Polyethylenimine- and lipid- based nanoparticles as gene and drug delivery systems for aerosol therapy to the lung

This thesis describes the development and characterization of a variety of polymeric and liposomal drug delivery systems for use in pulmonary aerosol therapy. A variety of polyethylenimines (PEIs) were studied in depth in an attempt to develop biocompatible gene vectors for efficient non-viral gene therapy for the lung (Chapter 2-5). Another study (Chapter 6) investigates a number of lipid-based nanoparticles that were specifically designed to act as sustained release formulations for pulmonary drug delivery. The investigations presented in Chapter 2 form the foundations of this thesis by evaluating the general suitability of PEI as a gene vector in an aerosol therapy. Four different PEI modifications (branched, linear, polyethylenglycol-grafted and biodegradable PEI) copolymer were utilized as carriers for plasmid DNA (pDNA), with the resulting polyplexes displaying diameters of approximately 100 nm. These were extensively characterized with respect to identifying any structural and/or physico-chemical alterations that occurred during aerosolization with both air-jet and ultrasonic nebulizer. A number of techniques including atomic force microscopy (AFM), dynamic light scattering (DLS) and laser Doppler anemometry (LDA) were employed to determine the various morphologies, diameters and zeta-potentials of the polyplexes pre- and post-aerosolization. The PEI modifications and displaying stronger DNA condensation were shown to form more stable polyplexes, due to the reduced level of damage observed post- aerosolization. The poly (ethylene glycol) PEI copolymer (PEGPEI) was shown to have the superior carrier properties regarding DNA condensation and protection. Furthermore, our results suggested that improved stability for PEI/DNA polyplexes can be attained using the ultrasonic nebulizer in preference to the air-jet apparatus. Thus, we concluded that ultrasonic nebulization is a milder aerosolization method for PEI-based gene delivery systems. Additionally, PEGPEI appeared to display the most promising properties as a pulmonary gene carrier, due to its stability. Therefore, we decided to employ the PEGPEI copolymer in addition to the commonly used branched 25 kDa PEI (BPEI) in the subsequent studies. Our primary aim in Chapter 3 was the development of a gene vector that achieves both high transgene expression and biocompatibility in the pulmonary epithelium. To accomplish this aim, a low molecular weight (5 kDa) PEI (LMWPEI) and a PEGPEI copolymer were employed as pDNA vectors and compared with the commonly utilized BPEI. Investigations of the polyplex morphologies (using AFM), sizes (by DLS) and zeta-potentials (using LDA) revealed that all three polymers are able to condense pDNA, forming small, positively charged particles of approximately 100 nm diameter. Cytotoxicity studies, performed using the MTT- and LDH-assay, indicated LMWPEI had the superior biocompatibility of the three polymers investigated. The transfection efficiencies of the three polyplexes were studied both in vitro (cell cultured lung epithelia) and in vivo (intratracheally instillation to the mouse lung). Whilst LMWPEI polyplexes were shown to be inefficient in transfecting lung epithelial cells in vitro, they caused the highest transfection rate in the mouse lung. Interestingly, the opposite behaviour was observed when PEGPEI polyplexes were investigated, with a high gene expression in vitro not being reproduced in vivo. We hypothesized that the polyplexes relative stability in the lung environment might explain this observation. In order to investigate the polyplex stability, natural lung lining fluid and lung surfactant were utilized, and a decreasing trend of DNA encapsulation in the order: PEGPEI > BPEI > LMWPEI was observed. Therefore, we concluded that stronger interactions between the carrier and the pDNA may hinder the DNA release under in vivo conditions, thus reducing the transfection efficiency. Our results indicated that LMWPEI fulfils the key requirements of low cytotoxicity and high gene expression in the mouse lung. Based upon the promising results of Chapter 3 the biocompatibility of LMWPEI/DNA in the mouse lung was further investigated, and the results are presented in Chapter 4. Polyplexes were applied via instillation, and after 48 hours lung lavages were performed. The bronchial alveolar lining fluid (BALF) obtained was subsequently investigated for total cell counts, quantity of neutrophils and macrophages as well as total protein and cytokine concentrations. These parameters are hallmarks of acute lung inflammation, and increased values were observed for all investigated systems in comparison to the control mice. In the case of pDNA and LMWPEI/DNA, only minor alterations were detected whereas BPEI/DNA caused stronger inflammation and surprisingly PEGPEI/DNA led to the most severe inflammation. When considering these findings together with the transfection results obtained in vivo (Chapter 3), we concluded that increasing cytotoxicity in the mouse lung (LMWPEI < BPEI < PEGPEI) causes decreasing transfection efficiency (LMWPEI > BPEI > PEGPEI). As such, LMWPEI was chosen as our preferred pDNA carrier for inhalation therapy. In parallel to this work, we developed a novel aerosol inhalation device for mice. Unfortunately, the transfection rate in the mouse lung decreased dramatically post- LMWPEI/DNA aerosolization in comparison to the instilled polyplexes. In an attempt to identify the reasons for this failure, we employed double-labeled polyplexes in order to compare the lung distribution of inhaled versus instilled polyplexes. The nebulized LMWPEI polyplexes were shown to be uniformly localized throughout the mouse lung in small quantities. In contrast, the instilled polyplexes were detected in much higher concentrations, in bronchial and alveolar regions, but were not evenly distributed. Interestingly, polyplexes were observed in epithelial and endothelial cells. Consequently, LMWPEI represents a highly efficient and biocompatible gene vector for the lung and is superior to the more commonly used BPEI. Another study (Chapter 5) describes the development and characterisation of a novel gene vector that is based upon BPEI covalently linked to a TAT peptide (a protein transduction domain) via a PEG spacer. The oligopeptide TAT was chosen to enhance cell uptake into lung cells, since reports had demonstrated high translocation ability of TAT by direct crossing biological membranes. In keeping with the two previous studies, the TAT-PEG-PEI conjugate was extensively investigated in terms of DNA condensation, DNA protection in the intra- and extracellular lung environment, polyplex size, stability, zeta-potential, in vitro and in vivo toxicity, transfection efficiency and polyplex distribution in the mouse lung. Since our key aim had been to develop a non-toxic, highly efficient carrier for the epithelial cells of the conducting and respiratory airways, this new carrier fulfilled the majority of our requirements. It was able to form very small and stable particles with pDNA. A ~600% improved gene expression in the mouse lung was observed for TAT-PEG-PEI polyplexes in comparison to BPEI and a ~300% improved gene expression in comparison to LMWPEI. Furthermore, only minor effects upon the lung function were observed, with no additional inflammation compared to pDNA instillation alone. A particular advantage of this carrier is its ability to transport DNA safely into the different cell types of the lung. Hence, it could be employed in the treatment of pulmonary diseases that attack the entire lung, such as lung cancer. Consequently, the TAT-PEG-PEI conjugate and to a lower extend also LMWPEI represent promising new approaches in pulmonary gene therapy of various lung disorders. Further work is necessary to decrease the cytotoxicity to a level where no inflammation is observed. This might be achieved by using either a highly branched PEGPEI copolymer or a LMWPEI in place of BPEI for the synthesis of TAT-PEG-PEI. Since a number of recent studies have reported conflicting results regarding the cellular uptake and pathway of TAT peptides, a study should be performed in order to track the pathway of TAT-PEG-PEI polyplexes into the cells and nucleus. Further work should focus on the development of a cell specific gene vector for pulmonary gene therapy. In this respect, the synthesis of LMWPEI modifications carrying target moieties such as lectin, folate, peptides (e.g. RGD) or antibodies would be of particular interest. The next major set of experiments should focus on the delivery of therapeutic genes such as IL-12, p53, prostacyclin or nitric oxide synthase, via optimized TAT-PEG-PEI or LMWPEI vehicles. Additionally, the challenge of efficient polyplex administration to mice via aerosol inhalation remains. Improvements of the nebulization system developed here may be achieved by employing a so called ‘drying spacer’, a development recently reported by Rudolph et al. Currently the drug iloprost is approved for the aerosol therapy of pulmonary hypertension. However, the effectiveness of this formulation is limited by the short half live of iloprost in vivo. As such, the aim of the study presented in Chapter 6 was to develop a sustained releasing formulation for iloprost. A variety of lipid combinations were studied in order to find a formulation that accomplish both, encapsulate high quantities of iloprost and is stable throughout the aerosolization process. Initially, the model drug Carboxyfluorescein was encapsulated in liposomes consisting of Dipalmitoyl-phosphatidylcholin (DPPC) and Cholesterol (CH), or DPPC, CH and PEG-dipalmitoyl-phoshatidylethanolamine (DPPE-PEG). Liposomal morphology was studied using AFM and the liposome phase transition temperatures were investigated using differential scanning calorimetry. Their stability during aerosolization was investigated using air-jet, ultrasonic and micro pump nebulizers. These nebulizers were compared in terms of mass output, aerosol droplet size and their effects upon the liposome stability. Regarding liposome size and drug loading pre- and post-nebulization, the DPPC/CH liposomes were shown to be the most stable formulation, particularly during ultrasonic and micro pump nebulization. In a second study, Iloprost-loaded liposomes were prepared and we were able to show that the DPPC/CH liposomes again displayed the highest encapsulation efficiency and stability during ultrasonic and micro pump nebulization. We concluded that DPPC/CH liposomes are well suited to act as a sustained release formulation for the treatment of pulmonary hypertension. This study highlights the clear possibilities that exist for the development of a pulmonary sustained release formulation for the treatment of pulmonary hypertension. The next steps would focus on drug release and cellular uptake studies in lung epithelial cells, followed by pharmacokinetic studies in an ex vivo animal model. The effectiveness of the liposomal iloprost formulation should be further investigated by employing an animal pulmonary hypertension model, e.g. holding mice under hypoxia conditions. The novel approaches and technologies described in this thesis represent small but important advances in the application of aerosol therapy to the treatment of acquired diseases. Hopefully formulations similar to those described here will one day offer significantly improved treatments for people suffering from a range of illnesses.