INVESTIGATION INTO SOFT TEMPLATED BIOACTIVE GLASS NANO PARTICLES FOR BIOMEDICAL APPLICATIONS

Abstract

Bioactive glass nanoparticles have numerous applications in the repair and regeneration of both hard and soft tissue. It has also used widely as drug delivery vehicle. Through the bio-inspired route of synthesis, bioactive glass nanoparticles can be synthesized with minimum non-green chemicals, time, energy, and cost-to-synthesis. Although surfactants are traditionally referred to as soft templates, we have used components like glycosaminoglycans (hyaluronic acid), drugs (doxorubicin) and biomolecules (gelatin) as alternative to the traditional soft templates. The bio-inspired route allows for the use of both hard and soft templates to easily initiate the synthesis and formation of nano-sized, mesoporous, amorphous, bioactive, and biocompatible bioactive glass nanoparticles. Additionally, the absence of a calcination step allows the template to be retained within the nanoparticles, providing specific properties to the resulting bioactive glass. In this thesis, we explore alternative templates, such as gelatin-calcium carbonate nanocomposites, doxorubicin, and hyaluronic acid, as substitutes for traditional surfactants for synthesizing bioactive glass. The goal is to harness the advantages that the bio-inspired route offers in terms of applications of range of templates that would ideally be difficult in case of traditional synthesis methods like sol-gel and melting quenching due to either high temperature involved in synthesis or calcination process or use of non-green solvents and synthetic surfactants. In the first study, we report a surfactant free route to synthesize hollow mesoporous bioactive glass nanoparticles at ambient atmospheric condition. Here, through the bio-inspired route, gelatin is utilized as soft template for synthesizing calcium carbonate nanocomposites which in turn gives rise to hollow bioactive glasses through multiple steps. The synthesized particles contain hollow core as a result of mild acid mediated removal of gelatin-calcium carbonate nanoparticles used as hard template. Removal of template was confirmed through FTIR and XRD while round morphology and sizes below 100 nm could be observed by TEM. In addition, N2 adsorption and desorption analysis confirmed hollow and mesoporous nature of the bioactive glass shell. Interestingly, post removal of template, particles reported higher surface area, pore volume and pore diameter along with decrease in surface charge. Deposition of hydroxyapatite on hollow bioactive glass in Simulated Buffer Fluid (SBF) could be observed from Day 7 of immersion while well-developed hydroxyapatite depositions could be observed by Day 30. This proved bioactivity of the material while cytotoxicity analysis on Human Osteosarcoma cell line (U2OS) through MTT assay proved the biocompatible nature of the hollow bioactive glass particle. In the second study, a common anti-cancer drug doxorubicin solution in Tris buffer acts as a soft template for preparing bioactive glass nanocomposites. Doxorubicin preparation with bioactive glass as a novel hybrid nanoparticle formulation was carried out through the bio inspired route where different precursors of bioactive glass are added to a Tris(hydroxymethyl)aminomethane buffer adjusted to slightly alkaline pH containing doxorubicin. With increasing concentration of doxorubicin, loading increased in quantity with comparatively higher drug release in acidic pH than neutral pH. Without synthetic surfactant or high temperature calcination, bioactive glass-ceramic nanoparticles demonstrated significantly superior cytotoxic behavior towards osteosarcoma cell line when compared to equivalent free drug or its action towards non-cancerous cell line. The particles exhibited hydroxayapatite deposition when immersed in simulated body fluid for 7 days. The bio inspired route for synthesis of doxorubicin-bioactive glass-ceramic hybrid nanoparticles was an efficient cost-effective synthesis mechanism which is environmentally friendly without hampering the stability or activity of the drug. Novel HA nano-formulations are constantly in demand due to its applications ranging from the medical to cosmeceutical industries. Therefore, in the third study, we explored a novel nano- composition of HA and bioactive glass (BG) for the delivery of HA across biological barriers. Using a bio-inspired method, HA, acting as soft template and mixed with Tris(hydroxymethyl)aminomethane buffer at mild alkaline pH was able to direct the synthesis of amorphous hyaluronic acid-bioactive glass nano-composites (BGHA) with step-wise addition of precursors. Unlike the traditional Stober’s method, the synthesis process does not require ethanol or ammonia, making it more environment friendly. The non-requirement of high-temperature calcination also makes the process energy efficient. The inherently mesoporous BGHA nano-composites demonstrate effective penetration across biological barriers, such as skin and bone cell membranes in in-vitro cell culture and human skin mimicking artificial skin membrane in ex-vivo studies. Further studies were conducted to analyze retention and penetration in keratinocytes, which form a viable barrier in the skin in addition to the lipid barrier. In addition, we externally conjugated HA to BGHA and observed similar results. Overall, nano-composition of high molecular weight hyaluronic acid (HA) and bioactive glass (BG) for efficient delivery of HA across biological barriers explored through the bio-inspired method can have potential medical and cosmeceutical applications if investigated further.