In Vitro Anticancer Activity of Mucoadhesive Oral Films Loaded with Usnea barbata (L.) F. H. Wigg Dry Acetone Extract, with Potential Applications in Oral Squamous Cell Carcinoma Complementary Therapy

Abstract

Oral squamous cell carcinoma (OSCC) is the most frequent oral malignancy, with a high death rate and an inadequate response to conventional chemotherapeutic drugs. Medical research explores plant extracts’ properties to obtain potential nanomaterial-based anticancer drugs. The present study aims to formulate, develop, and characterize mucoadhesive oral films loaded with Usnea barbata (L.) dry acetone extract (F-UBA) and to investigate their anticancer potential for possible use in oral cancer therapy. U. barbata dry acetone extract (UBA) was solubilized in ethanol: isopropanol mixture and loaded in a formulation containing hydroxypropyl methylcellulose (HPMC) K100 and polyethylene glycol 400 (PEG 400). The UBA influence on the F-UBA pharmaceutical characteristics was evidenced compared with the references, i.e., mucoadhesive oral films containing suitable excipients but no active ingredient loaded. Both films were subjected to a complex analysis using standard methods to evaluate their suitability for topical administration on the oral mucosa. Physico-chemical and structural characterization was achieved by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and atomic force microscopy (AFM). Pharmacotechnical evaluation (consisting of the measurement of specific parameters: weight uniformity, thickness, folding endurance, tensile strength, elongation, moisture content, pH, disintegration time, swelling rate, and ex vivo mucoadhesion time) proved that F-UBAs are suitable for oral mucosal administration. The brine shrimp lethality (BSL) assay was the F-UBA cytotoxicity prescreen. Cellular oxidative stress, caspase 3/7 activity, nuclear condensation, lysosomal activity, and DNA synthesis induced by F-UBA in blood cell cultures and oral epithelial squamous cell carcinoma (CLS-354) cell line were investigated through complex flow cytometry analyses. Moreover, F-UBA influence on both cell type division and proliferation was determined. Finally, using the resazurin-based 96-well plate microdilution method, the F-UBA antimicrobial potential was explored against Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27353, Candida albicans ATCC 10231, and Candida parapsilosis ATCC 22019. The results revealed that each UBA-loaded film contains 175 µg dry extract with a usnic acid (UA) content of 42.32 µg. F-UBAs are very thin (0.060 ± 0.002 mm), report a neutral pH (7.01 ± 0.01), a disintegration time of 146 ± 5.09 s, and an ex vivo mucoadhesion time of 85 ± 2.33 min, and they show a swelling ratio after 6 h of 211 ± 4.31%. They are suitable for topical administration on the oral mucosa. Like UA, they act on CLS-354 tumor cells, considerably increasing cellular oxidative stress, nuclear condensation, and autophagy and inducing cell cycle arrest in G0/G1. The F-UBAs inhibited the bacterial and fungal strains in a dose-dependent manner; they showed similar effects on both Candida sp. and higher inhibitory activity against P. aeruginosa than S. aureus. All these properties lead to considering the UBA-loaded mucoadhesive oral films suitable for potential application as a complementary therapy in OSCC.

ROS-Induced DNA-Damage and Autophagy in Oral Squamous Cell Carcinoma by Usnea barbata Oil Extract—An In Vitro Study

“…Data registered in the first level of Table 1 show the microdilutions [ 96 ] of standard antibiotic (CTR), antifungal (TRF) [ 46 ], P407 [ 44 ], and oil samples (UBO and CNO). All CTR microdilutions are over minimum inhibitory concentrations (MICs) against S. aureus and P. aeruginosa ; MIC is the minimal concentration of an antimicrobial agent that will inhibit the visible growth of a microorganism after 24 h of incubation [ 97 ].…”

“…Promega Corporation (Madison, WI, USA) produced 4 mg/mL RNase A. The following kits: EdU i-Fluor 488, Reactive Oxygen Species Detection Assay Kit, and Magic Red ® Caspase-3/7 Assay Kit were supplied by Abcam Plc (Cambridge, UK) [ 46 ].…”

“…U. barbata was harvested in March 2021 from the Călimani Mountains—900 m altitude and the following coordinates: 47°29′ N, 25°12′ E [ 46 ]. The Department of Pharmaceutical Botany (Faculty of Pharmacy, Ovidius University of Constanta) identified it through standard methods.…”

“…This study platform was the Attune™ Acoustic focusing cytometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) [ 165 ]. The Attune™ cytometric software version (v.) 1.2.5 (Thermo Fisher Scientific Inc.,Waltham, MA, USA) collected and processed the flow cytometry data [ 46 ]. All flow cytometry measurements and fluorescent microscopy images were achieved using suitable kits (mentioned in Section 4.1 ) in the previously detailed conditions [ 44 ].…”

“…Therefore, they are considered important bioactive representatives with pharmaceutical applications [ 37 ] as a potential source of anticancer [ 38 , 39 , 40 ] and antibiotic medicines [ 41 , 42 , 43 ]. Recently, a few authors performed lichens incorporation in different pharmaceutical formulations [ 44 , 45 , 46 , 47 , 48 , 49 ].…”