Cationic liposome-DNA Complexes Research Articles

Preparation and Physical Characterization of DNA Binding Cationic Liposomes.

Cationic liposomes are routinely employed as one of the major nonviral transfecting agents for intracellular delivery of hydrophilic molecules such as nucleic acids, peptides, and proteins. Cationic liposomes when complexed with DNA form a strong positively charged cationic liposome-DNA complex or lipoplex. The chapter discusses, primarily, the major preparation technique for cationic liposomes and its physical characterization, with a focus on SYBR Green I dye exclusion assay and DNA encapsulation enhancement by freeze-thaw technique. SYBR Green I dye exclusion assay is a technique to determine the total amount of liposomal lipids required to bind a unit weight of DNA, which is critical for transfection experiments. Freeze-thaw technique on the other hand is one of the major techniques to improve DNA encapsulation efficiency in liposomes.

In Vitro CRISPR/Cas9 Transfection and Gene-Editing Mediated by Multivalent Cationic Liposome-DNA Complexes.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease 9 (Cas9) gene-editing offers exciting new therapeutic possibilities for disease treatment with a genetic etiology such as cancer, cardiovascular, neuronal, and immune disorders. However, its clinical translation is being hampered by the lack of safe, versatile, and effective nonviral delivery systems. Herein we report on the preparation and application of two cationic liposome–DNA systems (i.e., lipoplexes) for CRISPR/Cas9 gene delivery. For that purpose, two types of cationic lipids are used (DOTAP, monovalent, and MVL5, multivalent with +5e nominal charge), along with three types of helper lipids (DOPC, DOPE, and monoolein (GMO)). We demonstrated that plasmids encoding Cas9 and single-guide RNA (sgRNA), which are typically hard to transfect due to their large size (>9 kb), can be successfully transfected into HEK 293T cells via MVL5-based lipoplexes. In contrast, DOTAP-based lipoplexes resulted in very low transfection rates. MVL5-based lipoplexes presented the ability to escape from lysosomes, which may explain the superior transfection efficiency. Regarding gene editing, MVL5-based lipoplexes achieved promising GFP knockout levels, reaching rates of knockout superior to 35% for charge ratios (+/−) of 10. Despite the knockout efficiency being comparable to that of Lipofectamine 3000® commercial reagent, the non-specific gene knockout is more pronounced in MVL5-based formulations, probably resulting from the considerable cytotoxicity of these formulations. Altogether, these results show that multivalent lipid-based lipoplexes are promising CRISPR/Cas9 plasmid delivery vehicles, which by further optimization and functionalization may become suitable in vivo delivery systems.

Artificial Protein Coronas Enable Controlled Interaction with Corneal Epithelial Cells: New Opportunities for Ocular Drug Delivery.

Topical administration is the most convenient route for ocular drug delivery, but only a minor fraction is retained in the precorneal pocket. To overcome this limitation, numerous drug delivery systems (DDS) have been developed. The protein corona (PC) is the layer of biomolecules (e.g., proteins, sugars, lipids, etc.) that forms around DDS in physiological environments by non-covalent interaction. The PC changes the DDS physical–chemical properties, providing them with a completely novel biological identity. The specific involvement of PC in ocular drug delivery has not been addressed so far. To fulfill this gap, here we explored the interaction between a library of four cationic liposome-DNA complexes (lipoplexes) and mucin (MUC), one of the main components of the tear film. We demonstrate that MUC binds to the lipoplex surface shifting both their size and surface charge and reducing their absorption by primary corneal epithelial cells. To surpass such restrictions, we coated lipoplexes with two different artificial PCs made of Fibronectin (FBN) and Val-Gly-Asp (VGA) tripeptide that are recognized by receptors expressed on the ocular surface. Both these functionalizations remarkedly boosted internalization in corneal epithelial cells with respect to pristine (i.e., uncoated) lipoplexes. This opens the gateway for the exploitation of artificial protein corona in targeted ocular delivery, which will significantly influence the development of novel nanomaterials.

A mechanistic explanation of the inhibitory role of the protein corona on liposomal gene expression

The past three decades have witnessed fast advances in the use of cationic liposome-DNA complexes (lipoplexes) for gene delivery applications. However, no lipoplex formulation has reached into the clinical practice so far. The primary drawback limiting clinical use of lipoplexes is the lack of mechanistic understanding of their low transfection efficiency (TE) in vivo. In physiological environments, lipoplexes are coated by a protein corona (PC) that mediates the interactions with the cell machinery. Here we show that the formation of PC can change the interactions of multicomponent (MC) lipoplexes with our cell model (i.e., HeLa). At the highest lipoplex concentration, the formation of PC can reduce the TE of MC lipoplexes from 60% to <5%. Combining dynamic light scattering and synchrotron small-angle X-ray scattering (SAXS), we clarify that the formation of PC modifies physical-chemical properties of MC lipoplexes so as to affect their TE. Moreover, we examined single transfection barriers by a combination of fluorescence-activated cell sorting, single-cell real-time fluorescence confocal microscopy, and synchrotron SAXS. We demonstrate that PC formation has the ability to modify the relative contribution of caveolae-mediated endocytosis and macropinocytosis in lipoplexes uptake, in favor of the latter, increasing accumulation of PC-decorated lipoplexes into degradative lysosomal compartments. Finally, we report evidences that PC reduces the structural stability of lipoplexes against solubilization by cellular lipids, likely favoring premature DNA release and cytosolic digestion by DNAase. These combined effects revealed here offer a comprehensive mechanistic explanation on the reason behind reduction in gene expression of MC lipoplexes.

3D Columnar Phase of Stacked Short DNA Organized by Coherent Membrane Undulations.

We report on the discovery of a new organized lipid-nucleic acid phase upon intercalation of blunt duplexes of short DNA (sDNA) within cationic multilayer fluid membranes. End-to-end interactions between sDNA leads to columnar stacks. At high membrane charge density, with the inter-sDNA column spacing (dsDNA) comparable but larger than the diameter of sDNA, a 2D columnar phase (i.e., a 2D smectic) is found similar to the phase in cationic liposome-DNA complexes with long lambda-phage DNA. Remarkably, with increasing dsDNA as the membrane charge density is lowered, a transition is observed to a 3D columnar phase of stacked sDNA. This occurs even though direct DNA-DNA electrostatic interactions across layers are screened by diffusing cationic lipids near the phosphate groups of sDNA. Softening of the membrane bending rigidity (κ), which further promotes membrane undulations, significantly enhances the 3D columnar phase. These observations are consistent with a model by Schiessel and Aranda-Espinoza where local membrane undulations, due to electrostatically induced membrane wrapping around sDNA columns, phase lock from layer-to-layer, thereby precipitating coherent "crystal-like" undulations coupled to sDNA columns with long-range position and orientation order. The finding that this new phase is stable at large dsDNA and enhanced with decreasing κ is further supportive of the model where the elastic cost of membrane deformation per unit area around sDNA columns (∝ κh2/dsDNA4, h2 = sum of square of amplitudes of the inner and outer monolayer undulations) is strongly reduced relative to the favorable electrostatic attractions of partially wrapped membrane around sDNA columns. The findings have broad implications in the design of membrane-mediated assembly of functional nanoparticles in 3D.

Immunization against full-length protein and peptides from the Lutzomyia longipalpis sand fly salivary component maxadilan protects against Leishmania major infection in a murine model.

Leishmaniasis is an arthropod vectored disease causing considerable human morbidity and mortality. Vaccination remains the most realistic and practical means to interrupt the growing number and diversity of sand fly vectors and reservoirs of Leishmania. Since transmission of Leishmania is achieved exclusively by sand fly vectors via immune-modulating salivary substances, conventional vaccination requiring an unmodified host immune response for success are potentially destined to fail unless immunomodulatory factors are somehow neutralized. Using cationic liposome DNA complexes (CLDC) as an adjuvant system along with Lu. longipalpis sand fly salivary component maxadilan (MAX) as antigen (Ag), we show that mice are protected from the MAX-induced exacerbation of infection with Leishmania major (Lm). The CLDC adjuvant and alum were comparable in terms of lesion induration and decreased parasite burden, however the alum adjuvant imposed more inflammation at the injection site. BALB/c, C3H and C57BL/6 mice vaccinated with MAX-CLDC containing either the full-length MAX or peptides spanning the N- and C-terminal regions of MAX are protected against footpad challenges with Lm co-injected with MAX. When compared to unvaccinated controls, all strains of mice immunized with CLDC containing either peptides encompassing the first 20 N-terminal AA or those spanning the last 15 AA of the C-terminal domain of MAX demonstrated decreased parasite burden after 9 or 18 weeks post challenge with Lm + MAX. MAX-CLDC immunized mice showed increased IFNγ-secreting and decreased IL-4-secreting CD4+ cells in footpad-draining lymph nodes. Antisera from C-terminal peptide (P11) MAX-CLDC-vaccinated animals was capable of recognizing FL-MAX and its C-terminal domain and also blocked MAX-mediated reprogramming of bone marrow-derived dendritic cells (BM-DC) in vitro. This peptide vaccine targeting sand fly MAX, improves host immunity against MAX-mediated immunomodulation.

Prevention of liver tumor formation in woodchucks with established hepatocellular carcinoma by treatment with cationic liposome-DNA complexes

BackgroundApproximately 250 million people worldwide are chronically infected with hepatitis B virus (HBV) and more than half of the hepatocellular carcinoma (HCC) cases are attributed to this infection. As HCC has a high mortality rate, and current treatment options are remarkably limited, the development of new therapeutic treatment strategies is warranted.MethodsIn this study, woodchucks infected with woodchuck hepatitis virus (WHV), and with pre-existing liver tumors, were used as a model to investigate if complexes of cationic liposomes and non-coding DNA (JVRS-100) were effective in treatment of HCC.ResultsIt was observed that the high serum viral load that is present in a typical chronic WHV infection (i.e., approximately 100-fold higher than human viral loads) results in immune suppression and resistance to treatment with JVRS-100. Treatment of woodchucks with lower serum viral load that more closely matched with the viral load usually seen in human HBV infection appears a better model for immunotherapeutic development based on the responsiveness to JVRS-100 treatment. In the latter case, marked declines in WHV DNA and WHV surface antigen were determined over the 12-week treatment period and WHV markers stayed suppressed during most time points of the 12-week follow-up period. Even more remarkably, the formation of new liver tumors was not observed in woodchucks treated with a well-tolerated dose of JVRS-100, as compared to several new tumors that developed in vehicle-treated control animals.ConclusionsAlthough there was little decrease in the volumes of the liver tumors existing at the time of treatment, it is generally accepted that preventing the spread and metastasis of almost always fatal cancers such as HCC and thus, reducing it to a chronic and treatable disease can also be a successful therapeutic approach. The results in woodchucks warrant the investigation of JVRS-100 as an intervention to prevent liver cancer in patients chronically infected with HBV and at high risk for HCC development.

A cationic liposome–DNA complexes adjuvant (JVRS-100) enhances the immunogenicity and cross-protective efficacy of pre-pandemic influenza A (H5N1) vaccine in ferrets

Influenza A (H5N1) viruses continue to pose a public health threat. As inactivated H5N1 vaccines are poorly immunogenic, adjuvants are needed to improve the immunogenicity of H5N1 vaccine in humans. Here, we investigated the immunogenicity and cross-protective efficacy in ferrets of a clade 2.2-derived vaccine with addition of JVRS-100, an adjuvant consisting of cationic liposome–DNA complexes (CLDC). After the first vaccination, significantly higher levels of hemagglutination-inhibition (HAI) and neutralizing antibody titers were detected in ferrets immunized with adjuvanted vaccine compared to unadjuvanted vaccine. Following a second dose of adjuvanted vaccine, HAI antibody titers of≥40 were detected against viruses from multiple H5N1 clades. HAI antibodies against newly isolated H5N2 and H5N8 viruses were also augmented by JVRS-100. Ferrets were challenged with a heterologous H5N1 virus. All ferrets that received two doses of adjuvanted vaccine exhibited mild illness, significantly reduced nasal wash virus titers and protection from lethal challenge. In contrast, ferrets that received unadjuvanted vaccine showed greater weight loss, high viral titers and 3 of 6 animals succumbed to the lethal challenge. Our results indicate that the addition of JVRS-100 to H5N1 vaccine enhanced immunogenicity and cross-protection against lethal H5N1 virus disease in ferrets. JVRS-100 warrants further investigation as a potential adjuvant for influenza vaccines.

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.

Liposomes as vaccine delivery systems: a review of the recent advances.

Liposomes and liposome-derived nanovesicles such as archaeosomes and virosomes have become important carrier systems in vaccine development and the interest for liposome-based vaccines has markedly increased. A key advantage of liposomes, archaeosomes and virosomes in general, and liposome-based vaccine delivery systems in particular, is their versatility and plasticity. Liposome composition and preparation can be chosen to achieve desired features such as selection of lipid, charge, size, size distribution, entrapment and location of antigens or adjuvants. Depending on the chemical properties, water-soluble antigens (proteins, peptides, nucleic acids, carbohydrates, haptens) are entrapped within the aqueous inner space of liposomes, whereas lipophilic compounds (lipopeptides, antigens, adjuvants, linker molecules) are intercalated into the lipid bilayer and antigens or adjuvants can be attached to the liposome surface either by adsorption or stable chemical linking. Coformulations containing different types of antigens or adjuvants can be combined with the parameters mentioned to tailor liposomal vaccines for individual applications. Special emphasis is given in this review to cationic adjuvant liposome vaccine formulations. Examples of vaccines made with CAF01, an adjuvant composed of the synthetic immune-stimulating mycobacterial cordfactor glycolipid trehalose dibehenate as immunomodulator and the cationic membrane forming molecule dimethyl dioctadecylammonium are presented. Other vaccines such as cationic liposome-DNA complexes (CLDCs) and other adjuvants like muramyl dipeptide, monophosphoryl lipid A and listeriolysin O are mentioned as well. The field of liposomes and liposome-based vaccines is vast. Therefore, this review concentrates on recent and relevant studies emphasizing current reports dealing with the most studied antigens and adjuvants, and pertinent examples of vaccines. Studies on liposome-based veterinary vaccines and experimental therapeutic cancer vaccines are also summarized.

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. We 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). In 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. Two-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.

The Role of Cytoskeleton Networks on lipid-mediated Delivery of DNA

Lipid-mediated delivery of DNA is hindered by extracellular and intracellular barriers that significantly reduce the transfection efficiency of synthetic nonviral vectors. In this study we investigated the role of the actin and microtubule networks on the uptake and cytoplasmic transport of multicomponent cationic liposome-DNA complexes in CHO-K1 live cells by means of confocal laser scanning microscopy and 3D single particle tracking. Treatment with actin (latrunculin B)- and microtubule-disrupting (nocodazole) reagents indicated that intracellular trafficking of complexes predominantly involves microtubule-dependent active transport. We found that the actin network has a major effect on the initial uptake of complexes, while the microtubule network is mainly responsible for the subsequent active transportation to the lysosomes. Collectively, a strategy to improve the efficiency of lipid gene vectors can be formulated. We could find a lipid formulation that allows the nanoparticles to avoid the microtubule pathway to lysosomes.

Structural evolution of environmentally responsive cationic liposome-DNA complexes with a reducible lipid linker.

Environmentally responsive materials (i.e., materials that respond to changes in their environment with a change in their properties or structure) are attracting increasing amounts of interest. We recently designed and synthesized a series of cleavable multivalent lipids (CMVLn, with n = 2-5 being the number of positive headgroup charges at full protonation) with a disulfide bond in the linker between their cationic headgroup and hydrophobic tails. The self-assembled complexes of the CMVLs and DNA are a prototypical environmentally responsive material, undergoing extensive structural rearrangement when exposed to reducing agents. We investigated the structural evolution of CMVL-DNA complexes at varied complex composition, temperature, and incubation time using small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS). A related lipid with a stable linker, TMVL4, was used as a control. In a nonreducing environment, CMVL-DNA complexes form the lamellar (L(α)(C)) phase, with DNA rods sandwiched between lipid bilayers. However, new self-assembled phases form when the disulfide linker is cleaved by dithiothreitol or the biologically relevant reducing agent glutathione. The released DNA and cleaved CMVL headgroups form a loosely organized phase, giving rise to a characteristic broad SAXS correlation profile. CMVLs with high headgroup charge also form condensed DNA bundles. Intriguingly, the cleaved hydrophobic tails of the CMVLs reassemble into tilted chain-ordered L(β') phases upon incubation at physiological temperature (37 °C), as indicated by characteristic WAXS peaks. X-ray scattering further reveals that two of the three phases (L(βF), L(βL), and L(βI)) constituting the L(β') phase coexist in these samples. The described system may have applications in lipid-based nanotechnologies.

Influence of multilayer rhBMP‐2 DNA coating on the proliferation and differentiation of MC3T3‐E1 cells seeded on roughed titanium surface

For bone morphogenetic protein (BMP) gene therapy to be a viable approach for enhancing implant osseointegration clinically, requires the development of efficient nonviral delivery vectors that can coat the implant. This study evaluated a multilayer cationic liposome-DNA complex (LDc) coating as a delivery vehicle for recombinant human BMP-2 (rhBMP-2). Multilayered coatings, comprising hyaluronic acid (HA) and LDc, were fabricated onto titanium using a layer-by-layer (LBL) assembly technique. Preosteoblastic MC3T3-E1 cells were cultured on the roughened titanium surfaces coated with multilayers of HA/LDc, or on uncoated or HA/liposome only surfaces as controls. The amount of rhBMP-2 secreted by the MC3T3-E1 cells and the effect of the various surfaces on cell viability, proliferation, alkaline phosphatase (ALP) activity, osteocalcin (OC) secretion, and calcium deposition were evaluated. Messenger RNA levels of OC, ALP, Runx2, and Osx were also investigated. The results demonstrated that rhBMP-2 protein secreted into culture medium at 3 days was significantly higher than control groups. MC3T3-E1 cells cultured on the HA/LDc coating displayed significantly higher ALP activity and OC secretion at 7 days and 14 days culture, respectively. MC3T3-E1 cells cultured on HA/LDc upregulated expression of the osteoblast differentiation markers, especially on days 12 for OC and on days 6 and 12 for ALP and Osx. In conclusion, MC3T3-E1 cell cultured on the multilayer HA/LDc coating surface can secret rhBMP-2 protein and the protein levels were effective in inducing early osteogenic differentiation.

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.

Intracellular trafficking of cationic liposome-DNA complexes in living cells.

Three-dimensional single-particle tracking (SPT) was used to calculate the mean square displacement (MSD) and the diffusion coefficients of multicomponent cationic liposome-DNA complexes (lipoplexes) in CHO-K1 living cells. In untreated (NT) control cells, we found that the intracellular lipoplex motion was either directed or Brownian with active transportation being definitely more frequent (more than 70%) than Brownian diffusion. The MSD analysis was supported by the calculation of the three-dimensional asphericity, A3, which was close to unity, denoting the preponderant occurrence of movement along a direction. To elucidate the role of the cytoskeleton structure in the lipoplex trafficking, cells were treated with cytoskeleton (actin microfilaments and microtubules) polymerization inhibitors (latrunculin B and nocodazole, respectively). When cells were treated with inhibitors, the lipoplex movement tended towards a random walk at the expense of directed motion. The disassembly of microtubules had a stronger effect on the reduction of directional movement than that of actin microfilaments. Relevance of the results for enhanced gene delivery is discussed.

Environmentally Responsive Cationic Liposome-DNA Complexes for Cell Delivery

Cationic lipids continue to attract attention as synthetic nucleic acid (NA) vectors, and are broadly used for gene transfection (with plasmid DNA) and silencing (with siRNA) including applications in clinical trials. This study elucidates the superior behavior of environmentally responsive multivalent cationic lipids containing degradable disulfide bond (labeled CMVLs). Remarkably, characterization of the complexes prepared with CMVLs containing degradable disulfide bond (compared to the non-degradable analogs) reveals the significant effects of stability control.

Cationic liposome–DNA complexes (CLDC) adjuvant enhances the immunogenicity and cross-protective efficacy of a pre-pandemic influenza A H5N1 vaccine in mice

The development of pre-pandemic influenza A H5N1 vaccines that confer both antigen-sparing and cross-clade protection are a high priority given the limited worldwide capacity for influenza vaccine production, and the antigenic and genetic heterogeneity of circulating H5N1 viruses. The inclusion of potent adjuvants in vaccine formulations may achieve both of these aims. Here we show that the addition of JVRS-100, an adjuvant consisting of cationic liposome-DNA complexes (CLDC) to a clade 1-derived H5N1 split vaccine induced significantly higher virus-specific antibody than unadjuvanted formulations, with a >30-fold dose-sparing effect and induction of increased antigen-specific CD4(+) T-cell responses in mice. All mice that received one dose of adjuvanted vaccine and subsequent H5N1 viral challenges exhibited mild illness, lower lung viral titers, undetectable spleen and brain viral titers, and 100% survival after either homologous clade 1 or heterologous clade 2 H5N1 viral challenges, whereas unadjuvanted vaccine recipients showed significantly increased weight loss, viral titers, and mortality. The protective immunity induced by JVRS-100 adjuvanted H5N1 vaccine was shown to last for over one year without significant waning. Thus, JVRS-100 adjuvanted H5N1 vaccine elicited enhanced humoral and T-cell responses, dose-sparing, and cross-clade protection in mice. CLDC holds promise as an adjuvant for human pre-pandemic inactivated H5N1 vaccines.

Two-Dimensional Packing of Short DNA with Nonpairing Overhangs in Cationic Liposome–DNA Complexes: From Onsager Nematics to Columnar Nematics with Finite-Length Columns

We report the formation of liquid crystalline (LC) phases of short double-stranded DNA with nonpairing (nonsticky) overhangs, confined between two-dimensional (2D) lipid bilayers of cationic liposome-DNA complexes. In a landmark study (Science2007, 318, 1276), Nakata et al. reported on the discovery of strong end-to-end stacking interactions between short DNAs (sDNAs) with blunt ends, leading to the formation of 3D nematic (N) and columnar LC phases. Employing synchrotron small-angle X-ray scattering, we have studied the interplay between shape anisotropy-induced and DNA end-to-end interaction-induced N ordering for 11, 24, and 48 bp sDNA rods with single-stranded oligo-thymine (T) overhangs modulating the end-to-end interactions. For suppressed stacking interactions with 10-T overhangs, the volume fraction of sDNA at which the 2D isotropic (I)-to-N transition occurs for 24 and 48 bp sDNA rods depended on their length-to-width (L/D) shape anisotropy, qualitatively consistent with Onsager's theory for the entropic alignment of rigid rods. As the overhang length is reduced from 10 to 5 and 2 T for 24 and 48 bp sDNA, the N-to-I transition occurs at lower volume fractions, indicating the onset of some degree of end-to-end stacking interactions. The 11 bp sDNA rods with 5- and 10-T overhangs remain in the I phase, consistent with their small shape anisotropy (L/D ≈ 1.9) below the limit for Onsager LC ordering. Unexpectedly, in contrast to the behavior of 24 and 48 bp sDNA, the end-to-end interactions between 11 bp sDNA rods with 2-T overhangs set in dramatically, and a novel 2D columnar N phase (N(C)) with finite-length columns formed. The building blocks of this phase are comprised of 1D stacks of (on average) four 11 bp DNA-2T rods with an effective L(stacked)/D ≈ 8.2. Our findings have implications for the DNA-directed assembly of nanoparticles on 2D platforms via end-to-end interactions and in designing optimally packed LC phases of short anisotropic biomolecules (such as peptides and short-interfering RNAs) on nanoparticle membranes, which are used in gene silencing and chemical delivery.