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Advanced Drug Delivery Reviews (v.57, #1)
Advances in fluorescence imaging: opportunities for pharmaceutical science
by Mark A. Jepson (Theme Editor) (pp. 1-4).
Coming out of the dark: the evolving role of fluorescence imaging in drug delivery research
by Mark Gumbleton; David J. Stephens (pp. 5-15).
Fluorescence techniques for drug delivery research: theory and practice by Nick S. White; Rachel J. Errington (pp. 17-42).
Advances in drug delivery require an understanding of drug design, drug stability and metabolism together with the complexities imposed by the biological system such as cell/tissue penetration, drug-target interaction, and the pharmacodynamic consequences. Fluorescence microscopy provides a comprehensive tool for investigating many of these aspects of drug delivery in single cells and whole tissue. This review presents the fundamental concepts of fluorescence-based methodologies. The core principles which underlie the fluorescence process and the interpretation of these events drives instrument design and the components required to illuminate and detect fluorescent probes. Many drugs are inherently auto-fluorescent and therefore can be tracked using microscopy techniques, while other more indirect approaches assay the consequences of drug perturbation. This review addresses the two principle aims in fluorescence microscopy; to generate and enhance fluorescence-derived contrast that may reveal a central process of drug delivery.
Keywords: Fluorescence probes; Fluorescence detection; Fluorescence imaging; Fluorescence microscopy; CCD camera imaging; Laser scanning microscopy; Confocal; Multi-photon; Image restoration; Deconvolution; PSF engineering
Intracellular trafficking pathways and drug delivery: fluorescence imaging of living and fixed cells by Peter Watson; Arwyn T. Jones; David J. Stephens (pp. 43-61).
Cellular processes depend on the fidelity of intracellular membrane traffic. Lipids, proteins, receptor ligands and solute molecules are trafficked to distinct compartments within the cell through both the biosynthetic and endocytic pathways. An appreciation of these pathways is vital for a complete understanding of intracellular drug delivery. Recent advances in fluorescence imaging have facilitated the analysis of these pathways in great detail. It is now possible to gain insight into the real-time dynamics of cellular components and macromolecular pharmacological agents as they are delivered into and traffic within single cells. Here, we discuss the analysis of intracellular drug delivery from the perspective of fluorescence imaging of both living and fixed cells. This review aims to cover trafficking pathways, markers for subcellular compartments, fluorescent labels for intracellular structures and pharmacological agents and relevant recent developments in imaging technology. In particular, we shall focus on the application of live cell imaging to the study of endocytic drug delivery.
Keywords: Endocytosis; Membrane traffic; Protein sorting; Live cell imaging; GFP
Real-time multiple-particle tracking: applications to drug and gene delivery by Junghae Suh; Michelle Dawson; Justin Hanes (pp. 63-78).
Complex biological environments, such as the cell cytoplasm or the mucus lining the airways of the lungs, can pose significant barriers to efficient therapeutic drug and gene delivery. Biological barriers are particularly important in controlled drug delivery applications that utilize a large carrier particle, such as a liposome or a polymer micro- or nanosphere. The dynamic transport of particulate drug and gene delivery vehicles through these barriers is poorly understood, having been primarily studied with static methods in the past. Recently, the transport of synthetic drug and gene carriers has been investigated quantitatively with real-time particle tracking technology, providing new insight into particle behavior in complex biological environments that is guiding rational improvements in particle design. This review briefly highlights basic principles of particle tracking and its application to elucidate important phenomena that limit effective particulate drug and gene delivery.
Keywords: Drug delivery; Gene delivery; Multiple-particle tracking; Intracellular; Mucus; Diffusion; Transport; Rheology
Imaging living central neurones using viral gene transfer by A.G. Teschemacher; J.F.R. Paton; S. Kasparov (pp. 79-93).
Studies of central neurones and other cellular components of the brain, such as glial and vascular cells, can be greatly advanced by the use of the modern optical techniques such as confocal live cell imaging. Fluorescent proteins have allowed imaging of particular cell types or intracellular elements to be visualised and distinguished from irrelevant background structures. To introduce the genetic information encoding for fluorescent proteins into relevant cellular targets, molecular tools are required. Viral vectors are one of the best ways of gene delivery into differentiated postnatal brain neurones and glia. Current progress in this field allows targeting of various cell types and therefore makes it possible to express a variety of fluorescent constructs in selected subpopulations of neurones, for example. In this review, we will discuss and compare the properties of the most popular viral gene delivery systems and the advantages of different brain cell preparations to illustrate how they can be used for high-resolution live cell confocal imaging in order to study new aspects of central nervous system (CNS) structure and function.
Keywords: Imaging; Neurones; Organotypic cultures; Gene expression; Viral vectors; Adenovirus; Lentivirus
Fluorescence microscopy to follow the targeting of liposomes and micelles to cells and their intracellular fate by Vladimir P. Torchilin (pp. 95-109).
Fluorescence microscopy may provide important information regarding interactions between nanoparticulate drugs carriers, such as liposomes and micelles, with target cells as well as their intracellular fate. Current paper describes various applications of fluorescence microscopy to investigate specific targeting of antibody-modified drug carriers to cancer cells. The enhanced antibody-mediated targeting of drug-loaded immunomicelles confirmed by fluorescence microscopy resulted in enhanced cancer cell killing compared to free drug or drug-loaded nontargeted micelles. Fluorescence microscopy was also used to prove the endosomal escape of properly assembled polymeric micelles (based on polyethylene glycol-phosphatidylethanolamine conjugate, PEG-PE) containing various additives destabilizing the endosomal membrane. When loaded with the anticancer drug (paclitaxel or vitamin K3), such micelles demonstrate increased cytotoxicity. Fluorescence microscopy was also applied to investigate the capture of cell-penetrating TAT peptide-modified liposomes by various cells and stability and intracellular trafficking of captured TAT-liposomes inside cells. It was also used to confirm the successful transfection of cells with TAT-liposomes bearing the plasmid encoding for the Green Fluorescent Protein (GFP).
Keywords: Fluorescence microscopy; Confocal microscopy; Micelles; Liposomes; Cell targeting; Internalization
Applications of imaging techniques to studies of epithelial tight junctions by Larry G. Johnson (pp. 111-121).
The intercellular junctional complex, which consists of the tight junction (TJ), adherens junction, and desmosomes, mediates cell–cell adhesion in epithelia and endothelia. The TJ forms the apical-most portion of this complex in epithelia, serving as a fence to lateral diffusion of apical and basolateral membrane components and as a semi-permeable barrier or gate to the flow of ions and solutes through the paracellular pathway. The TJ consists of a series of integral membrane and cytoplasmic plaque proteins with complex interactions. Included among the TJ proteins are the claudins, which play a major role in mediating the charge and solute selectivity of the junction. Yet, the profile of claudin and associated protein expression differs among epithelia and the function and regulation of many of the TJ proteins remain unknown. This review discusses the application of techniques to discern the function, localization, and regulation of epithelial TJs based on examples from published studies.
Keywords: Tight junctions; Epithelia; Immunofluorescence; Confocal microscopy; Permeability
Use of fluorescence imaging to investigate the structure and function of intestinal M cells by Andrea Buda; Caroline Sands; Mark A. Jepson (pp. 123-134).
Fluorescence imaging technology can be applied to many aspects of cell biology ranging from the analysis of specific markers in cells and tissues to the biological actions and distribution of fluorescent proteins or particles in living cells. In this review, we examine the role of fluorescence imaging, in conjunction with other microscopical techniques, to study sites of uptake of material across the gastrointestinal epithelium. We will focus primarily on intestinal M cells, specialised antigen-sampling cells in the epithelium of the gut-associated lymphoid tissue (GALT), including Peyer's patches. In addition to their importance as sites for uptake of inert material, and hence their potential as a route of delivery of vaccines, etc., M cells are also a major site of infection by a range of microbial pathogens. The application of new fluorescence imaging technologies has expanded our knowledge on the structure, development and function of these fascinating cells.
Keywords: M cells; Microparticles; Peyer's patches; Confocal microscopy; Fluorescence imaging
Intravital imaging of tumour vascular networks using multi-photon fluorescence microscopy by Gillian M. Tozer; Simon M. Ameer-Beg; Jennifer Baker; Paul R. Barber; Sally A. Hill; Richard J. Hodgkiss; Rosalind Locke; Vivien E. Prise; Ian Wilson; Borivoj Vojnovic (pp. 135-152).
The blood supply of solid tumours affects the outcome of treatment via its influence on the microenvironment of tumour cells and drug delivery. In addition, tumour blood vessels are an important target for cancer therapy. Intravital microscopy of tumours growing in ‘window chambers’ in animal models provides a means of directly investigating tumour angiogenesis and vascular response to treatment, in terms of both the morphology of blood vessel networks and the function of individual vessels. These techniques allow repeated measurements of the same tumour. Recently, multi-photon fluorescence microscopy techniques have been applied to these model systems to obtain 3D images of the tumour vasculature, whilst simultaneously avoiding some of the problems associated with the use of conventional fluorescence microscopy in living tissues. Here, we review the current status of this work and provide some examples of its use for studying the dynamics of tumour angiogenesis and vascular function.
Keywords: Vascular morphology; Vascular permeability; Vascular targeting; Tumour angiogenesis; Multi-photon microscopy; Intravital microscopy
Advanced microscopy solutions for monitoring the kinetics and dynamics of drug–DNA targeting in living cells by R.J. Errington; S.M. Ameer-beg; B. Vojnovic; L.H. Patterson; M. Zloh; P.J. Smith (pp. 153-167).
Many anticancer drugs require interaction with DNA or chromatin components of tumor cells to achieve therapeutic activity. Quantification and exploration of drug targeting dynamics can be highly informative in the rational development of new therapies and in the drug discovery pipeline. The problems faced include the potential infrequency and transient nature of critical events, the influence of micropharmacokinetics on the drug–target equilibria, the dependence on preserving cell function to demonstrate dynamic processes in situ, the need to map events in functional cells and the confounding effects of cell-to-cell heterogeneity. We demonstrate technological solutions in which we have integrated two-photon laser scanning microscopy (TPLSM) to track drug delivery in subcellular compartments, with the mapping of sites of critical molecular interactions. We address key design concepts for the development of modular tools used to uncover the complexity of drug targeting in single cells. First, we describe the combination of two-photon excitation with fluorescence lifetime imaging microscopy (FLIM) to map the nuclear docking of the anticancer drug topotecan (TPT) at a subset of DNA sites in nuclear structures of live breast tumor cells. Secondly, we demonstrate how we incorporate the smart design of a two-photon ‘dark’ DNA binding probe, such as DRAQ5, as a well-defined quenching probe to uncover sites of drug interaction. Finally, we discuss the future perspectives on introducing these modular kinetic assays in the high-content screening arena and the interlinking of the consequences of drug–target interactions with cellular stress responses.
Keywords: Minor groove ligands; Two-photon; Drug–target; Fluorescence lifetime imaging microscopy; Cell-based assays; Topotecan; DRAQ5
Fluorescence correlation spectroscopy of molecular motions and kinetics by Michael Gösch; Rudolf Rigler (pp. 169-190).
The foundations for fluorescence correlation spectroscopy (FCS) were already laid in the early 1970s, but this technique did not become widely used until single-molecule detection was established almost 20 years later with the use of diffraction-limited confocal volume element. The analysis of molecular noise from the GHz- to the Hz-region facilitates measurements over a large dynamic range covering photophysics, conformational transitions and interactions as well as transport properties of fluorescent biomolecules. From the Poissonian nature of the noise spectrum the absolute number of molecules is obtainable. Originally used for the analysis of molecular interactions in solutions, the strength of FCS lies also in its applicability to molecular processes at either the surface or interior of single cells. Examples for the analysis of surface kinetics including on and off rates of ligand–receptor interactions will be given. The possibility of obtaining this type of information by FCS will be of particular interest for cell-based drug screening.
Keywords: Fluorescence spectroscopy; Photophysics; FCS; FRAP; FRET; Parallel detection; Autocorrelation; PCH; TIR
Studying biophysical barriers to DNA delivery by advanced light microscopy by S.C. De Smedt; K. Remaut; B. Lucas; K. Braeckmans; N.N. Sanders; J. Demeester (pp. 191-210).
Advanced light microscopy (ALM) has been intensively employed by biophysicists to reveal cellular mechanisms. As described in this review, ALM clearly has potential to enhance our understanding of the mechanisms that affect macromolecular therapeutics or nanoscopic drug vectors in biological environments. However, while in recent years confocal microscopy and related techniques became rather routinely used in drug delivery it remains challenging to extract reliable information on the biophysical behaviour of drug delivery systems from ALM measurements. This review discusses studies in which confocal imaging, fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and fluorescence energy transfer were employed to reveal biophysical properties of DNA and DNA containing nanoparticles in extra- and intracellular media.
Keywords: Gene delivery; Antisense oligonucleotides; Light microscopy; Microphotolysis; Fluorescence recovery after photobleaching; Fluorescence correlation spectroscopy; Fluorescence fluctuation spectroscopy; Fluorescence resonance energy transfer; DNA degradation