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Colorimetric Nanoplasmonic Assay to Determine Purity and Titrate Extracellular Vesicles Daniele Maiolo, Lucia Paolini, Giuseppe Di Noto, Andrea Zendrini, Debora Berti, Paolo Bergese, and Doris Ricotta Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac504861d • Publication Date (Web): 12 Feb 2015 Downloaded from http://pubs.acs.org on February 16, 2015

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Analytical Chemistry

Colorimetric Nanoplasmonic Assay to Determine Purity and Titrate Extracellular Vesicles Daniele Maiolo,†1* Lucia Paolini, 2 Giuseppe Di Noto,2 Andrea Zendrini,2 Debora Berti,3 Paolo Bergese1 and Doris Ricotta2 1

Chemistry for Technologies Laboratory and INSTM, Department of Mechanical and Industrial Engi-

neering, University of Brescia, via Branze 38, 25123 Brescia, Italy 2

Department of Molecular and Translational Medicine, Faculty of Medicine, University of Brescia,

Brescia, Italy 3

Department of Chemistry “Ugo Schiff” and CSGI, University of Florence, via della Lastruccia 3,

50019 Sesto Fiorentino, Florence, Italy

KEYWORDS: Extracellular Vesicles, Separation, Nanoparticles, Protein Corona, Nanoplasmonics.

CORRESPONDING AUTHOR *Daniele Maiolo, e-mail: [email protected] †European Center for Nanomedicine CEN foundation, c/o Nanostructured Fluorinated Material laboratory, Department of Chemistry, Material and Chemical Engineering Milan Italian Polytechnic, Via Mancinelli 7, 20131 Milan.

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ABSTRACT

Extracellular Vesicles (EVs) - cell secreted vesicles that carry rich molecular information of the parental cell and constitute an important mode of intercellular communication- are becoming a primary topic in translational medicine. EVs (that comprise exosomes and microvesicles/microparticles) have a size ranging from 40 nm to 1 µm and share several physicochemical proprieties, including size, density, surface charge and light interaction, with other nano-objects present in body fluids, such as single and aggregated proteins. This makes separation, titration and characterization of EVs challenging and time consuming. Here we present a cost-effective and fast colorimetric assay for probing by eye protein contaminants and determine the concentration of EV preparations, which exploits the synergy between colloidal gold nanoplasmonics, nanoparticle-protein corona and nanoparticle-membrane interaction. The assay hits a limit of detection of protein contaminants of 5 ng/µl, and has a dynamic range of EV concentration ranging from 35 fM to 35 pM, which matches the typical range of EV concentration in body fluids. This work provides the first example of the exploitation of the nanoparticle-protein corona in analytical chemistry.


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INTRODUCTION Extracellular Vesicles (EVs) have been identified as mediators of intercellular communication in many pathophysiological processes throughout the body; for this role they have been the subject of a growing scientific interest over the last few years.1 These cellular secreted vesicles comprise microvesicles and ectosomes, budded from the plasma membrane with a size from 40 nm to 1 µm, and exosomes of intracellular origin with a size from 30 to 120 nm.2,3 EVs contain cell specific cargos such as lipids, proteins, DNA, mRNA and miRNA4 and populate different biological fluids (plasma, cerebrospinal fluid, urine, saliva, etc…), making them ideal candidates for biomarker studies.5–7 The role that EVs are believed to play in various pathological conditions is linked to the delivery of cargos to surrounding cells actuating a functional manipulation of the microenvironment, for example tumor derived EVs can impair antitumor immune response reducing lymphocyte activity.8 In addition several studies have focused on the role of EVs in cancer biology because their release is significantly altered in cancer cells;9 in the same way their activity in intercellular communication2 seems to be involved in neurodegenerative disorders.2,10–12 Therefore EVs are envisioned as captivating means for “remote” biopsies13,14 and theranostics.15–18 Unfortunately, understanding the molecular mechanisms of EV biogenesis, the precise identification of EV physiological relevance and role, and the implementation of EV based biotechnology are to date hampered by the poor physicochemical knowledge of these nanosized objects, due to inherent shortcomings of the current bioanalytical methods used for their separation and characterization.2,3,19,20 EVs have a low refractive index and are heterogeneous both in size and composition. In addition, the fact that protein complexes, especially insoluble immunocomplexes, share some critical biophysical properties with EVs − including size, surface charge and interaction with light− compromises their detection and isolation. This, in turn, affects EV quantification by conventional bioanalytical methods, such as flow cytometry, and purification by differential centrifugation, especially in diseases where 3 ACS Paragon Plus Environment


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formation of immunocomplexes is common, including autoimmune diseases, as well as hematologic disorders, infections, and cancer.19,21 Recently, the problem has been tackled from the analytical side by dedicated resistive pulse sensing22 and multiplex systems.13,14 In parallel, unedited biophysical methods, including nanoparticle tracking analysis, Raman microspectroscopy, micro nuclear magnetic resonance and small-angle X-ray scattering (SAXS) are under exploration.22 In the last year, excellent results have been obtained with commercial9,23 and nanostructured13,14 Surface Plasmon Resonance (SPR) sensors. The present work is a nanotechnological contribution to these efforts and reports about a costeffective and fast colorimetric assay for probing protein contaminants and determine the concentration of EV preparations. The assay working principle, sketched in Fig. 1, exploits colloidal gold nanoplasmonics24–29 and the fact that nanoparticle (NP) aggregation at lipid membranes30 is modulated by the presence of a protein corona (PC31–34) around the NPs. The assay kit simply consists of a probe solution of dispersed gold NPs of 15-20 nm size, which looks red due to the NP characteristic LSPR (localized SPR, red absorption spectrum). After the addition of a pure EV preparation to the colloidal dispersion, the NPs adsorb and cluster at the EV membrane causing a red-shift and a broadening of the LSPR (blue absorption spectrum), thus a change of the solution color into blue. Conversely, in the case of an EV preparation containing single and aggregated protein contaminants (hereafter referred as SAP) the NPs preferentially interact with those proteins to form a PC (EV+SAP, Extracellular Vesicles + Single and Aggregated Proteins).35 The PC both inhibits the interaction between NPs and EVs and prevents NPs from aggregating at the EV membrane, thus keeping unchanged the NP LSPR and in turn the solution color to the initial red. In this contribution we will present and discuss the proof of concept of the assay, as well as its implementation for the titration of pure EV preparations and the mechanism of signal generation.


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Figure 1. Nanoplasmonic assay for probing by eye protein contaminants (single and aggregated exogenous proteins, SAP) in EV preparations. See the main text for full explanation.

EXPERIMENTAL SECTION EV purification and characterization EVs were separated from human serum by performing differential centrifugation (DC) or DC and sucrose gradient fractionation (SGF) in sequence, adapting recently published protocols20,21 (see scheme and methods in SI). As demonstarted in this subsection, the preparations obtained by DC contain single and aggregated protein contaminants not associated to the EVs (hereafter referred as SAP) which can be subsequently removed by SGF. Therefore the samples obtained through DC can be considered as the model for SAP contaminated EV preparations (hereafter referred as EVs+SAP), while the samples purified by DC and SGF applied in sequence can be considered as the model for pure EV preparations (hereafter referred as EVs). EVs and EVs+SAP were first verified by routine biochemical and biophysical analysis21 (materials and methods are given as SI). Both preparations resulted to contain high concentrations of the same EV population, as evidenced by the characteristic phosphatidyl-choline and sfingomyelin lipid content, by 5 ACS Paragon Plus Environment


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the acetylcholine-esterase enzymatic activity by the presence of the typical exosomal and microvesicular markers (SI, Fig. S2). The morphology of the EVs was checked by Scanning Electron Microscopy (SEM) (SI, Fig S2) and Atomic Force Microscopy (AFM), which gave consistent results (SI, Fig S3S4). In particular, both EVs and EVs+SAP resulted to contain vesicles of the expected36 spherical shape and size distribution, centered at 160 nm The nature of the considered EV populations was further confirmed by the ζ-potential value, equal to −17.7 ± 2.3 mV for EVs while is lower for EV+SAP (-10.3 + 1.2 mV) due to a higher presence of proteins in the sample that lower the “apparent” ζ-potential.37,38 Finally the hydrodynamic sizes obtained from Photon Correlation Spectroscopy (PCS) (Figure 2-A), and the positivity to the TritonX-100 test (SI, Fig S3, panel C), are all in perfect agreement with the typical characteristics of purified EVs reported in the literature.19 The total protein content of EVs and of EVs+SAP derived from 1 ml of human serum was first determined through a Bradford quantification39 and resulted 0.3 µg/µl for EVs and 9 µg/µl for EVs+SAP, respectively (SI, Fig. S2). These values highlight a significant difference in protein content between the two samples. However, the Bradford assay routinely used to quantify the total proteins and often used to quantify EVs, is biased by the lipid amount in the sample, and therefore does not allow discriminating between EV associated proteins, SAP and lipids.20 This drawback, recurrent in literature,20,40 has been circumvented performing dedicated experiments of PCS, AFM and developing an ad hoc native agarose gel electrophoresis assay. PCS results are reported in Fig. 2A, where the scattered field autocorrelation curves for EVs (black triangles) and EVs+SAP (red circles) are reported. Here EVs+SAP displays a faster and steeper decay of the autocorrelation function, G(τ), with respect to EVs, indicating that the EVs+SAP sample contains a significant fraction of objects with a smaller size with respect to the EV sample (see also SI Fig S4).


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To confirm that these smaller particles are SAP, we developed and run an agarose gel electrophoretic assay in native conditions for the separation of a mixed colloidal solution.41,42 We fluorescently stained EVs derived from 1 ml of human serum, EVs+SAP derived from 1 ml of human serum, 3 ng of synthetic phosphatidylcholine (PCh) liposomes of 150 nm average diameter and 10 µg of Bovine Serum Albumin (BSA) protein with the marker PKH67, specific for lipid bilayers (all diluted in 10 mM PBS solution, see Supp Info for description of the experiment).

Figure 2. (A) Normalized autocorrelation curves obtained through photon correlation spectroscopy (PCS) for EV and EVs+SAP preparations. The lower decay time of the EVs+SAP is due to the presence of SAP. (B) Agarose electrophoretic separation of the EVs and of the EVs+SAP preparations. Left panel and right panel refer to PHK67 fluorescent staining and Comassie staining, respectively. PCh liposomes (PCh ves) and BSA were loaded as control. (C and D) AFM images of EVs and EVs+SAP adsorbed on mica (topographic mode). 7 ACS Paragon Plus Environment


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Note that PCh liposomes are a convenient synthetic mimic of EVs, since they have similar lipid composition and size distribution with respect to EVs;23 the PCh liposomes were prepared as described in Ref 30. The preparations were run on the gel shown in the left panel of Fig. 2B, where dark bands corresponding to the lipid vesicles appear at the same height in the lanes of the 150 nm liposomes (PCh ves), EVs and of the EVs+SAP, while they are not present in the BSA lane. The smearing of the EVs+SAP band can be attributed to the SAP contaminants present in the sample. The Coomassie staining, which marks both lipids and proteins, of the same gel in the right panel of Fig. 2B confirms this interpretation and evidences that the SAP content is negligible in the EV sample. Therefore the small amount of proteins measured in EVs by the Bradford assay consists of proteins and lipids associated to EVs. These salient characteristics are definitively confirmed by the AFM images of EVs and EVs+SAP deposited onto freshly cleaved mica. In fact, while the EVs sample shows isolated and well-defined EVs of 100-200 nm size lying onto the sub-nanometer smooth mica surface (Fig. 2C), the EVs+SAP sample (Fig. 2D) consists of a few EVs of the same size buried into a matrix of smaller SAP nanoparticles. All these observation confirm, as already shown,20 that EVs retrieved by DC or by DC-SGF share the same integrity and structure and that the preparations just differ for the presence or not of SAP contaminants. Synthesis and characterization of Au NPs 15 nm Au NPs were synthesized in milliQ water (nanomolar concentration) following the classical Turkevich protocol and characterized by UV/Vis/NIR spectroscopy, AFM and PCS. Full details are given in SI. UV/Vis/NIR spectroscopy UV-Vis spectra were measured with a JASCO UV/Vis/NIR spectrophotometer. Briefly, NP or PC were resuspended in the appropriated solvent (water or PBS) and the absorbance of the solution measured in the presence and absence of the different EVs preparations. 8 ACS Paragon Plus Environment

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Nanoplasmonic assay for detecting SAP in EV preparations EVs and EVs+SAP were prepared and characterized as described in the SI and then resuspended at the desired concentration using PBS. EVs and EVS+SAP were then incubated for 20 minutes, with gold NPs (concentration 6 nM) and the spectroscopic features of the solution were then evaluated using a JASCO spectrophotometer. Alternatively gold NPs after their purification from a 10% human serum solution were incubated with EVs. The same solutions were also measured using Photon Correlation Spectroscopy using a Brookhaven Instrument 90 Plus (Brookhaven, Holtsville, NY). 1 ml of human serum was processed by DC (see Results and discussion). The supernatant of the solution containing the soluble SAP was then collected discarding the pelleted fraction containing the EVs. SAP were quantified by Bradford assay and were spiked in SGF purified EVs at concentrations ranging from 5 ng/ml to 1 µg/ml. The PBS solutions containing increasing concentration of SAP and EVS were then incubated with gold NPs (concentration 6 nM).After twenty minutes of incubation 100 µl of each sample were loaded on a 96 wells plate and the absorbance of the solutions were measured by a plate reader at the wavelengths 540 nm and 650 nm. Plasmonic titration Increasing concentration of synthetic PCh vesicle were resuspended in Tris NaCl EDTA (same concentration used for EV sample) and they were then diluted in PBS (see main text for the concentration tested) and incubated with gold NP (concentration 6 nM). After twenty minutes of incubation samples were evaluated by UV/Vis/NIR spectroscopy using a JASCO UV/Vis/NIR spectrophotometer. Other experimental information A fully detailed Materials and Methods section is given in SI, which includes the description of chemicals and the standard methods and techniques used (e.g. immunoassays, synthesis of PCh liposomes, AFM and SEM microscopy, PCS experiments and data analysis), 9 ACS Paragon Plus Environment


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RESULTS AND DISCUSSION Probing the presence of SAP in EV preparation by the nanoplasmonic colorimetric assay The nanoplasmonic assay consists of 5 µl of a 6 nM water dispersion of 15 nm gold NPs, stabilized by citrate anions (the physicochemical characterization has been reported in SI Fig S5-S6). Fig. 3A shows the assay at work. When 10 µl of a 10 mM PBS solution containing EVs derived from 1 ml of human serum are added, the solution color turns into blue (right tube). Conversely, upon addition of the same amount of EVs+SAP the solution keeps the initial red color (left tube) (a thorough report about EV and EV+SAP preparations and characterization can be found in the experimental section and in the supplementary information). Therefore, the assay allows for probing EV preparation purity by eye. The assay optical proprieties were quantitatively investigated by UV/Vis/NIR spectroscopy.43 To ensure optimal conditions for collecting gold NPs LSPR absorption spectra, all the analyzed assay solutions were diluted to the same final NP molar concentration of 2 nM. From the spectra, reported in Fig. 3B, we learn that the addition of the EVs drives a red shift (blue triangles) of the initial LSPR peak (red squares). This well agrees with the observed color change of the solution from red to blue and mirrors the agglomeration and clustering of NPs at the EV surface.30 The hypothesis that the red shift is not merely due to NP aggregation caused by the addition of 10 mM PBS solution (through charge shielding) is ruled out by a control sample obtained by adding pure 10 mM PBS solution to the NP solution (and by other results presented in the next sections of the paper). The spectrum of this control sample (black circles) has a markedly different profile with respect to the one of EVs+NP, and is characterized by the unspecific broadening of the LSPR peak (see SI for replicates of the experiment). Fig. 3C shows the typical LSPR spectrum obtained upon addition of EVs+SAP to the NP solution. In contrast to what happens with the addition of EVs, here the LSPR peak undergoes a slight broadening and red shift with respect to bare NPs (black circles and red squares spectra, respectively). These features account for the unchanged color of the solution and are typical of gold NPs cloaked by a protein corona.44 This suggests the NPs are passivated (with respect to aggregation or clustering) by a 10 ACS Paragon Plus Environment

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preferential interaction with the SAP contained in the sample, eventually bringing to the formation of isolated NP-protein corona (NP-PC) complexes (see later in the text for a thorough discussion). We then investigated the dynamic range of the nanoplasmonic assay. 10 mM

PBS solutions

containing a fixed concentration of pure EVs were spiked in with increasing amounts of SAP varying from 5 ng/µl to 1 µg/µl molar concentrations (10 mM PBS solution), covering the typical SAP concentration range commonly found in EVs prepared from biological fluids (see “EV purification and characterization” Section and Ref. 19,21). The samples were then loaded in a multi-well plate and the probe NP solution added (again to a final NP concentration of 2 nM). The most relevant results are displayed in Fig 3D. The addition of the NP probe to the EVs with increasing SAP amount is mirrored by a gradual change of the solution color from blue to red, indicative of gradual NP aggregation inhibition. NP aggregation was quantified through the aggregation index (AI), which can be defined for spherical gold NPs as the ratio of the LSPR absorption peak of pure NPs and the LSPR at some significant red-shift wavelength.19,45 In view of Fig. 3B and 3C spectra, here we chose LSPR absorption at 540 nm and 650 nm (AI = A540/A650). The obtained AI values are plotted against the SAP concentrations in Fig. 3E to yield a calibration curve for the SAP detection range of the nanoplasmonic assay, which hits a lower limit of 5 ng/µl of SAP, far below the typical SAP content in EVs preparations.19,21 Additional tests of the assay effectiveness against pure EVs, high SAP concentration (4 µg/µl) and human serum are reported in SI, Fig. S7.



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(nm)

(nm)

Figure 3. (A) 15 µl of the nanoplasmonic assay, that is an aqueous dispersion of 15 nm gold NPs at 6 nM concentration, upon the addition of 10 µl of a 10 mM PBS solution containing EVs+SAP (left tube) or EVs (right tube) derived from 1 ml of human serum. (B) UV/Vis/NIR absorption spectra of the assay before and after the addition of EVs and pure 10 mM PBS . (C) UV/Vis/NIR absorption spectra of the assay before and after the addition of EVs+SAP. Spectra where acquired with 1 nm step size and a data point every 25 nm highlighted with a symbol to ease comparison. (D) and (E) SAP progressive inhibition of NP clustering visually probed with a multi welled plate and quantified by the NP aggregation index (AI), respectively. In panel (E) the errors bars indicate the standard error of three different replicates. 12 ACS Paragon Plus Environment

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Determining EV concentration in pure EV preparations. The nanoplasmonic assay can be implemented to titrate pure EV preparations (viz. preparations containing negligible amounts of SAP). Titration grounds on the finding that in pure EV solutions the LSPR red shift, viz. the AI, of the NPs has a linear dependence to the NP/EV molar ratio, as shown in Fig. 4A and 4B. Since the molar concentration of any EV preparation is unknown, we used the 150 nm synthetic phosphatidylcholine (PCh) liposomes introduced in the Experimental Section (Fig. 2B) – which are synthetic vesicles that mimic EVs in both size and lipid composition – to build a calibration line. PCh liposome molar concentration can be easily calculated from the stoichiometry of the prepared PCh solution.30 In particular, 10 mM PBS solutions of PCh liposomes with concentrations ranging from 2.1·107 to 2.1·1010 vesicles/ml, viz. from 35 fM to 35 pM, were prepared. This range was chosen as it includes the pathophysiological EV concentrations reported in the literature.46 Each PCh liposome solution was then incubated with the 6 nM solution of 15 nm gold NPs in 1:1 volume ratio. The collected LSPR spectra are shown in Fig. 4C. Here we see that at the highest liposome concentration the spectrum (blue squares) is slightly shifted with respect to the one of isolated gold NPs reported in Fig. 3B and 3C, with the LSPR peak centered at about 540 nm. Then, as the liposome concentration decreases, the LSPR peak undergoes a gradual red shift and a decrease of intensity (red circles). This suggests that NP clustering is linearly dependent to the liposome concentration and becomes more and more prominent along with the decrease of the vesicle concentration.47 Interestingly the LSPR spectra of the samples containing the lower EV or PCh liposome concentration (yellow triangles up and red circles, presented respectively in Figure 4 A and C) are characterized by the presence of a shoulder in the near IR region. This spectroscopic feature is very likely ascribable to the presence of nanoparticles structuring in dimers, that can couple the LSPR transverse mode proper of isolated nanoparticles to a longitudinal mode due to the presence of interconnected particles.48,49



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By applying the calculations presented in our recent study on the interaction of gold NPs with giant unilamellar vesicles30 (see SI), we were able to evaluate the dependence of the clustering to the NP cross section/EV surface area, finding that that at the highest liposome concentration the overall surface of the NPs matches the overall surface of the external bilayer leaflet of the liposomes, and that as the liposome amount decreases the overall surface of the NPs exceeds the liposome one up to a factor of 10. This suggests that at the highest liposome concentration the amount of NPs is too low to saturate the overall liposome surface and clustering is negligible; clustering is then progressively triggered as the liposome/NP ratio decreases and gets to plateau for low vesicle concentration.30 Indeed, as suggested by comparison of fig. 4A and 4C, analogous scenario and reasoning hold if one has EVs in place of PCh liposomes. In fig. 4D the AI from fig. 4C is plotted as a function of the liposome concentration, and, as evidenced by the linear regression line, in the investigated conditions the parameters are in linear dependence. This calibration line finally allows to extrapolate the unknown concentration of any pure EV solution, provided it falls in the calibration range. For example, we determined the concentration of the EV preparation purified from human plasma used throughout this work. The AI value was obtained from the UV/VIS/NIR spectrum reported in Fig. 3B. In particular the star point in Figure 4D highlights the intercept of the AI value with the regression line, namely x = 4.5 ·109 vesicles/ml. Since the original EV sample was diluted to 1:12 vol/vol in a 10 mM PBS solution for UV/Vis/NIR spectrometry, from the value of x and of the dilution proportion we obtain the original concentration of the sample is equal to 5.4·1010 vesicles /ml. Remarkably, this value is consistent with the typical EV concentrations in human plasma.20 Titration was successfully replicated on another EV preparation separated from a different donor plasma and by using a different batch of NPs and PCh liposomes, proofing the robustness and repeatability of the titration method (SI, Fig. S8).


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(nm)

Figure 4. (A) UV/Vis/NIR absorption spectra of PBS solutions containing a fixed concentration of NPs and increasing concentrations of EVs, obtained after the dilution of the EV stock preparation (EVin is the dilution ratio of the stock solution). Spectra where acquired with 1 nm step size and a data point every 25 nm highlighted with a symbol to ease comparison. (B) AI obtained from Fig. 4A plotted versus EVin and linear regression fit (red line). (C) UV/Vis/NIR absorption spectra of PBS solutions containing a fixed amount of NPs and increasing concentrations of PCh liposomes. Spectra where acquired and represented as in panel A. (D) Calibration line obtained by plotting the NP AI obtained from Fig. 4C versus the PCh liposome solution concentration (black circles) and linear regression best fit (red line, R2= 0.99). Errors bars indicate standard error of three different replicates. The star point highlights the intercept of the AI value of the EVs preparation (obtained from the spectra of Fig. 3B) with the regression line. The star point projection on the abscissa axis allows to extrapolate the unknown concentration (x = 4.5 ·109 vesicles/ml).



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Further assessment of the interaction between NPs, SAP and EVs. The (bio)chemical/(bio)physical mechanism we propose for explaining the signal generation of the nanoplasmonic assay (sketched in Fig. 1) relies on the extension to EVs of our recently reported findings about the interaction of gold NPs with synthetic Giant Unilamellar Vesicles.30 In essence, our experimental evidences are in agreement with the claim that bare (citrate capped) gold NPs cluster at the EV surface, while the formation of a protein corona (PC)31 around the NPs strongly attenuates this phenomenon − this for NP concentrations in excess with respect to the full coverage (upon adsorption) of the EV external bilayer leaflet, see Ref. 30 for a throughout discussion −. To substantiate this hypothesis, we compared the interaction of the assay NPs and the same NPs passivated with a protein corona from human serum (hereafter referred as PCs) with EVs. Fig. 5 shows the UV/Vis/NIR spectra of a 2 nM water solution of bare gold NP (red squares), of a 2 nM PBS solution (PBS 10 mM) of PC (blue triangles) and of the PC solution after incubation with a 10 mM PBS solution of EVs of concentration of 4.2·109 vesicles/ml EVs (black circles). All the three spectra share the same main features, showing a LSPR peak centered at 540-550 nm, indicating that all the samples contain isolated NPs. Remarkably, the spectra of the PC+EV sample (grey triangles) definitively matches the one obtained for the NP+EV+SAP sample reported in Fig. 3C. This indicates SAP inhibits the interaction between NPs and EVs as a protein corona formed ex situ does. Interaction between EVs and PC was further confirmed through an AFM morphological analysis of EVs+NP and EVs+PC deposited on mica. As the AFM phase image of EVs+NP shows (Figure 5B), the presence of areas of the lipid bilayers with different composition and stiffness, visible as dark clusters that decorate the EV membrane (see inset), is consistent with the presence of NPs onto the membrane. Conversely, for the PC+EV sample (AFM phase image, Figure 5C) the presence of the PC limits the formation of NP clusters, as most of the PC can be found on the background of the mica surface and very few NPs can be visualized adsorbed at the EV surface (inset). 16 ACS Paragon Plus Environment

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To further prove this hypothesis in situ, we performed some equilibrium binding experiments with photon correlation spectroscopy.50 Applying this method we were able to measure the diffusion coefficients of the NP and of the EV+NP sample, that are related to the hydrodynamic sizes of the diffusing objects through the Stokes-Einstein relationship. The average of 30 autocorrelation curves for the NPs before and after the formation of the PC have been evaluated. Figure 5-D shows that the characteristic diffusion time of NPs increased in the presence of PC. In particular, PC formation drives a shift of the autocorrelation curve to higher decay times τ, while when PC are dispersed in the presence of EVs there is a small increase of the decay time that can be attributed to a partial interaction of PC with EVs. On the contrary the clustering of NP at the EV surface drove an higher shift of the autocorrelation curve that is similar to the one obtained for EVs. This hints to the presence in solution of object containing NPs with size similar to the size of the EVs. The ξ-pot for these hybrid NP-EV objects is different (-11.1 ± 1.3 mV) from the one of the NPs alone in water (-31.6 ± 3.3 mV) or the NPs dispersed in PBS (-24.8 ± 0.5 mV) (See Supp Fig S9) suggesting the presence of NP-Membrane complexes than simple NP aggregates. Moreover the fact that NP clustering at EV membrane, that is mirrored by a variation of the AI, has a linear dependence on the EV concentration in the studied range (See Figure 4 A-D) represents another genuine control of the ability of the assay to sense EVs in pure EV preparations. Taken together, all the above data point to the mechanism of signal generation of the assay that is summarized and described in Figure 1. In the presence of pure EV preps, NPs can freely interact with EV membranes clustering at their surface, while in the presence of SAP the NP preferentially interact with them forming a SAP corona which inhibits membrane clustering. Therefore, the NPs can be exploited as nanoplasmonic transducer of the presence of SAP or as nanoplasmonic gauge for the evaluation of the EV concentration.



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Figure 5. (A) Normalized UV/Vis/NIR spectra obtained for an aqueous solution containing 15 nm gold NP, and a 10 mM PBS solution containing the same NP coated by a human serum PC in the presence or absence of EVs. AFM Phase imaging of 15 nm gold NP. Spectra where acquired with 1 nm step size and a data point every 35 nm highlighted with a symbol to ease comparison. (B) and the same NP coated by a human serum protein corona (C) after their interaction with EVs and their deposition on a mica substrate. Inset reports a magnification of the EV boxed. NP Clustering over extended EV zone can be detected (dark area on EVs in B) while PC adsorption at EV surface results a limited event. (D) Autocorrelation curves obtained for an aqueous solution of 15 nm gold NPs (NP) at nanomolar concentration, the same NP coated by a human serum protein corona (PC) and dispersed in 10 mM PBS, 10 mM PBS solution of EVs (EVs), and a 10 mM PBS solution containing EVs+PC. or EVs+NP. The amplitude of the autocorrelation curves is normalized to 1 for better comparison of the characteristic diffusion time, τ. 18 ACS Paragon Plus Environment

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CONCLUSIONS We presented a nanoplasmonic colorimetric assay for probing by eye the presence of SAP contaminants in EV preparations and to determine the concentration of EVs in pure preparations. The assay applies to the whole multiscale population of EVs in body fluids, which comprises vesicles from 40 nm to 1 µm in size. The working principle of the assay is based on the fact that gold NP clustering at the EV membrane and the related LSPR shift is directly gauged by the amount of SAP contained in the preparation. Bare gold NPs adsorb and aggregate at the EV membrane in pure EV preparations, while this phenomenon is inhibited in SAP contaminated preparations by the SAP-NP interaction that leads to the formation of a passivating protein corona that keeps the NPs dispersed, inhibiting NP-EV interactions. The two conditions are optically characterized by a specific LSPR shift, which therefore acts as a color switch, giving a naked eye colorimetric read out of the presence of protein contaminants, that is visible down to 5 ng/µl of SAP, far below the typical concentrations of SAP in EVs preparations. Remarkably, the LSPR red-shift driven by NP clustering at the EV membrane is directly proportional to the EV concentration. This discloses the opportunity to exploit the assay for titrating EV pure preparations, which is another fundamental issue in EV sample analysis.20 The titration method resulted robust and repeatable, with a dynamic range from 35 fM to 35 pM, matching the range of EV concentration in body fluids. These findings are an effective application of nanotechnology in biology with immediate impact in EV biology − which strives for effective analytical procedures that fit the requirements of low volumes, reduced operative costs, fast readout and compatibility with common laboratory practices51,52 − and complement the available information on the interaction of NPs with 19 ACS Paragon Plus Environment


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biological membranes.53 On a wider perspective, the fact that the assay applies invariably to micro- and nano-vesicles prompts its extensions to other bio membrane objects, such as membrane coated viruses or organelles isolated from cell materials. Finally, it is worth noticing that this work widen to clinical and biological analytical chemistry the applications − to date all focused on drug loading and delivery54–58 aimed at exploiting, rather than avoiding, the NP-protein corona.59

ASSOCIATED CONTENT Supplementary data, figures and a fully detailed Materials and Methods section are provided as Supporting Information (SI). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected] Present Address †European Center for Nanomedicine CEN foundation, c/o Nanostructured Fluorinated Material laboratory, Department of Chemistry, Material and Chemical Engineering Milan Italian Polytechnic, Via Mancinelli 7, 20131 Milan.

Author Contributions DR selected and collected serum for EV purification. DM, LP,AZ and GDN, performed the experiments, with the exception of PCS experiments, that were performed by DB. DM, DR and

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PB conceived and supervised the work. All authors contributed to the conception of the experiments and discussion of the results, and contributed to writing the manuscript. All authors have given approval to the final version of the manuscript. Funding Sources This work was supported by MIUR (Ministero Italiano dell'Università e della Ricerca) through the project Soft Matter Nanostrutturata: dall'indagine chimico-fisica allo sviluppodi applicazioni innovative, PRIN 2010–2011 grant 2010BJ23MN to DM, DB and PB and by University of Brescia Research found (ex 60%) and ‘‘Prima spes onlus’’ foundation to DR, LP, AZ and GDN. ACKNOWLEDGMENT The authors would like to thank Sara Busatto for technical assistance, Kimberly Hamad Schifferli, Francesca Baldelli Bombelli, Ciro Chiappini and Eugenio Monti for fruitful discussions and suggestions. We thank Marco Vitale and Davide Dallatana for SEM consulting and Roberta Giuliani for use of UV-Vis spectrometer. Dr. Di Noto received a fellowship "Bando Mecenati" from "Fondazione CEUR".DM is also grateful to the Academia Delle Nanoscienze di Gagliato (Nanogagliato) for the Salvatore Venuta fellowship. ABBREVIATIONS NP, Nanoparticle; PC, protein corona; EVs, extracellular vesicles; SAP, single and aggregated protein contaminants; DC, differential centrifugation; SGF, sucrose gradient fractionation; SPM, Scanning Probe Microscopy.


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Colorimetric Nanoplasmonic Assay To Determine ... - ACS Publications

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