Unveiling the in Vivo Protein Corona of Circulating Leukocyte-like

Unveiling the in Vivo Protein Corona of Circulating Leukocyte-like Carriers Claudia Corbo,*,†,‡ Roberto Molinaro,† Francesca Taraballi,† Naama E. Toledano Furman,† Kelly A. Hartman,† Michael B. Sherman,§ Enrica De Rosa,∥ Dickson K. Kirui,∥ Francesco Salvatore,‡,⊥ and Ennio Tasciotti*,†,# †

Center for Biomimetic Medicine and ∥Department of Nanomedicine, Houston Methodist Research Institute, 6670 Bertner Avenue, Houston, Texas 77002, United States ‡ CEINGE−Biotecnologie Avanzate s.c.a r.l., Via G. Salvatore 486, Naples, 80145, Italy § Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, Texas 77555, United States ⊥ Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Via Sergio Pansini 5, Naples, 80131, Italy # Department of Orthopedics and Sports Medicine, Houston Methodist Hospital, 6565 Fannin Street, Houston, Texas 77030, United States S Supporting Information *

ABSTRACT: Understanding interactions occurring at the interface between nanoparticles and biological components is an urgent challenge in nanomedicine due to their effect on the biological fate of nanoparticles. After the systemic injection of nanoparticles, a protein corona constructed by blood components surrounds the carrier’s surface and modulates its pharmacokinetics and biodistribution. Biomimicry-based approaches in nanotechnology attempt to imitate what happens in nature in order to transfer specific natural functionalities to synthetic nanoparticles. Several biomimetic formulations have been developed, showing superior in vivo features as a result of their cell-like identity. We have recently designed biomimetic liposomes, called leukosomes, which recapitulate the ability of leukocytes to target inflamed endothelium and escape clearance by the immune system. To gain insight into the properties of leukosomes, we decided to investigate their protein corona in vivo. So far, most information about the protein corona has been obtained using in vitro experiments, which have been shown to minimally reproduce in vivo phenomena. Here we directly show a time-dependent quantitative and qualitative analysis of the protein corona adsorbed in vivo on leukosomes and control liposomes. We observed that leukosomes absorb fewer proteins than liposomes, and we identified a group of proteins specifically adsorbed on leukosomes. Moreover, we hypothesize that the presence of macrophage receptors on leukosomes’ surface neutralizes their protein corona-meditated uptake by immune cells. This work unveils the protein corona of a biomimetic carrier and is one of the few studies on the corona performed in vivo. KEYWORDS: biomimicry, leukosome, in vivo corona, liposome, nanomedicine, protein corona, macrophages intestinal, or blood),12−14 protein source (human or murine),15,16 or physiological state of the donor’s blood (patient or healthy donors),17,18 (ii) physicochemical features of the NP, e.g., size,17 surface charge,19 shape,20 and type of functionalization/coating,21,22 and (iii) experimental conditions, e.g., incubation time,23 temperature,24 and choice of anticoagulant in plasma-based experiments.15 Despite earlier

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anoparticles (NPs) injected into the body interact with blood components that bind to their surface, giving rise to a layer of NP-surrounding biomolecules referred as the “protein corona” (PC).1 The PC induces changes in the physicochemical properties of NPs and strongly affects their biological fate in terms of cellular uptake, immune response, and targeting efficiency.2−4 For these reasons, characterization of PC composition has become a hot topic in the field of nanomedicine.5−11 We have learned that the PC composition changes in relation to (i) composition and type of surrounding milieu, i.e., type of biological fluid (pulmonary, © 2017 American Chemical Society

Received: January 17, 2017 Accepted: March 6, 2017 Published: March 6, 2017 3262

DOI: 10.1021/acsnano.7b00376 ACS Nano 2017, 11, 3262−3273

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Figure 1. Size and surface charge of nanoparticles at 10 min and 1 h postinjection. Dynamic light scattering distributions showing hydrodynamic diameter and zeta potential of liposomes and leukosomes without the corona and 10 min and 1 h following in vivo corona formation.

adsorption of specific plasma proteins to act as targeting molecules could induce NP distribution to selected organs, representing an alternative and elegant approach in nanomedicine.31 In our previous study, we have attributed the properties of leukosomes to the presence of leukocyte membrane proteins on their surface considering that nonfunctionalized liposomes did not exhibit the same capabilities. The aim of the present work is to characterize the composition of the PC of leukosomes and to gain insights into its impact on leukosomes’ interaction with cells to check if leukosome capabilities could be attributed also to specific PC effects. Liposomes have been used as a control.

studies that greatly improved our understanding of the principles and self-assembly mechanisms that lead to PC formation, caution should be exercised in extrapolating data from in vitro studies to predict the pharmacological fate of NPs or their potential clinical impact. The fact remains that in vitro studies cannot recapitulate all the conditions inherent to a whole biological system. These experiments are usually performed under a single set of environmental conditions featuring solutions at a constant ionic strength, protein concentration, and pH value. In contrast, NPs injected in vivo are exposed to solutions of varying ionic strengths, protein concentrations/compositions, and flows depending on the organ compartment they are traversing.25 As an example, Palchetti et al.26 demonstrated that the PC composition and structure of PEGylated liposomes changes considerably in dynamic versus static incubations, whereas most PC studies have been conducted in static conditions.26 On the other hand, one of the main obstacles of in vivo studies is the lack of wellestablished methods for the postinjection recovery of NPs. For this reason, very few studies about PC formation after in vivo administration of nanoparticles have been reported.27−29 We recently developed biomimetic leukocyte-like liposomes, termed leukosomes, that have a prolonged circulation time and accumulate at the site of localized inflammation in greater amounts than control liposomes.30 Leukosome synthesis involves the introduction of leukocyte membrane proteins in the liposomal lipid bilayer to obtain nanoparticles mimicking the activity of natural leukocytes. We hypothesized that the presence of leukocyte membrane proteins exposed on the leukosomes’ surface could induce changes in the adsorbed plasma proteins, for example, by specifically attracting serum proteins that are natural receptors of the leukosomes’ membrane proteins. Considering that functionalized NPs, when injected, can lose their targeting capabilities due to the presence of the PC, NPs able to selectively direct the

RESULTS AND DISCUSSION Characterization of Liposomes and Leukosomes. Physicochemical properties, morphology, and mechanical properties of NPs under investigation were studied by dynamic light scattering (DLS), ζ-potential measurements, cryo-electron microscopy (EM), and atomic force microscopy (AFM). Before in vivo administration, both liposomes and leukosomes were homogeneous in size (hydrodynamic diameter ≈ 170 nm) with a low polydispersity index (PDI) (≤0.08) (Figure 1a). The surface charge of liposomes and leukosomes was negative: − 34 and −40 mV, respectively (Figure 1b, Figures S1 and S2). Cryo-EM showed round, homogeneous, unilamellar vesicles and sizes consistent with those shown by DLS (Figure 2a). The NPs were then injected in BALB/c mice via the tail vein and maintained in circulation for 10 min and 1 h in order to study time-dependent PC formation around the NPs. At each time point, blood was withdrawn, plasma was isolated, and NPs were recovered using centrifugation followed by extensive washing, as previously described.26,32 DLS analysis of NPs after injection revealed the presence of heterogeneous populations of particles 3263


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that measured up to several micrometers (Figure S3). Peak force quantitative nanoscale mechanical (QNM) analysis (Figure 3e) revealed differences in the mechanical properties of bare NPs. Bare leukosomes were slightly stiffer than bare liposomes (Figure 3a,e), confirming our previous results.30 Once coated by a PC, we found particles exhibited nonhomogeneous mechanical properties, being stiffer in some areas and more flexible in others (Figure 3b,c). This was true for both liposomes and leukosomes at each time point. Taken together, these observations indicate that, after injection, (i) NPs lose their homogeneity in size and (ii) the PC coating is not always homogeneous, which is in agreement with findings reported by Hadjidemetriou et al.27,28 Once injected and recovered, both NP groups were populated by NPs not covered by a PC, while some were evenly covered and others, as we had hypothesized in vitro,32 were covered by nonhomogeneous layers of PC. Notably, these results represent an average of these different conditions. Quantitative and Qualitative Analyses of Proteins Constituting the Protein Corona. We quantified the proteins associated with recovered NPs to evaluate differences in protein-binding capability between liposomes and leukosomes. The amount of adsorbed proteins per micromole of lipid vesicles was slightly lower on leukosomes than on liposomes 10 min after injection. This difference in protein binding was more pronounced after 1 h in the bloodstream (Figure 4a). Proteins associated with equal quantities of lipids were resolved through gel electrophoresis (SDS-PAGE) and visualized with Coomassie Brilliant Blue staining (Figure 4b). A visual evaluation of SDS-PAGE for the analysis of protein band intensities (Figure 4b,c) confirmed that liposomes adsorbed more proteins than leukosomes. In addition, the protein adsorption profiles of both NPs are more abundant at 1 h than at 10 min. Bands not visualized at 10 min appeared, instead, at 1 h (Figure 4a, see arrows), which suggests that the PC in vivo continues to change over time. We also characterized the proteins constituting the PCs by mass spectrometry (MS). Protein bands were excised from the gel, subjected to in situ hydrolysis, and identified by MS. Table 1 lists the proteins of the two NPs identified in this study at the two time points. The number of proteins identified in the PC of liposomes and leukosomes at 10 min postinjection was similar, i.e., 38 and 35 proteins for liposomes and leukosomes, respectively. Instead, at 1 h postinjection, 54 proteins were identified in the PC of liposomes and 38 in the PC of leukosomes. Venn diagrams show common and exclusive proteins between liposomes and leukosomes at the two time points (Figure 5a,b). Protein adsorption profiles are qualitatively similar to 24 common proteins identified in the PC of the two NPs at 10 min and 27 proteins at 1 h. Eighteen proteins were common and present at both time points (Table 1, pink); three proteins were common at 10 min (Table 1, green), while seven proteins were common at 1 h (Table 1, blue). Exclusive sets of proteins were identified on leukosomes and liposomes at both time points; most of them changed between the two time points on the same NP formulation. For example, the 11 exclusive proteins adsorbed on leukosomes at 10 min are different from the 11 exclusive proteins on the same NP at 1 h (Table 1, violet and gray) except for clusterin, which is instead exclusively adsorbed by leukosomes and not by liposomes at both time points. To investigate the dynamic changes of the PC’s composition, we quantitatively analyzed the proteins. Table 1S lists the 25

Figure 2. Cryo-electron microscopy. Cryo-EM analysis reveals a spherical homogeneous shape for (a) bare liposomes and leukosomes. At (b) 10 min and (c) 1 h postinjection we observed the presence of vesicles not homogeneous in size.

as demonstrated by higher PDIs (>0.2). In general, both liposomes and leukosomes had increased hydrodynamic diameters after injection (Figure 1a, Figures S1 and S2) with main populations of ∼180 and ∼350 nm at 10 min and 1 h postinjection, respectively. The presence of populations with a hydrodynamic diameter smaller (∼80 nm) and larger (up to 3 μm) than bare NPs was revealed for both NPs, in agreement with other in vitro32,33 and in vivo27,28 studies. As shown in Figure 2b,c, similar to bare NPs, PC-coated NPs analyzed by cryo-EM were well dispersed and structurally intact. In addition, we observed that the PC was not homogeneously distributed on the NPs’ surface, and the presence of larger entities was confirmed (Figure 2b,c). We next performed AFM studies to investigate the mechanical properties of the NPs before and after injection. Bare liposomes and leukosomes were homogeneous in size with most populations in the same size range (100−200 nm) (Figure 3a,d). However, PC-coated NPs showed a heterogeneous scenario in which smaller PC-coated NPs coexisted with aggregates of NPs and proteins (Figure 3b,d) that seem to become bigger at 1 h postinjection (Figure 3c,d). Size distribution analysis revealed a wide size fluctuation ranging from hundreds of nanometers up to a few micrometers (Figure 3d). Intravital microscopy of leukosomes and liposomes in the skin microcirculation revealed particles (mainly leukosomes) 3264


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Figure 3. Atomic force microscopy. Representation of AFM topography maps and the corresponding Young’s modulus maps of (a) bare liposomes and leukosomes and (b) liposomes and leukosomes after 10 min and (c) 1 h incubation in vivo. (d) Size distribution of liposomes and leukosomes with and without corona. (e) Young’s modulus property quantification.

attributable to the effect of the PC, as stated and demonstrated by others.35 The identification of many common proteins in the PC of liposomes and leukosomes indicates that protein adsorption on the NP surface is mostly due to the lipid composition that in our two formulations is the same. On the other hand, differences in the amount of adsorbed proteins and the presence of exclusive sets of proteins in the PC of the two types of NPs suggest that the integration of leukocyte membrane proteins in the lipid bilayer reduces nonspecific interactions and can induce specific protein−protein interactions. Protein Classifications. We classified the protein data sets according to their molecular mass (Figure 5c). Overall, we observed that the PCs of liposomes and leukosomes were consistently enriched in low molecular weight proteins (≥65% with a MW < 60 kDa), irrespective of the injection time. This is particularly true in the case of proteins constituting the leukosome’s corona at 1 h (∼100% with a MW < 80 kDa). Despite this general common feature, we identified slight differences in the percent distributions of these protein groups (classified according to MW range) among the different NPs and time points (Figure 5c). For example, at 1 h, both PCs were mostly enriched in proteins with a MW of 40−60 kDa (∼50%). However, the second most abundant group of proteins at 1 h differed between the PC of the two particles:

most abundant proteins associated with liposomes and leukosomes at the two time points under investigation. Interestingly, vitronectin was the most abundant protein in the corona of both NPs 10 min after injection, while at 1 h, it was much less abundant in the PC of leukosomes and even less so for that of liposomes. Fibrinogen gamma had the exact opposite trend, as the most abundant protein at 1 h and over 10 times less abundant at 10 min (Table 1S). Among the exclusive proteins, clusterin was present only on leukosomes and not on liposomes, even if it is more abundant at 10 min than at 1 h. Clusterin, also known as apolipoprotein J, has attracted the attention of researchers because of its recent discovered role in the stealth effect of PEGylated nanocarriers.34 According to the study by Schottler et al., polystyrene NPs functionalized with PEG are surrounded by a PC mainly constituted of clusterin proteins whose presence has shown to be necessary for the stealth properties of the NPs. The revolutionary message of this recently published study is that the proteins adsorbed on NPs, which have long-been believed to only be responsible for their clearance, can also prevent nonspecific cellular uptake.34 Our finding that clusterin belongs to the group of proteins exclusive of leukosome’s PC allows us to hypothesize that the stealth properties of leukosomes previously solely attributed to their biomimetic nature (i.e., the presence of leukocyte membrane proteins in their surface) could possibly be additionally 3265


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Figure 4. Evaluation of in vivo protein corona profiles of liposomes and leukosomes. (a) Comparison of the amount of proteins adsorbed on liposomes and leukosomes 10 min and 1 h postinjection measured as the protein-binding capability (μg proteins/μmol lipids). (b) Nanoparticles were recovered and protein coronas associated with the same quantity of lipids were eluted in SDS loading buffer. The SDS PAGE (12% acrylamide) was carried out at 120 V for 1.5 h. (c) Intensity of bands relative to plasma proteins adsorbed onto nanoparticles’ surface analyzed with ImageJ software (y-axis: intensity, x-axis: molecular weight).

In Vivo Biodistribution and Interaction with Immune Cells. Then, we decided to investigate whether the groups of IgGs, complement, and coagulation factors identified in the PC of both liposomes and leukosomes could affect their clearance and uptake by immune cells and if the effect was different on the two NPs. We studied the biodistribution of leukosomes and liposomes in the liver and spleen through intravital microscopy. We have also checked their presence in the blood up to 1 h after injection (Figure 6). Long-term biodistribution (up to 24 h postinjection) has been previously described by us.30 The results confirmed our previous data,30 showing a lower accumulation of leukosomes in mononuclear phagocyte system organs (Figure 6a,b) compared to control liposomes. Furthermore, leukosomes also revealed a prolonged circulation time in blood (Figure 6c). These results could be due either to the differences between synthetic identities of leukosomes and liposomes or to the differences in the compositions of their PCs. To determine if the in vivo ability of leukosomes to avoid immune clearance could be due to the impact of their PC, we set up an in vitro experiment in which bare NPs and PC-coated NPs were incubated with J774 macrophages in serum-free conditions. If the proteins (e.g., complement, coagulation, IgGs) in the PC are in their correct orientation and not masked from others, they should be able to bind to their receptors on macrophages and promote uptake. We found that while the PC induced an increased macrophage uptake of liposomes,

on leukosomes, 30% of proteins had MWs of 60−80 kDa and on liposomes 15% of proteins had MWs of 150−300 kDa. Also, the MWs of proteins adsorbed on both liposomes and leukosomes changed their distribution over time, confirming that the PC changes over time.28 We then sorted the proteins based on their charge, in terms of pI values (Figure 5d). As we demonstrated in vitro,32 negatively charged proteins were preferentially bound by liposomes and leukosomes (∼60% with a pI < 6), irrespective of injection time. This value was slightly lower for proteins surrounding the leukosomes at 1 h postinjection, in which 50% of proteins had a pI < 6, negatively charged at physiological pH. These results are in line with previous work on the characterization of liposome PC36 and with other studies concerning inorganic NPs37,38 and confirm that electrostatic attraction is not the only force driving interaction between NPs and proteins in biological fluids. We also categorized the identified proteins on the basis of their biological function (Figure 5e). From this standpoint, proteins associated with liposomes and leukosomes maintain a very similar distribution trend. Notably, there was a strong fluctuation in function distribution at different time points. While tissue leakage was the most represented function at the early time point, the most frequent function after 1 h was coagulation, which confirms the PC’s evolution in function over time. 3266


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ACS Nano Table 1. Proteins Identified in the PC of Liposomes and Leukosomes 10 min and 1 h after in Vivo Injection

leukosomes’ PC induced a reduction in their uptake by macrophage (Figure 7a,b). This could be due to the different abundance of these groups of proteins in the PCs of leukosomes and liposomes, to a different orientation of the

proteins within the two PCs, or to a combination of these effects. The proteins in the corona are, in general, attached to the NPs mainly through electrostatic and hydrophobic interactions. 3267


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Figure 5. Proteins identified on the PC following the in vivo experiment. Venn diagrams showing common and exclusive proteins adsorbed (a) 10 min and (b) 1 h postinjection on liposomes (green) and leukosomes (red). Protein corona proteins were classified according to their (c) molecular weight, (d) isoelectric point, and (e) function. Values reported represent molar percentage.

Figure 6. Biodistribution of liposomes and leukosomes. Intravital microscopy (IVM) images of spleen, liver, and blood showing the presence of liposomes and leukosomes (red) together with macrophages (blue) within the vessels (green). The graphs below show the quantification of liposomes (black) and leukosomes (red) as area fraction in each image over time.

In the case of leukosomes, which expose macrophage receptors30 on their surface, the serum proteins in the PC

can also be adsorbed through a receptor-to-ligand binding. This implies that their recognition domain is oriented toward the 3268


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Figure 7. Macrophage uptake in vitro. Impact of protein corona on liposome and leukosome uptake by J774 macrophages. (a) Confocal images of J774 cells treated for 1 h with bare and PC-coated NPs. Cell membranes are WGA-Alexa Fluor 488 stained (green), nuclei are DAPI-stained (blue), and particles are rhodamine-labeled (red). (b) Flow cytometry. Histograms show the average fluorescence of three experiments ± SD. Liposomes with a PC had an increased uptake by J774 than bare liposomes. An opposite trend was observed for leukosomes. PC, protein corona.

Figure 8. Schematic of the macrophage uptake. Macrophage uptake of liposomes and leukosomes when the PC is adsorbed on their surface. Leukosomes are leukocyte-like liposomes with surface receptors for IgG, coagulation factors, and complement proteins. Liposomes are their non- biomimetic counterpart. IgG, coagulation factors, and complement proteins have been identified in the PC of both liposomes and leukosomes. However, the protein corona has an opposite impact on the macrophage uptake of these NPs: it increased the uptake of liposomes and decreased the uptake of leukosomes. Our hypothesis is that the presence on the leukosome’s surface of leukocyte receptors favors the interaction of IgG and other proteins with their receptors on the leukosomes, and this makes them unavailable to bind their receptors on macrophages. In this case of liposomes, instead, a regular opsonization process occurs. Elements are not drawn to scale.

CONCLUSIONS Given the potential of NPs as drug and gene delivery systems,39 attempts have been made to allow NPs to avoid the immune system and target diseased tissue. Among these, functionalization with poly(ethylene glycol) (PEG),40 i.e., PEGylation, is the

leukosomes and, as a consequence, not available to be recognized by macrophages. On the other hand, proteins adsorbed on liposomes can be randomly oriented so the liposomes are more subject to macrophage recognition and consequent uptake (Figure 8). 3269


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respectively. Smaller lipid vesicles were obtained by repeated extrusions through 200 nm polycarbonate filters. Animal Treatment and in Vivo Protein Corona Formation. The study was conducted according the Guide for the Care and Use of Laboratory Animals and was approved by the Houston Methodist Institutional Animal Care and Use Committee. Healthy BALB/c mice were purchased from Charles River Laboratories International, Inc. (Wilmington, MA, USA). After anesthetization, mice were intravenously injected via the tail vein with liposome and leukosome solutions at a lipid dose of 50 mg/kg body weight. Blood (∼800 μL) was withdrawn by cardiac puncture 10 min and 1 h after injection using 50 mM ethylenediaminetetraacetic acid as an anticoagulant. The blood volume withdrawn from each mouse was calculated according to the Lee and Blaufox equation: BV = 0.06 × BW + 0.77, where BV = blood volume (mL), BW = body weight (g), and plasma volume = BV/2.48 Recovery of Protein Corona-Coated Nanoparticles after in Vivo Injection. Blood collection tubes were inverted several times to mix blood with the anticoagulant. Plasma was isolated from blood by centrifugation for 15 min at 1000g. The plasma of three mice for each time point was pooled. The PC-coated NPs were then recovered by centrifugation at 15000g followed by washings in PBS, as previously described.32 The same procedure was applied to the blood isolated from PBS-injected mice used as controls to verify the absence of a pellet due to protein precipitation. All experiments were performed in triplicate. Size and Zeta Potential Experiments. Size and surface charge of NPs before and after injection in mice were measured with a Multisizer 4 Coulter Counter (Beckman Coulter Inc., Miami, FL, USA) and ZetaSizer Nano ZS (Malvern Instruments Ltd., Southborough, MA, USA), respectively. A 20 μL amount of samples was added to 1 mL of twice-distilled water. Three measurements per sample were collected. Cryo-electron Microscopy. Both liposomes and leukosomes were plunge-frozen on holey film grids (R2x2 Quantifoil; Micro Tools GmbH, Jena, Germany). A 626 cryo-specimen holder (Gatan, Inc., Pleasanton, CA, USA) was employed for imaging. Data were collected on a JEOL 2100 electron microscope (JEOL Ltd., 3-1-2 Musashino, Akishima, Tokyo, Japan). Images were recorded under low-electrondose conditions (5−20 electrons/Å2) using a 4096 × 4096 pixel CCD camera (UltraScan 895, Gatan, Inc.) at a nominal magnification of 20 000×. Atomic Force Microscopy. Peak force QNM enables quantitative measurement of modulus, adhesion, or deformation at the nanoscale. Quantitative mechanical properties of liposomes and leukosomes with or without PC were mapped at the molecular scale using the peak force QNM mode of a MultiMode 8 AFM from Bruker (Billerica, MA, USA) (resonance frequency 10 kHz, nominal tip radius of curvature 10 nm, force constant 0.04 N/m). Mean values from 50 random particles in three independent experiments are reported. We prepared the samples by coating the mica surface sample holder with 0.1% APTES to prevent NPs from collapsing on the mica surface. The elastic modulus was calculated with NanoScope software version 1.5 using the following equation, reported elsewhere:32

gold standard. It was long-believed that the antiopsinization effect of PEGylation was due to the hydrodynamic radius created by PEG that reduced adsorbed proteins;41−43 however, Schottler et al. recently discovered that the presence of specific proteins in the PC is required for the stealth effects of PEGylated NPs.34 Additionally, PEGylation has also been reported to impair the delivery of therapeutics due to the induced immune response subsequent to an enhanced serum protein binding, thus reducing cell targeting and increasing blood clearance.44 Biomimetic strategies have been developed to overcome the limitations of such approaches.45 The proofof-concept at the basis of this approach is to imitate cells and/ or pathogens that elude the immune system by inhibiting or reducing the complement activation. In 1989, Allen et al. developed liposomes whose membranes imitated the phospholipid composition of erythrocytes.46 Previously, we showed how to transfer the features of leukocytes (free circulation in the bloodstream and targeting of inflamed endothelium) onto synthetic vesicles by coating mesoporous silica nanoparticles.47 More recently, we developed biomimetic leukosomes, by directly combining purified leukocyte membranes with synthetic biocompatible phospholipids.30 Leukosomes showed prolonged circulation time and improved accumulation in inflamed tissues compared to control liposomes.30 Given the crucial role in determining NP functionalities recently attributed to the PC, we decided to investigate if the integration of leukocyte membrane proteins in the liposome bilayer could somehow affect the adsorption of proteins in the corona. Considering the gap existing between in vitro and in vivo results,27 we studied the time evolution of the PC formed in vivo. Our results revealed that the integration of leukocyte membrane proteins into the leukosome bilayer affected the number, amount, and type of plasma protein adsorbed. This could be due to a masking effect of the integrated proteins that induce a reduction in nonspecific interactions. On the other hand, we believe that the presence of proteins on the leukosome’s surface could promote the adsorption of specific proteins over others. Also, even when the same sets of proteins are adsorbed by both liposomes and leukosomes, their orientation could be different due to different binding mechanisms (Figure 8). Here we report that also biomimetic NPs adsorb a PC and that this corona, as for the other NPs, affects their biodistribution and interaction with cells. To the best of our knowledge, this is one of the few works studying the PC formed around NPs after in vivo administration and in which the PC of bioinspired NPs has been investigated. While this study helps to improve our understanding of the overall properties of leukosomes, further studies are needed to define the potential role of PC proteins in the capabilities of leukosomes. We aim also to study if the set of proteins adsorbed by leukosomes and not by liposomes have a role in the stealth properties of this platform.

F − Fadh = 4/3E* R(d − d0 )3 where F − Fadh is the force on the cantilever relative to the adhesion force, R is the tip end radius, d − d0 is the deformation of the sample, and E* is the reduced modulus. Quantification of Plasma Proteins Adsorbed on Nanoparticles. The amounts of lipids and proteins in PC-coated NPs recovered after in vivo administration were analyzed to calculate the protein-binding capability of NPs expressed as amount of proteins/ lipid concentration. First, liposomes and leukosomes were quantified using the phospholipid colorimetric assay kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s protocol. Lipid concentrations were calculated according to a standard curve. The amount of proteins within the corona was determined by Bradford assay (Bio-Rad) using bovine serum albumin at a known concentration as the standard to build a five-point standard curve (R2 = 0.99).

EXPERIMENTAL SECTION Liposome and Leukosome Preparation. Liposomes and leukosomes were prepared with the thin-layer evaporation method followed by hydration and extrusion, as described previously.30 Briefly, phospholipids and cholesterol (DPPC:DOPG:DSPC:CHOL) at a molar ratio of 5:3:1:1 (Avanti Polar Lipids, Alabaster, AL, USA) were dissolved in a chloroform/methanol mixture (3:1 v/v), and then the formation of the films was induced through a rotary evaporator (Büchi Labortechnik AG, Flawil, Switzerland) at 45 °C. Films were hydrated with phosphate-buffered saline (PBS) or PBS dispersed with leukocyte membrane proteins to formulate liposomes and leukosomes, 3270


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ACS Nano Protein binding values (μg protein/μM lipid) are recorded as an average of three experiments. Separation of Proteins in the Corona by One-Dimensional Gel Electrophoresis. Proteins surrounding the NPs were dissolved in Laemmli buffer and heated for 5 min at 90 °C before being loaded and resolved onto 10% Mini-PROTEAN TGX precast gels (Bio-Rad Laboratories, Hercules, CA, USA) for 1 h at 120 V. Proteins were stained with Coomassie Brilliant Blue (Fisher Scientific, Fair Lawn, NJ, USA) overnight followed by extensive washing in distilled water. Protein bands were excised from the gels, cut in small pieces, destained using acetonitrile, and then in situ digested as previously described.49−51 Briefly, destained protein bands were reduced and alkylated with 10 mM dithiothreitol for 1 h at 57 °C and 55 mM iodoacetamide for 45 min at room temperature, respectively. Gel pieces were then washed to remove all the excess of reagents and then digested with trypsin (Promega Corporation, Madison, WI, USA) overnight at 37 °C. Mass Spectrometry. The peptide mixtures derived from the digestion of each single band were analyzed by liquid chromatography MS/MS using a NanoAcquity UPLC system (Waters, Milford, MA, USA) coupled to a Synapt HDMS (G1) ESI mass spectrometer, as already described.52 Peptide mixtures were separated by liquid chromatography using the following gradient: from 97% of solvent A (0.1% formic acid in water) and 3% to 40% of solvent B (0.1% formic acid in acetonitrile), in 120 min at 300 nL/min, using a 75 μm × 250 mm BEH130 C18 (1.7 μm particle) analytical column (Waters). Proteins were identified using the Protein Lynx Global Server (PLGS v2.4; Waters Corporation) using Uniprot 2013_03 reviewed mouse proteome (16 614 entries) as the target database. One trypsin missed cleavage and oxidation of methionine were used as variable modifications, while carbamidomethylation of cysteins was the only allowed fixed modification. Quantitative values were obtained using 100 fmol/sample of yeast alcohol dehydrogenase as internal standard and analyzed with the Identity E algorithm.53 Intravital Confocal Microscopy. Intravital imaging was performed under protocol AUP 0611-0032 in accordance with the guidelines of the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals and The Houston Methodist Institutional Animal Care and Use Committee. Intravital imaging was performed on anesthetized mice after surgery to expose the tissue of interest. Anesthesia was maintained by inhalation of 2% isoflurane during surgical and imaging procedures. Under the microscope TEXAS-Redlabeled particles (liposomes and leukosomes) and tracer (FITClabeled 70 kDa dextran) were administered via retro-orbital injection. Tracer was used to delineate the vasculature. Images and videos were acquired using an upright Nikon A1R laser scanning confocal microscope equipped with a resonance scanner and motorized and heated stage. All settings including laser power, gain, offset, and pinhole diameter were maintained the same throughout each acquisition. All images were analyzed with NIS-Elements software to quantify the amount of particles in different tissues at different time points. Data were obtained by averaging results on at least three images from three mice. Macrophage Uptake. The J774 mouse macrophage cell line (ATCC TIB-67) was used for these studies. Cells were cultured using Dulbecco’s modified Eagle’s medium with 10% (v/v) fetal bovine serum and 1% (v/v) antibiotics solution. Cells were passaged at 80% confluence. Cellular uptake of liposomes and leukosomes was studied by confocal fluorescence microscopy and flow cytometry. For the confocal microscopy experiment, 15 000 cells/well were seeded onto an eight-well chamber slide and treated with 0.15 mM bare NPs and PC-coated NPs (rhodamine-labeled) in serum-free conditions for 1 h at 37 °C. At the end of the incubation time, cells were washed, fixed in 4% paraformaldehyde solution, and stained: nuclei (blue) were stained using DAPI, and cell membranes (green) were stained using WGAAlexaFluor 488 conjugated. We used a Nikon A1 confocal imaging system to take images and the NIS Elements AR to analyze them. Flow cytometry analysis was performed to quantitatively analyze the uptake. The day before the experiment, 200 000 cells/well were seeded

in a 12-well tissue culture plate. Then, cells were treated with the particles as described above. After treatment, cells were washed and then transferred into the tubes for the analysis. We used a Becton Dickinson LSR II instrument equipped with Diva software. Each sample was evaluated collecting 10 000 events. Results are the average of three independent experiments.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b00376. Additional data including DLS details, intensity, number and volume distributions, leukosomes’ and liposomes’ size in circulation within skin vessels, and top 25 most abundant proteins associated with the protein corona of liposomes and leukosomes at the two time points under investigation (PDF)

AUTHOR INFORMATION Corresponding Authors

*Tel (C. Corbo): +1 832-266-7235. E-mail: corbo@ceinge. unina.it. *Tel (E. Tasciotti): +1 713-441-7319. E-mail: etasciotti@ houstonmethodist.org. ORCID

Claudia Corbo: 0000-0001-6468-2571 Francesca Taraballi: 0000-0002-4959-1169 Ennio Tasciotti: 0000-0003-1187-3205 Author Contributions

C.C. designed, performed, and analyzed the experiments, interpreted the data, and wrote the paper. R.M. synthesized the nanoparticles and performed in vivo experiments; F.T. performed AFM; N.T.F. performed cell uptake; K.H. performed DLS; M.S. performed cryo-EM; E.D.R. and D.K. performed IVM; F.S. edited the paper; E.T. supervised the studies and edited the paper. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors thank D. A. Engler, R. K. Matsunami, and the HMRI Proteomics Programmatic Core Laboratory for mass spectrometry analyses and J. A. Gilder (Scientific Communication srl., Naples, Italy) and M. Livingston for editing the text. The authors acknowledge the Sealy Center for Structural Biology and Molecular Biophysics at the University of Texas Medical Branch at Galveston for providing research resources. This work was supported by grants RF-2010-2318372 and RF2010-2305526 from the Italian Ministry of Health, The Regenerative Medicine Program Cullen Trust for Health Care (Project ID 18130014), Brown Foundation (Project ID 18130011), the Hearst Foundation (Project ID 18130017), and Cancer Prevention & Research Institute of Texas (Project ID RP170466) to E.T. and by POR Campania FSE 2007−2013 Project DIAINTECH, Italy (to F.S.). The schematic in Figure 8,, has been kindly drawn by C. Boada (https://www.behance. net/JuanitoMalafacha). REFERENCES (1) Lundqvist, M.; Stigler, J.; Elia, G.; Lynch, I.; Cedervall, T.; Dawson, K. A. Nanoparticle Size and Surface Properties Determine the 3271


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