Protease Degradation of Protein Coronas and Its Impact on Cancer

Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Protease Degradation of Protein Coronas and Its Impact on Cancer Cells and Drug Payload Release Cristina Rodriguez-Quijada,† Helena de Puig,∥ Maria Sań chez-Purra,̀ † Chandra Yelleswarapu,‡ Jason J. Evans,§ Jonathan P. Celli,‡ and Kimberly Hamad-Schifferli*,†,∥

ACS Appl. Mater. Interfaces Downloaded from pubs.acs.org by UNIV OF LOUISIANA AT LAFAYETTE on 04/12/19. For personal use only.

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Department of Engineering, ‡Department of Physics, and §Department of Chemistry, University of Massachusetts Boston, Boston, Massachusetts 02125, United States ∥ Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States S Supporting Information *

ABSTRACT: The effect of matrix metalloproteinases (MMPs) on preformed protein coronas around spherical gold nanoparticles (AuNPs) was studied. Protein coronas of different compositions (human serum, human serum albumin, and collagen IV) were formed around AuNPs and characterized. The protease MMP-9 had different effects on the corona depending on the corona composition, resulting in different changes to the corona hydrodynamic diameter (DH). When incubated with PANC-1 cells, the corona showed evidence of both increases as well as decreases in DH. Varying the composition of the corona influenced the MMP-9 activity. Furthermore, the corona was influenced not only by the protease activity of the MMP-9 but also by its ability to exchange with proteins in the preformed corona. This exchange could also occur with proteins in the media. Thus, the net effect of the MMP-9 was a combination of the MMP-9 protease activity and also exchange. Time scales for the exchange varied depending on the nature that make up the protein corona (weakly vs strongly bound corona proteins). Mass spectrometry was used to probe the protein corona composition and supported the exchange and degradation model. Together, these results indicate that the mechanism of protease activity on AuNP coronas involves both rearrangement and exchange, followed by degradation. KEYWORDS: protein coronas, protease, nano-bio interface, nanoparticles, hydrodynamic diameter, cell uptake

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INTRODUCTION Biomolecules found in high concentration in biological fluids obscure the surface of functionalized nanoparticles (NPs) by the spontaneous formation of a cloud known as the protein corona.1−3 Because the protein corona is what ultimately interacts with cells and the biological environment, it strongly influences the NP uptake by cells, and as a result their biodistribution in organisms,4,5 and is thought to play a major role in nonspecific NP delivery and toxicity.6−8 Because the NP material, size, shape, and surface chemistry directly impact the formation of the protein corona, NPs can be engineered to improve their biological identity (ID).9−13 The composition of the protein corona that forms is now understood to be “personalized,” where its composition varies depending on the disease state of the individual, such as cancer versus healthy patients.14−16 Protein corona design is now a major factor in tailoring NPs for chemotherapeutic drug delivery, targeting strategies for cancer treatment and diagnosis, or to personalize liposomal cancer therapies.17−20 However, one aspect of protein coronas that has been relatively unexplored is the effect of proteases on their properties. This is particularly important for use of NPs in cancer therapies as there is a high protease expression in cancer cells, in particular, matrix metalloproteinases (MMPs).21 These © XXXX American Chemical Society

proteases help in the proliferation of cancer cells throughout the healthy tissue by degrading the extracellular matrix. Therefore, MMP activity will degrade the protein corona formed around the NP and thus change its biological performance in the tumor microenvironment.22 This degradation would depend on the corona composition and also the protease’s access to the proteins constituting the corona. Issues such as layering and crowding could obscure or hinder cleavage of the protein substrates. Furthermore, MMPs are proteins themselves and hence may exchange with proteins in the protein corona, giving rise to a complex net effect.1,23 Here, we study the effect of MMPs on protein coronas formed around spherical gold nanoparticles (AuNPs) using an established pancreatic ductal adenocarcinoma cell line (PANC1) (Figure 1). This cell line is known to express MMPs, particularly MMP-2 and MMP-9.24 We investigated the impact of proteases when changing the protein corona composition with different model proteins. Proteins were chosen based on their interaction with the NPs (weakly and strongly bound corona proteins) as well as with the proteases secreted by Received: January 15, 2019 Accepted: April 1, 2019

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DOI: 10.1021/acsami.9b00928 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Eagle’s medium (DMEM). Centrifugation and resuspension conditions were fixed for all experiments so that they are consistent throughout the results. NP and Corona Characterization. Both naked NPs and the corona conjugates were analyzed by ultraviolet−visible (UV−vis) spectrophotometry from 400 to 800 nm (Cary 100 UV−vis DualBeam, Varian Inc.). The concentration of NPs was calculated from the obtained UV−vis spectra and the known extinction coefficient. Changes in the NP morphology were characterized by TEM. Changes in the size and charge due to protein corona formation and degradation were determined by dynamic light scattering (DLS, DynaPro Titan, Wyatt Technology Corporation) and zeta potential (Malvern Zetasizer Nano ZS90) measurements. DLS data is plotted from the intensity distributions and the polydispersity index (PDI) of these DLS measurements and can be found in the Supporting Information (Table S1). It was confirmed that the predominant peak in the intensity distribution matches with the volume and number distributions to verify the homogeneity of the AuNP coronas and to distinguish them from aggregates, which can be observed in the volume distributions, as well as smaller sized species arising from the degradation of proteases, inferred from the number distribution.28 Cell Culture and Protein Corona Degradation Kinetics. PANC1 cells obtained from the American Type Culture Collection (ATCC, Manassas, VA) were grown according to ATCC guidelines. Cells were seeded in a 96-well plate at a cell concentration of 9 × 104 cells/mL with DMEM containing 10% fetal bovine serum, 100 IU/ mL penicillin, and 1% streptomycin at 37 °C in a humidified 5% CO2 incubator for 24 h. Reagents for cell media preparation were obtained from HyClone. Centrifuged NPs with preformed HS, HSA, or Coll protein corona were resuspended in fresh cell media. The cells were incubated for the indicated time points, and the protein corona was characterized as previously described. Protein Isolation for SDS PAGE or MS. Protein isolation was performed as described previously with minor modifications.10 In short, 1 mL of NP−protein corona complexes were centrifuged at 15 000g for 1 h 15 min at 4 °C. The supernatant was discarded, and the pellet was resuspended in washing solution phosphate-buffered saline (PBS)/TWEEN20 0.05% (w/v) and centrifuged at 15 000g for 1 h at 4 °C. This washing step was repeated twice for 45 min with the last resuspension in PBS without TWEEN20 (Sigma). The resulting pellets were incubated at 70 °C for 1 h with 8 μL of NuPAGE 4× LDS (Invitrogen) and 4 μL of 500 mM dithiothreitol (DTT) (Thermofisher) and centrifuged for 15 min at 15 000g. Supernatants with the isolated proteins were kept at −20 °C for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) or used for the next steps of mass spectrometry (MS) sample preparation. PBS was obtained from Life Technologies. No modifications were added to the Protein precipitation and Cleanup protocol from Walkey et al.10 For this protocol, trichloroacetic acid was purchased from FisherScientific, and sodium deoxycalate was purchased from Sigma. Then, MS sample preparation was conducted with minor modifications. In short, 95 μL of 100 mM aqueous ammonium bicarbonate (FisherScientific) and 5 μL of acetonitrile (FisherScientific) were used to solubilize the protein pellets. DTT (5 μL, 100 mM, Thermofisher) was added, and the samples were incubated at 80 °C for 45 min to reduce disulfide bonds. The samples were left to cool down at room temperature for 10 min, and 11.5 μL of 500 mM iodoacetamide (Thermofisher) in ammonium bicarbonate solution was added and incubated at room temperature in the dark for 1 h to alkylate-reduced disulfides. Prechilled MS grade 100% acetone (460 μL, Fisher Chemical) was added and kept at −20 °C for 1 h to precipitate the proteins. The samples were centrifuged for 10 min at 16 000g 4 °C, and the protein pellets were dried and resuspended in 100 μL of 50 mM ammonium bicarbonate pH 7−8. Trypsin/lysine mixture (4 μL, Promega) was added to the protein sample and incubated at 37 °C overnight. Protein digestion was stopped in 10 min at −80 °C, and the samples were stored at −20 °C for the MS analysis.

Figure 1. Effect of protein corona composition on the interaction with PANC-1 cells, which secrete MMPs, especially MMP-9.

pancreatic cancer cells. Human serum (HS) was chosen because it is a complex mixture containing both weakly and strongly bound corona proteins, in addition to small molecules and ions, and is close to the environment the NPs would be exposed to when introduced into a biological fluid. Different biological fluids can be used to study the proteolytic effect on a model of strongly bound proteins, such as plasma. HS was used in the present work to offer a simpler and well-studied system to determine the effects of proteases on the protein corona in a tumor microenvironment25,26 and to avoid the effect of additional anticoagulants present in the plasma. Pure human serum albumin (HSA) was studied to determine the behavior of a weakly bound corona protein when exposed to a proteolytic environment. Even though the abundance of HSA in coronas is low relative to its abundance in serum, it has been used to improve the carrier properties of NP coronas, facilitating drug release encapsulated in the coronas.18 Finally, collagen IV (Coll) was chosen as a corona protein because it is the substrate of MMP-9, a protease which is abundant in pancreatic cancer and specially associated with an aggressive and invasive phenotype. This enables us to study the impact of purposely seeding coronas with the MMP substrate. Interactions of the coronas with the proteins in the conditioned media were explored for the different protein corona compositions, and it was found that the coronas are impacted by a complex combination of exchange and its protease activity. We also examined the cell uptake, toxicity, and delivery of a loaded chemotherapeutic drug, doxorubicin. These results show that the effect of proteases on preformed coronas is complex.

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EXPERIMENTAL SECTION

NP Synthesis. Spherical AuNPs were synthesized by standard citrate and tannic acid reduction using literature methods.27 Briefly, a reducing mixture of 0.1 mL of tannic acid 1% (w/v), 1 mL of sodium carbonate 0.26% (w/v), and 4 mL of trisodium citrate 1% (w/v) diluted with 14.9 mL of pico-pure water was added to 80 mL of chloroauric acid solution 0.01% (w/v) preheated at 70 °C. Transmission electron microscopy (TEM) analysis showed that the spherical NPs had a diameter of 11.3 ± 0.3 nm. Protein Corona Formation. HS, HSA, and Coll were purchased from Sigma-Aldrich. As the relative amounts of protein vary between different individuals,27 a pool of HS was used to perform experiments with HS protein corona. The corona formation was carried out by resuspension of a 4 mL pellet of washed NPs with 1 mL of the corona cocktail. The protein corona cocktail was composed of 5% (v/v) of HS, HSA, or Collthe latter in a 1.5 Coll/HS ratio in 5 mM phosphate buffer (pH 7.6). All NPs were incubated overnight at 37 °C, centrifuged (HS and HSA at 10 000 rcf for 20 min and 16 000 rcf for 9 min, Coll 4000 rcf for 12 min, 7000 rcf for 14 min, and 16 000 rcf for 3 min), and resuspended in water or Dulbecco’s modified B


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Figure 2. NP and protein corona characterizations. (a) TEM image of AuNPs, (b) DLS of the AuNPs with coronas made of Coll, HS, and HSA, (c) UV−vis spectra of bare NPs (gray), with coronas made of Coll (green), HS (orange), and HSA (yellow), and (d) zeta potential of AuNPs and AuNP coronas. Error bars represent the standard deviation of three replicates. SDS PAGE. Protein supernatants were run in a 10% acrylamide gel following supplier instructions with a Mini-PROTEAN Tetra system (Biorad). MS. All MS experiments were performed using an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific). The mass spectrometer was coupled to an EASY-nLC 1200 system (Thermo Scientific). The high-performance liquid chromatography (HPLC) mobile phases were 96.1:3.9 water/acetonitrile with 0.1% formic acid (A) and 20.0:80.0 water/acetonitrile with 0.1% formic acid (B). The flow rate was 300 nL/min, and the following gradient was used for each run; 0% B for 5 min, 0−5% B for 10 min, 5−30% B for 150 min, 30−90% B for 30 min, and 90% B for 10 min. The tryptic peptide digests were diluted by a factor of 10 with 0.1% formic acid, and aliquots of 1 μL of the diluted digests were injected onto and separated by a PepMap RSLC, C18, 3 μm, 100 Å, 75 μm × 150 mm EASY-Spray column (Thermo Scientific). Electrospray ionization was performed at a voltage of 1.9 kV. The mass spectrometer was operated in the data-dependent mode. Survey scans were collected in a range of 400−1600 m/z in the orbitrap at a resolution of 120 000 and an AGC target of 2 × 105 (or maximum injection time of 100 ms). Precursor ions were filtered by charge state (2−6 z), dynamic exclusion for 30 s at a 20 ppm mass width, and monoisotopic precursor selection. For charge states 2 and 3, the precursors were isolated in the ion trap with a 1.2 m/z isolation window and an AGC target of 2 × 104 with a maximum injection time of 50 ms. Collision-induced dissociation was performed on the isolated parent ions using 35% collision energy and 10 ms activation time. EThCD was used to dissociate precursors having charge states of 3−6 using a 1.2 m/z isolation window and an AGC target of 2 × 104 (or maximum injection time of 50 ms). The Proteome Discoverer (PD) Software Version 2.1 (Thermo Scientific) was used for peptide sequencing and protein identification. The SEQUEST search algorithm was used to analyze the data against fasta files from a human protein database downloaded from Uniprot. The cleavage enzyme was set to trypsin (full), and the maximum missed cleavages was set to 2. Precursor mass tolerance was set to 10 ppm and fragment mass tolerance to 0.6 Da. The following peptide modifications were also set for the SEQUEST search: oxidation (methionine, dynamic), acetyl (N-terminus, dynamic), and carbamidomethyl (cysteine, static). For protein validation, the Percolator algorithm in PD was utilized. It was set to a false discovery rate (FDR) of 1%. All proteomic data presented in this paper is based on identifications that met this 1% FDR threshold. The protein

identification output from the PD search was downloaded into an Excel spreadsheet for further analysis. Further Analysis. Three biological replicates were conducted per each condition: NP−HS, NP−HS−DMEM, NP−HS−PANC1, and NP−HS−PANC1−protease inhibitor (Sigma). Two technical repetitions were used for the analysis. Intensity values were used to determine the top 50 proteins, with the highest peptide ion intensity values per each of the samples, leading to a list of 80 proteins in total. Heat maps were generated with R function heatmap.2 from RStudio Version 1.0.153. Student t-tests were conducted for protein corona isolates from NP−protein corona complexes from PANC-1 cell supernatants exposed or not to the protease inhibitor. Gene ontology (GO) analyses were used to address if protein exchange and proteolytic degradation modified the terms enriched. PANTHER Gene Ontology Classification System tool Version 13.129 was used to identify the type of proteins enriched in each sample, and GO terms were also defined for enrichment in the GO molecular function. Doxorubicin Uptake Determination by Confocal Imaging. To visualize the cellular uptake of doxorubicin loaded within the protein corona in PANC1, the cells were seeded on coverslips settled in 6-well plates at a concentration of 200 000 cells/well and incubated in the described cell media for 24 h. NPs with doxorubicin loaded at a concentration of 13 ± 1 μM were incubated for 1.5 h. The cells were then washed with PBS three times, fixed with 4% of paraformaldehyde, stained with ActinGreen 488 (Thermofisher), and mounted on microscope glass slides with (4′,6-diamidino-2-phenylindole) (DAPI) mounting medium. Images were obtained using an automated Zeiss AxioObserver Z1 confocal laser scanning microscope at 100× magnification. Cellular Uptake Determination by ICP MS. To determine the cell uptake of NPs with the preformed protein corona in PANC1, the cells were seeded at a concentration of 9 × 104 cells/mL for 24 h. The medium was then replaced with protein corona NPs resuspended in fresh cell media for 24 h. The cells were then washed with PBS 3× and trypsinized. The pellets from 2500 μL of the initial seeded cells were cleaned with DPBS, counted, and digested with 1 mL of aqua regia for 24 h. The Au content of each pellet was determined by inductively coupled plasma mass spectrometry (ICP−MS, ELAN DRC II PerkinElmer). MTS Cytotoxicity Assay. Cytotoxicity experiments were performed using PANC1 cells seeded in a 96-well plate at a concentration of 4000 cells/well and incubated for 48 h. The cell media were then replaced with doxorubicin-loaded NPs resuspended C


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RESULTS AND DISCUSSION Protein Corona Formation with Different Compositions. Spherical AuNPs were synthesized using literature approaches (see the Experimental Section). AuNPs were 11.3 ± 0.3 nm in diameter as measured by TEM (Figure 2a) and had a hydrodynamic diameter (DH) of 19.1 ± 1.7 nm as measured by DLS (Figure 2b). We formed protein coronas on the AuNPs using three different protein systems: HS, which has a mixture of weakly and strongly bound corona proteins, pure HSA, which has been found to be a weakly bound corona protein,30 and Coll, which is the substrate of one of the most abundant proteases secreted by PANC1 cells, MMP-9. Coronas were formed around AuNPs as previously described31 (Experimental Section). DLS measurements showed that protein corona formation resulted in an increase in size as seen with TEM and DLS (Figure 2 and Supporting Information Figure S1). AuNP-HS coronas were DH = 100.9 ± 8.3 nm in diameter, whereas HSA corona formation led to a smaller DH of 23.3 ± 1.9 nm.32 This could be due to the weaker interactions of HSA with the NPs and the small size of the pure HSA protein (66.4 kDa), compared to the wide range of protein sizes that compose the HS.33 AuNP-Coll coronas were the largest, with a DH of 156.7 ± 25.3 nm (Figure 2b). DLS cannot indicate the information about the number of AuNPs in a given corona. Thus, it is important to note that the number of AuNPs in each of the coronas could differ. Protein corona formation was also verified by the changes in the zeta potential (Figure 2d). Before protein corona formation, bare AuNPs had a zeta potential of −34.2 ± 12.9. Corona formation changed the zeta potential for each of the coronas, with the zeta potential of HS to be −21.7 ± 9.0 mV, pure HSA −39.5 ± 11.0 mV, and Coll −25.3 ± 8.1 mV (Figure 2d). The AuNP surface plasmon resonance showed a slight red shift and broadening after the formation of the protein coronas because of the change in the index of refraction around the AuNPs upon corona formation (Figure 2c). Characterization of Protein Exchange with Proteins in the Cell Media. The proteins in a corona continuously exchange with the proteins in the environment, which results in a composition and physicochemical properties that are dynamic. We explored the contribution of exchange with the cell media with a protease-free experiment to provide a baseline for when protease was present. Preformed AuNP coronas with HS, HSA, and Coll were incubated with DMEM for 24 h, and their DH values were measured before and after incubation in the media. We observed that protein exchange for each of the coronas occurred at different degrees depending on the preformed corona. AuNP-HS coronas exchanged with the biomolecules from the cell media, resulting in a decrease in DH of 61.9 ± 5.4 nm (Figure 3). This indicates that the weakly bound corona proteins had desorbed or exchanged with smaller species in the cell media. Similarly, AuNP-Coll decreased in size, resulting in a DH of 78.8 ± 0.0 nm. Pure HSA protein corona also exchanged with the proteins from

Figure 3. DH of the AuNP coronas before (blue) and after (red) incubation with DMEM. Error bars represent the standard deviation of three replicates.

DMEM, but resulted in a net increase in DH to 56.1 ± 4.6 nm. Therefore, the weakly bound HSA corona was exchanged with larger proteins from DMEM that adsorb more strongly to the AuNP surface. Protein Corona Degradation by Pure MMP-9 Activity. We then studied the capacity of the pure protease (MMP-9) to change the corona properties depending on the corona composition. We investigated the effect of incubating the different AuNP coronas for 1 h with pure MMP-9 both in active and inactive forms. Activation of MMP-9 was achieved by using 4-aminophenylmercuric acetate and resulted in an MMP-9 activity of 560 pmol/min/μg, which is the activity found in the PANC1 cell concentration used in the in vitro assays of the present paper, as measured with the SensoLyte 520 MMP-9 Assay kit. Degradation of the protein corona by MMP-9 was also monitored by measuring the DH of the coronas. AuNP-HS coronas decreased in size by 9% when either active or inactive MMP-9 was added to the solution (Figure 4a). For the AuNP-Coll coronas, the DH increased by 34% in the presence of active MMP-9 and by 22% in the presence of inactive MMP-9. Thus, two effects occurred: on

Figure 4. Effect of pure MMP-9 AuNP coronas. (a) DH of AuNPColl, AuNP-HS, and AuNP-HSA before MMP (green) and after incubation with active MMP (blue) and inactive MMP (red), where incubations were for 1 h and 560 pmol/min/μg MMP-9. DH of AuNP-HS as a function of MMP concentration in DMEM for 1 h (dark blue) and 24 h (light blue) for (b) AuNP-HS, (c) AuNP-HSA, and (d) AuNP-Coll. Error bars represent the standard deviation of three replicates. D


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ACS Applied Materials & Interfaces the one hand, there was protein exchange between the proteins from the protein corona and the MMP-9; in addition, there was protein degradation driven by the MMP-9 activity. Thus, the resulting corona change in size was a function of both effects. Interestingly, AuNP-HSA coronas aggregated solely when active MMP-9 was incubated in the colloidal solution. This was evidenced by a large size increase in the DLS measurements and higher PDI compared to the protein corona exposed to inactive MMP-9 (Table S1). Protein corona formation has shown to help stabilize NPs in biological environments such as blood34−36 because of steric stabilization. Because HSA is a small and weakly bound protein corona, it forms a smaller and more weakly bound protein layer that can be easily exchanged off the AuNP surface.25 Consequently, the digestion by the MMP-9 is facilitated, and degradation of the weakly bound corona proteins destabilizes the AuNPs, resulting in their aggregation. Furthermore, HSA forms a smaller protein corona compared to the other proteins, and thus AuNPs have a greater chance of being in proximity to one another when in solution. Taken together, these two effects accelerated the precipitation of the AuNP in solution. In contrast, the inactive protease contributed similar to any other protein solely in the protein-exchange effect, increasing the HSA corona from 21.0 ± 0.0 to 75.2 ± 6.2 nm and not causing aggregation. For HS and Coll coronas, this behavior was not observed as they were larger coronas, which helped stabilize the AuNPs to a further extent. The effect of DMEM was then added to the equation to study the protein corona exchange with both proteins in the surrounding cell media and pure MMP-9 themselves, in addition to the impact of the degradation by the MMP-9. DH was measured at different protease activities after 1 and 24 h (Figure 4b−d). The protein corona size at the 1 h time point decreased with increasing protease activity. Destabilization of the AuNP-HSA corona was prevented as proteins adsorbed from the cell media acted as stabilizing agents to the degraded protein corona. After 24 h, the corona degradation saturated and exhibited no trend with MMP-9 activity, regardless of the original corona composition and protease activity. These results suggest that the degradation by the MMP-9 was primarily in the outer layers of proteins of the corona that are loosely attached to the NP through protein−protein interactions. Behavior of AuNP Coronas in the Presence of PANC-1 Cells. We investigated the change in the AuNP corona size for the different corona compositions when exposed to PANC-1 cells, which secrete MMPs (Figure 5a). The three AuNP corona compositions were incubated with PANC-1 by addition to the cell media. AuNP coronas were collected at different time points from the cell supernatants, and their DH was measured. Under these conditions, protein exchange occurred with proteins both from the cell media and from those secreted by PANC-1 cells, which include the proteases. For AuNP-HS coronas, the DH first increased relative to its initial size (dotted line, t = 0 h) for the first hour when exposed to PANC-1 cells and then decreased to a final DH of 68.1 ± 0.0 nm (Figure 5b). This suggests that there is an initial period where the corona increases in size because of protein incorporation or exchange, followed by a decrease due to protease activity. This same behavior was also observed in the previous experiment with pure active MMP-9 (Figure 4b). However, in this experiment, the conditioned media have a different protein composition, and there are other oxidative

Figure 5. Protein corona evolution with the PANC1 cell line. (a) Model for the AuNP-corona behavior involves both rearrangement of the corona, where proteins from the media (including secreted MMPs) exchange with proteins in the preformed coronas (gray, filled), and protein degradation by proteases (gray, dashed). DH of AuNP coronas after incubation with PANC-1 cells as a function of time for (b) AuNP-HS, (c) AuNP-HSA, and (d) AuNP-Coll. Original corona size (t = 0) indicated by the dashed line. Error bars represent the standard deviation of three replicates. P value calculated as compared to 0 h time point, where P*** < 0.001; P** < 0.01; P* < 0.05.

molecules secreted by PANC-1 cells that may influence the protein corona integrity differently. As a result, these conditions lead to a greater size decrease after 24 h when compared to the pure active MMP-9 (68.1 ± 0.0 and 78.8 ± 0.0 nm, respectively). Student’s t-test indicated that all observed size changes were significantly different from the initial preformed coronas (t = 0, dashed line). The corona rearrangement or the exchange time span was shorter for the AuNP-HSA coronas. DH also increased relative to the initial size and then decreased with time, but the initial period was only 0.5 h (Figure 5c). This could be attributed to the fact that HSA is a weakly bound corona protein and thus has a more rapid protein exchange and is more accessible for degradation by the MMP-9. As a result, AuNP-HSA resulted in a smaller DH = 49.2 ± 1.3 nm. Differences could also be observed because of the abovementioned effects when compared with the saturated protein corona size with pure MMP-9 (Figure 4c). AuNP-Coll coronas exhibited the slowest kinetics. The protein exchange displayed a decrease in DH because of the protein corona desorption and exchange with proteins from DMEM (Figure 5d) as previously observed (Figure 3). Compared to AuNP-HS and AuNP-HSA, the exchanged Coll protein corona did not show a decrease in size due to the protease activity until more than 4 h after incubation. DH after 24 h was 81.6 ± 2.4 nm, showing the highest decrease in size compared to the already exchanged protein corona (from 119.0 to 81.6 nm), followed by HS (from 96.1 to 68.1 nm) and HSA (from 56.1 to 49.2 nm). Student’s t-test indicated that all observed size changes were significantly different from the initial preformed coronas. Therefore, protein coronas exposed to PANC-1 cells also displayed the two-effect phenomena seen in the previous sections with the pure protease (Figure 4b−d). Proteins found in the PANC-1 cell microenvironment exchange with the E


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Figure 6. Proteomic studies of the preformed protein corona show exchange and degradation of proteins of the preformed protein corona with proteins from cell media or secreted by PANC-1 cells. (a) Venn diagram of the proteins in the AuNP-HS corona before and after DMEM and PANC1 exposures, (b) heat map of the top 50 proteins found in the preformed HS-protein corona before (HS) and after DMEM exposures (DMEM) or incubated with PANC-1 cells (PANC1), (c) heat map of the proteins statistically different (P < 0.01) in peptide ion intensity values from preformed HS-NP isolated from the PANC-1 cell supernatant with and without protease inhibitor, and (d) PANTHER analysis for the enrichment in protein type for both. Colors indicate the most granular term (upper term from the class with same color) followed from the parent terms.

preformed protein coronas, inducing a rapid change in size. This intermediate size stage remained constant for different lapses of time depending on the nature of the preformed protein corona. Finally, protease degradation occurred, observed as a decrease in size with time. As a result, varying the composition of the protein coronas led to distinct degradation patterns. Different drug release capacities were observed at high concentrations of doxorubicin loaded for each of the protein coronas (Supporting Information, Figure S2). Different degradation patterns may have implications not only on the time-dependent release of drugs but also on the renal clearance and cell uptake of the nanomaterial. Protein Corona Composition with MS and SDS-PAGE. To probe how the protein corona composition changes upon exposure to the cell media and PANC-1 cells, we performed SDS-PAGE and MS on the NP coronas to complement the DLS data. First, SDS PAGE was performed on digested coronas as they underwent composition changes with exposure to the cell media and proteases, as seen from the differences in their pattern (Supporting Information Figure S3c). To better address these distinct compositions, reversedphase high performance liquid chromatography/nanospraytandem mass spectrometry (HPLC/NS-MS/MS was performed on the tryptic digests of the proteins that composed the AuNP-HS coronas to identify the proteins that were exchanged with fresh and conditioned media (Figure 6). The top 50 proteins with higher intensity values were mapped for the preformed protein corona (HS), incubated with fresh media (DMEM) and isolated from PANC-1 supernatants (PANC1) (Figure 6b). The different patterns observed between the preformed protein corona and fresh media showed that most proteins from this subset were exchanged with proteins found in fresh DMEM. Also, differences were observed between the protein corona isolated from DMEM and exposed to PANC-1 cells, which could be attributed either to exchange with cell-secreted proteins or to proteolytic degradation. Venn diagrams were obtained for the entire set of proteins detected and showed a more diverse protein corona

composition when exposed to PANC1 or fresh cell media (Figure 6a and Supporting Information, Figure S3a). Analysis of the protein type also showed changes in the enrichment of protein class for shared and unique proteins in each of the systems (Supporting Information, Figure S3a). Therefore, the observed size decrease in Figures 3 and 5 is due to a rearrangement of proteins. These results show that there are more protein types adsorbed to the corona when exposed to complex biological media and that different protein concentrations can also lead to a decrease in the overall size of the coronas. To better address which proteins were degraded by PANC-1 secreted proteases, a protease inhibitor was introduced in the cell media. The protein corona was isolated and analyzed by MS (Figure 6b,c). Proteins that were significantly different between PANC-1 with or without the protease inhibitor were plotted in a heat map. Most of these proteins (67.6%) had higher intensity values in the media in which the proteolytic effect was suppressed. When the protease inhibitor was present, it led to enrichment by proteins of a different type, as analyzed with PANTHER (Figure 6d). GO analyses were conducted with proteins that showed statistically significant differences between both samples. A high percentage of unmapped IDs was observed (56 and 66% for PI and PANC1, respectively), and the vast majority of those were isoforms (98 and 66%). Deletion of isoforms from the list led to similar enrichment patterns, and therefore, the raw list of proteins was used for the GO analysis (Figure 6b). GO enrichment based on molecular function also showed that the resultant protein corona after degradation is enriched with proteins with different molecular functions, which could lead to a different biological ID (Supporting Information, Figure S3b). Impact of Protein Corona on the Cell Uptake and Viability. The protein corona around a NP is the primary factor influencing the cellular uptake, often playing a more significant role than the NP physical properties. Therefore, we measured the cell uptake of the AuNP-HS, AuNP-HSA, and AuNP-Coll coronas.10,25,35,37−39 ICP−MS was conducted in PANC-1 pellets after incubation with preformed protein F


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Figure 7. AuNP corona internalization in PANC-1 cells. (a) ICP−MS after 24 h incubation. Error bars represent the standard deviation of three replicates. P value calculated between the three cell uptake results, where P** < 0.01. (b) Confocal microscopy showing AuNP, free Dox, and AuNP coronas showing actin (green), DAPI (blue), dox (red), and merged images. HS/Coll pval = 0.0002, HSA/Coll pval = 0.002, and HSA/HS pval = 0.2.

following the standard method in the literature.41 This relatively high cytotoxic response for the AuNP-Coll corona is consistent with the uptake data reported above, showing that this construct delivers more than 3 times the doxorubicin payload than either of the others tested. It is noteworthy, however, that the HSA corona achieves greater cytotoxic response than the HS corona while the payload of drug delivered is about the same for these two constructs. This suggests that in addition to the drug payload delivered, biological responses to individual corona components may be an important contributor to the overall cytotoxic efficacy of a given construct.

corona for 24 h (Figure 7a). AuNP-Coll coronas displayed 3.1 and 3.8× more cell uptake than those composed of HS and HSA protein coronas, respectively. It is important to note that the number of NPs per corona for each of the AuNP coronas varies; ICP−MS yields information about the number of NPs taken up by the cells but not the number of coronas. Confocal imaging was performed to explore the capability of coronas to deliver drug payloads into the nucleus of PANC-1 cells (Figure 7b). Doxorubicin was loaded into the protein corona via the combined method,31 and cellular internalization was probed by fluorescent imaging. All three coronas could deliver the doxorubicin payload into the cell nucleus after 1.5 h of incubation. Incubation of PANC-1 cells with free doxorubicin and no coronas or AuNPs also show internalization of the doxorubicin into the nucleus as expected with free Dox.40 Control experiments where no DAPI was incubated with the cells (Supporting Information, Figure S4) verified that the fluorescence signal shown in the red channel was due to doxorubicin and not spectral bleed through DAPI. MTS cytotoxicity assay measurements show that the cytotoxic response to doxorubicin delivered via AuNP-Coll (EC50 8.3 μM) was the highest, followed by AuNP-HSA (EC50 11.5 μM) and then AuNP-HS (EC50 24.3 μM) (Figure 8). EC50 values were obtained by a sigmoidal fit of PANC-1 viability incubated with the three systems at different doxorubicin payloads and

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SUMMARY AND CONCLUSIONS These results indicate that the activity of proteases such as MMP-9 in the cellular environment do impact the properties of the protein corona by either exchange, adsorption, or degradation and that these effects are dependent on the protein corona composition. The influence of proteases is complex because size changes in the corona can be due to both the media and the protease itself exchanging into the coronas on the NP. However, size changes in the corona are not straightforward, as exposure to MMP-9 does not result in just a decrease in the size because of digestion. Because MMP-9 is a protein itself, it can participate as a corona protein, where it can exchange on and off the AuNP. Also, the impact of the proteins in the cell media exchanging with the proteins in the corona can also change the size. Depending on the corona composition, both the exchange and protease activity have different effects, resulting in either size increases or decreases. Pure HSA tends to form a more labile but also smaller corona that first undergoes exchange before degradation. HS, which forms coronas that have a mixture of both strongly and weakly bound corona proteins, also undergoes exchange first but then is followed by degradation. However, protease degradation does not reduce the size of the corona below its initial size, indicating that there are strongly bound corona proteins that cannot be exchanged or degraded and that the epitopes of the proteins that can be digested by MMPs may not be exposed.42 Coll tends to form larger “stickier” coronas which are degraded by proteases from PANC-1. Whereas these results are for a set

Figure 8. Relative viability of PANC-1 cells incubated with AuNPprotein corona loaded with doxorubicin represented against the log10 of doxorubicin payloads (5, 10, 30, and 70 μM) for HS (red), HSA (blue), and Coll (green). The chi-squared test for the fitted sigmoidals are 0.80 (HS), 0.99 (HSA), and 0.89 (Coll). G



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of three preformed coronas in HS, they indicate that one can engineer preformed protein coronas to modify the biological performance of NPs.

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ASSOCIATED CONTENT

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b00928. TEM images, additional MS data, doxorubicin release, and confocal images of control experiments (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Kimberly Hamad-Schifferli: 0000-0002-4839-3179 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding

C.R.-Q. was supported by a Rafael del Pino Fellowship and a UMB Goranson award. This work was supported by UMB funding. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Cedervall, T.; Lynch, I.; Lindman, S.; Berggard, T.; Thulin, E.; Nilsson, H.; Dawson, K. A.; Linse, S. Understanding the nanoparticleprotein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles. Proc. Natl. Acad. Sci. U.S A. 2007, 104, 2050−2055. (2) Tenzer, S.; Docter, D.; Kuharev, J.; Musyanovych, A.; Fetz, V.; Hecht, R.; Schlenk, F.; Fischer, D.; Kiouptsi, K.; Reinhardt, C.; Landfester, K.; Schild, H.; Maskos, M.; Knauer, S. K.; Stauber, R. H. Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology. Nat. Nanotechnol. 2013, 8, 772−781. (3) Castagnola, V.; Zhao, W.; Boselli, L.; Lo Giudice, M. C.; Meder, F.; Polo, E.; Paton, K. R.; Backes, C.; Coleman, J. N.; Dawson, K. A. Biological recognition of graphene nanoflakes. Nat. Commun. 2018, 9, 1577. (4) Monopoli, M. P.; Åberg, C.; Salvati, A.; Dawson, K. A. Biomolecular coronas provide the biological identity of nanosized materials. Nat. Nanotechnol. 2012, 7, 779−786. (5) Hadjidemetriou, M.; Al-Ahmady, Z.; Kostarelos, K. Timeevolution of in vivo protein corona onto blood-circulating PEGylated liposomal doxorubicin (DOXIL) nanoparticles. Nanoscale 2016, 8, 6948−6957. (6) Ritz, S.; Schöttler, S.; Kotman, N.; Baier, G.; Musyanovych, A.; Kuharev, J.; Landfester, K.; Schild, H.; Jahn, O.; Tenzer, S.; Mailänder, V. Protein Corona of Nanoparticles: Distinct Proteins Regulate the Cellular Uptake. Biomacromolecules 2015, 16, 1311− 1321. (7) Walczyk, D.; Bombelli, F. B.; Monopoli, M. P.; Lynch, I.; Dawson, K. A. What the Cell “Sees” in Bionanoscience. J. Am. Chem. Soc. 2010, 132, 5761−5768. (8) Braun, N. J.; DeBrosse, M. C.; Hussain, S. M.; Comfort, K. K. Modification of the protein corona-nanoparticle complex by physiological factors. Mater. Sci. Eng., C 2016, 64, 34−42. (9) Caracciolo, G.; Pozzi, D.; Capriotti, A. L.; Cavaliere, C.; Piovesana, S.; La Barbera, G.; Amici, A.; Laganà, A. The liposomeprotein corona in mice and humans and its implications for in vivo delivery. J. Mater. Chem. B 2014, 2, 7419−7428. (10) Walkey, C. D.; Olsen, J. B.; Song, F.; Liu, R.; Guo, H.; Olsen, D. W. H.; Cohen, Y.; Emili, A.; Chan, W. C. W. Protein Corona Fingerprinting Predicts the Cellular Interaction of Gold and Silver Nanoparticles. ACS Nano 2014, 8, 2439−2455. (11) Barrán-Berdón, A. L.; Pozzi, D.; Caracciolo, G.; Capriotti, A. L.; Caruso, G.; Cavaliere, C.; Riccioli, A.; Palchetti, S.; Laganà, A. Time Evolution of Nanoparticle-Protein Corona in Human Plasma: Relevance for Targeted Drug Delivery. Langmuir 2013, 29, 6485− 6494. (12) Pearson, R. M.; Juettner, V. V.; Hong, S. Biomolecular corona on nanoparticles: a survey of recent literature and its implications in targeted drug delivery. Front. Chem. 2014, 2, 108. (13) Luby, A. O.; Breitner, E. K.; Comfort, K. K. Preliminary protein corona formation stabilizes gold nanoparticles and improves deposition efficiency. Appl Nanosci 2016, 6, 827−836. (14) Caputo, D.; Papi, M.; Coppola, R.; Palchetti, S.; Digiacomo, L.; Caracciolo, G.; Pozzi, D. A protein corona-enabled blood test for early cancer detection. Nanoscale 2017, 9, 349−354. (15) Hajipour, M. J.; Laurent, S.; Aghaie, A.; Rezaee, F.; Mahmoudi, M. Personalized protein coronas: a “key” factor at the nanobiointerface. Biomater. Sci. 2014, 2, 1210−1221. (16) Colapicchioni, V.; Tilio, M.; Digiacomo, L.; Gambini, V.; Palchetti, S.; Marchini, C.; Pozzi, D.; Occhipinti, S.; Amici, A.; Caracciolo, G. Personalized liposome-protein corona in the blood of breast, gastric and pancreatic cancer patients. Int. J. Biochem. Cell Biol. 2016, 75, 180−187. (17) Ke, P. C.; Lin, S.; Parak, W. J.; Davis, T. P.; Caruso, F. A Decade of the Protein Corona. ACS Nano 2017, 11, 11773−11776. (18) Cifuentes-Rius, A.; de Puig, H.; Kah, J. C. Y.; Borros, S.; Hamad-Schifferli, K. Optimizing the Properties of the Protein Corona Surrounding Nanoparticles for Tuning Payload Release. ACS Nano 2013, 7, 10066−10074.

S Supporting Information *

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Research Article

ACKNOWLEDGMENTS

This work was supported by the UMass Boston via Proposal Development Fund. C.R.-Q. was supported by a Fellowship from the Rafael del Pino Foundation and a Gormanson Award from the UMass Boston. H.d.P. was supported by a TATA fellowship and a Broshy fellowship. We thank Lee Gehrke for use of lab facilities. We thank Elena Paltrinieri for experimental assistance with confocal imaging. We thank Alan Stebbins, Alan Christian, and Amy Johnston and the SEF facility in the School for the Environment (SFE) at the UMass Boston. We thank the Center for Materials Science and Engineering (CMSE) facilities at MIT for use of TEM. We thank Alexey Veraska in the Department of Biology at the UMass Boston for use of confocal imaging facilities and Ljubica Petrovic for experimental advice.

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ABBREVIATIONS AuNP, gold nanoparticle Coll, collagen CID, Collision Induced Dissociation DH, hydrodynamic diameter EThcD, Electron-Transfer/Higher-Energy Collision Dissociation HS, human serum HSA, human serum albumin MMP, matrix metalloproteinase NP, nanoparticle Dox, doxorubicin RP-HPLC/NS-MS/MS, Reverse phase-high performance liquid chromatography/nanospray-tandem mass spectrometry H


Research Article


biological activity of silver nanoparticles. J. Mater. Chem. 2010, 20, 512−518. (37) Darabi Sahneh, F.; Scoglio, C.; Riviere, J. Dynamics of Nanoparticle-Protein Corona Complex Formation: Analytical Results from Population Balance Equations. PLoS One 2013, 8, No. e64690. (38) Aggarwal, P.; Hall, J. B.; McLeland, C. B.; Dobrovolskaia, M. A.; McNeil, S. E. Nanoparticle interaction with plasma proteins as it relates to particle biodistribution, biocompatibility and therapeutic efficacy. Adv. Drug Delivery Rev. 2009, 61, 428−437. (39) Salvati, A.; Pitek, A. S.; Monopoli, M. P.; Prapainop, K.; Bombelli, F. B.; Hristov, D. R.; Kelly, P. M.; Åberg, C.; Mahon, E.; Dawson, K. A. Transferrin-functionalized nanoparticles lose their targeting capabilities when a biomolecule corona adsorbs on the surface. Nat. Nanotechnol. 2013, 8, 137−143. (40) Upadhyay, K. K.; Bhatt, A. N.; Mishra, A. K.; Dwarakanath, B. S.; Jain, S.; Schatz, C.; Le Meins, J.-F.; Farooque, A.; Chandraiah, G.; Jain, A. K.; Misra, A.; Lecommandoux, S. The intracellular drug delivery and anti tumor activity of doxorubicin loaded poly(γ-benzyl lglutamate)-b-hyaluronan polymersomes. Biomaterials 2010, 31, 2882−2892. (41) Alexander, B.; Browse, D. J.; Reading, S. J.; Benjamin, I. S. A simple and accurate mathematical method for calculation of the EC50. J. Pharmacol. Toxicol. Methods 1999, 41, 55−58. (42) Herda, L. M.; Hristov, D. R.; Lo Giudice, M. C.; Polo, E.; Dawson, K. A. Mapping of Molecular Structure of the Nanoscale Surface in Bionanoparticles. J. Am. Chem. Soc. 2017, 139, 111−114.

(19) Yeo, E. L. L.; Cheah, J. U.-J.; Neo, D. J. H.; Goh, W. I.; Kanchanawong, P.; Soo, K. C.; Thong, P. S. P.; Kah, J. C. Y. Exploiting the protein corona around gold nanorods for low-dose combined photothermal and photodynamic therapy. J. Mater. Chem. B 2017, 5, 254−268. (20) Caracciolo, G. Clinically approved liposomal nanomedicines: lessons learned from the biomolecular corona. Nanoscale 2018, 10, 4167−4172. (21) Coussens, L. M.; Fingleton, B.; Matrisian, L. M. Matrix Metalloproteinase Inhibitors and Cancer–Trials and Tribulations. Science 2002, 295, 2387−2392. (22) Chanana, M.; RiveraGil, P.; Correa-Duarte, M. A.; Liz-Marzán, L. M.; Parak, W. J. Physicochemical properties of protein-coated gold nanoparticles in biological fluids and cells before and after proteolytic digestion. Angew. Chem., Int. Ed. 2013, 52, 4179−4183. (23) Capriotti, A. L.; Caracciolo, G.; Cavaliere, C.; Colapicchioni, V.; Piovesana, S.; Pozzi, D.; Laganà, A. Analytical Methods for Characterizing the Nanoparticle-Protein Corona. Chromatographia 2014, 77, 755−769. (24) Jakubowska, K.; Pryczynicz, A.; Januszewska, J.; Sidorkiewicz, I.; Kemona, A.; Niewiński, A.; Lewczuk, Ł.; Kędra, B.; GuzińskaUstymowicz, K. Expressions of Matrix Metalloproteinases 2, 7, and 9 in Carcinogenesis of Pancreatic Ductal Adenocarcinoma. Dis. Markers 2016, 2016, 9895721. (25) Fleischer, C. C.; Payne, C. K. Nanoparticle-Cell Interactions: Molecular Structure of the Protein Corona and Cellular Outcomes. Acc. Chem. Res. 2014, 47, 2651−2659. (26) Walkey, C. D.; Olsen, J. B.; Guo, H.; Emili, A.; Chan, W. C. W. Nanoparticle Size and Surface Chemistry Determine Serum Protein Adsorption and Macrophage Uptake. J. Am. Chem. Soc. 2012, 134, 2139−2147. (27) Slot, J. W.; Geuze, H. J. A new method of preparing gold probes for multiple-labeling cytochemistry. Eur. J. Cell. Biol. 1985, 38, 87−93. (28) Hühn, J.; Carrillo-Carrion, C.; Soliman, M. G.; Pfeiffer, C.; Valdeperez, D.; Masood, A.; Chakraborty, I.; Zhu, L.; Gallego, M.; Yue, Z.; Carril, M.; Feliu, N.; Escudero, A.; Alkilany, A. M.; Pelaz, B.; del Pino, P.; Parak, W. J. Selected Standard Protocols for the Synthesis, Phase Transfer, and Characterization of Inorganic Colloidal Nanoparticles. Chem. Mater. 2017, 29, 399−461. (29) Mi, H.; Muruganujan, A.; Casagrande, J. T.; Thomas, P. D. Large-scale gene function analysis with the PANTHER classification system. Nat. Protoc. 2013, 8, 1551−1566. (30) Winzen, S.; Schoettler, S.; Baier, G.; Rosenauer, C.; Mailaender, V.; Landfester, K.; Mohr, K. Complementary analysis of the hard and soft protein corona: sample preparation critically effects corona composition. Nanoscale 2015, 7, 2992−3001. (31) Kah, J. C. Y.; Chen, J.; Zubieta, A.; Hamad-Schifferli, K. Exploiting the Protein Corona around Gold Nanorods for Loading and Triggered Release. ACS Nano 2012, 6, 6730−6740. (32) Gebauer, J. S.; Malissek, M.; Simon, S.; Knauer, S. K.; Maskos, M.; Stauber, R. H.; Peukert, W.; Treuel, L. Impact of the nanoparticleprotein corona on colloidal stability and protein structure. Langmuir 2012, 28, 9673−9679. (33) Tirumalai, R. S.; Chan, K. C.; Prieto, D. A.; Issaq, H. J.; Conrads, T. P.; Veenstra, T. D. Characterization of the low molecular weight human serum proteome. Mol. Cell. Proteomics 2003, 2, 1096− 1103. (34) Monopoli, M. P.; Walczyk, D.; Campbell, A.; Elia, G.; Lynch, I.; Baldelli Bombelli, F.; Dawson, K. A. Physical−Chemical Aspects of Protein Corona: Relevance to in Vitro and in Vivo Biological Impacts of Nanoparticles. J. Am. Chem. Soc. 2011, 133, 2525−2534. (35) Lundqvist, M.; Stigler, J.; Elia, G.; Lynch, I.; Cedervall, T.; Dawson, K. A. Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 14265−14270. (36) Kittler, S.; Greulich, C.; Gebauer, J. S.; Diendorf, J.; Treuel, L.; Ruiz, L.; Gonzalez-Calbet, J. M.; Vallet-Regi, M.; Zellner, R.; Köller, M.; Epple, M. The influence of proteins on the dispersability and cellI


Protease Degradation of Protein Coronas and Its Impact on Cancer

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