Safe and effective cancer therapy requires a suitable nanocarrier that can target particular sites, such as cancer cells, in a selective manner. With the tremendous growth in nanotechnology, liposomes, among various competing nanocarriers, have shown promising advances in cancer therapy. Magnetic nanoparticles and metal ions are wide-reaching candidates for conferring magnetic properties and for incorporation into liposomes. Combining liposomes with magnetic structures enables construction of magnetoresponsive liposomes, allowing stimuli-responsiveness to an alternating magnetic field, magnetic targeting, and tracking by magnetic resonance imaging, which could all occur in parallel. This review presents a comprehensive analysis of the practical advances and novel aspects of design, synthesis and engineering magnetoresponsive liposomes, emphasizing their diverse properties for various applications. Our work explores the innovative uses of these structures, extending beyond drug delivery to include smart contrast agents, cell labeling, biosensing, separation, and filtering. By comparing new findings with earlier studies, we showcase significant improvements in efficiency and uncover new potentials, setting a new benchmark for future research in the field of magnetoresponsive liposomes.

In the past decade, nucleic acid therapies have seen a boon in development and clinical translation largely due to advances in nanotechnology that have enabled their safe and targeted delivery. Nanoparticles can protect nucleic acids from degradation by serum enzymes and can facilitate entry into cells. Still, achieving endosomal escape to allow nucleic acids to enter the cytoplasm has remained a significant barrier, where less than 5% of nanoparticles within the endo-lysosomal pathway are able to transfer their cargo to the cytosol. Lipid-based drug delivery vehicles, particularly lipid nanoparticles (LNPs), have been optimized to achieve potent endosomal escape, and thus have been the vector of choice in the clinic as demonstrated by their utilization in the COVID-19 mRNA vaccines. The success of LNPs is in large part due to the rational design of lipids that can specifically overcome endosomal barriers. In this review, we chart the evolution of lipid structure from cationic lipids to ionizable lipids, focusing on structure-function relationships, with a focus on how they relate to endosomal escape. Additionally, we examine recent advancements in ionizable lipid structure as well as discuss the future of lipid design.

Large-scale transient transfection has advanced significantly over the last 20 years, enabling the effective production of a diverse range of biopharmaceutical products, including viral vectors. However, a number of challenges specifically related to transfection reagent stability and transfection complex preparation times remain. New developments and improved transfection technologies are required to ensure that transient gene expression-based bioprocesses can meet the growing demand for viral vectors. In this paper, we demonstrate that the growth of cationic lipid-based liposomes, an essential step in many cationic lipid-based transfection processes, can be controlled through adoption of low pH (pH 6.40 to pH 6.75) and in low salt concentration (0.2× PBS) formulations, facilitating improved control over the nanoparticle growth kinetics and enhancing particle stability. Such complexes retain the ability to facilitate efficient transfection for prolonged periods compared with standard preparation methodologies. These findings have significant industrial applications for the large-scale manufacture of lentiviral vectors for two principal reasons. First, the alternative preparation strategy enables longer liposome incubation times to be used, facilitating effective control in a good manufacturing practices setting. Second, the improvement in particle stability facilitates the setting of wider process operating ranges, which will significantly improve process robustness and maximise batch-to-batch control and product consistency.

Non-viral vectors for in vivo delivery of plasmid DNA rely on optimized formulations to achieve robust transgene expression. Several cationic lipids have been developed to deliver nucleic acids, but most recent literature has focused on mRNA due to its increased expression profile and excluded plasmid DNA, which may have the advantage of being less immunogenic. In this study, we describe the in vivo evaluation of cationic triazine based lipids, previously prepared by our group. We identify one lipid with limited in vivo toxicity for studies to optimize the lipid formulations, which include an evaluation of the influence of PEG and helper lipids on transgene expression. We then demonstrate that lipoplexes, but not lipid nanoparticles, formed from triazine lipids achieve similar transgene expression levels as AAV vectors and offer enhanced expression as compared to a commercially available cationic lipid, DOTAP. Importantly, the lipid nanoparticles and lipoplexes induce minimal antibody profiles toward the expressed protein, while serving as a platform to induce robust antibody responses when directly delivering the protein. Collectively, these data demonstrate the potential for triazine based lipids as non-viral vectors for gene delivery, and highlights the need to optimize each formulation based on the exact contents to achieve enhanced transgene expression with plasmid DNA constructs.

According to global statistics, cancer is the second leading cause of death worldwide. Because of the heterogeneity of cancer, single-drug therapy has many limitations due to low efficacy. Therefore, combination therapy with two or more therapeutic agents is being arisen. One of the most important approaches in cancer therapy is the shot down of key genes involved in apoptotic processes and cell cycle. In this regard, siRNA is a good candidate, a highly attractive method to suppressing tumor growth and invasion. Combination therapy with siRNAs and chemotherapeutic agents can overcome the multidrug resistance and increase apoptosis. The efficient delivery of siRNA to the target cell/tissue/organ has been a challenge. To overcome these challenges, the presence of suitable delivery systems by using nanoparticles is interesting. In this review, we discuss the current challenges for successful RNA interference. Also, we suggested proper a strategy for delivering siRNA that can be useful in targeting therapy. Finally, the combination of a variety of anticancer drugs and siRNA through acceptable delivery systems and their effects on cell cycle and apoptosis will be evaluated.

With recent advances in the field of RNAi-based therapeutics, it is possible to make any target gene 'druggable', at least in principle. The present review focuses on aspects critical for pulmonary delivery of formulations of nucleic acid-based drugs. The first part introduces the therapeutic potential of RNAi-based drugs for the treatment of lung diseases. Subsequently, we discuss opportunities for formulation-enabled pulmonary delivery of RNAi drugs in light of key physicochemical properties and physiological barriers. In the following section, an overview is included of methodologies for imparting inhalable characteristics to nucleic acid formulations. Finally, we review one of the bottlenecks in the early preclinical testing of inhalable nucleic acid-based formulations, in other words, devices suitable for pulmonary administration of powder-based formulations in rodents.

The successful intracellular delivery of exogenous macromolecules is crucial for a variety of applications ranging from basic biology to the clinic. However, traditional intracellular delivery methods such as those relying on viral/non-viral nanocarriers or physical membrane disruptions suffer from low throughput, toxicity, and inconsistent delivery performance and are time-consuming and/or labor-intensive. In this study, we developed a single-step hydrodynamic cell deformation-induced intracellular delivery platform named "hydroporator" without the aid of vectors or a complicated/costly external apparatus. By utilizing only fluid inertia, the platform focuses, guides, and stretches cells robustly without clogging. This rapid hydrodynamic cell deformation leads to both convective and diffusive delivery of external (macro)molecules into the cell through transient plasma membrane discontinuities. Using this hydroporation approach, highly efficient (∼90%), high-throughput (>1 600 000 cells per min), and rapid delivery (∼1 min) of different (macro)molecules into a wide range of cell types was achieved while maintaining high cell viability. Taking advantage of the ability of this platform to rapidly deliver large molecules, we also systematically investigated the temporal biostability of vanilla DNA origami nanostructures in living cells for the first time. Experiments using two DNA origami (tube- and donut-shaped) nanostructures revealed that these nanostructures can maintain their structural integrity in living cells for approximately 1 h after delivery, providing new opportunities for the rapid characterization of intracellular DNA biostability.

3D bioprinting is a technique which enables the direct printing of biodegradable materials with cells into 3D tissue. So far there is no cell reprogramming in situ performed with the 3D bioprinting process. Forkhead box D3 (FoxD3) is a transcription factor and neural crest marker, which was reported to reprogram human fibroblasts into neural crest stem-like cells. In this study, we synthesized a new biodegradable thermo-responsive waterborne polyurethane (PU) gel as a bioink. FoxD3 plasmids and human fibroblasts were co-extruded with the PU hydrogel through the syringe needle tip for cell reprogramming. The rheological properties of the PU hydrogel including the modulus, gelation time, and shear thinning were optimized for the transfection effect of FoxD3 in situ. The corresponding shear rate and shear stress were examined. Results showed that human fibroblasts could be reprogrammed into neural crest stem-like cells with high cell viability during the extrusion process under an average shear stress ∼190 Pa. We further translated the method to the extrusion-based 3D bioprinting, and demonstrated that human fibroblasts co-printed with FoxD3 in the thermo-responsive PU hydrogel could be reprogrammed and differentiated into a neural-tissue like construct at 14 days after induction. The neural-like tissue construct produced by 3D bioprinting from human fibroblasts may be applied to personalized drug screening or neuroregeneration.

There is no study so far on cell reprogramming in situ with 3D bioprinting. In this manuscript, a new thermoresponsive polyurethane bioink was developed and employed to deliver FoxD3 plasmid into human fibroblasts by the extrusion-based bioprinting. When the polyurethane gel was extruded through the syringe tip, the shear stress generated may have caused the transient membrane permeability for transfection. The shear stress was optimized for transfection in situ by 3D bioprinting. We demonstrated that human fibroblasts could be reprogrammed into neural crest-like stem cells by 3D bioprinting with the gel, and the reprogrammed cells underwent neural differentiation in the printed structure after induction. The neural-like tissue engineering constructs fabricated by 3D bioprinting from human fibroblasts may be applied for neuroregeneration or further developed as mini-brain for basic research and drug screening.

Efficient delivery of nucleic acids into cells is of great interest in the field of cell biology and gene therapy. Despite a lot of research, transfection efficiency and structural diversity of gene-delivery vectors are still limited. A better understanding of the structure-function relationship of gene delivery vectors is also essential for the design of novel and intelligent delivery vectors, efficient in "difficult-to-transfect" cells and in vivo clinical applications. Most of the existing strategies for the synthesis of gene-delivery vectors require multiple steps and lengthy procedures. Here, we demonstrate a facile, three-component one-pot synthesis of a combinatorial library of 288 structurally diverse lipid-like molecules termed "lipidoids" via a thiolactone ring opening reaction. This strategy introduces the possibility to synthesize lipidoids with hydrophobic tails containing both unsaturated bonds and reducible disulfide groups. The whole synthesis and purification are convenient, extremely fast, and can be accomplished within a few hours. Screening of the produced lipidoids using HEK293T cells without addition of helper lipids resulted in identification of highly stable liposomes demonstrating ∼95% transfection efficiency with low toxicity.

Lipopolyamines (LPAs) are cationic lipids; they interact spontaneously with nucleic acids to form lipoplexes used for gene delivery. The main hurdle to using lipoplexes in gene therapy lies in their immunostimulatory properties, so far attributed to the nucleic acid cargo, while cationic lipids were considered as inert to the immune system. Here we demonstrate for the first time that di-C18 LPAs trigger pro-inflammatory responses through Toll-like receptor 2 (TLR2) activation, and this whether they are bound to nucleic acids or not. Molecular docking experiments suggest potential TLR2 binding modes reminiscent of bacterial lipopeptide sensing. The di-C18 LPAs share the ability of burying their lipid chains in the hydrophobic cavity of TLR2 and, in some cases, TLR1, at the vicinity of the dimerization interface; the cationic headgroups form multiple hydrogen bonds, thus crosslinking TLRs into functional complexes. Unravelling the molecular basis of TLR1 and TLR6-driven heterodimerization upon LPA binding underlines the highly collaborative and promiscuous ligand binding mechanism. The prevalence of non-specific main chain-mediated interactions demonstrates that potentially any saturated LPA currently used or proposed as transfection agent is likely to activate TLR2 during transfection. Hence our study emphasizes the urgent need to test the inflammatory properties of transfection agents and proposes the use of docking analysis as a preliminary screening tool for the synthesis of new non-immunostimulatory nanocarriers.

Nanoparticles based on cationic polymers, lipids or lipidoids are of great interest in the field of gene delivery applications. The research on these nanosystems is rapidly growing as they hold promise to treat wide variety of human diseases ranging from viral infections to genetic disorders and cancer. Recently, combinatorial design principles have been adopted for rapid generation of large numbers of chemically diverse polymers and lipids capable of forming multifunctional nanocarriers for the use in gene delivery applications. At the same time, current high-throughput screening systems as well as convenient cell assays and readout techniques allow for fast evaluation of cell transfection efficiencies and toxicities of libraries of novel gene delivery agents. This allows for a rapid evaluation of structure-function relationship as well as identification of novel efficient nanocarriers for cell transfection and gene therapy. Here, the recent contribution of high-throughput synthesis to the development of novel nanocarriers for gene delivery applications is described.

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) nuclease system represents an efficient tool for genome editing and gene function analysis. It consists of two components: single-guide RNA (sgRNA) and the enzyme Cas9. Typical sgRNA and Cas9 intracellular delivery techniques are limited by their reliance on cell type and exogenous materials as well as their toxic effects on cells (for example, electroporation). We introduce and optimize a microfluidic membrane deformation method to deliver sgRNA and Cas9 into different cell types and achieve successful genome editing. This approach uses rapid cell mechanical deformation to generate transient membrane holes to enable delivery of biomaterials in the medium. We achieved high delivery efficiency of different macromolecules into different cell types, including hard-to-transfect lymphoma cells and embryonic stem cells, while maintaining high cell viability. With the advantages of broad applicability across different cell types, particularly hard-to-transfect cells, and flexibility of application, this method could potentially enable new avenues of biomedical research and gene targeting therapy such as mutation correction of disease genes through combination of the CRISPR-Cas9-mediated knockin system.

Intracellular delivery of materials is a challenge in research and therapeutic applications. Physical methods of plasma membrane disruption have recently emerged as an approach to facilitate the delivery of a variety of macromolecules to a range of cell types. We use the microfluidic CellSqueeze delivery platform to examine the kinetics of plasma membrane recovery after disruption and its dependence on the calcium content of the surrounding buffer (recovery time ∼ 5 min without calcium vs. ∼ 30 s with calcium). Moreover, we illustrate that manipulation of the membrane repair kinetics can yield up to 5× improvement in delivery efficiency without significantly impacting cell viability. Membrane repair characteristics initially observed in HeLa cells are shown to translate to primary naïve murine T cells. Subsequent manipulation of membrane repair kinetics also enables the delivery of larger materials, such as antibodies, to these difficult to manipulate cells. This work provides insight into the membrane repair process in response to mechanical delivery and could potentially enable the development of improved delivery methods.

Short-interfering RNAs (siRNAs) offer a potential tool for the treatment of skin disorders. However, applications of siRNA for dermatological conditions are limited by their poor permeation across the stratum corneum of the skin and low penetration into the skin's viable cells. In this study, we report the use of SPACE-peptide in combination with a DOTAP-based ethosomal carrier system to enhance skin delivery of siRNA. A DOTAP-based SPACE Ethosomal System significantly enhanced siRNA penetration into porcine skin in vitro by 6.3±1.7-fold (p<0.01) with an approximately 10-fold (p<0.01) increase in epidermis accumulation of siRNA compared to that from an aqueous solution. Penetration of siRNA was also enhanced at the cellular level. Internalization of SPACE-peptide occurred in a concentration dependent manner marked by a shift in intracellular distribution from punctate spots to diffused cytoplasmic staining at a peptide concentration of 10mg/mL. In vitro delivery of GAPDH siRNA by SPACE peptide led to 83.3±3.0% knockdown relative to the control. In vivo experiments performed using female BALB/C mice also confirmed the efficacy of DOTAP-SES in delivering GAPDH-siRNA into skin. Topical application of DOTAP-SES on mice skin resulted in 63.2%±7.7% of GAPDH knockdown, which was significantly higher than that from GAPDH-siRNA PBS (p<0.05). DOTAP-SES formulation reported here may open new opportunities for cutaneous siRNA delivery.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

A series of benzothiazole based lipids (1-10) containing different derivatives of benzothiazole in the head group region were synthesized to determine the structure-activity relationship for gene delivery. The liposomes formulated were mixed with plasmid DNA encoding green fluorescent protein (α5GFP) or β-galactosidase (pCMV-SPORT-β-gal) and transfected into B16F10 (Human melanoma cancer cells), CHO (Chinese hamster ovary), A-549 (Human lung carcinoma cells) and MCF-7 (Human breast carcinoma cells) types of cell lines. The efficiencies of lipids 9 and 10 in particular, were found to be comparable and even more when compared to that of LipofectAmine-2000. The transfection profiles of the efficient lipids are proved to be maintained even in the presence of serum. Thus, the benzothiazole head group based lipids developed have the potential to be used as transfection reagents in vitro and in vivo.

We report principles for active, user-defined control over the locations and timing with which DNA is expressed in cells. Our approach exploits unique properties of a ferrocenyl cationic lipid that is inactive when oxidized, but active when chemically reduced. We show that methods that exert spatial control over the administration of reducing agents can lead to local activation of lipoplexes and spatial control over gene expression. The versatility of this approach is demonstrated using both soluble and solid-phase reducing agents. These methods provide control over cell transfection, including methods for remote activation and the patterning of expression using solid-phase redox agents, that are difficult to achieve using conventional lipoplexes.

The purpose of this study was to demonstrate the effectiveness of an integrin peptide ligand-labeled liposomal delivery system loaded with vascular endothelial growth factor (VEGF)-siRNA in a model study of gene therapy for retinopathy using human retinal pigment epithelial cells.

Arg(R)-Gly(G)-Asp(D) motif peptide conjugating polyethylene glycol modified (RGD-PEGylated) liposomes were prepared using a thin-film hydration method and optimized for surface charge, particle size, small interfering RNA (siRNA) load, and entrapment efficiency. Reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assays were used to determine VEGF levels in retinal pigment epithelial cells. Cytotoxicity was determined using the 3-[4, 5-dimethylthiazol-2-yl]-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and flow cytometry.

Physicochemical properties, including particle size, zeta potential, and siRNA load, of the prepared RGD-PEGylated liposomes and their entrapment efficiency were determined to be within the following ranges: 123.8-234.1 nm, 17.31-40.09 m V, 5.27%-6.33%, and >97%, respectively. RGD-PEGylated liposome-mediated fluorescent-labeled siRNA delivery demonstrated significantly enhanced cellular uptake, and 3 mol% RGD-PEGylated liposomes (having 3β-[N-(N', N'-dimethylaminoethane) carbamoyl] cholesterol (DC-cholesterol) DSPE and DSPE-PEG(2000)-RGD with molar ratio of 50/47/3) were shown to have better efficacy with regard to specificity for retinal pigment epithelial cells, reduced cytotoxicity, and knockdown of the target molecule.

By integrin receptor-mediated endocytosis, 3 mol% RGD-PEGylated liposomes were shown to be a suitable vector when loaded with VEGF-siRNA for efficient downregulation of VEGF in retinal pigment epithelial cells at both the protein and gene levels. This integrin ligand-modified liposomal delivery system has therapeutic potential for ocular gene therapy.

In the work described here, gene delivery using polymer microbubbles triggered by ultrasound in vitro was investigated. The effects of pressure amplitude (0-2 MPa), center frequency (1-5 MHz), pulse length (3-12,000 μs), pulse repetition frequency (5-20,000 Hz) and exposure time (0-30 s) on transfection efficiency and cell viability were examined. The effects of radiation force, calcium ion concentration and timing of treatments were also examined. Cells were successfully transfected with pressure amplitudes as low as 250 kPa. Transfection was most efficient at lower frequencies and longer pulse lengths, with a transfection efficiency of 24.2 ± 2.0% achieved using a center frequency of 1 MHz, pressure amplitude of 1 MPa, pulse length of 12,000 μs and pulse repetition frequency of 5 Hz. Gene delivery was also affected by the extracellular calcium ion concentration and the timing of treatments.

Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations, including their reliance on exogenous materials or electrical fields, which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30-80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material, such as carbon nanotubes, proteins, and siRNA, to 11 cell types, including embryonic stem cells and immune cells. When used for the delivery of transcription factors, the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed, its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications.

Therapeutic gene delivery systems offer the potential for the treatment of a range of inherited and acquired inherited diseases. In contrast with viral gene vectors, the nonviral gene vectors provide a safer alternative and additional advantage such as the improved delivery efficiency, low cost, and often unlimited capacity to package DNA. Here we describe preparation of nonviral gene delivery technique based on lipid:peptide:DNA (LPD) complexes. The size of LPD particles is in the nanometre range. The use of these nanoparticulate LPDs results in high efficiency transfections and a high level of gene expression in vitro. LPDs provide a convenient and efficient tool for gene therapy for the gene delivery.

siRNA therapeutics has developed rapidly and already there are clinical trials ongoing or planned; however, the delivery of siRNA into cells, tissues or organs remains to be a major obstacle. Lipid-based vectors hold the most promising position among non-viral vectors, as they have a similar structure to cell or organelle membranes. But when used in the form of liposomes, these vectors have shown some problems. Therefore, either the nature of lipids themselves or forms used should be improved. As a novel class of lipid like materials, lipidoids have the advantages of easy synthesis and the ability for delivering siRNA to obtain excellent silencing activity. However, the toxicities of lipidoids have not been thoroughly studied. pH responsive lipids have also gained great attention recently, though some of the amine-based lipids are not novel in terms of chemical structures. More complex self-assembly structures, such as LPD (LPH) and LCP, may provide a good solution to siRNA delivery. They have demonstrated controlled particle morphology and size and siRNA delivery activity for both in vitro and in vivo.

We report an approach to the chemical oxidation of a ferrocene-containing cationic lipid [bis(11-ferrocenylundecyl)dimethylammonium bromide, BFDMA] that provides redox-based control over the delivery of DNA to cells. We demonstrate that BFDMA can be oxidized rapidly and quantitatively by treatment with Fe(III)sulfate. This chemical approach, while offering practical advantages compared to electrochemical methods used in past studies, was found to yield BFDMA/DNA lipoplexes that behave differently in the context of cell transfection from lipoplexes formed using electrochemically oxidized BFDMA. Specifically, while lipoplexes of the latter do not transfect cells efficiently, lipoplexes of chemically oxidized BFDMA promoted high levels of transgene expression (similar to levels promoted by reduced BFDMA). Characterization by SANS and cryo-TEM revealed lipoplexes of chemically and electrochemically oxidized BFDMA to both have amorphous nanostructures, but these lipoplexes differed significantly in size and zeta potential. Our results suggest that differences in zeta potential arise from the presence of residual Fe(2+) and Fe(3+) ions in samples of chemically oxidized BFDMA. Addition of the iron chelating agent EDTA to solutions of chemically oxidized BFDMA produced samples functionally similar to electrochemically oxidized BFDMA. These EDTA-treated samples could also be chemically reduced by treatment with ascorbic acid to produce samples of reduced BFDMA that do promote transfection. Our results demonstrate that entirely chemical approaches to oxidation and reduction can be used to achieve redox-based 'on/off' control of cell transfection similar to that achieved using electrochemical methods.

In order to promote siRNA transfer in tumour cells, we used an original cationic lipid, synthesized in our laboratory, dimethyl-hydroxyethyl-aminopropane-carbamoyl-cholesterol (DMHAPC-Chol). Liposomes were prepared from this lipid and dioleoylphosphatidylethanolamine (DOPE) in equimolar proportion. Its transfecting capacity was evaluated using ELISA, cell cytometry, and RT-PCR in estimating the silencing effect of VEGF siRNA. This liposome efficiently delivered VEGF siRNA in two human cancer cell lines abundantly secreting VEGF, A431 and MDA-MB-231. Results showed that 50 nM of VEGF siRNA carried by DMHAPC-Chol/DOPE liposomes already silenced more than 90% of VEGF in these cells. A comparative study with two commercial carriers indicated that the inhibition induced by VEGF siRNA transported by cationic DMHAPC-Chol/DOPE liposomes was comparable to that induced by INTERFERin and better than lipofectamine 2000. Moreover, a transfection by a GFP plasmid followed by a GFP siRNA showed that DMHAPC-Chol/DOPE liposomes compared to lipofectamine were less efficient for plasmid but better for siRNA transport. Following one of our previous works concerning cell delivery of plasmid ( Percot et al., 2004 ), the main interest of results presented here resides in the double potential of DMHAPC-Chol/DOPE liposomes to deliver little-sized siRNA as well as large nucleic acids in cells.

Whilst there is a large body of evidence looking at the design of cationic liposomes as transfection agents, correlates of formulation to function remain elusive. In this research, we investigate if lipid packaging can give further insights into transfection efficacy. DNA lipoplexes composed of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) in combination with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-stearoyl-3-trimethylammonium-propane (DSTAP) were prepared by the lipid hydration method. Each of the formulations was prepared by hydration in dH2O or phosphate buffer saline (PBS) to investigate the effect of buffer salts on lipoplex physicochemical characteristics and in vitro transfection. In addition, Langmuir monolayer studies were performed to investigate any possible correlation between lipid packaging and liposome attributes. Using PBS, rather than dH2O, to prepare the lipoplexes increased the size of vesicles in most of formulations and resulted in variation in transfection efficacies. However, one combination of lipids (DSPE:DOTAP) could not form liposomes in PBS, whilst the DSPE:DSTAP combination could not form liposomes in either aqueous media. Monolayer studies demonstrated saturated lipid combinations offered dramatically closer molecular packing compared to the other combinations which could suggest why this lipid combination could not form vesicles. Of the lipoplexes prepared, those formulated with DSTAP showed higher transfection efficacy, however, the effect of buffer on transfection efficiency was formulation dependent.

Human retinal pigment epithelial cells are promising target sites for small interfering RNA (siRNA) that might be used for the prevention and/or treatment of choroidal neovascularization by inhibiting the expression of angiogenic factor; for example, by downregulating expression of the vascular endothelial growth factor gene.

A novel functional lipid, DSPE-PEG-RGD, a Arg(R)-Gly(G)-Asp(D) motif peptide conjugated to 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine- N-[maleimide (polyethylene glycol)-2000], was synthesized for the preparation of siRNA-loaded RGD-PEGylated liposomes to enhance uptake of encapsulated siRNA in retinal pigment epithelial cells. Various liposomes, with 1 mol% and 5 mol% PEGylated lipid or 1 mol% and 5 mol% RGD-PEGylated lipid, were fabricated.

Characterization of the liposomes, including siRNA entrapment efficiency, average particle size and ζ-potential, were determined to be as follows: >96%, 129.7 ± 51 to 230.7 ± 60.7 nm, and 17.3 ± 0.6 to 32 ± 1.3 mV, respectively. For the in vitro retinal pigment epithelial cell studies, the RGD-PEGylated liposomes had high delivery efficiency with siRNA delivery, about a four-fold increase compared with the PEGylated liposomes. Comparison of the various liposomes showed that the 1 mol% RGD-modified liposome had less cytotoxicity and higher siRNA delivery efficiency than the other liposomes. The antibody blocking assay confirmed that uptake of the 1 mol% RGD-PEGylated liposome was via integrin receptor- mediated endocytosis in retinal pigment epithelial cells.

The results of this study suggest that RGD-PEGylated liposomes might be useful for siRNA delivery into retinal pigment epithelial cells by integrin receptor-medicated endocytosis.

The level of cell transfection mediated by lipoplexes formed using the ferrocenyl lipid bis(11-ferrocenylundecyl)dimethylammonium bromide (BFDMA) depends strongly on the oxidation state of the two ferrocenyl groups of the lipid (reduced BFDMA generally mediates high levels of transfection, but oxidized BFDMA mediates very low levels of transfection). Here, we report that it is possible to chemically transform inactive lipoplexes (formed using oxidized BFMDA) to "active" lipoplexes that mediate high levels of transfection by treatment with the small-molecule reducing agent ascorbic acid (vitamin C). Our results demonstrate that this transformation can be conducted in cell culture media and in the presence of cells by addition of ascorbic acid to lipoplex-containing media in which cells are growing. Treatment of lipoplexes of oxidized BFDMA with ascorbic acid resulted in lipoplexes composed of reduced BFDMA, as characterized by UV/vis spectrophotometry, and lead to activated lipoplexes that mediated high levels of transgene expression in the COS-7, HEK 293T/17, HeLa, and NIH 3T3 cell lines. Characterization of internalization of DNA by confocal microscopy and measurements of the zeta potentials of lipoplexes suggested that these large differences in cell transfection result from (i) differences in the extents to which these lipoplexes are internalized by cells and (ii) changes in the oxidation state of BFDMA that occur in the extracellular environment (i.e., prior to internalization of lipoplexes by cells). Characterization of lipoplexes by small-angle neutron scattering (SANS) and by cryogenic transmission electron microscopy (cryo-TEM) revealed changes in the nanostructures of lipoplexes upon the addition of ascorbic acid, from aggregates that were generally amorphous, to aggregates with a more extensive multilamellar nanostructure. The results of this study provide guidance for the design of redox-active lipids that could lead to methods that enable spatial and/or temporal control of cell transfection.

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.

The prospects of gene therapy have generated unprecedented interest in the properties and structures of complexes of nucleic acids (NAs) with cationic liposomes (CLs), which are used as nonviral NA carriers in worldwide clinical trials. An improved understanding of the mechanisms of action of CL-NA complexes is required to enable their widespread therapeutic use. In prior studies of CL-mediated DNA delivery, membrane charge density (sigma(M)) was identified as a key parameter for transfection efficiency (TE) of lamellar (L(alpha)(C)) CL-DNA complexes. The TE of CL-DNA complexes containing cationic lipids with headgroup valencies from 1+ to 5+ follows a universal bell-shaped curve as a function of sigma(M). As we report here, the TE of CL-DNA complexes containing new multivalent lipids with dendritic headgroups (DLs) strongly deviates from this curve at high sigma(M). We have investigated four DLs, MVLG2 (4+), MVLG3 (8+), MVLBisG1 (8+), and MVLBisG2 (16+), in mixtures with neutral 1,2-dioleoyl-sn-glycerophosphatidyl-choline (DOPC). To understand the TE behavior, we have performed X-ray diffraction (XRD), optical microscopy, and cryo-TEM studies of the DL/DOPC mixtures and their DNA complexes. XRD reveals a complex phase behavior of DL-DNA complexes which strongly depends on the headgroup charge. MVLG2(4+)/DOPC-DNA complexes exhibit the lamellar phase at all molar fractions of DL, Phi(DL). In stark contrast, MVLBisG2(16+)/ DOPC-DNA complexes remain lamellar only for Phi(DL) < or = 0.2. In a narrow regime around Phi(DL) = 0.25, the hexagonal phase H(I)(C), consisting of a hexagonal lattice of cylindrical lipid micelles and a DNA honeycomb lattice, is formed. At Phi(DL) > 0.3, XRD suggests formation of a distorted H(I)(C) phase. For Phi(DL) > or = 0.5 under high salt conditions, this phase coexists with a bundle phase of DNA condensed by the depletion-attraction effect of DL micelles. The transitions at high sigma(M) from the lamellar phase to the new hexagonal phases of DL-DNA complexes coincide with the deviation from the universal TE behavior of lamellar complexes. The observed high TE, which is independent of sigma(M), strongly suggests a novel mechanism of action for these DL-DNA complex phases.

Nonviral vectors are safer than viral systems for gene therapy applications. However, the limited efficacy always prevents their being widely used in clinical practice. Aside from searching new gene nonviral vectors, many researchers focus on finding out new substances to improve the transfection efficiency of existent vectors. In this work, we found a transfection enhancer, nocodazole (NCZ), for dimethyldioctadecylammonium (DODAB, a cationic lipid) bilayer coated gold nanoparticles (AuNPs) mediated gene delivery. It was found that NCZ produces 3-fold transfection enhancement to HEK 293T cells assessed by flow cytometry (FCM). The result was further confirmed by luciferase assay, in which NCZ induced more than 5 times improvement in transfection efficiency after 48 h of transfection. The results from the inductively coupled plasma mass spectrometry (ICP-MS) and FCM showed that NCZ did not affect the internalization of DODAB-AuNPs/DNA complexes. The trafficking of the complexes by transmission electron microscopy (TEM) indicated that the interrupted transportation of the complexes to the lysosomes contributed greatly to the transfection enhancement. Therefore, NCZ can be used as a transfection enhancer in DODAB-AuNPs mediated transfection system. This work also gave an insight to improving the efficiency of lipid-mediated transfection: modifying lipid on gold nanoparticles and pre-treating cells by NCZ before the transfection.

We recently reported that the ferrocene-containing cationic lipid BFDMA [bis(11-ferrocenylundecyl)dimethylammonium bromide] can be used to mediate cell transfection, and that levels of transfection depend critically upon the oxidation state of the ferrocenyl groups of the lipid. Here, we report that the redox activity of BFDMA can be exploited to transform lipoplexes formed from oxidized BFDMA (which do not transfect cells) to lipoplexes that are "active" (and thus mediate high levels of transgene expression) by treatment with the chemical reducing agent glutathione (GSH). We demonstrate that GSH can be used to reduce the ferrocenium groups of oxidized BFDMA rapidly both (i) in solution and (ii) in lipoplexes formed by mixing oxidized BFDMA and DNA. Lipoplexes transformed in this manner mediate levels of cell transfection in vitro that are comparable to levels of transfection mediated by lipoplexes prepared by mixing DNA and reduced BFDMA. We demonstrate further that the chemical reduction of oxidized BFDMA leads to changes in the zeta potentials of these lipoplexes (e.g., from negative to positive). Characterization of lipoplex internalization using confocal microscopy demonstrated that these changes in zeta potential correlate to differences in the extents to which these lipoplexes are internalized by cells. These results provide a framework from which to interpret differences in cell transfection mediated by reduced and oxidized BFDMA. When combined, the results of this study suggest the basis of an approach that could be used to transform lipoplexes actively or "on-demand" and provide spatial and/or temporal control over the transfection of cells in a range of different fundamental and applied contexts.

In this paper, liposomes containing a lipopeptide bearing a ligand specifically recognized by neuropilin-1 (NRP-1) have been used to target a human breast cancer cell line overexpressing this receptor. The synthesis of this lipopeptide, C16-A7R, formed by the sequence of 7 amino acids ATWLPPR, linked to a palmitoyl fatty chain by an amide bond was described. After the characterisation of cationic liposomes formulated with the lipopeptide, the results obtained using various techniques showed that the lipopeptide-based liposomes were well accumulated in cells of the human breast cancer line MDA-MB-231 overexpressing NRP-1. Delivery of reporter genes expressing either beta-galactosidase (beta-gal) or green fluorescent protein (GFP) was selectively enhanced in these cells when compared with NRP-1-negative cells. In MDA-MB-231 cells, an increase by 250% in beta-gal activity was observed when delivered by lipopeptide-based liposomes compared to cationic liposomes alone.

The effect of lipid oxidation state on the physical properties of complexes formed by plasmid DNA and the redox-active lipid bis-(11-ferrocenylundecyl)dimethylammonium bromide (BFDMA) is reported. With increasing concentration of BFDMA, the hydrodynamic sizes of complexes formed by BFDMA and DNA (in the presence of 1 mM Li(2)SO(4)) pass through a maximum and the zeta-potential changes monotonically from -40 mV to +40 mV. In contrast, complexes formed by oxidized BFDMA and DNA exhibit a minimum in size and maintain a negative zeta-potential with increasing concentration of BFDMA. Angle-dependent dynamic light scattering measurements also reveal the presence of relaxation processes within complexes formed by DNA and oxidized BFDMA that are absent for complexes formed by DNA and reduced BFDMA. These results, when combined, reveal that the amphiphilic nature of reduced BFDMA leads to lipoplexes with physical properties resembling those formed by classical cationic lipids, whereas the interaction of oxidized BFDMA with DNA is similar to that of nonamphiphilic cationic molecules bearing multiple charges (e.g., spermidine). In particular, the negative zeta-potential and measurable presence of DNA chain dynamics within complexes formed by oxidized BFDMA and DNA indicate that these complexes are loosely packed with excess charge due to DNA in their outer regions. These results, when combined with additional measurements performed in OptiMEM reduced-serum cell culture medium, lead to the proposition that the strong dependence of transfection efficiency on the oxidation state of BFDMA, as reported previously, is largely a reflection of the substantial change in the zeta-potentials of these complexes with changes in the oxidation state of BFDMA.