Spheroidal and nanocrystal structures created from carbodiimide crosslinking reaction with RADA16

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-ter-[r]

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Original Article

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

Article history: Received May 2017 Received in revised form 16 May 2017

Accepted 17 May 2017 Available online 20 May 2017 Keywords:

RADA16 Crosslinking EDC Spherules

a b s t r a c t

RADA16 is a widely studied polypeptide known for its ability to self-assemble intob-sheets that 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 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/)

1 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 intob-sheets and

self-assembles into nanoﬁbers with diameters in the range of

3e10 nm [4,5]forming hydrogels when dissolved in water

Self-assembly 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

com-bination of RADA16-Bone morphogenic protein with poly(lactic-co-glycolic acid) via EDC for bone regeneration[7] Here we study self-crosslinking of the RADA16 peptide via EDC which could lead to an entirely new range of possible designed peptides with a myriad of functional characteristics Weﬁnd the formation of nanocrystals as a result of the crosslinking reaction The methods for nanocrystal formation described here, particularly for drug delivery applica-tions, are highly desirable as they constitute simple wet chemistry reactions at room temperature Such simplicity seems advanta-geous to current nanocrystal production methods such as milling, precipitation with colloidal stabilization, and homogenization for medical and clinical applications[8]

RADA16 studied here is acetylated with an amine N-terminus

Ac-[RADA]4-NH2 The arginine and aspartic acid amino acid

res-idues 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 mech-anism 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 in-termediates 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 containseCH3, the side reaction was not

ex-pected to occur here

2 Experimental

RADA16 was obtained from 3D Matrix as a lyophilized powder

prepared by exchanging TFA for HCl[10] The arginine had a

chlo-rine 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 pHz2e3

EDC was obtained from TCI America (USA) as a hydrochloride with a MW¼191.70 g mol1and of 98.0% purity It was dissolved in *Corresponding author

E-mail address:jmonreal@alum.mit.edu(J Monreal)

Peer review under responsibility of Vietnam National University, Hanoi

Contents lists available atScienceDirect

Journal of Science: Advanced Materials and Devices j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / j s a m d

http://dx.doi.org/10.1016/j.jsamd.2017.05.008

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deionized water to obtain a nominal 20% (w/v) solution with

pHz7.68 as measured with a Sensorex polymer electrode

The RADA16eEDC reaction proceeded as follows To 100mL of

2% (w/v) RADA16 gel we added 50mL 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 pH¼3.53 Mixing was carried out at 22C

Sample preparation for viewing under SEM consisted of 250mL

of 70% (w/v) ethanol added to reactant mixture Approximately

100mL of the solution was placed on a coverglass that had

previ-ously 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 90C A 10 nm layer of

AuePd was deposited on top of the dried RADA16/EDCﬁlm with a

Denton sputtering system

Preparation of samples for viewing under TEM required nominal

dilution factors (DF)¼1000 Samples were vacuum dried at 45C

and negatively died

Nanoparticle Tracking Analysis (NTA) equipment, Malvern In-struments Nanosight LM10 with capability of tracking particles in

the size range ofe2000 nm, required volumes in the range of

0.8e1 mL We used DF ¼1000 in deionized water to study the

distribution of particle sizes in our sample

FTIR studies on RADA16 were conducted at room temperature, 22C on a Jasco FT/IR 4100, at cm1resolution, equipped with a

multi-reﬂection Attenuated Total Reﬂectance (ATR) accessory

equipped with a ZnSe crystal Spectra for EDC and RADA16ỵEDC

product were measured on a Bruker Vertex 70 spectrometer with a single pass ATR accessory

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 ob-tained in collaboration with the Microscopy Core Facility

3 Results and discussion

Fig 1a shows a SEM picture of the resulting product from a re-action 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 theﬁlm surface To rule out

contamination from NaCl or other types of salts, we measured

elemental X-ray dispersion with the EDS detector on a 1mm1mm

ﬁeld of view at four different sample locations In addition to ele-ments typical of organic compounds EDS measureele-ments 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 preponder-ance 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>0.5mm Interestingly, one could also observe the presence of

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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 DF¼1000 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 orflakes 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 ofFig 2a withFig 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, well-formed orthorhombic-like structures NTA quantified size distribu-tions 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 DF¼1000 and

measured at 25C 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

N¼13 spherules yielded an average size D¼987 nm with standard

error¼59 nm The 95% conﬁdence interval in this region is [859, 1115] nm Therefore, we attribute the size distribution peaking at 902 nm inFig 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 RADAỵEDC after reaction.Fig 3a is an FTIR plot of RADA16 hydrogel prior to reaction with EDC It shows the distinctiveb-sheet peak at 1621 cm1[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 cm1as these distinctive peaks for EDC disappear after the crosslinking reaction with RADA16 The peak at 2130 cm1 is attributed to the N]C]N bonds of EDC[12] We attribute the peak at 1702 cm1to stretching of the cumulated C]N bonds since

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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 RADA16ỵEDC product in black Peaks at 2130

and 1702 cm1are conspicuously absent, conﬁrming that a

cross-linking reaction indeed took place Theb-sheet peak disappears

after crosslinking Evidently, the stableb-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 un-stable, 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 Fig 3.(a) FTIR spectra of 2% RADA16 The signiﬁcant peak at 1636 cm1is due to the stableb-sheets (b) Overlaid FTIR spectra of unreacted 20% EDC (magenta) and RADA16ỵEDC

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While the guanidinium cation is highly stable in an aqueous solu-tion, reactions stemming from a combination of both the N-ter-minus and possibly guanidinium groups cannot be ruled out

4 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-ter-minus and possibly the guanidinium group of the arginine sub-group 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

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