Formulation, physicochemical characterization and clinical evaluation of therapeutic potential of quercetin loaded chitosan nanoparticles in management of chronic periodontitis

Introduction: Periodontitis is a chronic inflammatory disease initiated by plaque biofilm affecting the supporting structures of the teeth. It is a disease of multifactorial etiology, with involvement of microbial, genetic, environmental and host factors. The aim of periodontal treatment is primarily focused on eliminating the microorganisms and their by-products by means of mechanical instrumentation like scaling and root planing (SRP). It has been observed that the local route of drug delivery can attain 100-fold higher concentrations of an antimicrobial agent in subgingival sites compared with a systemic drug regimen thereby reducing the total patient dose by over 400 fold avoiding development of drug-resistant at non oral body sites.

Quercetin is one of the bioflavonoid that is present in foods including vegetables, nuts, berries, legumes, green tea, onions and citrus. It has been approved by the United States Food and Drug Administration (FDA) as an ingredient in over-the-counter health supplement. To date, there has been no evidence in the literature of any adverse effects from consuming quercetin. Some of the antimicrobial effects of quercetin have been reported.

Amongst various semi-synthetic polymers, chitosan, a deacetylated product of chitin is widely used in drug delivery devices Since it exhibits favorable biological properties such as non-toxicity biocompatibility, biodegradability and wound healing traits, it has attracted great attention in the pharmaceutical and biomedical fields. Chitosan based drug delivery system fulfill all these properties; it stays for extended periods in the oral cavity, has adequate drug penetration, shows excellent antimicrobial activity, high efficiency and acceptability.

Aim & objectives: To formulate, characterize physicochemical and evaluate clinical therapeutic potential of quercetin loaded chitosan nanoparticles in management of chronic periodontitis and to test Anti-inflammatory activity of the formulated gel in subjects with chronic periodontitis using ELISA.

Methodology:

• Preparation of chitosan and quercetin loaded chitosan nanoparticles-

Will be prepared according to the study by Zhang et al based on the ionic gelation of chitosan with Tripolyphosphate anions.

• Physico-chemical characterization studies-

To determine the hydrodynamic diameter, morphology and particle size of nanoparticles using Transmission electron microscope.

To perform Spectroscopy characterization by UV-visible and Fourier transformed infrared technique (FTIR).

To determine antimicrobial activity of the synthesized QLC nanoparticle.

To determine water holding capacity of the QLC nanoparticles

To formulate gel with synthesized and characterized QLC nanoparticles using suitable polymer

•  Antioxidant potential of QLC nanoparticles- will be measured by 2,2-diphenyl-1-picrylhydazyl (DPPH) assay

• Anti-microbial activity of QLC nanoparticles- shall be studied on the microorganisms referred to as initial colonizers of periodontal disease by conducting assays such as MIC and MBC according to study by Munot et al with minor modifications

• Formulation of therapeutic quercetin chitosan gel (QCG) using chitosan and QLC nanoparticles-

An in-situ quercetin chitosan gel shall be prepared using suitable polymer post screening from the technique of gel forming injectable depot by Nair et al. The polymeric gelling agent shall be weighed to achieve a desired porosity and shall be placed for overnight mixing on magnetic stirrer. Post obtaining clear solution, the nanoparticles shall be added and will be mixed thoroughly. The gel will be allowed to set at room temperature and further gelation characteristics shall be studied.

• Anti-inflammatory activity assessment of QCG

In vivo study

The double blinded randomized controlled clinical study will be conducted from subjects selected from outpatient Department of Periodontology in the respective college. The selected 45 subjects to be divided into three equal groups (Group I, Group II and Group III). All the subjects meeting the inclusion criteria and willing to participate in the study with duly signed consent forms will be enrolled for the study. Group 1 and Group 2 subjects will be of chronic periodontitis and Group 3 subjects will be systemically healthy subjects without chronic gingivitis and periodontitis.

Group I:  After baseline UWS sampling & clinical parameters ultrasonic scaling, polishing and root planing with appropriate gracey curettes in a single appointment to be carried out in fifteen subjects. No chemotherapeutic agents to be prescribed after treatment. This is followed by subgingival local drug delivery administration of of quercetin chitosan gel (QCG) into the selected site.

Group II: After baseline UWS sampling & clinical parameters ultrasonic scaling, polishing & root planing with appropriate gracey curettes in a single appointment to all to be carried out in fifteen subjects. This is followed by sub gingival local drug delivery administration of placebo gel into the selected site

Group III:  Fifteen Healthy subjects after a session of scaling and polishing will be subjected to UWS sampling

Both UWS sampling and assessment of clinical parameters to be recorded preoperatively at baseline (0 day) and post operatively at 45th day for Group I and Group II.

The analysis and interpretation of results of the clinical study by Enzyme linked immunoassay will be conducted at Department of Biotechnology and Bioinformatics in respective college.

Unstimulated salivary sampling assay to be evaluated by ELISA

Data collected will be compiled on to a MS Office excel worksheet and will be subjected to statistical analysis using an appropriate package like SPSS software.

Expected outcome: Successful formulation of nanoparticles

Antimicrobial and antioxidant properties of nanoparticles

Successful formulation of quercetin chitosan nanogel and antiplaque effect of quercetin chitosan gel.