Evaluation of organophosphorus chemicals-degrading enzymes: a comparison of Escherichia coli and human cytosolic aminopeptidase P

An enzyme capable of hydrolyzing organophosphate compounds is of biological as well as environmental significance. We evaluated the possibility of human cytosolic aminopeptidase P (hcAMPP) as an attractive bioscavenger candidate by measuring the enzymatic rates of hydrolysis for a wide variety of organophosphorus compounds. The comparison of substrate specificity exhibited by hcAMPP and E. coli aminopeptidase P (E. coli AMPP) was studied. We cloned, expressed, and purified hcAMPP from HeLa cells and AMPP from Escherichia coli. The pH-rate profiles of hcAMPP were measured in the presence of organophosphate compound 3 or 5. All of the organophosphorus compounds, 1-19, were synthesized by using the approach of phosphorus chemistry described in a previous publication. The relative activity of hcAMPP and E. coli AMPP in hydrolyzing a series of organophosphorus analogues, 1-17, was evaluated in a spectrophotometric assay by monitoring the difference of accumulation of 4-nitrophenol at 400 nm. The overall substrate preference of hcAMPP is as follows: methylphosphonates>ethylphosphonates> or =organophosphates. Interestingly, the observed enhancement in the activity of hcAMPP with methyl phosphonates, 8, 10, 12, and 13, suggests that there is particularly special about the substructure of both methyl moiety and P=O ligand, since the values of specific activity with hcAMPP for the methylphosphonates 8, 10, 12, and 13 are 2- to 73-fold greater than those for the ethylphosphonates 14-17 and the organophosphates 1-7. Similarly, in E. coli AMPP toward ethylphosphonates 14-17, the results indicate that the regions of both MeO moiety and P=O ligand may be located in the vicinity of the substrate-binding site, which have not been altered within the active site of enzyme upon mutation of Trp88, Arg153, and Arg370. These studies demonstrate that E. coli AMPP and hcAMPP display different substrate preference toward organophosphorus compounds. Evidence here, therefore, represents the first example of hcAMPP that might serve as a valuable bioscavenger candidate as E. coli AMPP due to the promise from the hydrolysis of these toxic chemicals.     

Physicochemical evaluation of a stability-driven approach to drug entrapment in regular and in surface-modified liposomes

The traditional mode of encapsulating drugs in liposomes poses risks to drug stability, especially when recognition agents are attached to the liposomal surface to obtain targeted liposomes. To reduce such risks, we devised a simple, novel method to entrap drugs in liposomes, consisting of (i) preparation and lyophilization of drug-free regular and surface-modified liposomes and (ii) drug encapsulation in the course of liposome reconstitution through rehydration in an aqueous solution of the drug. In this paper, we report physicochemical studies in which we compared regular and surface-modified liposomes made by this novel approach (denoted N-liposomes) to respective liposomes made by the traditional mode (denoted T-liposomes). The studies were performed with fluorescein, sucrose, histidine, mitomycin C (MMC), and chloramphenicol (CAM) encapsulated (each) in regular and in bioadhesive liposomes, the latter having hyaluronic acid as the surface-bound ligand. Our major findings are as follows: (1) The drug-specific encapsulation efficiencies spanning the range of 10-90% were, excepting sucrose, either similar in the N- and T-liposomes or better in the N- than in the T-liposomes, for both regular and bioadhesive liposomes. (2) For all liposome types and methods of preparation, fluorescein, histidine, and MMC did not adsorb to the liposomal surface. Sucrose and MMC did adsorb to the liposomal surface irrespective of the liposome preparation mode, sucrose favoring bioadhesive over regular liposomes and MMC having the opposite trend. (3) For both regular and bioadhesive liposomes, the mechanism of drug efflux from the N-liposomes was found to be governed by a single rate constant, as previously found for the T-liposomes. The magnitudes obtained, ranging from 3.5(+/-0.2) x 10(-3) to 400(+/-17) x 10(-3) h(-1), were always drug specific and occasionally also liposome type (i.e., regular or bioadhesive) specific. For MMC and CAM, the novel approach rendered liposomes with improved sustained release. The results reported here attest, overall, to the potential of this novel approach, meriting further investigations. Studies currently underway with MMC indicate N-liposomes also have functional advantages.     

Efficiency of cytoplasmic delivery by non-cationic liposomes to cells in vitro: A confocal laser scanning microscopy study

It is necessary to understand liposomal uptake mechanisms and intracellular distribution in order to design more efficient gene (drug) carrier systems. Until now, a few studies have been carried out using confocal laser scanning microscopy (CLSM) to investigate the cellular uptake and transfection mediated with liposomes. So, by CLSM, we demonstrated that artificial virus-like envelope (AVE) vesicles labeled with rhodamine-PE (Rh-PE), carbocyanine (DiI) and carboxyfluorescein (CF) were investigated into the cytoplasm of two human cell lines, Mewo (human melanoma cell line) and HepG2 (human hepatoma cell line) cells grown in DMEM medium supplemented with different percentages (0%, 30%, and 100%) fetal calf serum (FCS). The liposome uptake was dependent on the cell line, in view that the whole process of liposomes associated with cells (uptake) is a two-step process involving binding and endocytosis. Based upon the various assays used to measure cellular uptake of liposomes, we conclude the efficacy of cytoplasmic delivery by AVE-liposomes to cells in culture.     

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.     

Targeting of liposomes by covalent coupling with ecto-NAD+-glycohydrolase ligands

We have tested the feasibility of targeting liposomes via interaction with specific ecto-enzymes, i.e., enzymes which have their active site oriented to the external surface of the cell. 3,4-Dimethylpyridine adenine dinucleotide, a competitive inhibitor of ecto-NAD⁺-glycohydrolase, was substituted at N⁶ with a hydrophilic spacer arm, functionalized with a sulfhydryl group, and covalently linked to preformed liposomes containing 4-(p-maleimidophenyl)butyryl phosphatidylethanolamine. We show that compared to control vesicles, the binding of the conjugated liposomes was greatly increased (up to 5-fold) to cells presenting ecto-NAD⁺-glycohydrolase activity (Swiss 3T3 fibroblasts, mouse peritoneal macrophages); in contrast, no specific binding was detected with hepatoma tissue culture cells, which lack this enzyme. Specific binding was found to depend on the ligand/lipid molar ratio of the vesicles and on the length of the arm. High concentrations of free 3,4-dimethylpyridine adenine dinucleotide virtually abolished the specific binding to cells of the targeted liposomes. Analysis of binding revealed that the ligand conjugated to the liposomes presented a functional affinity for 3T3 fibroblasts 15-fold superior to that of the free ligand.     

Antigen delivery to macrophages using liposomal nanoparticles targeting sialoadhesin/CD169

Sialoadhesin (Sn, Siglec-1, CD169) is a member of the sialic acid binding Ig-like lectin (siglec) family expressed on macrophages. Its macrophage specific expression makes it an attractive target for delivering antigens to tissue macrophages via Sn-mediated endocytosis. Here we describe a novel approach for delivering antigens to macrophages using liposomal nanoparticles displaying high affinity glycan ligands of Sn. The Sn-targeted liposomes selectively bind to and are internalized by Sn-expressing cells, and accumulate intracellularly over time. Our results show that ligand decorated liposomes are specific for Sn, since they are taken up by bone marrow derived macrophages that are derived from wild type but not Sn(-/-) mice. Importantly, the Sn-targeted liposomes dramatically enhance the delivery of antigens to macrophages for presentation to and proliferation of antigen-specific T cells. Together, these data provide insights into the potential of cell-specific targeting and delivery of antigens to intracellular organelles of macrophages using Sn-ligand decorated liposomal nanoparticles.     

Cyclooxygenase inhibition by diclofenac formulated in bioadhesive carriers

Adverse effects and gastrointestinal toxicity limit the use of Diclofenac, a frequently-used NSAID for treatments of rheumatic disorders and other chronic inflammatory diseases. Diclofenac-carrier formulations may alleviate adverse effects, increase efficacy and allow local administration. We report here our first step, biophysical and biochemical investigations of Diclofenac formulated in our previously-developed bioadhesive liposomes carrying hyaluronan (HA-BAL) or collagen (COL-BAL) on their surface. Both liposome types encapsulated Diclofenac at high efficiency, encapsulated doses reaching 13 mg drug/ml, and performed as sustained-release Diclofenac depots, half-lives of drug release (under fastest conditions) ranging from 1 to 3 days. Therapeutic activity of liposomal Diclofenac was evaluated in CT-26 cells that possess the CD44 hyaluronan receptors and integrins, and are a bench-mark for intracellular COX enzymes. HA-BAL and COL-BAL showed high cellular-affinity that was 40 fold and 6 fold over that of regular liposomes. Free, and liposome-encapsulated, Diclofenac showed similar activities. For example: 2-3nM Diclofenac given to intact cells generated COX-inhibition levels in the range of 60-70% for free drug and for encapsulated drug in COL-BAL and in HA-BAL. We propose these novel Diclofenac formulations possess key physicochemical and biochemical attributes for task performance, meriting the next step into in vivo studies.     

Cyclooxygenase inhibition by diclofenac formulated in bioadhesive carriers

Adverse effects and gastrointestinal toxicity limit the use of Diclofenac, a frequently-used NSAID for treatments of rheumatic disorders and other chronic inflammatory diseases. Diclofenac-carrier formulations may alleviate adverse effects, increase efficacy and allow local administration. We report here our first step, biophysical and biochemical investigations of Diclofenac formulated in our previously-developed bioadhesive liposomes carrying hyaluronan (HA-BAL) or collagen (COL-BAL) on their surface. Both liposome types encapsulated Diclofenac at high efficiency, encapsulated doses reaching 13mg drug/ml, and performed as sustained-release Diclofenac depots, half-lives of drug release (under fastest conditions) ranging from 1 to 3days. Therapeutic activity of liposomal Diclofenac was evaluated in CT-26 cells that possess the CD44 hyaluronan receptors and integrins, and are a bench-mark for intracellular COX enzymes. HA-BAL and COL-BAL showed high cellular-affinity that was 40 fold and 6 fold over that of regular liposomes. Free, and liposome-encapsulated, Diclofenac showed similar activities. For example: 2-3nM Diclofenac given to intact cells generated COX-inhibition levels in the range of 60-70% for free drug and for encapsulated drug in COL-BAL and in HA-BAL. We propose these novel Diclofenac formulations possess key physicochemical and biochemical attributes for task performance, meriting the next step into in vivo studies.     

Development of the novel PEG-PE-based polymer for the reversible attachment of specific ligands to liposomes: synthesis and in vitro characterization

Surface grafting of liposomes with the wide variety of ligands including antibodies and other proteins is a promising approach for targeted delivery of therapeutics. In this paper, we describe a simple method of synthesizing a hydrazine-functionalized poly(ethylene glycol)-phosphatidylethanolamine (PEG-PE)-based amphiphilic polymer which can conjugate a variety of ligands via a reversible, pH-cleavable bond. In this method, the targeting ligand is attached to the distal end of the PEG chain, which facilitates its easy access to the targeted site of interaction. The reversible attachment of targeting ligands is useful especially in multifunctional liposomal systems, whereafter successfully performing the function of targeting to the specific site, the bulky ligands, such as proteins or antibodies, are cleaved off in response to an environmental stimulus to expose some other functionalities such as ligands for intracellular penetration or organelle-specific targeting. To investigate the applicability of the protocol, the model ligands monoclonal antinucleosome antibody 2C5 and antimyosin antibody 2G4, and glycoproteins concanavalin A (Con-A) and avidin were conjugated to the synthesized polymer and incorporated into liposomes. In vitro assays including biochemical, enzyme-linked immunosorbent, fluorescence microscopy, and flow cytometry were used to confirm three key characteristics of the modified and/or liposome-attached proteins: successful conjugation of the targeting ligands to the polymer, preservation of specific activity of the ligands after the conjugation and liposome attachment, and the facile pH-sensitive ligand detachment. Monoclonal antibody 2C5 and 2G4, immobilized on the liposome surface, retained their binding affinity to corresponding antigens as confirmed by ELISA. The Con A-bearing liposomes showed significantly higher agglutination in the presence of its substrate mannan compared to plain liposomes (PL) and avidin-functionalized liposomes bound specifically with biotin-agarose. The study on the pH-dependence showed that almost 80% of the hydrazone bond was cleaved after rather brief preincubation of the immunoliposomes at pH 5 for 0.5 to 1 h. Fluorescence microscopy and flow cytometry analysis of cancer cells (HeLa and MCF-7) treated with cancer cell-specific targeting ligand mAb 2C5-bearing liposomes showed enhanced cellular binding. Studies at low pH clearly confirmed the easy cleavability of the targeting ligand from the liposomes resulting in significantly less or virtually no cellular association.     

Effect of sialyl Lewis X-glycoliposomes on the inhibition of E-selectin-mediated tumour cell adhesion in vitro

The aim of this study was to evaluate the potential of different types of sialyl Lewis X-conjugated liposomes as competitive inhibitors for tumour cell adhesion to endothelial E-selectin. Sterically stabilised liposomes with the sLeX ligand at the terminal end of the polyethyleneglycol (PEG) chain, as well as vesicles that had the ligand embedded within the PEG-layer, were compared to ligand-bearing liposomes without sterical stabilisation. First, 14 different tumour cell lines were characterised for their expression of sialyl Lewis X and/or A. Tumour cell adhesion was characterised in three static assays in vitro using: (i) immobilised E-selectin, (ii) CHO cells, transfected to express E-selectin and (iii) human umbilical vein endothelial cells (HUVEC). Sterically stabilised liposomes with the ligand at the terminal end of the polyethylene chain were the most effective inhibitors in all three assays and inhibited the adhesion of HT29 colon- and Lewis lung (LL) carcinoma cells by about 60-80%. The binding was not affected by a PEG-coating of the liposomes. Sterical stabilisation, on the other hand, completely prevented macrophage uptake (J774 cell line) independently of the presence of the ligand, while plain liposomes were taken up in an amount of 5.4 nmol liposomal lipids/10(6) macrophages.     

Probing the acyl binding site of acetylcholinesterase by protein engineering

Recombinant acetylcholinesterase from rat brain and two mutants were studied for their hydrolytic activity toward acetyl- and butyrylthiocholine substrates and for their sensitivity toward organophosphate and carbamate inhibitors. Both mutants, a point mutant where F295 was replaced by leucine, and a second mutant where loop PQES was replaced by SG, were designed for increased size of the acyl binding pocket. Wild type and mutant enzymes were expressed in baculovirus-infected insect cells and biochemically characterized. As expected, wild type rat brain acetylcholinesterase hydrolyzed acetylthiocholine, but not butyrylthiocholine. Sensitivity toward small- and medium-sized organophosphate inhibitors like paraoxon-methyl and paraoxon-ethyl was comparable, but bulky organophosphates like ethoprophos were less efficient inhibitors. This tendency applied to carbamates as well, since small carbamoyl moieties like carbofuran and aldicarb were stronger inhibitors than furathiocarb which features a bulky carbamoyl moiety. In contrast to wild type enzyme, both mutants were capable of hydrolyzing butyrylthiocholine. However, k_cat/K_m toward acetylthiocholine of the F295L mutant was reduced if compared to the wild type enzyme. All five organophosphate and three carbamate inhibitors inhibited mutant F295L more efficiently than the wild type enzyme.     

Biosensor system for continuous monitoring of organophosphate aerosols

An enzyme-based monitoring system provides the basis for continuous sampling of organophosphate contamination in air. The enzymes butyrylcholinesterase (BuChE) and organophosphate hydrolase (OPH) are stabilized by encapsulation in biomimetic silica nanoparticles, entrained within a packed bed column. The resulting immobilized enzyme reactors (IMERs) were integrated with an impinger-based aerosol sampling system for collection of chemical contaminants in air. The sampling system was operated continuously and organophosphate detection was performed in real-time by single wavelength analysis of enzyme hydrolysis products. The resulting sensor system detects organophosphates based on either enzyme inhibition (of BuChE) or substrate hydrolysis (by OPH). The detection limits of the IMERs for specific organophosphates are presented and discussed. The system proved suitable for detection of a range of organophosphates including paraoxon, demeton-S and malathion.     

Development of a liposomal delivery system for temperature-triggered release of a tumor targeting agent, Ln(III)-DOTA-phenylboronate

Liposomes, capable of temperature-triggered content release at the site of interest, can be of great importance for imaging and therapy of tumors. The delivery of imaging agents or therapeutics can be improved by application of liposomes with a gel-to-liquid phase-transition temperature suitable for mild hyperthermia (41-43 °C), and by prolonging their circulation time by incorporation of lipids containing polyethyleneglycol moieties. Still, the rapid wash out of the delivered material from the tumor tissue is a major obstacle for both imaging and therapy. In this study, we developed an optimized temperature sensitive liposomal system to be used with mild hyperthermia: highly stable at physiological temperature and with a sharp transition of the bilayer at 41.5 °C, with subsequent rapid release of entrapped compounds such as calcein or tumor cell-targeting contrast agents. Intravital microscopy on calcein/rhodamine containing liposomes was applied to demonstrate the applicability of this system in vivo. The calcein loaded liposomes were injected iv into nude mice with a human BLM melanoma tumor implanted in a dorsal skin-fold window chamber. Arrival of the liposomes at the tumor site and content release after temperature increase were monitored. The results demonstrated not only accumulation of the liposomes at the tumor site, but also a massive release of calcein after increase of the temperature to 41 °C. The versatility of the thermosensitive liposomes was further demonstrated by encapsulation of a tumor cell-targeting DOTA-phenylboronate conjugate and its release at elevated temperatures. The DOTA ligand in this system is able to chelate a variety of metals suitable for both diagnostic and therapeutic applications, whereas the phenylboronate function is able to target specifically to tumor cells through a covalent binding with sialic acid moieties over-expressed on their surface upon heat-triggered release from the liposomal carrier.     

Why acetylcholinesterase reactivators do not work in butyrylcholinesterase

The pyridinium oxime therapy for treatment of organophosphate poisoning is a well established, but not sufficient method. Recent trends also focus on prophylaxis as a way of preventing even the entrance of organophosphates into the nervous system. One of the possible prophylactic methods is increasing the concentration of butyrylcholinesterase in the blood with the simultaneous administration of butyrylcholinesterase reactivators, when the enzyme is continuously reactivated by oxime. This article summarizes and sets forth the structural differences between butyrylcholinesterase and acetylcholinesterase, essential for the future design of butyrylcholinesterase reactivators. Butyrylcholinesterase lacks the reactivator aromatic binding pocket found in acetylcholinesterase, which is itself a part of the acetylcholinesterase peripheral anionic site. This difference finally renders the current acetylcholinesterase reactivators, when used in butyrylcholinesterase, non-functional.     

Hyaluronic acid modified pH-sensitive liposomes for targeted intracellular delivery of doxorubicin

Context: Surface-modified pH-sensitive liposomal system may be useful for intracellular delivery of chemotherapeutics.

Objective: Achieving site-specific targeting with over-expressed hyaluronic acid (HA) receptors along with using pH sensitive liposome carrier for intracellular drug delivery was the aim of this study.

Materials and methods: Stealth HA-targeted pH-sensitive liposomes (SL-pH-HA) were developed and evaluated to achieve effective intracellular delivery of doxorubicin (DOX) vis–a-vis enhanced antitumor activity.

Results: The in vitro release studies demonstrated that the release of DOX from SL-pH-HA was pH-dependent, i.e. faster at mildly acidic pH ～5, compared to physiological pH ～7.4. SLpH-HA was evaluated for their cytotoxicity potential on CD44 receptor expressing MCF-7 cells. The half maximal inhibitory concentration (IC50) of SL-pH-HA and SL-HA were about 1.9 and 2.5?μM, respectively, after 48?h of incubation. The quantitative uptake study revealed higher localization of targeted liposomes in the receptor positive cells, which was further confirmed by fluorescent microscopy. The antitumor efficacy of the DOX-loaded HA-targeted pH-sensitive liposomes was also verified in a tumor xenograft mouse model.

Discussion: DOX was efficiently delivered to the tumor site by active targeting via HA and CD44 receptor interaction. The major side-effect of conventional DOX formulation, i.e. cardiotoxicity was also estimated by measuring serum enzyme levels of LDH and CPK and found to be minimized with developed formulation. Overall, HA targeted pH-sensitive liposomes were significantly more potent than the non-targeted liposomes in cells expressing high levels of CD44.

Conclusion: Results strongly implies the promise of such liposomal system as an intracellular drug delivery carrier developed for potential anticancer treatment.     

Properties of stearylamine liposomes containing tyrosine phenol-lyase

The activity of tyrosine phenol-layse a chemotherapeutic enzyme with a dissociable pyridoxal phosphate cofactor, was studied after incorporation into multilamellar positively charged liposomes. Tyrosine phenol-lyase activity was assessed in the presence and absence of exogenous pyridoxal phosphate. A maximum of 75% total enzyme activity was associated with liposomes when prepared from a molar lipid ratio of egg lecithin, cholesterol, stearylamine (7 : 2 : 1, w/w). The total tyrosine phenol-lyase activity was comprised of 25% membrane-associated enzyme and 50% encapsulated enzyme. Encapsulation increased the stability of the enzyme under the in vitro conditions of cold storage at 4°C for 3 weeks and under elevated temperatures up to 61°C. Liposomal encapsulation afforded little protection against trypsin and no protection against whole mouse plasma in vitro. Heat-treated plasma (100°C for 1 h) had little effect on the activity of free and encapsulated tyrosine phenol-lyase. These results indicated that whole plasma contained a heat-labile factor(s) which destroyed both the liposomal and free tyrosine phenol-lyase activity. Plasma clearance after intraperitoneal injection of tyrosine phenol-lyase in B6D2F₁ female mice was reduced by liposomal encapsulation, particularly when the animals were pre-treated with empty liposomes; however, only a small proportion of free and liposomal tyrosine phenol-lyase was absorbed. The free enzyme rapidly lost holoenzyme activity after absorption but the liposomes maintained holoenzyme activity. Even though liposomes preserved holo-tyrosine phenol-lyase activity, the holoenzyme was not present in sufficient concentration to sustain a reduced plasma tyrosine level.     

Activity of bile-salt-stimulated human milk lipase in the presence of liposomes and mixed taurocholate-phosphatidylcholine micelles

(1) The interaction of bile-salt-stimulated human milk lipase and liposomal membranes has been investigated in the presence or absence of sodium taurocholate. Freshly purified enzyme enhances the permeability of liposomal membranes but thermally inactivated enzyme does not. (2) The ability of the enzyme to catalyze the hydrolisys of a relatively hydrophilic substrate, 4-nitrophenyl acetate, and a more hydrophobic substrate, 4-nitrophenyl palmitate, has also been measured in media containing small unilamelar vesicles of egg phosphatidylcholine in both the absence and presence of taurocholate, and also in the presence of free taurocholate in the absence of liposomes. (3) The enzyme-catalyzed hydrolysis of 4-nitrophenyl acetate is enhanced in all of these systems, but 4-nitrophenyl palmitate is protected from enzymic attack in the phosphatidylcholine-bile salt systems. If free taurocholate be present in the system before 4-nitrophenyl palmitate is added, then, and only then, is enzymic activity observed. (4) These results have been interpreted in terms of the importance of the microenvironment around the substrate and the role played by the bile salt surfactant in stimulating the enzyme.     

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.