ruled out Conclusion We have provided evidence that crosslinking in RADA16 is activated by EDC It is likely that crosslinking proceeds through EDC activation of the carboxyl groups present in the aspartic acid amino acid residues reacting with primary amines either from the N-terminus and possibly the guanidinium group of the arginine subgroup The reaction produces nanocrystals and micron-sized spherules It is not immediately clear whether or not nanocrystal size can be tuned with the methods used here Additional studies must be conducted However, there is a possibility that spherules can be tuned with either pH, the degree of polymerization, or counterion choice Studies of hydrophobic polyelectrolytes have shown that ionic charge and solvent quality dictate the extent of necklace-like beading of polyelectrolyte chains [13] Control of spherule formation via solvent manipulation could lead to several medical applications Further studies are required to understand the mechanisms leading to crosslinking as well as formation of nanocrystals and spherules Acknowledgements This work has been supported in part by the Microscopy Core Facility in the Department of Integrative Biology at the University of South Florida We also like to thank Dr Haynie for very useful comments References [1] K Hamada, M Hirose, T Yamashita, H Ohgushi, Spatial distribution of mineralized bone matrix produced by marrow mesenchymal stem cells in self-assembling peptide hydrogel scaffold, J Biomed Mater Res Part A 84 (2008) 128e136 [2] A.L Sieminski, C Semino, H Gong, R Kamm, Primary sequence of ionic selfassembling peptide gels affects endothelial cell adhesion and capillary morphogenesis, J Biomed Mater Res Part A 87 (2008) 494e504 [3] C Cunha, S Panseri, O Villa, D Silva, F Gelain, 3D culture of adult mouse neural stem cells within functionalized self-assembling peptide scaffolds, Int J Nanomed (2011) 943e955 [4] A.R Cormier, C Ruiz-Orta, R.G Alamo, A.K 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EDC prior to reaction with EDC Of particular importance are the peaks at 2130 and 1702 cmÀ1 as these distinctive peaks for EDC disappear after the crosslinking reaction with RADA16 The peak at 2130... 20% EDC (magenta) and RADA16 þ EDC (black) after reaction N]C]N bonds in EDC produce two distinctive peaks at 2130 and 1702 cmÀ1, respectively, which disappear after crosslinking reaction C is an... either from the N-terminus and possibly the guanidinium group of the arginine subgroup The reaction produces nanocrystals and micron-sized spherules It is not immediately clear whether or not nanocrystal