Current Drug Discovery Technologies - Volume 8, Issue 4, 2011

Drug targeting is one of the most important phenomena for therapies of diseases. The phenomena have been divided into two categories, passive targeting in which drug carriers circulate in the bloodstream and accumulate in a target tissue without any specific interactions, and active targeting in which drug carriers with ligands accumulate in a target tissue with receptors through ligand-receptor interactions. The enhanced permeation and retention (EPR) effect in drug delivery has been the subject of many studies on passive targeting of drugs, and has paved the way for nanoparticles and poly(ethylene glycol)-coupled liposomes as drug delivery systems (DDS). However, the physicochemical basis of many active targeting phenomena, including targeting of nanoparticles to particular cells or tissues, has not always been understood well. Clarifying this physicochemical basis may help drug delivery become more systematic, efficient and successful. Whereas passive targeting by EPR may depend on a large number of factors, only a few of them, such as particle size and charge, are usually determined quantitatively. Other factors should be considered. Two such factors are introduced in this issue. When the physicochemical factors of active targeting are characterized thoroughly, colloidal systems may be formulated and specified to satisfy these factors. Therefore, a better knowledge of kinetics and thermodynamics, colloid and surface phenomena, polymer science and rheology, may result in more successful active DDS. In this issue, exciting recent studies on the physical chemistry of targeting phenomena are reviewed by expert contributions. I. Meerovich et al. have made an attempt to analyze the main factors that influence the outcome from tissue or subcellular targeting on the kinetic or thermodynamic level. The different physicochemical mechanisms of cell-nanocarrier interactions are elucidated in thermodynamic terms, including Gibbs free energy and the binding constant. The problems related to kinetic modeling of targeted drug nanocarriers are also discussed in the review. H. Nabika et al. have applied surface chemistry to targeting of a lipid particle to a specific cell surface with a self-spreading lipid bilayer at the solid / liquid interface. K. Akiyoshi et al. have reported that a polysaccharide nanogel acts as an artificial molecular chaperone. Here, they report that a complex of nanogels with cancer antigen proteins triggers an immune response, and is useful as a method of cancer immunotherapy. G. M. Pavan et al. have constructed a virtual bridge between the design of cationic dendrimers and anionic DNA and siRNA macromolecules. T. Sato et al., who have studied the enhancement of pDNA transfection efficiency of pDNA/chitosan complexes with lactose- modified chitosan, have found that the morphologies of pDNA/chitosan complexes were largely affected by the molecular weight of chitosan and the stoichiometry of pDNA:chitosan. They have also found that cell transfection and resistance of pDNA against DNase were also affected by the morphology of the complexes. Y. Sun, in connection with the morphology of nonviral vectors, has determined rheologically the shape parameters of lipoplexes, and has shown that a flow incubation during the formation of lipoplexes from cationic lipids and DNA can control the shape of the resulting lipoplex. It is known that poly(ethylene glycol) modification of liposomal membranes forms a fixed aqueous layer and prevents the attraction of opsonins to the liposome. I. Sugiyama et al. have determined the thickness of the fixed aqueous layer (FALT) around liposomes and have shown that doxorubicin-containing liposomes increased the concentration of the drug in plasma and tumors according to the increase of FALT. S. Sakuma et al. have described a design concept for a nanoprobe, a peanut agglutinin-immobilized polystyrene nanosphere with surface poly(N-vinylacetamide) chains, encapsulating coumarin 6. The nanosphere has thus become an imaging agent that enables real-time and accurate diagnosis of small-sized colorectal tumors. The editors believe that this special issue will serve as a stimulus for future investigations into this potentially powerful approach to drug discovery technologies.

The effectiveness of many promising drug candidates (e.g. anticancer agents) awaits the development of drug forms capable of delivery of their drug load specifically to particular sites of an organism or a cell. To make universal and efficient drug carriers, administered drug-loaded vehicles should be able to reach the pathologic zone, recognize and bind their targets at a therapeutic concentration before clearance from the organism. Numerous methods have been developed to couple drug vehicles with active targeting substances - including monoclonal antibodies and substrates or ligands for pathologic cell receptors. Other approaches have included the use of such factors as decreased pH and elevated activity of enzymes in tumor tissues and the hypoxic environment inside the tumor core. This review makes an attempt to analyze the main factors that influence targeting on the kinetic or thermodynamic level that may provide the basis for a strategy to develop and improve drug delivery systems.

Self-spreading lipid bilayers at the solid/liquid interface can be used as a molecular transport medium in targeting nano-devices such as drug delivery and micro-total analytical systems. To gain physico-chemical insight in the selfspreading lipid bilayer, we have characterized the distribution of dye-labeled lipids in the upper and lower monolayers of a self-spreading lipid bilayer on a hydrophobic substrate by fluorescence quenching experiments using KI as a quencher. TR-DHPE, a molecule with a dye moiety at the head group, was found to be distributed primarily in the upper layer and accumulated especially at the spreading edge because of high steric repulsion. This resulted in an asymmetric distribution of TR-DHPE in the self-spreading bilayer in both the vertical and lateral directions. By contrast, NBD-PC, bearing a dye moiety at the alkyl chain, was distributed almost symmetrically both vertically and laterally. The observed difference is attributed to the difference in interactions between these molecules in the lower layer and the substrate surface. We have also found that the self-spreading velocity was decreased by the addition of KI. Since the spreading dynamics are determined by the interaction energy between the bilayer and solid substrate, a part of the observed velocity decrease could be explained by the change in the lipid density resulting from the adsorption of the I- anion on the lipid head group, thereby reducing the van der Waals interaction energy.

Molecular chaperones selectively trap heat-denatured proteins or their intermediates, primarily by hydrophobic interactions, to prevent irreversible aggregation resulting from macromolecular host (molecular chaperone)-guest (protein) interactions. The molecular chaperone function is an important concept that is expected to lead to breakthroughs in drug delivery systems, especially for protein or peptide delivery in regenerative medicine, such as bone regeneration. We have reported that polysaccharide nanogels act as artificial molecular chaperones. To further clarify the molecular chaperone function of nanogels as protein carriers, the elucidation of nanogel-protein interactions are especially important. Here, we investigated the interaction of a protein with a polysaccharide nanogel using fluorescence correlation spectroscopy at variable temperatures, using fluorescence-labeled bovine serum albumin (BSA) as a model protein. In particular, thermodynamic parameters of the heat-induced complexation of protein with CHP nanogels were evaluated using the van't Hoff plot. The plot shows that the CHP nanogels strongly complexed with heat-denatured BSA. The increased hydrophobicity of the denatured, unfolded protein may prefer complexation with amphiphilic hydrogel nanoparticles over complexation with the completely folded native protein. Thermodynamic parameters suggest that the complexation is entropically driven, rather than enthalpically, under the conditions studied.

The design of macromolecules able to generate a stable binding with nucleic acids is of great interest for their possible application in gene delivery. During the last years particular attention has been addressed to the use of dendritic scaffolds as a base to construct efficient DNA and siRNA nano-carriers. Dendrimers and dendrons are hyperbranched polymers characterized by a well-defined structure and by the possibility to functionalize their surface in many different ways. In particular, their multivalent character allows the creation of multiple binding sites between the positively charged groups that decorate the surface of cationic dendrons and dendrimers and the negatively charged phosphate groups present on the strands of DNA and siRNA. The engineering of “ideal dendritic candidates” to deliver and release genetic materials into cells is, however, not trivial due to the huge distance that exists between the design phase and the real application of such molecules. A differentarchitecture of the dendritic scaffold (flexible or rigid) can strongly modify the binding efficiency, but, at the same time, is influenced by the interactions with the external solution. In this context, molecular simulation can represent a “virtual bridge” between the design and the comprehension of the real behavior of such macromolecules.

Successful gene therapy depends on the development of effective gene carriers. Naturally occurring chitosan has been employed widely as a non-viral gene carrier because of its low toxicity, low immunogenicity, biocompatibility, and biodegradability. In this review, we summarize the utilization of chitosan, modified chitosan, and chitosan-containing ternary complexes as gene carriers. In particular, we discuss the influence of the physicochemical features of pDNA/chitosan complexes on their functions, such as stability and gene transfer into cells.

Lipoplexes, the complexes of plasmid DNA with cationic lipids, are considered as an attractive alternative to viral delivery systems. However, synthesized lipoplexes showed several limitations including insufficient transfection, low reproducibility and low stability. Here we attempt to delineate the relationships between the synthesis process, morphology (e.g., shape and liquid crystal structure), and the transfection efficiency of lipoplexes with rheological technology. Mini-capillary viscometers with automatic measurement and control components were designed and used to study the morphology of lipoplexes at a macroscopical scale. In such a dilute macromolecule suspension system, the shape factor of lipoplex was correlated with the viscosity measurement. The results showed that the shape factors of lipoplexes were different with various molecular structures of cationic lipid and helper lipid. A quantitative relation was set up between the shape factors and the length of DNA/polyelectrolytes, which may help better explain lipoplexes formation. To improve the stability and reproducibility of lipoplexes, an incubation period was suggested before the use of lipoplex. A rheological method was introduced to fix the hydromechanical parameters so that the entire preparation and incubation process was carried out consistently. A laminar flow incubation environment was showed suitable for lipoplex preparation and helped improve lipoplex stability and minimize aggregation. Other flow incubations, such as turbulent flow or impinging flow, were more complicated and further study is necessary to fully understand them. In brief, the rheological methods can help reveal the mechanisms of lipoplex formation and advance the rational design of lipoplexes for pharmaceutical applications.

Liposomes are recognized as useful drug carriers, but have some problems to overcome. Liposomes are easily opsonized with serum proteins (opsonization) and taken up by the reticuloendothelial system (RES) cells, such as spleen and liver. Polyethyleneglycol (PEG) modification on the liposomal membrane forms a fixed aqueous layer and thus prevents opsonization and uptake by the RES. Our research indicates clearly that the electrical potential distributions near the membrane surfaces were different between doxorubicin (DOX)-containing liposomes with and without a PEG coating. Moreover, the value of the fixed aqueous layer thickness (FALT) around the liposome, formed by PEG modification, correlates with the circulation time and antitumor effect in a murine model. In this review, we introduce the observation that measurement of FALT as a physical characteristics is a useful method for demonstrating the antitumor effect of antitumor agent-containing PEG-modified liposomes. The use of this technique may preclude the performance of certain in vivo experiments. Our approach using FALT enables the rapid and reliable development of PEG-modified liposome formulations.

The goal of this research is to develop an imaging agent that enables real-time and accurate diagnosis of smallsized colorectal cancer. Since colorectal cancer initially develops in the mucous membrane of the large intestine, a nonabsorbable colonoscopic imaging agent capable of being administered intracolonically was designed. The imaging agent is peanut agglutinin (PNA)-immobilized polystyrene nanospheres with surface poly(N-vinylacetamide) (PNVA) chains encapsulating coumarin 6. PNA is a targeting moiety that binds to β-D-galactosyl-(1-3)-N-acetyl-Dgalactosamine, which is the terminal sugar of the Thomsen-Friedenreich antigen that is specifically expressed on the mucosal side of colorectal cancer cells. PNVA is immobilized with the aim of reducing nonspecific interactions between the imaging agent and normal tissues, because the initial tumor-derived change is very small throughout the entire large intestine. Coumarin 6 is encapsulated into nanosphere cores to provide endoscopically-detectable fluorescence intensity. It is anticipated that the intracolonically-administered imaging agent recognizes tumor-derived changes in the large intestinal mucosa with high affinity and specificity. Real-time and accurate diagnosis of small-sized early colorectal cancer can be achieved through an imaging agent providing clear fluorescence contrast between normal and cancer tissues observed with a florescence endoscope. This review describes the design concept of this nanoprobe from a physicochemical perspective.