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siRNA therapeutics are currently regarded as promising candidates to make a leap forward in the search for treatments of various hard to cure diseases. In order to exploit the full potential of siRNA based therapeutics, development of delivery systems that can efficiently guide the siRNA molecules to their target without major side effects will be the key to success. Lipid based delivery systems, originating from earlier research in the fields of gene delivery, are the most studied candidates for siRNA delivery. Here we discuss the requirements that need to be met by these siRNA delivery systems to ensure adequate stability after systemic application and subsequent deposition in the target tissue. The encountered hurdles in the blood stream and the solutions proposed in literature are discussed.

The adsorption of blood proteins onto liposomes and other colloidal particles is an important process influencing the circulation time. Proteins adsorbed to the surface of liposomes can mediate recognition of the liposomes by macrophages of the reticuloendothelial system (RES) facilitating their clearance from the circulation. Coating liposomes with poly(ethylene glycol) (PEG) decreases the blood clearance considerably, most likely due to reduced protein adsorption and/or liposome aggregation. By using the relation between clearance and protein binding, the present study introduces an in vitro assay measuring interactions of liposomes with proteins to predict their blood clearance in vivo. Such assay is valuable since it limits time and costs, and importantly reduces the number of animals required for pharmacokinetic investigations of new formulations. In the current study, Surface Plasmon Resonance (SPR) and fluorescence Single Particle Tracking (fSPT) were used to study liposome-protein interactions and blood induced liposome aggregation in vitro. By means of SPR the interactions between proteins and liposomes coated with PEG of different molecular weights and at different densities (PEG(2000) in 2.5%, 5% and 7%; PEG(5000) in 0.5%, 1.5% and 2.5%), were measured for several plasma proteins: human serum albumin (HSA), apolipoprotein E (ApoE), α2-macroglobulin (α2-M), β2-glycoprotein (β2-G) and fibronectin (Fn). Liposomes coated with PEG interacted less with all proteins, an effect which increased with the PEG surface density. In parallel, fSPT analysis showed that the exposure of liposomes to full blood did not change the liposome size, indicating that aggregation is not a strong attributive factor in the clearance of these liposomes. In addition, the SPR measurements of the interactions between liposomes and proteins were correlated with the blood clearance of the liposomes. For each protein, the degree of protein-liposome interaction as determined by SPR showed a moderate to strong positive correlation with the clearance of the liposome type.

While extremely relevant to many life science fields, such as biomedical diagnostics and drug delivery, studies on the size of nanoparticulate matter dispersed in biofluids are missing due to a lack of suitable methods. Here we report that fluorescence single particle tracking (fSPT) with maximum entropy analysis is the first technique suited for accurate sizing of nanoparticles dispersed in biofluids, such as whole blood. After a thorough validation, the fSPT sizing method was applied to liposomes that have been under investigation for decades as nanocarriers for drugs. The tendency of these liposomes to form aggregates in whole blood was tested in vitro and in vivo. In addition, we have demonstrated that the fSPT sizing technique can be used for identifying and sizing natural cell-derived microparticles directly in plasma. fSPT sizing opens up the possibility to systematically study the size and aggregation of endogenous or exogenous nanoparticles in biofluids.

SiRNA based therapeutics are currently under investigation for treatment of cancer and viral infections. Upon intravenous administration, the nanoscopic delivery systems which carry the siRNA need to be stable in serum, an aspect which is often overlooked in numerous publications on siRNA delivery systems. Techniques currently available for studying the dissociation of siRNA-liposome complexes are time consuming and incompatible with full serum. We therefore developed a fluorescence fluctuation spectroscopy (FFS) based method which allows to monitor the integrity of siRNA-carrier complexes. The method can very rapidly provide quantitative information on the complex integrity in biological media, like full human serum, and at very low siRNA concentrations (approximately 20 nM siRNA). Information on the integrity of intravenously injected siRNA nanoparticles in serum is crucial. Consequently, the FFS method reported in this work may find broad applicability in the field of siRNA-carrier design.

A large effort is currently put into the development of nano-scaled carrier systems that can guide siRNA molecules to their target cells after intravenous injection. One of the main issues in this research is the integrity of the siRNA containing nanoparticles in the blood stream. The integrity of the nanoparticles comprises both the particle size and the stable encapsulation of siRNA. Techniques currently available for studying the disassembly and size distribution of siRNA containing nanoparticles are time-consuming and incompatible with biological fluids. We initially developed a fluorescence fluctuation spectroscopy (FFS) based method which allows us to monitor the integrity of siRNA-carrier complexes in less than one minute in complex biological media and at very low siRNA concentrations. Second, while the size distribution of the complexes can be easily measured in a clear dispersion by dynamic light scattering or electron microscopy, it cannot be measured in more complex biological media such as plasma or whole blood, which contain several different interfering components. To address this issue, we have developed a novel technique, based on single particle tracking (SPT) microscopy, for studying the size distribution (and aggregation) of nanoscopic drug complexes in biological fluids. For stabilization of the particle size of cationic lipid based nanoparticles, inclusion of lipids conjugated with PEG is widely used to sterically hinder aggregate formation. We have demonstrated that in order to obtain remaining siRNA complexation to the cationic liposomes, effective encapsulation inside the liposome, or in between lipid multilayers is required, since siRNA electrostatically bound to the outer side of the liposomes is quickly pushed away by the ubiquitous albumin molecules in blood which leads to siRNA degradation and loss of effectiveness. Formation of siRNA protecting multilayers is hindered by inclusion of PEG-lipids, a hurdle that needs to be overcome either by post-insertion of the PEG-lipid into multilayer containing siRNA-liposome complexes, or by efficient encapsulation of the siRNA inside the aqueous core of the PEGylated liposome. Size stabilization in buffer can be easily achieved by inclusion of minor percentages (not, vert, similar1%) of PEG-lipids. In whole blood however, we demonstrate that much higher percentages of PEG–lipids (5–10%) are required to achieve size stabilization. This requirement has not been previously considered because of the lack of a suitable technique to study the aggregation phenomena in whole blood. In our work we demonstrate that assaying the physicochemical properties of siRNA encapsulating nanoparticles should always be carried out in the biological media they are designed to be employed in. Two novel microscopy based methods were developed that enable such characterization in biological fluids such as serum, plasma or even whole blood.