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Association and Internal Morphology of Self-Assembled HPPhOx/BSA Hybrid Nanoparticles in Aqueous Solutions Aristeidis Papagiannopoulos, Eleni Vlassi, Stergios Pispas, and Judith E. Houston J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b04364 • Publication Date (Web): 27 Jun 2018 Downloaded from http://pubs.acs.org on July 7, 2018

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The Journal of Physical Chemistry

Association and internal morphology of self-assembled HPPhOx/BSA hybrid nanoparticles in aqueous solutions Aristeidis Papagiannopoulos1,*, Eleni Vlassi1, Stergios Pispas1 and Judith Elizabeth Houston2 1

Theoretical and Physical Chemistry Institute, National Hellenic Research

Foundation, 48 Vassileos Constantinou Avenue, 11635 Athens, Greece. 2

Jülich Centre for Neutron Science (JCNS) at Heinz Maier Leibnitz-Zentrum (MLZ),

Forschungszentrum Jülich GmbH, Lichtenbergstrasse 1, 85748 Garching, Germany.

Email: [email protected]

Abstract We investigate the formation of hybrid polyelectrolyte/protein nanoparticles by associations between aggregates of partially hydrolyzed poly(2-phenyl-2-oxazoline) (HPPhOx) and bovine serum albumin (BSA) in aqueous solutions. Light scattering experiments show that at conditions of low salt BSA creates inter-aggregate bridges and increases the size of the HPPhOx nanoparticles. At high salt contents breaking of aggregates leads to well-defined nanoparticles. The interior of the formed nanoparticles is probed by small angle neutron scattering. At low salt, diffuse arrangements are observed whereas at high salt concentration, scattering is dominated by well-defined hydrophobic domains enhanced by the incorporation of BSA. This system shows that the combination of hydrophobic and electrostatic interactions in random-amphiphilic-polyelectrolyte/protein complexes can be used to determine the properties of self-assembled hybrid multifunctional nanoparticles.

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Introduction Functional nanoparticles produced by self-assembly of proteins and charged polymers offer unique possibilities in the fields of biotechnology1,

2

and nanodelivery of

proteins and drugs3, 4. Polysaccharides, that are mostly hydrophilic polyelectrolytes, have been used extensively in protein containing nanoassemblies because they are biocompartible and biodegradable5. In particular BSA has been used in polysaccharide-coated nanoparticles 6 and hierarchical polysaccharide-based scaffolds 7

for biomedical applications. Synthetic polyelectrolytes have well-defined properties

and versatile synthesis routes have been devised, but at the same time they raise questions regarding toxicity and biodegradability. At the same time they offer the ability to tune their hydrophilic (charged) in relation to their hydrophobic content. Polyoxazolines and polyoxazoline-containing polyelectrolytes are a promising class of polymers 8-10 for biomedical11 and pharmaceutical applications12 with the potential of satisfactorily low biotoxicity

13-15

. Polyoxazoline containing polyelectrolytes16,

17

may be synthesized in ways providing both hydrophobic and charged groups within their polymer chain and hence they can interact with proteins, DNA and surfactants. In thermoresponsive random copolymers of n-propyl-2-oxazoline and ethylenimine (PPrOx-PEI) the interaction of EI units with BSA was confirmed by increasing the EI content of the polymers. More importantly the authors illustrated that above the transition temperature globular protein-mimicking mesoglobules are formed with their positive charges exposed in their exterior leading to more effective interactions with DNA18. In partially hydrolyzed thermoresponsive polyoxazoline (POx-PEI) polyplexes were formed by mixing with DNA resulting to well-defined nanoparticles that could also be coated by a cross-linked poly(N-isopropylacrylamide shell) 19. The


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polyplexes had significantly lower cytotoxity in comparison to the standard PEI vectors. Complexation between proteins and polyelectrolytes is a subject under intense research in the last two decades 20, 21 aiming at basic understanding of interactions and structure formation, as well as in view of their potential applications in drug, protein/peptide and gene delivery. Different polyelectrolyte systems e.g. spherical polyelectrolyte brushes22, self-assembled core-shell micelles

23,

24

and linear

polyelectrolytes25, 26 lead to a vast range of possibilities for nanostructure formation. These systems are often studied by scattering techniques, like static and dynamic light scattering,27, 28 and by small angle neutron and X-ray scattering25, 29. Light scattering methods elucidate the overall size and shape of the formed nanoparticles and they are very sensitive to aggregation and self-association. On the other hand small angle scattering methods provide more details on length scales that are relevant to the internal morphology of aggregates (1-100 nm). In our previous work30 we have synthesized and characterized partially hydrolyzed poly(2-phenyl-2-oxazoline) (HPPhOx) leading to amphiphilic charged random copolymers. These macromolecules contain hydrophobic (PhOx) and hydrophilic ethylene imine (EI) units in a random sequence. We have previously observed that the HPPhOx copolymers form aggregates ~100 nm in size with positive surface charge and illustrated that they interact with proteins of fetal bovine serum. In this study we explore the associations of these aggregates with BSA at neutral pH where the two components are oppositely charged. We perform light scattering and small angle neutron scattering in order to resolve the overall structure and internal morphology of the HPPhOx/BSA nanoparticles at several solution conditions leading to intermolecular self-assembly. The system under study provides hybrid multifunctional



nanoparticles with the ability for complex hydrophobic and electrostatic interactions at the molecular level and has potential for hydrophobic drug loading, protein separation and tissue engineering applications. Materials and Methods Materials and sample preparation HPPhOx synthesis and solution properties have been described in detail elsewhere 30. HPPhOx with Mn=2.03x103 g·mol-1, Mw/Mn=1.13 (PPhOx precursor) and 67% molar content in PhOx was utilized for the present studies. This random copolymer comes from the partial hydrolysis of PPhOx and contains hydrophobic PhOx and cationic EI units (scheme 1). It was directly dissolved in D2O at the desired concentration and left overnight to equilibrate at 4 °C. BSA was supplied by Sigma-Aldrich and used without further treatment. BSA was dissolved in D2O and left overnight to equilibrate at 4 °C before mixing with any polymer solution. HPPhOx/BSA mixtures were prepared by mixing volumes of equilibrated (overnight at 4 °C) separate solutions. In salt containing solutions appropriate volumes of 1M NaCl were added.

Scheme 1: HPPhOx random copolymer (poly(EI-co-PhOx)).

Light scattering Light scattering (LS) was performed on an ALV/CGS-3 compact goniometer system (ALVGmbH, Germany) with an ALV-5000/EPP multi tau digital correlator and a HeNe laser (λ=632.8 nm). In static light scattering (SLS) the Rayleigh ratio31 was


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extracted and treated by the Guinier approximation

= ∙ −1/3 , so

that the molar mass and radius of gyration are obtained. The scattering wave vector is =

with θ the scattering angle and the solvent’s refractive

index while c is the particle mass concentration. K is given by ! =

" " # $ %

&/&'

where &/&' the refractive index increment of the scattering particles in the solvent ((&/&')*++,-. = 0.22 34567 , (&/&')9:; = 0.18 34567).

In dynamic light scattering (DLS) the field autocorrelation functions are obtained at a broad angular range and are converted to the field autocorrelation functions by the Siegert relation =|57 ?| = 5 ? − 1 where = is a normalization factor32. We used CONTIN analysis to Laplace-transform the field autocorrelation functions into timedistribution functions @?. The relaxation rate A was obtained by the characteristic time ?BC. of the maximum of the distribution function at every angle by A = 1/?BC. .

The diffusion coefficient D may be calculated by the slope of the A vs plots.

Eventually the hydrodynamic radius is taken by the Stokes-Einstein equation , =

EF/6HID . Size polydispersity index PDI is taken from cumulant analysis at θ=90°. Small angle neutron scattering

Experiments were performed on the KWS-2 high intensity / wide-q small angle neutron scattering diffractometer, at the Jülich Centre for Neutron Science (reactor FRM II). In small angle neutron scattering (SANS) the scattering vector (q) range was 0.002 to 0.1 Å-1 and was covered by three separate detection configurations; 2 m, 8 m and 20 m detection length, with neutron wavelength λ=4.5 Å. Raw data was treated by standard corrections and reduction and the collected isotropic 2-D raw data was transformed into 1-D scattered intensity J . All numerically calculated intensities



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mentioned in the results section are convoluted33 with a Gaussian function so that instrumental resolution is taken into account34, 35. Small angle neutron scattering data analysis A three-level hierarchical Beaucage model36,

37

was used to model the SANS data

described in equations 1, 2 and 3. Each level a and b consist of a Guinier regime that defines its forward scattering KL and radius of gyration ,L and a power-law regime

with a characteristic exponent ML . We assign level N as the large and level O as the

small hierarchical structure i.e. ,C > ,Q . Alternatively level-O may be denoted as “internal domains” and level-N as “aggregates” The error function in equation 2 67 constrains the power-law at > ,L . At higher , terms N and O are limited at high q 7

by a cut-off function R− TU,L V with TU,L = ,LW7 because at length scales S

lower than the gyration radius of the next smaller level the correlations within that smaller level dominate.

J = JC + JQ + J

(1)

Where 7

7

JL = KL ∙ R− S ,L V + YL ∙ R− S TU,L V ∙ Z [@ ,L /√6]

with = N, O (2)

S^_

∙ 6^_

6^

The prefactor YL = KL ML ,L _ (6ML 2 + ML 67 2 + 2ML 67 )^_ / AML /2 bridges the

two regimes smoothly38. S^`

J = Y ∙ Z [@ , /√6]

∙ 6^`

(3)

The third hierarchical level is described by equation 3. It is a power-law function that 67 contributes at > , and it is normally used for correlations within aggregates e.g.

scattering by individual chains within micelles24. This kind of scattering is also predicted by analytical core-shell models where the explicit conformations of


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macromolecular chains within micellar coronas are taken into account39 or modelled by empirical formulas29.

KL =

∙#_ ∙ab #$

7

∙ c^ ∙ de − df"- g

b

(4)

Forward scattering of a structural level i.e. JL = 0 = KL is connected to the number

of individual chains (hL ) inside the specific structure40 using equation 4. e is the

molecular weight of the chains, Me (≈1.1 g/cm3) the dry mass density and ' the mass solution concentration of the structures assumed here as the nominal solution concentration i.e. all polymer chains in the solution are incorporated in the hierarchical structures. The neutron scattering length densities of the polymer and D2O we used are de = 1.5 ∙ 106j Å

6

and de = 6.4 ∙ 106j Å

6

respectively.

Electrophoretic light scattering The experiments were performed on a Zetasizer Nano-ZS (Malvern Instruments Ltd). Zeta potential was calculated by Henry equation in the Smoluchowski approximation. Averages of 10-20 measurements at 173° angle are reported. All the experiments were performed at room temperature. Circular dichroism A Jasco J-815 CD spectrophotometer with a peltier model PTC-423S/15 thermo stabilizing system was used for circular dichroism (CD) measurements. The spectrums were accumulated by averaging six successive runs with 0.5 nm wavelength step at 100 nm/min rate, response time 1 s and bandwidth 1 nm. Fourier Transform Infrared spectroscopy Infrared spectra (FTIR) were recorded on a Bruker Equinox 55 Fourier Transform Instrument, equipped with an attenuated total reflectance (ATR) diamond accessory, from SENS-IR, and a press. The dried solutions were placed at the center of the



sample holding device and 64 scans were performed in the range 500-5,000 cm-1, at a resolution of 2 cm-1. Results and Discussion The size and shape of the HPPhOx/BSA complexes was addressed by DLS and SLS. Size-distribution functions at the tested solution conditions are shown in figure 1. At no added and intermediate amounts of salt the distributions are broad. In the case of no added NaCl at the highest amount of BSA there is a clear contribution of a small size population at 4.2 nm, which signifies the presence of free BSA in solution. The hydrodynamic radius of BSA reported in the literature41-43 is 3.5-3.7 nm. The increased size observed in our case can be related to some adsorption of free polymer on BSA monomers. At 0.03 and 0.15 M NaCl the signature of BSA globules at the maximum BSA content is not detected. This is because the molar mass of the complexes is much higher in these two cases (as it will be shown in the following) and hence dominates the scattered intensity. At 0.15 M NaCl the distributions are significantly narrower than in the other two salinities. This is a sign that at conditions where electrostatic interactions are highly screened, better-defined nanoparticles are formed. In complexes between BSA and quaternized poly(chloromethyl styrene) copolymers addition of the protein (at no added salt) resulted in an increase in molar mass and not in size, indicating the predominance of intra-aggregate hydrophobic interactions between protein and polymer26. In scheme 2 a representation of the main interactions considered in this work is presented.


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Scheme 2: (a) Hydrophobic interactions in HPPhOx self-assemblies. (b) Hydrophobic (i), electrostatic (ii) and bridging (iii) interactions in HPPhOx/BSA complexes. Gray circles denote the region of interaction. EI units, PhOx units and BSA are represented by green circles, black circles and red ellipsoids respectively.



Figure 1: Distribution of Rh extracted by CONTIN analysis at θ=90° for aqueous solutions of HPPhOx (0.75 mgml-1) mixed with BSA at 0.1 (black), 0.2 (red) and 0.4 (blue) mgml-1 protein concentration and for 0 M (a), 0.03 M (b) and 0.15 M (c) NaCl concentration.

The relaxation rate from HPPhOx/BSA complexes is shown in figure 2. The diffusion coefficient was obtained by fitting a quadratic formula A = D ∙ + ' ∙

because the trends were not linear. The cause of the curved A is most probably the ACS Paragon Plus Environment

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polydispersity of the complexes. Indeed at high salt content where the distribution functions appear narrow (figure 1) the corresponding A profiles are nearly linear (Figure 2 c). The SLS data are fitted by the Guinier model (figure 3). When is

relatively small they appear linear. When is high a low-q upturn appears because

the internal morphology of complexes is probed and because of polydispersity. In those cases the low-q linear part of the plots is fitted (figure 3 a and b).



Figure 2: Relaxation rate from aqueous solutions of HPPhOx (0.75 mgml-1) mixed with BSA at 0.1 (black), 0.2 (red) and 0.4 (blue) mgml-1 for 0 M (a), 0.03 M (b) and 0.15 M (c) NaCl. Lines are polynomial fits as explained in the main text.


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Figure 3: SLS data from aqueous solutions of HPPhOx (0.75 mgml-1) mixed with BSA at 0.1 (black), 0.2 (red) and 0.4 (blue) mgml-1 protein concentration and for 0 M (a), 0.03 M (b) and 0.15 M (c) NaCl concentration. Lines are fits with the Guinier approximation.

Table 1: Results from SLS and DLS from HPPhOx/BSA complexes with HPPhOx at 0.75 mgml-1.



[NaCl] 0M

0.03 M

0.15 M

cBSA (mgml-1)

M (105gmol-1)

Rg (nm)

Rh (nm)

ρ=Rg/Rh

0.1 0.2 0.4 0.1 0.2 0.4 0.1 0.2 0.4

2.49 2.08 1.50 1.57 2.15 39.1 2.49 452 47.4

143 146 75.5 45.9 53.1 220 75.9 78.4 64.1

111 107 51.7 33.2 36.4 60.3 76.3 88.8 55.6

1.29 1.36 1.46 1.38 1.46 3.65 0.99 0.88 1.15

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PDI ζ (mV) (θ=90°) 0.54 0.44 0.54 0.48 0.50 0.52 0.41 0.022 0.27

18.8±4 21.5±2 29.1±2 20.1±1 27.8±2 20.8±2 17.9±2 13.4±3 16.9±2

In table 1 the results from SLS, DLS and ELS are presented. The apparent molar mass, radius of gyration and hydrodynamic radius of the HPPhOx aggregates of this work in similar conditions (in H2O) have been reported previously 30. The reported values were = 0.32 ∙ 10l 53m4 67 , = 41 3 and , = 24 3 at 0 M (at HPPhOX 1 mgml-1), = 35 ∙ 10l 53m4 67, = 229 3 and , = 219 3 at 0.03

M and = 4.5 ∙ 10l 53m4 67 , = 84 3 and , = 89 3 at 0.15 M.

In the absence of salt the complexes have significantly higher mass and size than the BSA-free copolymer aggregates. Protein globules cause secondary aggregation to the initial bare copolymer aggregates because they create inter-aggregate bridges23. At the highest amount of BSA the smallest size and molecular mass of complexes is obtained with the maximum zeta potential. This shows that bridging is not so effective at high protein content. The increased number of protein globules in solution leads to small-size complexes with high apparent surface charge density. The enhanced presence of BSA introduces an increased number of hydrophobic sites that couple to the hydrophobic PhOx monomers. This apparently causes conformational rearrangements in the amphiphilic copolymer chains. A more pronounced exposion of charged EI units occurs in the outer surface of the nanoparticles increasing their surface charge.These hybrid nanoparticles are smaller than 100 nm in radius and can be ideal for drug delivery applications. At intermediate salt concentration


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nanoparticles are obtained with size in the range of 40-50 nm and molecular weights similar to 0 M NaCl. The exception is that, at 0.4 mgml-1 BSA and appear significantly larger and similar to the BSA-free aggregates. This originates from the strong low-q upturn and the related formation of large complexes. The smaller complexes at 0.1 and 0.2 mgml-1 BSA are formed by a mechanism where the presence of BSA most probably causes breaking of the initial large copolymer aggregates. At 0.15 M NaCl high molar masses are found while the sizes are below 100 nm. The shape factors d only at high salt and moderate protein content approach the ones for homogeneous spherical particles44 (~0.775) contrary to low and no added salt where more open/loose microgel-type morphologies are indicated45. In particular, at 0.15 M and 0.4 mgml-1 BSA high molar mass hybrid nanoparticles are formed with spherical shape. In general zeta potential measurements show that BSA’s negative charges do not decrease the positive surface charge of the complexes. HPPhOx contributes one charge per EI monomer while BSA (at pH 7) contributes about 9 net negative charges per globule46. At the concentrations studied the BSA/HPPhOx charge ratios are not higher than 1%. Hence the surface charge of the complexes is dominated by the presence of HPPhOx chains. Similarly in polyplexes between DNA and random copolymers of n-propyl-2-oxazoline and ethylenimine (PPrOx-PEI) positive surface charge is observed at high amine-to-phosphate groups (N/P) ratios18. It has to be mentioned that in PPrOx-PEI thermoresponsive copolymers the formation of globular aggregates with significant positive zeta potential occurred above the transition temperature i.e. at 37 °C 18. In HPPhOx formation of aggregates with positive surface charge occurs at room temperature30. This is possibly caused by the comparably higher content of EI units in HPPhOx and the hydrophobicity of PhOx which in our case although it does not induce thermoresponsive behaviour30 it can effectively drive



self-association at room temperature in contrast to PrOx. Additionally the presence of a cross-linked polymeric shell is needed to stabilize the complexes at room temperature while it is in the cases of PPrOx-PEI18 and POx-PEI19 polyplexes. PDI values are smaller than in the other salt contents in line with the CONTIN analysis of figure 1. The morphology of HPPhOx/BSA complexes at length scales between 1 and 100 nm in aqueous media were measured by SANS. These length scales correspond to the interior of the investigated nanoparticles especially the ones with , ~ 100 3 or higher. HPPhOx was at a concentration that provides adequately strong scattering in SANS. In complexes, BSA concentration was chosen so that the protein/polymer mass ratios were in the same range with the ones in LS experiments i.e. 39:; ⁄3*++,-. is between 0.13 and 0.5. In figure 4a SANS profiles at different salt contents in D2O are presented. In the case of 0M the separate contributions of equation 1 are also shown (figure 4b). The SANS profiles in the absence of salt are apparently dominated by associations with diffuse internal structure as they do not diverge significantly from power-law behavior. At 0.03 M a well-defined structure appears at intermediate length scales in the form of a Guinier regime between 0.01 and 0.04 Å-1. At 0.15 M the low-q scattering increases again but the Guinier regime is still evident. The extracted forward scattering from the hierarchical levels N and O follow these trends (table 2). In the absence of salt ,C is

higher than the values obtained by LS of our previous work30 ( ≈ 40 3). This possibly comes from the different concentration regime we test in SANS in comparison to LS. The higher hierarchical level indicates presence of clustered aggregates. As it will be discussed in the following the association in HPPhOx is evidently very sensitive to concentration.


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At 0.03 and 0.15 M the largest structural level has a characteristic size ,C clearly smaller than the one observed previously by SLS and DLS mentioned above and previously30. This means that internal structure is probed. Indeed the corresponding SANS profiles (figure 4a) show an upward trend at very low signifying clustering at even higher length scales. The internal domains’ contribution is dominant with ,Q ≈ 9 3 (figure 4a and table 2). It is related to well-defined domains made of associative hydrophobic polymeric segments of high local concentration. Their morphology does not change significantly upon further salt addition. Their fractal exponent MQ approaches the one of rough surface fractal in the presence of salt. This points out the compact chain associations under conditions of screened electrostatic interactions. Similarly the high-q, the power-law exponent, defined by M slightly increases from 1.54 to 1.76-1.77 revealing denser inter-chain associations and less extended chain conformations at the nm-scale. The relative contribution of levels N

and O change non-systematically mainly because of the abrupt increase in KC upon

addition of 0.15 M salt (table 2). Their aggregation numbers obtain their minimum value at 0.03 M NaCl. At high salt clustering between O-level structures is greatly pronounced.



Figure 4: (a) SANS profiles from HPPhOx 3.75 mgml-1 at 0 M (black), 0.03 M (red) and 0.15 M (blue) NaCl in D2O. Lines are fits to the experimental data by equations 1-3. (b) The separate contributions JC , JQ and J of equation 1 are illustrated (dashed lines) for the case of HPPhOx 3.75 mgml-1 at 0 M.

In figure 5 the SANS data at different concentrations show that concentration dependence is not trivial. Upon increasing polymer content both the scattering intensity and the shape of the curves change. A well-defined structure at intermediate is evident at 7.5 mgml-1, which corresponds to the internal hydrophobic domains. ACS Paragon Plus Environment

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On the other hand the forward scattering of the intermediate level KQ increases

systematically with concentration at 0 M NaCl resulting to aggregation numbers hQ

between 270 and 390. At 1.875 and 7.5 mgml-1 the O-structure is pronounced (figure 5). The HPPhOx aggregates show some structural similarities with casein micelles and other disordered protein aggregates. The amphiphilic nature of casein proteins allow their associations to interact and encapsulate hydrophobic substances47. Casein aggregates48 consist of submicelles (R~7 nm) into spherical particles (R~120 nm) and are covered by a “hairy layer” that stabilizes the particles49. In any case the non-trivial concentration dependence reveals soft and fluctuating interior of aggregates that should be susceptible to significant rearrangements under the influence of other objects e.g. protein globules and hydrophobic drugs.

Figure 5: SANS profiles from HPPhOx 1.875 (black), 3.75 (red) and 7.5 (blue) mgml1

in D2O with no added salt. Lines are fits to the experimental data by equations 1-3.

Table 2: Extracted parameters from SANS on HPPhOx solutions in D2O. cHPPhOx (mgml-1) [NaCl]

1.875

3.75 0M

7.5

3.75 0.03 M


0.15 M


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11.0±0.7

93±5

99±5

10.7±0.6

235±10

62±5

69±6

50.±4

51±4

72±6

2.38±0.07

3.09±0.08

3.21±0.08

Ga (cm-1) Rg,,a (nm) da Gb (cm-1) Rg,b (nm) db Bc (10-4cm-1Å-dc) Rg,c (nm) dc Na Nb

3.04±0.08

3.36±0.08

3.3±0.2 29±2

9.6±0.5 23±2

18.9±0.8

3.8±0.2

6.8±0.5

14±2

9±1

10±2

2.15±0.05 0.76±0.04 3.3±0.2

2.71±0.05 2.1±0.1

3.44±0.07 5.6±0.3

4.2±0.07 2.0±0.2

3.44±0.06 1.5±0.1

2.6±0.2

3.3±0.2

4.4±0.3

4.1±0.3

1.69±0.04 890±60 270±20

1.54±0.03 3800±200 390±20

1.65±0.03 2000±100 380±20

1.77±0.02 430±30 150±10

1.76±0.03 9500±400 280±20


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Figure 6: SANS profiles from HPPhOx (3.75 mgml-1) mixed with BSA at 0 (black), 0.469 (red), 0.938 (blue) and 1.875 (green) mgml-1 for 0 M (a), 0.03 M (b) and 0.15 M (c) NaCl in D2O. Lines are fits to the experimental data.

In figure 6 the fitted SANS profiles from HPPhOx/BSA complexes are depicted. The presence of protein does not seem to drastically change the morphology at 0 M NaCl. The resulting parameters (table 3) do not show any systematic trend either. From LS at 0 M, interactions with BSA were described as an effect on inter-aggregate association and dissociation. Consequently this takes place at length scales of the order of 100 nm or higher i.e. in the outer regions of the polymeric aggregates. This effect does not influence the internal morphology of the aggregates and hence the SANS picture. At 0.03 M NaCl (figure 6 b) the intermediate length-scale scattering is gradually taken over by the scattering at low-q. This means that BSA accumulates preferably at the exterior of the aggregates causing their inter-associations. The relative contributions from KC and KQ (table 3) are compatible with this conclusion. The extracted ,C can

be identified with the obtained by SLS at 0.1 and 0.2 mgml-1 BSA where breaking of the aggregates was concluded. At high salt content (0.15 M NaCl) (figure 6c) it is clear that protein reduces aggregation at large length scales in favour of the intermediate length-scale domains. This is a qualitatively different situation than in the absence of salt. BSA globules enter the aggregates and interact with the internal hydrophobic domains. Indeed KQ systematically increases, while ,Q also does so but marginally (table 3). This is compatible with the increased molar masses observed by SLS at high salt.


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Table 3: Extracted parameters from SANS on HPPhOx (3.75 mgml-1)/BSA complexes. cBSA (mgml1 ) [NaCl] Ga (cm1 ) Rg,,a (nm) da Gb (cm1 ) Rg,b (nm) db Bc (104 cm-1 Å-dc) Rg,c (nm) dc

0.469

0.938

1.875

0.469

0M

0.938

1.875

0.469

0.03 M

0.938

1.875

0.15 M

68±4

55±3

58±3

25±2

50±

60±3

10±1

10±1

10±1

51±4

47±4

46±4

62±5

83±7

75±7

53±6

51±5

51±5

1.93±0.05

2.55±0.05

2.83±0.04

1.70±0.05

2.03±0.06

1.89±0.04

3.13±0.08

3.47±0.08

3.46±0.09

6.9±0.4

7.3±0.4

7.2±0.4

4.3±0.2

6.7±0.3

6.6±0.3

3.9±0.2

5.0±0.4

6.3±0.4

22±2

21±2

20.0±2

12±1

16±2

17±2

8±1

9±1

11±1

2.49±0.05

2.56±0.05

2.54±0.05

2.83±0.05

2.81±0.06

2.48±0.05

4.20±0.08

3.67±0.06

2.87±0.05

2.0±0.1

2.5±0.1

2.2±0.1

2.9±0.1

2.8±0.2

2.9±0.2

1.6±0.2

1.8±0.2

1.7±0.1

2.5±0.3

3.2±0.3

3.6±0.2

3.3±0.3

2.0±0.2

1.9±0.2

5.5±0.4

6.4±0.5

5.9±0.5

1.57±0.03

1.50±0.03

1.66±0.04

1.68±0.03

1.79±0.04

1.61±0.04

1.76±0.05

1.77±0.04

1.79±0.04

In figure 7 the circular dichroism results show the expected signal for solutions of pure BSA. By increasing polymer concentration the noise level is enhanced and the wavelength range is restricted, probably due to spectral contributions from the phenyl groups of the copolymer. However we can assume that no significant change in BSA conformation is detected by CD after interaction with HPPhOx aggregates.



Figure 7: Circular dichroism from HPPhOx/BSA complexes. Concentration is at (0.2 mgml-1) and the one of HPPhOx is 0 (black), 0.5 (red) and 1.0 (blue) mgml-1 with no added salt.

ATR-FTIR was used in order to elucidate any possible secondary structure changes of BSA upon interaction with HPPhOx. In quaternized cloromethyl styrene cationic polyelectrolytes we have shown that the hydrophobic interaction led to significant denaturation of the protein’s native structure 26. Hence ATR-FTIR data of this work was analysed in the amide I region (1700-1600 cm-1). In figure 8a the data from pure BSA, pure HPPhOx and HPPhOx/BSA complex are presented. The difference between the polyelectrolyte/protein complex and the pure protein, which is the signal from the complexed protein, shows that there is a clear structural change in BSA. In denaturation studies of BSA this enhancement of signal at lower wavenumbers is a sign of denaturation of α-helix, which dominates its native conformation 50, 51.


Page 24 of 35

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Figure 8: (a) ATR-FTIR from HPPhOx (0.75 mgml-1)/BSA (0.4 mgml-1) complexes with no added salt (black), pure HPPhOx (0.75 mgml-1) (gray), difference of the two spectra (blue) and pure BSA (0.2 mgml-1) (red). Fitting of pure BSA (b) and BSA inside the HPPhOx complex (c) as described in the main text. Experimental data (blue) and fitting profiles are shown with continuous lines. The separate components



Page 26 of 35

representing different wavenumber regions (secondary structure assignments) are presented with black, red, blue and green dotted lines.

Table 4: Secondary structure of BSA determined by fitted ATR-FTIR data. Assignment

β-turn

α-helix

Random coil

Wavenumbers (cm-1) BSA HPPhOx/BSA1* HPPhOx/BSA2* HPPhOx/BSA3* HPPhOx/BSA4*

1675-1679 14.2% 18.5% 20.1% 23.2% 23.1%

1655-1658 63.9% 33.5% 38.9% 35.2% 33.7%

1640-1642 25.6% 23.6% 21.1% 22.3%

Short-segment chains connecting α-helical segments 1632-1639 17.9% 21.3% 17.4% 20.5% 20.9%

Intermolecular β-sheet 1618-1619 4.0% 1.0% -

*

From HPPhOx/BSA complexes at 0.75 mgml-1 HPPhOx. For 1, 3 and 4 BSA concentration at 0.2 mgml-1 and for 2 at 0.4 mgml-1. For 1 and 2 NaCl concentration at 0 M, for 3 at 0.03 M and for 4 at 0.15 M.

Data from uncomplexed and complexed BSA were quantified by fitting the experimental curves with a superposition of Gaussian profiles52,

53

with the band

assignments of table 4. The native structure is rich in α-helix conformation (16551658 cm-1), while upon interaction with HPPhOx the β-turn (1675-1679 cm-1) and short short-segment chains connecting α-helical segments (1632-1639) contributions increase. At the same time the (1640-1642 cm-1) random-coil conformation becomes a significant part of the spectrum whereas it was not detected in pure BSA. We conclude that ATR-FTIR measurements were capable of proving a significant conformational change in BSA from mostly α-helix to mostly random-coil, β-turn and short short-segment chains connecting α-helical segments. Conclusions Complexation of the protein BSA with aggregates from the amphiphilic polyelectrolyte HPPhOx was used for the formation of nanoparticles with controllable size and internal morphology. As both electrostatic and hydrophobic interactions drive associations in this system it was found that screening of electrostatic


Page 27 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


interactions by addition of salt weakens BSA-induced inter-aggregate bridging, leads to better defined nanoparticles and enhances hydrophobic domains in the interior of the complexes. The native protein conformation was significantly denatured upon interaction with the amphiphilic polyelectrolyte as it was found by ATR-FTIR. This copolymer/protein

combination

protein/hydrophobically

modified

enriches polyelectrolyte

our

understanding

interactions

and

of

structure

manipulation of resulting complexes and offers candidate stimuli-responsive, potentially biocompatible nanoformulations. It could find applications in protein delivery, as a hydrophobic drug container and as a more general guide for producing multi-functional hybrid macromolecular nanoparticles. Acknowledgements We acknowledge support of this work by the project “Advanced Materials and Devices” (MIS 5002409) which is implemented under the “Action for the Strategic Development on the Research and Technological Sector”, funded by the Operational Programme "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund). This work is based upon experiments performed at the KWS-2 instrument operated by JCNS at the Heinz Maier-Leibnitz Zentrum (MLZ), Garching, Germany. References 1.

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