Trajectories in nanotechnology: embracing complexity, seeking analogies.

This account comprises personal reflections on the field of nanosystems primarily designed for the delivery of biologically active agents. It emphasises the colloidal nature of nanoparticles obeying the same physical laws that dictate the behaviour of disperse systems. Research reveals not only intrinsic complexities but a variety of possible trajectories in vivo and ex vivo, issues of stability, interactions and behaviour in a range of often constrained environments. Such are the variations in the chemical and physical nature of the nanosystems and the active agents they carry, their putative "targets" and the many biological systems and models in which they are employed, it is not possible to generalise. Stochastic events may exclude precise prediction or extrapolation of outcomes, but embracing and studying complexity lead to new insights, often aided by consideration of analogies in cognate areas. This is part of the process of illumination. Unexpected results provide the true essence and excitement of scientific endeavour. Simplification is perhaps its antithesis.

Nanotechnologies for site specific drug delivery: Changing the narrative.

This account of issues in the field of nanotechnologies for specific drug delivery is concerned mainly with its diverse literature. It is a prelude to proposing guidelines to ensure that papers are written acknowledging first the complexity of the task, and second that as there is no such thing as a generic nanoparticle, nor a typical drug, nor standard targets, generalisations are rarely possible. The objective is to discuss some trends which have led to over-confident extrapolations of experimental work in animals to treatment. It is argued that a greater appreciation of the physics, biology and pharmaceutics involved could clarify the sense of any work done and the manner in which this is conveyed in publications. The essential content to be addressed in publications is outlined.

Towards more effective advanced drug delivery systems.

This position paper discusses progress made and to be made with so-called advanced drug delivery systems, particularly but not exclusively those in the nanometre domain. The paper has resulted from discussions with a number of international experts in the field who shared their views on aspects of the subject, from the nomenclature used for such systems, the sometimes overwrought claims made in the era of nanotechnology, the complex nature of targeting delivery systems to specific destinations in vivo, the need for setting standards for the choice and characterisation of cell lines used in in vitro studies, to attention to the manufacturability, stability and analytical profiling of systems and more relevant studies on toxicology. The historical background to the development of many systems is emphasised. So too is the stochastic nature of many of the steps to successful access to and action in targets. A lacuna in the field is the lack of availability of data on a variety of carrier systems using the same models in vitro and in vivo using standard controls. The paper asserts that greater emphasis must also be paid to the effective levels of active attained in target organs, for without such crucial data it will be difficult for many experimental systems to enter the clinic. This means the use of diagnostic/imaging technologies to monitor targeted drug delivery and stratify patient groups, identifying patients with optimum chances for successful therapy. Last, but not least, the critical importance of the development of science bases for regulatory policies, scientific platforms overseeing the field and new paradigms of financing are discussed.

"Targeting" nanoparticles: the constraints of physical laws and physical barriers.

In comparison to the complexities of the body, its organs, its normal and aberrant cells, many nanoparticles will appear to be relatively simple objects. This view is deceptive because the physicochemical properties of nanosystems, although quite well understood on the basis of material science, surface science and colloid theory, are far from simple in practice. While their properties are largely controllable in vitro, often purportedly "designed", their administration by any route changing environments conspires to produce additional layers of complexity. Some of the key physical laws and physicochemical parameters governing the fate of nanoparticles on their journey from point of intravenous administration to desired destinations such as tumors are discussed. Much of the science relevant to nanocarrier based targeting has been elaborated in studying purely physical phenomena, but there can be found therein many analogies with biological systems. These include factors that impede quantitative targeting: diffusion in complex media, aggregation and flocculation, hindered behavior of particles in confined spaces, jamming and dispersion in flow. All of these have the ability to influence fate and destination. Most of the critical processes are particle size dependent but not always linearly so. Virtually all processes in vivo involve an element of probability. Particle size and properties can be controlled to a large extent, but stochastic processes cannot by definition. Progress has been made, but the quantitative delivery of a nanocarrier to defined sites in tumors is neither inevitable nor yet predictable.

Reductionism and complexity in nanoparticle-vectored drug targeting.

This paper briefly discusses reductionism as a process for dissecting the complexities of drug targeting mediated by nanoparticulate carriers. While reductionism has been said to have been a drawback to enhanced appreciation and understanding of complex biological systems, it is concluded here that the dissection of the individual stages of the procession from injection to final destination in specific targets in a living complex organism is essential. It should allow a decrease in the empiricism from laudable and inventive efforts to achieve high levels of drug delivery to specific diseased targets such as tumours. At the stage of development of the field there have perhaps been fewer than desirable detailed experimental or theoretical investigations of these individual stages. However, there are frequently analogies in the literature from which to draw at least tentative conclusions about the physics, physical chemistry and biology which underpin the processes involved.

Anti-angiogenic poly-L-lysine dendrimer binds heparin and neutralizes its activity.

The interaction between heparin, a polyanion, and a polycationic dendrimer with a glycine core and lysine branches Gly-Lys63(NH2)64 has been investigated. Complexation was assessed by transmission electron microscopy, size and zeta potential measurements, methylene blue spectroscopy, and measuring the anti-coagulant activity of heparin in vitro and in vivo. Complete association between the heparin and the dendrimer occurred a 1:1 mass ratio (2:1 molar ratio or +/-charge ratio) with formation of quasi-spherical complexes in the size range of 99-147 nm with a negative zeta potential (-47 mV). Heparin-dendrimer (dendriplex) formation led to a concentration-dependent neutralization of the anticoagulant activity of heparin in human plasma in vitro, with complete loss of activity at a 1:1 mass ratio. The anticoagulant activity of the dendriplexes in Sprague-Dawley rats was also evaluated after subcutaneous administration with uncomplexed heparin as a comparator. The in vivo anticoagulant activity of heparin in plasma, evaluated using an antifactor Xa assay, was abolished after complexation. Measurement of [(3)H]-heparin showed that both free heparin and dendriplexes were present in plasma and in organs. Such data confirmed stably the formation of dendriplexes, which could be essential in developing novel dendrimer-based anti-angiogenic therapeutics suitable in combinatory therapeutics and theranostics.

Current-stimulated release of solutes solubilized in water-immiscible room temperature ionic liquids (RTILs).

Room temperature ionic liquids (RTILs), salts which are liquid at room temperature, may be water-soluble or water immiscible, depending on the combination of cation and anion. They are efficient solvents for a wide range of solutes including drugs. The water-immiscible RTILs studied in this paper (the 1-butyl, hexyl and octyl 3-methyl imidazolium (BMIM, HMIM, and OMIM) hexafluorophosphate (PF(6)(-)) salts) can act as drug reservoirs. Passage of an electric current through these immiscible liquids can enhance the release of some solutes into an aqueous medium. Current flow (over the range 1-5 mA) increased the release rate of a solubilized hydrophilic solute, (3)H-sucrose, and of a model hydrophobic drug, (3)H-dexametasone. A threefold increase in the release rate of both sucrose and dexamethasone into water was observed under some conditions although the effect of application of current was not always linear. OMIM[PF(6)] was the most responsive liquid. Some measurable physical properties of the ionic liquids change on the application of current. For example, the surface tension of the three RTILs studied decreased significantly on application of current for 15 min (from 47.8 mNm(-1) to 36.2 mNm(-1) for BMIM) but the effect on the surface tension of the OMIM salt was small. Only a small decrease in the viscosity of RTILs was observed. Although the mechanisms of the enhanced release are not yet elucidated, RTILs are potentially interesting depots for electrically controlled drug delivery.

Biphasic interactions between a cationic dendrimer and actin.

Gene delivery systems face the problem not only of the route toward the cell and tissues in question, but also of the molecularly crowded environment of both the cytoplasm and the nucleus itself. One of the physical barriers in the cytoplasm for diffusing nanoparticles is an actin network. Here, we describe the finding that a self-fluorescent sixth generation cationic dendrimer (6 nm in diameter) interacts reversibly and possibly electrostatically with actin filaments in vitro. Not only does this interaction slow the diffusion of the dendrimer but it also affects actin polymerization in a biphasic manner. At low concentrations the dendrimer behaves like a G-binding actin protein, retarding actin polymerization, whereas at high concentrations the dendrimer acts as a nucleating protein accelerating the polymerization. Thus in vivo the diffusion of a dendrimer carrier such as this has both physical and chemical elements: by decreasing polymerization it might accelerate its own transport, and by enhancing actin polymerization retard it. This finding suggests that such a dendrimer may have a role as an anticancer agent through its inhibitory effect on actin polymerization.

Dynamics of microparticles inside lipid vesicles: movement in confined spaces.

Microparticles and nanoparticles used in drug delivery frequently depend on their movement in confined spaces such as cells. Liposomes containing small numbers of 1-µm diameter polystyrene particles were used to study the dynamics of their movement within the confined space of the liposome interior. The analysis of the trajectories of single and multiple entrapped particles revealed that the particles were largely localized toward the periphery of the liposome with a rare presence in the centre. Interparticle interactions were studied by calculating interparticle distances, ranging from close to zero to around 8 µm with a mean of ∼4 µm. The diffusion coefficient of a single entrapped particle was D = 0.27 × 10(-9) cm(2) s(-1) when compared with 5.1 × 10(-9) cm(2) s(-1) free in water. When more than one particle was entrapped, the calculated diffusion coefficients were D = 0.61 × 10(-9) cm(2) s(-1) for two particles, D = 1.26 × 10(-9) cm(2) s(-1) for three particles, and D = 1.3 × 10(-9) cm(2) s(-1) for multiple particles). Particle movement was found to be distinctly faster at the periphery (average velocity 21.4 µm s(-)1) than at the centre of the vesicle (average velocity 14.2 µm s(-1)). These results demonstrate the significance of particle-particle interactions as well as particle-surface interactions, which is evident here in some systems by particle aggregation close to the liposome membrane.

Systemic antiangiogenic activity of cationic poly-L-lysine dendrimer delays tumor growth.

This study describes the previously unreported intrinsic capacity of poly-L-lysine (PLL) sixth generation (G(6)) dendrimer molecules to exhibit systemic antiangiogenic activity that could lead to solid tumor growth arrest. The PLL-dendrimer-inhibited tubule formation of SVEC4-10 murine endothelial cells and neovascularization in the chick embryo chick chorioallantoic membrane (CAM) assay. Intravenous administration of the PLL-dendrimer molecules into C57BL/6 mice inhibited vascularisation in Matrigel plugs implanted subcutaneously. Antiangiogenic activity was further evidenced using intravital microscopy of tumors grown within dorsal skinfold window chambers. Reduced vascularization of P22 rat sarcoma implanted in the dorsal window chamber of SCID mice was observed following tail vein administration (i.v.) of the PLL dendrimers. Also, the in vivo toxicological profile of the PLL-dendrimer molecules was shown to be safe at the dose regime studied. The antiangiogenic activity of the PLL dendrimer was further shown to be associated with significant suppression of B16F10 solid tumor volume and delayed tumor growth. Enhanced apoptosis/necrosis within tumors of PLL-dendrimer-treated animals only and reduction in the number of CD31 positive cells were observed in comparison to protamine treatment. This study suggests that PLL-dendrimer molecules can exhibit a systemic antiangiogenic activity that may be used for therapy of solid tumors, and in combination with their capacity to carry other therapeutic or diagnostic agents may potentially offer capabilities for the design of theranostic systems.

Nanosystem drug targeting: Facing up to complex realities.

This review considers some of the obstacles to successful drug targeting and delivery of therapeutic agents to desired target sites in the body, in the context of the sometimes overblown claims made for nanoparticle and nanosystem based delivery. It covers aspects of issues surrounding the instability of particles in vivo through flocculation and aggregation, their complex flow and adhesion patterns in the capillary network, particle jamming and bridging, the heterogeneity of access of drugs to some sites such as tumours even in their free molecular state, the diffusion of free drug and nanoparticles in tumour tissue and in single cells. There are the fundamental laws of physics and materials, especially in relation to diffusion, adsorption, adhesion and hydrodynamics, which apply and these cannot be denied in our attempts to target carriers to anatomically distant targets, tumours being the archetypal target experiencing most of the barriers which prevent quantitative carrier and hence drug uptake. The paper closes with a discussion of some of the unmet challenges which must be addressed before quantitative delivery and targeting is achieved in many disease states. It is clear that if progress is to be made an International System for testing nanoparticulate delivery systems should be established. In this way data from different laboratories will be comparable. The International protocol should cover both in vitro and in vivo testing.