The discovery and enhanced properties of trichain lipids in lipopolyplex gene delivery systems.

The formation of a novel trichain (TC) lipid was discovered when a cationic lipid possessing a terminal hydroxyl group and the helper lipid dioleoyl l-α-phosphatidylethanolamine (DOPE) were formulated as vesicles and stored. Importantly, the transfection efficacies of lipopolyplexes comprised of the TC lipid, a targeting peptide and DNA (LPDs) were found to be higher than when the corresponding dichain (DC) lipid was used. To explore this interesting discovery and determine if this concept can be more generally applied to improve gene delivery efficiencies, the design and synthesis of a series of novel TC cationic lipids and the corresponding DC lipids was undertaken. Transfection efficacies of the LPDs were found to be higher when using the TC lipids compared to the DC analogues, so experiments were carried out to investigate the reasons for this enhancement. Sizing experiments and transmission electron microscopy indicated that there were no major differences in the size and shape of the LPDs prepared using the TC and DC lipids, while circular dichroism spectroscopy showed that the presence of the third acyl chain did not influence the conformation of the DNA within the LPD. In contrast, small angle neutron scattering studies showed a considerable re-arrangement of lipid conformation upon formulation as LPDs, particularly of the TC lipids, while gel electrophoresis studies revealed that the use of a TC lipid in the LPD formulation resulted in enhanced DNA protection properties. Thus, the major enhancement in transfection performance of these novel TC lipids can be attributed to their ability to protect and subsequently release DNA. Importantly, the TC lipids described here highlight a valuable structural template for the generation of gene delivery vectors, based on the use of lipids with three hydrophobic chains.

Trichain cationic lipids: the potential of their lipoplexes for gene delivery.

Lipoplexes (LDs) have been prepared from DNA and positively charged vesicles composed of the helper lipid, dioleoyl l-α-phosphatidylethanolamine (DOPE) and either a dichain (DC) oxyethylated cationic lipid or their corresponding novel trichain (TC) counterpart. This is the first study using the TC lipids for the preparation of LDs and their application. Here the results of biophysical experiments characterising the LDs have been correlated with the in vitro transfection activity of the complexes. Photon correlation spectroscopy, zeta potential measurements and transmission electron microscopy studies indicated that, regardless of the presence of a third chain, there were little differences between the size and charge of the TC and DC containing LDs. Small angle neutron scattering studies established however that there was a significant conformational re-arrangement of the lipid bilayer when in the form of a LD complex as opposed to the parent vesicles. This re-arrangement was particularly noticeable in LDs containing TC lipids possessing a third chain of C12 or a longer chain. These results suggested that the presence of a third hydrophobic chain had a significant effect on lipid packing in the presence of DNA. Picogreen fluorescence and gel electrophoresis studies showed that the TC lipids containing a third acyl chain of at least C12 were most effective at complexing DNA while the TC lipids containing an octanoyl chain and the DC lipids were least effective. The transfection efficacies of the TC lipids in the form of LDs were found to be higher than for the DC analogues, particularly when the third acyl chain was an octanoyl or oleoyl moeity. Little or no increase in transfection efficiency was observed when the third chain was a methyl, acetyl or dodecanoyl group. The large enhancement in transfection performance of the TC lipids can be attributed to their ability to complex their DNA payload. These studies indicate that presence of a medium or long third acyl chain was especially beneficial for transfection.

Delivery of siRNA using ternary complexes containing branched cationic peptides: the role of peptide sequence, branching and targeting.

Ternary nanocomplexes, composed of bifunctional cationic peptides, lipids and siRNA, as delivery vehicles for siRNA have been investigated. The study is the first to determine the optimal sequence and architecture of the bifunctional cationic peptide used for siRNA packaging and delivery using lipopolyplexes. Specifically three series of cationic peptides of differing sequence, degrees of branching and cell-targeting sequences were co-formulated with siRNA and vesicles prepared from a 1 : 1 molar ratio of the cationic lipid DOTMA and the helper lipid, DOPE. The level of siRNA knockdown achieved in the human alveolar cell line, A549-luc cells, in both reduced serum and in serum supplemented media was evaluated, and the results correlated to the nanocomplex structure (established using a range of physico-chemical tools, namely small angle neutron scattering, transmission electron microscopy, dynamic light scattering and zeta potential measurement); the conformational properties of each component (circular dichroism); the degree of protection of the siRNA in the lipopolyplex (using gel shift assays) and to the cellular uptake, localisation and toxicity of the nanocomplexes (confocal microscopy). Although the size, charge, structure and stability of the various lipopolyplexes were broadly similar, it was clear that lipopolyplexes formulated from branched peptides containing His-Lys sequences perform best as siRNA delivery agents in serum, with protection of the siRNA in serum balanced against efficient release of the siRNA into the cytoplasm of the cell.

Multifunctional receptor-targeted nanocomplexes for the delivery of therapeutic nucleic acids to the brain.

Convection enhanced delivery (CED) is a method of direct injection to the brain that can achieve widespread dispersal of therapeutics, including gene therapies, from a single dose. Non-viral, nanocomplexes are of interest as vectors for gene therapy in the brain, but it is essential that administration should achieve maximal dispersal to minimise the number of injections required. We hypothesised that anionic nanocomplexes administered by CED should disperse more widely in rat brains than cationics of similar size, which bind electrostatically to cell-surface anionic moieties such as proteoglycans, limiting their spread. Anionic, receptor-targeted nanocomplexes (RTN) containing a neurotensin-targeting peptide were prepared with plasmid DNA and compared with cationic RTNs for dispersal and transfection efficiency. Both RTNs were labelled with gadolinium for localisation in the brain by MRI and in brain sections by LA-ICP-MS, as well as with rhodamine fluorophore for detection by fluorescence microscopy. MRI distribution studies confirmed that the anionic RTNs dispersed more widely than cationic RTNs, particularly in the corpus callosum. Gene expression levels from anionic formulations were similar to those of cationic RTNs. Thus, anionic RTN formulations can achieve both widespread dispersal and effective gene expression in brains after administration of a single dose by CED.

Gene delivery using ternary lipopolyplexes incorporating branched cationic peptides: the role of Peptide sequence and branching.

Cationic peptide sequences, whether linear, branched, or dendritic, are widely used to condense and protect DNA in both polyplex and lipopolyplex gene delivery vectors. How these peptides behave within these particles and the consequences this has on transfection efficiency remain poorly understood. We have compared, in parallel, a complete series of cationic peptides, both branched and linear, coformulated with plasmid DNA to give polyplexes, or with plasmid DNA and the cationic lipid, DOTMA, mixed with 50% of the neutral helper lipid, DOPE, to give lipopolyplexes, and correlated the transfection efficiencies of these complexes to their biophysical properties. Lipopolyplexes formulated from branched Arg-rich peptides, or linear Lys-rich peptides, show the best transfection efficiencies in an alveolar epithelial cell line, with His-rich peptides being relatively ineffective. The majority of the biophysical studies (circular dichroism, dynamic light scattering, zeta potential, small angle neutron scattering, and gel band shift assay) indicated that all of the formulations were similar in size, surface charge, and lipid bilayer structure, and longer cationic sequences, in general, gave better transfection efficiencies. Whereas lipopolyplexes formulated from branched Arg-containing peptides were more effective than those formulated from linear Arg-containing sequences, the reverse was true for Lys-containing sequences, which may be related to differences in DNA condensation between Arg-rich and Lys-rich peptides observed in the CD studies.

Multifunctional receptor-targeted nanocomplexes for magnetic resonance imaging and transfection of tumours.

The efficient targeted delivery of nucleic acids in vivo provides some of the greatest challenges to the development of genetic therapies. We aim to develop nanocomplex formulations that achieve targeted transfection of neuroblastoma tumours that can be monitored simultaneously by MRI. Here, we have compared nanocomplexes comprising self-assembling mixtures of liposomes, plasmid DNA and one of three different peptide ligands derived from ApoE, neurotensin and tetanus toxin for targeted transfection in vitro and in vivo. Neurotensin-targeted nanocomplexes produced the highest levels of transfection and showed a 4.7-fold increase in transfected luciferase expression over non-targeted nanocomplexes in Neuro-2A cells. Transfection of subcutaneous Neuro-2A tumours in vivo with neurotensin-targeted nanocomplexes produced a 9.3-fold increase in gene expression over non-targeted controls. Confocal microscopy analysis elucidated the time course of DNA delivery with fluorescently labelled nanocomplex formulations in cells. It was confirmed that addition of a gadolinium lipid conjugate contrast agent allowed real time in vivo monitoring of nanocomplex localisation in tumours by MRI, which was maintained for at least 24 h. The peptide-targeted nanocomplexes developed here allow for the specific enhancement of targeted gene therapy both in vitro and in vivo, whilst allowing real time monitoring of delivery with MRI.

Convection-enhanced delivery of neprilysin: a novel amyloid-ß-degrading therapeutic strategy.

Enzymatic degradation contributes to the control of intracerebral amyloid-ß (Aß) peptide levels. Previous studies have demonstrated the therapeutic potential of viral vector-mediated neprilysin (NEP) gene therapy in mouse models of Alzheimer's disease (AD). However, clinical translation of NEP gene therapy is limited by ethical and practical considerations. In this study we have assessed the potential of convection-enhanced delivery (CED) as a means of elevating intracerebral NEP level and activity and degrading endogenous Aß. We analyzed the interstitial and perivascular distribution of NEP following CED into rat striatum. We measured NEP protein level, clearance, activity, and toxicity by ELISA for NEP and synaptophysin, NEP-specific activity assay, and immunohistochemistry for NEP, NeuN, glial fibrillary acidic protein and Iba1. We subsequently performed CED of NEP in normal aged rats and measured endogenous Aß by ELISA. CED resulted in widespread distribution of NEP, and a 20-fold elevation of NEP protein level with preservation of enzyme activity and without evidence of toxicity. CED in normal, aged rats resulted in a significant reduction in endogenous Aß(40) (p = 0.04), despite rapid NEP clearance from the brain (half-life ~3 h). CED of NEP has therapeutic potential as a dynamically controllable Aß(40)-degrading therapeutic strategy for AD. Further studies are required to determine the longer term effects on Aß (including Aß(42)) and on cognitive function.

Lipopolyplex ternary delivery systems incorporating C14 glycerol-based lipids.

The structure, biophysical properties and biological behavior of lipopolyplex ternary gene delivery vectors incorporating novel C14 glycerol based lipids of varying alkyl chain geometry (containing cis, trans or alkyne double bonds) have been studied in the presence and absence of a bifunctional targeting peptide designed to both condense DNA and confer integrin-specific targeting. In vitro transfection studies in breast cancer MDA-MB-231 cells revealed that ternary formulations of lipid:peptide:DNA (LPD) complexes prepared using the aforementioned lipids possessed highly synergistic transfection activity up to 2500-fold higher than their respective lipid:DNA (LD) or peptide:DNA (PD) counterparts. Furthermore, the small structural differences in the lipid alkyl chain geometries also resulted in pronounced differences in transfection within each type of formulation, whereby the trans lipids showed best activity when formulated as LD complexes, whereas the cis lipids were superior in LPD formulations. Confocal fluorescence internalization studies using labeled components of the formulations showed both the lipid and the DNA of LD complexes to be trapped in endocytic compartments, whereas in the case of LPD complexes, the DNA was clearly released from the endosomal compartments and, together with the peptide, internalized within the cell nucleus. Physicochemical characterization of the formulations carried out by light and neutron scattering, zeta potential measurement, and negative staining electron microscopy detected major structural differences between LD and LPD complexes. Gel electrophoresis assays additionally showed differences between the individual lipids tested in each type of formulation. In conclusion, the superior transfection of the trans lipids in the LD complexes was thought to be attributed to superior DNA binding caused by a more closely matched charge distribution of the more rigid, trans lipids with the DNA. In the case of the LPD complexes, the DNA was thought to be predominantly condensed by the cationic portion of the peptide forming a central core surrounded by a lipid bilayer from which the targeting sequence partially protrudes. The more fluid, cis lipids were thought to confer better activity in this formulation due to allowing more of the targeting peptide sequence to protrude.

Protease sensing with nanoparticle based platforms.

Nanoparticulate systems in various unique configurations are highly effective at detecting protease activity both in vivo and in vitro. In this article, we have summarised the conventional modern methods for monitoring protease activity, and critically appraised recent advances in protease-responsive nanosensors.

A facile method to clickable sensing polymeric nanoparticles.

Clickable biocompatible nanoparticles were prepared in a one-pot process by microemulsion polymerization using acrylamide, N,N'-methylene bisacrylamide and either N-(11-azido-3,6,9-trioxaundecanyl)acrylamide or N-propargylacrylamide, which were then readily modified by CuAAC reaction to afford sensing nanomaterials.

Protease responsive nanoprobes with tethered fluorogenic peptidyl 3-arylcoumarin substrates.

Protease responsive nanosensors were obtained by the attachment of unique green fluorescent bifunctional 3-arylcoumarin-derived fluorogenic substrates to poly(acrylamide-co-N-(3-aminopropyl)methacrylamide) nanoparticles, in which proteolysis results in substantial signal amplification.