title: Processing biologically assembled biopolymers into advanced functional materials
Creators: name: Pande, Deeptee Chandrashekhar
Publisher: name: Griffith University
Year: 2024
Subjects: subject: biopolymers
subject: advanced functional materials
subject: biomedicine

description: In conditions of nutrient imbalance, bacteria and archaea produce polyhydroxybutyrate (PHB) as a carbon storage material. These naturally self-assembled biopolymers can be bioengineered for use in various biomedical applications. These biopolymer (BP) particles are biocompatible and biodegradable, and their production is environmentally friendly. As bacteria can be bioengineered to express the desired protein on the surface of the BP particles, these BPs have been explored for drug delivery, diagnosis, and bioseparation applications. One part of the thesis focuses on exploring the possibility of reformatting these BP particles into reformatted functional materials. Functional biomaterials are materials designed to possess functionality required for the applications including diagnostics, scaffolds for tissue engineering, wound healing, and vaccine delivery to name a few. Biomaterials in general can be functionalized by postproduction processing. However, this was found to affect the size and shape of the final biomaterials. This can be avoided by reformatting functionalized BPs. Reformatting is a process of changing the material's shape, size and physicochemical properties. The effect of reformatting on the functionality of the BP particles were studied. For this, BP particles displaying IgG binding domains of protein A (Z domain (BP-Z)) and protein G ((GB1)3 domain (BP-G)) were selected. BP-P (empty BP particles) and the green fluorescent protein (GFP (BPGFP)) were selected as negative controls and BP-Z and BP-G were selected as BP particles possessing functionality. BP particles were reformatted by dissolving them in solvent and forming new structures after solvent removal. Dissolution was carried out over a temperature range of 40°C to 90°C. For the dissolution, BP particles were freeze-dried to remove the moisture content. After formatting the BP particles into porous particles with PVA precipitation and electrospun mesh, the binding domain activity was studied analysing IgG binding capacity. This was compared with the respective BP particles prior to dissolution. It was observed that the IgG binding capacity was affected by the experimental conditions selected for the dissolution. It was observed that freeze-drying did not affect protein profiles of the BP particles and the functionality was retained for BP-Z and BP-G. Whereas, after dissolution, only the BP-Z protein and its functionality was retained. Retention of protein function was most efficient when dissolution was conducted at 50°C and was declined by 2-fold at 60°C and by 4.3-fold at 90°C compared to the retained functionality at 50°C. BP particles were found to have not dissolved at the dissolution temperature of 40°C. The IgG binding capacity of the BP-Z particles into hollow spheres by precipitating out the dissolved BP particles with PVA and electrospun mesh was influenced by the used dissolution temperature. BP-Z particles formatted into hollow spheres (with PVA precipitation) and electrospun mesh were found to have retained functionality when a dissolution temperature of 50° was selected. The second part of the thesis focuses on the formation of the core shell structure consisting of BP core and silica shell which was formed through post-production treatments of engineered BP particles. For this, the silica forming peptide RK1 ((RKK)4G3Y) was displayed on the BP particles by a one-step assembly inside the engineered endotoxin free Escherichia coli (E.coli) strain. The peptide was inserted at one of the surface exposed sites (K139) of the BP anchoring protein PhaC and was displayed on the surface of the BP particles. The amino acid site K139 was selected because of its proximity to the surface and the activity of the inserted functional peptide as previously shown. The BP particles isolated from bacterial cells were then treated with different silica precursors at different reaction times to study silica shell formation. It was observed that the precursor tetraethyl orthosilicate (TEOS) did lead to the formation of a consistent silica shell. The precursor tetramethyl orthosilicate (TMOS) formed the silica shell on the BP particles. The silica shell formation was confirmed with Fourier transformed infrared spectroscopy (FT-IR) and transmission electron microscopy (TEM) imaging. The effect of silica shell formation on physicochemical properties of the BP particles was examined using imaging, thermogravimetric analysis, differential scanning calorimetry. A study of changes in surface properties was conducted using curcumin encapsulation. The encapsulation efficiency of the TMOS treated BP particles was found to have been improved. Thermogravimetric analysis showed that 0.14 g of silica shell was formed per gram of the BP particles having higher RK1 peptide concentration per gram of BP particle. The effect of TMOS treatment on BP particle cytotoxicity was studied with HEK293T cells. While silica shell formation was observed to follow no specific trend for the TEOS precursor. For precursor TMOS, silica shell formation was uniform and controlled for higher reaction time-with lower concentration and lower reaction time-with higher concentration. Higher reaction time-with higher concentrations lead to uncontrolled reaction which was observed using TEM and analysed FT-IR data. The optimum reaction condition for the formation of silica shell formation was selected based on the results obtained for TEM and FT-IR characterization. When studied against HEK293T cells, TMOS treatment had no effect on BP particle cytotoxicity.