Novel Fusogenic Liposomes for Fluorescent Cell Labeling and

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Bioconjugate Chem. 2010, 21, 537–543

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Novel Fusogenic Liposomes for Fluorescent Cell Labeling and Membrane Modification Agnes Csisza´r,*,† Nils Hersch,† Sabine Dieluweit,† Ralf Biehl,‡ Rudolf Merkel,† and Bernd Hoffmann† Institute of Bio- and Nanosystems, IBN-4, Biomechanics, and Institute of Solid State Research, IFF-5, Neutron Scattering, Forschungszentrum Ju¨lich GmbH, 52425 Ju¨lich, Germany. Received October 28, 2009; Revised Manuscript Received January 20, 2010

Efficient delivery of biomolecules into membranes of living cells as well as cell surface modifications are major biotechnological challenges. Here, novel liposome systems based on neutral and cationic lipids in combination with lipids modified by aromatic groups are introduced for such applications. The fusion efficiency of these liposome systems was tested on single cells in culture like HEK293, myofibroblasts, cortical neurons, human macrophages, smooth muscle cells, and even on tissue. Fusogenic liposomes enabled highly efficient incorporation of molecules into mammalian cell membranes within 1 to 30 min at fully unchanged cell growth conditions and did not affect cell behavior. We hypothesize that membrane fusions were induced in all cases by the interaction of the positively charged lipids and the delocalized electron system of the aromatic group generating local dipoles and membrane instabilities. Selected applications ranging from basic research to biotechnology are envisaged here.

INTRODUCTION Phospholipid vesicles (liposomes) are widely used, e.g., for cell tracking (1, 2), for nonviral gene transfer (transfection) (3–5), or for drug delivery (6, 7). On the basis of their compositions, liposomes are more bioavailable than synthetic polymer capsules (8) or quantum dots (QD). Their cellular uptake has been intensely investigated and shows a strong preference of endocytotic cellular uptake over fusion or lipid mediated poration (9). Unfortunately, endocytotic pathways go along with limited efficiencies, often below 1%. Increasing efficiencies is therefore of major interest and approached by enhancing the uptake probability of whole liposomes by techniques as, e.g., electroporation (10) or microinjection (11). The drawback of these techniques is extreme cell stress, and most of them result in high cell death rates. Furthermore, electroporation can hardly be performed on tissues or adherent cells, and protocols as well as reagents still need to be optimized for every cell type. The more promising alternative for efficiency enhancement is the induction of membrane fusion between liposomes and the plasma membrane of the mammalian cell. Unfortunately, changes in the chemical composition of liposomes like addition of free fatty acids or alcohols (12) or increasing of the chain unsaturations in the hydrophobic region of lipids alone seemed not to be sufficient to develop a universally deployable incorporation system for molecules, just to slightly increase fusion efficiency. At the current state, the most effective factors promoting fusion have biological origins. For example, generation of membrane connecting tubuli (13) and integration of fusionproteins(14–17),aswellasviralmembranecomponents(18,19) in the particle surfaces, are effective biochemical techniques to yield high fusion efficiencies. Although effective, these methods are expensive and time-consuming. Here, a new, simple, and almost universal fusogenic liposome system containing neutral and positively charged lipid molecules, * Corresponding author. Forschungszentrum Ju¨lich GmbH, 52425 Ju¨lich, Germany, Tel.: ++49 (0)2461 61 1415, Fax: + +49 (0)2461 61 3907, e-mail: [email protected]. † Institute of Bio- and Nanosystems, IBN-4, Biomechanics. ‡ Institute of Solid State Research, IFF-5, Neutron Scattering.

as well as an additional lipid component with an extended conjugated π electron system, variable in the head or chain region of lipid molecules, has been introduced. The fusion efficacy and toxicity of these liposomes was tested on numerous cell types in culture and even on cell tissue. We furthermore present a possible mechanism underlying this constantly high fusion efficiency.

MATERIALS AND METHODS Materials. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-3-trimethylammonium-propane, chloride salt (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamineN-(lissamine rhodamine B sulfonyl) (ammonium salt) (LRDOPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(1pyrenesulfonyl) (ammonium salt) (Pyrene-DOPE), and 1,2dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3benzoxadiazol-4-yl) (ammonium salt) (NBD-DOPE), 1,2dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt) (capBio-DOPE), and L-R-lysophosphatidylinositol (sodium salt) (PI) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). All other modified lipids like 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (βPyrene-C10-HPC), DiOC18(3)3,3′-dioctadecyloxacarbocyanine perchlorate (DiO), 2-(4,4-difluoro-5-methyl-4-bora-3a,4a-diazas-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (β-BODIPY-C12HPC), N-(4,4-difluoro-5,7-dimethyl4-bora-3a,4a-diaza-s-indacene-3-propionyl)-1,2-dihexadecanoylsn-glycero-3-phosphoethanolamine (triethylammonium salt) (BODIPY FL-DHPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein) (ammonium salt) (FluoresceinDHPE), N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (triethylammonium salt) (TMR-DHPE), 2-(4,4-difluoro-5,7-diphenyl-4-bora-3a,4adiaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3phosphocholine (β-BODIPY-C5-HPC), and Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (triethylammonium salt) (TR-DHPE) were ordered from Invitrogen (Eugene, OR). Liposome Preparation. Lipid components like DOPE, DOTAP, and lipids bonded to aromatic groups were mixed in

10.1021/bc900470y  2010 American Chemical Society Published on Web 02/25/2010

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chloroform in a weight ratio of DOPE/DOTAP/aromatic lipid of 1/1/0.1. Chloroform was evaporated under vacuum for 0.5-1 h. Then, lipids were dispersed in 20 mM 2-(4-(2-hydroxyethyl)1-piperazinyl)-ethansulfonic acid (HEPES) buffer (VWR, Darmstadt, Germany) in a total lipid concentration of 2 mg/mL. The solution was vortexed for approximately 1-2 min to produce multilamellar liposomes. After homogenization in an ultrasonic bath for 10-20 min, mainly unilamellar vesicles or liposomes were formed. Dynamic Light Scattering (DLS). Liposomes made from DOPE/DOTAP (1/1 w/w), DOPE/DOTAP/TR-DHPE (1/1/0.1), and DOPE/DOTAP/β-BODIPY-C12HPC (1/1/0.1) mixtures were studied by dynamic light scattering (20) (DLS) in a concentration of 0.02 mg/mL. DLS measurements were performed on a DLS standard setup with an argon ion laser (514 nm) and an Avalanche Photo Diode (APD, ALV, Germany) mounted on an ALV-125 compact goniometer together with an ALV5000E digital correlator (both ALV, Germany). The temperature of the sample was fixed to 20 and 37 °C by a temperature-controlled environment. To account for polydispersity, we evaluated the data according to the CONTIN algorithm (21) provided by the ALV-correlator software. The resulting relaxation rate distribution was converted to a hydrodynamic radius distribution according to the Stokes-Einstein relation. A weighting of the relaxation distribution function according to a Rayleigh-Debye theory is not appropriate here because the micelle size is far above the limits of the Rayleigh-Debye theory. Freeze-Fracture/Transmission Electron Microscopy (TEM). Liposomes made from DOPE/DOTAP (1/1 w/w), DOPE/DOTAP/LR-DOPE (1/1/0.1), and DOPE/DOTAP/βBODIPY-C12HPC (1/1/0.1) mixtures were studied by freezefracture in a concentration of 1 mg/mL. Samples were incubated at 20 °C or at 37 °C for at least 10 min. After incubation, samples were rapidly frozen by plunging into liquid propane cooled by liquid nitrogen. Fracturing was performed at -150 °C in a freeze-fracture apparatus (BAF 400 D, Bal-Tec, Liechtenstein). Etching was achieved by warming the samples to -110 °C for 1 min followed by shadowing with platinum in an electron beam at an angle of 45° and subsequently with carbon at an angle of 90°. The replicas were cleaned with a methanol (VWR, Darmstadt, Germany)/water mixture (9/1 v/v). Replica were captured with copper grids and examined in an EM902 (Carl Zeiss SMT, Oberkochen, Germany) microscope. Light Microscopy. Samples were imaged at 37 °C using a laser scanning microscope LSM 710 (Carl Zeiss MicroImaging GmbH, Jena, Germany) equipped with an argon ion laser (488 nm) and a helium-neon laser (543 nm). To detect the fluorescent signal of TMR, LR, and TR (excitation 543 nm), a long pass filter LP 650 nm, for BODIPY, DiO, NBD, and fluorescein (excitation 488 nm), a band-pass filter BP 500-520 nm was used. The microscope was equipped with an oil immersion objective EC Plan-Neofluar 40×/1.30 Ph3 (Carl Zeiss). The images were analyzed with the LSM 710 ZEN software (Carl Zeiss). Fluorescent recovery after photobleaching (FRAP) was used to determine the diffusion constant of lipid components in living cell membranes. For these measurements, an intense, focused laser beam of appropriate wavelength (488 nm for BODIPY FL and 543 nm for LR-DOPE) bleached a small circular spot (ca. 0.5-1 µm2) on the cell membrane for 50-100 ms. Subsequently, the same beam with a strongly reduced intensity of approximately 3% monitored the reappearance of fluorescence within the circle due to the diffusion of unbleached molecules. The fluorescence recovery curve, I(t) (fluorescence intensity vs time after bleaching) was first corrected as described by Mo¨hl et al. (22). Diffusion constants (D) of fluorescent labeled lipids were calculated as follows (23):

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D ) 0.224r2 /t1/2

(1)

where r is radius of the bleached spot and t1/2 is half time of fluorescent recovery. Cell Culture. Isolation of Rat Embryonic Myofibroblasts. Fibroblasts were isolated from 18-day old Wistar rat embryos as described earlier (24). Cells were seeded on fibronectin coated glass surfaces (2.5 µg/cm2 human plasma fibronectin (BD Biosciences, San Jose, CA)). Cells were maintained in F10 Ham’s medium (Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum and a 1/100 dilution of antibiotics (10 000 units penicillin and 10 mg/mL streptomycin in 0.9% NaCl, (Sigma)). For additional isolation of pericardial tissue, the pericardium was carefully detached from embryonic heart, washed in cold Hanks Balanced Salt Solution (HBSS) (Sigma), and maintained as described above for myofibroblasts. Isolation of Rat Embryonic Cortical Neurons. Rat embryos were recovered from pregnant rats at day 18 of gestation as described in refs 25 and 26. Isolated cortical neurons were resuspended in 1 mL Neurobasal Medium (Invitrogen), 1% B27 supplement (Invitrogen), 0.5 mM L-glutamine per hemisphere isolated, and seeded on poly-D-lysine (Sigma) coated glass surfaces (1 µg/cm2). Additional cell types investigated in this study were purchased from different providers and cultivated in the recommended media (Table 1). All cell types listed in Table 1 were cultivated in a humidified incubator at 37 °C and 5% CO2. Isolation of Macrophages. Human monocytes were isolated according to the protocol from Noble and Cutts (27) using the Leucosep-System (Greiner bio-one, Kremsmu¨nster, Austria). Isolated leucocytes were resuspended in RPMI medium (Sigma) supplemented with 10% fetal bovine serum and a 1/100 dilution of an antibiotic solution (10,000 units penicillin and 10 mg streptomycin in 0.9% NaCl, (Sigma)) and seeded on fibronectin (BD Bioscience, San Jose, CA USA) coated glass surfaces. Nonadherent cells were washed away after four hours. Since monocytes attach to culture surfaces more rapidly than other lymphocytes, this step resulted in an enriched culture. After two days, medium was completely replaced by fresh RPMI containing 0.1 ng/mL granulocyte macrophage colony stimulating factor (G-MCF) (Sigma). After further 3-4 days, monocytes were differentiated to macrophages. Membrane-Fusion Experiments. For fusion experiments, 5 µL of liposome stock solution was diluted 1/100 with appropriate cell culture medium (Table 1) and gently shaken for 1-2 min at room temperature. Cells in a Petri dish (L ) 3.5 cm) (20 000-30 000 per dish) were incubated in 500 µL of fusogenic liposomes solution (pH 7.4) for 10-30 min at 37 °C. Subsequently, the fusion mixture was replaced by fresh medium, and cells were imaged by light microscopy.

RESULTS As lipid base, we used a 1:1 (w/w) ratio of a neutral phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), and a cationic lipid, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). Figure 1 shows the molecular structures of these lipids. This lipid mixture was not able to fuse with cell membranes resulting in low uptake efficiencies (data not shown). The addition of a third lipid component containing large delocalized π electron systems coupled to either the hydrophobic or the hydrophilic lipid parts completely changed the cellular uptake processes. Table 2 summarizes some of the tested aromatic lipid molecules. However, all of them are widely used for fluorescent membrane labeling and none of these molecules can be transferred spontaneously into membranes (28) (exception here are lipids of the Di- series, e.g., DiO). Only the synergetic interaction of the three components,

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Table 1. Summary of All Cell Types Investigated in This Study cell name

cell type

organism

provider

medium

myofibroblast keratinocyte cortical neuron macrophage HEK293 cell bronchial smooth muscle cell pericardial tissue

primary cell primary cell primary cell primary cell cell-line primary cell primary tissue

embryonic rat human embryonic rat human human human embryonic rat

isolated (24) purchased at Lonza isolated (25, 26) isolated (27) purchased at DSMZ purchased at Lonza isolated

F10 Ham’s medium, 10% FBS, 1× penicillin-streptomycin keratinocyte basal medium neurobasal medium, 1% B27 supplement RPMI-1640 medium, 10% FBS, 1× penicillin-streptomycin DMEM medium, 10% FBS, 1× penicillin-streptomycin BSMC, 10% FBS, 1× penicillin-streptomycin F10 Ham’s medium, 10% FBS, 1× penicillin-streptomycin

neutral lipid, cationic lipid, and aromatic lipid resulted in an effective fusogenic mixture. Characterization of the Fusogenic Liposomes. Although DOPE/DOTAP (1/1 w/w) liposomes did not fuse measurably with cell membranes, they were chosen as reference sample to compare their physicochemical behavior with that of the fusogenic liposomes. After preparation, the average radius of the DOPE/DOTAP liposomes was 0.72 ( 0.09 µm (Figure 2a) according to dynamic light scattering analyses. Freeze-fracture and subsequent electron microscopy further specified them as unilamellar vesicles with smooth surfaces at room temperature as well as at 37 °C (Figure 2b). The addition of aromatic lipid components to a DOPE/ DOTAP mixture, e.g., lissamine rhodamine B fluorescent dye coupled to DOPE (Table 2) or β-BODIPY coupled to C12-HPC (Figure 1 and Table 2), just slightly affected the size distributions of liposomes with an average radius of 0.78 ( 0.07 µm and 0.94 ( 0.10 µm, respectively. Smaller vesicles or larger aggregates of vesicles were not observed in the dynamic light scattering analyses (Figure 2a). Vesicles were observed as unilamellar, spherical structures with smooth, nonstructured surfaces at both temperatures similar to the reference sample (Figure 2b). Administration of Fusogenic Liposomes to Single Cells. In order to prove fusion, HEK293 cells were imaged during incubation with fusogenic liposomes made from DOPE/DOTAP/ BODIPY FL-DHPE (1/1/0.1 w/w) lipid mixture (Figure 1a). Immediately after vesicle addition (Figure 3a: 0 s), small green dots, i.e., vesicles, were observed in the BODIPY FL channel. After 100 s, the first fluorescently labeled cell membranes appeared, and 200 s later, most of the plasma membranes could

Table 2. Summary of Aromatic Lipids Used in This Study to Induce Membrane Fusion with Living Cellsa

a Rainbow scale (left) describes their corresponding emission spectra. Fusion efficiency is indicated by asterisks ranging from poor (*) to very good (***).

Figure 1. Molecular structure of (I) 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) as a neutral lipid component, (II) 1,2dioleoyl-trimethylammonium-propane (DOTAP) as a cationic lipid component, (III) 2-(4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (β-BODIPY C12-HPC) as a chain modified lipid, and (IV) N-(4,4-difluoro5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)-1,2dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (BODIPY FL-DHPE) as a head modified lipid.

be imaged in the green fluorescence channel (Figure 3a). Cellular plasma membranes were homogenously labeled by BODIPY FL without liposome clumps on the surface. Unchanged cell behavior and cell growth were detected in the phase contrast channel simultaneously with the appearance of the fluorescent signal in the cell membranes. After successful membrane fusion, excess liposomes were replaced by cell medium, and cell survival and clearance of fluorescent lipids from the plasma membrane were determined. Cell growth of HEK293 was not influenced by liposome fusion, and a healthy cell layer completely covered the glass surface 24 h after fusion. Most dye lipid disappeared from the cell membrane during this time period as shown in Figure 3b. Several aromatic phospholipid molecules were tested for fusion capacity. Although they all induced membrane fusion with living cells, clear differences were seen in fusion time,

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Csisza´r et al. Table 3. Lateral Diffusion Constant (D) and Fluorescence Recovery of Head and Chain-Labeled Lipids Measured in HEK293 Cell Membranes after Liposome Fusion (mean ( sd for n measurements)

Figure 2. Physico-chemical characterization of fusogenic liposomes. (a) Hydrodynamic radius distribution from dynamic light scattering after CONTIN analysis of DOPE/DOTAP (1/1 w/w), DOPE/ DOTAP/LR-DOPE (1/1/0.1 w/w), and DOPE/DOTAP/β-BODIPYC12HPC (1/1/0.1 w/w) mixtures at 20 µg/mL concentration in 20 mM HEPES buffer. Measurements were carried out at the indicated scattering angles. (b) The same fusogenic vesicles at 2 mg/mL concentration were freeze-fractured and subsequently analyzed by transmission electron microscopy. Note their unilamellar composition and smooth surface.

fusion efficiency, and signal homogeneity in the plasma membrane. These parameters together entered an admittedly subjective efficiency scale (Table 2). The most effective and fast fusion processes with homogeneously distributed fluorescence signals in the cell membrane were induced by large aromatic groups containing hetero atoms with a significantly higher electronegativity, e.g., oxygen, nitrogen, or boron coupled to fluorine (Table 2: ***). Dye molecules as pyrene with a

dye lipid

D (µm2/s)

recovery (%)

n

LR-DOPE BODIPY FL-DHPE β-BODIPY C12HPC

0.21 ( 0.14 0.16 ( 0.08 0.15 ( 0.05

84 ( 5 76 ( 6 75 ( 7

8 9 9

homogenously distributed π electron system without hetero atoms correspondingly were found to be less effective fusion promoters (Table 2: *). Cyclic groups coupled to lipid molecules that were not in compliance with Hu¨ckel’s rule were also investigated (e.g., capbiotin-DOPE or phosphatidylinositol), and no fusion with cell membranes was detected. Furthermore, the efficiency of pH-sensitive dyes like fluorescein depended on their ionic form. At low pH values where the dye group was deprotonated, they were less effective than at higher pH in the protonated form (Table 2: **). The successful incorporation of aromatic lipid molecules was furthermore controlled by detecting their lateral diffusion constants using fluorescence recovery after photobleaching technique (FRAP) (29, 30). Liposome fusion events were induced in HEK293 cells by LR-DOPE and BODIPY FL-DHPE as headgroup-modified lipids and β-BODIPY C12HPC as chainlabeled lipid. Plasma membranes were first localized by confocal imaging. Subsequently, diffusion constants (D) and fluorescence recovery of the dye lipids in the plasma membrane were determined. A typical recovery curve is shown in Figure 3b, and all results are summarized in Table 3. The resulting diffusion constants of all investigated dye lipids were found to be relatively similar with values around 0.18 µm2/s. Even the location of the aromatic group in the polar head or apolar chain region did not significantly influence the diffusion behavior. Fluorescence recovery, frequently called mobile fraction, was also determined, and the values also appeared statistically similar

Figure 3. Vesicle fusion with plasma membranes. (a) HEK293 cells were incubated with vesicles made from DOPE/DOTAP/BODIPY FL-DHPE (1/1/0.1 w/w) and analyzed over 300 s in fluorescence (left) and phase contrast (right). Within this time period, efficient membrane fusion was detected with unchanged cell behavior. Scale bars ) 50 µm. (b) Uninfluenced cell growth (phase contrast channel) and completely degraded liposomes (BODIPY FL channel) were observed 24 h after membrane fusion. Scale bars ) 80 µm (c). Fluorescence recovery curve of LR-DOPE in HEK293 plasma membrane after membrane fusion. The diffusion parameters of LR-DOPE calculated from the FRAP curve confirm the incorporation of fluorescently labeled lipid into the cell membrane.

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Figure 5. Isolated pericardial tissues were incubated for 1 h using DOPE/DOTAP/BODIPY FL-DHPE (1/1/0.1 w/w) mixture as fusogenic liposomes and subsequently imaged by confocal microscopy. Small micrographs below and on the right of the main image indicate the cross sections parallel to xz- and yz-planes respectively. Cross section images revealed several fluorescently labeled cell layers below the top layer. Scale bar ) 100 µm.

below the top layer were labeled with similar efficiency in spite of the relatively large liposomal size (Figure 5).

DISCUSSION

Figure 4. Cell membranes of HEK293, smooth muscle cells (SMC), rat myofibroblasts, human macrophages, and embryonal cortical neurons were fluorescently labeled by vesicle fusion. As aromatic lipids, either LR-DOPE (red) or BODIPY FL-DHPE (green) was used. Scale bars ) 10 µm.

to each other (around 78 ( 6%) independent from the dye and its location in the lipid molecule. The measured values were in good agreement with other values given for fluorescent tracer lipid diffusion in cell membranes (23, 31). The ubiquitous ability of our system to induce fusion was successfully proven by labeling many other cell types as human keratinocytes, human macrophages, rat embryonic cortical neurons, bronchial smooth muscle cells, and rat myofibroblasts. For all of these cell types, cell labeling efficiency reached an almost complete labeling of all cells. Only in the case of myocytes was an increased resistance against fusogenic liposomes observed (data not shown). Some labeled cells are presented in Figure 4. Administration of Fusogenic Liposomes on Cellular Tissue. As an exemplary tissue sample, rat embryonic pericardium, a multicelled layer of defined thickness around the contractile heart apparatus was chosen and incubated with fusogenic liposomes made from DOPE/DOTAP/BODIPY FLDHPE (1/1/0.1 w/w) lipid mixture for one hour. This prolonged incubation period was necessary due to the increased cell number and the cell stacks on top of each other. Confocal imaging revealed an almost complete staining of all pericardial surface cells. Additionally, almost all cell layers (three to four)

Classical liposomes used for DNA delivery are based on a composition of neutral and positively charged lipid molecules. The cellular uptake processes of these vesicles have been investigated earlier and have shown a strong preference of endocytotic pathways with a limited efficiency (32). Here, the addition of a third lipid component converted these liposomes to a nearly universal fusogenic system. The third lipid component needed to meet the criteria of (a) amphiphilic molecular character to be easily incorporated into biological or biomimetic membranes and (b) a delocalized conjugated π-electron system located in the polar or apolar molecular range. Interestingly, these criteria were fulfilled by almost all fluorescent labels when coupled to phospholipids. Table 2 summarized all successfully tested molecules. Other molecules meeting these criteria should work as well but have not been explicitly tested here since fluorescently labeled lipids had the strong advantage of direct visualization of fusion to unravel the functional mechanism. The synergistic interaction of the three components, neutral lipid, positively charged lipid, and lipids with an aromatic group resulted in an effective fusogenic mixture. Although the mechanism of action has not yet been elucidated, it is to assume that the neutral molecule DOPE has a cone-like effective shape and supports fusion intermediate states (33). The positive charge of DOTAP in the polar headgroup region interacts with the negatively charged glycocalyx of the cell membrane decreasing the distance between liposome and cell membrane and increasing the probability of fusion, respectively (3). Additionally, the strong positive charges in the headgroup of liposomes polarize the delocalized π electrons inducing local dipoles. These dipoles presumably yield local instabilities and disorders of molecular arrangements in the bilayer. Cevc and Richardsen reported that such effects could positively influence membrane fusion processes (12). Moreover, the presence of an additional heteroatom with high electronegativity, e.g., fluorine (χ ) 4 on the Pauling scale) in BODIPY dyes (Figure 1a) or oxygen (χ ) 3.5) and nitrogen (χ ) 3.0) increased the dipole character in the aromatic group resulting in a higher fusion

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Figure 6. Overview of the potential fields of application for fusogenic liposomes: (I), plasma membrane visualization (II), transmembrane protein incorporation into plasma membranes, (III) cell surface functionalization, and (IV) content delivery.

potential, while molecules with a large but homogenously distributed π electron system like pyrene (Table 2) revealed reduced fusion behavior. Cyclic groups without aromaticity and coupled to phospholipids did not induce fusion processes with membranes of living cells. Delocalized electron systems could be bound to the head as well as the chain regions of lipids (Table 2, e.g., pyrene or BODIPY) without any influence on the fusion efficiency. The most effective composition ratio between the three components was found from 1/1/0.05 to 1/1/0.2 (w/w) of DOPE/DOTAP/ aromatic lipid. A lower amount of aromatic lipids is often not sufficient to promote fusion, while higher concentrations can be toxic to cells. In general, an approximate weight ratio of 1/1/0.1 could be successfully used for the first experimental approaches. The digestion of lipids used in fusogenic liposomes in a weight ratio of 1/1/0.1 of DOPE/DOTAP/aromatic lipid by HEK293 was investigated, and their full degradation was observed in a time period of 24 h accompanied by completely unchanged cell growth. On the basis of these observations, we propose that administration of these novel liposomes yielded highly efficient cell membrane modifications without noticeable toxic side effects. Liposome fusion events were successfully observed in several cell types like HEK293 cells, human macrophages, rat embryonic cortical neurons, smooth muscle cells, and rat myofibroblasts (Figure 4). After membrane fusion, diffusion constants (D) of aromatic lipids were determined with 0.18 ( 0.14 µm2/s (Table 3) corresponding to other values given for lipid diffusion in cell membranes (23, 31). This value is approximately one magnitude lower in comparison to lipid diffusion in model membranes without hindering proteins where the diffusion constant of NBD-DOPE was found to be 2.5 ( 0.2 µm2/s (34, 35). This fact supports the hypothesis of membrane fusion against liposomal adhesion on cell membrane surfaces. Additionally, the selective fluorescent signal of the cell membrane as well as the short uptake time and the high efficiency suggested a membrane fusion against endocytosis as main uptake mechanism. These results open a large number of possible pharmaceutical and biotechnological application fields as schematically summarized in Figure 6. Fluorescent membrane labeling is only one of the numerous cell membrane modifications

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(Figure 6 I). With addition of other functionalized molecules like chelator lipids or biotinylated or antigenic lipids at an optional low concentration, surface functionalization of living cells can be achieved (Figure 6 III). This membrane modification can be completed in an additional step by protein coupling to the cell surface. Furthermore, it is assumed that membrane proteins in addition to functionalized lipids can also be incorporated into fusogenic liposomes and, corresponding to this, into cell membranes as well (Figure 6 II). Nevertheless, if the membrane mixing is accompanied by content mixing, highly efficient delivery of soluble molecules to living cells can be performed (Figure 6 IV). Fusogenic liposomes could easily be modified by tumor specific targeting and used for medical purposes. In conclusion, we have designed a new, simple and almost universal fusogenic liposome system containing neutral and positively charged lipid molecules as well as additional lipid components with aromatic molecular groups. The liposomes mediate highly effective fusion processes with living cell membranes without toxic side effects. As fusion mechanism, we hypothesize a synergistic interaction of the positively charged lipids and the delocalized electron system of the aromatic group inducing local dipoles and membrane instabilities. However, the exact mechanism has to be investigated in the future. This new type of fusogenic liposomes opens a large number of biotechnological application fields.

ACKNOWLEDGMENT We thank R. Fricke and K. Michael for isolation of rat cortical neurons. We are also grateful to J. Fleischhauer for technical support.

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