Periodic Reporting for period 1 - SoftNanoHybrid (Polymer-Nanoparticle co-assembly for drug targeting and release kinetics: Investigation on Structure, Morphology, Responsiveness, and Nanoparticle distribution)
Reporting period: 2018-07-01 to 2020-06-30
The soft nanohybrids formed by the bacterial amphiphiles in water are characterised with an aim to understand their role in various bacteriological processes e.g. bioadhesion, colonisation and antimicrobial resistance. A biopolymer (lipoteichoic acid, LTA) that is pH responsive has been used as a central building unit for the model systems for self-assembly. We have successfully formed and characterised the LTA self assemblies in water and examined their adsorption at the interfaces. In the actual bacteria membrane, the LTA is adhered directly via lipid-like chains with the phospholipid membrane, and accordingly could play a major role in the structural stability and integrity of the membrane. These possibilities were checked by designing mixed vesicles from phospholipids representing the actual composition of the bacteria membrane and LTA. These structures were analysed by SANS, DLS and CryoTEM, which provided conclusions on the structural effects caused by LTA on the size and distribution of the mixed vesicles.
According to National Health Services (NHS) UK, several strains of bacteria have reportedly developed resistance to many different types of antibiotics, including: MRSA (methicillin-resistant Staphylococcus aureus), Clostridium difficile and the bacteria that cause multi-drug-resistant tuberculosis. In order to encounter this challenge, the molecular interactions within bacteria membrane must be understood by detailed experimental approaches, which would presumably provide suggestions for the design and development of the antibiotics with specific structural properties to tackle the resistance caused by the bacteria. Our experiments have provided useful results in this direction and this field remains open with several unanswered questions about the physicochemical understanding of these bacteria membrane processes.
The objective of the project was to understand self-assembly behaviour of soft nanohybrids. Having examined the availability and costs of a number of block copolymers with different molecular weight distribution, we have focused on mixed liposomes/vesicles and their structural transformations on interactions with lipoteichoic acid (LTA).
According to National Health Services (NHS) UK, several strains of bacteria have reportedly developed resistance to many different types of antibiotics, including: MRSA (methicillin-resistant Staphylococcus aureus), Clostridium difficile and the bacteria that cause multi-drug-resistant tuberculosis. In order to encounter this challenge, the molecular interactions within bacteria membrane must be understood by detailed experimental approaches, which would presumably provide suggestions for the design and development of the antibiotics with specific structural properties to tackle the resistance caused by the bacteria. Our experiments have provided useful results in this direction and this field remains open with several unanswered questions about the physicochemical understanding of these bacteria membrane processes.
The objective of the project was to understand self-assembly behaviour of soft nanohybrids. Having examined the availability and costs of a number of block copolymers with different molecular weight distribution, we have focused on mixed liposomes/vesicles and their structural transformations on interactions with lipoteichoic acid (LTA).
1. The self-assembly of lipoteichoic acid (LTA) in aqueous solutions:
The LTA aggregates play a major role in the process of bioadhesion and colonisation, which are responsible for initiation of bacterial infections. Considering the amphiphilic structure of LTA, its aggregation behavior in water may reveal interesting structural features. The self-assembly of LTA in water and their adsorption behavior at the air-water and air-solid interfaces are interesting to understand their role in the post-infection sequalae. The microscopic images indicated formation of spherical aggregates that do not adsorb considerably on charged silica surface, while on rinsing with Ca2+ ions, the adsorption increases significantly indicating role of Ca2+ ions in LTA binding on charged surfaces. We also examined these structures by DLS and SANS methods, which indicated decrease in size of LTA aggregates on Ca2+ addition.
2. Effect of LTA on mixed vesicles containing phospholipids (soft nanohybrids):
The lipoteichoic acid (LTA) is tethered directly to the hydrocarbon chain of phospholipids in the actual membrane, and accordingly influences the structural stability and integrity of the phospholipid membrane. We investigated the effect of LTA on mixed vesicles (representing the bilayer phospholipid membrane) by scattering and microscopy methods. The mixed vesicles containing LTA and phospholipids, namely phosphatidyl glycerol (PG), phosphatidyl ethanolamine (PE) and cardiolipin (C16:0) were prepared by film drying method. LTA composition in the membrane is regulated by bacteria under different physiological conditions. How such LTA compositional variations modulate the membrane structural stability and integrity is poorly understood. Here, we have investigated structural changes in mixed liposomes mimicking the lipid composition of Gram-positive bacteria membranes, in which the concentration of Bacillus Subtilis LTA was varied between 0–15 mol%. Small-angle neutron scattering (SANS) and dynamic light scattering (DLS) measurements indicated formation of mixed unilamellar vesicles, presumably stabilized by the negatively charged LTA polyphosphates. The vesicle size increased with the LTA molar concentration up to ∼6.5 mol%, accompanied by a broadened size distribution, and further increasing the LTA concentration led to a decrease in the vesicle size. This work was published in Colloids and Surfaces B: Biointerfaces (Bhavesh Bharatiya, Gang Wang, Sarah E. Rogers, Jan Skov Pedersen, Stephen Mann, Wuge H. Briscoe*, Colloids and Surfaces B: Biointerfaces, 2021, 199, 111551. Gold Open Access.
The fellow also worked in collaborative projects with colleagues at the Bristol University and a research center in India.
3. The design and formulation of Exemestane formulation for breast cancer treatment :
We designed liquid crystalline (LC) formulation of Exemestane using Pluronic block copolymer P85/P84 and propylene glycol as cosolvents in a suitable oil media. This formulation containing thermoresponsive lamellar liquid crystal gels of EXE represented a viable option for hyperthermia induced enhanced drug release. This work was accomplished in collaboration with Dr V Shah at Shah-Schulman Research Center, India.
4. The effect of cations on self-assembly behaviour of Gram-negative bacterial lipopolysaccharide (LPS) :
Lipopolysaccharide (LPS) is a key structural component of the outer membrane of gram-negative bacteria. LPS is a macromolecule with six hydrophobic tails and a very long hydrophilic headgroup which shows a very complex aggregation behaviour forming a wide variety of structures in solution. Cations are known to be crucial for the integrity of the bacterial out membrane and can complex with LPS which affects bacterial membrane integrity and LPS virulence. To develop effective and alternative antibacterial agents, it is crucial to understand the structure of, and fundamental interactions at, the bacterial cell wall. Here, the effects of monovalent (Na+), divalent (Ca2+), and trivalent cations (La3+), as well as LPS carbohydrate head group length on the structure, size and electrostatic properties of LPS aggregates at near physiological conditions are studied. This work was done in collaboration with Dr Gang Wang, Marie Curie fellow, University of Bristol. A manuscript based on this work is under preparation.
The LTA aggregates play a major role in the process of bioadhesion and colonisation, which are responsible for initiation of bacterial infections. Considering the amphiphilic structure of LTA, its aggregation behavior in water may reveal interesting structural features. The self-assembly of LTA in water and their adsorption behavior at the air-water and air-solid interfaces are interesting to understand their role in the post-infection sequalae. The microscopic images indicated formation of spherical aggregates that do not adsorb considerably on charged silica surface, while on rinsing with Ca2+ ions, the adsorption increases significantly indicating role of Ca2+ ions in LTA binding on charged surfaces. We also examined these structures by DLS and SANS methods, which indicated decrease in size of LTA aggregates on Ca2+ addition.
2. Effect of LTA on mixed vesicles containing phospholipids (soft nanohybrids):
The lipoteichoic acid (LTA) is tethered directly to the hydrocarbon chain of phospholipids in the actual membrane, and accordingly influences the structural stability and integrity of the phospholipid membrane. We investigated the effect of LTA on mixed vesicles (representing the bilayer phospholipid membrane) by scattering and microscopy methods. The mixed vesicles containing LTA and phospholipids, namely phosphatidyl glycerol (PG), phosphatidyl ethanolamine (PE) and cardiolipin (C16:0) were prepared by film drying method. LTA composition in the membrane is regulated by bacteria under different physiological conditions. How such LTA compositional variations modulate the membrane structural stability and integrity is poorly understood. Here, we have investigated structural changes in mixed liposomes mimicking the lipid composition of Gram-positive bacteria membranes, in which the concentration of Bacillus Subtilis LTA was varied between 0–15 mol%. Small-angle neutron scattering (SANS) and dynamic light scattering (DLS) measurements indicated formation of mixed unilamellar vesicles, presumably stabilized by the negatively charged LTA polyphosphates. The vesicle size increased with the LTA molar concentration up to ∼6.5 mol%, accompanied by a broadened size distribution, and further increasing the LTA concentration led to a decrease in the vesicle size. This work was published in Colloids and Surfaces B: Biointerfaces (Bhavesh Bharatiya, Gang Wang, Sarah E. Rogers, Jan Skov Pedersen, Stephen Mann, Wuge H. Briscoe*, Colloids and Surfaces B: Biointerfaces, 2021, 199, 111551. Gold Open Access.
The fellow also worked in collaborative projects with colleagues at the Bristol University and a research center in India.
3. The design and formulation of Exemestane formulation for breast cancer treatment :
We designed liquid crystalline (LC) formulation of Exemestane using Pluronic block copolymer P85/P84 and propylene glycol as cosolvents in a suitable oil media. This formulation containing thermoresponsive lamellar liquid crystal gels of EXE represented a viable option for hyperthermia induced enhanced drug release. This work was accomplished in collaboration with Dr V Shah at Shah-Schulman Research Center, India.
4. The effect of cations on self-assembly behaviour of Gram-negative bacterial lipopolysaccharide (LPS) :
Lipopolysaccharide (LPS) is a key structural component of the outer membrane of gram-negative bacteria. LPS is a macromolecule with six hydrophobic tails and a very long hydrophilic headgroup which shows a very complex aggregation behaviour forming a wide variety of structures in solution. Cations are known to be crucial for the integrity of the bacterial out membrane and can complex with LPS which affects bacterial membrane integrity and LPS virulence. To develop effective and alternative antibacterial agents, it is crucial to understand the structure of, and fundamental interactions at, the bacterial cell wall. Here, the effects of monovalent (Na+), divalent (Ca2+), and trivalent cations (La3+), as well as LPS carbohydrate head group length on the structure, size and electrostatic properties of LPS aggregates at near physiological conditions are studied. This work was done in collaboration with Dr Gang Wang, Marie Curie fellow, University of Bristol. A manuscript based on this work is under preparation.
Our investigation featuring the LTA aggregates formed by Bacillus Subtilis is the first attempt to explore the self assembly behavior of these bacterial lipids. The SANS experiments on LTA mixed phospholipid vesicles was the first study on these soft nanohybrids representing the bacterial membrane composition. Our forthcoming NR and XRR experiments will be the first to investigate the interfacial adsorption of these soft nanohybrids.
According to NHS UK, the bacterial resistance is prevalent for various bacteria species e.g. MRSA (methicillin-resistant Staphylococcus aureus) and Clostridium difficile. Our experiments have provided molecular level understanding of the model softnanohybrids representing Bacillus subtilis bacteria membranes, and has provided useful suggestions for the design and development of the next generation of antimicrobial peptides.
According to NHS UK, the bacterial resistance is prevalent for various bacteria species e.g. MRSA (methicillin-resistant Staphylococcus aureus) and Clostridium difficile. Our experiments have provided molecular level understanding of the model softnanohybrids representing Bacillus subtilis bacteria membranes, and has provided useful suggestions for the design and development of the next generation of antimicrobial peptides.