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J. Phys. Chem. B 2009, 113, 1552–1558
ARTICLES Dielectrophoresis of Functionalized Lipid Unilamellar Vesicles (Liposomes) with Contrasting Surface Constructs Victoria E. Froude and Yingxi Zhu* Department of Chemical and Biomolecular Engineering, UniVersity of Notre Dame, Notre Dame, Indiana 46556 ReceiVed: September 23, 2008; ReVised Manuscript ReceiVed: December 4, 2008
Dielectrophoresis (DEP) has been exceedingly exploited recently as an effective technique for rapid manipulation, separation, and assembly of biological particles including cells, proteins, and DNAs. However, optimizing of the DEP process with biocolloidal suspensions remains challenging, mainly due to inadequate understanding of AC polarization of complex biological structures. In this paper, we examine the DEP behavior of model functionalized lipid unilamellar vesicles (liposomes) with distinct exterior surface properties and compare the measured crossover frequency, ωc, with the theoretical prediction based on the Maxwell-Wagner interfacial polarization mechanism for a shell structure. With a uniform shell coating of calcium phosphate, we observe a drastic decrease in ωc compared to the measured value for plain liposome particles. In sharp contrast, with the patchy surface created by the adsorption of oppositely charged nanoparticles on liposome outer surfaces, we observe that ωc is independent of nanoparticle surface coverage, despite the considerable change in liposome surface conductivity by increasing adsorbed nanoparticles. Our results indicate the particle construct, rather than surface conductivity, plays a critical role in the DEP behavior of a shell particle, which is remarkably different from a solid dielectric particle. 1. Introduction The application and integration of dielectrophoresis (DEP) in a nonuniform AC electric field with microfluidic techniques has recently emerged as a rapid and effective method to manipulate, separate, and assemble synthetic and biological colloids as well as cellular complexes. For instance, DEP-based separation protocols have been developed to discriminate E. coli and M. luteus bacteria1 or blood cells of varied starvation age.2 DEP alone or combined with other AC electrokinetic techniques over various complex microelectrode designs3-5 has been employed for the synthesis and assembly of functional biomaterials, such as biocomposites from assembling living cells with functionalized colloids6 or drug encapsulation and release in lipid vesicles under applied AC electric fields.7,8 However, the precise and controllable process with dielectrophoretic optimization remains one of the highly promising yet much unrealized goals for biocolloidal and cellular manipulation, mainly due to insufficient understanding of the DEP behaviors of complex biological systems, whose complexity ranges from biological constructs of multicomponents and varied chemistry to ionic aqueous environments, all sensitive to the imposed AC electric fields. In this work, we examine the DEP responses of a model biocolloidal suspension, lipid unilamellar vesicles (liposomes) of varied surface constructs suspended in aqueous media. Liposomes, synthetic lipid-based spherical vesicles, have been heavily studied for applications as model cell membranes, analytical markers, and therapeutic drug carriers due to their * To whom correspondence should be addressed.
hollow, aqueous interior core capable of encapsulating small volumes of various molecules and biomacromolecules such as proteins, DNAs, and drug molecules.9-11 Currently, liposome technology has expanded from therapeutic drug treatment to cosmetic and food processing, which has led to vital research in liposome stabilization and functionalization. For instance, various stabilization themes have been proposed and examined to stabilize liposomes by using antibodies,12 polymers,12,13 or charged nanocolloids14 as well as biodegradable, inorganic materials15 as exterior surface coatings to prevent fusion and encapsulate leakage. Therefore, liposome particles are of great interest to us as a model biocolloidal system to examine and understand the DEP behaviors of complex cellular membranes in response to applied AC fields, where distinct liposome constructs can be easily designed and synthesized to effectively tune the interfacial dielectric and conductive properties of functionalized liposome particles. In this work, we modify the outer liposome aqueous interface with two contrasting surface constructs, namely, patchy nanocolloidal attachment versus a uniform shell of calcium phosphate coating, and thereby examine the effects of interfacial conductivity and morphology on the AC-induced polarization and DEP behavior of liposome particles in comparison to the classical DEP shell model for cellular membranes. By using confocal laser microscopy for direct and in situ fluorescence imaging, we examine the assembly of liposome particles of varied surface constructs at varied AC field frequencies and determine their crossover frequency, ωc, upon the transition from positive DEP to negative DEP. The measured ωc of functionalized liposomes is thus examined against varied
10.1021/jp808454w CCC: $40.75 2009 American Chemical Society Published on Web 01/15/2009
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Figure 1. (a) Size distribution of unmodified plain liposomes as determined by light scattering, (b) schematic diagram of the DEP experimental setup, and (c) schematic diagram of quadrupole electrode arrays where high-field regions are located adjacent to the electrode post walls and the low-field region is located in the center of the electrode array.
surface conductivity and multilayer coatings and compared with the theoretical prediction based on the Maxwell-Wagner interfacial polarization mechanism for a shell structure. 2. Materials and Methods 2.1. Liposome Synthesis and Surface Modification. Liposome synthesis. Liposomes are synthesized via the commonly used extrusion technique as described in detail elsewhere.16 1,2Dioleoyl-sn-glycero-3-phosphate (DOPA) and fluorescencelabeled 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7nitro-2-1,3-benzoxadiazol-4-yl) (DOPE) (λex) 445 nm, λem) 507 nm) lipids are obtained in the form of lyophilized powders in sealed vials from Avanti Polar Lipids. The two lipids in a DOPA/DOPE powder weight ratio of 10:1 are well mixed in deionized water (Barnstead Nanopure II). After being stirred at 600 rpm for 24 h at room temperature, the suspension of hydrated lipids is subsequently extruded repeatedly five times through a microextruder (Avinti Polar Lipids) with a polycarbonate filter of 1 µm pore diameter. After extrusion, the liposome suspension is diluted in deionized water to approximately 0.05% in liposome particle volume fraction. The average liposome radius and its size distribution are determined by dynamic light scattering (Brookhaven Instruments, ZetaPALS) with typically three repeated runs. As shown in Figure 1a, liposomes of a ) 140 ( 23 nm with a polydispersity of 0.165, also confirmed from fluorescent micrographs as shown in Figure 3, are used in this work. The surface conductivity of liposomes is characterized by a zeta potential analyzer (Brookhaven Instruments, Zeta PALS), averaged over typically 20 repeated measurements. Liposome Functionalization. To stabilize liposomes from fusion, two stabilization strategies are employed, which also results in the distinct construction of liposomes of varied surface conductivity and morphology. Amine-functionalized latex nanoparticles of 20 nm in diameter (Molecular Probes Inc.) electrostatically adsorb onto the liposome exterior surface via a protocol described in detail elsewhere14 to stabilize liposome particles and also effectively vary surface conductivity with increased nanoparticle coverage up to 20%. In this work, the volume fraction of nanocolloidal particles added into a liposome suspension of ∼0.05% in
Figure 2. (a) Schematic illustration of the concentric multilayers of liposome particles and (b) the theoretical Clausius-Mossotti (CM) factor versus the AC field frequency, ω, for a plain liposome of a1)150 nm (squares) and one coated with a uniform calcium phosphate layer of 10 (circles), 25 (upper triangles), and 50 nm (down triangles) thick by using the values listed in Table 1 and eqs 4-6. (Inset) Theoretically predicted dependence of crossover frequency with calcium phosphate shell thickness.
liposome volume fraction is varied from 0.015 to 2.5%, which results in the considerable increase in the zeta potential of the liposome-nanocolloid complex (see Figure 5a below). The mixtures are vortexed for 1 min and allowed to rest for 24 h before the DEP experiments. A sample’s zeta potential measurement is an average of 20 cycles with the mean value and standard deviation in mV. Alternative to using nanoparticles for liposome stabilization, calcium phosphate is coated on the exterior surface to form a uniform thin shell,15 in contrast to the patchy surface coating by nanocolloids. The thickness of the calcium phosphate shell is controllable by varying the reaction time before capping by carboxyethylphosphonic acid (CEPA) through the following coating procedure with chemicals all purchased from Aldrich (purum grade >95%): 100 µL of 0.1 M CaCl2 and 150 µL of plain liposomes are simultaneously added to a solution of 50 mL deionized water, 10 µL of 1 M phosphoric acid, and 40 µL of 1 M NaOH. The suspension is stirred at room temperature for 10-150 min to vary the thickness of the calcium phosphate shell, and after the reaction is stopped, the shell is stabilized by adding 50 µL of 0.1 M CEPA. Subsequently, the solution is stirred for 24 h and then cleared of all extraneous salts by dialysis for 3 days in 10 000 MWCO dialysis membranes with
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Figure 3. (a) Representative fluorescence micrograph to illustrate the collection of liposome particles under pDEP after an AC field of 5 Vpp and 250 kHz is applied to plain liposomes suspended in deionized water for 30 s. Under pDEP, liposomes preferentially aggregate at the electrode edges with the formation of pearl chains, as shown in panel (b), with higher magnification at the early stage and subsequently fill in the gap between two microelectrodes over time.
refreshed deionized water. In this work, the reaction time is typically 60 min, which also results in modifying the surface zeta potential from -55 to -44 mV for plain and CP-coated liposomes, respectively. 2.2. Experimental Setup. The DEP experimental setup is schematically depicted in Figure 1b. A clean plastic sample cell, injected with functionalized liposome suspension, is sealed with UV optical glue onto a glass coverslip surface embedded with microelectrode arrays. As shown in Figure 1c, arrays of four microelectrodes in a quadrupole configuration are fabricated onto a glass coverslip (Fisher Scientific) using the photolithographic technique as described elsewhere.17 The glass substrate is first spin-coated with a thin layer of hexamethyldisilazane (HMDS) followed by a thin layer of photoresist (PR5413) coating before the microelectrode pattern is imposed by ultraviolet light. Subsequently, four triangular gold thin posts separated by 20 µm between each other are symmetrically deposited by the method of chemical vapor deposition onto a glass coverslip that was predeposited with a thin layer of titanium of ∼7.5 nm thick to enhance gold-glass bonding. The fabrication is completed by photoresist dissolution and metal liftoff in acetone. The center of the electrodes is connected to a function generator (Agilent 33220A) via copper tape and wire. The quadrupole electrode array produces an electric field of absolute minimum at the center of the array and absolute maximum along the edge of the electrodes.17 During pDEP, liposomes are expected to aggregate along the electrodes and between adjacent poles, leaving the center region void of particles where a field minimum is experienced. As liposomes exhibit zero or weak nDEP, as the AC field frequency approaches the crossover frequency according to the classical DEP theory described in section 3, they will be repelled from the electrode and redisperse into the bulk suspension. An AC electric field of constant 5 Vpp at varied frequencies, ω, from 25 kHz to 20 MHz, is applied across the array by the function generator. 2.3. Characterization and Analysis. We visualize the motions and structural evolution of DEP-induced liposome assembly by using confocal laser scanning microscopy (Zeiss LSM 5 Pascal) with a 100× objective lens (NA ) 1.4, oil immersion) at real-time and single-particle resolution. Time series fluorescent micrographs are taken at the resolution of 512 × 512 pixels2 over an area of 50 × 50 µm2. Liposome surface coverage at a fixed AC field frequency is determined based on the fluorescent intensity using the MetaMorph image analysis algorithm (Meta Imaging Series 7.0) in the area between the four electrodes
above the glass substrate to distinguish particle surface coverage at varied AC field frequencies. 3. Theoretical Prediction A dielectric particle suspended in a medium exposed to a nonuniform AC electric field can be polarized and moved toward the high or low field points, depending on its induced dipole with respect to the applied field, as described by dielectrophoresis (DEP). The DEP force, FDEP, exerted on a colloidal particle (p) of radius a and complex permittivity ε˜ p, suspended in a medium (m) of ε˜ M is developed from the interfacial polarization mechanism based on the classical Maxwell-Wagner (M-W) theory,3 and the time-averaged FDEP is given by
brms | 2 〈FDEP〉 ) 2πεMa3 Re[ fCM(ω)]∇|E
(1)
where the dipolar Clausius-Mossotti (CM) factor
fCM ) (ε˜ p - ε˜ m)/(ε˜ p + 2ε˜ m)
(2)
determines the orientation of induced colloidal dipoles and is frequency-dependent as ε˜ ) ε - iσ/ω, where ε is the static permittivity and σ is the conductivity.3 The particle moves toward the high-field region when Re[fCM(ω)] > 0, referred to as positive DEP (pDEP), and toward the low-field regions when Re[fCM(ω)] < 0, referred to as negative DEP (nDEP). Thus, a crossover frequency, ωc, exists as
ωc )
1 2π
(σp - σm)(σp + 2σm) (εm - εp)(εp + 2εm)
(3)
at Re[fCM(ω)] ) 0, where fCM changes its sign. For the shell structure of liposomes, as well as many other biological particles such as blood cells, the polarization and CM factor for the concentrically multilayered particle must be modified to account for the permittivity and conductivity of each layer in the particle construct. As depicted in Figure 2a, a plain liposome can be considered a single-shell sphere with a lipid bilayer membrane of ∼5 nm in thickness and finite radii, a1 and a2, permittivity, εIM and εLB, and conductivity, σIM and σLB, for the interior medium (IM) and lipid bilayer (LB), respectively. The multishell model combined with classical Maxwell-Wagner
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TABLE 1: Dielectric Properties for DEP Theoretical Calculation of Functionalized Liposome Particles σ (S · m-1)
ε inner medium (IM) lipid bilayer (LB) calcium phosphate (CP) outer medium (OM)
a (nm)
-2
1.4 × 10 4 × 10-9 6 × 10-6 5 × 10-5
80 10 50 80
145 150 200
4. Experimental Results and Discussion
18
theory, first described by Irimajiri, approximates the particle with one effective permittivity, εLB-IM, in terms of the dielectric properties of the IM and LB layers
( (
ε˜ LB-IM ) ε˜ LB γ312
) )
ε˜ IM - ε˜ LB ε˜ IM + 2ε˜ LB ε˜ IM - ε˜ LB ε˜ IM + 2ε˜ LB
γ312 + 2
(4)
with γ12 ) a1/a2 defined as a ratio of the outer to inner radius of the spherical shell.3 The CM factor is thus modified accordingly as
fCM )
ε˜ LB-IM - ε˜ OM ε˜ LB-IM + 2ε˜ OM
(5)
where ε˜ OM is defined as the complex permittivity of the outer aqueous medium. It is apparent in this shell model that each interface separating the dielectric regions can contribute a crossover frequency according to the Maxwell-Wagner interfacial polarization mechanism. In some cases of surface modification, for instance, coating the liposome particle with another uniform layer can once again modify fCM to account for the additional layer, and multiple crossover frequencies can be expected; yet, each depends on the relative permittivity and conductivity between each of two layers. Now, the previously described CM factor equation in eq 5 for shell models can still be used, but ε˜ LB-IM is replaced by ε˜ CP-LB that is given as
( (
ε˜ CP-LB ) ε˜ CP γ323
) )
ε˜ LB-IM - ε˜ CP ε˜ LB-IM + 2ε˜ CP ε˜ LB-IM - ε˜ CP ε˜ LB-IM + 2ε˜ CP
γ323 + 2
fCM factor of a shelled particle such as liposomes can be effectively modified by varying their surface functionality and the construct of liposomes as well as the conductivity of the inner medium by encapsulates.
(6)
where γ23 ) a2/a3, as illustrated in Figure 2a. Thus, each of the concentric multiple interfaces contributes to the overall DEP behavior of the particle in suspension and might result in additional crossover frequencies, which yet depends on the relative permittivity and conductivity between the adjacent interfaces. By combining eqs 4-6 and using the values listed in Table 1, the CM factors for plain liposomes and calciumphosphate-coated multilayer liposomes can be obtained versus the AC field frequency, as shown in Figure 2b. In addition, the polarization of multilayered biocolloidal particles strongly depends on the interfacial conductivity.19 For instance, it is reported that the shell model to describe the DEP behavior of a cell with highly active ion channels reduces to the classical Maxwell-Wagner theory for a solid dielectric sphere when the conductivity of the cell membrane approaches that of the inner cytoplasm medium; it thus suggests that the
Plain Liposomes. We first start with the DEP-induced assembly of plain liposome particles of a )150 nm suspended in deionized water of σm ) 5.6 × 10-5 S m-1 at varied AC field frequencies ranging from ω ) 100 kHz to 20 MHz. The typical assembly is shown in Figure 3a at applied AC field frequency ω ) 250 kHz and peak-to-peak voltage Vpp ) 5 V. We observe that liposome particles are rapidly collected at the edges of the four microelectrodes, that is, the high-field region, indicating dominant pDEP. Shortly after the AC field is applied to the liposome suspension, pearl chains of liposome particles are observed, as shown in high magnification in Figure 3b, suggesting that the mutual DEP attractive force, resulting from the parallel alignment of induced dipoles of particles to the axis of applied field lines, is strong enough to overcome interparticle electrostatic repulsion.20,21 With the continued accumulation of liposome particles near the microelectrodes under pDEP, pearl chains grow to bridge across the gap between two microelectrodes until the space is completely filled with fluorescent liposomes, except the center depletion region (i.e., see Figure 3a), which is the lowest field region according to the numerical analysis of the field strength distribution.3 The liposome accumulation reaches a nearly steady state after approximately 6-8 min at the applied field of Vpp ) 5 V, and all of the fluorescent micrographs are acquired over a period of at least 10 min to ensure the maturity of the liposome assembly with the presence of applied AC fields at a given frequency. Additionally, we wait at least 3-5 min between two consecutive applications of AC fields at varied frequencies to allow the liposome particles with a Brownian diffusivity, D, on the order of 0.5-2.5 µm2/s to dissemble and redisperse into the suspension. It is noted that upon pDEP-induced liposome assembly, single liposome particles can be spatially distinguished, as exhibited in Figure 3, suggesting no propensity to fusion with neighboring liposomes. We also are aware of colloidal assembly induced by other AC electrokinetic effects such as AC electroosmosis (ACEO),3,20-22 which is also often used to manipulate and concentrate particles in aqueous media with the presence of electrolytes by the movement of the suspension fluid. AC-EO flow is produced between two microelectrodes that can be polarized to form an AC-field-induced double layer that causes the fluid flow. According to the capacitive charging theories,2,22 the optimum frequency for AC-EO flow scales with D/λL, where D is ion diffusivity and ∼1.31 × 10-9 m2/s for Na+, which might exist in the aqueous media from the liposome synthesis, λ is the Debye length, and L is the spacing between two electrodes and equal to 20 µm for this work. As λ is constant at ∼1 µm in deionized water in this work, the optimum ACEO frequency is ∼70 Hz and far below the AC frequency range we apply. With this work focusing on the aqueous medium of low conductivity, electrothermal flow and the Joule heating effects, which, similar to AC-EO, result from the double-layer charging at the microelectrode interfaces, are also negligible. Below, we thereby focus on the DEP behavior of stabilized liposomes of varied surface chemistry. To understand the polarization of liposome particles in AC fields, we examine the ωc of functional liposomes stabilized with varied surface chemistry. The ωc is experimentally
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Figure 4. Fluorescence micrographs of the DEP-induced assembly of liposome particles at an applied AC field of constant amplitude at 5 Vpp and varied frequency of (a) 500 kHz, (b) 3 MHz, (c) 15 MHz, and (d) 18 MHz. Panels (a-d) indicate the decreased liposome coverage between microelectrodes with increasing AC frequency, where the transition from pDEP to nDEP takes place; (e) simulated AC electric field gradient between the quadruple microelectrodes by using Gauss’s Law with COMSOL;3 (f) the illustration of image analysis corresponding to the original fluorescence micrograph shown in panel (a) by using MetaMorph to determine the liposome coverage between microelectrodes based on the total areas of fluorescence intensity; (g) liposome surface coverage against the applied AC frequency for a plain liposome of a1 ) 150 nm. A crossover frequency, ωc, is thereby defined as the frequency extrapolated from the liposome coveragesfrequency plot where liposome coverage decreases to reach the saturation of typically less than 20%. The determined ωc for a plain liposome of a1 ) 150 nm is ∼18 MHz, in good agreement with the theoretical prediction as indicated in Figure 2b.
determined according to the frequency-dependent surface coverage of liposome accumulation in the space between microelectrodes, as illustrated in Figure 4. When the applied AC frequency is increased from ω ) 100 kHz to 20 MHz at constant Vpp ) 5 V, collected fluorescent liposomes become repelled away from the microelectrode edges and diffuse into the aqueous suspension, indicated by the reduced fluorescent intensity as shown in Figure 4a-d and consistent with the simulated AC electric field gradient between quadruple microelectrodes shown in Figure 4e. A representative image analysis is shown in Figure 4f, where the regions of high fluorescence intensity are determined by using MetaMorph on the original fluorescence micrograph in Figure 4a to determine the percentage of liposome coverage over the same scanning area at varied AC field frequency. As shown in Figure 4g, the fraction of liposome coverage over the gap between four microelectrodes exhibits a strong dependence on the applied AC field frequency. The frequency where coverage reaches less than 20% of the scanning area and saturates at higher frequency is determined as the ωc of liposomes of a give size and chemical construct. The ωc ) 18 ( 1.5 MHz is determined from Figure 4g for the plain liposomes of a )150 nm, which agrees well with the theoretical prediction of ∼19.7 Mhz as indicated in Figure 2b by using the values listed in Table 1. It should be noted that complexity in colloidal DEP behaviors as well as the deviation of measured ωc could arise when the presence of a neighboring colloidal assembly leads to variations in the mutual polarization of individual particles. In addition, the observed saturation in surface coverage, instead of a decrease at ω > ωc, confirms zero DEP as a hallmark of a shell structure, instead of strong nDEP usually observed for solid dielectric particles, as a result of the AC field penetration through the exterior and interior media of the same permittivity (εIM ≈ εOM ) 80) across the lipid bilayer interfaces at high AC frequency where permittivity, instead of conductivity, dominates the AC polarization of colloidal par-
ticles. It is also noted that we do not observe the low ωc of ∼10-20 kHz as theoretically predicted in Figure 2b for plain liposomes mainly because of its overlap with the frequency range of charge relaxation and AC-EO flow that we strive to avoid in this DEP experiment, as the applied AC frequency typically starts from 20-100 kHz in this work. Calcium-Phosphate-Coated Liposomes. We first examine the DEP behavior of liposomes coated with a uniform conductive shell of calcium phosphate (CP). With the CP shell coated on the outer layer of liposomes, it is expected that a concentrically multilayer shell model as predicted in eqs 4-6 can be employed. The theoretical CM factor of the CP-coated liposome is presented in Figure 2b with the values in Table 1, giving a new ωc ∼ 7.8 MHz for the liposome coated with a uniform CP layer of ∼50 nm thick; given the varied CP shell thickness, ωc can be significantly reduced with increasing CP shell thickness, as indicated in Figure 2b. Furthermore, the addition of the CP shell, with a higher permittivity and conductivity than those of the lipid bilayer, can significantly decrease the pDEP force at both the low and high ωc region. For liposomes coated with a CP shell of ∼50 nm thick, which is estimated from the reaction time,15 the experimentally measured high ωc is reduced to ∼3 MHz, in sharp contrast to the high ωc ∼18 MHz for plain liposomes, suggesting that the addition of a dielectric layer with different permittivity can greatly modify the DEP behavior of liposomes. Although the measured high ωc ∼ 3 MHz is lower than theoretically predicted, it qualitatively agrees with the predicted trend of lowering the high ωc with the additional CP shell coating. Moreover, we observe a low ωc of ∼100 kHz for CP-coated liposomes, in good agreement with the theoretical prediction. As the CP layer thickness is increased by lengthening the CP coating reaction time from 10 to 150 min, the low ωc is observed to shift to higher frequency. Therefore, with liposomes coated with a uniform CP shell, we observe that both low and high ωc are modified by the CP shell coating. However, due to
Dielectrophoresis of Functionalized Liposomes
J. Phys. Chem. B, Vol. 113, No. 6, 2009 1557 whose related double-layer effect on the DEP behaviors has been recently examined.23,24 In this work, we mix latex nanoparticles (NP) with the plain liposome aqueous suspension after repeated microextrusion with the volume fraction, φNP, of NPs in the mixed aqueous solution ranging from 0 to 2.5%. As shown in Figure 5a, the measured zeta potential, ζ, of liposomes in the suspension with oppositely charged NPs added increases greatly by adding a small amount of NPs; plain liposomes have a strongly negative zeta potential of ∼-55 mV due to the anionic head group of DOPA, in stark contrast to ζ ) -21 mV for a liposome with φNP ) 0.15% NP, which suggests the effective attachment of NPs on the outer shell interfaces of liposomes due to electrostatic attraction. Upon further increasing the NP concentration, ζ increases rapidly until reaching a saturation at φNP g 0.7%, possibly due to the maximum surface coverage of NPs coated on liposomes.14 It is noted that NPs are believed to disperse well over the entire outer surface of the liposomes as no NP aggregation is observed microscopically on a single liposome particle and no change in the measured medium conductivity, σm, is detected with increasing NP volume fraction. Furthermore, NPs are expected to behave as macro-ions adhered to the liposome surface, instead of an additional shell coating, as supported by the ωc measurement of NP-coated liposomes shown in Figure 5c below. On the basis of the measured ζ, the addition of NPs effectively reduces the surface conductance of the lipid bilayer membrane, as well the overall conductivity, σLB, by an order of magnitude from 10-9 to 10-8 S m-1, as estimated based on the diffuse layer conductance given by3
Ks,d ) Figure 5. The measured zeta potential, ζ, and crossover frequency, ωc, are plotted against the volume percentage, φNP of nanoparticles coated on plain liposome particles of a1 ) 150 (squares) and 225 nm (circles) in panels (a) and (b), respectively; (c) theoretical frequencydependent Clausius-Mossotti (CM) factors at varied surface conductivity, σ ) 10-6 (squares), 10-7 (circles), and 10-9 (triangles), and 10-12 S m-1 (stars) for liposomes of a1 ) 150 nm and σ )10-9 S m-1 (open triangles) for liposomes of a1 ) 225 nm.
the difficulty in accurately determining the shell thickness by TEM and DLS, the CP shell thickness reported above is estimated from the CP coating reaction data published elsewhere;15 thus, it is unfortunately impossible for us to accurately quantify the dependence of low and high ωc on CP layer thickness. Nanoparticle-Coated Liposomes. According to classical DEP theory with the shell model, the polarization and resultant DEP behaviors of liposome particles strongly depend on the shell conductivity and construct. In addition, recent experimental and theoretical work has demonstrated that dynamic doublelayer effects play a critical role in modulating the DEP response of functional colloidal suspensions in AC fields.23,24 We start by examining the crossover frequency of liposomes coated with charged nanocolloids on the outer shell of the lipid bilayer membrane. In this work, we focus on positively charged latex nanoparticles (NPs) of 20 nm in diameter to vary the outer conductivity of liposomes based on the following consideration: (1) it is reported that the positively charged nanocolloids can stabilize anionic liposomes more effectively than the negatively charged ones,25 and (2) the addition of negative nanocolloids appears equivalent to adding salts or macroions to effectively vary the medium conductivity, rather than liposome surfaces,
4q2n0z2D 3
10 kβTκ
(
1+
( ) )( [ ] )
2ε kβT z µη q 2
2
cosh
zqζ -1 2kβT
(7) where q is elementary charge (1.602 × 10-19 C), n0 is the bulk number density (m-3), z is the valency of the ions, D is the diffusion coefficient of the ions (m2 s-1), κ is the reciprocal Debye length (m-1), µ is the ion mobility (m2 V-1 s-1), and η is the solution viscosity (kg m-1 s-1). Despite the effective modification by NPs on the liposome surface charge, it is surprising to observe that the presence of NPs on liposome shells shows negligible effects on the measured crossover frequency, ωc, of modified liposomes in comparison to those of the plain ones, as show in Figure 5b; the ωc of NPcoated liposomes is nearly constant in the narrow range of 15-18 MHz, within the experimental uncertainty. The generality of the independence of high ωc on NP coverage on liposomes is verified with varied liposome size, as shown in Figure 5b with a liposome of a1 ) 225 nm. The observation is consistent with the theoretical estimation that a variation in the lipid bilayer conductivity results in no change in the high ωc and highfrequency DEP behavior of the spherical shells; yet, there is a marked change in the low-frequency DEP behavior, as illustrated in the Figure 5c. As suggested in Figure 5c, a low-frequency ωc could arise when the outer conductance of the lipid bilayer membrane is reduced below 10-7 S m-1, which explains why no second ωc is observed with NP-coated liposomes whose lowest conductivity remains on the order of 10-8 S m-1 at the maximum NP coverage. The independence of NP coverage on the ωc of liposomes is in stark contrast with the effect of the additional CP shell on modifying ωc, despite the considerable change in surface conductivity for both cases, suggesting that the surface construct, but not surface conductivity, is critical to
1558 J. Phys. Chem. B, Vol. 113, No. 6, 2009 modify the DEP behavior of liposomes. The unchanged response of liposomes stabilized by NPs in applied AC fields could have some practical implications for cellular manipulation and drug delivery by employing AC fields rather than other external perturbations, whereas variance in cellular responses by the docking of conductive biomacromolecules is the least desired. 5. Conclusion In this work, we examine the DEP responses of functionalized liposome particles of contrasting surface modifications to applied AC fields of varied frequency. With a uniform shell coating of calcium phosphate, we observe a drastic decrease in ωc compared to the one measured with plain liposome particles; in sharp contrast, with the patchy surface created by the adsorption of oppositely charged nanoparticles on liposome outer surfaces, we observe that ωc is independent of nanoparticle surface coverage, despite the considerable change in liposome surface conductivity by increasing adsorbed nanoparticles. Our observation is consistent with the AC polarization model for a shell structure. Because liposome particles studied in this work have a highly conductive interior, surface conductivity changes exhibit little effect on the DEP behavior of the suspensions even in the conductivity controlled low-frequency region; changes in the permittivity of multilayer coatings, on the other hand, can be used to effectively tune the polarizability of the particle to a much greater extent, significantly altering the highfrequency response of the suspension. Our results indicate that the particle surface construct, rather than surface conductivity, plays a critical role in the DEP behavior of a shell particle, which is much different from that of a solid dielectric particle. The true DEP behavioral switch of a shell particle can be effectively realized by the coating of an additional dielectric shell or the modification in both the permittivity and conductivity of a surface layer. Acknowledgment. We thank Hsueh-Chia Chang and Zachary Gagnon for useful discussions. We are grateful to Nicole Lapka and Rosary Abot for their assistance in liposome synthesis. We acknowledge financial support from the NSF (CBET-0730813)
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