Effectiveness of Epigallocatechin-3-Gallate (Green Tea Extract) as Adjunctive Locally Delivered Nanotherapeutic in the Treatment of Stage II Periodontitis

Introduction Periodontitis is a complex, chronic inflammatory disease affecting the gingiva and the periodontal tissues, ultimately leading to the destruction of the periodontal ligament and supporting bone. This condition is often triggered by a localized or generalized overgrowth of dysbiotic microorganisms within the dental biofilm. Their presence accelerates the formation of periodontal pockets and subsequent bone loss, ultimately leading to tooth loss. The presence of periodontal pockets acts as a biological niche, promoting the release of pro-inflammatory cytokines and proteolytic enzymes, which further enhance bone resorption and contribute to the progressive breakdown of the periodontal apparatus (1).

The initiation and progression of periodontitis are primarily driven by four interconnected events: periodontal pathogen infection, inflammation, oxidative stress, and autophagy (2). A critical pathogen-associated molecular pattern (PAMP) implicated in periodontitis pathogenesis is lipopolysaccharide (LPS), predominantly derived from Porphyromonas gingivalis. Upon exposure to P. gingivalis LPS, gingival epithelial cells release a spectrum of inflammatory mediators, including interleukin-1β (IL- 1β), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α), all of which exert significant immunomodulatory effects (3).

Crucially, the destructive processes observed in periodontal tissues are not directly caused by the pathogen itself. Instead, they are predominantly a consequence of the host's dysregulated immune response to the pathogenic challenge, compounded by damage induced by reactive oxygen species (ROS), resulting in oxidative protein damage and denaturation, as well as enzyme inhibition (2, 4, 5).

3 The first suggested method for managing periodontal disease is nonsurgical periodontal therapy (NSPT), which is the cornerstone of periodontal therapy (6). The primary goal of the initial phase of periodontal therapy is to reduce inflammation by eliminating local factors using scaling and root planing (SRP). Although, a new attachment is not frequently formed, this therapy reduces the probing pocket depth because of extended junctional epithelium growth or shrinking. The healing result appears to be sufficient currently but is susceptible to future disease progression (7).

While scaling and root planing (SRP) remains the gold standard for managing periodontitis, individuals with advanced forms of the disease may benefit from adjunctive therapeutic strategies. Systemic antibiotics are commonly employed as part of periodontal treatment to help suppress or eliminate residual pathogenic bacteria, serving as a complement to conventional mechanical debridement (8, 9).

Systemic antibiotics combined with non-surgical periodontal therapy (SRP) have shown mixed results in treating periodontitis, with success often depending on the severity of inflammation and bone loss. However, systemic antibiotic therapy comes with notable drawbacks: it can be toxic to the liver and kidneys, contribute to the development of antibiotic-resistant bacteria, and often requires high doses to effectively reach periodontal tissues (10, 11).

Administering antimicrobial drugs directly into periodontal pockets offers an alternative to systemic antibiotics (12). This in situ approach minimizes systemic side effects and does not rely on patient compliance. The concept of localized drug delivery for periodontal therapy was first introduced by Goodson et al. in 1979. Since then, many studies have explored various antimicrobial agents in different clinical situations (13).

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The management of periodontal disease currently involves the use of local antibiotics such as tetracycline, doxycycline, minocycline, metronidazole, chlorhexidine, azithromycin, clarithromycin, moxifloxacin, clindamycin, and satranidazole in a variety of drug delivery systems, including irrigations, fibers, films, injectables, gels, strips, compacts, vesicular liposomes, microparticles, and recently introduced nanoparticle systems (8). Moreover, local drug delivery systems (LDDs) have been explored for periodontitis treatment, using agents like non-steroidal anti- inflammatory drugs, statins and other herbal compounds like green tea extract (14).

In recent years, green tea has been used for daily health care in many countries due to its anti-inflammatory, antioxidant, and oral health-promoting properties. Green tea is one of the most popular beverages in the world and is known to be a rich source of polyphenols (15,16). Green tea catechins have many biological functions, including antibacterial, anticancer, and antioxidant effects. The main catechins of green tea include epicatechin (EC), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and epigallocatechin- 3-gallate (EGCG) (17, 18).

EGCG is the most abundant catechin in green tea, accounting for more than 50 % of the total components of green tea, followed by EGC and ECG (19). EGCG is known to exhibit the strongest antibacterial activity among all catechins, and it has been reported that this effect is derived from the galloyl groups in its structure (20). EGCG has also been reported to have antimicrobial activity against periodontal disease-associated bacteria (21). When used against Porphyromonas gingivalis, EGCG inhibited its 5

proliferation and adhesion to epithelial cells, destroyed its biofilms, and inhibited its collagenase activity (18).

The effects of EGCG on Prevotella intermedia have been reported to include the inhibition of protein tyrosine phosphatase (PTPase) activity and bactericidal effects. In addition, EGCG inhibited the adhesion of Fusobacterium nucleatum to gingival epithelial cells, reduced its biofilm formation, and had bactericidal effects in F. nucleatum biofilms (21).

It was demonstrated that EGCG exhibits superior bactericidal, growth-inhibiting, and metabolism-inhibiting effects against bacteria associated with periodontal disease compared to Streptococcus mutans. While EGCG showed similar bacterial aggregation-inducing effects on both periodontal pathogens and S. mutans, some inter-species differences were observed (18). Ultimately, EGCG could help prevent periodontal disease by directly killing bacteria, suppressing bacterial growth through metabolic inhibition, and aiding bacterial clearance from the oral cavity by promoting aggregation (22).

Bioadhesive polymer-based delivery methods have been widely researched to prevent the delivery system from being removed from the site of application as a result of saliva flow and tongue movement. These polymers aid in the system's attachment to the application site and enable medication release at the appropriate time (23, 24).

In the past decade, chitosan-based delivery systems have garnered significant attention. Chitosan, a naturally derived polymer obtained through the deacetylation of chitin, exhibits a range of desirable biological properties, including non-toxicity, anti-inflammatory activity, adaptability, biocompatibility, biodegradability, and bio-adhesiveness. These 6

characteristics support its osteoconductive capabilities and make it highly suitable for applications in tissue engineering, drug delivery, and wound healing (25). Previous studies have demonstrated that chitosan possesses several beneficial properties, including the ability to stimulate osteoblast activity, inhibit biofilm formation, promote neovascularization, and exhibit strong antimicrobial effects ( 26).