Summary and outlook

Supported with a strong literature background, this thesis elaborately describes the perspectives of an efficient, biocompatible delivery system capable of transfecting both in vitro and in vivo with minimal toxicity. A detailed study of the lipopolyplexes was performed to evaluate its efficacy and capabilities yielding consistent results.

The introduction part of the thesis deals with aspects such as gene delivery, RNA interference and vectors used for the delivery. Non-viral vectors, especially polymer and liposomal based gene delivery vehicles are reviewed. These formed the basis for the composite nanocarrier system, lipopolyplex used in this study. Advantages and disadvantages of liposomal and polymer based gene delivery systems are reviewed. Composition, structural characteristics and physicochemical properties of lipopolyplexes are discussed. Physical methods for enhancing the gene transfer using lipopolyplexes via photochemical internalisation and ultrasound enhanced gene transfer are described. A therapeutic anti-inflammatory model to evaluate the efficacy of the lipopolyplexes has been described. The necessity of toxicity and haemocompatibility studies for the evaluation of delivery vehicles have been summarised.

Chorioallantoic membrane model has been described with the aim to prove the biocompatibility and efficacy of the lipopolyplexes in vivo.

In the results part, preparation and formulation of the liposomes, polyplexes and lipopolyplexes has been described in detail. Physicochemical characterisation of the delivery vehicles using dynamic light scattering and laser Doppler velocimetry has revealed a stable, monodisperse formulation within the size range suitable for cellular internalisation. Influence of factors such as liposomal composition, N/P ratio of polyplexes, liposome/PEI mass ratio of lipopolyplexes and storage conditions on size and zeta potential have been described. The results obtained show that the lipopolyplexes are stable even after a month of storage with minimal loss in biological activity.

To give a deeper insight into the structure of the lipopolyplexes, extensive electron microscopic characterisation has been performed. Morphological characteristics were described using atomic force microscopy. The size of the lipopolyplexes obtained by dynamic light scattering has been confirmed using micrographs from electron and atomic force microscopy.

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Scanning electron micrographs show a defined spherical morphology of the lipopolyplexes which is a characteristic of their parent liposomes. The micrographs from the transmission electron microscopy using gold coupled PEI elucidate the multicomponent polyplex-in-liposome structure of the lipopolyplexes. Freeze fracture technique has been utilised to obtain replica images showing structural characteristics of the lipopolyplexes. Atomic force micrographs confirmed the shape of the lipopolyplexes.

Transfection studies have been performed to determine the efficiency of the complexes.

Compared to the polyplexes, lipopolyplexes, especially those formulated using linear PEI showed a tremendous increase in expression of luciferase gene. The increased gene expression has been confirmed using confocal laser scanning microscopy with green fluorescence protein expression. The effect of photochemical enhancement has been studied using curcumin loaded lipopolyplexes which showed improved transfection efficiencies followed by irradiation using a prototype LED device. Similar effects were observed upon application of ultrasound in ultrasound enhanced gene transfer experiments. The potential of lipopolyplexes to be delivered via different methods depending on the target has been explained using physical enhancement methods. Applicability of such methods for delivering genetic material into difficult to transfect primary cells using lipopolyplexes has been reported.

Toxicity studies describing the membrane damage and time dependent toxicity of the complexes have been discussed in detail. The membrane integrity studies compare the cytotoxic potential of polyplexes and lipopolyplexes. The decreased cytotoxic potential upon addition of liposomes to polyplexes helps understand the shielding effect of liposomes on PEI. Time dependent toxicity studies comparing the cell viability of cells transfected using polyplexes and lipopolyplexes show a substantial improvement in the cell viability upon liposomal encapsulation of polyplexes.

Haemocompatibility studies were performed to determine the biocompatibility and potential of the lipopolyplexes for in vivo use. Lipopolyplexes are shown to have a decreased haemolytic potential compared to the polyplexes. Similarly, aPTT studies show that lipopolyplexes, on contrary to polyplexes, especially bPEI polyplexes, do not interfere with coagulation. The haemocompatibility studies revealed the lipopolyplexes to be biocompatible and therefore safe for intravenous administration.

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In vivo chick chorioallantoic model (CAM) has been used as an alternative to animal models.

The CAM studies showed that the lipopolyplexes were also able to transfect in vivo with a considerable transfection efficiency. The GFP expression was visualised by confocal laser scanning microscopy of the CAM section. The in vivo toxicity assay, wherein the lipopolyplexes were injected into the blood stream, show no deleterious effect on the development of the embryo and the egg, confirming the safety profile of lipopolyplexes.

The investigations carried out in this work have shown that the combination of lPEI polyplexes with DOPE/DPPC/Cholesterol liposomes is a promising candidate in DNA and siRNA delivery both in vitro and in vivo. The liposomal shielding effect, which reduces the surface charge of the PEI, has been reported to be the key for the increased efficiency and low toxicity of the lipopolyplexes.

A clear understanding into the mechanisms of action regarding photochemical internalisation and ultrasound enhanced gene transfer is needed to further optimise the methods. This could be achieved using markers for cell death pathways. Use of fluorescence probes for specific cellular organelles together with confocal microscopic investigations would help understand the enhancement mechanisms involved. Further investigation into the beneficial properties of the lipopolyplexes for therapeutic gene delivery could lead to the development of a safe delivery system for clinical purposes.

Together with the reliable results and elaborate characterisation, this thesis work presents a potential composite nanocarrier as a worthy contender for gene therapy.

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