Biomacromolecules 2003, 4, 581-588
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Interaction of Liposome with Immobilized Chitosan during Main Phase Transition Ning Fang and Vincent Chan* Tissue Engineering Laboratory, School of Mechanical and Production Engineering, Nanyang Technological University, Singapore 639798 Received September 18, 2002; Revised Manuscript Received December 9, 2002
It has been recently demonstrated that chitosan in aqueous solution alters the phase behavior and structure of a phospholipid bilayer (Fang, N.; et al. Biomacromolecules 2001, 2, 1161-1168). Until now, the physical driving forces between chitosan and the phospholipid bilayer upon their initial encounter remains unknown. In this study, confocal reflectance interference contrast microscopy (C-RICM), phase contrast microscopy and bioadhesion modeling are concurrently applied to probe the interaction of phospholipid vesicle with immobilized chitosan at various temperatures, pH, and osmotic stress. First, the successful immobilization of chitosan on amino-silanized glass is indicated by the increases in both the degree of vesicle deformation and adhesion energy of vesicles adhering on chitosan modified substrate in comparison with those on aminosilanized glass. Second, the phase transition of a phospholipid bilayer does not modulate the adhesion strength at the chitosan-biomembrane interface at pH 7.4. With increase of the degree of protonation on the chitsoan backbone at pH 4, the adhesion energy is increased by 5-fold for vesicles of all sizes compared to that in pH 7.4. Furthermore, pH reduction amplifies the thermal-induced response of larger vesicles on the immobilized chitosan layer. Interestingly, a moderate increase of osmotic stress maximizes the degree of vesicle deformation and adhesion energy at 23 °C and dampens the effect of phase transition on vesicle adhesion. Overall, this study demonstrates the quantitation of chitosan-biomembrane interactions that will be critical for future applications of chitosan in biological systems. Introduction Chitosan is a polyelectrolyte derived from chitin and is readily soluble in slightly acidic solution (pH 5). The positively charged backbone of chitosan is known as the major physical determinant for chitosan interaction with other biomacromolecules and supramolecular structures.1 Several previous studies indicate that chitosan strongly binds with other negatively charged molecules.2 In particular, the strong electrostatic attraction between chitosan and negatively charged DNA induces coacervation between the two polyelectrolytes and leads to the formation of biodegradable DNA-chitosan nanoparticles.3 In the absence of ligand coupling on the nanoparticle surface, it has been shown that chitosan-DNA nanoparticles successfully overcome the cell membrane barrier and subsequently induce gene expressions in several biological models.4 Moreover, chitosan in its fully protonated state increases the paracellular permeability of peptide drugs across mucosal epithelia.5 In the area of tissue engineering, chitosan immobilized on biomaterial scaffold was shown to promote membrane adhesions and biological functions of chondrocyte6 and endothelial cells.7 The interactions between chitosan and cell membrane as mentioned above have prompted our efforts in investigating the phase behavior and structure of phospholipid vesicles/chitosan aqueous mixture.8,9 It is shown that * Address correspondence to Vincent Chan, Nanyang Technological University, MPE, 50 Nanyang Ave, Singapore 639798.
chitosan reduces the enthalpy of gel to liquid crystalline transition of dipalmitoylphosphocholine (DPPC) bilayer in a concentration-dependent manner and induces fusion of small DPPC vesicles to form larger lamellar structures.8 Moreover, the degree of membrane perturbation induced by chitosan is enhanced by the reduction of solution pH.9 However, the physical driving forces leading to membrane destabilization are still unclear. Intuitively, the quantitation of the interfacial energy between chitosan and a biomembrane upon initial chitosan association on a DPPC vesicle surface will provide new insights into the physical origins of chitosan-induced membrane perturbation. The recent development of bioconjucative chemistry enables the chemical coupling of chitosan onto amino-silanized substrate and provides a practical experimental model for studying the physical interaction between chitosan and other biomolecular structures.10 In this study, we simultaneously apply confocal interference contrast microscopy (C-RICM) and phase contrast microscopy to probe the contact mechanics of DPPC unilamellar vesicle (ULV) on the immobilized chitosan layer. The magnitude of the DPPC-chitosan interaction is eventually determined from the experimental data with the use of our proven bioadhesion theory.11 Also, the biophysical responses including the degree of vesicle deformation and adhesion energy of DPPC-ULV on chitosan-modified substrates are correlated with changes in pH, temperature, and ionic strength. Overall,
10.1021/bm025682s CCC: $25.00 © 2003 American Chemical Society Published on Web 02/26/2003
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our results provide quantitative parameters for manipulating chitosan-membrane interactions. Materials and Methods Materials. Chitosan with a molecular weight of 213 kDa and degree of deacetylation of 87.5% was obtained from Pronovo Biomedical (Oslo, Norway, a gift from Johns Hopkins Singapore Biomedical Research Centre) and was used as received. Dipalmitoylphosphocholine (DPPC) in powder form was obtained from Avanti Polar Lipids Inc. (USA) and was used as received. Sodium chloride (NaCl), dibasic sodium phosphate (Na2HPO4), monobasic potassium phosphate (KH2PO4), dibasic potassium phosphate (K2HPO4), monobasic sodium phosphate (NaH2PO4), potassium chloride (KCl), 1 N hydrochloric acid (HCl), glutardialdehyde, sodium azide, ethanolamine, 3-aminopropyltriethoxysilane (APTES), acetic acid, methanol, and chloroform were obtained from Fisher Chemicals Inc. (USA) and used as received. Water (18.2 MΩ) was obtained from Maxima water purification system (Elga, USA) and was used in the preparations of all solutions. 1X phosphate buffer saline (PBS) including 150 mM sodium chloride, 10 mM sodium phosphate, 50 mM potassium chloride. and 80 mM potassium phosphate was adjusted to pH 7.4 with 1 N hydrochloric acid. Chitosan-Modified Substrate and Liposome Preparations. Pure fused silica coverslips (Fisher Scientific Pte Ltd., Singapore) were cleaned as described previously.11 Aminemodified substrate is prepared from fused silica substrate by silanization with APTES as previously described.12 The coupling of chitosan onto APTES-modified substrate was based on a proven method as previously described.10 In brief, APTES-coated glasses are immersed in aqueous solution with 1 M NaCl, 1 wt % glutardialdehyde, 0.4 mg/mL chitosan, 0.25 mM sodium azide, and 0.1 M acetic acid (adjusted to pH 5 with NaOH) for 12 h. The residual aldehydes were terminated by incubating the modified substrate with 1 M ethanolamine (pH 5) for 4 h. Last, the substrates are washed with 18 MΩ water for three times. Under the experimental conditions mentioned above, the immobilized layer of chitosan has a thickness of 10 nm and a surface concentration of 1 µg/cm2.10 The presence of immobilized chitosan on APTES glass was further confirmed with atomic force microscopy (AFM) topographic imaging (Thermomicroscope, USA). Giant unilamellar vesicles (ULVs) were synthesized by a well-established method by rehydrating a DPPC film deposited on a roughened Teflon surface.13 The unilamellar structure was verified by phase contrast microscopy as shown previously.9 The average diameter of the unilamellar vesicle was 30 ( 10 µm. Cross-Polarized Light Microscopy. The detail of the instrument has been previously described.12 Vesicle solutions in 1X PBS (isotonic buffer) were incubated on either an amine-modified coverslip or a chitosan-modified substrate for an hour and was loaded in a temperature-controlled chamber for subsequent imaging under the microscope. Image analysis software, ZSM5 (Carl Zeiss, Germany), was used for determining the midplane diameter of adherent vesicles. All experiments for each lipid mixture were carried in triplicate.
Fang and Chan
Confocal Reflection Interference Contrast Microscopy (C-RICM). The detail of the instrument that is based on a laser scanning confocal microscope has been described elsewhere.15 An argon-ion laser with an excitation wavelength of 488 nm was used for sample illumination. A 63× oil immersion objective (Neofluar, N.A. 1.25) was used. The strong contact zone of the adhering vesicle appears as a dark region on the image. DPPC vesicle solution in 1X PBS buffer was incubated on a chitosan- or APTES-modified substrate for 45 min, and images were taken at temperatures of either 23 or 49 °C (at least 30 min of incubation time at each temperature). All experiments were carried in triplicate. ZSM5 software (Carl Zeiss, Germany) was used for all image analysis. The degree of vesicle deformation is the ratio of the contact zone radius and the midplane radius (from crosspolarized light microscopy) of vesicle at each temperature. Contact Mechanics Model. The contact mechanics model for a vesicle adhering on a rigid substrate has been reported in detail.11 A truncated sphere geometry of adherent vesicle has been extensively validated in several studies.12 The adhesion energy when the vesicle wall is under a uniform equi-biaxial stress, σ ) C is shown as W ) (1 - cos θ)C + C2
(1)
where C is equivalent to Eh/(1 - υ) in a linear system under small strain, with E and υ the elastic modulus and the Poisson’s ratio, respectively, and h the film thickness. On the basis of the experimental measurements of the midplane diameter R (cross-polarized light microscopy) and the radius of contact zone, a (C-RICM), W is determined by eq 1 since is a function of a/R. E of the phosphocholine bilayer vesicle composed of phosphatidylcholines in gel and liquid crystalline phase is taken as 28 800 and 16 000 N/m2, respectively, according to the experimental results obtained from optical dynamometry, membrane bending spectroscopy, and micropipet aspiration.16,17 Results and Discussions The interaction between a DPPC unilamellar vesicle (ULV) and immobilized chitosan is directly correlated with the biomechanical responses of the adherent vesicle upon the formation of adhesion contact with chitosan-modified substrate. Figure 1 shows the cross-polarized light image at 23 °C (i), the C-RICM image at 23 °C (ii), and the C-RICM image at 49 °C (iii) of a typical DPPC ULV adhering on chitosan-modified substrate in neutral buffer (pH 7.4). Crosspolarized light microscopy indicates that the midplane diameter of the adherent vesicle is 18.4 µm. Also, the vesicle has a strong affinity with chitosan-modified substrate since Brownian motion of the bound vesicle is absent during the course of imaging. C-RICM directly probes the chitosan/ membrane interface which is otherwise not observable under conventional light microscopy.18 The crosses on the C-RICM images represent the adhesion contact zone between vesicle surface and substrate which is defined as the area enclosed by the first Newtonian ring of the interference fringes. The rather uniform contact zone as shown on the C-RICM image supports the presence of lateral homogeneity at the micrometer-
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Figure 1. The cross-polarized light image at 23 °C (i), C-RICM image at 23 °C (ii), and C-RICM image at 49 °C (iii) of a typical DPPC ULV adhering on chitosan-modified substrate in neutral buffer (pH 7.4).
scale throughout the immobilized chitosan layer. Our preliminary AFM topographic image of chitosan-modified substrate further supports the lack of micrometer-scale roughness. The C-RICM results indicate that the contact area of the adherent vesicle on the substrate is 15 and 19 µm2 at 23 and 49 °C, respectively. The temperature dependency in the contact mechanics of adherent vesicle stems from the change of biomechanical property of phospholipid bilayer during the gel to liquid crystalline transition.15 On the basis of our simultaneous cross-polarized light/CRICM measurements, the mechanical deformation of adherent vesicle under the influence of immobilized chitosan layer is quantified. Figure 2A shows the degree of vesicle deformation (a/R) against vesicle diameter for DPPC ULV adhering on APTES- and chitosan-modified glasses in pH 7.4 buffer at 23 °C. Each error bar represents the standard deviation in the data of at least three vesicles of same size. In this study, APTES-modified substrate is used as a control as it is subsequently coupled with chitosan through a glutardialdehyde linker. In general, a/R is a reducing function of vesicle size on both substrates. This trend is commonly found in the adhesion of vesicle and polymeric microcapsules on nondeformable substrates.12,19 This result is caused by the different extent of increase in adhesion contact area and in vesicle surface area when vesicle diameter increases.15 It is found that the average magnitude of a/R is increased by 20% from 0.30 to 0.36 for vesicles with a midplane diameter ranging from 5 to 60 µm when chitosan is coupled on APTES-modified substrate. The successful immobilization of chitosn is supported by the increase of terrace height of
AFM measured topographic features from 9 to 33 nm upon the linking of chitosan molecules on the APTES substrate. The result shows that the immobilized chitosan layer strongly interacts with the DPPC bilayer in comparison with APTESmodified surface. The strong interaction between the DPPC bilayer and chitosan observed herein is supported by the phase behavior of DPPC/chitosan aqueous mixture and the disruption of mimic membrane caused by chitosan.8,9,20 On the basis of our contact mechanics model, the interfacial energy between the chitosan and DPPC bilayers is determined from our experimental data. Figure 2B shows the adhesion energy against vesicle diameter for DPPC ULV adhering on APTES- and chitosan-modified glasses in pH 7.4 buffer at 23 °C. Each error bar represents the standard deviation of the data for at least three vesicles of same size. On both substrates, adhesion energy spans 3 orders of magnitude against the increase of vesicle size. When chitosan is chemically coupled onto APTES-modified glass, the interfacial energy of adherent vesicle is substantially increased for vesicles for all sizes. For smaller vesicle (38 µm vesicle diameter) in the adhesion energy diagram when gel to liquid crystalline transition leads to opposing changes in the adhesion energy. For smaller vesicles (
