Development of “Intelligent Particles” for the treatment of dental caries
Date
2024
Authors
He, Yanping
Editors
Advisors
Zilm, Peter
Vasilev, Krasimir
Vasilev, Krasimir
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Thesis
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Abstract
Dental caries, a prevalent non-communicable disease worldwide, primarily arises from the growth of cariogenic bacteria within plaque biofilms which adhere to both soft and hard tissues in the oral cavity. Elevated levels of acidophilic and acidogenic bacteria such as Streptococcus mutans embed themselves within the exopolysaccharide (EPS) enriched biofilm matrix, producing lactic acid that demineralizes the mineral component (hydroxyapatite) of teeth resulting in dental caries. In recent decades, with the progress of nanotechnology, metallic nanoparticles such as silver, gold, and platinum have undergone extensive research and demonstrated promising antibacterial and antibiofilm properties. Among these, silver nanoparticles (AgNPs) have attracted significant attention due to their exceptional antimicrobial efficacy and low toxicity. It is widely acknowledged that smaller AgNPs (< 10 nm) possess notably higher antimicrobial potency, and positively charged AgNPs exhibit superior antibacterial effects against Gram-positive bacteria compared to their negatively charged counterparts. Importantly, unlike traditional antibiotics, silver nanoparticles demonstrate potent bactericidal properties without developing bacterial resistance. Furthermore, there is growing interest in the application of AgNPs in dentistry. However, conventional treatment practice still faces challenges in addressing two key issues: 1) the rapid clearance of therapeutic agents from the oral environment, and 2) the destruction of bacteria essential in maintaining a healthy oral microbiome. To address these problems, my project aimed to, 1. Synthesise polycationic silver nanoparticles that bind to the negatively charged bacterial surface, thereby increasing antibacterial effectiveness. Low molecular weight branched poly(ethyleneimine) (BPEI) with primary, secondary and tertiary amine groups were used as a stabilizer to confer positive charge to the silver nanoparticle surface to make BPEI capped silver nanoparticles (BPEI-AgNPs) 2. Encapsulate the developed BPEI-AgNPs in poly(D,L-lactic-co-glycolic acid) (PLGA) to make a delivery system with the aid of double surfactant for higher loading of BPEI-AgNPs . 3. Coat the BPEI-AgNPs encapsulated PLGA particles with a Fe(III) and tannic acid (TA) complex (Fe(III)-TA/PLGA@BPEI-AgNPs). We have termed the resultant construct “Intelligent Particles (IPs)” as they enable binding to the plaque biofilm thereby increasing substantivity in the oral cavity. The Fe(III)-TA complex also conferred pH responsiveness to the delivery system that responds “intelligently” to an acidic pH environment (caries conditions) by releasing BPEI-AgNPs to target the cariogenic biofilm. Firstly, we synthesized highly positively charged silver nanoparticles (AgNPs) by coating AgNPs with low molecular weight branched poly(ethyleneimine) to make BPEI-AgNPs. The BPEI-AgNPs were characterized using UV–vis absorption and transmission electron microscopy (TEM), revealing a size of approximately 7.5 nm. The antimicrobial efficacy and anti-biofilm properties of BPEI-AgNPs were then assessed against cariogenic bacteria. The findings demonstrated that BPEI-AgNPs exhibit potent clinical oral antiseptic properties. Additionally, the cytotoxicity of BPEI-AgNPs was evaluated against two mammalian cell lines, the results indicated their superior safety profile compared to commercially available dental antiseptics. For aims 2 and 3, we developed a pH responsive carrier for delivery of BPEI-AgNPs, by encapsulating BPEI-AgNPs in a tannic acid−Fe(III) complex-modified - PLGA particle (Fe(III)-TA/PLGA@BPEI-AgNPs), which we termed "intelligent particles" (IPs). Tannic acid was used to enhance binding to the plaque biofilm, while the Fe(III)-TA complex coating imparted "intelligence" functionality to the delivery system by releasing BPEI-AgNPs in acidic conditions associated with dental caries. The resulting IPs displayed notable binding to an axenic S. mutans biofilm grown on hydroxyapatite discs. They also exhibited accelerated release of silver ions at pH 4 (cariogenic pH) in comparison to pH 7. Furthermore, IPs significantly diminished both the volume and viability of S. mutans biofilm under acidic conditions, without demonstrating cytotoxic effects on differentiated Caco-2 cells and human gingival fibroblasts at 3 to 5 times the antibiofilm concentration. Finally, we prepared a dual-responsive (pH/temperature) formulation composed of a thermos-responsive polymer-Pluronic F-127 (PF127), a mucoadhesive polymer (Sodium carboxymethyl cellulose) and the IPs. All the components were crossed linked through physical mixing. The resulting formulation was liquid at low temperature but formed gel at 37°C within 3 minutes. Analysis of the microstructure via scanning electron microscopy (SEM) of freeze-dried formulations unveiled a characteristic multi-layer arrangement with a highly porous interconnected hydrogel network structure. Transmission electron microscopy (TEM) coupled with energy-dispersive X-ray spectroscopy (EDX) further confirmed the incorporation of IPs in the formulation. pH-responsive release of silver ion from the formulation was confirmed as at pH 4, a burst release of silver ions within the first hour was observed, notably more than that at pH 7. The antibiofilm efficacy was assessed using an in vitro flow cell biofilm model, wherein multispecies biofilms grown on hydroxyapatite discs in a flow cell system were examined via confocal laser scanning microscopy (CLSM), SEM imaging, and colony-forming unit (CFU) counts. Results indicated that sucrose substantially enhanced bacterial growth and biofilm formation. Following three cycles of formulation treatment (equivalent to a total of 14.6 μg of Ag+), the release of silver ions under physiological pH eradicated established biofilms but was insufficient for biofilms cultivated in the presence of sucrose. These findings underscored the need for further optimization in both the formulation design and the flow cell model. In summary, the findings from this research have great potential to contribute to the development of an innovative, safer and more effective, cariogenic biofilm-targeted oral care product for dental caries therapy.
School/Discipline
Adelaide Dental School
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, Adelaide Dental School, 2024
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