Small Surfactant Concentration Differences Influence Adsorption of

Subscriber access provided by The University of Liverpool

Article

Small Surfactant Concentration Differences Influence Adsorption of Human Serum Albumin on Polystyrene Nanoparticles Svenja Winzen, James Ciro Schwabacher, Julius Mueller, Katharina Landfester, and Kristin Mohr Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01503 • Publication Date (Web): 26 Oct 2016 Downloaded from http://pubs.acs.org on November 1, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 21

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

Biomacromolecules

Small Surfactant Concentration Differences Influence Adsorption of Human Serum Albumin on Polystyrene Nanoparticles Svenja Winzen, James C. Schwabacher,† Julius Müller, Katharina Landfester and Kristin Mohr* Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany KEYWORDS. Protein adsorption, surfactants, nanoparticles, isothermal titration calorimetry. Abstract: Surfactants, even in miniscule amounts, are often used for the synthesis and especially the stabilization of nanomaterials, which is essential for in vivo applications. In this study, we show that the interaction between nanoparticles and proteins strongly depends on the type of stabilizing surfactants and their (small) concentration changes. The reaction between human serum albumin (HSA) and polystyrene nanoparticles (PS-NPs) stabilized by an ionic or nonionic surfactant – sodium dodecyl sulfate (SDS) or Lutensol AT50, respectively – was monitored using isothermal titration calorimetry (ITC). It was found that the amount of surfactant molecules on the surface significantly determines the protein binding affinity and adsorption stoichiometry, which is important for all nanomaterials coming into contact with biological components such as blood plasma proteins. Thus, after synthesizing nanomaterials for in vivo applications as drug

ACS Paragon Plus Environment

1

Biomacromolecules

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

Page 2 of 21

delivery agents, it is crucial to perform a detailed analysis of the obtained surface chemistry that accounts for the presence of minimal amounts of stabilizing agents.

Introduction Nanomaterials are currently being developed as potential carrier devices or diagnostic tools.1-6 To control the behavior of the nanomaterials in vivo, an understanding of the interaction with biological components is critical. Since often the first contact with biological fluids is human blood, the interaction between nanoparticles and blood proteins has been extensively studied.7 Proteins adsorb to nanomaterial surfaces, forming a corona around the particle. Prior research conveys that this protein corona determines the biological identity of the nanomaterial and therefore influences body distribution and recognition.8-10 It has been illustrated that surface chemistry as well as hydrophobicity strongly influences adsorption processes, but some of the mechanisms remain poorly understood. For example it is well known that protein adsorption is generally promoted the more hydrophobic a nanomaterial surface is.11, 12 On the contrary, nanoparticles with different covalently linked functional groups were synthesized to evaluate the various structural effects.9,

13, 14

It can be seen that those

functional groups almost always influence the protein adsorption behavior, but there are no general concepts yet that could be applied to all kinds of materials. However, the surface functionalization is important for all types of nanoparticles since certain functional groups provide stability in aqueous solution. Nanoparticle aggregation can be prevented by either electrostatic or steric stabilization. Depending on the synthesis procedure, the stabilizing groups (e.g. surfactants) may not be covalently bound but physisorbed. Surfactants must be considered regarding particle-protein interactions because they are commonly utilized and actively influence the surface chemistry of nanoparticles. Isolated


2

Page 3 of 21

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

Biomacromolecules

surfactants interact with proteins, as demonstrated in analytical techniques such as SDS polyacrylamide gelelectrophoresis.15 Those interactions are mostly unspecific, meaning that all proteins form complexes with a given surfactant; but in some cases interactions are limited to specific proteins as reported for CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1propanesulfonate) with ApoA-I.16 The influence of different stabilization methods has been evaluated previously,17,

18

but currently there is no quantitative description of the effect on

protein adsorption. Despite the extensive purification procedures that nanoparticles undergo post-synthesis, small amounts of surfactants must remain on particle surfaces to ensure their colloidal stability. The effects of surfactant molecules on protein adsorption, therefore, require careful investigations similar to the rigorous studies of covalently attached functional groups. The aim of this study is to quantify the effect of two different surfactants on the protein adsorption on model nanoparticles. In order to investigate the dependency on the surfactant surface coverage, different surfactant concentrations are used. This should provide important information for the preparation of nanomaterials for biomedical applications.

Experimental Section Materials Human Serum Albumin (HSA) was purchased from Sigma Aldrich (St. Louis, USA; Product No. A3782) and used without further purification. All protein solutions were freshly prepared with water (Millipore, Milli-Q water with a conductivity 99%) and hexadecane (> 99%) were also purchased from Sigma Aldrich. Styrene was freshly purified before the synthesis by filtration through aluminum oxide to remove the stabilizer 4-tertbutylcatechol. 2,2'-Azobis(2-methylbutyronitrile) (V59) was purchased from Wako Chemicals


3

Biomacromolecules

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

Page 4 of 21

GmbH (Neuss, Germany). The non-ionic surfactant Lutensol® AT50 (poly(ethylene glycol)hexadecyl ether) was purchased from BASF AG (Ludwigshafen, Germany). The anionic surfactant sodium dodecyl sulfate (SDS) was purchased from Fluka (Sigma Aldrich).

Methods Sample preparation. Polystyrene (PS) nanoparticles were synthesized according to previously published procedures19, 20 with SDS or Lutensol AT50 as surfactant. Details on the materials and purification can be found in the supporting information. PS particles stabilized with SDS were purified using dialysis against deionized water, whereas Lutensol AT50stabilized nanoparticles were purified using repetitive centrifugation and resuspension in water. The procedures were performed until a constant surface tension of the air – water interface was reached. Different amounts of surfactant in deionized water were then added to the purified samples according to the maximum coverage as determined by ITC. The amounts added correspond to a final concentration of 0.04 mM, 0.08 mM and 0.13 mM of SDS and 0.01 mM, 0.02 mM and 0.03 mM of Lutensol AT50 in the respective nanoparticle samples as used in ITC experiments (c = 8.9·10-6 mM for SDS-prestabilized NPs, c = 1.88·10-5 mM for Lut-prestabilized NPs). Particle characterization. The size of the nanoparticles was measured by dynamic light scattering using an ALV instrument (ALV, Langen, Germany) with goniometer and ALV-5001 multiple-tau full-digital correlators with 320 channels. The light source consisted of a heliumneon laser (JDS Uniphase, Milpitas, USA) with 25 mW output power and a laser wavelength of 632.8 nm. Zeta potential measurements were performed in 0.001 M potassium chloride solution at pH 7 and 20 °C with a Zetasizer Nano Z (Malvern Instruments GmbH, Herrenberg, Germany).


4

Page 5 of 21

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

Biomacromolecules

The sample morphology was characterized by means of low voltage scanning electron microscopy (SEM) using a Zeiss 1530 Gemini (Oberkochen, Germany) at 0.2 kV landing voltage. Surface tension measurements were performed with a DCAT 21 tensiometer from DataPhysics Instruments GmbH (Filderstadt, Germany). Isothermal titration calorimetry (ITC). The calorimetric measurements were performed with a Nano ITC Low Volume (TA instruments, Eschborn, Germany) with an effective cell volume of 170 µL. For the titration of PS-NPs with SDS a purified PS-NP batch (SDS prestabilized) with a concentration of 2.5·10-5 mM (8.3 g L-1) along with an SDS concentration of 2 mM was used. For the titration of PS-NPs with Lutensol purified PS-NPs (Lut prestabilized) with a concentration of 1.71·10-6 mM (10.9 g L-1) and Lut with a concentration of 0.5 mM (1.23 g L-1) were used. For a protein adsorption experiment 50 µL of HSA solution (0.15 mM, 10 g L-1 in water) were titrated into the different PS nanoparticle suspensions. The concentration of nanoparticles was 8.9·10-6 mM (3.2 g L-1) for all SDS-stabilized samples in the ITC experiments. For Lut-stabilized nanoparticles the concentration was 1.69·10-6 mM (10.8 g L-1) for all titrations with HSA. For comparison, aqueous solutions of pure Lutensol (0.05 mM) and SDS (0.25 mM) were titrated with HSA (0.15 mM). The experimental temperature was kept constant at 25 °C. The heat of dilution for each experiment was determined by titrating the same amount of titrant into pure water. The number and injected volume of the titration steps were the same for all measurements (25 x 2 µL). The spacing between injections was set to 300 s. For the data analysis the heats of dilution were subtracted from the heats of adsorption experiments and the data was then analyzed according to an independent binding model (details see Supplementary Information).


5

Biomacromolecules

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

Page 6 of 21

Results and Discussions In this study, two types of model polystyrene (PS) nanoparticles with different stabilizing agents were synthesized via miniemulsion polymerization to investigate their interaction with proteins. An ionic (sodium dodecyl sulfate - SDS) and nonionic PEG-based (Lutensol AT50 Lut) surfactant were chosen as representative stabilizers. SDS features a negatively charged headgroup providing an electrostatic repulsion between particles (Table 1). Lut is a polymeric surfactant composed of a C16-C18 hydrophobic saturated fatty alcohol and a hydrophilic poly(ethyleneglycol) (PEG) block (Table 1), therefore functioning as a steric stabilization agent. Both surfactants were characterized with 1H-NMR spectroscopy ensuring their purity before use (see Figures S1 and S2). Table 1. Chemical structures of SDS and Lutensol AT50. surfactant

structure

SDS

O

Lut

x O

50

H

x = 15 - 17

The nanoparticles were prepared according to previously published procedures.19,

20

To

compare different surfactant coverages of the nanoparticle surfaces, an aliquot of each sample was thoroughly purified (details see Supplementary Information). However, the usual purification time was extended to ensure that the minimal surfactant concentration required for stabilized particles was obtained (surfactant levels below detection limit). As a control, the


6

Page 7 of 21

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

Biomacromolecules

surface tension of the air-water interface was measured and compared to the value for pure water (72.75 mN·m-1).21 For the SDS-stabilized nanoparticles a surface tension of 72.79 ± 0.10 mN·m-1 was obtained. The highest surface tension for Lut-stabilized particles was 54.01 ± 0.10 mN·m-1. Removing additional surfactant resulted in aggregate formation, indicating that the concentration of Lut was too low to stabilize all particles. Those highly purified samples (Figure 1A) were taken as reference samples because of minimal surfactant remaining and were thereby considered ‘surfactant-free’. To determine the maximum surfactant loading capacity of the two nanoparticle batches isothermal titration calorimetry experiments were performed. In those experiments the SDS-prestabilized NPs were titrated with SDS and Lut-prestabilized NPs titrated with Lut until no further binding was detected. The obtained binding isotherms are shown in Figure S3 together with an independent binding fit and the resulting adsorption parameters in Table S1. We found that in both cases the adsorption process was driven by an entropy gain which points to the fact that hydrophobic interactions between the particle surface and the hydrophobic surfactant parts play a key role since water molecules from the hydration shells are released. This is in good agreement with previous studies on the interaction of polystyrene surfaces with surfactant molecules.22-24 The maximum number of surfactant molecules on one particle was determined to be 10,514 ± 3,111 for SDS and 19,366 ± 1,620 for Lut. Compared to the theoretically possible maximum coverage numbers (61,300 for SDS assuming an area of 0.62 nm2 per molecule25 and 265,500 for Lut assuming an area of 0.80 nm2 per molecule26) those values are very small, indicating that already a fraction of the surface covered by surfactants is enough to sufficiently stabilize the nanoparticles. The numbers obtained from ITC were set as 100% nanoparticle surface coverage and used as a reference for further sample preparation. For both nanoparticle batches three additional samples were then prepared by adding defined amounts of each


7

Biomacromolecules

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

Page 8 of 21

surfactant to the corresponding surfactant-free samples (Figure 1B). The amounts added were chosen to cover the range of concentrations between the purified and unpurified nanoparticles.

Figure 1. Characterization of nanoparticle samples A) Scanning electron microscopy (SEM) images of SDS (top) and Lut (bottom) stabilized samples after purification. B) Sample preparation scheme for nanoparticles with different surfactant concentrations. All samples were characterized with regard their surface tension (Table 2, Figure S4) to ensure that different surface coverages of surfactant were obtained. The differences in surface tension of the air-water interface of the samples indicate that indeed different surfactant concentrations are present. Additionally, the same amounts of surfactant were dissolved in water and compared to the nanoparticle samples with added surfactant (Figure S4). It can clearly be seen that the


8

Page 9 of 21

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

Biomacromolecules

addition of both SDS and Lut into pure water results in a much lower surface tension. Therefore, we can be sure that most of the surfactant added in the nanoparticle samples indeed coats the nanoparticle surface. This is further confirmed by zeta potential measurements (Table 2). While those measurements are not suitable to conclude information about the surface coverage for Lut due its nonionic nature, the increasing coverages with SDS are manifested in a decreasing zeta potential. Subsequently, all samples were characterized with regard to size and size distribution by angle dependent dynamic light scattering (DLS) to ensure that neither was influenced by surfactant content. Obtained hydrodynamic sizes are displayed in Table 2 and size distributions are shown in Figures S5-S6. The unpurified Lut-stabilized NPs exhibit a lower mean Rh as all other Lut samples. This is a result of the excessive centrifugation purification steps where most likely some of the smaller particles stayed in solution and were discarded.

Table 2. Characterization of nanoparticle samples. sample

PS (SDS)

PS (Lut)

surface tension zeta / mN·m-1 potential mV

Rh / nm /

surfactant molecules particle[a]

surface per coverage with surfactant[b] / % min[c]

purified

72.79 ± 0.10

-20 ± 4

59 ± 6

0.04 mM

72.25 ± 0.05

-25 ± 2

55 ± 6

4,500

43

0.08 mM

71.78 ± 0.18

-28 ± 5

55 ± 6

9,001

86

0.13 mM

70.37 ± 0.18

-31 ± 2

60 ± 6

15,077

143

Unpurified (0.13 mM)

67.70 ± 0.03

-39 ± 4

61 ± 6

15,077

143

purified

54.01 ± 0.11

-4 ± 1

134 ± 13

0.01 mM

54.00 ± 0.10

-2 ± 1

122 ± 12

5810

30

0.02 mM

53.73 ± 0.15

-5 ± 1

128 ± 13

11,620

60

0.03 mM

52.88 ± 0.08

-4 ± 1

140 ± 14

17,429

90

min[c]


9

Biomacromolecules

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

unpurified (0.05 mM)

52.94 ± 0.10

2±1

86 ± 9

Page 10 of 21

29,824

154

[a] corresponding to amounts added after purification. [b] relative to the maximum coverage as determined by ITC. [c] below detection limit or not possible to determine.

To evaluate the influence of both surfactant type and concentration on nanoparticle interaction with plasma proteins, human serum albumin (HSA) was chosen as for binding studies. HSA is the most abundant protein in blood plasma27 and thus a suitable model protein. Since our study focused on the physico-chemical characterization, isothermal titration calorimetry (ITC) was used to determine the binding affinity, stoichiometry and binding enthalpy of HSA-nanoparticle interactions. Each nanoparticle sample was titrated with an aqueous solution of HSA (raw data see Figures S7-S8) and the resulting heats (Figure 2) were analyzed with an independent binding model (Equation S1). The obtained binding parameters for the different surfactant types and concentrations are summarized in Table 3 and Figure 3. The pure surfactants were each titrated with HSA for reference (Figure S9).


10

Page 11 of 21

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

Biomacromolecules

Figure 2. Isothermal titration calorimetry (ITC) binding isotherms obtained from the titration of all nanoparticle samples with HSA: A) SDS stabilized, B) Lut stabilized. The graphs display the integrated heats of each titration step minus the heat of dilution (symbols) with the corresponding independent binding model fit (straight lines).

Table 3. Parameters obtained from fits of ITC measurements of PS-NP / surfactants and HSA according to an independent binding model. Ka / 105 L mol-1

N

∆H / kJ mol-1

∆S / J K-1 mol-1

purified

2.4 ± 0.8

1,358 ± 135

-192 ± 45

-540 ± 151

0.04 mM

2.1 ± 0.1

2,180 ± 49

-225 ± 6

-648 ± 26

0.08 mM

1.8 ± 0.2

2,367 ± 301

-273 ± 45

-814 ± 153

0.13 mM

1.2 ± 0.2

3,431 ± 286

-283 ± 25

-851 ± 85

Unpurified (0.13 mM)

0.7 ± 0.1

3,556 ± 312

-406 ± 5

-1,269 ± 15

1.1 ± 0.3

0.12 ± 0.01

-291 ± 60

-880 ± 207

purified

0.6 ± 0.2

7,898 ± 1,189

-199 ± 54

-487 ± 74

0.01 mM

1.0 ± 0.1

7,657 ± 801

-228 ± 23

-668 ± 79

0.02 mM

1.7 ± 0.1

8,484 ± 546

-164 ± 15

-451 ± 52

0.03 mM

1.8 ± 0.3

8,599 ± 1,072

-177 ± 36

-493 ± 122

unpurified (0.05 mM)

1.9 ± 0.1

2,698 ± 226

-149 ± 14

-400 ± 48

20.6 ± 9.0

0.21 ± 0.01

-150 ± 16

-386 ± 59

sample PS (SDS)

SDS PS (Lutensol)

Lutensol

Errors represent the mean standard deviation of values obtained from 3 individual experiments


11

Biomacromolecules

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

Page 12 of 21

Figure 3. Illustration of observed trends for binding parameters dependency on surfactant concentration: A) binding affinity trends, B) adsorption enthalpy and stoichiometry trends. Our results indicate that both the SDS and Lut concentrations significantly influence the binding parameters for the nanoparticles studied. Interestingly, the trends differ between SDSand Lut-stabilized nanoparticles. The SDS-stabilized particles exhibit binding parameters between the ‘surfactant-free’ and the unpurified samples. This is expected since the unpurified nanoparticles contain the highest SDS concentration as obtained from the synthesis (higher than maximum surface coverage as determined from ITC). The binding enthalpy and number of interacting proteins per particle increase with increasing SDS content. In contrast, the calculated binding affinity decreases and approaches the value for pure SDS binding to HSA. This finding indicates that, though the particle surface is not completely covered, SDS alters the observed


12

Page 13 of 21

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

Biomacromolecules

interaction with HSA and significantly contributes to the adsorption behavior, resulting in adsorption parameters quite different from the initial surface material (PS) parameters. Prior research reports that, in an initial step, about 10 SDS molecules bind to positively charged amino groups of the HSA lysine residues.28, 29 This corroborates the stoichiometry of 8-9 SDS per HSA obtained via ITC. Most likely, the SDS-HSA interaction at the nanoparticle surface also consists of electrostatic interactions corresponding to the binding process described. This is underlined by the fact that the adsorption is definitely driven by enthalpic interactions since a significant entropy loss is observed in the ITC measurements. Thus, hydrophobic interactions cannot be the driving force of the protein adsorption because they generally result in an entropy gain due to water molecules being released from the hydration shells.30 As a conclusion we suggest that electrostatic interactions and hydrogen bonding are involved in the interaction with SDS on the nanoparticle surface rather than hydrophobic interactions. As a consequence it is unlikely that SDS molecules actually detach from the nanoparticle surface upon protein adsorption since that would most probably involve hydrophobic interactions between surfactant tail and pockets of the protein and the order of the system would be decreased. However that cannot be the only reason for such high entropy losses. Another process that probably also takes place is the interaction of albumin with the small amounts of free surfactant in solution since the coverage with surfactants is also an equilibrium. These surfactant molecules will also attach to the protein surfaces via electrostatic interactions first (as mentioned above) and cover the sides of the protein that are not in contact with the particle surface. Thus, the order in the system is further increased. Also, the interaction of pure SDS with albumin involves a rather high loss of entropy (see Table 3). This effect should be more pronounced for SDS since its solubility in water is much higher than the one of Lutensol and the entropy loss for the interaction with albumin is twice as high. Our most


13

Biomacromolecules

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

Page 14 of 21

important finding, though, is that a change in the number of SDS molecules per particle leads to significantly different protein binding affinities. With less SDS present the protein molecules are able to interact directly with the particle surface resulting in hydrophobic interactions with a higher affinity. The Lut-stabilized nanoparticles present a different protein binding behavior. While the HSA adsorption process is still exothermic the adsorption enthalpy and stoichiometry remain constant within the error. N is smaller for the unpurified NPs since the particles themselves are smaller in average so that the surface area available for adsorption is also smaller. In contrast to the SDSstabilized samples, Ka increases over a small range (around 2 fold) with higher Lut concentrations. With an excess of Lut present (more than maximum coverage) it seems that the binding affinity reaches a saturation point. Even though there is free Lut present, the binding parameters are significantly different then what is observed for pure Lut interacting with HSA. This suggests that as mentioned above, that the main driving forces include polar interactions resulting in an enthalpy driven process as well as probably the attachment of free Lutensol molecules to the protein surface. Interestingly, the entropy loss during the adsorption process remains independent of the Lut concentration, meaning that even at low coverage the long hydrophilic chains of Lut prevent direct hydrophobic interactions with the polystyrene surface. Additionally, it suggests that the Lutensol concentration in solution does not change significantly. As previously reported, the affinity of HSA towards Lut (low CMC of < 0.01 mM31) is around 50 times higher than towards SDS (high CMC of 8.6 mM31).32 It is known that PEG chains attached to a surface increase the blood circulation half-life of nanocarriers by reducing unspecific protein interactions.33 In our study, however, the existence of a PEG containing polymer actually promoted HSA interaction. The binding affinity of Lut to HSA is


14

Page 15 of 21

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

Biomacromolecules

around 5 times higher than the reported affinity for isolated PEG to HSA.34 Unlike pure PEG, Lut contains a hydrophobic alkyl chain, and may therefore prefer binding to hydrophobic pockets of HSA. This could explain the high binding affinity of pure Lut to HSA, since hydrophobic interactions usually result in much higher affinities than electrostatic interactions. However, this high affinity was not observed for the adsorption of HSA on Lut-coated NPs, even for the highest surface coverage and although a very small fraction of free Lut is present in every sample (due to the surfactant coating being an equilibrium). That means that probably the amount of free Lut is so small that the interaction of free Lut with HSA is not detected and that it does not contribute significantly to the observed adsorption isotherms. Conversely, this also implies that no significant amounts of Lut detaching from the particle surface are detected in those measurements. However, the calorimetric measurements do not provide a deep insight into simultaneously occurring processes, so that the question about the fate of the surfactant after protein interaction remains. There are three possible scenarios: i) formation of a protein-surfactant-particlecomplex, ii) formation of a protein-surfactant-complex detached from the particle and iii) formation of a protein-particle-complex where the surfactant is released into solution. To address this point, we studied the sample composition after ITC measurements with dynamic light scattering (DLS). The measurements suggest that minor aggregation occurs in Lut-stabilized samples after interaction with HSA, while SDS-stabilized particles do not form any larger structures (Figures S10-S11). In contrast, both pure surfactants form macroscopic aggregates when exposed to HSA. This gives some hints that Lut forms dissolved complexes with HSA after protein adsorption or the adsorbed HSA leads to bridging between smaller particles. Together with the ITC results this suggests that if the observed aggregates originated from Lut-


15

Biomacromolecules

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

Page 16 of 21

HSA complexes, the involved Lut molecules would have already been present in solution in very low concentrations. However, it is very difficult to determine the origin of the aggregation and therefore further investigations are required in the future to elucidate the fate of the surfactant in such systems.

Conclusions In conclusion, the type and amount of available groups on particle surfaces have significant effects on the protein binding properties of polystyrene model nanoparticles. Even small amounts of stabilizing agents remaining can have adverse effects on albumin adsorption and lead to different processes. It was not clear, until now, that small variations in the number of ‘functional’ groups on the particle surface change the protein binding affinities. We have observed that at high concentrations the surfactant determines the adsorption process, not the particle material. This is of great importance for both surfactant-stabilized nanomaterials and covalently attached functional surface groups, since the influence of surfactants on nanoparticle interactions with the environment is often considered to be negligible after extensive purification procedures. The answer to the remaining question, what happens to the surfactant during or after protein adsorption, is not fully clear and will be addressed in future studies.

ASSOCIATED CONTENT Supporting Information. Details on nanoparticle synthesis and purification, ITC data analysis, DLS sample preparation and measurement details, NMR data of surfactants, surface tension measurements, DLS data, and additional ITC data. This material is available free of charge via the Internet at http://pubs.acs.org.


16

Page 17 of 21

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

Biomacromolecules

AUTHOR INFORMATION Corresponding Author *Email: [email protected] Present Addresses † Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, IL 602083113, USA Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT Funding was provided by the DFG (Deutsche Forschungsgemeinschaft) within the collaborative research center SFB. J.S. thanks the DAAD (German Academic Exchange Service) for financial support. We thank A. Schoth for help with nanoparticle synthesis. E. Muth and G. Glasser are thanked for help with surface tension measurements and electron microscopy respectively.

ABBREVIATIONS HSA, human serum albumin; PS-NPs, polystyrene nanoparticles; SDS, sodium dodecyl sulfate; Lut, Lutensol AT50; ITC, isothermal titration calorimetry; DLS, dynamic light scattering. REFERENCES


17

Biomacromolecules

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

1.

Page 18 of 21

Brigger, I.; Dubernet, C.; Couvreur, P., Nanoparticles in cancer therapy and diagnosis. Adv. Drug Del. Rev. 2002, 54, (5), 631-651.

2.

Moghimi, S. M.; Hunter, A. C.; Murray, J. C., Long-Circulating and Target-Specific Nanoparticles: Theory to Practice. Pharmacol. Rev. 2001, 53, (2), 283-318.

3.

Duncan, R., Polymer conjugates as anticancer nanomedicines. Nature Rev. Cancer 2006, 6, (9), 688-701.

4.

Azzazy, H. M. E.; Mansour, M. M. H., In vitro diagnostic prospects of nanoparticles. Clin. Chim. Acta 2009, 403, (1–2), 1-8.

5.

Torchilin, V. P., Multifunctional, stimuli-sensitive nanoparticulate systems for drug delivery. Nature Rev. Drug Discov. 2014, 13, (11), 813-827.

6.

Zhang, M.; Kataoka, K., Nano-structured composites based on calcium phosphate for cellular delivery of therapeutic and diagnostic agents. Nano Today 2009, 4, (6), 508-517.

7.

Vogler, E. A., Protein adsorption in three dimensions. Biomaterials 2012, 33, (5), 12011237.

8.

Salvati, A.; Pitek, A. S.; Monopoli, M. P.; Prapainop, K.; Bombelli, F. B.; Hristov, D. R.; Kelly, P. M.; Aberg, C.; Mahon, E.; Dawson, K. A., Transferrin-functionalized nanoparticles lose their targeting capabilities when a biomolecule corona adsorbs on the surface. Nature Nanotech. 2013, 8, (2), 137-143.

9.

Tenzer, S.; Docter, D.; Kuharev, J.; Musyanovych, A.; Fetz, V.; Hecht, R.; Schlenk, F.; Fischer, D.; Kiouptsi, K.; Reinhardt, C.; Landfester, K.; Schild, H.; Maskos, M.; Knauer, S. K.; Stauber, R. H., Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology. Nature Nanotech. 2013, 8, (10), 772-781.

10.

Monopoli, M. P.; Aberg, C.; Salvati, A.; Dawson, K. A., Biomolecular coronas provide the biological identity of nanosized materials. Nat Nano 2012, 7, (12), 779-786.

11.

Cedervall, T.; Lynch, I.; Foy, M.; Berggård, T.; Donnelly, S. C.; Cagney, G.; Linse, S.; Dawson, K. A., Detailed Identification of Plasma Proteins Adsorbed on Copolymer Nanoparticles. Angew. Chem. Int. Ed. 2007, 46, (30), 5754-5756.

12.

Cedervall, T.; Lynch, I.; Lindman, S.; Berggård, T.; Thulin, E.; Nilsson, H.; Dawson, K. A.; Linse, S., Understanding the nanoparticle–protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles. Proc. Nat. Acad. Sci. 2007, 104, (7), 2050-2055.


18

Page 19 of 21

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

Biomacromolecules

13.

Gessner, A.; Lieske, A.; Paulke, B.-R.; Müller, R. H., Functional groups on polystyrene model nanoparticles: Influence on protein adsorption. J. Biomed. Mater. Res. Part A 2003, 65A, (3), 319-326.

14.

Lück, M.; Paulke, B. R.; Schröder, W.; Blunk, T.; Müller, R. H., Analysis of plasma protein adsorption on polymeric nanoparticles with different surface characteristics. J. Biomed. Mater. Res. 1998, 39, (3), 478-485.

15.

Laemmli, U. K., Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature 1970, 227, (5259), 680-685.

16.

Maiolo, D.; Bergese, P.; Mahon, E.; Dawson, K. A.; Monopoli, M. P., Surfactant Titration of Nanoparticle–Protein Corona. Anal. Chem. 2014.

17.

Podila, R.; Chen, R.; Ke, P. C.; Brown, J. M.; Rao, A. M., Effects of surface functional groups on the formation of nanoparticle-protein corona. Appl.Phys. Lett. 2012, 101, (26), 263701.

18.

Jedlovszky-Hajdú, A.; Bombelli, F. B.; Monopoli, M. P.; Tombácz, E.; Dawson, K. A., Surface Coatings Shape the Protein Corona of SPIONs with Relevance to Their Application in Vivo. Langmuir 2012, 28, (42), 14983-14991.

19.

Landfester, K.; Bechthold, N.; Tiarks, F.; Antonietti, M., Formulation and Stability Mechanisms of Polymerizable Miniemulsions. Macromolecules 1999, 32, (16), 52225228.

20.

Landfester, K.; Bechthold, N.; Tiarks, F.; Antonietti, M., Miniemulsion Polymerization with Cationic and Nonionic Surfactants: A Very Efficient Use of Surfactants for Heterophase Polymerization. Macromolecules 1999, 32, (8), 2679-2683.

21.

Vargaftik, N. B.; Volkov, B. N.; Voljak, L. D., International Tables of the Surface Tension of Water. J. Phys. Chem. Ref. Data 1983, 12, (3), 817-820.

22.

Wesemeyer, H.; Müller, B. W.; Müller, R. H., Adsorption of ethoxylated surfactants on nanoparticles. II. Determination of adsorption enthalpy by microcalorimetry. Int. J. Pharm. 1993, 89, (1), 33-40.

23.

Winnik, M. A.; Bystryak, S. M.; Odrobina, E., Interaction of PBMA Latex Particles with Nonionic Surfactants in Aqueous Solution. Langmuir 2000, 16, (15), 6118-6130.

24.

Stolnik, S.; Heald, C. R.; Garnett, M. G.; Illum, L.; Davis, S. S., Differences in the adsorption behaviour of poly(ethylene oxide) copolymers onto model polystyrene


19

Biomacromolecules

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

Page 20 of 21

nanoparticles assessed by isothermal titration microcalorimetry correspond to the biological differences. J. Drug Targeting 2005, 13, (8-9), 449-458. 25.

Myers, D., Surfaces, interfaces and colloids. Wiley-Vch New York etc.: 1990.

26.

Walkey, C. D.; Olsen, J. B.; Guo, H.; Emili, A.; Chan, W. C. W., Nanoparticle Size and Surface Chemistry Determine Serum Protein Adsorption and Macrophage Uptake. J. Am. Chem. Soc. 2012, 134, (4), 2139-2147.

27.

Le, T.; Bhushan, V.; Rao, D. A., First Aid for the USMLE Step 1: 2008. McGraw Hill Professional: 2007.

28.

Markus, G.; Karush, F., Structural Effects of the Interaction of Human Serum Albumin with Sodium Decyl Sulfate1. J. Am. Chem. Soc. 1957, 79, (12), 3264-3269.

29.

Nielsen, A. D.; Borch, K.; Westh, P., Thermochemistry of the specific binding of C12 surfactants to bovine serum albumin. BBA - Protein Struct. M. 2000, 1479, (1–2), 321331.

30.

Lu, D. R.; Lee, S. J.; Park, K., Calculation of solvation interaction energies for protein adsorption on polymer surfaces. J. Biomater. Sci., Polymer Edition 1992, 3, (2), 127-147.

31.

Hecht, L. L.; Schoth, A.; Muñoz-Espí, R.; Javadi, A.; Köhler, K.; Miller, R.; Landfester, K.; Schuchmann, H. P., Determination of the Ideal Surfactant Concentration in Miniemulsion Polymerization. Macromol. Chem. Phys. 2013, 214, (7), 812-823.

32.

Valstar, A.; Almgren, M.; Brown, W.; Vasilescu, M., The Interaction of Bovine Serum Albumin with Surfactants Studied by Light Scattering. Langmuir 2000, 16, (3), 922-927.

33.

Owens Iii, D. E.; Peppas, N. A., Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles. Int. J. Pharm. 2006, 307, (1), 93-102.

34.

Ragi, C.; Sedaghat-Herati, M. R.; Ouameur, A. A.; Tajmir-Riahi, H. A., The effects of poly(ethylene glycol) on the solution structure of human serum albumin. Biopolymers 2005, 78, (5), 231-236.


20

Page 21 of 21

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

Biomacromolecules

Small Surfactant Concentration Differences Influence Adsorption of Human Serum Albumin on Polystyrene Nanoparticles Svenja Winzen, James C. Schwabacher,† Julius Müller, Katharina Landfester and Kristin Mohr* Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany

For Table of Contents only:


Small Surfactant Concentration Differences Influence Adsorption of

Recommend Documents