Evaluation of Usnea barbata (L.) Weber ex F.H. Wigg Extract in Canola Oil Loaded in Bioadhesive Oral Films for Potential Applications in Oral Cavity Infections and Malignancy

Usnea lichens are known for their beneficial pharmacological effects with potential applications in oral medicine. This study aims to investigate the extract of Usnea barbata (L.) Weber ex F.H. Wigg from the Călimani Mountains in canola oil as an oral pharmaceutical formulation. In the present work, bioadhesive oral films (F-UBO) with U. barbata extract in canola oil (UBO) were formulated, characterized, and evaluated, evidencing their pharmacological potential. The UBO-loaded films were analyzed using standard methods regarding physicochemical and pharmacotechnical characteristics to verify their suitability for topical administration on the oral mucosa. F-UBO suitability confirmation allowed for the investigation of antimicrobial and anticancer potential. The antimicrobial properties against Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27353, Candida albicans ATCC 10231, and Candida parapsilosis ATCC 22019 were evaluated by a resazurin-based 96-well plate microdilution method. The brine shrimp lethality assay (BSL assay) was the animal model cytotoxicity prescreen, followed by flow cytometry analyses on normal blood cells and oral epithelial squamous cell carcinoma CLS-354 cell line, determining cellular apoptosis, caspase-3/7 activity, nuclear condensation and lysosomal activity, oxidative stress, cell cycle, and cell proliferation. The results indicate that a UBO-loaded bioadhesive film’s weight is 63 ± 1.79 mg. It contains 315 µg UBO, has a pH = 6.97 ± 0.01, a disintegration time of 124 ± 3.67 s, and a bioadhesion time of 86 ± 4.12 min, being suitable for topical administration on the oral mucosa. F-UBO showed moderate dose-dependent inhibitory effects on the growth of both bacterial and fungal strains. Moreover, in CLS-354 tumor cells, F-UBO increased oxidative stress, diminished DNA synthesis, and induced cell cycle arrest in G0/G1. All these properties led to considering UBO-loaded bioadhesive oral films as a suitable phytotherapeutic formulation with potential application in oral infections and neoplasia.


Formulation and Preparation of the UBO-Loaded Bioadhesive Oral Films
The U. barbata extract in canola oil (UBO) was obtained using a method adapted from that described by Basiouni et al. [29], from 20.2235 g dried and ground lichen and 500 mL cold-pressed canola seed oil [26] in darkness, at room temperature (21)(22) • C). The container with both components was shaken daily for three months; then, UBO was filtered in another brown vessel with a sealed plug and preserved in a plant room, sheltered from sun rays. Both oil samples had a pH of 4.
For the development of the bioadhesive oral films containing U. barbata extract in canola oil (F-UBO), HPMC K100 (with a viscosity of 100 mPa) was selected as the filmforming polymer. It displays excellent hydrophilicity, water-absorbing ability, good biocompatibility, and biodegradability [30][31][32]. PEG 400 was included in the films' formulations as an external plasticizer for its high hydrophilic character and non-toxicity [33].
The non-ionic surfactant poloxamer 407 (P407) was chosen for emulsifying the oily phase to ensure the uniform incorporation of UBO in the polymer matrix.
Bioadhesive films, containing suitable excipients but no active ingredient load, were prepared and used as a reference (R) to prove the UBO activity and influence on the F-UBO pharmaceutical characteristics.
The UBO amount was selected according to the emulsifying ability of the poloxamer 407 (P407) and film-forming polymer. HPMC was weighed using a Mettler Toledo AT261 balance (Marshall Scientific, Hampton, NH, USA) with 0.01 mg sensitivity for the polymeric matrix system. Then, it was dispersed in water by stirring at 700 rpm and room temperature, using an MR 3001K magnetic stirrer (Heidolph Instruments GmbH & Co. K.G., Schwabach, Germany) and mixed with PEG 400. P407 was dissolved in the matrix, and UBO was included under continuous stirring for 1 h.
References (R) were realized by mixing P407 with the base system, and the formed gels were left overnight at room temperature for deaeration. The viscous dispersions were poured in a thin layer into Petri glass plates and dried in ambient conditions for 24 h. Finally, the dried films were peeled off the plate surface and cut into 1.5 × 2 cm patches.
The manufacturing process led to defining the concentration of UBO in the film formulation: F-UBO encloses 315 µg UBO.

Physico-Chemical Analysis of Bioadhesive Oral Films 2.3.1. SEM Analysis
A scanning electron microscope (SEM) in a high-resolution Quanta3D FEG (Thermo Fisher Scientific, GmbH, Dreieich, Germany) was used to investigate the bioadhesive oral film morphology.

Atomic Force Microscopy (AFM)
The bioadhesive oral film's morphology was obtained via atomic force microscopy (AFM). The AFM measurements were registered with an AFM XE-100 (Park Systems Corporate, Suwon, Korea) assisted with flexure-guided, crosstalk eliminated scanners in non-contact mode to minimize the tip-sample interaction. AFM images were registered with sharp tips (PPP-NCLR, from NANOSENSORS™, Neuchatel, Switzerland) having the following characteristics: less than 10 nm radius of curvature, 225 mm mean length, 38 mm mean width,~48 N/m force constant, and a resonance frequency of 190 kHz. An XEI program (v 1.8.0-Park Systems Corporate, Suwon, Korea) was carried out to process the AFM images and to evaluate the roughness. The surface profile of the scanned samples (the dimensions of the selected particles indicated with red arrows along the selected line) shows the representative line scans presented below the AFM images in the so-called "enhanced contrast" mode.

FTIR Analysis
The infrared spectra of the materials (obtained as bioadhesive films) were registered using FTIR equipment (Nicolet Spectrometer 6700 FTIR from Thermo Electron Corporation, Waltham, MA, USA) assisted with a diamond-crystal ATR accessory. In transmittance mode, data were acquired in the spectral range of 400-4000 cm −1 (resolution of 4 cm −1 and a total of 32 scans per spectrum).

X-ray Diffraction Patterns
A Rigaku Ultima IV diffractometer (Rigaku Corporation, Tokyo, Japan) in parallel beam geometry was used to investigate the X-ray diffraction (XRD) patterns of bioadhesive films. A step size of 0.02 and a 2 • (2θ)/min speed over 10-80 • were used. The source of the X-rays was a CuKα tube (λ = 1.54056 Å) operating at 40 kV and 30 mA.

Thermogravimetric Analysis
The thermal investigations were conducted on a Mettler Toledo TGA/SDTA851e thermogravimetric analyzer (Mettler-Toledo GmbH, Greifensee, Switzerland). The nonisothermal measurements were performed in the 25-600 • C temperature range, under 80 mL min −1 synthetic air atmosphere at a constant heating rate of 10 • C min −1 .

Weight Uniformity
The weight uniformity was evaluated on 20 bioadhesive oral films of each formulation (F-UBO and R). They were individually weighed, and the average weight was determined.

Thickness
This parameter was also measured on 20 bioadhesive films of each type (F-UBO and R) using a Yato digital micrometer (Yato China Trading Co., Ltd., Shanghai, China) with a measuring range of 0-25 mm and a resolution of 0.001 mm. Then, the mean value was calculated.

Folding Endurance
The bioadhesive films were repeatedly folded and rolled until they broke, or up to 300 times [5]. The folding times were registered and expressed as folding endurance values.

Tensile Strength and Elongation Ability
The film's tensile strength and elongation ability were determined using an L.R. 10K Plus digital tensile force tester for universal materials (Lloyd Instruments Ltd., West Sussex, United Kingdom). The analysis was performed from a 30 mm distance with a speed of 30 mm/min. Therefore, each film was placed in a vertical position between the two braces, and the breakage force was registered. The measurements were achieved in triplicate. The following equations (Equations (1) and (2)) were used to calculate the tensile strength and the elongation at break: Tensile strength kg/mm 2 = Force at breakage (kg) Film thickness (mm) − Film width (mm) (1) Elongation % = Increase in film length Initial film length × 100

Moisture Content
The moisture content was assessed as the loss on drying by the thermogravimetric method using an H.R. 73 halogen humidity analyzer (Mettler-Toledo GmbH, Greifensee, Switzerland) [6]. Five bioadhesive oral films of each formulation (F-UBO and R) were analyzed for moisture content determination.

Surface pH
Five films of each formulation (F-UBO and R) were moistened with 1 mL of distilled water (pH 6.5 ± 0.5) for 5 min at room temperature. Then, the pH value was measured with the CONSORT P601 pH-meter (Consort bvba, Turnhout, Belgium).

In Vitro Disintegration Time
The time required to disintegrate the F-UBO and R bioadhesive oral films, with no residual mass completely, was measured in simulated saliva phosphate buffer pH of 6.8 at 37 ± 2 • C, using an Erweka DT 3 apparatus (Erweka ® GmbH, Langen, Germany) [34].

Swelling Rate
Six films of each formulation (F-UBO and R) were placed on 1.5% agar gel in Petri plates and incubated at 37 ± 1 • C. Every 30 min, for 6 h, the patches were weighed. The swelling rate was calculated according to Equation (3): where w t is the patch weight at time t after the incubation and w i is the initial weight [35][36][37][38].

Ex Vivo Bioadhesion Time
The bioadhesion time [39] was measured through the method described by Gupta et al. [40] on a detached porcine buccal mucosa by removing the fat layer and any residual tissue. The buccal mucosa was washed with distilled water and a phosphate buffer (pH 6.8) at 37 • C and fixed on a glass plate. Each bioadhesive film was hydrated in the center with 15 µL phosphate buffer and brought on the mucosa surface by pressing it for 30 s. The glass plate was placed in 200 mL phosphate buffer pH 6.8 and maintained at 37 • C for 2 min. The suitable simulation of the oral cavity conditions was ensured using a paddle with a stirring rate of 28 rpm. Then, the necessary time for the entire film's erosion or detachment from the buccal mucosa surface was recorded [41]. This registered time represents the film's residence time [42] on the oral mucosa, known as a bioadhesion time [43]. This test was realized in triplicate.

Inoculum Preparation
The bacterial inoculum was prepared by the direct colony suspension method [44]. Thus, bacterial colonies selected from a 24 h agar plate were suspended in M.H.A. medium, according to the 0.5 McFarland standard, measured at Densimat Densitometer (Biomerieux, Marcy-l'Étoile, France) with around 108 CFU/mL (CFU = colony-forming unit), The yeast inoculum was prepared using the same method, adjusting the RPMI 1640 with fungal colonies to the 1.0 McFarland standard, with 10 6 CFU/mL.

Samples and Standards
F-UBO was dissolved in 1 mL of diluted phosphate buffer. As standards, ceftriaxone (Cefort 1g Antibiotice SA, Iasi, Romania) solutions 30 mg/mL and 122 mg/mL in distilled water were used for bacteria. The Cefort powder was weighted at Partner Analytical balance (Fink & Partner GmbH, Goch, Germany) and dissolved in distilled water. Terbinafine solution 10.1 mg/mL (Rompharm Company SRL, Otopeni, Romania) was used as standard for Candida sp. As a positive control for antimicrobial activity evaluation, 5% P407 was selected, the emulsifier used for the UBO-loaded bioadhesive oral films formulation.

Microdilution Method
All successive steps were performed in an Aslair Vertical 700, laminar flow, microbiological protection cabinet (Asal Srl, Cernusco (MI), Italy). In four 96-well plates, we performed seven serial dilutions, adapting the protocol described by Fathi et al. [45].
All 96-well plates were incubated for 24 h at 37 • C for bacteria and 35 • C for yeasts in a My Temp mini Z763322 Digital Incubator (Benchmark Scientific Inc., Sayreville, NJ, USA).

Reading and Interpreting
After 24 h incubation, the plates were examined with a free eye to see the color differences between standard and samples [46]. The corresponding sample concentration activities were compared with the Standard antibiotic ones. For yeasts, the color chart of the resazurin dye reduction method was used [47,48].

Evaluation of UBO-Loaded Bioadhesive Oral Films Cytotoxicity on Animal Model
Aiming to evaluate the F-UBO cytotoxicity, we used Artemia salina as an animal model, adapting a previously described method [49].
F-UBO film was placed in a diluted buffer (1 mL) and incubated for 15 min at 37 • C, resulting in a homogenous dispersion.

Brine Shrimp Lethality Assay
The larvae were obtained from A. salina cysts through continuous light and aeration in a 0.35% saline solution at 20 • C. The brine shrimp larvae in the first stage (instar I) were introduced in 0.3% saline solution into experimental pots (with a volume of 1 mL) [26]. The analysis was compared to a blank (untreated nauplii) to obtain accurate results regarding the F-UBO cytotoxic effect. An amount of 5% P407 in water was a positive control. The nauplii have embryonic energy reserves as lipids, and they were not fed during the test, thus avoiding interference with the sample and positive control. Their evolution was analyzed after 24 h and 48 h, exploring the morphological changes induced by F-UBO and P407 [50,51].

Fluorescent Microscopy
The brine shrimp larvae were stained with 3% acridine orange (Merck Millipore, Burlington, MA, USA) for 5 min. The samples were subjected to drying for 15 min in darkness and placed on the microscope slides.
Fluorescent microscopy images were achieved using an OPTIKA B-350 microscope (Ponteranica, BG, Italy) blue filter (λex = 450-490 nm; λem = 515-520 nm) and green filter The present study platform for in vitro F-UBO cytotoxicity analysis was the Attune Acoustic focusing cytometer (Applied Biosystems, Bedford, MA, USA). Before cell analysis, the flow cytometer was first set by fluorescent beads-Attune performance tracking beads, labeling, and detection (Life Technologies, Europe BV, Bleiswijk, The Netherlands), with standard size (four intensity levels of beads population). The cell amount was established by counting cells below 1 µm [52]. Using forward scatter (FSC) and side scatter (SSC), more than 10,000 cells per sample for each analysis were gated.

Human Blood Cell Cultures
The blood samples were collected into heparin vacutainers, and the blood cell cultures were obtained according to a previously described method [53]. Then, the blood cells were treated with F-UBO and controls in Nunclon Vita Cell culture 6-well plates (Kisker Biotech GmbH & Co.KG, Steinfurt, Germany) and incubated in a Steri-Cycle™ i160 CO 2 Incubator (Thermo Fisher Scientific Inc., Waltham, MA, USA), at 37 • C, in 5% CO 2 for 24 h. All flow cytometry analyses were performed after this incubation time.

CLS-354 Cell Line
The human mouth squamous cell carcinoma cell line CLS-354 (CLS catalog number 300152) consists of epithelial cells established in vitro from the primary squamous carcinoma of a 51-year-old Caucasian male. The CLS-354 cells [54] were cultured in DMEM high glucose with 10% FBS, supplemented with antibiotic mix solution in humidity conditions of 5% CO 2 at 37 • C for 7 days. The cells were dissociated from the monolayer with trypsin-EDTA, centrifugated at 3000 rpm for 10 min in a Fisher Scientific GT1 centrifuge (Thermo Fisher Scientific Inc., Waltham, MA, USA), and distributed in Millicell™ 24-well cell culture microplates (Thermo Fisher Scientific Inc., Waltham, MA, USA). After treatment, they were incubated for 24 h in the same conditions. All the flow cytometry analyses were performed after this incubation period.

Samples and Control Solutions
F-UBOs were dissolved in the suitable culture media for both types of cells, with 1% DMSO. As positive controls, 5% P407 and usnic acid of 125 ug/mL in 1% DMSO were used, and as a negative control, 1% DMSO.

Annexin V-FITC Apoptosis Assay
The cells with annexin V-FITC and PI (20 µg/mL) were incubated in darkness, for 30 min, at room temperature [53]. Then, the viable, early apoptotic, late apoptotic, and necrotic cells were examined at a flow cytometer using a 488 nm excitation, green emission for annexin V-FITC (BL1 channel), and orange emission for PI (BL2 channel).

Evaluation of Nuclear Condensation and Lysosomal Activity
The cells were stained successively with Hoechst 33,342 and AO and incubated for 30 min at room temperature in darkness [53]. Then, they were examined at the flow cytometer, using UV excitation and blue emission for Hoechst 33,342 (VL2) at 488 nm and green emission for acridine orange (BL1 channel).

Cell Cycle Analysis
The cells with PI (20 µg/mL) and RNase A (30 µg/mL) were incubated at room temperature, into darkness, for 30 min [53]. Next, the cell cycle distribution was detected by flow cytometry, using a 488 nm excitation and orange emission for PI (BL2 channel) [56].

Evaluation of Total ROS Activity
ROS Assay Stain solution was well-mixed with cell cultures and incubated at 37 • C for 60 min [53]. Then, the cells were analyzed by flow cytometry, using a 488 nm excitation and green emission for ROS (BL1 channel).

Evaluation of Cell Proliferation
The cell cultures were incubated for 2 h with 50 µM EdU (500 µL) at 37 • C. Following a succession of previously described steps [53], they were prepared for flow cytometry examination at a 488 nm excitation and green emission for EdU-iFluor 488 (BL1).

Data Analysis
All analyses were effectuated in triplicate, and the data were registered as means values ± standard deviation (SD). The results are expressed as percent (%) in the case of cell apoptosis, caspase-3/7 activity, nuclear condensation, autophagy, cell cycle arrest, and DNA synthesis, and count (×10 4 ) for ROS levels. Data analysis was realized with SPSS v. 23 software, IBM, 2015. Paired t-test established the differences between F-UBO and controls, and p < 0.05 was considered statistically significant. The principal component analysis was performed with XLSTAT 2022.2.1. by Addinsoft (New York, NY, USA) and examined the correlations between variable parameters.

Organoleptic Characteristics of Bioadhesive Oral Films
The organoleptic characteristics of the F-UBO and R films depend highly on the active ingredient state. Both bioadhesive films (R and F-UBO) are white, with the typical appearance of emulsified systems (Figure 1a,b). The films withstand normal handling and cutting processes without air bubbles, cracks, or imperfections. All formulations lead to homogenous, thin, and easy-to-peel bioadhesive oral films, with a uniform, smooth, and glossy surface (Figure 1a

Morphology
Scanning electron microscopy (SEM) was performed to study the morphology of the bioadhesive films (Figure 1c

Atomic Force Microscopy
The AFM images are displayed in Figure 1e,f. The reference (R) is corrugated, with a surface exhibiting large protruding particles (ranging from tens of nm up to microns-for example, see the upper, middle-left elongated particle in Figure 1e). Therefore, R is characterized by a higher global RMS roughness of 8.5 nm and an Rpv parameter of 804.5 nm. The line scan exhibits a vertical gradient (∆z level difference) of~180 nm and more prominent surface features (Figure 1e). F-UBO displays a rougher surface, having an RMS roughness of 98.5 nm and a peakto-valley parameter of 480.6 nm (Figure 1f). The compact morphology is maintained similar to that of R; however, deep grooves and cavities are seen in the image, such as the one imaged along the red line, which is more than 300 nm deep. The surface features (such as pits, cavities, and grooves) create a clear surface corrugation, which could enhance the bioadhesive films' adherence to the targeted tissue.

FTIR Spectra
The FTIR spectra of R and F-UBO are illustrated in Figure 2. The literature data showed that the main absorption peaks characterize the FTIR spectrum of P407 at 2893 cm -1 due to C-H stretch aliphatic, 1355 cm -1 corresponding to in-plane O-H bend, and 1124 cm -1 due to C-O stretch [57]. In addition, the FTIR spectrum of pure HPMC shows an absorption band at 3444 cm −1 assigned to the stretching The prominent peaks of P407 and pure HPMC were shifted in R and F-UBO films due to the formation of bioadhesive film [58,59]. The main FTIR peaks of UBO [60] are superposed to the peaks of the polymer matrix.
On the other hand, the spectra exhibit the νO-H stretching vibration detected at 3460 cm −1 and νsim CH2 at 2853 cm −1 , characteristic of P407. The band observed at 1050 cm −1 was assigned to the C-O group [61] (Figure 2). These findings are in accord with the assumption that F-UBO bioadhesive films are formed through UBO dispersion in the polymer matrix.

X-ray Diffractograms
The X-ray diffractograms of bioadhesive oral films are presented in Figure 3a. The literature data showed that the main absorption peaks characterize the FTIR spectrum of P407 at 2893 cm -1 due to C-H stretch aliphatic, 1355 cm -1 corresponding to in-plane O-H bend, and 1124 cm -1 due to C-O stretch [57]. In addition, the FTIR spectrum of pure HPMC shows an absorption band at 3444 cm The prominent peaks of P407 and pure HPMC were shifted in R and F-UBO films due to the formation of bioadhesive film [58,59]. The main FTIR peaks of UBO [60] are superposed to the peaks of the polymer matrix.
On the other hand, the spectra exhibit the νO-H stretching vibration detected at 3460 cm −1 and νsim CH 2 at 2853 cm −1 , characteristic of P407. The band observed at 1050 cm −1 was assigned to the C-O group [61] (Figure 2). These findings are in accord with the assumption that F-UBO bioadhesive films are formed through UBO dispersion in the polymer matrix.

X-ray Diffractograms
The X-ray diffractograms of bioadhesive oral films are presented in Figure 3a. The X-ray diffractograms of R and F-UBO exhibited two peaks at 2θ = 8° and 2θ = 20°. They represent the prominent HPMC XRD peaks, portraying their semicrystalline structure [62,63]. The peak at 2θ = 22° is attributed to P407 [64]. The X-ray diffraction pattern of F-UBO shows higher intensity peaks than the reference, proving the influence of UBO dispersion in the polymer matrix and correlated with the bioadhesive behavior.

Thermogravimetric Analysis
Thermogravimetric analysis coupled with differential thermal analysis was performed to characterize the film's thermal behavior and stability. Both materials (reference and F-UBO films) exhibit a similar behavior upon heating from 25 to 600 °C ( Figure  3b). A 0.8-2.5% weight mass loss occurs on heating up to ~100 °C, which can be associated with the loss of residual solvent and physisorbed water. The decomposition process of the organic compounds takes place in two distinct steps, between 200-400 °C and 400-550 °C. Each decomposition step is accompanied by an exothermic thermal effect ( Figure 3b). The mass losses associated with the solvent loss and first and second organic decomposition steps are presented in Table 2. Table 2. Thermal parameters for the bioadhesive oral films (F-UBO and R) decomposition in air.  Figure 3b and Table 2 indicate that the first stage of both bioadhesive films (R and F-UBO) starts at a temperature below 100 °C due to the loss of adsorbed water. The second stage begins from 230 °C to 380 °C, corresponding to ~86% (for R) and ~87% (for F-UBO) weight loss. The third stage, with a maximum of 495.3 °C (for R) and 488.8 °C (for F-UBO) was due to the different organic part decomposition. The X-ray diffractograms of R and F-UBO exhibited two peaks at 2θ = 8 • and 2θ = 20 • . They represent the prominent HPMC XRD peaks, portraying their semicrystalline structure [62,63]. The peak at 2θ = 22 • is attributed to P407 [64]. The X-ray diffraction pattern of F-UBO shows higher intensity peaks than the reference, proving the influence of UBO dispersion in the polymer matrix and correlated with the bioadhesive behavior.

Thermogravimetric Analysis
Thermogravimetric analysis coupled with differential thermal analysis was performed to characterize the film's thermal behavior and stability. Both materials (reference and F-UBO films) exhibit a similar behavior upon heating from 25 to 600 • C ( Figure 3b). A 0.8-2.5% weight mass loss occurs on heating up to~100 • C, which can be associated with the loss of residual solvent and physisorbed water. The decomposition process of the organic compounds takes place in two distinct steps, between 200-400 • C and 400-550 • C. Each decomposition step is accompanied by an exothermic thermal effect ( Figure 3b). The mass losses associated with the solvent loss and first and second organic decomposition steps are presented in Table 2. Table 2. Thermal parameters for the bioadhesive oral films (F-UBO and R) decomposition in air.

Film Solvent Mass Loss (%) T ( • C)/Mass Loss 1st Decomposition Step (%) T ( • C)/Mass Loss 2nd Decomposition Step (%)
R-reference (films without UBO); F-UBO-UBO-loaded bioadhesive oral film; UBO-U. barbata extract in canola oil. Figure 3b and Table 2 indicate that the first stage of both bioadhesive films (R and F-UBO) starts at a temperature below 100 • C due to the loss of adsorbed water. The second stage begins from 230 • C to 380 • C, corresponding to~86% (for R) and~87% (for F-UBO) weight loss. The third stage, with a maximum of 495.3 • C (for R) and 488.8 • C (for F-UBO) was due to the different organic part decomposition.

Pharmacotechnical Evaluation of Bioadhesive Oral Films
The results of pharmacotechnical evaluation of F-UBO and R are presented in Table 3. The film's weight varies depending on the active ingredient state and dispersion method. No significant differences are registered between the UBO-loaded films and the reference films (63 ± 1.79 vs. 62 ± 3.27 mg, p > 0.05). The swelling rate over 6 h is presented in Figure 4.
3. The film's weight varies depending on the active ingredient state and dispersion method. No significant differences are registered between the UBO-loaded films and the reference films (63 ± 1.79 vs. 62 ± 3.27 mg, p > 0.05). The swelling rate over 6 h is presented in Figure 4.  indicates that the swelling index increases linearly in the first 4 h (approximately 20% to every 30 min); then, the growth becomes slower, the differences between 330 and 360 min being insignificant. No films were eroded after 6 h, and no swelling could be detected after this time. With the oily active ingredient emulsified in the matrix, F-UBO displays a lower swelling behavior than R due to UBO's state and dispersion (Table 3).

Antimicrobial Activity
Data registered in Table 4 show the standard antibiotic (CTR), antifungal drug (TRF), P407, and F-UBO initial concentrations and microdilutions (mg/mL). The results obtained after 24 h incubation at 37 • C are displayed in Tables S1-S3 from the Supplementary Material.
Data from Table S1 indicate that the color showed by the standard antibiotic correlates with its inhibiting power and varies in a manner directly proportional to its concentration. CTR induced a "moderate" to "good" inhibition of bacterial strains' growth; the microdilutions [67,68] (Table S1).
In the present study, 5% P407 had inhibitory effects against both bacteria tested (Table S2a- Table S3 shows that terbinafine of [0.500-0.007] mg/mL exhibited the highest antifungal activity, having a fungicidal effect on both Candida sp.
F-UBO inhibited both Candida sp. proliferation. Thus, F-UBO of 3.176 mg/mL had the most significant inhibitory activity on both species, higher on C. albicans than on C. parapsilosis. The following decreasing F-UBO concentrations moderately inhibited the Candida sp. proliferation.
The antifungal activity of P407 is displayed in Table S2e-h. P407 of [2.506-0.078] mg/mL had a significant inhibitory effect, partially inducing the death of both fungal species [48]. The lowest concentration (0.039 mg/mL) similarly affected C. albicans and produced a moderate to fast proliferation of C. parapsilosis [48]. Moreover, Tables S2 and S3 show that 5% P407 had considerably higher inhibitory activity on both Candida sp. than F-UBO.

Evaluation of UBO-Loaded Bioadhesive Oral Films Cytotoxicity on Animal Model
A. salina nauplii were examined under the microscope to detect morphological changes after 24 and 48 h of exposure, compared to a blank and positive control (5% P407). All these data are illustrated in Figure 5.
After 24 h, all larvae were alive, swimming, and showing normally visible movements. However, F-UBO cytotoxicity was revealed after the first 24 h, even if the larvae were alive and apparently normal. Compared to untreated larvae (Figure 5a-d), the images 400× (Figure 5e-h) show the following processes in the early stage: penetration of emulsified lipids into tissues, depletion of cellular structures, and minimal detachment of the cuticle from the terminal portion of the digestive tract. S3 show that 5% P407 had considerably higher inhibitory activity on both Candida sp. than F-UBO.

Evaluation of UBO-Loaded Bioadhesive Oral Films Cytotoxicity on Animal Model
A. salina nauplii were examined under the microscope to detect morphological changes after 24 and 48 h of exposure, compared to a blank and positive control (5% P407). All these data are illustrated in Figure 5.   After 24 h, all larvae were alive, swimming, and showing normally visible movements. However, F-UBO cytotoxicity was revealed after the first 24 h, even if the larvae were alive and apparently normal. Compared to untreated larvae (Figure 5a-d), the images 400× (Figure 5e-h) show the following processes in the early stage: penetration of emulsified lipids into tissues, depletion of cellular structures, and minimal detachment of the cuticle from the terminal portion of the digestive tract. These morphological changes were intensified over the next 24 h, becoming incompatible with the brine shrimp nauplii survival. Hence, after 48 h, 64.81% of larvae were alive, and 11.11% were in the sublethal stage; the registered mortality was 25.92%.
Compared to blank (Figure 5i-l), the exposed larvae (Figure 5m-p) had blocked digestive transit due to accumulated lipids, cell damage with large intercellular spaces, tissue destruction, and massive detachment of the cuticle from larval tissues.
The A. salina larvae were alive and had normal movements after 24 and 48 h exposure at 5% P407. Microscopic examination after 48 h revealed a significant digestive tube volume increase, especially in the superior part, and low penetration of emulsified lipids into tissue (Figure 5v-x). Moreover, at the intracellular level, FM images ( Figure 5A-F) show activated lysosomes in cell death processes in brine shrimp larvae exposed to F-UBO ( Figure 5F). The effects of F-UBO on normal blood cells and CLS-354 tumor cells based on morphology and cell membrane integrity are illustrated in Figure 6.
These morphological changes were intensified over the next 24 h, becoming incompatible with the brine shrimp nauplii survival. Hence, after 48 h, 64.81% of larvae were alive, and 11.11% were in the sublethal stage; the registered mortality was 25.92%.
Compared to blank (Figure 5i-l), the exposed larvae (Figure 5m-p) had blocked digestive transit due to accumulated lipids, cell damage with large intercellular spaces, tissue destruction, and massive detachment of the cuticle from larval tissues.
The A. salina larvae were alive and had normal movements after 24 and 48 h exposure at 5% P407. Microscopic examination after 48 h revealed a significant digestive tube volume increase, especially in the superior part, and low penetration of emulsified lipids into tissue (Figure 5v-x). Moreover, at the intracellular level, FM images ( Figure 5A-F) show activated lysosomes in cell death processes in brine shrimp larvae exposed to F-UBO ( Figure 5F).

Annexin V-FITC Apoptosis Assay
The effects of F-UBO on normal blood cells and CLS-354 tumor cells based on morphology and cell membrane integrity are illustrated in Figure 6. Moreover, Figure 6 shows that F-UBO did not induce early apoptosis in both cell types, thus reporting considerable differences compared to C3UA in normal blood cells (0.00 ± 0.00 vs. 37.04 ± 0.66, p < 0.01), and both positive controls in CLS-354 tumor cells (0.00 ± 0.00 vs. C2P: 5.88 ± 1.24, p < 0.05 and C3UA: 12.92 ± 1.35, p < 0.01).

Cell Cycle Analysis
The effects of F-UBO on the cell cycle of normal blood cells and CLS-354 tumor cells, evidenced with PI/RNase stain, are displayed in Figure 9.

Evaluation of Cell Proliferation
The F-UBO effects on DNA synthesis in normal blood cells and CLS-354 tumor cells were also assessed by EdU incorporation, and the results are presented in Figure 11. Antioxidants 2022, 11, x FOR PEER REVIEW 26 of 36 In normal blood cells, F-UBO blocked DNA synthesis recording considerable differences from C1 negative control: 0.00 ± 0.00 vs. 10.36 ± 1.21; p < 0.01 ( Figure 11A,C,I,K). As a result of DNA content diminution, the cell cycle arrest in subG0/G1 phase corresponds to apoptotic cell fraction; these cells have less DNA than healthy ones due to DNA fragmentation [72]. However, F-UBO induced apoptotic cell fraction (subG0/G1) [73] had significantly lower values compared with 1% DMSO (0.84 ± 0.09 vs. 2.01 ± 0.20, p < 0.05) and higher ones compared to UA (0.84 ± 0.09 vs. 0.00 ± 0.00, p < 0.01).

Principal Component Analysis
The principal component analysis (PCA) was performed for F-UBO and controls (C1-DMSO, C2P407, and C3UA). It correlates the variable parameters determined in both cell types (normal blood cells and CLS-354 OSCC tumor cells) according to the correlation matrix and PCA-correlation circle from the Supplementary Materials. The results are displayed in Figure 12. In normal blood cells, F-UBO blocked DNA synthesis recording considerable differences from C1 negative control: 0.00 ± 0.00 vs. 10.36 ± 1.21; p < 0.01 ( Figure 11A,C,I,K). As a result of DNA content diminution, the cell cycle arrest in subG0/G1 phase corresponds to apoptotic cell fraction; these cells have less DNA than healthy ones due to DNA fragmentation [72]. However, F-UBO induced apoptotic cell fraction (subG0/G1) [73] had significantly lower values compared with 1% DMSO (0.84 ± 0.09 vs. 2.01 ± 0.20, p < 0.05) and higher ones compared to UA (0.84 ± 0.09 vs. 0.00 ± 0.00, p < 0.01).

Principal Component Analysis
The principal component analysis (PCA) was performed for F-UBO and controls (C1-DMSO, C2P407, and C3UA). It correlates the variable parameters determined in both cell types (normal blood cells and CLS-354 OSCC tumor cells) according to the correlation matrix and PCA-correlation circle from the Supplementary Materials. The results are displayed in Figure 12.
The two principal components explained 83.36% of total data variance, with 51.50% attributed to the first (PC1) and 31.85% to the second (PC2). PC1 was associated with C3UA, caspase-3/7 activity in normal blood cells and CLS-354 tumor cells, and ROS levels in CLS-354 tumor cells. At the same time, PC2 was related to F-UBO bioadhesive oral films, C1DMSO, and ROS levels in normal blood cells (Figure 12). Antioxidants 2022, 11, x FOR PEER REVIEW 28 of 36 The two principal components explained 83.36% of total data variance, with 51.50% attributed to the first (PC1) and 31.85% to the second (PC2). PC1 was associated with C3UA, caspase-3/7 activity in normal blood cells and CLS-354 tumor cells, and ROS levels in CLS-354 tumor cells. At the same time, PC2 was related to F-UBO bioadhesive oral films, C1DMSO, and ROS levels in normal blood cells ( Figure 12).
In CLS-354 tumor cells, caspase-3/7 activation is highly positively correlated with cell cycle arrest in G0/G1 phase (r = 0.800, p > 0.05), and low with ROS level (r = 0.513, p > 0.05). However, oxidative stress (expressed as ROS level) shows a high and moderate positive correlation with the most damaging processes in OSCC cells. It displays a high correlation with late apoptosis and nuclear condensation (r = 0.812 and 0.802, p > 0.05), and a moderate one with early apoptosis and autophagy (r = 0.739 and 0.733, p > 0.05).
Data analysis shows that F-UBO acts on CLS-354 cells, inducing the highest levels of nuclear condensation and autophagy compared to all controls. Moreover, nuclear condensation and autophagy triggered by F-UBO display higher levels in OSCC cells than in normal blood ones. F-UBO also causes the most elevated oxidative stress in normal blood cells compared to controls. However, F-UBO significantly diminishes the apoptotic cell fraction (subG0/G1) and autophagy (A) and slowly decreases the cell cycle arrest in G0/G1, triggered in normal cells by 1% DMSO.
Usnic acid, the main secondary metabolite of U. barbata lichens, induced the highest oxidative stress and caspase-3/7 activation in OSCC cells, leading to the most substantial cellular apoptosis. It highlights a significant protective effect on normal blood cells, appreciably diminishing caspase-3/7 activation, nuclear condensation, and autophagy determined by 1% DMSO, thus reducing apoptotic cell fraction (subG0/G1).
In the present study, 5% P407, the emulsifier used in the F-UBO formulation, was selected as a positive control. It significantly acts on OSCC cells, determining cellular apoptosis by triggering all mechanisms that lead to cancer cell death. Moreover, it induces the highest DNA synthesis compared to F-UBO and controls. In normal blood cells, it generates oxidative stress and caspase-3/7 activation after 24 h of treatment, but the cell viability is not significantly affected.
By correlating and interpreting these data, the places of F-UBO and controls (C1DMSO, C2P407, and C3UA) in the PCA-correlation biplot ( Figure 12) were justified, evidencing the corresponding processes triggered in CLS-354 cancer cells and normal blood cells.

Discussion
The previous UBO analysis measured the heavy metals content, quantified the active constituents (UA content = 0.915 ± 0.018 mg/g UBO), explored the antioxidant, cytotoxic, and rheological properties, and then proved its suitability for pharmaceutical formulation [26].
The use of HPMC in a 15% aqueous dispersion ensured the suitable film toughness, while 5% PEG 400 provided an elegant, glossy, smooth appearance and high flexibility. The film's homogeneity proved that the active ingredients were adequately incorporated into the polymer matrix, emulsifying the oil extract. All formulations led to a thin and uniform film, suitable characteristics for bioadhesive performance, and a comfortable administration.
The low variation in weight and thickness guarantees the efficiency of the formulation and applied method and provides a certain uniformity of content. The results obtained for film thickness agree with other developed studies on HPMC films [74]. Both types of formulations proved to have weight and thickness suitable for application to the oral mucosa [36,74,75].
Regarding the mechanical properties of both bioadhesive oral films (F-UBO and R), the differences between formulations are not substantial because they contain identical amounts of HPMC and PEG 400.
Thus, the film's flexibility is mandatory for easy handling and administration. It is induced by the plasticizer used in the formulation and the film-forming polymer [76]. Semalty M. et al. [77] proved that mixing HPMC with PEG 400 in 30% of the weight of the polymer leads to low folding endurance and that the optimal plasticizer is PEG, used in low concentrations. In the present study, using PEG 400 in a 5% concentration led to the excellent flexibility of both film types. The plasticizer reduces the film's rigidity by decreasing the intermolecular forces [78]. Still, it was proven that high amounts of plasticizer might diminish the film adhesive properties by over-hydration [79].
F-UBO contains the active ingredient emulsified in the polymer matrix, leading to its physical interruption and a suitable elasticity. The disruption of polymer molecular chains induces higher chain mobility, augmenting flexibility and diminishing rigidity. Maher et al. [80] proved the influence of the polymer type (including HPMC) on the film's tensile strength. It was also confirmed that the tensile strength increases with the filmforming polymer concentration. It was observed that 15% HPMC water dispersion conducts to the development of a strong matrix with a sufficiently dense network. The obtained results show that even if the film-forming agent and the plasticizer have the primary influence on the film's mechanical attributes, other factors, such as the active ingredient nature or concentration and its dispersion type, affect the bioadhesive film's strength. The data obtained in this study show that F-UBO's elongation and tensile strength are adequate to resist stress during handling [81].
The moisture content ensures the suitable film's mechanical properties. It influences the film's friability; however, both formulations (F-UBO and R) display good resistance. The moisture can be due to the solvent system used in the formulation or to the ingredients' hygroscopic properties, especially the plasticizer ones [82]. PEG 400 presents high hygroscopicity due to its hydrophilic hydroxyl groups interacting with water [83], providing more sites for interactions and leading to moisture retention in the films. HPMC also has hydrophilic hydroxypropyl substituents, but contains hydrophobic methoxyl groups and does not maintain excessive moisture [84]. Bioadhesive oral films must have a moderate moisture content to ensure their elasticity and protection from being brittle, dry, and easy to break [85], and F-UBO and R show suitable humidity.
Each ingredient influences the film's pH value in the formulation. The purpose is to properly select the components to obtain bioadhesive films with a surface pH close to the buccal one. F-UBO shows an approximately neutral pH on the surface, close to the oral cavity, ensuring good tolerability with no possible irritation of the buccal mucosa. In this work, both films have similar pH values, proving that the active ingredients do not modify the pH of the matrix system.
The formulation's disintegration time strongly depends on the polymer matrix. Shen et al. [86] demonstrated that the film's disintegration time rises directly proportional to HPMC concentration. The results show that F-UBO's rapid disintegration allows a fast release of active ingredients, suitable for in vitro studies.
The film's swelling properties are essential for bioadhesion [87] and highly depend on water diffusivity into the polymer [88]. The residence time [41,42] also depends on the film-forming polymer and the plasticizer's retention properties, being highly controlled by the ratio between them [89]. The disturbance of the polymer chains by including active ingredients in the matrix decreases the water content [90] and considerably influences the bioadhesive behavior. Adhesion is enhanced with increasing hydration until it reaches an optimum point. Overhydration causes the breaking of the polymer/tissue interface, thus decreasing the bioadhesive force. The values registered for ex vivo residence time are strongly related to the film's in vivo bioadhesive performance, and results were satisfactory for F-UBO.
Generally, the differences between UBO-loaded bioadhesive oral films and references are not statistically significant, proving that UBO does not considerably influence these previously mentioned properties.
On the other hand, P407, the emulsifier from the F-UBO formulation, is most known for its use as a surfactant in various oral hygiene products (dentifrices, mouthwashes, breath fresheners) in a concentration range of 0.3-20% [91]. Furthermore, it is particularly interesting in clinical use for surgical application due to its thermoreversible gelation and bactericidal effects on S. aureus [92]. Veyries et al. [93] revealed the potential of P407 for inhibiting the attachment of S. aureus and S. epidermidis and increasing their susceptibility to antibiotics once they are adherent. This study results show the significant antifungal potential of 5% P407 in water against C. albicans and C. parapsilosis.
Teanpaisan et al. [94] proposed a mixture of P407 and Artocarpus lakoocha (Moraceae) for endodontic treatment, proving its antibacterial activity against E. fecalis. Recently, another study proved antifungal activity of a hydroethanolic extract from Astronium urundeuva leaves loaded into a nanostructured lipid system with 0.5% P407 ® against C. albicans and C. glabrata [95]. Previous studies [96,97] included the most common oral cavity pathogens responsible for various opportunistic infections in immunocompromised patients, S. aureus, P. aeruginosa, and C. albicans. Our F-UBO oral biofilms revealed a dose-dependent inhibitory activity against S. aureus, P. aeruginosa, C. albicans, and C. parapsilosis.
The BSL cytotoxicity assay is one of the most known methods, using Artemia sp.-A. salina [26] and A. franciscana [98]. Together with phytotoxicity assay on Triticum aestivum L.-which evaluates the Triticum radicle growth [99], the BSL assay is considered a rapid, simple, low-cost, and effective test to estimate various natural products' safety for human use [100]. Okumu et al. [49] recently used A. salina as an animal model for the preliminary evaluation of snake-venom-induced toxicity. Nazir et al. [101] proved that the BSL assay is a significant antitumor prescreening in anticancer drug discovery. Rajabi et al. [102] described Artemia salina as a model organism in the toxicity assessment of nanoparticles. The present study used the BSL assay to evaluate the F-UBO cytotoxic potential, and the obtained results could be extrapolated to those on tumor cells.
Previous studies regarding the various poloxamer types' cytotoxicity on tumor cells reported a dose-dependent action. Thus, a concentration of 30 ± 10 mg/mL of poloxamer 188 inhibited 50% of HeLa (cervical cancer) cell growth, whereas a dose 10 times lower (2-5 mg/mL) was necessary to inhibit 50% of B-16 (mouse melanoma) cell growth [91]. In this study, 50 mg/mL P407 reduced the viability of CLS-354 (oral cancer) cells to 72.51 ± 2.51% after 24 h incubation. F-UBO dispersion led to 315 µg/mL UBO and 3.15 mg/mL P407; UBO penetrated rapidly through the cell wall in emulsified form, then the onset of apoptotic processes after 24 h of treatment could be explained. The F-UBO complex composition suggests a synergy between U. barbata secondary metabolites with pharmacological properties [103,104], canola oil's bioactive constituents [105], and P407 [106,107].

Conclusions
This study evaluated the pharmacological potential of the U. barbata (L.) Weber ex F.H. Wigg extract in canola oil (UBO) as an oral pharmaceutical formulation.
The UBO-loaded bioadhesive oral films were manufactured using P407, HPMC, and PEG 400 for their formulation. F-UBO suitability for topical administration on buccal mucosa was confirmed through complex physicochemical and pharmacotechnical analyses.
F-UBO antimicrobial and anticancer properties were investigated using P407 as a positive control. Data obtained revealed F-UBO and P407 dose-dependent inhibitory activity against the most common bacterial and fungal pathogens implied in immunosuppressed patients' oral infections. Moreover, they highlighted in vitro antitumor effects on oral epithelial squamous cell carcinoma (CLS-354 cell line).
The present research suggests that bioadhesive oral films with U. barbata extract in canola oil can be considered a phytotherapeutic formulation with potential applications against oral cavity infections and neoplasia. In vivo and clinical studies could be further steps in F-UBO analysis to confirm their medical benefits.