Tethered Fibronectin Liposomes on Supported Lipid Bilayers as a

Jun 22, 2012 - The construct can serve as a multifunctional platform for cell attachment and drug release. The successful fabrication of the FN-liposo...
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Tethered Fibronectin Liposomes on Supported Lipid Bilayers as a Prepackaged Controlled-Release Platform for Cell-Based Assays Po-Yuan Tseng and Ying-Chih Chang* Genomics Research Center, Academia Sinica, 128, Sec 2, Academic Road Nankang, Taipei 115, Taiwan ABSTRACT: A biomimetic construct containing an extracellular matrix protein−liposome composite tethered on supported lipid bilayers (SLBs) was formed with fibronectin (FN), and polyethylene glycol (PEG) and cholesterol-containing liposomes. The construct can serve as a multifunctional platform for cell attachment and drug release. The successful fabrication of the FN-liposome-SLB model platform was analyzed in situ with a quartz crystal microbalance with dissipation. The long-term stability of the surface tethered liposomes was measured via an encapsulated fluorescent probe. Less than 20% of the fluorescent probe content was released in 8 days, which compared favorably to the release of 90% of the probe content in one day from a similar construct made without PEG and cholesterol. HeLa cells were used to study the cellular interactions with the model platform. The extracellular matrix composition, FN, was found to be essential to promote HeLa cell adhesion on the liposome−SLB surfaces. Upon cell adhesion, the liposomes were spatially reorganized and absorbed by the cells. The rate of HeLa cell apoptosis was correlated with the surface density of doxorubicin-loaded liposomes, confirming the effective drug delivery through liposomes. The multifunctional model platform could be useful as preadministered, controlled-release platforms for cell- and tissue-based assays.

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INTRODUCTION Liposomes are well-known as delivery vehicles of drug molecules, proteins, nucleotides, and other substances to cells.1,2 For the systematic study of drug delivery from liposomes to adhered cells, it would be helpful to have the liposomes tethered to a substrate, such that the passage and dosage of the drug could be preadministered and the quantity could be controlled better than freely suspended liposomes. One strategy to immobilize liposomes on a surface with minimal impact from steric hindrance or deformation is to tether liposomes onto a supported lipid bilayer (SLB). Previously, the architecture, fabrication, and characterization of liposomes tethered to SLBs have been exploited.3−9 For example, it was found that liposomes can be tethered to SLBs through DNA hybridization3,10 or biotin-streptavidin (SA) binding6 while maintaining the intactness of liposomes and the fluidity of the SLB. These tethered liposome systems were subsequently used for the encapsulation of proteins for single biomolecule detection,4 as well as the reconstitution of membrane proteins to study protein−protein and protein− lipid interactions.3,10 Similarly, lipid vesicles tethered to SLBs have been used to analyze quantitatively surface-bound antibodies.6 In another example of the utility of tethered liposomes, it has been shown that immobilized liposomes encapsulating fluorescent probes on hydrogel-based surfaces can be highly stable, with a very low, consistent release of the probes over time, a feature that would be useful for the use of surface-tethered liposomes in drug delivery.11 However, previous findings have shown that lipid-based surfaces generally © 2012 American Chemical Society

provide poor medium for cell adhesion, resulting in subsequent cell death.12,13 The need for the liposome-SLB surfaces to enable cell adhesion is just one of many important requirements for liposome-SLB surfaces to serve as effective localized delivery platforms. Other basic criteria for successful design of liposome compositions, including the leakage proof, such that the materials intended for delivery to the cell would not be lost prior to adsorption of liposome by the cell. Also, the “leakage proof” property has to be optimized so that the liposome could eventually release the content inside the designated location such as intracellular plasma or nucleus. Finally, the delivered material must have the desired consequence, such as drug efficacy, without being hindered by the presence of the liposome or the SLB. In this work, we formed a biomimetic liposome-SLB platform in which the surface was modified with biocompatible synthetic polymers or extracellular matrix proteins to provide sites for cell adhesion. We used in situ quartz crystal microbalance with dissipation mode (QCM-D) to monitor the SLB formation and the subsequent binding of liposomes and fibronectin (FN). Once the FN-liposome-SLB system was formed, we tested the stability of liposomes by measuring the release rates of fluorescent probe molecules encapsulated within the liposomes. Liposomes consisting of polyethylene glycol Received: March 19, 2012 Revised: June 8, 2012 Published: June 22, 2012 2254

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buffer to form a solution with a concentration of 1 mg/mL) was mixed with 10 mM Sulfo NHS-LC-biotin dissolved in Milli-Q water for 30 min at room temperature. A hundred times molar excess of biotin solution was used to react with FN to generate bFN with approximately 10 biotins per FN molecule.14 Excess biotin was removed by dialysis in phosphate buffered saline for 24 h at 4 °C. Preparation of Substrates for Fluorescence Microscopy. Glass microscope coverslips were purchased from Deckglaser (Freiburg, Germany) and glass-bottomed dishes (WillCo-dish, 22 mm I.D.) were purchased from WillCo Wells B.V. (Amsterdam, Netherlands). Before surface treatment, the substrates were cleaned with 10% DECON 90 (Decon Laboratories Limited, England), rinsed extensively with Milli-Q water, dried under nitrogen gas, and exposed to oxygen plasma in a plasma cleaner (Harrick Plasma, Ithaca, NY, U.S.A.) operating at 100 mTorr for 10 min (5 min twice). The substrates were used immediately after being briefly rinsed with ethanol and dried under a stream of nitrogen gas. Construction of FN-Liposomes-SLB on Glass. The method for forming the surface tethered liposome platform is outlined in Figure 1. First, a cleaned glass substrate was exposed to 0.25 mg/mL of POPC/ b-PE liposome solution to form a SLB, followed by extensively rinsing with phosphate buffer (10 mM PBS + 150 mM NaCl, pH 7.2) to remove excess liposomes. Next, the SLB-coated surface was incubated

(PEG) and cholesterol (“b-PEG liposomes”) were evaluated in comparison with the liposomes without them. We then tested the cellular interaction of the system with HeLa cells. We analyzed the adhesion of the cells, and the possible uptake of the liposomes by the cells. Finally, we studied HeLa cells adhered on a FN-doxorubicin (DOX)-loaded liposomes-SLB surface to demonstrate the effectiveness of drug delivery.

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EXPERIMENTAL SECTION

Materials. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethylene glycol)-2000) (DSPE-PEG2000), cholesterol, and 1,2dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-cap-biotinyl (b-PE) dissolved in chloroform were purchased (except cholesterol in powder form) from Avanti Polar Lipids (Alabaster, AL) and used without further purification. Human plasma FN was purchased from Gibco/ Invitrogen Life Technologies (Carlsbad, CA). For fluorescence work, lyophilized Alexa Fluor 488 (succinimidyl esters) and Texas Red 1,2dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (Texas Red-DHPE), were purchased from Invitrogen (Carlsbad, CA) and used for liposome preparation after being dissolved in chloroform. Lyophilized streptavidin (SA, affinitypurified) derived from Streptomyces avidinii and DOX hydrochloride were purchased from Sigma (St. Louis, MO) and used after being solubilized in phosphate buffer containing 10 mM phosphate buffered saline (PBS), 150 mM sodium chloride aqueous solution, and 0.02% (w/v) sodium azide (NaN3, Sigma-Aldrich), with pH adjusted to 7.2. Sodium dodecyl sulfate (SDS) was purchased from J.T. Baker (Phillipsburg, NJ) and 0.1 M of SDS (dissolved in Milli-Q water) was used for substrate cleaning and storage. The water used in all experiments was obtained from a Milli-Q reverse osmosis system (Millipore, Billerica, MA) with a product resistivity of 18.2 MΩ. Preparation of Lipid Vesicles. Two different compositions of liposomes were prepared for this study: one for SLB formation and one for the surface-tethered liposome vesicles. The liposome used for SLB formation was composed of POPC and b-PE (0.1−4 mol % of bPE in the binary mixture). Chloroform solutions of the lipids were prepared in clean glass tubes with a lipid concentration of 5 mg/mL and dried while vortexing under a slow stream of nitrogen to form a thin, uniform lipid film. The lipid film was subsequently dried in a vacuum chamber overnight to remove residual chloroform. The dried POPC/b-PE film was then hydrated with phosphate buffer (10 mM phosphate buffered saline + 150 mM NaCl, pH 7.2) while being mixed vigorously and then extruded through first a 100 nm membrane and then through 50 nm Nuclepore track-etched polycarbonate membranes (Whatman Schleicher and Schuell, Dassel, Germany). Extrusion through each size of membrane was performed for a minimum of 10 times under 150 psi at room temperature, using a LIPEX Extruder by Northern Lipids, Inc. (Burnaby, BC, Canada), to generate homogeneous population of unilamellar vesicles. The surface-tethered “b-PEG liposomes” were composed of 63.0 mol % DSPC, 31.5 mol % cholesterol, 5.0 mol % DSPE-PEG2000, and 0.5 mol % of biotin-PE. The b-PEG liposomes were prepared in the same manner as the POPC/b-PE liposomes, except that they underwent 3−4 freeze−thaw cycles (liquid N2 to 75 °C) in the vortex before extruding through 200 nm membranes at 75 °C. The sizes of the liposomes were in the range of 65 ± 3 nm (N = 5) for the POPC/b-PE liposomes and 154 ± 5 nm (N = 5) for the b-PEG liposomes, as determined by dynamic laser light scattering (Zetasizer Nano ZS, Malvern Instruments, Germany). For visualizing b-PEG liposomes with fluorescence microscopy, fluorescent-labeled molecules were introduced either by incorporating 0.5 mol % of Texas RedDHPE in the lipid mixture or by encapsulating fluorescent-labeled molecules (1 mg/mL of Alexa Fluor 488, or 0.5 mg/mL DOX) in the b-PEG liposomes. Biotinylated FN (bFN). FN was biotinylated with sulfosuccinimidyl-6-(biotin-amido)hexanoate (Sulfo NHS-LC-biotin, Pierce, Rockford, IL).14 A solution of FN (reconstituted by sterilized PBS

Figure 1. Schematic illustration (not to scale) of FN-liposomes functionalized surfaces based on biotinylated lipid bilayer (FNliposome-SLB). Construction of the model surface involves five steps: (I) formation of SLB, (II) first layer of SA binding, (III) immobilization of b-PEG liposomes, which may incorporate dyelabeled lipids, or encapsulate DOX/fluorescent dye, (IV) second layer of SA binding, and (V) immobilization of bFN. 2255

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Figure 2. Kinetics of the build-up of the FN-liposome composite on the SLB, monitored by QCM-D. Biotinylated lipid vesicles containing 2 mol % of biotin were introduced at point (I) and absorbed and ruptured to form a SLB on the SiO2 surface. An SA solution was injected at point (II) into the QCM-D chamber for specific binding on the SLB, followed by b-PEG liposomes were flowed in at point (III) to create immobilized liposomes. Then SA solution was injected at point (IV) into the QCM-D chamber for specific binding on b-PEG liposomes, followed by coupling of bFN by introducing a bFN solution at point (V). Extensive rinsing with pH 7.2 PBS buffer, denoted as *, was performed after each step. The blue line and the red line represent the response curves of normalized frequency and dissipation, respectively. with a 0.1 mg/mL solution of SA (in excess) to form SA-SLB via SAbiotin recognition, followed by rinsing extensively with buffer to remove excess SA. The SA-SLB was then sequentially incubated with 0.5 mg/mL of b-PEG liposomes, 0.1 mg/mL of SA, and 0.05 mg/mL of bFN, with an extensive phosphate buffer wash between each coating step to remove excess unbound materials, yielding the FN-liposomeSLB on glass surfaces. For cell culture studies, the FN-liposome-SLBcoated substrate was sterilized using UV lights in a laminar flow hood (Thermo) for 6 h. Surface-tethered liposomes encapsulating dyes, DOX, or incorporating Texas Red-PE was visually analyzed with a Leica TCS SP5 confocal microscope and multiphoton imaging system (Leica, Wetzlar, Germany). QCM-D Measurements. All QCM experiments were performed using QCM-D D300 (Biolin Scientific AB/Q-sense, Sweden) as previously described.12,13 Silicon dioxide (SiO2) coated QCM crystal chips (AT-cut quartz crystals, f 0 = 5 MHz, Biolin Scientific AB/Qsense, Sweden) were cleaned in 0.1 M sodium dodecyl sulfate, followed by rinsing with Milli-Q water, drying under nitrogen, and exposing to oxygen plasma for 10 min. The concentration and the washing conditions of each liposome coating step in the QCM-D chamber were identical to those described in the previous section. For QCM-D measurements, the chamber was temperature-stabilized to 25 °C. All measurements were recorded at the third overtone (15 MHz), and the data shown here were normalized to the fundamental frequency (5 MHz) by dividing by the overtone number. Leakage Test of SLB-Tethered Liposomes. SLB-tethered liposomes containing 1 mg/mL of Alexa Fluor 488 were fabricated on the SLB with 2.0 mol % biotin-PE on circular glass slides (2.0 cm in diameter) in 12-well tissue culture polystyrene plates (TCPS; Costar, Corning Incorporated, New York, U.S.A.). At timed intervals, the released fluorescent probes were measured at room temperature in the following manner. For each sample, 1 mL of buffer (0.01 M PBS + 0.15 M NaCl + 0.02% (w/v) sodium azide, pH 7.2) was collected after rinsing the surface, 0.2 mL of which was analyzed by detecting fluorescent intensity using a Fluoroskan Ascent FL microplate fluorometer and luminometer (Thermo Fisher Scientific Inc.). After 144 h, the surface was rinsed with 1 mL of 0.5% (w/v) Triton X-100 solution to disrupt surface liposomes for measuring residual fluorescent intensity. Cumulative release of fluorescent probe over

time up to 144 h was analyzed. Each surface sample at timed points was analyzed in five replicates. Fluorescence Recovery after Photobleaching (FRAP). Prior to FRAP, an SLB was formed from fusion of lipid vesicles containing 2 mol % b-PE and 0.5 mol % Texas Red-DHPE (TR-PE; Molecular Probe, Eugene, OR, U.S.A.) on cleaned, hydrophilic glasses. A Leica TCS SP5 (Leica, Wetzlar, Germany) was used to perform the FRAP measurement. The 561 nm laser of the confocal microscope was used to bleach a circular region (15 μm in diameter), and all images from prebleach, bleach, and postbleach (recovery) were captured with a 110 s time interval. The recovery curves were analyzed by Leica TCS SP5 offline software. Cell Culture Studies. For regular cell cultures, the HeLa human cervical cancer cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (FBS, Hyclone Laboratories, Logan, UT), 2 mM Lglutamine, 100 units/mL penicillin, and 100 g/mL streptomycin (Invitrogen), on 100 × 20 mm (D × H) cell culture dishes (Corning, NY), in a humidified incubator with 5% CO2 at 37 °C. Cells were treated with 1× 0.25% trypsin (Invitrogen) at 37 °C for 3 min to detach them from the TCPS surface. In each trial, approximately 5 × 105 cells were added to a glass-bottomed WillCo-dish coated with SLB only, liposomes-SLB, or FN-liposomes-SLB and incubated with 1 mL of serum-free DMEM in a humidified incubator. The dishes were then rinsed with 1× PBS (Hyclone) three times to remove unbound cells. Cells were cultured on TCPS as positive controls. For cell cultures on DOX-loaded FN-liposome-SLB surfaces, serumfree DMEM was initially used until 6 h after cell culture, then serumcontaining DMEM was used to maintain regular culture conditions. Cell population was observed at timed points and cell density (per mm2) was determined by counting cell number in the observation field of pictures taken under 20× objective (image size: 0.41 × 0.54 mm2).

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RESULTS AND DISCUSSION Fabrication and Characterization of the Delivery Platform. Our fabrication scheme for the construction of a liposome-tethered SLB surface that is suitable for cell culture and drug delivery, the FN-liposome-SLB, starts with the 2256

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The negative dissipation shift could be explained by proteininduced stiffening of the surface materials, as a result of multivalent interactions of SA with both b-PEG liposomes and bFN, similar to a previous study showing the decrease in dissipation shift when bivalent antibody binds onto antigenconjugated surface lipid vesicles.6 Regardless, the multiple binding sites altered the mechanical properties of the delivery platform as shown by the dissipation change. In summary, the QCM-D study characterized the binding kinetics of each step and confirmed the successful fabrication of surface FNliposome-SLB formation. Stability Afforded by Surface-Immobilized b-PEG Liposomes. We incorporated b-PEG liposomes into the delivery platform with the expectation that they would provide stability to the liposomes, as well as to any drug or chemical contained within the liposomes. It is commonly accepted that stabilization of b-PEG liposomes results from their molecular shape19 and steric properties, which minimize interactions with proteins and cells.20,21 We analyzed the stability of our platform via the release kinetics of Alexa Fluor 488, which had been encapsulated into the liposomes. The fluorescent intensity of a rinse of the FN-liposome-SLB provided information on the release kinetics of Alexa Fluor 488. As shown in Figure 3, the

deposition of a lipid vesicle solution containing POPC and bPE onto a glass substrate, as shown in Figure 1 (step I). The vesicle solution forms a SLB, with biotin distributed across the top surface. The biotin on the top surface of the SLB serves as binding sites for SA, which is dispensed from solution onto the SLB in step II of the process. In step III of the process, a b-PEG liposome solution, which can also contain the desired drug or analytical chemical, is dispensed on the SA-modified SLB, resulting in the formation of b-PEG liposomes attached to the SA sites on the SLB. After the SA is bound to the vesicles (step IV), a bFN solution was coated on the SA-modified vesicles (step V). The bFN bonded to the SA sites on the vesicle, and provided the surface with cell adhesive sites, thereby completing the formation of the delivery platform. The formation of the delivery platform on a quartz crystal was monitored via in situ QCM-D. The points (I−V) shown in the QCM-D response in Figure 2 correspond to the process steps shown in Figure 1. The lipid vesicle solution containing POPC and b-PE was dispensed into the QCM chamber at point I. The normalized frequency change ΔF and dissipation shift ΔD of −26.7 Hz and 0.2 × 10−6, respectively, were typical of the formation of a highly uniform SLB.15,16 Followed by two buffer washes (denoted as * in Figure 2), a solution of SA was dispensed at point II. The change of frequency from the previous equilibrium point, ΔF = −37.7 Hz, and the final dissipation change, ΔD = 1.9 × 10−6, were consistent with ΔF and ΔD observed in previous work,5,15,16 confirming SA binding through the SA-surface biotin recognition. At point III, a b-PEG liposome solution was dispensed onto the SAmodified SLB, leading to dramatic shifts in both frequency and dissipation. Binding of b-PEG liposomes to SA-SLB resulted in equilibrated shifts in ΔF of −357.6 Hz and ΔD of 47.4 × 10−6, but the process was slower to reach an equilibrated value than in the previous step. Shifts in frequency and dissipation required about 1 min to reach half of the equilibrated values, while SA binding to SLB reached equilibrium almost immediately after introducing the SA solution.5 Interestingly, dissipation reached a maximum ∼5 min after loading the liposomes and then gradually decreased to about 5 × 10−6, while the frequency continued to decrease until 40 min after loading the b-PEG liposomes. During the next 35 min, the frequency rebounded, with a slight decrease in dissipation (∼ 1 × 10−6). We suspect that the decrease in frequency was caused by net desorption of liposomes and that the decrease in dissipation was possibly due to slight vesicle deformation caused by multivalent binding of the biotinylated lipid to the surface SA.17,18 After rinsing with buffer, shifts in frequency and dissipation decreased, suggesting the removal of excess, unbounded vesicles. Overall, significant difference in energy dissipation on dispensing liposomes after steps I and III clearly indicated the formation of a rigid SLB structure, and the hydrated, viscous liposome layers.5,6 At point IV, a SA solution was introduced and the saturated frequency shift ΔF was −10.8 Hz. In addition, it required ∼3 min to reach half of the equilibrated frequency shift, which was significantly slower than the adsorption of the first layer of SA onto SLB. The decrease in the amount of bound protein, and longer binding process time, could have been caused by steric hindrance by PEG molecules. At point V, the bFN solution was introduced to the system. The shift of frequency of −27.7 Hz occurred almost instantaneously. Interestingly, the dissipation shift was negative after binding the second layer of SA and bFN (about −2 × 10−6 for each step), indicating an increased rigidity of the surface.

Figure 3. Remaining fraction of Alexa Fluor 488 in immobilized bPEG liposomes vs POPC/b-PE liposomes based on SLB surfaces at room temperature. The Alexa Fluor content was measured for up to 8 days. Each measurement was performed five times.

SLB tethered b-PEG liposomes were much more stable than the SLB tethered POPC/b-PE liposomes, which are nonPEGylated liposomes. Surface-tethered b-PEG liposomes containing the dye revealed an initial fast release of ∼10% of the dye within 2 h, and then the dye concentration stabilized after 48 h (showing less than 20% of release up to 192 h observation). In comparison, the dye release from the POPC/ b-PE liposomes was much more instantaneous (around 80% of dye was released within 2 h and more than 90% after 48 h). The significant difference of release kinetics suggests that bPEG liposomes possess better stability on our model surfaces than the non-PEGylated liposomes. Our results are comparable to previous studies regarding release of carboxyfluorescein from immobilized liposomes on polymer-based surfaces.11,22,23 Lipid Mobility. The maintenance of lipid mobility in the SLB is important because it determines the surface dynamics, which have been shown to promote the multivalency between the surface and the cell receptors.13,24 As a result, we anticipate 2257

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Figure 4. (A) Diffusivity and (B) mobility fraction (%) of lipids on SLBs with a range of surface additives: pure SLB, SLB + 1st SA, SLB + 1st SA + b-PEG liposomes, SLB + 1st SA + b-PEG liposomes, SLB + 1st SA + b-PEG liposomes + 2nd SA, SLB + 1st SA + b-PEG liposomes + 2nd SA + bFN, and cells on SLB + 1st SA + b-PEG liposomes + 2nd SA + bFN (N = 5).

Figure 5. Differential interference contrast (DIC) images of HeLa cells on surface biomimetic platform in the absence (A) and the presence (B) of bFN. Cells on TCPS are shown as a control (C). Schematic representations of cells on each surface construct are illustrated at the above of corresponding DIC images: scale bar = 50 μm.

was due to the added mass of tethered liposomes, as well as multiple linkages of some b-PEG liposomes with the surface SA. The multiple linkages were a more distinct possibility given the size of a single liposome (150 nm in diameter) and projected area (∼ 20 nm2) of a single SA,26−28 thus, leading to clustering and consequently increasing the immobile fraction of SLB. Interestingly, further modification of liposome surface, including binding of bFN and adhesion of HeLa cells on top, did not further reduce the diffusion constant or the mobility of the lipids in the SLB. For example, when using the FNliposome-SLB construct to culture HeLa cells, we observed that the lipid diffusion coefficient and mobile fraction of the SLB under cell-covered areas was 0.33 ± 0.03 μm2/s and 34 ± 5%, respectively, similar to the values of the SLB lipids in the areas absent of cells. Nevertheless, the maintenance of lipid mobility in the construct suggests that the bilayer structures of lipids were retained even after the cell adhesion. In summary, the most dominant factor in retarding the mobility of the lipids in the SLB is the tethering of the liposomes, and the subsequent FN binding and cell adhesion do not attribute further

that the liposome-lipid would be recruited more effectively than that of an immobilized liposome-tethered film. The mobility of the lipids in the SLB in the presence of various portions of the platform structure (Figure 1), up to the complete platform structure, including attached HeLa cells, were characterized using the FRAP technique. The results of the FRAP analysis, expressed both as diffusion coefficients and mobile fractions, are presented in Figure 4. The calculated diffusion coefficient of the lipids in the pure SLB, without any additional attached elements, was 1.22 ± 0.13 μm2/s, which was comparable to lipid diffusion coefficients in previous studies.5,25 Lipids in the pure SLB were highly mobile, with a calculated mobile fraction of 88 ± 3%. After binding SA to the SLB, the diffusion coefficient and mobile fraction remained high, at 1.1 ± 0.20 μm2/s and 84 ± 3%, respectively, showing that lateral diffusion of lipids in the SLB was not significantly affected upon SA binding. After the addition of b-PEG liposomes to bind onto the SA-SLB surface, the lipid diffusion coefficient and mobile fraction decreased significantly, to 0.42 ± 0.02 μm2/s and 45 ± 8%, respectively. The reduced motion of lipid molecules in SLB 2258

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Figure 6. Confocal fluorescent images of HeLa cells adhesion on functionalized surfaces containing bFN and Texas Red-DPPE incorporated b-PEG liposomes. Merged phase contrast (of cells) and confocal fluorescent images of fluorescent liposomes focused on (A) the plane of liposome-coated area and (B) the plane of 3 μm above the liposome-coated area. Confocal fluorescent images are magnified to show intracellular ((C) and (D) framed in green) and borderline/extracellular ((E) and (F) framed in blue) liposome distribution. (C and E) Top views from 3D construction of confocal images. (D and F) Side views of (C) and (E), respectively, by flipping the top views 90° toward the paper. Images were taken after 5 h of cell seeding. Scale bars: 50 μm for (A) and (B); 5 μm for (C) and (E), 8.7 μm for (D) and (F).

compromise the cell attachment to the delivery platform. The ability of the cells to adhere to the delivery platform allowed us to study cell−liposome interactions, as described below. Redistribution of Surface Liposomes upon Cell Adhesion. To track the surface liposomes after cell adhesion in the bFNliposome-SLB platform by fluorescent visualization, we predoped the liposomes with Texas Red-PE (0.5 mol % in the lipid mixture). As shown in Figure 6, the confocal fluorescent images present views of a cell on the delivery platform, taken at both the surface planes of liposome-SLB surface (Figure 6A) and 3 μm above the liposome surface (Figure 6B). The surface liposomes after cell attachment were clearly redistributed in the vicinity of cell adhesion areas, where the dark region surrounding cells indicated the removal of liposomes (Figure 6A). Figure 6B further suggested the liposome moving “upward” along the cell surface, as shown by the red image distributed along the edges of the cell. The confocal fluorescent images were magnified to show the intracellular (Figure 6C,D) and borderline/extracellular (Figure 6E,F) liposome distribution. Figure 6C and E are top views from the 3D construction of the confocal images. Figure 6D and F are side views of Figure 6C and E, respectively, formed by rotating the 3D constructions used for the top views 90° toward the paper. The magnified confocal views (Figure 6C,D) suggested that the liposomes were concentrated inside the cell. The concentration within the cell was likely due to “pull in” of liposomes along with bFN by the cells, followed by cellular uptake of the liposomes (red) and possibly the bFN. A previous study showed that the rupture force to break a single bond of biotin-SA is ∼0.05 nN.38,39 In our case, there might be multiple biotin molecules conjugated with FN14 and multiple biotin-SA

retardation. Future study of replacing currently used SA-biotin binding agents might be helpful for improving the lipid mobility. Interactions of FN-Liposome-SLB Platform and HeLa Cells. Presence of FN on Cell Adhesion. To study the interaction of the liposome-SLB materials with cells, we examined the adhesion of HeLa cells on delivery platforms made with and without a top layer of bFN. To exclude the effect of serum proteins on cell attachment, cells were cultured in serum-free medium and their morphology was observed after 5 h of incubation. In the absence of surface bFN, the HeLa cells did not adhere to the delivery platform, as shown in Figure 5A. We believe the lack of adhesion was primarily caused by the nonbiofouling properties of PEG and lipids.29−34 In contrast, the majority of cells in the presence of bFN (Figure 5B) were the same as those on regular TCPS plates (Figure 5C) with spread, irregular shapes, demonstrating that surface modification with FN was essential for cell adhesion. There were some nonadherent cells in Figure 5B possibly reside on the area where there is no or insufficient liposome associated FN due to competition on FN which has been adhered by other cells earlier. Therefore, cells contacting directly on cell-resisting materials including PEGylated liposome or SLB become nonadherent. It has been recognized that FN is one of the most important extracellular matrix (ECM) proteins for cell adhesion.35−37 Significantly, our findings are consistent with previous studies that demonstrated that on SLB-based surfaces, cell adhesion is poor on pure SLB but dramatically improved in the presence of exogenously added ECM proteins such as collagen and FN conjugated onto SLB.12,13 Our cell culture results also show that the b-PEG liposomes did not 2259

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Figure 7. Effect of surface immobilized b-PEG liposomes encapsulating DOX on HeLa cell population over culture time after cell seeding for 5 h (A, E, I), 24 h (B, F, J), 48 h (C, G, K), and 72 h (D, H, L). (A−D) Cells cultured on model constructs with surface liposomes containing no drug. (E− H and I−L) Cells cultured on model constructs with surface liposomes encapsulating drug tethered onto SLB formed by 2 and 0.1 mol % biotinylated vesicle fusion, respectively: scale bar = 200 μm. (M) Effect of drug-loaded b-PEG liposomes tethered onto a variety of bilayer biotinylated lipid concentration on HeLa cell surface density over culture time. Cell surface density was determined by counting cell number in the observation field of pictures taken under 20× objective (sample size = 5).

quantity of the DOX-loaded liposomes. Figure 7A−D are the representative images of HeLa cells grown on a drug-free FNliposome-SLB for comparison. As anticipated, drug-free surfaces had no effect on cell apoptosis, where cell density peaked after 2 days of incubation and decreased afterward, owing to insufficient nutrient from the medium when the cells were overpopulated (cell numbers were counted with time, as summarized in Figure 7M). In contrast, the cell survival rate was mediated by the density of DOX-loaded liposomes. Significantly, cells were eliminated after 3 days on the bFNDOX-loaded liposome-SLB (containing 2 mol % of b-PE; Figure 7E−H), while cells survived on bFN-DOX-loaded liposome−SLB (containing 0.1 mol % of b-PE; Figure 7I−L). As summarized in Figure 7M, HeLa cell viability is dependent on the amount of surface-bound DOX-loaded liposomes. For delivery platforms with surfaces based on SLB formed by more than 1 mol % biotinylated vesicle fusion, cell density was dramatically reduced (approximately 75% compared to drug-free systems) after initial seeding for 5 h, followed by a gradual decrease over time until 72 h. In contrast, cell density was generally high at all times for platforms containing less than 0.5 mol % of b-PE, indicating that the drug-loaded liposomes in those cases had no significant inhibitory effect on cell proliferation.

binding sites on the SLB, liposomes, and FN. Therefore, the combined strength of the bonds is expected to be possibly on the order of ∼1 nN or higher, based on studies of multivalent parallel antigen−antibody interactions.40 Maskarinec et al. investigated cellular traction forces by observing Swiss 3T3 fibroblasts migration on FN-modified polyacrylamide gels.41 They observed simultaneous push and pull behavior for migrating cells and quantified the traction force up to ∼102 nN. Although the traction force may be dependent on celladherent substrate and cell type, previous studies in strength of biological bonds and forces involved in cell adhesion and migration may provide an explanation for ripped surface materials by cells. Therapeutic Effect of Drug-Loaded Surface Liposomes. The fluorescent studies indicated that the bFN-liposome-SLB is a suitable platform for cell adhesion and subsequent liposome uptake. To further test the efficacy of drug delivery with the platform, we cultured and monitored the adhesion of HeLa cells directly on a bFN-liposomes-SLB platform in which the liposomes were loaded with 0.5 mg/mL of DOX. DOX is an anticancer drug that is known to cause HeLa cell apoptosis through DNA damage. PEGylated liposomes encapsulating DOX have been administrated to treat cancers.42−44 To study the DOX dosage effects, we varied the surface SLB compositions of b-PE from 0−0.1 to 2.0 mol % to adjust the 2260

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Figure 8. Confocal fluorescent images of HeLa cell adhesion on functionalized surfaces containing bFN and DOX-encapsulated b-PEG liposomes. Merged phase contrast (of cells) and confocal fluorescent images of liposomes focused on (A) the plane of liposome-coated area and (B) the plane of 3 μm above the liposome-coated area. (C and D) 3D construction of confocal fluorescent images of fluorescent doxorubicin from the cropped area in (A) or (B). (C and D) 3D construction of confocal images: (C) is top view and (D) is side view of (C) by flipping the top view 90° toward the paper. Images were taken after overnight of cell seeding: scale bars: 50 μm for (A) and (B); 5 μm for (C); 8.7 μm for (D).

excitation peak near 595 nm, shown as pseudo-orange color in Figure 8) was taken up by HeLa cells during cell culture on the model surface. Specifically, fluorescent signals inside a HeLa cell indicate that DOX molecules were mostly located in the cytoplasm and a small amount of them were inside the nucleus (arrows in Figure 8B,C). This decrease in the intensity of the fluorescent signal when going from the cytoplasm to the nucleus is to be expected. We suspect that DOX molecules in the nucleus were released free forms from liposomes because free DOX is positively charged and readily binds and diffuses through membranes to accumulate in nuclear domains.46,47 In contrast, DOX-loaded liposomes are believed to be taken up dominantly by endocytosis46,48 and consequently reside in the cytoplasm, although we do not exclude the possibility of free DOX existing in the cytoplasm due to membrane fusioninvolved liposomal delivery. Beyond our work shown here, there are many potential approaches for expanding the capabilities of SLB-tethered liposome platforms for cell assays and tissue engineering. For example, the fluidized liposome-SLB surfaces could be modified with a variety of functional groups, such as transmembrane proteins, antibodies, glycol receptors, or peptides, to provide biomimetic cellular surfaces for multivalent studies. Additionally, various active compositions such as drugs, growth factors, serums, and dyes, could be encapsulated in liposomes and delivered to adhered cells. Building on this work, the controlled-release rate of liposomes could be further mediated by control of the compositions of the lipid contents, thus, enabling long-term release. Furthermore, because the liposomes are tethered on the surfaces, one can freely exchange the cell cultured medium without altering liposomal contents.

The effectiveness of the DOX-loaded delivery platforms in reducing the HeLa cell population can be understood in terms of the surface DOX concentration in the liposomes. For our delivery platform, given the condition of using 1 mL of DOXencapsulated b-PEG liposomes (single liposome size ∼150 nm with volume of ∼1.4 × 10−15 ml and 0.5 mg/mL of drug concentration) for coating on a surface area of 3.8 cm2, and immobilized liposomes on SLB formed by vesicle fusion containing 1 mol % of biotinylated lipids, which provides biotin binding sites that could accommodate as high as 5.4 × 1012 liposomes on the surface, we estimate the surface DOX concentration to be 1.8 × 104 fmol/mm2 (Mw of DOX: 579.98 g/mol). For an initial cell surface density of ∼2000 cells/mm2, based on cell seeding on the drug-free surface, as shown in Figure 7M, the drug level in cells could be as high as 8.8 fmol/ cell on average. In this case, we observed that 10% of cells survived after 24 h of seeding, which is comparable to previous studies using free liposomes. For example, Eliaz et al. revealed the effect of free DOX-loaded liposomes on B16F10 melanoma cell viability and showed that less than 10% of cells (initial cell number was 2.5 × 105) survived after 27 h of a 3 h long exposure to 5 μg/mL of DOX encapsulated within hyaluronantargeted liposomes. In their work, the uptake drug level was 8.6 fmol/cell.45 For surfaces containing SLB formed by vesicle fusion containing less than 0.5 mol % of biotinylated lipids, especially for 0.1 mol %, the drug level in the cells could be less than 1 fmol/cell, resulting in more than 50% survival of cancer cells after 24 h of seeding (Figure 7M). Our observations suggest that the model surface with controllable amount of drug-loaded liposomes can be useful as an alternative for evaluating therapeutic efficiency of drug dosage on cancer cells. Fluorescent images further confirmed that the DOX-loaded liposome (fluorescent with excitation peak near 485 nm and 2261

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CONCLUSION In summary, we used a bFN-liposome-SLB composite system to demonstrate the feasibility of drug delivery through surfacetethered liposomes directly to adhered cells. We determined that the liposome needs to have cholesterol and PEG to be leakage-proof and that tethered biotinylated FN on liposomes is important for cell adhesion. After cells were attached to the composite surface, surface liposomes were observed to be locally redistributed, including surrounding the cell adhesion area, accumulating on the cell surface, and being internalized by the cells. Drug-loaded liposomes were used to evaluate the effect of surface drug dosage on cell survival and quantify the dosage required for cell death. While our work here focused on drug delivery, the FN-liposome-SLB composite could serve as a platform for tissue engineering or studying the effect of growth factor or other substances on cells. Additionally, the platform could be customized by varying ECM proteins and lipid compositions, including the specific receptors for targeting delivery, making the model surface versatile for studying cell− cell and cell−biomaterial interactions and desired biomedical applications.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +886-2-27871277. Fax: +886-2-27899931. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Genomics Research Center, Academia Sinica, Taiwan, for the financial support. REFERENCES

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