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Hierarchical assembly of building blocks via competing, orthogonal interactions is a hallmark of many of nature's composite materials that do not require highly specific ligand-receptor interactions. To mimic this assembly mechanism requires the development of building blocks capable of tunable interactions. In the present work, we explored the interplay between repulsive (steric and electrostatic) and attractive hydrophobic forces. The designed building blocks allow hydrophobic forces to effectively act at controlled, large distances, to create and tune the assembly of membrane-based building blocks under dilute conditions, and to affect their interactions with cellular membranes via physical cross-bridges. Specifically, we employed double-end-anchored poly(ethylene glycol)s (DEA-PEGs)-hydrophilic PEG tethers with hydrophobic tails on both ends. Using differential-interference-contrast optical microscopy, synchrotron small-angle X-ray scattering (SAXS), and cryogenic electron microscopy, we investigated the ability of DEA-PEGs to mediate assembly in the dilute regime on multiple length scales and on practical time scales. The PEG length, anchor hydrophobicity, and molar fraction of DEA-PEG molecules within a membrane strongly affect the assembly properties. Additional tuning of the intermembrane interactions can be achieved by adding repulsive interactions via PEG-lipids (steric) or cationic lipids to the DEA-PEG-mediated attractions. While the optical and electron microscopy imaging methods provided qualitative evidence of the ability of DEA-PEGs to assemble liposomes, the SAXS measurements and quantitative line-shape analysis in dilute preparations demonstrated that the ensemble average of loosely organized liposomal assemblies maintains DEA-PEG concentration-dependent tethering on defined nanometer length scales. For cationic liposome-DNA nanoparticles (CL-DNA NPs), aggregation induced by DEA-PEGs decreased internalization of NPs by cells, but tuning the DEA-PEG-induced attractions by adding repulsive steric interactions via PEG-lipids limited aggregation and increased NP uptake. Furthermore, confocal microscopy imaging together with colocalization studies with Rab11 and LysoTracker as markers of intracellular pathways showed that modifying CL-DNA NPs with DEA-PEGs alters their interactions with the plasma and endosomal membranes.

The free-solvent-based (FSB) model and osmotic pressure were used to probe the ion binding and protein hydration for self-crowded bovine serum albumin in 0.15 M NaF, NaCl, NaI, and NaSCN solutions. All experiments were conducted with solutions at pH 7.4. The regressed results of the FSB model behavior to the measured osmotic pressure were excellent, albeit, the osmotic pressure data for NaSCN were noisy. The resulting ion binding and hydration were realistic values and the covariance of the two parameters was exceptionally low, providing substantial credibility to the FSB model. The results showed that the kosmotropic F- and neutral Cl- solutions generated significantly higher ion binding and protein hydration than the chaotropic solutions of I- and SCN-. Further, the ionic strength ratio and resulting hydration implied that the chaotropic solutions had substantially higher aggregation than the other salts investigated. Overall, the FSB model provides an additional, complementary tool to contribute to the analysis of crowded protein solutions relative to anions in the Hofmeister series as it can interrogate crowded solutions directly; something that is not possible with many measurement techniques.

Cationic liposome-nucleic acid (CL-NA) complexes, which form spontaneously, are a highly modular gene delivery system. These complexes can be sterically stabilized via PEGylation [PEG: poly (ethylene glycol)] into nanoparticles (NPs) and targeted to specific tissues and cell types via the conjugation of an affinity ligand. However, there are currently no guidelines on how to effectively navigate the large space of compositional parameters that modulate the specific and nonspecific binding interactions of peptide-targeted NPs with cells. Such guidelines are desirable to accelerate the optimization of formulations with novel peptides. Using PEG-lipids functionalized with a library of prototypical tumor-homing peptides, we varied the peptide density and other parameters (binding motif, peptide charge, CL/DNA charge ratio) to study their effect on the binding and uptake of the corresponding NPs. We used flow cytometry to quantitatively assess binding as well as internalization of NPs by cultured cancer cells. Surprisingly, full peptide coverage resulted in less binding and internalization than intermediate coverage, with the optimum coverage varying between cell lines. In, addition, our data revealed that great care must be taken to prevent nonspecific electrostatic interactions from interfering with the desired specific binding and internalization. Importantly, such considerations must take into account the charge of the peptide ligand as well as the membrane charge density and the CL/DNA charge ratio. To test our guidelines, we evaluated the in vivo tumor selectivity of selected NP formulations in a mouse model of peritoneally disseminated human gastric cancer. Intraperitoneally administered peptide-tagged CL-DNA NPs showed tumor binding, minimal accumulation in healthy control tissues, and preferential penetration of smaller tumor nodules, a highly clinically relevant target known to drive recurrence of the peritoneal cancer.

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'.

Cationic liposomes (CLs) are widely studied as carriers of DNA and short-interfering RNA for gene delivery and silencing, and related clinical trials are ongoing. Optimization of transfection efficiency (TE) requires understanding of CL-nucleic acid nanoparticle (NP) interactions with cells, NP endosomal pathways, endosomal escape, and events leading to release of active nucleic acid from the lipid carrier. Here, we studied endosomal pathways and TE of surface-functionalized CL-DNA NPs in PC-3 prostate cancer cells displaying overexpressed integrin and neuropilin-1 receptors. The NPs contained RGD-PEG-lipid or RPARPAR-PEG-lipid, targeting integrin, and neuropilin-1 receptors, respectively, or control PEG-lipid. Fluorescence colocalization using Rab11-GFP and Lysotracker enabled simultaneous colocalization of NPs with recycling endosome (Rab11) and late endosome/lysosome (Rab7/Lysotracker) pathways at increasing mole fractions of pentavalent MVL5 (+5 e) at low (10 mol %), high (50 mol %), and very high (70 mol %) membrane charge density (σM). For these cationic NPs (lipid/DNA molar charge ratio, ρchg = 5), the influence of membrane charge density on pathway selection and transfection efficiency is similar for both peptide-PEG NPs, although, quantitatively, the effect is larger for RGD-PEG compared to RPARPAR-PEG NPs. At low σM, peptide-PEG NPs show preference for the recycling endosome over the late endosome/lysosome pathway. Increases in σM, from low to high, lead to decreases in colocalization with recycling endosomes and simultaneous increases in colocalization with the late endosome/lysosome pathway. Combining colocalization and functional TE data at low and high σM shows that higher TE correlates with a larger fraction of NPs colocalized with the late endosome/lysosome pathway while lower TE correlates with a larger fraction of NPs colocalized with the Rab11 recycling pathway. The findings lead to a hypothesis that increases in σM, leading to enhanced late endosome/lysosome pathway selection and higher TE, result from increased nonspecific electrostatic attractions between NPs and endosome luminal membranes, and conversely, enhanced recycling pathway for NPs and lower TE are due to weaker attractions. Surprisingly, at very high σM, the inverse relation between the two pathways observed at low and high σM breaks down, pointing to a more complex NP pathway behavior.

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.

Current activity in developing synthetic carriers of nucleic acids (NA) and small molecule drugs for therapeutic applications is unprecedented. One promising class of synthetic vectors for the delivery of therapeutic NA is PEGylated cationic liposome (CL)-NA nanoparticles (NPs). Chemically modified PEG-lipids can be used to surface-functionalize lipid-NA nanoparticles, allowing researchers to design active nanoparticles that can overcome the various intracellular and extracellular barriers to efficient delivery. Optimization of these functionalized vectors requires a comprehensive understanding of their intracellular pathways. In this chapter we present two distinct methods for investigating the intracellular activity of PEGylated CL-NA NPs using quantitative analysis with fluorescence microscopy.The first method, spatial localization, describes how to prepare fluorescently labeled CL-NA NPs, perform fluorescence microscopy and properly analyze the data to measure the intracellular distribution of nanoparticles and fluorescent signal. We provide software which allows data from multiple cells to be averaged together and yield statistically significant results. The second method, fluorescence colocalization, describes how to label endocytic organelles via Rab-GFPs and generate micrographs for software-assisted NP-endocytic marker colocalization measurements. These tools will allow researchers to study the endosomal trafficking of CL-NA NPs which can guide their design and improve their efficiency.

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.

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

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.

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.

BackgroundCationic 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.MethodsWe prepared CL-DNA complexes in which we varied the neutral lipid [1,2-dioleoyl-sn-glycerophosphatidylcholine, glycerol-monooleate (GMO), cholesterol], the headgroup charge and chemical structure of the cationic lipid, and the ratio of neutral to cationic lipid; we then measured the TE of these complexes as a function of serum content and assessed their cytotoxicity. We tested selected formulations in two human cancer cell lines (M21/melanoma and PC-3/prostate cancer).ResultsIn the absence of serum, all CL-DNA complexes of custom-synthesized multivalent lipids show high TE. Certain combinations of multivalent lipids and neutral lipids, such as MVL5(5+)/GMO-DNA complexes or complexes based on the dendritic-headgroup lipid TMVLG3(8+) exhibited high TE both in the absence and presence of serum. Although their TE still dropped to a small extent in the presence of serum, it reached or surpassed that of benchmark commercial transfection reagents, particularly at a high serum content.ConclusionsTwo-component vectors (one multivalent cationic lipid and one neutral lipid) can rival or surpass benchmark reagents at low and high serum contents (up to 50%, v/v). We propose guidelines for optimizing the serum resistance of CL-DNA complexes based on a given cationic 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.

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.