Use Of Cationic Lipids Research Articles

Protein-liposome interactions: the impact of surface charge and fluidisation effect on protein binding

At the dawn of a new nanotechnological era in the pharmaceutical field, it is very important to examine and understand all the aspects that influence in vivo behaviour of nanoparticles. In this point of view, the interactions between serum proteins and liposomes with incorporated anionic, cationic, and/or PEGylated lipids were investigated to elucidate the role of surface charge and bilayer fluidity in protein corona’s formation. 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), hydrogenated soybean phosphatidylcholine (HSPC), and 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine (DSPC) liposomes with the presence or absence of 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DPPG), 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (chloride salt) (DOTAP), and/or 1,2-dipalmitoylsn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (DPPE-PEG 5000) lipids were prepared by the thin-film hydration method. The evaluation of their biophysical characteristics was enabled by differential scanning calorimetry and dynamic and electrophoretic light scattering. The physicochemical characteristics of mixed liposomes were compared before and after exposure to foetal bovine serum (FBS) and were correlated to calorimetric data. Our results indicate protein binding to all liposomal formulations. However, it is highlighted the importance of surface charge and fluidisation effect to the extent of protein adsorption. Additionally, considering the extensive use of cationic lipids for innovative delivery platforms, we deem PEGylation a key parameter, because even in a small proportion can reduce protein binding, and thus fast clearance and extreme toxicity without affecting positive charge. This study is a continuation of our previous work about protein-liposome interactions and fraction of stealthiness (Fs) parameter, and hopefully a design road map for drug and gene delivery.

Tuning the Immunostimulation Properties of Cationic Lipid Nanocarriers for Nucleic Acid Delivery.

Nonviral systems, such as lipid nanoparticles, have emerged as reliable methods to enable nucleic acid intracellular delivery. The use of cationic lipids in various formulations of lipid nanoparticles enables the formation of complexes with nucleic acid cargo and facilitates their uptake by target cells. However, due to their small size and highly charged nature, these nanocarrier systems can interact in vivo with antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages. As this might prove to be a safety concern for developing therapies based on lipid nanocarriers, we sought to understand how they could affect the physiology of APCs. In the present study, we investigate the cellular and metabolic response of primary macrophages or DCs exposed to the neutral or cationic variant of the same lipid nanoparticle formulation. We demonstrate that macrophages are the cells affected most significantly and that the cationic nanocarrier has a substantial impact on their physiology, depending on the positive surface charge. Our study provides a first model explaining the impact of charged lipid materials on immune cells and demonstrates that the primary adverse effects observed can be prevented by fine-tuning the load of nucleic acid cargo. Finally, we bring rationale to calibrate the nucleic acid load of cationic lipid nanocarriers depending on whether immunostimulation is desirable with the intended therapeutic application, for instance, gene delivery or messenger RNA vaccines.

Efficient anti-tumor nano-lipoplexes with unsaturated or saturated lipid induce differential genotoxic effects in mice

Cationic lipids are well-known excipients for nanometric liposomal gene delivery systems. However, because of the suspected, collateral toxicity in normal cells, the use of cationic lipids for the treatment of human tumor is largely limited. Recently, we developed a glucocorticoid receptor (GR)-targeted liposomal, anticancer delivery system (DXE nano-lipoplex), which carried cationic lipid of saturated twin aliphatic chains. It exhibited efficient anti-tumor effect in aggressive and drug-resistant tumor models. Toward exploring lipoplex’s human clinical use, we incorporated another nano-lipoplex (D1XE) group that carried cationic lipid with one of its aliphatic chain carrying unsaturation and compared in vivo genotoxicological profiling-based safety assessment and the respective anti-tumor efficacy of the lipoplexes. Thus, both the lipoplexes differ only by the chemical identity of one of their constituent cationic lipid. Unsaturated aliphatic chains in lipid generally impart efficient cell surface fusogenic property in lipid formulations. Herein, we report that nanoplex with unsaturated cationic lipid (D1XE) exhibited better physical appearance with less flocculent behavior than nanoplex with saturated lipid (DXE). Upon multiple injections, D1XE nanoplex imparted better tumor regression but most importantly, exhibited much lower overall toxicity (e.g. genotoxicity, weight loss, etc.) than DXE nanoplex. With a higher antitumor effect but a lower genotoxic effect, D1XE is proved to be a better nanoplex than DXE for the potential clinical trial. Thus, this study clearly delineates the importance of incorporating a constituent lipid that carries a single unsaturated aliphatic chain toward developing efficient anti-tumor nano-lipoplexes with reduced genotoxicity.

Optimization of Peptide-Tagged Cationic Lipid Nanoparticles for Targeted Gene Delivery

A limitation in the use of cationic lipids as gene delivery vectors is lack of in vivo targeting. PEGylation (PEG; polyethylene glycol) sterically stabilizes cationic lipid-nucleic acid (CL-NA) nanoparticles but reduces their therapeutic efficacy through inhibition of cell-nanoparticle interactions [1]. To successfully deliver genetic material, nanoparticles must overcome barriers, including cellular binding and uptake, endosomal escape, and release of cargo. Both binding and endosomal escape require attractive interactions between the nanoparticle and cellular membranes. To recover cell binding and internalization, targeting ligands (e.g. peptides) must be attached to the distal end of PEG chains [2]. Peptide sequence and structure determine the receptor-ligand interaction strength, while mole fraction of ligand-PEG-lipid modulates efficacy due to competing factors of adequate cell binding and decreased stealth properties. We have also seen cell detachment due to blocking of receptors. Further, the addition of acid-labile PEG-lipids (HPEG-lipid) increases membrane interactions in late endosomes via shedding of PEG molecules [1]. A reduction in ligand-PEG-lipid allows for replacement with HPEG, promoting endosomal escape.We used CL-NA nanoparticles displaying linear and cyclic variations on RGD as well as C-end Rule motifs (e.g. iRGD and RPARPAR), which target integrin and neuropilin-1 receptors, respectively [3]. We determined the efficacy of CL-NA nanoparticles as a function of ligand type and ligand-tagging densities using flow cytometry, fluorescent colocalization with Rab proteins (GFP-Rab-GTPases; markers of endocytic organelles), and gene expression measurements. Our goal is to determine the relationship between different nanoparticle formulations and their ability to overcome the barriers in gene delivery and to identify regions in the nanoparticle formulation phase diagram that give optimized vectors.[1] Chan, C.L.et al.; Biomaterials, 33, 4928-4935, 2012[2] Majzoub, R. N. et al.; Biomaterials, 35, 4996-5005, 2014[3] Teesalu, T. et al.; Front Oncol, 3, 216, 2013

Composites of malonic acid diamides and phospholipids — Impact of lipoplex stability on transfection efficiency

The use of cationic lipids as gene delivery systems is a basic method in gene therapy. Through ongoing research, lipofection is currently the leader of non-viral vectors in clinical trials. However, in order to unleash the full potential of lipofection further intensive investigations are indispensable.In this study, various lipoplex formulations were compared regarding their ability to bind DNA. To obtain information about a possible premature release of DNA at the cell surface, heparin and chondroitin dependent lipoplex destabilization experiments were carried out. Complementary investigations in cell culture were performed to quantify DNA outside the cell. Additionally, DNase I stability was investigated. In this regard a multitude of methods, namely confocal laser scanning microscopy (CLSM), polymerase chain reaction (PCR), cell culture experiments, ethidium bromide assay, gel electrophoresis, Langmuir-isotherm experiments, infrared reflection absorption spectroscopy (IRRAS), Brewster angle microscopy (BAM), zeta-(ζ)-potential measurements, and dynamic light scattering (DLS), were applied. Although the complexation of DNA is a fundamental step, we show that the DNA release by biological agents (proteoglycans) and an unsuccessful cell attachment are major transfection limiting parameters.

Liposomal nanoparticles encapsulating iloprost exhibit enhanced vasodilation in pulmonary arteries.

Prostacyclin analogues are standard therapeutic options for vasoconstrictive diseases, including pulmonary hypertension and Raynaud’s phenomenon. Although effective, these treatment strategies are expensive and have several side effects. To improve drug efficiency, we tested liposomal nanoparticles as carrier systems. In this study, we synthesized liposomal nanoparticles tailored for the prostacyclin analogue iloprost and evaluated their pharmacologic efficacy on mouse intrapulmonary arteries, using a wire myograph. The use of cationic lipids, stearylamine, or 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (DOTAP) in liposomes promoted iloprost encapsulation to at least 50%. The addition of cholesterol modestly reduced iloprost encapsulation. The liposomal nanoparticle formulations were tested for toxicity and pharmacologic efficacy in vivo and ex vivo, respectively. The liposomes did not affect the viability of human pulmonary artery smooth muscle cells. Compared with an equivalent concentration of free iloprost, four out of the six polymer-coated liposomal formulations exhibited significantly enhanced vasodilation of mouse pulmonary arteries. Iloprost that was encapsulated in liposomes containing the polymer polyethylene glycol exhibited concentration-dependent relaxation of arteries. Strikingly, half the concentration of iloprost in liposomes elicited similar pharmacologic efficacy as nonencapsulated iloprost. Cationic liposomes can encapsulate iloprost with high efficacy and can serve as potential iloprost carriers to improve its therapeutic efficacy.

Cationic Lipids as Transfecting Agents of DNA in Gene Therapy

The use of cationic lipids (CLs) as transfecting agents of DNA has received an increasing attention in the last two decades. In order to improve the transfection efficiency with lower cytotoxicity, many CLs have been synthesized to be used as non-viral vectors, not only of DNA but also for other nucleic acids. Cationic lipids together with a helper lipid form mixed liposomes that compact DNA forming lipoplexes, gene vectors able to transport DNA into the cells without provoke an immune response. This review is focused in the progress and recent advances experimented in this area, mainly during last decade. Special attention has been paid: (a) to the biophysical characterization (electrostatics, structure, size and morphology) of the lipoplexes using a wide variety of experimental methods and, (b) to the biological studies (transfection efficiency and cell viability/cytotoxicity) addressed to confirm the viability and the optimum formulations of these DNA vectors to be used in gene therapy. Finally, and in order to take advantage towards a rational design of improved lipid gene vectors, the lipoplex structure-biological activity relationship has been also reviewed.

Preparation and characterization of vinculin-targeted polymer–lipid nanoparticle as intracellular delivery vehicle

Intracellular delivery vehicles have been extensively investigated as these can serve as an effective tool in studying the cellular mechanism, by delivering functional protein to specific locations of the cells. In the current study, a polymer–lipid nanoparticle (PLN) system was developed as an intracellular delivery vehicle specifically targeting vinculin, a focal adhesion protein associated with cellular adhesive structures, such as focal adhesions and adherens junctions. The PLNs possessed an average size of 106 nm and had a positively charged surface. With a lower encapsulation efficiency 32% compared with poly(lactic-co-glycolic) acid (PLGA) nanoparticles (46%), the PLNs showed the sustained release profile of model drug BSA, while PLGA nanoparticles demonstrated an initial burst-release property. Cell-uptake experiments using mouse embryonic fibroblasts cultured in fibrin–fibronectin gels observed, under confocal microscope, that the anti-vinculin conjugated PLNs could successfully ship the cargo to the cytoplasm of fibroblasts, adhered to fibronectin–fibrin. With the use of cationic lipid, the unconjugated PLNs were shown to have high gene transfection efficiency. Furthermore, the unconjugated PLNs had nuclear-targeting capability in the absence of nuclear-localization signals. Therefore, the PLNs could be manipulated easily via different type of targeting ligands and could potentially be used as a powerful tool for cellular mechanism study, by delivering drugs to specific cellular organelles.

Nonviral Vectors for The Delivery of Small Interfering RNAs to The CNS

While efficient methods for cell line transfection are well described, for primary neurons a high-yield method different from those relying on viral vectors is lacking. Viral vector-based primary neuronal infection has several drawbacks, including complexity of vector preparation, safety concerns and the generation of immune and inflammatory responses, when used in vivo. This article will cover the different approaches that are being used to efficiently deliver genetic material (both DNA and small interfering RNA) to neuronal tissue using nonviral vectors, including the use of cationic lipids, polyethylenimine derivatives, dendrimers, carbon nanotubes and the combination of carbon-made nanoparticles with dendrimers. The effectiveness, both in vivo and in vitro, of the different methods to deliver genetic material to neural tissue is discussed.

Advancing Nonviral Gene Delivery: Lipid- and Surfactant-Based Nanoparticle Design Strategies

Gene therapy is a technique utilized to treat diseases caused by missing, defective or overexpressing genes. Although viral vectors transfect cells efficiently, risks associated with their use limit their clinical applications. Nonviral delivery systems are safer, easier to manufacture, more versatile and cost effective. However, their transfection efficiency lags behind that of viral vectors. Many groups have dedicated considerable effort to improve the efficiency of nonviral gene delivery systems and are investigating complexes composed of DNA and soft materials such as lipids, polymers, peptides, dendrimers and gemini surfactants. The bottom-up approach in the design of these nanoparticles combines components essential for high levels of transfection, biocompatibility and tissue-targeting ability. This article provides an overview of the strategies employed to improve in vitro and in vivo transfection, focusing on the use of cationic lipids and surfactants as building blocks for nonviral gene delivery systems.

Efficient nucleic acid transduction with lipoplexes containing novel piperazine- and polyamine-conjugated cholesterol derivatives

To advance the use of cationic lipids for non-viral nucleic acid vector formulation, a panel of novel nitrogen heterocycle cholesteryl derivatives containing a biodegradable carbamate linker was synthesised. Optimally acting piperazine and cyclen compounds had nucleic acid-binding and lipoplex nanoparticle formation properties that were suitable for their use as non-viral vectors. It was found that the lipoplexes formed were capable of efficient non-toxic nucleic acid delivery to cells in culture. The chemical structure of individual cationic lipids, which is likely to influence lipoplex formation, affected efficiency of DNA or RNA transfection. The results indicated that the cyclen containing compound possessing two cholesteryl moieties resulted in efficient siRNA-mediated target gene silencing but was a poor reagent for DNA transfection.

Gene methylation for the diagnosis and prognosis of cancer

Initially, gene therapy was viewed as an approach for treating hereditary diseases, but its potential role in the treatment of acquired diseases such as cancer is now widely recognized. The understanding of the molecular mechanisms involved in cancer and the development of nucleic acid delivery systems are two concepts that have led to this development. Systemic gene delivery systems are needed for therapeutic application to cells inaccessible by percutaneous injection and for multi-located tumor sites, i.e. metastases. Non-viral vectors based on the use of cationic lipids or polymers appear to have promising potential, given the problems of safety encountered with viral vectors. Using these non-viral vectors, the current challenge is to obtain a similarly effective transfection to viral ones. Based on the advantages and disadvantages of existing vectors and on the hurdles encountered with these carriers, the aim of this review is to describe the “perfect vector” for systemic gene therapy against cancer.

DiC14-amidine confers new anti-inflammatory properties to phospholipids.

The inflammatory effect of unmethylated CpG DNA sequences represents a major obstacle to the use of cationic lipids for in vivo gene therapy. Although the mechanism of CpG-induced inflammatory response is rather well understood nowadays, few solutions have been designed to circumvent this effect in gene therapy experiments. Our previous work has shown that a refractory state towards inflammation can be elicited by preinjecting cationic liposomes. Here, we present evidence that diC14-amidine liposomes confer new anti-inflammatory properties to phospholipids from low-density lipoprotein (LDL) and even to synthetic phospholipids for which such an observation has not been reported so far. Whereas oxidation of LDL lipids was a prerequisite for any anti-inflammatory activity, lipid oxidation is no longer required in our experiments, suggesting that cationic lipids transport phospholipids through a different route and affect different pathways. This opens up new possibilities for manipulating inflammatory responses in gene therapy protocols but also in a general manner in immunological experiments.

Insulin Receptor Substrate 1 Knockdown in Human MCF7 ER+ Breast Cancer Cells by Nuclease-Resistant IRS1 siRNA Conjugated To a Disulfide-Bridged d-Peptide Analogue of Insulin-Like Growth Factor 1

IRS-1 overexpression has been associated with breast cancer development, hormone independence and antiestrogen resistance. IRS-1 is a major downstream signaling protein for insulin and IGF1 receptors, conveying signals to PI-3K/Akt and ERK1/2 pathways. In estrogen-sensitive breast cancer cell lines, the widely used antiestrogen tamoxifen treatment reduces IRS-1 expression and function, thereby inhibiting IRS-1/PI-3K signaling. IRS-1 may serve as an alternative target to overexpressed IGF1R in breast cancer. While siRNA technology has become commonplace in many laboratories for in vitro gene knockdown studies, and in vivo stability issues are largely solved, its use in vivo is limited by an inability to efficiently and specifically deliver it to the intended site of action. We previously reported reduced survival of human MCF7 estrogen receptor positive breast cancer cells treated with a normal IRS1 siRNA delivered by a cationic lipid, plus an additive effect in combination with tamoxifen. We now report enhanced cellular uptake, relative to the unconjugated serum-stabilized IRS1 siRNA, of a serum-stabilized IRS1 siRNA conjugated with our previously characterized peptide mimetic of IGF1, D-(Cys-Ser-Lys-Cys), without the use of cationic lipids or electroporation, in MCF7 cells that overexpress IGF1R. Excess native IGF1 blocked uptake. An IRS1 siRNA cholesterol conjugate, targeted universally to cell membranes, was taken up by MCF7 cells as much as the peptide mimetic conjugate. IRS1 mRNA knockdown and IRS-1 protein knockdown were comparable for the IGF1 peptide and cholesterol conjugates. The unconjugated serum-stabilized IRS1 siRNA control showed negligible effects. Viability assays showed additive effects of siRNA treatment in combination with tamoxifen. In summary, we have taken the first step in converting an siRNA into a pharmacologically active agent for breast cancer.

A Cationic Liposomal Vincristine Formulation with Improved Vincristine Retention, Extended Circulation Lifetime and Increased Anti-Tumor Activity

A critical characteristic of optimized liposomal delivery systems is controlled drug release. This report describes the use of the monoacyl cationic lipids stearylamine (SA) and sphingosine (SPH), in conjunction with a transmembrane pH gradient, to enhance retention of the anticancer agent vincristine within liposomes administered intravenously to mice. The addition of SA to liposomes composed of distearoylphosphatidylcholine (DSPC) and cholesterol (Chol), to achieve a final composition of DSPC/Chol/SA at ratios of 45/45/10 (mol/mol/mol), caused a significant improvement in vincristine retention in the circulation of mice when combined with a transmembrane pH gradient of 5 units. Specifically, the presence of SA in liposomes having an internal pH (pHi) of 2.0 had a plasma vincristine AUC of 41.8 mg.h/100 mL plasma, compared to a vincristine AUC in the plasma of 28.0 mg.h/100 mL for identical liposomes lacking SA; an increase of 49%. Similar effects were observed with 10 mol% of the cationic lipid SPH. Comparison to control formulations lacking SA or SPH, or with smaller pH gradients (3 units), showed that improved vincristine retention required both the cationic lipid and the larger pH gradient. The presence of SA or SPH significantly improved vincristine pharmacokinetics due to the increased drug retention, but did not adversely alter the liposome pharmacokinetics. Enhanced retention of vincristine using SPH and a transmembrane pH gradient was associated with significant increases in anti-tumor activity against the murine P388 leukemia. The results are directly relevant to the use of charged lipid components to improve the drug retention/release attributes and the use of cationic lipids to promote accumulation at tumor-associated vascular endothelium. Keywords: Liposome, Vincristine, Cationic lipid, Pharmacokinetics, Cancer

Recent Developments in Particulate-Based Vaccines

Vaccines remain a key tool in the defence against major diseases. However, in the development of vaccines a trade off between safety and efficacy is required with newer vaccines, based on sub-unit proteins and peptides, displaying improved safety profiles yet suffering from low efficacy. Adjuvants can be employed to improve their potency, but currently there are only a limited number of adjuvant systems licensed for clinical use. Of the new adjuvants being investigated, particulate systems offer several advantages including: passive targeting to the antigen-presenting cells within the immune system, protection against adjuvant degradation, and ability for sustained antigen release. There has been a range of particulate vaccine delivery systems outlined in recent patents including polymer-based microspheres (which are generally more focused on the use of synthetic polymers, in particular the polyesters) and surfactant-based vesicles. Within these formulations, several patented systems are exploiting the use of cationic lipids which, despite their limitations in gene therapy, clearly offer strong potential as adjuvants. Within this review, the current range of particulate system technologies being investigated as potential adjuvants are discussed with regard to both their respective advantages and the potential hurdles which must be overcome for such systems to be converted into successful pharmaceutical products.

Photochemically Induced Gene Silencing Using Small Interfering RNA Molecules in Combination with Lipid Carriers

Novel strategies for efficient delivery of small interfering RNA (siRNA) molecules with a potential for targeting are required for development of RNA interference (RNAi) therapeutics. Here, we present a strategy that is based on delivery of siRNA molecules through the endocytic pathway, in order to develop a method for site-specific gene silencing. To achieve this, we combined the use of cationic lipids and photochemical internalization (PCI). Using the human S100A4 gene as a model system, we obtained potent gene silencing in four tested human cancer cell lines following PCI induction when using the cationic lipid jetSI-ENDO. Gene silencing was shown at both the RNA and protein levels, with no observed PCI toxicity when using the jetSI reagent and an optimized PCI protocol. This novel induction method opens for in vivo site-specific delivery of siRNA molecules toward a sequence of interest.

덱사메타손이 봉입된 지질나노입자의 제조: 지질의 종류와 함량 변화에 따른 지질나노입자의 특성

the coricosteroid drug dexamethasone is an efficacious antiinflammatory drug, it is difficult to formulate in an injectable formulation due to its poor aqueous solubility. A lipid-based nanosphere formulation containing dexamethasone was designed for solubilization of the drug in aqueous solution and sustained release of the drug from the nanosphere. The lipid nanospheres, composed of phospholipid, cholesterol and cationic lipid, were prepared by self emulsification-solvent diffusion method followed by diafiltration. Physicochemical characteristics such as mean particle diameter, zeta potential and drug loading efficiency of the lipid nanospheres were investigated according to the variation of either the kind of lipid or the lipid composition. The lipid nanospheres had a mean diameter 80-120 nm and dexamethasone loading efficiency of greater than 80%. The drug loading efficiency increased with the increase of the length of aliphatic chain attached to the phospholipid. However, the drug loading efficiency was inversely proportional to the increase of cholesterol content in the lipid composition. The lipid nanosphere could not be prepared without the use of cationic lipid and the drug loading efficiency was proportional to the increase of cationic lipid content. The lipid nanospheres containing dexamethasone are a promising novel drug carrier for an injectable formulation of the poorly water-soluble drugs.

Linear DNA Low Efficiency Transfection by Liposome Can Be Improved by the Use of Cationic Lipid as Charge Neutralizer

A plasmid expressing the beta-galactosidase enzyme was used to transfect Vero cells in order to evaluate the efficiency of a liposome-mediated transfection by circular and linear DNA. The results obtained showed a low rate of transfection by linear DNA:liposome complexes. To explore whether the structure of the complexes was interfering with the transfection, atomic force microscopy (AFM) was used. It has confirmed the difference between the linear and circular condensates: whereas the circular DNA:liposome complexes presented compact spherical or cylindrical structures of about 100-800 nm, the linear DNA showed pearl necklace-like structures, with pearls varying from 250 to 400 nm. On the basis of the theory proposed by Kuhn et al. (1999), low concentrations of cationic amphihile were used to neutralize or reverse the DNA charge in order to improve the transfection efficiency of the linear DNA. Using this method, we were able to obtain the expression of the transgene without an associated toxicity observed with the linear DNA liposome delivery.

Cationic lipids for transfection.

Among other strategies, the use of cationic lipids as autoassembling vehicles for non viral DNA transfection has received considerable attention. An exponentially growing litterature has been published on this topic (over 700 hits for the past decade, including 400 in the last two years). The present review focuses on the main present strategies aiming at improving cationic lipids induced transfection, and on some of the frequently encountered problems that should be solved to apply these non-viral vectors for human health. The review contains several sections dealing with the chemistry, physico-chemistry, cell biology, in vivo biology, and targeting of cationic-lipid DNA complexes.