Layered nanoemulsions as mucoadhesive buccal systems for controlled delivery of oral cancer therapeutics

Abstract

Oral cavity and oropharyngeal cancers are considered the eighth most common cancer worldwide, with relatively poor prognosis (62% of patients surviving 5 years, after diagnosis). The aim of this study was to develop a proof-of-concept mucoadhesive lozenge/buccal tablet, as a potential platform for direct sustained delivery of therapeutic antimitotic nanomedicines. Our system would serve as an adjuvant therapy for oral cancer patients undergoing full-scale diagnostic and operative treatment plans. We utilized lipid-based nanocarriers, namely nanoemulsions (NEs), containing mixed-polyethoxylated emulsifiers and a tocopheryl moiety-enriched oil phase. Prototype NEs, loaded with the proapoptotic lipophilic drug genistein (Gen), were further processed into buccal tablet formulations. The chitosan polyelectrolyte solution overcoat rendered NE droplets cationic, by acting as a mucoadhesive interfacial NE layer. With approximate size of 110 nm, the positively charged chitosan-layered NE (+25 mV) vs negatively charged chitosan-free/primary aqueous NE (-28 mV) exhibited a controlled-release profile and effective mucoadhesion for liquid oral spray prototypes. When punch-pressed, porous NE-based buccal tablets were physically evaluated for hardness, friability, and swelling in addition to ex vivo tissue mucoadhesion force and retention time measurements. Chitosan-containing NE tablets were found equivalent to primary NE and placebo tablets in compression tests, yet significantly superior in all ex vivo adhesion and in vitro release assays (P≤0.05). Following biocompatibility screening of prototype chitosan-layered NEs, substantial anticancer activity of selected cationic Gen-loaded NE formulations, against two oropahryngeal carcinomas, was observed. The data strongly indicate the potential of such nanomucoadhesive systems as maintenance therapy for oral cancer patients awaiting surgical removal, or postresection of identified cancerous lesions.

Keywords: chitosan; genistein; isoflavone; squamous cell carcinomas.

Figure 1

Formulation process of genistein-loaded mucoadhesive buccal tablets. Notes: Representative scheme of preparation of chitosan-coated NEs (incorporating Gen as the active ingredient), which is pressed with excipients into solid tablets, following flow filtration and lyophilization. Included graphs demonstrate (A) average particle diameter (nm) and (B) surface charge (ζ-potential in mV), obtained for the prototype layered NE. Abbreviations: Gen, genistein; HAc, acetic acid; NE, nanoemulsion; PUFA, polyunsaturated fatty acid; TFF, tangential flow filtration.

Figure 2

Change in (A) mean droplet size and (B) electrical charge (measured as ζ-potential), of the various NE formulations, with the increasing concentration of cationic chitosan solution (0–0.5 wt%) as the external layer, at pH 5.7. Notes: Values are shown ± SD. n=6. Abbreviations: NE, nanoemulsion; SD, standard deviation; SHS15, Solutol^® HS; T80, Tween^® 80; TPGS, tocopheryl polyethylene glycol succinate.

Figure 3

Physical stability of the different test NE formulations. Notes: (A) The relation between formulation stability (indicated via sudden dilution turbidimetry %, and CI and overall HLB value, calculated for the surfactant blends (0, 40:60, 60:40, or 100 wt%) present in various prepared NE formulations, of both types – primary/AQ or layered/Chito NE. Values are shown with ± SD. Mean values with unlike superscripts (*, #, §, †, ‡) are statistically different (P≤0.05) n=4–6). (B) Photographs of chitosan-coated chia seed oil-based NEs after 72 hours of shelf-storage at room temperature: (1) NE with SHS15 as emulsifier at 100 wt%; (2) NE with SHS15/TPGS emulsifier at 60/40 wt%; (3) NE with SHS 15/TPGS emulsifier at 40/60 wt%; (4) NE with T80 as emulsifier at 100 wt%; (5) NE with T80/TPGS emulsifier at 60/40 wt%; (6) NE with T80/TPGS emulsifier at 40/60 wt%; and (7) NE with TPGS as emulsifier at 100 wt%. Abbreviations: AQ, aqueous; Chito, chitosan; CI, creaming index; HLB, hydrophilic–lipophilic balance; NE, nanoemulsion; SD, standard deviation; SHS15, Solutol^® HS-15; T80, Tween^® 80; TPGS, tocopheryl polyethylene glycol succinate.

Figure 4

Physicochemical characterization of model layered genistein-NE buccal formulations. Notes: (A) Stability results based on average particle size and electrical charge of selected layered NE liquid platforms, determined over the period of 90 days. (B) Postreconstitution measurements of both droplet size and surface charge, for lyophilized prototype-layered NE liquid formulations. In vitro release profile of lead Gen-loaded layered buccal NE formulations, as (C) liquid spray platform and (D) pressed into solid tablet form. Values are shown with ± SD. Mean values with unlike superscripts (*, †) are statistically different. (P≤0.05). n=5–7. Abbreviations: AQ, aqueous; Chito, chitosan; Gen, genistein; NE, nanoemulsion; SD, standard deviation; Sol, solution; SHS15, Solutol^® HS-15; T80, Tween^® 80; TPGS, tocopheryl polyethylene glycol succinate.

Figure 5

Physical characterization of prototype mucoadhesive Gen-loaded NE solid buccal tablets. Notes: (A) Ex vivo mucoadhesive force data, measured for both liquid and solid tablet dosage forms, either drug-free or Gen-incorporated into primary(AQ) or layered (Chito) NEs. (B) Ex vivo mucoadhesion residence time measured using a USP 2 dissolution apparatus, for both empty or Gen-loaded NE buccal tablets. (C) Qualitative photographs showing the physical appearance of selected primary (AQ) and Chito-layered NE-based tablets, immediately following the mucoadhesive force testing. C1, PBS (MCC+dextrose) Placebo; C2, Gen-T80/TPGS-Chito; C3, Gen-SHS15/TPGS-Chito; C4, Gen-T80/TPGS-AQ; C5, Gen-SHS15/TPGS-AQ. Values are shown with ± SD. Mean values with unlike superscripts (*, #, ‡, †) are statistically different (P≤0.05). n=5–6. Abbreviations: AQ, aqueous; Chito, chitosan; Gen, genistein; NE, nanoemulsion; PBS, phosphate-buffered saline; SD, standard deviation; SHS15, Solutol^® HS-15; T80, Tween^® 80; TPGS, tocopheryl polyethylene glycol succinate; USP 2, United States Pharmacopiea type-2.

Figure 6

Biocompatibility assay of primary and layered NEs. Notes: Cell viability assay for lead Chito-layered NE formulations, compared with primary aqueous NEs – employing untreated culture media as negative control – all coincubated with (A) murine areolar fibroblast (L929) cells. Values are shown with ± SE. n=4–5. (B) Qualitative transmitted light micrographs demonstrating the morphology of unstained L929 cells, following 24-hour coincubation with various treatments: (1) negative control serum-free EMEM medium, (2) T80/TPGS-Chito NE, (3) T80/TPGS-AQ NE, (4) SHS15/TPGS-Chito NE, and (5) SHS15/TPGS-AQ NE. The selected liquid NEs were added to confluent cells (as two hundred fold dilutions) in serum-free EMEM medium. Abbreviations: AQ, aqueous; EMEM, Eagle’s minimal essential medium; NE, nanoemulsion; SE, standard error; SHS15, Solutol^® HS-15; T80, Tween^® 80; TPGS, tocopheryl polyethylene glycol succinate.

Figure 7

In vitro anticancer activity of prototype Gen-layered NEs against oral carcinomas. Notes: Concentration-dependent cytotoxicity profiles of different Gen-loaded cationic/Chito-layered NEs compared with corresponding primary (AQ) NE controls, in (A) FaDu (pharyngeal) and (B) SCC-4 (tongue, human squamous cell carcinoma) cells. Plus, temporal cytotoxicity profile (over 48 hours of coincubation, at 1 mM equivalent Gen concentration), in test oral cancer cell lines, FaDu (C) and SCC-4 (D). Mean values with unlike superscripts (*, ‡, †) are statistically different (P≤0.05). n=4–6. Abbreviations: AQ, aqueous; Chito, chitosan; equiv conc, equivalent concentration; Gen, genistein; NE, nanoemulsion; SHS15, Solutol^® HS-15; T80, Tween^® 80; TPGS, tocopheryl polyethylene glycol succinate.