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Design of a Homogeneous Multifunctional Supported Lipid Membrane on LbL Coated Microcarriers Martin Göse, Paula Pescador, and Uta Reibetanz Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 02 Feb 2015 Downloaded from http://pubs.acs.org on February 3, 2015

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Design of a Homogeneous Multifunctional Supported Lipid Membrane on LbL Coated Microcarriers Martin Göse, Paula Pescador and Uta Reibetanz* Institute for Medical Physics and Biophysics, Faculty of Medicine, University of Leipzig, Germany

Layer-by-Layer, supported lipid membrane, microcarrier, biocompatible, multifunctional lipid, homogeneity

Abstract Key challenges in the development of drug delivery systems are the prevention of serum compartment interaction and the targeted delivery of the cargo. Layer-by-Layer microcarriers offer many advantages due to various options in drug assembly and multifunctional design. Surface modification with a supported lipid membrane enhances biocompatibility, drug protection ability and specific functionality. However, the integration of functionalized lipids strongly influences the membrane formation and is often accompanied with sub-micrometer irregularities: The accessibility of underlying polymers to serum components may change the carrier’s properties and enhances the susceptibility to opsonisation. Therefore, the formation of a tightly assembled multi-functional lipid membrane has been emphasized. A 1 ACS Paragon Plus Environment

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phosphatidylserine/phosphatidylcholine

(POPS/POPC)

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bilayer

equipped

with

phosphatidylethanolamine-polyethylene glycol-biotin (PE-PEG-Biotin) was used to facilitate a biotin/streptavidin binding site for a variable attachment of an additional function, such as antibodies for specific targeting. Thus a pre-functionalized carrier where only the outer functionality needs to be replaced without disturbing the underlying structure could be created.

Introduction The development of biofunctionalized drug delivery systems or drug carriers with specific cell-targeting surface modifications offers a huge potential for the delivery of active agents to specific cells. Several forms of nano- and micrometer sized carriers, such as nanoparticles,1 liposomes,2 dendrimers,3 polyplexes4 or polymer coated microparticles5 are under intense investigation which can be further surface functionalized with specific ligands in order to achieve a targeted, accelerated or eased uptake.6-10 One promising approach to fabricate a multifunctional drug delivery system is the assembly of a polyelectrolyte multilayer onto spherical microparticles by means of Layer-by-Layer (LbL) technique.11 LbL implies the stable attachment of a polyelectrolyte pair, consisting of biopolymers (proteins, peptides, polysaccharides or nucleic acids) when considered to be used in biomedical applications, onto a surface. The polyelectrolytes assemble in an alternating fashion, forming a multilayer structure. To achieve the LbL coating, several driving forces (electrostatic interaction,12 hydrogen bonding,13 covalent coupling14) can be used. The modular character of the resulting multilayer allows the integration of active agents into or onto its structure as well as into the underlying template material or the hollow core after template dissolution.15-21 Furthermore, functional materials can be assembled in the same way either to influence the carrier’s properties e.g. by triggered rupturing of the multilayer22,23 or to add specific features e.g. for intracellular localization24 or for enhanced or specified carrier-cell interaction.9,25 2 ACS Paragon Plus Environment

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The charged nature of the polyelectrolytes often results in a net charge after completing multilayer assembly, which can induce unwanted drawbacks in biological environments. Such drawbacks could arise from unspecific binding of multiple serum components (e.g. proteins or cells) and an increased probability to get located by immune cells due to opsonisation.18,26-28 To overcome this problem, multiple surface modifications have been investigated so far: On the one hand, polyethylene glycol (PEG) or zwitterionic peptides have been assembled to inhibit unspecific protein interaction or even avoid interaction with bacteria,26,29-31 on the other hand strategies using a modifiable supported lipid membrane assembly as a camouflage of the internal LbL-drug delivery system have been successfully applied.32-35 Hereby, the assembly of an artificial supported lipid membrane provides most convenient conditions, as it can be fast and easily prepared from appropriate lipid mixtures and equipped with components of native cell membranes and/or functional artificial compounds, while the modularity of the LbL technology is maintained. Furthermore, the assembly of a supported lipid membrane increases the stability of active agents incorporated into the underlying LbL multilayer36 and enhances the biocompatibility of the drug delivery system by mimicking a cell-like surface.37-39 Beside those characteristics, an additional feature enhances the application spectrum compared to simply surface assembly of molecules: the supported lipid membrane can be independently used as a platform for the integration of further functionalities, such as for surface targeting, without losing its biocompatibility and shielding properties. However, an inhomogeneous or homogeneous formation of a supported lipid membrane is strongly influenced by the applied lipid mixture, coating conditions as well as the amount of integrated functional lipids. Nevertheless, a homogeneous lipid membrane is of huge importance to prevent penetration and unwanted binding of serum proteins to multilayer components. Though the assembly of a supported lipid membrane on planar surfaces is under intensive investigation,40-42 far less investigation have been performed regarding the lipid membrane assembly on micrometer sized, LbL coated particles.43 3 ACS Paragon Plus Environment

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Therefore, in this study the influence of assembly conditions on the formation of a functionalized, artificial supported lipid membrane on polyelectrolyte covered microparticles (microcarriers) and its examination by conventional easy accomplishable fluorescence based techniques have been emphasized. Special focus was on the integration of a multifunctional lipid/polymer composite. Spherical silica microparticles (d = 4.99 µm) have been coated with biocompatible and biodegradable biopolymers protamine sulphate (PRM) and dextran sulphate (DXS)44,45 as a precursor for the functionalized membrane assembly. Supported lipid membrane has been then assembled via Liposome Spreading (LS), that means by adsorption and spreading of preprepared small unilamellar vesicles (liposomes)46,47 consisting of equal amounts of anionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and zwitterionic lipid 1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), onto the polyelectrolyte multilayer according to Fischlechner et al.48 As a lipid/polymer composite, phosphoethanolamine (PE) as “lipid anchor” was combined with PEG and biotin (PE-PEG-Biotin). This lipid/polymer composite combines several advantages, since the different components are intended to facilitate specific demands: PE provides the stable integration into the POPS/POPC membrane, PEG reduces unspecific binding of proteins and serves as a spacer between the microcarrier surface and biotin, and biotin is used as a specific binding site to enable a sandwich like binding strategy for streptavidin and biotinylated agents e.g. antibodies, cellpenetrating peptides or active agents. The emphasis here is on the fabrication of a preassembled functional basis system with an exchangeable outer functionalization without risking integrity disturbances of the inner structure. The assembled lipid membrane was investigated regarding the following aspects in order to detect convenient assembly conditions: (1) A homogenous lipid membrane formation on top of the microcarrier surface is required to reduce possibly unspecific multilayer binding of serum proteins to a minimum. 4 ACS Paragon Plus Environment

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(2) The presence of a multi-functional lipid/polymer composite within the lipid mixture may not disturb this homogeneity. (3) A sufficient amount of the multi-functional lipid/polymer composite has to be integrated and the specific binding part (biotin) has to remain accessible for the subsequent binding of specific agents e.g. targeting antibodies. In order to achieve those demands, the influence of PE-PEG-Biotin concentration in the POPS/POPC mixture as well as the LS incubation conditions on the lipid membrane formation has been investigated. The lipid membrane structure was mainly monitored by means of Flow Cytometry (FC), Confocal Laser Scanning Microscopy (CLSM) and multiple adsorption techniques. For the detection of irregularities indirect approaches (fluorescenceprobe as lipid membrane constituent) have been compared with direct approaches (differentsized molecules sensing irregularities by penetration) illustrating wide differences in lipid membrane formation depending on the above mentioned parameters. Finally, best parameters have been identified providing a homogeneous, functional lipid surface on top of an LbL biopolymer coated microcarrier. The assembly of specific antibodies proves the design as an effective combination of biocompatibility, shielding and targeting functionalities on a supported lipid membrane.

Materials and Methods (1) Materials Protamine sulphate salt from herring (PRM), fluorescein isothiocyanate-bovine serum albumin (BSA-FITC), bovine serum albumin (BSA), trypan blue (TB), streptavidin-Cy5 (strep-Cy5), sucrose and ascorbic acid have been purchased from Sigma-Aldrich (Taufkirchen bei München, Germany). 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), 1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

(POPC),

1,2-distearoyl-sn-glycero-3-

phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (PE-PEG-Biotin) and 1,25 ACS Paragon Plus Environment

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dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein) (PE-FL) were obtained from Avanti Polar Lipids (Alabaster, USA). Mouse monoclonal antibody [T21/8] against CCR5 (Biotin) (ABp) and goat polyclonal secondary antibody against mouse IgG – H&L (Cy5) (ABs-Cy5) have been purchased from Abcam (Cambridge, UK). Silica-microparticles d = 4.99 µm (SiO2) were purchased from microparticles GmbH (Berlin, Germany). Dextran sodium sulphate (DXS) (MW ~ 40.000 Da) was purchased from ICN Biochemicals (Irvine, USA). FITC Easy Calibration Kit was obtained from Spherotech Inc. (Lake Forest, USA). Phosphate buffered saline (PBS) was from PAA (Buckinghamshire, UK) and Sodium chloride was from Carl Roth (Karlsruhe, Germany). Polycarbonate membrane filter (d = 50 nm) were obtained from Avestin (Ottawa, USA). Perchloric acid was purchased from Riedel-de Haën (Seelze, Germany) and ammonium molybdate was from VEB Laborchemie Apolda (Apolda, Germany). (2) Methods Small Unilamellar Vesicle preparation: Small unilamellar vesicles (SUVs) of different lipid compositions were prepared directly before use. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoL-serine (POPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (PEPEG-Biotin) were mixed in 1:1:0 (0 mol% PE-PEG-Biotin), 1:1:0.002 (0.1 mol% PE-PEGBiotin), 1:1:0.01 (0.5 mol% PE-PEG-Biotin), 1:1:0.02 (1 mol% PE-PEG-Biotin), 1:1:0.1 (5 mol% PE-PEG-Biotin), 1:1:0.2 (10 mol% PE-PEG-Biotin) and 1:1:0.4 (20 mol% PE-PEGBiotin) molar ratio in chloroform. To perform lipid fluorescence experiments, 1,2-dioleoyl-snglycero-3-phosphoethanolamine-N-(carboxyfluorescein) (PE-FL) has been added to the lipid mixture with a final concentration of 0.1 mol%. The lipid mixtures were dried and redissolved in phosphate buffered saline (PBS) solution to obtain a final concentration of 10.2 mM. Lipid solutions were briefly vortexed and sonicated for 15 min to obtain multilamellar vesicles (MLV). MLVs were converted into small unilamellar vesicles by 6 ACS Paragon Plus Environment

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extruding the MLV suspension using a polycarbonate membrane (hole diameter = 50 nm) 20 times above phase transition temperature (ϑ = 37°C). Giant Unilamellar Vesicle preparation: Giant unilamellar vesicles (GUVs, d > 10 µm) were obtained by electroformation. Several lipid mixtures were prepared (POPS:POPC in 1:1 ratio; POPS:POPC:PE-PEG-Biotin

in

1:1:0.01

(0.5 mol%

PE-PEG-Biotin)

ratio

and

POPS:POPC:PE-PEG-Biotin in 1:1:0.2 ratio (10 mol% PE-PEG-Biotin)). The lipid solutions were adjusted to a total lipid concentration of 1.5 mM in chloroform. GUVs were obtained in 250 mM sucrose solution using titan electrodes and an alternating electric field for 3.5 h.49,50 GUV suspension was collected and stored at 4°C for maximum 24 h. For the investigation of unspecific protein interaction with lipid membranes, GUV suspension have been incubated in 0.15 mM (10 mg ml-1) BSA-FITC solution for 1 h. Afterwards, GUV BSA-FITC suspension was diluted with 250 mM sucrose solution. Layer-by-Layer coating of microparticles: Silica (SiO2)-microparticles with an average diameter of (4.99 ± 0.22) µm were coated with polyelectrolytes using the Layer-by-Layer (LbL) technique. 200 µl SiO2-microparticle stock suspension have been dispersed in 500 µl of a 1 mg ml-1 protamine sulphate solution (PRM) containing 0.1 M NaCl. After 10 min incubation under gentle shaking, the sample was centrifuged (1 min, 2000 g) and the supernatant and residual protamine sulphate have been removed by three times washing with 400 µl 0.1 M NaCl and coating procedure has been continued with dextran sodium sulphate solution (DXS, 1 mg ml-1 in 0.1 M NaCl). This procedure was repeated until the desired number of nine polyelectrolyte layers was deposited: [PRM/DXS]4/PRM. Polyelectrolyte covered microparticles are referred as microcarriers. Finally the microcarrier concentration has been adjusted to the prior stock solution concentration (≈ 3 × 105 microparticles µl-1). Lipid

coated

microcarriers:

Polyelectrolyte

covered

SiO2-microcarriers

([PRM/DXS]4/PRM) have been coated with an additional lipid membrane using liposome spreading (LS) technique.46 5 µl, 10 µl, 20 µl, 40 µl and 100 µl microcarrier suspension have 7 ACS Paragon Plus Environment

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been incubated with 80 µl SUV suspension in 250 µl PBS under constant shaking for 1 h at 37°C. In addition, 95 µl, 90 µl, 80 µl, 60 µl or 0 µl 0.1 M NaCl solution was added to the incubation mixture to adjust ionic strength and incubation volume, respectively. The coating conditions correspond to a liposome surface area to microcarrier surface area ratio (AL/AM) of 1600:1, 800:1, 400:1, 200:1 and 80:1, respectively. Residual SUVs were removed by three centrifugation (1 min, 2000 g) and washing steps using 400 µl PBS. Finally, the microcarriers were resuspended in 200 µl PBS. Streptavidin binding: Binding of streptavidin to the lipid coated microcarrier surface was observed using fluorescence labelled streptavidin-Cy5 (strep-Cy5). 100 µl of the microcarrier suspension (1 × 105 microparticles µl-1) was dispersed in 100 µl strep-Cy5 solution (0.15 mg ml-1). After 1 h gentle shaking, the microcarriers were collected by centrifugation and three washing steps with 200 µl PBS. The final volume has been adjusted to 100 µl. BSA binding: Binding of bovine serum albumin (BSA) to the lipid coated microcarriers was observed

using

fluorescence

labelled

BSA-FITC.

The

microcarriers

(100 µl,

1 × 105 microparticles µl-1) were dispersed in 400 µl of 0.15 mM (10 mg ml-1) BSA-FITC solution for 1 h under gentle shaking. Thereafter, residual BSA-FITC was removed by three centrifugation and washing steps with 400 µl PBS. The final volume was adjusted to 200 µl. The amount of assembled BSA-FITC molecules onto the microcarrier surface has been performed using standard fluorescence FITC beads. The beads have been measured at the same amplification as the microcarriers, allowing the determination of assembled FITC molecules. Based on a label degree of 7 FITC molecules per BSA molecule, the amount of assembled BSA-FITC molecules has been determined. Antibody binding: The specific and unspecific binding of the biotinylated primary antibody (mouse monoclonal antibody [T21/8] against CCR5 (Biotin), ABp) onto the microcarriers surface has been investigated using a secondary Cy5 fluorescence labelled antibody (goat polyclonal secondary antibody against mouse IgG – H&L (Cy5), ABs-Cy5). In a first step, the 8 ACS Paragon Plus Environment

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microcarriers (25 µl, 1 × 105 microparticles µl-1) have been incubated in 100 µl of 0.02 µg µl-1 ABp solution for 1 h under gentle shaking. Afterwards, unbound ABp have been removed by three centrifugation and washing steps with 200 µl PBS. As a second step, the microcarriers were dispersed in 100 µl of 0.05 µg µl-1 ABs-Cy5 solution for 1 h under constant shaking, followed by three centrifugation and washing steps with 200 µl PBS. The final volume was adjusted to 200 µl for further investigations. Fluorescence quenching assay: Lipid coated SiO2-microcarriers which were additionally doped with 0.1 mol% PE-FL have been mixed with trypan blue (TB)51 solution. 5 × 105 microcarriers have been dispersed in 220 µl 0.035 % TB solution and incubated for 10 min. Bartlett assay: The amount of assembled lipid onto the microcarrier surface as well as the bilayer character of POPS/POPC 1:1 SUVs were determined using Bartlett assay.52 1.5 × 107 lipid coated microcarriers or 4.2 × 1011 SUVs have been incubated with 200 µl 70 % perchloric acid solution at 230°C for 30 min leading to a destruction of the phospholipids to inorganic phosphate, respecively. Thereafter, 700 µl 3.6 mM ammonium molybdate with 12.5 % perchloric acid and 700 µl 3 % ascorbic acid have been added to the microcarriers and incubated for 5 min at 100°C. Thus, the inorganic phosphate is converted to phosphormolybdic acid, which is later reduced to a blue coloured compound. The solutions were allowed to cool down and absorbance at λ = 820 nm was measured using a Tecan Infinite 200 Pro. Confocal Laser Scanning Microscopy (CLSM): The fluorescence labelled microcarriers and GUVs were imaged with a Zeiss LSM 510 Meta, equipped with a 63x objective. Fluorescein labelled components (PE-FL and BSA-FITC) were excited using an Argon laser with λEx = 488 nm and fluorescence intensity was detected using a bandpass filter of λ = (505 – 550) nm. Cy5 labelled streptavidin and secondary antibody were excited using a Helium 9 ACS Paragon Plus Environment

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Neon laser with λEx = 633 nm and fluorescence was detected using a longpass filter of λ ≥ 650 nm. Flow Cytometry (FC): Quantitative analysis of the microcarrier coating procedure has been performed using a BD FACS Calibur Flow Cytometer. About 5 × 105 microcarriers have been suspended in 200 µl PBS. 1 × 104 events of single microcarriers has been collected and analysed according to standard procedures. The amplification of each measurement has been adjusted to the demands of the experiment. Electrophoretic mobility of SUVs: The electrophoretic mobility of SUVs was measured in 1 mM NaCl (pH = 6.5) solution at room temperature using a Brookhaven ZetaPALS. The zeta potential was calculated using the Smoluchowski equation.53 Hydrodynamic diameter and concentration of SUVs: The hydrodynamic diameter of SUVs and the SUV concentration were measured in 0.1 M NaCl solution at room temperature using a NanoSight LM10HS device. The final lipid concentration has been adjusted to 1-0.1 µM in order to enable single SUV detection.

Results and Discussion In a first step different amounts of PE-PEG-Biotin (0 mol%, 0.1 mol%, 0.5 mol%, 1 mol%, 5 mol%, 10 mol% and 20 mol%) were integrated into a POPS/POPC 1:1 lipid mixture. This range covering two decades has been used to determine the maximum PE-PEG-Biotin concentration of the lipid mixture without changing the negative net charge of the liposomes since the first process in LS is the electrostatic attraction of liposomes towards the oppositely charged surface.54 On the other hand, a minimum of specific binding sites is needed to facilitate the specific binding of adequate amounts of functionalized molecules. Thus, liposomes (d = 50 nm) of different lipid compositions were analysed regarding their electrophoretic mobility (Figure 1). As can be seen, pure POPS/POPC 1:1 lipid mixture 10 ACS Paragon Plus Environment

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(0 mol% PE-PEG-Biotin) exhibits a considerably high negative zeta potential of -(26.73 ± 1.45) mV. Low PE-PEG-Biotin concentrations of 0.1 mol%, 0.5 mol% and 1 mol% show only a minor influence on the zeta potential, represented by comparable equally low zeta

potentials

of

-(22.73 ± 1.43) mV,

-(22.98 ± 3.43) mV

and

-(24.25 ± 0.8) mV,

respectively. Nevertheless, a decrease of the zeta potential occurs with PE-PEG-Biotin concentrations above 1 mol%. Concentrations of 5 mol%, 10 mol% and 20 mol% exhibit a zeta potential of -(10.66 ± 0.54) mV, -(8.65 ± 1.57) mV and -(2.79 ± 0.65) mV, respectively. Further investigations of multiple liposome properties (size distribution, lipid composition after preparation, liposome/micelle ratio depending on PEG concentration, influence of the individual components of the composite system; see Supporting Information Figure S1-S3) show that this effect can be contributed to structural changes of the liposome surface, caused most likely by a transition of PEG from the ‘mushroom” state towards a ‘brush’ state,31,55 followed by ion condensation. Thus it can be concluded that high concentrations of PE-PEGBiotin might reduce effective liposome/surface interaction and finally impede a homogeneous lipid membrane formation.

Figure 1. Zeta potential measurement of small unilamellar vesicles (d = 50 nm). The vesicles are prepared of POPS/POPC ratio 1:1 with varying amounts of PE-PEG-Biotin. Based on those results, the membrane formation onto positively charged LbL-microcarriers ([PRM/DXS]4/PRM)

was

examined

by

investigating

two

distinct

PE-PEG-Biotin 11

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concentrations: 0.5 mol% (SiO20.5%: negative zeta potential, but low PE-PEG-Biotin concentration) and 10 mol% (SiO210%: neutral zeta potential, but high PE-PEG-Biotin concentration). To visualize the assembled membrane, the lipid mixtures were further equipped with 0.1 mol% of the fluorescent probe PE-Fluorescein (PE-FL). The effect of the two distinct PE-PEG-Biotin concentrations as well as the liposome- to microcarrier-surface ratio (AL/AM: surface area of the applied liposomes related to the surface area of the spherical SiO2-microcarriers) onto the lipid membrane formation have been investigated. AL/AM ratios of 1600:1, 800:1, 400:1, 200:1 and 80:1 have been applied, taking into account that all ratios represent an excess of lipids related to the overall microcarrier surface. FC based analysis of the resulting microcarrier fluorescence intensities are displayed in Figure 2a. As can be seen, the integration of low amounts of PE-PEG-Biotin (SiO20.5%) results in high fluorescence intensities independent on the AL/AM ratio (378 ± 41) a.u. In comparison, an increase in PEPEG-Biotin concentration to 10 mol% (SiO210%) shows an intensity reduction to (148 ± 28) a.u. (2.5 fold less signal) but retains the independence on AL/AM ratio. Those results were supported by CLSM images (Figure 2b-e) using the same amplification. Independent on the preparation conditions, SiO20.5% (Figure 2b and d) exhibit a higher fluorescence intensity compared to SiO210% (Figure 2c and e). In addition all preparations show a presumably homogeneous fluorescence distribution surrounding the microcarriers which is supposed to illustrate a sufficient homogeneity of the lipid membrane. But although the integration of a fluorescence label directly into the membrane (e.g. PE-FITC, PE-NBD, PE-Rhodamine) is often used to confirm membrane homogeneity,56 the lateral resolution of CLSM in the dimension of the applied wavelength may mislead the interpretation of the data especially regarding smaller but nonetheless important irregularities, as shown in subsequent experiments.

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Figure 2. (a) FC fluorescence intensity measurements of SiO2-microcarriers coated with a lipid membrane made of POPS/POPC 1:1 + 0.5 mol% or 10 mol% PE-PEG-Biotin doped with 0.1 mol% PE-FL in dependence of different AL/AM incubation conditions. Uncoated SiO2-microcarriers served as a non-fluorescent control. (b)-(e) CLSM images of lipid membrane coated SiO2-microcarriers prepared at AL/AM ratio of 80:1 (b and c) and 1600:1 (d and e). Images (b) and (d) represent a lipid membrane made of POPS/POPC 1:1 + 0.5 mol% PE-PEG-Biotin, whereas images (c) and (e) show a lipid membrane made of POPS/POPC 1:1 + 10 mol% PE-PEG-Biotin. Scale bars correspond to 10 µm. It can be assumed, that the differences in fluorescence intensities of SiO20.5% and SiO210% are caused by distinct differences in lipid membrane formation onto the microcarrier surface, since an equal concentration of the fluorescent lipid PE-FL has been applied. Due to the low


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concentration of PE-FL (0.1 mol%), self-quenching effects of neighbouring fluorescein molecules can be excluded. Thus, two explanations seem to be possible: (1) particular high fluorescence intensity of SiO20.5%-microcarriers: caused by a tendency towards lipid multilayer formation or (2) particular low fluorescence intensity of SiO210%-microcarriers: caused by incomplete surface coverage. In order to explore the nature of the assembled lipid membrane in dependence on the amount of functionalized lipid, that is the formation of multilayer, bilayer or irregular bilayer, several experiments were carried out. At first, Bartlett assay, detecting the amount of phosphate,52 has been performed to determine membrane formation based on the amount of the assembled phospholipids. Control measurements with SUVs composed of POPS/POPC 1:1 show a good determination of bilayer characteristics (1.07 ± 0.40 lipid bilayers per SUV). As shown in Table 1, presenting the amount of phosphate per 107 microcarriers (nPO4), the approximate amount of assembled lipid monolayers (nmono) has been estimated based on the assumption of perfect spherical microparticles with equal diameters of d = 4.99 µm and a lipid head group size of ALipid = 0.66 nm² according to the following equation: n mono =

n PO 4 N A ALipid

π d 2 10 7

To determine the overall influence of PE-PEG-Biotin on the lipid membrane formation, 0 mol% PE-PEG-Biotin (SiO20%) has been used as a reference. As can be seen, 0 mol% and 0.5 mol% PE-PEG-Biotin containing lipid membrane assemblies (AL/AM = 80:1) both show a comparable amount of phospholipids. In both cases, 3.12 ± 1.65 or 3.22 ± 0.93 lipid monolayers can be calculated. In comparison, a PE-PEG-Biotin concentration of 10 mol% (AL/AM = 80:1) results in a drastic decrease of the phospholipid amount, building up only 1.61 ± 0.40 lipid monolayers onto the microcarrier surface, which correlates with the intensity differences in Figure 2a. Nevertheless, this method implicates a considerably high standard 14 ACS Paragon Plus Environment

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deviation of (35 – 45) % due to counting procedure and diameter varieties of the microcarriers rendering an explicit estimation of multilayer or irregular bilayer formation impossible. Here, samples exhibiting 0 mol% and 0.5 mol% PE-PEG-Biotin develop two to four lipid monolayers, whereas 10 mol% PE-PEG-Biotin samples express at most two lipid monolayers. Thus, in case of SiO20.5% and SiO210% a (a) double bilayer/bilayer or (b) bilayer/irregular bilayer formation seems to be possible.

Table 1. Bartlett assay based determination of assembled phosphate/lipid monolayers onto microcarrier surface in dependency of the PE-PEG-Biotin concentration PE-PEG-Biotin concentration [mol%] 0 0.5 10

nPO4 [nmol]

nmono

6.19 ± 3.27 5.7 ± 1.65 3.19 ± 0.79

3.12 ± 1.65 3.22 ± 0.93 1.61 ± 0.4

To specify the lipid membrane formation characteristics of the three preparations further, a fluorescent quenching approach has been used. 0.1 mol% PE-FL has been integrated into the lipid mixture to be exposed to a fluorescence quencher (trypan blue, TB). TB is known to quench fluorescein molecules in close distance and is not able to penetrate intact lipid membranes. Considering no phase separation and therefore a homogeneous distribution of the fluorescence marker PE-FL within the lipid membrane, a quenching distance R0 between FL and TB of (3.8 - 4.2) nm,57,58 negligible inter-membrane exchange processes as flip-flops between lipid monolayers and a reversible quenching process, it can be assumed that in case of compact lipid membranes only surface fluorescence molecules are accessible to TB. Thus, perfect lipid bilayer formation would be represented at 50 % quenching while lipid multilayer formation would be characterized by lower quenching values (remaining fluorescence intensities > 50 %). Irregularities of sufficient size within the lipid membrane would allow the 15 ACS Paragon Plus Environment

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quencher to penetrate and gain access to the inner fluorophores and fluorescence intensities < 50 % can be expected. Cross sections from CLSM images of SiO20%-, SiO20.5%- and SiO210%-microcarriers, prepared at AL/AM ratio of 80:1, are displayed in Figure 3. Fluorescence intensities before quenching (w/o TB) and after quenching (w TB) have been compared, whereas the maximum of TB-untreated samples was set to 100 %. Observing SiO20%- and SiO20.5%-microcarriers, TB incubation results in an intensity reduction to approximately 50 % (57 % and 46 % respectively), which corresponds to a lipid bilayer formation at the microcarrier surface. In contrast, SiO210%-microcarriers show a drastic reduction of fluorescence intensity after TB incubation with the remaining signal only slightly above background noise. This behaviour indicates a porous, irregular structure of the assembled lipid membrane, allowing TB to reach FL-molecules in the inner lipid monolayer. In combination with the results obtained by the Bartlett assay it can be concluded that the assembled lipid membrane exhibits a bilayer structure at 0 mol% and 0.5 mol% PE-PEGBiotin, whereas an irregular, perforated bilayer is formed at 10 mol% PE-PEG-Biotin.


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Figure 3. Cross sections of CLSM images of SiO2-microcarrier coated with lipid mixtures of POPS/POPC 1:1 + (a) 0 mol% PE-PEG-Biotin, (b) 0.5 mol% PE-PEG-Biotin and (c) 10 mol% PE-PEG-Biotin at AL/AM ratio of 80:1. The lipid mixtures have been additionally doped with 0.1 mol% PE-FL. Fluorescence intensities were detected before (w/o TB, black curve) and after (w TB, red curve) the addition of fluorescence quencher TB. The insets show the corresponding CLSM images of SiO2-microcarriers. Another indication towards irregular lipid membrane formation at high PE-PEG-Biotin concentrations is shown in Figure 4a. Here, the specific binding of fluorescence labelled streptavidin (strep-Cy5) to the biotin compartment of the lipid/polymer composite was investigated and is exemplarily shown for AL/AM ratio of 80:1. SiO20%-, SiO20.5%-, SiO210%- as well as control polyelectrolyte covered microcarriers were incubated with equal amounts of strep-Cy5. As can be seen, polyelectrolyte covered microcarriers (PRM/DXS, grey curve, region M1) do not exhibit an increased strep-Cy5 fluorescence signal compared to the non17 ACS Paragon Plus Environment

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fluorescent control (control, black curve, region M1), indicating a negligible binding affinity of strep-Cy5 to the positively charged PRM surface. Also SiO20%-microcarriers (light grey curve, region M1) do not exhibit unspecifically bound strep-Cy5. It can be concluded, that strep-Cy5 binding behaviour in the presence of biotin exclusively visualizes the accessibility of biotin and thus the surface structure of the assembled functional lipid membrane. As expected, the integration of PE-PEG-Biotin leads to a drastic increase of strep-Cy5 binding. In case of SiO20.5% (blue curve) a high fluorescence intensity increase with a narrow distribution (geometric mean value of 360 a.u.) can be detected. In case of SiO210% (red curve) only a 3 fold higher signal of 1146 a.u. was detected despite of a 20 fold higher PE-PEG-Biotin concentration. This suggests a reduced accessibility of biotin. Furthermore, the distribution of the strep-Cy5 signal changes notably. Whereas the narrow fluorescence intensity distribution (Region M2) of SiO20.5%-microcarriers indicates a uniform coating of the microcarriers, SiO210%-microcarriers reveal a broad distribution with two binding populations: a comparable narrow part at high values (Region M3-2) and an additional broad shoulder at low fluorescence intensities (Region M3-1). Region M3-1 indicates the existence of microcarriers with reduced streptavidin binding abilities due to the inaccessibility or lack of biotin binding partners. The corresponding CLSM images show the strep-Cy5 binding pattern onto the microcarrier surface in more detail (Figure 4b and c). In correlation to the narrow FC intensity peak, SiO20.5%-microcarriers (Figure 4b) exhibit a uniform fluorescence distribution. Also SiO210%-microcarriers (Figure 4c) show an almost homogeneous fluorescence distribution with slight intensity fluctuation on the microcarrier surface (indicated by arrows) which indicates variations in lipid membrane formation.


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Figure 4. (a) FC based fluorescence intensity histograms of strep-Cy5 binding onto lipid membrane coated SiO2-microcarriers. Microcarriers have been assembled with lipid mixtures of POPS/POPC 1:1 + 0 mol%, 0.5 mol% or 10 mol% PE-PEG-Biotin at AL/AM ratio of 80:1. As a control PRM/DXS covered microcarriers have been measured before (control) and after (PRM/DXS) incubation with strep-Cy5. Region M1 corresponds to background fluorescence intensity of untreated microcarriers. Regions M2, M3-1 and M3-2 represent specific (M2 and M3-2) and restrained (M3-1) strep-Cy5 binding. Images (b) and (c) show CLSM images of strep-Cy5 fluorescence intensity distribution on the surface of (b) 0.5 mol% and (c) 10 mol% PE-PEG-Biotin covered microcarriers, whereas the arrows point on spots of inhomogeneous strep-Cy5 binding. Scale bars correspond to 10 µm. On the basis of these measurements, it can be concluded that the integration of an increasing amount of the lipid/polymer composite PE-PEG-Biotin indeed affect the formation of a lipid bilayer. In biological systems, such irregularities may play an important role since they would allow penetration, intercalation or assembly of serum proteins in the range of several kDa59 on the exposed polyelectrolyte multilayer which alters the surface properties of the microcarrier. 19 ACS Paragon Plus Environment

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However, CLSM imaging of fluorophores attached to the lipid membrane seems not to be appropriate for the detection of irregularities below micrometer dimension. Thus, the strategy was changed from indirect, that is detecting uncovered parts by using fluorophores in the lipid covered surface, towards a direct detection of irregularities by following the penetration of molecules through the lipid membrane. Two different molecules have been used inspired by the size range of serum components as shown schematically in Figure 5. The penetration of fluorescein isothiocyanate labelled bovine serum albumin (BSA-FITC) was followed to report larger irregularities in the range of ≥ 5 - 20 nm (MW BSA: 68 kDa, Figure 5a). The formation of small irregularities in the range of a few nanometres has been investigated using TB (MW about 1 kDa) as a reporter by quenching inner-membrane integrated PE-FL (Figure 5b). In a first step, BSA was used as a representative of a larger molecule as albumins are regular blood components. BSA has an ellipsoid hydrodynamic structure of about 14 nm × 4 nm × 4 nm60 and exhibits a negative net charge at physiological conditions (IP: 4.7).61 Although BSA is not known to bind permanently onto PRM,62 the attraction towards the positively charged microcarrier surface makes its suitable as a reporter molecule. To observe the binding, FITC fluorescence labelled BSA has been incubated with SiO20%-, SiO20.5%-

and

SiO210%-

as

well

as

with

polyelectrolyte

covered

microcarriers

(PRM/DXS+BSA). BSA-untreated microcarriers (PRM/DXS) served as a non-fluorescent control. The fluorescence intensities as well as the calculated concentration of assembled BSA-FITC are displayed in Figure 6a. Compared to the non-fluorescent control (PRM/DXS, (1.43 ± 0.12) a.u.), PRM/DXS+BSA microcarriers show a slightly increased BSA-FITC fluorescence intensity signal of (102.92 ± 38.12) a.u. (≙ 0.049 ng mm-2).


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Figure 5. Detection concepts of possible lipid membrane formation on SiO2-microcarriers at high PE-PEG-Biotin concentrations. Image (a) shows the detection of large irregularities (d ≥ (5-20) nm) during lipid membrane assembly by the penetration of larger molecules e.g. BSA-FITC (MW: 68 kDa), whereas image (b) illustrates the detection of small irregularities (d ≥ (1-2) nm) during lipid membrane assembly, by the penetration of TB (MW: 961 Da). SiO20%-microcarriers, prepared at AL/AM ratio of 80:1, exhibit a comparable amount of BSAFITC fluorescence intensity ((100.51 ± 50.17) a.u. ≙ 0.048 ng mm-2). A similar result can also be found for SiO20.5%-microcarriers, representing a slight, but not significant increase in fluorescence intensity ((170.61 ± 37.21) a.u. ≙ 0.075 ng mm-2). However, SiO210%-microcarriers show a completely different behaviour. They exhibit a significant

2.5 fold

increased

BSA-FITC

fluorescence

intensity

((383.23 ± 62.18) a.u. ≙ 0.2 ng mm-2). A similar behaviour can be obtained at AL/AM ratio of 1600:1. Beside a low but homogeneous BSA-FITC fluorescence intensity distribution on PRM/DXS, SiO20%- and SiO20.5%-microcarriers (data not shown), CLSM images of SiO210%microcarriers AL/AM ratio 80:1 (Figure 6b) and AL/AM ratio 1600:1 (Figure 6c) confirm the presence of parts of increased (Figure 6b) or reduced (Figure 6c) BSA-FITC fluorescence intensity (arrows). These findings suggest that besides a low unspecific binding of BSA onto lipid and polyelectrolyte covered surfaces another interaction mechanism in presence of high amounts of the lipid/polymer composite does exist. Two options seem to be possible:


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(1) BSA interacts with the functional part (PEG and/or biotin) of the lipid/polymer composite or (2) The homogeneity of the lipid bilayer is affected by the presence of the functionalized lipid allowing the intercalation of BSA into the interlayer between the polyelectrolyte covered surface and the assembled lipid membrane.

Figure 6. (a) FC investigations of the amount of assembled BSA-FITC onto lipid membrane coated SiO2-microcarriers incubated with different lipid compositions (POPS/POPC 1:1 + 0 mol%, 0.5 mol% and 10 mol% PE-PEG-Biotin) at AL/AM ratio of 80:1 and 1600:1. As a control polyelectrolyte covered microcarriers with an outer layer of positively charged PRM with (PRM/DXS + BSA) and without (PRM/DXS) BSA-FITC incubation are shown. The concentration of assembled BSA-FITC has been determined using a standard calibration kit. Statistical analysis was carried out using a two-tailored student`s t-test: ** p ≤ 0.01, n.s. not significant. Images (b) and (c) show CLSM images of BSA-FITC fluorescence intensity distributions on the surface of 10 mol% PE-PEG-Biotin (b) AL/AM = 80:1 and (c) AL/AM = 1600:1 covered microcarriers, whereas the arrows point on spots of inhomogeneous BSA-FITC binding. Scale bars correspond to 10 µm. 22 ACS Paragon Plus Environment

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To rule out the binding of BSA to the functional part of the lipid/polymer composite, giant unilamellar vesicles (GUVs) have been prepared of POPS/POPC 1:1 with varying amounts of PE-PEG-Biotin (0 mol%, 0.5 mol% and 10 mol%). They exhibit a continuous, homogeneous bilayer without irregularities and represent a convenient model system for binding observations. GUVs have been incubated with high amounts of BSA-FITC and investigated by means of CLSM (Figure 7). As shown in Figure 7a-c (GUVs composed of: a) POPS/POPC 1:1, b) POPS/POPC 1:1 + 0.5 mol% PE-PEG-Biotin and c) POPS/POPC 1:1 + 10 mol% PEPEG-Biotin), non-purified GUVs exhibit a strong fluorescence intensity in supernatant due to high concentration of BSA-FITC, but no internal fluorescence intensity, demonstrating the integrity of the lipid membrane. To detect possible BSA-FITC accumulation at the GUVs surface, supernatant BSA-FITC has been removed (purified GUVs, Figure 7d-f) nearly completely reducing background fluorescence intensity. High amplification was then used to enable detection of even low amounts of surface-attached BSA-FITC molecules. Nevertheless, neither unfunctionalized (Figure 7d) nor functionalized GUVs (Figure 7e-f) show an increase in fluorescence intensity at the surface, thus an at most minimal BSA-FITC binding can be assumed. It has to be mentioned, that images obtained by CLSM allow a quantitative analysis only to a limited degree. Thus, minor amounts of BSA-FITC still might be assembled onto the GUVs surface which is in good agreement with BSA-FITC signals detected by FC (Figure 6a).


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Figure 7. CLSM intensity images of GUVs composed of POPS/POPC 1:1 + (a and d) 0 mol% PE-PEG-Biotin, (b and e) 0.5 mol% PE-PEG-Biotin and (c and f) 10 mol% PE-PEGBiotin, after incubation in BSA-FITC solution. Images (a)-(c) have been recorded before sample purification (high amount of BSA-FITC in solution), whereas images (d)-(f) show GUVs after sample purification (insignificant amounts of BSA-FITC in solution). Since the binding of BSA-FITC to PRM, lipid components (POPS and POPC) and components of the lipid/polymer composite (PE, PEG and biotin) is rather low, a penetration of BSA-FITC through lipid irregularities and the intercalation into the water containing interlayer between the lipid membrane and the underlying polyelectrolyte multilayer can be assumed (Figure 5a). The formation of such an accessible interlayer driven by the integration of a PEGylated lipid into the membrane mixture has already been reported by Kaufmann et al. and Wagner et al.63,64 It was shown, that the dimension of such an interlayer is determined by the length of the PEG molecule and ranges a few nanometers. Thus, irregularities or holes within the lipid membrane in the size range of (5 – 20) nm would allow BSA-FITC or other serum proteins of similar sizes to intercalate into this interlayer. 24 ACS Paragon Plus Environment

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But also smaller molecules such as hormones65 are common serum components and would be able to affect surface properties of the microcarrier in the case of an irregular assembled lipid membrane. Thus, smaller irregularities have to be considered. As a reporter, TB was used for such investigations due to its low molecular weight and its nature not to be able to penetrate an intact lipid membrane66. Therefore, it has been used again as a fluorescence quencher of fluorescein which was assembled to PE as a lipid membrane constituent. As mentioned earlier, irregularities can be visualized by an intensity decrease below 50 %. SiO20%-, SiO20.5%- and SiO210%-microcarriers (AL/AM ratio of 80:1 and 1600:1) have been investigated. The fluorescence intensity of the microcarriers before (Iw/o) and after TB incubation (Iw) was measured by FC. The ratio of Iw/o/Iw is displayed in Figure 8. Compared to BSA-FITC investigations regarding larger irregularities (Figure 6), in those measurements significant differences in quenching behaviour can be observed not only at different PE-PEGBiotin concentrations (1) but also at different preparation conditions (AL/AM ratio) (2).

Figure 8. FC investigations of SiO2-microcarrier fluorescence intensity, which have been coated with a POPS/POPC 1:1 + 0.5 mol% or 10 mol% PE-PEG-Biotin lipid mixture at AL/AM ratio of 80:1 and 1600:1. The lipid mixtures have been additionally doped with 0.1 mol% PE-FL as fluorescence marker. Fluorescence intensities before (Iw/o) and after (Iw) incubation with the fluorescence quencher TB have been measured and their ratio is


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displayed. Statistical analysis was carried out using a two-tailored student`s t-test: * p ≤ 0.05, n.s. not significant. Both, SiO20%- and SiO20.5%-microcarriers show a similar behaviour of fluorescence quenching, achieving intensity values between 35 % (AL/AM of 1600:1) and 45 - 55 % (AL/AM of 80:1). In contrast, TB quenching of SiO210%-microcarriers results in a high decrease of fluorescence intensity. Compared to control polyelectrolyte covered microcarriers, providing a fluorescence intensity of (2.38 ± 0.01) a.u. (data not shown), SiO210%microcarriers exhibit a remaining fluorescence intensity of only (4.93 ± 1.48) a.u. at AL/AM ratio of 80:1 and (3.97 ± 0.48) a.u. at AL/AM ratio of 1600:1. This nearly total fluorescence quenching could be expected, since already larger molecules (BSA-FITC) have been observed to penetrate (Figure

6). Quenching experiments performed with PE-PEG-Biotin

concentrations between 0.5 mol% and 10 mol% show that the observed change in quenching behaviour is determined by a gradual reduction, which also correlates with the measured zeta potential of equivalent SUVs (see Supporting Information Figure S4). (2) On the other hand, different preparation conditions have to be considered in case of small irregularities, too. At AL/AM ratio of 80:1, SiO20%-microcarriers exhibit a fluorescence signal of (53.3 ± 6.9) %, whereas at AL/AM ratio of 1600:1 a significantly lower signal of (35.28 ± 1.45) % was detected. A similar behaviour can be found in case of 0.5 mol% PEPEG-Biotin, resulting in (44.25 ± 3.23) % and (36.16 ± 2.6) % remaining fluorescence intensity, respectively. This effect seems to be also present in SiO210%-microcarriers but there it is dominated by the overall accessibility of inner and outer PE-FL molecules to the quencher. Since liposome concentration is constant within the different AL/AM ratios, but microcarrier concentration changes, a different liposome adsorption and spreading behaviour can be assumed leading to the formation of regular/irregular lipid membrane structures.


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Those experiments show that the amount of PE-PEG-Biotin and AL/AM strongly influence the formation of the lipid bilayer on top of a polyelectrolyte coated LbL-microcarrier. Only low amounts of functional lipid (0.5 mol% PE-PEG-Biotin) combined with a low AL/AM ratio of 80:1 result in the formation of a tightly formed bilayer structure not allowing large (BSA) or even small (TB) molecules to penetrate. Considering the above mentioned parameters, a highly effective multi-functionalized drug delivery system can be designed. To prove the advantages of this microcarrier system, the surface was functionalized with a specific antibody. SiO20.5%- and SiO210%- (AL/AM 80:1) as well as polyelectrolyte covered microcarriers (PRM/DXS) have been incubated with a primary human CCR5-biotinylated antibody (ABp) to be verified with a fluorescent secondary antibody (ABs-Cy5). ABp is supposed to bind exclusively to PE-PEG-Biotin in the presence of the coupling partner streptavidin. Non-fluorescent microcarriers as well as ABs-Cy5 and ABp + ABs-Cy5 without streptavidin incubation served as controls. FC and CLSM data are displayed in Figure 9. As shown in FC measurements of polyelectrolyte covered microcarriers (Figure 9a), a high fluorescence signal of ABs-Cy5 (167 a.u.) on top of the microcarriers surface compared to the non-fluorescent control (1.9 a.u.) can be detected. Furthermore, an almost homogeneous fluorescence distribution surrounding the microcarrier with minor intensity variations (dots) occurs (CLSM inset). Incubation with ABp and ABs-Cy5 leads to a further, nearly 10 fold increase of the detected fluorescence signal to 1410 a.u. and a homogeneous fluorescence distribution at the microcarrier surface (CLSM inset). These findings show that both ABp and ABs-Cy5 exhibit a highly unspecific interaction with the polyelectrolyte covered surface. After the assembly of 0.5 mol% PE-PEG-Biotin containing lipid membrane (Figure 9b), the unspecific binding of ABs-Cy5 has nearly vanished, reducing the fluorescence signal to 3.8 a.u. The corresponding CLSM inset supports this finding, demonstrating the effective shielding of the underlying polyelectrolyte multilayer by the assembled lipid membrane. However, the application of ABp and ABs-Cy5 shows a slightly 27 ACS Paragon Plus Environment

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increased fluorescence signal to 56 a.u. with a spot-like distribution on the microcarrier surface. Due to the lack of unspecific ABs-Cy5 binding, this behaviour can be contributed to an unspecific interaction of ABp with the assembled lipid membrane. To confirm this assumption, BSA was applied previous to ABp to block possible unspecific lipid membrane binding sites. Figure 9c shows the clearly reduced fluorescence intensity after BSA pretreatment from (41.6 ± 5.4) a.u. to (8.6 ± 2.8) a.u. Nevertheless, the introduction of the specific coupling partner streptavidin (Figure 9b) leads to a 4 fold increase of the fluorescence intensity to 202 a.u. and a homogeneous fluorescence distribution on top of the microcarrier surface (CLSM inset).

Figure 9. FC based Cy5 fluorescence intensity histograms of secondary antibody-Cy5 (ABsCy5) binding onto lipid membrane coated SiO2-microcarriers. As a control (a) PRM/DXS covered microcarriers have been measured. Microcarriers have been covered with lipid mixture of POPS/POPC 1:1 + (b) 0.5 mol% and (d) 10 mol% PE-PEG-Biotin at AL/AM ratio of 80:1. In both investigations, the microcarriers have been former incubated with: secondary antibody (+ABs-Cy5), primary antibody and secondary antibody (+ABp+ABs-Cy5) and streptavidin, primary antibody and secondary antibody (+strep+ABp+ABs-Cy5). The insets 28 ACS Paragon Plus Environment

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represent the corresponding CLSM images of SiO2-microcarrier. The bar plot (c) shows Cy5 fluorescence intensities after the primary and secondary antibody binding (+ABp+ABs-Cy5) onto SiO20.5%- and SiO210%-microcarriers without (w/o) and with (w) prior BSA incubation. As expected, the increase of the PE-PEG-Biotin concentration to 10 mol% (Figure 9d) shows a different behaviour. In comparison to SiO20.5%-microcarriers, the fluorescence intensity after ABs-Cy5 incubation increases significantly (27 a.u.) compared to the control (1.9 a.u.). This is confirmed by the corresponding CLSM image, already showing an ABs-Cy5 coverage of the microcarrier surface, which can now bind to the underlying polyelectrolytes. The application of ABp and ABs-Cy5 shows then only a slightly increased but very broad fluorescence distribution with a geometric mean value of 74 a.u. compared to ABs-Cy5 alone. The CLSM inset confirms a highly irregular attachment with a spot like structure. This finding again shows that a high PE-PEG-Biotin concentration leads to the formation of irregularities in the lipid membrane, which can be observed even by huge molecules like antibodies (MW: 150 kDa). Also pre-treatment with BSA (Figure 9c) did not fully reduce this unspecific interaction as shown by only a marginal decrease of the detected ABs-Cy5 signal from (58.6 ± 38.7) a.u. to (35.9 ± 10.7) a.u., further indicating an irregular lipid membrane formation. The integration of the coupling partner streptavidin (Figure 9d) leads then to further increased fluorescence signal of 779 a.u., as expected. Even though the CLSM images reveal a homogeneous coverage of the microcarrier surface, the FC detected fluorescence distribution appears to be very similar to the streptavidin binding shown in Figure 4. Again the distribution consists of a high specific as well as a retained binding part, which can be further visualized by the two CLSM insets. In addition, the 20 fold increase in PE-PEG-Biotin (0.5 mol% to 10 mol%) leads only to a 4 fold increase in ABp binding which is also comparable

to

previously

shown

streptavidin

experiments

(Figure

4).


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Conclusion This study emphasises the establishment of a functional microcarrier system with exchangeable outer modification. The well-established basic principle of the Layer-by-Layer technique applied onto spherical SiO2-microparticles has been combined with the liposome spreading technique, resulting in a lipid membrane protected microcarrier system. To achieve an adaptable microcarrier, the lipid membrane composition has been expanded with the functional lipid/polymer composite PE-PEG-Biotin which necessitates the investigation of its influence on a homogeneous lipid membrane formation. It could be shown, that proper adjustment of the incubation parameters (low AL/AM incubation ratio as well as a low PEPEG-Biotin concentration of 0.5 mol%) is needed to create a homogeneous functionalized lipid membrane which exhibits at most minor interaction with possible serum components (e.g. BSA or antibodies), but on the other hand can be replaceable equipped with functional molecules via biotin-streptavidin coupling. For this purpose, the established microcarrier system can be easily functionalized e.g. with biotinylated antibodies for targeting specific cells. Therefore, the presented system will have future impact in biomedical applications, as drug delivery systems.

Supporting Information Available The Supporting Information (SI) comprise further detailed investigations of multiple liposome properties: size distribution (Figure S1), lipid composition after extrusion and liposome/micelle ratio in dependence of PEG concentration (Figure S2) and the influence of the individual components of the composite system regarding the zeta potential (Figure S3). Furthermore the quenching value IW/O/IW has been analysed for PE-PEG-Biotin concentrations between 0.5 mol% and 10 mol% (Figure S4). This material is available free of charge via the Internet at http://pubs.acs.org.


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AUTHOR INFORMATION Corresponding Author * Dr. Uta Reibetanz, Universität Leipzig, Institute for Medical Physics and Biophysics, Härtelstr. 16-18, 04107 Leipzig, Germany Present Addresses Paula Pescador, c-LEcta GmbH, Leipzig, Germany 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 thank Dr. Beate Fuchs and Dr. Jürgen Schiller for experimental assistance regarding 31P NMR measurements. The work presented in this paper was made possible by funding from German Research Foundation (DFG, RE 2681/2-1), the European Union and the Free State of Saxony and was supported by the DFG graduate school 185 “Leipzig School of Natural Sciences Building with Molecules and Nano-objects”(BuildMoNa).


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Reference List

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TOC

For Table of Contents Only


Biomacromolecules

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Zeta potential measurement of small unilamellar vesicles (d = 50 nm). The vesicles are prepared of POPS/POPC ratio 1:1 with varying amounts of PE-PEG-Biotin. 57x40mm (300 x 300 DPI)


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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

Biomacromolecules

(a) FC fluorescence intensity measurements of SiO2-microcarriers coated with a lipid membrane made of POPS/POPC 1:1 + 0.5 mol% or 10 mol% PE-PEG-Biotin doped with 0.1 mol% PE-FL in dependence of different AL/AM incubation conditions. Uncoated SiO2-microcarriers served as a non-fluorescent control. (b)(e) CLSM images of lipid membrane coated SiO2-microcarriers prepared at AL/AM ratio of 80:1 (b and c) and 1600:1 (d and e). Images (b) and (d) represent a lipid membrane made of POPS/POPC 1:1 + 0.5 mol% PEPEG-Biotin, whereas images (c) and (e) show a lipid membrane made of POPS/POPC 1:1 + 10 mol% PEPEG-Biotin. Scale bars correspond to 10 µm. 125x190mm (300 x 300 DPI)


Biomacromolecules

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Cross sections of CLSM images of SiO2-microcarrier coated with lipid mixtures of POPS/POPC 1:1 + (a) 0 mol% PE-PEG-Biotin, (b) 0.5 mol% PE-PEG-Biotin and (c) 10 mol% PE-PEG-Biotin at AL/AM ratio of 80:1. The lipid mixtures have been additionally doped with 0.1 mol% PE-FL. Fluorescence intensities were detected before (w/o TB, black curve) and after (w TB, red curve) the addition of fluorescence quencher TB. The insets show the corresponding CLSM images of SiO2-microcarriers. 132x99mm (300 x 300 DPI)


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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

Biomacromolecules

(a) FC based fluorescence intensity histograms of strep-Cy5 binding onto lipid membrane coated SiO2microcarriers. Microcarriers have been assembled with lipid mixtures of POPS/POPC 1:1 + 0 mol%, 0.5 mol% or 10 mol% PE-PEG-Biotin at AL/AM ratio of 80:1. As a control PRM/DXS covered microcarriers have been measured before (control) and after (PRM/DXS) incubation with strep-Cy5. Region M1 corresponds to background fluorescence intensity of untreated microcarriers. Regions M2, M3-1 and M3-2 represent specific (M2 and M3-2) and restrained (M3-1) strep-Cy5 binding. Images (b) and (c) show CLSM images of strepCy5 fluorescence intensity distribution on the surface of (b) 0.5 mol% and (c) 10 mol% PE-PEG-Biotin covered microcarriers, whereas the arrows point on spots of inhomogeneous strep-Cy5 binding. Scale bars correspond to 10 µm. 83x85mm (300 x 300 DPI)


Biomacromolecules

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Detection concepts of possible lipid membrane formation on SiO2-microcarriers at high PE-PEG-Biotin concentrations. Image (a) shows the detection of large irregularities (d ≥ (5-20) nm) during lipid membrane assembly by the penetration of larger molecules e.g. BSA-FITC (MW: 68 kDa), whereas image (b) illustrates the detection of small irregularities (d ≥ (1-2) nm) during lipid membrane assembly, by the penetration of TB (MW: 961 Da). 41x9mm (300 x 300 DPI)


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Biomacromolecules

(a) FC investigations of the amount of assembled BSA-FITC onto lipid membrane coated SiO2-microcarriers incubated with different lipid compositions (POPS/POPC 1:1 + 0 mol%, 0.5 mol% and 10 mol% PE-PEGBiotin) at AL/AM ratio of 80:1 and 1600:1. As a control polyelectrolyte covered microcarriers with an outer layer of positively charged PRM with (PRM/DXS + BSA) and without (PRM/DXS) BSA-FITC incubation are shown. The concentration of assembled BSA-FITC has been determined using a standard calibration kit. Statistical analysis was carried out using a two-tailored student`s t-test: ** p ≤ 0.01, n.s. not significant. Images (b) and (c) show CLSM images of BSA-FITC fluorescence intensity distributions on the surface of 10 mol% PE-PEG-Biotin (b) AL/AM = 80:1 and (c) AL/AM = 1600:1 covered microcarriers, whereas the arrows point on spots of inhomogeneous BSA-FITC binding. Scale bars correspond to 10 µm. 86x91mm (300 x 300 DPI)


Biomacromolecules

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CLSM intensity images of GUVs composed of POPS/POPC 1:1 + (a and d) 0 mol% PE-PEG-Biotin, (b and e) 0.5 mol% PE-PEG-Biotin and (c and f) 10 mol% PE-PEG-Biotin, after incubation in BSA-FITC solution. Images (a)-(c) have been recorded before sample purification (high amount of BSA-FITC in solution), whereas images (d)-(f) show GUVs after sample purification (insignificant amounts of BSA-FITC in solution). 98x118mm (300 x 300 DPI)


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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

Biomacromolecules

FC investigations of SiO2-microcarrier fluorescence intensity, which have been coated with a POPS/POPC 1:1 + 0.5 mol% or 10 mol% PE-PEG-Biotin lipid mixture at AL/AM ratio of 80:1 and 1600:1. The lipid mixtures have been additionally doped with 0.1 mol% PE-FL as fluorescence marker. Fluorescence intensities before (Iw/o) and after (Iw) incubation with the fluorescence quencher TB have been measured and their ratio is displayed. Statistical analysis was carried out using a two-tailored student`s t-test: * p ≤ 0.05, n.s. not significant. 57x40mm (300 x 300 DPI)


Biomacromolecules

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FC based Cy5 fluorescence intensity histograms of secondary antibody-Cy5 (ABs-Cy5) binding onto lipid membrane coated SiO2-microcarriers. As a control (a) PRM/DXS covered microcarriers have been measured. Microcarriers have been covered with lipid mixture of POPS/POPC 1:1 + (b) 0.5 mol% and (d) 10 mol% PEPEG-Biotin at AL/AM ratio of 80:1. In both investigations, the microcarriers have been former incubated with: secondary antibody (+ABs-Cy5), primary antibody and secondary antibody (+ABp+ABs-Cy5) and streptavidin, primary antibody and secondary antibody (+strep+ABp+ABs-Cy5). The insets represent the corresponding CLSM images of SiO2-microcarrier. The bar plot (c) shows Cy5 fluorescence intensities after the primary and secondary antibody binding (+ABp+ABs-Cy5) onto SiO20.5%- and SiO210%-microcarriers without (w/o) and with (w) prior BSA incubation. 105x62mm (300 x 300 DPI)


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