Protein Corona Formation on Colloidal Polymeric Nanoparticles and

Article pubs.acs.org/Biomac

Protein Corona Formation on Colloidal Polymeric Nanoparticles and Polymeric Nanogels: Impact on Cellular Uptake, Toxicity, Immunogenicity, and Drug Release Properties Katja Obst,†,⊥,¶ Guy Yealland,†,¶ Benjamin Balzus,‡ Enrico Miceli,§,⊥ Mathias Dimde,§ Christoph Weise,§ Murat Eravci,§ Roland Bodmeier,‡ Rainer Haag,§,⊥ Marcelo Calderón,§,⊥ Nada Charbaji,†,⊥ and Sarah Hedtrich*,†,⊥ †

Institute for Pharmacy, Freie Universität Berlin, Königin-Luise-Str. 2-4, 14195 Berlin, Germany Institute for Pharmacy, Freie Universität Berlin, Kelchstr. 31, 12169 Berlin, Germany § Institute for Chemistry and Biochemistry, Freie Universität Berlin, Takustr. 3, 14195 Berlin, Germany ⊥ Multifunctional Biomaterials for Medicine, Helmholtz Virtual Institute, Kantstr. 55, 14513 Teltow, Germany ‡

S Supporting Information *

ABSTRACT: The adsorption of biomolecules to the surface of nanoparticles (NPs) following administration into biological environments is widely recognized. In particular, the “protein corona” is well understood in terms of formation kinetics and impact upon the biological interactions of NPs. Its presence is an essential consideration in the design of therapeutic NPs. In the present study, the protein coronas of six polymeric nanoparticles of prospective therapeutic use were investigated. These included three colloidal NPssoft core−multishell (CMS) NPs, plus solid cationic Eudragit RS (EGRS), and anionic ethyl cellulose (EC) nanoparticlesand three nanogels (NGs)thermoresponsive dendritic-polyglycerol (dPG) nanogels (NGs) and two amino-functionalized dPG-NGs. Following incubation with human plasma, protein coronas were characterized and their biological interactions compared with pristine NPs. All NPs demonstrated protein adsorption and increased hydrodynamic diameters, although the solid EGRS and EC NPs bound notably more protein than the other tested particles. Shifts toward moderately negative surface charges were also observed for all corona bearing NPs, despite varied zeta potentials in their pristine states. While the uptake and cellular adhesion of the colloidal NPs in primary human keratinocytes and human umbilical vein endothelial cells were significantly decreased when bearing the protein corona, no obvious impact was seen in the NGs. By contrast, corona bearing NGs induced marked increases in cytokine release from primary human macrophages not seen with corona bearing colloidal NPs. Despite this, no apparent enhancement to in vitro toxicity was noted. Finally, drug release from EGRS and EC NPs was assessed, where a decrease was seen in the EGRS NPs alone. Together these results provide a direct comparison of the physical and biological impact the protein corona has on NPs of widely varied character and in particular highlights a distinction between the corona’s effects on NGs and colloidal NPs.

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INTRODUCTION The use of nanoparticles (NPs) holds great potential within the medical field in areas such as targeted drug delivery,1−4 vaccination,5 diagnostics, and imaging.6,7 Depending on the intended use, several routes of administration are pursued: oral,8,9 topical,10−12 intranasal,13 and most frequently, intravenous.14,15 The adsorption of biomolecules such as proteins and lipids, to the surfaces of synthetic NPs following administration into biological environments, is widely recognized and known to fundamentally alter a NP’s biological identity, affecting cellular interactions, immunogenicity, circulation half-life, and elimination from the body.16−21 The best studied aspect of this is the so-called protein corona. With few reported exceptions,22 all NPs develop protein coronas to some extent, even when in possession of antifouling surface properties.16,23,24 Owing to the necessity of purification © 2017 American Chemical Society

from unbound matter, studies have typically investigated the hard corona, those proteins that adhere strongly to NPs. Works detailing the assembly of proteins that associate more weakly, the so-called soft corona, are however emerging.25−27 NP characteristics such as surface charge, hydrophobicity, curvature, and surface area have all been found to impact on the formation of the hard protein corona.17,25 Notably, subminute assemblies have been demonstrated followed by varied and complex protein binding kinetics (Vroman effect).27−29 The biological impact of the protein corona has also been widely studied. Demonstrated effects include alteration to toxicity, immunogenicity, cell uptake, hemolysis, and NP biodistribuReceived: February 2, 2017 Revised: May 15, 2017 Published: May 16, 2017 1762

DOI: 10.1021/acs.biomac.7b00158 Biomacromolecules 2017, 18, 1762−1771

Article

Biomacromolecules tion.16,17,30 Given the apparent impact of the protein corona in mediating the biological interactions of NPs, insight into its formation, character, and biological effects is essential to the assessment of prospective nanotherapeutics both for their direct use and future development. Here, we assess and directly compare the protein corona formation and subsequent biological interactions of six soluble polymeric nanoparticles of markedly different chemical and physical properties.17,25,31 Core multishell (CMS) nanocarriers consist of a dendritic polyglycerol (dPG) core to which a hydrophobic and then hydrophilic polymer are sequentially bound, creating an unimolecular entity that self-organizes into a colloid on hydration, given its amphiphilic nature.32 They hold potential as drug delivery enhancers for topical and systemic administrations.33−36 Eudragit RS 100 (EGRS) is a cationic polymer, and ethylcellulose a neutral polymer, both of which are used as insoluble formulations for various pharmaceuticals to betow controlled drug release. The preparation of solid, dexamethasone-loaded, colloidal NP suspensions made by emulsificiation of EGRS and a commercially available ethylcellulose standard (EC4CP) have recently been reported, both of which were investigated for their ability to alter drug release profiles.37−40 Thermoresponsive nanogels (NG) are threedimensional polymer networks freely dispersed within water. Formed of dPG cross-linked with the thermoresponsive polymer poly(N-isopropylacrylamide) (pNIPAM), they reversibly swell with water below their lower critical transition temperature around 33−34 °C.10 They have been shown to encapsulate hydrophilic molecules ranging from small molecules to large molecular weight proteins with exceptional efficiency. They hold great potential for enhanced intradermal delivery applications. Similarly, positively-charged NGs were formed by cross-linking aminated dPG (10% or 30% amine wt) with acrylated dPG (10% wt) via a mild and surfactant free thiol-michael nanoprecipitation process (hereafter referred to as dPG-NH2 10% and dPG-NH2 30%).41 A recent report details their use in detecting the pH gradient found with hair follicles of the skin. These six NPs were incubated with human plasma to emulate the protein corona encountered on intravenous administrations. Subsequently, their protein coronas were characterized, and their interactions with primary normal human keratinocytes (NHKs), human umbilical vein endothelial cells (HUVECs), and primary human macrophages were investigated, as representative members of epithelia, endothelia, and innate immune cells. A distinction between the corona formation on the solid EGRS and EC4CP NPs and the nonfouling CMS NPs and NGs could be made. However, the biological impact of the protein corona clearly differed between the three NGs and the three colloidal NPs possessing a single continuous surface (CMS, EC4CP, and EGRS NPs).

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nanogels (10% and 30%) were synthesized under mild conditions using a surfactant and copper free nanoprecipitation method.41,43 Indocarbocyanine (ICC) was covalently bound to the CMS nanotransporters as described previously.34 Thermoresponsive and aminated nanogels were covalently labeled with RhB and RhB-ITC, respectively. Plasma Isolation and Protein Corona Formation. Human plasma was isolated and pooled from whole blood of three healthy donors (with permission, Deutsches Rotes Kreuz). Blood was centrifuged for 5 min at 400g, and supernatants were pooled and stored at −80 °C. NPs (5 mg/mL) were incubated with human plasma suspensions (1:1 v/v) for 1 or 24 h (37 °C). Nanoparticle Characterization. Dynamic light scattering (DLS) and zeta potential measurements were performed using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). Mean particle diameter (z-average) and polydispersity index (PDI) were calculated from 10 subruns using CONTIN analysis. Measurements were taken at a scattering angle of 173° and at a temperature of 25 °C. For zeta potential measurements, the NPs were suspended in 0.001 M KCl (pH 7). Electrophoretic mobility was then analyzed following application of a 20 V/cm electric field and converted into the zeta potential according to the Helmholtz-Smoluchowski equation. Corona-coated NPs were assessed after purification. Protein Corona Assessment. Corona-coated particles were loaded onto a 0.7 M sucrose cushion and centrifuged (12 000g, 20 min, 4 °C) to separate the particle−protein complexes from free protein.44 The resulting pellet was washed by three rounds of resuspension and centrifugation (15 min, 12 000g, 4 °C). Adherent proteins were quantified by the bicinchoninic acid (BCA) assay (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific, Darmstadt, Germany) according to the manufacturer’s protocol. Details of polyacrylamide gel electrophoresis (PAGE) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) can be found in the Supporting Information. Cell Culture. Normal human keratinocytes (NHK, passages 2−4) and human umbilical vein endothelial cells HUVECs (passages 3−9) were cultured at 37 °C with 5% CO2 using KGM and EGM, respectively. Primary human macrophages were generated from peripheral blood mononuclear cells (PBMCs), isolated from the buffy-coats (NycoPrep 1.077) of anonymous healthy volunteers with permission (Deutsches Rotes Kreuz, Berlin, Germany). After three washing steps using Ca2+/Mg2+ free phosphate buffered saline (PBS) (2 mM EDTA), monocytes were separated from the PBMCs using CD14+ MicroBeads (clone HI149; Miltenyi Biotech) and magnetic cell separation according to the manufacturer’s instructions. Subsequently, monocytes were cultured in complete growth media (RPMI-1640 supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, 10% heat-inactivated fetal calf serum, and 20 ng/mL MCSF). After 6 days, mature macrophages were obtained as confirmed by the expression of the cell surface markers CD11b, CD14, CD206, and absence of CD1a (flow cytometric analysis, FACSCalibur, Becton Dickinson, Heidelberg, Germany). Cytotoxicity. NHK and HUVEC viability were assessed by MTT assay following incubation with NPs diluted directly into culture media (50 μg/mL). Necrotic and apoptotic macrophage populations were identified by PI and Annexin V staining (flow cytometric analysis). Macrophage Activation. Release of interleukins (IL)-1β, IL-6, and IL-10 were assessed by enzyme-linked immunosorbent assay (ELISA) (ELISA Ready-Set-Go, eBioscience, Frankfurt, Germany). Quantification and Mechanism of Cellular Uptake. Fluorescently-labeled NPs were added to 100 000 NHK, HUVECs, or macrophages and incubated for 3 h (final NP concentration 50 μg/ mL) at 37 °C. Cells were then washed three times to remove unbound NPs with cold PBS, and fluorescence was quantified by flow cytometry. Nanoparticle Adhesion to Cell Membrane. Adhesion of NPs to the cellular membrane was assessed as previously described (modified from Yan et al. 201345). Cells were incubated with fluorescently labeled NPs (50 μg/mL) at 4 °C for 30 min. Afterward, cells were washed three times to remove unbound NPs and incubated in fresh

METHODS

Nanoparticle Formation. EGRS and E4C4P nanocarriers were prepared by the solvent evaporation method. Briefly, polymers, and where required Nile red labeled polymer (0.004% w/w), were dissolved in ethyl acetate and emulsified into an aqueous 2.5% (w/ v) poly(vinyl alcohol) solution by high-shear-homogenization (UltraTurrax T 25; IKA-Werke, Germany). Subsequently, the suspension was kept on ice and sonicated at 200 W for 3 min (Bandelin Sonopuls HD 200, Bandelin electronic, Berlin, Germany) then placed in a rotary evaporator to remove excess solvent. CMS nanotransporters and thermoresponsive polyglycerol nanogels were synthesized as described previously.32,42 Aminated polyglycerol 1763


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Biomacromolecules cell media for 3 h (37 °C). Flow cytometric analysis was then performed to determine NP adhesion. Drug Release. The drug release of dexamethasone loaded on EGRS and E4C4P nanocarriers was carried out using Franz diffusion cells under sink conditions (14% saturation). A dialysis membrane (Spectra/Por 2 Dialysis Membranes, Spectrum Laboratories, Inc., CA, USA) made of regenerated cellulose (MWCO 12−14 kDa) was fixed. At first, Franz cells were filled with PBS and after 1 h equilibration to 37 °C, dexamethasone loaded nanocarriers without or with corona were applied onto Franz cells. Drug release conditions over 24 h were set to 37 °C and 600 rpm stirring. Samples were taken out after 0.25 h, 0.75 h, 1.5 h 3 h, 5 h, 7 h, and 24 h. Therefore, 400 μL samples were taken from Franz cell, and the same removed volume was replaced with PBS. Collected samples were analyzed by high performance liquid chromatography (HPLC, HITACHI LaChrom, Detector L-7480, Autosampler L-7200, Merck KG, Darmstadt, Germany) to determine the amount of released dexamethasone. Statistical Analysis. Statistical significance of single experimental factors were assessed using one-way ANOVA with Dunnett’s post correction for multiple comparisons against control values. Statistical significance of two experimental factors was assessed by multiple t tests with the Holm-Sidak correction for multiple comparisons. The individual statistics used for each figure are noted in the figure legend. All stats were performed using Graphpad Prism 6.

(Figures 1 and S2).28 It should however be noted that the precise composition of the protein corona, that is, the proportion each protein occupies, is known to vary over minutes to hours, particularly on initial formation where sequential protein displacements can occur (“Vroman effect”).28,46 The greatest protein adsorptions in terms of both variety and magnitude were found on EGRS and EC4CP NPs (Figures 1 and S1). As pristine particles, both of these possessed high surface charges (Table 1), a property previously demonstrated to enhance interactions with plasma biomolecules.28,47 Despite their opposite charges, incubation with plasma resulted, as with all other tested NPs, in shifts toward moderately negative ζ potentials. This is notable given the greatly varied chemistries of the tested particles. Tenzer et al.28 demonstrated preferential adsorption of proteins possessing a negative charge at physiological pH, regardless of NP charge, suggesting surface charge plays only a partial role in determining protein adsorption. Unlike the other tested NPs, the EC4CP and EGRS NPs were formed from insoluble polymers by emulsification in a 2.5% PVA solution. As a result, these particles were the densest tested and in possession of the lowest surface hydrophilicities, factors that contribute to greater overall surface energies generated by weak forces and that favor the adhesion of biomolecules.27,29,48−51 Relative to the solid colloidal NPs formed of emulsified insoluble polymer, neither of the dPG-NH2 NGs bound protein to any great extent despite their positive surface charges in pristine form (Figure 1, Table 1). Both sets of NG consist of dPGs connected by soluble, flexible linkers making them softer, less dense, and more hydrophilic than the aforementioned NPs, properties less favorable to protein adsorption.27,29,48,50 It has previously been shown that dPG possesses excellent antifouling properties, though it should be noted that electrostatic and hydrophobic interactions with this chemistry should still be possible as seen with similar charge neutral hydrophilic chemistries such as PEG.52−56 Meanwhile, the free and covalently linked acrylate and amine groups found on these NGs will provide further polar-regions in addition to apolar, zwitterionic, and sulfide moieties, respectively permitting hydrophobic interactions, electrostatic interactions, and disulfide bonds to form. In this regard, it is interesting to note the increased protein adsorption capacity the dPG-NH2 (30%) NGs have at 24 h as compared to the dPG-NH2 (10%). It is also interesting to note that where the dPG-NH2 (10%) NGs showed only moderate increases in diameter following incubation with plasma, the dPG-NH2 (30%) NGs showed increases indicative of interparticle flocculation, in line with the notion of greater protein adsorption to the more highly aminated NG (Table 1). Interpretation of results from the dPG-pNIPAM NGs requires consideration of their thermoresponsive nature, wherein they reversibly expel and swell with water above and below their lower critical transition temperature of ∼33−34 °C.10,57 NP incubation with plasma was conducted at 37 °C where the NGs will exist in their shrunken, more hydrophobic conformation. However, in line with previously published literature,28 purification of the hard corona however was conducted at 4 °C and subsequent assessments performed at room temperature, allowing the corona-coated NGs to swell increasing in size and water content. The introduction of water into the NGs may have displaced certain protein−NP interactions, while others may be lost due to the decreased particle density, resulting in a diminished number of local

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RESULTS AND DISCUSSION Characterization of Protein Coronas. The formation and character of NP coronas are known to be influenced by NP characteristics including size, chemistry, charge density, and surface topology.17,25 Following incubation with plasma pooled from three healthy donors, NPs were purified by centrifugation through a sucrose gradient to separate the NPs and their hard coronas from components of the soft corona and other unbound material. SDS-PAGE was then performed to separate and visualize the major proteinaceous components of the hard corona (Figure S1), and the BCA assay performed to quantify these (Figure 1). NP diameters and charges were also assessed

Figure 1. Quantification of NP protein coronas. NPs were incubated with human plasma at 37 °C for 1 h (light gray) and 24 h (dark gray), purified, and protein adsorption quantified by the BCA assay (n = 3; mean ± SEM; statistical significance between time-points assessed by paired t test: ∗, p ≤ 0.05).

with and without the hard corona by dynamic light scattering (DLS) and zeta potential measurements, respectively (Table 1). Additionally, given the time frame over which this study was conducted, NP diameters and polydispersities were reassessed after one year storage at 4 °C, which confirmed their long-term stability (Table S1). In keeping with previous reports, the hard corona increased between 1 and 24 h in terms of protein quantity but not variety 1764


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Table 1. Size, Polydispersity Index (PDI), and Zeta Potential of the Polymeric NPs before and after Incubation with Bovine Serum Albumin (BSA) or Human Plasma ζ potential [mV]

Z-average [nm] (PDI) NP CMS EC4CP EGRS dPG-pNIPAM dPG-NH2 (10%) dPG-NH2 (30%)

pristine 17.5 175.3 61.7 127.4 115.5 163.0

± ± ± ± ± ±

0.1 1.0 0.5 0.9 2.1 3.3

(0.18) (0.13) (0.26) (0.01) (0.1) (0.20)

BSA 22.4 171.9 131.9 136.3 112.7 230.3

± ± ± ± ± ±

plasma

0.3 (0.39) 3.1 (0.13) 14.2 (0.39) 3.06 (0.31) 0.819 (0.158) 33.9 (0.37)

58.6 190.2 192.43 448.6 146 372.63

± ± ± ± ± ±

5.4 (0.53) 1.9 (0.24) 0.81 (0.46) 84.2 (0.45) 62.7 (0.46) 17.39 (0.72)

pristine 1.6 −24.1 30.2 −0.9 5.7 17.9

± ± ± ± ± ±

0.1 0.7 0.5 0.2 0.2 0.6

plasma −18.4 −9.3 −5.9 −14.4 −13.6 −6.5

± ± ± ± ± ±

0.3 0.3 0.4 0.6 1.5 0.2

pNIPAM NGs, and CMS nanocarriers were bound by human serum albumin (66.5 kDa) only, by far the most abundant protein in plasma.66 Of these particles, the particularly large shift in zeta potential from the pristine to corona bearing CMS nanocarriers is particularly notable given their minimal protein binding (Table 1, Figure 1). The greater density in negative surface charge may result from greater surface exposure and packing of HSA, in line with the continuous surface of the CMS nanocarriers relative to the discontinuous surfaces of the NGs. Another possibility could be contributions from other proteins that may have associated with these particles at levels undetectable by PAGE. Indeed, when these particles were incubated with BSA alone, notably different hydrodynamic diameters were measured (Table 1), suggesting other plasmaderived proteins or biomolecules such as lipids play a significant role in defining the NP coronas.18,46,67 EC4CP NPs were bound by human serum albumin, the innate immune system activator precursor complement C3 (188.5 kDa), and the HDL component apolipoprotein A1 (28.3 kDa). EGRS NPs were bound by more than 20 different species, ranging from 10 to 290 kDa, including several transport proteins, apolipoproteins such as clusterin, active complement components, but interestingly not human serum albumin (Table S1). Previous work has demonstrated hydrophobic NPs preferentially bind apolipoprotein, whereas more hydrophilic NPs tend to attain fibrinogen, IgG, and albumin.29,46,48 Thus, the adherence of highly abundant albumin to the EGRS NPs is likely displaced by stronger interactions with other proteins. Recently, the binding of clusterin to PEG coated nanocarriers was shown to play a key role in imparting the “stealth” effects imparted by PEG surface chemistries, making its absorption to the EGRS NPs of note.63 Nanoparticle Adhesion and Cellular Uptake. Using fluorescently labeled NPs, the effects of protein coronas formed following 1 h incubation with human plasma on the nanoparticle−cell interactions. NP uptake, a combination of NP adhesion to the cell surface and entry into the cell (Figure 2a), as well as cell surface adhesion alone (Figure 2b), was assessed by flow cytometry. Validation experiments to confirm the absence of significant changes to the nanoparticles fluorescent properties when bearing a protein corona (Figure S2) and that the washing steps used effectively removed unbound NPs (Figure S3) were performed. Un-normalized data from the uptake and adhesion assays can be found in Figure S4 of the Supporting Information. For clarity, it should be emphasized that the measure of NP uptake reported here indicates internalization into the cell as well as cell adhesion. Evidence of dPG-pNIPAM NG and CMS nanocarrier endocytosis is reported elsewhere.42,68,69 Cellular adhesion and potential internalization of coronacoated CMS nanocarriers were significantly reduced in NHKs

interactions, and increased hydrophilicity. Indeed there is evidence from other pNIPAM NGs that protein adsorption may be reversibly regulated by temperature manipulation.58 Thus, it is possible that some adsorbed protein was lost from the dPG-pNIPAM NGs during processing. Persistently bound protein still appears present, however, given the clear impact plasma incubation had upon the surface charge and hydrodynamic diameter of these NGs (Table 1).10 The low protein adsorption to the dPG-pNIPAM NGs is consistent with the results from the dPG-NH2 NGs and the highly effective antifouling properties of dPG (Figure 1).52−54 Of the tested NPs, the CMS nanocarriers possessed the smallest pristine diameter and therefore the greatest curvature, a factor known to limit protein−protein interactions within the corona and decrease corona thickness.59,60 Though individually the CMS nanocarriers presented a smaller surface area, a factor directly linked to decreased protein adsorption,27,28,48,51,61 at matched polymer concentrations, their total number will greatly exceed those of the larger or denser particles, presenting a larger total surface area. In this regard, their low total protein adsorption is particularly noteworthy. CMS nanocarriers are surrounded hydrophobic domains attached directly to outer polyethylene glycol (PEG) domains,32 a charge neutral and hydrophilic polymer used widely within pharmaceuticals to bestow antifouling properties.62 On hydration, this results in the self-assembly of a hydrophilic corona that buffers the CMS nanocarriers against interactions with plasma components, a strategy successfully used by many other NPs to diminish protein−NP interactions. Notably, however, protein coronas do still form on PEGylated NPs and have even been shown to play an important part in imparting the “stealth” related effects of this chemistry.24,55,56,63 Incubation with plasma resulted in increased hydrodynamic diameter of all purified hard corona-bearing NPs (Table 1). While this was only moderate for the EC4CP NPs and dPGNH2 (10%) NGs, the diameters of the CMS nanocarriers and EGRS NPs as well as the dPG-NH2 (30%) dPG-pNIPAM NGs more than doubled, indicative of NP flocculation. The formation of supramolecular aggregates of CMS nanocarriers has previously been observed on encapsulation of hydrophobic compounds and indicates a similar phenomena may be occurring here.64 Hydrodynamic diameters were also measured following NP incubation with negatively charged BSA alone, over 1 h (Table 1). Following purification, these particles demonstrated more moderate increases in diameter that appeared to correlate with pristine NP surface charge, in line with other work.65 To determine the major proteinaceous components of hard protein coronas, visible protein bands from PAGE (Figure S1) were excised and assessed by MALDI-TOF analysis. The low density hydrophilic dPG-NH2 NGs (10% and 30%), dPG1765


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Figure 2. Cellular uptake and adhesion of corona-coated nanoparticles. (a) Uptake and (b) adherence of corona-coated NPs to normal human keratinocytes (NHK, lined white), HUVECs (lined gray), and macrophages (lined black), expressed as a percentage of pristine NP uptake or cell adherence (n = 3; mean ± SEM; results are normalized against pristine NP control of each replicate; statistical differences against 100% control assessed by two-way ANOVA with Dunnett’s correction for multiple comparisons: ∗, p ≤ 0.05; ∗∗, p ≤ 0.01; ∗∗∗, p ≤ 0.001).

NHKs and HUVECs (Figure 2b), in agreement with other work that casts doubt on the relevance of direct noncovalent charged interactions between anionic cell membranes and corona-coated cationic NPs.28,47 This does however conflict with their decreased uptake in both cell types (Figure 2a). As noted earlier, EGRS NPs acquired the largest and most varied hard protein corona, which appeared to produce an increased propensity for NP flocculation. While increased NP diameter is one major cause for decreased cellular uptake by many endocytic pathways, it may also present an increased surface area over which cell−NP interactions may occur given the correct particle geometry.72,73 Interestingly, though EGRS NPs bound fibronectin, complement C4a, and complement C4b, active members of the complement cascade, no dramatic increase in macrophage uptake was seen as might be expected on complement activation.75,76 In this regard, it is notable that clusterin was also found in the hard corona, a protein recently attributed a key role in mediating the lowered NP−cell interactions seen in NPs with PEG surface chemistries.63 Adhesion and internalization of the dPG-NH2 (10% and 30%) NGs were not significantly impeded by the presence of a plasma-derived corona (Figure 2). Like the CMS nanocarriers, these were measurably bound by albumin alone and at comparable levels (Figure 1). Whether corona-bearing or not, un-normalized uptake and cell adhesion values indicate little measurable cellular interactions of the dPG-NH2 (10%) NGs with NHKs, HUVECs, or macrophages (Figure S4). Unnormalized values from the dPG-NH2 (30%) NGs on the other hand show large cell adhesion, in line with their greater surface charge (Figure S4). Previous work using porous NPs possessing surface topologies comparable to these NGs demonstrated the adsorption of albumin following incubation with fetal bovine serum.45 The albumin subsequently underwent unfolding that exposed misaligned epitopes, recognizable by scavenger receptors present on macrophages and resulting in macrophage activation and phagocytosis. In this regard, the unaffected dPG NH2 (30%) uptake is of note. The uptake of dPG-pNIPAM NGs too was unaffected by protein corona formation (Figure 2a). Though cellular adhesion of the corona bearing dPGpNIPAM NGs was significantly decreased in all cells, the temperature sensitivity of these NGs needs to be taken into account (Figure 2b). This assay was conducted at 4 °C, and the results reflect differences in the adhesion of water swollen NGs where the protein corona appears to have a clear impact. In the NG’s shrunken conformation at 37 °C, the corona appears to have no effect on our measure of uptake, a combination of NG internalization and cell adhesion, from which one could

relative to pristine NPs, reflected by significantly less adherent corona-coated CMS nanocarriers (Figure 2). This may relate to the absorption of albumin to the CMS nanocarriers and subsequent reductions in cell membrane binding avidity. Previous work has demonstrated albumin based coronas decreased NP internalization in phagocytic and nonphagocytic cells and has recently been indicated to mediate the stealth related properties of PEG surface coatings in at least some NPs.45,63,70,71 Size, density, and surface topology are also known to impact on the endocytosis of NPs by cells owing to the effects these characteristics have on the ability of NPs to deform the cell membrane and the energy with which they bind to it.72,73 The decreased uptake of particles may therefore also partly be caused by the increases to CMS nanocarriers hydrodynamic diameter (Table 1). It should however be noted that studies have typically indicated NP diameters around 40−60 nm as best suited for enhanced uptake, though this estimate does depend on multiple NP properties.45 Adhesion and potential internalization of CMS nanocarriers to HUVECs by contrast appeared only marginally impeded by the presence of the corona (Figure 2), though this might be explained by their particularly low total uptake and adherence values (Figure S3). While the overall adherence of CMS nanocarriers to macrophages was also relatively low (Figure S3), a statistically significant decrease of corona bearing particle adherence was detected that did not apparently affect their relatively high NP uptake (Figure 2). While the adherence of corona-bearing EC4CP NPs was decreased in all tested cells, their uptake onto, and potentially into the cell were only significantly decreased in NHKs and HUVECs (Figure 2). The prominence of phagocytosis in macrophages may perhaps explain this disparity (Figure 2). In addition to albumin, these NPs were also bound by complement C3, which too has been associated with decreased cell uptake in nonimmune cells.71 Though in the canonical pathway C3 must be broken-down to the C3a fragment to activate receptors found on macrophages, adsorption of the whole protein to nanoparticles has been found to induce macrophage activation.24,74,75 In this regard, it is notable that EC4CP uptake on and into macrophages was not increased. EC4CP NPs are also bound by Apolipoprotein A1, the major proteinaceous component of high density lipoproteins (HDL). Interestingly, previous work has indicated that HDL is capable of binding to NPs intact, and cells may subsequently recognize and process NPs as lipoproteins.46 Despite their cationic to anionic shift in surface charge, corona-coated EGRS NPs show largely unaltered adherence to 1766


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Figure 3. Cytotoxicity of pristine and corona-coated nanoparticles. Pristine (solid bars) and corona-bearing (lined bars) were incubated with (a) normal human keratinocytes (NHK, white) and HUVECS (gray) for 48 h as well as (b) with macrophages (black) for 4 h. Cell viability was assessed by (a) MTT assay and (b) annexin V and PI staining (n = 3, mean ± SEM; statistically significant differences between corona-bearing and pristine NPs assessed by multiple t tests with Holm-Sidak corrected multiple comparisons: ∗, p ≤ 0.05).

Interestingly, the cationic dPG-NH2 (30%) NGs proved moderately toxic to NHKs but not HUVECs or macrophages, an effect less pronounced with the less cationic dPG-NH2 NGs. This correlates well with the NG adhesion and uptake, which was greatest for dPG-NH2 (30%) in NHKs. The effect was unaltered by the protein corona, in keeping with unchanged uptake between the pristine and corona-coated NGs. Induction of Cytokine Release from Macrophages. The release of the cytokines IL-1ß, IL-6, and IL-10 from macrophages was measured following 4 h incubation with NPs (Figure 4). In all instances, the pristine dPG-NH2 (30%) NGs induced the greatest inherent cytokine release, significant increases relative to control being seen for all tested cytokines following treatment with the pristine NG. All corona bearing NGs induced significant increases in IL-6 and -10 release relative to control (Figure 4b,c). Between the pristine and corona-bearing NPs, significant increases of IL-6 release from macrophages were seen from dPG-NH2 NGs (Figure 4b) and significant releases in IL-10 release from dPG-pNIPAM NGs and dPG-NH2 (30%) NGs (Figure 4c). The release of IL-1ß by contrast appeared unaffected by the presence of the protein corona relative to pristine NPs (Figure 4a). No significant impact on cytokine release was seen in macrophages incubated with the colloidal NPs, whether pristine or corona-bearing. Though IL-10 is an immunosuppressive cytokine, it is typically released in tandem with other pro-inflammatory cytokines as a mechanism to limit and control the inflammatory response, thus could still signify immunogenic potential.30 Why the corona based dPG-based NGs induce increased IL6 and IL-8 release not seen with the tested corona bearing NPs is currently unclear. Work elsewhere has demonstrated the ability of dPG-sulfate to induce the release of several cytokines from chondrocytes, including IL-1ß and IL-10.77 Thus, the results seen here may partly stem from the shared dPG chemistry between the NGs, the differences in cytokine induction perhaps relating to differences in macrophage binding. Intriguingly, one study demonstrated a general positive correlation between cytokine expression and NP surface hydrophobicity in vitro.78 Given the relatively small abundance of protein adsorption to these particles, this perhaps represents a correlation between the strength of interaction between NPs and immune cells. While cell adhesion of other NPs tested here was reduced, the dPG-NH2 NGs showed unaltered macrophage adhesion following attainment of a plasma-derived corona, as is speculatively the case for dPG-pNIPAM NGs held above their transition temperature (Figure 2b). Albumin bound to the NGs may have undergone conformational changes that expose them

speculate adhesion would be unaffected by the presence of the protein corona, based on the uptake of dPG-pNIPAM NGs at 37 °C (Figure 2a). The lack of apparent change to the uptake of corona bearing dPG-NH2 and dPG-pNIPAM NGs is notable when compared to the clear decreases seen in the colloidal NPs in NHKs and HUVECs, particularly the CMS NPs, which attained protein coronas similar in size and composition to the NGs. It is interesting to consider that of the tested NPs, only the NGs present nonuniform surface chemistries and topologies. Given the antifouling properties of dPG, plasma proteins are likely to discriminately bind to the cross-linking chemistries that make up these NGs.52−54 It could be speculated that the dPG of the NGs primarily mediates NG−cell surface interactions and the absence of protein from this chemistry results in relatively unaltered uptake for corona-bearing NGs. Nanoparticle Toxicity. The toxicity of unlabeled NPs in NHKs and HUVECs following 24 (Figure S5) and 48 h (Figure 3a) incubation was assessed by MTT assay. Similarly, NP toxicity in macrophages following 4 h incubation with NPs was measured by Annexin V and propidium iodide (PI) staining, where absence of both indicated cell viability (Figure 3b, S6). Cell viabilities below 80% are considered as cytotoxic effects. Following 24 and 48 h incubation with NHKs or HUVECs, no significant differences were seen between the cytotoxicities of pristine or corona-coated NPs (Figures 3a and S5). This is with the exception of the EGRS NPs, which after 48 h incubation were significantly less toxic to NHKs and HUVECS when in possession of a plasma-derived corona (Figure 3a). The same significant effect is seen in macrophages after 4 h (Figure 3b). EGRS NPs are by far the most cationic NPs, a characteristic well known for its cytotoxic induction. The plasma-derived corona not only lowers the uptake of EGRS NPs, but also reverses its surface charge, providing two mechanisms by which toxicity may be reduced, assuming the corona remains intact on NP-cell interactions as seen with other NPs (Figure 2a; Table 1). It is notable however that these differences were not seen after 24 h incubation with NHKs and HUVECs (Figure S5). While EGRS NPs have previously proven nontoxic to primary dermal fibroblasts at concentrations and time-points matched to the present study, these differences likely reflect differences in the cell types used.39 For instance, macrophages demonstrate a far larger uptake of EGRS NPs relative to NHKs and HUVECs (Figure 2) and demonstrate a loss of viability at 4 h that is only achieved following 48 h incubation in the other two cell types (Figure 3). 1767


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Figure 4. Induction of interleukin release from macrophages by nanoparticles. Release of (a) IL-1ß, (b) IL-6, and (c) IL-10 from macrophages following 24 h incubation with pristine (solid bars) and protein corona-bearing (lined bars) NPs. Untreated macrophages served as control (n = 3, mean ± SEM; statistically significant differences between corona-bearing and pristine NPs assessed by multiple t tests with Holm-Sidak corrected multiple comparisons: ∗, p ≤ 0.05. Statistical significance from untreated control was assessed by two-way ANOVA with Dunnett’s correction for multiple comparisons: §, p ≤ 0.05; §§, p ≤ 0.01; §§§, p ≤ 0.001).

influences were anticipated. Moreover, the NGs are not readily suitable for encapsulation of the moderately hydrophobic dexamethasone given their hydrophilicity and the likeliness of small molecules to freely diffuse through and out of these 3D polymer networks. However, it is notable that the dPGpNIPAM NGs have been shown to encapsulate biomacromolecules such as proteins with exceptional efficiencies when coadministered on NG hydration and to efficiently release these in a temperature-dependent manner.10

to binding by scavenger receptors present on macrophages and which induce inflammatory responses such as enhanced cytokine release.45,65,79,80 The especially low level of protein adhesion found on the NGs may contribute further to this by limiting possible steric hindrance between denatured albumin and macrophages that excesses of NP associated protein may present to the macrophages. The pristine and corona-bearing CMS nanocarriers demonstrated little cytokine induction, in line with the mPEG-based surface chemistry.32,62 It is important to understand that while macrophages are major players in the innate immune response, assessment in a single immune cell type is limited given the full complexity of the in vivo immune response, and so results require careful interpretation.30 For instance, the EGRS NPs were bound by active members of the complement cascade fibronectin, complement C4a and complement C4b, which are known to induce features of acute inflammation that could not be detected within this in vitro setting, though this might be explained by the presence of clusterin and other apolipoproteins within the hard corona.63 Along with the EC4CP NPs, they were also bound by complement C3, a precursor of the complement cascade that has previously been found associated with corona-coated NP complement activity.81 Nonetheless, the large cytokine release from the dPG-NH2 (30%) NGs is a good indication of their potential immunogenic potential. Nanoparticle Drug Release. The effects of the plasmaderived corona on drug release from dexamethasone loaded EGRS and EC4CP NPs were assessed over 24 h by diffusion assay under sink conditions.38 While dexamethasone release from EC4CP NPs was unaffected, a marked decrease was seen for corona-bearing EGRS NPs (Figure 5). EGRS NPs possess a hard protein corona more than double the weight of that found on EC4CP NPs, possibly explaining the observed differences. In addition, EGRS NPs showed indications of an increased propensity to form interparticle aggregates that the EC4CP NPs did not, another potential mechanism by which drug release may be impeded. Previous work has also noted altered drug release from NPs as a result of the protein corona, and indeed has even been intentionally used to manipulate pay-load release.82,83 A significant difference between the dexamethasone release from EGRS NPs (two-way ANOVA, p < 0.001), but not the EC NPs, was detected. The drug release profiles were measured exclusively for the EC4CP and EGRS NPs since only these particles demonstrated large scale protein corona formation (Figure 1), and potential

Figure 5. Impact of the plasma corona on drug release. The release of dexamethasone from loaded EGRS (squares) and EC4CP (circles) nanoparticles when pristine (hollow shape) or bearing a plasma derived corona (black solid shapes) was assessed by Franz cell diffusion assay under sink conditions (n = 3, mean ± SEM).

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CONCLUSION The present study investigates the formation and impact of the protein corona on polymeric NPs of varied surface chemistry, size, architecture, and mechanical properties. The largest and most varied coronas were found on solid and charged EGRS and EC4CP NPs, and clear alterations to their cellular adhesion and uptake were seen. In the instance of the EGRS NPs, this also imparted reductions to their inherent in vitro cytotoxicity. Interestingly the cellular uptake of the comparatively low density and charge-neutral CMS nanocarriers was also notably impacted, despite only marginal protein adsorption by albumin only, which highlights the significance of even minimal protein 1768


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Core Facility BioSupraMol supported by the Deutsche Forschungsgemeinschaft (DFG). Moreover, we thank the German Research Foundation for financial support of G.Y. and S.H. (HE 7440/4-1) and the Collaborative Research Center (SFB) 1112 (S.H., B.B., R.B., R.H., M.C.).

adsorption. By contrast to these colloidal NPs, the low density dPG-NGs were essentially unaffected by the protein corona in terms of uptake, though they too were bound only by albumin in marginal amounts. Instead, the protein corona impacted markedly on their ability to induce cytokine release from macrophages. In this regard, it is notable that of the tested NPs, only the NGs possessed nonuniform surface chemistries and topologies as well as a greater inherent ability to undergo conformational changes. Finally, drug release from the EGRS but not EC4CP NPs was impaired by the presence of the protein corona, likely reflecting the different magnitudes of protein adsorption to these NPs and suggesting potential differences in drug release profiles assessed in vitro or in vivo. Together these results highlight a distinction in the biological impact of corona bearing polymeric dPG NGs and polymeric colloidal NPs, something that may relate to the discontinuous surface chemistries and topologies of the NGs or more specifically the dPG chemistry.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.7b00158. Materials and methods used; table listing protein components of EGRS hard protein corona; table detailing z averages of particles; representative SDSPAGE gels from corona bearing NPs; graph detailing impact of corona formation of NP fluorescence; validation of washing steps used in uptake and adherence assays; un-normalized data from uptake and adherence assays; NP toxicity following 24 h incubation with NHKs and HUVECs; representative dot plot from macrophage Annexin V and PE staining (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +49 30 838 55065. Fax: + 49 30 838 455065. ORCID

Rainer Haag: 0000-0003-3840-162X Sarah Hedtrich: 0000-0001-6770-3657 Author Contributions ¶

These authors contributed equally to this work. The NPs were prepared by E.M., M.D., and B.B. Protein corona assessment, NP characterization, cell uptake, cytototoxicity, and cytokine release studies were performed by K.O. Mass-spectrometric analysis was performed by K.O. and C.W. Drug release studies were performed by B.B., K.O., and G.Y. Experimental planning was performed by S.H., N.C., G.Y., R.B., and R.H. Data analysis was performed by K.O. and G.Y. Data interpretation was conducted by G.Y. and S.H. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant from the Helmholtz Virtual Institute (K.O., N.C., E.M., S.H., M.C.). For mass spectrometry (C.W.), we would like to acknowledge the assistance of the 1769


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