Nanoscale

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aIstituto Italiano di Tecnologia (IIT), Nan

16163 Genova, ItalybIstituto di Cristallograa – Consiglio N

Amendola 122/O, 70126 Bari, ItalycEuropean Synchrotron Radiation Facility, BdKAUST (King Abdullah University of Science

Jeddah, Saudi ArabiaeBIONEM lab University of Magna Graecia,

88100, Germaneto-Catanzaro, Italy. E-mail:

† Electronic supplementary informa10.1039/c3nr34032e

Cite this: Nanoscale, 2013, 5, 2295

Received 10th December 2012Accepted 2nd February 2013

DOI: 10.1039/c3nr34032e

www.rsc.org/nanoscale

This journal is ª The Royal Society of

Superhydrophobic surfaces allow probing of exosomeself organization using X-ray scattering†

Angelo Accardo,a Luca Tirinato,a Davide Altamura,b Teresa Sibillano,b

Cinzia Giannini,b Christian Riekelc and Enzo Di Fabrizio*de

Drops of exosome dispersions from healthy epithelial colon cell line

and colorectal cancer cells were dried on a superhydrophobic PMMA

substrate. The residues were studied by small- and wide-angle X-ray

scattering using both a synchrotron radiation micrometric beam and

a high-flux table-top X-ray source. Structural differences between

healthy and cancerous cells were detected in the lamellar lattices of

the exosome macro-aggregates.

Ultralow interactions of droplets with superhydrophobicsubstrates provide novel possibilities for studying variousbiological processes such as single protein molecule detec-tion,1 protein conformational changes,2 peptide amyloid-osis,3,13,14 biomineralization4 and cell growth.5 Indeed, theconvective ows induced by the homogeneous evaporationrate during droplet drying favors conformational transitions2

and brillar alignment.3,13,14 In this work we present X-raydiffraction investigation of exosomes which are small extra-cellular membrane vesicles (40–100 nm in diameter)homogenous in shape (cup-shaped)21 and secreted by a widerange of mammalian cell types. Their typical structure iscomposed of a lipid bilayer containing several proteins (i.e.adhesion and immunomodulatory proteins) and RNA. Theirmain functions can be summarized as follows: in cell–cellcommunication for the transport of proteases, growthfactors, adhesion molecules, signaling molecules as well asDNA, mRNA and microRNA sequences.22 Tumor cells release

ostructures Department, via Morego 30,

azionale delle Ricerche (IC-CNR), Via

.P.220, F-38043 Grenoble Cedex, France

and Technology) PSE and BESE divisions,

Campus Salvatore Venuta, Viale Europa

enzo.difabrizio@iit.it

tion (ESI) available. See DOI:

Chemistry 2013

large quantities of exosomes containing different types ofbiomolecules such as procoagulant, growth regulatory andoncogenic load, which can be transferred throughout thecancer cell population and to non-transformed stromal cells,endothelial cells and possibly to the inammatory inl-trates.22 Since they are derived from multivesicular bodies,their molecular composition might provide clues to themechanism of protein and lipid sorting in endosomes.23

Depending on their origin, exosomes can play roles in severalphysiological processes. Their release was indeed found to beassociated with the secretion of Ab peptides in the plaques ofbrains of patients with Alzheimer's disease,24 to affectangiogenesis evolution25 and to have a fundamental role inthe trophic support of neurons.26 Further, since these“microvesicles” are implicated in cell–cell communication,recent works have also shown that cancer exosomes areemerging as mediators in the tumorigenesis and metastasisphenomena.6 Efforts to identify and monitor the presenceand the characteristics of such microvesicles are therefore ofparamount importance in the cancer detection and preven-tion and also to better understand their underlying mecha-nisms in several biological processes.

The general aim of this study was to assess the sensitivity ofX-ray scattering techniques for probing microstructuralchanges due to differences in the biochemical composition ofexosomes related to their origin, namely, from healthy tocancerous cells.

X-ray scattering data at small and wide-angles were acquiredusing a synchrotron radiation (SR) microbeam as well as a table-top setup. The full agreement between the results obtained bythese two approaches suggest that X-ray laboratory techniquescan be used for probing exosomes routinely prior to specicstructural studies at higher brilliance SR-sources.

We enhanced the X-ray probing discrimination capability byinducing an alignment of exosomes during their drying on asuperhydrophobic substrate.2 In order to maximize the signal-to-noise ratio we used SR microbeam Small and Wide AngleX-ray Scattering (SAXS/WAXS) techniques. Indeed, the results

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obtained in this work could open new possibilities for revealingand discriminating exosomes during clinical cancer biopsy.

Materials

Experiments were performed on exosomes derived from twodifferent colon cell lines: CCD841-CoN (healthy epithelial coloncell line) and HCT116 (colorectal cancer cells). The exosomeisolation procedure is described elsewhere.17 Drops of about5 ml exosome dispersion were deposited using a pipette on ananostructured superhydrophobic PMMA surface2,3 (inset ofFig. 1A). Aer drying in air, the solid residue was attached by asmall amount of fast glue (Loctite) to a glass capillary tip forX-ray scattering experiments. The high resolution SEM images(ESI, Fig. S1†) reveal some agglomerated particles in the 100 nmrange as expected for single exosomes.9,10 Aer cutting theresidues (following superhydrophobic drying) into two hemi-spheres, a well-ordered lamellar structure is revealed (Fig. 1A–D). The residuals show a hollow conguration as alreadyobserved in a previous work2 (Fig. 1A). Closer inspection of thelamellar structure reveals a periodic substructure on top of thelamellae (Fig. 1D).

X-ray scattering experiments

SR-experiments were performed at the ESRF ID13-beamline.8 Amonochromatic beam of l¼ 0.0995 nm was focused on a 1.3 (h)� 1.5 (v) mm2 spot with �1 mrad divergence at the sampleposition. A Frelon CCD camera11 was used for data collection.Raster-diffraction scans were performed at room temperature inair with a typical exposure time of 1 second per pattern.MicroWAXS patterns were collected at a calibrated12 sample-to-detector distance of 114.8 mm and microSAXS patterns at adistance of 511.7 mm (ESI†).

Fig. 1 (A and B) SEM images of exosome cross-cut residue from the HCT116 cellline attached to a glass capillary tip (inset: two droplets placed on a PMMAsuperhydrophobic substrate) and (C and D) close-up SEM images of exosomecross-cut residue of (A).

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The laboratory X-ray experiments were performed using anX-ray MicroImaging (XMI) table-top X-ray setup.7 A 200 mm2

circular monochromatic beam was used at l ¼ 0.154 nm. ATriton 20 gas lled proportional counter and an image-plate (IP)detector were respectively used for SAXS and WAXS dataacquisition. The SAXS-range was explored at a detector-to-sample distance of 2200mmwhile theWAXS one at 27 mm. Theexposure times were around 1 hour per pattern.

ResultsWAXS

MicroWAXS patterns of the CCD and HCT residues suggest highcrystallinity as shown in Fig. 2A, B and E by the strong Braggpeaks and the low diffuse scattering background. An estimationof crystallinity for the CCD sample, based on tting Gaussianproles to the Bragg peaks and the diffuse scattering back-ground, suggests about 40% crystallinity. A composite diffrac-tion image8 based on a raster-diffraction scan with a 10 mm stepincrement across the CCD-residue is shown in the ESI(Fig. S2B†). The composite diffraction image reveals, notably,the hollow structure visible in the SEM image (Fig. 1A).

The appearance of textured patterns at the edge of the resi-dues (Fig. 2A and B) is attributed to convective shearing inducedby evaporation of the drop.13 The azimuthal integration of theWAXS-patterns from the center of the residues is very similar forCCD and HCT residues (Fig. 2E) suggesting the same micro-structure. WAXS-reections with the same lattice-spacings(d-values; with d ¼ 2p/Q) were revealed for the same samples atthe XMI Laboratory (Fig. 2F) for the corresponding angular rangewhich is a remarkable result considering the difference in bril-liance between the SR-source and table-top X-ray source.Further, the use of the SR microbeam allowed the detection of adiffracted intensity along the azimuthal direction, as shown inFig. 2A, B and S2A.† This behavior, could not be observed withthe larger beam when a table-top setup was used (Fig. 2C and D).

SAXS

The microSAXS patterns from the CCD- and HCT-residues showone-dimensional (1D) scattering with several periodic orders(multiple of the rst ones observed at 13.2 nm and 15.1 nm,respectively for CCD- and HCT-residues) which is attributed to alamellar morphology (Fig. 3A and D). The azimuthal integrationof the patterns is shown in Fig. 3E. The CCD-pattern reveals 2well dened orders and Q-dependant peak broadening which isparticularly strong for the 3rd order. The HCT-pattern shows incontrast up to 5 orders without Q-dependant peak broadening.The periodicity is dened as d ¼ 13.5 � 0.5 nm for several CCD-patterns and d ¼ 15.0 � 0.5 nm for several HCT-patterns. Thepeak broadening of the CCD-pattern is attributed to size uc-tuations of the lamellar lattice. The HCT-pattern suggests incontrast a regular lamellar packing although we observed alsoless ordered patterns in the residue. To further conrm thesedata, we repeated the analysis under the same experimentalconditions using the table top X-ray setup. The SAXS patternsshow less order as compared to the microSAXS patterns in

This journal is ª The Royal Society of Chemistry 2013

Fig. 2 (A) CCDmicroWAXS pattern collected at the ID13 beamline; (B) the same for HCT residue; (C) CCDWAXS pattern collected at XMI Lab; (D) the same for the HCTdiffraction pattern; (E) azimuthally averaged CCD and HCT patterns of selected reflections collected at the ID13 beamline. The averaged patterns were recorded at thecentres of the residues in order to avoid texture effects; (F) azimuthally averaged CCD and HCT patterns acquired at XMI Lab. Q ¼ 4psin Ql�1¼2p/d where Q is theBragg angle, l the wavelength and d the lattice spacing. Note that the angular range of (F) is limited to that explored in the XMI Lab experiments. The additionalreflection at 22.5 nm was also observed in several microWAXS patterns for both CCD and HCT exosomes (ESI, Fig. S2†).

Fig. 3 (A) CCD microSAXS-pattern (log-scale) collected at the ID13 beamline. (B) CCD-SAXS pattern (log-scale) collected at the XMI Lab; (C) the same for HCT exo-somes; (D) HCT microSAXS-pattern (log-scale). (E) azimuthally integrated CCD and HCT microSAXS patterns acquired at the ID13 beamline. The black and red barshighlight respectively the number of the HCT and CCD peak orders; (F) azimuthally integrated CCD & HCT SAXS patterns (treated with the restoration algorithm16)collected at the XMI Lab showing the periodicity discrepancy in the lamellar morphology. Q ¼ 4psin Q/l where Q is the Bragg angle and l the wavelength.

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Fig. 3B and C. We attribute this to a lower X-ray beam intensityand a larger beam size (200 mm versus 1 mm beam size), thusresulting in an averaging over a wider part of the residueincluding also the disordered parts. This result is supported bythe higher orientational ordering observed at the edge of theresidues by microSAXS. A novel restoration algorithm,16

opportunely modied for SAXS geometry, allowed improvementof the peak visibility. Indeed, the rst order SAXS-peaks (Fig. 3F)showing a probable lamellar periodicity for the CCD andHCT exosomes of, respectively, d ¼ 14.6 � 0.5 nm and d ¼15.7 � 0.5 nm conrm the data acquired by microSAXS.

Conclusions

Exosomes derived from HCT116 (colorectal cancer cells) andCCD841-CoN (healthy epithelial colon cell line) form crystallineresidues when dried upon a superhydrophobic PMMAsubstrate. The convective ow during drying induces orderingeffects which are reected in the microSAXS/WAXS patternsfrom the edge of the residuals. The microstructures of HCT116and CCD841-CoN residues appear to be the same, as evidencedby almost identical WAXS patterns obtained by SR- and table-top X-ray techniques. The analysis of the microSAXS patternssuggests a difference between both cell types, which will befurther explored in future experiments. Indeed, the lamellarmorphology revealed by SAXS analysis shows differences in thenumber of orders, their periodicities and peak broadening. Thiscould be due to a higher disorder degree for the CCD-residueand a more regular organization for the HCT-residue. Theobserved variations could depend on different compositions ofthe exosome membrane due to the variability of its inner lipidmolecules17,18 and, consequently, inuencing a different orga-nization/distance that occurs between hydrophilic and hydro-phobic regions of the membrane.19 At the present stage it is,however, not completely clear whether the ultrastructures of theresidues are composed of aggregated exosome particles orwhether particle recombination or fusion into a lamellarmorphology happened, as observed for lipid vesicles.15

Further studies could also look at the transition of dispersedexosomes9,10 into a lamellar solid phase by in situ microSAXSduring their drying from drops on a superhydrophobic surface2

or with the aim of novel EWOD devices.20 This could be com-plemented by high resolution SAXS experiments looking at theshape and size of single exosome particles to better understandthe mechanisms underlying the self assembly of these vesicles.Finally, the same measurements have been conducted using ahigh-ux table-top X-ray source conrming scattering fromexosomes. Both scattering techniques are highly complemen-tary in terms of local probing capability and availability. Theneed to include SAXS data, collected with a table-top setup, andto compare their quality with synchrotron data, is relevant inview of the possible future use of these methods to histologyscreening, where a table-top set-up is conceivable.

In future, this study can be extended to exosomes comingfrom other cell lines in order to detect possible commonfeatures, both in their crystalline structure and in their macro-aggregation order.

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Acknowledgements

The authors are grateful for the SEED project “X-ray synchrotronclass rotating anode microsource for the structural microimaging of nanomaterials and engineered biotissues (XMI-LAB)” – IIT Protocol no. 21537 of 23/12/2009 and the FIRB 2009/2010 project “Rete integrata per la Nano Medicina (RINAME)” –RBAP114AMK_006. Rocco Lassandro and Giuseppe Chita areacknowledged for the technical support.

Notes and references

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2 A. Accardo, F. Gentile, F. Mecarini, F. De Angelis,M. Burghammer, E. Di Fabrizio and C. Riekel, In situ X-rayscattering studies of protein solution droplets drying onmicro- and nanopatterned superhydrophobic PMMAsurfaces, Langmuir, 2010, 26(18), 15057–15064.

3 A. Accardo, M. Burghammer, E. Di Cola, M. Reynolds, E. DiFabrizio and C. Riekel, Lysozyme brillation induced byconvective ow under quasi contact free conditions, SoMatter, 2011, 7(15), 6792–6796.

4 A. Accardo, M. Burghammer, E. Di Cola, M. Reynolds, E. DiFabrizio and C. Riekel, Calcium Carbonate Mineralization:X-ray Microdiffraction Probing of the Interface of anEvaporating Drop on a Superhydrophobic Surface,Langmuir, 2011, 27(13), 8216–8222.

5 T. Limongi, F. Cesca, F. Gentile, R. Marotta, R. Ruffilli,A. Barberis, M. Dal Maschio, E. M. Petrini, S. Santoriello,F. Benfenati and E. Di Fabrizio, NanostructuredSuperhydrophobic Substrates Trigger the Development of3D Neuronal Networks, Small, 2013, 9(3), 402–412.

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8 C. Riekel, M. Burghammer, R. Davies, R. Gebhardt andD. Popov, Fundaments of So Condensed MatterScattering and Diffraction with Microfocus Techniques, inApplications of Synchrotron Light to Scattering and Diffractionin Materials, ed. T. A. Ezquerra, M. Garcia-Gutierrez, A.Nogales and M. Gomez, Springer, Heidelberg, 2009, vol.776, pp. 91–104.

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