Ramsey N. Majzoub

Endosomal escape and transfection efficiency of PEGylated cationic liposome-DNA complexes prepared with an acid-labile PEG-lipid.

Cationic liposome-DNA (CL-DNA) complexes are being pursued as nonviral gene delivery systems for use in applications that include clinic trials. However, to compete with viral vectors for systemic delivery in vivo, their efficiencies and pharmacokinetics need to be improved. The addition of poly (ethylene glycol)-lipids (PEGylation) prolongs circulation lifetimes of liposomes, but inhibits cellular uptake and endosomal escape of CL-DNA complexes. We show that this limits their transfection efficiency (TE) in a manner dependent on the amount of PEG-lipid, the lipid/DNA charge ratio, and the lipid membrane charge density. To improve endosomal escape of PEGylated CL-DNA complexes, we prepared an acid-labile PEG-lipid (HPEG2K-lipid, PEG MW 2000) which is designed to lose its PEG chains at the pH of late endosomes. The HPEG2K-lipid and a similar but acid-stable PEG-lipid were used to prepare PEGylated CL-DNA complexes. TLC and dynamic light scattering showed that HPEG2K-CL-DNA complexes are stable at pH 7.4 for more than 24 h, but the PEG chains are cleaved at pH 5 within 1 h, leading to complex aggregation. The acid-labile HPEG2K-CL-DNA complexes showed enhanced TE over complexes stabilized with the acid-stable PEG-lipid. Live-cell imaging showed that both types of complexes were internalized to quantitatively similar particle distributions within the first 2 h of incubation with cells. Thus, we attribute the increased TE of the HPEG2K-CL-DNA complexes to efficient endosomal escape, enabled by the acid-labile HPEG2K-lipid which sheds its PEG chains in the low pH environment of late endosomes, effectively switching on the electrostatic interactions that promote fusion of the membranes of complex and endosome.

Synthesis and Characterization of Degradable Multivalent Cationic Lipids with Disulfide-Bond Spacers for Gene Delivery

Gene therapy provides powerful new approaches to curing a large variety of diseases, which are being explored in ongoing worldwide clinical trials. To overcome the limitations of viral gene delivery systems, synthetic nonviral vectors such as cationic liposomes (CLs) are desirable. However, improvements of their efficiency at reduced toxicity and a better understanding of their mechanism of action are required. We present the efficient synthesis of a series of degradable multivalent cationic lipids (CMVLn, n=2 to 5) containing a disulfide bond spacer between headgroup and lipophilic tails. This spacer is designed to be cleaved in the reducing milieu of the cytoplasm and thus decrease lipid toxicity. Small angle X-ray scattering demonstrates that the initially formed lamellar phase of CMVLn-DNA complexes completely disappears when reducing agents such as DTT or the biologically relevant reducing peptide glutathione are added to mimic the intracellular milieu. The CMVLs (n=3 to 5) exhibit reduced cytotoxicity and transfect mammalian cells with efficiencies comparable to those of highly efficient non-degradable analogs and benchmark commercial reagents such as Lipofectamine 2000. Thus, our results demonstrate that degradable disulfide spacers may be used to reduce the cytotoxicity of synthetic nonviral gene delivery carriers without compromising their transfection efficiency.

Cationic liposome–nucleic acid complexes for gene delivery and gene silencing

Cationic liposomes (CLs) are studied worldwide as carriers of DNA and short interfering RNA (siRNA) for gene delivery and gene silencing, and related clinical trials are ongoing. Optimization of transfection efficiency and silencing efficiency by cationic liposome carriers requires a comprehensive understanding of the structures of CL-nucleic acid complexes and the nature of their interactions with cell membranes as well as events leading to release of active nucleic acids within the cytoplasm. Synchrotron x-ray scattering has revealed that CL-nucleic acid complexes spontaneously assemble into distinct liquid crystalline phases including the lamellar, inverse hexagonal, hexagonal, and gyroid cubic phases, and fluorescence microscopy has revealed CL-DNA pathways and interactions with cells. The combining of custom synthesis with characterization techniques and gene expression and silencing assays has begun to unveil structure-function relations in vitro. As a recent example, this review will briefly describe experiments with surface-functionalized PEGylated CL-DNA nanoparticles. The functionalization, which is achieved through custom synthesis, is intended to address and overcome cell targeting and endosomal escape barriers to nucleic acid delivery faced by PEGylated nanoparticles designed for in vivo applications.

Uptake and transfection efficiency of PEGylated cationic liposome–DNA complexes with and without RGD-tagging

Steric stabilization of cationic liposome-DNA (CL-DNA) complexes is required for in vivo applications such as gene therapy. PEGylation (PEG: poly(ethylene glycol)) of CL-DNA complexes by addition of PEG2000-lipids yields sterically stabilized nanoparticles but strongly reduces their gene delivery efficacy. PEGylation-induced weakening of the electrostatic binding of CL-DNA nanoparticles to cells (leading to reduced uptake) has been considered as a possible cause, but experimental results have been ambiguous. Using quantitative live-cell imaging in vitro, we have investigated cell attachment and uptake of PEGylated CL-DNA nanoparticles with and without a custom synthesized RGD-peptide grafted to the distal ends of PEG2000-lipids. The RGD-tagged nanoparticles exhibit strongly increased cellular attachment as well as uptake compared to nanoparticles without grafted peptide. Transfection efficiency of RGD-tagged PEGylated CL-DNA NPs increases by about an order of magnitude between NPs with low and high membrane charge density (σM; the average charge per unit area of the membrane; controlled by the molar ratio of cationic to neutral lipid), even though imaging data show that uptake of RGD-tagged particles is only slightly enhanced by high σM. This suggests that endosomal escape and, as a result, transfection efficiency of RGD-tagged NPs is facilitated by high σM. We present a model describing the interactions between PEGylated CL-DNA nanoparticles and the anionic cell membrane which shows how the PEG grafting density and membrane charge density affect adhesion of nanoparticles to the cell surface.

Optimizing Cationic and Neutral Lipids for Efficient Gene Delivery at High Serum Content

Cationic liposome (CL)–DNA complexes are promising gene delivery vectors with potential application in gene therapy. A key challenge in creating CL–DNA complexes for application is that their transfection efficiency (TE) is adversely affected by serum. In particular, little is known about the effects of a high serum content on TE, even though this may provide design guidelines for application in vivo.

Cationic liposome-nucleic acid nanoparticle assemblies with applications in gene delivery and gene silencing.

Cationic liposomes (CLs) are synthetic carriers of nucleic acids in gene delivery and gene silencing therapeutics. The introduction will describe the structures of distinct liquid crystalline phases of CL–nucleic acid complexes, which were revealed in earlier synchrotron small-angle X-ray scattering experiments. When mixed with plasmid DNA, CLs containing lipids with distinct shapes spontaneously undergo topological transitions into self-assembled lamellar, inverse hexagonal, and hexagonal CL–DNA phases. CLs containing cubic phase lipids are observed to readily mix with short interfering RNA (siRNA) molecules creating double gyroid CL–siRNA phases for gene silencing. Custom synthesis of multivalent lipids and a range of novel polyethylene glycol (PEG)-lipids with attached targeting ligands and hydrolysable moieties have led to functionalized equilibrium nanoparticles (NPs) optimized for cell targeting, uptake or endosomal escape. Very recent experiments are described with surface-functionalized PEGylated CL–DNA NPs, including fluorescence microscopy colocalization with members of the Rab family of GTPases, which directly reveal interactions with cell membranes and NP pathways. In vitro optimization of CL–DNA and CL–siRNA NPs with relevant primary cancer cells is expected to impact nucleic acid therapeutics in vivo. This article is part of the themed issue ‘Soft interfacial materials: from fundamentals to formulation’.

Fluorescence microscopy colocalization of lipid-nucleic acid nanoparticles with wildtype and mutant Rab5-GFP: A platform for investigating early endosomal events

Endosomal entrapment is known to be a major bottleneck to successful cytoplasmic delivery of nucleic acids (NAs) using cationic liposome-NA nanoparticles (NPs). Quantitative measurements of distributions of NPs within early endosomes (EEs) have proven difficult due to the sub-resolution size and short lifetime of wildtype EEs. In this study we used Rab5-GFP, a member of the large family of GTPases which cycles between the plasma membrane and early endosomes, to fluorescently label early endosomes. Using fluorescence microscopy and quantitative image analysis of cells expressing Rab5-GFP, we found that at early time points (t<1h), only a fraction (≈35%) of RGD-tagged NPs (which target cell surface integrins) colocalize with wildtype EEs, independent of the NPs membrane charge density. In comparison, a GTP-hydrolysis deficient mutant, Rab5-Q79L, which extends the size and lifetime of EEs yielding giant early endosomes (GEEs), enabled us to resolve and localize individual NPs found within the GEE lumen. Remarkably, nearly all intracellular NPs are found to be trapped within GEEs implying little or no escape at early time points. The observed small degree of colocalization of NPs and wildtype Rab5 is consistent with recycling of Rab5-GDP to the plasma membrane and not indicative of NP escape from EEs. Taken together, our results show that endosomal escape of PEGylated nanoparticles occurs downstream of EEs i.e., from late endosomes/lysosomes. Our studies also suggest that Rab5-Q79L could be used in a robust imaging assay which allows for direct visualization of NP interactions with the luminal membrane of early endosomes.

PEGylated Cationic Liposome - DNA Complexation in Brine is Pathway-Dependent.

Cationic liposome-DNA (CL-DNA) complexes, are regarded as promising materials for safe and efficient delivery of genes for therapeutical applications. In order to be used in vivo, these complexes may be coated with a hydrophilic polymer (e.g. polyethylene-glycol, PEG) that provides steric stabilization towards adhesion of proteins and removal by the immune system. In this work we study the influence of the initial salt concentration (Cs) - which modulates the electrostatic interaction between oppositely charged vesicles and DNA - on the structure and stability of PEGylated CL-DNA particles. Previous small-angle X-ray scattering has shown that if non-PEGylated or PEGylated CL-DNA lamellar complexes are prepared in water, their structure is well defined with a high number of lipid membrane-DNA layers (larger than 20). Here we show that if these complexes are transferred to saline media (150mM NaCl or DMEM, both near physiological conditions), this structure remains nearly unchanged. Conversely, if PEGylated complexes are prepared in saline media, their lamellar structure is much looser, with fewer number of layers. This pathway dependent behavior of PEGylated complex formation in brine is modulated by the liposome membrane charge density and the mole fraction of PEG 2000 in the membranes, with the average number of layers decreasing with increasing Cs and in going from 5mol% to 10mol% PEG-lipid. Each of these structures (high and low number of layers) is stable with time, suggesting that despite complex formation being thermodynamically favored, the complexation process in PEGylated membranes, which determines the number of layers per particle, is kinetically controlled. In the extreme case (when polymer repulsions from 10mol% PEG-lipid are maximized and electrostatic attraction between PEGylated CLs and DNA are minimized at low membrane charge density) complex formation is suppressed at high Cs=150mM.

Synthesis of linear and cyclic peptide-PEG-lipids for stabilization and targeting of cationic liposome-DNA complexes.

Because nucleic acids (NAs) have immense potential value as therapeutics, the development of safe and effective synthetic NA vectors continues to attract much attention. In vivo applications of NA vectors require stabilized, nanometer-scale particles, but the commonly used approaches of steric stabilization with a polymer coat (e.g., PEGylation; PEG=poly(ethylene glycol)) interfere with attachment to cells, uptake, and endosomal escape. Conjugation of peptides to PEG-lipids can improve cell attachment and uptake for cationic liposome-DNA (CL-DNA) complexes. We present several synthetic approaches to peptide-PEG-lipids and discuss their merits and drawbacks. A lipid-PEG-amine building block served as the common key intermediate in all synthetic routes. Assembling the entire peptide-PEG-lipid by manual solid phase peptide synthesis (employing a lipid-PEG-carboxylic acid) allowed gram-scale synthesis but is mostly applicable to linear peptides connected via their N-terminus. Conjugation via thiol-maleimide or strain-promoted (copper-free) azide-alkyne cycloaddition chemistry is highly amenable to on-demand preparation of peptide-PEG-lipids, and the appropriate PEG-lipid precursors are available in a single chemical step from the lipid-PEG-amine building block. Azide-alkyne cycloaddition is especially suitable for disulfide-bridged peptides such as iRGD (cyclic CRGDKGPDC). Added at 10 mol% of a cationic/neutral lipid mixture, the peptide-PEG-lipids stabilize the size of CL-DNA complexes. They also affect cell attachment and uptake of nanoparticles in a peptide-dependent manner, thereby providing a platform for preparing stabilized, affinity-targeted CL-DNA nanoparticles.

Patterned Threadlike Micelles and DNA-Tethered Nanoparticles: A Structural Study of PEGylated Cationic Liposome–DNA Assemblies

The self-assembly of oppositely charged biomacromolecules has been extensively studied due to its pertinence in the design of functional nanomaterials. Using cryo electron microscopy (cryo-EM), optical light scattering, and fluorescence microscopy, we investigated the structure and phase behavior of PEGylated (PEG: poly(ethylene glycol)) cationic liposome-DNA nanoparticles (CL-DNA NPs) as a function of DNA length, topology (linear and circular), and ρ(chg) (the molar charge ratio of cationic lipid to anionic DNA). Although all NPs studied exhibited lamellar internal nanostructure, NPs formed with short (∼2 kbps), linear, polydisperse DNA were defect-rich and contained smaller domains. Unexpectedly, we found distinctly different equilibrium structures away from the isoelectric point. At ρ(chg) > 1, in the excess cationic lipid regime, threadlike micelles rich in PEG-lipid were found to coexist with NPs, cationic liposomes, and spherical micelles. At high concentrations these PEGylated threadlike micelles formed a well-ordered, patterned morphology with highly uniform intermicellar spacing. At ρ(chg) < 1, in the excess DNA regime and with no added salt, individual NPs were tethered together via long, linear DNA (48 kbps λ-phage DNA) into a biopolymer-mediated floc. Our results provide insight into what equilibrium nanostructures can form when oppositely charged macromolecules self-assemble in aqueous media. Self-assembled, well-ordered threadlike micelles and tethered nanoparticles may have a broad range of applications in bionanotechnology, including nanoscale lithograpy and the development of lipid-based multifunctional nanoparticle networks.