Biosensor-based evaluation of liposomal behavior in the target binding process

In this study we evaluated the quartz crystal microbalance (QCM) as a biosensor for a real-time investigation of liposomal binding, under dynamic flow conditions, onto target proteins immobilized at the sensor. The mass-sensitive frequency changes of quartz sensors allow for a quantification of the liposomal binding process. Furthermore, simultaneous damping analysis gives an insight into liposomal behavior, such as the degree of liposomal deformation or spreading at the target surface. In this study a series of liposomes was evaluated, differing in the kind and concentration of ligands interacting with appropriate target proteins. It became evident that an increase in homing device concentration accelerated deformation and flattening of liposomes, triggering a fusion process. Furthermore, liposomal deformation corresponded with the binding affinity of target molecules, comparing biotin/avidin with E-selectin/ligand interactions. Deformation could be emphasized using dioleoylphosphatidylethanolamine (DOPE) as a fusiogenic membrane component, while sterical stabilization by polyethylenglycol (PEG-PE) appeared in a low degree of deformation. Consequently, the online detection of liposomal target binding by QCM is an excellent facility to control and predict the liposomal behavior at the target site for increasing therapeutic potency.     

Plasma membrane-mediated leakage of liposomes induced by interaction with murine thymocytic leukemia cells

The interaction of liposomes with BW 5147 murine thymocytic leukemia cells was studied using fluorescent probes (entrapped carboxyfluorescein and fluorescent phosphatidylethanolamine) in conjunction with a Ficoll-Paque discontinous gradient system for rapid separation of liposomes from cells. Reversible liposomal binding to discrete sites on the BW cell surface was found to represent the major form of interaction; uptake of intact liposomal contents by a process such as liposome-BW cell membrane fusion was found to apparently represent a minor pathway of interaction (2%). Liposomal lysis was found to be associated with the process of liposomal binding (perhaps as a result of the binding itself). Lysis was followed by release of the entrapped carboxyfluorescein into the media and its subsequent uptake by the cells. This lysis was shown to be dependent upon discrete membrane-associated sites that have some of the properties of proteins. The results of these studies suggest that liposomal binding to the cells, subsequent lysis of the liposomes and cellular uptake of their contents should be seriously considered in all studies of liposome-cell interactions as an alternate mode of interaction to the four modes (fusion, endocytosis, adsorption and lipid exchange) previously emphasized in the literature.     

Comparison of surface plasmon resonance spectroscopy and quartz crystal microbalance techniques for studying DNA assembly and hybridization

In this study we evaluate the strengths and weaknesses of surface plasmon resonance (SPR) spectroscopy and quartz crystal microbalance (QCM) technique for studying DNA assembly and hybridization reactions. Specifically, we apply in parallel an SPR instrument and a 5 MHz QCM device with dissipation monitoring (QCM-D) to monitor the assembly of biotinylated DNA (biotin-DNA) on a streptavidin-modified surface and the subsequent target DNA hybridization. Through the parallel measurements, we demonstrate that SPR is more suitable for quantitative analysis of DNA binding amount, which is essential for interfacial DNA probe density control and for the analysis of its effect on hybridization efficiency and kinetics. Although the QCM is not quantitative to the same extent as SPR (QCM measures the total mass of the bound DNA molecules together with the associated water), the dissipation factor of the QCM provides a qualitative measure of the viscoelastic properties of DNA films and the conformation of the bound DNA molecules. The complexity in mass measurement does not impair QCM's potential for a kinetic evaluation of the hybridization processes. For quantification of target DNA, the biotin-DNA modified SPR and QCM sensors are exposed to target DNA with increasing concentration. The plots of SPR/QCM signals versus target DNA concentration show that water entrapment between DNA strands make the QCM sensitivity for the hybridization assay well comparable with that of the SPR, although the intrinsic mass sensitivity of the 5 MHz QCM is approximately 20 times lower.     

Evaluation of a high-affinity QCM immunosensor using antibody fragmentation and 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer

This study evaluated construction of a highly affinitive quartz crystal microbalance (QCM) immunosensor using anti-C-reactive protein (CRP) antibody and its fragments for CRP detection. Three types of antibody were immobilized on the surface of a QCM via covalent-bounding. Then affinity was evaluated through antigen-antibody binding between CRP and its antibody. Affinity between antigen-antibody was shown to be highest when anti-CRP F(ab')2-IgG antibody (70 microg/mL) was immobilized on the QCM. In case of anti-CRP F(ab')2-IgG antibody, affinity which was attributable to antigen-antibody binding was almost twice that of anti-CRP IgG antibody, which is used conventionally for QCM immunosensors. In addition, when it was treated with 2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate, so-called MPC polymer, highly affinitive and selective immunosensing for CRP was achieved without non-specific binding from plasma proteins in human serum. When anti-CRP F(ab')2-IgG antibody was immobilized on the QCM, the detection limit and the linearity of CRP calibration curve were achieved at concentrations from 0.001 to 100 microg/dL even during investigation in serum samples. Experimental results verified the successful construction of a highly affinitive and selective QCM-immunosensor which was modified with anti-CRP F(ab')2-IgG antibody and MPC polymer.     

Specific adhesion of vesicles monitored by scanning force microscopy and quartz crystal microbalance

The specific adhesion of unilamellar vesicles with an average diameter of 100 nm on functionalized surfaces mediated by molecular recognition was investigated in detail. Two complementary techniques, scanning force microscopy (SFM) and quartz crystal microbalance (QCM) were used to study adhesion of liposomes consisting of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine and varying concentrations of N-((6-biotinoyl)amino)hexanoyl)-1, 2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (biotin-X-DHPE). Monitoring the adhesion of the receptor-doped vesicles to avidin-coated gold surfaces by QCM (f(0) = 5 MHz) revealed an increased shift in resonance frequency with increasing biotin concentration up to 10 mol% biotin-X-DHPE. To address the question of how the morphology of the liposomes changes upon adhesion and how that contributes to the resonator's frequency response, we performed a detailed analysis of the liposome morphology by SFM. We found that, with increasing biotin-concentration, the height of the liposomes decreases considerably up to the point where vesicle rupture occurs. Thus, we conclude that the unexpected high frequency shifts of the quartz crystal (>500 Hz) can be attributed to a firm attachment of the spread bilayers, in which the number of contacts is responsible for the signal. These findings are compared with one of our recent studies on cell adhesion monitored by QCM.     

Nonspecific hydrophobic interactions of a repressor protein, PhaR, with poly[(R)-3-hydroxybutyrate] film studied with a quartz crystal microbalance

The gene expression for phasins (PhaP), which are predominantly polyhydroxyalkanoates (PHAs) granule-associated proteins, is regulated by a repressor protein of PhaR through the dual binding abilities of PhaR to the target DNAs and the granules. In this study, the binding functions of PhaR to poly[(R)-3-hydroxybutyrate] (P(3HB)) were investigated quantitatively by using a quartz crystal microbalance (QCM) technique. Adsorption of PhaR onto a melt-crystallized film of P(3HB) (cr-P(3HB)) was detected as a negative frequency shift of the QCM. The time course of the frequency changes observed for PhaR adsorption was composed of a quick frequency decrease at an initial stage and a subsequent slower frequency decrease for several hours, indicating multilayered adsorption of PhaR molecules onto cr-P(3HB). The initial rapid adsorption, which corresponds to direct adsorption of PhaR molecules onto a bare surface of cr-P(3HB), was a diffusion-controlled process. Strong interactions between PhaR and cr-P(3HB) were also observed as apparently irreversible adsorption. The comparative QCM measurement of PhaR adsorption onto various types of polymers with different aliphatic chemical structures revealed that PhaR was adsorbed onto the surfaces of polymers, including cr-P(3HB), mainly by nonspecific hydrophobic interactions. These results illustrate the high affinity and low specificity for adsorption of PhaR to P(3HB).     

Synthesis of bilirubin imprinted polymer thin film for the continuous detection of bilirubin in an MIP/QCM/FIA system

A bilirubin imprinted polymer (BIP) was coated on a thiol pretreated Au electrode on a quartz crystal microbalance (QCM) chip. The BIP thin film was synthesized using 4-vinylpyridine (4-Vpy) as the monomer, divinylbenzene (DVB) as the cross-linker, and benzophenone as the initiator. By using a photo-graft surface polymerization technique with irradiation by ultra-violet (UV) light, a thin BIP film was prepared, from which a biomimetic sensor for the detection of bilirubin was developed. The sensor was able to discriminate bilirubin in solution owing to the specific binding of the imprinted sites. The BIP/QCM chip has been repeatedly used for more than 7 months in many continuous experiments. The detection signal of bilirubin from the BIP thin film/QCM was compared with the non-BIP thin film/QCM. Biliverdin, an analogue of bilirubin, was used for comparison. The analogue comparison confirmed the binding specificity of the BIP film toward bilirubin. The selectivity can be as high as 31.2. The effect of pH on the detection of bilirubin is also discussed. With proper solvent for elution and recovery, flow injection analysis (FIA) could be applied to the system. The performance of the BIP/QCM chip was evaluated. A linear calibration of the bilirubin concentration with respect to the frequency shift was successfully obtained. The reproducibility of measurements from the same BIP/QCM chip was confirmed. In addition, repeatability of detection was also confirmed from different BIP/QCM chips. In conclusion, a combined BIP thin film/QCM/FIA method was successfully established for the detection of bilirubin concentration using a molecularly imprinted film.     

Antibodies induced by liposomal protein exhibit dual binding to protein and lipid epitopes

Natural polyreactive antibodies can accommodate chemically unrelated epitopes, such as lipids and proteins, in a single antigen binding site. Because liposomes containing lipid A as an adjuvant can induce antibodies directed against specific lipids, we immunized mice with liposomes containing lipid A together with a protein or peptide antigen to determine whether monoclonal antibodies generated after immunization would be specifically directed both to the liposomal lipid (either cholesterol or galactosylceramide) and also to the accompanying liposomal protein or peptide. Monoclonal antibodies were obtained that bound, by ELISA, to cholesterol and to recombinant gp140 envelope protein from HIV-1, or to galactosylceramide and to an HIV-1 envelope peptide. Surface plasmon resonance studies with the former antibody showed that the liposomal cholesterol and liposomal gp140 each contributed to the overall binding energy of the antibody to liposomes containing cholesterol and protein.     

Adsorption of chitosan on PET films monitored by quartz crystal microbalance

The adsorption behavior of chitosan on poly(ethylene terephthalate) (PET) model film surface was studied using the quartz crystal microbalance (QCM) technique. QCM with a dissipation unit (QCM-D) represents a very sensitive technique for adsorption studies at the solid/liquid interface in situ, with capability of detecting a submonolayer of adsorbate on the quartz crystal surface. Chitosan as well as PET were chosen for this study due to their promising biocompatible properties and numerous possibilities to be used in biomedical applications. As a first step, PET foils were activated by alkaline hydrolysis in order to increase their hydrophilicity. Model thin films were prepared from PET foils by the spin coating technique. The chemical composition of the obtained model PET films was analyzed using X-ray photoelectron spectroscopy (XPS) and their morphology was characterized by atomic force microscopy (AFM). Furthermore, the adsorption behavior of chitosan on these activated PET films and the influence of adsorption parameters (pH, ionic strength and chitosan solution concentration) were investigated in detail. Additionally, the surface chemistry and morphology of the PET films and the chitosan coated PET films were analyzed with XPS and AFM.     

Novel quartz crystal microbalance based biosensor for detection of oral epithelial cell-microparticle interaction in real-time

Recent applications of quartz crystal resonant sensor technology to monitor cell adhesion and specific ligand interaction processes has triggered the development of a new category of quartz crystal microbalance (QCM) based biosensors. In this study human oral epithelial cells (H376) were cultured on quartz sensors and their response to microspheres investigated in situ using the QCM technique. The results demonstrated that this novel biosensor was able to follow cell-microsphere interactions in real-time and under conditions of flow as would occur in the oral cavity. Unique frequency profiles generated in response to the microspheres were postulated to be due to phases of mass addition and altered cellular rigidity. Supporting microscopic evidence demonstrated that the unique frequency responses obtained to these interactions were in part due to binding between the cell surface and the microspheres. Furthermore, a cellular uptake process, in response to microsphere loading was identified and this, by influencing the rigidity of the cellular cytoskeleton, was also detectable through the frequency responses obtained.     

The role of beta2-glycoprotein I in liposome-hepatocyte interaction

Adsorption of serum proteins to the liposomal surface plays a critical role in liposome clearance from the blood. The aim of this study was to investigate the role of liposome-adsorbed serum proteins in the interaction of liposomes with hepatocytes. We analyzed the serum proteins adsorbing to the surface of differently composed small unilamellar liposomes during incubation with human or rat serum, and found that one protein, with a molecular weight of around 55 kDa, adsorbed in a large amount to negatively charged liposomes containing phosphatidylserine (PS) or phosphatidylglycerol (PG). The binding was dependent on the liposomal charge density. The approximately 55-kDa protein was identified as beta2-glycoprotein I (beta2GPI) by Western blotting. Despite the high affinity of beta2GPI for strongly negatively charged liposomes, in vitro uptake and binding experiments with isolated rat hepatocytes, Kupffer cells or liver endothelial cells, and with HepG2 cells showed no enhancing effect of this protein on the association of negatively charged liposomes with any of these cells. On the contrary, an inhibitory effect was observed. We conclude that despite abundant adsorption to negatively charged liposomes, beta2GP1 inhibits, rather than enhances, liposome uptake by liver cells.     

Interaction of N-acylated and N-alkylated chitosans included in liposomes with lipopolysaccharide of gram-negative bacteria

The interactions of lipopolysaccharide (LPS) with the polycation chitosan and its derivatives — high molecular weight chitosans (300 kDa) with different degree of N-alkylation, its quaternized derivatives, N-monoacylated low molecular weight chitosans (5.5 kDa) — entrapped in anionic liposomes were studied. It was found that the addition of chitosans changes the surface potential and size of negatively charged liposomes, the magnitudes of which depend on the chitosan concentration. Acylated low molecular weight chitosan interacts with liposomes most effectively. The binding of alkylated high molecular weight chitosan with liposomes increases with the degree of its alkylation. The analysis of interaction of LPS with chitoliposomes has shown that LPS-binding activity decreased in the following order: liposomes coated with a hydrophobic chitosan derivatives > coated with chitosan > free liposomes. Liposomes with N-acylated low molecular weight chitosan bind LPS more effectively than liposomes coated with N-alkylated high molecular weight chitosans. The increase in positive charge on the molecules of N-alkylated high molecular weight chitosans at the cost of quaternization does not lead to useful increase in efficiency of binding chitosan with LPS. It was found that increase in LPS concentration leads to a change in surface ζ-potential of liposomes, an increase in average hydrodynamic diameter, and polydispersity of liposomes coated with N-acylated low molecular weight chitosan. The affinity of the interaction of LPS with a liposomal form of N-acylated chitosan increases in comparison with free liposomes. Computer simulation showed that the modification of the lipid bilayer of liposomes with N-acylated low molecular weight chitosan increases the binding of lipopolysaccharide without an O-specific polysaccharide with liposomes due to the formation of additional hydrogen and ionic bonds between the molecules of chitosan and LPS.     

G‐protein coupled receptor array technologies: Site directed immobilisation of liposomes containing the H1‐histamine or M2‐muscarinic receptors

This paper describes a novel strategy to create a microarray of G‐protein coupled receptors (GPCRs), an important group of membrane proteins both physiologically and pharmacologically. The H₁‐histamine receptor and the M₂‐muscarinic receptor were both used as model GPCRs in this study. The receptor proteins were embedded in liposomes created from the cellular membrane extracts of Spodoptera frugiperda (Sf9) insect cell culture line with its accompanying baculovirus protein insert used for overexpression of the receptors. Once captured onto a surface these liposomes provide a favourable lipidic environment for the integral membrane proteins. Site directed immobilisation of these liposomes was achieved by introduction of cholesterol‐modified oligonucleotides (oligos). These oligo/cholesterol conjugates incorporate within the lipid bilayer and were captured by the complementary oligo strand exposed on the surface. Sequence specific immobilisation was demonstrated using a quartz crystal microbalance with dissipation (QCM‐D). Confirmatory results were also obtained by monitoring fluorescent ligand binding to GPCRs captured on a spotted oligo microarray using Confocal Laser Scanning Microscopy and the ZeptoREADER microarray imaging system. Sequence specific immobilisation of such biologically important membrane proteins could lead to the development of a heterogeneous self‐sorting liposome array of GPCRs which would underpin a variety of future novel applications.     

Influence of glycolipids on immune reactions of phospholipid antigens in liposomes

Complement-dependent immune damage to liposomes mediated by a murine monoclonal antibody to the liposomal bilayer was completely inhibited by ceramide tetrasaccharide (globoside) at an 8% concentration in the liposomes. Lower concentrations of globoside, or higher concentrations of ceramide tri-, di-, or monohexoside, were not inhibitory. At a globoside concentration of 5.8%, inhibition of the monoclonal antibody activity was reduced and inhibition was related to antibody concentration; but at 2% globoside, suppression of antibody activity was not observed at all. Analysis of space-filling models revealed that at 8% globoside the distance between adjacent tetrasaccharides of globoside approached the dimensions of the antigen-binding end of a 7S immunoglobulin molecule. We therefore propose that globoside, and perhaps other glycolipids, can exert steric hindrance to the binding of extracellular proteins to nonglycolipid constituents of the lipid bilayer. We conclude that microheterogeneity among polar groups of glycolipids may be a novel mechanism for allowing selective access of proteins to phospholipids on the lipid bilayer.     

A quartz crystal microbalance method for rapid detection and differentiation of shiga toxins by applying a monoalkyl globobioside as the toxin ligand

A simple globobiosyl (Gb2) ceramide mimic carrying a monoalkyl chain (C18) was applied for a monolayer Langmuir-Blodgett (L-B) technique to detect Shiga toxins (Stxs) by a quartz crystal microbalance (QCM) method. The artificial glycolipid, synthesized from penta-O-acetyl-D-galactopyranose via a conventional glycosidation pathway, was developed at the air-water surface for the formation of the monolayer film. Then, the film was transferred onto a QCM cell surface modified with alkanethiols. Upon the addition of each of Stx-1 and Stx-2, the decrease of frequency reached saturation within 45 min at a few nanogram order per quartz cell. Binding constants (Ka) estimated for each of Stx-1 and Stx-2 showed little difference between the two toxins. On the other hand, in the presence of an artificial acrylamido Gb2 copolymer as a competitive inhibitor, the two toxins showed a large difference in the binding behavior to the L-B monolayer.     

Adhesion characteristics and stability assessment of lectin-modified liposomes for site-specific drug delivery

Carbohydrate moieties of the cellular glycocalyx have been suggested to play an important role in biological recognition processes during pathologic conditions, such as inflammation and cancer. Herein, we describe lectin-modified liposomes which might have potential for site-specific drug delivery during the therapy of such diseases. Specific interactions of plain (i.e., unmodified) and PEGylated, lectin-grafted liposomes with model membranes were investigated under real-time flow conditions using a quartz crystal microbalance. In addition, the morphology of the liposomal systems was assessed by atomic force microscopy. Plain liposomes exhibited only unspecific adhesion to glycolipid membranes and had a tendency to coalesce. The degree of membrane interaction was significantly increased when plain liposomes were modified with the lectin, Concanavalin A. However, vesicle fusion also markedly increased as a result of lectin modification. Additional PEGylation of liposomes reduced unspecific adhesion phenomena, as well as coalescence. Moreover, our studies enabled us to establish quartz crystal microbalance and atomic force microscopy as powerful and complementary methods to characterize adhesion properties of targeted drug delivery systems.     

Influence of glycolipids on immune reactions of phospholipid antigens in liposomes

Complement-dependent immune damage to liposomes mediated by a murine monoclonal antibody to the liposomal bilayer was completely inhibited by ceramide tetrasaccharide (globoside) at an 8% concentration in the liposomes. Lower concentrations of globoside, or higher concentrations of ceramide tri-, di-, or monohexoside, were not inhibitory. At a globoside concentration of 5.8%, inhibition of the monoclonal antibody activity was reduced and inhibition was related to antibody concentration; but at 2% globoside, suppression of antibody activity was not observed at all. Analysis of space-filling models revealed that at 8% globoside the distance between adjacent tetrasaccharides of globoside approached the dimensions of the antigen-binding end of a 7S immunoglobulin molecule. We therefore propose that globoside, and perhaps other glycolipids, can exert steric hindrance to the binding of extracellular proteins to nonglycolipid constituents of the lipid bilayer. We conclude that microheterogeneity among polar groups of glycolipids may be a novel mechanism for allowing selective access of proteins to phospholipids on the lipid bilayer.     

Bioadhesion of supramolecular structures at supported planar bilayers as studied by the quartz crystal microbalance

A quartz crystal microbalance (QCM) was used to study the adhesion behavior of supramolecular aggregates at supported planar bilayers (SPBs). The QCM technique is a suitable method to detect the adsorption of biomolecules at the quartz surface owing to its sensitivity for changes in mass and viscoelastic properties. To simulate biomembranes, the quartz plates were coated with highly ordered lipid films. Therefore, a combination of self-assembled monolayers and Langmuir-Blodgett films was used. Firstly, the adsorption of liposomes coupled with the lectin concanavalin A was investigated at glycolipid-containing model membranes. Using different carbohydrates, it was possible to determine specific and nonspecific parts of the interactions. The adhesion occurred owing to specific lectin-carbohydrate interactions (about 20%) and to nonspecific interactions (about 80%). The composition of the liposomes was changed to simulate the structure of a native biomembrane consisting of the glycocalix, the lipid-protein bilayer, and the cytoskeleton. An artificial glycocalix was created by incorporating poly(ethylene glycol) into the liposomes. Liposomes which were intravesicular polymerized with polyacrylamide or polyacrylcholate simulated the cytoskeleton. It was determined that the modified liposomes had significant lower interactions with SPBs. The adsorption was reduced by approximately 80% compared to unmodified liposomes. Secondly, a model was developed for the detection of interactions between simple or mixed bile salt micelles and model membranes. It was found that simple bile salts did not adsorb at model membranes. Binary systems consisting of bile salt and phospholipid induced only small interactions. On the other hand, ternary systems consisting of bile salt, phospholipid, and fatty acid showed strong interactions. A dependence on the chain length of the fatty acid was observed. Thirdly, the interaction between ganglioside-containing model membranes and cholera toxin (beta-subunit) was investigated. Different ganglioside fractions showed varying adsorption in the following sequence: GM1 > GD1a > GD1b > GT1b.     

Adhesion characteristics and stability assessment of lectin-modified liposomes for site-specific drug delivery

Carbohydrate moieties of the cellular glycocalyx have been suggested to play an important role in biological recognition processes during pathologic conditions, such as inflammation and cancer. Herein, we describe lectin-modified liposomes which might have potential for site-specific drug delivery during the therapy of such diseases. Specific interactions of plain (i.e., unmodified) and PEGylated, lectin-grafted liposomes with model membranes were investigated under real-time flow conditions using a quartz crystal microbalance. In addition, the morphology of the liposomal systems was assessed by atomic force microscopy. Plain liposomes exhibited only unspecific adhesion to glycolipid membranes and had a tendency to coalesce. The degree of membrane interaction was significantly increased when plain liposomes were modified with the lectin, Concanavalin A. However, vesicle fusion also markedly increased as a result of lectin modification. Additional PEGylation of liposomes reduced unspecific adhesion phenomena, as well as coalescence. Moreover, our studies enabled us to establish quartz crystal microbalance and atomic force microscopy as powerful and complementary methods to characterize adhesion properties of targeted drug delivery systems.     

OVCAR-3 cells internalize TAT-peptide modified liposomes by endocytosis

For cytosolic delivery of liposomes containing macromolecular drugs, such as proteins or nucleic acids, it would be beneficial to bypass endocytosis to prevent degradation in the lysosomes. Recent reports pointed to the possibility that coupling of TAT-peptides to the outer surface of liposome particles would enable translocation over the cellular plasma membrane. Here, we demonstrate that cellular uptake of TAT-liposomes occurs via endocytosis rather than plasma membrane translocation. The coupling of HIV-1 derived TAT-peptide to liposomes enhances their binding to ovarian carcinoma cells. The binding was inhibited by the presence of heparin or dextran sulfate, indicating that cell surface proteoglycans are involved in the binding interaction. Furthermore, living confocal microscopy studies revealed that binding of the TAT-liposomes to the plasma membrane is followed by intracellular uptake in vesicular structures. Staining the endosomes and lysosomes demonstrated that fluorescent liposomal labels are present within the endosomal and lysosomal compartments. Furthermore, incubation at low temperature or addition of a metabolic or an endocytosis inhibitor blocked cellular uptake. In conclusion, coupling TAT-peptide to the outer surface of liposomes leads to enhanced endocytosis of the liposomes by ovarian carcinoma cells, rather than direct cytosolic delivery by plasma membrane translocation.