Neonatal Fc Receptor Binding Tolerance toward the Covalent

Brief Article pubs.acs.org/molecularpharmaceutics

Neonatal Fc Receptor Binding Tolerance toward the Covalent Conjugation of Payloads to Cysteine 34 of Human Albumin Variants Steffan S. Petersen,†,∥ Eva Klan̈ ing,†,∥ Morten F. Ebbesen,†,∥ Birgitte Andersen,‡ Jason Cameron,§ Esben S. Sørensen,∥ and Kenneth A. Howard*,†,∥ †

Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK-8000 Aarhus, Denmark Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus, Denmark ‡ Novozymes A/S, Krogshøjvej 36, DK-2880 Bagsværd, Denmark § Novozymes Biopharma UK Ltd., Castle Court, 59 Castle Boulevard, NG7 1FD Nottingham, United Kingdom ∥

S Supporting Information *

ABSTRACT: The long circulatory half-life of albumin facilitated by the interaction with the cellular recycling neonatal Fc receptor (FcRn) is utilized for drug half-life extension. FcRn engagement effects following covalent attachment of cargo to cysteine 34, however, have not been investigated. Poly(ethylene glycol) polymers were used to study the influence of cargo molecular weight on human FcRn engagement of recombinant wild type (WT) albumin and an albumin variant engineered for increased FcRn binding. Decreased affinity was observed for all conjugates; however, the engineered albumin maintained an affinity above that of unmodified wild type albumin that promotes it as an attractive drug delivery platform. KEYWORDS: albumin, cysteine 34, neonatal Fc receptor (FcRn), covalent conjugation, poly(ethylene glycol), biolayer interferometry

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INTRODUCTION Albumin is the most abundant plasma protein and is responsible for supporting the colloidal osmotic pressure homeostasis, buffering the blood pH, and ligand binding transport of small molecules such as fatty acids.1,2 Human serum albumin (HSA) consists of 3 domains (DI− III, where each domain is composed of two subdomains, a and b, e.g., DIa or DIb) showing considerable homology, and has a long circulatory half-life of ∼19 days in humans facilitated by receptor-mediated megalin−cubilin renal rescue and engagement with the cellular neonatal Fc receptor.3−5 FcRn is a major histocompatibility class I receptor that comprises a transmembrane heavy chain (HC), which noncovalently associates with the common β2m subunit. The HC consists of three extracellular domains (α1, α2, and α3).6 The receptor is found in many tissues and cell types, including vascular endothelial and hematopoietic cells.7 FcRn engagement of albumin has been shown to exhibit a pH-dependent binding profile with an increased binding strength at acidic pH.3,8 Structural insights into the binding region show that the DIII of HSA exhibits an © XXXX American Chemical Society

extensive interface of contact with human FcRn (hFcRn), whereas DI is important for optimal binding FcRn binding.8−11 FcRn regulates the serum half-life of IgG and HSA by rescue from lysosomal degradation as a consequence of high affinity binding in the acidic endosomal compartments, and subsequent trafficking by a recycling pathway to the extracellular surface and consequent release at physiological pH.3,12 Direct evidence for the intracellular transport of albumin is lacking in the literature; nonetheless, recombinant human albumins with an enhanced FcRn binding region have been shown to alter the blood circulation profile of human albumin variants in mice and nonhuman primates, which results in an increased circulation time.11,13 Harnessing the physiological capacity of HSA to transport a range of molecules is an attractive feature that has been utilized Received: August 6, 2015 Revised: November 8, 2015 Accepted: December 9, 2015

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DOI: 10.1021/acs.molpharmaceut.5b00605 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Brief Article

Molecular Pharmaceutics

(0−5 min, 0% buffer B; 5−50 min, 0−100% buffer B) at a flow rate of 0.85 mL/min. All the collected fractions were dissolved in Milli-Q water and evaluated by SDS−PAGE. The homogeneous fractions containing the conjugated albumin variant PEG products were used for subsequent binding studies and molecular weights determined using MALDI-TOF (Brüker) using the dried droplet method with sinapinic acid. Additionally, albumin variants (4 mg/mL) containing equimolar amounts of either 2 or 8 kDa mPEG (Sigma-Aldrich) were produced, and evaluated by SDS−PAGE and BLI. Determination of hFcRn Binding by Biolayer Interferometry. An Octet RED96 system (FortéBio) was used with a soluble nonglycosylated hFcRn expressed in Escherichia coli17 (Immunitrack, Denmark) coupled using biotin−streptavidin chemistry. Briefly, streptavidin-coated biosensors were first washed in PBS pH 7.4 with 0.01% Tween-20 (T-20) before the addition of biotinylated hFcRn receptor in PBS pH 7.4 with 0.01% T-20. The sensors were then washed in PBS pH 7.4 with 0.01% T-20. Kinetic titration series were performed in the interaction buffer (25 mM sodium acetate, 25 mM NaH2PO4, 150 mM NaCl 0.01% T-20, pH 5.0−7.0). Regeneration was performed in 10 mM PBS, 0.01% T-20, pH 7.4. In total, seven sensors were used to measure, in parallel, seven different analyte concentrations, while one sensor was used to measure the buffer reference. Two baselines were made between each measurement of 60 and 30 s. Kinetic measurements were performed with the concentrations 3030, 1515, 757.6, 378.8, 185.4, 34.7, and 47.35 nM in parallel at 30 °C. To measure the interaction between the conjugates and hFcRn, the association and dissociation phases were recorded for 120 and 300 s, respectively, with an agitation speed of 1000 rpm. All binding profiles were zero-adjusted, and the buffer reference value was subtracted. The seven binding profiles were fitted using predefined 1:1 binding models provided by Octet software v. Eight (FortéBio) to calculate the binding kinetic constants.

for drug half-life extension for prolonged therapeutic effects.14,15 The role of HSA in ligand binding transport has been exploited in the design of albumin-binding drugs; however, a commonly employed approach is direct chemical coupling of the drug.15 Covalent conjugation through the single free thiol in cysteine 34 (Cys34) is a standard strategy that facilitates site-specific coupling distant from the main hFcRnbinding region.8,16 The effect of covalent conjugation of Cys34 in albumin on hFcRn engagement, however, has not been addressed. In this work, the influence of payload conjugation to Cys34 on albumin−FcRn engagement was investigated. A range of poly(ethylene glycol) (PEG) polymers with defined molecular weight (Mw) were used as a model drug and conjugated by maleimide chemistry to the thiol of Cys34 of recombinant human wild type albumin (WT) and a human high binding FcRn variant (HB) K573P, where Lys573 was replaced by a proline within the C-terminal helix of DIII.13 The conjugated samples were HPLC purified and characterized, and the hFcRn binding affinity was determined using biolayer interferometry (BLI). A decrease in affinity with increasing PEG Mw was observed for all conjugates; however, HB albumin conjugates maintained affinities well above that of the WT counterpart. This work suggests that the hFcRn interaction is tolerant to drug payload conjugation to HB albumin and promotes the application of HB albumin to control the pharmacokinetics and tune the therapeutic profile of Cys34 covalently conjugated drugs.

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EXPERIMENTAL SECTION Materials. Monofunctional mPEG-maleimide polymers of 5, 10, and 30 kDa were purchased from Laysan Bio, USA. Recombinant human wild type (WT) albumin and hFcRn highbinding (HB) albumin variant (a single Lys573Pro (K573P) amino acid substitution) were provided by Novozymes Biopharma UK. When referring to the albumin variants or conjugates, the recombinant albumin is referred to as WT or high binder (HB). Albumin−Drug Conjugation, Purification, and Analysis. The available thiol content in albumin was determined by Ellman’s assay. The albumin variants were diluted to 4 mg/mL in 0.1 M Tris-HCl, 0.01 M EDTA, pH 8, and to 100 μL was added 50 μL of DTNB solution (0.01 M 5,5′-dithiobis(2nitrobenzoic acid) in 0.05 M sodium phosphate buffer pH 7.0). The mixture was incubated for 10 min and the absorbance measured at 412 nm with a plate reader (Biotek PowerWave XS2). A cysteine hydrochloride standard curve was used to calculate the thiol concentration. The respective mPEG-maleimide polymers were conjugated to the albumin variants (4 mg/mL) with a molar ratio of 5:1 in Dulbecco’s PBS, pH 7 (Life Technologies), by overnight shaking at room temperature. Conjugation of the polymers to the albumin variants was confirmed by 10% SDS−PAGE using NuPAGE MES SDS running buffer (Life Technologies) and stained with Coomassie Brilliant Blue (Thermo Scientific). The conjugation resulted in a heterogeneous solution of nonconjugated and conjugated albumin variant PEG products. The albumin−PEG conjugates were purified from the heterogeneous solution by RP-HPLC on a Vydac C4 column (The Separations Group) connected to a Pharmacia LKB system (Pharmacia). Separation was carried out at 40 °C in 0.1% aq TFA (buffer A) and eluted with a gradient of 75% propan-2-ol in 0.1% aq TFA (buffer B) developed over 50 min

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RESULTS Sixty percent of free thiol, as determined by the Ellman’s assay, was found to be available for conjugation for both the WT and HB albumin variants. A range of well-defined albumin−PEG conjugates with a single PEG of 5, 10, or 30 kDa were covalently coupled (Figure 1 and Figures S1 and S2). Additionally, the Mw of the conjugates were identified by MALDI-TOF (Table 1). No albumin−PEG conjugates were

Figure 1. SDS−PAGE analysis of the covalent attachment of PEG to WT albumin. A well-defined shift is observed after addition of mPEGmaleimide (shown by arrows), whereas no shifts are observed after addition of either mPEG (WT-nc2k and WT-nc8k). B


Brief Article

Molecular Pharmaceutics Table 1. Recombinant Albumin and Conjugate Variants albumin or conjugate variants WT WT-5k WT-10k WT-30k HB HB-5k HB-10k HB-30k a

substitution

PEG [kDa] 5 10 30

K573P K573P K573P K573P

5 10 30

Table 2. Biolayer Interferometry (BLI) of Recombinant Albumin Binding to hFcRna

measd Mwa [kDa]

kinetic parameters from the measurement in Figure 2

66.4 72.5 77.6 98 66.4 72.5 77.6 98

Molecular weight determined using MALDI-TOF.

formed with the albumin variants containing equimolar amounts of either 2 or 8 kDa mPEG, i.e., nc2k or nc8k (nonconjugated (nc) mPEG), thus confirming specific conjugation using maleimide chemistry to albumin (Figure 1, WT-nc2k and WT-nc8k). The influence of the covalent attachment of PEG on albumin variant−hFcRn binding was first investigated with WT albumin using biolayer interferometry. Equal concentrations of each conjugate were analyzed against hFcRn immobilized on the biosensor tip at pH 5.5. As albumin associates and dissociates from the biosensor tip surface, the interference pattern of the reflected light from the surface of the biosensor tip shifts compared to the interference pattern of a reference surface. The difference between the two interference patterns is recorded and gives a classical association/dissociation curve from which the binding affinities, KD, can be calculated. The WT albumin conjugates showed reversible binding at pH 5.5, but with reduced binding capacity (Figure 2A−D). The kinetic calculations indicate that the affinity of unmodified WT albumin for hFcRn was 811 nM, while attachment of 10 kDa PEG lowered the affinity to 2225 nM (WT-10k) (Table 2). The conjugation of PEG to Cys34 had a negative effect on the binding affinity toward hFcRn for all the conjugated WT variants, which could be attributed to slower association rates as the dissociation rates were unaffected (Table 2). These results prompted us to investigate the effect of covalent attachment to a HB albumin variant (K573P), which previously had shown an extended circulatory half-life due to improved FcRn affinity.13 In contrast to WT albumin, the HB albumin exhibited a considerably stronger binding (Figure

KD av [nM]

ka × 103 [1/Ms]

kd × 10−3 [1/ s]

KD [nM]

R2c

± ± ± ± ± ± ± ± ± ± ± ±

13.0 4.5 5.0 2.8 18.8 20.8 18.6 7.6 6.9 3.8 21.3 23.1

17.6 20.2 16.9 15.1 17.7 17.5 1.0 1.2 1.1 1.0 2.5 1.6

1351.0 4509.7 3347.9 5472.7 941.5 841.3 55.1 156.8 164.1 260.4 87.0 68.7

0.997 0.990 0.981 0.972 0.995 0.996 0.999 0.999 0.999 0.999 0.998 0.998

albumin or conjugate variants WT WT-5k WT-10k WT-30k WT-nc2kd WT-nc8kd HB HB-5k HB-10k HB-30k HB-nc2kd HB-nc8kd

811 5722 2225 2400 828 753 52 189 156 316 78 75

b

613 2020 1119 2249 120 80 18 42 16 52 8 6

a

The kinetic rate constants at pH 5.5 are obtained using a 1:1 binding model with the relation KD = kd/ka. bThe KD represents the average of 3−6 replicates each consisting of a 7-step dilution series. cR2 values resulting from curve fitting using the using the 1:1 binding model from Octet software v. Eight (FortéBio). dKinetic values represent equimolar amounts of nonconjugated mPEG to albumin in the interaction buffer.

2A,E). Reversible binding of the HB conjugates was observed at pH 5.5, however, with a reduction in affinity (Figure 2E−H). The attachment of 10 kDa PEG lowered the hFcRn affinity from 52 to 156 nM, HB and HB-10k, respectively. The most prominent effect was observed for the 30 kDa PEG attachment (HB-30k) (Table 2). The effect was due to slower association rates. The HB conjugates still exhibited a much slower dissociation rate compared to unmodified WT albumin due to the amino acid substitution. We attempted to investigate whether the presence of free PEG in the interaction buffer could affect the albumin−FcRn engagement, therefore, the nc mPEG albumin samples were investigated using BLI to mimic conditions comparable to those of the albumin−PEG conjugates. Free mPEG was not found to affect the binding affinity for WT or HB (WT/HBnc2/8k), as indicated by the KD (Table 2). When normalized KD values are compared (Figure 3), it is clear that the HB

Figure 2. Representative binding profiles showing hFcRn binding of PEG-conjugated WT and HB albumin variants obtained by BLI at pH 5.5. C


Brief Article


used as a well-defined model to evaluate the Mw influence of the payload. We showed that the attachment of PEG resulted in a 2−3fold lowered hFcRn affinity of the WT albumin. A lower affinity than endogenous albumin for the hFcRn receptor could restrict the effectiveness of albumin−drug conjugates. The unmodified HB albumin variant showed up to 25 times higher affinity toward hFcRn at pH 5.5 than unmodified WT albumin, and this engineered improvement was translated into a 5−9 times improved affinity of the HB conjugates with up to 30 kDa covalently bound (Figure 3). Furthermore, this data indicates that the HB conjugates maintain a binding affinity higher than unmodified WT albumin at relevant pH that suggests a capability for prolonged blood circulation mediated by hFcRn recycling. Additionally, all PEG conjugates maintained the necessary pH-dependent binding required for intracellular transport of albumin recycling by hFcRn (Figure 4). According to the crystal structure complex of HSA bound to hFcRn (Figure 5A),8 Cys34 lies close to the binding interface between DI of HSA and α1−α2 of hFcRn (Figure 5B). Previous work has highlighted the importance of DI for optimal FcRn binding.8,11,18 It seems likely that the covalently attachment of PEG to Cys34 in albumin introduces interference, by either a slight conformational change in DI or steric hindrance, and thereby disrupts the suggested interactions (Figure 5B).8 Both types of interferences could be a possible explanation for the significant reduction in the association rate of all the albumin conjugates. This suggests a slower recognition between the albumin conjugates and hFcRn. Considering the pharmacokinetics of albumin−drug conjugates, the FcRn recycling ensures the return into the bloodstream and a slower recognition might decrease the circulatory half-life of the conjugates. The utilization of engineered albumin variants with improved affinity may compensate for slower recognition.11,13 On the other hand, if the dissociation is too slow, the conjugates cannot be released. As albumin binds to FcRn in acidified endosomes before being sorted and recycled back and exposed to the neutral pH extracellularly, the pH-dependent process is highly important.3,12 All the HB PEG conjugates maintained the pH-dependent affinity (Figure 4B), which suggests that the slower dissociation does not alter the pHdependent FcRn recycling. As all HB conjugates maintained an affinity toward hFcRn above the affinity of unmodified WT albumin, we investigated the crystal structure of hFcRn bound to HSA for a possible explanation.8 The amino acid substitution, K573P, in the HB variant is located in the C-terminal part of albumin, which is close to the β2m chain of hFcRn8,13 (Figure 5C). DIII is the principal binding site in HSA, and a mutation in this region could promote the formation of a stronger hydrophobic interface with hFcRn and β2m, which may compensate for the otherwise important binding site in DI for optimal binding and, thereby, rescue the lowered affinity introduced by the covalent attachment of drug cargo to Cys34 of albumin.10,11,13 This has been suggested to be similarly beneficial for peptide fusions to the C- or N-terminal part of albumin.9,13 PEG has been suggested to behave as a random coil adjacent to the protein rather than protein wrapping.19 We observed a very significant drop in affinity when 5 kDa PEG was attached, which highlights the need for albumin variants with improved affinity for the hFcRn (Figure 3). In particular, 5 kDa showed the largest effect for WT albumin. As the 5 kDa PEG is smaller, and perhaps more rigid than the larger PEG polymers, we

Figure 3. Relative affinity mean ± standard deviation of PEGconjugated albumin variants to hFcRn. Affinity constants for all conjugates were normalized to the affinity of WT in the corresponding measurements.

albumin conjugates maintained a considerable improvement in binding affinity to hFcRn over the WT albumin conjugates. HB-30k showed a 4-fold improved binding strength to hFcRn as compared to unmodified WT albumin. The high affinity of albumin to FcRn at low pH and subsequent endosomal recycling and consequent release at physiological pH is seemingly required for an extended half-life of an albumin−drug conjugate. Using BLI as above, the panel of WT and HB albumin with PEG attachments were screened from pH 5.0 to 6.5. The KD of all conjugates maintained the same pH-dependent binding affinity as unmodified WT and HB albumin (Figure 4). The conjugation of PEG to Cys34, therefore, did not disrupt the pH dependency of the hFcRn interaction.

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DISCUSSION In this work, we investigate the effect on hFcRn engagement of covalent conjugation to the thiol of Cys34 of wild type albumin and an albumin variant engineered for improved hFcRn binding. PEG polymers in the range from 5 to 30 kDa were

Figure 4. pH−affinity profiles for hFcRn engagement of the WT (A) and HB (B) albumin variants and conjugates. (*) No binding at pH 6.5. D


Brief Article


Figure 5. (A) An overview of the crystal structure complex between hFcRn and HSA. The three domains of albumin are shown in purple while the HC of the hFcRn is shown in green with the β2m subunit in gray. (B) Close-up of Cys34 in domain DIa in the vicinity of the possible charged interactions indicated by black dotted lines between the interface of HSA DI (purple) and the hFcRn α-domains (green). (C) Close-up of the K573P mutation in WT albumin to generate HB albumin with less charged interaction with the β2m subunit. Illustrations made with PyMOL (PDB entry 4N0U from ref 6).

speculate that it may fit better between into the groove close to Cys34 to disrupt the charged interactions between HSA DI and the hFcRn α-domains to a higher degree than the larger PEGs8,10,11 (Figure 5B). This leads to lower interactions in this region, and the overall binding is less in this case. This may account for a size-independent affinity observed for WT albumin. The suggested formation of the stronger hydrophobic interface between DIII of albumin and hFcRn and β2m with the HB variant supports the binding, and consequently the HB conjugates exhibited a more size-dependent lowered affinity with increasing Mw of conjugated PEG (Figure 3). It has previously been suggested that the C-terminal part of albumin has some conformational flexibility and might stabilize the binding to hFcRn.13,20,21 Our kinetic measurements resulted in very narrow standard deviations for all the HB conjugates, whereas the deviations are considerably larger for the WT albumin with PEG attached (Table 2 and Figure 3). Furthermore, the improved affinity over unmodified albumin of the HB conjugates could further suggest a stabilizing effect on the engagement of the C-terminal part of albumin. The effect of polymer Mw was investigated in this work. Biologics such as recombinant protein drugs, e.g., insulin (Mw ∼ 5000 Da) and nucleic acid based drugs such as small interfering RNA (Mw ∼ 13.000 Da), are expected to be within the range of investigated Mw cargo. The hydrodynamic radius is, however, a relevant parameter to consider. The hydrodynamic radius calculated by intrinsic viscosity can vary

between polymers and alternative materials such as proteins or nucleic acids of similar Mw.22 This requires a case-by-case investigation; however, we demonstrate that a PEG model payload has an influence on hFcRn binding and that investigation into the effect of payload on hFcRn engagement is an important requirement in the development of albuminbased drug delivery systems. Many therapeutic peptides and proteins have a short circulatory half-life, and several strategies have been developed to improve the pharmacokinetic profile. PEGylation is a common approach that reduces kidney clearance as a consequence of increased molecular weight; however, tissue accumulation of high molecular weight PEG and possible reduction in efficacy and nonfavored altered drug biodistribution are potential drawbacks.23,24 Albumin for half-life extension is a highly attractive alternative technology gaining great interest due to its physiological transport properties that can be utilized for drug delivery applications.14,15,25,26 Conjugation to the free cysteine residue within DI of WT albumin is a common approach to attach agents of interest; however, our findings suggest that this may negatively influence binding to hFcRn responsible for its cellular recycling and certainly needs to be assessed. We show that this can be compensated for by the introduction of amino acid substitutions distant from Cys34 and, consequently, yield engineered albumin variants with retained binding to FcRn after payload conjugation suitable for tailored pharmacokinetic applications. E


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Turnover: FcRn-Mediated Recycling Saves as Much Albumin from Degradation as the Liver Produces. Am. J. Physiol. Gastrointest. Liver Physiol. 2006, 290 (2), G352−G360. (13) Andersen, J. T.; Dalhus, B.; Viuff, D.; Ravn, B. T.; Gunnarsen, K. S.; Plumridge, A.; Bunting, K.; Antunes, F.; Williamson, R.; Athwal, S.; Allan, E.; Evans, L.; Bjørås, M.; Kjærulff, S.; Sleep, D.; Sandlie, I.; Cameron, J. Extending Serum Half-Life of Albumin by Engineering Neonatal Fc Receptor (FcRn) Binding. J. Biol. Chem. 2014, 289 (19), 13492−13502. (14) Sleep, D.; Cameron, J.; Evans, L. R. Albumin as a Versatile Platform for Drug Half-Life Extension. Biochim. Biophys. Acta, Gen. Subj. 2013, 1830 (12), 5526−5534. (15) Kratz, F. A Clinical Update of Using Albumin as a Drug Vehicle - A Commentary. J. Controlled Release 2014, 190, 331−336. (16) Kratz, F. DOXO-EMCH (INNO-206): The First AlbuminBinding Prodrug of Doxorubicin to Enter Clinical Trials. Expert Opin. Invest. Drugs 2007, 16 (6), 855−866. (17) Andersen, J. T.; Justesen, S.; Berntzen, G.; Michaelsen, T. E.; Lauvrak, V.; Fleckenstein, B.; Buus, S.; Sandlie, I. A Strategy for Bacterial Production of a Soluble Functional Human Neonatal Fc Receptor. J. Immunol. Methods 2008, 331 (1−2), 39−49. (18) Andersen, J. T.; Dalhus, B.; Cameron, J.; Daba, M. B.; Plumridge, A.; Evans, L.; Brennan, S. O.; Gunnarsen, K. S.; Bjørås, M.; Sleep, D.; Sandlie, I. Structure-Based Mutagenesis Reveals the Albumin-Binding Site of the Neonatal Fc Receptor. Nat. Commun. 2012, 3, 610. (19) Pai, S. S.; Hammouda, B.; Hong, K.; Pozzo, D. C.; Przybycien, T. M.; Tilton, R. D. The Conformation of the Poly(ethylene Glycol) Chain in Mono-PEGylated Lysozyme and Mono-PEGylated Human Growth Hormone. Bioconjugate Chem. 2011, 22 (11), 2317−2323. (20) Sugio, S.; Kashima, a; Mochizuki, S.; Noda, M.; Kobayashi, K. Crystal Structure of Human Serum Albumin at 2.5 A Resolution. Protein Eng., Des. Sel. 1999, 12 (6), 439−446. (21) Curry, S.; Mandelkow, H.; Brick, P.; Franks, N. Crystal Structure of Human Serum Albumin Complexed with Fatty Acid Reveals an Asymmetric Distribution of Binding Sites. Nat. Struct. Biol. 1998, 5 (9), 827−835. (22) Armstrong, J. K.; Wenby, R. B.; Meiselman, H. J.; Fisher, T. C. The Hydrodynamic Radii of Macromolecules and Their Effect on Red Blood Cell Aggregation. Biophys. J. 2004, 87 (6), 4259−4270. (23) Veronese, F. M.; Pasut, G. PEGylation, Successful Approach to Drug Delivery. Drug Discovery Today 2005, 10 (21), 1451−1458. (24) Knop, K.; Hoogenboom, R.; Fischer, D.; Schubert, U. S. Poly(ethylene Glycol) in Drug Delivery: Pros and Cons as Well as Potential Alternatives. Angew. Chem., Int. Ed. 2010, 49 (36), 6288− 6308. (25) Elsadek, B.; Kratz, F. Impact of Albumin on Drug Delivery–New Applications on the Horizon. J. Controlled Release 2012, 157 (1), 4− 28. (26) Howard, K. A. Albumin: the next-generation delivery technology. Ther. Delivery 2015, 6, 265−268.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.5b00605. RP-HPLC chromatogram and SDS−PAGE analysis (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Interdisciplinary Nanoscience Center (iNANO), Gustav Wieds Vej 14, Aarhus University, DK-8000 Aarhus, Denmark. Fax: +45 87154041. Phone: +45 87155831. E-mail: kenh@ inano.au.dk. Notes

The authors declare no competing financial interest.

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REFERENCES

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Neonatal Fc Receptor Binding Tolerance toward the Covalent

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