Organization and Compartmentalization by Lipid Membranes Promote Reactions Related to the Origin of Cellular Life.

Liquid crystals have certain physical properties that promote chemical reactions which cannot occur in bulk phase media. These properties are displayed, among other molecules, by amphiphilic compounds which assemble into membrane structures then concentrate and organize biologically relevant monomers within their confined spaces. When mixtures of lipids and nucleotides are cycled multiple times between hydrated and anhydrous conditions, the monomers polymerize in the dry phase into oligonucleotides. Upon rehydration, mixtures of the polymers are encapsulated in lipid-bounded compartments called protocells. Reactions in liquid crystalline organizing matrices represent a promising approach for future research on how primitive cells could emerge on the early Earth and other habitable planets.

Drug Delivery Systems Based on Hydroxyethyl Starch.

The advantageous biological properties of hydroxyethyl starch (HES) triggered research interest toward the design and synthesis of drug delivery systems (DDSs) based on this polysaccharide. Convenient reaction schemes, including one-step reactions, led to the synthesis of HES conjugates with selected anticancer molecules or therapeutic proteins. Nanocapsules and hydrogels based on HES were also prepared and studied as prospective drug delivery systems. Formulations originating from these drug conjugates and also from nanocapsules and hydrogels loaded with drugs were characterized, highlighting the extension of their half-life in plasma, which is a critical property as far as their efficacy is concerned. Results obtained in vitro and in vivo proved promising, justifying the undertaking of additional experiments with such systems, including their multifunctionalization. The promising formulations that are discussed in this Topical Review is expected to further increase interest in applying HES for molecular constructing novel DDSs with enhanced efficacy, which may, in the future, find clinical applications.

Triphenylphosphonium Decorated Liposomes and Dendritic Polymers: Prospective Second Generation Drug Delivery Systems for Targeting Mitochondria.

Targeting specific intracellular organelles has been a biological process of significant interest. Specifically, for mitochondrial targeting, conventional liposomal and dendritic polymer nanoparticles were selected to be presented in this miniperspective. Both types of nanoparticles were decorated on their external surface with triphenylphosphonium cation (TPP), a well-known and effective mitochondrial targeting moiety. Due to their advantageous specificity toward mitochondria, these nanoparticles may be considered as prospective second generation drug delivery systems (DDSs). Functionalized liposomal and dendritic nanoparticles are conveniently prepared, and although they encounter several hurdles on their route from the extracellular environment to the interior of mitochondria, they manage to be accumulated inside them in experiments in vitro. Therefore, the TPP-functionalized nanoparticles presented in this miniperspective can prove effective DDSs and efforts should be continued to obtain results that will trigger further studies including clinical studies, hopefully leading to effective drugs for mitochondrial diseases. In fact, since these DDSs enter and act at the site where the dysfunction exists, a new medicine subspecialty is emerging, the so-called mitochondrial medicine.

Carboxylated hydroxyethyl starch: a novel polysaccharide for the delivery of doxorubicin.

Hydroxyethyl starch (HES) was interacted with succinic anhydride affording a carboxylated derivative which has proved to be a promising polymeric drug delivery system. Specifically, this polymer is conveniently prepared, is biodegradable, non-immunogenic, and can encapsulate doxorubicin due to the protonation of the primary amino group of doxorubicin by the carboxylic group located on the branched scaffold of the polysaccharide. In addition, due to the polyhydroxylated character of the polysaccharide, the latter can act as a protective coating in an analogous manner to the PEG-chains ensuring prolonged circulation in vivo. In vitro experiments showed controlled release of doxorubicin to the nuclei of DU145 prostate cancer cells when the anticancer drug is incorporated in the carboxylated hydroxyethyl starch.

Molecular recognition and organizational and polyvalent effects in vesicles induce the formation of artificial multicompartment cells as model systems of eukaryotes.

Researchers have become increasingly interested in the preparation and characterization of artificial cells based on amphiphilic molecules. In particular, artificial cells with multiple compartments are primitive mimics of the structure of eukaryotic cells. Endosymbiotic theory, widely accepted among biologists, states that eukaryotic cells arose from the assembly of prokaryotic cells inside other cells. Therefore, replicating this process in a synthetic system could allow researchers to model molecular and supramolecular processes that occur in living cells, shed light on mass and energy transport through cell membranes, and provide a unique, isolated space for conducting chemical reactions. In addition, such structures can serve as drug delivery systems that encapsulate both bioactive and nonbiocompatible compounds. In this Account, we present various coating, incubation, and electrofusion strategies for forming multicompartment vesicle systems, and we are focusing on strategies that rely on involving molecular recognition of complementary vesicles. All these methods afforded multicompartment systems with similar structures, and these nanoparticles have potential applications as drug delivery systems or nanoreactors for conducting diverse reactions. The complementarity of interacting vesicles allows these artificial cells to form, and the organization and polyvalency of these interacting vesicles further promote their formation. The incorporation of cholesterol in the bilayer membrane and the introduction of PEG chains at the surface of the interacting vesicles also support the structure of these multicompartment systems. PEG chains appear to destabilize the bilayers, which facilitates the fusion and transport of the small vesicles to the larger ones. Potential applications of these well-structured and reproducibly produced multicompartment systems include drug delivery, where researchers could load a cocktail of drugs within the encapsulated vesicles, a process that could enhance the bioavailability of these substances. In addition, the production of artificial cells with multiple compartments provides a platform where researchers could carry out individual reactions in small, isolated spaces. Such a reactive space can avoid problems that occur when the environment can be destructive to reactants or products or when a diverse set of compounds difficult to obtain in a conventional reactor space are produced. Our work on these artificial cells with multicompartment structures also led us to formulate a hypothesis on the processes that possibly generated eukaryotic cells. We hope both that our research efforts will excite interest in these nanoparticles and that this research could lead to systems designed for specific scientific and technological applications and further insights into the evolution of eukaryotic cells.

Formation of artificial multicompartment vesosome and dendrosome as prospected drug and gene delivery carriers.

Extensive studies in the last fifty years on the development of multifunctional liposomes have improved their drug delivery potential. Specifically, they fulfill to a significant degree the requirements which an effective drug carrier should exhibit, i.e. biocompatibility, biodegradability, drug encapsulation and protection of the drug, targeting to specific cells, reasonable stability in the biological milieu, transport through cell membranes and controlled drug release. However, despite these properties which have been achieved to a significant degree through molecular engineering of the liposome bilayers, a universal liposomal carrier has not yet been developed since it is rather difficult for the above properties to be simultaneously fulfilled. For this purpose a multicompartmentalization strategy was applied through which liposomes encapsulating smaller ones in their aqueous core were prepared. Multicompartment systems have also been prepared by encapsulation of dendrimers in the aqueous core of liposomes. In this manner drug delivery systems were prepared providing a double protection to drugs encapsulated inside the core of the small liposomes or incorporated in dendrimers. The external liposomal bilayer is also susceptible to multifunctionalization while their drug release can more effectively been tuned compared to single-compartment systems. Modular-type strategies have been employed for the preparation of these multicompartment systems. Thus, unilamellar liposomes and mono-dispersed dendrimers are selected as the drug delivery modules from which universal multifunctional and multicompartment drug delivery systems have been obtained i.e. vesosomes, which are liposomes encapsulating smaller liposomes and dendrosomes, which are liposomes encapsulating dendrimers. Examples of the application of drug and gene delivery employing vesosomes and dendrosomes as carriers are critically discussed.

Insulin complexes with PEGylated basic oligopeptides.

Biodegradable oligolysine and oligoarginine-type homopeptides functionalized with PEG of two different molecular weights interact with insulin, at physiological pH, affording complexes studied by dynamic light scattering, ζ-potential, circular dichroism, FTIR spectroscopy, and isothermal titration calorimetry (ITC). High levels of insulin complexation efficiencies (>99.5%) were determined for all derivatives. FTIR spectra suggest that the positively charged homo-oligopeptide derivatives interact with B chain C-terminus of insulin leading to the formation of nanoparticles than can be traced even at low oligopeptide/insulin molar ratios. The ITC profiles are complex, displaying significant endothermic and exothermic contributions. Oligoarginine-type derivatives exhibit the strongest interactions, while PEGylation of either oligopeptide with the high molecular weight chains significantly affects the ITC profiles and leads to larger enthalpy changes. This may be attributed to PEG-induced aggregation of insulin due to the depletion attraction effect leading to the formation of stable nanocomplexes. Stabilization of complexed insulin against enzymatic degradation by trypsin and α-chymotrypsin is observed especially for the high molecular weight PEGylated arginine-based derivative. Insulin release rates in simulated intestinal fluid are controlled by the length of PEG chains and the presence of arginine end-groups. Released insulin retains its secondary structure as established by circular dichroism spectroscopy.

Cholesteryl-functionalized ADNF-9 peptide: enhanced membrane transport through mouse neuroblastoma Neuro-2a cells.

A cholesteryl-functionalized derivative of activity dependent neurotrophic factor-9 peptide (a nine amino acid core peptide of activity-dependent neurotrophic factor, acting against Alzheimer's disease) was synthesized aiming at the improvement of its bioavailability. Therefore, its uptake was comparatively investigated with that of its parent peptide by employing mouse neuroblastoma Neuro-2a cells. Owing to the hydrophobic character of this cholesteryl-functionalized peptide, it exhibited enhanced permeability and intracellular uptake while it also retained its low cytotoxicity at concentrations up to 1 µM. FACS analysis also revealed that when Neuro-2a cells were treated with this activity dependent neurotrophic factor-9 derivative, at a concentration of 50 nM, an almost 100% uptake was obtained. In addition, in vitro biological activity experiments showed that the functionalized peptide retained its neurotrophic activity at femtomolar concentration range.

Preparation of multicompartment lipid-based systems based on vesicle interactions.

Various strategies for constructing artificial multicompartment vesicular systems, which primitively mimic the structure of eukaryotic cells, are presented. These model systems are appropriate for addressing several issues such as the understanding of cell processes, the development of nanoreactors and novel multicompartment delivery systems for specific drug applications, the transport through bilayer membranes, and also hypothesizing on the evolution of eukaryotic cells as originating from the symbiotic association of prokaryotes.

A novel mitotropic oligolysine nanocarrier: Targeted delivery of covalently bound D-Luciferin to cell mitochondria.

New and emerging therapeutic approaches focus on the targeted delivery of therapeutic agents to cell mitochondria with high specificity. Herein we present a novel mitotropic nanocarrier based on an oligolysine scaffold by addition of two triphenylphosphonium cations per oligomer. Although the parent oligolysine failed to enter healthy cells, the triphenylphosphonium modified carrier, with or without D-Luciferin, attached as cargo molecule, demonstrated striking mitochondrial specificity. Furthermore, the oligolysine bound d-Luciferin exhibited chemiluminescence, of lower intensity than free d-Luciferin, yet of remarkably longer steady-state temporal profile.

Interaction of vesicles: adhesion, fusion and multicompartment systems formation.

In this review, interactions of selected vesicles, induced either by molecular recognition or by electrostatic interactions, for the simulation of cell-cell and cell-drug interaction processes are discussed. In order of increasing complexity, examples of vesicles adhesion are presented at first, followed by recognition experiments in which fusion takes place. This differentiation in behavior was primarily attributed to the structural features of interacting vesicular pairs primarily affected by concentration and lateral phase separation of the anchored recognizable groups. In certain cases, fusion is accompanied by multicompartmentalization of the obtained aggregates. In connection with the formation of these multicompartment systems, it was proposed that an analogous mechanism could be operating in the evolution of eukaryotes during the symbiosis of prokaryotes.

Drug delivery using multifunctional dendrimers and hyperbranched polymers.

IMPORTANCE OF THE FIELD: The review presents the design strategy and synthesis of multifunctional dendrimers and hyperbranched polymers with the objective to develop effective drug delivery systems. AREAS COVERED IN THIS REVIEW: Well-characterized, commercially available dendritic polymers were subjected to functionalization for preparing drug delivery systems of low toxicity, high loading capacity, ability to target specific cells and transport through their membranes. This has been achieved by surface targeting ligands, which render the carriers specific to certain cells and polyethylene glycol groups, securing water solubility, stability and prolonged circulation. Moreover, transport agents facilitate transport through cell membranes while fluorescent probes detect their intracellular localization. A common feature of surface groups is multivalency, which considerably enhances their binding strength with complementary cell receptors. To these properties, one should also add the property of attaining high loading of active ingredients coupled with controlled and/or triggered release. WHAT THE READER WILL GAIN: Readers will be exposed to the strategy of synthesizing multifunctional polymers, aimed at the development of effective drug delivery systems. TAKE HOME MESSAGE: Multifunctional systems upgrade the therapeutic potential of drugs and, in certain cases, may even lead to the application of new bioactive compounds that would otherwise not be feasible.

Synthesis of a folate functionalized PEGylated poly(propylene imine) dendrimer as prospective targeted drug delivery system.

Based on fourth generation diaminobutane poly(propylene imine) dendrimer, a novel targeted drug nanocarrier was prepared, bearing protective PEG chains and a folate targeting ligand. As a control a PEGylated derivative without folate was also synthesized. The encapsulation and release properties of these PEGylated derivatives were investigated employing etoposide, an anticancer hydrophobic drug. Enhanced solubility of etoposide was achieved inside the dendrimeric scaffold which was subsequently released in a controlled manner. These properties coupled with specificity towards the folate receptor and the low toxicity render folate functionalized PEGylated poly(propylene imine) dendrimer promising candidate for targeted drug delivery.

Arginine end-functionalized poly(L-lysine) dendrigrafts for the stabilization and controlled release of insulin.

Second generation biodegradable poly(l-lysine) dendrigrafts functionalized with 12-48 arginine end-groups interact, at physiological pH, with insulin affording dendrigraft/insulin complexes as established by dynamic light scattering, ζ-potential, circular dichroism and isothermal titration calorimetry. Binding occurs in two steps; at low dendrigraft/insulin molar ratios (< or = 0.07) interaction is accompanied with the endothermic dissociation of insulin dimers, while at higher molar ratios, complexation of insulin monomers with dendrigraft derivatives occurs exothermically. High levels of insulin complexation efficiencies (>99%) were determined for all derivatives. Stabilization of complexed insulin against enzymatic degradation by trypsin and α-chymotrypsin is observed especially for the highly arginine end-functionalized dendrigrafts. Insulin release rates in simulated intestinal fluid are being controlled by the number of arginine end-groups and released insulin retains its conformation.

Synthesis and characterization of multifunctional hyperbranched polyesters as prospective contrast agents for targeted MRI.

Based on a commercially available hyperbranched aliphatic polyester, novel multifunctional gadolinium complexes were prepared bearing protective PEG chains, a folate targeting ligand and EDTA or DTPA chelate moieties. Their relatively high water relaxivity values coupled with biodegradability of the hyperbranched scaffold, folate receptor specificity render these non-toxic dendritic polymers promising candidates for MRI applications.

Structural features of interacting complementary liposomes promoting formation of multicompartment structures.

The structural features of complementary liposomes and factors favoring formation of multicompartment systems are investigated. Specifically, liposomal formulations consisting of PEGylated unilamellar liposomes with guanidinium moieties located at the distal end of polyethylene glycol (PEG) chains interact with complementary multilamellar liposomes bearing phosphate moieties. Furthermore, the number of PEG chains attached to the unilamellar interface of the liposomes is enhanced by incorporating PEGylated cholesterol in their bilayer. While molecular recognition of the liposomes is the driving force for initiating multicompartmentalization, it is the enhanced PEGylation at the liposomal interface that synergistically promotes fusion resulting in large and well-formed multicompartment systems. A mechanism is proposed according to which initial adhesion of the liposomes, followed by reorganization of their membrane lipids, leads to giant bilayer aggregates incorporating large liposomes.

Gene delivery using functional dendritic polymers.

OBJECTIVE: This review describes a strategy for the development of multifunctional dendritic polymers for application as gene delivery systems. These polymers can address the low transfection efficiency usually encountered by synthetic non-viral vectors. METHODS: Employing appropriate, well-characterized and mainly commercially available dendritic polymers, the emphasis is placed primarily on step-wise molecular engineering of their surface for providing gene carriers of low toxicity, specificity to certain cells and transport ability through their membranes, with the ultimate objective of enhanced transfection efficiency. Cationic dendritic polymers interact with appropriate genetic material, affording complexes that are employed for cell transfection. CONCLUSION: Multifunctionalization of dendritic polymers provides gene vectors of low toxicity, significant transfection efficiency, specificity to certain biological cells and transport ability through their membranes.

Multifunctional dendritic drug delivery systems: design, synthesis, controlled and triggered release.

This review describes the strategy for the development of multifunctional dendrimeric and hyperbranched polymers, collectively named dendritic polymers, aiming at their application as drug and gene delivery systems. Employing well-characterized and mainly commercially available dendritic polymers, the functionalization of these polymers is aimed at providing drug carriers of low toxicity, high encapsulating capacity, specificity to certain type of cells and transport ability through their membranes. Following a step-wise functionalization strategy of the starting dendritic polymers one has the option to prepare products that fulfill one or more of these requirements. In particular, in addition to polyvalency which is a common feature of the dendritic polymers, these carriers bearing a number of targeting ligands exhibit specificity to certain cells, another type of groups secures stability in biological milieu and prolonged circulation, while other moieties facilitate their transport through cell membranes. Furthermore, dendritic polymers applied for gene delivery should be or become cationic in the biological environment for the formation of complexes with the negatively charged genetic material.

Novel amiodarone-doxorubicin cocktail liposomes enhance doxorubicin retention and cytotoxicity in DU145 human prostate carcinoma cells.

We have developed novel cocktail liposomes bearing doxorubicin in their hydrophilic cores, and amiodarone, a potent multidrug resistance inhibitor, in their lipid bilayers. The efficacy of these liposomes was studied in DU145 human prostate carcinoma cells. Intracellular calcein retention, which is inversely proportional to multidrug resistance activity, significantly increased following cell incubation with amiodarone loaded liposomes. Fluorescence confocal microscopy on cells incubated with the cocktail liposomes revealed enhanced intranuclear doxorubicin accumulation. Two liposomal drug concentration combinations were employed to assess the differential cytotoxicity of the cocktail liposomes, doxorubicin (1.4 microM)-amiodarone (15 microM) and doxorubicin 3 (microM)-amiodarone (45 microM), and two incubation times, 5 and 19 h. Cell toxicity was determined by XTT assays at 24, 48, and 72 h following incubation and was significantly enhanced for incubation with the cocktail liposomes. On the whole, we believe that these liposomes will greatly contribute to the cancer chemotherapy arena.