Patchy Layersomes Formed by Layer-by-Layer Coating of Liposomes

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Patchy layersomes formed by layer-by-layer coating of liposomes with strong biopolyelectrolytes Yaser Kashcooli, Keunhan Park, Arijit Bose, Michael Lewis Greenfield, and Geoffrey D. Bothun Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01467 • Publication Date (Web): 10 Oct 2016 Downloaded from http://pubs.acs.org on October 17, 2016

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Patchy layersomes formed by layer-by-layer coating of liposomes with strong biopolyelectrolytes

Yaser Kashcooli1, Keunhan Park2, Arijit Bose1, Michael Greenfield1, and Geoffrey D. Bothun1,*

1

Department of Chemical Engineering, University of Rhode Island, 16 Greenhouse Road,

Kingston, RI 02881 2

Department of Mechanical Engineering, University of Utah, 1495 E 100 S, Salt Lake City, UT

84112 *Corresponding author: [email protected], +1-401-874-9518

1 ACS Paragon Plus Environment

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Abstract Layer-by-layer deposition of polyelectrolytes (PEs) onto self-assembled liposomes represents an alternative to PE deposition on solid particles for the formation of hollow nanoscale capsules. This work examines how competition between PE-liposome and inter-PE interactions drives the structure and colloidal stability of layersomes. Unlike solid particles, liposomes respond to adsorbed material through lipid reorganization and changes in size and shape. This responsive nature could yield new types of layered PE structures. We show that sequential deposition of strong biopolyelectrolytes, dextran sulfate-sodium salt (DxS-) and polyL-arginine (PA+), onto cationic liposomes in water yields the expected charge inversion behavior commonly observed for dispersed particles. However, cryogenic transmission electron microscopy (cryo-TEM) results show that the layersomes formed and their PE coatings were heterogeneous. The PE coatings contained PE complexes (PECs) that were formed when an even number of layers (2 or 4) were deposited. PECs remained attached as patches that were spatially distinguishable. This behavior was confirmed through fluorescence anisotropy measurements of liposome bilayer fluidity, where PA+ counteracted the ordering effects of DxS- on the lipid bilayer through charge neutralization and local PEC desorption. With increased charge screening, DxS- desorbed from the layersomes, while the patchy layersomes terminating in PA+ retained their PE coatings and colloidal stability at higher salt concentrations. To our knowledge this is the first time such patchy layersome structures have been observed.

Keywords: Layersome; layer-by-layer; polyelectrolyte adsorption; liposome stability.


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Introduction Layer–by–layer (LbL) deposition is a versatile technique for creating multilayer nanostructured materials. The LbL technique typically involves the sequential adsorption of oppositely charged polyelectrolytes (PEs) and other bio/macromolecules onto the surface of a substrate or template to create a self-assembled coating. By using particle templates, it is possible to create polymeric capsules with tailored surface functionality and barrier properties.1-6 The advantages of using the LbL technique include the ability to control the chemical, physical, and mechanical properties of the capsules by using different materials in the capsule wall1; the ability to tailor the capsule wall charge by varying the terminal layer or the assembly conditions (for example, temperature, pH, and salt concentration);7-9 and the ability to encapsulate cargo.10, 11 Capsules prepared by the LbL technique are typically formed by adsorbing PEs on solid or porous inorganic particles as sacrificial templates, which are dissolved under acidic or basic conditions to leave a PE shell encapsulating an aqueous core. An alternative capsule template is liposomes, which have been used extensively in drug delivery.12,

13

Liposomes are self-

assembled phospholipid vesicles that have a bilayer membrane structure with an internal aqueous core. They can encapsulate both hydrophilic and hydrophobic compounds and, when used as capsule templates, liposomes are layered with PEs (the structures are referred to as layersomes) and can remain a functional component of the capsule wall.14-16 This approach has been used to create layersomes for drug delivery.17,

18

When formed with biomacromolecules, including

naturally derived PEs16, 19 and polypeptides,14, 15, 20 layersomes can provide a nanoscale colloidal capsule that is biocompatible and biodegradable. Adding each PE layer leads to charge reversal and increases layersome size, capsule wall thickness and stiffness, and capsule barrier properties that resist spontaneous leakage.21, 22


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A key aspect to layersome formation is determining how the competition between PE– liposome and inter–PE interactions affects layersome structure and colloidal stability. Volodkin et

al.20

investigated

the

interaction

of

poly-L-lysine

(PLL)

coated

dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol/cholesterol (DPPC/DPPG/CL, 10wt% anionic lipid DPPG) liposomes with polyanions of varying charge density; poly-(4styrenesulfonate) (PSS), poly-L-glutamic acid (PGA), and hyaluronic acid (HA). PSS with a strong sulfonic group and a high charge density led to complete PLL desorption from the liposomes, while PGA with a weak carboxyl group and a high charge density led to partial PLL desorption (10 mM Tris, 15 mM NaCl, pH 7.4). Partial PLL desorption led to surface charge heterogeneity and layersome aggregation driven by patch-charge attraction, which has been used to create stable clusters of single-layered layersomes in electrolyte solutions.23-26 In contrast, HA with a weak carboxyl group and a low charge density yielded stable layersome capsules because the PLL–liposome interaction was stronger than the PLL–HA interaction. The competition between PE–liposome and inter–PE interactions has not been thoroughly investigated for other PEs or multilayered structures. An understanding of this competition could be used to create new layersome structures and control layersome behavior. In this work layersome structures were formed using cationic liposomes composed of dioleoylphosphatidylcholine (DOPC) and dioleoyltrimethylammonium-propane (DOTAP), coated with alternating layers of dextran sulfate (DxS-) and poly-L-arginine (PA+). DxS- and PA+, both strong polyelectrolytes (pKa ~ 2 and 9.5, respectively), were chosen because they have been used to create capsules for therapeutic applications via LbL deposition on solid particles. DxS-/PA+ microcapsules prepared using calcium carbonate particle templates are reported to be biodegradable and biocompatible in vivo27, and are capable of encapsulating proteins.11 They


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have also been shown to activate pulmonary antigen presenting cells28 and achieve immuneactivity by targeting antigens to dendritic cells.29 This work extends DxS-/PA+ multi-layering to soft liposome templates. The linear charge densities of DxS- and PA+ used in this study were 0.46 Å-1 (2.2 Å charge spacing)30 and 0.1 Å-1 (10.3 Å charge spacing)31 based on complete dissociation or association of the charged groups, respectively: –OSO3Na ➞ –OSO3- + Na+ for DxS- and =NH2 + H+ ➞ =NH3+ for PA+. The layersome structures differ from those typically reported using inorganic or polymeric particle templates, or for layersomes formed with other PEs. While layersome hydrodynamic diameter and zeta potential measurements are consistent with ‘typical’ results for PE multilayers on particle templates, structural characterization shows that the multilayers are heterogeneous due to the formation polyelectrolyte complexes (PECs) when PA+ was adsorbed. The structures reflect the relative strength of PE-liposome and inter-PE interactions; strong inter-PE interactions drove PEC formation. However, by using a liposome template with high charge density (50 mol% cationic DOTAP), the PECs remained attached to the layersome surfaces. This approach reflects the emerging interests in controlling the size, shape, and ‘patchiness’ or surface heterogeneity of PE capsules,32 and forming clusters of soft macromolecules and nanoscale assemblies.

Materials and methods Materials. DOPC and DOTAP were purchased from Avanti Polar Lipids, Inc. (Alabama, US). DxS- (6,500–10,000 MW), PA+ (5,000–15,000 MW) and 1,6-diphenyl-1,3,5-hexatriene (DPH, 98%) were purchased from Sigma-Aldrich Company (Missouri, US). The specification for the average number of sulfate (SO3-) groups per glucose unit in DxS- was 2.3. All materials were used as received.


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Liposome preparation. Liposomes were prepared in deionized water at 10 mM total lipid at a DOPC/DOTAP molar ratio of 1:1 using a rotary evaporator. The lipids in chloroform were placed in a round-bottom flask and the chloroform was evaporated at 50 °C at 450 mbar for 30 min, then 300 mbar for 30 min, and finally 200 mbar for 30 min. The flask containing a dry lipid film was placed under vacuum for 8 h to remove any residual solvent. The film was hydrated with deionized water and the formed liposomes were diluted by a factor of 10 and extruded through 100 nm track-etched polycarbonate membranes to yield small monodispersed unilamellar liposomes. For the samples which include DPH for fluorescence anisotropy measurements, DPH in tetrahydrofuran was added to DOPC and DOTAP in chloroform at a DPH:lipid molar ratio of 1:500 before placing in the rotary evaporator.

Layersome preparation. A washless method was used to form the layersomes using PE solutions prepared in DI water at 0.05% w/w. This method has been used previously for solid particles.33 Layersomes were formed by sequentially adding PE to the liposomes (layer 1) with an initial lipid concentration of 1 mM, or layersomes (layers 2–4) with the respective PE solutions (Figure 1). Each layer was coated under stirring for 5 min at room temperature. For each layer, titrations were first carried out beforehand to determine the correct amount of PE to add at each step that would yield a complete coating based on charge reversal, similar to the technique used by Cuomo et al.19 During each titration, the zeta potential was measured as a function of the PE concentration. The PE concentrations were chosen before the zeta potential plateaued to minimize the presence of unbound PE in solution.


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Dynamic light scattering (DLS). Hydrodynamic diameter (dh) and zeta potential (ζ) were measured using a Malvern Instruments Zetasizer Nano ZS with a detector angle of 173° and a 4 mW, 633 nm He-Ne laser. To determine dh, 1 ml of the sample was placed in an optical grade polystyrene cuvette at 25 oC. The reported hydrodynamic diameters are based on 15 scans measured in triplicate for each sample. Zeta potential was measured by combined Doppler electrophoretic velocimetry and phase angle light scattering. Measurements were performed using 1 mL samples at 25 oC. The dh and ζ interpretations were based on layersome diffusion assuming spherical particles. Layersome shape and the conformation of the PE coatings can strongly affect these measurements by altering the slip plane and leading to anisotropic diffusion coefficients. Hence, dh and ζ reflect the average values for equivalent spheres.

Cryogenic Transmission Electron Microscopy (cryo-TEM). Cryo-TEM samples were prepared at 25 °C using a Vitrobot (FEI Company), which is a PC-controlled robotic assembly for sample vitrification. Quantifoil grids were used with 2 µm carbon holes on 200 square mesh copper grids (Electron Microscopy Sciences, Hatfield, PA). The sample was first equilibrated within the Vitrobot at 25 oC and 100% humidity for 30 min. After immersing the grid into the sample, it was then removed, blotted to reduce film thickness, and vitrified in liquid ethane. The sample was then transferred to liquid nitrogen for storage. Imaging was performed at -170 oC in a cooled stage (model 626 DH, Gatan Inc., Pleasanton, CA) at 200 kV using a JEOL JEM-2100 TEM (Peabody, MA).

Bilayer fluidity measurements. DPH anisotropy of the samples as a function of temperature was measured by using a Perkin Elmer LS 55 fluorescence spectrometer. The excitation


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wavelength and the detection wavelength were set at λ = 350 nm and λ = 452 nm, respectively, and the excitation and emission slit widths were set at 10 nm. Anisotropy, , was calculated from the following equation < > =

−

+

where I is the fluorescence emission intensity and subscripts V and H represent the vertical and horizontal orientation, respectively, of the excitation and emission polarizers.34 G is a grating factor (IHV/IVH) that accounts for differences in sensitivity to horizontal and vertical polarized light.

Results and Discussion Layersome formation and characterization. The cationic DOPC/DOTAP liposome templates had an average hydrodynamic diameter of 133 nm and a zeta potential of +53 mV. A low polydispersity index of 0.08 indicated that the liposomes had a narrow size distribution. The total charge ratio for the layersomes, n-:n+, based on charged lipid and PEs are shown for each layer Ln (n = 1 to 4) corresponding to the final PE concentrations based on the titration experiments (Figure 1a). Each layer led to a reversal of surface charge, and the high zeta potential values are consistent with electrostatic repulsion between the layersomes (Figure 1b) that stabilized the dispersion and prevented aggregation over 30 days (Figure 1c).


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Figure 1. (a) Polyelectrolyte titration experiments to determine the concentration of DxS- (L1, L3) or PA+ (L2, L4) for layersome formation using DOPC/DOTAP liposome templates. The arrows depict the concentration used based on the point at which ζ began to plateau. The term n:n+ corresponds to the total charge ratio for each layersomes (lipids + PEs). (b) Layersome zeta potential, ζ, and (c) hydrodynamic diameter, dh, as a function of layer number (Ln) depicting 9 ACS Paragon Plus Environment

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charge reversal at the PE concentrations determined from titration 1 h after formation and after 30 days. Error bars in (b) and (c) represent standard error of duplicate samples, and some of the error bars are smaller than the symbols.

Layersome structure was examined by cryo-TEM. Representative micrographs of the liposome template (Figure 2a) confirm that the liposomes were unilamellar with a bilayer thickness of approximately 4 nm. The first layer, DxS-/L1, yielded layersomes with a uniform coating approximately 9 nm thick (Figure 2b). Based on previous work for polyelectrolytes adsorbing onto oppositely charged liposomes,35, 36 DxS- adsorption led to lipid exchange, also referred to as lipid flip-flop, where the cationic DOTAP lipids accumulated in the outer liposome leaflet in contact with DxS- and the zwitterionic DOPC lipids accumulated in the inner liposome leaflet. The second layer, PA+/L2, produced patches of DxS-:PA+ PECs on the layersome surfaces (Figure 2c1). Layersome clusters, defined as layersomes linked by or sharing a common PE coating, were also observed. The layersomes were no longer uniformly coated and in some cases the underlying liposome template can be observed, indicating that some of the PECs may have desorbed (Figure 2c2). Using anionic liposomes with an adsorbed polycation, Yaroslavov et al.37 have shown that a polyanion can displace the polycation and form PECs in solution. However in this case the anionic lipid content was 20 mol% and desorption was driven by strong inter-PE attraction. For our layersomes the DOTAP content was 50 mol%, which yielded a stronger interaction with the DxS- and kept the majority of the PECs bound to the layersome. The third layer, DxS-/L3, coated the structures and there was evidence of this layer covering the PECs resulting from the second layer, PA+/L2 (Figure 2d). For the final coating, PA+/L4, PEC


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patches were again observed (Figure 2e1) and there was evidence of desorbed PECs for this final PA+ layer (Figure 2e2).

(b)

(c1)

(d)

(e1)

(a)

(f)

1

35

y Ka

0.8

30 25

0.6

20 15

0.4

(e2)

Ka (mM2)

(c2)

y

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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10 0.2 0

5 0 1

2

3

4

layer

(g)

-SO3- (DxS-) Na+ (DxS-) + DOTAP

=NH2+ (PA+) Cl- (DOTAP, PA+)

+ + + + L1

+ + + +

+ + + +

+ + + + L2

L3

+ + + + L4

Figure 2. (a-e) Representative cryo-TEM images of the liposome and layersome structures formed in deionized water: (a) DOPC/DOTAP liposomes, (b) DxS-/L1 layersomes, (c1, c2) 11 ACS Paragon Plus Environment

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PA+/L2 layersomes, (d) DxS-/L3 layersomes, and (e1, e2) PA+/L4 layersomes. PECs adsorbed on layersome surfaces are denoted with black arrows and dashed circles are shown for select PECs to guide the eye. Labels (c2) and (e2) denote images at lower magnification where desorbed PECs (white arrows) were observed. Scale bars = 100 nm. (f) The fraction of counterion-compensated DxS- and PA+ (y) within the layersomes or PECs based on the charge ratio n-:n+ (Figure 1a) and the equilibrium association constant, Ka, between counterioncompensated and ion-paired states. (g) A schematic depicting layersome formation and associated counterion release with PE adsorption (not to scale). Only the outer DOTAP lipid leaflet is shown. Dextran sulfate (DxS-) is shown in blue and poly-l-arginine (PA+) and DOTAP are shown in red. Sodium (Na+) and chloride (Cl-) counterions are shown in yellow and green, respectively.

We extend the recent “ion-pairing” model from Lu and Schlenoff38 to describe the balance between ion-paired and counterion-compensated repeat units (PE monomers and DOTAP molecules) within the layersomes. Our approach differs in that we do not assume that complete PE ion-pairing occurred for the layersomes prepared in deionized water. In deionized water, Na+ and Cl- counterions were present in the ranges of 1.8 to 2.9 mM and 0.3 to 1.3 mM, respectively, based on the lipid and PE concentrations. Furthermore, the layersome components had different linear charge densities that, as discussed below, prevented complete ion-pairing at the conditions employed. Hence, layersome formation is depicted as a series of reactions (eqs. 14) where the reactants are counterion-compensated components within the layersomes and the products are ion-paired components within the layersomes and released (dissociated) counterions.


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L1: DOTAP+Cl- + DxS-Na+ ⇋ DOTAP+DxS- + Cl- + Na+

(1)

L2: DOTAP+Cl- + DxS-Na+ + PA+Cl- + ⇋ DOTAP+DxS-PA+ + Cl- + Na+

(2)

L3: DOTAP+Cl- + DxS-Na+ + PA+Cl- + ⇋ DOTAP+DxS-PA+DxS- + Cl- + Na+

(3)

L4: DOTAP+Cl- + DxS-Na+ + PA+Cl- + ⇋ DOTAP+DxS-PA+DxS-PA++ Cl- + Na+ (4) The fraction of counterion-compensated PEs (y) within the layersomes or PECs was calculated as (n-:n+ - 1)/n-:n+ for DxS- and [(1/n-:n+) - 1)]/(1/n-:n+) for PA+ (n-:n+ shown in Figure 1a) based on the assumption that the amount of adsorbed PE was much greater than the amount of soluble or ‘free’ PE. Values calculated for y indicate that approximately 65% and 40% of the DxS- in layers L1 and L3 were counterion-compensated, respectively, compared to approximately 20% of the PA+ in L2 and L4 (Figure 2f). The equilibrium association constant Ka, which denotes the balance between counterion-compensated and ion-paired states in equations 1 and 2, was determined from Ka = (1-y)aMA2/y2 where aM is the mean activity of Na+ and Cl-. aM was calculated from the concentrations of Na+ and Cl-, determined by a mole balance based on n-:n+, and an activity coefficient of 0.96. The association constants for layersomes terminating with PA+ were an order of magnitude larger than those terminating with DxSconsistent with greater PA+/DxS- ion-pairing when PA+ was adsorbed in L2 (eq. 2) and L4 (eq. 4). This behavior is shown schematically in Figure 2g corresponding to reaction eqs. 1-4. The extent of counter-compensation can be further analyzed by counterion condensation theory based on a linear charge density parameter, ξ = λB/b, where λB is the Bjerrum length (7 Å in water) and b is the average linear distance between charges in the PEs.39 Counterion condensation occurs when ξ > 1 for monovalent ions. For DxS- and for PA+, ξ = 3.3 and 0.7, respectively. Based on ξ and y, it is clear that DxS- was counterion-compensated within the PE layers when it was the terminating layer (L1 and L3). For ion-pairing to occur in L2 and L4, the


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Na+ counterions on DxS- would have had to have been displaced, which would have led to an increase in entropy when PA+ adsorbed. This entropic gain, coupled with the enthalpic energy associated with ion-pairing, led to strong inter-PE attraction that was greater than the interaction between the PEs and the liposome template and drove the formation of PEC patches that partially desorbed. This proposed mechanism was further examined using fluorescence anisotropy and DLS experiments with increasing salt concentration as described below.

PE-liposome interactions based on lipid bilayer fluidity. Lipid monolayers and bilayers restructure when exposed to oppositely charged PEs to optimize their interactions with the PEs.40,

41

This concept was extended to the layersomes based on the ordering effects of PE

adsorption on lipid bilayers. Lipid ordering was determined based on DPH anisotropy, , where is inversely proportional to fluidity; a high reflects a high degree of lipid ordering or a low degree of bilayer fluidity, and vice versa. Measured values for reflect an average membrane fluidity based on previous work that has shown that the distribution of DPH within the liposomes is independent of lipid ordering.42 Results are shown as the change in anisotropy at 25 oC between the layersomes and the liposome template; ∆ = layersome – liposome, where liposome ≈ 0.1. The first DxS- layer (L1) led to a two-fold increase in due to lipid ordering driven by electrostatic attraction between DxS- and DOTAP+ (Figure 3a). The adsorption of PA+ (L2) counteracted the effects of DxS- on lipid ordering by reducing the attraction between DxS- and the liposomes; DxS- interacted more favorably with PA+ than with the lipids to form PECs. The decrease in is attributed PEC formation and partial desorption that, based on the cryo-TEM results, led to ‘bare’ regions on the liposome templates. The fact that ∆ is > 0 for L2 (and L4)


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confirms that PECs remained bound to the layersome surface. Layers 3 and 4 followed this same pattern where DxS- (L3) increased lipid ordering by adsorbing onto the layersomes, and PA+ (L4) decreased lipid ordering through PEC formation. Hence, an even number of layers with strong inter–PE interactions and extensive ion-pairing (1-y; Figure 2f, g) coincided with the formation of PEC patches that partially desorbed. The effect of temperature on bilayer fluidity was also examined. DPH anisotropy did not change upon heating from 25 to 39 oC, confirming that the layersome structures remained intact within this temperature range (Figure 3b). (a) 0.3

25 oC

+ +++ + +++

∆

0.2

0.1

0 0

1

2 layer

3

4

(b) 0.3 3 4

0.2

1 2

0.1

liposome

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0

25

30

35 T (ºC)

40

45

Figure 3. (a) Changes in the lipid bilayer fluidity of layersomes based on DPH anisotropy as a function of layer number, Ln. Inset: cartoon schematic of DPH within a lipid bilayer containing 15 ACS Paragon Plus Environment

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cationic (red) and zwitterionic (black) lipids (not to scale). DOTAP is depicted in the outer lipid leaflet in contact with DxS- based on previous work. (b) DPH anisotropy as a function of layer number and temperature. Error bars in (a) and (b) represent the standard error of duplicate samples.

Layersome response to increasing ionic strength. Based on the ion-pairing analysis, a significant amount of DxS- was counterion-compensated when it was the terminating layer. Increasing NaCl concentration would further increase counterion-compensation for these layersomes, reducing the surface charge and extent of ion-pairing. Layersome zeta potential, hydrodynamic diameter, and fluidity were examined as a function of [NaCl] (Figure 4). Layersomes terminating with DxS- (L1, L3) showed a significant reduction in surface charge with increasing [NaCl] (Figure 4a) and aggregated above 1 mM [NaCl] based on dh (Figure 3b). Aggregation yielded large clusters ranging from approximately 400-3,000 nm with PDIs from 0.4-0.9. In contrast, layersomes terminating in PA+ (L2, L4) retained a high surface charge compared to DxS-, similar to the liposome template. These layersomes were more stable against aggregation over the range of [NaCl] examined (Figure 4c). However, there was evidence of clustering based on dh due to charge screening and possible patch-charge attraction. All layersomes were in an unstable cluster phase and flocculated above the aggregation [NaCl], which is consistent with recent work showing that DOPC/DOTAP liposomes with adsorbed sodium polyacrylate form this same phase at similar [NaCl] and charge ratios.43 DPH anisotropy was used to examine the effects of increasing [NaCl] on PE-lipid interactions and PEC desorption. Results for 0, 10, and 100 mM [NaCl] are shown in Figure 4d.


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With increasing [NaCl], ∆ decreased when DxS- was the terminating layer. At 100 mM [NaCl] ∆ for L1 is close to zero, indicating that the liposome-DxS- interaction was negligible and DxS- desorbed. However, there was no significant change in when PA+ was the terminating layer (L2 and L4).

Figure 4. (a) Layersome zeta potential, ζ, and (b) hydrodynamic diameter, dh, as a function of NaCl concentration, [NaCl]. Cationic liposomes and layersomes are shown in red and anionic layersomes are shown in blue. (c) Summary of [NaCl] where layersomes exhibited aggregation. (d) Changes in DPH anisotropy as a function of layer number and [NaCl]. Error bars represent


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standard error of duplicate samples. In (b), layersomes with PDI > 0.3 that exhibited significant aggregation are not shown.

Layersomes terminating in DxS-, L1 and L3, were prone to surface charge neutralization and aggregation in the presence of NaCl. The reduction in surface charge up to 10 mM NaCl followed by a plateau with increasing [NaCl] is consistent with previous work attributing DxSneutralization to Na+ condensation.44 The anisotropy results further show that increasing [NaCl] reduced the ordering effect of the PEs when DxS- was the terminating layer, and led to DxSdesorption when it was the lone PE layer (L1). When PA+ is the terminating layer, the counterions appear unable to penetrate into the PE multilayers and overcome ion-pairing, and lead to further PE or PEC desorption over the times scales associated with this work.

Conclusions We have shown that a washless method can be used to create multilayered layersomes with DxS- and PA+ polyelectrolytes with ‘high’ and ‘low’ charge density, respectively. Four key conclusions can be made. First, while the LbL process appears to behave as expected for multilayer formation on particles based on layersome size and charge reversal, the layers were not ‘uniform’ (with the exception of the first layer), but rather they were heterogeneous and comprised of PEC patches, some of which desorbed from the layersome surface. Hence, tuning PE and template charge densities could give rise to new self-assembled structures. Second, an ion-pairing model revealed appreciable counterion-compensation when DxS- was the terminating layer. Ion-pairing and counterion-release when PA+ was adsorbed onto DxS- PECs led to strong inter-PE attraction that drove PEC formation. Third, counterion condensation on DxS- led to


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surface neutralization and layersome aggregation when DxS- was the terminating layer. However, when PA+ was the terminating layer the layersomes remained dispersed despite the propensity for DxS- counterion condensation. Fourth, an anisotropic probe in the liposome was used to examine the structure of the template with PE adsorption. Template structure can strongly influence LbL deposition, but for soft colloidal templates direct structural analyses are lacking. Feedback between template structure and LbL deposition can further be used to design new templates and new LbL colloids. Additional work is needed to determine the kinetics of PE adsorption and how this influences layersome structure, and to directly analyze changes in layersome structure in the presence of salt.

Acknowledgements This work was supported by the National Science Foundation under Grant No. CBET-1337061. We would like to thank Dr. Richard Kingsley for assistance with cryo-TEM imaging, and the core facilities of the Rhode Island Consortium for Nanoscience and Nanotechnology.

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Strong interactions between polyelectrolytes adsorbed onto liposomes yields heterogeneous coatings with polyelectrolyte complexes. 40x18mm (300 x 300 DPI)

ACS Paragon Plus Environment

Patchy Layersomes Formed by Layer-by-Layer Coating of Liposomes

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