Membrane Condensation and Curvature Induced by SARS-CoV-2 Envelope Protein.

The envelope (E) protein of SARS-CoV-2 participates in virion encapsulation and budding at the membrane of the endoplasmic reticulum Golgi intermediate compartment (ERGIC). The positively curved membrane topology required to fit an 80 nm viral particle is energetically unfavorable; therefore, viral proteins must facilitate ERGIC membrane curvature alteration. To study the possible role of the E protein in this mechanism, we examined the structural modification of the host lipid membrane by the SARS-CoV-2 E protein using synchrotron-based X-ray methods. Our reflectometry results on solid-supported planar bilayers show that E protein markedly condenses the surrounding lipid bilayer. For vesicles, this condensation effect differs between the two leaflets such that the membrane becomes asymmetric and increases its curvature. The formation of such a curved and condensed membrane is consistent with the requirements to stably encapsulate a viral core and supports a role for E protein in budding during SARS-CoV-2 virion assembly.

Modern Photodynamic Glioblastoma Therapy Using Curcumin- or Parietin-Loaded Lipid Nanoparticles in a CAM Model Study.

Natural photosensitizers, such as curcumin or parietin, play a vital role in photodynamic therapy (PDT), causing a light-mediated reaction that kills cancer cells. PDT is a promising treatment option for glioblastoma, especially when combined with nanoscale drug delivery systems. The curcumin- or parietin-loaded lipid nanoparticles were prepared via dual asymmetric centrifugation and subsequently characterized through physicochemical analyses including dynamic light scattering, laser Doppler velocimetry, and atomic force microscopy. The combination of PDT and lipid nanoparticles has been evaluated in vitro regarding uptake, safety, and efficacy. The extensive and well-vascularized chorioallantois membrane (CAM) of fertilized hen's eggs offers an optimal platform for three-dimensional cell culture, which has been used in this study to evaluate the photodynamic efficacy of lipid nanoparticles against glioblastoma cells. In contrast to other animal models, the CAM model lacks a mature immune system in an early stage, facilitating the growth of xenografts without rejection. Treatment of xenografted U87 glioblastoma cells on CAM was performed to assess the effects on tumor viability, growth, and angiogenesis. The xenografts and the surrounding blood vessels were targeted through topical application, and the effects of photodynamic therapy have been confirmed microscopically and via positron emission tomography and X-ray computed tomography. Finally, the excised xenografts embedded in the CAM were analyzed histologically by hematoxylin and eosin and KI67 staining.

Investigating 3R In Vivo Approaches for Bio-Distribution and Efficacy Evaluation of Nucleic Acid Nanocarriers: Studies on Peptide-Mimicking Ionizable Lipid.

Formulations based on ionizable amino-lipids have been put into focus as nucleic acid delivery systems. Recently, the in vitro efficacy of the lipid formulation OH4:DOPE has been explored. However, in vitro performance of nanomedicines cannot correctly predict in vivo efficacy, thereby considerably limiting pre-clinical translation. This is further exacerbated by limited access to mammalian models. The present work proposes to close this gap by investigating in vivo nucleic acid delivery within simpler models, but which still offers physiologically complex environments and also adheres to the 3R guidelines (replace/reduce/refine) to improve animal experiments. The efficacy of OH4:DOPE as a delivery system for nucleic acids is demonstrated using in vivo approaches. It is shown that the formulation is able to transfect complex tissues using the chicken chorioallantoic membrane model. The efficacy of DNA and mRNA lipoplexes is tested extensively in the zebra fish (Danio rerio) embryo which allows the screening of biodistribution and transfection efficiency. Effective transfection of blood vessel endothelial cells is seen, especially in the endocardium. Both model systems allow an efficacy screening according to the 3R guidelines bypassing the in vitro-in vivo gap. Pilot studies in mice are performed to correlate the efficacy of in vivo transfection.

Amphiphilic Anionic Oligomer-Stabilized Calcium Phosphate Nanoparticles with Prospects in siRNA Delivery via Convection-Enhanced Delivery.

Convection-enhanced delivery (CED) has been introduced as a concept in cancer treatment to generate high local concentrations of anticancer therapeutics and overcome the limited diffusional distribution, e.g., in the brain. RNA interference provides interesting therapeutic options to fight cancer cells but requires nanoparticulate (NP) carriers with a size below 100 nm as well as a low zeta potential for CED application. In this study, we investigated calcium phosphate NPs (CaP-NPs) as siRNA carriers for CED application. Since CaP-NPs tend to aggregate, we introduced a new terpolymer (o14PEGMA(1:1:2.5) NH3) for stabilization of CaP-NPs intended for delivery of siRNA to brain cancer cells. This small terpolymer provides PEG chains for steric stabilization, and a fat alcohol to improve interfacial activity, as well as maleic anhydrides that allow for both labeling and high affinity to Ca(II) in the hydrolyzed state. In a systematic approach, we varied the Ca/P ratio as well as the terpolymer concentration and successfully stabilized NPs with the desired properties. Labeling of the terpolymer with the fluorescent dye Cy5 revealed the terpolymer's high affinity to CaP. Importantly, we also determined a high efficiency of siRNA binding to the NPs that caused very effective survivin siRNA silencing in F98 rat brain cancer cells. Cytotoxicity investigations with a standard cell line resulted in minor and transient effects; no adverse effects were observed in organotypic brain slice cultures. However, more specific cytotoxicity investigations are required. This study provides a systematic and mechanistic analysis characterizing the effects of the first oligomer of a new class of stabilizers for siRNA-loaded CaP-NPs.

Extracellular matrix-inspired surface coatings functionalized with dexamethasone-loaded liposomes to induce osteo- and chondrogenic differentiation of multipotent stem cells.

Biomimetic surface coatings can be combined with conventional implants to mimic the extracellular matrix (ECM) of the surrounding tissue to make them more biocompatible. Layer-by-layer technique (LbL) can be used for making surface coatings by alternating adsorption of polyanions and polycations from aqueous solutions without need of chemical reactions. Here, polyelectrolyte multilayer (PEM) systems is made of hyaluronic acid (HA) as polyanion and Collagen I (Col) as polycation to mimic the ECM of connective tissue. The PEM are combined with dexamethasone (Dex)-loaded liposomes to achieve a local delivery and protection of this drug for stimulation of osteo- and chondrogenic differentiation of multipotent stem cells. The liposomes possess a positive surface charge that is required for immobilization on the PEM. The surface properties of PEM system show a positive zeta potential after liposome adsorption and a decrease in wettability, both promoting cell adhesion and spreading of C3H10T1/2 multipotent embryonic mouse fibroblasts. Differentiation of C3H10T1/2 was more prominent on the PEM system with embedded Dex-loaded liposomes compared to the basal PEM system and the use of free Dex-loaded liposomes in the supernatant. This was evident by immunohistochemical staining and an upregulation of the expression of genes, which play a key role in osteogenesis (RunX2, ALP, Osteocalcin (OCN)) and chondrogenesis (Sox9, aggrecan (ACAN), collagen type II), determined by quantitative Real-time polymerase chain reaction (qRT-PCR) after 21 days. These findings indicate that the designed liposome-loaded PEM system have high potential for use as drug delivery systems for implant coatings that can induce bone and cartilage differentiation needed for example in osteochondral implants.

Ultrasound-Responsive Smart Drug Delivery System of Lipid Coated Mesoporous Silica Nanoparticles.

The immediate release of chemotherapeutics at the target site, along with no premature release in circulation is always challenging. The purpose of this study was to develop a stimuli responsive drug delivery system, composed of lipid supported mesoporous silica nanoparticles (MSNPs) for triggered drug release at the target site and simultaneously avoiding the premature release. MSNPs with a higher drug loading capacity and very slow release were designed so as to enhance release by FDA approved US-irradiation. Doxorubicin, as a model drug, and perfluoropentane (PFP) as a US responsive material, were entrapped in the porous structure of MSNPs. Lipid coating enhanced the cellular uptake and in addition provided a gatekeeping effect at the pore opening to reduce premature release. The mechanical and thermal effects of US induced the conversion of liquid PFP to a gaseous form that was able to rupture the lipid layer, resulting in triggered drug release. The prolonged stability profile and non-toxic behavior made them suitable candidate for the delivery of anticancer drugs. This smart system, with the abilities of better cellular uptake and higher cytotoxic effects on US-irradiation, would be a good addition to the applied side of chemotherapeutic advanced drug delivery systems.

Enhanced efficacy and drug delivery with lipid coated mesoporous silica nanoparticles in cancer therapy.

The exposure of cancer cells to subtherapeutic drug concentrations results in multidrug resistance (MDR). The uniqueness of mesoporous silica nanoparticles (MSNPs) with larger surface area for higher drug loading can solve the issue by delivering higher amounts of chemotherapeutics to the cancer cells. However, premature drug release and lower biocompatibility remain challenging. Lipid coating of MSNPs at the same time, can enhance the stability and biocompatibility of nanocarriers. Furthermore, the lipid coating can reduce the systemic drug release and deliver higher amounts to the tumor site. Herein, lipid coated MSNPs were prepared by utilizing cationic liposomes and further investigations were made. Our studies have shown the higher entrapment of doxorubicin (Dox) to MSNPs due to availability of porous structure. Lipid coating could provide a barrier to sustain the release of drug along with reduced premature leakage. In addition, the biocompatibility and enhanced interaction of cationic liposomes to cell membranes resulted in better cellular uptake. Lipid coated silica nanoparticles have shown higher cellular toxicity as compared to non-lipid coated particles. The increase in cytotoxicity with time supports the hypothesis of sustained release of drug from lipid coated MSNPs. We propose the Lip-Dox-MSNPs as an effective approach to treat cancer by delivering and maintaining effective concentration of drugs to the tumor site without systemic side effects.

Magnetic resonance activatable thermosensitive liposomes for controlled doxorubicin delivery.

To limit the massive cytotoxicity of chemotherapeutic agents, it is desirable to establish an appropriate subtle blend of formulation design based on a dual-responsive strategy. In this study, a combined therapeutic platform based on magnetic thermosensitive liposomes (LipTS-GD) was developed. The incorporation of chelated-gadolinium imparted magnetic properties to thermosensitive liposomes (LipTS). The application of an ultra high field magnetic resonance imaging (UHF-MRI) induced hyperthermia, thus provided an improved chemotherapeutic effect of Doxorubicin (DOX). The paramagnetic platform demonstrated thermal sensitivity over a narrow temperature range starting at 37.8 °C, hence the release of DOX from LipTS-GD can be well triggered by inducing hyperthermia using UHF-MRI application. The prepared LipTS-GD were below 200 nm in diameter and an adequate release of DOX reaching 68% was obtained after 1 h UHF-MRI exposure. Profoundly, triple-negative breast cancer (TNBC) cells that were treated with LipTS-GD and subjected thereafter to UHF-MRI exposure for 60 min showed 36% viability. Hemocompatibility studies of LipTS-GD showed a physiological coagulation time and minimal hemolytic potential. Conclusively, LipTS-GD guided local delivery of DOX to solid tumors will potentially raise the therapeutic index, thus reducing the required dose and frequency of DOX administered systemically without influencing the adjacent tissues.

Wavelength dependent photo-cytotoxicity to ovarian carcinoma cells using temoporfin loaded tetraether liposomes as efficient drug delivery system.

5,10,15,20-Tetrakis(3-hydroxyphenyl)chlorin (mTHPC; temoporfin) is one of the most potent second-generation photosensitizers available today for the treatment of a variety of clinical disorders and has a unique capability of being activated at different wavelengths. However, due to its highly lipophilic nature, poor solubility in the aqueous media and poor bioavailability limits its application in anticancer therapies. To overcome these potential issues, we developed three different liposomal formulations with mTHPC encapsulated in hydrophobic milieu thus increasing the bioavailability of the drug. The prepared formulations were characterized in terms of hydrodynamic diameter, surface charge, encapsulation efficiency, and stability studies. The mean size of the liposomes was found to be in the nanoscale range (about 100 nm) with zeta potential ranging from -6.0 to -13.7 mV. mTHPC loaded liposomes were also evaluated for morphology using atomic force microscopy (AFM) and cryo-transmission electron microscopy (cryo-TEM). Data obtained from the hemocompatibility experiments showed that these formulations were compatible with blood showing less than 10% hemolysis and coagulation time lower than 40 s. The results obtained from the single-cell gel electrophoresis assay also demonstrated no incidence of genotoxicity. Photodynamic destruction of SK-OV-3 cells using mTHPC loaded liposomes showed a dose-response relationship upon irradiation with two different wavelength lights (blue λ = 457 nm & red λ = 652 nm). A 10-fold pronounced effect was produced when liposomal formulations were irradiated at 652 nm as compared to 457 nm. This was also evaluated by the quantitative assessment of reactive oxygen production (ROS) using fluorescence microscopy. The qualitative assessment of PDT pre- and post-irradiation was visualized using confocal laser scanning microscopy (CLSM) which demonstrated an intense localization of mTHPC liposomes in the perinuclear region. Chick chorioallantoic membrane assay (CAM) was used as an alternative in-ovo model to demonstrate the localized destruction of tumor microvasculature. Overall, the prepared nanoformulation is a biocompatible, efficient and well characterized delivery system for mTHPC for the safe and effective PDT.

The Impact of Alkyl-Chain Purity on Lipid-Based Nucleic Acid Delivery Systems - Is the Utilization of Lipid Components with Technical Grade Justified?

The physicochemical properties and transfection efficacies of two samples of a cationic lipid have been investigated and compared in 2D (monolayers at the air/liquid interface) and 3D (aqueous bulk dispersions) model systems using different techniques. The samples differ only in their chain composition due to the purity of the oleylamine (chain precursor). Lipid 8 (using the oleylamine of technical grade for cost-efficient synthesis) shows lateral phase separation in the Langmuir layers. However, the amount of attached DNA, determined by IRRAS, is for both samples the same. In 3D systems, lipid 8 p forms cubic phases, which disappear after addition of DNA. At physiological temperatures, both lipids (alone and in mixture with cholesterol) assemble to lamellar aggregates and exhibit comparable DNA delivery efficiency. This study demonstrates that non-lamellar structures are not compulsory for high transfection rates. The results legitimate the utilization of oleyl chains of technical grade in the synthesis of cationic transfection lipids.

Lysine-based amino-functionalized lipids for gene transfection: the protonation state in monolayers at the air-liquid interface.

Cationic lipids are considered as non-viral carriers for genetic material used in gene therapy. They have no carcinogenic potential and cause low immune response compared to existing viral systems. The protonation degree of these cationic lipids is a crucial parameter for the binding behavior of polynucleotides (e.g., DNA). Newly synthesized peptide-mimic lysine-based amino-functionalized lipids have been investigated in 2D models as monolayers at the air-liquid interface. Standard surface pressure - area isotherms have been measured to prove the layer stability. Total reflection X-ray fluorescence (TRXF) has been used as a surface sensitive analytical method to estimate the amount of counterions at the head groups. Using a standard sample as a reference, the protonation degree of these cationic lipids can be quantified on buffers with different pH values. It is found that the protonation degree depends linearly on the packing density of the lipid monolayer.

Fast therapeutic DNA internalization - A high potential transfection system based on a peptide mimicking cationic lipid.

The delivery of nucleic acids into cells is a determining factor for successful gene therapy. In this study we investigate the uptake and time dependent processing of a lipid-based non-viral nucleic acid delivery system composed of a peptide-mimicking cationic lipid (N-{6-amino-1-[N-(9Z)-octadec-9-enylamino]-1-oxohexan-(2S)-2-yl}-N'-{2-[N,N-bis(2-aminoethyl)amino]ethyl}-2-hexadecylpropandiamide - OH4) and a phospholipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine - DOPE). Studies by confocal laser scanning microscopy (CLSM) indicate a rapid internalization of fluorescent labelled DNA within 1h. Furthermore, vesicular structures on the lipid surface were reported, which are associated with the application of the lipid-based non-viral vector. Time dependent investigations of the gene expression of a reporter gene encoding for enhanced green fluorescent protein (eGFP) or luciferase in 4 different cell lines demonstrate an initial gene expression soon after 4h followed by a boost in gene expression beginning from 12h to 24h. Investigations with selective blocking of endocytic pathways using low molecular weight inhibitors suggested clathrin-mediated endocytosis as main internalization route in 3 cell lines. Our research presents a new horizon in rapid gene therapy using non-viral vectors; due to the modifications of the lipid components, fast nucleic acid internalizations could be achieved using our delivery systems.

Structures of malonic acid diamide/phospholipid composites and their lipoplexes.

As a continuation of previous work, the self-assembly process of cationic lipid formulations in the presence and absence of DNA was investigated with respect to binary lipid mixtures suitable as polynucleotide carrier systems. The lipid blends consist of one malonic-acid-based cationic lipid with a varying alkyl chain pattern, either N-{6-amino-1-[N-(9Z)-octadec-9-enylamino]-1-oxohexan-(2S)-2-yl}-N'-{2-[N,N-bis(2-aminoethyl)amino]ethyl}-2-hexadecylpropandiamide () or N-[6-amino-1-oxo-1-(N-tetradecylamino)hexan-(2S)-2-yl]-N'-{2-[N,N-bis(2-aminoethyl)amino]ethyl}-2-hexadecylpropandiamide (), and one neutral co-lipid, either 1,2-di-[(9Z)-octadec-9-enoyl]-sn-glycero-3-phosphocholine (DOPE) or 1,2-di-(hexadecanoyl)-sn-glycero-3-phosphocholine (DPPC). Although the cationic lipids exhibit only slight differences in their structure, the DNA transfer efficiency varies drastically. Therefore, self-assembly was studied in 3D systems by small- and wide-angle X-ray scattering (SAXS and WAXS) and transmission electron microscopy (TEM) as well as in 2D systems by infrared reflection-absorption spectroscopy (IRRAS) on Langmuir films. The investigated lipid mixtures show quite different self-assembly in the absence of DNA, with varying structures from vesicles (/DOPE; /DOPE) and tubes (/DOPE) to discoid structures (/DPPC; /DPPC). Twisted ribbons and sheets, which were stabilized due to hydrogen-bond networks, were found in all investigated lipid mixtures in the absence of DNA. The addition of DNA leads to the formation of lamellar lipoplexes for all the investigated lipid compositions. The lipoplexes differ in crucial parameters, such as the lamellar repeat distance and the spacing between the DNA strands, indicating differences in the binding strength between DNA and the lipid composition. The formation of associates with an ideal charge density might emerge as a key parameter for efficient DNA transfer. Furthermore, the structures observed for the different lipid compositions in the absence of DNA prepare the way for other applications besides gene therapy.

The Directional Observation of Highly Dynamic Membrane Tubule Formation Induced by Engulfed Liposomes.

Highly dynamic tubular structures in cells are responsible for exchanges between organelles. Compared with bacterial invasion, the most affordable and least toxic lipids were found in this study to be gentle and safe exogenous stimuli for the triggering of membrane tubules. A specific lipid system was internalized by NIH3T3 cells. Following cellular uptake, the constructed liposomes traveled towards the nucleus in aggregations and were gradually distributed into moving vesicles and tubules in the cytosol. The triggered tubules proceeded, retreated or fluctuated along the cytoskeleton and were highly dynamic, moving quickly (up to several microns per second), and breaking and fusing frequently. These elongated tubules could also fuse with one another, giving rise to polygonal membrane networks. These lipid systems, with the novel property of accelerating intracellular transport, provide a new paradigm for investigating cellular dynamics.

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.

Tris(2-aminoethyl)amine-based α-branched fatty acid amides - Synthesis of lipids and comparative study of transfection efficiency of their lipid formulations.

The synthesis of a new class of cationic lipids, tris(2-aminoethyl)amine-based α-branched fatty acid amides, is described resulting in a series of lipids with specific variations in the lipophilic as well as the hydrophilic part of the lipids. In-vitro structure/transfection relationships were established by application of complexes of these lipids with plasmid DNA (pDNA) to different cell lines. The α-branched fatty acid amide bearing two tetradecyl chains and two lysine molecules (T14diLys) in mixture with the co-lipid 1,2-di-[(9Z)-octadec-9-enoyl]-sn-glycero-3-phosphoethanolamine (DOPE) (1/2, n/n) exhibits effective pDNA transfer in three different cell lines, namely Hep-G2, A549, and COS-7. The presence of 10% serum during lipoplex incubation of the cells did not affect the transfection efficiency. Based on that, detailed investigations of the complexation of pDNA with the lipid formulation T14diLys/DOPE 1/2 (n/n) were carried out with respect to particle size and charge using dynamic light scattering (DLS), ζ-potential measurements, and transmission electron microscopy (TEM). Additionally, the lipoplex uptake was investigated by confocal laser scanning microscopy (CLSM). Overall, lipoplexes prepared from T14diLys/DOPE 1/2 (n/n) offer large potential as lipid-based polynucleotide carriers and further justify advanced examinations.

Lamellar versus Micellar Structures-Aggregation Behavior of a Three-Chain Cationic Lipid Designed for Nonviral Polynucleotide Transfer.

The aggregation behavior of a cationic lipid, N-[6-amino-1-oxo-1-(N-tetradecylamino)hexan-(2S)-2-yl]-N'-{2-[N,N-bis(2-aminoethyl)amino]ethyl}-2,2-ditetradecylpropandiamide (DiTT4), is investigated in aqueous dispersions at different pH values (5, 7.3, and 10). An unusual aggregation behavior is observed whereby DiTT4 forms bilayer structures at pH 10 and 7.3. At pH 5, rod-like micelles are the dominant aggregate form. The thermotropic and lyotropic behavior is studied using differential scanning calorimetry, small-angle X-ray scattering, and FTIR spectroscopy. In addition, investigations at the air-water interface are performed by recording area-pressure-isotherms and infrared reflection-absorption (IRRA) spectra. Complementary dynamic light scattering experiments and transmission electron microscopy (TEM and cryoTEM) are also used. The ability of DiTT4 to complex plasmid DNA is investigated using fluorescence techniques and zeta potential measurements. Cell culture experiments demonstrate the ability of DiTT4 to enhance plasmid transfer in A549 cells.

General synthesis and physicochemical characterisation of a series of peptide-mimic lysine-based amino-functionalised lipids.

A series of novel malonic acid diamides (second generation) with two long hydrophobic alkyl chains and an alkaline polar head group was synthesised and characterised as a new class of amino-functionalised lipids. These peptide-mimic lipids are suitable for polynucleotide transfer. The lipids bear a novel backbone consisting of a lysine unit and a malonic acid unit. Six different head-group structures, which vary in size and number of amino groups that can be protonated, were attached to the backbone structure. Furthermore, different alkyl chains were used to build the lipophilic part (namely tetradecyl, hexadecyl, and oleyl). Phase transitions of the new compounds in aqueous dispersions at pH 10 were analysed and discussed in terms of head group and alkyl chain variations. The shape and size of the formed aggregates of selected lipid dispersions were investigated by dynamic light scattering and transmission electron microscopy.

Structure-function relationships of new lipids designed for DNA transfection.

Cationic liposome/DNA complexes can be used as nonviral vectors for direct delivery of DNA-based biopharmaceuticals to damaged cells and tissues. To obtain more effective and safer liposome-based gene transfection systems, two cationic lipids with identical head groups but different chain structures are investigated with respect to their in vitro gene-transfer activity, their cell-damaging characteristics, and their physicochemical properties. The gene-transfer activities of the two lipids are very different. Differential scanning calorimetry and synchrotron small- and wide-angle X-ray scattering give valuable structural insight. A subgel-like structure with high packing density and high phase-transition temperature from gel to liquid-crystalline state are found for lipid 7 (N'-2-[(2,6-diamino-1-oxohexyl)amino]ethyl-2,N-bis(hexadecyl)propanediamide) containing two saturated chains. Additionally, an ordered head-group lattice based on formation of a hydrogen-bond network is present. In contrast, lipid 8 (N'-2-[(2,6-diamino-1-oxohexyl)amino]ethyl-2-hexadecyl-N-[(9Z)-octadec-9-enyl]propanediamide) with one unsaturated and one saturated chain shows a lower phase-transition temperature and a reduced packing density. These properties enhance incorporation of the helper lipid cholesterol needed for gene transfection. Both lipids, either pure or in mixtures with cholesterol, form lamellar phases, which are preserved after addition of DNA. However, the system separates into phases containing DNA and phases without DNA. On increasing the temperature, DNA is released and only a lipid phase without intercalated DNA strands is observed. The conversion temperatures are very different in the two systems studied. The important parameter seems to be the charge density of the lipid membranes, which is a result of different solubility of cholesterol in the two lipid membranes. Therefore, different binding affinities of the DNA to the lipid mixtures are achieved.

Synthesis and DNA transfection properties of new head group modified malonic acid diamides.

Malonic acid diamides with two long hydrophobic alkyl chains and a basic polar head group as a new class of non-viral gene transferring compounds have shown high transfection efficiency and moderate toxicity. Based on the results obtained with saturated and unsaturated alkyl residues new derivatives with a more complex head group structure have been synthesized. For this purpose, cationic respectively basic groups were introduced by one or two lysine residues bound via tris(aminoethyl)amine spacer to the malonic acid diamide backbone. By studying in vitro gene delivery an increase of transfection efficacy was observed when using lipids with at least one unsaturated alkyl chain. This leads to cationic lipids exhibiting comparable or even higher transfection efficacies compared to the commercially available transfection agents LipofectAmine™ and SuperFect™. Phase transitions and phase structures of selected compounds have been analyzed and discussed in terms of transfection abilities. Particle size and zeta potential of liposomes and lipoplexes were also determined.