Time Evolution of Nanoparticle–Protein Corona in Human Plasma

Article pubs.acs.org/Langmuir

Time Evolution of Nanoparticle−Protein Corona in Human Plasma: Relevance for Targeted Drug Delivery Ana Lilia Barrán-Berdón,†,‡,§,# Daniela Pozzi,†,# Giulio Caracciolo,†,* Anna Laura Capriotti,‡ Giuseppe Caruso,‡ Chiara Cavaliere,‡ Anna Riccioli,⊥ Sara Palchetti,⊥ and Aldo Laganà‡ †

Department of Molecular Medicine, “Sapienza” University of Rome, Viale Regina Elena 291, 00161 Rome, Italy Department of Chemistry, “Sapienza” University of Rome, P.le A. Moro 5, 00185 Rome, Italy § Departamento de Química Física I, Universidad Complutense de Madrid, Viale Complutense s/n, 28040 Madrid, Spain ⊥ Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, Section of Histology and Medical Embryology, “Sapienza” University of Rome, Via A. Scarpa 14, 00161 Rome, Italy ‡

S Supporting Information *

ABSTRACT: When nanoparticles (NPs) enter a biological fluid (e.g., human plasma (HP)), proteins and other biomolecules adsorb on the surface leading to formation of a rich protein shell, referred to as “protein corona”. This corona is dynamic in nature and its composition varies over time due to continuous protein association and dissociation events. Understanding the time evolution of the protein corona on the time-scales of a particle’s lifetime in blood is fundamental to predict its fate in vivo. In this study, we used lipid NPs, the cationic lipid 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl] (DC-Chol) and the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE), that are among the most promising nanocarriers both in vitro and in vivo. Here, we investigated the time evolution of DC-Chol−DOPE NPs upon exposure to HP. On time scales between 1 and 60 minutes, nanoliquid tandem mass spectrometry revealed that the protein corona of DC-Chol− DOPE NPs is mainly constituted of apolipoproteins (Apo A-I, Apo C−II, Apo D, and Apo E are the most enriched). Since the total apolipoprotein content is relevant, we exploited the protein corona to target PC3 prostate carcinoma cell line that expresses high levels of scavenger receptor class B type 1 receptor, which mediates the bidirectional lipid transfer between low-density lipoproteins, high-density lipoproteins, and cells. Combining laser scanning confocal microscopy experiments with flow cytometry we demonstrated that DC-Chol−DOPE/HP complexes enter PC3 cells by a receptor-mediated endocytosis mechanism.

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INTRODUCTION Over the past few decades, there has been extensive interest in developing nanoparticles (NPs) as effective devices in view of their applications in the delivery of drugs, proteins, peptides,, and nucleic acids.1−5 Among existing ones, lipid NPs, vesicular systems in which the drug is confined to a water cavity surrounded by a lipid envelope, exhibit unique advantages. They include increased encapsulation capacity, stability of drugs, and protection of drugs from degradation, ability to target the drug to the site of action and controlled release properties. When lipid NPs enter a biological fluid (e.g., human plasma (HP)), biomolecules, especially proteins, compete for binding to the NP surface.6−12 Rapidly the identity of the bare NP is lost, while its fate is dominated by the layer of adsorbed proteins, the so-called “protein corona”. Unfortunately, opsonins such as immunoglobulins (Ig), fibrinogen, and complement proteins promote phagocytosis with rapid clearance of the NPs from the bloodstream. Despite undesirable implications, the “protein corona” possesses © 2013 American Chemical Society

favorable properties that can be exploited. First, adsorption of dysopsonins like human serum albumin (HSA), apolipoproteins etc. may promote prolonged bloodstream circulation. Furthermore, since corona formation on NPs is unavoidable, this could be used in a positive way for targeted drug delivery. Very promising results11 have given credence to the proof-ofconcept that (i) dysopsonins associated with the NP−protein corona provide a “do not eat me” signal to the macrophages; (ii) target cells can actually be activated via proteins associated to the NP-corona. Despite its feasibility, some aspects limit the application of the “protein corona” nanotechnology. One of the major drawbacks is that the NP-corona composition is not constant, but it changes with time due to continuous protein binding and unbinding events up to when final equilibrium is reached typically within a few hours.13−15 Therefore, the Received: March 30, 2013 Revised: April 15, 2013 Published: April 30, 2013 6485

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sulfate (SDS)-PAGE sample buffer to the pellet and boiling the solution. A 12% polyacrylamide gel was employed to separate the proteins as reported elsewhere.18 Coomassie PhastGel Blue R-350 was used to stain the gels with gentle agitation, in accordance with the manufacturer’s manual (GE Healthcare, Milan, Italy). All experiments were conducted three times to ensure the reproducibility of the NP− protein complex pellet sizes, general pattern, and band intensities on the 1D gels. To determine the molecular weights (MWs) of proteins after an electrophoretic run, protein MW markers were used. The MWs were finally obtained by means of Kodak dedicated software (Rochester, NY, USA). Protein Digestion and Peptide Desalting. The NP−protein complex pellet was redissolved in 40 μL of 8 mol/L urea in 50 mmol/ L NH4HCO3 (pH = 7.8). Afterward, protein tryptic digestion was conducted following the protocol already described elsewhere,18 employing 2 μg of trypsin. The digestion was stopped by adding TFA. Then, the resulting peptide mixture was passed through a solid phase extraction C18 silica cartridge for desalting, washed, and eluted with 500 μL of H2O:ACN (50:50, v/v) 0.05% TFA. After solvent removal in a Speed-Vac apparatus (mod. SC 250 Express; Thermo Savant, Holbrook, NY, USA), sample residue was reconstituted with 100 μL H2O 0.1% (v/v) HCOOH. All samples were stored at −80 °C until analysis. Nanoliquid Tandem Mass Spectrometry (NanoLC−MS−MS) and Data Analysis. NanoLC system was Dionex Ultimate 3000 (Dionex, Sunnyvale, CA, USA), equipped with a degasser and a thermostatted microwell-plate autosampler. Peptides were preconcentrated injecting 5 μL aliquot of sample onto a 300 μm i.d. × 5 mm Acclaim PepMap 100 C18 (5 μm particle size, 100 Å pore size) μprecolumn (Dionex); the loading pump was then operated for 5 min with H2O:ACN 98:2 (v/v) containing 0.1% (v/v) HCOOH at flowrate of 10 μL/min. The peptide mixture was separated on an in-house manufactured fritless silica column, 75 μm i.d. × 100 mm, packed with ReproSil-Pur C18-AQ 3 μm resin (Dr. Maisch GmbH), operated at a flow rate of 250 nL/min. Phase A was H2O and phase B was ACN, both with 0.1% (v/v) HCOOH. After an isocratic step at 5% B for 5 min, B was linearly increased to 30% within 75 min; then, B was increased to 80% within 5 min, and to 95% within the following 10 min to rinse the column. Mass spectrometry detection was performed by an LTQ-Orbitrap XL instrument (ThermoFisher Scientific, Bremen, Germany) with a nanospray source, operated in positive ion mode. Full MS spectra were acquired in the m/z range 350−1800 in the Orbitrap with the resolution set at 60 000, whereas the datadependent MS/MS scan of the five most intense monoisotopic peaks in the spectra was operated with collision-induced dissociation activation at low resolution in the LTQ. All MS/MS spectra were collected using a normalized collision energy of 35%, and an isolation window of 2 m/z. The whole LC−MS system was managed by Xcalibur software (v.2.07, ThermoFisher Scientific). Five technical replicates per sample were performed. Raw data files, obtained from Xcalibur software, were submitted to Proteome Discoverer (1.2 version, Thermo Scientific) for database search using Mascot (version 2.3.2 Matrix Science). Data were searched against human entries in the SwissProt protein database (57.15 version, 20266 sequences) selecting the built-in decoy option. Trypsin was specified as the proteolytic enzyme with up to two missed cleavages. Carbamidomethylation of cysteine and oxidation of methionine were set as fixed and variable modification, respectively. The monoisotopic mass tolerance for precursor ions and fragmentation ions were set to 10 ppm and 0.8 Da, respectively. To validate protein identifications derived from MS/ MS sequencing results, the Mascot output files (.dat) were submitted in the commercial software Scaffold (v3.1.2, Proteome Software, Portland, Oregon, USA; http://www.proteomesoftware.com/). The scaffold tool to integrate Mascot identification results with X!Tandem search engine results (performed in automatic with the same parameters settled for Mascot) was used. Only protein identification based on mass spectra correlating to at least two unique tryptic peptides were considered; minimum peptide identification probability was set at 95%, whereas protein identification probability was set at 99%. For protein quantitative analysis, Scaffold software allows the

understanding of the time evolution of the NP−protein corona is fundamental to address the challenges and future directions of drug delivery, in hopes of bringing it a step closer toward an effective clinical reality. Since the surface properties of lipid NPs are largely determined by their lipid envelope,16 cationic liposomes (CLs) are commonly used as model systems of lipid NPs. In this work, we used lipid NPs, the cationic lipid 3β-[N(N′,N′-dimethylaminoethane)-carbamoyl] (DC-Chol) and the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE), which are among the most promising nanocarriers both in vitro and in vivo. Here, we investigated the time evolution of DCChol−DOPE NPs upon exposure to HP. Most of the kinetic studies conclude that the length of incubation is an important determinant of the type and amount of absorbed protein13−15 and that final composition is finally reached after 1 h incubation. According to these indications, in this study incubation time ranged from 1 to 60 min. Remarkably, while a few minutes incubation is mimetic of blood-NP interactions when NPs are administered locally to tumors, 60 min exposure is a reasonable time to study interactions experienced by NPs in the systemic treatment of diseases.

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EXPERIMENTAL SECTION

Human Plasma: Human Plasma Collection, Preparation, and Storage. Human whole blood was provided by the Department of Experimental Medicine of “Sapienza” University of Rome. Mixed plasma was aliquoted into 200 μL volumes and stored at −80 °C in labeled Protein LoBind tubes (Eppendorf, Milan, Italy) to ensure plasma stability until further use. When used, aliquots were thawed at 4 °C and then left to warm at room temperature. Cationic Liposomes Preparation. Cationic lipid 3β-[N-(N′,N′dimethylaminoethane)-carbamoyl] (DC-Chol), zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). 1,2-Dioleoyl-sn-glycero-3phosphoethanolamine, 7-nitrobenzofurazan-labeled (NBD-DOPE) was purchased from Sigma Aldrich (Milan, Italy). The films of mixed lipids were prepared according to standard protocols,17 by dissolving appropriate amounts of lipids at ϕ = neutral lipid/total lipid (mol/mol) = 0.5. Lipid films were hydrated (final lipid concentration 1 mg/mL) with ultrapure water and stored at 4 °C. For size, zetapotential, and proteomics experiments, unlabeled lipids were used. For laser scanning confocal microscopy (LSCM) and flow cytometry experiments, NBD-DOPE was mixed with unlabeled DOPE to obtain labeled DC-Chol−DOPE CLs. For flow cytometry experiments, NBDDOPE was mixed with DC-Chol to obtain labeled DC-Chol CLs. In all the samples the concentration of fluorescently labeled NBD-DOPE was fixed at 7 × 10−3 mg/mL (fluorescent lipid/total lipid molar ratio ≈ 5/1000). Cationic Liposome-HP Complexes. DC-Chol−DOPE CLs were mixed with human plasma (1:1 v/v) and were incubated at 37 °C for 1, 30, and 60 min. This volume ratio was chosen because it is mimetic of in vivo condition.10,11 After incubation, the samples were centrifuged 15 min at 14000 rpm followed by pellet resuspension; this procedure was repeated three times to wash the sample and remove all the molecules not bound to the CLs. Size and Zeta-Potential Measurements. All size and zetapotential measurements were made on a Zetasizer Nano ZS90 (Malvern, U.K.) at 25 °C. Size measurements were made on the neat vesicle dispersions, whereas the samples were diluted 1:50 with distilled water for the zeta potential experiments to obtain reliable and accurate measurements. For all of the samples investigated, the data show a unimodal distribution and represent the average of at least five different measurements carried out for each sample. Results are given as means ± standard deviation of the five replicates. Proteomics Experiments. For separation by one-dimension polyacrilamide gel electrophoresis (1D-PAGE), the proteins of the samples were eluted from the particles by adding sodium dodecyl 6486

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normalization of the spectral countings (normalized spectral countings, NSCs) and offers various statistical tests to identify significant abundance differences in two or more categories. The mean value of NSCs obtained in the three experimental replicates for each protein was further normalized to the protein molecular weight (MWNSC) and expressed as the relative protein quantity by applying the following equation:

MWNSCk =

(NSC/MW)k N

∑i = 1 (NSC/MW)i

and extensive washing to remove the unbound proteins. After 1 min of exposure to HP, adsorption of plasma proteins leads to larger complexes (D ≈ 250 nm). After 60 min incubation, the size of NP−protein complexes reached its plateau value (D ≈ 470 nm). A marked drop in zeta-potential (from ca. 55 mV to ca. −14 mV) shows that the shortest incubation time (t = 1 min) is enough to form a “hard protein corona”,6−11 that is, a layer of strongly bound proteins, which are not washed away from the NP−protein complex during centrifugation and following resuspension in the original buffer. The “soft” corona is made of loosely associated (or unbound) proteins in dynamical exchange with the medium. This component does not precipitate “pelleting” and is removed together with the supernatant.7 One Dimensional SDS-PAGE Results. Figure 2A shows 1D SDS-PAGE of plasma proteins retrieved from DC-Chol−

100 (1)

where MWNSCk is the percentage molecular weight normalized NSC for protein k, and MW is the molecular weight in kDa for protein k. This correction takes into account the protein size and evaluates the actual contribution of each protein reflecting its relative protein abundance (RPA) in the “hard corona”.9 Cell Line. Human prostate cancer (PC3) cell line, derived from human bone prostate cancer metastasis, was purchased from ATCC (Manassas, VA, USA). PC3 cells were maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 IU/mL penicillinstreptomycin, 1 mM sodium pyruvate, 10 mM hepes, 1.5 mg/L sodium bicarbonate, and 10% fetal bovine serum (FBS) (SigmaAldrich, St. Louis, MO, USA). Laser Scanning Confocal Microscopy (LSCM). PC3 cells were seeded onto 24-mm round glass coverlips and incubated with fluorescently labeled DC-Chol−DOPE and DC-Chol−DOPE/HP NPs for 3 h. Then cells were fixed in paraformaldehyde 4% in phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO, USA) for 20 min. LSCM experiments were performed with a Leica TCS SP2 (Leica Microsystems Heidelberg GmbH, Germany). Flow Cytometry. An amount of 200.000 cells/mL/well were plated in 12-well dishes. After 24 h, fluorescently labeled DC-Chol− DOPE and DC-Chol−DOPE/HP were incubated with PC3 cells for 3 h. After the treatment the cells were detached with trypsin/ ethylenediaminetetraacetic acid (EDTA), washed two times with cold PBS, and run on a cyan cytometer (Beckman Coulter, Fullerton, CA, USA). Data were analyzed using FCS3 express software (De Novo Software, Los Angeles, CA, USA).

Figure 2. (panel A) 1D SDS-PAGE indicating (1) MW standard, (2) DC-Chol−DOPE NPs incubated 1 min with human plasma, (3) DCChol−DOPE NPs incubated 30 min with human plasma, (4) DCChol−DOPE NPs incubated 60 min with human plasma. (panel B) Histograms representing the total lane intensity of proteins recovered from DC-Chol−DOPE NPs incubated 1, 30, and 60 min with human plasma.

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RESULTS Size and Zeta-Potential. Size and zeta-potential experiments (Figure 1) showed that bare DC-Chol−DOPE NPs are

DOPE NPs after 1, 30, and 60 min of incubation with HP (lanes 2, 3, and 4, respectively). We observe that the protein pattern changes with increasing incubation time. To roughly estimate the time evolution of the whole protein content, we calculated the total lane intensities9 (Figure 2B). Even though high-abundance spots are likely underestimated due to staining saturation, total lane intensity is a good marker to compare protein contents. As evident, the total amount of proteins at 1 min of exposure is higher than those obtained at 30 and 60 min. However, we observe that the time evolution of single bands does not follow the same general trend. Within the limits of gel separation methodologies, content of proteins of similar molecular weight is compared by their relative band intensities. In Figure 3, we show the relative densitometry results of relevant bands from the gels in Figure 2A. To clarify, proteins were divided in three groups by their MW (0−100, 100−200, and 200−300 kDa). The following score of abundance, irrespective of exposure time, was found: 0−100 KDa > 200−300 KDa > 100−200 KDa. This finding suggests that DC-Chol−DOPE/HP complexes are rich in low MW proteins. One group of proteins (0−100 KDa) showed an initial decrease at 30 min of incubation followed by a minor increase at t = 60 min. This time behavior is typical of those proteins that are abundant in HP. In fact, upon exposure to biological fluids, the most abundant and the highest mobility plasma proteins are the first to cover NPs (Vroman effect), but are subsequently replaced by less motile proteins that have a higher affinity for the surface.

Figure 1. Size and zeta-potential of DC-Chol−DOPE NPs (t = 0 min) and DC-Chol−DOPE/human plasma NPs as a function of incubation time. Results are given as means ± standard deviation of five independent replicates.

positively charged (zeta-potential ≈ 55 mV) and small (hydrodynamic diameter, D ≈ 130 nm) vesicles quite homogeneous in size (pdi = 0.2) (representative size distributions are reported in Figure S1 in the Supporting Information). DC-Chol−DOPE NPs were incubated in HP for 1, 30, and 60 min, respectively, and the resulting NP−protein complexes were separated from excess plasma by centrifugation 6487

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proteins identified in the three coronas were calculated (see Table S1 in the Supporting Information). NanoLC−MS−MS results allowed us to roughly estimate the charge on the protein corona. This is a three-step calculation (Table S1 in the Supporting Information): (i) The charge at pH 7.4 of all the associated proteins is calculated by a dedicated software (http://www. scripps.edu/∼cdputnam/protcalc.html); (ii) The relative charge of each protein is calculated by multiplying the total charge on the protein k, qk, for its relative protein abundance, RPA;29 (iii) The total charge on the protein corona is calculated by Q = ∑k N= 1 qk, where N is the number of identified proteins. Interestingly, the calculated charge (Table S1 in the Supporting Information) shows a time evolution similar to that of the zeta-potential. To address the specific role of identified proteins, we restricted to classes of proteins (i.e., apolipoproteins, immunoglobulins, complement proteins, etc.) and single proteins with RPA ≥ 1 in, at least, on of the coronas (Table 1). We found that the most abundant proteins present in the corona of DC-Chol−DOPE/HP NPs are apolipoproteins, imnumoglobulins (Ig), complement proteins, fibrinogen, human serum albumin (HSA), and vitronectin. In particular, apolipoproteins were definitely the most abundant class of HP proteins associated with DC-Chol−DOPE NPs. We observe that the RPAs of IgG and complement proteins slightly decreased with incubation time, while that of fibrinogen remained roughly constant. On the other side, the RPAs of apolipoproteins and HSA rose with increasing incubation time. Since the total apolipoprotein content is relevant, we seek to exploit the protein corona to target PC3 prostate carcinoma cells that express high levels of scavenger receptor class B type 1 (SR-BI) receptor,30,31 which mediates the bidirectional lipid transfer between low-density lipoproteins, high-density lipoproteins, and cells. LSCM Results. To exploit the protein corona effect11,20 we treated PC3 prostate carcinoma cells with both DC-Chol− DOPE and DC-Chol−DOPE/HP NPs. To stress any possible difference, we compared the cellular uptake of complexes incubated for 1 and 60 min in HP. After 3 h of incubation with lipid NPs, cells were robustly washed and fixed. We find that DC-Chol−DOPE fluorescent NPs were found at the apical cell surface exclusively. As Figure 5A clearly shows, the introduced label is more frequently found throughout the cell (with exception of the nucleus). These results suggest that DCChol−DOPE NPs bind to and fuse with the plasma membrane, while the endocytic activity of the cells is negligible. Alternatively, if endocytosis occurs, LSCM results indicate that after adsorbing on the cell surface and being engulfed into endosomes, bare cationic liposomes largely fuse with the membranes of endosomes with the result that fluorescent molecules diffuse in the cytoplasm. In contrast, DC-Chol− DOPE/HP complexes are taken up as intact vesicles by PC3 cells (Figure 5B,C). In summary, LSCM experiments provided the following body of evidence: (i) in the absence of protein corona, lipid NPs fuse before being internalized (i.e., with the plasma membrane) or immediately after; (ii) in the presence of protein corona complexes are uptaken as intact vesicles; (iii) the cellular uptake seems to be irrespective of the incubation time in HP. Another question still to be answered is whether the protein layer on the nanoparticle surface mediates the binding to cells through a specific active mechanism or via nonspecific interactions with the cell surface, behaving as a simple coating. To answer this question, our strategy was to find out a correlation between the RPA of apoliproteins and the

Figure 3. Histograms representing the total band intensity of proteins recovered from DC-Chol−DOPE/human plasma NPs at different incubation times. Proteins are grouped by molecular weight.

On the other side, a single group of proteins (200−300 kDa) showed a monotonic increase in the band intensity. This trend is usually found for those proteins that are poorly abundant in HP but with high affinity for the NP surface. Taken together our results confirm previous observations that NPs bind different amounts of plasma proteins in a time-dependent fashion. Accurate identification of both major and minor NPassociated proteins is fundamental to decipher the biological role of the protein corona.6,18−29 To this end, we applied highresolution NanoLC−MS−MS analysis that allows a very precise quantification of adsorbed plasma proteins. NanoLC−MS−MS Results. Supplementary Table S1 summarizes the identification of all plasma proteins adhering to DC-Chol−DOPE NPs after incubation with HP. Remarkably, the repertoire of identified proteins was found to be rather limited (160, 200, and 134 for 1, 30, and 60 min, respectively). As a first step toward clarifying the time evolution of the protein corona, the numbers of both unique and common proteins for each formulation are generally considered. This can be visualized by the Venn diagram of Figure 4 that provides a

Figure 4. Venn diagram reporting the number of proteins identified onto the surface of DC-Chol−DOPE NPs incubated 1, 30, and 60 min with human plasma.

clear overview of the relationship existing between the proteins identified in the corona at different incubation times. The Venn diagram shows that the largest amount of proteins found in the protein corona of NP−HP complexes (127 proteins) binds to the lipid surface just after 1 min of incubation with HP. After 30 min of incubation, 46 new proteins are adsorbed, five of which remain bound after a further 30 min of incubation (t = 60 min). By using eq 1,9 the relative protein abundance, RPA, of all the 6488

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Table 1. Representative Corona Proteinsa Associated with DC-Chol−DOPE Nanoparticles Incubated 1, 30, and 60 min with Human Plasma, As Identified by NanoLC−MS−MS RPA identified proteins Apolipoprotein Apolipoprotein Apolipoprotein Apolipoprotein Apolipoprotein Apolipoprotein Apolipoprotein Apolipoprotein Apolipoprotein Apolipoprotein Apolipoprotein Apolipoprotein Apolipoprotein Complement Complement Complement Complement Complement Complement Complement Complement Complement Complement Complement Complement Complement Complement Complement Complement Complement

accession number

A-I OS = Homo sapiens GN = APOA1 PE = 1 SV = 1 A-II OS = Homo sapiens GN = APOA2 PE = 1 SV = 1 A-IV OS = Homo sapiens GN = APOA4 PE = 1 SV = 3 B-100 OS = Homo sapiens GN = APOB PE = 1 SV = 2 C−I OS = Homo sapiens GN = APOC1 PE = 1 SV = 1 C−II OS = Homo sapiens GN = APOC2 PE = 1 SV = 1 C−III OS = Homo sapiens GN = APOC3 PE = 1 SV = 1 C−IV OS = Homo sapiens GN = APOC4 PE = 1 SV = 1 D OS = Homo sapiens GN = APOD PE = 1 SV = 1 E OS = Homo sapiens GN = APOE PE = 1 SV = 1 F OS = Homo sapiens GN = APOF PE = 1 SV = 2 L1 OS = Homo sapiens GN = APOL1 PE = 1 SV = 5 M OS = Homo sapiens GN = APOM PE = 1 SV = 2

APOA1_HUMAN APOA2_HUMAN APOA4_HUMAN APOB_HUMAN APOC1_HUMAN APOC2_HUMAN APOC3_HUMAN APOC4_HUMAN APOD_HUMAN APOE_HUMAN APOF_HUMAN APOL1_HUMAN APOM_HUMAN

C1q subcomponent subunit A OS = Homo sapiens GN = C1QA PE = 1 SV = 2 C1q subcomponent subunit B OS = Homo sapiens GN = C1QB PE = 1 SV = 3 C1q subcomponent subunit C OS = Homo sapiens GN = C1QC PE = 1 SV = 3 C1r subcomponent OS = Homo sapiens GN = C1R PE = 1 SV = 2 C1s subcomponent OS = Homo sapiens GN = C1S PE = 1 SV = 1 C3 OS = Homo sapiens GN = C3 PE = 1 SV = C4−B OS = Homo sapiens GN = C4B PE = 1 SV = 1 C5 OS = Homo sapiens GN = C5 PE = 1 SV = 4 component C6 OS = Homo sapiens GN = C6 PE = 1 SV = 3 component C7 OS = Homo sapiens GN = C7 PE = 1 SV = 2 component C8 alpha chain OS = Homo sapiens GN = C8A PE = 1 SV = 2 component C8 beta chain OS = Homo sapiens GN = C8B PE = 1 SV = 3 component C8 gamma chain OS = Homo sapiens GN = C8G PE = 1 SV = 3 component C9 OS = Homo sapiens GN = C9 PE = 1 SV = 2 factor B OS = Homo sapiens GN = CFB PE = 1 SV = 2 factor H OS = Homo sapiens GN = CFH PE = 1 SV = 4 factor I OS = Homo sapiens GN = CFI PE = 1 SV = 2

Fibrinogen alpha chain OS = Homo sapiens GN = FGA PE = 1 SV = 2 Fibrinogen beta chain OS = Homo sapiens GN = FGB PE = 1 SV = 2 Fibrinogen gamma chain OS = Homo sapiens GN = FGG PE = 1 SV = 3 Ig Ig Ig Ig Ig Ig Ig Ig Ig Ig Ig Ig Ig Ig

C1QA_HUMAN C1QB_HUMAN C1QC_HUMAN C1R_HUMAN C1S_HUMAN CO3_HUMAN CO4B_HUMAN CO5_HUMAN CO6_HUMAN CO7_HUMAN CO8A_HUMAN CO8B_HUMAN CO8G_HUMAN CO9_HUMAN CFAB_HUMAN CFAH_HUMAN CFAI_HUMAN FIBA_HUMAN FIBB_HUMAN FIBG_HUMAN

alpha-1 chain C region OS = Homo sapiens GN = IGHA1 PE = 1 SV = 2 gamma-1 chain C region OS = Homo sapiens GN = IGHG1 PE = 1 SV = 1 gamma-2 chain C region OS = Homo sapiens GN = IGHG2 PE = 1 SV = 2 heavy chain V−III region VH26 OS = Homo sapiens PE = 1 SV = 1 kappa chain C region OS = Homo sapiens GN = IGKC PE = 1 SV = 1 kappa chain V−I region AG OS = Homo sapiens PE = 1 SV = 1 kappa chain V−II region GM607 (Fragment) OS = Homo sapiens PE = 4 SV = 1 kappa chain V−III region HAH OS = Homo sapiens PE = 2 SV = 1 kappa chain V−IV region B17 OS = Homo sapiens PE = 2 SV = 1 lambda chain V−I region HA OS = Homo sapiens PE = 1 SV = 1 lambda chain V−III region LOI OS = Homo sapiens PE = 1 SV = 1 lambda-2 chain C regions OS = Homo sapiens GN = IGLC2 PE = 1 SV = 1 mu chain C region OS = Homo sapiens GN = IGHM PE = 1 SV = 3 lambda-like polypeptide 5 OS = Homo sapiens GN = IGLL5 PE = 2 SV = 2

Serum albumin OS = Homo sapiens GN = ALB PE = 1 SV = 2 Serotransferrin OS = Homo sapiens GN = TF PE = 1 SV = 3 Vitronectin OS = Homo sapiens GN = VTN PE = 1 SV = 1

IGHA1_HUMAN IGHG1_HUMAN GHG2_HUMAN HV303_HUMAN IGKC_HUMAN KV101_HUMAN KV205_HUMAN KV312_HUMAN (+1) KV404_HUMAN LV102_HUMAN (+1) LV302_HUMAN LAC2_HUMAN IGHM_HUMAN IGLL5_HUMAN ALBU_HUMAN TRFE_HUMAN VTNC_HUMAN

t = 1 min

t = 30 min

t = 60 min

3.08 1.55 0.98 1.25 0.33 2.37 2.10 0.27 2.77 1.95 0.06 0.34 0.19 17.24 0.15 0.22 0.23 0.06 0.03 2.27 0.91 0.36 0.19 0.13 0.06 0.16 0.96 0.67 0.06 0.07 0.06 6.60 0.89 1.88 1.49 4.26 0.74 1.48 0.64 0.54 2.43 0.50 0.00 0.72 0.33 0.00 0.17 2.92 1.11 0.52 12.10 4.61 0.77 2.60

3.35 1.12 1.13 1.21 1.62 1.33 2.37 0.36 2.16 2.20 0.31 0.26 0.07 17.49 0.00 0.23 0.30 0.07 0.02 1.77 0.72 0.42 0.16 0.17 0.17 0.07 0.66 0.50 0.16 0.14 0.02 5.58 1.11 1.89 1.62 4.63 0.59 1.62 1.2 0.65 2.75 0.13 0.41 0.77 0.26 0.13 0.00 2.65 0.91 0.87 12.95 6.19 1.35 1.55

4.09 2.97 1.24 1.61 2.73 2.85 3.10 0.36 2.73 2.69 0.35 0.37 0.00 25.09 0.00 0.00 0.10 0.00 0.00 2.30 0.56 0.41 0.18 0.14 0.15 0.10 0.56 0.61 0.06 0.05 0.00 5.23 1.12 1.58 1.94 4.64 0.47 1.86 0.76 0.42 2.39 0.00 0.21 0.58 0.00 0.00 0.00 2.23 0.72 0.30 9.93 5.55 0.97 1.79

a

The table contains only the most significant hits, while the full list of the most abundant proteins identified by NanoLC−MS−MS is given in Table S1 in the Supporting Information. 6489

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Figure 5. Representative confocal images of DC-Chol−DOPE NPs (green) uptake by PC3 cells in the absence (A) and in the presence of protein corona after exposure to human plasma for 1 (B) and 60 min (C). Scale bars, 50 μm. High-magnification images of cells indicated by white arrows are shown to the right (scale bars, 10 μm). DC-Chol−DOPE fluorescent NPs were found at the apical cell surface exclusively. In contrast, when PC3 cells are treated with DC-Chol−DOPE/human plasma NPs a massive endocytosis of intact vesicles occurs.

incubated with the different formulations for 3 h at 37 °C. A low level of background fluorescence was demonstrated. In cellular uptake tests, DC-Chol−DOPE/HP complexes showed significantly higher uptake by PC3 cells compared to DC-Chol/ HP ones. In addition, the mean fluorescence intensity (Figure 6B) of DC-Chol−DOPE/HP complexes showed an intensity increase of about 44% with respect to DC-Chol/HP ones.

level of cell uptake in PC3 cells. To this end, we used DCChol/HP complexes as a reference. Indeed, DC-Chol/HP complexes exhibit virtually identical size, zeta-potential, and corona composition than DC-Chol−DOPE/HP complexes (data not reported), but a consistently low level of apolipoproteins (RPA < 8%). Flow Cytometry Results. The internalization of DCChol−DOPE/HP and DC-Chol/HP NPs by PC3 cells was determined by flow cytometry. Figure 6A and B show the cellular uptake of NP−protein complexes after cells were

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DISCUSSION Upon administration, NPs are exposed to biological fluids from which they adsorb proteins and other biomolecules to form a “protein corona”. Even though biological fluids contain a large variety of proteins, typical coronas contain only a few hundred proteins organized in a tightly bound immobile layer formed by the proteins with higher affinities for the particle surface (the hard corona) and a weakly associated mobile layer (the soft corona). One of the major drawbacks is that the NP-corona composition is not constant, but it changes with time due to continuous protein binding and unbinding events up until the final equilibrium is reached.13−15 As pointed out in the literature, the competitive adsorption of a complex mixture of plasma proteins for a NP surface may take from a few minutes up to several hours. While a few minutes incubation is mimetic of blood−NP interactions when NPs are administered locally to tumors, 1 h exposure is a reasonable time to study interactions experienced by NPs in systemic treatment of diseases. According to these indications, in this study we investigated the time evolution of DC-Chol−DOPE/HP complexes with an incubation time from 1 to 60 min. The DC-Chol−DOPE NP system was chosen because it is among the most efficient lipid formulations both in vitro and in vivo. First, NP−HP complexes have been characterized by dynamic light scattering (DLS), and zeta-potential once isolated from excess plasma (Figure 1). At the shortest incubation time (t = 1 min) initial formation of the hard corona leads to a significantly larger hydrodynamic diameter. Zeta-potential values for NP−protein complexes indicate that the relatively high zeta-potential (+ 55 mV) is reduced (to approximately −12 mV) even at short incubation time, suggesting that the protein coating itself is rapidly formed in plasma. Time evolution of both size and zetapotential of NP−HP complexes are consistent with a mechanism of protein-induced NP aggregation.9 Indeed, when a protein corona adsorbs on the NP surface, the decrease in zeta-potential weakens electrostatic repulsions with the result

Figure 6. (A) Flow cytometry charts showing the cellular uptake of DC-Chol−DOPE/human plasma and DC-Chol/human plasma NPs by PC3 cells. (B) The hystograms represent fluorescence values, which are expressed as the mean of triplicate samples ± SD within a single experiment. Asterisk (∗) denotes p < 0.05, Student’s paired t test. 6490

dx.doi.org/10.1021/la401192x | Langmuir 2013, 29, 6485−6494

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that aggregation occurs. According to literature,9 we can interpret the data to mean that lipid NPs insert themselves within plasma protein clusters leading to formation of NP− protein complexes. Despite the intrinsic complexity of the system, results for populations of multimers, aggregates, and the proteins extracted from their surfaces are highly reproducible.9 It has been shown that the size of nanoscale objects is highly influential in their biointeractions. In particular, knowledge of complex size is important for predicting the in vivo fate of the NP, since clearance cells recognize objects somewhat larger than ∼300 nm, while smaller NPs circulate longer, interact with nonspecialized cells, and cross biological barriers (e.g., the blood brain barrier). In addition, there is a general consensus that, once at the site of action, the most significant role of NP size is (co)determining the nature of the entry pathway of complexes into the cells.19 Related to that, we asked whether the protein corona of NP−protein complexes evolved over 1 h incubation with HP. As a first step to answer this question, 1D SDS-PAGE analysis is commonly employed. Although its resolution is low, 1D SDS-PAGE is particularly suitable for proteins with very high molecular mass, and/or with very high or low isoelectric points that are normally excluded from more accurate 2D-PAGE analysis. In particular, the total lane intensities provide a good estimation of the corona thicknesses (Figure 2 B); interestingly, Figure 2B is likely to indicate that the corona thickness of small size NPcomplexes (t = 1 min, Figure 1) is larger than that of larger size ones (t = 60 min). This is, at first sight, inconsistent with size evolution of complexes. However, this apparent contradiction can be resolved as soon as one recognizes that the formation of the protein corona is more likely not a property of the isolated proteins alone but a collective phenomenon. In this view, we suggest that larger size complexes are aggregates of NPs covered by thinner coronas.9 In Figure 3, we show the relative densitometry results of relevant bands from the gels in Figure 2A. We find that NPs bind different amounts of plasma proteins in a time-dependent fashion. In summary, combining size and zeta-potential results with 1D SDS-PAGE findings we showed that: (i) Adsorption of plasma proteins results in immediate formation of a protein corona on the NP surface whose thickness decreases with incubation time; (ii) NP− protein complexes aggregate to form clusters whose equilibrium size is reached within 1 h of incubation; (iii) The protein pattern changes with increasing incubation time, suggesting that less plentiful proteins act as competitive binders and facilitate the desorption of proteins with lower binding affinity. Accurate identification of both major and minor NP-associated proteins is fundamental to decipher the biological role of the protein corona.6,18,24−29 To this end, we applied high-resolution NanoLC−MS−MS analysis that allows very precise identification and quantification of adsorbed plasma proteins (Table 1). This allows us to illustrate more quantitatively the degree to which the biomolecule corona can change, depending on the NP residence time in the biological environment. The Venn diagram reported in Figure 4 shows that the largest amount of proteins found in the protein corona of NP−HP complexes (127 proteins) binds to the lipid surface just after 1 min of incubation with HP. In Table 1 we have reported classes of proteins (i.e., apolipoproteins, immunoglobulins, complement proteins, etc.) and single proteins with RPA ≥ 1 in at least one corona. NanoLC−MS−MS results suggest that the decrease in the intensity of the protein bands at 0−100 kDa (Figure 3) at t = 30 min is predominantly due to a decrease of the Apo C−II

and Apo D content as well as from a diminution in the content of proteins such as C4-b binding protein, prothrombin, vitronectin, and vitamin K. The slight increase in the intensity of the protein bands at 200−300 kDa (Figure 3) with incubation time is essentially due to an increase of the Apo B content. We found that the total apolipoprotein content is relevant (Apo A-I, Apo C−II, Apo D, and Apo-E are the most enriched) and increases with incubation time (RPA=18 and 25% at t = 1 and 60 min, respectively). It is quite remarkable to notice that, fibrinogen, which is one the most abundant proteins in the plasma (10−27 μmol/L), is less favored to adsorb with respect to proteins whose concentration is significantly lower. A clear example is given by vitronectin, whose concentration in human plasma is estimated to be around 3−5 μmol/L; at t = 1 min it is more abundant (∼2.6%) than any other fibrinogen chain (