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pH responsive adhesion of phospholipid vesicle on poly(acrylic acid) cushion grafted to poly(ethylene terephthalate) surface
Colloids and Surfaces B: Biointerfaces 42 (2005) 245–252
pH responsive adhesion of phospholipid vesicle on poly(acrylic acid) cushion grafted to poly(ethylene terephthalate) surface Ning Fanga , Wee Jin Tanb , Kam W. Leongb,c , Hai-Quan Maob,d , Vincent Chana,∗ a
c
Center of Biotechnology, Division of Chemical and Biomolecular Engineering, Nanyang Technological University, Singapore 639798, Singapore b The Division of Biomedical Sciences, Johns Hopkins in Singapore, Singapore 138669, Singapore Johns Hopkins University School of Medicine, Department of Biomedical Engineering, Baltimore, MD 21205, USA d Johns Hopkins University, Department of Material Sciences and Engineering, Baltimore, MD 21218, USA Received 15 September 2004; accepted 1 November 2004 Available online 28 April 2005
Abstract Polymer-supported lipid bilayer is a key enabling technology for the design and fabrication of novel biomimetic devices. To date, the physical driving force underlying the formation of polymer-supported lipid bilayer remains to be determined. In this study, the interaction between dipalmitoylphosphocholine (DPPC) vesicle and poly(ethylene terephthalate) [PET] surface with or without grafted poly(acrylic acid) [PAA] layer is examined with several biophysical techniques. First, vesicle deformation analysis shows that the geometry of adherent vesicle on either plain PET or PAA-grafted PET surface is best described by a truncated sphere model. At neutral pH, the degree of deformation and adhesion energy are unaltered by the grafted polymerization of acrylic acid on PET surface. Interestingly, the average magnitude of adhesion energy is increased by 185% and −43% on PAA-grated PET and plain PET surface, respectively, towards an increase of pH at room temperature. Our results demonstrate the possibility of tuning the adhesive interaction between vesicle and polymer cushion through the control of polyelectrolyte ionization on the solid support. © 2004 Elsevier B.V. All rights reserved. Keywords: Phospholipid vesicle; Dipalmitoylphosphocholine; Poly(ethylene terephthalate)
1. Introduction Supported lipid bilayer has emerged as the standard experimental model for probing the structure-function relationship of biomembrane [1]. Traditionally, supported lipid bilayer has been prepared by either vesicle fusion or Langmuir–Blodgett deposition on rigid glass and mica substrates. Despite the presence of an ultrathin water layer between the supported lipid bilayer and conventional rigid substrates, the fluidic properties of native biomembrane are significantly suppressed (e.g., lower diffusion coefficient of lipid) [2]. As a result, mica or glass-supported lipid bilayer does not serve as
∗
Corresponding author. Tel.: +65 67906739, fax: +65 63971320. E-mail address: [email protected] (V. Chan).
0927-7765/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2004.11.002
an ideal environment for preserving the biological functions of transmembrane proteins [3]. Recently, polymer layer immobilized on rigid substrates has been applied as a biomimetic support for lipid bilayer [4]. In general, the polymer layer as mentioned above acts as a cushion and aids in preserving the biophysical properties of biomembrane. For example, the two-dimensional diffusion coefficient of phospholipids supported on polymer cushion approaches that in intact cell membrane [5]. On the other hand, little work has been done on the elucidation of the physical driving forces underlying the formation of supported lipid bilayer on polymer cushion. Particularly, the quantitation of vesicle-polymer interaction would allow researchers to engineer the fabrication of supported bilayer through vesicle fusion for applications in protein chip, biosensor and biomaterials [6].
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Poly(acrylic acid) [PAA] is a weak polyelectrolyte and has been recently exploited for potential applications as a solid support in the fabrication of supported lipid bilayer [7]. However, the lack of physical understanding in the interaction of lipid bilayer with immobilized PAA has impaired the design of molecularly designed film with optimized bio-functional characteristics. In this study, the interaction between phospholipid bilayer and poly(acrylic acid) cushion immobilized on poly(ethylene terephthalate) [PET] was thoroughly investigated. First, confocal-reflectance interference contrast microscopy (C-RICM) probes the contact zone (membrane-substrate distance <30 nm) of adherent phospholipid vesicle on the PAA cushion [8]. By simultaneous application of cross-polarization microscopy with C-RICM and contact mechanics modeling, the adhesion energy at membrane-polymer interface was determined. Furthermore, the effect of PAA ionization induced by pH change on the adhesion energy is elucidated. Quantitative analysis of the vesicle adhesion on polymer cushion will lead to the development of tailored polymer-supported lipid bilayer for biosensor and biotechnology applications.
2. Experimental methods 2.1. Materials Amorphous poly(ethylene terephthalate) film was obtained from Goodfellows (UK). Dipalmitoylphosphocholine (DPPC) in powder form was obtained from Avanti Polar Lipids Inc. (USA) and was used as received. Toluidine Blue-O (TBO), acrylic acid, sodium periodate, sodium carbonate, calcium chloride, methanol, acetic acid, Dulbecco’s modified Eagle medium (DMEM), HEPES, HBSS and sodium hydroxide were obtained from Sigma Chemical (Singapore). 18.2 M water was obtained from Maxima water purification system (Elga, USA) and was used in the preparation of all solutions. Monobasic potassium phosphate (KH2 PO4 ); dibasic potassium phosphate (K2 HPO4 ); sodium chloride (NaCl); monobasic sodium phosphate (NaH2 PO4 ); potassium chloride (KCl); dibasic sodium phosphate (Na2 HPO4 ); 1 N hydrochloric acid (HCl) were obtained from Fisher Chemicals (Singapore). 1X phosphate buffer saline (PBS) was prepared with 150 mM sodium chloride, 10 mM sodium phosphate, 50 mM potassium chloride and 80 mM potassium phosphate and was adjusted to pH 7.4 with 1 N hydrochloric acid. Micro BCA protein assay reagent kit was obtained from Pierce Chemical (USA). 2.2. Sample preparations PET disc with a diameter of 22 mm was cleaned with 100% methanol for 10 min in ultrasonic bath. A piece of cleaned PET film was placed in a glass container with 80 mL solution containing 1% (v/v) aqueous acrylic acid and 20 mg of sodium periodate. The grafting reaction was carried out in a
custom built water flow-bath under a 400 W UV light course (Dymax 5000-EC, Dymax corporation). After the reaction, the amount of COOH grafted onto the film was confirmed and characterized by the Toluidine Blue-O assay, in which the dye stains the deprotonated acid groups through ionic interaction. It is assumed that one mole of TBO binds to one mole of COOH (along the PAA chains). In brief, a solution of 5 × 10−4 M TBO was prepared by dissolving TBO powder in sodium hydroxide solution (pH 10). PET disc (diameter: 0.55 cm) grafted with acrylic acid was placed in a 1.5 mLEppendorf tube. One milliliter of TBO solution was added on the PET surface inside the Eppendorf tube and the sample was shaken for 16 h. After TBO binding, the supernatant was removed from Eppendorf Tube and the stained PET surface was washed with NaOH solution (pH 10) following by pure water for two times. One milliliter of 50% acetic acid was added to remove the TBO dye from the carboxyl groups on PAA–PET. Five hundred microliters of the washed solution from each sample was loaded into 96-well-plate and the optical density at 633 nm is measured with a 96-well-plate reader (Molecular Devices, USA). The concentration of the carboxyl groups in acetic acid is determined with a calibration plot containing several samples of acetic acid with different concentrations. Giant unilamellar vesicles (ULV) were synthesized by a well-accepted method [8]. Briefly, 1 mg of DPPC was dissolved in methanol/chloroform co-solvent (2:1 by volume) at a concentration of 1 mg/100 L and the mixture was, subsequently, added on the surface of a roughened Teflon disc. A thin film of DPPC was left on the Teflon surface following the evaporation of solvent and was dried in vacuum for 12 h. Then the Teflon disc was covered with 1X PBS buffer and was hydrated at 50 ◦ C for 16 h. A hydrated layer of lipid film was developed on the disc after a few hours. Finally, opaque suspension of vesicles was dispersed in solution by gentle shaking of the sample. 2.3. Phase contrast microscopy The detailed setup of the instrument has been previously described [9]. DPPC vesicles in 1X PBS (pH 7 or pH 9) were loaded on the plain or PAA grafted PET surfaces. All experiments were carried out at 22 ◦ C in a temperature control chamber (SEC, Korea). Phase contrast images for a representative group of vesicles were taken after 30 min of incubation. All experiments of vesicle adhesion on each PET-based surface were performed in triplicate. An image analysis software, ZSM5 (Carl Zeiss, Germany) was used for measuring the mid-plane diameter of adherent vesicles. 2.4. Confocal-reflection interference contrast microscopy (C-RICM) The instrument is based on a laser scanning confocal microscope and has been described elsewhere in detail [9]. An
N. Fang et al. / Colloids and Surfaces B: Biointerfaces 42 (2005) 245–252
Argon-ion laser with an excitation wavelength of 488 nm was used. A 63× oil immersion objective (Neofluar, N.A.: 1.25) was used for all measurements. The protocols for sample preparation, and image analysis were identical to those described in the previous section. The degree of vesicle deformation is defined as the ratio of contact zone radius (from C-RICM) and mid-plane radius (from cross-polarized light microscopy) of an adherent vesicle [10]. An adherent vesicle has been modeled as a truncated capsule with a thin wall, assumed as a sphere with a planar contact area of radius a. The degree of vesicle deformation is sin θ = (a/R) = α, where R is the mid-plane radius. The adhesion energy of the biomembrane under a uniform equi-biaxial stress, σ = Cε is shown as W = (1 − cosθ)Cε + Cε2
(1)
where C is equivalent to Eh/(1–υ) in a linear system under small strain where E, h and υ the elastic modulus, film thickness and the Poisson’s ratio, respectively [10]. Based on the experimental measurements of the mid-plane diameter, R (cross-polarized light microscopy) and the radius of contact zone, a (C-RICM); W is found by Eq. (1).
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3. Results and discussions Poly(acrylic acid) immobilized on poly(ethylene terephthalate) surface is a potential solid support for phospholipid bilayer due to the proven affinity of PAA with biomembrane [11]. However, the contact mechanics of phospholipid vesicles on a polymer substrate like PET has not been previously interrogated due to the lack of experimental technique for visualizing the membrane-polymer interface. PET can be chemically functionalized with UV radiation and enables the subsequent grafting of PAA chains [12]. In order to probe PAA-vesicle interaction accurately, the biomechanical responses of phospholipid vesicles upon adhesion on a plain PET surface in the absence of poly(acrylic acid) layer must be determined. Fig. 1 shows the cross-polarized light image (i) and C-RICM image (ii) of a representative DPPC vesicle adhering on plain PET surface at 23 ◦ C in 1X PBS solution. Cross-polarized light image shows that the vesicle has a mid-plane diameter of 16.1 m. In parallel, C-RICM image demonstrates the formation of adhesion contact (cross on Fig. 1A (i)) with an area of 14.5 m2 along the centroid of the vesicle. It is critical to show that our contact mechanics model is applicable to phospholipid vesicle adhering on a polymeric material surface. The light intensity profile of
Fig. 1. (A) The cross-polarized light image (i) and C-RICM image (ii) of a representative DPPC vesicle on a plain PET surface at 23 ◦ C in 1X PBS solution. (B) The vesicle-substrate separation profile of the adherent DPPC vesicle on plain PET.
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interference fringe pattern propagating (red line on Fig. 1A (ii)) can be converted into membrane-substrate separation distance through the inverse cosine transformation [13]. Fig. 1B shows the experimental vesicle-substrate separation profile (dotted line) of the adherent DPPC vesicle on plain PET. Based on the measured adhesion contact and mid-plane diameter, the cell-substrate separation profile of the adherent vesicle is simulated with Eq. (1) (solid line on Fig. 1B). The agreement between the experimental membrane-substrate separation profile and the simulated profile as described above indicates that the geometry of the adherent vesicle on plain PET surface approaches that of a truncated sphere [14]. The coupling of PAA cushion on plain PET surface may lead to the change of the optical properties of plain PET surface. Thus it is critical to elucidate the applicability of our biophysical approach on the PAA-grafted PET surface. First, the success of PPA grafting on PET surface is independently verified with Toluidine Blue-O test. It is shown that density of acrylic acid on PET is 0.53 nmole/cm2 . Furthermore, AFM topographic image indicates that the root-mean-square roughness of PET is increased from 0.6 to 1.5 nm upon the immobilization of PAA chain. Fig. 2A shows the cross-polarized light image (i) and C-RICM image (ii) of a typical DPPC vesicle bound on the PAA-grafted PET at 23 ◦ C in 1X PBS solution. Cross-polarized light microscopy demonstrates that the mid-plane diameter of the
adherent vesicle is 35 m. C-RICM shows that the contact area of adherent vesicle is 27.2 m2 . It is indicated that the modification of PET surface with PAA chains does not impair the sensitivity of C-RICM for revealing the adhesion contact and interference fringes. Fig. 2B shows the vesicle -substrate separation profile (obtained from C-RICM) and the simulated profile (predicted with a and R) of the DPPC vesicle adhering on PAA–PET surface. The result demonstrates that the geometry of adherent vesicle is correctly modeled as a truncated sphere. Therefore, the PAA coupling has not modulated the adhesion mechanism of DPPC vesicle. The biomechanical responses of DPPC vesicles towards adhesive interaction on the polymeric surfaces can be quantified by the ratio of adhesion contact radius and mid-plane radius. Fig. 3A shows the degree of vesicle deformation (a/R) against vesicle diameter for DPPC vesicles on plain PET and PAA–PET at 23 ◦ C in 1X PBS solution (pH 7). The dotted line represents the best fit of the experimental data. The result indicates that average a/R for vesicle of all sizes on plain PET surface is 0.16. Interestingly, the inclusion of PAA layer on PET surface imposes negligible effect on the average magnitude of a/R as shown by the overlap of two sets of data. The trend as mentioned above implies that the biomechanical response of DPPC vesicles upon adhesion on PET is similar to that on PAA–PET. It is useful to determine the adhesion energy between the vesicle surface and polymer surface. Fig. 3B shows the adhesion energy of DPPC vesicles of various sizes
Fig. 2. (A) The cross-polarized light image (i) and C-RICM image (ii) of a typical DPPC vesicle bound on the PAA-grafted PET at 23 ◦ C in 1X PBS solution. (B) The membrane-substrate separation profile (obtained from C-RICM) and the simulated profile (predicted with a and R) of the vesicle adhering on PAA–PET surface.
N. Fang et al. / Colloids and Surfaces B: Biointerfaces 42 (2005) 245–252
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Fig. 3. (A) The degree of vesicle deformation (a/R) against the vesicle diameter for DPPC vesicles on plain PET and PAA–PET at 23 ◦ C in 1X PBS solution (pH 7) (B) The adhesion energy of DPPC vesicles of various sizes on plain PET and PAA–PET in 1X PBS solution at 22 ◦ C.
on plain PET and PAA–PET in 1X PBS solution at 22 ◦ C. The result indicates that the average adhesion energy for vesicle of all sizes on plain PET is 1 × 10−10 J/m2 . Also, the average adhesion energy remains unchanged upon the modification of plain PET with PAA layer. The trends as mentioned above imply that the interfacial force between vesicle surface and plain PET is not affected by PAA inclusion at pH 7. The surface of plain PET is mainly composed of the ester carbonyl, methylene, and phenyl groups. In general, the ester groups in the polyester chain of plain PET are hydrophilic and polar due to the presence carbonyl oxygen atom and the carbonyl carbon atom [15]. Therefore, the major physical driving force of DPPC bilayer adhesion on plain PET surface is likely originated from the hydrogen bond formation between the amino group on DPPC and the ester carbonyl group of PET. Our hypothesis was supported by the shift of the ester carbonyl stretch upon the interaction of PET film with the primary amine of 3-aminopropyltrimethoxysilane [16]. Under acrylic acid density of 0.53 nmole/cm2 , the hydrophilicity of PAA–PET approaches that of plain PET
as shown by the similar contact angle [15]. Furthermore, the PAA chains grafted on PET surface is partially ionized due to the limited deprotonation of carboxylic acid at pH 7 [17]. The two factors as mentioned above have led to negligible enhancement in adhesion energy on PAA–PET compared to that induced by hydrogen bonding on plain PET at pH 7. It is known that the degree of carboxylic group ionization on PAA is correlated with the change of solution pH. The elucidation of pH effect on the adhesion of DPPC vesicle on plain PET serves as a necessary control experiment for PAAlipid bilayer interaction. Fig. 4A shows a/R against vesicle diameter for DPPC vesicle on plain PET at 22 ◦ C under pH 7 and pH 9 medium. The result indicates that average magnitude of a/R for DPPC vesicles of all sizes is 0.14 and 0.16 at pH 9 and pH 7, respectively. Fig. 4 B shows the adhesion energy of DPPC vesicles on plain PET at 22 ◦ C under pH 7 and pH 9 medium. It is shown that the average magnitude of adhesion energy for vesicles of all sizes is decreased from 1 × 10−10 J/m2 to 3 × 10−11 J/m2 against the increase of solution pH. Overall, the result implicates that the interfacial
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Fig. 4. (A) a/R against vesicle diameter for DPPC vesicle on plain PET at 22 ◦ C under pH 7 and pH 9 medium. (B) The adhesion energy of DPPC vesicles on plain PET at 22 ◦ C under pH 7 and pH 9 medium.
force between DPPC vesicle and plain PET is slightly dampened under slightly basic condition. The headgroup of DPPC lipid composes of both a quaternary amine and phosphate which carries a permanent positive charge and a transitional negative charge, respectively. Under the slightly basic pH used in this study, the phosphate groups of DPPC mostly remain deprotonated and negatively charged while the PET surface without any electrostatic charge is hydrophilic in nature under all pH. Therefore, the slight reduction of adhesion energy induced by pH increase is likely caused by the disruption of hydrogen bonding between DPPC and plain PET. Our result is supported by the fact that the strength of hydrogen bonds between carboxylic acid-functionalized AFM tips and hydrophilic substrate is dependent on the pH of solution [18]. The pH-induced responses of DPPC vesicle adhesion on PAA–PET would shed light on the fundamental properties of polymer cushion-supported bilayer. Fig. 5A shows a/R
against vesicle diameter for DPPC vesicles adhering on PAA–PET at pH 7 and pH 9 under 22 ◦ C. Obviously, the increase from pH 7 to pH 9 leads to the shift up of the a/R vs. 2R curve. Moreover, the average magnitude of a/R for vesicle of all sizes is increased by 18.8% from 0.16 and 0.19 at pH 7 and pH 9, respectively. Fig. 5B shows the adhesion energy against vesicle diameter for adherent DPPC vesicles on PAA–PET at pH 7 and pH 9 under 22 ◦ C. In contrast to the trend observed on plain PET, the average magnitude of adhesion energy for DPPC vesicles of all sizes on PAA–PET is 1 × 10−10 J/m2 and 1.8 × 10−10 J/m2 at pH 7 and pH 9, respectively. The results indicate that adhesion energy is increased by 80% towards pH increase. Interestingly, the modification of plain PET with PAA chain exemplifies the pH induced enhancement of vesicle adhesion on the PET support. The observed effect is brought about by the enhanced electrostatic interaction between the
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Fig. 5. (A) a/R against vesicle diameter for DPPC vesicles adhering on PAA–PET at pH 7 and pH 9 under 22 ◦ C. (B) The adhesion energy against vesicle diameter for adherent DPPC vesicles on PAA–PET at pH 7 and pH 9 under 22 ◦ C.
quaternary amine (positive charge) of DPPC headgroup and the carboxylate ions (negative charge) of PAA. It is because the degree of PAA ionization is increased from 40 to 100% and the degree of deprotonation of phosphate group on DPPC remains constant when pH is increased from 7 to 9 [17]. To the best of our knowledge, it is the first time that the biomechanical response of adherent phospholipid vesicles on a polymer cushion is shown to be correlated with solution pH. Most important, the approach demonstrated herein provides new physical insights into the control of membrane-polymer interaction during supported membrane fabrication.
4. Conclusion Polymer-supported lipid bilayer has emerged as novel platform for biosensor technology and biochemistry research.
In this study, PAA cushion grafted on PET surface has been used as a model system for interrogating the interfacial driving forces involved in the initial stage of DPPC vesicle fusion on polymer cushion. Specifically, the pH induced response of adherent vesicle on PET surface is only obvious in the presence of PAA cushion. Overall, this study demonstrates the possibility of applying our biophysical approach for the design of novel polymer-supported lipid bilayer system through the tuning of adhesive interaction.
Acknowledgements VC and NF were supported by a Tissue Engineering Initiative of Professor Lim Mong King (NTU) and NTU AcRF 00/15. CL, HQM and KWL were partially supported by NIH grant (5P01CA79862-020003) and A*STAR (Singapore).
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References [1] H. Mozsolits, W.G. Thomas, M.I. Aguilar, J. Pept. Sci. 9 (2003) 77–89. [2] R.N. Orth, J. Kameoka, W.R. Zipfel, B. Ilic, W.W. Webb, T.G. Clark, H.G. Craighead, Biophys. J. 85 (2003) 3066–3073. [3] T. Buranda, J. Huang, G.V. Ramarao, L.K. Ista, R.S. Larson, T.L. Ward, L.A. Sklar, G.P. Lopez, Langmuir 19 (2003) 1654–1663. [4] P. Theato, R. Zentel, Langmuir 16 (2000) 1801–1805. [5] M.L. Wagner, L.K. Tamm, Biophys. J. 79 (2000) 1400–1414. [6] M. Halter, Y. Nogata, O. Dannenberger, T. Sasaki, V. Vogel, Langmuir 20 (2004) 2416–2423. [7] P. Vermette, T. Gengenbach, U. Divisekera, P.A. Kambouris, H.J. Griesser, L. Meagher, J. Colloid Interface Sci. 259 (2003) 13–26. [8] V. Chan, K.T. Wan, Langmuir 18 (2002) 3134–3141. [9] J.J. Foo, V. Chan, K.K. Liu, Ann. Biomed. Eng. 31 (2003) 1279–1286.
[10] K.T. Wan, K.K. Liu, Med. Biol. Eng. Comput. 39 (2001) 605–608. [11] C. Ladaviere, C. Tribet, S. Cribier, Langmuir 18 (2002) 7320–7327. [12] J.Y. Zhang, I.W. Boyd, H. Esrom, Surf. Interface Anal. 24 (1996) 718–722. [13] W.J. Parak, J. Domke, M. George, A. Kardinal, M. Radmacher, H.E. Gaub, A.D.G. de Roos, A.P.R. Theuvenet, G. Wiegand, E. Sackmann, J.C. Behrends, Biophys. J. 76 (1999) 1659–1667. [14] N. Elsner, F. Dubreuil, A. Fery, Phys. Rev. E (2004) 69, Art. No. 031802, Part 1. [15] H. Mirzadeh, M. Dadsetan, N. Sharifi-Sanjani, J. Appl. Polym. Sci. 86 (2002) 3191–3196. [16] C.L. Loch, D. Ahn, C. Chen, J. Wang, Z. Chen, Langmuir (2004), ACS ASAP ALERT EDITION. [17] A. Malgat, J.-P. Boisvert, C. Daneault, J. Colloid Interface Sci. 269 (2004) 320–328. [18] D.A. Smith, M.L. Wallwork, J. Zhang, J. Kirkham, C. Robinson, A. Marsh, M. Wong, J. Phys. Chem. B 104 (2000) 8862–8870.
pH responsive adhesion of phospholipid vesicle on poly(acrylic acid) cushion grafted to poly(ethylene terephthalate) surface Ning Fanga , Wee Jin Tanb , Kam W. Leongb,c , Hai-Quan Maob,d , Vincent Chana,∗ a
c
Center of Biotechnology, Division of Chemical and Biomolecular Engineering, Nanyang Technological University, Singapore 639798, Singapore b The Division of Biomedical Sciences, Johns Hopkins in Singapore, Singapore 138669, Singapore Johns Hopkins University School of Medicine, Department of Biomedical Engineering, Baltimore, MD 21205, USA d Johns Hopkins University, Department of Material Sciences and Engineering, Baltimore, MD 21218, USA Received 15 September 2004; accepted 1 November 2004 Available online 28 April 2005
Abstract Polymer-supported lipid bilayer is a key enabling technology for the design and fabrication of novel biomimetic devices. To date, the physical driving force underlying the formation of polymer-supported lipid bilayer remains to be determined. In this study, the interaction between dipalmitoylphosphocholine (DPPC) vesicle and poly(ethylene terephthalate) [PET] surface with or without grafted poly(acrylic acid) [PAA] layer is examined with several biophysical techniques. First, vesicle deformation analysis shows that the geometry of adherent vesicle on either plain PET or PAA-grafted PET surface is best described by a truncated sphere model. At neutral pH, the degree of deformation and adhesion energy are unaltered by the grafted polymerization of acrylic acid on PET surface. Interestingly, the average magnitude of adhesion energy is increased by 185% and −43% on PAA-grated PET and plain PET surface, respectively, towards an increase of pH at room temperature. Our results demonstrate the possibility of tuning the adhesive interaction between vesicle and polymer cushion through the control of polyelectrolyte ionization on the solid support. © 2004 Elsevier B.V. All rights reserved. Keywords: Phospholipid vesicle; Dipalmitoylphosphocholine; Poly(ethylene terephthalate)
1. Introduction Supported lipid bilayer has emerged as the standard experimental model for probing the structure-function relationship of biomembrane [1]. Traditionally, supported lipid bilayer has been prepared by either vesicle fusion or Langmuir–Blodgett deposition on rigid glass and mica substrates. Despite the presence of an ultrathin water layer between the supported lipid bilayer and conventional rigid substrates, the fluidic properties of native biomembrane are significantly suppressed (e.g., lower diffusion coefficient of lipid) [2]. As a result, mica or glass-supported lipid bilayer does not serve as
∗
Corresponding author. Tel.: +65 67906739, fax: +65 63971320. E-mail address: [email protected] (V. Chan).
0927-7765/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2004.11.002
an ideal environment for preserving the biological functions of transmembrane proteins [3]. Recently, polymer layer immobilized on rigid substrates has been applied as a biomimetic support for lipid bilayer [4]. In general, the polymer layer as mentioned above acts as a cushion and aids in preserving the biophysical properties of biomembrane. For example, the two-dimensional diffusion coefficient of phospholipids supported on polymer cushion approaches that in intact cell membrane [5]. On the other hand, little work has been done on the elucidation of the physical driving forces underlying the formation of supported lipid bilayer on polymer cushion. Particularly, the quantitation of vesicle-polymer interaction would allow researchers to engineer the fabrication of supported bilayer through vesicle fusion for applications in protein chip, biosensor and biomaterials [6].
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Poly(acrylic acid) [PAA] is a weak polyelectrolyte and has been recently exploited for potential applications as a solid support in the fabrication of supported lipid bilayer [7]. However, the lack of physical understanding in the interaction of lipid bilayer with immobilized PAA has impaired the design of molecularly designed film with optimized bio-functional characteristics. In this study, the interaction between phospholipid bilayer and poly(acrylic acid) cushion immobilized on poly(ethylene terephthalate) [PET] was thoroughly investigated. First, confocal-reflectance interference contrast microscopy (C-RICM) probes the contact zone (membrane-substrate distance <30 nm) of adherent phospholipid vesicle on the PAA cushion [8]. By simultaneous application of cross-polarization microscopy with C-RICM and contact mechanics modeling, the adhesion energy at membrane-polymer interface was determined. Furthermore, the effect of PAA ionization induced by pH change on the adhesion energy is elucidated. Quantitative analysis of the vesicle adhesion on polymer cushion will lead to the development of tailored polymer-supported lipid bilayer for biosensor and biotechnology applications.
2. Experimental methods 2.1. Materials Amorphous poly(ethylene terephthalate) film was obtained from Goodfellows (UK). Dipalmitoylphosphocholine (DPPC) in powder form was obtained from Avanti Polar Lipids Inc. (USA) and was used as received. Toluidine Blue-O (TBO), acrylic acid, sodium periodate, sodium carbonate, calcium chloride, methanol, acetic acid, Dulbecco’s modified Eagle medium (DMEM), HEPES, HBSS and sodium hydroxide were obtained from Sigma Chemical (Singapore). 18.2 M water was obtained from Maxima water purification system (Elga, USA) and was used in the preparation of all solutions. Monobasic potassium phosphate (KH2 PO4 ); dibasic potassium phosphate (K2 HPO4 ); sodium chloride (NaCl); monobasic sodium phosphate (NaH2 PO4 ); potassium chloride (KCl); dibasic sodium phosphate (Na2 HPO4 ); 1 N hydrochloric acid (HCl) were obtained from Fisher Chemicals (Singapore). 1X phosphate buffer saline (PBS) was prepared with 150 mM sodium chloride, 10 mM sodium phosphate, 50 mM potassium chloride and 80 mM potassium phosphate and was adjusted to pH 7.4 with 1 N hydrochloric acid. Micro BCA protein assay reagent kit was obtained from Pierce Chemical (USA). 2.2. Sample preparations PET disc with a diameter of 22 mm was cleaned with 100% methanol for 10 min in ultrasonic bath. A piece of cleaned PET film was placed in a glass container with 80 mL solution containing 1% (v/v) aqueous acrylic acid and 20 mg of sodium periodate. The grafting reaction was carried out in a
custom built water flow-bath under a 400 W UV light course (Dymax 5000-EC, Dymax corporation). After the reaction, the amount of COOH grafted onto the film was confirmed and characterized by the Toluidine Blue-O assay, in which the dye stains the deprotonated acid groups through ionic interaction. It is assumed that one mole of TBO binds to one mole of COOH (along the PAA chains). In brief, a solution of 5 × 10−4 M TBO was prepared by dissolving TBO powder in sodium hydroxide solution (pH 10). PET disc (diameter: 0.55 cm) grafted with acrylic acid was placed in a 1.5 mLEppendorf tube. One milliliter of TBO solution was added on the PET surface inside the Eppendorf tube and the sample was shaken for 16 h. After TBO binding, the supernatant was removed from Eppendorf Tube and the stained PET surface was washed with NaOH solution (pH 10) following by pure water for two times. One milliliter of 50% acetic acid was added to remove the TBO dye from the carboxyl groups on PAA–PET. Five hundred microliters of the washed solution from each sample was loaded into 96-well-plate and the optical density at 633 nm is measured with a 96-well-plate reader (Molecular Devices, USA). The concentration of the carboxyl groups in acetic acid is determined with a calibration plot containing several samples of acetic acid with different concentrations. Giant unilamellar vesicles (ULV) were synthesized by a well-accepted method [8]. Briefly, 1 mg of DPPC was dissolved in methanol/chloroform co-solvent (2:1 by volume) at a concentration of 1 mg/100 L and the mixture was, subsequently, added on the surface of a roughened Teflon disc. A thin film of DPPC was left on the Teflon surface following the evaporation of solvent and was dried in vacuum for 12 h. Then the Teflon disc was covered with 1X PBS buffer and was hydrated at 50 ◦ C for 16 h. A hydrated layer of lipid film was developed on the disc after a few hours. Finally, opaque suspension of vesicles was dispersed in solution by gentle shaking of the sample. 2.3. Phase contrast microscopy The detailed setup of the instrument has been previously described [9]. DPPC vesicles in 1X PBS (pH 7 or pH 9) were loaded on the plain or PAA grafted PET surfaces. All experiments were carried out at 22 ◦ C in a temperature control chamber (SEC, Korea). Phase contrast images for a representative group of vesicles were taken after 30 min of incubation. All experiments of vesicle adhesion on each PET-based surface were performed in triplicate. An image analysis software, ZSM5 (Carl Zeiss, Germany) was used for measuring the mid-plane diameter of adherent vesicles. 2.4. Confocal-reflection interference contrast microscopy (C-RICM) The instrument is based on a laser scanning confocal microscope and has been described elsewhere in detail [9]. An
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Argon-ion laser with an excitation wavelength of 488 nm was used. A 63× oil immersion objective (Neofluar, N.A.: 1.25) was used for all measurements. The protocols for sample preparation, and image analysis were identical to those described in the previous section. The degree of vesicle deformation is defined as the ratio of contact zone radius (from C-RICM) and mid-plane radius (from cross-polarized light microscopy) of an adherent vesicle [10]. An adherent vesicle has been modeled as a truncated capsule with a thin wall, assumed as a sphere with a planar contact area of radius a. The degree of vesicle deformation is sin θ = (a/R) = α, where R is the mid-plane radius. The adhesion energy of the biomembrane under a uniform equi-biaxial stress, σ = Cε is shown as W = (1 − cosθ)Cε + Cε2
(1)
where C is equivalent to Eh/(1–υ) in a linear system under small strain where E, h and υ the elastic modulus, film thickness and the Poisson’s ratio, respectively [10]. Based on the experimental measurements of the mid-plane diameter, R (cross-polarized light microscopy) and the radius of contact zone, a (C-RICM); W is found by Eq. (1).
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3. Results and discussions Poly(acrylic acid) immobilized on poly(ethylene terephthalate) surface is a potential solid support for phospholipid bilayer due to the proven affinity of PAA with biomembrane [11]. However, the contact mechanics of phospholipid vesicles on a polymer substrate like PET has not been previously interrogated due to the lack of experimental technique for visualizing the membrane-polymer interface. PET can be chemically functionalized with UV radiation and enables the subsequent grafting of PAA chains [12]. In order to probe PAA-vesicle interaction accurately, the biomechanical responses of phospholipid vesicles upon adhesion on a plain PET surface in the absence of poly(acrylic acid) layer must be determined. Fig. 1 shows the cross-polarized light image (i) and C-RICM image (ii) of a representative DPPC vesicle adhering on plain PET surface at 23 ◦ C in 1X PBS solution. Cross-polarized light image shows that the vesicle has a mid-plane diameter of 16.1 m. In parallel, C-RICM image demonstrates the formation of adhesion contact (cross on Fig. 1A (i)) with an area of 14.5 m2 along the centroid of the vesicle. It is critical to show that our contact mechanics model is applicable to phospholipid vesicle adhering on a polymeric material surface. The light intensity profile of
Fig. 1. (A) The cross-polarized light image (i) and C-RICM image (ii) of a representative DPPC vesicle on a plain PET surface at 23 ◦ C in 1X PBS solution. (B) The vesicle-substrate separation profile of the adherent DPPC vesicle on plain PET.
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interference fringe pattern propagating (red line on Fig. 1A (ii)) can be converted into membrane-substrate separation distance through the inverse cosine transformation [13]. Fig. 1B shows the experimental vesicle-substrate separation profile (dotted line) of the adherent DPPC vesicle on plain PET. Based on the measured adhesion contact and mid-plane diameter, the cell-substrate separation profile of the adherent vesicle is simulated with Eq. (1) (solid line on Fig. 1B). The agreement between the experimental membrane-substrate separation profile and the simulated profile as described above indicates that the geometry of the adherent vesicle on plain PET surface approaches that of a truncated sphere [14]. The coupling of PAA cushion on plain PET surface may lead to the change of the optical properties of plain PET surface. Thus it is critical to elucidate the applicability of our biophysical approach on the PAA-grafted PET surface. First, the success of PPA grafting on PET surface is independently verified with Toluidine Blue-O test. It is shown that density of acrylic acid on PET is 0.53 nmole/cm2 . Furthermore, AFM topographic image indicates that the root-mean-square roughness of PET is increased from 0.6 to 1.5 nm upon the immobilization of PAA chain. Fig. 2A shows the cross-polarized light image (i) and C-RICM image (ii) of a typical DPPC vesicle bound on the PAA-grafted PET at 23 ◦ C in 1X PBS solution. Cross-polarized light microscopy demonstrates that the mid-plane diameter of the
adherent vesicle is 35 m. C-RICM shows that the contact area of adherent vesicle is 27.2 m2 . It is indicated that the modification of PET surface with PAA chains does not impair the sensitivity of C-RICM for revealing the adhesion contact and interference fringes. Fig. 2B shows the vesicle -substrate separation profile (obtained from C-RICM) and the simulated profile (predicted with a and R) of the DPPC vesicle adhering on PAA–PET surface. The result demonstrates that the geometry of adherent vesicle is correctly modeled as a truncated sphere. Therefore, the PAA coupling has not modulated the adhesion mechanism of DPPC vesicle. The biomechanical responses of DPPC vesicles towards adhesive interaction on the polymeric surfaces can be quantified by the ratio of adhesion contact radius and mid-plane radius. Fig. 3A shows the degree of vesicle deformation (a/R) against vesicle diameter for DPPC vesicles on plain PET and PAA–PET at 23 ◦ C in 1X PBS solution (pH 7). The dotted line represents the best fit of the experimental data. The result indicates that average a/R for vesicle of all sizes on plain PET surface is 0.16. Interestingly, the inclusion of PAA layer on PET surface imposes negligible effect on the average magnitude of a/R as shown by the overlap of two sets of data. The trend as mentioned above implies that the biomechanical response of DPPC vesicles upon adhesion on PET is similar to that on PAA–PET. It is useful to determine the adhesion energy between the vesicle surface and polymer surface. Fig. 3B shows the adhesion energy of DPPC vesicles of various sizes
Fig. 2. (A) The cross-polarized light image (i) and C-RICM image (ii) of a typical DPPC vesicle bound on the PAA-grafted PET at 23 ◦ C in 1X PBS solution. (B) The membrane-substrate separation profile (obtained from C-RICM) and the simulated profile (predicted with a and R) of the vesicle adhering on PAA–PET surface.
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Fig. 3. (A) The degree of vesicle deformation (a/R) against the vesicle diameter for DPPC vesicles on plain PET and PAA–PET at 23 ◦ C in 1X PBS solution (pH 7) (B) The adhesion energy of DPPC vesicles of various sizes on plain PET and PAA–PET in 1X PBS solution at 22 ◦ C.
on plain PET and PAA–PET in 1X PBS solution at 22 ◦ C. The result indicates that the average adhesion energy for vesicle of all sizes on plain PET is 1 × 10−10 J/m2 . Also, the average adhesion energy remains unchanged upon the modification of plain PET with PAA layer. The trends as mentioned above imply that the interfacial force between vesicle surface and plain PET is not affected by PAA inclusion at pH 7. The surface of plain PET is mainly composed of the ester carbonyl, methylene, and phenyl groups. In general, the ester groups in the polyester chain of plain PET are hydrophilic and polar due to the presence carbonyl oxygen atom and the carbonyl carbon atom [15]. Therefore, the major physical driving force of DPPC bilayer adhesion on plain PET surface is likely originated from the hydrogen bond formation between the amino group on DPPC and the ester carbonyl group of PET. Our hypothesis was supported by the shift of the ester carbonyl stretch upon the interaction of PET film with the primary amine of 3-aminopropyltrimethoxysilane [16]. Under acrylic acid density of 0.53 nmole/cm2 , the hydrophilicity of PAA–PET approaches that of plain PET
as shown by the similar contact angle [15]. Furthermore, the PAA chains grafted on PET surface is partially ionized due to the limited deprotonation of carboxylic acid at pH 7 [17]. The two factors as mentioned above have led to negligible enhancement in adhesion energy on PAA–PET compared to that induced by hydrogen bonding on plain PET at pH 7. It is known that the degree of carboxylic group ionization on PAA is correlated with the change of solution pH. The elucidation of pH effect on the adhesion of DPPC vesicle on plain PET serves as a necessary control experiment for PAAlipid bilayer interaction. Fig. 4A shows a/R against vesicle diameter for DPPC vesicle on plain PET at 22 ◦ C under pH 7 and pH 9 medium. The result indicates that average magnitude of a/R for DPPC vesicles of all sizes is 0.14 and 0.16 at pH 9 and pH 7, respectively. Fig. 4 B shows the adhesion energy of DPPC vesicles on plain PET at 22 ◦ C under pH 7 and pH 9 medium. It is shown that the average magnitude of adhesion energy for vesicles of all sizes is decreased from 1 × 10−10 J/m2 to 3 × 10−11 J/m2 against the increase of solution pH. Overall, the result implicates that the interfacial
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Fig. 4. (A) a/R against vesicle diameter for DPPC vesicle on plain PET at 22 ◦ C under pH 7 and pH 9 medium. (B) The adhesion energy of DPPC vesicles on plain PET at 22 ◦ C under pH 7 and pH 9 medium.
force between DPPC vesicle and plain PET is slightly dampened under slightly basic condition. The headgroup of DPPC lipid composes of both a quaternary amine and phosphate which carries a permanent positive charge and a transitional negative charge, respectively. Under the slightly basic pH used in this study, the phosphate groups of DPPC mostly remain deprotonated and negatively charged while the PET surface without any electrostatic charge is hydrophilic in nature under all pH. Therefore, the slight reduction of adhesion energy induced by pH increase is likely caused by the disruption of hydrogen bonding between DPPC and plain PET. Our result is supported by the fact that the strength of hydrogen bonds between carboxylic acid-functionalized AFM tips and hydrophilic substrate is dependent on the pH of solution [18]. The pH-induced responses of DPPC vesicle adhesion on PAA–PET would shed light on the fundamental properties of polymer cushion-supported bilayer. Fig. 5A shows a/R
against vesicle diameter for DPPC vesicles adhering on PAA–PET at pH 7 and pH 9 under 22 ◦ C. Obviously, the increase from pH 7 to pH 9 leads to the shift up of the a/R vs. 2R curve. Moreover, the average magnitude of a/R for vesicle of all sizes is increased by 18.8% from 0.16 and 0.19 at pH 7 and pH 9, respectively. Fig. 5B shows the adhesion energy against vesicle diameter for adherent DPPC vesicles on PAA–PET at pH 7 and pH 9 under 22 ◦ C. In contrast to the trend observed on plain PET, the average magnitude of adhesion energy for DPPC vesicles of all sizes on PAA–PET is 1 × 10−10 J/m2 and 1.8 × 10−10 J/m2 at pH 7 and pH 9, respectively. The results indicate that adhesion energy is increased by 80% towards pH increase. Interestingly, the modification of plain PET with PAA chain exemplifies the pH induced enhancement of vesicle adhesion on the PET support. The observed effect is brought about by the enhanced electrostatic interaction between the
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Fig. 5. (A) a/R against vesicle diameter for DPPC vesicles adhering on PAA–PET at pH 7 and pH 9 under 22 ◦ C. (B) The adhesion energy against vesicle diameter for adherent DPPC vesicles on PAA–PET at pH 7 and pH 9 under 22 ◦ C.
quaternary amine (positive charge) of DPPC headgroup and the carboxylate ions (negative charge) of PAA. It is because the degree of PAA ionization is increased from 40 to 100% and the degree of deprotonation of phosphate group on DPPC remains constant when pH is increased from 7 to 9 [17]. To the best of our knowledge, it is the first time that the biomechanical response of adherent phospholipid vesicles on a polymer cushion is shown to be correlated with solution pH. Most important, the approach demonstrated herein provides new physical insights into the control of membrane-polymer interaction during supported membrane fabrication.
4. Conclusion Polymer-supported lipid bilayer has emerged as novel platform for biosensor technology and biochemistry research.
In this study, PAA cushion grafted on PET surface has been used as a model system for interrogating the interfacial driving forces involved in the initial stage of DPPC vesicle fusion on polymer cushion. Specifically, the pH induced response of adherent vesicle on PET surface is only obvious in the presence of PAA cushion. Overall, this study demonstrates the possibility of applying our biophysical approach for the design of novel polymer-supported lipid bilayer system through the tuning of adhesive interaction.
Acknowledgements VC and NF were supported by a Tissue Engineering Initiative of Professor Lim Mong King (NTU) and NTU AcRF 00/15. CL, HQM and KWL were partially supported by NIH grant (5P01CA79862-020003) and A*STAR (Singapore).
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