POPE supported lipid bilayers modified with hydrophobic quantum dots on polyelectrolyte cushions

Colloids and Surfaces B: Biointerfaces 158 (2017) 667–674

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Properties of POPC/POPE supported lipid bilayers modified with hydrophobic quantum dots on polyelectrolyte cushions Marta Kolasinska-Sojka a,∗ , Magdalena Wlodek a , Michal Szuwarzynski b,c , Sami Kereiche d , Lubomir Kovacik d,1 , Piotr Warszynski a a

Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, PL-30239 Krakow, Poland Faculty of Chemistry, Jagiellonian University, Ingardena 3, PL-30-060 Krakow, Poland c AGH University of Science and Technology, Academic Centre for Materials and Nanotechnology, al. A. Mickiewicza 30, PL-30059 Krakow, Poland d Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University, Albertov 4, 128 01 Prague, Czech Republic b

a r t i c l e

i n f o

Article history: Received 7 February 2017 Received in revised form 19 June 2017 Accepted 19 July 2017 Available online 23 July 2017 Keywords: Supported lipid bilayer Quantum dots Quantum dots-liposome complex Cryo-TEM Theranostic nanocontainers

a b s t r a c t The formation and properties of supported lipid bilayers (SLB) containing hydrophobic nanoparticles (NP) was studied in relation to underlying cushion obtained from selected polyelectrolyte multilayers. Lipid vesicles were formed from zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) in phosphate buffer (PBS). As hydrophobic nanoparticles − quantum dots (QD) with size of 3.8 nm (emission wavelength of 420 nm) were used. Polyelectrolyte multilayers (PEM) were constructed by the sequential, i.e., layer-by-layer (LbL) adsorption of alternately charged polyelectrolytes from their solutions. Liposomes and Liposome-QDs complexes were studied with Transmission Cryo-Electron Microscopy (Cryo-TEM) to verify the quality of vesicles and the position of QD within lipid bilayer. Deposition of liposomes and liposomes with quantum dots on polyelectrolyte films was studied in situ using quartz crystal microbalance with dissipation (QCM-D) technique. The fluorescence emission spectra were analyzed for both: suspension of liposomes with nanoparticles and for supported lipid bilayers containing QD on PEM. It was demonstrated that quantum dots are located in the hydrophobic part of lipid bilayer. Moreover, we proved that such QD-modified liposomes formed supported lipid bilayers and their final structure depended on the type of underlying cushion. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Lipid vesicles as biodegradable, biocompatible, immunogenic species of low toxicity represent very effective, nanotechnologybased drug delivery platform [1–3]. They can fuse with cells facilitating the transport of drugs across biomembranes [4]. The monitoring of liposomal distribution within tissues of interest is done by incorporation of fluorescent dyes into liposomes [5]. However, since they are photo unstable and have relatively low brightness, quantum dots (QDs) have been used to overcome the drawbacks of usual organic fluorophores [6]. QDs are nanocrystals made of semiconductors with light-emitting properties dependent on their composition and size [3]. Due to their unique photophys-

∗ Corresponding author. E-mail address: [email protected] (M. Kolasinska-Sojka). 1 Current address: Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Mattenstrasse 26, CH-, Basel, 4058, Switzerland. http://dx.doi.org/10.1016/j.colsurfb.2017.07.046 0927-7765/© 2017 Elsevier B.V. All rights reserved.

ical properties, including high photostability, a broad excitation wavelength range, size–tunable, symmetric and narrow emission spectra, ranging from 400 to 2000 nm [7,8] – they are great alternative to classical fluorescent dyes. Almost all of highly luminescent QDs are originally synthesized in organic solvents and capped with hydrophobic ligands [9,10]. Their hydrophobic surface introduces serious limitations for the biomedical and clinical applications, however, coating them with amphiphilic molecules, such as surfactants or phospholipids helps to overcome this problem [11]. One of the strategies proposed is the formation of quantum dots-liposome complexes. They are liposomes with QD embedded within the lipophilic part of the lipid bilayer. Hydrophobic QDs within liposomes not only show better fluorescent characteristics, but also better mechanical stability [12,13]. Moreover, their cytotoxicity is reduced. Such hybrid nanostructures have application potential for medical imaging, theranostics [5,14], targeted therapy [14], drug delivery and biosensing [15,16]. They are studies showing that the presence of QD within lipid bilayer of theranostic liposomes used to treat brain disorders

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increased the distribution of liposomes in the brain, decreasing their accumulation in the liver [5]. Thus, lipid vesicles with hydrophobic quantum dots embedded within bilayer membrane are very promising systems offering not only drug delivery and imaging/diagnostics simultaneously, but also improvement of liposomes’ distribution inside the organ of interest. The most common preparation pathway to embed the functional hydrophobic nanoparticles (NPs) into lipid bilayers is to dry out a chloroform suspension of hydrophobic particles and phospholipids to obtain a NP-lipid film, which, subsequently is hydrated with aqueous buffer solution. Vesicle formation in the presence of buffer solution is promoted by the sonication. Further extrusion allows obtaining the final vesicle-NP monodisperse suspension [17]. It is known that nanoparticles can be inserted into the nonpolar interior of the lipid bilayer under certain conditions. First, the size of NPs must be small enough to fit within lipid bilayer and second, they must possess a hydrophobic surface [18]. It was observed that nanoparticles could be incorporated within lipid membrane, or they could cause the deformation of membranes by the formation of nanoscale holes [19–21]. According to many experimental reports [12,13,17–21], QDs with a diameter comparable to or smaller than the typical thickness of the lipid bilayer, i.e., 4–5 nm [22], are easily embedded in the part of hydrophobic tails of the bilayer. On the other hand, it was found that hydrophobic nanoparticles with diameters greater than 6.5 nm forced micelles’ formation due to the high local curvature strain on the lipid bilayer [23]. Hydrophobic NPs embedded inside bilayers may cause some changes in lipid packing and they may disrupt lipid–lipid interactions amongst the head groups and/or alkyl tails. Disruption of such interlipid interactions can result in changes in lipid bilayer phase behavior, which is related to the degree of lipid ordering and bilayer viscosity [24–26]. There exist studies on lipid vesicles containing hydrophobic quantum dots, such as CdSe [11], CdSe/ZnS [27,28] and CdSe/CdZnS [29]. However, little is known about the influence of incorporated nanoparticles on physicochemical properties of lipid vesicles such as stability, membrane fluidity and bilayer phase transition. In the case of such hybrid structures, there are many basic questions to be answered. Some of them concern the process of embedding of QDs in the lipid bilayer during the self-assembly process, the change of SLB physicochemical properties before and after QD-loading or how lipid composition affects the stabilization of the QDs inside the lipid bilayer, etc. The main goal of presented studies was to obtain model lipid-QD bilayers and to prove their applicability in fluorescent visualization in order to look for new solutions in theranostic systems. For that reason supported 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine and negatively charged 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine, (POPC/POPE) lipid bilayers with hydrophobic cadmium sulphide quantum dots of size 3.8 nm, incorporated into alkyl tails region of lipids were designed. Structure and stability of such hybrid materials was investigated. Selected bilayers were formed on solid support modified with various polyelectrolyte multilayers (PEMs) according to the previously established conditions [30,31] (details showed in the next section). Polyelectrolyte cushions with different number of layers, applied as support for lipid bilayer with quantum dots, were constructed using the layer-by-layer deposition technique [32–34]. Quantum dots were inserted directly to the lipid during preparation of lipid vesicles. Deposition of liposomes with QDs on the top of positively charged, polycation–terminated polyelectrolyte films was studied in situ using QCM-D measurements. Fluorimetric experiments were performed to confirm the presence of fluorescent nanoparticles within liposome structure in their suspension and in the supported lipid bilayer deposited on the top of polyelectrolyte multilayers. We believe that such nanoparticle-vesicles hybrids are promising tools

in biotechnology. Use of quantum dot-vesicles allows tracking of the vesicle fusion on surfaces by the visualization of the resulting patches on the cell membrane by fluorescence. 2. Experimental section 2.1. Materials The polyelectrolytes (PE) used were: branched poly(ethyleneimine) (PEI) of molecular weight c.a. 750 kDa, poly(diallyldimethylammonium)chloride (PDADMAC) of molecular weight in the range of 100–200 kDa, poly–L–lysine hydrobromide (PLL) of molecular weight about 30 kDa as polycations and polysodium 4-styrenesulfonate (PSS) of 70 kDa, poly–L–glutamic acid sodium salt (PGA) of molecular weight about 50 kDa as polyanions. All polyelectrolytes were purchased from Sigma-Aldrich (Poland). Lipids used for bilayer formation were: zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and negatively charged 1-palmitoyl-2-oleoyl-sn-glycero3-phosphoethanolamine (POPE) both from Avanti Polar Lipids (Instruchemie B.V., Netherlands). As quantum dots (QD), commercially available cadmium sulphide (CdS) with concentration of 5 mg/ml in toluene (Aldrich, Poland) were used. According to the manufacturer’s specification the typical nanocrystal size was 3.8 nm with corresponding fluorescence emission maximum of 420 nm. They were surface-stabilized with oleic acid coating. Sodium chloride (NaCl) (99,5%) sodium hydrogen phosphate (Na2 HPO4 ), sodium dihydrogen phosphate (NaH2 PO4 ), chloroform (ChCl3 ) and hydrogen chloride (HCl) – were obtained from Fluka, Poland, sulfuric acid, hydrogen peroxide (H2 O2 ) and sodium hydroxide (NaOH) – from Aldrich. Phosphate buffer (PBS) was made of NaCl, Na2 HPO4 and NaH2 PO4 , with pH adjusted to 9.5 by NaOH. Ultrapure, water (Milli-Q water) with resistivity over 18 M/cm (Millipore, Poland) was used for all prepared solutions. As a support material for the PEM/(lipid + QDs), natural ruby mica (from Dean Transted, Great Britain), standard gold/quartz sensors QSX 301 (Q-Sense, Sweden) and Si wafers (On semiconductor, Czech Republic) were used. Mica was freshly cleaved before each sample deposition. Gold/quartz crystals and silicon wafers were cleaned by washing in piranha solution, which is a mixture of equivalent volumes of concentrated sulfuric acid and perhydrol. (Precaution! This solution is a very strong oxidizing agent and should be handled carefully). Substrates were dipped into piranha solution for 30 min and then carefully rinsed with Milli-Q water followed by 30 min of incubation in hot (c.a. 70 ◦ C) Milli-Q water. Polyelectrolyte multilayers used as cushions for lipid bilayers were prepared by the layer-by layer deposition technique. Adsorption of polyelectrolytes on mica, gold (in the case of QCM experiments) or Si wafers was performed from 0.15 M NaCl solutions. PDADMAC, PSS, PGA were used in their natural pH. PLL solution was adjusted to pH = 10. Such a choice of polyions and deposition conditions promoted the formation of complete supported lipid bilayers as was described in details in our previous studies [30,31]. PEI was always used as the first, anchoring layer for the build-up of cushion films to get more stable and homogenous multilayers [35]. Each deposition step took 10 min and rinsing in between was done in water three times for 1 min. The process was repeated until 7-polyelectrolyte-layer-film was obtained. Polycation terminated samples were chosen to investigate the deposition of liposome-quantum dots, which were negatively charged. 2.2. Preparation of liposomes with quantum dots Liposomes with quantum dots (L-QDs) were prepared using the thin lipid film hydration method, which is as follows: 150 ␮l of com-

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mercial suspension of CdS in toluene (concentration: 5 mg/ml) was mixed with 5 mg of POPC and 5 mg of POPE phospholipids in 850 ␮l of chloroform. The organic solvent was evaporated using vacuum pump overnight. Hydration of the lipids-quantum dots film was then performed with 2 ml phosphate buffer of ionic strength 0.2 M and pH value 9.5. Large multilamellar vesicles with quantum dots (LMV-QD) were generated using sonication with a bath–type sonicator for 20 min at room temperature. The resulting suspensions were then extruded in two series, each of 15 times through polycarbonate membranes with nominally 200 nm pores (first series) and 100 nm (second one) using mini-extruder (Avanti Polar Lipids, USA) and then diluted to the final concentration of 0.4 mg/ml lipid small unilamellar vesicles–QDs suspension (dilution assumed no losses due to extrusion). 2.3. Liposomes size and zeta potential The size distribution of (POPC/POPE) and (POPC/POPE with QD) liposomes in aqueous suspension was determined by the dynamic light scattering (Zetasizer Nano Series, Malvern Instruments, Great Britain) at scattering angle of 90◦ . The measurements were performed at 25 ◦ C. The obtained hydrodynamic diameter for vesicles without QD in solutions was approximately 91 ± 6 nm, which was in agreement with pore size of membranes applied by extrusion. The size of liposomes with QD was around 110 nm. The zeta potential measured by electrophoretic mobility was −19.0 ± 1.1 mV, the same for both types of liposomes: without and with QD. 2.4. Fluorescence emission spectra The fluorescence emission spectra of quantum dots in different environments were investigated using spectrofluorometer Fluoro Log-3, Horiba, Jobin Yvon (Japan). Xenon lamp was used for excitation. Spectra of CdS with size of 3.8 nm in toluene and within bilayer of POPC/POPE liposomes in buffer solution were obtained with a 1 cm path length quartz cuvette, with excitation and emission slit widths 3 and 4.5 nm respectively, depending on the sample fluorescence. In the case of films with quantum dots the experiments were performed with excitation and emission slit widths 5 nm and films were formed on mica sheets and measured in a dedicated solidsample holder designed for thin films. Each sample was excited using light of 300 nm wavelength. 2.5. Transmission cryo-electron microscopy (cryo-TEM) Cryo-TEM measurements were carried out on a Tecnai G2 Sphera 20 electron microscope (FEI Company, Hillsboro, OR, USA) equipped with a Gatan 626 cryo-specimen holder (Gatan, Pleasanton, CA, USA) and a LaB6 gun. The samples for cryo-TEM were prepared by plunge freezing. Briefly, 3 ␮l of the sample solution were applied to a copper electron microscopy grid covered with a perforated carbon film forming woven-mesh-like openings of different sizes and shapes (the lacey carbon grids #LC-200 Cu, Electron Microscopy Sciences, Hatfield, PA, USA), which was glow discharged for 40 s with 5 mA current prior to specimen application. Most of the sample was removed by blotting (Whatman no. 1 filter paper) for approximately 1 s, and the grid was immediately plunged into liquid ethane held at −183 ◦ C. The grid was then transferred without rewarming into the microscope. Images were recorded at the accelerating voltage of 120 kV and with magnifications ranging from 11500× to 50000× using a GatanUltraScan 1000 slow scan CCD camera in the low-dose imaging mode, with the electron dose not exceeding 1500 electrons per nm2 . The magnifications resulted in final pixel size ranging from 0.9 to 0.2 nm, the typical value of applied under focus ranged between 0.5–2.5 ␮m. The applied blotting conditions resulted in the specimen thickness varying between

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100 to ca. 300 nm. All cryo-TEM pictures were carefully inspected for possible artifacts such as radiation damage and ice crystals, and high-quality images were CTF-corrected and band-pass filtered in order to suppress both ice thickness variations and noise below 1 nm detail size. 2.6. Quartz crystal microbalance The quartz crystal microbalance with dissipation control (QCMD) is based on the measurements of oscillation frequency of a disc-shaped piezoelectric quartz crystal with metal electrodes deposited on its two sides. For stiff films, small mass of adsorbate added to the electrodes induces a decrease in resonant frequency (f), which is proportional to the mass deposited (m), according to the Sauerbrey’s relationship: m = −C

f n

where: C is the constant that depends on the physical properties of quartz crystal (in our system it is equal to 17.7 ng/cm2 Hz), and n – the crystal oscillation overtone number, n = 1,3,5,7. . . m – the adsorbed mass. The Sauerbrey’s relationship can be used only when the difference between dissipation values for measured overtones does not exceed 10−6 . In other case the viscoelastic models of the film need to be used as the dissipation increment (D) is related to the viscoelastic properties of adsorbed multilayers [36]. The QCMD technique was applied to determine adsorption of liposomes with QDs on polyelectrolyte films and their stability. In particular, general properties of the liposome-quantum dots deposit can be determined by monitoring dissipation that allows distinguishing between intact, adsorbed vesicles with nanoparticles (high dissipation) and nanoparticles within planar lipid bilayer (low dissipation). The deposition of POPC/POPE vesicles with QDs on positively charged polycation-terminated polyelectrolyte multilayers was monitored by quartz crystal microbalance, Q-Sense AB Gothenburg, Sweden (present name: Biolin Scientific, Sweden). AT-cut gold layer coated quartz crystals with a fundamental resonance frequency of 5 MHz (Q-Sense AB, Sweden) were covered with polyelectrolyte films using the LbL assembly. The crystals modified with PEMs were mounted in the liquid cell with one side exposed to the solution. L-QDs’ adsorption from their dispersion (0.4 mg/ml) was monitored in situ. After obtaining a constant value of resonance frequency, the liquid was exchanged with phosphate buffer in order to rinse the whole setup. The Maxwell viscoelastic model, implemented in QTools 3 software, Q-Sense AB, Gothenburg [36] was used to interpret the experimental data. The detailed description of the implemented model can be found elsewhere [37]. The measurements were performed at 25 ◦ C. The deviation among repeated experiments has never exceeded 10%. 2.7. AFM technique Atomic force microscopy was used to study the complete supported lipid bilayers with 3.8 nm hydrophobic quantum dots on the polyelectrolyte films. Images were obtained with Dimension Icon atomic force microscope (Bruker, Santa Barbara, CA, USA) working in the fluid in the Peak Force TappingTM (PFT) mode. Standard silicon cantilevers for PFT in fluids (Bruker, USA) with nominal spring constant of 0.7 N/m and tip radius <10 nm were used for these measurements. The liposome-quantum dots complexes were deposited on silicon with PEMs for approximately 2 h then rinsed with phosphate buffer solution and measured directly after deposition in the PBS.

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Fig. 1. Emission spectra of quantum dots of diameter = 3.8 nm (a) in toluene and (b) POPC/POPE vesicles with QD (suspension).

Fig. 2. CryoTem micrographs of (a) POPC/POPE vesicle; (b) POPC/POPE vesicles with quantum dots of diameter = 3.8 nm.

3. Results and discussion 3.1. Incorporation of quantum dots into POPC/POPE lipid bilayers The presence of quantum dots within the liposomes’ structure was proved by the fluorescence experiments. First the suspensions of non-modified QD in toluene were studied to find the fluorescence of the selected QDs (see Fig. 1a). Next, the fluorescence emission spectra of QDs incorporated into lipid bilayer of studied liposomes in buffer solution were collected (Fig. 2b). In all cases emission spectra were taken after excitation with the light of 300 nm wavelength. The concentration of QD in studied samples in toluene was similar to the initial concentration of QD taken for liposomes-QD hybrids in phosphate buffer, i.e., 0.75 mg/ml. As a background – spectrum of liposomes without quantum dots was subtracted from each spectrum of liposomes-QD suspension. One can see that incorporation of QD into lipid bilayer of selected liposomes was successful, leading to the formation of hybrid (lipid-QD) materials. It has to be point out that hydrophobic QD can be placed only within hydrophobic part of lipid bilayer and due to the preparation procedure they would be separated from the final liposomes’ dispersion if they were not inside the lipid bilayer forming liposomes. Incorporation of QDs within the lipid bilayer led to the reduction of fluorescence intensity compared to QDs in toluene, although the initial concentration of quantum dots was the same. Nevertheless, the intensity is still high enough to be easily detected. One can observe that the emission maximum of QD within liposomes is slightly shifted (c.a. 4 nm) towards the violet emission wavelength, comparing to the spectrum of QD in toluene, which could be caused by the interactions between the entrapped QDs and the lipid bilayer

[38]. Since the known concentration of QD in toluene gives the maximum intensity of 2.18 × 107 and 3.50 × 106 in liposomes, one can estimate the final concentration of QD in liposomes by comparing the maximum intensities of both dispersions: in toluene and in liposomes. For quantum dots in toluene, one can calculate also the number of particles per unit volume, nCdS . It is based on the relation: nCdS = cCdS /mp where: nCdS is number of particles per unit volume, cCdS – concentration of particles in the suspension expressed as mass per unit volume and mp – mass of a single CdS particle. The mass of the single CdS particle is given by the formula:

 3

4 d mp = ϕp V = ϕp  3 2

where: ϕp – density of single CdS particle equal to 4.82 g/ml [39], V – volume of single CdS particle; d – diameter of CdS quantum dots = 3.8 nm. It gives the mass of the single particle equal to 1.385 × 10−19 g, which leads to the number of QD particles: 5.42 × 1015 /cm3 . Assuming that the calculated number of particles corresponds to the measured maximum intensity of quantum dots in toluene, the number of QD in liposomes was estimated comparing the maximum intensities of both dispersions: in toluene and in liposomes, giving the number of 8.70 × 1014 QD particles in cm3 of liposome suspension. The number of particles in liposomes was c.a. 6 times lower than in toluene, which indicated some QD losses during preparation due to the extrusion of liposomes through polycarbonate membranes.

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Fig. 3. Emission spectra of POPC/POPE vesicles with 3.8 nm QDs deposited on selected PEM supporting cushions: PEI; PEI(PSS/PEI)3 ; PEI(PSS/PDADMAC)3 and PEI(PGA/PLL)3 .

To prove the concept of embedded quantum dots into alkyl part of lipid bilayer, series of cryo-TEM micrographs was taken (see Fig. 2). Liposomes without quantum dots have sizes about 100 nm and a very well defined lipid membrane bilayer, about 4–5 nm in thickness (Fig. 2a). As far as liposomes with quantum dots are concerned (see Fig. 2b), their membrane thickness and sharpness appears to be similar to the control sample, i.e., the liposomes without QDs. This indicates that the quantum dots do interact with the lipid bilayer of the liposomes. Moreover, the fact that the liposomes don’t have their cavity darker then the membrane leads us to think that the quantum dots are embedded within the lipid bilayers as we could see it clearly in Fig. 3b. We could also observe that 3.8 nm QDs have suitable size to be embedded within POPC/POPE membrane bilayers. 3.2. Properties of POPC/POPE lipid bilayers with quantum dots after deposition on polyelectrolyte films The fluorescence emission spectra of POPC/POPE vesicles with QDs deposited on the following polyelectrolyte films: PEI, PEI(PSS/PEI)3 , PEI(PSS/PDADMAC)3 and PEI(PGA/PLL)3 were shown in Fig. 3. The spectral characteristics of QDs was the same for all compared supports. In all studied cases, i.e., for all types of support materials we were able to bind liposomes-QD to the selected surfaces successfully, which has proved the concept of the forma-

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tion of nanocomposite films possessing components of different hydrophobicity. From fluorimetric experiments one can prove the presence of QD within studied systems, but no information on the structure of the studied sample is given. Thus, adsorption kinetics of POPC/POPE liposomes containing hydrophobic quantum dots on polycationterminated selected PEMs was studied in situ using the QCM-D measurements. The results for the frequency and dissipation shifts as a function of deposition time of POPC/POPE vesicles with 3.8 nm QDs on the top of PEI, PEI(PSS/PEI)3 , PEI(PSS/PDADMAC)3 , PEI(PGA/PLL)3 are shown in Fig. 4. One can see that the deposition of vesicles modified with 3.8 nm hydrophobic quantum dots leads to the formation of lipid bilayer (at least partially) via vesicle fusion for all selected PEM cushions, however the process of bilayer formation differs significantly among studied multilayer supports, which confirms our previous results on formation of supported lipids bilayers deposited on selected polyelectrolyte multilayers [30]. The formation of POPC/POPE bilayer on the film terminated with PLL polycation differs fundamentally from other systems studied. Its kinetics is faster, the critical liposomes coverage is much lower, i.e.: 3 times lower than for PEI monolayer as well as PDADMAC terminated films and 4.5 times lower than for PSS/PEI multilayer. Moreover, a very shallow short lasting minimum (the difference in frequency at critical liposomes coverage and reached plateau is very low comparing to other systems measured, c.a. 6 Hz vs. 50–95 Hz) in the frequency shift accompanied with small maximum of dissipation suggests that fusion of liposomes practically doesn’t occur. The explanation of such behavior is probably the formation of hydrogen bonds between the head group of 1-palmitoyl-2-oleoylsn-glycero-3-phosphatidylethanolamine (POPE) and the amine group of poly-l-lysine [40]. There exist studies on the stabilization of liposomes by PLL [41], which confirm strong interactions between lipids and PLL and resulting vesicle adsorption without their further fusion. Another interesting aspect is the comparison of QCM results for PEI monolayer and PEI(PSS/PEI)3 film. Although in both cases the outermost layer is PEI, they show different critical liposomes coverage and the kinetics of liposomes fusion. For the PEI(PSS/PEI)3 one can observe well indicated frequency minimum at 180 Hz, which corresponds to the critical liposome coverage while for PEI monolayer the peak is much wider and flatter with the minimum value at 130 Hz. As far as the kinetics of the fusion is concerned (the part of the curve from the frequency minimum till plateau), one can see that it is faster for PEI(PSS/PEI)3 than for PEI monolayer, which leads to the conclusion that surrounding of the outermost layer, i.e. film or solid support underneath, affects its properties and thus, the

Fig. 4. Frequency (a) and dissipation (b) shifts upon the deposition of POPC/POPE vesicles with 3.8 nm CdS onto selected PEM cushions: PEI, PEI(PSS/PEI)3 , PEI(PSS/PDADMAC)3 , PEI(PGA/PLL)3 .

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Fig. 5. Frequency shifts upon the deposition of POPC/POPE vesicles without or with 3.8 nm CdS onto selected PEM cushions: (a) PEI, (b) PEI(PSS/PEI)3 , (c) PEI(PSS/PDADMAC)3 , (d) PEI(PGA/PLL)3 .

structure of formed lipid bilayer. When adsorption of liposomes on PEI(PSS/PDADMAC)3 is taken into considerations, one can observe that the amount of released water due to the vesicles rupture and fusion is the biggest one. One can expect that bilayer formed on PEI(PSS/PDADMAC)3 has a continuous structure with the lowest number of liposomes. To analyze the impact of QD on the vesicle adsorption and fusion the changes of QCM frequency on the deposition of POPC/POPE liposomes without QD and similar liposomes containing QD were compared for all selected polyelectrolyte cushions. That comparison is depicted in Fig. 5. One can see that the effect of QD incorporation into bilayer structure of liposomes is rather ambiguous but strongly related to the type of the PEM cushion. The kinetics of reaching of the maximum of liposomal coverage is either faster (on PEI and PEI/(PSS/PEI)3 film) or the same (for PEI/(PSS/PDADMAC)3 and PEI/(PGA/PLL)3 film) as for liposomes without QD. The liposomes start to rupture faster on PEI and PEI/(PSS/PEI)3 and PEI/(PGA/PLL)3 film when they possess incorporated QD comparing to ones without QD, thus, they do not stabilize liposomes. To verify our hypothesis concerning the effect of polyelectrolyte cushion, the morphology of the lipid bilayers with QD on all PEM supports was studied with atomic force microscopy (Fig. 6). The results of AFM analysis confirmed that the type of underlying polyelectrolyte multilayer has a dominant impact on the structure of the lipid bilayer with QD. Firstly, one can observe the big fraction of stable liposomes deposited on PLL terminated multilayer, which is in agreement with the QCM measurements. In the case of PEI monolayer used as the support, patches of lipid bilayer are seen introducing some roughness at the surface. In the case of PEI(PSS/PEI)3 and PEI(PSS/PDADMAC)3 surfaces are more homo-

geneous with smaller roughness than for PEI monolayer, which also confirms the QCM results that those films showed biggest frequency shift upon vesicles rupture and formation of large area of supported lipid bilayer.

4. Conclusions The ability to design and manufacture the hydrophilic polyelectrolyte-lipid films with embedded hydrophobic nanoparticles determines the formation of functional, hybrid systems. Our studies show that commercially available CdS quantum dots can be successively embedded in the hydrophobic region of the POPC/POPE bilayers and we proved it by fluorimetric measurements and cryo-TEM micrographs of hybrid QD-lipid liposomes. We demonstrated that liposomes with QDs could form the supported lipid bilayers on selected polyelectrolyte multilayers and resulting films were still fluorescent. We found that the type of the underlying PEM played the crucial role in a mechanism of bilayer formation via vesicle fusion. Depending on the PEM it could either promote SLB formation (PEI or PDADMAC terminated films) or stabilize deposited liposomes as PLL terminated multilayers, which was an important conclusion for selecting a proper material of programmed functions. On the other hand, presence of hydrophobic particles embedded in the liposome bilayer (with the size smaller than bilayer thickness) did not seem to influence the liposomes stability and the mechanism of SLB formation. Such quantum dotvesicles hybrids are very promising systems offering not only drug delivery and imaging/diagnostics simultaneously (theranostics), but also improvement of liposomes’ distribution inside the organ of interest, as well as biosensing. Thus, the practical impact of the work and application potential cause a big interest in such systems.

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Fig. 6. AFM images and profiles of deposited POPC/POPE vesicles with 3.8 nm CdS on selected PEM cushions: (a) PEI, (b) PEI(PSS/PEI)3 , (c) PEI(PSS/PDADMAC)3 , (d) PEI(PGA/PLL)3 .

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