On the synergistic effects of ligand-mediated and phage-intrinsic properties during in vivo selection.
ABSTRACT Phage display has been used as a powerful tool in the discovery and characterization of ligand-receptor complexes that can be utilized for therapeutic applications as well as to elucidate disease mechanisms. While the basic properties of phage itself have been well described, the behavior of phage in an in vivo setting is not as well understood due to the complexity of the system. Here, we take a dual approach in describing the biophysical mechanisms and properties that contribute to the efficacy of in vivo phage targeting. We begin by considering the interaction between phage and target by applying a kinetic model of ligand-receptor complexation and internalization. The multivalent display of peptides on the pIII capsid of phage is also discussed as an augmenting factor in the binding affinity of phage-displayed peptides to cellular targets accessible in a microenvironment of interest. Lastly, we examine the physical properties of the total phage particle that facilitate improved delivery and targeting in vivo compared to free peptides.
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ABSTRACT: In cancer therapy and imaging, the systemic passive delivery of particulate systems has relied on the enhanced permeability and retention (EPR) effect: sufficiently small particles can cross the endothelial fenestrations and accumulate in the tumor parenchyma. The vast majority of man-made particulates exhibit a spherical shape as a result of surface energy minimization during their synthesis. The advent of phage display libraries, which are revealing the extraordinary molecular diversity of endothelial cells, and the development of processes for fabricating particles with shapes other than spherical are opening the path to new design solutions for systemically administered targeted particulates. In this paper, the role of particle geometry (i.e., size and shape) is discussed at the tissue and cellular scales. Emphasis is placed on how the synergistic effect of particle geometry and molecular targeting can enhance the specificity of delivery. The intravascular delivery process has been broken into three events: margination, firm adhesion and control of internalization. Predictions from mathematical models and observations from in-vitro experiments were used to show the relevance of particle geometry in systemic delivery. Rational design of particulate systems should consider, beside the physico-chemical properties of the surface coatings, geometrical features as size and shape. The integration of mathematical modeling with in-vitro and in-vivo testing provides the tools for establishing a rational design of nanoparticles.Pharmaceutical Research 09/2008; 26(1):235-43. DOI:10.1007/s11095-008-9697-x · 3.95 Impact Factor
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ABSTRACT: Cell-penetrating peptides (CPPs) traverse cell membranes of cultured cells very efficiently by a mechanism not yet identified. Recent theories for the translocation suggest either the binding of the CPPs to extracellular glycosaminoglycans or the formation of inverted micelles with negatively charged lipids. In the present study, the binding of the protein transduction domains (PTD) of human (HIV-1) and simian immunodeficiency virus (SIV) TAT peptide (amino acid residues 47-57, electric charge z(p) = +8) to membranes containing various proportions of negatively charged lipid (POPG) is characterized. Monolayer expansion measurements demonstrate that TAT-PTD insertion between lipids requires loosely packed monolayer films. For densely packed monolayers (pi > 29 mN/m) and lipid bilayers, no insertion is possible, and binding occurs via electrostatic adsorption to the membrane surface. Light scattering experiments show an aggregation of anionic lipid vesicles when the electric surface charge is neutralized by TAT-PTD, the observed stoichiometry being close to the theoretical value of 1:8. Membrane binding was quantitated with isothermal titration calorimetry and three further methods. The reaction enthalpy is Delta H degrees approximately equal to -1.5 kcal/mol peptide and is almost temperature-independent with Delta C(p) degrees approximately 0 kcal/(mol K), indicating equal contributions of polar and hydrophobic interactions to the reaction heat capacity. The binding of TAT-PTD to the anionic membrane is described by an electrostatic attraction/chemical partition model. The electrostatic attraction energy, calculated with the Gouy-Chapman theory, accounts for approximately 80% of the binding energy. The overall binding constant, K(app), is approximately 10(3)-10(4) M(-1). The intrinsic binding constant (K(p)), corrected for electrostatic effects and describing the partitioning of the peptide between the lipid-water interface and the membrane, is small and is K(p) approximately 1-10 M(-1). Deuterium and phosphorus-31 nuclear magnetic resonance demonstrate that the lipid bilayer remains intact upon TAT-PTD binding. The NMR data provide no evidence for nonbilayer structures and also not for domain formation. This is further supported by the absence of dye efflux from single-walled lipid vesicles. The electrostatic interaction between TAT-PTD and anionic phosphatidylglycerol is strong enough to induce a change in the headgroup conformation of the anionic lipid, indicating a short-lived but distinct correlation between the TAT-PTD and the anionic lipids on the membrane outside. TAT-PTD has a much lower affinity for lipid membranes than for glycosaminoglycans, making the latter interaction a more probable pathway for CPP binding to biological membranes.Biochemistry 08/2003; 42(30):9185-94. DOI:10.1021/bi0346805 · 3.19 Impact Factor
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ABSTRACT: Societal expectations about drug safety and efficacy are rising while productivity in the pharmaceutical industry is falling. In 2004, the US Food and Drug Administration introduced the Critical Path Initiative with the intent of modernizing drug development by incorporating recent scientific advances, such as genomics and advanced imaging technologies, into the process. An important part of the initiative is the use of public-private partnerships and consortia to accomplish the needed research. This article explicates the reasoning behind the Critical Path Initiative and discusses examples of successful consortia.Annual Review of Medicine 02/2008; 59:1-12. DOI:10.1146/annurev.med.59.090506.155819 · 15.48 Impact Factor