1. We studied the effect of intracellular angiotensin II (Ang II) and related peptides on rat aortic contraction, whether this effect is pharmacologically distinguishable from that induced by extracellular stimulation, and determined the Ca2+ source involved. 2. Compounds were delivered into the cytoplasm of de-endothelized aorta rings using multilamellar liposomes. Contractions were normalized to the maximum obtained with phenylephrine (10-5 M). 3. Intracellular administration of Ang II (incorporation range: 0.01-300 nmol mg-1) resulted in a dose-dependent contraction, insensitive to extracellular administration (10-6 M) of the AT1 receptor antagonist CV11947, the AT2 receptor antagonist PD 123319, or the non-selective AT receptor antagonist and partial agonist saralasin ([Sar1,Val5,Ala8]-Ang II (P < 0.05). 4. Intracellular administration of CV11947 or PD 123319 right shifted the dose-response curve about 1000 fold or 20 fold, respectively. PD 123319 was only effective if less than 30 nmol mg-1 Ang II was incorporated. 5. Contraction was partially desensitized to a second intracellular Ang II addition after 45 min (P < 0.05). 6. Intracellular administration of Ang I and saralasin also induced contraction (P < 0.05). Both responses were sensitive to intracellular CV11947 (P < 0.05), but insensitive to PD 123319. The response to Ang I was independent of intracellular captopril. 7. Contraction induced by extracellular application of Ang II and of Ang I was abolished by extracellular pre-treatment with saralasin or CV11947 (P < 0.05), but not with PD 123319. Extracellular saralasin induced no contraction. 8. Intracellular Ang II induced contraction was not affected by pre-treatment with heparin filled liposomes, but completely abolished in Ca2+-free external medium. 9. These results support the existence of an intracellular binding site for Ang II in rat aorta. Intracellular stimulation induces contraction dependent on Ca2+-influx but not on Ins(1,4,5)P3 mediated release from intracellular Ca2+-stores. Intracellular Ang I and saralasin induce contraction, possibly via the same binding site. Pharmacological properties of this putative intracellular receptor are clearly different from extracellular stimulated AT1 receptors or intracellular angiotensin receptors postulated in other tissue.

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Combretastatin A4 phosphate (CA4P) is a potent vascular disrupting agent utilized in the treatment of cancer. The observed rapid vascular shutdown post administration as well as its potency at 1/10th of the established maximum tolerated dose (MTD) have made it one of the most prevalent tubulin binding agents. CA4P is currently involved in 19 clinical trials. Unfortunately, as is the case with most forms of chemotherapy, the off target effects associated with its use can be prohibitive for a large percentage of cancer patients. The advantages associated with the liposomal encapsulation of chemotherapeutic agents have been established for over 20 years and are shown to decrease off target effects whilst increasing stability, bioavailability, and circulation time. Liposomes comprised of conventional phospholipids, such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), are usually stabilized by the incorporation of cholesterol. However, the addition of cholesterol to liposomal formulations is concerning due to the capability of cholesterol to form oxysterol molecules and exacerbate other preexisting conditions such as hypertension or cardiovascular disease. With this study we aim to enhance the usage of CA4P through liposomal encapsulation in order to reduce some of the associated off target effects and increase bioavailability and overall efficacy. We also aimed to enhance the stability of our lipid vesicles through the incorporation of bipolar tetraether lipids isolated from the thermoacidophilic archaea S. acidocaldarius. The polar lipid fraction E (PLFE) lipids studied here have previously been shown to generate highly stable lipid vesicles. The liposomal formulation studied here included the encapsulation of the anti-cancer drug CA4P in PLFE liposomes. With this work we characterized our liposomes to optimize their drug loading, membrane stability, size, colloidal stability, and membrane surface charge. We also identified a photochemical isomerization reaction occurring in our CA4P samples and then proceeded to characterize the fluorescence and aggregation behavior of our CA4P isoforms. From our studies of CA4P in solution we observed a red shift in the excitation spectra of CA4P with increasing concentrations. This bathochromic shift is characteristic with the formation of j-aggregates. The CA4P concentrations with the most dramatic red shift corresponded exactly with the drug concentrations associated with self-quenching behavior. From these studies we determined the effects of increased CA4P concentration on fluorescence intensity, drug aggregation and how these phenomena can be utilized and exploited to maximize liposomal drug loading and decrease rate constants of drug leakage and cytotoxicity. The end goal of liposomal chemotherapeutic formulations is a stable, controlled release of as much encapsulated drug as possible. With the thorough understanding of our membrane system, drug fluorescence and CA4P aggregation behavior; we can maximize our encapsulated drug loading as well as create a stable liposomal formulation with a predictable CA4P release. / Biomedical Sciences

Liposomes have the ability to carry and deliver both hydrophilic and hydrophobic drugs and to protect them when injected into the circulatory system. They thus provide an attractive vehicle for drug delivery. However, problems of rapid clearance and inability to target liposomes to specific cells and tissues remain unresolved. Rapid clearance has been attributed to adsorption of opsonins, and one approach to reduce such adsorption is to create sterically stabilized liposomes by modifying the surface with polyethylene glycol (PEG) or dextran. To deliver their drug "payload" liposomes must interact with the membranes of target cells. Interactions with cellular components of the vascular walls have been observed for various sulfated polysaccharides such as heparin and functionalised dextrans. Based on the above considerations, the purpose of this work was to investigate the ability of various polymeric modifiers on liposomes to reduce protein adsorption and promote incorporation into target cells. Liposomes of composition PC/PEI cholesterol (70/10/20 mol %) were surface modified with PEG, dextran, heparin, and functionalised dextran. Protein adsorption was studied from solutions of IgG in buffer and from plasma. Adsorption from buffer was measured by radio labelling methods. For the plasma work, a total protein assay was used to determine the amount of protein adsorbed to the liposome surface, while gel electrophoresis and immunoblotting methods were used to examine the profiles of protein binding. Liposome incorporation into vascular smooth muscle and endothelial cells was evaluated using fluorescent labelling and radio labelling techniques. The IgG adsorption studies showed reduced adsorption on all polymer-modified liposomes. Plasma adsorption data showed that adsorbed protein layer compositions on the different liposome types were similar, but different from that of the plasma itself, showing that the plasma was fractionated on the liposome surfaces. Cell interaction studies showed that liposomes modified with dextran and sulfated dextran were incorporated into both cell types. The unmodified, PEG-and heparin-modified liposomes were not incorporated to any significant extent. / Thesis / Master of Engineering (ME)

No / This work evaluated the in vitro transdermal iontophoretic delivery of tritiated estradiol from ultradeformable liposomes compared with saturated aqueous solution (control). Effects of current density and application time on tritium exchange with water were also determined. Penetration studies used three Protocols. Protocol I involved occluded passive steady state estradiol penetration from ultradeformable liposomes and control. The effect of current densities on drug penetration rates was also assessed (Protocol II). In Protocol III, three consecutive stages of drug penetration (first passive, iontophoresis and second passive) through the same human epidermal membranes were monitored. Such an experimental design investigated the possible effect of high current density (0.8 mA/cm2) on skin integrity. The tritium exchange study showed that extent of exchange correlated well with current density and time of application, with some shielding of estradiol by the liposomal structure. Liposomes enhanced estradiol passive penetration after occlusion. Protocol II showed that estradiol flux increased linearly with current density, although being delivered against electroosmotic flow. In Protocol III, reduction in flux of the second passive stage to near that of the first reflected a reversibility of the structural changes induced in skin by current.

No / Electrically assisted skin delivery of liposomal estradiol; phospholipid as damage retardant.

Yes / Lipid membrane-water partitions (e.g., immobilized artificial membrane systems where the lipid membrane is a neutral phospholipid monolayer bound to gel beads) were compared to various organic solvent-water partitions using linear free energy relationships. To this end, we also measured the retention factors of 36 compounds (including neutral and ionic species) from water to liposomes made up of 3-sn-phosphatidylcholine and 3-sn-phosphatidyl-l-serine (80:20, mol/mol), employing liposome electrokinetic chromatography in this work. The results show that lipid membranes exhibit a considerably different chemical environment from those of organic solvents. For both neutral species and ionic species, partitions into the more polar hydroxylic solvents are chemically closer to partition into the lipid membrane as compared to partitions into the less polar hydroxylic solvents and into aprotic solvents. This means that solutes partition into the polar parts of lipid membranes, regardless of whether they are charged or not. In addition, cerasome (i.e., liposome composed mainly of stratum corneum lipids) was compared with regular phospholipid liposomes as a possible model for human stratum corneum in partitions. It was found that the cerasome-water partition exhibits a better chemical similarity to skin permeation. This is probably due to the unique structures of ceramides that occur in cerasome and in the stratum corneum lipid domain. We further show that membranes in membrane-water partitions exhibit very different properties.