Osmotic shock-triggered assembly of highly-charged, nanoparticle

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Osmotic shock-triggered assembly of highlycharged, nanoparticle-supported membranes Peter J. Chung, Hyeondo Luke Hwang, Kinjal Dasbiswas, Alessandra Leong, and Ka Yee C. Lee Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03026 • Publication Date (Web): 10 Oct 2018 Downloaded from http://pubs.acs.org on October 16, 2018

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Osmotic shock-triggered assembly of highly-charged, nanoparticle-supported membranes Peter J. Chung,†‡‖ Hyeondo Luke Hwang,‡ Kinjal Dasbiswas,† Alessandra Leong,‡ and Ka Yee C. Lee*†‡‖ †

James Franck Institute, ‡Department of Chemistry, ‖Institute of Biophysical Dynamics, The

University of Chicago, Chicago, IL 60637

Keywords: osmotic shock, silica nanoparticle, supported-lipid bilayers, transmission electron microscopy, cryogenic transmission electron microscopy

Abstract Spherical-nanoparticle supported lipid bilayers (SSLBs) combine precision nanoparticle engineering with biocompatible interfaces for various applications, ranging from drug delivery platforms to structural probes for membrane proteins. While the bulk, spontaneous assembly of vesicles and larger silica nanoparticles (>100 nm) robustly yields SSLBs, it will only occur with low charge-density vesicles for smaller nanoparticles (100 nm),6,7 with even lower charge-density (1 hour, with lipid mixture mass checked via analytical balance. Lipid mixtures were then rehydrated into desired buffer of choice (CBS, or 10 mM citrate pH 6.0, 150 mM NaCl and, if applicable, sucrose) and shaken for 1 hour at 40°C to form multilamellar vesicles. Unilamellar vesicles were formed from multilamellar vesicles by five subsequent freeze-thaw cycles in dry ice-ethanol bath and heated water bath. Vesicles were then extruded using a Lipex Extruder purchased from Evonik Transferra Nanosciences (Burnaby, BC, Canada) utilizing 25 mm Whatman Nuclepore membranes with either 50 or 80 nm pore sizes (50 nm pore size for use with 40 nm nanoparticles, 80 nm pore size for use with 50 and 60 nanoparticles) at 400 PSI for 50 nm pores and at 250 PSI for 80 nm pores. Vesicles were used within 24 hours of extrusion.

Spherical-Nanoparticle Supported Lipid Bilayer (SSLB) Production: For amine-functionalized silica nanoparticles, ethanol was exchanged for DI water by dialyzing nanoparticles in 0.5-3 mL capacity, 3.5k MWCO Slide-A-Lyzer Dialysis Cassette in DI Water for 14 hours (with DI water replaced 1 and 2 hours into dialysis) at room temperature. Both amine- and hydroxyl-

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functionalized nanoparticles were diluted to 2.5 mg/mL in DI water, with nanoparticles subsequently bath sonicated for 15 minutes. Monodispersity and nanoparticle size was checked via dynamic light scattering. Concentrated 5x CBS (50 mM citrate pH 6.0, 750 mM NaCl) was then added and mixed to dilute nanoparticles to 2 mg/mL to a final concentration of 1x CBS.

An equal volume of extruded vesicles (with vesicle number concentration three times the number of nanoparticles) was mixed with nanoparticle solution thoroughly via repeated pipetting. After incubating solution for 1 hour at 40°C to maximize SSLB formation, excess vesicles were removed via 3 cycles of sample centrifugation at 1700g for 15 minutes with supernatant replaced with buffer of choice (usually HB7, or 10 mM HEPES pH 7.0, 100 mM NaCl for EM measurements or DI water, for dynamic light scattering and ζ-potential measurements, see Supporting Information, Figure S1). Prior to measurement, samples were allowed to settle for ~6 hours with supernatant sample removed from any settled aggregates.

Dynamic Light Scattering and ζ-potential Measurements: Dynamic light scattering and ζ-potential measurements were performed on a Malvern Zetasizer NanoZS at a 1:50 sample dilution into buffer or DI water, respectively.

Whole Mount Transmission Electron Microscopy (TEM): 1.5 μL droplet of SSLB sample (at nominal silica nanoparticle concentration of 2 mg/mL) in HB7 buffer (10 mM HEPES pH 7.0, 100 mM NaCl) was deposited onto a plasma-treated, formvar/carbon film supported on a copper grid


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(Electron Microscopy Sciences). The sample droplet was left on grid for one minute before wicking it away with filter paper. Next, grid was negative stained with 2 μL of 1.66 wt/vol % ammonium molybdate solution for 30 seconds, after which the stain was wicked away with filter paper and the grid fully air dried before imaging. All transmission electron micrographs were taken using FEI Spirit microscope operating at 120 kV at the Advanced Electron Microscopy Facility at the University of Chicago.

Cryogenic Transmission Electron Microscopy (Cryo-TEM): SSLB sample at 1 mg/mL silica concentration was vitrified in liquid ethane using FEI Vitrobot on a plasma treated, C-Flat holey carbon grid. The ideal Vitrobot setting was found to be blotting force “2” and blotting time for 1 s. The sample was stored in liquid nitrogen for two days prior to imaging, in order to remove residual solid ethane formed on the grid while performing the vitrification step. Cryo-TEM images were taken using 200 kV FEI Talos microscope equipped with a Falcon 3 direct electron detector at the Advanced Electron Microscopy Facility at the University of Chicago.

Results Here we examine highly charged SSLB formation with donor vesicles experiencing increasing transmembrane osmotic gradients that are subsequently mixed with surface-modified (with amine or hydroxyl group) silica nanoparticles. Initially (and in the absence of an osmotic gradient), extruded vesicles (4.8 mM total lipid through 80 nm pores) made up of 50% anionic lipid DOPA (1,2-dioleoyl-sn-glycero-3-phosphate) and 50% zwitterionic lipid DOPC (1,2dioleoyl-sn-glycero-3-phosphocholine) hydrated in citrate buffered saline (CBS, or 10 mM citrate


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pH 6.0, 150 mM NaCl) were mixed with an equal volume of 60 nm cationic amine-functionalized silica nanoparticles (2 mg/mL) suspended in similar buffer conditions. After separating out excess vesicles via centrifugation, samples were stained for whole mount transmission electron microscopy (TEM). TEM revealed unruptured vesicles adhering to nanoparticles but no formation of SSLBs (Figure 1a), similar to previous results for highly-charged vesicles and silica nanoparticles.7

Figure 1. Transmission electron microscopy reveals SSLB formation via an increase in osmotic pressure of highly-charged donor vesicles mixed with spherical-nanoparticles. a-c, highly-charged SSLB formation was verified via whole mount transmission electron microscopy (TEM) after incubating 60 nm amine-functionalized silica nanoparticles in citrate salt buffer (10


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mM citrate pH 6.0, 150 mM NaCl) with highly-charged 50% DOPA/50% DOPC vesicles suspended in CBS (a), CBS and increasing sucrose concentrations, ΔCSucrose = 250 mM (b) and ΔCSucrose = 325 mM (c). d-e, to verify that SSLB formation was mediated via an osmotic gradient and not sucrose-specific effect, SSLB preparation was performed as before but with vesicles suspended in CBS and increasing glycerol concentrations, ΔCGlycerol = 300 mM (d) and ΔCGlycerol = 400 mM (e). Scale bar: 50 nm.

However, in the presence of a transmembrane osmotic gradient we saw dramatically different results. To induce a hypoosmotic gradient across donor vesicle membranes (Pout < Pin, where P refers to osmotic pressure), lipid vesicles were instead formed in CBS and sucrose (a membrane impermeable osmolyte, permeability coefficient ~ 10-12 cm s-1)13 and mixed with an equal volume suspension of nanoparticles in just CBS. Upon complete mixing of the two solutions, the sucrose concentration (CSuc. Soln.) exterior to vesicles is halved, but within the vesicle lumens the sucrose concentration remains constant, leaving osmotically-pressurized vesicles with concentration difference ΔCSucrose (=CSuc. Soln./2). Strikingly, with increasing sucrose (CSuc. Soln. = 500 mM, ΔCSucrose = 250 mM), TEM reveals an increasing proportion of nanoparticles surrounded by lipid material that appears as a 4-5 nm white ring on electron dense background, commensurate with a supported lipid bilayer (Figure 1b). This SSLB formation is driven to completion at higher sucrose concentrations (CSuc. Soln. = 650 mM, ΔCSucrose = 325 mM, Figure 1c). To rule out a sucrosespecific effect driving SSLB formation, another osmolyte, glycerol, was used in a similar manner. Osmotic gradients driven by glycerol, a membrane-permeable osmolyte (permeability coefficient ~ 10-6 cm s-1),13 also increased SSLB formation (CGlyc. Soln. = 600 mM, ΔCGlycerol = 300 mM, Figure 1d) with completion at higher glycerol concentrations (CGly. Soln. = 800 mM, ΔCGlycerol = 400 mM,


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Figure 1e). With either osmolyte, the SSLB formation process and the resulting structure observed appear to be similar. As using the membrane-permeable glycerol quantitatively yields as many SSLBs compared to using sucrose (see Supporting Information, Note S1), the negligible impact of glycerol diffusion indicates not only an osmotically-controlled process but a relatively rapid one as well. To obtain higher-resolution images and rule out artifacts from TEM staining or sample preparation, cryogenic transmission electron microscopy (cryo-TEM) was also used to examine SSLB formation (Figure 2). In the absence of a transmembrane osmotic gradient, cryo-TEM revealed 50% DOPA/50% DOPC vesicles mixed with 60 nm amine-functionalized sphericalnanoparticles result in vesicles adhering to nanoparticles with no SSLB formation (Figure 2a). However, with an osmotic gradient (ΔCSucrose = 325 mM), spherical-nanoparticles are surrounded by a continuous, concentric ring of electron-dense material (thickness ~4-5 nm) faithfully following nanoparticle topology (Figure 2b), consistent with a supported lipid bilayer. Although previous measurements indicated difficulty in assessing SSLB formation with TEM (possibly due to dehydration damage during sample preparation), the homology of structures observed via cryoTEM and TEM suggests an interchangeability in the two techniques in confirming supported lipid bilayers. While cryo-TEM allows for high precision and time-dependent characterization of SSLB formation, TEM allows for rapid optimization of osmolyte concentrations required for complete SSLB formation for a given lipid composition and nanoparticle size/surface functionalization.


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Figure 2. Highly-charged SSLBs were confirmed via cryogenic transmission electron microscopy. To preclude artifacts due to sample preparation for whole mount transmission electron microscopy, cryogenic transmission electron microscopy (cryo-TEM) was used to validate solution structures. a, In the absence of a transmembrane osmotic gradient, highlycharged vesicles adhered to oppositely-charged amine-functionalized nanoparticles without rupturing. b, In the presence of an optimal transmembrane osmotic gradient (ΔCSucrose = 325 mM), 50% DOPA/50% DOPC SSLBs were formed with contiguous (i.e. defect-free) supported lipid bilayers on top of 60 nm amine-functionalized nanoparticles. Scale bar: 50 nm.

With rapid validation via TEM in hand, we sought to demonstrate the tunability of osmotically-induced SSLB formation. SSLBs were formed for a variety of small sphericalnanoparticles (diameters of 40, 50, and 60 nm) for 50% DOPA/50% DOPC mixtures (Figure 3ac, details in Supporting Information, Table S1). Additionally, to demonstrate the generalizability


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of this technique, SSLBs were also formed for mixtures of another physiological anionic lipid, DOPS (1,2-dioleoyl-sn-glycero-3-phosphoserine) and DOPC for different spherical-nanoparticle diameters (Figure 3d-f, details in Supporting Information, Table S1). Intriguingly, the optimal osmolyte concentration required for SSLB formation on 60 nm spherical-nanoparticles with 50% DOPS/50% DOPC mixtures (ΔCSucrose = 300 mM, Figure 3a) nearly matched the osmolyte concentration needed for 50% DOPA/DOPC mixtures (ΔCSucrose = 325 mM, Figure 1c), suggesting that overall membrane charge density may be a key parameter in driving SSLB formation as opposed to the molecular details of each lipid headgroup.

Figure 3. SSLBs of various sizes can be obtained by modulating the osmotic gradient across donor vesicle membranes. a-c, mixing highly-charged (50% DOPA/50% DOPC) vesicles with


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60 (a), 50 (b), and 40 nm (c) amine-functionalized spherical-nanoparticles yields SSLBs at optimal osmotic pressures (see details in Supporting Information, Table S2). d-e, replacing DOPA with another physiological anionic lipid, DOPS, also yields SSLBs when mixed with 60 (d), 50 (e), and 40 nm (f) amine-functionalized spherical-nanoparticles, suggesting that the SSLB formation via transmembrane osmotic gradient is independent of specific lipids. Scale Bar: 50 nm.

To that end, we explored the relationship between membrane charge density and osmolyte concentration necessary for SSLB formation on 60 nm spherical-nanoparticles through two different tacks. First, we tracked the osmolyte concentration required for SSLB formation as a function of increasing charge density, using mixtures of anionic DOPA and zwitterionic DOPC with amine-functionalized nanoparticles. Second, to reinforce the lack of lipid specificity in our process, we concurrently examined SSLB formation after inverting the charge of nanoparticles and membranes by using anionic hydroxyl-functionalized nanoparticles and vesicle membranes made of increasingly cationic lipid, DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) and DOPC. While one might expect that increasing membrane charge density would increase the strength of interactions between vesicles and nanoparticles with opposite charge (thus driving SSLB formation), surprisingly, the osmolyte concentrations required for increasing membrane charge density indicated the very opposite (Table 1, additional characterization in Supporting Information, Table S2). For lipid compositions up to 60% DOPA/40% DOPC and 50% DOTAP/50% DOPC, the osmolyte concentration required for optimal SSLB formation varies


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nonlinearly with increasing charge density. Perhaps even more striking, osmolyte concentrations tracked with the absolute membrane charge density and not with specific lipid headgroup chemistry of both cationic and anionic membranes or the charge density differences for either amine- or hydroxyl- functionalized nanoparticles. These data suggest that our osmotically controlled process can be parameterized by a generalized membrane charge density and the formation process is controlled by membrane properties rather than vesicle-nanoparticle interactions.

Table 1. Osmolyte Concentration required for Charged Lipid/DOPC SSLB formation with vesicles extruded through 80 nm pores and 60 nm spherical-nanoparticles (aminefunctionalized for DOPA and DOPS samples; hydroxyl-functionalized for DOTAP samples) Charged Lipid Charged ΔCSucrose ζ-Potential [mV] (Charge) Lipid [%] [mM] DOPA (-1)

10

200

-16.7 ± .611

DOPS (-1)

10

200

-26.0 ± 1.79

DOTAP (+1)

10

200

+24.6 ± .924

DOPA

25

200

-20.8 ± .800

DOPS

25

200

-39.4 ± 4.34

DOTAP

25

200

+29.1 ± 1.15

DOPA

50

325

-45.1 ± 1.45

DOPS

50

300

-48.0 ± 1.88

DOTAP

50

300

+32.6 ± 1.27

DOPA

60

350

-51.4 ± 1.61


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To understand how highly-charged, small nanoparticle ( Pout) to form SSLBs indicates a mechanism not previously observed, as earlier experiments suggest hypoosmotic gradients inhibits planar supported lipid bilayer formation.16,17 Furthermore, smaller nanoparticles become increasingly harder to wrap, a problem exacerbated by increasing membrane rigidity due to increasing charge (see Supporting Information, Note S2). Accordingly, we propose that the strong interaction between highly-charged lipid vesicles and oppositely-charged nanoparticles triggers an increase in membrane tension due to vesicle deformation (Figure 1a, Figure 2a), which, with the addition of a hypoosmotic gradient, causes membrane defects to form and drive SSLB formation. At high membrane tensions, membranes have been known to form pores,18 with the threshold for pore formation/closure on highly-charged vesicles measured to occur at ~20 mN m-1.19 Akin to high-energy point defects that lower material fracture strengths, these pores should lower the barrier for vesicle rupture. Indeed, for osmotically-pressurized vesicles at 50% charge density, the osmolyte concentration difference (ΔCSucrose ~ 300-325 mM) corresponds to a membrane tension of ~ 15 mN m-1 using the Laplace pressure equation. When these vesicles adhere to a nanoparticle


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surface, the subsequent deformation would cause a jump in osmotic pressure (and membrane tension), likely leading to pore formation. This osmotic shock is thus a function of vesicle deformation which, in it of itself, will be controlled by the membrane bending rigidity (e.g. by the addition of cosurfactants to lower bending rigidity, Supporting Information, Figure S2) and the vesicle-nanoparticle interaction strength. While a more extensive theoretical treatment may be needed to more precisely relate highly-charged vesicle deformation to vesicle mechanical changes and nanoparticle/vesicle interaction energies20, we have clearly demonstrated highly-charged SSLB formation using hypoosmotic gradients and established an empirical relationship for osmolyte concentration required to membrane charge density.

Conclusions The robust and tunable control in forming bulk amounts of highly-charged, highly-curved supported lipid bilayers via a transmembrane osmotic gradient bypasses issues associated with other techniques. By using vesicles larger than our nanoparticles, we only observed contiguous supported lipid bilayers and avoided membrane “patches” associated with techniques utilizing electrostatics alone.21 In exploiting the natural mechanical behavior of membranes, our supported lipid bilayers carried more charged lipids than other supported membranes that required specific nanoparticle/lipid linkers.22

Finally, protein denaturation and drug/DNA incompatibility

associated with SSLB formation through organic solvent exchange allows SSLBs to be used as a platform for membrane protein measurements and therapeutic delivery vehicles.23,24 Through the manipulation of membrane physical properties and use of spherical-nanoparticles as a template for directed assembly, we have advanced the boundary of attainable, highly-charged nanostructures.


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ASSOCIATED CONTENT Supporting Information. Additional details on experimental methods, notes, and details. (PDF) AUTHOR INFORMATION Corresponding Author *Email: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT We would like to thank T.A. Witten for his initial suggestion on defect-mediated membrane rupture, and Jotham Austin and Tera Lavoie of the Advanced Electron Microscopy Facility for their advice and help with cryo-TEM imaging. This research was supported by NSF MCB 1413613 and Chicago MRSEC, which is funded by NSF through grant DMR 1420709. P.J.C. acknowledges support from a Kadanoff-Rice Fellowship from Chicago MRSEC. ABBREVIATIONS SSLB, spherical-nanoparticle supported lipid bilayer; TEM, transmission electron microscopy; cryo-TEM, cryogenic transmission electron microscopy; DOPA, 1,2-dioleoyl-sn-glycero-3-


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phosphate; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPS, 1,2-dioleoyl-sn-glycero-3phosphoserine; DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane REFERENCES (1)

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TABLE OF CONTENTS GRAPHIC


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Figure 1. Transmission electron microscopy reveals SSLB formation via an increase in osmotic pressure of highly-charged donor vesicles mixed with spherical-nanoparticles. a-c, highly-charged SSLB formation was verified via whole mount transmission electron microscopy (TEM) after incubating 60 nm aminefunctionalized silica nanoparticles in citrate salt buffer (10 mM citrate pH 6.0, 150 mM NaCl) with highlycharged 50% DOPA/50% DOPC vesicles suspended in CBS (a), CBS and increasing sucrose concentrations, ΔCSucrose = 250 mM (b) and ΔCSucrose = 325 mM (c). d-e, to verify that SSLB formation was mediated via an osmotic gradient and not sucrose-specific effect, SSLB preparation was performed as before but with vesicles suspended in CBS and increasing glycerol concentrations, ΔCGlycerol = 300 mM (d) and ΔCGlycerol = 400 mM (e). Scale bar: 50 nm. 156x106mm (220 x 220 DPI)


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Figure 2. Highly-charged SSLBs were confirmed via cryogenic transmission electron microscopy. To preclude artifacts due to sample preparation for whole mount transmission electron microscopy, cryogenic transmission electron microscopy (cryo-TEM) was used to validate solution structures. a, In the absence of a transmembrane osmotic gradient, highly-charged vesicles adhered to oppositely-charged aminefunctionalized nanoparticles without rupturing. b, In the presence of an optimal transmembrane osmotic gradient (ΔCSucrose = 325 mM), 50% DOPA/50% DOPC SSLBs were formed with contiguous (i.e. defectfree) supported lipid bilayers on top of 60 nm amine-functionalized nanoparticles. Scale bar: 50 nm. 154x76mm (220 x 220 DPI)


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Figure 3. SSLBs of various sizes can be obtained by modulating the osmotic gradient across donor vesicle membranes. a-c, mixing highly-charged (50% DOPA/50% DOPC) vesicles with 60 (a), 50 (b), and 40 nm (c) amine-functionalized spherical-nanoparticles yields SSLBs at optimal osmotic pressures (see details in Supporting Information, Table S2). d-e, replacing DOPA with another physiological anionic lipid, DOPS, also yields SSLBs when mixed with 60 (d), 50 (e), and 40 nm (f) amine-functionalized spherical-nanoparticles, suggesting that the SSLB formation via transmembrane osmotic gradient is independent of specific lipids. Scale Bar: 50 nm. 154x108mm (220 x 220 DPI)


Osmotic shock-triggered assembly of highly-charged, nanoparticle

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