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Complexation of Lysozyme with Adsorbed PtBS-bSCPI Block Polyelectrolyte Micelles on Silver Surface Aristeidis Papagiannopoulos, Anastasia Christoulaki, Nikolaos Spiliopoulos, Alexandros A Vradis, Chris Toprakcioglu, and Stergios Pispas Langmuir, Just Accepted Manuscript • DOI: 10.1021/la504873h • Publication Date (Web): 19 Dec 2014 Downloaded from http://pubs.acs.org on December 24, 2014
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Complexation of Lysozyme with Adsorbed PtBS-b-SCPI Block Polyelectrolyte Micelles on Silver Surface Aristeidis Papagiannopoulos1*, Anastasia Christoulaki2, Nikolaos Spiliopoulos2, Alexandros Vradis2, Chris Toprakcioglu2, Stergios Pispas1* 1
Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, 48 Vassileos
Constantinou Avenue, 11635 Athens, Greece 2
Department of Physics, University of Patras, GR 26 500, Greece
*
[email protected] *
[email protected] ABSTRACT We present a study of the interaction of the positively charged model protein lysozyme with the negatively charged
amphiphilic
diblock
polyelectrolyte
micelles
of
poly(tert-butylstyrene)-b-sodium
(sulfamate/carboxylate)isoprene) (PtBS-b-SCPI) on the silver/water interface. The adsorption kinetics are monitored by surface plasmon resonance and the surface morphology is probed by atomic force microscopy. The micellar adsorption is described by stretched-exponential kinetics and the micellar layer morphology shows that the micelles do not lose their integrity upon adsorption. The complexation of lysozyme with the adsorbed micellar layers depends on the micelles arrangement and density in the underlying layer and lysozyme follows the local morphology of the underlying roughness. When the micellar adsorbed amount is small, the layers show low capacity in protein complexation and low resistance in loading. When the micellar adsorbed amount is high the situation is reversed. The adsorbed layers both with or without added protein are found to be irreversibly adsorbed on the Ag surface. INTRODUCTION Adsorption of synthetic and biological macromolecules on surfaces and interfaces is a field of wide interest due to its variety of applications1 and its great impact on the physicochemical properties2 of the newly created interface i.e. biocompatibility, stimuli-responsiveness and stability3. Physical adsorption of polyelectrolytes on
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flat surfaces can be achieved by a variety of driving forces. These can act individually or in combination and may result from electrostatic interactions between the adsorbed macromolecules and the surface and/or hydrophobic effects4. Driving forces of this kind may also induce self-assembly in solution with extremely attractive structures, as for example micelles of diblock polyelectrolytes where the hydrophobic parts of the diblock chains aggregate to form a dense hydrophobic core whereas the hydrophilic polyelectrolyte blocks extend in solution to form a highly hydrated corona5. Polyelectrolyte micelles containing both hydrophobic and charged groups may interact with surface groups to form physical bonds. An adsorbed polyelectrolyte micelle may keep its solution structure and integrity upon adsorption so that a rough layer forms6, when the hydrophobic blocks in its core are in a glassy state and they are kinetically frozen. So even if it is energetically favorable for them to release their mutual contacts and create new ones with the surface they remain inside the micelle’s core. In the case of a core in a liquid state, the micellar structure may dissolve, so that the cores spread out on the surface and the hydrophilic blocks extend away from the surface to form a flat layer 7. Proteins interact with polyelectrolytes, both in solution and on surfaces8 mainly due to electrostatic interactions. This kind of interactions opens a field for research towards applications in engineering and medicine, including encapsulation and delivery of drugs, proteins and DNA
9, 10
with oppositely charged macromolecular chains belonging to spherical micelles
. Proteins normally complex 11
in solution, which affects
critically the chain conformation even causing intermicellar aggregations12. Similarly, on a planar surface coated with a polyelectrolyte layer, incorporation of protein globules is expected to cause conformational changes upon the adsorbed chains13. Surface Plasmon Resonance (SPR) is a high sensitivity tool for surface studies and it has been widely utilized for biosensing applications14. SPR has also been successfully used for adsorption studies of polyelectrolytes15 and proteins16. In this article we present the adsorption kinetics of poly(tert-butylstyrene)-b-sodium (sulfamate/carboxylate)isoprene) (PtBS-b-SCPI) micelles on the silver/water interface and the interactions of the formed layers with the oppositely charged lysozyme. PtBS-b-SCPI block polyelectrolyte is a novel diblock 2 ACS Paragon Plus Environment
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copolymer that combines a hydrophobic block, with a polyelectrolyte block that contains one weak and one strong charge group and which at the same time has an intrinsically hydrophobic backbone. We have studied the formation of micelles by PtBS-b-SCPI and its complexation with lysozyme in solution12 in the past. In this work we continue to study the behavior of this system on surfaces. The morphology of the adsorbed layers is investigated by Atomic Force Microscopy (AFM). We are primarily interested in understanding the physicochemical and self-organization processes taking place in such synthetic/biological hybrid nanosystems. MATERIALS AND METHODS Materials The diblock polyelectrolyte poly-(tert-butylstyrene-b-sodium (sulfamate/carboxylate) isoprene), PtBS-b-SCPI, was synthesized by anionic polymerization high vacuum techniques and block selective post-polymerization functionalization reactions17, 18. The resulting Mw is = 164 · 10 / (I=1.03) and the respective weight contents are 12% for PtBS and 88% for SCPI. PtBS is a hydrophobic polymer with high glass transition temperature and SCPI is a highly charged polyelectrolyte that contains both strong ( ) and weak ( ) charged groups neutralized by Na. At pH7 both groups are dissociated. The PI backbone contains 75% of chargeable and 25% of hydrophobic units in a random sequence. In aqueous solution, PtBS-b-SCPI forms micelles with a core of hydrophobic PtBS blocks and a corona of hydrophilic negatively charged SCPI blocks. A diblock polyelectrolyte solution (1 mg/ml) was prepared in a pH7 0.01M (NaCl) aqueous solution (the same conditions were used to prepare lysozyme solutions) by heating at 60 °C overnight. The pH of the deionized water used was 5.5-6 and hence NaOH was added to the solutions to raise pH to 7. This stock solution was used to prepare solutions of the preferred lower concentrations by diluting with water at pH7 and 0.01M NaCl. The hydrophobic PtBS blocks form a solid “frozen” core (because of the strong hydrophobic nature of PtBS and its high19 Tg≈130°C), which is surrounded by the water-soluble hydrophilic polyelectrolyte SCPI blocks. The PtBS-b-SCPI diblock copolymer in aqueous solutions has been studied thoroughly 12 in the past and welldefined core-shell micelles were found to form in aqueous solutions of similar conditions. The hydrodynamic
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radius of the micelles was = 100 and the molecular weight = 1.6 · 10 / , which corresponds to about 10 diblock copolymer chains per micelle. Lysozyme (HEWL) with molecular weight = 14.7 · 10 / was purchased from Fluka and used without further purification. HEWL was dissolved at 0.1 mg/ml (at pH7 and 0.01M NaCl) and left overnight to equilibrate. The target concentrations were produced from the parent solution by dilution in water with pH7 and 0.01M NaCl. Strong electrostatic attractions between the polyelectrolyte micelles and the proteins are expected to occur on the surface as it has been proved to happen in solution12. Surface Plasmon Resonance Experiments The Kretschmann20 configuration was realized by silver (Ag) films formed on the optically flat face of SF10 equilateral prisms (nSF10=1.723). The glass films were cleaned by exposure to fresh mixed nitric and hydrochloric acid (1:3 by volume) and then rinsed with water and further washed with ethanol. The silver layers (48-52nm) were deposited on the glass surface by thermal evaporation of 99.999% pure silver wire at a base pressure of 1·10−6Torr and at a deposition rate of 0.15 nm/s. The prism remains in vacuum for at least half an hour before being removed from the evaporation chamber. The light source used for SPR was a He-Ne laser beam (λ=632.8 nm). A polarizer is used for the p-polarization of the beam since surface plasmons are excited only by the p-component of electromagnetic waves, parallel to the metal surface21. The solutions are loaded in a PTFE cell which is sealed on the metal-deposited surface of the film. The temperature of the solution is monitored by a Teflon-coated thermocouple immersed into the solution. The experiments were performed at room temperature. Surface Plasmon Resonance Data Analysis The dispersion relation of a surface plasmon formed on a thin metal film is influenced by both the characteristics of the film (thickness and dielectric constant) and the characteristics of any substances in the vicinity of the film. Consequently, any adsorbed layer will cause the reflectivity vs. angle of incidence curve to change from its original form in the absence of adsorption
21
. In more detail, a system of layers with
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different dielectric constants ( , , … , ) can be described22 by an equal number of characteristic matrices (equation 1). " #$%& ' ( ) " #* +
-
− . #0$%& ' ( ) " #* + / =! 2 for = 1, … , (1) −01 #0$%& ' ( ) " #* + " #$%& ' ( ) " #* + 4
Where 1 = " #* /35/ for = 0, … , + 1 and ) is the magnetic permittivity of layer which is taken /
equal to unity for all the materials used in this study. When = 0 and = + 1 the two semi-infinite
mediums (glass and water respectively) are considered. ' is the thickness of layer . %& is the wave number corresponding to the wavelength (7& ) of the incident light (%& =
8 ). 9:
The angle of incidence (* ) is given by
4
" #* = 31 − 4; #0 *, where * is the angle of incidence on the silver surface. /
Eventually a 2x2 matrix that represents the whole stack of layers is calculated by equation 2.
