Final Report Summary - SMASE LIPOSOME (Targeting atherosclerosis: Raman-spectroscopy validated, triggered release of drugs from de novo phospholipid-incorporating liposomes by sphingomyelinase enzymatic degradation of the membrane bilayer.)
Cardiovascular diseases, including atherosclerosis, are the world’s number one killer, accounting for 43% of all deaths and costing the EU 192 billion Euros annually. Early diagnosis of atherosclerotic plaques and delaying their progression are therefore two highly important avenues of research. Over recent decades, significant inroads have been made into designing smart delivery systems which enable injected drugs to be delivered locally within the body to the site of disease. This overcomes a range of negative side effects that such drugs might have in other, unaffected, areas of the body. Such approaches typically involve encapsulating drugs into nanometre-sized containers, termed vesicles, which break down in response to specific chemical or physical disease environments. Within the project SMase LIPOSOME, important progress has been in made into designing such vesicles from lipid compositions that are sensitive to the enzyme sphingomyelinase (SMase), which has recently been found to be highly active in atherosclerotic plaques. SMase-targeting drugs that decrease the enzyme activity are an emerging research topic to treat not only atherosclerosis but also endothelial dysfunction, ischemic heart disease, emphysema, cancers, cystic fibrosis and depression. The work carried out within this project presents the backbone for new localised delivery vectors to increase local concentrations of drugs at the site of disease, and overcome negative systemic side effects of SMase-targeting drugs. In particular, it marks two world-firsts: using SMase as a target for drug delivery, and a liposome formulation from which release is not only targeted but endogenously triggered by the target enzyme.
The following significant results were obtained during this Marie Skłodowska-Curie Fellowship:
1. Thorough characterisation of novel SMase-cleavable vesicle formulations including the effect of varying lipid composition.
2. Development of an assay to screen SMase activity that is faster and more sensitive than current laboratory techniques, and can be interpreted by simple observation of a change in colour.
3. Application of techniques developed in SMase-cleavable vesicle formulations to understand the implications for vesicles that can target other phospholipase-type enzymes such as phospholipase C and D.
4. Formulation of non-spherical vesicles, presenting potentially interesting implications for biodistribution and efficacy of drug delivery.
The following paragraphs provide a more detailed description of each result.
1. Thorough characterisation of novel SMase-cleavable vesicle formulations including the effect of varying lipid composition:
The phospholipid sphingomyelin (SM) is the substrate for SMase and represents a class of molecules with a phosphocholine head group, sphingosine and a fatty acid. Additionally, naturally derived SM, such as egg sphingomyelin (Egg SM) extracted from chicken egg yolk, brain sphingomyelin (Brain SM) extracted from porcine brain, and milk sphingomyelin (Milk SM) extracted from bovine milk, is actually a mixture of SMs with differing chain lengths. Combination with other lipids is required to form stable vesicles. Specifically, mixtures rich in SM and cholesterol are found in lipid rafts in cell membranes, which serve as signalling platforms and regulate protein localisation and interactions. Within the scope of this project, small angle neutron scattering (SANS) was used to show that vesicles formulated from mixtures of SM and cholesterol were highly active towards SMase activity, breaking down to form large scale aggregates. The effect of SMase on the organization of the molecules within the vesicle membranes was monitored using small angle X-ray scattering (SAXS) of mixtures of lipid molecules representing different amounts of conversion of SM by SMase to its reaction product, ceramide. The structure of vesicles after treatment with SMase was also characterised using cryo-TEM, which enables individual vesicles immobilised in vitreous ice to be imaged. Finally, Raman spectroscopy and surface plasmon resonance techniques confirmed that the optimised vesicles were not degraded by other phospholipase enzymes such as phospholipase A2, C and D.
2. Development of an assay to screen SMase activity that is faster and more sensitive than current laboratory techniques, and can be interpreted by simple observation of a change in colour.
Vesicles sensitive to SMase were loaded with the small molecule cysteine which can cause aggregation of gold nanoparticles (AuNPs) in low (µM) concentrations. By incubating vesicles with SMase it was possible to release enough cysteine to cause aggregation of AuNPs in solution. Due to the surface plasmon resonance effect, AuNPs of less than 100 nm diameter in solution appear red by eye and blue when aggregated. Therefore, aggregation caused by the presence of very low amounts of SMase was observed easily by the naked eye. Importantly, the limit of detection was lower than in commercially available kits. This assay was also applied to detect drugs that can inhibit the activity of SMase, a highly important application for both screening SMase inhibitor drug candidates for treatment of SMase-dysregulating diseases, and for screening the concentration of SMase-inhibitor drugs in the blood of patients. This second application has important implications for detecting toxic concentrations of SMase inhibitor drugs frequently used to treat depression, where overdose is a risk. The findings reported here are in preparation for submission for publication in the coming weeks.
3. Formulation of non-spherical vesicles, presenting potentially interesting implications for biodistribution and efficacy of drug delivery.
After having established cryo-TEM protocols that are now available for the entire Imperial College Department of Materials, I was able to image a substantial number of vesicle formulations both before and after incubation with SMase. Unusually, it was observed that some vesicle formulations did not form spherical morphologies but took on an elongated structure previously observed in polymer-based vesicles of similar length scales. The ability to form highly deformable, non-spherical large unilamellar vesicles has huge implications for the field of targeted drug delivery and more generally membrane biophysics.
4. Application of techniques developed in SMase-cleavable vesicle formulations to understand the implications for phospholipase C and D based vesicle formulations.
The enzyme SMase is one of several reported phospholipases. These classes of enzymes cleave phospholipids and are implicated in a range of cell signalling pathways, and their dysregulation is observed in several diseases. They are therefore of high interest as biomarkers for disease detection and drug delivery. I applied the tools used in the characterisation of SMase activity on SMase-sensitive vesicles to study the effect of two such phospholipases, phospholipase C and D, on the stability of vesicle formulations. Using SANS I observed significant degradation of the spherical vesicle structure in the presence of phospholipase C, whereas vesicles remain unchanged even after incubation with phospholipase D over 12 hours. SAXS of concentrated lipid mixtures corresponding to partial lipid substrate conversion by the enzyme confirmed that in the case of phospholipase C there is a change in lipid packing whereas in the case of phospholipase D the packing remains largely unchanged even at 90 % conversion. Such changes in lipid organisation are highly indicative of vesicle stability. Additionally, all-atom simulations on these systems carried out in collaboration with Prof. Irene Yarovsky’s group at RMIT, Melbourne, Australia, have provided important insights into how hydrogen bonding and salt coordination affects the packing of lipids on a molecular scale. The combined data suggest that vesicle-based diagnostic and drug delivery platforms analogous to those developed in this Fellowship for SMase are also directly applicable to phospholipase C-, but not phospholipase D-dysregulating conditions. This data is currently being prepared for publication.
The following significant results were obtained during this Marie Skłodowska-Curie Fellowship:
1. Thorough characterisation of novel SMase-cleavable vesicle formulations including the effect of varying lipid composition.
2. Development of an assay to screen SMase activity that is faster and more sensitive than current laboratory techniques, and can be interpreted by simple observation of a change in colour.
3. Application of techniques developed in SMase-cleavable vesicle formulations to understand the implications for vesicles that can target other phospholipase-type enzymes such as phospholipase C and D.
4. Formulation of non-spherical vesicles, presenting potentially interesting implications for biodistribution and efficacy of drug delivery.
The following paragraphs provide a more detailed description of each result.
1. Thorough characterisation of novel SMase-cleavable vesicle formulations including the effect of varying lipid composition:
The phospholipid sphingomyelin (SM) is the substrate for SMase and represents a class of molecules with a phosphocholine head group, sphingosine and a fatty acid. Additionally, naturally derived SM, such as egg sphingomyelin (Egg SM) extracted from chicken egg yolk, brain sphingomyelin (Brain SM) extracted from porcine brain, and milk sphingomyelin (Milk SM) extracted from bovine milk, is actually a mixture of SMs with differing chain lengths. Combination with other lipids is required to form stable vesicles. Specifically, mixtures rich in SM and cholesterol are found in lipid rafts in cell membranes, which serve as signalling platforms and regulate protein localisation and interactions. Within the scope of this project, small angle neutron scattering (SANS) was used to show that vesicles formulated from mixtures of SM and cholesterol were highly active towards SMase activity, breaking down to form large scale aggregates. The effect of SMase on the organization of the molecules within the vesicle membranes was monitored using small angle X-ray scattering (SAXS) of mixtures of lipid molecules representing different amounts of conversion of SM by SMase to its reaction product, ceramide. The structure of vesicles after treatment with SMase was also characterised using cryo-TEM, which enables individual vesicles immobilised in vitreous ice to be imaged. Finally, Raman spectroscopy and surface plasmon resonance techniques confirmed that the optimised vesicles were not degraded by other phospholipase enzymes such as phospholipase A2, C and D.
2. Development of an assay to screen SMase activity that is faster and more sensitive than current laboratory techniques, and can be interpreted by simple observation of a change in colour.
Vesicles sensitive to SMase were loaded with the small molecule cysteine which can cause aggregation of gold nanoparticles (AuNPs) in low (µM) concentrations. By incubating vesicles with SMase it was possible to release enough cysteine to cause aggregation of AuNPs in solution. Due to the surface plasmon resonance effect, AuNPs of less than 100 nm diameter in solution appear red by eye and blue when aggregated. Therefore, aggregation caused by the presence of very low amounts of SMase was observed easily by the naked eye. Importantly, the limit of detection was lower than in commercially available kits. This assay was also applied to detect drugs that can inhibit the activity of SMase, a highly important application for both screening SMase inhibitor drug candidates for treatment of SMase-dysregulating diseases, and for screening the concentration of SMase-inhibitor drugs in the blood of patients. This second application has important implications for detecting toxic concentrations of SMase inhibitor drugs frequently used to treat depression, where overdose is a risk. The findings reported here are in preparation for submission for publication in the coming weeks.
3. Formulation of non-spherical vesicles, presenting potentially interesting implications for biodistribution and efficacy of drug delivery.
After having established cryo-TEM protocols that are now available for the entire Imperial College Department of Materials, I was able to image a substantial number of vesicle formulations both before and after incubation with SMase. Unusually, it was observed that some vesicle formulations did not form spherical morphologies but took on an elongated structure previously observed in polymer-based vesicles of similar length scales. The ability to form highly deformable, non-spherical large unilamellar vesicles has huge implications for the field of targeted drug delivery and more generally membrane biophysics.
4. Application of techniques developed in SMase-cleavable vesicle formulations to understand the implications for phospholipase C and D based vesicle formulations.
The enzyme SMase is one of several reported phospholipases. These classes of enzymes cleave phospholipids and are implicated in a range of cell signalling pathways, and their dysregulation is observed in several diseases. They are therefore of high interest as biomarkers for disease detection and drug delivery. I applied the tools used in the characterisation of SMase activity on SMase-sensitive vesicles to study the effect of two such phospholipases, phospholipase C and D, on the stability of vesicle formulations. Using SANS I observed significant degradation of the spherical vesicle structure in the presence of phospholipase C, whereas vesicles remain unchanged even after incubation with phospholipase D over 12 hours. SAXS of concentrated lipid mixtures corresponding to partial lipid substrate conversion by the enzyme confirmed that in the case of phospholipase C there is a change in lipid packing whereas in the case of phospholipase D the packing remains largely unchanged even at 90 % conversion. Such changes in lipid organisation are highly indicative of vesicle stability. Additionally, all-atom simulations on these systems carried out in collaboration with Prof. Irene Yarovsky’s group at RMIT, Melbourne, Australia, have provided important insights into how hydrogen bonding and salt coordination affects the packing of lipids on a molecular scale. The combined data suggest that vesicle-based diagnostic and drug delivery platforms analogous to those developed in this Fellowship for SMase are also directly applicable to phospholipase C-, but not phospholipase D-dysregulating conditions. This data is currently being prepared for publication.