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dc.contributor.advisorRizwan, Shakila
dc.contributor.advisorClarkson, Andrew
dc.contributor.advisorBoyd, Benjamin
dc.contributor.authorMohammad, Younus
dc.date.available2019-09-16T21:31:38Z
dc.date.copyright2019
dc.identifier.citationMohammad, Y. (2019). Non-Lamellar Liquid Crystalline Nanoparticles for Targeted Delivery of Endogenous Lipids to the Brain (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/9599en
dc.identifier.urihttp://hdl.handle.net/10523/9599
dc.description.abstractPurpose: Bioactive endogenous lipids such as N-acylethanolamines (NAEs) and alkylglycerols have shown therapeutic potential in various neurological diseases and targeted brain drug delivery, respectively. However, their delivery at therapeutic doses using physiologically suitable dosage forms (vehicles) still represent challenges. The present thesis explores the possibility of formulating endogenous lipids, specifically oleoylethanolamide (OEA), linoleoylethanolamide (LEA) (both NAEs) and selachyl alcohol (an alkylglycerol), into functionalised non-lamellar liquid crystalline nanoparticles which are inherently bioactive (cubosomes and hexosomes) and are also capable of targeted delivery of drugs to brain delivery. Methods: Cubosomes and hexosomes were prepared using the solvent precursor method. OEA was co-formulated with phyantriol and LEA and selachyl alcohol were used as core cubosome forming lipids. Three different steric stabilisers, Pluronic F127 (control) and ligands that can target the blood-brain barrier, Tween 80 and Pluronic F68 (in the case of LEA) were used to functionalise the nanoparticles. Particle size analysis was performed using dynamic light scattering. Morphology and internal structure of nanoparticles was interrogated using cryo-TEM and small angle X-ray scattering respectively. The chemical stability and encapsulation efficiency of NAEs and phenytoin in nanoparticles were determined using a combination of HPLC, infrared spectroscopy and size exclusion chromatography. The resistance of NAEs encapsulated in nanoparticles to enzymatic hydrolysis was measured using LCMS. Cytotoxicity of nanoparticles towards human cerebral microvascular endothelial cells (hCMEC/D3) was investigated using a flow cytometer-based propidium iodide assay and an MTT assay. Results: Depending upon its structural characteristics and self-assembly behaviour, OEA was co-formulated with a synthetic lipid, phytantriol, into cubosomes. Cubosomes containing OEA had a mean particle size of less than 200 nm with low polydispersity (polydispersity index <0.25) and there was no significant difference found in particle size after a week. Up to 30% w/w OEA (relative to phytantriol) was able to be incorporated into phytantriol cubosomes without any significant disruption to the nanostructure of the cubosomes. OEA was found to be chemically stable and retained in nanoparticles for at least a week after formulation. OEA-loaded cubosomes showed increased resistance to in vitro enzymatic hydrolysis compared to free OEA solution and this was dependent on internal structure and amount of OEA in the cubosomes. Total lipid concentrations of up to 30 μg/mL were found to be non-toxic towards hCMEC/D3 cells. Inclusion of OEA into the cubosomes did not induce any additional toxicity compared to phytantriol cubosomes. There was no significant difference found between any of the above parameters for the cubosomes stabilised with either stabiliser Pluronic F127 and Tween 80. In studies with LEA as a core cubosome forming lipid, stabiliser type was found to influence the colloidal stability of cubosomes. A higher concentration of Tween 80 (25% of LEA content) was required compared to Pluronic F127 and Pluronic F68 to stabilise cubosomes. All cubosome formulations had a mean particle size of less than 250 nm with low PDIs (<0.2) indicating uniform distribution within a day after formulation. Cubosomes produced had a bicontinuous cubic internal structure with an Im3m space group but different lattice parameters, indicating the different modes of interaction between the stabilisers and LEA. Short-term stability studies indicated that there was no significant difference in particle size distribution and chemical stability of LEA in Pluronic-stabilised cubosomes (F127 and F68) a week after formulation. In contrast, Tween 80-stabilised cubosomes showed significantly higher particle size, PDIs and a lower amount of LEA present in the cubosomes a week after formulation. Cubosomes prepared in the presence of all the three stabilisers (Pluronic F127, Pluronic F68 and Tween 80) showed less resistance to in vitro enzymatic hydrolysis compared to free LEA solution. Cubosomes were found to be non-toxic to hCMEC/D3 cells at concentrations of up to 20 µg/mL LEA in the presence of all the stabilisers. In studies with the alkylglycerol (selachyl alcohol), the addition of Tween 80 to selachyl alcohol bulk phase samples triggered concentration-dependent phase changes with a sequence evolving from an inverse hexagonal phase (H2) to a mixed H2 and reverse bicontinuous cubic (V2) then V2 phase alone. In contrast, addition of Pluronic F127 resulted in a phase change from H2 phase to a mixed lamellar and H2 phase system. The dispersions showed a mean particle size of 125-190 nm with low polydispersity indices (0.1–0.2) and retained the bulk phase internal structure in the presence of Tween 80, whereas in the presence of Pluronic F127, the additional lamellar phase that formed in bulk phase systems was non-existent. The model neuroactive drug phenytoin showed higher solubility in selachyl alcohol compared to phytantriol (control). Consequently, selachyl alcohol nanoparticles showed significantly higher encapsulation efficiencies compared to phytantriol nanoparticles. Inclusion of phenytoin in nanoparticles did not induce any significant change in internal structure of the nanoparticles. The nanoparticles were found to be non-toxic to hCMEC/D3 cells up to lipid concentrations of 30 μg/mL with or without phenytoin and there was no significant difference in toxicity between selachyl alcohol cubosomes and hexosomes. Conclusions: These findings demonstrate that hydrolysis susceptible and insoluble endogenous lipids, NAEs and insoluble lipids alkylglycerols can be encapsulated into, or used to form, liquid crystalline nanoparticles. The surface of the nanoparticles can be functionalised with targeting ligands and possibly influence the in vivo distribution and targeting capacity of endogenous lipid nanoparticles.
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectEndogenous
dc.subjectlipids
dc.subjectCubosomes
dc.subjectHexosomes
dc.titleNon-Lamellar Liquid Crystalline Nanoparticles for Targeted Delivery of Endogenous Lipids to the Brain
dc.typeThesis
dc.date.updated2019-09-16T04:25:53Z
dc.language.rfc3066en
thesis.degree.disciplineSchool of Pharmacy
thesis.degree.nameDoctor of Philosophy
thesis.degree.grantorUniversity of Otago
thesis.degree.levelDoctoral
otago.interloanyes
otago.openaccessAbstract Only
otago.evidence.presentYes
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