Due to the immuncompromised status of AIDS patients, secondary infections and malignancies are common. Conditions secondary to AIDS for which patients require treatment include Karposi's sarcoma (treated with methotrexate), varicella-zoster (treated with antivirals such as acyclovir) and herpes simplex (also treated with antivirals like acyclovir or idoxuridme). However the clinical efficacy of these drugs is limited by poor skin permeability. Few reports, however, have dealt with the delivery of low molecular weight hydrophilic drugs from these vesicles (El Maghraby et al, 2000). The aim of our study was to investigate in vitro permeation of methotrexate, acyclovir and idoxuridine across human epidermal membrane from elastic liposomes. The intent was to establish whether formulation of these hydrophilic drugs into elastic liposomes would enhance their skin permeation parameters. We developed and validated high-performance liquid chromatographic techniques for quantitative analysis of methotrexate, idoxuridine and acyclovir. Elastic liposomes were prepared from various phospholipids- phosphatidylcholine 78.6%; phosphatidylcholine 50%; hydrogenated phosphatidylcholine 90%; phosphatidylcholine 95% and surfactants - sodium cholate, sodium deoxycholate, Span 20, 40, 60, 80. These vesicles were characterised by transmission electron microscopy. The solubilities of methotrexate, acyclovir and idoxuridine were determined. Phospholipon G (95% phosphatidylcholine) was chosen for the preparation of the liposomes with different surfactants. Permeation of methotrexate, acyclovir and idoxuridme from these vesicles across human epidermal membrane was investigated. Flux values for methotrexate, acyclovir and idoxuridine values (J) obtained by curve-fitting of data using Easyplot were compared to those obtained by linear regression. We used Student's t-test to determine statistically significant differences in the flux values of the formulations. A computer program http://www.physics.csbsju.edu/stats/ttest- bulk-form.html was used for this purpose. Our results indicate that there are no statistically significant differences between flux values from elastic liposomes and saturated aqueous solutions. / Thesis (Ph.D. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2004.

The thermodynamic properties of bioactive peptides determine how they interact with cellular assemblies. Ultrasonic velocity and density measurements were used to analyse the volumetric properties in aqueous solution of 3 different materials: KCl, bioactive peptides (from hemp seed and dairy proteins), and liposomes (cell membrane models), as well as the interaction between peptides and liposomes. Serial dilutions of the three different materials showed linear relationships between density and concentration and between ultrasonic velocity and concentration. The apparent specific volume and apparent specific compressibility in solution of all materials showed concentration dependence as a result of increased electrostriction as solutions were diluted. The experimental ultrasonic velocities of liposome-dairy peptide mixes were higher than the theoretical additive value, due to interactions between liposomes and peptides. My research demonstrates the benefits of precise volumetric assessments in biological assays.

The high gene knockdown specificity and efficiency of RNA interference (RNAi) provides a potentially viable avenue for the development of a new class of nucleic acid therapeutics for gene-based disease conditions. However, serum instability, inefficient cellular trafficking and non-specific effects of small interfering RNAs (siRNAs), one of the functional mediators of RNAi, has necessitated the development of carriers to facilitate targeted cell delivery. The decline of viral vectors in human gene therapy as a consequence of safety issues has intensified the importance of non-viral vector development. Among the non-viral vectors available for siRNA delivery, cationic liposomes have emerged as an attractive option owing to their simplicity, versatility, relatively low toxicity and potential for cell-specific targeting. Although existing cationic lipids and liposomes traditionally used for DNA delivery have also been used for siRNAs, there still exists a need to develop cationic lipids tailored towards siRNA transfection for improved gene silencing efficiency. Among the cell specific targets available for RNAi therapeutics, hepatocytes expressing the asialoglycoprotein receptor (ASGP-R) are an ideal choice due to the large number of disease targets present for treatment. In this investigation four novel cationic liposome formulations were prepared from equi-molar quantities of either the cationic cholesterol derivative 3β [N-(N’,N’- dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) or 3β [N-(N’, N’, N’- trimethylammoniumpropyl)-carbamoyl] cholesterol iodide (Chol-Q) and DOPE, with and without the hepatotropic ligand, cholesteryl-β-D-galactopyranoside. Electrophoretic gel analysis and SYBR®green displacement assays were employed to determine siRNA binding and condensation efficiencies for all cationic liposomes; while liposome and lipoplex size measurements were made by cryoTEM. SiRNAlipoplex stability in serum was determined by the nuclease protection assay. Cell studies performed on the ASGP-R+ human hepatoma cells, HepG2 and the ASGP-Rembryonic kidney cells, HEK293, to determine lipoplex toxicity and transfection efficiencies were also undertaken. We show that the cationic liposomes formulated for this investigation were able to bind synthetic siRNA optimally at a positive to negative charge ratio of ± 1 : 6. In addition, the cationic liposomes were able to afford siRNA duplexes substantial protection from ribonuclease digestion in serum. From the results obtained in this study, it appears that the cationic liposomes are well tolerated by both the HEK293 and HepG2 cells in vitro. More importantly, the results obtained demonstrated higher transfection efficiencies for the targeted lipoplexes compared with the untargeted controls, strongly supporting the notion that incorporation of the cholestryl-β-D-galactopyranoside into the liposome structure increases transfection efficiency to the targeted HepG2 cells in culture via ASGP receptor mediation. Comparative studies in the HEK293 cell line yielded low transfection effeciences in the order of 20%, with no significant difference being recorded between galactosylated and non-galactosylated lipoplexes. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2011.

Due to the immuncompromised status of AIDS patients, secondary infections and malignancies are common. Conditions secondary to AIDS for which patients require treatment include Karposi's sarcoma (treated with methotrexate), varicella-zoster (treated with antivirals such as acyclovir) and herpes simplex (also treated with antivirals like acyclovir or idoxuridme). However the clinical efficacy of these drugs is limited by poor skin permeability. Few reports, however, have dealt with the delivery of low molecular weight hydrophilic drugs from these vesicles (El Maghraby et al, 2000). The aim of our study was to investigate in vitro permeation of methotrexate, acyclovir and idoxuridine across human epidermal membrane from elastic liposomes. The intent was to establish whether formulation of these hydrophilic drugs into elastic liposomes would enhance their skin permeation parameters. We developed and validated high-performance liquid chromatographic techniques for quantitative analysis of methotrexate, idoxuridine and acyclovir. Elastic liposomes were prepared from various phospholipids- phosphatidylcholine 78.6%; phosphatidylcholine 50%; hydrogenated phosphatidylcholine 90%; phosphatidylcholine 95% and surfactants - sodium cholate, sodium deoxycholate, Span 20, 40, 60, 80. These vesicles were characterised by transmission electron microscopy. The solubilities of methotrexate, acyclovir and idoxuridine were determined. Phospholipon G (95% phosphatidylcholine) was chosen for the preparation of the liposomes with different surfactants. Permeation of methotrexate, acyclovir and idoxuridme from these vesicles across human epidermal membrane was investigated. Flux values for methotrexate, acyclovir and idoxuridine values (J) obtained by curve-fitting of data using Easyplot were compared to those obtained by linear regression. We used Student's t-test to determine statistically significant differences in the flux values of the formulations. A computer program http://www.physics.csbsju.edu/stats/ttest- bulk-form.html was used for this purpose. Our results indicate that there are no statistically significant differences between flux values from elastic liposomes and saturated aqueous solutions. / Thesis (Ph.D. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2004.

The thermodynamic properties of bioactive peptides determine how they interact with cellular assemblies. Ultrasonic velocity and density measurements were used to analyse the volumetric properties in aqueous solution of 3 different materials: KCl, bioactive peptides (from hemp seed and dairy proteins), and liposomes (cell membrane models), as well as the interaction between peptides and liposomes. Serial dilutions of the three different materials showed linear relationships between density and concentration and between ultrasonic velocity and concentration. The apparent specific volume and apparent specific compressibility in solution of all materials showed concentration dependence as a result of increased electrostriction as solutions were diluted. The experimental ultrasonic velocities of liposome-dairy peptide mixes were higher than the theoretical additive value, due to interactions between liposomes and peptides. My research demonstrates the benefits of precise volumetric assessments in biological assays.

Bio-nanotechnology is the use of biomolecules to control both the structure and property of nanomaterials. No biomolecule has been employed more often than DNA as exemplified in the numerous demonstrations of DNA-directed assembly of nanomaterials. DNA has been used to covalently functionalize and assemble soft nanoparticles (e.g. liposomes) and hard nanoparticles (e.g. gold and silica nanoparticles) into a variety of hierarchical nanostructures. The majority of previous work however has focused on the latter, i.e., the assembly of “hard” nanoparticles such as gold nanoparticles (AuNPs) as oppose to the assembly of soft materials. The primary focus of this thesis is to add to the growing field of DNA-directed assembly of soft materials owing to the promise of such materials in a variety of analytical and biomedical applications. The first class of soft materials considered are liposomes which interestingly can be deformed by relatively weak intermolecular forces. In addition, DNA anchored to its surface can readily diffuse laterally within the lipid bilayer while DNA attached to inorganic nanoparticles remain fixed in position. We systematically consider the effect of varying the liposome structure, size, charge, and fluidity on liposome assemblies, in chapter 2. In addition, the interesting properties of liposomes are highlighted by a side-by-side comparison to DNA-functionalized gold nanoparticles, offering fundamental insights into DNA-directed assembly. Furthermore, hybrid DNA-directed assemblies composed of both AuNPs and liposomes are described in Chapter 3. In particular, the photothermal effects of such DNA-coupled liposome and AuNP assemblies were modulated by controlling the distance between liposome and AuNP allowing such systems to have potential application in drug-delivery. In chapter 4, the utility of liposomes is demonstrated as we exploit the fluidity of its diffuse bilayer with split aptamer functionalization for the rapid and selective detection of metabolites. The second class of soft material of interest in this thesis are hydrogels, which are cross-linked hydrophilic polymers. Because hydrogels are swollen in water, they can be used to immobilize biomolecules such as DNA for a myriad of applications. In chapter 5, the preparation and characterization of DNA-functionalized polyacrylamide hydrogels are presented. The use of such a DNA-modified hydrogel for the simultaneous detection and removal of mercury from water is subsequently demonstrated.

For the treatment of cancer, Auger-electron emitting radionuclides are strongly dependent on their close proximity to DNA to utilize the local therapeutic potential of the Auger electrons. This thesis investigates a two-step targeting approach that uses targeting liposomes for the delivery of an Auger-electron emitter, 125I, coupled to a DNA-binding compound, Comp1, to the tumour-cell DNA. In the first step the liposome targets overexpressed cell-surface receptors. Receptors belonging to epidermal growth factor receptor (EGFR) family are overexpressed in a number of different cancers and are therefore suitable targets. The second step is transportation of the radionuclide to the cell nucleus utilizing a DNA-binding compound. The DNA-binder used in this thesis is a daunorubicin derivative called Comp1. Papers I and II are in vitro characterizations of the targeting liposomes. Both EGFR- and HER2-targeting liposomes delivered 125I-Comp1 receptor specifically to tumour cells, and were efficient in decreasing growth of cultured tumour cells. Paper II also included a biodistribution of 125I-Comp1 delivered by HER2-targeting liposomes in tumour-bearing mice. The results gave a time-dependent uptake in tumours differed from when non-targeting liposomes encapsulating 125I-Comp1 were given. Paper III investigates the therapeutic effect of 125I-Comp1 delivered by HER2-targeting liposomes, in an animal model that mimics a situation of disseminated tumour cells in the abdomen. 125I-Comp1 delivered by HER2-targeting liposomes effectively prolonged survival of the mice in a dose-dependent relation. Several mice in the groups receiving the highest doses were tumour-free at the end of the study. Paper IV compares different lipid compositions of the liposomes with respect to leakage, cellular uptake and therapeutic efficacy of delivered 125I-Comp1on cultured cells. Liposomes containing sphingomyelin or dihydrosphingomyelin retained drug more efficiently and exhibited more receptor specific delivery properties than distearoylglycerophosphatidylcholine (DSPC) containing liposomes. However, it was the DSPC-containing liposomes that displayed best growth inhibition on cultured tumour cells. The thesis concludes that 125I-Comp1 delivered by targeting liposomes is a promising candidate for effective radionuclide therapy.

Liposomes are used as biocompatible carriers of drugs, peptides, proteins, plasmic DNA, antisense oligonucleotides or ribozymes, for pharmaceutical, cosmetic, and biochemical purposes. They are considered as effective drug delivery systems due to their colloidal size, easily controllable surface and membrane properties, large carrying capacity of drugs and biocompatibility. / This study was concerned with the stability of liposomes, their interaction at solid-liquid interfaces and their release of hydrophilic drug compounds. Either conventional liposomes containing lecithin and cholesterol, and Stealth® liposomes containing polyethylene glycol (PEG) 5000-lipids or PEG2000-lipids were investigated. Steric hindrance introduced by PEG chains proved to be influential in controlling liposome stability, interaction and drug release processes. Electrostatic forces were shown to be essential to the mechanisms and kinetics of liposome adsorption and colloidal stability, but not influential for the release of guanosine, used as a model hydrophilic drug. / Findings from this research improve the understanding of liposome interaction during drug delivery, give insight into the actions of liposomes in the body and may form the basis for improved liposome formulation. In addition, this research has developed a more comprehensive understanding of the role of PEG in controlling the colloidal stability, interfacial interactions of liposomes and drug transport kinetics across the lipid bilayers of liposomes. / Thesis (PhDAppliedScience)--University of South Australia, 2005.

The ability of small interfering RNA (siRNA) to silence specific target genes offers not only a tool to study gene function but also represents a novel approach for the treatment of various human diseases, including cancers. The clinical use of siRNA, however, has been severely hampered by the inefficient delivery of these molecules to target cell populations due to their instability, inefficient cell entry, and poor pharmacokinetic profile. Much effort has therefore been devoted to the development of efficient in vivo siRNA delivery systems, with liposomes being the most widely employed vector. The traditional methods of packaging siRNA into liposomes, however, are often quite complex and labour-intensive, with the resulting products also being unstable at room temperature which limits their wide spread application in the clinic. The main aim of this research was to develop a simple, yet efficient, formulation technique to prepare stable siRNA-loaded liposomes which could be utilized as an efficient therapy for cancer treatment. Throughout this study, cervical cancer was used as the model system to assess the efficiency of various delivery systems. It is an ideal disease for siRNA therapy due to the cancer’s reliance on the expression of a single messenger RNA sequence which encodes two essential viral oncogenes, E6 and E7. Previous research has shown that targeting E6 and E7 by siRNA in cervical cancer cells in vitro results in either cell senescence or apoptosis. This thesis investigates the feasibility of applying E6/7 siRNA both intravaginally and intravenously to model the treatment of early-stage and end-stage cervical cancer, respectively. The practicability of applying E6/7 siRNA intravaginally for the treatment of localised cervical cancer tumours was firstly evaluated by administrating liposome-complexed siRNAs directly into the vaginal cavity of transgenic E7 mice. As no knockdown of E7 in cervical epithelium was observed for mice which received repeated treatments of E6/7 siRNA, the vaginal delivery efficiency of liposomes was further examined using fluorescently-labelled oligonucleotides. Contrary to previous reports, no delivery of lipoplexes into cervicovaginal tissues was detected irrespective of the dosage, type of lipid vector used, or the mouse estrus state at the time of administration. This lack of delivery was likely due to the poor retention of lipoplexes in the vaginal cavity as well as the inefficient penetration of lipoplexes across the mucosal layer lining the cervicovaginal epithelium. Overall, these findings indicated the necessity of developing more suitable and clinically acceptable vaginal siRNA delivery systems to enable this treatment strategy to become a reality. Despite the challenges of using liposomes to deliver siRNA via vaginal administration, their successful use in delivering siRNA intravenously to tumours was demonstrated in a subcutaneous cervical cancer mouse model. These experiments were carried out using PEGylated siRNA-loaded liposomes which were formulated using a novel Hydration-of-Freeze-Dried-Matrix (HFDM) technique. Compared to the existing formulation strategies, this method of preparation is less labour-intensive and the end product is also freeze-dried, ensuring product stability. It was found that the liposomes prepared using the HFDM method were stable in the presence of serum and they also possessed high siRNA entrapment and gene-silencing efficiencies. Following intravenous administration to mice, these particles were also found to accumulate in subcutaneous tumours to a similar degree compared to formulations prepared using a previously established technique. Importantly, these HFDM-formulated preparations showed superior stability over ones prepared using the traditional formulation method, with the particles still retaining 100% of their gene-silencing ability after storage for one month at room temperature. Using HFDM-formulated liposomes loaded with siRNA against Green Fluorescence Protein (GFP), a 50% knockdown of the GFP expression was achieved in tumours following intravenous administration. Additionally, the use of E6/7-targeted siRNA also resulted in a 50% reduction in tumour size when the siRNAs were delivered using HFDM-formulated liposomes. Importantly, this level of tumour growth suppression was comparable to that achieved from cisplatin, a clinically used chemotherapeutic for cervical cancer, at the clinically used dose. Overall, this research demonstrated that while there are still some challenges to overcome for siRNA to be used vaginally for cervical cancer treatment, HFDM-formulated PEGylated liposomes showed promise in bringing E6/7 siRNA forward as a treatment option for end-stage cervical cancer. In addition, the simplicity of preparation procedure along with superior product stability obtained from the HFDM method developed in this thesis will likely facilitate the translation of siRNA technology from laboratory to clinics for a range of other medical applications.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.