DOCSPER [1,3-Dioleoyloxy-2-(N5-carbamoyl-spermine)-propane] is a cationic amphiphile consisting of a hydrophobic 1,3 dioleylglycerol moiety and threefold positively charged spermine head group ( ). We optimised the 5-step-synthesis of the lipospermine and after up-scaling we have obtained sufficient amounts to initiate preclinical investigations. DOCSPER was tested for its ability to transfect eukaryotic cells in vitro. It has proven to possess high transfection efficiency in comparison to commercially available liposomal transfection agents. Furthermore, DOCSPER was extensively tested in several in vivo studies ( ). These studies revealed a high transfection efficiency, whereas very low toxicity levels were detected. Thus, the results clearly indicate that the cationic lipid DOCSPER is a reliable, low-risk system for broad applications in gene therapy. Cancer chemotherapy targeted on angiogenic vessels is expected to cause indirect tumor regression through the damage of the neovasculature without the induction of drug resistance. To develop a new tool for neovasculature-specific drug delivery, we isolated novel peptides homing to angiogenic vessels from a phage-displayed peptide library. After the determination of the epitope sequences in isolated peptides, liposomes composed of distearoylphosphatidylcholine and cholesterol were modified with penta-peptides. When 14C-labeled liposomes were injected into tumor-bearing mice, liposomes modified with Ala-Pro-Arg-Pro-Gly (APRPG) showed the highest accumulation in murine tumor xenografts. Similar results were also demonstrated by using positron emission tomography. APRPG-modified liposomes (PRP-Lip) encapsulating adriamycin (ADM) effectively suppressed experimental tumor growth with reducing side effects compared with control liposomes encapsulating ADM and with free ADM. Furthermore, these liposomes markedly damaged angiogenic vessels in dorsal air sac model. Next, we investigated whether the peptides selected in murine angiogenic model have affinity for angiogenic endothelium in human cancer. Confocal observation demonstrated that APRPG-modified liposome specifically bound to human umbilical vein endothelial cells (HUVECs) only when HUVECs were activated by vascular endothelial growth factor (VEGF). Furthermore, histochemical analysis demonstrated that biotinylated PRP-containing peptide specifically bound to angiogenic endothelium in human insulinoma and glioma specimens. These data indicate that PRP-containing peptides may be useful for human cancer treatment. The present study indicates the usefulness of APRPG-modified liposomes as a tool for anti-neovascular therapy, a novel modality of cancer treatment. The aim of this study was to check the adjuvanticity of squalene in a model consisting of liposomal vaccine, containing an antigenic peptide from the V3 loop of gp120 of HIV. The liposomes were composed of dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol, with or without cholesterol, squalene, or lipid A. We characterized the interaction between the squalene and the liposomes and measured the effect of squalene on liposome formation and stability. The CGP18 was found at least partially on the surface of the liposomes as shown by flow-cytometricanalysis using monoclonal anti-P18 antibodies. Balb/c mice were injected i.p. with 10 µg of CGP18 with 7 mole%, 43 mole% or 71 mole% squalene, with or without lipid A. Mice injected with liposomal CGP18 containing either lipid A or squalene, developed a moderate anti-CGP18 antibody response at a similar level. Liposomes containing CGP18 without lipid A or squalene failed to develop an antibody response. Splenocytes from mice immunized with liposomes containing CGP18 were tested for INF-γ secretion in an ELISPOT assay. Lipid A enhanced the IFN-γ production 15–50 fold depending on the formulation used, indicating an induction of Th1-response. No increased IL-4 secretion was detected by ELISPOT in any of the groups. In conclusion, liposomes containing squalene show moderate adjuvanticity for induction of a humoral response against CGP18. Addition of lipid A to the liposomes is necessary for IFN-γ secretion by the splenocytes. We conclude further, that squalene itself has a distinctive adjuvant activity upon incorporation into liposomes. Antibody-targeted liposomes (immunoliposomes/IL) have been regarded as very promising targeting vehicles for systemic drug delivery. A liposomal drug therapy necessarily implies a repeated application of liposomes within a certain periode. Despite a clear indication for immunological drawbacks of coupled antibodies, strongly affecting the pharmacokinetic profile, the behavior of IL upon repeated injection has attracted nearly no attention up to now. The present study investigates the pharmacokinetics as well as organ distribution of repeatedly injected IL of different design as indicator for immunogenicity in rats. To correlate IL structure with immunogenicity, the amount of conjugated antibodies was comparable in each case at about 30 µg per μmol total lipid. When injected into naive rats, an increase in elimination can clearly be attributed to the presence of antibodies, since plain pegylated liposomes display the longest circulation half-life (12.8 h). A second injection of identical liposomes into the rats after 14 days has a strong influence on the pharmacokinetic parameters. The circulation time of plain pegylated liposomes drastically droped (t1/2 5.5 h), which indicates that a sterical barrier of the liposomes seems to be less efficient to reduce opsonization and immune reactions. An active role of PEG-PE in provoking liposomal immunogenicity could not be found. Surprisingly, the circulation time of the IL was only sligthly reduced or, in case of terminally coupled IL nearly unaffected. The differences among the various IL can neither be correlated to the accessibility of the coupled antibodies nor to the intensity of the sterical barrier. However, this study confirms that the immunogenicity of IL cannot be generalized to be very high, but depend on several structural parameters such as antibody coupling technique, liposomal size and composition. Although this study for the first time directly compares different types of IL on repeated injections, the immunological parameters can not totally be cleared at this point. These findings might have important implication for the repeated application of IL as drug carriers. Endothelial cells play an active role in various diseases such as inflammation or cancer metastasis. They undergo phenotypic modulations to an activated state which is especially marked by expression of several cell surface adhesion molecules. These adhesion receptors are attractive targets for cell selective pharmacological intervention employing drug targeting strategies since they are easily accessible due to the direct contact with the blood. Recently, we could demonstrate that immunoliposomes (IL) bearing anti-E-Selectin antibodies specifically accumulate at activated endothelial cells (HUVEC) in vitro. In order to derive therapeutical strategies from liposomal targeting, we analyzed route and degree of cellular uptake of the targeted liposomes. Results from several spectroscopical and microscopical techniques display that about 25% of targeted IL were internalized by active as well as passive mechanisms. The internalization was correlated with several liposomal parameters such as type of antibody, coupling strategy or sterical stabilization. Following the intracellular trafficking of liposomes, we modified the liposomal lipid composition in order to avoid lysosomal degradation. Therefore, we established pH-sensitive, sterically stabilized immunoliposomes that deliver their content into the cytoplasm due to liposome destabilization in the late endosomes followed by liposome-endosome fusion. Therefore, these drug carriers offer a great potential for therapeutical approaches in the treatment of inflammation, such as liposomal gene therapy. First results will be introduced. Parkinson's disease is characterized by selective loss of neurons in the substantia nigra pars compacta and significant reduction of neostriatal content of dopamine (DA) and its major acidic metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA); parkinsonian symptoms are relieved by administration of l-dihydroxyphenylalanine (l-DOPA), which is converted by neuronal aromatic l-amino acid decarboxylase (AADC) into DA, hence restoring DA levels in surviving neurons. In order to improve the bioavailability and to minimize unfavourable side effects we studied new dimeric l-Dopa derivatives, as potential prodrugs encapsulated in unilamellar liposomes (PC-CHOL). For better evaluate the influence of geometric conformation of prodrugs on entrapment efficiency and “in vivo” activity, we synthesized new maleic and fumaric amides of l-Dopa. The new compounds and drug loaded vesicular systems were characterized evaluating chemical and physical stability, entrapment efficiency and pharmacokinetic parameters. The striatal l-Dopa and DA concentrations, after i.p. administration, of new delivery systems was performed by means of a new HPLC-EC method2. The use of polyethylene glycol (PEG)-modified lipids is well established for liposome encapsulated drugs and their ability to enhance the delivery of anti-cancer drugs to tumor sites has been demonstrated. We examined the effect PEG-lipid incorporation has on the fundamental in vitro and in vivo characteristics of cationic lipid based Lipid-Protamine-DNA (LPD) formulations. Pegylated lipids allowed for a higher final DNA concentration (280 µg/mL vs. 150 µg/mL for non-PEG formulations), prevented serum-induced aggregation or size increases, and effectively shielded DNA from serum nucleases. The in vivo manifestation of these properties were addressed in experiments directed at examining transfection characteristics, lipid deposition, and acute toxicity responses after systemic administration in murine models. Incorporation of pegylated lipid into LPD resulted in a 3-log reduction of luciferase expression in the lung as compared to non-PEG controls. This reduction was reflective of a trend in all organs examined. Biodistribution studies using 3H-cholesteryl-hexadecyl ether as a lipid tracer indicated that luciferase expression correlated with lipid deposition. Further, an enhanced circulatory half-life was observed for the PEG bearing formulations. Peak serum levels of TNF-α induced by PEG-LPD were up to158 fold lower than non-PEG controls in C57BL6 mice. In three murine xenograft models tumor expression generated by systemic LPD or pegylated-LPD administration was examined. Luciferase expression was evaluated at 6 or 16 h post-administration with one set of experimental groups receiving ultrasound treatment at the tumor site immediately following formulations administration. Non-PEG formulations had a peak expression 6 h post administration unaffected by ultrasound treatment. PEG-bearing LPD formulations did not generate a measurable signal at the 6 hr. time point. At 16 h post administration, expression was minimal for each formulation type, in non-ultrasound treated tumors, but an 8–150 fold enhancement was observed for PEG bearing over non-PEG formulations in ultrasound treated tumors. This data suggested greater passive accumulation of transfection competent PEG bearing formulations at the tumor site with ultrasound treatment, enhancing transit through, and into, cells resulting in enhanced expression levels. This data suggests that DSPE-PEG5k addition to LPD formulations modulates LPD behavior in vivo generating a formulation profile with reduced non-specific deposition and transfection characteristics, coupled with reduced inflammatory effects. PEG-LPD may have clear utility for systemic treatment of cancer. This work was supported by Emerald Gene Systems. Magnetoliposomes were prepared by coating magnetite nanoparticles with a phospholipid bilayer, made of zwitterionic dimyristoylphosphatidylcholine and anionic dimyristoylphosphatidylglycerol. Since their discovery in the late 1960s, these magnetic fluids have continued to attract attention. They can be used to deliver drugs to a target organ in the body whether or not combined with electromagnetically induced hyperthermal treatment of this organ. Because these so-called magnetoliposomes can be effectively captured by magnetic forces, these colloids are very suitable material to study the sorption behaviour of e.g., medical compounds into or onto liposomal bilayers. Unlike other separation techniques, such as gel permeation chromatography, equilibrium conditions are obtained during the complete separation of bound molecules from unbound molecules. The sorption behaviour of propranolol, a nonselective beta-adrenergic receptor blocking agent, in magnetoliposomes was assessed. A clear negative influence of electrolyte addition on the sorption behaviour of this molecule in the magnetoliposomes at pH 7 was found, which indicates that the electrostatic attraction between the negatively charged phospholipid bilayer and the positively charged propranolol is at least partly governing this sorption phenomenon. The knowledge that infections caused by Mycobacterium avium Complex (MAC) affect up to 50% of AIDS patients has led to the development of efficient prophylactic strategies. Rifabutin (RFB) was approved by FDA, as a single agent, for prophylaxis of MAC. With the aim to enhance its therapeutic index, the delivery of the drug to the more vulnerable organs namely liver Kupffer cells and spleen macrophages, was tested using liposomes as a carrier system. RFB was incorporated in conventional liposomes made from phosphatidylcholine/phosphatidilglycerol (PC/PG) or phosphatidylcholine/phosphatidylserine (PC/PS) and in stealth liposomes. The prophylactic effect of liposomal formulations was tested in a Mycobacterium avium infection model. Doses of 5 and 10 mg/kg of the formulations were daily injected, starting one day before the infection induction. The reduction of the infection degree of RFB incorporated in stealth liposomes was not significantly different from the non-incorporated drug. On the contrary, conventional liposomes incorporating RFB were able to reduce to a higher extent the bacterial levels in comparison to the free drug, in a dose dependent manner. Stronger reduction on bacterial load was observed for the lipid composition PC: PG compared to PC: PS either in liver or in spleen, for the 10 mg/kg administered dose. These results indicate a different intrahepatic distribution of PG and PS liposomes and a higher affinity of PG versus PS liposomes for the spleen. Conventional liposomes, including PG in their composition, seem to be promising delivery system for RFB as a prophylactic M. avium agent. These systems will be tried in future for prophylaxis of M. tuberculosis. Small arginine- and tryptophan-rich motifs have been identified in antimicrobial peptides with different secondary structure. To analyze the role of the specific side chains and of conformational constraints we synthesized linear and cyclic hexa-peptides with substitutions of arginine (R) by lysine (K) and of tryptophan (W) by tyrosine (Y) and naphthylalanine (Nal). Measurements of the growth inhibiting activity against Gram positive and Gram negative bacteria revealed a complex relationship between peptide structure and biological activity. No hemolytic and antimicrobial activity was observed for K- and Y-containing linear and cyclic peptides. Cyclization of the R- and W-bearing hexapeptide induced pronounced antimicrobial activity while conserving low activity against red blood cells. Substitution of W by the bulky Nal conferred pronounced activity on the linear as well as cyclic peptides towards bacteria and induced moderate activity towards erythrocytes. Conformational studies of SDS- and lipid-bound peptides and investigation of the lipid vesicle permeabilizing activity served to clarify the biophysical principles involved in the modulation of the biological effects. Comparable activity pattern of the all-l peptides and their corresponding d-enantiomers point to a membrane disturbing mode of action. We previously described ion gradient loading of idarubicin driven by creation of a transmembrane pH gradient in distearoyl phophatidylcholine:distearoyl phosphatidylethanolamine conjugated polyethylene glycol (95:5 molar ratio) and demonstrated that idarubicin was retained better in cholesterol-free liposomes as compared to identical liposomes with cholesterol. When investigating the application of these cholesterol-free liposomes as carriers for other drugs, we observed that some drugs, like doxorubicin, could not be loaded below the phase transition temperature of the bulk phospholipid. We proposed to utilize ethanol incorporation in the lipid bilayer to increase the rates of drug loading in liposomes. Doxorubicin loading rates in cholesterol-free liposomes was 6.6 fold higher in the presence of ethanol. We demonstrated that >90% doxorubicin was encapsulated at 37°C within 2 h, 2.4 fold higher than in the absence of ethanol. Optimal ethanol concentration was 10–15% (vol/vol), a concentration that did not affect either liposome size, trapped volume or, most importantly, the stability of the pH gradient. At 30% (vol/vol), we observed interdigitation characterized by gelling of the sample. Ethanol-induced increases in drug loading was temperature, lipid composition and lipid concentration dependent. Collectively, our results suggest that ethanol addition during drug loading may be an improved method to incorporate high levels of entrapped drug in the absence of high temperatures. A novel approach to prophylaxis against poisoning by organophosphates (OP), the toxic components of nerve gases, is offered by combination of new OP-scavenging formulations positioned at major gates through which the OP gain entry into living systems. The new formulations consist of OP-scavenging enzymes entrapped in bioadhesive liposomes. The liposomes binding with high affinity to recognition sites at the locations desired for scavenger positioning, and remaining bound there for prolonged periods in active (enzyme) form. To provide efficient protection against a wide repertoire of OP, we propose a multi-enzyme approach, operating in more than one mechanism, in which OP is an irreversible inhibitor (Cholinesterases) or a substrate (Organophosphorous Acid Anhydrolase (OPAA), Paraoxonase (PON) and others). Feasibility of our conceptual approach was previously shown with one formulation: burytylcholinesterase (BChE) entrapped in bioadhesive liposomes that had collagen as the bioadhesive ligand. In this communication we report on: (1) optimization of BChE-encapsulating systems, through testing the relationships between the magnitude of the bioadhesive coat (9, 12 and 26 mg collagen/mmol lipid) and system properties and (2) first steps in obtaining OPAA-encapsulating liposomal systems. Our major findings with liposomal BChE are: (i) All three formulations were similar in retention of enzymatic activity, with Km = 0.48 ± 0.18 mM and comparable Vmax values. (ii) Liposomal diameter slightly decreased with the increase in coat size (1.0 to 0.6 cm) (iii) All three formulations were similar in protection of liposome-associated BChE from hostile proteolytic environment. (iv) All formulations showed good binding to recognition sites but with differences in capacity and affinity. (v) Most important all tested formulations performed well as stoichiometric OP scavengers. The cumulative data favor the liposomes with the intermediate-sized bioadhesive coat. Our preliminary findings with OPAA show that the enzyme can be entrapped in liposomes with retention of activity and that, using paraoxon as the test OP/substrate, Intact OPAA-encapsulating liposomes perform well as catalytic OP scavengers. In conclusion, the data reported here is further support for the feasibility of our prophylaxis approach, confirming the multi-enzyme approach and showing that the magnitude of the bioadhesive coat is a tool for system optimization. Liposomes were formed of highly purified membrane-spanning archaeal phospho tetraether lipid by (a) French pressure cell extrusion, (b) extrusion through polycarbonate filters, or (c) detergent dialysis resulting in unilamellar vesicles of 100–200 nm (a,b) or 300–400 nm (c) in diameter. Shelf life stability was tested at 4°C over two years: during this time, these liposomes are stable as determined by electron microscopy and size distribution measurements. Leakiness of the liposomal membrane for electrolytes, alcohols etc. is much lower than that of lecithin bilayer liposomes; permeability of protons is even about two magnitudes lower. Release of insulin and carboxyfluorescein depends on pH, from absolutely no release in acidic milieu liberation increases with rising pH. Rapid intermembrane exchange of lipophilic bromobimane and Nile red from the liposomal membrane to erythrocyte ghosts was observed. The affinity of the liposomes towards T84 colon carcinoma cells in culture was much higher than that of lecithin liposomes as measured by carboxyfluorescein fluorescence. This phenomenon may be ascribed to the sugar component, the biologically rare gulose. No cytotoxicity, no mutagenicity of the liposomes and no toxicity in mice could be determined. The calculated LD50 is above 561 mg/kg BW and immunosuppressed mice lived even longer than controls if liposomes (or the lipid) were admixed to the diet. Spin labelled retinoic acid was incorporated into the liposomes and penetration into the skin of hairless mice measured by electron paramagnetic tomography (MOSS) in comparison to a hydrogel formulation. The extraordinary characteristics of these liposomes can be ascribed to the unique membrane-spanning bipolar main phospholipid from Thermoplasma acidophilum. On the one hand its conformation is much more dynamic than formerly assumed and on the other hand it forms the most stable and impermeable membranes from biological material investigated so far. The most important function of the skin is the formation and maintenance of a barrier that protects the organism against environmental damage and from desiccation. This barrier is located in the stratum corneum (SC). The SC consists of at least two phases: the proteinaceous, highly cross-linked cornified cell envelopes, (corneocytes) and the extracellular, lamellar lipid phase. The corneocytes - imbedded in the lipid phase - have a size of 20–30 micron (pig) and are mainly oriented parallel to the surface of the skin. The lipid matrix is composed of cholesterol, free fatty acids and ceramides. A number of biophysical techniques are used to study the components of the SC ( ). Most studies focuses on the molecular organization of the lipid matrix and the interactions of the different ceramides with cholesterol, fatty acids or water molecules. However, less is known of the molecular organization and distribution of the protein and lipid fraction in the SC tissue. Traditional Fourier transform infrared (FTIR) spectroscopy provides information concerning lipid structure, the formation of micro-domains and thermotropic alterations in macroscopic samples. We have used a new FTIR technology, which is based on a focal plane array detector (FPAD). This technique combines FTIR spectroscopic chemical identification and quantification with microscopic imaging technology ( ). FPAD technology performs much like a camera. It consists of an array of detectors, which are working in parallel to produce spectra from different portions of the tissue sample, and thus collecting spectra simultaneously from all the detector pixels. Any spectral parameters obtained by FTIR microscopy may be monitored, scaled, and mapped across the array, thus providing a true IR vibrational spectroscopic imaging of the analysed tissue area at a spatial resolution approaching the IR diffraction limit (˜5 micron). The images obtained from pig skin SC clearly demonstrate that the SC is a rather inhomogeneous tissue consisting of protein domains embedded in a lamellar lipid matrix. Isothermal titration calorimetry (ITC) is used to determine the critical micelle concentration (cmc) and the demicellization enthalpy (ΔHdemic) of a variety of surfactants as a function of temperature. ITC has the advantage that the cmc and the thermodynamic parameter ΔHdemic can be directly determined from one and the same experiment. In addition, the experimentally observed quantity, the reaction heat, is already a differential quantity, because changes in enthalpy due to changes in concentration are measured. This improves the precision of the cmc determination as long as ΔHdemic is sufficiently large. ΔHdemic, the cmc, and the thermodynamic parameters ΔGdemic (change in Gibbs energy) and ΔSdemic (change in entropy) are usually calculated using the pseudo-phase separation model ( ). This facilitates the comparison between different detergents and with literature data. The experimental data show that ΔHdemic is clearly temperature dependent. The heat capacity change Δcpdemic, obtained from the temperature dependence of ΔHdemic, contains information on changes in exposed hydrophobic surface area when the aggregates dissociate. This finding can be used for making suggestions about the structure of the micelles in terms of water-exposed hydrophobic surface area in the aggregates. This is a critical information for the formulation of drugs in micellar systems. For the low aggregation numbers found for different surfactants, the pseudo-phase-separation model is a crude approximation. Therefore, the aggregation process can better be described using a mass action model ( ). To derive the thermodynamic parameters for the aggregation process, the ITC titration curves have to be simulated with the aggregation number n as one of the variable parameters. We will show that with this model additional information on the aggregation number n as a function of salt concentration and temperature can be obtained. Gene transfer methodologies are emerging strategies for the treatment of both genetic and acquired diseases. The advances in understanding the mechanisms involved in the disease development have stimulated gene therapy research. CNS diseases, as many others, are also a potential target for gene therapy. Work in this area has mainly used viral vectors, mainly due to their enhanced efficiency over non-viral vectors. However, viruses can produce inflammatory and immune responses. Non-viral strategies have become increasingly important and cationic liposomes have shown to be particularly promising. In this work we conducted experiments to evaluate the potential application of cationic liposomes for gene transfection of brain cells. For this purpose, we performed in vitro studies using rat embryo hippocampal and cortical neurons as experimental models, and luciferase and GFP as reporter genes. Several parameters affecting transfection activity were examined, including liposome composition, cationic lipid/DNA charge ratio, association of the ligand transferrin, and degree of cell differentiation. The lipoplexes internalization pathway was also investigated. Our results showed that, independently of the experimental model used, complexes prepared with DOTAP: Chol liposomes were the most efficient to transfect the cells. Moreover, the presence of transferrin proved to be crucial to enhance the transfection activity mediated by these systems. The efficient transfer of genes into specific cell type remains an unresolved problem for gene therapy. In the past, several gene transport systems were tested to deliver DNA pieces into specific target cell types. Toward this goal, we have generated a new liposomal vector which is based on the composition of anionic retroviral envelopes. The packaging of plasmid DNA into these Artificial Viral Envelopes (AVE) in the presence of a suitable condensing cationic polymer yields serum-resistant liposomes of high stability. These liposomes were endowed with a specificity for avβ3-integrin expressing cells (such as melanoma and tumor ECs) by attaching a cyclic RGD-containing peptide to the liposomal surface. These targeted particles are non-toxic, exhibit a very high transduction efficiency for primary ECs of >80%, and are target cell-selective. The in vivo performance of these liposomes as well as their applicability for the targeted delivery of small molecule drugs is currently under investigation. Lipid-Protamine-DNA (LPD) lipopolyplexes have successfully been used for systemic gene transfer. The use of polyethylene glycol (PEG)-modified lipids is well established for liposome encapsulated drugs and their ability to enhance the delivery of anti-cancer drugs to tumor sites has been proven (Lasic, (1998) Trends in Biotechnology 16; 307–332). Pegylated lipids are also known to control surface properties of cationic lipid-based gene transfer systems. However, pegylated lipid incorporation into lipid-DNA complexes causes a concentration dependent reduction of in vitro transfection activity, a result that can be partially attributed to a reduction in particle binding to cells (Harvie et al. J. Phar Sci. (2000) 89;5 652–663). In order to restore particle binding and target LPDs to tumor cells, we incorporated a folate-lipid-conjugate, DSPE-PEG5k-Folate, into the formulations and compared such formulations with those containing DSPE-PEG5k. As expected, addition of pegylated lipid without targeting functions altered the LPD transfection characteristics resulting in a concentration dependent reduction of in vitro transfection activity in KB cells. In contrast, particle binding and transfection were enhanced by the incorporation of the DSPE-PEG5k-Folate conjugate. Addition of such lipid-conjugated-ligands was found to be compatible with LPD formation and results in a dose-dependent enhancement of luciferase expression following in vitro transfection of KB cells. A maximum of 20–50 fold enhancement of luciferase expression over the corresponding DSPE-PEG5k base formulation was observed when 2 mol percent lipid-conjugated-ligand was incorporated into the LPD formulation. FACS analysis also demonstrated DSPE-PEG5k-Folate increased LPD-cell binding by 2 fold over the base formulations. Moreover, transfection activity of LPD bearing DSPE-PEG5k-Folate can be partially abolished in a competition assay using 5000 fold excess of free folate. More interestingly, folate mediated transfection enhancement can be totally inhibited using DOPC:CHOL:DSPE-PEG5k-Folate liposomes as competition agent, while DOPC:CHOL:DSPE-PEG5k control liposomes formulation did not block the transfection enhancement. These data suggest that this new systemic gene delivery formulation will lead to more effective in vivo cell specific targeting. This work was supported by Emerald Gene Systems. Systemic chemotherapy does not result in adequate intratumoral drug levels in the treatment of many solid tumors. High interstitial fluid pressure (IFP) and other pathophysiologic conditions at the tumor site are responsible for this. Some of these barriers may be overcome (in part) by using long-circulating liposomal formulations. We have demonstrated that systemic administration of low dose Tumor Necrosis Factor-α (TNFα) plus doxorubicin liposomes (DOXIL) results in an increased intratumoral uptake of liposomes and a significantly enhanced antitumor efficacy. We also have shown that in an isolated limb perfusion (ILP) the addition of TNFα to the perfusate resulted in significant enhanced accumulation of doxorubicin in rat sarcomas. In vitro studies demonstrated that addition of TNFα had no additive effect when tumor cells were exposed to cytostatic agents. When human umbilical vein endothelial cells (HUVEC) are exposed to TNFα a change in morphology and some gaps between the cells were observed. Apparently TNFα has an indirect effect on the tumor vasculature resulting in an increased permeability and a higher uptake of cytostatic agents. Studies are performed examining the role of TNFα on the antitumor activity of stealth DOXIL in other animal studies. In addition the effect of TNFα on Stealth liposomal Cisplatin (CDDP-SL) uptake in tumor tissue is investigated. Phospholipase A2 (PLA2) hydrolyses phospholipids, rendering fatty acids and lysophospholipids. Secretory PLA2 is found in inflammatory tissue and venom from snakes. PLA2 from snake venom readily catalyses the hydrolysis of both zwitterionic and anionic lipids. However, it has previously been shown, that the activity of secretory PLA2 type IIA towards anionic phospholipids is significantly higher than the activity towards zwitterionic phospholipids. In the present study human tear fluid has been employed to study the activity of PLA2 type IIA towards zwitterionic phospholipids (dipalmitoyl-phosphatidylcholine and dipalmitoyl-phosphatidylethanolamine) and anionic phospholipids (dipalmitoyl-phosphatidylglycerol and dipalmitoyl-phosphatidylserine). The degree of hydrolysis of each phospholipid component in multi-component large unilamellar liposomes was determined. In agreement with previous work (Yang et al., Anal Biochem 1990; 269:278–288) the overall activity of PLA2 was high towards liposomes with a negatively charged surface as compared to neutral liposomes. Interestingly, the activity of PLA2 towards a specific lipid component in multi-component liposomes exhibits a non-trivial dependence on surface charge. The results presented in this study have applications in drug-delivery research in relation to the site-specific degradation of liposomes at desired pathological sites where the concentration of PLA2 is significantly increased. Our laboratory has developed a method to isolate and identify both transcytosing and non-transcytosing, luminal exposed endothelial integral membrane proteins. These proteins can be used to derive novel and tissue-specific targeting ligands for use in drug-delivery. One of the antibodies (LVET) derived by this technique was shown following iv injection and three-color histology of rat lung tissue, to recognize a lung-specific, transcytosing vascular endothelial target. We have developed a LVET-targeted, liposomal formulation of gentamicin which may have clinical utility. Aminoglycoside antibiotics are limited in their clinical use due to their severe renal- and oto-toxicities. However, they remain an important alternative for treating nosocomial pneumonia and chronic lung infection in cystic fibrosis patients. The use of targeted-liposomal formulations of gentamicin may ameliorate some toxic side effects by rapidly distributing the drug to the lung, keeping the serum concentration low and allowing dose reduction. In our laboratory, the amount of gentamicin delivered to healthy rat lung by LVET-targeted, gentamicin-loaded liposomes prepared with egg phosphatidyl choline reached approximately 11 µg/g tissue at 30 min, exceeding the minimum inhibitory concentration for Pseudomonas aeruginosa. Importantly, the gentamicin dose was only 0.75 mg/kg, which is 26 times lower than the standard dose in rats. In contrast, the lung uptake was approximately 0 and 1 µg/g tissue for free and non-targeted liposomal gentamicin, respectively. Liposomes, sterically stabilized by surface grafted polyethylene glycol (sterically stabilized liposomes (SL)) have been shown to be attractive carriers for anti-cancer agents. The accelerated clearance on multiple injection of SL, have been recently reported ( ), could be one of problems to further develop drug-containing SL for clinical applications. Although understanding the mechanism is very important, limited data are available on the effect. In this study, therefore, we investigated in more detail biodistribution of SL in rats and mice after repeated injection. SL indicated long circulation time in both animals in the absence of first-injection. The accelerated clearance occurred in rats and most highly at 5 days after first injection, whereas it occurred in mice at 10 days after first injection. Highly increased hepatic accumulation of SL and hepatic clearance was observed in rats at 5 days post-injection and in mice at 10 days post-injection. At 14 days (2 weeks) after pre-injection, the biodistribution of SL had almost returned normal. It appears that a species difference on the innate immunity for SL clearance exist, although the underlying mechanism is uncertain in this stage. The results obtained in this study may have important implications for the design of liposomal anticancer formulations for clinical application based on animal scale up. The capacity of four different cytostatic drugs to generate free radicals was investigated and correlated to their capacity to cause PPE (palmar-plantar erythrodysestesia) or Hand-Foot Syndrome. This particular dermatologic disorder occurs during chemotherapy with liposome-encapsulated or free doxorubicin and 5-FU, less severe forms are observed with daunorubicin and no PPE occurs with mitoxantrone ( ). The production of reactive oxygen species (ROS) and organic radicals are strongly thought to be involved in the occurrence of the PPE ( ). In the present study the hydroxyl radical production as well as the lipid peroxidation were investigated in the presence of cytostatics. The production of hydroxyl radicals (·OH) was analysed by ESR spectroscopy, using a spin-trap method: OH radicals produced by Fenton reaction or by an azo-compound AAPH were trapped with DMPO in the presence of cytostatics and the ESR signal intensity was analysed. Lipid peroxidation was initiated by the AAPH system in spin labeled liposomes and red blood cells from rats. The signal intensity decay of the 5-, and 16-DXSA spin labels was analysed and the lineshapes of the obtained ESR spectra were simulated. In the presence of doxorubicin the highest amounts of ·OH radicals were produced. Lipid peroxidation was significantly increased in the presence of doxorubicin, daunorubicin, and 5-FU. The addition of glutathione inhibits lipid peroxidation in red blood cells, indicating the possibility to attenuate the PPE by supplementing the chemotherapy with liposome-encapsulated antioxidants. Entrapment of anticancer drugs in liposomes has been shown to decrease their side-effects while increasing or preserving therapeutic activity by drug targeting and elongation of the blood circulation time. Vesicular phospholipid gels (VPG)—highly concentrated, semisolid matrices of densely packed liposomes—are suitable to entrap substances with particular high encapsulation efficiencies up to 70%. In order to increase the tolerability and the clinical efficacy we are developing a VPG formulation of the anticancer drug 5-FU (FU-VPG). We have studied different formulations of VPG and analysed their main in vitro characteristics—encapsulation efficiency and 5-FU-release—with regard to the desired application that can be intratumoral as semisolid VPG or intravenous as vesicular redispersion of VPG. Encapsulation efficiency was found to depend on both the lipid amount and the lipid composition. Using mixtures of soy phosphatidylcholine and cholesterol, an increasing amount up to the upper limit of 45 mol% of cholesterol decreased the encapsulation efficiency. Also changing the pH of the FU-stock-solution influenced the encapsulation efficiency, whereby an increase of the pH led to increased encapsulation efficiency. We found that 5-FU release from redispersed FU-VPG occurs rapidly with a half-life time of approximately one hour. So the formulation would have to be injected quickly after redispersion to ensure high encapsulation efficiency. The half life time of the release of 5-FU from non-redispersed FU-VPG was found to be in the order of a few hours. In vitro data of these different 5-FU loaded VPG suggest an applicability of them as implants with controlled release properties or, after redispersion, as intravenously injected liposomal formulations. This study discloses a novel formulation of paclitaxel-liposome. A liposome made of two major phospholipids with distinct transition temperatures was prepared and characterized. The liposome showed a remarkable capacity for increasing drug loading, especially for hydrophobic drugs which are incorporated in the phospholipid bilayer. The formulated liposome can incorporate as much as 20% paclitaxel to lipid molar ratio, which has been below 3% in most of the previous literature. Shelf stability was found to depend on paclitaxel and/or lipid concentration. Liposome remained stable in liquid form at 4°C for at least 12 months when the drug/lipid molar ratio below 15%. Cell culture test displayed a similar growth inhibition efficacy as the cremophorEL/alcohol formulation (Taxol®). The AUC in rats at a dose of 5 mg/kg was comparable as well. Animal tests indicated that liposomal paclitaxel exhibited considerably lower acute toxicity than did Taxol®. Taken together, the novel liposome formulation developed in this study can incorporate a high content of hydrophobic paclitaxel. These results demonstrate that the liposomal paclitaxel is highly promising for clinical use. By the “oxidative burst” phagocytes are able to kill ingested bacteria and fungi after phagocytosis. Phagocytes of patients suffering from chronic granulomatous disease (CGD) have lost this ability due to a defective enzyme, a NADPH-oxidase. Aim of our project is to reconstitute this ability by glucose-oxidase (GOD) encapsulated in liposomes. The liposomes should be taken up by granulocytes and H2O2, produced from GOD, can be used for the oxidative burst. In order to follow up the uptake and the fate of liposomes inside of granulocytes, we applied different microscopic methods. Confocal laser microscopy was used to determine the intracellular location of liposomes and the potential co-localization of ingested bacteria together with liposomes in the same phagosome. For electron microscopy, ferritin was encapsulated into liposomes to increase their electron density. Spectral imaging, using different liposomal and cellular fluorescence labels, was additionally used to follow the fate of the liposomes in granulocytes. With all three methods the intracellular uptake of the liposomes and their co-localisation with ingested bacteria (S. aureus) was shown and no evidence of liposomes remaining on the cell surface was found. This work was supported by the Reinhold-Beitlich-Stiftung and the fortüne-Programm, Tübingen, Germany. Many papers have shown that immunoliposomes can deliver a large amount of encapsulated drugs into tumor cells when the liposomes were targeted to internalizable receptors. But it is unclear which liposome property affects the cytotoxicity of anti-cancer agent containing immunoliposomes. We investigated effect of membrane fluidity of immunoliposomes on cytotoxicity and their interaction of the liposomes with tumor cells. Transferrin-grafted immunoliposomes were composed by egg phosphatidylcholine (high fluidity) or hydrogenated soy phosphatidylcholine (low fluidity), cholesterol and N-[3-(2-pyridyldithio)propionyl] phosphtidylethanolamine (2:1:0.02, mol/mol). Doxorubicin was loaded into the liposomes by using ammonium-sulfate gradient method. There was significant difference between high and low fluid immunoliposomes on the cytotoxicity of liposomal doxorubicin. High fluid immunoliposomes showed much higher cytotoxicity against both cell lines. Then we examined amount of liposomes associated with the cells as well as intracellular doxorubicin level. High fluid immunoliposomes delivered a large amount of doxorubicin into not only drug sensitive but also drug resistant cell lines, while low fluid immunoliposomes did the same amount of doxorubicin as non-targetted liposomes. Our results showed that immunoliposomes having high membrane fluidity could deliver a large amount of liposomal doxorubicin into both cell lines, resulting in increased cytotoxicity. The presence of large hydrophilic polymers such as polyethyleneglycol on the liposomal surface retards elimination of liposomes from the circulation and increases accumulation in the tumor tissue. We prepared pegylated liposomes containing novel cationic lipid TRX-20, which bind preferentially to chondroitin sulfates (CS). The liposomal composition was optimized to 8 mol% of TRX-20 and 0.75 mol% of PEG-PE to achieve long retention in the circulation and enhanced binding to metastatic tumor cells such as LM8G5 and ACHN that express high levels of CS on the cell surface. In vitro, TRX-20 liposome-entrapped cisplatin showed anti-tumor cell cytotoxicity equivalent to free cisplatin. In vivo, they suppressed the subcutaneous growth of LM8G5 tumor cells more effectively than free-cisplatin or cisplatin encapsulated in pegylated liposomes without TRX-20. Furthermore, cisplatin-loaded TRX-20 liposomes markedly suppressed metastatic spreading of the LM8G5 tumor cells to the liver, and thus significantly prolonged survival of the tumor bearing mice. TRX-20 liposomes loaded with chemotherapeutic drugs may be a useful tool to prevent the growth and metastatic spreading of tumor cells that have enhanced expression of CS. Matrix metalloproteinases (MMPs) are involved in angiogenesis. Several MMPs are known to abundantly present on tumor-induced neovasculature. Especially, membrane-type (MT) MMPs may provide potential targeting guide for selective delivery of agents such as cytotoxic agents and angiogenesis inhibitor. In this study, we attempted to develop novel liposomes targeted on MT1-MMP. The peptide-ligands specific for MT1-MMP was recently obtained by use of phage displayed peptide library, and the epitope sequences were determined as a targeting probe to neovasculature. In here, we prepared liposomes modified with three kinds of candidate 6 mer peptides. In vivo behaviors of these liposomes were determined by a non-invasive method using positron emission tomography (PET). We also performed usual biodistribution study of them by labeling liposomes with [3H] cholesteryl ether. As a result, all peptide-modified liposomes accumulated in tumor compared with control liposomes after intravenous administration. These liposomes had tendency to avoid the reticuloendothelial system (RES)-trapping. We are now investigating the inhibitory effect of them on activation of MMP-2 and invasion of tumor cells. Novel liposomal formulations modified with MT1-MMP-targeted peptides would be useful for anti-neovascular therapy. Vascular endothelial cells (VEC) play a key role in various inflammatory processes. VEC are therefore an important target cell population for active drug delivery strategies. We target adhesion molecules (AM) expressed on VEC involved in the two major processes occurring in (chronic) inflammation; leukocyte recruitment and angiogenesis. Recruitment of leukocytes into inflamed areas is one of the key issues in maintaining an inflammation. Angiogenesis is known to enhance an inflammation by providing means to transport of leukocytes, oxygen and nutrients into the inflamed area. Active drug targeting strategies, specifically blocking or modulating these AM may provide a major therapeutic approach in anti-inflammatory therapy. Therefore we are developing liposomes targeted to AM on VEC for the delivery of anti-inflammatory drugs and DNA. Liposomes are targeted by attaching antibodies specific for E-selectin or ICAM-1, AM involved in leukocyte recruitment, or by coupling an RGD-peptide specific for αvβ3-integrin, an angiogenesis-associated integrin. In vitro studies demonstrated the possibility of using these liposomes with either one of the three types of ligands for intracellular drug delivery to VEC. Transfection of VEC using stabilized plasmid lipid particles (SPLP) ( ) was enhanced up to ten-fold after attachment of RGD peptides. This targeted system provides a means for active gene delivery to VEC at pathological sites. In vivo studies using fluorescently labeled liposomes and intravital microscopy demonstrated extensive binding of E-selectin targeted liposomes to the blood vessel wall in an LPS-induced inflammation in mice. In a rat model for adjuvant arthritis as well as in control animals the pharmacokinetics and biodistribution of RGD-liposomes were assessed. Decreased circulation time, increased uptake in liver and spleen and, surprisingly, equal target to blood ratios in inflamed paws of arthritic rats were observed for targeted compared to non-targeted liposomes. RGD-liposomes encapsulating an anti-inflammatory drug showed enhanced efficacy compared to free drug or non-targeted liposomal drug in a rat adjuvant arthritis model. In conclusion, VEC at inflamed sites provide a target cell population with high accessibility that can be reached by AM-targeted liposomes. Delivery of an anti-inflammatory drug by such liposomes strongly inhibited ongoing arthritis and suggests an important contribution of endothelial cell modulation on the development of this inflammatory disease. Targeting drugs or DNA to VEC at inflamed sites opens new possibilities for therapeutic intervention in (chronic) inflammatory disorders. Many studies showed significantly higher absorption rates as well as a greater pharmacological effect for drugs applied to the skin entrapped in liposomes as compared to conventional topical formulations, which depend on liposome bilayer properties and their size. Information about the role of size and composition are based primarily on measurements of drugs in different skin layers in vitro. In this work electron paramagnetic resonance (EPR) oximetry in vivo is used to measure directly the vasodilatory response produced by local application of benzylnicotinate in liposomal formulations to the skin. It increases the blood flow in skin and consequently the oxygen concentration increases. Partial oxygen pressure pO2 in skin is measured with time after topical application of benzylnicotinate to the skin of mice. The liposomes with the entrapped benzylnicotinate (1.25 w/w%) composed of hydrogenated (HSL) or nonhydrogenated soy lecithin (NSL) and 30 w/w% cholesterol (25 mg/mL lipids) were prepared by thin film method and dispersed in hydroxyethylcellulose hydrogel. Measurements were performed on MLV with mean diameter 350 nm for NSL and 770 nm for HSL and on extruded liposomes with mean diameter 250 nm and 289 nm, respectively. It was found that the efficacy of the drug measured as the area under the curve (AUC) is about the same for all the preparations, but the time, when the oxygen concentration increases as well as the time of action depends strongly on liposome composition and their size. It is well established that over the course of HIV infection, viral particles accumulate and replicate actively in lymphoid organs. The high viral load observed in lymphoid tissues was reported to be associated with trapped HIV-1 particles on the follicular dendritic cells (FDC) located in the germinal centers. FDC, antigen presenting cells such as macrophages and activated CD4+ T-cells are abundant in lymphoid tissues and all express substantial levels of the HLA-DR determinant of MHC-II. In addition, the HLA-DR determinant is physically present on the virion surface and constitutes one of the most abundant host-derived molecules carried by HIV-1. Liposomes bearing surface-attached anti-HLA-DR antibodies thus constitute an interesting approach to target specifically HIV-1 cellular reservoirs as well as free HIV-1 particles. Our previous studies have demonstrated that sterically stabilized anti-HLA-DR immunoliposomes were efficient in delivering high concentrations of drugs to lymphoid tissues leading to a 126-fold increased drug accumulation in lymph nodes. On the other hand, we showed that incubation of HIV-1 particles with anti-HLA-DR immunoliposomes containing amphotericin B (2.5 µg/mL) completely inhibited viral replication. In this study, we have evaluated the efficacy of amphotericin B incorporated in anti-HLA-DR immunoliposomes to inhibit HIV-1 replication in an ex vivo human tonsil model. Preliminary results showed that lymphoid tissue cultures constitute an appropriate model to evaluate the efficacy of immunoliposomal drugs in a human microenvironment. Fluorescence resonance energy transfer (FRET) is a potential method for the characterisation of DNA-cationic lipid complexes. Most of the studies involving fluorescence probes are essentially qualitative. In this work we are using the methodology of resonance energy transfer within the context of parameterised models previously derived in studies of membrane biophysics. The application of these models to DNA-cationic lipid complexes allows the measurement of the distances between the fluorescent intercalator on the DNA and the membrane dye on the lipid, or to evaluate encapsulation efficiencies of this liposomal vehicle. The experiments were carried out both in steady and transient state, with DOTAP/pUC19 complexes with charge ratios (+/−) of 0.5, 2 and 4, in 30 mM Tris/HCl. We used DPH and BODIPY-PC as membranes dyes and etidium bromide and a cyanine dye as DNA intercalators. In cationic complexes (Charge ratios (+/−) ≥ 2), we verified that the cyanine dye remains bounded to DNA and energy transfer occurs to the membrane dye. This was also confirmed by anisotropy and lifetime measurements. In complexes with all the DNA bounded to the lipid, by the application of the referred biophysical models, we determined the distance between both dyes. In complexes with DNA unbound (Charge ratio (+/−)mePhe-D-Trp-Leu-Met-NH2, known as antagonist G, coupled to the liposome surface (SLG). SLG showed 30–44 fold higher binding to H69 cells harvested from H69 xenografts than SL. At 48 and 72 h post-injection, tumor accumulation of [125I]-tyraminylinulin-containing liposomes was shown to be independent of the presence of the targeting ligand. Maximum tumor uptake of either SLG or SL ranged from 2–4% of injected dose/g of tissue. In therapeutic studies, mice received 3 weekly injections of 3 or 6 mg free DXR/kg, or 3 or 10 mg liposomal DXR/kg, at initial tumor volumes of either 7 or 33 mm3. The therapeutic efficacy of DXR-containing SL or SLG was significantly improved over free DXR, but SLG did not improve anti-tumor efficacy relative to SL. Stealth liposomes containing DXR have potential as a therapy against human SCLC tumors. In the context of a research project for gene therapy of glioma, novel cationic liposomes have been designed and prepared for an efficient transport of genetic material into cells. These new liposomes have been prepared from novel lipids, whose polar head group is constituted by morpholinic ring (MM43; MM45; MM73), piperazinic ring (MM46; MM69; MM76) and dimethilamine group, respectively. The non-polar moiety is constituted by different fat acid residues or triterpenic units. Liposomes (cationic lipid:DOPE = 1:1 mixtures) have been prepared by sonication and by injection methods. The diameter of vesicles, measured by dinamic light scattering (Brookhaven Spectrometer), is in the range between 100 and 200 nm. The toxicity of cationic liposomes to C6 rat glioma cells has been determined by XTT cell proliferation test. Lipoplexes were formed by adding cationic liposomes to pGL3 Luciferase Reporter Vector. Size analysis of the resulting complexes has been performed by the deconvolution of the light intensity correlation function, using CONTIN program. The formation of complexes has been demonstrated by agarose gel electrophoresis. The transfection efficiency of these new cationic liposomes in C6 rat glioma cells has been measured by Luciferase Reporter Gene Assay, using pGL3 Luciferase Reporter Vector. The transfection efficiency has been measured as function of different cationic liposome to plasmid concentration ratios and compared with that of Fugene. The cationic liposomes MM54 have a good transfection efficiency, which increases with increasing liposome: plasmid concentration ratio. We here for the first time describe the encapsulation of single liposomes within individual networks made from polymers or proteins. The resulting structures resemble biological cells, with a watery interior being surrounded by an almost impermeable lipid bilayer, that is in turn packed into a meshlike and self stable network. We here show construction and characteristics of the novel material. In particular we will emphasize the ability of the shell to protect the inner liposome against lytic components. Liposomal nanocapsules are expected being useful in the development of novel drug delivery systems that are in particular well suited for the transport of biological macromolecules such as DNA, proteins or peptides. Of particular advantage for biomedical applications is the fact, that the entire structure can be constructed from biological materials. Melanoma is a highly malignant and increasingly common tumor. Since metastatic melanoma remains incurable, new treatment approaches are needed. We previously reported that coated cationic liposomes targeted with a monoclonal antibody (mAb) against GD2 and containing c-myc as ODN (anti-GD2-CCL [c-myc]) resulted in a selective inhibition of the proliferation of melanoma cell lines. Further, treatments with anti-GD2-CCL[c-myc] also reduced levels of c-myc protein in melanoma cells. In this study we show the pharmacokinetic and biodistribution results obtained after intravenous injection of 3H-c-myc-asODN, free or encapsulated in non-targeted or anti-GD2-targeted CCLs. Free c-myc-asODN was rapidly cleared, with less than 10% of the injected dose remaining in blood at 30 m post-injection. C-myc-asODN encapsulated within either CCL or anti-GD2-targeted CCL demonstrated a more favorable profile in blood, with around 45% of the dose of either preparations remaining in vivo at 24 h post-injection. The free and the encapsulated c-myc-asODN had different biodistribution profiles in vivo. While free c-myc-asODN was widely distributed (mainly liver, kidney and spleen) as soon as 30 m post-injection, c-myc-asODN entrapped in CCL or anti-GD2-CCL only accumulated in the spleen to a low degree even after 24 h. These results indicate that anti-GD2-CCL[c-myc] are long-circulating. Preliminary studies in an in vivo melanoma metastatic model show that anti-GD2-CCL[c-myc] inhibited the development of microscopic metastasis in the lung compared to animals treated with free c-myc asODN or free anti-GD2. Further experiments, using CCL[c-myc] and sense c-myc, either free or in a CCL-encapsulated formulation as controls, will define the efficiency of this liposomal formulation as a new therapeutic strategy for treatment of melanoma. Supported by Inex Pharmaceuticals, Burnaby, BC, Canada and Costa Crociere. Design of novel vaginal delivery systems for local drug delivery and also, systemic administration of peptides and proteins has been extensively investigated over the last decade ( ). Due to increasing incidence of sexually-transmitted diseases, especially genital HSV infections ( ), appropriate local therapy of antiviral agents has been recently given more attention. Here we report development of a liposomal delivery system for vaginal administration of acyclovir, able to provide sustained release and improved bioavailability of drug. Acyclovir was entrapped in liposomes prepared by polyol dilution method ( ), whereby different phospholipid compositions were used (EPC, EPC/SA = 9/3 and EPC/EPG = 9/1, molar ratio). All liposomal preparations were characterised for particle size, entrapment efficiency and tested for in vitro stability in buffer pH 4.5 (corresponding to normal human vaginal pH), as well as in vaginal fluid simulant (medium developed to simulate the fluid produced in the human vagina) ( ). To be closer to in vivo application of liposomes and to achieve further improvement of their stability, liposomes were incorporated in vehicles suitable for vaginal self-administration. Bioadhesive hydrogel made of Carbopol 974P NF resin with adequate pH value and desirable viscosity was chosen as vehicle for liposomes containing acyclovir. In vitro release studies of liposomes incorporated in gel confirmed their applicability as a novel vaginal delivery system in localised and sustained release of entrapped acyclovir. Even after 24 h of incubation in vaginal fluid simulant more than 35% of originally entrapped drug was retained in liposomal gel. Cationic liposomes are among the most efficient nonviral vectors used for gene delivery purposes. In our laboratory, we have explored different strategies to improve the biological activity of cationic liposome/DNA complexes (lipoplexes), namely through their association to transferrin (Tf) (ternary complexes). In this work a comparison between plain lipoplexes and ternary complexes prepared under different experimental conditions was performed. The effect of the ionic strength of the medium where the complexes are prepared and its relation to their final size and transfection activity was evaluated. In addition, the ability of the obtained complexes to condense and protect DNA was investigated. Transfection activity was drastically reduced when plain lipoplexes were produced in dextrose solution as compared to that in saline buffer (HBS), whereas no significant effect was observed for the ternary complexes. On the other hand, ternary complexes were shown to be more susceptible to DNase degradation than lipoplexes, independently of the lipid/DNA charge ratio tested, suggesting that association of Tf weakens the interaction between DNA and cationic liposomes. However, ternary complexes produced in dextrose appear to protect DNA more efficiently than those produced in HBS. A correlation between the size of the complexes and their ability to mediate transfection will also be presented. Several preparations of different carnitine liposome derivatives were evaluated both in in vitro and in in vivo models for their ability to gene trasfection. Using these approaches we identified ST983 (also named PUCE) as a liposome with optimal characteristics for gene delivery both in in vitro and in vivo assay. In addition to gene delivery, PUCE liposome was tested for its ability to deliver lipophilic antitumoral drug. To this end, using the lipid film method, we encapsulated with high efficiency a new sigma-tau camptothecin derivative, named Gimatecan (ratio 1:23 drug to PUCE respectively) in PUCE liposome. The intravenous administration of Gimatecan/PUCE liposome reduces the tumor growth of 91% respect to the control group in murine carcinoma 3LL Lewis Lung tumor-bearing mice. In addition, Gymatecan/PUCE liposome induced a good antitumoral responses in Human ovaric carcinoma IGROV-1 and human non small cell lung carcinoma H-460 xenografted in nude mice.Taken together, our results suggest that cationic PUCE liposome could be a new delivery system useful for clinical application. In order to develop a new cationic liposome for drug delivery, we synthesized a series of acyl-derivatives of l-carnitine (general formula (CH3)3-N+-CH2-CH(OCOR1)-CH2COOR2 (Patent US 5498633). In particular, we selected ST983 (where R1 = (CH2)14CH3, R2 = (CH2)10CH3) for its intrinsic ability to generate cationic liposomes in aqueous medium. In this work we present our results on ST983 (also named PUCE) and its ability to entrap antitumoral substances like Taxol and a new proprietary derivative of camptothecin named Gimatecan. Among different feasible methods to entrap drugs into liposomes, for our liposomal preparation, we selected the lipidic film method which gives an high entrapment rate (drug/lipid = 1/23 mol/mol). Moreover, we analyzed the preparation of PUCE by physical-chemical methods and as a result we obtained a homogeneous dispersion of SUV liposome with a size of 100 nm. Finally, we tested cryoprotecting agents, such as lactose, sucrose and trehalose, to support the step of lyophilization. Interestingly, after liposome reconstitution in physiological solution, all tested disaccharides induced a preservation of liposomes size analyzed by QLS method. Taken together our experiments led us to set up a new injectable formulation ready to use for clinical applications. Targeted drug delivery through the blood brain barrier could distinctly improve the therapy of many diseases and reduce peripheral side effects. The recognition and cellular uptake of apolipoprotein E bearing lipoproteins is governed by LDL receptor mediated transcytosis. Here we report the investigation of a potential drug delivery system designed as an artificial lipoprotein with the function to activate an transcytotic process in brain capillary endothelia cells. The model carrier system consists of liposomes and α-helical amphipathic vector peptides containing the LDL receptor binding sequence from human apo E (residues 141–150) and different lipid associating domains, such as (I) transmembrane helices, (II) amphipathic helices or (III) long chain carboxylic acids. Structural studies done by circular dichroism spectroscopy revealed the α-helicity of these peptides and confirmed required binding to POPC SUVs. To assess the binding efficiency and to obtain binding constants isothermal titration calorimetry (ITC) and surface plasmon resonance (BIACORE®) experiments were performed. Membrane lytic properties of the peptides were estimated in release experiments using liposomes with encapsulated fluorescent dye. The well characterised peptide-loaded liposomes provide a promising tool for further receptor binding and translocation studies. Plasmid DNA—actually in gene therapy and DNA vaccination ( ) a pharmaceutical substance by itself ( )—has a topological structure that can now be characterised by capillary gel electrophoresis (CGE) for quality control and stability assessments in storage or in application. Any process development for the manufacturing of plasmid DNA has to be accompanied by a powerful in-process-control (IPC) system to generate data on the characteristics of the plasmid molecules. In particular, methods are required for obtaining supercoiled covalently closed circular (ccc) plasmid DNA in pure form. Commonly, other plasmid topologies appear as well, which have to be separated from the desired product ( ). The innovative technology of CGE (see www.CGEservice.com for further information) is a necessary tool to be added to the actually short list of applicable quality control assays for clinical grade plasmid DNA because it is superior to simple agarose gel electrophoresis (AGE) assays ( ). Here we will focus on an aspect that turned out to be of significant importance for the formulation of plasmid based therapeutics. It allows the establishment of quality assurance standards for plasmid DNA form distribution. In comparison to AGE, CGE offers high resolution and high sensitivity. The data on plasmid stability in storage assessments or those, generated by the analysis of shear effects influencing the plasmid quality in DNA drug delivery formulation and application (e.g. gene gun or jet injection), will heavily influence the QA performed for clinical studies. The aspect for manufacturers of DNA pharmaceutical is, that now the in-line monitoring for the plasmid production ( ) staring from fermentation control until the formulated and filled product (see www.PlasmidFactory.com) will be performed the first time in a way conforming to GMP. Our attempts to design interface targeting inhibitors for HIV-1 Protease (PR) are reviewed: (a) Modified peptides: Short lipopeptides, structurally derived from the terminal segments of PR show good inhibitory activities against PR and act as interface targeting “dimerization inhibitors” (1). They have low metabolic stability and bio-availability. Therefore, the structures had to be modified either by backbone modifications or by side-chain modifications (e.g. as pro-drugs). Modified peptides were synthesised and tested (König, S., et al., accepted, Dumond, J. et al. in preparation). (d). Examples: Esterification of inhibitory peptides may even improve the activity of the parent peptide while neither peptoids nor retro-inverso compounds show strong improvement. In the case of the ß-sheet targeting “dimerization inhibitors” of PR, it may be difficult to obtain good inhibitory constants with backbone modified compounds, since the hydrogen binding of the interface ß-sheet is disturbed. (b) Receptor targeting? The use of thyronine (1), thyroxine and amino-palmitic acid as amino acids in the 3-mer lipopetide structure Pam-YEX has revealed further improvement in binding. Interface targeting anti-HIV agents should be active also against therapy mutants since the mutated active site is not involved in binding. Natural interface mutations are very rare. (c) Computer modelling suggests for ß-sheet structures targeting other modifications, e.g., D-amino acids, substituted aminoglycins, aza-glycins, depending on the position. (d) Other composite enzymes: The principle of creating potent allosteric inhibitors is applicable to many enzymes. The steps are: identification of weakly binding peptides, transformation into ‘modified peptides’ or peptidomimetics with improved activity and bio-availability, attachment of lipids as auxiliary binding groups. (e) Some PR dimerization inhibitors also interfere with other ß-sheet interactions, e.g., ß-secretase, amyloid formation (Aß(1-42) aggregation, Alzheimer). On the other hand, some serpine (ß-sheet) insertion peptides inhibit PR. The questions of specificity and toxicity arise. The results may be helpful for formulation of a ß-sheet rule for inhibitor design, e.g., adding “10” H-bonds to “rule of 5”. We present a novel colorimetric biosensor for studying a broad spectrum of biochemical processes that lead to chemical or physical disruption of membranes, such as peptide-membrane association; changes in pH, temperature, ionic strength and their effects on natural lipid bilayers. The assay is based upon generation of rapid visible color changes in liposomes composed of lipids and polymerized polydiacetylene (PDA). We have applied the assay for studying antimicrobial peptides of the α-defensin family [cryptdins] and analogs with altered antibacterial activity. We demonstrate the existence of direct relationships between the chromatic transitions and the mechanism of peptide-membrane association. A correlation is observed between the colorimetric response of the assay and the extent of membrane penetration by the peptides. The applicability of the assay for determination of membrane properties is shown by analysis of PDA vesicles containing natural lipid components, extracted from diverse sources, such as erythrocytes, yeast and E.Coli mutants with various lipid compositions. The aim of our present study is to develop an enhanced liposomal formulation containing linoleic acid in order to prevent skin hyperpigmentation disorder. For these issues, an inclusive complex of liposomes composed of soya lecithin and linoleic acid has been investigated. We have previously shown that linoleic acid decreases melanin synthesis. Further, clinical trial for melasma showed that the topical application of liposomes is the most suitable formulation for applying linoleic acid to skin. In order to clarify the mechanism of effect of liposomal formulation, the evaluation of both penetration of linoleic acid and long-time existence of the linoleic acid around melanocytes are required. The cutaneous absorption of 14C-labeled linoleic acid with liposomal formula and conventional formula has been evaluated by using brownish guinea pig. In the case of using once frozen skin, no difference in the cutaneous absorption was observed between both formulae. On the other hand, in the case of using freshly extracted skin, the topical application of liposomal formula was more remarkable for imparting the long-lasting effect of linoleic acid in the epidermis than conventional one. The influence of ultraviolet (UV) light irradiation against the permeation of linoleic acid with several type of liposomes was evaluated by 3D reconstructed skin model. Further, the distribution of linoleic acid in the epidermis was determined by the micro-autoradiography method. It was found that 14C-linoleic acid was retained in the epidermis even after 24 h by the topical application of liposomal formula. Back ground: Cancer chemotherapy, in general, accompanies side effects. To reduce them and to enhance the therapeutic efficacy, many applications have been made by using DDS technology. In this study, we challenged to encapsulate TOP-53, a novel and effective topoisomerase inhibitor, into liposomes and investigated its properties in vitro and anti-tumor efficacy in vivo. Methods: TOP-53 was encapsulated into liposomes by remote-loading method and the properties of liposomal TOP-53, such as the stability at 4 and the agglutinability in 50% serum, were examined. Anti-tumor activity of liposomal TOP-53 against solid tumor was examined in vivo by using colon 26 NL-17 carcinoma model mice. Results and Discussion: TOP-53 was effectively entrapped into liposomes, stable at 4, and acceptable aggregation in the presence of serum. Liposomal TOP-53 showed potent cytotoxic activity against tumor cell in vitro, and significant anti-tumor activity against solid tumor as well as the increase in life span of tumor-bearing mice. Improved reduction of side effects will also be discussed. Mechanisms of cationic lipid-based nucleic acid delivery are receiving increasing attention, but despite this the factors that determine high or low activity of lipoplexes are poorly understood. This study is focused on the fine structure of cationic lipid–DNA complexes (lipoplexes) and its relevance to transfection efficiency. Monocationic (DOTAP, DMRIE) and polycationic (DOSPA) lipid-based assemblies, with or without neutral lipid (DOPE, DOPC, cholesterol) were used to prepare lipoplexes of different L+/DNA− charge ratios. Circular dichroism,cryogenic-transmission electron microscopy and static light scattering were used for lipoplex characterization, while expression of human growth hormone or green fluorescent protein was used to quantify transfection efficiency. All monocationic lipids in the presence of inverted hexagonal phase-promoting helper lipids (DOPE, cholesterol) induced appearance of Ψ-DNA, a chiral tertiary DNA structure. The formation of Ψ-DNA was also dependent on cationic lipid–DNA charge ratio. On the other hand, monocationic lipids either alone or with DOPC as helper lipid, or polycationic DOSPA-based assemblies, neither of which promotes a lipid–DNA hexagonal phase, did not induce the formation of Ψ-DNA. Parallel transfection studies reveal that the size and phase instability of the lipoplexes, and not the formation of Ψ-DNA structure, correlate with optimal transfection. This study was supported in part by two grants from the “Center of Excellence” of Israel Science Foundation of the Israel Academy of Sciences and Humanities (to Y.T. and Y.B.), and the Canada-Israel Industrial Research and Development Foundation (CIIRDF) grant. Back ground: A novel quinoline derivative, TAS-103, is known to show high anti-tumor activity through the inhibition of topoisomerases. To enhance the bioavairability of the agent, liposomalization was attempted. Methods: TAS-103 was encapsulated by remote-loading method into liposomes composed of DPPC and cholesterol. Liposomal TAS-103 was evaluated for the stability, agglutinability in the presence of 50% serum, and cytotoxic activity against Lewis lung carcinoma in vitro. Then, the anti-tumor activity against Lewis lung carcinoma-bearing mice was examined in vivo. Result and Discussion: TAS-103 was entrapped into liposomes effectively. Liposomal TAS-103 was stable for storage and in the presence of serum, and showed potent anti-tumor activity like as free TAS-103. In In vivo study, liposomal TAS-103 showed a significant anti-tumor activity and increased in the life span of Lewis lung carcinoma-bearing mice after three dose-administration. These result indicate that liposomal TAS-103 is useful for cancer therapy. Recently, it is clear that anti-angiogenic photodynamic therapy (PDT) is induced intensive suppression of tumor growth in comparison to the general tumor cell-targeted phototherapy. In this study, we developed benzoporphyrin derivative monoacid ring A (BPD-MA)-entrapped polycation liposome (PCL) as a novel angiogenic vessel-targeted phosensitizer. PCL is constructed that cationic polymer, cetylated polyethylenimine (cetyl-PEI), is located on the surface of liposome. We report here that BPD-MA-entrapped PCL induces remarkable phototoxicity for vascular endothelial cells, specific damage to angiogenic vessels for angiogenesis-model mice induced by dorsal air sac technique, and intensive suppression of tumor growth for Meth-A sarcoma-bearing mice at low BPD-MA dose (only 0.25 mg/kg in BPD-MA) following PDT treatment. The photodynamic efficacy of BPD-MA-entrapped PCL is elucidated by facilitation of photosensitizer uptake efficiency due to the strong electrostatic interaction between PCL and plasma membrane and subsequent rapid intracellular transport of liposomal photosensitizers via endocytosis. Furthermore, some degrees of photosensitizers mediated with PCL were accumulated in nuclei, so that the intranuclear photosensitization is one of the factors for induction of enhanced phototoxicity. A-fect™ is a novel liposomal drug delivery system with unique properties. Special constituents of A-fect are chemically modified membrane spanning tetraether lipids (TEL) derived from archaea. Due to their bipolar structure the membrane spanning TEL exhibit a clearly higher stability than conventional bilayer forming lipids. In addition, TEL considerably enhance the stability of conventional liposomes. These unique features allow a continuous adjustment of the release characteristics of A-fect™ liposomes in biological fluids. Release experiments using carboxyfluorescein were performed to study the stability of A-fect™ liposomes in serum. Compared to conventional liposomes A-fect™ liposomes exhibited a high stability with a slow and nearly linear release over several days at 37°C. The release rate could be adjusted continously by the amount of TEL in the A-fect™ liposomes. In vitro and in vivo studies were carried out to evaluate the potential of A-fect™ as an oral drug delivery system. Compared with conventional liposomes consisting of phosphatidylcholine (PC) A-fect™ liposomes demonstrated a significant higher stability under conditions mimicking the gastrointestinal environment. Additionally, multilamellar A-fect™ liposomes are substantially more stable towards bile salts and in low pH media than unilamellar A-fects™. Somatostatin and vasopressin were used as model compounds for oral uptake studies in rats. Peptide formulations were administered by oral gavage, subsequently the plasma levels of peptides were determined at different time points. The A-fect™ formulations led to a significant improvement of the oral resorption of both peptides in comparison to conventional liposomes or to free peptide administration. Both the in vitro data as well as the in vivo results clearly indicate that A-fect™ formulations could be developed into powerful tools for parenteral controlled release systems and for the oral delivery of compounds. Folate-diplasmenylcholine (1,2-di-O-(Z-1′-hexadecenyl)-sn-glycero-3-phosphocholine; DPPlsC) liposomes have been shown to greatly enhance the potency of water-soluble antitumor agents via a selective folate-mediated uptake and acid-catalyzed endosomal escape mechanism. We report an adaptation of this strategy for the delivery of chloroaluminum phthalocyanine tetrasulfonate ( ), a water-soluble sensitizer used in photodynamic therapy, in a binary role as tumor imaging beacon and phototoxic agent. /DPPlsC: folate liposomes (9.8 µM bulk concentration, 2.5 mM intraliposomal concentration) were substantially more phototoxic to folate-deficient KB cells than 12.5 µM free after a 30 m irradiation (630–910 nm). Experiments with /DPPC:folate and folate-free /DPPlsC liposomes (acid-insensitive and non-targeted controls, respectively) showed significantly reduced phototoxicities under the same illumination conditions. Confocal microscopy experiments indicate that these differences are due to 1) increased concentrations of water-soluble sensitizers that can be delivered to target cells using the folate receptor-mediated pathway and 2) changes in the biodistribution and intracellular localization of the beacon when acid-labile DPPlsC liposomes are used as the delivery vehicle. Preliminary results in a subcutaneous murine tumor model suggest that this is a promising in vivo detection and ablation tool for folate-positive tumor tissues. Potential advantages of this approach include the use of lower bulk concentrations, rapid plasma clearance of free , and better phototoxic responses—due to higher intracellular concentrations combined with reduced collateral photodamage arising from misguided sensitizer accumulation—thereby enhancing the selective phototoxicity of PDT treatments. The predictable pharmacokinetics and flexible formulation properties of this targeted liposomal beacon offer significant advantages over related polymer-based conjugates. Self-assembling peptides present attractive platforms for engineering stimulus-responsive materials with controlled nano- and microstructures. Here, we demonstrate the stimuli-triggered self-assembly of fluid peptide and protein precursors into highly crosslinked peptide hydrogels. Our system takes advantage of thermal or light triggered release of encapsulated salts from liposomes to trigger self-assembly of either a 16-amino acid peptide or an enzyme-crosslinkable protein substrate. The peptide consists of alternating hydrophobic and hydrophilic residues and is known to self-assemble into β-sheet structures in the presence of salts. We show that temperature and light-triggered release of calcium salts from liposomes results in rapid gelation of peptide/liposome suspensions into highly crosslinked, fibrillar, β-sheet hydrogels. The enzymatically-crosslinked system forms hydrogels upon near-infrared irradiation of calcium-containing liposomes in the presence of transglutaminase and fibrinogen. These solutions also form patterned hydrogels upon photolithographic processing. This approach may lead to useful nanomaterials that are able to undergo drastic changes in physical properties upon activation by mild triggering stimuli. The efficacy of polyethyleneglycol (PEG)-modified liposomal doxorubicin (PEG-LDOX) against tissue distribution and antitumor activity has been demonstrated. Our results on the fixed aqueous layer thickness (FALT) have suggested that sing le PEG-modification by the PEG-lipid, which had a large molecular weight, couldn't display sufficient ability, whereas the mixture of PEGs, which had a long and short polyoxyethylene chain, exhibited a stronger effect on PEG-modification. We recognized the most suitable mixed rate (PEG2000:500 = 2:1), showed the maximum FALT. In this study, we examined the effects of this mixed PEG-modification on tissue distribution and antitumor activity. With increase of FALT, the DOX concentration increased in the plasma and decreased in the livers. This result suggested that the ability to avoid trapping by RES was caused by increase of FALT. Furthermore, The administration of PEG(2:1)-LDOX caused a reduction of tumor weight as compared with PEG2000-LDOX and PEG500- LDOX. Moreover, the DOX concentration in the tumor showed PEG(2:1)-LDOX > PEG2000-LDOX > PEG500-LDOX, this order agreeing with that of FALT. Namely, the connection between increase of FALT and the enhancement of antitumor activity of these liposomes was suggested. In conclusion, it was confirmed that this mix ed PEG- modification of liposome was superior. The overproduction of biochemical mediators, and the activation of leucocytes and endothelial cells, generated in thermally injured tissue, gives rise to both local and distant effects. Highly reactive metabolites, such as oxygen derived free radicals are involved in the postburn phase of thermally damaged skin, leading to enhanced tissue loss an delayed wound healing. To capture these radicals, enzymes such as superoxide dismutase (SOD) are of great therapeutical interest. But the nature of the SOD, with a high molecular weight, the hydrophilic characteristic and the extremely short biological half life diminish its therapeutical application. The aim of our studies was to evaluate the effects of topically applied recombinant human SOD in different formulations, whereas special attention was payed on the liposomal encapsulated SOD. To evaluate the effects edema formation in the inflammatory stage and the wound healing properties were examined by histological and macroscopical observations. In all tested parameters the liposomal formulation showed statistically relevant data compared to all other groups. Therefore, we believe that the liposomal application of SOD may compensate its unfavourable characteristics. In the case of topical application, an alternative non invasive strategy, liposomes may act as drug carrier to localize the drug in the affected tissue and furthermore, the sustained release of the protein enhances its potential in radical scavenging. Liposomes represent one of the major tools for modern drug delivery strategies. We aimed to establish a novel liposome production procedure, where several features, such as simplicity, robustness, easy handling in sterilization procedures and especially scalability were of major concern. Based on the principles of the ethanol injection technique (Batzri and Korn, 1973) we developed a new technique, whereby a substantial progress was achieved, leading from the conventional batch process to a novel continuous procedure. Herein, the principal item is the crossflow injection module, especially designed for this purpose. This specially conceived unit has the benefit of liposome manufacture independent of production scale, as scale is determined only by the free disposable vessel volumes. All devices such as vessels, valves, pumps, manometers and tubes, were designed to be suitable for sterile operations. Manufacture of liposomes using the newly invented crossflow module in the multiple injection technique resulted in reproducibility regarding vesicle size and size distribution as well as high protein entrapment. Variation of lipid concentration and injection pressures resulted in narrow distributed vesicles at any desired vesicle. Subsequent continuous crossflow filtration within the same closed containment separates unentrapped material from the liposome suspension. Stability testing was performed until now for more than one year and up to date the liposomes remained stable. In addition no loss of protein activity could be detected, due to the mild and optimized preparation and filtration conditions. In previous work we reported that liposomes surface-modified with AcoHSA are massively targeted to the hepatic endothelial cells ( ). In order to modulate the function of sinusoidal endothelial cells in pathological conditions we modified stabilized lipid plasmid particles (SPLPs) developed by Cullis et al. ( ) with AcoHSA for the selective targeting of genes to these cells. SPLPs were prepared by solving PEG-lipids, cationic lipids, neutral lipids and DNA in high detergent solution. Lipid particles were formed during detergent dialysis. The DNA encoded for the reporter genes Enhanced Green Fluorescent Protein or luciferase. SPLPs of varying composition were prepared and characterized. AcoHSA was covalently coupled directly to the SPLP surface or to the distal end of a PEG-chain. The size of the SPLPs ranged from 80–120 nm and up to 60% of the DNA was encapsulated. The amount of AcoHSA coupled to the surface of the SPLPs was approximately 10 µg/µmol total lipid, but could be increased to 22 µg/µmol total lipid by coupling the AcoHSA to the distal end of the PEG-chain. Transfection efficiency in vitro with COS-7 cells (as control cells) is low but is increased in the presence of CaCl2. In conclusion, we are able to make stable SPLPs containing high amounts of DNA that can be targeted to the hepatic endothelial cells by coupling of AcoHSA. In vivo transfection experiments are currently under investigation. consisting of phosphatidylcholine (PC), cholesterol, and (MPB-PE) in the ratio of 6:4:0.4:0.32. AcoHSA was covalently coupled to the MPB-PE present in the lipid coating. After extrusion the coated cationic lipoplexes (CCL's) were 144 ± 20 nm. CCL's containing plasmid DNA could be separated from empty vesicles by using a sucrose gradient. The CCL's were further characterised by measuring the amount of AcoHSA coupled to the CCL's, the amount of incorporated DNA, and transfection efficiency. The amount of DNA associated with the CCL's varied from 0.35 to 0.81 µg/µmol total lipid. Transfection of COS cells with the coated lipoplexes was observed after a 96 h incubation with these particles (96 h). The number of transfected cells is low. In conclusion, we are able to make CCL's containing DNA that are stable for at least several weeks, and have a low transfection efficiency in vitro and which can be used for in vivo studies. The resulting SPLPs consisted of 1,2-diacyl-sn-glycero-3-phosphoethanol-amine (DOPE), cholesterol, poly(ethylene-glycol)2000C14-ceramide (PEG-Cer14) or 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene-glycol)2000] (DSPE-PEG2000), 1,2-diacyl-3-trimethylammonium-propane (DOTAP), and 1,2-dioleoyl-sn-glycero-3-phophoethanolamine-N-[4-(p-maleimidophenyl)butyramide] (MPB-PE) in the molar ratio of 37.5:40:10:12:2.5. The amounts of DOPE and cationic lipid varied. To improve liposomal gene transfer we investigated the influence of alkyl phospholipids (APLs) on gene transfer efficiency in human tumour cell lines. To approach this, liposomes were employed which consist of APLs of varying chain lengths, saturation of the chain and head groups combined with dioleylphosphoethanolamine and dioctadecyldimethylamine bromide. The membrane-interacting activity of APLs in these liposomal preparations was utilised to enhance DNA transfer into targeted cells. The transfer of the β-galactosidase (LacZ) expressing plasmid pCMV by these APL-liposomes containing the short chain tetradecylcholine led to an up to 3-fold increase in transfer efficiency in HCT15 and HCT116 human colon carcinoma cells compared to lipofectin-mediated transfection. We further evaluated the transfection potency in vivo using the LacZ reporter gene and a plasmid encoding the cytosine deaminase (CD)- gene. It was found that the LacZ activity was comparable after transfection with the TPC-lipoplex and with lipofectin. By contrast, a significantly improved therapeutic effect was observed in mice treated with the TPC-lipoplexes for intratoumoral CD-gene transfer compared to animals, which received lipofectin-lioplexes. It was found that the best in vivo transfection efficiency correlates with a higher brutto charge as it was found in vitro. The studies demonstrated that utilisation of short chain APL for the preparation of lipoplexes may be of significant benefit to generate more efficient lipid-DNA complexes for gene transfer in vitro and in vivo. The analysis of high- and low-density lipoproteins merits huge interest in the diagnosis and understanding of atherosclerosis. Significant changes are detectable towards protein as well as lipid composition under pathological conditions. Since damages of lipids lead to the formation of well-defined products like lysophospholipids or chlorohydrins, analytical methods which allow the fast and reliable determination of lipid composition of lipoproteins are strongly needed. MALDI-TOF mass spectrometry was applied to the analysis of the lipid composition of human lipoproteins. Differences between LDL and HDL in sphingomyeline and phosphatidylcholine content could be easily monitored by mass spectrometry and differences towards the extraction efficiency of different solvent systems could also be found by MALDI-TOF MS. Additionally, treatment of LDL with hypochlorite and phospholipase A2 resulted in marked changes (formation of chlorohydrines and lysolipids) which were detectable to a different extent by mass spectrometry. Using MALDI-TOF MS the chlorohydrine of cholesterol was detectable. We conclude that MALDI-TOF mass spectrometry is able to provide fastly a reliable lipid profile of human lipoproteins, and may be useful in clinical investigations of LDL and HDL. The transfection efficiency of DOTAP and DOTAP-Cholesterol complexed with DNA encoding the luciferase reporter gene was optimized in cell culture in the presence or absence of protamine sulfate. From 164 different complexes four were selected for further analyses. Although the four selected complexes had a similar high transfection efficiency in cell culture 22-fold differences in transduction efficiencies of the complexes were observed in mice. In vivo transduction efficiency seemed to correlate with resistance to inhibition of transfection by high serum concentrations. Physical properties, such as zeta-potential, turbidity, degree of resonance energy transfer between lipids and accessability of DNA were determined to better understand the underlying cause for serum resistance. Control complexes, that contained the same compounds at the same final concentration as the selected complexes but that differed in transfection efficiency due to a change in the order of addition in compounds were also used to detect structural differences in complexes correlating with transfection efficiency. The Zeta potential and changes in turbidity or resonance energy transfer after addition of serum did not seem to correlate with transfection efficiency or resistance to serum. The percent inhibition of transfection by serum was dependent on the lipid used and the lipid concentration but not on the order of addition of components. Protamine sulfate when added to the DNA before addition to lipid did only decrease the degree of lipid membrane pertubation but also increased the capture efficiency of the complexes there by reducing the accessability of their DNA to PicoGreen and DNAse.