Abstract
Oral mucositis is an inflammation and ulceration of oral mucosa suffered by cancer patients undergoing radiation and chemotherapy. The lesions compromise nutrition, interrupt dosing, increase infection risk and mortality, requiring parenteral nutrition, extended hospital stays and cost. Gold nanoparticles (AuNPs) (<10 nm diameter) with documented immunomodulatory properties and low toxicity offer a potential therapeutic approach. AuNPs combined with the antioxidant α-lipoic acid (LA), show synergistic anti-inflammatory effects. Hence, the present study evaluated LA-capped AuNPs (LA-AuNP) (< 10 nm) as a potential topical therapeutic agent for treating oral mucositis. The primary aims of the study were to assess the cytotoxicity, anti-inflammatory, and antimicrobial activities of LA-AuNPs (<10 nm) against oral epithelial cells, and microorganisms relevant to oral mucositis. Additionally, as long-term efficacy of topically administered medications is a challenge in the oral cavity due to salivary flow, LA-AuNPs (<10 nm) were incorporated into a nontoxic and mucoadhesive hydrogel (carboxy methyl cellulose) and assessed for oral application.
To evaluate the effects of size and capping agent on AuNPs activity, two distinct sizes (3 and 15 nm diameter) of AuNPs capped with either LA or citrate were purchased and compared. Antimicrobial efficacy of LA-AuNPs were assessed against LA-capped silver nanoparticles (LA-AgNPs), an established antimicrobial agent, of equivalent size ranges. The effect of AuNPs synthesis method was also assessed by comparing purchased gold and silver nanoparticles of < 10 nm diameter with laboratory-synthesized particles of similar size. Nanoparticles dimensions were confirmed by transmission electron microscopy (TEM) and UV-vis spectroscopy. Cytotoxicity was assessed using cultured human oral epithelial cells (H400). Cellular uptake of AuNPs were evaluated by TEM and inductively coupled plasma-mass spectroscopy (ICP-MS). Anti-inflammatory response to AuNPs was measured using inflammatory stimulation of H400 cells. Antimicrobial and antibiofilm activities were tested against the common opportunistic micro-organisms associated with oral mucositis: Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Candida albicans.
TEM visualization confirmed that all nanoparticles whether purchased or synthesized were spherical and well-dispersed. Epithelial cells remained viable (>80%) following exposure to 25 μg/mL AuNPs for 72 hours. LA-AuNPs (<10 nm) were less cytotoxic than Cit-AuNPs of similar size even at the highest concentration (100 μg/mL), while larger particles were nontoxic irrespective of their size and capping agent. Although, cells exposed to AuNPs displayed higher viability, they suppressed cell proliferation in a size- and time-dependent manner, however the inhibitory effect was temporary, and cells recover with time. Cellular internalization of AuNPs by epithelial cells was governed by endocytosis in a size- and time-dependent manner, without nuclear penetration. Uptake of LA-AuNPs (<10 nm) was greater than other particles suggesting cellular uptake is also influenced by the capping agent. LA-AuNPs (< 10 nm) displayed significantly greater anti-inflammatory effects measured as IL-1β, IL-6, and IL-8 secretion by stimulated H400 cells than similar sized Cit-AuNPs, while larger particles were less suppressive. AuNPs exhibited no antimicrobial activity against tested species (MIC >100 μg/ml), but inhibited biofilm formation in a concentration- and size-dependent manner. LA-AuNP (< 10 nm) at 25 μg/ml reduced more than 80% bacterial biofilm formation by all tested species. The AuNPs (< 10 nm) were also effective (>80%) against Candida biofilms at 6.25 μg/ml, while AuNPs > 10 nm in diameter were ineffective. Furthermore, LA-AuNP incorporated into carboxy methyl cellulose (6%) showed effective release of nanoparticles and increased mechanical strength, suggesting carboxy methyl cellulose hydrogel could facilitate oral delivery of AuNPs, justifying further investigation to optimize the formulation.
In conclusion, the study demonstrates that LA-AuNP (<10 nm) are non-toxic, display anti-inflammatory and biofilm inhibitory effects, thus show promise as a topical therapeutic agent for oral mucositis. Additionally, AuNP-carboxy methyl cellulose hydrogel could mediate an effective oral delivery.