Microbubbling and microencapsulation by co-axial electrohydrodynamic atomization

Microbubbles coated with polymers or surfactants have been used in medical imaging for several years as ultrasound contrast agent particles and are now being investigated by researchers as drug and gene delivery vehicles and blood substitutes. Current methods available for the preparation of microbubbles are insufficient as they result in microbubbles with a wide size distribution and as such filtration is necessary before their use. With a view to fill the above demand, a detailed investigation has been carried out in this research to learn the viability of co-axial electrohydrodynamic atomization (CEHDA) technique to prepare microbubbles. The research also focuses on the effects of the process parameters such as flow rates, applied voltage and material parameters such as electrical conductivity, surface tension and viscosity with the objective of preparing polymer or surfactant coated stabilized microbubbles with diameters < 8 μm and with a narrow size distribution. A model glycerol-air system was used so that the CEHDA technique was modified to generate suspensions of microbubbles to a diameter < 8 μm with a narrow size distribution and then to characterise the CEHDA microbubbling process in terms of size and stability with varying process parameters and material parameters. Construction of a parametric plot between the air flow rate and the liquid flow rate was extremely useful in identifying the flow rate regime of air and liquid or suspension or solution for the continuous microbubbling of the system used. With further investigations into the CEHDA microbubbling technique, it was possible to develop strategies, first, to prepare suspensions of stabilized phospholipids-coated microbubbles with a mean diameter of ~ 5 μm and a polydispersivity index of 9%, and second, polymeric microspheres with a mean diameter of 400 nm and a polydispersivity index of 8% using a biocompatible polymer

Investigation of microbubble-cell interaction and development of an ultrasound delivery system

Microbubbles have been used for several decades as ultrasound contrast agents in diagnostic ultrasound imaging. However, their application in gene therapy as delivery vehicles has only recently been realised. The presence of microbubbles in close proximity to cells during ultrasound insonation can increase the efficacy of drug or gene delivery by inducing formation of transient, non-lethal perforations in the cell membrane, a process termed sonoporation. In order to develop techniques for successful delivery of therapeutic agents, it is necessary to quantify the composition and physical characteristics of microbubbles in order to be able to determine how these affect the sonoporation process as required. Although several microbubbles are available commercially, the components of the shell of these proprietary microbubbles have not been disclosed. In order to study sonoporation and the possibility of delivering drugs and genes it became necessary to develop a formulation for in-house experimental microbubbles. These experimental in-house microbubbles have not been previously investigated with regard to their interaction with cells, their potential for sonoporation and / or their bioeffects. Characterisation of the in-house microbubbles was necessary prior to any attempts to use them as delivery vehicles in vitro, or indeed, in vivo. Confocal laser scanning microscopy (CLSM) was used in order to determine the size distribution of both in-house microbubbles and Definity® a commercially available contrast agent. Confocal imaging and 3-D reconstruction of in-house microbubbles indicated the structure, morphology and size-distribution of these membrane-bound microbodies. Microbubbles were later separated according to size using a density gradient. It was concluded that the distribution of sizes of the microbubbles was in part due to the multi-lamellar nature of the microbubble shell. Cells were initially cultured in Petri dishes and insonated in the presence and absence of in-house microbubbles, in order to assess any bioeffects emerging from the application of ultrasound alone or in the presence of the microbubble constructs. Cells were cultured subsequently on an acoustically-transparent Mylar membrane, which was then “sandwiched” between two acetal homopolymer (Derlin) rings and placed in a specially designed ultrasound tank. Ultimately, cells were grown in an OptiCell™, an acoustically-transparent parallel membrane environment, where delivery of molecules of various sizes, in the presence of both in-house and Definity® microbubbles was investigated. Sonoporation was achieved with insonication of SK Hep-1 cells with a “physiotherapy machine” applying a power of 2.54 W / cm2 for 2-3 secs in the presence of Definity® microbubbles and passage of Calcein, an impermeable molecule, into the cells was detected using flow cytometric analysis. In addition, expression of enhanced green fluorescent protein (EGFP) was also detected 24 hours after insonication of SK Hep-1 cells in the presence of Definity® microbubbles and a linearised plasmid pCS2, encoding EGFP, under the same ultrasonic conditions. Sonoporation was also investigated with the use of a diagnostic ultrasound scanner, since it is more clinically relevant. Although several acoustic and non-acoustic parameters were investigated, sufficient sonoporation was not attained using this scanner. The bioeffects of ultrasound on cells both in vivo and in vitro have been extensively investigated. However, the exact cellular mechanisms that are affected by the application of ultrasound waves are not understood. In this study, the effects of ultrasound on a number of pathways were investigated. Expression of Hsp70, a cell stress protein often associated with heat-shock, during application of continuous wave ultrasound, suggests that cells may undergo heat stress. During application of continuous wave ultrasound in the presence of Definity® microbubbles, expression of Hsp70 was shown to decrease compared to when ultrasound was applied in the absence of Definity® microbubbles. In addition, expression of HO-1, a protein associated with hypoxic pathways was also present during application of ultrasound in the absence of microbubbles. These results suggest that in the absence of ultrasound contrast agents, insonation can cause the expression of proteins associated with different forms of cell stress such as heat-shock and hypoxia, thus initiating the apoptotic process. In this thesis, it has been shown that the mean size of the in-house microbubbles is comparable to that of commercially available microbubbles such as Definity®. In addition, it has been shown that sonoporation and successful delivery of small molecules in the presence of Definity® microbubbles is achievable with the equipment and the specific system which was developed. This reinforces the promising role of in-house microbubbles as delivery vehicles for therapeutic agents. Finally, an investigation on the possible bioeffects of ultrasound in the presence and absence of ultrasound contrast agents, revealed that under acoustic conditions identical to those used for achieving sonoporation, cells experience stress, instigating pathways that could potentially lead to cell death

Liposomes:Formulation and characterisation as contrast agents and as vaccine delivery systems

Liposome systems are well reported for their activity as vaccine adjuvants; however novel lipid-based microbubbles have also been reported to enhance the targeting of antigens into dendritic cells (DCs) in cancer immunotherapy (Suzuki et al 2009). This research initially focused on the formulation of gas-filled lipid coated microbubbles and their potential activation of macrophages using in vitro models. Further studies in the thesis concentrated on aqueous-filled liposomes as vaccine delivery systems. Initial work involved formulating and characterising four different methods of producing lipid-coated microbubbles (sometimes referred to as gas-filled liposomes), by homogenisation, sonication, a gas-releasing chemical reaction and agitation/pressurisation in terms of stability and physico-chemical characteristics. Two of the preparations were tested as pressure probes in MRI studies. The first preparation composed of a standard phospholipid (DSPC) filled with air or nitrogen (N2), whilst in the second method the microbubbles were composed of a fluorinated phospholipid (F-GPC) filled with a fluorocarbon saturated gas. The studies showed that whilst maintaining high sensitivity, a novel contrast agent which allows stable MRI measurements of fluid pressure over time, could be produced using lipid-coated microbubbles. The F-GPC microbubbles were found to withstand pressures up to 2.6 bar with minimal damage as opposed to the DSPC microbubbles, which were damaged at above 1.3 bar. However, it was also found that DSPC-filled with N2 microbubbles were also extremely robust to pressure and their performance was similar to that of F-GPC based microbubbles. Following on from the MRI studies, the DSPC-air and N2 filled lipid-based microbubbles were assessed for their potential activation of macrophages using in vitro models and compared to equivalent aqueous-filled liposomes. The microbubble formulations did not stimulate macrophage uptake, so studies thereafter focused on aqueous-filled liposomes. Further studies concentrated on formulating and characterising, both physico-chemically and immunologically, cationic liposomes based on the potent adjuvant dimethyldioctadecylammonium (DDA) and immunomodulatory trehalose dibehenate (TDB) with the addition of polyethylene glycol (PEG). One of the proposed hypotheses for the mechanism behind the immunostimulatory effect obtained with DDA:TDB is the ‘depot effect’ in which the liposomal carrier helps to retain the antigen at the injection site thereby increasing the time of vaccine exposure to the immune cells. The depot effect has been suggested to be primarily due to their cationic nature. Results reported within this thesis demonstrate that higher levels of PEG i.e. 25 % were able to significantly inhibit the formation of a liposome depot at the injection site and also severely limit the retention of antigen at the site. This therefore resulted in a faster drainage of the liposomes from the site of injection. The versatility of cationic liposomes based on DDA:TDB in combination with different immunostimulatory ligands including, polyinosinic-polycytidylic acid (poly (I:C), TLR 3 ligand), and CpG (TLR 9 ligand) either entrapped within the vesicles or adsorbed onto the liposome surface was investigated for immunogenic capacity as vaccine adjuvants. Small unilamellar (SUV) DDA:TDB vesicles (20-100 nm native size) with protein antigen adsorbed to the vesicle surface were the most potent in inducing both T cell (7-fold increase) and antibody (up to 2 log increase) antigen specific responses. The addition of TLR agonists poly(I:C) and CpG to SUV liposomes had small or no effect on their adjuvanticity. Finally, threitol ceramide (ThrCer), a new mmunostimulatory agent, was incorporated into the bilayers of liposomes composed of DDA or DSPC to investigate the uptake of ThrCer, by dendritic cells (DCs), and presentation on CD1d molecules to invariant natural killer T cells. These systems were prepared both as multilamellar vesicles (MLV) and Small unilamellar (SUV). It was demonstrated that the IFN-g secretion was higher for DDA SUV liposome formulation (p<0.05), suggesting that ThrCer encapsulation in this liposome formulation resulted in a higher uptake by DCs

Advanced characterisation study of ultrasound contrast bubbles in their natural hydrated state

This study presents a systematic investigation on in-house ultrasonic contrast agents, known as microbubbles (MBs), in their natural hydrated state. Contrast microbubbles have strong acoustic scatter profiles that significantly enhance ultrasonic visualisation of the human vasculature. Understanding and characterising the behaviour and morphological properties of these microbubbles is of interest and is the main research aims of this thesis - currently there are only a few clinically approved microbubbles. To manufacture clinically translatable theranostic vehicles, it is imperative to understand the mechanical and nanostructural properties of these bubbles in vitro; this will enrich the understanding of how their structural, biophysical and chemical properties impact their functionality in vivo. The behaviour and morphological properties of microbubbles have not been fully explored, this includes the lipid arrangement of the shell membrane. Hence, the work of this thesis is centred around exploring the physical properties of the bubbles. In particular, the microbubble shell is investigated in detail by applying complementary, state-of-the-art, experimental techniques such as atomic force microscopy and cryogenic focused-ion-beam scanning electron microscopy. The phospholipid microbubbles used throughout have been manufactured using microfluidic technology; the gaseous phase would intersect at a T-junction with the hydrophilic liquid phase, producing contrast bubbles by exploiting the amphiphilic properties of phospholipids. Subsequent ultrasound investigations have investigated their attenuation capabilities (dB cm-1 ) over the 12 – 55 MHz frequency range. The results from these studies indicated a sub-population of sub-micron bubbles due to the higher attenuation seen at higher frequencies. Then, using nanoparticle tracking analysis, resonant mass measurements and optical brightfield microscopy, data has been generated to reveal the complete size distribution of bubbles produced using the patented microfluidic method for generating ultrasound contrast bubbles. One of the key findings of this thesis was the discovery of sub-micron (<1000 nm) and micron-sized bubbles in solution. This finding then allowed for both the distributions to be investigated using atomic force microscopy and cryogenic focused ion-beam scanning electron microscopy. Atomic force microscopy (in combination with an optical microscopy set up) is used extensively to directly visualise the shell membrane and its nanostructural components. This led to the quantification of the thickness of shell membranes as well as the observation of morphological changes that occur during bubble deformation and, ultimately, their collapse. AFM imaging mode techniques such as tapping mode and quantitative imaging mode have allowed for the thickness and lipid configuration of phospholipid-shelled MBs to be quantified for the first time, a key finding in this thesis. A shell thickness of ~6.5 nm has been found using atomic force microscopy, leading to the proposal of the membranes being tri-layered; undertaking a {hydrophilic head-hydrophobic tail}-{hydrophobic tail - hydrophilic head}-{hydrophilic head hydrophobic tail} – configuration. Further work using force-curve mode AFM has also been conducted to measure mechanical forces at the nanoscale. This AFM mode generated force spectroscopy data with enough force resolution that, when combined with elastic models, gave insight into the interactions, and mechanical responses that these bubbles elicit when undergoing force compression. Bubble stiffness and Young’s Modulus have been calculated using different mechanical theories to evaluate which is most appropriate for analyses on the soft matter bubble systems. In this context, it has been shown for the first time comprehensively (accounting for the now measured shell thickness) that the linear elastic Reissner Model is not a suitable one as it can overestimate (GPa range) or underestimate (KPa range) if the correct shell thickness value is not used. The high force resolution allowed for investigating the polyethylene glycol brushes end-grafted to the phospholipid microbubbles. Using the Alexander-de Gennes polymer brush theory revealed overestimated PEG- brush thickness values, which could also be affected by ionic/DVLO forces due to the curvature of the bubble shell. Cryogenic focused-ion beam scanning electron microscopy is a valuable technique for the detailed study of soft matter systems. The focused-ion-beam allowed for probing deep into the cryopreserved sample by milling through the upper surface to expose suspended bubbles. Cryo imaging through focused ion beam-scanning electron microscopy allowed for probing sub-micron contrast bubbles under conditions that are close to in vivo. This work corroborated the results highlighting a trilayer configuration of lipids (~8 nm when taking into account the PEG brush), and provided novel information on the structure of the shell membrane and heterogenic lipid domain formation, which could have implications in drug/gene loading capabilities. In conclusion, this study provides systematic characterisation of in-house phospholipid-shelled contrast bubbles using various advanced techniques to characterise mechanical, nanostructural, and size properties across the nano and microscale. This study is the first to offer such a comprehensive report on the properties of phospholipid contrast bubbles in their natural hydrated states. Thus, cutting edge techniques and improved methods for bubble imaging are presented, which can be used to propel the development of theranostic contrast bubbles

Liposomes : Formulation and characterisation as contrast agents and as vaccine delivery systems

Liposome systems are well reported for their activity as vaccine adjuvants; however novel lipid-based microbubbles have also been reported to enhance the targeting of antigens into dendritic cells (DCs) in cancer immunotherapy (Suzuki et al 2009). This research initially focused on the formulation of gas-filled lipid coated microbubbles and their potential activation of macrophages using in vitro models. Further studies in the thesis concentrated on aqueous-filled liposomes as vaccine delivery systems. Initial work involved formulating and characterising four different methods of producing lipid-coated microbubbles (sometimes referred to as gas-filled liposomes), by homogenisation, sonication, a gas-releasing chemical reaction and agitation/pressurisation in terms of stability and physico-chemical characteristics. Two of the preparations were tested as pressure probes in MRI studies. The first preparation composed of a standard phospholipid (DSPC) filled with air or nitrogen (N2), whilst in the second method the microbubbles were composed of a fluorinated phospholipid (F-GPC) filled with a fluorocarbon saturated gas. The studies showed that whilst maintaining high sensitivity, a novel contrast agent which allows stable MRI measurements of fluid pressure over time, could be produced using lipid-coated microbubbles. The F-GPC microbubbles were found to withstand pressures up to 2.6 bar with minimal damage as opposed to the DSPC microbubbles, which were damaged at above 1.3 bar. However, it was also found that DSPC-filled with N2 microbubbles were also extremely robust to pressure and their performance was similar to that of F-GPC based microbubbles. Following on from the MRI studies, the DSPC-air and N2 filled lipid-based microbubbles were assessed for their potential activation of macrophages using in vitro models and compared to equivalent aqueous-filled liposomes. The microbubble formulations did not stimulate macrophage uptake, so studies thereafter focused on aqueous-filled liposomes. Further studies concentrated on formulating and characterising, both physico-chemically and immunologically, cationic liposomes based on the potent adjuvant dimethyldioctadecylammonium (DDA) and immunomodulatory trehalose dibehenate (TDB) with the addition of polyethylene glycol (PEG). One of the proposed hypotheses for the mechanism behind the immunostimulatory effect obtained with DDA:TDB is the ‘depot effect’ in which the liposomal carrier helps to retain the antigen at the injection site thereby increasing the time of vaccine exposure to the immune cells. The depot effect has been suggested to be primarily due to their cationic nature. Results reported within this thesis demonstrate that higher levels of PEG i.e. 25 % were able to significantly inhibit the formation of a liposome depot at the injection site and also severely limit the retention of antigen at the site. This therefore resulted in a faster drainage of the liposomes from the site of injection. The versatility of cationic liposomes based on DDA:TDB in combination with different immunostimulatory ligands including, polyinosinic-polycytidylic acid (poly (I:C), TLR 3 ligand), and CpG (TLR 9 ligand) either entrapped within the vesicles or adsorbed onto the liposome surface was investigated for immunogenic capacity as vaccine adjuvants. Small unilamellar (SUV) DDA:TDB vesicles (20-100 nm native size) with protein antigen adsorbed to the vesicle surface were the most potent in inducing both T cell (7-fold increase) and antibody (up to 2 log increase) antigen specific responses. The addition of TLR agonists poly(I:C) and CpG to SUV liposomes had small or no effect on their adjuvanticity. Finally, threitol ceramide (ThrCer), a new mmunostimulatory agent, was incorporated into the bilayers of liposomes composed of DDA or DSPC to investigate the uptake of ThrCer, by dendritic cells (DCs), and presentation on CD1d molecules to invariant natural killer T cells. These systems were prepared both as multilamellar vesicles (MLV) and Small unilamellar (SUV). It was demonstrated that the IFN-g secretion was higher for DDA SUV liposome formulation (p<0.05), suggesting that ThrCer encapsulation in this liposome formulation resulted in a higher uptake by DCs.EThOS - Electronic Theses Online ServiceGBUnited Kingdo