Confocal Reflectance Interference Contrast Microscopy Research Articles

Modulating Cell Adhesion Dynamics on Carbon Nanotube Monolayer Engineered with Extracellular Matrix Proteins

Although it has been demonstrated that carbon nanotubes (CNTs) may have potentials for tissue engineering applications because of their unparalleled physical properties, little has been known on the cell adhesion mechanisms on model CNT monolayer pertaining to the design of novel cell therapeutics device. In this study, the adhesion dynamics of primary porcine esophageal fibroblasts (PEFs) on CNT monolayer were elucidated with confocal reflectance interference contrast microscopy (C-RICM) integrating with phase contrast microscopy. Moreover, CNT monolayer (CNT-ML) was functionalized with two typical extracellular matrix (ECM) proteins including collagen type I (COL) and fibronectin (FN) in order to promote its biocompatibility. First, it is shown by atomic force microscopy that the topographical features of CNT-ML were dependent on the types of immobilized ECM protein. Second, significant time lag in adhesion contact evolution (around 10 min) for PEFs was found on both CNT-ML and CNT-COL compared to the negligible time lag on CNT-FN. It was found that adhesion energy of PEFs on the CNT-COL and CNT-FN surfaces reached steady state at 60 and 30 min after cell seeding compared to 70 min on CNT-ML surface. At steady state, the adhesion energy of PEFs on the CNT-COL and CNT-FN surfaces was about twice as much than that on the CNT-ML surface. Moreover, immobilization of collagen or fibronectin on CNT monolayer led to an increase in seeding efficiency and proliferation rate of PEFs. Scanning electron microscopy and immunostaining together demonstrated that PEFs displayed an elongated morphology and highly polarized actin network on both CNT-COL and CNT-FN surfaces, whereas PEFs displayed nonuniform cell morphology and actin organization on the CNT-ML surface. Overall, our results demonstrated that the biophysical responses and biological behavior of PEFs on unmodified or functionalized CNT monolayer were different. Functionalization of CNT through extracellular matrix protein immobilization effectively promotes cell adhesion and proliferation, which may provide information for designing CNT-based biomaterials or novel cell therapeutics devices in biomedical engineering.

Effect of cytoskeleton inhibitors on deadhesion kinetics of HepG2 cells on biomimetic surface

Cytochalasin-D (Cyto-D) and latrunculin-A (Lat-A) are known inhibitors of actin microfilaments and adversely affect the physiological functions of anchorage-dependent cells. Alternatively, doxorubicin (Dox), a chemotherapeutic drug is known to induce apoptosis and cell detachment of tumor cells. However, the intricate interplay between drug administration, cytoskeletal rearrangement and biophysical responses of live cells on immobilized layer of extracellular matrix (ECM) protein remains unknown. In this study, the deadhesion kinetics and actin remodeling of live HepG2 cells following the addition of the three drugs are probed with confocal reflectance interference contrast microscopy (C-RICM) and fluorescence confocal microscopy. First, it is shown that the reduction in two-dimensional spread area of HepG2 cells is 10.5%, 15.4% and 21.9% under the influence of 5 μM of Lat-A, Cyto-D and Dox, respectively. Secondly, C-RICM demonstrates the recession of strong adhesion contact against time of cell seeding upon the addition of the three drugs. Thirdly, the initial cell detachment rate and extent of reduction in the degree of cell deformation ( a/ R) are dependent on both the drug types and concentration. Lastly, oscillation-like responses of a/ R and adhesion energy are uniquely found in Lat-A induced cell detachment. Overall, our biophysical approaches have been proven as a highly quantitative platform for elucidating the interfacial properties of adherent cells on biomimetic surfaces under cytoskeleton disruption.

The effect of adhesive ligands on bacterial and fibroblast adhesions to surfaces

The modification of medical device surface with adhesive ligands has been recently shown to be an effective means for making a bioselective surface which can inhibit bacterial adhesion while promoting host cell adhesion on device materials. Currently, the lack of quantitative correlation between the adhesion strength of bacteria, nature of adhesive ligand and adhesion kinetics of mammalian cells hinders the development of such device surface. In this study, the biophysical responses of bacteria and mammalian cells towards adhesive ligand on model device surfaces formed by the chemisorption of dopamine (a moderate antibiotic) on glass are elucidated. The effects of RGD, collagen and dopamine modification on the adhesion strength of two clinically significant bacteria including Escherichia coli ( E. coli) and Staphylococcus aureus ( S. aureus) were investigated by the determination of minimum lateral forces for bacterial detachment and the density of adhering bacteria. The result indicates that RGD has no apparent effect on E. coli and S. aureus adhesion, while collagen reduces E. coli but enhances S. aureus. In order to assess the degree of host cell integration, the adhesion kinetics of 3T3 fibroblasts on the four surfaces was examined by confocal reflectance interference contrast microscopy (C-RICM). In contrast to the difference found in bacterial adhesion, the result indicates that both collagen and RGD significantly enhance the initial rate of deformation and adhesion energy for fibroblasts compared to those on glass and dopamine–glass. Overall, it is demonstrated that the choice of adhesive ligand is critical for designing a device surface which simultaneously minimizes bacterial adhesion and enhances host cell integrations.

Adhesion dynamics of porcine esophageal fibroblasts on extracellular matrix protein-functionalized poly(lactic acid)

Effective attachment of esophageal cells on biomaterials is one important requirement in designing engineered esophagus substitute for esophageal cancer treatment. In this study, poly(lactic acid) (PLA) was subjected to surface modification by coupling extracellular matrix (ECM) proteins on its surface to promote cell adhesion. Two typical ECM proteins, collagen type I (COL) and fibronectin (FN), were immobilized on the PLA surface with the aid of glutaraldehyde as a cross linker between aminolyzed PLA and ECM proteins. By using confocal reflectance interference contrast microscopy (C-RICM) integrating with phase contrast microscopy, the long-term adhesion dynamics of porcine esophageal fibroblasts (PEFs) on four types of surfaces (unmodified PLA, PLA-COOH, PLA-COL and PLA-FN) was investigated during 24 h of culture. It is demonstrated by C-RICM results that PEFs form strong adhesion contact on all four types of surfaces at different stages of cell seeding. Among the four surfaces, PEFs on the PLA-FN surface reach the maximum adhesion energy (9.5 × 10−7 J m−2) in the shortest time (20 min) during the initial stage of cell seeding. After adhesion energy reaches the maximum value, PEFs maintain their highly deformed geometries till they reached a steady state after 20 h of culture. F-actin immunostaining results show that the evolvement of spatial organization of F-actin is tightly correlated with the formation of adhesion contact and cell spreading. Furthermore, the cell attachment ratio of PEFs on PLA in 2 h is only 26% compared with 88% on PLA-FN, 73% on PLA-COL and 36% on PLA-COOH. All the results demonstrate the effect of surface functionalization on the biophysical responses of PEFs in cell adhesion. Fibronectin-immobilized PLA demonstrates promising potential for application as an engineered esophagus substitute.

Dual requirements of extracellular matrix protein and chitosan for inducing adhesion contacte volution of esophageal epithelia

It has been recently shown that chitosan (CHI)/collagen prostheses induced epithelization at the esophagus site of animal model. However, little is known on the biophysical mechanisms of cell adhesion on CHI-based material pertaining to esophagus tissue engineering. In this study, the adhesion contact dynamics of porcine esophageal epithelial cells seeded on CHI surface is probed using confocal-reflectance interference contrast microscopy in conjunction with phase-contrast microscopy. First of all, cells fail to form any adhesion contact on either CHI or elastin (ES)-coated surface. On CHI coated with fibronectin (CHI-FN) or elastin (CHI-ES), strong adhesion contact of cells evolved over time until they reached a steady-state level. The initial cell deformation rates of cells on CHI-FN and CHI-ES are 0.0138 and 0.0151 min(-1), respectively. Interestingly, cells on fibronectin (FN) coated substrate transiently form strong adhesion contact and eventually undergo deadhesion. Moreover, the steady-state adhesion energy of epithelial cells on CHI-FN is 1.73 and 148 times larger than that on CHI-ES and FN, respectively. The actin of cells on CHI-FN transforms from microfilament meshes at cell periphery to stress fibers throughout the cytoplasm during cell seeding. At the same time, vinculin staining demonstrated the evolution of focal adhesion complexes in cells on CHI-FN after 130 min of seeding. Interestingly, CHI-ES induces the formation of focal adhesion complexes in a lesser extent in cell but fails to lead to stress fiber formation. Overall, our study reveals that long-term adhesion contact evolution of esophageal epithelia is only triggered by both extracellular matrix protein and chitosan.

A Novel Optical Technique to Measure Pad-Wafer Contact Area in Chemical Mechanical Planarization

AbstractReal contact area between a CMP pad and wafer is a key factor in local contact pressure, friction and pad wear, all of which impact material removal and defect formation. A new optical method is introduced that quantifies the real contact area during polishing. Confocal reflectance interference contrast microscopy (C-RICM) uses a single focal plane to image the pad-wafer contact interface. A sapphire cover slip is used to provide optical transparency and to match the refractive index of the pad. Imaging the pad surface through the cover slip reveals areas of no reflection (pad-cover slip contact), areas of reflection (non-contact), and interference fringes (near contact). The C-RICM method was validated using micro-fabricated pads having uniform arrays of cylindrical surface structures of known contact area. Experiments conducted on porous polyurethane pads revealed that the real contact area is less than 10% of the total presented area. However Greenwood-Williamson (G-W) theory, widely used in CMP material removal models, predicts a contact area at least a factor of ten smaller. The discrepancy was found to result at least in part because the individual contact zones are not elliptical as assumed in G-W theory. In fact many contacting structures are crescents located at the perimeter of individual pores. These findings underscore the need for accurate control of pore density and morphology in polishing pads, in both initial manufacture and surface conditioning during CMP. The C-RICM method allows non-destructive benchmarking of polishing media in terms of pad-wafer contact, essential for developing improved pad architectures that achieve lower CMP defect levels.

Adhesion contact dynamics of 3T3 fibroblasts on poly (lactide- co-glycolide acid) surface modified by photochemical immobilization of biomacromolecules

A simple and effective method of biomacromolecule immobilization on biomaterial surface for direct tuning of biophysical parameters such as the initial cell deformation rate, degree of cell spreading and adhesion kinetics is important for tissue engineering. The photochemical immobilization of azide-chitosan (Az-CS) on poly (lactide- co-glycolide) acid (PLGA) is applied here. Chitosan immobilization on PLGA through the photoactive azide group further facilitates subsequent grafting of other biocompatible biomacromolecules like gelatin (Gel) through the active amine groups on CS. This study quantitatively compares the 3T3 fibroblast adhesion dynamics on three PLGA surfaces (Gel-CS-PLGA, CS-PLGA and unmodified PLGA surfaces) using Confocal-Reflectance Interference Contrast Microscopy (C-RICM) together with phase contrast imaging. CS-PLGA and Gel-CS-PLGA surfaces developed were confirmed by X-ray photoelectron spectroscopy, atomic force microscopy and water contact angle and cell adhesion contact dynamics measurements. The cell adhesion was strongest on the Gel-CS-PLGA surface and lowest on unmodified PLGA. The steady state adhesion energy attained by the cells on gelatin modified PLGA surface is determined as 4.0×10 −8 J/m 2, which is about 400 times higher than that on PLGA surface (1.1×10 −10 J/m 2). Significantly increased cell adhesion with Gel-CS-PLGA is postulated to result in increased cell spreading. Our integrated biophysical method can quantify the transient contact dynamics and is sufficiently accurate to discriminate even between Gel and CS modified surfaces.

Adhesion Contact Dynamics of Fibroblasts on Biomacromolecular Surfaces

Biomacromolecules like gelatin and chitosan have emerged as highly versatile biomimetic coatings for applications in tissue engineering. The elucidation of the interfacial kinetics of cell adhesion on biomacromolecular surfaces will pave the way for the rational design of chitosan/gelatin-based systems for cell regeneration. Biomacromolecular ultra-thin films, chemically immobilized on fused silica are ideal experimental models for determining the effect of surface properties on the biophysical cascades following cell seeding. In this study, confocal reflectance interference contrast microscopy (C-RICM), in conjunction with phase contrast microscopy and fluorescence confocal microscopy, was applied to detect the adhesion contact dynamics of 3T3 fibroblasts on chitosan and gelatin ultrathin films. X-ray photoelectron spectroscopy (XPS) confirmed the immobilization of chitosan or gelatin on the silanized glass surface. Both the initial cell deformation rate and the change of two-dimensional spread area of the 3T3 fibroblasts are higher on gelatin-modified surfaces than on chitosan surfaces. The steady-state adhesion energy of 3T3 fibroblasts on gelatin film is three times higher than that on chitosan film. Immuno-staining of actin further demonstrates the different organization of cytoskeleton, likely induced by the change in cell signaling mechanism on the two biomacromolecular surfaces. The better attachment of 3T3 fibroblast to gelatin is postulated to be caused by the presence of adhesive domains on gelatin.

Adhesion contact dynamics of primary hepatocytes on poly(ethylene terephthalate) surface

The design of bioartificial liver assist device requires an effective attachment of primary hepatocytes on polymeric biomaterials. A better understanding of this cell-surface interaction would aid the optimal choice of biomaterials. In this study, the adhesion contact dynamics of primary hepatocytes on poly(ethylene terephthalate) (PET) surface with grafted poly(acrylic acid) (PAA) and coated collagen is probed with confocal reflectance interference contrast microscopy (C-RICM) in conjunction with phase contrast microscopy. An increase of acrylic acid density from 0 to 12 nmole/cm 2 raises both the root-mean-square surface roughness and amount of adsorbed collagen of PET surface. C-RICM demonstrates that hepatocytes form tight adhesion contacts upon seeding on both plain PET and PAA-grafted PET (both with collagen coating) despite the insignificant two-dimensional cell spreading. At two hours after cell seeding, the normalized contact area and adhesion energy of hepatocytes on 12 nmole/cm 2 PAA-grafted-PET (with collagen coating) is 27% and 114% higher, respectively, than that on collagen coated plain PET. Interestingly, the growth kinetics of adhesion patch for hepatocyte on PAA-grafted PET with collagen coating is best fitted by R∝ t 0.5 and is significantly different from that on collagen coated plain PET, which is best fitted by R∝ t 0.25. Overall, this study demonstrates the modulation of biophysical response of adherent hepatocytes through the control of the biomaterial surface properties.

Adhesion contact dynamics of primary hepatocytes on poly(ethylene terephthalate) surface

The design of bioartificial liver assist device requires an effective attachment of primary hepatocytes on polymeric biomaterials. A better understanding of this cell-surface interaction would aid the optimal choice of biomaterials. In this study, the adhesion contact dynamics of primary hepatocytes on poly(ethylene terephthalate) (PET) surface with grafted poly(acrylic acid) (PAA) and coated collagen is probed with confocal reflectance interference contrast microscopy (C-RICM) in conjunction with phase contrast microscopy. An increase of acrylic acid density from 0 to 12 nmole/cm2 raises both the root-mean-square surface roughness and amount of adsorbed collagen of PET surface. C-RICM demonstrates that hepatocytes form tight adhesion contacts upon seeding on both plain PET and PAA-grafted PET (both with collagen coating) despite the insignificant two-dimensional cell spreading. At two hours after cell seeding, the normalized contact area and adhesion energy of hepatocytes on 12 nmole/cm2 PAA-grafted-PET (with collagen coating) is 27% and 114% higher, respectively, than that on collagen coated plain PET. Interestingly, the growth kinetics of adhesion patch for hepatocyte on PAA-grafted PET with collagen coating is best fitted by R∝t0.5 and is significantly different from that on collagen coated plain PET, which is best fitted by R∝t0.25. Overall, this study demonstrates the modulation of biophysical response of adherent hepatocytes through the control of the biomaterial surface properties.

A quantitative contour analysis of axisymmetric vesicles spontaneously adhering onto a substrate

The determination of membrane-substrate profile for adherent vesicle using confocal reflectance interference contrast microscopy (C-RICM) has pushed for the need of advanced mechanics model for interpreting adhesion mechanisms. In this work, a model for vesicles or cells adhesion is established, the governing equation is derived from the variation of the potential energy at the cohesive zone. A closed-form solution is found for vesicle spontaneously adheres to a substrate when its shear modulus, μ vanishes. Based on the model and C-RICM experiments the magnitude of the adhesion force is calculated for a lipid vesicle adheres to a glass substrate.

Bioadhesive characterization of poly(methylidene malonate 2.12) microparticle on model extracellular matrix

The efficacy of a drug delivery system is predicated on its retention in the target tissue. Microparticle is one of the most popular and effective drug delivery configurations. Recently, it has been shown that the interaction between drug-loaded microparticles and tissues is related to the effectiveness of paclitaxel delivery to the bladder wall of mice for treating superficial bladder cancer. In this study, the adhesive interaction between poly(methylidene malonate 2.12) or PMM 2.1.2 microparticles and collagen, which serves as the model extracellular matrix for bladder wall, was probed with confocal reflectance interference contrast microscopy (C-RICM), single-particle compressive force measurement and contact mechanics theory. Young's modulus of single PMM 2.1.2 microparticle was determined as 1.56±0.25×10 4 N/m 2. For plain PMM 2.1.2 microparticle in water (pH 5.5), the degree of deformation ( a/ R) on collagen coated substrate decreased from 0.77 to 0.26 against the increase of mid-plane diameter from 2 to 18 μm. The adhesion energy of PMM 2.1.2 microparticle was determined from Maguis-JKR theory and remained at around 1.5 mJ/m 2 against the increase of particle diameter. At pH 4, the average degree of particle deformation and adhesion energy was increased by 11% and 32%, respectively, in comparison with that at pH 5.5. The loading of paclitaxel in PMM 2.1.2 microspheres enhanced the deformation and adhesion of microspheres at pH 5.5. It is hypothesized that the electrostatic repulsion between paclitaxel and collagen at pH 4 reduces the adhesion energy of PMM 2.1.2-paclitaxel microsphere. This study may offer insight for design of future microparticulate delivery systems by providing the experimental and theoretical tools to study the bioadhesive interaction between drug-loaded microparticles and model extracellular matrices.

Effect of acyl chain mismatch on the contact mechanics of two-component phospholipid vesicle during main phase transition

It has been recently demonstrated that acyl chain mismatch of phospholipid bilayer composed of a binary lipid mixture induces component formation on the lateral plane of the bilayer [Biophys. J. 83 (2002) 1820–1883]. In this report, the contact mechanics of unilamellar vesicles composed of binary dimyristoyl-phosphatidylcholine (DMPC)/dipalmitoyl-phosphocholine (DPPC) mixtures on fused silica and amino-modified substrates is simultaneously probed by confocal-reflectance interference contrast microscopy (C-RICM) and cross-polarized light microscopy during gel to liquid crystalline transition of the lipid bilayer. C-RICM results indicate that the average degree of vesicle deformation for DMPC-rich and DPPC-rich vesicles adhering on fused silica substrate is increased by 30% and 14%, respectively, in comparison with that in pure DMPC and DPPC vesicles. Also, lateral heterogeneity induced by acyl chain mismatch increases the average magnitude of adhesion energy in DMPC-rich and DPPC-rich vesicles of all sizes by 6.4 times and 2.3 times, respectively. Similar modulation of adhesion mechanics induced by carbon chain difference is obtained on amino-modified substrate. Most importantly, the thermotropic transition of the mixed bilayer from gel (below T m ) to fluid phase (above T m ) further exemplifies the effect of acyl chain mismatch on the increases of degree of vesicle deformation and adhesion energy.

Interaction of liposome with immobilized chitosan during main phase transition.

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 amino-silanized 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 degrees 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.

Adhesion contact dynamics of HepG2 cells on galactose-immobilized substrates

The specific recognition between asialoglycoprotein receptor and galactose ligand at cell–substrate interfaces has been shown to mediate hepatocyte adhesion and maintain liver specific functions of hepatocytes. Conventionally, the success of hepatocyte attachment on engineered tissue scaffold is inferred from the degree of two-dimensional cell spreading that is measured by transmitted light microscopy. However, the actual contact mechanics and adhesion strength of hepatocytes during two-dimensional cell spreading has not been elucidated due to lack of biophysical probe. In this study, a novel biophysical technique known as confocal reflectance interference contrast microscopy (C-RICM) in conjunction with phase contrast microscopy is utilized to probe the adhesion dynamics, contact mechanics and two-dimensional spreading kinetics of HepG2 cells on galactose immobilized and collagen gel coated substrates. C-RICM demonstrates that HepG2 cells form strong adhesion contacts with both galactose-immobilized surfaces and collagen gel coated substrates. Moreover, HepG2 cells maintain their compact shapes in the presence of asialoglycoprotein receptor-mediated recognition while they become exceedingly spread under integrin-mediated adhesion on collagen gel coated substrate. The initial rate of adhesion contact formation and the steady-state adhesion energy of HepG2 cell population are highest on substrate conjugated with galactose ligand via a longer spacer. The adhesion dynamics and final adhesion energy of HepG2 cells depends both on the type of ligand–receptor interaction and the length of spacer between the ligand and substrate. Most importantly, new biophysical insights into the initial hepatocyte attachment that are critical for hepatocyte culture are provided through the decomposition of two-dimensional spreading and adhesion contact formation on bio-functional substrates.

Substrate-induced deformation and adhesion of phospholipid vesicles at the main phase transition

The physiochemical properties of phospholipid vesicle, e.g. permeability, elasticity, etc., are directly modulated by the chain-melting transition of the lipid bilayer. Currently, there is a lack of understanding in the relationship between thermotropic transition, mechanical deformation and adhesion strength for an adherent vesicle at temperature close to main phase transition temperature T m. In this study, the contact mechanics of dimyristoyl-phosphatidylcholine (DMPC) vesicle at the main phase transition are probed by confocal reflectance interference contrast microscopy in combination with phase contrast microscopy. It is shown that DMPC vesicles strongly adhere on pure fused silica substrate at T m and the degree of deformation as well as the adhesion energy is a decreasing function against the mid-plane diameter of the vesicles. Furthermore, an increase of osmotic pressure at the gel/liquid crystalline phase co-existence imposes insignificant changes in both the degree of deformation and adhesion energy of adherent vesicles when the lipid bilayer permeability is maximized. With the reverse of substrate charge, the mechanical deformation and adhesion strength for larger vesicles (mid-plane diameter >18 μm) are significantly reduced. By monitoring the parametric response of substrate-induced vesicle adhesion during main phase transition, it is shown that the degree of deformation and adhesion energy of adhering vesicle is increased and unchanged, respectively, against the increase of temperature.

Thermal induced modification of the contact mechanics of adhering liposomes on cationic substrate

The correlation between the mechanical property and the thermotropic transition of the phospholipid bilayer has been recently demonstrated (Chem. Phys. Lipids 110 (2001) 27). However, the role of thermal induced mechanical responses of phospholipid bilayer on the contact mechanics of liposome adhering on a cationic substrate has not been determined. In this study, confocal-reflectance interference contrast microscopy, phase contrast microscopy and contact mechanics modeling are applied to probe the adhesion mechanisms of liposomes in the presence of electrostatic interactions during the thermotropic transition of the lipid bilayer. When temperature increases from 23 to 49 °C at pH 7.4, the degree of liposome deformation ( a/ R) and adhesion energy of dipalmitoyl-sn-glycero-3-phosphocholine liposome increases by 10% and remains constant, respectively, on 3-amino-propyl-triethoxy-silane (APTES) modified substrate. The extents of increase in these two parameters are highly dependent on the physicochemical properties of the rigid substrate. At pH 4, the adhesion energies above and below the phase transition temperature ( T m) are increased by one order of magnitude due to the formation of the free silanol groups on APTES substrate. In hypotonic condition, the degree of vesicle deformation remains constant and the adhesion energy reduces by 20% during sample heating. Under all conditions, the adhesion energy of the adhering liposome spans a few orders of magnitude against the increase of liposome size as the surface area to volume ratio is maximized in smallest vesicle.