Regine Peschka-Süss

Transfection Efficiency and Cytotoxicity of Nonviral Gene Transfer Reagents in Human Smooth Muscle and Endothelial Cells

AbstractPurpose. Evaluation of a nonviral transfection reagent with respect to efficient gene transfer into primary human vascular cells. Methods. Complexes consisting of seven commercially available transfection reagents (DAC-30, DC-30, Lipofectin, LipofectAMINE PLUS, Effectene, FuGene 6 and Superfect) and EGFP encoding plasmid DNA were studied. The in vitro transfection efficiency and cytotoxicity in human aorta smooth muscle cells (HASMCs) and endothelial cells (HAECs) and rat smooth muscle cells (A-10 SMCs) were assayed in the presence of serum using flow cytometric analysis and ATP-quantitation assay, respectively. Results. Human primary cells were transfected less efficiently compared to the rat smooth muscle cell line. Transfection efficiency depended on the type of reagent, the reagent/DNA ratio, and, most importantly, on the cell type used. Determination of cytotoxicity showed that the effects of transfection on cell viability did not significantly differ from one another depending on the cell type. The exception to this was Superfect, which obviously reduced cell viability in all cell types. Conclusions. Our experiments showed that DAC-30 is the preferred transfection reagent for HASMCs and HAECs, exhibiting an improved efficiency combined with an acceptable cytotoxicity. Therefore, it might offer a therapeutic option for the treatment of cardiovascular disease and prove suitable for further drug development.

Angiogenesis as a target in neuroblastoma

Several research investigations on neuroblastoma (NB) have shown the important dependency of this embryonic tumour on angiogenesis, especially in the advanced and aggressive stages. However, the first pre-clinical data on anti-angiogenic drugs in NB have not been published until recently and clinical trials with anti-angiogenic agents in NB treatment protocols are still missing. Here, we summarise current knowledge on the important role and mechanisms of angiogenesis in NB, and report available pre-clinical results of anti-angiogenic agents used to treat NB. This review clearly shows that angiogenesis is a target in NB and that clinical trials are urgently needed to bring forward promising anti-angiogenesis treatment strategies into NB therapy.

Parameters influencing the determination of liposome lamellarity by 31P-NMR.

The lamellarity of liposomes influences to a great extent the encapsulation efficiency, the efflux rate of liposomally encapsulated material, and the fate of a drug after cellular uptake. 31P-NMR in combination with the use of chemical shift reagents has been described for the determination of lamellarity of liposomes and this study was performed to evaluate the applicability of 31P-NMR analysis as published in the past. To date, very few details about the required conditions throughout the measurements are known. In this study the influence of various parameters on the measurement, such as different buffers with changing ion concentrations, varying pH and different shift reagents at increasing concentrations was investigated. Results were discussed by using cryo-electronmicroscopy as a reference method. The data of this study show that 31P-NMR might not result in the correct determination of liposome lamellarity, depending on the experimental settings and the shape of the liposomes.

Simple and precise detection of lipid compounds present within liposomal formulations using a charged aerosol detector.

In recent decades the use of liposomal preparations as drug delivery systems has become very attractive in pharmaceutical development. Therefore, thorough characterization and quantification of the lipids which form liposomes is wished from both investigators and regulatory authorities when the application in humans is being considered. In this study a new HPLC method for the detection of lipids in liposomal formulations was established using corona charged aerosol detection (CAD) which has the advantage to be independent of the chemical properties of the analytes. The superiority of this method over UV detection was demonstrated. Compared to UV detection no absorption effects of the organic solvent in the mobile phase interfering with the lipid signals were observed with CAD. CAD showed good linearity (R(2)>0.990) for all liposomal compounds. The acceptance criteria for precision including repeatability were met. The average recovery for each of the excipients of the liposomal formulation was in the range of 90.0-110%.

Fourier Transformed Spectral Bio-imaging for Studying the Intracellular Fate of Liposomes

To improve the efficiency of liposomal drug targeting systems, it is necessary to understand the mechanism of liposome uptake by the cell and to follow the intracellular fate of internalized liposomes and their contents.

Receptor-Specific Targeting with Liposomes In Vitro Based on Sterol-PEG1300 Anchors

PurposeThe challenge in developing liposomes to be used in active drug targeting is to design a method that can be used for modifying liposomal membranes that is applicable for a number of different specific ligands. In this study, the post insertion technique was used with activated sterol-PEG1300 anchors and was evaluated with regard to its effectiveness in active targeting in vitro. The key advantage of these anchors is that the insertion step into the liposomal membrane takes place at room temperature and is very fast.Materials and MethodsFor in vitro experiments, neuroblastoma cell lines overexpressing GD2 antigen on their surface as a target structure were chosen. This allowed the use of anti-GD2 antibodies coupled to the liposomal surface for testing of specific binding. These modified liposomes were labelled with rhodamine-PE and their cellular association was analyzed by flow cytometry.ResultsIt was shown that the activated sterol-PEG1300 anchors allow specific and significant interactions of the modified liposomes with GD2 positive cells.ConclusionCoupling using sterol-PEG1300 anchors is both simple and rapid. It is reproducible and applicable for all ligands bearing amino groups. This method demonstrates the advantage of a ready-to-use system for the modification of pre-formed liposomes with different ligands.

Pyridinium Lipids with the Dodecaborate Cluster as Polar Headgroup: Synthesis, Characterization of the Physical-Chemical Behavior, and Toxicity in Cell Culture

We have prepared nine new dodecaborate cluster lipids with potential use in boron neutron capture therapy of tumors. This new generation of boron lipids is only singly negatively charged and consists of a pyridinium core with C(12), C(14), and C(16) chains as lipid backbone, connected through the nitrogen atom via a butylene, pentylene, or ethyleneoxyethylene linker to the oxygen atom on the dodecaborate cluster as headgroup. The lipids were obtained by nucleophilic attack of 4-(bisalkylmethyl)pyridine on the tetrahydrofurane, the dioxane, and a newly prepared tetrahydropyrane derivative, respectively, of closo-dodecaborate. All of these boron lipids are able to form closed vesicles in addition to some bilayers in the pure state and in the presence of helper lipids. The thermotropic behavior was found to be increasingly complex and polymorphic with increasing alkyl chain length. Except for two lipids, all lipids have low in vitro toxicity, and longer alkyl chains lead to a significant decrease in toxicity. The choice of the linker plays no major role with respect to their ability to form liposomes and their thermotropic properties, but the toxicity is influenced by the linkers in the case of short alkyl chains.

Development of Highly Stable and Low Toxic Cationic Liposomes for Gene Therapy

Cationic liposomes with high stability and low cytotoxicity for gene therapy have been developed. Luciferase plasmid DNA (pLuc) was used as a model gene. The empty liposomes and niosomes were prepared by freeze dried empty liposomes (FDEL) method. The entrapment of pLuc in the liposomes was by reconstitution of the lyophilized dried vesicles with the plasmid solution. The morphology of the vesicles showing multilamellar structure was characterized by transmission electron microscope (TEM) and cryo-TEM. Cytotoxicity of the vesicular formulations was investigated on mouse melanoma cell lines (B16F10) by MTT assay. Cationic lposomes and niosomes containing the cationic lipid DDAB were less cytotoxic than other bilayer vesicular formulations. The pLuc entrapped in the cationic DPPC/Chol/DDAB liposomes (at 1:1:1 molar ratio) exhibited higher stability than other vesicular formulations and the pLuc in solution when stored at 4, 30 and 50 degrees C for 8 weeks. The entrapment efficiency determined by gel electrophoresis and gel documentation of the pLuc in this liposomal formulation was 100%. Luciferase gene expression of pLuc-loaded in cationic liposomes (lipoplexes) in HeLa cell lines was evaluated from luciferase activity determined by a luminometer at 24 and 48 h incubation. Percentages of cell proliferation of the lipoplexes on HeLa cell line at 24 and 48 h incubation were evaluated by the WST-1 assay. When the amount of DPPC or cholesterol was increased in the lipoplexes, the higher amount of DDAB was needed to protect pLuc from enzymatic degradation. However, DPPC and cholesterol exceeded 33 and 50% mol, respectively gave no gene expression. The DPPC/Chol/DDAB (at 1:1:1 molar ratio) lipoplex has demonstrated moderately lower luciferase gene expression and low cytoxicity. This lipoplex with the DDAB/pLuc weight ratio of 14:1 was the most desirable formulation for gene therapy because of its high stability, high luciferase gene expression and low cytotoxicity.

The Association of Plain and Ligand- Bearing Neutral and Ph-Sensitive Liposomes with Various Cells

Abstract The majority of commonly used liposomes are supposed to enter cells through an endocytotic pathway and to be delivered to the lysosomes where enzymatic degradation of the lipids and the encapsulated material can occur. Therefore liposomes that do not end up in the lysosomes have been designed. One strategy is the application of pH-sensitive liposomes. Because of their lipid composition these liposomes show increased sensitivity to reduced pH which leads to fusion with endosomal membranes and to release of encapsulated material in the cytosol before degradation in the lysosome. One more step forward is receptor-mediated targeting through specific ligands coupled onto the liposomal surface. The exact mechanism by which liposomes are taken up by cells is still under investigation. It has become obvious in the past that it is necessary to gain individual information for every liposomal preparation and to study the effect on each cell line and primary cell culture. We have investigated the association of liposome preparations composed of the neutral lipid SPC (soy lecithin) and pH-sensitive lipids DOPE/CHEMS 3:2 mol/mol (dioleoylphos-phatidylethanolamine and cholesteryl hemisuccinate) with different cell lines: HeLa (human cervix carcinoma), COS-7 (monkey kidney, SV 40 transformed) and LMH (chicken hepatoma). Serum stability and influence of serum on the association efficiency was studied. Our data show that the presence of 10% fetal calf serum (FCS) during incubation did not reduce association efficiency significantly. Liposome encapsulated marker (FITC-dextran) was effectively delivered to HeLa and COS-7 cells by means of pH-sensitive liposomes. Receptor-mediated delivery could be achieved to LMH cells by coupling specilic ligand N-Ac-BSA to pH-sensitive liposomes. Some of the experiments were performed at 37°C and 4°C to study the involvement of phagocytosis, a process which does not occur below 10°C.

Expression of luciferase plasmid (pCMVLuc) entrapped in DPPC/Cholesterol/DDAB liposomes in HeLa cell lines

The luciferase gene expression of lipoplexes, a liposome containing luciferase plasmid (pCMVLuc), in HeLa cell lines, was investigated. Cationic liposomes were prepared by the chloroform film method with sonication. The lipoplex was formed by loading the liposome with pCMVLuc. The lipoplex with an optimal weight ratio of dimethyl dioctadecyl ammonium bromide (DDAB)/pCMVLuc protected from DNaseI was determined by an agarose gel electrophoresis. The selected lipoplexes were assayed for luciferaase activity by using a luminometer. The effect on cell proliferation was evaluated by WST-1 assay. The highest luciferase activity of 1.5 × 106 RLU was observed in the cholesterol (Chol)/DDAB (2:1 molar ratio) lipoplex at the DDAB/pCMVLuc weight ratio of 10:1 at 48 hours, which was about 10, 100, and 1,000 times higher than the DDAB, L-alpha-dipalmitoyl phosphatidylcholine (DPPC)/Chol/DDAB (1:2:1 molar ratio), and DPPC/Chol/DDAB (2:2:1 molar ratio) lipoplexes, respectively. The liposome with the smallest particle size was obtained from the cationic liposome composed of DPPC/Chol/DDAB (7:1:1 molar ratio) with the ζ potential of 7.17 ± 0.73. The optimal weight ratio of DDAB/pCMVLuc that protected pCMVLuc from DNaseI digestion was 4:1 in the DDAB formulation. The Chol/DDAB (2:1 molar ratio) lipoplex with the DDAB/pCMVLuc of 10:1 showed the highest luciferase activity of 1.5 × 106 RLU and the highest cytotoxicity as well. DPPC/Chol/DDAB (1:1:1 molar ratio)-lipoplex (DDAB/pCMVLuc = 14:1), which had the amount of DPPC and cholesterol not exceeding 33 and 50% mol, respectively, gave the lower gene expression of about 4 times, but lower cytoxicity of about 14 times, than the Chol/DDAB lipoplex (2:1 molar ratio) and was considered to be the most suitable formulation. The results from this study can be applied as a model for the development of a gene-therapeutic dosage form.