Spheroidal and Nanocrystal Structures From Carbodiimide Crosslinking Reaction With RADA16
SSpheroidal and Nanocrystal Structures From CarbodiimideCrosslinking Reaction With RADA16
Jorge Monreal ∗ and Robert Hyde Department of Physics, University of South Florida, Tampa, Florida, USA
Abstract
RADA16 is a widely studied polypeptide known for its ability to self-assemble into β -sheetsthat form nanofibers. Here we show that it is possible to self-crosslink the molecule via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) as aqueous solutions. The productresults in a mix of nanocrystals and near micron-size spherules. SEM and TEM pictures providea view of the structures and nano tracking analysis give their size distributions. FTIR analysisprovides evidence for the existence of a crosslinking reaction. Keywords: RADA16, Crosslinking, EDC, Spherules ∗ [email protected] a r X i v : . [ c ond - m a t . s o f t ] A ug he ability of RADA16 to self-assemble into nanofibers has been studied extensivelyfor use as cell culture scaffolding and drug delivery Zhao and Zhang [1], Hamada et al. [2], Sieminski et al. [3], Gelain et al. [4], Cunha et al. [5]. It is known that RADA16conforms into β -sheets and self-assembles into nano-fibers with widths in the range of 3-10nm in diameter [6–9] forming hydrogels when dissolved in water. Self -assembly producestwo distinctive sides: one side hydrophobic due to alanine, the other hydrophilic due toarginine and aspartic acid [9]. At least one study has crosslinked a peptide made of acombination of RADA16-Bone morphogenic protein with poly(lactic-co-glycolic acid) viaEDC for bone regeneration [10]. Here we study self-crosslinking of the RADA16 peptide viaEDC, which could lead to an entirely new range of possible designed peptides with a myriadof functional characteristics. FIG. 1.
Acetylated RADA16 with N-terminus [11].
RADA16 studied here is acetylated with an amine N-terminus Ac-[RADA] -NH . Thearginine (R) and aspartic acid (D) amino acid residues are positively and negatively charged,respectively. There are a total of 17 peptide bonds: 15 between RADA amino groups; oneat the acetyl end; one at the primary amine. It contains 17 C=O and 16 N-H bonds inthe backbone. A total of nine sp hybridized C bonds stemming from the acetyl end andalanine (A) amino acid subgroups are also present in RADA16. Side chains in aspartic acidprovide a total of four carboxyl groups on the hydrophilic side available for crosslinkingby a carbodiimide reaction mechanism. The N-terminus primary amine is available forcrosslinking. In addition, there are four amines in the arginine guanidinium group that couldpossibly take part of a crosslinking reaction. EDC is a zero-length crosslinker which reactswith carboxyl groups to form amine reactive intermediates. These react with amino groups toform peptide bonds. An N-substituted urea forms when the intermediate fails to react withthe amine [12]. 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. Given2hat alanine, which forms the hydrophobic region of RADA16 and only contains -CH , theside reaction was not expected to occur here.Figure 1a shows an SEM picture of the resulting product from a reaction betweenRADA16 hydrogel and EDC prepared as detailed in the Experimental section, both pre-viously dissolved in deionized water. Nanoparticles of approximately 70-80 nm are readilyvisible and randomly dispersed throughout the film surface. To rule out contamination fromNaCl or other types of salts, we measured elemental X-ray dispersion with the EDS detectoron a 1 µ m x 1 µ m field of view at four different sample locations. In addition to elementstypical of organic compounds such as carbon, oxygen, nitrogen and hydrogen, EDS mea-surements showed significant traces of chlorine. No other elements were found. We attributethe presence of chlorine to counterions in the RADA16 arginine amino acid residues as wellas the hydrochloride from EDC. Higher magnifications of nanoparticles resulted in picturesthat were very fuzzy due to surface charge build-up. TEM pictures provided better details.Figure 2b shows the sample viewed under TEM at 28.7 kX magnification and exhibits asimilar nanoparticle monodispersity as seen under SEM. Under TEM, it is readily apparentthat nanoparticles appear to be crystalline in nature and randomly located. Figure 2c showsa close-up picture taken with TEM at 824 kX magnification of one of these nanocrystals.This particle appears to have either an orthorhombic or tetragonal crystal structure. Stud-ies of additional TEM pictures, led us to believe there is a preponderance of orthorhombicstructures with regards to the nanocrystals. Mixed in with the nanocrystals, and somewhathidden in Figures 2b and 3c, are larger sized spherules. Figure 2d presents these spherules,which are bigger in size and in general tend to be > µ m. Interestingly, one could alsoobserve the presence of crosslinked RADA16 nanofibers in process of agglomeration in figure2d at the middle and lower left corner of the picture.To ensure the nanocrystals were not due to unreacted EDC, we measured particle dis-tribution of the reactant using NTA on a sample at DF=1000 in deionized water. Weadditionally viewed the same dilution sample under TEM. Figure 3a, TEM picture at 78.7kX magnification, shows that indeed there are “plate-like” square particles or flakes withinthe EDC solution. NTA showed particles to be typically in the range of 46 - 300 nm. Lessprobable were particles of sizes ranging between 500-700 nm. Figure 3b presents data forone set of measurements at 21 o C. A visual comparison of figure 3a with figure 3c, alsotaken with TEM at 78.7 kX magnification and showing reaction product nanocrystals, re-3
IG. 2. a.) SEM picture of 2% w/v RADA16 reacted with 20% EDC at 10 kX magnification.Monodisperse particles seen throughout sample. In addition to typical organic elements, EDS mea-surements showed significant traces of chlorine. ; b.) Same sample viewed under TEM at 28.7 kXmagnification. Approximately monodisperse orthorhombic crystals visible; c.) TEM close-up viewof a ≈
70 nm nanocrystal at 824 kX magnification. ; d.) Spherules were also present in sample.TEM view of spherules at 10.9 kX. Crosslinked RADA16 nanofibers in process of agglomerationare visible in the middle of picture and lower left corner. veals crystal morphologies are different. Whereas crystals in EDC are “plate-like” flakes atvarious stages of dissolution, product nanocrystals are solid, well-formed orthorhombic-likestructures. NTA quantified the size distribution of the mix of nanocrystals and spherules inthe product solution. Figure 3d presents data obtained for one set of measurements froma sample of product solution diluted in deionized water at DF=1000 and measured at 25 o C. It shows particles present in the 100-600 nm range within which the majority appearto be nanocrystals. Larger sizes, >
900 nm, most likely stem from spherules. Indeed, in arepresentative area covered primarily with spherules, 3e, a manual count of N=13 spherules4
IG. 3. a.) TEM view of plate-like crystals present in 20% EDC dissolved in deionized waterat 78.7 kX magnification.; b.) NTA measurement of 20% EDC only, DF=1000, in deionizedwater measured at 21 o C. ; c.) TEM view of solution made with 2% RADA16+20% EDC at 78.7kX magnification. Visual inspection shows post-reaction crystals have different morphology thanEDC crystals. Additionally, spherules appear in the mix. ; d.) NTA measurement of 2% w/vRADA16+20% EDC, DF=1000, in deionized water at 25 o C.; e.) TEM view of spherules fromdifferent location than figure 2d at 28.7 kX magnification. ; c.) Spherule size distribution statisticsof e.) as measured with the TEM measuring tool. β -sheet peak at 1636 cm-1 [13]. The broad peak at 2116 cm-1covers the range of alkyne (C ≡ C) and nitrile (C ≡ N) stretches, which are not thought tobe present in RADA16. Therefore, this broad peak is unknown as of this writing. Figure4b shows FTIR data for EDC prior to reaction with EDC. Of particular importance arethe peaks at 2130 and 1702 cm-1 as these distinctive peaks for EDC disappear after thecrosslinking reaction with RADA16. The peak at 2130 cm-1 is attributed to the N=C=Nbonds of EDC [14]. We attribute the peak at 1702 cm-1 to stretching of the cumulated C=Nbonds since C is an sp hybridized carbon. It is expected that these bonds would no longerbe present after reaction of the primary amine with the unstable intermediate o-acylisourea.That is in fact what we found. Figure 4c shows overlaid plots of EDC reactant (magenta)with the RADA16+EDC product (black). Peaks at 2130 and 1702 cm-1 are conspicuouslyabsent, confirming that a crosslinking reaction indeed took place. It is also clearly obviousthat the β -sheet peak at 1636 cm-1 present in RADA16 no longer appears after crosslinking.Evidently, the stable β -sheet structure of RADA16 has been disrupted by the crosslinkingmechanism. The two most prominent peaks of the RADA16+EDC product (black curve)appear at 1619 and 1571 cm-1. Both peaks could be attributed to different modes of N-Hbending vibrations of primary amine groups such as those present in urea. Given the relativesignal strength of the peaks, it is also possible that they are produced by bending vibrationsof amide groups. Crosslinking confirmed, we would expect the peaks to be produced bythe presence of both amide bonds from crosslinked peptides and amine bonds from theurea by-product. The peak at 1571 cm-1 could also be produced by a nitro group (-NO )asymmetric stretch, though we believe this type of bond is less likely to occur in the current6 IG. 4. a.) FTIR spectra of 2% RADA16. The significant peak at 1636 cm-1 is due to the stable β -sheets. ; b.) FTIR spectra of 20% EDC. N=C=N bonds produce two distinctive peaks at 2130and 1702 cm-1, respectively. These disappear after a crosslinking reaction. ; c.) Overlaid FTIRspectra of unreacted 20% EDC (magenta) and RADA16+EDC (black) after reaction, diluted indeionized water. ;CH ) are attributed to 1481 cm-1. It seems likely that the C-N stretch of an amine groupcauses the peak at 1281 cm-1. The low energy peaks at 878, 849 and 813 cm-1 which appearon the product curve, but not on the EDC curve could be due to C-Cl bond stretching.However, that needs to be studied in more detail and we will not mention them further. Onthe higher energy side of the product spectrum we attribute the peak at 2982 cm-1 to sp hybridized C-H bonds signifying the presence of acetyl groups. It seems the 2754 cm-1 peakon the RADA16/EDC product curve could be produced by the O-H stretch of regeneratedcarboxylic acids that did not react with a primary amine. It is not likely that the peak isproduced by the C-H stretch of aldehydes.FTIR data, thus, lends support to the existence of a proposed crosslinking reaction ofRADA16 activated by EDC. It is likely that crosslinking proceeds through EDC activation ofthe carboxyl groups present in the aspartic acid amino acid residues. The unstable, amine-reactive O-acylisourea intermediate that results from activation of the arboxyl groups thenreacts with available primary amines. Primary amines available for reaction either come fromthe N-terminus or the guanidinium group of the arginine subgroup. While the guanidiniumcation is highly stable in an aqueous solution, reactions stemming from a combination ofboth the N-terminus and possibly guanidinium groups cannot be ruled out.It should be pointed out that Powder XRD analysis of the product yielded inconclusiveresults. It was not possible to obtain significant readings with the amount of raw materialat hand. Currently available TEM is not equipped with XRD capability to further analyzenanocrystalline structures.RADA16 was obtained from 3D Matrix as a lyophilized powder that was prepared byexchanging TFA for HCl [11] so that the arginine had a chlorine counterion and the asparticacid was protonated. It was reconstituted in deionized water at a nominal 2.0% (w/v) togive a solution with pH ≈ µ L of 2% (w/v)RADA16 gel we added 50 µ L of 20% (w/v) EDC. The mixture was shaken vigorously for8pproximately 5 minutes on a Vortex Genie mixer at setting 7 then placed in a lab benchFisher-Scientific centrifuge for two minutes . To improve mixing, we let the mixture sitovernight for approximately 24 hrs. The resulting aqueous solution had a pH=3.53. Allreactions were carried out at 22 o C.In preparation for viewing the sample under SEM, 250 µ L of 70% (w/v) ethanol wasadded to reactant mixture to both dissolve any unreacted polymer and aid in evaporationof the solution. Approximately 100 µ L of the solution was placed on a coverglass that wascleaned by immersion in ethanol and sonicated for 10 minutes. The product solution on thecoverglass was then evaporated for about 6 minutes on top of a hotplate set at 90 o C. Toview under SEM, an approximately 10 nm layer of Au-Pd was deposited on top of the driedRADA16/EDC film with a Denton sputtering system.Preparation of samples for viewing under TEM required nominal dilution factors ofDF=1000. Samples were vacuum dried at 45 o C and negatively died.Nanoparticle Tracking Analysis equipment required volumes in the range of 0.8-1 mL.We used dilution factors of DF=1000 in deionized water to study the distribution of particlesizes in our sample.FTIR studies were conducted at room temperature, 22 o C, using the same dilution factor.A JEOL JSM-63900LV SEM equipped with an energy-dispersive X-ray spectroscopy(EDS) detector from Oxford Instruments was used to obtain SEM pictures and materialcomposition data. RADA16 FTIR spectra were obtained at 4 cm-1 resolution on a JascoFT/IR 4100 with a multi-reflection Attenuated Total Reflectance (ATR) accessory equippedwith a ZnSe crystal. FTIR spectra for EDC and RADA16+EDC product were measured ona Bruker Vertex 70 spectrometer with a single pass ATR accessory. Nanoparticle TrackingAnalysis (NTA) was performed on a Malvern Instruments Nanosight LM10 with capability oftracking particles in the size range of 10 - 2000 nm. TEM data was obtained in collaborationwith the Microscopy Core Facility.We have provided evidence that crosslinking in RADA16 is activated by EDC. It is likelythat crosslinking proceeds through EDC activation of the carboxyl groups present in theaspartic acid amino acid residues reacting with primary amines either from the N-terminusand possibly the guanidinium group of the argininine subgroup. The reaction producesnanocrystals and micron-sized spherules. Further studies are required to understand themechanisms leading to crosslinking as well as formation of nanocrystals and spherules.9his work has been supported in part by the Microscopy Core Facility in the Departmentof Integrative Biology at the University of South Florida. We also like to thank Dr. Hayniefor very useful comments. [1] X. Zhao and S. Zhang, Chem. Soc. Rev , 1105 (2006).[2] K. Hamada, M. Hirose, T. Yamashita, and H. Ohgushi, J. Biomed. Mater. Res. Part A ,128 (2008).[3] A. L. Sieminski, C. Semino, H. Gong, and R. Kamm, J. Biomed. Mater. Res. Part A , 494(2008).[4] F. Gelain, A. Horii, and S. Zhang, Macromol. Biosci. , 544 (2007).[5] C. Cunha, S. Panseri, O. Villa, D. Silva, and F. Gelain, Int. J. Nanomed. , 943 (2011).[6] A. R. Cormier, C. Ruiz-Orta, R. G. Alamo, and A. K. Paravastu, Biomacromolecules ,1794 (2012).[7] A. R. Cormier, X. Pang, M. I. Zimmerman, H.-X. Zhou, and A. K. Paravastu, ACS Nano ,7562 (2013).[8] H. Yokoi, T. Kinoshita, and S. 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