Lipophilic Interactions Research Articles

Lipophilic and electrostatic forces encoded in IAM-HPLC indexes of basic drugs: Their role in membrane partition and their relationships with BBB passage data

The membrane phospholipid affinity data, logkwIAM, for 14 basic drugs spanning a wide lipophilicity range were measured by HPLC on two different phospholipid stationary phases, i.e. IAM.PC.MG and IAM.PC.DD2. These data related weakly with logPN values, the n-octanol/water partition coefficients of the neutral forms; poorer relationships were found with logD7.0 values, the n-octanol/water partition coefficients of the mixtures of neutral and ionized forms at pH 7.0. The lack of collinearity confirms that, differently from partition in n-octanol/water, partition in phospholipids encodes not only lipophilic/hydrophobic intermolecular recognition forces but also ionic bonds, due to electrostatic interactions between electrically charged species and phospholipids, according to the “pH-piston hypothesis”. This component of interaction was parameterized by ΔlogkwIAM values; they are the differences between the logkwIAM values experimentally measured and the values expected for neutral isolipophilic compounds. ΔlogkwIAM values of the various analytes changed almost linearly from positive to negative values at increasing lipophilicity. This behavior is consistent with an interaction mechanism with membrane phospholipids including two intermolecular interaction forces: (i) lipophilic/hydrophobic interactions, which decrease on ionization proportionally to the lipophilicity of the neutral forms, and (ii) electrostatic interactions, which increase on ionization and are quite constant for all the analytes at a given ionization degree. Since BBB passage of the considered compounds is supposed to be based on passive mechanisms, we investigated the possible relationships between log BB values, i.e. the logarithms of the ratio between brain and blood concentrations, and three physico-chemical parameters, i.e. (i) logPN (lipophilic interaction of the neutral form), (ii) logkwIAM (global interaction with phospholipids), and (iii) ΔlogkwIAM (electrostatic component of interaction with phospholipids). The results suggest that the electrostatic interactions encoded in logkwIAM values might act as trapping forces in a phospholipid barrier. Actually, we observed an inverse linear dependence of log BB on ΔlogkwIAM values, but only for the compounds showing positive ΔlogkwIAM values. We conclude that the driving force for BBB passage is the lipophilicity of the neutral forms, logPN, and not the lipophilicity actually displayed at the experimental pH, logD7.0. Indeed, the latter does not adequately take into account the role played by protonation in the analyte/membrane interactions because protonation, although hindering membrane passage, can either reduce or enhance partition in phospholipids, depending on analyte lipophilicity.

Lipophilic and cationic triphenylphosphonium grafted linear polyethylenimine polymers for efficient gene delivery to mammalian cells

Synthetic chemical vectors have recently provided a versatile and robust platform for the safe and efficient delivery of exogenous genes. Here, for the first time, a small series of N-butyltriphenylphosphonium bromide-grafted-linear polyethylenimine (BTP-g-lP) polymers (N–P hybrid polymers) have been evaluated for their ability to deliver genes into mammalian cell lines, viz., MCF-7 and A549 cells. Biophysical characterization revealed that the projected polymers efficiently interacted with plasmid DNA, and the resulting complexes displayed hydrodynamic diameters in the range of 249–307 nm with relatively higher zeta potential values of +31 to +34 mV (cf. lPEI, +26 mV). The tethering of lipophilic and cationic triphenylphosphonium moieties to linear PEI (lPEI) addressed several limitations associated with lPEI, such as solubility, the stability of the pDNA complexes and the timely release of pDNA for nuclear localization as assessed by protection and release assays. Also, the lipophilic interactions between cellular membranes and the pDNA complexes mediated the efficient cellular uptake and internalization of the pDNA complexes, resulting in significantly higher transfection efficiency in these cell lines, outperforming the GenePORTER 2™, Lipofectamine™ and Superfect™ used in the study for comparison purposes. Confocal studies using dual-labeled TMR-BTP-g-lP3/YOYO-1-pDNA complex in MCF-7 cells confirmed that the complex behaved more or less like native lPEI, as the substitution of the phosphonium moiety was too small to affect the intracellular trafficking. Furthermore, the versatility of the BTP-g-lP3 vector was established by GFP specific siRNA delivery, which resulted in ∼79% suppression of targeted gene expression (cf. Lipofectamine™, ∼55%). Altogether, the study demonstrates the potential of these hybrid polymers for the efficient delivery of nucleic acids for future gene therapy applications.

Vitamin A Metabolism: Challenges and Perspectives

Vitamins set an unprecedented example of the essential environment-gene interactions that are necessary to sustain life. Seventeen Nobel prizes were awarded in the last century for discoveries related to vitamins’ structures, physiology, and functions [1]. These discoveries help to abolish diseases related to vitamin deficiencies in the developed world and offer solutions to eradicate these disorders worldwide [2]. For example, embryonic malformations, night blindness, and immune deficiency in children were effectively treated by vitamin A supplementation [2,3]. In spite of encouraging results in vitamin-deficient subjects, wide applications of vitamin supplements in the nutrient-sufficient populations have been partially discouraging. Many clinical and supplementation trials reported increased mortality in subjects on long-term lipophilic vitamin A and E supplementations [4,5]. Especially striking deleterious effects were reported for lipophilic vitamin A and provitamin A (β-carotene) [4,6]. The answer to safe application of lipophilic vitamins for the treatment and prevention of diseases may lie in the better understanding of their interaction with genes. There are two principal levels of lipophilic vitamin interactions with genes: 1) genes control metabolism of dietary vitamin into derivatives with hormone-like properties, and 2) vitamin-derived metabolites regulate specific gene programs through signaling and transcription pathways (Figure1).

Excitation energy transfer from non-aggregated molecules to perylenediimide nanoribbons via ionic interactions in water

Two energy donor–acceptor self-assembly systems have been constructed by using π–π, lipophilic, and ionic interactions in water. π-stacked N,N′-ditridecylperylenediimide (PDI), which forms nanoribbons, has been dispersed in water in the presence of myristyltrimethylammonium bromide (MTAB) through lipophilic interactions of tridecyl groups of PDIs with long tails of MTAB molecules. Cationic heads of MTAB molecules, anchored on the bulk of the side-chains of the nanoribbons, attract water-soluble zinc tetra(4-sulfonatophenyl)porphyrin tetrapotassium salt (ZnTPPSK4) and lucifer yellow CH dipotassium salt (LY). By this design, efficient photosensitization of non-aggregated energy donors, ZnTPPSK4 and LY, has been achieved while retaining the one-dimensional order at nanoscale, resulting in the efficient excitation energy transfer to PDI nanoribbons in each system.

Design of 7-amino-6-chloro-3H-imidazo[4,5-b]pyridine scaffold from 5-chloro-2,4-diaminopyrimidine pharmacophore: identification of potent inhibitors of anaplastic lymphoma kinase

A series of potent anaplastic lymphoma kinase (ALK) inhibitors based on a 7-amino-6-chloro-3H-imidazo[4,5-b]pyridine scaffold were identified through rational design from a 5-chloro-2,4-diaminopyrimidine pharmacophore, maintaining key binding elements, favourable lipophilic interactions and orienting the side chains into favoured trajectories. Importantly, potency and selectivity determinants from the parent series were directly applicable to the new scaffold. This highly focused strategy led to the identification of several lead inhibitors that displayed potent activity in enzyme and cellular assays as well as pronounced oral bioavailability.

Editorial [Hot Topic: Lipophilic and Amphiphilic Interactions in Glycoprotein 41:Targets for Therapy (Guest Editor: Miriam Gochin)

Editorial [Hot Topic: Lipophilic and Amphiphilic Interactions in Glycoprotein 41:Targets for Therapy (Guest Editor: Miriam Gochin)

Solubility improvement of an anthelmintic benzimidazole carbamate by association with dendrimers

The improvement of aqueous solubility of methyl (5-[propylthio]-1H-benzimidazol-2-yl) carbamate, albendazole (ABZ) using polyamidoamine (PAMAM) dendrimers as solubility enhancers was investigated. Full generation PAMAM dendrimers with amine terminal groups, (G3), with hydroxyl terminal groups (G3OH) and half generation PAMAM dendrimers with carboxylate terminal groups (G2.5 and G3.5), were chosen for this study. The nature of dendrimer-ABZ association was investigated by UV absorption, fluorescence emission measurements and by ¹H-NMR spectroscopy. The results obtained show that these polymeric structures have the capacity to enhance the solubility of ABZ, both lipophilic and specific hydrogen bond interactions contributing to the guest-host association. Although all studied dendrimers have hydrophobic internal nanoenvironments with similar dimensions, their surfaces differ significantly and the nature and the localization of the interactions involved in ABZ-dendrimer association depend on the type of terminal groups.

Metabolism-Directed Design of Oxetane-Containing Arylsulfonamide Derivatives as γ-Secretase Inhibitors

A metabolism-based approach toward the optimization of a series of N-arylsulfonamide-based γ-secretase inhibitors is reported. The lead cyclohexyl analogue 6 suffered from extensive oxidation on the cycloalkyl motif by cytochrome P450 3A4, translating into poor human liver microsomal stability. Knowledge of the metabolic pathways of 6 triggered a structure-activity relationship study aimed at lowering lipophilicity through the introduction of polarity. This effort led to several tetrahydropyran and tetrahydrofuran analogues, wherein the 3- and 4-substituted variants exhibited greater microsomal stability relative to their 2-substituted counterparts. Further reduction in lipophilicity led to the potent γ-secretase inhibitor and 3-substituted oxetane 1 with a reduced propensity toward oxidative metabolism, relative to its 2-substituted isomer. The slower rates of metabolism with 3-substituted cyclic ethers most likely originate from reductions in lipophilicity and/or unfavorable CYP active site interactions with the heteroatom. Preliminary animal pharmacology studies with a representative oxetane indicate that the series is generally capable of lowering Aβ in vivo. As such, the study also illustrates the improvement in druglikeness of molecules through the use of the oxetane motif.

Combination of Two Pharmacophoric Systems: Synthesis and Pharmacological Evaluation of Spirocyclic Pyranopyrazoles with High σ1 Receptor Affinity

The novel class of spirocyclic σ(1) ligands 3 (6',7'-dihydro-1'H-spiro[piperidine-4,4'-pyrano[4,3-c]pyrazoles]) was designed by the combination of the potent σ(1) ligands 1 and 2 in one molecule. Thorough structure affinity relationships were derived by the variation of the substituents in position 1', 1, and 6'. Whereas the small electron rich methylpyrazole heterocycle was less tolerated by the σ(1) receptor protein, the introduction of a phenyl substituent instead of the methyl group led to ligands with a high σ(1) affinity. It is postulated that the additional phenyl substituent occupies a previously unrecognized hydrophobic region of the σ(1) receptor resulting in additional lipophilic interactions. The spirocyclic pyranopyrazoles are very selective against the σ(2) subtype, the PCP binding site of the NMDA receptor, and further targets. Despite high σ(1) affinity, the cyclohexylmethyl derivative 17i (K(i) (σ(1)) = 0.55 nM) and the isopentenyl derivative 17p (K(i) (σ(1)) = 1.6 nM) showed only low antiallodynic activity in the capsaicin assay.

Molecular docking studies of 1-(substituted phenyl)-3-(naphtha [1, 2-d] thiazol-2-yl) urea/thiourea derivatives with human adenosine A(2A) receptor.

Computational assessment of the binding interactions of drugs is an important component of computer-aided drug design paradigms. In this perspective, a set of 30 1-(substituted phenyl)-3-(naphtha[1, 2-d] thiazol-2-yl) urea/thiourea derivatives showing antiparkinsonian activity were docked into inhibitor binding cavity of human adenosine A(2A) receptor (AA2AR) to understand their mode of binding interactions in silico. Lamarckian genetic algorithm methodology was employed for docking simulations using AutoDock 4.2 program. The results signify that the molecular docking approach is reliable and produces a good correlation coefficient (r(2) = 0.483) between docking score and antiparkinsonian activity (in terms of % reduction in catalepsy score). Potent antiparkinsonian agents carried methoxy group in the phenyl ring, exhibited both hydrophilic and lipophilic interactions with lower energy of binding at the AA(2A)R. These molecular docking analyses should, in our view, contribute for further development of selective AA(2A)R antagonists for the treatment of Parkinson's disease.

Modification of uptake and subcellular distribution of doxorubicin by N-acylhydrazone residues as visualised by intrinsic fluorescence

Doxorubicin (1) is commonly used in the treatment of a wide range of cancers. Some N-acylhydrazones of 1 were previously found to have an improved tumour and organ selectivity. In order to clarify the molecular basis for this effect, the cellular uptake into various cancer cells and the localisation in PtK(2) potoroo kidney cells of 1 and its N-acylhydrazones derived from heptadecanoic acid (2) and 11-(menthoxycarbonyl)undecanoic acid (3) were studied drawing on their intrinsic fluorescence. The uptake of compounds 1-3 into human cells of HL-60 leukaemia, 518A2 melanoma, HT-29 colon, and resistant KB-V1/Vbl and MCF-7/Topo breast carcinomas was determined fluorometrically from their residual amounts in the supernatant. Their time-dependent accumulation in PtK(2) potoroo kidney cells was visualised by fluorescence microscopy. The uptake, though not the cytotoxicity, of 2 in multi-drug resistant MCF-7/Topo breast cancer cells was conspicuously greater than that of 1 and 3, probably due to an attractive lipophilic interaction with the lipid-rich membranes of these cells. In non-malignant PtK(2) cells, both 1 and 3 accumulated initially in the nuclei. Upon prolonged incubation, their fluorescent metabolites were visualised in lysosomes neighbouring the nuclei. In contrast, conjugate 2 was not observed in the nuclei at any time. After 2h, it had accumulated in vesicles scattered all over the cells, and upon prolonged incubation, its fluorescent metabolites were concentrated in the cellular membrane. Long unbranched fatty acyl residues when attached to doxorubicin via a hydrazone can act as lipophilic membrane anchors. This allows an increased uptake of such derivatives into lipid-rich membranes especially of multi-drug resistant cancer cells, a retarded release from there into the cytosol and the eventual storage of their metabolites again in the cell membrane rather than in lysosomes.

Comparison of the polymorphs and solvates of two analogous fungicides—a case study of the applicability of a supramolecular synthon approach in crystal engineering

The polymorphism and solvate formation of thiophanate-ethyl (TE), a fungicidal active, were investigated by solvent crystallization and compared to a close analogue, thiophanate-methyl (TM). Four polymorphs and seven solvates of TE were found and structurally compared with the previously found two polymorphs and fourteen solvates of TM by analyzing the hydrogen bonding patterns and using fingerprint plots, packing coefficients and lattice energies. TE and TM have the same functional groups that can build identical supramolecular synthons. Despite the strong similarities, the polymorphs and solvates of the two actives show significant differences in hydrogen bonding and packing. The results demonstrate the challenges in using a supramolecular synthon approach, and promote the importance in finding methods to also make use of packing effects and lipophilic interactions in crystal engineering.

Thermotropic liquid crystals based on 1,8,9,16-tetrasubstituted tetraphenylenes and their structure–property relationship studies

Twelve 1,8,9,16-tetraalkoxytetraphenylenes were synthesized and characterized, five of which exhibiting thermotropic liquid crystal properties were examined by differential scanning calorimetry (DSC), polarizing-light optical microscopy (POM) and variable-temperature X-ray diffractometry (VTXRD). The structure–property relationship of each mesomorphic solid is discussed in the context of the racemic/non-racemic nature of the sample, different chain length of n-alkyl substituents, effect of chain lipophilicity and intermolecular interactions.

The pharmacophore of thapsigargin

A derivative of thapsigargin (1) is currently in clinical phase 1 trials for treatment of prostate, bladder, and breast cancer. Thapsigargin has been targeted towards solid tumours by coupling the natural product to a peptide, which selectively is cleaved by enzymes present in excess in the cell walls of the blood vessels nourishing solid tumours. Human kallikrein 2 is an example of such a proteolytic enzyme [1]. The cytoxicity of thapsigargin relates to the ability to inhibit the sarco- or endoplasmic Ca2+ ATPase (SERCA). A pharmacophore model suggest that lipophilic interactions between the acyl groups at O-3, O-8, O-10 and CH3–15 are of major importance for the affinity to the binding cavity. Analysis of the X-ray structure of the complex of thapsigargin and SERCA [2] reveals that the segment F(834)FRY(837) andthe segment A(306)IPEGL(311) form the sides of the cavity.

Corrections to A Medicinal Chemist’s Guide to Molecular Interactions

Page 5063. The first sentence of the second paragraph in the left column should be replaced by the following: “In an impressively comprehensive study on several series of thrombin inhibitors, the Hangauer and Klebe groups have analyzed the sources of cooperativity between a lipophilic interaction and a hydrogen bond”.30,31 Page 5075. Figure 16 caption should read as follows: “Radial distribution of carbon atoms around phenyl rings: (a) CH3 bound to O or N (CSD statistics); (b) CH3 bound to sp3 C (CSD statistics); (c) CH3 bound to O or N (PDB statistics). Darker gray corresponds to higher density; peaks above a numerical value of 90 are colored red”. Page 5083. Reference 159 should be replaced by the following: Manley P. W.; Cowan-Jacob S. W.; Fendrich G.; Mestan J.Molecular interactions between the highly selective pan-Bcr-Abl inhibitor, AMN107, and the tyrosine kinase domain of Abl. Blood 2005, 53, 940a−941a.

A Medicinal Chemist’s Guide to Molecular Interactions

Molecular recognition in biological systems relies on the existence of specific attractive interactions between two partner molecules. Structure-based drug design seeks to identify and optimize such interactions between ligands and their host molecules, typically proteins, given their three-dimensional structures. This optimization process requires knowledge about interaction geometries and approximate affinity contributions of attractive interactions that can be gleaned from crystal structure and associated affinity data. Here we compile and review the literature on molecular interactions as it pertains to medicinal chemistry through a combination of careful statistical analysis of the large body of publicly available X-ray structure data and experimental and theoretical studies of specific model systems. We attempt to extract key messages of practical value and complement references with our own searches of the CSDa,(1) and PDB databases.(2) The focus is on direct contacts between ligand and protein functional groups, and we restrict ourselves to those interactions that are most frequent in medicinal chemistry applications. Examples from supramolecular chemistry and quantum mechanical or molecular mechanics calculations are cited where they illustrate a specific point. The application of automated design processes is not covered nor is design of physicochemical properties of molecules such as permeability or solubility. Throughout this article, we wish to raise the readers’ awareness that formulating rules for molecular interactions is only possible within certain boundaries. The combination of 3D structure analysis with binding free energies does not yield a complete understanding of the energetic contributions of individual interactions. The reasons for this are widely known but not always fully appreciated. While it would be desirable to associate observed interactions with energy terms, we have to accept that molecular interactions behave in a highly nonadditive fashion.3,4 The same interaction may be worth different amounts of free energy in different contexts, and it is very hard to find an objective frame of reference for an interaction, since any change of a molecular structure will have multiple effects. One can easily fall victim to confirmation bias, focusing on what one has observed before and building causal relationships on too few observations. In reality, the multiplicity of interactions present in a single protein−ligand complex is a compromise of attractive and repulsive interactions that is almost impossible to deconvolute. By focusing on observed interactions, one neglects a large part of the thermodynamic cycle represented by a binding free energy: solvation processes, long-range interactions, conformational changes. Also, crystal structure coordinates give misleadingly static views of interactions. In reality a macromolecular complex is not characterized by a single structure but by an ensemble of structures. Changes in the degrees of freedom of both partners during the binding event have a large impact on binding free energy. The text is organized in the following way. The first section treats general aspects of molecular design: enthalpic and entropic components of binding free energy, flexibility, solvation, and the treatment of individual water molecules, as well as repulsive interactions. The second half of the article is devoted to specific types of interactions, beginning with hydrogen bonds, moving on to weaker polar interactions, and ending with lipophilic interactions between aliphatic and aromatic systems. We show many examples of structure−activity relationships; these are meant as helpful illustrations but individually can never confirm a rule.

Physicochemical interactions between drugs and superdisintegrants

Abstract We have evaluated the interactions between superdisintegrants and drugs with different physicochemical characteristics, which may affect the in-vivo absorption e.g. after mucosal administration. The binding of sodium salicylate, naproxen, methyl hydroxybenzoate (methylparaben), ethyl hydroxybenzoate (ethylparaben), propyl hydroxybenzoate (propylparaben), atenolol, alprenolol, diphenhydramine, verapamil, amitriptyline and cetylpyridinium chloride monohydrate (CPC) to different superdisintegrants (sodium starch glycolate (SSG), croscarmellose sodium (CCS) and crospovidone) and one unsubstituted comparator (starch) was studied spectrophotometrically. An indication of the in-vivo effect was obtained by measuring the interactions at physiological salt concentrations. SSG was investigated more thoroughly to obtain release profiles and correlation between binding and ionic strength. The results showed that the main interactions with the anionic hydrogels formed by SSG and CCS were caused by ion exchange, whereas the neutral crospovidone exhibited lipophilic interactions with the non-ionic substances. The effect of increased ionic strength was most pronounced at low salt concentrations and the ion exchange interactions were almost completely eradicated at physiological conditions. The release profile of diphenhydramine was significantly affected by the addition of salt. It was thus concluded that the choice of buffer was of great importance for in-vitro experiments with ionic drugs. At physiological salt concentrations the interactions did not appear to be strong enough to influence the in-vivo bioavailability of any of the drug molecules.

A multimedia activity model for ionizable compounds: Validation study with 2,4‐dichlorophenoxyacetic acid, aniline, and trimethoprim

Fugacity models are widely adopted for the environmental exposure assessment of organic chemicals but are inconvenient for nonvolatile substances, such as ionizable chemicals. The activity approach is a robust alternative to the fugacity concept and provides the thermodynamically exact equations to describe the behavior of neutral and ionizable molecules in nonideal systems. A multimedia activity model applicable to neutral and ionizable molecules (MAMI) was developed and tested for 2,4-dichlorophenoxyacetic acid and the bases aniline and trimethoprim. The model features pH and ionic strength dependence and species-specific estimations of partition coefficients from physicochemical properties. Sorption estimates consider both lipophilic and electrical interactions. A realistic regional exposure scenario was simulated for the three test compounds, and model results were compared with results obtained with a conventional fugacity model and with monitoring data. The better performance of MAMI indicates that the activity approach can enlarge the applicability domain and improve model predictions of existing regional models. Model results, supported by experimental evidence, showed the importance of dissociation, electrical interactions in solids, humidity in air, and to a lesser extent salinity in seawater to describe the environmental fate of ionizable organic chemicals.

Comparative Characterization of Experimental and Calculated Lipophilicity and Anti-Tumour Activity of Isochromanone Derivatives

Compound lipophilicity connected to ADME(T)(a) has great importance in drug development and it has to be evaluated by the generally used drug developmental process. In addition to the importance of lipophilicity in ADMET, recently it has been reported that lipophilicity of small molecules correlates with their antiproliferative activity because of certain specific hydrophobic and lipophilic interactions. Due to the complexity of ADME(T) parameters an efficient and fast method is needed to characterize the many promising candidate lead molecules as a preselection in order not to be rejected from the latter phase of drug development. In the present paper we provide an overview of the importance of lipophilicity of drug candidates for biological action and for ADME(T) and describe a novel approach for drug-likeness characterization of a molecular library using correlation study between lipophilicity and biological activity. Lipophilicity and molecular characteristics have been measured, predicted and optimized for a diverse library from which the best members have been selected to describe their biological, chemical and drug-likeness properties. Molecules were selected from the family of alpha,beta-unsaturated ketones and thorough HPLC characterization for lipophilicity and morphological, antiproliferative and flow cytometric studies were carried out on them. Based on the results 17 member isochromanone library including E and Z geometric isomers were selected for further characterization. In this focused library linear correlation has been found between the calculated and measured lipophilicity and significant parabolic correlation was found between the antiproliferative effect and lipophilicity. Using our efficient and fast method, from a diverse library, we identified an outstandingly effective inhibitor of A431 tumour cell growth via a PARP(a) cleavage dependent apoptosis. In summary the optimized HPLC analyses of lipophilicity combined with the cell-culture assay, introduced above, resulted in the determination of an optimal lipophilicity range. This optimized lipophilicity range should be used in designing novel antiproliferative compounds.

Preparation of Targeting Proteoliposome by Postinsertion of a Linker Molecule Conjugated with Recombinant Human Epidermal Growth Factor

To develop a sterically stable, targeting proteoliposome, a postinsertion method was employed to biofunctionalize the liposome surface with a biocompatible anchor molecule (BAM) linker conjugated with epidermal growth factor (EGF) as a homing molecule. In this method, EGF was first conjugated with BAM that consisted of a hydrophilic reactive group at one end and oleic acid chains at the other end. The EGF-BAM complex was then inserted into the liposomal phospholipid bilayer through lipophilic interaction. When compared with the traditional surface modification method by amine coupling, the modification efficiency with BAM at 25 degrees C was about 2.5-fold higher, and the 24 h stability of the inserted BAM at 25 degrees C was also about 2.5-fold higher. The insertion affinity and stability of the BAM to the liposomal bilayer was not influenced by an increase in cholesterol concentration, a component in liposome preparation. Confocal microscopy studies showed that the proteoliposomes biofunctionalized with the BAM-EGF complex encapsulating Cy5 fluorescent dye were selectively bound to the surface of MDA-MB-231 cells overexpressing EGFR (epidermal growth factor receptor), but not to MCF-7 cells, which did not express EGFR. The same proteolipome encapsulating doxorubicin was selectively targeted to MDA-MB-231 cells and killed them. In sum, BAM could be used as a suitable postinsertion linker for biofunctionalization of liposome surface with high modification efficiency and stability. Furthermore, the protein used as a homing molecule maintained its bioactivity after the biofunctionalization.