Conformation of the Poly(ethylene Glycol) Chains in DiPEGylated

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Conformation of the Poly(ethylene Glycol) Chains in DiPEGylated Hemoglobin Specifically Probed by SANS: Correlation with PEG Length and in Vivo Efficiency Clémence Le Cœur,†,§,* Sophie Combet,*,† Géraldine Carrot,†,∥ Peter Busch,‡ José Teixeira,† and Stéphane Longeville† †

Laboratoire Léon-Brillouin (LLB), UMR 12 CEA-CNRS, CEA-Saclay, F-91191 Gif-sur-Yvette CEDEX, France Forschungszentrum Jülich GmbH, Jülich Centre for Neutron Science (JCNS) at Heinz Maier-Leibnitz Zentrum (MLZ), Lichtenbergstrasse 1, D-85747 Garching, Germany

‡

S Supporting Information *

ABSTRACT: Cell-free hemoglobin (Hb)-based oxygen carriers have long been proposed as blood substitutes but their clinical use remains tricky due to problems of inefficiency and/or toxicity. Conjugation of Hb with the biocompatible polymer poly(ethylene glycol) (PEG) greatly improved their performance. However, physiological data suggested a polymer molecular weight (Mw) threshold of about 10 kDa, beyond which the grafting of two PEG chains no longer improves efficiency and nontoxicity of diPEG/Hb conjugates. We used small-angle neutron scattering and contrast variation, which are the only techniques able to probe separately the conformation of PEG chains and Hb protein within the complex, to investigate the role of PEG chain conformation in diPEGylated Hb conjugates as a function of the polymer Mw. We found out that the structure of Hb tetramer is not modified by the polymer grafting. Similarly, with a constant grafting of two chains per protein, there is no significant change of the Gaussian conformation between free and grafted PEG below ∼10 kDa, the complex being well described by the “dumbbell” model. However, beyond that threshold, the radius of gyration of grafted PEG is significantly smaller than that of the free polymer, showing a compaction of the PEG chains, either in the “dumbbell” model or in the “shroud” one. In the latter model, the polymer may be wrapped on the surface of the protein spreading a protective “shielding” effect over a larger fraction of the protein. Both proposed models are in good agreement with the physiological data reported in the literature.

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INTRODUCTION Hemoglobin (Hb)-based oxygen carriers have long been developed to correct oxygen deficit due to ischemia in a variety of clinical settings, while addressing the shortage of blood for transfusion and the associated potential risk of infectious agent transmission.1−3 However, cell-free Hb, that is, Hb outside its natural protective environment, the red blood cell, is toxic since it causes vasoconstriction and consequent severe hypertension, attributed to the scavenging of nitric oxide (NO) by the protein.1,4 A route to overcome these undesired effects is to chemically modify Hb, by cross-linking or by chemical grafting onto Hb.1−3 Among these chemical modifications, the conjugation of Hb with biocompatible poly(ethylene glycol) (PEG), called “PEGylation”, largely used in many pharmaceutical and biotechnical fields, has been proposed for animal and clinical trials.5,6 PEGylation increases the apparent size of Hb protein, thus reducing its renal filtration and increasing its retention time in blood.7−9 Above all, PEGylation of Hb can reduce the vasoconstrictive effect and the consequent severe hypertension observed for cell-free Hb.1,4,9,10 However, clinical trials with diPEG/Hb conjugates have presented side effects, especially © 2015 American Chemical Society

because these complexes do not deliver oxygen efficiently due to both their lack of cooperativity and their high oxygen affinity.11 Conformation of diPEG/Hb conjugates, depending on PEG molecular weight (Mw), have been suggested to play a key role in such physiological changes. Nevertheless, they still remain misunderstood.10 For instance, blood pressure decreases with the increase of PEG Mw, whereas there is a threshold in PEG Mw, beyond which the efficiency of diPEG/ Hb oxygen-carrying decreases and its toxicity increases.10 The effect of PEG grafting on the three-dimensional structure of Hb, as well as the conformation of the polymer chains and their interaction with the protein, may therefore give key information to understand the physiological properties of grafted Hb and try to improve Hb PEGylation.12 The relatively large, highly flexible PEG chains prevent crystallization and structural determination of diPEG/Hb conjugates by protein crystallography. Recently, some studies have tried to elucidate a more precise structure of PEGylated complexes by dynamic Received: March 30, 2015 Revised: June 19, 2015 Published: July 7, 2015 8402

DOI: 10.1021/acs.langmuir.5b01121 Langmuir 2015, 31, 8402−8410

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Figure 1. PEG functionalization of hemoglobin (Hb) protein. (A) General synthetic route used for the preparation of (3) maleimido-poly(ethylene glycol) (Mal-PEG) using (1) monomethoxy-PEG and (2) 3-maleimido propionic acid (MPA), in the presence of dicyclohexylcarbodiimide (DCC) and dichloromethane (CH2Cl2) at 40 °C. (B) Condensation reaction of Mal-PEG with the thiol group (SH−) of the β 93 cysteine contained in each of the two β subunits (in blue) of Hb protein. The resulting covalent bond is detailed in the zoomed frames. See the Experimental Section for more details.

light scattering13 or electrophoresis.14 The three-dimensional structure of the PEGylated Hb grafted with two or six short PEG molecules (5 kDa) have been determined by small-angle X-ray scattering (SAXS), revealing that the major part of PEG chain protrudes away from Hb and that PEG chains interact with Hb.15 However, SAXS experiments do not permit to separately study each component of the complex and therefore need a high number of parameters for the three-dimensional reconstruction of the complex, which is nothing wrong as long as the reconstructions are physically justified. We report here a small-angle neutron scattering (SANS) study of the structure of diPEG/Hb conjugates made with two PEG chains attached per Hb tetramer. The use of SANS and contrast variation techniques enabled to separately probe at least partially the conformation of the protein and the grafted polymer chains. Indeed, this approach takes particular advantage of the unique ability to contrast match the aqueous buffer with portions of the conjugate (Hb or PEG), thus probing exclusively the conformation of the remainder of the conjugate. We performed these measurements for different PEG masses, ranging from 5 to 35 kDa, to investigate a correlation between the conformation of diPEG/Hb global complexes and the reported physiological properties. In particular, based on these data, we propose a conformation of diPEG/Hb conjugates depending on two possible models, the “dumbbell” or “shroud” ones, with the latter corresponding to attached PEG chains wrapping around the protein and generating a “shielding” effect.16,17

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phosphate (pH 6.8), followed by a centrifugation at 40 000g for 30 min at 4 °C. Hb is removed (supernatant) and dialyzed for 24 h against pure water at 4 °C. To remove 2,3-diphosphoglycerate, the solution is finally passed through a mixed-bed (AG 501, Bio-Rad, France) ion-exchange column19 and centrifugated at 45 000g for 15 min at 4 °C to get the purified native Hb solution (supernatant). The Hb protein concentration is measured before the SANS experiments by UV−vis spectrophotometry, by using the technique described by Antonini and Brunori.20 Briefly, the protein samples are diluted in buffer and an excess of potassium cyanide (KCN, Sigma-Aldrich) is added to the solution to complex the protein heme with the same ligand, before measurement at 540 nm. At that wavelength, the absorption by the protein/heme complex is not dependent on buffer or pH. Hemoglobin concentration is determined by the Beer− Lambert law with the absorption coefficient ελ=540 nm = 12 500 M−1 cm−1. PEG Functionalization. To be able to graft different molecular weights (Mw) of hydrogenated PEG (H-PEG) onto the Hb protein, and to perform the same reaction with deuterated PEG (D-PEG), monofunctional maleimido derivatives have been synthesized via an esterification reaction between (1) monomethoxy PEG (CH3O-PEG, Aldrich, or CD3O-PEG, Polymersource) and (2) 3-maleimido propionic acid (MPA, Aldrich) (see Figure 1A). Prior to the reaction, CH3O-PEG is freeze-dried under tetrahydrofuran. Typically, the reaction is performed as follows: dicyclohexylcarbodiimide (DCC, 0.16 g, 1.5 equiv) and MPA (0.12 g, 2 equiv) are added to the solution of CH3O-PEG (3 g, 1 equiv) in dried dichloromethane (CH2Cl2) (Figure 1A). The reaction is stirred for 24 h at 40 °C (Figure 1A). Then, the solution is filtered to remove urea, and the functionalized Mal-PEG (3) is recovered after two precipitations in cold ether and recrystallization from ethanol. Purity of the compound has been checked by 1H NMR (in CDCl3 solvent): δ (ppm) and, in parentheses, relative integration and assignment: 2.7 (2H, CO2CH2), 3.4 (3H, CH3O), 3.8 (2H, CH2N), 4.2 (2H, CH2CO2), and 6.75 (2H, COCHCHCO). PEG Grafting. Grafting of Mal-PEG on Hb has been performed following a method already described:10 the condensation of the terminal maleimido group of Mal-PEG onto the Hb protein via the thiol group of the accessible cysteine present in each β-subunit of Hb (Cys-β 93, Figure 1B). Mal-PEG is mixed in large excess (10 mol equiv of cysteine grafting sites) with Hb protein in a phosphatebuffered saline (PBS) solution (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KHPO4), pH 7, so that to ensure at least one polymer grafting on each protein. The solution is stirred at ambient temperature for one night. To remove the excess of unreacted Mal-

EXPERIMENTAL SECTION

Human Hemoglobin Preparation. Hb (approximately 64 000 g· mol−1) is a globular tetrameric protein constituted by two α and two β subunits. Each tetramer has six cysteine amino acids, whose only two, located on each β chain (Cys-β 93 residues), are accessible for chemical reactions. Hb is prepared from fresh adult human blood provided by the Institut Français du Sang (IFS, France) following a method adapted from Perutz.18 Briefly, after centrifugation of human blood at 3000g for 5 min at 4 °C, the supernatant (mainly plasma) is removed. The red blood cells (pellet) are washed three times with NaCl solution (9 g·L−1), and then lysed and stirred with pure water to release Hb. Membranes are precipitated by addition of 2.8 M 8403


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virtually matched in 100% D2O, the Hb form factor is fitted by the form factor of a solid sphere of radius R0:25

PEG chains and purify the PEGylated Hb, an extensive dialysis in PBS is performed (MWCO dialysis 3000). This process was repeated for different Mw of H-PEG (5, 10, 20, and 30 kDa) and D-PEG (5, 16, and 35 kDa). The concentration of diPEG/Hb conjugates was deduced by adjusting, if necessary, the ratio of grafted chains (relative to the number of grafting sites), which is not known but taken at first approximation equal to 100%. In such a condition, if the apparent molecular weight of the grafted PEG (i.e., the approximation to zero concentration of the PEG Mw as a function of PEG concentration, see Results and Discussion section), which is known, is not satisfied, this ratio is reduced. Small-Angle Neutron Scattering (SANS) Experiments. SANS experiments were performed at the spectrometers PACE (Laboratoire Léon-Brillouin, Saclay, France) and KWS-2 (JCNS-MLZ, Garching, Germany). The incident wavelength was λ = 6 Å on PACE and λ = 4.5 Å on KWS-2, with a wavelength spread of Δλ/λ = 0.1 and 0.2, on PACE and KWS-2 respectively. We used two instrumental setups in order to cover the wavevector range q = 0.01−0.4 Å−1 (q = 4π(sin θ)/ λ where 2θ is the scattering angle). The samples were extensively dialyzed against the appropriate aqueous medium (H2O, D2O, or H2O/D2O mixture) in cold chamber and poured into quartz cells (Hellma, France) before measurements. Due to the very different neutron coherent scattering lengths of hydrogen (H) and deuterium (D), the scattering length density difference between hydrogenated Hb (ρHb = 2.13 × 1010 cm−2) and deuterated PEG (ρD‑PEG = 6.45 × 1010 cm−2) is very significant, whereas this difference is lower between hydrogenated Hb and hydrogenated PEG (ρH‑PEG = 0.64 × 1010 cm−2). The scattering length density of H2O/D2O solvent is close to xρD2O + (1 − x)ρH2O, with ρH2O (−0.56 × 1010 cm−2) and ρD2O (6.34 × 1010 cm−2) being the coherent scattering length densities of H2O and D2O, respectively, and x the mass fraction of D2O in the solvent. The expected x value to match hydrogenated proteins in D2O/H2O solvent is about 40%, as previously described by Jacrot.21 Previously to the grafting studies, we determined experimentally the exact mass fraction that perfectly matched Hb signal in the solvent, and got exactly x = 0.39 (see Figure S1 in the Supporting Information). In such a condition, only D-PEG contributes to the q-dependent scattered intensity, I(q). The deuterated PEG cannot stricto sensu be totally matched, since its scattering length density is slightly higher than that of 100% D2O (x = 1). Nevertheless, in such a solvent, the contribution of D-PEG is strongly reduced since the difference between the coherent scattering length density of D2O and D-PEG is only a few percents. We assume that, in our 100% D2O experiments, we measure virtually only the hydrogenated Hb. This assumption is verified experimentally, as shown in Figure S2 (in the Supporting Information). Moreover, in the measurements of Hb/D-PEG conjugates, we checked that the characteristic PEG signal in q−2 is not observed (see Results and Discussion section). Therefore, in the following of the text, we will use for this condition the expression “matching of D-PEG”. Analysis of the intensity scattered by a completely hydrogenated diPEG/Hb complex in D2O gives the conformation of the complex for different polymer Mw. Polymer concentrations are chosen in order to stay far from the overlap concentration (“diluted regime”). SANS Analysis. SANS data were analyzed using the LLB in-house software PAsiNET.22,23 The scattered intensity I(q) is proportional to the form factor of the scatterers. In a preliminary analysis of the small q domain, in the range where qRg ≤ 1, called the “Guinier” domain, the scattered intensity has a general form which depends only on the radius of gyration Rg of the scattering objects:24 ⎛ q 2R 2 ⎞ g ⎟ I(q) = I(0)exp⎜⎜ − ⎟ 3 ⎝ ⎠

⎛ sin(qR ) − qR cos(qR ) ⎞2 0 0 0 ⎟ Ps(q , R 0) = 9⎜ (qR 0)3 ⎝ ⎠

(2)

Contrast Matching of Hb: Conformation of D-PEG. By matching Hb protein in 39% D2O, conformation of D-PEG chains can be measured independently. For polymer chains with Gaussian statistics, the form factor is given by the “Debye” function:26 Pp(x) =

2 (x − 1 + exp(− x)) x2

(3)

with x = q2Rg2. The diPEG/Hb complexes have been measured for at least five different concentrations for each mass of D-PEG (5, 16, and 35 kDa) in order to draw “Zimm-type plots” (scattering intensity over scatterer concentration vs scatterer concentration) and radius of gyration vs concentration. The scattered intensity can be written:27,28

I(q) = φΔρ2 vsP(q) S(q)

(4)

with φ being the volume fraction, vs being the partial specific volume of the scattering “objects,” Δρ being the neutron contrast factor, P(q) being the single object form factor, and S(q) being the interobject structure factor. At zero angle, we have:29

I(0) =

c pΔρp2 vp2 NA

⎛ 2c pa 2 ⎞ ⎟ NcM p⎜⎜1 − NcM p ⎟⎠ ⎝

(5) −1

where I(q) is the scattered intensity (cm ), cp is the concentration of D-PEG (g.cm−3), Δρ is the neutron scattering length density contrast between D-PEG and the solvent (cm−2), vp is the D-PEG partial specific volume (0.89 cm3·g−1), NA is the Avogadro number (mol−1), Mp is the apparent molecular weight of D-PEG (g·mol−1), P(q) is its molecular form factor (unitless), a2 is its second virial coefficient (cm3· mol−1), and Nc is the number (unitless) of polymer chains grafted on Hb. Conformation of the Complex. Equation 4 can be adapted and simplified for both cs (the scatterer concentration) and q approaching zero (at q → 0, P(q) → 1, and S(q) → 1) by using eq 5, for a diPEG/ Hb global system, constituted by one Hb protein and Nc chains of HPEG grafted on it:

⎡ I(q → 0) ⎤ 1 = (MhΔρh v h + NcM pΔρp vp)2 ⎢ ⎥ cs NA(NcM p + Mh) ⎣ ⎦c → 0 s

(6) where vh and vp are, respectively, the partial specific volumes of Hb (0.735 cm3·g−1) and H-PEG (0.88 cm3·g−1), Mh and Mp are their molecular weights, and Δρp and Δρh are the scattering density length differences between H-PEG chains and Hb with the solvent, respectively. This expression allows the evaluation of NcMp, the molecular weight of the grafted polymer chains on Hb. We used the model proposed by Pedersen30 to calculate the form factor of the global conjugate:

Pc(q) =

1 (NcΔρp2 Pq(q) + Nc(Nc − 1)Δρp2 Spp(q) (Δρh + NcΔρp )2 + 2Nc 2Δρp Δρh Sph(q) + Δρh 2 Ps 2(q , R 0))

(7)

where c, h, and p are used, respectively, for the global diPEG/Hb complex, Hb, and PEG. Pp(q) is the Debye function eq 3, R0 is the solid sphere radius of Hb, and Ps(q,R0) is the form factor of the solid sphere model eq 2. Two functions, Spp and Sph, representing the structure factors between the grafted PEG chains themselves and between PEG and Hb, respectively, are introduced (with Rg being the radius of gyration of the grafted chains):

(1)

This expression implies noninteracting scatterers, a good assumption in our experiments due to low concentrations and buffered conditions (low electrostatic interactions). Contrast Matching of D-PEG: Conformation of Hb. To study both free Hb and Hb bounded to D-PEG, with polymer chains 8404


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Langmuir ⎛ sin(qR 0) ⎞2 Spp(q) = Ψ2(q , R g)⎜ ⎟ ⎝ qR 0 ⎠

Sph(q) = Φ(q , R 0)Ψ(q , R g)

sin(qR 0) qR 0

(8)

(9)

where Ψ(x) = (1 − exp(−x))/x is the form factor amplitude of the chain31 with x = q2Rg2, and Φ(q,R0) is defined as Φ(q,R0) = [Ps(q,R0)]1/2 with Ps being the form factor of a solid sphere eq 2 and R0 being the radius of a solid sphere.

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RESULTS AND DISCUSSION Conformation of Hb (Matching of Grafted D-PEG). In order to check if D-PEG grafting does not induce a modification in the conformation of Hb tetramer, we probed Hb by matching the grafted D-PEG (Mw = 16 kDa) in 100% D2O. In such a contrast matching condition, only Hb protein form factor within the complex is measured. By using the form factor of a solid sphere eq 2, we compared D-PEG-grafted Hb protein with the protein free in solution, both at low concentration (ch = 1.8 g·L−1, corresponding to a concentration of 2.7 g·L−1 for the Hb/D-PEG conjugate). For q ≥ 0.025 Å−1, within the limit of our experimental resolution, we cannot distinguish between free and PEGylated Hb quaternary structure (Figure 2), as suggested previously in a SAXS

Figure 3. SANS intensity of D-PEG: comparison between Hb-grafted (solid squares) and free (open circles) D-PEG. Due to contrast matching conditions (39% D2O), only the D-PEG chains (Mw = 16 kDa, polymer concentration cp ≈ 18 g·L−1) within the diPEG/Hb complex are observed. Free D-PEG is measured in 100% H2O. The fits of the form factor of each sample are shown (solid and dash dot lines for, respectively, grafted and free D-PEG). The error bars for grafted PEG are smaller than the size of the solid squares.

no significant difference for the tetramer conformation between free and grafted Hb. This value of radius of gyration is also in agreement with oxyHb crystallographic models (23.5 Å).32 Conformation of D-PEG Chains (Matching of Hb Protein). We matched Hb with 39% D2O mass fraction to measure specifically D-PEG chains. We compared the form factor P(q) of D-PEG free in H2O with that of the polymer conjugated to Hb, using the Debye function. As shown in Figure 3, in the case of 16 kDa D-PEG, the form factors are significantly different for free and grafted PEG chains. We obtain the same significant difference for 5 and 35 kDa PEG (see Figure S3 in the Supporting Information). Depending on the PEG Mw, the degree of grafting is Nc ≈ 2 for 5 and 16 kDa PEG, showing an efficient grafting for low Mw. However, for 35 kDa PEG, the degree of grafting decreases (Nc ≈ 1.5) so we conclude that the grafting on Hb is not complete in that case. The second virial coefficient, revealing interactions between the macromolecules, and the apparent mass of the polymer are determined for each free and grafted D-PEG, using eq 5. The D-PEG apparent mass, as a function of D-PEG concentration, is shown in Figure 4A, from a “Zimm” plot analysis. D-PEG chains bounded to the same Hb protein have more steric repulsion, compared to free D-PEG, due to their grafting on two opposite sites on the protein (Figure 4A, Table 1). Moreover, Hb proteins themselves induce a higher excluded volume for the grafted D-PEG chains, compared to the free chains. We plotted, in Figure 4B, the radius of gyration of D-PEG as a function of its concentration. Free D-PEG adopts the conformation of a Gaussian chain, with a radius of gyration proportional to the square root of the length of the polymer chain (Table 1). Water is known to be a θ-type solvent for DPEG at ambiant temperature, as we assume this is the case in our study. The radius of gyration of grafted D-PEG is significantly different with respect to the free one (Table 1): at 5 kDa, the grafting to Hb induces an increase of the radius of gyration of D-PEG, whereas, at 16 kDa, Rg of grafted D-PEG is smaller than that of free D-PEG. For the highest Mw (35 kDa), D-PEG compaction is even more important, as compared to free polymer.

Figure 2. SANS intensity of hemoglobin (Hb): comparison between free (open circles) and D-PEGylated (solid squares) Hb. Due to contrast matching conditions (100% D2O), only Hb protein (protein concentration of 1.8 g·L−1) within the diPEG/Hb complex is observed. Each measurement is fitted from q = 0.025 Å−1 (marked by the dotted line) by the form factor of a solid sphere (solid and dash dot lines for grafted and free Hb, respectively). For each measurement, the error bars are displayed. The anticipated SANS curve (regardless of the spectrometer resolution) produced by an in-house software from the protein data bank for Hb, i.e., the 1HHO.pdb corresponding to the human oxyhemoglobin at 2.1 Å resolution, is also shown (in red) for comparison with the experimental data.

study.15 At the smallest q values, we observe an increase of the intensity in the case of grafted Hb, which may be due to a small amount of aggregation between complexes. However, this aggregation was present only for this sample and was not observed in the other contrasts (e.g., in Figure 3). For q ≥ 0.025 Å−1, the fitting of the scattering intensity gives hard sphere radii of 31 ± 1 Å and 31 ± 3 Å for free and grafted Hb, respectively, corresponding to radii of gyration of 24 ± 1 Å and 24 ± 2 Å. In the limit of our experimental resolution, there is 8405


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Langmuir Table 1.

a

within D-PEG/Hb conjugates in 39% D2O

in 100% H2O

Mw of D-PEG (kDa)

Ma,p of D-PEG (kDa)

a2,p of grafted DPEG (×103 Å3)

Rg,p of grafted D-PEG (Å)

Rg,p of free D-PEG (Å)

5 16 35

4.8 ± 0.4 15 ± 1 36 ± 4

72 ± 12 428 ± 56 1570 ± 140

36.9 ± 0.8 48 ± 2 58 ± 3 within H-PEG/ Hb conjugates in 100% D2O

31 ± 2 54 ± 1 73 ± 4

in 100% D2O

Mw of H-PEG (kDa) 0 (free Hb) 5 10 20 30

Ma,c of conjugates (kDa) 72 86 99 111

64 ±6 ±5 ±7 ±8

Rg,c of H-PEG/Hb conjugates (Å) 24 40.1 47 52.2 b

± ± ± ±

1 0.8 2 0.3

Rg,p of grafted H-PEG (Å) 34 43 47 51

± ± ± ±

2 4 2 3

in 100% D2 O Rg,p of free H-PEG (Å) 30 40 61 75

± ± ± ±

2 2 1 3

a Top: Apparent mass (Ma,p), second virial coefficient (a2,p), and radius of gyration (Rg,p) of grafted PEG within D-PEG/Hb conjugates, in “contrast matching of Hb” condition (39% D2O), as a function of different D-PEG molecular weights (Mw). The radii of gyration are compared to that of free D-PEG measured in 100% H2O. Bottom: Apparent mass (Ma,c) and radius of gyration (Rg,c) of H-PEG/Hb conjugates in 100% D2O for different H-PEG molecular weights (Mw). The radii of gyration are compared to that of free H-PEG measured in 100% D2O. The radius of gyration (24 ± 1 Å) of free Hb corresponds to the sphere radius R0 = 31 ± 1 Å. bRg,c is not given because the valid Guinier domain is too short to correctly fit the data.

Figure 4. Apparent mass and radius of gyration of D-PEG. The apparent mass (A) and the radius of gyration (B) are obtained by fitting the SANS data with the Debye function (see text and Figure 3 for details) and plotted as a function of D-PEG concentration (“Zimm” plot) for three different PEG molecular weights: 5 kDa (triangles), 16 kDa (circles), and 35 kDa (squares) for free (open symbols) and grafted (solid symbols) D-PEG to give the extrapolation to zero concentration. Due to the contrast matching condition (39% D2O), only the grafted D-PEG chains within the complex are observed. Free D-PEG is measured in 100% H2O.

We tried to elucidate the conformation of the grafted H-PEG chains on the two available cysteines (Cys-β 93) of Hb, that is, approximately at opposite sides on the protein, spaced about 22 Å. As reported before, within the limit of our experimental resolution, the conformation of the protein is not modified by D-PEG grafting. Therefore, we modeled Hb in the global complex with H-PEG by a solid sphere with the radius obtained in the experiment matching D-PEG contribution (see above). We added Nc Gaussian chains of grafted H-PEG. The form factor of the complex has then been calculated by using the model of Pedersen for micelles with spherical cores eq 7.30 In this way, we got the radii of gyration of the grafted H-PEG chains for different concentrations, which were extrapolated to zero concentration. The obtained radii of gyration were compared to the radii of gyration of D-PEG, either grafted on Hb (in 39% D2O to contrast match Hb), or free in solution (in 100% H2O) (Table 1 and Figure 6). As already observed for the deuterated polymer (Table 1), the radius of gyration of grafted H-PEG of low Mw (5 and 10 kDa), using the Pedersen model, is higher than that of the corresponding free H-PEG (Table 1, Figure 6). In contrast, for two larger Mw (20 and 30 kDa), the radius of gyration is smaller for grafted H-PEG as compared to free ones (Table 1, Figure 6). Our study allowed us to propose different structures for diPEG/Hb complexes, as a function of the polymer molecular weight. For short polymer chains (Mw < 10 kDa), the radius of gyration of H- and D-PEG is close or slightly larger compared to that of free PEG in solution (Table 1, Figure 6). Our data are consistent, within the Pedersen model, with the “dumbbell”

Conformation of diPEG/Hb Conjugates. We compared our results obtained for each component of Hb/D-PEG complexes with the structure of the whole hydrogenated complex. For four different Mw of H-PEG, we measured the scattered intensity at four concentrations of the complex and compared them with free Hb. We used the “Kratky” representation (i.e., the scattered intensity multiplied by q2 as a function of the wavevector q) of the data of each sample for a complex concentration of 2 g·L−1. For q ≈ 0.08 Å−1, each graph shows a peak characteristic of the globular shape of the protein (Figure 5). At higher q values, the contribution to the scattered intensity due to Gaussian chains of H-PEG (proportional to q−2) is dominant, as compared to protein contribution (proportional to q−4). Scattered intensity data are first analyzed by the Guinier function eq 1. We obtain the apparent radius of gyration of the complex (Rg,c), that increases with the Mw of the grafted H-PEG chain (Table 1), as expected. The increase is proportionally less important for the longer chains than for the shorter ones (Table 1). Extrapolation to zero concentration gives the apparent mass Ma,c of the complexes by using eq 6. We find that, for the three smallest H-PEG Mw (5, 10, and 20 kDa), the global Ma,c of the complex is consistent with the grafting of two H-PEG chains on each Hb (Table). In contrast, for 30 kDa H-PEG, the grafting is less efficient, as we already observed with the 35 kDa D-PEG (see above): there is approximately 1.4 H-PEG chain per Hb tetramer. This confirms that the grafting is less efficient for larger polymer masses. 8406


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conformation (Figure 7A): the Gaussian conformation adopted by both PEG chains grafted at opposite sides of Hb is slighly extended possibly due to steric repulsions with the protein (Table 1, Figure 6). Our data are comparable to the “mushroom” model previously described in the literature by SAXS and ab initio reconstruction15 and by molecular dynamics simulations.10 These articles report indeed a conformation for small PEG (5 kDa) of chains folding upon themselves to form a distinct loosely folded domain on the surface of the Hb protein. For higher polymer Mw (Mw > 10 kDa), The Pedersen model allowed us to suggest a compaction of the grafted chain conformation, compared to free PEG, as shown by the decreased Rg (Table 1, Figure 6). This behavior occurs whereas the polymer chains retain their random coil nature at local scale, as shown by the decreased intensity in q−2(I(q)q2 tends to a constant value) at high q values (Figure 5). We propose that the compaction might be due to (i) attractive interaction within the polymer chain itself (Figure 7B) or to (ii) attractive interactions between PEG and Hb (Figure 7C). The first assumption would be consistent with that reported by molecular dynamics simulations, where the polymer chains protrude away from the protein.10 The second one might be consistent with the “shroud” model previously described in the literature,16,33 in which the PEG polymer might wrap on the surface of the protein. This is in agreement with another study made on cytochrome c protein, showing that PEG could interact with the nonpolar residues on the protein surface.34,35 Between these two models, the entropy loss may be compensated by enthalpy benefit. Indeed, the Gaussian polymer chains, which are free in the “dumbbell” model, are in interaction with the protein in the “shroud” model (Figure 7). In the case of very high PEG Mw (Mw > 30 kDa), the lower grafting efficiency may be explained by the grafting of one polymer chain on the protein masking by “wrapping” the second grafting site on Hb, as suggested in Figure 7D. This assumption is consistent with the distance between the cysteine residues (Cys-β 93), which has been evaluated (using the PDB file 1HHO.pdb corresponding to the human oxyhemoglobin at 2.1 Å resolution used in Figure 7) to be about 22 Å. This is a relatively small distance compared to the radius of gyration found for high Mw polymers (about 50 Å, Table). Another assumption for the lower grafting efficiency may be the lower reactivity of the maleimido end group for high PEG Mw: these polymer chains may diffuse more slowly or may hide more easily their reactive site than small PEG chains. We have already reported the decrease of PEG radius of gyration depending on the polymer Mw but in the case of macromolecular crowding, thus with a completely different physical mechanism.36 In that previous study, similarly to what is presently reported, the higher the polymer molecular weight, the more compact is the PEG.37 The data we report on PEG conformation within diPEG/Hb conjugates are consistent with the physiological data described in the literature.1−3,10 The “dumbbell” configuration leaves part of the protein “unprotected”, but provides locally a steric repulsion force, strong enough to protect the conjugated parts of the complex. In the “shroud” model, the polymer chains spread their protective “shielding” effect over a larger fraction of the protein. In both cases, the layer surrounding the PEGylated Hb may induce protection against macrophages and improve immunogenicity: the immuno-response is decreased, as well as the subsequent proteolytic degradation, through repulsions

Figure 5. “Kratky” plots (SANS intensity multiplied by the squared modulus of the scattering vector) for free and PEGylated Hb. Free Hb (solid squares) and diPEG/Hb conjugates with different H-PEG molecular weights are represented: 5 kDa (open squares, [Hb] = 9.6 g· L−1), 10 kDa (solid circles, [Hb] = 8.7 g·L−1), 20 kDa (open diamonds, [Hb] = 7.0 g·L−1), and 30 kDa (solid triangles, [Hb] = 17.7 g·L−1). For diPEG/Hb conjugates, the measurements are fitted according to the Pedersen model (solid curves). Free Hb and PEGylated conjugates are hydrogenated therefore all measurements were performed in an 100% D2O solvent to maximize the contrast. For clarity, the intensities are shifted along the y-axis by one log unit with respect to each other.

Figure 6. Evolution of the radius of gyration (Rg) for free and grafted PEG. The radius of gyration of free H- and D-PEG (open circles) and grafted H- and D-PEG (respectively, solid triangles and squares), in 39% D2O (to contrast match the protein), is represented as a function of the square root of the polymer molecular weight. The continuous line represents the theoretical evolution of the radius of gyration of PEG (H-PEG or D-PEG) free in water (respectively in 100% D2O or 100% H2O): indeed, for a Gaussian chain in a θ-type solvent, Rg being proportional to N1/2, with N being the number of monomers, Rg is proportional to Mw1/2. 8407


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Figure 7. Representation of the proposed models for diPEG/Hb conjugates. The hemoglobin tetramer is represented with the β subunits in light blue and the α ones in dark gray, without the iron-containing heme groups. The two available cysteines are in red, whereas the other ones (nonavailable) are colored in yellow. The PEG chains are depicted in red and gray, with a length proportional to their molecular weight. They are grafted on the available cysteines located on each side of Hb protein. (A) For our data on PEG below 10 kDa, the global structure of the diPEG/Hb complex may be described by the “dumbbell” (or “mushroom”) model: both PEG chains grafted at opposite sides of the protein adopt a Gaussian conformation both locally and globally. (B) A possible representation for grafted PEG with higher Mw (above 10 kDa), with a higher compaction of the chains compared to the free ones (reduced Rg compared to free polymer). Like the conformation in (A), this representation follows the “dumbbell” model. Note that the Gaussian character of the chains is locally maintained. (C) A variant of the (B) representation with the assumption that the conformation of the compacted grafted chains follows the “shroud” model. In this representation, the polymer chains are (more or less) wrapped on the surface of Hb protein. (D) A variant of the (C) model in the case of very high PEG Mw (30−35 kDa), for which only one polymer chain is grafted on the protein. Such “shielding” model, in which the first grafted chain hides the second grafting site, is then consistent with the less grafting efficiency observed for such high molecular weights.

totally “protected”. The therapeutic trend is then toward the grafting of a small amount of large PEG chains, such as 20 kDa linear PEG or 40 kDa branched PEG, that improves performance of PEGylated Hb conjugates, as compared to the repeatedly PEGylation of the protein with small chains.38

between PEGylated Hb and activity-degrading proteins.7,8 The resulted increase in PEGylated Hb molecular size is also consistent with the enhanced retention time found in physiological studies,10 through decreasing glomerular filtration, with the size range for the pores of the glomerular basement membrane being 30−50 Å. Moreover, the enhanced size of PEGylated Hb, as compared to free Hb, may reduce the interaction of the protein with endothelium and would prevent diPEG/Hb conjugates to enter the interstitial space of blood vessels, where Hb would bind to NO, reducing the vasoconstriction effect and hypertension observed for cell-free Hb blood substitutes.1,4,9,10 The structural change of the grafted chains occurs at the same Mw threshold, between 10 and 20 kDa, beyond which the efficiency of PEG-conjugated oxygen carriers starts to decrease.10 The functional capillary density, which is an indicator of vasoactivity, has been reported to not directly correlate with the PEG Mw: it increases with polymer length but decreases significantly beyond 20 kDa.10 This suggests that the modulation of the vasoactivity of Hb by PEGylation could rather be a function of the surface shielding afforded by PEG grafting, the latter being a function of the arrangement of PEG chains on the protein surface.10 Such assumptions, based on physiological data, correlate well with our data and models: beyond a certain Mw threshold (between 10 and 30 kDa), following our assumptions (see above and Figure 7), the protein surface could be wrapped by the PEG chain(s) (like in Figure 7C,D). This is consistent with the fact that, above a certain threshold, adding more polymer provides no additional physiological/therapeutic benefits, the protein being already

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CONCLUSIONS The determination of the detailed three-dimensional structure of diPEG/Hb conjugates, especially the conformation of the grafted PEG chains, is a key point to better understand how PEGylated Hb can lead to blood substitutes in therapeutic applications. PEGylation has indeed been reported to be correlated with a higher efficiency and a lower toxicity, in particular through an increased intravascular retention time and a reduced NO scavenging.1−3 The efficiency of diPEGylated-Hb blood substitutes is known to be related to a threshold of PEG Mw of ∼10 kDa.10 In the present study, we took advantage of SANS and its associated contrast variation techniques to specifically and separately probe the conformation of both the polymer chains and the protein within the diPEG/Hb conjugates. First, we confirm that the tetrameric conformation of Hb is not modified by the PEG grafting. Our experiments show that the conformation of PEG is a function of its length, and a PEG Mw threshold of ∼10 kDa is consistent with the physiological data. If the PEG Mw is lower than ∼10 kDa, we report no significant change between free and grafted PEG conformations, and the Hb/PEG conjugates follow the “dumbbell” model. On the opposite, if the PEG Mw is higher than ∼10 kDa, 8408


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blood pressure responses to cell-free hemoglobin solutions and the reaction with nitric oxide. J. Biol. Chem. 1998, 273, 12128−12134. (5) Zhang, J.; Cao, S. Y.; Kwansa, H.; Crafa, D.; Kibler, K. K.; Koehler, R. C. Transfusion of hemoglobin-based oxygen carriers in the carboxy state is beneficial during transient focal cerebral ischemia. J. Appl. Physiol. 2012, 113, 1709−1717. (6) Vandegriff, K. D.; Winslow, R. M. Hemospan: Design Principles for a New Class of Oxygen Therapeutic. Artif. Organs 2009, 33, 133− 138. (7) Veronese, F. M. Peptide and protein PEGylation: a review of problems and solutions. Biomaterials 2001, 22, 405−417. (8) Roberts, M. J.; Bentley, M. D.; Harris, J. M. Chemistry for peptide and protein PEGylation. Adv. Drug Delivery Rev. 2002, 54, 459−476. (9) Rogers, M. S.; Ryan, B. B.; Cashon, R. E.; Alayash, A. I. Effects of polymerization on the oxygen-carrying and redox properties of diaspirin cross-linked hemoglobin. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1995, 1248, 135−142. (10) Manjula, B. N.; Tsai, S.; Upadhya, R.; Perumalsamy, K.; Smith, P. K.; Malavalli, A.; Vandegriff, K.; Winslow, R. M.; Intaglietta, M.; Prabhakaran, M.; Friedman, J. M.; Acharya, A. S. Site-specific PEGylation of hemoglobin at cys-93(beta): Correlation between the colligative properties of the PEGylated protein and the length of the conjugated PEG chain. Bioconjugate Chem. 2003, 14, 464−472. (11) Portörö, I.; Kocsis, L.; Herman, P.; Caccia, D.; Perrella, M.; Ronda, L.; Bruno, S.; Bettati, S.; Micalella, C.; Mozzarelli, A.; Varga, A.; Vas, M.; Lowe, K. C.; Eke, A. Towards a novel haemoglobin-based oxygen carrier: Euro-PEG-Hb, physico-chemical properties, vasoactivity and renal filtration. Biochim. Biophys. Acta, Proteins Proteomics 2008, 1784, 1402−1409. (12) Manjula, B. N.; Tsai, A. G.; Intaglietta, M.; Tsai, C. H.; Ho, C.; Smith, P. K.; Perumalsamy, K.; Kanika, N. D.; Friedman, J. M.; Acharya, S. A. Conjugation of multiple copies of polyethylene glycol to hemoglobin facilitated through thiolation: Influence on hemoglobin structure and function. Protein J. 2005, 24, 133. (13) Caccia, D.; Ronda, L.; Frassi, R.; Perrella, M.; Del Favero, E.; Bruno, S.; Pioselli, B.; Abbruzzetti, S.; Viappiani, C.; Mozzarelli, A. PEGylation Promotes Hemoglobin Tetramer Dissociation. Bioconjugate Chem. 2009, 20, 1356−1366. (14) Ronda, L.; Pioselli, B.; Bruno, S.; Faggiano, S.; Mozzarelli, A. Electrophoretic analysis of PEGylated hemoglobin-based blood substitutes. Anal. Biochem. 2011, 408, 118−123. (15) Svergun, D. I.; Ekstrom, F.; Vandegriff, K. D.; Malavalli, A.; Baker, D. A.; Nilsson, C.; Winslow, R. M. Solution structure of poly(ethylene) glycol-conjugated hemoglobin revealed by small-angle x-ray scattering: Implications for a new oxygen therapeutic. Biophys. J. 2008, 94, 173−181. (16) 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, 2317−2323. (17) He, L.; Wang, H.; Garamus, V. M.; Hanley, T.; Lensch, M.; Gabius, H.-J.; Fee, C. J.; Middelberg, A. Analysis of MonoPEGylated Human Galectin-2 by Small-Angle X-ray and Neutron Scattering: Concentration Dependence of PEG Conformation in the Conjugate. Biomacromolecules 2010, 11, 3504−3510. (18) Perutz, M. F. Preparation of Haemoglobin crystals. J. Cryst. Growth 1968, 2, 54−56. (19) Jelkmann, W.; Bauer, C. What is best method to remove 2,3diphosphoglycerate from hemoglobin. Anal. Biochem. 1976, 75, 382− 388. (20) Antonini, E.; Brunori, M. Hemoglobin and myoglobin in their reactions with ligands. In Frontiers of Biology; Neuberger, A., and Tatum, E. L., Eds.; North-Holland Publishing Company: Amsterdam, 1971. (21) Jacrot, B. Study of biological structures by neutron scattering from solution. Rep. Prog. Phys. 1976, 39, 911−953. (22) Brulet, A.; Lairez, D.; Lapp, A.; Cotton, J.-P. Improvement of data treatment in small-angle neutron scattering. J. Appl. Crystallogr. 2007, 40, 165−177.

we observe a compaction of the polymer chains in correlation with a lower in vivo efficiency of the conjugates. The same strategy (SANS and contrast variation techniques) we used in the present study could be applied to probe the conformation of the different components in other PEGylated proteins, as already done for monoPEGylated lysozyme and growth hormone,16 or in other PEGylated complexes, such as “drug delivery” systems.

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ASSOCIATED CONTENT

S Supporting Information *

Extinction of hemoglobin SANS signal in H2O/D2O solvent; extinction of D-PEG SANS signal in 100% D2O solvent; conformation of 5 and 35 kDa D-PEG chains (matching of Hb protein). The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.langmuir.5b01121.

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AUTHOR INFORMATION

Corresponding Authors

*Sophie COMBET Laboratoire Léon-Brillouin, UMR 12 CEACNRS CEA-Saclay, F-91191 Gif-sur-Yvette CEDEX, France. Email: [email protected]; Tel.: +33 1 69 08 67 20; Fax: +33 1 69 08 82 61. *Clémence LE COEUR Institut de Chimie et des Matériaux Paris-Est, UMR 7182, 2-8 rue Henri-Dunant, F-94320 Thiais, France. Email: [email protected]; Tel: +33 1 49 78 12 86; +33 1 49 78 12 08. Present Addresses §

C.L.C.: Institut de Chimie et des Matériaux Paris-Est, UMR 7182, 2-8 rue Henri-Dunant, F-94320 Thiais, France. ∥ G.C.: Laboratoire d’Innovation en Chimie des Surfaces et Nanosciences (LICSEN), IRAMIS/NIMBE, CEA-Saclay, F91191 Gif-sur-Yvette, France. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The Laboratoire Léon-Brillouin (LLB, Saclay, France) and Jülich Centre for Neutron Science (JCNS-MLZ, Garching, Germany) neutron facilities are acknowledged for beam time allocation on, respectively, PACE and KWS-2 SANS spectrometers. SANS experiments at JCNS-MLZ were supported by the European Commission under the 7th Framework Programme through the “Research Infrastructures” action of the “Capacities” Programme, NMI3-II Grant number 283883.

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ABBREVIATIONS Hb, hemoglobin; PEG, poly(ethylene glycol); Mal-PEG, maleimido-PEG; SANS, small-angle neutron scattering; Mw, molecular weight

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REFERENCES

(1) Alayash, A. I. Oxygen therapeutics: Can we tame haemoglobin? Nat. Rev. Drug Discovery 2004, 3, 152−159. (2) Winslow, R. M. Cell-free oxygen carriers: Scientific foundations, clinical development, and new directions. Biochim. Biophys. Acta, Proteins Proteomics 2008, 1784, 1382−1386. (3) Kluger, R. Red cell substitutes from hemoglobin - Do we start all over again? Curr. Opin. Chem. Biol. 2010, 14, 538−543. (4) Rohlfs, R. J.; Bruner, E.; Chiu, A.; Gonzales, A.; Gonzales, M. L.; Magde, D.; Magde, M. D.; Vandegriff, K. D.; Winslow, R. M. Arterial 8409


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Conformation of the Poly(ethylene Glycol) Chains in DiPEGylated

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