A supramolecular route for reversible protein-polymer conjugation

Abstract

The supramolecular formation of a PEGylated bovine serum albumin (BSA) protein-polymer bio-conjugate in water has been demonstrated through a selective host–guest interaction with the macrocycle cucurbit[8]uril (CB[8]). Both BSA and poly(ethylene glycol) were functionalised with either an electron-deficient first guest viologen or an electron-rich second guest naphthalene for the formation of the CB[8] ternary complex. With the help of spectroscopic (NMR, DOSY-NMR, DLS, UV/vis, fluorescence) and calorimetric (ITC) techniques, it was shown that a strong and specific binding interaction took place between the complementary labeled polymer and protein only in the presence of the macrocyclic host CB[8]. Moreover, we demonstrated that controlled formation of a supramolecular protein-protein complex was also possible through the use of CB[8] ternary formation.

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Introduction

Merging the biological activity of proteins with desirable properties of synthetic polymers in the form of bioconjugates has gained considerable interest and has found a wide variety of applications ranging from bio- and wet-nanotechnology to medicine.^1–11 Typically, two routes have been employed to achieve polymer-protein conjugate formation; either polymerization is initiated from an activatedprotein,^12–18 or the more widely employed synthetic route utilises a post-polymerization functionalization approach linking proteins to functionalised polymer end-groups.^19–21 The synthetic polymers used are almost always hydrophilic, and poly(ethylene glycol) (PEG) is the most popular choice for bioconjugations and its covalent attachment (known as PEGylation) to proteins has been known for several decades.^22,23 PEGylated proteins show distinct physicochemical properties such as alterations in bioavailability, in vitro and in vivo biological activity, biodistribution, pharmacokinetics, receptor affinity and a typically reduced immunogenicity/toxicity all of which resulted in their FDA approval as therapeutic drugs in several instances.^3,4,24–30

Chemical synthesis of PEGylated proteins is usually conducted by reaction of an activated PEGylation reagent with functional groups such as amino, carboxylate, hydroxy, thiol, and disulfide groups that are sterically accessible on the surface of proteins. Historically, the functionalisation of lysine side chains by activated PEGylation reagents containing chlorotriazine, succinimidyl succinate, succinimidyl carbonate and N-hydroxy-succinimide esters have gained considerable interest as a result of synthetic ease.³¹ However, bioconjugation relying upon modification of lysine residues results in complex mixtures of randomly modified proteins as the reagents mentioned above can also be reactive towards other amino acid residues.

As a consequence, the current trend is towards site-specific protein modification in order to obtain more well defined protein-polymer conjugates.²⁴ This may be carried out through the reductive amination of proteins by PEG-aldehyde reagents^32,33 and through thiol-specific reactions such as disulfide bond formation (pyridyl disulfide)¹² or thiol-ene reactions (maleimide,^34,35vinyl sulfone,³⁶ acrylates^37,38). Cysteine residues in proteins usually participate in disulfide bridges in their native state, reinforcing the biologically active structure of the protein, several examples of PEGylated proteins through free thiol groups have been described such as bovine serum albumin (BSA),^15,22 β-interferon³³ and the growth factor G-CSF.³⁹ Furthermore, genetically introduced cysteines can also act as the desired anchor unit.⁴⁰ Novel site-specific approaches such as PEGylation via bisalkylation of disulfide bonds⁴¹ and enzymatic PEGylations⁴² have also been suggested.

A drawback which is frequently encountered with covalent protein PEGylation is reduced biological activity on account of interactions between PEG and the protein's active site, and often tempers any improved pharmacokinetic properties in vivo.²⁴ To overcome this obstacle, reversible PEGylation has been suggested as an alternative. This would both allow for the temporarily reduced immunogenicity/toxicity and increased solubility of the protein and the subsequent release at the desired site of action restoring any enzymatic activity. Current approaches to reversible PEGylation mainly focus on the incorporation of labile or hydrolysable covalent bonds such as esters, oligo-lactic acid and disulfides.^43–46 Through the use of non-covalent host–guest interactions the reversible attachment of proteins to surfaces has been recently accomplished using supramolecular “glue” such as the biotin-streptavidin system,^17,47–49cucurbit[7]uril^50,51 and guest encapsulation with cyclodextrins (CDs).^8,52 However, it is known that CDs can also complex with unmodified proteins such as BSA⁵³ and insulin⁵⁴ and lead to alterations in folding which can impair or inhibit protein function.⁵⁵ Thus, the lack of selectivity in host–guest complexation with CDs prohibits their use in the formation of reversible PEGylated proteins. To the best of our knowledge, reversible, host–guest-complex mediated polymer-protein conjugate formation has not yet been reported; herein we describe the reversible PEGylation of proteins through the use of supramolecular host–guest chemistry with cucurbit[8]uril (CB[8]).

Cucurbit[n]urils (n = 5–8,10)^56–62 are a family of macrocyclic host molecules that allow for host–guest complexation in aqueous media but generally show much higher complexation binding strength and specificity towards their guests than the family of CDs.⁶³ The larger member of the family, cucurbit[8]uril,⁶⁴ can accommodate up to two organic guest molecules simultaneously inside its cavity as shown in Fig. 1a, commonly an electron-poor first guest and an electron-rich second guest. In aqueous solutions, this results in the formation of 1 : 1 : 1 ternary complexes in a stepwise fashion with high association constants (K_a up to 10¹²M⁻² in phosphate buffer).^65–68 Therefore, it can be considered as a supramolecular handcuff that ties together two different (macro)molecular units to form non-covalently bound supramolecular assemblies. As a result, CB[8] has recently been employed in areas as diverse as the design of molecular machines,⁶⁹peptide sensors⁶⁸ and polymeric materials.^70,71 Most commonly, viologen-containing first guest molecules are used which can be readily reduced to the radical cation form and consequently lead to release of the second guest and formation of a 2 : 1 ternary complex.^69,72–75 In this work, we report the synthesis of first- and second guest- labeled BSA and PEG monomethyl ether (5000 g mol⁻¹) and characterize their protein-polymer conjugate that selectively forms in the presence of CB[8] host by means of NMR, UV/vis, DLS, CD, fluorescence and ITC studies.

Fig. 1 The macrocyclic host CB[8] can “handcuff” together an electron-poor first guest and electron-rich second guest forming a stable ternary complex both for small molecules (a) and functionalised macromolecules (b) in aqueous solution with high binding specificity. First and second guest functionalised BSA proteins (c) were prepared by maleimide-thiol-eneclick chemistry with a variety of linkers for CB[8] supramolecular bio-conjugation with complementary functionalised PEGs (d).

Results and discussion

Materials and methods

We have chosen the medium-sized bovine serum albumin (“Fraction V”) as a model protein for bio-conjugation through its only free cysteine, residue 34, which has been well investigated in the literature (see point of attached yellow ‘linker’ in Fig. 1b).^15,21,22,76 In order to demonstrate the maximum versatility in the formation of the reversible supramolecular bio-conjugates, both the protein and polymer were each prepared bearing both first (viologen) and second (naphthalene) guests. Following standard protocols for maleimide-thiol-eneclick chemistry,^15,16,19 functionalised BSA proteins Np-BSA1, Np-BSA2 and MV-BSA3 were prepared from the appropriate maleimide small molecule linkers and BSA as depicted in Fig. 1c. Subsequently, the functional BSAs were purified from any remaining small molecule reagents by dialysis and lyophilisation. The purity of these functionlised BSA proteins was confirmed by aqueous gel permeation chromatography (GPC).

A similar strategy relying on maleimide-thiol chemistry was utilised to obtain the 5000 g mol⁻¹ viologen (first guest) terminated polyethylene glycol monomethyl ether (MV-PEG4) as depicted in Fig. 1d. Alternatively, PEGs (5000 g mol⁻¹) bearing second guests, naphthalene (Np-PEG5) and anthracene (Ant-PEG6), were synthesised from the appropriate isocyanate (see Fig. 1d).

Supramolecular protein-polymer bio-conjugation

There are a variety of experimental techniques capable of observing the selective formation of a supramolecular 1 : 1 : 1 ternary complex mediated by CB[8] in aqueous solutions including both spectroscopic and calorimetric experiments. As the formation of CB[8] mediated ternary complexes is highly selective, it was expected that complex formation would be observed only when all three of the necessary components (first and second guests and CB[8]) were present in solution. The chemical shifts stemming from the protons on both the first and second guests experience a substantial upfield shift in the ¹H NMR upon ternary complex formation inside the hydrophobic cucuribit[8]uril cavity.⁶⁶ In agreement with reported small molecule ternary complexes,⁶⁶¹H NMR experiments of an aqueous solution (0.05 mM in D₂O) of Np-BSA1 and the model small molecule viologen MV7 in the presence of CB[8] indeed displayed an upfield chemical shift and broadening of the viologen proton peaks upon ternary complex formation as shown in Fig. 2. Moreover, an upfield shift for the CB[8] protons occurred along with a decrease in spectral resolution as the CB[8] was now part of a much larger entity (CB[8]·MV7·Np-BSA1) thereby reducing its tumbling rate. The high selectivity of the binding is illustrated by the absence of any interaction between native (unfunctionalised) BSA and MV7 and CB[8] as both the viologen and CB[8] protons remain sharp and well resolved.

Fig. 2 ¹H NMR spectra of CB[8]·MV7·Np-BSA1 (a), CB[8]·MV7 and native BSA (b) and native BSA (c) at 0.05 mM concentration in D₂O. The inset shows two of the three different CB[8] protons in the CB[8]·MV7 complex in the presence (d) and absence (e) of Np-BSA1. The protons of MV7 (red circle) and the CB[8] protons (blue square) are highlighted.

Similar findings were observed when MV7 was replaced with MV-PEG4 to probe the formation of a supramolecular polymer-protein conjugate (ESI Fig. S2†). While the ¹H NMR spectrum for MV-PEG4 with NP-BSA1 in the presence of CB[8] was appreciably complex, there are two specific items to highlight, namely, the CB[8] protons were considerably broadened and the N-methyl-group singlet (appearing at 4.45 ppm) of the viologen-terminated MV-PEG4 vanished completely upon ternary complex formation. Moreover, the N-methyl-group proton signal of MV-PEG4 was not affected by the presence of Np-BSA1 when the host CB[8] was absent. Essentially the same observations were made when an alternative functionalised BSA, Np-BSA2, was employed (ESI Fig. S3†).

NMR diffusion experiments (DOSY) were performed on an equimolar mixture of MV7 and Np-BSA1 in both the absence and presence of the host CB[8] to provide further evidence for the formation of the ternary complex as well as to quantify the reduced tumbling rate of CB[8]·MV7 upon formation of the large supramolecular complex with Np-BSA1. The results are depicted in Fig. 3 and show the expected reduction of the diffusion coefficient of CB[8]·MV7 by one order of magnitude in the presence of Np-BSA1. In addition, all three components CB[8], MV7 and Np-BSA1 moved at the same rate, which further supports that a supramolecular protein-polymer complex has been formed. On the contrary, the absence of one of the three necessary components yielded no marked decrease on the tumbling rate and therefore diffusion rates. A significant, albeit somewhat smaller, reduction of the diffusion rate of the first guest species was also observed in the equimolar mixture of MV-PEG4 and Np-BSA1 when CB[8] was present as shown in ESI Fig. S5†. The smaller reduction in the diffusion rate of MV-PEG4 upon ternary formation is on account of two reasons, first it has a substantially higher molecular weight and thus size compared to small molecule MV7, and second, DLS measurements indicated that CB[8]·MV-PEG4 formed higher-ordered aggregates on its own in aqueous solution (see Fig. 4). When Np-BSA1 was present, however, the ternary complex formed preferentially and any aggregate containing CB[8]·MV-PEG4 alone was no longer present, serving as another indication for the successful formation of the supramolecular PEGylated BSA.

Fig. 3 DOSY NMR spectra for equimolar mixtures of MV7 and Np-BSA1 (a), CB[8], MV7 and native BSA (b) and CB[8], MV7 and Np-BSA1 (c) at 0.05 mM concentration in D₂O. The aromatic protons of MV7 (red circle) and the CB[8] protons (blue square) are highlighted.

Fig. 4 MV-PEG4·CB[8] aggregates into particles with average diameters (D_avg) = 105 nm in aqueous solution (0.05 mM at 25 °C) as measured by dynamic light scattering. Upon addition of Np-BSA1, the ternary complex forms and the measured D_avg significantly decreases to 8 nm, which is roughly the same as for Np-BSA1 alone or the binary solution mixture of Np-BSA1 and MV-PEG4.

Optical measurements such as UV/vis and fluorescence have also been employed widely to study CB[8] guest encapsulation as ternary complexes containing viologen first guests with electron-rich aromatic second guests show distinct features in their electronic spectra, most noticeably an the emergence of a charge-transfer (CT) absorbance band in the visible region of the spectra.⁶⁶ As illustrated in Fig. 5a and b, aqueous solutions (0.5 mM in each component) containing different mixtures of first guests (MV7, MV-PEG4), second guests (Np8, Np-BSA1) and CB[8] only display a distinct CT band in the region of 400–600 nm when all three components (first guest, second guest and CB[8]) are present at the same time. In the absence of CB[8], however, a much weaker electronic interaction between a viologen-containing first guest and a naphthalene-derivative second guest is observed;⁷⁰ this is also the case for an equimolar mixture of Np-BSA1 and MV-PEG4. Similar observations were made for complexation of MV-PEG4 with the aromatic-containing maleimide linker in Np-BSA2 with CB[8] (see ESI Fig. S7†). Furthermore, replacing Np-BSA1 with native BSA does not lead to any additional UV/vis CT features; therefore, UV/vis studies give further evidence for the formation of the ternary polymer-protein conjugate. Additionally, fluorescence quenching (of either the first⁶⁷ or second⁶⁸ guest) upon ternary complex formation serves as a valuable tool to monitor the binding process in substantially lower concentration ranges than are possible with UV/vis spectroscopy. When Np-BSA1 was excited at 310 nm, only the naphthalene moiety gave rise to a fluorescent signal with a λ_max = 350 nm whereas the two tryptophan chromophores present in the core of BSA do not fluoresce at this excitation wavelength (see ESI Fig. S10b†). Upon addition of 1 equivalent of MV7, the fluorescence was not significantly perturbed; however, when an equivalent of CB[8] (optically transparent below 250 nm) was also present, formation of the 1 : 1 : 1 ternary complex of Np-BSA1, MV and CB[8] led to quantifiable reduction in the fluorescence intensity.

Fig. 5 UV/vis spectra for Np-BSA1 in different combinations with MV-PEG4 and CB[8] (a) in comparison to the control experiments with small molecule first guest MV7 and second guest Np8 (b), all at 0.5 mM concentration in each component.

After successfully demonstrating the formation of the supramolecular protein-polymer bio-conjugate with a variety of spectroscopic techniques, we set about to quantify the strength of complexation in solution; this was achieved by carrying out isothermal titration calorimetry in buffered aqueous solutions. An exothermic binding isotherm was observed for the titration of Np-BSA1 with a 1 : 1 complex of CB[8]·MV-PEG4 as illustrated in Fig. 6a, however, no heat signal was observed in the absence of CB[8] (Fig. 6b). Fitting the binding isotherm in Fig. 6a to a ‘single site’ binding model yielded a binding constant (K_a2) of 3.9 × 10⁴ M⁻¹. When K_a2 is combined with the binding constant for first guest complexation with CB[8] (K_a1 = 3.8 × 10⁴ M⁻¹ for MV-PEG4·CB[8] complexation in the same buffer, also determined by ITC), an overall binding constant, K_a, of 1.5 × 10⁹M⁻² is obtained, which would be 1–2 orders of magnitude higher in pure water and sufficient for use in the mM concentration range.^58,77

Fig. 6 ITC binding isotherms for the titration of Np-BSA1 with CB[8]·MV-PEG4 (a) and with MV-PEG4 (b) in buffered aqueous solution at 25 °C.

It is important to note at this point that in the instance of the complementary conjugation strategy, i.e. a BSA bearing a first guest viologen unit (MV-BSA3) and a naphthalene-terminated PEG moiety (Np-PEG5), the observed behaviour was slightly more complex, but remained indicative for the formation of a CB[8] mediated supramolecular polymer-protein conjugate. The ¹H NMR spectrum for the CB[8]·MV-BSA3·Np-PEG5 ternary complex depicted both a broadening of the naphthalene and CB[8] proton peaks and the disappearance of the methylene unit at 4.33 ppm adjacent to the naphthalene unit on Np-PEG5 (ESI Fig. S4†), similar to the findings for Np-BSA1 or Np-BSA2 and MV-PEG4. In contrast to the experiments with Np-BSA1 and MV-PEG4, however, some interaction appeared to also take place between Np-PEG5 and MV-BSA3 in the absence of CB[8]. These findings are in agreement with and can be rationalised on account of earlier published studies showing that native BSA has at least two different binding sites (Sudlow site I and II) and can bind both hydrophobic small molecules⁷⁸ and hydrophobic ‘head groups’ of polymers,⁷⁹ of which naphthalene certainly qualifies. Additionally, the UV/vis spectrum (see ESI Fig. S8†) for the equimolar mixture of MV-BSA3 and Np-PEG5 displayed a lower absorbance in the region of 350–450 nm than the sum of the individual components (at identical concentrations), further suggesting that there was in fact an interaction of some sort between MV-BSA3 and Np-PEG5 in the absence of any CB[8] host. However, in the presence of CB[8] the absorbance in this region of the UV/vis spectrum was restored and a broad CT absorbance emerged around 500 nm, suggesting that a ternary complex was formed, as discussed previously.

Fluorescence studies incorporating Ant-PEG6, which possesses an even more hydrophobic end group than Np-PEG5, were carried out to further investigate the difference between the hydrophobic binding interactions of Ant-PEG6 to MV-BSA3 and the selective ternary complex formation in the presence CB[8]. Use of the excellent fluorophore anthracene enables the excitation wavelength of 360 nm, ensuring that the molar absorptivity of MV-BSA3 will be relatively low. When CB[8]·MV-BSA3 was titrated into Ant-PEG6 (5 μM), the expected reduction in the fluorescence intensity was observed in Fig. 7a which followed the trend for a titration of Ant-PEG6 with the small molecule complex CB[8]·MV7. This can be explained by the formation of a CB[8]-mediated supramolecular polymer-protein ternary complex with the viologen acting as a fluorescence quencher being held in close spacial proximity to the fluorescent dye. Moreover, to demonstrate the complete reversibility of this supramolecular approach, when a 1 : 1 : 1 ternary complex consisting of CB[8]·MV-BSA3·Ant-PEG6 was exposed to a competitive guest such as indole,⁶⁵ the fluorescence of Ant-PEG6 was fully recovered (ESI Fig. S11†). In contrast to what was observed for the polymer-protein conjugate, control experiments led to markedly different results as illustrated in Fig. 7b. Specifically, when MV-BSA3 was titrated into a solution of Ant-PEG6, the fluorescence intensity actually increased, whereas the small control molecule of MV7 being added into a solution of Ant-PEG6 displayed only a slight decrease in fluorescence intensity. The latter of these control experiments was straightforward to understand as small molecules such as MV7 easily diffuse in solution and serve to quench the fluorophore; however, the increase in fluorescence in the former control experiment remained a somewhat puzzling result. Interestingly, as an increase in fluorescence intensity of Ant-PEG6 was also observed upon addition of native BSA, the control experiment could then be rationalised as a protection of the hydrophobic anthracene unit from fluorescence quenchers in solution (e.g.oxygen or the MV moiety on MV-BSA3) upon binding one of two hydrophobic pockets of BSA.

Fig. 7 Fluorescence intensity of Ant-PEG6 (5 μM, 360 nm excitation) as a function of increasing concentration of CB[8]·MV-BSA3 (a) and MV-BSA3 (b).

Calorimetric binding studies by ITC further supported the binding hypothesis detailed above for Np-PEG5 (and Ant-PEG6) and MV-BSA3 in both the presence and absence of CB[8], albeit with tremendous differences in selectivity. In contrast to the findings for Np-BSA1 and MV-PEG4, the titration of MV-BSA3 with Np-PEG5 showed a noticeable, yet constant heat response throughout the titration (ESI Fig. S12†). This suggested that the interaction was either not very strong, that the stoichiometry substantially differed from 1 : 1 binding, or a combination of both. However, in the presence of one equivalent of CB[8], the binding isotherm was much greater (see ESI Fig. S13†) and in fact suggestive of a 1 : 1 complexation stoichiometry between Np-PEG5 and CB[8]·MV-BSA3. Fitting the binding isotherm data to a ‘single site’ model yielded a K_a2 value of 1.0 × 10⁴ M⁻¹ for the complexation of Np-PEG5 by CB[8]·MV-BSA3, which is in the same order of magnitude as for the complementary supramolecular bio-conjugation approach described previously. The numeric K_a2 value obtained from ITC experiments would only hold true for a 1 : 1 : 1 CB[8]·MV-BSA3·Np-PEG5 ternary complex, thus, it was only possible to rule out any non-specific Np-PEG5 binding in the presence of CB[8] with all of the correlating spectroscopic evidence. We were encouraged that CB[8]-mediated ternary complex formation was capable of redirecting complexation of hydrophobic moieties with BSA from the naturally occurring protein pocket to the synthetic receptor cavity, resulting in the same spectroscopic properties as for the complementary supramolecular polymer-protein linking strategy. Additionally, it was critical to demonstrate that the formation of a CB[8]-mediated polymer-protein conjugate did not strongly affect protein folding. The circular dichroism (CD) spectra (0.067 mM in 10 mM phosphate buffer) of the functional BSAs and their respective ternary complexes with the corresponding functionalised PEGs in the presence of CB[8] were essentially identical to that of native BSA (ESI Fig. S15†). Finally, our strategy for supramolecular bio-conjugation was applied to the selective, non-covalent formation of protein-protein conjugates, which is of great importance in signal transduction, modification of enzyme activity and antigen-antibody interactions.^80–82 While expected small changes in ¹H NMR spectra and large heats of dilution in the ITC hampered the use of NMR and ITC techniques, UV/vis and fluorescence spectroscopies proved to be a useful tool to monitor the ternary complex formation and thus the supramolecular protein-protein dimer. As can be seen in ESI Fig. S9†, there was a distinct increase in absorbance for the equimolar mixture of Np-BSA1 and MV-BSA3 in the presence of CB[8] compared to the control spectra. Furthermore, a decrease in fluorescence intensity of the a solution containing Np-BSA1 and MV-BSA3 in the presence of CB[8] was also observed as shown in ESI Fig. S10†. These observations offer compelling evidence that controlled, 1 : 1 : 1 ternary complexes can be formed by the use of CB[8] as a host with complementary labeled proteins. We believe that the stoichiometrically controlled manner of the ternary complexation described above allows for the exclusive formation of heteroprotein-protein conjugates which can be easily monitored by both UV/vis and fluorescence spectroscopy and represents an alternative non-statistical supramolecular approach to that which was recently reported.⁸³

Conclusions

The covalent functionalisation of proteins with polymers has proven to be a common strategy in chemical biology research as a result of the improved and novel properties such conjugates display and has led to considerable advances in both the laboratory and clinical use. While polymers have generally been employed to increase the solubility of the bio-conjugate as well as to protect the protein from biodegradation pathways, the covalent linking strategy has typically led to a decrease in protein activity, often as a result of blocking the active site. Therefore, controlled and reversible protein conjugation to polymers has gained appreciable interest. We have demonstrated that non-covalent protein-polymer bio-conjugation can be achieved with the help of a highly selective supramolecular host–guest interaction mediated by CB[8] in water that is inherently reversible. This selective method only requires that the protein carries (or is modified with) a small molecule recognition moiety; as the first and second guests for CB[8] are stable, small molecule aromatic compounds, this methodology can be easily translated to proteins that do not contain an accessible cysteine residue by means of other well-documented amino acid modification routes (see introduction). By means of spectroscopic (NMR, DOSY-NMR, DLS, UV/vis, fluorescence) and calorimetric (ITC) methods, it was shown that a strong and specific binding between the complementarily labeled polymers and proteins only occurred in the presence of the host CB[8]. In the absence of one of the three critical components (host, first guest and second guest labeled species) no binding interaction was observed. Additionally, we believe that this straightforward mix-and-match supramolecular approach will lead to a wide variety of protein-polymer bio-conjugates starting from simple building blocks which can be independently evaluated in toxicology studies prior to bio-conjugation. Finally, it was demonstrated that controlled formation of a supramolecular protein-protein complex was possible through the use of complementary CB[8] ternary formation and this can be extended to the controlled, reversible assembly of heteroprotein-protein complexes in aqueous solutions.

Acknowledgements

The authors would like to thank the EPSRC, ERC and the Walters-Kundert Trust for funding. F.B. thanks the German Academic Exchange Service (DAAD) for financial support.

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Footnote

† Electronic supplementary information (ESI) available: Experimental details, synthesis of Np-BSA1, Np-BSA2, MV-BSA3, MV-PEG4, Np-PEG5 and Ant-PEG6, NMR (Fig. S2-S4) and DOSY (Fig. S5) spectra of the different polymer-protein complexes, as well as DLS (Fig. S6), UV/vis (Fig. S7–S9), fluorescence (Fig. S10), ITC studies (Fig. S12–S14) and CD (Fig. S15). See DOI: 10.1039/c0sc00435a

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